不同介质环境中生物表面活性剂强化降解生物质的作用
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摘要
环境中大量存在的废生物质是一类难降解的有机物质,同时也是一种宝贵的可再生资源。强化废生物质的降解是加速废物资源化利用过程的重要环节,也是有效减缓环境中废生物质累积和污染的必要手段。自然界中生物质的降解大多依靠微生物作用来完成,表面活性剂的介入,可以促进微生物对有效降解酶的分泌,并改善降解体系的微环境,促进微生物的生长繁殖和代谢,从而使降解过程得到强化。生物表面活性剂作为表面活性剂的一种,与一般化学表面活性剂具有类似的结构,能够降低空气-水、油-水和固体-水的表(界)面张力。同时,由于生物表面活性剂具备低毒性、可降解性和生态相容性等诸多优势,被视为一种清洁而高效的“绿色试剂”,具有良好的应用前景。
本文对几种不同介质环境中生物表面活性剂强化降解生物质的作用进行研究,以堆肥体系难降解的木质纤维素生物质及污水污泥的堆肥化处理过程为例,在酶法降解、微生物降解及堆肥化处置过程中,通过引入生物表面活性剂,考察在典型的水介质环境、逆胶束介质环境、固态微生物发酵环境和复杂堆肥环境中,生物质降解过程的不同特征,并探讨生物表面活性剂对强化底物降解的作用。研究结果有助于深入了解生物表面活性剂在不同介质环境中的行为及界面效应原理,为生物表面活性剂在不同环境条件下的应用可行性提供科学依据,推动污染物强化降解及外源促进剂的技术发展。论文总体试验工作分为四个部分：
第一部分研究液态水介质环境中生物表面活性剂强化酶解纤维素生物质的作用。采用铜绿假单胞菌发酵生产生物表面活性剂鼠李糖脂,并将其应用于酶法降解纤维素生物质的过程,通过在低酶负荷条件下,添加单鼠李糖脂、皂角苷和Tween80表面活性剂进行对比,考察在稻草纤维素酶解转化过程中生物表面活性剂的优越性,证实在相同酶解反应条件下,生物表面活性剂单鼠李糖脂与Tween80和皂角苷相比具有更大的优势,能显著地缓解纤维素酶的失活过程,促进纤维素的转化；将生物表面活性剂鼠李糖脂二糖脂用于磁性固定化纤维素酶的制备,并将该固定化酶用于降解纤维素生物质,含生物表面活性剂的磁性固定化酶稳定性得到提高,能更有效地发挥酶的催化能力。
第二部分进行液态非水介质环境中生物表面活性剂强化酶解纤维素的可行性研究。首次将生物表面活性剂引入逆胶束酶反应系统,通过构建“鼠李糖脂/异辛烷/正已醇/水”逆胶束体系,并对其进行电导行为分析,获得最佳含水率(%)值条件,采用荧光探针罗丹明B(Rh-B)对表面活性剂在异辛烷/正己醇(1/1,v/v)溶剂中的临界胶束浓度(CMC)进行测定,在最佳W0条件下对纤维素模式物质进行酶法降解,与不同类型表面活性剂作用进行对比,研究结果表明阴离子表面活性剂SDS和鼠李糖脂的逆胶束体系最大增溶水量高于阳离子表面活性剂CTAB和非离子表面活性剂Tween80逆胶束体系,各表面活性剂在1倍CMC浓度下对纤维素酶解促进作用最强,并且同等条件下在逆胶束体系中的酶解效率高于相应水介质体系中的酶解效率,生物表面活性剂鼠李糖脂所构建的逆胶束体系在各浓度条件下对酶解过程的促进作用均优于CTAB、SDS和Tween80这3种化学表面活性剂所构建的逆胶束体系。
第三部分对固-液介质环境中生物表面活性剂强化降解生物质的作用进行研究。采用白腐菌典型代表菌种黄胞原毛平革菌(Phanerochaete chrysosporium)对固态发酵介质环境中稻草木质素进行降解,在生物表面活性剂二鼠李糖脂的介入下,P. chrysosporium的生长得到促进,同时,二鼠李糖脂对木素过氧化物酶(LiP)活性的保持以及水溶性有机碳(WSOC)的释放起到促进作用,在浓度为0.007%的二鼠李糖脂作用下,LiP活性最大值提高86%,木质素降解率提高54%,经方差分解分析(VPA)统计显示,0.007%二鼠李糖脂对该生物除木质素过程的强化作用具有显著性；通过在剩余污泥静态强制通风好氧堆肥过程中添加鼠李糖脂,对该堆肥化过程的物理化学参数的变化、微生物菌群动态和毒理学指标进行监测,考察了鼠李糖脂对污泥堆肥过程的强化作用,同时与外源复合微生物菌剂的促进作用进行比较,分析生物表面活性剂作用与微生物作用的区别。
第四部分为表面活性剂胶束化过程与生物质降解酶的缔合作用解析。通过稳态荧光技术,采用非极性的荧光探针芘(Py),对水介质中表面活性剂胶束化过程与酶分子的缔合作用进行解析,以纤维素酶和漆酶为例,探讨生物表面活性剂鼠李糖脂与化学表面活性剂SDS、CTAB和Tween80胶束体系的不同特征,芘的荧光行为可以反映出其所处微环境极性的变化,不同结构表面活性剂对芘的荧光作用各有不同,纤维素酶与表面活性剂的缔合,使芘的荧光行为随表面活性剂浓度的变化特征相对弱化,而漆酶与表面活性剂的缔合,减缓了水介质中表面活性剂的胶束化过程,鼠李糖脂在高于临界胶束浓度时,稳定性优于阴离子表面活性剂SDS；采用极性荧光探针ANS和Rh-B对“鼠李糖脂/异辛烷/正己醇/水”逆胶束体系中酶与表面活性剂的作用进行研究,两种探针的荧光行为均能有效指示出逆胶束体系W0的变化过程酶分子附近微环境极性的变化,同时也表征了不同的酶分子与表面活性剂结合的位点及其迁移趋势。
The large quantity of waste biomass produced all over the world is a kind of organic materials which is difficult to be degraded, while it represents a renewable resource that has attracted more and more attentions. To enhance the degradation of biomass is significant for improving the resource utilization and wastes minimization. Still the waste biomass degradation is important for environmental pollution control. As the biomass degradation is mainly carried out by the microorganisms in nature, the process can be enhanced by the presence of surfactant through increasing the degradable enzymes excretion, improving the microbial growth and the reacting micro-environment. The bio surfactants are typical surfactants which have the similar characteristics in molecular structure compared with synthetic surfactants. They can reduce the surface/interface tension of air-water, oil-water or solid-water phases. In special, the biosurfactants are believe to have a good prospect as they have several advantages such as lower toxicity, easier to be degraded and higher environmental compatibility, thus they are regarded as "green chemicals" which showing high efficient during utilization.
This dessertation investigated the biosurfactant-enhanced degradation of biomass in several typical media environment, and used the lignocellulosic wastes and sewage sludge in composting system as substrates. The experimental performances of biomass degradation included enzymatic hydrolysis, biodegradation and composting process. While the effects of biosurfactants in aqueous environment, in reversed micellar media, in solid-state fermentation and in composting matrix are studied. This research helps to understand the behaviors and interfacial influences of biosurfactants in different reacting media, and provides scientific data for biosurfactant application in different medium conditions. As a result, this work is significant for the development of contaminants enhanced-degradation technologies and the application of extrinsic accelerants.
The first section describes the research on the effects of biosurfactants on enzymatic hydrolysis of cellulosic biomass in aqueous liquid media. The rhamnolipid biosurfactant was produced by Pseudomonas aeruginosa and was used in the following experiments. The effects of three surfactants (Tween80, saponin and monorhamnolipid) on the hydrolysis of NaOH-pretreated rice straw by low dosage of cellulase were studied, and the results indicated that at the same condition, all surfactants were able to enhance the enzymatic hydrolysis by reducing the cellulase denaturalization, while the biosurfactant monorhamnolipid was demonstrated to be more active than Tween80and saponin. In addition, a new magnetic immobilized cellulase was developed by the presence of dirhamnolipid. It was applied in the hydrolysis of cellulosic biomass and showed capable in improving the enzyme stability.
The second section focuses on the feasibility of biosurfactant on enzymatic hydrolysis of cellulose in non-aqueous liquid media. For the first time, the biosurfactant was introduced into the enzyme-containing reversed micellar system. The'rhamnolipid/isooctane/n-hexanol/water' reversed micellar system was formed, and its electrical conductivity was measured to determine the maximum water solubilization W0. The critical micelle concentrations of surfactant rhamnolipid, SDS, CTAB and Tween80in isooctane/n-hexanol (1/1, v/v) were determined by steady-state fluorescence used the rhodamine B (Rh-B) as a probe. The degradation of cellulose model substrate in reversed micelle was conducted in the condition of W0=W0, max, while the effects of different surfactants were compared. The results showed that the W0, max of anionic surfactant (rhamnolipid and SDS) reversed micellar system was higher than it was in cationic surfactant (CTAB) and in nonionic surfactant (Tween80) system. The peak conversion rates of substrate were obtained when the surfactants concentration was at1CMC of their each. The enzymatic hydrolysis efficiency in reversed micelles was higher than that in aqueous reaction media at the same conditions, independent of surfactant types. While the rhamnolipid showed more effective than the other three synthetic surfactants.
The third section focuses on the effects of biosurfactant rhamnolipid on biomass degradation at solid-liquid interface. The influence of dirhamnolipid biosurfactant on biodegradation of rice straw by Phanerochaete chrysosporium was investigated. The results showed that the biodelignification of rice straw can be significantly enhanced by the presence of dirhamnolipid biosurfactant. In particular, the dirhamnolipid at the concentration of0.007%increased the peak activity of lignin peroxidase (LiP) by86%. The water-soluble organic carbon (WSOC) contents in the straw substrates as well as the microbial growth and activity were effectively improved by dirhamnolipid, while the degradation rate of lignin increased by54%with dirhamnolipid of0.007%. Variation partitioning analysis revealed that the improvement by dirhamnolipid addition is significant. To investigate the effect of biosurfactant on composting of sewage sludge, a static forced-aeration composting matrix was constructed by the presence of rhamnolipid. The physicochemical factors, microbial parameters and the biological toxicity were analysis. In addition, the influence of biosurfactant addition was compared with that of the microbial inoculants.
The fourth section demonstrates the interaction between enzyme and surfactant molecules during the micelle or reversed micelle formation. The cellulase and the laccase were taken as an example, while the rhamnolipid, SDS, CTAB and Tween80were used in the analysis of their interactions with enzymes, respectively, in the aqueous media. According to the fluorescence behavior of pyrene, the characterization of interactions depends on the type of surfactant and as well as the enzyme. The changes of pyrene fluorescence intensity along with the concentration of surfactants were reduced by the presence of cellulase, while the laccase seemed to inhibit the surfactant micelle formation in aqueous. Otherwise, the rhamnolipid showed higher stability than SDS at concentrations above CMC. The ANS and Rh-B probes were employed in analysis of interactions between rhamnolipid and enzymes in reversed micellar system. The fluorescence behavior of each probe demonstrated the micro-environmental polarity changes along with the W0, and it described the combining sites of enzymes and surfactant molecules as well as their trends.
本文对几种不同介质环境中生物表面活性剂强化降解生物质的作用进行研究,以堆肥体系难降解的木质纤维素生物质及污水污泥的堆肥化处理过程为例,在酶法降解、微生物降解及堆肥化处置过程中,通过引入生物表面活性剂,考察在典型的水介质环境、逆胶束介质环境、固态微生物发酵环境和复杂堆肥环境中,生物质降解过程的不同特征,并探讨生物表面活性剂对强化底物降解的作用。研究结果有助于深入了解生物表面活性剂在不同介质环境中的行为及界面效应原理,为生物表面活性剂在不同环境条件下的应用可行性提供科学依据,推动污染物强化降解及外源促进剂的技术发展。论文总体试验工作分为四个部分：
第一部分研究液态水介质环境中生物表面活性剂强化酶解纤维素生物质的作用。采用铜绿假单胞菌发酵生产生物表面活性剂鼠李糖脂,并将其应用于酶法降解纤维素生物质的过程,通过在低酶负荷条件下,添加单鼠李糖脂、皂角苷和Tween80表面活性剂进行对比,考察在稻草纤维素酶解转化过程中生物表面活性剂的优越性,证实在相同酶解反应条件下,生物表面活性剂单鼠李糖脂与Tween80和皂角苷相比具有更大的优势,能显著地缓解纤维素酶的失活过程,促进纤维素的转化；将生物表面活性剂鼠李糖脂二糖脂用于磁性固定化纤维素酶的制备,并将该固定化酶用于降解纤维素生物质,含生物表面活性剂的磁性固定化酶稳定性得到提高,能更有效地发挥酶的催化能力。
第二部分进行液态非水介质环境中生物表面活性剂强化酶解纤维素的可行性研究。首次将生物表面活性剂引入逆胶束酶反应系统,通过构建“鼠李糖脂/异辛烷/正已醇/水”逆胶束体系,并对其进行电导行为分析,获得最佳含水率(%)值条件,采用荧光探针罗丹明B(Rh-B)对表面活性剂在异辛烷/正己醇(1/1,v/v)溶剂中的临界胶束浓度(CMC)进行测定,在最佳W0条件下对纤维素模式物质进行酶法降解,与不同类型表面活性剂作用进行对比,研究结果表明阴离子表面活性剂SDS和鼠李糖脂的逆胶束体系最大增溶水量高于阳离子表面活性剂CTAB和非离子表面活性剂Tween80逆胶束体系,各表面活性剂在1倍CMC浓度下对纤维素酶解促进作用最强,并且同等条件下在逆胶束体系中的酶解效率高于相应水介质体系中的酶解效率,生物表面活性剂鼠李糖脂所构建的逆胶束体系在各浓度条件下对酶解过程的促进作用均优于CTAB、SDS和Tween80这3种化学表面活性剂所构建的逆胶束体系。
第三部分对固-液介质环境中生物表面活性剂强化降解生物质的作用进行研究。采用白腐菌典型代表菌种黄胞原毛平革菌(Phanerochaete chrysosporium)对固态发酵介质环境中稻草木质素进行降解,在生物表面活性剂二鼠李糖脂的介入下,P. chrysosporium的生长得到促进,同时,二鼠李糖脂对木素过氧化物酶(LiP)活性的保持以及水溶性有机碳(WSOC)的释放起到促进作用,在浓度为0.007%的二鼠李糖脂作用下,LiP活性最大值提高86%,木质素降解率提高54%,经方差分解分析(VPA)统计显示,0.007%二鼠李糖脂对该生物除木质素过程的强化作用具有显著性；通过在剩余污泥静态强制通风好氧堆肥过程中添加鼠李糖脂,对该堆肥化过程的物理化学参数的变化、微生物菌群动态和毒理学指标进行监测,考察了鼠李糖脂对污泥堆肥过程的强化作用,同时与外源复合微生物菌剂的促进作用进行比较,分析生物表面活性剂作用与微生物作用的区别。
第四部分为表面活性剂胶束化过程与生物质降解酶的缔合作用解析。通过稳态荧光技术,采用非极性的荧光探针芘(Py),对水介质中表面活性剂胶束化过程与酶分子的缔合作用进行解析,以纤维素酶和漆酶为例,探讨生物表面活性剂鼠李糖脂与化学表面活性剂SDS、CTAB和Tween80胶束体系的不同特征,芘的荧光行为可以反映出其所处微环境极性的变化,不同结构表面活性剂对芘的荧光作用各有不同,纤维素酶与表面活性剂的缔合,使芘的荧光行为随表面活性剂浓度的变化特征相对弱化,而漆酶与表面活性剂的缔合,减缓了水介质中表面活性剂的胶束化过程,鼠李糖脂在高于临界胶束浓度时,稳定性优于阴离子表面活性剂SDS；采用极性荧光探针ANS和Rh-B对“鼠李糖脂/异辛烷/正己醇/水”逆胶束体系中酶与表面活性剂的作用进行研究,两种探针的荧光行为均能有效指示出逆胶束体系W0的变化过程酶分子附近微环境极性的变化,同时也表征了不同的酶分子与表面活性剂结合的位点及其迁移趋势。
The large quantity of waste biomass produced all over the world is a kind of organic materials which is difficult to be degraded, while it represents a renewable resource that has attracted more and more attentions. To enhance the degradation of biomass is significant for improving the resource utilization and wastes minimization. Still the waste biomass degradation is important for environmental pollution control. As the biomass degradation is mainly carried out by the microorganisms in nature, the process can be enhanced by the presence of surfactant through increasing the degradable enzymes excretion, improving the microbial growth and the reacting micro-environment. The bio surfactants are typical surfactants which have the similar characteristics in molecular structure compared with synthetic surfactants. They can reduce the surface/interface tension of air-water, oil-water or solid-water phases. In special, the biosurfactants are believe to have a good prospect as they have several advantages such as lower toxicity, easier to be degraded and higher environmental compatibility, thus they are regarded as "green chemicals" which showing high efficient during utilization.
This dessertation investigated the biosurfactant-enhanced degradation of biomass in several typical media environment, and used the lignocellulosic wastes and sewage sludge in composting system as substrates. The experimental performances of biomass degradation included enzymatic hydrolysis, biodegradation and composting process. While the effects of biosurfactants in aqueous environment, in reversed micellar media, in solid-state fermentation and in composting matrix are studied. This research helps to understand the behaviors and interfacial influences of biosurfactants in different reacting media, and provides scientific data for biosurfactant application in different medium conditions. As a result, this work is significant for the development of contaminants enhanced-degradation technologies and the application of extrinsic accelerants.
The first section describes the research on the effects of biosurfactants on enzymatic hydrolysis of cellulosic biomass in aqueous liquid media. The rhamnolipid biosurfactant was produced by Pseudomonas aeruginosa and was used in the following experiments. The effects of three surfactants (Tween80, saponin and monorhamnolipid) on the hydrolysis of NaOH-pretreated rice straw by low dosage of cellulase were studied, and the results indicated that at the same condition, all surfactants were able to enhance the enzymatic hydrolysis by reducing the cellulase denaturalization, while the biosurfactant monorhamnolipid was demonstrated to be more active than Tween80and saponin. In addition, a new magnetic immobilized cellulase was developed by the presence of dirhamnolipid. It was applied in the hydrolysis of cellulosic biomass and showed capable in improving the enzyme stability.
The second section focuses on the feasibility of biosurfactant on enzymatic hydrolysis of cellulose in non-aqueous liquid media. For the first time, the biosurfactant was introduced into the enzyme-containing reversed micellar system. The'rhamnolipid/isooctane/n-hexanol/water' reversed micellar system was formed, and its electrical conductivity was measured to determine the maximum water solubilization W0. The critical micelle concentrations of surfactant rhamnolipid, SDS, CTAB and Tween80in isooctane/n-hexanol (1/1, v/v) were determined by steady-state fluorescence used the rhodamine B (Rh-B) as a probe. The degradation of cellulose model substrate in reversed micelle was conducted in the condition of W0=W0, max, while the effects of different surfactants were compared. The results showed that the W0, max of anionic surfactant (rhamnolipid and SDS) reversed micellar system was higher than it was in cationic surfactant (CTAB) and in nonionic surfactant (Tween80) system. The peak conversion rates of substrate were obtained when the surfactants concentration was at1CMC of their each. The enzymatic hydrolysis efficiency in reversed micelles was higher than that in aqueous reaction media at the same conditions, independent of surfactant types. While the rhamnolipid showed more effective than the other three synthetic surfactants.
The third section focuses on the effects of biosurfactant rhamnolipid on biomass degradation at solid-liquid interface. The influence of dirhamnolipid biosurfactant on biodegradation of rice straw by Phanerochaete chrysosporium was investigated. The results showed that the biodelignification of rice straw can be significantly enhanced by the presence of dirhamnolipid biosurfactant. In particular, the dirhamnolipid at the concentration of0.007%increased the peak activity of lignin peroxidase (LiP) by86%. The water-soluble organic carbon (WSOC) contents in the straw substrates as well as the microbial growth and activity were effectively improved by dirhamnolipid, while the degradation rate of lignin increased by54%with dirhamnolipid of0.007%. Variation partitioning analysis revealed that the improvement by dirhamnolipid addition is significant. To investigate the effect of biosurfactant on composting of sewage sludge, a static forced-aeration composting matrix was constructed by the presence of rhamnolipid. The physicochemical factors, microbial parameters and the biological toxicity were analysis. In addition, the influence of biosurfactant addition was compared with that of the microbial inoculants.
The fourth section demonstrates the interaction between enzyme and surfactant molecules during the micelle or reversed micelle formation. The cellulase and the laccase were taken as an example, while the rhamnolipid, SDS, CTAB and Tween80were used in the analysis of their interactions with enzymes, respectively, in the aqueous media. According to the fluorescence behavior of pyrene, the characterization of interactions depends on the type of surfactant and as well as the enzyme. The changes of pyrene fluorescence intensity along with the concentration of surfactants were reduced by the presence of cellulase, while the laccase seemed to inhibit the surfactant micelle formation in aqueous. Otherwise, the rhamnolipid showed higher stability than SDS at concentrations above CMC. The ANS and Rh-B probes were employed in analysis of interactions between rhamnolipid and enzymes in reversed micellar system. The fluorescence behavior of each probe demonstrated the micro-environmental polarity changes along with the W0, and it described the combining sites of enzymes and surfactant molecules as well as their trends.
引文
[1]朱清时,阎立峰,郭庆祥.生物质洁净能源.北京：化学工业出版社,2002,35-74
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