Laboratory Studies

This subcontract focused on the collection and characterization of picopleustonic algae, which are defined as algae (including the prokaryotic cyanophytes) that are very small (1-5 ^m) and that live on the surface of the water. In February 1985, water samples were taken from various sites in the Caribbean Sea, including sites near the U.S. Virgin Islands (St. John, St. Thomas, and St. Croix), Tortola, Puerto Rico, Curaçao, Panama, and the Florida Keys. 130 samples (250 mL each) were collected and filtered through a 3-8-^m filter to remove larger cells. Smaller cells were collected on a 0.45 ^m nitrocellulose filter, which was rolled up and placed in the original sampling water that had passed through the filter. These samples were placed under natural lighting at 20°C to 25°C until transported to the laboratory. The filters were then transferred into a tube of sterile enriched seawater (containing additional N and other nutrients) and incubated at 25° C under continuous

illumination from a fluorescent lamp at 30 ^E- m • s . In an attempt to stimulate lipid accumulation via nutrient deficiency, a portion of each culture was transferred after 4 weeks of growth to a fresh tube of unenriched seawater and then allowed to grow under the same conditions. After 4 more weeks, a film of cells was often observed floating on the surface of the cultures. Small samples of these cells were transferred to fresh enriched seawater. After incubation for an additional 2 weeks, the cells in these cultures were microscopically examined, and cultures that were dominated by diatoms, cyanophytes, and flagellates were

discarded, leaving approximately 60 cultures of small (1-5-^m) green cells. Unialgal cultures were established from these cells by isolating colonies on agar plates. Of these purified cultures, there were 14 isolates of Stichococcus, 21 isolates of

Nannochloris, four strains of Chlorella, and several representatives of other genera.

Stichococcus, Nannochloris, and Chlorella are all chlorophytes. Because cyanophytes typically do not accumulate lipids, they were eliminated from further The isolated strains were tested for the ability to grow in freshwater; all the study in this subcontract. The researchers anticipated that isolating strains in this Stichococcus and Chlorella strains grew well in freshwater, suggesting perhaps a manner would enrich for lipid-accumulating microalgae.

brackish water origin for these strains. Only six of the 21 Nannochloris strains

TOuquiadwadReR5n-lí§íffliam cICFISC content in the isolated strains, 1-L cultures were

grown for 3 weeks in enriched seawater under continuous illumination at 30 ^E • m

• s . The cultures were bubbled with 0.5% CO2 in air. Cells were harvested, frozen and lyophilized, and then extracted three times with a chloroform/methanol mixture (2:1 v/v). After the solvents evaporated, the lipid mass was determined gravimetrically and normalized to the cellular AFDW. The 13 Stichococcus strains had lipid contents ranging from 9% to 59% of the AFDW, with an average of 33%. The lipid contents of the 21 Nannochloris strains ranged from 6% to 63%, with an average of 31%. Data were not presented for the lipid contents of the four Chlorella sCíI?laspS(ltllIClaSíythíXl|leS})ísia:Ss °wil? susowtouCtytf Nafladcllillío^o^stia(CfwtlílS

suMfopv Qgniilag Cl?ngrwwti ^mrf msopeiyui' NiialapC!iC°j°psis rsRSia NsaaO-Q%° otde 6C80%}heviQiagaa°v:eííSias)^f 46% site ^hsCaeeSid0!« eofrdw s^awai^is^ an bthcySi? wwr5 wsiffp mtati sMiRtiy romvw§omew]ise p^lsr gnaw ris la hhiiMnsavii esis owm a ears cs? d^NMscidlaa awuidum mainum, satnedp .the lipid content rose substantially when the N source was initially supplied at levels below 200 ^M (as determined by the percentage of cells that were floating due to elevated lipid levels). A number of Nannochloropsis strains that had been obtained primarily from the Culture Collection of Marine Phytoplankton at Bigelow

Tl^sut°í(yn}WíScstlB;ofl}abdyaI|aoU^oMftlnrd)awp'? tlaílyse<dnwc'¿tli(íllvllcw1dcmaximum aiti a^iati? AsweuRtil (dla}^pwwtilt•at Theismst ltslZlP?íaíuc°FSleiuSts}aiRf alg^duM had hlndllíhSuveSíiFtlflífS(iecícyfli°WIm;5s0sCullUtll°fscili°yifwSllrsirwclulCi iiavibgeifegsoyvcte1 amps Loon Ill1 nd° Nmi Yddk hoi í?SgmF?íaBBíí° °PtiH°wfe°e r? 3° the c ells could be made to consistently float due to high lipid levels, this property might facilitate harvesting. Outdoor testing of the most promising strains would help to evaluate this group of microalgae. Publications:

Lewin, R.A. (1985) "Production of hydrocarbons by micro-algae; isolation and characterization of new and potentially useful algal strains." Aquatic Species Program

Review: Proceedings of the March 1985 Principal Investigators' Meeting, Solar

Energy Research Institute, golden, ColoradonSERI/CP-231-2700, pp. 43-51. „ Lewin, R.A.; Burrascano, C; Cheng, L. (1987) "Some picopleuston algae from the

Caribbean region." FY 1986 Aquatic Species Program Annual Report, Solar Energy

Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 105-121.

II.A.2.h. Collection of High Energy Yielding Strains of Saline Microalgae from South

Florida

Subcontractor: Harbor Branch Foundation Principal Investigator: John H. Ryther Period of Performance: 1985 - 1986 Subcontract Number: N/A

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 ) was somewhat higher than that of the Everglades (25.7 mmho- cm ), whereas the pH values were similar (~8). To select for the pífsti9lilíiícywt«HiustiO0acii:n§tga^lii«ctinhe^sIFa^s^IDll^slrilMis mignkwagm^wifl e^rI<sn(tubw?thoRt§IiningteicIc1?§ct§lSivaitirv1SímíiTyparíd2l?lcUmfIeCÍ Weffiont^iuous and Type IIM0 media. -The tesLtubeswire incubated at 30°C, under constant, light (880 mE- m • s , or 45% of full sunlight) at 30 C. The strains that became dofflnMon ah <300l tures%ere%oGrow totgn sup du^eu'rgSrri^ct mi cmo^spa&ghierial Siui^fivefiyddspreoriihiC aytoang-athp lM c uAuaecoftnsqy enae measure (IatsímsWsly6•l OffialgjnCuecurisi wee^aulu&pg 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- d ) was observed with a strain of Hymenomonas HB152 (HYMEN3)

in Type II/25 medium. Seven strains had growth rates that exceeded 2 doublings-

d in at least one media type; included in this group were Dunaliella HB37

SDUNfalpf ,tNa mmcsihPopISi H3nr4s (NArsrwei 1x11111121 cT rn en orr clitaHB5ty^Wi£Ngi4Cwn Cn^premaHix^ (CHLPRa)fft2i2tiBSS^imiiitsm(m-^s mmhp3 (PHAMI , srK HYM5N3

Nimp erasing Sis(d igro-wth' rStiibyf tSttl east orfesaputolpSTatidre issiSiriitiyeTffstCitsnti ncbidi mgs CeScrSiidH^SSHiLiORgc tsrias^Ar7aiElfiLíSR9i,síWsnnmpchíodllSlH^ASP(-í2i^Id^i? cirnaiSil vaesmTimmpiirtS pnfedi tgtslltlirvifs tttri svpssrissirSpfh reXciSil top HYM2ti3)The cultures were

exposed to constant illumination at 180 ^E- m • s . Each of the four strains tested

(Tetraselmis HB47 [TETRA4], PYRAM2, UNKNO4, and an olive green unicell HB154

LUNKNO5J) exhibited excellent growth pver fa ,wide range pf conditions. . All these A visual assessment of the lipid contents of the most rapidly growing strains was cOnSduc ttsabyg5lS^2si^ltS^gi#SSewJlRaN ioltn S)slO,utRl^ltg:si2ledt(eS1>Wst ent8e Stx4laí2lalUsC2g Ba^ed on this assessment (which was not carried out with nutrient-starved cells},

^.rfaCir^WWNeOíy^1 am<y 5,ighCesUNKKiNmii14ecnSCi i dTrpownt&nfiStSSERlTRilr hRme^, PoubAinT^'aKd bNlwNen5laíssraJp°al®a^lo•agC0lmiunatbilwistgnr(a° quancCi tieis oCi i pid.

Publications:

Rhyther, J.H.; Carlson, R.D.; Pendoley, P.D.; Jensen, P.R. (1987) "Collection and characterization of saline microalgae from South Florida." FY 1986 Aquatic Species fgpTram Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 122

II.A.2.i. Collection and Selection of High Energy Thermophilic Strains of Microalgae

Subcontractor: Montana State University Principal Investigator: Keith E. Cooksey Period of Performance: 3/86 - 4/87 Subcontract Number: 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. 1 .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 I PubiikstlohS to lerate extremes of temperature and pH variation. In these cultures, Nile Red wyrglluned! Do CCfoifSi3i,hg . - ^Ooksiymply - Pfisiuite^rf 'SícairetilíhshEl iSEcrlSewnfrfy oO?EfT ic:t•cltl•¿tEEdfoFlLlifdEd plndd^&i]foTnP 01x1^11) Med cfoo meowfOr ripe ars!ySipid ptdd•9etidreqljsEtiStrisinsiefsffrd^rweerrrdwvtiíypslfl? §C lar5 EryrgoKbSEritci itsHM1

GOnldiyh11ScOílOf0|PdLd4SE:ES^nSiIS2 3d Jthe 7pfcppE rtl 1s- 5oOf o il-producing algae isolated by other mydkOiy;, K.E. (1987) "Collection and Screening microalgae for lipid production."

Final Subcontract Report to the Solar Energy Research Institute, Solar Energy

Research Institute, Golden, Colorado, May 1987, 42 pp.

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," Journal of Microbiological Methods 6:333-345.

II.A.3. The SERI Microalgae Culture Collection II.A.3.a. History of SERI Microalgae Culture Collection

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

Tifo rmrigiiiai ^©84-1$ 85 tMes^st"?^!!!"; iO0uaognpCatoteicf0fgaphse p°°°wia]gi chgmiaafcc°mp°sti°iin°f lspM iconosibis ponflcgidisi igrhw ntatnaígre(V apioMi^ eMronmn ntiiil dlgatai1idli1l^ powthf hdlrdcrtgrnCiiCs in different media types and different temperatures, and?the reSE11ttSifyT yie1?' igsred1^1? ffltSb xrspggfKietieiepOnd trials. Furthermore, media com?positioii^^ee°p ifUVidgic°ldUClS:dV^iIlh1°ffii0 m 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

Foomse qhrcnc epam fne wureimacoaece0 r^unw^enotto ffi9i8t0y, cSoc¿tlnse fffe ^hr c0lecal!i0ogwere 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

Tht sEer i Ores gshoeM tube conideoef1 cSMsMmosn ^XeMk? rop maryfe ^gato» wmc roiagad . ^Hcpi.oeviidiçg thu tyfnase ctoon u^memtbes oh ^mMarigm per§iss(r (wuthtia ^hFeAdom wnaai-fc e ^chlteofthyte ^grUen 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

Mlt® cweee tC°lno(r°tà1í°|geSl 980% 1W5 ^stems^» wchFcysoifihyiteesd ane ê%l°r'°pel^,l^o(a1âyoms, Tin -(o1]^ys°pllyitseg esní(^t°tt11e etl^sitiîgsrc"t°(iî¥lyeê(. insomh oofi thesth setfEactïJstUrerê °iib1(atiV°ítiCf^c5ïlmO g°tlP:lit>(íASl1tUÎ(ec(Cl^iíFlC ttt°ns, ciHcsuGfingt^teiASFniversity of lexas algaLculture collection. i Thei strains listed.in the Toriginal 1984-198.5catalog Table II:A.6. Microalgal strains listed in the Tirst SERI Culture Collection waetrael oa|,fote1985.

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