环境工程外文翻译--进脱氯的性能的上流式厌氧污泥反应器.doc

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1、改进脱氯的性能的上流式厌氧污泥反应器摘要脱氯作用的氯乙脂，又名全氯乙烯(PCE)，作为被研究在一上流厌氧污泥床(上流式厌氧污泥层)反应堆从后面的结合的厌氧的，减少的脱氯的细菌入粒状淤渣。这些反应堆与那参考1(R1)反应器相比，是颗粒高压灭菌器用以在接种以前充分脱氯，和作为参考2(R2)反应堆，包含在活性粒状淤渣。所有的三个反应堆是投放矿物媒介包含3到57MPCE，2毫升甲酸盐，和0.5毫升醋酸盐和被控制在标准以下不活跃条件下。在这次测试的反应器，平均93%的那流出物氯乙是双氯(DCE)，和99%R1反应器对比，R2反应器，没有接种，只产生三氯乙(TCE)，平均43%的流出物氯乙。没有脱氯作用的

2、PCE被测试在无接种体的无接种体不活跃颗粒的非生物防治。在随着逐渐减少液压传动装置保留时间(HRTs)连续操作的过程中，两个测试的反应器和R1反应器显示氯乙向二氯乙的转化，甚至在均匀试验性反应比最大比生长速率的密度低得多，指示这些细菌是固定不动在那活跃的和高压灭菌过的粒状淤渣中。相反的，R2反应器，没有接种微生物，只在相同的条件下将氯乙转化成三氯乙。固定可以通过利用萤光素-标记抗体探测微生物的发酵来证实。从R1反应器获得的颗粒，细菌微生物的发育主要在集中位于那颗粒的中心，而在测试的反应器，那细菌主要覆盖在那颗粒的表面。1介绍氯乙，又名全氯乙烯(PCE)，已经广泛的用作脱脂剂为金属和作为干洗剂。

3、由于不适当的存储和处理，数量巨大的氯乙烯化合物是传遍全世界。氯乙烯的微生物转化已经证明是缺氧状态经过减少的机制的脱氯作用。氯乙烯的脱氯作用的减少当量已经包含在厌氧性的混合物和营养丰富的土壤。最近，厌氧菌种系族PCE1，哪些有效减少的脱氯作用的氯乙烯，已经有结论。生物资源人为操纵可以是实质的在技术系统为最理想的生物体内异物混合物的降解。在早期的研究中，我们已经证明通过引入粒状淤渣利用一厌氧菌活跃丧失活性的那目标化合物新活动；一种脱氯的细菌，是接种入那粒状淤渣的一上流厌氧污泥床(上流式厌氧污泥层)反应器，结果淤泥能力丧失，那些从前没有这些能力。这是进一步检验那细菌是固定不动在那粒状淤渣表面，表示它

4、的脱氯的活动。通过免疫荧光显微术在颗粒薄片的利用，可以表明这种细菌在粒状污泥制造小集落。在深入的研究中，我们介绍不活跃的粒状污泥中脱氯五氯苯酚的细菌。这些细菌被发现是固定不动在网络中-类似颗粒内部的组织，也就是它显示它的原-脱氯的糖酵解途径。目前研究的目的是将调查PCE的脱氯作用在上流式厌氧污泥层反应器和没有额外的PCE-降解的细菌此外在颗粒是否是活跃上比较性能。2所用材料和方法2.1反应器设计有三个有效容积为204ml、预留空间为30ml的玻璃UASB反应器被用在实验当中（见图1）。氟橡胶、不锈钢，以及玻璃管被用作使氯乙的吸附和挥发最小化的连接装置。在向反应器中加入颗粒剂之前，先检验了反应器

5、系统氯乙的非生物损失。反应器是连续运行的，其水力停留时间（HRT）由2天（流速为100ml/day）逐步降低到14.4h、7.2h，最后降到1.9h。与此同时，我们通过向混合容器中连续添加780m的PCE水溶液来提供PCE，PCE通过混合容器进入反应器。实验所用的试样是从取样气门1和2中获得，分别代表反应器的流入和流出。所有取样气门都用聚四氟乙烯丁基线纹状隔膜封闭。反应器在暗处以及室温（2025C）下操作。2.2接种物来源粒状淤渣从添加PCE以及乙醇试剂达一年左右的实验室UASB反应器中获取。最初的颗粒物取自处理造纸厂废水的UASB反应器。在运用之前，颗粒物在4C下储存大约两个月。2号参比反应

6、器（R2）由20ml湿颗粒物、N2CO2（4:1）气体混合物以及厌氧培养基媒介充满。1号参比反应器（R1）也填充了20ml湿颗粒物，所不同的是，它在用无菌N2CO2（4:1）供气和填充厌氧培养基媒介之前必须在140C下采用三倍热压处理。R1反应器中分别包含有生命的和经过热压处理的颗粒物，被进一步注入50ml纯人工培养的多主寄生物（由H.Scholz-Muramatsu提供），这些寄生物在反应器运行之前允许被驯化3天。作为一种非生物控制的手段，用一个118ml的药瓶装满5ml的湿润颗粒物和50ml的厌氧培养基媒介。先向瓶内吹气（N2CO2 1:4），再经过三次热压处理，然后再添加甲酸酯和醋酸盐来

7、使其终止浓度与反应系统的平衡。PCE是作为一种无菌水溶液添加的。在实验期间，每隔4到5天都要做批量实验来测试脱氯活性。2.3无菌性测试热压处理反应器的无菌性测试是通过每天灌输D.multivorans来测验的，而且无菌控制瓶每隔4到5天就要用光显微镜检查液样的方式检验一次。2.4媒介物除开0.5mg刃天青/升，0.48gNa2S 7 9H2O/升，0.5mM醋酸盐，以及从厌氧储备溶液中添加的2mM甲酸盐之外，基本媒介物的准备方法在前面已经提及。媒介物最终的pH值为7.07.5。2.5免疫的方法在反应器运行了1.9h的水力停留时间之后，颗粒物从各个反应器内取出。颗粒物和接种物经过4福尔马林的处理

8、之后，嵌入石蜡，再切成5m的切片，然后放入玻片之中。石蜡用二甲苯去除，颗粒物在递减的乙醇浓缩物中水合。标着FLUOS的抵抗D.multivorans的兔抗血清由德国卫生工程研究所提供。水合颗粒物在普通的兔抗血清内培养，用牛血清蛋白在磷酸盐（pH7.6）缓冲溶液中冲洗，并用抗血清培养25min。作为一个控制手段，颗粒物在没有抗血清培养的条件下配制。玻片用免疫荧光显微镜法研究。2.6分析方法PCE、三氯乙烯（TCE）和二氯甲烷（DCE）通过质谱仪来检测，所用方法为薄膜入口质谱仪分析法（MIMS），PCE、TCE和DCE分别在原子量为165.8、129.9和61.0的质荷比下分析。MIMS的探测极限

9、是1g/L以下。PCE、TCE和DCE的浓度测试范围是1500m，相当于标准溶液浓度。标准溶液的配制方法是：称量过量的氯乙加入双蒸馏水，搅拌一晚使其完全混合，然后倒入血清瓶稀释。然后测定液相浓度。为了检测未知的化合物，反应器内的液体会从氟橡胶管中经由MIMS反应器抽出并丢弃。测量标准溶液的时候也会用到与上相同的流速（60ml/h）。甲酸盐用高压液相色谱法分析。酸化试样（20L）被注入HPX-87-H型多孔性阴离子交换树脂柱并加热至60C。流动相是流速为0.7mL/min的0.01N的硫酸。甲酸盐用190nm的紫外线检测。标准溶液的测试浓度范围是0.110mM。而测试极限是0.05mM。试样和标

10、准溶液用20L10的硫酸酸化至pH为2.0并离心分离。再将上清液过滤然后注入。在记录数据之前，醋酸盐由气相色谱法测量。DCE的立体异构组成由气相色谱法确定。氯乙通过加入150L含4.2mg二溴三氯甲烷/L的正戊烷萃取，作为1.5ml试样的内部标准。然后再将三微升戊烷注入气相色谱仪，气相色谱仪连接在电子俘获探测器上。探测极限是500g cis-1,2-DCE/L。未知化合物通过从反应器中提取的液体试样测定，取样前必须确定反应器内不含DCE。3结果3.1反应器研究这次测试反应器的接种随着微生物进来的氯乙烯变换平均96%，直到氯乙烯的负荷率为1.4毫摩尔每升污泥每天。反应器的主要产物是氯乙烯，流出物

11、氯乙烯平均93%(mole /mole )。三氯乙烯排放浓度曾经超过氯乙烯的流入的浓度。在流出物中少量的氯乙烯和三氯乙烯(5M)可以临时的检出。第61天所取的样品显示立体异构组成是100%单独-1，2-二氯乙烯。氯乙烯向二氯乙烯的转化几乎是当量分子的在不同的均匀试验性反应器。然而，当从7.2到1.9小时减少均匀试验性反应器时间，氯乙烯排放浓度增强到在流出物的总数的氯乙烯的25%(mole/ mole )最大量。均匀试验性反应器减少到1.9小时的九天后，氯乙烯下降到比7(mole /mole )氯乙烯总量还要少。R2反应器，包含粒状淤渣，显示氯乙脱氯作用活动伴随三氯乙烯作为唯一产物。三氯乙烯平均

12、起来为在流出物的氯乙烯的43%(mole /mole )；其余是氯乙烯。任何时候没有二氯乙烯被检出。R1反应器，包含接种微生物的高压灭菌器颗粒，能变换平均100%进来的氯乙烯，直到二氯乙烯的负荷率为1.9毫摩尔每升污泥每天。在连续运转持续的过程中，二氯乙烯是主要的氯乙在取样气门地方的产物，表现流出物的氯乙总量的平均99%(mole/ mole )。氯乙烯和三氯乙烯在均匀试验性反应器2天后在痕量(1M)流出物被检出，从7.2每小时减少到1.9每小时。三氯乙烯在流出物中浓度为氯乙烯流入的浓度平均数150%。非生物控制实验随着反应器进行，显示氯乙烯浓度在取样气门1和2偏离精确MIMS 测量(标准偏差

13、，5%)。排除可测量的氯乙烯的非生物在两个反应器系统的损失。在非生物控制高压灭菌粒状淤渣，添加500M氯乙烯没有导致任何的三氯乙烯或者二氯乙烯的产生。氯乙烯浓度是上下摇动但是趋向随次数而减少，与氯乙烯总数数量减少的经过取样相当。任何时候通过光-显微检验对来自不活跃接种的反应器和不活跃控制瓶的液体样品无菌状态下的核对没有显示任何的细菌除了这种微生物。通过这次测试和R2反应器醋酸盐的消耗量在均匀试验性反应器为14。4，7.2，和1.9小时的情况下为3.4，6.7，和27.0毫摩尔每天每升污泥。据观察没有醋酸盐随着R1反应器消耗或产生。这次测试和R2反应器，甲酸盐是完全的耗费的，在均匀试验性反应堆为

14、14.4，7.2，和1.9小时等于一平均数21.8，43.7，和175.5毫摩尔每升污泥每天，而在均匀试验性反应堆为14.4，7.2，和1.9小时R1反应器为8.4，16.8，和67.2毫摩尔每升每天，结果在0.3毫米的排放浓度。3.2免疫研究通过免疫荧光显微术调查反应器内颗粒中微生物的分布。来自这次测试反应器的颗粒是随着微生物表面的网络-类似组织象征性的密布。聚合体在表面出现，但是很少。来自R2反应器有些颗粒的表面和通常的颗粒被用来实验，那些单细胞发光的明亮的能被看见(资料没显示)。然而，来自双方的证据显示没有发光信号但是看来像控制滑动(资料没显示)。从R1反应器里来的颗粒能发现微生物小集落

15、的增加主要位于颗粒的中部。小集落也出现在那的边缘(资料没显示）。控制滑动准备从活跃接种的反应器无抗血清偶合器没有给任何的发光信号但是显示一相似的深色(资料没显示)。4讨论在包含氯乙烯的媒介，甲酸盐，和醋酸盐，相应的微生物的最大生长速率在20C大概是10小时。实际操作反应器在均匀试验性反应器比相对最大比生长速率要低，因此，将导致微生物的自由的活细胞的被冲刷。这两次测试和R1反应器脱氯氯乙烯变成二氯乙烯，甚至在均匀试验性反应器的比微生物的相对最大比生长速率的低得多。那活跃的粒状淤渣R2反应器中也能脱氯氯乙烯；然而，氯乙烯的去除率以比在这次测试的反应器在恒等的均匀试验性反应器低超过两倍。而且，氯乙烯

16、只在R2反应器被脱氯，而二氯乙烯绝对不被检出。在这次测试的反应器中检测不到非生物的损失，R2反应器，或者那颗粒非生物控制，这是那氯化乙烯质量平衡超过那反应堆器系统在通常范围之内析仪器确定的标准偏差。这个结果表明在这次测试中微生物在R1反应器中是固定不动的，这是相对在R2反应器中提高脱氯作用活跃性造成的结果。微生物在R1反应器的固定被证实是通过免疫荧光显微术。在密集的类似组织的小集落的微生物的繁殖增加在高压灭菌器颗粒内部来自于在均匀试验性反应器的1。9小时反应器操作(资料没显示)。微生物的颗粒菌种的微观调查和来自R2反应器显示的一些零星的微生物在粒状表面检出细菌的抗体。从细胞检出的不太像是微生物

17、的脱氯系族，自从这些细菌可能显示快速的增殖以来，归究于大量的个体数，如这次测试和R1反应器所示。这是在早期的实验的例子，类似于R2反应器的反应器受到微生物的影响意外的变成污染。细菌在数天之内传遍粒状淤渣。氯乙烯去除率随着这次测试的反应器和R1反应器越来越多的氯乙烯负荷呈线性的增长，两者都通过微生物的接种，表明在氯乙烯负荷率的测试没有抑制效果。这次测试反应器的相对轻微的较低的氯乙烯去除率被看作是在均匀试验性反应器一种缓慢地适应能力的下降。这归究于在流出物中氯乙烯和三氯乙烯的临时的形态。相反的，R1反应器的脱氯的性能没有与在均匀试验性反应器的变化交换。这效果可以应归于微生物在每台设备相对较少的细胞

18、数目，这次测试反应器比R1反应器更明显，在均匀试验性反应器中使不活跃的接种系统对变化更加敏感。和在39%甲酸盐被加到反应器R1的消耗量相比，甲酸盐的消耗量的总数的增加是为了R2反应器。这表明甲酸盐是用于细菌，而不适用于不活跃的粒状淤渣。在这次测试反应器的流出物中从没有检出甲酸盐，这反应器可能缺少这种给电子体。氯乙烯脱氯作用是一种为微生物能量保存处理的方式，因此给电子体的缺少可能缩短能量增益和导致细胞数目的减少，除非甲酸盐供应是充分的。这种被介绍的细菌的放置对比早先的研究更符合这次研究。脱氯的细菌的固定在不活跃微生物颗粒导致一个类似统一的网络的有机体的增加，而在微生物在活跃的结果形成小集落。这个

19、结果表明文中介绍的细菌地放置通过单独的因素控制在特殊系统之内。自从微生物的条件，例如，关于甲酸盐的陈列，在不活跃颗粒将与那些活跃的颗粒不同，在放置的位置差异是可以预期的。就目前研究来说，我们第一次证明微生物可以是固定不动在粒状淤渣中，那是它提高氯乙烯脱氯作用活动重要的地方。此外，微生物结合的结果使产物形成接近混合的化合物（二氯乙烯），二氯乙烯比通常的产物（三氯乙烯）更加依赖在好氧后处理深层的转化。这些结果对那些氯乙烯-污染含水层的生物纠正有很大的重要性。致谢我们衷心感谢Jacob Rasmussen的技术帮助。这次研究由欧盟理事会基金和Danish科学技术理事会基金资助，基金号分别为EV5V-

20、CT92-0239 (BIODEC 项目)和no. 9502657-28813。Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket ReactorsChristine Hrber, Nina Christiansen, Erik Arvin, and Birgitte K. Ahring* Department of Environmental Science and Engineering, Technical University of Denmark, 2800Lyngby, Denmark Rec

21、eived 7 August 1997/Accepted 9 March 1998 ABSTRACTDechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium Dehalospirillum mult

22、ivorans into granular sludge. This reactor was compared to the reference 1(R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2(R2) reactor, containing only living granular sludge. All three reactors were fed mineral medium

23、 containing 3to 57M PCE, 2 mM formate, and 0.5mM acetate and were operated under sterile conditions. In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor. The R2 reactor, with no inoculation, produced

24、 only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes. No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum. During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor

25、 and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of D.multivorans, indicating that this bacterium was immobilized in the living and autoclaved granular sludge. In contrast, the R2 reactor, with no inoculation of D.multivora

26、ns, only converted PCE to TCE under the same conditions. Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against D.multivorans. In granules obtained from the R1 reactor, D.multivorans grew mainly in microcolonies located in the centers of the granules, while in

27、the test reactor, the bacterium mainly covered the surfaces of granules. INTRODUCTIONTetrachloroethene, also known as perchloroethylene (PCE), has commonly been used as a degreasing agent for metals and as a solvent for dry cleaning. Due to improper storage and disposal, significant amounts of PCE a

28、re spread throughout the environment worldwide. Microbial transformation of PCE has been demonstrated exclusively under anaerobic conditions by the mechanism of reductive dechlorination (13). Reductive dechlorination of PCE has been observed in anaerobic mixed and enrichment cultures (5, 6, 8). Rece

29、ntly, the anaerobic bacteria Dehalospirillum multivorans, Dehalobacter restrictus, and Desulfitobacterium sp. strain PCE1, which perform reductive dechlorination of PCE (9, 10, 13), have been isolated. Manipulation of the biomass can be essential in technical systems for optimal degradation of xenob

30、iotic compounds. In earlier studies, we have shown that de novo activity can be introduced into granular sludge by use of an anaerobic bacterium actively degrading the target compound; Desulfomonile tiedjei, a 3-chlorobenzoate-dechlorinating bacterium, was inoculated into the granular sludge of an u

31、pflow anaerobic sludge blanket (UASB) reactor, resulting in 3-chlorobenzoate-degrading capability of the sludge, which previously did not have this capability (1). It was further verified that the bacterium was immobilized in the granular sludge layer, where it expressed its dechlorinating activity.

32、 By the use of immunofluorescence microscopy on slices of granules, it was shown that the bacteria made microcolonies in the granular sludge. In a further study, we introduced the pentachlorophenol-dechlorinating bacterium Desulfitobacterium hafniense into sterile granular sludge (2, 3). This bacter

33、ium was found to be immobilized in a net-like structure inside the granules, where it expressed its ortho-dechlorinating pathway. The purpose of the present study was to investigate dechlorination of PCE in UASB reactors with and without addition of the PCE-degrading bacterium D.multivorans and furt

34、her to compare the performance depending on whether the granules were active or sterile. MATERIALS AND METHODSReactor design. Three glass UASB reactors with an active volume of 204ml and 31ml of headspace were used for the experiments (Fig. 1). Viton, stainless steel, and glass tubing were used for

35、connections to minimize chloroethene adsorption and evaporation. The reactor systems were checked for abiotic loss of chloroethenes before addition of granules to the reactors. The reactors were operated continuously, with a hydraulic retention time (HRT) decreasing stepwise from 2days (flow rate, 1

36、00ml/day) to 14.4,7.2,and finally 1.9h. PCE was supplied by continuously injecting a 780 M aqueous PCE solution into the mixing vessel, from which it flowed into the reactor. Samples were taken from sample ports 1and 2,representing the reactor influent and effluent, respectively. The samples were an

37、alyzed for chloroethenes, formate, and acetate. All sample ports were closed with Teflon-lined butyl septa. The reactors were operated at room temperature (22to 25C) and kept in the dark. View larger version (19K): in this window in a new window FIG. 1. Reactor setup. A and I, gas bags; B, medium bo

38、ttle; C, pump; D, PCE injection syringe; E, stirred mixing vessel; F, UASB reactor; G, granular sludge blanket; H, waste bottle; 1and 2,sample ports 1and 2. Source of inoculum. The granular sludge was taken from a lab scale UASB reactor fed PCE and ethanol for 1year (4). Originally, the granules cam

39、e from a full-scale UASB reactor treating paper mill effluent. The granules were stored at 4C for approximately 2months prior to use. The test and reference 2 (R2) reactors were filled with 20ml of wet granules, gassed with N2-CO2 (4:1), and filled with anaerobic medium. The reference 1 (R1) reactor

40、 was also filled with 20ml of wet granules but was autoclaved three times at 140C before being gassed with sterile N2-CO2 (4:1) and filled with anaerobic medium. The test and R1 reactors, containing living and autoclaved granules, respectively, were further inoculated with 50ml of a pure culture of

41、D.multivorans, kindly provided by H.Scholz-Muramatsu (Institute for Sanitary Engineering, Department of Biology, University of Stuttgart, Stuttgart, Germany) and allowed to acclimate for 3days before feeding of the reactors was initiated. As an abiotic control, a 118-ml serum vial was filled with 5m

42、l of wet granules and 50ml of anaerobic medium. The bottle was gassed (with N2-CO2 4:1), autoclaved three times, and supplemented with formate and acetate to final concentrations equivalent to those in the reactor system. PCE was added as an aqueous sterile solution. The batch was tested for dechlor

43、ination activity every 4to 5days during the experimental period. Sterility check. The sterility of the autoclaved reactor inoculated with D.multivorans was checked daily, and that of the sterile control bottle was checked every 4to 5days, by light-microscopic inspection of a liquid sample. Medium. A

44、 basal medium was prepared as previously described (12), except that 0.5mg of resazurin/liter, 0.48g of Na2S 7H2O to 9H2O/liter, 0.5mM acetate, and 2mM formate were added from anaerobic stock solutions. The final pH of the medium ranged from 7.0to 7.5. Immunological methods. Granules were sampled fr

45、om each reactor after reactors were operated at an HRT of 1.9h. The granules and inoculum were fixed in 4% formalin, embedded in paraffin, cut into 5-m slices, and placed on glass slides. Paraffin was removed with xylene, and the granules were hydrated in decreasing concentrations of ethanol. Polycl

46、onal rabbit antiserum against D.multivorans labeled with FLUOS (5-6-carboxyfluorescein-N-hydroxysuccinimide ester) was obtained from the Institute for Sanitary Engineering, Germany. The hydrated granules were incubated with normal rabbit serum, washed with bovine serum albumin in phosphate-buffered

47、saline (pH 7.6), and incubated with the antiserum for 25min. As a control, granules were prepared without antiserum incubation. The slides were investigated by immunofluorescence microscopy. Analytical methods. PCE, trichloroethene (TCE), and dichloroethene (DCE) were measured with a mass spectromet

48、er (MS QMG 421-1; Balzers, Liechtenstein) for membrane inlet mass spectrometry (MIMS) (11). PCE, TCE, and DCE were analyzed at mass-to-charge ratios (m/z) of 165.8, 129.9,and 61.0atomic mass units, respectively. The detection limit for the MIMS is 1 g/liter (11a). The range of concentrations tested was 1to 500M for PCE, TCE, and DCE, corresponding to the range of the standards. Standards were prepared by weighing exact amounts of chloroethenes into double-distilled water, stirring overnight fo