【作者】 张庆芳；

【导师】 陈吉祥；

【作者基本信息】 兰州理工大学 ， 可再生能源与环境工程， 2019， 博士

【摘要】 预处理可以改善餐厨垃圾原料结构,提高产甲烷潜力。纤维素降解困难是影响餐厨垃圾厌氧发酵产甲烷效率的限制性因素之一。本文筛选出了3株纤维素降解菌株,分别标记为Z1、Z2、Z3。利用细胞培养、形态学特征及18sr DNA序列分析,确定3株菌分别属于米根霉（Rhizopus oryzae）、黑曲霉（Aspergillus niger）和米曲霉（Aspergillus oryzae）。命名为米根霉（Rhizopus oryzae）Z1、黑曲霉（Aspergillus niger）Z2和米曲霉（Aspergillus oryzae）Z3。3株菌均能产生高活力纤维素酶。米根霉（Rhizopus oryzae）Z1还能产生较高活力的糖化酶,米曲霉（Aspergillus oryzae）Z3还能产生较高活力的蛋白酶。对3株菌的生长特性及产酶条件进行了研究,根据单因素实验结果对产酶条件进行优化,优化后纤维素降解菌Z1、Z2、Z3产酶活力较单因素最高酶活力分别提高了54.00%、11.80%和42.60%。甘蓝（Brassica oleracea L）、芹菜（Apium graveolens）、白菜（Brassica rapa pekinensis）是厨余垃圾中常见的纤维素含量较丰富的蔬菜,为了验证3株菌对纤维素的降解能力,分别将3株纤维素降解菌按一定比例接种到3种蔬菜原料中,以TS为评价指标,确定了不同菌株对几种蔬菜的最佳降解条件。比较了降解前后蔬菜的红外图谱,发现降解后红外谱图在不同波数处百分透光度均大于降解前的百分透光度。其中芹菜经Z1+Z2+Z3降解后,红外谱图显示在波数为1070cm^-1-1380cm^-1之间出现了较多新振动峰。白菜经菌株Z2降解后,红外谱图显示在波数为1250cm^-1处出现新振动峰,说明纤维素降解菌可以改变原料结构。不同菌株对几种蔬菜降解动力学拟合结果发现,纤维素降解菌Z1、Z2、Z3对甘蓝、芹菜和白菜降解前12h拟合效果均优于18h。米根霉Z1对甘蓝和芹菜的降解过程均符合片状模型,对白菜降解过程符合柱状模型。黑曲霉Z2对甘蓝、白菜和芹菜的降解过程分别符合片状模型、柱状模型和片状模型。米曲霉Z3对甘蓝、芹菜和白菜的降解过程分别符合片状模型、柱状模型和球状模型。基于以上研究结果认为,3株菌能够对纤维素结构产生破坏作用,不同菌株对不同蔬菜的降解方式不同。将Z1、Z2、Z3接种到餐厨垃圾与牛粪混合发酵系统中,通过测定日产甲烷率、总挥发性脂肪酸TVFA（Total volatile atty acids）、p H、F₄₂₀辅酶、丙酸浓度的变化,研究了3株菌对混合原料厌氧发酵产甲烷效率的协同效应。发现纤维素降解菌预处理能促进混合原料厌氧发酵产甲烷过程。Z1、Z2、Z3预处理组日产甲烷率第1个峰值出现时间均较对照组提前了5d,日产甲烷率分别提高60.00%、40.00%和23.00%。米根霉Z1在发酵初期具有更明显的促进作用,黑曲霉Z2在发酵后期具有较强的促进作用。预处理组总挥发性脂肪酸TVFA浓度均高于对照组,三次产甲烷率峰值出现之前总挥发性脂肪酸TVFA均有一定的累积,但其浓度不超过30.00g/L。F₄₂₀浓度变化趋势与日产甲烷率变化趋势一致。米曲霉Z3预处理组发酵液中F₄₂₀浓度波动幅度较大。与对照组相比,纤维素降解菌Z1、Z2、Z3预处理组均有较高的挥发性固体浓度VS（Volatile solid）去除率。预处理阶段较对照组分别提高了375.00%、300.00%和325.00%,整个厌氧消化阶段分别提高15.00%、13.70%和17.50%。在总挥发性脂肪酸TVFA中,丙酸转化成乙酸的ΔG₀大于丁酸和甲酸,而预处理组发酵液中丙酸浓度均较对照组低,从热力学角度证实预处理组具有较高产甲烷率。餐厨垃圾与牛粪混合原料产甲烷潜力较单独餐厨垃圾、单独牛粪产甲烷潜力及甲烷产率均有一定的提高。对混合垃圾经纤维素降解菌Z1、Z2、Z3预处理,经厌氧发酵产甲烷动力学分析发现,混合原料甲烷潜力分别提高了173.00%、177.00%和119.00%,甲烷产率分别提高了193.00%、158.00%和134.00%,产甲烷延滞期分别提前了1.74d、1.74d和1.91d,从而可以提高产甲烷效率。纤维素降解菌Z1、Z2、Z3预处理对混合原料厌氧发酵产甲烷协同效应明显。米根霉Z1具有最明显的促进作用。对菌株Z2、Z3、Z1预处理组、原料未经预处理但经自身水解12h、未经预处理且未自身水解而直接进行厌氧发酵的五个处理组（编号为A、B、C、D、E）进行高通量测序发现,预处理组微生物相对丰度均高于未经预处理组,C处理组物种相对丰度最高、物种多样性及均匀性均较高。预处理组优势菌群的丰度均高于未经预处理组。不同处理组中丰度高于1%的细菌类群共有7个门,7个优势菌门在不同预处理组中的丰度存在差异。热袍菌门（Thermotogae）和变形菌门（Proteobacteria）A处理组中丰度最高,互养菌门（Synergistetes）和广古菌门（Euryarchaeota）B处理组中丰度最高。厚壁菌门（Firmicutes）及未分类的细菌在C处理组中丰度最高。拟杆菌门（Bacteroidetes）E处理组中丰度最高。处理组中乳酸杆菌属（Lactobacillus）、梭杆菌属（Clostridium）、芽孢杆菌属（Bacillus）和Defluviitoga丰度均最高。但A处理组中Defluviitoga的丰度最高（42.51%）。厌气杆菌属（Anaerobaculum）B处理组最高。预处理组中栖热粪杆菌属（Coprothermobacter）丰度与对照组明显减少,甲烷囊菌属（Methanoculleus）丰度明显增加。更多还原

【Abstract】 Pretreatment of food waste can change the raw material structure and increase the potential of methane production.The difficulty of cellulose degradation is one of the factors that limit the rate of hydrolysis.In order to change the raw material structure of food waste and improve the capacity of methane production,three cellulose degradation strains were disolated in this paper,which were labeled as Z1,Z2 and Z3 respectively.Using cell culture,morphological characteristics and 18sr DNA sequence analysis,it was determined that the three strains belonged to Rhizopus oryzae,Aspergillus Niger and Aspergillus oryzae,respectively.They were named as Rhizopus oryzae Z1,Aspergillus Niger Z2 and Aspergillus oryzae Z3.It was found that the three strains could produce high activity cellulase.Rhizopus oryzae Z1 also had high activity of mashing enzyme and Aspergillus oryzae Z3 had high activity of protease.The growth characteristics and enzyme production conditions of the three strains were studied.According to the results of single factor experiment,the enzyme production conditions of different strains were optimized by using design-expert software.The cellulolytic activities of strains Z1,Z2 and Z3 were increased by 54.00%,11.80%and42.60%,respectively.Cabbage（Brassica oleracea L）,celery（Apium graveolens）and chinese cabbage（Brassica rapa pekinensis）are common cellulose rich vegetables in food waste.In order to verify the cellulose degradation ability of the three strains,the cultures of three cellulose decomposing strains were inoculated into several different vegetables containing rich cellulose at certain proportions.Taking TS as the evaluation index,the optimal degradation conditions of different strains on different vegetables were determined through experiments.Infrared spectroscopy showed that the structural of these vegetables changed significantly after degradation.The infrared spectra of the celery showed that the transmissibility at all peaks was higher than that of the celery after degradation by strains of Z1,Z2,Z3 and Z1+Z2+Z3,respectively.Many new absorption peaks appeared range the wave number numbers from 1070cm^-1to 1380cm^-1.The transmissibility from Z1+Z2+Z3 experienced the biggest growth.The infrared spectra of the chinese cabbage showed that many new absorption peaks appeared at the wave number 1250cm^-1 after degradation by strain Z2.The transmissibility from strain Z2 experienced the biggest growth.The kinetic model was used to study the degradation process of different strains on several vegetables.It was showed that the strains Z1,Z2 and Z3 had better fitting effect on the cabbage,celery and chinese cabbage after 12h degradation than 18h.For Z1,the degradation process of the cabbage and celery were in line with the flaky model and the degradation process of the chinese cabbage was in line with the columnar model.For Z2,the degradation process on the cabbage,celery and chinese cabbage conforms to flaky model,columnar model and flaky model,respectively.For Z3,the degradation process of on the cabbage,celery and chinese cabbage conforms to the flaky model,column model and spherical model,respectively.The synergistic effect of the three strains on the anaerobic fermentation of the co-digestion of food waste and cow dung was studied.The changes in the daily methane rate,TVFA(Total volatile atty acids,p H,coenzyme F₄₂₀ and propionic acid concentration were determined and the results showed that the biological pretreatment from the strains can promote the process of the anaerobic fermentation of the co-digestion to produce more methane.Compared with the control group,the time of the first peak of daily methane production rate of the pretreated groups of strains Z1,Z2 and Z3 were all 5 days earlier than that of the control group,and the daily methane production rates were increased by 60.00%,40.00%and 23.00%respectively.Rhizopus oryzae Z1 had more obvious promoting effect at the early stage of fermentation,while the Aspergillus Niger Z2 had stronger promoting effect at the later stage.The concentrations of TVFA in the pretreatment groups were all higher than that in the control group.The TVFA accumulated before three peaks of methane production appear,however,the TVFA concentration did not exceed 30.00g/L.The concentration of F₄₂₀ was consistent with that of daily methane rate.For Aspergillus oryzae Z3,the concentration of F₄₂₀ was fluctuated greatly.Compared with the control group,the VS（Volatile solid）removal rates were higher in the pretreatment groups.In the pretreatment stage,the VS removal rates were increased by 375.00%,300.00%and 325.00%respectively.In the whole anaerobic digestion stage,they were increased by 15.00%,13.70%and 17.50%respectively.The concentrations of propionic acid in the pretreatment groups were significantly decreased than that of the control group.TheΔG₀ of the propionic acid is greater than the butyric acid,acetic acid and formic acid.From the thermodynamics view,it was confirmed that pretreatment group has high methane production rate than the control group.The methane production rates and the methane potentials were all higher than those of the single food waste and cow dung group.After pretreatment by strains Z1,Z2,Z3,the methane potentials were increased 173.00%,177.00%and 119.00%respectively,The methane production rates were increased 193.00%,158.00%,134.00%respectively.The dynamic fitting results showed that the methane production demurrage times of co-digestion after pretreatment by the strains Z1,Z2,Z3 were ahead of the 1.74d,1.74d,1.91d,respectively.Compared with control group,the synergistic effect of biological pretreatment groups was obvious.Among them,Z1 had the most obvious promoting effect.High-throughput sequencing was used to analysis the bacterial communities of the cellulose degradation strain Z2,Z3 and Z1 pretreated groups.The group containing food waste and cow dung without addition of the strains but hydrolysis for 12h and the group of food waste and cow dung were used as the control groups.They were labeled as A,B,C,D,E groups respectively.The relative bacterial abundances in the pretreatment groups were higher than that of the control groups.The C pretreated group has the highest relative abundance,with higher species diversity and evenness.The abundance of dominant bacteria in the pretreatment groups was higher than that in the control group.There were seven dominant phyla with the abundance greater than 1%in different groups.The abundance of the dominant phyla changed in the pretreatment groups.Thermotogae and Proteobacteria had the highest abundance in group A,Synergistetes and Euryarchaeota had the highest abundance in group B.Firmicutes and unclassified bacteria had the highest abundance in group C,the abundance of Bacteroidetes was the highest in group E.Lactobacillus,Clostridium,Bacillus and Defluviitoga were the most abundant dominant genus in all the pretreatment groups,but the abundances were changed in the different pretreated groups.The abundance of Defluviititoga was 42.51%in group A.The abundance of Aerobaculum was the highest in B group.Compared with the control group,the abundance of Coprothermobacter in pretreatment groups was obviously decreased,and the abundance of Methanocytoleus was obviously increased.更多还原