硫酸盐还原菌及其还原解毒Cr(Ⅵ)的研究进展

硫酸盐还原菌是一类分布广泛, 能进行硫酸盐异化还原反应的严格厌氧菌。利用硫酸盐还原菌可去除环境中的许多污染物, 因而该类细菌在环境污染治理中具有广阔的应用前景。本文介绍了硫酸盐还原菌的生物学特性和代谢特征及其在环境污染治理中的应用, 并对硫酸盐还原菌还原解毒Cr(Ⅵ)及应用于含Cr(Ⅵ)废水处理的研究进展作了综述, 分析了其未来的研究方向。     

蜡样芽孢杆菌CP-1对Cr(Ⅵ)还原效果研究

为了提高蜡样芽孢杆菌CP-1菌株对Cr(Ⅵ)的还原效果,采用单因素和正交试验,通过摇瓶发酵培养,对影响蜡样芽孢杆菌CP-1菌株还原Cr(Ⅵ)的发酵培养基成分和培养条件进行了优化,并研究了最佳发酵条件下的蜡样芽孢杆菌CP-1对Cr(Ⅵ)的还原效果。结果表明,蜡样芽孢杆菌CP-1菌株还原Cr(Ⅵ)的最佳培养基组成为:1%甘露醇, 3%的大豆蛋白胨, 0.05%KCl, 0.1%CuSO4,在此基础上的最佳培养条件为:pH7.0、6%接种量、45℃培养3 d,在此条件下,Cr(Ⅵ)初始浓度为100mg·L^-1时,对Cr(Ⅵ)的还原率达99.75%。在Cr(Ⅵ)污染的土壤中添加蜡样芽孢杆菌CP-190d后,土壤中的Cr(Ⅵ)含量降低55.15%左右。     

一株李氏禾内生细菌去除Cr(Ⅵ)的特性

李氏禾(Leersia hexandra)是中国境内发现的第一种铬超积累植物,该文对李氏禾内生菌及其除铬性能进行了研究。采用添加Cr(VI)的牛肉膏蛋白胨固体平板培养方法,从李氏禾根部分离筛选获得一株具有较强Cr(VI)抗性的内生细菌G04,分子生物学鉴定结果表明该菌株属于阴沟肠杆菌(Enterobacter cloacae)。采用摇瓶培养方法,以Cr(VI)去除率、总Cr(铬)的去除率以及菌体生物量为指标,考察了pH、温度、底物浓度、装液量、接种量、摇床转速以及反应时间等因素对Cr(VI)去除率、总铬去除率和菌株生长的影响。结果表明:在牛肉膏蛋白胨液体培养基中,菌株E. cloacae G04去除Cr(VI)的较优反应条件为初始pH5. 0、温度37℃、Cr(VI)浓度为100 mg·L~(-1)、装液量80 mL(250 mL三角瓶)、接种量15%、摇床转速为120r·min~(-1)、反应时间48 h。在此条件下,菌株E. cloacae G04对Cr(VI)和总铬的去除率分别为84%和8%。根据Cr(VI)去除率和总铬去除率的结果推测该菌株去除Cr(VI)的机制可能是以还原为主、吸附为辅。这表明李氏禾内生细菌E. cloacae G04菌株具有较好的应用潜力,既有可能直接用于土壤、水环境铬污染的修复,也有可能作为促植物修复铬污染的后备菌株,另外可为深入研究李氏禾的铬积累作用机制提供参考。     

真菌还原Cr(Ⅵ)的研究

从不同来源的样品中分离筛选出几株抗Cr（Ⅵ）的真菌,他们能在含300—500mg／LK2Cr2O7的蔗糖合成培养基中生长,其中BS－1菌株抗K2Cr2O7达900mg／L．BS－1等4株真菌在含200mg／LK2Cr2O7的培养基中生长4-6d后,培养液中的Cr（Ⅵ）已全部消失。这些真菌经鉴定为青霉菌（Penicilliumsp．）BS－1和BS－3,黑曲霉（Aspergillusniger）BR-4和黄曲霉（Aspergillusflavus）BX－1。经紫外可见光扫描及化学分析证实,高毒的Cr（Ⅵ）可被真菌还原成为低毒的Cr（Ⅲ）。BS－1菌株细胞还原Cr（Ⅵ）的最适温度为30℃,最适pH7．0。葡萄糖（0．25％）对细胞还原Cr（Ⅵ）有促进作用,但高浓度的Cr（Ⅵ）则抑制细胞对Cr（Ⅵ）的还原。     

微生物还原Cr(VI)的研究进展

随着现代工业的发展, 水环境中的重金属对人类健康和环境带来严重的危害, 其中的Cr(VI)具有强烈的毒性。微生物在代谢过程中可以将Cr(VI)还原为Cr(III), 有效降低Cr(VI)的毒性。本文从可还原Cr(VI)的微生物、微生物还原Cr(VI)的机理、还原过程中存在的问题及发展方向等方面进行了综述。     

苏云金芽胞杆菌还原Cr(Ⅵ)的转座子突变体库构建和分析

将高毒性的Cr(Ⅵ)还原成低毒的Cr (Ⅲ),是处理含Cr(Ⅵ)废水常用的方法之一.以高效还原Cr(Ⅵ)的苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)407为研究对象,将转座子随机突变载体pIC333转化Bt407,构建容量为1 500株的随机突变体库,从中筛选出Cr(Ⅵ)还原能力极显著差异(P＜0.01)的突变株14株.通过扩增并测定转座子插入位点的侧翼序列,确定Bt407-Cr244突变株的转座子插入位点为细胞色素氧化酶亚单位Ⅰ.Bt 407及其14株突变株的生长曲线十分相近,表明突变株Cr(Ⅵ)还原能力发生改变不是由菌株繁殖能力提高引起的.在培养24 h后,Bt 407-Cr1 49和Bt 407-Cr285培养液中的总络浓度比Bt 407极显著降低(P＜0.01),说明这2株突变株在解毒Cr(Ⅵ)过程中除了还原作用,可能还具有生物吸附,而其余12株突变子主要通过还原Cr(Ⅵ)起作用.     

Cr(Ⅵ)还原菌Microbacterium sp.BD6的分离鉴定及还原特性

【目的】从电镀厂下水道的淤泥中分离筛选Cr(Ⅵ)高效还原菌,并对其生长和还原特性进行研究,以期为Cr(Ⅵ)污染的生物修复提供优质的菌种资源和应用参考。【方法】采用富集培养法从淤泥中分离、筛选出Cr(Ⅵ)还原菌,通过生理生化及16S rRNA基因序列分析进行初步鉴定。采用单因素实验确定菌株的最佳培养条件和抵抗胁迫环境的能力,利用外加电子供体改善菌株的Cr(Ⅵ)还原能力,筛选出最佳电子供体研究对菌株还原的影响。【结果】经分离筛选得到1株Cr(Ⅵ)耐受还原菌,初步鉴定为微杆菌属(Microbacterium sp.),命名为BD6。菌株BD6适宜在中温、偏碱性的环境条件下生长,能耐受50.0 g/L NaCl的高盐环境。Mn^2+对菌种的生长表现出较高的抑制,Ni^2+、Zn^2+、Cd^2+的抑制作用较小,Cu^2)产生了一定的促进作用。Cr(Ⅵ)对BD6的最低抑菌浓度为1700 mg/L。添加甘油、果糖、乳糖、葡萄糖、丙酮酸钠作为电子供体促进了菌株对Cr(Ⅵ)的还原。选择甘油作为菌株还原Cr(Ⅵ)的最佳电子供体,无电子供体添加时菌株96 h内对100 mg/L Cr(Ⅵ)的还原率仅为69.63%,添加2 g/L的甘油菌株在36 h内的还原率达到了100%。通过加大甘油的添加量可以促进菌株对初始浓度较高Cr(Ⅵ)的还原,但要受到Cr(Ⅵ)的毒性限制。菌株的最适还原条件和最适生长条件吻合,在50.0 g/L NaCl的高盐条件和50 mg/L Cd^2+的毒性环境中,添加2 g/L的甘油,菌株对100 mg/L Cr(Ⅵ)的还原率分别为72 h 96.79%、54 h 99.86%。【结论】分离筛选得到的Microbacterium sp.BD6是一株潜在的可用于Cr(Ⅵ)污染生物还原修复的候选菌株。     

黄麻吸附重金属Cr(Ⅵ)专用种质筛选

首次以国内外23个代表性黄麻种质为研究对象,用主茎嫩梢和一、二级分枝嫩梢制成天然重金属吸附剂,测定其对溶液中Cr(Ⅵ)离子的去除率,并对与黄麻吸附能力及产量密切相关的功能性状:生育期动态、株高、分枝习性、各级分枝嫩梢的产量进行调查、方差分析和相关性分析。结果表明,不同黄麻种质制成的生物吸附剂对Cr(Ⅵ)的吸附能力不同,去除率在85.25%~96.88%之间;不同级次分枝嫩梢的产量及对Cr(Ⅵ)的去除率不同;种质间除出苗速度外所有调查性状均存在较大差异;去除率与株高呈极显著负相关(P<0.01)。从产量和去除率方面综合考虑,J001和J011 2个种质表现优良,适宜作为吸附重金属专用品种推广种植。     

微生物还原Cr(VI)的研究进展

随着现代工业的发展,水环境中的重金属对人类健康和环境带来严重的危害,其中的Cr(VI)具有强烈的毒性.微生物在代谢过程中可以将Cr(VI)还原为Cr(Ⅲ),有效降低Cr(VI)的毒性.本文从可还原Cr(VI)的微生物、微生物还原Cr(VI)的机理、还原过程中存在的问题及发展方向等方面进行了综述.     

氨氮和硝酸盐氮对沙雷氏菌S2还原Cr(Ⅵ)能力的影响

【目的】研究氨氮(AN)与硝酸盐氮(NN)对沙雷氏菌S2还原Cr(Ⅵ)能力的影响。【方法】在实验室中模拟常见的环境中氮污染,S2在含Cr(Ⅵ)培养的同时在培养体系中加入不同剂量的AN或/和NN,每隔一定时间测定培养体系的菌量(A600)、Cr(Ⅵ)还原率、AN含量、NN含量。【结果】低、中浓度AN能缓解Cr(Ⅵ)对S2生长的抑制作用;高浓度NN和AN可加快S2的衰亡。AN独立作用时,各组间Cr(Ⅵ)去除率和氨氮含量无显著关联。NN独立作用时,S2的Cr(Ⅵ)去除率在低浓度组降低10.0%以上(P0.05),在高浓度组增高7.1%(P0.05);S2能在4 h内使200 mg/L的NN降至对照组水平。双氮联合作用时,低浓度组对菌株除Cr(Ⅵ)能力的影响与AN单独作用类似,而高浓度组则类似NN单独作用。【结论】AN的存在对S2的Cr(Ⅵ)还原能力无明显影响,NN浓度高低对S2的Cr(Ⅵ)还原能力有不同影响,S2具有很强的除NN能力,可同时去除环境中Cr(Ⅵ)和硝酸盐氮污染。     

Reduction of Cr(VI) by a Consortium of Sulfate-Reducing Bacteria (SRB III)

A consortium of bacteria with tolerance to high concentrations of Cr(VI) (up to 2,500 ppm) and other toxic heavy metals has been obtained from metal-refinishing wastewaters in Chengdu, People's Republic of China. This consortium consists of a range of gram-positive and gram-negative rods and has the capacity to reduce Cr(VI) to Cr(III) as amorphous precipitates which are associated with the bacterial surfaces. An endospore-producing, gram-positive rod and a gram-negative rod accumulate the most metallic precipitates, and, over time, 80 to 95% of Cr can be removed from concentrations ranging from 50 to 2,000 ppm (0.96 to 38.45 mM). Kinetic studies revealed a first-order constant for Cr removal of 0.1518 h^-1 for an initial concentration of 1,000 ppm (19.3 mM), and the sorption isothermal data could be interpreted by the Freundlich relationship. The sorption was not entirely due to a passive interaction with reactive sites on the bacterial surfaces since gamma-irradiated, killed cells could not immobilize as much metal. When U or Zn was added with the Cr, it was also removed and could even increase the total amount of Cr immobilized. The consortium was tolerant to small amounts of oxygen in the headspace of tubes, but active growth of the bacteria was a requirement for Cr immobilization through Cr(VI) reduction, resulting in the lowering of E_h. Our data suggest that the reduction was via H₂S. This consortium has been named SRB III, and it may be useful for the bioremediation of fluid metal-refining wastes.     

The Reduction of Cr(VI) by Bacteria of the Genus Pseudomonas

Non-nitrate-reducing collection bacteria from the genus Pseudomonas were found to be able to use hexavalent chromium as a terminal electron acceptor. The reduction of Cr(VI) was accompanied by an increase in the cell biomass. At Cr(VI) concentrations in the medium lower than 15 mg/l, the non-nitrate-reducing pseudomonads reduced Cr(VI) less efficiently than did denitrifying pseudomonads. In contrast, at Cr(VI) concentrations higher than 30 mg/l, Cr(VI) was reduced more efficiently by the non-nitrate-reducing pseudomonads than by the denitrifying pseudomonads.     

Aerobic Cr(VI) Reduction by Bacteria in Culture and Soil Conditions

We compared the performance of aerobic Cr(VI)-reducing bacteria isolated from Cr(VI)-contaminated soil in pure and mixed cultures of five isolated strains. The mixed culture had increased reduction rates compared to individual cultures. Cr(VI) reduction was observed in sterile soil inoculated with Pseudomonas fluorescens and in non-sterile soil with and without inoculation with P. fluorescens at initial pore water concentrations up to 1,600 mg Cr(VI)/L, whereas in culture the maximum inhibitory concentration was 500 mg Cr(VI)/L. Linear rates of Cr(VI) reduction in non-sterile soil amended with peptone were ～5 to 8 times higher than those observed in the mixed culture. Inoculation of non-sterile soil with P. fluorescens did not further enhance Cr(VI) reduction rates. Our results indicate that evaluation of Cr(VI) reduction capacity in Cr(VI)-contaminated soil for in-situ bioremediation purposes should not be done solely in pure culture. Although the latter may be used initially to assess the effects of process parameters (e.g., pH, temperature), the rate and extent of Cr(VI) reduction should be determined in soil for bioremediation design purposes.     

Characterization of Cr(VI)-Resistant Bacteria Isolated from Chromium-Contaminated Soil by Tannery Activity

Bacterial strains, previously isolated from a chromium-polluted soil, were identified on the basis of Gram reaction and biochemical characteristics (Biolog system). Moreover, chromate MICs, chromate reduction capability, multiple heavy metal tolerance, and antibiotic susceptibility were tested for each isolate. All strains but one were Gram-positive and resistant to high concentrations of chromate. The most Cr(VI)-resistant isolate (22mM) was identified as Corynebacterium hoagii. All Cr(VI)-resistant strains except the isolate ChrC20 were capable of catalyzing the reduction of Cr(VI) to Cr(III), a less toxic and less water-soluble form of chromium. The only isolate Cr(VI)-sensitive, attributed to the Pseudomonas genus, also exhibited Cr(VI)-reduction. Isolates were also screened for the presence of plasmid DNA. The strains ChrC20 and ChrB20 harbored one and two plasmids of high molecular mass, respectively. This approach permitted selection of some bacterial strains, which could be used for bioremediation of Cr(VI)-polluted environments. Received: 21 February 2002 / Accepted: 27 March 2002     

Ochrobactrum tritici strain 5bvl1 - characterization of a Cr(VI)-resistant and Cr(VI)-reducing strain

Bacterial strain 5bvl1, isolated from a chromium-contaminated wastewater treatment plant and identified as Ochrobactrum tritici, was resistant to a broad range of antibiotics, to Cr(VI), Ni(II), Co(II), Cd(II), and Zn(II), and was able to grow in the presence of 5% NaCl and within the pH range 4-10. Characterization showed that strain 5bvl1 could be considered a halotolerant and alkalitolerant microorganism resistant to high concentrations of Cr(VI). This strain was able to grow aerobically in up to 10 mmolxL(-1) Cr(VI). Cr(VI) resistance was independent of sulphate concentration. Under aerobic conditions strain 5bvl1 was also able to reduce high Cr(VI) concentrations (up to 1.7 mmolxL(-1)). Increasing concentrations of Cr(VI) in the medium lowered the growth rate of strain 5bv11 but the reduction in growth rate could not be directly correlated with the amount of Cr(VI) reduced. Unlike the type strain, which was only able to reduce Cr(VI), strain 5bvl1 was resistant to Cr(VI) and able to reduce it. Moreover, in strain 5bvl1, the rate and extent of Cr(VI)-reduction were higher than in the other strains of the genus Ochrobactrum. Ochrobactrum strain 5bvl1 resists high Cr(VI) concentrations and has a high Cr(VI)-reducing ability, making it a valuable tool in bioremediation.     

Environmental and Kinetic Parameters for Cr(VI) Bioreduction by a Bacterial Monoculture Purified from Cr(VI)-Resistant Consortium

Hexavalent chromium, Cr(VI), is toxic to living systems. Widespread contamination of water and soil by Cr(VI) present a serious public health problem. Chromium-resistant bacteria can reduce and detoxify Cr(VI). Twelve bacteria resistant to high concentrations of Cr(VI) were isolated from soil enrichment cultures. Environmental parameters and kinetic parameters of Cr(VI) bioreduction by one monoculture isolate, identified by 16S rRNA gene sequence as Bacillus sp. PB2, were studied. The optimal temperature for growth and Cr(VI) reduction was 35 degrees C. The isolate grew luxuriantly and substantially reduced Cr(VI) at initial pH 7.5 to 9. Maximal Cr(VI) bioreduction occurred at initial pH 8.0. Substantial Cr(VI) bioreduction was observed in salt media, but removal efficiency was inversely related to salt concentration (1-9%). Michaelis-Menten hyperbolic equation and the Lineweaver-Burk double reciprocal plot were comparatively employed to determine the k (m) and V (max) of Cr(VI) bioreduction. A k (m) of 82.5 microg mL(-1) and V (max) of 7.78 microg mL(-1) h(-1) were calculated by nonlinear regression analysis of the hyperbola curve. Linear regression analysis of the double reciprocal plot revealed k (m) and V (max) of 80.9 microg mL(-1) and 10.6 microg mL(-1) h(-1), respectively. Time course studies displayed about 90% reduction of Cr(VI) at an initial concentration of 8,000 microg L(-1) in 8 h, with an estimated t (1/2) of 4 h. Data from time course analysis of the rate of Cr(VI) bioreduction fitted zero-order model, and the kinetic constant k was calculated to be 840 microg L(-1) h(-1). The monoculture isolate, Bacillus sp. PB2, strongly reduces Cr(VI) and could be used for bioremediation of Cr(VI)-contaminated aquatic and terrestrial environments.     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III).

An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme. Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonezymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(III) with at least two reaction steps via Cr(V) as an intermediate.     

Bacterial diversity in Cr(VI) and Cr(III)-contaminated industrial wastewaters

The bacterial community structure of a chromium water bath, a chromium drainage waste system, a chromium pretreatment tank, and a trivalent chromium precipitation tank from the Hellenic Aerospace Industry S.A. was assessed using 16S rRNA libraries and a high-density DNA microarray (PhyloChip). 16S rRNA libraries revealed a bacterial diversity consisting of 14 distinct operational taxonomic units belonging to five bacterial phyla: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacteroidetes. However, employing a novel microarray-based approach (PhyloChip), a high bacterial diversity consisting of 30 different phyla was revealed, with representatives of 181 different families. This made it possible to identify a core set of genera present in all wastewater treatment stages examined, consisting of members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Bacteroidetes. In the chromium pretreatment tank, where the concentration of Cr(VI) is high (2.3 mg/l), we identified the presence of Pseudomonadales, Actinomycetales, and Enterobacteriales in abundance. In the chromium precipitation tank, where the concentration of Cr(III) is high, the dominant bacteria consortia were replaced by members of Rhodocyclales and Chloroflexi. The bacterial community structure changed significantly with changes in the chromium concentration. This in-depth analysis should prove useful for the design and development of improved bioremediation strategies.