The screening and characterization protocols used by SERI researchers were refined for the 1984 collecting season. Included in these refinements was the development of a modified “rotary screening apparatus”, a standard type of motorized culture mixing wheel for 16×150-mm culture tubes. The rotating wheel was constructed of Plexiglas to allow better light exposure (see Figure II. A.1). The wheel was typically illuminated with a high-intensity tungsten stage lamp, and

could be placed inside a box behind a CuSO4-water heat filter for temperature control. The Plexiglas wheel allowed all the cultures to receive equal illumination. Another technological advance used a temperature-salinity gradient table to characterize the thermal and salinity preferences and tolerances of the isolates.

Development of artificial saline media.

One of the most significant contributions made by SERI researchers during 1984 was the development of media that mimicked the saline water in shallow aquifers in the southwestern United States. This was an important undertaking because it allowed algal strains to be screened for growth in the types of water that would likely be available in an outdoor mass culture facility. To identify the major water types available in the southwestern United States, state and federal reports that described the chemical characteristics of water from 85 saline wells in New Mexico were studied. The data were statistically analyzed to identify the relationships between the various ionic constituents. (Data from wells deeper than 83 m was not used in this analysis, because the cost of pumping water from those depths was prohibitive.) R-mode factor analysis indicated that two factors were largely responsible for the differences between the waters examined (Barclay et al. 1988). The first factor, monovalent ion concentration, was responsible for 40% of the variance; the second factor, divalent ion concentration, for 30%. A plot of these factors against each other clearly delineated two primary water types, referred to as “Type I” and “Type II”. Type I waters were characterized by a low monovalent-to-divalent ion ratio (average value = 0.4), whereas Type II waters had a higher level of monovalent ions (monovalent-to — divalent ion ratio of 9.4). The major ions present in Type I water were Na+, Cl-, Mg2+, and Ca2+. The major ions of Type II water were Na+, Cl-, SO42-, and HCO3-. Type II water is consequently termed a “sodium bicarbonate class” of water. Approximately three-fourths of the saline well waters were of the Type II variety, and one-fourth could be characterized as Type I.

The survey indicated that both types of water exhibited a range of conductivities; the researchers believed that the higher-conductivity waters resulted from evaporation of the lower conductivity waters. In addition, they recognized that the conductivity of the water in an outdoor production pond would increase with time because of the high rates of evaporation in the southwestern United States (as high as 1 cm^day-1). Therefore, artificial media that covered a wide range of conductivities had to be developed. To this end, an experiment was conducted in which media that contained the salts typically present in low-conductivity Type I and Type II waters were allowed to evaporate with stirring at 35°C. Samples were removed at various times and filtered. The ions still dissolved in the waters were quantified using an inductively coupled plasma spectrometer and a high-performance liquid chromatograph. In this manner, media formulations were derived at SERI that covered a range of conductivities (from 10 to 70 mmho^cm-1) for both media types. The media most commonly used were designated SERI Type I/10, Type I/25, Type I/55, Type I/70, Type II/10, Type II/25, Type II/55, and Type II/70, in which the number following the slash indicates the specific conductivity of the medium. The compositions of these media are given in Figure II. A.2.

In order to assess whether these media formulations accurately reflected the types of water in desert region surface waters, samples of the water at numerous algal collection sites in the southwestern United States were chemically analyzed. The relative compositions of the anionic and cationic constituents were then plotted on separate trilinear plots, which allowed a graphical representation of the various water samples relative to SERI Type I and Type II media (Figure II. A.3). This analysis indicated that Type I water has higher proportions of Mg2+ and Ca2+ than most surface waters examined, whereas Type II water was fairly representative of the sampled waters with respect to these cations. On the other hand, natural surface waters often had an anion composition similar to both SERI Type I and Type II media. The researchers concluded that these artificial media would serve well as standardized media for testing newly acquired strains, thereby allowing all ASP researchers (both in-house personnel and subcontractors) to screen strains for growth potential in waters similar to those that would be available for commercial production.

Oak Ridge National Laboratory, Oak Ridge, Tennessee Jean A. Solomon 12/86 — 11/87 DK-4-04142-01

The purpose of this project was to determine if flow cytometry could be used to select for subpopulations of high lipid-producing algae within an algal culture. Flow cytometry is a method that measures the light scattered or emitted by particles as they pass through a laser beam. Scattered light is believed to reflect the size, shape, and refractive properties of cells. Dr. Solomon initially used exponentially growing and nutrient-stressed cells of the chrysophycean alga Boekelovia to demonstrate that the extent of right-angle scatter, which indicates changes in internal cell morphology, can be correlated to the lipid content of microalgal cells.

In subsequent studies, a lipid-specific fluorescent dye, Nile Red (see work by Dr. Cooksey, described in Section II. A.l. f.), was used to stain intracellular lipids. Nile Red is excited at a wavelength of 488 nm, and emits yellow-green light at 520-580 nm. In contrast, chlorophyll autofluorescence can be measured at wavelengths greater than 630 nm. Therefore, in contrast to the scattered light data mentioned above, flow cytometric analyses of cells stained with Nile Red would be more specific for changes in lipid content. Preliminary experiments in which cells of Boekelovia were stained with Nile Red demonstrated that increased yellow green fluorescence could be correlated with increased numbers of lipid droplets in the cells, suggesting that this method could work to screen for cells with high lipid contents.

Of the three microalgal species analyzed by TEM (Section II. B.l. d.), only Isochrysis was found to be appropriate for flow cytometric analysis. The cells of this strain are small and spherical, the optimal shape for flow cytometric analysis, and take up Nile Red well. In contrast, Ankistrodesmus cells are long and thin (40 nm x 4 nm). Nannochloropsis did not take up the Nile Red dye, possibly because of cell wall properties that also prevented good chemical fixation for microsopy.

In the initial experiments, cells were screened for lipid content based on Nile Red fluorescence alone. Several improvements to this procedure were implemented during the course of the study. First, efforts were made to optimize the Nile Red staining protocol. The best staining was achieved using a concentration of 1 mg Nile Red in 1 mL of cell suspension. The solvent for the Nile Red stain was changed from heptane to acetone, due to interfering fluorescence from undissolved heptane droplets. Finally, the researchers found that the fluorescence signal from Nile Red is unstable and decays rapidly. However, the fluorescence level stabilizes after about 45 minutes, so all readings were taken at least 45 minutes after staining the cells with Nile Red.

Another important change was to measure the chlorophyll autofluorescence as well as Nile Red fluorescence. This ensured that only viable cells containing lipid and intact chloroplasts would

be analyzed. In addition, the amount of chlorophyll is an indication of cell size. Cell sorting based on the ratio of chlorophyll fluorescence to Nile Red fluorescence would normalize the results to account for differences in cell size and age and allow detection of individual cells with unusually high lipid levels resulting from natural genetic variation. A decrease in the ratio of chlorophyll to Nile Red fluorescence would indicate lipid accumulation.

In one set of experiments, Isochrysis cultures were stressed by transferring the cells into N — deficient media, then screened for lipid content using flow cytometry, either by lipid content alone (Nile Red fluorescence) or by monitoring the chlorophyll to Nile Red fluorescence ratios. The daughter cells containing high or low levels of lipid were recultured in N-replete medium for

1 week or 1 month, then subjected again to N deprivation, and resorted. The lipid content of the daughter population was compared to that of the parent cells. These experiments produced inconsistent results. In some cases, the population of daughter cells selected for their high lipid content showed a wider range of lipid contents than the parent cells; other sorts produced daughters without significant differences in lipid content from the parents. One interesting observation was the bimodal distribution of cells in all populations subjected to N stress. Cells fell into two classes with low or high chlorophyll-to-lipid ratios. This again supports the author’s theory, discussed in the section on ultrastructural analysis of lipid accumulation, that cells respond as individuals to a lipid trigger, rather than gradually increasing the lipid content of the entire culture.

These results suggested that flow cytometry might be used to select for populations of high lipid algae if more was understood about the relationship of the physiological state of parent cells to lipid accumulation. Analysis of the growth of Isochrysis in N-replete media showed several phases, including a period of exponential growth that declined to a stationary phase. After about

2 weeks, the nutrients were depleted and the cells entered a stressed phase. A series of experiments was performed in which cells in various growth phases were stained with Nile Red and sorted based on lipid content alone (no chlorophyll measurements). The sorts on cells in exponential phase were usually not successful, but if the parent cells were in stationary phase or from very old cultures (stressed), the mean lipid content of the daughter population was about 20% higher than that of the parental cells. These results suggested that successful screening for high-lipid cells using flow cytometry was related to the cell cycle. The exponential cultures contained cells in all stages of cell growth and division. Cells that were preparing to divide would be larger and would be selected as high-lipid, so that the set of high lipid cells selected would actually contain only the largest and oldest cells, rather than high lipid genetic variants. Stationary or stressed populations are not actively dividing, so the cells are more uniform in size and sorting of the cells for high lipid should be more likely to identify true high lipid variants within the population.

The experiments using Isochrysis cultures in various growth stages as the parental population were repeated to test these assumptions. Cells were sorted based either on lipid levels alone (Nile Red fluorescence only) or using the ratio of chlorophyll to Nile Red fluorescence to control for cell size. The results presented in the available reports support the hypotheses. Sorting cells based solely on lipid content produced high-lipid daughter populations if the parent population

was in a stationary or stressed growth phase. Exponential cultures produced variable results. If the parental cells were sorted based on ratios of chlorophyll-to-lipid fluorescence, high-lipid populations could be produced from exponentially growing parent cultures.

These conclusions were based on very limited data. Only a few experiments were performed. In addition, several daughter cultures did not grow up after the sorting process, and failure of a growth chamber resulted in the loss of some cultures. The data were written up in only two technical reports to SERI, and it was difficult to determine the exact protocol used for each sorting experiment. However, the results of these studies are intriquing, and suggest that flow cytometry might be a viable method for screening for high lipid genetic variants within (or between) strains of oleaginous microalgae. The procedure would be limited to strains in which the cells are small and spherical. Dr. Solomon’s data suggest the best results would be achieved by using stationary or stressed cells as the parent population. In addition, cells should be selected based on a low chlorophyll-to-Nile Red fluorescence ratio, which would indicate high-lipid levels with respect to cell size. However, it is interesting that high-lipid daughter strains were not produced in the experiments in which exponentially growing cells were transferred directly to N — deficient media; yet, cells allowed to gradually deplete their N supply to induce the stressed condition could be used successfully in a flow cytometric screen. This suggests either that lipid accumulation occurs by different mechanisms under these two conditions, or, more likely, that the stressed cells from very old cultures had all entered a similar metabolic state so that size and lipid contents would be more indicative of genetic differences.

I Publications:

Solomon, J. A (1985) “Ultrastructure evaluation of lipid accumulation in microalgae.” Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators ’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 71-82.

Solomon, J. A. (1987) “Flow cytometry techniques for species improvement.” FY1986Aquatic Species Program: Annual Report (abstr.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 252.

Solomon, J. A.; Hand, R. E.; Mann, R. C. (1986a) “Ultrastructural and flow cytometric analyses of lipid accumulation in microalgae.” Annual Report, Oak Ridge National Laboratory, Oak Ridge, Tennessee, ORNL/M-258, 50 pp.

Solomon, J. A.; Hand, R. E.; Mann, R. C. (1986b) “Ultrastructural and Flow Cytometric Analyses of Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute, Golden, Colorado, SERI/STR-231-3089.

Solomon, J. A.; Palumbo, A. V. (1987) “Improvement of microalgal strains for lipid production.” FY 1987 Aquatic Species Program: Annual report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206.

II. A.2.a. Introduction

Included in this section are summaries of the research conducted by various subcontractors within the ASP who contributed to the collection, screening, and characterization of microalgal strains for potential use in biofuel production facilities. Initially, a variety of strain isolation and screening procedures were carried out by the various research groups, as there was no established protocol. This lack of uniformity in the screening protocols made comparing the results from one laboratory with those of another difficult, and meant that the criteria for selecting the best strains differed between the laboratories. At the same time, however, this arrangement provided the opportunity for new ideas regarding collecting and screening to be pursued, thereby allowing individual creativity in a manner that might be beneficial to the entire program.

Several subcontractors participated in the strain collection and screening effort. Dr. Bill Thomas and colleagues (Scripps Institution of Oceanography) collected a large number of strains from the desert regions of eastern California and western Nevada. Additional microalgal strains from desert waters in Arizona, New Mexico, California, Nevada, Utah, and Texas were obtained through the efforts of Dr. Milt Sommerfeld’s laboratory at Arizona State University. Dr. Mahasin T adros (Alabama A&M University) collected strains habitats in the southeastern United States (Alabama, Mississippi, and Florida). Additional strains from the Florida Keys and Everglades were collected by John Rhyther (Harbor Branch Foundation); Richard York (Hawaii Institute of Marine Biology) isolated a number of strains from the Hawaiian islands and surrounding waters. Certain environmental niches were focused on as well; Dr. Keith Cooksey (Montana State University) isolated several strains from thermal springs in Yellowstone National

Park; Dr. Ralph Lewin (Scripps Institution of Oceanography) focused on picopleustonic algae (“floating” algae at the air-sea interface). The results of these efforts are described below.

SERI researchers Lien and Roessler (1986) tried a somewhat different approach to understand the processes affecting lipid accumulation (Lien and Roessler 1986). A recently published technical evaluation (Hill et al. 1984) identified two major requirements for economic feasibility of biodiesel production:

1. Photosynthetic efficiency (which can simply be thought of as the percentage of incident radiation that is converted into biomass) needs to be 18%, and

2. Algal biomass needs to consist of 60% lipid.

Because very high lipid production is usually correlated with stress conditions (nutrient deprivation) that result in decreased photosynthetic efficiency and decreased growth, the two conditions of high lipid and high productivity seemed to be mutually exclusive. To overcome this technical hurdle, Lien and Roessler initiated a study to help understand the effects of nitrogen deprivation and lipid accumulation on photosynthetic efficiency.

Three strains of oleaginous algae were used in this study: Chlorella CHLSO1, Ankistrodesmus sp., and a newly isolated chrysophyte strain Chryso/F-1. The cells were grown in batch culture and monitored for nitrate concentration, light levels in the culture, chlorophyll concentration, and yield of cell mass and lipid (including total, neutral, and polar lipids). Maximum energy efficiency occurred as the culture approached N depletion. At this point, the culture showed a maximum density of photosynthetic pigments (before chlorophyll degradation and after N depletion), but the light energy reaching the cells was decreased due to the higher culture density. Thus, photosynthetic efficiency (biomass produced per light energy input) was maximized and the individual cells suffered less photooxidative damage due to lower light exposure. After the N in the culture was depleted, cell mass continued to increase for a time, eventually leveling off. All cultures experienced a two — to three-fold increase in total lipid, primarily as non-polar lipid. The photosynthetic efficiency decreased over the duration of the batch culture. However, in the early stages after the N was depleted, the cultures showed a decrease in energy efficiency with respect to total cell mass (AFDW) and with respect to the non lipid cell components, while photosynthetic efficiency remained constant or increased slightly with respect to lipid accumulation. In addition, N deprivation caused an increase in the efficiency of neutral, storage lipid production and suppressed the efficiency of polar structural lipid production.

These studies provided interesting preliminary data on the energetics of cell mass and lipid accumulation in algae. Follow-up experiments were proposed, including investigations of the relationship between initial N concentration and photosynthetic efficiency and lipid production after N depletion, and studying the effects of N resupply after depletion to attempt to extend the period of lipid production. These experiments were not continued; however, the results described earlier suggest that understanding the timing or kinetics of lipid accumlation in microalgae will be essential to maximize lipid production in a mass culture facility. If N starvation is used to trigger lipid accumulation, the data suggest that maximal photosynthetic

efficiency with respect to lipid production (and probably the best time for harvesting lipid- producing cells), occurs just after the N is depleted from the cultures.

Another set of experiments directed at optimizating photosynthetic efficiency in algal ponds was performed by SERI researcher Dr. Ken Terry. Previous studies had indicated that algal cells grown under high-intensity flashing light can use that light energy more efficiently than cells grown under the same intensity under constant illumination. The evidence suggests that an algal cell can integrate absorbed light energy such that the photosynthetic efficiency achieved under intermittent light conditions is similar to that attained under constant light of the same average intensity. This flashing light, or photomodulation, effect can be mimicked in vertically-mixed algal ponds, as cells circulate to the surface and back down to the lower levels in the pond where they receive minimal light. Thus, the photosynthetic efficiency of algal cells grown in ponds may be increased in high light by using mixing strategies that optimize this photomodulation effect.

In order to better understand the effects of intermittent light on photosynthetic efficiency of microalgal cultures, Dr. Terry set up a system to measure photosynthetic rates and oxygen evolution in laboratory cultures of Chlorellapyrenoidosa and Phaeodactylum tricorutum under flashing light conditions. Intermittent light conditions were simulated by placing sectored disks in front of a light source, and using this to illuminate exponentially growing cultures that had been placed in an oxygen electrode chamber. Photosynthesis was then measured under varying light/dark ratios (generated by changing the configuration of the disk) and light intensities. The data generated were used to calculate the percent “integration” of the incident light by the algal cultures. More rapid flashing led to greater integration, although lower flash frequencies produced higher levels of integration as the percentage of time the cells spent in the light decreased. Although these data were preliminary, they supported the proposal that photosynthetic efficiency in microalgal ponds could be enhanced by optimized vertical mixing strategies. However, increased photosynthetic efficiency might be compromised by increased losses to respiration as the cells spend increased time away from the surface, and the energy costs to achieve optimal mixing could be prohibitive.

Although Dr. Terry proposed follow-up studies using modulated light regimes that more closely mimic those seen in algal ponds, little further research on understanding photosynthetic efficiency in algal cultures was performed at SERI. Instead, the emphasis of the in-house research shifted to understanding the biochemistry and molecular biology of lipid accumulation.

CO2 is recognized as the most important (at least in quantity) of the atmospheric pollutants that contribute to the “greenhouse effect,” a term coined by the French mathematician Fourier in the mid-1800s to describe the trapping of heat in the Earth’s atmosphere by gases capable of absorbing radiation. By the end of the last century, scientists were already speculating on the potential impacts of anthropogenic

CO2. The watershed event that brought the question of global warming to the forefront in the scientific community was the publication of Revelle’s data in 1957, which quantified the geologically unprecedented build-up of atmospheric CO2 that began with the advent of the industrial revolution. Revelle14 characterized the potential risk of global climate change this way:

“Human beings are carrying out a large scale geophysical experiment of a kind that could not have happened in the past nor be produced in the future. Within a few centuries, we are returning to the atmosphere and the oceans the concentrated organic carbon stored in sedimentary rocks over hundreds of millions of years.”

Despite 40 years of research since Revelle first identified the potential risk of global warming, the debate over the real impacts of the increased CO2 levels still rages. We may never be able to scientifically predict the climatic effects of increasing carbon dioxide levels due to the complexity of atmospheric and meteorological modeling. Indeed, Revelle’s concise statement of the risks at play in global climate change remains the best framing of the issue available for policy makers today. The question we face as a nation is how much risk we are willing to take on an issue like this. That debate has never properly taken place with the American public.

As Revelle’s statement implies, the burning of fossil fuels is the major source of the current build up of atmospheric CO2. Thus, identifying alternatives to fossil fuels must be a key strategy in reducing greenhouse gas emissions. While no one single fuel can substitute for fossil fuels in an all of the energy sectors, we believe that biodiesel made from algal oils is a fuel which can make a major contribution to the reduction of CO2 generated by power plants and commercial diesel engines.

The SERI Microalgae Culture Collection was first established in 1984 by Dr. Bill Barclay to provide a central repository for strains that were believed to have potential as biomass fuel production organisms. The intent was to provide documented and partially characterized microalgal strains to researchers interested in conducting biofuels research or in developing algal mass culture technologies. The publicly available collection was described in a series of Culture Collection Catalogs published between 1984 and 1987. It was initially limited to strains that had been characterized quite extensively with respect to growth properties and chemical composition, and that were believed to hold the most promise. These catalogs contain a wealth of information for many of these strains, often including photomicrographs, proximate chemical compositions, lipid contents of cells grown under various environmental conditions, growth characteristics in different media types and different temperatures, and the results of small-scale outdoor production pond trials. Furthermore, media compositions are provided in these catalogs.

The original 1984-1985 Microalgae Culture Collection Catalog listed the following criteria for selection of strains to be placed in the cataloged public collection (in descending order of importance):

• Energy yield (growth rate x energy content)

• Type of fuel products available from biomass (hydrocarbon, diesel, alcohol, methanol)

• Environmental tolerance range (temperature, salinity, pH)

• Performance in mass culture (highly competitive, predator resistant)

• Media supplementation requirements (addition of vitamins, trace minerals)

• Amount of culture and composition data available on the clone or strain

• Budget for the culture collection

Although conceptually sound, these criteria carried with them the requirement to characterize the strains fairly extensively before a decision could be made as to whether they should be included in the collection. This detailed characterization became increasingly difficult as the number of strains available increased. As a consequence, many strains were maintained that were not officially documented in the catalogs.

From the inception of the culture collection until the late 1980s, strains in the collection were provided free of charge to anyone who requested them, with the hope that the research conducted (and published) using these strains would increase the overall understanding of these organisms.

Many laboratories took advantage of this. In the first year after the publication of the SERI Microalgae Culture Collection Catalog, more than 100 cultures were shipped to various groups studying biofuels production, natural product discovery, aquaculture, and the general physiology, biochemistry, and molecular biology of microalgae. In the ensuing years, hundreds of additional cultures were provided to researchers free of charge. Then, in the early 1990s, concerns were raised by the SERI Legal Department that the cultures should be considered as valuable intellectual property, and a moratorium was placed on providing cultures to outside parties; this moratorium persisted until the ASP were eliminated in late 1995.

The SERI Microalgae Culture Collection consists almost exclusively of eukaryotic, single-celled microalgae. Included in the collection are members of several algal classes, with a predominance of chlorophytes (green algae) and diatoms. These two groups tended to dominate under the high temperature/high light screening and selection regimes used to identify good production strain candidates. Of the strains present in the final culture catalog produced (published in 1987, including an addendum), 26% were chlorophytes, 60% were diatoms, 8% were chrysophytes, and 6% were eustigmatophytes. The following pages list the strains described in each of the three culture collection catalogs published during the course of the ASP.

The first culture collection catalog (1984-1985) listed 11 strains, which included five chlorophytes, four diatoms, one chrysophyte, and one eustigmatophyte. Some of these strains were obtained from other culture collections, including the University of Texas algal culture collection. The strains listed in the original 1984-1985catalog were as follows:

Collecting trips made by SERI researchers in 1984 focused on shallow saline habitats, including ephemeral ponds, playas, and springs in the arid regions of Colorado and Utah. After collection, the water and sediment samples were kept under cool, dark conditions for 1 to 3 days until they could be further treated in the laboratory. The pH, temperature, conductivity, redox potential, and alkalinity of the collection site waters were determined, and water samples were taken for subsequent ion analysis. In the laboratory, the samples were enriched with 300 pM urea, 30 pM PO4, 36 pM Na2SiO3, 3 pM NaFeEDTA, trace metals (5 mL/L PII stock, see Figure II. A.2), and vitamins. The enrichment tubes were then placed in the rotary screening apparatus (maintained at 25°C or 30°C) and illuminated at ~400 pE^m-2^s-1. Over a 5-day period, the illumination provided by the stage lamp was gradually increased to 1,000 pE^m-2^s-1. The predominant strains present in the tubes were isolated as unialgal cultures by agar plating or by serial dilution in liquid media.

The isolated strains were then tested for their ability to grow in incubators at 25°C at 150-200 pE^m-2^s-1 in the standard media types described above. and in artificial seawater (termed “Rila Salts ASW,” using Rila Marine Mix, an artificial sea salt mixture produced by Rila Products, Teaneck, NJ. The strains that grew well in at least one of these media were further characterized with respect to growth on a temperature-salinity gradient table at a light intensity of 200 pE^m-2^s-1. Thirty combinations of temperature (10° to 35°C) and salinity (10 to 70 mmho’cm’1) were included in this analysis, representing the ranges that might be expected in actual outdoor production systems. Once again, the cultures were enriched with nutrients to maximize growth rates. The cultures used to inoculate the test cultures were preconditioned by growth in the media at 17° and 27°C. The optical density at 750 nm (OD750) of the cultures was measured twice daily for 5 days, and the growth rates were calculated from the increase in culture density during the exponential phase of growth. A refinement of this method was to measure the growth rates in semicontinuous cultures, wherein the cultures were periodically diluted by half with fresh medium; this method provided more reproducible results than the batch mode experiments.

Figure II. A.3 gives an example of the type of growth data generated by the use of temperature — salinity gradient tables. The contour lines in the plot are interpolations indicating where a particular combination of temperature and salinity would result in a given growth rate. Many such plots were generated for various strains, and are shown in the culture collection catalogs and ASP annual reports.

Approximately 300 strains were collected from the 1984 trips to Utah and Colorado. Of these, only 15 grew well at temperatures >30°C and conductivities greater than 5 mmho^cm-1. Nine were diatoms, including the genera Amphora, Cymbella, Amphipleura, Chaetoceros, Nitzschia, Hantzschia, and Diploneis. Several chlorophytes (Chlorella, Scenedesmus, Ankistrodesmus, and Chlorococcum) were also identified as promising strains, along with one chrysophyte (Boekelovia).

Two strains isolated as a result of the 1984 collecting effort (Ankistrodesmus sp. and Boekelovia sp.) were characterized in greater detail using the temperature-salinity matrix described earlier. Boekelovia exhibited a wide range of temperature and salinity tolerance, and grew faster than one doubling^day-1 from 10 to 70 mmho^cm-1 conductivity and from 10° to 32°C, exhibiting maximal growth of 3.5 doublings^day-1 in Type II/25 medium. Reasonable growth rates were also achieved in SERI Type I and ASW-Rila salts media (as many as 1.73 and 2.6 doublings^day-1, respectively). Ankistrodesmus was also able to grow well in a wide range of salinities and temperatures, with maximal growth rates occurring in Type II/25 medium (3.0 doublings^day-1).

Boekelovia and Ankistrodesmus were also examined with regard to their lipid accumulation potential. Two-liter cultures were grown in media that contained high (600 pM) and low (300 pM) urea concentrations at a light intensity of 200 pE^m-2^s-1. Half of each culture was harvested 2 days after the low-N culture entered stationary phase to determine the lipid content of N — sufficient cells and cells that were just entering N-deficient growth. After 10 days of N-limited growth, the remainder of the low-N culture was harvested. Lipids were extracted via a modification of the method of Bligh and Dyer (1959) and lipid mass was determined gravimetrically. The lipid content of Boekelovia was 27% of the organic mass in N-sufficient cells, increasing to 42% and 59% after 2 days and 10 days of N-deficiency, respectively. There was less effect of N starvation on the lipid content of Ankistrodesmus; the lipid content only increased from 23% in N-sufficient cells to 29% in cells that were N-deficient for 10 days.

In conclusion, research at SERI in 1984 led to the development of artificial media that mimicked the saline groundwater typically found in the desert regions of the southwestern United States. This allowed the strains isolated during collecting trips at various ionic concentrations to be systematically screened and provided standardized media that could be used in different laboratories performing ASP-sponsored research. Numerous strains were characterized with respect to growth at several temperatures and salinities using these new media.

I Publications:

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae Culture Collection 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP — 232-3079, 147 pp.

Barclay, B.; Nagle, N.; Terry, K. (1987) “Screening microalgae for biomass production potential: Protocol modification and evaluation.” FY1986Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 23-40.

Barclay, B.; Nagle, N.; Terry, K.; Roessler, P. (1985) “Collecting and screening microalgae from shallow, inland saline habitats.” Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators ’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700; pp. 52-68.

Barclay, W. R.; Nagle, N. J.; Terry, K. L.; Ellingson, S. B.; Sommerfeld, M. R. (1988) “Characterization of saline groundwater resource quality for aquatic biomass production: A statistically-based approach.” Wat. Res. 22:373-379.

Sommerfeld, M. R.; Ellingson, S. B. (1987) “Collection of high energy yielding strains of saline microalgae from southwestern states.” FY 1986Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 53-66.

I Additional References:

Bligh, E. G.; Dyer, D. J. (1959) “A rapid method for total lipid extraction and purification.” Can. J. Biochem. Physiol. 37:911-917.

Siver, P. (1983) “A new thermal gradient device for culturing algae.” British J. Phycol. 18:159­163.

SERI Type I

Artificial Inland Saline Water

SERI Type П

Artificial Inland Saline Water

Conductivity (mmho cm’1)

Salt

CaCl,

NajSOj,.

KCt….

iidnwj.

NajCOj <

Suggested enrichments (mL/L) are:

Nitrogen source* (0.6M N). KH2PO^(0.6M) a

Bj2<l mg L’1) і……………………………………………………

Thiamine-HCI (i mg L’*)…………………………………..

Biotin (2 mg L’1)………………………………………………..

•Nitrogen source indicated for individual species, ammonium as NH^Cl, nitrate as KNO3. 230-500 mg L’* Na2Si03.9H20 should be added when cultivating diatoms in this medium.

РП trace element stock (lor 1 U:

Na2EDTA…………………………………………………. 6.0 g

FeCtj-CHjO………………………………………………. 0.29 s

H3B03……………………………………………………… 6Л4&

MnCyWjO……………………………………………….. Q. S6 I

ZnCl2 …………………………….. …………………… 0.0({

CoCl^tHjO……………………………………………….. 0.026*

Adjust trace element stock solution to pH 7.8-S. O with NaOH.

Figure II. A.2. Formulations for SERI Type I and Type II artificial inland saline waters.

Recipes for the preparation of Type I and Type II media at five different salinities, expressed as conductivity of the final solution. Formulas for these media were developed by statistical analysis of saline groundwater data for the state of New Mexico. For each salt, necessary additions in mg/L are listed. (Source: Barclay et al. 1986).

Figure II. A.3. Trilinear plots showing the ionic constituents of various water samples relative to SERI Type I and SERI Type II artificial saline media. (Source: Sommerfeld and Ellingson 1987.)

Figure II. A.4. Growth contour plots. Examples of growth contour plots generated from data obtained by the use of a temperature-gradient table. The contour lines represent interpolated values indicating where a particular combination of temperature and salinity would result in a given growth rate. The data shown, given as doublings»day) represent the exponential growth ofMonoraphidium sp. (S/MONOR-2) in semicontinuous culture. Each point represents the mean of at least five separate daily growth rate determinations. (Source: Barclay et al. 1987).

A: Type I inland saline water B: Type II inland saline water C: Seawater

Subcontractor: Principal Investigator:

Montana State University Keith E. Cooksey

Period of Performance: 1/86 — 10/87

Subcontract Numbers: XK-5-05073-1; XK-6-05073-1

The goal of this research was to understand the biochemistry of lipid accumulation in microalgae, in particular, to provide information on the biochemical triggers that induce lipid synthesis. The Nile Red fluorescence technique developed by Dr. Cooksey’s laboratory and at SERI (described above in Sections ILA. Lf. and III. B.1.e.) was used to study lipid accumulation in microalgae in response to N or Si depletion. Nile Red fluorescence was used to monitor the lipid levels in batch cultures of Chlorella over time. As the N became depleted, lipids accumulated in the cultures, predominately as triglycerides. The triglyceride levels began to increase before N was totally depleted from the medium. Microscopic examination showed that individual cells within the population began to accumulate lipid at different times, similar to results obtained by Dr. Solomon in Isochrysis. Dr. Cooksey concluded that lipid accumulation begins as the cells enter stationary phase and cell division ceases; the timing of this event would be different for individual cells within a population.

Dr. Cooksey’s laboratory next performed a complex series of experiments designed to correlate media factors, (i. e., nitrate concentrations, pH, and carbon availability), with lipid accumulation in CHLOR-1. Cell growth and lipid accumulation were monitored in batch cultures, with cells grown in unbuffered Bold’s medium, or media buffered at pH 7, 9, or 10. In unbuffered Bold’s medium, the initial pH was 7, and increased to pH 8 by day 6, and up to pH 9.5 by day 9. The cells grew in all conditions, with the best growth at pH 9. Under all growth conditions, the level of nitrate in the media decreased, but never fell below 25% of the initial levels.

Accumulation of neutral lipids was monitored by Nile Red fluorescence. There was no lipid accumulation in cultures maintained at or below pH 9. However, in buffered medium with a pH> 10, or in unbuffered medium that experienced an increase in pH during the growth period, the cultures generally showed a significant increase in lipid levels that was accompanied by a decrease in cellular growth rates.

Nutrient limitation, generally nitrate or silica, can trigger lipid accumulation in microalgae. Nutrient deprivation can cause a decrease in cell division, which presumably results in “targeting” of excess fixed carbon into storage lipids. The data obtained by Dr. Cooksey’s laboratory suggested that a shift in pH, which has been correlated with decreased rates of cell division, could also trigger lipid accumulation. These data suggested that nutrient limitation might not directly affect biochemical pathways to enhance lipid synthesis; rather, lipid accumulation may be an indirect consequence of inhibition of a stage in the cell cycle. In other photosynthetic systems studied, the data indicated that cells synthesize triglycerides in the light and utilize these lipids as energy stores in the dark and during cell division. If division were

blocked, the rate of neutral lipid utilization would be slower than the rate of synthesis, so triglycerides would accumulate in the cells. To help test this hypothesis, Dr. Cooksey used light microscopy to examine cells grown in media with different pH ranges. Cultures grown at pH 7-9 consisted almost entirely of small, single cells. However, at pH 10 and higher, a large proportion of the cells was in the form of autosporangial complexes, i. e., their nucleii had divided, but the autospores had not separated. The specific effect of increased pH on cell division is not clear, although some evidence suggests that increased pH can lead to increased flexibility of the autospore wall, preventing individual cells from breaking free. Alternatively, increased pH could affect precipitation of media components, indirectly affecting the cell cycle.

Although the data presented here suggest that nutrient deprivation or increased pH may affect lipid levels simply as a consequence of decreased cell division, additional research by Dr. Cooksey’s laboratory suggested that treatments that increase lipid accumulation may also affect the biochemistry of lipid biosynthesis. Analysis of the lipid classes present in the cells at the end of the 10-day growth period showed accumulation of triglycerides in cells at high pH, with a decrease in glycolipids and polar lipids. The nonpolar storage lipids predominantly contain 16- and 18-carbon saturated or monounsaturated fatty acids (16:0 and 18:1), which are considered “precursor” fatty acids in lipid biosynthesis. The polar lipids and glycolipids usually contain a higher proportion of polyunsaturated fatty acids. However, analysis of the fatty acid composition of the storage lipids showed that at higher pH, more of these precursor lipids were seen in the polar lipids and glycolipids. This suggests a switch in the lipid synthesis patterns that results in less desaturation of the fatty acids esterified to the polar lipids.

In summary, the finding that an increase in pH can also lead to lipid accumulation in cells before N is depleted suggested a method to uncouple lipid accumulation from nutrient deprivation, and provided another method to study the biochemistry of lipid accumulation in microalgae. The data from Dr. Cooksey’s laboratory supported the premise that lipid triggers such as nutrient deprivation or pH increase affect lipid accumulation in microalgae by similar mechanisms, i. e., inhibition of cell division, leading to decreased utilization of storage lipid while new synthesis of lipid continues. However, he also proposed that different stresses may affect different stages of the cell cycle. As there is evidence that the different lipid classes (neutral lipids versus polar lipids) may be synthesized at different times during the cell cycle, this could affect the quality and the quantity of the lipids synthesized. For example, pH stress appears to block release of autospores (after DNA replication); N deprivation could have multiple effects on the photosynthetic machinery or on a number of biochemical pathways in the cell that could directly or indirectly affect lipid synthesis. R. Thomas, a graduate student working with Dr. Cooksey, found that treating the cells with monofluoroacetate (MFA) also decreased cell growth and caused neutral lipid accumulation. MFA inhibits the TCA cycle, presumably preventing TCA oxidation of fatty acids and thus increasing the pool of acetyl CoA for synthesis of new fatty acids. (Thomas suggested that MFA could be used as a trigger for lipid accumulation in algal ponds, with the caveat that MFA is toxic to all living systems).

Dr. Cooksey concluded that to understand the biochemistry of neutral lipid accumulation in microalgae, it would be necessary to understand cellular cycles of lipid synthesis and utilization

that are coupled to cell growth and division. It will also be important to consider not only factors that affect synthesis of storage lipid, but also to understand the metabolic shifts that result in production of membrane lipids or storage lipids.

I Publications:

Cooksey, K. E. (1987) “Collection and screening of microalgae for lipid production.” Final Subcontract Report, Solar Energy Research Institute, Golden, Colorado, May 1987, 42 pp.

Cooksey, K. E.; Guckert, J. B.; Thomas, R. (1989) “Triglyceride accumulation and the cell cycle in microalgae.” Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, pp. 139-158.

Cooksey, K. E.; Guckert, J. B.; Williams, S. A.; Collis, P. R.(1987) Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red, J. of Microbiol. Methods 6:333-345.

Cooksey, K. E.; Williams, S. A.; Collis, P. R. (1987) “Nile Red, a fluorophore useful in assessing the relative lipid content of single cells,” In The Metabolism, Structure and Function of Plant Lipids, (Stumpf, P. K; Williams, S. A.; Callis, R. P., eds.), Plenum Press, N. Y., pp. 645-647.

Guckert, J. B.; Cooksey, K. E.; Jackson, L. L. (1987) “Lipid solvent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga, Chlorella.” Unpublished manuscript.

Guckert, J. B.; Cooksey, K. E. (1990) “Triglyceride accumulation and fatty aid profile changes in Chlorella (Chlorophyta) during high pH-induced cell cycle inhibition.” J. Phycol. 26:72-79.

Thomas, R. M. (1990) “Triglyceride accumulation and the cell cycle in Chlorella.” Masters Thesis, Montana State University, July 1990.

University of California, San Diego William H. Thomas 1980 — 1983 XK-0-9111-1

Work carried out under this subcontract represented one of the first attempts by an ASP subcontractor to characterize the productivity and lipid yields of various microalgae. Six algal strains (B. braunii, Dunaliellaprimolecta, Isochrysis sp., Monallanthus salina, Phaeodactylum tricornutum, and Tetraselmis sueica) were obtained from existing culture collections and analyzed with respect to lipid, protein, and carbohydrate content under various growth conditions. For these experiments, all cultures except for B. braunii were grown in natural seawater that was enriched with N, P, and trace metals. B. braunii was grown in an artificial seawater medium. Initial experiments to determine productivities of these species were performed using batch cultures in 9-L serum bottles. Of the strains tested, the highest growth rates were observed with P. tricornutum (Thomas strain) and M. salina.

Additional experiments were performed in plexiglas vessels that were 5 cm thick, 39 cm deep, and 24 cm wide (surface area ~940 cm2). The cultures were illuminated from the side with a 2,000-watt tungsten-halide lamp, which was placed behind a water/CuSO4 thermal filter. In these experiments, the cultures were typically maintained for 40 to 90 days. In the early stages of an experiment, the cultures were maintained in a batch mode, and then converted to a continuous or semi-continuous dilution mode. V arious culture parameters (including light intensity, dilution rate, and N status) were manipulated during the course of these experiments to determine their effects on the productivities and proximate chemical composition of the strains. The results of these experiments with each species tested are discussed below. These experiments are difficult to compare because the experiments were all carried out slightly differently (i. e., different light intensities, different culturing methods [batch, semi-continuous, and continuous], different means of obtaining N-deficient cultures, and inconsistent use of a CuSO4 heat filter, which resulted in differences in light quality and culture temperature). Nonetheless, the general conclusions of this study are of interest.

A note added to a chapter of the 1986 Annual Report (Lien and Roessler 1986) described preliminary data on the use of Si deficiency to trigger lipid accumulation in diatoms. Silicon is major component of diatom cell walls. Similar to the lipid trigger effect produced by N — deficiency, Si depletion also results in a decrease in cell growth and often is accompanied by an accumulation of lipid within the cells. However, Si (unlike N) is not a component of other cellular macromolecules (enzymes, membranes) or cell structures such as the photosynthetic

apparatus. Therefore, any changes in cellular biochemistry and lipid accumulation induced by Si deficiency might be more easily interpreted than changes induced by N starvation. This work initiated a series of experiments by Paul Roessler during the late 1980s and early 1990s on the biochemistry and molecular biology of lipid accumulation in Si-deficient diatoms.

The first set of experiments compared the effects of Si deficiency on lipid accumulation and cell physiology in several species of diatoms, including C. cryptica T13L, Thalassiosirapseudonana, and Cylindrothecafusiformis. Exponentially growing cultures were transferred to media that contained either excess Si or limited levels of Si so that the media became Si deficient while the cells were still growing exponentially. Cell growth, chlorophyll a content, AFDW, lipid, and photosynthetic capacity were monitored under both conditions. In all three species, cell division decreased as soon as the Si was depleted in the media. However the species responded differently with respect to other physiological parameters. In C. cryptica, chlorophyll a synthesis was almost completely inhibited after 12 hrs in Si-depleted media; C. fusiformis showed little change in chlorophyll a synthesis after 72 hrs. T. pseudonana exhibited an intermediate effect, with some decrease in chlorophyll a synthesis noted after 36 hours without Si. The effect on photosynthetic capacity, measured as O2 evolution, also varied between the three species. In C. fusiformis and C. cryptica, photosynthetic capacity decreased 33% and 58%, respectively, after 12 hours; T. pseudonana showed a steady decline in photosynthetic capability following Si — depletion. (However, photosynthetic capacity decreased in Si-replete cultures as well during the 72 hours time course of the experiment, presumably due to the increased ratio of antenna chlorophyll molecules versus reaction center molecules in the self-shaded, dense cultures).

The three species were also analyzed for accumulation of total biomass and lipid (Figure II. B.3.). In C. fusiformis, biomass accumulation (measured as AFDW) for the duration of the experiment was similar in cultures with or without sufficient Si, although lipids made up a higher percentage of the AFDW in the Si-deficient cultures (26% versus 21% in Si-replete cells). In T. pseudonana, synthesis of cell mass and lipid was not affected until 36 hours after Si depletion. At this point, biomass and lipid accumulation rates decreased; however, there was little difference in the percentage of total lipid in the cells with or without Si at the end of the 72 hours experimental period. The situation with C. cryptica was very different. Twelve hours following Si depletion, there was a 38% decrease in the growth rate of these cells compared to the Si — replete culture. However, lipid synthesis continued at the same rate in the Si-deficient cells as in the Si-replete cells, resulting in a significant increase in the lipid content of the Si-starved cells. Interestingly, after these initial changes, the Si deficient cultures of C. cryptica showed little gain in total AFDW or lipid during the remaining 72 hours of the experiment.

In order to determine if Si deprivation affected the composition of the lipids produced, the lipids were extracted and analyzed for the percentage of polar versus neutral lipids present. In all three species, the Si-deficient cultures showed a significant increase in the level of neutral lipids, primarily TAGs. For example, the percentage of neutral lipids in Si-deficient cultures of C. cryptica was 64%, compared to 32% in Si-replete cultures. In C. fusiformis, the percentage of neutral lipids increased from 17%-20% to 57% in Si-deprived cultures.

Based on these studies, C. cryptica was identified as the best candidate for further studies on the biochemistry of lipid accumulation. To determine the effects of Si deficiency on the synthesis of the cell components, the levels of protein, carbohydrate, and lipid were examined at various times after Si was depleted in the cultures. During the first 12 hours, protein and carbohydrate synthesis decreased. Lipid accumulation continued at a rate similar to that of the Si-replete cultures. This resulted in an increase in lipid content of the Si deficient cells from 19% to 27%. This observation was confirmed in subsequent studies that followed the incorporation of newly assimilated carbon (as H14CO3-) into the various cell components. Si depletion resulted in a net decrease in the rate of photosynthesis and carbon assimilation, but the individual cell fractions were affected differently. For example, the rate of 14C accumulation into lipids decreased by 48% in the first 4 hours of Si-deprivation; the uptake of 14C into chrysolaminarin, the major carbohydrate storage product in diatoms, decreased 84%. Therefore, the increase in lipid content of Si-deficient cells was not due to an increase in the rate of lipid synthesis, but to a relative decrease in the rate of synthesis of protein and carbohydrate.

Pulse-chase experiments were performed to test whether Si deficiency also caused the conversion of non-lipid cellular components into lipids. In these experiments, Si-replete cells were labeled with H14CO3- for 1 hour, then transferred into Si-deficient media without labeled bicarbonate. The amount of labeled carbon in the lipid fraction was determined at various times following transfer to Si-free media. This experiment showed that carbon was slowly redistributed from the nonlipid components of the cells into lipid under Si-deficient conditions, but not under Si-replete conditions. Therefore, the accumulation of lipids in diatoms in response to Si-deficiency is apparently due to two factors:

1. An increase in the proportion (but not the net amount) of newly assimilated carbon that is incorporated into lipids, resulting from a disproportionate decrease in the rate of lipid synthesis versus carbohydrate synthesis, and

2. A slow conversion of nonlipid cell material into lipids.

Fractionation of the lipids produced demonstrated that Si deprivation resulted in an increase in the proportion of total lipid as neutral lipids, primarily TAGs, from 43% to 63% after only 4 hours of Si deficiency. Analysis of the fatty acid composition of the accumulated lipids also showed changes induced by Si starvation. In Si-deficient cells, there was an increase in the proportions of mono — and unsaturated fatty acids (16:1, palmitoleic acid; 16:0, palmitic acid; and 14:0, myristic acid), and a reduction in the proportions of the three major polyunsaturated fatty acids, (16:3, 20:5, and 22:6). These results are consistent with the finding that the predominant fatty acids found in triacylglycerol storage lipids in C. cryptica are 16:1, 16:0, and 14:1. These shorter, more highly saturated fatty acids are also the most desirable substrates for conversion into fatty acid methyl esters (biodiesel), as they would be less likely to polymerize during combustion and “gum up” an engine.

Although Si depletion causes all diatoms tested to stop dividing, species responded differently with repsect to continued accumulation of biomass and lipid. C. cryptica showed a rapid

response to Si-depletion, with a decrease in growth accompanied by a significant increase in the proportion of the biomass as lipid within 12 hours (the response to N starvation was usually much slower, as the cells could utilize internal N stores). This result again emphasizes the need to understand the kinetics of lipid accumulation in individual species under specific conditions for cost-effective lipid production in the ponds.

Figure II. B.3. Changes in lipid mass, ash-free dry mass, and lipid content in Si-deficient cultures of three diatoms.

A. C. fusiformis B. C. cryptica C. T. pseudonana. Symbols: (■) Si-deficient cultures; (•) Si-replete cultures.

To better understand the processes involved in lipid accumulation in microalgae, and to identify potential molecular targets for genetic manipulation, studies were initiated to examine the effects of Si deficiency on the enzymatic pathways involved in lipid and carbohydrate synthesis in C. cryptica. One possibility is that the increased levels of storage lipid in cells exposed to Si starvation could result from shifts in the relative activities of one or more enzymes in the lipid biosynthesis pathway. Acetyl-coenzyme A (acetyl-CoA) is known to be the immediate precursor of fatty acid synthesis, but the source of this compound varies in different organisms. For example, in mammalian cells, acetyl-CoA used in cytosolic fatty acid synthesis is produced from citrate via the action of ATP citrate lyase. In plants, acetyl-CoA can be produced in the chloroplasts from pyruvate, catalyzed by pyruvate dehydrogenase. Alternatively, acetyl-CoA could be produced by the mitochondrial pyruvate dehydrogenase. In this case, the acetyl-CoA (which cannot diffuse across the organellar membranes) would be broken down to acetate and free CoA by acetyl-CoA hydrolase. Acetate would diffuse to the chloroplast and become incorporated into acetyl-CoA by the action of acetyl-CoA synthetase. Once acetyl-CoA is produced, it is then used as a substrate by acetyl-CoA carboxylase (ACCase) to produce malonyl CoA. Malonyl-CoA is a substrate for fatty acid synthase and this reaction is considered to be the first committed step in fatty acid synthesis.

These pathways had not previously been well-characterized in diatoms. To better understand the lipid synthesis pathways, Roessler first looked for the presence of these enzymes in extracts of C. cryptica, but found no citrate lyase activity. However, acetyl-CoA hydrolase, acetyl-CoA synthetase, and ACCase activity were all present. Enzyme activities were studied in Si-replete and Si-deficient cells (Figure II. B.4). The level of acetyl-CoA synthetase activity was similar under both conditions; however, the level of ACCase activity was two fold higher in Si-deficient cells after 4 hours, and four fold higher after 15 hours. Based on subsequent studies using protein synthesis inhibitors, the increased specific activity of the ACCase was believed to result from an increase in expression of the ACCase gene (Roessler 1988a; 1988c).

ACCase is a biotin-containing enzyme that catalyses the carboxylation of acetyl-CoA to form malonyl-CoA. This reaction entails two partial reactions: the carboxylation of biotin, followed by the transfer of the carboxyl group from biotin to acetyl-CoA. In bacteria, the enzyme is composed of four non-identical subunits. However, in eukaryotes, biotin binding, biotin carboxylation, and carboxyl-transfer all occur on a single large multifunctional protein; the functional ACCase occurs as a multimer of this polypeptide. ACCase had previously been shown to play a key regulatory role in the rates of fatty acid synthesis in both animal and plant systems. A project was initiated to isolate and characterize ACCase from C. cryptica to clarify the role of this enzyme in lipid accumulation induced by Si starvation, and to compare the microalgal enzyme with those isolated from plants, animals, yeast, and bacteria.

The enzyme was purified from C. cryptica by a combination of (NH4)2SO4 precipitation, gel filtration chromatography, and affinity chromatography based on the affinity of biotin to avidin. Consistent with ACCase enzymes isolated from other eukaryotes, C. cryptica ACCase was found
to consist of a homo-tetramer of 185 kDa subunits. The activity of the enzyme was assayed by the incorporation of 14C bicarbonate into malonyl-CoA, and other factors were identified that affect the stability and activity of the enzyme. As seen for other ACCases, the enzyme required a slightly alkaline pH for optimum activity (pH 8.2), although the enzyme was most stable when stored at pH 6.5. The enzyme was also stabilized by sulfhydryl reductants (i. e., dithiothreitol), citrate, NaCl, and KCl; divalent metal cations (Mg2+ or Mn2+) were required for activity. A number of cellular metabolites were also tested for their affects on ACCase activity. The enzyme was inhibited by products of the ACCase reaction, including malonyl-CoA, ADP, and NaH2PO4, and also by palmitoyl-CoA, but it was not affected by various glycolytic or photosynthetic intermediates or by free CoA. Two herbicides that inhibit ACCases from monocot plants were also had little or no effect on C. cryptica ACCase. Thus, the ACCase from this diatom was found to be similar to higher plant ACCase enzymes in that it is composed of multiple, identical, multifunctional subunits. In addition, the Kms for the ACCase substrates (acetyl-CoA, MgATP, and bicarbonate) in C. cryptica were similar to those found in plant ACCase enzymes (Roessler 1989; 1990).