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