Syntrophic acetate oxidation (SAO) is the predominant pathway for methane production in high ammonia anaerobic digestion processes. The bacteria (SAOB) occupying this niche and the metabolic pathway are poorly understood. Phylogenetic diversity and strict cultivation requirements hinder comprehensive research and discovery of novel SAOB. Most SAOB characterised to date are affiliated to the physiological group of acetogens. Formyltetrahydrofolate synthetase is a key enzyme of both acetogenic and SAO metabolism. The encoding fhs gene has therefore been identified as a suitable functional marker, using a newly designed primer pair. In this comparative study, we used a combination of terminal restriction fragment length polymorphism profiling, clone-based comparison, qPCR and Illumina amplicon sequencing to assess the bacterial community and acetogenic sub-community prevailing in high- and low-ammonia laboratory-scale digesters in order to delineate potential SAOB communities. Potential candidates identified were further tracked in a number of low-ammonia and high-ammonia laboratory-scale and large-scale digesters in order to reveal a potential function in SAO. All methodical approaches revealed significant changes in the bacterial community composition concurrently with increasing ammonia and predominance of SAO. The acetogenic community under high ammonia conditions was revealed to be generally heterogeneous, but formed distinct phylogenetic clusters. The clusters differed clearly from those found under low-ammonia conditions and represented an acetogenic assemblage unique for biogas processes and recurring in a number of high-ammonia processes, indicating potential involvement in SAO. The phylogenetic affiliation and population dynamics observed point to a key community, belonging mainly to the Clostridia class, in particular to the orders Clostridiales and Thermoanaerobacterales, which appear to specialise in SAO rather than being metabolically versatile. Overall, the results reported here provide evidence of functional importance of the bacterial families identified in high-ammonia systems and extend existing knowledge of bacterial and acetogenic assemblages at low and high ammonia levels. This information will be of help in monitoring and assessing the impacts on the SAOB community in order to identify characteristics of robust and productive high ammonia biogas processes.

Materials rich in lignocellulose, such as straw, are abundant, cheap and highly interesting for biogas production. However, the complex structure of lignocellulose is difficult for microbial cellulolytic enzymes to access, limiting degradation. The rate of degradation depends on the activity of members of the microbial community, but the knowledge of this community in the biogas process is rather limited. This study, therefore, investigated the degradation rate of cellulose and straw in batch cultivation test initiated with inoculums from four co-digestion biogas plants (CD) and six wastewater treatment plants (WWTP). The results were correlated to the bacterial community by 454-pyrosequencing targeting 16S rRNA gene and by T-RFLP analysis targeting genes of glycoside hydrolase families 5 (cel5) and 48 (cel48), combined with construction of clone libraries UniFrac principal coordinate analysis of 16S rRNA gene amplicons revealed a clustering of WWTPs, while the CDs were more separated from each other. Bacteroidetes and Firmicutes dominated the community with a comparably higher abundance of the latter in the processes operating at high ammonia levels. Sequences obtained from the cel5 and cel 48 clone libraries were also mainly related to the phyla Firmicutes and Bacteroidetes and here ammonia was a parameter with a strong impact on the cel5 community. The results from the batch cultivation showed similar degradation pattern for eight of the biogas plants, while two characterised by high ammonia level and low bacterial diversity, showed a clear lower degradation rate. Interestingly, two T-RFs from the cel5 community were positively correlated to high degradation rates of both straw and cellulose. One of the respective partial cel5 sequences shared 100 % identity to Clostridium cellulolyticum. The degradation rate of cellulose and straw varied in the batch tests dependent on the origin of the inoculum and was negatively correlated with the ammonia level. The cellulose-degrading community, targeted by analysis of the glycoside hydrolase families 5 (cel5) and 48 (cel48), showed a dominance of bacteria belonging the Firmicutes and Bacteriodetes, and a positive correlation was found between the cellulose degradation rate of wheat straw with the level of C. cellulolyticum.

This study investigated whether biogas reactor performance, including microbial community development, in response to a change in substrate composition is influenced by initial inoculum source. For the study, reactors previously operated with the same grass–manure mixture for more than 120 days and started with two different inocula were used. These reactors initially showed great differences depending on inoculum source, but eventually showed similar performance and overall microbial community structure. At the start of the present experiment, the substrate was complemented with milled feed wheat, added all at once or divided into two portions. The starting hypothesis was that process performance depends on initial inoculum source and microbial diversity, and thus that reactor performance is influenced by the feeding regime. In response to the substrate change, all reactors showed increases and decreases in volumetric and specific methane production, respectively. However, specific methane yield and development of the microbial community showed differences related to the initial inoculum source, confirming the hypothesis. However, the different feeding regimes had only minor effects on process performance and overall community structure, but still induced differences in the cellulose-degrading community and in cellulose degradation.

In this study, we investigated the variation in the microbial community present in bulk tank milk samples and the potential effect of different farm management factors. Bulk tank milk samples were collected repeatedly over one year from 42 farms located in northern Sweden. Total and thermoresistant bacteria counts and 16S rRNA gene-based amplicon sequencing were used to characterize microbial community composition. The microbial community was in general heterogeneous both within and between different farms and the community composition in the bulk tank milk was commonly dominated by Pseudomonas, Acinetobacter, Streptococcus, unclassified Peptostreptococcaceae, and Staphylococcus. Principal component analysis including farm factor variables and microbial taxa data revealed that the microbial community in milk was affected by type of milking system. Milk from farms using an automatic (robot) milking system (AMS) and loose housing showed different microbial community composition compared with milk from tiestall farms. A discriminant analysis model revealed that this difference was dependent on several microbial taxa. Among farms using an automatic milking system, there were further differences in the microbial community composition depending on the brand of the milking robot used. On tiestall farms, routines for teat preparation and cleaning of the milking equipment affected the microbial community composition in milk. Total bacteria count (TBC) in milk differed between the farm types, and TBC were higher on AMS than tiestall farms (log 4.05 vs. log 3.79 TBC/mL for AMS and tiestalls, respectively). Among tiestall farms, milk from farms using a chemical agent in connection to teat preparation and a more frequent use of acid to clean the milking equipment had lower TBC in milk, than milk from farms using water for teat preparation and a less frequent use of acid to clean the milking equipment (log 3.68 vs. 4.02 TBC/mL). There were no significant differences in the number of thermoresistant bacteria between farm types. The evaluated factors explained only a small proportion of total variation in the microbiota data, however, despite this, the study highlights the effect of routines associated with teat preparation and cleaning of the milking equipment on raw milk microbiota, irrespective of type of milking system used.