Bioresource Technology 193 (2015) 42–52 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Impact of the substrate loading regime and phosphoric acid supplementation on performance of biogas reactors and microbial community dynamics during anaerobic digestion of chicken wastes Dmitry E. Belostotskiy a, Elvira E. Ziganshina b, Maria Siniagina c, Eugenia A. Boulygina c, ⇑ Vasili A. Miluykov a, Ayrat M. Ziganshin b, a Department of Technologies, A.E. Arbuzov Institute of Organic and Physical Chemistry, RAN, Kazan 420088, The Republic of Tatarstan, Russia b Department of Microbiology, Kazan (Volga Region) Federal University, Kazan 420008, The Republic of Tatarstan, Russia c Laboratory of Omics Technologies, Kazan (Volga Region) Federal University, Kazan 420008, The Republic of Tatarstan, Russia highlights Anaerobic digestion of chicken wastes under various conditions was investigated. Phosphoric acid at moderate level positively affected the anaerobic digestion process. 454 pyrosequencing approach was used to evaluate the microbial community diversity. Bacteroidales, Erysipelotrichaceae, Clostridium, Methanosarcina were the abundant taxa. The major process parameter shaping community structure was the high ammonia level. article info abstract Article history: This study evaluates the effects of increasing organic loading rate (OLR) and decreasing hydraulic retention Received 24 April 2015 time (HRT) as well as phosphoric acid addition on mesophilic reactors’ performance and biogas production Received in revised form 9 June 2015 from chicken wastes. Furthermore, microbial community composition in reactors was characterized by a Accepted 13 June 2015 16S rRNA gene-based pyrosequencing analysis. Each step of increasing OLR impacted on the activity of Available online 18 June 2015 microorganisms what caused a temporary decrease in biogas production. The addition of phosphoric acid À1 resulted in the increased biogas production with values between 361 and 447 mL gVS from day 61 to day 74 Keywords: compared to control reactor (309–350 mL gÀ1). With reactors’ operation, Bacteroidetes phylotypes were Biogas VS noticeably replaced with Firmicutes representatives, and significant increase of Clostridium sp. was identi- Chicken wastes Phosphoric acid fied. Within Euryarchaeota, Methanosarcina sp. dominated in all analyzed samples, in which high + À1 Ammonia inhibition ammonium levels were detected (3.4–4.9 NH4-N g L ). These results can help in better understanding Microbial diversity the anaerobic digestion process of simultaneously ammonium/phosphate-rich substrates. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Research activities in the field of anaerobic digestion processes have recently received much attention, particularly focusing on Anaerobic digestion has received significant importance within the influence of different operational and environmental parame- the last 20–30 years. Biogas as a representative of renewable ters on different types of biogas reactors’ performance, optimiza- energy sources can be obtained via biological degradation of vari- tion of anaerobic digestion processes and microbiology of ous organic waste materials (e.g. agricultural residues and munic- anaerobic digesters (Niu et al., 2013a). ipal solid wastes) under anaerobic conditions. This anaerobic Anaerobic treatment of poultry manure is more problematic process leads to organic waste utilization simultaneously with than anaerobic digestion of manure from other farm animals, since the energy generation, thus solving society’s most urgent needs, poultry wastes contain high amounts of nitrogen and their anaer- effective wastes disposal and alternative clean energy production. obic conversion can result in ammonia inhibition process. Anaerobic digestion of chicken wastes with high content of uric acid and undigested proteins results in the production of toxic ⇑ Corresponding author. Tel.: +7 843 233 7872. un-ionized free ammonia (NH3, FAN) and ionized ammonium ions E-mail address: [email protected] (A.M. Ziganshin). http://dx.doi.org/10.1016/j.biortech.2015.06.066 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved. D.E. Belostotskiy et al. / Bioresource Technology 193 (2015) 42–52 43 + (NH4). Nitrogen is a macronutrient that is required in large quanti- Except nitrogen, phosphorous is a macronutrient that is ties by bacteria and methanogenic archaea. However, even some required in relatively large quantities by bacteria and methano- ammonium compounds are beneficial for microbial growth, free genic archaea, playing an important role in their metabolism. It ammonia above critical concentrations has been considered as was previously established in batch tests that the addition of phos- the cause of inhibition of microorganisms during anaerobic diges- phate at values within 414–465 mg P LÀ1 accelerated the anaerobic tion of nitrogen-rich substrates (Abouelenien et al., 2010). digestion processes (Lei et al., 2010; Wang et al., 2015). However, Especially ammonia is the most significant inhibitor of the last these experiments were performed in the presence of low ammo- stage, methanogenesis, and therefore high amounts of NH3 can nia concentrations, indicating the necessity to conduct additional decrease overall specific methane production (Niu et al., 2013b; experiments devoted to anaerobic conversion of simultaneously Lv et al., 2014). Different environmental parameters, mostly rising phosphorus-rich and ammonium-rich substrates. In addition, it is + temperature and pH, impact on balance of NH3/NH4 (total ammo- significant to evaluate the effect of phosphate/phosphoric acid nia nitrogen, TAN) and can lead to the increased effect of toxicity of addition on the activity of microbial communities during anaerobic ammonia on anaerobic microorganisms (Garcia and Angenent, digestion of such wastes. Furthermore, co-digestion of various 2009). In addition to toxicity, chicken wastes contain a fraction ammonium-rich and phosphorus-rich substrates can be used to of wood chips and shavings used as the bedding materials, and improve the quality of fertilizers. Since free ammonia levels drop therefore such wastes with high NH3 and lignocellulosic content in parallel with pH fall, the addition of phosphoric acid to digesting are less amenable to anaerobic destruction with biogas production mixture can be considered as another strategy to stabilize pH and (Costa et al., 2012; Ziganshina et al., 2014a). also to reduce FAN concentrations in the ammonium-rich anaero- Anaerobic digestion depends on the coordinated activity of bic digestion systems. various microbial groups and involves several consecutive In the present study, the first goal was to study the effect of destruction stages, such as hydrolysis, acidogenesis, acetogenesis increasing OLR and decreasing HRT on mesophilic continuously and finally methanogenesis. The activity and fate of distinct stirred tank laboratory reactors’ performance during anaerobic groups of microorganisms make the study of anaerobic digestion digestion of ammonium-rich chicken wastes. The second goal more challenging. The last step of anaerobic digestion consists of was to gain insight regarding the influence of phosphoric acid addi- aceticlastic, hydrogenotrophic and methylotrophic methanogene- tion on the efficiency of biogas production from chicken wastes. sis (Demirel and Scherer, 2008). Since aceticlastic methanogenic Finally, bacterial and archaeal community structure and dynamics archaea are more susceptible to process disturbances during in lab-scale reactors were characterized using a 16S rRNA gene anaerobic digestion, hydrogenotrophic methanogenesis is consid- amplicon 454 pyrosequencing approach. The main factors affecting ered to be the main methanogenic pathway at high ammonia microbial community shifts were also evaluated by nonmetric concentrations. The released acetate is biodegraded via syn- multidimensional scaling analysis. trophic acetate oxidation (SAO) pathway, and SAO coupled with hydrogenotrophic methanogenesis has been reported to be the 2. Methods predominant pathway at high ammonia levels in distinct anaero- bic reactor systems (Schnürer and Nordberg, 2008; Fotidis et al., 2.1. Chicken wastes properties 2014). On the contrary, aceticlastic methanogenesis performed by the activity of acclimatized Methanosarcinaceae at high ammo- + À1 À1 The chicken wastes with total solids (TS) of 78.28 ± 0.4%, vola- nium (5–7 NH4-N g L ) and acetate (9 g L ) concentrations was tile solids (VS) of 69.79 ± 0.7%, total nitrogen of 3.35 ± 0.11% and recently demonstrated in batch tests (Fotidis et al., 2013). total phosphorous of 3.04 ± 0.12% were collected from a poultry Therefore, the influence of different ammonia concentrations on farm located in the Zelenodolsky district, the Republic of the methanogenic pathways and microbial community structure Tatarstan (Russia) and stored in a refrigerator at 4 °C. These wastes should be further evaluated. In addition, it is important to enrich contained chicken manure and a fraction of chicken feathers with or construct highly adapted bacterial and archaeal communities wood shavings. After calculating the necessary OLR and HRT, the which can be used as remedy in case of inhibition of anaerobic desired amount of chicken wastes were diluted with tap water, digestion process. thoroughly stirred and transferred into continuously stirred tank To avoid inhibition effects during anaerobic digestion of laboratory reactors