Acetogenesis and acetotrophy in a hexanoate-catabolizing microbial association isolated from anoxic landfill

Metabolism of the key intermediates, acetate and hydrogen, in anaerobic hexanoate catabolism by an interacting microbial association isolated from a landfill was examined in the presence of sulphate. Hydrogen (the β-oxidation product of hexanoate and butyrate), was competitively utilized by the component sulphate-reducing bacteria whereas in the absence of sulphate an interaction between H₂-utilizing acetogenic and methanogenic bacteria facilitated H₂ removal, with acetate catabolism restricted to methanogenic bacteria. A possible mechanism for energy conservation was suggested in which excess electrons were stored as acetate for subsequent usage as an energy source.     

SULPHIDE PRODUCTION FROM SULPHATE-ENRICHED SEWAGE SLUDGES

SUMMARY: Sterilized raw sewage sludge enriched with sulphate and inoculated with pure strains of Desulphovibrio desulphuricans produced negligible sulphide. Unsterilized sludge supplemented with 7% (w/v) CaSO₄.2H₂O and inoculated with crude cultures of sulphate-reducing bacteria obtained from sewage yielded 1·0% S^2- (wt S^2- produced as H₂S/vol. of raw sludge) in 6 months at 30°. By repeated subculture more active cultures developed which produced 1% S^2- in 7 days and 1·2–1·9% in 28 days. Digested sludge yielded only 0·1% S^2-. In semicontinuous fermentations at 30°, raw sludge without added sulphate produced 20 times its own volume of gas containing 70% CH₄ and 30% CO₂. When 5% CaSO₄.2H₂O and an active crude culture of sulphate reducers were added, gas production decreased steadily to zero. There were no differences in pH, temperature and redox potential in sludges producing methane or sulphide. The chief cause of inhibition appeared to be the action of sulphide: 0·02% soluble sulphide (S^2-) totally inhibited methane formation; 0·01% S^2- initially decreased gas production by one-quarter but there was a slow recovery to normal, suggesting acclimatization of the methane-producing organisms to sulphide.
Linked fermentations, in which gas from a methane fermentation swept H₂S from a sulphide fermentation, gave a final gas mixture of about 60% CH₄, 30% CO₂ and 5–10% H₂S. The yield of sulphide depended on the rate of sweeping.     

Carbaryl-induced effects on glutathione content, glutathione reductase and superoxide dismutase activity of the cyanobacterium Nostoc muscorum

The carbamate insecticide carbaryl, at concentrations of 10 mg/l and above, significantly stimulated glutathione reductase (GR) and superoxide dismutase (SOD) activity in the cyanobacterium Nostoc muscorum. A low content of total glutathione (GSH + GSSG), decreased photosynthetic activity, and an increased level of H₂O₂ was observed in pesticide treated cyanobacteria. As no glutathione peroxidase was observed in this species, stimulation of GR and SOD activity, higher production of H₂O₂, and low glutathione level was attributed to the utilization of GSH to remove H₂O₂ spontaneously and nonenzymatically under conditions of pesticide toxicity.     

Characterization of populations of aerobic hydrogen-oxidizing soil bacteria

Abstract Freshly isolated soil bacteria were screened for different characteristics of the H₂ metabolism without prior selection for growth on H₂. The bacteria were isolated from different grain size fractions of a neutral meadow cambisol and an acidic forest cambisol, and then tested (1) for the ability to oxidize H₂, (2) for chemolithoautotrophic growth on H₂ as sole electron donor and energy source, (3) for DNA-DNA-hybridization with two hydrogenase gene fragments from Alcaligenes eutrophus and Rhizobium leguminosarum , and (4) for reduction of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC) in the presence of H₂. Many (65–90%) of the isolates were able to reduce TTC, but only 30–65% were actually able to oxidize H₂ indicating that the TTC test was not a specific characteristic for H₂ oxidation ability. The TTC test was only reliable in pure cultures of known bacteria with optimized test conditions, here shown for Alcaligenes eutrophus, Bradyrhizobium japonicum and Nocardia opaca , but not in mixed cultures of unknown bacteria. Still less (< 30%) of the isolates were able to grow chemolithoautotrophically indicating that culturable aerobic bacteria with the ability for H₂ oxidation are more abundant than bacteria with the ability for chemolithoautotrophic growth. The DNA-DNA-hybridization test failed to detect many of the bacteria with H₂ oxidation activity, probably since the hydrogenase genes present in the isolates were too diverse to be all detected by the DNA probes applied.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .     

H2 and CO2 production from methanol or formaldehyde by the methanogenic bacterium strain Gö1 treated with 2-bromoethanesulfonic acid

Abstract In cell suspensions of the methanogenic bacterium strain Gö1 or Methanosarcina barkeri H₂ formation from methanol in the presence of 2-bromoethanesulfonic acid (BES) was strictly dependent on sodium ions; apparent K _S for Na⁺, 1.3±0.3 mM.H₂ formation was inhibited by the uncoupler tetrachlorosalicylanilide (TCS), but this inhibition could be temporarily overcome, when a sodium pulse (100 mM) was given to the cell suspension. On the other hand, H₂ formation from formaldehyde in the presence of BES (rate: 300 nmol H₂/h·mg protein as compared to 25 nmol H₂/h·mg protein from methanol) was not sodium-dependent, not TCS-sensitive and not inhibited by addition of monensin. H₂ formation was accompanied by CO₂ formation in stoichiometric amounts, 3 H₂:1 CO₂ for methanol and 2 H₂:1 CO₂ for formaldehyde oxidation.     

The presence of oxygen in rumen liquor and its effects on methanogenesis

In situ measurement of O₂ in the rumen liquor of cows, sheep and goats using a membrane-covered O₂ electrode revealed the presence of up to 1630 nmol/l O₂; O₂ became undetectable immediately after feeding of animals. The effects of O₂ on H₂ production and methanogenesis in samples of rumen liquor were investigated using a mass spectrometer fitted with a membrane inlet system. Methanogenesis was totally and irreversibly inhibited after short term exposure (about 10 min) to 5 KPa (0·05 atm) O₂; H₂ production was unaffected. Glucose additions produced rapid transient increases in H₂ levels and increased O₂ uptake.     

Behaviour of Nif− mutants of Rhodobacter capsulatus in photosynthetic, ammonia-limited chemostat cultures

Abstract Nif⁻ mutants of Rhodobacter capsulatus carrying mutations either in the nifR4 regulatory gene or in the nifH structural gene both outgrew the wild-type strain B10 in mixed chemostat cultures under conditions favouring nitrogenase-mediated H₂ production by the wild-type (ammonia as limiting nutrient, inert argon atmosphere, light as energy source), whereas under aerobic conditions in the dark, or in batch culture, the growth of Nif⁻ mutants was not favoured. Nitrogenase-mediated H₂ production therefore appears to be detrimental to the growth of R. capsulatus in nitrogen-limited continuous culture, as may also be the case for other nitrogen fixers.     

On the screening of hydrogen peroxide-generating lactic acid bacteria

The published plate methods for the detection of hydrogen peroxide-producing lactic acid bacteria, which employ horseradish peroxidase and a chromogen, clearly fail to detect all the organisms that produce H₂O₂. Whilst keeping the same principle, the use of a novel growth medium and of the chromogen tetramethyl-benzidine allows the detection of H₂O₂ production by strains previously classified as non-producers.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

Most probable number enumeration of H2-utilizing acetogenic bacteria from the digestive tract of animals and man

Abstract A method is proposed that allows the enrichment and most probable number estimation of H₂/CO₂-utilizing acetogenic bacteria. It is based on the difference in acetate production for serial dilutions incubated under either a test H₂/CO₂ (4:1), or a control N₂/CO₂ (4:1) headspace atmosphere. A nutritionally non-selective medium was used, containing bromoethane-sulfonic acid as inhibitor of methanogenic archaea and 10% pre-incubated clarified rumen fluid. Acetogenic bacteria were enumerated in rumen and hindgut contents of animals and in human feces. They ranged from below 10² to above 10⁸ per gram wet weight gut content and their population levels were the highest in the absence of methanogenesis. The method described therein should prove useful to better understand the diversity and ecological importance of dominant gut acetogens.     

Sulphate reduction in oxic and sub-oxic North-East Atlantic sediments

Abstract Oxic and sub-oxic N.-E. Atlantic sediments were examined for sulphate-reducing activity. Oxygen and/or nitrate reduction are probably the dominant mineralisation processes in the abyssal plain sediment studied. A low rate of sulphate reduction (0.1 nmol SO²⁻₄/ml/day) was recorded in the surface 5 cm of the continental slope sediment, together with the presence of a range of sulphate-reducing bacteria (SRB). A higher activity of sulphate reduction (2.2 nmol SO²⁻₄/ml/day) occurred in the continental shelf sediment which led to a small decrease in pore water sulphate and an increase in titration alkalinity. This sediment contained approx. 10²–10³ acetate, lactate and propionate oxidising SRB/ml. No low- M _r organic acids were detected in these sediments. However, amendment with 75 μM acetate stimulated sulphate-reducing activity in the shelf sediment.     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Abstract The effect of temperature on CH₄ production, turnover of dissolved H₂, and enrichment of H₂-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H₂/CO₂, rates of CH₄ production increased 25°C, but decreased at temperatures lower than 20°C. Chloroform completely inhibited methano-genesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H₂, especially at low incubation temperatures. Cultures with H₂ as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacteria whenever incubation temperatures were lower than 20°C. Hydrogenotrophic methanogens could only be enriched at 30°C from anoxic paddy soil. A homoacetogen     

The metabolism of hydrogen by extremely thermophilic, sulfur-dependent bacteria

Abstract In just the last few years, a group of bacteria have been discovered that have the remarkable property of growing near and above 100°C. These extremely thermophilic organisms, defined here as having the ability to grow at 90°C with optimum growth at 80°C and above, have been isolated mainly from sulfur-rich, marine geothermal environments, both shallow and deep sea. They comprise over a dozen different genera, and except for one novel eubacterium, all may be classified as archaebacteria. The majority of the extremely thermophilic genera metabolize elemental sulfur (S°) and a survey of the various organisms reveals that most of them also depend upon the oxidation of hydrogen gas (H₂) as an energy source. In addition, two extremely thermophilic genera are known that actively produce H₂ as end-products of novel fermentative metabolisms. The enzyme hydrogenase, which is responsible for catalysing H₂ activation and H₂ production, appears to play several roles in electron and energy transfer during the growth of these organisms. Hydrogenase has so far been purified from only one extremely thermophilic species, from Pyrococcus furiosus ( T _opt = 100°C), and hydrogenase activity has been exmained in cell-free extracts of only a few others. However, a comparison of their properties with those of hydrogenases from mesophilic bacteria suggests that (a) the hydrogenase responsible for catalysing H₂ oxidation in extremely thermophilic organisms may be an extremely thermostable version of the mesophilic enzyme, and (b) a new type of 'evolution' hydrogenase, lacking the Ni-S or Fe-S catalytic sites of the mesophilic enzymes, is required for catalysing H₂ evolution at temperatures near and above 100°C.     

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Abstract Interspecies H₂ transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of ¹⁴CO₂ to ¹⁴CH₄ in anoxic paddy soil. Only 3–5% of the ¹⁴CH₄ were produced from the turnover of dissolved H₂. The H₂-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H₂-dependent methanogenesis and the turnover of dissolved H₂ did not change significantly for up to 7 months of incubation. However, while the rates of H₂-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H₂-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H₂ by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH₄ production was almost completely restricted to H₂-syntrophic MBA. The activity of MBA, as measured by the conversion of ¹⁴CO₂ to ¹⁴CH₄, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H₂. Propionate and acetate had no effect.