Cellulose Fermentation Research Articles

Disruption of phosphate metabolism and sterol transport-related genes conferring yeast resistance to vanillin and rapid ethanol production

Vanillin is a potent growth-inhibiting factor in Saccharomyces cerevisiae during lignocellulose biorefineries. Here, a haploid gene-deletion library was screened to search for vanillin-tolerant mutants and explain the possible tolerance mechanisms. Twenty-two deletion mutants were identified. The deleted genes in these mutants were involved in phosphate and inositol polyphosphate metabolism and intracellular sterol transport. Activation of the phosphate signaling pathway is not conducive to yeast against the pressure of vanillin. Furthermore, the findings indicate the role of inositol polyphosphates in altering vanillin tolerance by regulating phosphate metabolism. Meanwhile, reducing the transport of sterols from the plasma membrane enhanced tolerance to vanillin. In the presence of vanillin, the representative yeast deletions, pho84Δ and lam3Δ, showed good growth performance and promoted rapid ethanol production. Overall, this study identifies robust yeast strain alternatives for ethanol fermentation of cellulose and provides guidance for further genomic reconstruction of yeast strains.

Pretreatment with fermentable and recyclable deep eutectic solvent (DES) for improving resource utilization of biomass

A series of acidic FDES were prepared to pretreat lignocellulose. After pretreatment, solid product containing 82% cellulose, high quality lignin and liquid product containing hemicellulose were obtained, respectively. The enzymatic hydrolysis of the solid product could reach 84.5%. The enzymatic hydrolysate with the was used as feedstock for ethanol fermentation using commercial Saccharomyces cerevisiae live preparation instead of genetically engineered strains that can use 5-carbon sugars. The pretreatment solution was used as a raw material to culture Rhodotorula glutinis for microbial lipid production. phosphoric acid (PA) as a pretreatment catalyst could ensure the enzymatic efficiency compared with other acids, while more polysaccharide fractions could be formed. At the end of fermentation, the biomass could reach 30 g/L, and the lipid concentration could reach 7 g/L. In addition, the lignin prepared in this study has a consistent structure and low molecular weight, showing high potential for bio-based chemicals and materials conversion. According to the results of FDES reutilization, the pretreatment effect and fermentation effect of the FDES prepared here did not decrease significantly with the increase in the reutilization. The biomass pretreatment process developed in this study realized the full utilization of cellulose, hemicellulose, lignin and pretreatment solvent. It is innovative compared with the genetically engineered strain which mainly rely on the co-fermentation of glucose and xylose for cellulosic ethanol fermentation.

Bioconversion of cellulose into bisabolene using Ruminococcus flavefaciens and Rhodosporidium toruloides

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 ± 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production.

Characterization of Thermotoga neapolitana Alcohol Dehydrogenases in the Ethanol Fermentation Pathway.

Simple SummaryHyperthermophilic Thermotoga spp. are promising candidates for the metabolic engineering of cellulosic ethanol producing strains. However, Thermotoga spp. are hydrogen producers, and none of them have been found to produce significant amount of ethanol. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from ac-CoA to ethanol. Aldehyde dehydrogenases (Aldhs) and alcohol dehydrogenases (Adhs) are the vital enzymes involved in the metabolism of ethanol in Thermotoga spp.. However, the biochemical properties of a number of Adhs related to ethanol formation and consumption have not been identified until now. The aim of this study was to determine the role of putative Adhs in T. neapolitana (Tne) in the pathway from ac-CoA to ethanol. We found that the Fe-AAdh (encoded by CTN_0580) and Fe-Adh2 (encoded by CTN_1756) were the main enzymes responsible for ethanol production in Tne. While Zn-Adh (encoded by CTN_0257) was identified as the main protein responsible for ethanol consumption in the acid environment and Fe-Adh1 (encoded by CTN_1655) played a minor role in ethonal production and consumption in Tne. Basing on these results, it will be helpful to construct and assemble a novel hyperthermophilic strain to produce cellulosic ethanol.Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.

Improvement of bacterial cellulose fermentation by metabolic perturbation with mixed carbon sources

Bacterial cellulose (BC) has been widely used in many fields because of its nano-structure and excellent biomaterial performance. This work studied fermentation production of bacterial cellulose by Gluconacetobacter xylinus ATCC53524 using mixed carbon sources of glucose and glycerol. The results indicated that with 20 g/L of total carbon source, in medium at the ratio of glucose to glycerol being 1: 3 (w/w), the highest BC titer of 8.42 g/L was reached, which was 1.92 times higher than in conventional medium (20 g/L glucose). Metabolic analysis indicated that 57.1 % of the carbon source was converted into BC in the medium of glucose: glycerol at 1: 3, while it was only 29.6 % in the glucose medium. More ATP was also produced at 1: 3 of glucose: glycerol. The activity of two key enzymes - phosphoglucomutase and UDP-glucose pyrophosphorylase was 1.98-fold and 2.27-fold higher at 1: 3 of glucose: glycerol than those in the glucose medium, respectively. Furthermore, the BC produced in the former case showed higher Young’s modulus, better crystallinity, thicker fibers and lower porosity. The work demonstrated the use of glucose and glycerol at 1: 3 (w/w) was an effective strategy in improving the BC-producing strain’s fermentation performance and BC properties.

Conversion of golden oyster mushroom, Pleurotus citrinopileatus to sugar syrup using enzymatic hydrolysis as a substrate for novel bacterial cellulose (Nata) fermentation.

Enzymatic hydrolysis of the golden oyster mushroom (Pleurotus citrinopileatus) generated a new bacterial cellulose (BC). The sugar syrup obtained from the hydrolysis of mushroom powder by commercial enzymes gave maximum total soluble solids (TSS) content at 8.83 ± 0.29°Brix, while 8.82 ± 0.06mg GAE/g substrate of total phenolic content(TPC) was obtained when using initial substrate and enzyme concentrations at 125g/L and 5.0%, respectively. Glutamic acid, aspartic acid, alanine and valine were determined as the main amino acids found in P. citrinopileatus hydrolysis at 524.74 ± 0.03, 247.09 ± 0.04, 176.82 ± 0.07 and 174.57 ± 0.01mg/100g sample, respectively. Thin-layer chromatography revealed that the obtained sugar syrup was glucose. The hydrolyzed mushroom fermented with Komagataeibacter xylinus AGR 60 at 30 ± 2°C for 9days produced optimal conditions at 4.0°Brix of the initial mushroom syrup and 12.0% (v/v) of the starter culture. Maximum BC thickness was 0.88 ± 0.03cm with 7.90 ± 0.07g dry weight, equivalent to 39.50 ± 0.35g/L and 4.39 ± 0.04g/L/day for BC production (P) and BC production rate (R p), respectively. The obtained BC was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, small-angle X-ray scattering and wide-angle X-ray diffraction. These showed the structure and functional properties as a natural source of fiber from the fermentation of a novel substrate.

Emerging technologies for the production of biojet fuels from wood—can greenhouse gas emission reductions meet policy requirements?

The transition from fossil jet fuel to biojet fuel is an important step towards reducing greenhouse gas (GHG) emissions from aviation. To enable such a fuel shift, the Swedish Government introduced a GHG emission reduction mandate of 27% by 2030 for aviation fuel sold in Sweden, forcing fuel suppliers to blend in biojet fuel in fossil jet fuel. A similar policy instrument is being discussed within the EU. Biojet fuels with life cycle GHG emissions 90% lower than those for fossil jet fuel are projected to be available by 2025, which by far exceeds the requirement of 65% lower emissions in the EU Renewable Energy Directive. The purpose of this study was to carry out life cycle assessments for a number of wood-fuel-based production chains near commercialization and to determine whether they meet the Swedish projection and the EU requirement. The study illustrates what can be achieved in a region with high availability of wood fuels and access to heat and power with low GHG emissions. The production chains studied include the production of hydrocarbon intermediates via (i) fast pyrolysis, (ii) hydrothermal liquefaction, (iii) thermal gasification followed by Fischer–Tropsch-synthesis, and (iv) cellulosic ethanol fermentation followed by upgrading of these four intermediates to biojet fuel and other liquid biofuels. The results show that all the production chains studied can deliver biojet fuels with 89–91% lower GHG emissions than fossil jet fuels. Non-fossil hydrogen is required to achieve low emissions in the upgrading of intermediates from fast pyrolysis and hydrothermal liquefaction.

Antioxidant and antibacterial activities of Pandanus amaryllifolius Roxb. (Pandanaceae) prop roots and its application for a novel bacterial cellulose (Nata) fermentation by enzymatic hydrolysis

Nanocomposites based on bacterial cellulose in combination with osteogenic growth peptide for bone repair: cytotoxic, genotoxic and mutagenic evaluationsRaquel Mantuaneli Scarel-Caminagaa, Sybele Saskab, Leonardo Pereira Franchic, Raquel A. Santose, Ana Maria Minarelli Gaspara, Ticiana S.O. Capotea, Sidney José Lima Ribeirob, Younés Messaddeqb, Reinaldo Marchetto b, Catarina S. Takahashic d

O8. Net portal and faecal total and individual short-chain fatty acids as influenced by fermentation of cellulose and arabinoxylan rich diets

O8. Net portal and faecal total and individual short-chain fatty acids as influenced by fermentation of cellulose and arabinoxylan rich diets

Coupled Biohydrogen Production and Bio-Nanocatalysis for Dual Energy from Cellulose: Towards Cellulosic Waste Up-Conversion into Biofuels

Hydrogen, an emergent alternative energy vector to fossil fuels, can be produced sustainably by fermentation of cellulose following hydrolysis. Fermentation feedstock was produced hydrolytically using hot compressed water. The addition of CO2 enhanced hydrolysis by ~26% between 240 and 260 °C with comparable hydrolysis products as obtained under N2 but at a 10 °C lower temperature. Co-production of inhibitory 5-hydromethyl furfural was mitigated via activated carbon sorption, facilitating fermentative biohydrogen production from the hydrolysate by Escherichia coli. Post-fermentation E. coli cells were recycled to biomanufacture supported Pd/Ru nanocatalyst to up-convert liquid-extracted 5-HMF to 2,5-dimethyl furan, a precursor of ‘drop in’ liquid fuel, in a one-pot reaction. This side stream up-valorisation mitigates against the high ‘parasitic’ energy demand of cellulose bioenergy, potentially increasing process viability via the coupled generation of two biofuels. This is discussed with respect to example data obtained via a hydrogen biotechnology with catalytic side stream up-conversion from cellulose feedstock.

Re-examination of dilute acid hydrolysis of lignocellulose for production of cellulosic ethanol after de-bottlenecking the inhibitor barrier

Dilute acid hydrolysis of lignocellulose biomass had been used for production of cellulosic ethanol since 1940 s. The major technical barrier is the acid catalyzed dehydration of monosaccharides to furan aldehydes (furfural and 5-hydroxymethylfurfural), resulting in the high loss of fermentable sugars and significant inhibition on the fermentability of ethanologenic strains. This study re-examined the dilute acid hydrolysis of corn stover and cellulosic ethanol fermentation after a novel biodetoxification approach was introduced to de-bottleneck the inhibitor barrier. The cocktail of sulfuric acid, phosphoric acid and oxalic acid hydrolyzed corn stover to the 51.1 g/L of glucose (0.50 g/g cellulose) and 18.1 g/L of xylose (0.22 g/g xylan). The furfural, 5-hydroxymethylfurfural and acetic acid in the corn stover hydrolysate were completely removed by Paecilomyces variotii FN89, leading to the successful ethanol fermentation of 24.2 g/L, corresponding to 72.6 kg per metric ton of dry corn stover. No wastewater streams, solid wastes and toxic compounds were generated in hydrolysis, biodetoxification and fermentation. The techno-economic evaluations suggest that the cost reduction of replacing cellulase enzyme with cheap acid catalysts compensated the partial ethanol loss of sugar conversion to inhibitors (21.5–89.1%). The re-examination of acid hydrolysis process reveals that a substantial breakthrough in highly active and selective acid catalyst is required for acid hydrolysis to compete with enzymic hydrolysis for cellulosic ethanol fermentation.

Comparison of the Unfolded Protein Response in Cellobiose Utilization of Recombinant Angel- and W303-1A-Derived Yeast Expressing β-Glucosidase.

The unfolded protein response (UPR) is one of the most important protein quality control mechanisms in cells. At least, three factors are predicted to activate the UPR in yeast cells during fermentation. Using UPRE-lacZ as a reporter, we constructed two indicator strains, KZ and WZ, based on Angel-derived K-a and W303-1A strains, respectively, and investigated their UPR response to tunicamycin, ethanol, and acetic acid. Then, four strains carrying plasmids BG-cwp2 and BG were obtained to realize the displaying and secretion of β-glucosidase, respectively. The results of cellobiose utilization assays indicated interactions between the UPR and the metabolic burden between the strain source, anchoring moiety, oxygen supply, and cellobiose concentration. Meanwhile, as expected, growth (OD600), β-glucosidase, and β-galactosidase activities were shown to have a positive inter-relationship, in which the values of the KZ-derived strains were far lower than those of the WZ-derived strains. Additionally, extra metabolic burden by displaying over secreting was also much more serious in strain KZ than in strain WZ. The maximum ethanol titer of the four strains (KZ (BG-cwp2), KZ (BG), WZ (BG-cwp2), and WZ (BG)) in oxygen-limited 10% cellobiose fermentation was 3.173, 5.307, 5.495, and 5.486% (v/v), respectively, and the acetic acid titer ranged from 0.038 to 0.060% (v/v). The corresponding maximum values of the ratio of β-galactosidase activity to that of the control were 3.30, 5.29, 6.45, and 8.72, respectively. Under aerobic conditions with 2% cellobiose, those values were 3.79, 4.97, 6.99, and 7.67, respectively. A comparison of the results implied that β-glucosidase expression durably induced the UPR, and the effect of ethanol and acetic acid depended on the titer produced. Further study is necessary to identify ethanol- or acid-specific target gene expression. Taken together, our results indicated that the host strain W303-1A is a better secretory protein producer, and the first step to modify strain K-a for cellulosic ethanol fermentation would be to relieve the bottleneck of UPR capacity. The results of the present study will help to identify candidate host strains and optimize expression and fermentation by quantifying UPR induction.

Waste-Based Second-Generation Bioethanol: A Solution for Future Energy Crisis

The demand for more environmentally friendly alternative renewable fuels is growing as fossil fuel resources are depleting significantly. Consequently, bioethanol has attracted interest as a potentially viable fuel. The key steps in second-generation bioethanol production include pretreatment, saccharification, and fermentation. The present study employed simultaneous saccharification and fermentation (SSF) of cellulose through bacterial pathways to generate second-generation bioethanol utilizing corncobs and paper waste as lignocellulosic biomass. Mechanical and chemical pretreatments were applied to both biomasses. Then, two bacterial strains, Bacillus sp. and Norcadiopsis sp., hydrolysed the pretreated biomass and fermented it along with Achromobacter sp., which was isolated and characterized from a previous study. Bioethanol production followed by 72 h of biomass hydrolysis employing Bacillus sp. and Norcadiopsis sp., and then 72 h of fermentation using Achromobacter sp. Using solid phase micro extraction combined with GCMS the ethanol content was quantified. SSF of alkaline pretreated paper waste hydrolysed by Bacillus sp. following the fermentation by Achromobacter sp. showed the maximum ethanol percentage of 0.734±0.154. Alkaline pretreated corncobs hydrolyzed by Norcadiopsis sp. yielded the lowest ethanol percentage of 0.155±0.154. The results of the study revealed that paper waste is the preferred feedstock for generating second-generation bioethanol. To study the possible use of ethanol-diesel blends as an alternative biofuel E2, E5, E7, and E10 blend emulsions were prepared mixing commercially available diesel with ethanol. The evaluated physico-chemical characteristics of the ethanol-diesel emulsions fulfilled the Ceypetco requirements except for the flashpoint revealing that the lower ethanol-diesel blends are a promising alternative to transport fuels. As a result, the current study suggests that second generation bioethanol could be used as a renewable energy source to help alleviate the energy crisis..

Effects of Engineered Saccharomyces cerevisiae Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation.

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular β-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular β-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

Renewable Biofuel Production Using Red Ginseng Marc in Microbial Fuel Cells

Microbial fuel cell (MFC) is one of the clean and sustainable energy technologies, often referred to as renewable energy, and directly chemical energy contained in organic matter into electrical energy by using the catalytic activity of microorganisms. Cellulosic biomass is a particularly attractive renewable resource for its abundant supply at low cost and its neutral carbon balance. However, methanogenesis had been negatively linked to anaerobic cellulosic power generation in MFCs. Ginseng root is a saponin-rich plant material and red ginseng marc (RGM) has not been reused as a high-value resource for industry although its residue contained both electron donors and saponin, the potential power generation enhancers for MFC. In this study, RGM was supplemented into MFC to evaluate its effects on methanogenesis and power generation. Two-chamber H-type MFCs were established using rumen fluid as anolyte to ferment cellulose at 2% (w/v). RGM, the residue from the steam and press process for red ginseng beverage preparation, was freeze-dried and ground to pass 0.5 mm sieve and added to the anode of MFC at 1% (w/v; Exp. 1) or 0.1% (Exp. 2) dose for treatment. Open circuit voltage, voltage and current across an external resistor were measured daily for 10d. On d10 of operation, collected biogases were measured for total gas production and analyzed for its components. In Exp. 1, power density was between 44.0 and 97.2 with an average of 83.8 mW/m2 in 1% RGM MFCs and was between 45.2 and 76.3 with an average of 61.5 mW/m2 in control. In Exp. 2, power density was between 44.8 and 75.6 with an average of 60.9 mW/m2 in 0.1% RGM MFCs and was between 45.1 and 54.1 with an average of 49.7 mW/m2 in control. Total gas production for 10d was 563 and 523 mL for RGM and control, respectively, in Exp 1, and was 546 and 477 mL for RGM and control, respectively, in Exp 2. Methane took up 58.6 and 67.9% of total gas for RGM and control, respectively, in Exp 1, and 59.1 and 67.3% of total gas for RGM and control, respectively, in Exp 2. Both greater (P

Absolute abundance values reveal microbial shifts and co-occurrence patterns during gut microbiota fermentation of dietary fibres in vitro

The compositional alterations of human colonic microbiota when fermenting dietary fibres (DF) have been well studied. However, there is a challenge in quantifying microbiota responses to DF in absolute terms, and thus the extents to which these microbes grow on diverse DF are unclear. This study aimed to compare relative and absolute abundance datasets, to investigate how human faecal microbiota altered during the in vitro fermentation of arabinoxylan, mixed-linkage beta-glucan and bacterial cellulose (representing major wheat cell wall constituents), and a physical mixture of the three. Different microbial shifts occurred per DF, where the absolute abundance of total bacteria was observed to peak at 12 h, while this trend was not observed for the relative abundance data. The DF mixtures more efficiently supported microbial growth than the individual components. Additionally, the absolute abundance of the genera Faecalibacterium, Roseburia, Anaerostipes and the Eubacterium hallii group positively correlated to butyrate production, but no significant (P < 0.05) correlations were found based on the relative abundance data. While 95 genera were common to the DF types, interestingly, their co-occurrence patterns in absolute terms vastly differed, with 45.6% specific edges unique to the BC co-occurrence network. Therefore, applying absolute quantification is recommended for experiments focusing on how microbiota respond to DF.

Coordinately express hemicellulolytic enzymes in Kluyveromycesmarxianus to improve the saccharification and ethanol production from corncobs

BackgroundHemicellulose acts as one factor contributing to the recalcitrance of lignocellulose that prevents cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance the performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianus is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K.marxianus through a 2A-mediated ribosome skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs.ResultsTwo polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from the Foot-and-Mouth Disease virus (FMDV) in K.marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn. In the IMPαX gene, there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of the IMPX and IMPαX strains were 21.34 and 15.50 U/mL, respectively, but the extracellular β-xylanase activity of IMPαX strain was much higher than that of the IMPX strain, which was 136.17 and 42.07 U/mL, respectively. Subsequently, two recombinant strains, the IXPαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes, Xyn-CDBFV and β-D-xylosidase RuXyn1, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentation, extracellular activities of β-xylanase and β-xylosidase in the IXPαR strain were 1664.2 and 0.90 U/mL. Similarly, the IMPαXPαR strain secreted the three enzymes, β-mannanase, β-xylanase, and β-xylosidase, with the activities of 159.8, 2210.5, and 1.25 U/mL, respectively. Hemicellulolases of both strains enhanced the yields of glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yield by 8.7% at 144 h compared with the control. However, both ethanol and xylose yields were increased by 12.7 and 18.2%, respectively, at 120 h in HSF of aqueous ammonia pretreated (AAP) corncobs with this strain. Our results indicated that coordinate expression of hemicellulolytic enzymes in K. marxianus promoted the saccharification and ethanol production from corncobs.ConclusionsThe FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus and could be used for secretory expression of multiple enzymes in the presence of their signal sequences. The IMPαXPαR strain coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

In vitro fermentation of onion cell walls and model polysaccharides using human faecal inoculum: Effects of molecular interactions and cell wall architecture

Plant primary cell walls provide a primary dietary source of fermentable carbohydrates. They are typically based on a cellulose network cross-linked by xyloglucan, with pectin incorporated, but the relative contributions of components and their architectural arrangement to gut fermentation performance is incompletely understood. Onion cell walls (OCW) were isolated and used as a primary cell wall model. OCW, models for their main constituents (xyloglucan, pectin, cellulose) and the physical mixture of these model polysaccharides (Mix) were fermented in vitro up to 48h, using a human faecal inoculum. Each constituent in Mix was fermented to a similar extent as single-component substrates, with comparable gas and short chain fatty acid (SCFAs) production. The microbiota responded differently and specifically to each polysaccharide, with the microbial community for Mix reflecting both pectin and xyloglucan. OCW was degraded more slowly at the early stage of fermentation, with less SCFAs produced compared with Mix, though a more extensive fermentation occurred by the end of fermentation, due to the slow but essentially complete fermentation of cellulose in OCW but not Mix. Microbiota shifts for OCW were different to those for Mix. The architecture of OCW as well as polysaccharide composition determined both fermentation outcomes and microbial community shifts.

Ultrasound-assisted dilute acid hydrolysis for production of essential oils, pectin and bacterial cellulose via a citrus processing waste biorefinery

An orange peel waste biorefinery was developed employing a design of experiments approach to optimize the ultrasound-assisted dilute acid hydrolysis process applied for production of useful commodities. Central composite design-based response surface methodology was used to approximate the combined effects of process parameters in simultaneous production of essential oils, pectin and a sugar-rich hydrolyzate. Application of a desirability function determined the optimal conditions required for maximal production efficiency of essential oils, pectin and sugars as 5.75% solid loading, 1.21% acid concentration and 34.2 min duration. Maximum production yields of 0.12% w/w essential oils, 45% w/w pectin and 40% w/w sugars were achieved under optimized conditions in lab- and pilot-scale facilities. The hydrolyzate formed was applied in bacterial cellulose fermentations producing 5.82 g biopolymer per 100 g waste. Design of experiments was efficient for process analysis and optimization providing a systems platform for the study of biomass-based biorefineries.

High yield biorefinery products from sugarcane bagasse: Prebiotic xylooligosaccharides, cellulosic ethanol, cellulose nanofibrils and lignin nanoparticles

An integrated biorefining strategy was applied to fractionate Sugarcane bagasse (SCB) into its major constituents, enabling high-yield conversion of the fractionated materials into high-value coproducts alongside cellulosic ethanol. Pilot-scale steam explosion produced a hydrolysate rich in low molecular weight xylooligosaccharides that had a high in vitro efficacy as a prebiotic towards different bifidobacteria. Lignin recovered after alkaline treatment of the steam-exploded SCB was converted into uniform spherical lignin nanoparticles (11.3 nm in diameter) by a green mechanical method. The resulting cellulose was hydrolyzed at 17.5% (w/v) consistency and low enzyme loading (17.5 mg/g) to yield a pure glucose hydrolysate at a high concentration (100 g/L) and a cellulosic solid residue that was defibrillated by disc ultra-refining into homogeneous cellulose nanofibrils (20.5 nm in diameter). Statistical optimization of the cellulosic hydrolysate fermentation led to ethanol production of 67.1 g/L, with a conversion yield of 0.48 g/g and productivity of 1.40 g/L.h.