This project was a continuation of the project described in III. B.3.b. It was performed by a new company, Microbial Products, Inc. (EnBio was dissolved when John Benemann left in 1983 for the Georgia Institute of T echnology). The pond system described earlier continued to be used for this project. The objective was to obtain long-term productivity data with a pilot-scale system and generally demonstrate the requirements of large-scale algal mass cultivation (Weissman 1984; Weissman and Goebel 1985, 1986).

The first challenge was to obtain microalgal species that could be grown on the fresh to slightly brackish water available at the site. The common experience is that either inoculated strains from

culture collections fail to grow in the outdoor ponds, or that they grow initially but become rapidly outcompeted by indigenous strains. A common practice is to make the best of a bad situation and cultivate the invading organisms. This was also the experience and approach of this project.

After inoculation of Scenedesmus obliquus strain 1450 from the SERI Culture Collection, a strain of Scenedesmus quadricuada invaded. This turned out to be the most successful organism, cultivated for 13 months in fresh water and an additional 3 months in brackish. After an Oocystis sp. (Walker Lake isolate) was inoculated, a Chlorella sp. became dominant and was maintained (or maintained itself) for 2 months under semi-continuous dilution. However, some strains provided by SERI researchers could be grown for at least a few months outdoors, including an Ankistrodesmus falcatus and a freshwater Scenedesmus sp. So2a.

Productivity for S. quadricuada grown semi-continuously which is harvested every few days (a “sequential batch” growth mode), averaged about 15 g/m2/d for the 8 month period of March through October, with monthly averaged solar conversion efficiency ranging from 1.2% to 2.2% (Figure III. B.6.). “Typical” real biomass density just before harvest ranged from 60 to 100 g/m2, except for May, which recorded the highest standing biomass (160 g/m2) and productivity (20 g/m2/d). The continuously diluted cultures (diluted during the entire light period) exhibited approximately 20% higher productivity.

From a large number (39) of experiments, a correlation of Tmax, Tmin, and total insolation with productivity reduced the variance in the prediction of productivity by about 50% when using any single variable, but not in combination. This suggested that one of these three factors generally dominated (e. g., too high or too low a temperature or too little insolation). Similar experiments were carried out with the other microalgae in combination with the study of variables such as mixing speed, O2 outgassing, CO2 addition, and N limitation (for lipid induction).

The main conclusions of the extensive experimental program were:

1. Productivities of 15 to 25 g/m2/d were routinely obtained during the 8-month growing season at this location. However, higher numbers were rarely seen.

2. Continuous operations are about 20% more productive than semi-continuous cultures, but the latter densities are much higher, a factor in harvesting.

3. Culture collection strains fare poorly in competition with wild types.

4. Temperature effects are important in species selection and culture collapses, including grazer development.

5. Nighttime productivity losses increased to 10% to 20 % in July, when grazers were present; nighttime respiratory losses were high only at high temperatures.

6. There is a significant decrease in productivity in the afternoons, compared to the mornings, in the algal ponds.

7. Oxygen levels can increase as much as 40 mg/L, over 450% of saturation, and high oxygen levels limit productivity in some strains but not others. Oxygen inhibition was synergistic with other limiting factors (e. g., temperature).

8. Increasing TDS from 0.4 to 4 ppt decreased productivity, depending on strains.

9. Mixing power inputs were small at low mixing velocities (e. g., 15 cm/s) but increased exponentially. Productivity was independent of mixing speed.

10. The strains investigated in this study did not exhibit high lipid contents even upon N limitation.

11. The transfer of CO2 into the ponds was more than 60% efficient, even though the CO2 was transferred through only the 20-cm depth of the pond.

12. Harvesting by sedimentation has promise, but was strain specific and was increased by N limitation.

13. Initial experiments demonstrated that media recycle is feasible.

14. Project end input operating costs for large-scale production (@ $50/mt CO2,

70% use efficiency, etc.) was $130/mt of algae, of which half was for CO2 and one-third for other nutrients, with pumping and mixing power only about $10/mt.

This project answered a number of issues that had been raised about this process. One initially controversial observation was the finding that mixing speed had no effect on productivity (Figure III. B.7.). However, this experiment used a strain of Chlorella that did not settle, and care was taken to keep other parameters identical (in particular pH and pO2 levels). Thus, the increased productivities seen in some experiments (e. g., those of Hawaii), could possibly be accounted for by differences other than those of mixing, such as changes in outgassing of O2.

From the perspective of large-scale biomass production, one conclusion from this research was that mixing power inputs make any mixing speed much above about 30 cm/s impractical, as the energy consumed would rapidly exceed that produced. The rate of mixing should only be between about 15 and 25 cm/s, sufficient to keep cells in suspension and transfer the cultures to the CO2 supply stations in time to avoid C limitations in large-scale (>1-ha) ponds.

For low-cost production higher productivities would reduce capital, labor, and some other costs, but nutrient (e. g., CO2) related costs would not change. This suggested the need for low-cost CO2, and other nutrients, as well as a high CO2 utilization efficiency. Efficient utilization of CO2 appeared feasible based on the results obtained with even this unoptimized system.

Another major conclusion was that competitive strains would be required to maintain monocultures. The need for feedback from the outdoor studies to development of laboratory screening protocols was a major recommendation. Specifically, the relatively controllable parameters of CO2, pH, and O2 were of interest in determining species survival and culture productivity. Also, harvesting was identified as a specific area for further research. Finally, lipid induction remained to be demonstrated. These were the general objectives during the final year of this project, described in Section III. B.3.d.

MS*

Sustained	Maximum
Numbers above bars indicate photosynthetic efficiency (% of total)
XvX
“Ш!
Ш
Oct	NOV	May
Mar

Month

Figure III. B.6. Long-term productivity of S. quadricauda in freshwater (100-m2 pond). (Source: Weissman and Goebel 1985.)

Min/max temp., *C: 20/30 (15/25 for data’at 60 cm/sec)

Average linear mixing velocity (cm/sec)

Figure III. B.7. Chlorella mixing velocity experiments.

Data from 3.5-m2 ponds operated under constant pH and pO2 levels and compared to a control culture operated at 30 cm/s mixing velocity. Numbers above the data points indicate productivity of the control. Max/min temperatures 20/30 oC (15/25 oC for the 60 cm/s data set). (Source: Weissman and Goebel 1986.)