National Renewable Energy Laboratory 

Innovation for Our Energy Future 

A national laboratory of the U.S. Department of Energy

Office of Energy Efficiency & Renewable Energy

NREL is operated by Midwest Research Institute ● Battelle Contract No. DE-AC36-99-GO10337 

Analysis of Coconut-Derived 

Biodiesel and Conventional 

Diesel Fuel Samples from the 

Philippines 

Task 2 Final Report 

T.L. Alleman and R.L. McCormick 

Milestone Report 

NREL/MP-540-38643 

January 2006 
National Renewable Energy Laboratory 

1617 Cole Boulevard, Golden, Colorado 80401-3393 

303-275-3000 • www.nrel.gov 

Operated for the U.S. Department of Energy 

Office of Energy Efficiency and Renewable Energy 

by Midwest Research Institute • Battelle 

Contract No. DE-AC36-99-GO10337

Analysis of CoconutDerived Biodiesel and 

Conventional Diesel Fuel 

Samples from the 

Philippines 

Task 2 Final Report 

T.L. Alleman and R.L. McCormick 

Prepared under Task Nos. WF3Y.1000 and FC02.0800 under an 

agreement between the U.S. Agency for International Development 

and the U.S. Department of Energy’s Policy and International Affairs 

Office 

Milestone Report 

NREL/MP-540-38643 

January 2006 
NOTICE

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Table of Contents 

Table of Contents............................................................................................................................ 1 

List of Abbreviations...................................................................................................................... 1 

Executive Summary........................................................................................................................ 2 

Background..................................................................................................................................... 3 

Results and Discussion ................................................................................................................... 5 

Fuel Properties............................................................................................................................ 5 

Stability....................................................................................................................................... 9 

Microbial Degradation.............................................................................................................. 11 

Blend Level Determination....................................................................................................... 12 

Summary and Recommendations ................................................................................................. 14 

References..................................................................................................................................... 14 

Appendix 1. Subcontractor Report on Microbial Degradation 

Appendix 2. FTIR/ATR Method for Biodiesel Blend Determination 

List of Abbreviations 

 

AET Atmospheric Equivalent Temperature 

AOCS American Oil Chemists Society 

ASTM American Society for Testing and Materials 

ATP Adenosine triphosphate 

ATR Attenuated Total Reflectance 

oC degrees Celsius 

cm

-1 wavenumber 

CME coconut methyl ester 

DA additized diesel fuel 

DUA unadditized diesel fuel 

FTIR Fourier Transform infrared 

g gram 

hr hour 

IQT Ignition Quality TesterTM

mass% percent by mass 

mg KOH/g milligrams of Potassium Hydroxide per gram 

mg/100ml milligrams per 100 milliliters 

mm

2

/s millimeters squared per second 

NREL National Renewable Energy Laboratory 

PNS Philippine National Standard 

ppm parts per million 

USDOE U.S. Department of Energy 

vol% percent by volume 

1
2

Executive Summary 

NREL has provided technical assistance to the Philippines in the area of biodiesel property 

testing and utilization. Three fuel samples were provided to NREL from the Philippines. These 

fuel samples were an unadditized diesel fuel, an additized diesel fuel, and a coconut derived 

biodiesel (coconut methyl ester or CME) fuel. Each diesel fuel sample was analyzed neat and as 

a blend with CME. The CME-diesel fuel blend samples were 1 volume% and 5 volume% CME 

in the additized and unadditized diesel fuels. Fuel property testing was also performed on the 

neat CME sample. 

Results from the fuel property testing show that the unadditized diesel fuel and the additized 

diesel fuel samples met the Philippine National Standards for diesel fuel quality. Results from 

the fuel property testing for the CME sample shows that the current fuel quality standards were 

met. The 1% and 5% blends of CME in the diesel fuels also met the current Philippine National 

Standard for diesel fuel quality. The 5% blend of CME in diesel fuel did increase the cetane 

number slightly for each blend. 

The water separability was gauged to determine if the diesel fuels or the CME-diesel fuel blends 

mixed with salt water. Test results show that the diesel fuels and the CME-diesel blends did not 

take up significant amounts of water, nor were stable emulsions formed for any of the fuels or 

fuel blends tested. 

The stability of CME and CME-diesel fuel blends was determined through several test methods. 

In all cases, the CME sample, the diesel fuel samples, and the 5% CME-diesel blends exhibited a 

similar level of stability. The test results showed few insolubles were generated during the tests, 

which under storage conditions, may contribute to poor engine performance due to plugged fuel 

filters or clogged injectors. 

Sixteen indicators of microbial degradation were measured over the additized and unadditized 

diesel fuel samples and the CME fuel sample. The results from these sixteen indicators were 

somewhat equivocal, but show that the CME sample and the neat diesel fuel samples have 

similar resistance to microbial degradation, although the mechanisms for degradation may vary. 

A Fourier Transform infrared technique for determining the percentage of biodiesel in a blend 

was demonstrated on CME-diesel fuel blend samples. The technique was highly linear and can 

be used to quantitatively determine the percentage of CME in a diesel fuel sample. 
Background 

Biodiesel is a renewable diesel fuel produced from feedstocks like vegetable oils or animal fats. 

To produce biodiesel, the starting oil is reacted with alcohol in the presence of a catalyst. The 

products of these reactions are the fatty acid methyl esters that compose biodiesel. 

Because the Philippines imports a majority of its petroleum, the Philippine government has 

begun to explore domestically produced biodiesel to promote energy security. The Philippine 

government has recently directed all government offices to use 1% biodiesel in diesel fuel where 

the biodiesel is produced from domestically grown coconuts. The coconut-derived biodiesel 

(CME or coconut methyl ester) will aid in the reduction of petroleum imports and improve fuel 

lubricity and may reduce exhaust emissions. 

The U.S. Department of Energy’s (USDOE) National Renewable Energy Laboratory (NREL) is 

a world leader in the effort to develop and advance renewable energy and improve energy 

efficiency. NREL’s fuels research activities include fuels utilization impacts and the impact of 

fuel properties on engine and vehicle performance and emissions. 

NREL has provided technical assistance to the Philippines in the area of biodiesel property 

testing. This report covers fuel property testing results for coconut-derived biodiesel, 

conventional diesel fuel samples from the Philippines, as well as 1% and 5% blends of CME in 

the conventional diesel fuels. 

Test Matrix 

Three fuel samples were provided to NREL from the Philippines. These fuel samples were an 

unadditized diesel fuel (DUA), an additized diesel fuel (DA), and a CME fuel. Each diesel fuel 

sample was analyzed neat and as a blend with CME (1 volume% and 5 volume% CME in diesel 

fuel). Fuel property testing was also performed on the neat CME sample. Table 1, which follows, 

lists the tests performed for each fuel and fuel blend. In addition, samples of CME in DA fuel 

were examined using an existing method for determining the percentage of biodiesel. 

3
Table 1. Fuel Property Tests for Philippine Diesel Fuel and CME Samples. 

An “X” indicates that the test was performed. 

Fuel ASTM D975 ASTM D6751 ASTM D4625 EN 14112 ASTM D6468 ASTM D2274 ASTM D1401 

Standard 

Specification 

for Diesel 

Fuels 

Standard 

Specification 

for Biodiesel 

Fuel (B100) 

Blend Stock 

for Distillate 

Fuels 

Standard 

Test 

Method for 

Distillate 

Fuel 

Storage 

Stability at 

43oC 

Fatty Acid 

Methyl Esters 

(FAME) 

Determination 

of Oxidation 

Stability 

(Accelerated 

Oxidation Test) 

Standard Test 

Method for 

High 

Temperature 

Stability of 

Distillate 

Fuels 

Standard 

Test Method 

for Oxidation 

Stability of 

Distillate Fuel 

Oil 

(Accelerated 

Method) 

Standard Test 

Method for 

Water 

Separability of 

Petroleum 

Oils and 

Synthetic 

Fluids 

DA X X X X X X DUA X X X X X X 

CME X X X X X X 

CME + 

microbicide 

1%CME in DA X X X X X 

1% CME in 

DUA X X X X X

5% CME in 

DA X X X X X X

5% CME in 

DUA X X X X X X

4
Results and Discussion 

Fuel Properties 

The fuel property results from the CME sample are given in Table 2, along with the current 

Philippine National Standard (PNS2020:2003) and comparable results from a soy-derived 

biodiesel in the United States. 

Table 2. Physicochemical Property Results from CME Testing. 

Property Method CME Results PNS2020:2003 Soy 

Biodiesel1

Flash Point, oC ASTM D93 107 100.0, min 157 

Water & Sediment, vol% ASTM D2709 0.0 0.050, max <0.05 

Kinematic Viscosity, mm2

/s 

@ 40oC ASTM D445 2.656 2.0-4.5 4.2 

Sulfated Ash, mass% ASTM D874 0.002 0.020 0.002 

Sulfur, ppm ASTM D5453 3 50, max 9 

Copper Corrosion, 3hr 

@50oC ASTM D130 1A No. 3, max 1A 

Cetane Number ASTM D613 70 42, min 55 

Cloud Point, oC ASTM D2500 -5 Report 0.4 

Carbon Residue, mass% ASTM D4530 N/A** 0.050, max Not Performed 

ASTM D524 <0.010 N/A 0.02 

Acid Number, mg KOH/g ASTM D664 0.17 0.50, max 0.35 

Free Glycerin, mass% ASTM D6584 N/A No Standard 0.006 

Monoglyceride, mass% ASTM D6584 N/A N/A 0.40 

Diglyceride, mass% ASTM D6584 N/A N/A 0.21 

Triglyceride, mass% ASTM D6584 N/A N/A 0.18 

Total Glycerin, mass% ASTM D6584 0.043 N/A 0.16 

Free Glycerin, mass% AOCS Ea6-94* 0.02 0.02, max Not Used 

Total Glycerin, mass% AOCS Ca14-56 0.145 0.24, max Not Used 

Phosphorus, mass% ASTM D4951 0.000 0.001, max 0.0006 

Distillation, AET 90% 

recovered, oC ASTM D1160 327 360, max 352 

*PNS2020:2003 references AOCS Ea6-51, which has been replaced by AOCS Ea6-94 

**Not applicable 

As shown in Table 2, the CME sample tested in this study met all the PNS 2020:2003 

requirements. The carbon residue was tested using ASTM D524, rather than ASTM D4530. The 

ASTM guidelines allow for substitution of ASTM D524 in place of ASTM D4530 for carbon 

5
residue determination. Using the correlation provided in ASTM D524, the results for carbon 

residue are equivalent to those obtained for ASTM D4530 for these low levels of carbon residue. 

The results from the diesel fuel property testing are given in Table 3. The results from the DA 

and DUA fuel samples are given along with the PNS/DOE QS 004:2003, where applicable. As 

with the CME sample, the DUA and DA fuel samples met the Philippine fuel quality standards. 

The cetane number was measured through ASTM D6890 using an ignition quality tester or 

IQTTM. The IQT measures cetane number through combustion techniques and produces results 

comparable to the ASTM D613 cetane engine.2 The cetane results from the IQT measurement 

were above the minimum standard for both DA and DUA samples. 

At the request of Philippine stakeholders, the separability of diesel fuel and water was tested due 

to the practice in the Philippines of pushing diesel fuels through the pipeline with water. The test 

measures how known volumes of diesel fuel and water separate after mixing for 5 minutes at 

elevated temperatures. The results from this testing show that miscibility is low and very little 

water mixes with the diesel fuel sample during testing. 

6
Table 3. Physicochemical Property Results from 

Additized and Unadditized Diesel Fuels. 

Property Method DA DUA 

PNS/DOE QS 

004:2003 for 

On-Road 

Vehicles 

U.S. 

No.2 Diesel 

Fuel Sample 

Kinematic Viscosity, 

mm2

/s @40oC ASTM D445 3.570 3.608 2.0-4.5 2.622 

Flash Point, oC ASTM D93 85 79 55.0, min 63 

Distillation, oC ASTM D86 N/A* N/A N/A N/A 

 IBP 192.4 183.3 N/A 170.7 

 T10 218.2 214.9 N/A 208.6 

 T50 280.9 281.1 N/A 263 

 T90 355.8 355.2 370, max 318.2 

 FBP 389.2 386.7 N/A 347.1 

Sulfated Ash, mass% ASTM D482 <0.001 <0.001 No Standard <0.001 

Sulfur, ppm ASTM D2622 382 355 500, max 388 

Cloud Point, oC ASTM D2500 3 4 No Standard -22 

Water & Sediment, 

vol% ASTM D2709 0.01 0.01 0.10, max 0.01 

Copper Corrosion ASTM D130 1B 1B No Standard 1B 

Cetane Number ASTM D613 Not performed Not performed 50, min 44.5 

ASTM D6890 55 56 No Standard No Standard 

Carbon Residue, 10% 

Bottoms, mass% ASTM D524 0.08 0.09 0.15, max 0.11 

Water Separability, 

 Saline Water, 54oC ASTM D1401 N/A N/A No Standard N/A 

 Oil Layer 

 Appearance Cloudy Cloudy N/A N/A 

 Emulsion 

 Appearance N/A N/A N/A N/A 

 Water Layer 

 Appearance Clear Clear N/A N/A 

 Test Duration 5 minutes 5 minutes N/A N/A 

 Volume of Layers 40-40-0 40-40-0 N/A N/A 

 Oil/Emulsion 

Interface N/A N/A N/A N/A 

 Water/Emulsion 

 Interface N/A N/A N/A N/A 

* Not applicable 

The results for the CME-diesel fuel blends are presented in Table 4. CME was blended in the DA 

and DUA fuels at the 1 volume% and 5 volume% levels. The diesel fuel properties were not 

altered significantly by blending with 1% CME. A slight increase in the cetane number was 

noted with the 5% blends of CME. Both blends continued to meet the PNS/DOE QS 004:2003 

standards. 

7
The ASTM D1401 results show that little water is taken up by the fuel blends. A slight volume 

increase was recorded in the aqueous layer of the 1% CME in DA, with a lacy (bubbles present) 

appearance, although no emulsion layer was recorded. In contrast, no volume change in the 

layers was recorded for the 1% CME in DUA sample, although the aqueous layer was hazy 

(translucent). For both the 5% CME blends, no changes were noted in either the appearance of 

the layers or in the volumes of each layer. 

Table 4. Physicochemical Property Results from Blends of CME 

and Unadditized and Additized Diesel Fuels.

Property Method 

1%CME in 

Additized 

Diesel Fuel 

1%CME in 

Unadditized 

Diesel Fuel 

5%CME in 

Additized 

Diesel Fuel 

5%CME in 

Unadditized 

Diesel Fuel 

Kinematic Viscosity, 

mm2

/s @40oC ASTM D445 3.548 3.535 3.494 3.503 

Flash Point, oC ASTM D93 86 78 86 78 

Distillation, oC ASTM D86 

 IBP 199.8 185.4 190.2 182.3 

 T10 221.6 213.2 211.9 219.4 

 T50 282.7 278.5 276.9 280.9 

 T90 356.7 353.1 349.9 354.9 

 FBP 391.5 387.9 388.5 387.9 

Sulfated Ash, mass% ASTM D482 <0.001 <0.001 <0.001 <0.001 

Sulfur, ppm ASTM D2622 Not Requested Not Requested 366 350 

Cloud Point, oC ASTM D2500 4 5 4 4 

Water & Sediment, 

vol% ASTM D2709 0.01 0.01 0.01 0.01 

High Temperature 

Stability, 180 

minutes, Avg. % 

Reflectance 

ASTM D6468 90 98 96 98 

Copper Corrosion ASTM D130 1B 1B 1B 1B 

Cetane Number ASTM D6890 55 55 58 57 

Carbon Residue, 10% 

Bottoms, mass% ASTM D524 0.09 0.10 0.08 0.08 

Water Separability, 

Saline Water, 54oC ASTM D1401 

 Oil Layer 

 Appearance Cloudy Cloudy Cloudy Cloudy 

 Emulsion 

 Appearance N/A* N/A N/A N/A 

 Water Layer 

 Appearance Lacy Hazy Clear Clear 

 Test Duration 5 minutes 5 minutes 5 minutes 5 minutes 

 Volume of Layers 41-39-0 40-40-0 40-40-0 40-40-0 

 Oil/Emulsion 

 Interface N/A N/A N/A N/A 

 Water/Emulsion 

 Interface N/A N/A N/A N/A 

*Not Applicable 

8
Stability 

Fuel stability, while not part of current fuel property standards in the United States, is 

nevertheless important so tests were performed to understand how CME blending affects fuel 

property changes during storage. Fuel stability testing was performed using accelerated methods 

to approximate field aging. The methods use varying degrees of acceleration, from mild to 

severe. Table 5 shows the stability results from all the fuels and fuel blends. 

All the neat fuel and fuel blend samples exceeded 8 hours on the EN14112 oxidation stability 

test. The European standard (EN14214) for biodiesel (B100) on this test is 6 hours minimum. 

There is currently no oxidation stability standard for biodiesel in the United States. 

The neat fuels and the 5% blends of CME in the DA and DUA fuel samples were tested using 

the ASTM D2274 and D6468 methods for accelerated and high temperature stability, 

respectively. The 1% blends of CME in the diesel fuels were not tested because the 5% CME 

blend represented the most extreme case in terms of stability. The neat fuels and the 5% CME 

blends had very high percent reflectance on the D6468 test, thus indicating few solids were 

present in the fuels at the conclusion of the test. The insolubles for D2274 were lowest for the 

neat diesel fuels. The highest insoluble result was recorded with the neat CME fuel sample. 

Although the CME sample results were an order of magnitude greater than the neat diesel fuels 

(1.55 mg/100mL compared to 0.12 mg/100mL), the absolute level was low. For example, a 

typical pipeline specification for diesel fuel in the U.S. is 2.5mg/100 mL maximum for the 

ASTM D2274 test. All fuels tested in this study produced deposits well below this level. The 

influence of the CME sample on the blend results is evident, with the results increasing to 0.63 

mg/100mL and 0.54 mg/100mL for the DA and DUA blends, respectively. 

 

Storage stability testing was conducted using ASTM D4625 for the DUA and DA fuel samples, 

the CME sample, and a blend of 5% CME in the DUA fuel. In this method, the fuel storage 

stability is determined using mildly accelerated conditions (43oC). For petroleum fuels, there is 

an excellent correlation between D4625 and performance of fuels in storage. One week on the 

D4625 test approximates one month of storage in an underground tank. At specific intervals 

during testing, the insolubles in the sample are measured and classified as either filterable or 

adherent. Figure 1 illustrates how the insoluble levels varied over the test period (the data are in 

Table 6). 

The DA and DUA fuel samples had very low levels of insolubles over the test period. The 

variability observed in these levels is likely due to the very low levels measured. In contrast, the 

CME sample recorded relatively higher levels of insolubles over the test period. These levels are 

still quite low and suggest that storage of neat CME or CME blend samples for periods up to 12 

months is feasible. However if B100 CME is to be stored for 12 months, the inclusion of an 

antioxidant additive may be prudent. Again, the influence of the CME sample on the blend is 

evident in the results. The insolubles measured over the test period are increased relative to the 

base diesel fuels, although are still very low. 

9
Table 5. Fuel Stability Test Results. 

Property Method CME DUA DA 1% CME in 

DA 

1%CME in 

DUA 

5% 

Oxidative Stability EN14112 Time Exceeded 

8 hours 

Exceeded 

8 hours 

Exceeded 

8 hours 

Exceeded 

8 hours 

Exceeded 

8 hours 

Ex

8 

 Temperature 110oC 110oC 110oC 110oC 110oC High Temperature Stability,180 minutes, Avg % Reflectance 

ASTM D6468 100 97 94 Not tested Not tested 

Accelerated Stability, 

mg/100mL 

ASTM D2274 1.55 0.12 0.12 Not tested Not tested 

Table 6. ASTM D4625 Fuel Stability Results. 

5% CME in 

DUA 

(Sample 1)

5% CME in 

DUA 

(Sample 2) 

DA (Sample 1) DA (Sample 2) DUA (Sample 1) DUA (Sample 2) 

Week 0 

Filterable 0.00 0.17 0.00 0.00 0.00 0.00 

Adherent 0.03 0.06 0.03 0.03 0.11 0.03 

Total 0.03 0.23 0.03 0.03 0.11 0.03 

Week 4 

Filterable 0.11 0.00 0.00 0.03 0.00 0.14 

Adherent 0.00 0.09 0.00 0.06 0.09 0.00 

Total 0.11 0.09 0.00 0.09 0.09 0.14 

Week 8

Filterable 0.26 0.26 0.03 0.09 0.03 0.17 

Adherent 0.06 0.14 0.17 0.14 0.00 0.11 

Total 0.32 0.40 0.20 0.23 0.03 0.28 

Week 12 

Filterable 0.26 0.23 0.00 0.00 0.00 0.00 

Adherent 0.03 0.17 0.17 0.11 0.14 0.11 

Total 0.29 0.40 0.17 0.11 0.14 0.11 

10
Insolubles, mg/100mL

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Filterable

Adherent

Week 0

Week 4

Week 8

DUA Week 12

DA

5% CME in DUA

CME

DUA

DA

5% CME in DUA

CME

DUA

DA

5% CME in DUA

CME

DUA

DA

5% CME in DUA

CME

Figure 1. ASTM D4625 Results for Philippine Diesel Fuels, CME, and CME-Diesel 

Fuel Blends. 

Microbial Degradation 

The susceptibility to microbial degradation was tested through ASTM E1259. Sixteen indicators 

for biodegradability were examined for the neat diesel fuel and the neat CME samples. The null 

hypothesis for this testing was that CME and conventional diesel fuel samples do not differ 

significantly for biodeterioration risk. Table 7 lists each of the indicators and whether the null 

hypothesis is supported or refuted. The results show that the microbial degradation of neat CME 

sample is likely equivalent to that of conventional diesel fuel sample, but may occur by different 

mechanisms. The complete biodegradation report is attached as Appendix 1. 

Experience in the United States and other countries had shown that microbial degradation results 

for neat fuels should not be extrapolated to blended fuels. Because of this, the results presented 

here for the neat CME, DA, and DUA fuel samples may not be representative of the results for 

CME blend samples. Additional testing is required to fully understand the influence of low levels 

of CME in diesel fuel samples on biodeterioration susceptibility. However, it is not anticipated 

that blending of CME into diesel fuel at 5 volume% or lower will significantly impact 

biodeterioration susceptibility. 

11
Table 7. Results from Indicators for ASTM E1259 Biodegradability for CME 

Compared to Conventional Diesel Fuel. 

Indicator Support or Refute Null 

Hypothesis 

Fuel More Susceptible to 

Microbial Degradation 

Gross Appearance Supports Equivalent 

Fuel Chemistry 

 Entrained Water Refutes CME 

 Total Acid Number Refutes CME 

 Corrosivity Refutes CME 

Bottom Water Chemistry 

 pH Refutes Diesel Fuel 

 Alkalinity/Acidity Refutes Diesel Fuel 

 Hardness Refutes Diesel Fuel 

 Total Dissolved Solids Supports Equivalent 

 Total Organic Carbon Supports Equivalent 

Bottom Water Microbiology 

 Adenosine Triphosphate (ATP) Refutes Diesel Fuel 

 Oxygen Demand Refutes Diesel Fuel 

 Culturable Bacteria Refutes Diesel Fuel 

 Culturable Fungi Refutes Diesel Fuel 

Fuel Microbiology 

 ATP Equivocal 

 Culturable Bacteria Refutes Diesel Fuel 

 Culturable Fungi Refutes Diesel Fuel 

Blend Level Determination 

To determine the percentage of biodiesel in a sample of diesel fuel, a Fourier Transform infrared 

(FTIR) method has been applied. The method utilizes an Attenuated Thermal Reflectance (ATR) 

liquid cell and approximately 2 mL of sample. The spectra were collected from 48 scans together 

at a slow mirror speed. Spectra were taken from 2,000 cm-1 to 600 cm-1 and software baseline 

corrected (Nicolet supplied software) to bring the baseline to zero at 2,000 cm-1. The peak at 

1,745 cm-1 was used to determine the percent biodiesel in the sample. A small absorbance was 

noted from diesel fuel at this intensity and was subtracted from the peak intensity for the 

biodiesel blends. This technique is currently being discussed as an ASTM method to be included 

in future ASTM specifications for biodiesel blends. A copy of the method is in Appendix 2. 

Multiple blends were produced using the CME in a typical US diesel fuel meeting the 500 ppm 

sulfur standard. A calibration curve was produced using blends with varying percentages of 

CME in diesel fuel. A Beer’s law fit of the curve had a very high correlation coefficient (>0.99) 

and is illustrated in Figure 2. The robustness of the technique will allow for quantitative 

determination of unknown concentrations of biodiesel in diesel fuel. 

12
Weight % Biodiesel

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Absorbance

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

CME blends

Linear regression

r

2 = 0.9959

Figure 2. Calibration Curve of CME-Diesel Fuel Blends as Measured by FTIR 

Spectroscopy. 

13
Summary and Recommendations 

Fuel property, stability, and microbial deterioration tests were conducted on neat CME, neat DA 

and DUA fuel samples, and 1% and 5% blends of CME in each diesel fuel. Results from the fuel 

property testing for these samples show that the current fuel quality standards were met. 

Fuel stability testing revealed that all the samples tested had adequate storage stability. The 

microbial deterioration testing proved more complex to interpret. The results show that CME and 

the DA and DUA samples have similar susceptibility to microbial degradation, although the 

mechanisms vary. In order to fully assess the microbial susceptibility of the biodiesel blends 

likely to be used in the Philippines, additional testing on blended fuels with several replicates is 

recommended. 

The Philippines uses salt water to push fuels through their pipeline system. The tendency of fuels 

to take up water was examined for the neat diesel fuels and the 1% and 5% blends of CME in 

diesel fuel. The tests showed little miscibility of the fuel samples with water. FTIR testing of 

CME-diesel fuel blends showed the technique is applicable to coconut-derived biodiesel and 

could be used for quantitative determination of biodiesel percentage in blends. 

References 

1. “Survey of the Quality and Stability of Biodiesel and Biodiesel Blends in the United States in 

2004”, NREL-TP-540-38836, http://www.nrel.gov/docs/fy06osti/38836.pdf, October 2005. 

2. Diesel Fuel Ignition Quality Tester, online, http://www.aet.ca/IQT_Info_Brochure.pdf, 

Accessed August 2005. 

14
Appendix 1. 

Subcontractor Report on Microbial Degradation
NREL SUBCONTRACT NO. ADK-5-55501-01 

EVALUATION OF BIODIESEL FUEL BIODETERIORATION 

SUSCEPTIBILITY 

FINAL REPORT 

 
NREL SUBCONTRACT NO. ADK-5-55501-01 

August 12, 2005 

Page 2 of 41 

THIS PAGE LEFT BLANK INTENTIONALLY 
NREL SUBCONTRACT NO. ADK-5-55501-01 

August 12, 2005 

Page 3 of 41 

Table of Contents 

NREL SUBCONTRACT NO. ADK-5-55501-01 EVALUATION OF BIODIESEL FUEL 

BIODETERIORATION SUSCEPTIBILITY..................................................................... 1 

FINAL REPORT ................................................................................................................ 1 

Table of Contents................................................................................................................ 3 

Executive Summary............................................................................................................ 5 

Background......................................................................................................................... 6 

Materials and methods........................................................................................................ 6 

Facilities.......................................................................................................................... 6 

Challenge Population...................................................................................................... 7 

Stage 1 Microcosms.................................................................................................... 7 

Stage 2 Microcosm ..................................................................................................... 7 

Test Microcosms............................................................................................................. 9 

Sampling ......................................................................................................................... 9 

Testing............................................................................................................................. 9 

Gross Observations..................................................................................................... 9 

Microbiology............................................................................................................. 10 

Chemistry...................................................................................................................... 11 

Fuel Corrosivity ............................................................................................................ 12 

Results............................................................................................................................... 12 

Challenge Population.................................................................................................... 12 

Test Microcosms........................................................................................................... 15 

Gross Observations................................................................................................... 15 

Fuel Chemistry.......................................................................................................... 21 

Fuel Corrosivity ........................................................................................................ 22 

Bottom-water Chemistry........................................................................................... 23 

Bottom-water Microbiology ..................................................................................... 28 

Discussion......................................................................................................................... 33 

Conclusions....................................................................................................................... 38 

Recommendations............................................................................................................. 39 

APPENDIX A – BCA TEST METHODS...................................................................... A-1 

Modified Oxygen Demand for Monitoring Biocontamination of Fuel System Bottomswater............................................................................................................................ A-3 

ATP in Fuel System Bottoms-water ........................................................................... A-7 

APPENDIX B– LABORATORY REPORTS ................................................................ B-1 

Clark Laboratories Report 23878 of 3/1/05 – CME-100 Chemistry .......................... B-3 

Clark Laboratories Report 24834 of 6/9/05 – Microcosm A Fuel chemistry............. B-4 

Clark Laboratories Report 24835 of 6/9/05 – Microcosm B Fuel chemistry ............. B-5 

Clark Laboratories Report 24836 of 6/9/05 – Microcosm C Fuel chemistry ............. B-6 

Clark Laboratories Report 24838 of 6/9/05 – Microcosm E Fuel chemistry ............. B-8 

EMSL Analytical Case No: 360500219 of 03/09/05 Modified NACE TM0172 ....... B-9 

EMSL Analytical Case No: 360500555 of 06/08/05 Modified NACE TM0172 ..... B-12 

EMSL Analytical Case No: 370501236 of 03/28/05 Water Chemistry ................... B-15 
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EMSL Analytical Case No: 370501236 of 06/24/05 Water Chemistry ................... B-17 

EMSL Analytical Case No: 370501576 of 03/07/05 Water and Fuel MicrobiologyB-20 

EMSL Analytical Case No: 370501576 of 06/02/05 Water and Fuel MicrobiologyB-24 
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Executive Summary 

This study was undertaken in order to evaluate the biodegradability of 100% coconut 

methyl ester (CME-100) relative to conventional, non additized low sulfur diesel 

(NALSD) and additized low sulfur diesel (ALSD) fuels. The null hypothesis was: 

CME-100 biodegradation risk is not significantly different from conventional 

LSD biodeterioration risk when products are stored under typical above ground 

storage tank conditions for up to three months. 

Five microcosm test systems were set up; each containing 2 L fuel over 500 mL synthetic 

bottom-water. The microcosms were designated as follows: A) NALSD; B) ALSD; C) 

CME-100; D) microbicide-treated CME-100; and E) filter sterilized CME-100 over 

sterile bottom-water (sterile control). The microcosm exposure period was 90-days. At 

T0, microcosms A, B, C and D were inoculated with an uncharacterized mixed culture 

that had been preconditioned to grow in fuel over water. Microcosms were incubated in 

the dark at 23 ± 0.5 °C. 

Gross observations were made on all microcosms at T0 and monthly thereafter. Fuel and 

bottom-water samples were collected from microcosms A & E for chemical testing at T0. 

Microcosm A and E bottom-water adenosine triphosphate (ATP) concentration was 

tested monthly. All fuel and bottom-water tests were run on all microcosms at T3-months. 

The results of this study were somewhat equivocal, but generally did not support the null 

hypothesis. Sixteen parameters were used to compare CME-100 biodegradability against 

both additized and non-additized LSD. Data for six of the 16 parameters supported the 

null hypothesis. Although CME-100 was more hazy than LSD at T1-month and T-2 months, 

the CME-100 could not be differentiated from the ALSD or NALSD on the basis of gross 

appearance, corrosivity; bottom-water alkalinity, TDS, TOC, or O.D.; or fuel-phase ATP 

concentration at T3-months. Karl Fischer water, TAN, bottom-water pH, and bottom-water 

hardness data indicated that CME-100 was significantly more susceptible than LSD to 

biodeterioration. Fuel and bottom-water ATP and culturable bacteria and fungi data 

indicated that CME-100 was less likely than LSD to support culturable microbes. 

However, the ∼ 5 Log CFU bacteria/mL recoveries from CME-100 bottom-water made 

microcosms C, D, and E high biodeterioration risk systems. 

The overall results suggest that CME-100 and LSD have comparable but somewhat 

different biodeterioration susceptibilities. CME-100 may be less susceptible than 

LSD to biodeterioration. 
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Background 

NREL is working in cooperation with the Philippines biodiesel commercial development 

effort. As part of that effort, stakeholders are interested in understanding how CME-100 

susceptibility to biodeterioration compares with that of conventional diesel fuel. 

The current draft proposed revision to ASTM E1259, Practice for Evaluation of 

Antimicrobials in Liquid Fuels Boiling Below 390°C, provides a protocol for 

investigating both product biodegradability and microbicide performance in laboratory 

scale fuel systems (microcosms). 

BCA originally proposed a testing program that included replicate microcosms, incubated 

for a period of six-months. The proposed test parameter list included carbon-number 

distribution and distillation profile; two parameters that reflect substantial fuel chemistry 

changes. Through a series of teleconference discussions, the final testing program as 

reported in this document was defined. The three-month test duration reflected 

stakeholder estimates of the maximum storage period for CME-100. Cost considerations 

necessitated the elimination of carbon-number distribution and distillation profiles from 

the test battery. Additionally, cost considerations resulted in the designation of 

microcosms A & E as surrogates (most and least biodegraded) for the other three 

microcosms for microbiological tests run a T0, T1-month, and T2-months. These test design 

compromises were made with the mutual understanding of all stakeholders that the 

resulting data matrix, though somewhat limited, would probably be sufficient to test the 

study’s primary hypothesis: 

Hypothesis: CME-100 biodegradation risk is not significantly different from 

conventional LSD biodeterioration risk when products are stored under typical 

above ground storage tank conditions for up to three months. 

The final test plan was not designed to provide data that would support detailed 

explanations for differences in the relative biodeterioration of CME-100 and LSD in the 

test microcosms. 

Materials and methods 

Facilities 

All microcosms were assembled and maintained at EMSL Analytical, Inc., 107 Haddon 

Avenue, Westmont, NJ 08108. Fuel chemistry tests were performed by Clark 

Laboratories, LLC, 4000 Tech Center Drive, Monroeville, PA 15146. 
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Challenge Population 

Stage 1 Microcosms 

Triplicate Stage 1 microcosms were prepared by dispensing 100 mL synthetic bottomwater (SBW) into 1.0 L Mason jars and dispensing 500 mL NALSD1

 on top of the 

bottom-water. Bacto Minimal Broth Davis without Dextrose (Becton-Dickinson, 

Baltimore, MD) – 0.1 % w/v in deionized water (10.6 g/L) – was used as synthetic 

bottom-water. The SBW was sterilized by filtering it through a 0.2 μm cellulose acetate 

filter. 

Each Stage 1 microcosm was inoculated with 1 mL of uncharacterized contaminated 

bottom-water that had been collected from fuel storage tanks. Contaminated bottomwater samples were provided by Fuel Quality Services, Inc., Flowery Branch, GA. After 

inoculation, microcosms were incubated in the dark at 25 ± 2 °C for three weeks. 

Microcosms were then tested for culturable bacteria and fungal counts Each Stage 1

microcosm was reinoculated with an additional 1 mL of contaminated bottom-water and 

with 10 μL of a suspension of fungal biomass prepared from fungal colonies that had 

grown on the media used to enumerate microcosm fungi (see below). 

Stage 1 microcosms were incubated for an additional two-weeks then examined visually 

and tested for ATP concentration. A second round of Stage 1 microcosms was prepared. 

Each of the triplicate Stage 1, Round 2 microcosms was inoculated with 10 mL of pooled 

Stage 1, Round 1 bottom-water. Stage 1, Round 2 microcosms were incubated for two 

weeks then tested for robust growth. For the purposes of this project, robust was defined 

as: 

• ≥ 1 x 106

 CFU bacteria/mL bottom-water by viable count method (spread plate) 

• ≥ 1 x 105 CFU fungi/bottom-water mL by viable count method (spread plate) 

• ≥ 1 x 103

 RLU (log10 RLU ≥ 3) bottom-water ATP by BCA SOP 006 (Appendix A) 

After two-weeks and confirmation of robust growth in the Stage 1, Round 2 microcosms, 

67 mL bottom-waters from each of these microcosms was pooled to be used as the 

inoculum for the Stage 2 microcosm. 

Stage 2 Microcosm 

The Stage 2 microcosm was used as the microbial challenge source for the Test 

microcosms. A 3.78 L wide-mouthed glass jar was used for Stage 2. An 800 mL portion 

of SBW was dispensed into the glass jar. Next, the microcosm was inoculated with 200 

mL of pooled Stage 1, Round 2 microcosm bottom-water (figure 1). Finally, 2.0 L 

NALSD was decanted into the Stage 2 microcosm (figure 2). The microcosm was 

1

 All fuels were provided by NREL, Golden, CO. NREL provide approximately 15 L of LSD, additized 

LSD and CME-100 in 5-gal (18.9) polypropylene pails. Fuel was stored in the same room, under the same 

conditions as the microcosms throughout the study. 
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incubated in the dark at 25 ± 2 °C for one month. The robust microbial population in the 

Stage 2 microcosm was used to inoculate Test microcosms A, B, C & D. 

Figure 1. Stage 2 microcosm bottom-water. 200 mL pooled bottom-water 

from Stage 1, Round 2 microcosms have been added to 800 mL SBW. 

Figure 2. Stage 2 microcosm just after set-up: NALSD over SBW 

depicted in figure 1.
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Test Microcosms 

Five 3.78 L unused, wide-mouth glass jars were set up as Test microcosms. Each jar 

received 200 mL sterile SBW. Fuel was added to Test Microcosms A through D as 

follows: 

Microcosm Fuel 

A NALSD 

B ALSD 

C CME-100 

D CME-100 + microbicide 

Each microcosm received 2.0 L fuel. Microcosm D was treated with 220 μL Kathon®

FP1.5 to give 100 μL/L (220 ppm v/v) dose, as supplied (1.5 ppm active ingredient). 

A 2.0 L volume of CME-100 was filter sterilized using a 0.2 μm pore size cellulose 

acetate filter. The filter sterilized CME-100 was then dispensed into microcosm E. 

Except during monthly observations and sampling evolutions, all microcosms were 

stored in the dark. Incubation, observations and sampling were all completed in a climate 

controlled room with the temperature at 25 ± 2 °C. 

Sampling 

All samples were collected using 10 mL sterile serological pipets. Fluid transfers were 

made using a pipetter bulb. Samples for microbiological testing were transferred to 50 

mL, screw-cap, sterile, disposable, polyethylene centrifuge tubes. Samples for chemical 

testing were transferred to 250 mL wide-mouth, brown-glass bottles. Fuel samples were 

then packaged in accordance with DOT regulations and shipped to Clark Laboratories for 

analysis. Water samples for chemical analysis and all microbiological tests except ATP 

and O.D. were transferred to the appropriate lab at EMSL. 

Testing 

Gross Observations 

Gross observations were made on all Test microcosms at T0, T1-month, T2-months, and T3-

months. The fuel phase in each microcosm was rated for haze (ASTM D 4176-02, 

Standard Test Method for Free Water and Particulate Contamination in Distillate Fuels 

(Visual Inspection Procedures)) and color (ASTM D1500-04a Standard Test Method for 

ASTM Color of Petroleum Products (ASTM Color Scale)). 

® Kathon is a registered trademark of Rohm & Haas Company, Philadelphia, PA. 

 
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The fuel-water interface was rated for the presence of an invert emulsion layer, pellicle 

(membrane) or both. The following interface characteristics were reported: 

• Presence (Y or N) 

• Thickness (mm) 

• Stalactites (intrusions into the water phase; Y or N) 

• Stalagmites (intrusions into the fuel phase; Y or N) 

• Consistency (flocculent, membranous, dispersible, non-dispersible) 

• Adherence to glass jar surface 

The water-phase was rated for turbidity, color and the presence of sediment. Water 

turbidity was rate using the ASTM D4176 haze rating. Color was rated according to 

ASTM D1500 and sediment was rated as percentage of bottom coverage. 

A photographic record was made of each microcosm immediately after gross 

observations were recorded. Photographs were taken using a Nikon Coolpix 995 digital 

camera. 

Microbiology 

ATP 

Adenosine Triphosphate (ATP) was determined using a New Horizons Diagnostics 

(NHD, Columbia, MD) Model 4560 Bioluminometer. The Protocol is detailed in 

Appendix A. In summary, 50 μL bottom-water samples were concentrated onto a filter 

and rinsed twice with a mild surfactant solution. A strong surfactant was then used to 

lyse cells and extract ATP. The released ATP was then transferred onto a LuciferinLuciferase impregnated pad which was then placed into the Bioluminometer. Light 

emitted from the reaction of ATP with the Luciferin-Luciferase enzyme-substrate pair 

was recorded as Log10 relative light units2

 (RLU). 

For fuel samples, cells were concentrated by filtering 25 mL of fuel through a 0.45 μm 

pore-size filter, rinsing twice with a mild surfactant and extracting the ATP with a strong 

surfactant, as described above for bottom-water samples. 

Culturable Bacteria and Fungi 

Bottom-water culturable bacteria and fungal viable counts were performed by EMSL 

Analytical. Culturable bacteria were enumerated by the standard plate count method 

(Method 9125C Spread Plate Method, Standard Methods for the Examination of Water 

and Wastewater; APHA, Washington, DC). Aliquants (0.1 mL) of serial ten-fold 

dilutions were plated onto tryptic soy agar (TSA; Becton Dickenson, Baltimore, MD). 

Cultural fungi were enumerated similarly, except that 0.1 mL aliquants of serial dilutions 

2

 RLU: 1.0 RLU = 1 pg ATP. Typical ATP concentration/cell = 1 to 5 fg (10-15 g). 
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were plated onto malt dextrose agar (MDA; Becton Dickenson, Baltimore, MD). For 

both bacteria and fungi, inoculated plates were incubated inverted at 25 ± 0.5 °C. 

Bacterial colonies were counted after 24 to 76 hours incubation. Fungal colonies were 

counted after five days. 

Fuel-phase culturable bacteria and fungi were enumerated in accordance with ASTM 

D6974 Practice for Enumerating Viable Bacteria and Fungi in Liquid Fuels – Filtration 

and Culture Procedures. 

Oxygen Demand 

Oxygen demand (O.D.) was determined in accordance with BCA SOP 004 Modified 

Oxygen Demand for Monitoring biocontamination of Fuel System Bottoms-Water. This 

SOP is included in Appendix A. In summary, a 25 mL sample of bottom-water is 

transferred from a microcosm to a 50 mL disposable centrifuge tube and shaken 

vigorously for 30 sec. A T0 dissolve oxygen (DO) reading is taken, the centrifuge tube is 

sealed and allowed to stand for two-hours. After two-hours, a second DO reading is 

taken. The O.D. is the percent difference between the DO at T0 and the DO at T2h. 

Chemistry 

Bottom-water chemistry tests were performed by EMSL Analytical. 

Table 1 lists the tests and the methods used. 

Table 1. Water chemistry test methods used for the CME-100 biodeterioration study 

Parameter EMSL Method ASTM Method 

Alkalinity 310.1 D1067 

Hardness 130.2 D1126 

pH 150.1 D1293 

Total dissolved solids (TDS) 160.1 N/A 

Total organic carbon (TOC) 415.1 D4839 

Fuel Chemistry tests were performed by Clark Laboratories. The following Table 2 lists 

the tests and methods used. 

Table 2. Fuel chemistry test methods used for the CME-100 biodeterioration study 

Parameter ASTM Method

Water by Karl Fischer D6304 

Total acid number (TAN) D664 

Total base number (TBN) D4739 
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Fuel Corrosivity 

Fuel corrosivity testing was performed by EMSL Analytical in accordance with NACE 

Standard TM0172 modified as follows. 

The test specimens were 5.01 mm dia x 59 mm polished carbon steel cylinders. For 

corrosivity testing, 300 mL of fuel was dispensed into a 400 mL glass beaker with a 

magnetic stirring bar. The beaker was placed into a water bath which was then placed 

onto a hotplate magnetic stirrer. The fuel temperature was maintained at 38 °C and 

stirring was maintained at a speed sufficient to keep the fuel sample homogeneous, but 

not cause visible turbulence on the fuel surface. 

Once the fuel was at 38 °C, the test specimen was suspended into the beaker so that is 

was completely submerged and its lower end was ∼ 15 mm above the beaker’s bottom. 

After 30 minutes, 30 mL SBW was injected into the beaker. The exposure period 

continued for an additional 3.5 h, during which the beaker was covered to prevent fluid 

loss. 

After 4.0h total exposure, the specimen was removed form the fuel-water mixture, 

cleaned with acetone and inspected for evidence of corrosion. 

Results 

Challenge Population 

Stage 1 round 1 microcosms we set up on 30 November 2004. The original inoculum 

source was Water sample D from Ed’s microcosm started 9-17-043

 (received by BCA on 

23 November 2004). A robust community should yield ≥ 1,000 RLU (log10 RLU ≥ 3). 

The FQS sample had 104 ± 11 RLU (log10 RLU = 2.02). To compensate for the 

relatively low ATP concentration, 10 mL of sample was used to inoculate each of three 

Stage 1 round 1 microcosms. The BACTO Minimal Broth Davis without Dextrose that 

had been ordered, had not yet arrived at EMSL. Consequently, an alternative bottomwater preparation was formulated by dispersing 1 g of local garden soil into 1.0 L 

deionized water. 

As of 20 December, there was no gross evidence of growth in any of the three 

microcosms. EMSL resuspended colonies from a Sani-CheckTM AB test strip into tryptic 

soy broth (TSB) and colonies from a Sani-Check YM test strip onto (MDA). Within 48h, 

the TSB had become turbid and fungal colonies had grown on the MDA. Each of the 

3

 Microbially contaminated bottom-water was provided by Fuel Quality Services, Inc., Flowery Branch, 

GA. 

TM Sani-Check is a trademark of Biosan Laboratories, Warren, MI. AB and YM test strips are growth media 

impregnated filter pads. When they are dipped into an aqueous solution, the pads rehydrate. The AB test 

strips support bacterial growth and the YM test strips support fungal growth. 
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Stage 1 microcosms was inoculated with 10 mL of TSB culture and 10 mL of 

resuspended growth from an MDA plate. 

When observed on 10 January 2005, all three Stage 1 round 1 microcosms showed gross 

evidence of microbial activity (Table 3). 

Table 3. Stage 1, round 1 microcosm gross observations; 10 January 2005 

Parameter Microcosm 

A B C 

Fuel 

Haze Rating 1 1 1 

ASTM Color 3 3 3 

Interface

Visible? Y Y Y 

Thickness (mm) < 1 mm < 1mm < 1mm 

Stalactites? N N N 

Stalagmites? N N N 

Consistency Membranous 

pellicle 

Membranous 

pellicle 

Membranous 

pellicle 

Adheres to glass? Y Y Y 

Bottom-water 

Turbidity Haze 3 Haze 3 Haze 3 

Color 1 1 1 

Sediment N N N 

Figure 3. Stage 1, Round 1 microcosms 60 days after first inoculation and 18 days 

after reinoculation. 
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Bottom-water was drawn from each microcosm and tested for ATP concentration. 

Average Log10 RLU = 2.8 ± 0.07 (630 RLU). Based on the ATP results, a second round 

of Stage 1 microcosms was set up and inoculated. A 10 mL aliquant was drawn from 

each Stage 1 round 1 microcosm and pooled into a 50 mL disposable centrifuge tube. 

Each aliquant included membranous flocs of biomass. The pooled bottom-water samples 

were shaken vigorously for 30 sec then dispensed as 10 mL aliquots into the three Stage 1 

round 2 microcosms. 

On 31 January, the Stage 1 round 2 microcosms were observed and tested for ATP 

concentration. All three microcosms show gross symptoms of microbial activity (Table 

3). The average ATP concentration (log10 RLU) for the three microcosms was 3.2 ± 0.1. 

The ATP concentration met the robust growth criterion. Consequently 66.8 mL of 

bottom-water from each Stage 1 round 2 microcosm was pooled to create a 200 mL 

inoculum for the Stage 2 microcosm. The pooled inoculum was tested for ATP 

concentration, culturable bacterial count and cultural fungal count. The test results were: 

 

Parameter Result 

ATP (log10 RLU/50 μL) 3.5 

Log10 bacteria CFU/mL 4.60 

Log10 fungi CFU/mL 1.83 

The Stage 2 microcosm was retested on 01 March, just before bottom-water from this 

microcosm was used to challenge Test microcosms A through D. Duplicate ATP analysis 

yielded log10 3.3 ± 0.14. Figure 4 shows the substantial invert emulsion layer that had 

formed at the fuel-water interface of the Stage 2 microcosm. 

Figure 4. Stage 2 microcosm fuel-water interface 5-weeks post-challenge. 
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Test Microcosms 

Gross Observations 

Effect of Filtration on CME-100 

As noted under Materials and Methods CME-100 was filter sterilized for use in the 

sterile control (microcosm E). The filtration process affected CME-100’s appearance, 

imparting a slight green opalescent color to the product (figure 5). 

Figure 5. Color comparison; unfiltered (left) and filtered (right) CME-100. 

The filter sterilized CME-100 retained this green tint for the duration of the test. 

Effect of Exposure to Microbial Contamination 

Table 4 summarizes the gross observation data for Test microcosm A. Interestingly, 

between months 2 and 3, the fuel lost some of its color and the haze diminished. Also, 

the morphology of the fuel-water interface changed from a well defined, continuous 

membranous layer into a flocculent discontinuous zone. This change may have 

reflected the impact of microbial activity. Figure 6 illustrates the change in microcosm 

A’s appearance between months zero and three. 

Table 5 presents the gross observation data for Test microcosm B. Similarly to 

microcosm A, the ALSD microcosm’s gross appearance changed between months two 

and three (figure 7). The membranous layer at the fuel-water interface become flocculent 

and the fuel color decreased from ASTM 7 to ASTM 5. Moreover, the bottom-water 

color lightened from ASTM 4 to ASTM 1. 

Microcosm C (CME-100) gross observations are listed in Table 6 and illustrated in figure 

8. The primary change in this microcosm was the loss of haze between months two and 

three. 

Gross observation data for the Kathon FP1.5 treated microcosm (D) appear in Table 7. 

Figure 9 presents photographs of Microcosm D. At T3-months, the microbicide treated 

microcosm had a non-dimensional (< 1mm thick), adherent membrane layer that covered 
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approximately 98 percent of the fuel-water interface. There was no other gross 

indication of microbial activity in this microcosm. 

Figure 6. Microcosm A (NALSD) at T0 (left) and T3-months (right) 

Table 4. Microcosm A, NALSD, gross observations. 

0 1-month 2-months 3-months

Fuel

Haze Rating 1 1 4 2

ASTM Color 4 6 7 5

Interface

Visible? NYYY

Thickness (mm) N/A < 1 mm < 1 mm < 1mm

Stalactites? N N N N

Stalagmites? N Y (globules) N N

Consistency N/A Membranous 

pellicle

Membranous 

pellicle; adherent

Flocculent, 

partially 

dispersible, not 

continuous; not 

adherent

Adheres to glass? N/A N N Y

Bottom-water

Turbidity Haze 2 Haze 6 Haze 6 Haze 6

Color Water White 4 4 1

Sediment N N 20%; flocculent 30%; flocculent

Parameter Time
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Table 5. Microcosm B, ALSD, gross observations. 

0 1-month 2-months 3-months

Fuel

Haze Rating 1 1 2 2

ASTM Color 4 675

Interface

Visible? NYYY

Thickness (mm) N/A < 1 mm < 1 mm < 1mm

Stalactites? N N N N

Stalagmites? N Y (globules) N N

Consistency N/A Membranous 

pellicle

Membranous 

pellicle; adherent

Flocculent, 

partially 

dispersible, not 

continuous; 

adherent

Adheres to glass? N/AY Y Y

Bottom-water

Turbidity Haze 2 Haze 6 Haze 6 Haze 6

Color Water White 4 4 1

Sediment N N 10%; flocculent 20%; flocculent

Parameter Time

Figure 7. Microcosm B (ALSD) left to right: T0, T1-month, T2-months & T3 months 
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Table 6. Microcosm C, CME-100, gross observations. 

0 1-month 2-months 3-months

Fuel

Haze Rating 2 6 6 1

ASTM Color 2 2 2 (green tint) 2

Interface

Visible? NYYY

Thickness (mm) N/A < 1 mm < 1 mm < 1mm

Stalactites? N N N N

Stalagmites? NNNN

Consistency N/A Flat, uniform 

pellicle

Membranous 

pellicle

Membrancous 

pellicle

Adheres to glass? N/A Y Y Y

Bottom-water

Turbidity Haze 2 Clear Clear Haze 2

Color Water White Water-white Water-white 1

Sediment NNNN

Parameter Time

Figure 8. Microcosm C (CME-100) left to right: T0, T1-month, T2-months & T3 months 
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Table 7. Microcosm D, Kathon FP1.5 treated CME-100, gross observations. 

0 1-month 2-months 3-months

Fuel

Haze Rating 6 6 6 2

ASTM Color 2 2 2 2

Interface

Visible? NY/N Y/N Y

Thickness (mm) N/A 0 mm 0 mm < 1 mm

Stalactites? N N N N

Stalagmites? NNNN

Consistency N/A Flat, nondimensional; may 

be optical 

illusion

Some particles at 

fuel-water 

interface

Flat, nondimensional, 

adherent

Adheres to glass? N/A N N Y

Bottom-water

Turbidity Haze 6 Clear Clear Haze 2

Color Water-white Water-white Water-white Water-white

Sediment N N N N

Parameter Time

Figure 9. Microcosm D (Microbicide treated CME-100) left to right: 

T0, T1-month, T2-months & T3-months 

At T3-months microcosm E’s appearance was very similar to microcosm D’s. The haze that 

had characterized the CME-100 in microcosm E through the first two months had cleared 

by the end of the testing period (Table 8 and figure 10) although the filter-sterilized 
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CME-100 still had a darker green tint than unfiltered CME-100. Microcosm E had 

developed a non-dimensional, non-adherent fuel-water interface membrane between 

months two and three. Also, the bottom-water had become slightly translucent by the end 

of the study. 

Table 8. Microcosm E, filter-sterilized CME-100, gross observations. 

0 1-month 2-months 3-months

Fuel

Haze Rating 6 6 6 1

ASTM Color 2 (green) 2 (green) 2 (green) 1

Interface

Visible? NNNY

Thickness (mm) N/A 0 mm 0 mm < 1 mm

Stalactites? N N/A N/A N

Stalagmites? NN/A N/A N

Consistency N/A N/A Trace of oily 

film, not a 

complete layer

Flat, nondimensional, 

adherent

Adheres to glass? N/A N/A N/A Y

Bottom-water

Turbidity Haze 6 Clear Clear Haze 2

Color Water-white Water-white Water-white Water-white

Sediment N N N N

Parameter Time

Figure 10. Microcosm E (filter-sterilized CME-100) left to right: 

T0, T1-month, T2-months & T3-months 
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ge

Fuel Chemistry 

Fuel chemistry analyses were performed by Clark Laboratories. Data reports from Clark 

are provided in Appendix B. 

Initially, three chemical parameters were to be monitored during the test period: KarlFischer water, total acid number (TAN) and total base number (TBN). A T0-month CME100 sample was tested for water, TAN and TBN, but the CME-100 caused instrument 

problems when TBN was analyzed by ASTM Method D4739. Consequently, the TBN of 

microcosm C, D and E fuels was not determined at T3-months. 

Entrained Water 

Table 9 presents the Karl-Fischer water data. The percent change of water in CME-100 

was computed as: 

(1) [(W Microcosm X@ T3-mo - W Microcosm E@ T0-mo) ÷ W Microcosm E@ T0-mo] x 100 

Where W is Karl Fischer water content and X is microcosm C, D or E, respectively. The 

water content of microcosm C, D and E increased by > 330% during the course of the 

study. 

Table 9. Water content of microcosm fuels (mg water/kg fuel) 

Test time (months) %

0 3 chan

A n.d. < 50 n.d

B n.d. 86 n.d.

C n.d. 2,258 331%

D n.d. 2,256 331%

E 2,311 524 341%

Microcosm

Microbial activity did not seem to affect water partitioning. The microcosm B additive 

package may have been responsible for some water dispersion into microcosm B. The 

below detection limit (BDL) result for microcosm A and the very similar amounts of 

water in all three CME-100 microcosms suggest strongly that CME-100 has a 

substantially greater tendency than LSD to retain water. Had water dispersion been 

biologically mediated, the water content of CME-100 from microcosms D and E would 

have been significantly lower than that of CME-100 from microcosm C. 
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Total Acid Number 

The CME-100 TAN is approximately 10x that of the LSD. Moreover, the T3-month TAN 

for untreated, microbially challenged CME-100 (microcosm C) was slightly higher than it 

was for either microbicide treated or sterile control CME-100 (Table 10). 

Without replicate data, it is unclear whether the TAN differences amongst microcosms C, 

D and E was significantly different, or within the normal range of test variability. The 

TAN of microcosm C was at T3-months was 12 percent greater than the average TAN for 

CME-100 from microcosms D and E at T3-months, and 31% greater than the microcosm E 

T0-months TAN. 

Table 10. Total acid number of microcosm fuels (mg KOH/g fuel) 

Test time (months)

0 3

A n.d. 0.05 

B n.d. 0.06 

C n.d. 0.51 

D n.d. 0.45 

E 0.46 0.39

Microcosm

Total Base Number 

As noted above, CME-100 TBN was not testable. The T0-months microcosm E value (1.0) 

was substantially greater than the T3-months values for either LSD from microcosms A and 

B (both < 0.1 mg KOH/g fuel). Table 11 presents the TBN data. 

Table 11. Total base number of microcosm fuels (mg KOH/g fuel) 

Test time (months)

0 3

A n.d. <1.0

B n.d. <1.0

C n.d. n.d.

D n.d. n.d.

E n.d. 1.0

Microcosm

Fuel Corrosivity 

Microbial activity can increase the corrosivity of both fuel and fuel associated water. The 

primary mechanisms for this effect are identified below under pH. Biosurfactants are 

detergent molecules produced by microbes. Biosurfactants will increase water dispersion 

into fuel. The corrosive properties of the dispersed water will contribute to the fuel’s 
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corrosivity. Moreover, bioconversion of non-polar fuel molecules into charged species 

will affect the fuel’s corrosivity. 

The corrosivity data are presented in Table 12. Table 10a lists the NACE TM0172 

corrosivity ratings for the microcosm fuel samples. Table 10b captured the extent of 

coupon corrosion at the end of the exposure period. There was no evidence of corrosion 

on either T0 or T3-months coupons. Consequently, fuel corrosivity testing did not 

differentiate between conventional LSD and CME-100. 

Table 12. Fuel corrosivity 

a. NACE TM0172 corrosivity rating 

Test time (months) Microcosm 

0 1 2 3 

A n.d. n.d. n.d. A 

B n.d. n.d. n.d. A 

C n.d. n.d. n.d. A 

D n.d. n.d. n.d. A 

E B++ n.d. n.d. A 

b. Percent of test coupon surface corroded 

Test time (months) Microcosm 

0 1 2 3 

A n.d. n.d. n.d. < 0.1 

B n.d. n.d. n.d. < 0.1 

C n.d. n.d. n.d. < 0.1 

D n.d. n.d. n.d. < 0.1 

E < 0.1 n.d. n.d. < 0.1 

Bottom-water Chemistry 

Bottom-water chemistry was tested to evaluate the impact of fuel type and microbial 

activity on the primary characteristics of the bottom-waters in the different microcosms. 

Bottom-water chemistry testing was completed by EMSL. Their data reports are 

provided in Appendix B. 
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Any pH drop reflects the net production of acidic molecules. Many microbial metabolites 

are weak organic acids. These acids may, in turn react with chloride, sulfate and nitrate 

salts in the water-phase, producing weak organic bases and strong inorganic acids 

(hydrochloric, sulfuric and nitric acids, respectively). These biogenic strong inorganic 

acids play a major role in microbially influenced corrosion (MIC). The pH data from 

microcosm bottom-water samples appears in Table 13. 

The microcosm E bottom-water pH dropped substantially during the test period. The 

drop was both absolute and relative to all other microcosms. Were it not for the low 

alkalinity test results (Table 12), it might be assumed that the microcosm E pH result 

reflected analytical error. There is no apparent cause for the pH in the filter-sterilized 

control to have fallen by > 3 units during the test period. 

At T3-months, bottom-water from the two challenged CME-100 microcosms were 

significantly different (F-ratio[1,2] = 63.8; F-critical 0.01 [1,2] = 18.5). The substantial pH 

drop in the filter-sterilized control, combined with the significant differences between the 

LSD and CME-100 microcosms mean that the pH data did not support the null 

hypothesis.

Table 13. Microcosm bottom-water pH 

Test time 

Microcosm (months) 

0 3 

A n.d. 6.79 

B n.d. 6.86 

C n.d. 6.21 

D n.d. 6.33 

E 7.13 4.70 

Alkalinity 

Alkalinity was tested to determine whether microbial production of organic acids reduced 

the alkalinity of microcosm bottom-waters. Most often pH changes will occur only after 

the alkalinity has decreased substantially. Table 14 shows the alkalinity data for the test 

microcosms. As discussed in the preceding paragraph, Microcosm E bottom-water was 

substantially different from the other four microcosms. Instead of having reserve 

alkalinity, microcosm E bottom-water was strongly acidic (acidity = 3,700 mg CaCO3/ 

L). The alkalinity of microcosm B (ALSD) bottom-water was also substantially different 

from all of the other microcosms. The most likely explanation for this was additive 

partioning into the water-phase. This type of partitioning is common in fuel storage 

pH 
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systems. Generally, additive depletion from the fuel-phase is immeasurably small (less 

than the variability due to experimental error of the test method), but may reach 

saturation in the water-phase. Typically, this additive partitioning phenomenon is 

reflected in alkalinity, TDS and TOC results. It may occur abiotically or be microbially 

mediated. In the latter case, biosurfactants and other metabolites may alter additive 

solubility characteristics, partition-coefficients or both. Since microcosm C was the only 

additized fuel used in this study, it’s not possible to differentiate between biotic and 

abiotic processes that may have contributed to this partitioning. Since microcosm C and 

D bottom-water alkalinities were in the same range as microcosm A, the alkalinity data 

supported the null hypothesis. It is possible that the energy imparted to the CME-100 

during the filter-sterilization process contributed to the unexpected pH and alkalinity 

decreases in this microcosm. 

Table 14. Microcosm bottom-water alkalinity (mg CaCO3/L water) 

Test time 

Microcosm (months) 

0 3 

A n.d. 1,800 

B n.d. 3,500 

C n.d. 1,500 

D n.d. 1,000 

E 1,800 <20 

Hardness 

As water hardness increases, microbial populations tend to become more robust. The 

dissolved salts are essential nutrients for microbes. In static microcosms such as the ones 

set up for this test, the only source of CaCO3 and the other salts captured in the hardness 

titration would be those previously suspended or dissolved in the fuel. Consequently, 

there should be negligible changes in hardness during the course of the test. 

The hardness data are presented in Table 15. Bottom-water hardness under LSD was 

significantly less than the hardness under CME-100. One way analysis of variance 

(ANOVA) gave an observed F-ratio [1,3] = 30.7 (F-critical 0.01 [1,3] = 7.7). The average 

hardness of CME-100 bottom-water (108 mg CaCO3/L) was approximately three-times 

(2.8x) as high as the average hardness of bottom-water under LSD (40 mg CaCO3/L). 

Since the T0 hardness determination was unlikely to have been affected by partitioning 

between the fuel and water, the most likely explanation for the difference is that an 

interaction between the LSD and bottom-water in microcosms A and B caused the 

hardness to decrease in those systems. Alternatively, since there were no replicate 
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microcosms, the differences may have reflected microcosm to microcosm variability. 

The difference between LSD and CME-100 microcosm bottom-water hardness does 

not support the null hypothesis.

Table 15. Microcosm bottom-water hardness (mg CaCO3/L water) 

Test time 

Microcosm (months) 

0 3 

A n.d. 32.3 

B n.d. 48.6 

C n.d. 130.0 

D n.d. 93.6 

E 2.0 100.0 

Total Dissolved Solids 

The TDS data reflect dissolve organic and inorganic solids in the water-phase. Microbial 

activity will cause TDS to increase by a combination of one or more mechanisms. New 

biomass, resulting from cell growth and proliferation increases both TOC and TDS. 

Waste metabolites excreted by active microorganisms also contribute to both TOC and 

TDS. Biosurfactants and organic acids produced by the contaminant population will 

cause fuel constituents to partition into the water-phase. This process also contributes to 

both TDS and TOC increases. Consequently, microcosms A, B and C would be expected 

to have greater TDS and TOC concentrations than microcosms D and E. Differences 

between A and B would reflect the impact of the additive used in the ALSD. Differences 

between A and C would reflect the relative tendency for TDS to develop in bottomwaters associated with NALSD and CME-100, respectively. Data from microcosm D 

would reflect the impact of microbicide treatment on the net TDS change. 

Table 16 presents the TDS data. The dissolved solids contribution of the Davis Minimal 

Broth medium used to formulate synthetic bottom-water was 10g/L. During the threemonth test period, bottom-water TDS increased by an average of 31% relative to TDS at 

T0. It is likely that this increase was not biogenic, since the highest T3-months TDS value 

was in the filter sterilized CME-100 microcosm. As noted before, without replication, it 

is impossible to differentiate experimental variation from variation due to fuel type. 

There is no apparent difference in bottom-water TDS amongst the microcosms. 

Consequently, the TDS data support the null hypothesis. 
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Table 16. Microcosm bottom-water total dissolved solids (g dissolved solids/L water) 

Test time 

Microcosm (months) 

0 3 

A n.d. 12 

B n.d. 13 

C n.d. 13 

D n.d. 12 

E 9.8 14 

Total Organic Carbon 

As discussed in the preceding subsection, TOC changes are affected by many of the same 

processes that drive TDS changes. Additionally, TOC analysis captures both dissolved 

and particulate organic carbon. As noted before, changes in bottom-water TOC 

concentrations are generally proportional to microbial activity. 

However, non-microbially mediated partitioning of fuel constituents into the water and 

oxidation byproduct particles settling out of the fuel phase will also contribute to TOC. 

Data from the untreated CME-100, microbicide treated and filter sterilized microcosms 

will differentiate abiotic TOC generation from biologically mediated TOC generation. 

The results (Table 17) from these three microcosms were sufficiently close to indicate 

that the TOC increases were not microbially mediated. 

The data show that TOC increased an average of 13-fold during the three-month storage 

period. Test results are from microcosms A and B are counter-intuitive. Additive 

partitioning would have caused microcosm B bottom-water TOC to be substantially 

greater than the value for microcosm A. The pH, alkalinity and hardness data suggest that 

additive did partition from the ALSD. However, the microcosm B TOC concentration 

was only 25% that of microcosm A. These results suggest that significant additive 

portioning did not occur in microcosm B. The TOC concentrations in microcosm C, D 

and E were indistinguishable form the concentration in microcosm A; supporting the 

null hypothesis. 
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Table 17. Microcosm bottom-water total organic carbon (mg TOC/L water) 

Test time 

Microcosm (months) 

0 3 

A n.d. 1,400 

B n.d. 350 

C n.d. 1,300 

D n.d. 1,400 

E 97.8 1,300 

Bottom-water Microbiology 

No single parameter captures an adequate profile of microbial contamination in fuel 

systems. The basis for this statement will be addressed in the Discussion section, below. 

To capture the critical aspects of both biomass accumulation and microbial activity, three 

types of data were collected. Culture data were used to estimate changes in culturable 

bacteria and fungi in both fuel and water-phase samples. Since many microbes are not 

culturable, ATP data provide an alternative measure of total biomass. Oxygen demand 

(O.D.) is proportional to metabolic activity. High numbers of dormant or moribund 

microbes consume less oxygen than do metabolically active communities. Consequently, 

O.D. is an excellent measure of a population’s biodeteriogenic activity. 

Adenosine Triphosphate 

Bottom-water ATP data appear in Table 18. Microcosm A and E bottom-water ATP was 

determined monthly (figure 11). For the other three microcosms, bottom-water ATP was 

tested only at T3-months. 

Table 18. Microcosm bottom-water ATP (log RLU/50 μL water) 

Test time (months) Microcosm 

0 1 2 3 

A 1.4 4.1 3.6 4.7 

B n.d. n.d. n.d. 4.1 

C n.d. n.d. n.d. 1.8 

D n.d. n.d. n.d. 2.0 

E 0.2 - 0.4 0.9 
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-

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0123

Months

Log RLU

Microcosm A

Microcosm E

Figure 11. ATP concentration as a function of exposure time in NALSD (microcosm 

A) and filter sterilized CME-100 (microcosm E)

The ATP data show that during the first month after inoculation, the ATP concentration 

in the challenged NALSD control bottom-water increased by 2.7 orders of magnitude, 

and remained high through out the remaining two months of the study. In contrast, the 

ATP concentration in the filter-sterilized CME-100 control microcosm, as Log RLU 

remained at <1.0 throughout the test. The bottom-water ATP concentrations in the 

challenged, untreated and microbicide treated CME-100 microcosms were 

indistinguishable at T3 –months. Both the NALSD and ALSD bottom-waters had high ATP 

concentrations at T3-months. These data suggested that CME-100 did not support bottomwater microbial proliferation as well as LSD. The F-ratio [1,2] = 46.7 (F-critical 0.01 [1,2]

= 18.5) where the treatments (fuel type) were LSD or CME-100. The bottom-water 

ATP data do not support the null hypothesis. 

Fuel-phase ATP data are shown in Table 19. Microcosms A, B and C all have 

considerable ATP concentrations. The difference between fuel ATP in microcosms A 

(NALSD) and C (CME-100) is significant. However, the difference between fuel ATP in 

microcosms B (ALSD) and C (CME-100) is not significant. Nor is the difference 

between microcosms D (microbicide treated CME-100) and E (filter-sterilized CME100). The fuel-phase ATP test results do not support nor refute the null hypothesis 

although differences between CME-100 and LSD are affected by LSD additive use. 

The results suggest that ALSD and CME-100 may be less likely than NALSD to support 

microbial transport. Interestingly, fuel-phase ATP concentration does not appear to 

covary with the fuel’s water content. 
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Table 19. Microcosm fuel ATP (log RLU/25 mL fuel) 

ATP

Log RLU

A 3.1

B 1.7

C 2.0

D 0.9

E -

Microcosm

Oxygen Demand 

Table 20 presents the D.O. (mg O2/L) and O.D. test results. The T0h and T2h data for 

microcosm bottom-water O.D. at one and three-months are shown in 20a and 20b 

respectively. Summary O.D. data are shown in 20c. The D.O. of the inoculated NALSD 

microcosm bottom-water was 77% at the end of the first month and 91% at the end of the 

test. These results are consistent with the ATP test results, showing a substantial and 

metabolically active biomass. Similarly, the filter-sterilized CME-100 bottom-water had 

a negligible D.O. throughout the study; consistent with the low ATP concentration data. 

The O.D. data for microcosms B, C and D also paralleled the bottom-water ATP data 

from those systems. The O.D. in microcosm B was 16%. This was substantially lower 

than that in the NALSD microcosm, but still significant. In contrast, O.D. in challenged, 

untreated CME-100, microbicide treated CME-100 and filter-sterilized CME-100 were 

all negligible. The O.D. data do not support the null hypothesis. CME-100 appears 

to be substantially more bioresistant than LSD as reflected in bottom-water O.D. 

Culturable Bacteria 

Bacterial enumeration data are shown in Table 214. At T0-months log colony forming units 

(CFU) of bacteria/mL was below the method’s lower detection limits (Log CFU 

bacteria/mL < 3.0). 

By the end of the storage period, the bottom water culturable bacteria population had 

increased to > 108.5 CFU bacteria/mL. Although the bottom-water cultural bacteria 

populations were also quite dense in the three CME-100 microcosm bottom-waters, there 

was a 3.5 to 3.7 log suppression of growth relative to the LSD microcosms. Neither filter 

sterilization nor microbicide treatment affected the T3-months CME-100 bottom-water 

4

 Culturable bacteria and fungi testing were performed at EMSL. Data reports are provided in Appendix B. 

 
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Table 20. Microcosm bottom-water oxygen demand (% ∆ mgO2/L/2hours)

a. T1-month

T 0 T2h ∆D.O. % Change

A 1.17 5.01 3.84 77%

B n.d n.d n.d n.d

C n.d n.d n.d n.d

D n.d n.d n.d n.d

E 5.09 5.54 0.45 8%

Microcosm D.O. (mg O2/L) @ T0

b. T3-months 

T 0 T2h ∆D.O. % Change

A 0.54 5.72 5.18 91%

B 4.85 5.76 0.91 16%

C 5.76 5.98 0.22 4%

D 5.94 6.00 0.06 1%

E 6.69 6.67 (0.02) 0%

Microcosm D.O. (mg O2/L) @ T3

c. Summary 

012 3

A n.d. 77% n.d. 91%

B n.d. n.d. n.d. 16%

C n.d. n.d. n.d. 4%

D n.d. n.d. n.d. 1%

E n.d. 8% n.d. 0%

Microcosm Test time (months)

culturable bacteria population density. The high population density in microcosm E may 

have reflected the proliferation of bacteria that had been introduced during sampling. 

However the most likely explanation is that it is a reflection of the inadequacy of vacuum 

filtration for filter sterilization. The bottom-water cultural bacteria data did not 

support the null hypothesis. 
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Table 21. Microcosm bottom-water culturable bacteria counts (Log CFU/mL)

Test time (months) Microcosm 

0 1 2 3 

A n.d. n.d. n.d. > 8.5 

B n.d. n.d. n.d. >8.5 

C n.d. n.d. n.d. 4.8 

D n.d. n.d. n.d. 4.9 

E < 3.0 n.d. n.d. 5.0 

Fuel-phase bacterial culturability test results are shown in Table 22. At T0-months, log10

CFU bacteria/L < 1.0. By the end of the storage period, fuel phase culturable counts in 

microcosms A and B were 3.4 and 3.1 log10 CFU bacteria/L respectively. Without To

data from NALSD and ALSD, it is impossible to determine whether the bacterial 

population densities had remained stable or had increased in the LSD fuels. The positive 

microcosm E test results support the hypothesis posed in the previous paragraph. 

Vacuum filtration was probably not an adequate process for sterilizing the CME-100. 

However the negligible ATP concentrations detected in microcosm E bottom-water 

(Table 18) and fuel (Table 19), coupled with the zero O.D. (Table 20) indicate that 

culturable bacteria recovered from microcosm E were not metabolically active in the 

microcosm. 

The fuel-phase cultural bacteria data did not support the null hypothesis. 

Table 22. Microcosm fuel-phase culturable bacteria counts (log10 CFU/L) 

Test time (months) Microcosm 

0 1 2 3 

A n.d. n.d. n.d. 3.4 

B n.d. n.d. n.d. 3.1 

C n.d. n.d. n.d. - 

D n.d. n.d. n.d. - 

E < 1.0 n.d. n.d. 2.0 

Tables 23 and 24 present bottom-water and fuel-phase fungal data. At T0-months the filtersterilized CME-100 microcosms did not have detectible fungi in either the fuel or 
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bottom-water. Bottom-water culturable fungi data paralleled the bacterial results. The 

NALSD and ALSD microcosm bottom-waters had 3.0 and 5.6 log10 CFU fungi/mL 

respectively. Colony counts from microcosms C and D were below detection limits, but 

E had 2.0 Log CFU fungi/mL. 

Significant numbers of fungi were recovered from both LSD microcosms, but not from 

any of the CME-100 microcosms. Consequently, the culturable fungi data from both 

fuel and bottom-water samples did not support the null hypothesis. 

Table 23. Microcosm bottom-water culturable fungi counts (log10 CFU/mL) 

Test time (months) Microcosm 

0 1 2 3 

A n.d. n.d. n.d. 3.4 

B n.d. n.d. n.d. 3.1 

C n.d. n.d. n.d. <0 

D n.d. n.d. n.d. <0 

E < 3.0 n.d. n.d. 2.0 

Table 24. Microcosm fuel-phase culturable fungi counts (log10 CFU/L)

Test time (months) Microcosm 

0 1 2 3 

A n.d. n.d. n.d. 3.0 

B n.d. n.d. n.d. 1.7 

C n.d. n.d. n.d. <0 

D n.d. n.d. n.d. <0 

E < 1.0 n.d. n.d. <0 

Discussion 

Biodiesel biodeterioration has been reported routinely in trade meetings and non-peer 

reviewed literature, but has not been described in the peer reviewed literature. Typical 

anecdotal reports suggest that biodiesel fuel systems experience filter plugging more 

frequently than similar systems containing conventional diesel fuel. Increased 

component corrosion has been noted as well, although this phenomenon has been 

reported less frequently. 
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Typically, biodiesel fuels are marketed as blends comprised of 10 to 20 percent biodiesel 

stock in conventional petroleum distillate. Biodiesel stocks are methyl esters of 

vegetable oils or animal fats. Currently, rapeseed (Brassica napus) and soy (Glycine 

max) are the two predominant biodiesel source crops. However, biodiesel is being 

produced from an increasing number of corps. 

Uncut biodiesel stocks (B-100) must meet specifications established by the International 

Standards Organization (ISO) or ASTM International (ISO 14214 and ASTM D6751, 

respectively). Neither standard addresses B-100 or biodiesel blend biodegradability. 

Biodiesel biodeterioration susceptibility is a function of numerous factors. ASTM 

Manual 47 and ASTM D-6469 discuss the various factors contributing to fuel and fuel 

system biodeterioration. For the purposes of this report, it’s sufficient to note that 

microbial contamination, product chemistry, water availability and temperature are the 

four dominant factors affecting fuel biodeterioration. 

The present study focused on CME-100 biodegradability during storage over water at 

moderate temperature (25°C). The data generated during this study cannot be 

extrapolated to assess the likelihood of CME blend (CME-20, etc.) biodegradability or 

CME-100 biodegradability at warmer temperatures (e.g. > 30°C). Although replicate 

tests were not performed, normal variability for each of the methods used is known, and 

assumed to apply to the data reported herein. Consequently, only differences greater than 

five-times the expected range of experimental error were considered significant. 

The experimental matrix was designed to test: 

Null Hypothesis: CME-100 biodegradation risk is not significantly 

different from conventional LSD biodeterioration risk when products are 

stored under typical above ground storage tank conditions for up to three 

months. 

The hypothesis was tested on the basis of microcosm gross appearance, fuel corrosivity, 

fuel chemistry, fuel microbiology, bottom-water chemistry and bottom-water 

microbiology. Data from microcosms A (microbially challenged NALSD) and C 

(microbially challenged CME-100) were used to test the null hypothesis. Comparison of 

data from microcosms A and B reflected differences between NALSD and ALSD 

biodeterioration risk. Comparison of data from microcosm C and D permitted 

assessment of microbicide treatment effectiveness against biodeterioration. 

BCA has a standard guideline for scoring biodeterioration risk based on classes of 

parameters (gross observations, fuel chemistry, etc.). The decision tree for gross 

observations is provided in Appendix A. Although there were specific differences 

amongst the microcosms (see Tables 5 through 8 and figures 6 through 10), there was not 

significant difference in T3-months risk scores for the five test microcosms: 
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Microcosm Risk Score 

A 19 

B 20 

C 20 

D 17 

E 16 

The presence of a defined third layer between the fuel and water phases accounted for 8 

points of the 17 and 16 point scores for microcosms D and E, respectively. The 

microbiological data suggest that the apparent invert emulsion layer in these two 

microcosms was not produced microbially. Instead, they may have resulted from an 

abiotic interaction between CME-100 and water. Gross observation scores for A, B and 

C were indistinguishable. Consequently, the gross observation data support the null 

hypothesis. 

Without chemical analysis, it’s impossible to determine the extent to which the 

microcosm A though C invert-emulsion layers were biogenic. However, microcosms D 

and E appeared distinctly different form the others. This suggested that microbicide 

treatment did inhibit gross appearance changes to some degree. 

The only fuel chemistry parameter that this study shares with BCA’s biodeterioration risk 

survey parameter matrix is Karl-Fisher water. The BCA risk score for this parameter are: 

% water 

(

v

/v) 

Risk Score 

< 0.01 0 

0.01 – 0.1 1 

0.1 – 0.2 3 

> 0.2 5 

All of the fuels, including the microcosm E CME-100, had > 0.2% water at T3-months. 

Consequently all would be scored as fuels at high risk for biodeterioration. However, all 

CME-100 fuels (microcosms C, D and E) had water values three orders of magnitude 

greater than those detected in either the NALSD or ALSD. Thus, fuel-phase water 

content did not support the null hypothesis. 

All TAN results were below the ASTM D6751 specification upper control limit (0.8 mg 

KOH/g), however, TAN values for CME-100 samples from microcosms C through E 

were all an order of magnitude greater than either the NALSD or ALSD TAN values. 

Consequently, TAN data did not support the null hypothesis. 
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Page 36 of 41 

None of the microcosm fuels were corrosive. Consequently, the corrosivity data 

supported the null hypothesis. 

Table 25 summarizes BCA’s risk scores for the bottom-water microbiological parameters 

tested in this study. 

Table 25. Biodeterioration risk decision tree; bottom-water microbiology 

Parameter Criterion Risk Score 

Oxygen Demand < 10 % 1 

(O..D.) 10 to 50% 2 

 > 50 % 5 

ATP (Log RLU) < 2.5 1 

2.5 to 3.5 2 

3.5 to 4.0 3 

 > 4.0 5 

Bacteria (log 

CFU) <2 1 

2 to 4 2 

 > 4 5 

Fungi (log CFU) < 2 1 

2 to 3 2 

 > 3 5 

At T3-moths, the O.D. risk scores for both conventional diesel fuel microcosms were high 

(5). In contrast O.D. risk for all CME-100 microcosms was low (1). The O.D. results 

did not supporte the null hypothesis. Similarly, risk scores for CME-100 microcosm 

bottom-water ATP concentrations were all 1 and for the conventional diesel microcosms 

were both 5. Thus ATP data did not support the null hypothesis. The culturable 

bacteria and fungi data also did not support the null hypothesis. Bacterial and fungal 

recoveries from the three CME-100 microcosms were consistently lower than recoveries 

from the LSD microcosms. 

Table 26 summarizes the null hypothesis assessment for each parameter measured. 

Interestingly, most of the chemical data suggest that CME-100 is more susceptible than 

LSD to biodeterioration. However, all of the microbiologic data suggested that LSD was 

more susceptible than the CME-100. 

The use of replicate microcosms facilitates differentiation between normal system 

variability and variation attributable to the treatment (initial fuel chemistry). The data 

spread between LSD microcosm and CME-100 microcosm results were substantial (≥
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10x) for the parameters that for which data did not support the null hypothesis. This 

suggests that the observed differences between LSD and CME-100 were real. However, 

the results from a single microcosm may not be representative of the typical dynamics 

within a commercial operation. Triplicate microcosms address the need to minimize 

testing costs balanced against the risk of making incorrect interpretations on the basis of a 

single microcosm. 

Table 26. Summary of null hypothesis testing; relative LSD and CME-100 relative 

biodeterioration risk. 

Parameter Results Support 

Null Hypothesis (Y or N) 

More Susceptible Product 

(CME-100 or LSD) 

Gross observations Y - 

Fuel Chemistry 

Entrained water N CME-100 

Total Acid Number N CME-100 

Corrosivity N CME-100 

Bottom-water Chemistry 

pH N LSD 

Alkalinity/Acidity N LSD 

Hardness N LSD 

TDS Y - 

TOC Y - 

Bottom-water Microbiology 

ATP N LSD 

O.D. N LSD 

CFU bacteria/mL N LSD 

CFU fungi/mL N LSD 

Fuel Microbiology 

ATP Equivocal Result - 

CFU bacteria/L N LSD 

CFU fungi/L N LSD 

In this study, filtration imparted a green tint to the CME-100. Moreover, the filter 

sterilized CME-100 microcosm bottom-water pH plummeted during the test period. 

Initial total alkalinity of 1,800 mg CaCO3/L fell to 3,700 mg CaCO3/L acidity between T0

and T3-months. No change approaching this magnitude occurred in any of the other 

microcosms. Without replicate microcosms, it was impossible to determine whether this 

phenomenon was somehow related to the filtration process, or was just an aberration in 

this single microcosm. 

The original test design included simulated distillation and carbon number distribution. 

These two parameters reflect chemical changes in the fuel more precisely than the TAN, 

TBN and water data that were collected. They may also have provided some clues 
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regarding the major changes in microcosm E. Complete sets of tests at T1-month and T2-

months would also have provided rate information that would have facilitated more 

thorough interpretation of the test results. 

Rate data are important for designing cost-effective condition monitoring programs. 

Testing frequency should be based on the anticipated rate at which system change is 

expected to occur. The test results demonstrated that significant changes occur in CME100 storage systems during a period less than the three-month test period used for this 

project. 

Typically, O.D. and ATP concentration data are more sensitive than culture tests as 

indicators of microbial activity. Bacteria and fungi unable to elaborate into colonies on 

the growth media used may be active within the environment from which the sample was 

collected. Less frequently, microbes that are moribund or dormant in the sampled 

environment recover and flourish on the nutrient media. This appears to be the case in 

this study. Despite low ATP concentrations and O.D., microcosm C, D and E bottomwaters all yielded substantial numbers of culturable bacteria and fungi. Without replicate 

microcosms, it’s impossible to determine if this phenomenon is representative of CME100 stored over incidental water. Notwithstanding the weak correlation between nonconventional microbiological data and culture tests results, the data show that CME-100 

is no more likely than LSD to support microbial growth, and seems substantially less 

likely to support metabolic activity – the engine of biodeterioration. 

The testing reported herein considered only CME-100. Research that I’ve performed in 

the past has demonstrated that chemistries that are inhibitory at high concentrations may 

not be at lower concentrations. Testing performed with a variety of one to four –carbon 

alcohols, for example, demonstrated that methanol, ethanol, propanol (normal and iso) 

and butanol (normal, secondary and tertiary) all inhibited microbial growth at 

concentrations down to ∼ 17%. The effects between 10% and 17% varied with the 

alcohol tested, but at 10% all stimulated growth. This dynamic of inhibiting growth at 

high concentrations but stimulating growth at lower concentrations is well documented. 

Hormonisis is the term used for this dose-dependant effect. In South America, where 

gasoline is blended with 20% ethanol, microbial contamination problems are nonexistent. In the U.S. and U.K., where ethanol is blended into gasoline at 10%, 

biodeterioration problems occur routinely. Consequently, it is reasonable to speculate 

that despite the apparent bioresistance of CME-100, CME-5, CME-10 and CME-20 may 

be substantially less bioresistant. 

Conclusions 

The results of this study were somewhat equivocal, but generally did not support the null 

hypothesis. Sixteen parameters were used to compare CME-100 biodegradability against 

both additized and non-additized LSD. Data for six of the 16 parameters supported the 
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null hypothesis. Although CME-100 was more hazy than LSD at T1-month and T-2 months, 

the CME-100 could not be differentiated from the ALSD or NALSD on the basis of gross 

appearance, corrosivity; bottom-water alkalinity, TDS, TOC, or O.D.; or fuel-phase ATP 

concentration at T3-months. Karl Fischer water, TAN, bottom-water pH, and bottom-water 

hardness data indicated that CME-100 was significantly more susceptible than LSD to 

biodeterioration. Fuel and bottom-water ATP and culturable bacteria and fungi data 

indicated that CME-100 was less likely than LSD to support culturable microbes. 

However, the ∼ 5 log10 CFU bacteria/mL recoveries from CME-100 bottom-water made 

microcosms C, D, and E high biodeterioration risk systems. 

The overall results suggest that CME-100 and LSD have comparable but somewhat 

different biodeterioration susceptibilities. Although the CME-100 microcosms had 

lower microbial population densities, biomass and metabolic activity, CME-100’s 

chemical properties are characteristic of high biodeterioration risk fuels. It’s possible that 

CME-100’s high lauric (n-dodecanoic) acid content gives the product antimicrobial 

properties that compensate for the increased biodeterioration risk due to high TAN and 

water content. The microbiological data all support the hypothesis that CME-100 is 

less suitable than LSD as a medium for microbial activity.

Since CME-100 is most probably going to be used as a blend stock, it’s important to 

understand the relationship between blend concentration and biodeterioration resistance. 

Testing CME-10, CME-20 and CME-40 would provide important market application 

information. Comparing these blends with similar blends from outer common methyl 

ester stocks – particularly rapeseed and soy – would provide important competitive 

performance information. 

Recommendations 

The data from this study were inconclusive but very encouraging. I recommend that 

based on the generally positive results reported in this document, a second phase of 

testing be undertaken. 

I recommend the following test plan: 

Fuels: 

• Conventional Low Sulfur Diesel 

• Conventional Ultra Low Sulfur Diesel 

• Biodiesel Blend Stock (B-100) from: 

o Coconut 

o Rapeseed 

o Soy 

• Fuel blends from each stock: 

o B-40 

o B-20 
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o B-10 

Sampling times (all microcosms): 

• T0

• T1-month

• T2-months

• T3-months

Test parameters: the same parameters used in this study modified as follows: 

• Fuel corrosivity: immerse coupons for the duration of the study and compare 

coupons at T1, 2 and 3-months. 

• Fuel chemistry: add simulated distillation and carbon number distribution at times 

T0 and T3-months. Also use a methyl-ester compatible modification5

 of the TBN 

test. 

I further recommend that all fuels be tested in triplicate microcosms. As discussed 

earlier, replicates will provide the essential basis for differentiating between the effects of 

fuel chemistry and normal experimental variation. 

Favorable results from the proposed expanded study are expected to have a dramatic 

effect on CME marketability. Differentiating CME blends from other biodiesel on the 

basis of biodeterioration resistance would create unique pricing opportunities and give 

CME lends a distinct advantage in markets where climate in particularly conducive to 

biodeterioration. 

5

 S. Westbrook of SWRI has advised me that his lab has developed a modified TBN test for B-100 stocks 

that circumvents the equipment incompatibility problems that Clark Laboratories encountered during this 

project. 
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EVALUATION OF BIODIESEL FUEL 

BIODETERIORATION SUSCEPTIBILITY 

FINAL REPORT 

APPENDIX A – BCA TEST METHODS 

• Modified Oxygen Demand for Monitoring Biocontamination 

of Fuel System Bottoms-water 

• ATP in Fuel System Bottoms-water 

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APPENDIX A

Modified Oxygen Demand for Monitoring Biocontamination of 

Fuel System Bottoms-water 

Objective: 

To use dissolved oxygen measurements in order to estimate bioactivity in water-miscible 

metalworking fluid (MWF) samples. 

Theory: 

At typical MWF temperatures, oxygen concentrations ([O2]) range from 6 to 11 mg 

O2/liter of fluid. 

Active aerobic bacteria and fungi consume oxygen. Consequently, they will deplete [O2] 

rapidly if they are active in a bottoms-water sample allowed to stand without aeration. 

By measuring [O2] immediately after agitating a sample, and again after two-hours of 

standing, we can compute the rate of oxygen consumption. The traditional Biochemical 

Oxygen Demand test (BOD5) is a five-day procedure used to estimate the concentration 

of biodegradable organic matter in wastewater. This 2-hour modification of the BOD5

enables system operators to estimate microbial loads, or bioburdens. The greater the 

difference (∆ DO) between [O2] at time zero (T0) and two-hours (T2h), the greater the 

bioburden. 

Active microbial communities will deplete [O2] more quickly than dormant communities 

will. This means that ∆DO test results may not always correlate with viable count (dipslide) results. A small, active community (fewer CFU/mL) will have a greater ∆DO than 

a larger (more CFU/mL), less active community. However, the former will be more 

likely to cause biodeterioration problems than the latter. 

Materials: 

Dissolved oxygen meter – for example, Corning CheckmateTM Dissolve Oxygen System 

Sensor and accessories (electrolyte solution and replacement membranes) 

50 mL screw-cap disposable centrifuge tube 

Zero oxygen solution 

Distilled water in wash bottle 

Lab wipes 

250 mL beaker 

500 mL Boston round sample bottle 

10 mL volumetric or serological pipette 

Pipette pump 

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APPENDIX A

Procedure 

Meter calibration 

Follow dissolved oxygen (D.O.) meter manufacturer’s instructions for maintenance and 

calibration. 

Testing 

1. Collect MWF sample using an appropriate bottom sampling device, or directly 

into 50 mL centrifuge tube. 

2. Use 10 mL pipette to draw 35 to 40 mL water from sample, and transfer to clean 

centrifuge tube. If sample was collected in centrifuge tube ensure that volume is 

35 to 40 mL. 

3. Within 15 minutes of sampling, or as soon as sample temperature equilibrates to 

room temperature, tighten screw cap and agitate bottle vigorously for 30 seconds. 

4. Immediately remove cap and immerse D. O. probe into water and measure [O2] in 

mg O2/liter and percent D.O. Also record sample temperature. 

5. Rinse D.O. probe with distilled water, rinsing residual bottoms-water into the 250 

mL beaker and drying the probe gently with a lab wipe. Place the probe in 

distilled water until next use. 

6. Cover the centrifuge tube, securing the screw-cap lid tightly. Don’t over-torque 

the lid, or lid may split. 

7. Let sample stand for 2-hours. 

8. At the end of 2-hours, carefully remove sample centrifuge tube cap, and repeat 

steps 3 and 4. 

Reporting 

Enter data into a table similar to the example below: 

Sample ID Time T0 Time T2h ∆DO 

mg 

O2/L 

% O2 T °C mg 

O2/L 

% O2 T °C mg 

O2/L

% 

Change

Compute % change as follows: 

1) ∆DO = [O2]T2h – [O2]T0

2) % Change = (∆DO ÷ [O2]T0) x 100 

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APPENDIX A

Interpretation 

% DO Change Risk 

Descriptor 

≤ 10 % Low 

10% < DO Change ≤ 50% Moderate 

50% < DO Change High 

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APPENDIX A

ATP in Fuel System Bottoms-water 

Objective: 

To use a Luciferin-Luciferase enzyme-fluorochrome complex that will react specifically 

with the molecule, adenosine triphosphate (ATP), to create a bioluminescence reaction 

that can be measured quantitatively using a sensitive, accurate, bioluminometer. 

Theory: 

Adenosine triphosphate (ATP) is the primary energy transfer molecule in all living cells. 

The principle energy generating metabolic pathways – the Krebs Cycle and EmbdenMyerhoff Pathway react adenosine diphosphate (ADP) with polyphosphate to produce 

ATP. This ATP is then used to drive those metabolic pathways that consume energy. 

Luciferin is a bioluminescent molecule found in glow-worms and fireflies. In the 

presence of the enzyme, luciferase, and ATP, luciferin emits light at a characteristic 

wavelength. This light is seen with the naked eye as the glow of fireflies and glowworms. The intensity of the light emission is proportional to the ATP concentration 

available to react with the luciferin-luciferase complex. 

Consequently, Profile 1 bioluminescence reading, in lumens, is directly proportional to 

the ATP concentration within the sample tested. 

The ATP concentration in a sample depends on the total bioburden. Generally speaking, 

ATP concentration increases as the number of cells per mL increases. However, the 

amount of ATP per cell depends on both genetics and physiology. Different species have 

different typical specific ATP concentrations (average ATP per cell). For a given 

species, metabolically active cells will have greater specific ATP concentrations than will 

dormant cells. Consequently, ATP can be used as an indicator of total biomass and the 

metabolic potential of a contaminant population. 

Hydrocarbons and other chemical substances likely to be found in bottom-water may 

interfere with the ATP assay. This protocol includes preliminary sample preparation 

steps that eliminate these interferences. 

Materials 

Profile 1 Model 4560 Bioluminometer (New Horizons, Inc., Columbia, MD) 

Profile 1 Filtervette, 1 per test 

Profile 1 Filtervette 5-place holder, 2 

Profile 1 Filtervette blotters, 4 per 5 tests 

Profile 1 Filtervette filtration plunger 

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APPENDIX A

Profile 1 freeze-dried luciferin-luciferase reaction pads (tickets), 1 per test 

New Horizons SRA reagent, one vial per 50 samples 

New Horizons BRA reagent, one vial per 50 samples 

New Horizons ATP Standard, one vial 

Pipette, microliter, 50 μL 

Pipette, microliter, 15 μL 

Microliter pipette tips, disposable, sterile for 50 μL and 15 μL pipettes, 1 each per 

sample. 

Photo 1. Model 4560 Bioluminometer and supplies. 

Procedure 

Bioluminometer calibration 

1. With sample drawer closed, bioluminometer should read 0 (see Photo 1). 

2. Place 15 μL of BRA onto an unused ticket, place ticket into its position in sample 

drawer, close drawer and read bioluminescence. Value should be < 50 lumens. 

3. Place 50 μL ATP standard onto unused ticket, place ticket into its position in 

sample drawer, close drawer and read bioluminescence. Value should be 10,000 

± 4,000 lumens. 

4. If unit fails any of these three calibration/quality assurance tests, re-run test. If 

unit fails on second attempt, refer to manufacturer’s manual for further guidance. 

Testing 

1. Set up two Filtervette 5-place holders; place one or two Filtervette blotters onto 

base of each holder. 

2. Place a Filtervette cuvette into one of the holders in the Filtervette 5-place holder. 

3. Attach an unused pipette tip into the 50 μL pipette. 

4. Draw a 50 μL sub-sample from the sample container. NOTE: if the sample bottle 

contains fuel over water, use a sterile, glass (1 to 10 mL) pipette to transfer 

bottom-water to a sterile container (for example a 50 mL centrifuge tube), then 

draw the required 50 μL water sample. 

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APPENDIX A

5. Dispense the 50 μL sample portion into the Filtervette cuvette. 

6. Add 3 to 5 drops of SRA reagent. 

7. Draw the Filtervette filtration plunger’s plunger out approximately 3⁄4 from the 

plunger’s barrel, place the barrel’s rubber seal onto the top of the Filtervette 

cuvette and apply pressure gently to the plunger in order to create sufficient 

pressure within the cuvette to drive the fluid through the cuvette’s filter base. 

8. Repeat Steps 6 & 7 once more. 

9. Remove a ticket from the 5-ticket pouch, open the ticket and place the open ticket 

under one of the openings of the second Filtravette 5-place holders. NOTE: the 

ticket should be between the blotter and the cuvette holder opening. 

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APPENDIX A

10. Remove the cuvette from the first holder and place it into the opening under 

which the ticket has been positioned. Ensure that the cuvette’s base surrounds the 

pad of freeze-dried luciferin-luciferase reagent located near the center of the 

bottom half of the ticket. 

11. Place a pipette tip onto the 15 μL pipet. 

12. Draw a 15 μL portion of BRA reagent and pipet it into the cuvette, just above the 

cuvette’s bottom-filter pad. 

13. Repeat Step 7. 

14. As quickly as possible, remover the ticket from beneath the cuvette, refold the 

ticket, place the ticket into its position in the bioluminometer’s sample drawer and 

close the drawer. 

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APPENDIX A

15. Observe the digital display on the bioluminometer. 

16. When the numbers on the display remain constant (approximately 5 sec) record 

the value displayed. 

Precautions: 

1. The Filtravette’s base is made of a filtration medium that is used to concentrate 

the sample and separate interferences from ATP. When using the micropipettes 

to dispense sample or BRA reagent, position the pipette-tip near the cuvette’s 

base so that fluid is delivered to the base and not the Filtravette’s walls. 

2. Steps 13 and 14 deliver a droplet of BRA reagent to the ticket surface. Avoid 

allowing the droplet to come into contact with the Filtervette 5-place holder. 

Such contact may reduce the apparent ATP concentration (the ATP in the BRA 

fluid left behind on the holder won’t be measured) and may contaminate the next 

sample. If BRA from the ticket adheres to the Filtravette holder, dry the wetted 

surface and retest the sample. 

3. To prevent cross contamination, do not reuse pipette tips. If a sample is being 

measured in replicate, a single tip may be used for all of the replicate subsamples. 

4. Test variability depends on several factors including: 

a. Analytical technique 

i. Consistency of sample portion size (pipeting technique) 

ii. Precautionary items 1 through 3 listed above 

iii. Consistency and brevity of time lapse between completion of step 

13 and completion of step 14 

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APPENDIX A

b. Biomass distribution within sample. Biomass flocs may contribute to 

considerable variability amongst sub-samples. Optimally each sample 

should be tested in triplicate. Alternatively, triplicate analyses should be 

performed on 10% of the samples tested. Compute the average standard 

deviation amongst the triplicate analysis data sets and report all data as 

observed value ± this standard deviation. 

Interpretation 

Bioluminometer Reading 

Linear Scale Log10 Scale 

Risk 

Descriptor 

≤ 100 ≤ 2 Low 

100 < ATP ≤ 500 2 < ATP ≥ 2.7 Medium 

>500 > 2.7 High 

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EVALUATION OF BIODIESEL FUEL 

BIODETERIORATION SUSCEPTIBILITY 

FINAL REPORT 

APPENDIX B– LABORATORY REPORTS 

• Clark Laboratories Report 23878 of 3/1/05 – CME-100 Chemistry 

• Clark Laboratories Report 24834 of 6/9/05 – Microcosm A Fuel chemistry 

• Clark Laboratories Report 24835 of 6/9/05 – Microcosm B Fuel Chemistry 

• Clark Laboratories Report 24836 of 6/9/05 – Microcosm C Fuel Chemistry 

• Clark Laboratories Report 24837 of 6/9/05 – Microcosm D Fuel Chemistry 

• Clark Laboratories Report 24838 of 6/9/05 – Microcosm E Fuel Chemistry 

• EMSL Analytical Case No: 360500219 of 03/09/05 Modified NACE TM0172 

• EMSL Analytical Case No: 360500555 of 06/08/05 Modified NACE TM0172 

• EMSL Analytical Case No: 370501236 of 03/28/05 Water Chemistry 

• EMSL Analytical Case No: 370501236 of 06/24 /05 Water Chemistry 

• EMSL Analytical Case No: 370501576 of 03/7/05 Water and Fuel Microbiology 

• EMSL Analytical Case No: 370501576 of 06/20/05 Water and Fuel Microbiology 

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APPENDIX B

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Clark Laboratories Report 23878 of 3/1/05 – CME-100 Chemistry 
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APPENDIX B

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Clark Laboratories Report 24834 of 6/9/05 – Microcosm A Fuel chemistry 
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APPENDIX B

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APPENDIX B

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Clark Laboratories Report 24836 of 6/9/05 – Microcosm C Fuel 

chemistry 
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APPENDIX B

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Clark Laboratories Report 24837 of 6/9/05 – Microcosm D Fuel 

chemistry 
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APPENDIX B

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APPENDIX B

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TM0172 
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EMSL Analytical Case No: 370501576 of 03/07/05 Water and Fuel 

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Appendix 2. 

FTIR/ATR Method for 

Biodiesel Blend Determination
Standard Test Method for Determination of Biodiesel in Diesel Fuel Oil Using Mid 

Infrared Spectroscopy 

This method is derivative to ASTM D 6277 Standard Test Method for Determination of 

Benzene in Spark-Ignition Engine Fuels Using Mid Infrared Spectroscopy 

Summary of Test Method 

A sample of diesel fuel is introduced into a liquid sample cell. A beam of infrared light is 

imaged through the sample onto a detector, and the detector response is determined. 

Wavelengths of the spectrum that correlates highly with biodiesel or interferences are 

selected for analysis by mathematically selecting areas of the whole spectrum. A 

multivariate mathematical analysis converts the detector response for the selected areas 

of the spectrum of an unknown to a concentration of biodiesel 

Instrument: 

Nicolet Magna 550 Fourier Transform Mid-Infrared Spectrometer, 4000 to 650 cm-1, 

resolution 4 cm-1

Cell: 

Axiom Analytical, Inc, FT-IR Attenuated Total Reflectance (ATR) Tunnel Cell, TNL120A, ZnSe Element 60 degree, housing with 0.125 inch Swagelok connections. 

ATR element material ZnSe 

beam condensing optics conical, non-focusing optics 

integral to cell body 

element configuration circular cross section with 

coaxial conical ends 

cone half angle 60° 

element length 1.55 in. 

element diameter 0.125 in. 

angle of incidence at 

sample interface 53.8° 

maximum range of 

incidence angles 6 1.5° 

standard absorbance 

(1428 cm-1 band of acetone) 0.38 6 0.02 AU 

material of construction 316 stainless steel 

seals Chemraz or Kalraz o-rings 
Calibration 

Analysis type: Partial Least Squares 

Pathlength type: Constant 

Component name: Biodiesel 

Standards: biodiesel in diesel fuel (0-25% biodiesel) 

 Number of standards and concentrations to be determined 

Spectrum Range: 1810.8-1669.5 

Baseline type: Two points, point1 1838.0, point2 1547.0 

Measurement location: 1810.8-1669.5 

Corrections: No corrections 

Other: use mean centering technique 

Factors used: 1 

Preliminary Results:Based on two soy-based sources of B100, this method will 

measure the biodiesel +/-7% relative (@20% +/-1.4%). Better accuracy is expected with 

known source of biodiesel. 
F1147-E(12/2004) 

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1. REPORT DATE (DD-MM-YYYY)

January 2006 

2. REPORT TYPE

Milestone Report 

3. DATES COVERED (From - To)

November 2004 - June 2005 

5a. CONTRACT NUMBER

DE-AC36-99-GO10337 

5b. GRANT NUMBER

4. TITLE AND SUBTITLE

Analysis of Coconut-Derived Biodiesel and Conventional Diesel Fuel 

Samples from the Philippines: Task 2 Final Report 

5c. PROGRAM ELEMENT NUMBER

5d. PROJECT NUMBER

NREL/MP-540-38643 

5e. TASK NUMBER

WF3Y.1000 and FC020800 

6. AUTHOR(S)

T.L. Alleman and R.L. McCormick 

5f. WORK UNIT NUMBER

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)

National Renewable Energy Laboratory 

1617 Cole Blvd. 

Golden, CO 80401-3393 

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REPORT NUMBER

 NREL/MP-540-38643 

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NREL 

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National Technical Information Service 

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14. ABSTRACT (Maximum 200 Words)

NREL tested Philippines coconut biodiesel samples of neat and blended fuels. Results show that the current fuel 

quality standards were met with very few exceptions. Additional testing is recommended. 

15. SUBJECT TERMS

coconut; biodiesel; Philippines; fuel quality standards; fuel property; fuel stability; microbial deterioration; CME; 

coconut methy ester; DA; additized diesel fuel; DUA; unadditized diesel fuel 

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