Anaerobic digestion of energy crops, residues, and wastes is of increasing interest in order to reduce the greenhouse gas emissions and to facilitate a sustainable development of energy supply. Production of biogas provides a versatile carrier of renewable energy, as methane can be used for replacement of fossil fuels in both heat and power generation and as a vehicle fuel. For biogas production, various process types are applied which can be classified in wet and dry fermentation systems. Most often applied are wet digester systems using vertical stirred tank digester with different stirrer types dependent on the origin of the feedstock. Biogas is mainly utilized in engine-based combined heat and power plants, whereas microgas turbines and fuel cells are expensive alternatives which need further development work for reducing the costs and increasing their reliability. Gas upgrading and utilization as renewable vehicle fuel or injection into the natural gas grid is of increasing interest because the gas can be used in a more efficient way. The digestate from anaerobic fermentation is a valuable fertilizer due to the increased availability of nitrogen and the better short-term fertilization effect. Anaerobic treatment minimizes the survival of pathogens which is important for using the digested residue as fertilizer. This paper reviews the current state and perspectives of biogas production, including the biochemical parameters and feedstocks which influence the efficiency and reliability of the microbial conversion and gas yield.

Biogas production: current state and perspectives

Transfer of Intestinal Microbiota From Lean Donors Increases Insulin Sensitivity in Individuals With Metabolic Syndrome

Recent evidence suggests that the microbial community in the human intestine may play an important role in the pathogenesis of obesity. We examined 184,094 sequences of microbial 16S rRNA genes from PCR amplicons by using the 454 pyrosequencing technology to compare the microbial community structures of 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric-bypass surgery. Phylogenetic analysis demonstrated that although the Bacteria in the human intestinal community were highly diverse, they fell mainly into 6 bacterial divisions that had distinct differences in the 3 study groups. Specifically, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of Gammaproteobacteria. Numbers of the H2-producing Prevotellaceae were highly enriched in the obese individuals. Unlike the highly diverse Bacteria, the Archaea comprised mainly members of the order Methanobacteriales, which are H2-oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H2-utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H2-producing bacteria with relatively high numbers of H2-utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H2 transfer between bacterial and archaeal species is an important mechanism for increasing energy uptake by the human large intestine in obese persons. The large bacterial population shift seen in the post-gastric-bypass individuals may reflect the double impact of the gut alteration caused by the surgical procedure and the consequent changes in food ingestion and digestion.

https://www.pnas.org/content/pnas/106/7/2365.full.pdf

Human gut microbiota in obesity and after gastric bypass

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.

/pdf/development-of-a-prokaryotic-universal-primer-for-2ue0cno65x.pdf

Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.

Mass production and use of antibiotics and antimicrobials in medicine and agriculture have existed for over 60 years, and has substantially benefited public health and agricultural productivity throughout the world. However, there is growing evidence that resistance to antibiotics (AR) is increasing both in benign and pathogenic bacteria, posing an emerging threat to public and environmental health in the future. Although evidence has existed for years from clinical data of increasing AR, almost no quantitative environmental data exist that span increased industrial antibiotic production in the 1950s to the present; i.e., data that might delineate trends in AR potentially valuable for epidemiological studies. To address this critical knowledge gap, we speculated that AR levels might be apparent in historic soil archives as evidenced by antibiotic resistance gene (ARG) abundances over time. Accordingly, DNA was extracted from five long-term soil-series from different locations in The Netherlands that spanned 1940 to 2008, and 16S rRNA gene and 18 ARG abundances from different major antibiotic classes were quantified. Results show that ARG from all classes of antibiotics tested have significantly increased since 1940, but especially within the tetracyclines, with some individual ARG being >15 times more abundant now than in the 1970s. This is noteworthy because waste management procedures have broadly improved and stricter rules on nontherapeutic antibiotic use in agriculture are being promulgated. Although these data are local to The Netherlands, they suggest basal environmental levels of ARG still might be increasing, which has implications to similar locations around the world.

http://www.hunzeenaas.com/pdf/Link%204b%20Evidence%20of%20Increasing%20Antibiotic%20Resistance%20Gene%20.....pdf

Evidence of Increasing Antibiotic Resistance Gene Abundances in Archived Soils since 1940

Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.

Group-Specific Primer and Probe Sets to Detect Methanogenic Communities Using Quantitative Real-Time Polymerase Chain Reaction

Anaerobic digestion (AD) is an effective biological treatment for stabilizing organic compounds in waste/wastewater and in simultaneously producing biogas. However, it is often limited by the slow reaction rates of different microorganisms’ syntrophic biological metabolisms. Stable and fast interspecies electron transfer (IET) between volatile fatty acid-oxidizing bacteria and hydrogenotrophic methanogens is crucial for efficient methanogenesis. In this syntrophic interaction, electrons are exchanged via redox mediators such as hydrogen and formate. Recently, direct IET (DIET) has been revealed as an important IET route for AD. Microorganisms undergoing DIET form interspecies electrical connections via membrane-associated cytochromes and conductive pili; thus, redox mediators are not required for electron exchange. This indicates that DIET is more thermodynamically favorable than indirect IET. Recent studies have shown that conductive materials (e.g., iron oxides, activated carbon, biochar, and carbon fibers) can mediate direct electrical connections for DIET. Microorganisms attach to conductive materials’ surfaces or vice versa according to particle size, and form conductive biofilms or aggregates. Different conductive materials promote DIET and improve AD performance in digesters treating different feedstocks, potentially suggesting a new approach to enhancing AD performance. This review discusses the role and potential of DIET in methanogenic systems, especially with conductive materials for promoting DIET.

/pdf/role-and-potential-of-direct-interspecies-electron-transfer-50i25i6let.pdf

Role and Potential of Direct Interspecies Electron Transfer in Anaerobic Digestion

The TaqMan quantitative PCR (QPCR) method was used to detect and quantify the 16S rRNA genes of aceticlastic methanogens at different taxonomic levels. Three different sets of primers coupled with a TaqMan probe for QPCR assays to detect the 16S rRNA genes of the order Methanosarcinales, as well as the families Methanosarcinaceae and Methanosaetaceae, were separately used. Using these primer and probe sets, the 16S rRNA genes of aceticlastic methanogens in samples from various anaerobic processes (i.e., nine pure cultures, batch experiment, and three different continuous processes including a full-scale digester), were monitored and quantified by QPCR assays. A batch experiment cultivating a mixture of aceticlastic methanogens, was conducted to monitor their population dynamics. Using this group-specific quantification method, the dynamics of a competition between two aceticlastic populations, as modulated by the acetate concentration, could well be described. The target 16S rRNA genes in environmental samples, collected from three different anaerobic processes treating sludge, cheese whey, and synthetic wastewaters, were additionally quantified. The quantified 16S rRNA gene concentrations for all samples successfully represented the community structures of the target methanogens, which were correlated accurately with the operational parameters of the anaerobic processes. It was also successful to demonstrate probe nesting of aceticlastic methanogens at the levels of order and family.

Use of Real-Time PCR for Group-Specific Quantification of Aceticlastic Methanogens in Anaerobic Processes: Population Dynamics and Community Structures

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: Applications and considerations

The effect of biostimulation with ferric oxides, semiconductive ferric oxyhydroxide, and conductive magnetite on the anaerobic digestion of dairy wastewater was examined in a batch mode. The reactors supplemented with ferric oxyhydroxide (R2) and magnetite (R3) showed significantly enhanced biomethanation performance compared with the control (R1). The removal of chemical oxygen demand (COD) after 30 days was 31.9, 59.3, and 82.5% in R1, R2, and R3, respectively. The consumed COD was almost fully recovered as biogas in R2 and R3, while only 79% was recovered in R1. The total energy production as biogas was accordingly 32.2, 71.0, and 97.7 kJ in R1, R2, and R3, respectively. The reactors also differed in the acid formation profile with more propionate and butyrate found in R1 and more acetate found in R3. The enhanced biomethanation seems to be associated with variations in the bacterial community structure supposedly induced by the ferric oxides added. In contrast, no evident variation was observed in the archaeal community structure among the reactors. The potential electric syntrophy formed between Methanosaeta concilii-like methanogens and electroactive iron-reducing bacteria, particularly Trichococcus, was likely responsible for the enhanced performance. The stimulated growth of fermentative iron reducers may also have contributed by altering the metabolic characteristics of the bacterial communities to produce more favorable acidogenic products for methanogenesis. The overall results suggest the potential of biostimulation with (semi)conductive ferric oxides to enhance the rate and efficiency of the biomethanation of organic wastes. This seems to be potentially attractive, as increasing attention is being paid to the energy self-sufficiency of waste/wastewater treatment processes today.

Jaai Kim

Papers

Group-Specific Primer and Probe Sets to Detect Methanogenic Communities Using Quantitative Real-Time Polymerase Chain Reaction

Role and Potential of Direct Interspecies Electron Transfer in Anaerobic Digestion

Use of Real-Time PCR for Group-Specific Quantification of Aceticlastic Methanogens in Anaerobic Processes: Population Dynamics and Community Structures

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: Applications and considerations

The biostimulation of anaerobic digestion with (semi)conductive ferric oxides: their potential for enhanced biomethanation.