Characterization of the Cr(VI)-resistant isolated bacterial strains.
1.1. Characteristics of chromium
Chromium (Cr) is a naturally occurring element with atomic number 24 and atomic mass of 51.996 amu. The element belongs to the group of transition metals and in the oxidation state elementary presents an electronic configuration (Ar) 4d5s1. Chromium is naturally present in the environment, it is widespread in rocks, animal, plants and soil, and is the seventh most abundant element on Earth's crust, at concentrations ranging from 100 to 300 µg g-1. In nature, Cr is found in the form of its compounds, and the most important chromium ore is chromite, (Fe, Mn)Cr2O4 [1,2].
Chromium exists in different oxidation states, the most stable and common forms are the trivalent [Cr(III)] and the hexavalent [Cr(VI)] species, which display quite different chemical properties . Cr(III) in the form of oxides, hydroxides or sulfates, exists mostly bound to organic matter in soil and aquatic environments. Cr(VI) is usually associated with oxygen as chromate (CrO2-4) or dichromate (Cr2O2-7) ions . Cr(VI) is a strong oxidizing agent and in the presence of organic matter is reduced to Cr(III); this transformation is faster in acid environments such as acidic soils . However, high levels of Cr(VI) may overcome the reducing capacity of the environment and thus persist in this form .
Chromium represents an essential micronutrient for living organisms, considering that Cr(III) is an essential trace element known for its particular role in the maintenance of normal carbohydrate metabolism in mammals and yeasts . Moreover, it has also been suggested that Cr(III) is involved in the tertiary structure of proteins and in the conformation of cell RNA and DNA [5,6].
1.2. Toxicity of chromium
Cr(VI) exposure in humans can induce allergies, irritations, eczema, ulceration, nasal and skin irritations, perforation of eardrum, respiratory track disorders and lung carcinoma [7,8,9]. Moreover, Cr(VI) evidences the capability to accumulate in the placenta, damaging fetal development . Cr(VI) pollution in the environment alters the structure of soil microbial communities , reducing microbial growth and related enzymatic activities, with a consequent persistence of organic matter in soils and accumulation of Cr(VI) .
The toxic action of Cr(VI) is due to its capability to easily penetrate cellular membranes, and cell membrane damages caused by oxidative stress induced by Cr(VI) have been extensively reported, both in eukaryotic and prokaryotic cells, with effects such as loss of membrane integrity or inhibition of the electron transport chain [13,14]. Moreover, Cr(VI) enters cells using the sulfate transport system of the membrane in cells of organisms that are able to use sulfate [15,16,17,18,19,20].
Once Cr(VI) entered into cells, spontaneous reactions occur with the intracellular reductants as ascorbate and glutathione, generating the short-lived intermediates Cr(V) and/or Cr(IV), free radicals and the end-product Cr(III) [21,22,23]. In the cytoplasm, Cr(V) is oxidized to Cr(VI) and the process produces a reactive oxygen species, referred as ROS, that easily combines with DNA–protein complexes. On the other hand, Cr(IV) is able to bind to cellular materials, altering their normal physiological functions [24,25]. It is known that Cr(VI) species and hydroxyl radicals cause DNA lesions in vivo . The intermediates that originated from the action of Cr(VI) are dangerous to cell organelles, proteins and nucleic acids [27,28,29]. Cr(VI) is a very dangerous chemical form on biological systems as it can induce mutagenic, carcinogenic and teratogenic effects. Moreover, Cr(VI) is able to induce oxidative stress in cells, damaging its DNA . Inside of cells, the Cr(III)-DNA adducts and related hydroxyl radical oxidative DNA damages have a central role in originating the genotoxic and mutagenic effects . Moreover, the formation of Cr(III)-DNA binary adducts and L-cysteine-Cr(III)-DNA and ascorbate-Cr(III)-DNA ternary adducts likely increase both genotoxicity and mutagenicity in human cells [32,33]. Again the formation of DNA protein cross-linking, a process favoured by Cr(VI), induces a significant promutagenic effect .
Considering the dangerous effects Cr(VI) can cause to human health, Cr(VI) has been comprised among priority pollutants and listed as a class A human carcinogen by the US Environmental Protection Agency (USEPA) .
The cell membrane is nearly impermeable to Cr(III), Cr(III) has thus only about one thousandth of the toxicity of Cr(VI) [35,36]. Taking into account these considerations, it is possible to conclude that, depending on its oxidation state, chromium can have different biological effects, with Cr(VI) that is highly toxic to most organisms, and Cr(III) that is relatively innocuous [37,38].
2. Use of chromium and environmental contamination
Chromium enters in the anthropogenic activities, it is used in stainless steel plant, preparation of alloys, chrome plating, leather tanning, production of refractories, dye industry, industrial water cooling, paper pulp production, petroleum refining, wood preservation and nuclear power [1,39].
As consequence of its broad use, chromium is present in effluents originated from the different activities and represents a serious pollutant of sediments, soil, water and air . Wastewaters have resulted in significant quantities of Cr(VI) in the environment, which may constitute toxicological risk to humans, animals, and plants . Cr(VI) is introduced in the environment mainly as a consequence of its industrial use, while chromium in its trivalent form, Cr(III), naturally predominates in the environment .
Cr(VI) is highly dispersed in sediments and surface waters, and it is characterized by a much greater solubility, mobility and bioavailability than Cr(III) and all the forms of chromium [43,44]. As consequence of this high water solubility and elevated mobility, Cr(VI) diffuses easily away from the native site of contamination. Moreover, the increase in soil pH increases the leachability of Cr(VI). Cr(III) shows a low mobility and is relatively inert, and easily absorbable on mineral surfaces and solid-phase organic ligands, thus resulting less bioavailable in the environment. Additionally, Cr(III) is quite insoluble at environmentally significant pH values, since in these conditions there are formation of insoluble hydroxide and oxide compounds. Mobility of Cr(III) decreases with absorption of clays and oxide minerals below pH 5. Binding of Cr(III) by iron oxides can be considered an example of these mechanisms, as this feature can decrease the solubility of this form of chromium [43,45]. Again, the characteristic of insolubility of Cr(III) diminish its bioavailability and mobility of Cr(III) toxicity in saltwater exposures .
In the presence of oxidizing conditions Cr(VI), in forms of the anions chromate (CrO42–) and bichromate (HCrO4–), is extremely soluble and mobile (Barnhart 1997). In anaerobic environments, under reducing conditions, in the presence of reducing agent as sulfides, ferrous iron, and organic matter, that are several of the organic and inorganic constituents, Cr(VI) may rapidly convert to Cr(III) . Again, bacterially mediated reduction of Cr(VI) has also been considered in the chromium biogeochemical cycle .
Cr(III) is stable in aquatic environments and its oxidation to Cr(VI) is improbable, even in the presence of dissolved oxygen [49,50,51]. Different factors affect Cr(III) oxidation to Cr(VI), depending on the presence and mineralogy of Mn(III, IV) hydroxides, pH, and the form and solubility of Cr(III) . Oxidation of Cr(III) is improbable to occur in aquatic environments because aged waste materials containing Cr(III) are typically less soluble and more inert to oxidation, and Cr(OH)3 precipitates may form on surfaces of Mn(III, IV) hydroxide . Besides, possible Cr(III) oxidants are scarcer and less abundant than potential Cr(VI) reductants in natural sediments, and Cr(III) oxidation is slower than Cr(VI) reduction .
3. Microbial resistance to Cr(VI) and microbial Cr(VI)-reduction
Despite the toxicity of Cr(VI), some microorganisms evidence resistance to this heavy metal, showing the capability to reduce Cr(VI) to Cr(III), as was first reported for
Both aerobic and anaerobic microorganisms are able to reduce Cr(VI) to Cr(III). In aerobic conditions it is possible to observe the bio-reduction of Cr(VI) that can be obtained directly as a result of microbial metabolism . In the presence of oxygen, microbial reduction of Cr(VI) is commonly catalyzed by soluble enzymes, except in
4. Bioremediation of Cr(VI) by microorganisms
Conventional methods for removing metals from contaminated sites include chemical precipitation, oxidation/reduction, ion exchange, filtration, use of membranes, evaporation and adsorption on activated coal, alum, kaolinite, and ash [15,41]. However, most of these methods require high energy or large quantities of chemical reagents, with possible production of secondary pollution [76,77]. Concerning removal of Cr(VI), conventional approaches include chemical reduction followed by precipitation, ion exchange and adsorption on activated carbon, alum, kaolinite and of ashes, and most of these methods require a high energy and large amounts of chemical reagents . Moreover, costly safe disposal of toxic sludge, incomplete reduction of Cr(VI) and high cost for Cr(VI) reduction, especially for the removal of relatively low concentrations of Cr(VI) are non-convenient from the economical point of view [78,79].
An innovative technology is represented by bioremediation, which uses the metabolic potential of microorganisms to remove toxic metals, in order to decontaminate the polluted areas. Bioremediation techniques can be classified as
Cr(VI)-resistant microorganisms represent an important opportunity to have safe, economical and environmentally friendly methods for reducing Cr(VI) to Cr(III), for possible bioremediation applications . The reduction of Cr(VI) to Cr(III) is then a potential useful process for the recovery of sites contaminated by Cr(VI) . Cr(VI)-removal based on microorganisms is now considered to be an effective alternative method to the conventional processes, and is receiving great attention for potential application in bioremediation [76,80]. Taking into account that the insolubility of Cr(III) facilitates its precipitation and removal, the biotransformation of Cr(VI) to Cr(III) has been considered as an alternative process for treating Cr(VI)-contaminated wastes [81,82]. Among biotechnological approaches, microbial reduction of Cr(VI) is cost-effective and eco-friendly and can offer a viable alternative [80,83,84].
Chromium resistant microorganisms are responsible of the biological reduction of Cr(VI) into the less mobile Cr(III), and its consequent precipitation, could represent an effective method for detoxification of Cr(VI) contaminated sites and have a potential use in bioremediation .
Included in the bioremediation technologies, phycoremediation is the use of photosynthetic microorganisms as microalgae, macroalgae and cyanobacteria for the removal of pollutants as metals. Furthermore, it is essential to understand the distribution of the metal adsorbed onto the surface in relation to the metal accumulated inside the cell, in order to understand the predominant removal mechanisms and to make decisions of the viability of the recovery of the adsorbed metals .
Biosorption and bioaccumulation of chromium for bioremediation purposes have been demonstrated. Yeasts and especially molds have been most widely investigated from this aspect, and the mechanisms of chromium tolerance or resistance of selected microbes are of particular importance in bioremediation technologies. The mechanisms of chromium toxicity and detoxification have been studied extensively in yeasts and fungi, and some promising results have emerged in this area .
The ability existing in a number of environmental microorganisms, known for their capability to bind metals, can be evidenced in human gastrointestinal bacteria. Bacterial species belonging to the genus
A method for bioremediation of sites contaminated by metals, including chromium, is represented by bioaugmentation-assisted phytoextraction, in which bacteria and fungi, associated with plants able to accumulate metals were analyzed on the basis of a proposed as bioprocess for a bioremediation approach. The implementation of bioaugmentation to favour the microbial survival, was suggested in order to enhance the microbial-plant association and the efficiency of the process .
The process of biomineralization is a process by which microorganisms transform aqueous metal ions, including chromium, into amorphous or crystalline precipitates. Biomineralization is regarded as a promising and cost-effective strategy for remediating chromium contamination. An example of arsenic precipitation was considered as a possible mechanism for arsenic bioremediation of sediments contaminated by arsenic . Biologically mediated transformation, immobilization, and mineralization of toxic metals may represent an important perspective for bioremediation .
5. Case study: Cr(VI)-reduction by Actinobacteria isolated from polluted sediments near a stainless steel plant
Wastes from stainless steel plants produce soluble Cr(VI) contaminating sediments, soils and water bodies. Chromium at high concentrations are widespread in sediments of industrialized areas because of industrial discharges . In a previous study, carried out from polluted marine sediments near a stainless steel plant in Southern Italy, near the industrialized area of Taranto, an halophilic Cr(VI)-resistant bacterial strain
The aim of the present investigation was the isolation of bacterial strains from chromium-polluted sediments and their characterization in terms of phylogenetic and physiological features. The description of two Cr(VI)-reducing microorganisms isolated from polluted sediments and included into Actinobacteria was carried out, for their possible use in bioremediation applications.
6. Materials and methods
6.1. Study area
The microbiological study with the isolation of the bacterial strains investigated in this study was conducted in sediment samples collected from a polluted site near a stainless steel plant in the Bagnoli area, Naples (Southern Italy). The site was characterized by a total chromium content corresponding to 34 ± 0.23 mg kg-1.
6.2. Sediment sampling
Sediment samples for microbiological analyses were collected manually using Plexiglas tubes (i.d. 10 cm), in June 2008. Collected samples were maintained at 4 °C and transported to the laboratory. Sterile sediment subsamples (0-10 cm) were collected and processed within twelve hours for microbiological analyses.
6.3. Enrichment cultures and isolation of the bacterial strain
Enrichment cultures were grown in flasks containing the complex YPEG medium, containing 5.0 g of tryptone, 2.5 g of yeast extract, and 1.0 g of D-glucose per litre of distilled water, in the presence of 5.0 mM of Cr(VI), inoculated with 0.5 g of sediment samples, and incubated at 28 °C in the dark. From flasks showing turbidity, a 100 µl aliquot was spread on Petri dishes containing the complex solid medium in the presence of the same initial concentration of Cr(VI) and incubated at 28°C for 48 hours. Colonies showing different morphologies were selected and subcultured at least three times. Isolated strains were stored in the presence of 30% sterile glycerol (v/v) in liquid nitrogen.
6.4. Isolates characterization and identification by 16S rRNA gene sequencing
The bacterial isolates were observed under a stereomicroscope (Optika, mod 620). Gram reactions were determined following the standardized method of bacterial cells staining (Gram stain kit, Carlo Erba). Catalase and oxidase activities were determined following Smibert and Krieg . For 16S rDNA sequencing of the isolated bacterial strain, a single colony was suspended in 50 µl double-distilled water and treated for 5 min at 100°C. Amplification and sequencing of 16S rRNA gene was performed as previously reported . Partial 16S rDNA sequences were determined for the bacterial isolates chr 2 and chr 3, and the sequences were deposited in the GenBank database with the accession numbers: HQ609600 and HQ609601, respectively. The consensus sequences of the isolates were compared with those deposited in GenBank using the BLAST program .
6.5. Analysis of sequence data
The 16S partial sequences were compared at the prokaryotic small subunit rDNA on the Ribosomal Database Project II website . The 16S rDNA sequences retrieved from the databases were aligned using ClustalW included in the MEGA software, version 4.1 . The phylogenetic trees were inferred by MEGA 4.1 (neighbour-joining method) . Sequence divergences between strains were quantified using the Kimura-2-parameter distance model . The ‘‘Complete Deletion’’ option was chosen to deal with gaps. Bootstrap analysis (1000 replicates) was used to test the topology of the neighbour-joining method data. The trees were unrooted.
6.6. Minimum Inhibitory Concentrations (MICs)
One ml aliquots of overnight cultures were incubated in 99.0 ml of YEPG-NaCl broth, and 10 ml were distributed in 18 ml test tubes sealed with radial caps. MIC tests were carried out at different concentrations of Cr(VI). Tubes were incubated in a rotary drum at 30°C for 24 hours. The optical density of the cultures, used as a measure of microbial growth, was detected at a wavelength of 600 nm by an UV-visible spectrophotometer (Jenway, mod. AC30); a blank with the culture medium alone (without bacteria) was also analysed. Experiments were carried out in duplicate.
6.7. Chromium (VI) assay
Hexavalent chromium was determined colorimetrically using the 1,5-diphenylcarbazide (DPC) (Sigma-Aldrich, Milan, Italy) method .
6.8. Effect of chromium concentration on bacterial growth and Cr(VI)-reduction
The Cr(VI)-resistant isolates were grown over-night in YEPG-NaCl medium, in the presence of 0.2 mM Cr(VI). The pre-culture were used for inocula in different cultures at the same conditions, and incubated at 28°C in the presence of Cr(VI) concentrations: 0, 10, 25, 50, 75, 10 and 150 µg ml-1. At different times (0, 0.5, 3, 6, 12, 18 and 24 hours), aliquots were harvested in order to measure the absorbance at 600 nm spectrophotometrically, and to evaluate Cr(VI) reduction according to the DPC method. For each series, experiments were conducted in triplicate.
6.9. Effect of temperature on bacterial growth and Cr(VI)-reduction
Cultures of the isolates were incubated in a temperature range from 4 to 42°C, with an inoculum prepared by an overnight pre-culture in YEPG medium containing 25 µg ml-1 of Cr(VI). After different times of incubation of (0, 6, 12 and 24 hours), the effect of different temperatures was detected by harvesting two aliquots of 1 ml for each series, one to evaluate the biomass, revealing absorbance at 600 nm spectrophotometrically, the other to estimate Cr(VI) reduction according to the method of DPC. Experiments were conducted in triplicate.
6.10. Extraction of plasmids from the cells of the bacterial strains Cr (VI)-resistant isolates
The two isolated bacterial strains Cr(VI)-resistant were grown in liquid medium YEPG-NaCl in the presence of 25 µg ml-1 of Cr(VI). Aliquots of 2 ml were centrifuged at 15,000 ×
7. Results and discussion
From enrichment cultures arranged from samples of polluted sediments collected near the industrial area, including metallurgical plants, of Bagnoli (Naples, Italy), two Cr(VI)-resistant bacterial strains were isolated and named chr2 and chr3. A microbiological characterization of the isolated strains is reported in Table 1.
|chr2||positive||+||+||Ø 1.0 mm; beige color; regular margins; flat; moist|
|chr3||positive||-||+||Ø 1.5 mm; yellow color; regular margins; convex; mat|
BLAST analysis evidenced a similarity of 100% for strain chr2 with strains
Phylogenetic analysis of the strain
The Cr(VI)-resistant bacterial strains isolated in this study were assigned to the genera
The two genera
The bacterial strains showed MIC values in the presence of Cr(VI) of 150 µg ml-1 for the bacterial strain
The mechanism of resistance to Cr(VI) was investigated in the isolated bacterial strains, evidencing their capability to reduce Cr(VI), the most toxic and extremely soluble form of chromium, as revealed by the tests evidencing the depletion of the Cr(VI) content in cultures in conjunction with the increase of the bacterial biomass. The bacterial strain Cr(VI)-resistant
The bacterial strain
The isolated bacterial strain
The two isolated Cr(VI)-resistant strains belonging to Actinobacteria were investigated for their capability to grow and to reduce Cr(VI) added at different concentrations, tested in a range of temperatures included from 4°C to 42°C. Concerning the strain
Cr(VI)-reduction in the same cultures of the isolate
The isolated strain
This experiment was conducted in the presence of a fixed concentration of Cr(VI) corresponding to 25 mg ml-1, and in this case strain
The capability to reduce Cr(VI) to Cr(III) was evidenced in both the bacterial strains isolated in this study. It is known that different bacterial strains were isolated from polluted sites, as a strain of
The ability of the isolates to grow at different temperatures evidenced the range of the use of the Cr(VI)-reducing bacterial strains in eventual bioremediation processes, even in conditions of non-controlled temperature.
Recently, the bio-reduction of Cr(VI) to Cr(III) focused more attention for possible use in bioremediation processes of sites contaminated by Cr(VI). This strategy represents an environmentally friendly technology, to be applied
The two isolates evidenced the presence of plasmids when tested with primers specific for the presence of genes of Cr(VI)-resistance included into plasmids (data not shown). The bacterial strains capable of expressing both the resistance and the reduction of chromate are very useful for bioremediation. Plasmids involved both in resistance and in the reduction of Cr(VI) have been described in a strain of
The potentiality of the Cr(VI)-resistant microorganisms in bioremediation of polluted sites was evidenced in this chapter. A case study was moreover reported with the description of bacterial strains isolated from sediments contaminated by Cr(VI), and tests of Cr(VI)-reduction were included. The isolated bacterial strains showed resistance to Cr(VI) and phylogenetic analyses of the 16S rRNA gene assigned them to the genera
Further studies including investigations on mechanisms of resistance to Cr(VI) in autochthonous microorganisms isolated from polluted sites, and on the adaptability of microorganisms to contaminants, could give insights for new researches, favoring the development of new technologies for environmental recovery.