Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

Anaerobic Microbial Communities in Lake Pavin, a Unique Meromictic Lake in France

The Bacteria and Archaea from the meromictic Lake Pavin were analyzed in samples collected along a vertical profile in the anoxic monimolimnion and were compared to those in samples from the oxic mixolimnion. Nine targeted 16S rRNA oligonucleotide probes were used to assess the distribution of Bacteria and Archaea and to investigate the in situ occurrence of sulfate-reducing bacteria and methane-producing Archaea involved in the terminal steps of the anaerobic degradation of organic material. The diversity of the complex microbial communities was assessed from the 16S rRNA polymorphisms present in terminal restriction fragment (TRF) depth patterns. The densities of the microbial community increased in the anoxic layer, and Archaea detected with probe ARCH915 represented the largest microbial group in the water column, with a mean Archaea/Eubacteria ratio of 1.5. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed an elevated archaeal and bacterial phylotype richness in anoxic bottom-water samples. The structure of the Archaea community remained rather homogeneous, while TRFLP patterns for the eubacterial community revealed a heterogeneous distribution of eubacterial TRFs.     

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.     

Microbial Communities in the World's Largest Acidic Volcanic Lake, Kawah Ijen in Indonesia, and in the Banyupahit River Originating from It

A first study was made on the microbial community composition of the Indonesian crater lake Kawah Ijen (pH < 0.3) and the Banyupahit–Banyuputih river (pH 0.4–3.5) originating from it. Culture-independent, rRNA gene-based denaturing gradient gel electrophoresis was used to profile microbial communities in this natural and ancient, extremely acidic environment. Similarity in community profiles of the different sampling locations was low, indicating heterogeneity in community composition. Archaea were present at all sampling locations; archaeal diversity was low at the most acidic locations and increased at pH >2.6. Bacteria were not detected in the water column of the crater lake, but were found at all locations along the acidic river. Bacterial diversity increased with increasing pH. Eukarya were only present at pH >2.6. Retrieved rRNA gene sequences of Bacteria and Archaea were not closely related to known acidophilic species. It is concluded that tolerance to extreme acidity in this system is developed most extensively among Archaea. The acidity gradient of the Banyupahit–Banyuputih river has a clear effect on microbial community composition and biodiversity.     

Vertical Distribution of Archaea and Bacteria in a Meromictic Lake as Determined by Fluorescent In Situ Hybridization

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A “red-water” layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Influence of support material properties on the potential selection of Archaea during initial adhesion of a methanogenic consortium

In anaerobic wastewater treatment systems, the complex microbial biomass including Archaea and Bacteria can be retained as a biofilm attached to solid supports. The aim of this study was to evaluate the impact of specific properties of support material on early microbial adhesion. Seven different substrata are described in terms of topography and surface energy. Adhesion of a methanogenic consortium to these substrata was tested, the adhesion was quantified as a percentage of the surface area covered and the bacterial and archaeal community structures was assessed by molecular fingerprinting profiles (CE-SSCP). As expected, the overall adhesion on the supports was influenced mainly by total surface energy. Moreover, the adhered communities were different from the parent inocula, including the Archaea/Bacteria ratio. This could have a significant impact on the start-up of anaerobic digesters for which supports favoring Archaea adhesion, responsible for the limiting reaction of the process, should be preferred.     

Degradation of oxytetracycline and its impacts on biogas-producing microbial community structure

The effect of veterinary antibiotics in anaerobic digesters is a concern where methane production efficiency is highly dependent on microbial community structure. In this study, both anaerobic degradation of a common veterinary antibiotic, oxytetracycline (OTC), and its effects on an anaerobic digester microbial community were investigated. Qualitative and quantitative molecular tools were used to monitor changes in microbial community structure during a 60-day batch incubation period of cow manure with the addition of different concentrations of the antibiotic. Molecular data were interpreted by a further redundancy analysis as a multivariate statistics approach. At the end of the experiment, approximately 48, 33, and 17 % of the initially added 50, 100, and 200 mg l^?1 of OTC was still present in the serum bottles which reduced the biogas production via accumulation of some of the volatile fatty acids (VFAs). Biogas production was highly correlated with Methanobacteriales and Methanosarcinales gene copy numbers, and those parameters were negatively affected with oxytetracycline and VFA concentrations.     

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C₃–C₆), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.     

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.     

Volatile fatty acids (VFAs) and methane from food waste and cow slurry: Comparison of biogas and VFA fermentation processes

The potential of various biomasses for the production of green chemicals is currently one of the key topics in the field of the circular economy. Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and they can be produced in similar reactors as biogas to increase the productivity of a digestion plant, as VFAs have more varying end uses compared to biogas and methane. In this study, the aim was to assess the biogas and VFA production of food waste (FW) and cow slurry (CS) using the anaerobic biogas plant inoculum treating the corresponding substrates. The biogas and VFA production of both biomasses were studied in identical batch scale laboratory conditions while the process performance was assessed with chemical and microbial analyses. As a result, FW and CS were shown to have different chemical performances and microbial dynamics in both VFA and biogas processes. FW as a substrate showed higher yields in both processes (435 ml CH₄/g VS_fed and 434 mg VFA/g VS_fed) due to its characteristics (pH, organic composition, microbial communities), and thus, the vast volume of CS makes it also a relevant substrate for VFA and biogas production. In this study, VFA profiles were highly dependent on the substrate and inoculum characteristics, while orders Clostridiales and Lactobacillales were connected with high VFA and butyric acid production with FW as a substrate. In conclusion, anaerobic digestion supports the implementation of the waste management hierarchy as it enables the production of renewable green chemicals from both urban and rural waste materials.     

Microbial population in the biomass adhering to supporting material in a packed-bed reactor degrading organic solid waste

An anaerobic packed-bed reactor using carbon fiber textiles (CFT) as the supporting material was continuously operated using an artificial garbage slurry. 16S rRNA gene analysis showed that many bacteria in the biomass adhering to CFT were closely related to those observed from other anaerobic environments, although a wide variety of unidentified bacteria were also found. Dot blot hybridization results clarified that 16S rRNA levels of methanogens in the adhering biomass were higher than those in the effluent. Based on microscopic observation, the adhering biomass consisted of microorganisms, organic material, and void areas. Bacteria and Archaea detected by fluorescence in situ hybridization were distributed from the surface to the inner regions of the adhering biomass. Methanosarcina sp. tended to be more abundant in the inner part of the adhering biomass than at the surface. This is the first report to elucidate the structure of the microbial community on CFT in a packed-bed reactor.     

Persistence of bacterial and archaeal communities in sea ice through an Arctic winter

The structure of bacterial communities in first‐year spring and summer sea ice differs from that in source seawaters, suggesting selection during ice formation in autumn or taxon‐specific mortality in the ice during winter. We tested these hypotheses by weekly sampling (January–March 2004) of first‐year winter sea ice (Franklin Bay, Western Arctic) that experienced temperatures from ?9°C to ?26°C, generating community fingerprints and clone libraries for Bacteria and Archaea. Despite severe conditions and significant decreases in microbial abundance, no significant changes in richness or community structure were detected in the ice. Communities of Bacteria and Archaea in the ice, as in under‐ice seawater, were dominated by SAR11 clade Alphaproteobacteria and Marine Group I Crenarchaeota, neither of which is known from later season sea ice. The bacterial ice library contained clones of Gammaproteobacteria from oligotrophic seawater clades (e.g. OM60, OM182) but no clones from gammaproteobacterial genera commonly detected in later season sea ice by similar methods (e.g. Colwellia, Psychrobacter). The only common sea ice bacterial genus detected in winter ice was Polaribacter. Overall, selection during ice formation and mortality during winter appear to play minor roles in the process of microbial succession that leads to distinctive spring and summer sea ice communities.     

Single-stage, batch, leach-bed, thermophilic anaerobic digestion of spent sugar beet pulp

Spent sugar beet pulp as received was digested in a single-stage, batch, unmixed, leach-bed, laboratory scale thermophilic anaerobic digester. Biogasification of each 0.450 kg (wet weight) batch of spent pulp was initiated by inoculating with anaerobically digested liquor from previous run. The average methane yield was 0.336 m3 CH4 at STP (kgVS)(-1), the maximum methane production rate was 0.087 m3 CH4 at STP (kgVS)(-1)d(-1), average lag time to initiate methanogenesis was only 0.44 days and time required to achieve 95% methane yield was 8 days. The pH in the digesters ranged between 8.0 and 9.5. High rates of methane generation were sustained even at high pH values. The equivalent organic loading rate in the batch digesters was 4 kgCODm(-3)d(-1). The digestion process used here offers significant improvements over one-stage and two-stage systems reported in the literature with comparable performance as it is a single-stage system where the feedstock does not require size reduction, and mixing is not required in the digester.     

Methane- and Sulfur-Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem

Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.     

Relating MEC population dynamics to anode performance from DGGE and electrical data

The microbial electrolysis cell (MEC) is a promising system for H₂ production, but little is known about the active microbial population in MEC systems. Therefore, the microbial community of five different MEC graphite felt anodes was analyzed using denaturing gradient gel electrophoresis (DGGE) profiling. The results showed that the bacterial population was very diverse and there were substantial differences between microorganisms in anolyte and anode samples. The archaeal population in the anolyte and at the anodes, and between the different MEC anodes, was very similar. SEM and FISH imaging showed that Archaea were mainly present in the spaces between the electrode fibers and Bacteria were present at the fiber surface, which suggested that Bacteria were the main microorganisms involved in MEC electrochemical activity. Redundancy analysis (RDA) and QR factorization-based estimation (QRE) were used to link the composition of the bacterial community to electrochemical performance of the MEC. The operational mode of the MECs and their consequent effects on current density and anode resistance on the populations were significant. The results showed that the community composition was most strongly correlated with current density. The DGGE band mostly correlated with current represented a Clostridium sticklandii strain, suggesting that this species had a major role in current from acetate generation at the MEC anodes. The combination of RDA and QRE seemed especially promising for obtaining an insight into the part of the microbial population actively involved in electrode interaction in the MEC.     

Anaerobic cellulolytic microbial communities decomposing the biomass of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

Four previously isolated methanogenic anaerobic consortia, which were originally cultivated on a cellulose-containing substrate (filter paper), were used as inocula for the anaerobic conversion of the biomass of Anabaena variabilis into biogas at 55°C. The cumulative methane yield in the biogas produced by the most active consortia reached 64%. However, the biotransformation was only efficient in the course of the single inoculation and pretreatment of the cyanobacterial biomass by its concentration and freeze-thawing. The DGGE analysis of the structure of the selected microbial consortia, cultivated on the filter paper, revealed qualitative variations in the biodiversity of predominant Bacteria, showing differences in band number and intensity. The composition of methanogenic Archaea in these consortia was similar, with the presence of the genera Methanoculleus and Methanosarcina. The efficiency of the microbial consortia selection, and the role of the various microbial trophic groups in bioconversion of the substrates, such as cellulose and the biomass of phototrophic microorganisms are discussed.     

Performance and molecular evaluation of an anaerobic system with suspended biomass for treating wastewater with high fat content after enzymatic hydrolysis

The effect of a lipase-rich fungal enzymatic preparation, produced by a Penicillium sp. during solid-state fermentation, was evaluated in an anaerobic digester treating dairy wastewater with 1200 mg of oil and grease/L. The oil and grease hydrolysis step was carried out with 0.1% (w/v) of solid enzymatic preparation at 30 °C for 24 h, and resulted in a final free acid concentration eight times higher than the initial value. The digester operated in sequential batches of 48 h at 30 °C for 245 days, and had high chemical oxygen demand (COD) removal efficiencies (around 90%) when fed with pre-hydrolyzed wastewater. However, when the pre-hydrolysis step was removed, the anaerobic digester performed poorly (with an average COD removal of 32%), as the oil and grease accumulated in the biomass and effluent oil and grease concentration increased throughout the operational period. PCR-DGGE analysis of the Bacteria and Archaea domains revealed remarkable differences in the microbial profiles in trials conducted with and without the pre-hydrolysis step, indicating that differences observed in overall parameters were intrinsically related to the microbial diversity of the anaerobic sludge.     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.