Genome-centric metagenomics revealed the effect of pH on the microbiome involved in short-chain fatty acids and ethanol production.

Added-value chemicals production via food waste (FWs) valorization using open-mixed cultures is an emerging approach to replace petrochemical-based compounds. Nevertheless, the effects of operational parameters on the product spectrum remain uncertain given the wide number of co-occurring species and metabolisms. In this study, the identification of 58 metagenome-assembled genomes and their investigation assessed the effect of slight pH variations on microbial dynamics and the corresponding functions when FWs were subjected to anaerobic fermentation (AF) in 1-L continuous stirred tank reactors at 25 °C. The initial pH of 6.5 promoted a microbial community involved in acetate, butyrate and ethanol production, mediated by Bifidobacterium subtile IE007 and Eubacteriaceae IE027 as main species. A slight pH decrease to 6.1 shaped microbial functions that resulted in caproate and H2 production, increasing the relevance of Eubacteriaceae IE037 role. This study elucidated the strong pH effect on product outputs when minimal variations take place in AF.

Integrating metagenomic binning with flux balance analysis to unravel syntrophies in anaerobic CO2 methanation.

BACKGROUND: Carbon fixation through biological methanation has emerged as a promising technology to produce renewable energy in the context of the circular economy. The anaerobic digestion microbiome is the fundamental biological system operating biogas upgrading and is paramount in power-to-gas conversion. Carbon dioxide (CO2) methanation is frequently performed by microbiota attached to solid supports generating biofilms. Despite the apparent simplicity of the microbial community involved in biogas upgrading, the dynamics behind most of the interspecies interaction remain obscure. To understand the role of the microbial species in CO2 fixation, the biofilm generated during the biogas upgrading process has been selected as a case study. The present work investigates via genome-centric metagenomics, based on a hybrid Nanopore-Illumina approach the biofilm developed on the diffusion devices of four ex situ biogas upgrading reactors. Moreover, genome-guided metabolic reconstruction and flux balance analysis were used to propose a biological role for the dominant microbes. RESULTS: The combined microbiome was composed of 59 species, with five being dominant (> 70% of total abundance); the metagenome-assembled genomes representing these species were refined to reach a high level of completeness. Genome-guided metabolic analysis appointed Firmicutes sp. GSMM966 as the main responsible for biofilm formation. Additionally, species interactions were investigated considering their co-occurrence in 134 samples, and in terms of metabolic exchanges through flux balance simulation in a simplified medium. Some of the most abundant species (e.g., Limnochordia sp. GSMM975) were widespread (~ 67% of tested experiments), while others (e.g., Methanothermobacter wolfeii GSMM957) had a scattered distribution. Genome-scale metabolic models of the microbial community were built with boundary conditions taken from the biochemical data and showed the presence of a flexible interaction network mainly based on hydrogen and carbon dioxide uptake and formate exchange. CONCLUSIONS: Our work investigated the interplay between five dominant species within the biofilm and showed their importance in a large spectrum of anaerobic biogas reactor samples. Flux balance analysis provided a deeper insight into the potential syntrophic interaction between species, especially Limnochordia sp. GSMM975 and Methanothermobacter wolfeii GSMM957. Finally, it suggested species interactions to be based on formate and amino acids exchanges. Video Abstract.

Preliminary investigation of microorganisms potentially involved in microplastics degradation using an integrated metagenomic and biochemical approach.

Plastic pollution is becoming an emerging environmental issue due to inappropriate disposal at the end of the materials life cycle. When plastics are released, they undergo physical and chemical corrosion, leading to the formation of small particles, commonly referred to as microplastics. In this study, a microbial community derived from the leachate of a bioreactor containing a mixture of soil and plastic collected during a landfill mining process underwent an enrichment protocol in order to select the microbial species specifically involved in plastic degradation. The procedure was set up and tested on polyethylene, polyvinyl chloride, and polyethylene terephthalate, both in anaerobic and aerobic conditions. The evolution of the microbiome has been monitored using a combined approach based on microscopy, marker-gene amplicon sequencing, genome-centric metagenomics, degradation assays, and GC-MS analyses. This procedure permitted us to deeply investigate the metabolic pathways potentially involved in plastic degradation and to depict the route for microplastics metabolization from the enriched microbial community. Six enzymes, among the ones already identified, were found in our samples (alkane 1-monooxygenase, cutinase, feruloyl esterase, triacylglycerol lipase, medium-chain acyl-CoA dehydrogenase, and protocatechuate 4,5-dioxygenase) and new enzymes, addressed as MHETases most probably for the presence of the catalytic triad (His-Asp-Ser), were detected. Among the enzymes involved in plastics degradation, alkane 1-monooxygenase was found in high copy number (between ten and 62 copies) in the metagenomes that resulted most abundant in the microbiome enriched with polyethylene, while protocatechuate 4,5-dioxygenase was found between one and eight copies in the most abundant metagenomes of the microbial culture enriched with polyethylene terephthalate. Degradation assays, performed using both bacterial lysates and supernatants, revealed interesting results on polyethylene terephthalate degradation. Moreover, this study demonstrates to what extent different types of microplastics can affect the microbial community composition. The results obtained significantly increase the knowledge of the plastic degradation process.

Effect of pressure on biomethanation process and spatial stratification of microbial communities in trickle bed reactors under decreasing gas retention time.

The current study investigated the effect of elevating gas pressure on biomethanation in trickle-bed reactors (TBRs). The increased pressure led to successful biomethanation (CH4 > 90 %) at a gas retention time (GRT) of 21 min, due to the improved transfer rates of H2 and CO2. On the contrary, the non-pressurized TBR performance was reduced at GRTs shorter than 40 min. Metagenomic analysis revealed that the microbial populations collected from the lower and middle parts of the reactor under the same GRT were more homogeneous compared with those developed in the upper layer. Comparison with previous experiments suggest that microbial stratification is mainly driven by the nutrient provision strategy. Methanobacterium species was the most dominant methanogen and it was mainly associated with the bottom and middle parts of TBRs. Overall, the increased pressure did not affect markedly the microbial composition, while the GRT was the most important parameter shaping the microbiomes.

Large-scale sequencing and comparative analysis of oenological Saccharomyces cerevisiae strains supported by nanopore refinement of key genomes.

Saccharomyces cerevisiae has long been part of human activities related to the production of food and wine. The industrial demand for fermented beverages with well-defined and stable characteristics boosted the isolation and selection of strains conferring a distinctive aroma profile to the final product. To uncover variants characterizing oenological strains, the sequencing of 65 new S. cerevisiae isolates, and the comparison with other 503 publicly available genomes were performed. A hybrid approach based on short Illumina and long Oxford Nanopore reads allowed the in-depth investigation of eleven genomes and the identification of putative laterally transferred regions and structural variants. A comparative analysis between clusters of strains belonging to different datasets allowed the identification of novel relevant genetic features including single nucleotide polymorphisms, insertions and structural variants. Detection of oenological single nucleotide variants shed light on the existence of different levels of modulation for the mevalonate pathway relevant for the biosynthesis of aromatic compounds.

Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.