Also absenecainiTipfpfeoniimatesubistialsy ecimorere dimited to Abe w existenceonsT ngr

promotes the accumulation of lipids in certain species. Green algae are the evolutionary progenitors of higher plants, and, as such, have received more attention than other groups of algae. A member of this group, Chlamydomonas reinhardtii (and closely related species) has been studied very extensively, in part because of its ability to control sexual reproducaiOhh-^raU$n Ajpwi This (geOlipl o f ai;gh§,ticamlna^yi:í•efe rreIh(4ae|(s

Cfteys®yjhym®hass wwams ilathe tofirdtatote wo tiberegphetictaiypigmehafmlhll Hiowchevmiicifl dallSpnaiti§hculAUil|iEaxliiftiliill^arl(;00isu§ p«iienirecahsWrilhfoh use found pASfiar ilAniOltii?eshomitíia niaKnus . thitpi1lis ah§en hsysdiimifaifih

11x$eh§ií;Silyiri(C ^b^the major carbon storage form in this group. Some chysopfffyffiftlSiapihhtgy• siThfsedgeu walisf. algae, also known as the haptophytes, consists of approximately 500 species. They are primarily marine organisms, and can account for a substantial proportion of the primary productivity of tropical oceans. As with the diatoms and chrysophytes, fucoxanthin imparts a brown color to the cells, and lipids is common in the world's oceans. Chlorophyll a is the only chlorophyll present in the cells, although several xanthophylls serve as accessory photosynthetic pigments.

• Cyanobacteria. This group is prokaryotic, and therefore very different from all other groups of microalgae. They contain no nucleus, no chloroplasts, and have a different gene structure. There are approximately 2,000 species of cyanobacteria, which occur in many habitats. Although this group is distinguished by having members that can assimilate atmospheric N (thus eliminating the need to provide fixed N to the cells), no member of this class produces significant quantities of storage lipid; therefore, this group was not deemed useful to the ASP.

Collection and Screening of Microalgae: Programmatic Rationale

The in-house collection effort was focused on collecting strains from inland saline habitats, particularly in Colorado, New Mexico, and Utah. The reasoning behind collecting strains from these habitats was that the strains would be adapted to at least some of the environmental conditions in mass culture facilities in the southwestern

United States (i.e., high light intensity and high temperatures). They would also be well suited for growth in the saline waters available for use in such facilities. In addition, many of the aquatic habitats in this region are shallow, and therefore subject to large variations in temperature and salinity; thus, the strains collected in this region might be expected to better withstand the fluctuations that would occur in a

Thgnstateialoippriciivijoia f pgntSERICciinibacol^iectighryiiaphcieesniran'e frttoms often we®in§te inland saline habitats. The latter were of particular interest to the program

? Assemble and maintain a set of viable mono-specific algal cultures because of their propensity to accumulate lipids. There had never been a large-scale stored under conditions best suited to the maintenance of their original effort to collect strains with this combination of characteristics; therefore, they were physiological and, biochemical characteristics. not available from culture collections.

? Develop storage techniques that will help maintain the genetic variability and physiological adaptability of the species.

? Collect single species cultures of microalgae from the arid regions of

Colorado, Utah, and New Mexico for product and performance screening.

? Develop media which are suitable for their growth.

? Evaluate each species for its temperature and salinity tolerances, and quantify growth rates and proximate chemical composition for each species over the range of tolerated conditions.

2 Taken from the Proceedings of the April 1984 Aquatic Species Program Principal Investigators' Meeting.

Each objective was met during the course of research within the ASP. The following pages describe in detail the major findings of the work conducted by SERI tleAefebc hkrtte ction and Screening Activities - 1983

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 cBltelonoraC8ryiiies^1rfar?ger^drooTiso19teTimhd§mi5fiinta(di§ooii¥i Cany^ Sp^ed water samples. Cyanobacteria and other contaminants were removed primarily with 85.0 mmhos- cm (nearly, three _times ,the conductivity .of seawater) , at Piceance

AgftnainlihtengAiA®)iproX,iiogE§ii6 is1 C^amusiiy^iscuiiitCmbiseiiain^ nwm© nesolatybsigmhe

Spring. . Water temperature at the time ofcollection ranged from 11° to 46 °C. Moral Navpiltr Nfoun!iwe?fe urosigma, 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. 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. II.A.1.c. Collection and Screening Activities - 1984

The screening and characterization protocols used by SERI researchers were refined for the 1984 collecting season. Included in these refinements was the development of a modified "rotary screening apparatus", a standard type of motorized culture mixing wheel for 16x150-mm culture tubes. The rotating wheel was constructed of Plexiglas to allow better light exposure (see Figure II.A.1). The wheel was typically illuminated with a high-intensity tungsten stage lamp, and could be placed inside a box behind a CuSO4-water heat filter for temperature control.

The Plexiglas wheel allowed all the cultures to receive equal illumination. Another technological advance used a temperature-salinity gradient table to characterize the thermal and salinity preferences and tolerances of the isolates. Development of artificial saline media.

One of the most significant contributions made by SERI researchers during 1984 was the development of media that mimicked the saline water in shallow aquifers in the southwestern United States. This was an important undertaking because it allowed algal strains to be screened for growth in the types of water that would likely be available in an outdoor mass culture facility. To identify the major water types available in the southwestern United States, state and federal reports that described the chemical characteristics of water from 85 saline wells in New Mexico were studied. The data were statistically analyzed to identify the relationships between the various ionic constituents. (Data from wells deeper than 83 m was not used in this analysis, because the cost of pumping water from those depths was prohibitive.) R-mode factor analysis indicated that two factors were largely responsible for the differences between the waters examined (Barclay et al. 1988). The first factor, monovalent ion concentration, was responsible for 40% of the variance; the second factor, divalent ion concentration, for 30%. A plot of these factors against each other Tea tlu rdey nndtedtewohptibMhytwate oftypster rgforbitd' do aangTypfe cBhdndtivTtype the ffyeaiIciwrtg rsell03tedch:aa:1aCl1leriZ^(gllletyc^ ridwctivíh,owile^tt5■t:oB■^IVBEldrft(joh evaporatier'age vaeulower0c4)h ductheerre awaTrpe En wMeuisnhahdeyi rhohhr zEEelVEEhaOfthm oonvl^eiiit ityons (mohovateih- tod ivufeddooropiroduotiofi 9p4hd wou lchírr(Cteioa^s witfesenii ei nbeicaiMs eI Cate®

togfe ratees Clf' eMapo ranio iC an .the s omaofeiolh^roUnypd Htstesetaw erg hNas' C lcnSOdayn)d HBeOtefoilrep ea rIIi ficct^i rmise Gtianshquecdve rEEetmedvBde' srahgei Mcaohdua1levClls5s'ha)d wafers AebpErloíble:::a tee ly tiilEeeFf:O,utíhsx:bBlfim^nliKlewelOn,d:uC;lrsdvih rwioicthmType tHavatiattaiahd dme S:oUtftiy]^ol2llldyt^ereh0I:íailtE rizw-fc asductevi-ty Type I and Type II waters were allowed to evaporate with stirring at 35 °C. Samples were removed at various times and filtered. The ions still dissolved in the waters were quantified using an inductively coupled plasma spectrometer and a high-performance liquid chromatograph. In this manner, media formulations were derived at SERI that covered a range of conductivities (from 10 to 70 mmho • cm ) for both media types. The media most commonly used were designated SERI Type I/10, Type I/25, Type I/55, Type I/70, Type II/10, Type II/25, Type II/55, and Type II/70, in which the number following the slash indicates the specific conductivity of the medium. The compositions of these media are given in Figure II.A.2.

In order to assess whether these media formulations accurately reflected the types of water in desert region surface waters, samples of the water at numerous algal collection sites in the southwestern United States were chemically analyzed. The relative compositions of the anionic and cationic constituents were then plotted on separate trilinear plots, which allowed a graphical representation of the various water samples relative to SERI Type I and Type II media (Figure II.A.3). This analysis

indicated that Type I water has higher proportions of Mg and Ca than most surface waters examined, whereas Type II water was fairly representative of the sampled waters with respect to these cations. On the other hand, natural surface waters often had an anion composition similar to both SERI Type I and Type II media. C8|gecltg0naietivitiesc oncluded that these artificial media would serve well as coMlmdlgt edi pmmfee br serI ¥gaillifwhi ra cquiG®4 aoeun§d 0?gsel^ ow owing hiJib itas rfiaiuaelgi Igplhgothtain{:honds pBnyaopsflli andlnphcpffiid&tesreg0ona(roigc osorms aHor urowth ATe?nS0lleeti0¥l| tes§ wati? raKa temenia tsMulgshWeav|kigpt eundfec omonelaairk

1 to 3 days until they could be further treated in the laboratory. The pH, temperature, conductivity, redox potential, and alkalinity of the collection site waters were determined, and water samples were taken for subsequent ion analysis. In the laboratory, the samples were enriched with 300 p M urea, 30 p M PO4, 36 p M Na2SiO3, 3 p M NaFeEDTA, trace metals (5 mL/L PII stock, see Figure II.A.2), and vitamins. The enrichment tubes were then placed in the rotary screening apparatus

Tha iistaialgd atr2inSCw(e^e3tlll<eCl) tend dd fnntthiete dbaitl ity40£gpEw in incuhatOvat 2 5-Cayt

?sr0o200hp ^llufflinatsc3ni p rohei dStdndíl th enelgg types w§selrhgd( above wd ^rtifM

pEawater stermThe ":R§aloS[llllna n&$W"nupilgs eRil an Ifchsritub eMwe ransolatfidt as ueaasrit miXUuea ]hryoal|ilcg (p lby nRgt o rPhydnetsl ailaliioekl lNiUq'Uiafe istrdins that grew well in at least one of these media were further characterized with respect to growth on a

temperature-salinity gradient table at a light intensity of 200 pE- m • s . Thirty

combinations of temperature (10° to 35°C) and salinity (10 to 70 mmho- cm ) were included in this analysis, representing the ranges that might be expected in actual outdoor production systems. Once again, the cultures were enriched with nutrients to maximize growth rates. The cultures used to inoculate the test cultures were preconditioned by growth in the media at 17° and 27° C. The optical density at 750 nm (OD750) of the cultures was measured twice daily for 5 days, and the growth rates were calculated from the increase in culture density during the exponential phase of growth. A refinement of this method was to measure the growth rates in semicontinuous cultures, wherein the cultures were periodically diluted by half with fresh medium; this method provided more reproducible results than the batch mode experiments.

Figure II.A.3 gives an example of the type of growth data generated by the use of temperature-salinity gradient tables. The contour lines in the plot are interpolations indicating where a particular combination of temperature and salinity would result in a given growth rate. Many such plots were generated for various strains, and are AFip'0Ximal§iy^3tU^esCPiln§t\0nrga¿ol^fa31(írAA§p Mpts .to Utah and Colorado.

Of these, only 15 grew well at temperatures >30°C and conductivities greater than 5

-i mmho • cm . Nine were diatoms, including the genera Amphora, Cymbella, Amphipleura, Chaetoceros, Nitzschia, Hantzschia, and Diploneis. Several chlorophytes

(Chlorella, Scenedesmus, Ankistrodesmus, and Chlorococcum) were also identified as

Two strains isolated as a result of the 1984 collecting effort (Ankistrodesmus sp. and romising strains, along with one chrysopnyte (BoeKerovia).

oekelovia sp.) were characterized in greater detail using the temperature-salinity matrix described earlier. Boekelovia exhibited a wide range of temperature and

salinity tolerance, and grew faster than one doubling- day from 10 to 70 mmho-

cm conductivity and from 10° to 32°C, exhibiting maximal growth of 3.5 doublings

• day in Type II/25 medium. Reasonable growth rates were also achieved in SERI Boekelovia and Ankistrodesmus were also examined with regard to their lipid

Tccumula9dn A^oieTitiial . swo -lmedculfesemnye giown3 name diet^thdao uCo]^i^t^ílSleddhig^, respectively). .Ankistrodesmus was also able to grow well in a wide range ,of salinit2es (600 pM) and low (300 pM) urea concentrations at a light intensity of 200 pE- m •

and temperatures, with maximal growth rates occurring in Type 1I/25 medium (3.0 s . Half of each culture was harvested 2 days after the low-N culture entered stationary piiaye )to determine the lipid content of N-sufficient cells and cells that were just entering N-deficient growth. After 10 days of N-limited growth, the remainder of the low-N culture was harvested. Lipids were extracted via a modification of the method of Bligh and Dyer (1959) and lipid mass was determined gravimetrically.

Theo]IrpiusiP»ltentea)fcB oekERiian \1ss842 7%d of the, cosrveafiicmienitsso ff nrtificufficiiritaceigi, mCleakeT|i toe42% iaftdg50)Uírd\\tlle2 days and fou rccfeySf ofhee -dtesieiencegionp eofivthlye. ThethwsselessJnfieclt 0le£s>tTrVllta0pwndthee istf(aiCTSTf^at(§SAdu^ís^icCpeSlctfS;gf]trfiISFi(C various, ionly c0iccefiSgdtiPf0iI0 ^be^systemasui'cfiyie nc reens dt©n2 9%o^ece ls$aThardW9ce I|ieC4ificieMtfoCOl.0ddays. used in different laboratories performing ASP-sponsored research. Numerous strains were characterized with respect to growth at several temperatures and salinities using these new media.

Publication..

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.

Barclay, B.; Nagle, N.; Terry, K. (1987) "Screening microalgae for biomass production potential: Protocol modification and evaluation." FY 1986 Aquatic Species Program

Annual Report, Solar Energy Research Institute, Golden, Colorado, BgRc/y-231-NOe.,• pp 2T?-i0i', K.; Roessler, P. (1985) "Collecting and screening microalgae from shallow, inland saline habitats." Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators' Meeting, Solar Energy

"Characterization of saline groundwater resource quality for aquatic biomass production: A statistically-based approach." Wat. Res. 22:373-379.

Sommerfeld, M.R.; Ellingson, S.B. (1987) "Collection of high energy yielding strains of saline microalgae from southwestern states." FY 1986 Aquatic Species Program

Solar Energy Research Institute, Golden, Colorado,

Bligh, E.G.; Dyer, D.J. (1959) "A rapid method for total lipid extraction and purification." Can. J. Biochem. Physiol. 37:911-917.

Siver, P. (1983) "A new thermal gradient device for culturing algae." British J. Phycol.

'layh^:lRitute^gioeldei1J.^oloe]raryo, iSEI^I/Ëlîin2g3s1on2 7§.0B,pîSo5m2^l8rfeld, M.R. (1988)

Figure II.A. 1. Rotary screening apparatus used for microalgal screening.

SERI Type I Artificial Inland Saline Water

Salt

CaCl2

MgCl2-6H20

NsjSO^

KCl

NaHCOj

Conductivity (mmho

cm"1)

10

25

40

55

70

0

3,932

5,612

7,610

M 30

»,114

il, m

22,789

35,305

«,230

0

2,925

3,310

3,705

3,620

m

407

662

960

1,126

m

162

168

162

16$

2,1 u

3,m

9,132

13,023

16,039

1,686

0

0

0

0

SERITypeD Artificial Inland Saline Water

Conductivity immho cm"1)

Salt

10

25

40

35

70

2S

22

28

22

22

MgClj-iH^

1,953

3,026

3,920

4,362

4,230

2,671

5470

15,720

23,305

22,360

466

965

2,022

3,0tU

3,673

1,202

2,315

2,235

3,234

3,245

231

276

1,234

1,492

1,527

1,511

8,072

12,963

20,522

26,075

Suggested enrichments (inL/L)

are:

PD Trace Metals

♦Nitrogen source indicated tor individual species, ammonium as NH^Cl, nitrate as KNOj.

250-300 mg L~l NajSÎOySf^O should be added when cultivating diatoms in this medium.

FeClj.SHjO 0.29 t

MnCij-WljO 046 %

Adjust trace element stock solution to pH 7.8-ä.O with N*OH.

Figure II.A.2. Formulations for SERI Type I and Type II artificial inland saline waters.

Recipes for the preparation of Type I and Type II media at five different salinities, expressed as conductivity of the final solution. Formulas for these media were developed by statistical analysis of saline groundwater data for the state of New Mexico. For each salt, necessary additions in mg/L are listed. (Source: Barclay et al. 1986).

ao oh»

Relative anion composition for waters sampled for microalgae. Each dot represents a sampling site. Asterisks indicate relative anion composition of SE&I Type I and II Media.

sa »to

Relative cation composition for waters sampled for microalgae. Each dot represents a sampling site. Asterisks indicate relative cation composition of SEKI Type I and II Media.

Figure II.A.3. Trilinear plots showing the ionic constitutents of various water samples relative to SERI Type I and SERI Type II artificial saline media. (Source: Sommerfeld and Ellingson 1987.)

Figure II.A.4. Growth contour plots. Examples of growth contour plots generated from data obtained by the use of a temperature-gradient table. The contour lines represent interpolated values indicating where a particular combination of temperature and salinity would result in a given growth rate. The data shown, given as doublings- day) represent the exponential growth of Monoraphidium sp. (S/MONOR-2) in semicontinuous culture. Each point represents the mean of at

^'afyppteItniP!mdcSlanneWiatirth rate determinations. (Source: Barclay et al. 1987). C: Seawater

II.A.1 .d. Collection and Screening Activities - 1985

In 1985, the strain enrichment procedure utilizing the rotary screening apparatus described previously was modified to include incubation of samples in SERI Type I

and Type II media (25 and 55 mmho- cm conductivity) and in artificial seawater, in addition to the original site water. The cultures that exhibited substantial algal growth were further treated to isolate the predominant strains as unialgal (clonal) isolates. Coifec tionansivwee then tested for growth using the temperature-salinity matrix

Cmten efforts by SERI researchers in 1985 again focused on shallow inland saline habitats. This time collecting trips were also made to New Mexico and Nebraska, in addition to Colorado and Utah. Eighty-six sites were sampled during the year, 53 of which were sampled in the spring. From these 53 sites, 17 promising strains were isolated. An analysis was conducted comparing the results of the new protocol with those that would have resulted from the protocol used in prior years. This analysis indicated that the revised protocol was in fact superior to the older protocol. For example, only six of the 17 strains selected via the new protocol would also have been selected using the old protocol. Only three of the 17 strains grew best in the artificial medium type that most closely resembled the collection site water; in fact, only six strains were even considered to grow well in the collection site water relative to growth in at least one of the artificial medium. This analysis clearly indicated the value of performing the initial screening and enrichment in a variety of relevant Growth Thtes-esults suggest that the shallow saline environments sampled probably

SOR0MS1RP tRumbwerf an^g^dw^ii(SgRmfyfbPl!sfyiie rand &$wawenng me tempfeuwoialsiity1 eg»n t qusteed puehi osisy. vafe e grnsife ed TOdffi&oms CfeepMesi muewoCMorff) pnMU^RMICW oC;yfee^íeilny(CPecies2P,re§mjR)1h;síía aAMPtogr§fflrM#HeRy , inv(flíheim!íi•oro^PhlfteaiMfons^p^elap¿aPntiReItffilde^f(9NOR2í.

sNAVtCf ianti ^mffluer- eoac¥iíelíybyo!mscte <o fthm ethe alori da keys; the remaining strains were collected in Colorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in at least two media types. Furthermore, all strains exhibited temperature optima of 30 °C or higher. Maximal growth rates of these strains, along with the optimal temperature, conductivity, and media type determined in these experiments are shown in Table II.A.1. (Higher growth rates were determined for some of these strains in subsequent experiments; see results presented in Barclay et al. [1987]). Temperature-salinity growth contours are provided for these strains in the 1986 ASP Annual Report (Barclay et al. 1986).

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