| 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. |
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