Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two-phase system

Cellulose was hydrolyzed in the attrition bioreactor (ABR) with enzyme recycling by employing an aqueous two-phase system (composed of dextran and polyethylene glycol) and an ultrafiltration unit. The ABR combines wet ball milling and enzymatic hydrolysis in one process step. The cellulase enzymes were more stable in the two-phase system than in the normal buffer solution. With the initial substrate concentration (Solka Floe BW200) of 40 g/L and intermittent addition of cellulose, sugar was semicontinuously produced at dilution rates of 0.06 h(-1) and productivities of 2.1 g/L h, which is approximately a 10-fold increase of the previously reported values performed in a regular stirred reactor with an aqueous two-phase system. The conversion of the substrate was 86%.     

Semicontinuous enzymatic hydrolysis of lignocelluloses

Lignocelluloses (steamed hardwood and hardwood kraft pulp) were semicontinuously hydrolyzed on a large scale [2-2. 5 kg of substrate vs. 20, 000 IU filter paperase (FPase)] using a 10-L hydrolysis reactor with an ultrafiltration unit for the recovery and reuse of cellulases. The substrate was added to the reactor at appropriate intervals to keep a concentration of approximately 5% (w/v). All of the enzyme was added at the beginning and no further addition was done. The ultrafiltration unit was operated intermittently rather than continuously due to its enough capacity (dilution rate of 2.5 h(-1)) and making the enzyme durable. The enzyme required to produce one gram of reducing sugar in this reactor was 27.3 FPase IU/g RS for steamed hardwood and 7.4 FPase IU/g RS for hardwood kraft pulp. The sugar composition of hydrolyzate was unaltered virtually from beginning to end of the hydrolysis in spite of the progressive loss of enzyme activities. The analysis of the enzyme composition in the hydrolyzate during hydrolysis revealed that an exo-beta-D-glucanase component was adsorbed selectively at the stages of advanced hydrolysis extent.     

(R)-phenylacetylcarbinol production in aqueous/organic two-phase systems using partially purified pyruvate decarboxylase from Candida utilis

Aqueous/organic two-phase systems have been evaluated for enhanced production of (R)-phenylacetylcarbinol (PAC) from pyruvate and benzaldehyde using partially purified pyruvate decarboxylase (PDC) from Candida utilis. In a solvent screen, octanol was identified as the most suitable solvent for PAC production in the two-phase system in comparison to butanol, pentanol, nonanol, hexane, heptane, octane, nonane, dodecane, methylcyclohexane, methyl tert butyl ether, and toluene. The high partitioning coefficient of the toxic substrate benzaldehyde in octanol allowed delivery of large amounts of benzaldehyde into the aqueous phase at a concentration less than 50 mM. PDC catalyzed the biotransformation of benzaldehyde and pyruvate to PAC in the aqueous phase, and continuous extraction of PAC and byproducts acetoin and acetaldehyde into the octanol phase further minimized enzyme inactivation, and inhibition due to acetaldehyde. For the rapidly stirred two-phase system with a 1:1 phase ratio and 8.5 U/mL carboligase activity, 937 mM (141 g/L) PAC was produced in the octanol phase in 49 h with an additional 127 mM (19 g/L) in the aqueous phase. Similar concentrations of PAC could be produced in the slowly stirred phase separated system at this enzyme level, although at a much slower rate. However at lower enzyme concentration very high specific PAC production (128 mg PAC/U carboligase at 0.9 U/mL) was achieved in the phase separated system, while still reaching final PAC levels of 102 g/L in octanol and 13 g/L in the aqueous phase. By comparison with previously published data by our group for a benzaldehyde emulsion system without octanol (50 g/L PAC, 6 mg PAC/U carboligase), significantly higher PAC concentrations and specific PAC production can be achieved in an octanol/aqueous two-phase system.     

Semicontinuous cellulase production in an aqueous two-phase system with Trichoderma reesei rutgers C30

Cellulases [see 1,4(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma reesei, Rutgers C30, can be semicontinuously produced in an aqueous two-phase system composed of dextran and poly(ethylene glycol) using Solka Floc BW 200 as substrate. When substrate was intermittently added along with fresh top phase, which replaced the withdrawn top phase containing the produced enzymes, a yield of 1740 U endo-β-d-glucanase/g cellulose and 59.3 FPU/g cellulose was extracted with the top phase. Without fresh substrate added, a yield of 3920 U endo-β-d-glucanase/g cellulose and 127.7 FPU/g cellulose was extracted after five runs.     

Enzymatic hydrolysis of lime-pretreated corn stover and investigation of the HCH-1 Model: inhibition pattern, degree of inhibition, validity of simplified HCH-1 Model

The inhibition pattern was identified for a reaction system composed of Trichoderma reesei cellulase enzyme complex and lime-pretreated corn stover. Also, the glucose inhibition effect was quantified for the aforementioned reaction system over a range of enzyme loadings and substrate concentrations. Lastly, the range of substrate concentrations and enzyme loadings were identified in which the linear form of the simplified HCH-1 Model is valid. The HCH-1 Model is a modified Michaelis-Menton Model with non-competitive inhibition and the fraction of insoluble substrate available to bind with enzyme. With a high enzyme loading, the HCH-1 Model can be integrated and simplified in such a way that sugar conversion is linearly proportional to the logarithm of enzyme loading. A wide range of enzyme loadings (0.25-50 FPU/g dry biomass) and substrate concentrations (10-100g/L) were investigated. All experiments were conducted with an excess cellobiase loading to ensure the experimental results were not influenced by cellobiose inhibition. A non-competitive inhibition pattern was identified for the corn stover-cellulase reaction system, thereby validating the assumptions of the HCH-1 Model. At a substrate concentration of 10 g/L, glucose inhibition parameters of 0.986 and 0.979 were measured for enzyme loadings of 2 FPU/g dry biomass and 50 FPU/g dry biomass, respectively. At 5 FPU/g dry biomass, glucose inhibition parameters of 0.985 and 0.853 were measured for substrate concentrations of 10 and 100g/L, respectively. The linear form of the HCH-1 Model predicted biomass digestibility for lime-pretreated corn stover over an enzyme loading range of 0.25-50 FPU/g dry biomass and substrate concentration range of 10-100g/L.     

Production of fructose and ethanol from sugar beet molasses using Saccharomyces cerevisiae ATCC 36858

The production of enriched fructose syrups and ethanol from beet molasses using Saccharomyces cerevisiae ATCC 36858 was studied. In batch experiments with a total sugar concentration between 94.9 and 312.4 g/L, the fructose yield was above 93% of the theoretical value. The ethanol yield and volumetric productivity in the beet molasses media with sugar concentration below 276.2 g/L were in the range of 59-76% of theoretical value and between 0.48 and 2.97 g of ethanol/(L x h), respectively. The fructose fraction in the carbohydrates content of the produced syrups was more than 95% when the total initial sugar concentration in the medium was below 242.0 g/L. Some oligosaccharides and glycerol were also produced in all tested media. Raffinose and the produced oligosaccharides were completely consumed by the end of the fermentation process when the total initial sugar concentration was below 190.1 g/L. The glycerol concentration was below 16.1 g/L. The results could be useful for a potential industrial production of ethanol and high-fructose syrup from sugar beet molasses.     

Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.     

Acetone–butanol–ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment

In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone–butanol–ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.     

自絮凝颗粒酵母乙醇连续发酵耦合酵母回用工艺的研究

模拟现有酒精发酵行业普遍采用的多级发酵罐串联系统,建立了一套由三级串联操作的搅拌式发酵罐和两个沉降罐组成的反应器系统,以脱胚脱皮玉米粉双酶法制备的糖化液为发酵底物,培养基初始还原糖浓度为220g/L,添加(NH4)2HPO41.5g/L和KH2PO42.5g/L,以0.057h-1的恒定稀释速率流加,将自沉降浓缩后的酵母乳先后经活化和不活化两种方式处理并循环至第一级发酵罐,系统在两种操作条件下分别达到了拟稳态。实验结果表明活化处理对改善发酵工艺技术指标方面发挥了显著的作用,发酵终点乙醇浓度达到101g/L,还原糖和残总糖分别在3.2和7.7g/L左右,发酵系统的设备生产强度指标为5.77g/(L.h),与无酵母回用的搅拌式反应器系统中自絮凝颗粒酵母乙醇发酵工艺相比,提高了70%。     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Three-stage enzymatic hydrolysis of steam-exploded corn stover at high substrate concentration

The feasibility of three-stage hydrolysis of steam-exploded corn stover at high-substrate concentration was investigated. When substrate concentration was 30% and enzyme loading was 15-30 FPU/g cellulose, three-stage (9+9+12 h) hydrolysis could reach a hydrolysis yield of 59.9-81.4% in 30 h. Compared with one-stage hydrolysis for 72 h, an increase of 34-37% in hydrolysis yield could be achieved. When steam-exploded corn stover was used as the substrate for enzyme synthesis and hydrolysis was conducted at a substrate concentration of 25% with an enzyme loading of 20 FPU/g cellulose, a hydrolysis yield of 85.1% was obtained, 19% higher than that the commercial cellulase could reach under the same conditions. The removal of end products was suggested to improve the adsorption of cellulase on the substrate and enhance the productivity of enzymatic hydrolysis.     

High-rate continuous production of lactic acid by Lactobacillus rhamnosus in a two-stage membrane cell-recycle bioreactor

It is important to produce L(+)-lactic acid at the lowest cost possible for lactic acid to become a candidate monomer material for promising biodegradable polylactic acid. In an effort to develop a high-rate bioreactor that provides high productivity along with a high concentration of lactic acid, the performance of membrane cell-recycle bioreactor (MCRB) was investigated via experimental studies and simulation optimization. Due to greatly increased cell density, high lactic acid productivity, 21.6 g L(-1) h(-1), was obtained in the reactor. The lactic acid concentration, however, could not be increased higher than 83 g/L. When an additional continuous stirred tank reactor (CSTR) was attached next to the MCRB a higher lactic acid concentration of 87 g/L was produced at significant productivity expense. When the two MCRBs were connected in series, 92 g/L lactic acid could be produced with a productivity of 57 g L(-1) h(-1), the highest productivity among the reports of L(+)-lactic acid that obtained lactic acid concentration higher than 85 g/L using glucose substrate. Additionally, the investigation of lactic acid fermentation kinetics resulted in a successful model that represents the characteristics of lactic acid fermentation by Lactobacillus rhamnosus. The model was found to be applicable to most of the existing data with MCRBs and was in good agreement with Levenspiel's product-inhibition model, and the Luedeking-Piret equation for product-formation kinetics appeared to be effective in representing the fermentation kinetics. There was a distinctive difference in the production potential of cells (cell-density-related parameter in Luedeking-Piret equation) as lactic acid concentration increases over 55 g/L, and this finding led to a more precise estimation of bioreactor performance.     

沙柳原料高浓度底物酶解发酵乙醇工艺

为了提高沙柳生物转化过程的经济可行性,考察了沙柳原料经过蒸爆、超微粉碎+稀酸、超微粉碎+稀碱预处理后高浓度底物补料酶解的效果,并对其高浓度水解糖液进行了乙醇发酵。结果表明:蒸爆处理法水解效果最好,通过补料酶解,底物质量分数可以达到30%,酶解液中总糖质量浓度达到132 g/L,葡萄糖质量浓度105 g/L;超微粉碎+稀酸预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度达到123 g/L,葡萄糖质量浓度73 g/L;超微粉碎+稀碱预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度133 g/L,葡萄糖质量浓度77 g/L。3种预处理使沙柳原料的酶解糖液都可以较好地被酿酒酵母利用发酵产乙醇,蒸爆处理原料的酶解糖液乙醇发酵效果最好,乙醇质量浓度达到47 g/L。     

Biological hydrogen production in a UASB reactor with granules. II: Reactor performance in 3-year operation

The experiment was conducted to evaluate the performance of an upflow anaerobic sludge blanket (UASB) with granules for H(2) production from a sucrose-rich synthetic wastewater at various substrate concentrations (5.33-28.07 g-COD/L) and hydraulic retention times (HRTs) (3-30 h) for over 3 years. The kinetics of H(2) production was evaluated, and the sludge yield and endogenous decay coefficient of the H(2)-producing granules were estimated to be 0.334 g-VSS/g-COD and 0.004/h, respectively. Based on Gibbs free energy calculations, the formation thermodynamics of caproate, an important aqueous product, were analyzed. Experimental results show that the H(2) partial pressure in biogas decreased with increasing substrate concentration, but was not sensitive to the variation of HRT in a range of 6-22 h. The H(2) production rate increased with increasing substrate concentration, but decreased with increasing HRT. The H(2) yield was in the range of 0.49-1.44 mol-H(2)/mol-glucose. Acetate, butyrate, caporate, and ethanol were the main aqueous products in the reactor, and their concentrations were dependent on both substrate concentration and HRT. An elevated substrate concentration resulted in a shift of fermentation from butyrate- to caporate-type in the reactor and the formation of caproate was dependent on the H(2) partial pressure. The 3-year experimental results demonstrate that H(2) could be produced continuously and stably from the acidogenic-granule-based UASB reactor.     

3-Hydroxypropionaldehyde guided glycerol feeding strategy in aerobic 1,3-propanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

3-Hydroxypropionaldehyde (3-HPA) is a toxic intermediary metabolite in the biological route of 1,3-propanediol biosynthesis from glycerol. 3-HPA accumulated in culture medium would arouse an irreversible cessation of the fermentation process. The role of substrate (glycerol) on 3-HPA accumulation in aerobic fermentation was investigated in this paper. 1,3-Propanediol oxidoreductase and glycerol dehydratase, two key enzyme catalyzing reactions of 3-HPA formation and consumption, were sensitive to high concentration of 3-HPA. When the concentration of 3-HPA increased to a higher level in medium (ac 10 mmol/L), the activity of 1,3-propanediol oxidoreductase in cell decreased correspondingly, which led to decrease of the 3-HPA conversion rate, then the 3-HPA concentration increasing was accelerated furthermore. 3-HPA accumulation in culture medium was triggered by this positive feedback mechanism. In the cell exponential growth phase, the reaction catalyzed by 1,3-propanediol oxidoreductase was the rate limiting step in 1,3-propanediol production. The level of 3-HPA in culture medium could be controlled by the substrate (glycerol) concentration, and lower level of glycerol could avoid 3-HPA accumulating to a high, lethal concentration. In fed batch fermentation, under the condition of initial glycerol concentration 30 g/L, and keeping glycerol concentration lower than 7–8 g/L in cell exponential growth phase, 3-HPA accumulation could not be incurred. Based on this result, a glycerol feeding strategy was set up in fed batch fermentation. Under the optimized condition, 50.1 g/L of 1,3-propanediol was produced in 24 h, and 73.1 g/L of final 1,3-propanediol concentration was obtained in 54 h.     

Dehydrogenation of ribitol with Gluconobacter oxydans: production and stability of L-ribulose

l-Ribulose is an important chiral lead molecule used for the synthesis of, among others, l-ribose, a high-value rare sugar used in the preparation of antiviral drugs. These drugs--nucleoside-analogues--gain importance in the treatment of severe viral diseases, like those caused by the HIV or hepatitis virus. In this study, factors that may have an impact on l-ribulose production with Gluconobacter oxydans and on the stability of l-ribulose were investigated. A bioconversion-type process, using washed resting cells, was chosen to produce l-ribulose from ribitol. In this process, the cell production and bioconversion phase were separated. The former was first optimized and a maximum cell mass of 1.5 g CDWL(-1) could be produced. For the bioconversion phase, the aeration level of the system proved to be one of the most critical factors; a maximal production rate of 15.7 g L(-1)h(-1) or 5.9 g(g CDW)(-1)h(-1) of l-ribulose could be reached. Furthermore, resting cells were found capable of completely converting ribitol solutions of up to 300 g L(-1) within 30 h, although the kinetics indicated a rather low affinity of the dehydrogenase enzymes for the substrate.     

酵母发酵蔗渣半纤维素水解物生产木糖酶

采用二次正交旋转组合设计研究了蔗渣半纤维素水解过程中硫酸浓度与液 固比对木糖收率的影响。回归分析表明 ,这两个因素与木糖的收率之间存在显著的回归关系。通过回归方程优化水解条件 ,当硫酸浓度 2 .4g L ,液 固 =6 .2 ,在蒸汽压力 2 .5× 10 4Pa的条件下水解 2 .5h ,10 0g蔗渣可水解生成木糖约 2 4g。大孔树脂吸附层析处理蔗渣半纤维素水解物 ,能有效地减少其中的酵母生长抑制物含量 ,显著改善水解物的发酵性能。用大孔树脂在pH 2条件下处理过的蔗渣半纤维素水解物作基质 ,含木糖 2 0 0g L ,产木糖醇酵母菌株CandidatropicalisAS2 .1776发酵 110h耗完基质中的木糖 ,生成木糖醇 12 7g L ,产物转化率 0 .6 4(木糖醇g 木糖g) ,产物生成速率 1.15g L·h .     

Production of L-phenylacetyl carbinol by immobilized yeast cells: II. Semicontinuous fermentation

The cyclic, semicontinuous production of L-phenylacetyl carbinol (L-PAC) from a benzaldehyde substrate by Saccharomyces cerevisiae ATCC 834 immobilized in calcium alginate beads was substantially enhanced to about 4.5 g/L in a second cycle by reactivation in fresh medium for 24 h, following an earlier 24-h period of production from substrate. Intermittent feeding of benzaldehyde was employed (four doses in 3 h). In subsequent similar cycles, however, the production returned to that produced in the first cycle, viz. L-PAC concentration of 2-3 g/L in the medium. Production of L-PAC was also increased by adaptation of the cells over 200 h of exposure to the benzaldehyde substrate (compared to wild-type cells) and by continuous (as compared to intermittent) feeding of the substrate. A liter as great as 10 g/L was obtained with wild-type cells by continuous feeding of benzaldehyde over 6 h. Immobilization not only protected the cells from toxic effects of substrate but also permitted them to be used during 7 cycles of semicontinuous operation over more than 200 h.     

固定化纤维二糖酶的研究

黑曲霉 (AspergillusnigerLORRE 0 12 )的孢子中富含纤维二糖酶 ,将这些孢子用海藻酸钙凝胶包埋后 ,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定 ,半衰期为 38d ,耐热性和适宜的pH范围均比固定化前有所增加 ,其Km 和Vmax值分别为 6 .0 1mmol L和 7.0 6mmol (min·L)。利用固定化纤维二糖酶重复分批酶解10g L的纤维二糖 ,连续 10批的酶解得率均可保持在 97%以上 ;采用连续酶解工艺 ,当稀释率为 0 .4h- 1 ,酶解得率可达 98.5 %。玉米芯经稀酸预处理后 ,其纤维残渣用里氏木霉 (Trichodermareesei)纤维素酶降解 ,酶解得率为6 9.5 % ;通过固定化纤维二糖酶的进一步作用 ,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除 ,酶解得率提高到 84.2 % ,还原糖中葡萄糖的比例由 5 3 .6 %升至 89.5 % ,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

亚硫酸盐甘蔗渣浆酶解液发酵制备乙醇

以亚硫酸盐甘蔗渣浆酶解液作为原料,利用C. shehatae发酵制取燃料乙醇。结果表明：还原糖最适初始质量浓度为葡萄糖140 g/L、木糖60 g/L、酶解液总糖80 g/L。利用初始葡萄糖55.06 g/L、木糖11.18 g/L、纤维二糖4.51 g/L的亚硫酸盐甘蔗渣浆酶解液发酵,经18 h获得乙醇22.98 g/L。乙醇得率为67.23%,葡萄糖利用率为99.27%,木糖利用率为32.96%,C. shehatae适合作为蔗渣为原料的乙醇发酵菌株。