Metaproteome analysis of the microbial communities in agricultural biogas plants

In biogas plants agricultural waste and energy crops are converted by complex microbial communities to methane for the production of renewable energy. In Germany, this process is widely applied namely in context of agricultural production systems. However, process disturbances, are one of the major causes for economic losses. In addition, the conversion of biomass, in particular of cellulose, is in most cases incomplete and, hence, insufficient. Besides technical aspects, a more profound characterization concerning the functionality of the microbial communities involved would strongly support the improvement of yield and stability in biogas production. To monitor these communities on the functional level, metaproteome analysis was applied in this study to full-scale agricultural biogas plants. Proteins were extracted directly from sludge for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and subsequent identification with mass spectrometry. Protein profiles obtained with SDS–PAGE were specific for different biogas plants and often stable for several months. Differences of protein profiles were visualized by clustering, which allowed not only the discrimination between mesophilic and thermophilic operated biogas plants but also the detection of process disturbances such as acidification. In particular, acidification of a biogas plant was detected in advance by disappearance of major bands in SDS–PAGE. Identification of proteins from SDS–PAGE gels revealed that methyl CoM reductase, which is responsible for the release of methane during methanogenesis, from the order Methanosarcinales was significantly decreased.

Hence, it is assumed that this enzyme might be a promising candidate to serve as a predictive biomarker for acidification.

Introduction

Methane can be produced from organic waste and agricultural biomass by anaerobic digestion. More than 7000 biogas plants (BGP) contribute to the production of methane containing biogas in Germany.

Although this process is widely applied at an industrial scale it suffers from several problems. Because of persistence of cellulose and lignin, the biomass is not completely converted to methane [1]. Furthermore, anaerobic digestion can be disturbed by sudden changes in feedstock, lack of essential nutrients or intoxication by herbicides or mycotoxins causing problems such as foaming or acidification [2]. As a consequence, the methane production is decreased which results in economic losses. Better insight into functional interactions of involved microbial communities might allow overcoming parts of these problems. Not only process stability but also biogas yield could be increased. Furthermore, those findings should support identification of process failures, leading to an optimized anaerobic digestion process.

The conversion of biomass into methane is catalyzed by a complex microbial community comprising a huge number of varying Bacteria and Archaea. Several species involved in this conversion process were already characterized in detail using cultivation-dependent approaches. However, a major fraction of microbes remains uncultivable as pure cultures in laboratory [3]. Reasons might be unknown growth conditions or syntrophic interactions. Sequencing of 16S rRNA genes [4], [5], [6] of microbial communities in BGP revealed that noncultivable species are a substantial part of the community and appear to contribute to its performance. To estimate the genetic potential of noncultivable species in BGP pyro-sequencing of metagenomes [7], [8] was applied. However, it remains unresolved which genes leading to distinct metabolic activities are actually expressed.

To improve the characterization of the community's functioning, methods for the analysis and monitoring of the microbial metaproteome were established recently [9], [10]. A major challenge is in particular the extraction of proteins from matrices, which are often enriched by humic acids and other residues from microbial digestion of biomass. Thus, many studies focused on less contaminated ecosystems [11], [12] or circumvented this problem by analyzing enrichment cultures [13], [14].

Metaproteome analysis has already been applied successfully to anaerobic digestion in lab-scale reactors [15], [16]. In particular, the study of Hanreich et al. [16] reflects the analytical problems caused by the specific conditions prevalent in biogas reactors, namely the high content of interfering substances. Their protocol for purification of microbial proteins was based on sonication of cells and subsequent extraction of proteins with liquid phenol. Subsequently, proteins were separated by two-dimensional gel electrophoresis (2D-PAGE) [17], [18]. As a result, several enzymes involved in methanogenesis could be identified by nanoflow high performance liquid chromatography coupled to tandem mass spectrometry (nanoHPLC–MS/MS) methods.

The aim of the present study was to establish a protocol that is less time-consuming than 2D-PAGE and which can be easily applied to a larger number of samples from BGP. Therefore protein extraction was carried out in a ball mill and proteins were only separated by one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [19]. The resulting protein profiles were clustered for further analysis [20]. Afterwards, the lanes of SDS–PAGE gels were cut in slices and submitted to nanoHPLC–MS/MS for protein identification. As a first application of this newly developed approach, six industrial-scale BGP were sampled and monitored for up to nine months. Obtained proteins were analyzed with respect to the established chemical parameters for the process efficiency to determine microbiological indicators for process imbalances.