Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

Wheat straw was pretreated by wet explosion using three different oxidizing agents (H₂O₂, O₂, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O₂ as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15–20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.     

Fermentation of Biologically Pretreated Wheat Straw for Ethanol Production: Comparison of Fermentative Microorganisms and Process Configurations

The pretreatment of lignocellulosic biomass with white-rot fungi to produce bioethanol is an environmentally friendly alternative to the commonly used physico-chemical processes. After biological pretreatment, a solid substrate composed of cellulose, hemicellulose and lignin, the two latter with a composition lower than that of the initial substrate, is obtained. In this study, six microorganisms and four process configurations were utilised to ferment a hydrolysate obtained from wheat straw pretreated with the white-rot fungus Irpex lacteus. To enhance total sugars utilisation, five of these microorganisms are able to metabolise, in addition to glucose, most of the pentoses obtained after the hydrolysis of wheat straw by the application of a mixture of hemicellulolytic and cellulolytic enzymes. The highest overall ethanol yield was obtained with the yeast Pachysolen tannophilus. Its application in combination with the best process configuration yielded 163 mg ethanol per gram of raw wheat straw, which was between 23 and 35 % greater than the yields typically obtained with a conventional bioethanol process, in which wheat straw is pretreated using steam explosion and fermented with the yeast Saccharomyces cerevisiae.     

Production of Ethanol and Feed by High Dry Matter Hydrolysis and Fermentation of Palm Kernel Press Cake

Palm kernel press cake (PKC) is a residue from palm oil extraction presently only used as a low protein feed supplement. PKC contains 50% fermentable hexose sugars present in the form of glucan and mainly galactomannan. This makes PKC an interesting feedstock for processing into bioethanol or in other biorefinery processes. Using a combination of mannanase, β-mannosidase, and cellulases, it was possible without any pretreatment to hydrolyze PKC at solid concentrations of 35% dry matter with mannose yields up to 88% of theoretical. Fermentation was tested using Saccharomyces cerevisiae in both a separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) setup. The hydrolysates could readily be fermented without addition of nutrients and with average fermentation yields of 0.43?±?0.02 g/g based on consumed mannose and glucose. Employing SSF, final ethanol concentrations of 70 g/kg was achieved in 216 h, corresponding to an ethanol yield of 70% of theoretical or 200 g ethanol/kg PKC. Testing various enzyme mixtures revealed that including cellulases in combination with mannanases significantly improved ethanol yields. Processing PKC to ethanol resulted in a solid residue enriched in protein from 17% to 28%, a 70% increase, thereby potentially making a high-protein containing feed supplement.     

Effect and Modeling of Glucose Inhibition and In Situ Glucose Removal During Enzymatic Hydrolysis of Pretreated Wheat Straw

The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw with the cellulolytic enzyme system, Celluclast? 1.5 L, from Trichoderma reesei, supplemented with a β-glucosidase, Novozym? 188, from Aspergillus niger. Addition of glucose (0–40 g/L) significantly decreased the enzyme-catalyzed glucose formation rates and final glucose yields, in a dose-dependent manner, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48–72 h of reaction recovered from 20–40% to become ≈70% of the rate recorded during 6–24 h of reaction. Although Michaelis–Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis–Menten inhibition models without great significance of the inhibition mechanism. Moreover, the experimental in situ removal of glucose could be simulated by a Michaelis–Menten inhibition model. The data provide an important base for design of novel reactors and operating regimes which include continuous product removal during enzymatic hydrolysis of lignocellulose.     

Effects of Propionic Acid and pH on Ethanol Fermentation by Saccharomyces cerevisiae in Cassava Mash

The effects of propionic acid on ethanol and glycerol production by Saccharomyces cerevisiae in cassava mash were examined along with the influence of pH (4.0, 5.0, and 6.0) and of dissolved solids content (22%, 25%, and 27%). Inhibition by propionic acid increased as solids content increased and medium pH declined. Complete inhibition of ethanol fermentation was observed in mashes at pH 4.0 (60 mM propionic acid for 22% solids and 45 mM for 25% and 27%). Glycerol production linearly decreased with increased undissociated propionic acid concentration in all mashes at all pH levels, which partly contributed to increased final ethanol production when propionic acid concentration in mashes was low (≤ 30 mM).     

Effect of Fermentation Conditions on the Flocculation of Recombinant Saccharomyces cerevisiae Capable of Co-fermenting Glucose and Xylose

Flocculation is a desirable property in industrial yeasts and is particularly important in the fuel ethanol industry because it provides a simple and cost-free way to separate yeast cells from fermentation products. In the present study, the effect of pH and lignocellulose-derived sugars on yeast flocculation was investigated using a flocculent Saccharomyces cerevisiae, MA-R4, which has been recombinantly engineered to simultaneously co-ferment glucose and xylose to ethanol with high productivity. The flocculation level of MA-R4 dramatically decreased at pH values below 3.0 during co-fermentation of glucose and xylose. Sedimentation and microscopic observation revealed that flocculation was induced in MA-R4 when it fermented glucose, a glucose/xylose mixture, or mannose, whereas attempts to ferment xylose, galactose, and arabinose led to the loss of flocculation. MA-R4 fermented xylose and galactose more slowly than glucose and mannose. Therefore, the various flocculation behaviors shown by MA-R4 should be useful in the control of ethanol fermentation processes.     

Ethanol Production by Fermentation Using Immobilized Cells of Saccharomyces cerevisiae in Cashew Apple Bagasse

In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82–37.83 g L^?1 in average value) and ethanol productivities (about 3.30–6.31 g L^?1 h^?1) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L^?1) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30–98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.     

Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

拉曼光谱分析有机氮源促进乙醇发酵的机制

应用拉曼光谱和单细胞分析技术监测有机氮源尿素和酵母粉、无机氮源硝酸铵和硫酸铵对酿酒酵母乙醇发酵的影响及发酵过程胞内主要生物大分子的变化动态,以期从光谱学角度获知有机氮源促进乙醇发酵的机制。结果表明,利用酵母粉和尿素的乙醇发酵速度最快,14~18 h即可达到乙醇浓度的最大值;在有机氮源下,酵母细胞的RNA合成无明显的迟滞期,发酵前期,782 cm!1峰平均强度比无机氮源的高,其最大峰强是初始强度的1.9~2.1倍,而无机氮源仅为1.2~1.4倍;以酵母粉为氮源的不同发酵阶段,部分细胞的蛋白质二级结构以β折叠为主,而其它氮源的细胞则是以α螺旋为绝对主导。因此,尿素、酵母粉等有机氮源促进乙醇发酵的可能原因是缩短酵母的迟滞期,促进胞内RNA的快速合成,促进相关基因的快速转录和表达。     

The Effect of Intracellular Amino Acids on GSH Production by High-cell-density Cultivation of Saccharomyces cerevisiae

The present paper studies the effects of precursor amino acids, i.e., L-glutamic acid (Glu), L-glycine (Gly), and L-cysteine (Cys), on the glutathione (GSH) production. The three amino acids were added during the fermentations. The GSH production was analyzed by gas chromatography-mass spectrometry (GC-MS). It was observed that the cell content of Cys reduced continually, Gly maintained a fairly constant concentration, while Glu remained at a high concentration compared with Cys and Gly. The synthesis of GSH was found to significantly increase after 28?h of fermentation upon addition of 6?mmol?l(-1) of each of the three amino acids. Under these conditions, the GSH yields reached 2,250?±?50?mg?l(-1) at 34?h from 1,050?±?50?mg?l(-1) at 28?h. The GC-MS analyses on the effect caused by the addition of amino acids indicated that the addition of Glu was not necessary to improve the GSH production by high-cell-density cultivation of Saccharomyces cerevisiae. The addition of Cys or Gly individually enhances the production of GSH.     

The Effect of Different Ethoxylations for Sorbitan Monolaurate on Enhancing Simultaneous Saccharification and Fermentation (SSF) of Wheat Straw to Ethanol

In this paper, four nonionic surfactants with different hydrophilic–lipophilic balance (HLB) based on sorbitan monolaurate were synthesized by introducing ethylene oxide gas (n = 20, 40, 60, and 80 ethylene oxide units). The chemical structure of the prepared ethoxylated surfactants was confirmed using Fourier transform-infrared and ¹H NMR spectroscopes. The surface tension and thermodynamic properties of the prepared surfactants have been studied. The simultaneous saccharification and fermentation (SSF) process for ethanol production from microwave/alkali pretreated wheat straw has been assayed using nonionic surfactants have different ethylene oxide units. Ethanol yield was 82% and 61% for Kluyveromyces marxianus and Saccharomyces cerevisiae, respectively, with the addition of 2.5 g/l of the prepared nonionic surfactant (HLB = 18.2). Results show that the production of ethanol from microwave/alkali pretreated wheat straw increased with increasing the (HLB) value of the nonionic surfactant.     

Effect of Initial Cell Concentration on Ethanol Production by Flocculent Saccharomyces cerevisiae with Xylose-Fermenting Ability

Different initial cell concentrations of a recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated for their effects on xylose fermentation and glucose–xylose cofermentation. A high initial cell concentration greatly increased both the substrate utilization and ethanol production rates. During xylose fermentation, the highest rates of xylose consumption (2.58 g/L h) and ethanol production (0.83 g/L h) were obtained at an initial cell concentration of 13.1 g/L. During cofermentation, the highest rates of glucose consumption (14.4 g/L h), xylose consumption (2.79 g/L h), and ethanol production (6.68 g/L h) were obtained at an initial cell concentration of 12.7 g/L. However, a high initial cell density had no positive effect on the maximum ethanol concentration and ethanol yield mainly due to the increased amount of by-products including xylitol. The ethanol yield remained almost constant (0.34 g/g) throughout xylose fermentation (initial cell concentration range, 1.81–13.1 g/L), while it was slightly lower at high initial cell concentrations (9.87 and 12.7 g/L) during cofermentation. The determination of the appropriate initial cell concentration is necessary for the improvement of substrate utilization and ethanol yield.     

Effect of Hanseniaspora uvarum–Saccharomyces cerevisiae Mixed Fermentation on Aroma Characteristics of Rosa roxburghii Tratt,Blueberry, and Plum Wines

Hanseniaspora uvarum, a non-Saccharomyces cerevisiae species, has a crucial effect on the aroma characteristics of fruit wines, thus, attracting significant research interest in recent years. In this study, H. uvarum–Saccharomyces cerevisiae mixed fermentation was used to ferment Rosa roxburghii Tratt, blueberry fruit wine, and plum fruit wines using either a co-inoculated or a sequentially inoculated approach. The three fruit wines’ volatile aroma characteristics were analyzed by headspace–solid-phase microextraction–gas chromatography-mass spectrometry (HS–SPME–GC–MS). The results showed that the mixed inoculation of H. uvarum and S. cerevisiae reduced the alcoholic content of Kongxinli fruit wine. Moreover, H. uvarum–S. cerevisiae fermented Rosa roxburghii Tratt, blueberry, and plum fruit wines and further enriched their flavor compounds. The overall flavor characteristics of sequentially inoculated fruit wines differed significantly from those fermented with S. cerevisiae alone, although several similarities were also observed. Sequential inoculation of H. uvarum and S. cerevisiae positively affected the mellowness of the wine and achieved a better harmony of the overall wine flavors. Therefore, H. uvarum–Saccharomyces cerevisiae mixed fermentation can improve the complexity of the wines’ aromatic composition and empower them with a unique identity. In particular, H. uvarum–Saccharomyces cerevisiae blueberry wine produced by mixed fermentation had the widest variety and content of aroma compounds among the fermented wines. Therefore, H. uvarum–Saccharomyces cerevisiae mixed-fermentation inoculation in the three fermented fruit wines significantly increased the aroma compound variety and content, thus, enriching their aroma richness and complexity. This study is the first comparative evaluation of the aroma characteristics of different fruit wines fermented with a mixed inoculation of H. uvarum and S. cerevisiae and provides a preliminary guide for these fruit wines produced with non-Saccharomyces yeast.     

Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

The concentration of ethanol produced from lignocellulosic biomass should be at least 40 g l^?1 [about 5 % (v/v)] to minimize the cost of distillation process. In this study, the conditions for the simultaneous saccharification and fermentation (SSF) at fed-batch mode for the production of ethanol from alkali-pretreated empty palm fruit bunch fibers (EFB) were investigated. Optimal conditions for the production of ethanol were identified as temperature, 30 °C; enzyme loading, 15 filter paper unit g^?1 biomass; and yeast (Saccharomyces cerevisiae) loading, 5 g l^?1 of dry cell weight. Under these conditions, an economical ethanol concentration was achieved within 17 h, which further increased up to 62.5 g l^?1 after 95 h with 70.6 % of the theoretical yield. To our knowledge, this is the first report to evaluate the economic ethanol production from alkali-pretreated EFB in fed-batch SSF using S. cerevisiae.     

The influence of inert solids on ethanol production by Saccharomyces cerevisiae

The catalytic role of various inert solid supports on acceleration of alcohol fermentation by Saccharomyces cerevisiae was investigated. The enhanced rate of alcohol production was dependent on the nature of the support as well as on the amount used. Among all the tested supports, chitosan flakes showed the maximum yield of alcohol (93% of theoretical yield). This higher rate of alcohol production was associated with the twofold increase in the activity of alcohol dehydrogenase over control. Our results suggest that the addition of a small fraction of solids in submerged fermentations to facilitate cell anchorage for enhanced metabolic activity is easier and more economical compared to cell immobilization processes. IICT Communication No. 4266. Some of the results in this article are covered under a patent.     

Examining the Potential of Plasma-Assisted Pretreated Wheat Straw for Enzyme Production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml⁻¹) and cellulases (0.5 filter paper units (FPU) ml⁻¹). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml⁻¹) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml⁻¹) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.     

铕对小麦根可溶性钙含量的影响

研究了Ｅｕ对小麦细胞内Ｃａ＾２＋含量的影响方式,用原子吸收光谱法对根的可溶性钙,总钙含量进行了测定,结果表明,短时间内Ｅｕ＾３＋促进根细胞可溶性钙含量增加,而随着处理时间的延长,可溶性钙含量下降,但总钙含量不断增加;     

Effect of High Hydrostatic Pressure in the Storage of Spanish-Style Table Olive Fermented with Olive Leaf Extract and Saccharomyces cerevisiae

Olives treated according to the Spanish-style are firstly treated with caustic soda and then fermented in brine to reduce phenols. Next, olives are packed and subjected to pasteurization. The effect of different high hydrostatic pressure treatments (400 MPa, 4 and 6 min) was evaluated in Spanish-style table olives fermented with olive leaf extract (OLE) and S. cerevisiae compared with thermal pasteurization (P) at 80 °C for 15 min. HHP and P led to a significant reduction in yeast and aerobic mesophiles after the conservation treatment and during storage (300 days). The physical–chemical properties changed slightly during storage, except for olive hardness; olives treated with HHP presented a higher hardness than pasteurized ones. The CIELAB parameter L* decreased until day 300 in most of the treatments, as well as phenols. The HHP treatment led to significantly higher contents of phenolics (even during storage) than olives submitted to P. Some sensory attributes (colour, aspect, hardness, and overall evaluation) decreased during storage. P treatment caused a decrease in appearance, aroma, hardness, and overall evaluation compared to olives treated with HHP. Thus, the application of HHP in table olives to increase the shelf-life can be considered a valid alternative to P.