Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw

Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Alkaline H(2)O(2) pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H(2)O(2), v/v; pH 11.5; 35 degrees C; 24 h) and enzymatic saccharification (45 degrees C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, beta-glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 +/- 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 degrees C in 48 h was 18.9 +/- 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 +/- 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 degrees C in 48 h.     

粗糙脉孢菌中甾醇还原酶基因erg24对麦角甾醇合成的影响

粗糙脉孢菌为子囊菌中的高效纤维素降解菌,可以直接以纤维素为营养源进行生长.本研究以粗糙脉孢菌为实验对象,利用基因工程技术构建甾醇还原酶基因erg24的高表达菌株,分别以蔗糖、麦麸、玉米秸秆、小麦秸秆、杨树木屑、水稻秸秆6种物质的粉末为碳源培养野生型粗糙脉孢菌和erg24高表达菌株,利用半定量RT-PCR测定在不同培养条...     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen.

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

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.     

Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis

The inhibitory effect of the main inhibitors (acetic acid, furfural and 5-hydroxymethylfurfural) formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis. Experimental results showed that an increase in acetic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF together with solid fraction. When using only the solid fraction from steam explosion, hydrolysates presented 0.5 g/L of acetic acid, whose fermentations have submitted promising results, providing an ethanol yield of 0.45 g ethanol/g sugars and the final ethanol concentration reached was 12.2 g/L (10.9 g ethanol/100 g DM).     

Optimization of solid substrate fermentation of wheat straw

Optimal conditions for solid substrate fermentation of wheat straw with Chaetomium cellulolyticum in laboratory-scale stationary layer fermenters were developed. The best pretreatment for wheat straw was ammonia freeze explosion, followed by steam treatment, alkali treatment, and simple autoclaving. The optimal fermentation conditions were 80% (w/w) moisture content; incubation temperature of 37 degrees C; 2% (w/w) unwashed mycelial inoculum; aeration at 0.12 L/h/g; substrate thickness of 1 to 2 cm; and duration of three days. Technical parameters for this optimized fermentation were: degree of substance utilization, 27.2%; protein yield/substrate, 0.09 g; biomass yield/bioconverted substrate, 0.40 g; degree of bioconversion of total available sugars in the substrate, 60.5%; specific efficiency of bioconversion, 70.8%; and overall efficiency of biomass production from substrate, 42.7%. Mixed culturing of Candida utilis further increased biomass production by 20%. The best mode of fermentation was a semicontinuous fed-batch fermentation where one-half of the fermented material was removed at three-day intervals and replaced by fresh substrate. In this mode, protein production was 20% higher than in batch mode, protein productivity was maintained over 12 days, and sporulation was prevented.     

秸秆降解放线菌GC的筛选及其应用基础研究

运用循环流化技术,从土壤样品中筛选出1株具有单独降解秸秆能力的菌株GC,考察了该菌的生长特性及产纤维素酶和木质素酶能力,验证了该菌对小麦秸秆的处理效果。结果表明,该菌为放线菌的左式链霉菌(Streptomyces drozdowiczii);可在LB等基础培养基中快速繁殖;纤维素内切酶活和滤纸酶活分别可达67.57 U/mL和19.69 U/mL,并且具备木质素降解能力;该菌单独处理小麦秸秆20 d的秸秆失重率为11.52%;处理产物含多种石油烃、有机醇和植物甾醇等,表明该菌在秸秆等农业面源污染物的资源化利用方面具有良好的开发应用前景。     

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Summary Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.     

Liquefaction of lignocellulose at high-solids concentrations

To improve process economics of the lignocellulose to ethanol process a reactor system for enzymatic liquefaction and saccharification at high-solids concentrations was developed. The technology is based on free fall mixing employing a horizontally placed drum with a horizontal rotating shaft mounted with paddlers for mixing. Enzymatic liquefaction and saccharification of pretreated wheat straw was tested with up to 40% (w/w) initial DM. In less than 10 h, the structure of the material was changed from intact straw particles (length 1-5 cm) into a paste/liquid that could be pumped. Tests revealed no significant effect of mixing speed in the range 3.3-11.5 rpm on the glucose conversion after 24 h and ethanol yield after subsequent fermentation for 48 h. Low-power inputs for mixing are therefore possible. Liquefaction and saccharification for 96 h using an enzyme loading of 7 FPU/g.DM and 40% DM resulted in a glucose concentration of 86 g/kg. Experiments conducted at 2%-40% (w/w) initial DM revealed that cellulose and hemicellulose conversion decreased almost linearly with increasing DM. Performing the experiments as simultaneous saccharification and fermentation also revealed a decrease in ethanol yield at increasing initial DM. Saccharomyces cerevisiae was capable of fermenting hydrolysates up to 40% DM. The highest ethanol concentration, 48 g/kg, was obtained using 35% (w/w) DM. Liquefaction of biomass with this reactor system unlocks the possibility of 10% (w/w) ethanol in the fermentation broth in future lignocellulose to ethanol plants.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Enzymatic Saccharification of Pretreated Wheat Straw by T. Reesei Cellulases and A. Niger β-Glucosidase

Three methods of wheat straw treatment (with NaOH, H₂O₂ and butylamine) and its subsequent saccharification by Trichoderma reesei cellulases and Aspergillus niger β-glucosidase are reported. The treatment of straw with NaOH for 1 h in the autoclave (120°C) caused a great loss of the hemicellulose content and a partial removal of lignin, provoking an increase of the cellulose content (from 24% to 69%) in the residue. When the straw was pre-treated with H₂O₂ at 25°C for 20 h, the relative content of cellulose in the straw increased (from 24% to 52%) due to the solubilisation of hemicellulose.

The effect of varying the hydrolysis parameters (enzyme and substrate concentration, temperature and pH) was studied in order to maximise the yield of sugars. Under the best conditions and after 48 h with a mixture of 2:1 (w/w) cellulase/β-glucosidase (with a concentration of 7 and 0.1 U ml^-1, respectively) the native, NaOH-treated and H₂O₂-treated straw material were degraded to reducing sugars for 28%, 89% and 97% respectively.     

The suitability of a faecal suspension of sheep as inocula for the estimation of utilizable crude protein of feeds by in vitro incubation

The objective of the experiments was to study the suitability of using a faecal suspension of sheep for the estimation of the utilizable crude protein (uCP) of feeds for sheep by an in vitro incubation. Twenty-four single feeds and eight feed mixtures were used as incubation substrates. In Experiment 1, the gas production after the in vitro incubation with rumen fluid or with a faecal suspension of a sheep were compared using the Hohenheim gas test. It was found that there were significant linear regression between the 24, 48 and 72 h gas production with rumen fluid and those with faecal suspensions of 35, 50, 100 and 150 g wet faeces of sheep (which were 18.6, 23.5, 52.0 and 70.5 g faeces DM, respectively) per litre McDougall's buffer (P < 0.0001). The highest regression coefficient (r2) was calculated between the gas production after inoculation with a suspension of 100 g wet faeces per litre McDougall's buffer (x, ml x 200 mg (-1) feed DM) for 48 h and the gas production after inoculation with rumen fluid (y, ml x 200 mg (-1) feed DM) for 24 h: y = 0.82 (+/- 0.07)x + 9.87 (+/-3.83), r2 = 0.82, n = 32, P < 0.0001. Based on these results, in Experiment 2 the estimation of utilizable crude protein (uCP) of feeds was compared by using the in vitro incubation technique of Zhao and Lebzien (2000), where feeds were inoculated either with rumen fluid or with a faecal suspension (100 g wet faeces of sheep, i.e. 52 g faeces DM per litre McDougall's buffer). The results indicated that there were no significant differences of the estimated uCP after inoculation with rumen fluid or the faecal suspension (P > 0.05). A significant regression was found between the uCP after incubation for 48 h with 100 g wet faeces (x, g x kg (-1) DM) and the uCP after incubation for 24 h with rumen fluid (y, g x kg(-1) DM): y = 0.95 (+/-0.10)x - 4.90 (+/-26.70), r2 = 0.75, n = 32, Although this regression was significant, the coefficient r2 was not high. Therefore, further research is needed before sheep faeces could replace rumen fluid as an inocula for the estimation of uCP by the in vitro incubation technique.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

Pollution control of swine manure and straw by conversion to Chaetomium cellulolyticum SCP feed

Swine manure has a very high pollution potential and obnoxious odor. Large farms particularly are confronted with a manure disposal problem since environmentally acceptable solutions are now required by government regulations. Swine manure was found to be a good source of supplementary nutrients to ferment wheat straw into single-cell protein (SCP) with Chaetomium cellulolyticum when 0.13g (NH₄)₂SO₄/g solid was used as an additional source of N. In batch fermentations, inhibitory effects, possibly due to soluble released from the straw during alkali or acid pretreatment, were overcome by starting the fermentation at about pH 7.0 and then reducing it to 5.0 during growth. An overall protein productivity of up to 66 mg/L h was obtained from a slurry mixture of 1% w/v solids of manure and straw. This compares favorably with 99 mg/L h when manure was fermented with glucose instead of straw as the main carbon source. A high protein productivity of 200 mg/L h was obtained from a slurry mixture containing anaerobically prefermented swine manure liquor and 1.5% w/v solids from straw. The final products of the manure and straw fermentations contained 25–30% DW crude protein and 6–20% DW cellulose and the materials were free of the original obnoxious odor and undesirable microbial contamination.     

The single-batch bioconversion of wheat straw to ethanol employing the fungusTrichoderma viride and the yeastPachysolen tannophylus

We have developed and optimized a single-batch process for the production of ethanol from wheat straw employing the fungusTrichoderma viride and the yeastPachysolen tannophylus. T. viride andAspergillus niger were examined for their ability to produce fermentable sugars from cellulosic waste materials, e.g. different kinds of straw and wood waste.T. viride most efficiently saccharified delignified wheat straw within 3 days at 25–30°C with a yield of reducing sugars of 27 g from 50 g delignified wheat straw. The resulting wheat straw hydrolysates contained xylose and glucose in a 1:1.6 molar ratio. After heat inactivation of fungal activities the sugars were converted to ethanol by the oxygen-tolerant yeastP. tannophylus in the same batch. Under the optimized conditions developed (all weights are per liter) 70 g natural untreated wheat straw (100%) yielded 50 g delignified straw (71.4%), which was saccharified to 27 g reducing sugars (38.6%). Fermentation of the sugars yielded 11.8 g ethanol (16.9%) and followed the molar equation: 1 xylose + 1.6 glucose 5.3 ethanol + 5.6 CO₂.     

Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.47, 0.38 and 1.62 g/g DW/h, with an ethanol titre of 41.07 g/L and yield of 0.42 g/g. Industrial wheat straw hydrolysate fermentation resulted in maximal specific rates of glucose and xylose consumption, and ethanol production of 2.61, 0.54 and 1.38 g/g DW/h, respectively, with an ethanol titre of 54.11 g/L and yield of 0.44 g/g. These are among the best for wheat straw hydrolysate fermentation through separate hydrolysis and cofermentation.

     

Submerged fermentation of cellolignin materials (compared with solid-state fermentation and other alternatives)

The review characterizes the submerged and solid state fermentation processes (SF and SSF) for obtaining of microbial biomass protein (MBP) on cellulose- and lignin-containing (CL) agricultural wastes (straw, wine, branches of fruit trees). There are discussed other alternatives as well. The main technological parametres are presented (protein (biomass) yield, degree of available substrate utilization, process efficiency, etc.). The use of a special stirring system of the authors' design for mycelial cultures results in an increase of CL substrate concentration in the initial nutrient medium from 2% to 8%of dry matter (DM) and in a threefold increase of the specific productivity (from 0.07 g biomass/l · h to 0.21 g biomass/l · h). The technological parametres can be increased also in the processes with substrate addition. The obtained preparations are analyzed as to their usefulness for the feeds of ruminants.     

Succinic acid production from orange peel and wheat straw by batch fermentations of Fibrobacter succinogenes S85

Succinic acid is a platform molecule that has recently generated considerable interests. Production of succinate from waste orange peel and wheat straw by consolidated bioprocessing that combines cellulose hydrolysis and sugar fermentation, using a cellulolytic bacterium, Fibrobacter succinogenes S85, was studied. Orange peel contains d-limonene, which is a well-known antibacterial agent. Its effects on batch cultures of F. succinogenes S85 were examined. The minimal concentrations of limonene found to inhibit succinate and acetate generation and bacterial growth were 0.01%, 0.1%, and 0.06% (v/v), respectively. Both pre-treated orange peel by steam distillation to remove d-limonene and intact wheat straw were used as feedstocks. Increasing the substrate concentrations of both feedstocks, from 5 to 60 g/L, elevated succinate concentration and productivity but lowered the yield. In addition, pre-treated orange peel generated greater succinate productivities than wheat straw but had similar resultant titres. The greatest succinate titres were 1.9 and 2.0 g/L for pre-treated orange peel and wheat straw, respectively. This work demonstrated that agricultural waste such as wheat straw and orange peel can be biotransformed to succinic acid by a one-step consolidated bioprocessing. Measures to increase fermentation efficiency are also discussed.