March 1985 

Production of Liquid 

Fuels and Chemicals by 

Microalgae 

A Final Subcontract Report 

J. C. Weissman 

R. P. Goebel 

Microbial Products, Inc. 

Fairfield, California 

Prepared under Subcontract No. XK-3-03136 
SERI/STR-231-2649 

UC Category: 61c 

DE85008780 

Production of Liquid Fuels and 

Chemicals by Microalgae 

A Final Subcontract Report 

J. C. Weissman 

R. P. Goebel 

Microbial Products, Inc. 

Fairfield, California 

March 1985 

Prepared under Subcontract No. XK-3-03136 

SERl Technical Monitor: Robins Mclntosh 

Solar Energy Research lnstitute 

A Division of Midwest Research Institute 

1617 Cole Boulevard 

Golden, Colorado 80401 

Prepared for the 

U.S. Department of Energy 

Contract No. DE-AC02-83CH10093 
NOTICE 

This report was prepared as an account of work sponsored by the United States Government. Neither the 

United States nor the United States Department of Energy, nor any of their employees, nor any of their 

contractors, subcontractors, or their employees, makes any warranty, expressed or implied, or assumes any 

legal liability or responsibility for the accuracy, completeness or usefulness of any information, apparatus, 

product or process disclosed, or represents that its use would not infringe privately owned rights. 

Printed in the United States of America 

Available from: 

National Technical Information Service 

U.S. Department of Commerce 

5285 Port Royal Road 

Springfield, VA 22161 

Price: Microfiche A01 

Printed Copy A06 

Codes are used for pricing all publications. The code is determined by the number of pages in the publication. 

lnformation pertaining to the pricing codes can be found in the current issue of the following publications, 

which are generally available in most libraries: Energy Research Abstracts. (ERA); Government Reports 

Announcements and Index (GRA and I); Scientific and Technical Abstract Reports (STAR); and publication, 

NTIS-PR-360 available from NTIS at the above address. 
FOREWORD 

This is the final report on work completed under FY 1983 and FY 1984 

funding. The work was performed under subcontract to SERI with funds 

provided by the Biomass Energy Technology Division of the U.S. Department of Energy under a program to investigate the mass culture of microalgae as a source of renewable fuels. 

Robins P. McIntosh 

Aquatic Species Program 

Approved for 

SOLAR ENERGY RESEARCH INSTITUTE 

Stapley R. Bull, Meting Director 

Solar Fuels ~esehch Division 
SUMMARY 

OBJECTIVES 

fin overall objective of the project was to conceptually determine if simple 

open pond systems have application for the production of fuels from 

microalgae. To demonstrate the overall objective, work concentrated on 

showing the potential nicroalgal yields that are possible from an open pond 

system on a sustained basis. Furthermore, problems (pond management, design) 

associated with this experimental system were documented and reported so that 

future endeavors shall benefit. Finally, operational costs were documented 

to permit preliminary economic analysis of the system. Specific objectives 

that were set for fulfillment of this contract were: 

- demonstrate the outdoor monoculture potential of at least four species 

of promising oleagenous microalgae. 

- demonstrate sustained yields (4 mo.) of at least 2 species of promising 

oleagenous microalgae. 

- identify specific management strategies that are applicable to the open 

pond system, 

- identify cost centers, and report operational expenses associated with 

the present operations, 

- conduct experimental work aimed at reducing the operational costs 

associated with the system. 

Under this contract, the species selected to be grown was deemed very 

important. 4 primary objective of this work was to determine the suitability 

and productivities of microalgae designated as "promising" by other 

subcontractors working on this project. Thus species selection for outdoor 

work was determined with inputs and accord of other subcontractors and the 

SERI program coordinator. Once species were identified that grew well 

outdoors, work was initiated so as to establish management strategies for 

maximization of productivities, Specifically, this work compared batch vs. 

continuous culture and mixing speeds. Cost centers were identified and 

operational costs recorded and reported for subsequent economic analyses and 

comparisons. 

The specific tasks set for this project were! 

TASK I. Inoculate and maintain in outdoor ponds at least 2 species of 

microalgae that have been identified as "promisingt by ASP researchers 

involved in screening and development. 

TASK 11. Compare the productivities from two pond management strategies; 

batch and continuous culture. 
TASK 111. Acclimation of izncvieis ggldaea from a salinity of seawater (35 

ppt) to a slightly brackish salinity of 5 ppt. 

TASK IV. Determine the cast-productivity trade-o+fs of chemicals fchelators, 

trace metals) presently added to the medium. 

TASK V, Growth of microalgae on recycled media. 

TASK VI. Maintain two species of the previously selected microalgae in the 

outdoor ponds for a period of at least four months in a produrti~n mode that 

will be~t demonstrate the potential o+ the system. 

DISCUSSION 

All of the tasks outlined for this project were addressed during project 

performance, Of the strains of microalgae provided by other ASP 

subcontractors, none could outcompete invading wildtype algae. Nonetheless, 

two strains, Ankistr!des!us fal~atus and sgenedegmys So23 were grown ~utdoors 

in mass culture, one for two months, the other far one month. Results 

obtained using these strains did provide information useful to the screening 

program in the future. Two wildtype organisms, Scenmgesmu: qgg$ru~ag$g and 

Chlorella se, provided most of the outdoor results. The Scgngdgs~yg was 

maintained for thirteen months, averaging about 13 gm/n2/day. It was used 

to compare batch vs. continuous cultivation, for mixing speed experiments, 

+or media recycling, for storage product induction tests, and for the 

determination of operational costs. The CMgrglLg was grown for two months 

at over 20 gm/r2/day, and was used to determine the relationship between 

mixing velocity and productivity. 

The maintenance of a monoculture was not difficult when climatic conditions 

were relatively constant. Changes of species, from 5ice~giezggg to Chi~ce/la, 

occurred when pond temperatures rose above 3S°C and reversed when the 

temperatures dropped. Both wildtypes tolerated a range of TDS and mineral 

composition. finkjqi~odgq!uz tolerated most medium composition variations, 

except low K', but was sensitive to high irrrdiance when in dilute 

suspension, None of the organisms required chelator, trace supplementation 

(beyond that available in the water used), or vitamin supplementation. The 

results indicate that a monoculture can be maintained if either the seasonal 

temperature variations are small or a campetitive strain is used which has a 

broad temperature optimum. A competitive strain is one which does not 

exhibit significant photoinhibition, oxygen inhibition or respiratory 

losses. Of course, TDS will exert an overall selection criterion, 

As mentioned above, the productivity results ~btained with the "summer" 

ghig[glls and the Scgfi~dggmgg during the remainder of the year, indicate that 

s year round average of 15-20 gmlm2/day is attainable in this northern 

California climate with the strains used. A yegrly average of over 20-25 

gm/mZ/day could probably be obtained 500 miles south, where ambient 
temperatures are higher during spring, fall and winter. Continuous 

cultivation increased production relative to sequential batch operation, by 

30% during summer. However, the typical density of the culture at harvest 

time was much lower under continuous cultivation. In addition, induction of 

storage products required one to three days of batch growth after a 

continuous or batch active growth stage. 

Nixing velocity experiments revealed four factors of importance in 

considering optimum mixing speed; C02 outgassing which increases quickly 

with mixing speed, power input which increases even more quickly, suspension 

of cells, and productivity response. It was found that 15-20 cmlsec mixing 

speed was sufficient to keep even the largest (1-2mm) clumps of Sggngdgq4us 

suspended. It was also found that, from 1 to 60 cmlsec, mixing speed had no 

effect on productivity. 

Experimental results indicated that organisms respond very differently to pH 

and C02 concentration, as well as to dissolved oxygen. The latter may turn 

out to be an important screening criterion, as high DO is endemic to large 

systems. The response to pH and C02 determines the range of pH (given 

water alkalinity) in which a system can be operated. The pH should be as low 

as po6sible to obtain the highest dissolved C02 required for maximizing 

avwage production. But this must be balanced against outgassing loss of 

C02 which increases with concentration. Mixing speed affects the surface 

mass transfer coefficient. 

The data obtained from one month of operation of a Sgg~pdggqug culture with 

effluent recycle was used to evaluate the cost of the inputs to the system. 

Cell harvesting was inexpensively achieved due to the very good sedimentation 

properties of this strain which grew in large clumps. The nutrient, C02, 

and ! ower inputs were monitored. The harvested biomass output averaged 15 

gm/m /day. Initial input costs were $1.09/kg AFDW and were dominated by 

C02 costs 175% of the total). Realistic assumptions concerning prices of 

C02 and nutrients, bought in bulk, led to a 75% reduction in projected 

cost. Another 50% reduction would be necessary and would have to come from 

the assumption of a source of C02 at below present market price. Increased 

productivity, although important in terms of distributing annualized capital 

costs and labor costs, has little impact on the inputs considered here since 

most are nutrients that are proportional to biomass output. 

CONCLUSIONS 

The major conclusions of this project can be summarized as follows: 

1) Using two wildtype species in northern California a yearly average 

productivity of 15 gm/m2/day, or 24 tons/acre/yr can be obtained in 

water with TDS = 4-8 ppt. 

2) This can probably be increased to 20-25 gm/m2/day or 32-40 

tonsIacre/yr in southern California. 
3) Productivity can probably be further increased by using competitive 

strains screened for 

a) low respiration rates 

b) tolerances to high levels of dissolved oxygen 

c) broad temperature optima 

d) resistance to photoinhibition. 

4) In systems with randomized, turbulent mixing, productivity is 

independent of channel velocity at least far productivities up to 

25-30 gm/m2/day and veloci t ie5 from 1-30 cm/sec. 

9) Storage product induction requires one to three days of growth in 

batch mode under N-depleted conditions. 

61 Critical cost centers include C02 input, harvesting and system 

capital cost. Increased mixing speed increases C02 losses, power 

input, and system costs and must be adequately offset by very large 

increases in productivity. 

7) Media recycling, necessary for water conservation, has no adverse 

effects, at least in the short term for strains which do not excrete 

organics, and when the harvesting method is at least moderately 

effective for all algal forms which may be present. 

TABLE OF CONTENTS (CONT.) 

SECTION 5.0 

S. 1 

5. 2 

5.3 

5.4 

5.5 

SECTION 6.0 

6.1 

6.2 

6.3 

6.4 

h" 5 

6.6 

SECTION 7.0 

7.1 

7.2 

7.3 

7.4 

7. 5 

7 . t, 

SECTIQN 8.0 

8.1 

0 . 2 

!?egg 

Salinity. Mineral Composition. and Recycling ...... 44 

Introduction ....... .'. ............. 44 

Total Dissolved Solids Tolerance ............ 44 

Medium Composition and Culture Requirements far 

!jeki;irgdgyya .................... 46 

Recycling Clarified Effluents ............. 51 

Conclusions ....................... 54 

Mixing Velocity Experiments ............... 35 

Introduction ...................... 55 

Methods ........................ 55 

Results and Discussion ................. 59 

Mixing . Productivity Experiments ............ 61 

Mass Transfer Through the Surface ............ 64 

Summary ......................... 65 

Carbonation. ...................... &7 

Introduction ...................... b7 

Injection Efficiency .................. 67 

GO2 Utilization Efficiency ............... 60 

Physiological Aspects of C02 Utilization ........ 70 

GO2 Outgassing .....ma............ 73 

Conclusions8 ....................... 75 

Storage Product induction ................ 76 

Outdoor Cultures .................... 7% 

Indoor Cultures .................... 79 

LIST OF TABLES 

Media for Indoor Cultivation ............... 5 

Media for Outdoor Cultivation .............. 9 

Periods of Cultivation of Microalgae .......... 12 

S~e~edgggg~ gggdciggu#a Productivity in Freshwater . 8 . a 13 

Sgg~eggygs gggd.tLgpuig Continuous Culture Experiments . . 16 

S8 qygdyicaydp Continuous VS Sequential Cultivation . . 8 17 

S . guad~jgauda Saline 4 ppt Weekly Productivity ..... 19 

Statistical Parameters of . quadricauda Data 

A . Sustained Productivity ................ 20 

0 . Maximum Productivity ................. 21 

@lgyglla Productivity Summary .............. 22 

Statistical Parameters of Chlgfeiig Data 

A . Sustained Production ................. 25 

0. Maximum Production ................. 25 

fink~gf[gdes~u~ Productivity Summary: Weekly Averages . . 27 

2 555!~$!~!!!6 SgZg Cultivation: lOOm ........... 28 

Lipid Content of Mass Cultures .............. 28 

8 . gusdricagg$: Overnight Losses ............ 30 

2 S. qggdcjcauda: 1.4a Pond Results 1984 ......... 31 

Chigcglll: Overnight Losses ............... 32 

2 Chlg~plla: 4 Pond Results .............. 33 

ggcystiq: Overnight Respiration ............. 33 

2 kgysfip: 1.4~ Pond Results .............. 33 

Productivity Profile 4/25/84 8 . gysd~L~ggd5 100inZ ... 34 
LIST OF TABLES (CONT.) 

Productivity Profile 6/13/84 5 . qgadrigagga 

A, 1.4m2 Unheated .................... 35 

B . 1.4m2 Heated ..................... 36 

Productivity Profile 6/15/84 3 . ggg@.mjc~gulp 

A . 1.4m2 Unheated .................... 37 

B . 1 . 4m2 Heated ..................... 38 

Split-Day Productivities ................. 39 

Roux Bottle Cultures . Controls ............. 40 

Productivity and TDS 100-200m2 1904 ........... 45 

......... Isochrysir Productivity and TDS 1.4m2 .......... 45 

i2g~h~~giZ Productivity and TDS 1L Cultures ....... 45 

2 Losses from 1.4s Tank Cultures ............. 46 

Light Intensity-pH8 enkigtygdegeyy ........... 47 

i4nkistrodesmus: .............. Medium Composition Experiments ..... 49 

flnkiqtrgdeamug: Media Recycling ............. 52 

flnkistygdgseygr Media Recycling . Excretion Products . . 53 

2 S . qgadiicauda: Biomass Harvest-Effluent Recycle 200m . . 54 

Mixing Power and Velocity Results: 100m2 ........ 60 

Roughness Coefficient .................. 61 

Scenedes!us Mixing Speed Experiments . 100mZ Ponds .... 62 

Eh!eygl!r Mixing Velocity Experiments . 100m2 Ponds ... 64 

Outgassing Coefficient Vs . Mixing Velocity ........ 66 

C02 Losses at Injection ................. 67 

Overall Carbon Dioxide Utilization Efficiency ...... 68 

C02 Partitioning ..................... 69 
Case 

CD2 Partitioning with Oxygen Desorber .......... b9 

Chlqrliia Productivity Vs . pC02, pHI Alkalinity ... 72 

ChLgyella Maximum Specific Growth rate Vs . pC02. 

pH. hlkalinity. .................... 72 

pC02 and Chlorella Productivity: 1.4m2 Ponds .. 73 

Batch Induction af Continuous Pond Effluents, ...... 78 

S . qua@ricaydg Batch Induction .............. 78 

Storage Product Accumulation in Laboratory Cultures ... 79 

Sedimentation Rates ..,................ 81 

Sedimented Slurry Biomass Densities ........... 82 

Harvesting Mass Cultures, ................ a3 

Initial 4lgal Biomass Production Costs .......... 85 

200m2 Effluent Recycle . Media Component Inputs ... 85 

Cost Reduction Possibilitiee .............. 8& 

Climate Data: Monthly Averages 1983 ........... 31 

Climate Data! Monthly Averages 1984 ........... 91 

xiii 

LIST OF FIGURES (CONT.) 

Eisu~ Ease 

A- 1 Insolation and Temperature: Pleasante Valley 1983. . , . 92 

4-2 Insolation and Temperature: Pleasants Valley 1984. . , , 93 

A-3-5 Scatter Plots: Scengdg~mus Productivity. . . . . . . 94-96 

A-6-8 Scatter Plots: GhigMeLlg Productivity. , , . . . . . 97-99 
SECTION 1"Q 

INTRODUCTION 

1.1 BACKGROUND 

This report is a continuation of a project performed by EnSio, Inc. under 

subcontract XK-3-03000-1. It was performed parallel to USP species 

screening efforts and an outdoor project performed in Hawaii. The goal of 

the combined efforte is to advance the technology for producing lipid 

containing microalgae with the highest yields at the lowest cost, This 

specific effort was geared to identifying critical cost centers and 

elucidating methods for lowering their casts, 

The project utilized both naturally invading algae and organisms pravided 

from the species screening efforts of other ASP subcontracatars. The 

media used in this effort spanned the range from fresh to brackish waters 

- 0.4 to 8.5 ppt total dissolved solids. The pond system used, built 

under the previous subcontract, consisted of plastic-1 ined 100 n2 algal 

growth ponds, operated at 20 cm depth, and mixed by paddlewheel to provide 

slow to moderate channel velocities. Continuous and semi-cantinuau5 

operational modee were tested, and batch nitrogen deprivation studied. 

1,2 PROJECT GOALS AND TASKS 

One oC the major tasks set forth for the project was the determination of 

yisldr (gm/m2/day) averaged over an extended period of time. It was 

hoped that specifically screened strains, provided by other ASP 

subcontractors, would be used in generating the data. As it turned out, 

these strains did not compete well with indigenous algae, and only one, 

finkiptr~dgsgus faiggtgg, was successfully cultivat~d at the large scale 

for more than one month. ft was grown far two months on two different 

occasions, each time it was eventually outcompe.ted by indigenous algae, 

Two species of the latter were cultivated for two months or more. I4 

S~pn~deb~u~ g~ld~i~l~dl lp was grown for thirteen months and a Cbip~ella 

ae for two months. ?lost of the outdoor productivity data, and directed 

experimental results were obtained with these organisms, Much indoor data 

however, was obtained using the screened species. 

Another major goal of the project was to try to learn what factors limited 

the praductivities as measured and in addition, what factors were decisive 

in determining species dominance. Factors investigated in relation to 
yield limitation included pH, C02 levels, dissolved oxygen levels, 

mixing intensity, temperature and light input. Species competition was 

studied in relation to tolerance to high and low temperature, high light, 

high DO, and mineral composition of the various growth media. 

Besides the obvious impact of increased productivity on cost, the 

operational variables were examined for their impact on the cost of 

biomass production. Thus mixing intensity was studied in terms of yield 

enhancement, power utilization, and C02 outgassing. Media recycling was 

examined since it is essential for water conservation. The physiology of 

C02 utilization, and its relation to pH and alkalinity, were studied. 

Sedimentation, unaided by chemical flocculant addition was investigated as 

a means of harvesting biomass. Although no concrete rules can be 

formulated, as strains vary in their requirements and characteristics 

(e,g. sedimentation rates, pH optima) specific coets can be attached to 

any procedure or operation aimed at increasing yield and hence its 

cost-effectiveness can be evaluated. The results of the analyses in this 

report were formulated to serve this kind of function. 

1.3 ORGANIZATION OF THE REPORT 

The following report is broken down into eleven sections including this 

introduction. In Section 2.0, materials and methods general to the report 

ae a whole are outlined. However, some sections contain descriptions of 

those procedures which were specific to data collection reported therein. 

Section 3.0 presents the observational data obtained from extended 

cultivation of several strains. Thus, in addition to productivity data 

and correlations with climatic inputs, the characteristics of the 

organisms and the problems involved in maintaining prolonged cultivation 

are described. In Section 4.0 results are discussed from experiments that 

were aimed at taking a closer look at the physiology of yield dependence 

on factors like temperature, oxygen, light input and respiration. In 

Section 5.0 this discussion is extended to include mineral composition and 

salinity. Recycling of clarified culture effluents (water recycling) was 

also studied, Results from two six-week experiments are presented. In 

Sections 6.0 and 7.0 two of the most critical cost centers, mixing and 

carbonation are discussed in terms of experimental results. Two important 

operations, storage product induction and harvesting are treated 

respectively in Sections 8.0 and 9.0. Finally, all of the results are 

brought together in Selction 10.0 in an analysis of the operational costs 

of algal biomass production. Here the actual costs incurred during this 

project are summarized and from these, costs of production at the present 

state-of-the-art are projected for a large scale system. The major 

conclusions from the project are summarized in Section 11.0. 
SECTICSN 2.0 

METHODS 

2.1 LABORATORY METHODS 

fi~y-@gighiz. Volatile suspended solid5 were concentrated either by 

filtration (washed with 4 x H20) or by centrifuging 10-35 ml at 17,000 

rpm on a SS34 rotor in a Sorvall RCSB centrifuge tapp. 20,000 xg) and 

discarding the supernatant. The filter ar pellet was transferred to a 

preweighed weighing dish, heated at lOf0C overnight, weighed, ashed at 

55ooC for fifteen minutes, and reweighed to obtain the v~latile solids 

by difference. The data was calculated ae mg/t of ealida, For ash 

content the sample was either filtered through a Whatman GF/A paper and 

rinsed with 3-4 volumes of distilled water or the centrifuged pellet was 

washed with 10 ml distilled water. 

Igtfil-~~sg~l~g~,~g~~~~-jI~s~. Total dissolved salids were determined by 

placing 10-15 mls of clarified culture (centrifuge supernatent) in a 

pre-ashed, pre-weighed aluminum weighing dish, drying overnight at 

103OC, weighing, ashing at 550°C for fifteen minutes, and reweighing. 

The inorganic total dissolved solids were calculated as the difference 

between the final and initial weights, 

!g~~fi~~~-~ll. 50 ml of sample (clarified culture or water) was placed in 

a 100-150 nl beaker, 0.5 ml EDTh-NH3 buffer wal added, then one scoop of 

indicator powder (0,5 q Eriochrome Black T mixed with 100 g AR grade NnC1) 

was added and the solution stirred. The mixture was titrated with EDTA 

titrant (3.72 g Na2EDTA in 1 L distilled water standardized against r 

ltrndard CaC03 solution) until the solution turned from obvious pink to 

obvious blue, but not purple. Hardness was expressed as mg ca2+/~ or ng 

CaCO3/L. 

flikali~jty. Alkalinity was determined by titrating samples with 

standardized HCI or H2S04 down to pH 4.20. The alkalinity was 

expressed as mmules OH' consumed per liter sample. 

Generally a 100 ml sample volume was titrated with 0-20 ml O.lN HC1. The 

solution was gently stirred with a magnetic stir bar, and the addition of 

acid was moderately rapid below pH 7 to minimize the effect of C02 

escape. 

&~p~~-~g~i~~~-~2~. Algal suspensions were concentrated by 

centrifugation. The pellet was used for both ash-+ree dry weight 

determination and lipid determination by the modified Bligh and Qyer 

method. 
~ot2C-CSrb~h~drete-cgCteCi~L3~. 1.0 nl of 5% phenol solution was added to 

1.0 m1 sample containing 0.015 to 0.130 mg carbohydrate in an 18 x 150 mm 

tube. After mixing, 5.0 ml of 0.5% hydrazine sulfate in concentrated 

H2S04 was added, covered, and mixed well with a vortex mixer. After 

cooling to room temperature, absorbence at 490 nm was determined and total 

carbohydrate concentration was calculated from a glucose standard curve. 

!Eea-L4:. Clarified culture liquid was diluted appropriately and then 

treated with a buffered urease reagent to liberate ammonia. The ammonia 

was detected as below. 

fi!~!Cj+. Ammonia was determined from clarified culture liquid using the 

phenol-hypochlorite reaction to form blue indephenol at high pH, according 

to the method of Solarzano 151. 

........................... 

1 Liter Roux Flask Cultures. A low light, continuously illuminated 

experimental apparatus was used for initial studies and bioassays. One 

liter Roux flasks (5 cm thick) were filled with 750 ml culture, sparged at 

20-100 V/V/hr with a 98:2 air/C02, incubated at 20: ~OC at pH = 7.5: 

0.3. The bottles were placed in a light bank of cool-white fluorescent 

lamps at light intensities between 25-40 wmm2 per side (see data tables 

for specific conditions). Productivities from these experiments were 

determined from ash-free dry weights and expressed as gm/L/day or 

gr/m2/day, or percent visible light utilization efficiency. 

~:~-gqL-fi-ip~og~-lggk-cy1tg[eg. A high light, diurnally illuminated 

experimental apparatus was used to produce innoculum for outdoor work, for 

comparative productivity data, and for media optimization and recycling 

experiments. Generally 1.4 m2 tanks were operated as sequential batches 

at 10 cm depth (140 L) and mixed by paddlewheels ('1.5 fps). Light 

intensity varied depending on the experiment from 50-275 ~rn'? (see data 

tables for specific data) and was provided by two HSlOOQ metal halide 

lamps per tank. Temperatures were generally 25-3Q°C. pH was adjusted 

by intermittent addition of l00X C02 controlled by pH regulators. Grab 

samples were removed daily at 1000 hours. 

Sequential batches were operated by removing a percentage of the pond 

volume on a given schedule and replacing with an equal amount of fresh 

medium plus additional amounts of consumed nutrients in proportion to the 

biomass removed. When media recycling was practiced, a given volume of 

the pond was centrifuged through a Sharples T1 continuous separator, the 

effluent returned to the pond and consumed nutrients replaced. 

The cultivation media used varied with the organism being grown. Table 

2-1 lists the media compositions. 
Table 2-1. Media For Indoor Cultivation 

Chlorella 

Scenedesmws Ankistrodesmus f sochyses 

SP 9 Qocystis 

8t1 RI M mn .......................................................................... 

NaHC03 5,0-10.0 20.0 3.0-10.0 

K2H-KH2PD4 Buffer 2.0 1.0 0,2 

Trace Soln 

Urea 5- 12 6-12 - 

Biotin - 0 or 0.5 ug/L 0.5 ug/L 

Thi ami ne - 0 or 0.1 mg/t 0.1 mg/L 

Trace Solnr 

Element PPm Element PPm 

..------ --- ------- --- 

Hn 0,s V 0.01 

Ma 0.1 Co 0.01 

Z n 0.05 Ni 0.01 

Cu 0.02 Cr 0,Ol 

0 0.5 

5 
2.2 OUTDOOR POND OPERATIONS 

Outdoor ponds were operated as sequential batches, continuous cultures, and 

batch induction cultures. A schematic of the system is shown in Figure 2-1, 

and some photographs in Figure 2-2. 

!il~!ig!'!,g!d,!gdii A1 1 batch ponds were operated f ram initial inoculation or 

by emptying 75-902 of an operating pond culture and refilling with fresh 

medium. Density increased four to twelve fold prior to restart of the batch 

cultures. The basic medium components used were the same for all cultures and 

are shown in Table 2-2. Trace metals were available in sufficient amounts in 

the irrigation water used. Addition of chelated trace had no effect. 

Continuous ponds were diluted automatically over a 12-hour period from 7AM to 

7PM. Inflow was metered through a calibrated water meter. Outflow was via an 

overflow pipe and flowed by gravity to the harvesting ponds which were emptied 

automatically on a daily basis, Nutrients were fed to the ponds automatically 

from a nutrient mix tank (Urea, NaHC03, FeS04, EDTA) over a ten hour period 

or added manually each morning (P and Mg). Urea was added to reach a final 

concentration of 20-30 ppm urea-nitrogen. 

Bddi~fgg-gf-cgrCgn,~~gx_~~g. C02 was added from 65 lb. tanks and, after 5/84 

from a 6 ton tank via diffusers. Generally pH was kept between 7.0 and 9.5, 

occasionally it went as high as 10.0. Sodium bicarbonate was added to increase 

the C02 storage capacity. Alkalinity was measured to be between 5.0 and 20 

meq/L. At 10 meq/L, approximately 00 mg/L of algal biomass can be grown during 

a pH rise of 8.5 to 9.5, excluding C02 loss due to outgassing. C02 

additions were made automatic, slowly diffusing in C02 over an eight-ten hour 

period. 

!ggelieg,gnd,Meggyrggen_t,s Batch ponds were sampled manually (grab samples) 

each morning from four prescribed sampling points and composited. Samples were 

taken to the laboratory for solids, pH, and lipid analysis (if required). 

Effluents from continuous ponds were sampled every forty minutes from 9AM to 

7PM using an ISCO compositor. Each night the compositor samples were collected 

and stored in a refrigerator until the next day. The compositor was not 

refrigerated. Measurements of fresh samples ys unrefrigerated samples 

indicated that on the warmest days as much as 15% of the biomass may have been 

respired away during compositing. Productivity values were not corrected for 

this, but recirculation of cooling water through the ISCO sampler was 

implemented starting 6/14/03. 

Temperatures were recorded daily from maxfmin thermometers placed in each 

pond. Throughout, max/min ambient temperatures were recorded. pH was recorded 

manually twice a day. 

A LiCor Solar Monitor automatically measured, recorded and stored daily solar 

input totals as watt-hour/m2/day which was converted to Langleys per day. 


Depths were measured daily and evaporative water loss recorded. Productivity 

for batch ponds was calculated using daily ash free weight differences 

(corrected for evaporative concentration) and expressed as gms ash free dry wt 

per m2 per dry. For continuously operated ponds, the weight of the 

composited sample (gm/L) was multiplied by the corrected outflow per day 

ILIday) and the product divided by the pond area (a2)# 

Table 2-2. Media for Outdoar Cultivation 

Nutrient Added mM mM -------------1-a---------------------------------------------------- 

NaHC03 3.5 3.5-20 

Urea Oa35-0.70 0.35-0" 70 

Water Contains! 1.0-1.5 mH Rlkalinity 

0.1-0.2 mH K 

0.5-1.0 mH Ca 

1.0-1.5 mM Mg 

0.2-0.5 ppm Fe 

0.5-2.0 mM Na 

0.5 mfl C1 

0.2 mH SO4 

Trace: 

I3 

Zn 

Mn 

Cu 

Cr 

F 

Pb 

Hg 

Se 
2.3 SEDIMENTATION ANALYSIS 

Sedimentation rates were measured using 500 ml graduated cylinders filled to 

525 ml with well-mixed culture liquid. Samples were withdrawn from the 225 

sl mark (15 cm below the 500 a1 mark, 12.5 cm above the bottom) by siphoning 

through a thin line made of glass and plastic tubing. The line volume was 

about 6 ml. The density at the sampling point was measured in Klett units 

over a one hour period, starting at time zero, and then after 3, 6, 9, 15, 

20, 30 and 60 minutes. In an idealized system the fraction of particles 

(NO-Nt)/NO (NnKlett density), missing after a given time, say one hour, 

corresponds to the fraction of particles in the initial culture sample which 

have settling velocities greater than or equal to 13 cm/l hr = 15 cm/hr. In 

order for this to be strictly true, several conditions must be met: 11) the 

initial mixture must be uniform, (2) the extent of the cylinder must be great 

enough so that accumulation of sedimenting (or floating) cells does not 

interfere with the further settling of cells, i.e., no bulking problems, (3) 

there are no other interactions which hinder removal, (4) no convection, (5) 

the particles must not flocculate prior to settling, and 16) the liquid level 

must not change significantly. None of these criterion can be rigorously 

met, except the first. The sampling point was taken near the midpoint of the 

cylinder to allow ample space for compaction (or bulking) of both floating 

and settling particles. 
SECTION 3,0 

EXTENDED CULTIVATIDN RESULTS 

3.1 INTRODUCTIDN 

Three species of algae were cultivated in 100 m2 (and 200 aZ) ponds 

for at least one month. Two of the speeies were wild types that invaded 

the pond during cultivation of inoculated ipecies. One, a Sggngdgadgg 

qygdriraudg invaded during freshwater cultivation of a laboratory grown 

strain, gbllguye 1450. The S. guad~i~gudg was cultivated for thirteen 

months in freshwater and for three months in brackish water (4 ppt TIE), 

In freshwater it was grown both continuousXy (continuous dilution over 

daylight hours) and semi-continuously (sequential batches diluted every 

three to seven days), During the winter af 1983-84 (Dec., Jan,, Feb.) it 

was maintained in a 200 m2 pond but no data were taken. Another wild 

strain, ChlpcrlHg 5p. arose during attempts to grow Qggystiq (Walker take 

isolate) in brackish water (4.5 ppt TDS), during July 1984, It was 

cultivated far two months, hug,-Sept. 1984, semi-continuausly, The S. 

quaugicagda provided both long-term productivity data Ithis section) and, 

due to its pronounced tendency to form large settleable clumps, data on 

water recycling and biomass removal, The Chiqy~ila, on the other hand, 

grew as non-settleable single cells which afforded the opportunity to 

measure productivity as a function of channel mixing velocity even at the 

very low end of the scale (Section 6.0). 

Two strains, provided by species screening efforts oS ~ther 

subcontractors, were also cultivated, l4nkiqtya$esfiuz ffigatga was grown 

for two months at a time in the Fall of 1983 and Spring of 1984 in 

brackish water (4-8 ppt TDS). A freshwater Sgegedgwngg sp, So2a was grown 

during October 1983 both continuously and semi-continuously far a short 

period of time, However, neither of these strains could outcompete 

wildtype organisms in the long run. Two other pre-screened strains, an 

@EYsL~~ and Chjg~glig Soia were introduced into the outdoor environment 

but could not be grown long enough to inoculate the large-scale (1100n2) 

ponds. 

Table 3-1 outlines the species grown at the mass culture level, the 

duration of cultivation, and the time of the year in which they were 

cultivated, In the data tables presented in this report, productivity is 

cited as both "sustained" and "maximum" productivities. Sustained 

productivities are averages aver a specific period of time during which 

the cultivation was carried out, but it does not include data from very 

dilute suepensions ((50 ppm) or stationary cultures. Both of the 

situations arose often, as we tried to determine both the minimum and 

maximum densities which afforded good production. These changed uith 

climatic conditions. Maximum productivity refers to an average of the 

maximum production value from each batch measured during the averaging 

period. 
Table 3-1. Periods of Cultivation of Microalgae 

in 100m2, 200m2 Ponds 

1983 1984 

JASONDJFMAHJJASO 

S. quadricauda 

Freshwater 

semi-continuous ------------- 

Freshwater 

continuous 

Brackish Water 

semi -cont. 

Chlor. sp. Brackish Water 

Semi -cont. 

Ankistrodesmus Brackish 

Water semi -cont. - - .. - - - - - - 

S, So2a Brackish Water 

semi-cont. 

S, So2a Brackish Water 

continuous 

3.2 SCENEDESMUS QUADRICAUDA WILDTYPE 

Ten months of productivity data obtained from semi-continuous cultivation of S. 

qy!d~lcgudg in freshwater are shown in Table 3-2 and Figure 3-1, along with 

climatic summaries. The sustained average productivity for nine months 

(excluding Nov. 1983 - February 1984 when productivity war about 3-5 

gm/m2/day) was 14 gm/a2/day. Exclusion of one more month, July 1904, 

during which high temperatures eventually led to the death of the cultures, 

sustained productivity averaged almost 16 gm/m2/day. Photosynthetic 

efficiencies, based on total insolation varied between 1.2 and 2.2%. The 

lowest efficiencies were recorded during the warmest months. Hid to late 

spring appeared to be optimal for cultivating this organism. 

The cultivation media is listed in Table 2-2. The major chemical constituents 

from the irrigation water used are included; the minor constituents were also 

listed in Table 2-2. 
Table 3-2. Scgnedeegus gugdcjicau&a Productivity 

in Freshwater 

Typical 

Final 

Average Productivity Maximum Productivity T,OC Density 

Month/Yr gn/m2/d Lgly/d X Eff. gm/m2/d Lgly/d Nin/Nax ppm ------1----1--1-11---1------------------------------------------------------"--- 

Jul 83 15 658 1.2 10 664 20-32 275 

Aug 83 14 568 1.4 17 584 18-32 3 0 0 

Sep 83 14 4 13 1.8 18 504 16-29 3 0 0 

Nov 83 5 150 1.6 4 248 5-13 250 

Mar 84 13 419 1.6 15 41 1 9-22 400 

Apr 84 15 456 1.0 17 549 11-26 500 

May 84 20 656 1.7 26 67 1 17-31 800 

Jun 84 17 673 1.4 25 688 20-32 500 

Ave. 8 mo. 16 

Ave. 9 mo. 14 

The productivities from three months of cantinuous cultivation are depicted in 

Figure 3-2 and Table 3-3 and compared to concurrent semi-continuous values in 

'fable 3-4. the difference in productivity was greater during the summer, 

Maximum pond temperatures were also lower during continuous cultivation as 

expected due to the significant dilution with water at 20-25~C. Unother 

important distinction is the maximal density achievable., i.e., the density 

typical at harvest time. Here semi-continuous (batch) cultivation is 

advantageous since densities were twice as great when productivity was reduced 

only 25%. As shown in Table 3-2, typical batch densities (and productivities) 

were even greater in 1904 than 1983 reaching 700-800 ppm at time of dilution. 

A further distinction between continuous and semi-continuous operation is 

notable. The organism exhibited an extremely pronounced tendency to #orm 

colonies and for colonies to agglomerate into clumps. This occurred shortly 

after the initial invasive bloom (the lack of clumping upon initial blaoming 

was observed three times). The colonies were predominantly four-celled, 

although two and eight celled colonies were present in significant numbers. 


Table 3-3. Scenedesmus quadricauda Continuous Culture Experiments 

Det . Ave. Ave. Ave. Max. 

Time Insol. Density Prod. Eff. Prod, T OC 

Dates days Lglyld pH Range PPm gm1m2ld X gmlm21d Min-Max ................................................................................ 

6126- 1.70 645 7.5-8.5 115-200 17.2 1.2 19.1 18-29 

7/15 
Table 3-4. 9. gyad~lcayda Continuous vs. Sequential Cultivation 

Cont. Prod, Typical Seq. Batch Typical Max. Productivity 

gr/m2/day Density Production Batch Density X Increase 

Monthlyr PPm ga/m2/day PPm .......................................................................... 

7/83 18.6 170 14.8 275 2b 

8/83 18.8 1 SO 14 350 34 

9/83 15 140 14 350 7 

During continuous cultivation several colonies associated forming clumps of 

20-100 um in typical dimension. However, during batch growth, hundreds of 

colonies clumped into flocs of one hundred to several thousand microns. Figure 

3-3 shows a typical clump. Except during July 1984 when growth of the strain 

was tentative, these clumps were dense in cells and sparse in cell debr~s and 

detritus. It was typical that as temperature exceeded the optimum af 

25-30°C, more and more of the biomass of a clump was composed -of cellular 

debris, 

It was also observed that the S. qyad~igauda cultures were pleiomorphic and may 

not have been composed of one pure strain. Rather two morphologically distinct 

types of cells were always present, each in two size ranges. The dominant form 

resembled mature coenobia of S. gygd~igagdg end comprised about 80X of the 

populations. It occurred in a large (5 x 13 urn) and small (3 x 10 urn) body 

site. The other form resembled reproducing S. gygdcigguda and occurred in 5 x 

7 urn and 4 x 6 um cell sizes. 

8. gyid~iggydg was also cultivated in water containing 4.0 and 8.0 ppt TDS 

lflarch through May 19841, of compoeiti~n shown in Table 2-2, This medium, 

although high in NaCl was low in bath K+ and ng2+ compared to the medium 

optimal for growth 04 other brackish water strains (figkigt~pdey~yy, ggg~zfis~ 

&~~g[~~~wildtype). Productivity summaries for 4.0 ppt growth are shown in 

Table 3-5, as weekly averages. In all cases productivities were similar. 

However, as will be discussed in Section 4.0, the temperature optimum was 

shifted downward as salinity increased. 

fable 3-54 S. ggpdrigggda Saline 4 ppt 

Weekly Productivity Summaries 

Max 

Average Productivity Prod, T,OC 

Mo. /wk gm/r2/d Lply/d X Eff. gm/m2/d MinlMax PH Dens1 ty 

Table 3-6 presents another way of summarizing the freshwater sequential-batch 

growth of this organism. Here the values obtained for sustained 

productivity, maximum productivity and the environmental c~nditions of 

minimum pond temperature, maximum pond temperature, and total insolation are 

analyzed statistically. The data are from 39 sequential batches, 

weighted by batch duration (as was done in previous productivity summary 

tables). It can be seen from Table 3-6 that the variance in the prediction 

of productivity is reduced about 50% by using a knowledge of any cne of the 

three variables, in a linear regression. Adding more than one variable to 

the linear estimate adds little to the predictability of productivity. 

Better fits may have been obtained by, e.g., using two linear regions in a 

piecewise arrangement with a steeper slope at lower values of the 

temperatures and insolation. However, the sparseness of experimental paints 

in this lower end renders such an analysis of questionable value. 4s will be 

discussed in Section 4.0, temperature optima seem to exhibit sharp cut-offs 

at the high and low end. 
Table 3-6 

A: Statistical Parameters of S. gypdrigpyda Data: Sustained 

Variable Symbol Mean Variance S.D. n = 39 ...................................................................... 

Productivity P 14.7 15.8 4.0 

Total Insolation I T 526 26,600 163 

s2 = Variance of productivity measurement 

E = Estimate 

sE2 = Variance of productivity about the regression 1 ine. 
Table 3-6 1Cont.I 

0: Statistical Parameters of g. quadyigagdg Data: Maximum 

Mean Var i ance S. D. n = 38 !arlable .................................................... 

The wildtype S. qugdrjcauda was the naturally campetitive organism in 

freshwater and some brackish water formulations given the clrmatic 

conditions prevailing. Its natural tendency was to agglomerate into 

clumps. This tendency was most pronounced in freshwater semi-continuous 

operations, still pronounced in brackish water semi-continuous operations, 

and least evident in treshwater continuously diluted ponds. The clumping 

allowed easy harvesting, The longterm cu3tivation demonstrated that 

productivities of about 15 qm/n2/day longters average should be easily 

obtained. However, the organism was not sustainable in the ponds at the 

warmest time of the year when maximum pand temperatures rose above 3S0~. 
3.3 CHLORELLA WILDTYPE 

The wildtype ghig~glil was the naturally dominant organism during the 

summer in medium containing 4.5 ppt TDS, 2.5mM Mg2+ and 3mM K'. 

fi5kilt~~!!eS!L!S and Qggytie were the organisms originally intended to be 

grown in thie medium. However, the Chlgreiia was only sustainable during 

the warmest time of the year. As temperatures dropped in October 1984 to 

below 15OC min. and 25OC max. the organism died. 

Table 3-7 and Figure 3-4 show average weekly productivities measured with 

the Chig[qlia sp. Levels of dissolved oxygen were kept below those 

normally reached in the ponds. Section 6.0 has details. Sustained and 

maximum productivities were higher with ChNprgilg during the summer, than 

with 2. qgadrjgagda. 

Table 3-7. Chlprglig Productivity Summary 

Aver age Max i mum Typical batch 

Productivity Eff. Prod . T,OC Ending Density 

MoIWk gm/m2/day Lglyld % Tot gm/2/day Lglyld MinIMax PPm .......................................................................... 

81 1 23 64 1 2.0 30 64 1 19-32 560 

4 2 2 525 2.3 2 6 536 19-30 625 

- - - - 

Mo. ave. 21 2 7 

4 11 427 1.4 15 417 

- - - - 

Mo. ave. 16 20 

The data obtained from the Chlqrgiiq cultures were subjected to linear 

regression analyses as well. These data, however, were taken from a much more 

restricted period of time than the data from the Sggngdgsmne cultures. In 

addition, the Chlprpt-ig data were obtained during performance of a mixing speed 

experiment. Hence, operations and protocol were much more uniform in running 

the Chloreliq cultures relative to the gggnedgsgus cultures. As Tables 3-8A,b 

show, the correlation coefficients computed are much higher than those 

calculated for Sggngdggtys. The average (sustained) productivity correlated 

only fairly with maximum temperature and poorly with total insolation. In 

fact, little decrease in the unaccountable variance of the predicted 

productivity is gained by adding knowledge of these variables to knowledge of 

minimum temperature. Of course, the variability of all parameters was fairly 

small (10-207. of the means) during these experiments. 

The best correlations were found in the maximum production data set (Table 

3-BB). Linear models explained most of the variability in productivity, mainly 

in terms of maximum temperature and insolation. About 80% of the variance 

could be accounted for by the changes in these variables. Partial 

correlations, however, did not indicate that the correlation between production 

and either maximum temperature or insolation was due to the linear correlation 

of either of these variables on the other. 

The lack of correlation of some of the data may again be due to poor choice of 

model. A glance at the scatter diagrams (see Appendix) reveals that piecewise 

linear fits may have been a better choice of model for the sustained production 

data set and for the minimum temperature-maximum productivity data. 

Better correlations were expected using maximum productivity data than average 

productivity. The latter includes points which were most likely limited by 

suboptimal cell density. Even the high correlation of maximum production with 

insolation and maximum temperasture is not evidence that no other variable 

limited productivity. hlmost all productivity values were below 30 

gm/m2/day. 

flnkistrqdesmup faiggtus was obtained from W.Thomas as a result of his species 

screening effort. It was cultivated during the late summer and fall of 1983 

and again during the spring of 1984. Although the original medium recommended 

by Dr. Thomas called for vitamin supplementation, indoor experimental results 

indicated that this was not necessary. Consequently, after 9/28/83 vitamins 

were not included in the outdoor medium. 
'Table 3-0 

A: Statistical Parameters of GNg~sila Data: Sustained Production 

Variable Mean Var i ance S.D. n = 10 *-1--1--------------.-----------------0--.-------------------- 

P 18.7 21.6 4.6 

0: Statistical Parameters of El7157gl12 Data: Maximum Production 

Variable Mean Variance S. D. r! = 14 I-----~-"~LICC-C~C-----C----3----d------------------ 

P 23.5 40.7 6.4 

Coeff. vector R~ R II-II*CI"-IL- ----- 0--0 

(-5.3, 1.53) 0,324 0.57 

Partial Correlations: Based on P vs T,,,, IT 

rpT.1 = 0.357, 1 held constant 

rp1.T = 0.375, T held constant 
Productivity data is summarized in Table 3-9. Productivity of this strain was 

lower than that of both wildtypes except in Nov. 1983 at low insolation and 

temperature. Until late Sept, 1983, the cultivation was characterized by a 

browning of the cultures after the first batch growth. It was determined (via 

bioassay) that insufficient potassium was the cause. Potassium deficient 

cultures greened quickly when brought indoors and supplemented with potassium, 

Outdoor brown cultures, after supplementation uith potassium, greened very 

slowly and incompletely. The same problem with regreening occurred after 

nitrogen starvation. 

The growth of this organism, when healthy, was characterized by a deep green 

color and a complete dispersing of cells. As any one of a number of nutrient 

deficiencies set it, the cells clumped (with scores of cells to a clump). The 

cells themselves were almost straight or sickle-shaped, with sizes varying 

between 12-40 u in length and 2-4 u in width (Figure 3-31, In addition to 

nutrient deficiency, clumping of cells was a consistent consequence of extended 

cultivation of this organism. The gentle mixing caused by paddlewheels appears 

to have enhanced clumping. 

Another consistently observed characateristic of this organism was the tendency 

to bleach in high light or due to nutrient defiency. The bleaching, once 

advanced, was not reversible as was the case with the wildtype fgg~gdqsfig~ 

strain. When dilute cultures were initiated under sunlight, this bleaching was 

typical, but chlorophyll content increased as density increased due to growth. 

Cultivation of this strain was limited both due to competition from wild 

strains (initially gglgnqstyu! under low ~9~' or K' conditions, gcgngdggfiug 

Or Chlpygllg otherwise) and to a temperature optimum lower than summer pond 

temperatures. 

Average production from this strain was only 12 gm/m2/day. It was difficult 

to prevent contamination by S. gutdrlgaudg during April 1984 and impossible 

during May 1984. Increasing the salinity from 4.5 to 8.5 ppt in April did not 

lead to faster growth, higher productivity or increased competitiveness. 

During the summer months of 1984, growth of Chlprells prevented the 

establishment of even a small-scale outdoor Qnkiztrpdeqmys culture. 

3.5 SCENEDESHUS So24 CULTIVATION 

A strain of Scenedesmus, designated So2a by its isolator (S. Lien) was grown 

for a short period of time in the 100 m2 ponds. The duration of cultivation 

was limited by climate (cultures were inoculated in Sept. 1983) and 

contamination by the wildtype hgngdgsmn~. Table 3-10 summarizes the data. As 

the Fall progressed productivity declined. Since the organism was neither 

highly competitive, nor a lipid producer (Section 3.6) it was not studied 

further in 1984. 
Table 3-9 

Ankistrodesmus Productivity Summary: Weekly Uverages 

Average Productivity Max Prod. T,OC Densi t y 

Ho/Yr ,Wk gm/m2/day Lgly/d X Tot. gm/m2/d Min/Hax PH PPm --e-"-"-----II--I-I-----IC*L-I"-I.I-L--------I------------------------------- 

8/83,1 12 654 1.0 16 20-33 0.0-9. 0 235-425 

2 10.5 615 0.9 13.5 19-33 a. 0-0,7 50-225 

3,4 0 K+ deficient 

9/83,1 13 545 1.3 i 3 20-30 7.2-8.5 65-330 

2 14 526 1.5 16 20-31 7.9-8.5 167-306 

3 10 456 1.2 14 16-29 7.8-8.5 178-420 

4 15.5 402 2.4 18 17-27 7,b-6.8 75-300 

-- -- 

Uver age 13 15 

4 13 

-- 

Average 12 

* 2X salinity 
Table 3-10. Scenedesmus So2a Cultivation: 100m2 1983 

Ave. Productivity Eff. Nax.Prod. T,OC Density 0 

Dates gm/m2/day Lglyld X Tot, gm/m2/d minlmax pH PPm days 

Batch Start-up 

9/27-29 10 355 1.5 16 15-25 8.0-0.5 35-195 

Cont:inuous Operation 

10/ 1-7 15 405 2.0 17 19-25 7.2-8-9 130-180 2.3 

1018-11 10 37 1 1.5 12 14-29 7.0-8.8 120-155 3.2 

3.6 LIPID CONTENT 

None of the strains tested in the outdoor system produced lipids in large 

amounts, either when N-sufficient or starved. Table 3-11 lists lipid content of 

the strains used outdoors. 

Table 3-11. Lipid Content of Mass Cultures 

Organism N-Suf f icient N-St arved 

# tests % lipid S.D. # tests X lipid S.D. 

-------------------------------.------------------------------------ 

S, quadricuada 8 20.2 2.4 2 13.8 0.1 

Anki strodesmus 9 26.3 3.2 

3.7 CONCLUSIONS 

o Productivi ties of 15-25 gm/m2/day were routine1 y obtained 

from wildtype strains throughout the 7-8 month "growing season." 

However, higher values were rarely seen. Thus a "barrier" of 

unknown origin appears. 

o Continuous operation was about 20% more productive than semicontinuous operation, but "harvesting" densities were much lower. 

o Pre-selected strains fared poorly in competition with wildtypes. 

If species screening is to be effective, more attention must be 

paid to the climatic conditions prevailing at the mass culture site. 
SECTION 4.0 

PHYSIOLOGXCAL FACTORS AFFECTING MET PHOTOSYNTHETIC YIELD 

4.1 INTRODUCTION 

A noticeable aspect of the cultivation data is the consistency with which 

productivitiee were below 25 gm/m2/day. In this section the fcl lowing 

factors, which may have limited yield are discussed: grazing, temperature 

and levels of dissolved oxygen. It will be seen that each strain responds 

to a different extent to high levels of these parameters, 

4.2 GRAZING, TEMPERfiTURE AND DISSOLVED OXYGEN IN PONDS 

Experiments were conducted to t r y t o ascertain the reasons far low 

productivity in specific cases, and the appearance and disappearance of 

particular strains at certain times of the year. The 3. gggdyigauda 

faltered during the hottest months, but dominated ponds at other times. 

The results below indicate that i) this strain is respiratory at high 

temperatures, 2) that it is susceptible ta grazing at high temperatures, 

i.e., when grazing activity increases, but 3) it is relatively tolerant 

to very high levels o+ dissolved oxygen. 

Table 4-1 shows overnight respiratory and grazing losses measured in 5. 

gggfl~~ggJ~~ cultures, Grazers were totally absent from ponds throughout 

1983 and most of 1984. Only when pond temperatures reached 25°C 

overnight and 35OC during the day, for extended periods of time, did 

grazers bloom. This occurred in June 1984. The threat of grazing 

remained until Sept. 1904. During July-August 1984, even with heavy 

additions of aqua-ammonia, cultivation of S. qudy.mcagda could not be 

maintained. The @l!rglig cultures, on the other hand thrived, although 

each hatch needed to be operated on the final day, at high pH 09.31 with 

30 ppm free NH3, for twenty-four hours, to eliminate grazing. As shown 

in Table 4-1 overnight los6@~ of g. qggdywgagda biomass were absent in 

April when temperatures were low, and highest in June and July. The 2. 

qudyiggudp clumps underwent a change from the end of May to mid-June from 

being densely packed with cells to being SOX composed of cell debris and 

detritus, Grazing increased during this period with typical rotifer 

levels of O/ml in April, 0-l/ml in Hay and 10-:100/ml in June, The 

overnight losses became nearly equal to daytime production when heavy 

grazing accompanied respiration. It should be noted that at low to 

moderate overnight loss ((10% of daytime gross) the precise value of the 

loss was difficult to resolve. The calculation involves subtraction of 

two large numbers [evening and morning dry wts,), each of which was 

subject to a 1-4% error. As overnight losses got large, these differences 

became more and mare accurately resolvable. 
Table 4-1. 1. quqd~&cagcla Overnight Losses 

Gross Overnight Loss Pond 

Product. 7. of TO,C Size Hot. 

Date gm/m2/day gm/m2/day Gross min am max m2 #/ml ........................................................................ 

Table 4-2 shows results fromm sequential batches in the 1.4mZ ponds, one of 

which was heated. The collapse of the S.-quad[icayda occurred more quickly 

in the heated pond, which could not be grown to high density in late June. 

No grazers were present. By July when temperatures were above 35OC, it 

could not be grown at all (no data shown). At this time temperatures in the 

unheated pond were also high. As can be gleaned from the table, higher 

temperatures first impacted production at high cell density. 

Overnight losses for Chlprellg are presented in Table 4-3. Here losses were 

low except in a heated 1.4m2 pond, and in the 100m2 pond (1-1) when 

overnight temperature was highest. fit prevailing temperatures respiratory 

losses were 0-10%. Grazing increased losses and was most severe at higher 

temperatures. Productivity results from the 1.4m2 ponds (Table 4-41 

indicate that this organism tolerates temperatures from 12-15OC at night 

and 25-3e0C during the day. Low respiratory losses were measured from 

&cy:~~s-cultures as well (Table 4-51, The organisn grew well in the 1.4m2 

ponds for short periods of time after inoculation (Table 4-61, but did not 

compete well enough to successfully inoculate the larger ponds. 
Table 4-2. S. qgadriggydg 1.4m2 Pond Results 1984 

Dates Days Tmin Tma, pH TDS Density Ave.Prod. Max,Prod, Heated 

OC OC PP~ PPm gm/m2/day gm/m2/day ............................................................................. 

6/12-14 3 13 28 7.5 0.4 116-332 2 2 2 7 - 

6/15-17 3 15 29 7.5 0.4 332-490 14 19 - 

BATCH #2 

BATCH #3 

*Culture 50% dead 

4.2.2 Productivity Profiles 

In several instances productivity profiles were calculated in an attempt to 

determine how the biomass increase was distributed over the daytime. The 

first such experiment was carried out in April 1984 in a S. quadricagda 

culture (Table 4-71, Although production was not even over the day, 

afternoon cloud cover is easily seen to be the reason. The last row of the 

table gives an efficiency calculation, with errors. The errors are actual 

calculations based on the duplicate analysis of cell density. Thus although 

high efficiencies appear to have been obtained in the afternoon (and even 

overnight), the errors are so large that the values calculated are 

meaningless. Half-day averages shown in a later table indicate that in fact 

efficiency dropped in the afternoon. The data indicate that irradiance must 

be greater than 200 ~m'~ to obtain much biomass increase in a dense culture 

0700 ppm). Pond temperatures were below 20°C, but even at low temperature 

and insolation, pond DO was quite high. 
Table 4-3. Chlp~p/la Overnight Losses 

Gross Overnight Loss 

product. % of T,OC RO~. 

Date gm/m2/day ge/m2/day Gross min max 1900 0700 #/ml - 

1 a 4m2 unheated ....................................................................... 

8/3,4 25.5 -1.6 6 +6-3 15 27 24 15 0 

10,11 26.9 -3.7 14 +8-6 16 30 26 16 0 ....................................................................... 1, 4m2 heated ....................................................................... 

8/4,5 17.6 2.1 - 15 30 24 15 0 
Table 4-4. Ch!pyeWha: 1.4m2 Pond Results 

bin bax Ave.Prod. Max.Prod. Density 

Dates Days OC PC pH gm/m2/d gm/m2/d Heated 

Table 4-5. Qpcy.tigi Overnight Respiration - 1 .4m2 P~nd 

Gross Respiratory Loss 

Product, % of T, OC 

Date gr/m2/day gm/m2/day Gross min max 1900 0700 

Table 4-6. Ppgl&ll: 1. 4m2 Pond Results 

Aver.Prod. Nax.Prod. 

Dates Days in bax pH gm/m2/dry gn/2/day Heated 
Table 4-7 

Productivity Prof i 1 e 4125184 2. qytdr jgggdfl 100m2 Pond 

6 PP~ ------C---------------------------------------------------------------- 

4/26 

Time 0700 1100 1300 1500 1700 1900 0700 

Temp, OC 8 12 17 1 0 17 14.5 7 

D 0 

X sat 160 - - - 450 400 310 235 - 

P Pm 18 40 37 2 8 23 - - - 

Density 

P P * 660 698 721 726 732 734 736 

+PP~ 2 2 3 3 3 3 3 

I T 

Lrngleys 16 208 378 488 523 539 55 1 

% Total 3 38 6 9 8 8 9 5 98 100 

differential - 192 170 110 3 5 16 12 

Productivity 

cum. gm/m2 - 7.8 12.4 13.4 14.7 15.1 15.4 

Y. Total - 50 8 1 87 9 5 9 8 100 

differential - 7.6 4.7 1.0 1.2 0.4 0.3 

-------------- diff. Prod. xlo3 4024 2825 9+9 34234 25225 25+25 

diff. Langleys 

fin afternoon productivity depression was observed on 6/13 and 6/15, 1984 in 

the 1.4m2 ponds (Tables 4-BAB, 940). In these ponds mixing intensity 

(~m'~) was much higher than in the large ponds. This resulted in much 

increased gas transfer through the surface and hence lower DO levels. Pond 

temperatures were higher than in April. A disproportionate amount of the 

total yield was obtained prior to 1300 hrs. The effect was greater in the 

heated pond, as best evidenced from Tables 4-9AB, in which overnight losses 

are accounted for, Respiration is a possible cause, increasing as pond 

temperature increased. The effects of DO are uncertain, but as will be shown 

below, prolonged exposure to high DO only moderately reduced productivity of 

this strain in the laboratory. 
Table 4-8A 

Productivity Profile 6/13/84 i. guadrlgggda 

l.4m2 Pond - Unheated 

0.4 ppt 

'Fi me 0700 

Temp, OC 12 

Uensi ty 

PPm 182 

+PP~ 1 

IT 

Langleys 0 

XTotal 

differential 

Productivity 

cum. gm/m2 o 

%Total 

differential 

~~ftl-~rp~r_x lo3 

diff. Langleys 
Table 4-80 

Productivity Profile 6/13/04 9. gLtt@t-i~ag@fi 

1.4r2 Pond - Heated 

0.4 ppt .................................................................. 

Ti me 0700 1200 1400 (6/14) 0700 

Temp, OC 17 31.5 3 1 18 

Density 

P P 264 300 310 

?PP~ 2 3 1 

1 T 

Langleys 0 266 426 

%Total 3 0 6 9 

differential 266 160 

Product i vi t y 

cum. gm/mi! 0 9.7 14.5 

%Total 0 53 7 9 

differential 9.7 4.0 

diff. Prod. x103 3625 30:b 15+5 

diff, Langleys 
Productivity Profile b/15/04 S. gyad~icgyda 

1.4m2 P~nd - Unheated 

0.4 ppt -11------1111-11--1-------"----CCICI-ILLL------"------------~------1-- 

Time 0730 1300 1700 1900 (6-16) 0700 

Temp, OC 14 27 28 27 14 

Density 

PPm 332 372 395 394 - 

tFJpm 2 2 4 0.5 

IT 

Langleys 17 336 623 683 69 1 

%Total 2.5 49 90 99 100 

differential - 319 287 60 8 

Productivity 

cum. gm/m2 - 10.6 - 16.7 lb,5 

%Total - 63 - 100 

differential - 10.6 - 6.1 

diff. Prod. #lo3 33t4 

diff. Langleys 
Table 4-9B 

Productivity Profile 6/15/84 5, guadrjcayda 

1.4m2 Pond - Heated 

0.4 ppt 

Temp, OC 25 

DO 

%sat 130 

PPm 10.6 

Density 

PPm 428 

!PP~ 6 

1 T 

Langleys 17 

%Total 2.5 

differential - 

Productivity 

~um,~m/m2 - 

XTotal 

differential 

------------- diff. Prod. xlo3 

diff. Langleys 
In tables 4-8A0, the last row indicates that photosynthetic efficiency did 

not decrease at peak irradiance (1200-1400 hrs) but did decrease 30% 

thereafter. A halving mf efficiency in the afternoon relative to the 

morning is again evident on 6/13 (Tables 9AB). Table 4-10 summarizes the 

it prof i l@~ from 6. ggsdricgudg cultures (and one finkish~gdgsnyi 

culture). PM depression appears to correlate with pond temperature, with a 

greater decrease in efficiency occurring during warmest afternoons. 

Table 4-10. Split-Day Praductivities 

1st Half Photosynthesis 2nd Half Phatosynthesis 

Date T, OC IT P (PII~)X~O~ iT P (P/I~)x~o~ 

0700 1300 1900 lang. p/n2 gm/m2 lang. gm/n2 gm/m2 

lang. lang. 

" cloudy afternoon 

* @nki~tydgpfiug culture. All others S. quad,migauda 

x Heated pond 

4-3. LABORATORY TEMPERATURE AND DISSOLVED OXYGEN EXPERIMENTS 

Experiments were conducted in the laboratory in 1L Roux +lasks both with 

strains cultivated outdoors and with strains of importance to the ASP, In 

this section, the effects of temperature and DO on net productivity are 

discussed. 

Table 4-11 shows culture conditions and results of "control" experiments. 

Reproducibility of these experiments was good as evidenced by the small 

standard deviations relative to the means. The relative yields from cultures 

indoors was similar to outdoor results. 
Table 4-11. Roux Bottle Cultures - Controls 

Organism pC02 blk,mM pH T,OC ~m'~ gm/m2/day Eff ,% TDS,ppt n ............................................................................. 

S.quad. 1 5 8.0t.3 30 2 0 13.5i0.5 12.0 0.4 2 

Ankis. 1 20 8.22.3 25 2 5 10.0+1.4 11.3 4.5 6 

Isochysis 1 5 8.0+.3 25 25 8.0+0.5 9.1 2 8 5 

Docystis 1 20 8.32.3 30 25 10.0+0.5 11.3 4.5 2 

Chlorella 3 2-30 7.5-8.2 30 3 5 16.4+0.3 10.3 4.5 3 

Figure 4-1 depicts laboratory results using S. ggadriiagdg. The results are 

presented in order of descending productivity relative to the control. At 

0.4 ppt TDS (freshwater), productivity was highest at 30°, reduced 207. at 

25OC, and practically nil at 35O (data not shown: cultures were not 

sustainable at 3S°C, constant temperature). DO in these airIC02 grown 

cultures was 100-125% of saturating. Increasing salinity to 4 or 9 ppt, 

reduced productivity 15% at 25OC, and also lowered the temperature 

optimum. Productivity was reduced another 15% at 30°c, 4 ppt relative to 

the control. hgain, at elevated salinity and 3S°C, cultivation was not 

sustainable. Sparging with 02/C02 brought DO to a little above 500% of 

saturation. It reduced productivity over 20% at 30O~, 0.4 ppt but had 

little further affect on yield at elevated salinity and 30O~. Thus we can 

summarize by saying that only small effects were measured when salinity was 

increased and/or DO was high, and/or temperatures maintained between 

25-30°C. The most relevant result is that the strain would not grow at 

35Oc. 

Figure 4-2 shows results' from ChigMglia cultures. The pH effects will be 

discussed in the next section. With this organism, DO had a much greater 

effect, reducing productivity 50% during the first batch, 85% during growth 

of the culture after dilution, and (data not shown), killing the culture 

thereafter. However, switching back from 02 to air, led to swift and 

almost total recovery. Figure 4-3 shows the same results with 

flnkjstrgdpssgs. However, one important difference was the failure of this 

strain to recover when switched back to air. With both organisms, partial 

bleaching at pigmentation accompanied growth under pure oxygen. The 

flnkistrodgpfiuq failed to "regreen" when switched back to air, as the 

Chl~rella did. 

Figure 4-4 shows a similar experiment with Gp~ygtj~. 02 inhibition of 

yield was faster, occurring to greater extent during one cycle. This 

organism grew at 35OC but not at reduced pCO2. 


4.4 CONCLUSIDNS 

The strains responded differently to various temperature and oxygen 

stresses. 5. gyadrjcagda tolerated high DO, but exhibited a temperature 

limit of around 30°C. In the outdoor ponds it grew well a11 year except 

when pond temperatures were highest in July and August 1984. The ch~pr~~~a 

tolerated high temperature (up to 40°C in outdoor 1.4m2 ponds), was 

inhibited by constantly high DO, but recovered in fluctuating DO regimes. 

The ggcystip was greatly inhibited by DO. It was cultivatable in 1.4n2 

ponds (DO = 200X sat.) but not in 100n2 ponds (DO = 500% sat.). The 

flqkjgtygdeggug was also sensitive to DO and exhibited poor ability to recover 

[resynthesize pigment) when switched from stressed to non-stressed 

conditions. Its performance in outdoor growth units was variable and subject 

to upset. It competed poorly, 

Cultivating 2. gg$dricaydg at temperatures greater than 30% led to an 

accumulation of dead cell debris in the clumpe. Thie accumulation of 

detritus, which is known to be substrate for rotifer growth, along with 

higher rotifer activity at increased temperatures may explain the lack of 

resistance of the 2. ggid~i'gu~g to rcttifer attack. The Chig~ellg was much 

less easily grazed upon. 

Respiration and grazing appear to significantly limit net yield from some 

algae. High DO and photooxidation may also limit yieldg in mass culture. 

Screening under fluctuating conditions in which stressful conditions are 

realistically tested should eliminate strains that respire too much at high 

temperatures, exhibit low resistance to grazers (which may be related to high 

respiration rates), or are inhibited by high DO and light levels. Many of 

the pre-selected strains tested during this project did not tolerate high 

temperature, high DO, and/or high light intensity. 
SECTION 5.0 

SALINITY, MINERAL COMPOSITION, AND RECYCLING 

5.1 INTRODUCTION 

The mineral composition of the medium is an important factor in terms of 

species competition and suitability of strains in systems in which the 

composition may vary in time and space (i.e., well by well). Several of the 

strains were investigated for their tolerance to changes in total salinity 

and in composition of minerals. In addition two experiments were performed 

with recycling of clarified effluents to detect an short-term effects. One 1 of these experiments used hkjsfktodgsfiuz in a 1.4m pond indoors. The 

other used S, qupd~icagda in a 200m2 pond outdoors. 

5.2 TOTAL DISSOLVED SOLIDS TOLERANCE 

quadricauda, enkistrgdesmu~, and Ispchryqig qaibqna IUTEX 987) were tested 

for tolerance to different levels of overall salinity. Each displayed 

tolerance to the levels tested, however this did not guarantee a high level 

of competitiveness at each TDS level. For instance, it was difficult to 

culture lggghryssg in open, 1.4m2 tanks at 7 ppt TDS due to the rapid 

invasion by figkiqfrgdqq~gs. Of course the low productivity of Ippchrysjs 

even at 28 ppt would result in a saltwater strain taking over in intensive 

open culture. 

Table 5-1 shows mass culture result5 from 2. qgqgri-qgga and enkigfrpggdmg3 

cultures during the spring of 1984. The S~gfigdepgus grew about as well at 

TDS 4.0 as in freshwater, and somewhat less well at 8.5 ppt. As was shown in 

Section 4.0, however, its high temperature tolerance decreased as salinity 

increased. This was evidenced in the outdoor work by the order in which 

culture productivity decreased with time, leading to subsequent loss of the 

culture. As summer arrived, first the 8.5 ppt culture died, then the 4.0 ppt 

culture and then the freshwater culture. The !nkfgtrgdggmup grew as well at 

4.0 ppt as 8.5 ppt, but could not outcompete the Sgqfigdggmgg at either. It 

took over both finksgflodgggg? cultures within four weeks. 

Salinity tolerance of I~och~y~s~ is shown in Table 5-2AB. n represents the 

number of sequential batches. At all TDS levels, photosynthetic efficiency 

dropped as irradiance increased. The closed, axenic 1L cultures were 

relatively easy to keep going. However, lag times upon reinoculation of each 

successive batch were long unless initial densities were kept high 0250 

ppm). The open, 1.4m2 ponds were very difficult to operate due to lags, 

erratic growth, and contamination. 
Table 5-1. Productivity and TDS - 100-200m2 Ponds, 1984 

Month Organism 0.4 ppt 4.0 ppt 8.5 ppt 

March Scenedesmus 

Clpr i 1 Scenedesmus 15 

bnkististradesmus I) 

Hay Scenedesmus 20 1 b 13 

Table 5-2A. lsochrysi~ Productivity and TDS - 1.4m2 Ponds 

IDS Aver. Prod. Eff. Max . Prod, Max. Density 

gn/n2/dry % Vi5 gm/m2/day P Prn -----------------------------------------------*--------------- 

n = 2 20 6.32 .8 2.8 ltim i 550 

n=2 7 6.9t1.7 3.0 9.8tU.4 700 

I. = 125 wmW2 = 125 Lanqleys/day, L:D:112:12 

Table 5-20. 1:~chyysis Productivity and TDS - IL Cultures 

TDE Prod. tSD Eff. Iu 

gn/r2/day % vis wmm2 L------L"III--------------------------o-------------"~-~--"-~~-v"---~-- 

n=9 28 8.6+1.4 9.1 25 

n=7 2 1 8.3+2.1 0.8 25 

n=? 7 5.8to.9 6.1 25 
The maximum density that 1.4m2 pond cultures of hgchrygjq reached was less 

than that of other algae grown under similar conditions, Data were taken to 

measure loss of biomass during the twelve hour dark period, and excretion of 

products into the medium. Table 5-3 shows these data from an hgchyyqsg 

culture (28 ppt TDS, 125 illumination), and an fiqkrgtrigdgqmys culture 

for comparison. Both nighttime respiration and excretion of metabolic 

products were much greater in the 1. gglbpng culture. Both cultures were 

grown under N-sufficient conditions. When particulate biomass (ash-free) of 

1- qglba~g reached 400 ppm, the dissolved levels of protein, CHO and TOA 

(total organic acids) all together were more than 40% of that. During this 

time, along with production of 100 pprn of particulate biomass, almost 60 pprn 

of carbohydrate and organic acids were excreted into the medium. As shown in 

Table 5-3, no measurable protein was excreted by either organism. Although 

extracellular levels of carbohydrate, produced by 1, qglbana constantly 

increased, organic acid levels increased and decreased with time. 

Table 5-3. Losses From 1.4 m2 Pond Cultures 

,fiehjst~oggszug --- I. qalbaria --- 

(125 wmm2) (125 ~m'~) ......................................................................... 

Production during light period 16.2 4.7 

Loss during dark period* -3.6 -2.2 

Net 24 hr production* 12.6 2.5 

Biomass density, VSS ppm 600 400 

Dissolved protein, ppm 0 0 

Dissolved CHO, ppm 2 6 0 

Dissolved organic acids, ppm 2-30 90-1 15 

TOA/Biomass 0.01 0.40 

CHO/Biomass 0.01 0.20 

Protein/Biomass 0 0 

5.3 MEDIUM COMPOSITION AND CULTURE REQUIREMENTS FOR hNKISTRODESMUS 

The figkiqfrpdeq!ys strain was grown in 1.4 m2 tanks to determine the 

requirements for adequate growth and thus the cost - productivity trade-offs 

for media constituents. The data obtained indicates that expensive media 

inclusions, such as trace metals, EDTA, and alkalinity need not be added in 

large amounts. Indeed trace requirements can be supplied by the water 

resource, EDTA is necessary in only small amounts ((10 uM) or not at all, and 

alkalinity tolerance is broad (at least 5-20 mM). Problems were encountered 

in attempting to study long-term growth of this organism: as semi-batch 

cultivation endured the culture degenerated, losing its productivity and 

competitiveness and as light intensity was raised to 40% solar, this 

deterioration accelerated. 
Ihe first series af experiments compared growth at ~everal light intensities 

and pH regimes. Table 5-4 shows productivity data vr. light input and pH for 

healthy, dispersed cultures, From the table it is evident that cultivation 

at pH 7 resulted in the highest productivity. H~wever cycling pH between 

6.5-9.5 was nearly a5 satisfactory, Productivity was lower at pH 0.7. 

Figure 5-1 illustrates productivity vs. light input for healthy, dispersed 

cultures at optimal pH and at temperatures equal to 26t2O~. The average 

productivities (averaged over the entire batch cycle) show the saturation 

that would be expected in a turbulent, but not intensely mixed system (mixing 

speed approximately 45 cm/sec, 1.5 fps), Maximal productivities (averages of 

the maximal production of each batch) show this to a lesser extent with 

basically a steeper linear increase at low light vs, high light. This 

leveling of productivity vs. light input became very pronounced for both 

average and maximum productivity as the irradiance was increased above 200 

urnm2. In addition, clumped @fikigirgdg:nug cells competed very poorly with 

contaminants at the higher light intensity, Table 5-5 shows productivity 

data a5 a function of media composition, Lowering EDT4 to 13 uM l.2 mole 

EDTA : 1 mole Fe) from 130 uM (2 mole EDTA : f male Fe) had little effect. 

It might not be expected that such a short-term lawering (1 sequential batch) 

would have much effect on availability of trace metals, but even over the 

long-term (5 sequential batches) lowering the EDTA did not lower productivity 

in conparition to a long-tern control (fable 2-11, Since iron was near yield 

limiting levels in the medium (4 ppm as Fe for 1500 ppm biomass), even 

short-term reduction in EDTA, if important, should have had an effect. 

Table 5-4. tight intensity - pH: finkiztrgdggg~g 

n Langleys Avg Avg . Avg . Max. 

Prod. Eff. Prod. PH Media Cond. ----------------cc------------------------------------------------------------- 

4 00 60 12(10) 8.2 14(12) 6.5-9.5 Basal D 

3 80 60 8(7) 5.5 14(12) 8.7 Basal D 

4 120 100 lb(14) 7.3 23(20) 63-93 Basal D 

1 120 100 19(161 8.7 23 (20) 7.1, Basal D 

4 120 100 14(12) 6.4 19(16) 8.7 Basal D 

2 125 125 15 6.6 20 7.0 Basal+ 80% D 

2 175 175 20 5.5 30 7.0 Basal D 

1 225 225 20 4.7 25 7.0 Basal+ D 

See Table 5-5 for media used. D n dispersed. 

Values in ( ) were normalized from L:D cycle of 14:10 to 12:U 

Table 5-5. finkistrodesmus: Medium Composition Experiments 

Sequen. Lang./day Ave.Prod. five.€+ f. Max. Prod, Culture 

Batch (~m-~) gm/m2/day X vis gm/m2/day Medi um ~ondi ti on* 11---1-------------~--~----------111111111111111----------0~~---111--111-----"---"----~--~ 

EDT4 Experiments ............................................................................ 

Initial: Control: 

1 175 20 6.2 30 125um Na2EDTA D 

1 175 20 6.2 28 i3uM NazEDTA 

Long-term: Control : 

2-6 175 14.2 4.4 20.0 125uM 75XC 

2-6 175 13.b 4.3 17.4 13uM 75%C 

----------_---------------------*------------------------------------------- 

N-Addition Experiments -"-------11-----1-1-----------------"--------------------------------------- 

1 175 16 5.0 20 Control 25 

1 175 15 4.7 2 1 Dosed Urea-daily 25 

i 179 12 3.0 22 Dosad NO3 daily 25 Illl--CI""-oI-----l-----------------------"-----m--"------------------------ 

TDS Experiments ............................................................................ 175 15 4.7 25 Control 90XC 

175 15 4.7 2 1 Zx salts, Urea 30%C 

175 12 3.8 22 28 salts NO3 90XC 

Trace Metals Experiment 

15 b. 6 20 Control D 

14.3 6.3 19 10% trace D 

13,O 5.7 17 4X trace D 

*D = dispersed C + clumped 

Elevating baron and floride levels (to levels in Pyramid Lake from which the 

fl~~~~V~[g~~~~g' was isolated) also shcwed no difference from the control. 

Bioassays als~ indicated that low levels of these elements were not 

responsible for culture deterioration. Adding urea incrementally (1-2 RM per 

day) was no better or worse than adding it all at once (10 mM urea-N 

initially). Nitrate could be substituted for urea. Daubling the levels of 

NaC1, KC1, CaC12 and MgCl2 had no short-term effect. Neither did lowering 

only Hg levels. Growth of cells which were obtained from the outdoor ponds 

and thus grown for a prolonged period of time on trace metals available in 

the water supply only (10-50% of lab levels) was as good as controls. 

Reduction of alkalinity levels from 20 mM to 5 mM did not change 

productivity. In the short term firtki-ttygdesgus grew equally well over a 

range of salinity, mineral composition, alkalinity, trace metal and chelator 

concentrations. In addition, growth in outdoor ponds and at low 
light (30 continuous) with and without vitamin supplementation 

indicated that vitamins are not required for productivity up to 20 

gm/m2/day. 

As culture degeneration appeared to be a serious problem, data was obtained 

to document it and bioassays were performed (under low lights in 1 L bottles) 

to determine whether it was reversible. 

Decreases in productivity were associated in every instance with cell 

aggregation into clumps. As indicated in Table 5-5, as clumping increased, 

productivities of control cultures decreased. Eventually the cultures 

failed, i.e., productivity declined precipitously and contaminants grew. 

These events became typical, No significant levels of extracellular products 

were measured. 

Table 5-5 (Continued) 

Sequen. Lang./day Ave,Prod. Ave.Eff. Max.Prod. Culture 

Batch l~rn'*) gm/m2/day 7. vis gmlm2/day Medium ~ondi tion" .............................................................................. Alkalinity Experiments .............................................................................. 

1-2 125 15 6.6 20 20mH Alk (Control D 

1-2 125 15.6 6.8 2 1 5mM Alk D 

1 125 13.3 5.8 15 1.5mM Alk .............................................................................. High Light Intensity Experiments .............................................................................. 1 225 18.6 4.5 23.5 5mM Alk (Control) Good 

2 225 13 3.2 2 1 5mM Alk Bleaching 

3 225 Dead --- 5mM Alk 

1 225 14.4 3.5 2 3 10mM Alk (Control) Good 

2 225 16.3 4.0 2 2 10mN Fllk Bleaching 

Clumping, 

Debris 

3 225 Dead 10mM Alk .............................................................................. Magnesium Experiments .............................................................................. 

1 225 16 3.9 24.4 lOmM Alk, GOO~ 

0.5mM Mg 

2 225 14.3 3.5 22.6 l0mM Alk, Bleaching 

0.5mM Mg Clumping , Debt i s 

3 225 Dying --- l0mM Alk, 

0.5mM Mg 
4 new culture of the fiekiztrgggg!g~, was obtained tfrom Dr. W8 Thomas) and 

grown. It was, as before, initially mostly dispersed. Idhen grown at 25 

wmm2 continuous illumination, the cultures became about 20-30X clumped. 

However, when grown at 150-250 wma2 with a 12 hr. light cycle, the cell5 

became more clumped ae the number of sequential batches increased, The same 

medium was used in both cases. Thus, it appears that high light intensity 

may be responsible for deterioration of the culture, Cultures grown at 225 

wmM2 deteriorated faster and further than those grown at lower light 

intensity (Table 5-51. By the second batch, bleaching was evident and by the 

third batch the cultures were dead, In order to determine if a nutrient 

deficiency was also involved in the culture deterioration, bioassays were 

perjormed. The bioassay5 consisted of (1) growing healthy (dispersed) cells 

in basal laboratory medium, outdoor pond water, and clarified liquid from an 

unhealthy pond! 12) growing unhealthy (clumped) cells in the same media; (3) 

spiking the deteriorating culture liquor with several different nutrients; 

and (4) spiking a healthy outdoor culture liquor with the samE set of 

nutrients, Detailed results of the bioassays can be found in our Interim 

Report to SERI. To summarize the following conclusions were drawn: (1) iron 

was not growth-rate limiting in unhealthy indoor pond media, but it was the 

nutrient that first limited yield; (2) the healthy cells increased themselves 

4-10 fold at similar rates in all media and remained 80% dispersed; (3) the 

unhealthy cells grew as well in all media tested, but remained aggregated! 

(4) no nutrient deficiency was evident from the nutrient-spiking experiments; 

(5) initially dispersed cells farmed clumps during the nutrient bioassays, 

with more clumping evident (90% of the biomass) with added CaC12, but 

increased clumping (50%) under all treatments. Thue, under low light during 

short term assays (7 days), both dispersed and clumped cultures grew the same 

on all media. Furthermore clumping was not found to be reversible by adding 

any particular nutrient, (Basal medium contained 0.25 mH Ca, 1 mM P, 20 mfl 

alkalinity, and pH was 7,2-7.8.) A low light culture was grown with 0.1 mM 

P. It clumped as well8 

Although several factors may contribute to culture deterioration, including 

changes in salt balance especially with relation to KCI, NaC1, and CaC12, 

photoinhibition appears to be the primary cause. Deterioration was fastest 

at high light intensity. Even under low light, very dilute cultures 

exhibited an initial period of pigment bleaching before growth began, 

5.4 RECYCLING CLARIFIED EFFLUENTS 

4~k~~frpdg~~y~ was grown in the 1.4m2 ponds indoors with and without 

recycle of the medium. The pH was kept at 7.553 and the ~llumination was 

diurnal (12112) at 125 W~II-~. The effluent was clarified by centrifugation 

(Sharpies TI, 20,000 rpml, On average 48% of the effluent as returned to 

the pond with fresh medium used as make-up. The culture volume was adjusted 

far evaporation with distilled water prior to removal of the effluent. 
'lable 5-6 shows productivity from the recycled pond and a control pond, as a 

function of sequential batch (cycle) number. The productivity is shown 

graphically in Figure 5-2. As with all of the @ki:t~~dg?gu: cultures, 

productivity from the recycle and control ponds declined slowly with time. 

lable 5-6. flnkigtbodeggus Media Recycling 

P- 1 P-3 

Average Maxi mum Density fiver age Maxi mum Density 

Prod. Prod. Range Prod. Prod. Range 

~yc 1 e gm/m2/d gm/m2/d P pm gm/m2/d gm/m2/d PP~ ............................................................................ 1 2 0 24 660-7 10 2 0 30 160-1300 

Eventually both cultures became contaminated, the recycle pond somewhat more 

slowly. Pond characteristics are shown in Table 5-7. Even after eight 

cycles of recycling, little organic matter accumulated in the medium. 

Although this experiment lasted only five weeks, a relatively short period of 

time, it demonstrates that when excretion of organics into the medium is 

minimal, recycling most of the clarified effluents is feasible, Of course, 

the organism must be, by far, the most competitive in the given medium, since 

more practical means of harvesting the biomass will lend a competitive 

advantage to these organisms which harvest the most poorly. 

An effluent recycle experiment was performed with a mass culture of 

S~e~edeggug for six weeks during June-July 1984. The experiment was 

terminated when this species failed to grow, in non-recycled ponds as well, 

during July. 
In this experiment, pond effluents were clarified via sedimentation in 2m 

deep settling ponds. This could be done because of the large size of the 

flocs (1-2 mm) formed in the growth ponds. Settling pond5 were filled by 

gravity Slow from the growth pond. This took about 2 hrs, Sedimentation 

times varied from b to 12 hrs. Clarified supernatents were pumped back to 

the growth pond using a floating intake. The settled biomass compacted well 

enough to allow return of about ?OX of the efflulent. Fresh medium was 

Table 5-7. Media Recycle Dissolved Species 

Start Finish 

( 12-6-03) (1-13-841 Unit5 

A1 kalini ty 20 23 mM 

Hardness 400 200 ppm CaC03 

CHD 0 14 ppm Glucose equiv. 

Protein 

TDA 

14 ppm Folin-Lowry USA 

equi v, 

--- 

used to make up the difference. Irrigation water, without mineral 

supplementation, was used for evaporative make up water. Urea, phosphate, 

and iron were added to replenish nutrients taken up by algal hiomass 

production. The operational data, productivity, harvested weight, and 

e+ficiencies of harvest are shown in Table 5-8. Clarification efficiency was 

determined by compositing the effluent recycled, measuring hFDW and dividing 

by pond 4FDW. Slurry density was calculated from the biomass differences 

between settling pond input and output and the volume left in the settling 

pond, The culture showed no signs of contamination during the experiment. 

However, as pointed out above, unless the method of separating cells from the 

medium is universally efficient, organisms which are moderately competitive 

but not harvestable would be expected to dominate over the long term. 

5.5 CONCLUSIONS 

Host of the organisms tested exhibited broad tolerance to changes in total 

salinity and mineral composition. Although the chemical composition of the 

medium exerts a strong general selection pressure, it appears that a number 

of strains can grow well over non-exotic ranges of compcsition. Thus the 

maJor selection forces, in a given general medium, will be those conditions, 

and changing conditions of climatic inputs and stresses developed under 

intensive cultivation conditions. 
Table 5-8. 3. ggad~igagda Biomass Harvest--Effluent Recycle 

200 mZ Pond 

Harv. Sed. Effl. Evap. Ave. Harv. 

Harv. Eff. Time Dens. Blowdown loss TDS Hd Alk Prod. wt. 

Cycle Date % hrs, ppm 7, % ppm ppm mM gm/m2/d Kg ............................................................................. 

0 5/27 99 9 9 12 2 2 - - -- -- 2 0 25.1 

* Rotifer bloom, pH raised from 8.3 to 9.3 

TDS, Hd, Alk - initial values for succeeding cycle 

Hd - ppm as CaC03 

The recycling experiments did not show any deleterious short-term effects like 

the fast build-up of autoinhibitors, And no unusual problems of contamination 

were encountered. However, these kinds of questions can only be answered with 

assurance from much longer scale tests. The contamination problems will be 

closely tied to the harvesting technique. 
SECTION 6.0 

MIXING VELOCITY EXPERIMENTS 

6.1 INTRODUCTION 

Nixing isi a critical cost center of any hydraulic syaten. The provision for 

8omr movrmrnt of the rlgnl auaprnoion ia s naelrsity in ardgr te trks 

advantage of the cost savings obtained by limiting the number of paints of 

nutrient input and product recovery, Mixing has also been implicated a5 a 

determinant of photosynthetic productivity. Thus the major question i5 how 

low can mixing power be before productivity is impacted, or more precisely 

what is the optimum of the mixing power-productivity relationship, In this 

section the relationship between randomized, turbulent mixing and biomass 

productivity is discussed. First the methods used to measure channel 

velocity and pawer input are presented along with the results from the 

experimental ponds. Then mixing speed experiments using ggengdgg~us and 

Chigrellg are discussed. Finally the results of mixing vs gas transfer 

through the surface are presented, 

6.2 METHODS 

The total power required to mix a pond at a given velocity was determined by 

measuring both the voltage and current to the DC gearmotor (Badine #185, 1/4 

hp, 6O:l) which drives the paddle wheel, For a given speed setting, the DC 

motor controller provides an almost constant voltage to the mator's armature, 

via its feedback circuitry. The armature current varies directly with motor 

torque, The product of voltage and current represents the power into the 

motor, while the product of speed and torque represents the power out of the 

motor, which is transmitted through the geartrain to the paddle shaft. The 

load (torque) an the motor, especially at higher nixing speeds, is not 

constant, but fluctuates six times for each paddle revolution as each of the 

six paddle blades lifts a new slug of water. The fluctuating current was 

measured with a chart recorder, which monitored the voltage across the 0.1 

ohm resistor placed in series with the motors armature. (The load 

represented by the resistor was in all cases less than 1% of the motor 

load,) The average current was later determined from the chart recordings by 

graphical means, The full armature voltage was measured simultaneou~ly with 

a Beckman Tech 330 voltmeter, which measures the true RMS value of the 

voltage. This was important since DC motor controllers put out a 

non-sinusoidal waveform tespecially at low speeds) which are measured 

incorrectly on non-RMS type meters. 

The total power is useful in calculating the overall mixing efficiency, but 

such an efficiency is strangly dependent on the gearmotor characteristics. 

Small gearmotors are quite inefficient, so the overall mixing efficiencies 

calculated will not be representative of what can be achieved on a larger 
scale. If the actual paddle wheel shaft power were known, then "paddle 

efficiencies" could be calculated. Such a number would be a quite useful 

design tool. Two approaches were taken in estimating the "Net Shaft Power." 

The first was to simply calculate an efficiency for the gearmotor (motor plus 

speed reducer) from the manufacturer's nameplate data. Strictly speaking, 

such a value applies only at full load, and generally declines at partial 

load. The second approach was to measure the power under "no-load" 

conditions, i.e., in an empty pond, and to subtract this from the power 

measured under a load. A comparison of these two methods for calculating net 

shaft power is shown in Figure 6-1, for paddle wheel speeds ranging from 4.3 

to 0.3 rpm, which corresponds to velocities of 15 to 33 cm/sec. 

Since the two agree fairly well, and since the former method tends to 

overestimate net shaft power at lower speeds, the latter approach (using 

"no-load" power) was used in subsequent calculations. A plot of no-load 

power VS. paddle wheel speed for the 100 m2 pond is shown in Figure 6-2. 

Velocity measurements were taken with a Nixon Instrumentation Ltd. Steamflow 

#442 meter equipped with a low speed probe. The probe consists of a very 

small 1'5 am) propeller which turns on a low-friction spindle. The motion of 

each propeller blade past a sensor creates an impedance change which is 

detected electronically. The pulse frequency is averaged over 10 seconds and 

displayed. Readings were later converted to velocities using the calibration 

chart suppliled. (The relation is linear for velocities greater than 5 

cmfsec.) The measurements were taken at the far end of the pond, right 

before the first bend, in the center of the channel. It had been found 

previously that this point was representative of the average velocity in the 

channel. Readings were made at the bottom, middle, and top of the water 

column, and generally increased with distance from the bottom as expected in 

open channel flow. Although some fouling difficulties were encountered, 

especially at velocities below 15 cm/sec, performance was, for the most part, 

satisfactory Ithe instrument is designed for fresh water). Fouling was 

always accompanied by an abrupt drop in pulse frequency, so it did not occur 

undetected. In general, the availability of suitable instruments for use at 

velocities less than 30 cmfsec in shallow water is quite limited. 

Given the channel velocity, the total flow rate can be calculated readily, 

The product of density, flow rate, and head loss is the hydraulic power, or 

energy per unit time required to keep the water in motion. Given the 

hydraulic power, total efficiency, paddle efficiency, and equivalent 

roughness can be calculated. The total head loss is the difference in water 

levels before and after the paddle wheel. Accurate measurements were 

difficult, because the levels fluctuate (again six times per revolution), and 

because the head loss is quite small at low flows. The technique involved 

clamping rulers to an beam spanning the channel, both before and after the 

paddle wheel. With the mixing off, the rulers were adjusted to a convenient 

reference level. The mixing was then turned on, and allowed to come to 

steady state, at which point the mean deviations from the reference levels 

were determined visually and recorded. The differences were then averaged to 

produce a single figure. The paddle wheel RPM was used as a measure of 

channel velocity, since the two correlate quite well, as shown in Figure 

6-3. The minimum velocity for which the total head loss could be confidently 

measured using this technique was 15 cm/sec 10.4 cm). 


6.3 RESULTS 4ND DISCUSSION 

The power and flow measuremente for the 100 m2 pond are summarized in Table 

6-1 and shown graphically in Figure 6-4, Total power measurements were made 

over the motor's entire speed range, corresponding to channel velocities of 

3.8 - 33.1 cm/sec. Values below 15 cm/sec were taken at a different time, 

which may explain the discontinuity between 4.00 and 4.28 rpm. Velocities 

followed by an "i" are interpolated from Figure 6-2. No-load and net shaft 

power are only listed for velocities of 15 cmlsec and greater, Below this, 

the calculated net shaft power approached the probable error in the 

measurements. Part 0 of the table lists the values o+ hydraulic power 

derived from the velocity and head measurements. It is interesting to note 

that the hydraulic power varies as the velocity to the 2.5 power (somewhat 

less than the 3.0 power predicted by open channel flow equations), whereas 

net shaft power increases roughly as the velocity squared. The discrepancy 

is due in part to a drop in paddle efficiency at the lower speeds, as shown 

in part C, The relativelly large paddle clearances (2-3 cm) may account far 

this decline. The total efficiency dropped even more dramatically at lower 

velocities, but thia is due primarily to the inefficiency of the drivetrain 

at partial load. At 33 cm/sec the no-load power is 41X of the total power, 

whereas at 15 cm/sec, it is 57%. 

A final quantity that can be derived from the measurements is an equivalent 

Manning's "n" for open channel flow, which is a measure of channel 

roughness. Typical values vary from 0,012 for smooth concrete to ,029 for 

gravel, The governing equation is: 

where: h~ = head loss, meters (Table 6.1) 

V = Velocity, m sec-I (Table b. 1) 

n = Manning's n, sec m-0a167 

R =Hydraulic radius= 0.17m (d=0.2mf 

L = Channel length = 45 m 

Note: There is no 1.49 factor in SI units, 

Two sets of n values calculated from this equation are shown in Table 6-2, 

In the first, the head losses are used directly, while in the second, they 

are reduced by a factor of ~~/2~ to account for channel bend losses. This 

value was chosen somewhat arbitrarily, since no value of head loss for sharp 

bends in open channel flow could be found in available literature. It is 

consistent with literature values for sharp bends in piping systems. In 

large scale designs, where channel length to width ratios are high, the bend 

losses will be a small percentage of the total head loss. However, the 

100m2 ponds have a low L/W ratio of about 0.3/1, and the bend losses 

probably are a significant fraction. In theory, Manning's n is not a 

function of velocity, while the calculated values shown in Table 6-2 clearly 

are. The value shown for 15.0 cm/sec is probably the least accurate, as the 

uncertainty in the head loss was at its greatest. The values fall within tho 

expected range for a plastic lined pond, 
Table 6- I Mixing Power and Velocity Results 

A. POWER MEASUREMENTS 

Paddl e 

RPM 

----- 

1.07 

1.79 

2.45 

3.09 

4.00 

4.28 

5.66 

7.10 

8.33 

Ave Vel 

cm/sec 

------- 

3.8 

6.3i 

8.6i 

10.9i 

14. 0i 

15.0 

20.6 

26.9 

33.1 

DC 

Volts 

----- 

11.7 

18.5 

24.3 

30.2 

38.2 

42.0 

55.0 

68.0 

82.0 

DC 

Amps 

---- 

0.16 

0.19 

0.20 

0.22 

0.28 

0.34 

0.42 

0. 50 

0.60 

Power 

Watts 

----- 

1.9 

3.5 

4.9 

6.6 

10.7 

14.3 

23.1 

34.0 

49.2 

Total 

Power 

w/sq m 

------ 

0.02 

0.04 

0.05 

0.07 

0.11 

0. 14 

0.23 

0.34 

0.49 

+ 

No-load + 

Power + 

w/sq m + 

-------- + 

+ 

+ 

+ 

+ 

+ 

0.08 + 

0.12 + 

0.16 + 

0.20 + 

Net 

Shaft 

Power 

w/sq m 

------ 

i = interpolated value 

No-Load Power was measured at the same speed setting in an empty pond. 

Net Shaft Power = Total Power - (No-Load Power) 

B. FLOW MEASUREMENTS: 

Average ...................... Paddl e Head Velocity Flow 

RPM cm cm/sec m /sec 

Hydraul i c 

--------------- 

Power 

watts 

----- 

0. 0 1 e 

2.64 

5.80 

10.42 

18.26 

Power 

w/sq m 

0.0001e 

0.026 

0. 058 

0. 104 

0. 183 

Flow is calculated from Velocity x cross-sectional area. 

Hydraulic Power is calculated from the measured Head & Flow 

e = extrapolated value 

C. EFFICIENCIES Ave Ef f i ci ency Velocity --------------- 

cm/sec Total Paddl e 

------ ----- --me-- 

15. 0 19% 44% 

20.6 25% 5 1 % 

26.9 31% IZ 9 ~9/. 

33.1 37% 64% 

Total Efficiency = Hydraulic Power / Total Power 

Paddle Efficiency = Hydraulic Power / Net Shaft Power 
Table 6-2. Roughness Coefficient 

hve Adj. Cldj. 

Velocity Head Planning's Head Manning's 

cm/sec cm " n " c m 'I n I' 

......................................................................... 

15.0 0.44 0.019 0.29 0,016 

20.6 0.64 0.01B 0.42 0,010 

26.9 0.07 0,016 0.50 0,016 

33, 1 1.24 0,015 0.62 0.015 

hdjusted Head = Measured Head - v2/zg (see text) 

b single power and velocity measurement was made in one of the 200 m2 

ponds. At 33 cn/sec, the total power was 96 watts, or 0.48 natts/a2. This 

is consistent with the value of 0.49 watts/m2 obtained in the 100 m2 

pond. The corresponding no-load power wa5 not measured. 

A final point should be made with regard to the data in this section. While 

the channel velocities measured span the normal range for practical pond 

operation, the corresponding head losses all reflect the relatively small 

scale of the system, Production systems will have much larger L/W ~atios, 

with greater head losse9 for any given velocity. This situation could be 

simulated in a small scale pond through the use of obstacles which induce head 

losses. While such a procedure would invalidate the Manning's roughness 

calculations, it would be useful in evaluating paddle wheel performance at 

higher 1 if ts. Any such studies should also incorporate improved paddle wheel 

design, such as those suggested by Dodd C63. These include an increase in the 

number of blades and the offset of adjacent paddle wheel sections to reduce 

pulsations, and a reduction in backflow through carefully controlled 

clearances and the use of a depression under the paddle wheel. 

6.4 MIXING - PRODUCTIVITY EXPERIMENTS 

THO 100n2 ponds were operated at different mixing speeds during May and 

early June 1904, The results are given in Table 6-3, No difterence in 

productivity was observed for the range of mixing speed from 15-27 cn/sec. In 

experiments 1 and 2, the use of turbulence generating obstacles (bricks placed 

across the channel every eight ft.), which partially organized the mixing into 

rolling swirls, did not increase productivity. It did, however, greatly 

increase the power input by increasing the effective Manning's roughness. The 

lower limit of mixing speeds used in this experiment was 14 cm/sec. When 

lower speeds were attempted, the clumped biomass settled in the ponds. Thus, 

about 15 cm/sec appears to be requird to keep flocced material suspended. As 

experiment 3 shows, randomized mixing regimes at high speed (27 cm/sec) did 

not increase the productivity of dense cultures either. 
Table 6-3. +engiegmui Mixing Speed ~x~eriments-100m~ Ponds 

Exp't. Dates T,OC P H Tot. Head Hydr. Prod. Days Vel. TDS 

max /mi n ~/m~ cm w/m2 gm/m2/d 

cm/sec 

............................................................................. 

191-2 5/6-11 30114 8.1-8.7 .34 .87 .11 18 ti 27 3.9 

1, I-l* I1 I1 7.8-8.5 .86 --- ('32) 20 6 15 3.9 

* Turbulence generating obstacles in pond 

( Interpolated values 

A more controlled, broader mixing experiment was conducted in August and 

September 1984 when the Chlgrglla was being cultivated. This organism lent 

itself well to mixing experiments since its small size (3-5u) precluded 

settling, even in practically still water. Thus mixing speeds from <1 to 60 

cmlsec were tested. Also, the level of DO, which decreases as mixing speed 

is increased in this range, was kept constant. This was accompli5hed by 

recirculating the suspension at one location in each pond, through a 113 hp 

pump and back into the pond via a two inch PVC pipe across the channel bored 

with 318 inch holes on six inch centers. The water leaving the pipe was 

directed straight downward to minimize the effect on average mixing speed. 

The use of this deoxygenatinq device, wind mixing, and twice a day mixing of 

all ponds for sampling, explains why the lowest speed used was (1 cmlsec 

rather than zero. 

Table 6-4 gives all the pertinent data from the experiments. The major 

result, productivity vs. mixing speed is shown in Figure 6-5. It's clear 

that for productivity up to 25 gm/m2/day and mixing speeds from 0 to 30 

cmlsec there is no dependence of productivity on mixing speed. This result 

really is not surprising for slow uncorrelated movement of cells in the water 

column. Each cell responds to the slowly changing irradiance it is 

receiving. The turnover of cells even at the lowest mixing speed was 

apparently sufficient to prevent cell death due either to prolonged exposure 

to the highest irradiance or to total respiratory decay in the darkest zones 

at the pond bottom. These cultures grew to maximal densities of about 600 

PP'". 

Table 6-4. Ctilgrgllg Nixing Velocity Experiments 100m2 Ponds 

Mixing Hyd. Aver, Max. fiver. 

Vel. Power Prod. Prod. Insolation T, OC Duration 

Exp't cm/sec ~m'~ gm/m2/d gm/m2/d Langleys/d min-max Days ......................................................................... 

Maximum DO at 30 cmlsec 220% sat., 14.5ppm 

at 3.7cm/sec 285% sat., 19.0ppm 

The low productivities at 60 cmlsec were due to rather abrupt decrease in 

pond temperatures which occurred in late Sept. and early Oct. 1984. 

6.5 MASS TRANSFER THROUGH THE SURFACE 

The mass transfer coefficient, KL, was measured at two mixing velocities: 

15 and 30 cm/sec. The measurements were done by measuring the rate of 

decrease of total carbon from a 100 m2 pond with known alkalinity. The 

carbon level was calculated from the carbonate equilibrium equations with 

constants adjusted for temperature and ionic strength. pH was measured using 

a Radiometer pH meter capable of accurately measuring .01 pH units. The pH 

measurements were monotonically decreasing. The range of pH, 7.4 to 7.2, was 

chosen to preclude effects from back pressure. In the given system, the 
driving force of C02 in the bulk liquid was about 0.61 mmolar. Air 

equilibrates to about 0,01 mmoler, The resistance coefficient was calculat~d 

a5: 

KL = transfer rate I(C10-10): meters per hour 

for 30 cm/sec,, KL = 0.1 a/hr. For 15 ca/sec, KL = 0.030 m/hr. 

Calculations +or the mass transfer caefficients under pond conditions were 

made by Weissman 171, using empirical correiatians derived from channel and 

stream aeration studies C83. The results yielded an equation of the 

following form: 

S hydraulic slope 

V = mixing speed, ft/sec 

d = depth, ft, 

Substitution of the relevant numb~rs yields the following values for KL at 

30 and 15 cm/sec respectively: 0.027 and 0.0124. Although the theoretical 

values are high by a factor of 3.453 the relative ratio is off only by 20%. 

Assuming this relationship to be true, Table 6-5 gives outgassing 

coefficients as a function cf mlxing velocity, It can be readily appreciated 

that high mixing velocities (or equivalently high turnover rates of bulk 

liquid to the surface) will result in very high outgassing coefficients, The 

relationship between mixing and C02 loss will be explored in the next 

section in which the expected Levels of dissolved CU2 are discussed, 

6.6 SUMMARY 

Channel velocity and mixing power measurements were made over the range oC 

3.8 to 33 cm/sec, A flanning's roughness of about 0.01b was calculated far 

the large, plastic-lined ponds. The overall efficiency of the paddlewheel 

system varied with speed, being almost 40% at 33.1 cm/sec and 20% at 15 

cm/sec. 40-50% efficiency is considwed close to the maximum practically 

achievable. 

The dependence of hydraulic power on channel velocity was just less than 

cubic, basically confirming the steep increase found in "ideal" open channel 

flow, 
Table 6-5. Outgassing Coefficient vs Mixing Velocity (d = 20 cm) 

KL, m/hr V, cm/sec 

,012 5 

,026 10 

,040 15 

,063 2 0 

.lo 30 

.22 6 0 

The production of biomass was found not to depend on channel velocity over a 

large range of the latter. Thus other considerations, like carbonation and 

particle suspension will determine mixing speed. The cost of mixing is very 

low at low mixing speed, but increases quickly. At 15 cm/sec, mixing power 

required (at 100% efficiency) was 0.026 ~m'~. At 24 hr/day mixing and 20 

gm/m2/day algal productivity, this is equal to about 0.03 kw.hr/kg algae or 

about $.003/kg algae produced, On an energy basis the mixing power required 

represents only 0.5% of the heat of combustion of the algae output. At 30 

cm/sec these numbers changed to $.02/kq algae and 3.3%. 
SECTION 7.0 

CARBONAT ION 

7.1 INTRODUCTION 

Carbon dioxide input is another critical cost center of algal biomass 

production. The cost is determined by the actual cost of COZl the capital 

cost of distribution and injection, and the efficiency of its use. ?he 

subjects of this section are the factors which determine this efficiency. 

They include injection efficiency, outgassing loss, and the physiological 

response of the organism to pC02, pH, and alkalinity. 

7.2 INJECTION EFFICIENCY 

Injection lo~ses were based on three experiments detailed in Table 7-1. The 

procedure used included carbonating the ponds and recording initial and final 

pH, temperature, and alkalinity. After calculating the difference in total 

inorganic carbon, the outgassing loss, based on mass transfer coefficients 

measured previously was subtracted. The experiments were performed in 

irrigation water with alkalinity added, attaining the total alkalinity 

specified in the table. Injection efficiencies of 57%, 571, and 66% were 

measured. For subsequent calculations the value of 66% was used since 

conditions most closely resembled the ones which yielded this result, The 

injection efficiencies obtained are much high~r than one would normally 

expect sparging into a water column of only 20 cm. The mass transfer across 

a bubbke surface is very sensitive to bubble size. Although an equilibrium 

size of 1 cm is usually attained, it takes about 1 m of rise for equilibrium 

to be reached. Thus shallow sumps are more efficient, per meter o+ depth 

than deeper ones. 

Table 7-1. C02 Losses at Injection 

Pond Inflow 

Size 7 Alkn pHi pH+ CTt-CTi Rate Cin* Effic. 

mz OC rn~ 1101 es LPM moles x ---II--II"C-------L------I-------.-.---------------"----------------------- 

*Corrected for outgassing 
7.3 C02 UTILIZATION EFFICIENCY 

Ihe overall C02 utilization efficiency was measured by two methods. In the 

first method, the biomass production from 2 X 100m2 plus 1 X 200 m2 was 

totalled over 42 days of operation (518-bl20) and then divided by the total 

C02 used, as measured by the difference in the CU2 meter readings on the 

six ton C02 tank (Table 7-21, The 3.3 kg C02/kg AFDW corresponds to a 

30% efficiency, or 54% of that theoretically achievable. This includes 

injection losses and outgassing losses, but does not account for urea-derived 

C02. The latter only contributes 5% of the carbon input and thus could 

maximally reduce the efficiency to 29% overall or 52% of theoretical. 

Table 7-2. Overall Carbon Dioxide Utilization Efficiency 

518-6/20, 1984 

1. Algal biomass produced in 400 mZ of ponds: 242 kg AFDW 

2. C02 level drop in six ton tank 795 kg C02 

3. Urea carbon: (242/10/0.46) X 12/60 = 10.5 

Equivalent COZ: 10.5 X 44/12 39 kg C2 

4. (Algal biomassICO2 input) X 100 29% 

5 Theoretical 1/1.8 55.6% 

6. % Theoretical attained 52% 

7. C02 Requirement: 7971242 3.3 kg/kg 

In the second method, C02 meters and timers, which were installed on the 

100 m2 ponds in August 1984, were used to tally the total C02 input to 

the ponds. The equivalent algal biomass that the total daily CU2 input 

could have supported (based on 1.0 kg C02/kg algae) was calculated. The 

actual productivity was divided by this number, The results are shown in 

Tables 7-3,-4. Also shown in these tables are the losses used for injection 

and calculated for outgassing under the stated conditions. 

There was substantial variability in the overall efficiency (SD 10-25% of 

mean values). Thus the total accounting of C02 (into algae, lost upon 

injection, and outgassed) is only very approximate. The injection loss was 

large, but this should be reduced easily to 5% or so by proper design of a 

carbonation station. Not unexpectedly, overall efficiencies were greatest at 

lower mixing speeds where outgassing is reduced. Thus the interplay between 

algal productivity, mixing velocity, and dissolved C02 concentration will 

determine the economics of LO2 utilization. 
Table 7-3. C02 Partitioning 100m2 Ponds 

Overall 

Mixing Aver, CU2 in Effic, In j, Outgassing Tot. 

cn/rec qm/mZ/ci gm/m2/d x LOSS x Loss x x ~-~----C-~--~~-~~~l~I~..LLI"r1*I-.c.CCCICC~C~~~,

30 22.9 49.8 48.4 34 20.7 103.1 

n=7 2 "J4 t12.9 t13.7 

pH 7.5 Alk = 5 mH 

KL a 0.1 m/hr for 30 cm/~ecl 0,030 for 13 cm/zec 

Table 7-4. C02 Partitioning with Oxygen Desorber 

Overall 

Hi x i ng hver C02 in Effic. Injl Outgassing Tot, 

cm/sec gm/m2/d gm/m2/d X loss % Loss Y, % 11---1-,---w------1~1-"-----""-----""-~~-w------------ww~~-w,,----------- 

30 18.9 42.9 43.8 34 22.4 100.2 

n=5 4 4.9 : 4.9 tiO.0 

pH = 7..5 Alk = 5 mM 

KL = 0.1 m/hr for 30 cn/sec, 0.02 for 3.7 cm/sec 

An interesting feature of the data is that the mechanism used for dcsarbinq 

oxygen had apparently little effect an carbon mass transfer. This, too, was 

expected, since it was obseved that the oxygen desorbed was mainly from 

nucleated release of supersaturated U2 and not mass transfsr. 

Measurements of DO before and after the operation ~f the desorber fur 10 

minutes showed that DO decreased 15X, but pH measurements showed no decrease. 
7.4 PHYSIOLOGICAL ASPECTS OF C02 UTILIZATION 

Since outgassing of C02 is so dependent on dissolved C02 levels, and thus 

can be very high or very low, experiments were conducted to investigate the 

growth response of the Chlg~glia as a function of dissolved C02, pH, and 

alkalinity. Since alkalinity is usually a given, the pH of operation will 

determine the C02 level. But experiments with differing alkalinities were 

required to yield specific C02 levels independent of pH. Fortunately this 

organiam exhibited a very broad range of tolerance to alkalinity, as 

discussed below. 

The iL Roux bottle apparatus was used in these experiments. Temperature was 

30+1°c. Illumination was continuous at 40 ~m'~ but only from one side of 

the bottle. Gas transfer rates were measured and made sufficient to keep 

dissolved C02 essentially in equilibrium with the inflowing gas phase for 

PCO~) 0.025. That is C02 input was an order of magnitude greater than 

maximal uptake at these pCOZ levels, For experiments in which air was 

used, even at 3 LPH with several bubblers, input could not keep up with 

demand. Of course this was observed as an increase in measured pH. The 

actual concentration of dissolved C02 was estimated by input minus demand. 

The demand of the cultures was quite constant over a 3 to 4 day period. Only 

at the beginning, when cultures were dilute, was input much greater than 

demand. Thus maximum specific growth rate results and average production 

results obtained from the same culture are referenced to different C02 

levels, e.g. ,0331 and ,017. respectively. 

The results of over 30 experimental runs are shown in Tables 7-5, 6, and 

Figures 7-1, 2. Reproducibility was quite good. Neither maximum specific 

growth rate nor average production depended on alkalinity. Both were 

dependent on pCOZ and pH. However, production was more sensitive than 

Average production was more dependent on C02 level than pH. At 22 C02, 

pH hardly mattered between 7.0 and 9.0. At 0.5% C02 there is some dropoff 

at pH >9.0. There is no significant difference between productivity at 0.5% 

and 2%. The lowest COZ, 0.01% (one third air equilibrium) resulted in the 

lowest production at all pH, but still showed (as in the case of 0.5% COZ) 

a 25% decrease at pH )9 relation to pH 7, An important conclusion is that pH 

cannot be used as a variable independent of pC02, unless care is taken to 

vary the two independently. This is not usually done in "pH optima" 

experiments. In this experiment the effect of pC02 on productivity is seen 

to be more pronounced than that of pH, 

The same was found to be true of umax, but to an even greater extent. 

There was no correlation between um and pH, so all of the data is grouped 

by pC02 only. Even the dependence on C02 level is less pronounced, with 

only a 25% reduction in umax compared to a 40% reduction in productivity as 

pC02 is decreased. Maximum specific growth rate determinations are not 

good predictors of productivity. 

70 

Table 7-5. Chlg~glla Product~vitv vs pCU2, pH, Alk. 

oLb2 P H kver. Prod. S, G. klk. n 

7. gm/ m2/ aav m M 

......................................................................... 

2 7.0-9.0 19.1 2.1 2-15v 9 

2 1.0-7.3 20. 4 3.3 2-5.3 3 

2 8.0-6.5 19.4 0.4 5,25 2 

2 9. 0 17.9 1.1 100-150 4 

Table 7-6. Chlq~elia Maximum Specific Growth Rate vs pC02, pHe Ulk. 

PCOJ P H Urnax 5. D. Alk. n 

7. d - m N -------------------------------------------------*---------------------- 

1,2 7.0-Y.31 2. 80 U.22 1 - 100 4 
Some CuZ. pH experiments were pertormed in the 1.4 m2 ponds outdoors 

uslno the ghig[glh {fable 7-71. Pr~ductivdy was equal to narmallv operated 

cultures (pH 7.5, Alk = 5 mH) a5 long as dissolved CU2 was kept hiqh 

enough. In experiment #I, CU2 was kept low by controllinq pH at 8.3 and 

9.U. Averaqe production was reduced about 25-39%. Low pH combined with low 

alkalinrty, or high pH and high alkal~nity (#Zj yielded essentially equal 

productivity. In both cases dissolved ED2 was high (equivalent to 

equilibrium with a 1-ZX Cog gas phase). 

lable 7-7. pCU2 and Cbt~o-gdig Productivi tv: 1. 4m2 Ponds 

# Days T ,OC pC02 PH Alk. Uver. Prod, Max. Prod. 

min-max mM nH emlm21day qm/m2/day --"-C----------------------I-----------~--*------"-d----"-----~----~-w~-*--- 

1 5 14-28 0.04 13. 3 5 16 3ir 

b 15-30 0.007 9.0 5 15 - 

7.5 LOg OUTGASSING 

Unce the response ot an alga to to2 level and pH is known (ar better yet to 

cycfes ot chanqinq CU2 and pH) the specitic eronomlcs ot the use ot that 

strain can be determined. Choice ot an orqanlsm wlll depend an the trade-a+t 

between productivity at increased COZ level and C02 loss due to increased 

out gas sin^. As an example, the equivalent algal biomass prDdUCtiOn potential 

ot outgassed LO2 1s plotted in Figures /-3 and 1-4 as a function o+ pH and 

alkal~n~ty (that 15, dissolved C02 cuncentration) tor two d~tferent types 

of waters. 1he tlrst 1s sim~lar to the medium used +or qrowth at t3igrg&la 

in thrs project. The second is a hypothetical water with the same 

equilibrium constants (tor the carbonate svstem) as seawater. It lsn t 

seawater slnce the addition o+ the rnd~rated amounts o+ alkalinltv would 

result In CaCOS precipitation from seawater. it IS simplv a high IDS case. 

if a given medlum, atter evaporative concentration contains, for example, LO 

mN alkalinity then the ranqe of dissolved LO2 (pH1 at whlch outqassinq 15 

minimal is restricted. The best approach 1s to screen tor oraanisms whicn 

are highly praductive withln this acceptable C02 range. 1t productrvity 

Increases as CD2 level increases, the increase must ~ustity the larger 

C02 outaasslng 1085. At about ZO mM alkalinitv losses are not too severe 

In low TDS waters down to pH 8 or so, In high TDS waters down to pH "7.1. 

The corresp~ndinq dissolved LO1 concentrat~on is about .3 mM (equivalent to 

equilibrium with a IX pas phase) which does not hmit pruductiv~ty ot 

the ~&~~~gllg8 However, pH would rise In between carbonation stat~ons, 

lowerino EDL to .07 mM (at pH 9.3 or 6.7) which might impact prctd~~tivit~. 

However, operating closer to pH I becomes uneconomical unless alkalinity is 

very low as In a seawater system without recycle. Even so, the lack ot 

alkalinitv lowers the carbon storage capacity so much that the distribution 

sy5tem tor CU2 in~ection would become prohibitively expenswe, 

7he above dlscusston is based on a mixing speed o+ 15 cm/sec. it this is 

increased to 30 cm/sec, then operation below pH b becomes expensive. sxnce 

outqassinq increases L.5 +old. If the pond 1s op~rated at lu cm instead at 

20 cm, the outqassino is increased another +actor oi about 1.3. In add~tion, 

any mechanism whlch increases the eftlclency ot vertical movement ot water 

will further increase outgassino lasses. 

ihe malor losses ot Lo2 were rncurred upon injection and due to outpassrnq 

through the pond surface. The former 15 easily reduced to below 5% bv 

sparqing into the bottom of a one meter deep sump, (he latter can onlv be 

reduced by operatino at lower concentrations o+ dissolved CU2, by lowerinq 

channel mixinq velocity, and by increasinq pond depth, Thus an ootimization 

problem arises, particularly with respect to blomass production as a tunction 

of CD1 rancentratlon. Slnce alkalinity can be assumed to be moderate to 

hlgh trequired tor adequate carbon storaqe and a given in most groundwarers 

aiter evaporative concentrationl, pH must also be moderate to high, The 

growth characterlstirs ot the arpanism in terms ot LLJ2 must be known In 

order to evaluate the economics of the system. ihe usual pH optlmum 

experiments are not satistactory, In the tlnal analysis, the strain 

screening protocol should include the type oi experiment described aoove, 

bicarbonate uptake, which was not signi+icant In the work reported here, 

should also be measured since it may siqni+icantlv increase productivitv at 

low CU2 (h~oh pH). 
bECTION 8.0 

STORAGE PRODUCl 1NDUCTION 

8.1 OUTDOOR CULTURES 

In order to induce accumulation of storaqe products, cultures were qrown past 

their nitrogen-su++icient growth potential. Effluents +rom continuousl~ 

operated Ponds of both S. guadr~cauda and S. S02a ----were diluted wlth n~trooen 

+ree medium and allowed to 'induce" in batch. Batch cultures of both 

Scqnqdgpmgq strains and the finklst~odeqmgs were grown on yield-limitinq 

nitrogen levels, In general none o+ the organisms tested increased in lipla 

content atter nitrogen-starved arowth, Iota1 carbohydrate content did 

Increase. lhe h. quagrlg-gdg strain was productive atter nitrogen depletion 

and after readdition at nrtrogen. The h. SgiIa grew after nitrogen depletlon 

but ~roductivity declined compared to N-sufticient growth. The 

Apkl~trodesqns grew poorly when N-starved and did not recover: it dld not 

resynthesize pigment and eventually died, after readdition ot urea-nitroqen. 

Effluents trom continuously grown cultures ot #. qyadr:~auda were used tor 

growth, in batch, on N-deficient media. Sable 0-1 snows total, Iipid, and 

carbohydrate productivities ot those ettluents that actuallv became nitrouen 

depleted. The effluents were diluted with nitrooen-tree medium to lower 

initial nitroqen levels. Ihe time course ot one ot the runs is shown in 

Fioure B-1. 

In oeneral, productivities ot nitrogen-starved cultures were as high as 

non-starved batch cultaures operated at the same time. lhis was especially 

true it starvation did not proceed past L-5 days, Severe drop otts in 

productivity occurred thereafter. There o+ten was a day ot lowered 

productivity as nltroqen depletlon set In, although variable insulation make 

this uncertain. Carbohydrate content doubled and lipid content dropped 25%. 

batch cultures ot three strains were started with minimal leveis ot urea to 

result in depletion ot nltrooen trom the medium. For two-tnree days atter h 

de~letlon induced culture5 ot S. qggcjt-lcagda were as productive a5 uninduced 

cultures Ilable 8-21. Huain, lipid content dropped 25%. Larbohydrate 

content was not routinelv measured in these experiments. The cells used in 

runs Y-11 were N-deticient betore additlon of lu Qpm urea-N at the start o+ 

each run. his well, durinq Hun #lu luu porn ot urea-N was added atter dav 4. 

Ihe culture increased in productivitv (to 16 gm/m2/dav, over tne next day 

and a halt, lhese results indicate that th~s stra~n recovers qulcklv trom 

N-starvation. H typical time course o+ induction is shown in Fiuure 8-L. 

Sable d-1. Batch Induction of Continuous Pond Effluents 

7, Lipid 7. CHO Iota1 Lulv/aav 

Date Uav Lipid Prod, CHO Prod. Prod. lotal 

S~-gk!Sd~i~ak!d$ 

Yill 0 26.6 3.5 2 5 3.5 14.0 530 

9/12 -- 1 - - - - 7.4 514 

9/ 13 1 16.3 (m.4) 4 2 (6.6) lO(0.1) 53 1 

91 14 3 14.7 -. 3 4 7 4.3 3.4 522 

5/ 15 4 14.u -. 5 55 4.0 U 514 

l~nitlal values at becjinnlng ot batch growth. 

\ ) indicate an averaqe value. Productivities in qmlmLld 

-- indicate depletion of N from med~um. 

Table 8-2. s. gpadclcagcia Batch Induction 

Prod. beiore Prod. Utter 

N-depletion N-depletion 

qmlm2/day gm/mz/day ............................................................................ 

Run # Max. Hvq. (Days) flax. fivq. (Davs) Hvg. {Uays) ............................................................................ 

9 17.6 13(2) 19. I 18(2) 12t5) 

The nitrooen-starved cultures of S,-SgLli lncreasea trom 25% to 60% 

carbohvdrate content, but productivity was lowered. The l4n4~s$tgdegmgs grew 

poorlv after depletion of N trom the medium (7 gm/m2/day~ and carbohydrate 

content onlv increased to 32%. 
Tests tor storage product induction were pertormed in the lab wlth I. 

q$bba?!!, !!fikash~!O~s!!!u~, and 5. gu.idrlcayda, to determine the potential tor 

increases rn +uel precursors under more easily controlled conditions, batch 

cultures were grown in It Roux tlasks, under contlnuaus illurninatlon of 25 

~rn-~ at pH 1.5-.5 and ~328t2'~. Ihe results are shown in Table 8-3. 

alono wrth some data previously taken by the author for comparrson. 

fable t?-3. Storaqe Product hccumulation in Laboratorv Cultures 

Productivity Storage Product 

qm/m2/ day ---------------9--"----"9-----""----"--------------------------------------- 

Rax. hax kve 

HLljA N-Su++ic, N-Starved Cont, Prod, Prod. 

hvq. Max. ~vg. ax. ~ype x vss am/mL/d am/rnL/d 

---------------------------------------------------------------------------- 

10 14 t3 lo LHU 50 -- wnklsi, 4 

CHO 

S,-!GWS 10 15 12 LO CHO 75 15 9 

CHO 

kl,tg[ti 5'99 

Seawater i I 17 13 20 CHO 7 5 - - - - 

2.4 H NaC1 6 6 - - - - LHCI -- -- - - 

--- I. qalbana -----.- 

Hi density 7 lii 7 9 Lipid 40 4 

LO density 1 10 I g - Lipid 63 g - 
SECTION 9.0 

BIOMASS HARVESTING 

Harvesting microalgal biomass from dilute suspension has been one ot the most 

persistent problems in the development ot production technoloav. It is 

particularly dlfticult when the size of the organism is small and the value 

of the b~omass is low. In this section results from sedimentation tests are 

presented. Ihe S. gt.iad.i~gatcla, due to its agglomeration into large clumps, 

was unusually easy to harvest via sedimentation without flocculants. 

However, data is presented which indicates that nitroqen-starved sinqle cells 

sediment more rapidly than nitrogen-sufficient cells, lhus the sedimentation 

ot nitrogen-starved biomass may be teasible. 

9.2 SEDIMENTATION HATES - LQBOHATORY RESULTS 

Samples from the outdoor ponds were taken to the laboratory to measure 

biomass sedimentation rates. 7he rates of clari+ication were based on 

turbidometric assays, Ihe tests pertormed were not aided by chemicallv 

induced +locculation. Flocculants can iead to areatly increased rates ot 

claritication, but must be evaluated in terms of cost per unit biomass 

concentrated. Also, the effects of res~dual flocculants in the media. which 

must be recvcled many times, are unknown. 

The data trom sedimentation tests are summarlzed in lable 4-1. It 1% clear 

that the naturally tlocculant culture, S. gyadrl.tauda, grown in batch, 

sedimented much more qulckly and completely than any other culture. Nitroaen 

starvation ot th~s clumped culture did not have much ettect on claritlcation 

rates. When this orqanism was qrown in continuous culture, it did not 

aoqreqate. The sedimentation rates retlect this with onlv 40-60% ot the 

biomass settllnq faster than 15 cmlsec. Effluents from continuous ponds, 

"induced" in batch, settled somewhat faster than uninduced b~omass with 

65-757. sedimentina faster than 1b cmlsec. Sedimentation of finkistyg@eggyg 

and 5. SLlJa cells was not very rapid. Haain, induction {nitrogen starvation) 

increased the rates. 
lable 9-1. Sedimentation Rates 

% ot b~omass Sedimenting at Rates V cm/hr -1-----1-1---11---------1----------------------"--------------------"-"----- 

5. quad. batch S, quad S. quad S.SoLa Batch Hnkist. 

(clumped) 2 stage Contin. Batch 

llni nd. Induc. Induc. Unind. Unind. fnduc, Unind. --------------_--"---------------------------------------------------------- 

V 5'-ib 9-El 

cm/ hr 9- 1 9-8 9-25 9-25 10-3 10-7 10-7 

Since a sedimentation rate of 3u tmrsec may allow tor economlc aiqae removal, 

the Increase in the amount ot biomass settling at thls rate when 

nltroven-starved, could be srgn~+icant. 

Slurry density is another sedimentation parameter that was measured in 

certain instances, Table 9-2 lists culture, sedimentation alurrv and 

supernatent densltles measured durinq some a+ the sedimentation tests. 

Concentratian facators were 50-10U told, indicating that compaction o+ the 

biomass was good. 
Table Y-2. Sedimented Slurry Biomass Densities 

super - 

Cult. Slur, natent 

V Dens, ben s. Dens. 

EFlZEE gmll, ~CLL cir?~L ........................................................................... 

b. gqua!CLgauda batch 

Uninducea Y-1 6 u U. L75 25.2 . VL~ 

9-14 b 0 0.680 38.5 .1 U3 

2-13 6 U 0.405 25.0 . 1 UO 

lnduced '5-0 6 U 0.515 29.6 ,070 

S. qquadrlcaytia continuous 

9-8 15 0.14U 14.3 . V8U 

9-25 15 0.170 15.0 ,073 

9-30 9 0 0.174 13.0 . I U5 

5. quadcLcaud!! continuous ett luent 

9-15 15 0.325 22.5 ,055 

9-2b 15 0,398 14.0 ,140 

9.3 HARVESTS OF MAGS CULTURES 

In several instances, cultures trom 1UU mr and 200 mL ponds were oravitv 

ted Into the dm deep sedimentation ponds, allowed to settle tor 8-L4 hrs. and 

then decanted with a floatinq pump. lable 9-3 shows results ot these tests, 

The cultures used were all highly clumped. Settling times could have been as 

short a5 4-6 hrs. with no decrease in e+ficiency. A ten hour settllna tlme 

1s equivalent to a rate o+ LO-30 cm/hr. The slurry densities listed are 

based on the measurements taken immediately upon pumpino slurries out ot the 

sedimentation ponds and into a ,250 gallon tank. Further compaction upon 

settlinq would easily double the densities shown. 

Ihe Chlorellg cultures in general did not settle well at all. However, if a 

population o+ rotiters was allowed to develop, the cells clumped. The 

cultures, when harvested, then exhibited the efficiency ot settling shown in 

the table. 
Table 9-3. Harvesting Mass Cultures 

Pond E++1 . Slurry Sed. Harv, 

Ciraanlsm Density Uengi t y Oensi t y Ti me Ett'. 

PPm PPm PP~ hrs. 7. I----L-L--C----C"L----------------------------C-""----------"--------------- 
SECTION 10.0 

OPERATING COSTS 

10.1 INTRODUCTION 

The data shown in Section J.0, indicated that an average production ot about 

15 gmlm2/dav was sustainable throughout most of the year. hlthough thls 

fiqure would probably be higher with better strains qrown in the U.5. 

bouthwestern environment, we will use 15 gmlmL/day as a baseline. During 

the recyclinq experiment described in Section 5.0, the lnputs to the svstem, 

i.e., nutrients, water, and power, were monitored tor about a month, durinq 

which harvested productivity also averaqed 15 qm/m21day, lhus the cost per 

unit biomass, ot these inputs, can be calculated. Rnnualized caoital costs. 

costs of labor, and biomass processinq are not included in the discussion 

be1 ow. 

10.2 CURRENT COSTS OF INPUTS 

Table 10-1 summarizes the costs ot inputs during the recycle experiment that 

is beinq taken as representative o+ system performance. Durin~ this 

experiment, the depth ot the pond was Lu cm, the mixing speed was 90 cm/sec, 

and the loss of C02 at inject~on was about 34%. An overall input cost of 

$1.09/kq, hFDW resulted. Most of this was due to the hiqh cost ot C02 and 

its inetflcient use. Nitrogen contributed 12% ot the total cost, at a 

biomass N content ot 12%. The other nutrient costs, detailed in Table 10-2, 

are hiqher than would be expected in a large-scale svstem slnce 1) many ot 

these chemicals would have to be available in the water resource and 2) bulk 

purchaslnq would decrease unit costs of phosphate and iron. Mlxinq power 

cost is based on a high channel velocity that, as discussed in Section 6.0, 

is not necessary when less than 25 qm/m2/day are being produced. 

10.3 PROJECTED COSTS OF INPUTS 

Table 1U-3, shows the costs ot the same tactors, but modltied to take into 

account cost reduction possibilities. COL is still the largest cost 

factor, and is, in +act, still too expensive even at the price ot $.US/ko. 

the best commercial price available now. hn increased utilization efficiency 

is used: onlv 5% loss at injection and outqassina losses equivalent to 5 

qm/mL/day algal production. Both should be easily achieved when a sump is 

used tor carbonation, when mixlnq speed 1s 5 20 cm~sec, and when the pH ranoe 

1s chosen to avoid excessively hiah dissolved COL levels. Even so, LO2 

comprises 50% of the total input costs, at $.125/kq aloae produced. Since 

this is about all that the biomass tuel precursor is worth, COi cost must 

be cut further, at least 50% more. Findinq large amounts of low cost COL 

is probablv the greatest sinqle obstacle to lowest cost algal biomass 

production. 
Table 10-1. Initial Alqal Biomass Production Costs 

243 days4 5. qggd~igwd$, effluent recycle, treshwater, 2Wm2 -c-c------------------3C----I--I------------------------------------------------- 

Component Unit Price,$ Rmt. Used Cost ,Z % total cost .......................................................................... 

GO2 .215/Ko SKafKq alg. 68.31 '7SB'i 

Urea .51/Kq .L6Kq/Kq alg, 10.78 12.u 

Other Nutr . ---- ---I 6.71 7.4 

Pump power , Ub/Kw. hr 5,2Kw,hr . $1 0. S 

Mlxlng power . ObIKw. hr . 42kl/mZ 3.3B 3. b 

Water i1.51U ft 91m2/day 0.48 0.5 

1 otal biomass harvested: 82.8Kq (15.0 ~nlrn~/day av) 

Cost: tl.oY/Kq AFDW $.49/lb 

Tabi e 10-2 20umL Effluent Recycle--Medi a Component Input 

Biomass NaHC03 MaSb4 DkP DSP FeS04 Urea Water 

Date Harv, ,Kg $O.bblKq 80.55/Ka $2.73/ka $1.70Kq $.88/ka $a51/kq ,000 L ----------------------------------"------------------------------w----------- 

5/27 24,b 1.33 .I25 ,225 ,225 . 3 6.0 14.4 

88 .07 .61 .38 .26 3.04 

TOTAL 82.8 1.33 1.25 . 975 ,675 1.5 21.3 50.4 

.€I8 .70 2.66 1.i6 11 10.78 

lop entry: ko used 

Bottom entry: cost,$ 
Table 10-3. Cost Reduction Possibilities 

Pro lected 

Component Cost heduction Cost,$ % Total cl'kg alg. c/kg alg. ............................................................................ 

Urea .Zb kg/kq --.I3 kqikg 

5.39 26.6 6.5 3.3 

Other chemicals 

A11 but P, Fe available 

in water, bulk pricing 

3.50 16.3 4.0 

Pump Power - 0.31 1.5 0.37 0.4 

Mixing Pwr. 0.42 w/m2--0. 14w/m2 

Effic.: 50% 0.45 2.2 0.54 0.54 

Water - 0.48 2.4 0.69 0.6 

----- ---- ----- ---- 

TOTAL 20.28 100 24.6 13.0 

Ihe cost o+ nitrogen has been lowered in the projection due mainlv to 

production of nitroqen limited biomass. If the biomass contained only 6% N, 

then the ura costs $.065ikq of alqae. if the content is lowered further, and 

some nitrogen is recycled trom the biomass processing, then this cost can be 

halved again, lhe other chemical costs are limited to iron and phosphate. 

These must be replenished as algae is produced on recycled water. 

Power costs can be lowered by lowering the channel velocity from 30 cm/sec to 

lScm/sec. 7he system must be designed to maintain an overall e+ticiencv of 

40-SOX for the mixing systems. 

10.4 CONCLUSIONS 

The total cost of the inputs considered is 13-25 cents per kq AFDW, with the 

cost of C02 making most of the difference withln this range. increased 

productivity would have little impact on this cost since the productivity 

related inputs - nutrients - comprise over 90% of the total. With increased 

productivitv C02 cost would decrease due to an improved ratio of carbon 

going.into algae vs outqassing (if the mixing speed and C02 level are not 

increased to achieve the higher productivitv). but this would still only 

lead to a sliqht decrease in C02 use, e.g,, from 2.5 to 2.2 kg/kg algae 

produced it productivity were doubled. The major impact of increased 

productivity comes in annualizing capital casts per unit biomass produced and 

in distributinq labor costs, Since these costs would need to be less than 

that of the 
lowest total costikg in Table 10-3 lotherwise the biomass is too expensive 

for fuel), the ~mpact a+ increasing productivity is still limited by nutrient 

costs. hny cost savings trom increasing productivity can be gained only when 

the increases are not accompanied by major increases in capital costs or 

operating costs, 
SECTION 11.0 

SUMMARY AND CONCLUSIONS 

11.1 PRODUCTIVITY RELATED RESULTS 

With a two species rotation - a high temperature ChlgHella 2~ in the summer 

and a S~gqgdgsgyq-sp in fall, winter, and spring - a yearly average ot 15 

gm/m2/day was obtained in sequential batch, nitrogen suf ficient 

cultivation. 

At a locatlon where maxlmum ambient temperatures are above 2S°C for 9-10 

months, and minlmum ambient temperatures above 10-15~, 20-25 omlmLldav 

could be achieved. 

Higher productivities mav result using strains pre-screened for: 

tolerance to hlph DO 

low respiratory rates at high temperature 

tolerance to high irradiance 

broad temperature optlmum 

Induction of storage products, under nitrogen-depleted conditions has little 

negative lmpact on productivity if accomplished withln one to three days. 

Productivitv is independent of mixinq speed, +or mlxing regimes characterized 

by random turbulence, at least in the range from 1-30 cm/sec up to 

productivi ties of at least 25-30 qm/m2/day. 

11.2 COST RELATED RESULTS 

Mixinq must be sutficient to keep cells in suspension (15-20 cm/sec at most). 

Higher mixinq speeds require substantlallv more power input and increase 

CO2 losses due to outgassing. 

Media recycle is a teasible method tor conserving water. 

CU2 is the largest cost tactor, Culture operation must be optrmlzed for 

high productivitv (whlch increases with COL level) and reduced C02 

outqasslnq (which Increases with CU2 level and mlxlnq speed). Sources of 

C02 must be found which are both plenti+ul and below present market prlces. 

Depending on how it is achieved, increaslnq product~vit~, e.g., by a tactor 

of two can only be expected to lower the overall cost of biomass ~y, e.o., 

10-SO%, because nutrient Input is proportional to blomass output, as are most 

processlnq costs. For the most part only annualized capltal costs and labor 

do not necessarily increase with productlvlty. 
11.3 SPECIES SCREENING, MONOCULTURE MAINTENANCE 

The more constant the environment, the easier it is to maintain a 

mon~culture. 

Verv competitive strains will be required to maintain monocultures. IDS and 

temperature tolerance exert the greatest selection on species, with tolerance 

to DO and light also important. 

Determination of growth response to pH, Cop and DO is important in 

optimizing system output and costs, 

Systems must be managed for biomass harvesting, preferably bv 

autoflocculation, or other methods which harvest small cells, Unless a 

strain is the mast competitive by a wide margin, harvesting methods which are 

not universal will increase the relative competitiveness ot non-harvestable 

organisms when media recycling 1s practiced. 
REFERENCES 

I. American Public Health Assoc., ~tg~@a~~-~p~hgdp-f~~-~~e_-~x_a_~in_gt,ign_-gf~ 

!ater-a!d_Wasteyater, 14th edition, Washington, D.C.: hPHA, 1976. 

2. Bliqh, E.G., and W.J. Dyer, "A Rapid Method of Total Lipid Extraction and 

Purification," Cgpgdl-J,-~ioghg~,-fh_ygigI.., 32, 1959, pp. 911-917. 

3, Eolterman, H.L., R.S. Clume, and H.A.M. Ohnstad, Methgdp-for-Fhy!~~aL-aC!dLhegigaL-fipaLysis,~f-Er_egh-_W$g_ers, 10P Handbook No. 8, London: Blackwell 

Scientific Publications, 1978. 

4. Sigma Chemical Company, Technical Bulletin No. 640, 1980. 

3, Solarzano, L., Ugggl-gcpagqr. 14, 1969, pp. 799-801. 

4. Dodd, J.C., Personal Communicatron. 

7. Weissman, J.C., "Carbon Uioxide rransfer," in J.R. Benemann, et.al., 

~~crgg~qgg-fls-a-sgyrc_geg~-L~gy~d-~y_e~p, D.U.E. Final Technical Report, 

DEACOS 81 ER 30014, 1982, pp. 123-148. 

0, Wilson, G.T., and N. HacCleod, "A Critical Appraisal of Empirical 

Equations and Models for the Prediction of the Coefficient of Reaeration 

of Deoxygenated Water, Watey-Rgs., ;, 1974, pp. 341-366. 
Table 14-1. Climate Datar Monthly hvetages - 1983 

MONTH INSDLAT3ON TEUPERATUHL (degrees C) 

Lgy/Day Std.Dev, MAX Std.Dev. MIN Std.Dev. 

Jan-83 

Feb-83 

Mar -83 

Apr-BJ 

May-83 

Jun-83 

Jul-83 

hug-83 

Sep-83 

Oc t -63 

Nov-83 

Dec-83 

Table A-2. Monthly Averages - 1984 

MONTH INSOLATION TEMPERATURE ldeqreee C) 

Lgy/Day Std.Dev. MAX Std.Dev. MIN Std.Dev. 

Jan-84 

Feb-84 

Mar -84 

Hpr-84 

May -84 

Jun-84 

Jul-84 

Aug-04 

Sep-84 

Oct -04 








1 Document Control 11. SERI Report No. 1 2. NTlS Accession No. 1 3. Recipient's Accession No. '-1 

Production of Liquid Fuels and Chemicals by 

Mi croal gae 

Page I SERI /STR-231-2649 I 

4. Title and Subtitle 

March 1985 I 5. Publication Date 

I 

7. Author(s) 1 8. Performing Organization Rept. No. 

J. C. Weissman, R. P. Goebel I 

9. Performing Organization Name and Address ( 10. Project/Task/Work Unit No. 

Microbial Products, Inc. I Fairfield, California 

4625.10 

11. Contract (C) or Grant (G) No. 

16. Abstract (Limit: 200 words) Of the strains of microalqae provided b.y other Aquatic Species 

12. Sponsoring Organization Name and Address 

Solar Energy Research Institute 

161 7 Cole Boulevard 

Go1 den, Colorado 80401 

Program subcontractors, none could out-compete invading wild type aigae. Nonethe1 ess , two strai ns--Anki strodesmus fa1 catus and Scenedesmus S02a--were grown outdoors in mass culture, one for two months and the other for one month. Two wild 

13. Type of Report & Period Covered 

Technical Report 

14. 

type organisms, Scenedesmus quadricauda and Chlorella sp.,provided most of the outdoor rpsults. The Scenedesmus- was maintained for thirteen months, averaging about 

13 g/m day. It was used to compare batch and continuous cultivation for mixing 

speed experiments, media recycling, storage product induction tests, and the determinatign of operational costs. Chlorella was grown for two months at over 

20 glm day and was used to determine the relationship between mixing velocity and 

productivity. Experimental results indicated that organisms responded very differently to pH and C02 concentration, as well as to dissolved oxygen. The latter 

may turn out to be an important screenin? criterion since high dissolved oxygen is 

endemic to large systems. The pH should ,be as low as possible to obtain the 

highest dissolved CO required for maximizing average production. But this must 

be balanced against gutgassing loss of CO , which increases with concentration. 

Mixing speed affects the surface mass tra6sfer coefficient. 

15. Supplementary Notes 

- 

17. Document Analysis 

a.Descripton Algae ; Carbon Dioxide ; Chlorella ; Cost ; Cultivation ; Ponds ; 

Productivity ; Scenedesmus ; Sedimentation ; Unicellular Algae 

b. Identifiers/Open-Ended Terms 

I c. UC Categories I 

18. Availability Statement 1 19. No. of Pages 1 

( National Technical Information Service I I 

U .S. Department of Commerce 

5285 Port Royal Road 

Springfield, Virginia 22161 20. Price 1-1 

I I I 

Form No. 0069 (3-25-82)