【作者】 时进钢；

【导师】 曾光明； 袁兴中；

【作者基本信息】 湖南大学 ， 环境工程， 2007， 博士

【摘要】 堆肥过程是由大量微生物活动组成的一个复杂的生物化学过程,堆肥的物料成分复杂,除一小部分小分子的物质可以被微生物直接利用以外,大部分的有机物是以高聚物的形式存在,只有通过微生物所分泌的胞外酶将其分解为溶解性有机物质,才能渗入到细胞中供微生物代谢利用。由于堆肥物料中高聚物的存在形式是十分丰富的,因此,各种微生物产生的胞外酶也是多种多样的。如果能提高堆肥过程中一系列胞外酶的酶活,必将加快堆肥的进程,提高堆肥的效率。本研究将一种生物表面活性剂鼠李糖脂和一种化学合成的“温和型”非离子表面活性剂吐温80用于堆肥过程中,采用模拟堆肥实验、固态发酵实验、酶的内源荧光法及荧光探针等方法,从多个方面研究了两种表面活性剂对堆肥过程中微生物胞外酶的作用及其机理。在富含纤维素的废物稻草和麸皮的堆肥过程中,鼠李糖脂和吐温80对纤维素酶和木聚糖酶的活力有不同程度的增强和稳定作用,相应的,加速了纤维素和半纤维素的降解。堆肥过程中,主要来自于纤维素和半纤维素降解产生的水溶性有机碳,在含有表面活性剂的堆体中的含量要高于对照堆体,这有利于堆肥中微生物的生长,使得含有表面活性剂的堆体中微生物的种群数量要高于对照堆体。通过对比吐温80和鼠李糖脂对堆肥过程中的一系列参数的效果可以看出,鼠李糖脂对堆肥过程的促进作用要略好于吐温80。本实验的结果表明,“温和型”的表面活性剂,如吐温80和鼠李糖脂,对于富含纤维素废物的堆肥过程具有明显的促进作用。吐温80和鼠李糖脂对真菌简青霉(Penicillium simplicissimum),放线菌栗褐链霉菌(Streptomyces badius),细菌铜绿假单胞菌(Pseudomonas aeruginosa)和枯草芽孢杆菌(Bacillus subtilis)胞外酶有不同的作用效果,这和表面活性剂的种类、微生物的种类、酶的种类都有一定的关系。总体来看,吐温80对于大多数微生物和酶来说都是一种提高酶活的促进剂,其适用条件更加广泛,而鼠李糖脂起作用的范围要相对小一些,但对一些酶却有突出的作用效果,例如木聚糖酶。吐温80和鼠李糖脂对微生物的生长没有抑制作用。酶的稳定性随着pH值的变化是不同的,纤维素酶在pH值3~7的条件下都较为稳定,基本不失活,而木聚糖酶在pH值7~9的条件下较为稳定,失活速度缓慢。稻草基质对纤维素酶的吸附失活作用随着pH值的增大而增强,而对木聚糖酶的吸附失活作用受pH值的影响不是很大;表面活性剂吐温80和鼠李糖脂在整体上都表现出对两种酶的稳定作用,但在细节上有一些不同。在酸性及中性(pH值3~7)条件下,吐温80对木聚糖酶的稳定作用要好于鼠李糖脂,而在碱性条件下,鼠李糖脂的效果要明显好于吐温80。通过内源荧光法研究酶蛋白分子构型的变化,得出了淀粉酶、蛋白酶、纤维素酶和木聚糖酶在不同pH值条件下的稳定性,表面活性剂吐温80和鼠李糖脂对淀粉酶、纤维素酶和木聚糖酶都有不同程度的稳定作用,扩大了它们保持酶分子稳定的pH值范围,而对蛋白酶的作用不明显。通过采用芘作为荧光探针,研究鼠李糖脂和吐温80与4种微生物胞外酶,淀粉酶、蛋白酶、纤维素酶和木聚糖酶之间的作用关系。鼠李糖脂和木聚糖酶之间有较强的作用力,纤维素酶次之,而淀粉酶和蛋白酶与鼠李糖脂之间的作用力不明显。吐温80与纤维素酶和木聚糖酶之间的作用力能使它们较好的结合,而与淀粉酶、蛋白酶之间的作用力反而不利于吐温80胶束的形成。和鼠李糖脂相比,吐温80和酶之间的作用力较小,随着吐温80的浓度的增大,它和酶之间的作用力逐渐越大,尤其在其浓度超过临界胶束浓度时。综上所述,一方面,表面活性剂吐温80和鼠李糖对堆肥过程中微生物的各种胞外酶具有不同程度的促进作用和稳定作用,从而加快了有机质的降解,提高了堆肥的效率;另一方面,这种促进产酶和稳定酶活的作用的大小和表面活性剂的种类、微生物的种类、酶的种类、环境pH值、堆肥物料等条件有一定的关系,不能从一而论。由于生物表面活性剂的多样性、可以由微生物原位产生、无污染等众多优点,使其在堆肥化处理有机固体废物中具有非常好的应用前景。更多还原

【Abstract】 Composting is considered as a useful process for the disposal of municipal and agricultural solid waste. During the composting, the raw material is transformed through a variety of biological and biochemical processes in which enzymes play an important role. Enzymes that catalyze the degradation of biomacromolecules, such as starch, protein, cellulose, hemicellulose and lignin, are extracellular enzymes since the biomacromolecules are too large to be transported across the cellular membrane. Therefore, it is obvious that the enhancement of the extracellular enzyme activities will promote the degradation of the biomacromolecules, which will in turn speed up the composting process.In the present study, a biosurfactant rhamnolipid and a soft chemical surfactant Tween 80 were added into the composting process. The composting experiment, solid state fermentation, endogenetic fluorescence of enzymes and fluorescence probe were used to investigate the effects and mechanism of the two surfactants on the extracellular enzymes from the microorganism during the composting process.During the composting of waste rich in cellulose content, both rhamnolipid and Tween 80 had the increasing and stabilizing effects on CMCase and xylanase activities to different extents, and accordingly speeded up the decomposition of cellulose and hemicellulose. Therefore, the water soluble carbon (WSC) contents, mainly containing glucose and xylose released from the decomposition of cellulose and hemicellulose, in the treatments with surfactants were higher than that in the control during the whole composting process, which partially contributed to the lager microbial community populations than the control. The comparison between the effects of Tween 80 and rhamnolipid on enzyme activities, WSC contents and decomposition of substrate indicated that rhamnolipid is a little superior to Tween 80 during the composting process. The present study revealed that soft surfactants, such as rhamnolipid and Tween 80, might be used in the composting of waste rich in cellulose in order to speed up the composting process.The effect of surfactants Tweeen 80 and rhamnolipid on extracellular enzymes of fungi Penicillium simplicissimum, actinomycetes Streptomyces badius, bacteria Pseudomonas aeruginosa and Bacillus subtilis were related to the types of surfactants, kinds of microorganisms and classification of enzymes. In the whole, Tween 80 is a stimulatory agent for most of the microorganisms and enzymes, while the stimulatory effects of rhamnolipid is confined to a smaller range but it has a prominent effect on some specific enzymes, such as xylanase. Neither Tween 80 nor rhamnolipid had a prohibitory effect on the growth of microorganism.The stability of enzymes varied with pH values. Cellulase was the most stable between pH 3 and 7 and almost did not lose its activity, while xylanase was the most stable between pH 7 and 9 and the activity decreased slowly. The activity cut-down of enzymes due to adsorption by the substrate straw varied with enzymes and pH values. The adsorption of cellulase on straw increased with pH value going higher, while pH value did not have an obvious effect on the adsorption of xylanase. Both Tween 80 and rhamnolipid decreased the adsorption of enzymes on straw and stabilized the activities of cellulase and xylanase.The stabilities of amylase, protease, cellulase and xylanse in different pH conditions were investigated by endogenetic fluorescence of enzymes. Tween 80 and rhamnolipd increased the stability of amylase, cellulase and xylanase to different extends and widened the pH range in which the enzymes kept their molecule structure, but did not have an obvious effect on protease.The actions between surfactants, Tween 80 and rhamnolipid, and four enzymes, amylase, protease, cellulase and xylanase, were studied by fluorescence probe pyrene. The action between rhamnolipid and xylanase was the most strong, with cellulase taking a second place, but the action between rhamnolipid and amylase and protease was not distinct. The actions between Tween 80 and cellulase and xylanase made them combining together well, but amylase and protease went against the micelle formation of Tween 80. Compared with rhamnolipid, the action force between Tween 80 and enzymes was on the small side, but with the increasing of concentration, the action force increased, especially the concentration going over the critical micelle concentration.In conclusion, surfactants Tween 80 and rhamnolipid promoted and stabilized the extracellular enzymes of microorganism in the composting process, which accelerate the degradation of organic matter and improved the composting efficiency, on the other hand, the promoting and stabiling effects were related to type of surfactants, kind of microorganisms, classification of enzymes, pH values and the substrate. In addition, biosurfactant, with the advantage of diversity, being produced in situ and non-toxicity, has a perspective in composting of solid waste.更多还原