Biogas is an important renewable energy carrier. It is a product of stepwise anaerobic degradation of organic materials by highly diverse microbial communities forming complex interlinking metabolic networks. Knowledge about the microbial background of long-term stable process performance in full-scale reactors is crucial for rationally improving the efficiency and reliability of biogas plants. To generate such knowledge, in the present study three parallel mesophilic full-scale reactors fed exclusively with energy crops were sampled weekly over one year. Physicochemical process parameters were determined and the microbial communities were analysed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and 454-amplicon sequencing. For investigating the methanogenic community, a high-resolution T-RFLP approach based on the mcrA gene was developed by selecting restriction enzymes with improved taxonomic resolution compared to previous studies. Interestingly, no Methanosarcina-related generalists, but rather specialized hydrogenotrophic and acetoclastic methanogenic taxa were detected. In general, the microbial communities in the non-connected reactors were remarkably stable and highly similar indicating that identical environmental and process parameters resulted in identical microbial assemblages and dynamics. Practical implications such as flexible operation schemes comprising controlled variations of process parameters for an efficient microbial resource management under fluctuating process conditions are discussed.

Microbiome based anaerobic digestion combined with microbial electrochemical technologies exploits biomass efficiently and flexibly by concurrent conversion to methane and electrons.

Geobacter spp. enrichment biofilms were cultivated in batch using one-chamber and twochamber bioelectrochemical reactors. Time-resolved substrate quantification was performed to derive physiological parameters as well as incremental coulombic efficiency (i.e., coulombic efficiency during one batch cycle, here every 6h) during early stage biofilm development. The results of one-chamber reactors revealed an intermediate acetate increase putatively due to the presence of acetogens. Total coulombic efficiencies of two-chamber reactors were considerable lower (19.6±8.3% and 49.3±13.2% for 1 st and 2 nd batch cycle, respectively) compared to usually reported values of mature Geobacter spp. enrichment biofilms presumably reflecting energetic requirements for biomass production (i.e., cells and extracellular polymeric substances) during early stages of biofilm development. The incremental coulombic efficiency exhibits considerable changes during batch cycles indicating shifts between phases of maximizing metabolic rates and maximizing biomass yield. Analysis based on Michaelis-Menten kinetics yielded maximum substrate uptake rates (v max,Ac , v max,I) and half-saturation concentration coefficients (K M,Ac ,K M,I) based on acetate uptake or current production, respectively. The latter is usually reported in literature but neglects energy demands for biofilm growth and maintenance as well as acetate and electron storage. From 1 st to 2 nd batch cycle, v max,Ac and K M,Ac , decreased from 0.0042-0.0051 mmol Ac − h −1 cm −2 to 0.0031-0.0037 mmol Ac − h −1 cm −2 and 1.02-2.61 mM Ac − to 0.28-0.42 mM Ac − , respectively. Furthermore, differences between K M,Ac /K M,I and v max,Ac /v max,I were observed providing insights into the physiology of Geobacter spp. enrichment biofilms. Notably, K M,I considerably scattered while v max,Ac /v max,I and K M,Ac remained rather stable indicating that acetate transport within biofilm only marginally affects reaction rates. The observed data variation mandates the requirement of a more detailed analysis with an improved experimental system, e.g., using flow conditions and a comparison with Geobacter spp. pure cultures.

Biofilm anodes based on Geobacter enrichment in one-chamber and two-chamber reactors are qualitatively and quantitatively investigated. The methanogenic community of biofilms in both types of reactors consists exclusively of hydrogenotrophs, mainly the genus Methanobacterium. As qPCR demonstrates in one-chamber reactors, the abundance of Geobacter and methanogens increases due to the presence of cathodically produced hydrogen. In two-chamber reactors, the abundance of methanogens is decreased by 98 % compared to one-chamber reactors. Adding hydrogen gas to the anodic compartment of two-chamber reactors recovers the abundance of methanogens and Geobacter. Ratios of methanogens/bacteria are confirmed by using the cofactor F 420 autofluorescence of methanogens. The impact of the reactor setup for developing primary microbial electrochemical technologies is discussed, reasoning that two-chamber reactors have to be generally favored. Yet, their use inheres a trade-off when cultivating biofilm anodes, as the indicated syntrophic interactions between methanogens and Geobacter are reduced, but pH decrease might influence microbial processes.

Microbial electrosynthesis is a highly promising application of microbial electrochemical technologies for the sustainable production of organic compounds. At the same time a multitude of questions need to be answered and challenges to be met. Central for its further development is using appropriate electroactive microorganisms and their efficient extracellular electron transfer (EET) as well as wiring of the metabolism to EET. Among others, Clostridia are believed to represent electroactive microbes being highly promising for microbial electrosynthesis. We investigated the potential steps and challenges for the bio-electrochemical fermentation (electro-fermentation) of mid-chain organic acids using Clostridium kluyveri. Starting from a metabolic model the potential limitations of the metabolism as well as beneficial scenarios for electrochemical stimulation were identified and experimentally investigated. C. kluyveri was shown to not be able to exchange electrons with an electrode directly. Therefore, exogenous mediators (2-hydroxy-1,4-naphthoquinone, potassium ferrocyanide, neutral red, methyl viologen, methylene blue, and the macrocyclic cobalt hexaamine [Co(trans-diammac)]) were tested for their toxicity and electro-fermentations were performed in 1L bioreactors covering 38 biotic and 8 abiotic runs. When using C. kluyveri and mediators, maximum absolute current densities higher than the abiotic controls were detected for all runs. At the same time, no significant impact on the cell metabolism (product formation, carbon recovery, growth rate) was found. From this observation, we deduce general potential limitations of electro-fermentations with C. kluyveri and discuss strategies to successfully overcome them.