Abstract

The main objective of the present research is to identify and characterize the essential genes responsible for Cr removal by the highly resistant bacterial strain Pseudomonas stutzeri Strain M15-10-3 isolated from leather tanning industrial wastewater to optimize its capacity and to be exploited in the development of chromium bioaccumulation capacity in other microorganisms. Pseudomonas stutzeri (PS) was previously investigated among a total of 20 bacterial isolates (17 indigenous and 3 exogenous) to decontaminate heavily polluted leather tannery wastewater using batch and fixed (biofilm) mode where it exhibited remarkable efficiency in removing Cr, the highly toxic and main contaminant in tannery effluent. PS and 3 sub strains of Bacillus cereus ATCC 14579 were subjected to 4 elevated Cr levels (2000-5000 mg/l) for 9 days to confirm the PS affinity for Cr bioaccumulation. Results proved the highest affinity of PS (≈ 80%) to bioaccumulate Cr from polluted media even at very high concentration (5000 mg/l). Moreover, Cr has remarkable inhibition activity on the growth of Bacillus cereus strains (75 to 99.7%) even at the lowest tested Cr concentration without any stimulation at all the tested concentrations. However, PS exhibited the superior acclimatization ability against Cr even at the highest Cr concentration reflecting its high Cr resistance with 25% growth stimulation at 4000 mg Cr/l and the lowest growth inhibition (37.1% at 2000 mg Cr/l). Therefore, PS was considered highly efficient candidate for Cr removal and was selected to be molecularly investigated to characterize Cr resistance genes. Results demonstrated that ChrT gene responsible for chromate reduction ability was most probably present in Pseudomonas stutzeri genome.

1. Introduction

As a result of industrialization, metal pollutants such as chromium, mercury, lead, zinc, uranium and selenium are introduced to the environment posing a real threat to the human race ¹. Among heavy metals, chromium (Cr) is described as a highly toxic metal that severely affects human health as well as the surrounding environment ^{2, 3}. The harmful health effects of chromium ions include skin allergy, vomiting, lung and nervous system damage, severe diarrhea and human hemorrhage. Moreover, it possess mutagenic and carcinogenic properties ^{4, 5}. Chromium enters the environment through wastes of many different industries such as mining and refining raw materials, leather tanning, industrial and household sludge, fly ash from holocausts, radioactive materials, pesticides or preservatives and metal plating ^{6, 7, 8}. Therefore, it is a must to remove, in particular, Cr⁶⁺, from drinking and wastewater due to its high toxicity.

There are several chemical methods for the removal of toxic metals such as solvent extraction, ion exchange, chemical precipitation, membrane separation process, oxidation-reduction, filtration, adsorption, incineration as well as electrochemical treatment ^{9, 10}. However, such methods have many disadvantages among which the generation of toxic sediment or waste products which in turn require safe disposal ¹¹. Many microorganisms (viable and dead; free or fixed) proved to act as biological remediants and absorbers to remove heavy metals from wastewater ¹² especially the highly toxic such as Cr⁶⁺, thus, considered as viable, eco-friendly and cost effective technology for cleanup of chromium (VI). Chromium detoxification includes transformation of a highly toxic, mutagenic and carcinogenic hexavalent Cr⁶⁺ into its non-toxic reduced trivalent form Cr³⁺. Recently, two soluble Cr⁶⁺ reductases, ChrR and YieF, have been isolated from Pseudomonas putida MK1 and Escherichia coli, correspondingly. The direct use of Cr⁶⁺ reductases may be a favorable way for bioremediation of wide range of Cr⁶⁺- contaminated environments ¹³.

Pseudomonas aeruginosa Rb-1 and Ochrobactrum intermedium Rb-2 enhanced wheat seed germination under chromium (III and IV) stress compared with non-bacterial inoculated control ¹⁴. Strains Rb-1 and Rb-2 that are able to survive in chromium-contaminated environment could improve wheat growth along with decreasing the toxicity of chromate by different direct and indirect mechanisms ¹⁵. Not only bacteria, but viable cells of the white rot fungus Phanerochaete chrysosporium (MTCC787) was evaluated to remediate chromium from fortified solution where it removed 99.7% Cr (VI) after 72 h and considered highly potential for decontaminated polluted media ¹⁶.

2. Materials & Methods

Pseudomonas stutzeri Strain M15-10-3 (PS) isolated from leather tanning industrial wastewater, and for comparison, 3 sub strains of Bacillus cereus ATCC 14579, a well-known metals bio-accumulator Gram +ve bacteria were investigated for Cr bioaccumulation to confirm PS activity. Bacillus cereus strains were isolated from pesticide contaminated - agricultural soil. Selected strains were grown in nutrient agar (NA) or broth (NB) supplied by Oxoid LTD (Basingstoke, Hampshire, England) as dehydrated media. Prior to each experiment, cultures were reactivated overnight.

The four selected isolates were activated from 24-h agar cultures in 100 ml NB. Cultures were incubated for 24 h at 37 °C to obtain dense inocula. Four sets of four 250 ml flasks (16 flasks total) were prepared with 100 ml sterilized NB. Each set was emended with 4 elevated Cr levels (2000-5000 mg/l final concentration). Total viable count of bacteria (TVC) of the 24 h - liquid culture of each strain was determined after appropriate serial dilution and divided into 4 aliquots (25 ml) and added under aseptic conditions to the Cr- amended NB in the four 250 ml flasks. After inoculation, all cultures were incubated at 37°C. After 9 exposure days 6 ml sample from each culture were aseptically drawn for TVC and Cr residuals determination.

TBVC (CFU/ml) of the selected strains before and after exposure to Cr were serially diluted (up to 10^-8), and 1000 µl of the appropriate dilution was cultured (3 replica each) under aseptic conditions on NA plates and incubated for 24 h at 37°C using the pour plate technique of the standard heterotrophic plate count method ¹⁷. Colony forming units (CFU) of the total viable bacterial counts (TVC) were recorded (Colony Counter Stuart Colony Counter Protected by Bio Cote) and averages were calculated.

Chromium levels were determined in the stock solution as well as in the broth cultures amended with Cr before and after treatment with the selected bacteria using Atomic Absorption Spectrophotometer, ASS {Thermo Scientific, S SERIES, AA Spectrophotometer, England, (European Union)}. After incubation, 5 ml of each culture was aseptically drawn, centrifuged (6000 rpm) for 10 min, bacterial pellets was discarded and Cr residues in the broth were determined using the AAS. Removal efficiency of Cr by the selected bacteria was calculated to determine the effectiveness of the remediation process according to the following equation:

Where C0 = Initial Concentration before Treatment (Zero Time);

RC= Residual Concentration after Treatment at each Exposure Time.

Extraction of total DNA was carried out using bacterial DNA extraction kit; GeneJET Genomic DNA Purification Kit (Thermo scientific co., Molecular biology, EU). DNA was extracted and purified for PCR amplification by the kit protocol.

PCR reactions were carried out using specific primers for Cr resistance ¹⁸. The PCR amplifications were performed in a final volume of 25 µl with 2x MyTaq Red Mix PCR Master Mix (Bioline Co.), according to the manufacturer's instructions. The reaction mixtures included 0.5µl (10 PM) of each of the specific primers and 1µl template DNA ¹⁹. Two pairs of primers, Scfmn1forward (F), Scfmn1reverse (R), Scfmn forward (F) and Scfmn -reverse (R) are shown in Table 1 (Biosearch technologies Co., USA), were designed and used to amplify the fragment of the Pseudomonas stutzeri gene (ChrT) which concerning to Cr reduction ¹⁸. The fulllength ChrT gene was obtained using specific primers (ScfmnF and ScfmnR) corresponding to the 5'and 3'ends of the ChrT gene. Amplification has been performed with thermal Cycler (TECHNE, TC-3000) using the following program: denaturation for 1 min at 95 °C; then 35 cycles consisting of 95 °C for 30 sec, annealing temperatures of 54 °C for 30 sec and 72 °C for 1 min; as well as a final extension step at 72 °C for 5 min.

The PCR product was purified by Gene JET Gel extraction Kit (Thermo scientific Co., Fermentas, EU) after slicing the specific band from the loaded gel. The purified PCR product was stored in -20°C for further need of sequencing.

PCR amplification products were analyzed using electrophoresis in 1 % (w/v) agarose (Sigma) gel and run in 1X TBE buffer, pH 8.3 ²⁰. The ethidium bromide stained gels to visualize DNA bands, and gels were photographed using transmitted UV in a gel documentation system (Syngene Ingenius). Gene size was determined by comparison with a DNA ladder (100 bp DNA ladder, Solis BioDyne Co.) ²¹.

3. Results

In a comparative study, PS and 3 sub strains of Bacillus cereus ATCC 14579 were subjected to 4 elevated Cr levels (2000-5000 mg/l) for 9 days to confirm the PS affinity and superiority for Cr bioaccumulation. Table 2 represents comparison between Pseudomonas stutzeri strain M15-10-3 (PS) and Bacillus cereus ATCC 14579 sub strains for the removal efficiencies of Cr.

Results revealed general unexpected trend by all the tested strains where they exhibited +ve relation between Cr concentrations and its RE. In that respect, Cr removal by all strains increased with increasing the concentration reaching their highest RE at 5000 mg/l.

Removal efficiency (RE%) of Cr by the selected strains confirmed the superiority of Pseudomonas stutzeri strain M15-10-3 compared to the other tested strains where it exhibited the highest RE% at 2000 and 4000 mg Cr/l (70.7 and 77.81% respectively). It almost achieved similar REs at 3000 and 5000 mg Cr/l as Bacillus cereus ATCC 14579 (23). Bacillus cereus ATCC 14579 strain 13 showed the lowest RE of Cr at all the tested concentrations (58.9- 69.72%) while Bacillus cereus ATCC 14579 strain 12 showed intermediate RE% of Cr. These results proved the highest affinity PS to bioaccumulate Cr from polluted media even at very high concentration (5000 mg/l) and for wide range. Therefore, PS was selected to be molecularly investigated to determine Cr resistance genes.

As shown in Table 3 Cr has remarkable inhibition activity on the growth of almost all the selected bacteria except PS. Inhibition% for Bacillus cereus strains ranged from 75 to 99.7% even at the lowest tested Cr concentration. None of them exhibited any stimulation at all the tested concentrations. However, PS exhibited superior acclimatization ability against Cr even at the highest Cr concentration reflecting its high Cr resistance. Moreover, it showed 25% growth stimulation at 4000 mg Cr/l which considered marvelous ability. Compared with Bacillus cereus strains, PS showed the lowest growth inhibition (37.1%) at 2000 mg Cr/l and almost similar inhibition at 3000 and 5000 mg Cr/l. Therefore, PS is considered highly efficient candidate for Cr removal. Studying PS genes responsible for this ability is of high importance not only for optimizing this ability but also to be exploited in the development of chromium bioaccumulation capacity in other microorganisms for the ultimate target of decontaminating polluted media.

The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr (VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). Firstly, two pairs of specific primers were synthesized on the basis of the FMN red gene sequence of Serratia sp. AS13. When the genomic DNA from the Pseudomonas stutzeri cells was used as a template, an expected 305bp fragment of the ChrT gene was amplified using the Scfmn1F/R primers, which was subsequently sequenced. Then, a fulllength ChrT gene of 400 bp and 650 bp were obtained with the specific ScfmnF/R primers (Figure 1). This result demonstrated that the ChrT gene was most probably present in Pseudomonas stutzeri genome which is specific gene responsible for chromate reduction ability ¹⁸.

4. Discussion

Among heavy metals, chromate Cr(VI) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), thus, leads to effective bioremediation. As mentioned above, many microorganisms (bacteria, fungi, yeast….etc.) can efficiently act as biosorbents for heavy metals from contaminated media either in viable or dead form and either as free or fixed forms ^{12, 13, 14, 15, 16}. Cr especially Cr⁶⁺ considered highly toxic posing both acute and chronic threats on the different compartments of the environment, thus, it is a must to remove, in particular, Cr⁶⁺, from drinking and wastewater. Microbial reduction of Cr⁶⁺ to Cr³⁺ is well known and effective mechanism to reduce Cr toxicity and an important step in its removal from contaminated media. Among bacteria, various genera were found highly capable of reducing Cr (VI) including Arthrobacter ²², Bacillus ²³, Microbacterium ²⁴, Brucella ²⁵ and Pseudomonas ^{13, 14, 15, 26}. This highly supports the present study where P. stutzeri and Bacillus cereus exhibited superior ability for Cr removal (79.3 and 81.1% respectively) at 5000 mg Cr/l due to the included genes.

Pseudomonas spp., in particular, characterize by effective biodegradation and bioaccumulation capability towards wide range of environmental organic and inorganic pollutants including heavy metals. P. stutzeri strain M15-10-3 examined in the present study was selected based on its ability to achieve more than 98% Cr from tannery effluent with an initial concentration of 3500 mg/l ^{27, 28}. Generally, many researches are concerned with isolation and identification of naturally occurring microorganisms from contaminated environments with Cr removal capability ²⁹, while few have focused on genes and proteins responsible for Cr reduction ³⁰. The direct use of Cr⁶⁺ reductases such as ChrR and YieF, isolated from Pseudomonas putida MK1 and Escherichia coli may be a favorable way for bioremediation of wide range of Cr⁶⁺-contaminated environments ¹³. Moreover, Pseudomonas aeruginosa Rb-1 and Ochrobactrum intermedium Rb-2 enhanced wheat seed germination under chromium (III and IV) stress compared with non-bacterial inoculated control ¹⁴. They could decrease chromate toxicity by different direct and indirect mechanisms ¹⁵.

Genetic and protein engineering of suitable enzymes improve bacterial bioremediation. The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr (VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red) ¹⁸. This gene was in the Serratia sp. strain S2 and was successfully constructed in other bacteria using genetic engineering technology ³¹. The engineered strain, which contained the chromate reductase ChrT gene from Serratia sp. S2, was studied for its Cr (VI) reduction efficiency, optimal culture conditions and chromate reductase activity in metal-contaminated soil and water ecosystems. It could achieve up to 40% Cr (VI) reduction rate of at a concentration of 50 mg/l after 48 h exposure with optimal culture conditions of pH 7.0, 37˚C. and chromate reductase reached 14.83 U/mg ³².

Many bacterial enzymes can reduce Cr(VI) to Cr(V), generating excessive reactive oxygen species (ROS) and making those enzymes not appropriate for bioremediation, as they harm the bacteria and do not produce Cr(III) as their primary end product. Thus, not every Cr reductase enzyme is suitable for Cr reduction. For example, pure soluble bacterial flavoproteins; ChrR (from Pseudomonas putida) and YieF (from Escherichia coli) were examined. ChrR transferred >25% of the NADH electrons to ROS and probably generates Cr(V), but only transiently. ChrR Pseudomonas putida mutants protects against chromate toxicity by minimizing ROS generation. However, YieF Cr reductase transferred only 25% of the NADH electrons to ROS. Therefore, it was suggested that YieF may be an even more suitable candidate for further studies than ChrR ³³.

Although several bacterial Cr resistance mechanisms were reported, the best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). For example, efflux of chromate by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy dependent process driven by the membrane potential. However, the CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life can reduce chromate by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms ³⁴.

5. Conclusion

Pseudomonas stutzeri (PS) and 3 sub strains of Bacillus cereus ATCC 14579 were subjected to 4 elevated Cr levels (2000-5000 mg/l) for 9 days. Results proved the highest affinity of PS (≈ 80%) to bioaccumulate Cr at initial concentration of 5000 mg/l. Moreover, PS exhibited the superior acclimatization ability against Cr even at the highest Cr concentration reflecting its high Cr resistance with 25% growth stimulation at 4000 mg Cr/l and the lowest growth inhibition (37.1% at 2000 mg Cr/l). On the other hand, Cr has remarkable inhibition activity (75 to 99.7%) on the growth of Bacillus cereus strains even at the lowest tested Cr concentration without any stimulation at all the tested concentrations. Therefore, PS was considered highly efficient candidate for Cr removal and was selected to be molecularly characterized. Results demonstrated that ChrT gene responsible for chromate reduction ability was most probably present in Pseudomonas stutzeri genome.

Conflict of Interest

The authors declare that there is no conflict of interest.

Acknowledgments

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant No. (D-100-247-1437). The authors, therefore, acknowledge with thanks DSR technical and financial support.

References