The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid     

Temperature effects on solute accumulation by Candida utilis

Summary A study was made of the effect of temperature on accumulation of glucosamine and 2-aminoisobutyrate by Candida utilis NCYC 321 grown at 30° C or 10° C. Exponential-phase cells contained greater proportions of C_16:1 and C_18:3 acids, and smaller proportions of C_13:1 and C_18:2 acids, when grown in a defined medium at 10° C compared with 30° C. Cells grown at 30° C or 10° C were able to accumulate extracellular (10 mM) glucosamine and 2-aminoisobutyrate against concentration gradients. 2-Aminoisobutyrate was not metabolised by the cells; glucosamine was accumulated probably as a mixture of glucosamine 1- and 6-phosphates. Rates of accumulation of glucosamine and 2-aminoisobutyrate by cells grown at 30° C or 10° C decreased markedly when the test temperature was decreased from 30° C to 15° C. The rate of accumulation of glucosamine by cells grown at 10° C was considerably lower at each of the test temperatures compared with the corresponding rates for cells grown at 30° C; the rate of accumulation of 2-aminoisobutyrate was much less affected by the temperature at which the cells were grown and then only when measured at temperatures below about 20° C. Apparent K _m values for accumulation of glucosamine by cells grown at 30° C or 10° C decreased considerably when the test temperature was lowered from 20° C to 15° C. The extent of the decrease in K _m value was approximately the same for cells grown at 30° C or 10° C. Apparent K _m values for accumulation of 2-aminoisobutyrate were hardly affected by test temperature. Apparent V _max values for accumulation of glucosamine or 2-aminoisobutyrate were much lower when measured at 15° C than at 30° C. When measured at 30° C, apparent V _max values for accumulation of either solute were slightly lower with cells grown at 10° C compared with cells grown at 30° C; when measured at 15° C, the values were slightly greater with cells grown at 10° C. Net accumulation of glucosamine, at 30° C or 20° C, by cells grown at 30° C or 10° C ceased after 4–6 h. Cells grown at either temperature continued to accumulate 2-aminoisobutyrate at 30° C or 20° C for at least 12 h. The rate of efflux of glucosamine by cells grown at 30° C was slower when measured at 20° C compared with 30° C. With cells grown at 10° C, the rate of efflux at 30° C was slower than with cells grown at 30° C; when measured at 20° C, the rates were about equal. The temperature at which the cells were grown did not affect the ability of d-glucose, d-mannose or d-ribose to compete with d-glucosamine, or with the ability of l-alanine to compete with 2-aminoisobutyrate, when tested at 30° C or 20° C. Cells grown 30° C or 10° C had very similar ATP contents. The results are discussed in relation to the effect of temperature on the rate of solute accumulation by micro-organisms.Abbreviation AIB 2-Aminoisobutyrate     

Thermophilic amylase-producing bacteria from Vietnamese soils

Of 25 bacterial isolates from Vietnamese soils, two were identified asBacillus stearothermophilus and one asThermoactinomyces thalpophilus, both thermophilic, amylase-producing bacteria. Amylase activity was highest in the presence of cassava starch as carbon source and (NH₄)₂HPO₄ as nitrogen source. The strains exhibit a high amylase productivity within the first 5 to 7 h of cultivation at 55°C. The crude enzyme had optima of pH 6.5 and 70°C.     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.     

Nodulation and N2 fixation of guar at high root temperature

Guar (Cyamopsis tetregonoloba (L.) may be grown when soil temperatures are potentially high enough at the time of planting to inhibit nodulation and N₂ fixation. An experiment was conducted using controlled conditions to determine the influence of high root temperature on growth and N₂ fixation of guar. The experiment included two strains of rhizobia, two varieties of guar, two mineral N treatments, and root temperatures of 34, 37, and 40°C. Plants were grown for 44 days. The root temperature of 40°C reduced N fixation by at least 80% and nodule weight by more than 50%. Significant interactions occurred between most factors in influencing nodulation, N₂ fixation and dry matter production. Guar, nodulated by rhizobial strain GAR022-1 and fully dependent on N₂ fixation or provided with starter mineral N (25 mg pot^–1), was not influenced by the root temperature of 37°C as compared to 34°C. Nodulation and N₂ fixation by strain 32H1 was reduced by at least 40% when no starter mineral N was provided and the root temperature was 37°C. Providing starter mineral N to one variety of guar doubled the quantity of N₂ fixed by strain 32H1 at both 34 and 37°C but N₂ fixation was lower at the higher root temperature. It appears that root temperatures between 37° and 40°C bracketed the critical root temperature for N₂ fixation by nodulated guar and that the critical root temperature for guar dependent on mineral N was above 40°C.     

A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase

Summary [C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25°, but which is deficient in cytochromes b and aa ₃ when grown at 37° (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37°, the rate of mitochondrial protein synthesis is decreased to approximately 25% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37°-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rates of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37°-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.     

Temperature dependence of membrane-bound enzymes of the energy metabolism in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides

Rhodospirillum rubrum grown either chemotrophically or phototrophically at 14°C and 30°C, was employed to study the effect of temperature on fatty acid composition as well as on several membrane bound functions involved in energy metabolism. Upon growth at both temperatures the fatty acid composition of membranes showed differences, which could be attributed to an incomplete formation of photosynthetically active membranes rather than specifically to the growth temperature. Activities of NADH dependent respiration and light induced proton extrusion by cells did not show discontinuities in Arrhenius plots down to temperatures of 15°C and 5°C, respectively. In contrast, coupling factor Mg²⁺- and Ca²⁺-ATPase as well as succinate cytochrome c oxidoreductase showed significant breaks at 20°C and 18°C, respectively. Similarly, in Rhodopseudomonas sphaeroides. NADH dependent respiration and light induced proton extrusion by cells was continuous over the entire range of temperatures applied. ATPase as well as succinate cytochrome c oxidoreductase, on the other hand, featured discontinuities in Arrhenius plots at 20°C and 19°C. The implication of the data on growth rates and membrane structure are discussed.Abbreviation Bchl baceteriochlorophyll     

Altered heat resistance in spores and vegetative cells of a mutant fromBacillus subtilis

The relationship between sporulation temperature and spore killing temperature is described.Bacillus subtilis YB886, grown and sporulated at 25°, 30°, 37°, and 45°C, produced spores having D₉₀ values of 63.5, 76.3, 89.0, and 106 min respectively. In addition, the vegetative cells of this strain also demonstrated resistance to heat killing when grown at elevated temperatures (D₅₀ of 26.6, 32.5, 39.0, and >50 min for cells grown at 25°, 30°, 37°, and 45°C). A transposon-generated mutant of strain YB886, designated as BUL786, which is missing a heat shock-induced protein (97 kDa) (Qoronfleh MW and Streips UN, BBRC, 138:526–532, 1986 and FEMS 1987), was tested for thermotolerance under similar conditions. The cells failed to respond to growth at high temperature by producing heat-resistant spores or vegetative cells. For strain BUL786 the D₉₀ of spores generated at 20°, 25°, 30°, 37°, and 45°C was 9.4, 11.3, 12.8, 14.1, and 20 min, respectively. Similarly, the D₅₀ of vegetative cells was 15, 16.8, 17.8, 19.0, and 22.3 min when the cells were grown at 20°, 25°, 30°, 37°, and 45°C. Also, sporulation of YB886 cells in the presence of cadmium chloride increased the D₉₀ values for the resulting spores (5µM CdCl₂ resulted in a D₉₀ of 160 min). Strain BUL786 failed to produce spores with any elevated D₉₀ when grown in the presence of CdCl₂.     

Thermostability of enzymes of the tricarboxylic acid cycle ofBacillus coagulans

The thermostability of four enzymes of the tricarboxylic acid cycle has been studied in the facultative thermophile,Bacillus coagulans. Although isocitrate dehydrogenase appeared to be more temperature-sensitive in whole-cell extracts of cultures grown at 30°C compared with that in cultures grown at 55°C, this difference could be largely eliminated by the removal of cell-wall material. The specific activity of each of the enzymes examined was approximately threefold higher in cultures grown at 55°C than in those grown at 30°C. The maximum temperature, Arrhenius plot and effect of stabilizing agents for each enzyme were examined and found to be independent of growth temperature. Sodium chloride (10% w/v) was an effective protective agent for fumarase, aconitase and malate dehydrogenase. Protection from thermal denaturation of isocitrate dehydrogenase, aconitase and fumarase but not malate dehydrogenase was also given when the enzymes were heated in the presence of their substrates. These results are discussed in light of the generalized theories of facultative thermophily which have been proposed.     

Changes in Parameters of the Plasmalemma ATPase during Peach Vegetative Bud Dormancy

Plant dormancy and dormancy breaking depend, at least partially, on close relationships between buds and tissues underlying bud (bud stands). In Prunus persica, the dormancy was related to high nutrient absorption in bud stands linked to high plasmalemma ATPase (EC 3.6.1.3) activity. Two plasmalemma fractions was isolated from peach vegetative buds and bud stands using aqueous phase partitioning and ultracentrifugation. Results of markers enzyme assays indicated that both plasmalemma enriched fractions obtained were highly purified. During the dormancy period plasma membrane ATPase amount and activity were higher in bud stands than in buds. Moreover, assays performed at different temperatures (4, 18, 30 °C) indicated modifications of kinetic parameters (K_m, V_m) in both tissues during dormancy release. In buds, from November to February, K_m declined at 4°C and increased at 30 °C whereas no changes was measured at 18 °C and V_m increased at all temperature. In bud stands, no changes of K_m was measured at 4 °C and 18 °C whereas an increase occurred at 30 °C and V_m decreased at all temperature. According to the results, it can be postulated that dormancy release in peach-tree could be related to modifications of plasma membrane ATPase properties, in buds and bud stands, during winter time.     

Effect of temperature on rice growth in nutrient solution and in acid sulphate soils from Vietnam

Climatic and soil factors are limiting rice growth in many countries. In Vietnam, a steep gradient of temperature is observed from the North to the South, and acid sulphate soils are frequently devoted to rice production. We have therefore attempted to understand how temperature affects rice growth in these problem soils, by comparison with rice grown in nutrient solution. Two varieties of rice, IR64 and X2, were cultivated in phytotrons at 19/21°C and 28/32°C (day/night) for 56 days, after 3 weeks preculture in optimal conditions. Two soils from the Mekong Delta were tested. Parallel with the growing experiments, these two soils were incubated in order to monitor redox potential (E _h ), pH, soluble Al and Fe, soluble, and available P. Tillering retardation at 20°C compared to 30°C was similar in nutrient solutions and in soils. The effect of temperature on increasing plant biomass was more marked in solutions than in soils. The P concentrations in roots and shoots were higher at 20°C than at 30°C, to such an extent that detrimental effect was suspected in plants grown in solution at the lowest temperature. The translocation of Fe from roots to shoots was stimulated upon rising temperature, both in solutions and in soils. This led to plant death on the most acid soil at 30°C. Indeed, the accumulation of Fe in plants grown on soils was enhanced by the release of Fe²⁺ due to reduction of Fe(III)-oxihydroxides. Severe reducing conditions were created at 30°C: redox potential (E _h ) dropped rapidly down to about 0 V. At 20°C, E _h did not drop below about 0.2 V, which is a value well in the range of Fe(III)/Fe(II) buffering. Parallel to E _h drop, pH increased up to about 6–6.5 at 30°C, which prevented plants from Al toxicity, even in the most acid soil. Phosphate behavior was obviously related to Fe-dynamics: more reducing conditions at 30°C have resulted in enhancement of available P, especially in the most acid soil.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

Extracellular protease from Bacillus coagulans,a psychrotrophic,Antarctic bacterium

Bacillus coagulans, when grown on casein at 20°C, produced an inducible, metalloprotease of 28 kDa at 1.6 U/mg cell protein. (NH₄)₂SO₄ at 2 g/l decreased enzyme production irrespective of carbon source.The authors are with the Defence R & D Establishment, Tansen Road, Gwalior-474 002, India.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Effects of temperature on acaricidal and insecticidal activities of the benzoylureas flucycloxuron and diflubenzuron

Effects of temperature on the activity of flucycloxuron on larval stages of Panonychus ulmi (Koch), based on LC₅₀ values, were highly significant (P < 0.001) with temperature coefficients of-1.7 in both the ranges of 15° to 25°C and 20° to 30°C. The slopes of probit regression lines at 15° and 20°C were significantly steeper than those at 25° and 30°C. As a consequence the temperature coefficients based on LC₉₀ values were-4.4 and-2.2, for the 2 temperature ranges. The ovicidal activity of flucycloxuron on P. ulmi was low and was only statistically detectable at 20°C (LC₉₀ of 84 mg a.i./l). In studies with larvae of Aedes aegypti (Linnaeus), Leptinotarsa decemlineata (Say), Plutella xylostella (Linnaeus), Spodeptera exigua (Hübner) and Spodoptera littoralis (Boisduval) probit regression lines were parallel over temperature. The activity of flucycloxuron on these five insect species was not affected by temperature. Based on LC₅₀ values, diflubenzuron showed positive temperature coefficients on P. xylostella of + 2.1 at 15° to 25°C and + 2.5 at 20° to 30°C. For S. littoralis the temperature coefficient was positive (+ 2.4) at 15° to 25°C but negative (-1.9) at the 20° to 30°C range. Temperature coefficients of diflubenzuron were neutral for A. aegypti, L. decemlineata and S. exigua. In the design and analysis of these studies special allowance was made for date effects and variation in natural mortality over temperature.     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

Production of a thermophilic,extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl₂. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India     

Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus<Emphasis Type="Italic"> Aspergillus phoenicis</Emphasis>

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and -xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 °C or 42 °C. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 °C; and levels were three- to five-fold higher than at 25 °C. Secretion of xylanase and -xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 °C for extracellular and 90 °C for intracellular -xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 °C to 55 °C when the fungus was cultivated at 42 °C. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermostability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE–cellulose and Biogel P-60 columns.