Wen-Tso Liu

Nontuberculous mycobacteria (NTM) are any mycobacteria that do not cause tuberculosis or leprosy. While the majority of NTM are harmless and some of them are considered probiotic, a growing number of people are being diagnosed with NTM infections. Therefore, their detection in the environment is of interest to clinicians, environmental microbiologists, and water quality researchers alike. This review provides a tutorial on the foundational approaches for taxonomic classifications, with a focus on the phylogenetic relationships among NTM revealed by the 16S rRNA gene, rpoB gene, and hsp65 gene, and by genome-based approaches. Recent updates on the Mycobacterium genus taxonomy are also provided. A synthesis on the habitats of 189 mycobacterial species in a genome-based taxonomy framework was performed, with attention paid to environmental sources (e.g., drinking water, aquatic environments, and soil). The 16S rRNA gene-based classification accuracy for various regions was evaluated (V3, V3–V4, V3–V5, V4, V4–V5, and V1–V9), revealing overall excellent genus-level classification (up to 100% accuracy) yet only modest performance (up to 63.5% accuracy) at the species level. Future research quantifying NTM species in water systems, determining the effects of water treatment and plumbing conditions on their variations, developing high throughput species-level characterization tools for use in the environment, and incorporating the characterization of functions in a phylogenetic framework will likely fill critical knowledge gaps. We believe this tutorial will be useful for researchers new to the field of molecular or genome-based taxonomic profiling of environmental microbiomes. Experts may also find this review useful in terms of the selected key findings of the past 30 years, recent updates on phylogenomic analyses, as well as a synthesis of the ecology of NTM in a phylogenetic framework.

Background Prevalent viral particles are found in activated sludge (AS) and anaerobic digester (AD) during wastewater treatment processes. In contrast to the conventional approaches, culture-independent metagenomic method opens up the possibility to characterize uncultured viruses, which were previously inaccessible. In this study, we aimed to investigate the involvement of viruses in AS, mesophilic AD, and thermophilic AD using metagenomic sequences. We constructed the viromes, predicted the potential host, and profiled the functional genes in the viral genome. Results From a total of 64 samples, 42,548 viruses were detected. Among them, 43.6% were classified to Uncultivated Viral Genomes (UViGs) by the IMG/VR database and 28.5% were clustered into viral clusters (VCs) using vConTACT2. In total, only 18.2% of the viruses were taxonomically assigned to known viral families, with Siphoviridae, Myoviridae, Inoviridae, and Podoviridae being the most commonly observed. Most of the detected viruses were associated with engineered ecosystem, and more viruses in AS were associated with environmental ecosystem than in mesophilic and thermophilic AD. Diversity analysis suggested that the local habitat has a greater impact on the AD viromes than the immigration effect. Host prediction revealed that viruses in AD were mostly linked to site-specific active populations involved in syntrophic fatty acid oxidation, methanogenesis, hydrolysis, and fermentation. Functional gene annotation revealed that the viral gene profile follows the microbial gene profile, suggesting that viruses in AD not only tend to infect site-specific active hosts but also acquire functional genes transferred from the hosts. Conclusions Overall, this study provided insights of the viral diversity in the wastewater treatment process and uncovered the potential viral involvement in AD.

Interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation, indicating Ca. G. eutrophica’s EET ability. The high-quality draft genome further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. A Bayesian network was trained with the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis from dominant fermentative bacteria, Geobacter, and Methanobacterium. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

Fermentation of waste activated sludge (WAS) is an alternative approach to reduce solid wastes while providing valuable soluble products, such as volatile fatty acids and alcohols. This study systematically identified optimal fermentation conditions and key microbial populations by conducting two sets of experiments under different combinations of biochemical and physical parameters. Based on fermentation product concentrations, methane production, and solid removal, fermentation performance was enhanced under the combined treatments of inoculum heat shock (>60°C), pH 5, 55°C, and short solid retention time (SyntrophomonasandParabacteroidesincreased with a decrease in SRT. In contrast, treatment conditions, such as inoculum heat shock and high incubation temperature inhibited the growth of WAS-associated populations, such asTerrimonasandBryobacter.There were also populations insensitive to the treatment conditions, such as those related toMicrobacterandRikenellaceae. Overall, the EGMB approach clearly revealed the ecological roles of important microbial guilds in the WAS fermentation system, and guided the selection of optimal conditions for WAS fermentation in future pilot-scale operation.

Current understanding of amino acid (AA) degraders in anaerobic digesters is mainly based on cultured species, whereas microorganisms that play important roles in a complex microbial community remain poorly characterized. This study investigated short-term enrichments degrading single AAs using metagenomics and metatranscriptomics. Metagenomic analysis revealed that populations related to cultured AA degraders had an abundance 35% of the sequences. Phylogenetic analyses suggested that these Bacteroidales populations represented a yet-to-be characterized family lineage, i.e., Bacteroidetes vadinHA17. The bins possessed the genetic capacity related to protein degradation, including surface adhesion (3–7 genes), secreted peptidase (52–77 genes), and polypeptide-specific transporters (2–5 genes). Furthermore, metatranscriptomics revealed that these Bacteroidales populations expressed the complete metabolic pathways for degrading 16 to 17 types of AAs in enrichments fed with respective substrates. These characteristics were distinct from cultured AA degraders including Acidaminobacter and Peptoclostridium, suggesting the uncultured Bacteroidales were the major protein-hydrolyzing and AA-degrading populations. These uncultured Bacteroidales were further found to be dominant and active in full-scale anaerobic digesters, indicating their important ecological roles in the native habitats. “Candidatus Aminobacteroidaceae” was proposed to represent the previously uncharted family Bacteroidetes vadinHA17.

Background: Current understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H₂ transfer between organotrophs and methanogens. However, many metabolic processes are thermodynamically sensitive to H₂ accumulation and can be inhibited by H₂ produced from co-occurring metabolisms. Strategies for driving thermodynamically competing metabolisms in methanogenic environments remain unexplored. Results: To uncover how anaerobes combat this H₂ conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology—methanogenic bioreactors. Through analysis of 17 anaerobic digesters, we recovered 1343 high-quality metagenome-assembled genomes and corresponding gene expression profiles for uncultured lineages spanning 66 phyla and reconstructed their metabolic capacities. We discovered that diverse uncultured populations can drive H₂-sensitive metabolisms through (i) metabolic coupling with concurrent H₂-tolerant catabolism, (ii) forgoing H₂ generation in favor of interspecies transfer of formate and electrons (cytochrome- and pili-mediated) to avoid thermodynamic conflict, and (iii) integration of low-concentration O₂ metabolism as an ancillary thermodynamics-enhancing electron sink. Archaeal populations support these processes through unique methanogenic metabolisms—highly favorable H₂ oxidation driven by methyl-reducing methanogenesis and tripartite uptake of formate, electrons, and acetate. Conclusion: Integration of omics and eco-thermodynamics revealed overlooked behavior and interactions of uncultured organisms, including coupling favorable and unfavorable metabolisms, shifting from H₂ to formate transfer, respiring low-concentration O₂, performing direct interspecies electron transfer, and interacting with high H₂-affinity methanogenesis. These findings shed light on how microorganisms overcome a critical obstacle in methanogenic carbon cycles we had hitherto disregarded and provide foundational insight into anaerobic microbial ecology.

Changes in the sequence of an organism’s genome, i.e. mutations, are the raw material of evolution ¹ . The frequency and location of mutations can be constrained by specific molecular mechanisms, such as Diversity-generating retroelements (DGRs) ^2–4 . DGRs introduce mutations in specific target genes, and were characterized from several cultivated bacteria and bacteriophages ² . Whilst a larger diversity of DGR loci has been identified in genomic data from environmental samples, i.e. metagenomes, the ecological role of these DGRs and their associated evolutionary drivers remain poorly understood ^5–7 . Here we built and analyzed an extensive dataset of >30,000 metagenome-derived DGRs, and determine that DGRs have a single evolutionary origin and a universal bias towards adenine mutations. We further identified six major lineages of DGRs, each associated with a specific ecological niche defined as a genome type, i.e. whether the DGR is encoded on a viral or cellular genome, a limited set of taxa and environments, and a distinct type of target. Finally, we leverage read mapping and metagenomic time series to demonstrate that DGRs are consistently and broadly active, and responsible for >10% of all amino acid changes in some organisms at a conservative estimate. Overall, these results highlight the strong constraints under which DGRs diversify and expand, and elucidate several distinct roles these elements play in natural communities and in shaping microbial community structure and function in our environment.