With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

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Background Lignin intermediates resulting from lignocellulose degradation have been suspected to hinder anaerobic mineralisation of organic materials to biogas. Phenyl acids like phenylacetate (PAA) are early detectable intermediates during anaerobic digestion (AD) of aromatic compounds. Studying the phenyl acid formation dynamics and concomitant microbial community shifts can help to understand the microbial interdependencies during AD of aromatic compounds and may be beneficial to counteract disturbances. Results The length of the aliphatic side chain and chemical structure of the benzene side group(s) had an influence on the methanogenic system. PAA, phenylpropionate (PPA), and phenylbutyrate (PBA) accumulations showed that the respective lignin intermediate was degraded but that there were metabolic restrictions as the phenyl acids were not effectively processed. Metagenomic analyses confirmed that mesophilic genera like Fastidiosipila or Syntrophomonas and thermophilic genera like Lactobacillus, Bacillus, Geobacillus, and Tissierella are associated with phenyl acid formation. Acetoclastic methanogenesis was prevalent in mesophilic samples at low and medium overload conditions, whereas Methanoculleus spp. dominated at high overload conditions when methane production was restricted. In medium carbon load reactors under thermophilic conditions, syntrophic acetate oxidation (SAO)-induced hydrogenotrophic methanogenesis was the most important process despite the fact that acetoclastic methanogenesis would thermodynamically be more favourable. As acetoclastic methanogens were restricted at medium and high overload conditions, syntrophic acetate oxidising bacteria and their hydrogenotrophic partners could step in for acetate consumption. Conclusions PAA, PPA, and PBA were early indicators for upcoming process failures. Acetoclastic methanogens were one of the first microorganisms to be impaired by aromatic compounds, and shifts to syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in thermophilic reactors. Previously assumed associations of specific meso- and thermophilic genera with anaerobic phenyl acid formation could be confirmed.