Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 microg K2CrO4 mL(-1), and an inoculum size of 9.6 x 10(7) cells mL(-1), B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 microg K2CrO4 mL(-1), Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30 degrees C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.     

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.     

In-situ Cr(VI) reduction with electrogenerated hydrogen peroxide driven by iron-reducing bacteria

Cr(VI) was reduced in-situ at a carbon felt cathode in an air-cathode dual-chamber microbial fuel cell (MFC). The reduction of Cr(VI) was proven to be strongly associated with the electrogenerated H₂O₂ at the cathode driven by iron-reducing bacteria. At pH 2.0, only 42.5% of Cr(VI) was reduced after 12 h in the nitrogen-bubbling-cathode MFC, while complete reduction of Cr(VI) was achieved in 4 h in the air-bubbling-cathode MFC in which the reduction of oxygen to H₂O₂ was confirmed. Conditions that affected the efficiency of the reduction of Cr(VI) were evaluated experimentally, including the cathodic electrolyte pH, the type of iron-reducing species, and the addition of redox mediators. The results showed that the efficient reduction of Cr(VI) could be achieved with an air-bubbling-cathode MFC.     

Cr(VI)还原菌的筛选、鉴定及其还原物质分析

【背景】铬污染土壤是我国土壤污染修复的重点治理对象,在众多修复技术中,微生物法因具有简单、经济、无二次污染等特性已成为研究热点,而微生物法中筛选出既能适应污染场地环境又能高效还原Cr(VI)的菌株尤为重要。【目的】筛选适应西北寒旱区高效还原Cr(VI)的菌株,丰富铬还原菌资源库,为铬污染土壤修复奠定基础。【方法】采用富集驯化、分离纯化法进行筛菌;通过形态学和分子生物学相结合的方法对目的菌株进行鉴定;采用傅里叶变换红外光谱法对还原机理进行研究。【结果】菌株G-13有较强的Cr(VI)还原能力,pH 9.0、温度为30°C条件下,60 h对Cr(VI)(100 mg/L)的还原率达到82.8%。经形态学和分子生物学鉴定,菌株G-13为Micrococcus luteus。反应中Cr(VI)的降低伴随着Cr(III)的增加,说明以还原反应为主,并且还原能力与细菌生长呈依赖型关系。对细胞各组分及变性研究表明,胞外酶在还原反应中占主要作用。除Pd~(2+)、Cd~(2+)外,其余金属离子对酶活性无明显抑制作用。通过傅里叶变换红外光谱分析,发现G-13与Cr(VI)结合位点主要为羟基、羰基、羧基、–CH、酰胺基等。【结论】菌株G-13有较强的Cr(VI)还原能力,能为西北寒旱区铬污染土壤修复丰富菌种资源。     

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.     

Chromate reduction and 16S rRNA identification of bacteria isolated from a Cr(VI)-contaminated site

A gram-positive, hexavalent chromium [chromate: Cr(VI)]-tolerant bacterium, isolated from tannery waste from Pakistan, was identified as a Microbacterium sp. by 16S rRNA gene sequence homology. The strain (designated as MP30) reduced toxic Cr(VI) only under anaerobic conditions at the expense of acetate as the electron donor. The bacterium was able to grow aerobically in L-broth supplemented with 15 mM CrO4(2-) but then did not reduce Cr(VI). At a concentration of 2.4x10(9) cells/ml, 100 microM sodium chromate was reduced within 30 h; however, the maximum specific reduction rate was obtained at lower initial cell concentrations.     

Evaluation of in vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 microM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 microM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 degrees C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 microM Cr(VI) within 100 min. The apparent K(m) and V(max) of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 microM Cr(VI) and 5.5 micromol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg(2+) and Ag(+).     

Chromium sorption and Cr(VI) reduction to Cr(III) by grape stalks and yohimbe bark

In this work, two low cost sorbents, grape stalks and yohimbe bark wastes were used to remove Cr(VI) and Cr(III) from aqueous solutions. Batch experiments were designed to obtain Cr(VI) and Cr(III) sorption data. The mechanism of Cr(III) and Cr(VI) removal and Cr(VI) reduction to Cr(III) by the two vegetable wastes, has been investigated. Fourier transform infrared rays (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis on solid phase were performed to determine the main functional groups that might be involved in metal uptake and to confirm the presence of Cr(III) on the sorbent, respectively. Results put into evidence that both sorbents are able to reduce Cr(VI) to its trivalent form.     

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.     

Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).     

Aerobic Cr(VI) reduction by Thermus scotoductus strain SA-01

AIM: To evaluate Thermus scotoductus SA-01's ability to reduce Cr(VI) aerobically. METHODS AND RESULTS: T. scotoductus SA-01 is able to reduce Cr(VI) aerobically when grown in a complex organic medium containing Cr(VI) concentrations up to 0.5 mmol l(-1). Suspension of T. scotoductus SA-01 cells also reduced Cr(VI) aerobically under nongrowth conditions using a variety of electron donors as well as in the absence of an exogenous electron donor. The optimum temperature and pH for Cr(VI) reduction under nongrowth conditions were found to be 80 degrees C and 7, respectively. It was also found that the Cr(VI) reduction was catalysed by a cytoplasmic, constitutively expressed enzyme. CONCLUSIONS: Apart from SA-01's ability to reduce Cr(VI) through a strictly anaerobic membrane-bound mechanism (unpublished data), it also has a second enzyme localized in the cytoplasm that can reduce Cr(VI) aerobically. As this enzyme is constitutively expressed and not induced by Cr(VI), it remains to be determined whether it has any other physiological functions. SIGNIFICANCE AND IMPACT OF THE Study: This is the first report of a Thermus species able to reduce Cr(VI) aerobically and extends the knowledge of parameters associated with Cr(VI) reduction. Employing thermophiles in bioremediation using industrial bioreactors would cancel the need for expensive cooling systems.     

Bacterial mechanisms for Cr(VI) resistance and reduction: an overview and recent advances

Chromium pollution is increasing incessantly due to continuing industrialization. Of various oxidation states, Cr⁶⁺ is very toxic due to its carcinogenic and mutagenic nature. It also has deleterious effects on different microorganisms as well as on plants. Many species of bacteria thriving in the Cr⁶⁺-contaminated environments have evolved novel strategies to cope with Cr⁶⁺ toxicity. Generally, decreased uptake or exclusion of Cr⁶⁺ compounds through the membranes, biosorption, and the upregulation of genes associated with oxidative stress response are some of the resistance mechanisms in bacterial cells to overcome the Cr⁶⁺ stress. In addition, bacterial Cr⁶⁺ reduction into Cr³⁺ is also a mechanism of specific significance as it transforms toxic and mobile chromium derivatives into reduced species which are innocuous and immobile. Ecologically, the bacterial trait of reductive immobilization of Cr⁶⁺ derivatives is of great advantage in bioremediation. The present review is an effort to underline the bacterial resistance and reducing mechanisms to Cr⁶⁺ compounds with recent development in order to garner a broad perspective.     

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.     

Effect of Ni(II) on the reduction of Cr(VI) by Ecklonia biomass

In this study, the removal of Cr(VI) was examined in a binary aqueous system containing Ni(II), and the competitive interaction between them was successfully modeled. The removal rate of Cr(VI) was unaffected by the presence of Ni(II). However, in an equilibrium state, the uptake of total Cr or Ni(II) was decreased in the presence of the other metal. The mono- and multi-component Langmuir adsorption models clearly represented the uptake behavior of these metals.     

Bioremediation of chromate: thermodynamic analysis of the effects of Cr(VI) on sulfate-reducing bacteria

Developing new bioremediation processes for soils and effluents polluted by Cr(VI) requires the selection of the most efficient and the most heavy-metal-resistant bacteria. The effects of Cr(VI) on bioenergetic metabolism in two sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris Hildenborough and Desulfomicrobium norvegicum, were monitored using isothermal microcalorimetry. The complete reduction of Cr(VI) to Cr(III) was studied by spectrophotometry and by speciation using a combination of high-performance liquid chromatography and inductively coupled plasma-mass spectrometry. Results revealed that Cr(VI) induces an inhibition of growth with concomitant production of energy, which can be compared to the reaction of the bacteria to a stress such as oxidative stress. Moreover, the sensitivity of bacteria towards this metal is as a characteristic of the strain, which leads to differences in the kinetics of Cr(VI) reduction. The study by microcalorimetry of heavy metal effects on SRB bioenergetic metabolism thus appears an appropriate tool to identify better strains to be used for industrial bioremediation process development.     

Comparison of in vitro Cr(VI) reduction by CFEs of chromate resistant bacteria isolated from chromate contaminated soil

Chromate resistant and reducing strains were isolated from chromium contaminated soil and identified as Bacillus sp. (KCH2 and KCH3), Leucobacter sp. (KCH4) and Exiguobacterium sp. (KCH5). KCH3 and KCH4 showed higher Cr(VI) tolerance (2 mM) and Cr(VI) reduction (1.5 mM) than KCH5 (1.5 mM and 0.75 mM, respectively). Cr(VI) reduction by CFEs of KCH3 and KCH4 showed NAD(P)H dependence, optimum activity at pH 5.5, low K(m) (45-55 microM) and substrate inhibition by Cr(VI) (>75 microM), whereas that of KCH5 showed NADH dependence, pH optimum at 6.0, high K(m) (200 microM) and no inhibition by Cr(VI). Cr(VI) reduction was optimum at 35 degrees C for CFEs of KCH3 and KCH5 and 30 degrees C for that of KCH3. Cr(VI) reduction by CFEs of all the strains were inhibited by Hg(2+) and enhanced by Cu(2+). Activity enhancement by Cu(2+) was more predominant (290%) for KCH4. The characterization of Cr(VI) reduction by CFEs of chromate resistant isolates of different genera is useful for development of Cr(VI) bioremediation.     

Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential

Microbial metabolites are of huge biotechnological potential and their production can be coupled with detoxification of environmental pollutants and wastewater treatment mediated by the versatile microorganisms. The consortia of cyanobacteria/microalgae and bacteria can be efficient in detoxification of organic and inorganic pollutants, and removal of nutrients from wastewaters, compared to the individual microorganisms. Cyanobacterial/algal photosynthesis provides oxygen, a key electron acceptor to the pollutant-degrading heterotrophic bacteria. In turn, bacteria support photoautotrophic growth of the partners by providing carbon dioxide and other stimulatory means. Competition for resources and cooperation for pollutant abatement between these two guilds of microorganisms will determine the success of consortium engineering while harnessing the biotechnological potential of the partners. Relative to the introduction of gene(s) in a single organism wherein the genes depend on the regulatory- and metabolic network for proper expression, microbial consortium engineering is easier and achievable. The currently available biotechnological tools such as metabolic profiling and functional genomics can aid in the consortium engineering. The present review examines the current status of research on the consortia, and emphasizes the construction of consortia with desired partners to serve a dual mission of pollutant removal and commercial production of microbial metabolites.     

A dicyclic-type electrode-based biofilm reactor for simultaneous nitrate and Cr(VI) reduction

Bioprocess and Biosystems Engineering - A dicyclic-type electrode-based biofilm-electrode reactor (BER) was investigated for simultaneous removal of nitrate and Cr(VI). In the absence of Cr(VI),...     

The effect of carbon source on microbial community structure and Cr(VI) reduction rate

In the present work, the effect of the carbon source on microbial community structure in batch cultures derived from industrial sludge and hexavalent chromium reduction was studied. Experiments in aerobic batch reactors were carried out by amending industrial sludge with two different carbon sources: sodium acetate and sucrose. In each of the experiments performed, four different initial Cr(VI) concentrations of: 6, 13, 30 and 115 mg/L were tested. The change of carbon source in the batch reactor from sodium acetate to sucrose led to a 1.3–2.1 fold increase in chromium reduction rate and to a 5‐ to 9.5‐fold increase in biomass. Analysis of the microbial structure in the batch reactor showed that the dominant communities were bacterial species (Acinetobacter lwoffii, Defluvibacter lusatiensis, Pseudoxanthomonas japonensis, Mesorhizium chacoense, and Flavobacterium suncheonense) when sodium acetate was used as carbon source and fungal strains (Trichoderma viride and Pichia jadinii), when sodium acetate was replaced by sucrose. These results indicate that the carbon source is a key parameter for microbial dynamics and enhanced chromium reduction and should be taken into account for efficient bioreactor design. Biotechnol. Bioeng. 2010;107: 478–487. © 2010 Wiley Periodicals, Inc.     

Treatment of Cr(VI)-containing wastewaters with exopolysaccharide-producing cyanobacteria in pilot flow through and batch systems

Seven exopolysaccharide-producing cyanobacteria were tested with regard to their capability to remove Cr(VI) from the wastewater of a plating industry. The cyanobacterium which showed, under lab conditions, the most promising features with regard to both Cr(VI) removal (about 12 mg of Cr(VI) removed per gram of dry biomass) and growth characteristics (highest growth rate and simplest culture medium) was Nostoc PCC7936. Furthermore, in lab experiments, it was also found that a HCl pretreatment is essential to abate the concentration of Cr(VI) in solution and that the viability of the biomass is not necessary. Subsequently, three pilot devices were tested, one batch (a dialysis cell) and two flow-through systems (a filter press and a column filled with quartz grain). The best performances were obtained with the filter press, where it was observed a sharp decrease in the concentration of Cr(VI), partly due to the adsorption of the metal by the biomass (about 50%) and partly due to its reduction to Cr(III). The results are discussed in terms of the role played by the different components (biomass and polysaccharide) of the cyanobacterial cultures in the removal of Cr(VI).