Laboratory Studies

standardized conditions in various

"Zzyzx medium" before algal isolation (see Thomas et al. [1986] for media compositions).

artificial media that were designed to mimic the water from which the strains were originally obtained. The pH of these various media formulations was typically high because of the presence of high levels of carbonate and bicarbonate, and the total

dissolved solids ranged from approximately 1.5 g- L to over 260 g- L . The growth of the cultures was visually scored, and nine of the fastest growing strains For larger scale cultures, 6 L of medium that was enriched with N (nitrate, urea, or were further analyzed with respect to growth under scaled-up conditions.

ammonium), phosphate, trace metals, and vitamins were placed in 9-L serum bottles, and the cultures were illuminated with fluorescent bulbs at a light intensity of 18% full sunlight. To enhance growth, the cultures were bubbled with 1% CO2 in air, and more nutrients were added as the cell density of the cultures increased. The strains tested in this manner included Nitzschia, Ankistrodesmus, Nannochloris, Oocystis (two strains), Chlorella (three strains), and Selenastrum. The estimated productivities

ranged from 8.8 g dry weight- m • d for for Nitzschia S-16 (NITZS1 ) grown in the

presence of urea to 45.8 g dry weight- m • d for Oocystis pusilla 32-1, which was also grown with urea as the N source. (These productivity values were considered overestimates in that there was incidental side lighting of the flasks under the incubation conditions.) Also the productivity values did not always correlate with

AsaitboftmasgpiiiMevftSjwi reidasteugouhatogsssitfe she^f§mííbiriwgs^ffgcíhecCt#mpiíft^EUP^e anUusM iiliysoii stho rgthevva roousever&pof tTfeeismaemu m bourns geld i wtstsbta ws wish Oor fussaswimens^ whyc hwh!h!Gub%io nrggopesulus^ thxsstaexpsiioifefts taCieaveiieitoiwiffl sUM han^ a^aP pfnitiii ooorthiihgreure aweiivrarti tthivN SouremiBic alonguM oShifgWfS: aas^uess asxfwnsiVil stthan ^iraaiu rthiwi tusk ti a^ing ec on omatg i impgcatcofti rewllhc(?iSb)iaatioclsalcfaltefflíssra$u^tu rand However, Gnowtfe swuas coming c pre fepence corFpamusurNmoutsceawi riontourwa^rpriddswe1. upon a matrix chart of the various temperature/salinity combinations. This approach was later used by other subcontractors and SERI in-house researchers to determine the optimal growth characteristics of numerous promising algal strains. Results from this Thalfe cw eof lnporltllldnscryBOghf idífgrr©nth> of An.kIntgiCiS1aílushil ktatonS^gr-IwiANiiS 1at frigmerPyafimildes, Hindi canI¿lg^EhpUrs1ii^^ o§£-11c (fr(ítQíeWWanCehaCalsi}npIliSltUf^opti?ad for 00r8»w-th cutweintha0 ewers nUacese aC .vaQfngi I istancss fedtia titongMofloi ampesouolaie rNi?;í^ail^ilrQSlll:i|is if^luilus:ii(iiclfi:c[tlítu r:ii?ilIiInildisIsn:;gb^Wi(afi3E^(gffiCe36ehi;n.

relevant.

placed between the light source and the cultures. This arrangement provided between 30% and 70% of full sunlight. For ANKIS1, maximum productivity (21.9 g

dry weight- m • d) was attained when the cells were subjected to 50% full sunlight.

At 30% full sunlight, the productivity fell off to 14.7 g dry weight- m • d (because of light limitation), and-2 at 7-10% full sunlight, the productivity was reduced to 19.0 g 23.2g dry weight- m ' d , respectively. These productivity values are believed to tofefcrswthdn test rlpofeSiaausi polcphiRo isRlcibot^cee•caUSer liglltPUlf^illlb is

maxnmumhp rcduuriv itysoe l2 5c•íSl3gfedm wig htdem Thd r els0SvalSdg1l:t<easdtisS 50thiull guRBfti (wh e|íe<!ísyP^e?UfCfdViLy1 fp3Pi%i ttnd SlftM sunoatHwHl igil7r light intensities

Experiments were also conducted to determine the effects of varying the culture than would be possible in less dense cultures, because of the self-shading of the cells.

vessel width (i.e., culture depth) on overall productivities. ANKIS1 was grown in containers that were 5, 10, and 15 cm wide. The containers were illuminated with a tungsten lamp at 50% full sunlight, and were bubbled vigorously with 1% CO2 in air.

The results of these experiments indicated that growth rate, when expressed as g dry

weight- L • d , was highest in the 5-cm thick culture and lowest in the 15-cm thick

culture. However, when productivities were expressed as g dry weightd m d d , which takes into account the actual surface area that is illuminated, the thicker cultures were more productive. For example, the volumetric productivities over 10

days were 0.72, 0.35 and 0.31 g dry weight- L • d ) for the 5-, 10-, and 15-cm thick cultures, respectively, whereas the corresponding areal productivities for these irultueeHRifeye nof , % . 2u1scoatí27, g^ry^g^m^g ir^ecsus^tea eednfe moMrmMs^Vdittfaa aSuiseaigssmdatenB p^oducm3 fah§kyladcfei§r)hicfectecc durlffg pond- ersystpmsweffie scaiiaedprooui( tfvayojste eshafr ebefige. mIc^ethi?npdvtsfnc cdresltidrsp?OR(? íslSh(dhill3l€§jnwsteríeiarRldb(jidR)Secqltfe rrlli^[email protected]ís lWg1l1)errllSRSll)ili||l liR<reíquíiíer wmperauriRg swRctlthIIlOlí»il itiesio! thul turssas toigrow dRuSvHi esandpo<tlcldlsí " twise experiments. mTi$ es eCieíidal maocec(dlílvlílsutillde intrlins isolutioa cdfevi iadoutloasr mrslasuMtebscsualng Oils tcrpaiiis iiz©cdiMixci ngcubstinrstloaisogims . at 25°C and 30° C under 40% full sunlight (provided by a 2000-W tungsten-halide lamp) in the SERI

standard medium that most closely resembled the water from which they were

The • development of„the, SERI .standard media is discussed , in Chapter II.A. 1. The originally isolated. Twelve of the strains that grew best under these conditions were compositions of these media are given in Table II.A.r.

then tested under the same temperature and light conditions in an early version of standard SERI media (Type I and Type II at low, medium, and high salinities; see media compositions in Thomas et al. [1985]).

The results indicated that most of the strains had a definite preference for a particular medium type and level of salinity. The results also indicated some inconsistencies in the growth rates of cells grown in the two experiments. For example, Chlorella BL-6 (CHLOR2) grew very well in the preliminary experiments in Type II/low salinity medium (2.48 doublings- d ), but grew much more poorly when grown in all five SERI media (including Type II/low salinity) in the second set of experiments. Conversely, Chlamydomonas HL-9 grew much more quickly in the second set of experiments than in the first set. The reasons for these discrepancies are unclear, as the culture conditions were essentially the same, and underscore the need to perform

Tebeli00tKblxBe^irIrii0ts. oevte&p^r§^uce mBdro>slpywBe % (grewfe rorrf Eebgiity oo grow neeEs IcoliíBiiadd smi . waeoaeorbUKitrb}oCbu turn, o;fl§eteIcrepe.sagcrfol^i,i bBh gradient table. In general,, the strains grew best -tin the range of salinity that was Platymonas all grew well (>1.25 doublings • d ) in at least one SERI medium. similar to that of the water from which they were originally isolated. The optimal Isochrysis T-ISO was unable to grow in any SERI medium, however. temperature for growth was generally in the 25°C to 35° C range, although one

Chlorella strain from Salt Creek grew well at 40° C. Based on the results of these experiments, two strains were selected for analysis of growth characteristics in larger

Tcase siuc oBi tcuctureprts e50%d 70% ou lthufirstfOCHLO Igl2s aAh^vo d( oal lpro ductiscty: eof swaga} swep^m^^ lSB¿itecbtbbse, fucBdlcocBsc ^ sMnS)■chIO^ie MoMuM e many of the screening and characterization protocols were still being developed; productivity of 31.9 g dry weight- m • d .

therefore, there is a substantial lack of uniformity in the testing of the various strains isolated. Nonetheless, a number of promising strains were isolated during the course PubOf( atiisnsesearch, and several methods were developed that helped establish standard TCC(eKie|Síl8WPCOt;OG(alBUS,ea. Íby(1t9b8e2 )AíeFi|la§eeCCcberS^e arid southwestern United States: an annotated bibliography." Report for Subcontract XK-2-0270-01. Solar Energy Research Institute, Golden, Colorado, October 1982.

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P.; Neori, A.; Gaines, S. (1983b)

"Selection of high-yielding microalgae from desert saline environments." Aquatic

Species Program Review: Proceedings of the March 1983 Principal Investigators'

Meeting, Solar Energy: Research Institute, Golden, Colorado, SERI/CP-231-,1946, pp. Thomas, W.H. (19oob) "Microalgae from desert saline waters as potential biomass 97-122.

producers." Progress in Solar Energy 6:143-145.

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1984c) "Cultural requirements, yields, and light utilization efficiencies of some desert saline microalgae." Aquatic

Species Program Review: Proceedings of the April 1984 Principal Investigators'

Meeting, Solar Energy Research Institute, Golden,!Colorado, SERI/CP-231 -2341, m ThomaS, W.H.; Torrfabene, TX.; Weissman, J. (1984d) "Screening for lipia yielding 7-63

microalgae: Activities for 1983." Final Subcontract Report. Solar Energy Research Institute, Golden, Colorado, SERI/STR-231-2207.

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1985) "Selection of desert saline microalgae for high yields at elevated temperatures and light intensities and in SERI Standard artificial media." Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators' Meeting, Solar Energy Research Institute, Go0CflaB,CWHSciO5lbe?i/<D?L2R?;1-íí7^, M7dge, P. (1986) "Cultural requirements, yields and light utilization efficiencies of some desert saline microalgae." Nova MActw.tiigia 83:60-69ning and Characterizing Oleaginous Microalgal Species from theSoutheastern United States Subcontractor: Alabama A&M University Principal Investigator: Mahasin Tadros Period of Performance: 1983 Subcontract Number: XK-3-03-50-1

The goal of this subcontract was to isolate and characterize strains of microalgae from the southeastern United States that have attributes desirable for a biodiesel production strain. During the first year of this work, field trips were made to several sites in Alabama to collect microalgal strains from a variety of habitats. Freshwater and brackish water strains were collected from rivers, lakes, estuaries, and ponds, and marine strains were collected from the waters surrounding Dauphin Island in the Gulf of Mexico. Collected samples were inoculated into various artificial media, including Bold's Basal Medium, Chu no. 10, and "f/2" (Barclay et al. 1986). Artificial sea salts were used in place of seawater for the saltwater media. For initial strain selection, the cultures were incubated at 29-30° C with shaking at a light intensity of 100 to

125 |mE- m • s provided by cool white fluorescent bulbs with a 14 h:10 h light:dark cycle. The fastest growing strains were isolated via micropipetting or by spreading samples on agar plates. In these preliminary experiments, the marine strains exhibiting the fastest growth were Cyclotella DI-35 (CYCLO1), Hantzschia DI-160 (NITZS2), and Chlorococcum DI-34. The freshwater strains exhibiting the fastest growth rates were Chlorella MB-31, Scenedesmus TR-84, Ankistrodesmus TR-87, and Nitzschia TR-114.

CYCLO1, Nitzschia TR-114, and Scenedesmus TR-84 were selected for more detailed growth analyses under various combinations of temperature, salinity, and light intensity. A temperature gradient table was employed for these experiments that was similar in design to the tables used by Dr. William Thomas (discussed earlier) and SERI researchers for screening purposes. Growth of standing cultures was determined by measuring final cell densities after 12 days of incubation. CYCLO1

achieved maximum cell density at a temperature of 30° C, a light intensity of 100 mE

• m • s , and a salinity of 15 ppt (parts per thousand). Growth was nearly as good

at a light intensity of 200 mE- m • s and a temperature of 35 C, and substantial growth occurred at a salinity of 32 ppt. Growth did not occur at 15° to 20° C.

Nhtzsthid TRn1¿Tt!iidlfiyvvd rM&ih:? eSO:MtdUtiT!g) °thC . ^pp lcC?lEfntiTgannífPS 00yrE

demrmined.grtowt11 4wlsy-OVereyu:TEhitbhid witf anploit§dpptNsMlmtfyi (fithttuhhwas ivfe. iwgniM dOME^m^1 sfr thy SMI elcffidntlsh te augffifynghg intfhsity dfrgsífld IssssTcwiafeaSl^fd UCWSSY WetanSe1 %Of ]NttihiSCiii!!sl^R^lhe4 ffe§hWS:tECfhloflilln MeBlnnEfillc,nrS28•S6!?%l■I:in2-^Wy ríddS|i77ll:W^sehk:iEStlrscndg|!yswslE!líEW:^t^ ^cCl%ií

SC!idslE!^^CSTndCi%Ee6S)fí]]hTSsWWSl^ íCrrii^sSEEfEE?i iminary screening experiments indicated that five strains (all of which were diatoms) had the best growth rates and lipid accumulation potential: Navicula acceptata (two strains, NAVIC6 and NAVIC8), N. saprophila (NAVIC7), Nitzschia dissipata (NITZS13), and Amphiprora hyalina (ENTOM3). These strains and CYCLO1 were grown semi-continuously in media with six different salinities at 25°, 30°, and 35° C. Cells were grown at light intensities

-2 -1 -2 -1 of 80 mE- m • s and 160 mE • m • s (approximately 4% and 8% of full sunlight, respectively). The media were produced by adding various quantities of artificial sea

ESNTSCIOI^¿weSum- Ct^to 2f!UdOn6 lC^CtVayS cwetf in1mesia2wit3 5o4:dU(íinVitfTe§

m-mhl-60i nn1mhCdtEc me1awGMWlhlywilSllby t35- 45)fflnU?Oa smhitra^ l :ssrainfs NxhOUfyd nnOotef rraip^ gtiow tinumdlnruIm dME iipicd cohtiBU mfra•ttdTfTteBU fftclih .mEe llsnwass2,2a1nd lCfYítiac^íOltlillTIi:IC(w::^!!i.rEa tl|igfg®^^1:lEgr^f|dííílovli:^!i1fnn . gatntla in3f^2l%dtiírni:nE|irTíEi-d^fSitfT3ti °mCt

N-de ficiehttCondilion^, respectivyly.

bytWe-¿T)W ahm 35 Snmho •cm . Cells grew best with nitrate as a N source, followed by ammonium and then urea. The highest lipid content was observed in N-deficient cells (42.1%), but was also elevated in Si-deficient cells (38.6%) relative to nutrient-sufficient cells (13.2%).

NAVIC8 grew most rapidly (3.8 doublings • d ) at 35° C and 45 mmho- cm . Nitrate and ammonium were more suitable N sources than urea. Lipid contents of 21.8%, 48.5%, and 32.4% were observed for cells grown under nutrient-sufficient,

Duf W fifiSi iy-iiarfipiie£Hii? piubicpfflSaetii ^ccmigna l promising strains were isolated. Included in this group was Navicula BB-324 (NAVIC9), which had a growth rate exceeding 2.5 doublings- d at 30° C in artificial seawater and SERI Types I/10, I/25, I/40, II/10, II/25, II/40, and II/55 media. Navicula SB-304 (NAVIC8) also exhibited excellent growth (1.5-3.0 doublings • d ) in each medium. These two strains had Si starvation-induced lipid contents of 42.5% and 47.2%, respectively. Other notable strains were Nitzschia SB-307 (NITZS13), which had a maximal growth rate cpfncliiS iQi^uHianysprQ^it^ it tripiCS Wififen?P^:ale4l5% -4 7SS uMS rhniitufe2P2lisefeed conditTOriSruAmi^riipiOtUS fR® 3el(ENTOMw)?s Cl1>alSlaCanpHPPilS:2° rpCeAET6l6), and

.accumulation. . .Furthermore, the nature of the N spurce included in .the medium,had , Cyrindrotneca AB-204 also grew rapidly (2.3-BjD doublings- a ), with stress-induced

Publip aCuOrnSnnal M OrCgnOWlh)% 37t1cu.ltures. Several or these strains were further tested in outdoor mass culture, as described in Section III.

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986)

"Microalgae Culture Collection 1986-1987." Solar Energy Research Institute, Golden,

Colorado, SERI/SP232-3079, 147 pp.

Tadros, M.G. (1985) "Screening and characterizing oleaginous microalgal species from the southeastern United States." Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators' Meeting, Solar Energy Research Institute,

Cpaipni Mm8S^a^i/'^Pr2e3eni2ng7 0Sra ^haratcterizing oleaginous microalgal species from the southeastern United States." FY 1986 Aquatic Species Program Annual

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 67C<gOS , M.G. (1987b) "Conclusion of the warm-water algae collection and screening efforts conducted in the southeastern United States." FY 1987 Aquatic Species

Program Annual Report, Solar Energy Research Institute, SERI/SP-231-3206, pp. 58174, M.G.; Johansen, J.R. (1988) "Physiological characterization of six lipid-producing diatoms from the southeastern United States." J. Phycol. 24:445-452.

II.A.2.e. Collection of High Energy Strains of Saline Microalgae from Southwestern

V.A. SERI/NREL/DOE REPORTS AND PUBLICATIONS 263

V.B. ADDITIONAL REFERENCES 293

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