Hydrolysis Of Sugarcane Bagasse Research Articles

High yield recovery of 2,3-butanediol from fermented broth accumulated on xylose rich sugarcane bagasse hydrolysate using aqueous two-phase extraction system

Downstream processing of chemicals obtained from fermentative route is challenging and cost-determining factor of any bioprocess. 2,3-Butanediol (BDO) is a promising chemical building block with myriad applications in the polymer, food, pharmaceuticals, and fuel sector. The current study focuses on the recovery and purification of BDO produced (68.2 g/L) from detoxified xylose-rich sugarcane bagasse hydrolysate by a mutant strain of Enterobacter ludwigii. Studies involving screening and optimization of aqueous-two phase system (ATPS) revealed that 30% w/v (NH4)2SO4 addition to clarified fermented broth facilitated BDO extraction in isopropanol (0.5 v/v), with maximum recovery and partition coefficient being 97.9 ± 4.6% and 45.5 ± 3.5, respectively. The optimized protocol was repeated with unfiltered broth containing 68.2 g/L BDO, cell biomass, and unspent protein, which led to the partitioning of 66.7 g/L BDO, 2.0 g/L xylose and 9.0 g/L acetic acid into organic phase with similar BDO recovery (97%) and partition coefficient (45).

Engineering Zymomonas mobilis for the Production of Xylonic Acid from Sugarcane Bagasse Hydrolysate.

Sugarcane bagasse is an agricultural residue rich in xylose, which may be used as a feedstock for the production of high-value-added chemicals, such as xylonic acid, an organic acid listed as one of the top 30 value-added chemicals on a NREL report. Here, Zymomonas mobilis was engineered for the first time to produce xylonic acid from sugarcane bagasse hydrolysate. Seven coding genes for xylose dehydrogenase (XDH) were tested. The expression of XDH gene from Paraburkholderia xenovorans allowed the highest production of xylonic acid (26.17 ± 0.58 g L−1) from 50 g L−1 xylose in shake flasks, with a productivity of 1.85 ± 0.06 g L−1 h−1 and a yield of 1.04 ± 0.04 gAX/gX. Deletion of the xylose reductase gene further increased the production of xylonic acid to 56.44 ± 1.93 g L−1 from 54.27 ± 0.26 g L−1 xylose in a bioreactor. Strain performance was also evaluated in sugarcane bagasse hydrolysate as a cheap feedstock, which resulted in the production of 11.13 g L−1 xylonic acid from 10 g L−1 xylose. The results show that Z. mobilis may be regarded as a potential platform for the production of organic acids from cheap lignocellulosic biomass in the context of biorefineries.

The Effect of Xylan Removal on the High-Solid Enzymatic Hydrolysis of Sugarcane Bagasse

The Effect of Xylan Removal on the High-Solid Enzymatic Hydrolysis of Sugarcane Bagasse

Multi-efficient thermostable endoxylanase from Bacillus velezensis AG20 and its production of xylooligosaccharides as efficient prebiotics with anticancer activity

In this study, we have unveiled the novel potential of xylanase production by Bacillus velezensis, previously known only for its PGPR traits. According to our investigations, Bacillus velezensis AG20 produced extracellular xylanase with a fold purification of 5.3 and a yield of 21 %. The purified xylanase (45 kDa) aptly cleaved linear β-(1→4)-xylan with a maximum velocity of 21.0 ± 3.0 U/mL, a turnover of 1.75/s, and a catalytic cycle of 0.571 at optimum pH 7. The temperature of 50 °C was found optimum for enzyme activity. Purified endoxylanase was thermostable and retained its significant residual activities at 50 °C and 60 °C. Metal cations such as Ca2+ and Mg2+ enhanced the substrate catalysis up to 1.5 fold. The purified endoxylanase also demonstrated multi-substrate hydrolyzing properties. However, the enzymatic hydrolysis of sugarcane bagasse produced xylooligosaccharides (XOS), including xylobiose, xylotriose, and xylotetrose. The mixture of released XOS has profound stability in gastric juice, intestinal fluid, and α-amylase and facilitates probiotic bacteria Bifidobacterium. The mixed XOS (300 μg/mL) significantly inhibited the HT-29 and Caco-2 cell proliferation by 90 % and 75 %, respectively, at 48 h. The possible mechanism of XOS uptake through the BIAXP receptor of Bifidobacterium has been contemplated and established using the caver analysis, Molecular Dynamics (MD) simulation, and Density Functional Theory (DFT) studies. Thus, here we reveal the utility of this novel bacterial strain in industries as high-temperature catalysts.

Improvement of cellulase and xylanase production in Penicillium oxalicum under solid-state fermentation by flippase recombination enzyme/ recognition target-mediated genetic engineering of transcription repressors

Penicillium oxalicum has received increasing attention as a potential cellulase-producer. In this study, a copper-controlled flippase recombination enzyme/recognition target (FLP/FRT)-mediated recombination system was constructed in P. oxalicum, to overcome limited availability of antibiotic resistance markers. Using this system, two crucial transcription repressor genes atf1 and cxrC for the production of cellulase and xylanase under solid-state fermentation (SSF) were simultaneously deleted, thereby leading to 2.4- to 29.1-fold higher cellulase and 78.9% to 130.8% higher xylanase production than the parental strain under SSF, respectively. Glucose and xylose released from hydrolysis of pretreated sugarcane bagasse achieved 10.6%–13.5% improvement by using the crude enzymes from the engineered strain Δatf1ΔcxrC::flp under SSF in comparison with that of the parental strain. Consequently, these results provide a feasible strategy for improved cellulase and xylanase production by filamentous fungi.

Process optimization for deep eutectic solvent pretreatment and enzymatic hydrolysis of sugar cane bagasse for cellulosic ethanol fermentation

This study investigated cellulosic ethanol production from sugar cane bagasse (SCB) pretreated by triethylbenzyl ammonium chloride/lactic acid (TEBAC/LA) deep eutectic solvent (DES). The results showed that the pretreatment of SCB with TEBAC/LA DES at 120 °C for 4 h with 1:15 (solid to liquid ratio) resulted in the best cellulose digestibility (88.23 ± 1.24%), which was approximately 228% higher than that of untreated SCB. Furthermore, the maximum cellulosic ethanol concentration of 16.84 g/L was achieved using glucose (36.06 g/L) and xylose (7.36 g/L). Moreover, the ethanol productivity and yield were 0.70 g/(L·h) and 0.42 g/g fermentable sugar, respectively. The efficient bioconversion was ascribed to the remarkable delignification (88.72 ± 1.63%), xylan removal (73.93 ± 0.17%), along with optimum cellulose recovery (95.89 ± 1.54%). Importantly, the enzymatic hydrolysis digestibility remained unchanged after 5 times DES recycling process. Overall, it also provided an insight into the efficient SCB biorefinery of DES systems.

Xylooligosaccharides production by optimized autohydrolysis, sulfuric and acetic acid hydrolysis for minimum sugar degradation production

Xylooligosaccharides (XOS) acts as a prebiotic, a food component that is not digested but stimulates the growth of beneficial microorganisms present in the intestine, such as bifidobacteria and lactobacilli, improving the health of the host. This study obtained XOS by comparing optimized conditions for acid hydrolysis and autohydrolysis of sugarcane bagasse, showing that is possible to produce XOS with minimum degradation of sugras. The acid hydrolysis process varied the parameters applying a 23 factorial design using 1–3% (m/v) sulfuric or acetic acid; a temperature of 100–160 °C and a reaction time of 15–55 min. The autohydrolysis was performed by 22 factorial design using the same range of temperature and time. The acetic acid hydrolysis of the sugarcane bagasse resulted in the conversion of xylan into XOS of 18.41% (m/m) with 1% (m/v) acid at 100 °C for 15 min, with xylopentaose/xylohexaose predominance. The hydrolysis of sugarcane bagasse with sulfuric acid resulted in 90.13% (m/m) of XOS with 2% (m/v) of acid at 79.55 °C for 35 min, with xylobiose prevalence. Autohydrolysis of bagasse resulted in 13.67% (m/m) xylan conversion into XOS at 172.4 °C for 35 min, with xylotetraose prevalence. The degradation sugar products were low for the conditions studied, with 0.31%, 0.16%, and 0.01% (m/m) of furfural using sulfuric, acetic, and autohydrolysis, successively. The results demonstrated that XOS with different degrees of polymerization can be produced applying specific pretreatment conditions of autohydrolysis, sulfuric, and acetic acid, with the minimum content of sugar degradation.

Sequential process of solid-state cultivation with fungal consortium and ethanol fermentation by Saccharomyces cerevisiae from sugarcane bagasse.

Solid-state cultivation (SSC) is the microbial growth on solid supports, producing a nutrient-rich solution by cell enzymes that may be further used as a generic microbial medium. "Second-generation" ethanol is obtained by fermentation from mainly the acid hydrolysates of lignocellulosic wastes, generating several microbial growth inhibitors. Thus, this research aimed at evaluating the feasibility of ethanol fermentation from sugarcane bagasse hydrolysate after SSC with vinasse as the impregnating solution by a consortium of A. niger and T. reesei as opposed to the conventional method of acid hydrolysis. Fermentation of the hydrolysate from SSC leading to the yield of 0.40gg-1, i.e., about 78% of maximum stoichiometric indicating that the nonconventional process allowed the use of two by-products from sugarcane processing in addition to ethanol production from glucose release.

Simultaneous production of green hydrogen and bioethanol from segregated sugarcane bagasse hydrolysate streams with circular biorefinery design

This study represents an integrated and sustainable approach to mine fullest potential of sugarcane bagasse (SCB) for the production of bio-H2 and bioethanol along with organic farming. Experiments were designed in three phases, where feasibility of bio-H2 production was initially evaluated with pure xylose and further validated with real field feedstock (i.e, SCB). Acid pretreatment of SCB with 2% H2SO4 (v/v) yielded xylose rich hydrolysate (21.8 g xylose/100 g SCB), which was subjected to dark fermentation for bio-H2 and VFA production. Heat-pretreated inoculum at pH 7.0 showed maximum bio-H2 fraction of 34.2% (v/v) with corresponding volumetric yield of 204.5 mL H2/g xylose. 5200 mg/L of VFA was noted with butyrate and acetate as the major fermentative metabolites. The unhydrolysed biomass recovered after acid hydrolysis of SCB was further subjected to simultaneous saccharification and fermentation (SSF) for bioethanol production. SSF of cellulose rich recovered biomass with cellulase enzyme showed 86.56% (w/w) depolymerization of cellulose into glucose and production of 241.2 mg ethanol/g treated SCB by Saccharomyces cerevisiae. Lastly acidogenic effluent (AE) from bio-H2 production process was utilized as phosphate solubilizing organic fertilizer for the cultivation of chick pea (Cicer arietinum). Regular supplementation of AE showed beneficial effect on phosphate solubilization in soil and uptake by plants. The obtained results confirm the feasible utilization of segregated streams of SCB for bio-H2 and bioethanol production with integrated organic farming in a closed loop approach. This integrated strategy would extract maximum potential of the agri-biomass feedstock with net-zero waste discharge.

Enhanced biohydrogen production by an ammonium-tolerant Rhodobacter capsulatus from sugarcane bagasse

Lignocellulosic biomass is an abundant and renewable resource from plants, and has great potential as an alternative to fossil resources to produce second-generation biofuels. Biohydrogen production from lignocellulosic biomass is an attractive approach to generate clean energy. Here a new ammonium-tolerant mutant named Rhodobacter capsulatus WH02 was isolated by transposon mutagenesis. Batch experiments were conducted to investigate the effects of key parameters such as inoculum concentration (5%–60%, v/v), glucose concentration (10–50 mmol/L), and ammonium concentration (2–8 mmol/L) on photo-fermentation of WH02. The results showed that the hydrogen yield of WH02 was about 3.74 folds of the wild type strain at 8 mmol/L ammonium chloride addition, suggesting the mutant strain WH02 exhibited improved biohydrogen production performance under ammonium suppression conditions compared with the wild type strain. Sugarcane bagasse (SCB) hydrolysate with high ammonium concentration (87.94 ± 2.22 mmol/L) was used as substrate to evaluate the ammonium-tolerant performance of WH02. The maximum hydrogen production was up to 153.96 ± 1.98 mL-H2/g-SCB by WH02 from SCB hydrolysate with the energy conversion efficiency of hydrogen of 10.95%, demonstrating the feasibility of producing biohydrogen from lignocellulosic wastes containing high ammonium by the ammonium-tolerant strain WH02.

Optimization of high-solid enzymatic hydrolysis of two-step alkaline and dilute acid-pretreated sugarcane bagasse at low enzyme loadings by response surface methodology

Optimization of high-solid enzymatic hydrolysis of two-step alkaline and dilute acid-pretreated sugarcane bagasse at low enzyme loadings by response surface methodology

β-Glucosidase produced by Moniliophthora perniciosa: Characterization and application in the hydrolysis of sugarcane bagasse.

β-Glucosidases (BGLs) belong to the group of enzymes of cellulases and act in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of BGL from Moniliophthora perniciosa in the hydrolysis of pretreated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein.

Salting-out assisted solvent extraction of L (+) lactic acid obtained after fermentation of sugarcane bagasse hydrolysate

Lactic acid is among the twelve platform chemicals produced from inexpensive and renewable feedstocks such as lignocellulosic biomass. The present study illustrates salting-out assisted solvent extraction of L (+) lactic acid, derived from sugarcane bagasse hydrolysate. Screening studies with various extractants and diluents revealed that a combination of tri-n-butyl phosphate and ethyl acetate yielded the best results and extracted 59.63 ± 1.28% lactic acid from the dilute fermentation broth adjusted to a pH of 1.6 ± 0.2. Various inorganic salts were screened to enhance extraction efficiency further. The addition of 60% (w/v) ammonium sulfate improved the lactic acid extraction by 36.17%. This salt concentration successfully extracted 85.95 ± 0.44% lactic acid to the organic phase under optimized conditions from the fermentation broth at a pH of ~ 2.5 containing 40–100 g L−1 lactic acid. Recovery of > 80% salt is also shown using chilled acetone, which upon reuse showed a nominal decline of 3.3% during extraction. Thus an eco-friendly approach using a green solvent like ethyl acetate with mild operating conditions is demonstrated in the present study.

Co-fermentation of sugarcane bagasse hydrolysate and molasses by Clostridium saccharoperbutylacetonicum: Effect on sugar consumption and butanol production

Co-fermentation of mixed sugars to produce butanol is an attractive route in sucrochemical production chains. Herein, high-level mixed sugars from sugarcane bagasse hemicellulosic hydrolysate (HH) and molasses (SCM) were investigated as potential substrates for acetone-butanol-ethanol (ABE) production in batch fermentation by Clostridium saccharoperbutylacetonicum DSM 14923. HH produced after hydrothermal pretreatment was concentrated 5-fold and was called concentrated hemicellulosic hydrolysate (CHH). The fermentation media that were investigated consisted solely of CHH and SCM and three CHH-to-SCM ratios were used to provide 30 g/L initial sugar concentrations and furan derivatives lower than 0.1 g/L. For CHH50/SCM50 and CHH75/SCM25, diluting CHH to concentrations of 15 g/L sugars and 22.5 g/L sugars, respectively, which were supplemented with SCM and nutrients, suffered growth inhibition as a function of the concentration of undissociated acid in the medium. The best-performing medium, CHH25/SCM75 (7.5 g/L and 22.5 g/L sugars from CHH and SCM, respectively and about 0.017 g/L furan derivatives), showed 97 % sugar consumption, and in the pH range of 5.5–6.5, undissociated acetic acid was not an inhibitory molecular form to C. saccharoperbutylacetonicum. After 30 h of fermentation, 7.8 and 9.8 g/L butanol and ABE were produced, respectively, which yielded 0.28 and 0.36 g/g. Based on our findings, C. saccharoperbutylacetonicum exhibits its potential and effective application for renewable butanol production by co-fermenting mixed sugars.

Co-production of fermentable glucose, xylose equivalents, and HBS-lignin from sugarcane bagasse through a FeCl3-catalyzed EG/H2O pretreatment

In this study, a FeCl3-catalyzed ethylene glycol/water (FeCl3-EG/H2O) pretreatment was developed and applied for the enzymatic hydrolysis of sugarcane bagasse. The results indicated that FeCl3-EG/H2O pretreatment showed excellent selectivity for the removal of both hemicellulose (∼100 %) and lignin (∼65.3 %), leading to the residual pretreated solid with high cellulose content (∼74.7 %) that could be easily for subsequent enzymatic saccharification. The yield of glucose after enzymatic hydrolysis was almost linearly positively correlated with the removal of lignin and xylan, and the maximum glucose yield obtained the using method was 91.5 %, which was 4.1-fold as large as that without pretreatment. The SEM, FT-IR, and XRD analysis indicated that the improvement of enzyme digestibility of sugarcane bagasse was not only related to the selectively remove of some component barriers, but was also correlated with the changes of surface morphology and internal structure. Furthermore, part of the hemicellulose and lignin in sugarcane bagasse also can be recovered in xylose equivalents, furfural, and high boiling solvent lignin (HBS-lignin) with the yields of 84.0 %, 12.5 %, and 60.1 %, respectively. It can be concluded that the FeCl3-EG/H2O pretreatment developed in this study was effective in achieving the hierarchic conversion and utilization of bagasse.

Improving the productivity of Candida glycerinogenes in the fermentation of ethanol from non-detoxified sugarcane bagasse hydrolysate by a hexose transporter mutant.

In this study, we attempted to increase the productivity of Candida glycerinogenes yeast for ethanol production from non-detoxified sugarcane bagasse hydrolysates (NDSBH) by identifying the hexose transporter in this yeast that makes a high contribution to glucose consumption, and by adding additional copies of this transporter and enhancing its membrane localisation stability (MLS). Based on the knockout and overexpression of key hexose transporter genes and the characterisation of their promoter properties, we found that Cghxt4 and Cghxt6 play major roles in the early and late stages of fermentation, respectively, with Cghxt4 contributing most to glucose consumption. Next, subcellular localisation analysis revealed that a common mutation of two ubiquitination sites (K9 and K538) in Cghxt4 improved its MLS. Finally, we overexpressed this Cghxt4 mutant (Cghxt4.2A) using a strong promoter, PCgGAP , which resulted in a significant increase in the ethanol productivity of C. glycerinogenes in the NDSBH medium. Specifically, the recombinant strain showed 18 and 25% higher ethanol productivity than the control in two kinds of YP-NDSBH medium (YP-NDSBH1G160 and YP-NDSBH2G160 ), respectively. The hexose transporter mutant Cghxt4.2A (Cghxt4K9A,K538A ) with multiple copies and high MLS was able to significantly increase the ethanol productivity of C. glycerinogenes in NDSBH. Our results provide a promising strategy for constructing efficient strains for ethanol production.

Enhanced Tolerance of Spathaspora passalidarum to Sugarcane Bagasse Hydrolysate for Ethanol Production from Xylose.

During the pretreatment and hydrolysis of lignocellulosic biomass to obtain a hydrolysate rich in fermentable sugars, furaldehydes (furfural and hydroxymethylfurfural), phenolic compounds, and organic acids are formed and released. These compounds inhibit yeast metabolism, reducing fermentation yields and productivity. This study initially confirmed the ability of Spathaspora passalidarum to ferment xylose and demonstrated its sensibility to the inhibitors present in the hemicellulosic sugarcane bagasse hydrolysate. Then, an adaptive laboratory evolution, with progressive increments of hydrolysate concentration, was employed to select a strain more resistant to hydrolysate inhibitors. Afterward, a central composite design was performed to maximize ethanol production using hydrolysate as substrate. At optimized conditions (initial cell concentration of 30 g/L), S. passalidarum was able to produce 19.4 g/L of ethanol with productivity, yield, and xylose consumption rate of 0.8 g/L.h and 0.4 g/g, respectively, in a sugarcane bagasse hemicellulosic hydrolysate. A kinetic model was developed to describe the inhibition of fermentation by substrate and product. The values obtained for substrate saturation and inhibition constant were Ks = 120.4 g/L and Ki = 1293.4 g/L. Ethanol concentration that stops cell growth was 30.1 g/L. There was an agreement between simulated and experimental results, with a residual standard deviation lower than 6%.

Bioprocess Optimization for Enhanced Production of Bacterial Cellulase and Hydrolysis of Sugarcane Bagasse

Cellulolytic enzymes have gained considerable importance as potential candidates for the efficient bioconversion of agricultural biomass into second-generation biofuel. This study aimed to improve cellulase production from a bacterial strain isolated from cow dung. The biochemical and molecular identification was performed, and the capacity of the isolated bacterium to hydrolyze sugarcane bagasse was also determined. Response surface methodology (RSM) and one-variable-at-a-time (OVAT) designs were employed to maximize the cellulase production. The bacterium identified as Bacillus subtilis gave the highest cellulase yield at temperature of 45 °C, pH-7.0, and 72 h of incubation time. Optimization by the Plackett–Burman design consisting of 12 experimental runs at two levels of nine independent variables displayed the significant contribution of galactose (22%) and sodium nitrate (16%) in cellulase production. The responses in the form of contour and 3D plots (generated by Box-Behnken design) predicted maximum cellulase production with an activity of 0.8 U/mL/min in a medium containing 3.0 g/L carboxymethyl cellulose, 3.0 g/L galactose, 0.5 g/L sodium nitrate, 0.75 g/L KH2PO4, and 0.375 g/L MgSO4·7H2O. The experimentally deduced activity value was 0.698 U/mL/min, showing 90% validity of the predicted value and gave a threefold increase in cellulase production. The process adequately represented by the quadratic model (p < 0.0343) for all the responses was significant. Results obtained show that optimization of fermentation medium favored the increased production of cellulase by the bacterium. The bacterial isolate B. subtilis CD001 was found to be a potential candidate to hydrolyze the lignocellulosic feedstock, sugarcane bagasse.

Impact of Alkaline Pretreatment Condition on Enzymatic Hydrolysis of Sugarcane Bagasse and Pretreatment Cost.

A combined severity factor (RCSF) which is usually used to evaluate the effectiveness of hydrothermal pretreatment at above 100 °C had been developed to assess the influence of temperature, time, and alkali loading on pretreatment and enzymatic hydrolysis of lignocellulose. It is not suitable for evaluating alkaline pretreatment effectiveness at lower than 100 °C. According to the reported deducing process, this study modified the expression of [Formula: see text] as [Formula: see text] which is easier and more reasonable to assess the effectiveness of alkaline pretreatment. It showed that RCSF exhibited linear trend with lignin removal, and quadratic curve relation with enzymatic hydrolysis efficiency (EHE) at the same temperature. The EHE of alkali-treated SCB could attain the maximum value at lower RCSF, which indicated that it was not necessary to continuously enhance strength of alkaline pretreatment for improving EHE. Within a certain temperature range, the alkali loading was more important than temperature and time to influence pretreatment effectiveness and EHE. Furthermore, the contribution of temperature, time, and alkali loading to pretreatment cost which was seldom concerned was investigated in this work. The alkali loading contributed more than 70% to the pretreatment cost. This study laid the foundation of further optimizing alkaline pretreatment to reduce cost for its practical application.

Assessing oleaginous yeasts for their potentials on microbial lipid production from sugarcane bagasse and the effects of physical changes on lipid production

We assess oleaginous yeast strains from five genera to identify the strain suitable to use sugarcane bagasse as a feedstock for lipid production. Upon initial assessment in glucose or xylose media, five potential strains were chosen for further assessment in sugarcane bagasse hydrolysate. All strains exhibited improved growth and lipid production due to a buffering capacity of the hydrolysate, with R. glutinis showing the best lipid production. The yeast growth and lipid production were similar over the pH range between 5.5 and 7.5. It performed best at 30 °C. Using baffled flask at 200 rpm resulted in the highest cell dry weight of 40 g/L and 20 g/L of lipid, while using 100 rpm or normal flasks resulted in a much lower growth and lipid production. The results from this study suggest that sugarcane bagasse could be a raw material for microbial lipid production, providing the control criteria are suitable.