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Producing scalable, economically viable, low-carbon biofuels or biochemicals hinges on more efficient bioconversion processes. While microbial conversion can offer robust solutions, the native microbial growth process often redirects a large fraction of carbon to CO2 and cell mass. By integrating genome-scale metabolic models with techno-economic and life cycle assessment models, this study analyzes the effects of converting cell mass lipids to hydrocarbon fuels, and CO2 to methanol on the facility’s costs and life-cycle carbon footprint. Results show that upgrading microbial lipids or both microbial lipids and CO2 using renewable hydrogen produces carbon-negative bisabolene. Additionally, on-site electrolytic hydrogen production offers a supply of pure oxygen to use in place of air for bioconversion and fuel combustion in the boiler. To reach cost parity with conventional jet fuel, renewable hydrogen needs to be produced at less than $2.2 to $3.1/kg, with a bisabolene yield of 80% of the theoretical yield, along with cell mass and CO2 yields of 22 wt% and 54 wt%, respectively. The economic combination of cell mass, CO2, and bisabolene yields demonstrated in this study provides practical insights for prioritizing research, selecting suitable hosts, and determining necessary engineered production levels.

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

Abstract Pseudomonas putida has emerged as a promising host for the conversion of biomass‐derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non‐native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass‐derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non‐native genes was achieved by using chassis‐independent recombinase‐assisted genome engineering (CRAGE) for single‐step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

Laccases from white-rot fungi catalyze lignin depolymerization, a critical first step to upgrading lignin to valuable biodiesel fuels and chemicals. In this study, a wildtype laccase from the basidiomycete Fomitiporia mediterranea (Fom_lac) and a variant engineered to have a carbohydrate-binding module (Fom_CBM) were studied for their ability to catalyze cleavage of β-O-4′ ether and C–C bonds in phenolic and non-phenolic lignin dimers using a nanostructure-initiator mass spectrometry-based assay. Fom_lac and Fom_CBM catalyze β-O-4′ ether and C–C bond breaking, with higher activity under acidic conditions (pH < 6). The potential of Fom_lac and Fom_CBM to enhance saccharification yields from untreated and ionic liquid pretreated pine was also investigated. Adding Fom_CBM to mixtures of cellulases and hemicellulases improved sugar yields by 140% on untreated pine and 32% on cholinium lysinate pretreated pine when compared to the inclusion of Fom_lac to the same mixtures. Adding either Fom_lac or Fom_CBM to mixtures of cellulases and hemicellulases effectively accelerates enzymatic hydrolysis, demonstrating its potential applications for lignocellulose valorization. We postulate that additional increases in sugar yields for the Fom_CBM enzyme mixtures were due to Fom_CBM being brought more proximal to lignin through binding to either cellulose or lignin itself.

Abstract Background Plant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities. Results To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes. Conclusions The two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance. Video Abstract

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (Plac, Ptac, PtetR/tetA and Pbad) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

Abstract Background Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results A fed-batch system with plant biomass-derived sugars d-xylose, l-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion This work has identified two additional CAZyme inducers for T. aurantiacus, l-arabinose and cellobiose, along with d-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.

Biorefinery processes for converting lignocellulosic biomass to fuels and chemicals proceed via an integrated series of steps. Biomass is first pretreated and deconstructed using chemical catalysts and/or enzymes to liberate sugar monomers and lignin fragments. Deconstruction is followed by a conversion step in which engineered host organisms assimilate the released sugar monomers and lignin fragments, and produce value-added fuels and chemicals. Over the past couple of decades, a significant amount of work has been done to develop innovative biomass deconstruction and conversion processes that efficiently solubilize biomass, separate lignin from the biomass, maximize yields of bioavailable sugars and lignin fragments and convert the majority of these carbon sources into fuels, commodity chemicals, and materials. Herein, we advocate that advanced in silico approaches provide a theoretical framework for developing efficient processes for lignocellulosic biomass valorization and maximizing yields of sugars and lignin fragments during deconstruction and fuel and chemical titers during conversion. This manuscript surveys the latest developments in lignocellulosic biomass valorization with special attention given to highlighting computational approaches used in process optimization for lignocellulose pretreatment; enzyme engineering for enhanced saccharification and delignification; and prediction of the genome modification necessary for desired pathway fine-tuning to upgrade products from biomass deconstruction into value-added products. Physics-based modeling approaches such as density functional theory calculations and molecular dynamics simulations have been most impactful in studies aimed at exploring the molecular level details of solvent-biomass interactions, reaction mechanisms occurring in biomass-solvent systems, and the catalytic mechanisms and engineering of enzymes involved in biomass degradation. More recently, with ever increasing amounts of data from, for example, advanced mutli-omics experiments, machine learning approaches have begun to make important contributions in synthetic biology and optimization of metabolic pathways for production of biofuels and chemicals.

Abstract Background Lignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ ether bonds and C–C bonds in lignin, both of which are essential for depolymerizing lignin into fragments amendable to biological or chemical upgrading to valuable products. Studies of the specificity of lignin peroxidases to catalyze these various reactions and the role reaction conditions such as pH play have been limited by the lack of assays that allow quantification of specific bond-breaking events. The subsequent theoretical understanding of the underlying mechanisms by which pH modulates the activity of lignin peroxidases remains nascent. Here, we report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-O-4′ ether bonds and of C–C bonds by a lignin peroxidase isozyme H8 from Phanerochaete chrysosporium and an acid stabilized variant of the same enzyme. Results Using a nanostructure initiator mass spectrometry assay that provides quantification of bond breaking in a phenolic model lignin dimer we found that catalysis of degradation of the dimer to products by an acid-stabilized variant of lignin peroxidase isozyme H8 increased from 38.4% at pH 5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution resulted from 65.5% β-O-4′ ether bond cleavage, 27.0% Cα-C1 carbon bond cleavage, and 3.6% Cα-oxidation as by-product. Using ab initio molecular dynamic simulations and climbing-image Nudge Elastic Band based transition state searches, we suggest the effect of lower pH is via protonation of aliphatic hydroxyl groups under which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, particularly β-O-4′ bonds. Conclusion These coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved in lignin depolymerization should include targeting stability at low pH.

Abstract Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

Summary: Single-cell sequencing of environmental microorganisms is an essential component of the microbial ecology toolkit. However, large-scale targeted single-cell sequencing for the whole-genome recovery of uncultivated eukaryotes is lagging. The key challenges are low abundance in environmental communities, large complex genomes, and cell walls that are difficult to break. We describe a pipeline composed of state-of-the art single-cell genomics tools and protocols optimized for poorly studied and uncultivated eukaryotic microorganisms that are found at low abundance. This pipeline consists of seven distinct steps, beginning with sample collection and ending with genome annotation, each equipped with quality review steps to ensure high genome quality at low cost. We tested and evaluated each step on environmental samples and cultures of early-diverging lineages of fungi and Chromista/SAR. We show that genomes produced using this pipeline are almost as good as complete reference genomes for functional and comparative genomics for environmental microbial eukaryotes.

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) individually and in combination (pCA ​+ ​FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L pCA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of pCA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in pCA ​+ ​FA TALE-derived strains but did not affect growth in pCA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

Tightly regulated promoters with strong inducibility and scalability are highly desirable for biological applications. Here the authors describe ‘Jungle Express’, a EilR repressor-based broad host system activated by cationic dyes.

Abstract Background There is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass. Methods To generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing. Results The most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc]. Conclusion The generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.

Abstract Background Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. Results Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. Conclusions Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

Abstract Background Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts. Results In this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline α-ketoglutarate, which produced 261 mg/L of bisabolene at bench scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high-gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars. Conclusions This study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.

ABSTRACT Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses. IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.

The conversion of biomass to biofuels presents a solution to one of the largest global challenges of our era, climate change. A critical part of this pipeline is the process of breaking down cellulosic sugars from plant matter to be used by microbes containing biosynthetic pathways that produce biofuels or bioproducts. In this inquiry-based course, students complete a research project that isolates cellulase-producing bacteria from samples collected from the environment. After obtaining isolates, the students characterize the production of cellulases. Students then amplify and sequence the 16S rRNA genes of confirmed cellulase producers and use bioinformatic methods to identify the bacterial isolates. Throughout the course, students learn about the process of generating biofuels and bioproducts through the deconstruction of cellulosic biomass to form monosaccharides from the biopolymers in plant matter. The program relies heavily on active learning and enables students to connect microbiology with issues of sustainability. In addition, it provides exposure to basic microbiology, molecular biology, and biotechnology laboratory techniques and concepts. The described activity was initially developed for the Introductory College Level Experience in Microbiology (iCLEM) program, a research-based immersive laboratory course at the US Department of Energy Joint BioEnergy Institute. Originally designed as an accelerated program for high-potential, low-income, high school students (11th–12th grade), this curriculum could also be implemented for undergraduate coursework in a research-intensive laboratory course at a two- or four-year college or university.

Salinity is one of the strongest environmental drivers of microbial evolution and community composition. Here we aimed to determine the impact of salt concentrations (2.5, 7.5, and 33.2%) on the microbial community structure of reclaimed saltern ponds near San Francisco, California, and to discover prospective enzymes with potential biotechnological applications. Community compositions were determined by 16S rRNA amplicon sequencing revealing both higher richness and evenness in the pond sediments compared to the water columns. Co-occurrence network analysis additionally uncovered the presence of microbial seed bank communities, potentially primed to respond to rapid changes in salinity. In addition, functional annotation of shotgun metagenomic DNA showed different capabilities if the microbial communities at different salinities for methanogenesis, amino acid metabolism, and carbohydrate-active enzymes. There was an overall shift with increasing salinity in the functional potential for starch degradation, and a decrease in degradation of cellulose and other oligosaccharides. Further, many carbohydrate-active enzymes identified have acidic isoelectric points that have potential biotechnological applications, including deconstruction of biofuel feedstocks under high ionic conditions. Metagenome-assembled genomes (MAGs) of individual halotolerant and halophilic microbes were binned revealing a variety of carbohydrate-degrading potential of individual pond inhabitants.

ABSTRACT Ionic liquid (IL) pretreatment is a promising approach for the conversion of lignocellulose to biofuels. The toxicity of residual IL, however, negatively impacts the performance of industrial enzymes and microorganisms in hydrolysis and fermentation. In this study, a thermophilic microbial community was cultured on switchgrass amended with various levels of the ionic liquid 1-ethyl-3-methylimidazolium acetate. Changes in the microbial community composition and transcription of genes relevant to IL tolerance and lignocellulose hydrolysis were quantified. Increasing the level of IL to 0.1% (wt) led to increased levels of relative abundance and transcription in organisms of the phylum Firmicutes. Interestingly, IL concentrations of up to 1% (wt) also resulted in greater xylanase transcription and enzyme activity as well as increased transcription of endoglucanase, beta-glucosidase, and IL tolerance genes compared to communities without IL. IL levels above 1% (wt) resulted in decreased enzyme activity and transcription of genes involved in lignocellulose hydrolysis. The results indicate that moderate levels of IL select for thermophilic microorganisms that not only tolerate IL but also effectively hydrolyze lignocellulose from switchgrass. Discovery of IL-tolerant organisms and enzymes is critical for the development of biological processes that convert IL-pretreated biomass to biofuels and chemicals. Employing metatranscriptomic analysis of enrichment cultures can facilitate the discovery of microorganisms and enzymes that may be active in the presence of toxic compounds such as ionic liquids. IMPORTANCE Pretreatment using ionic liquids (IL) is a promising approach for the conversion of lignocellulose to biofuels. Because IL can be inhibitory to enzymes and microorganisms involved in downstream hydrolysis and fermentation steps, discovery of IL-tolerant organisms and enzymes is critical for advancing this technology. Employing metatranscriptomics in the analysis of IL-enriched cultures facilitated tracking of dynamic changes in a complex microbial community at the level of gene transcription and doing so with genome resolution. Specific organisms were discovered that could simultaneously tolerate a moderate IL concentration and transcribe a diverse array of cellulolytic enzymes. Gene sequences of cellulolytic enzymes and efflux pumps from those same organisms were also identified, providing important resources for future research on engineering IL-tolerant organisms and enzymes.

ABSTRACT Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCE Cellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.

In the original version of this Article, an incorrect URL was provided in the Data Availability Statement regarding the deposition of plasmids listed in Supplementary Table 4. The correct URL is https://public-registry.jbei.org/folders/378. This error has been corrected in both the PDF and HTML versions of the Article.

Soil biosolarization (SBS) is a pest control technique that combines passive solar heating and fermentation of amended organic matter. The extreme soil conditions generated during SBS could decrease microbial biomass and restructure the soil microbiome, which could impact soil quality. Digestates from anaerobic digesters may harbor microbial communities tolerant of the oxygen, moisture, and temperature stresses encountered during SBS as these conditions may also occur in digesters. Digestate microbial communities may contribute to soil fermentation during SBS and affect organic matter turnover in soils treated with SBS. The objective of this study was to assess the effect of SBS on soil microbial diversity and quantity when solid digestates from thermophilic (TD) and mesophilic (MD) anaerobic digesters were used as soil amendments. In the soils amended with TD, communities showed the greatest divergence from the initial soil state whereas MD amendment resulted in a microbiome more similar to the non-amended soil. The microbial biomass of the biosolarized soils was significantly greater than the non-amended, solar-heated soil. The microbial biomass in the biosolarized soils was dominated by K-strategic or “native” species. Solar heating of the non-amended soil mainly affected “native” species, leading to conditions where other opportunistic species become more dominant. Further studies are needed to elucidate whether the persistent microbes in the soil are benign or pathogenic and to understand their roles in pest inactivation and nutrient cycling during and following SBS.