Improved Design of Anaerobic Digesters for Household Biogas Production in Indonesia: One Cow, One Digester, and One Hour of Cooking per Day

A government-sponsored initiative in Indonesia to design and implement low-cost anaerobic digestion systems resulted in 21 full-scale systems with the aim to satisfy the cooking fuel demands of rural households owning at least one cow. The full-scale design consisted of a 0.3 m diameter PVC pipe, which was operated as a conventional plug-flow system. The system generated enough methane to power a cooking stove for ∼1 h. However, eventual clogging from solids accumulation inside the bioreactor proved to be a major drawback. Here, we improved the digester configuration to remedy clogging while maintaining system performance. Controlled experiments were performed using four 9-L laboratory-scale digesters operated at a temperature of 27±1°C, a volatile solids loading rate of 2.0 g VS·L−1·day−1, and a 21-day hydraulic retention time. Two of the digesters were replicates of the original design (control digesters), while the other two digesters included internal mixing or effluent recycle (experimental digesters). The performance of each digester was compared based on methane yields, VS removal efficiencies, and steady-state solids concentrations during an operating period of 311 days. Statistical analyses revealed that internal mixing and effluent recycling resulted in reduced solids accumulation compared to the controls without diminishing methane yields or solids removal efficiencies

Inoculum selection influences the biochemical methane potential of agro-industrial substrates

Obtaining a reliable estimation of the methane potential of organic waste streams in anaerobic digestion, for which a biochemical methane potential (BMP) test is often used, is of high importance. Standardization of this BMP test is required to ensure inter-laboratory repeatability and accuracy of the BMP results. Therefore, guidelines were set out; yet, these do not provide sufficient information concerning origin of and the microbial community in the test inoculum. Here, the specific contribution of the methanogenic community on the BMP test results was evaluated. The biomethane potential of four different substrates (molasses, bio-refinery waste, liquid manure and high-rate activated sludge) was determined by means of four different inocula from full-scale anaerobic digestion plants. A significant effect of the selected inoculum on the BMP result was observed for two out of four substrates. This inoculum effect could be attributed to the abundance of methanogens and a potential inhibiting effect in the inoculum itself, demonstrating the importance of inoculum selection for BMP testing. We recommend the application of granular sludge as an inoculum, because of its higher methanogenic abundance and activity, and protection from bulk solutions, compared with other inocula

Chain elongation in anaerobic reactor microbiomes to recover resources from waste

Different microbial pathways can elongate the carbon chains of molecules in open cultures of microbial populations (i.e. reactor microbiomes) under anaerobic conditions. Here, we discuss three such pathways: 1. homoacetogenesis to combine two carbon dioxide molecules into acetate; 2. succinate formation to elongate glycerol with one carbon from carbon dioxide; and 3. reverse β oxidation to elongate short-chain carboxylates with two carbons into medium-chain carboxylates, leading to more energy-dense and insoluble products (e.g. easier to separate from solution). The ability to use reactor microbiomes to treat complex substrates can simultaneously address two pressing issues: 1. providing proper waste management; and 2. producing renewable chemicals and fuels.The authors thank Wolfgang Bucket (MPI Marburg) for assistance with Figure 1. C.M.S. and L.T.A. were supported by the U. S. Army Research Laboratory and the U. S. Army Research Office under contract/grant number W911NF-12-1-0555. H.R. was supported for this work by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-123452 received from the National Institutes for Food and Agriculture (NIFA), U.S. Department of Agriculture. K.R. was supported by the European Research Council Starter Grant Electrotalk and the Multidisciplinary Research Partnership Ghent Bio-Economy. A.J.M.S. was supported by the Chemical Sciences division of the Netherlands Science Foundation (CW-TOP 700.55.343) and the European Research Council (ERC grant 323009)

Development of a Bioelectrochemical System as a Tool to Enrich H2-Producing Syntrophic Bacteria

Syntrophic microbial partnerships are found in many environments and play critical roles in wastewater treatment, global nutrient cycles, and gut systems. An important type of syntrophy for the anaerobic conversion of carboxylic acids is H2 syntrophy. In this type of microbial partnership, dissolved H2 is produced by a bacterium and rapidly consumed by an archeon (methanogen), resulting in methane gas. This is referred to as interspecies H2 transfer, and some conversions rely on this mechanism to become thermodynamically feasible. For this reason, syntrophic partners are often not possible to separate in the lab, which hampers the full understanding of their physiology. Bioelectrochemical systems (BESs) may show promise to ultimately separate and study the behavior of the syntrophic bacterium by employing an abiotic H2 oxidation reaction at the anode, actively removing dissolved H2. Here, we performed a proof-of-concept study to ascertain whether an H2-removing anode can: (1) provide a growth advantage for the syntrophic bacterium; and (2) compete with the methanogenic partner. A mathematical model was developed to design a BES to perform competition experiments. Indeed, the operated BES demonstrated the ability to provide a growth advantage to the syntrophic bacterium Syntrophus aciditrophicus compared to its methanogenic partner Methanospirillum hungatei when grown in co-culture. Further, the BES provided the never-before isolated Syntrophomonas zehnderi with a growth advantage compared to Methanobacterium formicicum. Our results demonstrate a potential to use this BES to enrich H2-sensitive syntrophic bacteria, and gives prospects for the development of an effective method for the separation of obligate syntrophs

Effect of Shear on Performance and Microbial Ecology of Continuously Stirred Anaerobic Digesters Treating Animal Manure

We Determined the Effect of Different Mixing Intensities on the Performance, Methanogenic Population Dynamics, and Juxtaposition of Syntrophic Microbes in Anaerobic Digesters Treating Cow Manure from a Dairy Farm. Computer Automated Radioactive Particle Tracking in Conjunction with Computational Fluid Dynamics Was Performed to Quantify the Shear Levels Locally. Four Continuously Stirred Anaerobic Digesters Were Operated at Different Mixing Intensities of 1,500, 500, 250, and 50 Revolutions Per Min (RPM) over a 260-Day Period at a Temperature of 34 ± 1°C. Animal Manure at a Volatile Solids (VS) Concentration of 50 G/L Was Fed into the Digesters Daily at Five Different Organic Loading Rates between 0.6 and 3.5 G vs./L Day. the Different Mixing Intensities Had No Effect on the Biogas Production Rates and Yields at Steady-State Conditions. a Methane Yield of 0.241 ± 0.007 L CH 4/g vs. Fed Was Obtained by Pooling the Data of All Four Digesters during Steady-State Periods. However, Digester Performance Was Affected Negatively by Mixing Intensity during Startup of the Digesters, with Lower Biogas Production Rates and Higher Volatile Fatty Acids Concentrations Observed for the 1,500-RPM Digester. Despite Similar Methane Production Yields and Rates, the Acetoclastic Methanogenic Populations Were Different for the High- and Low-Intensity Mixed Digesters with Methanosarcina Spp. and Methanosaeta Concilii as the Predominant Methanogens, Respectively. for All Four Digesters, Epifluorescence Microscopy Revealed Decreasing Microbial Floc Sizes Beginning at Week 4 and Continuing through Week 26 after Which No Microbial Flocs Remained. This Decrease in Size, and Subsequent Loss of Microbial Flocs Did Not, However, Produce Any Long-Term Upsets in Digester Performance. © 2007 Wiley Periodicals, Inc

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

ABSTRACT Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro . The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate

Microbial community dynamics and stability during an ammonia-induced shift to syntrophic acetate oxidation

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity

Transcriptional Analysis of Shewanella oneidensis MR-1 with an Electrode Compared to Fe(III)Citrate or Oxygen as Terminal Electron Acceptor

Shewanella oneidensis is a target of extensive research in the fields of bioelectrochemical systems and bioremediation because of its versatile metabolic capabilities, especially with regard to respiration with extracellular electron acceptors. The physiological activity of S. oneidensis to respire at electrodes is of great interest, but the growth conditions in thin-layer biofilms make physiological analyses experimentally challenging. Here, we took a global approach to evaluate physiological activity with an electrode as terminal electron acceptor for the generation of electric current. We performed expression analysis with DNA microarrays to compare the overall gene expression with an electrode to that with soluble iron(III) or oxygen as the electron acceptor and applied new hierarchical model-based statistics for the differential expression analysis. We confirmed the differential expression of many genes that have previously been reported to be involved in electrode respiration, such as the entire mtr operon. We also formulate hypotheses on other possible gene involvements in electrode respiration, for example, a role of ScyA in inter-protein electron transfer and a regulatory role of the cbb3-type cytochrome c oxidase under anaerobic conditions. Further, we hypothesize that electrode respiration imposes a significant stress on S. oneidensis, resulting in higher energetic costs for electrode respiration than for soluble iron(III) respiration, which fosters a higher metabolic turnover to cover energy needs. Our hypotheses now require experimental verification, but this expression analysis provides a fundamental platform for further studies into the molecular mechanisms of S. oneidensis electron transfer and the physiologically special situation of growth on a poised-potential surface