Convergent Microbial Community Formation in Replicate Anaerobic Reactors Inoculated from Different Sources and Treating Ersatz Crew Waste.

Future manned space travel will require efficient recycling of nutrients from organic waste back into food production. Microbial systems are a low-energy, efficient means of nutrient recycling, but their use in a life support system requires predictability and reproducibility in community formation and reactor performance. To assess the reproducibility of microbial community formation in fixed-film reactors, we inoculated replicate anaerobic reactors from two methanogenic inocula: a lab-scale fixed-film, plug-flow anaerobic reactor and an acidic transitional fen. Reactors were operated under identical conditions, and we assessed reactor performance and used 16s rDNA amplicon sequencing to determine microbial community formation. Reactor microbial communities were dominated by similar groups, but differences in community membership persisted in reactors inoculated from different sources. Reactor performance overlapped, suggesting a convergence of both reactor communities and organic matter mineralization. The results of this study suggest an optimized microbial community could be preserved and used to start new, or restart failed, anaerobic reactors in a life support system with predictable reactor performance.

Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs.

Methylotrophy describes the ability of organisms to utilize reduced one-carbon compounds, notably methane and methanol, as growth and energy sources. Abundant natural gas supplies, composed primarily of methane, have prompted interest in using these compounds, which are more reduced than sugars, as substrates to improve product titers and yields of bioprocesses. Engineering native methylotophs or developing synthetic methylotrophs are emerging fields to convert methane and methanol into fuels and chemicals under aerobic and anaerobic conditions. This review discusses recent progress made toward engineering native methanotrophs for aerobic and anaerobic methane utilization and synthetic methylotrophs for methanol utilization. Finally, strategies to overcome the limitations involved with synthetic methanol utilization, notably methanol dehydrogenase kinetics and ribulose 5-phosphate regeneration, are discussed.

Coupling of anaerobic waste treatment to produce protein- and lipid-rich bacterial biomass.

Future long-term manned space missions will require effective recycling of water and nutrients as part of a life support system. Biological waste treatment is less energy intensive than physicochemical treatment methods, yet anaerobic methanogenic waste treatment has been largely avoided due to slow treatment rates and safety issues concerning methane production. However, methane is generated during atmosphere regeneration on the ISS. Here we propose waste treatment via anaerobic digestion followed by methanotrophic growth of Methylococcus capsulatus to produce a protein- and lipid-rich biomass that can be directly consumed, or used to produce other high-protein food sources such as fish. To achieve more rapid methanogenic waste treatment, we built and tested a fixed-film, flow-through, anaerobic reactor to treat an ersatz wastewater. During steady-state operation, the reactor achieved a 97% chemical oxygen demand (COD) removal rate with an organic loading rate of 1740 g d-1 m-3 and a hydraulic retention time of 12.25 d. The reactor was also tested on three occasions by feeding ca. 500 g COD in less than 12 h, representing 50x the daily feeding rate, with COD removal rates ranging from 56-70%, demonstrating the ability of the reactor to respond to overfeeding events. While investigating the storage of treated reactor effluent at a pH of 12, we isolated a strain of Halomonas desiderata capable of acetate degradation under high pH conditions. We then tested the nutritional content of the alkaliphilic Halomonas desiderata strain, as well as the thermophile Thermus aquaticus, as supplemental protein and lipid sources that grow in conditions that should preclude pathogens. The M. capsulatus biomass consisted of 52% protein and 36% lipids, the H. desiderata biomass consisted of 15% protein and 7% lipids, and the Thermus aquaticus biomass consisted of 61% protein and 16% lipids. This work demonstrates the feasibility of rapid waste treatment in a compact reactor design, and proposes recycling of nutrients back into foodstuffs via heterotrophic (including methanotrophic, acetotrophic, and thermophilic) microbial growth.

In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex.

Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel-precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243-1249, 2017.

Response of lab-scale methanogenic reactors inoculated from different sources to organic loading rate shocks.

Anaerobic digester failure due to a pH drop may be overcome with the use of an acidotolerant methanogenic community. To test this, lab-scale reactors were inoculated from acidic bog sediments, a municipal sludge digester, or a combination of these inocula and challenged with glucose pulses without pH control. Only the bog reactor survived the first glucose shock, and the methanogen community was dominated by members of the acidic Fen Cluster. After restarting the digester and hybrid reactors, two subsequent glucose shocks were applied. Methanogenic communities converged in all reactors and were dominated by Methanosarcina and Methanobacteriaceae. The Fen Cluster was eventually nondetectable in bog and hybrid reactors, presumably due to periods of circumneutral pH with only intermittent periods of low pH following glucose shocks. Although the resultant communities required base addition, an increase in Methanosarcina numbers after glucose pulses resulted in decreased acetate and increased reactor pH and methane production.

mcrA-targeted real-time quantitative PCR method to examine methanogen communities.

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase alpha-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4 degrees C or 20 degrees C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.

Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge.

Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro- and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.

Enzymes responsible for chlorate reduction by Pseudomonas sp. are different from those used for perchlorate reduction by Azospira sp.

Pseudomonas sp. PDA is an unusual bacterium due to its ability to respire using chlorate under aerobic conditions. The chlorate reductase produced by PDA was shown to be intrinsically different from the enzyme responsible for chlorate and perchlorate [(per)chlorate] reduction produced by Azospira sp. KJ based on subunit composition and other enzyme properties. The perchlorate reductase from strain KJ appeared to have two subunits (100 and 40 kDa) while the chlorate reductase from PDA had three subunits (60, 48, and 27 kDa). N-terminal amino acid sequencing of the 100 kDa protein from strain KJ showed a 77% similarity with the perchlorate reductase alpha subunit from another perchlorate-respiring bacterium, Dechloromonas agitata, while the N-terminus amino acid sequence of the 60 kDa protein from strain PDA did not show a similarity to previously isolated chlorate or perchlorate reductases.

Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336.

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of omega-hydroxy fatty acids to omega-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K(m) values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes omega-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not omega-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K(m) values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.

Chlorate and nitrate reduction pathways are separately induced in the perchlorate-respiring bacterium Dechlorosoma sp. KJ and the chlorate-respiring bacterium Pseudomonas sp. PDA.

The effect of nitrate on perchlorate and chlorate reduction by perchlorate-respiring bacteria (PRB), and on chlorate reduction by chlorate-respiring bacteria (CRB), is not well understood, particularly with respect to the induction of pathways used to degrade these different chemicals. Based on kinetic data obtained in a series of batch tests, we determined that perchlorate respiratory enzymes were inducible (by chlorate or perchlorate) and separate from those used for denitrification by PRB strain Dechlorosoma sp. KJ. Aerobically grown cultures of KJ had lag times of greater than 0.3-2 days when transferred to a medium containing only perchlorate, chlorate, or nitrate as an electron acceptor. There were no lag times for transfers between identical media. Washed cells reduced very little nitrate (<10%) when grown only on chlorate or perchlorate. When grown on nitrate, they degraded little chlorate or perchlorate. The same lack of activity with these electron acceptors was also observed using cell extracts and methyl viologen as an electron carrier, indicating a lack of reactivity was not due to failure of the chemical to diffuse into the cell. Taken together, these results indicated that enzymes for perchlorate and nitrate reduction are separately expressed in strain KJ. The presence of small amounts of nitrate in contaminated groundwater may actually help to increase rates of perchlorate reduction once the nitrate is completely removed. When strain KJ was pre-grown on nitrate and perchlorate, perchlorate degradation (in the absence of nitrate) was more rapid compared to cells grown only on perchlorate. Pseudomonas sp. PDA was unable to degrade perchlorate or grow using nitrate, and the induction of enzymes necessary for chlorate respiration differed for strains KJ and PDA. While chlorate reductase and chlorite dismutase activity were induced in KJ by chlorate or perchlorate under anaerobic conditions, these two enzymes were constitutively expressed by PDA under anaerobic and aerobic conditions independent of the presence of chlorate. To our knowledge, this is the first report of constitutive expression of both chlorate reductase and chlorite dismutase in a bacterium.