The thermostable tau-muurolol synthase from Roseiflexus sp.RS-1 (RoseRS_3509) has been converted, via single residue mutations, into both beta-farnesene, and germacrene D-4-ol synthases with turnover numbers similar to other bacterial terpene synthases (TPS). In the absence of a crystal structure, potential sites for site-directed mutagenesis were predicted by a combination of structural modelling and sequence comparison based on critical residues identified in other TPSs. Twelve mutants were created and initially characterized for activity and product formation profiles. Three mutants which formed a single dominant product, differing from the wild type, were then characterized kinetically. Surprisingly, the Y310A mutation of a residue normally associated with initial binding of the farnesene pyrophosphate (FPP) substrate together with Mg2+ produced a germacrene D-4-ol synthase. Mutations of the aromatic residues F78 and W296 to alanine both produced beta-farnesene synthases, in accordance with previous studies suggesting that aromatic residues at the base of the binding cavity determine the binding conformation of the substrate, which dictates product formation.

Ethanol concentrations above 4% (v/v) are required for economic bioethanol production due to the cost of recovery from dilute solutions. Although thermophilic bacteria have many potential advantages over Saccharomyces cerevisiae as process organisms for second generation bioethanol production, they are known to be less tolerant to ethanol, typically to concentrations less than 4% (v/v). To address this issue we have investigated the application of in situ gas-stripping of ethanol using microbubbles to increase the surface area per unit volume of gas, using fed-batch and continuous cultures of the engineered ethanologenic thermophile Parageobacillus thermoglucosidasius TM242. By using microbubbles generated at room temperature using a Desai-Zimmerman Fluid Oscillator, we initially operated a mixed batch and fed-batch fermentation, followed by a continuous fermentation and finally a chemostat fermentation, under conditions which would have generated in excess of 4% (v/v) ethanol. In all cases, gas stripping maintained the actual dissolved ethanol concentration below, or close to toxic levels. As the focus of this study was on demonstrating the efficiency of in situ microbubble gas stripping, to simplify the operation the latter two processes involved a combination of produced and supplemented ethanol, with the chemostat culture producing a nominal maximum 7.1% v/v based on glucose used (5.1-5.3% (v/v) based on ethanol recovered). This offers a practical way to produce second generation bio-ethanol from thermophiles.

Abstract 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.

Improved technologies are needed for sustainable conversion of cellulosic waste to valuable products. Here we demonstrate the successful integration of a synthetic microbial consortium (SynCONS) based consolidated bioprocessing with pyrolysis to produce commodity chemicals from cellulose. Promising microbial partners were rationally identified from 7626 organisms via comparative metabolic mapping which led to establishing two promising SynCONS with abilities to convert cellulose to ethanol and lactate in bioreactors. The partners in the two SynCONS were a) the mesophilic fungus Trichoderma reesei grown sequentially with the thermophilic bacterium Parageobacillus thermoglucosidasius NCIMB 11955 (TrPt) and b) a thermophilic bacterium Thermobifida fusca grown together with Parageobacillus thermoglucosidasius NCIMB 11955 (TfPt). TrPt sequential bioprocessing resulted in 39% (g/g) cellulose consumption with product yields up to 9.3% g/g (ethanol + lactate). The TfPt co-cultures demonstrated a cellulose consumption of 30% (g/g) and combined yields of ethanol and lactic acid up to 23.7% g/g of consumed cellulose. The total product yields were further enhanced (51% g/g cellulose) when commercially available cellulases were used in place of T. fusca. Furthermore, when the metabolically engineered ethanol-producing strain of P. thermoglucosidasius TM242 (TfPt242) was substituted in the thermophilic TfPt co-culture consortium, ethanol yields were substantially higher (32.7% g/g of consumed cellulose). Finally, subjecting the residual cellulose and microbial biomass to pyrolysis resulted in carbon material with physicochemical properties similar to commercially available activated carbon as analysed using Scanning Electron Microscopy, X-Ray Diffraction and Raman spectroscopy. Overall, the integration of this synthetic microbial consortia-based bioprocessing strategy with pyrolysis demonstrated a promising strategy for conversion of waste cellulose to chemicals, biofuels, and industrial carbon potentially suitable for several industrial applications.

The exploration of residual lignocellulosic biomass for biofuel production is crucial to achieve significant greenhouse gas (GHG) emissions mitigation within the following decades, translating to diminished agricultural environmental impact and land-use change dynamics. On the other hand, cellulosic ethanol production pathways are typically resource-intensive, in energy and chemical terms, which directly influence its carbon intensity. Pretreatment choice, to this end, is key, since it dictates the overall process performance and yield, and may include crucial flows, under the life-cycle perspective, such as solvents and other chemicals. This work, then, aims to evaluate the effect of pretreatment choice, namely hydrothermal (HT), steam explosion (SE), and alkaline (AK), in the technical and environmental performance of cellulosic ethanol production from sugarcane straw (SCS), extending this analysis to the final GHG emissions mitigation potential for gasoline and fossil-generated electricity substitution, under the Brazilian context in São Paulo. Results show that, while AK provided the highest ethanol yield, this pretreatment option gave the lowest electricity generation surplus, and its sodium hydroxide usage was identified as an important environmental hotspot in most impact categories, which narrowed down its GHG emission mitigation gap for gasoline substitution. HT and SE presented similar ethanol and electricity yields, with SE being the most balanced option in terms of productivity and environmental impact profile. By selecting the SE route, all of the available SCS in São Paulo could be converted into 10% of the Brazilian annual ethanol production, and mitigate 5.4 MtCO2e of gasoline emissions, 15% of the Brazilian Biofuel Policy (RenovaBio) target for 2022.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two β-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa , yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1 , that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 ( sor1 Δ gre3 Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

The engineering of xylo-oligosaccharide-consuming Saccharomyces cerevisiae strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in S. cerevisiae strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by S. cerevisiae strains. The suitability of S. cerevisiae for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.

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).

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.

This study investigated the production of xylo-oligosaccharides (XOS) from sugarcane straw (SCS) using steam explosion (SE) pretreatment at pilot-scale, as well as co-production of fermentable sugars and lignin-rich residues for bioethanol and bioenergy, respectively. SE conditions 200 degrees C; 15 bar; 10 min led to 1) soluble XOS yields of up to 35 % (w/w) of initial xylan with similar to 50 % of the recovered XOS corresponding to xylobiose and xylotriose, considered the most valuable sugars for prebiotic applications; 2) fermentable glucose yields from the enzymatic hydrolysis of SE-pretreated SCS of up to similar to 78 %; 3) increase in the energy content of saccharified SCS residues (16 %) compared to the untreated material. From an integrated biorefinery perspective, it demonstrated the potential use of SCS for the production of value-added XOS ingredients as well as liquid and solid biofuel products.

Abstract 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.

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 cataboliteresponsive 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 (Ser46P) complexes to the cre(s) in these operons.

With the advent of RenovaBio, Brazil has cast a new light towards the life cycle of cellulosic ethanol. Once considered a resource intensive alternative pathway to achieve the same biofuel, second-generation approaches can now provide an economic advantage due to their potentially lower carbon footprint. The exploration of lignocellulosic harvest residues to this end can be beneficial, since productivity can be increased while not expanding cultivated areas. Eucalyptus forest residues are an example, result of logging and harvest procedures, being a low-cost and readily available biomass. Through an integrated biorefinery process simulation and a Life Cycle Assessment of the coproduction of ethanol and electricity, it was analyzed whether forestry burden is truly relevant when exploring this material, identifying technical and environmental bottlenecks. The biorefinery design implementation of anaerobic digestion and energy integration allowed a productivity boost of 20% for ethanol and 115% for electricity. With a 80 km collection radius, an annual production capacity of 30.3 ML could be achieved in the Campinas region. Enzymes were identified as the main environmental hotspot, but inconsistent published datasets and lack of transparency lead to inconclusive results regarding this input. While the burden associated with the lignocellulosic feedstock is relevant in most impact categories, the main bottleneck resides within the biorefinery itself, with inputs related to pretreatment and hydrolysis, boiler emissions and water consumption. Nevertheless, eucalyptus harvest residues cannot be considered a burden-free resource, since additional operations such as retrieval and transportation cannot be dismissed and often surpasses the impact potential of the aforementioned forestry activities.& nbsp; (c) 2021 Elsevier Ltd. All rights reserved.

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 (similar to 120 degrees C) by fluidic oscillation was used to remove ethanol from similar to 2% (v/v) ethanol-water mixtures maintained at 60 degrees 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 (similar to 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(-1)h(-1) continuously.

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.

Producers look for co‐products to increase the value chain and production flexibility. With a high market aggregate value, the production of cello‐oligosaccharides (COS) as functional oligosaccharides from cellulosic substrates has been investigated. As COS/cellopentaose production process are rarely described in the literature, this study estimated the cellopentaose production cost and performed a life‐cycle assessment of the cellopentaose production process previously developed using sugarcane straw as a substrate. The results demonstrated that, through the six scenarios studied, it was possible to obtain a cellopentaose unit production cost varying between USD 0.40 and 1.15/mg, and that optimization in the upstream sector can reduce the total plant direct cost and the overall life‐cycle impact. The results also suggested that a better understanding of the hydrolysis solid/liquid proportion is necessary to reduce the bulk material cost. An environmental impact reduction of between 16.2% and 19.9% was also observed. In terms of the integrated biorefinery concept, sugarcane straw fermentation represents a prospective technology for the production of COS. © 2021 Society of Chemical Industry and John Wiley & Sons, Ltd

Acidogenic fermentation is attractive for food waste valorisation. A better understanding is required on how operation affects product selectivity. This study demonstrated that the hydraulic retention time (HRT) and organic loading rate (OLR) selected fermentation pathways in a single-stage, semi-continuous stirred tank reactor. Three combinations of HRT and OLR were tested to distinguish the effect of each parameter. Three fermentation profiles with distinct microbial communities were obtained. Predominantly n-butyric acid (13 ± 2 gCOD L-1, 55 ± 14% of carboxylates) was produced at an HRT of 8.5 days and OLR around 12 gCOD L-1d-1. Operating at an HRT two days longer, yet with similar OLR, stimulated chain elongation (up to 13.6 gCOD L-1 of n-caproic acid). This was reflected by a microbial community twice as diverse at longer HRT as indicated by first and second order Hill number (1D = 24 ± 4, 2D = 12 ± 3) and by a higher relative abundance of genera related to secondary fermentation, such as the VFA-elongating Caproiciproducens spp., and secondary lactic acid fermenter Secundilactobacillus spp.. Operating at a higher OLR (20 gCOD L-1d-1) but HRT of 8.5 days, resulted in typical lactic acid fermentation (34 ± 5 gCOD L-1) harbouring a less diverse community (1D = 8.0 ± 0.7, 2D = 5.7 ± 0.9) rich in acid-resistant homofermentative Lactobacillus spp. These findings demonstrate that a flexible product portfolio can be achieved by small adjustments in two key operating conditions. This improves the economic potential of acidogenic fermentation for food waste valorisation.

Pretreatment strategies are fundamental to effectively deconstruct lignocellulosic biomass and economically produce biofuels, biomaterials and bio-based chemicals. This study evaluated individual and combinatorial steam explosion (SE) and ionic liquid (IL) pretreatments for production of high-value oligosaccharides from a novel seed-based Miscanthus hybrid (Mx2779). The two ILs used for pretreatment were triethylammonium hydrogen sulphate [TEA][HSO4] and 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. The results showed that each pretreatment leads to distinct effects on the fragmentation (cellulose and xylan dissolution, delignification, deacetylation) and physicochemical modification (cellulose and lignin properties) of lignocellulose. This, in turn, dictated enzymatic hydrolysis efficiencies of the cellulose pulp to glucose or gluco-oligosaccharides for downstream applications. Our findings suggest that the stand-alone SE or [C2mim][OAc] pretreatments may offer cost advantages over [TEA][HSO4] through the production of oligosaccharides such as xylo- and gluco-oligosaccharides. This study also highlights technical and economic pretreatment process challenges related to the production of oligosaccharides from Miscanthus Mx2779 biomass.

Enzymatic hydrolysis of lignocellulosic biomass accounts for 20-30% of the total cost of second-generation bioethanol production and many efforts have been made in recent years to overcome the high cost of enzymes. Using cello-oligosaccharides (COS), intermediates in cellulose conversion to glucose, may provide advantages over monomeric glucose fermentation, such as lower risk of growth of process contaminants, shorter fermentation time and limited process inhibition by high concentrations of glucose. In addition, COS are also useful as functional oligosaccharides in the food and feed sectors. This study aimed to optimize COS production for further industrial applications. To the best of our knowledge, this is the first study that has used a design of experiments approach to analyze the synergism between endoglucanases, lytic polysaccharide monooxygenase (LPMO), cellobiose dehydmgenase (CDH) and different additives during the hydrolysis of a pretreated sugarcane straw for COS production. After optimization of enzymatic hydrolysis, a combination of the endoglucanases CaCel and CcCel9m, the LPMO TrCe161A, the CDH NcCDHIIa, with lactose and copper as additives, produced 60.49 mg of COS per g of pretreated sugarcane straw, 1.8-2.7-fold more than the commercial enzyme cocktails Cellic (R) Ctec2 and Celluclast (R) 1.5 L. The COS/glucose ratio achieved was 298.31, an increase of 3314 and 2294-fold over the commercial enzymatic cocktails, respectively. These results open a new perspective regarding COS production and its industrial application.

High molecular weight renewable polyesters are synthesised from an unsaturated fatty acid and structurally unmodified, hemicellulosic sugars, with post-polymerisation modification inducing semicrystallinity and allowing casting of transparent films.