拜氏梭菌13-2发酵甘蔗渣水解液生产丁醇的研究

目的:蔗渣是一种重要的可再生生物质资源,蔗渣原料生产丁醇将大大降低丁醇的成本.方法:实验利用0.25 ～3.0%不同浓度稀H2SO4对蔗渣进行121℃的高温作用1h,以水解液为碳源,进行丁醇的发酵实验.结果:相对于8052菌株,13 -2菌株对甘蔗渣水解液具有更高的发酵效率,在0.5%硫酸用量条件下,13 -2菌株的丁醇发酵量最高,达到4.5g/L.而8052只有2.3g/L的丁醇发酵量.结论:在同等条件下,拜氏梭菌菌株13 -2比模式菌株8052具有更高的溶剂产量和抑制物耐受能力,最佳的蔗渣水解条件为1.5%硫酸用量,丁醇发酵量和总溶剂分别为4.57g/L和5.41 g/L.     

种泥预处理和发酵温度对暗发酵产氢的影响研究进展

生物质暗发酵产氢不仅可以处理有机废物,同时可以获得清洁能源,实现了废物资源化利用。然而产氢种泥中大量耗氢菌的存在会导致暗发酵氢气产量低等问题,因此种泥预处理是暗发酵产氢的必需条件。随着暗发酵产氢基质的多样化,产氢种泥的预处理方法也不断发展。对近十年来产氢种泥预处理方法的发展进行了综述,并且结合发酵温度,讨论了种泥预处理方法和发酵温度两方面条件对暗发酵产氢的影响,并对该研究方向提出了展望,以期为后续暗发酵产氢的进一步研究提供参考。     

种泥预处理和发酵温度对暗发酵产氢的影响研究进展

生物质暗发酵产氢不仅可以处理有机废物,同时可以获得清洁能源,实现了废物资源化利用。然而产氢种泥中大量耗氢菌的存在会导致暗发酵氢气产量低等问题,因此种泥预处理是暗发酵产氢的必需条件。随着暗发酵产氢基质的多样化,产氢种泥的预处理方法也不断发展。本文对近十年来产氢种泥预处理方法的发展进行了综述,并且结合发酵温度,讨论了种泥预处理方法和发酵温度两方面条件对暗发酵产氢的影响,并对该研究方向提出了展望,以期为后续暗发酵产氢的进一步研究提供参考。     

灵芝菌株固体发酵对枇杷叶主要活性成分的影响

采用灵芝固体发酵枇杷叶,筛选出了适合发酵的灵芝菌株并分析了固体发酵前后枇杷叶中主要活性成分的变化。本文选用8个灵芝菌株,根据不同灵芝菌株在枇杷叶基质中的生长情况选出最适合发酵的菌株,并通过灵芝菌株固体发酵三个品种的枇杷叶,研究发酵前后三种枇杷叶中多糖、总黄酮、三萜、总蛋白等活性成分的变化。结果表明:适宜发酵的灵芝菌株为松杉灵芝,经过灵芝菌株发酵枇杷叶后,不同品种的枇杷叶中多糖平均增长率为15.40%、总蛋白平均增长率为43.60%、三萜含量平均降低30.08%、黄酮含量大幅度降低视为未检出。     

发酵白酒糟生产饲料蛋白的优良菌种的筛选

采用常规方法从1000多株菌(包括丝状真菌、酵母菌、链霉菌、细菌)中筛选到一批优良菌种,并进行了单菌发酵、多菌株组合发酵,不同原料配方发酵试验。在实验室条件下,发酵产物的粗蛋白含量高达35.9％,比原料本身的粗蛋白含量高50％以上,比所用培养基的粗蛋白含量高30％,发酵产物的粗纤维含量降低率为15％;粗脂肪含量为5.5％左右;产率达80％以上。结果证明,筛选到的菌株确是发酵白酒糟生产饲料蛋白的优良菌种。     

利用甘蔗糖蜜发酵生产花生四烯酸的研究

通过培养高山被孢霉利用糖蜜来发酵生产花生四烯酸(ARA),研究了不同甘蔗糖蜜预处理方法对ARA发酵生产的影响。研究表明:H2SO4法是最利于ARA发酵生产的糖蜜预处理方法。利用预处理的甘蔗糖蜜发酵生产ARA,通过单因素实验设计,确定了最优的培养条件,包括初始还原糖80 g/L,N源6 g/L,接种量20%,初始pH6.0和培养温度25℃,在此条件下发酵,干细胞质量、油脂含量、ARA产量和糖利用率分别达到28.5 g/L、11.7g/L、3.68 g/L和94.5%。     

基因工程菌的发酵研究

本文对大肠杆菌表达的ｒｈＧＭ－ＣＳＦ工程菌的发酵条件进行了详细的研究,探讨了发酵条件对工程菌表达外源蛋白量的影响,优化了影响发酵的各种条件,形成了一套工程菌发酵表达外源蛋白的工艺,并从工业化角度对工程菌的高密度高表达间的关系进行了探讨。     

基因工程菌的发酵研究

本文对大肠杆菌表达的rhGM - CSF 工程菌的发酵条件进行了详细的研究, 探讨了发酵条件对工程菌表达外源蛋白量的影响, 优化了影响发酵的各种条件, 形成了一套工程菌发酵表达外源蛋白的工艺, 并从工业化角度对工程菌的高密度高表达间的关系进行了探讨。     

响应曲面优化秸秆稀酸水解工艺用于发酵产壳聚糖

为实现利用秸秆水解产生的五碳糖发酵产壳聚糖,以米根霉为研究对象,研究水解温度、水解时间、酸浓度等不同预处理方式获得的半纤维素水解液对米根霉发酵产壳聚糖的影响。结果表明:水解温度、水解时间对水解液中木糖含量以及甲酸、乙酸、糠醛等抑制剂浓度具有显著影响,并进一步影响后续发酵产壳聚糖的生成量。利用响应曲面对稀酸水解预处理条件进行优化,获得最佳工艺条件:H_2SO_413.6 g/L,99.5℃,水解时间1.91 h,在此条件下预测壳聚糖发酵产量为0.79 g/L,实验验证产量为0.82 g/L,占菌体生物量的15%~18%。研究结果为秸秆资源的高效利用及发酵生产壳聚糖提供新思路。     

基因重组Echistatin发酵工艺的优化

目的:利用基因工程方法对一种蛇毒锯鳞蝰素蛋白的发酵纯化工艺进行优化,以提高目的蛋白的产量和纯度。方法:对工程菌进行发酵培养并诱导表达,研究不同的培养基、不同补料方式、溶解氧浓度、培养和诱导时间对工程菌产量和目的蛋白表达量的影响,利用几丁质亲和层析纯化Ecs融合蛋白,通过合适温度和pH裂解融合蛋白得到Ecs纯品,并鉴定和检测Ecs活性。结果:经过高密度发酵优化后,菌体湿重可达110g/L,目的蛋白表达量约占总蛋白的40%;亲和层析纯化后,得到Ecs单体,得率为68mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。结论:通过发酵和纯化工艺优化,大大提高了目的蛋白产量,为进一步规模化研究和生产奠定了基础。     

Chemical characteristics and ethanol fermentation of the cellulose component in autohydrolyzed bagasse

The chemical characteristics, enzymatic saccharification, and ethanol fermentation of autohydrolyzed lignocellulosic material that was exposed to steam explosion were investigated using bagasse as the sample. The effects of the steam explosion on the change in pH, organic acids production, degrees of polymerization and crystallinity of the cellulose component, and the amount of extractive components in the autohydrolyzated bagasse were examined. The steam explosion decreased the degree of polymerzation up to about 700 but increased the degree of crystallinity and the micelle width of the cellulose component in the bagasse. The steam explosion, at a pressure of 2.55 MPa for 3 mins, was the most effective for the delignification of bagasse. 40 g/L of glucose and 20 g/L of xylose were produced from 100 g/L of the autohydrolyzed bagasse by the enzymatic saccharification using mixed cellulases, acucelase and meicelase. The maximum ethanol concentration, 20 g/L, was obtained from the enzymatic hydrolyzate of 100 g/L of the autohydrolyzed bagasse by the ethanol fermentation usingPichia stipitis CBS 5773; the ethanol yield from sugars was 0.33 g/g sugars.     

Radiation and fermentation treatment of cellulosic wastes

The effect of radiation pasteurization of sugar cane bagasse and rice straw and fermentation using various strains of fungi were studied for upgrading of cellulosic wastes. The initial contamination by fungi and aerobic bacteria both in bagasse and straw was high. The doses of 30 kGy for sterilization and 8 kGy for elimination of fungi were required. Irradiation effect showed that rice straw contained comparatively radioresistant microorganisms. It was observed that all the fungi (Hericium erinacium, Pleurotus djamor, Ganoderma lucidum, Auricularia auricula, Lentinus sajor-caju, Coriolus versicolor, Polyporus arcularius, Coprinus cinereus) grow extending over the entire substrates during one month after inoculation in irradiated bagasse and rice straw with 3% rice bran and 65% moisture content incubated at 30°C. Initially, sugar cane bagasse and rice straw substrates contained 39.4% and 25.9% of cellulose, 22.9% and 26.9% of hemicellulose, and 19.6% and 13.9% of lignin + cutin, respectively. Neutral detergent fibre (NDF) values decreased significantly in sugar cane bagasse fermented byG. lucidum, A. auricula andP. arcularius, and in rice straw fermented by all the 8 strains of fungi. Acid detergent fibre (ADF) values also decreased in bagasse and rice straw fermented by all the fungi.P. arcularius, H. erinacium, G. lucidum andC. cinereus were found to be the most effective strains for delignification of sugar cane bagasse.     

Production of SCP and cellulase by Aspergillus terreus from bagasse substrate

The fermentation of 1.0% untreated bagasse under optimum cultural and nutritional conditions with Aspergillus terreus GN1 indicated that the maximum rate of protein and cellulase production could be obtained during three days of submerged fermentation. Even though 16.4% protein recovery, 0.55 units CMCase/mL, and 0.027 FPase units/mL were obtained on the seventh day, the rates of increase in protein recovery and cellulase production were slower than those obtained up to these days, which were 14.3% protein recovery, 0.45 units CMCase/mL, and 0.019 units FPase/mL. There was an initial lag in the utilization of cellulose up to two days due to the utilization of the water-soluble carbohydrate present in untreated bagasse. Cellulose utilization and water-soluble carbohydrate content during fermentation were correlated with protein recovery and enzyme production. The protein and cellulase production during three days fermentation with 1.0% untreated and treated bagasse were compared and the protein content of the total biomass was calculated and treated bagasse were compared and the protein content of the biomass was calculated into constituent protein contributed by the fungal mycelium and the under graded bagasse. The total biomass recovered with untreated and treated bagasse was 1020 and 820 mg/g bagasse substrate, respectively, and contained 14.3 and 20.6% crude protein, respectively. The contribution of fungal biomass and under graded bagasse was 309 and 711, and 373 and 447 mg/g untreated and treated bagasse substrates, respectively. In an 8-L-flask trial during three days of fermentation, the recovery of SCP and cellulase were 66 g and 32,400 units (Sigma) for treated bagasse and 82 g and 8200 units (Sigma) for untreated bagasse, respectively.     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.     

Effect of nutritional factors on cellulase enzyme and microbial protein production by Aspergillus terreus and its evaluation

The biomass yield, cellulolytic activity, and protein recovery using Aspergillus terreus GN1 with alkali-treated sugarcane bagasse was studied using different levels (250-600 mg of N/L of broth) of organic and inorganic nitrogen sources. e.g., cattle urine, urea, cornsteep liquor, ammonium sulfate, ammonium nitrate, ammonium iron sulfate, ammonium chloride, and sodium nitrate. Among different levels of alkali-treated bagasse substrate concentrations (0.5-4.0% w/v) tested, 1.0% substrate yielded the highest crude protein content, protein recovery, and cellulolytic activity. The biomass recovery with 1.0% substrate ranged from 290-380 mg/500 mg bagasse substrate in a 50-mL broth with a nitrogen level of 250-600 mg of N/L in all the sources except ammonium iron sulfate, which yielded 402-439 mg/500 mg bagasse substrate. However, crude protein content of biomass obtained with an ammonium iron sulfate nitrogen source was the lowest. Cornsteep liquor nitrogen source at the rate of 600 mg of N/L yielded the maximum crude protein of 32.9%, protein recovery of 22.2 g/100 g of bagasse, and carboxymethyl cellulase and filter paper enzyme activities of 1.1 and 0.09 units/mL, among the organic and inorganic nitrogen sources studied. In general, the organic nitrogen sources and inorganic nonammonium nitrogen sources were utilized preferentially for protein production over the inorganic ammonium nitrogen sources. The fermentation time required under optimum cultural and nutritional conditions for A. terreus GN1 was also evaluated. The crude protein content of the biomass increased gradually up to the seventh day of fermentation, but the protein recovery rate was high up to two or three days. It was observed that the cellulose utilization rate increased after an initial lag of one day up to the third day and gradually increased further, which corresponded positively with protein content, biomass protein recovery, and cellulase enzyme activity. On the seventh day of fermentation, the crude protein content, biomass protein recovery, water-soluble carbohydrate, bagasse cellulose utilization, CMCase, and FPase activities were 32.8%, 20.1 g/100 g of bagasse, 6.2%, 82.7%, 1.0. and 0.08 U/mL, respectively. The final biomass recovered contained 32.8% crude protein content and had an in vitro rumen digestibility (IVRD) coefficient of 68.8%. The biomass contained almost all the essential and nonessential amino acids and was comparable with FAO reference protein. It is concluded that a fermentation time of 72 h gave a faster rate of protein production of 16.9 g/100 g of bagasse with 69.8% bagasse cellulose utilization with 76.0% IVRD. and contained almost all the essential and nonessential amino acids.     

Enzyme hydrolysis and ethanol fermentation of dilute ammonia pretreated energy cane

This study is the first one ever to report on the use of high fiber sugarcane (a.k.a. energy cane) bagasse as feedstock for the production of cellulosic ethanol. Energy cane bagasse was pretreated with ammonium hydroxide (28% v/v solution), and water at a ratio of 1:0.5:8 at 160 °C for 1 h under 0.9-1.1 MPa. Approximately, 55% lignin, 30% hemicellulose, 9% cellulose, and 6% other (e.g., ash, proteins) were removed during the process. The maximum glucan conversion of dilute ammonia treated energy cane bagasse by cellulases was 87% with an ethanol yield (glucose only) of 23 g ethanol/100 g dry biomass. The enzymatic digestibility was related to the removal of lignin and hemicellulose, perhaps due to increased surface area and porosity resulting in the deformation and swelling of exposed fibers as shown in the SEM pictures.     

Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.     

Sugarcane Internode Composition During Crop Development

Sugarcane sugar and bagasse can be utilized for the production of ethanol or other biofuels. A better understanding of the changes in composition with development along the stalk and with crop development will maximize the usage of sugarcane for this purpose. Two experiments were designed to elucidate internode composition changes during the growing season. In experiment 1, an internode of stalks of 5 modern cultivars were marked at the start of elongation, and then sampled every 1 to 2?weeks from July until October. Sugars were extracted and assayed, and a sequential detergent method was used to estimate hemicellulose, cellulose, and lignin contents. In experiment 2, internodes 1, 3, 5, 7, 9, and 11 down the stalk were sampled in late July (grand growth) and late September (ripening). Internode length, fresh weight, dry weight, water content, and sugar contents were determined as well as cell wall composition. Both experiments were repeated in 2?years. As internodes elongated, total sugar increased, and hemicellulose decreased as a proportion of neutral detergent fiber, while cellulose and lignin increased. After elongation, sucrose and lignin increased, and cellulose content decreased with internode age. The variability in cell wall composition among the five cultivars suggests that selection for desirable composition may be possible. In Experiment 2, hemicellulose contents were lower, and lignin and ash contents were higher at ripening than during grand growth. Delaying sugarcane harvest to maximize sucrose content may decrease bagasse suitability for cellulosic ethanol production because of the increased lignin content.     

Production of bioactive polysaccharides by Inonotus obliquus under submerged fermentation supplemented with lignocellulosic biomass and their antioxidant activity

The effect of lignocellulose degradation in wheat straw, rice straw, and sugarcane bagasse on the accumulation and antioxidant activity of extra- (EPS) and intracellular polysaccharides (IPS) of Inonotus obliquus under submerged fermentation were first evaluated. The wheat straw, rice straw, and sugarcane bagasse increased the EPS accumulation by 91.4, 78.6, and 74.3 % compared with control, respectively. The EPS and IPS extracts from the three lignocellulose media had significantly higher hydroxyl radical- and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the control medium. Of the three materials, wheat straw was the most effective lignocellulose in enhancing the mycelia growth, accumulation and antioxidant activity of I. obliquus polysaccharides (PS). The carbohydrate and protein content, as well as the monosaccharide compositions of the EPS and IPS extracts, were correlated with sugar compositions and dynamic contents during fermentation of individual lignocellulosic materials. The enhanced accumulation of bioactive PS of cultured I. obliquus supplemented with rice straw, wheat straw, and bagasse was evident.     

Preparation and properties of HDPE/sugarcane bagasse cellulose composites obtained for thermokinetic mixer

The use of natural fibers as reinforcement for thermoplastics has generated much interest due to their low cost, possibility of environmental protection and use of locally available renewable resources. In this work the mechanical and morphological properties of high density polyethylene/pre-treated and modified residues from sugarcane bagasse cellulose composites were analyzed. Composites were produced by a thermokinetic mixer. The microstructural analyses of fracture surface from composites can be easily evaluated by microscopic techniques. Results showed that the modification of sugarcane bagasse cellulose with zirconium oxychloride was successfully accomplished and that this reinforcement material with high density polyethylene showed tensile strength higher than non-modified sugarcane bagasse cellulose. Modification in the sugarcane bagasse cellulose influenced directly in mechanical properties of the composite material. This can be observed by the fracture surface, which showed that modified cellulose sugarcane bagasse improved interfacial adhesion between fiber and matrix.