Production of xylanase under solid-state fermentation by <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H₂SO₄) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.     

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.     

Hemicellulose sugar recovery from steam-exploded wheat straw for microbial oil production

There are currently few successful examples of using straw hemicellulose as a carbon source in the fermentation industry. In this paper, hemicellulose hydrolysates were recovered from steam-exploded wheat straw (SEWS) and used to produce microbial oil. The effects of the steam explosion treatment conditions, the elution temperature and the ratio of elution water to SEWS on sugar recovery were examined. A broth with 3.8 g l^?1 of reducing sugar and 22.3 g l^?1 of total soluble sugars was obtained with a 10-fold excess (w/w) of water at 40 °C to wash the SEWS treated under steam explosion conditions at 200 °C for 5 min. This broth was used to produce microbial oil by the oleaginous fungus Microsphaeropsis sp., which was able to secrete xylanase to degrade oligosaccharides from straw hemicellulose and accumulate microbial oil. Under optimized conditions, the oil concentration was 2.6 g l^?1. The yield of oil from sugar consumed was 0.14 g g^?1. The microbial oil produced by this research could be used as feedstock for biodiesel production because the microbial oil was primarily composed of neutral lipids. This research establishes a novel protocol for microbial oil production from straw hemicellulose.     

Production of a cellulolytic enzyme system in mixed-culture solid-state fermentation of soybean hulls supplemented with wheat bran

Solid-state fermentation of soybean hulls supplemented with wheat bran using a co-culture of Trichoderma reesei and Aspergillus oryzae was performed. Three parameters — initial moisture content, incubation temperature, and initial pH — were optimized in culture flasks using response surface methodology. Parameter optimization was carried out with respect to filter paper activity and β-glucosidase activity in the culture. Temperature of 30 °C, pH of 5, and moisture content of 70% were found to be optimum. Optimized parameters were used for laboratory scale-up in static tray fermenters. The maximum filter paper activity of 10.7 FPU/g-ds and β-glucosidase of 10.7 IU/g-ds were obtained after 96-h incubation period in static tray fermenters in agreement with optimized activities at shake flask level. The results of static tray fermentation also highlighted the importance of mixed-culture fermentation. Both enzyme activities and volumetric productivities of enzyme produced were significantly higher in mixed-culture fermentation as compared to mono-culture static tray fermentation. Expression profile of cellulase system was characterized using SDS-PAGE and it indicated the presence of all the five major activities corresponding to β-glucosidase, CBH I, CBH II, EG I and xylanase. Enzyme broth was centrifuged and concentrated in an ultrafiltration cell. The concentrate was used for enzymatic saccharification of pretreated wheat straw and the potential of an indigenously developed enzyme concoction was reported in terms of saccharification efficiency. Pretreatment using both acid and alkali was carried out, and differences in sugar yield due to differences in composition as a result of pretreatment were reported. Results showed that alkali treatment generated higher sugars as compared to acid pretreatment. This was due to lignin removal and concentration of the cellulosic fraction. Present work showed that solid-state fermentation in a static tray bioreactor is a valuable technique for producing a system of enzymes with balanced activities that can efficiently saccharify lignocellulosic biomass like wheat straw.     

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.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

An economical approach for d-lactic acid production utilizing unpolished rice from aging paddy as major nutrient source

In order to reduce the raw material cost of d-lactic acid fermentation, the unpolished rice from aging paddy was used as major nutrient source in this study. The unpolished rice saccharificate, wheat bran powder and yeast extract were employed as carbon source, nitrogen source and growth factors, respectively. Response surface methodology (RSM) was applied to optimize the dosages of medium compositions. As a result, when the fermentation was carried out under the optimal conditions for wheat bran powder (29.10 g/l) and yeast extract (2.50 g/l), the d-lactic acid yield reached 731.50 g/kg unpolished rice with a volumetric production rate of 1.50 g/(l h). In comparison with fresh corn and polished rice, the d-lactic acid yield increased by 5.79% and 8.71%, and the raw material cost decreased by 65% and 52%, respectively, when the unpolished rice was used as a major nutrient source. These results might provide a reference for the industrial production of d-lactic acid.     

Influence of wheat cultivars on straw quality and Pleurotus ostreatus cultivation

The main raw material for Pleurotus ostreatus (oyster mushroom) cultivation is wheat straw. Estimation of straw biodegradability from 15 different spring wheat cultivars under irrigation in South Africa was determined using linear discriminant analysis to discriminate or group the 15 cultivars by combining chemical analysis and in vitro enzymatic hydrolysis. Significant differences (P < 0.01) were found between ash, nitrogen, reducing sugars, anthrone reactive-carbohydrates, water-soluble dry matter, and oyster mushroom yields. The significance of these measurements was investigated and discussed.     

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.     

Feruloyl esterase production by Aspergillus terreus CECT 2808 and subsequent application to enzymatic hydrolysis

Ferulic acid esterases (FAE) were produced by Aspergillus terreus CECT 2808 from vine trimming shoots (VTS) and corn cob. Later, the fungal extracts thus obtained were used to enzymatically release ferulic acid (FA) from both substrates. Our findings showed a higher FAE activity in the enzymatic extracts produced on corn cob (0.070 ± 0.004 U/mL). Nevertheless, the enzymatic extracts produced on VTS demonstrated a better performance for FA release from both corn cob (2.05 ± 0.01 mg/g) and VTS (0.19 ± 0.003 mg/g). This result was probably because of the higher xylanase/FAE ratio determined in VTS extract. Therefore, an additional assay was carried out by supplementing corn cob extract with a commercial xylanase to test the influence of FAE/xylanase ratio in FA release. The results revealed the relevance of the FAE/xylanase ratio for an optimal FA release.     

Bioethanol,biohydrogen and biogas production from wheat straw in a biorefinery concept

The production of bioethanol, biohydrogen and biogas from wheat straw was investigated within a biorefinery framework. Initially, wheat straw was hydrothermally liberated to a cellulose rich fiber fraction and a hemicellulose rich liquid fraction (hydrolysate). Enzymatic hydrolysis and subsequent fermentation of cellulose yielded 0.41 g-ethanol/g-glucose, while dark fermentation of hydrolysate produced 178.0 ml-H₂/g-sugars. The effluents from both bioethanol and biohydrogen processes were further used to produce methane with the yields of 0.324 and 0.381 m³/kg volatile solids (VS)_added, respectively. Additionally, evaluation of six different wheat straw-to-biofuel production scenaria showed that either use of wheat straw for biogas production or multi-fuel production were the energetically most efficient processes compared to production of mono-fuel such as bioethanol when fermenting C6 sugars alone. Thus, multiple biofuels production from wheat straw can increase the efficiency for material and energy and can presumably be more economical process for biomass utilization.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali–peracetic acid pretreatment

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)–peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 °C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 °C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali–PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali–PAA pretreated bagasse after enzymatic hydrolysis for 120 h.     

Nutrient composition,digestion and rumen fermentation in sheep of wheat straw treated with calcium oxide,sodium hydroxide and alkaline hydrogen peroxide

This study tested the effect of calcium oxide (CaO), sodium hydroxide (NaOH) and NaOH plus hydrogen peroxide (H₂O₂; AHP) on cell wall composition, digestion and fermentation of wheat straw (straw) in sheep. Treated straws were prepared by mixing straw either with water followed by dusting with CaO at 160 g kg⁻¹ DM or with a NaOH solution alone at 3 l kg⁻¹ DM to supply 80 g NaOH kg⁻¹ DM (Na) or pre-soaked with Na exactly as in the previous treatment for 27 h followed by mixing with 130 g H₂O₂ kg⁻¹ DM (AHP) for 6 h. After 14 days of storage, the treated straws and an untreated straw (U) were fed automatically every 2 h to four individually housed sheep together with a supplement in a 4×4 latin square experiment. Each kilogram supplement DM contained 422 g CP and 10.8 MJ ME. NDF (p<0.001) and hemicellulose (p<0.01) contents were significantly reduced whereas cellulose was increased (p<0.001) in treated compared to untreated straw. ADL was reduced in Ca (p<0.05) but increased (p<0.05) in Na and AHP compared with U. The rumen and total tract digestibility were significantly (p<0.001) greater in sheep fed treated compared with untreated straw. Significant differences (p<0.05) between treatments for pH, NH₃ and VFA were also observed. All treatments improved the nutritive value of straws compared with untreated through modification of cell wall with a subsequent increase in digestibility by sheep. Although the digestibility for Ca was lower than that for Na despite reduction in cell wall, its use to treat straws may be more safe and cost effective than Na. AHP was the most effective and could also improve the energy value of other low quality forages for ruminants. However, the need of AHP for a large amount of NaOH to achieve highly alkaline pH limits its farm scale application. Therefore, further studies should either consider reducing the amount of NaOH or finding alternative alkalis that are cost effective and user-friendly.     

Enzymatic hydrolysis of rice straw and corn stalks for monosugars production

Rice straw and corn stalks were used as fermentation substrate for the evaluation of cellulases activity secreted by different organisms. The substrates were pretreated with alkaline hydrogen peroxide (AHP) for 6 h at 30 and 60 °C. From the fermentation studies, rice straw and corn stalks substrates showed the highest cellulases activity after 96 h at 60 °C of pre-treatment.     

Characterizing the ensiling properties of sugarbeets with dry feedstuffs

A study was conducted to evaluate the ensiling characteristics of chopped sugarbeets with dry feedstuffs and the corresponding change in the nutritive composition of the silages with the addition of dry substrates. Pre-calculated amounts of each feedstuff were weighed individually to achieve desired proportions of each silage product and thoroughly mixed for 5 min. After mixing, the silage was distributed evenly into three 19-L buckets and sealed to provide an anaerobic environment. The treatments for this study were arranged in a 4 × 4 + 1 factorial design to determine the effects of DM level and source of dry feedstuff on the ensiling properties of sugarbeets following a 42-d fermentation period. Treatments were ensiled sugarbeets alone (250 g/kg) or based on (1) formulated silage DM concentrations of 275, 350, 425, and 500 g/kg and (2) the inclusion of dry feedstuffs (alfalfa hay, dry-rolled corn, wheat middlings, and wheat straw). Fermentation and nutritive characteristics of ensiled sugarbeets were influenced with the addition of dry substrates. A linear increase (P<0.001) in silage pH was observed with the addition of alfalfa, dry-rolled corn, wheat middlings, and wheat straw to ensiled sugarbeets. Lactic acid increased (P<0.001) with the addition of wheat middlings. Alfalfa addition to sugarbeet silage did not alter (P<0.001) lactate concentration. Concentration of lactate decreased (P=0.01) when corn was added, while wheat straw addition did not influence (P=0.37) lactate. A contrast was used to compare ensiling characteristics of sugarbeets alone (250 g/kg DM) to 350 g/kg DM (sugarbeets with dry substrates). Results indicated fermentative parameters were altered; pH increased (P<0.001) for all dry substrates while lactate was lower (P=0.003) for the sugarbeets ensiled with dry-rolled corn compared with sugarbeets ensiled alone. Alfalfa, wheat straw, and wheat middlings decreased (P<0.001) while dry-rolled corn did not affect (P=0.54) in vitro DM digestion. These results indicate the inclusion of dry feedstuffs with sugarbeets altered fermentation and with the exception of corn, decreased in vitro DM digestion. Nutrient composition and DM content of ensiled sugarbeets was altered with the addition of dry substrates.     

Enhanced ethanol production via fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC 13803

Neutralized hydrolysate and pretreated rice straw obtained from a 2% (w/v) sulfuric acid pretreatment were mixed at 10% (w/v) and subjected to simultaneous saccharification and co-fermentation (SSCF), with cellulase, β-glucosidase, and Candida tropicalis cells at 15 FPU/g-ds, 15 IU/g-ds and 1 × 10⁹ cells/ml, respectively. A 36-h SSCF with adapted cells resulted in YP/S and ethanol volumetric productivity of 0.36 g/g and 0.57 g/l/h, respectively. In addition to ethanol, insignificant amounts of glycerol and xylitol were also produced. Adapted C. tropicalis cells produced nearly 1.6 times more ethanol than non-adapted cells. Ethanol yield (Yp/s), ethanol volumetric productivity and a xylitol concentration of 0.48 g/g, 0.33 g/l/h and 0.89 g/l, respectively, were produced from fermentation of remaining hydrolysate with adapted C. tropicalis cells. The 0.20 g/g ethanol yield and 77% production efficiency from SSCF of pretreated rice straw indicate scale-up potential for the process. This study demonstrated that C. tropicalis produced ethanol and xylitol from a mixed-sugar stream, although cell adaptation affected ethanol and xylitol yields. Scanning electron microscopy indicated agglomeration of cellulose microfibrils and globular deposition of lignin in acid-pretreated rice straw.     

Production of xylanase by Thermoascus aurantiacus from sugar cane bagasse in an aerated growth fermentor

In recent years, the use of xylanases has been adopted by many processing industries, such as pulp and paper, food and textile factories. This study demonstrates that Thermoascus aurantiacus ATCC 204492 is able to produce a high level of thermostable xylanase when sugar cane bagasse is used as a substrate. Fermentations were performed in a glass-column reactor with forced aeration. A xylanase activity of 1597 U/g was attained after 10 days of solid-state fermentation. The effects of different airflow rates (0, 3.0, 6.0 l/(h g) bagasse) and initial mass of bagasse (8, 12.5, 17 g) on the production of xylanase were investigated using a statistical experimental design. The airflow rates had a significant effect on enzyme activity, whereas initial mass of bagasse had no significant effect on enzyme activity. 6 l/(h g) airflow rate and 8 g substrate resulted in the highest yields of xylanase (1597 U/g).     

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.     

In vitro and in sacco digestibility of wheat straw treated with calcium oxide and sodium hydroxide alone or with hydrogen peroxide

A series of five factorial experiments examined the effects of sodium hydroxide (NaOH) and calcium oxide (CaO) alone or together with hydrogen peroxide (H₂O₂, 27.5% w/w) at pH of about 11.5 (AHP) on in vitro (IVDMD) and in sacco (ISDMD) dry matter digestibility of wheat straw. The effects of different temperatures (20°C, 40°C and 60°C), various times (2, 3, 4, 6 and 27 h), pre-soaking, filtration and washing on the efficacy of the above levels of chemicals in improving IVDMD and ISDMD were tested in separate experiments. AHP improved IVDMD (P<0.001) of straws when pH was regulated to around 11.5 using NaOH. In contrast, AHP was ineffective or depressive (P<0.001) when CaO was used to regulate pH to around 11.5. However, CaO alone increased IVDMD to a similar extent as did NaOH. Washing, filtration and temperature were ineffective in improving the IVDMD of CaO-treated straw. AHP was most effective when 130 g H₂O₂ was applied to each kg DM of straw after soaking it with 3 l solution containing 80 g NaOH for a period of 27 h. The nutritional value of low quality forages can be enhanced for ruminants by using alkalis provided conditions as described above are maintained during alkali treatments.