Growth and activity of ANME clades with different sulfate and sulfide concentrations in presence of methane

Extensive geochemical data showed that significant methane oxidation activity exists in marine sediments. The organisms responsible for this activity are anaerobic methane-oxidizing archaea (ANME) that occur in consortia with sulfate-reducing bacteria. A distinct zonation of different clades of ANME (ANME-1, ANME-2a/b and ANME-2c) exists in marine sediments, which could be related to the localized concentrations of methane, sulfate and sulfide. In order to test this hypothesis we performed long-term incubation of marine sediments under defined conditions with methane as a headspace gas: low or high sulfate (?4 and ?21 mM, respectively) in combination with low or high sulfide (?0.1 and ?4 mM, respectively) concentrations. Control incubations were also performed, with only methane, high sulfate or high sulfide. Methane oxidation was monitored and growth of subtypes ANME-1, ANME-2a/b, and ANME-2c assessed using qPCR analysis. A preliminary archaeal community analysis was performed to gain insight into the ecological and taxonomic diversity. Almost all of the incubations with methane had methane oxidation activity, with the exception of the incubations with combined low sulfate and high sulfide concentrations. Sulfide inhibition occurred only with low sulfate concentrations, which could be due to the lower Gibbs free energy available as well as sulfide toxicity. ANME-2a/b appear to mainly grow in incubations which had high sulfate levels and methane oxidation activity, whereas ANME-1 did not show this distinction. ANME-2c only grew in incubations with only sulfate addition. These findings are consistent with previously published in situ profiling analysis of ANME subclusters in different marine sediments. Interestingly, since all ANME subtypes also grew in incubations with only methane or sulfate addition, ANME may also be able to perform anaerobic methane oxidation under substrate limited conditions or alternatively perform additional metabolic processes.We want to thank all reviewers for constructive comments, Joan Edwards (Laboratory of Microbiology, WUR) for extensive proof-reading, Bartholomeus van den Bogert (Laboratory of Microbiology, WUR) for help with the MiSeq sequencing and Diego A. Suarez-Zuluaga (Environmental Technology, WUR) for help with carbon dioxide calculations. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

Identification of syntrophic butyrate degrading community with stable isotope probing technique in anaerobic bioreactors

Bütirat, metanojenik şartlar altında organik madde dönüşümünde önemli bir ara ürün olup havasız biyoreaktörlerde metanojenezin %60’ını kapsayabilen bir maddedir. Organik maddenin ayrışması sırasında oluşan hidrojen ve/veya format, ortamdan uzaklaştırılmadığı sürece bütiratın ayrışması termodinamik açıdan mümkün değildir. Bütiratın ayrışması hidrojen tüketen organizmalarla yapılan sintrofik etkileşimlere dayalı olup ayrışma mekanizması ile ilgili bilgiler saf kültür çalışmalarıyla sınırlıdır. Bu açıdan bakıldığında, bütirat ayrışmasını yapan sintrofik bakterilerin çeşitliliği ve ekolojisi birçok bilinmeyen özelliği içermektedir Bu kapsamda yeni bir teknik olan stabil izotop işaretlemesi kullanılarak havasız ortamlardaki bütiratı ayrıştıran aktif mikroorganizmaların kimliği tespit edilmiştir. Sintrofik bütirat ayrıştıran bu aktif türlerin, filogenetik olarak bir gruba ait olmadığı ve 9 farklı filum içerisinde yer aldığı bulunmuştur. Ayrıca bu çalışmada saf kültür tanımlaması yapılmamış türlerin varlığına rastlanmıştır. 16S ribozomal ribo nükleik asit sekans analizleri belirlenen klonların, veri bankalarında yapılan karşılaştırmalı analizinde bakteriyel türlerin çok çeşitli metabolik aktivitelere sahip olabileceklerini ve büyük bir kısmının havasız ortamlardan izole edilmiş klonlara benzerlik gösterdiği bulunmuştur. Özellikle, Proteobacteria filumunda yer alan klonlara benzerlik gösteren türlerin baskın olduğu belirlenmiştir. Bunun yanında sintrofik bütirat ayrışmasında başlıca Syntrophus sp. türünün önemli rol aldığı tespit edilmiştir. Bu bulgu, şimdiye kadar kabul gören bütiratı ayrıştıran Syntrophomonas türlerinin ait olduğu Firmicutes’ten farklı olduğunu göstermiştir. Bununla birlikte sintrofik propiyonat oksitleyen Syntrophobacter türünün de bütirat gideriminde aktif rol oynadığı tespit edilmiştir. Anahtar kelimeler: Havasız arıtma, moleküler biyolojik teknikler, stabil izotop işaretlemesi.Anaerobic treatment is presently accepted as a sustainable technology for a wide range of wastewater and waste types; and its applicability is growing each year. An important intermediate of organic matter conversion under methanogenic conditions is butyrate; which may account for up to 60 % of methanogenesis in anaerobic bioreactors. The degradation of butyrate is thermodynamically not favorable unless the H2 and/or formate can be removed by one of the hydrogen consumers. To proceed this reaction, the hydrogen level should be kept below 10-4to 10-5 atm. Butyrate oxidation requires syntrophic interactions between -oxidizing, hydrogen producing bacteria and hydyrogen and/or acetate utilizers. To date, several butyrate as well as some long-chain fatty acids (up to C18) oxidizing bacteria have been isolated in co-culture with hydrogen utilizing partner. This could be either methanogenic or sulfidogenic micro-organisms. The information on the degradation of butyrate is limited to the pure cultures. Diversity and ecology of syntrophic butyrate- degrading bacteria is sharing this unknown characteristic and is waiting to be explored. In this concept, the novel SIP technique was used in this study to identify the key microorganisms of the syntrophic butyrate degrading communities. The SIP incubation with 13C labeled butyrate was carried out on the wild anaerobic granular sludge of Eerbeek paper mill wastewater treatment plant, in the presence of sulfate (3 mM). It is very difficult to assess the best method for observing the most active microorganisms in mixed cultures. In this study, to reveal the genetic diversity of the complex microbial diversity, the conceptual design of the experiments were conducted to mimic in situ conditions to approach more real conditions as much as possible. For example, the 13C labelled butyrate was fed together with the actual wastewater, which was fed to the full scale anaerobic bioreactor. Since the feeding conditions were not changed from the actual situation, the syntrophic butyrate degraders would be selectively separated by the density of the nucleic acids. These results can be assigned directly to natural systems, assuming the same environmental conditions. Subsequently applied Denaturing Gradient Gel Electrophoresis (DGGE) profiling method was also useful for following the changes of the presence of species along the centrifugation gradient, and was also helpful for observing the heavy and light fraction differences. The composition of the bacteria and archaea community in the syntrophic butyrate degradation environment in the full-scale upflow anaerobic sludge blanket reactor of paper mill wastewater was determined by the 16S rRNA phylogenetic analyses of clone libraries derived from RNA extracted from the density resolved gradient of the SIP. Around 120 bacterial and 24 archaeal clones from each heavy and light fraction of the enrichment 16S rRNA gene libraries constructed from the original sludge were analyzed by comparing the DGGE and Terminal-Restriction Fragment Length Polymorphism (T-RFLP) fragment patterns of the amplified 16S rRNA genes. This diverse active member of the syntrophic butyrate degraders were represented by grouping in 9 different phyla, which showed more diversity than recent studies of the bacteria capable of syntrophic metabolism in terms of both phylogenetics and physiology. This functional group of organisms did not fall into the phylogenetically consistent groups, rather, it spread out into several lineages. In most cases, the closest uncultured relatives have been identified from anaerobic ecosystems. These were the majority of the microbial community associated with deep subsurface aquifer, anaerobic dechlorinating mixed cultures equine fecal contaminated sites and bioreactors. Sequence representatives of several bacterial divisions have been identified in a wide range of habitats, suggesting the sophisticated distribution of the corresponding organisms in the environment and, potentially, their wide metabolic capabilities. The 16S rRNA gene clone library showed that the largest groups of clones belonged to the members of the Proteobacteria, which were not what was expected from the community of syntrophic butyrate degradation that belong to the phyla of Firmicutes. The main possible role of the butyrate degradation was attained to the Syntrophus sp., Sequence types associated with the genus Syntrophus sp. can produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. However, it was also found that the Syntrophobacter sp., known as propionate degrader, also played an active role in the butyrate degradation. By using these techniques, potential roles of the strain specific microorganisms involved in the syntrophic butyrate degradation were achieved. Keywords: Anaerobic treatment, molecular biological techniques, stable isotope probing

Butyrate conversion by sulfate-reducing and methanogenic communities from anoxic sediments of Aarhus Bay, Denmark

The conventional perception that the zone of sulfate reduction and methanogenesis are separated in high-and low-sulfate-containing marine sediments has recently been changed by studies demonstrating their co-occurrence in sediments. The presence of methanogens was linked to the presence of substrates that are not used by sulfate reducers. In the current study, we hypothesized that both groups can co-exist, consuming common substrates (H2 and/or acetate) in sediments. We enriched butyrate-degrading communities in sediment slurries originating from the sulfate, sulfate–methane transition, and methane zone of Aarhus Bay, Denmark. Sulfate was added at different concentrations (0, 3, 20 mM), and the slurries were incubated at 10◦ C and 25◦ C. During butyrate conversion, sulfate reduction and methanogenesis occurred simultaneously. The syntrophic butyrate degrader Syntrophomonas was enriched both in sulfate-amended and in sulfate-free slurries, indicating the occurrence of syntrophic conversions at both conditions. Archaeal community analysis revealed a dominance of Methanomicrobiaceae. The acetoclastic Methanosaetaceae reached high relative abundance in the absence of sulfate, while presence of acetoclastic Methanosarcinaceae was independent of the sulfate concentration, temperature, and the initial zone of the sediment. This study shows that there is no vertical separation of sulfate reducers, syntrophs, and methanogens in the sediment and that they all participate in the conversion of butyrate.</p

Multi-production of high added market value metabolites from diluted methane emissions via methanotrophic extremophiles

Producción CientíficaThis study constitutes the first-proof-of-concept of a methane biorefinery based on the multi-production of high profit margin substances (ectoine, hydroxyectoine, polyhydroxyalkanoates (PHAs) and exopolysaccharides (EPS)) using methane as the sole carbon and energy source. Two bubble column bioreactors were operated under different magnesium concentrations (0.2, 0.02 and 0.002 g L−1) to validate and optimize this innovative strategy for valorization of CH4 emissions. High Mg2+ concentrations promoted the accumulation of ectoine (79.7–94.2 mg g biomass−1), together with high hydroxyectoine yields (up to 13 mg g biomass−1) and EPS concentrations (up to 2.6 g L culture broth−1). Unfortunately, PHA synthesis was almost negligible (14.3 mg L−1) and only found at the lowest Mg2+ concentration tested. Halomonas, Marinobacter, Methylophaga and Methylomicrobium, previously described as ectoine producers, were dominant in both bioreactors, Methylomicrobium being the only described methanotroph. This study encourages further research on CH4 biorefineries capable of creating value out of GHG mitigation.Ministerio de Economía, Industria y Competitividad (Project CTM2015-70442-RNOVEDAR)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. E-47-2014-0140696UIC71

Long-term acclimation of anaerobic sludges for high-rate methanogenesis from LCFA

Inhibition of methanogens by long chain fatty acids (LCFA) and the low numbers of LCFA-degrading bacteria are limitations to exploit biogas production from fat-rich wastewaters. Generally reactors fail due to excessive LCFA accumulation onto the sludge. Here, long-term acclimation and bioaugmentation with a LCFA-degrading coculture were hypothesized as strategies to enhance methanogenic conversion of these compounds. Anaerobic sludges previously exposed to LCFA for more than 100 days converted a specific biomass-associated substrate of (3.2 ± 0.1) kg·kg−1 with very short lag phases (<1 day), whereas non-acclimated sludges showed lag phases of 11–15 days for metabolizing (1.6–1.8) kg·kg−1. Addition of a coculture of Syntrophomonas zehnderi and Methanobacterium formicicum to sludges previously loaded with LCFA and containing different amounts of biomass-associated substrate (from (0.5–3.2) kg·kg−1) did not improve methane production neither lag phases were shortened, indicating that the endogenous microbiota are not a limiting factor. Clearly, we show that long-term sludge acclimation to LCFA is essential for high rate methanogenesis from LCFA.The authors acknowledge the financial support by the European Regional Development Fund - ERDF, through the Operational Program Thematic Factors of Competitiveness - COMPETE, and by Portuguese funds, through the Portuguese Foundation for Science and Technology (FCT), in the frame of the project FCOMP-01-0124-FEDER-014784. FCT Strategic Project PEst-OE/EQB/LA0023/2013 is also acknowledged. A.J. Cavaleiro thanks FCT for the post-doctoral fellowship ref. SFRH/BPD/75247/2010. A.J.M. Stams has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. [323009]

Novel haloalkaliphilic methanotrophic bacteria: An attempt for enhancing methane bio-refinery

Producción CientíficaMethane bioconversion into products with a high market value, such as ectoine or hydroxyectoine, can be optimized via isolation of more efficient novel methanotrophic bacteria. The research here presented focused on the enrichment of methanotrophic consortia able to co-produce different ectoines during CH4 metabolism. Four different enrichments (Cow3, Slu3, Cow6 and Slu6) were carried out in basal media supplemented with 3 and 6% NaCl, and using methane as the sole carbon and energy source. The highest ectoine accumulation (∼20 mg ectoine g biomass−1) was recorded in the two consortia enriched at 6% NaCl (Cow6 and Slu6). Moreover, hydroxyectoine was detected for the first time using methane as a feedstock in Cow6 and Slu6 (∼5 mg g biomass−1). The majority of the haloalkaliphilic bacteria identified by 16S rRNA community profiling in both consortia have not been previously described as methanotrophs. From these enrichments, two novel strains (representing novel species) capable of using methane as the sole carbon and energy source were isolated: Alishewanella sp. strain RM1 and Halomonas sp. strain PGE1. Halomonas sp. strain PGE1 showed higher ectoine yields (70–92 mg ectoine g biomass−1) than those previously described for other methanotrophs under continuous cultivation mode (∼37–70 mg ectoine g biomass−1). The results here obtained highlight the potential of isolating novel methanotrophs in order to boost the competitiveness of industrial CH4-based ectoine production.Ministerio de Economía, Industria y Competitividad (Project CTM2015-70442-R project and Red NOVEDAR)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. UIC71)European Union through the FEDER Funding Program (PhD Grant contract Nº E-47-2014-0140696

Enrichment of anaerobic syngas-converting bacteria from thermophilic bioreactor sludge

Thermophilic (55 °C) anaerobic microbial communities were enriched with a synthetic syngas mixture (composed of CO, H2, and CO2) or with CO alone. Cultures T-Syn and T-CO were incubated and successively transferred with syngas (16 transfers) or CO (9 transfers), respectively, with increasing CO partial pressures from 0.09 to 0.88 bar. Culture T-Syn, after 4 successive transfers with syngas, was also incubated with CO and subsequently transferred (9 transfers) with solely this substrate – cultures T-Syn-CO. Incubation with syngas and CO caused a rapid decrease in the microbial diversity of the anaerobic consortium. T-Syn and T-Syn-CO showed identical microbial composition and were dominated by Desulfotomaculum and Caloribacterium species. Incubation initiated with CO resulted in the enrichment of bacteria from the genera Thermincola and Thermoanaerobacter. Methane was detected in the first two to three transfers of T-Syn, but production ceased afterward. Acetate was the main product formed by T-Syn and T-Syn-CO. Enriched T-CO cultures showed a two-phase conversion, in which H2 was formed first and then converted to acetate. This research provides insight into how thermophilic anaerobic communities develop using syngas/CO as sole energy and carbon source can be steered for specific end products and subsequent microbial synthesis of chemicals.This study has been financially supported by FEDER funds through the Operational Competitiveness Programme (COMPETE) and by national funds through the Portuguese Foundation for Science and Technology (FCT) in the frame of the project FCOMP-01-0124-FEDER-027894. Financial support from FCT and the European Social Fund (POPH-QREN) through the PhD grant SFRH/BD/48965/2008 attributed to J.I.A. is gratefully acknowledged. A.J.M. Stams acknowledges grants from CW-TOP 700.55.343 and ALW 819.02.014 of the Netherlands Science Foundation (NWO), Consolider-CSD 2007-00055 and ERC (323009)

A genomic view on syntrophic versus non-syntrophic lifestyle in anaerobic fatty acid degrading communities

In sulfate-reducing and methanogenic environments complex biopolymers are hydrolyzed and degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate-reduction. Here we study from a genome perspective why some of these micro-organisms are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases (InterPro domain number; IPR006443), including their maturation protein FdhE (IPR024064 and IPR006452) is a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production (IPR019079) and a second in cell division, shape-determination or sporulation (IPR018365). The sulfate-reducing bacteria Desulfobacterium autotrophicum HRM2, Desulfomonile tiedjei and Desulfosporosinus meridiei were never tested for syntrophic growth, but all crucial domains were found in their genomes, which suggests their possible ability to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnf-complex and the formate transporter in syntrophy, and indicate that DUF224 may have a role in electron transfer in bacteria other than Syntrophomonas wolfei as well. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.This research was financed by grants of BE-Basic (project 7.2.3.), the Technology Foundation, the Applied Science Division (STW) (project 11603) and the Divisions CW and ALW (projects 700.55.343 and 819.02.014) of the Netherlands Science Foundation (NWO) and ERC (project 323009). Furthermore, this work was carried out on the Dutch national e-infrastructure with the support of

Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFAaccumulation/ degradation cycles.Fundação para a Ciência e a Tecnologia (FCT); Fundo Social Europeu (FSE)

Ecology and application of haloalkaliphilic anaerobic microbial communities

Haloalkaliphilic microorganisms that grow optimally at high-pH and high-salinity conditions can be found in natural environments such as soda lakes. These globally spread lakes harbour interesting anaerobic microorganisms that have the potential of being applied in existing technologies or create new opportunities. In this review, we discuss the potential application of haloalkaliphilic anaerobic microbial communities in the fermentation of lignocellulosic feedstocks material subjected to an alkaline pre-treatment, methane production and sulfur removal technology. Also, the general advantages of operation at haloalkaline conditions, such as low volatile fatty acid and sulfide toxicity, are addressed. Finally, an outlook into the main challenges like ammonia toxicity and lack of aggregation is provided.This work was performed in the TTIW- cooperation framework of Wetsus, European Centre of Excel- lence for Sustainable Water Technology (www.wetsus.nl). Wetsus is funded by the Dutch Ministry of Economic Affairs, the European Union Regional Development Fund, the Province of Fryslân, the City of Leeuwarden and the EZ/Kompas program of the“ Samenwerkingsverband Noord-Nederland”. The authors would like to thank the participants of the research theme "Sulfur", namely Paqell, for fruitful discussions and financial suppor