II. B.1. Physiology, Biochemistry, and Molecular Biology of Lipid

Production: Work by SERI Subcontractors

II. B.1 .a. Introduction

Eukaryotic algae, like all photosynthetic organisms, efficiently convert solar energy into biomass. The algal research program at SERI was designed as a long-term basic research effort to adapt or use photosynthesis and related metabolic pathways to produce renewable fuels and chemicals. Research at SERI under the Aquatic Species Program (Biodiesel) has focused on ways to increase the yield of oil from microalgae for cost-effective liquid fuel production. Initially, a large component of the research performed both by subcontractors and by SERI researchers was the collection of microalgal strains from saline environments in the desert southwest of the United States (a region targeted as a feasible location for large-scale microalgal culture), marine environments, and established culture collections. These organisms were then screened and numerous species were identified as candidates for biodiesel production; this research was described in Sections II. A. and II. B. of this report. However, no one species was identified that displayed the ideal combination of rapid growth, environmental tolerance, and high lipid production. Subsequent research efforts were directed toward understanding the biochemistry and physiology of lipid production in oleaginous microalgal strains, with the idea of using strain improvement technologies (breeding, cell fusion, genetic engineering, mutagenesis and selection) to develop algal strains with optimized traits for biodiesel production.

Early in the research program it became obvious the maximal lipid accumulation in the algae usually occurred in cells that were undergoing physiological stresses, such as nutrient deprivation or other conditions that inhibited cell division. Unfortunately, these conditions are the opposite of those that promote maximum biomass production. Thus, the conditions required for inexpensive biodiesel production, high productivity and high lipid content, appeared to be mutually exclusive. To overcome this problem, research efforts were focused on understanding the biochemistry and physiology of lipid accumulation, with emphasis on understanding the “lipid trigger”, a mechanism that could induce production of large quantities of lipid under nutrient deprivation. In addition, research was directed toward understanding genetic variation within microalgal populations and to develop methods to screen for high-lipid subpopulations within algal cultures. The knowledge of the biochemistry and physiology of lipid synthesis, combined with basic studies on microalgal molecular biology, was used in the later years of the project in attempts to use genetic engineering to develop microalgal strains with optimal properties of growth and lipid production.

Part II. B.2. of this report describes work by ASP subcontractors to understand the biochemistry and physiology of lipid accumulation in microalgae, including ultrastructural studies, the development of methods for screening for high lipid strains, and attempts to understand the biochemical lipid trigger. The research performed by SERI/NREL subcontractors on the physiology, biochemistry, and molecular biology of lipid production in oleaginous microalgae

took place during the second half of the 1980s and is presented here, roughly chronologically, according to the work performed by the individual subcontractors.

Eight additional strains collected previously from warm-water sites that grew well during the initial screening procedures were characterized with respect to temperature and salinity tolerances, growth rates, and lipid content under various conditions. These strains were Chaetoceros muelleri (strains CHAET6, CHAET9, CHAET10, CHAET15, and CHAET39), Cyclotella cryptica (CYCLO4), Pleurochrysis carterae (PLEUR1), and Thalassiosira weissflogii (THALA2). Each strain was grown in a variety of temperature-salinity combinations by the use of a temperature-salinity gradient table. The maximal growth rate achieved under these conditions occurred with CHAET9, which exhibited a growth rate of 4.0 doublings^day-1. The remaining strains all had maximum growth rates that exceeded 1.4 doublings^day-1, and several grew at rates exceeding 2.5 doublings^day-1 (i. e., CHAET6, CHAET10, and CHAET39). All had an optimal temperature of 30°C or higher, except for PLEUR1 and THALA2, which had optimal temperatures of 25°C and 28°C, respectively. Most of the strains grew well in a wide range of salinities (e. g., five of the eight strains exhibited a growth rate greater than one doubling^day-1 at

conductivities between 10 and 70 mmho^cm’1). With respect to the effect of water type on growth, CHAET39, CYCLO4, and PLEUR1 grew best on SERI Type I medium. On the other hand, CHAET6, CHAET9, and CHAET10 grew best in SERI Type II medium, but also exhibited good growth on Type I medium and artificial seawater. CHAET15 and THALA2 achieved maximal growth rates on artificial seawater, and, along with PLEUR1, grew very poorly on Type II medium. These results again highlight the need to have a variety of algal strains available for the specific water resources that would be available for mass culture in various locations.

The lipid contents of these 10 strains were also determined for exponentially growing cells, as well as for cells that were grown under nutrient-limited conditions. Nitrogen deficiency led to an increase in the lipid contents of CHAET6, CHAET9, CHAET10, CHAET15, CHAET39, and PLEUR1. The mean lipid content of these strains increased from 11.2% (of the total organic mass) in nutrient-sufficient cells to 22.7% after 4 days of N deficiency. Silicon deficiency led to an increase in the lipid content of all strains (although in some cases the increase was small and probably not statistically significant). The mean lipid content of the eight strains increased from 12.2% in nutrient-sufficient cells to 23.4% in Si-deficient cells. A few strains were poor lipid producers, such as CHAET6, CYCLO4, and PLEUR1, which did not produce more than 20% lipid under any growth conditions.

Dr. Meints received a number of algal strains from Dr. Bill Barclay at SERI that were believed to have potential for biodiesel production. These included Chlorella 501 and A. falcatus (A record

of the precise number and identity of the strains sent to Dr. Meints could not be found). Growth conditions for these strains were optimized, and the algae were then tested for infection by the algal viruses. None of the SERI strains were susceptible to infection by any virus isolate tested. This project was discontinued in early 1987.

Land, water and CO2 resources can support substantial biodiesel production and CO2 savings.

The ASP regularly revisited the question of available resources for producing biodiesel from microalgae. This is not a trivial effort. Such resource assessments require a combined evaluation of appropriate climate, land and resource availability. These analyses indicate that significant potential land, water and CO2 resources exist to support this technology. Algal biodiesel could easily supply several “quads” of biodiesel—substantially more than existing oilseed crops could provide. Microalgae systems use far less water than traditional oilseed crops. Land is hardly a limitation. Two hundred thousand hectares (less than 0.1% of climatically suitable land areas in the U. S.) could produce one quad of fuel. Thus, though the technology faces many R&D hurdles before it can be practicable, it is clear that resource limitations are not an argument against the technology.

A Look Back at the U. S. Department of Energy’s Aquatic Species Program:

Part I:

Program Summary

Background

Origins of the Program

This year marks the 20th anniversary of the National Renewable Energy Laboratory (NREL). In 1978, the Carter Administration established what was then called the Solar Energy Research Institute (SERI) in Golden, CO. This was a first-of-its kind federal laboratory dedicated to the development of solar energy. The formation of this lab came in response to the energy crises of the early and mid 1970s. At the same time, the Carter Administration consolidated all federal energy activities under the auspices of the newly established U. S. Department of Energy (DOE).

Among its various programs established to develop all forms of solar energy, DOE initiated research on the use of plant life as a source of transportation fuels. Today, this program—known as the Biofuels Program—is funded and managed by the Office of Fuels Development (OFD) within the Office of Transportation Technologies under the Assistant Secretary for Energy Efficiency and Renewable Energy at DOE. The program has, over the years, focused on a broad range of alternative fuels, including ethanol and methanol (alcohol fuel substitutes for gasoline), biogas (methane derived from plant materials) and biodiesel (a natural oil — derived diesel fuel substitute). The Aquatic Species Program (ASP) was just one component of research within the Biofuels Program aimed at developing alternative sources of natural oil for biodiesel production.

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

Arizona State University Milton Sommerfeld 1985 — 1987 N/A

The objectives of this subcontract were to collect microalgal strains from a variety of locations in the desert regions of the Southwestern United States and to screen them for their ability to grow under conditions in a commercial microalgal biodiesel facility. Studies were also conducted to optimize a fluorometric procedure for estimating cellular lipid content, and to use this method to screen some of the strains.

Collecting trips took place between April 1985 and June 1986. Water samples containing microalgae were collected from 125 sites in Arizona, California, Nevada, New Mexico, Texas, and Utah. Some samples were taken from saline surface waters in the regions of Arizona and New Mexico that were deemed suitable for microalgal mass culture, based in part on the availability of large quantities of saline groundwater (Lansford et al. 1987). Researchers believed that strains from these areas would be well adapted to the indigenous waters available for mass culture. These areas included the Palo Verde Irrigation District in Arizona, and the Pecos River Basin, the Crow Flats area, and the Tularosa Basin in New Mexico. The temperature, pH, specific conductance, and water depth were recorded at each collection site. Most of the waters sampled had a specific conductance exceeding 2 mmho^cm-1. T emperatures ranged from 18°C to 45°C (mean = 26.9°C), pH ranged from 6.1 to 10.2 (mean = 8.0), and specific conductance ranged from 0.45 mmho^cm-1 to 474 mmho^cm-1 (mean = 22.7 mmho^cm-1).

Planktonic algae were the primary type of alga collected, although neustonic and benthic forms were also collected when algal growth in such habitats was clearly visible. From these samples, more than 1,700 strains of microalgae were obtained. From these strains, approximately 700 unialgal cultures were established. Initial strain isolations were performed by streaking out samples onto 1.5% agar plates containing seawater, sterilized collection site water, or SERI Type I or Type II medium having a conductivity similar to that of the collection site water. In some cases, an enrichment step was performed before streaking out the cells, wherein the samples were placed in tubes on a rotary agitation wheel in liquid media at a light intensity of 1500 pE^m-2^s-1 (~75% of full sunlight) using a 12h:12h light:dark cycle.

Of the 700 unialgal cultures, 120 were identified taxonomically; 24 genera were represented in this group, including 60 chlorophytes, 40 diatoms, and 20 cyanophytes. The most common genera were Dunaliella, Chlorococcum, Chlorosarcina, Amphora, Nitzschia, Navicula, Oscillatoria, and Chroococcus. Initial screening typically involved visual assessment of growth at 25°C at 200 pE^m-2^s-1 in tubes containing SERI Type I/40 and Type II/40 media. Strains that grew the most rapidly were subjected to further characterization, including analysis of the effects

of temperature, salinity, light intensity, and N source on growth rates. Cultures were grown in several different media (SERI Types I and II media at various conductivities, and artificial seawater) at 30°C and 500 pE^m-2^s-1 using a 12h:12h light:dark cycle on a rotary screening apparatus. Thirty-one diatoms were tested under these conditions, and 11 exhibited growth rates exceeding one doubling^-1. The highest growth rate observed for a diatom under these conditions was 1.96 doublings^-1 for Amphora ASU0308. Of the 50 chlorophyte species tested, 17 strains exhibited growth rates exceeding one doubling^-1; the highest growth rate (2.58 doublings^-1) was observed for a strain of Dunaliella (ASU0038). Of the strains that were tested for growth in all seven standard media, 80% of the cultures grew in the low salinity SERI Type I and Type II media and more than 50% of the strains grew in seawater. The highest growth rates were typically observed in SERI Type I/10 and Type II/10 media and seawater, although the mean growth rates of all strains combined at the highest salinities (70 mmho^cm-1) were 60% to 80% of the mean growth rates obtained at the lower salinities. Most of the strains were isolated from waters with specific conductances below 40 mmho^cm-1, which may explain the lower growth rates in the media having higher salinities. A few strains, however, grew quite well in the higher salinity media. Amphora ASU0032 (AMPHO27), Synechococcus ASU0071 (CHLOC5), also referred to by the subcontractor as Chroococcus, and Navicula ASU0267 were the only strains that had a growth rate that exceeded one doubling^-1 in Type II/70 medium. Certain strains had high growth rates in both Type I/70 and Type II/70 media; included in this group were the strains mentioned earlier along with Synechococcus ASU0075 (CHROC2) and Dunaliella ASU0038. Some strains were clearly euryhaline (i. e., able to tolerate a wide range of salinities), and could grow in all media tested (e. g., CHLOC5 had a growth rate that exceeded one doubling^-1 in each of the seven media tested). Other strains were stenohaline, and grew much better at one particular salinity. Certain strains showed no real preference for SERI Type I versus SERI Type II medium, despite the very different ionic composition of these media types. Other strains exhibited a clear preference for one media type over the other (e. g., Dunaliella ASU0038 grew much better in SERI Type I medium or seawater than in SERI Type II medium). Twenty-eight of the strains tested had growth rates exceeding one doubling^-1 in at least one media type, three strains had growth rates that exceeded two doublings^-1, and one strain (either Eremosphaera or Chlorococcum ASU0132 [CHLOC6] or ASU0048 [CHLOC4]) had a growth rate higher than three doublings^-1.

The proximate chemical compositions of 11 isolates were also determined in this study. Total lipid was determined by the Bligh-Dyer procedure (Bligh and Dyer 1959). Protein was determined by the heated biuret-Folin assay, and total carbohydrate was estimated by the phenol- sulfuric acid method (see Sommerfeld et al. [1987b] for details). Of the newly isolated strains tested, Franceia ASU0146 (FRANC1) had the highest lipid content under normal growth conditions (26.5% of the AFDW). The strains were not evaluated under nutrient-deficient conditions, which often increases the lipid content of microalgae. Because analyzing the lipid content of all the strains that had been isolated by this type of procedure would be very difficult, the strains were examined by the use of lipophilic dyes. The dyes Nile Blue A, Sudan Black B, Oil Red O, and Nile Red were used in conjunction with fluorescence microscopy to check for oil droplets within the cells. Inconsistent results were obtained when using the first three stains, but Nile Red appeared to give more reliable results. The stained cells were visually scored for the

presence of fluorescing lipid droplets. Additional work was carried out to develop a Nile Red staining procedure that could (in theory) provide a quantitative measure of lipid content by the use of a fluorometer. This latter work was discussed in Section II. A.1. along with the results of similar efforts carried out by SERI researchers and other subcontractors.

This subcontract was somewhat unusual in that many of the strains were collected from the actual areas in which the commercial microalgal biodiesel facilities could be. As a consequence, many of the strains that were isolated had characteristics that could make them good candidates for production strains. Additional results from this subcontract are presented in Section II. A.1.

Engineering

II. B.3.a. Introduction

The overall goal of the ASP was to cost-effectively produce biodiesel fuel from microalgal lipids. The early laboratory efforts focused on the characterization of microalgae with regard to traits deemed desirable for mass culture and fuel production, i. e., rapid growth, tolerance to environmental fluxes, and high production of TAG storage lipids. Although numerous promising organisms were identified, no individual strain demonstrated rapid growth with constitutively high lipid production. Although high lipid levels could be induced in many strains by starving the cells for an essential nutrient such as N or Si, the increase in lipid was accompanied by a decrease in cell division and total productivity.

During the late 1980s and 1990s, the direction of the laboratory research efforts at NREL shifted to the study of the biochemical pathways involved in lipid synthesis, with the goal of identifying targets for genetic manipulation. As discussed earlier, the desirable traits for biodiesel production (high productivity and high lipid content) were found to be mutually exclusive conditions in the organisms studied. Therefore, it was decided to use mutagenesis or genetic engineering to manipulate the algal biosynthetic pathways to produce algal strains with constitutively high lipid levels. Another possibility would be to engineer an organism in which lipid synthesis could be regulated by inducing or repressing key genes. Very little was known about the molecular biology of oleaginous microalgae and the genetic regulation of lipid biosynthesis pathways, so a concentrated research effort in this direction was deemed critical to

the success of the biodiesel project. Another reason for the shift to research on genetic manipulation of algae was more practical. Funding levels for ASP decreased during this period from the high funding levels in the mid-1980s that had allowed large numbers of subcontractors and the development of the Outdoor Test Facility in Roswell, New Mexico (Section II. B.5.). Laboratory experiments emphasizing biochemistry, molecular biology, and genetic engineering could be performed with a limited budget and few personnel.

This section of the report will describe the in-house research efforts at NREL to develop high lipid algae by genetically manipulating selected oleaginous strains. Microalgae generally reproduce asexually by simple fission. Many strains can also produce sexually, but the conditions required to induce algal sexual reproduction in the laboratory are not known for most species. Thus, genetic manipulation by classical “breeding” was not an option for the algae. The approaches explored at NREL were (1) mutagenesis and selection; and (2) genetic engineering. The information summarized in this report was taken from annual reports, scientific publications and meeting reports. No annual reports were generated by the ASP after 1993, and no quarterly reports after September 1995, so some of the most recent information presented was derived from the personal experience of Terri Dunahay and discussions with former coworkers (Paul Roessler and Eric Jarvis).

Like all of the renewable fuels programs, the ASP has always been on a fiscal roller coaster

In its heyday, this program leaped to levels of $2 to $2.75 million in annual funding. In most cases, these peaks came in sudden bursts in which the funding level of the program would double from one year to the next. After the boom years of 1984 and 1985, funding fell rapidly to its low of $250,000 in 1991. The last three years of the program saw a steady level of $500,000 (not counting FY 1996, which were mostly used to cover the cost of employee terminations). Ironically, these last three years were among the most productive in the history of the program (given the breakthroughs that occurred in genetic engineering). Though funding levels were relatively low, they were at least steady—providing a desperately needed stability for the program. The years of higher spending are, for the most part, dominated by costly demonstration work (the tests carried out in California, Hawaii and culminating in New Mexico), engineering analysis and culture collection activities.

High Return for a Small Investment of DOE Funds

The total cost of the Aquatic Species Program is $25.05 million over a twenty-year period. Compared to the total spending under the Biofuels Program ($458 million over the same period), this has not been a high cost research program. At its peak, ASP accounted for 14% of the annual Biofuels budget; while, on average, it represented only 5.5% of the total budget. Given that relatively small investment, DOE has seen a tremendous return on its research dollars.

The purpose of the research performed by Dr. Sriharan and coworkers was to study the effects of nutrient deprivation and temperature on growth and lipid production in microalgae with potential for liquid fuel production. All the reports generated by this laboratory during the subcontract describe a virtually identical set of experiments performed on several species of microalgae. The benefit of this approach is that the productivity data can be compared between the species. However, the flaws in experimental design and reporting were carried through all experiments and reports.

The basic design for these experiments was to grow the algal cells in batch cultures in media that contained either “sufficient” or “deficient” levels of N or Si, and to test for algal growth rate, productivity, and lipid production. Cultures were grown at 20°C and at 30°C to test for the effect of temperature on growth and lipid induction. Exponentially growing cells were inoculated into fresh media containing high or low levels of Si or N. Growth was monitored by measuring the optical density of the culture, and the growth rate was reported as the number of cell doublings per day. The cells were harvested and processed to determine lipid content (reported as a percentage of the AFDW), and fatty acid composition under the various growth conditions.

The organisms studied were all diatoms, except for the chlorophyte M. minutum (which the authors initially reported to be a diatom, but they corrected this error in a later report). The diatoms tested were Chaetoceros SS-14, C. muelleri var subsalsum, Navicula saprophila, obtained from SERI, Cyclotella DI-35, Cyclotella cryptica Reimann, Lewin, and Guillard, and Hantzchia DI-60, obtained from M. Tadros at Alabama A&M University. All the organisms tested grew more rapidly at 30°C (versus 20°C) and in nutrient-sufficient media. A decrease in the total AFDW was reported for all strains grown in nutrient-deficient media compared to nutrient-sufficient cultures, and this was accompanied by an increase in the percentage of the AFDW made up of lipids.

The most dramatic increases in the lipid content of the cultures were seen under N-deficient conditions in cells grown at 30°C. In C. cryptica, the total lipids, as a percentage of AFDW, increased from 15% to 44%. In Hantzschia, lipids increased from 29% to 53%, and in Navicula saprophila, lipids increased from 26% to 44%. In all cases, the increase in total lipids was due to increases in both the neutral lipid and polar lipid fractions. In several cases, the ratio of neutral

lipids to polar lipids increased significantly in the nutrient-stressed cells (i. e., in Hantzschia and C. muelleri grown in Si-deficient media, and in Navicula grown in N-deficient media). Dr. Sriharan also presented data comparing the fatty acid profiles of lipids from diatoms grown under nutrient-sufficient and nutrient-deficient conditions. Although the data was incomplete, it indicated that changes in the fatty acid composition (lipid quality) did occur in nutrient-stressed cells, suggesting that nutrient deprivation can affect the lipid biosynthetic pathways.

In all cases nutrient-deficiency resulted in a decreased rate of cell growth and a decrease in total cell productivity. Therefore, an increase in lipid as a percentage of cell mass may not be economically advantageous for liquid fuel production from mass-cultured algae if the conditions that induce lipid accumulation also result in a significant drop in total biomass, and thus in total lipid produced. Although this was not discussed by Dr. Sriharan, the total effect of nutrient limitation on lipid content of the algal cultures could be estimated by multiplying the total biomass (AFDW) produced by the percentage of the AFDW attributable to lipid under nutrient — sufficient or nutrient-deficient conditions. In general, these calculations demonstrated an increase in lipid content of the cultures induced by nutrient stress, in the range of a 20% to 30% increase in total lipid.

The results reported here clearly suggest that algal productivity is increased under nutrient — sufficient conditions and at elevated temperatures. However, it is difficult to determine the validity of the data presented regarding nutrient-deprivation as a lipid trigger. Growth curves were not presented for the organisms studied. Therefore, it cannot be determined if the low nutrient levels limited growth throughout the period of the experiment, or whether the nutrients became depleted and the lipid effects correlated with a decrease in cell division, as reported elsewhere. It is also not clear at what point in the cell cycle the cells were harvested for determination of AFDW and lipid content, and how to compare the data for nutrient-sufficient and nutrient-deficient cells. In several experiments, the authors reported that the cultures were only harvested when lipid droplets were seen in the cells, although this would seem to bias experiments designed to test nutrient effects on lipid production. All in all, it is difficult to determine whether the experiments were badly performed, or just poorly reported.

In summary, the data presented by Dr. Sriharan was difficult to interpret for these reasons; however, several general conclusions can be made. Diatoms seem to be promising candidates for neutral lipid production. Many species produce constitutively high levels of lipid, and the level of lipid as a percentage of biomass can be increased by growing the cells under nutrient-limited conditions (the data presented here suggests that N-limitation may be more effective than Si — limitation). In addition, Dr. Sriharan’s results suggest that nutrient-limitation may alter the lipid biosynthetic pathways in diatoms to increase lipid production and possibly affect lipid composition.

Most microalgal collection efforts carried out under the auspices of the ASP before 1987 focused on sites that were expected to naturally experience high temperatures; indeed, one subcontractor, Keith Cooksey (Montana State University) specifically searched for thermophilic strains isolated from hot springs. This was because the temperatures of production ponds in the southwestern United States during the prime growing season were expected to reach high levels; thus the production strains would have to thrive under such conditions. However, temperatures in this region are quite cool for several months of the year and can drop to below freezing at night. Consequently, an effort was initiated by SERI researchers to collect, screen, and characterize strains from cold-water habitats.

Four collecting trips were made between October 1986 and March 1987 to various inland saline water sites in Utah and eastern Washington, and to the coastal lagoon waters in southern California. Water samples were enriched with N, P, trace metals, and vitamins; artificial media were not used in the initial selection protocol for these experiments. The rotary screening apparatus was maintained at 15 °C for the duration of the screening process by including a copper cooling coil inside the screening chamber. The cultures were incubated for 5-10 days, which is longer than for warm-water strains because of the slower growth at the cooler temperature. This procedure created a problem, however, in that many more strains survived the selection process than when 30°C was used as the selection temperature. As a consequence, separating strains from each other and identifying which were the best for further characterization were more difficult.

An interesting finding from the cold water strain collection project was that many species that predominate after the enrichment procedure were the same as the warm water species selected in previous collection efforts. The genera and species that were commonly found in both the cold water and warm water screening projects were C. muelleri, Amphora coffeiformis, Cyclotella, Navicula, and Nitzschia. However, some ochromonids and green coccoid algae were also isolated from the cold water collection effort; these types of alga were less commonly isolated during the warm-water selection procedures. Additional work would be needed to characterize these strains with respect to lipid production potential. Future work should look at the fatty acid profiles of oil found in the cold-water strains. Such cold-water organisms often contain high levels of polyunsaturated fatty acids, which would perform poorly as a feedstock for biodiesel because of their low oxidative stability and tendency to polymerize during combustion (Harrington et al. 1986).

I Publications:

Johansen, J. R.; Doucette, G. J.; Barclay, W. R.; Bull, J. D. (1988) “The morphology and ecology of Pleurochrysis carterae var dentata nov. (Prymnesiophyceae), a new coccolithophorid from an inland saline pond in New Mexico, USA.” Phycologica 27:78-88.

Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987) “Collection, screening, and characterization of lipid producing microalgae: Progress during Fiscal Year 1987.” FY 1987 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231 -3206, pp. 27-42.

Johansen, J. R.; Theriot, E. (1987) “The relationship between valve diameter and number of central fultoportulae in Thalassiosira weissflogii (Bacillariophyceae).” J. Phycol. 23:663-665.

Tadros, M. G.; Johansen, J. R. (1988) “Physiological characterization of six lipid-producing diatoms from the southeastern United States.” J. Phycol. 24:445-452.

I Additional References:

Harrington, K. J. (1986) Biomass 9:1-17.

Infection of Chlorella N1a or NC64A by the algal viruses resulted in rapid lysis of the algal cells. Dr. Meints’ laboratory developed methods to isolate the lytic enzymes and to use the lysin preparation to produce algal protoplasts (cells without cell walls). The protoplasts could be used in studies of somatic cell fusion (genetic improvement by fusion of two individuals with useful traits such as pH tolerance and high lipid production). Alternatively, the protoplasts could be used as targets in a genetic transformation system in which DNA plasmids are taken up directly into cells without walls.

A crude lysin preparation was produced by infecting Chlorella N1a with PBCV -1. After several lytic cycles (approximately 24 hours), the sample was centrifuged to remove cell debris and virus. Initially, the supernatant from this preparation was used directly to produce protoplasts from Chlorella N1a cells. Alternatively, lysin activity was precipitated from the supernatant with 65% ammonium sulfate. Cells were exposed to lysin in the presence of 1 M sorbitol as an osmoticum. (One interesting result from the protoplast studies is that algal strains exhibited significant differences in their sensitivities to osmotica commonly used for higher plant cells; i. e. mannitol, but not sorbitol, was toxic to Chlorella N1a. The sensitivity of individual algal strains to different osmotica will need to be determined empirically.) The cell wall of algal cells exposed to lysin was rapidly degraded over the entire cell surface, as judged by electron microscopy and staining of the cells with calcofluor white, which stains plant cell walls. The lysin preparation could be purified further by exposing the crude sample to an affinity matrix composed of algal cell wall fragments. Lysin activity was eluted by salt washes. Exposure of Chlorella N1a cells to this lysin sample resulted in degradation of the algal wall at specific sites; when the osmoticum was reduced to half strength, the alga protoplast was released through discrete holes in the wall. This result suggested the presence of more than one enzymatic activity in the crude lysin preparation.

Somewhat surprisingly, the protoplasts did not lyse when transferred to water. However, exposure of lysin-treated cells, but not untreated cells, to low concentrations of detergent caused the release of chlorophyll. The amounts of chlorophyll released from lysin-treated cells was used as a measure of the extent of protoplast formation in a cell sample. The protoplasts were viable, as judged by staining with fluorescein diacetate. Unfortunately, although some regeneration of the cell wall occurred, the lysin-treated cells never formed new colonies. Attempts to use the viral-lysin to produce protoplasts from other microalgal strains met with little success. A manuscript was prepared and submitted to SERI that described the progress made on the use of lysin to produce algal protoplasts, but the article was never published in a technical journal.