III. A.1. Introduction

The concept of microalgae biomass production for conversion to fuels (biogas) was first suggested in the early 1950s (Meier 1955). Shortly thereafter, Golueke and coworkers at the University of California-Berkeley demonstrated, at the laboratory scale, the concept of using microalgae as a substrate for anaerobic digestion, and the reuse of the digester effluent as a source of nutrients (Golueke et al. 1957; Golueke and Oswald 1959).

Oswald and Golueke (1960) presented a conceptual analysis of this process, in which large (40- ha) ponds would be used to grow microalgae. The algae would be digested to methane gas used to produce electricity. The residues of the digestions and the flue gas from the power plant would be recycled to the ponds to grow additional batches of algal biomass. Wastewaters would provide makeup water and nutrients. The authors predicted that microalgae biomass production of electricity could be cost-competitive with nuclear energy.

This concept was revived in the early 1970s with the start of the energy crisis. The National Science Foundation-Research Applied to National Needs Program (NSF-RANN) supported a laboratory study of microalgae fermentations to methane gas (Uziel et al. 1975). Using both fresh and dried biomass of six algal species, roughly 60% of algal biomass energy content converted to methane gas.

With the establishment of ERDA, the NSF-RANN activities were transferred to this new agency, which initiated a program in biomass fuels production. The Fuels from Biomass Program at ERDA funded a new project at Berkeley to develop a microalgae wastewater treatment and fuel production process. This project, started in 1976, was carried out at the Richmond Field Station of the University of California-Berkeley, and continued for about 4 years in parallel with several related projects. These projects included an ERDA-funded biophotolysis project (reviewed in Benemann et al. 1980), a NSF-RANN project on N-fixing blue-green algae (cyanobacteria) for fertilizer production (Benemann et al. 1977), and an EPA-funded project on algal bioflocculation in oxidation ponds (Koopman et al.1978; 1980).

The initial objective of the ERDA microalgae fuels project was to develop methods by which particular species of microalgae could be maintained in open ponds used for wastewater treatment. There are many large (>100 ha) and many hundreds of smaller wastewater treatment pond systems in California and elsewhere in the United States. The problem addressed by this project was the removal (harvesting) of the algal biomass from the effluents. Not only did the algal biomass represent a potential resource for the production of biogas, but the algal solids discharged from the ponds were pollutants that resulted in eutrophication and dissolved O2 reduction in the receiving bodies of waters. Thus, there was considerable interest in lower-cost and less energy-intensive microalgae harvesting technologies and wastewater treatment processesn general. In the absence of cost-effective microalgae harvesting technologies, microalgae pond systems, although widely used, could not meet the increasingly stringent wastewater treatment plant discharge standards, specifically in regards to suspended solids (mainly algal cells). A process that could reduce algal solids in pond effluents would have a ready market and potential near-term applications.

This was the justification for the initial emphasis on wastewater treatment processes from microalgae production. However, the ERDA/DOE Fuels from Biomass Program soon shifted its emphasis towards large-scale biomass production systems, having multi-quad (quad = 1015 Btu) impacts. This accounted, in part, for the early emphasis by this program on large-scale biomass “energy plantations” and even immense ocean energy farms, which some thought would provide solutions for the perceived U. S. energy crisis (Benemann 1980).

Large effects on U. S. energy supplies would probably not be plausible with wastewater treatment systems, which in aggregate represent a maximum potential of perhaps only one- or two-tenths of a quad of fuels (Benemann et al.1998, in preparation), a fraction of 1% of U. S. energy needs. However, wastewater systems can arguably serve as an initial step in the long-term development of larger, stand-alone systems. Although this argument was controversial at the time, the Univeristy of California-Berkeley project continued to emphasize wastewater treatment systems in its R&D. However, the supporting economic analyses carried out by the ASP (see Section

III. D.), started to focus on very large-scale, stand-alone systems. Section III. A. of this report reviews the algal mass-culture projects that were supported by ERDA and DOE before the ASP was initiated.

This report (Nelson 1982) was the first analysis of the availability of CO2 specifically for microalgal mass culture. There was, and still is, considerable interest in CO2 sources for EOR (enhanced oil recovery). Thus, a significant body of literature had developed in the preceding 5 years, particularly for the southwestern United States, where several small pipelines were built to deliver CO2 for EOR. One study even considered the production of CO2 from a power plant flue gas for such a purpose. The Argonne report concluded that there would be little extra available CO2 for microalgae production until the time for EOR had passed, about the year 2020. Of course, since then declining oil prices, and increasing interest in CO2 mitigation, have changed this situation. The report also concluded that flue gas sources would be a poor source for CO2 for the microalgae ponds, as power plants were not generally located in a suitable area for microalgae cultivation. The authors also concluded that the delivery of pure CO2 would be expensive, even after CO2 became available after the EOR era.

I Publications:

Nelson. (1982) “An Investigation of the availability of carbon dioxide for the production of microalgae lipids in the southwest.” Report to the Argonne National Laboratory, unpublished.

Laws and Terry (1983; see also Laws 1984a) reported on the further development of the ARPS. Four 9.2 m2 experimental raceways were built to allow replication of experiments and testing of variables, again using P. tricornutum. These raceways were used in a multifactorial experimental design testing eight variables at two levels each:

1. depth (5 and 10 cm),

2. dilution rate (0.2 and 0.4 d-1),

3. night time temperatures (15°or 20°C),

4. flow rates (15 and 30 cm/s),

5. CuSO4 filters (present and absence),

6. pH (8 and 9),

7. N source (ammonia and urea), and

8. salinity (15 and 35 ppt).

Sixteen runs (of 256 possible) in four sequential blocks were carried out, with the assumption apparently being that these variables are non-interacting and additive (probably a poor assumption for biological processes). No block-to-block controls were provided, which could have been affected by light intensity and other variables.

The ponds had been equipped with six sets of mixing foils, but rather suprisingly the presence or absence of the foils was not a variable tested. A complex data evaluation, in terms of “factor effects”, was presented, but no actual productivity data for any of the experiments are available. The authors concluded that “by far the most significant factor affecting biomass production” was culture depth, arguing that the “self-shading effects were more than offset by higher areal standing crops.” This was a rather puzzling conclusion as it is contrary to both theory and experience, which assumes that, everything else being equal, depth should not affect productivity. Of course, depth can affect pH, temperature, pO2, and other variables, which if not held constant will indeed affect productivity. But these should have been second-order effects, not the overriding factor in determining productivity.

Although no actual productivity data were reported, Laws (1984) stated that this factorial experiment demonstrated maximum productivities of about 25 g/m2/d, corresponding to an approximately 5% light conversion efficiency. The author claimed that this was 50% to 100% better than achieved with “conventional culture techniques,” though the basis for such extrapolations or comparisons was unclear.

In recent years, there has been increasing interest in greenhouse gas mitigation technologies. As a consequence, there has been renewed interest in microalgae mass culture and fuels production from the perspective of CO2 utilization. This is not a new concept, as Oswald and Golueke (1960) had previously emphasized the potential for microalgae systems to reduce and avoid CO2 emissions and thus reduce the potential for global warming. Indeed, microalgae have a rather unique attribute: they can utilize concentrated CO2 for growth, rather than the air-levels of CO2 used by higher plants. This could allow the culture of microalgae on power plant flue gases, probably the only method for directly using such CO2 sources. Of course, once the microalgae biomass is converted to, and used as fuel, this CO2 is again released. However, an equivalent amount of fossil fuel is not burned and fossil CO2 released into the atmosphere, reducing overall CO2 emissions.

In the early 1990s, some ASP work at NREL was supported by the Pittsburgh Energy Technology Center, PETC (now FETC, Federal Energy Technology Center, see Section III. D.9.). PETC also contracted with the University of California-Berkeley to analyze microalgae systems for power plant flue gas utilization and CO2 mitigation (Benemann and Oswald 1996). This report updated and extended the earlier cost analysis reviewed above. In particular it reanalyzed the assumptions on which these studies were based, and the costs for the various system components. For example, the costs of laser grading and earthworks were independently cost estimated through contacts with agricultural engineers with expertise in constructing rice paddies in northern California. Paddle wheel costs were based on the experience of the principal investigator (WJO) with large unit designs for waste treatment ponds. Pond walls and dividers were simple earthworks, much cheaper than the Weissman and Goebel (1987) design. Among the process innovations introduced was the use of a three-phase centrifuge to separate the algal lipids from the water and other biomass fractions. This provides a relatively straightforward method for lipid recovery (a major issue in prior studies) at only marginally higher costs than the centrifuge earlier specified for final concentration. However, overall this analysis was derivative of the prior studies.

Table III. D.6. summarizes the costs projected by this analysis. Both 20 and 60 g/m2/d productivities were assumed, with a high (40%) lipid biomass, equivalent to a 10% total solar conversion efficiency. Such very high productivities would clearly require a major R&D breakthrough. The theoretical approaches to such advances were reviewed. A reduction in light harvesting (“antenna”) pigments would increase the photosynthetic efficiencies at high light intensities. Microalgae with reduced antenna pigments would, however, not be very competitive in large-scale algal pond systems, and thus would be subject to contamination. However, nutrient limitation, required in any event to maximize lipid content in the algal cells, could be used as a strategy to limit such contaminants.

Both direct flue-gas utilization near the power plant and remote use of CO2 captured from flue gas and piped to the algal ponds were considered. With projected oil prices of $25/bbl and productivities of 5% solar conversion and 30 g/m2/d assumed to be achievable in the near-term, projected cost were $77/mt to $100/mt CO2 avoided, similar to other direct flue gas mitigation options. With higher productivities (60 g/m2/d) and oil prices ($35/bbl), CO2 avoidance with microalgae costs could drop below $ 10/mt CO2, a very competitive cost compared to other direct CO2 mitigation options. The estimates for CO2 mitigation are summarized in Table III. D.7.

This report also estimated the costs of providing a significant inoculum for the culture systems, taking as the example the cultivation of B. braunii. This would involve producing small amounts of inoculum under highly controlled laboratory conditions, then amplifying the cultures using increasingly larger, but less controlled and less expensive photobioreactors. Such a process would add some 10% to 30% to overall costs, depending on the amount and control over inoculum production desired. The microalgae industry and harvesting technologies were also reviewed in some detail.

A major emphasis in this report was the potential of microalgae CO2 utilization during wastewater treatment, recapitulating the work since the 1950s by this group (Section III. B.). Indeed, with CO2 mitigation being now the primary goal, rather than fuel production, what was before a cost could now add to the waste disposal credits of such systems. Microalgae wastewater treatment uses less energy, and thus fossil fuels, than conventional treatment processes, resulting in a reduction of greenhouse gas emissions. Wastewater treatment processes could provide a near-term pathway to developing large-scale microalgae production processes and could find applications around the world. With climate change a global problem, this now allows consideration of such international perspectives, even within a DOE-funded R&D program.

The applications of microalgae to CO2 mitigation from power plants became a major focus of the ASP during the 1990s, as briefly reviewed in the following section.

I Publications:

Benemann, J. R. (1993) “Alternative recommendation regarding biological CO2 utilization R&D.” In The Capture, Utilization and Disposal of Carbon Dioxide from Fossil Fuel Power Plants (Herzog, H., et al., eds.), U. S. Dept. of Energy, DOE/ER-30194/1, 56 pp.

Benemann, J. R.; Oswald, W. J. (1966) “Systems and economic analysis of microalgae ponds for conversion of CO2 to biomass.” Final Report, Pittsburgh Energy Technology Center, Grant No. DE-FG22-93PC93204.

Herzog, H., et al. (1993) “The capture, utilization and disposal of carbon dioxide from fossil fuel power plants.” Report, U. S. Dept. of Energy, DOE/ER-30194, vol. 1 and 2.

Table III. D.6. Summary of cost analysis of microalgae CO2 mitigation.

400-ha system, @ $0.065/kWh for power sales.

(Source: Benemann and Oswald 1996.)

Table III. D.7. Greenhouse gas balances and mitigation costs.

400-ha system, @$0.065/kWh.

(Source: Benemann and Oswald 1996.)

Productivity Assumptions 30 g/m^/day £0 ghn^/day

CO2 Flue Gas CO2 Flue Gas

1. Gross Power Produced kWhr/ha-yr 52,000

2. Net Power Exported kWhr/ha-yr 26,500 10Д00

3. CO2 mitigated from #2, mt/ha-yr 23 9

4. CO; due to fertilizers, etc, mt/ha/yr

5. CO2 mitigated before oil mt/ha/yr

5. Algal oil outputs barrel/ha-yr

6. CO2 mitigated from oQ, mt/ha-yr

7. Net CXZ>2 avoided, mt/ha-yr

8. Net CX>2 avoided, mt/barrel oil

9. $/barrel algal oil (from Table 83)

10. Net cost of CO2 avoided S/mt

for $25/barrel ofl for $35/barrel ой

5 10

18 4 63 35

Several oleaginous microalgal strains had been identified as potential candidates for biodiesel fuel production. These organisms became the target of genetic engineering efforts to manipulate the lipid biosynthetic pathways. Before the work on genetic transformation of algae at NREL, very little information was available on the molecular biology of these organisms. One of the first steps was to develop techniques to isolate and purify DNA from these organisms. A desirable protocol would disrupt the cell wall using methods gentle enough to prevent shearing of the genomic DNA. This was not trivial for some species, such as Monoraphidium, which has a very resistant wall that contains sporopollenin. A method that worked for most species tested (described in Jarvis et al. 1992) was developed based on a protocol used to isolate yeast DNA (Hoffman and Winston 1987). The cells were suspended in buffer that contained 2% Triton X — 100 and 1% SDS, then added to a tube that contained glass beads and an equal volume of phenol:cholorform:isoamyl alcohol (PCI). The cells were agitated for 1 minute using a vortex mixer. The DNA in the aqueous phase was purified by re-extraction with PCI, ethanol precipitation, and treated with RNase A. For some species, the DNA had to be purified further by using precipitation with hexadecyltrimethylammonium bromide (CTAB; Murray and Thompson 1980) to remove contaminating carbohydrates or by purifying the DNA on CsCl gradients. This procedure produced DNA that digested well with many common restriction endonucleases, but even highly purified DNA would not digest well with all restriction enzymes.

NREL researcher Eric Jarvis theorized that poor digestion of the DNA by some enzymes could be attributable to characteristics of the DNA. All DNA is composed of four nucleosides; deoxycytidine, deoxyguanosine, deoxythymidine, and deoxyadenosine, (abbreviated dC, dG, dT, dA); in double stranded DNA, dC is always paired with dG, and dT with dA. The percentage of each nucleoside (often represented as %GC) is variable between species. Restriction enzymes cut DNA at specific nucleotide sequences, generally recognizing 4-6 bp motifs. Therefore, the frequency of cutting by a particular enzyme will be affected by the total nucleotide composition of the DNA (i. e., an enzyme that recognizes CCGG would cut infrequently in an organism with a low %GC). The GC content is also reflected in the codon usage by each organism, as DNA with a high GC content would show a bias toward codons ending with G or C in the variable third position. DNA can also contain unusual modified nucleosides, including 5- hydroxymethyldeoxycytidine (hm5dC) and 5-hydroxymethyldeoxyuridine (hm5dU), although the biological significance is unclear. Another common modification is the presence of methylated nucleosides, in particular 5-methyldeoxycytidine (m5dC) and 6-methyldeoxyadenosine (m6dA). The degree of methylation has been associated with levels of gene expression. In addition, some microorganisms use DNA methylation as a defense mechanism, in that methylated DNA sequences are often not recognized by endonucleases from invading pathogens. Although the presence of methylated nucleosides is characteristic for some species, the degree of methylation can vary on a short time scale with changing environmental conditions. In contrast, the %GC and presence of modified nucleosides are characteristic for a particular organism. These characteristics only on an evolutionary time scale.

DNA was isolated from microalgae strains, including 10 species from 5 classes. The nucleoside composition was analyzed by reverse-phase HPLC and by digestion with restriction endonucleases. The results of the HPLC analysis are summarized in Table I. B.4-1. Although the diatoms showed a GC content typical for most eukaryotes (42%-48% GC), the GC content of the green algae (excepting Stichococcus) was significantly higher. In particular, Monoraphidium DNA contains 71% GC. The table also shows the presence of m5dC in the algal DNA. All species tested contained some level of this modified base, although once again Monoraphidium stands out with approximately 11% m5dC. The only other unusual feature was the presence of 12% hm5dU in the dinoflagellate C. cohnii (data not shown); dinoflagellates were not considered to be good candidates for biodiesel fuel production, so this observation was not explored further.

These data provided a good background for developing genetic transformation systems for these organisms. As mentioned above, the GC content of an organism can be reflected in the codon usage, suggesting that an organism with a high GC content such as Monoraphidium may not successfully express heterologous marker genes. This was found to be true for the green alga Chlamydomonas; successful transformation of this organism was achieved only by the use of homologous selectable markers (discussed in more detail later). Also, GC content should be considered when designing synthetic DNA probes based on protein sequences, i. e., for isolation of algal genes by PCR. In addition, DNA methylation can affect the ability to construct DNA libraries and to clone algal DNA, and may require the use of bacterial host strains that are insensitive to DNA methylation.

III. B.5.a. Facility Design and Construction.

After the above noted projects carried out in California, the ASP decided to hold a competition for the development of a larger process development outdoor test facility (OTF) located in the southwestern United States. Two independent designs and proposals were commissioned, one consisting of enclosed production units (Aquasearch, Inc., Dr. Mark Huntley, Principal Investigator); the other of open ponds, similar to the design tested in California (Microbial Products, Inc., J. C. Weissman, Principal Investigator). Microbial Products, Inc., won this competition, with a proposed facility consisting of two 1,000-m2 ponds, one plastic lined and another unlined, as well as supporting R&D using six small, 3-m2 ponds, continuing and extending the work carried out in the prior projects in California (Weissman and Goebel 1987. See Section III. D. for a discussion of this engineering /cost analysis. No report for the Aquasearch proposal is available.).

Although the proposal recommended establishing this facility in Southern California, the ASP selected a site in Roswell, New Mexico to establish the OTF. The project was located at an abandoned water research facility. Roswell has high insolation, abundant available flatland and supplies of saline groundwaters. The primary limitation of this site was temperature, which, in retrospect, turned out to be too low for more than 5 months of the year for the more productive species identified during the prior project.

The objective of the first year of the research at this new site was to initiate a species screening effort at this site with the small 3-m2 ponds, which were installed while designing and constructing the larger facility. A major objective of this project was to identify cold weather — adapted strains (Weissman et al. 1987).

Building the large system required installation of two water pipelines of 1,300-m in length (15 and 7.5 cm, for brackish and fresh waters). The ponds were about 14 x 77 m, with concrete block walls and a central wooden divider. The paddle wheels were approximately 5-m wide, with a nominal mixing speed of 20 cm/s, and a maximum of 40 cm/s. Carbonation was achieved with a sump that allowed counterflow injection of CO2, to achieve high (90%+) absorption of CO2. One pond was plastic lined; the other had a crushed rock layer. The walls were cinder block. A 50-m2 inoculum production pond was included. Figure III. B.9. shows an overview of the layout of the completed facility.

IV. B.2 a. Biodiesel Production and Algal Mass Culture for Wastewater Treatment

The laboratory work outlined earlier will be a relatively longterm effort. Even after the demonstration at the laboratory and small-scale (e. g., 1 m2) of the ability of genetically improved microalgae strains to exhibit high lipid productivities, many other factors and abilities are still required in algal mass culture. These, however, cannot be demonstrated in the laboratory. They include competitiveness, predation resistance, and harvestability. The issue thus arises of the need to carry out such algal mass culture research in parallel with the laboratory studies.

The outdoor projects of the ASP demonstrated the ability to mass culture microalgae under relatively unrestricted conditions (e. g., without a highly selective chemical environment), and to do so potentially at relatively low cost, as the inputs for power, nutrients, and water are rather modest. Considerable advances were also made in developing techniques for managing microalgae species in ponds, and demonstrating increasing biomass productivities. Of course, these subjects still require much more work. However, in the absence of improved microalgae strains, with the high total and lipid productivities required by the cost analyses, it would not be possible to make significant advances in this technology. Thus, a continuing emphasis on outdoor algal mass cultures, or on cost and resource analyses, is not recommended at this time. These accomplishments of the ASP now allow research to be focussed on the genetic work, as outlined above and in Section II, and to allow confident prediction of the ability to apply this research to mass culture systems. This is not to exclude some supporting outdoor studies, for example to verify the selection criteria used for the competitive strains for the genetic development. But, in general, the emphasis should be on genetics and strain improvements, not on outdoor culture technology development.

However, a strictly laboratory-based R&D program, may rapidly loose touch with the realities of the eventual applications. Thus, some outdoor mass culture R&D is recommended, specifically for near-term development and demonstration of a combined microalgae wastewater treatment— biodiesel production process. This recommendation is based on the potential for such systems to be developed and demonstrated rather quickly, and at relatively low cost. They would provide an early practical application of this technology, and justify the larger effort that would be required for the development of a significant microalgae-biodiesel industry. Benemann and Oswald (1996) present a detailed discussion of this approach.

IV. B.3. Conclusions

A microalgae biodiesel production system must be a solar conversion device, which operates at high efficiency and with minimal inputs at overall low cost. Cost constraints restrict consideration of such systems to the simplest possible devices, which are large unlined, open, mixed raceway ponds. Several decades of R&D in this field, in particular by the ASP, have revealed no plausible alternative to this basic design. Even some of the design details, such as the mixing devices (paddle wheels), depth (15-25 cm), mixing velocity (15-25 cm/s), CO2 transfer (countercurrent sumps), and others are fixed by the engineering and economic constraints. The commercial experience with open mass culture ponds suggests that such systems require relatively little further engineering development. Certainly, it would be of interest to determine the practical limits of such systems. Can single raceway pond scales be larger than 5 ha? What are the wind effects in such large systems? But overall, the engineering and hardware for the low-cost mass culture of microalgae cannot be considered a major R&D need in this field.

Any effort toward the development of closed photobioreactors is probably too high risk in the present context. Although such devices could have a role in the buildup and production of

inoculum (starter cultures), they are not likely to be an essential or crucial component of large-scale, low-cost microalgae culture processes for energy production.

Any future R&D program for microalgae CO2 capture and biofuels production must start with the development of the microalgae “biocatalysts.” The goal will be to construct strains via genetic engineering or other strain improvement methods that achieve very high solar conversion efficiencies and yield high lipid (oil) microalgal biomass, as required by the economic analyses. The central recommendation for a future R&D program is to emphasize such a biocatalyst development effort, building on the knowledge developed by the ASP. For a more near-term approach, there is a significant opportunity to develop and demonstrate microalgae biodiesel production as part of a wastewater treatment process; R&D in this area is also recommended. Finally, the international nature of the global warming problem now allows consideration of global impacts of such technologies. This could help justify a U. S., and an international, R&D effort, even if the impacts of microalgae biodiesel to future U. S. energy supplies were perceived to be modest.

[1] The Solar Energy Research Institute (SERI) became the National Renewable Energy Laboratory (NREL) in 1990. In this report, the laboratory may be referred to as either SERI or NREL, depending on the time period during which the work being described was performed.

[2] Diatoms. Diatoms are among the most common and widely distributed groups of algae in existence; about 100,000 species are known. This group tends to dominate the phytoplankton of the oceans, but is commonly found in fresh — and brackish-water habitats as well. The cells are golden-brown because of the presence of high levels of fucoxanthin, a photosynthetic accessory pigment.

Several other xanthophylls are present at lower levels, as well as P-carotene, chlorophyll a and chlorophyll c. The main storage compounds of diatoms are lipids (TAGs) and a P-1,3-linked carbohydrate known as chrysolaminarin. A distinguishing feature of diatoms is the presence of a cell wall that contains substantial quantities of polymerized Si. This has implications for media costs in a commercial production facility, because silicate is a relatively expensive chemical. On the other hand, Si deficiency is known to promote storage lipid

[3] Eustigmatophytes. This group represents an important component of the “picoplankton”, which are very small cells (2-4 pm in diameter). The genus Nannochloropsis is one of the few marine species in this class, and is common in the world’s oceans. Chlorophyll a is the only chlorophyll present in the cells, although several xanthophylls serve as accessory photosynthetic pigments.

2 T aken from the Proceedings of the April 1984 Aquatic Species Program Principal Investigators’ Meeting.

[4] hexane: acyclic hydrocarbons

[5]

NREL Microalgae Culture Collection strain designations are provided when relevant.

[6]The development of the SERI standard media is discussed in Chapter II. A. 1. The compositions of these media are given in T able II. A. 1.

[7] The viruses are highly infectious and grow rapidly within the algal cells. Algal growth is inhibited rapidly following viral infection. Synthesis of host DNA and RNA is shut down, and the organellar and genomic DNA is degraded. Viral gene expression entails transcription of early and late genes, and may include

[8] Imhoff Cone removals are 24-h laboratory settling tests, indicating percentage of algal biomass that spontaneously flocculate and settle (“bioflocculation”) under quiescent conditions after the culture is removed from the mixed ponds.

[9] Oocystis sp. (S/00CYS-1)

[10] Isolate and study strains suitable for mass cultures, then apply the productivity enhancement techniques developed under laboratory conditions to these strains.

This project addressed the problem of the uncontrolled nature of the algal populations in wastewater treatment ponds. Because the dominant algal species found in a pond could range from small unicellular to large colonial or filamentous species, harvesting of the algae for biomass conversion would require a universally applicable harvesting technology, such as centrifugation or chemical flocculation, to enable the recovery of any algal type. However, these processes are very expensive (Golueke and Oswald 1965; see also Shelef 1984 for an ASP supported assessment, and Benemann and Oswald, 1996 for a recent review). If, however, algal species could be controlled in the ponds, then filamentous microalgae species might be grown that would be easier and cheaper to harvest using microstrainers. Microstrainers, which are rotating screens (typically 25 to 50 pm openings) with a backwash, are already widely used for removing filamentous algae, mainly filamentous cyanobacteria (blue-green algae) from potable water supplies. Thus, the first objective of this project, initiated in 1976, was to investigate how to selectively cultivate filamentous microalgal species in waste treatment ponds (Benemann et al. 1977).

The first issue was that conventional waste treatment ponds are generally deep (2 m), and unmixed. Such ponds do not maximize algal productivity, nor do they provide a uniform hydraulic flow or physical-chemical environment. Thus, this project focused on the use of shallow, mixed, raceway-type ponds, the “high rate pond” of Oswald (1963), for microalgae

roduction and demonstration of algal species control in wastewater treatment. Initially four small (approximately 3 cm2) and, four larger (12 m2) rectangular, paddle wheel-mixed ponds were built and used. The Richmond Field station also had a large pilot-scale (0.25 ha) shallow high rate pond that was fed settled (primary effluent) municipal wastewater, available for this research.

A fundamental theory of species control was developed based on selective recycle of harvestable biomass (Figure III. A. 1.). The concept is that harvesting filamentous algae and recycling part of the biomass back to the ponds (similar to the process of activated sludge), would favor the slower-growing filamentous algae over the faster-growing unicellular types (which would thus get washed out of the system). This theory was both mathematically proven and experimentally demonstrated in the laboratory in competition experiments with mixed cultures of Chlorella (a unicellular green alga) and Spirulina (a filamentous blue-green alga). Without biomass recycle Chlorella always out-competed Spirulina; with biomass recycle Spirulina could be made to dominate the culture (see also Weissman and Benemann 1978).

This process was also tested in outdoor ponds. Experiments with Spirulina grown on wastewater were not encouraging, as this species dominates only at high salinities and alkalinities. Thus, the first issue was the source of algal species for the experiments. A pond in the city of Woodland was found to have a dominant culture of the filamentous cyanobacterium Oscillatoria. It was decided to use this alga in the initial tests. First the alga was isolated from a small pond sample and grown in the laboratory. It was then inoculated into a small circular pond fed with settled Richmond algae. The results of the first experiment are shown in Figure III. A.2. The Oscillatoria culture grew, but the unicellular organisms grew faster. Only by completely harvesting the entire culture and recycling all of the biomass retained in the microstrainer (almost pure Oscillatoria), could the culture be maintained.

It was thought that perhaps the laboratory cultivation stage had selected for a laboratory-adapted strain that did not do well outdoors. A small microstrainer was taken to Woodland, and enough Oscillatoria biomass was collected and returned to the Richmond Field Station to inoculate one of the small circular ponds. The pond was diluted about one-third per day with wastewater, with about 40% of the algae harvest recycled. However, after a couple of weeks, a new algal species became dominant. This species, Micractinium, a colonial organism covered with large spines (thought to be a protection against zooplankton grazers), also was captured by the (26-pm opening) fabric of the microstrainers and recycled. This gave a Micractinium competitive advantage over the unicellular forms, but also over the Oscillatoria. Several more inoculations of Oscillatoria gave similar results, with Micractinium dominating.

However, even Micractinium could not be stably cultured with recycling, as during apparently unfavorable conditions it washed out (probably due to loss of spines) and then was replaced by Scenedesmus. On the other hand, when conditions were favorable for Micractinium, biomass recycle helped the culture dominate the ponds faster, but, regardless of recycling, this alga replaced Scenedesmus. Thus, the theory worked in principle, but in practice selective biomass

recycle could be only one, and not a sufficient, tool for controlling microalgal species in such ponds.

Biomass productivities equivalent to a total solar energy conversion efficiency of about 2% (about 15 g/m2/d) were achieved for about 1-week periods. Effluents from the more harvestable cultures were below the EPA wastewater treatment system discharge limits of 30 ppm suspended solids (SS), with reductions of over 80% for ammonia and 50% for total organic C. A preliminary economic analysis was also presented (Benemann et al. 1977).

This study (Maxwell et al. 1985) assessed the availability and suitability of land, brackish water resources, and climate in the southwestern United States. The objective was to “stratify the Southwestern United States into zones of varying suitability for such [microalgal] systems.” The Battelle Columbus report discussed earlier (Vignon et al. 1982) was identified as a companion effort, although it was “not directly supportive of the stratification effort… ” Climate, land resources, and water resources maps based on various process constraints and characteristics (e. g., freeze-free period, land slope, water depth) were overlayed to develop suitability maps. Because of insufficient water data, water supply was excluded from the final analysis. Available maps were also inadequate for land classification for slopes <10%.

This report includes an excellent discussion of the sources of maps and information about water, water rights, land ownerships, slopes, topography, climate, etc. Of course, in the dozen (or more) years since this research was carried out, the computer revolution has made access to this data easier or at least different. The report provides a very comprehensive review of the problem, and supplies a large amount of specific information. One interesting point is that microalgae systems will likely encroach on pasture and non-irrigated agricultural land; much of this land has low productivity and value, but is generally accessible and already generally serviced by roads and power.

A relative productivity map was developed for the southwestern United States, by combining frost-free days with insolation values. Many other such maps, including land suitability and water availability, were prepared, including a final overall suitability assessment (Figure III. C.1.). The authors proposed continuing such an assessment, pointing out the many limitations of the present study (such as water laws and rights issues) that required further studies. However, a point of diminishing returns is likely to be reached. It may be best to evaluate some very specific areas, even sites, for actual suitability for such a process. Indeed, any generic analysis may miss important details. For example, as indicated in Section III. B. from the experience at Roswell, New Mexico, that location is quite unsuitable for microalgae production, due to its short (approximately 200-day) growing season, although it appears in the overall suitability map (Figure III. C. 1.). This was likely due to insufficiently restrictive temperature criteria (freeze-free days, etc.).

I Publications:

Maxwell, E. L; Folger, A. G.; Hogg, S. E. (1985) “Resource evaluation and site selection for microalgae production systems.” Report, Solar Energy Research Institute, Golden, Colorado, SERI/TR-215-2484.

Figure III. C.1. Overall suitability map for microalgae culture in the Southwestern United States.

Zones of relative suitability for microalgae biomass production based on compositing of climate, land and water suitability maps. (Source: Maxwell et al. 1985.)

During this year a number of experiments were carried out to determine productivity of the cultures as a function of several variables, this time studied independently (Laws 1984b, c; see also Laws et al. 1985). One task was to screen candidate species in the laboratory for possible growth outdoors. However, as the author concludes, “the fact that a given species grows well in the laboratory is no guarantee that it will perform well in an outdoor culture system.” One reason the project switched to different algal species was that the P. tricornutum strain used in the experiments described above was quite sensitive to even moderate (above 25°C) temperatures, and required temperature control (cooling) of the reactors. A Platymonas sp. was thus tested without temperature control in the outdoor flumes, at several dilution rates and maximal pH levels of 7 to 8. This strain showed a maximum productivity of about 26 g/m2/d, about the same as observed with P. tricornutum with temperature control.

Another question raised was the reproducibility of the data obtained with the flumes. During this year all four flumes were operated under identical conditions for two periods, resulting in very similar productivities (5% and 10% standard deviations in the two separate experiments). The Platymonas sp. strain was then inoculated into the larger (48 m2) flume which was operated at various depths. A depth of about 12-cm gave the best results, producing about 26 g/m2/d during February 1984.

One important experiment carried out, again with the Platymonas sp., was to determine the effects of N and P limitation on algal lipid content. Lipids increased from less than 20% to almost 40% of dry weight upon combined N and P limitation. However, no actual productivity data were reported for this 2-week experiment.

The major experiment carried out this year (Laws 1984b, c), was the cultivation of Platymonas in the 48-m2 flume, over most of the year. One major variable was dilution, with the culture diluted every 2, 3, or 4 days. As seen in Figure III. B.4., maximal productivity (50 g/m2/d) was achieved by diluting the culture every 3 days. Further investigation of this phenomenon suggested that this was not an artifact, but a reproducible effect. Indeed, such a culture diluted every third day had twice the productivity on the third as on the first 2 days. This was a most unexpected, and controversial, result, and a major focus for the following years of this project.

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Figure III. B.4. Productivity as a function of dilution. (Source: Laws 1984.)