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This study aimed to examine the bacterial, methanogenic archaeal, and eukaryotic community structure in both the midgut and hindgut of Pachnoda marginata larvae using an amplicon sequencing approach. The goal was to investigate how various diets and the soil affect the composition of these three-domain microbial communities within the gut of insect larvae. The results indicated a notable variation in the microbial community composition among the gut compartments. The majority of the bacterial community in the hindgut was composed of Ruminococcaceae and Christensenellaceae. Nocardiaceae, Microbacteriaceae, and Lachnospiraceae were detected in midgut samples from larvae feeding on the leaf diet, whereas Sphingomonadaceae, Rhodobacteraceae, and Promicromonasporaceae dominated the bacterial community of midgut of larvae feeding on the straw diet. The diet was a significant factor that influenced the methanogenic archaeal and eukaryotic community patterns. The methanogenic communities in the two gut compartments significantly differed from each other, with the midgut communities being more similar to those in the soil. A higher diversity of methanogens was observed in the midgut samples of both diets compared to the hindgut. Overall, the microbiota of the hindgut was more host-specific, while the assembly of the midgut was more influenced by the environmental microorganisms., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

With the increasing use of renewable energy resources for the power grid, the need for long-term storage technologies, such as power-to-gas systems, is growing. Biomethanation provides the opportunity to store energy in the form of the natural gas-equivalent biomethane. This study investigates a novel plug flow reactor that employs a helical static mixer for the biological methanation of hydrogen and carbon dioxide. In tests, the reactor achieved an average methane production rate of 2.5 LCH4LR∗d (methane production [L CH4 ] per liter of reactor volume [L R ] per day [d]) with a maximum methane content of 94%. It demonstrated good flexibilization properties, as repeated 12 h downtimes did not negatively impact the process. The genera Methanothermobacter and Methanobacterium were predominant during the initial phase, along with volatile organic acid-producing, hydrogenotrophic, and proteolytic bacteria. The average ratio of volatile organic acid to total inorganic carbon increased to 0.52 ± 0.04, while the pH remained stable at an average of pH 8.1 ± 0.25 from day 32 to 98, spanning stable and flexible operation modes. This study contributes to the development of efficient flexible biological methanation systems for sustainable energy storage and management.

Ecological role of the viral community on the fate of antibiotic resistance genes (ARGs) (reduction vs proliferation) remains unclear in anaerobic digestion (AD). Metagenomics revealed a dominance of Siphoviridae and Podoviridae among 13,895 identified viral operational taxonomic units (vOTUs) within AD, and only 21 of the vOTUs carried ARGs, which only accounted for 0.57 ± 0.43% of AD antibiotic resistome. Conversely, ARGs locating on plasmids and integrative and conjugative elements accounted for above 61.0%, indicating a substantial potential for conjugation in driving horizontal gene transfer of ARGs within AD. Virus-host prediction based on CRISPR spacer, tRNA, and homology matches indicated that most viruses (80.2%) could not infect across genera. Among 480 high-quality metagenome assembly genomes, 95 carried ARGs and were considered as putative antibiotic-resistant bacteria (pARB). Furthermore, lytic phages of 66 pARBs were identified and devoid of ARGs, and virus/host abundance ratios with an average value of 71.7 indicated extensive viral activity and lysis. The infectivity of lytic phage was also elucidated through laboratory experiments concerning changes of the phage-to-host ratio, pH, and temperature. Although metagenomic evidence for dissemination of ARGs by phage transduction was found, the higher proportion of lytic phages infecting pARBs suggested that the viral community played a greater role in reducing ARB numbers than spreading ARGs in AD.

Power-to-X (P2X) technologies will play a more important role in the conversion of electric power to storable energy carriers, commodity chemicals and even food and feed. Among the different P2X technologies, microbial components form cornerstones of individual process steps. This review comprehensively presents the state-of-the-art of different P2X technologies from a microbiological standpoint. We are focusing on microbial conversions of hydrogen from water electrolysis to methane, other chemicals and proteins. We present the microbial toolbox needed to gain access to these products of interest, assess its current status and research needs, and discuss potential future developments that are needed to turn todays P2X concepts into tomorrow's technologies., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

The effects of the inoculum origin, temperature or operational changes on ex situ biomethanation by complex microbial communities have been investigated; however, it remains unclear how the diversity of the inoculum influences the process and its stability. We explored the effect of microbial diversity of four inocula (coded as PF, WW, S37 and Nrich) on methane production, process stability and the formation of volatile fatty acids as by-products. The highest methane amounts produced were 3.38 ± 0.37 mmol, 3.20 ± 0.07 mmol, 3.07 ± 0.27 mmol and 3.14 ± 0.06 mmol for PF, WW, S37 and Nrich, respectively. The highest acetate concentration was found in less diverse cultures (1679 mg L -1 and 1397 mg L -1 for S37 and Nrich, respectively), whereas the acetate concentrations remained below 30 mg L -1 in the more diverse cultures. The maximum concentration of propionate was observed in less diverse cultures (240 mg L -1 and 37 mg L -1 for S37 and Nrich cultures, respectively). The highly diverse cultures outperformed the medium and low diversity cultures in the long-term operation. Methanogenic communities were mainly composed of hydrogenotrophic methanogens in all cultures. Aceticlastic methanogenesis was only active in the highly diverse sludge community throughout the experiment. The more diverse the inocula, the more methane was produced and the less volatile fatty acids accumulated, which could be attributed to the high number of microbial functions working together to keep a stable and balanced process. It is concluded that the inoculum origin and its diversity are very important factors to consider when the biomethanation process is performed with complex microbial communities.

The Pachnoda marginata larva have complex gut microbiota capable of the effective conversion of lignocellulosic biomass. Biotechnological utilization of these microorganisms in an engineered system can be achieved by establishing enrichment cultures using a lignocellulosic substrate. We established enrichment cultures from contents of the midgut and hindgut of the beetle larva using wheat straw in an alkaline medium at mesophilic conditions. Two different inoculation preparations were used: procedure 1 (P1) was performed in a sterile bench under oxic conditions using 0.4% inoculum and small gauge needles. Procedure 2 (P2) was carried out under anoxic conditions using more inoculum (4%) and bigger gauge needles. Higher methane production was achieved with P2, while the highest acetic acid concentrations were observed with P1. In the enrichment cultures, the most abundant bacterial families were Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae, and Marinilabiliaceae. Further, the most abundant methanogenic genera were Methanobrevibacter , Methanoculleus, and Methanosarcina . Our observations suggest that in samples processed with P1, the volatile fatty acids were not completely converted to methane. This is supported by the finding that enrichment cultures obtained with P2 included acetoclastic methanogens, which might have prevented the accumulation of acetic acid. We conclude that differences in the inoculum preparation may have a major influence on the outcome of enrichment cultures from the P. marginata larvae gut.

Mixed or pure cultures can be used for biomethanation of hydrogen. Sodium 2-bromoethanesulfonate (BES) is an inhibitor of methanogenesis used to investigate competing reactions like homoacetogenesis in mixed cultures. To understand the effect of BES on the hydrogenotrophic metabolism in a biomethanation process, anaerobic granules from a wastewater treatment plant, a hydrogenotrophic enrichment culture, and pure cultures of Methanococcus maripaludis and Methanobacterium formicicum were incubated under H 2 /CO 2 headspace in the presence or absence of BES, and the turnover of H 2 , CO 2 , CH 4 , formate and acetate was analyzed. Anaerobic granules produced the highest amount of formate after 24 h of incubation in the presence of BES. Treating the enrichment culture with BES led to the accumulation of formate. M. maripaludis produced more formate than M. formicicum when treated with BES. The non-inhibited methanogenic communities produced small amounts of formate whereas the pure cultures did not. The highest amount of acetate was produced by the anaerobic granules concomitantly with formate consumption. These results indicate that formate is an important intermediate of hydrogenotrophic metabolism accumulating upon methanogenesis inhibition.

Livestock manure emits reduced sulfur compounds and methane, which affect nature and the climate. These gases are efficiently mitigated by addition of a tannic acid-sodium fluoride combination inhibitor (TA-NaF), and to some extent by acidification. In this paper, TA-NaF treatment was performed on swine manure to study the treatment influence on methanogenic pathways and sulfur transformation pathways in various laboratory experiments. Stable carbon isotope labeling revealed that both untreated and TA-NaF treated swine manures were dominated by hydrogenotrophic methanogenesis. However, in supplementary experiments in wastewater sludge, TA-NaF clearly inhibited acetoclastic methanogenesis, whereas acidification inhibited hydrogenotrophic methanogenesis. In swine manure, TA-NaF inhibited s-amino acid catabolism to a larger extent than sulfate reduction. Conversely, acidification reduced sulfate reduction activity more than s-amino acid degradation. TA-NaF treatment had no significant effect on methanogenic community structure, which was surprising considering clear effects on isotope ratios of methane and carbon dioxide. Halophile sulfate reducers adapted well to TA-NaF treatment, but the community change also depended on temperature. The combined experimental work resulted in a proposed inhibition scheme for sulfur transformations and methanogenic pathways as affected by TA-NaF and acidification in swine manure and in other inocula., Competing Interests: A patent application, "Mitigation of ammonia, odor and greenhouse gases", was filed by University of Southern Denmark on 12th of June 2019 to the European Patent Office under Application No/Patent No 19179648.1 – 1105. The patent inventors who are also authors of this paper are: Frederik R. Dalby, Michael J. Hansen, and Anders Feilberg. The patent covers the application of tannic acid and sodium fluoride for abatement of gaseous emission from manure slurry. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Ex situ biomethanation allows the conversion of hydrogen produced from surplus electricity to methane. The flexibility of the process was recently demonstrated, yet it is unknown how intermittent hydrogen feeding impacts the functionality of the microbial communities. We investigated the effect of starvation events on the hydrogen consumption and methane production rates (MPRs) of two different methanogenic communities that were fed with hydrogen and carbon dioxide. Both communities showed functional resilience in terms of hydrogen consumption and MPRs upon starvation periods of up to 14 days. The origin of the inoculum, community structure and dominant methanogens were decisive for high gas conversion rates. Thus, pre-screening a well performing inoculum is essential to ensure the efficiency of biomethanation systems operating under flexible gas feeding regimes. Our results suggest that the type of the predominant hydrogenotrophic methanogen (here: Methanobacterium ) is important for an efficient process. We also show that flexible biomethanation of hydrogen and carbon dioxide with complex microbiota is possible while avoiding the accumulation of acetate, which is relevant for practical implementation. In our study, the inoculum from an upflow anaerobic sludge blanket reactor treating wastewater from paper industry performed better compared to the inoculum from a plug flow reactor treating cow manure and corn silage. Therefore, the implementation of the power-to-gas concept in wastewater treatment plants of the paper industry, where biocatalytic biomass is readily available, may be a viable option to reduce the carbon footprint of the paper industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Logroño, Popp, Nikolausz, Kluge, Harms and Kleinsteuber.)

Livestock waste is stored and used as soil fertilizer or directly as substrate for biogas production. Methane emissions from manure storages and ammonia inhibition of anaerobic digesters fed with manure, are well-known problems related to manure management. This study examines the effect of adding tannic acid with fluoride (TA-NaF) and lignosulfonic acid (LS) on methanogenic activity in batch reactors with ammonia inhibited maize silage digestate and in batch reactors with manure. Lignosulfonic acid counteracted urea induced ammonia inhibition of methanogenesis, whereas TA-NaF inhibited methanogenesis itself. Stable carbon isotope ratio analysis and methanogen community analysis suggested that TA-NaF affected acetoclastic methanogens the most. The combined findings suggest that TA-NaF could be used to reduce methane emissions from stored manure. Conversely, LS could be used as supplement in anaerobic digesters prone to urea induced ammonia inhibition., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following personal financial interests: A patent application, “Mitigation of ammonia, odor and greenhouse gases”, was filed by University of Southern Denmark and Aarhus University on 12th of June 2019 to the European Patent Office under Application No/Patent No 19179648.1 – 1105. The patent authors included in this manuscript are, Frederik R. Dalby, Michael J. Hansen, Anders Feilberg., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biomethanation is a promising solution to convert H 2 (produced from surplus electricity) and CO 2 to CH 4 by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H 2 /CO 2 . We enriched a hydrogenotrophic microbiota on CO 2 and H 2 as sole carbon and energy sources, respectively, to investigate these competing reactions. The microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched, as acetate was concomitantly produced alongside CH 4 . By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH 4 (≥97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities ≥ 1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus . The bacterial community was dominated by Lutispora . The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards the selective production of CH 4 from H 2 /CO 2 by adapting the media composition, the reducing agent and the stirring intensity.

Lindane, the γ-hexachlorocyclohexane (HCH) isomer, was among the most used pesticides worldwide. Although it was banned in 2009, residues of Lindane and other HCH-isomers are still found with high concentrations in contaminated fields. For clean-up, phytoremediation combined with anaerobic digestion (AD) of contaminated biomass to produce biogas and fertilizer could be a promising strategy and was tested in two 15 L laboratory-scale continuous stirred tank reactors. During operation over one year by adding HCH isomers (γ, α and β) consecutively, no negative influence on conventional reactor parameters was observed. The γ- and α-HCH isomers were transformed to chlorobenzene and benzene, and transformation became faster along with time, while β-HCH was not removed. Genus Methanosaeta and order Clostridiales, showing significant enhancement on abundance with HCH addition, may be used as bioindicators for HCH dehalogenation in AD process. The potential for HCH degradation in AD system was restricted to axial Cl atoms of HCH and it showed slight enantioselective preference towards transformation of (+) α-HCH. Moreover, metabolite benzene was mineralized to CO 2 and methane, deducing from tracer experiments with benzene- 13 C 6 . Overall, AD appears to be a feasible option for treatment of γ and α-HCHs contaminated biomass., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of this review is to give a summary of natural lignocellulose-degrading systems focusing mainly on animal digestive tracts of wood-feeding insects and ruminants in order to find effective strategies that can be applied to improve anaerobic digestion processes in engineered systems. Wood-feeding animals co-evolved with symbiotic microorganisms to digest lignocellulose-rich biomass in a very successful way. Considering the similarities between these animal gut systems and the lignocellulose-based biotechnological processes, the gut with its microbial consortium can be a perfect model for an advanced lignocellulose-degrading biorefinery. The physicochemical properties and structure of the gut may provide a scheme for the process design, and the microbial consortium may be applied as genetic resource for the up-scaled bioreactor communities. Manipulation of the gut microbiota is also discussed in relation to the management of the reactor communities.

The ability of anaerobic digestion (AD) to eliminate organophosphorus model compounds (OPs) with structural elements of phosphate, phosphorothioate and phosphorodithioate esters was studied. The enzymatic mechanism of the first irreversible degradation reaction was characterized using metabolite pattern and kinetic 2 H/ 13 C-isotope effect in original, cell-free and heat sterilized biogas slurry. The isotope fractionation study suggests different modes of degradation reactions. Representatives for phosphate ester, tris(2-chloroethyl) phosphate and tris(1,3-dichloro-2-propyl) phosphate, were hydrolyzed in biogas slurry without carbon or hydrogen isotope fractionation. Representatives for phosphorodithioate, Dimethoate and Malathion, were degraded in original slurry yielding carbon enrichment factor (ε C ) of -0.6 ± 0.1‰ and -5.5 ± 0.1‰ (-0.9 ± 0.1‰ and -7.2 ± 0.5‰ in cell-free slurry), without hydrogen isotope fractionation. Phosphorothioate degradation represented by Parathion and Parathion-methyl yielded surprisingly different ε C (-0.7 ± 0.2 and -3.6 ± 0.4‰) and ε H (-33 ± 5 and -5 ± 1‰) in original slurry compared to cell-free slurry (ε C  = -2.5 ± 0.5 and -8.6 ± 1.4‰; ε H  = -61 ± 10 and -10 ± 3‰) suggesting H-C bond cleavage. Degradation of Parathion and Parathion-methyl in sterilized slurry gave carbon but not hydrogen fractionation implying relative thermostable enzymatic activity with different mechanism. The correlation of 2 H and 13 C stable isotope fractionation of Parathion in biogas slurry showed distinct pattern (Λ original  = 31 ± 11, Λ cell-free  = 20 ± 2), indicating different mechanism from chemical hydrolysis. Overall, AD can be a potential treatment for OPs contaminated biomass or contaminated organic waste material., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Anaerobic digestion of nitrogen-rich substrate often causes process inhibition due to the susceptibility of the microbial community facing ammonia accumulation. However, the precise response of the microbial community has remained largely unknown. To explore the reasons, bacterial communities in ammonia-stressed reactors and control reactors were studied by amplicon pyrosequencing of 16S rRNA genes and the active methanogens were followed by terminal restriction fragment length polymorphism (T-RFLP) analyses of mcrA/mrtA gene transcripts. The results showed that the diversity of bacterial communities decreased in two parallel ammonia-inhibited reactors compared with two control reactors, but different levels of inhibitions coinciding with different community shifts were observed. In one reactor, the process was completely inhibited, which was preceded by a decreasing relative abundance of the phylum Firmicutes. Despite the same operating conditions, the process was stabilized in the parallel, partially inhibited reactor, in which the relative abundance of Firmicutes greatly increased. In particular, both ammonia-inhibited reactors lacked taxa assumed to be syntrophic bacteria (Thermoanaerobacteraceae, Syntrophomonadaceae, and Synergistaceae). Besides the predominance of the hydrogenotrophic methanogens Methanoculleus and Methanobacterium, activity of Methanosarcina and even of the strictly aceticlastic genus Methanosaeta were found to contribute at very high ammonia levels (> 9 g NH 4 -N L -1 ) in the stabilized reactor (partial inhibition). In contrast, the lack of aceticlastic activity in the parallel reactor might have led to acetate accumulation and thus process failure (complete inhibition). Collectively, ammonia was found to be a general inhibitor while accumulating acetate and thus acidification might be the key factor of complete process failure.

Three different bioaugmentation cultures enriched from natural and engineered cellulolytic environments (cow and goat rumen, a biogas reactor digesting sorghum biomass) were compared for their enhancement potential on the anaerobic digestion of wheat straw. Methane yields were determined in batch tests using the Automatic Methane Potential Test System operated for 30 days under mesophilic conditions. All cultures had positive effects on substrate degradation, and higher methane yields were observed in the bioaugmented reactors compared to control reactors set up with standard inoculum. However, the level of enhancement differed according to the type of the enrichment culture. Methane yield in batch reactors augmented with 2% cow rumen derived enrichment culture was increased by only 6%. In contrast, reactors amended with 2% goat rumen derived enrichment culture or with the bioaugmentation culture obtained from the biogas reactor digesting sorghum biomass produced 27 and 20% more methane, respectively. The highest methane yield was recorded in reactors amended with 6% goat rumen derived enrichment culture, which represented an increase by 36%. The microbial communities were quite similar at the end of the batch tests independently of the bioaugmentation sources, indicating that the introduced microbial communities of the enrichment cultures did not dominate the reactors., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hexachlorocyclohexane (HCH) production for pesticides was banned by Stockholm Convention (2009) due to its harmful and adverse effects on the environment. Despite this measure, many areas contaminated with former HCH production-waste products still require management. As a potential solution contributing to clean-up of these sites, anaerobic digestion (AD) of pesticide-contaminated biomass to produce biogas is a promising strategy. High pesticide concentrations, however, may inhibit biogas production. Therefore, laboratory-scale batch reactors were set up to investigate biogas reactor performance in presence of HCH. Inhibitory effects on biogas yield was observed with concentrations of HCH ≥ 150 mg/L. Carbon isotope composition of methane (δ 13 C CH4 ) showed significant fluctuation after an inhibition phase, indicating that HCH toxicity can affect the activity of acetoclastic methanogens. Furthermore, combined results of metabolites and carbon isotope fractionation factors (ε c ) demonstrated that α- and γ-HCH can be degraded to chlorobenzene and benzene via anaerobic reductive dechlorination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.

Anaerobic digestion of sugarcane straw co-digested with sugarcane filter cake was investigated with a special focus on macronutrients supplementation for an optimized conversion process. Experimental data from batch tests and a semi-continuous experiment operated in different supplementation phases were used for modeling the conversion kinetics based on continuous stirred-tank reactors. The semi-continuous experiment showed an overall decrease in the performance along the inoculum washout from the reactors. By supplementing nitrogen alone or in combination to phosphorus and sulfur the specific methane production significantly increased (P<0.05) by 17% and 44%, respectively. Although the two-pool one-step model has fitted well to the batch experimental data (R 2 >0.99), the use of the depicted kinetics did not provide a good estimation for process simulation of the semi-continuous process (in any supplementation phase), possibly due to the different feeding modes and inoculum source, activity and adaptation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mL N CH 4 g VS -1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Comparative analysis of methanogenic archaea compositions and dynamics in 11 laboratory-scale continuous stirred tank reactors fed with different agricultural materials (chicken manure, cattle manure, maize straw, maize silage, distillers grains, and Jatropha press cake) was carried out by analysis of the methyl coenzyme-M reductase α -subunit ( mcrA ) gene. Various taxa within Methanomicrobiales, Methanobacteriaceae, Methanosarcinaceae, Methanosaetaceae, and Methanomassiliicoccales were detected in the biogas reactors but in different proportions depending on the substrate type utilized as well as various process parameters. Improved coverage and higher taxonomic resolution of methanogens were obtained compared to a previous 16S rRNA gene based study of the same reactors. Some members of the genus Methanoculleus positively correlated with the relative methane content, whereas opposite correlations were found for Methanobacterium . Specific biogas production was found to be significantly correlating with Methanosarcinaceae. Statistical analysis also disclosed that some members of the genus Methanoculleus positively correlated with the ammonia level, whereas the prevalence of Methanocorpusculum , Methanobacterium , and Methanosaeta was negatively correlated with this parameter. These results suggest that the application of methanogenic archaea adapted to specific feedstock might enhance the anaerobic digestion of such waste materials in full-scale biogas reactors., Competing Interests: The authors declare that there are no competing interests regarding the publication of this manuscript.

The effects of hydraulic retention time (HRT) reduction at constant high organic loading rate on the activity of hydrogen-producing bacteria and methanogens were investigated in reactors digesting thin stillage. Stable isotope fingerprinting was additionally applied to assess methanogenic pathways. Based on hydA gene transcripts, Clostridiales was the most active hydrogen-producing order in continuous stirred tank reactor (CSTR), fixed-bed reactor (FBR) and anaerobic sequencing batch reactor (ASBR), but shorter HRT stimulated the activity of Spirochaetales. Further decreasing HRT diminished Spirochaetales activity in systems with biomass retention. Based on mcrA gene transcripts, Methanoculleus and Methanosarcina were the predominantly active in CSTR and ASBR, whereas Methanosaeta and Methanospirillum activity was more significant in stably performing FBR. Isotope values indicated the predominance of aceticlastic pathway in FBR. Interestingly, an increased activity of Methanosaeta was observed during shortening HRT in CSTR and ASBR despite high organic acids concentrations, what was supported by stable isotope data., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, the effects of nitrogen, phosphate and trace elements supplementation were investigated in a semi-continuously operated upflow anaerobic sludge blanket system to enhance process stability and biogas production from sugarcane vinasse. Phosphate in form of KH2PO4 induced volatile fatty acids accumulation possibly due to potassium inhibition of the methanogenesis. Although nitrogen in form of urea increased the reactor's alkalinity, the process was overloaded with an organic loading rate of 6.1gCODL(-1)d(-1) and a hydraulic retention time of 3.6days. However, by supplementing urea and trace elements a stable operation even at an organic loading rate of 9.6gCODL(-1)d(-1) and a hydraulic retention time of 2.5days was possible, resulting in 79% higher methane production rate with a stable specific methane production of 239mLgCOD(-1)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The effect of pig manure mixed with rice straw on methane yield and the microbial community involved in a thermophilic (55°C) anaerobic digestion process was investigated. Three substrates composed of mixed pig manure and rice straw at different ratios (95:5; 78:22 and 65:35 w/w, which resulted in C/N ratios of 10:1, 20:1 and 30:1) were used for the experiment. The substrate type had a major influence on the total bacterial community, while the methanogens were less affected. The members of the class Clostridia (phylum Firmicutes) were predominant regardless of mixture ratio (C/N ratio), but at species level there was a major difference between the low and high C/N ratio samples. The hydrogenotrophic methanogenic genus of Methanothermobacter was predominant in all samples but higher C/N ratio sequences affiliated to the genus Methanosarcina were also detected. The appearance of Methanosarcina sp. is most likely due to the less inhibition of ammonia during the anaerobic digestion., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: During strategic planning of a biogas plant, the local availability of resources for start-up and operation should be taken into consideration for a cost-efficient process. Because most bioethanol/sugar industries in Brazil are located in remote areas, the use of fresh cattle manure from local farms could be a solution for the inoculation of the biogas process. This study investigated the diversity and dynamics of bacterial and archaeal communities and the performance of biogas reactors inoculated with manure and a mixed inoculum from different biogas reactors as for a controlled start-up until steady state., Results: Laboratory-scale biogas reactors were fed semi-continuously with sugarcane filter cake alone (mono-digestion) or together with bagasse (co-digestion). At the initial start-up, the reactors inoculated with the mixed inoculum displayed a less diverse taxonomic composition, but with higher presence of significant abundances compared to reactors inoculated with manure. However, in the final steady state, the communities of the differently inoculated reactors were very similarly characterized by predominance of the methanogenic genera Methanosarcina and Methanobacterium, the bacterial families Bacteroidaceae, Prevotellaceae and Porphyromonadaceae (phylum Bacteroidetes) and Synergistaceae (phylum Synergistetes). In the mono-digestion reactors, the methanogenic communities varied greater than in the co-digestion reactors independently of the inoculation strategy., Conclusion: The microbial communities involved in the biogas production from waste products of the Brazilian bioethanol/sugar industry were relatively similar and stable at the reactor's steady phase independently of the inoculum source (manure or mixed inoculum). Therefore, the locally available manure can be used as inoculum for start-up of the biogas process, since it also contains the microbial resources needed. The strong fluctuation of methanogenic communities in mono-digestion reactors indicates higher risk of process instability than in co-digestion reactors.

We present a simple protocol for the cost- and time-efficient profiling of methanogens based on T-RFLP fingerprinting of mcrA amplicons. Sequence data were compiled from mesophilic lab-scale and full-scale biogas reactors operated under various conditions and fed with various substrates. The database facilitates the rapid identification of methanogens, thus reducing the need of cloning and sequencing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The anaerobic digestion of sugarcane filter cake and the option of co-digestion with bagasse were investigated in a semi-continuous feeding regime to assess the main parameters used for large-scale process designing. Moreover, fresh cattle manure was considered as alternative inoculum for the start-up of biogas reactors in cases where digestate from a biogas plant would not be available in remote rural areas. Experiments were carried out in 6 lab-scale semi-continuous stirred-tank reactors at mesophilic conditions (38±1°C) while the main anaerobic digestion process parameters monitored. Fresh cattle manure demonstrated to be appropriate for the start-up process. However, an acclimation period was required due to the high initial volatile fatty acids concentration (8.5gL(-1)). Regardless the mono-digestion of filter cake presented 50% higher biogas yield (480mLgVS(-1)) than co-digestion with bagasse (320mLgVS(-1)) during steady state conditions. A large-scale co-digestion system would produce 58% more biogas (1008m(3)h(-1)) than mono-digestion of filter cake (634m(3)h(-1)) due to its higher biomass availability for biogas conversion. Considering that the biogas production rate was the technical parameter that displayed the most relevant differences between the analyzed substrate options (0.99-1.45m(3)biogasm(3)d(-1)). The decision of which substrate option should be implemented in practice would be mainly driven by the available construction techniques, since economically efficient tanks could compensate the lower biogas production rate of co-digestion option., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Different methods for optimization the anaerobic digestion (AD) of sugarcane filter cake (FC) with a special focus on volatile fatty acids (VFA) production were studied. Sodium hydroxide (NaOH) pretreatment at different concentrations was investigated in batch experiments and the cumulative methane yields fitted to a dual-pool two-step model to provide an initial assessment on AD. The effects of nitrogen supplementation in form of urea and NaOH pretreatment for improved VFA production were evaluated in a semi-continuously operated reactor as well. The results indicated that higher NaOH concentrations during pretreatment accelerated the AD process and increased methane production in batch experiments. Nitrogen supplementation resulted in a VFA loss due to methane formation by buffering the pH value at nearly neutral conditions (∼ 6.7). However, the alkaline pretreatment with 6g NaOH/100g FCFM improved both the COD solubilization and the VFA yield by 37%, mainly consisted by n-butyric and acetic acids., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Flexible biogas production that adapts biogas output to energy demand can be regulated by changing feeding regimes. In this study, the effect of changes in feeding intervals on process performance, microbial community structure, and the methanogenesis pathway was investigated. Three different feeding regimes (once daily, every second day, and every 2 h) at the same organic loading rate were studied in continuously stirred tank reactors treating distiller's dried grains with solubles. A larger amount of biogas was produced after feeding in the reactors fed less frequently (once per day and every second day), whereas the amount remained constant in the reactor fed more frequently (every 2 h), indicating the suitability of the former for the flexible production of biogas. Compared to the conventional more frequent feeding regimes, a methane yield that was up to 14% higher and an improved stability of the process against organic overloading were achieved by employing less frequent feeding regimes. The community structures of bacteria and methanogenic archaea were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA and mcrA genes, respectively. The results showed that the composition of the bacterial community varied under the different feeding regimes, and the observed T-RFLP patterns were best explained by the differences in the total ammonia nitrogen concentrations, H2 levels, and pH values. However, the methanogenic community remained stable under all feeding regimes, with the dominance of the Methanosarcina genus followed by that of the Methanobacterium genus. Stable isotope analysis showed that the average amount of methane produced during each feeding event by acetoclastic and hydrogenotrophic methanogenesis was not influenced by the three different feeding regimes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The anaerobic digestion of filter cake and its co-digestion with bagasse, and the effect of gradual increase of the organic loading rate (OLR) from start-up to overload were investigated. Understanding the influence of environmental and technical parameters on the development of particular methanogenic pathway in the biogas process was an important aim for the prediction and prevention of process failure. The rapid accumulation of volatile organic acids at high OLR of 3.0 to 4.0 gvs·L⁻¹·day⁻¹ indicated strong process inhibition. Methanogenic community dynamics of the reactors was monitored by stable isotope composition of biogas and molecular biological analysis. A potential shift toward the aceticlastic methanogenesis was observed along with the OLR increase under stable reactor operating conditions. Reactor overloading and process failure were indicated by the tendency to return to a predominance of hydrogenotrophic methanogenesis with rising abundances of the orders Methanobacteriales and Methanomicrobiales and drop of the genus Methanosarcina abundance.

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion., (© 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Biogas production from sugarcane waste has large potential for energy generation, however, to enable the optimization of the anaerobic digestion (AD) process each substrate characteristic should be carefully evaluated. In this study, the kinetic challenges for biogas production from different types of sugarcane waste were assessed. Samples of vinasse, filter cake, bagasse, and straw were analyzed in terms of total and volatile solids, chemical oxygen demand, macronutrients, trace elements, and nutritional value. Biochemical methane potential assays were performed to evaluate the energy potential of the substrates according to different types of sugarcane plants. Methane yields varied considerably (5-181 Nm³·tonFM(-1)), mainly due to the different substrate characteristics and sugar and/or ethanol production processes. Therefore, for the optimization of AD on a large-scale, continuous stirred-tank reactor with long hydraulic retention times (>35 days) should be used for biogas production from bagasse and straw, coupled with pre-treatment process to enhance the degradation of the fibrous carbohydrates. Biomass immobilization systems are recommended in case vinasse is used as substrate, due to its low solid content, while filter cake could complement the biogas production from vinasse during the sugarcane offseason, providing a higher utilization of the biogas system during the entire year.

The potential of two alkali-tolerant, lignocellulolytic environmental enrichment cultures to improve the anaerobic fermentation of Ca(OH)₂-pre-treated wheat straw was studied. The biomethane potential of pre-treated straw was 36% higher than that of untreated straw. The bioaugmentation of pre-treated straw with the enrichment cultures did not enhance the methane yield, but accelerated the methane production during the first week. In acidogenic leach-bed fermenters, a 61% higher volatile fatty acid (VFA) production and a 112% higher gas production, mainly CO₂, were observed when pre-treated instead of untreated straw was used. With one of the two enrichment cultures as the inoculum, instead of the standard inoculum, the VFA production increased by an additional 36% and the gas production by an additional 110%, again mainly CO₂. Analysis of the microbial communities in the leach-bed processes revealed similar bacterial compositions in the fermenters with pre-treated straw, which developed independently of the used inoculum. It was suggested that the positive metabolic effects with the enrichment cultures observed in both systems were due to initial activities of the alkali-tolerant microorganisms tackling the alkaline conditions better than the standard inocula, whereas the latter dominated in the long term.

Four 15-L lab-scale continuous stirred tank reactors were operated under mesophilic conditions to investigate the effect of ammonia inhibition. Stable isotope fingerprinting of biogas was applied as a process monitoring tool. Ammonia inhibition was initiated by amendment of chicken manure to maize silage fed reactors. During the accumulation of ammonia, the concentration of volatile fatty acids increased while the biogas production and pH decreased. However, in one reactor, an inhibited steady state with stable gas production even at high ammonia levels was achieved, while the other reactor proceeded to complete process failure. A depletion of the δ(13)CH4 and δ(13)CO2 values preceded the process inhibition. Moreover, the stable isotope composition of biogas also forecasted the complete process failure earlier than other standard parameters. The stable isotope analyses of biogas have a potential for mechanistic insights in anaerobic processes, and may be used to pre-warn process failure under stress conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The effect of several easily degradable substrates, such as protein, starch and sunflower oil was investigated on the bacterial community of a laboratory-scale biogas model system. Besides measuring gas yield, Denaturing Gradient Gel Electrophoresis (DGGE), Phospholipids Fatty Acid Analysis (PLFA) for Bacteria and T-RFLP analysis of the mcrA gene for Archaea were used. The community of the examined biogas reactors adapted to the new substrates through a robust physiological reaction followed by moderate community abundance shifts. Gas yield data clearly demonstrated the physiological adaptation to substrate shifts. Statistical analysis of DNA and chemotaxonomic biomarkers revealed community abundance changes. Sequences gained from DGGE bands showed the dominance of the phyla Bacteroidetes and the presence of Firmicutes (Clostridia) and Thermotogae. This was supported by the detection of large amounts of branched 15-carbon non-hydroxy fatty acids in PLFA profiles, as common PLFA markers of the Bacteroidetes group. Minor abundance ratios changes were observed in the case of Archaea in accordance with changes of the fed substrates.

An enrichment culture dominated by one type of Dehalococcoides sp. (83% of clones) was characterised. This culture, originally derived from contaminated groundwater from the area of Bitterfeld-Wolfen (Saxony-Anhalt, Germany), dehalogenates chlorinated ethenes to ethene. Further, the culture also dehalogenated vinyl bromide (VB) and 1,2-dichloroethane (DCA) to ethene, 1,2,3,4- and 1,2,3,5-tetrachlorobenzene (TeCB), penta- and hexachlorobenzene (PeCB and HCB) to trichlorobenzenes (TCB), lindane to monochlorobenzene (MCB) and pentachlorophenol (PCP) to 2,3,4,6-tetrachlorophenol (TeCP). Growth was proven by quantitative PCR for all active cultures, except for those with TeCB, lindane and PCP. The growth yields obtained ranged from (2.9 ± 0.7) × 10(7) cells μmol(-1) Br(-) released on VB to (34.8 ± 5.4) × 10(7) cells μmol(-1) Cl(-) released on VC. Genes coding for nine putative reductive dehalogenases, the enzymes that mediate the respiratory process of dehalogenation, were identified. Phylogenetic analysis revealed eight reductive dehalogenases with similar sequences in other Dehalococcoides strains and one unique sequence., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The description of microbial community structure is always biased by the selectivity of the methods applied. Although TA cloning of PCR amplified community DNA is one of the most widely used techniques in bacterial community analysis, no thorough comparative testing has been carried out on different TA cloning systems. In this study, we measured and compared the selectivity of two widely used TA-cloning kits in experimental setups where the length heterogeneity of the inserts modeled the natural length variation of the 16S rRNA gene and the 16S-23S intergenic spacer region. Both TOPO TA (Invitrogen, CA USA) and pGem-T vector system (Promega, WI USA) cloning kits showed significant and reproducible insert size related selectivity. The effect of ligation time and temperature was also studied in case of the pGem-T vector system. We compared the performance of the two cloning kits on an environmental sample, along with a semiquantitative community fingerprinting method to gain reference data free of cloning bias. The two clone libraries showed significantly different compositions, and were also differing from the community structure revealed by length heterogeneity PCR., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A Dehalococcoides-dominated culture coupling reductive dechlorination of tetrachloroethene (PCE) to ethene to growth was enriched from a European field site for the first time. Microcosms were set up using groundwater from a chlorinated ethene-contaminated anaerobic aquifer in Bitterfeld (Germany). Active, lactate-amended microcosms capable of PCE dechlorination to ethene without the accumulation of intermediates were used for further enrichment. After three transfers on lactate as an electron donor and PCE as an electron acceptor, the enrichment was transferred to parallel cultures with one of the chlorinated ethenes as an electron acceptor and acetate and hydrogen as the carbon and energy source, respectively. After three more transfers, a highly purified culture was derived that was capable of dechlorinating PCE with hydrogen and acetate as the electron donor and carbon source, respectively. PCR, followed by denaturing gradient gel electrophoresis, cloning and sequencing revealed that this culture was dominated by a Dehalococcoides sp. belonging to the Pinellas group. Investigation of substrate specificity in the parallel cultures suggested the presence of a novel Dehalococcoides that can couple all dechlorination steps, from PCE to ethene, to energy conservation. Quantitative real-time PCR confirmed growth with PCE, cis-dichloroethene, 1,1-dichloroethene or vinyl chloride as electron acceptors. The culture was designated BTF08 due to its origin in Bitterfeld.

The dynamics and composition of microbial communities in the aqueous phase of a model wetland supplied with cis- and trans-1,2-dichloroethenes (DCE)-contaminated groundwater was characterized. PCR-denaturing gradient gel electrophoresis analysis of water samples obtained from different parts of the wetland revealed that changes of the bacterial community structure coincided with a succession of the hydrochemical conditions in the wetland, from oxic towards anoxic conditions. During this transition phase, the appearance of vinyl chloride and ethene correlated with the presence of putative dechlorinating bacteria (Dehalococcoides spp., Geobacter spp. and Dehalobacter spp.). Additionally, a shift of the DCE isotopic composition indicated the progressive prevalence of reductive dechlorination in the wetland. Although the DCE degradation processes varied over time, biodegradation activity was maintained in the wetland system. 16S rRNA gene libraries revealed that Proteobacteria accounted for >50% of 16S rRNA genes clone libraries, whereas approximately 17% of the sequences from the wetland were related to sulphate reducers. Based on a multiple-method approach, this study illustrates the linkage between microbial community dynamics and composition, changes of hydrochemical conditions and processes of DCE degradation in a wetland system.

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats., (Copyright 2009 Elsevier B.V. All rights reserved.)

A simple, fast and cost-effective method for the taxon-specific separation of various 16S rRNAs was developed. This primer extension mediated separation of different RNA species was obtained on agarose gels. The method provides an easy way for the investigation of substrate mediated radioisotope incorporation into the RNA.

Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3'-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.

Methods based on SNuPE (single-nucleotide primer extension) have become invaluable tools for the rapid and highly specific detection of point mutations and single-nucleotide polymorphisms in the field of human genetics. In the primer extension reaction, a DNA polymerase is used to label a specific primer hybridized to the target sequence by incorporating a single labelled ddNTP (dideoxynucleotide). This labelling provides not only information about the complementary nucleotide of interest in the opposite strand but also a semiquantitative analysis of the sequence targeted by the primer. Since several subdisciplines of microbiology increasingly require cultivation-independent molecular screening tools for elucidating differences between either strains or community structures based on sequence variations of marker genes, SNuPE offers a promising alternative to the existing tool box. The present review describes the method in detail and reports the state-of-the-art applications of this technique both in the field of nucleic acid detections in human genetics and in microbiology.

Strain XA05 and FG03 with high biodegradation activity of phenol were isolated from the activated sludge and phenol-contaminated soils in Northwest of China, respectively. DNA sequencing and homologous analysis of 16s rRNA gene identified that XA05 belonged to an Acinetobacter sp. and FG03 was closely related to the Sphingomonas sp. Cells of strain XA05 and FG03 were mixed at the ratio of 1:1, and polyvinyl alcohol (PVA) was used as a gel matrix to immobilize mixed cells by repeated freezing and thawing. Biodegradation was evaluated by determining phenol. Detoxication was evaluated by using Daphnia magna toxicity tests. The removal effciency of phenol and factors affecting phenol degradation were investigated, the stability of the immobilized cells was also reported. Experimental values indicated that both free cells and immobilized cells showed high phenol degradation effciencies, higher than 95% within 35 h with an initial concentration of 800 mg/L phenol, and the immobilized cells showed better performance than that of the suspended-culture cells. These results indicate that immobilized Acinetobncter sp. XA05 and Sphingomonas sp. FG03 possesses a good application potential in the treatment of phenol-containing wastewater.

The removal of dichloromethane (DCM) from polluted air was studied both in biotrickling filters and in continuous stirred tank bioreactors, using either a single-liquid aqueous phase or a combination of an aqueous-organic liquid phase. The presence of the organic phase, i.e. silicone oil, at a volume ratio of 10% of the liquid phase, increased the maximum EC by about 25% in the BTF, reaching 200 gm(3)/h, and by as much as 300% in the CSTB, reaching 350 gm(3)/h. Based on data of chloride release in the aqueous phase and carbon dioxide production in the gas phase, complete dechlorination and mineralization of the pollutant could be confirmed. When applying shock loads, a more stable behaviour was observed in the presence of the organic phase. Generally, the completely mixed reactors were also more stable than the plug-flow biotrickling filters, irrespective of the presence of the organic phase. The use of molecular techniques allowed showing that the originally inoculated DCM-degrading Hyphomicrobium strains remained present, although not dominant, after long-term bioreactor operation. Different new bacterial populations did also appear in the systems, some of which were unable to degrade DCM.

The occurrence of in situ degradation of chlorinated ethenes was investigated using an integrated approach in a complex groundwater system consisting of several geological units. The assessment of hydrogeochemistry and chlorinated ethenes distribution using principal component analysis (PCA) in combination with carbon stable isotope analysis revealed that chlorinated ethenes were subjected to substantial biodegradation. Shifts in isotopic values up to 20.4 per thousand, 13.9 per thousand, 20.1 per thousand and 31.4 per thousand were observed between geological units for tetrachloroethene (PCE), trichloroethene (TCE), cis-dichloroethene (cDCE) and vinyl chloride (VC), respectively. The use of specific biomarkers (16S rRNA gene) indicated the presence of Dehalococcoides sp. DNA in 20 of the 33 evaluated samples. In parallel, the analysis of changes in the bacterial community composition in the aquifers using canonical correspondence analysis (CCA) indicated the predominant influence of the chlorinated ethene concentrations (56.3% of the variance, P=0.005). The integrated approach may open new prospects for the assessment of spatial and temporal functioning of bioattenuation in contaminated groundwater systems.

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.

A novel concept was developed applying radioisotope-labelled substrate incorporation into the biomass. The resulting radiolabelled RNA was used both as an indicator of activity and as a template for gaining structural and functional information about a substrate-utilizing microbial community. Sequences of PCR products are separated via cloning or using molecular fingerprinting techniques. Nucleic acids from predominant clones or the whole molecular fingerprinting pattern are transferred to a membrane and hybridized with the radiolabelled sample RNA. Scanning of the hybridized blots for radioactivity indicates the members involved in the utilization of the substrate. This novel 'random walk' approach using radioisotope probing was evaluated in a model community experiment.

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.