Impact of chromium-contaminated wastewaters on the microbial community of a river

The influence of chromium on the microbial community structure was analyzed in a river system subjected to long-term chromium contamination, by plating and by sequencing 16S rRNA genes cloned from DNA extracted from the river sediments. We also analyzed the influence of chromium on the ability of the microbial community to resist and reduce Cr(VI) and on its resistance to antibiotics. Shifts in the microbial community structure were analyzed by amplified ribosomal DNA restriction analysis fingerprinting. The isolates obtained were phylogenetically related to Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria, whereas Acidobacteria and Deltaproteobacteria were only revealed by clone analyses. Cr(VI)-resistant and Cr(VI)-reducing strains were isolated in all sites examined. However, each sample site had a microbial community with a different antibiotic resistance pattern. Our study seems to indicate that in this river ecosystem chromium influenced the microbial communities, altering some of their functional characteristics, such as the percentage of the microbial community able to resist or to reduce Cr(VI) and the phylogenetic groups isolated, but it did not affect the structural diversity. Furthermore, the concentration of Cr(VI) in the sediments could not be correlated with a lower number of bacteria or lower index of generic diversity, neither with the ability of the microbial community to resist or to reduce higher Cr(VI) concentrations.     

Characterization of chromate-resistant and -reducing bacteria by traditional means and by a high-throughput phenomic technique for bioremediation purposes

To select strains for the bioremediation of Cr(VI)-polluted environments, four highly Cr(VI)-resistant bacterial isolates were identified and characterized using both traditional techniques and a novel approach called phenotype microarrays. The isolates were identified as members of Pseudomonas mendocina (strains 34 and 56) and members of Pseudomonas corrugata (strains 22 and 28). Results showed that it was possible, by varying the carbon/energy source, to decouple bacterial growth and Cr(VI) reduction, inasmuch as some carbon/energy sources were more effective electron donors for chromate reduction, whereas other sources supported growth but not an effective chromate reduction. The isolates were characterized by a novel high-throughput technique, phenotype microarrays (PM)-Biolog, which can test up to 2000 cellular phenotypes simultaneously. The isolates belonging to P. corrugata had PM profiles different from those of the isolates belonging to P. mendocina. Such differences were related to the capacity of the isolates to resist various chemicals, pH values, and osmolytic substances. With the PM technique a very large amount of information about the fitness of isolates in the presence of different stressors could be obtained.     

Effect of Zn(II) on the reduction and accumulation of Cr(VI) by <Emphasis Type="Italic">Arthrobacter</Emphasis> species

Natural habitats are often characterized by the coexistence of Zn and Cr. This study assessed the potential of two Gram-positive, Cr(VI)-reducing, aerobic bacterial strains belonging to Arthrobacter genera, which were isolated from basalt samples taken from the most polluted region of the Republic of Georgia, to remediate Cr(VI) in environments in the presence of Zn(II). Our batch experiments revealed that the addition of Zn(II) to the tested bacterial cells significantly enhanced the accumulation of Cr. According to electron spin resonance (ESR) measurements, the presence of Zn(II) ions did not change the nature of Cr(V) and Cr(III) complexes generated during the microbial reduction of Cr(VI). The efficiency of Cr(VI) reduction also remained unchanged after the addition of 50 mg/l of Zn(II) to the bacterial cells. However, at high concentrations of Zn(II) (higher than 200 mg/l), the transformation of Cr(VI) to Cr(V) and Cr(III) complexes decreases significantly. In addition, it was shown that the accumulation pattern of Zn in the tested bacterial species in the presence of 100 mg/l of Cr(VI) fits the Langmuir–Freundlich model well. The two tested bacterial strains exhibited different characteristics of Zn accumulation.     

Aerobic Reduction of Hexavalent Chromium in Soil by Indigenous Microorganisms

Surface soil containing 25,100 mg/kg total Cr [12,400 mg/kg Cr(VI)] obtained from a Superfund site was used in laboratory microcosm studies to evaluate the potential for aerobic reduction of Cr(VI) by the indigenous soil microbial community. Hexavalent chromium in soil was reduced by as much as 33% (from 1840 to 1240 mg/L) within 21 days under enrichment conditions. Reduction of Cr(VI) in this system was biologically mediated and depended on the availability of usable energy sources. Mass balance studies suggested that the microbial populations removed Cr(VI) from the soil solutions by reduction. Indigenous microbial soil communities even with no history of Cr(VI) contamination were capable of mediating this process. However, Cr(VI) removal was not observed when microbial populations from a sewage sludge sample were added to the soil microcosms. The results suggest that Cr(VI)-reducing microbial populations are widespread in soil, and thus the potential exists for in situ remediation of environmentally significant levels of Cr( VI) contamination.     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

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.     

铬还原菌的分离筛选及其在微生物燃料电池生物阴极中的应用

【目的】水溶性的Cr(Ⅵ)对环境及人类造成的危害是社会亟待解决的问题。Cr(Ⅵ)还原菌株的分离筛选、还原特性的分析和在微生物燃料电池中的应用为六价铬污染水体的微生物修复提供科学依据和新的方法。【方法】从黄河兰州段排污口采集样本,用平板法分离筛选获得具有Cr(Ⅵ)还原能力的菌株,并将Cr(Ⅵ)还原能力最强的LZU-26菌株应用到微生物燃料电池中,检测其产电能力和Cr(Ⅵ)还原特性。【结果】共分离得到21株具有Cr(Ⅵ)还原能力的菌株,其中LZU-26菌株Cr(Ⅵ)还原能力最强,属于Cellulosimicrobium cellilans。0.4 mmol/L初始Cr(Ⅵ)在LZU-26的作用下24 h铬还原率可达到95.89%,在48 h后达99.97%。将LZU-26运用在微生物燃料电池生物阴极,所获得的最大电压和最大功率密度分别为68 mV和6.8 W/cm~2。生物阴极Cr(Ⅵ)还原率(68.9%)也远高于化学阴极(14.7%)和对照组(2.7%)。【结论】利用Cr(Ⅵ)还原菌作为微生物燃料电池生物阴极处理含铬废水,将会是一种高效、节能和环境友好的方法。     

Bioremediation Potential of Cr(VI)-Contaminated Soil Using Indigenous Microorganisms

Amendment of Cr(VI)-contaminated soil (approx. 200 mg/kg) with various treatments resulted in greater CO₂ evolution and Cr(VI) reduction with organic amendments relative to controls receiving no organics, indicating bacterial reduction of Cr(VI) under anaerobic conditions. Isolation of Cr(VI)-reducing, indigenous bacteria, representative of the dominant soil population, further indicated Cr(VI) reduction by indigenous bacteria. Although desorption of Cr(VI) was evident with some treatments, its reduction was not affected.     

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 the 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 the 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.     

Chromium(VI)-resistant yeast isolated from a sewage treatment plant receiving tannery wastes

A Cr(VI)-resistant yeast, designated strain DBVPG 6502, was isolated from a sewage treatment plant receiving wastes from tannery industries in Italy. The strain was tentatively identified as a species of Candida based on morphological and physiological analyses. This strain was highly resistant to Cr(VI) when compared with eight other yeast species, growing at Cr(VI) concentrations of up to 500 micrograms/ml (10 mM). This resistance was constitutive. The Cr(VI)-resistant yeast did not reduce Cr(VI) to Cr(III) species under aerobic conditions. The yeast showed very little accumulation of Cr(VI). Consequently, the mechanism of resistance of the yeast to Cr(VI) appears to involve reduced accumulation of Cr, as has been shown in Cr(VI)-resistant bacteria.     

耐盐菌Staphylococcus sp. YZ-1和Bacillus cereus CC-1的Cr(VI)脱毒特性与机理

[背景]高盐含铬废水的去除过程中,Cr(Ⅵ)还原菌是研究者关注的重点,但目前对耐盐菌株的Cr(Ⅵ)脱毒特性及机理的分析仍较少。[目的]比较两株耐盐菌株的Cr(Ⅵ)移除特性,并区分Cr(Ⅵ)耐受机制的差异;通过基因组测序分析,从基因层面推测铬耐受相关基因;构建铬还原菌的混菌体系,考察两者对去除污染物的协同作用。[方法]从青海茶卡盐湖分离耐盐菌Staphylococcus sp.YZ-1,与Bacillus cereus CC-1进行基础特性和Cr(Ⅵ)去除性能的比较,并通过全基因组序列的分析验证特性测试的结果。[结果]两株菌都具有铬移除特性,但CC-1的铬移除效率更高,在初始Cr(Ⅵ)浓度为0.1 mmol/L情况下,CC-1能在12h内移除95.3%的Cr(Ⅵ),而YZ-1只能移除40.1%。在进一步实验中发现YZ-1只能对Cr(Ⅵ)进行还原,将其转化为可溶的有机态Cr(Ⅲ),而CC-1能同时对Cr(Ⅵ)进行还原和吸附。全基因组分析发现YZ-1具有编码外排泵蛋白的基因和编码NAD(P)H氧化还原酶的基因,而CC-1具有编码铬转运蛋白ChrA和细胞色素C氧化还原酶的基因。两株菌的混菌体系在处理含Cr(Ⅵ)、Te(Ⅳ)的废水时,菌群能将还原产物聚集成团并沉淀到底部。[结论]菌株YZ-1和CC-1均为耐盐铬还原菌,但YZ-1中的铬还原酶为诱导型酶,CC-1则为组成型酶。基因组数据分析鉴别出两者可能同时存在多种铬耐受机制相关编码基因。混合菌群可以结合YZ-1的自絮凝特性和两者均有的Te(Ⅳ)/Cr(Ⅵ)还原活性,具有潜在的实用价值。     

Chromium(VI)-resistant yeast isolated from a sewage treatment plant receiving tannery wastes.

A Cr(VI)-resistant yeast, designated strain DBVPG 6502, was isolated from a sewage treatment plant receiving wastes from tannery industries in Italy. The strain was tentatively identified as a species of Candida based on morphological and physiological analyses. This strain was highly resistant to Cr(VI) when compared with eight other yeast species, growing at Cr(VI) concentrations of up to 500 micrograms/ml (10 mM). This resistance was constitutive. The Cr(VI)-resistant yeast did not reduce Cr(VI) to Cr(III) species under aerobic conditions. The yeast showed very little accumulation of Cr(VI). Consequently, the mechanism of resistance of the yeast to Cr(VI) appears to involve reduced accumulation of Cr, as has been shown in Cr(VI)-resistant bacteria.     

Isolation of hexavalent chromium-reducing anaerobes from hexavalent-chromium-contaminated and noncontaminated environments

Hexavalent chromium [Cr(VI)], is a toxic, water-soluble contaminant present in many soils and industrial effluents. Bacteria from various soils were examined for Cr(VI) resistance and reducing potential. Microbes selected from both Cr(VI)-contaminated and-noncontaminated soils and sediments were capable of catalyzing the reduction of Cr(VI) to Cr(III) a less toxic, less water-soluble form of Cr, demonstrating the utility of using a selection strategy for indigenous Cr(VI)-reducing bacteria in a bioprocess. As a result, indigenous Cr(VI)- reducing microbes from contaminated sites should provide the means for developing a bioprocess to reduce Cr(VI) to Cr(III) in nonsterile effluents such as those from soil washes. This approach also avoids the contamination problems associated with pure cultures of allochthonous microorganisms. In addition the apparent ubiquity of Cr(VI)-reducing bacteria in soil and sediments indicates potential for in situ bioremediation of Cr(VI)-contaminated soils and ground water.     

Cr(VI) reduction by sulfidogenic and nonsulfidogenic microbial consortia

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.     

Cr(VI) Reduction by Sulfidogenic and Nonsulfidogenic Microbial Consortia

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

A low-GC Gram-positive Thermoanaerobacter-like bacterium isolated from an Indian hot spring contains Cr(VI) reduction activity both in the membrane and cytoplasm

Aim:  Characterization of an anaerobic thermophilic bacterium and subcellular localization of its Cr(VI)-reducing activity for potential bioremediation applications.
Methods and Results:  16S rRNA gene sequence-based analyses of bacterial strains isolated from sediment samples of a Bakreshwar (India) hot spring, enriched anaerobically in iron-reducing medium, found them to be 86–96% similar to reported Thermoanaerobacter strains. The most efficient iron reducer among these, BSB-33, could also reduce Cr(VI) at an optimum temperature of 60°C and pH 6·5. Filtered culture medium could reduce Cr(VI) but not Fe(III). Cell-free extracts reduced Cr(VI) inefficiently under aerobic conditions but efficiently anaerobically. Fractionation of the cell-free extracts showed that chromium reduction activity was present in both the cytoplasm and membrane.
Conclusions:  BSB-33 reduced Fe(III) and Cr(VI) anaerobically at 60°C optimally. After fractionation, the reducing activity of Cr(VI) was found in both cytoplasmic and membrane fractions.
Significance and Impact of the Study:  To the best of our knowledge, this is the first systematic study of anaerobic Cr(VI) reduction by a gram-positive thermophilic micro-organism and, in contrast to our results, none of the earlier reports has mentioned Cr(VI)-reducing activity to be present both in the cytoplasm and membrane of an organism. The strain may offer itself as a potential candidate for bioremediation.     

Global transcriptional profiling of Shewanella oneidensis MR-1 during Cr(VI) and U(VI) reduction

Whole-genome DNA microarrays were used to examine the gene expression profile of Shewanella oneidensis MR-1 during U(VI) and Cr(VI) reduction. The same control, cells pregrown with nitrate and incubated with no electron acceptor, was used for the two time points considered and for both metals. U(VI)-reducing conditions resulted in the upregulation (> or = 3-fold) of 121 genes, while 83 genes were upregulated under Cr(VI)-reducing conditions. A large fraction of the genes upregulated [34% for U(VI) and 29% for Cr(VI)] encode hypothetical proteins of unknown function. Genes encoding proteins known to reduce alternative electron acceptors [fumarate, dimethyl sulfoxide, Mn(IV), or soluble Fe(III)] were upregulated under both U(VI)- and Cr(VI)-reducing conditions. The involvement of these upregulated genes in the reduction of U(VI) and Cr(VI) was tested using mutants lacking one or several of the gene products. Mutant testing confirmed the involvement of several genes in the reduction of both metals: mtrA, mtrB, mtrC, and menC, all of which are involved in Fe(III) citrate reduction by MR-1. Genes encoding efflux pumps were upregulated under Cr(VI)- but not under U(VI)-reducing conditions. Genes encoding proteins associated with general (e.g., groL and dnaJ) and membrane (e.g., pspBC) stress were also upregulated, particularly under U(VI)-reducing conditions, pointing to membrane damage by the solid-phase reduced U(IV) and Cr(III) and/or the direct effect of the oxidized forms of the metals. This study sheds light on the multifaceted response of MR-1 to U(VI) and Cr(VI) under anaerobic conditions and suggests that the same electron transport pathway can be used for more than one electron acceptor.     

Bacterial Reduction of Cr(VI) and Fe(III) in In Vitro Conditions

ABSTRACT Chemical reduction of Cr(VI) can be a strategy to detoxify toxic metals in oxidized states, whereas reduction of Fe(III) could enhance the availability of Fe in the form of Fe(II) to boost plant growth. However, it creates another problem of chemical sludge disposal. Hence, microbial conversion of Cr(VI) to Cr(III) and Fe(III) to Fe(II) is preferred over the chemical method. Out of 11 bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dump sites, four isolates were selected for the reduction of Cr(VI) and Fe(III) and were identified as Micrococcus roseus NBRFT2 (MTCC 9018), Bacillus endophyticus NBRFT4 (MTCC 9021), Paenibacillus macerans NBRFT5 (MTCC 8912), and Bacillus pumilus NBRFT9 (MTCC 8913). These strains were individually tested for survival at different concentrations of Cr(VI) and Fe(III), pH, and temperature, and then, their ability for reduction of both metals was evaluated at optimum pH 8.0 and temperature 35°C. The results indicated that NBRFT5 was able to reduce the maximum amount, 99% Cr(VI) and 98% Fe(III). Other strains also reduced these metals to different levels, but less than NBRFT5. Hence, these strains may be used for decontamination of metal-contaminated sites, particularly with Cr(VI) and Fe(III) through the reduction process.