菜粕与蓝藻混合高温发酵过程中的物质转化及光谱学特性
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摘要
解决农业固体有机废弃物问题的根本途径就在于开展其资源化利用。目前,资源化利用农业固体有机废弃物的最有效和最经济的途径是好氧堆肥。快速高效堆肥技术资源化利用固体有机废弃物的关键是,理解堆肥过程中的物质降解情况及选择合适的接种微生物。本论文以菜粕为主要原料生产氨基酸有机肥,通过添加微生物菌剂和蓝藻,建立了3个不同的堆体：CK,菜粕堆肥；AA,菜粕+微生物菌剂；LA,菜粕+蓝藻+微生物菌剂。本论文主要研究了不同堆体在堆肥过程中的物理、化学、生物学指标以及光谱学特征的动态变化,揭示了菜粕和蓝藻共发酵过程中物质变化的基本规律；研究了添加微生物菌剂和蓝藻对菜粕堆肥光谱学性质变化的影响,运用二维相关光谱方法研究菜粕堆肥过程中的物质降解顺序；研究了三维荧光光谱-荧光区域指数(EEM-FRI)方法评价堆肥腐熟度的可行性；应用多重荧光标记-激光共聚焦显微镜(CLSM)方法,研究了添加微生物菌剂和蓝藻对菜粕堆肥中的大分子有机物降解和微生物空间分布的影响,揭示堆肥腐熟机制。本论文获得了如下的研究成果。
1)加入微生物菌剂可以明显缩短堆体升温时间,AA较CK堆体提前5天达到高温期；提高堆体的最高温度,AA较CK堆体平均温度高10℃以上；加速发酵进程。发酵结束时,3个堆体pH和EC差异不显著,均达到国家有机肥标准；微生物菌剂的添加明显促进了有机碳的降解,总碳(TC)含量下降,水溶性总碳(DOC)含量上升；堆肥总氮含量变化不大,但水溶性总氮和游离氨基酸含量升高,发酵结束时,AA和LA处理游离氨基酸含量达到50.00g g kg-1左右；发酵结束时,LA处理检测不到藻毒素。该结果表明,蓝藻和菜粕混合高温发酵处理不但使蓝藻无害化处理,而且也达到了蓝藻得资源化利用目的。该研究表明蓝藻与菜粕混合高温发酵是资源化利用蓝藻的一个重要途径。
2)在发酵过程中,3个堆体不同天数的堆肥样品间的一维红外光谱(FTIR)谱图中的峰位置十分相似,仅峰强度上有所变化。运用二维相关光谱分析方法,发现堆肥样品中有机物降解特征差异明显。具体而言,在1800-900cm-1波数范围内的二维FTIR相关光谱显示,在堆肥过程中,纤维素、蛋白质和异质多糖降解之间有协同关系；接种微生物菌剂能促进多糖的降解,但蓝藻却抑制了多糖的降解；纤维素、蛋白质和异质多糖的降解顺序不同,具体为CK堆体中物质降解顺序为异质多糖>氨基酸化合物Ⅰ>氨基酸化合物Ⅱ>纤维素,AA和LA堆体中物质降解顺序为纤维素>异质多糖>氨基酸化合物Ⅱ>氨基酸化合物Ⅰ。在3600-3100cm-1波数范围内的二维FTIR相关光谱谱图,进一步揭示了接种微生物菌剂能够改变堆肥中的纤维素和异质多糖中的OH键的降解顺序。因此,二维FTIR相关光谱方法可以用于研究接种加速堆肥腐熟机制的一种工具。
3)三维荧光光谱结果显示,在发酵过程中,类蛋白质物质含量逐步下降,而腐殖酸类物质和富里酸类物质含量均呈整体上升趋势；接种微生物菌剂能增强类富里酸和类胡敏酸物质含量。通过对三维荧光峰强度和区域面积积分与化学、生物学指标的皮尔森相关性分析确定,三维荧光光谱可以用于表征堆肥腐殖化程度,其中中类富里酸和类胡敏酸峰强度和类蛋白类荧光区域面积积分更适用于表征堆肥的腐熟度。
4)菜粕原料接种微生物菌剂和蓝藻影响了木质纤维素的降解和纤维素结晶度的变化。添加微生物菌剂可以明显促进发酵过程中半纤维素和木质素的降解,使纤维素的含量增加。蓝藻与菜粕混合发酵,则在一定程度上影响了微生物菌剂作用的发挥。堆体材料主体菜粕纤维结晶体为典型的纤维素Ⅰ型,整个堆肥过程没有改变纤维素的晶型；Si02和碳酸钙的衍射强度峰随着堆肥的进行逐步增强；各堆体的纤维素结晶度先上升后下降,其中,以AA处理下降的幅度最大。
5)通过变性梯度凝胶电泳方法,发现堆肥过程中所有堆体的细菌种类逐渐增多,而随着高温期的延续,堆体的细菌种类逐步下降,堆肥前期和后期细菌类型差异较大。多重荧光标记-激光共聚焦显微镜(CLSM)原位观察结果表明,菜粕原料中的纤维素形成完整的网络结构,蛋白质、α-多糖、脂肪镶嵌在网格中；发酵结束时,镶嵌在网格内的蛋白质、α-多糖、脂肪被大量降解,荧光强度减弱,而纤维素的荧光强度增加。
Aerobic composting is a cheap, efficient and sustainable treatment for agricultural organic solid wastes, since it not only solves the pollution problem of agricultural organic solid wastes but also applies organic solid wastes as a resource. The key issue of rapid composting is to understand the degradation of organic matters and selection of composting inoculation. In this paper, the three piles were build by rapeseed meal mixed with blue algae and microbial agent, including rapeseed meal pile (CK treatment), rapeseed meal+microbial agent (AA treatment), and rapeseed meal+blue algae+microbial agent (LA treatment). The purposes of this study were1) to investigate the dynamic evolution of physical, chemical and biological indices as well as spectroscopy in order to explore the fate of organic matters during the fermentation;2) to identify the influence of microbial agent and blue algae in the spectroscopic characteristics of rapeseed meal composting;3) to explore the feasibility of two dimensional FTIR correlation spectroscopy on the mechanism of the degradation of rapeseed meal during compositng and assessment of compost maturity by fluorescence excitation-emmision matrix combined with fluorescence regional integrity (EEM-FRI) technique;4) to study the distribution of macromolecules and cells by multiple fluorescence labeling-confocal laser scanning microscopy (CLSM) method during the fermentation in order to reveal the mechanism of compost maturity. The main orginal conclusions of this work are drawn.
1) Addition of microbial agent during the fermentation could significantly shorten the duration of start-up (i.e.5day), improve the maximum temperature (about10℃), and speed up composting. At the end of composting, pH and electricity conductivity (EC) had no markedly differences among the three piles. Meanwhile, the quality of compost products was compliant to the provisions of the relevant national standards. Moreover, the degradation of organic carbon was enhanced after the addition of microbial agent. Specifically, the total carbon content decreased and the dissolved carbon content enhanced with the addition of compost inoculation. More importantly, no cyanobacterial toxin was detected in the compost at the end of LA treatment, suggesting that blue algae being treated to harmless extent during the fermentation.
2) The results of one dimensional FTIR of compost showed that for the compost from the three piles, the peak position of FTIR was similar but the intensity of peak was slightly distinct. The results of two dimensional FTIR correlation spectroscopy revealed the marked difference among the peak intensity among the three piles. Through2D FTIR correlation spectroscopy in the range of1800-900cm-1, the following trend in the ease of the degradation of organic compounds was observed:proteins, heteropolysaccharides and cellulose co-vary in theirdegradation during the fermentation; heteropolysaccharides> amide Ⅰ in proteins> amide Ⅱ in proteins> cellulose in proteins in the CK treatment; cellulose> heteropolysaccharides> amide Ⅱ in proteins> amide Ⅰ in the AA and LA treatment. The above-mentioned results revealed that addition of microbial agent could speed up the degradation of heteropolysaccharides. As a result, heteropolysaccharides play an important role in the priming effect which speeds up the degradation of other organic matters. Howevetr, blue algae could slow down the degradation of heteropolysaccharides and restain the role of compost inoculation. The2D FTIR correlation spectroscopy in the range of3600-3100cm-1further demonstrated that addition of microbial agent could change the degradation sequencing of OH bond. However, the role of compost inoculation was impressed in LA treatment but the performance of LA treatment was better than that of CK treatment. Therefore, the2D FTIR correlation spectroscopy can be applied as a tool to explore the mechanism of speeding up of the compost maturity with the addition of inoculant.
3) Fluorescence EEM spectroscopy was applied to assess the maturity of compost. The results showed that during the three piles, proteins-like substances decreased but humic-and fulvic-like substances increased during the fermentation, revealing the increase of humification process. The fluorescence intensity of humic-like substances was enhanced in the pile with the addition of inoculation. Pearson correlation analysis between fluorescence indices and physical, chemical and biological indices demonstrated that the peak intensity of humic-like substances and fluorescence integrity indices of proteins-like substances were more suitable to assess the maturity of compost.
4) Both the microbial agent and blue algae could influence the degradation and the crystallinity of lignocellulose. Specifically, the addition of microbial agent could significantly improve the degradation of hemicellulose and lignin and increase the content of cellulose. However, the addition of blue algae to composting affected the role of microbial agent to some extent. X-ray diffraction peak intensity of SiO2and CaCO3increased with the duration of composting. The crystallinity of lignocellulose for all the three piles increased first and then decreased. Among the three piles, the crystallinity of lignocellulose in AA pile decreased the most.
5) DGGE profiles showed that bacterial communities were rich during compost start-up period and dominant communities were not obvious. In the thermophilic period bacterial communities decreased and dominant communities were significant. In the cooling period the structure of the bacterial population remained stable. Therefore, temperature played an important screening role in the composition of bacteria population. Multiple fluorescent labeling combined with CLSM observation clearly demonstrate that cellulose protects other biopolymers (i.e. proteins, a-polysccharides and lipids) from enzymatic attack and its successive aerobic fermentation by forming a network structure not accessible to enzymes. In the end of the fermentation, proteins, a-polysaccharides, lipids and eDNA were degraded and fluorescence intensity of those decreased significantly while the fluorescence intensity of cellulose increased markedly.
1)加入微生物菌剂可以明显缩短堆体升温时间,AA较CK堆体提前5天达到高温期；提高堆体的最高温度,AA较CK堆体平均温度高10℃以上；加速发酵进程。发酵结束时,3个堆体pH和EC差异不显著,均达到国家有机肥标准；微生物菌剂的添加明显促进了有机碳的降解,总碳(TC)含量下降,水溶性总碳(DOC)含量上升；堆肥总氮含量变化不大,但水溶性总氮和游离氨基酸含量升高,发酵结束时,AA和LA处理游离氨基酸含量达到50.00g g kg-1左右；发酵结束时,LA处理检测不到藻毒素。该结果表明,蓝藻和菜粕混合高温发酵处理不但使蓝藻无害化处理,而且也达到了蓝藻得资源化利用目的。该研究表明蓝藻与菜粕混合高温发酵是资源化利用蓝藻的一个重要途径。
2)在发酵过程中,3个堆体不同天数的堆肥样品间的一维红外光谱(FTIR)谱图中的峰位置十分相似,仅峰强度上有所变化。运用二维相关光谱分析方法,发现堆肥样品中有机物降解特征差异明显。具体而言,在1800-900cm-1波数范围内的二维FTIR相关光谱显示,在堆肥过程中,纤维素、蛋白质和异质多糖降解之间有协同关系；接种微生物菌剂能促进多糖的降解,但蓝藻却抑制了多糖的降解；纤维素、蛋白质和异质多糖的降解顺序不同,具体为CK堆体中物质降解顺序为异质多糖>氨基酸化合物Ⅰ>氨基酸化合物Ⅱ>纤维素,AA和LA堆体中物质降解顺序为纤维素>异质多糖>氨基酸化合物Ⅱ>氨基酸化合物Ⅰ。在3600-3100cm-1波数范围内的二维FTIR相关光谱谱图,进一步揭示了接种微生物菌剂能够改变堆肥中的纤维素和异质多糖中的OH键的降解顺序。因此,二维FTIR相关光谱方法可以用于研究接种加速堆肥腐熟机制的一种工具。
3)三维荧光光谱结果显示,在发酵过程中,类蛋白质物质含量逐步下降,而腐殖酸类物质和富里酸类物质含量均呈整体上升趋势；接种微生物菌剂能增强类富里酸和类胡敏酸物质含量。通过对三维荧光峰强度和区域面积积分与化学、生物学指标的皮尔森相关性分析确定,三维荧光光谱可以用于表征堆肥腐殖化程度,其中中类富里酸和类胡敏酸峰强度和类蛋白类荧光区域面积积分更适用于表征堆肥的腐熟度。
4)菜粕原料接种微生物菌剂和蓝藻影响了木质纤维素的降解和纤维素结晶度的变化。添加微生物菌剂可以明显促进发酵过程中半纤维素和木质素的降解,使纤维素的含量增加。蓝藻与菜粕混合发酵,则在一定程度上影响了微生物菌剂作用的发挥。堆体材料主体菜粕纤维结晶体为典型的纤维素Ⅰ型,整个堆肥过程没有改变纤维素的晶型；Si02和碳酸钙的衍射强度峰随着堆肥的进行逐步增强；各堆体的纤维素结晶度先上升后下降,其中,以AA处理下降的幅度最大。
5)通过变性梯度凝胶电泳方法,发现堆肥过程中所有堆体的细菌种类逐渐增多,而随着高温期的延续,堆体的细菌种类逐步下降,堆肥前期和后期细菌类型差异较大。多重荧光标记-激光共聚焦显微镜(CLSM)原位观察结果表明,菜粕原料中的纤维素形成完整的网络结构,蛋白质、α-多糖、脂肪镶嵌在网格中；发酵结束时,镶嵌在网格内的蛋白质、α-多糖、脂肪被大量降解,荧光强度减弱,而纤维素的荧光强度增加。
Aerobic composting is a cheap, efficient and sustainable treatment for agricultural organic solid wastes, since it not only solves the pollution problem of agricultural organic solid wastes but also applies organic solid wastes as a resource. The key issue of rapid composting is to understand the degradation of organic matters and selection of composting inoculation. In this paper, the three piles were build by rapeseed meal mixed with blue algae and microbial agent, including rapeseed meal pile (CK treatment), rapeseed meal+microbial agent (AA treatment), and rapeseed meal+blue algae+microbial agent (LA treatment). The purposes of this study were1) to investigate the dynamic evolution of physical, chemical and biological indices as well as spectroscopy in order to explore the fate of organic matters during the fermentation;2) to identify the influence of microbial agent and blue algae in the spectroscopic characteristics of rapeseed meal composting;3) to explore the feasibility of two dimensional FTIR correlation spectroscopy on the mechanism of the degradation of rapeseed meal during compositng and assessment of compost maturity by fluorescence excitation-emmision matrix combined with fluorescence regional integrity (EEM-FRI) technique;4) to study the distribution of macromolecules and cells by multiple fluorescence labeling-confocal laser scanning microscopy (CLSM) method during the fermentation in order to reveal the mechanism of compost maturity. The main orginal conclusions of this work are drawn.
1) Addition of microbial agent during the fermentation could significantly shorten the duration of start-up (i.e.5day), improve the maximum temperature (about10℃), and speed up composting. At the end of composting, pH and electricity conductivity (EC) had no markedly differences among the three piles. Meanwhile, the quality of compost products was compliant to the provisions of the relevant national standards. Moreover, the degradation of organic carbon was enhanced after the addition of microbial agent. Specifically, the total carbon content decreased and the dissolved carbon content enhanced with the addition of compost inoculation. More importantly, no cyanobacterial toxin was detected in the compost at the end of LA treatment, suggesting that blue algae being treated to harmless extent during the fermentation.
2) The results of one dimensional FTIR of compost showed that for the compost from the three piles, the peak position of FTIR was similar but the intensity of peak was slightly distinct. The results of two dimensional FTIR correlation spectroscopy revealed the marked difference among the peak intensity among the three piles. Through2D FTIR correlation spectroscopy in the range of1800-900cm-1, the following trend in the ease of the degradation of organic compounds was observed:proteins, heteropolysaccharides and cellulose co-vary in theirdegradation during the fermentation; heteropolysaccharides> amide Ⅰ in proteins> amide Ⅱ in proteins> cellulose in proteins in the CK treatment; cellulose> heteropolysaccharides> amide Ⅱ in proteins> amide Ⅰ in the AA and LA treatment. The above-mentioned results revealed that addition of microbial agent could speed up the degradation of heteropolysaccharides. As a result, heteropolysaccharides play an important role in the priming effect which speeds up the degradation of other organic matters. Howevetr, blue algae could slow down the degradation of heteropolysaccharides and restain the role of compost inoculation. The2D FTIR correlation spectroscopy in the range of3600-3100cm-1further demonstrated that addition of microbial agent could change the degradation sequencing of OH bond. However, the role of compost inoculation was impressed in LA treatment but the performance of LA treatment was better than that of CK treatment. Therefore, the2D FTIR correlation spectroscopy can be applied as a tool to explore the mechanism of speeding up of the compost maturity with the addition of inoculant.
3) Fluorescence EEM spectroscopy was applied to assess the maturity of compost. The results showed that during the three piles, proteins-like substances decreased but humic-and fulvic-like substances increased during the fermentation, revealing the increase of humification process. The fluorescence intensity of humic-like substances was enhanced in the pile with the addition of inoculation. Pearson correlation analysis between fluorescence indices and physical, chemical and biological indices demonstrated that the peak intensity of humic-like substances and fluorescence integrity indices of proteins-like substances were more suitable to assess the maturity of compost.
4) Both the microbial agent and blue algae could influence the degradation and the crystallinity of lignocellulose. Specifically, the addition of microbial agent could significantly improve the degradation of hemicellulose and lignin and increase the content of cellulose. However, the addition of blue algae to composting affected the role of microbial agent to some extent. X-ray diffraction peak intensity of SiO2and CaCO3increased with the duration of composting. The crystallinity of lignocellulose for all the three piles increased first and then decreased. Among the three piles, the crystallinity of lignocellulose in AA pile decreased the most.
5) DGGE profiles showed that bacterial communities were rich during compost start-up period and dominant communities were not obvious. In the thermophilic period bacterial communities decreased and dominant communities were significant. In the cooling period the structure of the bacterial population remained stable. Therefore, temperature played an important screening role in the composition of bacteria population. Multiple fluorescent labeling combined with CLSM observation clearly demonstrate that cellulose protects other biopolymers (i.e. proteins, a-polysccharides and lipids) from enzymatic attack and its successive aerobic fermentation by forming a network structure not accessible to enzymes. In the end of the fermentation, proteins, a-polysaccharides, lipids and eDNA were degraded and fluorescence intensity of those decreased significantly while the fluorescence intensity of cellulose increased markedly.
引文
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