The last major engineering-related activity carried out by the ASP was a PETC-funded study, both laboratory and process design, for microalgae biodiesel production using power plant flue gases. The general arguments for use of microalgae in CO2 mitigation, from high productivity to relatively low water use were reviewed in several reports (Brown et al. 1991; Chelf et al. 1991). The authors concluded that the SOx and NOx impurities in flue gas would likely not have a major effect on algal cultures, and, indeed, that the NOx could not even provide the N requirements of the cultures. Prior work indicating an inhibitory effect of flue gases (for example Negoro et al. 1991) was probably due to the acidification of the media and resulting pH drop, because of excessive flue gas transfer.

To demonstrate that flue gases can be used for microalgae culture, NREL set up an experimental apparatus to supply controlled and measured amounts of such gases to the algal cultures. Figure III. D.1. shows a typical result, with no detectable difference between the simulated flue gas culture and the control gas (similar CO2 levels, but without SOx and NOx). With almost 1,000 times more CO2 than SOx in flue gas, alkalinity neutralization would not be a major problem except where high water reuse and low alkalinities in the water coincide. In general, flue gas supply to algal cultures should not present a major problem.

Aside from the experimental work, the ASP also carried out work on systems design and analysis for microalgae biodiesel production using power plant flue gases (Kadam 1994, 1995). The analysis was based on the production of essentially pure (liquified) CO2 from the flue gases of a 500-MW power plant, using conventional amine scrubbing processes, and its supply to a 100-km remote microalgae production facility. Delivered costs were estimated at $40.5/mt CO2. The microalgae pond system design was based on the prior effort of Neenan et al. (1986; see Section III. D.6.) and included site selection criteria for specific power plants in New Mexico. The summary of the system model inputs and outputs are provided in Table III. D.8., which summarizes current and long-term projections for such a process. Although there are some significant differences between this and other cost analyses (prior sections), overall these results agree that through long-term productivity increases such processes could achieve CO2 mitigation costs competitive with other options. This places the focus on long-term efforts for productivity enhancements.

I Publications:

Brown, L. M.; Zeiler, K. G. (1993) “Aquatic biomass and carbon dioxide trapping.” Energy Conv. Mgmt. 34:1005-1013.

Chelf, P.; Brown, L. M.; Wyman, C. E. (1991) “Aquatic biomass resources and carbon dioxide trapping.” Biomass and Bioenergy 4:175-183.

Kadam, K. L. (1994) “Bioutilization of coal combustion gases.” Draft Milestone Completion Report, Recovery & Delivery, National Renewable Energy Laboratory, Golden, Colorado.

Kadam, K. L. (1995) “Power plant flue gas as a source of CO2 for microalgae cultivation: technology and economics of CO2 recovery & delivery.” Draft Report, National Renewable Energy Laboratory, Golden, Colorado.

Kadam, K. L. (1997) “Power plant flue gas as a source of CO2 for microalgae cultivation: economic impact of different process options.” Energ. Convers. Mgmt. 38:S505-S510.

Zeiler, K. G.; Kadam, K. L. (1994) “Biological trapping of carbon dioxide.” Draft Milestone Completion Report, National Renewable Energy Laboratory, Golden, Colorado.

Table III. D.8. Summary of costs for microalgae CO2 mitigation.

‘COj recovery cost = $40/t ZC02 credit = $50Л CO2

3Based on credit at the following rate: lipid «= $240/t, protein = $120/t, carbohydrate = $ 120/t

III. D.10. Conclusions.

The cost analyses for large-scale microalgae production for fuels reviewed earlier evolved from the rather superficial analysis of the 1970s to the much more detailed and sophisticated studies during the 1980s, with some updates and advances during the present decade. The basic process did not change significantly from the conceptual designs first suggested by Oswald and Golueke (1960): very large open, shallow, unlined, mixed, raceway ponds. However, the design details have evolved significantly, and current engineering and cost analyses are much more realistic.

There are, of course, still some major uncertainties with these engineering studies. For the fundamental raceway design these are the issues of scale and the need for some type of pond lining. Current commercial microalgae production ponds are typically 0.25-0.5-ha in size, and are lined with plastics to prevent percolation and silt suspension and to allow pond cleaning. However, there are also examples of much larger and unlined raceway ponds in a commercial production facility, specifically the Earthrise Farms Spirulina plant in southern California, where two large (approximately 5-ha) unlined ponds are currently operating. Similar systems are also used in wastewater treatment. The City of Hollister wastewater treatment plant includes a single 7-ha raceway unlined pond mixed with an Archimedes screw. Even larger (>50 ha) unlined, unmixed ponds are also used for microalgae production in Australia for commercial production of Dunaliella, and in several countries in wastewater treatment. Thus, although some uncertainties remain (such as allowable channel width and wind fetch effects), in general the basic engineering designs and assumptions for the microalgae cultivation ponds appear well established.

For the harvesting, fuel processing, and media/nutrient recycling subsystem designs the cost analyses are perhaps less robust, based on often untested assumptions. However, overall, none of these appear to provide a likely major show stopper. Still, most of these issues require more R&D. One area where little work has been done is in the extraction of the algal oils. Although in the most recent studies the use of large three-phase centrifuges was recommended (Benemann and Oswald 1996), this requires further study.

Although no single design component or unit process in these engineering analyses has an overwhelming effect on costs, the cost projections are optimistic; therefore, there is relatively little scope for any further cost reductions. In most cases, engineering designs and specifications were based on the cheapest possible design and likely lowest costs. Also, the engineering design and system construction approaches were based on agricultural engineering practices, rather than those of chemical engineering, as agricultural materials and construction methods are more applicable, in addition to being of lower cost.

A major conclusion from the cost analyses is that there is little prospect for any alternative designs for microalgae production systems that would be able to meet the requirements of microalgae production for fuels. This is particularly true of closed photobioreactors, in which the culture is entirely enclosed, in greenhouses, plastic tubes or bags, or other transparent enclosures. The costs of even the simplest such system would likely be well above what is affordable for

fuel production processes. Even the simplest plastic sheeting cover over the ponds would much more than double total systems capital and operating costs. The simplest tubular photobioreactors are projected to have capital costs some ten times higher (e. g., $50/m2) than open pond designs (Benemann 1998). And, despite many proponents of such closed photobioreactors, current commercial microalgae production systems use exclusively open pond cultures, even for very high-value microalgae products. The few attempts at large-scale (> 1 t/yr) production of microalgae in closed systems have failed.

Of course, closed photobioreactors could have benefits in areas such as better control over environmental conditions (pH, temperature) and biological contaminants, and higher cell concentrations, reducing liquid handling and harvesting costs. Thus, it would be theoretically possible to grow algal strains not able to dominate in open ponds, at higher productivities and reduced harvesting costs, thereby making up for the higher costs of closed photobioreactors (which proponents assume to be only marginally higher than open pond systems). Closed systems of various types may find important applications in the production of the “starter culture” or inoculum that will be required to initiate and maintain large-scale open pond operations. This could be particularly important when genetically improved or genetically engineered algal strains are used.

At the other engineering design extreme are the very large (up to 100 ha) unmixed ponds used in the production of Dunaliella in Australia (and, until recently, also used for Spirulina production in Mexico). Such production processes are of even lower cost than the mixed raceway designs. However, due to hydraulic and CO2 supply limitations (among others), productivities are maximally only a few g/m2/d, a small fraction of those required for microalgae fuels production. Thus, there seem to be few practical choices in the basic engineering design of a raceway pond system. Even the mixing options are restricted; paddle wheels are overall more economical, flexible, and suitable than the alternatives (e. g., Archimedes screws, recirculation pumps, or air­lifts).

However, the most important issues raised in these economic and engineering analyses are not the engineering designs, or even the cost estimates, but the biological assumptions on which such designs are based. These have changed dramatically during the past 2 decades in one major aspect: productivity. Productivity projections have escalated from less than 50 mt/ha/y in the initial studies (e. g., Benemann et al. 1977), to almost 300 mt/ha/y (on an equivalent heat of combustion basis) in the most recent extrapolations (Benemann et al. 1993). In terms of photosynthetic efficiency, these improvements are from about 2% total solar energy conversion to a near-theoretical 10% efficiency. This dramatic increase in projected productivities was based on two main factors: first the significant advances in the state-of-the-art during these 2 decades, with significantly higher productivities than originally anticipated being measured in outdoor systems. And second, the clear necessity to achieve very high efficiencies for any sunlight-to-fuels process. Although there are theoretical, and practical, approaches to achieving such high efficiencies, they will without a doubt require relatively long-term R&D efforts (see Section IV. A.2.).

Productivity, in terms of solar conversion efficiency, is only one of the objectives of future R&D in this field. A related issue is that much of this productivity must be in the form of algal lipids, suitable for utilization and upgrading to fuels. Although some progress was made in this area in the laboratory, through physiological and genetic means (see Section III. B.5.d.; also Section II), this still will require considerable research. Another area that will require significant research is the development of a low-cost harvesting process. Again, the engineering and economic realities constrict the choices to the lowest-cost option, which would appear to be a simple settling process, followed by further mechanical concentration and processing.

The major conclusion of these analyses is that microalgae production for fuels is currently not limited by engineering designs, but by the many microalgae cultivation issues, from species control in large outdoor systems to harvesting and lipid accumulation to overall productivity. Future R&D must focus on these biological issues as a primary research objective, in the quest for low-cost production processes.

During the first year of the proj ect (Weissman et al. 1988), all experimental work was carried out using the small ponds, which allowed essentially fully automatic operation and continuous dilution, as well as heating if needed. The objectives were to determine long-term productivity and stability for this site with previously studied and new species. Five of the strains inoculated into the 3-m2 ponds were successfully cultivated, including two that derived from local isolates (one of which had invaded these ponds). Three of the culture collection strains could not be cultivated stably in the small ponds. Reproducibility of the experiments was tested, with the conclusion that differences between the different treatments should be judged significant only if these approached 20%.

Productivities in the summer month of August reached 3 0 g/m2/d for C. cryptica CYCLO1, but decreased to about half this level in September and October. At this point, M. minutum (MONOR2) was used, as this is a more cold-tolerant organism. By November productivity of MONOR2 fell to about 10 g/m2/d, and was very low (3.5 g/m2/d) in December in unheated ponds. Remarkably, despite these ponds freezing over repeatedly, the culture survived and exhibited some productivity. During August and September, productivities for CYCLO1 and Amphora sp. exhibited short-term excursions above 40 g/m2/d. Faulty data are not suspected.

A physical model of the pond environment developed by David Tillett at the Georgia Institute of Technology, combining climatic, design, operation, physico-chemical and biological process characteristics, was validated with temperature data from ponds in Roswell (see Section III. B.6).

The large-scale system was completed by the second year. Some problems were encountered: the spongy clay at the site did not compact well, resulting in an uneven pond bottom. This made it difficult to clean and drain the ponds, and resulted in settling and sedimentation of solids. Significant differences were noted between the lined (north) and unlined (south) ponds, in terms of mixing velocities, head losses, and roughness coefficients. In any case, power inputs at low mixing velocities (<30 cm/s) were relatively low (<0.1 w/m2). The efficiency of CO2 injection into the ponds through the carbonation sumps (at approximately 0.6 to 0.9-m depth) was estimated at close to 90%. From the measured gas transfer coefficient, outgassing losses from the lined pond were estimated as approximately 10% depending on pH levels. Also, the unlined pond lost 0.3-0.4 cm/d more water due to percolation.

A theoretical model of selective cell harvesting and recycling resulting in dominance of the slower-growing over the faster-growing species. (Source: Benemann et al. 1978.)

Figure III. A.2. Initial experiment demonstrating the maintenance of Oscillatoria in outdoor ponds.

Maintenance of a culture of Napa Oscillatoria using selective recycle. Bars indicate the relative volume, concentration and filament length distribution of Oscillatoria (white bar height indicates filaments shorter than 150 pm; vertical stripes indicate filaments between 150 and 375 pm; slashes indicate filaments longer than 375 pm). Volumes of single algal cells were calculated on an equivalent concentration basis and are shown as triangles (shaded area, top). Vertical arrows marked R indicate when the culture was harvested with a microstrainer (30-pm opening fabric) and the captured algae (almost all Oscillatoria) returned to the pond. Experiments were carried out in a 3-m2 circular pond mixed continuously at 3 cm/s. Settled raw sewage, supplemented with 100 ppm of bicarbonate was used as substrate. (Source: Benemann et al. 1977).

The objective of this study (Feinberg and Karpurk 1990) was to examine CO2 resources for microalgae production in the year 2010 and beyond. This report was a very comprehensive and authoritative source of information on this subject, from merchant CO2 supplies and costs to potential competition from EOR for CO2 sources. CO2 recovery from existing processes was judged to be relatively low cost from ethanol and ammonia plants, and much more expensive from cement, refineries, or power plants.

After a detailed review of the options, the authors estimated that the potential CO2 resource base was sufficient to support the annual production of roughly 2 to 7 quads of algal fuels. This corresponds to as much as 1.1 billion tons of CO2 per year, at prices ranging from about $9 to $90/t CO2. However, this analysis lacks the spatial resolution of the earlier study; thus, the actual CO2 availability (particularly of the low-cost supplies) was somewhat more speculative. Certainly CO2 resources will be a major limiting factor in microalgae production technology. However, as CO2 utilization has become a central objective of microalgae production systems, perhaps rather than looking at CO2 as a limitation it should be considered a site-specific opportunity, where the other requirements for microalgae production are met (e. g., land, climate, water, infrastructure). Table III. C.1. summarizes the conclusions of this report regarding CO2 costs and supplies.

I Publications:

Feinberg, D. A.; Karpuk, M. E. (1990) “CO2 sources for microalgae based liquid fuel production.” Report, Solar Energy Research Institute, Golden, Colorado, SERI /TP-232-3820.

Karpuk, M. (1987) “CO2 sources for fuels synthesis.” FY1986Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 269-275.

Table III. C.1. Summary of Availability and Cost of CO2 Sources

In FY1984-85 (Laws 1985), the research was directed toward the study of more thermotolerant species. Algal strains collected by the ASP researchers in the southwestern United States were evaluated using the Type I and II waters (see Section II. A. 1.for a description of SERI Type I and Type II media). Several species, including Platymonas sp. (used previously), Amphora sp., C. gracilis, and Boekelovia sp. were grown in the two water types, each at two salinities and at four temperatures (25° to 32°C), with the data reported as the number of doublings per day. One interesting, but unexplained, observation was that at higher temperatures there was a consistent shift, among all four algae, of maximum doubling rates to the higher salinity and Type II waters.

The small outdoor flumes were used to test this cultivation strategy. The cultures were diluted each third day, to a concentration of 2 x 106 cells. The results were “consistent with those of earlier studies,” with solar conversion (PAR) efficiencies close to 10% (5% of total solar). The C. gracilis species was also tested, though at a 2-day dilution rate (requiring a one per day doubling time), with somewhat lower efficiencies (8%), though still rather high productivities. Also, Tetraselmis suecica was cultivated in the ponds with good results. Over a 78-day cycle, in spring 1984 and summer 1985, productivity was 37+5 g/m2/d, with a corresponding PAR efficiency of 9.1%.

Removing half the arrays had no significant effect on productivity; removing all foils reduced light conversion efficiencies from 8% to 5.5%. However, a major variable in such systems is the pO2 in the ponds, which may well account for the difference observed. The higher mixing (e. g., power inputs) caused by the foils may have increased outgassing of O2 from the pond enough to increase productivity, rather than to any flashing light effect. The reports of increased productivities caused by 3-day batch dilutions and foils remained controversial, and continued to be the major focus of this project.

IV. A.1. Conclusions

From the early 1980s through the mid-1990s, there was a major effort by ASP researchers and contractors to identify or develop microalgal strains that demonstrated properties conducive to cost-effective biomass and lipid production. The characteristics deemed desirable in these strains include high productivity, high lipid content, competitiveness in outdoor culture, and tolerance to fluctuations in temperature and salinity. Although a number of strains were identified as possible candidates, no one strain was found to possess the optimal characteristics. As discussed elsewhere in this report, perhaps the most significant observation is that the conditions that promote high productivity and rapid growth (nutrient sufficiency) and the conditions that induce lipid accumulation (nutrient limitation) are mutually exclusive. Further research will be needed to overcome this barrier, probably in the area of genetic manipulation of algal strains to increase photosynthetic efficiency or to increase constitutive levels of lipid synthesis in algal strains.

The collection and screening efforts produced a number of significant findings. The SERI/NREL Microalgae Culture Collection was established as a valuable genetic resource and was the first microalgal collection that focused on organisms from brackish or saline environments. The organisms remaining in this collection (see Section II. A.3.) are being transferred to the University of Hawaii and should be available to interested researchers.

Although a number of algal strains were investigated for growth and lipid production properties, the best candidates were found in two classes, the Chlorophyceae (green algae) and the Bacilliarophyceae (diatoms). Organisms were identified in both classes that showed high productivity, ability to grow in large-scale culture, and lipid accumulation upon nutrient stress. However, in some ways the diatoms may turn out to be better candidate organisms for biofuels production. The highest lipid levels (40%-60% of the AFDW) were found in diatoms. Limiting the availability of Si, a major component of the diatom cell wall, can induce lipid accumulation in diatoms. In green algae, lipid accumulation is induced by N starvation. N is a component of many cellular molecules, and N limitation would induce a complex response, affecting photosynthesis, protein and nucleic acid synthesis, and other biochemical processes. In contrast, Si is not involved in most intracellular processes, so the response to Si limitation should be simpler to interpret and control. The disadvantage to diatom cultivation is the added cost of Si supplementation in the medium for optimal growth, although this could be minimized by the use of strains with lightly silicified walls. Some green algal strains, for example M. minutum, are advantageous for mass culture applications in that they can survive temperature fluctuations that are lethal for diatoms. We found however, that the properties that make these organisms very hardy, such as a very tough cell wall, also make their biochemical and molecular studies problematic. Despite these generalizations, the ideal organism(s) for a biofuels production facility will likely be different for each location, particularly for growth in outdoor ponds. The best approach will likely be to screen for highly productive, oleaginous strains at selected sites,

optimize growth conditions for large-scale culture, and optimize productivity and lipid production through genetic manipulation or biochemical manipulation of the timing of lipid accumulation in the selected strains. It is also likely that more than one strain will be used at a site, to maximize productivity at different times of the year.

Significant progress was made during the last 15 years in the understanding of lipid accumulation in the microalgae, although there is still much to be learned. Clearly microalgal cells can be induced to accumulate significant quantities of lipid when the medium is limited for an essential nutrient. However, the actual mechanism that triggers the accumulation is unclear. Lipid accumulation is correlated with the cessation of cell division. A simple explanation is that lipid synthesis continues in the non-dividing cells, but since no new membranes are being synthesized, the lipid is shunted into storage lipids. Alternatively, non-dividing cells are not utilizing cellular energy reserves as rapidly as dividing cells, so lipid accumulates as synthesis occurs more rapidly than utilization. Nutrient deprivation affects specific biochemical pathways, as lipid accumulation is accompanied by an increase in the proportion of storage lipids (TAGs) to polar membrane lipids, and Si deprivation in diatoms increases the expression of at least one gene involved in lipid synthesis, acetyl-CoA carboxylase. In general, nutrient deprivation induces lipid accumulation in cells and is accompanied by a decrease in total (and total lipid) productivity. However, studies of lipid accumulation suggest that an understanding of the kinetics of the process could be critical and could allow the identification of a stage where biomass productivity and lipid levels are optimal for maximal lipid accumulation.

Significant progress was also made in understanding the molecular biology of microalgae. Many of the green algae were found to contain DNA with unusually high GC ratios, and often with unusual modifications that would make these organisms more difficult as targets for genetic engineering. In contrast, the DNA content of diatoms is more typical of other eukaryotes. Work at NREL by ASP researchers resulted in the cloning and characterization of several genes involved in lipid and carbohydrate accumulation in diatoms, including the ACCase gene and a “fused” gene encoding the enzymes UDPglucose pyrophosphorylase and phosphoglucomutase. Isolation of these genes facilitated the development of a genetic transformation system for the diatoms. These genes were used in preliminary attempts to manipulate lipid production in these organisms. The successful development of the transformation system led to an increased understanding of the factors involved in introducing and expressing foreign genes in these organisms, and should facilitate the development of similar methods for other algal strains.

There is still much to be done in the area of microalgal strain development for lipid or biofuels production. A number of suggestions for possible research areas will be discussed in the following sections.

The first step in transformating any organism is getting the foreign DNA inside the cell. For organisms with a cell wall, methods must be devised to either remove or permeabilize the wall, or to get DNA into the cell through the intact wall. Bacterial cell walls do not seem to represent a significant barrier to DNA uptake, and can be induced to take up foreign DNA simply by being washed in low osmotic medium and glycerol, followed by a brief heat shock. Cell walls can be removed enzymatically from yeast cells to form spheroplasts, or from plant cells to form protoplasts. These wall-less cells can be induced to take up DNA by chemically permeabilizing the cell membrane with polyethylene glycol and/or calcium. Alternatively, DNA can enter yeast spheroplasts or plant protoplasts via electroporation, a method in which a rapid, high voltage electric pulse is used to produce transient pores in a cell membrane.

Based on their research backgrounds, NREL researchers tended to view microalgae as either single cell plants, or pigmented yeasts. In either case, the initial tendency was to try to produce wall-less algal cells as targets for transformation. There had previously been some reports of protoplast production in green microalgae of the genus Chlorella (Braun and Aach 1975; Berliner 1977). NREL researcher Eric Jarvis decided to attempt to introduce foreign DNA into Chlorella protoplasts, with the eventual goal of adapting these protocols for other algal strains with biodiesel production potential.

The production of a stably transformed line of cells involves several steps, including introducing the foreign DNA into the target cell, expressing the foreign gene, stabilizating (replicating) the new DNA by the host cell, and survival and proliferation of the genetically altered cells. Transient expression assays can be used to monitor and optimize just the first two of these processes, i. e., DNA entry and expressing a foreign gene in a population of cells, and thus can be useful intermediate steps in developing genetic transformation systems. Transient assays usually involve the introduction of a gene that codes for an enzyme detectable by a simple biochemical assay (often referred to as a reporter gene). Dr. Jarvis decided to use one such gene, the firefly luciferase gene, to monitor the entry and expression of foreign DNA into Chlorella protoplasts.

The alga used for these studies was C. ellipsoidea (strain CCAP 211/1 a, obtained from the Culture Collection of Algae and Protozoa, Freshwater Biological Association, United Kingdom). Protoplasts were produced using a protocol adapted from Global and Aach (1985). The cells were grown to early stationary phase, then incubated overnight in 10 mg/mL Cellulysin, a crude commercial preparation of the cellulose-degrading enzyme cellulase. Protoplast production was monitored by sonication of the treated cells in water; generally about 80% of the cells were disrupted by this treatment and were considered to be protoplasts. A plasmid containing the luciferase gene driven by plant regulatory sequences was introduced in the protoplasts by mixing the cells with the plasmid DNA for 30 minutes in the presence of 50 mM CaCl2 and 13% polyethylene glycol (mw 4000). The cells were washed and incubated in a regeneration medium overnight. The cells were then harvested and luciferase activity was monitored in crude protein extracts. Luciferase catalyzes the oxidation of luciferin with the production of a photon of light via the following reaction:

luciferase, Mg2+, O2

LUCIFERIN + ATP………………………. > OXYLUCIFERIN + AMP + CO2 + hv

The light produced can be monitored using a scintillation counter or a luminometer.

The results of these experiments are shown in Figure II. B.6. Luciferase activity was detectable in protoplasts treated with the luciferase plasmid, but not in protoplasts that had not been exposed to plasmid or to polyethylene glycol. Intact cells did not take up the DNA. There was a significant decrease in luciferase expression when carrier DNA was left out of the transformation reaction (“carrier DNA” is usually sheared genomic DNA from calf thymus or salmon sperm that is added to reduce the effects of cellular nucleases on the added plasmid DNA). Monitoring of the luciferase activity over time showed that the activity was maximal at about 24 hours after exposing the protoplasts to the plasmid; expression decreased over time and was virtually undetectable after 80-100 hours. Unfortunately, attempts to regenerate the protoplasts into viable walled cells were unsuccessful.

These results were important as they demonstrated the first successful steps in developing a genetic transformation system for microalga, including the production of viable protoplasts, the introduction of DNA into the protoplasts, and the expression of a foreign gene by the algal cells. This last point was very significant, as homologous genes were required to achieve transformation in another green alga (Chlamydomonas). The dogma in the field was that heterologous gene expression in green algae would likely be unsuccessful due to codon biases resulting from high GC contents. The work resulted in a publication (Jarvis and Brown 1991), and was the basis for later studies in which the luciferase gene was used to monitor promoter activities in Cyclotella (discussed later). However, attempts to adapt this procedure to algal strains with significance to the biodiesel project were unsuccessful. The composition of microalgal cell walls is highly variable between species and even between isolates of the same species. Some unsuccessful efforts were made to determine the enzymatic conditions for wall degradation for several oleaginous algal strains. However, the conclusion, in the words of the

project manager at the time, was that this was “an endless pit of fruitless endeavor”, and the decision was made to explore other methods of introducing DNA into microalgal cells. In addition, although low levels of luciferase expression were acheived in Chlorella, the decision was made to pursue the development of selectable marker systems that would allow the isolation of very rare individual transformants within a population of microalgal cells. This will be discussed in the following section.

Operation of the two large ponds (Weissman et al. 1989; Weissman and Tillett 1990, 1992) was initiated in August 1988. Ponds were inoculated with T. suecica and operated at relatively high mixing velocities (30 cm/s in the lined pond, 22 cm/s in the unlined pond) to reduce sedimentation. Productivities were only 11 and 10 g/m2/d, respectively, lower than in the small ponds, but with an unknown amount of algal biomass settling out. After loss of this alga, M. minutum was inoculated, and productivities were, again, somewhat lower in the larger than the smaller ponds.

Experiments were also carried out in the small pond, primarily to determine the best operating pH and pCO2 range to help minimize CO2 outgassing while maximizing productivity. At reduced CO2 levels (higher pH) a decrease of 10% to 15% in productivity was observed with three algal species tested. Another variable tested was a 2- versus 3-day dilution routine, which had no significant effect. In addition, six cultures were examined for productivity in Si — or N — deficient media. Only one strain exhibited significantly higher total (AFDW) productivities with nutrient deficiency, but no lipid data were collected.

The conclusions from this work were, in brief:

1. Power for pond mixing is within the expected range, and quite low (< 1 kW/ha).

2. Pond mixing should be in the 15-25 cm/s range, and pond depth 15-25 cm.

3. CO2 utilization efficiencies of near 90% overall should be achievable with little compromise of productivity, through operation at an optimal pH/pCO2 range.

4. Only preliminary 1,000-m2 pond operations were carried out during this year, hampered by design and operational problems, which lowered expected productivities.

5. Large-scale pond productivities of 70 mt/ha/yr are realistic goals for this process, though probably not at this site because of low seasonal productivities.

6. Very high, 50 g/m2/d, single day, productivities were observed on some occasions.

7. The small-scale ponds can be used to screen strains and optimize conditions.

The final report (Weissman and Tillett 1992) in this series on the New Mexico OTF operations, reported on the demonstration of productivity for the two large ponds for 1 full year, continuation of the small-scale pond operations, and improvements in mixing and carbonation. One major improvement in the system was an automated data recording and operations system.

Mixing was improved by improving the flow deflectors and increasing operating depths from 15 to 22.5 cm, which is probably a better depth for large-scale systems. Culture instability was a problem, particularly in spring because of greater temperature fluctuations, and resulted in low average productivity of only 7 g/m2/d for March through May. In contrast, the average productivity was 18 g/m2/d for June through October, decreasing to 5-10 g/m2/d in November (depending on onset of cold weather), and only about 3 g/m2/d in the winter months.

Overall productivity, including 10%-15% down-time for the ponds for repairs and modifications, was 10 g/m2/d, only one-third of ASP goals (Table III. B.3.). Clearly the major limiting factor was temperature, as smaller systems in warm climates have achieved annual yields two to three

times as high. A major conclusion from this work is that scale-up is not a limitation with such systems. Climatic factors are the primary ones that must be considered in their siting.

A countercurrent flow injection system was installed in the sumps resulting in a carbonation system that was essentially 100% efficient in CO2 transfer. Overall CO2 utilization was higher than 90%. The unlined pond performed nearly as well as the lined pond, with minor decreases in productivity (10%-20%), CO2 utilization efficiency (5%-10%) and a small increase in mixing power. The unlined pond consumed only 0.04 w/m2, allowing the entire 1,000-m2pond to be powered by the equivalent of a 40-w light bulb. Species stability in the lined and unlined pond exhibited no significant difference. This work clearly established the feasibility of using unlined ponds in microalgae cultivation. This was a critical issue, as plastic lining of ponds is not economically feasible for low-cost production.

In the small 3-m2 systems, two variables were investigated: Si supply and pH. Both are major cost factors in pond operation, due to sodium silicate costs and CO2 outgassing. They affect overall productivity as well as lipid production. For Cyclotella, for example, productivity was about 20 g/m2/d at pH 7.2 or 8.3, but only 15 g/m2/d at pH 6.2. As the higher pH range is preferred, where CO2 outgassing is minimal, this demonstrates the feasibility of operating such cultures within the constraints of a large-scale production system. Si additions could be halved with only a modest decrease in productivity, suggesting that Si supply could be reduced, particularly if low Si-containing diatoms are cultivated. Also Si limitation can be used to induce lipid production, as was demonstrated during this project, with lipid biosynthesis increasing as soon as intracellular Si content dropped, with a 40% lipid content being achieved. However, overall, lipid productivity did not increase as CO2 fixation limitation also set in. This remains as a major issue for the future (See also Section III. B.5.d.).

Table III. B.3. Long Term OTF Results from 0.1-Ha Raceways.

gm/afdw/m2/d: grams of ash-free dry mass per square meter per day.

Pond liner: YES indicates a plastic lined pond; NO indicates an unlined (dirt bottom) pond.

III. B.5.d. Conclusions

The performance of the large-scale system improved considerably in all aspects during the 2 years of operations. The parallel use of the smaller-scale ponds helped guide this research, in particular in selecting algal strains and identifying operating characteristics. The high CO2 utilization efficiency demonstrated in the small and large-scale ponds was another major accomplishment of this project.

The major limitation of this project was the overall low productivity in the large-scale ponds. This was due in large part to the adverse climatic conditions at this location, and the initial suboptimal nature of the large-scale pond operations. Even so, productivities were lower than anticipated, with annual average productivities only about one-third the projected productivities by the ASP that would be required for minimal economics (see Section III. D.). This must be a major ongoing objective for future research, first in terms of overcoming the lower temperature limitations on productivity, and second by relocating this type of process development to more favorable climatic sites. (See Section III. B.6. for a discussion of temperature effects.)

But perhaps the major limitation of this project was that it did not carry out a longer-term process development effort. Although 2 years of data were collected for the large-scale ponds, the rapid advances made suggested that further research would have allowed continued improvements in performance and increased understanding of the overall process in specific critical areas of culture maintenance.

The engineering evaluation of the operation of the 0.1-ha raceway ponds showed these systems to be potentially very efficient in terms of energy, water, nutrient and CO2 utilization, and even basic construction cost inputs. Most important, the absence of liners did not significantly reduce pond performance (e. g., productivity). This was a major observation of this project, giving greater confidence in the engineering analysis and cost projections carried out by the ASP and DOE, discussed again in Section III. C.

A major uncertainty in this project was the nature of the species control achieved. A review of the data would suggest considerable success with species control, with several species cultivated successfully for relatively long periods. However, considerably more research will be required on this subject, as the tools were not available to allow a closer study of possible population dynamics (e. g., strain selection and even replacement) within the ponds. Thus, the subject of species control still requires considerable effort, as discussed further in the following section.

During FY1976-1977, the project described in Section III. A.2. continued with the same overall objective: to determine what pond operating factors could allow control over algal species, and thus permit cultivation of algal types that allow low-cost harvesting by microstrainers. The biomass recycling process described earlier continued to be studied, using the 12-m2 rectangular ponds and various pond operating strategies (mixing speeds, retention times, biomass recycle fraction), to test for their influence on microalgae species composition and productivity. Mainly the four 12-m2 ponds were used, with some initial experiments with the large 0.25-ha pilot pond.

An extensive series of experiments was carried out in the small ponds, with daily analysis of suspended solids (SS) concentrations (the best measure of algal biomass, although some, 10%- 15%, contributions from wastewater and bacterial solids could not be avoided). Other parameters measured, less frequently, were chlorophyll concentrations, BOD5, P, ammonia, and, microscopic algal counts, including cell dimensions. Experimental pond operating parameters tested included retention time (hydraulic dilution) and depth (though this was typically 25 cm), mixing speed, and biomass recycle ratios.

Both at short and long retention times the algal cultures invariably became unharvestable with microstrainers. Intermediate hydraulic retention times selected for larger colonial algal species that were more readily harvestable. However, long retention times also resulted in low productivities. There was an optimum residence time, which varied with depth of the culture and climatic variables that selected for harvestable cultures. However, biomass recycling was only marginally effective in improving biomass harvestability by microstraining. Mixing speeds >15 cm/s also improved harvestability by microstraining. Mixing speeds of 15 to 30 cm/s tended to induce algal flocculation.

Problems were encountered with zooplankton grazing of the algal cultures. Coarse (150-pm) screens did not effectively remove the grazers. Shorter retention times reduced grazer pressures, but also made the cultures less harvestable by microstrainers. In all the ponds, Scenedesmus dominated in the winter and spring, and then was replaced with Microactinium. Loss of dominance correlated with the breakup of the colonies, which may have been related to zooplankton grazing. The best productivity was 13.4 g/m2/d, during a 10-month period, irrespective of harvest efficiency. For the most harvestable pond, productivity was only 8.5 g/m2/d (of which only 7.2 g/m2/d was harvested by the microstrainers). Clearly, optimizing for productivity and harvestability required quite different operating conditions. It was concluded that the use of microstrainer harvesting and biomass recycling was unlikely to lead to both a high algal productivity and effective harvesting process.

After growing and harvesting an algal culture on sewage, enough nutrients remain to grow a second crop of microalgae. Such a second crop would then deplete available N. Due to excess inorganic and organic phosphates in sewage, sufficient P remains after harvest of the second algal crop to allow cultivation of additional batches of N-fixing microalgae. Of course, due to C

limitation, CO2 must be supplied to these cultures. Such a process, which would achieve so-called tertiary wastewater treatment (nutrient removal) is shown schematically in Figure

III. A.3., and was demonstrated during this project. Considerable problems were encountered with the secondary ponds (shown as a small box labeled “green algae” in Figure III. A.3.), due to culture instabilities, lack of sufficient algal removal in the first stage, grazers, etc. Figure III. A.4. shows a composite of the general results obtained, with a 7-day batch cultivation, at which point the culture settled quite well and, from its yellowish color was apparently N limited. The final, N-fixing, stage in this process was demonstrated under the following year’s project and a NSF-RANN funded project (Benemann et al. 1977).

In May 1977, cultures were started in the 0.25-ha high rate pond. That pond, mixed (poorly) with centrifugal pumps located in a 2.5-m deep sump, exhibited rather poor hydraulics. Sewage supply limitations resulted in longer retention times than desired. Despite these and other operational problems, the results were “reasonably consistent with the smaller 12-m2 ponds, both in productivity and harvestability responses to detention time” (Benemann et al. 1978). Productivities of 19 g/m2/d were observed over an 18-day campaign in summer, with a retention time of 3.5 days. Interestingly, zooplankton grazing was not as big a problem as with the smaller ponds.

These 0.25 ha pond algal effluents were also tested for settling, in a “pond isolation” experiment, in which cultures were removed from the pond and held for as long as 3 weeks in a settling pond. In one experiment, when the culture had been grown at a dilution rate at 0.22 day-1, it settled more than 90% in one day, while a culture grown at a dilution rate of 0.5 day-1 required 3 weeks to settle. This suggested another approach to algal harvesting, which became the focus of the project described in Section III. A.4.

Algal 8iomass