Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium

ABSTRACT Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial factors economically challenging to its use. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge, enrichment strategies can be applied to develop microbial communities with an efficient CAZyme arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of the ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S rRNA sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of the ERAC disclosed 41 metagenome-assembled genomes (MAGs) harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The amino acid sequences of the majority of the predicted CAZymes (60% of the total) shared less than 90% identity with the sequences found in public databases. Additionally, a clostridial MAG identified in this study produced proteins during consortium development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism. IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries.

Anaerobic Dechlorination by a Humin-Dependent Pentachlorophenol-Dechlorinating Consortium under Autotrophic Conditions Induced by Homoacetogenesis

Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H2) for various electron-accepting processes. This deficiency often results in the accumulation of persistent organic pollutants, where bioremediation using organic compounds often leads to secondary contamination. This study involves the reductive dechlorination of pentachlorophenol (PCP) by dechlorinators that do not use H2 directly, but rather through a reduced state of humin—a solid-phase humic substance—as the extracellular electron donor, which requires an organic donor such as formate, lactate, etc. This shortcoming was addressed by the development of an anaerobic mixed culture that was capable of reductively dechlorinating PCP using humin under autotrophic conditions induced by homoacetogenesis. Here, H2 was used for carbon-dioxide fixation to acetate; the acetate produced was used for the reduction of humin; and consequently used for dechlorination through reduced humin. The 16SrRNA gene sequencing analysis showed Dehalobacter and Dehalobacterium as the possible dechlorinators, while Clostridium and Oxobacter were identified as the homoacetogens. Thus, this work contributes to the development of an anaerobic consortium that balanced H2 dependency, where efficiency of humin reduction extends the applicability of anaerobic microbial remediation in aquifers through autotrophy, syntrophy, and reductive dechlorination.

Long-Term Storage and Use of Artificially Immobilized Anaerobic Sludge as a Powerful Biocatalyst for Conversion of Various Wastes Including Those Containing Xenobiotics to Biogas

The aim of this paper is to demonstrate the possibilities of anaerobic sludge cells immobilized into poly(vinyl alcohol) cryogel for the methanogenic conversion of various lignocellulosic waste and other media containing antibiotics (ampicillin, kanamycin, benzylpenicillin) or pesticides (chlorpyrifos or methiocarb and its derivatives). It was established that the immobilized cells of the anaerobic consortium can be stored frozen for at least three years while preserving a high level of metabolic activity. The cells after the long-term storage in an immobilized and frozen state were applied for the methanogenesis of a wide number of wastes, and an increase in both methane yield and methane portion in the produced biogas as compared to the conventionally used suspended anaerobic sludge cells, was ensured. It was shown that the “additional” introduction of bacterial Clostridium acetobutylicum, Pseudomonas sp., Enterococcus faecalis cells (also immobilized using same support) improves characteristics of methanogenesis catalyzed by immobilized anaerobic sludge.