Similar Methanogenic Shift but Divergent Syntrophic Partners in Anaerobic Digesters Exposed to Direct versus Successive Ammonium Additions.

During anaerobic digestion (AD) of protein-rich wastewater, ammonium (NH4+) is released by amino acid degradation. High NH4+ concentrations disturb the AD microbiome balance, leading to process impairments. The sensitivity of the AD microbiome to NH4+ and the inhibition threshold depend on multiple parameters, especially the previous microbial acclimation to ammonium stress. However, little is known about the effect of different NH4+ acclimation strategies on the differential expression of key active microbial taxa. Here, we applied NH4+ inputs of increasing intensity (from 1.7 to 15.2 g N-NH4+ liters-1) in batch assays fed with synthetic wastewater, according to two different strategies: (i) direct independent inputs at a unique target concentration and (ii) successive inputs in a stepwise manner. In both strategies, along the NH4+ gradient, the active methanogens shifted from acetoclastic Methanosaeta to Methanosarcina and eventually hydrogenotrophic Methanoculleus. Despite shorter latency times, the successive input modality led to lower methane production rate, lower soluble chemical oxygen demand (sCOD) removal efficiency, and lower half maximal inhibitory concentration, together with higher volatile fatty acid (VFA) accumulation, compared to the independent input modality. These differential performances were associated with a drastically distinct succession pattern of the active bacterial partners in both experiments. In particular, the direct exposure modality was characterized by a progressive enrichment of VFA producers (mainly Tepidimicrobium) and syntrophic VFA oxidizers (mainly Syntrophaceticus) with increasing NH4+ concentration, while the successive exposure modality was characterized by a more dynamic succession of VFA producers (mainly Clostridium, Sporanaerobacter, Terrisporobacter) and syntrophic VFA oxidizers (mainly Tepidanaerobacter, Syntrophomonas). These results bring relevant insights for improved process management through inoculum adaptation, bioaugmentation, or community-driven optimization. IMPORTANCE Anaerobic digestion (AD) is an attractive biotechnological process for wastewater bioremediation and bioenergy production in the form of methane-rich biogas. However, AD can be inhibited by ammonium generated by protein-rich effluent, commonly found in agro-industrial activities. Insights in the microbial community composition and identification of AD key players are crucial for anticipating process impairments in response to ammonium stress. They can also help in defining an optimal microbiome adapted to high ammonium levels. Here, we compared two strategies for acclimation of AD microbiome to increasing ammonium concentration to better understand the effect of this stress on the methanogens and their bacterial partners. Our results suggest that long-term cumulative exposure to ammonia disrupted the AD microbiome more strongly than direct (independent) ammonium additions. We identified bioindicators with different NH4+ tolerance capacity among VFA producers and syntrophic VFA oxidizers.

Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing.

BACKGROUND: Wastewater surveillance was proposed as an epidemiological tool to define the prevalence and evolution of the SARS-CoV-2 epidemics. However, most implemented SARS-CoV-2 wastewater surveillance projects were based on qPCR measurement of virus titers and did not address the mutational spectrum of SARS-CoV-2 circulating in the population. METHODS: We have implemented a nanopore RNA sequencing monitoring system in the city of Nice (France, 550,000 inhabitants). Between October 2020 and March 2021, we monthly analyzed the SARS-CoV-2 variants in 113 wastewater samples collected in the main wastewater treatment plant and 20 neighborhoods. FINDINGS: We initially detected the lineages predominant in Europe at the end of 2020 (B.1.160, B.1.177, B.1.367, B.1.474, and B.1.221). In January, a localized emergence of a variant (Spike:A522S) of the B.1.1.7 lineage occurred in one neighborhood. It rapidly spread and became dominant all over the city. Other variants of concern (B.1.351, P.1) were also detected in some neighborhoods, but at low frequency. Comparison with individual clinical samples collected during the same week showed that wastewater sequencing correctly identified the same lineages as those found in COVID-19 patients. INTERPRETATION: Wastewater sequencing allowed to document the diversity of SARS-CoV-2 sequences within the different neighborhoods of the city of Nice. Our results illustrate how sequencing of sewage samples can be used to track pathogen sequence diversity in the current pandemics and in future infectious disease outbreaks. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Anaerobic lignocellulolytic microbial consortium derived from termite gut: enrichment, lignocellulose degradation and community dynamics.

BACKGROUND: Lignocellulose is the most abundant renewable carbon resource that can be used for biofuels and commodity chemicals production. The ability of complex microbial communities present in natural environments that are specialized in biomass deconstruction can be exploited to develop lignocellulose bioconversion processes. Termites are among the most abundant insects on earth and play an important role in lignocellulose decomposition. Although their digestive microbiome is recognized as a potential reservoir of microorganisms producing lignocellulolytic enzymes, the potential to enrich and maintain the lignocellulolytic activity of microbial consortia derived from termite gut useful for lignocellulose biorefinery has not been assessed. Here, we assessed the possibility of enriching a microbial consortium from termite gut and maintaining its lignocellulose degradation ability in controlled anaerobic bioreactors. RESULTS: We enriched a termite gut-derived consortium able to transform lignocellulose into carboxylates under anaerobic conditions. To assess the impact of substrate natural microbiome on the enrichment and the maintenance of termite gut microbiome, the enrichment process was performed using both sterilized and non-sterilized straw. The enrichment process was carried out in bioreactors operating under industrially relevant aseptic conditions. Two termite gut-derived microbial consortia were obtained from Nasutitermes ephratae by sequential batch culture on raw wheat straw as the sole carbon source. Analysis of substrate loss, carboxylate production and microbial diversity showed that regardless of the substrate sterility, the diversity of communities selected by the enrichment process strongly changed compared to that observed in the termite gut. Nevertheless, the community obtained on sterile straw displayed higher lignocellulose degradation capacity; it showed a high xylanase activity and an initial preference for hemicellulose. CONCLUSIONS: This study demonstrates that it is possible to enrich and maintain a microbial consortium derived from termite gut microbiome in controlled anaerobic bioreactors, producing useful carboxylates from raw biomass. Our results suggest that the microbial community is shaped both by the substrate and the conditions that prevail during enrichment. However, when aseptic conditions are applied, it is also affected by the biotic pressure exerted by microorganisms naturally present in the substrate and in the surrounding environment. Besides the efficient lignocellulolytic consortium enriched in this study, our results revealed high levels of xylanase activity that can now be further explored for enzyme identification and overexpression for biorefinery purposes.

Ecofriendly lignocellulose pretreatment to enhance the carboxylate production of a rumen-derived microbial consortium.

Innovative dry chemo- and chemo-mechanical pretreatments form an interesting approach for modifying the native physico-chemical composition of lignocellulose facilitating its microbial conversion to carboxylates. Here, the impact of four dry-pretreatment conditions on the microbial transformation of wheat straw was assessed: milling to 2mm and 100µm, and NaOH chemical impregnation at high substrate concentrations combined with milling at 2mm and 100µm. Pretreatment effect was assessed in the light of substrate structure and composition, its impact on the acidogenic potential and the major enzyme activities of a rumen-derived microbial consortium RWS. Chemo-mechanical pretreatment strongly modified the substrate macroporosity. The highest carboxylate production rate was reached after dry chemo-mechanical treatment with NaOH at 100µm. A positive impact of the dry chemo-mechanical treatment on xylanase activity was observed also. These results underline that increasing substrate macroporosity by dry chemo-mechanical pretreatment had a positive impact on the microbial acidogenic potential.

Uncovering the Potential of Termite Gut Microbiome for Lignocellulose Bioconversion in Anaerobic Batch Bioreactors.

Termites are xylophages, being able to digest a wide variety of lignocellulosic biomass including wood with high lignin content. This ability to feed on recalcitrant plant material is the result of complex symbiotic relationships, which involve termite-specific gut microbiomes. Therefore, these represent a potential source of microorganisms for the bioconversion of lignocellulose in bioprocesses targeting the production of carboxylates. In this study, gut microbiomes of four termite species were studied for their capacity to degrade wheat straw and produce carboxylates in controlled bioreactors. All of the gut microbiomes successfully degraded lignocellulose and up to 45% w/w of wheat straw degradation was observed, with the Nasutitermes ephratae gut-microbiome displaying the highest levels of wheat straw degradation, carboxylate production and enzymatic activity. Comparing the 16S rRNA gene diversity of the initial gut inocula to the bacterial communities in lignocellulose degradation bioreactors revealed important changes in community diversity. In particular, taxa such as Spirochaetes and Fibrobacteres that were highly abundant in the initial gut inocula were replaced by Firmicutes and Proteobacteria at the end of incubation in wheat straw bioreactors. Overall, this study demonstrates that termite-gut microbiomes constitute a reservoir of lignocellulose-degrading bacteria that can be harnessed in artificial conditions for biomass conversion processes that lead to the production of useful molecules.

CAZyChip: dynamic assessment of exploration of glycoside hydrolases in microbial ecosystems.

BACKGROUND: Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans. RESULTS: This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares, and microarrays were directly synthesized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known GHs RNA extracted from recombinant E. coli strains, which were previously identified by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota. CONCLUSIONS: The CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems.

Efficient anaerobic transformation of raw wheat straw by a robust cow rumen-derived microbial consortium.

A rumen-derived microbial consortium was enriched on raw wheat straw as sole carbon source in a sequential batch-reactor (SBR) process under strict mesophilic anaerobic conditions. After five cycles of enrichment the procedure enabled to select a stable and efficient lignocellulolytic microbial consortium, mainly constituted by members of Firmicutes and Bacteroidetes phyla. The enriched community, designed rumen-wheat straw-derived consortium (RWS) efficiently hydrolyzed lignocellulosic biomass, degrading 55.5% w/w of raw wheat straw over 15days at 35°C and accumulating carboxylates as main products. Cellulolytic and hemicellulolytic activities, mainly detected on the cell bound fraction, were produced in the earlier steps of degradation, their production being correlated with the maximal lignocellulose degradation rates. Overall, these results demonstrate the potential of RWS to convert unpretreated lignocellulosic substrates into useful chemicals.