Dilution-to-Stimulation/Extinction Method: a Combination Enrichment Strategy To Develop a Minimal and Versatile Lignocellulolytic Bacterial Consortium.

The engineering of complex communities can be a successful path to understand the ecology of microbial systems and improve biotechnological processes. Here, we developed a strategy to assemble a minimal and effective lignocellulolytic microbial consortium (MELMC) using a sequential combination of dilution-to-stimulation and dilution-to-extinction approaches. The consortium was retrieved from Andean forest soil and selected through incubation in liquid medium with a mixture of three types of agricultural plant residues. After the dilution-to-stimulation phase, approximately 50 bacterial sequence types, mostly belonging to the Sphingobacteriaceae, Enterobacteriaceae, Pseudomonadaceae, and Paenibacillaceae, were significantly enriched. The dilution-to-extinction method demonstrated that only eight of the bacterial sequence types were necessary to maintain microbial growth and plant biomass consumption. After subsequent stabilization, only two bacterial species (Pseudomonas sp. and Paenibacillus sp.) became highly abundant (>99%) within the MELMC, indicating that these are the key players in degradation. Differences in the composition of bacterial communities between biological replicates indicated that selection, sampling, and/or priority effects could shape the consortium structure. The MELMC can degrade up to ∼13% of corn stover, consuming mostly its (hemi)cellulosic fraction. Tests with chromogenic substrates showed that the MELMC secretes an array of endoenzymes able to degrade xylan, arabinoxylan, carboxymethyl cellulose, and wheat straw. Additionally, the metagenomic profile inferred from the phylogenetic composition along with an analysis of carbohydrate-active enzymes of 20 bacterial genomes support the potential of the MELMC to deconstruct plant polysaccharides. This capacity was mainly attributed to the presence of Paenibacillus sp.IMPORTANCE The significance of our study mainly lies in the development of a combined top-down enrichment strategy (i.e., dilution to stimulation coupled to dilution to extinction) to build a minimal and versatile lignocellulolytic microbial consortium. We demonstrated that mainly two selectively enriched bacterial species (Pseudomonas sp. and Paenibacillus sp.) are required to drive the effective degradation of plant polymers. Our findings can guide the design of a synthetic bacterial consortium that could improve saccharification (i.e., the release of sugars from agricultural plant residues) processes in biorefineries. In addition, they can help to expand our ecological understanding of plant biomass degradation in enriched bacterial systems.

Exploring the Lignin Catabolism Potential of Soil-Derived Lignocellulolytic Microbial Consortia by a Gene-Centric Metagenomic Approach.

An exploration of the ligninolytic potential of lignocellulolytic microbial consortia can improve our understanding of the eco-enzymology of lignin conversion in nature. In this study, we aimed to detect enriched lignin-transforming enzymes on metagenomes from three soil-derived microbial consortia that were cultivated on "pre-digested" plant biomass (wheat straw, WS1-M; switchgrass, SG-M; and corn stover, CS-M). Of 60 selected enzyme-encoding genes putatively involved in lignin catabolism, 20 genes were significantly abundant in WS1-M, CS-M, and/or SG-M consortia compared with the initial forest soil inoculum metagenome (FS1). These genes could be involved in lignin oxidation (e.g., superoxide dismutases), oxidative stress responses (e.g., catalase/peroxidases), generation of protocatechuate (e.g., vanAB genes), catabolism of gentisate, catechol and 3-phenylpropionic acid (e.g., gentisate 1,2-dioxygenases, muconate cycloisomerases, and hcaAB genes), the beta-ketoadipate pathway (e.g., pcaIJ genes), and tolerance to lignocellulose-derived inhibitors (e.g., thymidylate synthases). The taxonomic affiliation of 22 selected lignin-transforming enzymes from WS1-M and CS-M consortia metagenomes revealed that Pseudomonadaceae, Alcaligenaceae, Sphingomonadaceae, Caulobacteraceae, Comamonadaceae, and Xanthomonadaceae are the key bacterial families in the catabolism of lignin. A predictive "model" was sketched out, where each microbial population has the potential to metabolize an array of aromatic compounds through different pathways, suggesting that lignin catabolism can follow a "task division" strategy. Here, we have established an association between functions and taxonomy, allowing a better understanding of lignin transformations in soil-derived lignocellulolytic microbial consortia, and pinpointing some bacterial taxa and catabolic genes as ligninolytic trait-markers.

Andean soil-derived lignocellulolytic bacterial consortium as a source of novel taxa and putative plastic-active enzymes.

An easy and straightforward way to engineer microbial environmental communities is by setting up liquid enrichment cultures containing a specific substrate as the sole source of carbon. Here, we analyzed twenty single-contig high-quality metagenome-assembled genomes (MAGs) retrieved from a microbial consortium (T6) that was selected by the dilution-to-stimulation approach using Andean soil as inoculum and lignocellulose as a selection pressure. Based on genomic metrics (e.g., average nucleotide and amino acid identities) and phylogenomic analyses, 15 out of 20 MAGs were found to represent novel bacterial species, with one of those (MAG_26) belonging to a novel genus closely related to Caenibius spp. (Sphingomonadaceae). Following the rules and requirements of the SeqCode, we propose the name Andeanibacterium colombiense gen. nov., sp. nov. for this taxon. A subsequent functional annotation of all MAGs revealed that MAG_7 (Pseudobacter hemicellulosilyticus sp. nov.) contains 20, 19 and 16 predicted genes from carbohydrate-active enzymes families GH43, GH2 and GH92, respectively. Its lignocellulolytic gene profile resembles that of MAG_2 (the most abundant member) and MAG_3858, both of which belong to the Sphingobacteriaceae family. Using a database that contains experimentally verified plastic-active enzymes (PAZymes), twenty-seven putative bacterial polyethylene terephthalate (PET)-active enzymes (i.e., alpha/beta-fold hydrolases) were detected in all MAGs. A maximum of five putative PETases were found in MAG_3858, and two PETases were found to be encoded by A. colombiense. In conclusion, we demonstrate that lignocellulose-enriched liquid cultures coupled with genome-resolved metagenomics are suitable approaches to unveil the hidden bacterial diversity and its polymer-degrading potential in Andean soil ecosystems.

Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations.

Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving Cupriavidus necator H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD600 value of 56.0 and a dry cell weight of 15.2 g L-1, compared to the wild type (WT) strain's final OD600 of 39.1 and dry cell weight of 8.4 g L-1. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD600 of 48.9 and a dry cell weight of 12.7 g L-1, in contrast to the WT strain's final OD600 of 9.0 and dry cell weight of 3.1 g L-1. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

Changes in Lolium perenne L. rhizosphere microbiome during phytoremediation of Cd- and Hg-contaminated soils.

The contamination of soil and water by metals such as mercury (Hg) and cadmium (Cd) has been increasing in recent years, because of anthropogenic activities such as mining and agriculture, respectively. In this work, the changes in the rhizosphere microbiome of Lolium perenne L. during the phytoremediation of soils contaminated with Hg and Cd were evaluated. For this, two soil types were sampled, one inoculated with mycorrhizae and one without. The soils were contaminated with Hg and Cd, and L. perenne seeds were sown and harvested after 30 days. To assess changes in the microbiome, DNA isolation tests were performed, for which samples were subjected to two-step PCR amplification with specific 16S rDNA V3-V4 primers (337F and 805R). With mycorrhizae, changes had been found in the absorption processes of metals and a new distribution. While with respect to microorganisms, families such as the Enterobacteriaceae have been shown to have biosorption and efflux effects on metals such as Hg and Cd. Mycorrhizae then improve the efficiency of removal and allow the plant to better distribute the absorbed concentrations. Overall, L. perenne is a species with a high potential for phytoremediation of Cd- and Hg-contaminated soils in the tropics. Inoculation with mycorrhizae modifies the phytoremediation mechanisms of the plant and the composition of microorganisms in the rhizosphere. Mycorrhizal inoculation and changes in the microbiome were associated with increased plant tolerance to Cd and Hg. Microorganism-assisted phytoremediation is an appropriate alternative for L. perenne.

Novel bacterial taxa in a minimal lignocellulolytic consortium and their potential for lignin and plastics transformation.

The understanding and manipulation of microbial communities toward the conversion of lignocellulose and plastics are topics of interest in microbial ecology and biotechnology. In this study, the polymer-degrading capability of a minimal lignocellulolytic microbial consortium (MELMC) was explored by genome-resolved metagenomics. The MELMC was mostly composed (>90%) of three bacterial members (Pseudomonas protegens; Pristimantibacillus lignocellulolyticus gen. nov., sp. nov; and Ochrobactrum gambitense sp. nov) recognized by their high-quality metagenome-assembled genomes (MAGs). Functional annotation of these MAGs revealed that Pr. lignocellulolyticus could be involved in cellulose and xylan deconstruction, whereas Ps. protegens could catabolize lignin-derived chemical compounds. The capacity of the MELMC to transform synthetic plastics was assessed by two strategies: (i) annotation of MAGs against databases containing plastic-transforming enzymes; and (ii) predicting enzymatic activity based on chemical structural similarities between lignin- and plastics-derived chemical compounds, using Simplified Molecular-Input Line-Entry System and Tanimoto coefficients. Enzymes involved in the depolymerization of polyurethane and polybutylene adipate terephthalate were found to be encoded by Ps. protegens, which could catabolize phthalates and terephthalic acid. The axenic culture of Ps. protegens grew on polyhydroxyalkanoate (PHA) nanoparticles and might be a suitable species for the industrial production of PHAs in the context of lignin and plastic upcycling.

Top-Down Enrichment Strategy to Co-cultivate Lactic Acid and Lignocellulolytic Bacteria From the Megathyrsus maximus Phyllosphere.

Traditionally, starting inoculants have been applied to improve ensiling of forage used for livestock feed. Here, we aimed to build up a bioinoculant composed of lactic acid-producing and lignocellulolytic bacteria (LB) derived from the Megathyrsus maximus (guinea grass) phyllosphere. For this, the dilution-to-stimulation approach was used, including a sequential modification of the starting culture medium [Man, Rogosa, and Sharpe (MRS) broth] by addition of plant biomass (PB) and elimination of labile carbon sources. Along 10 growth-dilution steps (T1-T10), slight differences were observed in terms of bacterial diversity and composition. After the sixth subculture, the consortium started to degrade PB, decreasing its growth rate. The co-existence of Enterobacteriales (fast growers and highly abundance), Actinomycetales, Bacillales, and Lactobacillales species was observed at the end of the selection process. However, a significant structural change was noticed when the mixed consortium was cultivated in higher volume (500ml) for 8days, mainly increasing the proportion of Paenibacillaceae populations. Interestingly, Actinomycetales, Bacillales, and Lactobacillales respond positively to a pH decrease (4-5), suggesting a relevant role within a further silage process. Moreover, gene-centric metagenomic analysis showed an increase of (hemi)cellulose-degrading enzymes (HDEs) during the enrichment strategy. Reconstruction of metagenome-assembled genomes (MAGs) revealed that Paenibacillus, Cellulosimicrobium, and Sphingomonas appear as key (hemi)cellulolytic members (harboring endo-glucanases/xylanases, arabinofuranosidases, and esterases), whereas Enterococcus and Cellulosimicrobium have the potential to degrade oligosaccharides, metabolize xylose and might produce lactic acid through the phosphoketolase (PK) pathway. Based on this evidence, we conclude that our innovative top-down strategy enriched a unique bacterial consortium that could be useful in biotechnological applications, including the development/design of a synthetic bioinoculant to improve silage processes.