【作者】 周伶俐；

【导师】 杨树林； 孙东平；

【作者基本信息】 南京理工大学 ， 生物化工， 2008， 博士

【摘要】 细菌纤维素是由微生物合成的一种生物纳米材料,与植物纤维相比,具有独特的三维网络结构和超细、纯度高、结晶度高、与水结合能力强和生物合成时的可调控等特点,成功应用于很多领域,是当今国内外生物材料研究的热点之一。目前国内大多数研究限于实验室水平,与国外工业化生产相差甚远。作者选育出细菌纤维素产生菌,并较为系统地研究了逐步放大的发酵工艺,考察了不同培养方式下得到的细菌纤维素在造纸和固定化细胞方面的初步应用。从残次水果样中筛选出产纤维素野生菌NUST1292,16S rDNA序列同源性分析结果结合形态、生理生化特征可鉴定其为Acetobacter属;后采用紫外诱变改良,选育出高产且生产性能稳定的菌株Acetobacter NUST4,采用TEM、AFM、GC-MS、FT-IR、XRD和CP/MAS ¹³C-NMR对菌体形态和发酵产物进行表征。较为系统地研究了Acetobacter NUST4静置培养和从摇瓶振荡培养、13L发酵罐振荡培养到500L罐培养的逐步放大的发酵工艺优化试验。结果是:①经优化条件后静置培养7d产9.87g·L^-1干纤维素;②从发酵罐中驯化分离到的AcetobacterNUST4.1,优化条件后摇瓶振荡培养5d产7.16g·L^-1干纤维素,同步静置产量为10.9g·L^-1,该摇瓶培养基更适合今后的工业化生产;考察了不同培养方式下的细菌纤维素的结构和性能的差异;③添加羧甲基纤维素钠、海藻酸钠和琼脂不仅能有效解决发酵罐中严重结团和菌株逐级放大后生产稳定性差的两大难题,并能显著提高纤维素产量,影响产物结构、热和机械性能;其中添加0.06%羧甲基纤维素钠后效果最明显,由此优化发酵罐基本工艺,能获得3.35g·L^-1干纤维素,是未优化的3.1倍,采用动静二步发酵法后纤维素产量提高到8.44g·L^-1;④Acetobacter NUST4.1在500L罐中的生产性能较稳定。将匀浆处理的发酵罐振荡培养得到的细菌纤维素（Y-ABC）添加到废纸浆中抄纸,发现Y-ABC能提高废纸纤维的机械性能和防水性;将匀浆处理的静置培养得到的细菌纤维素（Y-SBC）、Y-ABC分别添加到苇木浆中抄纸,发现与苇木浆配合抄的纸中添加Y-ABC后对纸张性能改善优于添加Y-SBC后。将动静二步发酵法得到的细菌纤维素直接固定化白腐真菌Z-4降解不同种类染料,发现游离菌和固定化菌能有效降解三苯甲烷类染料,尤其是孔雀石绿。白腐真菌Z-4经固定化后能耐高浓度孔雀石绿,在pH4.0～7.0时脱色降解时间比游离菌球短,固定化菌的强度较游离菌球高,重复稳定性好,可操作性强,显示细菌纤维素膜是一优良的固定化载体。更多还原

【Abstract】 Bacterial cellulose （BC）, a kind of nano-biomaterial, is produced by some microorganisms. It is of particular network structure composed of ultra-fine ribbon-shaped fibers. In comparison with plant cellulose, BC displays high purity, high crystallinity, high water-holding capacity, high mechanical strength and controllable during the course of biosynthesis and so on. Therefore, BC can be applied in areas where plant cellulose can hardly be used. Nowadays BC has been one of the most active topics in the materials field. But domestic research on BC mostly rests on the level of lab, which lags behind far away from abroad. In this dissertation, the screening of suitable cellulose-producing strains, systematical research on fermentation technologies of successive enlargement and preliminary application in paper-making and immobilization of microorganism were reported.Twelve cellulose-producing strains were efficiently isolated from rotten fruits. Strain NUST1292 belonged to Acetobacter according to morphologic, biochemical and physiological characteristic and the 16S rDNA sequence analysis. Then it was mutagenized with UV. The mutant Acetobacter NUST4 had higher productivity of cellulose. The morphology, structural units, chemical and crystal structure of fermentation products were investigated using TEM, AFM, GC-MS, FT-IR, XRD and CP/MAS ¹³C-NMR.The optimum fermentation technologies of successive enlargement from static culture and shake flask culture, 13L stirred-tank reactor to 500L reactor were systematically researched. The main results were as follows: （1） Using optimum technology, BC production by Acetobacter NUST4 reached 9.87g·L^-1 in stationary culture for 7 days; （2） Acetobacter NUST4 was repeatedly domesticated in the stirred-tank reactor. Acetobacter NUST4.1, a high BC producer under the shake condition was efficiently isolated. Using optimum technology, BC production by Acetobacter NUST4.1 reached 7.16g·L^-1 in shake flask culture for 5 days. In the same conditions, BC yield reached 10.9g·L^-1 in stationary culture for 5 days. Influence of culture modes on BC structure and properties was characterized by SEM, FT-IR, XRD, purity and water-absorption capacity measurement, TG-DTA and mechanical tensile test; （3） In order to solve two problems on serious clump forming and spontaneous appearance of cellulose nonproducers in agitated culture, carboxymethyl cellulose sodium （CMC-Na）, alginate sodium （NaAlg） and agar were added into the medium in shake flask and 13L reactor. It’s surprising that the addition of these polymers contributed to forming uniform suspension and enhancing BC production. Influence of addition of three polymers on BC structure and properties in the shake flask was characterized by SEM, FT-IR, XRD, TG-DTG and mechanical tensile test. With the addition of 0.06% CMC-Na in the stirred-tank reactor, BC production reached the maximum. Then using optimum fermentation technology, the production of 3.35g·L^-1 was achieved, which was 3.1-fold higher than the control. Later, using two-step fermentation technology, BC production was 8.44g·L^-1; （4） Acetobacter NUST4.1 had stable cellulose-producing performance in enlargement test with 500L reactor.Application of BC produced in static （Y-SBC） and stirred-tank reactor （Y-ABC） by homogenate treatment adding to the different kinds of pulp in paper-making was investigated. The fiber structures of different papers were observed by SEM and paper properties were analyzed. The results showed that adding Y-ABC into waste paper pulp could enhance mechanical and water-resistant properties, and adding Y-SBC and Y-ABC into unbleached reed-wood pulp could also enhance mechanical and water-resistant properties, and paper properties adding ABC was better than that adding SBC.Application of BC produced by two-step fermentation method in immobilization carrier of white rot fungi Z-4 to decolorize and degrade five dyes with different structures was investigated. Free and immobilized cells could efficiently degrade triarylmethane dyes. Especially they could thoroughly degrade malachite green. Comparing with free cells, immobilization could enhance the ability to decolorize and degrade dyes, endure dye with higher concentration of malachite green, keep the stable rate of decolorize in the condition of different pH value, efficiently decolorize under the condition when immobilized-cells was added into the medium in company with dye but free cells needed culture for 3 days beforehand. The method can reduce the medium consumption and the possibility of contamination, showing high efficient and great economic benefit. It shows that BC is a fine immobilization carrier.更多还原