Ethanologenic fermentation by Parageobacillus thermoglucosidasius with continuous hot microbubble gas-stripping.

The increased use of biofuels in place of fossil fuels is one strategy to support the transition to net-zero carbon emissions, particularly in transport applications. However, expansion of the use of 1st generation crops as feedstocks is unsustainable due to the conflict with food use. The use of the lignocellulosic fractions from plants and/or co-products from food production including food wastes could satisfy the demand for biofuels without affecting the use of land and the availability of food, but organisms which can readily ferment all the carbohydrates present in these feedstocks often suffer from more severe bioethanol inhibition effects than yeast. This paper demonstrates the potential of hot gas microbubbles to strip ethanol from a thermophilic fermentation process using Parageobacillus thermoglucosidasius TM333, thereby reducing product inhibition and allowing production to continue beyond the nominal toxic ethanol concentrations of ≤ 2% v/v. Using an experimental rig in which cells were grown in fed-batch cultures on sugars derived from waste bread, and the broth continuously cycled through a purpose-built microbubble stripping unit, it was shown that non/low-inhibitory dissolved ethanol concentrations could be maintained throughout, despite reaching productivities equivalent to 4.7% v/v dissolved ethanol. Ethanol recovered in the condensate was at a concentration appropriate for dewatering to be cost effective and not prohibitively energy intensive. This suggests that hot microbubble stripping could be a valuable technology for the continuous production of bioethanol from fermentation processes which suffer from product inhibition before reaching economically viable titres, which is typical of most thermophilic ethanologenic bacteria.

Simultaneous saccharification and ethanologenic fermentation (SSF) of waste bread by an amylolytic Parageobacillus thermoglucosidasius strain TM333.

The starch in waste bread (WB) from industrial sandwich production was directly converted to ethanol by an amylolytic, ethanologenic thermophile (Parageobacillus thermoglucosidasius strain TM333) under 5 different simultaneous saccharification and fermentation (SSF) regimes. Crude α-amylase from TM333 was used alone or in the presence of amyloglucosidase (AMG), a starch monomerizing enzyme used in industry, with/without prior gelatinisation/liquefaction treatments and P. thermoglucosidasius TM333 fermentation compared with Saccharomyces cerevisiae as a control. Results suggest that TM333 can ferment WB using SSF with yields of 94-100% of theoretical (based on all sugars in WB) in 48 h without the need for AMG addition or any form of heat pre-treatment. This indicates that TM333 can transport and ferment all of the malto-oligosaccharides generated by its α-amylase. In the yeast control experiments, addition of AMG together with the crude α-amylase was necessary for full fermentation over the same time period. This suggests that industrial fermentation of WB starch to bio-ethanol or other products using an enhanced amylolytic P. thermoglucosidasius strain could offer significant cost savings compared to alternatives requiring enzyme supplementation.

Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism.

Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.

The heterologous production of terpenes by the thermophile Parageobacillus thermoglucosidasius in a consolidated bioprocess using waste bread.

Parageobacillus thermoglucosidasius is a genetically tractable thermophile that grows rapidly at elevated temperatures, with a doubling time at 65 °C comparable to the shortest doubling times of Escherichia coli. It is capable of using a wide variety of substrates, including carbohydrate oligomers, and has been developed for the industrial production of ethanol. In this study, P. thermoglucosidasius NCIMB11955 has been engineered to produce the sesquiterpene τ-muurolol by introduction of a heterologous mevalonate pathway constructed using genes from several thermophilic archaea together with a recently characterised thermostable τ-muurolol synthase. P. thermoglucosidasius naturally uses the methylerythritol phosphate pathway for production of the terpene precursor, isopentenyl pyrophosphate, while archaea use a version of the mevalonate pathway. By introducing the orthogonal archaeal pathway it was possible to increase the flux through to sesquiterpene biosynthesis. Construction of such a large metabolic pathway created problems with genetic vector introduction and stability, so recombinant plasmids were introduced by conjugation, and a thermostable serine integrase system was developed for integration of large pathways onto the chromosome. Finally, by making the heterologous pathway maltose-inducible we demonstrate that the new strain is capable of using waste bread directly as an autoinduction carbon source for the production of terpenes in a consolidated bioprocess.

Simultaneous saccharification and lactic acid fermentation of the cellulosic fraction of municipal solid waste using Bacillus smithii.

OBJECTIVE: A primary drawback to simultaneous saccharification and fermentation (SSF) processes is the incompatibility of the temperature and pH optima for the hydrolysis and fermentation steps-with the former working best at 50-55 °C and pH 4.5-5.5. Here, nine thermophilic Bacillus and Parageobacillus spp. were evaluated for growth and lactic acid fermentation at high temperature and low pH. The most promising candidate was then carried forward to demonstrate SSF using the cellulosic fraction from municipal solid waste (MSW) as a feedstock. RESULTS: B. smithii SA8Eth was identified as the most promising candidate and in a batch SSF maintained at 55 °C and pH 5.0, using a cellulase dose of 5 FPU/g glucan, it produced 5.1 g/L lactic acid from 2% (w/v) MSW cellulosic pulp in TSB media. CONCLUSION: This work has both scientific and industrial relevance, as it evaluates a number of previously untrialled bacterial hosts for their compatibility with lignocellulosic SSF for lactic acid production and successfully identifies B. smithii as a potential candidate for such a process.

Continuous removal of ethanol from dilute ethanol-water mixtures using hot microbubbles.

Product inhibition is a barrier to many fermentation processes, including bioethanol production, and is responsible for dilute product streams which are energy intensive to purify. The main purpose of this study was to investigate whether hot microbubble stripping could be used to remove ethanol continuously from dilute ethanol-water mixtures expected in a bioreactor and maintain ethanol concentrations below the inhibitory levels for the thermophile Parageobacillus thermoglucosidasius (TM242), that can utilize a range of sugars derived from lignocellulosic biomass. A custom-made microbubble stripping unit that produces clouds of hot microbubbles (~120 °C) by fluidic oscillation was used to remove ethanol from ~2% (v/v) ethanol-water mixtures maintained at 60 °C. Ethanol was continuously added to the unit to simulate microbial metabolism. The initial liquid height and the ethanol addition rate were varied from 10 to 50 mm and 2.1-21.2 g h-1 respectively. In all the experiments, ethanol concentration was maintained well below the inhibition threshold of the target organism (~2% [v/v]). This microbubble stripping unit has the potential to operate in conjunction with a 0.5-1.0 L fermenter to allow an ethanol productivity of 14.9-7.8 g L- 1h-1 continuously.

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli.

BACKGROUND: Geraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds-which are often utilised for anti-microbial and anti-fungal functions in their host plant. RESULTS: In this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed. CONCLUSION: In this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production.

Medium Chain Carboxylic Acids from Complex Organic Feedstocks by Mixed Culture Fermentation.

Environmental pressures caused by population growth and consumerism require the development of resource recovery from waste, hence a circular economy approach. The production of chemicals and fuels from organic waste using mixed microbial cultures (MMC) has become promising. MMC use the synergy of bio-catalytic activities from different microorganisms to transform complex organic feedstock, such as by-products from food production and food waste. In the absence of oxygen, the feedstock can be converted into biogas through the established anaerobic digestion (AD) approach. The potential of MMC has shifted to production of intermediate AD compounds as precursors for renewable chemicals. A particular set of anaerobic pathways in MMC fermentation, known as chain elongation, can occur under specific conditions producing medium chain carboxylic acids (MCCAs) with higher value than biogas and broader applicability. This review introduces the chain elongation pathway and other bio-reactions occurring during MMC fermentation. We present an overview of the complex feedstocks used, and pinpoint the main operational parameters for MCCAs production such as temperature, pH, loading rates, inoculum, head space composition, and reactor design. The review evaluates the key findings of MCCA production using MMC, and concludes by identifying critical research targets to drive forward this promising technology as a valorisation method for complex organic waste.

An integrated biorefinery concept for conversion of sugar beet pulp into value-added chemicals and pharmaceutical intermediates.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

BACKGROUND: Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process. RESULTS: Our technique incorporates counter-selection using ß-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation. CONCLUSION: The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris.

Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.

Rapid screening of cellular stress responses in recombinant Pichia pastoris strains using metabolite profiling.

Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P. pastoris clones.

PathwayBooster: a tool to support the curation of metabolic pathways.

BACKGROUND: Despite several recent advances in the automated generation of draft metabolic reconstructions, the manual curation of these networks to produce high quality genome-scale metabolic models remains a labour-intensive and challenging task. RESULTS: We present PathwayBooster, an open-source software tool to support the manual comparison and curation of metabolic models. It combines gene annotations from GenBank files and other sources with information retrieved from the metabolic databases BRENDA and KEGG to produce a set of pathway diagrams and reports summarising the evidence for the presence of a reaction in a given organism's metabolic network. By comparing multiple sources of evidence within a common framework, PathwayBooster assists the curator in the identification of likely false positive (misannotated enzyme) and false negative (pathway hole) reactions. Reaction evidence may be taken from alternative annotations of the same genome and/or a set of closely related organisms. CONCLUSIONS: By integrating and visualising evidence from multiple sources, PathwayBooster reduces the manual effort required in the curation of a metabolic model. The software is available online at http://www.theosysbio.bio.ic.ac.uk/resources/pathwaybooster/ .

The genus Geobacillus and their biotechnological potential.

The genus Geobacillus comprises a group of Gram-positive thermophilic bacteria, including obligate aerobes, denitrifiers, and facultative anaerobes that can grow over a range of 45-75°C. Originally classified as group five Bacillus spp., strains of Bacillus stearothermophilus came to prominence as contaminants of canned food and soon became the organism of choice for comparative studies of metabolism and enzymology between mesophiles and thermophiles. More recently, their catabolic versatility, particularly in the degradation of hemicellulose and starch, and rapid growth rates have raised their profile as organisms with potential for second-generation (lignocellulosic) biorefineries for biofuel or chemical production. The continued development of genetic tools to facilitate both fundamental investigation and metabolic engineering is now helping to realize this potential, for both metabolite production and optimized catabolism. In addition, this catabolic versatility provides a range of useful thermostable enzymes for industrial application. A number of genome-sequencing projects have been completed or are underway allowing comparative studies. These reveal a significant amount of genome rearrangement within the genus, the presence of large genomic islands encompassing all the hemicellulose utilization genes and a genomic island incorporating a set of long chain alkane monooxygenase genes. With G+C contents of 45-55%, thermostability appears to derive in part from the ability to synthesize protamine and spermine, which can condense DNA and raise its Tm.

Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius.

The facultatively anaerobic, thermophilic bacterium Geobacillus thermoglucosidasius is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in G. thermoglucosidasius. Initially, a minimal three-part E. coli-Geobacillus sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 E. coli origin of replication and multiple cloning site (MCS) of pUC18, the Geobacillus sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (knt), respectively. G. thermoglucosidasius could be transformed with pUCG3.8 at an increased efficiency [2.8×10(5) c.f.u. (µg DNA)(-1)] compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in G. thermoglucosidasius, designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from Thermotoga maritima as a secretion target. Expression of cel5A was placed under the control of a cellobiose-inducible promoter (Pßglu) together with a signal peptide encoding sequence from a G. thermoglucosidasius C56-YS93 endo-ß-1,4-xylanase. The interchange of parts was demonstrated by exchanging the cel5A gene with the 3' region of a gene with homology to celA from Caldicellulosiruptor saccharolyticus and substituting Pßglu for the synthetic, constitutive promoter PUp2n38, which increased Cel5A activity five-fold. Cel5A and CelA activities were detected in culture supernatants indicating successful expression and secretion. N-terminal protein sequencing of Cel5A carrying a C-terminal FLAG epitope confirmed processing of the signal peptide sequence.

OnpA, an unusual flavin-dependent monooxygenase containing a cytochrome b(5) domain.

ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b(5) domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b(5) domain were associated with binding of the heme, and the cytochrome b(5) domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b(5) domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b(5) and flavin-dependent monooxygenases.

Application of pheB as a reporter gene for Geobacillus spp., enabling qualitative colony screening and quantitative analysis of promoter strength.

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism.

Removing carbon catabolite repression in Parageobacillus thermoglucosidasius DSM 2542.

Parageobacillus thermoglucosidasius is a thermophilic bacterium of interest for lignocellulosic biomass fermentation. However, carbon catabolite repression (CCR) hinders co-utilization of pentoses and hexoses in the biomass substrate. Hence, to optimize the fermentation process, it is critical to remove CCR in the fermentation strains with minimal fitness cost. In this study, we investigated whether CCR could be removed from P. thermoglucosidasius DSM 2542 by mutating the Ser46 regulatory sites on HPr and Crh to a non-reactive alanine residue. It was found that neither the ptsH1 (HPr-S46A) nor the crh1 (Crh-S46A) mutation individually eliminated CCR in P. thermoglucosidasius DSM 2542. However, it was not possible to generate a ptsH1 crh1 double mutant. While the Crh-S46A mutation had no obvious fitness effect in DSM 2542, the ptsH1 mutation had a negative impact on cell growth and sugar utilization under fermentative conditions. Under these conditions, the ptsH1 mutation was associated with the production of a brown pigment, believed to arise from methylglyoxal production, which is harmful to cells. Subsequently, a less directed adaptive evolution approach was employed, in which DSM 2542 was grown in a mixture of 2-deoxy-D-glucose(2-DG) and xylose. This successfully removed CCR from P. thermoglucosidasius DSM 2542. Two selection strategies were applied to optimize the phenotypes of evolved strains. Genome sequencing identified key mutations affecting the PTS components PtsI and PtsG, the ribose operon repressor RbsR and adenine phosphoribosyltransferase APRT. Genetic complementation and bioinformatics analysis revealed that the presence of wild type rbsR and apt inhibited xylose uptake or utilization, while ptsI and ptsG might play a role in the regulation of CCR in P. thermoglucosidasius DSM 2542.

Relaxed control of sugar utilization in Parageobacillus thermoglucosidasius DSM 2542.

Though carbon catabolite repression (CCR) has been intensively studied in some more characterised organisms, there is a lack of information of CCR in thermophiles. In this work, CCR in the thermophile, Parageobacillus thermoglucosidasius DSM 2542 has been studied during growth on pentose sugars in the presence of glucose. Physiological studies under fermentative conditions revealed a loosely controlled CCR when DSM 2542 was grown in minimal medium supplemented with a mixture of glucose and xylose. This atypical CCR pattern was also confirmed by studying xylose isomerase expression level by qRT-PCR. Fortuitously, the pheB gene, which encodes catechol 2, 3-dioxygenase was found to have a cre site highly similar to the consensus catabolite-responsive element (cre) at its 3' end and was used to confirm that expression of pheB from a plasmid was under stringent CCR control. Bioinformatic analysis suggested that the CCR regulation of xylose metabolism in P. thermoglucosidasius DSM 2542 might occur primarily via control of expression of pentose transporter operons. Relaxed control of sugar utilization might reflect a lower affinity of the CcpA-HPr (Ser46-P) or CcpA-Crh (Ser46-P) complexes to the cre(s) in these operons.

Enzymatic generation of short chain cello-oligosaccharides from Miscanthus using different pretreatments.

Enzyme combinations producing short-chain cello-oligosaccharides (COS) as major bio-products from cellulose of Miscanthus Mx2779 accessed through different pretreatment methods were compared. Over short hydrolysis times, processive endoglucanase TfCel9a produced a high percentage of cellotetraose and cellopentaose and is synergistic with endoglucanase CcCel9m for producing short oligomers from amorphous cellulose but had low activity on untreated Miscanthus. Hydrolysis of the latter improved when these were combined with a mutant cellobio/triohydrolase OsCelC7(-105) and a lytic polysaccharide monooxygenase TrCel61a, a combination which also produced the highest COS yields from phosphoric acid swollen cellulose. Steam explosion pretreatment of Miscanthus increased COS yields, with/without phosphoric acid swelling, while increased swelling time (from 20 to 45 min) also increased yields but decreased the need for TrCel61a. The highest COS yields (933 mg/g glucan) and most stable product profile were obtained using ionic liquid [C2mim][OAc] pretreatment and the three enzyme mixture TfCel9a, Cel9m and OsCel7a(-105).