High levels of ammonia and the presence of sulphide have major impacts on microbial communities a... more High levels of ammonia and the presence of sulphide have major impacts on microbial communities and are known to cause operating problems in anaerobic degradation of protein-rich material. Operating strategies that can improve process performance in such conditions have been reported. The microbiological impacts of these are not fully understood, but their determination could help identify important factors for balanced, efficient operation. This study investigated the correlations between microbial community structure, operating parameters and digester performance in high-ammonia conditions. Continuous anaerobic co-digestion of household waste and albumin was carried out in laboratory-scale digesters at high ammonia concentrations (0.5-0.9 g NH3/L). The digesters operated for 320 days at 37 or 42 °C, with or without addition of a trace element mixture including iron (TE). Abundance and composition of syntrophic acetate-oxidising bacteria (SAOB) and of methanogenic and acetogenic communities were investigated throughout the study using 16S rRNA and functional gene-based molecular methods. Syntrophic acetate oxidation dominated methane formation in all digesters, where a substantial enhancement in digester performance and influence on microbial community by addition of TE was shown dependent on temperature. At 37 °C, TE addition supported dominance and strain richness of Methanoculleus bourgensis and altered the acetogenic community, whereas the same supplementation at 42 °C had a low impact on microbial community structure. Both with and without TE addition operation at 42 °C instead of 37 °C had low impact on digester performance, but considerably restricted acetogenic and methanogenic community structure, evenness and richness. The abundance of known SAOB was higher in digesters without TE addition and in digesters operating at 42 °C. No synergistic effect on digester performance or microbial community structure was observed on combining increased temperature with TE addition. Our identification of prominent populations related to enhanced performance within methanogenic (high dominance and richness of M. bourgensis) and acetogenic communities are valuable for continued research and engineering to improve methane production in high-ammonia conditions. We also show that a temperature increase of only 5 °C within the mesophilic range results in an extreme dominance of one or a few species within these communities, independent of TE addition. Furthermore, functional stable operation was possible despite low microbial temporal dynamics, evenness and richness at the higher temperature.
Changes in the acetogenic population were investigated in an experimental laboratory-scale biogas... more Changes in the acetogenic population were investigated in an experimental laboratory-scale biogas reactor (37°C) subjected to gradually elevated ammonia levels (0.8 to 6.9 g NH(4)(+)-N L(-1)). A shift from aceticlastic acetate degradation to syntrophic acetate oxidation had previously been confirmed in this reactor. In a parallel control reactor, operating at constant ammonia levels (0.65-0.90 g NH(4)(+)-N L(-1)), acetate degradation proceeded via the aceticlastic pathway throughout the operating period (660 d). The acetogenic populations in the reactors were analysed using degenerated primers designed to target the functional gene encoding a key enzyme of the acetyl-CoA pathway, 10-formyltetrahydrofolate synthetase (FTHFS). The analysis consisted of terminal restriction fragment length polymorphism (T-RFLP) analysis coupled with the construction of clone libraries, and quantitative PCR (qPCR) analysis. The T-RFLP data obtained were statistically analysed by non-metric multidimensional scaling. The most abundant FTHFS genes recovered in the clone libraries were assigned to terminal restriction fragments of the T-RFLP profile. The results of the investigation clearly indicated that increased ammonia concentration substantially influenced the putative acetogenic population structure and caused two distinct shifts of the most abundant members; however, the identity of the dominating species remains unknown, as none of the genes had been identified previously. Despite the shifts in the population, the qPCR analysis revealed a relatively stable abundance of the acetogenic population throughout the operation.
Four anaerobic syntrophic acetate-oxidizing bacteria, the thermotolerant strains Re1(T), Re2, T1 ... more Four anaerobic syntrophic acetate-oxidizing bacteria, the thermotolerant strains Re1(T), Re2, T1 and T2, were isolated from two different mesophilic methanogenic systems. The strains originate from sludge of a continuously stirred laboratory-scale reactor containing high levels of ammonium and from a high ammonium enrichment culture. Comparative 16S rRNA gene sequence analysis confirmed that the strains belong to the Firmicutes-Clostridia class. The most closely related species to strains Re1(T), Re2, T1 and T2 was Tepidanaerobacter syntrophicus, with a 16S rRNA gene sequence identity of 96%. The DNA-DNA relatedness of strains Re2, T1 and T2 to strain Re1(T) was 92, 102, 81%, respectively. The gene encoding the acetogen key enzyme formyltetrahydrofolate synthetase (FTHFS) was detected and partly sequenced from the strains. In pure culture the bacteria used different organic compounds as carbon and energy source, such as organic acids, alcohols, sugars and amino acids. Furthermore, acetate-oxidizing ability was observed during co-cultivation with a hydrogen-consuming Methanoculleus sp. The bacteria were found to be spore-forming, rod-shaped and motile, and possessed Gram-positive cell walls. The four strains were thermotolerant and grew at temperatures between 25 and 55°C. Strain Re1(T) had a DNA G+C content of 38.4% and the major fatty acids were C(18:1) w7c, C(18:1) w9c, anteiso-C(17:0), C(16:1) w7c and C(18:0). The genetic and phenotypic properties of strains Re1(T), Re2, T1 and T2 suggest classification as representatives of a novel species of the genus Tepidanaerobacter; the name Tepidanaerobacter acetatoxydans sp. nov. is suggested. The type strain of T. acetatoxydans is Re1(T) (=DSM 21804(T)=JCM 16047(T)).
The importance of syntrophic acetate oxidation for process stability in methanogenic systems oper... more The importance of syntrophic acetate oxidation for process stability in methanogenic systems operating at high ammonia concentrations has previously been emphasized. In this study we investigated bioaugmentation of syntrophic acetate-oxidizing (SAO) cultures as a possible method for decreasing the adaptation period of biogas reactors operating at gradually increased ammonia concentrations (1.5 to 11 g NH(4)(+)-N/liter). Whole stillage and cattle manure were codigested semicontinuously for about 460 days in four mesophilic anaerobic laboratory-scale reactors, and a fixed volume of SAO culture was added daily to two of the reactors. Reactor performance was evaluated in terms of biogas productivity, methane content, pH, alkalinity, and volatile fatty acid (VFA) content. The decomposition pathway of acetate was analyzed by isotopic tracer experiments, and population dynamics were monitored by quantitative PCR analyses. A shift in dominance from aceticlastic methanogenesis to SAO occurred simultaneously in all reactors, indicating no influence by bioaugmentation on the prevailing pathway. Higher abundances of Clostridium ultunense and Tepidanaerobacter acetatoxydans were associated with bioaugmentation, but no influence on Syntrophaceticus schinkii or the methanogenic population was distinguished. Overloading or accumulation of VFA did not cause notable dynamic effects on the population. Instead, the ammonia concentration had a substantial impact on the abundance level of the microorganisms surveyed. The addition of SAO culture did not affect process performance or stability against ammonia inhibition, and all four reactors deteriorated at high ammonia concentrations. Consequently, these findings further demonstrate the strong influence of ammonia on the methane-producing consortia and on the representative methanization pathway in mesophilic biogas reactors.
Changes in communities of syntrophic acetate-oxidizing bacteria (SAOB) and methanogens caused by ... more Changes in communities of syntrophic acetate-oxidizing bacteria (SAOB) and methanogens caused by elevated ammonia levels were quantified in laboratory-scale methanogenic biogas reactors operating at moderate temperature (37°C) using quantitative polymerase chain reaction (qPCR). The experimental reactor was subjected to gradually increasing ammonia levels (0.8-6.9 g NH4 (+) -N l(-1) ), whereas the level of ammonia in the control reactor was kept low (0.65-0.90 g NH4 (+) -N l(-1) ) during the entire period of operation (660 days). Acetate oxidation in the experimental reactor, indicated by increased production of (14) CO2 from acetate labelled in the methyl carbon, occurred when ammonia levels reached 5.5 and 6.9 g NH4 (+) -N l(-1) . Syntrophic acetate oxidizers targeted by newly designed qPCR primers were Thermacetogenium phaeum, Clostridium ultunense, Syntrophaceticus schinkii and Tepidanaerobacter acetatoxydans. The results showed a significant increase in abundance of all these bacteria except T. phaeum in the ammonia-stressed reactor, coincident with the shift to syntrophic acetate oxidation. As the abundance of the bacteria increased, a simultaneous decrease was observed in the abundance of aceticlastic methanogens from the families Methanosaetaceae and Methanosarcinaceae. qPCR analyses of sludge from two additional high ammonia processes, in which methane production from acetate proceeded through syntrophic acetate oxidation (reactor SB) or through aceticlastic degradation (reactor DVX), demonstrated that SAOB were significantly more abundant in the SB reactor than in the DVX reactor.
High levels of ammonia and the presence of sulphide have major impacts on microbial communities a... more High levels of ammonia and the presence of sulphide have major impacts on microbial communities and are known to cause operating problems in anaerobic degradation of protein-rich material. Operating strategies that can improve process performance in such conditions have been reported. The microbiological impacts of these are not fully understood, but their determination could help identify important factors for balanced, efficient operation. This study investigated the correlations between microbial community structure, operating parameters and digester performance in high-ammonia conditions. Continuous anaerobic co-digestion of household waste and albumin was carried out in laboratory-scale digesters at high ammonia concentrations (0.5-0.9 g NH3/L). The digesters operated for 320 days at 37 or 42 °C, with or without addition of a trace element mixture including iron (TE). Abundance and composition of syntrophic acetate-oxidising bacteria (SAOB) and of methanogenic and acetogenic communities were investigated throughout the study using 16S rRNA and functional gene-based molecular methods. Syntrophic acetate oxidation dominated methane formation in all digesters, where a substantial enhancement in digester performance and influence on microbial community by addition of TE was shown dependent on temperature. At 37 °C, TE addition supported dominance and strain richness of Methanoculleus bourgensis and altered the acetogenic community, whereas the same supplementation at 42 °C had a low impact on microbial community structure. Both with and without TE addition operation at 42 °C instead of 37 °C had low impact on digester performance, but considerably restricted acetogenic and methanogenic community structure, evenness and richness. The abundance of known SAOB was higher in digesters without TE addition and in digesters operating at 42 °C. No synergistic effect on digester performance or microbial community structure was observed on combining increased temperature with TE addition. Our identification of prominent populations related to enhanced performance within methanogenic (high dominance and richness of M. bourgensis) and acetogenic communities are valuable for continued research and engineering to improve methane production in high-ammonia conditions. We also show that a temperature increase of only 5 °C within the mesophilic range results in an extreme dominance of one or a few species within these communities, independent of TE addition. Furthermore, functional stable operation was possible despite low microbial temporal dynamics, evenness and richness at the higher temperature.
Changes in the acetogenic population were investigated in an experimental laboratory-scale biogas... more Changes in the acetogenic population were investigated in an experimental laboratory-scale biogas reactor (37°C) subjected to gradually elevated ammonia levels (0.8 to 6.9 g NH(4)(+)-N L(-1)). A shift from aceticlastic acetate degradation to syntrophic acetate oxidation had previously been confirmed in this reactor. In a parallel control reactor, operating at constant ammonia levels (0.65-0.90 g NH(4)(+)-N L(-1)), acetate degradation proceeded via the aceticlastic pathway throughout the operating period (660 d). The acetogenic populations in the reactors were analysed using degenerated primers designed to target the functional gene encoding a key enzyme of the acetyl-CoA pathway, 10-formyltetrahydrofolate synthetase (FTHFS). The analysis consisted of terminal restriction fragment length polymorphism (T-RFLP) analysis coupled with the construction of clone libraries, and quantitative PCR (qPCR) analysis. The T-RFLP data obtained were statistically analysed by non-metric multidimensional scaling. The most abundant FTHFS genes recovered in the clone libraries were assigned to terminal restriction fragments of the T-RFLP profile. The results of the investigation clearly indicated that increased ammonia concentration substantially influenced the putative acetogenic population structure and caused two distinct shifts of the most abundant members; however, the identity of the dominating species remains unknown, as none of the genes had been identified previously. Despite the shifts in the population, the qPCR analysis revealed a relatively stable abundance of the acetogenic population throughout the operation.
Four anaerobic syntrophic acetate-oxidizing bacteria, the thermotolerant strains Re1(T), Re2, T1 ... more Four anaerobic syntrophic acetate-oxidizing bacteria, the thermotolerant strains Re1(T), Re2, T1 and T2, were isolated from two different mesophilic methanogenic systems. The strains originate from sludge of a continuously stirred laboratory-scale reactor containing high levels of ammonium and from a high ammonium enrichment culture. Comparative 16S rRNA gene sequence analysis confirmed that the strains belong to the Firmicutes-Clostridia class. The most closely related species to strains Re1(T), Re2, T1 and T2 was Tepidanaerobacter syntrophicus, with a 16S rRNA gene sequence identity of 96%. The DNA-DNA relatedness of strains Re2, T1 and T2 to strain Re1(T) was 92, 102, 81%, respectively. The gene encoding the acetogen key enzyme formyltetrahydrofolate synthetase (FTHFS) was detected and partly sequenced from the strains. In pure culture the bacteria used different organic compounds as carbon and energy source, such as organic acids, alcohols, sugars and amino acids. Furthermore, acetate-oxidizing ability was observed during co-cultivation with a hydrogen-consuming Methanoculleus sp. The bacteria were found to be spore-forming, rod-shaped and motile, and possessed Gram-positive cell walls. The four strains were thermotolerant and grew at temperatures between 25 and 55°C. Strain Re1(T) had a DNA G+C content of 38.4% and the major fatty acids were C(18:1) w7c, C(18:1) w9c, anteiso-C(17:0), C(16:1) w7c and C(18:0). The genetic and phenotypic properties of strains Re1(T), Re2, T1 and T2 suggest classification as representatives of a novel species of the genus Tepidanaerobacter; the name Tepidanaerobacter acetatoxydans sp. nov. is suggested. The type strain of T. acetatoxydans is Re1(T) (=DSM 21804(T)=JCM 16047(T)).
The importance of syntrophic acetate oxidation for process stability in methanogenic systems oper... more The importance of syntrophic acetate oxidation for process stability in methanogenic systems operating at high ammonia concentrations has previously been emphasized. In this study we investigated bioaugmentation of syntrophic acetate-oxidizing (SAO) cultures as a possible method for decreasing the adaptation period of biogas reactors operating at gradually increased ammonia concentrations (1.5 to 11 g NH(4)(+)-N/liter). Whole stillage and cattle manure were codigested semicontinuously for about 460 days in four mesophilic anaerobic laboratory-scale reactors, and a fixed volume of SAO culture was added daily to two of the reactors. Reactor performance was evaluated in terms of biogas productivity, methane content, pH, alkalinity, and volatile fatty acid (VFA) content. The decomposition pathway of acetate was analyzed by isotopic tracer experiments, and population dynamics were monitored by quantitative PCR analyses. A shift in dominance from aceticlastic methanogenesis to SAO occurred simultaneously in all reactors, indicating no influence by bioaugmentation on the prevailing pathway. Higher abundances of Clostridium ultunense and Tepidanaerobacter acetatoxydans were associated with bioaugmentation, but no influence on Syntrophaceticus schinkii or the methanogenic population was distinguished. Overloading or accumulation of VFA did not cause notable dynamic effects on the population. Instead, the ammonia concentration had a substantial impact on the abundance level of the microorganisms surveyed. The addition of SAO culture did not affect process performance or stability against ammonia inhibition, and all four reactors deteriorated at high ammonia concentrations. Consequently, these findings further demonstrate the strong influence of ammonia on the methane-producing consortia and on the representative methanization pathway in mesophilic biogas reactors.
Changes in communities of syntrophic acetate-oxidizing bacteria (SAOB) and methanogens caused by ... more Changes in communities of syntrophic acetate-oxidizing bacteria (SAOB) and methanogens caused by elevated ammonia levels were quantified in laboratory-scale methanogenic biogas reactors operating at moderate temperature (37°C) using quantitative polymerase chain reaction (qPCR). The experimental reactor was subjected to gradually increasing ammonia levels (0.8-6.9 g NH4 (+) -N l(-1) ), whereas the level of ammonia in the control reactor was kept low (0.65-0.90 g NH4 (+) -N l(-1) ) during the entire period of operation (660 days). Acetate oxidation in the experimental reactor, indicated by increased production of (14) CO2 from acetate labelled in the methyl carbon, occurred when ammonia levels reached 5.5 and 6.9 g NH4 (+) -N l(-1) . Syntrophic acetate oxidizers targeted by newly designed qPCR primers were Thermacetogenium phaeum, Clostridium ultunense, Syntrophaceticus schinkii and Tepidanaerobacter acetatoxydans. The results showed a significant increase in abundance of all these bacteria except T. phaeum in the ammonia-stressed reactor, coincident with the shift to syntrophic acetate oxidation. As the abundance of the bacteria increased, a simultaneous decrease was observed in the abundance of aceticlastic methanogens from the families Methanosaetaceae and Methanosarcinaceae. qPCR analyses of sludge from two additional high ammonia processes, in which methane production from acetate proceeded through syntrophic acetate oxidation (reactor SB) or through aceticlastic degradation (reactor DVX), demonstrated that SAOB were significantly more abundant in the SB reactor than in the DVX reactor.
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Papers by Maria Westerholm