摘 要
黄连素被应用于治疗多种疾病，导致对黄连素的需求不断增长，大量黄连素制药废水因此产生。经研究，物化—生物耦合工艺是处理黄连素废水的最佳方法，但是黄连素容易被污泥吸附，进入污泥处理环节。厌氧消化是目前处理污泥的主要方式之一，黄连素具有广谱抗生素性质，可能会影响污泥厌氧消化性能以及厌氧消化过程中抗性基因的变化。
本研究通过4组序批式厌氧反应器考察了黄连素浓度对污泥厌氧消化性能的影响规律。结果表明，黄连素会促进污泥减量化，增加污泥固体有机物的溶解；500 mg/L黄连素会显着促进氨氮的释放和VFAs的积累；20 mg/L黄连素轻微促进甲烷产生，但是500 mg/L黄连素会严重抑制累计甲烷产量。然而，由于污泥中铁的大量存在，使得磷沉淀于污泥中，导致上清液中磷的含量极少。在厌氧消化后期，研究了黄连素浓度对污泥性质的影响。结果表明，厌氧消化处理会大量减少污泥EPS，并且TB-EPS的减少量明显多于LB-EPS；相比于蛋白质，EPS中腐殖质和多糖更易于降解，但黄连素会抑制多糖的降解；消化污泥EPS中主要存在酪氨酸类和色氨酸类物质，TB-EPS中物质含量明显高于LB-EPS，此外，500 mg/L黄连素会显着增加酪氨酸类和色氨酸类物质的含量。从红外光谱分析可知，黄连素不会改变消化污泥表面的官能团结构。
在黄连素影响机理部分，首先研究黄连素浓度对厌氧消化四个阶段的影响。结果表明，在溶解阶段，黄连素会促进污泥蛋白质和多糖的溶解，且黄连素会促进TB-EPS中蛋白质的释放。在水解阶段，黄连素会抑制蛋白质的水解，当黄连素浓度过高时，系统难以恢复对蛋白质的水解能力；多糖的水解情况稍有不同，只有500 mg/L黄连素会显着抑制多糖的水解，且系统能迅速恢复对多糖的水解能力。在酸化阶段，20 mg/L黄连素轻微促进VFAs的产生，但是500 mg/L黄连素会显着抑制VFAs的产生。在产甲烷阶段，20 mg/L黄连素能够显着促进甲烷的产生，但100 mg/L及以上的黄连素会严重抑制产甲烷过程。
在机理研究部分还探究了黄连素的降解情况。结果显示，活性污泥对黄连素有很强的吸附能力，导致体系中黄连素几乎全部留存在泥相中；黄连素的降解速率为先快后慢，短时间内可以降解大量黄连素，最终，黄连素的降解率可达到98%以上。通过微生物群落进一步探究黄连素的影响机理。研究发现，厌氧消化会减少微生物的丰富度和多样性，而黄连素的添加会进一步减少微生物的丰富度和多样性。经厌氧消化处理后，Firmicutes、Patescibacteria和Bacteroidetes这三种发酵细菌菌门的丰度明显增长，其中Patescibacteria会被500 mg/L黄连素抑制，相反，Firmicutes和Bacteroidetes可以耐受高浓度黄连素的压力。从属水平分析，Acetoanaerobium和Petrimonas是两种产乙酸功能细菌，在BH500组中均处于优势地位，因此BH500组中VFAs产量更高。产甲烷过程涉及到很多古菌的参与，因此分析了古菌群落结构。结果表明，氢营养型产甲烷古菌，如Methanobacteria（纲水平）、Methanobacterium（属水平）能够耐受高浓度黄连素的压力，因此在BH500组中占主导地位。
最后通过宏基因组数据研究了黄连素对厌氧消化过程中抗性基因（ARGs）的影响。结果表明，黄连素对不同耐药类型和不同耐药机制ARGs的组成分布没有影响，但是500 mg/L黄连素会轻微增加多重耐药性、四环素类和肽类等ARGs的丰度；消化污泥中留存的ARGs能够对37种抗生素产生耐药性，其中，具有多重耐药性的ARGs数量最多，其次是四环素抗性基因（TC-ARGs）；体系中最主要的耐药机制为抗生素外排泵机制。选择五种TC-ARGs做进一步分析，可以发现绝大多数TC-ARGs都存在于细菌中，有少量TC-ARGs会存在病毒中。几种TC-ARGs的主要宿主细菌为Proteobacteria、Actinobacteria、Chloroflexi、Bacteroidetes和Firmicutes。这几类菌门同样也是体系中的优势微生物。
100 mg/L黄连素增加了TC-ARGs（tet X除外）在Chloroflexi中的占比，同时500 mg/L黄连素也增加了TC-ARGs在Firmicutes中的占比。
关键词 ： 黄连素；厌氧消化；活性污泥；微生物群落；抗性基因。
ABSTRACT
Berberine is used to treat a variety of diseases, leading to an increasing demand for berberine,and a large amount of berberine pharmaceutical wastewater is produced. After research, thephysicochemical-biological coupling process is the best method to treat berberine wastewater, butberberine is easily adsorbed by sludge and enters the sludge treatment process. Anaerobicdigestion is currently one of the main ways to treat sludge. Berberine has broad-spectrumantibiotic properties, which may affect the anaerobic digestion performance of sludge and thechanges in antibiotic resistance genes during anaerobic digestion.
In this study, the influence of berberine concentration on the anaerobic digestion performanceof sludge was investigated through 4 sets of sequential batch anaerobic reactors. The resultsshowed that berberine could promote sludge reduction and increase the dissolution of sludge solidorganic matter; 500 mg/L berberine could significantly promote the release of ammonia nitrogenand the accumulation of VFAs; 20 mg/L berberine slightly promoted methane production, but 500mg/L berberine would severely inhibit cumulative methane production. However, due to the largeamount of iron in the sludge, phosphorus was precipitated in the sludge, resulting in extremelylow phosphorus content in the supernatant. In the later stage of anaerobic digestion, the effect ofberberine concentration on the properties of sludge was studied. The results showed that anaerobicdigestion treatment would greatly reduce the sludge EPS, and the reduction of TB-EPS wassignificantly greater than that of LB-EPS; Compared with protein, humus and polysaccharides inEPS were easier to degrade, but berberine could inhibit the degradation of polysaccharides; TheEPS of digested sludge mainly contained tyrosine and tryptophan. The content of TB-EPS wassignificantly higher than that of LB-EPS. In addition, 500 mg/L berberine would significantlyincrease tyrosine and tryptophan. From the analysis of infrared spectroscopy, it was known thatberberine did not change the structure of functional groups on the surface of digested sludge.
In the part of influence mechanism, firstly, the influence of berberine concentration on thefour stages of anaerobic digestion was studied. The results showed that in the dissolution stage, berberine promoted the dissolution of sludge proteins and polysaccharides, and berberinepromoted the release of proteins in TB-EPS. In the hydrolysis stage, berberine would inhibit thehydrolysis of protein. When the concentration of berberine was too high, the system was difficultto recover the ability to hydrolyze proteins; the hydrolysis of polysaccharides was slightlydifferent, only 500 mg/L berberine would significantly inhibit the hydrolysis of polysaccharides ,And the system could quickly restore the ability to hydrolyze polysaccharides. In the acidificationstage, 20 mg/L berberine slightly promoted the production of VFAs, but 500 mg/L berberine couldsignificantly inhibit the production of VFAs. In the methanogenesis stage, 20 mg/L berberinecould significantly promote the production of methane, but 100 mg/L and above berberine couldseverely inhibit the process of methane production.
In the mechanism research part, the degradation of berberine was also explored. The resultsshowed that activated sludge had a strong adsorption capacity for berberine, which resulted inalmost all berberine in the system remaining in the mud phase; the degradation rate of berberinewas fast and then slow, and a large amount of berberine could be degraded in a short time, andfinally , The degradation rate of berberine could reach more than 98%. Further explore theinfluence mechanism of berberine through the microbial community. Studies had found thatanaerobic digestion would reduce the abundance and diversity of microorganisms, and theaddition of berberine would further reduce the abundance and diversity of microorganisms. Afteranaerobic digestion, the abundance of Firmicutes, Patescibacteria, and Bacteroidetes all increasedsignificantly. Among them, Patescibacteria would be inhibited by 500 mg/L berberine. On thecontrary, Firmicutes and Bacteroidetes could tolerate high concentrations of berberine. pressure.
According to genus level analysis, Acetoanaerobium and Petrimonas were two acetogenicfunctional bacteria, which were in a dominant position in the BH500 group, so the production ofVFAs in the BH500 group was higher. The process of methanogenesis involved the participationof many archaea, so the structure of the archaeal community was analyzed. The results showedthat hydrotrophic methanogenic archaea, such as Methanobacteria (class level) andMethanobacterium (genus level) can tolerate the pressure of high concentrations of berberine, andtherefore dominated the BH500 group.
Finally, the effect of berberine on resistance genes (ARGs) during anaerobic digestion wasstudied through metagenomic data. The results showed that berberine had no effect on thecomposition and distribution of ARGs with different types of resistance and different resistancemechanisms, but 500 mg/L berberine would slightly increase the abundance of ARGs such asmultidrug resistance, tetracyclines and peptides; The ARGs remaining in the digested sludge coulddevelop resistance to 37 antibiotics. Among them, the ARGs with multi-drug resistance were themost numerous, followed by tetracycline resistance genes (TC-ARGs); the main resistance mechanism in the system was antibiotic efflux pump mechanism. Selecting five TC-ARGs forfurther analysis, it could be found that the vast majority of TC-ARGs were present in bacteria, anda small amount of TC-ARGs would be present in viruses. The main host bacteria of severalTC-ARGs were Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes and Firmicutes. Thesetypes of bacteria phyla were also the dominant microorganisms in the system. 100 mg/L berberineincreased the proportion of TC-ARGs (except tet X) in Chloroflexi, and 500 mg/L berberine alsoincreased the proportion of TC-ARGs in Firmicutes.
KEY WORDS : berberine; anaerobic digestion; wasted activated sludge; microbialcommunity; antibiotic resistance gene。
第一章 绪论
1.1、盐酸黄连素的性质。
1.1.1、理化性质。
盐酸黄连素，又名盐酸小檗碱，分子式为：
C20H18Cl NO4·2H2O，结构如图1-1所示。外形为黄色针状结晶或粉末，无臭、味极苦。在160℃-220 ℃时会分解，可溶于热水和乙醇中，具有很强的抗氧化降解性能[1]。是一种从黄连等小檗属植物中分离得到的异喹啉类生物碱。
作为一种广谱抗生素，盐酸黄连素对大多数微生物均有抑制作用，低剂量时抑菌，高剂量时杀菌[2]。
【2】
1.1.2、药用价值。
盐酸黄连素具有广泛的药理特性，可用于治疗多种疾病。盐酸黄连素能抑制毒素和包括幽门螺杆菌在内的细菌[3]，保护肠道上皮屏障免受损伤，改善肝脏损伤[4, 5]；还能抑制各种癌细胞的增殖[6]，阻止癌细胞的侵袭和转移[7]；还有证据证实盐酸黄连素能提高放化疗的疗效和安全性[8]；此外，盐酸黄连素可调节糖代谢[9]和脂代谢，改善能量消耗，减轻体重[10]，减轻非酒精性脂肪性肝病；另外还可改善心血管血流动力，抑制缺血性心律失常，减缓动脉粥样硬化的发展，降低高血压[11, 12]；在神经系统方面，盐酸黄连素也体现出强大的保护作用，包括抗氧化、抗凋亡和抗缺血。
【由于本篇文章为硕士论文，如需全文请点击底部下载全文链接】
1.2 盐酸黄连素废水的处理
1.2.1 物化法
1.2.2化学法
1.2.3生物法
1.3 活性污泥厌氧消化技术.
1.3.1原理.
1.3.2 影响因素
1.4抗生素对厌氧消化的影响.
1.4.1 抗生素对厌氧消化各阶段的影响
1.4.2抗生素对厌氧消化菌群的影响.
1.5抗性基因在厌氧消化过程中的变化
1.5.1温度
1.5.2固体停留时间
1.5.3 pH
1.6课题研究背景、意义及内容
1.6.1研究背景及意义
1.6.2研究内容及技术路线.
1.6.3创新点
第二章实验材料与测定方法.
2.1实验材料及装置.
2.1.1实验材料
2.1.2实验装置
2.2分析方法
2.2.1 CH4测定
2.2.2溶解性多糖测定
2.2.3溶解性蛋白质测定
2.2.4腐殖酸测定
2.2.5三维荧光光谱
2.2.6 VFAs测定
2.2.7铁离子测定.
2.2.8胞外聚合物的提取
2.2.9黄连素的测定
2.2.10环境扫描电镜
2.2.11傅里叶红外光谱扫描.
2.2.12微生物种群结构分析.
2.2.13其他指标测定方法
第三章黄连素浓度对污泥厌氧消化的影响.
3.1实验材料与方.
3.2黄连素对甲烷产量的影响.
3.3黄连素对污泥减量的影响.
3.4黄连素对上清液有机物的影响.
3.4.1 SCOD浓度.
3.4.2蛋白质、多糖浓度
3.5黄连素对挥发性脂肪酸的影响.
3.6黄连素对氨氮积累的影响.
3.7黄连素对磷释放的影响.
3.8黄连素对污泥性质的影响.
3.8.1黄连素对EPS含量及成分的影响.
3.8.2黄连素对污泥表面官能团的影.响
3.9本章小结
第四章黄连素对污泥厌氧消化的影响机理.
4.1实验材料与方
4.1.1黄连素对污泥厌氧消化各阶段的影响.
4.1.2黄连素的降解情况
4.1.3细菌和古菌群落结构分析.
4.2黄连素对厌氧消化各阶段的影响.
4.2.1溶解阶段
4.2.2水解阶段
4.1.4产甲烷阶段.
4.3黄连素在厌氧消化过程中的降解.
4.4微生物种群分析.
4.4.1 Alpha多样性分析
4.4.2细菌组成分析
4.4.3古菌组成分析
4.5本章小结
第五章黄连素对污泥厌氧消化过程中抗性基因的影响
5.1实验材料与方法.
5.2黄连素对ARGs的影响.
5.2.1黄连素对ARGs类型的影响
5.2.2黄连素对ARGs耐药机制类型的影响.
5.3黄连素对四环素抗性基因丰度的影响.
5.3.1黄连素对TC-ARGs丰度的影响
5.3.2黄连素对细菌中TC-ARGs丰度的影响
5.3.3微生物群落结构组成.
5.4本章小结
第六章 结论
本课题首先研究黄连素对污泥厌氧消化性能的影响，通过分析累计甲烷产量、VFAs浓度、上清液有机物浓度及组成、污泥胞外聚合物含量及组成、污泥理化性质等指标得到黄连素的影响规律。在影响机理方面，研究黄连素对污泥厌氧消化四阶段（溶解、水解、酸化和产甲烷）的影响，同时探究黄连素在厌氧消化过程中降解情况，还会分析微生物（细菌、古菌）群落结构的变化。最后，通过宏基因组数据研究黄连素对厌氧消化过程中ARGs的影响规律。得到结论如下：
（1）黄连素浓度对污泥厌氧消化的影响。
①黄连素对累计甲烷产量的影响效果与浓度相关，20 mg/L黄连素会轻微促进甲烷产生，但是100 mg/L黄连素会抑制甲烷生成，当浓度继续增加到500 mg/L时，累计甲烷产量比空白组减少83.89%。
②添加黄连素会促进污泥减量化，增强厌氧消化过程中固体有机物的溶解，在厌氧消化第22天，BH500组中污泥TSS和VSS分别减少了33.07%和41.46%，液相中SCOD浓度达到3435.92±251.08 mg/L。此外，500 mg/L黄连素会显着促进蛋白质、多糖的溶解，氨氮的释放和VFAs的积累。
③理论上，在厌氧消化过程中，污泥会释放大量的磷到消化液中，但是由于污泥含有大量的铁，导致磷沉淀于污泥中。
④经厌氧消化处理，污泥LB-EPS和TB-EPS均明显减少，其中TB-EPS的下降幅度更显着。相比于蛋白质，EPS中腐殖质和多糖的降解率更高。此外，添加黄连素会抑制EPS中多糖的降解，但是不会影响蛋白质和腐殖酸的降解率。
在消化污泥EPS中主要存在酪氨酸类和色氨酸类物质，消化污泥LB-EPS和TB-EPS具有相似的组成物质，但是TB-EPS中物质含量明显高于LB-EPS。
500mg/L黄连素使得消化污泥LB-EPS中酪氨酸类和色氨酸类物质的含量明显增加，但是不会影响TB-EPS中物质含量。此外，通过红外光谱可知，黄连素不会改变消化污泥表面的官能团结构。
（2）黄连素对污泥厌氧消化的影响机理。
①在溶解阶段，黄连素会促进蛋白质和多糖的溶解，且黄连素浓度越高，溶出的蛋白质和多糖越多。污泥胞外聚合物的溶解可以贡献部分溶解性蛋白质和多糖，经过2天的厌氧发酵，污泥LB-EPS和TB-EPS均明显减少，同时，添加黄连素促进了TB-EPS中蛋白质的释放。水解过程中，100 mg/L和500 mg/L黄连素会抑制BSA的水解，在这两组中，分别只有33.66±1.37%和30.12±3.71%的蛋白质得到降解。500 mg/L黄连素同样会抑制Dextran的水解，但是系统对Dextran的水解能力可以很快恢复。在酸化过程中，20 mg/L黄连素能够促进L-谷氨酸和葡萄糖的酸化，产生更多的VFAs，500 mg/L黄连素却显着抑制了酸化过程。在产甲烷阶段，20 mg/L黄连素会促进产甲烷过程，但是高浓度（100 mg/L以上）黄连素显着抑制了乙酸钠的甲烷化。
②活性污泥对黄连素具有很强的吸附作用，导致体系中黄连素几乎全部存在于污泥中。黄连素的降解速率特点为先快后慢，短时间内可以降解大部分黄连素，因此体系中很多功能可以快速恢复。此外，污泥厌氧消化处理可以去除98%以上的黄连素。
③在厌氧消化反应前期，添加适量黄连素会增加污泥厌氧消化过程中微生物的丰富度和多样性，但是，在厌氧消化反应后期，黄连素会减少微生物的丰富度及多样性。细菌群落中，经过厌氧消化处理，Firmicutes、Patescibacteria和Bacteroidetes这三种常见的发酵细菌菌门丰度明显增长，其中Firmicutes和Bacteroidetes可以耐受高浓度黄连素的压力，但是Patescibacteria会被500 mg/L黄连素抑制。从属水平上分析，Acetoanaerobium和Petrimonas是两种产乙酸功能菌，在BH500组中均处于优势地位，因此BH500组中VFAs产量提高。
④在古菌群落中，从纲水平分析可知，虽然产甲烷菌Methanobacteria和Methanomicrobia在BH500组中总占比为96.18%-99.46%，但可能存在较高比例的死菌，从而导致累计甲烷产量较低。从属水平分析可知，高浓度黄连素会抑制Methanosaeta、Methanosarcina和Candidatus_Methanofastidiosum的生长，但是能够促进Methanobacterium的生长。在产甲烷期间，Methanobacterium和Methanosarcina为优势菌属，表明氢营养型产甲烷是体系中产生沼气的主要途径。
（3）黄连素对污泥厌氧消化过程中抗性基因的影响。
①厌氧消化污泥中检测到的ARGs能够对37种抗生素产生耐药性，其中，具有多重耐药性的ARGs数量最多，其次是TC-ARGs。添加黄连素不影响各类ARGs的占比，但是500 mg/L黄连素会明显增加单个ARGs的丰度。在消化污泥中ARGs中共检测到6种耐药机制，其中抗生素外排泵机制为最主要的耐药机制。某些ARGs还具有多种（两种及以上）耐药机制。黄连素的添加不会改变污泥体系中ARGs耐药机制类型的分布。
②由于TC-ARGs含量较高，选取5种TC-ARGs（tet A、tet B、tet M、tet O、tet X）进一步研究。绝大多数TC-ARGs都存在于细菌中，有少量TC-ARGs出现在病毒中。几种TC-ARGs的丰度大小表现为：tet A>tet B>tet M≈tet O>tet X。黄连素的添加使得BH100组中tet M和tet O基因的丰度显着增加。
③几种TC-ARGs的主要宿主细菌是Proteobacteria、Actinobacteria、Chloroflexi、Bacteroidetes和Firmicutes。添加黄连素会改变TC-ARGs在细菌菌门中的分布：相比于空白组，BH100组中Chloroflexi和BH500组中Firmicutes对tet A、tet B、tet M和tet O这四种TC-ARGs的贡献显着提高，另外，添加黄连素减少了tet X在Proteobacteria中的占比，同时增加了其在Firmicutes中的占比。
④联系微生物群落组成TC-ARGs进行分析，可以发现体系中几种优势微生物——Proteobacteria、Firmicutes、Bacteroidetes、Actinobacteria和Chloroflexi同时也是几种TC-ARGs的主要宿主细菌。
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