J. Microbiol. Biotechnol. (2014), 24(11), 1464–1472 http://dx.doi.org/10.4014/jmb.1404.04043 Research Article Review jmb Microbial Community Diversity in Anaerobic Reactors Digesting Turkey, Chicken, and Swine Wastes Elvira E. Ziganshina1, Dmitry E. Belostotskiy2, Roman V. Shushlyaev2, Vasili A. Miluykov2, Petr Y. Vankov1, and Ayrat M. Ziganshin1* 1Department of Microbiology, Kazan (Volga Region) Federal University, Kazan 420008, Republic of Tatarstan, Russia 2Department of Technologies, A.E. Arbuzov Institute of Organic and Physical Chemistry, Russian Academy of Sciences, Kazan 420088, Republic of Tatarstan, Russia Received: April 24, 2014 Revised: June 11, 2014 The microbial community structures of two continuous stirred tank reactors digesting turkey Accepted: July 11, 2014 manure with pine wood shavings as well as chicken and swine manure were investigated. The First published online reactor fed with chicken/swine wastes displayed the highest organic acids concentration (up to July 15, 2014 15.2 g/l) and ammonia concentration (up to 3.7 g/l ammonium nitrogen) and generated a higher *Corresponding author biogas yield (up to 366 ml/gVS) compared with the reactor supplied with turkey wastes (1.5– Phone: +7-843-233-7872; 1.8 g/l of organic acids and 1.6–1.7 g/l of ammonium levels; biogas yield was up to 195 ml/gVS). Fax: +7-843-238-7121; E-mail: a.ziganshin06@fulbright- The microbial community diversity was assessed using both sequencing and profiling terminal mail.org restriction fragment length polymorphisms of 16S rRNA genes. Additionally, methanogens were analyzed using methyl coenzyme M reductase alpha subunit (mcrA) genes. The bacterial community was dominated by members of unclassified Clostridiales with the prevalence of specific clostridial phylotypes in each reactor, indicating the effect of the substrate type on the community structure. Of the methanogenic archaea, methanogens of the genus Methanosarcina were found in high proportions in both reactors with specific methanosarcinas in each reactor, whereas the strict hydrogenotrophic methanogens of Methanoculleus sp. were found at significant levels only in the reactor fed with chicken/swine manure (based on the analyses of 16S rRNA gene). This suggests that among methanogenic archaea, Methanosarcina species which have different metabolic capabilities, including aceticlastic and hydrogenotrophic pISSN 1017-7825, eISSN 1738-8872 methanogenesis, were mainly involved in anaerobic digestion of turkey wastes. Copyright© 2014 by The Korean Society for Microbiology Keywords: Anaerobic digestion, agricultural wastes, 16S rRNA genes, mcrA genes and Biotechnology Introduction with European objectives regarding the partial substitution of fossil fuels with alternative energy sources and limitation The livestock industry is the active sector with generation of global warming, the bioenergy production from renewable of large amounts of wastes. The existing ecological problems sources, including biogas, is among the main priorities [4]. dictate the need for the development of safe and at the However, in order to reduce gas emissions, it is also same time effective methods for the residual biomass recommended to focus on digestates storage and exhaust disposal. Undoubtedly, one of such ways avoiding the of the co-generation [27]. accumulation of wastes in the environment is the anaerobic Various agricultural and industrial waste materials can digestion of biomass with concomitant production of be anaerobically converted to energy-rich biogas by methane, and after the digestion process the effluent can be complex microbial consortia [8, 15, 29, 36]. The first three used as a fertilizer [2, 8, 15]. Anaerobic digestion of stages of anaerobic digestion – hydrolysis, acidogenesis multifarious organic waste materials is also a promising and acetogenesis – are mediated by various functional way of reducing greenhouse gas emissions. In accordance groups of bacteria, whereas the last step – methanogenesis November 2014 ⎪ Vol. 24⎪ No. 11 1465 Ziganshina et al. – is performed by methanogenic archaea. Many research individually is not effective for the full-scale production of works in the field of biogas generation process are devoted methane [13, 37]. Poultry manure is a good source for to the investigation of the diversity and abundance of biogas production; however, excess of ammonia in the bacterial and archaeal communities, which play the key biogas reactors treating poultry dung may represent a role in the anaerobic treatment of a variety of organic digestion inhibition risk as well [1, 31]. Co-digestion of materials. Many different molecular techniques allow the poultry dung with some other livestock manures can reliable evaluation of biodiversity in anaerobic digesters as improve the biogas production [18, 29]. well as the monitoring of community shifts. Thus, Firmicutes, Therefore, for the efficacious production of biogas, we Bacteroidetes, Actinobacteria, Synergistetes, Proteobacteria, Tenericutes, have to deepen our knowledge in the area of anaerobic Spirochaetes, and Chloroflexi as well as some other taxa are digestion process optimization. This involves the study of found as the abundant bacterial phyla in various anaerobic the main biotechnological parameters of biogas generation digesters. Of the Euryarchaeota, methanogens of the orders as well as the investigation of microbes responsible for the Methanosarcinales, Methanomicrobiales, Methanobacteriales, and effective anaerobic transformation of distinct organic materials Methanococcales are known to be present in different biogas into methane. In this work, bacteria and methanogenic reactors [23, 30-33, 38, 40, 43]. In addition, metaproteome archaea of two continuous stirred tank reactors treating analysis is also used to characterize microbial community farm residues, namely turkey manure with pine wood changes in biogas reactors [19, 21]. Monitoring of the microbial shavings as well as chicken manure and swine manure, community structure and dynamics during anaerobic were investigated by sequencing and profiling terminal treatment of biowastes can lead to the development of restriction fragment length polymorphisms of 16S rRNA stable and adapted communities, what can finally increase genes. Additionally, methanogens were analyzed based on the maximum methane yield. the mcrA (methyl coenzyme M reductase) gene profiles. Bioconversion of recalcitrant substrates (e.g., lignocellulosic biomass, wood chips, and wood shavings used as the Materials and Methods bedding materials) under anaerobic conditions in most cases is limited; what is required is the development and Performance of the Biogas Reactors and Analytical Methods implementation of new technologies for the production of Mesophilic (38oC) anaerobic experiments were carried out in biogas from such materials [35, 42]. Enhancement of the two laboratory-scale continuous stirred tank reactors with a anaerobic digestion process can be achieved through co- working volume of 10 L (Table 1). The reactor R.1 was supplied digestion of several substrates [15, 22, 36, 45]. Swine with wastes containing turkey manure and pine wood shavings, manure is often used as the feedstock for many large-scale whereas the reactor R.2 was fed with chicken manure and swine biogas plants to produce methane for energy purposes. manure. Turkey wastes were received from a poultry farm located However, owing to the low carbon/nitrogen ratio (deficiency in the Zelenodolsky district, Republic of Tatarstan. Chicken and of carbon), the anaerobic treatment of swine manure swine wastes were obtained from poultry and pig farms located in Table 1. Operating conditions and process parameters of the two laboratory-scale biogas reactors. Organic Inlet, g/day (VS, g/day) Biogas Acid + Hydraulic loading Methane NH4 -N, Reactor Sample Turkey Chicken Swine yield, pH capacity, retention rate, Total content, % g/l –1 –1 wastes manure manure ml/gVS g/l time, days gVS l day R.1 1 2.60 71.5 – – 360.5a 193 ± 14 60.6 ± 0.6 7.90 ± 0.08 1.47 ± 0.09 1.71 ± 0.06 27.7 2 (26.0) (26.0) 195 ± 21 61.6 ± 0.5 7.91 ± 0.07 1.82 ± 0.11 1.70 ± 0.08 27.7 3 189 ± 12 61.9 ± 0.6 7.92 ± 0.07 1.68 ± 0.09 1.63 ± 0.09 27.7 R.2 1 3.53 – 79.6 365.9 445.5 342 ± 37 56.3 ± 0.6 8.09 ± 0.07 15.2 ± 0.14 3.71 ± 0.12 22.4 2 (29.0) (6.3) (35.3) 366 ± 41 56.8 ± 0.7 8.04 ± 0.06 14.8 ± 0.17 3.26 ± 0.14 22.4 3 345 ± 32 58.1 ± 0.6 8.05 ± 0.06 13.9 ± 0.17 3.32 ± 0.17 22.4 Parameters at three distinct sampling times are presented (19 February 2013; 26 February 2013; 5 March 2013). + Biogas yield, methane content, and pH are shown as weekly mean values (3 days before and 3 days after sampling; n = 7). Acid capacity and NH4 -N data are presented as the average values of a sample measured three times (n = 3). aWater was added to a final concentration of 360.5 g/day. J. Microbiol. Biotechnol. Microbial Community Diversity in Anaerobic Reactors 1466 the Tukayevsky district, Republic of Tatarstan. Substrates were genes were cleaved with the restriction endonucleases HaeIII and added to the digesters every day; digestates were removed from RsaI, archaeal 16S rRNA genes were digested with HaeIII and the reactors every day as well. Biogas yield, methane content, and MseI, and mcrA genes were cut with HaeIII and MspI. T-RFLP + pH were analyzed every day, while organic acids and NH4 -N was performed with three replicates for each restriction analysis levels were measured twice a week. All parameters were analyzed to ensure reproducibility. Noise removal, peak binning, and as described by us previously [43-46]. Briefly, biogas production normalization of signal intensity were done using an R script was analyzed with milligascounters MGC-1 (Ritter, Germany), (R ver. 2.12.2; http://www.r-project.org). methane content was measured by a gas analyzer GA 94 (Ansyco, Germany), NH +-N levels were analyzed with Nessler’s reagent 4 Results and Discussion (Sigma-Aldrich, Germany) in a SPECOL 1300 spectrophotometer (Analytik Jena, Germany), and acid capacity was estimated by a Performance of the Reactors titration method with 0.025-0.1 M H2SO4 in a pH of 4.5 and 3.5.