Many of our fossil fuel reserves, but especially coal, are going to play significant roles for years to come. On a worldwide basis, coal is, by far, the largest fossil energy resource available. About one-fourth of the world’s coal reserves reside in the United States. To put this in perspective, consider the fact that, at current rates of consumption, coal reserves could last for over 200 years.

Regardless of how much faith you put in future fossil energy projections, it is clear that coal will continue to play an important role in our energy future—especially given the relatively large amounts of coal that we control within our own borders. DOE’s Energy Information Administration estimates that electricity will become an increasingly large contributor to future U. S. energy demand. How will this new demand be met? Initially, low cost natural gas will grow in use. Inevitably, the demand for electricity will have to be met by coal. Coal will remain the mainstay of U. S. baseline electricity generation, accounting for half of electricity generation by the year 2010.

The long term demand for coal brings with it a demand for technologies that can mitigate the environmental problems associated with coal. While control technologies will be used to reduce air pollutants associated with acid rain, no technologies exist today which address the problem of greenhouse gas emissions. Coal is the most carbon-intensive of the fossil fuels. In other words, for every Btu of energy liberated by combustion, coal emits more CO2 than either petroleum or natural gas. As pressure to reduce carbon emissions grows, this will become an increasingly acute problem for the U. S.

One measure of how serious this problem could be is the absurdity of some of the proposals being developed for handling carbon emissions from power plants. The preferred option offered by researchers at MIT is ocean disposal, despite the expense and uncertainty of piping CO2 from power plants and injecting the CO2 in the ocean15.

Commonsense suggests that recycling of carbon would be more efficacious than deep ocean disposal. No one clearly understands the long-term effects of injecting large amounts of CO2 into our oceans. Beyond these environmental concerns, such large — scale disposal schemes represent an economic sinkhole. Huge amounts of capital and operating dollars would be spent simply to dispose of carbon. While such Draconian measures may ultimately be needed, it makes more sense to first re-use stationary sources of carbon as much as possible. Algae technology is unique in its ability to produce a useful, high-volume product from waste CO2.

Consumption of coal, an abundant domestic fuel source for electricity generation, will continue to grow over the coming decades, both in the U. S. and abroad. Algae technology can extend the useful energy we get from coal combustion and reduce carbon emissions by recycling waste CO2 from power plants into clean-burning biodiesel. When compared to the extreme measures proposed for disposing of power plant carbon emissions, algal recycling of carbon simply makes sense.

The next edition of the SERI Microalgae Culture Collection Catalog (1985-1986) included the following additional strains:

Table II. A.7. Microalgal strains added to the SERI Culture Collection Catalog, 1985­1986.

The 1986-1987 SERI Microalgae Culture Collection Catalog (including an addendum) added 29 more strains, bringing the total number of strains in the collection to 50. The 1986-1987 catalog included the following additional strains:

Table II. A.8. Microalgal strains added to the SERI Culture Collection Catalog, 1986­1987.

In 1985, the strain enrichment procedure utilizing the rotary screening apparatus described previously was modified to include incubation of samples in SERI Type I and Type II media (25 and 55 mmho^cm-1 conductivity) and in artificial seawater, in addition to the original site water. The cultures that exhibited substantial algal growth were further treated to isolate the predominant strains as unialgal (clonal) isolates. These strains were then tested for growth using the temperature-salinity matrix described earlier.

Collection activities.

Collection efforts by SERI researchers in 1985 again focused on shallow inland saline habitats. This time collecting trips were also made to New Mexico and Nebraska, in addition to Colorado and Utah. Eighty-six sites were sampled during the year, 53 of which were sampled in the spring. From these 53 sites, 17 promising strains were isolated. An analysis was conducted comparing the results of the new protocol with those that would have resulted from the protocol used in prior years. This analysis indicated that the revised protocol was in fact superior to the older protocol. For example, only six of the 17 strains selected via the new protocol would also have been selected using the old protocol. Only three of the 17 strains grew best in the artificial medium type that most closely resembled the collection site water; in fact, only six strains were even considered to grow well in the collection site water relative to growth in at least one of the artificial medium. This analysis clearly indicated the value of performing the initial screening and enrichment in a variety of relevant media. The results suggest that the shallow saline environments sampled probably contain a large number of species whose metabolism is arrested at any given time. In other words, the water quality of such sites varies greatly, depending on precipitation and evaporation, so probably only a few of the many species present are actively growing at any given time. This also may explain the wide range of salinities and temperatures tolerated by many of these strains.

The goal of the research performed by Dr. Schwartzbach and coworkers was to understand the biochemistry and physiology of lipid accumulation in microalgae, in particular the biochemical responses to N deficiency as a trigger for lipid accumulation. Lipid biosynthesis is dependent on the availability of fixed carbon and the activity of enzymes involved in lipid synthesis. These experiments were directed at understanding how these processes are affected by N limitation in the algal cells.

The first set of experiments analyzed lipid synthesis in the eustigmatophytes Nannochloropsis salina and Nanno Q, two oleaginous strains from the SERI Culture Collection. Similar results were obtained for the two strains. The basic protocol was to innoculate the algal cells into media containing either non-limiting levels or low levels of nitrogen (0.1 mM NaNO3), and to monitor cell growth, chlorophyll content, and the lipid levels per cell and per culture volume. In cultures containing low N, cell division ceased after 50-60 hours, and the cells entered stationary phase as the N was depleted. In contrast, cells grown with sufficient N continued to divide. In the N — replete cultures, the lipid content of the individual cells remained constant, and there was a steady increase in the amount of lipid per mL of culture as the cell number increased. In contrast, the N-deficient culture showed a significant increase in the level of lipid per cell. However, the lipid content of the culture per mL (or percentage of the AFDW composed of lipid) did not change. In N. salina, lipid made up 26%-32% of the AFDW, and in Nanno Q, lipid was 23%- 24% of the AFDW in cultures grown under N-replete and N-depleted conditions. These results indicate that N depletion causes the cells to stop dividing, while lipid synthesis continues. However, there is no net increase in lipid synthesis, and the trigger in these cells does not change the activity of enzymes involved in lipid biosynthesis. One caveat to these studies is that Dr. Schwartzbach measured only total lipid produced in the cells, including polar membrane lipids and nonpolar storage lipids; it is unclear from these studies and those described later whether N deficiency could differentially affect accumulation of the nonpolar lipids in these algae.

Another result from the studies on Nannochloropsis was that the level of chlorophyll in the cells declined rapidly in N depleted cells. Thus, N depletion would also presumably decrease photosynthetic efficiency and the availability of fixed carbon. The next set of experiments was designed to separate the effects of reduced photosynthetic efficiency from direct effects of N limitation on biosynthetic enzyme activities. To accomplish this, a series of experiments was performed using the eukaryotic green alga Euglena gracilis var. bacillaris Cori. Euglena can grow heterotrophically using ethanol as the sole carbon source. The growth of cells in the presence of externally supplied carbon (ethanol) should not be limited by decreased photosynthesis, so the rate of lipid synthesis would be solely limited by lipid biosynthetic capabilities.

Euglena is unique compared to most algae of interest to the ASP as potential producers of biodiesel. Euglena produces both lipid (primarily in form of the wax ester myristyl-miristate) and carbohydrate (the major product is paramylum, a P-1,3-glucan) as storage products. Using Euglena, a complicated series of experiments was conducted comparing the growth, lipid and carbohydrate content, and chlorophyll levels in algae under photosynthetic and heterotrophic growth conditions, as well as under aerobic and anaerobic conditions (Coleman et al. 1988b). Basically, cells were grown to N deficiency, then resuspended in fresh media containing either sufficient or limiting amounts of N. The new media also did, or did not, contain ethanol as a carbon source, and the cells were grown in the dark or in light.

As was seen with the Nannochloropsis strains, cell growth under N deficient conditions caused an increase in the levels of storage products (in this case, lipid plus carbohydrate) per cell. However, there was no net increase in total lipid/carbohydrate when measured as a percentage of
dry cell weight. This was true in cells grown autotrophically or heterotrophically. Nitrogen depletion caused the cells to stop dividing, but the storage products continued to accumulate in the cells at the same rate as in non-nitrogen limited cells. In addition, the proportion of carbohydrate and lipid was unchanged, thus there did not appear to be a N trigger effect, either directly or indirectly via carbon limitation, on the enzymes of the lipid or carbohydrate synthetic pathways. One caveat to this result was that in very old cultures, (i. e., 12 days after transfer of the cells to N-deficient media), the lipid as a percentage of the dry cell weight increased in all cultures. However, this was accompanied by a decrease in the total cell mass, and the lipids are apparently more stable than other cell components.

Growth of Euglena under N-deficient conditions resulted in loss of chlorophyll, as seen for Nannochloropsis. Dr. Schwartzbach also used two-dimensional gel electrophoresis to monitor changes in the levels of chloroplast and mitochondrial proteins under N-deficient conditions (Coleman et al., 1988a). Under photosynthetic growth conditions (high light), exposure of the cells to N-deficient conditions resulted in a decrease in the levels of 37 proteins identified as components of the chloroplast. Under low light conditions, there was little change in the population of chloroplast proteins. The degradation of the chloroplasts under low N conditions was presumably due to photooxidation of chlorophyll, accompanied by degradation of newly synthesized photosynthetic membrane proteins that could not assemble properly into the unstable chloroplast. Synthesis of chlorophyll requires N to form 5-aminolevulinic acid, a chlorophyll precursor. Although photooxidation of chlorophyll occurs constantly in the light, synthesis of new chlorophyll molecules also occurs to replace the degraded molecules. However, if N levels are depleted, new chlorophyll cannot be produced, and photosynthetic efficiency decreases. This result is important with regard to biodiesel production. It suggests that there would be limitations on the amount of lipid that could be produced in outdoor ponds using N limitation as a trigger for lipid accumulation even if carbon was not limiting (i. e., for cells grown in outdoor ponds).

One process that affected the biosynthetic pathways in Euglena and resulted in an increase in the total lipid in the cultures was cell growth under anaerobic conditions with ethanol as a carbon source (lipids increased from 5 -10% to 45% of the AFDW). Growth via anaerobiosis caused the activation of the oxygen-sensitive pyruvate dehydrogenase in the mitochondria. This led to increased levels of acetyl CoA in the mitochondria, which activates the mitochondrial fatty acid synthesis pathways. However, the increased flow of carbon to lipid synthetic pathways was accompanied by degradation of non-lipid components under anaerobic condition, including paramylum, the main storage carbohydrate, which resulted in a decrease in total cell mass. Dr. Schwartzbach estimated that if the anaerobic cells had increased in cell mass to the same extent as cells grown aerobically, the lipids would only compose 15% of the dry weight.

This observation that anaerobiosis could result in increased lipid yields by actually affecting the lipid biosynthetic pathway suggested that lipid synthesis could be increased by increasing the levels of the lipid precursors acetyl CoA and malonyl CoA. Little is known about lipid synthesis in algae, but data from other organisms suggested that pyruvate dehydrogenase and acetyl CoA carboxylase could function as regulatory enzymes in algal lipid synthesis. Understanding the biochemical factors that limit production of the lipid precursors could lead to biochemical or

genetic engineering strategies to increase the activity of these enzymes that could produce an organism with the ability to produce very high lipid levels. To this end, Dr. Schwartzbach initiated a project to isolate and characterize these enzymes from several algae, including Euglena, N. salina, Nanno Q, and Monoraphidium 2 (Smith and Schwartzbach 1988). They reported some very preliminary information on protein extraction techniques and assay techniques for these enzymes. This work was a precursor to a major effort at SERI/NREL in the late 1980s through the end of project in 1996 to identify key enzymes in the algal biosynthetic pathways and to increase lipid levels by manipulating these pathways through genetic engineering (see Sections II. B.2. and II. B.3.).

In summary, although Euglena is not typical of the oleaginous microalgae targeted as potential biodiesel producers by the ASP, the data from Dr. Schwartzbach’s laboratory point out the importanance of understanding the biochemical mechanisms by which algae accumulate lipids. For Euglena and the Nannochloropsis strains described here, N deprivation does not seem to function as an actual trigger to induce biosynthesis of lipid. Rather, it acts as a block to cell division. Lipid synthesis continues by normal pathways, and lipid levels increase per cell, with no net accumulation in the culture. This result confirms the conclusions of Cooksey and coworkers. In addition, N deficiency also affects other cell processes, such as photosynthetic efficiency, which could affect lipid accumulation as the availability of fixed carbon is decreased.

I Publications:

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1987a) “Environmental control of lipid accumulation in Nannochloropsis salina, Nanno Q and Euglena.” FY 1987 Aquatic Species Program Annual Report (Johnson, D. A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 190-206.

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1987b) “Biochemistry of neutral lipid synthesis in microalgae.” FY 1986Aquatic Species Program Annual Report (Johnson, D. A., ed.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 255.

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1988a) “Preferential loss of chloroplast proteins in nitrogen deficient Euglena.” Plant Cell Physiol 29:1007-101. (Note: a preprint of this article was also submitted as a SERI Report, 47pp.)

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1988b) “Environmental control of carbohydrate and lipid synthesis in Euglena” Plant Cell Physiol. 29:423-432.

Smith, C. W.; Schwartzbach, S. D. (1988) “Preliminary characterization of pyruvate dehydrogenase and acetyl-CoA synthetase.” Manuscript submitted as a report to SERI, 6 pp.

This strain has been used for several past studies, and was concomitantly being tested in outdoor mass culture by another subcontractor (the University of Hawaii; principal investigator Dr. Edward Laws; discussed in Section III). Therefore, this strain was subjected to more extensive testing than the other strains in this subcontract. In one experiment reported for this strain, the

effects of light intensity on productivity were determined in batch cultures (i. e., in the Plexiglas culture apparatus described earlier without culture replacement and dilution). The maximum productivity observed for this strain (21 to 22 g dry weighPm — •d-) was observed at a total daily illumination of 63-95 kcal (representing approximately 40%-60% of full sunlight in southern California during the summer). This value was slightly higher than the productivity observed with a total daily illumination of 70% full sunlight (17.1 g dry weighHm-2^d-1). Productivities under N-limiting, continuous growth mode conditions were between 7 and 11 g dry weighHm-2^d-1. Likewise, productivities under N-sufficient, continuous growth mode conditions were reduced relative to batch cultures.

In addition to measuring overall productivities, the levels of protein, carbohydrate, lipid, and ash were determined for cells grown under the various conditions described earlier. Illumination of the cultures from 40% to 70% of full sunlight did not have a large impact on the cellular composition. Growth of P. tricornutum cells under N-deficient conditions resulted in a reduction of the protein content from 55% (in N-sufficient cells) to 25% of the cellular dry weight. Carbohydrate content increased from 10.5% to 15.1%, and the mean lipid content increased from 19.8% to 22.2%, although these differences in carbohydrate and lipid contents did not appear to be statistically significant. At one stage of the experiment, however, a time course of N deficiency led to a consistent rise in lipid content from 19.9% to 30.8% over the course of 7 days. The actual rate of lipid production did not increase, however, because the overall productivity of the cultures was reduced under N-deficient growth.

A. (Top) — Activities of several enzymes in Si-replete and Si-deficient C. cryptica cells. There is no significant difference in the activities of UDPglucose pyrophosphorylase, acetyl-CoA synthetase, or citrate synthase in the cells under the two conditions. However, there is a relative increase in acetyl-CoA carboxylase activity, and a decrease in chrysolaminarin synthase activity in Si-deficient cells.

B (Bottom) — Graph showing the activity of ACCase in C. cryptica cells. Exponential-phase cells were transferred into Si-free media at 0 hr. At 6 hr, (arrow), the culture was split and 1.8 mM Na2SiO3 was added to one culture. (•) Si-deficient cells; (■) Si-replete cells.

(Source: Roessler 1988a).

Algae grow in aquatic environments. In that sense, algae technology will not compete for the land already being eyed by proponents of other biomass-based fuel technologies. Biomass power and bioethanol both compete for the same land and for similar feedstocks—trees and grasses specifically grown for energy production. More importantly, many of the algal species studied in this program can grow in brackish water—that is, water that contains high levels of salt. This means that algae technology will not put additional demand on freshwater supplies needed for domestic, industrial and agricultural use.

The unique ability of algae to grow in saline water means that we can target areas of the country in which saline groundwater supplies prevent any other useful application of water or land resources. If we were to draw a map showing areas best suited for energy crop production (based on climate and resource needs), we would see that algae technology needs complement the needs of both agriculture and other biomass — based energy technologies.

In a world of ever more limited natural resources, algae technology offers the opportunity to utilize land and water resources that are, today, unsuited for any other use. Land use needs for microalgae complement, rather than compete, with other biomass-based fuel technologies.

Of the strains included in the most recent Culture Collection Catalog (the 1986-1987 edition, including the addendum), 37 are still viable. In addition, approximately 260 additional strains are part of the collection, but were never characterized well enough to be included in the catalog. All of these strains are in the process of being transferred to the University of Hawaii, where they will be maintained within the Center for Marine Biotechnology. The university intends to again make these strains available to the research community. A complete list of the strains still being maintained is included below:

Table II. A.9. Current list of microalgal strains in the SERI Culture Collection

I Publications:

Microalgae Culture Collection 1984-1985. Solar Energy Research Institute, SERI/SP-231-2486; 59 pp.

Microalgae Culture Collection 1985-1986. Solar Energy Research Institute, SERI/SP-232-2863, 97 pp.

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae Culture Collection 1986-1987.” Solar Energy Research Institute, SERI/SP-232-3079, 149 pp.

Johansen, J.; Lemke, P.; Nagle, N.; Chelf, P.; Roessler, R.; Galloway, R.; Toon, S. (1987) “Addendum to Microalgae Culture Collection 1986-1987.” Solar Energy Research Institute, SERI/SP-232-3079a, 23 pp.

Six promising strains were analyzed in SERI Type I, Type II, and ASW (Rila) using the temperature-salinity gradient described previously. These included the diatoms Chaetoceros muelleri (CHAET14), Navicula (NAVIC1), Cyclotella (CYCLO2), Amphora (AMPHO1 and AMPHO2), and the chlorophyte Monoraphidium minutum (MONOR2). (NAVIC1 and CYCLO2 were actually collected from the Florida keys; the remaining strains were collected in Colorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in at least two media types. Furthermore, all strains exhibited temperature optima of 30°C or higher. Maximal growth rates of these strains, along with the optimal temperature, conductivity, and media type determined in these experiments are shown in Table II. A.1. (Higher growth rates were determined for some of these strains in subsequent experiments; see results presented in Barclay et al. [1987]). Temperature-salinity growth contours are provided for these strains in the 1986 ASP Annual Report (Barclay et al. 1986).

Experiments were also conducted in an attempt to identify the chemical components of SERI Type I and Type II media most important for controlling the growth of the various algal strains. Bicarbonate and divalent cation concentrations were found to be important determinants in controlling the growth of Boekelovia sp. (BOEKE1) and Monoraphidium (MONOR2). The growth rate of MONOR2 increased by more than five-fold as the bicarbonate concentration of Type II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 by approximately 60% over this range. These results make sense, since media enriched in bicarbonate would have more dissolved carbon available for photosynthesis. An unexpected finding was that there was a decrease of nearly 50% in the growth rate of BOEKE1 as the divalent cation concentration increased from 5 mM to 95 mM (in Type I/10 medium containing altered amounts of calcium and magnesium). The effects of magnesium and calcium concentration on the growth of MONOR2 were less pronounced. These results indicate that matching the chosen strain for a particular production site to the type of water available for mass cultivation will be important.

Production in Microalgae

University of Nebraska; Oregon State University Russel H. Meints 1/86 — 12/89 XK-5-05073-2

By the mid-1980s, SERI researchers became convinced that optimal lipid production in microalgae could be achieved only by genetic manipulation of the cells to produce the desired traits. The overall goal of the research performed by Dr. Meints for SERI was to develop an algal virus system as a tool for the genetic manipulation of microalgae with potential for liquid fuel production. Viral vector systems had been used successfully to transfer DNA into other cell types. Dr. Meints’ laboratory was preeminent in the field of algal viruses. The work performed from 1986 to 1989 was based on earlier studies from this laboratory on isolating and characterizing a unique group of viruses from symbiotic algae. Funding from SERI contributed to ongoing research on the algal viral system by Dr. Meints; much of this work was done in collaboration with Dr. James Van Etten, in the Department of Plant Pathology at the University of Nebraska. The list of publications at the end of this section includes articles produced before and after the period of the SERI subcontract. This information was included to give interested readers an overview of the work performed on this topic.