Xylitol production from non-detoxified corncob hemicellulose acid hydrolysate by Candida tropicalis

The interest on use of lignocellulose for producing chemicals is increasing as these feedstocks are low cost, renewable and widespread sources of sugars. Corncob is an attractive raw material for xylitol production due to its high content of xylan. In this study, hemicellulose hydrolysate from corncobs without detoxification was used for xylitol production by Candida tropicalis CCTCC M2012462. Compared with prepared xylose medium, xylitol production with dilute acid hydrolysate medium does not seem to influence specific xylose reductase activity. The decrease in xylitol productivity with dilute acid hydrolysate medium is a result of a lower biomass concentration and lag-phase time. It appears that biomass growth rate is essential for xylitol production. In xylitol fermentation with a low initial inhibitors concentration and substrate feeding strategy, a maximal xylitol concentration of 38.8 g l⁻¹ was obtained after 84 h of fermentation, giving a yield of 0.7 g g⁻¹ xylose and a productivity of 0.46 g l⁻¹ h⁻¹.     

Evaluation of biocomposite-based supports for immobilized-cell xylitol production compared with a free-cell system

This study was conducted to evaluate the importance of aeration in free and immobilized cell systems in an aerated bioreactor for xylitol production from an oat hull hemicellulosic hydrolysate using an integrated process. The aeration rate (AR) or oxygen mass transfer coefficient (k_La) demonstrated a significant role in controlling cell (Candida guilliermondii FTI 20037) regeneration and bioconversion performance in free and immobilized cell systems. In the free cell system, an aeration rate of 1.25 vvm corresponding to k_La of 15.8 1/h resulted in maximum values of product yield (Y_p/s: 0.87 g/g), productivity (Q_p: 0.57 g/l/h), and final xylitol concentration (P_f: 55 g/l) from the hydrolysate with a 74.5 g/l xylose concentration. However, in the aerated immobilized cell system, maximum and almost similar results (almost P_f: 54 g/l, Q_p: 0.57 g/l/h and Y_p/s: 0.84 g/g) were obtained with aeration rates from 1.25 to 1.5 vvm using composites based on polypropylene (PP) and partially delignified fiber (PDF). Composites based on acid treated fiber (ATF) containing a high amount of lignin showed some inhibitory impact on xylose uptake and xylitol formation (P_f: 47 g/l and Q_p < 0.49 g/l/h) with the optimal aeration rate of 1.5 vvm in the initial cycle of the bioconversion; this inhibition impact could be resolved in the next consecutive cycles. The surface modifier polyethyleneimine (PEI) slightly enhanced cell retention in the immobilized form on the ATF-based cell support. This investigation helps fill in the knowledge gaps existing on the integrated processing of the lignocellulosic biomass for xylitol bioproduction and biorefinery industry; however, more scale-up studies are recommended for commercialization.     

A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures

An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol production from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple process, employing two different organisms for the fermentation of the two sugars. An innovative fermentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of 30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and P. stipitis. Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z. mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P. stipitis.     

The hydrolysate of barley straw containing inhibitors can be used to produce cephalosporin C by solvent extraction using ethyl acetate

When the solvent extraction of the hydrolysate from barley straw was performed using ethyl acetate (EA), the logarithm of the partition coefficient (log P) of the phenols and furans for EA was found to be more than 1.00, which means that more than 90% of the inhibitors were removed from the hydrolysate layer. Cephalosporin C (CPC) was produced from the hydrolysate of dilute acid pretreatment (DAP) by Acremonium chrysogenum M35. A. chrysogenum M35 was cultured using the hydrolysate and the amount of CPC produced was found to be 10.35 g/L at 144 h. Also, the dry cell weight was about 101.5 g/L at 120 h. The utilization of the hydrolysate for CPC production was effective and the solvent extraction method for the removal of inhibitory substances could contribute to the biorefinery process.     

Ability of a perfluoropolymer membrane to tolerate by-products of ethanol fermentation broth from dilute acid-pretreated rice straw

A perfluoropolymer (PFP) membrane has been prepared for use in vapor permeation to separate aqueous ethanol mixtures produced from rice straw with xylose-assimilating recombinant Saccharomyces cerevisiae. PFP membranes commonly have been used for dehydration process and possess good selectivity and high permeances. The effects of by-products during dilute acid pretreatment, addition of yeast extract, and ethanol fermentation on PFP membrane performance were investigated. While feeding mixtures of ethanol (90 wt%) in water, to which individual by-products (0.1–2 g/L) were added, the PFP membrane demonstrated no clear change in permeation rate (439–507 g m⁻² h⁻¹) or separation factor (14.9–23.5) from 2 to 4 h of the process. The PFP membrane also showed no clear change in permeation rate (751–859 g m⁻² h⁻¹) or separation factor (12.5–13.8) while feeding the mixture (final ethanol conc.: 61 wt%) of ethanol and distillation of the fermentation broth using a suspended fraction of dilute acid-pretreated rice straw for 20 h. These results suggest that the PFP membrane can tolerate actual distillation liquids from ethanol fermentation broth obtained from lignocellulosic biomass pretreated with dilute acid.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species

Chitin, which is a polymer of β-(1–4) linked N-acetyl-d-glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.     

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.     

Single cell oil production from hydrolysates of inulin and extract of tubers of Jerusalem artichoke by Rhodotorula mucilaginosa TJY15a

In this study, we found that Rhodotorula mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.     

Conversion of cassava flour to fuel ethanol by sequential solid state and submerged cultures

A process for conversion of cassava flour to ethanol was developed. This involved direct inoculation of Aspergillus awamori spores into a cassava flour paste and incubation for some period during which hydrolytic enzymes are produced (solid state culture or koji production) and subsequent addition of water and yeast cells, during which there is simultaneous hydrolysis and ethanol production (submerged culture). When cassava flour alone was used for the solid state phase, the paste was very sticky, making mixing and aeration difficult. However, addition of rice bran improved the texture and enzyme production. The optima rice bran concentration, spore inoculum concentration, and duration of solid state culture before submerged culture were 20%, 6.16 × 10⁶ spores/100 g, and 2 days, respectively. Under these optimum conditions, a high ethanol concentration of 120 g/L and ethanol yield of 0.309 g-ethanol/g-cassava flour were obtained. This ethanol yield corresponds to 0.44 g-ethanol/g-cassava starch.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

Production of microbial lipid by Cryptococcus curvatus on rice straw hydrolysates

Microbial biolipids/biodiesels derived from volatile fatty acids (VFAs) can be a valuable alternative to plant oils if optimum fermentation conditions are determined. VFAs were used for cell mass and microbial lipid production by Cryptococcus curvatus. The lipid content in the cells increased up to 48% and 28% in batch cultures with the use of 20 g/L glucose and 6 g/L of VFAs as the carbon source, respectively. In this study, C. curvatus used VFAs as a carbon source via anaerobic digestion of rice straw hydrolysates. VFAs produced from rice straw resulted in yield of 0.43 g VFAs/g substrate and 40% higher specific growth rate(0.305 h⁻¹) than synthetic VFAs. The highest fatty acid composition observed was C18:1, was obtained using glucose and VFAs as the carbon source to yield a cetane number of 56–59, which is suitable for biodiesel production. The cost of microbial lipids was estimated to be 0.30–1.15 USD/L given 0–150 USD/ton of VFAs cost for a yield of 0.17 g/g of lipids. Thus, VFAs can be a suitable carbon source for economical biodiesel production.     

Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca

Corncob acid hydrolysate, detoxed by sequently boiling, overliming and activated charcoal adsorption, was used for 2,3-butanediol production by Klebsiella oxytoca ACCC 10370. The effects of acetate in hydrolysate and pH on 2,3-butanediol production were investigated. It was found that acetic acid in hydrolysate inhibited the growth of K. oxytoca while benefited the 2,3-butanediol yield. With the increase in acetic acid concentration in medium from 0 to 4 g/l, the lag phase was prolonged and the specific growth rate decreased. The acetic acid inhibition on cell growth can be alleviated by adjusting pH to 6.3 prior to fermentation and a substrate fed-batch strategy with a low initial acetic acid concentration. Under the optimum condition, a maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing sugar and a productivity of 0.59 g/h l.     

Xylitol production in a bubble column bioreactor: Influence of the aeration rate and immobilized system concentration

This work evaluated the xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a bubble column bioreactor using cells of the yeast Candida guilliermondii immobilized in calcium-alginate. The fermentation runs were performed according to a 2² full factorial design with three replicates at the center point in order to determine the effect of the variables: aeration rate (0.66–1.33 vvm) and immobilized system concentration (20–40% v/v), on the efficiency of xylose-to-xylitol conversion and on the xylitol volumetric productivity. The results indicated a significant influence of both variables on xylitol production. The highest conversion efficiency (41%) was attained using 1.33 vvm aeration rate and 40% immobilized system. Under these conditions, the volumetric productivity was 0.21 g l⁻¹ h⁻¹.     

Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass

Sequential-co-culture technique was investigated in this study for the production of bioethanol from relatively cheaper lignocellulosic biomass of Kans grass (Saccharum spontaneum). The consortium of Pichia stipitis and Zymomonas mobilis was used to develop a suitable sequential-co-culture system. The Kans grass biomass was hydrolyzed in such a manner that the two sugar fractions, xylose rich and glucose rich were generated (a separate study). The P. stipitis cells and respective fermentation media (xylose rich) were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media were fed to the same vessel. Different strategies have been followed and experiments were conducted initially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentation media and Kans grass hydrolysate media to compare the kinetic parameters. The sequential addition of cultures with their respective media and imposed process conditions, showed better utilization of total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic condition for Z. mobilis fermentation were found significant. The average ethanol yield (Y_p/s) and overall volumetric productivity (r_po) were found as 0.453 g_p/g_s and 1.580 g/l/h respectively for Kans grass hydrolysate media and 0.474 g_p/g_s and 2.901 g/l/h respectively for synthetic fermentation media.     

Two-step acid and alkaline ethanolysis/alkaline peroxide fractionation of sugarcane bagasse and rice straw for production of polylactic acid precursor

Sugarcane bagasse and rice straw were subjected to acid and alkaline ethanolysis and sequential enzymatic hydrolysis to produce glucose for lactic acid production. Influence of physico-chemical treatments using ultrasonic bath and ultrasonic probe was studied compared with mechanical stirring. The results showed that the highest glucose yield with least contamination of xylose was obtained from acid ethanolysis fractionation (5 N H₂SO₄ + 50%, v/v ethanol) when stirred at 90 °C for 4 h. Alkaline ethanolysis accomplished high amount of both glucose and xylose released, however it was not favorable substrate for homofermentative lactic acid bacteria. In order to enhance enzymatic hydrolysis of acid ethanolysis fractionated samples, lignin was subsequently removed by the second step alkaline/peroxide delignification. The maximum lactic acid was obtained at 23.6 ± 0.2 g/L from Lactobacillus casei fermentation after 72 h when hydrolysate from two-step acid hydrolysis and alkaline/peroxide fractionated sugarcane bagasse containing 24.6 g/L initial glucose concentration was used as substrate.     

High efficiency bioethanol production from barley straw using a continuous pretreatment reactor

We developed a new pretreatment process for producing high-efficiency bioethanol from a lignocellulosic biomass. Barley straw was pretreated with sodium hydroxide in a twin-screw extruder for continuous pretreatment. The biomass to ethanol ratio (BTER) for optimal pretreatment conditions was evaluated by response surface methodology. Simultaneous saccharification and fermentation (SSF) was conducted to investigate the BTER with 30 FPU/g cellulose of enzyme and 7% (v/v) yeast (Saccharomyces cerevisiae CHY 1011) using 10% (w/v) pretreated biomass under various pretreatment conditions. The maximum BTER was 73.00% under optimal pretreatment conditions (86.61 °C, 0.58 M, and 84.79 mL/min for temperature, sodium hydroxide concentration, and solution flow rate, respectively) and the experimental BTER was 70.01 ± 0.59%. SSF was performed to investigate the optimal enzyme and biomass dosage. As a result, maximum ethanol concentration and ethanol yield were 46.00 g/L and 77.36% at a loading pretreated biomass of 20% with 30 FPU/g cellulose of the enzyme dosage for barley straw to bioethanol. These results are a significant contribution to the production of bioethanol from barley straw.     

Decanter cake waste as a renewable substrate for biobutanol production by Clostridium beijerinckii

The aim of this study was to determine if decanter cake waste from a palm oil mill could be used as a renewable substrate for biobutanol production. Decanter cake waste was first hydrolyzed to fermentable sugars by nitric acid and detoxified by activated-charcoal. The detoxified hydrolysate supplemented with whey protein and ammonium sulfate as cheap nitrogen sources, was used for butanol production by growing cells of Clostridium beijerinckii. The detoxified hydrolysate was also used as a co-substrate for direct conversion of butyric acid to butanol in a nitrogen-free medium. By these two steps, C. beijerinckii produced 3.42 g/L of butanol with a yield of 0.28 C-mol butanol/C-mol carbon in the first step and produced 6.94 g/L of butanol with a yield of 0.47 C-mol butanol/C-mol carbon in the second step. This study has showed that decanter cake waste could serve as a low-cost substrate for biobutanol production.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.