Effect of inorganic sulfide on the growth and metabolism of Methanosarcina barkeri strain DM.

Minimal growth of Methanosarcina barkeri strain DM occurred when sulfide was omitted fromthe growth medium, and addition of either sodium sulfate or coenzyme M to sulfide-depleted media failed to restore growth. Optimal growth occurred in the presence of 1.25 mM added sulfide, giving a molar growth yield (YCH4) of 4.4 mg (dry weight) of cells per mmol of CH4 produced. Increasing sulfide to 12.5 mM led to decrease in YCH4 (1.9 mg [dry weight]/mmol of CH4), in the specific growth rate and in be intracellular levels of adenosine triphosphate. However, the specific rate of methane production increased. The data suggested that at elevated sulfide levels (12.5 mM) the decrease in YCH4 might be a result of an increase in the relative energy needed for maintnenace and of uncoupling of growth from energy production.     

Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge.

Tests were made to determine the effects of inorganic and organic sulfur sources on the degradation of cellulose to methane in a chemically defined medium with sulfur-poor inoculum prepared from sewage sludge. The results show that a sulfur source of about a 0.85 mM concentration is essential for the degradation of cellulose to CH4. However, the production of CH4 from CO2 and H2 provided in the headspace occurred with 0.1 mM sulfate or sulfide. At a 9 mM concentration, all inorganic sulfur compounds other than sulfate inhibited both cellulose degradation and methane formation, and this inhibition increased in the order thiosulfate less than sulfite less than sulfide less than H2S. It appears that the degradation of cellulose to CH4 in a sulfate-free medium by inoculum maintained in a low-sulfur medium is inhibited because of the lack of availability of sulfur for growth of bacteria and synthesis of cell materials and sulfur-containing cofactors involved in cellulose degradation and methanogenesis. The reduction of methanogenesis by higher levels of sulfate probably occurs as a result of stimulation of reactions converting acetate and H2 to end products other than CH4.     

Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge

Tests were made to determine the effects of inorganic and organic sulfur sources on the degradation of cellulose to methane in a chemically defined medium with sulfur-poor inoculum prepared from sewage sludge. The results show that a sulfur source of about a 0.85 mM concentration is essential for the degradation of cellulose to CH4. However, the production of CH4 from CO2 and H2 provided in the headspace occurred with 0.1 mM sulfate or sulfide. At a 9 mM concentration, all inorganic sulfur compounds other than sulfate inhibited both cellulose degradation and methane formation, and this inhibition increased in the order thiosulfate less than sulfite less than sulfide less than H2S. It appears that the degradation of cellulose to CH4 in a sulfate-free medium by inoculum maintained in a low-sulfur medium is inhibited because of the lack of availability of sulfur for growth of bacteria and synthesis of cell materials and sulfur-containing cofactors involved in cellulose degradation and methanogenesis. The reduction of methanogenesis by higher levels of sulfate probably occurs as a result of stimulation of reactions converting acetate and H2 to end products other than CH4.     

Effect of amino acids on the heat production and growth efficiency of Streptococcus bovis: balance of anabolic and catabolic rates.

Streptococcus bovis JB1 grew nearly twice as fast (0.9 versus 1.6 h-1) and had a 40% greater growth yield (18 versus 12.5 mg of protein per mmol of glucose) when an ammonia-based medium was supplemented with amino acids, but the glucose consumption rate (88 mumol mg of protein-1 h-1) and specific rate of heat production (2.1 mW/mg of protein) were unaffected. Amino acid availability had little effect on the catabolic rate, but the specific heat decreased 40% (8.8 to 5.2 J/mg of protein). These growth rate-dependent changes in metabolic efficiency were fivefold greater than the maintenance energy. Chloramphenicol (100 mg/l), an inhibitor of protein synthesis, caused a gradual decrease in anabolic (growth) rate, but there was little change in the rate of glucose consumption and the specific heat increased. When growth was inhibited by iodoacetate, the catabolic and anabolic rates both declined and there was not increase in specific heat. On the basis of these results, the benefit of amino acid supplementation was largely explained by the balance of anabolic and catabolic rates. When amino acids were available, the anabolic and catabolic rates were more closely matched and less energy was spilled as heat.     

正交设计法优化三岛柴胡愈伤组织培养基

目的:优化三岛柴胡愈伤组织培养条件。方法:采用正交设计法,以相对生长速率、鲜重和干重为考察指标,对三岛柴胡愈伤组织生长的MS培养基成分进行单因素多水平和多因素多水平考察。结果:最佳培养基为:MS+3%蔗糖+10/40mmol.L-1NH4+/NO3-+3.75mmol.L-1HPO24-+NAA(0.005mg.ml-1)+6-BA(0.010mg.ml-1)。最佳培养条件下,愈伤组织相对生长速率为0.1548,干重达0.1668g.瓶-1。结论:2,4-D与NAA配合使用可以促进三岛柴胡愈伤组织快速生长,L9(34)正交设计实验中愈伤组织干重明显高于单因子实验设计的。     

Coupling of Methanothermobacter thermautotrophicus methane formation and growth in fed-batch and continuous cultures under different H2 gassing regimens

In nature, H2- and CO2-utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth, Methanothermobacter thermautotrophicus was cultured in a fed-batch fermentor and in a chemostat under different 80% H(2)-20% CO2 gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (pH2). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (YCH4 max), notably approximately 3 and 7 g (dry weight) of methane formed mol-1, for growth under low (pH2 < 12 kPa)- and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the "specific maintenance" requirements or, more likely, the degree of proton leakage and proton slippage processes. At low pH2 values, the "specific maintenance" diminished and the specific growth yields approached YCH4 max, indicating that growth and methanogenesis became fully coupled.     

Vancomycin production in batch and continuous culture

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Y(p/x)) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h(-1)), specific vancomycin production rate (q(vancomycin)) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, q(vancomycin) was a function of specific growth rate; the maximum value was observed at D = 0.087 h(-1) (52% of the maximum specific growth rate). Under phosphate limited growth conditions, q(vancomycin) was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). (c) 1996 John Wiley & Sons, Inc.     

Transport of coenzyme M (2-mercaptoethanesulfonic acid) in Methanobacterium ruminantium.

A system for transport of coenzyme M, 2-mercaptoethanesulfonic acid (HS--CoM), in Methanobacterium ruminatium strain M1 required energy, showed saturation kinetics, and concentrated the coenzyme against a gradient. The process was sensitive to temperature and was maximally active at pH 7.1. Cells took up HS--CoM at a linear rate, with a Vmax of 312 pmol/min per mg (dry weight) and an apparent Km of 73 nM. An intracellular pool of up to 5 mM accumulated which was not exchangeable with the medium. Uptake required both hydrogen and carbon dioxide; it was inhibited by O2. Bromoethanesulfonic acid (BrCH2CH2SO3-), a potent inhibitor of methanogenesis in cell-free extracts, inhibited both uptake and methane production. Results of inhibitor studies with derivatives and analogs of the coenzyme showed that the specificity of the carrier is restricted to a limited range of thioether, thioester, and thiocarbonate derivatives. 2-(Methylthio)ethanesulfonic acid (CH3--S--CoM) showed an apparent Ki for HS--CoM uptake of 15 nM, being taken up itself with a Vmax of 320 pmol/min per mg (dry weight) and an apparent Km of 50 nM. An analysis of intracellular pools after HS--CoM uptake indicated that the predominant forms are a heterodisulfide of unknown composition and CH3--S--CoM.     

pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1

Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight(-1) h(-1), whereas 18.5 g L(-1) cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility.     

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.     

Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation.

A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Bock, A. Prieger-Kraft, and P. Schönheit, Arch. Microbiol. 161:33-46, 1994). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonate (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Yacetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis. Dicyclohexylcarbodiimide, an inhibitor of H(+)-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethenogenic conditions contained pyruvate: ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosphorylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.     

Growth kinetics of the photosynthetic bacterium Chlorobium thiosulfatophilum in a fed-batch reactor

Hydrogen sulfide dissolved in water can be converted to elementary sulfur or sulfate by the photosynthetic bacterium Chlorobium thiosulfatophilum. Substrate inhibition occurred at sulfide concentrations above 5.7 mM. Light inhibition was found at average light intensities of 40,000 lux in a sulfide concentration of 5 mM, where no substrate inhibition occurred. Light intensity, the most important growth parameter, was attenuated through both scattering by sulfur particles and absorption by the cells. Average cell and sulfur particle sizes were 1.1 and 9.4 mum, respectively. Cells contributed 10 times as much to the turbidity as sulfur particles of the same weight concentration. The light attenuation factor was mathematically modeled, considering both the absorption and scattering effects based on the Beer-Lambert law and the Rayleigh theory, which were introduced to the cell growth model. Optimal operational conditions relating feed rate vs. light intensity were obtained to suppress the accumulation of sulfate and sulfide and save light energy for 2- and 4-L fed-batch reactors. Light intensity should be greater for the same performance (H(2)S removal rate/unit cell concentration) in larger reactors due to the scaleup effect on light transmission. Knowledge of appropriate growth kinetics in photosynthetic fed-batch reactors was essential to increase feed rate and light intensity and therefore cell growth. A mathematical model was developed that describes the cell growth by considering the light attenuation factor due to scattering and absorption and the crowding effect of the cells. This model was in good agreement with the experimental results. (c) 1992 John Wiley & Sons, Inc.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.     

Modeling reduction of uranium U(VI) under variable sulfate concentrations by sulfate-reducing bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 +/- 3 degrees C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V(max), ranged from 2.4 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB. h(-1)) at 0.25 mM sulfate to 5.0 +/- 1.1 micromol of sulfate/mg (dry weight) of SRB. h(-1) at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V(max) was 1.6 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB. h(-1) at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 +/- 0.003 mg (dry weight) of cells/ml. min(-1) for the mixed culture and 0.137 +/- 0.016 mg (dry weight) of cells/ml. min(-1) (U(0) = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Simultaneous methanogenesis and oxygen reduction by Methanobrevibacter cuticularis at low oxygen fluxes

Methanogenic Archaea are often encountered in habitats that are not entirely anoxic in space or time. Recent biochemical and genomic studies have revealed the capacity of methanogens to reduce molecular oxygen. O(2) reduction by Methanobrevibacter species was investigated. Cell suspensions incubated in agar tubes under a headspace of H(2)-CO(2) and increasing concentrations of O(2) formed a distinct growth band, which coincided with the oxic-anoxic interface and indicated that the influx of O(2) into the band was balanced by its consumption. However, in batch cultures methanogenesis ceased as soon as traces of O(2) were added. Focusing on Methanobrevibacter cuticularis, a species colonizing the microoxic gut epithelium of termites, a diffusion-limited setup was used that allowed the exposure of dense cell suspensions to controlled O(2) fluxes. Here, Methanobrevibacter cuticularis was capable of simultaneous CH(4) production and O(2) consumption. Low O(2) fluxes (10% of the CH(4) production rate) had virtually no influence on methanogenesis [4.5 micromol CH(4) (mg dry wt)(-1) h(-1)], whereas higher O(2) fluxes (up to 30% of the initial CH(4) production rate) caused a reversible decrease in methanogenesis, which was accompanied by a reversible, partial conversion of coenzyme F(420) to factor F(390). The maximum O(2) reduction rate [4.8 micromol O(2) (mg dry wt)(-1) h(-1)] that could be maintained over extended time periods (>30 min) was similar to the CH(4) production rate under anoxic conditions.     

Effect of sulfide on growth of marine bacteria

Severe hypoxia leads to excess production of hydrogen sulfide in marine environments. In this study, we examined the effect of sulfide on growth of four facultative anaerobic marine bacteria in minimal media under anaerobic conditions. The Gram-negative chemolithoautotrophic Marinobacter sp. tolerated sulfide concentrations up to 0.60 mM, with doubling and lag times increasing as a function of increasing sulfide concentration but with no change in maximum culture yields; growth did not occur at 1.2 mM sulfide. Similar results were obtained for the metabolically diverse Gram-negative denitrifying Pseudomonas stutzeri, except that growth occurred at 1.2 mM and culture yields at 0.60 and 1.2 mM sulfide were approximately 10-fold lower than at sulfide concentrations between 0 and 0.30 mM. Increases in doubling and lag times accompanied by an overall 10-fold decrease in maximum culture yields were found for the Gram-negative chemoheterotrophic Vibrio sp. at all sulfide concentrations tested. In contrast, growth of a Gram-positive chemoheterotrophic Bacillus sp. was resistant to all sulfide concentrations tested (0.15–1.2 mM). Our results highlight the variable responses of marine bacteria to sulfide and provide some insight into shifts that may occur in microbial community structure and diversity as a consequence of changes in sulfide levels that are the result of hypoxia.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.