As discussed earlier, environmental variables (particularly nutrient status) can have great effects on growth and the quantity and quality of lipids in microalgae. To determine the effects of several environmental variables alone and in combination on the growth and lipid contents of microalgae, a multivariate, fractional factorial design experiment was carried out with two promising diatoms, Navicula saprophila (NAVIC1) and C. muelleri (CHAET9). For these experiments, cells were grown in modified SERI Type II/25 medium in which the alkalinity was adjusted by adding sodium carbonate and sodium bicarbonate and the conductivity was adjusted by adding sodium chloride. Cultures were grown on the temperature gradient table described previously at 200 qE^m-2^s-1. The following variables were tested in the multivariate analysis: conductivity (20 to 80 mmho^cm-1), temperature (17° to 32°C), N (urea) concentration (0 to 144 mg^L-1), sodium silicate concentration (0 to 500 mg-L-1), and alkalinity (8.8 to 88 meq^L-1). In these experiments, growth was measured by changes in AFDW and lipid was measured by the use of the Nile Red fluorometric assay.

The results indicated that the N content and conductivity of the medium were the most important variables affecting lipid content (Nile Red fluorescence) of both NAVIC1 and CHAET9. As N levels and conductivity increased, the amount of neutral lipid per mg of AFDW decreased. The interaction of N and conductivity was an important determinant of lipid content as well. Silicon level and alkalinity were more important factors in determining the lipid content for CHAET9 than for NAVIC 1. N concentration was by far the most important factor in determining final cell mass for NAVIC 1, and was a major factor for cell mass yield in CHAET9 (along with the interaction of conductivity and alkalinity, which had a large negative impact on growth). Alkalinity was a major factor for growth of both NAVIC 1 and CHAET9. However, these experiments did not determine actual growth rates, but only the final cell yields; thus, how actual cell division rates compared with each other is not known.

These experiments indicate the importance of examining the interactions of environmental variables in determining the effects on growth and lipid production. However, the models generated by these kinds of experiments are specific for the strains being studied, and the results cannot necessarily be used to predict the effects of these variables on other strains. Furthermore, for such models to be truly predictive of growth and lipid production in an actual mass culture, much more sophiticated (and realistic) experimental setups would be required.

I Publications:

Chelf, P. (1990) “Environmental control of lipid and biomass production in two diatom species.” J. Appl. Phycol. 2:121-129.

Production: NREL In-House Researchers

II. B.2.a. Introduction

During the first few years of the ASP, in-house research efforts in the area of lipid biosynthesis focused on understanding the lipid trigger and the effects of N starvation on lipid synthesis and photosynthetic efficiency. It was also shown that lipid accumulation can be induced in diatoms by Si starvation, a major component of the diatom cell wall. During the second half of the 1980s

and early 1990s, a major component of the research at SERI/NREL was the study of lipid biosynthesis in the oleaginous diatom C. cryptica. This was primarily the work of Paul Roessler, who identified a key enzyme involved in lipid accumulation and isolated and characterized both the protein and the gene for the enzyme acetyl-CoA carboxylase from Cyclotella. Other research efforts at NREL examined related biosynthetic pathways, including synthesis of chrysolaminarin, a storage carbohydrate, and lipid processing reactions such as fatty acid desaturation. The basic biochemistry and molecular biology research formed the basis of the efforts to manipulate microalgal lipids by genetic engineering, which will be described in Section II. B.3.

One aspect of the algal research at SERI was the possibility of producing hydrogen by microalgae, for use as a gaseous fuel. During photosynthetic electron transport, electrons from reduced ferredoxin can be transferred to hydrogen ions to produce H2. This reaction is catalyzed by the enzyme hydrogenase. Unfortunately, hydrogenase is inhibited by molecular oxygen, a by­product of the photosynthetic reaction, making the practical application of this process difficult. There was a significant research effort at SERI in the early 1980s, primarily by Dr. Steve Lien and Dr. Paul Roessler, to understand the biochemistry of hydrogen production by microalgae. The work on hydrogenase was funded by the Hydrogen Program at DOE, not the ASP, and will not be included in this report. However, studies on hydrogen production by microalgae are ongoing at NREL (SERI) in the Center for Basic Sciences, and interested readers should contact Dr. Michael Seibert for more information.

The previous sections have alluded to a number of potential fuel products from algae.

The ASP considered three main options for fuel production:

• Production of methane gas via biological or thermal gasification.

• Production of ethanol via fermentation

Production of biodiesel

A fourth option is the direct combustion of the algal biomass for production of steam or electricity. Because the Office of Fuels Development has a mandate to work on transportation fuels, the ASP did not focus much attention on direct combustion. The concept of algal biomass as a fuel extender in coal-fired power plants was evaluated under a separate program funded by DOE’s Office of Fossil Fuels. The Japanese have been the most aggressive in pursuing this application. They have sponsored demonstrations of algae production and use at a Japanese power plant.

Algal biomass contains three main components:

• Carbohydrates

• Protein

• Natural Oils

The economics of fuel production from algae (or from any biomass, for that matter) demands that we utilize all the biomass as efficiently as possible. To achieve this, the three fuel production options listed previously can be used in a number of combinations. The most simplistic approach is to produce methane gas, since the both the biological and thermal processes involved are not very sensitive to what form the biomass is in. Gasification is a somewhat brute force technology in the sense that it involves the breakdown of any form of organic carbon into methane. Ethanol production, by contrast, is most effective for conversion of the carbohydrate fraction. Biodiesel production applies exclusively to the natural oil fraction. Some combination of all three components can also be utilized as an animal feed. Process design models developed under the program considered a combination of animal feed production, biological gasification and biodiesel production.

The main product of interest in the ASP was biodiesel. In its most general sense, biodiesel is any biomass-derived diesel fuel substitute. Today, biodiesel has come to mean a very specific chemical modification of natural oils. Oilseed crops such as rapeseed (in Europe) and soybean oil (in the U. S.) have been extensively evaluated as sources of biodiesel. Biodiesel made from rapeseed oil is now a substantial commercial enterprise in Europe. Commercialization of biodiesel in the U. S. is still in its nascent stage.

The bulk of the natural oil made by oilseed crops is in the form of triacylglycerols (TAGs). TAGs consist of three long chains of fatty acids attached to a glycerol backbone. The algae species studied in this program can produce up to 60% of their body weight in the form of TAGs. Thus, algae represent an alternative source of biodiesel, one that does not compete with the existing oilseed market.

As a matter of historical interest, Rudolph Diesel first used peanut oil (which is mostly in the form of TAGs) at the turn of the century to demonstrate his patented diesel engine2. The rapid introduction of cheap petroleum quickly made petroleum the preferred source of diesel fuel, so much so that today’s diesel engines do not operate well when operated on unmodified TAGs. Natural oils, it turns out, are too viscous to be used in modern diesel engines.

In the 1980s, a chemical modification of natural oils was introduced that helped to bring the viscosity of the oils within the range of current petroleum diesel3. By reacting these TAGs with simple alcohols (a chemical reaction known as “transesterification” already commonplace in the oleochemicals industry), we can create a chemical compound known as an alkyl ester4, but which is known more generically as biodiesel (see the figure below). Its properties are very close to those of petroleum diesel fuel.

+

HCOH

I

HCOH

I

HCOH

1 molecule of glycerol

Commercial experience with biodiesel has been very promising5. Biodiesel performs as well as petroleum diesel, while reducing emissions of particulate matter, CO, hydrocarbons and SOx. Emissions of NOx are, however, higher for biodiesel in many engines. Biodiesel virtually eliminates the notorious black soot emissions associated with diesel engines. Total particulate matter emissions are also much lower6,7,8. Other environmental benefits of biodiesel include the fact that it is highly biodegradable9 and that it appears to reduce emissions of air toxics and carcinogens (relative to petroleum diesel)10. A proper discussion of biodiesel would require much more space than can be accommodated here. Suffice it to say that, given many of its environmental benefits and the emerging success of the fuel in Europe, biodiesel is a very promising fuel product.

High oil-producing algae can be used to produce biodiesel, a chemically modified natural oil that is emerging as an exciting new option for diesel engines. At the same time, algae technology provides a means for recycling waste carbon from fossil fuel combustion. Algal biodiesel is one of the only avenues available for high-volume re-use of CO2 generated in power plants. It is a technology that marries the potential need for carbon disposal in the electric utility industry with the need for clean-burning alternatives to petroleum in the transportation sector.

The goal of the work performed under this subcontract was to collect and screen microalgal species from southern Florida. It emphasized collecting chromophytic algae (e. g., diatoms, chrysophytes, and prymnesiophytes), because this group of algae was known to often accumulate lipids. Collection trips were made in June and September 1985, and in February 1986 to the Florida Keys and the Everglades. Samples were taken from 123 sites, including various mangrove swamps, salt flats, canals, ditches, and shallow ponds. The basic physicochemical characteristics of the collection site waters were determined. The mean temperature was 29°- 30°C both for sites in the Florida Keys and the Everglades. The mean conductivity of the water from the Keys (35.6 mmho^cm-1) was somewhat higher than that of the Everglades (25.7 mmho^cm-1), whereas the pH values were similar (~8). To select for the fastest growing microalgal strains in the water samples, the original samples were enriched with nitrate, trace metals, and vitamins, and incubated under continuous light (880 pE^m-2^s-1, or 45% of full sunlight) at 30°C. The strains that became dominant in the cultures were isolated into unialgal cultures via micropipetting, serial dilution, and spreading onto agar plates. As a consequence of these experiments, 61 unialgal cultures were produced.

Preliminary evaluation of the growth of these strains in various media was performed in test tubes containing enriched seawater, SERI Type I/25, Type I/40, Type II/25, and Type II/40 media. The test tubes were incubated at 30°C under constant illumination at 300 pE^m-2^s-1. Growth rates were determined by measuring the OD750 every day for 5 days, and the final culture density was measured after 10 days. One hundred ten strains (including some strains already in

the Harbor Branch algal collection) were screened in this manner. In general, the strains that were newly isolated under the selection scheme outlined above grew more rapidly than the culture collection strains. Members of the Prymnesiophyceae, particularly coccolithophorids and ochromonads, tended to grow well in most media types, but the dinoflagellates isolated via these procedures did not grow well in the SERI standard media. Most species grew better in Type II medium than in Type I medium, although there were certainly exceptions to this. The highest growth rate (3.26 doublings^-1) was observed with a strain of Hymenomonas HB152 (HYMEN3) in Type II/25 medium. Seven strains had growth rates that exceeded 2 doublings^-1 in at least one media type; included in this group were Dunaliella HB37 (DUNAL2), Nannochloris HB44 (NANNO2), a yellow green unicell HB54 (UNKNO4), Chlorella HB82 (CHLOR7) and HB87, Pyramimonas HB133 (PYRAM2), and HYMEN3. Nine strains had growth rates of at least one doubling^-1 in all five media (including Chlorella HB84 (CHLOR8) and HB97 (CHLOR9), Nannochloris HB85 (NANO3), and all the strains mentioned earlier in this paragraph except for HYMEN3).

Several of the most promising strains were examined in more detail; they were grown in a matrix of five different salinities (8-60 mmho^cm-1) at five different temperatures (15°-35°C) by the use of a temperature-salinity gradient table, as described in previous sections. An artificial seawater medium (ASP-2) diluted with varying amounts of distilled water was used for these experiments. The cultures were exposed to constant illumination at 180 pE^m-2^s-1. Each of the four strains tested (Tetraselmis HB47 [TETRA4], PYRAM2, UNKNO4, and an olive green unicell HB154 [UNKNO5]) exhibited excellent growth over a wide range of conditions. All these strains had a growth rate greater than one doubling^-1 between 8 and 60 mmho^cm-1 and between 20° and 35°C. UNKNO4 had growth rates higher than 1.5 doublings^-1 between 15 and 60 mmho^cm-1 and between 20° and 35°C.

A visual assessment of the lipid contents of the most rapidly growing strains was conducted by staining the cells with Nile Red, the stained cells were examined using fluorescence microscopy. Based on this assessment (which was not carried out with nutrient-starved cells), TETRA5 and UNKNO4 had the highest estimated lipid contents. TETRA4, HYMEN2, PYRAM2, and UNKNO5 also appeared to accumulate substantial quantities of lipid.

The first collecting trips made by SERI researchers took place in the fall of 1983. Five saline hot springs in western Colorado were selected for sampling because of their abundant diatom populations, and because a variety of water types was represented. Water samples were used to inoculate natural collection site water that had been enriched with N (ammonium and nitrate) and phosphate (P) and then filter sterilized. Water samples were also taken for subsequent chemical analyses. The temperature and conductivity of the site water were determined at the time of collection. Conductivity ranged from 1.9 mmhos^cm-2 at South Canyon Spring to 85.0 mmhos^cm-2 (nearly three times the conductivity of seawater) at Piceance Spring. Water temperature at the time of collection ranged from 11° to 46°C.

In the laboratory, researchers tried to isolate the dominant diatoms from the enriched water samples. Cyanobacteria and other contaminants were removed primarily with agar plating. Approximately 125 unialgal diatom strains were isolated. The predominant genera found were Achnanthes, Amphora, Caloneis, Camphylodiscus, Cymbella, Entomoneis, Gyrosigma, Melosira, Navicula, Nitzschia, Pleurosigma, and Surirella.

A standardized lipid analysis protocol was not yet in place to screen these strains. However, many algal strains were known to accumulate lipids under conditions of nutrient stress. Microscopic analysis of cells grown under N-deficient conditions revealed lipid droplets in several of the strains, particularly in Amphora and Cymbella.

In addition to yielding several promising algal strains, this initial collection trip was useful for identifying areas for improving the collection and screening protocols. Some of these improvements were implemented for the 1984-collecting season, and are described in the next section.

I Publications:

Barclay, W. R. (1984) “Microalgal technology and research at SERI: Species collection and characterization.” Aquatic Species Program Review: Proceedings of the April 1984 Principal Investigators ’Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341; pp. 152-159.

Oak Ridge National Laboratory, Oak Ridge, Tennessee Jean A. Solomon 10/84 — 11/86 N/A

The goal of this project was to gain further understanding of the physiology of lipid accumulation in microalgae by examination of the ultrastructure of cells containing high levels of storage lipids. The questions that Dr. Solomon addressed were:

1. Where does the lipid accumulate within the cells; and

2. What other ultrastructual changes are seen in microalgae induced to accumulate lipid?

Three oleaginous microalgal strains were used in this study, Ankistrodesmus fulcatus (SERI strain ANKIS1; class Chlorophyceae), Isochrysis aff. glabana (ISOCH1, class Prymnesiophyceae), and Nannochloropsis salina (NANNO1, class Eustigmatophyceae). Ultrastructural changes were monitored by transmission electron microscopy (TEM). In this technique, cells are chemically fixed and embedded in a plastic resin. The resin is then cut into thin sections (70-100nm), stained with heavy metals, and viewed in an electron microscope. The first step was to develop adequate fixation and embedding techniques for the algal species to be studied. This is often problematic for microalgae, presumably due to the chemical and physical properties of the algal cell wall, which can act as barriers to penetration of the fixatives or resin. Dr. Solomon tested five fixation protocols (see Solomon 1985, p.74, Table 1), all variations of standard methods of fixation using glutaraldehyde and osmium tetroxide, dehydration with an organic solvent, embedding of the cells in an acrylic resin, and poststaining of the sections with uranyl acetate and lead citrate. Initially, Dr. Solomon reported that the best fixation of Ankistrodesmis and Isochrysis was achieved by exposing the cells briefly to glutaraldehyde and osmium simultaneously, followed by dehydration in acetone and embedding in Spurrs resin. However, a later report stated that Ankistrodesmis was better preserved by exposing the cells sequentially to glutaraldehyde and then osmium (in cacodylate buffer supplemented with sucrose as an osmoticum). Also, Araldite/Embed12 resin was used, as it appeared to provide better penetration into the cells. For Isochrysis, the initial protocol was also modified by adding sucrose. Fixation of Nannochloropsis was poor with any method used; the scaley cell wall of this organism seemed to provide a significant barrier to adequate penetration of fixatives and resins.

Nitrogen deprivation was used to trigger the production of lipids in the cells. The cells were grown in N-replete medium, then collected by centrifugation and resuspended in growth medium without added N. Samples were fixed immediately and at regular intervals during the following 13 days, and thin sections were cut and examined for ultrastructual changes by TEM.

As expected, N deprivation resulted in the accumulation of lipid within the cells of all three microalgal species. The lipid appeared primarily as droplets within the cytoplasm, not within the chloroplast or other cellular organelles. The lipid droplets often appeared adjacent to a mitochondrion. In Ankistrodesmus, N-deficiency also produced an increased number of starch granules within the chloroplasts, and resulted in the formation of unusual membrane structures consisting of packed, concentric layers of double membranes within the cytoplasm. Whether these unusual structures were the site of excess lipid accumulation, or were structural artifacts of the fixation process, was unclear.

It is difficult to conclude much more about ultrastructural changes that might have been induced in these cells following N deprivation. The sample size was very small. One hundred-nm thick sections may represent less than 1/100th of the volume of a microalgal cell. In addition, only a few cells within a population can be examined easily by this technique. Finally, there is a high likelihood that the chemical fixation methods used in the study can create artifacts that are not related to actual cell structure. However, these studies supported the observation that significant levels of storage lipids can accumulate in the cytoplasm of microalgal cells exposed to N deficiency. Dr. Solomon’s microscopic observations in Ankistrodesmus also suggested the presence of a discrete lipid trigger mechanism within each cell, as lipid did not appear to accumulate gradually within all cells of a population after N deprivation. Instead, individual cells appeared to accumulate large amounts of lipid during a 1-2 day period. This result was supported by the flow cytometric data also performed in Dr. Solomon’s laboratory, which is described below.

I Publications:

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.

Figure II. B.2. Electron micrographs of nitrogen-sufficient (top) and nitrogen-deficient (bottom) cells of Nannochlorposis salina.

Note the accumulation of large lipid droplets (L) in the cytoplasm in the nitrogen-deficient cells. The lipid often appeared adjacent to a mitochondrion (M). N: nucleus. C: chloroplast. Scale bars = 0.5 pm. The numbers in the lower left corner of each figure are from the original publication (Solomon et al. 1986b).

In addition to the in-house research being conducted in the area of strain collection and screening, there was an effort by Dr. Thomas Tornabene and others to characterize various strains via detailed lipid compositional analyses. Dr. Tornabene’s laboratory at SERI (and later at the Georgia Institute of Technology) served as the focal point for the analysis of lipids in algal samples supplied by various researchers in the ASP. This section will describe the results of these analyses, and will provide details about the analytical methods used, as these methods were the most comprehensive used in the program. An early report by Tornabene et al. (1980) described the lipids that were present in the halophilic alga Dunaliella that had been isolated from the Great Salt Lake in Utah. The cells were grown to late logarithmic phase, harvested, and extracted with chloroform/methanol via the method of Bligh and Dyer (1959). Additional extraction by acetate buffer, followed by refluxing with an alkaline methanol/water mixture was then performed, followed by partitioning of lipids into petroleum ether. The extracted lipids were fractionated on the basis of polarity using silicic acid columns via differential elution with hexane, benzene, chloroform, acetone, and methanol. In this procedure, the lipids are eluted as follows: [4]

2. benzene: cyclic hydrocarbons, polyunsaturated acyclic hydrocarbons, sterols, and xanthophylls

3. chloroform: mono-, di — and triacylglycerols, free fatty acids, and phaeophytin a

4. acetone: glycolipids, carotenoids, and chlorophyll a and b; and

5. methanol: phospholipids and chlorophyll c.

The various lipid classes were further analyzed via Si gel thin layer chromatography (both one — and two-dimensional), wherein lipids were detected via the use of iodine vapors (and autoradiography in the case of 14C-labeled lipids). In addition, lipids containing amino groups were detected via the ninhydrin reagent, and phospholipids were detected by the use of molybdate/H2SO4. Fatty acids were analyzed via gas chromatography using either flame ionization or mass spectroscopic detection after being converted to their methyl ester derivatives in the presence of methanolic HCl. The head groups of the polar lipids were identified via gas chromatography after being converted to alditol acetates. These and related methods were described by Tornabene et al. (1982).

These analyses indicated that lipids comprised 45%-55% of the total organic mass of Dunaliella cells. Based on the distribution of 14C after labeling the cells with 14C-bicarbonate, neutral lipids accounted for 58.5% of the lipid mass, whereas phospholipids and galactolipids were 22.9% and 10.9% of the lipid mass, respectively. Isoprenoid hydrocarbons (including p-carotene) and aliphatic hydrocarbons (in which the major components were tentatively identified as straight — chain and methyl-branched C17 and C19 hydrocarbons with various degrees of unsaturation) represented 7.0% and 5.2% of the lipids, respectively. The major fatty acids present were palmitic (20.6%), linolenic (12.5%), linoleic (10.7%) and palmitoleic (7.8%), but no attempt was made to ascertain whether any of these fatty acids predominated a particular lipid class. The high hydrocarbon content of this alga is rather atypical of most of the strains characterized in the ASP. These types of hydrocarbons would probably require catalytic conversion into a usable fuel source, which would perhaps limit their utility as a production organism.

A detailed analysis of the lipids present in the green alga Neochloris oleoabundans was also carried out by Tornabene (who was later to hold a position at the Georgia Institute of Technology), along with G. Holzer (Colorado School of Mines), S. Lien and N. Burris (SERI) (Tornabene et al. 1983). The strain used in this study was obtained from the University of Texas Algal Culture Collection, and reportedly contained substantial quantities of lipid when grown under N-deficient conditions. (However, this is a freshwater strain). Exponentially growing cells were transferred into a low-N medium, and after 5 to 7 days of growth in stirred cultures that were bubbled with 1% CO2 in air, the cells were harvested and the lipids were extracted. Analytical methods were similar to those described earlier, and included the use of pyrrolidine — acetic acid/mass spectrometry to determine the position of double bonds in the fatty acids. These analyses indicated that 35%-54% of the cellular dry weight was in the form of lipids in N — deficient cells. Neutral lipids accounted for more than 80% of the total lipids, and were

predominantly in the form of TAGs. Small amounts of straight-chain hydrocarbons and sterols were also found (one sterol was identified as a Д7 sterol, but low quantities of material made identification of the sterols difficult). A number of polar lipids were also quantified, but all polar lipids combined accounted for less than 10% of the lipid mass. The fatty acids that comprised the TAGs were present in the following proportions: 36% oleic (18:1 Д9), 15% palmitic (16:0), 11% stearic (18:0), 8.4% iso-17:0 (an unusual fatty acid for microalgae), and 7.4% linoleic (18:2 Д9,12). Other saturated and monounsaturated fatty acids were present in TAGs, but represented less than 5% each of the total fatty acids present. The high proportion of saturated and monounsaturated fatty acids in this alga is considered optimal from a fuel quality standpoint, in that fuel polymerization during combustion would be substantially less than what would occur with polyunsaturated fatty acid-derived fuel (Harrington, 1986).

Additional research carried out in Tornabene’s laboratory (Ben-Amotz et al. 1985) examined the lipid composition of 7 algal species. Some were from existing culture collections and others were isolated by ASP researchers. The lipid contents of these strains were determined under conditions of N sufficiency, after 10 days of N deficiency, and under different salinity levels.

Botryocooccus braunii has received considerable interest as a fuel production organism in other laboratories because of its high lipid content. This study confirmed the high lipid levels (55% of the organic mass for N-deficient cells). Most of this lipid was in the form of hydrocarbons, including C29 to C34 aliphatic hydrocarbons and a variety of branched and unsaturated isoprenoids. Glycerolipids were less abundant than the hydrocarbons, and were composed primarily of 16:0 and various CJ8 fatty acids. These data, coupled with the fact that this species grows very slowly (one doubling per 72 hours), indicated that Botryococcus would not function well as a feedstock for lipid-based fuel production.

The other species examined in this study were the chlorophytes Ankistrodesmus, Dunaliella, and Nannochloris, the diatom Nitzschia, and the chrysophyte Isochrysis. N deficiency led to an increase in the lipid content of Ankistrodesmus (from 24.5% to 40.3%), Isochrysis (from 7.1% to 26.0%), and Nannochloris (from 20.8% to 35.5%), but resulted in a decrease in the lipid content of Dunaliella (from 25.3% to 9.2%). Elevating the NaCl concentration of the medium had little effect on the lipid content of Botryococcus cells, but caused a slight decrease in the lipid content of Dunaliella salina (from 25.3% to 18.5% with an increase in [NaCl] from 0.5 to 2 M). Conversely, the lipid content of Isochrysis increased from 7.1% to 15.3% as the NaCl increased from 0.5 to 1 M. These results once again highlight the impact of culture conditions on the quantities of lipids present. However, as stated before, the most important characteristic of a lipid production strain is the overall lipid productivity for a given amount of time, which was not examined in this study.

The polar lipid composition of the strains examined in this study were typical of photosynthetic microalgae, and included phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol.

Table II. A.5 indicates the major fatty acids (those at levels exceeding 5% of the total) present in these strains, both under N-sufficient and N-deficient growth conditions.

In conclusion, the work carried out by Tornabene’s laboratory provided a detailed characterization of the lipids present in a variety of microalgae. No general conclusions could be made from the work except that the lipid composition of various microalgal strains can differ quite substantially. Because the nature of the lipids can have a large impact on the quality of the fuel product, characterizing the potential production strains is important to ensure that deleterious lipids (e. g., highly polyunsaturated fatty acids in the case of biodiesel fuel) are not present at high levels.

Table II. A.5. Major fatty acids of various microalgae. (Fatty acids in bold are present at levels of 15% or higher)

As a result of the algal screening efforts by SERI subcontractors and in-house researchers, several algal species were identified as good candidates for biodiesel production during the early 1980s. This was facilitated by the development of a cytochemical staining technique for intracellular lipids that allowed researchers to visualize storage lipid droplets in algal cells (see Section II. A.Lf. and Lien 1981a). Two of the most promising candidates were the green alga N. oleoabundans, which showed a high lipid content and rapid growth, and a Chlorella strain (CHLS01) isolated from a local site.

First, a sensitive method to monitor nitrate levels in liquid cultures using ion chromatography was developed to study the effects of N limitation on lipid accumulation in these organisms. Algal growth, lipid content, and chlorophyll a content were measured in batch cultures of N. oleoabundans and CHLS01. Cell division and chlorophyll accumulation occurred rapidly in the cultures as long as N was present. When N was depleted, cell division stopped, although biomass accumulation continued for several days. The major portion of the new biomass was composed of lipids and storage oils. N depletion resulted in a rapid decrease in the level of chlorophyll a in the cultures, suggesting that the cells might metabolize chlorophyll during periods of nitrogen stress. There was also an increase in the ratio of carotenoid to chlorophyll and a significant decrease in the complexity of the intracellular membranes in N-starved cells. These last three observations indicated that the photophysiology of the cells was affected, suggesting that the lipid trigger could also directly or indirectly alter photosynthetic efficiency in the treated cells (discussed in more detail below).

There are a number of benefits that serve as driving forces for developing and deploying algae technology. Some of these benefits have already been mentioned. Four key areas are outlined here. The first two address national and international issues that continue to grow in importance—energy security and climate change. The

remaining areas address aspects of algae technology that differentiate it from other technology options being pursued by DOE.

Energy Security

Energy security is the number one driving force behind DOE’s Biofuels Program. The U. S. transportation sector is at the heart of this security issue. Cheap oil prices during the 1980s and 1990s have driven foreign oil imports to all time highs. In 1996, imports reached an important milestone—imported oil consumption exceeded domestic oil consumption. DOE’s Energy Information Administration paints a dismal picture of our growing dependence on foreign oil. Consider these basic points11:

• Petroleum demand is increasing, especially due to new demand from Asian markets.

• New demand for oil will come primarily from the Persian Gulf.

• As long as prices for petroleum remain low, we can expect our imports to exceed 60% of our total consumption ten years from now.

• U. S. domestic supplies will likewise remain low as long as prices for petroleum remain low.

Not everyone shares this view of the future, or sees it as a reason for concern. The American Petroleum Institute12 does not see foreign imports as a matter of national security. Others have argued that the prediction of increasing Mideast oil dependence worldwide is wrong. But the concern about our foreign oil addiction is widely held by a broad range of political and commercial perspectives13.

While there may be uncertainty and even contention over when and if there is a national security issue, there is one more piece to the puzzle that influences our perspective on this issue. This is the fact that, quite simply, 98% of the transportation sector in the U. S. relies on petroleum (mostly in the form of gasoline and diesel fuel). The implication of this indisputable observation is that even minor hiccups in the supply of oil could have crippling effects on our nation. This lends special significance to the Biofuels Program as a means of diversifying the fuel base in our transportation sector.

Our almost complete reliance on petroleum in transportation comes from the demand for gasoline in passenger vehicles and the demand for diesel fuel in commerce. Bioethanol made from terrestrial energy crops offers a future alternative to gasoline, biodiesel made from algal oils could do the same for diesel fuel.

Subcontractor: Principal Investigator: Period of Performance: Subcontract Number:

Montana State University Keith E. Cooksey 3/86 — 4/87 XK-4-04136-04

The goal of this research was to develop a technique for rapidly screening microalgae for high lipid content, and to use this method to select microalgae with potential for liquid fuel production. Dr. Cooksey’s laboratory initiated the development of the Nile Red lipid staining procedure, which is fully described in Section II. A.l. f. The Nile Red staining procedure was used to screen for high lipid strains of microalgae, first using cultures collected mainly from Florida and maintained at Montana State University, and in cultures containing diatoms freshly isolated from hot springs in Yellowstone National Park. Because algae to be used in outdoor mass culture in the desert southwest would be subject to high temperatures, the Florida strains, isolated at 28°C, were first tested for growth and lipid production at 35°C. Although some strains produced fairly high levels of lipid, most grew poorly. Some diatom strains were then isolated from the hot springs, based on the premise that they would be more likely to tolerate extremes of temperature and pH variation. In these cultures, Nile Red was used to screen the initial sample for lipid-producing strains. These cells were then cultured, made unialgal and axenic, and tested for growth rate and lipid production. The strains tested showed growth rates of 0.5 to 2 doublings/d and lipid contents of 9%-54%, similar to the properties of oil-producing algae isolated by other methods.