The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol

Autohydrolysis and ethanol-alkali pulping were used as pretreatment methods of wheat straw for its subsequent saccharification by Trichoderma reesei cellulase. The basic hydrolysis parameters, i.e., reaction time, pH, temperature, and enzyme and substrate concentration, were optimized to maximize sugar yields from ethanol-alkali modified straw. Thus, a 93% conversion of 2.5% straw material to sugar syrup containing 73% glucose was reached in 48 h using 40 filter paper units/g hydrolyzed substrate. The pretreated wheat straw was then fermented to ethanol at 43 degrees C in the simultaneous saccharification and fermentation (SSF) process using T. reesei cellulase and Kluyveromyces fragilis cells. From 10% (w/v) of chemically treated straw (dry matter), 2.4% (w/v) ethanol was obtained after 48 h. When the T. reesei cellulase system was supplemented with beta-glucosidase from Aspergillus niger, the ethanol yield in the SSF process increased to 3% (w/v) and the reaction time was shortened to 24 h.     

内切葡聚糖酶高产菌株的诱变选育

【目的】建立里氏木霉(Trichoderma reesei)高产突变菌株的快速筛选方法,选育出高产内切葡聚糖酶的突变株。【方法】对里氏木霉T306菌株的初筛培养基进行优化,建立快速筛选方法;通过紫外诱变手段选育内切葡聚糖酶高产突变菌株,并对突变菌株的产酶培养基进行优化。【结果】在初筛培养基中添加浓度为0.1%(W/V)的乳糖、蛋白胨及脱氧胆酸钠有利于菌株的筛选。诱变后筛选出菌落形态发生明显变化的内切葡聚糖酶高产突变株0516,其羧甲基纤维素酶活力(CMC酶)较出发菌株提高了38.9%。其产酶培养基经优化后,得到最适碳、氮源分别为:乳糖1.50%、硫酸铵0.14%、尿素0.05%、蛋白胨0.10%,优化后CMC酶活力达64.2 U/mL,较优化前提高了2.3倍。【结论】建立了里氏木霉高产突变菌株的快速筛选方法,通过紫外诱变育种获得了产内切葡聚糖酶能力高且遗传稳定的突变株0516。     

Comparison of SHF and SSF processes from steam-exploded wheat straw for ethanol production by xylose-fermenting and robust glucose-fermenting Saccharomyces cerevisiae strains

In this study, bioethanol production from steam-exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentoses. Red Star is a robust hexose-fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. The highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) in an SSF process, it showed an ethanol yield on consumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/L h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furthermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced.     

Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875

Simultaneous saccharification and fermentation (SSF) process for ethanol production from various lignocellulosic woody (poplar and eucalyptus) and herbaceous (Sorghum sp. bagasse, wheat straw and Brassica carinata residue) materials has been assayed using the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875. Biomass samples were previously treated in a steam explosion pilot plant to provide pretreated biomass with increased cellulose content relative to untreated materials and to enhance cellulase accessibility. SSF experiments were performed in laboratory conditions at 42 °C, 10% (w/v) substrate concentration and 15 FPU/g substrate of commercial cellulase. The results indicate that it is possible to reach SSF yields in the range of 50–72% of the maximum theoretical SSF yield, based on the glucose available in pretreated materials, in 72–82 h. Maximum ethanol contents from 16 to 19 g/l were obtained in fermentation media, depending on the material tested.     

Combined submerged and solid substrate fermentation for the bioconversion of lignocellulose

A novel two-stage bioreactor has been designed for a combined submerged (SF) and solid substrate fermentation (SSF) of wheat straw. The straw was pretreated with steam, and cellulases from the culture fluid of Trichoderma reesei were adsorbed on it for increased bioconvertibility. SSF was conducted in the top part of the bioreactor by inoculating the straw with a 36-h mycelial culture of T. reesei, or Coriolus versicolor. In the bottom part of the fermenter, Endomycopsis fibuliger was grown in SF. The SF liquor was recirculated through the SSF stage at 24 h intervals to remove glucose and other metabolites that may inhibit growth, and to maintain optimum moisture level and temperature. The removed glucose and other metabolites provided nutrients for the yeast in the SF stage. The combined fermentation resulted in overall higher biomass yield, increased bioconversion, increased cellulase production, and increased digestibility compared with single SSF or SF.     

Solid substrate fermentation of wheat straw to fungal protein

Steam-treated wheat straw at a 70% (w/w) moisture level was subjected to solid substrate fermentation (SSF) with Trichoderma reesei (Riga, USSR) or a mixed culture of T. reesei and Endomycopsis fibuliger (R-574) in fermentation equipment of various design: some with mixing, some with stationary layers, including a mixedlayer 1.5-m(3) pilot plant scale fermenter. The best protein productivity was obtained in stationary layer fermenters with a product containing 13% protein. The main limitations of lignocellulose SSF, such as hindrance of fungal growth, limiting accessibility and availability of substrate, and difficulty in moisture and heat control, were analyzed. The technological parameters of SSF, submerged fermentation, and alternate lignocellulose conversion processes were compared. The SSF had lower overall efficiency but higher product concentration per reaction volume than other conversion schemes.     

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.     

Utilization of dry distiller’s grain and solubles as nutrient supplement in the simultaneous saccharification and ethanol fermentation at high solids loading of corn stover

Dry distiller’s grain and solubles (DDGS) is a major by-product of corn-based ethanol production and is usually used as animal feed. Here, it was added to the simultaneous saccharification and ethanol fermentation (SSF) carried out at high solids loading of steam explosion pretreated corn stover using a mutant strain Saccharomyces cerevisiae DQ1. The performance of SSF process with DDGS was comparable to those using the expensive yeast extract supplementation. With 30% (w/w) solids plus the addition of cellulase and 1 g DDGS l⁻¹, the final ethanol reached 55 g l⁻¹ (7% v/v). The results indicated that the expensive supplement of yeast extract could be replaced by DDGS.     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was grown at 45°C on media containing 2, 4 and 6 % (w/v) pulverized barley straw and supplemented with 2% (v/v) cellulase. Maximum ethanol concentrations produced were 2, 3 and 3.6g/l, respectively. When the pulverized straw was replaced by NaOH pretreated straw (at 2, 4 and 6% (w/v); based on original untreated straw), ethanol concentrations increased to maxima of 3.9, 8, and 12g/l, respectively. The ethanol yields amount to 20g ethanol from 100g of straw.     

SSF of steam-pretreated wheat straw with the addition of saccharified or fermented wheat meal in integrated bioethanol production

Background

Integration of second-generation (2G) bioethanol production with existing first-generation (1G) production may facilitate commercial production of ethanol from cellulosic material. Since 2G hydrolysates have a low sugar concentration and 1G streams often have to be diluted prior to fermentation, mixing of streams is beneficial. Improved ethanol concentrations in the 2G production process lowers energy demand in distillation, improves overall energy efficiency and thus lower production cost. There is also a potential to reach higher ethanol yields, which is required in economically feasible ethanol production. Integrated process scenarios with addition of saccharified wheat meal (SWM) or fermented wheat meal (FWM) were investigated in simultaneous saccharification and (co-)fermentation (SSF or SSCF) of steam-pretreated wheat straw, while the possibility of recovering the valuable protein-rich fibre residue from the wheat was also studied.

Results

The addition of SWM to SSF of steam-pretreated wheat straw, using commercially used dried baker’s yeast, S. cerevisiae, resulted in ethanol concentrations of about 60 g/L, equivalent to ethanol yields of about 90% of the theoretical. The addition of FWM in batch mode SSF was toxic to baker’s yeast, due to the ethanol content of FWM, resulting in a very low yield and high accumulation of glucose. The addition of FWM in fed-batch mode still caused a slight accumulation of glucose, but the ethanol concentration was fairly high, 51.2 g/L, corresponding to an ethanol yield of 90%, based on the amount of glucose added.In batch mode of SSCF using the xylose-fermenting, genetically modified S. cerevisiae strain KE6-12, no improvement was observed in ethanol yield or concentration, compared with baker’s yeast, despite the increased xylose utilization, probably due to the considerable increase in glycerol production. A slight increase in xylose consumption was seen when glucose from SWM was fed at a low feed rate, after 48 hours, compared with batch SSCF. However, the ethanol yield and concentration remained in the same range as in batch mode.

Conclusion

Ethanol concentrations of about 6% (w/v) were obtained, which will result in a significant reduction in the cost of downstream processing, compared with SSF of the lignocellulosic substrate alone. As an additional benefit, it is also possible to recover the protein-rich residue from the SWM in the process configurations presented, providing a valuable co-product.

     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

Production of multiple extracellular enzyme activities by novel submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis> for ethanol production from raw cassava flour

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.     

湿氧化爆破稻秆的同步糖化发酵条件研究

探讨了木质纤维素经过湿氧化爆破后在同步糖化发酵过程中酵母产乙醇的基本规律.采用单因素方法对湿氧化爆破条件、酶系组成和添加量以及预酶解时间和温度进行了优化.不同湿氧化爆破预处理条件下的稻秆对同步糖化发酵工艺的影响较大,在预处理温度160 ℃,进氧压力为4×105 Pa,碱用量为6%(w/w),反应时间为20 min的条件...     

Cellulosic ethanol production on temperature-shift simultaneous saccharification and fermentation using the thermostable yeast Kluyveromyces marxianus CHY1612

In cellulosic ethanol production, use of simultaneous saccharification and fermentation (SSF) has been suggested as the favorable strategy to reduce process costs. Although SSF has many advantages, a significant discrepancy still exists between the appropriate temperature for saccharification (45-50 °C) and fermentation (30-35 °C). In the present study, the potential of temperature-shift as a tool for SSF optimization for bioethanol production from cellulosic biomass was examined. Cellulosic ethanol production of the temperature-shift SSF (TS-SSF) from 16 w/v% biomass increased from 22.2 g/L to 34.3 g/L following a temperature shift from 45 to 35 °C compared with the constant temperature of 45 °C. The glucose conversion yield and ethanol production yield in the TS-SSF were 89.3% and 90.6%, respectively. At higher biomass loading (18 w/v%), ethanol production increased to 40.2 g/L with temperature-shift time within 24 h. These results demonstrated that the temperature-shift process enhances the saccharification ratio and the ethanol production yield in SSF, and the temperature-shift time for TS-SSF process can be changed according to the fermentation condition within 24 h.     

基因组重排技术选育乙醇高产菌株

里氏木霉（Trichoderma reesei）被认为是最合适联合生物加工（consolidated bioprocessing）的微生物之一。原始里氏木霉菌株产乙醇能力太低,需要进一步提高其产酒量。我们通过基因组重排技术提高了里氏木霉菌株产乙醇能力和乙醇耐受力。首先对CICC40360菌株孢子进行NTG诱变得到正向突变菌株,再以此为出发菌株进行基因组重排。进行基因组重排后,重组菌株在含不同乙醇浓度的原生质体再生培养基上进行筛选。突变菌株和原始菌株一起做摇瓶发酵实验进行比较以确定产乙醇能力的提高。经过两轮基因组重排后,筛选获得表现最优异的重组菌S2-254。该菌株能在利用50g/l葡萄糖发酵出6.2g/l乙醇,同时能耐受3.5% （v/v）浓度乙醇。上述结果表明,本实验采用的基因组重排技术能够有效而且快速获得具有目的性状的优良菌株。     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Selection and adaptation of Saccharomyces cerevisae to increased ethanol tolerance and production

A total of 24 yeast strains were tested for their capacity to produce ethanol, and of these, 8 were characterized by the best ethanol yields (73.11-8 1.78%). The most active mutant Saccharomyce s cerevisiae ER-A, resistant to ethanol stress, was characterized by high resistance to acidic (pH 1.0 and 2.0), oxidative (1 and 2% of H2O2), and high temperature (45 and 52 degrees C) stresses. During cultivation under all stress conditions, the mutants showed a considerably increased viability ranging widely from about 1.04 to 3.94-fold in comparison with the parent strain S. cerevisiae ER. At an initial sucrose concentration of 150 g/l in basal medium A containing yeast extract and mineral salts, at 300C and within 72 h, the most active strain, S. cerevisiae ER-A, reached an ethanol concentration of 80 g/1, ethanol productivity of 1.1 g/Il/h, and an ethanol yield (% of theoretical) of 99.13. Those values were significantly higher in comparison with parent strain (ethanol concentration 71 g/1 and productivity of 0,99 g/l/h). The present study seems to confirm the high effectiveness of selection of ethanol-resistant yeast strains by adaptation to high ethanol concentrations, for increased ethanol production.     

The effect of glucose on the expression of extracellular protein genes by Staphylococcus aureus strain V8

The bacteria from overnight cultures (20 h) of S. aureus V8 and exp negative mutant K6812-1, grown, aerobically, in 3% (w/v) Tryptone Soya Broth, at 37 degrees C, were resuspended in fresh medium, in the case of the parent strain +/- 1% (w/v) glucose, without change in bacterial density. During a 6 h incubation period there was an approximate doubling of bacterial density, to the same level, in each case. However, exoprotein production by the mutant was only 20% that of the parent whilst the addition of glucose to the V8 strain resulted in a tenfold reduction in the exoprotein formed. SDS-polyacrylamide gel electrophoresis showed that the exoprotein patterns of both organisms after 6 h incubation were the same as those observed in the overnight cultures whilst the presence of 1% (w/v) extra glucose changed the pattern produced by the parent to one similar to that of the mutant. The results showed that conditions which lead to the rapid formation of glucose catabolites produced an effect consistent with the inhibition of the activity of the exp gene product.     

Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading

Ethanol production by a recombinant bacterium from wheat straw (WS) at high solid loading by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid pretreated WS (150 g/L) after enzymatic saccharification was 86.3 ± 1.5 g/L. The pretreated WS was bio-abated by growing a fungal strain aerobically in the liquid portion for 16 h. The recombinant Escherichia coli strain FBR5 produced 41.1 ± 1.1 g ethanol/L from non-abated WS hydrolyzate (total sugars, 86.6 ± 0.3 g/L) in 168 h at pH 7.0 and 35 °C. The bacterium produced 41.8 ± 0.0 g ethanol/L in 120 h from the bioabated WS by SHF. It produced 41.6 ± 0.7 g ethanol/L in 120 h from bioabated WS by fed-batch SSF. This is the first report of the production of above 4% ethanol from a lignocellulosic hydrolyzate by the recombinant bacterium.