Validation of Bacteroidales-based microbial source tracking markers for pig fecal pollution and their application in two rivers of North China

• Pig feces is the predominant excrement produced by animal husbandry in China. • The PF, Pig-1-Bac^TaqMan, and Pig-2-Bac^TaqMan MST assays showed better performance. • The pig-specific MST assays can contribute to managing the pig fecal pollution. In China, pig feces is the predominant source of excrement produced by animal husbandry. Improper use or direct discharge of pig feces can result in contamination of natural water systems. Microbial source tracking (MST) technology can identify the sources of fecal pollution in environmental water, and contribute to the management of pig fecal pollution by local environmental protection agencies. However, the accuracy of such assays can be context-dependent, and they have not been comprehensively evaluated under Chinese conditions. We aimed to compare the performance of five previously reported pig-specific MST assays (PF, Pig-Bac1^SYBR, Pig-Bac2^SYBR, Pig-1-Bac^TaqMan, and Pig-2-Bac^TaqMan, which are based on Bacteroidales 16S rRNA gene markers) and apply them in two rivers of North China. We collected a total of 173 fecal samples from pigs, cows, goats, chickens, humans, and horses across China. The PF assay optimized in this study showed outstanding qualitative performance and achieved 100% specificity and sensitivity. However, the two SYBR green qPCR assays (Pig-Bac1^SYBR and Pig-Bac2^SYBR) cross-reacted with most non-pig fecal samples. In contrast, both the Pig-1-Bac^TaqMan and Pig-2-Bac^TaqMan assays gave 100% specificity and sensitivity. Of these, the Pig-2-Bac^TaqMan assay showed higher reproducibility. Our results regarding the specificity of these pig-specific MST assays differ from those reported in Thailand, Japan, and America. Using the PF and Pig-2-Bac^TaqMan assays, a field test comparing the levels of pig fecal pollution in rivers near a pig farm before and after comprehensive environmental pollution governance indicated that pig fecal pollution was effectively controlled at this location.     

二苯并噻吩降解菌筛选及16S rDNA序列分析

以DBT(dibenzothiophene,二苯并噻吩)为模型化合物,分离得到了一株能降解DBT的微生物HB2,应用HPLC对其脱硫特性进行了检测。应用PCR技术克隆到16SrDNA片段,核苷酸序列分析结果表明,该菌的16SrDNA全序列与假单胞菌存在98%的同源性,初步确定该菌在微生物系统发育学上的地位。     

一株反硝化菌的分离鉴定和系统发育分析

从反硝化反应器中分离到一株异养型兼氧反硝化细菌,命名为菌株JIN1,分离菌株为革兰氏染色阴性杆状。茵落颜色为乳白色。该菌株能以硝酸钠为唯一氮源进行反硝化作用并产生亚硝酸。部分长度的16SrDNA序列分析表明,分离菌株JIN1与Aquaspirillum菌具有99%相似,与Microvirgulaaerodenitrificans菌具有99%相似。其生化性状与Aquaspirillum菌不相符。系统发育学分析表明菌株JIN1与Microvirgulaaerodenitrificans菌关系最近。故确认菌株JIN1属Microvirgulaaerodenitrificans菌。     

从不同反应器筛选、鉴别好氧反硝化菌

采用2个序批式反应器A和B,以硝态氮为唯一氮源,采用间歇曝气,以驯化、富集耐氧脱氮污泥.反应器A,其pH约为6.3,ρ(DO)为2.2～6.1 mg/L,碳氮比(ρ(C)/ρ(N),ρ(C)以ρ(COD_Cr)计)约为9;反应器B,其pH约为6.8～7.8,ρ(DO)为 2.2～3.0 mg/L,ρ(C)/ρ(N)约为15.2个反应器的ρ(NO₃－-N)均保持为80 mg/L.当2个反应器的总氮去除率达到60%以上,则认为完成好氧反硝化菌的富集.从2个反应器中共筛选得到20株BTB阳性菌,其中8株菌株的DNA样品经PCR成功扩增,进行16S rRNA测序.测序结果提交GenBank进行Blast同源性检索,并分析比对鉴别,判断8株菌株分属于假单胞菌(Pseudomonas),戴尔福特菌(Delftia),草螺菌(Herbaspirillum)和丛毛单胞菌(Comamonas)菌属.反硝化性能测定证实8株菌株均为好氧反硝化菌.      

两株溶藻细菌的分离鉴定及其溶藻特性

从富营养化池塘中筛选分离出两株溶藻细菌L7和L18,通过生理生化实验及16SrDNA测序进行鉴定,对其单一以及混合菌液去除铜绿微囊藻(Microcystis aeruginosa)、水华鱼腥藻(Anabaena flos-aquae)的效果进行研究,并考查其溶藻方式。结果表明:菌液的滤液有溶藻效果,溶藻细菌具有间接溶藻特性;细菌需要一定的初始浓度才能有明显的溶藻效果,初始浓度越高,溶藻效果越好;溶藻效果大致趋势为混合菌液>L18>L7;不同生长时期溶藻菌,其滤液的溶藻效果差异很大,衰减期的滤液溶藻效果好于对数生长期和稳定期的;滤液投加体积分数与溶藻效果成正相关;细菌鉴定结果表明,L7为蜡状芽孢杆菌(Bacillus cereus),L18为短小芽孢杆菌(Bacilluspumilus)。     

Radionuclides in cigarettes may lead to carcinogenesis via p16 inactivation

It is widely accepted that tobacco smoke is responsible for the vast majority of lung cancers worldwide. There are many known and suspected carcinogens present in cigarette smoke, including α-emitting radioisotopes. Epidemiologic studies have shown that increased lung cancer risk is associated with exposure to ionizing radiation, and it is estimated that the majority of smoking-induced lung cancers may be at least partly attributable to the inhaled and deposited radiation dose from radioisotopes in the cigarette smoke itself. Recent research shows that silencing of the tumor suppressor gene p16^INK4a (p16) by promoter methylation plays a role in smoking-related lung cancer. Inactivation of p16 has also been associated with lung cancer incidence in radiation-exposed workers, suggesting that radionuclides in cigarette smoke may be acting with other compounds to cause smoking-induced lung cancer. We evaluated the mechanism of ionizing radiation as an accepted cause of lung cancer in terms of its dose from tobacco smoke and silencing of p16. Because both radiation and cigarette smoking are associated with inactivation of p16, and p16 inactivation has been shown to play a major role in carcinogenesis, ionizing radiation from cigarette smoke likely plays a role in lung cancer risk. How large a role it plays, relative to chemical carcinogens and other modes of action, remains to be elucidated.     

一株高效脱硫脱氨氮菌的分离、鉴定及系统发育分析

从运行稳定的能同步脱臭的曝气生物滤池中采集样品,富集分离获得一株能高效脱硫脱氨氮的菌株TS-1。对分离菌株进行形态观察、生理生化试验及16S rRNA基因序列分析,结果表明,该菌株TS-1为革兰氏阳性菌,杆状;菌落在营养琼脂培养基上呈圆形,表面光滑,乳白色半透明;V-P试验阴性,能水解淀粉和明胶,利用柠檬酸盐生长;对菌株进行16S rRNA的PCR扩增,扩增产物测序结果表明分离菌株TS-1与巨大芽孢杆菌（Bacillus megaterium）同源性达到99%;以16S rRNA同源性为基础构建了包括24株相关种属细菌在内的系统发育树,在系统发育树中,分离菌株TS1与Bacillus megaterium在同一分支。结合形态观察、生理生化试验及16S rRNA基因序列分析结果,将其初步鉴定为巨大芽孢杆菌（Bacillus megaterium）。在常温（30±2）℃、转速为150 r/min的条件下,处理pH 7、S^2-和NH⁺₄-N分别为80 mg/L和88 mg/L的水样40 h,硫化物和氨氮的脱除率分别为91.8%和96.6%。     

活性污泥体系中聚糖菌的富集与鉴定

活性污泥体系中,聚糖菌(GAOs)在厌氧环境下与聚磷菌(PAOs)形成对底物的竞争关系,对聚糖菌的研究对于优化生物除磷工艺有重要意义。以葡萄糖为惟一碳源,在磷限制条件下,利用特殊运行方式对活性污泥进行驯化培养出了稳定的聚糖菌颗粒污泥,厌氧阶段磷释放量与有机物吸收量浓度(mg/L)比从7.4%下降为0.25%。从培养好的活性污泥反应器中分离获得2株聚糖菌,经菌落PCR和16S rRNA序列分析确定了所得聚糖菌菌株G1和菌株G2分别是枯草芽孢杆菌(Bacillus subtilis)和解鸟氨酸克雷伯氏菌(Klebsiella ornithinolytica)。     

高效降解环己酮红球菌JDM-3-12的分离及其系统发育分析

从岳阳巴陵石化公司环己酮生产车间总出水口的污泥中筛选到一株环己酮降解菌株,菌号为JDM-3-12;该菌能以环己酮为唯一碳源且能忍受5000 mg/L的环己酮,当环己酮的质量浓度为2000 mg/L时,在温度为30℃,转速为150 r/m in,pH=7的条件下,72小时内该菌株对环己酮的降解率达到97.91%。通过形态观察、生理生化特征检测和基于16S rRNA基因序列的系统发育分析,初步鉴定其为赤红球菌(Rhodococcus ruber)中的一个菌株,该菌最适生长温度为35℃,最适生长pH 7。     

渤海排污口邻近海域异养细菌的组成与分布

对渤海4个重点监测排污口（北塘、大蒲河、弥河和虞河）邻近海域异养细菌的组成和分布进行了调查研究。结果表明,分离到的各排污口邻近海域的细菌隶属于变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、放线菌门（Acti-nobacteria）和拟杆菌门（Bacteroidetes）4个细菌类群,其中变形菌门的细菌最丰富。各排污口的优势菌有所不同,北塘、弥河2个采样点的优势菌分别为Bacillus sp．、Pseudoalteromonas sp．,大蒲河采样点的优势菌为Vibrio sp．和Oceanospirillum sp．,虞河采样点的优势菌为 Shewanella sp．和 Bacillus sp．。多维尺度分析表明,北塘和虞河采样点的细菌种群特征较相近,细菌种群分布特征可能与排污口的海洋环境有一定的相关性。