【作者】 郭香；

【导师】 洪枫；

【作者基本信息】 东华大学 ， 纺织化学与染整工程， 2014， 博士

【摘要】 细菌纤维素(bacterial cellulose,简称BC)是一种由微生物产生的具有纳米结构的纤维素,因此,也称为细菌纳米纤维素。与植物纤维素相比它具有许多优良的特性,譬如：高持水性、高聚合度、高结晶度、高纯度、良好的生物相容性和高机械强度,因此在生物医学材料、食品、高级音响振动膜、燃料电池、造纸和纺织等领域具有广阔的应用前景。由于细菌纤维素生产成本偏高,尤其是培养基成本偏高,限制了其规模化工业生产和商业应用。纤维素废料是地球上最丰富的可再生聚糖类资源,可作为生物炼制的碳源。由于其来源广、价格低,因此最有希望用于细菌纤维素的规模化低成本发酵生产。本文从细菌纤维素规模化工业生产和纤维素废料资源化利用的多角度出发,利用不同来源的纤维素废料开发细菌纤维素的生物炼制技术。由于原料的结构和化学组成不同,在生物炼制过程中所面临的问题也不尽相同。因此,本文针对高效生物利用木质纤维素资源的共性关键技术,分别以纤维素行业(纺织和造纸)中产生的三种不同纤维素废料为原料模型,研究探讨这三种原料制备细菌纤维素的可行性,重点研究制备细菌纤维素过程中所遇到的关键技术问题。主要包括原料水解前预处理、水解液脱毒、降低纤维素酶水解成本、发酵废液资源化利用和废液减排等核心问题。通过对以上问题的研究,开发针对不同类型纤维素废料制备细菌纤维素的共性关键技术,为今后的细菌纤维素规模化工业生产提供必需的技术路线,并倡导资源节约型理念。本文的主要研究内容和结果如下：1、以云杉木屑为原料,经SO2预处理、酶水解获得可发酵糖以制备细菌纤维素。重点比较了多种脱毒方法对水解液中抑制物的去除效果,并研究了水解液脱毒前后对木醋杆菌生长和细菌纤维素产量的影响。结果显示活性炭吸附脱毒对抑制物去除效果最好,在四大类主要抑制物的去除中效率最高,分别去除了94%的醛类、88%的酚类、39%的甲酸和28%的乙酸,因此BC产量也最高。木醋杆菌主要利用葡萄糖,少量利用木糖和甘露糖,几乎不利用阿拉伯糖和半乳糖。乙酸比甲酸更易被细菌代谢利用,糠醛和5-羟甲基糠醛除了挥发外还应该可以被细菌消耗或转化为其它化合物。通过酚氧化酶的专一性去除酚类抑制物的实验结果表明,水解液中的酚类相对于弱酸和呋喃醛类抑制物有更强的抑菌性,是今后木质纤维素水解液制备细菌纤维素工艺中必须重点除去的抑制物。2、以硫酸盐和亚硫酸盐纸浆废料为原料,经直接酶水解获得可发酵糖以制备细菌纤维素。重点探讨以酶水解液为碳源制备细菌纤维素及其发酵废液资源化利用制备木质纤维素水解酶的可行性。结果显示,两种纸浆废料经水解后都能很好地作为碳源生产细菌纤维素。硫酸盐和亚硫酸盐纸浆酶解液制备的细菌纤维素产量分别可达11和10g/L,但是里氏木霉利用两种发酵废液的产酶情况差异巨大。硫酸盐纸浆发酵废液为速效碳源,补充硫酸盐纸浆为诱导物时,纤维素酶活力可达5.2U/mL,与葡萄糖为速效碳源获得的活力(5.1U/mL)相当,然而木聚糖酶活力高达74.7U/mL,比葡萄糖对照组的22.6U/mL局了3倍多。以亚硫酸盐纸浆发酵废液为速效碳源,亚硫酸盐纸浆为诱导物时,纤维素酶和木聚糖酶的活力都很低,但是分别以发酵废液和亚硫酸盐纸浆与其它碳源结合时都能获得接近于葡萄糖对照组的活力。综合分析后发现,可能是亚硫酸盐纸浆废料发酵液结合亚硫酸盐纸浆废料会产生强抑制,不利于产酶。为寻找其中可能的抑制物,探讨在培养基中添加小于100mM的Na2SO4和小于10mM的Na2SO3对里氏木霉产酶的影响。结果发现100mM浓度以内的硫酸钠对提升纤维素酶和木聚糖酶活力都有一定作用,尤其促木聚糖酶活力明显。5mM以内的亚硫酸盐也具有一定的促产酶效果,但是当浓度达到10mM时会强烈抑制产酶。生物炼制企业的发酵废液资源化利用产纤维素水解酶,不仅可以减少废液排放,而且可以充分利用原料资源,获得的水解酶用于补充木质纤维素原料水解用酶以降低酶解成本。3、以废弃棉织物为原料,通过新型绿色溶剂——离子液体[AMIM]Cl预处理以提高酶水解效率和得糖率,成功制备细菌纤维素。获最佳酶解效果所需的处理工艺为：1g棉布在110℃下溶于10g离子液体。再生棉纤维素中残余[AMIM]Cl浓度高于20mg/mL时将抑制纤维素酶的活性；浓度高于0.5mg/mL时会降低BC产量。研究发现,当活性染料浓度超过5g/L时也会抑制纤维素酶活力,降低酶水解效率；当浓度大于1g/L时,则会抑制BC产量。预处理后棉织物的酶解得糖率可达95%以上,是未处理的3-6倍。棉织物酶解液可以成功制备BC,产量比葡萄糖对照组高出约2倍。离子液体预处理显著提高酶解效率和得糖率,为木质纤维素资源的高效利用提供了新途径。4、为了进一步研究发酵废液产酶的机理,本文研究了不同微生物源的发酵废液对里氏木霉生产木质纤维素水解酶的影响。结果表明利用木醋杆菌、大肠杆菌和金黄色葡萄球菌的发酵废液都可以作为里氏木霉的培养基组分生产纤维素酶和木聚糖酶。纤维素酶活力与葡萄糖对照组的相当,而木聚糖酶活力比对照组的高3-15倍。促进产酶的原因可能包括：发酵废液中剩余的原培养基成分(譬如：氮源和无机盐等),以及微生物的代谢物(譬如：寡糖等)。更多还原

【Abstract】 Bacterial cellulose (BC) is a nanostructured polymer product of some bacteria, which is sometimes named bacterial nanocellulose. Compared to plant cellulose, the nanofibril network of BC has unique properties, such as excellent water-holding capacity, high degree of polymerization, high crystallinity, high purity, good biocompatibility, and excellent mechanical properties. Therefore, BC has a great potential in wide applicationas including biomedical materials, health foods, high-quality audio membranes, functional paper, fuel cell membranes, and textiles. However, the cost of BC production is very high, principally due to the high cost of culture medium, which heavily prevents the large-scale industrial production and commercial application of BC. Cellulosic waste is the most abundant renewable polysaccharide resource in the world, and it can be developed as feedstock for biorefinery. Because of its abundant sources and relatively cheap price, it is the most promising raw material for large-scale and low-cost production of BC. From the perspective of the large-scale industrial production of BC and the utilization of waste cellulosic resource, in this work biorefinery technologies of Gluconacetobacter xylinus for production of BC was studied by using three different cellulosic wastes as fermentation feedstocks.Because the structure and chemical composition of cellulosic wastes are different, the key problems faced in the biorefinery process are also different. Therefore, for the common key technologies of the utilization of lignocellulosic resources, in this thesis the feasibility of the bacterial cellulose production by using three different cellulosic wastes that were used as models of raw materials for lignocellulose feedstocks, such as spruce chips, waste fiber sludges and waste cotton-based fabrics from textile and paper-making industry was investigated, and the key technical problems were focused on for those encountered in the process of BC production by using the cellulosic wastes. The problems contain four parts,(i) raw materials pretreatment before enzymatic hydrolysate,(ii) detoxification of lignocellulosic hydrolysate,(iii) decreasing the cost of cellulase,(iv) utilization of spent fermentation liquid and waste stream emission reduction. Through the study of the key technical problems, common key technologies in BC production were developed in order to use different types of cellulose waste as feedstock. The study provided the necessary technical routes for large-scale industrial production of BC in the future and pioneered the resource-saving concept. The major contents and results of the dissertation are summarized as follows:1. For spruce wood chips as raw material, an enzymatic hydrolysate of spruce wood was prepared after SO2-pretreatment to obtain fermentable sugars for BC production. The effects of different detoxification methods were compared by investigating the impact on the concentrations of potential fermentation inhibitors, as well as on the growth of G. xylinus and BC production in the spruce hydrolysate before and after detoxification. Among the different treatments, the activated charcoal treatment was most efficient, removing94%furans,88%total phenolics,39%formic acid and28%acetic acid, and therefore the highest yield of BC was obtained. Glucose was the main nutrient source and it was consumed efficiently in all cultivation. Some of xylose and mannose was also consumed, but arabinose and galactose with used little. It is expected that acetic acid would be more easily metabolized than formic acid by G. xylinus. The decrease in the concentration of furfural and5-hydroxymethyl-furfural in the cultures that obtained BC is probably due to a combination of bioconversion and evaporation. Through the detoxification experiments with phenol-oxidizing enzymes that specifically remove phenolic compounds, phenolic compounds were identified to be key fermentation inhibitors in the production of BC by G. xylinus, while furan aldehydes and aliphatic acids probably play a less important role. Therefore, phenolic compounds must be removed for the BC production with lignocellulose hydrolysate.2. For sulfate fiber sludge (SAFS) and sulfite fiber sludge (SIFS) as raw materials, the fiber sludge were hydrolyzed enzymatically without prior thermochemical pretreatment and the resulting hydrolysates were used for BC production. The objectives of this study were to investigate the feasibility of using waste fiber sludge for BC production, and the possibility to use the fermentation broth (spent hydrolysate) after harvesting BC to produce lignocellulose hydrolytic enzymes. It was shown that sulfate and sulfite fiber sludges were suitable raw materials for BC production. The highest volumetric yield of BC from the enzymatic hydrolysate of SAFS and SIFS was11g/L and10g/L (DW), respectively. But the enzyme production by Trichoderma reesei was different between SAFS and SIFS spent hydrolysates. The cellulase activity reached5.2U/mL when the SAFS spent hydrolysate supplemented with2%sulfate fiber sludge. And the cellulase activity was the same as the reference medium with glucose as carbon source (5.1U/mL). The xylanase activity from SAFS spent hydrolysate reached74.7U/mL, which was3-times higher than that of reference medium (22.6U/mL). The activity of cellulase and xylanase was very low when the SIFS spent hydrolysate was supplemented with2%sulfite fiber sludge. If the SIFS spent hydrolysate and SIFS were used to produce enzyme separately, the enzyme production was the same as the reference medium. It is concluded that sulfite fiber sludge would make some inhibition on the enzymes production by T. reesei only when combined with its spent fermentation broth. In order to identify possible inhibitors, the effects of Na2SO4(10-100mM) and Na2SO3(1-10mM) on enzyme production by T. reesei were investigated. It was shown that addition of Na2SO4would promote the cellulase and xylanase activity, especially the xylanase activity. Na2SO3of less than5mM could also promote the enzymes production, but it would make inhibition when increasing to10mM. That the hydrolytic enzymes were produced by using spent fermentation broth from biorefinery enterprises may not only reduce waste emissions but also take full advantage of resources. And the obtained hydrolytic enzymes can be supplemented to for the hydrolytic process of lignocellulosic feedstock in order to decrease the cost of enzymatic hydrolysis.3. For the waste cotton fabrics as raw material, a new green solvent [AMIM]Cl ionic liquid (IL) was chosen to dissolve cotton fabrics as a pretreatment to enhance the efficiency of enzymatic hydrolysis and sugar yield for bacterial cellulose production. The optimal pretreatment condition for enzymatic hydrolysis is as follows:1g cotton fabric was selected for the pretreatment at110℃in10g [AMIM]Cl. The cellulase activity would be partial inhibited when the concentration of residual [AMIM]Cl was higher than20mg/mL in regenerated cellulose, and the BC production was decreased when the concentration of [AMIM]Cl was higher than0.5mg/mL. It was indicated that the cellulase activity would be inhibited when the concentration of reactive dyes was higher than5g/L and reactive dyes of more than1g/L could decrease the BC production. IL-treated cotton fabrics exhibited higher enzymatic hydrolysis rate of up to95%and gave3-6times larger yield of reducing sugar. The enzymatic hydrolysates of cotton fabrics can be used as the carbon source for BC production and the yield of BC from cotton hydrolysate is2-times higher than that from reference medium with glucose. IL pretreatment can significantly enhance the efficiency of enzymatic hydrolysis and sugar yields, which would provide a new approach for efficient utilization of lignocellulosic resources.4. In order to get further study on the mechanisms of enzymes production by spent fermentation broth, in this work, the hydrolytic enzyme production by T. reesei was investigated by using various spent fermentation broth. It was shown that the spent fermentation broth from G. xylinum, Escherichia. coil, Staphylococcus. aureus served as good media for cellulase and xylanase production with T. reesei. The cellulase activities were the same as reference medium with glucose as carbon source, but the xylanase activities were much higher (3-15times) than those of reference medium. The possible reasons behind improvement of enzyme production may be ascribed to the residual medium composition in the spent fermentation broth, such as nitrogen sources and inorganic salts, and to the microbial metabolites, such as oligosaccharides.更多还原