Paul G. Roessler

Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae; Close-Out Report

The Aquatic Species Program was a relatively small research effort intended to look at the use of aquatic plants as sources of energy. Its history dates back to 1978, but much of the research from 1978 to 1982 focused on using algae to produce hydrogen. The program switched emphasis to other transportation fuels, particularly biodiesel, beginning in the early 1980s. This report summarizes the research activities carried out from 1980 to 1996, with an emphasis on algae for biodiesel production.

GENETIC TRANSFORMATION OF THE DIATOMS CYCLOTELLA CRYPTICA AND NAVICULA SAPROPHILA

Two species of diatoms were genetically transformed by introducing plasmid vectors containing the Escherichia coli neomycin phosphotransferase II (nptII)gene. Expression of the bacterial nptII gene in the diatoms was achieved using the putative promoter and terminator sequences from the acetyl‐CoA carboxylase gene from the centric diatom Cyclotella cryptica T13L Reimann, Lewin, and Guillard. The vectors were introduced into C. cryptica and the pennate diatom Navicula saprophila NAVIC1 Lange‐Bertalot and Bonik by microprojectile bombardment. Putative transformants were selected based on their ability to grow in the presence of the antibiotic G418, and production of the neomycin phosphotransferase protein by the transformed cells was confirmed by western blotting. The foreign DNA integrated into one or more random sites within the genome of the transformed algal cells, often in the form of tandem repeats. This is the first report of reproducible, stable genetic transformation of a chlorophyll c‐containing alga.

Manipulation of microalgal lipid production using genetic engineering

Genetic transformation of two species of diatoms has been accomplished by introducing chimeric plasmid vectors containing a bacterial antibiotic resistance gene driven by regulatory sequences from the acetyl-CoA carboxylase (ACCase) gene from the diatom Cyclotella cryptica. The recombinant DNA integrated into one or more random sites within the algal genome and the foreign protein was produced by the algal transformants. This is the first report of genetic transformation of any chlorophyll c-containing microalgal strain. We are using this system to introduce additional copies of the ACCase gene into diatoms in an attempt to manipulate lipid accumulation in transformed strains.

Changes in the activities of various lipid and carbohydrate biosynthetic enzymes in the diatom Cyclotella cryptica in response to silicon deficiency

The effects of silicon deficiency on the activities of several enzymes involved in lipid and storage carbohydrate synthesis in the diatom Cyclotella cryptica were determined. The activity of UDPglucose pyrophosphorylase was not affected after 4 h of silicon-deficient growth, but the activity of UDPglucose: beta-(1----3)-glucan-beta-3-glucosyltransferase (chrysolaminarin synthase) was reduced by 31% during this period. Acetyl-CoA synthetase, acetyl-CoA hydrolase, and citrate synthase activities were present in cell-free extracts of C. cryptica, but did not change in response to 4 h of silicon deficiency. However, the activity of acetyl-CoA carboxylase increased approximately two- and fourfold after 4 and 15 h of silicon-deficient growth, respectively. This induction could be blocked by cycloheximide (20 micrograms/ml) and actinomycin D (10 micrograms/ml), suggesting that silicon deficiency may induce an increase in the rate of acetyl-CoA carboxylase synthesis. These changes in enzymatic activity may be partially responsible for the accumulation of lipids that has been observed in C. cryptica and other diatoms in response to silicon deficiency.

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [14C]‐bicarbonate or [14C]‐acetate. [14C]‐bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [14C]‐acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [14C]‐acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5‐h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C20 fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C18 fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C20 fatty acids.

A mutant of Nannochloropsis deficient in eicosapentaenoic acid production

The most abundant fatty acids found in Nannochloropsis are 16:0, 16:1 (n-7) and 20:5 (n-3). By analysing the fatty acid composition of mutagenized cells by gas chromatography, we discovered a mutant strain of Nannochloropsis that is completely devoid of 5,8,11,14,17-cis-eicosapentaenoic acid [20:5 (n-3)]. An analysis of total fatty acids from whole cells showed that the loss of 20:5 (n-3) in mutant cells was accompanied by a concomitant increase in the relative amount of 20:4 (n-6). However, the increase in 20:4 (n-6) was not proportional to the loss of 20:5 (n-3) within individual lipid classes. The percentage of 20:4 (n-6) in monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol and phosphatidylglycerol (PG) in the mutant strain was lower than the wild type levels of 20:5 (n-3) in these lipids. Conversely, the percentage of 20:4 (n-6) in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the mutant cells was higher than the wild type levels of 20:5 (n-3). Molecular species analysis of MGDG showed that, compared to the wild type strain, the mutant strain contained more 16:116:0 MGDG, and less of the MGDG species containing C20 fatty acids. Relatively low levels of 20:420:4 MGDG were found in the mutant, although 20:520:5 MGDG was abundant in wild type cells. Relative membrane lipid levels were altered in the mutant compared to the wild type strain, particularly PG, which showed a fourfold decrease. The mutant cells grow more slowly than the wild type cells on both solid and in liquid media. Electron micrographs showed that the mutant strain had fewer thylakoid stacks per chloroplast, and fewer thylakoids per stack than wild type cells. We concluded that the mutation affects an extrachloroplastic (n-3) desaturase that is required for the synthesis of 20:5 (n-3), which is found in both chloroplast and extrachloroplast lipids.

Anionic modulation of the catalytic activity of hydrogenase from Chlamydomonas reinhardtii.

Abstract Hydrogen production by cell-free extracts of Chlamydomonas reinhardtii is stimulated by anions when methyl viologen, reduced by dithionite, is used as the electron donor to hydrogenase. The increasing effectiveness of various anions closely follows their position in the Hofmeister chaotropic sequence. The most stimulatory anion tested, I − , gives a six-fold increase in activity at a concentration of 0.5 n . The K m of the enzyme for methyl viologen is not affected by anions, while the V is greatly increased. H 2 oxidation coupled to methyl viologen reduction is also greatly stimulated by anions. However, when reduced ferredoxin is used as the electron donor to hydrogenase, there is a very strong inhibition of H 2 production by salts. In this case, the V of the enzyme is unaffected, but there is a large increase in the K m of the enzyme for ferredoxin. The most inhibitory salt tested, KI, decreases hydrogenase activity by 93% at a concentration of 0.2 n .

Effects of electron mediator charge properties on the reaction kinetics of hydrogenase from Chlamydomonas

Anions modulate hydrogenase activity in cell-free preparations of Chlamydomonas reinhardtii, and this modulation is greatly influenced by the charge properties of the redox agent included to mediate electron transfer to hydrogenase. With cationic methyl viologen as the electron mediator, anions stimulate the maximum velocity of H2 production (e.g., a 320% increase in the presence of 1 M NaCl) but have little effect on the Km for methyl viologen. Conversely, when hydrogenase activity is mediated by polyanionic metatungstate or ferredoxin, H2 production is strongly inhibited by anions (e.g., 70-77% inhibition by 0.2 M NaCl). This inhibition is primarily due to a reduced affinity of hydrogenase for these mediators (as evidenced by a large increase in Km values), rather than a change in the maximum velocity of the reaction. Anions have little effect on the kinetics of hydrogenase activity mediated by zwitterionic sulfonatopropyl viologen, a redox agent with a nearly neutral net charge. These results suggest the presence of a cationic region near the active site of hydrogenase. This cationic region, probably due to lysine and/or arginine residues, may serve in vivo to facilitate the interaction between hydrogenase and ferredoxin, the polyanionic, physiological electron mediator.

PHOTOSYNTHETIC CHARACTERIZATION OF A MUTANT OF NANNOCHLOROPSIS DEFICIENT IN THE SYNTHESIS OF EICOSAPENTAENOIC ACID

ABSTRACT Photosynthetic performance of an eicosapentaenoic acid (EPA; 20:5ω3) deficient mutant of the eustigmatophyte Nannochloropsis sp. was compared to the wild type (Wt) strain in order to evaluate the effect of fatty acid composition on the function of the photosynthetic apparatus. Cellular photosynthetic capacity and the cellular pool of pigments and of reaction centers were reduced in the mutant concomitant with a reduction in the amount of thylakoid membranes and their volume-specific density. Despite the changes observed in photosynthetic activity, the fluorescence properties of the mutant were virtually the same as those of the wild type, although the phase transition of thylakoid membrane was recorded at higher temperature in the mutant than in the Wt. The results suggest that the change in one double bond in a very long chain fatty acid of the thylakoid lipids plays a minor role in regulating photosynthetic electron transport, but that the mutation modified the ability of the mutant to acclimat...

A Novel Acyltransferase Activity in an Oleaginous Alga

Nannochloropsis sp. is a unicellular marine alga that accumulates fatty acids in triacylglycerols (TAGs) as well as in membrane lipids [1], Whereas TAG contains mostly 16:0 and 16:1 (n-7) fatty acids, the phospholipids and galactolipids contain a large proportion of eicosapentaenoic acid [20:5 (n-3)]. The accumulation of this fatty acid is regulated by environmental conditions such as light intensity [2]. We have previously developed a preliminary model of polyunsaturated fatty acid (PUFA) synthesis based on pulse-chase radiolabeling studies [3] and analysis of a mutant strain deficient in 20:5 (n-3) (data not shown). The model proposes that C18 fatty acids are desaturated while attached to phosphatidylcholine (PC), whereas C20 fatty acids are desaturated when attached to phosphatidylethanolamine (PE). The PUFAs formed on these extrachloroplastic lipids are the sole source for PUFAs that accumulate in monogalactosyldiacylglycerol (MGDG).