Analysis of propionate‐degrading consortia from agricultural biogas plants

Abstract In order to investigate the propionate‐degrading community of agricultural biogas plants, four propionate‐degrading consortia (Ap1a, N12, G12, and Wp2a) were established from different biogas plants which were fed with renewable resources. The consortia were cultivated in a batch for a period of 2–4 years and then analyzed in an 8‐week batch experiment for microbial succession during propionate degradation. Community shifts showed considerable propagation of Syntrophobacter sulfatireducens, Cryptanaerobacter sp./Pelotomaculum sp., and “Candidatus Cloacamonas sp.” in the course of decreasing propionate concentration. Methanogenic species belonged mainly to the genera Methanosarcina, Methanosaeta, and Methanoculleus. Due to the prevalent presence of the syntrophic acetate‐oxidizing species Tepidanaerobacter acetatoxydans and potentially autotrophic homoacetogenic bacteria (Moorella sp., Thermacetogenium sp.), a theoretical involvement of syntrophic acetate oxidation and autotrophic homoacetogenesis in stable and efficient propionate degradation was indicated. Considering theoretical Gibbs free energy values at different hydrogen partial pressures, it is noticeable that syntrophic acetate oxidation and autotrophic homoacetogenesis have the potential to counterbalance adverse hydrogen partial pressure fluctuations, stabilizing most probably continuous and stable propionate degradation.

biogas-producing biomass degradation (Krakat, Westphal, Schmidt, & Scherer, 2010;Theuerl et al., 2015;Wirth et al., 2012). In this respect, one of the challenges is the control of the propionate concentration by investigating microbial propionate degradation. Up to now, studies concentrating on propionate degradation in agricultural biogas plants have been underrepresented.
In addition, the complete conversion of propionate to methane and carbon dioxide (ΔG 0 ′ = −56.6 kJ per reaction) requires the formation of methane from acetate by acetoclastic methanogens (Stams, 1994).

| Nucleic acid extraction and domain-specific 16S rRNA gene amplification
Consortia samples of 2.5 ml were concentrated to <200 μl via centrifugation (5 min, 17,000 g) and transferred to bead tubes of the reactants from VWR International GmbH, Erlangen, Germany) according to the following program: 10 min initial denaturation at 95°C was followed by 40 constant cycles (1 min at 94°C, 1 min at °C, 2 min 72°C) and 10 min final elongation at 72°C.

| ARDRA analysis, DNA sequencing, and removal of chimeric sequences
The 16S rRNA clones were phylogenetically grouped via ARDRA analysis. All restriction enzymes (10 U μl −1 ) and respective buffers utilized in the ARDRA analysis were obtained from Thermo Fisher Scientific, Waltham, USA. Bacterial 16S rRNA gene clone amplicons derived from colony PCR were cut in two separate reactions with HhaI and HinfI restriction enzymes. Bacterial PCR amplicons (8.7 μl) were mixed with 0.3 μl restriction enzyme in 1 μl green buffer and incubated for 5 hr at 37°C. Archaeal ARDRA analysis was conducted according to (Stantscheff, 2013

| Domain-specific quantitative real-time PCR (qPCR)
Quantification of total bacteria and total archaea was determined according to (May et al., 2015), using an artificial DNA fragment for standard preparation and the primer combinations BAC338F/BAC805R and 931F/M1100R for bacterial and archaeal 16S rRNA gene-fragment amplification. The qPCR assays were performed using a realplex2 ep gradient S Mastercycler (Eppendorf AG, Hamburg, Germany) supported by the evaluation software realplex 2.2. Reactions were carried out using the iQ ™ SYBR ® Green Supermix (BioRad, Hercules, USA) applied into white EasyStrip snap tubes (Thermo Fisher Scientific).  Furthermore, the total bacterial and archaeal cell titer of the samples were analyzed using quantitative PCR.

| Propionate-oxidizing bacteria
Consortium Ap1a included Syntrophobacter sulfatireducens, a sulfate-reducing δ-proteobacterium, known for its syntrophic propionate-oxidizing activity . Its proportion of bacterial diversity rose in the course of progressing propionate degradation (Table 2). "Candidatus Cloacamonas sp." was also affiliated with propionate degradation. Its nearest species relation was "Candidatus Cloacamonas acidaminovorans" (92%-93% sequence identity), a so far uncultivated but genomically analyzed species, whose genome featured all the genes involved in propionate oxidation (Pelletier et al., 2008). It showed a considerable propagation in consortium Wp2a (Table 2) and might, therefore, have been involved in the propionate degradation of this consortium. A potentially propionateoxidizing key species of consortia N12 and Wp2a was Cryptanaerobacter sp./Pelotomaculum sp., whose sequences were related to
The latter was described as a syntrophic propionate-oxidizing species (De Bok et al., 2005). As the sequences did not exceed 97% sequence identity to any of the three species, it might have been a so far unknown species.
Tepidanaerobacter acetatoxydans was profoundly abundant throughout this analysis and could be detected in all samples (t 1 -t 3 ) of the four consortia (Table 2). Its proportion of the species composition declined constantly during cultivation in all four consortia. Its potential function in propionate degradation could be its capability to degrade acetate in syntrophy with hydrogenotrophic archaea, forming H 2 and CO 2 under very low hydrogen partial pressure. This species was also prevalent in negative consortium G12, possibly feeding on complex substrates of the added biomass filtrate or its degradation products (e.g., also acetate). In addition, consortium N12 exhibited a putative SAOB, whose 16S rRNA gene sequence was closely related to Syntrophaceticus schinkii and Thermacetogenium phaeum, however, it has below 97% 16S rRNA gene sequence identity.

| Hydrogen-oxidizing bacteria
H 2 consumption is essential for propionate degradation, due to its endergonic nature under elevated hydrogen partial pressure.
Thermacetogenium phaeum is even able to perform the reaction in both directions (Hattori et al., 2005) and was, therefore, mentioned above already. The potential role of AHA in propionate degradation may be the disposal of H 2 under rising H 2 partial pressure (e.g., if H 2 consumption drops behind H 2 formation).

| Propionate-forming bacteria
Since the positive consortia (Ap1a, N12, and Wp2a) degraded propionate efficiently, it was not surprising to find species which are able to form propionate. Aminobacterium colombiense is known for its syntrophic amino acid metabolism in coculture with methaneforming hydrogenotrophic methanogens. Syntrophic glutamate and α-ketoglutarate oxidation resulting in propionate formation were observed (Baena, Fardeau, Labat, Ollivier, Thomas et al., 1998).
Interestingly, within our analysis, A. colombiense was detected as a main cluster only in successfully propionate-degrading consortia ( Table 2). As transcriptomic analysis revealed potential amino acid transfer in syntrophic propionate-oxidizing cocultures (Kato et al., 2009;Sieber et al., 2012), A. colombiense might be affiliated in this respect. The nearest species relations of Sedimentibacter sp.
are S. hydroxybenzoicus and S. saalensis. These two species form propionate from acetate and pyruvate, respectively. They are involved in amino acid degradation as much as A. colombiense (Breitenstein et al., 2002;Zhang, Mandelco, & Wiegel, 1994).
T A B L E 3 Composition of archaeal 16S rRNA gene clones of the consortia Ap1a, G12, N12, and Wp2a after 39 days of incubation

| Methanogenic archaea
Archaeal species compositions of the four consortia were determined for samples t 2 after 39 d of incubation. Up to 12 archaeal 16S rRNA gene clones were analyzed as the species diversity was expected to be substantially lower compared to bacterial diversity ( These Methanosarcina species are able to utilize all propionate oxidation end products, H 2 , CO 2 , and acetate (Maestrojuán & Boone, 1991).
T A B L E 4 Gibbs free energy calculations of anaerobic metabolic reactions according to Zinder, 1984 In contrast, acetoclastic Methanosaeta and hydrogenotrophic Methanospirillum were the dominant methanogenic genera in an upflow anaerobic sludge blanket reactor running on molasses wastewater (Ban et al., 2013). A similar composition propagated within our propionate-degrading consortium Ap1a, whose propionate-degrading key species was Syntrophobacter sulfatireducens. Here, Methanosaeta concilii and Methanosaeta harundinacea were found with Methanoculleus receptaculi, whose electron donor usage is identical to that from Methanospirillum spp. (Kim & Gadd, 2008). Furthermore, our studies reveal, that genetically putative propionate-oxidizing Cloacimonete "Candidatus Cloacamonas sp." (Pelletier et al., 2008) actually propagates in propionate-degrading communities.
In addition to the identification of the propionate-oxidizing and methanogenic key species, our goal was to identify further bacterial species which might be part of the propionate degradation community, but have been hitherto neglected. With respect to our findings, acetateand H 2 -consuming bacteria came under consideration. The ubiquitous occurrence of the syntrophic acetate-oxidizing species Tepidanaerobacter acetatoxydans and the detection of putative autotrophic homoacetogenic Moorella and Thermacetogenium-related species, as well as further genera, which can be linked to SAO (Syntrophaceticus, Mesotoga) and AHA (Treponema), indicate an involvement of SAO and AHA in propionate degradation. Although repeatedly detected in methanogenic ecosystems, information about the ecological roles of SAO and AHA are currently limited (Saady, 2013;Westerholm, Leven, & Schnurer, 2012).
Since this reaction can act as a sink as well as a source of hydrogen, it offers the potential to adjust and stabilize the hydrogen partial pressure in anaerobic biomass digestion systems, such as syntrophic propionate degradation in biogas plants. Regarding the Gibbs free energy of propionate oxidation, SAO, AHA, acetoclastic, and hydrogenotrophic methanogenesis (Table 4), it is noticeable that SAO and AHA will not occur if acetoclastic and hydrogenotrophic methanogenesis are equally efficient (at 5 × 10 −5 atm pH 2 ). However, if pH 2 increases or decreases significantly, propionate oxidation or hydrogenotrophic methanogenesis, respectively, lose free energy (Table 4), most probably resulting in propionate degradation instability due to product formation/disposal imbalance. Therefore, SAO and AHA may counterbalance severe hydrogen input, excess hydrogen formation or hydrogen deficiency, leading to increased process balance and stability (Fig. 1). Neither AHA nor SAO reduce the methane yield, because either product serves as a methanogenic precursor. Furthermore, AHA and SAO performing species (e.g., Moorella thermoacetica, Tepidanaerobacter acetatoxydans) can be competent sugar metabolizers (Pierce et al., 2008;Westerholm et al., 2011), which do not depend on the low energy yield of AHA or SOA at low pH 2 ; however, they depend on a stable biotope with efficient propionate degradation and biogas formation. In conclusion, stable and efficient propionate degradation might rely not only on propionate oxidation, acetoclastic, and hydrogenotrophic methanogenesis, but also on pH 2 -adjusting SAO and AHA.