72 VITTORIA DI MAR D. H. 1988, RUBP limitation of photosynthetic carbon fix: ation during NH, assimilation, Interactions between pho- tosynthesis, respiration, and ammonium assimilation in Nelimited green algae. Plant Physiol. 87:395-40 1 Fini, LR. & Turpin, D. H. 1986. Nitrate and ammonium in- ‘duced photosynthetic suppression in N-limited Selenastrum rminutwm, Plant Physiol. 81:273-9, Genthner, B. R,'S. & Wall, J. D. 1985, Ammonium uptake in Rhodepseudomonas enpsulata, Arch, Microbiol. 141:219-24 Heber, U. 1974, Metabolite exchange between chloroplasts and ‘cytoplasm. Annu. Rev. Plant Physiol. 25:803-421 F. & Kanazawa, T. 1982, ‘Transient changes in the energy state of adenylates and the redox states of pyridine hiucleotides in Chlorella cells induced by environmental changes, Plant Cell Physiol. 28:775-83, chi, S. & Miyachi, S. 1985. Ammonia induces starch deg- radation in Chloreita cells. Plant Cell Physiol. 26:245~52 ‘Ohmori, M. 1981. Effect of ammonia on dark CO, fixation by Anabaena. Plant Cell Physiol. 29:709-16. Okabe, K. & Miyachi, 8. 1984, Effects Adenylate levels, amino acid syn ‘cells of Chlorella vulgaris 11h in Plant Call Physiol. 25:749-56. Rai, A. Nv Rowell, P. & Stewart, W. D. P. Kaw 1984, Evidence for J. Phycol. 26, 72-79 (1990) TRIGLYCERIDE ACCUMULA CHLORELLA (CHLOROPHY ION AND FATTY ACID PROFILE CHANG! ‘A) DURING HIGH pH-INDUCED CELL, INO RIGANO ETT AL, jum transport system in free-living and symbiotic Cyanobacteria, Arch, Microbiol. 137:241-6, Rapp. B.J.. Landrum, D.C. Wall, D, 1986. Methylamm fim uptake by Rhodobacter capsulatus. Arch, Microbiol. 146: 134-41 Syrett, P.J. 1956, ‘The assimiation of ammonia and nitrate by hitrogen-starved cells of Chlorella vulgaris, 1V. The dark fix ation of carbon dioxide. Physiol. Plant. 9:105-71 Syrett, P.., Flynn, K. J. Molloy, C. J., Dixon, G. k., Peplinska, ALM.& Creswell, B.C. 1986. Effeetsof nitrogen deprivation ‘on rates of uptake of nitrogenous compounds by the diatom Phacodartylum tricornutum Bohlin. New Phytol. 102:39-44, hacker, A.& Syrett, P. J. 1972. The assimilation of nitra ‘ammonium by Chlamydomonas reinhardit, New Phytol. 71423. 33, Ullrich, W. R., Larsson, M., Larsson, C. M., Lesch, 8. & Novacky, ‘A. 1984. Ammonium uptake in Lemna gibia Gl, related ‘membrane potential changes, and inhibition of anion uptake. Physiol. Plant. 61:369-76, Weger, H. G, Birch, D. G., Elvi, LR. & Turpin, D-H. 1988. ‘Ammonium assimilation requires mitochondrial respiration inthe light_A study with the green alga Selenasirum minut Plant Physiol. 86:688-92 CYCLE INHIBITION! James B. Guckert® and Keith E. Cooksey? Department of Microbiology, Montana State University, Bozeman, Montana 59717 ABSTRACT Alkaline pH stress resulted in triglyceride (TG) accu- mulation in Chlorella CHLORI and was independent of medium nitrogen or carbon levels. Based on morpho logical observations, alkaline pH inhibited autospore re- lease, thus increasing the time for cell cycle completion. ‘Autospore release has been postulated to coincide with TG. utilization within the microalgal cell division cycle. The alkaline pH stress affected lipid accumulation by inhib~ iting the cell division cycle prior to autospore release and, therefore, prior to TG utilization. Cells inhibited in this manner showed an increase in TG accumulation but a decrease in bath membrane lipid classes (glycolipid and polar lipid), Unlike TG fatty acid profiles, membrane lipid fatty acid profiles were not stable during TG accumula tion. The membrane profiles became similar to the TG, ive, less unsaturated than in the membrane lipids of un- stressed control cells. * Received 20 June 1989. Accepted 6 November 1989. ? Address for reprint requests and present address: Insti for Applied Microbiology, University of Tennessee, 10515 search Drive, Suite 300, Knoxsille, Tennessee 37982-2567, “Present address: U.S. Office of Naval Research, Department Key index. words: alkaline pH; autosporangial com- plexes; cell cycle inhibition; Chlorella; fatty acids; tvi- ‘glyceride acciumulation Under optimal conditions of growth, microorga isms synthesize fatty acids principally for este cation into membrane lipids: phospholipids for pl: ma and many organelle membranes and glycolipids, for chloroplast membranes of the photosynthetic microalgae. Many of these fatty acids are polyun- saturated and derived through aerobic desaturation and chain elongation from the ‘precursor’ fatty acids palmitic (16:0) and oleic (18:19e) acids (Erwin 1973), When microorganisms are stressed by nutrient-lim- iting conditions which still permit carbon incorpo- ration, lipid biosynthesis patterns change. In mi- crocukaryotic algae, triglyceride (TG) lipid droplets enriched in the precursor fatty acids 16:0 and 18:1w9c (Shaw 1966) form in the cytoplasm (Cook- sey et al. 1987). Limiting concentrations of a variety of medium constituents cause lipid accumulation in microalgae. The most common limiting nutrient is, nitrogen (Suen et al. 1987) along with silicate in diatoms (Taguchi et al. 1987). ‘Although lipid accumulation during nutrient lim- itation is well-documented, no direct biochemicalLipips mechanism for this change in metabolism has been determined, Nutrient limitations which result in mi- croalgal TG accumulation generally increase the time Fequired to complete the cell cycle (Olson et al. 1986). tis possible, therefore, that nutrient limitation does, not directly influence “TG accumulation biochem- istry. Instead, the nutrient limitation might indi- rectly affect TG accumulation by inhibiting a spe- cific portion of the microalgal cell cycle, at which time TG synthesis is greater than its utilization (Fish- er and Schwarzenbach 1978). ‘The most common nutrient limitation studied with respect to microalgal ‘TG accumulation is nitrate/ nitrogen (e.g. Fogg 1956, Newman 1966, Richard- son et al. 1969, Opute 1974, Klyachko-Gurvich et al. 1981, Shifrin and Chisholm 1981, Piorreck et al 1984, Ben-Amotz et al. 1985, Suen et al. 1987). rogen is assimilated principally during the initial growth phase of the cell eycle (Tamiya 1963). When the expansion in time of various cell cycle stages was, quantified in response to sub-optimal supplies of ni- trate for two marine phytoplankton species, the first growth phase of the cell (G1) specifically lagged (Ol- son et al. 1986), Contrary to most published studies, our initial studies of microalgal lipid biochemistry indicated that Chlorella CHLORI accumulated TG prior to nitrogen limitation. In addition, the medium we used, for the growth of the algae (Bold’s basal medium from Nichols and Bold 1965) is poorly buffered so that a culture with an initial pH of 6.8 reaches a pH greater than 9.0 within 5 days of photosynthetic growth. The following experiments were designed to uncouple rising pH condition: tion and microalgal cell growth from ‘TG accumu- lation and investigate shifts in microalgal lipid bio- chemistry during TG accumulation. MATERIALS AND METHODS, Solar Energy Research Institute culture on, Golden, Colorado) was grown in axenic cultures on Bold’s basal medium, initial pH 6.7 (Nichols and Bold 1965). This ‘medium was modified as needed with a biological buffer at final concentration of 25 mM. ological buffers used were: Hepes (N-2-hydronyethylpiperarine-N’-2-cthanve-sulfonie acid, pKa 7-5). Chey (2:{N-cyclohexylaminoJethane-sulfonie acid, pk 9.3), and Caps (3-{eyclohexylaminoL-propanesulfonic acid, pKa L04) (Sigma Chemical Company), Sodium bicarbonate additions (final 0 previously-autoclaved Bold’s medium. Experiments were conducted sith 100 mL. cultures in 250 mL. flasks shaken at 100 rpm. The cells were grown at 26° under continuous illumination (200 teem *s-'), Cell eo trations were determined using a hemocytometer: Trigheride aerumulatio experiment. Comparisons of cell growth, culture pH, medium nitrate levels and triglyceride accurmalation made for Chlorella cultures inoculated into the following lasks: unbulfered Bold’s medium, unbuffered Boles nedium + 5: mM sodium bicarbonate, Bold's medium buffered ial pH 7.0-7.4) either with 5 mM sodium biear out, Bold's medium buffered with Ches (pH 9.0 ‘or without, Bold’s (pH 10.0-10.4) either with 5 mM. Sodium bicarbonate or without, and unbuffered Bold’s medium IN HIGH-PH STRESSED CHLORELLA 73 to which 5 mM sodium bicarbonate was added after 7 days of growth, Three separate experiments were conducted each com Siting of four independent replicates for cach of four catare (restients,Thowetrestments repented between experimen were thereforesan indicator of betscenrexperiiment variation Analysis of media components ‘The concentration of nitrate (as nitrate nitrogen. sg mL) was estimated by the eadmiim re tluction metho! using a commercially available reagent test kit (tach, Ine. Loveland, Colorado). pH was measured on media supernatant samples, Lipid anal sing ile Red. ‘The Nite Red extimation of ti glvceride lipid was performed according to methods developed by Cooksey etal. (1987), To monitor triglyceride accumulation Fer time. 5 mL aliquots were removed from the algal ealsre and avayed directly with Nile Red. The fluorescent response was “quantifed by fvorometry 8 min after the scqucutal addition of Nite Red 20 gl. of 250 nga. in acetone) and acetone (180 AL), Nile Red @-iethstamino-54 One) was obtained fom Molecular Probes i br Kodak (Rochester, New Vork). The standard used was 2 8 gm Nile Red-atained monodisperse latex particles (agit of Dr. C. Wang, Pandex Laboravores, Mundelein, Iino). The working standard was made up to a concentration of 388 * 10 beads nL". Five mL of thivstandard was used to lade 10 fluorometer ats setting of 5.0 (net scale expansion = 3.16) The fiers were a 480+ 10:am excation filter and a 380 + 9.8 nm bandpae emission fier. The Nile Red data were ex- pressed in arbitrary units defined sich thatthe working standard Frprescnts 100 ents Microvpy and photomicrographs. Photomicragraphic records of alltreatment flasks were taken a each sampling time thoughout cach 10 day experiment. The algal ces were always viewed at ‘hei undiurbed ell densities at 400% and recorded both with light and epi-tuorescent lamination with the appropriate filters for viewing the Nile Red response duc ta TG droplets (Cooksey etal 1987} Photomicrograph were taken witha Nikon Optiph ‘nicroscope uilizing Ektachrome fli (160 ASA, tungsten) set a ASA 80, Colorslde positives were used to make black and white internegatives Analyial lipid analysis, Algal cells were harvested at the end of the experiments by centrifugation at $000 g for 15 min Alter wet weighn dearreiunions the céllcwere quiefromrn sed Iyophilinss Col dry welts were recovered porto lipid ex tration. Deals of the modied Bligh and Dyer (1989) thlov form/methanol/phosphate buffer lipid extraction procedure are dleserbed in Guckert etal (1988). ‘The recovered tral extract abl ipds were separated into three general lipid clawesly aici acid column chromatography (SAG) utilizing 1 series of mobile phases of increasing polarity chloroform tociue the trigheeride- Containing neutral pid fraction, acetone for ghscolipids and ‘methanol for polar lipids (Guckert tal. 1988). Preparative thine layer chromatography was used to sole the trighcerides from the SAC neutral pad fraction Suen eral 1987). Fay ail meth twers (FAME) were prepared from the ested lipids in the trighecride, ghcolipl and polar pid factions by mild alkaline tnethanolictranssterifcation as reported in Guckert etal (1983) piration,quantifation and identification of the FAME. from cath ip clase by capillary gas chromatography and ma spee- trometry are described in Cooksey etal (1987), calibrate the Turner RESULTS ‘The buffered media maintained the desired pH over the 10 day experiments (Fig. 1). Chlorella grew under all conditions (Fig. 24-C). Hepes maintained pH 6.8-7.1, and cell number doublings averaged 3.5 over 10 days of growth. pH increased to 7.1— 7.4 with 3.8 cell number doublings when 5 mM bi- carbonate was added (Figs. 1C, 2C). Ches main-74 JAMES 8, GUCKERT AND KEITH E, COOKSEY Fic. IA-C. Average pH of four independent flasks for each, treatment over a ten day incubation. A) Ches (0), Ches + bicar- (@). unbuffered Bold’ (4), and unbuffered Bold’sto which Pbonate was added at Day 7 (a). B) Caps (C), Caps + bica donate Ml mate (0, 2separa parate experiments). ication of the variability of the data by showing lard deviation from the data for selected values tained pH 8.8-9.1 with an average of 3.9 cell num- ber doublings and pH 9.0-9.7 with 4.4 cell number doublings when 5 mM bicarbonate was added (Figs. 1A, 2A). Caps maintained pH 10-10.4 and 3.0 cell number doublings over 10 days; 3.4 cell number doublings and pH 9.8-10.6 occurred when 5 mM. bicarbonate was added (Figs. 1B, 2B). Unbuffered Bold’s medium ranged from an initial pH of 6.7 to a final pH of 9.7 after 10 days with the Chlorella cell number doubling 3.2 times (Figs. 1A, 2A). When, Chlorella was inoculated directly into unbuffered Bold’s medium plus 5 mM bicarbonate, pH rose from an initial 7.8-8.3 to pH 11 by the third day of i cubation (Fig. 1B), and the average number of cell number doublings declined to 2.6 during 10 days, (Fig. 2B). The pH remained high in these cultures for the remainder of the experiment. When 5 mM. bicarbonate was added to unbuffered Bold’s medi- um after 7 days of growth (Fig. 1A), there was an initial pH rise (7.9 to 9.2). The following day, the 22 6 0 2 Average cell density (10° cells ml RI of four independent flasks for each 1 tetvday incubation. A-C and symbol designat in Figs | 2A-C ') of Cherelle nate the same as Fic, 3A-C, by Nile Red response (in Average triglyceride accumulation as measured som. ‘on a log scale) of four in- pH had risen to over pH 11 and remained at this, level for the remainder of the experiment. ‘The total cell yield for this treatment was 3.4 doublings over 10 days (Fig. 2A). ‘The accumulation of triglyceride (TG) lipid per volume of Chlorella culture was measured by the Nile Red technique for each treatment during the ex- periment’s time course (Fig. 3). The results of this technique were verified analytically (see Table 1 for Day 10 data). All treatments were begun with Nile Red values =10 units-mL~' (Fig. 3). Cells in the Hepes buffered media (with and without bicarbon- ate) accumulated little TG by the final day of the incubation (9 + 0.4 and 16 + 3 units:mL-', re- spectively) (Fig. 3C). In unbuffered Bold’s medium as well as Ches buffered Bold’s medium (with and without bicarbonate) a Nile Red response twice as great as that for the Hepes treatments was mea- 1, Total lipid per gram dryserightof Day 10 cultures organized 1 group: pH = 10-= Bold’, Hepes. Hepes + bicarbonate, Ches, Chet + bicarbonate; pH 10-11 = Caps, Caps + bicarbonate; pH > 11 = Holds + bicarbonate, Bold’ with bicarbonate added at Day 7. Significant differences from the unsteesed, pH =< 10 group. are shown with ant ® Sigmsieance determined by a Scheffe multiple companion of nan with an ervor of O03. Triglyceride pl < 10 22 37420 pH 10-11 5 68420 pH >t 7 205 +78" Glycolipid <10 26 haa 10-1 8 922 >a u sia lpia pH < 10 26 47.0 8 114 lot 8 679 > " 272 otal lipid pH < 10 2 171 + 38 pH 10-11 5 It 15, pH >t 7 103 + 18¢LIPIDS IN HIGH-6H 75 ESSED CHLORELLA pH units plot matrix displaying interve ing in the medium (ug nitrogen for any particular row or calum sured, je. 23 + 7,25 4 4and 27 + 4 units -mL-', respectively (Fig. 3A). On the other hand, when cells were inoculated directly into Bold’s medium plus 5 mM bicarbonate, the Nile Red response rose to ap- proximately 100 units:mL.~! within 3 days and after 10 days had increased to over 470 units ‘mL (Fig. 8B). When Bold’s medium was buffered with Caps (with and without bicarbonate) the response was an increase to 71 + 7 and 89. 11 unitssmL~! spectively) within two days of algal inoculation. For the remainder of the 10 day experiment, this value remained around 100 units mL! (Fig. 3B). When 5 mM bicarbonate was added to cells which were incubated in unbuffered Bold’s medium and pro- ducing little Nile Red response (17 + 5 units:mL~* at day 7, Fig. 3A), the cells responded with an in- crease to 69 19 units:mL-! within 2 days of bi- Garbonate addition, and 108 + 26 units:mL "after 3 days, A seatterplot matrix (Fig. 4) displays the inter- Telationships of media and cell variables in a concise manner. Although nitrate was utilized during algal cell growth (Fig. 4€), only 50% of the initial nitrate concentration in Bold’s basal medium was used over the 10 day experiments, leaving approximately 20 ugemL. ' nitrate nitrogen available for the cultures. The nitrate/Nile Red scatterplot (Fig. 4B) indicates cultures clustered into two groups based on nitrate Utilization: the cultures in one cluster did not ac- cumulate TG (as measured by Nile Red response) above 100 units:mL.! whereas the other cultures lowing cross-comparison NILE RED CELL DENSITY Tt b8, ] i - ie Bho see a |]! Bie F a . z Z x105 cells ml~1 € dnits mint progressively increased their Nile Red response to approximately 500 units:mL~? as nitrate was uti- lized. Algal cell production varied in response to the medium pH (Fig. 4D). The cell density scatterplot (Fig. 4D) indicates a general decline in algal cell density above pH 10 with a possible maximum at pH 9 and similar densities at pH 7. The Nile Red/ pH scatterplot (Fig. 4F) indicates that no cultures incubated below pH 9.5 accumulated TG lipid over the 10 day experiment. Generally, all cultures began at Day 0 with equivalent lipid contents as measured by the Nile Red technique (Fig. 3). The cultures grown at higher pH accumulated more TG, as in- dicated by increased Nile Red responses (Figs. 3, 4F). The cell density /Nile Red scatterplot (Fig. 4E) has three distinet clusters of points, The cultures within the first cluster had little TG accumulation (Nile Red <50 units:mL-') with cell growth to 5 x 10® cellssmL. *. ‘The second cluster was character- ized by a Nile Red response of approximately 100 units-mL-' with cell densities of 4 x 10° cells mL. !. ‘The last group of cultures accumulated TG (Nile Red >100 units:mL-), but its growth rate lagged with cell densities of 2 x 10° cells:mL-! as Cells grown in Hepes (pH 6.8-7.1) or Ches (pH 8.8-9.1) were predominantly individual, small cells (Fig. 5A, B). Cells grown in Caps (pH '10.0-10.4) showed an increased number of autosporangial com- plexes (Fig. 5C) in which individual autospores had76 JAMES B. GUCKERTT AND KEITH FE, COOKSEY. Fie. 5A-D. Chlorella CHLOR cell ) Hepes, final pH 7.1: B) Che, fi = 10 am: pH 9.1; ©) Caps, final ph not yet separated following nuclear division. Cells, incubated in unbuffered Bold’s medium with 5 mM bicarbonate continued the trend noted for the Caps, cultures to the degree that the autosporangial com- plex was the dominant, and sometimes exclusively, morphology noted (Fig. 5D). The analytical lipid results from day 10 cultures are organized into three groups based on the final (Day 10) pH of the culture media (see Fig. 1) as described in Table 1. As indicated by the Nile Red technique (Fig. 3), the triglyceride fatty acids (TGFA), increased with higher pH (Table 1). The membrane lipids, which include the glycolipids (GLFA) and po- lar lipids (PLFA), declined at higher pH values. Overall, the total lipid in the microalgal cells (per gram dry weight) declined at pH values greater than, i The three pH groups had similar TGFA profiles, but as pH increased, the GLFA and PLFA profiles, fer 10 days incubation indifferent media at the undisturbed final cell densities. ‘The treatments 0.5: D) unbuffered Bold's + bicarbonate, final pH 11.1. Seale bar did not remain stable (Table 2). ‘The higher pH incubated cells had more of the ‘precursor’ fatty acids such as 16:0 (i.e. 16:0 GLFA, pH < 10 = 18.3 + 2.8 mole%, pH 10-11 = 22.5 + 2.8 mole%, pH 11 = 29.2 + 8.4 mole%) and fewer polyunsatur- ated fatty acids (i.e. 18:303 GLFA, pH < 10 = 44.0 + 3.4 mole%, pH 10-11 = 41.0 + 1.3 mole%, pH 11 = 31.6 * 5.0 mole%) To evaluate these changes, two ratios (Table 3) were developed. Precursor fatty acids were 16:0 + 18:1w9c. Most of the other fatty acids in the profile were biochemically derived from these precursors. The unsaturated 16-carbon fatty acids = 16:1w13t + 16:206 + 16:303 + 16:3u6 + 16:403 while the polyunsaturated 18-carbon fatty acids include 18:26 + 18:303 + 18:306 + 18:403. There were no sig- nificant differences in these ratios between the pH groups for the TGFA profiles. The ratios for the GLFA and PLFA were higher than the TGFA ratiosLIPLDS IN HIGH Tams 2 ofthe total lipid prope, PRESSED CHLORELLA 77 Esenfed fatty acid profiles for lipid clases by pH groups as explained in Table 1, Values are given as average mole percent (1 SD) Chet Tea Tho 05 = 08 04204 OLS On 16:0 304475 9 183 © 228 Wl “0.6 = 07 O82 01 06201 VGletst 0.2 + 03 £03 07401 16206 =o. a+ 04 16:80 04 to 15 £03, 16:306 214 02 +06 Vesaas, 0.1 17813 18:0 : 3 03202 1s:1ade + 3 Bb 208 Is:ta7e + 25 14 £03 18206 . BA Ta 10 18348 . M0234 aloe Ls. le 26203 LT 203 Number of asks 2 5 7 26 8 u 26 8 u (Table 3). As incubation pH increased, the values for the membrane lipid ratios (GLFA, PLFA) de- creased to values significantly lower than those of the pH < 10 group (Table 3). DISCUSSION When Chlorella CHLORL is incubated in an un- buffered algal medium such as Bold’s basal (Nichols id Bold 1965), its growth and photosynthet ity drive the pH of the medium from value of less than 7 to above 9 within 10 days. Over two decades ago Galloway and Krauss (1961) warned, that several standard algal media were improperly buffered and that pH changes during experimental incubation could confound interpretation of results. Even so, such media are widely used. ‘The nonme- tabolizable biological buffers used in the present study permitted the uncoupling of the rising pH conditions from other media changes during algal growth. The similarities among Nile Red responses, final cell densities, and morphology for unbuffered and buffered Bold’s culture at similar media pH’s indicate that the buffered media pose few artifacts and were representative model systems for this work. This buffering allowed the description of a nitrogen- independent, high pH-related stress which results, in cell-cycle inhibition and indirectly in ‘TG accu- mulation in Chlorella CHLOR1. When the cell division rate declines due to a par- ticular stress imposed by the medium, presumably one stage of the algal cell cycle is more sensitive to the stress than others. The stress will, therefore, result in a preferential lengthening of that stage of the cell eycle (Olson et al. 1986). One assay to iden- tify the sensitive cell cycle stage is to determine at which stage cell growth is arrested when population Browth ceases due to the stress (Olson et al. 1986). Our morphological observations indicate that the Chlorella CHLORI autosporangial cell stage (i.e. prior to autospore release but after nuclear division) sensitive to high pH medium. Previous work has proposed a pH-dependent increased flexibility of Chlorella mother cell walls preventing its rupture and, therefore, inhibiting autospore release (Malis- Arad and McGowan 1982a, b). It is also likely that the interaction of pH shifts and medium component precipitation could influence the microalgal cell cycle (Galloway and Krauss 1961, Malis-Arad and Mc Gowan 1982a, b). The similarity of buffered treat- ments with and without bicarbonate supplements indicates that this effect is not likely to involve me- dium carbon concentrations. In addition, available nitrogen concentrations were never limiting over the 10-day incubation. Tests 3. Ratios of unsaturated product fatty acids to preeurso fatty acids: precursor fatty acids = 16:0°+ 18:19: unsaturated T6-carbon fairy acids = 10:18131 + 16:246 + 16:303 + 16.300 + 10r4a. olvunsaturated 18carbon faty acids ™ 18:2u6 + 18383 + 18°56 + 18:4}. Average (+1 SD) rato given for pH groups as in Table 1 * indicates significant diference from pH < 10 group bx a Scheffe multiple comparison of means tet with rrrors of 0.08, Where the ratio becomes smaller, the relative degre of unvaturation becomes less wear fay a recut ne Trigheerde pl < 10 0.30 + 0.14 0.04 + 0.03 pH 10-11 O31 + 0.10 0.06 + 0.08 pH > 11 O31 + 0.07 0.02 = 0.01 Glycolipid pH = 10 oat 0.91 + 0.15, pH 10-1 og 0.63 = 0.08* pH > 1 033" 0.36 = 0.13% Polar lipid pH < 10 0.18 = 0.03 pit 0.08 = 0. pet 0.05 = 0.78 JAMES B, GUCKER'TT AND KEITH E. COOKSEY Regardless of whether high pH-stress has a direct or indirect (through medium component precipi- tation, etc.) effect on the microalgal cell cycle, this study has shown that high pH results in a repro- ducible inhibition of a particular stage in Chlorella’s cell cycle. Otsuka and Morimura (1966) predicted that with a balance between TG synthesis and uti- lization in a normal cell eycle, no net accumulati of TG will occur. High pH stress in Chlorel CHLOR1 ultimately results in a lag of the cell cycle stage just prior to TG utilization (Otsuka and Mori- mura’ 1966, Thomas and Cooksey, unpubl. data) and, therefore, a net increase in TG accumulation. The accumulation of TG may be either a) a function of having more cells synchronized at the cell cycle stage rich in TG, or b) the result of a lag at a cell cycle stage in which TG synthesis continues and, there- fore, exceeds the utilization requirements for the completion of cell division (Cohen and Parnas 1976). The three clusters in the cell density/Nile Red scat- terplot show a gradient of TG accumulation with lower cell densities. The lower cell densities are a result of longer cell cycles but indicate that the cells were still dividing under the pH stress. This suggests, the second mechanism of TG accumulation for Chlo- rella CHLOR under these conditions. Since high pH-stress apparently affects the cell cycle, itis likely an indirect cause of TG accumulation, just as other nutrientlimitations which inhibit the development of the cell cycle would be. Our research is currently addressing these hypotheses utilizing synchronous cultures and cell cycle inhibitors. ‘The accumulation of TG as a function of the pH of the growth medium cannot be explained simply by an interference with the progression of the cell cycle and a concomitant oversynthesis of triglycer- ide. During this accumulation of triglyceride, the fatty acid profiles of the membrane lipids, as well as their quantity, changed also. ‘To explain these changes we must consider the synthetic pathways and their activity within the cell cycle for all three classes of lipid, i.e. storage triglycerides and mem- brane polar and glycolipids. TG and membrane lip- ids are synthesized sequentially during the cell eycle (Otsuka and Morimura 1966, Beck and Levine 1977, Klyachko-Gurvich et al. 1981), Because of this se- quential synthesis, Chlamydomonas reinhardi, for ex- ample, was shown to be phospholipid and glycolipid rich at early stages of its cell cycle, probably because synthetic rates for these membrane lipids declined later in the cycle (Beck and Levine 1977). In Chlo- rella sp. K,a partial decomposition of glycolipids was shown to occur immediately prior to autospore re- lease (Klyachko-Gurvich et al. 1981). Similarly, membrane lipids (glycolipids and polar lipids) d clined in Chlorella CHLOR1 stressed at high pH. In fact, the total lipid in Chlorella declined during high pH ‘stress, an observation that would have been, missed if individual lipid classes had not been ana- lyzed. “The fatty acid profiles of Chlorella CHLORI had distinctive lipid class patterns similar to other algae and higher plants (Frentzen 1986, Roughan 1987). In non-stressed cells (pH < 10), the GLFA of the chloroplast membranes were the precursor fatty acids 16:0/18:149, the unsaturated 16-carbon fatty acids, as well as the polyunsaturated 18-carbon fatty acids, with 18:303, 16:4w3 and 16:0 dominating the pro- file. The membrane PLFA were predominately pre- cursors and 18:303. Unlike the membrane GLFA and PLFA, the GFA profiles are dominated by the precursor fatty acids. This results in the higher ratios of unsaturated product fatty acids to precursors for the unstressed (pH < 10) GLFA and PLFA profiles compared to TGFA (Table 3). ICappears from the low ratios for TGFA that TG is synthesized in Chlorella CHLOR1 with little further modification of the precursor fatty acids, regardless of the amount of TG accumulated in the culture. If the sole function of algal TG is, carbon /energy storage during the cell cycle (Otsuka and Morimura 1966), further desaturation is un- jecessary and, in fact, detrimental due to loss of nergy yield during 8-oxidation from the introduc- of an unsaturation (Schulz 1985). Other algae, especially the marine diatoms (Opute 1974), have TGFA profiles which are highly polyunsaturated in- dicating that their TG biosynthesis is more like that in developing oil seeds (Frentzen 1986). Our results suggest that the previously reported enrichment of precursor fatty acids during TG ac- cumulation (Fisher and Schwarzenbach 1978, Pio reck et al, 1984, Ben-Amotz et al. 1985, Millie 1986, Suen et al. 1987, Sicko-Goad et al. 1988) may not be due solely to an increase in TGFA, Algal lipid biochemistry apparently shifted so that significantly less desaturation of precursor fatty acids ring in membrane lipid fractions during ‘TG accu- mulation, However, if TG accumulation functioned merely as storage for excess carbon fixed by the cell, these changes in membrane fatty acid profiles would not be predicted. Instead, these data suggest a reg- ulation point in ‘TG synthesis with a possible inhi- bition of the algal desaturation enzyme system in Chlorella CHLORL These results emphasize the importance of a com- plete model for a shift in lipid metabolism from membrane lipid synthesis to storage lipid synthesis. ‘A model which only argues for an increased syn- thesis of fatty acids is incomplete. A biochemical model for lipid accumulation must take into acc that fatty acids can be esterified to cither storage lipids (triglycerides) or membrane lipids (glycolipids, and polar lipids), but only triglycerides accumulate during stresses which inhibit the microalgal cell di- vision cycle. This research was supported by the Solar Energy Research In- stitute, Golden, Colorado, USA, through contract number XK: 5:05073-1. This work was greatly enhanced by the technLIPIDS IN HIGH-PH sistance of Nick Cooksey Jimmy Cutler Statistical and of the work, aration of the f The authors also wish to thank Dr w the use of the GC, Dr. Martin Hamilton for iphical advice, Dr. Larry Jackson for discussions Dr. Kennedy Gauger for assistance in the prep Beck, J.C. & Levine, R. P. 1977. Synthesis of chloroplast mem- bbrane lipids and chlorophyll in synchronous cultures of Chlamydomonas reinkardi. Biochim, Biophys. Acta 489:360-9. motz, A., ‘Tornabene, TG. & Thomas, W. H. 1985, themical profile of selected species of microalgae with em- phasis on lipids. . Phycol. 21:72-81 Bligh, E. G. & Dyer, W. J. 1959. rapid method for total lipid ‘extraction and purilication. Can. J. Biochem. Phys. 37:911-7 Cohen, D.& Parnas, H. 1976. An optimal policy for the metab- olism of storage materials in unicellular algae. J. Theor. Bio 56:1-18, Cooksey, K. E., Guckert, J. Bo, Williams, 8. A. & Calis, P. R. 1987. Fluorometric determinations of the neutral lipid com: tent of microalgal cells using Nile Red. J. Microbiol. Meth 6 145, 1 J. A. 1973. Comparative biochemistry of fatty acids in ‘eukaryotic microorganisms. Jn Erwin, J. A. [Ed.] Lipids and Biomembranes of Eukaryotic Microorganions, Academic Press, New Vork, pp. 41-143. Fisher, N. S, & Schwarzenbach, R. P. 1978, Fatty acid dynamics in Thalassiosira pseudonana (Bacillariophyceae): implications for physiological ecology. , Phycol 14: 143-50. Fogg, G. E. 1956. Photosynthesis and formation of fats ina ‘diatom. Ain, Bot, NS. 10:265-85, Frentzen, M. 1986. Biosynthesis and desaturation of the differ- iacylglycerol moieties in higher plants. J. Plant Physiol 13-209, Eri uss, RW. 1961. The effect of CO, on ulture media for algae. Plant Cell Physiol. 2:381-7, ert, J.B, Antworth, C, Ps, Nichols, P. D, & White, D. C. 1985. Phospholipid, esterlinked fatty acids profiles as re- producible assays for changes in prokaryotic community structure of estuarine sediments ‘Microbiol. Fool, SY 147-58, Guckert, J. B.. Cooksey, K. FE. & Jackson, L. L. 1988. Lipid solvent systems are not equivalent for analysis of lipid classes in the microeukaryotie green alga, Chlorella, J. Microbiol, Meth 8139-49, ichko-Gurvich, G. L., Tsoglin, L. N. & Mozhaitseva, G. 1981. Lipid metabolism during the course of ontogenesis of Chlorella in connection with activity of the photosynthetic apparatus, Sov. Plant Physiol. 28:304-11. MalisArad, $. & McGowan, R. E. 1982, Alkalinity.induced ag- RESSED CHLORELLA 79 sgregation in Chlorella vulgaris 11. Changes in the cell wall, during the cell eycle, Plant Geil Physiol. 28:11~7. 1982b. A “point of no return’ in the cell eycle of Chio- rella. Plant Cell Physiol. 23:397-401 Millie, D.F. 1986. Nutrientlimitation effectson the biochemical composition of Cjelotrlla meneghiniana (Bacillariophyta). an, experimental and statistical analysis, Can. J. Bol. 64:19-26, Newman, D.W. 1966. Chloroplast fatty acid transformations in nitrogen-deficient and senescent tissues. Plant Physiol. 41 828-3 Nichols, H.W. Bold, H.C. 1965, Trichosarrine polymorpha gen ‘et sp. nov. J Phycol, 134-8, Oson, R. J, Vaulot, D. & Chisholm, SW. 1986. Effects of environmental stresses on the cell cycle of two marine phy= toplankton species. Plant Physiol. 80:918-25. pute, F. 1. 1974. Lipid and fatty-acid composition of diatoms. J. Exp. Botany 25:823-5, Otsitka, H.& Morimura, Y. 1966. Change in fatty acid com- position of Chlorella ellipsoidea during its cell cycle, Plant Cel, Physiol. 71663~70. Piorreck, M., Basch, K-H. 8 Pohl, P, 1984. Biomass production, total protein, chlorophyil, ipidsand fatty acids of freshwater sgreen and blue-green algae under different nitrogen re- Eimes, Phytochemistry 28:207-16. Richardson, B., Orcutt, D. M., Schwertner, H. A., Martine, €. L. & Wickline, H. E. 1969, Effects of nitrogen limitation on the growth and composition of unicellular algae in contin- uous culture. Appl. Microbiol, 18:245-50, Roughan, P.G. 1987. On the control of fatty acid compositions ‘of plant glycerolipids. Jv Stumpf, P. K., Mudd, J. B. & Nes, W. D. [Fds.] The Metabolion, Structure, and Function of Plant Lipids. Plenum Press, New York, pp. 247-54, Schulz, H. 1985. Oxidation of faty acids. In Vance, D. E. & Vance, J. F[Eds.] Bichemisty of Lipids and Membranes. Bene jamin/Cummings, Menlo Park, California, pp. 116-42. Shaw. R. 1966. ‘The polyunsaturated fatty acids of microorgan- ims. Adv. Lipid Res. 4:107-73, Shifrin, N. S. & Chisholm, 8. W. 1981. Phytoplankton lipids interspecific differences and effects of nitrate, silicate and, light-dark eyeles. - Phycol. 17:374-84, Sicko-Goad, L., Simmons, M. S., Lazinsky, D. & Hall, J. 1988, Effect of light cycle on diatom fatty acid composition and quantitative morphology. J. Phyol. 24: J-S., Holzer, G. & Tornabene, T. G. 1987. tion of the green alga Nannochloropss sp. QH under different nitrogen regimes. J. Phycol. 23:289-96. Taguchi, S., Hirata, JA. & Laws. E. A. 1987. Silicate deficiency and lipid synthesis of marine diatoms. J. Phycol. 28:260-7. Vamiya, H. 1963. Control of cell division in microalgae. J. Cel Comp. Physiol. 62(Suppl. 1: 157-74,This document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.