• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.42-48
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• 2009

An in vitro incubation study was conducted to investigate effects of 2-bromoethanesulfonic acid (BES) on ruminal fermentation characteristics and methanogen population. BES at the final concentration of 0, 1 and 5 mM with two different substrates having a different ratio of timothy and concentrate (100% timothy vs. 40% timothy-60% concentrate) was incubated for 0, 24, 48 and 72 h in a $39^{\circ}C$ incubator. Total DNA extracted from culture fluid was used as a template for real-time PCR to measure the population of methanogens. Four different primer sets were used for amplification of total bacteria, total methanogens, the order Methanobacteriales and the order Methanomicrobiales. BES reduced (p<0.01) total gas and methane production in a dose-dependent manner. BES at 5 mM inhibited methane production by more than 95% compared to the control. An interaction between substrate and level of BES in total gas and methane was detected (p<0.01). The decrease of methane production with increasing BES level was more pronounced on mixed substrate than on timothy alone. However, hydrogen production was increased by BES treatment (p<0.01). Total VFA concentration was not affected, but molar percentage of propionate and butyrate was increased and acetate to propionate ratio was reduced by BES treatment (p<0.01). BES did not affect the population density of total bacteria but reduced (p<0.01) the population of total methanogens, the order Methanobacteriales and the order Methanomicrobiales in a dose-dependent manner. The type of substrate did not influence the trend, although the magnitude of response was different between all-roughage and 40% roughage substrate.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1464-1472
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• 2014

The microbial community structures of two continuous stirred tank reactors digesting turkey manure with pine wood shavings as well as chicken and swine manure were investigated. The reactor fed with chicken/swine wastes displayed the highest organic acids concentration (up to 15.2 g/l) and ammonia concentration (up to 3.7 g/l ammonium nitrogen) and generated a higher biogas yield (up to $366ml/g_{VS}$) compared with the reactor supplied with turkey wastes (1.5-1.8 g/l of organic acids and 1.6-1.7 g/l of ammonium levels; biogas yield was up to $195ml/g_{VS}$). The microbial community diversity was assessed using both sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Additionally, methanogens were analyzed using methyl coenzyme M reductase alpha subunit (mcrA) genes. The bacterial community was dominated by members of unclassified Clostridiales with the prevalence of specific clostridial phylotypes in each reactor, indicating the effect of the substrate type on the community structure. Of the methanogenic archaea, methanogens of the genus Methanosarcina were found in high proportions in both reactors with specific methanosarcinas in each reactor, whereas the strict hydrogenotrophic methanogens of Methanoculleus sp. were found at significant levels only in the reactor fed with chicken/swine manure (based on the analyses of 16S rRNA gene). This suggests that among methanogenic archaea, Methanosarcina species which have different metabolic capabilities, including aceticlastic and hydrogenotrophic methanogenesis, were mainly involved in anaerobic digestion of turkey wastes.

• Journal of Environmental Science International
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• v.31 no.12
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• pp.1051-1059
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• 2022

This study investigated microbial communities and their diversity in a full-scale mesophilic anaerobic digester treating sewage sludge. Influent sewage sludge and anaerobic digester samples collected from a wastewater treatment plant in Busan were analyzed using high-throughput sequencing. It was found that the microbial community structure and diversity in the anaerobic digester could be affected by inoculation effect with influent sewage sludge. Nevertheless, distinct microbial communities were identified as the dominant microbial communities in the anaerobic digester. Twelve genera were identified as abundant bacterial communities, which included several groups of syntrophic bacteria communities, such as Candidatus Cloacimonas, Cloacimonadaceae W5, Smithella, which are (potential) syntrophic-propionate-oxidizing bacteria and Mesotoga and Thermovigra, which are (potential) syntrophic-acetate-oxidizing bacteria. Lentimicrobium, the most abundant genus in the anaerobic digester, may contribute to the decomposition of carbohydrates and the production of volatile fatty acids during the anaerobic digestion of sewage sludge. Of the methanogens identified, Methanollinea, Candidatus Methanofastidiosum, Methanospirillum, and Methanoculleus were the dominant hydrogenotrophic methanogens, and Methanosaeta was the dominant aceticlastic methanogens. The findings may be used as a reference for developing microbial indicators to evaluate the process stability and process efficiency of the anaerobic digestion of sewage sludge.

• Asian-Australasian Journal of Animal Sciences
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• v.5 no.3
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• pp.511-517
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• 1992

Ethanol was utilized by mixed rumen microbes, but addition of pentachlorophenol (25 mg/l), a methanogen inhibitor, suppressed the utilization of ethanol. Carbon monoxide (50% of the gas phase), a hydrogenase inhibitor, more strongly suppressed the utilization of ethanol, propanol, and butanol. These results suggest that the major ethanol utilizers are $H_2$ producers. Ethanol utilization was depressed at low pH (below 6.0). Since methanogens were shown to be relatively resistant to low pH, it appears that ethanol utilizers are particularly sensitive to low pH. Ruminococcus albus and R. flavefaciens in mono-culture produced ethanol from carbohydrate (glucose and cellobiose), even when a high level (170 mM) of ethanol was present. Ethanol was not utilized even in the absence of carbohydrate, but the co-culture of these bacteria with methanogens resulted in the utilization of ethanol, i.e., when $H_2$ was rapidly converted to $CH_4$, R. albus and R. flavefaciens utilized ethanol. These results suggest that ethanol is utilized when the electrons liberated by the oxidation of ethanol are rapidly removed, and ready electron disposal in ethanol-utilizing, $H_2$-producing bacteria is accomplished by the interspecies transfer of $H_2$.

• Journal of Microbiology
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• v.39 no.3
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• pp.195-201
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• 2001

The effect of moisture content an the reductive dechlorination of polychlorinated biphenyls and population dynamics of dechlorinating microorganisms was investigated in sediments spiked with Aroclor 1248. In sediment slurry with an overlying water layer, dechlorination ensued after a 4-week lag period and reduced the average number of chlorines per biphenyl from 3.91 to 3.15 after 48 weeks. In the sediments of reduced moisture content, however, dechlorination occurred after a lag period of 12 weeks and decreased the average number of chlorines per biphenyl to only 3.62, and the dechlorination rate was also slower. When the population size of dechlorinators, methanogens, and sulfate-reducing bacteria was determined by the most probable number techniques, however, no difference was found between the slurry and the low-moisture sediments, except for methanogens. The growth of dechlorinating populations coincided with the end of the lag period and they then increased by 3 orders of magnitude in two conditions. Specific growth rate of dechlorinators showed little difference between the slurry and the low-moisture sediments; however, growth yield was high in the sediments of reduced moisture content. The reduction of sediment moisture decreased the dechlorination rate and extent of PCBs but did not inhibit the growth of PCB dechlorinators.

• Journal of Korean Society on Water Environment
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• v.23 no.6
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• pp.993-997
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• 2007

The upflow anaerobic sludge blanket (UASB) reactor can be effective for treating simple organic compounds containing high concentration of ammonia nitrogen. The chemical oxygen demand (COD) removal efficiency was about 80% at ammonia nitrogen concentration up to 6,000 mg-N/L. This result also showed that it would be possible to treat propionate effectively at free ammonia nitrogen concentration up to 724 mg-N/L if sufficient time was allowed for adaptation. However the specific methanogenic activity (SMA) of granule was lower than that of granule in the reactor with lower ammonia nitrogen concentration. At 8,000 mg-N/L, the inhibition of high ammonia concentration was observed with evidence of increase of the volatile suspended solids (VSS) concentration in the effluent. It might be ascribed to the decrease in the content of extracellular polymer (ECP), which resulted to the sloughing off of obligated proton-reducing acetogens and heterogenotrophic methanogens from the exterior of granular sludge. This caused a great portion of the finely sludge to be easily washed out. Therefore, failure to maintain the balance between these two groups of microorganism cause accumulation of the hydrogen partial pressure in the reactor, which could have inhibited the growth of acetate utilizing methanogens.

• Journal of Environmental Science International
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• v.30 no.12
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• pp.1093-1100
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• 2021

The present study investigated the effect of ammonia load on microbial communities in mesophilic anaerobic digestion of propionic acid. A laboratory-scale continuous anaerobic digester treating propionic acid as a sole organic substrate was operated under non-inhibitory condition and inhibitory conditions with ammonia (1.5 g and 3.5 g ammonia-N/L, respectively), and bacterial and archaeal communities in the steady states of each ammonia condition were analyzed using high-throughput sequencing. Thirteen bacterial families were detected as abundant bacterial groups in mesophilic anaerobic digestion of propionic acid. Increase in ammonia concentration resulted in significant shifts in microbial community structures. Syntorophobacter, Pelotomaculum, and Thermovigra were determined as the dominant groups of (potential) propionate oxidizing bacteria in the non-inhibitory condition, whereas Cryptanaerobacter and Aminobacterium were the dominant groups of (potential) propionate oxidizing bacteria in the ammonia-inhibitory condition. Methanoculleus and Methanosaeta were the dominant methanogens. Acetate-oxidation coupled with hydrogenotrophic methanogenesis might be enhanced with increases in the relative abundances of Methanoculleus and Tepidanaerobacter acetatoxydans under the ammonia-inhibitory condition. The results of the present study could be a valuable reference for microbial management of anaerobic digestion systems that are exposed to ammonia inhibition and propionic acid accumulation.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1198-1205
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• 2017

Objective: An experiment was conducted to isolate and identify new methanogens in Korea from an anaerobic digester that uses pig slurry. Methods: An anaerobic digestate sample was collected from an anaerobic digester using pig slurry. Pre-reduced media were used for the growth and isolation of methanogens. Growth temperature range, pH range, NaCl concentration range, substrate utilization, and antibiotic tolerance were investigated to determine the physiological characteristics of isolated methanogens. The isolates were also examined microscopically for their morphology and Gram-stained. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification purpose. Results: Four strains, designated KOR-3, -4, -5, and -6, were isolated and were non-motile, irregular coccoid, and 0.5 to $1.5{\mu}m$ in diameter. Moreover, the cell walls of isolated strains were Gram-negative. KOR-3 and KOR-4 strains used acetate for methane production but did not use $H_2+CO_2$, formate, or methanol as a growth substrate KOR-5 and KOR-6 strains utilized acetate, methanol, and trimethylamine for methanogenesis but did not use $H_2+CO_2$ or formate as a growth substrate. The optimum temperature and pH for growth of four strains were $39^{\circ}C$ and 6.8 to 7.2, respectively. The optimum concentration of NaCl for growth of KOR-3, KOR-5, and KOR-6 were 1.0% (w/v). The optimum NaCl concentration for KOR-4 was 0.5% (w/v). All of the strains tolerated ampicillin, penicillin G, kanamycin, streptomycin, and tetracycline; however, chloramphenicol inhibited cell growth. Phylogenetic analysis of 16S rRNA and mcrA genes demonstrated that strains KOR-3, -4, -5, and -6 are related to Methanosarcina mazei (M. mazei, 99% sequence similarity). Conclusion: On the basis of physiological and phylogenetic characteristics, strains KOR-3, -4, -5, and -6 are proposed to be new strains within the genus Methanosarcina, named M. mazei KOR-3, -4, -5, and -6.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.4
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• pp.517-522
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• 2013

The effect on methanogens attached to the surface of rumen ciliate protozoa by the addition of plant extracts (pine needles and ginkgo leaves) was studied with particular reference to their effectiveness for decreasing methane emission. The plant extracts (pine needles and ginkgo leaves) were added to an in vitro fermentation incubated with rumen fluid. The microbial population including bacteria, ciliated-associated methanogen, four different groups of methanogens and Fibrobacter succinogenes were quantified by using the real-time PCR. Gas profiles including methane, carbon dioxide and hydrogen, and runinal fermentation characteristics were observed in vitro. The methane emission from samples with an addition of individual juices from pine needles, ginkgo leaves and 70% ethanol extract from ginko leaves was significantly lower (p<0.05, 27.1, 28.1 and 28.1 vs 34.0 ml/g DM) than that of the control, respectively. Total VFAs in samples with an addition of any of the plant extracts were significantly lower than that of the control (p<0.05) as well. The order Methanococcales and the order Methanosarcinales were not detected by using PCR in any incubated mixtures. The ciliate-associated methanogens population decreased from 25% to 49% in the plant extacts as compared to control. We speculate that the supplementation of juice from pine needles and ginkgo leaves extract (70% ethanol extract) decreased the protozoa population resulting in a reduction of methane emission in the rumen and thus inhibiting methanogenesis. The order Methanobacteriales community was affected by addition of all plant extracts and decreased to less than the control, while the order Methanomicrobiales population showed an increase to more than that of the control. The F. succinogenes, the major fibrolytic microorganism, population in all added plant extracts was increased to greater than that of the control. In conclusion, pine needles and ginkgo leaves extracts appear to have properties that decrease methanogenesis by inhibiting protozoa species and may have a potential for use as additives for ruminants.

• KSBB Journal
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• v.20 no.6
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• pp.431-437
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• 2005

Molecular methods were employed to investigate microorganisms in a thermophilic continuous stirred tank reactor(CSTR) used for continuous $H_2$ production. The reactor was inoculated with heat-treated anaerobic sludge and fed with a glucose-based medium. Denaturing gradient gel electrophoresis showed dynamic changes of bacterial populations in the reactor during 43 days of operation. Gas composition was constant from approximately 14 days but population shift still occurred. Populations affiliated with Fervidobactrium gondwanens and Thermoanaerobacterium thermosaccharolyticum were dominant on 21 and 41 days, respectively. Keeping pH of the medium at 5.0 could suppress methanogenic activity that was detected during initial operation period. $CH_4$ and mcrA detected in the samples obtained from the reactor or inoculum suggested the heat treatment condition employed in this study is not enough to remove methanogens in the inoculum. PCR using primer sets specific to 4 main orders of methanogens suggested that major $H_2$-consuming methanogens in the CSTR belong to the order Methanobacteriales.