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Concept: Methanogen

191

The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn’s icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H2) and methane (CH4) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH4under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH4conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H2gas production to serve as a substrate for CH4production on Enceladus. We conclude that some of the CH4detected in the plume of Enceladus might, in principle, be produced by methanogens.

Concepts: Oxygen, Atom, Carbon, Methane, Jupiter, Methanogen, Titan, Enceladus

171

Microbial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD.

Concepts: DNA, Archaea, Bacteria, Microbiology, Anaerobic digestion, Methane, Methanogen, Thermophile

40

Context:Colonization of the gastrointestinal tract with methanogenic archaea (methanogens) significantly affects host metabolism and weight gain in animal models, and breath methane is associated with a greater body mass index (BMI) among obese human subjects.Objective:The objective of the study was to characterize the relationship between methane and hydrogen on breath test (as a surrogate for colonization with the hydrogen requiring methanogen, Methanobrevibacter smithii), body weight, and percent body fat in a general population cohort.Design and Subjects:This was a prospective study (n = 792) of consecutive subjects presenting for breath testing.Setting:The study was conducted at a tertiary care center.Outcome Measurements:BMI and percent body fat were measured.Results:Subjects were classified into 4 groups based on breath testing: normal (N) (methane <3 ppm and hydrogen <20 ppm at or before 90 minutes); hydrogen positive only (H+) [methane <3 ppm and hydrogen ≥20 ppm); methane positive only (M+) (methane ≥3 ppm and hydrogen <20 ppm), or methane and hydrogen positive (M+/H+) (methane ≥3 ppm and hydrogen ≥20 ppm]. There were significant differences in age but not in gender across the groups. After controlling for age as a confounding variable, M+/H+ subjects had significantly higher BMI than other groups (N: 24.1 ± 5.2 kg/m(2); H+: 24.2 ± 4.5 kg/m(2); M+: 24.0 ± 3.75 kg/m(2); M+/H+: 26.5 ± 7.1 kg/m(2), P < .02) and also had significantly higher percent body fat (N: 28.3 ± 10.0%; H+: 27.5 ± 9.0%; M+: 28.0 ± 8.9%; M+/H+; 34.1 ± 10.9%, P < .001).Conclusions:The presence of both methane and hydrogen on breath testing is associated with increased BMI and percent body fat in humans. We hypothesize that this is due to colonization with the hydrogen-requiring M smithii, which affects nutrient availability for the host and may contribute to weight gain.

Concepts: Archaea, Nutrition, Obesity, Mass, Body mass index, Anaerobic digestion, Body weight, Methanogen

28

The effects of replacing 50% of cereals-based concentrate with feed blocks (FB) including waste fruits of tomato, cucumber or barley grain on nutrient utilization, ruminal fermentation, microbial N flow to the duodenum, methane emissions and abundances of total bacteria and methanogen were studied in goats. Four adult, dry, non pregnant, rumen-fistulated Granadina goats (32.1 ± 5.52 kg BW) were used and 4 diets were studied in 4 trials by following a 4 x 4 Latin square experimental design. Diets consisted of alfalfa hay plus concentrate in a 1:1 ratio (AC) or 1:0.5 plus feed blocks (FB) including wastes of tomato fruit (ACT), cucumber (ACC) or barley (ACB). In each trial, goats were randomly assigned to one of the diets. Intake of each FB including tomato, cucumber, and barley were 203 ± 73, 179 ± 39, and 144 ± 68 g•animal(-1)•d(-1), respectively. The replacement of 50% of concentrate with FB including wastes of tomato or cucumber fruits did not (P > 0.05) compromise energy digestive and metabolic utilization, but reduced N retention by up to 29% (P = 0.03). Cucumber-based feed blocks decreased the purine bases (PB):N ratio (P = 0.02) in total bacterial pellets isolated from the rumen content. Tomato-based feed blocks decreased purine derivatives urinary excretion (P = 0.04) and microbial N flow (P = 0.03) without affecting the efficiency of synthesis, but decreased methane emission by 28% (P < 0.001) compared to the other diets. Tomato and cucumber-based FB resulted in greater (P < 0.001) rumen VFA concentration and molar proportions of propionate and butyrate (P ≤ 0.02) than AC and ACB diets. No effect (P = 0.07) of diet on total bacteria abundance was observed while the abundance of methanogens increased (P = 0.01) with wastes-based FB. Our study suggests that cucumber-based FB could replace half of the amount of concentrate in goat diet without compromising rumen fermentation, nutrient and energy utilization and without increasing in methane emissions. Blocks including tomato waste fruits showed antimethanogenic effect but reduced microbial N flow to the duodenum. Further research is needed to improve both N and energy utilisation of diets including tomato- and cucumber-based FB.

Concepts: Archaea, Bacteria, Metabolism, Hydrogen, Fruit, Vegetable, Methane, Methanogen

28

Biogas is an energy source that is produced via the anaerobic digestion of various organic materials, including waste-water sludge and organic urban wastes. Among the microorganisms involved in digestion, methanogens are the major microbiological group responsible for methane production. To study the microbiological equilibrium in an anaerobic reactor, we detected the methanogen concentration during wet digestion processes fed with pre-treated urban organic waste and waste-water sludge. Two different pre-treatments were used in successive experimental digestions: pressure-extrusion and turbo-mixing. Chemical parameters were collected to describe the process and its production. The method used is based on real-time quantitative PCR (RT-qPCR) with the functional gene mcrA as target. First, we evaluated the validity of the analyses. Next, we applied this method to 50 digestate samples and then we performed a statistical analysis. A positive and significant correlation between the biogas production rate and methanogen abundance was observed (r = 0.579, p < 0.001). This correlation holds both when considering all of the collected data and when the two data sets are separated. The pressure-extrusion pre-treatment allowed to obtain the higher methane amount and also the higher methanogen presence (F = 41.190, p < 0.01). Moreover a higher mean methanogen concentration was observed for production rate above than of 0.6 m(3) biogas/kg TVS (F = 7.053; p < 0.05). The applied method is suitable to describe microbiome into the anaerobic reactor, moreover methanogen concentration may have potential for use as a digestion optimisation tool.

Concepts: Scientific method, Archaea, Bacteria, Anaerobic digestion, Waste management, Biogas, Methane, Methanogen

27

The present work reports the first ever evaluation of the biological CH4 potential (BMP) of starfish, classified as invasive species. Since starfish contain a large amount of inorganic matter, only the supernatant obtained through grinding and centrifugation was used for BMP test. By applying response surface methodology, the individual and interactive effects of three parameters, inoculum/substrate ratios, substrate concentrations, and buffer capacities on CH4 production were investigated, and the maximum CH4 yield of 334mL CH4/g COD was estimated. In addition, continuous CH4 production was attempted using a two-stage (acidogenic sequencing batch reactor+methanogenic up-flow anaerobic sludge blanket reactor (UASBr)) fermentation process. Acidification efficiency was maximized at 2days of hydraulic retention time with valerate, butyrate, and acetate as main acids, and these were converted to CH4 with showing 296mL CH4/g CODadded. Overall, the two-stage fermentation process could convert 44% of organic content in whole starfish to CH4.

Concepts: Protein, Anaerobic digestion, Biogas, Methane, Methanogen, Landfill, Upflow anaerobic sludge blanket digestion, Methanogenesis

25

Anaerobic methane oxidation (AMO) is considered to be an important sink of CH4 in habitats as marine sediments. But, few studies focused on AMO in landfills which may be an important sink of CH4 derived from waste fermentation. To show evidence of AMO and to uncover function anaerobic methanotroph (ANME) community in landfill, different age waste samples were collected in Jinqianpu landfill located in north China. Through high-throughput sequencing, Methanomicrobiales and Methanosarcinales archaea associated with ANME and reverse methanogenic archaea of Methanosarcina and Methanobacterium were detected. Sulfate-reducing bacteria (SRB) (Desulfobulbus and Desulfococcus) which could couple with ANME-conducting AMO were also found. But, the community structure of ANME had no significant difference with depths. From the results of investigation, we can come to a conclusion that sulfate-dependent anaerobic methane oxidation (SR-DAMO) would be the dominant AMO process in the landfill, while iron-dependent anaerobic methane oxidation (M/IR-DAMO) process was weak though concentration of ferric iron was large in the landfill. Denitrification-dependent anaerobic methane oxidation (NR-DAMO) was negative because of lack of nitrate and relevant function microorganisms in the landfill. Results also indicate that CH4 mitigation would have higher potential by increasing electron acceptor contents and promoting the growth of relevant function microorganisms.

Concepts: Archaea, Bacteria, Microbiology, Anaerobic digestion, Methane, Methanotroph, Methanogen, Landfill

24

Anaerobic digestion is an effective method for reducing the by-product of waste-activated sludge (WAS) from wastewater treatment plants and for producing bioenergy from WAS. However, only a limited number of studies have attempted to improve anaerobic digestion by targeting the microbial interactions in WAS. In this study, we examined whether different antibiotics positively, negatively, or neutrally influence methane fermentation by evaluating changes in the microbial community and functions in WAS. Addition of azithromycin promoted the microbial communities related to the acidogenic and acetogenic stages, and a high concentration of soluble proteins and a high activity of methanogens were detected. Chloramphenicol inhibited methane production but did not affect the bacteria that contribute to the hydrolysis, acidogenesis, and acetogenesis digestion stages. The addition of kanamycin, which exhibits the same methane productivity as a control (antibiotic-free WAS), did not affect all of the microbial communities during anaerobic digestion. This study demonstrates the simultaneous functions and interactions of diverse bacteria and methanogenic Archaea in different stages of the anaerobic digestion of WAS. The ratio of Caldilinea, Methanosarcina, and Clostridium may correspond closely to the trend of methane production in each antibiotic. The changes in microbial activities and function by antibiotics facilitate a better understanding of bioenergy production.

Concepts: Archaea, Bacteria, Microbiology, Sewage treatment, Anaerobic digestion, Methane, Methanogen, Acetogenesis

18

Thawing permafrost in the Canadian Arctic tundra leads to peat erosion and slumping in narrow and shallow runnel ponds that surround more commonly studied polygonal ponds. Here we compared the methane production between runnel and polygonal ponds using stable isotope ratios, (14)C signatures, and investigated potential methanogenic communities through high-throughput sequencing archaeal 16S rRNA genes. We found that runnel ponds had significantly higher methane and carbon dioxide emissions, produced from a slightly larger fraction of old carbon, compared to polygonal ponds. The methane stable isotopic signature indicated production through acetoclastic methanogenesis, but gene signatures from acetoclastic and hydrogenotrophic methanogenic Archaea were detected in both polygonal and runnel ponds. We conclude that runnel ponds represent a source of methane from potentially older C, and that they contain methanogenic communities able to use diverse sources of carbon, increasing the risk of augmented methane release under a warmer climate.

Concepts: Carbon dioxide, Archaea, Ribosomal RNA, 16S ribosomal RNA, Anaerobic digestion, Methane, Methanogen, Global warming

17

Human-associated archaea remain understudied in the field of microbiome research, although in particular methanogenic archaea were found to be regular commensals of the human gut, where they represent keystone species in metabolic processes. Knowledge on the abundance and diversity of human-associated archaea is extremely limited, and little is known about their function(s), their overall role in human health, or their association with parts of the human body other than the gastrointestinal tract and oral cavity. Currently, methodological issues impede the full assessment of the human archaeome, as bacteria-targeting protocols are unsuitable for characterization of the full spectrum of Archaea The goal of this study was to establish conservative protocols based on specifically archaea-targeting, PCR-based methods to retrieve first insights into the archaeomes of the human gastrointestinal tract, lung, nose, and skin. Detection of Archaea was highly dependent on primer selection and the sequence processing pipeline used. Our results enabled us to retrieve a novel picture of the human archaeome, as we found for the first time Methanobacterium and Woesearchaeota (DPANN superphylum) to be associated with the human gastrointestinal tract and the human lung, respectively. Similar to bacteria, human-associated archaeal communities were found to group biogeographically, forming (i) the thaumarchaeal skin landscape, (ii) the (methano)euryarchaeal gastrointestinal tract, (iii) a mixed skin-gastrointestinal tract landscape for the nose, and (iv) a woesearchaeal lung landscape. On the basis of the protocols we used, we were able to detect unexpectedly high diversity of archaea associated with different body parts.IMPORTANCE In summary, our study highlights the importance of the primers and data processing pipeline used to study the human archaeome. We were able to establish protocols that revealed the presence of previously undetected Archaea in all of the tissue samples investigated and to detect biogeographic patterns of the human archaeome in the gastrointestinal tract and on the skin and for the first time in the respiratory tract, i.e., the nose and lungs. Our results are a solid basis for further investigation of the human archaeome and, in the long term, discovery of the potential role of archaea in human health and disease.

Concepts: Archaea, Bacteria, Lung, Human gastrointestinal tract, Digestion, Cofactor, Methanogen, Routes of administration