Physiological adaptations of anaerobic bacteria to low pH: metabolic control of proton motive force in Sarcina ventriculi.

Detailed physiological studies were done to compare the influence of environmental pH and fermentation end product formation on metabolism, growth, and proton motive force in Sarcina ventriculi. The kinetics of end product formation during glucose fermentation in unbuffered batch cultures shifted from hydrogen-acetate production to ethanol production as the medium pH dropped from 7.0 to 3.3. At a constant pH of 3.0, the production of acetate ceased when the accumulation of acetate in the medium reached 40 mmol/liter. At a constant pH of 7.0, acetate production continued throughout the entire growth time course. The in vivo hydrogenase activity was much higher in cells grown at pH 7.0 than at pH 3.0. The magnitude of the proton motive force increased in relation to a decrease of the medium pH from 7.5 to 3.0. When the organism was grown at pH 3.0, the cytoplasmic pH was 4.25 and the organism was unable to exclude acetic acid or butyric acid from the cytoplasm. Addition of acetic acid, but not hydrogen or ethanol, inhibited growth and resulted in proton motive force dissipation and the accumulation of acetic acid in the cytoplasm. The results indicate that S. ventriculi is an acidophile that can continue to produce ethanol at low cytoplasmic pH values. Both the ability to shift to ethanol production and the ability to continue to ferment glucose while cytoplasmic pH values are low adapt S. ventriculi for growth at low pH.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Isolation and characterization of an obligately anaerobic,pectinolytic, member of the genus Eubacterium from mullet gut

A gram positive, motile rod-shaped strictly anaerobic non sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C at pH 6.5 and at a salinity of 10/10³. Out of a variety of mono-, di-, and polysaccharides tested only pectin, cellobiose and starch actively supported growth in either semi defined medium or peptone-yeast extract (PY) medium. Galacturonic acid and maltose were less effective as substrates. Mol product per 100 mol of pectin monomer degraded were: acetate, 163; ethanol, 30; methanol, 88 and formate, 48. Per 100 mol of hexose in cellobiose or starch degraded, the amounts were acetate, 39; ethanol, 128 and formate, 41. Hydrogen was not detectable in the incubations (detection limit, <10^-5 atm) and propionate, butyrate, lactate or succinate were not produced as fermentation end-products (<2 mol per 100 mol monomer). The guanine plus cytosine content of DNA from the bacterium was 31 mol%, and the cell walls contained meso-diaminopimelic acid. A phylogenetic analysis of the organism by 16S rDNA sequencing and DNA-DNA homology indicated that the organism grouped more closely with several species of Clostridium than with Eubacterium. The phenotypic characteristics of the organism indicated that it did not fit within the genus Clostridium and more closely resembled Eubacterium. The organism is therefore designated as a species of Eubacterium; the type strain is P-1 (DSM 6788).     

Effects of o-cresol co-disposal and pH on the methanogenic degradation of refuse

SULISTI, I.A. WATSON-CRAIK AND E. SENIOR. 1996. Both maximum o -cresol degradation and activity of sulphate-reducing bacteria (SRB) were observed at refuse pH values between 7.0 and 8.0. Optimum pH values for methane release were between 6.5 and 7.5. Partial inhibition of methane production was recorded at pH 5.7, 6.0 and 8.0, whilst sulphate reduction was inhibited partially at pH values 5.7–6.5. Both sulphate reduction and methanogenesis were completely inhibited in refuse with initial pH 4.0. The catabolism of acetate occurred under similar conditions to methane production, and was promoted at pH 6.5–7.5. It appeared that propionate oxidation depended upon the activities of SRB. Optimum conditions for the metabolism of propionate and other volatile fatty acids were between pH 7.0 and 8.0.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).     

In Vitro Lactate Metabolism by Ruminal Ingesta

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.     

Selection of a Mixed Culture of Cellulolytic Thermophilic Anaerobes from Various Natural Sources

Microbial associations capable of converting cellulose-containing substrates to ethanol and organic acids were isolated from natural sources. The resulting mixed cultures utilized cellulose, cellobiose, glucose, maize residue, cotton, and flax boon producing ethanol (up to 0.9 g/l) and acetic acid (up to 0.8 g/l). The most complete conversion of cellulose-containing substrates occurred at 60°C and pH 7.0. The selected association of thermophilic anaerobic bacteria produced 0.64 g of ethanol per g substrate utilized at the ethanol/acetate ratio 4.7 : 1.     

Selection of a mixed culture of cellulosolytic thermophilic anaerobes from various natural sources

Microbial associations capable of converting cellulose-containing substrates to ethanol and organic acids were isolated from natural sources. The resulting mixed cultures utilized cellulose, cellobiose, glucose, maize residue, cotton, and flax boon producing ethanol (up to 0.9 g/l) and acetic acid (up to 0.8 g/l). The most complete conversion of cellulose-containing substrates occurred at 60 degrees C, pH 7.0. The selected association of thermophilic anaerobic bacteria produced 0.64 g ethanol per g substrate utilized at the ethanol/acetate ratio 4.7:1.     

Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature

The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 degrees C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was observed. The results were also compared with the 30 degrees C fermentation.     

Staphylococcus aureus Enterotoxin B Release (Excretion) Under Controlled Conditions of Fermentation

Release of Staphylococcus aureus enterotoxin B (SEB) into the culture medium was initiated during the mid-log phase of growth. A medium consisting of 4% N-Z Amine A (Sheffield), 0.2% dextrose, and 1% yeast extract supported maximum production of SEB. Although pH of the medium during cultivation did not significantly affect the growth curve of the organism, the time required for detectable excretion was affected, as was the final yield. Optimal conditions for SEB production were achieved with pH control at 7.0; alkaline control (pH 8.0) produced only minimal amounts of toxin, whereas acid control (pH 6.0) resulted in 50% reduction in yield. Slightly less SEB was produced when there was no extrinsic pH control, and cultures were buffered only by media constituents and by-products of growth. With pH control at 7.0, deletion of 0.2% dextrose from the medium resulted in 40% reduction in the 8-h yield. There was also a delay in production during early stages of fermentation.     

High-rate sulfate reduction at high salinity (up to 90 mS.cm(-1)) in mesophilic UASB reactors

Sulfate reduction in salt-rich wastewaters using unadapted granular sludge was investigated in 0.9 L UASB reactors (pH 7.0 +/- 0.2; hydraulic retention time from 8-14 h) fed with acetate, propionate, or ethanol at organic loading rates up to 10 gCOD x L(-1) x day(-1) and in excess sulfate (COD/SO(4) (2-) of 0.5). High-rate sulfate reduction rates (up to 3.7 gSO(4) (2-) x L(-1).day(-1)) were achieved at salinities exceeding 50 gNaCl.L(-1) and 1 gMgCl(2) x L(-1). Sulfate reduction proceeded at a salinity of up to 70 gNaCl x L(-1) and 1 gMgCl(2) x L(-1) (corresponding to a conductivity of about 85-90 mS x cm(-1)), although at lower rates compared to a conductivity of 60-70 mS x cm(-1). Ethanol as well as propionate were suitable substrates for sulfate reduction, with acetate and sulfide as the end products. The successful high-rate treatment was due to the proliferation of a halotolerant incomplete oxidizing SRB population present in the unadapted inoculum sludge. Bioaugmentation of this sludge with the acetate oxidizing halotolerant SRB Desulfobacter halotolerans was unsuccessful, as the strain washed out from the UASB reactor without colonizing the UASB granules.     

Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values.

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.     

Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Competitive exclusion of Salmonella enterica serovar Enteritidis by Lactobacillus crispatus and Clostridium lactatifermentans in a sequencing fed-batch culture

Competitive exclusion of Salmonella enterica serovar Enteritidis by a mixed culture of Lactobacillus crispatus and Clostridium lactatifermentans was studied in a sequencing fed-batch reactor mimicking the cecal ecophysiology of broiler chickens. Growth of serovar Enteritidis was inhibited by a mixed culture of L. crispatus and C. lactatifermentans at pH 5.8 but not by a monoculture of L. crispatus at the same pH. Moreover, experiments performed at pH 7.0 did not show growth inhibition of serovar Enteritidis. L. crispatus fermented lactose to lactate, and C. lactatifermentans fermented the lactate to acetate and propionate in a mixed culture of L. crispatus and C. lactatifermentans growing on lactose. In contrast, only lactate was produced from lactose by a monoculture of L. crispatus. At pH 5.8 considerable concentrations of acetate and propionate were present as undissociated acids, whereas only trace levels of undissociated lactate were present at pH 5.8 due to the low pK(a) of lactate. At pH 7.0 all three acids were present in their dissociated forms. We conclude that a mixed culture of L. crispatus and C. lactatifermentans inhibits growth of serovar Enteritidis under cecal growth conditions. The undissociated forms of acetate and propionate produced in the mixed culture inhibited the growth of serovar Enteritidis.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Regulation of carbon and electron flow in Propionispira arboris: physiological function of hydrogenase and its role in homopropionate formation

Abstract Hydrogenase activity was characterized in cell extracts of Propionispira arboris that consumed or produced H₂, coupled to methyl viologen reduction, and displayed highest levels (2.6 μmol/min/mg protein) in extracts prepared from fumarate-grown cells. Reversible hydrogenase activity in cell extracts correlated with the production of low levels of hydrogen during the growth phase and its subsequent consumption during the stationary phase of cells grown on glucose or lactate as the carbon and energy source. The addition of exogenous hydrogen to glucose, lactate or fumarate-grown cells dramatically increased propionate production at the expense of acetate formation. This accounted for the formation of propionate as nearly the sole end product of glucose fermentation under two atmospheres of hydrogen. The physiological function of hydrogenase in regulation of carbon and electron flow, and the significance of the results in applied and environmental microbiology are discussed.     

Evidence for involvement of an electron shuttle in electricity generation by Geothrix fermentans

In experiments performed using graphite electrodes poised by a potentiostat (+200 mV versus Ag/AgCl) or in a microbial fuel cell (with oxygen as the electron acceptor), the Fe(III)-reducing organism Geothrix fermentans conserved energy to support growth by coupling the complete oxidation of acetate to reduction of a graphite electrode. Other organic compounds, such as lactate, malate, propionate, and succinate as well as components of peptone and yeast extract, were utilized for electricity production. However, electrical characteristics and the results of shuttling assays indicated that unlike previously described electrode-reducing microorganisms, G. fermentans produced a compound that promoted electrode reduction. This is the first report of complete oxidation of organic compounds linked to electrode reduction by an isolate outside of the Proteobacteria.