The effect of substituting energy crop with agricultural waste on the dynamics of bacterial communities in a two-stage anaerobic digester.

The replacement of energy crops with agricultural waste in biogas production through anaerobic digestion (AD) is both an environmentally sustainable and economically profitable strategy. However, the change of feeding mix in AD might result in nutrient imbalance or increase of the ammonium concentration, negatively affecting the activity of the microbes responsible for the process. In the present study the structure and dynamics of the bacterial communities of a full-scale two-stage AD plant, composed of a hydrolysis/acidogenesis (H) and an acetogenesis/methanogenesis (M) tanks, was monitored during feedstock substitution. Energy crop (triticale) was replaced by poultry manure litter and olive mill pomace. The increase percentage of poultry manure litter (up to 8.6%) and olive mill pomace (up to 30.5%) in the recipe incremented the total solids (up to 21% in H) and, consequently, the nitrogen content in the digestate (6.7 g N/kg in the solid fraction in H and 4-5 g NH4+-N/L in the liquid fraction). This favored the growth of Lactococcus sp. with consequent increment of lactate production (∼ 1 mg L-1 last two days of the survey) and the establishment of Weissella and Lactobacillus spp. Syntrophic acetate-oxidizers, including Syntrophaceticus (6% ± 1.7%), were detected manly in M but were negatively affected by the addition of the poultry manure litter, while the sulfate-reducing bacteria correlated with the variations of the volatile fatty acids. Planctomycetes putatively capable of anammox process were also found in the H during the first two days of the survey and accounted for 0.3 ± 0.01% of the total bacterial community. The stability of the process during feedstock change is the result of the shift of bacterial populations of different functional groups that showed peculiar adaptation patterns in the two stages of the plant.

Syntrophic acetate oxidation replaces acetoclastic methanogenesis during thermophilic digestion of biowaste.

BACKGROUND: Anaerobic digestion (AD) is a globally important technology for effective waste and wastewater management. In AD, microorganisms interact in a complex food web for the production of biogas. Here, acetoclastic methanogens and syntrophic acetate-oxidizing bacteria (SAOB) compete for acetate, a major intermediate in the mineralization of organic matter. Although evidence is emerging that syntrophic acetate oxidation is an important pathway for methane production, knowledge about the SAOB is still very limited. RESULTS: A metabolic reconstruction of metagenome-assembled genomes (MAGs) from a thermophilic solid state biowaste digester covered the basic functions of the biogas microbial community. Firmicutes was the most abundant phylum in the metagenome (53%) harboring species that take place in various functions ranging from the hydrolysis of polymers to syntrophic acetate oxidation. The Wood-Ljungdahl pathway for syntrophic acetate oxidation and corresponding genes for energy conservation were identified in a Dethiobacteraceae MAG that is phylogenetically related to known SAOB. 16S rRNA gene amplicon sequencing and enrichment cultivation consistently identified the uncultured Dethiobacteraceae together with Syntrophaceticus, Tepidanaerobacter, and unclassified Clostridia as members of a potential acetate-oxidizing core community in nine full-scare digesters, whereas acetoclastic methanogens were barely detected. CONCLUSIONS: Results presented here provide new insights into a remarkable anaerobic digestion ecosystem where acetate catabolism is mainly realized by Bacteria. Metagenomics and enrichment cultivation revealed a core community of diverse and novel uncultured acetate-oxidizing bacteria and point to a particular niche for them in dry fermentation of biowaste. Their genomic repertoire suggests metabolic plasticity besides the potential for syntrophic acetate oxidation. Video Abstract.

Identification of Novel Butyrate- and Acetate-Oxidizing Bacteria in Butyrate-Fed Mesophilic Anaerobic Chemostats by DNA-Based Stable Isotope Probing.

Butyrate is one of the most important intermediates during anaerobic digestion of protein wastewater, and its oxidization is considered as a rate-limiting step during methane production. However, information on syntrophic butyrate-oxidizing bacteria (SBOB) is limited due to the difficulty in isolation of pure cultures. In this study, two anaerobic chemostats fed with butyrate as the sole carbon source were operated at different dilution rates (0.01/day and 0.05/day). Butyrate- and acetate-oxidizing bacteria in both chemostats were investigated, combining DNA-Stable Isotope Probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results showed that, in addition to known SBOB, Syntrophomonas, other species of unclassified Syntrophomonadaceae were putative butyrate-oxidizing bacteria. Species of Mesotoga, Aminivibrio, Acetivibrio, Desulfovibrio, Petrimonas, Sedimentibacter, unclassified Anaerolineae, unclassified Synergistaceae, unclassified Spirochaetaceae, and unclassified bacteria may contribute to acetate oxidation from butyrate metabolism. Among them, the ability of butyrate oxidation was unclear for species of Sedimentibacter, unclassified Synergistaceae, unclassified Spirochaetaceae, and unclassified bacteria. These results suggested that more unknown species participated in the degradation of butyrate. However, the corresponding function and pathway for butyrate or acetate oxidization of these labeled species need to be further investigated.

Identification of novel potential acetate-oxidizing bacteria in thermophilic methanogenic chemostats by DNA stable isotope probing.

Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day-1) and ATH (0.05 day-1) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day-1) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA stable-isotope probing (SIP). The results showed that, in addition to Tepidanaerobacter, a known SAOB, species of Thauera, Thermodesulfovibrio, Anaerobaculum, Ruminiclostridium, Comamonas, and uncultured bacteria belonging to Lentimicrobiaceae, o_MBA03, Thermoanaerobacteraceae, Anaerolineaceae, Clostridiales, and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based on the intense fluorescence of coenzyme F420, majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.

Differences of methanogenesis between mesophilic and thermophilic in situ biogas-upgrading systems by hydrogen addition.

To investigate the differences in microbial community structure between mesophilic and thermophilic in situ biogas-upgrading systems by H2 addition, two reactors (35 °C and 55 °C) were run for four stages according to different H2 addition rates (H2/CO2 of 0:1, 1:1, and 4:1) and mixing mode (intermittent and continuous). 16S rRNA gene-sequencing technology was applied to analyze microbial community structure. The results showed that the temperature is a crucial factor in impacting succession of microbial community structure and the H2 utilization pathway. For mesophilic digestion, most of added H2 was consumed indirectly by the combination of homoacetogens and strict aceticlastic methanogens. In the thermophilic system, most of added H2 may be used for microbial cell growth, and part of H2 was utilized directly by strict hydrogenotrophic methanogens and facultative aceticlastic methanogens. Continuous stirring was harmful to the stabilization of mesophilic system, but not to the thermophilic one.

Identification of novel potential acetate-oxidizing bacteria in an acetate-fed methanogenic chemostat based on DNA stable isotope probing.

Acetate is a significant intermediate of anaerobic fermentation. There are two pathways for converting acetate to CH4 and CO2: acetoclastic methanogenesis by acetoclastic methanogens, and syntrophic acetate oxidation by acetate-oxidizing bacteria (AOB) and hydrogenotrophic methanogens. Detailed investigations of syntrophic acetate-oxidizing bacteria (SAOB) should contribute to the elucidation of the microbial mechanisms of methanogenesis. In this study, we investigated the major phylogenetic groups of acetate-utilizing bacteria (AUB) in a mesophilic methanogenic chemostat fed with acetate as the sole carbon source by using DNA stable isotope probing (SIP) technology. The results indicated that acetoclastic methanogenesis and acetate oxidization/hydrogenotrophic methanogenesis coexisted in the mesophilic chemostat fed with acetate, operated at a dilution rate of 0.1 d-1. OTU Ace13(9-17) (KU869530), Ace13(9-4) (KU667241), and Ace13(9-23) (KU667236), assigned to the phyla Firmicutes and Bacteroidetes, were probably potential SAOB in the chemostat, which needs further investigation. Species in the phyla Proteobacteria, Deferribacteres, Acidobacteria, Spirochaetes and Actinobacteria were probably capable of utilizing acetate for their growth. Methanoculleus was likely to be the preferred hydrogenotrophic methanogen for syntrophy with AOB in the chemostat.

A Combination of Stable Isotope Probing, Illumina Sequencing, and Co-occurrence Network to Investigate Thermophilic Acetate- and Lactate-Utilizing Bacteria.

Anaerobic digestion is a complicated microbiological process that involves a wide diversity of microorganisms. Acetate is one of the most important intermediates, and interactions between acetate-oxidizing bacteria and archaea could play an important role in the formation of methane in anoxic environments. Anaerobic digestion at thermophilic temperatures is known to increase methane production, but the effects on the microbial community are largely unknown. In the current study, stable isotope probing was used to characterize acetate- and lactate-oxidizing bacteria in thermophilic anaerobic digestion. In microcosms fed 13C-acetate, bacteria related to members of Clostridium, Hydrogenophaga, Fervidobacterium, Spirochaeta, Limnohabitans, and Rhodococcus demonstrated elevated abundances of 13C-DNA fractions, suggesting their activities in acetate oxidation. In the treatments fed 13C-lactate, Anaeromyxobacter, Desulfobulbus, Syntrophus, Cystobacterineae, and Azospira were found to be the potential thermophilic lactate utilizers. PICRUSt predicted that enzymes related to nitrate and nitrite reduction would be enriched in 13C-DNA fractions, suggesting that the acetate and lactate oxidation may be coupled with nitrate and/or nitrite reduction. Co-occurrence network analysis indicated bacterial taxa not enriched in 13C-DNA fractions that may also play a critical role in thermophilic anaerobic digestion.

Metabolism and Occurrence of Methanogenic and Sulfate-Reducing Syntrophic Acetate Oxidizing Communities in Haloalkaline Environments.

Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (M-SAO) and sulfate-reducing (S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that H2 and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was 'Candidatus Syntrophonatronum acetioxidans,' and a near-complete draft genome of this SAOB was reconstructed. 'Ca. S. acetioxidans' has all genes necessary for operating the Wood-Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na+-dependent ATP synthase and marker genes for 'salt-out' strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of 'Ca. S. acetioxidans' showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA gene amplicon datasets of the soda lake sediments showed a high similarity of reads to 'Ca. S. acetioxidans' with abundance as high as 1.3% of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers were not abundant (≤0.1%) or could not be detected. These combined results indicate that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline conditions performed by haloalkaliphilic syntrophic consortia.

Microbial community shifts in a farm-scale anaerobic digester treating swine waste: Correlations between bacteria communities associated with hydrogenotrophic methanogens and environmental conditions.

Microbial community structure in a farm-scale anaerobic digester treating swine manure was investigated during three process events: 1) prolonged starvation, and changes of 2) operating temperature (between meso- and thermophilic) and 3) hydraulic retention time (HRT). Except during the initial period, the digester was dominated by hydrogenotrophic methanogens (HMs). The bacterial community structure significantly shifted with operating temperature and HRT but not with long-term starvation. Clostridiales (26.5-54.4%) and Bacteroidales (2.5-13.7%) became dominant orders in the digester during the period of HM dominance. Abundance of diverse meso- and thermophilic bacteria increased during the same period; many of these species may be H2 producers, and/or syntrophic acetate oxidizers. Some of these species showed positive correlations with [NH4+-N] (p<0.1); this relationship suggests that ammonia was a significant parameter for bacterial selection. The bacterial niche information reported in this study can be useful to understand the ecophysiology of anaerobic digesters treating swine manure that contains high ammonia content.

Working draft genome sequence of the mesophilic acetate oxidizing bacterium Syntrophaceticus schinkii strain Sp3.

Syntrophaceticus schinkii strain Sp3 is a mesophilic syntrophic acetate oxidizing bacterium, belonging to the Clostridia class within the phylum Firmicutes, originally isolated from a mesophilic methanogenic digester. It has been shown to oxidize acetate in co-cultivation with hydrogenotrophic methanogens forming methane. The draft genome shows a total size of 3,196,921 bp, encoding 3,688 open reading frames, which includes 3,445 predicted protein-encoding genes and 55 RNA genes. Here, we are presenting assembly and annotation features as well as basic genomic properties of the type strain Sp3.