Kyohei Kuroda

Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen

The ecophysiology of one candidate methanogen class WSA2 (or Arc I) remains largely uncharacterized, despite the long history of research on Euryarchaeota methanogenesis. To expand our understanding of methanogen diversity and evolution, we metagenomically recover eight draft genomes for four WSA2 populations. Taxonomic analyses indicate that WSA2 is a distinct class from other Euryarchaeota. None of genomes harbor pathways for CO2-reducing and aceticlastic methanogenesis, but all possess H2 and CO oxidation and energy conservation through H2-oxidizing electron confurcation and internal H2 cycling. As the only discernible methanogenic outlet, they consistently encode a methylated thiol coenzyme M methyltransferase. Although incomplete, all draft genomes point to the proposition that WSA2 is the first discovered methanogen restricted to methanogenesis through methylated thiol reduction. In addition, the genomes lack pathways for carbon fixation, nitrogen fixation and biosynthesis of many amino acids. Acetate, malonate and propionate may serve as carbon sources. Using methylated thiol reduction, WSA2 may not only bridge the carbon and sulfur cycles in eutrophic methanogenic environments, but also potentially compete with CO2-reducing methanogens and even sulfate reducers. These findings reveal a remarkably unique methanogen ‘Candidatus Methanofastidiosum methylthiophilus’ as the first insight into the sixth class of methanogens ‘Candidatus Methanofastidiosa’.

Community composition of known and uncultured archaeal lineages in anaerobic or anoxic wastewater treatment sludge.

Microbial systems are widely used to treat different types of wastewater from domestic, agricultural, and industrial sources. Community composition is an important factor in determining the successful performance of microbial treatment systems; however, a variety of uncultured and unknown lineages exist in sludge that requires identification and characterization. The present study examined the archaeal community composition in methanogenic, denitrifying, and nitrogen-/phosphate-removing wastewater treatment sludge by Archaea-specific 16S rRNA gene sequencing analysis using Illumina sequencing technology. Phylotypes belonging to Euryarchaeota, including methanogens, were most abundant in all samples except for nitrogen-/phosphate-removing wastewater treatment sludge. High levels of Deep Sea Hydrothermal Vent Group 6 (DHVEG-6), WSA2, Terrestrial Miscellaneous Euryarchaeotal Group, and Miscellaneous Crenarchaeotic Group were also detected. Interestingly, DHVEG-6 was dominant in nitrogen-/phosphate-removing wastewater treatment sludge, indicating that unclear lineages of Archaea still exist in the anaerobic wastewater treatment sludges. These results reveal a previously unknown diversity of Archaea in sludge that can potentially be exploited for the development of more efficient wastewater treatment strategies.

High organic loading treatment for industrial molasses wastewater and microbial community shifts corresponding to system development

Molasses wastewater contains high levels of organic compounds, cations, and anions, causing operational problems for anaerobic biological treatment. To establish a high organic loading treatment system for industrial molasses wastewater, this study designed a combined system comprising an acidification tank, a thermophilic multi-stage (MS)-upflow anaerobic sludge blanket (UASB) reactor, mesophilic UASB reactor, and down-flow hanging sponge reactor. The average total chemical oxygen demand (COD) and biochemical oxygen demand removal rates were 85%±3% and 95%±2%, respectively, at an organic loading rate of 42kgCODcrm(-3)d(-1) in the MS-UASB reactor. By installation of the acidification tank, the MS-UASB reactor achieved low H2-partial pressure. The abundance of syntrophs such as fatty acid-degrading bacteria increased in the MS-UASB and 2nd-UASB reactors. Thus, the acidification tank contributed to maintaining a favorable environment for syntrophic associations. This study provides new information regarding microbial community composition in a molasses wastewater treatment system.

Development of a BR–UASB–DHS system for natural rubber processing wastewater treatment

Natural rubber processing wastewater contains high concentrations of organic compounds, nitrogen, and other contaminants. In this study, a treatment system composed of a baffled reactor (BR), an upflow anaerobic sludge blanket (UASB) reactor, and a downflow hanging sponge (DHS) reactor was used to treat natural rubber processing wastewater in Vietnam. The BR showed good total suspended solids (TSS) removal of 47.6%, as well as acidification of wastewater. The UASB reactor achieved a high chemical oxygen demand (COD) removal efficiency of 92.7 ± 2.3% and energy recovery in the form of methane with an organic loading rate of 12.2 ± 6.6 kg-COD m−3 day−1. The DHS reactor showed high performance in residual organic matter removal from UASB effluent. In total, the system achieved high-level total COD removal of 98.6% ± 1.2% and TSS removal of 98.0% ± 1.4%. Massive parallel 16S rRNA gene sequencing of the retained sludge in the UASB reactor showed the predominant microbial phyla to be Bacteroidetes, Firmicutes, Proteobacteria, WWE1, and Euryarchaeota. Uncultured bacteria belonging to the phylum Bacteroidetes and Phylum WWE1 were predominant in the UASB reactor. This microbial assemblage utilizes the organic compounds contained in natural rubber processing wastewater. In addition, the methane-producing archaea Methanosaeta sp. and Methanolinea sp. were detected.

Evaluating digestion efficiency in full-scale anaerobic digesters by identifying active microbial populations through the lens of microbial activity

Anaerobic digestion is a common technology to biologically stabilize wasted solids produced in municipal wastewater treatment. Its efficiency is usually evaluated by calculating the reduction in volatile solids, which assumes no biomass growth associated with digestion. To determine whether this assumption is valid and further evaluate digestion efficiency, this study sampled 35 digester sludge from different reactors at multiple time points together with the feed biomass in a full-scale water reclamation plant at Chicago, Illinois. The microbial communities were characterized using Illumina sequencing technology based on 16S rRNA and 16S rRNA gene (rDNA). 74 core microbial populations were identified and represented 58.7% of the entire digester community. Among them, active populations were first identified using the ratio of 16S rRNA and 16S rDNA (rRNA/rDNA) for individual populations, but this approach failed to generate consistent results. Subsequently, a recently proposed mass balance model was applied to calculate the specific growth rate (μ), and this approach successfully identified active microbial populations in digester (positive μ) that could play important roles than those with negative μ. It was further estimated that 82% of microbial populations in the feed sludge were digested in comparison with less than 50% calculated using current equations.

Identification and Detection of Prokaryotic Symbionts in the Ciliate Metopus from Anaerobic Granular Sludge

The aim of the present study was to investigate the prokaryotic community structure of the anaerobic ciliate, Metopus sp. using rRNA sequencing, fluorescence in situ hybridization (FISH), and transmission electron microscopy (TEM). Metopus sp. was physically separated from anaerobic granular sludge in a domestic wastewater treatment plant and anoxically cultivated for 7 d. 16S rRNA gene sequences from the prokaryotes Methanoregula boonei and Clostridium aminobutyricum were abundantly detected in Metopus ciliates. The FISH analysis using the oligonucleotide probes Mg1200b and Cla568 demonstrated that these prokaryotes were localized within Metopus cells. These results identify M. boonei- and C. aminobutyricum-like prokaryotes as novel endosymbionts of Metopus ciliates.

Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge

Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates—ciliates frequently found in anoxic ecosystems—on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885–3,190 and 2,387–2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

Operation-driven heterogeneity and overlooked feed-associated populations in global anaerobic digester microbiome

Anaerobic digester (AD) microbiomes harbor complex, interacting microbial populations to achieve biomass reduction and biogas production, however how they are influenced by operating conditions and feed sludge microorganisms remain unclear. These were addressed by analyzing the microbial communities of 90 full-scale digesters at 51 municipal wastewater treatment plants from five countries. Heterogeneity detected in community structures suggested that no single AD microbiome could be defined. Instead, the AD microbiomes were classified into eight clusters driven by operating conditions (e.g., pretreatment, temperature range, and salinity), whereas geographic location of the digesters did not have significant impacts. Comparing digesters populations with those present in the corresponding feed sludge led to the identification of a hitherto overlooked feed-associated microbial group (i.e., the residue populations). They accounted for up to 21.4% of total sequences in ADs operated at low temperature, presumably due to ineffective digestion, and as low as 0.8% in ADs with pretreatment. Within each cluster, a core microbiome was defined, including methanogens, syntrophic metabolizers, fermenters, and the newly described residue populations. Our work provides insights into the key factors shaping full-scale AD microbiomes in a global scale, and draws attentions to the overlooked residue populations.

Diversity Profile of Microbes Associated with Anaerobic Sulfur Oxidation in an Upflow Anaerobic Sludge Blanket Reactor Treating Municipal Sewage

We herein analyzed the diversity of microbes involved in anaerobic sulfur oxidation in an upflow anaerobic sludge blanket (UASB) reactor used for treating municipal sewage under low-temperature conditions. Anaerobic sulfur oxidation occurred in the absence of oxygen, with nitrite and nitrate as electron acceptors; however, reactor performance parameters demonstrated that anaerobic conditions were maintained. In order to gain insights into the underlying basis of anaerobic sulfur oxidation, the microbial diversity that exists in the UASB sludge was analyzed comprehensively to determine their identities and contribution to sulfur oxidation. Sludge samples were collected from the UASB reactor over a period of 2 years and used for bacterial 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and next-generation sequencing analyses. T-RFLP and sequencing results both showed that microbial community patterns changed markedly from day 537 onwards. Bacteria belonging to the genus Desulforhabdus within the phylum Proteobacteria and uncultured bacteria within the phylum Fusobacteria were the main groups observed during the period of anaerobic sulfur oxidation. Their abundance correlated with temperature, suggesting that these bacterial groups played roles in anaerobic sulfur oxidation in UASB reactors.

A single-granule-level approach reveals ecological heterogeneity in an upflow anaerobic sludge blanket reactor

Upflow anaerobic sludge blanket (UASB) reactor has served as an effective process to treat industrial wastewater such as purified terephthalic acid (PTA) wastewater. For optimal UASB performance, balanced ecological interactions between syntrophs, methanogens, and fermenters are critical. However, much of the interactions remain unclear because UASB have been studied at a “macro”-level perspective of the reactor ecosystem. In reality, such reactors are composed of a suite of granules, each forming individual micro-ecosystems treating wastewater. Thus, typical approaches may be oversimplifying the complexity of the microbial ecology and granular development. To identify critical microbial interactions at both macro- and micro- level ecosystem ecology, we perform community and network analyses on 300 PTA–degrading granules from a lab-scale UASB reactor and two full-scale reactors. Based on MiSeq-based 16S rRNA gene sequencing of individual granules, different granule-types co-exist in both full-scale reactors regardless of granule size and reactor sampling depth, suggesting that distinct microbial interactions occur in different granules throughout the reactor. In addition, we identify novel networks of syntrophic metabolic interactions in different granules, perhaps caused by distinct thermodynamic conditions. Moreover, unseen methanogenic relationships (e.g. “Candidatus Aminicenantes” and Methanosaeta) are observed in UASB reactors. In total, we discover unexpected microbial interactions in granular micro-ecosystems supporting UASB ecology and treatment through a unique single-granule level approach.