Sulfidogenic污泥海洋沉积物和三氯乙烯减少在上流式厌氧污泥床反应器开发
Summary
Microbial sulfate reduction is a process of great importance in environmental biotechnology. The success of the sulfidogenic reactors depends among other factors on the microbial composition of the sludge. Here, we present a protocol to develop sulfidogenic sludge from hydrothermal vents sediments in a UASB reactor for reductive dechlorination purposes.
Abstract
The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide.
This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.
Introduction
一对环境生物技术的最重要的贡献是生物反应器，其中使用（接种物）的污泥能够硫酸盐还原条件下进行的设计。硫酸盐还原（SR）允许含有除同时除去COD，重金属和有机污染物，这一事实使SR中的污泥1的期望特性的高浓度硫酸的废水流的治疗。废水污染硫酸的一些例子来自制革，造纸，医药，化工等制造行业1。然而，大多数的文献是指sulfidogenic污泥时产甲烷颗粒污泥已适应sulfidogenesis 2。这种适应是通过操纵COD / SO 4 2-比在生物反应器和添加的化学品，以抑制在污泥2,3甲烷通常达到。除了长的时间是m唉要求sulfidogenic颗粒的形成中，甲烷和硫酸盐还原和污泥的高浓度硫化物的公差之间的竞争，是一些主要问题，如果是从适应获得在生物反应器中使用的sulfidogenic污泥可能出现主要产甲烷污泥硫酸盐还原条件。在这项工作中，我们描述的方法在上流式厌氧污泥床反应器（UASB），以获得从热液喷口沉积物（蓬美达，纳亚里特，墨西哥）主要为sulfidogenic污泥，然后我们评估它的硫酸盐还原活性随着时间的推移，进行了一个实验以评估其对还原脱氯的应用。被选择的沉积物的位置，因为它已被报道，在该网站有形成硫化物由于通过微生物群落栖息该特定地方4呈现的硫酸盐还原活性。
有SEVER人的优势获得沉积物这sulfidogenic污泥在适应产甲烷颗粒污泥sulfidogenesis。其中的一些优点是：（1）这是没有必要以形成颗粒的生物反应器进行操作，（2）将污泥容忍较高浓度硫化物的相比更为UASB与适于甲烷污泥操作，和（3）有用即使乙酸的用量为包含在培养基中以促进污泥的形成挥发性脂肪酸的混合物甲烷为基片没有竞争。
此步骤，以促进sulfidogenesis因为海洋沉积物有各种各样，如硫酸盐还原菌，发酵菌和细菌脱卤，只是仅举几例5,6微生物的天然泳池。财团从海洋沉积物开发利用这一协议的类型可能会出现在硫酸盐还原，因此，高的工作效率 ulfate还原活性随着时间的推移和更高的耐受性硫化物的浓度比报告为有毒的产甲烷菌和硫酸盐还原菌更高。另一方面，它很可能是该脱卤能力由以下此处协议提议的，但它可以取决于原始微生物群落也示于沉积物。这种假设是基于这样还原脱氯可以通过呼吸或共代谢发生的事实完成的，这两个条件，可能会在海洋微生物群落7得到提升。沉积物以获得污泥的培养物通过使用乙酸盐，丙酸盐和丁酸盐的混合物作为底物，因为这些挥发性脂肪酸所使用的几株硫酸盐还原细菌的进行。这些酸也经常发现在海洋沉积物碳化合物的种类，根据在文献中关于碳质材料在海沉积物5,6-若干报告。
内容“>最后，一些被发现在地下水和其他水体，世界各地的最有毒化合物是氯化溶剂，如三氯乙烯（TCE）或全氯乙烯（PCE）。这些化合物是有毒的，不仅对人，而是还对微生物，特别是三氯乙烯，这仍然是在美国被认为是优先的污染物由环境保护局8。在这项工作中，我们提出，是在一个实验，其中sulfidogenic污泥减少TCE浓度的其能力进行测试系列报道的甲烷条件下，9,10含氯化合物生物降解性。值得一提的是，大多数的含氯化合物的生物降解研究已经甲烷条件9,10下进行的。我们认为，实验TCE在该协议提出的是一个的污泥的潜在应用很好的例子。该实验的目的是到e计价污泥的三氯乙烯和对硫酸盐还原活性的三氯乙烯效果的耐受性。考虑到最上氯化化合物的生物降解研究的是甲烷条件下进行的，该协议建议一个油泥的产生可以被用来同时：（1）除去硫酸，（2）除去COD和（3）除去氯化合物。进一步的步骤可以是评价对同时除去三氯乙烯和重金属的污泥（除了硫酸盐和COD），这是不能被甲烷条件下评价两个条件。Protocol
Representative Results
Discussion
有sulfidogenesis的环境生物技术多个应用程序，硫酸盐还原细菌的代谢的最常用的应用程序在财团与发酵菌是在废水处理中的一个。 UASB反应器是主要的设计方法，以工业废水处理与硫酸盐高浓度之间。在这项工作中，我们提出了一个协议，以获得从海洋沉积物sulfidogenic污泥的UASB反应器。在协议中的关键步骤，以从海洋沉积物获得sulfidogenic污泥是：（1）促进沉积物样品的的同源被放置在反应器中，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors are grateful for the financial support provided by Instituto Politécnico Nacional grants 20120110, 20130399 and 20140239 SIP and also by Instituto de Ciencia y Tecnología del Distrito Federal Mexico (PICS 08-79, ICYT-DF, 2009-2012). Thanks also to CONACYT – Mexico for the graduate scholarship (225806) awarded to Selene Montserrat García-Solares and for the financial support provided by grant 82627.
Materials
| trichloroethylene | sigma Aldrich | 251402 | |
| cis- 1,2 dichlorotehylene | sigma Aldrich | ||
| trans-1,2 dichloroethylene | sigma Aldrich | D-62209 | |
| vinyl chloride scotty standard | supelco | 1000 ppm v/v in nitrogen | |
| ethene scotty standard | supelco | 99% purity | |
| pump | Masterflex | Model 7553-75 | |
| spectrophotometer | any | ||
| microcentrifuge | any | ||
| gas tight syringes | any | 100 and 200 microliters | |
| UASB glass reactor | any | under design | |
| gas chromatograph | any | FID detector | |
| capillary column SPB-624 | supelco | ||
| pH meter | any | ||
| viton tubing | Masterflex | ||
| basal medium reagents | any | ||
| trace metals reagents | any | ||
| vitamins solution reagents | any | ||
| sodium sulfate | any | ||
| volatile fatty acids | any | ||
| COD determination kit | HACH | range 0-15000 mg/L | |
| TOPO-TA cloning kit pCR®4.0 | Invitrogen, US | ||
| S.N.A.P. TM Miniprep Kit | Invitrogen, UK | ||
| Pure link TM Quick Plasmid Miniprep kit | Invitrogen |
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