Hydrogenase was funded by the Hydrogen Program at DOE not the ASP and will not IIBK Lipid Accumulation Induced by Nitrogen Limitation

be included in this report. However, studies on hydrogen production by microalgae

As a result of .the i aJgaLTicregninT efforts by SERI .subcontractors and jn-hoise are ongoing at NREL (SERI) in the Center fOrBasic Sciences, and lntgrestSa readirs researchers, several algal species were identified as good candidates for biodiesel shouia contact Dr. MicHSel Silbert for more information.

production during the early 1980s. This was facilitated by the development of a cytochemical staining technique for intracellular lipids that allowed researchers to visualize storage lipid droplets in algal cells (see Section II.A.l.f. and Lien 1981a).

Two of the most promising candidates were the green alga N. oleoabundans, which First, a sensitive method to monitor nitrate levels in liquid cultures using ion showed a high lipid content and rapid growth, and a Chlorella strain (CHLS01) chromatography was developed to study the effects of N limitation on lipid isolated from a local site.

accumulation in these organisms. Algal growth, lipid content, and chlorophyll a content were measured in batch cultures of N. oleoabundans and CHLS01. Cell division and chlorophyll accumulation occurred rapidly in the cultures as long as N was present. When N was depleted, cell division stopped, although biomass accumulation continued for several days. The major portion of the new biomass was composed of lipids and storage oils. N depletion resulted in a rapid decrease in the level of chlorophyll a in the cultures, suggesting that the cells might metabolize chlorophyll during periods of nitrogen stress. There was also an increase in the ratio of carotenoid to chlorophyll and a significant decrease in the complexity of the intracellular membranes in N-starved cells. These last three observations indicated that the photophysiology of the cells was affected, suggesting that the lipid trigger could also directly or indirectly alter photosynthetic efficiency in the treated cells (discussed in more detail below).

II.B.2.c. Studies on Photosynthetic Efficiency in Oleaginous Algae

SERI researchers Lien and Roessler (1986) tried a somewhat different approach to understand the processes affecting lipid accumulation (Lien and Roessler 1986). A

recently published technical evaluation (Hill et al. 1984) identified two major requirements for economic feasibility, of bi°diese1 production:

1 Photosynthetic efficiency (which can simply be thought of as the percentage of incident radiation that is converted into biomass) needs to be

(nutrient deprivation) that result in decreased photosynthetic efficiency and decreased growth, the two conditions of high lipid and high productivity seemed to be mutually exclusive. To overcome this technical hurdle, Lien and Roessler initiated a

Thugs totrairs urdeoaidririCug rffars wSri trograi dig pdvetioni aid Cipidr race uCHLstioii

ArlfihCfC0ar!SitinSliepr,ff^drtylew1y isolated chrysophyte strain Chryso/F-1. The cells were grown in batch culture and monitored for nitrate concentration, light levels in the culture, chlorophyll concentration, and yield of cell mass and lipid (including total, neutral, and polar lipids). Maximum energy efficiency occurred as the culture approached N depletion. At this point, the culture showed a maximum density of photosynthetic pigments (before chlorophyll degradation and after N depletion), but the light energy reaching the cells was decreased due to the higher culture density. Thus, photosynthetic efficiency (biomass produced per light energy input) was maximized and the individual cells suffered less photooxidative damage due to lower light exposure. After the N in the culture was depleted, cell mass continued to increase for a time, eventually leveling off. All cultures experienced a two- to essential to maximize1 lipid production in a mass culture facility. If N starvation is used to trigger lipid accumulation, the data suggest that maximal photosynthetic

D 2 Algal biomass, needs to .consist of 60% lipid. , . ,...

Because very high lipid production is usually correlated with stress conditions

efficiency with respect to lipid production (and probably the best time for harvesting lipid-producing cells), occurs just after the N is depleted from the cultures.

Another set of experiments directed at optimizating photosynthetic efficiency in algal ponds was performed by SERI researcher Dr. Ken Terry. Previous studies had indicated that algal cells grown under high-intensity flashing light can use that light energy more efficiently than cells grown under the same intensity under constant illumination. The evidence suggests that an algal cell can integrate absorbed light energy such that the photosynthetic efficiency achieved under intermittent light conditions is similar to that attained under constant light of the same average intensity. This flashing light, or photomodulation, effect can be mimicked in vertically-mixed algal ponds, as cells circulate to the surface and back down to the In order to better understand the effects of intermittent light on photosynthetic lower levels in the pond where they receive minimal light. Thus, the photosynthetic efficiency of microalgal cultures, Dr. Terry set up a system to measure photosynthetic efficiency of algal cells grown in ponds may be increased in high light by using rates and oxygen evolution in laboratory cultures of Chlorella pyrenoidosa and mixing, strategies that optimize this Dhotomodulftion effect: . t ,

Pnae0aactylUm tricorutum under nasning ngnt conditions. Intermittent light conditions were simulated by placing sectored disks in front of a light source, and using this to illuminate exponentially growing cultures that had been placed in an oxygen electrode chamber. Photosynthesis was then measured under varying light/dark ratios (generated by changing the configuration of the disk) and light intensities. The data generated were used to calculate the percent "integration" of the incident light by the algal cultures. More rapid flashing led to greater integration, although lower flash frequencies produced higher levels of integration as the

Aitheugige^ Ti®y ^fopO-Sl cs PíIonfcA- upe stuM eusfng ef^odA1fbecu^ig hbe?egdffl? AAlIfrn

Hf(friimiInl?ryfy ibLeml((utli^e?ii{i5^ etnifrffilgDf(p?fl1 rrcnatietie ifllli3{RiIrfiifSiflíif^ionf]UIíncffSíffinaíIrg1 pnec0syre,uIccticbn ffirrfef^ eih tfgf piiuiit^eics vfaS^permrM stra;Seijiis' InsHdAevne. ercpfsid (PRliiíí|SffhLiheUiff elífififhfb^ jRifteic ttto cffwsimI?ílrCg bthii hffdCRffIli?is?|Sl nce

II.B.2.d.iLipid Accumulation in Silicon-Deficient Diatoms r ^ r , ^

nfsniguitfii'biesien^ efip idiccumuraiioep time awfy from the surface' and the energy

AdSOetadfchi itovia (haH0fiixinigp8Ouic3nbiapfReh^iifilvien and Roessler 1986) described preliminary data on the use of Si deficiency to trigger lipid accumulation in diatoms. Silicon is major component of diatom cell walls. Similar to the lipid trigger effect produced by N-deficiency, Si depletion also results in a decrease in cell growth and often is accompanied by an accumulation of lipid within the cells. However, Si (unlike N) is not a component of other cellular macromolecules (enzymes, membranes) or cell structures such as the photosynthetic apparatus. Therefore, any changes in cellular biochemistry and lipid accumulation induced by Si deficiency might be more easily interpreted than changes induced by N starvation. This work initiated a series of experiments by Paul Roessler during the late 1980s and early 1990s on the biochemistry and molecular biology of lipid accumulation in Si-deficient diatoms.

The first set of experiments compared the effects of Si deficiency on lipid accumulation and cell physiology in several species of diatoms, including C. cryptica

T13L, Thalassiosira pseudonana, and Cylindrotheca fusiformis. Exponentially growing cultures were transferred to media that contained either excess Si or limited levels of

Si so that the media became Si deficient while the cells were still growing exponentially. Cell growth, chlorophyll a content, AFDW, lipid, and photosynthetic capacity were monitored under both conditions. In all three species, cell division decreased as soon as the Si was depleted in the media. However the species responded differently with respect to other physiological parameters. In C. cryptica, chlorophyll a synthesis was almost completely inhibited after 12 hrs in Si-depleted media; C. fusiformis showed little change in chlorophyll a synthesis after 72 hrs. T.

pseudonana exhibited an intermediate effect, with some decrease in chlorophyll a

Synthesis noted after 36 hours without Si. The effect on photosynthetic capacity, Tie _thre? _spicies iwir! .also_snalxzidfor .sccumu lstlon _ofi total Tbic fepmecat what0isyiraheirícc5ltaacstyviihcoeawethc330/sufo^ieot0/sl,r§a:l\ací|^llyp idtey^ tpihigp ^nnnagefd Ait3WynW S-dn- fptooscultheeic caf/abteo Wos Si-dfe]P[lfetéo8ells)^0nwTvp5eàd)ta;s^o,tlsyriChêaiaaaiflyceiifcna^aednio lSpidfwas^ifgctad wntli 3w Mit! e fter Soup lem . couth^ pctte- bwaàmeflii ppped unnât?I^adauía noates ilecreaaeil ,rhSwaVerll|esna who roped mta^ vtífTSllaeFeantaoge c0¥rí0Talnnopedui[r^stin

Che[SaeWIlhadPdwdeMt ^^W^e end of the 72 hours experimental period. The situation with C. cryptica was very different. Twelve hours following Si depletion, there was a 380 decrease in the growth rate of these cells compared to the Si-replete culturel r Howevermipid siy n^h edèspTSlra11iil0lreca fafe chd same dalmpaahiaO i-de fHientipelS

praou1Csi,S|[email protected] plptdslW^arerexrarlgldnaodigoiidzlol for reaes epëTCtllrítíaigpí doc (prtlartveT^ nèîutraveii peis. iJTleêeOltilIglyala ftefirtfeea§pioílesl cttangSisdete iSind edaíefflledu[Sliawesf C dTg®tída^nthaw$ealaetlin gtain igvto toll AfQtWilori pips prtrtniritye^AíSsailrílTge7a2îipîll,r:|hsf peTcexPlaTimao|le utral lipids in Si-deficient cultures of C. cryptica was 64%, compared to 32% in Si-replete cultures. In C. fusiformis, the percentage of neutral lipids increased from 17%-20% to 57% in Si-deprived cultures.

Based on these studies, C. cryptica was identified as the best candidate for further studies on the biochemistry of lipid accumulation. To determine the effects of Si deficiency on the synthesis of the cell components, the levels of protein, carbohydrate, and lipid were examined at various times after Si was depleted in the cultures. During the first 12 hours, protein and carbohydrate synthesis decreased. Lipid accumulation continued at a rate similar to that of the Si-replete cultures. This resulted in an increase in lipid content of the Si deficient cells from 19% to 27%. This observation was confirmed in subsequent studies that followed the incorporation of

14 A

newly assimilated carbon (as H CO3) into the various cell components. Si depletion resulted in a net decrease in the rate of photosynthesis and carbon assimilation, but

Ptdstadhdduskperi rftetctsowg rweIerMeeíddtolifl6tewtiy.tli® SaxMpfe,ncheara[tec0use(C aceucotivatios i® itnocf lipids ipec reas.udaby: 4rS%oiBetltelfl^tlllpida.rsInof:lSe-ses|ePiV6ri(Dn?rttss

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