OCS Study 

MMS 2002-048 

 

Coastal Marine Institute 

Development of Bioremediation 

for Oil Spill Cleanup in Coastal Wetlands 

Cooperative Agreement

Coastal Marine Institute 

Louisiana State University 

U.S. Department of the Interior

Minerals Management Service 

Gulf of Mexico OCS Region 
U.S. Department of the Interior Cooperative Agreement 

Minerals Management Service Coastal Marine Institute 

Gulf of Mexico OCS Region Louisiana State University 

OCS Study 

 MMS 2002-048 

 

Coastal Marine Institute 

Development of Bioremediation 

for Oil Spill Cleanup in Coastal Wetlands 

Editors 

Irving A. Mendelssohn 

Qianxin Lin 

August 2003 

Prepared under MMS Contract 

14-35-0001-30660-19909 

by 

Louisiana State University 

Wetland Biogeochemistry Institute 

Center for Coastal, Energy and Environmental Resources 

Baton Rouge, Louisiana 70803 

Published by 
iii 

DISCLAIMER 

This report was prepared under contract between the Minerals Management Service (MMS) and Louisiana 

State University. This report has been technically reviewed by the MMS and approved for publication. Approval 

does not signify that the contents necessarily reflect the views or policies of the Service, nor does mention of trade 

names or commercial products constitute endorsement or recommendation for use. It is, however, exempt from 

review and compliance with MMS editorial standards. 

REPORT AVAILABILITY 

Extra copies of this report may be obtained from the Public Information Office (MS 5034) at the following address: 

U.S. Department of the Interior 

Minerals Management Service 

Gulf of Mexico OCS Region 

Public Information Office (MS 5034) 

1201 Elmwood Park Boulevard 

New Orleans, Louisiana 70123-2394 

Telephone Number: (504) 736-2519 

 1-800-200-GULF 

CITATION 

Suggested citation: 

Mendelssohn, I.A. and L. Oianxin (ed.). 2003. The development of bioremediation for oil spill cleanup in coastal 

wetlands. U.S. Dept. of the Interior, Minerals Management Service, Gulf of Mexico OCS Region, New 

Orleans, LA. OCS Study MMS 2002-048. 84 pp. 
v 

CONTRIBUTING AUTHORS 

Irving A. Mendelssohn, Editor Qianzin Lin, Co-editor 

Wetland Biogeochemistry Institute Wetland Biogeochemistry Institute 

School of the Coast and Environment School of the Coast and Environment 

Louisiana State University Louisiana State University 

Nazan Atilla Charles B. Henry, Jr. 

Louisiana Universities Marine Consortium Institute for Environmental Studies 

8124 Highway 56 School of the Coast and 

Environment 

Chauvin, Louisiana Louisiana State University 

Karolien Debusschere Edward B. Overton 

Coastal Environments, Inc. Institute for Environmental Studies 

1260 Main Street School of the Coast and Environment 

Baton Rouge, Louisiana Louisiana State University 

M. Scott Miles Nancy N. Rabalais 

Institute for Environmental Studies Louisiana Universities Marine Consortium 

School of the Coast and Environment 8124 Highway 56 

Louisiana State University Chauvin, Louisiana 

Ralph J. Portier Maud M. Walsh 

Institute for Environmental Studies Institute for Environmental Studies 

School of the Coast and Environment School of the Coast and Environment 

Louisiana State University Louisiana State University 

Pauline O. Roberts 

Institute for Environmental Studies 

School of the Coast and Environment 

Louisiana State University 

ACKNOWLEDGMENTS 

This project was supported by Minerals Management Service (MMS), US Department of the Interior 

(Contract No. 1435-0001-30660 /19909) and Exxon Company, USA. We thank Michele Young of Baton Rouge 

Refinery of Exxon for the Louisiana crude oil; Randy Drake of Polybac for Petrobac; Brain Birrenkott of Grace 

Sierra for Osmocote; FMC Corp. for PermeOx; and Paul Frederick of Exxon and Pat Roscigno of MMS for their 

invaluable counsel throughout this research. 
vii 

TABLE OF CONTENTS 

Page 

FIGURES .....................................................................................................................................................................xi

TABLES.....................................................................................................................................................................xiii

EXECUTIVE SUMMARY .........................................................................................................................................xv

INTRODUCTION.........................................................................................................................................................1

Overall Goal and Objectives ..................................................................................................................................2

Project Organization...............................................................................................................................................2

CHAPTER 1. AN INVESTIGATION OF THE POTENTIAL TOXICITY OF BIOREMEDIATION AGENTS 

IN WETLAND MESOCOSMS.............................................................................................................3

Materials and Methods ...........................................................................................................................................4

Experimental Design.......................................................................................................................................4

Experimental Procedures.................................................................................................................................4

Methods...........................................................................................................................................................5

Plant Response .........................................................................................................................................5

Photosynthetic Rate. .........................................................................................................................5

Plant Stem Density............................................................................................................................5

Average and Maximum Shoot Height. .............................................................................................5

Aboveground Biomass of Dominant Marsh Plants...........................................................................5

Soil Response...........................................................................................................................................5

Soil Respiration Rate. .......................................................................................................................5

Soil Redox Potential .........................................................................................................................5

Soil Nutrient Concentrations.............................................................................................................5

Interstitial pH and Salinity. ...............................................................................................................5

Microbial Response..................................................................................................................................6

Infaunal Response....................................................................................................................................6

Macroinfauna. ...................................................................................................................................6

Meiofauna. ........................................................................................................................................6 

Residual Oil Chemistry in the Soil...........................................................................................................7 

Results.............................................................................................................................................................7

Responses of Spartina alterniflora ..........................................................................................................7

Responses of Microbial Community......................................................................................................11

Responses of Infauna to the Treatments ................................................................................................17

Macrofauna. ....................................................................................................................................17

Meiofauna. ......................................................................................................................................17

Copepod Taxa ........................................................................................................................................18

Oil Chemistry..................................................................................................................................18

Normal Hydrocarbon (alkane) Profile. ...........................................................................................18

Composition of Selected Aromatic Hydrocarbons. ........................................................................21

Discussion ............................................................................................................................................................23

Summary ..............................................................................................................................................................24

CHAPTER 2. THE EFFECT OF BIOREMEDIATION AGENTS ON CRUDE OIL DEGRADATION AND 

BIOTIC RESPONSE UNDER DRAINED AND FLOODED CONDITIONS IN MARSH 

MESOCOSMS ....................................................................................................................................27

Materials and Methods .........................................................................................................................................27

Experimental Design.....................................................................................................................................27

Experimental Procedures...............................................................................................................................28

Methods.........................................................................................................................................................28

Plant Response .......................................................................................................................................28

Photosynthetic Rate. .......................................................................................................................28

Plant Stem Density..........................................................................................................................28
 viii

Average and Maximum Shoot Height. ..........................................................................................29

Aboveground Biomass of Dominant Marsh Plants.........................................................................29

Soil Response.........................................................................................................................................29

Soil Respiration Rate. .....................................................................................................................29

Soil Redox Potential. ......................................................................................................................29

Soil Nutrient Concentrations...........................................................................................................29

Interstitial pH and Salinity. .............................................................................................................29

Microbial Response................................................................................................................................29

Residual Oil Chemistry in the Soil.........................................................................................................30

Results...........................................................................................................................................................30

Responses of S. alterniflora to the Treatments ......................................................................................30

Responses of Microbes to the Treatments..............................................................................................35

Oil Morphology.............................................................................................................................................36

Efficacy of the Bioremediation Products ...............................................................................................36

Oil Chemistry.........................................................................................................................................41

Alkane Degradation. .......................................................................................................................41

Aromatic Hydrocarbon Degradation...............................................................................................41

Discussion ............................................................................................................................................................43

Summary ..............................................................................................................................................................44

CHAPTER 3 EFFECTS OF BIOREMEDIATION AGENTS ON OIL DEGRADATION ON MINERAL 

AND SANDY SALT MARSH SEDIMENTS ....................................................................................47

Materials and Methods .........................................................................................................................................47

Marsh Sod .....................................................................................................................................................47

Experimental Design.....................................................................................................................................48

Experimental Procedures...............................................................................................................................48

Plant and Soil Responses .......................................................................................................................48

Aboveground Biomass of Dominant Marsh Plants.........................................................................48

Soil Respiration Rate. .....................................................................................................................48

Soil Redox Potential. ......................................................................................................................49

Soil Nutrient Concentrations...........................................................................................................49

Microbial Populations.....................................................................................................................49

Residual Oil Chemistry in the Soil..................................................................................................49

Results ..................................................................................................................................................................49

Plant Growth Response .................................................................................................................................49

Microbial Response.......................................................................................................................................50

Soil Fertility ..................................................................................................................................................52

Oil Biodegradation ........................................................................................................................................52

Discussion ............................................................................................................................................................54

Summary ..............................................................................................................................................................57

CHAPTER 4 A COMPARISON OF BIOSTIMULATION AND PHYTOREMEDIATION OF CRUDE OIL 

IN WETLAND MESOCOSMS...........................................................................................................59

Materials and Methods .........................................................................................................................................59

Experimental Design.....................................................................................................................................59

Experimental Procedures...............................................................................................................................60

Methods.........................................................................................................................................................60

Plant Response .......................................................................................................................................60

Photosynthetic Rate ........................................................................................................................60 

Plant Stem Density..........................................................................................................................60 

Average and Maximum Shoot Height. ...........................................................................................60

Aboveground Biomass....................................................................................................................60

Belowground Biomass of the Transplant........................................................................................61

Water Evapo-Transpirational Rate..................................................................................................61

Soil Response.........................................................................................................................................61

Soil Respiration Rate. .....................................................................................................................61

Soil Redox Potential. ......................................................................................................................61
 ix

Soil Nutrient Concentrations...........................................................................................................61

Oil-Degrading Microbes. ................................................................................................................61

Residual Oil Chemistry in the Soil..................................................................................................61

Results...........................................................................................................................................................62

Responses of S. alterniflora to the Treatments ......................................................................................62

Responses of Soil Variables...................................................................................................................65

Oil Chemistry ................................................................................................................................................69

Alkane Degradation ...............................................................................................................................69

Aromatic Hydrocarbon Degradation......................................................................................................69

Discussion ............................................................................................................................................................70

Summary ..............................................................................................................................................................72

CHAPTER 5 PROJECT CONCLUSIONS ...............................................................................................................73

REFERENCES............................................................................................................................................................75

APPENDIX A. EFFECTS OF BIOREMEDIATION ON MICROBIAL BIOMASS .............................................83

APPENDIX B OIL MORPHOLOGY DEFINITIONS...........................................................................................87
xi 

FIGURES 

Page 

Figure 1. Effect of bioremediation agents on leaf elongation rate of Spartina alterniflora. ...................................8

Figure 2. Effect of bioremediation agents on photosynthetic rate of Spartina alterniflora. ...................................9

Figure 3. Effect of bioremediation agents on the relative growth based on stem density (A) and cumulative 

shoot height (B) of Spartina alterniflora. ................................................................................................9

Figure 4. Effect of bioremediation agents on aboveground biomass of Spartina alterniflora..............................10

Figure 5. Effect of bioremediation agents on interstitial pH. ................................................................................11

Figure 6. Effect of bioremediation agents on soil redox potential.........................................................................11

Figure 7. Effect of bioremediation agents on soil respiration rate.........................................................................12

Figure 8. Effect of bioremediation agents and oil application on heterotrophic bacteria 28 days after 

treatment.................................................................................................................................................13

Figure 9. Effect of bioremediation agents and oil application on petroleum-degrading bacteria 28 days after 

treatment.................................................................................................................................................13

Figure 10. Effect of bioremediation agents and oil application on chitin-degrading bacteria 28 days after 

treatment.................................................................................................................................................14

Figure 11. Effect of bioremediation agents and oil application on cellulose-degrading actinomycetes and 

bacteria 28 days after treatment. ............................................................................................................14

Figure 12. Effect of bioremediation agents and oil application on yeast populations 28 days after treatment. ......15

Figure 13. Effect of bioremediation agents and oil application on filamentous fungi 28 days after treatment.......15

Figure 14. Effect of bioremediation agents and oil application on microbial biomass 28 days after treatment......16

Figure 15. Effect of bioremediation agents and oil application on 15-minute readings of microtox analysis 90 

days after treatment................................................................................................................................17

Figure 16. Chromatographic comparison of unweathered south Louisiana crude oil to the laboratory 

weathered south Louisiana crude oil......................................................................................................19

Figure 17. Chromatographic comparison of the reduced crude oil used in this experiment to the laboratory 

weathered crude oil. ...............................................................................................................................20

Figure 18. Chromatographic comparison of the normal hydrocarbon profile in the oiled control plot, C+, 

compared to the unoiled control plot, C-, at Day 1. ...............................................................................21

Figure 19. Aromatic hydrocarbon profile comparison between the reduced crude oil and the mean C+ Day 1 

values. ....................................................................................................................................................22

Figure 20. Aromatic hydrocarbon profile comparison between the mean C+ Day 1 values and the C-, control....23

Figure 21. Effect of bioremediation agents on leaf elongation of Spartina alterniflora .........................................30

Figure 22. Effect of bioremediation agents on photosynthetic rate of Spartina alterniflora. .................................31

Figure 23. Effect of bioremediation agents on stem growth rate (A) and cumulative shoot growth rate (B) of

Spartina alterniflora.. ............................................................................................................................31

Figure 24. Effect of bioremediation agents on aboveground biomass of Spartina alterniflora..............................32

Figure 25. Effect of inundation regime on soil redox potential at the depth of 2 cm (A) and 12 cm (B) below 

the soil surface. ......................................................................................................................................33

Figure 26. Effect of bioremediation agents on interstitial pH of Spartina alterniflora...........................................34

Figure 27. Effect of bioremediation agents on interstitial inorganic nitrogen (A) and phosphorus (B)..................34

Figure 28. Effect of bioremediation agents on soil respiration rate 2 (A), 4 (B), 8 (C), and 12 (D) weeks after 

the bioremediation treatment..................................................................................................................35

Figure 29. Effect of bioremediation agents on heterotrophic bacteria 6 hours after the bioremediation 

treatment.................................................................................................................................................36

Figure 30. Effect of bioremediation agents on petroleum-degrading bacteria 6 hours after the bioremediation 

treatment.................................................................................................................................................37

Figure 31. Effect of bioremediation agents on heterotrophic bacteria 3 weeks after the bioremediation 

treatment.................................................................................................................................................37

Figure 32. Effect of bioremediation agents on petroleum-degrading bacteria 3 weeks after the bioremediation 

treatment.................................................................................................................................................37

Figure 33. Effect of bioremediation agents on heterotrophic bacteria 12 weeks after the bioremediation 

treatment.................................................................................................................................................38

Figure 34. Effect of bioremediation agents on petroleum-degrading bacteria 12 weeks after the 

bioremediation treatment........................................................................................................................38

Figure 35. Effect of bioremediation agents on microbial biomass 3 weeks after the bioremediation treatment.....39
 xii

FIGURES 

(continued) 

Figure 36. Effect of bioremediation agents on microbial biomass 12 weeks after the bioremediation 

treatment.................................................................................................................................................39

Figure 37. Effect of bioremediation agents on alkane degradation 3 and 5 weeks after the bioremediation 

treatment.................................................................................................................................................41

Figure 38. Effect of bioremediation agents on the residual total targeted aromatic hydrocarbon concentrations 

3, 5, and 12 weeks after the bioremediation treatment...........................................................................42

Figure 39. Effect of bioremediation agents on the residual total targeted aromatic hydrocarbon normalized to 

hopane 3, 5, and 12 weeks after the bioremediation treatment. .............................................................42

Figure 40. The effect of bioremediation products on aboveground biomass of Spartina alterniflora, averaged 

over marsh type, after a 4-month experimental period. .........................................................................50

Figure 41. Soil microbial respiration responses, averaged over marsh type, to bioremediation product 

treatments 7 weeks after application. .....................................................................................................51

Figure 42. Effect of the bioremediation products on total targeted normal hydrocarbons......................................53

Figure 43. Total targeted aromatic hydrocarbons (TTAH) remaining in the soil, averaged over marsh type, as 

a function of the bioremediation product treatments..............................................................................53

Figure 44. Effect of nitrogen and phosphorus application on photosynthetic rate of Spartina alterniflora six 

months after the treatments. ...................................................................................................................62

Figure 45. Effect of nitrogen and phosphorus application on stem density of Spartina alterniflora six months 

after the treatments.................................................................................................................................62

Figure 46. Effect of nitrogen and phosphorus application on canopy height of Spartina alterniflora six 

months after the treatments. ...................................................................................................................63

Figure 47. Effect of nitrogen and phosphorus application on aboveground biomass of Spartina alterniflora

six months after the treatments...............................................................................................................63

Figure 48. Effect of nitrogen and phosphorus application on belowground biomass of Spartina alterniflora

six months after the treatments...............................................................................................................64

Figure 49. Effect of phytoremediation and nitrogen and phosphorus application on evapo-transpiration rate 

six months after the treatments...............................................................................................................65

Figure 50. Effect of phytoremediation and nitrogen and phosphorus application on soil redox potential at 3 

cm below the soil surface six months after the treatments.....................................................................66

Figure 51. Effect of phytoremediation and nitrogen and phosphorus application on soil redox potential at 12 

cm below the soil surface six months after the treatments.....................................................................66

Figure 52. Effect of phytoremediation and nitrogen and phosphorus application on interstitial inorganic 

nitrogen six months after the treatments. ...............................................................................................67

Figure 53. Effect of phytoremediation and nitrogen and phosphorus application on interstitial phosphate 

concentration six months after the treatments........................................................................................67

Figure 54. Effect of phytoremediation and nitrogen and phosphorus application on soil respiration rate six 

months after the treatments. ...................................................................................................................68

Figure 55. Effect of phytoremediation and nitrogen and phosphorus application on oil-degrading microbial 

populations six months after the treatments...........................................................................................68

Figure 56. Effect of phytoremediation and nitrogen and phosphorus application on total targeted normal 

hydrocarbons (TTNH) six months after the treatments..........................................................................69

Figure 57. Effect of phytoremediation and nitrogen and phosphorus application on total targeted aromatic 

hydrocarbons (TTNH) six months after the treatments..........................................................................70

Figure A1. Effect of bioremediation agents and oil application on heterotrophic bacteria three days after the 

treatment.................................................................................................................................................83

Figure A2. Effect of bioremediation agents and oil on petroleum-degrading bacteria three days after 

treatment.................................................................................................................................................83

Figure A3. Effect of bioremediation agents and oil on chitin-degrading bacteria 3 days after treatment................83

Figure A4. Effect of bioremediation agents and oil on cellulose-degrading actinomycetes and bacteria 3 days 

after treatment. .......................................................................................................................................84

Figure A5. Effect of bioremediation agents and oil on yeast populations 3 days after treatment. ...........................84

Figure A6. Effect of bioremediation agents and oil on filamentous fungi 3 days after treatment............................85

Figure A7. Effect of bioremediation agents and oil on microbial biomass three days after treatment.....................85
xiii 

TABLES 

Page 

Table 1. Targeted Compounds Assessed by GC/MS .............................................................................................8

Table 2. Statistical Table with P-Value of Response of Marsh Plant and Soil Variables to Bioremediation 

Agent and Oil Application .....................................................................................................................10

Table 3. Statistical Table with P-Value for Response of Microbial Populations to Bioremediation Agent 

and Oil Application................................................................................................................................16

Table 4. Summary of Multiple Analysis of Variance on Four Taxa of Meiofauna and Nematode: Copepod 

Ratio with P-Values for Main Factors and Their Interactions ...............................................................18

Table 5. Statistical Table for Plant and Soil Variables with P-Values Provided for Main Factors and Their 

Interactions.............................................................................................................................................32

Table 6. Relative Degree of Degradation for Each of the Observations by Inundation Regime and Product 

Application (*) Estimated by Oil Morphology. .....................................................................................40

Table 7. Relative Degree of Degradation for Each of the Observations Periods and Total Score of 

Degradation-Effectiveness by Product-Application (**).......................................................................40

Table 8. Statistical Table for the Oil Chemistry Variables with P-Values Provided for Main Factors and 

Their Interactions ...................................................................................................................................43

Table 9. Target Compounds Assessed by GC/MS...............................................................................................50

Table 10. Effect of Bioremediation Products on Soil Heterotrophic Populations and Petroleum Degraders 

After a 4-Month Experimental Period....................................................................................................51

Table 11. Effect of Bioremediation Products on the Inorganic Interstitial Nutrient Concentrations Before 

and 2 Weeks After the Third Product Application.................................................................................52

Table 12. Effect of Bioremediation Product Application on Relative Oil Degradation, the Percentage 

Decrease of Petroleum Components Normalized to Each Soil Type's Control (Control 100%). ..........54

Table 13. Statistical Table for the Transplant Variables with P-Values Provided for Main Factors and Their 

Interactions.............................................................................................................................................64

Table 14. Statistical Table for the Soil Variables and Residual Oil Concentration with P-Values Provided for 

Main Factors and Their Interactions ......................................................................................................65
xv 

EXECUTIVE SUMMARY 

 Although bioremediation for oil spill cleanup has received considerable attention in recent years, its 

satisfactory use in the cleanup of oil spills in the wetland environment is still questionable and generally untested. 

We have conducted a multi-disciplinary experimental program to evaluate the use of various bioremediation 

products, including microbial seeding, inorganic fertilizer, and soil oxidant, as a means of enhancing oil 

biodegradation in coastal salt marshes. The overall goal was to determine the potential for oil bioremediation in 

coastal marshes. The specific objectives of this research were to determine (1) the toxicity and ecological safety of 

some common biodegradation agents, (2) the effect of these bioremediation agents on crude oil degradation under 

the most common marsh inundation environments, (3) the effect of biostimulants on crude oil degradation as a 

function of soil texture, and (4) the comparative efficacy of bioremediation and phytoremediation of oil. Chapters 1 

through 4 address the above objectives. 

 The experiment described in Chapter 1 was designed to determine the toxicity and ecological safety of 

common biodegradation agents. In the greenhouse, the following bioremediation treatments were applied to salt 

marsh sods with intact Spartina alterniflora: (1) fertilizer, (2) microbial seeding, and (3) no bioremediation agent 

addition (control). The experimental design was a randomized block with a 3 x 2 factorial treatment arrangement 

(the three bioremediation types mentioned above and two oil dosage levels (oiled with 1 L/m2

 and no oil addition)). 

The results of experiment 1 indicated that all bioremediation agents used in this research, including microbial 

seeding and inorganic fertilizer, were not toxic to plants, microbes, and infauna animals in a salt marsh. The 

bioremediation agents did not adversely impact the dominant marsh macrophyte, Spartina alterniflora. Microbial 

seeding did not significantly affect the photosynthetic rate, plant stem density, leaf elongation rate, and aboveground 

biomass of S. alterniflora. However, inorganic fertilizer significantly enhanced these variables. Oil application had 

no significant effect on plant growth. Furthermore, bioremediation agents did not adversely affect various microbial 

populations, such as microbial heterotrophic populations, fungi, petroleum-utilizing populations, and overall 

microbial biomass. However, fertilization significantly increased soil respiration, but microbial seeding had no 

significant effect on soil respiration. Bioremediation agents did not adversely affect various infauna animals such as 

macrofauna and meiofauna. There were no significant differences in bioremediation agent treatments for either 

number of individuals or number of species for macrofauna and meiofauna. Gas chromatography-mass 

spectrometry indicated that both reduced crude oil used in this experiment and laboratory weathered crude oil 

appear to be acceptable since they contain a wide range of middle distillate normal hydrocarbons. Overall, the 

bioremediation agents used in this experiment are safe to marsh communities. 

 Experiment 2, which is described in Chapter 2, was designed to determine the effect of bioremediation 

agents on oil degradation and biotic response under drained and flooded conditions in marsh mesocosms. In the 

greenhouse, the following bioremediation treatments were applied to salt marsh sods with intact Spartina 

alterniflora: (1) fertilizer, (2) microbial seeding, (3) oxidant plus fertilizer, and (4) no bioremediation agent 

addition (control). The experimental design was a randomized block with a 4 x 2 factorial treatment arrangement. 

The four bioremediation types mentioned above and two inundation regimes (flooded with 3 cm of standing water 

and drained with the water table 10 cm below the soil surface) were included in this experiment. Reduced crude oil 

was applied to the surface at the rate of 2 L/m2

. The bioremediation agents were applied to the marsh sod surface. 

Fertilizer application enhanced plant growth with significantly higher photosynthetic rates, stem growth rates, and 

aboveground biomass of S. alterniflora, but microbial seeding application did not affect these variables compared to 

the control. Oil application and inundation regime did not have a differential effect on S. alterniflora. 

Bioremediation with a soil oxidant combined with fertilizer significantly increased interstitial phosphorus 

concentration, soil respiration, soil heterotrophic microbial populations, oil-degrading microbial populations, and 

degradation rate of alkane and aromatic hydrocarbons, especially in the drained conditions. However, the role of the 

soil oxidant needs further study because the soil oxidant treatment also contained extra KH2PO4 to buffer a high pH 

value created by the oxidant. Fertilizer application also significantly increased plant and soil variables and oil 

degradation. Microbial seeding did not significantly affect soil microbes or oil degradation. Oil degradation was 

greater in the drained condition than in the flooded condition. 

 Chapter 3 compares three major categories of bioremediation agents, fertilizer, microbial seeding and soil 

oxidant, as a means of enhancing oil biodegradation in coastal mineral and sandy marsh substrates under controlled 

greenhouse conditions. Artificially weathered south Louisiana crude oil was applied to sods of marsh (soil and 

vegetation intact) at the rate of 2 L/m2

. Four months after the bioremediation treatment, fertilizer application 
 xvi

enhanced marsh plant growth, soil microbial populations, and oil biodegradation rate. Live aboveground biomass 

of S. alterniflora with fertilizer application was higher than that without fertilizer. Fertilizer significantly increased 

soil microbial respiration rates and the population of heterotrophic bacteria, indicating an enhanced oil 

biodegradation potential. Bioremediation with fertilizer significantly reduced total targeted normal hydrocarbons 

(TTNH) and total targeted aromatic hydrocarbons (TTAH) remaining in the soil, with concentrations of 82% and 

48%, respectively, lower than those in the treatments without fertilizer. TTNH/hopane and TTAH/hopane showed 

similar scales of reduction, further demonstrating the enhancement of oil biodegradation by fertilization. However, 

soil type did not affect oil bioremediation; the extent of fertilizer-enhanced oil biodegradation was similar for sandy 

and mineral marsh types. Furthermore, microbial seeding and soil oxidant application had no positive effects on the 

variables mentioned above under the present experimental conditions. These results support the conclusion that 

bioremediation with inorganic nutrient additions is a promising methodology for promoting oil spill cleanup in 

coastal wetlands. 

 Chapter 4 compares the effect of phytoremediation and bioremediation on oil degradation. In the 

greenhouse, the following treatments were applied to the experimental units: (1) phytoremediation with Spartina 

alterniflora or without, (2) three nitrogen levels, and (3) two phosphorus level. The experimental design was a 

randomized block with a 2 x 3 x 2 factorial treatment arrangement. Six months after the treatments, both nitrogen 

and phosphorus application significantly enhanced plant growth with higher photosynthetic rate, stem density, and 

above- and below-ground biomass. Phytoremediation by the salt marsh plant S. alterniflora significantly increased 

soil redox potential, indicating a more oxidized soil environment that may enhance aerobic oil degradation. 

Phytoremediation by S. alterniflora enhanced oil degradation. Concentrations of residual alkane (TTNH) were 

significantly lower in the treatment receiving S. alterniflora than without phytoremediation. In addition, 

concentrations of residual aromatic hydrocarbons (TTAH) were significantly lower in the treatment receiving S. 

alterniflora compared to the treatment without S. alterniflora, and were lowest in the treatment receiving S. 

alterniflora and high doses of nitrogen. Thus, nitrogen addition enhanced the efficacy of phytoremediation on the 

degradation of aromatic hydrocarbons. However, bioremediation by nitrogen and phosphorus additions did not 

significantly affect residual oil concentration compared to no fertilizer additions in the absence of S. alterniflora. 

Concentrations of residual alkane and aromatic hydrocarbons were not significantly different between nitrogen and 

phosphorus treatments when S. alterniflora was absent in the experimental units.
1 

INTRODUCTION 

 The northern Gulf Coast of the United States is a region of intense oil exploration, production, 

transmission, and refining. Consequently, coastal states, such as Louisiana, are subject to oil spills resulting from 

shipping accidents, production-related incidents, and pipeline ruptures. Since these incidents often occur in the 

nearshore environment, coastal salt marshes are frequently the first wetland habitat to be subjected to the oil. As a 

result, a large number of investigations have documented the effect of petroleum hydrocarbon spills on the 

dominant salt marsh plant species, especially Spartina alterniflora (Hershner and Lake 1980; Lee et al. 1981; 

Alexander and Webb 1983; Ferrell et al. 1984; Mendelssohn et al. 1990; Lin and Mendelssohn 1996 and others). In 

addition, some investigators (DeLaune et al. 1984; Mendelssohn et al. 1993; Lin et al. 1999a) have evaluated the 

impact of oil cleanup procedures in salt marshes. Not only can petroleum hydrocarbons have a detrimental impact 

on coastal marshes, but, additionally, the cleanup of the oil from these highly sensitive environments is often more 

damaging than the oil itself. Hence, it is important to develop less intrusive oil spill cleanup procedures that exert 

little to no impact on wetland ecosystems. 

 Bioremediation is the act of adding materials to contaminated environments, such as oil spill sites, to cause 

an acceleration of the natural biodegradation process (U.S. Congress 1991). It is a promising means by which oil 

released into salt marshes, as well as other wetland types, can be removed with little impact to the habitat. Bacteria, 

cultured and selected for high rates of oil degradation, and fertilizers, which enhance native microbial activity, are 

two types of bioremediation products that can be added to oil-contaminated wetlands. 

 Inorganic fertilizer, which enhances native microbial activity, is one of the most common bioremediation 

agents applied to oil contaminated wetlands. A number of studies have demonstrated the potential for enhanced oil 

degradation as a result of bioremediation, especially through nutrient additions (Lee and Levy 1987; Tabak et al. 

1991; Safferman 1991; Lee and Levy 1991; Bragg et al. 1993; Lee et al. 1993). Specifically for wetlands, Scherrer 

and Mille (1990) confirmed enhanced degradation of oil in a West Indies mangrove swamp after the addition of an 

oleophilic fertilizer. Similarly, Lee and Levy (1991) found enhanced degradation of oil, this time in salt marsh 

sediments, treated with inorganic nutrients. However, critical evaluations of oil bioremediation potential in wetland 

environments, based on oil chemical analyses that can unequivocally identify enhanced biodegradation, is sparse in 

the published literature. 

 Microbial seeding as a means of enhancing oil biodegradation, has even greater uncertainties associated 

with it, especially in systems such as wetlands where hydrocarbon degrading bacteria are naturally prevalent. For 

example, microbial seeding was used in an experimental mode to test its effectiveness in cleaning up an oil spill in a 

marsh (Marrow Marsh) in Galveston Bay. The reported results did not indicate that the microbial seeding 

significantly degraded oil at this marsh site (Mearns 1991). In a more recent investigation (Venosa et al. 1992), two 

microbial products, which exhibited enhanced biodegradation of Alaska North Slope crude oil in shaker flask tests, 

did not accelerate biodegradation in a field experiment conducted on an oiled beach in Prince William Sound. The 

high variability in the data, the highly weathered nature of the oil, and a lack of sufficient time for biodegradation 

were cited as possible reasons for the lack of response. Regardless of these equivocal results, many microbial 

products have been commercialized. If added microbes, per se, are not effective in increasing oil degradation, the 

high costs of microbial amendments may not be warranted. Oil response agencies, both public and private, require a 

critical evaluation of microbial seeding in enhancing oil biodegradation. 

 Soil oxidation status is another important factor influencing oil biodegradation in wetland environments. 

Generally, wetland soils are saturated with water and exhibit biochemically reduced soil conditions, which may 

limit oil degradation (Hambrick et al. 1980). Therefore, procedures that increase the oxidation status of sediments 

may favor bioremediation (Lin and Mendelssohn 1997). The use of soil oxidants to increase oil biodegradation in 

the wetland environment has received little attention (McKee and Mendelssohn 1995). 

 Finally, the ecological impacts of application of these bioremediation agents to wetland environments, if 

any, must be identified before a large scale bioremediation is conducted. 

 This report describes the results from a multi-disciplinary, multi-investigator research program initiated to 

address the question: Is bioremediation, via fertilization, microbial seeding or soil oxidant, an effective and 

ecologically safe means of oil spill cleanup in coastal wetlands?
 2

Overall Goal and Objectives 

 The overall goal of the research was to determine the potential use of bioremediation as an oil-spill cleanup 

technique in wetlands. The specific objectives of this study were to 

(1) determine the potential toxicity and ecological impacts, if any, of bioremediation agents on wetlands, 

(2) determine the effectiveness of bioremediation by application of fertilizer, microbial seeding, or soil 

oxidant, singly or in combination, on oil degradation, 

(3) determine the bioremediation effectiveness under different marsh inundation conditions, 

(4) determine the effectiveness of bioremediation of oil under different marsh types, 

(5) identify the role of nitrogen or phosphorus, singly or in combination, in bioremediation of oil, and 

(6) separate the effectiveness of bioremediation and phytoremediation of oil. 

To accomplish the preceding goal and objectives, we divided the project into four separate experiments as reported 

in each chapter. 

Project Organization 

 The effectiveness of bioremediation and its ecological safety were assessed in the experiments described 

below by evaluating the following: (1) petroleum hydrocarbon chemistry to identify and quantify the degree of oil 

biodegradation, (2) oil morphology, which is related to oil chemistry, as an inexpensive means of evaluating oil 

biodegradation, (3) soil microbial response to determine the effect of the bioremediation products on the microbial 

communities that are performing the oil biodegradation, (4) soil chemistry to determine the effect of the 

bioremediation products on those factors that limit the growth of microbes and plants (e.g. nutrients, soil reducing 

conditions and soil toxins), (5) plant response to evaluate the combined effects of the oil and products on plant 

components of the marsh system, and (6) infaunal response to evaluate the combined effects of the oil and products 

on animal components of the marsh system. 

Chapter 1 

The first experiment was designed to determine the potential toxicity and ecological impacts, if any, of 

bioremediation agents on wetland plants, infaunal animals, and microbial communities. This experiment is required 

to ensure that the product loading rates suggested by the manufacturer are not toxic to wetland plants and estuarine 

animals. Only products on the National Contingency Plan (NCP) list, with defined maximum loading rates, were 

used in this study. 

Chapter 2 

The second experiment was designed (a) to evaluate the effectiveness of bioremediation by application of 

fertilizer, microbial seeding, or soil oxidant on oil degradation and (b) to determine the bioremediation effectiveness 

under different marsh inundation conditions. Since large-scale field demonstration projects are exceedingly 

expensive to initiate and complete, it is prudent to first evaluate bioremediation in smaller and less complicated 

greenhouse marsh-mesocosm experiments before scaling-up to field demonstration trials. 

Chapter 3 

The third experiment was designed (a) to determine the effectiveness of bioremediation by application of 

fertilizer, microbial seeding, and soil oxidant, singly and in combination, on oil degradation and (b) to determine the 

effectiveness of bioremediation of oil for different marsh types. Salt marsh soils, depending on their texture and 

specific microbial communities, may exhibit different capacities for bioremediation, which must be quantified to 

access the variability in bioremediation potential of salt marshes. 

Chapter 4 

 The fourth experiment was designed (a) to identify the role of nitrogen and phosphorus, singly and in 

combination, in bioremediation of oil and (b) to identify the relative effectiveness of bioremediation and 

phytoremediation of oil. 
3 

CHAPTER 1 

AN INVESTIGATION OF THE POTENTIAL TOXICITY 

OF BIOREMEDIATION AGENTS IN WETLAND MESOCOSMS 

by Irving A. Mendelssohn, Qianxin Lin, and Charles B. Henry, Edward B. Overton, 

 Ralph J. Portier, Nazan Atilla, Nancy N. Rabalais, Pauline O. Roberts, and Maud M. Walsh 

 Efficacy and success of bioremediation of oil by adding materials to contaminated environments to 

accelerate the biodegradation process depend on the extent of contaminant removal and on the ecological safety of 

the bioremediation agents. Thus, bioremediation agents for oil spill cleanup must not negatively impact wetland 

structure and function. Generally, common bioremediation agents such as inorganic fertilizer and selected 

microbial agents are relatively safe to environments. Microbial inocula are selected from natural soils that have the 

potential for enhancing oil degradation. Nitrogen and phosphorus fertilizers are widely used in agriculture to 

enhance the growth of crops and other plants. However, little information is available concerning the impact of 

bioremediation agents on the biotic components of wetlands. 

 The microbial community is an important wetland component in affecting organic matter decomposition 

and nutrient transformations, and it is particularly important in the carbon-rich salt marsh environments (Hood and 

Meyers 1978). Microbial degradation is one of the most important processes to cleanup organic contaminants 

(Bragg et al. 1993). Many microbial products have been commercialized to enhance bioremediation, especially for 

oil degradation. However, microbial seeding as a means of enhancing oil biodegradation has considerable 

uncertainties because in areas commonly exposed to a certain contaminant, such as oil, there usually exists 

populations of microorganisms that is well-adapted to tolerating and/or degrading the contaminant. In addition, soil 

fertility may affect microbial community. A number of studies (Thirukkumaran and Parkinson 2000; Arnebrant et 

al. 1996; Nohrstedt et al. 1989) have found that addition of ammonium nitrate, ammonium sulfate, and urea 

suppressed soil microbial respiration. However, increased soil microbial respiration by the addition of urea and 

ammonium nitrate was also noted in short term incubations (Roberge 1976; Thirukkumaran and Parkinson 2000). 

Effects of phosphorus fertilizer on soil microbes have also been reported as negative, neutral, or positive 

(Thirukkumaran and Parkinson 2000; Amador and Jones 1993). The tracking of various components of the 

microbial community is important in predicting the overall response of the ecosystem. The enumeration of these 

populations provides a direct measure of microbial response. 

 Studies of the effects of chemical discharges and oil spills have also focused on the benthos because 

organisms living or feeding at the sediment-water interface, such as meiofauna and macrofauna, may be exposed to 

particularly high concentrations of contaminants. Typical responses of benthic communities to the addition of toxic 

materials from chemical discharges or spills may include the reduction or elimination of species or individuals 

(Boesch and Rabalais 1989; Rabalais et al. 1992) or increases in populations of opportunistic species and reduction 

in species diversity (Addy et al. 1984; Boesch and Rabalais 1989; Nance 1991). The benthos is composed of 

macrobenthos, meiobenthos, and microbenthos, operationally defined by size and, often, taxonomic and functional 

groups (e.g., life history stage). However, few studies have focused on both, probably due to the high labor 

requirements for thorough benthic community analyses in these groups, and limitations of taxonomic expertise to 

select groups. Each component provides important information with regards to environmental impacts (Sanders et 

al. 1980; Hampson and Moul 1978; Lee et al. 1981; Boucher 1980; Fricke et al. 1981; Wornald,1976; Holt et al. 

1978; Spies 1987). 

 Plants are the important wetland primary producers providing food and habitat for consumer and 

decomposer. Environmental change may influence productivity and alter wetland structure and function. Oil spills 

may detrimentally affect wetland plants (Ferrell et al. 1984; Alexander and Webb 1987; Mendelssohn et al. 1990, 

Lin and Mendelssohn 1996) or do little plant damage (Burk 1977; Hershner and Moore 1977; Li et al. 1990, Lin 

and Mendelssohn 1996), depending upon such things as oil type, oil amount or plant species. Fertilization, such as 

application of nitrogen and phosphorus, generally increases marsh plant growth (Wilsey et al. 1992; Lin and 

Mendelssohn 1998a; Lin et al. 1999b). However, little information is available on the effects of microbial seeding 

on wetland plants since most of microbial inoculation studies focus on the response of the microbial community. 
 4

 The objectives of this study were to determine if the bioremediation agents, inorganic fertilizer and a 

commercial microbial product, generate adverse impacts to wetland plants, infaunal animals, and microbial 

communities. We specifically asked the question: Can bioremediation be used for oil spill cleanup without causing 

negative impacts to wetland structure and function? 

Materials and Methods 

Experimental Design 

 Sods of marsh (soil and vegetation intact), approximately 28 cm in diameter (0.06 m2

) and 30 cm deep, 

were collected from the inland zone (approximately 5 m from the creekbank natural levee) of a Spartina 

alterniflora-dominated salt marsh located west of Cocodrie, Louisiana, and used as the experimental units. Inland 

sods were chosen because the inland zone comprises the largest aerial extent of most salt marshes. We recognize 

that soil types will likely influence bioremediation, and, thus, this factor will be examined in future research. 

Spartina alterniflora is the dominant inter-tidal salt marsh grass along the Atlantic and Gulf Coasts of the United 

States, and thus results from this study generally will be applicable to many other salt marshes. 

 In the greenhouse, the following treatments were randomly assigned to the collected sods: (1) fertilizer 

product, (2) microbial seeding agent, and (3) no bioremediation agent addition (control). The experimental design 

was a randomized block with a 3 x 2 factorial treatment arrangement (three bioremediation types mentioned above 

and two oil dosage levels: oiled with 1 L/m2

 and no oil addition as a control). Each treatment combination was 

replicated 5 times for a total of 30 marsh sods. 

Experimental Procedures 

 A reduced crude (with nC-13 and below absent) was added to the surface water of the mesocosms at a dose 

of 1 L/m2

 (1 mm of oil thickness) of 15 experimental units (marsh sods in 28 cm x 30 cm buckets fitted with water 

level controls). An additional 15 experimental units without oil served as controls. The reduced crude simulated 

the kind of oil that might impact a marsh after the oil had been spilled in open water and subsequently transported 

into the salt marsh by winds or tides. After the applied oil was evenly spread over the surface water in the buckets, 

the water was drained from the bottom of the buckets to allow the oil to come in contact with and penetrate the soil 

of each sod. The bioremediation products utilized were those proven to be most successful in enhancing oil 

biodegradation from marsh sediment-microcosm experiments performed by Ms. Sara McMillen of Exxon 

Production Research, Houston, Texas, as part of this project. This work employed both respirometry and oil 

chemistry (GC-FID) to identify enhanced oil biodegradation. The results indicated that Customblen, a fertilizer 

product used during the Valdez Spill and Petrobac, a microbial product, show promise as bioremediation agents 

(personal communication, Sara McMillen, Exxon Production Research). Thus, we used these two products in the 

present experiment. 

 The Customblen used in this study contained 28% N and 8% P as ammonium nitrate, calcium phosphate 

and ammonium phosphate (Bragg et al. 1992). Petrobac contained microbes, without any fertilizer, selected for 

hydrocarbon degradation in a saline medium (personal communicatiaon, R. Drake, Polybac Corp.). The 

bioremediation treatments (fertilizer, microbial seeding and control) were applied to both oiled and unoiled marsh 

sods. The bioremediation agents were applied to the soil surface in a manner similar to that during a field 

application and following the manufacturer's specifications (Customblen: 93 g/m2

; Petrobac: 0.833 L/m2 of 

inoculum (46 g of Petrobac/ L of de-ionized water)). The sod-mesocosms were kept moist, but drained by 

maintaining an average water level at 5 cm below the soil surface. Water levels were allowed to fluctuate due to 

evapo-transpiration, but they were re-watered daily to 5 cm below the soil surface to maintain relatively constant 

salinity. 

 Statistical analysis was conducted with the SAS system (SAS 1990). General Linear Model (GLM) was 

used to test for statistically significant differences (P<0.05) among the treatments, and Duncan's multiple range test 

was used to determine significant differences among the main factors. Treatment-level combination differences, if 

interactions of main factors were significant, were tested with least square means. 
 5

Methods 

Plant Response

 Photosynthetic Rate. Leaf photosynthetic rate was measured to indicate plant growth status. A portable 

photosynthesis system, including an infrared gas analyzer (IRGA) (The Analytical Development Co. Ltd, (ADC) 

model LCA-2), an ADC air flow control unit, and an ADC Parkinson leaf chamber, was used. Sample air, taken 5 

m aboveground to obtain relatively stable CO2 concentrations, was led through the ADC air flow control unit at a 

flow rate of 5 ml/s during photosynthetic rate measurements. Measurements were conducted at a quantum flux 

density of 2000 μmol/m2

/s1 provided by a Kodak projector lamp. An intact, attached and fully expanded young leaf 

was enclosed in the leaf chamber and the difference in CO2 concentration and humidity between inlet and outlet air 

was measured. Photosynthetic rate (CO2 exchange) was calculated in accordance with von Caemmerer and 

Farquhar (1981) and expressed as μmol CO2/m2

/s. 

 Plant Stem Density. Plant stem density was measured by direct counting of stem number in each 

experimental unit and expressed as the number of stems per pot. 

 Average and Maximum Shoot Height. The average shoot height and maximum shoot height of the 

transplants were measured to the nearest centimeter. 

 Aboveground Biomass of Dominant Marsh Plants. Plant aboveground biomass was analyzed at the end of a 

four-month experimental period to determine the effect of product addition on plant growth. Plant aboveground 

material was clipped at the soil surface. Live and dead components were separated, dried in an oven at 65°C to 

constant weight, and weighed. 

Soil Response

 Soil Respiration Rate. In-situ measurements of soil respiration rate, an indicator of soil microbial activity, 

were made with an infra-red gas analyzer (IRGA) by measuring carbon dioxide production from the soil. A PVC 

chamber (4 cm in diameter and 8 cm in height) with one open end was equipped with an inlet and an outlet for air 

flow through the chamber. The open end of the chamber was inserted into the soil 4 cm below the soil surface. An 

air flow rate through the chamber was held constant at 300 ml per minute by an ADC mass flow controller. The 

respiratory CO2 produced from the soil resulted in a difference in CO2 concentration between inlet and outlet that 

was measured by an ADC infra-red gas analyzer. Soil respiration was calculated based on the CO2 exchange rate 

from the soil per unit surface area. 

 Soil Redox Potential . Soil redox potentials at 2 and 10 cm depths were determined with bright platinum 

electrodes and a calomel reference electrode. Readings were taken with a portable pH/mV digital meter. The 

potential of a calomel reference electrode (+244 mV) was added to each value to calculate Eh (Patrick et al. 1996). 

 Soil Nutrient Concentrations. Interstitial water samples were withdrawn from soil sods with a simple 

apparatus as described by McKee et al. (1988). This consisted of a small diameter (3 mm inside diameter) rigid 

plastic tube, containing numerous holes (ca. 0.5 mm diameter) covered with 3 to 4 layers of cheesecloth, connected 

to a 30 ml syringe. The collected interstitial water was filtered through a 0.45-syringe filter. Inorganic nutrients in 

the filtered interstitial water were analyzed to determine the effect of product application on soil fertility and other 

variables. Ammonium-nitrogen (NH4) and nitrate-nitrogen (NO3 + NO2) were analyzed with an auto-analyzer. 

Total inorganic nitrogen was determined as the sum of NH4, NO3, and NO2-nitrogen concentrations. Phosphorus 

and potassium concentrations in the interstitial water were analyzed with ICP (inductively coupled argon-plasma 

emission spectrometer). 

 Interstitial pH and Salinity. Interstitial water samples were withdrawn from soil sods as described below. 

Interstitial pH was measured with a digital pH meter (Model 420A, Orion) and interstitial salinity was measured 

with a salt refractometer. 
 6

Microbial Response

 Determining the response of the salt marsh microbial component of the ecosystem was accomplished by 

measuring general microbial heterotrophic populations, cellulose-utilizing populations, chitin-utilizing populations, 

yeast and fungi, petroleum-utilizing populations, and overall microbial biomass. Samples were collected at the 

termination of the experiment for the Microtox toxicity assay. 

 Samples of marsh soil were collected with a 5 cc syringe with the end cut off and the remaining edge 

sharpened to facilitate penetration into the soil. Samples for microbial analysis were taken from the top 3 cm in two 

locations in the marsh sod for each sampling episode. The two samples were mixed to form a composite sample. 

Samples were taken from each mesocosm on days 3, 14, 28, and 90. On day 90, 100 gm of soil to a depth of the top 

5 cm were collected from each mesocosm for Microtox analysis. General microbial populations and several key 

components were tracked using plate count methods on a variety of selective agars. Nutrient agar (Difco) was used 

to grow general heterotrophic populations. Chitin agar was made according to modifications of recipes in Hood and 

Meyers (1978) and Scherbarth (1984). Martin's agar (Martin, 1950) was used for detection for yeast and 

filamentous fungi and Jensen's agar (Jensen 1930), modified by the addition of cellulose, for the enumeration of 

cellulose-degrading bacteria and actinomycetes. A minimal salts agar to which 50 ppm naphthalene and 50 ppm 

cresol were added as the sole carbon source was used for the selective culturing of petroleum-degrading organisms. 

General microbial biomass was measured using the adenosine triphosphate method (Bianchini et al. 1988). All 

microbial population measurements and ATP values are reported per gram dry weight. The Microtox assay was 

performed on the samples from Day 90; the samples were centrifuged to separate the interstitial water, and the water 

was analyzed according to the procedure outlined for toxicity measurements on hazardous waste land treatment 

demonstrations (USEPA 1984). 

Infaunal Response

 Macroinfauna. Because of the size of the macroinfaunal cores (7.6 cm diameter), macroinfaunal samples 

were collected only at the termination of the experiment. A 7.6-cm diameter area was marked in each mesocosm at 

the beginning of the experiment to be left intact for eventual macroinfaunal sampling. Five additional marsh sods 

not used in the experiment were sampled for macroinfauna at the beginning of the experiment to determine the pretreatment status of the community. Macroinfauna cores were taken to a depth of 10 cm, preserved in 10% buffered 

formalin stained with Rose Bengal, and transported to the laboratory at Louisiana Universities Marine Consortium 

(LUMCON) 

 Within 48 hours, the formalin in the benthic samples was decanted over a 0.5-mm sieve, the sample rinsed 

with water, and the sample returned to 75% ethanol for storage until analysis. Organisms were sorted in water from 

gridded dishes under a dissecting microscope. The debris from a sample was rechecked for any missed organisms. 

If any were found, the sample was resorted. Organisms were counted and identified to the lowest possible taxon. 

Because of the difficulty in identification of certain marsh organisms, taxonomy for oligochaetes and insect larvae 

were less specific than for other macroinfauna, (e.g., polychaetes, bivalves, gastropods, pericaridean crustaceans). 

 Meiofauna. A series of meiofauna collections was taken during the experiment. Fifteen meiofauna cores 

(2.54 cm diameter) were taken from the salt marsh where the cores were collected to compare to cores taken from 9-

week acclimated sods in the greenhouse and to determine the effects of the acclimation period on the meiofauna 

community. 

 Samples were collected from pre-treatment mesocosms to determine where the majority of the meiofauna 

were distributed vertically in a core. Splits of 2.54-cm diameter cores were made at 0-1, 1-2, 2-3, and 3-4 cm on a 

precision core extruder. We determined that most of the meiofauna were in the upper 3 cm of the cores (see 

Results); this protocol was used for the remainder of the experiment. At the same time the macroinfauna pretreatment samples were collected, three replicates from each of five mesocosms were sampled for meiofauna. 

 For the experiment, meiofauna were collected with a bulk density coring device (2.3 cm diameter) to a 

depth of 3 cm at days 0 and 1, weeks 2, 4, and 12. The majority of meiofaunal organisms (74-99%) were 

distributed in the upper 3 cm of the sediment. All cores were taken between Spartina culms. When a meiofauna 

core was removed from a sod, a core from a similarly treated spare sod was used to replace it. The replaced core 

was marked, and the area was not re-sampled on subsequent days. 
 7

 Meiofauna samples were preserved in 10% formalin stained with Rose Bengal. The samples were sieved 

through two different mesh sizes: 500 μm to retain the root mass and 63 μm to retain the meiofauna. After sieving, 

Ludox AM centrifugation was used to extract the meiofauna. The supernatant was examined for organisms. The 

pellet from the centrifuge tube was examined to see if the extraction method was efficient. Nematodes, copepods, 

copepod nauplii and oligochaetes were counted under a dissection microscope. Only copepods were preserved for 

further identification. Numbers per core were converted to numbers per 10 cm2

. 

 All meiofauna samples contained dense root mass. To remove the mass, samples were sieved through a 

500-μm mesh sieve and examined for any retained larger meiofauna, such as oligochaetes. Few, if any, were 

observed, and we concluded that most meiofauna passed through the 500-μm sieve and were retained on the 63-μm 

sieve. The meiofauna retained on the 63-μm sieve were extracted with Ludox AM by centrifugation. Meiofauna 

were identified to major taxa, which included nematodes, harpacticoid copepods, oligochaetes, copepod nauplii and 

miscellaneous organisms. Miscellaneous organisms included some isopods, polychaetes and some other groups of 

benthic meiofauna. The harpacticoid copepod adults were identified to family or species. 

 Abundances were calculated as number of organisms per 10 cm2

. The copepod:nematode ratio was 

determined rather than the standard nematode:copepod ratio because the number of copepods was frequently zero. 

The ratio was multiplied by 1000 for plotting results. 

Residual Oil Chemistry in the Soil. 

The top five cm of soil from each sod was collected with the same technique as for the microbial samples, 

thoroughly homogenized, and extracted using a modified EPA method 3550. Approximately 20 grams of wet 

sediment were extracted using dichloromethane and sodium sulfate as a chemical drying agent (Venosa et al. 1996;

Sauer and Boehm 1991; Henry and Overton 1993; Henry et al. 1993), then reduced to a final volume of 10 ml. A 

field treatment composite was then analyzed by gas chromatography-mass spectrometry (GC/MS) operated in the 

Selective Ion Monitoring (SIM) mode to characterize compositional changes in targeted normal hydrocarbons (NH) 

and aromatic hydrocarbons (AH). Although these targeted AH generally represent less than 5% of the bulk oil 

composition, they are essential to characterize the petroleum source, identify potential biological effects, determine 

exposure pathways, and monitor weathering trends and degradation of the oil (Sauer and Boehm 1991, Roques et al. 

1994). The targeted aromatic hydrocarbons are listed in Table 1. 

Results 

Responses of Spartina alterniflora

The effect of the bioremediation agents and oil on the growth response of Spartina alterniflora was 

assessed by determining plant leaf elongation rate, photosynthetic rate, stem density, and aboveground biomass. 

There was no adverse effect of microbial seeding on plant leaf elongation rate (Fig. 1), photosynthetic rate (Fig. 2), 

relative growth rate based on plant stem density and cumulative shoot height (Fig. 3), and live and total plant 

aboveground biomass (Fig. 4). Furthermore, fertilization significantly enhanced plant growth as evidenced by 

higher plant leaf elongation rate (Fig. 1), photosynthetic rate (Fig. 2), relative growth rate (Fig. 3), and live and total 

plant aboveground biomass (Fig. 4) compared to the control. The addition of the reduced crude oil to the marsh 

mesocosms had no significant impact on plant response, and there was no significant interaction between 

bioremediation agents and the oil addition (Table 2). Thus neither the bioremediation agents nor the oil had 

negative impacts on the vegetation. 
 8

Table 1. Targeted Compounds Assessed by GC/MS 

______________________________________________________________________________ 

Compound ion mass Compound ion mass

alkanes* (nC-10 thru nC-31) 85 fluoranthrene/pyrene 202 

decalin* 138 naphthobenzothiophene 234 

C-1 decalin* 152 C-1 pyrenes 216 

C-2 decalin* 166 C-2 pyrenes 230 

C-3 decalin* 180 chrysene 228 

naphthalene 128 C-1 chrysenes 242 

C-1 naphthalenes 142 C-2 chrysenes 256 

C-2 naphthalenes 156 phenanthrene 178 

C-3 naphthalenes 170 C-1 phenanthrenes 192 

C-4 naphthalenes 184 C-2 phenanthrenes 206 

fluorene 166 C-3 phenanthrenes 220 

C-1 fluorenes 180 C-1 naphthobenzothiophenes 248 

C-2 fluorenes 194 C-2 naphthobenzothiophenes 262 

C-3 fluorenes 208 C-3 naphthobenzothiophenes 276 

dibenzothiophene 184 benzo(b)fluoranthene 252 

C-1 dibenzothiophenes 198 benzo(k)fluoranthene 252 

C-2 dibenzothiophenes 212 benzo(e)pyrene 252 

C-3 dibenzothiophenes 226 benzo(a)pyrene 252 

perylene 252 benzo(g,h,i)perylene 276 

indeno(1,2,3-cd)pyrene 276 hopanes (191 family)* 191 

dibenzo(a,h)anthracene 278 sterenes (217 family)* 217 

Sum of these compounds excluding those identified with a * is the TTAH value. 

______________________________________________________________________________ 

 

0.5

1

1.5

2

2.5

Leaf Elongation (cm/day)

0 4 8 12

TIME (Weeks)

Control Microbe Fertilizer

Figure 1. Effect of bioremediation agents on leaf elongation rate of Spartina alterniflora. Values are means 

averaged over oil treatments (n=10) with standard errors. 
 9

 

5

10

15

20

25

Photosynthetic Rate ( umol m -2 s-1)

0 4 8 12

TIME (Weeks)

Control Microbe Fertilizer

Figure 2. Effect of bioremediation agents on photosynthetic rate of Spartina alterniflora. Values are means 

averaged over oil treatments (n=10) with standard errors. 

 

Figure 3. Effect of bioremediation agents on the relative growth based on stem density (A) and cumulative shoot 

height (B) of Spartina alterniflora. Values are means averaged over oil treatments (n=10) with standard 

errors. 

0

0.05

0.1

0.15

0.2

 

Control Microbial Seeding Fertilizer

0

0.1

0.2

0.3

Control Microbial Seeding Fertilizer

BIOREMEDIATION AGENTS

B

A

Relative Growth Rate Based on Stem Density (stems month-1)

Relative Growth Rate Based on Cumulative Shoot Hei9ght (cm month-1)
 10

 

0

50

100

150

Aboveground Biomass (g/pot)

Control Microbial Seeding Fertilizer

BIOREMEDIATION PRODUCTS

Total

Dead

Live

Figure 4. Effect of bioremediation agents on aboveground biomass of Spartina alterniflora. Values are means 

averaged over oil treatments (n=10) with standard errors. 

Table 2. Statistical Table with P-Value of Response of Marsh Plant and Soil Variables 

to Bioremediation Agent and Oil Application 

Variables Bioremediation Agent Oil Agent * oil 

Live Biomass 0.0001 0.7487 0.6852 

Dead Biomass 0.0959 0.9703 0.3807 

Total Biomass 0.0001 0.4827 0.8486 

Stem density 

increase in 3 Months 0.0021 0.1169 0.1525 

Cumulative height 

increase in 3 months 0.0001 0.2317 0.7999 

Soil respiration 4 wk 0.0056 0.032 0.5137 

 12 wk 0.0003 0.0442 0.7452 

Leaf Elongation rate 

2 wk 0.0001 0.7541 0.4710 

4 wk 0.0001 0.5830 0.0889 

12 wk 0.0001 0.4706 0.0664 

Photosynthetic rate 

0 wk 0.3866 0.3950 0.4659 

2 wk 0.0001 0.2673 0.3098 

4 wk 0.0001 0.2956 0.8369 

8 wk 0.0001 0.5601 0.6226 

12 wk 0.0001 0.0356 0.2699 

 Soil physico-chemical parameters did not greatly change with the application of oil and bioremediation 

agents. Interstitial pH did not significantly change with the application of bioremediation agents (Fig. 5), although 

the pH values at weeks 8 and 12 showed minor decreases (0.4 pH differential) that were not likely of biological 

significance. 
 11

 

0

1

2

3

4

5

6

7

Interstitial pH

0 4 8 12

TIME (Weeks)

Control Microbe Fertilizer

Figure 5. Effect of bioremediation agents on interstitial pH. Values are means averaged over oil treatments (n=10) 

with standard errors. 

The soil redox potential (Eh) at the 2 cm depth was significantly higher in the fertilizer treatment compared to 

control (Fig. 6). The soil redox potential at 12 cm was not significantly different with either bioremediation agent 

or oil (Fig. 6). Interstitial salinity was not significantly affected by either the bioremediation agents or the oil and 

was in the range of 10 to 11.5 parts per thousand during the 3-month experimental period (data not shown). 

Concentrations of interstitial sulfide, inorganic nitrogen and phosphorus measured 0, 2, 4, 8, and 12 weeks after the 

treatment were not significantly different with the application of either the bioremediation agents or the oil (data not 

shown). Overall, the applications of the bioremediation agents and the reduced oil did not adversely affect soil 

parameters. 

 

-100

0

100

200

300

400

Eh in the Soil (mV)

control Microbial Seeding Fertilizer

Bioremediation Agents

n=50 Eh at 2 cm Eh at 12 cm

Figure 6. Effect of bioremediation agents on soil redox potential. Values (n=50) are means averaged over oil 

levels and 5 repeated measurements (weeks 0, 2, 4, 8, and 12) with standard errors. 

Responses of Microbial Community

In-situ soil respiration was measured to identify whether the bioremediation agents and oil affected the 

metabolism of the soil community. The fertilizer application had a significantly positive effect on soil respiration 
 12

compared to both the control and the microbial treatments (Fig. 7). Soil respiration in the microbial treatment, 

however, was not significantly different from that of the control, suggesting that microbial seeding had no adverse 

effect on the activity of the soil community. 

 

0

0.5

1

1.5

2

Soil Respiration Rate (umol/m 2/s)

Control Microbial Seeding Fertilizer

Bioremediation Agents

12 weeks

4 weeks

Figure 7. Effect of bioremediation agents on soil respiration rate. Values are means averaged over oil treatments 

(n=10) with standard errors. 

 A number of soil microbial components, such as the general heterotrophic microbial populations (Fig. 8), 

cellulose-utilizing populations (Fig. 9), chitin-utilizing populations (Fig. 10), yeast (Fig. 11), fungi (Fig. 12), 

petroleum-utilizing populations (Fig. 13), and overall microbial biomass (Fig. 14), were measured 3 and 28 days 

after bioremediation agent addition. Overall, there were no significant differences among the bioremediation 

agents. After three days, there were no statistically significant differences in the various microbial components 

(Figs. A1-1 to A1-7 in Appendix A) among the bioremediation agents and oil addition (Table 3). The short-term 

results suggest no acute toxicity of the bioremediation agents and oil to the general microbial populations. After 28 

days, the bioremediation agents did not significantly affect the various microbial parameters (Figs. 13) except 

microbial ATP (Fig 14); ATP was higher in the fertilizer treatment compared to the control and the inoculated 

treatments (Table 3). After 28 days, oil addition adversely affected the microbial heterotrophic populations (Fig. 8), 

fungi (Fig. 13), petroleum-utilizing populations (Fig. 9), and overall microbial biomass (Fig. 14), but did not 

significantly affect cellulose-utilizing populations, chitin-utilizing populations, and yeast (Table 3). A significant 

interaction between bioremediation agent and oil application on ATP indicated that fertilizer enhanced ATP only in 

treatments without oil application (Fig. 14). Microtox analyses conducted on samples from day 90 show no 

significant difference in toxicity (measured as EC 50 at 15 minutes) between samples treated with either fertilizer or 

microbial seeding for either the oiled or the unoiled samples (Fig 15). 
 13

 

1.0000E+06

1.0000E+07

1.0000E+08

1.0000E+09

1.0000E+10

Heterotrophic bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure 8. Effect of bioremediation agents and oil application on heterotrophic bacteria 28 days after treatment. 

Values are reported in colony-forming units per gram (dry weight) of soil with standard errors. C: 

control; M: microbial seeding; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+06

1.0000E+07

1.0000E+08

1.0000E+09

Petroleum-degrading bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure 9. Effect of bioremediation agents and oil application on petroleum-degrading bacteria 28 days after 

treatment. Values are reported in colony-forming units per gram (dry weight) of soil with standard 

errors. C: control; M: microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no 

oiling. 
 14

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

1.0000E+08

Chitin-degrading bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure 10. Effect of bioremediation agents and oil application on chitin-degrading bacteria 28 days after treatment. 

Values are reported in colony-forming units per gram (dry weight) of soil with standard errors. C: 

control; M: microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

1.0000E+08

1.0000E+09

Actinomycetes and bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure 11. Effect of bioremediation agents and oil application on cellulose-degrading actinomycetes and bacteria 28 

days after treatment. Values are reported in colony-forming units per gram (dry weight) of soil with 

standard errors. C: control; M: microbial addition; F: fertilizer. Plus signs indicate oiling and minus 

signs no oiling. 

 
 15

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

1.0000E+08

Yeast(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure 12. Effect of bioremediation agents and oil application on yeast populations 28 days after treatment. Values 

are reported in colony-forming units per gram (dry weight) of soil with standard errors. C: control; M: 

microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

1.0000E+08

Filamentous Fungi(Colony-forming Units)

CC+

MM+

FF+

Treatment

Figure 13. Effect of bioremediation agents and oil application on filamentous fungi 28 days after treatment. Values 

are reported in colony-forming units per gram (dry weight) of soil with standard errors. C: control; M: 

microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 
 16

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

ATP (pg)

CC+

MM+

FF+

Treatment

Figure 14. Effect of bioremediation agents and oil application on microbial biomass 28 days after treatment. Values 

are reported in picograms of ATP per gram (dry weight) of soil with standard errors. C: control; M: 

microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

Table 3. Statistical Table with P-Value for Response of Microbial Populations 

to Bioremediation Agent and Oil Application 

Variables Bioremediation Agent Oil Agent * oil 

Heterotrophic 

3 days 0.2317 0.9245 0.8564 

28 days 0.5306 0.0253 0.4367 

Cellulose-utilizing 

3 days 0.1904 0.6170 0.1025 

28 days 0.1707 0.1770 0.1789 

Chitin-utilizing 

 3 days 0.1017 0.4716 0.7436 

28 days 0.3979 0.1959 0.3269 

Yeast 

3 days 0.3580 0.3721 0.6048 

28 days 0.3498 0.1207 0.4486 

Fungi 

3 days 0.2452 0.2673 0.3263 

28 days 0.1273 0.0406 0.3034 

Petroleum-degrading 

3 days 0.3905 0.2779 0.5707 

28 days 0.7704 0.0263 0.7469 

ATP as biomass 

3 days 0.1190 0.3286 0.4179 

28 days 0.0019 0.0027 0.0022 
 17

Figure 15. Effect of bioremediation agents and oil application on 15-minute readings of microtox analysis 90 days 

after treatment. Values are expressed as EC50 with standard errors, so higher values are less toxic than 

lower values. C: control; M: microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs 

no oiling. 

Responses of Infauna to the Treatments

Macrofauna. The makeup of the benthic community at the beginning of the experiment and at the end of the 

12 weeks was documented. The fauna was composed primarily of oligochaetes, gastropods and insects. Decapods 

(fiddler crabs), polychaetes, and amphipods were also represented. There were no significant differences in 

bioremediation agent treatments or oil levels, nor in their interactions for either number of individuals or number of 

species 12 weeks after the treatments. Twelve weeks after the treatments the average number of individuals was 

about 25/sod, and the average number of species was less than 3. The only difference was that some macrofauna 

populations changed with time but not due to the treatments. There was no difference in the number of individuals, 

but the number of species dropped significantly over the experimental period of 12 weeks. For example, the 

number of species decreased from 10.2 to 3.0 for the control.

 Because oligochaetes often respond to organic enrichment in sediments, we tested for differences in the 

percentage of oligochaetes in the macrofauna community. There were no significant differences between the 

bioremediation agent treatments or oil levels nor in their interactions. However, by the end of the experiment, 

richness was significantly reduced and oligochaetes were by far the dominant fauna. 

 MeiofaunA. There were no significant differences for any of the meiofaunal groups or the copepod:nematode 

ratio due to the bioremediation agents (Table 4). Overall, the numbers of meiofauna were usually lower in the oiled 

mesocosms, but the effect of oil was significant only for the number of oligochaetes and for the copepod:nematode 

ratio. The only overall significant effect was for time (Table 4). There was a significant time effect on number of 

copepods, copepod nauplii and oligochaetes, but not on the number of nematodes (Table 4). There were no effects 

of bioremediation agent treatments or oil application on them except for those specifically mentioned (Table 4). 

0

50

100

150

EC 50 (%)

CC+

MM+

FF+

Treatment
 18

Table 4. Summary of Multiple Analysis of Variance on Four Taxa of Meiofauna and Nematode: 

Copepod Ratio with P-Values Provided for Main Factors and Their Interactions. 

Experiment 

component 

 Main 

Factors 

 Interaction 

and interactions Agents Oil Time Agent* 

Oil 

Agent* 

time 

Oil* 

Time 

Agent* 

Oil* 

Time 

Nematodes 0.91 0.32 0.98 0.73 0.99 0.99 1.00 

Copepods 0.18 0.09 0.0001 0.40 0.04 0.16 0.20 

Copepod 

Nauplii 

0.15 0.07 0.0001 0.98 0.02 0.01 0.10 

Oligochaetes 0.06 0.07 0.0001 0.29 0.07 0.79 0.89 

Nematode: 

Copepod Ratio 0.07 0.003 0.0001 0.14 0.15 0.02 0.30 

Data Were Natural Log Transformed for the Analysis. Time = 1-Week, 2-Week, 4-Week and 12-Week; 

Agents = Control, Fertilizer, and Microbial Seeding; Oil = Oiled and Unoiled. 

Copepod Taxa

 There were 11 identifiable taxa of copepods in the bioremediation samples. Some of these copepods were 

identified to species and family level, while some could not be identified. The most abundant groups were those 

associated with the Spartina alterniflora culms and roots (Rutledge and Fleeger 1993). Mudflat species and 

plankton species were rarely observed. Calanoids and other planktonic forms were not included in the data. The 

four most abundant copepod taxa were members of the Laophontidae, Nannopus spp., Mesocra mexicana, and 

Nitocra spp. None were affected significantly by either oil or treatment (data not shown). 

Oil Chemistry. This study was primarily designed to evaluate toxicity of the bioremediation agents. Thus, only a 

limited number of samples were analyzed by detailed GC/MS for quantifying the alkane profile and specific 

aromatic hydrocarbons (AH) 

Normal Hydrocarbon (alkane) Profile. Figure 16 compares the chromatographic profile of unweathered south 

Louisiana crude oil and laboratory weathered crude oil. Weathered oil had lost most of the lower molecular weight 

components through nC-13 by simple evaporation compared to the unweathered crude oil. The first normal 

hydrocarbon detected is nC-11 for the laboratory weathered crude oil. 

 Figure 17 is a chromatographic comparison of the reduced crude oil used in this experiment to the 

laboratory weathered crude oil. The lowest molecular weight alkane found for both oils was nC-11. The effect of 

vacuum distillation significantly altered the distribution pattern for the reduced crude oil, which is typical for 

naturally weathered crude oil. The laboratory weathered oil was altered by simple evaporation only. 

 Figure 18 is a chromatographic comparison of the alkane distribution for the oiled control (C+) and unoiled 

control (C-). Note the presence of hydrocarbons in the unoiled control. The hydrocarbon distribution is indicative 

of biogenic hydrocarbons, probably derived from the waxy coating covering the leaves of Spartina alterniflora. 
 19

 

Figure 16. Chromatographic comparison of unweathered south Louisiana crude oil (top) to the laboratory 

weathered south Louisiana crude oil (bottom). 
 20

Figure 17. Chromatographic comparison of the reduced crude oil (top) used in this experiment to the laboratory 

weathered crude oil (bottom). 
 21

 

Figure 18. Chromatographic comparison of the normal hydrocarbon profile in the oiled control plot, C+ (top) 

compared to the unoiled control plot, C- (bottom) at Day 1. 

Composition of Selected Aromatic Hydrocarbons. Figure 19 is a comparison of the AH profile for the reduced 

crude (RC) used in this experiment and the Day 1 oiled control (C+). Generally, the composition of the AH profile 

of RC and C+ one day after oil addition was very similar. A slight reduction in the concentration of the naphthalene 

and fluorene constituents is shown. The loss can probably be attributed to a combination of evaporation and 

dissolution between day 0 and day 1. 
 22

0

250

500

750

1000

ng/mg (wet)

NAPH

C-1 NAPHC-2 NAPHC-3 NAPHC-4 NAPH

FLU

C-1 FLUC-2 FLUC-3 FLU

DBT

C-1 DBTC-2 DBTC-3 DBT

PHEN

C-1 PHENC-2 PHENC-3 PHEN

ANT

NBTP

C-1 NBTPC-2 NBTPC-3 NBTP

FLURANT

PYR

C-1 PYRC-2 PYR

B(a)ANT

CHRY

C-1 CHRYC-2 CHRY

B(b)F

B(e)PB(a)P

PERYL

INDPYR

BENZPER

BIBZANT 

0

5

10

15

20

ng/mg (wet)

NAPH

C-1 NAPHC-2 NAPHC-3 NAPHC-4 NAPH

FLU

C-1 FLUC-2 FLUC-3 FLU

DBT

C-1 DBTC-2 DBTC-3 DBT

PHEN

C-1 PHENC-2 PHENC-3 PHEN

ANT

NBTP

C-1 NBTPC-2 NBTPC-3 NBTP

FLURANT

PYR

C-1 PYRC-2 PYR

B(a)ANT

CHRY

C-1 CHRYC-2 CHRY

B(b)F

B(e)PB(a)P

PERYL

INDPYR

BENZPER

BIBZANT

Figure 19. Aromatic hydrocarbon profile comparison between the reduced crude oil (top) and the mean C+ Day 1 

values (bottom). The C+ values are derived from the mean of 3 replicate plots. 

 Figure 1-20 is the day 1 oiled control compared to the unoiled control for the target aromatic hydrocarbons. 

While the control sample does contain evidence of AH exposure, the source of the AH was combustion byproducts, 

not petroleum. AH derived from the incomplete combustion of fossil fuels and organic hydrocarbons are ubiquitous 

in the marine environment. Combustion-sourced AH is characterized by the dominance of unalkylated 3-, 4-, and 5-

ring AH as opposed to petroleum-sourced AH which is dominated by 1-, 2- and 3-ring, and highly alkylated AH as 

exhibited in the oiled control in Figure 1-20. 
 23

0

5

10

15

20

ng/mg (wet)

NAPH

C-1 NAPHC-2 NAPHC-3 NAPHC-4 NAPH

FLU

C-1 FLUC-2 FLUC-3 FLU

DBT

C-1 DBTC-2 DBTC-3 DBT

PHEN

C-1 PHENC-2 PHENC-3 PHEN

ANT

NBTP

C-1 NBTPC-2 NBTPC-3 NBTP

FLURANT

PYR

C-1 PYRC-2 PYR

B(a)ANT

CHRY

C-1 CHRYC-2 CHRY

B(b)F

B(e)PB(a)P

PERYL

INDPYR

BENZPER

BIBZANT

0.00

0.01

0.02

0.03

0.04

0.05

0.06

ng/mg (wet)

NAPH

C-1 NAPHC-2 NAPHC-3 NAPHC-4 NAPH

FLU

C-1 FLUC-2 FLUC-3 FLU

DBT

C-1 DBTC-2 DBTC-3 DBT

PHEN

C-1 PHENC-2 PHENC-3 PHEN

ANT

NBTP

C-1 NBTPC-2 NBTPC-3 NBTP

FLURANT

PYR

C-1 PYRC-2 PYR

B(a)ANT

CHRY

C-1 CHRYC-2 CHRY

B(b)F

B(e)PB(a)P

PERYL

INDPYR

BENZPER

BIBZANT

Figure 20. Aromatic hydrocarbon profile comparison between the mean C+ Day 1 values (top) and the C-, control 

(bottom). The C- sample is dominated by combustion derived AH while the C+ plots are dominated by 

the characteristic petroleum AH profile. 

Discussion 

 These results demonstrated that the bioremediation agents applied in this study were not toxic to the salt 

marsh organisms investigated. Application of bioremediation agents did not impact the macrophyte Spartina 

alterniflora, various soil microbial populations, macroinfauna, or meiofauna. 

 The microbial agent, Petrobac, did not adversely affect marsh plant, infaunal animals and soil microbes. 

Microbial seeding used in this study contains cultivated microbes and no fertilizer, and it was selected for 

hydrocarbon degradation in a saline medium. The marsh sods used in this study were intact natural salt marsh 

sections dominated by S. alterniflora, which contain a variety of native microbial populations naturally, including 

indigenous hydrocarbon-degrading microbes (Atlas 1993). When mesocosms were inoculated with Petrobac in this 

study, a microbial consortium that was proven to degrade petroleum in the laboratory was applied. However, the 
 24

inoculated microbial populations were probably in lower abundance than the indigenous microbes in the marsh soil; 

thus the inoculation may not have been able to influence the microbial community composition in the sods. 

 Fertilizer application did not adversely affect marsh plants, infaunal animals, and microbes, but it did 

positively enhance the marsh plants. The Customblen (fertilizer) used in this study contained 28% N and 8% P. It 

is well known that nitrogen and phosphorus are essential to plants. Inorganic nutrients in salt marshes are generally 

not high enough for maximum plant growth. Fertilizer addition, especially nitrogen, usually enhances marsh plants 

(Lin and Mendelssohn 1998, 1999a; Wilsey et al. 1992). In fact, the fertilizer product, Customblen, stimulated 

plant growth, a response that was not unexpected. 

 The living soil community is composed of bacteria, fungi, invertebrates, and roots of Spartina alterniflora. 

The increase in soil respiration caused by the fertilizer and oil treatments could be a response to either increased 

microbial activity, greater root density within the soil, or both. However, the belowground live root densities of 

cores taken between the plants (where the respiration chamber was inserted to measure the soil respiration rate) 

were not significantly different among either the bioremediation agents or the oil application (data not shown). This 

suggests that the increased soil respiration by the application of fertilizer and oil was mainly caused by an increase 

in microbial activity, not by an increase in plant root respiration. 

 Oil application did not adversely affect the marsh plants. Oil spills may cause various acute and chronic 

damages, including reduced stem heights, stem density, and aboveground biomass, and increased mortality and 

impaired growth and regrowth (Krebs and Tanner 1981; Ferrell et al. 1984; Alexander & Webb 1987; Mendelssohn 

et al. 1990). In contrast, there is also evidence that oil may do little plant damage or may even stimulate growth 

(Burk 1977; Hershner and Moore 1977; Delaune et al. 1979; Li et al. 1990). In the present study, the absence of an 

adverse effect of the oil on Spartina alterniflora was most likely due to a low oil dosage (1 L/m2

) and the relatively 

low toxicity of the reduced crude oil. 

 The typical response of benthic communities to the addition of toxic materials from chemical discharges or 

spills is the reduction or elimination of species or individuals (e.g., Boesch and Rabalais 1989; Rabalais et al. 1992). 

Where toxicity is not a factor, but organic enrichment of the sediments is the environmental change, a range of 

benthic community responses may be observed (Pearson and Rosenberg 1978). These include increases in 

populations of opportunistic species and reduced species diversity (e.g., Addy et al. 1984; Boesch and Rabalais 

1989; Nance 1991). Copepod reductions during the experiment appear to be the result of mesocosm effects and 

elimination of potential recruitment mechanisms. Nematodes appear to be responding to organic enrichment of the 

sediments, from either hydrocarbons or microbial enhancement. There was no evidence of a reduced number of 

macro- and meio-fauna due to application of either bioremediation products. Field studies are needed to better 

evaluate the effect of these bioremedation products on the faunal community. 

 The first normal hydrocarbon detected was nC-11 for both the reduced crude oil used in this experiment 

and the laboratory weathered crude oil commonly used in many oil bioremediation studies. The reduced crude oil 

and the laboratory weathered crude oil are representative of samples collected during the first 10 days of the 

Greenhill Blowout near East Timbalier Island, Louisiana, for both hydrocarbon distribution pattern and degree of 

evaporation. Of the 22 samples collected between 3 and 13 October, 1992, the first detectable normal hydrocarbon 

ranged between nC-9 and nC-17 (mean nC-13, and medium nC-13). For the purpose of a bioremediation study, 

both reduced crude oil and laboratory weathered crude oil are acceptable since they contain a wide range of middle 

distillate normal hydrocarbons. 

Summary 

 The bioremediation agents used in this investigation, including microbial seeding and inorganic fertilizer, 

had little toxicity to biota in the selected salt marsh sods. The bioremediation agents did not adversely affect the 

dominant marsh macrophyte, Spartina alterniflora, as evidenced by the plant photosynthetic rate, plant stem 

density, leaf elongation rate, and aboveground biomass. As expected, inorganic fertilizer application enhanced the 

growth of S. alterniflora, with significantly higher plant parameters as mentioned above. Microbial seeding had no 

significant effect on plant growth, and oil application also had no significant effect on plant growth. Furthermore, 

the bioremediation agents did not adversely affect various microbial populations, with no significant difference 

between the bioremediation treatments and the control for the various microbial parameters (heterotrophic microbial 

populations, fungi, petroleum-utilizing populations, and overall microbial biomass). In addition, the bioremediation 
 25

agents did not adversely affect various infauna animals, as evidenced by no significant differences in bioremediation 

agent addition on either number of individuals or number of species for macrofauna and meiofauna. Overall, the 

bioremediation agents used at the rates in this experiment are safe to salt marsh communities, with little potential 

ecological impact to wetland plants, infaunal animals and microbial communities. Both reduced crude oil and 

laboratory weathered crude oil appear to be acceptable for bioremediation studies because they contain a wide range 

of middle distillate normal hydrocarbons. 
27 

CHAPTER 2 

THE EFFECT OF BIOREMEDIATION AGENTS 

ON CRUDE OIL DEGRADATION AND BIOTIC RESPONSE 

UNDER DRAINED AND FLOODED CONDITIONS IN MARSH MESOCOSMS 

by Irving A. Mendelssohn, Qianxin Lin, Karolien Debusschere, Charles B. Henry, Jr., 

Edward B. Overton, Ralph J. Portier, Paulene O. Roberts, and Maud M. Walsh 

 Many factors, especially soil physico-chemical parameters, such as oxidization status, fertility, pH, 

concentration of oil, and the presence of hydrocarbon degrading microbes (Alexander 1989; Blaba et al. 1991; Lin 

and Mendelssohn 1997 and 1998b; Lin et al. 1999c) may affect oil degradation. Oxygen and nutrients in wetland 

soils are generally not sufficient for maximum biological activity. In the application of bioremediation, the most 

common agents shown to enhance oil degradation are inorganic nutrients and soil aeration where, O2 is deficient; 

microbial seeding has also been frequently used for bioremediation (Mikesell et al. 1991; Altas 1993; Bragg et al. 

1993; Prince et al. 1993; Venosa et al. 1996; Lin et al. 1999c). 

 Previous studies (Lindstrom et al. 1991; Banks and Schwab 1993; Venosa et al. 1996; Lin and 

Mendelssohn 1998a and 1998b; Lin et al. 1999c) indicated that fertilization with inorganic nitrogen and phosphorus 

enhanced petroleum hydrocarbon degradation. Oxidants have shown to enhance oil degradation in some cases, 

especially for the bioremediation of petroleum-contaminated aquifers (Wang and Latchaw 1990; Lovley et al. 1994;

Chapelle 1999). In addition, many microbial products have been commercialized for use in oil bioremediation, 

although controversial results regarding the value of microbial inocula on bioremediation have been reported (Atlas 

1995a; Cassidy et al. 1997; Jorgensen et al. 2000). 

 Hydrology and associated inundation regimes are major-forcing functions in wetlands. Oil degradation rate 

can be much slower in anaerobic, water saturated soils compared to wetland soils that experience daily drainage and 

are less biochemically reduced (Hambrick et al. 1980; Lin and Mendelssohn 1998a). The effectiveness of 

bioremediation for oil spill cleanup could be greatly different under different inundation conditions. However, the 

effect of different inundation regimes on bioremediation has received little investigation. 

 The objectives of this experiment were to determine (a) the effectiveness of bioremediation with an 

inorganic fertilizer, a commercial microbial product, and a soil oxidant on oil degradation and (b) if inundation 

regime influences the effectiveness of oil bioremediation. This experiment will answer the question: Does 

bioremediation enhance oil degradation in intact salt marsh soil systems? 

Materials and Methods 

Experimental Design 

Sixty sods of marsh (soil and vegetation intact), approximately 28 cm in diameter (0.06 m2) and 30 cm 

deep, were collected from the inland zone (approximately 5 m from the creekbank natural levee) of a Spartina 

alterniflora dominated salt marsh located west of Cocodrie, Louisiana and used as the experimental units (see 

Chapter 1). The following treatments were randomly applied to the marsh sods: (1) microbial seeding (Petrobac), 

(2) fertilizer (Customblen), (3) oxidant application (Permeox) plus fertilizer (Customblen), and (4) control (no 

agents applied). The experimental design was a randomized block with a 4 x 2 factorial treatment arrangement (the 

4 bioremediation types mentioned above) and 2 soil inundation regimes (drained and flooded conditions); all of 

these treatment combinations received 2 L/m2 of oil and were replicated 5 times. In addition, drained and flooded 

controls, each replicated five times, that received neither oil nor bioremediation products were included in the 

experimental design so that statistical contrasts could be conducted between any of the treatment combinations 

within the 4 x 2 factorial design and these controls. A total of 50 sod mesocosms were used in this experiment (40 

sods for the 4 x 2 factorial design and 10 additional sods for the drained and flood controls that received no oil or 

products). 
 28

Experimental Procedures 

 In the greenhouse, a reduced Louisiana crude with nC-13 and below removed was added to the surface 

water of the mesocosms at a dose of 2 L/m2

 (2 mm oil thickness) to the surface of 40 oiled experimental units. The 

other 10 experimental units received no oil or bioremediation agents and served as controls. After the applied oil 

was evenly spread over the surface water in the buckets, the water was drained from the bottom of the buckets to 

allow the oil to come in contact with and penetrate the soil of each sod. 

 The Customblen fertilizer used in this study contained 28% N and 8% P2O5 as ammonium nitrate, calcium 

phosphate and ammonium phosphate (Bragg et al. 1992). Petrobac contained microbial populations, without any 

fertilizer, selected for hydrocarbon degradation in a saline medium. PermeOx hydrolyzes when wet and converts, 

slowly, to Ca(OH)2, CaO and H2O2 (hydrogen peroxide). The oxygen from the hydrogen peroxide is then 

catalytically released, thus, potentially accelerating aerobic microbial activity. The four bioremediation treatments 

(fertilizer, microbial seeding, soil oxidant plus fertilizer, and control) were applied to appropriate sods from all 40 

oiled sods. The bioremediation agents were applied to the soil surface in a manner similar to that during a field 

application and following the manufacturer's specifications (Customblen: 93 g/m2

; Petrobac: 0.833 L/m2 of 

inoculum (46 g of Petrobac/L of deionized water). In addition, 13.7 g/pot of an oxidation product (PermeOx, a 

product of FMC Corp., Philadelphia, PA), combined with 93 g/m2 of Customblen plus 6.85 g/pot of KH2PO4 were 

applied as the oxidant treatment; KH2PO4 was applied to buffer the high pH that might be caused by the PermeOx 

according to the manufacture’s instruction. Interstitial water salinity was maintained at the ambient salt marsh level 

(about 10 ppt). The sod-mesocosms were kept in appropriate inundation regimes: 25 sods with water table 10 cm 

below the soil surface, and 25 sods with 3 cm standing water over the soil surface. Water level in each experimental 

unit was monitored and maintained by re-watering daily. 

 The experiment was terminated after three months. This duration was chosen based on a review of oil spill 

bioremediation in saline wetland soils (Scherrer and Mille 1990; Lee and Levy 1991). During the three-month 

experimental period, the mesocosms were sampled for (1) petroleum hydrocarbon chemistry to identify and quantify 

the degree of oil biodegradation, (2) oil morphology, which was tested as an inexpensive means of evaluating oil 

biodegradation, (3) soil microbial response to determine the effect of the bioremediation products on the microbial 

communities that are performing the oil biodegradation, (4) soil chemistry to determine the effect of the 

bioremediation products on those factors that may limit the growth of the microbes and plants (e.g., nutrients, soil 

reducing conditions and soil toxins) and (5) plant response to evaluate the effects of the oil and products on plant 

vigor and growth. 

 Statistical analysis was conducted with the SAS system (SAS 1990). General Linear Model (GLM) was 

used to test for statistically significant differences (P<0.05) among the treatments and Duncan's multiple range test 

was used to determine significant differences among the main factors. Treatment-level combination differences, if 

interactions of main factors were significant, were tested with least square means. 

Methods 

Plant Response

Photosynthetic Rate. Leaf photosynthetic rate was measured to indicate plant growth status. A portable 

photosynthesis system, including an infrared gas analyzer (IRGA) (The Analytical Developmemt Co. Ltd, (ADC) 

model LCA-2), an ADC air flow control unit, and an ADC Parkinson leaf chamber, was used. Sample air, taken 5 

m aboveground to obtain relatively stable CO2 concentrations, was led through the ADC air flow control unit at the 

flow rate of 5 ml/s during photosynthetic rate measurements. Measurements were conducted at a quantum flux 

density of 2000 μmol/m2

/s provided by a Kodak projector lamp. An intact, attached and fully expanded young leaf 

was enclosed in the leaf chamber and the difference in CO2 concentration and humidity between inlet and outlet air 

was measured. Photosynthetic rate (CO2 exchange) was calculated in accordance with von Caemmerer and 

Farquhar (1981) and expressed as μmol CO2/m2

/s. 

 Plant Stem Density. Plant stem density was measured by counting the number of stems in each 

experimental unit, expressed as the number of stems per pot. 
 29

 Average and Maximum Shoot Height. The average shoot height and maximum shoot height of the 

transplants were measured to the nearest centimeter. 

Aboveground Biomass of Dominant Marsh Plants. Plant aboveground biomass was analyzed at the end of 

a three-month experimental period to determine the effect of product addition on plant growth. Plant aboveground 

material was clipped at the soil surface. Live and dead components were separated, dried in an oven at 65°C to 

constant weight, and weighed. 

Soil Response

Soil Respiration Rate. In-situ measurements of soil respiration rate, an indicator of soil microbial activity, 

were made with an infra-red gas analyzer (IRGA) by measuring carbon dioxide production from soil. A PVC 

chamber (4 cm in diameter and 8 cm in height) with one open end was equipped with an inlet and an outlet for air 

flow through the chamber. The open end of the chamber was inserted into the soil 4 cm below the soil surface. An 

air flow rate through the chamber was held constant at 300 ml/minute by an ADC mass flow controller. The 

respiratory CO2 produced from the soil resulted in a difference in CO2 concentration between inlet and outlet that 

was measured by an ADC infra-red gas analyzer. Soil respiration was calculated based on the CO2 exchange rate 

from the soil per unit surface area. 

Soil Redox Potential. Soil redox potentials at 2 and 10 cm depths were determined with bright platinum 

electrodes and a calomel reference electrode. Readings were taken with a portable pH/mV digital meter. The 

potential of a calomel reference electrode (+244 mV) was added to each value to calculate Eh (Patrick et al. 1996). 

Soil Nutrient Concentrations. Interstitial water samples were withdrawn from soil sods with a simple 

apparatus as described by McKee et al. (1988). This consisted of a small diameter (3 mm inside diameter) rigid 

plastic tube, containing numerous 0.5 mm diameter holes covered with 3 to 4 layers of cheesecloth, connected to a 

30 ml syringe. The collected interstitial water was filtered through a 0.45 μm syringe filter. Inorganic nutrients in 

the filtered interstitial water were analyzed to determine the effect of product application on soil fertility and other 

variables. Ammonium-nitrogen (NH4) and nitrate-nitrogen (NO3 + NO2) were analyzed with an auto-analyzer. 

Total inorganic nitrogen was determined as the sum of NH4, NO3, and NO2-nitrogen concentrations. Phosphorus 

concentrations in the interstitial water were analyzed with ICP (inductively coupled argon-plasma emission 

spectrometer). 

Interstitial pH and Salinity. Interstitial water samples were withdrawn from soil as previously described. 

Interstitial pH was measured with a digital pH meter and interstitial salinity was measured with a salt refractometer. 

Microbial Response

 Determining the response of the salt marsh microbial component of the ecosystem was accomplished by 

measuring general microbial heterotrophic populations, cellulose-utilizing populations, chitin-utilizing populations, 

yeast and fungi, petroleum-utilizing populations, and overall microbial biomass. Samples were collected at the end 

of the experiment for the Microtox toxicity assay. 

 Samples of marsh soil were collected with a 5 cc syringe with the end cut off and the remaining edge 

sharpened to facilitate penetration into the soil. Samples for microbial analysis were taken from the top 3 cm in two 

locations in the pot for each sampling episode. The two samples were mixed to form a composite sample. General 

microbial populations and oil-degrading were tracked using plate count methods on a variety of selective agars. 

Nutrient agar (Difco) was used to grow general heterotrophic populations. A minimal salts agar to which 50 ppm 

naphthalene and 50 ppm cresol were added as the sole carbon source was used for the selective culturing of 

petroleum-degrading organisms. General microbial biomass was measured using the adenosine triphosphate 

method (Bianchini et al. 1988). All microbial population measurements were reported per gram dry weight gram. 
 30

Residual Oil Chemistry in the Soil. 

The top 5 cm of soil from each sod was collected with the same technique as for the microbial samples, 

thoroughly homogenized, and extracted using a modified EPA method 3550. Approximately 20 grams of wet 

sediment were extracted using dichloromethane and sodium sulfate as a chemical drying agent (Venosa et al. 1996;

Sauer and Boehm 1991; Henry and Overton 1993; Henry et al. 1993), then reduced to a final volume of 10 ml. A 

field treatment composite was then analyzed by gas chromatography-mass spectrometry (GC/MS) operated in the 

Selective Ion Monitoring (SIM) Mode to characterize compositional changes in targeted normal hydrocarbons (NH) 

and aromatic hydrocarbons (AH). Although these targeted AH generally represent less than 5% of the bulk oil 

composition, they are essential to characterize petroleum sources, identify potential biological effects, determine 

exposure pathways, and monitor weathering trends and degradation of the oil (Sauer and Boehm 1991; Roques et al. 

1994). The targeted aromatic hydrocarbons are listed in Table 1 in Chapter 1. 

 In addition, oil morphology was documented in this experiment. The detailed methods of oil morphology 

are attached in Appendix B. 

Results 

Responses of S. alterniflora to the Treatments

 The effect of the bioremediation agents and oil on Spartina alterniflora were estimated by measuring leaf 

elongation rate, photosynthetic rate, relative growth rates based on stem density and cumulative shoot height, and 

aboveground biomass. Similar to our observations in the toxicity experiment in Chapter 1, the fertilizer had a 

significant positive effect on leaf elongation (Fig. 21), photosynthetic rate (Fig. 22), relative growth rates (Figs. 23A 

and 23B), and live aboveground biomass (Fig. 24) (Table 5). However, the addition of the oxidant (PermeOx) 

somewhat counteracted the enhancing effect of the fertilizer on live aboveground biomass (Fig. 24) although the 

treatment of the soil oxidant plus fertilizer still significantly increased plant growth responses compared with the 

control. In addition, microbial seeding had no significant effect on plant growth responses (Figs. 21 to 24) 

compared to the controls. The addition of oil did not significantly affect plant growth responses (Figs. 21 to 24). 

The inundation treatment also had no significant effect on plant growth responses (Table 5). 

 

0

0.5

1

1.5

2

Leaf Elongation Rate (cm day -1)

C- C+ M+ F+ OF+

BIOREMEDIATION AGENTS

leaf

Figure 21. Effect of bioremediation agents on leaf elongation of Spartina alterniflora. Values are means averaged 

over inundation treatments and times of 4, 8, and 12 weeks after the bioremediation treatment (n=30). 

Standard errors are presented. C=control; M=microbial seeding; F=fertilizer; OF=oxidant plus fertilizer. 

Plus signs indicate oiling, and minus signs no oiling. 
 31

 

0

5

10

15

20

Photosynthetic Rate (umol m -2 s-1 )

C- C+ M+ F+ OF+

BIOREMEDIATION AGENTS

Figure 22. Effect of bioremediation agents on photosynthetic rate of Spartina alterniflora. Values are means 

averaged over inundation treatments and times of 4 and 12 weeks after the bioremediation treatment 

(n=20). Standard errors are presented. C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus 

fertilizer. Plus signs indicate oiling, and minus signs no oiling. 

 

0

0.1

0.2

0.3

0.4

Relative Growth Rate Based on Stem Density (stem stem -1 month-1)

C- C+ M+ F+ OF+

0

0.1

0.2

0.3

0.4

0.5

Relative Growth Rate Based on Cumulative  Shoot Height (cm cm-1 month-1)

C- C+ M+ F+ OF+

BIOREMEDIATION PRODUCTS

A

B

Figure 23. Effect of bioremediation agents on stem growth rate (A) and cumulative shoot growth rate (B) of

Spartina alterniflora. Values are means averaged over inundation treatments (n=10). Standard errors 

are presented. C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs 

indicate oiling, and minus signs no oiling. 
 32

 

0

10

20

30

40

50

60

Aboveground Biomass (g/pot)

C- C+ M+ F+ OF+

BIOREMEDIATION AGENTS

Dead

Live 

Figure 24. Effect of bioremediation agents on aboveground biomass of Spartina alterniflora. Values are means 

averaged over inundation treatments (n=10). Standard errors are presented. C=control; M=microbial 

seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, and minus signs no oiling. 

Table 5. Statistical Table for Plant and Soil Variables with P-Values Provided 

for Main Factors and Their Interactions. 

Parameter Main 

Factors 

 Interaction 

 Agents Inund Time Agents* 

Inund 

Agent* 

Time 

Inundation* 

Time 

Agent* 

Inundation*

Time 

Leaf Elongation 

 (4, 8, 12-wks) 

0.0001 0.8699 0.0001 0.2333 0.0787 0.3158 0.5933 

RGR Stem 

 0-12 wk 

0.0003 0.4742 0.7068 

RGR of Height 

 0-12 

0.0001 0.8684 0.9067 

Photosynthetic rate 

 (4 and 12 wk) 

0.0001 0.8823 0.0001 0.9296 0.6851 0.9408 0.1674 

Live Aboveground 

 Biomass 

0.0001 0.3566 0.1389 

Dead Aboveground 

 Biomass 

0.7825 0.5720 0.3986 

Total Aboveground 

 Biomass 

0.0001 0.3566 0.1389 

Resp 

 (2,4,8,12) 

0.0001 0.0001 0.0672 0.0001 0.0107 0.0001 0.0006 

Eh at 2 cm 

 (1,2,4,12 wks) 

0.2595 0.0001 0.0598 0.3346 0.5154 0.0060 0.4725 

Eh at 12 cm 

 1,2,4,12 wks) 

0.8033 0.0004 0.0001 0.8828 0.1547 0.0499 0.1997 

Agents: Bioremediation Agents; Inund: Inundation regimes; Time: Sampling Time; Agent*Inund: Bioremediation Agents by 

Inundation Regime Interaction 
 33

 Soil parameters such as Eh, interstitial sulfide, salinity, pH and nutrients were measured to determine if the 

bioremediation products, oil, and inundation regimes had any effect on soil condition. The addition of the products 

and oil did not significantly affect soil redox potential (Eh) at 2 and 12 cm depths. However, as expected, Eh was 

significantly higher in the drained inundation regime compared with the flood regime (Figs. 25A and B) (Table 5). 

The application of the bioremediation agents and oil did not significantly affect interstitial salinity (data not shown). 

Generally, the products and oil, and the inundation regimes did not significantly affect interstitial pH (Fig 26). 

 Fertilization, as expected, increased interstitial inorganic nitrogen (ammonia plus nitrate) concentration 

throughout the three-month experimental period, with significantly higher nitrogen concentration in the fertilizer 

and oxidant plus fertilizer treatments (Fig. 27A) compared to the control and microbial seeding treatments. 

Microbial seeding did not significantly affect interstitial inorganic nitrogen concentration compared to the control. 

The interstitial phosphorus concentrations in the oxidant treatment were significantly higher than those of the other 

treatments most likely due to the use of KH2PO4 in the oxidant treatment to buffer a possible pH increase by 

PermeOx (Fig. 27B). However, the fertilizer and microbial treatments did not affect interstitial phosphorus 

concentrations compared to the control. 

 

-100

0

100

200

300

400

Soil Eh (mV) at the 2 cm depth

1 2 4 12

TIME (Weeks)

A Drained Flooded

 

-100

0

100

200

300

400

Soil Eh (mV) at 12 cm depth

1 2 4 12

TIME (Weeks)

B Drained Flooded

Figure 25. Effect of inundation regime on soil redox potential at the depth of 2 cm (A) and 12 cm (B) below the soil 

surface. Values are means averaged over bioremediation agents and oil application (n=25). Standard 

errors are presented. 
 34

 

0

1

2

3

4

5

6

7

8

Interstitial pH 

C- C+ M+ F+ OF+

BIOREMEDIATION PRODUCTS

Figure 26. Effect of bioremediation agents on interstitial pH of Spartina alterniflora. Values are means averaged 

over inundation treatments and time of 2, 4, 8, and 12 weeks after the treatment (n=40). C=control; 

M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, and minus 

signs no oiling. Note: Standard errors are small, and bars not apparent. 

 

0

0.5

1

1.5

2

2.5

Interstitial Inorganic Nitrogen (ppm)

C- C+ M+ F+ OF+

BIOREMEDIATION PRODUCTS

A

 

0

1

2

3

4

Interstitial Phosphorus (ppm)

C- C+ M+ F+ OF+

BIOREMEDIATION PRODUCTS

B

P

Figure 27. Effect of bioremediation agents on interstitial inorganic nitrogen (A) and phosphorus (B). Values are 

means averaged over inundation treatments and time of 1, 2, 4, and 12 weeks after the treatment (n=40). 

Standard errors are presented. C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus 

fertilizer. Plus signs indicate oiling, and minus signs no oiling. 
 35

Responses of Microbes to the Treatments

 Soil respiration was measured at 2, 4, 8 and 12 weeks after the treatments to determine the effects of the 

bioremediation products, oil, and inundation regimes on the metabolism of the soil community (Fig. 28). Generally, 

soil respiration rates in the drained regime were significantly higher than those in the flooded regime (Figs. 28 A, B, 

C and D) (Table 5). Surprisingly, the addition of the oxidant plus fertilizer to oiled pots in the drained regime 

(OF+L) sharply increased soil respiration. Soil respiration of the OF+L treatment was significantly (p < 0.0001) 

higher than that of the other treatments 2, 4 and 8 weeks after bioremediation with the peak soil respiration at week 

2 and 4 (Figs. 28 A, B, and C). This dramatic increase in soil respiration was most likely due to an increase in soil 

microbial activity. In addition, fertilizer significantly increased soil respiration 12 weeks after the bioremediation 

treatment (Fig. 28 D). 

 

Figure 28. Effect of bioremediation agents on soil respiration rate 2 (A), 4 (B), 8 (C), and 12 (D) weeks after the 

bioremediation treatment. Values are means (n=5) with standard errors. C=control; M=microbial 

seeding; F=fertilizer; OF=oxidant plus fertilizer. Plus signs indicate oiling, and minus signs no oiling. 

 Bioremediation stimulated soil heterotrophic bacteria, especially in the oxidant plus fertilizer treatment. 

Even at the start of the experiment, treatment effects were noted in some cases; a sampling was several hours after 

the treatment application and because bacteria generally have a doubling time on the order of minutes or hours, 

bacterial responses to the treatments occurred early in the experiment. Soil oxidant plus fertilizer addition showed 

statistically higher populations of heterotrophic bacteria than all other treatments (Fig. 29) in both flooded and 

drained inundation regimes, but no significant difference in petroleum-degrading bacteria (Fig. 30). 

 At week 3, both heterotrophic (Fig. 31) and petroleum-degrading bacteria (Fig. 32) had significantly higher 

population counts in the flooded, oxidizer plus fertilizer treatment than for any other treatment. Under drained 

conditions, week 3 showed significantly higher petroleum-degrading populations in mesocosms treated with 

oxidizer and fertilizer. Neither ATP values (Fig. 35) nor heterotrophic populations showed significant differences 

among bioremediation treatments for the drained treatment. 

 At week 12, heterotrophic bacteria in the flooded and drained treatment again showed significantly higher 

numbers for the oxidant and fertilizer combination than in the other treatments (Fig. 33). There were higher 

numbers of petroleum-degrading bacteria for the oxidant plus fertilizer treatment in the flooded regime compared to 

the other treatments (Fig. 34). ATP values showed no significant differences (Fig. 36). 
 36

Oil Morphology 

Efficacy of the Bioremediation Products

 The presence or absence and extent of different oil morphologies indicative of biodegradation in the 

various application series were used to compare relative degrees of degradation between the various productapplications throughout the experiment. The results of this evaluation are presented in Tables 5 and 6 and 

summarized below. 

 Oil morphological observations indicated that six days after product-application, the oxidant plus fertilizer 

marsh sods started to exhibit oil characteristics that were indicative of biodegradation (paste). By the eighth day, oil 

in the drained oxidant plus fertilizer treatment showed an enhancement of biodegradation (organic coat) as 

compared to the other product-applications and flooding regimes. The only other marsh sods in which oil 

morphologies indicative of biodegradation were observed were the drained fertilized treatments. 

 While other treatments also were characterized by some degradation at this time, the process was not as 

advanced as in the oxidant plus fertilizer treatment or even the fertilizer treatment. In the control and microbial 

seeding treatments, no obvious differences in oil morphologies could be recognized to determine relative degrees of 

biodegradation between the two flooding regimes. Oil was generally moist and creamy during these first 

observations and oil degradation was generally most advanced in the oxidant plus fertilizer followed by the 

fertilizer, control, and microbial treatments (Tables 6 and 7). 

 

1.0000E+08

1.0000E+09

1.0000E+10

Heterotrophic bacteria(Colony-forming units)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 29. Effect of bioremediation agents on heterotrophic bacteria 6 hours after the bioremediation treatment. 

Values are means in colony-forming units per gram (dry weight) of soil (n=5) with standard errors. 

C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, 

and minus signs no oiling. H indicates flooded mesocosms and L Drained. 
 37

 

1.0000E+06

1.0000E+07

1.0000E+08

1.0000E+09

1.0000E+10

Petroleum-degrading bacteria(Colony-forming units)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 30. Effect of bioremediation agents on petroleum-degrading bacteria 6 hours after the bioremediation 

treatment. Values are means in colony-forming units per gram (dry weight) of soil (n=5) with standard 

errors. C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate 

oiling, and minus signs no oiling. H indicates flooded mesocosms and L Drained. 

 

1.0000E+08

1.0000E+09

1.0000E+10

Heterotrophic bacteria(Colony-forming units)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 31. Effect of bioremediation agents on heterotrophic bacteria 3 weeks after the bioremediation treatment. 

Values are means in colony-forming units per gram (dry weight) of soil (n=5) with standard errors. 

C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, 

and minus signs no oiling. H indicates flooded mesocosms and L Drained. 

 

1.0000E+06

1.0000E+07

1.0000E+08

1.0000E+09

Petroleum-degrading bacteria(Colony-forming units)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 32. Effect of bioremediation agents on petroleum-degrading bacteria 3 weeks after the bioremediation 

treatment. Values are means in colony-forming units per gram (dry weight) of soil (n=5) with standard 

errors. C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate 

oiling, and minus signs no oiling. H indicates flooded mesocosms and L Drained. 
 38

 

1.0000E+08

1.0000E+09

Heterotrophic bacteria(Colony-forming units)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 33. Effect of bioremediation agents on heterotrophic bacteria 12 weeks after the bioremediation treatment. 

Values are means in colony-forming units per gram (dry weight) of soil (n=5) with standard errors. 

C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, 

and minus signs no oiling. H indicates flooded mesocosms and L Drained. 

 

1.0000E+08

1.0000E+09

Petroleum-degrading bacteria(Colony-forming units)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 34. Effect of bioremediation agents on petroleum-degrading bacteria 12 weeks after the bioremediation 

treatment. Values are means in colony-forming units per gram (dry weight) of soil (n=5) with standard 

errors. C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate 

oiling, and minus signs no oiling. H indicates flooded mesocosms and L Drained. 
 39

 

1.0000E+04

1.0000E+05

1.0000E+06

ATP (pg)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 35. Effect of bioremediation agents on microbial biomass 3 weeks after the bioremediation treatment. 

Values are means in picograms of ATP per gram (dry weight) of soil (n=5) with standard errors. 

C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, 

and minus signs no oiling. H indicates flooded mesocosms and L Drained. 

 

1.0000E+05

1.0000E+06

1.0000E+07

ATP (pg)

C-L

C-HC+L

C+HM+L

M+H

F+LF+H

OF+L OF+H 

Treatment

Figure 36. Effect of bioremediation agents on microbial biomass 12 weeks after the bioremediation treatment. 

Values are means in picograms of ATP per gram (dry weight) of soil (n=5) with standard errors. 

C=control; M=microbial seeding; F=fertilizer; OF= oxidant plus fertilizer. Plus signs indicate oiling, 

and minus signs no oiling. H indicates flooded mesocosms and L Drained. 
 40

Table 6. Relative Degree of Degradation for Each of the Observations 

by Inundation Regime and Product Application (*) Estimated by Oil Morphology. 

 Day 2 Day 6 Day 8 Day 21 Day 35 Day 49 Day 93

 L H L H L H L H L H L H L H

C+ 1 4 3 3 3 3/2 3/4 3 3 3 4 3 3 4 

F+ 1 2 2 2 2 2/3 2 1/2 1/2 1 2/3 2/1 4 2 

M+ 1 3 2 2 4 4 4/3 4 4 4 1 4 2 1 

OF+ 1 1 1 1 1 1 1 2/1 2/1 2 3/2 1/2 1 3 

Product applications are ranked on a scale of 1 to 4 on relative degradation effectiveness (1=highest, 4=lowest).

Table 7. Relative Degree of Degradation for Each of the Observations Periods 

and Total Score of Degradation-Effectiveness by Product-Application (**). 

 Day 2 Day 6 Day 8 Day 21 Day 35 Day 49 Day 93 TOTAL

C 5 6 5.5 6.5 6 7 7 43.0 

F 4 4 4.5 3.5 2.5 4 6 28.5 

M 4 4 8 7.5 8 5 3 39.5 

OF 2 2 2 2.5 3.5 4 4 20.0 

 

A small number in total indicates a high degradation effectiveness.

 (*) If differences between applications were observed to be minimal, the score consisted of a range with the first 

score being the dominant rating (e.g., 2/1 or 1/2). If no differences were noted, applications were given the 

same score. 

 (**) Scores are derived by adding individual scores for the L and H inundation regimes from Table 2-1 for each 

observation. These composite scores were subsequently added to provide the total score for each 

bioremediation product. 

C = Control 

F = Fertilizer 

M = Microbial seeding 

OF = Oxidant and fertilizer 

L = Drained Regime 

H = Flood Regime 

 The overall effectiveness to enhance biodegradation for each of the treatments is presented in the last 

column of Table 7. This overall-score was derived by adding the individual scores for the each of the observations 

by product-application. The results indicate that the oxidant plus fertilizer was the most effective of all 

bioremediation products in enhancing biodegradation throughout the 12 week period while the microbial seeding 

was the least effective. 
 41

Oil Chemistry

Alkane Degradation. The nC-18/phytane ratio was used, in part, to evaluate normal alkane degradation. 

Bioremediation enhanced alkane degradation. Both the fertilizer and the soil oxidant plus fertilizer treatments 

significantly increased alkane degradation, with significantly lower nC-18/phytane in these treatments compared to 

the controls 3 and 5 weeks after the treatment (Fig. 37) (Table 8). Furthermore, alkane degradation was 

significantly higher in the drained treatment than in the flooded treatment three and five weeks after the treatment. 

However, microbial seeding application did not significantly affect alkane degradation. All of the treatments 

including the controls reduced the resolvable alkane profile such that few resolvable alkanes remained, 

predominately isoprenoids. Since the alkane constituents are so easily degraded, even without bioremediation, they 

should not be a primary parameter to assess efficacy. 

Aromatic Hydrocarbon Degradation. The effect of bioremediation on aromatic hydrocarbon degradation 

appeared to increase with the time after the treatment. Three weeks after treatment application, neither 

bioremediation agents nor inundation treatment had a significant effect on aromatic hydrocarbon degradation (Fig. 

38). However, five weeks after application, TTAH concentration in the treatment receiving soil oxidant plus 

fertilizer under the drained condition was significantly lower than the other treatments (Fig. 38). However, the 

other bioremediation agents had no significant effect on TTAH concentration compared to the control. TTAH 

concentration was significantly lower in the drained treatment than in the flooded treatment 5 weeks after the 

bioremediation application. Twelve weeks after the bioremediation application, TTAH concentrations in the 

treatments receiving fertilizer and treatments receiving oxidant plus fertilizer were significantly lower than the 

control (Fig. 38) (Table 8). 

Variance usually can be reduced by normalizing the data to degradative-resistant compounds contained 

within the oil itself. One technique is the use of hopane as an internal standard. The ratio of TTAH/hopane was 

significantly lower in the treatment receiving the soil oxidant plus fertilizer under the drained condition compared to 

the control 5 and 12 weeks after the treatment (Fig. 39) (Table 8). The ratio of TTAH/hopane was significantly 

lower in the treatment receiving fertilizer compared to the control 3 and 5 weeks after the treatment, but not for 12 

weeks after the treatment. Microbial seeding did not significantly affect the ratio of TTAH/hopane. A large 

variability in the hydrocarbon data due to a patchy oil distribution resulting from an uneven soil surface made it 

difficult to detect treatment differences. 

 

0

0.5

1

1.5

2

nC-18/phytane

C+ M+ F+ OF+ C+ M+ F+ OF+

TREATMENTS

3 weeks 5 weeks

Drained Flooded

Figure 37. Effect of bioremediation agents on alkane degradation 3 and 5 weeks after the bioremediation treatment. 

Values are means (n=5) with standard errors. C=control; M=microbial seeding; F=fertilizer; OF= 

oxidant plus fertilizer. Plus signs indicate 2 l m-2 of oil addition at the beginning of the experiment. 
 42

 

0

100

200

300

400

TTAH Conccentrations (ug/g dry soil)

C+ M+ F+ OF+ C+ M+ F+ OF+

TREATMENTS

3 weeks 5 weeks 12 weeks

Drained Flooded

Figure 38. Effect of bioremediation agents on the residual total targeted aromatic hydrocarbon concentrations 3, 5, 

and 12 weeks after the bioremediation treatment. Values are means (n=5) with standard errors. 

C=control; M=microbial seeding; F=fertilizer; OF=oxidant plus fertilizer. Plus signs indicate 2 L/m2

 of 

oil addition at the beginning of the experiment. 

 

0

10

20

30

40

 TTAH/Hopane 

C+ M+ F+ OF+ C+ M+ F+ OF+

TREATMENTS

12 weeks

5 weeks

3 weeks

Drained Flooded

Figure 39. Effect of bioremediation agents on the residual total targeted aromatic hydrocarbon normalized to 

hopane 3, 5, and 12 weeks after the bioremediation treatment. Values are means (n=5) with standard 

errors. C=control; M=microbial seeding; F=fertilizer; OF=oxidant plus fertilizer. Plus signs indicate 2 

L/m2

 of oil addition at the beginning of the experiment. 
 43

Table 8. Statistical Table for the Oil Chemistry Variables 

 with P-Values Provided for Main Factors and Their Interactions 

Parameter Agents Inund Agent*Inund 

n-C18/phytane 

 (3 week) 

00021 0.0001 0.0001 

n-C18/phytane 

 (5 week) 

0.0305 0.0067 0.7947 

TTAH 

 (3 week) 

0.7966 0.6023 0.8166 

TTAH 

 (5 week) 

0.0955 0.0123 0.3095 

TTAH 

 (12 week) 

0.0874 0.4138 0.8799 

TTAH/Hopane 

 (3 week) 

0.0356 0.4750 0.0401 

TTAH/Hopane 

 (5 week) 

0.0300 0.7025 0.3003 

TTAH/Hopane 

 (12 week) 

0.0067 0.5295 0.2073 

Agents: Bioremediation Agents; Inund: Inundation Regimes; Time: Sampling Time; Agent*Inund: Bioremediation Agents by 

Inundation Regime Interaction 

Discussion 

 In the present study, fertilizer application significantly enhanced plant growth in the treatments receiving 

fertilizer and the soil oxidant plus fertilizer, but microbial seeding application had no effect. Soil oxidant 

application combined with fertilization and the pH buffer, KH2PO4, significantly enhanced soil parameters such as 

soil interstitial phosphorus concentration, soil respiration rate, and soil microbial counts. More importantly, 

concentrations of residual aromatic hydrocarbons were significantly lower in the treatment receiving soil oxidant 

plus fertilizer compared to the control. Generally, microbial seeding did not affect oil degradation compared to the 

control. 

 We used a slow-release fertilizer containing 28% N, 8% P2O5 (or 3% P), and 0% K in the present 

experiment. As a result, fertilizer application significantly increased interstitial nitrogen concentration in the 

treatments receiving fertilizer and soil oxidant plus fertilizer. The increased nitrogen was primarily responsible for 

greater plant growth as evidenced by higher leaf elongation rate, photosynthetic rate, stem density, and live 

aboveground biomass. It is well known that nitrogen limits the growth of Spartina alterniflora. Fertilizer 

application also increased soil microbial respiration 8 and 12 weeks after the bioremediation treatment. Increased 

microbial activity appeared to be responsible for the increased TTAH and alkane degradation. Previous studies 

(Scherrer and Mille 1990; Lindstrom et al. 1991; Banks and Schwab 1993; Atlas 1995 a and b; Venosa et al. 1996; 

Lin and Mendelssohn 1998a, 1998b; Lin et al. 1999c, Williams et al. 1999) indicated that fertilization with 

inorganic nitrogen and phosphorus and organic fertilizer such as poultry litter enhanced degradation of petroleum 

hydrocarbons. In the present study, fertilizer application may directly enhance soil microbial populations and 

activity, thus increasing oil degradation. Additionally, fertilizer may increase oil degradation through a 

phytoremediation effect since fertilization enhanced plant growth, thus potentially promoting oil phytoremediation 

(Lin and Mendelssohn 1998b). However, in this study we can not separate the effect of fertilizer on oil degradation 

resulting from increased microbial activity, per se, from that due to plant-induced degradation and uptake. 

 The soil oxidant treatment had the greatest effect on soil variables and oil degradation rate. The greatest 

increases in the interstitial phosphorus concentration, soil respiration rate, soil heterotrophic microbial counts, oildegrading microbial population, and degradation rate of alkane and aromatic hydrocarbons occurred in the treatment 

receiving soil oxidant and fertilizer compared to the other treatments. Soil respiration measures CO2 release from 

all living organisms, thus increased soil respiration may be also due to CO2 efflux from plant roots. However, if the 

increase in soil respiration were primarily caused by an increase in root respiration, the soil respiration in other 
 44

fertilization treatments (e.g. F+H, F+L and OF+H) would have been expected to increase to a similar extent as the 

OF+L treatment. In addition, greater soil microbial populations in this treatment support a microbially mediated 

effect. Soil oxidation status is important for oil degradation (Lin et al., 1997, 1988a: Hambrick et al. 1980). The 

purpose of the soil oxidant is to oxidize the soil, thus increasing aerobic oil degradation. A handful of studies have 

reported the enhancements of oil degradation by oxidants although they were not in wetland environments 

(Lipczynskakochany 1992; Wang and Latchaw 1990; Lovley et al. 1994). The addition of hydrogen peroxide 

significantly increased degradation of the pollutants, aqueous nitrobenzene and nitrophenols, in a homogeneous 

phase (Lipczynskakochany 1992). In batch methanogenic cultures, omicron-cresol (phenolic compounds) under 

high doses of hydrogen peroxide was biodegradable to methane and the biodegradable fraction increased with 

increasing dose of hydrogen peroxide (Wang, and Latchaw 1990). Lovley et al. (1994) suggest that increasing the 

bioavailability of Fe(III) by adding suitable ligands provides a potential alternative to oxygen addition for the rapid 

bioremediation of petroleum-contaminated aquifers. 

 In the present study, increased oil degradation in the treatment receiving the soil oxidant plus fertilizer was 

caused by the soil oxidant or the nitrogen and phosphorus fertilizer added with the oxidant. As mentioned in 

Materials and Methods, in addition to 13.7 g/pot of the soil oxidant (PermeOx), 6.85 g/pot of KH2PO4 was applied 

to this treatment to buffer the high pH of the PermeOx. The applied KH2PO4 in the soil oxidant treatment is 

equivalent to application of 253 kg P/ha, almost 10-fold higher than the amount of phosphorus contained in the 

fertilizer (Customblen) treatment. Thus, the higher oil degradation may also be due to the applied phosphorus 

contained in the pH buffer KH2PO4. Wright et al. (1997) reported that the addition of phosphorus and nitrogen in a 

mesocosm experiment significantly increased oil degradation, but nitrogen alone did not. This suggests that 

phosphorus may limit microbial degradation of oil in some wetlands. In the present study, interstitial phosphorus 

concentration was about 0.1 ppm in the control. This low phosphorus concentration may limit microbial activity. 

Application of the pH buffer KH2PO4 increased interstitial P to about 3 ppm (30-fold higher than the control), and 

thus may have enhanced microbial oil degradation. Other evidence also suggests that the increased oil degradation 

was not due to the soil oxidant. Soil oxidant application did not increase the redox potential in this experiment. In 

addition, heterotrophic microbial populations increased immediately after the oxidant treatment in both flooded and 

drained conditions. The soil in the drained inundation regime was quite oxidized. The soil oxidant did not increase 

soil redox potential above that in other drained treatments, indicating that something other than the soil oxidant may 

have played a role in enhancing microbial counts. The addition of KH2PO4 with the oxidant was most likely the 

cause. Maki et al. (1999) also reported sharp increases in bacterial number and the oxygen consumption 

immediately after addition of sludge containing high concentrations of phosphorus. 

 Microbial seeding did not significantly affect the marsh plants, soil variables, or oil degradation. 

Interstitial nitrogen and phosphorus concentrations were quite low in the control and microbial seeding treatments. 

Microbial seeding did not increase soil fertility, and did not affect plant response. Addition of microbial 

populations did not significantly increase the microbial counts nor the activity of the soil microbes and oil 

degradation rate. Previous studies have indicated controversial results of microbial inoculum on bioremediation. 

Jorgensen et al. (2000) reported no particular effect of added inoculum for soil microbial respiration and oil 

degradation in a field investigation of bioremediation of diesel-contaminated soil. In a review paper, Atlas (1995b) 

concluded that although seeding with adapted non-indigenous microbial hydrocarbon degraders has been tested on 

smaller spills, bioremediation to remove petroleum pollutants by microbial seeding has yet to be demonstrated as 

efficacious in field trials. However, in a bench-top scale experiment, Cassidy et al. (1997) showed that microbial 

seeding encapsulated in kappa-carrageenan can enhance pollutant mineralization in a chemically contaminated soil 

containing pentachlorophenol and petroleum hydrocarbons, but native cells did not. In the present study, microbial 

seeding may have had no effect due to the natural prevalence of microbes in wetlands or due to the low inorganic 

nutrient concentrations in the soil, limiting inoculum activity in this study. 

Summary 

 Efficacy of bioremediation varied with the different agents applied. Fertilization significantly enhanced 

plant growth in the treatments receiving fertilizer and the soil oxidant plus fertilizer, but microbial seeding did not 

affect plant growth. Oil application and inundation regime did not affect growth of S. alterniflora. More 

importantly, bioremediation with the soil oxidant plus fertilizer combined with the pH buffer, KH2PO4,

significantly affected soil nutrient concentrations, soil microbes, and oil degradation rate, especially in the drained 

condition. However, the effect of soil oxidant cannot be clearly separated in the present study from the effects of 

extra phosphorus contained in the pH buffer of KH2PO4. A future experiment will be designed to determine the 
 45

role of soil oxidant and phosphorus in bioremediation. Fertilizer application also significantly increased oil 

degradation. The effect may be due to the fertilizer itself or through a phytoremediation effect; this needs further 

investigation. Microbial seeding did not significantly affect the plant, soil microbes, or oil degradation, but the low 

soil nutrient concentrations may have limited the inoculated microbial seeding activity. Future research should 

investigate the microbial inoculum in a higher fertility soil environment before eliminating its application for 

bioremediation in the marsh environments. In addition, inundation regime affected bioremediation. Oil degradation 

was greater in the drained condition than in the flooded condition. The current study shows the potential of 

bioremediation for oil spill cleanup in coastal wetlands. 
47 

CHAPTER 3 

EFFECTS OF BIOREMEDIATION AGENTS ON OIL DEGRADATION ON MINERAL AND SANDY 

SALT MARSH SEDIMENTS 

by Qianxin Lin, Irving A. Mendelssohn, Charles B. Henry, Jr., Edward B. Overton, 

Ralph J. Portier, Paulene O. Roberts, and Maud M. Walsh 

Microbial products, cultured and selected to enhance oil degradation rates, have great uncertainties, 

especially in systems such as wetlands where hydrocarbon degrading bacteria are naturally prevalent (see 

Introduction for a background discussion). For example, a microbial product used in an experimental mode to test 

its effectiveness in oil spill cleanup in a marsh in Galveston Bay did not significantly enhance oil degradation 

(Mearns 1991). Also, two microbial products, which exhibited enhanced biodegradation of Alaska North Slope 

crude oil in shaker flask tests, did not accelerate biodegradation in a field experiment conducted on an oiled beach 

in Prince William Sound, although the high variability in the data, the highly weathered nature of the oil, and a lack 

of sufficient time for biodegradation were cited as possible reasons for the lack of response (Venosa et al. 1992). In 

a more recent study, Venosa et al. (1996) found that an indigenous microbial inoculum did not increase oil 

biodegradation in a beach environment. Regardless of these equivocal results, many microbial products have been 

commercialized. If added microbes, per se, are not effective in increasing oil degradation, the high costs of 

microbial amendments may not be warranted. Recently, Wright et al. (1997) reported that the addition of 

phosphorus and nitrogen in a mesocosm experiment significantly increased oil degradation, but nitrogen alone did 

not. However, comparisons of the effectiveness of different bioremediation agents in wetland environments, based 

on oil chemical analyses that can identify enhanced biodegradation, are depauperate in the published literature. 

 Soil oxidation state is another important factor influencing oil biodegradation in wetland environments. 

Generally, wetland soils are saturated with water and exhibit biochemically reduced soil conditions, which inhibit 

oil degradation (Hambrick et al. 1980). Therefore, procedures that increase the oxidation status of sediments may 

favor bioremediation (Lin and Mendelssohn 1997). The use of soil oxidants to increase oil biodegradation in the 

wetland environment has received little attention (McKee and Mendelssohn 1995). 

 We initiated a multi-disciplinary investigation to address the question: Is bioremediation, via fertilization, 

microbial seeding or soil oxidant application, an effective and ecologically safe means of oil spill cleanup in coastal 

wetlands? Our overall goal was to determine the potential for the use of bioremediation as an oil-spill cleanup 

technique in wetlands. Specifically, we determined the effects of fertilization, microbial seeding and soil oxidant 

application on (Hershner and Lake 1977) petroleum hydrocarbon degradation, (Lee et al. 1981) the extent to which 

the effectiveness of these products is modified by marsh soil type, and (Alexander and Webb 1987) the adverse 

impacts, if any, of these bioremediation agents on wetland vegetation and microbial activity. 

Materials and Methods 

Marsh Sod 

 Thirty mineral marsh sods (soil and intact vegetation), approximately 28 cm in diameter (0.062 m2

) and 25 

cm deep, were extracted from the inland zone (approximately 5-10 m from the creekbank natural levee) of a 

Spartina alterniflora- dominated salt marsh located west of Cocodrie, Louisiana. In addition, 30 sods of the same 

size were collected from a sandy backbarrier marsh dominated by S. alterniflora located approximately 100 meters 

from the shoreline of the Gulf of Mexico near Fourchon, Louisiana. All 30 sods from each marsh type had similar 

plant biomasses. Marsh sods from the inland zone were chosen because the inland zone comprises the largest aerial 

extent of most salt marshes, and is generally of relatively high mineral and organic content. In contrast, the 

backbarrier marsh is sandy and of low mineral and organic content. The mineral marsh soil was composed of 

63.8% clay, 31% silt and 5.2% fine sand, with 18.6% soil organic matter and 0.312 g/cm3

 soil bulk density. The 

sandy marsh soil was composed of 2.5% clay, 6.3% silt and 91.2% fine sand, with 0.5% soil organic matter and 

1.35 g/cm3 soil bulk density. We recognize that soil types will likely influence bioremediation, and, thus, this factor 
 48

was examined in this research. Spartina alterniflora is the dominant intertidal salt marsh grass along the Atlantic 

and Gulf Coasts of the United States, and results from this study should be generally applicable to many other salt 

marshes. 

Experimental Design 

 In the greenhouse, artificially weathered Louisiana crude oil (25% weathered by volume) was applied at a 

dose of 2 L/m2

 (2 mm oil thickness) to the surface of all experimental units (marsh sods in 28 cm x 32 cm buckets 

fitted with water level controls). After the applied oil was evenly spread over the surface water in the buckets, the 

water was drained from the bottom of the buckets to allow the oil to come in contact with and penetrate the soil of 

each sod. The initial oil concentration in the top 5 cm of soil after the application of the oil was 20.8 mg/g dry soil 

(s.e. 0.85) in the sandy sediment and 57.9 mg/g dry soil (s.e. 9.7) in the mineral sediment as determined by the 

gravimetric method with dichloromethane (DCM) extraction (Lin and Mendelssohn 1996). The difference in the 

initial oil concentration between two types of sediment was primarily due to their different soil bulk densities. The 

oil was artificially weathered to remove hydrocarbons nC-10 and below, thereby simulating oil spilled in open water 

and subsequently transported into a salt marsh by winds or tides. The following treatments were randomly assigned 

to both mineral and sandy marsh sods with applied oil: (1) slow release fertilizer, (2) microbial seeding plus slow 

release fertilizer, (3) soil oxidant, (4) soil oxidant plus slow release fertilizer, (5) soluble fertilizer, and (6) control 

(no application of products). The experimental design was a randomized block by the location within greenhouse 

with a 6 x 2 factorial treatment arrangement (the six bioremediation types mentioned above and two soil types—

mineral and sandy). Each treatment combination was replicated five times for a total of 60 marsh sods. Statistical 

analysis was conducted with the SAS system (SAS 1990). General Linear Model (GLM) was used to test for 

statistically significant differences (P<0.05) among the treatments, and Duncan's multiple range test was used to 

determine significant differences among the main factors. Treatment-level combination differences, if interactions 

of main factors were significant, were tested with least square means. 

Experimental Procedures 

 The products were applied to the soil surface in a manner similar to that during a field application and 

following the manufacturer's specifications. Slow release fertilizer (Osmocote 14-14-14, product of Grace Sierra, 

Milpitas, CA) was applied at the rate of 93 g/m2 each application (130 kg N as NH4 and NO3, 57 kg P as P2O5 and 

108 kg K as K2O per hectare). The microbial product (Petrobac, selected for hydrocarbon degradation in a saline 

medium, a product of Polybac Corp., Bethlehem, PA) was applied at the rate of 0.833 L/m2 of inoculum (46 g of 

Petrobac/L of deionized water). A soil oxidant (PermeOx, a product of FMC Corp., Philadelphia, PA) was applied 

at the rate of 111 g/m2

. All products were applied 3 times (i.e., at days 0, 40, and 80 after application of oil) during 

the low tide over a 4-month experimental period (8/96-12/96). The soluble fertilizer treatment (NH4NO3 and 

NaH2PO4) was implemented by applying nitrogen and/or phosphorus to maintain concentrations above 5 ppm in the 

interstitial water throughout the experiment to insure high and likely non-limiting concentrations for bioremediation. 

Semi-quantitative measurements of ammonium, nitrate and phosphate (CHEMets Kit, products of CHEMetrics, 

Inc., Calverton, VA) were used weekly to determine when application of soluble fertilizer was needed. Also, 

quantitative determinations of ammonium, nitrate and phosphate were conducted as described below. Water levels 

were manipulated to simulate a diurnal tide regime with 12 hours of night-time high tide (5-cm deep surface water) 

and 12 hours of day-time low tide (water drained from the bottom of the pots to individual reservoirs and reused to 

create high tide). Tapwater was used to replace evapotranspirational water loss. 

Plant and Soil Responses

Aboveground Biomass of Dominant Marsh Plants. Plant aboveground biomass was analyzed at the end of 

a four-month experimental period to determine the effect, if any, of product addition on plant growth. Plant 

aboveground material was clipped at the soil surface. Live and dead components were separated, dried in a oven at 

65°C to constant weight, and weighed. 

Soil Respiration Rate. In-situ measurement of soil respiration rate, an indicator of soil microbial activity, 

was conducted with an ADC (the Analytical Development Co. LTD) infra-red gas analyzer (IRGA) by measuring 

carbon dioxide production from soil. A PVC chamber (4 cm in diameter and 8 cm in height) with one open end was 

equipped with an inlet and an outlet for air flow through the chamber. The open end of the chamber was inserted 

into the soil 4 cm below the soil surface. An air flow rate through the chamber was held constant at 300 ml per 
 49

minute by an ADC mass flow controller. The respiratory CO2 produced from the soil resulted in a difference in 

CO2 concentration between inlet and outlet that was measured by an ADC infra-red gas analyzer. Soil respiration 

was calculated based on the CO2 exchange rate from the soil per unit surface. 

Soil Redox Potential. Soil redox potentials at 2 and 10 cm depths were determined with bright platinum 

electrodes and a calomel reference electrode. Readings were taken with a portable pH/mV digital meter. The 

potential of a calomel reference electrode (+244 mV) was added to each value to calculate Eh (Patrick et al., 1996). 

Soil Nutrient Concentrations. Interstitial water samples were withdrawn from soil sods with a simple 

apparatus as described by McKee et al. (1988). This consisted of a small diameter (3 mm inside diameter) rigid 

plastic tube, containing numerous 0.5 mm diameter holes covered with 3 to 4 layers of cheesecloth, connected to a 

30 ml syringe. The collected interstitial water was filtered through a 0.45 μ syringe filter. Inorganic nutrients in the 

filtered interstitial water were analyzed to determine the effect of product application on soil fertility and other 

variables. Ammonium-nitrogen (NH4) and nitrate-nitrogen (NO3 + NO2) were analyzed with an auto-analyzer 

before and two weeks after the third product application. Nutrient levels were analyzed at these specific times to 

determine to what extent available nutrients were depleted before the next nutrient application and whether the 

nutrient levels remained high after addition (over a two-week period). Total inorganic nitrogen was determined as 

the sum of NH4, NO3, and NO2-nitrogen concentrations. Phosphorus and potassium concentrations in the 

interstitial water were analyzed with ICP (inductively coupled argon-plasma emission spectrometer). Phosphate 

concentrations were estimated from regression equations relating ICP phosphorus to methylene blue determined 

phosphate measured in a subset of marsh sods (Y = 1.195x-0.718; r2

=0.996; n=20). 

Microbial Populations. The top 5 cm of the soil in each sod was collected for oil chemistry and microbial 

analyses. Heterotrophic bacterial populations and petroleum-degrading organisms were enumerated to determine 

the relationship between microbes and oil degradation. Soils for microbial analyses were subsampled from 

thoroughly mixed surface soil samples. Standard plate counts were performed according to the method described in 

Wollum (1982). Approximately 1.25 g of sample was added to 99 ml of normal saline (0.85 weight % NaCl). 

Several drops of sterile Tween 80 were added as a surfactant. The bottles were mixed on a vortex mixer, then 

shaken vigorously. Subsequent serial dilutions in 99 ml of normal saline were made up to 10-6 g/ml dilution and 

plated by the pour plate method. Nutrient agar (Difco) was used to grow general heterotrophic bacteria. A minimal 

salt agar to which 50 ppm kerosene was added as the sole carbon source was used for the selective culturing for 

petroleum-degrading organisms. Plates were incubated at 30 ̊C for 24-26 hours, then colony-forming units of 

bacteria were enumerated. Values were calculated for colony-forming units/g dry weight of soil. 

Residual Oil Chemistry in the Soil. The top 5 cm of soil from each sod was collected, thoroughly 

homogenized, subsampled and extracted using a modified EPA method 3550. Approximately 20 grams of wet 

sediment were extracted using dichloromethane and sodium sulfate as a chemical drying agent (Venosa et al. 1996; 

Sauer and Boehm 1993; Henry and Overton 1993; Henry et al. 1993), then reduced to a final volume of 10 ml. A 

field treatment composite was then analyzed by gas chromatography-mass spectrometry (GC/MS) operated in the 

Selective Ion Monitoring (SIM) Mode to characterize compositional changes in selected aromatic hydrocarbons 

(AH) and normal hydrocarbons (NH). Although these selected targeted AH generally represent less than 5% of the 

bulk oil composition, they are essential to characterize petroleum source, identify potential biological effects, 

determine exposure pathways, and monitor weathering trends and degradation of the oil (Sauer and Boehm 1991;

Roques et al. 1994). The targeted aromatic hydrocarbons are listed in Table 9. 

Results 

Plant Growth Response 

 The effect of the bioremediation products on the growth response of the salt marsh grass Spartina 

alterniflora was assessed by determining aboveground biomass. Fertilizer had a positive effect on aboveground 

biomass (Fig. 40) regardless of soil type (no significant treatment by soil type interaction) with live aboveground 

biomass significantly higher (p <0.0001) in the treatment receiving soluble fertilizer than without. Live 

aboveground biomass of S. alterniflora in all treatments receiving slow release fertilizer tended to be greater than 

that without fertilizer, but differences were not statistically significant. As expected, application of the microbial 

product had no significant effect on plant growth response; live aboveground biomass in the microbial product plus 

fertilizer treatment was not significantly different from that receiving fertilizer alone. Also, application of the soil 
 50

oxidant had no significant effect on plant growth. Live aboveground biomass in the treatment receiving the soil 

oxidant alone was not significantly different from the control, and live aboveground biomass in the treatment 

receiving the soil oxidant plus fertilizer was not significantly different than that receiving fertilizer alone. Dead and 

total aboveground biomass did not significantly differ with product application, and there was no significant 

difference in aboveground biomass between marsh types. 

Table 9. Target Compounds Assessed by GC/MS 

Compound ion mass Compound ion mass Compound ion mass

alkanes* (nC-10 thru nC-31) 85 C-3 fluorenes 208 fluoranthrene/pyrene 202 

decalin* 138 dibenzothiophene 184 C-1 pyrenes 216 

C-1 decalin* 152 C-1 dibenzothiophenes 198 C-2 pyrenes 230 

C-2 decalin* 166 C-2 dibenzothiophenes 212 chrysene 228 

C-3 decalin* 180 C-3 dibenzothiophenes 226 C-1 chrysenes 242 

naphthalene 128 phenanthrene 178 C-2 chrysenes 256 

C-1 naphthalenes 142 C-1 phenanthrenes 192 benzo(b)fluoranthene 252 

C-2 naphthalenes 156 C-2 phenanthrenes 206 benzo(k)fluoranthene 252 

C-3 naphthalenes 170 C-3 phenanthrenes 220 benzo(e)pyrene 252 

C-4 naphthalenes 184 naphthobenzothiophene 234 benzo(a)pyrene 252 

fluorene 166 C-1 naphthobenzothiophenes 248 perylene 252 

C-1 fluorenes 180 C-2 naphthobenzothiophenes 262 indeno(1,2,3-cd)pyrene 276 

C-2 fluorenes 194 C-3 naphthobenzothiophenes 276 sterenes (217 family)* 217 

dibenzo(a,h)anthracene 278 benzo(g,h,i)perylene 276 hopanes (191 family)* 191 

Sum of these compounds excluding those identified with a * is the TTAH value. 

 

0

25

50

75

100

125

BIOREMEDIATION AGENTS

Total

Dead

Live

a a

ab ab

ab

b

Figure 40. The effect of bioremediation products on aboveground biomass of Spartina alterniflora, averaged over 

marsh type, after a 4-month experimental period (n=10, means ± standard errors). Means with the same 

letter are not significantly different within each biomass component; for example, means with the letter a 

are significantly different from means with the letters bc, but not from means with the letters ab. Cont: 

control; Ox: soil oxidant; SRF: slow release fertilizer; Ox+SRF: soil oxidant plus slow release fertilizer; 

Mic+SRF: microbial agent plus slow release fertilizer; SolFert: soluble fertilizer. 

Microbial Response 

 In-situ soil respiration was measured to identify if the application of the bioremediation agents affected the 

metabolism of the soil microbial community. The fertilizer treatments had significant positive effects on soil 
 51

respiration compared to those treatments without fertilizer for both soil types (Fig. 41, no significant treatment by 

soil type interaction). However, addition of the microbial product or the soil oxidant had no significant effect on 

soil respiration. Soil respiration rates of the treatment receiving microbial seeding plus slow release fertilizer was 

not significantly different from that of the fertilizer alone (Fig. 41), suggesting that the microbial agent had no effect 

on soil respiration. Soil respiration rates of the treatments receiving the soil oxidant alone and the soil oxidant plus 

fertilizer were also not significantly different from those of the control and fertilizer alone, respectively (Fig. 41). 

Overall, soil respiration was significantly higher for the sandy soil (7.04 μmol/m2/s ± 0.54 s.e., n=30) than for the 

mineral marsh soil (5.9 μmol/m2/s ± 0.33 s.e, n=30). 

 

0

2.5

5

7.5

10

BIOREMEDIATION AGENTS 

a

ab

bc

c c c

Figure 41. Soil microbial respiration responses, averaged over marsh type, to bioremediation product treatments 7 

weeks after application (n=10, mean ± s.e). Means with the same letter are not significantly different. 

Cont: control; Ox: soil oxidant; SRF: slow release fertilizer; Ox+SRF: soil oxidant plus slow release 

fertilizer; Mic+SRF: microbial agent plus slow release fertilizer; SolFert: soluble fertilizer. 

 Soil microbial response was also evaluated by determining the colony-forming units of heterotrophic 

bacteria and petroleum-degrading organisms (Table 10). Fertilizer application tended to increase the populations of 

heterotrophic bacteria (Table 10), which is likely correlated to increased microbial degradation of residual oil. 

However, the differences in heterotrophic bacterial numbers between the treatments with and without fertilizer were 

not statistically significant (p=0.39) because of large within-treatment variation. The application of the microbial 

product and the soil oxidant had no significant effects on heterotrophic bacterial numbers. Similar trends were 

observed for petroleum-degrading organisms, with no significant difference among product treatments (p=0.35) 

(Table 10). Heterotrophic bacterial numbers (p=0.1) and petroleum-degrading organisms (p=0.308) did not 

significantly differ between soil type, thus means are averages over the two soil types (Table 10). 

Table 10. Effect of Bioremediation Products on Soil Heterotrophic Populations 

and Petroleum Degraders After a 4-Month Experimental Period 

____________________________________________________________________________________________

 Cont Ox SRF Ox+SRF Mic+SRF SolFert 

Hetero- 1.76 × 108

 3.76 × 107 1.60 × 109 2.92 × 109 4.92 × 109 1.44 × 109

bacteria (3.49 × 107

) (1.09 × 107) (1.16 × 109) (1.04 × 109) (4.1 × 109) (5.43 × 108) 

Petro- 3.32 × 108

 2.28 × 107 7.15 × 107 4.48 × 109 1.92 × 108 2.29 × 108

degrader (1.98 × 108

) (1.46 × 107) (2.69 × 107) (3.99 × 109) (1.06 × 108) (1.26 × 108) 

____________________________________________________________________________________________ 

Cont: control; Ox: soil oxidant; SRF: slow release fertilizer; Ox+SRF: soil oxidant plus slow release fertilizer; Mic+SRF: 

microbial agent plus slow release fertilizer; SolFert: soluble fertilizer. Values Are Means (the colony-forming units/g dry soil), 

averaged over the two soil types, with standard errors in parentheses (n=10). 
 52

Soil Fertility 

 The effect of fertilizer addition on soil fertility was determined by analyzing the concentrations of 

inorganic nutrients in the soil interstitial water. The application of soluble fertilizer significantly (p<0.0001) 

increased the concentrations of ammonium, nitrate and phosphate in the interstitial water (Table 11). The 

ammonium, nitrate and total inorganic nitrogen concentrations tended to be higher in all treatments with slow 

release fertilizer than without, but the differences were not statistically significant. Marsh types did not affect the 

interstitial nitrogen concentration. The interstitial phosphate concentration (Table 11) was significantly higher in 

treatments with fertilizer than without (p<0.0001) and higher in the sandy soil type than in the mineral soil (p 

<0.0001). A significant interaction (p<0.0001) between product treatment and soil type indicated that fertilizer 

application increased interstitial phosphate concentration more in the sandy soil than in mineral soils. Potassium 

concentration did not significantly vary among bioremediation treatments.

Table 11. Effect of Bioremediation Products on the Inorganic Interstitial Nutrient Concentrations 

Before and 2 Weeks After the Third Product Application. 

______________________________________________________________________________ 

Nutrients Mineral Marsh Sandy Marsh 

 Cont Ox SRF O+F M+F SF Cont Ox SRF O+F M+F SF 

NH4 0.07 0.09 0.47 0.34 0.17 5.66*

 0.06 0.07 0.43 0.11 0.21 5.82*

 (0.03) (0.05) (0.18) (0.16) (0.07) (0.67) (0.01) (0.02) (0.13) (0.03) (0.12) (1.85) 

NO3 0.10 0.05 0.13 0.04 0.11 2.35*

 0.11 0.10 0.06 0.18 0.13 1.42*

 (0.01) (0.01) (0.03) (0.01) (0.03) (1.22) (0.01) (0.02) (0.02) (0.04) (0.06) (1.04) 

Total 0.17 0.147 0.60 0.38 0.28 8.01*

 0.17 0.17 0.50 0.29 0.34 7.32*

inorganic N (0.05) (0.06) (0.20) (0.15) (0.09) (1.75) (0.02) (0.02) (0.15) (0.06) (0.18) (2.68) 

PO4 0.44 0.40 1.245* 1.96* 1.23*

 1.75* 0.67 0.34 9.60* 4.12* 11.2* 20.25*

 (0.04) (0.06) (0.26) (0.36) (0.24) (0.32) (0.14) (0.12) (1.47) (1.02) (2.31) (5.91) 

_______________________________________________________________________________________________ 

Values are means (ppm), averaged over the two sampling events, with standard errors in parentheses (n=5). Cont: control; Ox: 

soil oxidant; SRF: slow release fertilizer; O+F: soil oxidant plus slow release fertilizer; M+F: microbial agent plus slow release 

fertilizer; SF: Soluble fertilizer. Values with asterisks (*) are significantly higher than their controls. 

Oil Biodegradation 

 In general, fertilizer application enhanced oil biodegradation in the soil. The concentrations of both total 

targeted normal hydrocarbons (TTNH) and total targeted aromatic hydrocarbons (TTAH) remaining in the soil were 

significantly reduced (p<0.0001) by all remediation treatments receiving the fertilizer application (Figs. 42 and 43), 

Table 12). Concentration of TTNH remaining in marsh soils of the treatments receiving fertilizer was only 16% of 

those without fertilizer (averaged over all fertilizer treatments). Concentration of TTNH remaining in sandy soil 

was significantly higher (p<0.0001) than in mineral soil, with 177 μg/g dry soil (± 35, n = 30) in the former versus 

the latter. A significant interaction (p<0.0001) between product addition and marsh type indicated that the soil 

oxidant addition in the mineral soil appeared to have an adverse effect on TTNH degradation as evidenced by 

higher TTNH concentrations with the soil oxidant compared to the control in the mineral soil, and no difference in 

the sandy soil. Also, soil type significantly affected the percentage of fertilizer-enhanced degradation of TTNH 

(Table 12) compared to the control (mineral soil: 26% ± 5 (n=20), averaged over treatments receiving fertilizer 

relative to the mean of the mineral control; sandy soil: 13% ± 3, n=20), suggesting that fertilizer application was 

more effective in enhancing TTNH degradation in the sandy soil than in the mineral soil. 
 53

 

0

100

200

300

400

500

TREATMENTS 

Mineral marsh Sandy marsh

 a a

 a a

 ab

 ab ab ab

 b

 c

 cd d

 a

b

Figure 42. Effect of the bioremediation products on total targeted normal hydrocarbons (TTNH) (n=5, mean ± s.e). 

Means with the same letter are not significantly different. Cont: control; Ox: soil oxidant; SRF: slow 

release fertilizer; Ox+SRF: soil oxidant plus slow release fertilizer; Mic+SRF: microbial agent plus slow 

release fertilizer; SolFert: soluble fertilizer. 

 

0

25

50

75

100

BIOREMEDIATION AGENTS

a

b

bc

bcd cd

d

Figure 43. Total targeted aromatic hydrocarbons (TTAH) remaining in the soil, averaged over marsh type, as a 

function of the bioremediation product treatments (n=10, mean ± s.e.). Means with the same letter are 

not significantly different. Cont: control; Ox: soil oxidant; SRF: slow release fertilizer; Ox+SRF: soil 

oxidant plus slow release fertilizer; Mic+SRF: microbial agent plus slow release fertilizer; SolFert: 

soluble fertilizer. 
 54

Table 12. Effect of Bioremediation Product Application on Relative Oil Degradation, 

the Percentage Decrease of Petroleum Components Normalized to Each Soil Type's Control (Control 100%). 

______________________________________________________________________________________________ 

 Mineral Marsh Sandy Marsh __

 Cont Ox SRF O+F M+F SF Cont Ox SRF O+F M+F SF

TTNH 100b

 167a 7c 36c 43c 18c 100b 90b 8c 12c 22c 9c

 (11.1) (14) (2) (11) (10) (5) (7) (14) (4) (9) (10) (3) 

TTAH 100bc 271a

 54c 138b 123bc 83bc 100bc 123bc 58c 77bc 66c 70c

 (15) (30) (14) (16) (14) (14) (5) (16) (8) (12) (80) (8) 

 Cont Ox SRF O+F M+F SF __

TTNH/ 100a

 78a 10b 14b 22b 11b

Hopane (6) (8) (3) (4) (4) (2) 

TTAH/ 100a

 115a 60b 69b 66b 64b

Hopane (4) (9) (5) (3) (3) (4) 

____________________________________________________________________________________________ 

Values are means (% of the control) with standard errors in parentheses (n=5 for TTNH and TTAH, n=10 for TTNH/Hopane and 

TTAH/Hopane, averaged over marsh type). Values lower than 100% indicate enhanced biodegradation, and values higher than 

100% indicate inhibited biodegradation. Cont: control; Ox: soil oxidant; SRF: slow release fertilizer; O+F: soil oxidant plus 

slow release fertilizer; M+F: microbial agent plus slow release fertilizer; SF: Soluble fertilizer. Means with the same letter are 

not significantly different within each hydrocarbon component. 

 Bioremediation product application significantly (p<0.0001) affected TTAH concentrations in the 

sediments (Fig. 12). Concentrations of TTAH remaining in marsh soils receiving fertilizer were 57% lower than 

those without fertilizer (averaged over all fertilizer treatments vs. no fertilizer treatments). TTAH concentrations in 

the treatments receiving slow release fertilizer and soluble fertilizer were significantly lower than the control. In 

addition, TTAH concentration in the sandy substrate was significantly higher than in mineral substrate, with 59 μg/g 

dry soil (± 4, n=30) in the former and 32 μg/g dry soil (± 4, n=30) in the latter. TTAH concentrations in the 

treatments receiving the soil oxidant alone were significantly higher than that of the control. Also, soil type 

significantly affected the percentage of fertilizer-enhanced degradation of TTAH (Table 12) compared to the control 

(mineral soil: 99% ± 10% [n=20], averaged over treatments receiving fertilizer relative to the mean of the mineral 

control; sandy soil: 68% ± 4 [n=20]), suggesting that the fertilizer application was more effective in enhancing 

TTAH degradation in the sandy soil than in the mineral soil. 

 The TTNH/Hopane and TTAH/Hopane ratios were consistently significantly lower (p<0.0001) in the 

treatments with fertilizer than without, supporting the conclusion of a fertilizer-enhanced biodegradation (Table 12). 

In addition, the percentage change of TTNH/Hopane and TTNH/Hopane for the fertilizer treatments compared to 

the control's (Table 12) showed no effects of the microbial product and the soil oxidant. However, a fertilizer effect 

on oil biodegradation was evident. Overall, the addition of the microbial product did not significantly lower the 

values of TTNH, TTAH, TTNH/Hopane and TTAH/Hopane in the soil relative to controls (Figs. 42 and 43, Table 

12). Also, the soil oxidant did not significantly affect the values of TTNH, TTAH, TTNH/Hopane and 

TTAH/Hopane in the soil relative to controls except with the increase in TTNH and TTAH in the mineral soil by the 

soil oxidant compared to the control. 

Discussion 

 Biostimulation, the enhancement of biodegradation by increasing indigenous microbial activity, 

was demonstrated in both mineral and sandy marsh sods with concurrent stimulation in microbial activity 

and plant growth with fertilizer addition (a type of biostimulation). In contrast, microbial seeding 

(bioaugmentation) and soil oxidant application (a type of biostimulation) had no positive effect on plant 

growth, microbial activity, or petroleum hydrocarbon degradation. 

 Application of fertilizer increased live aboveground biomass, especially in the treatment receiving the 

soluble fertilizer. Salt marshes are usually deficient in inorganic nutrients (Wilsey et al. 1992). The application of 
 55

fertilizer provided essential macronutrients (N and P) to marsh plants. As a result, plants were visually more 

vigorous and had higher live aboveground biomass in the treatments with fertilizer than without. When this 

experiment started in August, the marsh plants in the sods had almost reached their peak biomass. Little 

aboveground growth occurred during the following 4-month experimental period. Thus, biomass differences 

between fertilizer and non-fertilizer treatments were not great. It is not surprising that the microbial product had no 

significant effect on plant growth because it only contained cultured bacteria, with no inorganic nutrients. Also, the 

soil oxidant elicited no positive effect on plant growth, suggesting that soil oxygen availability was not limiting 

plant growth. Because the inundation regime in this experiment was 12-hours drained and 12-hours flooded every 

24-hour period, the sods were relatively aerobic (average soil redox potential (Eh) of +121 mV at the depth of 2 cm 

below the soil surface and +31 mV at 10 cm depth even when flooded). Also, the soil oxidant did not significantly 

increase soil Eh relative to control. Thus, the application of the soil oxidant had no measurable effect on soil 

oxidation status. The Eh level in this experiment was not low enough to stress S. alterniflora, which adapts to 

flooding with a well-developed aerenchyma system that ventilates the roots with air. Spartina alterniflora can grow 

vigorously under moderately reducing condition (Mendelssohn and McKee 1987). 

 Microbial activity was enhanced by bioremediation as evidenced by increased soil respiration. The living 

soil community is composed of bacteria, fungi, invertebrates and roots of S. alterniflora. The greater soil 

respiration with fertilization could have resulted from an increase in microbial activity, from enhanced root biomass 

and root respiration, or from both. However, our on-going studies show no significant relationship between the 

mass of living roots of S. alterniflora and the soil respiration rate (r2

=0.03, p=0.802). Therefore, the fertilizerinduced increase in soil respiration was most likely due to increased soil microbial activity. This conclusion is also 

supported by the heterotrophic bacteria data, showing an increase, albeit not significant, in colony counts with 

fertilization. A number of studies have demonstrated the effects of inorganic nutrient addition on microbial activity 

and population counts. In the Exxon Valdes oil spill, fertilizer application increased microbial number and activity 

(Chianelli et al. 1991), and fertilizer application increased the populations of alkane and aromatic degraders in an 

experimental oil spill in Delaware beach (Venosa et al. 1996). Microbial population and activity were also 

enhanced by nutrient addition to laboratory microcosms containing hydrocarbon-contaminated arctic soils 

(Braddock et al. 1997). 

 Soil inorganic nutrient concentrations were increased by fertilizer application, especially by the soluble 

nutrient addition. The objective of the soluble fertilizer treatment was to maintain elevated interstitial nutrient levels 

with nutrient additions when needed on a weekly-basis. This objective was met. The interstitial nitrogen 

concentrations in the treatments receiving slow release fertilizer tended to be higher than that without fertilizer 

although the difference was not statistically significant. In a comparison of bioremediation with and without marsh 

plants, fertilizer application did not increase the interstitial nitrogen pools when marsh plants were present, but 

significantly increased nitrogen concentrations in the absence of plants (Lin and Mendelssohn 1997). Thus, plant 

uptake can regulate the interstitial nitrogen concentration levels. Therefore, soil nitrogen pools cannot solely be 

used as indicators of fertilizer application rate during bioremediation trials when plants are present. The fertilizer 

applications also significantly raised phosphate concentrations compared to the control, with greater increase in the 

sandy marsh sediment than in the more mineral soil. 

 Fertilizer application changed the soil environment by increasing nutrient concentrations and microbial 

activity, responses that should favor oil biodegradation. The concentrations of TTNH and TTAH in both types of 

marsh sediments with fertilizer application were significantly lower than without fertilizer application. The extent 

that fertilizer enhanced the reduction in TTNH concentration over no-fertilizer was much greater than for TTAH. 

For example, the residual TTNH in the sediment receiving slow release fertilizer was only 8% of the control (no 

bioremediation products added), while the TTAH in the same treatment was 56% of the control, suggesting that 

TTNH was more biodegradable than TTAH when fertilized. An experimental oil spill on a Delaware beach also 

found that the degradation rate of alkanes was greater than that for aromatic hydrocarbons (Venosa et al. 1996). 

Also, nutrient addition stimulated saturate degradation to a greater extent than aromatic degradation under shakeflask conditions (Fedorak and Westlake 1981). In the present study, although interstitial nitrogen and phosphorus 

concentrations were much higher in treatments receiving soluble fertilizer than in those with slow release fertilizer, 

the petroleum hydrocarbons (TTNH and TTAH) remaining in the sediment of the former were greater than in the 

latter. This indicates that maintaining constant and high (7-8 ppm or above) nitrogen and phosphorus 

concentrations in the interstitial water in this study was not necessary for maximum oil biodegradation. Fertilizer 

addition to hydrocarbon-contaminated arctic soils showed that high nutrient levels (N>61 ppm and P>106 ppm of 

dry soil) did not enhance microbial activity and hydrocarbon degradation more than at lower concentrations (22.2 
 56

ppm N and 37.6 ppm P) (Braddock et al. 1997). In a mesocosm experiment (Wright et al. 1997), addition of 

phosphorus and nitrogen significantly increased oil degradation, but nitrogen alone did not. This suggests that 

phosphorus may limit microbial degradation of oil in some wetlands. In an experiment associated with the Exxon 

Valdez oil spill (Bragg et al. 1993), effectiveness was determined to depend primarily on the amount of nitrogen 

fertilizer delivered to the sediment per unit oil present. The effectiveness of nitrogen or phosphorus addition on oil 

bioremediation most likely depends on their background concentrations and availability in the soil. 

 Hopane, a saturated, high molecular weight alkane in crude oil, has been shown to be highly resistant to 

biodegradation and is often used as an internal reference marker for oil biodegradation experiments (Venosa et al. 

1996; Atlas 1995). In the present study, when the analytes were normalized to hopane (TTNH/Hopane and 

TTAH/Hopane), an enhancement of oil biodegradation by fertilizer was also observed. All treatments with fertilizer 

application had consistently lower ratios of TTNH/Hopane and TTAH/Hopane compared to those without fertilizer. 

The differences among treatments receiving fertilizer was small, regardless of whether other products (oxidant or 

microbial agent) were included, further suggesting the importance of fertilization in stimulating oil biodegradation. 

 The addition of the microbial product to the slow release fertilizer did not affect soil respiration rate, 

heterotrophic microbial populations, petroleum-degrading microbes, or TTNH and TTAH concentrations in the 

sediment any more than the application of slow release fertilizer alone. TTNH/hopane and TTAH/hopane ratios 

further indicated no significant positive effect of microbial seeding on oil biodegradation. Our results suggest that 

presence of microbial hydrocarbon degraders in Louisiana coastal marshes is not limiting biodegradation. The 

present study supports a number of other studies showing the ineffectiveness of microbial inoculents on oil 

biodegradation in wetlands (Mearns 1991; Venosa et al. 1992). Virtually all marine ecosystems harbor indigenous 

hydrocarbon-degrading microbes. The hydrocarbon degraders, which usually comprise <1% of the bacterial 

community in unpolluted environments, often exceed 10% following petroleum contamination (Atlas 1993). Thus, 

microbial seeding usually is not an important limiting factor for oil degradation in wetlands (Prince et al. 1993). 

This study suggests that the high cost of microbial amendments for oil bioremediation may not be warranted in 

coastal marshes. 

 The soil oxidant also did not significantly enhance oil degradation rate in this study. Generally, petroleum 

biodegradation rate is low in anaerobic sediments because molecular oxygen is required by most microorganisms 

for the initial step in hydrocarbon metabolism (Atlas 1995). In the present experiment, the marsh sods were drained 

daily to simulate a diurnal tidal regime. During the 12-hour daily low tide period, interstitial water drained from soil 

pores and atmospheric air was able to enter the soil via direct diffusion from the soil surface. Thus, the soil was 

relatively oxidized (+388 mV and +326 mV at 2 and 10 cm below soil surface, respectively) when drained. The 

effect of direct atmospheric oxygen diffusion into the soil on soil oxidation status likely overwhelmed the relatively 

minor effect of the soil oxidant. Even during the 12-hour high tide period, the inundation was not long enough to 

cause severely reducing conditions as mentioned previously. The redox potential was not significantly different 

between treatments with and without the soil oxidant. Also, interstitial pH was not significantly affected by the soil 

oxidant or the other product applications (7.7 ± 0.04, n=120). Interestingly, the soil oxidant appeared to have a 

negative effect on oil degradation in this experiment when the soil oxidant was applied without fertilizer. TTNH 

and TTAH in the mineral marsh receiving the soil oxidant alone were significantly higher than those in the control 

(no product added), but the differences in concentrations of TTNH and TTAH between the treatments receiving the 

soil oxidant plus slow release fertilizer and slow release fertilizer only were not significantly different (Figs. 42 and 

43). However, the TTNH/hopane and TTAH/hopane ratios (Table 12) suggest that the soil oxidant had neither 

positive nor negative effects on oil biodegradation. 

 Marsh soil type affected oil biodegradation. In the present study, TTNH, TTAH, TTNH/Hopane and 

TTAH/Hopane were higher in the sandy marsh than in the mineral marsh soil. The differences in these values were 

primarily controlled by the no fertilizer treatments (control and soil oxidant alone). Higher TTNH and 

TTNH/hopane values in the controls of the sandy soil than in the mineral soil may indicate lower natural microbial 

biodegradation rates in sandy soil than in mineral soil. More importantly, however, the percentage of decrease in 

TTNH in treatments receiving fertilizer compared to the related control (Table 12) was greater in sandy soil (87%) 

than in mineral soil (74%), suggesting that biostimulation with fertilizer application was more effective in the 

former than in the latter, although fertilizer significantly enhanced TTNH biodegradation in both soil types. 
 57

Summary 

Oil bioremediation with fertilizer application has shown great potential for oil spill cleanup in coastal 

marshes in the present study. Inorganic nutrient addition was the most effective bioremediation agent tested in this 

study. The ambient inorganic nutrient concentrations in the marsh sods, ca. 0.2 ppm nitrogen and 0.5 ppm 

phosphate in the sediment interstitial water, were not high enough for optimum microbial degradation of petroleum. 

Fertilizer addition increased soil nutrient concentration and/or availability which, in turn, stimulated soil microbial 

activity. As a result, fertilizer application significantly increased biodegradation of normal alkane and aromatic 

hydrocarbons. The concentrations of total targeted normal hydrocarbons remaining in sediments with fertilizer were 

26% of the control in the mineral marsh, and 13% of the control in the sandy marsh. For both alkanes and aromatic 

hydrocarbons, enhanced biodegradation was observed with the hopane normalization method in treatments with 

fertilizer compared to treatments without fertilizer. However, raising nitrogen (up to 8 ppm) and phosphorus (up to 

20 ppm) concentrations above that created with slow release fertilizer did not further increase oil biodegradation. 

Soil type influenced bioremediation. Fertilizer was more effective in enhancing oil biodegradation in sandy soils 

than mineral soils; the percentages of TTNH and TTAH remaining in treatments receiving fertilizer compared to 

controls were lower in sandy than in mineral soils, although natural (unfertilized) degradation rates of TTNH and 

TTAH may be lower in the sandy soils than in the mineral soils. Microbial seeding and soil oxidant addition did not 

enhance petroleum biodegradation rates, even when combined with slow release fertilizer, more than that resulting 

from the slow release fertilizer alone. These results suggest that the high costs of microbial amendments during 

bioremediation trials may not be warranted in coastal marshes where oil-adapted microbes occur. Although the soil 

oxidant did not accelerate oil biodegradation, under other experimental conditions or with the application of other 

oxidants, these products, in combination with fertilizer, may yet prove to be valuable. The results of this 

investigation indicate that bioremediation with fertilizers is a promising oil spill cleanup technique in wetland 

environments. 
 59

CHAPTER 4 

A COMPARISON OF BIOSTIMULATION AND PHYTOREMEDIATION 

OF CRUDE OIL IN WETLAND MESOCOSMS 

by Qianxin Lin, Irving A. Mendelssohn, Charles B. Henry, Jr., Scott Miles, 

Edward B. Overton, Ralph J. Portier, and Maud M. Walsh 

 Biostimulation attempts to accelerate the natural degradation process of contaminants, such as petroleum 

hydrocarbons, by adding non-bacterial agents to overcome factors that limit bacterial hydrocarbon degradation. The 

most common biostimulants, shown to enhance oil degradation, are inorganic nutrients (Mikesell et al. 1991; Altas 

1993; Bragg et al. 1993; Prince et al. 1993; Venosa et al. 1992, 1996; Lin et al. 1999b). Previous studies (Scherrer 

and Mille 1990; Lindstrom et al. 1991; McKee and Mendelssohn 1995; Venosa et al. 1996; Wright et al. 1997; Lin 

and Mendelssohn 1998b; Lin et al. 1999c) indicated that fertilization with inorganic nitrogen and phosphorus 

enhanced degradation of petroleum hydrocarbons. In a wetland mesocosm experiment, addition of phosphorus and 

nitrogen significantly increased oil degradation, but nitrogen alone did not (Wright et al. 1997), suggesting that 

phosphorus may limit microbial degradation of oil in some wetlands. However, biostimulation effectiveness on an 

oiled shoreline associated with the Exxon Valdez oil spill (Bragg et al. 1993) was determined to depend primarily 

on the amount of nitrogen fertilizer delivered to the substrate. Thus, the role of N versus P in oil bioremediation has 

not been clarified. 

 Phytoremediation, the use of vegetation for the in-situ treatment of contaminated soil and sediment, is an 

emerging technology that promises effective and inexpensive cleanup of pollutants (Stomp et al. 1993; Schnoor et 

al. 1995). Phytoremediation applies to all plant-influenced biological, chemical, and physical processes that aid in 

the remediation of contaminants (Cunningham and Breti 1993), such as petroleum hydrocarbons. Phytoremediation 

has already been shown to have potential for the cleanup of both inorganic pollutants, such as heavy metals, and 

organic pollutants, including polycyclic aromatic hydrocarbons (PAHs) (Anderson and Walton 1992; Bell 1992; 

Brown et al. 1995; Salt et al. 1995; Reilley et al. 1996; Kling 1997; Lin and Mendelssohn 1997, 1998a and 1998b; 

Lin et al. 1999b; Banks et al. 1999; Liste and Alexander 2000b). However, few studies have focused on the 

efficacy of phytoremediation for oil spill cleanup in wetland environments. In addition, most biostimulation studies 

in wetland environments have assumed that the primary effect of fertilization is the direct stimulation of bacterial 

activity. However, the fertilization may accelerate oil degradation through plant-associated processes, e.g., uptake 

or plant-stimulated microbial activity. The importance of the vegetation, per se, in the remediation of wetlands with 

fertilizers has not been investigated. 

 The objectives of this study were to (a) determine the effectiveness of biostimulation with inorganic 

nitrogen and phosphorus, singly and in combination, on oil degradation and (b) separate the effect of 

phytoremediation from biostimulation in accelerating oil degradation. 

Materials and Methods 

Experimental Design 

 In the greenhouse, the following treatments were randomly applied and their effects on oil biodegradation 

determined: (1) phytoremediation with Spartina alterniflora or no plants, (2) nitrogen fertilization, and (3) 

phosphorus fertilization. The experimental design was a completely randomized block with a 2 x 3 x 2 factorial 

treatment arrangement. We compared phytoremediation by S. alterniflora with the soil without plants. We also 

evaluated the effect of biostimulation of oil at three nitrogen dosage rates (high = 466 kg N/ha;, low = 233 kg N/ha; 

and 0 kg N/ha nitrogen fertilizer) on oil degradation. In addition, we compared the biostimulation effect of nitrogen 

fertilizer with that of phosphorus fertilization (175 and 0 kg P/ha). Treatment-level combinations were replicated 

five times. A total of 60 experimental units were used in this experiment. 
 60

Experimental Procedures 

 Artificially weathered (25% by volume) south Louisiana crude oil was applied at a level of 2 L/m2

 to a 

mixture of two potting soils (50% by volume of Jiffy Mix, Jiffy Products of America, Chicago, IL and 50% of 

VitaHume potting soil, Hyponex Corporation, Marysville, OH). This oil dosage is equivalent to about 20 mg oil/g 

dry soil. The soil and the applied oil were well mixed, then placed into individual 8-liter buckets. Directly mixing 

the oil with the soil was done to reduce the variation in the initial oil concentrations among experimental units, a 

common condition when oil is added to the soil surface (Chapters 2 and 3). Fifteen (15) culms (shoots and intact 

roots and rhizomes) of Spartina alterniflora, free of soil surrounding the roots, were transplanted into each bucket. 

The experiment was conducted in the greenhouse. High (133 kg N/ha) and low (67 kg N/ha) rates of soluble 

nitrogen fertilizer (NH4Cl) and soluble phosphorus fertilizer (50 kg P/ha of NaH2PO4) were dissolved in water and 

applied to the soil surface of the appropriate fertilization experimental units 1, 8, and 16 weeks after transplanting S. 

alterniflora. Half of the previous amounts of N and P were applied to the appropriate experimental units after 20 

weeks to ensure reasonable concentrations of inorganic nutrients in the final four weeks of the experiment. The 

four fertilizer applications resulted in a total of 466 kg N/ha and 233 kg N/ha for the high and low nitrogen 

treatments, respectively, and 175 kg P/ha for the phosphorus application treatment. The water levels in each 

experimental unit were kept at 0.5 cm above the soil surface by daily watering the pots with tap water. 

 At the termination of the six-month experiment, the mesocosms were sampled for (1) petroleum 

hydrocarbon chemistry, such as total targeted aromatic hydrocarbons (TTAH) and total targeted normal 

hydrocarbons (TTNH) to identify and quantify the degree of oil biodegradation; (2) soil microbial responses, such 

as oil-degrading microbes and microbial respiration rates to determine the effect of the bioremediation products on 

the microbial communities that are performing the oil biodegradation; (3) soil chemistry to determine the effect of 

the bioremediation products on those factors that limit the growth of microbes and plants; and, (4) plant response, 

such as plant photosynthetic rate, plant stem density, and plant biomass to evaluate the effects of fertilizer on plant 

growth and its relationship to oil degradation. 

 Statistical analysis was conducted with the SAS system (SAS 1990). General Linear Model (GLM) was 

used to test for statistically significant differences (P<0.05) among the treatments and Duncan's multiple range test 

was used to determine significant differences among the main factors. Treatment-level combination differences, if 

interactions of main factors were significant, were tested with least square means. 

Methods 

Plant Response

 Photosynthetic Rate. Leaf photosynthetic rate was measured to indicate plant growth status. A portable 

photosynthesis system, including an infrared gas analyzer (IRGA) (The Analytical Developmemt Co. Ltd, (ADC) 

model LCA-2), an ADC air flow control unit, and an ADC Parkinson leaf chamber, was used. Sample air, taken 5 

m aboveground to obtain relatively stable CO2 concentrations, was led through the ADC air flow control unit at the 

flow rate of 5 ml/s during photosynthetic rate measurements. Measurements were conducted at a quantum flux 

density of 2000 μmol/m2

/s provided by a Kodak projector lamp. An intact, attached and fully expanded young leaf 

was enclosed in the leaf chamber and the difference in CO2 concentration and humidity between inlet and outlet air 

was measured. Photosynthetic rate (CO2 exchange) was calculated in accordance with von Caemmerer and 

Farquhar (1981) and expressed as μmol CO2/m2

/s.

Plant Stem Density. Plant stem density was measured by direct counting of stem number in each 

experimental unit and expressed as the number of stems per pot. 

 Average and Maximum Shoot Height. Canopy height of the transplants was measured to the nearest 

centimeter. 

Aboveground Biomass. Plant aboveground biomass of S. alterniflora was analyzed at the end of a sixmonth experimental period to determine the treatment effect on plant growth. Plant aboveground material was 

clipped at the soil surface, dried in an oven at 65°C to constant weight, and weighed. 
 61

Belowground Biomass of the Transplant. Plant belowground biomass of S. alterniflora was determined by 

washing all soil from the belowground plant material at the end of the experiment, dring in an oven at 65°C to 

constant weight, and weighing. 

Water Evapo-Transpirational Rate. Evapo-transpirational water losses, the combination of water loss by 

transpiration from plant leaves and by evaporation from the water surface, were determined by measuring the mass 

water loss from the each experimental unit in a 24-hour period. First, the water table in the container was raised to 

0.5 cm above the soil surface and the experimental unit weighed. After 24 hours, the mass of the experiment unit 

was again determined. The difference in mass over a 24-hour period was defined as the evapo-transpirational water 

loss. 

Soil Response

Soil Respiration Rate. In-situ measurements of soil respiration rate, an indicator of soil microbial activity, 

were made with an infra-red gas analyzer (IRGA) by measuring carbon dioxide production from soil. A PVC 

chamber (4 cm in diameter and 8 cm in height) with one open end was equipped with an inlet and an outlet for air 

flow through the chamber. The open end of the chamber was inserted into the soil 4 cm below the soil surface. The 

air flow rate through the chamber was held constant at 300 ml per minute by an ADC mass flow controller. The 

respiratory CO2 produced from the soil resulted in a difference in CO2 concentration between inlet and outlet that 

was measured by an ADC infra-red gas analyzer. Soil respiration was calculated based on the CO2 exchange rate 

from the soil per unit surface area. 

Soil Redox Potential. Soil redox potentials at 3 and 12 cm depths were determined with bright platinum 

electrodes and a calomel reference electrode. Readings were taken with a portable pH/mV digital meter. The 

potential of a calomel reference electrode (+244 mV) was added to each value to calculate Eh (Patrick et al. 1996). 

Soil Nutrient Concentrations. Interstitial water samples were withdrawn from soil sods with a simple 

apparatus as described by McKee et al. (1988). This consisted of a small diameter (3 mm inside diameter) rigid 

plastic tube, containing numerous ca. 0.5 mm diameter holes covered with 3 to 4 layers of cheesecloth, connected to 

a 30 ml syringe. The collected interstitial water was filtered through a 0.45 μm syringe filter. Inorganic nutrients in 

the filtered interstitial water were analyzed to determine the effect of product application on soil fertility and other 

variables. Ammonium-nitrogen (NH4) and nitrate-nitrogen (NO3 + NO2) were analyzed with an auto-analyzer. 

Total inorganic nitrogen was determined as the sum of NH4, NO3, and NO2-nitrogen concentrations. Phosphate was 

measured by the Murphy-Riley procedure (Parsons et al. 1984). 

Oil-Degrading Microbes. Petroleum-utilizing populations were determined at the end of a six-month 

experimental period. Soil samples were collected from 0 to 8 cm below the soil surface at the termination of the 

experiment. Oil-degrading population were tracked using plate count methods. A minimal salts agar to which 50 

ppm naphthalene and 50 ppm cresol were added as the sole carbon source was used for the selective culturing of 

petroleum-degrading organisms. All microbial population measurements were reported per gram dry weight gram. 

Residual Oil Chemistry in the Soil. Total targeted normal hydrocarbons (TTNH) and total targeted 

aromatic hydrocarbons (TTAH) were analyzed to determine the oil degradation rates. . Soil samples were collected 

from the soil surface to the bottom (about 16 cm below the soil surface) at the termination of the experiment from 

each experimental unit, thoroughly homogenized, and extracted using a modified EPA method 3550. 

Approximately 20 grams of wet soil were extracted using dichloromethane and sodium sulfate as a chemical drying 

agent (Venosa et al. 1996; Sauer and Boehm 1991; Henry and Overton 1993; Henry et al. 1993), then reduced to a 

final volume of 10 ml. A field treatment composite was then analyzed by gas chromatography-mass spectrometry 

(GC/MS) operated in the Selective Ion Monitoring (SIM) Mode to characterize compositional changes in targeted 

normal hydrocarbons (NH) and aromatic hydrocarbons (AH). Although these targeted AH generally represent less 

than 5% of the bulk oil composition, they are essential to characterize petroleum source, identify potential biological 

effects, determine exposure pathways, and monitor weathering trends and degradation of the oil (Sauer and Boehm 

1991; Roques et al. 1994). The targeted aromatic hydrocarbons are listed in Table 1 (Chapter 1). 
 62

Results 

Responses of S. alterniflora to the Treatments

 The effects of biostimulants on Spartina alterniflora, the dominant plant in Atlantic and Gulf coast salt 

marshes of the United States, were estimated by measuring photosynthetic rate, stem density, plant shoot height, and 

above- and belowground biomass. Nitrogen application significantly increased photosynthetic rate (Fig. 44), plant 

stem density (Fig. 45), and plant shoot height (Fig. 46) of S. alterniflora. They were significantly higher in the 

treatments with a low dosage of nitrogen than without nitrogen application, and significantly higher in the 

treatments with a high dosage of nitrogen than the low dosage (Fig. 44 to 463). Phosphorus application increased 

photosynthetic rate (Fig. 44), plant stem density (Fig. 45), and plant shoot height (Fig. 46) of S. alterniflora in 

addition to nitrogen. 

 

0

5

10

15

Photosynthetic Rate (umol m-2 s-1)

0N-0P 0N-P LN-0P LN-P HN-0P HN-P

Treatments

Figure 44. Effect of nitrogen and phosphorus application on photosynthetic rate of Spartina alterniflora six months 

after the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 0P: no 

phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 

 

0

20

40

60

80

Stem Density (number/pot)

0N-0P 0N-P LN-0P LN-P HN-0P HN-P

Treatments

Figure 45. Effect of nitrogen and phosphorus application on stem density of Spartina alterniflora six months after 

the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 0P: no 

phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 
 63

 

0

20

40

60

80

Canopy Height (cm)

0N-0P 0N-P LN-0P LN-P HN-0P HN-P

Treatments

Figure 46. Effect of nitrogen and phosphorus application on canopy height of Spartina alterniflora six months after 

the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 0P: no 

phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 

 Both nitrogen and phosphorus applications significantly increased aboveground and belowground biomass 

of S. alterniflora. At the termination of the experiment 6 months after the treatment, aboveground and belowground 

biomass of S. alterniflora were significantly higher in the treatment with a low dosage of nitrogen than without 

nitrogen application. Also, aboveground and belowground biomass of S alterniflora were significantly higher in the 

treatment with a high dosage of nitrogen than the low dosage (Figs. 47 and 48, Table 13). In addition, phosphorus 

application significantly increased aboveground and belowground biomass of S. alterniflora compared to the 

treatments without phosphorus application. 

 

0

50

100

150

 Aboveground Biomass (g/pot)

0N-0P 0N-P LN-0P LN-P HN-0P HN-P

 Treatments

Figure 47. Effect of nitrogen and phosphorus application on aboveground biomass of Spartina alterniflora six 

months after the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 

0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 
 64

 

0

20

40

60

80

 Below-ground Biomass (g/pot)

0N-0P 0N-P LN-0P LN-P HN-0P HN-P

 Treatments

Figure 48. Effect of nitrogen and phosphorus application on belowground biomass of Spartina alterniflora six 

months after the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 

0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 

Table 13. Statistical Table for the Transplant Variables 

with P-Values Provided for Main Factors and Their Interactions 

Plant Variables N P N*P 

Photosynthetic rate 0.0001 0.0114 0.8135

Stem density 0.0001 0.0007 0.5338

Canopy height 0.0001 0.0001 0.2179

Aboveground Biomass 0.0001 0.0001 0.0003

Belowground Biomass 0.0001 0.0015 0.0741

N: nitrogen application; P: phosphorus application; 

N*P: interaction of nitrogen and phosphorus applications. 

 Plant evapo-transpiration rate was measured to determine the effect of the transplants and fertilizer 

application on water loss rate from the experimental units. Evapo-transpiration rates in the treatments with 

phytoremediation by S. alterniflora were significantly higher than in those without transplants (Fig. 49, Table 14). 

Water evapo-transpiration rates in the treatments receiving nitrogen and phosphorus fertilizer were significantly 

higher than treatment without fertilizer (Fig. 49, Table 14). A significant interaction between phytoremediation and 

nitrogen fertilizer application (Table 14) indicated that the higher evapo-transpiration rate in the treatments with 

nitrogen occurred only in the presence of transplants. 
 65

 

0

100

200

300

400

500

Evapotranspiration Rate (g/pot/day)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

Treatments

Without Phytoremediation With Phytoremediation 

Figure 49. Effect of phytoremediation and nitrogen and phosphorus application on evapo-transpiration rate six 

months after the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 

0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 

Table 14. Statistical Table for the Soil Variables and Residual Oil Concentration 

with P-Values Provided for Main Factors and Their Interactions 

Parameter Main 

Factors 

 Interaction 

 Phyto N P Phyto*N Phyto*P* N*P Phyto* 

N*P 

EH 3cm 0.0001 0.2291 0.6799 0.3920 0.3133 0.7384 0.5100 

EH 12cm 0.0266 0.4366 0.4571 0.7476 0.1439 0.7873 0.3173 

Evapo-transpiration 0.0001 0.0001 0.0073 0.0001 0.0628 0.5042 0.2831 

Soil Respiration 0.5471 0.6594 0.7499 0.6611 0.8600 0.8239 0.9629 

Interstitial Inorganic 

 Nitrogen 

0.0198 0.0344 0.2928 0.0297 0.3125 0.2256 0.2325 

Interstitial Phosphate 0.0002 0.3603 0.8841 0.1597 0.4555 0.5933 0.3528 

TTNH 0.0001 0.7023 0.1853 0.8279 0.4521 0.4927 0.1533 

TTAH 0.0146 0.0608 0.6141 0.0010 0.8791 0.9966 0.6107 

Phyto: phytoremediation treatment; N: nitrogen application; P: phosphorus application; N*P: interaction of nitrogen and 

phosphorus applications, etc. 

Responses of Soil Variables

 Soil parameters (e.g., soil Eh and nutrients) were measured to determine if addition of the fertilizer and 

transplants had any effect on the soil environment. Soil redox potential is a measure of the degree to which a soil is 

oxidized or reduced. Higher redox potentials signify more aerated soils. Soil redox potential was significantly 

higher in the treatments receiving transplants than those without transplants for both 3 and 12 cm below the soil 
 66

surface (Figs. 50 and 51), indicating that transplants aerated the soil by transporting atmospheric air to their 

rhizosphere. For example, average redox potential at the 3 cm soil depth was +168 mV (± 16 standard error, n=30) 

for the treatment receiving S. alterniflora and -54 mV (± 15 standard error, n=30) for the treatment without 

transplants (Fig 50). Average redox potential at the 12 cm soil depth was +12 mV (± 18 standard error, n=30) for 

the treatment receiving S. alterniflora and -46 (± 17 standard error, n=30) mV for the treatment without transplants 

(Fig 51). The addition of nitrogen and phosphorus did not significantly affect soil redox potential at 3 and 12 cm 

depth. 

 

-100

0

100

200

300

Redox Potential (mV)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

At 3 cm depth

Without Phytoremediation With Phytoremediation 

Figure 50. Effect of phytoremediation and nitrogen and phosphorus application on soil redox potential at 3 cm 

below the soil surface six months after the treatments. Values are means (n=5) with standard errors. 0N: 

no nitrogen application, 0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg 

N/ha. 

 

-150

-100

-50

0

50

100

150

Redox Potential (mV)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

Treatments

At 12 cm depth

Without Phytoremediation With Phytoremediation 

Figure 51. Effect of phytoremediation and nitrogen and phosphorus application on soil redox potential at 12 cm 

below the soil surface six months after the treatments. Values are means (n=5) with standard errors. 0N: 

no nitrogen application, 0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg 

N/ha. 

 Interstitial inorganic nitrogen and phosphorus were analyzed to determine the available nutrient status of 

the experimental units and their effect on phytoremediation. Phytoremediation by S. alterniflora and nitrogen 

addition significantly affected interstitial inorganic nitrogen concentration (Fig. 52). A significant interaction 

between phytoremediation and nitrogen fertilizer application showed that the higher interstitial nitrogen in the 
 67

treatments receiving nitrogen fertilizer occurred only in the absence of phytoremediation by S. alterniflora. In the 

presence of the plants, plant uptake of nitrogen may control the interstitial nitrogen concentration. Phosphorus 

addition did not affect interstitial inorganic nitrogen. Furthermore, phytoremediation by S. alterniflora significantly 

affected interstitial phosphate concentration (Fig. 53). Average interstitial phosphate concentration was 

significantly higher in the treatments without plants than those with plants, with 9.66 mg/L in the former versus 7.45 

mg/L in the latter six months after treatment initiation (Figs. 50B). However, the addition of nitrogen and 

phosphorus fertilizer did not affect interstitial phosphorus concentrations compared to the treatments without 

fertilization. 

 

0

0.25

0.5

0.75

1

Interstitial Inorganic Nitrogen (mg/L)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

 Treatment

Without Phytoremediation With Phytoremediation

Figure 52. Effect of phytoremediation and nitrogen and phosphorus application on interstitial inorganic nitrogen six 

months after the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 

0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 

 

0

5

10

15

 Interstitial Phosphate (mg/L)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

 Treatment

Without Phytoremediation With Phytoremediation

Figure 53. Effect of phytoremediation and nitrogen and phosphorus application on interstitial phosphate 

concentration six months after the treatments. Values are means (n=5) with standard errors. 0N: no 

nitrogen application, 0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 
 68

 Microbial soil respiration and oil-degrading microbial populations were measured to determine the 

treatment effects and their relationship to oil degradation. Generally, neither phytoremediation by S. alterniflora

nor addition of nitrogen and phosphorus fertilizer significantly affected microbial soil respiration rate (Fig. 54) and 

oil-degrading microbial populations (Fig. 55). A large variation was observed for these two variables. 

 

0

5

10

15

20

Soil Respiration Rate (umol/m2/s)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

Treatments

Without Phytoremediation With Phytoremediation 

Figure 54. Effect of phytoremediation and nitrogen and phosphorus application on soil respiration rate six months 

after the treatments. Values are means (n=5) with standard errors. 0N: no nitrogen application, 0P: no 

phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha. 

 

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

 Oil Degraders (counts/g dry soil)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

 Treatments

Without Phytoremediation With Phytoremediation 

Figure 55. Effect of phytoremediation and nitrogen and phosphorus application on oil-degrading microbial 

populations six months after the treatments. Values are means (n=5) with standard errors. 0N: no 

nitrogen application, 0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 kg N/ha 
 69

Oil Chemistry 

Alkane Degradation 

Phytoremediation by S. alterniflora enhanced alkane degradation in the present study. The concentration 

of residual total targeted normal hydrocarbons (TTNH) was significantly lower (p<0.0001) in the treatments with S. 

alterniflora than in those without S. alterniflora, with 4597 μg/g dry soil (± 241 standard error, n=30) in the former 

versus 10124 μg/g (± 979 standard error, n=30) after six months of treatment (Fig. 56). However, addition of 

nitrogen and phosphorus did not significantly affect concentrations of residual total targeted normal hydrocarbons. 

 

0

5000

10000

15000

20000

TTNH Concentration (ug/g dry soil)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

Treatments

Without Phytoremediation With Phytoremediation 

Figure 56. Effect of phytoremediation and nitrogen and phosphorus application on total targeted normal 

hydrocarbons (TTNH) six months after the treatments. Values are means (n=5) with standard errors. 

0N: no nitrogen application, 0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 

kg N/ha. 

Aromatic Hydrocarbon Degradation

Phytoremediation by S. alterniflora enhanced aromatic hydrocarbon degradation. Concentrations of 

residual total targeted aromatic hydrocarbons (TTAH) were significantly lower in the treatments with S. alterniflora

than in those without S. alterniflora, with 43.4 μg/g dry soil (± 3.7 standard error, n=30) in the former versus 55.7 

μg/g (± 3.8 standard error, n=30) after six months of treatment (Figs. 57, Table 14). Application of nitrogen 

fertilizer tended to decrease (p=0.0608) the concentration of residual total targeted aromatic hydrocarbons compared 

to those without nitrogen application. A significant interaction (p<0.001) between phytoremediation and nitrogen 

fertilizer application demonstrated that nitrogen only had a significant effect in decreasing residual total targeted 

aromatic hydrocarbons when plants were present. For example, the concentration of residual total targeted aromatic 

hydrocarbons was 22.6 μg/g dry soil (± 2.7 standard error, n=10) in the treatment receiving high nitrogen dosage 

(with and without applying phosphorus) combined with S. alterniflora, compared to 61.8 μg/g dry soil (± 10.4 

standard error, n=10) in the treatment receiving high nitrogen dosage (with and without applying phosphorus) but 

without S. alterniflora. However, the addition of phosphorus alone did not significantly affect the concentration of 

residual total targeted aromatic hydrocarbons. 
 70

 

0

25

50

75

100

TTAH Concentrations (ug/g dry soil)

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

0N-0P

0N-P

LN-0P

LN-P

HN-0P

HN-P

Treatments

Without Phytoremediation With Phytoremediation 

Figure 57. Effect of phytoremediation and nitrogen and phosphorus application on total targeted aromatic 

hydrocarbons (TTNH) six months after the treatments. Values are means (n=5) with standard errors. 

0N: no nitrogen application, 0P: no phosphorus application, P: 175 kg P/ha, LN: 233 kg N/ha, HN: 466 

kg N/ha. 

Discussion 

 In the present study, both nitrogen and phosphorus application significantly enhanced plant growth. 

Phytoremediation by the salt marsh plant S. alterniflora significantly increased soil redox potential, indicating a 

more oxidized soil environment that may enhance aerobic oil degradation. More importantly, concentrations of 

residual alkane and aromatic hydrocarbons were significantly lower in the treatments receiving phytoremediation, 

especially when combined with phosphorus and the high nitrogen dosage. 

 Phytoremediation by S. alterniflora enhanced oil degradation, with a significant lower concentration of 

total targeted normal hydrocarbons (TTNH) and total targeted aromatic hydrocarbons (TTAH) in the treatments 

with phytoremediation compared to those without phytoremediation. A number of studies have found 

phytoremediation enhancement of hydrocarbon degradation. Liste and Alexander (2000b) reported that more 

pyrene was degraded in the presence of roots of all nine plant species tested than in un-planted soil. Within 

approximately 8 weeks, as much as 74% of the pyrene disappeared from vegetated soil compared to 40% or less 

from unplanted soil. Phytoremediation by Typha latifolia reduced 90% of the hydrocarbons in a artificial wetland 

over a 360-day period (Salmon et al. 1998). Banks et al. (1999) reported that the presence of plants of Festuca 

arandinacea enhanced the degradation of highly adsorbed, recalcitrant benzo(a)pyrene in soils in a greenhouse 

experiment. Guenther et al. (1996) reported that in a rhizosphere sod system with growing ryegrass in the 

laboratory, aliphatic hydrocarbons disappeared faster than in unvegetated columns. These results indicate that 

biodegradation of hydrocarbons in the rhizosphere is stimulated by plant roots. The present study supported these 

findings, and again suggested the promise and potential of phytoremediation of oil. 

 Higher plants are being used to enhance the remediation of soils contaminated with recalcitrant organic 

compounds, such as petroleum hydrocarbons in oil spills. Several possible mechanisms of enhanced oil degradation 

via phytoremediation have been cited although mechanisms of action are poorly understood. The soil physicochemical environment can effect soil microbial activity and plant condition, thus affecting microbial degradation 

rate. Oxygen is an important electron acceptor for microbial oil degradation (Mikesell et al. 1991; Lee and 

Swindoll 1993; Hurst et al. 1996; Wilson et al. 1999). Oil degradation rate is generally much slower in a water 

saturated anaerobic condition than an aerobic environment (Hambrick et al. 1980, Lin and Mendelssohn 1998a). 

Hurst et al. (1996) reported that 45% and 55% of pyrene and PAH compounds were degraded under soil gas oxygen 

concentrations between 2% and 21%. No statistically significant mineralization was found to occur at 0% oxygen. 

In a laboratory bioremediation study of hydrocarbon contaminated groundwater and sediment, Wilson et al. (1999) 

found that only toluene was degraded at initial oxygen levels ranging from 0 to 1 mg O2/L. Naphthalene 
 71

degradation was observed in addition to toluene removal at initial oxygen levels ranging from 1.5 to 2.0 mg O2/L. 

This range represents a threshold level below which naphthalene could not be degraded under the experimental 

conditions. At oxygen levels greater than or equal to 7 mg O2/L, some removal of all aromatic compounds was 

observed. It is well known that many wetland plants can transport oxygen from the atmosphere through plant airspace tissue to aerate the soil rhizosphere (Armstrong 1978; Smirnoff and Crawford 1983; Teal and Kanwisher 

1966). In the present study, phytoremediation by S. alterniflora significantly increased soil redox potential, 

indicating a more oxidized soil environment in the treatments receiving transplants. Therefore, increased oxygen 

supply in the rhizosphere likely increased aerobic oil degradation in the treatments with phytoremediation. 

 Another possible mechanism of action in the phytoremediation process is adsorption of the organic 

contaminant onto the surface of the roots and subsequent uptake and/or degradation, thus reducing its concentration 

in the soil. By comparing affinity and adsorption of plant roots for naphthalene, a polycyclic aromatic hydrocarbon, 

for tall fescue and alfalfa, Schwab et al. (1998) found that alfalfa roots had approximately twice the affinity for 

naphthalene than fescue roots and concluded that lipid content is a controlling factor in adsorption of naphthalene 

onto plant roots. In a study determining PAH concentrations in the rhizosphere of plants grown in soil containing 

phenanthrene or pyrene, Liste and Alexander (2000a) reported that the rhizosphere of several plant species 

temporarily contained appreciably more phenanthrene and pyrene than unplanted soil, but those PAHs were 

degraded with time, suggesting that plants accumulate such hydrophobic compounds in the rhizosphere after 

facilitating their transport toward the roots. Lytle and Lytle (1987) reported that Juncus roemerianus uptakes 

petroleum hydrocarbons from marsh sediments, thus reducing oil concentration. Burken and Schnoor (1998) 

reported that translocation and subsequent transpiration of volatile organic compounds from the leaves to the 

atmosphere was shown to be a significant pathway of hydrocarbon loss. In the present study, phytoremediation 

with S. alterniflora had up to a seven-fold higher transpirational water loss from the plant leaf surface. The 

transpiration stream of S. alterniflora would have likely facilitated the hydrocarbon transport toward the roots, 

uptake by the plants, metabolism in the plants, and/or transpiration of volatile hydrocarbons to the atmosphere, thus 

reducing the concentration of TTNH and TTAH in the soil. 

Many studies have indicated that bioremediation with fertilizers, such as nitrogen and phosphorus, 

increases oil degradation. Swannell et al. (1999) reported that bioremediation of a mixture of Forties Crude Oil and 

Heavy Fuel Oil stranded on shorelines of Bullwell Bay, Milford Haven, UK, after the grounding of the Sea Empress 

in 1996. Both a weekly application of mineral nutrients dissolved in seawater and a single application of a slowrelease fertilizer supplied the proportional amount of nitrogen and phosphorus enhanced oil degradation. In a 

mesocosm experiment, addition of phosphorus and nitrogen significantly increased oil degradation, but nitrogen 

alone did not (Wright et al. 1997). In a laboratory study on the effects of nitrogen and phosphorus levels on 

petroleum bioremediation, Walworth and Reynolds, (1995) reported that at 10 ̊ C, degradation rates of diesel fuel 

were not affected by fertility treatments. At 20 ̊ C, the rates were increased by the addition of P, but unaffected by 

N. These results suggest that phosphorus may limit microbial degradation of oil in some cases. However, in an 

experiment associated with the Exxon Valdez oil spill (Bragg et al. 1993), effectiveness was determined to depend 

primarily on the amount of nitrogen fertilizer delivered to the sediment. In a laboratory bioremediation study of 

crude oil, Chang et al. (1996) reported that at 3% oil in the soil, addition of P, without N, generally did not enhance 

biodegradation. Addition of N, without P, approximately tripled the quantity of oil degraded. Addition of P and N 

together did not increase biodegradation of oil more than the addition of N alone, suggesting the importance of N in 

bioremediation of oil. At 6 and 9% oil concentrations, CO2 evolution increased by adding P and N together in 

comparison to adding N alone, and total petroleum hydrocarbon (TPH) biodegradation increased by 30% in 60 

days. These results suggest that nitrogen may be more important in microbial degradation of oil than phosphorus, 

but phosphorus can increase oil degradation once nitrogen limitations are relieved. 

 In the present study, in the absence of the marsh plant of S. alterniflora, application of nitrogen and 

phosphorus, singly or in combination, did not affect soil oil-degrading microbial populations, soil respiration, and 

residual concentrations of TTNH and TTAH. High concentrations of the background phosphorus level mute the 

effect of the added P on oil degradation in the current study. Interstitial phosphorus concentration was fairly high, 

with more than 7 ppm in the control treatment even after 6 months. Thus, addition of phosphorus did not increase 

interstitial phosphate concentration. Also, the addition of nitrogen, when transplants were absent, did not affect the 

residual TTNH and TTAH, although interstitial nitrogen concentration increased. This suggests that, in the absence 

of plants, the role of N and P fertilization in oil degradation may be limited, especially in water-saturated soils. In 

the presence of the marsh plant of S. alterniflora, high nitrogen, with or without phosphorus, significantly decreased 

TTAH by more than 2 fold. Nitrogen and phosphorus addition significantly increased plant growth with higher 
 72

photosynthetic rate, stem density, and above- and belowground biomass. Increased plant growth was responsible 

for the increased evapo-transpiration rate and soil redox potential. Thus, fertilizer-increased plant growth enhanced 

the degradation of the oil due possibly to higher aerobic oil degradation, plant uptake, metabolism in the plants, and 

transpiration of volatile hydrocarbons to the atmosphere. These results suggest that the effectiveness of fertilization 

on oil degradation may be dependent on phytoremediation with fertilizer-enhanced plants. 

 The effect of fertilizer on oil degradation was also related to phytoremediation in several other studies 

(Reynolds et al. 1999; Wright et al. 1997; Lin and Mendelssohn 1997 and 1998). Reynolds et al. (1999) reported 

that in a 34-week field study, total petroleum hydrocarbon (TPH) concentrations of a diesel-contaminated soil 

decreased significantly more in the rhizosphere plus nutrient treatment compared to the control that was not 

vegetated with bahiagrass or fertilized. In a mesocosm system containing S. alterniflora, Wright et al. (1997) 

reported that addition of phosphorus and nitrogen significantly increased oil degradation. Lin and Mendelssohn 

(1997, 1998a) reported that in the absence of plants, fertilization with nitrogen and phosphorus did not significantly 

affect residual oil degradation. However, fertilizer significantly enhanced the effect of phytoremediation on oil 

degradation by plants such as J. roemerianus, S. alterniflora, and S. patens. All these results suggest that 

fertilization may increase oil degradation through phytoremediation, hence the important of vegetation in 

accelerating oil degradation. 

Summary 

 Both nitrogen and phosphorus application significantly enhanced plant growth. Plant photosynthetic rate, 

stem density, and above- and belowground biomass of S. alterniflora increased with increasing nitrogen dosages. 

Phosphorus application additionally increased plant growth in each nitrogen application level. Fertilizer-increased 

plant growth was responsible for increased evapo-transpiration rate and soil redox potential. Phytoremediation by 

the salt marsh plant S. alterniflora significantly increased soil redox potential, indicating a more oxidized soil 

environment that may enhance aerobic oil degradation. More importantly, concentrations of residual alkanes 

(TTNH) were significantly lower in the treatment receiving phytoremediation by S. alterniflora compared to those 

without the plants. In addition, concentrations of residual aromatic hydrocarbons (TTAH) were significantly lower 

in the treatment receiving phytoremediation by S. alterniflora compared to those without S. alterniflora, and were 

lowest in the treatment receiving S. alterniflora in combination with application of high doses of nitrogen. This 

result suggests that high levels of nitrogen additionally increase the effect of phytoremediation on TTAH 

degradation. However, bioremediation by nitrogen and phosphorus addition, singly or in combination, did not 

significantly reduce residual oil concentration in the absence of S. alterniflora. The current study indicates the 

potential of phytoremediation for the cleanup of hydrocarbon contaminated soil in coastal wetlands. In addition, 

bioremediation by N and P fertilization enhances the efficacy of oil phytoremediation. 
73 

CHAPTER 5 

PROJECT CONCLUSIONS 

Bioremediation by adding non-bacterial agents to enhance natural oil degradation has considerable 

potential in wetland environments. Efficacy and success of bioremediation of oil to accelerate the biodegradation 

process depend on the extent of contaminant removal and on the ecological safety of the bioremediation agents. 

The bioremediation agents used in this research, including a microbial product and inorganic fertilizers, had no 

negative impacts to the plant, animal, and microbial responses evaluated in this study (Chapter 1). Application of 

the bioremediation agents at the specified application rates to the salt marsh sods did not adversely affect the marsh 

macrophyte, Spartina alterniflora, various microbial populations and soil respiration, and various infaunal animals 

including macrofauna and meiofauna. Overall, the bioremediation agents used in this study proved safe to marsh 

communities. 

Application of fertilizer, either in the form of slow release or water soluble, significantly enhanced plant 

growth with significantly higher photosynthetic rates, stem growth rates, and above- and below-ground biomass of 

S. alterniflora (Chapter 4). In addition, application of fertilizer significantly reduced the concentrations of both the 

reduced crude oil (Chapter 2) and the artificially weathered crude oil (Chapters 3 and 4). Total targeted normal 

hydrocarbons (TTNH) and total targeted aromatic hydrocarbons (TTAH), TTNH/hopane and TTAH/hopane, and 

the nC-18/phytane index for evaluation of normal alkane degradation were significantly lower in the treatments with 

fertilizer compared to those without fertilizer. These results suggest that oil biodegradation in marsh systems may 

be enhanced by fertilization (biostimulation). Phosphorus application to marsh sods with low interstitial phosphorus 

significantly increased soil heterotrophic microbial population, oil-degrading microbial populations and soil 

microbial respiration immediately, and increased TTNH and TTAH degradation, but not in the soils with high initial 

interstitial phosphorus concentration. The fertilizer-enhanced oil degradation mentioned above occurred only in the 

presence of the marsh macrophyte, S. alterniflora. 

The effect of fertilization on oil degradation by directly enhancing soil microbial activity was separated 

from the phytoremediation effect by comparing bioremediation by added N and P in the absence of plants with that 

in the presence of S. alterniflora. Phytoremediation significantly increased oil degradation. Concentrations of 

TTNH and TTAH were significantly lower in the treatments receiving S. alterniflora than without 

phytoremediation. In addition, high application rates of nitrogen significantly enhanced the efficacy of 

phytoremediation on TTAH degradation. In the absence of S. alterniflora, however, bioremediation by nitrogen 

and/or phosphorus addition did not significantly affect residual oil concentration. Thus, the effect of nitrogen on oil 

degradation in vegetated marshes is most likely through plant-mediated processes. Phosphorus application can be 

important in stimulating the microbial activity and phytoremediation effect. 

Application of the soil oxidant, PermeOx, did not significantly affect oil degradation in the marsh sods. 

The alkane (TTNH) and aromatic hydrocarbons (TTAH) degradation rates in the treatments receiving the soil 

oxidant were not significantly higher than the control (Chapter 3). Furthermore, the TTNH and TTAH degradation 

rates in the treatments receiving the soil oxidant plus fertilizer were not significantly higher than in the treatment 

receiving fertilizer alone (Chapter 3). These results suggest that there is no positive effect of soil oxidant on oil 

degradation. Higher degradation rates of alkane (TTNH) and aromatic hydrocarbons (TTAH) in the treatment 

receiving the soil oxidant plus fertilizer combined with KH2PO4 (a pH buffer) compared to the fertilizer alone were 

not due to the soil oxidant, but mainly due to the effect of phosphorus in KH2PO4 (Chapter 2). 

The microbial product did not significantly affect oil degradation. The addition of the microbial product 

alone and the microbial product with slow release fertilizer did not decrease TTNH and TTAH concentrations in the 

sediment any more than the control and the slow-release fertilizer, respectively. TTNH/hopane and TTAH/hopane 

ratios further showed no significant positive effect of microbial product on oil biodegradation. Our results suggest 

that the presence of microbial hydrocarbon degraders in Louisiana coastal marshes is not limiting oil 

biodegradation. Thus, this study suggests that the high cost of microbial amendments and soil oxidant application 

for oil bioremediation may not be warranted in coastal marshes. 
 74

Bioremediation with fertilizer enhanced oil degradation in both drained and flooded inundation conditions 

although oil degradation was greater in drained conditions than in flooded conditions (Chapter 2). In addition, 

bioremediation with fertilizer enhanced oil degradation in both sandy marsh with low organic matter (<1%) and clay 

marsh with 19% organic matter. Thus, bioremediation with inorganic nutrient additions has potential for aiding oil 

spill cleanup efforts in coastal wetlands with various inundation regimes and soil types. 
75 

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83 

APPENDIX A 

EFFECTS OF BIOREMEDIATION ON MICROBIAL BIOMASS 

 

1.0000E+06

1.0000E+07

1.0000E+08

Heterotrophic bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure A1. Effect of bioremediation agents and oil application on heterotrophic bacteria three days after the 

treatment. Values are reported in colony-forming units per gram (dry weight) of soil with standard error. 

C: control; M: microbial seeding; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+06

1.0000E+07

1.0000E+08

Petroleum-degrading bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure A2. Effect of bioremediation agents and oil on petroleum-degrading bacteria three days after treatment. 

Values are reported in colony-forming units per gram (dry weight) of soil with standard error. C: control; 

M: microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+06

1.0000E+07

1.0000E+08

1.0000E+09

Chitin-degrading bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure A3. Effect of bioremediation agents and oil on chitin-degrading bacteria 3 days after treatment. Values are 

reported in colony-forming units per gram (dry weight) of soil with standard error. C: control; M: 

microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 
 84

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

1.0000E+08

Actinomycetes and bacteria(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure A4. Effect of bioremediation agents and oil on cellulose-degrading actinomycetes and bacteria 3 days after 

treatment. Values are reported in colony-forming units per gram (dry weight) of soil with standard error. 

C: control; M: microbial addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+05

1.0000E+06

1.0000E+07

Yeast(Colony-forming units)

CC+

MM+

FF+

Treatment

Figure A5. Effect of bioremediation agents and oil on yeast populations 3 days after treatment. Values are reported 

in colony-forming units per gram (dry weight) of soil with standard error. C: control; M: microbial 

addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+04

1.0000E+05

1.0000E+06

1.0000E+07

1.0000E+08

Filamentous fungi(Colony-forming units)

CC+

MM+

FF+

Treatment
 85

Figure A6. Effect of bioremediation agents and oil on filamentous fungi 3 days after treatment. Values are reported 

in colony-forming units per gram (dry weight) of soil with standard error. C: control; M: microbial 

addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 

 

1.0000E+05

1.0000E+06

ATP (pg)

CC+

MM+

FF+

Treatment

Figure A7. Effect of bioremediation agents and oil on microbial biomass three days after treatment. Values are 

reported in picograms of ATP per gram (dry weight) of soil with standard error. C: control; M: microbial 

addition; F: fertilizer. Plus signs indicate oiling and minus signs no oiling. 
87 

APPENDIX B 

OIL MORPHOLOGY DEFINITIONS 

 Surface oil in the marsh sods expressed changes in surface oil character, which could not be attributed to 

biodegradation: 

(1) Oil Mousse: The term refers to the brown to light brown coat or cover which wipes off easily and 

has a creamy consistency. This Oil Mousse is sometimes a little tacky. In the flooded series this 

creamy texture was most likely enhanced by the mixing of oil and water, hence "Oil Mousse." 

 A similar oil morphology was observed during the Quality Control Monitoring Program, was referred 

to as Oil Cream, and was shown not to indicate biodegradation but rather a chemical reaction with the 

bioremediation product. 

 Oil Mousse was a common change in surface oil characteristics during the first observations in the 

flooded and drained M-treated series and in the flooded C-, F-, and OF-treated series. However, Oil 

Mousse was found up to seven weeks after product-application. Because of its unweathered 

character, the presence of this oil morphology several weeks after oil-application was attributed to 

renewed exposure of fresher oil after a layer of the surface oil had been degraded. 

(2) White Surface Coating: This coating appears as a white film covering oil and surface sediments. 

The presence of this surface coating was unique to the OF-treated marsh sods and was attributed to 

the application of the oxidant. 

 White surface coating was observed on the surface of all marsh sods after OF-application. This coat 

was present in both flooded and drained marsh sods but was better mixed into the sediments in the 

flooded series. In the drained series, this coating was observed up to five weeks after productapplication. In the flooded series, the last observation of this coating was observed on the eighth day 

after product-application. 

 A change in surface oil character indicative of biodegradation was observed in all treatments and replicates 

(subsurface oil did not occur in any of the marsh sods). Surface oil character changes were often first detected 

through a change in color of the oil, usually from black to brown or darker to lighter shades of brown. In addition 

to this initial indication of oil change, color changes in surface oiling were commonly associated with a change in 

the consistency of the oil. The latter was the most important oil morphology indicator of degradation. 

 Unfortunately, samples of altered oil were not taken for microbiological and/or detailed oil analyses to 

confirm enhanced microbial activity and enhanced biodegradation. However, the oil morphologies observed in the 

current study and defined below were largely consistent with some of the oil changes observed during the Exxon 

Valdez Quality Control Monitoring Program for which such samples were taken. For example, samples of oil 

defined as Organic Coat and Paste during this monitoring program were the most highly biodegraded oil samples 

collected throughout the area of oil impact at the time and confirmed that the visual and tactile changes in surface 

oil observed during the surveys were indicative of biodegradation (Debusschere et al. 1991; McMillen 1991). 

Other oil morphologies indicative of advanced biodegradation resulting from bioremediation-applications observed 

during this monitoring program were not observed in the current study. However, oil morphologies were noted to 

be partly dictated by the substrate and pre-application oil character (Debusschere et al. 1991). As the current study 

focused on salt marsh habitats and marsh sods that are relatively uniform in substrate, the limited number of oil 

changes indicative of enhanced biodegradation in the current study are most likely the result of the similarities in 

marsh sod substrate. 
 88

The following provides a list of definitions of oil morphologies observed during the current study. Reference to 

previously identified oil morphologies and associated oil chemistry and microbial analyses (Debusschere et al. 

1991, McMillen 1991) are made when applicable. 

(1) Organic Coat: The oil is dark to light-brown and rubs off very easily in a semi-liquid consistency 

without leaving any oily residue. In some cases only a surface layer of the oil had changed to an 

Organic Coat. Only this surface layer then constitutes the Organic Coat, which, when wiped off, 

leaves a tacky and tarry residue on the sediment surface. 

 Several samples of Organic Coat were analyzed during the Quality Control Monitoring Program. The 

oil analysis of these samples substantiated that all were extremely biodegraded. Dense populations of 

algae, fungi, and/or bacteria were present in samples of Organic Coat. 

 Organic Coat was a common oil change throughout the duration of this study and occurred in drained 

and flooded series for all four treatments. The first occurrence of Organic Coat in the drained 

flooding regime was observed on day 8 in the OF-treated marsh sods. In the flooded series, Organic 

Coat was first observed three weeks after product-application in the OF-, M-, and C-series. 

(2) Paste: As is the case for Organic Coat, the oil comes off when rubbed but leaves a tacky residue of a 

more solid consistency. This layer of degrading oil sometimes occurs as thin flakes and can be rolled 

into a tubular clayey mass. 

 Paste was found to be substantially biodegraded during the Quality Control Monitoring Program. 

Dense populations of algae, fungi, and/or bacteria were found in Paste samples. 

 Paste was most commonly associated with the drained series. The first occurrence of Paste in the 

drained flooding regime was observed on day 6 in the OF-treated marsh sods. In the flooded series, 

Paste was not observed until seven weeks after product-application. At that time, only the F- and Cseries were characterized by this oil change. Paste never was observed in the flooded OF-application. 

(3) Organic Film: The oil is brown and rubs off very easily without leaving any residue. The 

consistency of this film is more viscous when compared with the semi-liquid consistency of Organic 

Coat. 

 During the Quality Control Monitoring Program, this oil character was usually found in the 

subsurface. No samples of Organic Film were available for analysis. 

 Organic Film was observed in all flooded product-applications. First occurrence of the oil character 

was noted three weeks into the experiment in the flooded OF- and F-treated marsh sods. In the Mand C- treated sods, Organic Film was first observed seven weeks after product-application. In the 

drained flooding regime, Organic Film was only observed in the OF-series (first at week 5) and the 

M-series (only at week 12). 

(4) Powder: The term refers to a layer of surface sediments in the marsh sods with a powdery 

consistency. The sediments rub off very easily without leaving any residue and do not create a sheen 

when submerged in water. This oil change was termed Powder because of similarities with Powder 

found during the Quality Control Monitoring Program where it was described as a brown dry coat, 

which also rubbed off very easily without leaving any residue. This dry coat had a consistency very 

much like a "chocolate cake-mix" (Debusschere et al. 1991). 

 During the Quality Control Monitoring Program, Powder was observed on boulders and bedrock 

from which it could be easily blown away or washed away with water. These observations indicated 

that Powder was substantially biodegraded. Unfortunately, because of the transient nature of this oil 

morphology, no samples of Powder were available for analysis. 

 Powder only was observed in the drained OF-treated marsh sods at week 12. Although Paste, 

Organic Coat, and Organic Film sometimes had a powdery feel in the OF-treated series during earlier 
 89

observations, this consistency was attributed to the presence of the oxidant rather than advanced 

biodegradation. 

(5) Brown Flakes: The term is used in association with water. Dark-Brown Flakes are composed of 

biomass and oil floating on the water surface. Brown Flakes are usually characterized by a jagged 

edge. 

 An identical oil character was observed during the Quality Control Monitoring Program and was 

identified as an indicator of enhanced biodegradation for subsurface oil. 

 The only occurrence of this oil character during the current study was in association with an oil sheen 

in the flooded OF-treated marsh sods at day 2 of the experiment. 

During the Quality Control Monitoring Program, Paste, Organic Coat, and Organic Film sometimes had a silty feel. 

The siltiness of these surface oil characters was noted to be most likely the result of the presence of fine sediments 

mixed in the oil, which can be observed by touch after the oil has reached a certain degree of biodegradation. This 

observation was not repeated during the current study. However, the lack of coarser sediments in marsh sediments 

may have prevented this characteristic to occur. 
The Department of the Interior Mission

As the Nation's principal conservation agency, the Department of the Interior has responsibility 

for most of our nationally owned public lands and natural resources. This includes fostering 

sound use of our land and water resources; protecting our fish, wildlife, and biological diversity; 

preserving the environmental and cultural values of our national parks and historical places; 

and providing for the enjoyment of life through outdoor recreation. The Department assesses 

our energy and mineral resources and works to ensure that their development is in the best 

interests of all our people by encouraging stewardship and citizen participation in their care. 

The Department also has a major responsibility for American Indian reservation communities 

and for people who live in island territories under U.S. administration. 

The Minerals Management Service Mission 

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) 

primary responsibilities are to manage the mineral resources located on the Nation's Outer 

Continental Shelf (OCS), collect revenue from the Federal OCS and onshore Federal and Indian 

lands, and distribute those revenues. 

Moreover, in working to meet its responsibilities, the Offshore Minerals Management Program 

administers the OCS competitive leasing program and oversees the safe and environmentally 

sound exploration and production of our Nation's offshore natural gas, oil and other mineral 

resources. The MMS Minerals Revenue Management meets its responsibilities by ensuring the 

efficient, timely and accurate collection and disbursement of revenue from mineral leasing and 

production due to Indian tribes and allottees, States and the U.S. Treasury. 

The MMS strives to fulfill its responsibilities through the general guiding principles of: (1) being 

responsive to the public's concerns and interests by maintaining a dialogue with all potentially 

affected parties and (2) carrying out its programs with an emphasis on working to enhance the 

quality of life for all Americans by lending MMS assistance and expertise to economic 

development and environmental protection.