First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Relation of xylitol formation and lignocellulose degradation in yeast.

One of the critical steps of the biotechnological production of xylitol from lignocellulosic biomass is the deconstruction of the plant cell wall. This step is crucial to the bioprocess once the solubilization of xylose from hemicellulose is allowed, which can be easily converted to xylitol by pentose-assimilating yeasts in a microaerobic environment. However, lignocellulosic toxic compounds formed/released during plant cell wall pretreatment, such as aliphatic acids, furans, and phenolic compounds, inhibit xylitol production during fermentation, reducing the fermentative performance of yeasts and impairing the bioprocess productivity. Although the toxicity of lignocellulosic inhibitors is one of the biggest bottlenecks of the biotechnological production of xylitol, most of the studies focus on how much xylitol production is inhibited but not how and where cells are affected. Understanding this mechanism is important in order to develop strategies to overcome lignocellulosic inhibitor toxicity. In this mini-review, we addressed how these inhibitors affect both yeast physiology and metabolism and consequently xylose-to-xylitol bioconversion. In addition, this work also addresses about cellular adaptation, one of the most relevant strategies to overcome lignocellulosic inhibitors toxicity, once it allows the development of robust and tolerant strains, contributing to the improvement of the microbial performance against hemicellulosic hydrolysates toxicity. KEY POINTS: • Impact of lignocellulosic inhibitors on the xylitol production by yeasts • Physiological and metabolic alterations provoked by lignocellulosic inhibitors • Cell adaptation as an efficient strategy to improve yeast's robustness.

Genetically Engineering Escherichia coli to Produce Xylitol from Corncob Hydrolysate without Lime Detoxification.

Before fermentation with hemicellulosic hydrolysate as a substrate, it is generally necessary to detoxify the toxic substances that are harmful to microorganism growth. Cyclic AMP receptor protein (CRP) is a global regulator, and mutation of its key sites may have an important impact on E. coli virulence tolerance. Using corncob hydrolysate without ion-exchange or lime detoxification as the substrate, shake flask fermentation experiments showed that CRP mutant IS5-dG (I112L, T127G, A144T) produced 18.4 g/L of xylitol within 34 h, and the OD600 was 9.7 at 24 h; these values were 41.5% and 21.3% higher than those of the starting strain, IS5-d, respectively. This mutant produced 82 g/L of xylitol from corncob hydrolysate without ion-exchange or lime detoxification during fed-batch fermentation in a 15-L bioreactor, with a productivity of 1.04 g/L/h; these values were 173% and 174% higher than the starting strain, respectively. To our knowledge, this is the highest xylitol concentration and productivity produced by microbial fermentation using completely non-detoxified hemicellulosic hydrolysate as the substrate to date. This study also showed that alkali neutralization, high temperature sterilization, and fermentation of the hydrolysate had important effects on the xylose loss rate and xylitol production.

Metabolomic profiling of Spathaspora passalidarum fermentations reveals mechanisms that overcome hemicellulose hydrolysate inhibitors.

Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production. Here, we used ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and MetaboAnalyst 4.0 for analysis of MS data to examine the changes in the metabolic profile of the yeast Spathaspora passalidarum during early fermentation of hemicellulosic hydrolysates containing high or low levels of inhibitors (referred to as control hydrolysate or CH and strategy hydrolysate or SH, respectively). During fermentation of SH, the maximum ethanol production was 16 g L-1 with a yield of 0.28 g g-1 and productivity of 0.22 g L-1 h-1, whereas maximum ethanol production in CH fermentation was 1.74 g L-1 with a yield of 0.11 g g-1 and productivity of 0.01 g L-1 h-1. The high level of inhibitors in CH induced complex physiological and biochemical responses related to stress tolerance in S. passalidarum. This yeast converted compounds with aldehyde groups (hydroxymethylfurfural, furfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) into less toxic compounds, and inhibitors were found to reduce cell viability and ethanol production. Intracellularly, high levels of inhibitors altered the energy homeostasis and redox balance, resulting in lower levels of ATP and NADPH, while that of glycolytic, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways were the most affected, being the catabolism of glucogenic amino acids, the main cellular response to inhibitor-induced stress. This metabolomic investigation reveals interesting targets for metabolic engineering of ethanologenic yeast strains tolerant against multiple inhibitors for E2G production. KEY POINTS: • Inhibitors in the hydrolysates affected the yeast's redox balance and energy status. • Inhibitors altered the glycolytic, pentose phosphate, TCA cycle and amino acid pathways. • S. passalidarum converted aldehyde groups into less toxic compounds.

Particle size influence on the composition of sugars in corncob hemicellulose hydrolysate for xylose fermentation by Meyerozyma caribbica.

The xylitol production was performed with acidophilic Meyerozyma caribbica. The particle size of 0.02 ± 0.01 to 0.1 ± 0.05 mm was rich in glucose (12.0 ± 0.5 g/L), whereas 0.5 ± 0.25 to 2.0 ± 0.5 mm had a high content of xylose (8.0 ± 0.5 g/L). The xylitol production in the synthetic, non-detoxified and detoxified hydrolysate media was studied (50 ± 0.5 g/L) using 10% v/v non - induced cells of M. caribbica for 120 h. At the end of fermentation with the specific growth rate of 0.056 ± 0.01 (µ), xylitol yields of 45.00 ± 1.00%, 10.00 ± 1.00% and 54.00 ± 1.00% were obtained. The detoxification of the hydrolysate prepared using an identified corncob particle size of 0.5 ± 0.25 to 2.0 ± 0.5 mm could be used as the prospective pretreatment process for ecofriendly and industrial scale production of xylitol with M. caribbica.

Screening and Growth Characterization of Non-conventional Yeasts in a Hemicellulosic Hydrolysate.

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h-1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h-1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL-1 and 1.72 ± 0.04 mgL-1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

Improvement of enzymatic bioxylitol production from sawdust hemicellulose: optimization of parameters.

Enzymatic production of bioxylitol from lignocellulosic biomass (LCB) provides a promising alternative to both chemical and fermentative routes. This study aimed to assess the impacts of catalytic variables on bioxylitol production from wood sawdust using xylose reductase (XR) enzyme and to optimize the bioprocess. Enzyme-based xylitol production was carried out in batch cultivation under various experimental conditions to obtain maximum xylitol yield and productivity. The response surface methodology (RSM) was followed to fine-tune the most significant variables such as reaction time, temperature, and pH, which influence the synthesis of bioxylitol from sawdust hydrolysate and to optimize them. The optimum time, temperature, and pH became were 12.25 h, 35 °C, and 6.5, respectively, with initial xylose 18.8 g/L, NADPH 2.83 g/L, XR 0.027 U/mg, and agitation 100 rpm. The maximum xylitol production was attained at 16.28 g/L with a yield and productivity of 86.6% (w/w) and 1.33 g/L·h, respectively. Optimization of catalytic parameters using sequential strategies resulted in 1.55-fold improvement in overall xylitol production. This study explores a novel strategy for using sawdust hemicellulose in bioxylitol production by enzyme technology.

Optimization of activated charcoal detoxification and concentration of chestnut shell hydrolysate for xylitol production.

OBJECTIVES: To increase xylose concentration of the chestnut shell hemicellulosic hydrolysate with an acceptable phenolic compound level in order to enhance xylitol production by Candida tropicalis M43. RESULTS: The xylose concentration and total phenolic compound concentration of the hydrolysate were obtained as 33.68 g/L and 77.38 mg gallic acid equivalent/L, respectively by optimization of detoxification parameters and concentration level (60 °C, 115 min contact time, 5.942% (w/v) dosage of activated charcoal, 120 strokes/min shaking rate and 0.2 volume ratio). Xylitol production was achieved in the hydrolysate by using Candida tropicalis M43. The maximum xylitol concentration was 6.30 g/L and productivity, yield and percentage of substrate conversion were calculated as 0.11 g/L h, 19.13% and 97.79%, respectively. In addition, the chestnut shell hydrolysate fortified with xylose and the maximum xylitol concentration increased to 18.08 g/L in the hydrolysate-based medium containing 80 g/L xylose. CONCLUSIONS: Optimizing detoxification conditions with concentration level was found to be useful for enhancing xylitol production. In addition, fortification of the hydrolysate caused a three fold increase in maximum xylitol concentration.

Hydrolysis of Corncob Hemicellulose by Solid Acid Sulfated Zirconia and Its Evaluation in Xylitol Production.

Corncob is an abundant agricultural residue containing high content of hemicellulose. In this paper, the hemicellulosic hydrolysate was prepared from the hydrolysis of corncob using the solid acid sulfated zirconia as a catalyst. According to response surface analysis experiments, the optimum conditions for preparing hemicellulosic hydrolysate catalyzed by sulfated zirconia were determined as follows: solid (sulfated zirconia)-solid (corncob) ratio was 0.33, solid (corncob)-liquid (water) ratio was 0.09, temperature was 153 °C, and time was 5.3 h. Under the optimized conditions, the soluble sugar concentration was 30.12 g/L with a yield of 033 g/g corncob. Subsequently, xylitol production from the resulting hemicellulosic hydrolysate was demonstrated by Candida tropicalis, and results showed that the yield of xylitol from the hemicellulosic hydrolysate could be significantly improved on a basis of decolorization and detoxification before fermentation. The maximum yield of xylitol from the hemicellulosic hydrolysate fermented by C. tropicalis was 0.76 g/g. This study provides a new attempt for xylitol production from the hemicellulosic hydrolysate.

Valorization of apple pomace using bio-based technology for the production of xylitol and 2G ethanol.

Apple pomace was studied as a raw material for the production of xylitol and 2G ethanol, since this agroindustrial residue has a high concentration of carbohydrate macromolecules, but is still poorly studied for the production of fermentation bioproducts, such as polyols. The dry biomass was subjected to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and fermented. The hydrolyzate after characterization was submitted to submerged fermentations, which were carried out in Erlenmeyer flasks using, separately, the yeasts Candida guilliermondii and Kluyveromyces marxianus. High cellulose (32.62%) and hemicellulose (23.60%) contents were found in this biomass, and the chemical hydrolysis yielded appreciable quantities of fermentable sugars, especially xylose. Both yeasts were able to metabolize xylose, but Candida guilliermondii produced only xylitol (9.35 g L-1 in 96 h), while K. marxianus produced ethanol as the main product (10.47 g L-1 in 24 h) and xylitol as byproduct (9.10 g L-1 xylitol in 96 h). Maximum activities of xylose reductase and xylitol dehydrogenase were verified after 24 h of fermentation with C. guilliermondii (0.23 and 0.53 U/mgprot, respectively) and with K. marxianus (0.08 e 0.08 U/mgprot, respectively). Apple pomace has shown potential as a raw material for the fermentation process, and the development of a biotechnological platform for the integrated use of both the hemicellulosic and cellulosic fraction could add value to this residue and the apple production chain.

Combination of the CRP mutation and ptsG deletion in Escherichia coli to efficiently synthesize xylitol from corncob hydrolysates.

The biotechnology-based production of xylitol has received widespread attention because it can use cheap and renewable lignocellulose as a raw material, thereby decreasing costs and pollution. The simultaneous use of various sugars in lignocellulose hydrolysates is a primary prerequisite for efficient xylitol production. In this study, a ΔptsG and crp* combinatorial strategy was used to generate Escherichia coli W3110 strain IS5-dI, which completely eliminated glucose repression and simultaneously used glucose and xylose. This strain produced 164 g/L xylitol from detoxified corncob hydrolysates during a fed-batch fermentation in a 15-L bioreactor, which was 14.7% higher than the xylitol produced by the starting strain, IS5-d (143 g/L), and the xylitol productivity was 3.04 g/L/h. These results represent the highest xylitol concentration and productivity reported to date for bacteria and hemicellulosic sugars. Additionally, strain IS5-dG, which differs from IS5-dI at CRP amino acid residue 127 (I127G), was tolerant to the toxins in corncob hydrolysates. In a fed-batch fermentation experiment involving a 15-L bioreactor, IS5-dG produced 137 g/L xylitol from non-detoxified corncob hydrolysates, with a productivity of 1.76 g/L/h. On the basis of these results, we believe that IS5-dI and IS5-dG may be useful host strains for the industrial-scale production of xylitol from detoxified or non-detoxified corncob hydrolysates.

Efficient production of xylitol by the integration of multiple copies of xylose reductase gene and the deletion of Embden-Meyerhof-Parnas pathway-associated genes to enhance NADPH regeneration in Escherichia coli.

Cofactor supply is a rate-limiting step in the bioconversion of xylose to xylitol. Strain WZ04 was first constructed by a novel simultaneous deletion-insertion strategy, replacing ptsG, xylAB and ptsF in wild-type Escherichia coli W3110 with three mutated xylose reductase genes (xr) from Neurospora crassa. Then, the pfkA, pfkB, pgi and/or sthA genes were deleted and replaced by xr to investigate the influence of carbon flux toward the pentose phosphate pathway and/or transhydrogenase activity on NADPH generation. The deletion of pfkA/pfkB significantly improved NADPH supply, but minimally influenced cell growth. The effects of insertion position and copy number of xr were examined by a quantitative real-time PCR and a shake-flask fermentation experiment. In a fed-batch fermentation experiment with a 15-L bioreactor, strain WZ51 produced 131.6 g L-1 xylitol from hemicellulosic hydrolysate (xylitol productivity: 2.09 g L-1 h-1). This study provided a potential approach for industrial-scale production of xylitol from hemicellulosic hydrolysate.

Xylitol production by Debaryomyces hansenii and Candida guilliermondii from rapeseed straw hemicellulosic hydrolysate.

This study evaluated the possibility of using rapeseed straw hemicellulosic hydrolysate as a fermentation medium for xylitol production. Two yeast strains, namely Debaryomyces hansenii and Candida guilliermondii, were used for this bioconversion process and their performance to convert xylose into xylitol was compared. Additionally, different strategies were evaluated for the hydrolysate detoxification before its use as a fermentation medium. Assays in semi-defined media were also performed to verify the influence of hexose sugars on xylose metabolism by the yeasts. C. guilliermondii exhibited higher tolerance to toxic compounds than D. hansenii. Not only the toxic compounds present in the hydrolysate affected the yeast's performance, but glucose also had a negative impact on their performance. It was not necessary to completely eliminate the toxic compounds to obtain an efficient conversion of xylose into xylitol, mainly by C. guilliermondii (YP/S=0.55g/g and 0.45g/g for C. guilliermondii and D. hansenii, respectively).

Xylitol production from lignocellulosic whole slurry corn cob by engineered industrial Saccharomyces cerevisiae PE-2.

In this work, the industrial Saccharomyces cerevisiae PE-2 strain, presenting innate capacity for xylitol accumulation, was engineered for xylitol production by overexpression of the endogenous GRE3 gene and expression of different xylose reductases from Pichia stipitis. The best-performing GRE3-overexpressing strain was capable to produce 148.5 g/L of xylitol from high xylose-containing media, with a 0.95 g/g yield, and maintained close to maximum theoretical yields (0.89 g/g) when tested in non-detoxified corn cob hydrolysates. Furthermore, a successful integrated strategy was developed for the production of xylitol from whole slurry corn cob in a presaccharification and simultaneous saccharification and fermentation process (15% solid loading and 36 FPU) reaching xylitol yield of 0.93 g/g and a productivity of 0.54 g/L·h. This novel approach results in an intensified valorization of lignocellulosic biomass for xylitol production in a fully integrated process and represents an advance towards a circular economy.

Production of bioethanol in sugarcane bagasse hemicellulosic hydrolysate by Scheffersomyces parashehatae, Scheffersomyces illinoinensis and Spathaspora arborariae isolated from Brazilian ecosystems.

AIMS: This study aimed to evaluate new d-xylose-fermenting yeasts from Brazilian ecosystems for the production of second-generation ethanol. METHODS AND RESULTS: d-xylose-fermenting yeasts isolated from rotting wood and wood-boring insects were identified as the species Scheffersomyces parashehatae, Scheffersomyces illinoinensis, Spathaspora arborariae and Wickerhamomyces rabaulensis. Among the yeasts tested, those of Sc. parashehatae exhibited the highest ethanol production when cultivated on complex medium (Yp/set  = 0·437 g g-1 ). Sheffersomyces illinoinensis and Sp. arborariae showed similar ethanol production in this assay (Yp/set up to 0·295 g g-1 ). In contrast, in sugarcane bagasse hemicellulosic hydrolysate, Sc. parashehatae and Sc. illinoinensis exhibited similar ethanol production (Yp/set up to 0·254 g g-1 ), whereas Sp. arborariae showed the lowest results (peak Yp/set  = 0·160 g g-1 ). Wickerhamomyces rabaulensis exhibited a remarkable xylitol production (Yp/sxyl  = 0·681  g g-1 ), but producing low levels of ethanol (Yp/set  = 0·042 g g-1 ). CONCLUSIONS: The novel d-xylose-fermenting yeasts showed promising metabolic characteristics for use in fermentation processes for second-generation ethanol production, highlighting the importance of bioprospecting research of micro-organisms for biotechnological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study widens the scope for future researches that may examine the native yeasts presented, as limited studies have investigated these species previously.

Xylitol production by genetically modified industrial strain of Saccharomyces cerevisiae using glycerol as co-substrate.

Xylitol is commercially used in chewing gum and dental care products as a low calorie sweetener having medicinal properties. Industrial yeast strain of S. cerevisiae was genetically modified to overexpress an endogenous aldose reductase gene GRE3 and a xylose transporter gene SUT1 for the production of xylitol. The recombinant strain (XP-RTK) carried the expression cassettes of both the genes and the G418 resistance marker cassette KanMX integrated into the genome of S. cerevisiae. Short segments from the 5' and 3' delta regions of the Ty1 retrotransposons were used as homology regions for integration of the cassettes. Xylitol production by the industrial recombinant strain was evaluated using hemicellulosic hydrolysate of the corn cob with glucose as the cosubstrate. The recombinant strain XP-RTK showed significantly higher xylitol productivity (212 mg L-1 h-1) over the control strain XP (81 mg L-1 h-1). Glucose was successfully replaced by glycerol as a co-substrate for xylitol production by S. cerevisiae. Strain XP-RTK showed the highest xylitol productivity of 318.6 mg L-1 h-1 and titre of 47 g L-1 of xylitol at 12 g L-1 initial DCW using glycerol as cosubstrate. The amount of glycerol consumed per amount of xylitol produced (0.47 mol mol-1) was significantly lower than glucose (23.7 mol mol-1). Fermentation strategies such as cell recycle and use of the industrial nitrogen sources were demonstrated using hemicellulosic hydrolysate for xylitol production.

Xylitol production and furfural consumption by a wild type Geotrichum sp

Background: Xylitol is a five carbons polyol with promising medical applications. It can be obtained from chemical D-xylose reduction or by microbial fermentation of Sugarcane Bagasse Hemicellulosic Hydrolysate. For this last process, some microbial inhibitors, as furfural, constitute severe bottleneck. In this case, the use of strains able to produce xylitol simultaneously to furfural neutralization is an interesting alternative. A wild-type strain of Geotrichum sp. was detected with this ability, and its performance in xylitol production and furfural consumption was evaluated. Furthermore, were analyzed its degradation products. Results: Geotrichum sp. produced xylitol from D-xylose fermentation with a yield of 0.44 g-g-1. Furfural was fully consumed in fermentation assay and when provided in the medium until concentration of 6 g-L-1. The furfural degradation product is not an identified molecule, presenting a molecular weight of 161 g-mol-1, an uncommon feature for the microbial metabolism of this product. Conclusion: This strain presents most remarkable potential in performing furfural consumption simultaneous to xylitol production. Subsequent efforts must be employed to establish bioprocess to simultaneous detoxification and xylitol production by Geotrichum sp.

Evaluation of xylitol production using corncob hemicellulosic hydrolysate by combining tetrabutylammonium hydroxide extraction with dilute acid hydrolysis.

In this paper, we produced hemicellulosic hydrolysate from corncob by tetrabutylammonium hydroxide (TBAH) extraction and dilute acid hydrolysis combined, further evaluating the feasibility of the resultant corncob hemicellulosic hydrolysate used in xylitol production by Candida tropicalis. Optimized conditions for corncob hemicellulose extraction by TBAH was obtained via response surface methodology: time of 90min, temperature of 60°C, liquid/solid ratio of 12 (v/w), and TBAH concentration of 55%, resulting in a hemicellulose extraction of 80.07% under these conditions. The FT-IR spectrum of the extracted corncob hemicellulose is consistent with that of birchwood hemicellulose and exhibits specific absorbance of hemicelluloses at 1380, 1168, 1050, and 900cm(-1). In addition, we found that C. tropicalis can ferment the resulting corncob hemicellulosic hydrolysate with pH adjustment and activated charcoal treatment leading to a high xylitol yield and productivity of 0.77g/g and 2.45g/(Lh), respectively.

Butanol production by a Clostridium beijerinckii mutant with high ferulic acid tolerance.

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production.

Enhanced xylitol production using immobilized Candida tropicalis with non-detoxified corn cob hemicellulosic hydrolysate.

This study reports an industrially applicable non-sterile xylitol fermentation process to produce xylitol from a low-cost feedstock like corn cob hydrolysate as pentose source without any detoxification. Different immobilization matrices/mediums (alginate, polyvinyl alcohol, agarose gel, polyacrylamide, gelatin, and κ-carrageenan) were studied to immobilize Candida tropicalis NCIM 3123 cells for xylitol production. Amongst this calcium alginate, immobilized cells produced maximum amount of xylitol with titer of 11.1 g/L and yield of 0.34 g/g. Hence, the process for immobilization using calcium alginate beads was optimized using a statistical method with sodium alginate (20, 30 and 40 g/L), calcium chloride (10, 20 and 30 g/L) and number of freezing-thawing cycles (2, 3 and 4) as the parameters. Using optimized conditions (calcium chloride 10 g/L, sodium alginate 20 g/L and 4 number of freezing-thawing cycles) for immobilization, xylitol production increased significantly to 41.0 g/L (4 times the initial production) with corn cob hydrolysate as sole carbon source and urea as minimal nutrient source. Reuse of immobilized biomass showed sustained xylitol production even after five cycles.