The methanotrophic interactome: microbial partnerships for sustainable methane cycling

Methane (CH4) is an important greenhouse gas, and the majority (ca. 60%) of its emission originates from anthropogenic sources. Methane oxidizing bacteria (MOB) are characterized by their unique ability to use CH4 as a sole carbon and energy source. Recently, accumulating evidence demonstrated that methane oxidation is stimulated when MOB are interacting with non-methanotrophic microbes. These interactions can be very specific, although it is not yet entirely elucidated what the determining factors to a successful partnership are. Hence, it seems that a methanotrophic interactome is required for effective biological aerobic methane oxidation, rather than individual obligate methanotrophic bacteria. In this dissertation, we focused on bacterial interactions between MOB and non-MOB and try to assess what are the determinants for non-MOB partner selection in the methanotrophic interactome. We focus specifically on representatives of alpha- and gammaproteobacterial MOB, which are known to exhibit differential functional traits which can in turn be conceptualized as distinct life strategies. The impact of these differences in MOB life strategies on interactions with non-MOB has not been studied. An increased insight in the co-existence and the extent of co-dependence of MOB and non-MOB partners is required to fully understand the essential ecosystem service of biological methane oxidation. In PART 1 (TOP-DOWN APPROACH), we dissected a methanotrophic enrichment culture (optimal methanotrophic interactome) by means of time-resolved stable isotope probing combined with 454-pyrosequencing of the 16S rRNA genes to assess which interactome partners are most intricately involved in the assimilation of 13CH4-derived carbon. In PART 2 (BOTTOM-UP APPROACH), we artificially assembled an optimal methanotrophic interactome by piecing together multiple non-MOB strains with MOB (co-cultivation). Firstly, we supervised the selection of MOB for non-MOB partners by establishing compatibility and matching selected MOB with non-MOB partners. Microbial adaptation and abundance of all partners was investigated using q-PCR. Secondly, we allowed an unsupervised selection for non-MOB partners by the MOB and these interactions were monitored by DGGE. In PART 3 (CRYOPRESERVETION), we develop a cryopreservation protocol based on DMSO (and DMSO+TT) as a cryopretactant, which can used to adequately preserve optimal interactomes (preservation of key functional characteristics as well as composition) to allow possible biotechnological applications. In PART 1, we found that 13CH4-derived carbon is differentially distributed among interactome partners through time. We suggested that the most intricately associated non-MOB partners would be the first to assimilate 13CH4-C and could hence be considered “primary consumers” of whereas organisms labeled later in the time-course SIP experiment are more loosely associated with the MOB and may be “secondary consumers” of organic carbon derived from both the MOB and the primary consumers. In PART 2a, we could not observe adaptation of a moderately compatible partner to MOB (or vice versa) during repeated co-cultivation with a gammaproteobacterial MOB. Conversely, clear (though limited) adaptation was observed with an alphaproteobacterial MOB. Regardless of its initial compatibility with the MOB, a third partner nearly always completely obliterated the non-MOB partner in the existing MOB:non-MOB interactome. In PART 2b, we showed a clear partner selection where some of the persisting partners were “promiscuous” and could persist regardless of MOB type , whereas others were more specifically associated with either MOB type. Additionally, we observed that alpha- and gammaproteobacterial MOB differentially route CH4-derived carbon to the interactome. Finally, while repeated co-cultivation did not significantly impact the magnitude of overall methane oxidation rates (MOR), it did appear to stabilize the biological variability in MOR as compared to acenically grown MOB. In PART 3, the optimized cryopreservation protocol for storage of mixed microbial cultures adequately preserved both community structure and functionality of a methanotrophic interactome (among others) for 3 months at -80°C. These findings may be of significance for methanotrophic interactome Microbial Resource Management, if biotechnological application of non-MOB partners is the ultimate goal. Overall, unsupervised synthetic interactome assembly approaches should be preferred, as they specifically restrict only non-MOB partners that can persist with the MOB from other non-MOB partners. If specific biotechnological applications can be found for such an optimal combination of interactome partners, it is of great interest to preserve a reproducible sample of it for extended periods of time. Exploitation and engineering of these methanotrophic interactomes could lead to improved and sustainable mitigation and recovery of CH4 in the form of metabolic energy or CH4-derived carbon

Efficient molasses fermentation under high salinity by inocula of marine and terrestrial origin

BACKGROUND: Molasses is a dense and saline by-product of the sugar agroindustry. Its high organic content potentially fuels a myriad of renewable products of industrial interest. However, the biotechnological exploitation of molasses is mainly hampered by the high concentration of salts, an issue that is nowadays tackled through dilution. In the present study, the performance of microbial communities derived from marine sediment was compared to that of communities from a terrestrial environment (anaerobic digester sludge). The aim was to test whether adaptation to salinity represented an advantage for fermenting molasses into renewable chemicals such as volatile fatty acids (VFAs) although high sugar concentrations are uncommon to marine sediment, contrary to anaerobic digesters. RESULTS: Terrestrial and marine microbial communities were enriched in consecutive batches at different initial pH values (pH(i); either 6 or 7) and molasses dilutions (equivalent to organic loading rates (OLRs) of 1 or 5 g(COD) L(−1) d(−1)) to determine the best VFA production conditions. Marine communities were supplied with NaCl to maintain their native salinity. Due to molasses inherent salinity, terrestrial communities experienced conditions comparable to brackish or saline waters (20–47 mS cm(−1)), while marine conditions resembled brine waters (>47 mS cm(−1)). Enrichments at optimal conditions of OLR 5 g(COD) L(-1) d(-1) and pH(i) 7 were transferred into packed-bed biofilm reactors operated continuously. The reactors were first operated at 5 g(COD) L(-1) d(-1), which was later increased to OLR 10 g(COD) L(−1) d(−1). Terrestrial and marine reactors had different gas production and community structures but identical, remarkably high VFA bioconversion yields (above 85%) which were obtained with conductivities up to 90 mS cm(−1). COD-to-VFA conversion rates were comparable to the highest reported in literature while processing other organic leftovers at much lower salinities. CONCLUSIONS: Although salinity represents a major driver for microbial community structure, proper acclimation yielded highly efficient systems treating molasses, irrespective of the inoculum origin. Selection of equivalent pathways in communities derived from different environments suggests that culture conditions select for specific functionalities rather than microbial representatives. Mass balances, microbial community composition, and biochemical analysis indicate that biomass turnover rather than methanogenesis represents the main limitation to further increasing VFA production with molasses. This information is relevant to moving towards molasses fermentation to industrial application. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0701-8) contains supplementary material, which is available to authorized users

A unified framework for unconstrained and constrained ordination of microbiome read count data

Explorative visualization techniques provide a first summary of microbiome read count datasets through dimension reduction. A plethora of dimension reduction methods exists, but many of them focus primarily on sample ordination, failing to elucidate the role of the bacterial species. Moreover, implicit but often unrealistic assumptions underlying these methods fail to account for overdispersion and differences in sequencing depth, which are two typical characteristics of sequencing data. We combine log-linear models with a dispersion estimation algorithm and flexible response function modelling into a framework for unconstrained and constrained ordination. The method is able to cope with differences in dispersion between taxa and varying sequencing depths, to yield meaningful biological patterns. Moreover, it can correct for observed technical confounders, whereas other methods are adversely affected by these artefacts. Unlike distance-based ordination methods, the assumptions underlying our method are stated explicitly and can be verified using simple diagnostics. The combination of unconstrained and constrained ordination in the same framework is unique in the field and facilitates microbiome data exploration. We illustrate the advantages of our method on simulated and real datasets, while pointing out flaws in existing methods. The algorithms for fitting and plotting are available in the R-package RCM

Bacterial exchange in household washing machines

Household washing machines (WMs) launder soiled clothes and textiles, but do not sterilize them. We investigated the microbial exchange occurring in five household WMs. Samples from a new cotton T-shirt were laundered together with a normal laundry load. Analyses were performed on the influent water and the ingoing cotton samples, as well as the greywater and the washed cotton samples. The number of living bacteria was generally not lower in the WM effluent water as compared to the influent water. The laundering process caused a microbial exchange of influent water bacteria, skin-, and clothes related bacteria and biofilm-related bacteria in the WM. A variety of biofilm-producing bacteria were enriched in the effluent after laundering, although their presence in the cotton sample was low. Nearly all bacterial genera detected on the initial cotton sample were still present in the washed cotton samples. A selection for typical skin- and clothes related microbial species occurred in the cotton samples after laundering. Accordingly, malodour-causing microbial species might be further distributed to other clothes. The bacteria on the ingoing textiles contributed for a large part to the microbiome found in the textiles after laundering

Microbial protein out of thin air : fixation of nitrogen gas by an autotrophic hydrogen-oxidizing bacterial enrichment

For the production of edible microbial protein (MP), ammonia generated by the Haber-Bosch process or reclaimed ammonia from waste streams is typically considered as the nitrogen source. These processes for ammonia production are highly energy intensive. In this study, the potential for using nitrogen gas (N-2) as a direct nitrogen source for MP production by hydrogen-oxidizing bacteria (HOB) was evaluated. The use of N-2 versus ammonium as nitrogen source during the enrichment process resulted in differentiation of the bacterial community composition of the enrichments. A few previously unknown potential N-2-fixing HOB taxa (i.e., representatives of the genus Azonexus and the family Comamonadaceae) dominated the enrichments. The biomass yield of a N-2-fixing HOB enrichment was 30-50% lower than that of the ammonium-based HOB enrichment from the same inoculum source. The dried biomass of N-2-fixing HOB had a high protein content (62.0 +/- 6.3%) and an essential amino acid profile comparable to MP from ammonium-based HOB. MP from N-2-fixing HOB could potentially be produced in situ without entailing the emissions caused by ammonia production and transportation by conventional means. It could be a promising substitute for N-2-fixing protein-rich soybean because it has 70% higher protein content and double energy conversion efficiency from solar energy to biomass

In vitro increased respiratory activity of selected oral bacteria may explain competitive and collaborative interactions in the oral microbiome

Understanding the driving forces behind the shifts in the ecological balance of the oral microbiota will become essential for the future management and treatment of periodontitis. As the use of competitive approaches for modulating bacterial outgrowth is unexplored in the oral ecosystem, our study aimed to investigate both the associations among groups of functional compounds and the impact of individual substrates on selected members of the oral microbiome. We employed the Phenotype Microarray high-throughput technology to analyse the microbial cellular phenotypes of 15 oral bacteria. Multivariate statistical analysis was used to detect respiratory activity triggers and to assess similar metabolic activities. Carbon and nitrogen were relevant for the respiration of health-associated bacteria, explaining competitive interactions when grown in biofilms. Carbon, nitrogen, and peptides tended to decrease the respiratory activity of all pathobionts, but not significantly. None of the evaluated compounds significantly increased activity of pathobionts at both 24 and 48 h. Additionally, metabolite requirements of pathobionts were dissimilar, suggesting that collective modulation of their respiratory activity may be challenging. Flow cytometry indicated that the metabolic activity detected in the Biolog plates may not be a direct result of the number of bacterial cells. In addition, damage to the cell membrane may not influence overall respiratory activity. Our methodology confirmed previously reported competitive and collaborative interactions among bacterial groups, which could be used either as marker of health status or as targets for modulation of the oral environment

Reconciliation between operational taxonomic units and species boundaries

The development of high-throughput sequencing technologies has revolutionised the field of microbial ecology via 16S rRNA gene amplicon sequencing approaches. Clustering those amplicon sequencing reads into operational taxonomic units (OTUs) using a fixed cut-off is a commonly used approach to estimate microbial diversity. A 97% threshold was chosen with the intended purpose that resulting OTUs could be interpreted as a proxy for bacterial species. Our results show that the robustness of such a generalised cut-off is questionable when applied to short amplicons only covering one or two variable regions of the 16S rRNA gene. It will lead to biases in diversity metrics and makes it hard to compare results obtained with amplicons derived with different primer sets. The method introduced within this work takes into account the differential evolutional rates of taxonomic lineages in order to define a dynamic and taxonomic-dependent OTU clustering cut-off score. For a taxonomic family consisting of species showing high evolutionary conservation in the amplified variable regions, the cut-off will be more stringent than 97%. By taking into consideration the amplified variable regions and the taxonomic family when defining this cut-off, such a threshold will lead to more robust results and closer correspondence between OTUs and species. This approach has been implemented in a publicly available software package called DynamiC

Gut microbiota of migrating wild rabbit fish (Siganus guttatus) larvae have low spatial and temporal variability

We investigated the gut microbiota of rabbit fish larvae at three locations in Vietnam (ThuanAn-northern, QuangNam-intermediate, BinhDinh-southern sampling site) over a three-year period. In the wild, the first food for rabbit fish larvae remains unknown, while the juveniles and adults are herbivores, forming schools near the coasts, lagoons, and river mouths, and feeding mainly on filamentous algae. This is the first study on the gut microbiota of the wild fish larvae and with a large number of individuals analyzed spatially and temporally. The Clostridiales order was the most predominant in the gut, and location-by-location alpha diversity showed significant differences in Chao-1, Hill number 1, and evenness. Analysis of beta diversity indicated that the location, not year, had an effect on the composition of the microbiota. In 2014, the gut microbiota of fish from QuangNam was different from that in BinhDinh; in 2015, the gut microbiota was different for all locations; and, in 2016, the gut microbiota in ThuanAn was different from that in the other locations. There was a time-dependent trend in the north-south axis for the gut microbiota, which is considered to be tentative awaiting larger datasets. We found limited variation in the gut microbiota geographically and in time and strong indications for a core microbiome. Five and fifteen OTUs were found in 100 and 99% of the individuals, respectively. This suggests that at this life stage the gut microbiota is under strong selection due to a combination of fish-microbe and microbe-microbe interactions