Physiological,Biochemical, and Cytological Characteristics of a Haloalkalitolerant Methanotroph Grown on Methanol

The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M). At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h^–1) was twice as high as on methane (0.1 h^–1). The maximum growth rate increased with increasing medium salinity and pH. The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively. The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars. The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine. The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold. The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells. The data obtained suggest that the high methanol tolerance of M. buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs

The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline. M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

Regulation of glycogen metabolism in astrocytoma and neuroblastoma cells in culture.

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in aprallel with the medium glucose. The critical glucose concentration is 2.5 mM for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations. When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E1 is effective with the neuroblastoma cells.     

Regulation of glycogen synthesis in rat-hepatocyte cultures by glucose, insulin and glucocorticoids.

The changes in glycogen content and in its rate of synthesis in two-day-old primary cultures of rat hepatocytes were assessed under various conditions. Hepatocytes cultivated in serum-free and hormone-free medium switch from glycogen degradation to glycogen deposition at 10.3 mM glucose. After pretreatment of the cells with glucocorticoids this threshold was reduced, in the absence or presence of insulin, to 5.4 or 1.2 mM glucose, respectively. The rate of glycogen synthesis in the presence of 10 mM glucose was amplified from 5 nmol x h-1 x mg protein-1 to 20 nmol glucose x h-1 x mg protein-1 after pretreatment with triamcinolone. Glucagon pretreatment also significantly increased the subsequent glycogen synthesis rate. Insulin addition accelerated glycogen synthesis about twofold regardless of the pretreatment. The dose-response relationship between insulin concentration and glycogen synthesis rate showed half-maximal effect at 0.62 +/- 0.22 nM (mean +/- S.D.) insulin. Pretreatment of hepatocytes with glucocorticoids, glucagon, insulin or combinations of these hormones did not significantly change the concentration which gives the half-maximal effect.     

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Synthesis of osmoprotectants by halophilic and alkaliphilic methanotrophs

The¹H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5–1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium ofMethylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-contaihing osmoprotectants, ectoine and 5-oxo-1-proline.M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH ₄ ⁺ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

The ATP pool in relation to the production of glycerol and heat during growth of the halotolerant yeast Debaryomyces hansenii

In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Y _m-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.Abbreviations CFU colony-forming units - PCA perchloric acid - TCA trichloroacetic acid     

Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.     

Continuous cultivation in a chemostat of the phototrophic procaryote,anacystis nidulans,under nitrogen-limiting conditions

Anacystis nidulans was grown photoautotrophically in a chemostat in the presence of light, air and CO2 as the sole carbon source. Either the amount of the nitrogen source in the medium or light intensity were used as growth-limiting parameters. 1. Cells of high glycogen content obtained by pre-incubation under nitrogen starvation conditions maintained their glycogen content during continuous cultivation. Both growth rate and the amount of cell-mass and of glycogen depended on the nitrate content of the medium and the light intensity. The values for the growth rate, the maximal rates of glycogen synthesis and of cell mass formation were 0.1 h-1, 6 mg/l.h and 17 mg/l.h, respectively. 2. Cells without glycogen which had been transferred from an exponentially growing batch culture to chemostat conditions showed increasing rates of growth and of cell mass formation when the light intensity was increased. A determination of specific values resulted in 0.15 h-1 for growth rate and 23 mg/1.h for cell mass formation. 3. The chemostat apparatus is described in detail.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

Formaldehyde production with heat-treated cells of methanol yeast

Chemostat-grown cells of a methanol yeast, Candida boidini S2 AOU-1, were investigated for formaldehyde production. The productivity and catalytic stability were improved by preincubation of the cells at 37°C for 24 h in 0.1 M potassium phosphate buffer (pH 7.5) containing 50 mM NaN₃. These cells produced 1150 mM formaldehyde after a 10-h reaction.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Optimization of lab scale methanol production by <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Methylosinus trichosporium OB3b is a methanotrophic bacterium containing particulate methane monooxygenase (MMO), which catalyzes the hydroxylation of methane to methanol. The methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). We developed a novel compulsory circulation diffusion system for cell cultivation. A methane/air mixture (1:1, v/v) was prepared in a tightly sealed gas reservoir and pumped into a nitrate mineral salt culture medium under optimal conditions (5 μM CuSO₄, pH 7.0, 30°C). Cells were harvested, washed, and resuspended (0.6 mg dry cells/mL) in a 500 mL flask in 100 mL of 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA as MDH inhibitors, and 20 mM sodium formate. A single 12 h batch reaction at 25°C yielded a final concentration of 13.2 mM methanol. The use of a repeated batch mode, in which the accumulated methanol was removed after each of three 8 h cycles over a 24 h period, showed a productivity of 2.17 μmol methanol/h/mg dry cell wt. Finally, a lab-scale reaction performed using a 3 L cylindrical reactor with a working volume of 1 L produced 13.7 mM methanol after 16 h. Our results identify a simple process for improving the productivity of biologically derived methanol and, therefore the utility of methane as an energy source.     

Cyanophycin and glycogen synthesis in a cyanobacterial Scytonema species in response to salt stress

The response to salinity of a Scytonema species isolated from the central Australian desert was studied. Under nitrogen-fixing conditions the addition of increasing concentrations of salt (NaCl) caused progressive inhibition of growth, with growth ceasing at 150 mM NaCl. This correlated with a progressive loss of nitrogenase activity, a low level of activity being retained at 150 mM NaCl. The inhibition of growth was overcome when KNO₃ (10 mM) was added to the growth medium. In response to the salt stress, cells accumulated the reserve compounds cyanophycin and glycogen. Time course experiments showed that they were steadily synthesized over 48 h, after which the concentrations stabilized. Cyanophycin synthesis was enhanced in salt-stressed cells grown in nitrate. When cells were restored to their normal growth medium the content of these substances decreased towards control levels.     

Properties of modified amperometric biosensors based on methanol dehydrogenase and cells Methylobacterium nodulans

The properties of amperometric biosensors based on methanol dehydrogenase (MDH) Methylobacterium nodulans, cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition of hydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135–0.5 and 0.032–1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 μM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.     

Initial microbial adhesion is a determinant for the strength of biofilm adhesion

Abstract A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica , methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.