Hydrogen-using bacteria in a methanogenic acetate enrichment culture

A rcher , D.B. 1984. Hydrogen-using bacteria in a methanogenic acetate enrichment culture. Journal of Applied Bacteriology 56 , 125–129.
In a study of the anaerobic utilization of acetate, an enrichment culture of sewage sludge organisms was initiated with calcium acetate as the sole carbon and energy source. A mixed bacterial population became established from which 14 anaerobic species were isolated. Two of the isolates were methanogenic bacteria but only one of these, Methanosarcina barkeri , utilised acetate as an energy source in axenic culture. The other methanogenic isolate, a Methanobacterium sp., utilised H₂/CO₂ but not acetate. A third methanogen, which was morphologically identical to Methanothrix soehngenii , was detected in the enrichment but was not obtained in monoculture. 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis. completely inhibited the enrichment at a concentration of 10 μmol/1. Addition of H₂ formate or methanol to the enrichment did not affect the rate of methanogenesis. An H₂-utilizing Desulfovibrio sp. was also isolated from the enrichment.     

Random mutagenesis of Legionella pneumophila with mini-Tn10

Abstract The degradation of sheets of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (BIOPOL^®) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2×10⁵ aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8×10³ per ml sludge of a fresh-water lake, 9.2×10⁵ per g garden-soil, 1.3×10⁶ per g field-soil and 4.3×10⁶ per g compost.     

Inhibition of methanogenesis by ethylene and other unsaturated hydrocarbons

Abstract Ethylene (ethene) was found to inhibit methane formation in slurries from sewage sludge and sediment samples taken from freshwater and marine sources. Methane formation from sediment contents was inhibited by 50% at 0.07% ethylene concentration in the gas phase (approx. 5 μmol · 1⁻¹ in the aqueous phase) and by 94% at ≥0.05% ethylene in the gas phase (≥36 μ mol · 1⁻¹ in the aqueous phase). Sulphate reduction was not impaired. Methane formation from added acetate, hydrogen or methanol was inhibited by ≥98%, from lactate by about 90%. The inhibition was reversible, and methanogenic activity recuperated completely after ethylene removal. Cyclopentadiene and cycloheptatriene led to strong inhibition; benzene, toluene, isoprene, and 1-hexine to moderate inhibition of methanogenesis; several unsaturated linear hydrocarbons were without effect. Pure cultures of Methanospirillum hungatei, Methanothrix soehngenii , and Methanosarcina barkeri were all inhibited by 50% at 0.05–0.1% ethylene concentration in the gas phase (3.6–7.2 μmol · 1⁻¹ in the aqueous phase). Pure cultures of Acetobacterium woodii, Halobacterium halobium and Sulfolobus acidocaldarius were not significantly inhibited by either ethylene or acetylene. Ethylene is recommended as a selective inhibitor of methanogenesis for physiological and enrichment experiments with sediment and sludge samples.     

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.     

Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor

Abstract Until now, oxidation of ammonium has only been known to proceed under aerobic conditions. Recently, we observed that NH₄⁺ was disappearing from a denitrifying fluidized bed reactor treating effluent from a methanogenic reactor. Both nitrate and ammonium consumption increased with concomitant gas production. A maximum ammonium removal rate of 0.4 kg N · m⁻³ · d⁻¹ (1.2 mM/h) was observed. The evidence for this anaerobic ammonium oxidation was based on nitrogen and redox balances in continuous-flow experiments. It was shown that for the oxidation of 5 mol ammonium, 3 mol nitrate were required, resulting in the formation of 4 mol dinitrogen gas. Subsequent batch experiments confirmed that the NH₄⁺ conversion was nitrate dependent. It was concluded that anaerobic ammonium oxidation is a new process in which ammonium is oxidized with nitrate serving as the electron acceptor under anaerobic conditions, producing dinitrogen gas. This biological process has been given the name ‘Anammox” (anaerobic ammonium oxidation), and has been patented.     

The influence of pH and growth rate on production of the bacteriocin, bavaricin MN, in batch and continuous fermentations

Bavaricin MN, a bacteriocin produced by Lactobacillus bavaricus MN, reached titres of 2000 AU ml^-1 in APT broth maintained at pH 6.0, 30°C in a batch fermenter. Levels of bavaricin MN at pH 5.5 and 6.5 were lower despite comparable levels of producer cells. The addition of 3.0 g l^-1 beef extract to APT broth resulted in increases in both the growth rate of the culture and the production of bavaracin MN. The titre of bavaricin MN in batch fermenters controlled at pH 6.0 in APT broth plus 3 g l^-1 beef extract reached 3200 AU ml^-1 at 30°C. This level was reduced to 800 AU ml^-1 by 76 h. Glucose-limited continuous culture of Lact. bavaricus MN under the same conditions resulted in an increase in the titre of bavaricin MN to 6400 AU ml^-1. This level was maintained, independent of growth rate, for 345 h. Growth rates of 0.205, 0.118, 0.169 and 0.058 h^-1 were examined.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33°56'N, 102°52'E) region and under the annual temperature around 1°C. Previous studies indicated that Zoige wetland was one of the CH₄ emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales , could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10⁷ cells per gram of soil, accounting for about 30% of the total Archeae . By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO₂, methanol or trimethylamine was determined to increase to 10⁸ cells ml⁻¹, but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30°C and 15°C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae , and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.     

Acid phosphatase production by Aspergillus niger N402A in continuous flow culture

The production of acid phosphatases (E.C.3.1.3.2, ACPs) by Aspergillus niger N402A is regulated by specific growth rate, as well as phosphate availability and pH, as demonstrated by studies in continuous flow culture. Specific ACP activity was highest when A. niger was grown at pH 6.3 (64±8 U g⁻¹) or pH 2.8 (99±11 U g⁻¹), at a dilution rate of 0.07 h⁻¹ and phosphate concentrations below 0.46 mM. ACP production was growth correlated for specific growth rates between 0.07 and 0.13 h⁻¹. Four different ACPs, including two phytases, were produced by A. niger N402A. The ACP and the phytase with maximal activities at pH 5.5 were differentially expressed at different culture pH values, with greater production at low pH.     

Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate

Abstract A defined 3-chlorobenzoate-degrading methanogenic consortium was constructed by recombining key organisms isolated from a 3-chlorobenzoate-degrading methanogenic sludge enrichment. The organisms comprise a three-tiered food chain which includes: (1) reductive dechlorination of 3-chlorobenzoate; (2) oxidation of benzoate to acetate, H₂ and CO₂; (3) removal of H₂ plus CO₂ by conversion into methane. The defined consortium, consisting of a dechlorinating organism (DCB-1), a benzoate degrader (BZ-1) and a lithotrophic methanogen ( Methanospirillum strain PM-1) grew well in a basal salts medium supplemented with 3-chlorobenzoate (3.2 mM) as the sole energy source. The chlorine released from the aromatic ringe was recovered in stoichiometric amounts as the chloride ion. The reducing power required for reductive dechlorination was obtained from the hydrogen produced in the acetogenic oxidation of benzoate. One-third of the benzoate-derived hydrogen was recycled via the reductive dechlorination of 3-chlorobenzoate, indicating that the consortium operated as a food web rather than a food chain.     

Hydrophobicities and Electrophoretic Mobilities of Anaerobic Bacterial Isolates from Methanogenic Granular Sludge

The hydrophobicities and electrophoretic mobilities of isolates from methanogenic anaerobic granular sludge were measured and compared with those of strains from culture collections. All new isolates were highly hydrophobic, indicating that the upflow anaerobic sludge blanket reactor concept selects for hydrophobic bacteria. Methanothrix soehngenii, a methanogen often observed in methanogenic granular sludge, was highly hydrophobic and showed low electrophoretic mobility at pH 7. The role of this strain in the formation of methanogenic granular sludge is discussed.     

Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures.

The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied. The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days. Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor. The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection. Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium. Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent. However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture. In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM. However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated. All experiments were conducted at pH 7.0 to 7.7. The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature. Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid dehalogenation of 2,4,6-trichlorophenol at alkaline pH by an anaerobic enrichment culture

An enrichment culture, derived from the anaerobic stage of a two-step sequential anaerobic-aerobic reactor system which mineralized 2,4,6-trichlorophenol, stoichiometrically converted 2,4,6-trichlorophenol to 4-chlorophenol. Dehalogenation occurred only in alkaline media (pH 8–9) at concentrations of substrate up to 1 mmol 1¹. Formate plus acetate or trypticase could serve as electron donors. Neither vitamins nor trace elements were required in a chloride-free defined medium. The dehalogenating organism was oxygen-resistant, but was not active in media which were oxidized with respect to resazurin indicator dye. Most probable number counts of the dehalogenating cultures showed that the dehalogenating organisms were present in very small numbers, yet catalysed dehalogenation at rates considerably faster than other dehalogenating organisms described in the literature.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

Synergistic mineralization of biphenyl by Alcaligenes faecalis type II BPSI-2 and Sphingomonas paucimobilis BPSI-3

Polychlorinated biphenyls (PCBs) are important environmental pollutants and have been found to have adverse effects on a number of different organisms. Aerobic biodegradation of PCBs occurs through direct oxidation of the biphenyl nucleus. Biphenyl degraders are instrumental in the mineralization of PCBs to CO₂ and water. Here two bacteria, Alcaligenes faecalis type II strain BPSI-2 and Sphingomonas paucimobilis strain BPSI-3, are described that exhibit synergistic mineralization of biphenyl (using ¹⁴C-UL-biphenyl) when grown as a co-culture. Mineralization rates (23·7 and 9·1 nmol nmol⁻¹ h⁻¹, respectively) and extent of mineralization (38·1% and 24·4%, respectively) were significantly different between the strains as well as when compared to the co-culture (35·2 nmol nmol⁻¹ h⁻¹ and 45·2%). Both strains were originally isolated from an enrichment culture, BSEN-2. The co-culture of BPSI-2 and 3 showed a threefold increase in mineralization rate compared with the parent culture and a decrease in the time taken for ¹⁴CO₂ evolution to occur. There was no significant difference in the extent of mineralization between the co-culture and BSEN-2. Examination of enrichment cultures at the community level may play a role in optimizing bioremediation programmes.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Abundance and distribution of anaerobic protozoa and their contribution to methane production in Lake Cadagno (Switzerland)

Abstract In the uupermost layers of the anoxic sediment in Lake Cadagno, 9 different species of anaerobic protozoa were identified. The total number of these organisms was about 580 cells·ml⁻¹ sediment. Most pf these protozoa contained endosymbiotic methanogenic bacteria which in total amounted to 10⁶ methanogens·ml⁻¹ sediment. In addition to the methanogenic endosymbionts, cells of Metopus setosus and Caenomorpha lata also contained a non-fluorescent bacterial rod inside the cytoplasm. In some individual cells of C. lata this second type of endosymbiotic bacterium was sometimes the only endosymbiont observed. Contrary to earlier suggestions, anaerobic protozoa do not seem to play a major role in methane production at least in Lake Cadagno. No significant methane production due to the anaerobic protozoa and their methanogenic endosymbionts was found in situ. Isolated ciliates and amoebae produced methane at 12°C, but not at 6°C, probably as a result of temperature limitation. In the sediment of Lake Cadagno sulfate reduction seemed to be the dominant terminal degradation process.     

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.     

Methanogenesis in the hypersaline Solar Lake (Sinai)

Abstract Enrichment studies on microbial mat sediments (potential stromatolites) from the hypersaline Solar Lake (Sinai) indicated high numbers of methanogenic bacteria (up to 10⁵ ml⁻¹ sediment) in spite of the high sulfate reduction rate, sulfate concentration and salinity. Among H₂/CO₂, acetate and monomethylamine, the methylated amine was the preferred substrate. The predominant species enriched was a Methanosarcina sp. The findings indicate that methanogenic bacteria play an important role in hypersaline sulfate-enriched anoxic sediments and stromatolithic microbial mats.