Lipid metabolism and cell composition of the oleaginous yeast Apiotrichum curvatum grown at different carbon to nitrogen ratios

Apiotrichum curvatum ATCC 20509, an oleaginous yeast that can accumulate up to 60% of its cellular dry weight as intracellular lipid when grown with excess carbon, was grown in nitrogen-limited, balanced, and lactose-free medium with asparagine as nitrogen source and lactose as carbon source. Biomass and lipid accumulation were measured, cell composition was analyzed, and catalase activity was followed as marker enzyme for peroxisomes. The organism accumulated 54% of its dry weight as total cellular lipid when grown under nitrogen limitation and accumulated only 20-25% of its dry weight as lipid when grown in balanced medium. When starved for carbon, cells utilized endogenous lipid and carbohydrate as carbon and energy sources; the intracellular contents of lipid and carbohydrate decreased by 31 and 26%, respectively. Intracellular carbohydrates also seemed to be used as intermediates for lipid accumulation and lipid turnover. Catalase activity was strongly induced (over 10-fold increase in specific activity) when cells metabolized endogenous lipid. The lipid content of cells was inversely related to catalase activity and to intracellular protein or total nitrogen content. Lipid content showed no correlation with intracellular carbohydrate content.     

Polysaccharide production and the possible occurrence of GDP-d-mannose dehydrogenase inAzotobacter vinelandii

During the growth ofAzotobacter vinelandii in batch culture in Burk's 2% glucose medium supplemented with 50mg EDTA per litre, water-insoluble capsular polysaccharide material accumulated in cultures prior to the appearance of water-soluble polysaccharide in the culture medium. On isolation, hydrolysis and chromatography, both these polysaccharides were observed to be composed of carbohydrate monomers having the same chromatographic mobilities as glucose, rhamnose, guluronic acid and mannuronic acid. The activity of GDP-d-mannose dehydrogenase recorded in crude cell-free extracts fromAzotobacter vinelandii, when these polysaccharides were produced, may indicate a close similarity between the biosynthetic pathway of alginate synthesis in marine Phaeophyceae and this soil microorganism.     

Carbohydrate constituents of Lessonia trabeculata

D-mannitol was the only low-molecular weight carbohydrate isolated from ethanolic extracts of Lessonia trabeculata blades. After sequential extraction with water, acid and alkali, laminaran, fucose-containing polysaccharides and alginic acid were also isolated. Fucose-containing polysaccharide from the acidic extract was separated into three fractions by ion exchange chromatography. Alginic acid was the major polysaccharide obtained in the sequential extraction.     

Physiological parameters of growth in Saprolegnia parasitica coker

Persistent bacteria were separated fromS. parasitica by means of the oligodynamic effect of a silver ring in a modification ofRaper's technique. Inoculation of fungal cultures was by means of mycelial macerate. Growth was measured by mycelial dry weight. A chemically defined medium (standard medium) was developed which consisted of a mineral base (chlorides of magnesium, manganese, zinc, calcium, and iron) chelated with EDTA, supplemented with glucose, sodium glutamate, and methionine, and buffered at pH 7.0 with 0.01 M. KH₂PO₄. Shaking culture methods supported increased growth rates and higher dry weight yields compared to stationary methods. Excellent growth occurred between 15 to 30°C. in the standard medium and between pH 4.0 and 8.0 in the standard medium plus 0.01 M. sodium succinate and 0.01 M. TRIS used as additional buffers. Significant phosphate toxicity was demonstrated at concentrations exceeding 0.05 M. Sodium succinate and TRIS, used as buffers at 0.01 M. each, were compatible withS. parasitica, whereas boric acid, sodium barbital, and sodium citrate inhibited growth under similar conditions. Substitution of other carbon sources for glucose in the standard medium (on an equal carbon basis where possible) indicated that cellobiose, dextrin, fructose, glycerine, glycogen, sodium lactate, and soluble starch supported significantly heavier growth than did the standard medium minus glucose; glycogen had a greater yield than did the standard medium minus glucose; glycogen had a greater yield than the standard medium. Arabinose, dulcitol, galactose, inulin, lactose, mannitol, mannose, raffinose, rhamnose, sorbitol, sucrose, and xylose neither stimulated nor inhibited growth; however, growth inhibition was produced by α-ketoglutaric acid, sodium citrate, and sodium succinate. When fatty acids and lipids were substituted for glucose (on an equal carbon basis where possible), only butter, lard, oleo, and palmitic acid supported heavier growth ofS. parasitica than the standard medium minus glucose. Stearicacid neither stimulated nor inhibited growth; acetic acid, butyric acid, formic acid, octanoic acid, and propionic acid significantly inhibited the growth of the fungus. Various nitrogen sources were substituted for sodium glutamate in the standard medium (on an equal nitrogen basis where possible). Casein hydrolysate and gelatin produced yields higher than that developed in the standard medium; other nitrogen sources produced lesser yields but still greater than those from the standard medium minus sodium glutamate:

1. Alanine, arginine, aspartic acid, and histidine (good nitrogen sources).
2. Ammonium chloride, cysteine, leucine, serine, and urea (fair nitrogen sources).
3. Glycine, isoleucine, lysine, methionine, phenylalanine, potassium nitrate, sodium nitrate, threonine, tryptophan, and valine (poor nitrogen sources).

When various sulfur sources were substituted for methionine in the standard medium (on an equal sulfur basis), only cysteine and cystine produced dry weights comparable to that which developed in the standard medium. The following were very poor sulfur sources yet supported more growth than did the standard medium minus methionine: sodium sulfide, sodium thiosulfate, and thiourea. The ability of the other sulfur sources to support growth was questionable: potassium persulfate, sodium bisulfite, sodium dithionate, sodium hydrosulfite, sodium sulfate, sodium sulfite, and sodium thiocyanate. The standard medium contained only two nitrogen sources: sodium glutamate and methionine. Sodium glutamate served as a carbon source as well as a nitrogen source, but methionine could serve only as a source of sulfur.     

Use of Carbowax as a high molecular weight nonprotein substance for cultivation of lymphoid cell and study of their morphology

Lymphoid cells prepared from rabbit popliteal lymph nodes were cultivated in medium in which the high molecular weight component was replaced by Carbowax 20 M. This substance, in 0.2% concentration, proved to be a substitute for the high molecular weight component in short-term cultivation, and the viability results were similar to those in medium containing normal rabbit serum or 0.5% human serum albumin. In addition, mitoses and the formation of large basophilic cells resembling the blastoid cells induced during cultivation with phytohaemagglutinin were observed in this medium. The results are discussed.     

Optimization of lipid production in the oleaginous yeastApiotrichum curvatum in wheypermeate

Summary Lipid production of the oleaginous yeastApiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h^-1. At C/N-ratios higher than 25–30 lipid content gradually increased to nearly 50% at C/N=70 and the maximal obtainable dilution rate decreased to 0.02 h^-1 at C/N=70. From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30–35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates ofA. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes with high cell densities. A lipid production rate of nearly 1 g/l/h was reached in a partial recycling culture. It was calculated that by using this cultivation technique lipid production rates of even 2.9 g/l/h may be reached when the supply of oxygen can be optimized.Nomenclature C/N-ratio carbon to nitrogen ratio of the growth medium (g/g) - C/N_crit C/N-ratio at which there is just enough nitrogen to allow all carbon source to be converted to biomass - D dilution rate=volume of incoming medium per unit time/volume of medium in the culture vessel (h^-1) - D_max maximum dilution rate (h^-1) - DW cell dry weight - L lipid yield (g storage lipid/g carbon source) - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1) - Q_L lipid production rate (g/l/h) - Y_i molecular fraction of carbon substrate that is converted to storage carbohydrate (C-mol/C-mol) - Y_ls maximal amount of storage lipid that can be produced per mol carbon source (C-mol/C-mol)     

A Laboratory Case Study of Efficient Polyhydoxyalkonates Production by Bacillus cereus,a Contaminant in Saccharophagus degradans ATCC 43961 in Minimal Sea Salt Media

A contaminating bacterium growing along with the stock culture of Saccharophagus degradans ATCC 43961 (Sde 2-40) on marine agar plate was isolated and investigated for its ability to produce polyhydoxyalkonates (PHA). Preliminary screening by Sudan black B and Nile blue A staining indicated positive characteristic of the isolate to produce PHA. The isolate was able to grow and produce PHA in minimal sea salt medium broth. PHA quantification studies with gas chromatographic analyses of the dry cells derived from culture broths revealed accumulation of PHA in bacterial cells. PHA production started after 20 h and increased with cell growth and attained maximum values of 61 % of dry cell weight at 70 h of cultivation. After 70 h, a slight decrease in the level of PHA content was observed. The nature/type of PHA was found to be poly(3-hydroxybutyraye) by Fourier transform-infrared spectroscopy. Microbiological and 16S rRNA gene sequencing analyses suggested that the PHA producing bacterial isolate belongs to Bacillus genera and shows 100 % nucleotide sequence similarity with Bacillus cereus species in GenBank. This study is a first report for ability of Bacillus species to grow in marine sea salt media and produce PHA. The media used for the polymer production was novel in the context of the genus Bacillus and the production of PHA was three-fold higher than Sde 2-40 using same growth medium. This study shows that the contaminant bacteria once properly investigated can be used for advantageous characteristic of metabolites production in place of original cultures.     

Transversion of cell polarity from bi- to multipolarity is the mechanism determining multiple spore formation in Anaerobacter polyendosporus PS-1T

The number of spores formed in a single cell of Anaerobacter polyendosporus PS-1^T is significantly influenced by the composition of nutrient media. Depending on carbohydrate concentration in synthetic medium, the number of spores may vary from one or two to as many as five to seven. Investigation of spore formation process by fluorescence and electron microscopy revealed that on media with 0.5–1.0% glucose or galactose most of vegetative cells remained rod-shaped after cessation of cell division in the culture. The nucleoids of these cells were localized at cell poles close to the polar site of the cytoplasmic membrane. Fore-spores were formed at one or both of these poles. A satellite nucleoid (operator) was observed close to each forespore. In the variant with bipolar organization of mother cells, only one or two spores per cell were formed. In the second variant of culture development, when the cells were grown at low galactose concentrations (0.1–0.3%), most of vegetative cells increased in volume and became oval or spherical after cessation of cell division in the culture. Epifluorescence microscopy with nucleic acid-specific fluorochromes (DAPI and acridine orange) revealed the presence of multiple (six to nine) nucleoids in these cells. The nucleoids were located at the cell periphery in close contact with the cytoplasmic membrane. These nucleoids became the centers (poles) for forespore formation. Thus, in the early stationary phase transversion from bipolar to multipolar cells occurred. Cessation of cell division combined with continuing replication of the nucleoids resulted in formation on multinuclear cells. The multiplicity of nucleoides and multipolarity of these cells were prerequisites determining endogenous polysporogenesis, occurring as synchronous formation of three to seven twin spores in many of the oval and spherical cells.     

多变鱼腥藻在不同氮源培养条件下光能转换效率的研究

依公式PE=KY,其中K等于生物量的热值(每克干重千卡),Y等于产率即每吸收千卡光能所产生的生物量的干重,测试了在无氮和有结合氮培养下的多变鱼腥藻(Anabaena variabilis)的光能转化效率。结果指出在无氮培养下的最高PEN2为8．1％,在结合氮(NH4Cl)培养下PENH =4+为5.8％。对这种差异性作了简要讨论。     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

Summary The effect of the nature and concentration of the nitrogen source on respiratory activity and removal of carbohydrate from the medium in suspension cultured sycamore (Acer pseudoplatanus L.) cells was determined. Comparison was also made of the rates of uptake of the two alternative nitrogen sources, nitrate and glutamate, at differing initial nitrogen concentrations within the range 7–14 mM. The initial pH of the culture medium before inoculation was 5.2; after inoculation the pH of both nitrate and glutamate cultures rose to reach an eventual level in the range 7.0–7.1. Glutamate was removed from the medium more slowly than nitrate. Under the particular conditions of culture used the growth of the cells was nitrogen limited. Sugar uptake from the medium continued for some time after the nitrogen in the medium was depleted. The data show that although cell division and protein content are nitrogen-limited, dry weight and fresh weight yields may also be determined in a complex interaction through carbohydrate availability. There were no obvious differences in respiratory activity between cultures grown on nitrate or glutamate.     

Nitrogen Effect on Water-Soluble Polysaccharide Accumulation in <Emphasis Type="Italic">Streblonema</Emphasis> sp. (Ectocarpales,Phaeophyceae)

The water-soluble polysaccharides of brown algae attract the increasing attention of researchers as an important class of polymeric materials of biotechnological interest. The sole source for production of these polysaccharides has been large brown seaweeds such as members of Laminariales and Fucales. A new source of water-soluble polysaccharides is suggested here: it is a filamentous brown alga Streblonema sp., which can be cultivated under controlled conditions in photobioreactors that allow obtaining algal biomass with reproducible content and quality of polysaccharides. The accumulation of water-soluble polysaccharides can be stimulated by macronutrient limitation. In response to nitrogen deficiency, Streblonema sp. accumulated water-soluble polysaccharides (WSPs) rich in laminaran. WSP accumulation started after 3–4 days following nitrate depletion and reached a plateau at around day 7. Polysaccharide accumulation was related to cellular nitrogen content. The critical internal N level that triggered the onset of polysaccharide accumulation was 2.3% dry weight (DW); at a cellular N concentration less than 1.4% DW, the polysaccharide synthesis stopped. Upon nitrate re-supply, mobilization of WSP occurred after 3 days. These results suggest that a two-stage cultivation process could be used to obtain large algal biomass with high water-soluble polysaccharide production: a first cultivation stage using nitrate-supplemented medium to accumulate algal biomass followed by a second cultivation stage in a nitrate-free medium for 3 to 7 days to enhance polysaccharide content in the alga.     

多变鱼腥藻在不同氮源培养条件下光能转换效率的研究

依公式PE=KY,其中K等于生物量的热值(每克干重千卡),Y等于产率即每吸收千卡光能所产生的生物量的干重,测试了在无氮和有结合氮培养下的多变鱼腥藻(Anabaena variabilis)的光能转化效率。结果指出在无氮培养下的最高PE_(N2)为8.1%,在结合氮(NH_4Cl)培养下PE_(NH_4~ )为5.8%。对这种差异性作了简要讨论。     

Comparative studies on sporulation-promotive actions of cyclic AMP,theophylline and caffeine in Saccharomyces cerevisiae

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylling did significantly when added to a presporulation medium containing glucose Caffeine inhibited the vegetative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Changes in non-cellulosic cell-wall polysaccharides during the growth of carrot cells in suspension cultures

The cell-wall composition of carrot (Daucus carota L.) cells has been studied during their growth in suspension culture. Pectic and hemicellulosic polymers were fractionated according to molecular size by a Sepharose 4B column. Polyuronides in the pectic fraction were resolved into high- and low-molecular-weight components. The low-molecular-weight polyuronides were relatively free of neutral sugars and showed a marked increase during the growth of the cell wall. Hemicellulosic polysaccharides were of disperse molecular size. As cell expansion proceeded, the contents of glucose and xylose in the high-molecular-weight region increased while those in the low-molecular-weight fraction decreased. Removal of auxin from the medium apparently caused degradation of high-molecular-weight polymers in both the pectic and hemicellulosic fractions.     

Osmotically-regulated trehalose accumulation and cyclic β-(1,2)-glucan excretion by Rhizobium leguminosarum biovar trifolii TA-1

Rhizobium leguminosarum biovar trifolii TA-1 produced high molecular weight extracellular (EPS) and capsular polysaccharides (CPS) as the main carbohydrate products in a medium (10 g of mannitol and 1 g of glutamic acid per liter) with low osmotic pressure of 0.20 MPa. By increasing the osmotic pressure of the medium with the addition of NaCl or other osmolytes up to 1.44 MPa, the synthesis of EPS and CPS was suppressed. Cyclic -(1,2)-glucans were excreted instead. Concentrations of over 1500 mg of glucans/l medium were produced by a biomass of 520 mg protein at 200 mM NaCl (1.20 MPa). Intracellular cyclic -(1,2)-glucan concentrations remained at 45 to 100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells as osmo-protectant. Concentrations of up to 130 mg/g protein were reached. Strain TA-1 could tolerate 350 mM NaCl.Abbreviations CPS capsular polysaccharide - EPS extracellular polysaccharide - LM_r low molecular weight - HM_r high molecular weight     

Glutamine synthetase,glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH ₄ ⁺ (90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60–85%). The enzyme was adenylylated in bacteroids (43–75%) and in the nodule tissues (52–68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of ¹⁵N-labelled (NH₄)₂SO₄ into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on ¹⁵N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.     

The influence of EDTA on the composition of alginate synthesized by Azotobacter vinelandii

1. During an investigation of the physiology of Azotobacter vinelandii with particular reference to polysaccharide formation, a suitable medium which was precipitate-free was developed by adding EDTA at a concentration of 50 mg/l to a basal medium containing one of eight different carbohydrates as sole carbon source.
2. Acetylated alginate was always produced by the organism when cultured under defined conditions, regardless of the carbohydrate source incorporated in the basal medium.
3. When EDTA was added to the medium, the bacteria produced acetylated polyuronides with a preponderance of mannuronic acid residues.
4. A comparison of the infrared spectra of the alginate produced by Azotobacter vinelandii and the affect of EDTA upon the mannuronic acid/guluronic acid ratios of the alginate are reported.

     

Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover

Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX?)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.     

The growth of Pseudomonas putida on m-toluic acid and on toluene in batch and in chemostat cultures

Summary The present study describes the growth of Pseudomonas putida cells (ATCC 33015) in batch and continuous cultures on two toxic substrates; toluene and m-toluic acid as sole carbon and energy sources. In fed-batch cultures on m-toluic acid up to 3.55 g cell dry weight/1 were achieved with a maximal specific growth rate (_max) of 0.1 h^-1. The average cellular yield was 1.42 g cell dry weight/g m-toluic acid utilized. When liquid toluene was added to shake-flask cultures in the presence of 0.7 g/1 m-toluic acid, the average cellular yield obtained was 1.3 g cell dry weight/g toluene utilized and the _max was 0.13 h^-1. Growth on toluene vapour in the presence of 0.7 g/l m-toluic acid in batch cultures resulted in a cellular yield of 1.28 g cell dry weight/g toluene utilized, with growth kinetics almost identical to those with liquid toluene (_max liquid=0.13 h^-1, _max vapour=0.12 h^-1). The maximal biomass concentration was 3.8 g cell dry weight/l, obtained in both cases after 100 h of incubation. Pseudomonas putida was grown in a chemostat initially on 0.7 g/l m-toluic acid and vapour toluene and then in the steady state on toluene as the sole source of carbon and energy. Toluene was added continuously to the culture as vapour with the inflowing airstream. Chemostat cultures could be maintained at steady state for several months on toluene. The maximal biomass concentration obtained in the chemostat culture was 3.2 g cell dry weight/l. The maximum specific growth rate was 0.13 h^-1, with a cellular yield of 1.05 g cell dry weight/g toluene utilized. Approximately 70% of the toluene consumed was converted into biomass, and the remainder was converted to CO₂ and unidentified byproducts.     

pH and CO<Subscript>2</Subscript> effects on <Emphasis Type="Italic">Coelastrella</Emphasis> (<Emphasis Type="Italic">Scotiellopsis</Emphasis>) <Emphasis Type="Italic">rubescens</Emphasis> growth and metabolism

We studied effects of рН and СО₂ enrichment on the physiological condition and biochemical composition of a carotenogenic microalga Coelastrella (Scotiellopsis Vinatzer) rubescens Kaufnerová et Eliás (Scenedesmaceae, Sphaeropleales, Chlorophyceae), a promising source of natural astaxanthin. The microalga was grown at a constant pH (5, 6, 7 or 8) maintained by direct СО₂ injection. The air-sparged culture served as the control. Cell division rate and size, dry biomass productivity, the rates of nitrogen and phosphorus uptake as well as photosynthetic pigment and total lipid content and fatty acid composition were followed. С. rubescens possessed a narrow-range рН tolerance (the optimum рН 6–7). Under these conditions, the highest values of the maximum (1.0–1.1 1/day) and average (0.3–0.35 1/day) specific growth rate, chlorophyll а (4.8–4.9%) and total carotenoid dry weight percentages (1.7–1.8%) were recorded. Cell lipid fatty acid unsaturation index (1.851) and polyunsaturated fatty acid percentage (36–39%) and С18:3 ω3/С18:1 ω9 ratio (3.8–4.5) were also the highest under these conditions. A decline of рН to 5 brought about severe stress manifesting itself as a cell division cessation, photosynthetic apparatus reduction, two-fold increase in cell volume, accumulation of dry weight and lipids and a considerable decline in fatty acid unsaturation. Cultivation of С. rubescens without СО₂ enrichment resulted in a rapid alkalization of the medium to рН 9.5–10.5 impairing the physiological condition of the cells. Reasons of the deteriorative effects of suboptimal pH values on the physiological condition of C. rubescens are discussed.