Enzyme production by wood-rot and soft-rot fungi cultivated on corn fiber followed by simultaneous saccharification and fermentation

This research aims at developing a biorefinery platform to convert lignocellulosic corn fiber into fermentable sugars at a moderate temperature (37 °C) with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum), and soft-rot (Trichoderma reesei) fungi were used for in situ enzyme production to hydrolyze cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Solid-substrate fermentation of corn fiber by either white- or brown-rot fungi followed by simultaneous saccharification and fermentation (SSF) with coculture of Saccharomyces cerevisiae has shown a possibility of enhancing wood rot saccharification of corn fiber for ethanol fermentation. The laboratory-scale fungal saccharification and fermentation process incorporated in situ cellulolytic enzyme induction, which enhanced overall enzymatic hydrolysis of hemi/cellulose components of corn fiber into simple sugars (mono-, di-, and trisaccharides). The yeast fermentation of the hydrolyzate yielded 7.8, 8.6, and 4.9 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest ethanol yield (8.6 g ethanol per 100 g initial corn fiber) is equivalent to 35% of the theoretical ethanol yield from starch and cellulose in corn fiber. This research has significant commercial potential to increase net ethanol production per bushel of corn through the utilization of corn fiber. There is also a great research opportunity to evaluate the remaining biomass residue (enriched with fungal protein) as animal feed.     

Solid-state fermentation of soybean and corn processing coproducts for potential feed improvement

Two agro-industrial coproducts, soybean cotyledon fiber and distiller's dried grains with solubles (DDGS), were used as substrates to evaluate the effect of coculturing three different fungi, Aspergillus oryzae , Trichoderma reesei , and Phanerochaete chrysosporium , on enzyme production by solid-state fermentation (SSF). When soybean fiber was used as the substrate, a maximum xylanase activity of 757.4 IU/g and a cellulase activity of 3.2 IU/g were achieved with the inoculation and incubation of T. reesei and P. chrysosporium for 36 h, followed by A. oryzae for an additional 108 h. This inoculation scheme also resulted in the highest xylanase activity of 399.2 IU/g compared to other fungi combinations in the SSF of DDGS. A large-scale SSF by this fungus combination produced fermented products that had xylanase and cellulase activities of 35.9-57.0 and 0.4-1.2 IU/g, respectively. These products also had 3.5-15.1% lower fiber and 1.3-4.2% higher protein contents, suggesting a potential feed quality improvement.     

蒸汽爆破玉米芯水解液脱毒及其发酵生产燃料丁醇

为探究以玉米芯为原料生产燃料丁醇的最佳工艺技术,该研究对蒸汽爆破玉米芯水解液的脱毒方式及脱毒后的水解液的丙酮丁醇发酵进行了研究。结果表明:D301树脂对玉米芯水解液进行脱毒的综合效果最好,甲酸、乙酸和总酚的脱除率分别达到60%、46.04%和56.31%,香草醛脱除率为100%,对糠醛和5-HMF的脱除率分别达到了82.95%和87.52%;同时总糖的损失率为4.38%。D301树脂脱毒后的水解液经C.acetobutylicum CICC 8016发酵丁醇和总溶剂产量分别为5.2和7.5 g/L,葡萄糖和总糖的利用率分别达到100%和73.67%。当D301树脂脱毒的玉米芯水解液初始糖的质量浓度为50 g/L时,丁醇和总溶剂(丙酮、丁醇和乙醇)的质量浓度分别达到最大9.7和14.6 g/L。该研究为利用玉米芯工业化生产燃料丁醇提供了可靠的技术支持。     

Optimization of simultaneous saccharification and fermentation for the production of ethanol from lignocellulosic biomass

Simultaneous saccharification and fermentation (SSF) of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized using cellulase from Trichoderma reesei QM-9414 (Celluclast from Novo) and Saccharomyces cerevisiae NRRL-Y-132 cells. Response surface methodology (RSM) and a three-level four-variable design were employed to evaluate the effects of SSF process variables such as cellulase concentration (20-100 FPU/g of substrate), substrate concentration (5-15% w/v), incubation time (24-72 h), and temperature (35-45 degrees C) on ethanol production efficiency. Cellulase and substrate concentrations were found to be the most significant variables. The optimum conditions arrived at are as follows: cellulase = 100 FPU/g of substrate, substrate = 15% (w/v), incubation time = 57.2 h, and temperature = 38.5 degrees C. At these conditions, the predicted ethanol yield was 3.02% (w/v) and the actual experimental value was 3.0% (w/v).     

前处理对玉米秸秆蒸汽爆破效果的影响

为提高纤维乙醇生产过程中秸秆的预处理效果,该文研究了水预浸和CaO前处理对蒸汽爆破和酶解糖化的影响,并利用场发射扫描电镜(FE-SEM)、X-射线衍射仪(XRD)及傅里叶红外光谱仪(FTIR)对其影响机制进行了分析。结果表明:玉米秸秆经30%水(水料质量比30:100)预浸5d、经2%CaO(CaO与秸秆质量比2:100)处理3d或经30%水和2%CaO协同处理1d后再进行蒸汽爆破均可显著提高蒸汽爆破对木质素的降解,降解率由单独蒸汽爆破的20.6%分别提高到27.8%、35.1%和30.9%。玉米秸秆经3种复合预处理和酶解糖化后总糖浓度分别为3.81、3.59和3.46g/100mL,糖得率分别为42.2%、39.8%和38.3%,比单独蒸汽爆破预处理分别提高了23.7%、16.6%和12.3%。水预浸或CaO复合蒸汽爆破预处理后秸秆结构破坏严重,秸秆相对结晶度由单独蒸汽爆破的42.6%分别提高到47.0%和54.5%。水浸泡或CaO前处理可提高蒸汽爆破预处理效果和后期糖化效果,且所用试剂价格低廉,可以应用推广。     

蒸汽爆破预处理和微生物发酵对玉米秸秆降解率的影响

为了提高玉米秸秆的利用效率,首先对玉米秸秆进行蒸汽爆破预处理（压力2.5 Mpa,维压200 s）,然后再进行米曲霉发酵,研究物理和生物学处理对秸秆成分及相关酶活变化的影响。结果表明,蒸汽爆破使秸秆中纤维素、半纤维素和木质素的降解率分别达到8.47%、50.45% 和36.65% （p＜0.05）。爆破预处理的秸秆再经米曲霉发酵6 d后,秸秆中纤维素和半纤维素的降解率分别为27.89%和64.80% （p＜0.05）,发酵秸秆中的滤纸酶、羧甲基纤维素酶、淀粉酶和蛋白酶活力分别达到335.10、1138.92、1954.20和201.99 U/g。爆破预处理后进行米曲霉发酵,对于提高玉米秸秆的降解率具有非常重要的意义。     

Hydrolysis and Fermentation of Pericarp and Endosperm Fibers Recovered from Enzymatic Corn Dry‐Grind Process

A modified dry‐grind corn process has been developed that allows recovery of both pericarp and endosperm fibers as coproducts at the front end of the process before fermentation. The modified process is called enzymatic milling (E‐Mill) dry‐grind process. In a conventional dry‐grind corn process, only the starch component of the corn kernel is converted into ethanol. Additional ethanol can be produced from corn if the fiber component can also be converted into ethanol. In this study, pericarp and endosperm fibers recovered in the E‐Mill dry‐grind process were evaluated as a potential ethanol feedstock. Both fractions were tested for fermentability and potential ethanol yield. Total ethanol yield recovered from corn by fermenting starch, pericarp, and endosperm fibers was also determined. Results show that endosperm fiber produced 20.5% more ethanol than pericarp fiber on a g/100 g of fiber basis. Total ethanol yield obtained by fermenting starch and both fiber fractions was 0.370 L/kg compared with ethanol yield of 0.334 L/kg obtained by fermenting starch alone.     

酶法复合脱毒提高玉米秸秆水解液丁醇发酵效率

利用玉米秸秆发酵产丁醇在生物质转化领域具有明显优势。为解除玉米秸秆水解液中多种有毒物质对微生物生长的抑制及对发酵产量的影响,该研究摒除常用的理化脱毒法,选择高效环保的酶法脱毒以实现溶剂高产。研究结果表明:通过优化漆酶和甲酸脱氢酶添加量以去除水解液中酚类和甲酸,单独添加漆酶5 U/m L、甲酸脱氢酶1 U/m L,水解液发酵的丙酮-丁醇-乙醇(acetone-butanol-ethanol,ABE,总溶剂)产量分别为1.03和1.11 g/L。再在活性炭的辅助下形成高效酶法复合脱毒体系,经复合脱毒处理的水解液发酵后丁醇产量达2.90 g/L,总溶剂ABE产量达到4.4 g/L,比未作处理的对照组发酵产量高出约5倍,实现了生物质的高效转化。可为玉米秸秆水解液发酵生产燃料丁醇提供参考。     

不同糖质原料和菌株固态发酵制取乙醇的特性比较

利用固态发酵技术直接将糖质原料转化为乙醇,过程简单且糖利用率高,已经成为近年来的一个研发热点。为寻求最适原料及菌种,该文对不同原料和菌株进行固态发酵产乙醇特性进行了比较研究:2种菌株为实验室自行筛选酵母菌株TSH和市场上常见的安琪酿酒酵母菌株AGL;3种糖料作物为甘蔗、甜菜和甜高粱。结果表明,无论对于TSH或者AGL,投入相同质量的原料,由于可发酵总糖初始含量的不同,最终乙醇产量最高为甜菜,甜高粱次之,甘蔗最低。为消除原料初始糖含量不同造成的影响,该文比较了消耗单位质量糖分所生成的乙醇质量,发现对于2种菌株甜高粱都具有最高的乙醇/糖转化率,说明甜高粱茎秆中糖分用于生成乙醇的比率较多,而用于维持酵母菌自身生长繁殖及副产物生成的糖分较少。比较2菌株的发酵能力,对于甜高粱TSH表现出较为明显的发酵优势:发酵周期比AGL缩短了近6h,且乙醇/糖转化率略高于AGL;而对于甘蔗和甜菜原料,TSH的乙醇产率与AGL相差不大;说明自行筛选的TSH菌种对多种糖料发酵底物具有广泛适用性,具有良好的工业应用前景。该文对利用糖质原料固态发酵生产燃料乙醇工艺的工业化实现及改进具有一定的借鉴意义。     

Pericarp Fiber Separation from Corn Flour Using Sieving and Air Classification

In the dry‐grind process, starch in ground corn (flour) is converted to ethanol, and the remaining corn components (protein, fat, fiber, and ash) form a coproduct called distillers dried grains with solubles (DDGS). Fiber separation from corn flour would produce fiber as an additional coproduct that could be used as combustion fuel, cattle feed, and as feedstock for producing valuable products such as “cellulosic” ethanol, corn fiber gum, oligosaccharides, phytosterols, and polyols. Fiber is not fermented in the dry‐grind corn process. Its separation before fermentation would increase ethanol productivity in the fermenter. Recently, we showed that the elusieve process, a combination of sieving and elutriation (air flow), was effective in fiber separation from DDGS. In this study, we evaluated the elusieve process for separating pericarp fiber from corn flour. Corn flour remaining after fiber separation was termed “enhanced corn flour”. Of the total weight of corn flour, 3.8% was obtained as fiber and 96.2% was obtained as enhanced corn flour. Neutral detergent fiber (NDF) of corn flour, fiber, and enhanced corn flour (dry basis) were 9.0, 61.5, and 5.7%, respectively. Starch content of corn flour, fiber, and enhanced corn flour (dry basis) were 68.8, 23.5, and 71.3%, respectively. Final ethanol concentration from enhanced corn flour (14.12% v/v) was marginally higher than corn flour (13.72% v/v). No difference in ethanol yields from corn flour and enhanced corn flour was observed. The combination of sieving and air classification can be used to separate pericarp fiber from corn flour. The economics of fiber separation from corn flour using the elusieve process would be governed by the production of valuable products from fiber and the revenues generated from the valuable products.     

以菊芋粉为原料同步糖化发酵生产燃料乙醇

利用粟酒裂殖酵母（Schizosaccharomyces pombe）能发酵菊芋未水解糖液高产乙醇的特点提出了以菊芋粉为原料,同步糖化发酵生产燃料乙醇的新工艺。在摇瓶中考察了原料预处理方法、原料浓度和初始pH值对乙醇发酵的影响,进而在5 L发酵罐中考察了未调控pH值和恒定pH值与通气情况对乙醇发酵的影响。结果表明：该菌株最适pH值为4.0;100目筛分的菊芋粉发酵效果良好,115℃灭菌处理优于121℃,在此条件下,菊芋粉浓度200 g/L时,乙醇产量达到66.58 g/L,理论转化率为85.88%;发酵液pH值下降对乙醇发酵没有影响,通入适量氧气会导致乙醇产量的下降,这表明粟酒裂殖酵母进行乙醇发酵时不需要供氧;通入氮气保持厌氧环境不能显著提高乙醇产量,不通气进行乙醇发酵也达到高的转化率,因此在工业生产中,不必保持厌氧发酵环境。在此基础上,对菊芋粉补料发酵进行了试验,补料至菊芋粉终浓度为300 g/L,发酵终点乙醇浓度为94.81 g/L,理论转化率为81.54%。这些研究工作,为以菊芋为原料的燃料乙醇工业化生产提供技术依据。     

甜高粱茎秆固态发酵制取燃料乙醇中试项目能耗分析

对以甜高粱茎秆为原料燃料乙醇中试项目的工艺进行了描述,对乙醇及副产品生产进行了能耗分析。该项目采用固态发酵工艺,乙醇转化率达到理论值的95.8%,并对剩余的茎秆渣进行综合利用,实现了余热回收利用,具有低排放的环保特性。项目年产无水乙醇1000t/a、发酵秸秆蛋白饲料1500t/a和秸秆纤维纸浆5000t/a。能耗分析表明,在考虑余热回收情况下,系统全年生产总能耗为4.31×106kW·h/a,无水乙醇的单位生产能耗为2759.67kW·h/t,蛋白饲料的单位生产能耗为36.86kW·h/t,秸秆纤维纸浆的单位生产能耗为298.41kW·h/t。无水乙醇生产工艺中回收余热量8.9×105kW·h/a。该系统中乙醇生产能量回收率为62.9%,高于以玉米等粮食原料生产乙醇的能量回收率。     

Enzymatic and fungal treatments on sugarcane bagasse for the production of mechanical pulps

Crude ligninolytic enzyme extracts from Phanerochaete chrysosporium fungi were applied to sugarcane bagasse, prior to thermomechanical (TMP) and chemithermomechanical pulping (CTMP), and their properties were compared with the normal TMP and CTMP and also with TMP and CTMP pretreated with Ceriporiopsis subvermispora and P. chrysosporium fungi. The sugarcane bagasse was impregnated with the crude enzyme extract containing lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac). The results show that pretreatment with enzyme crude extract is an advantageous way to produce TMP and CTMP from sugarcane bagasse, as compared with only fungal pretreatment. Enzymatic pretreatments need only hours to enhance pulping and paper properties, compared with the weeks necessary for fungal treatments. Higher pulp yields were obtained compared with the fungal pretreatments. Enzymatic pretreatment reduced the energy consumption in a proportion similar to that of C. subvermispora fungal pretreatment and increased the pulp tensile index compared with the normal TMP and CTMP pulps, although the tensile strength was somewhat lower than that for pulps from C. subvermispora fungal pretreatment before CTMP processing. An advantage of enzymatic pretreatment is that brightness is increased compared with normal TMP and CTMP processes, whereas fungal pretreatments reduce the brightness.     

Dry‐Grind Processing of Corn with Endogenous Liquefaction Enzymes

An amylase corn has been developed that produces an α‐amylase enzyme that is activated in the presence of water at elevated temperatures (>70°C). Amylase corn in the dry‐grind process was evaluated and compared with the performance of exogenous amylases used in dry‐grind processing. Amylase corn (1–10% by weight) was added to dent corn (of the same genetic background as the amylase corn) as treatments and resulting samples were evaluated for dry‐grind ethanol fermentation using 150‐g and 3‐kg laboratory procedures. Ethanol concentrations during fermentation were compared with the control treatment (0% amylase corn addition or 100% dent corn) which was processed with a conventional amount of exogenous α‐amylase enzymes used in the dry‐grind corn process. The 1% amylase corn treatment (adding 1% amylase corn to dent corn) was sufficient to liquefy starch into dextrins. Following fermentation, ethanol concentrations from the 1% amylase corn treatment were similar to that of the control. Peak and breakdown viscosities of liquefied slurries for all amylase corn treatments were significantly higher than the control treatment. In contrast, final viscosities of liquefied slurries for all amylase corn treatments were lower than those of the control. Protein, fat, ash, and crude fiber contents of DDGS samples from the 3% amylase corn treatment and control were similar.     

Detoxification of corn stover and corn starch pyrolysis liquors by ligninolytic enzymes of Phanerochaete chrysosporium

Phanerochaete chrysosporium (ATCC 24725) shake flask culture with 3 mM veratryl alcohol addition on day 3 was able to grow and detoxify different concentrations of diluted corn stover (Dcs) and diluted corn starch (Dst) pyrolysis liquors [10, 25, and 50% (v/v)] in defined media. GC-MS analysis of reaction products showed a decrease and change in some compounds. In addition, the total phenolic assay with Dcs samples demonstrated a decrease in the phenolic compounds. A bioassay employing Lactobacillus casei growth and lactic acid production was developed to confirm the removal of toxic compounds from 10 and 25% (v/v) Dcs and Dst by the lignolytic enzymes, but not from 50% (v/v) Dcs and Dst. The removal did not occur when sodium azide or cycloheximide was added to Ph. chrysosporium culture media, confirming the participation of lignolytic enzymes in the detoxification process. A concentrated enzyme preparation decreased the phenolic compounds in 10% (v/v) corn stover and corn starch pyrolysis liquors to the same extent as the fungal cultures.     

Continuous whey fermentation using kefir yeast immobilized on delignified cellulosic material

Delignified cellulosic-supported biocatalyst, prepared by immobilization of kefir yeast on delignified cellulosic material (DCM), was found to be suitable for continuous, modified whey fermentation. The modified whey contained 1% raisin extract and molasses. Ethanol productivities ranged from 3.6 to 8.3 g L(-1)day(-1), whereas parameters such as ethanol concentration, residual sugars, and daily fermented whey productivity were acceptable for the production of potable alcohol and alcoholic drinks in industrial fermentations. The continuous fermentation bioreactor was operated for 39 days, stored for 18 days at 4 degrees C, and operated again for another 15 days without any diminution of the ethanol productivity. The concentrations of higher alcohols (propanol-1, isobutyl alcohol, and amyl alcohols) were low. The main volatile byproducts formed in the continuous process were similar to those observed in alcoholic beverages, and the fermented whey had a good aroma. The concentrations of higher alcohols were very low when compared to that of ethyl acetate, therefore resulting in a quality product. The possibility of using such a process for the production of potable alcohol or a novel, low-alcohol content drink is proposed.     

Effects of Thermomechanical Extrusion and Particle Size Reduction on Bioconversion Rate of Corn Fiber for Ethanol Production

The objective of this research was to evaluate the effect of thermomechanical extrusion and particle size (PS) reduction on the bioconversion rate of corn fiber for ethanol production. Extrusion was conducted at a screw speed of 300 rpm, feed rate of 120 g/min, feed moisture content of 30%, melt temperature of 140°C, and die diameter of 3 mm. Raw and extruded corn fiber were separated into three different PSs (1 > PS ≥ 0.5, 0.5 > PS ≥ 0.3, and 0.3 > PS ≥ 0.15 mm) with a wire sieve. Extrusion pretreatment and PS reduction resulted in a significant (P < 0.05) difference in physical properties and color values of extruded corn fiber as a result of accelerated degradation of corn fiber structure. Significant increase in water solubility index of extruded corn fiber at 0.3 > PS ≥ 0.15 mm was an indication of high degradation of starch during extrusion for higher release of polysaccharides. Moreover, extruded corn fiber at PS reduction 0.3 > PS ≥ 0.15 mm also significantly increased (P < 0.05) ethanol yield (69.11 g/L) and conversion (68.18%) by increasing protein digestibility and free amino nitrogen, which are essential for higher fermentation efficiency.     

Corn Endosperm Fermentation Using Endogenous Amino Nitrogen Generated by a Fungal Protease

Fractionating the corn kernel to separate endosperm from germ and pericarp improves corn ethanol processing by increasing fermentation throughput and generating salable coproducts. One fractionation technology, dry fractionation (DF), suffers from loss of germ‐derived nutrients and amino acids, resulting in poor fermentation performance. Such deficiencies may be addressed by increasing nitrogen and other nutritional supplementation. As an alternative to exogenous nitrogen source, we investigated the use of a fungal protease to generate free amino nitrogen (FAN) from corn endosperm. Incubation of endosperm with protease did not affect subsequent liquefaction and saccharification. FAN supplementation through proteolysis resulted in fermentation being 99% complete in 48 hr, compared to 93% maximum with urea supplementation. Viable cell growth rates were similar in FAN and urea‐supplemented fermentations. Urea and FAN addition resulted in similar fermentation characteristics and similar FAN consumption rates as with FAN alone, which was indicative that FAN was assimilated preferentially. Increased amounts of maltose remaining after fermentation were correlated with initial FAN concentrations in mash. This observed trend was implicated in ethanol yield reduction of 2 g/L at high protease loading (generating 1.6 mg of FAN/g of glucose substrate) compared to a urea control. Using a glucose and maltose solution, we confirmed higher residual maltose in fermentations supplemented with high FAN concentrations. Use of protease to generate optimal FAN concentration in mash (1.2 mg of FAN/g of glucose substrate) could improve economics of dry fractionated corn ethanol production by increasing fermentation rates and, consequently, reducing fermentation time.     

补料发酵法生产玫瑰醋工艺优化及风味分析

为了提高传统玫瑰醋的生产效率,以装料量为500 kg的陶缸作发酵容器,在自然发酵条件下,分别在1.5、2.5、3.5 g/100 mL的初始酸度进行补料,补料体积比(原醋液:黄酒醪)设置为2:1和1:1两种情况,完成玫瑰醋补料发酵工艺的研究。结果表明:在初始酸度2.5 g/100 mL时补加等体积酒醪,发酵过程酸度最高上升到(5.59±0.27)g/100 mL(对照组为(5.19±0.23)g/100 mL),醋酸发酵周期从90 d缩短至78 d;非挥发性有机酸积累量达(27.15±1.11)mg/mL(对照组为(24.57±0.69)mg/mL),样品酸甜适口,酸味柔和;补料成品色率达到1.8×105(对照组色率为1.9×105),色泽接近传统玫瑰醋。该工艺的完成对玫瑰醋生产企业扩大产量、提高效率有积极意义。     

Comparison of Enzymatic (E‐Mill) and Conventional Dry‐Grind Corn Processes Using a Granular Starch Hydrolyzing Enzyme

A new low temperature liquefaction and saccharification enzyme STARGEN 001 (Genencor International, Palo Alto, CA) with high granular starch hydrolyzing activity was used in enzymatic dry‐grind corn process to improve recovery of germ and pericarp fiber before fermentation. Enzymatic dry‐grind corn process was compared with conventional dry‐grind corn process using STARGEN 001 with same process parameters of dry solid content, pH, temperature, enzyme and yeast usage, and time. Sugar, ethanol, glycerol and organic acid profiles, fermentation rate, ethanol and coproducts yields were investigated. Final ethanol concentration of enzymatic dry‐grind corn process was 15.5 ± 0.2% (v/v), which was 9.2% higher than conventional process. Fermentation rate was also higher for enzymatic dry‐grind corn process. Ethanol yields of enzymatic and conventional dry‐grind corn processes were 0.395 ± 0.006 and 0.417 ± 0.002 L/kg (2.65 ± 0.04 and 2.80 ± 0.01 gal/bu), respectively. Three additional coproducts, germ 8.0 ± 0.4% (db), pericarp fiber 7.7 ± 0.4% (db), and endosperm fiber 5.2 ± 0.6% (db) were produced in addition to DDGS with enzymatic dry‐grind corn process. DDGS generated from enzymatic dry‐grind corn process was 66% less than conventional process.