Removal of hexavalent chromium [Cr(VI)] from contaminated effluent using Bacillus sp. and its secondary metabolites: a lab-scale bioreactor approach

Removal of Cr(VI) from synthetic aqueous solutions by filamentous green algae Spirogyra porticalis

BACKGROUND: Bacterial strains belonging to the genus Bacillus, isolated from Cr‐ polluted soil (tannery sludge) were employed as consortium for Cr(III) removal from tannery effluents. Kaolin clay, a natural adsorbent, was used as supporting material for bacterial biofilm formation. The use of clay‐supported bacterial biofilm has not previously been employed for the treatment of tannery effluents containing Cr(III) salt.RESULTS: Commercial tannery effluent containing 1000 ppm initial metal ion concentration was treated in stages. The initial Cr(III) concentration of 1000 ppm was brought down to 2 ppm, a permissible level for discharge, after the fourth stage. The bacterial isolates were found to be Bacillus subtilis VITSCCr01 and Bacillus cereus VITSCCr02 by 16s rRNA gene sequencing. Batch assay and confocal laser scanning microscopy results revealed the role of kaolin as a support material in biofilm formation. Best fit was obtained with the Freundlich adsorption isotherm. The mechanism of sorption was confirmed by Fourier transform infrared (FT‐IR) spectroscopy and scanning electron microscopy–energy dispersive X‐ray spectroscopy (SEM‐EDS).CONCLUSION: Cr(III) removal from tannery effluent using low cost adsorbents such as kaolin and bacteria proved to be effective for metal concentrations ⩽1000 ppm; this is normally not possible using conventional treatment methods. This work has demonstrated feasible sorption of Cr(III), especially during post‐tanning operations. Copyright © 2011 Society of Chemical Industry

Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.

Contamination of agricultural soils with heavy metals (HMs) poses a significant environmental threat, especially because industrial discharges often irrigate agricultural lands. A prominent source of HM(s) pollution occurs from tannery effluents containing high concentrations of chromium (Cr) in both Cr3+ and Cr6+ forms along with other toxic materials. Cr is known for its carcinogenic and mutagenic properties in biological systems. Microbe-assisted phytoremediation has emerged as a promising and environmentally friendly approach for detoxifying Cr-contaminated environments. This study aimed to evaluate the performance of citric acid (CA) and a Cr-reducing bacterial strain (Staphylococcus aureus) on the phytoextraction potential of Lemna minor within a Constructed Wetland System treated with tannery wastewater. Various combinations of tannery wastewater (0, 50, and 100 %), CA (0, 5 and 10 mM), and microbial inoculants were applied to the test plants. The mitigative effects of Staphylococcus aureus strain K1 were examined in combination with different concentrations of CA (0, 5, 10 mM). Data on growth and yield attributes highlighted the beneficial effects of bacterial inoculation and CA in ameliorating Cr toxicity in L. minor, as evidenced by increased foliar chlorophyll and carotenoid contents, enhanced antioxidant enzyme activities (SOD, POD, APX, CAT), and improved nutrient uptake. Specifically, CA application resulted in an enhancement of Cr ranging from 12% to 15% and 23%–31% in concentration, and 134%–141% and 322%–337% in Cr accumulation, respectively. When combined with the S. aureus inoculation treatment, CA application (5 and 10 mM) further increased the concentration and accumulation of Cr in L. minor. The enhancement in Cr ranged from 12% to 23% and 27%–41% in concentration, 68%–75%, and 179%–185% in accumulation, respectively. These results demonstrated that L. minor is an effective choice for environmentally friendly Cr remediation due to its continued ability to grow in polluted wastewater. This study suggested that microbial-assisted phytoextraction combined with chelating agents such as CA could be a practical and effective approach for remediating tannery effluents.

A commercially important alkaline protease, produced by Bacillus sp. RRM1 isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex Silva, was first recognized and characterized in the present study. Identification of the isolated bacterium was done using both biochemical characterization as well as 16S rRNA gene sequencing. The bacterial strain, Bacillus sp. RRM1, produced a high level of protease using easily available, inexpensive agricultural residues solid-state fermentation (SSF). Among them, wheat bran was found to be the best substrate. Influences of process parameters such as moistening agents, moisture level, temperature, inoculum concentration, and co-carbon and co-nitrogen sources on the fermentation were also evaluated. Under optimized conditions, maximum protease production (i.e., 2081 U/g) was obtained from wheat bran, which is about 2-fold greater than the initial conditions. The protease enzyme was stable over a temperature range of 30-60 degrees C and pH 6-12, with maximum activity at 50 degrees C and pH 9.0. Whereas the metal ions Na+, Ca2+, and K+ enhanced the activity of the enzyme, others such as Hg2+, Cu2+, Fe2+, Co2+, and Zn2+ had rendered negative effects. The activity of the enzyme was inhibited by EDTA and enhanced by Cu2+ ions, thus indicating the nature of the enzyme as a metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents, surfactants, and organic solvents. Moreover, the present findings opened new vistas in the utilization of wheat bran, a cheap, abundantly available, and effective waste as a substrate for SSF.

The tannery industry effluents are the major source for the Cr(VI) production in wastewater streams. The present work deals with the determination of Cr(VI) removal capacity from synthetically prepared industrial effluent of and tannery industries using sawdust which is a low -cost adsorbent. In the present study, batch experiments were carried out for an initial Cr(VI) concentration ranging from 10 – 50 mg/l. Experimental results demonstrated that the sawdust adsorbent has a significant capacity for adsorption of Cr(VI) from tannery effluent. The effect of various parameters such as pH, temperature, adsorbent concentration, adsorbate concentration and retention time was investigated. The maximum adsorption of Cr(VI) on sawdust is obtained at pH 6 and 27 0 C. In the present study it was also clear that the higher absorbance was recorded in 50 ppm concentration of tannery effluent at 50g of saw dust.

<p>Sulfide-modified nanoscale zerovalent iron (S-nZVI) is attracting a lot of attention due to its ease of production and high reactivity with hexavalent chromium (Cr (VI)). However, until now, the most commonly used is the use of NaS2O4 sulfide nano zero valent iron. The study on the removal of hexavalent chromium from water by nanometer zero-valent iron with calcium sulfide is not comprehensive enough. Herein, the removal of high concentration of hexavalent chromium from wastewater by nanometer zero-valent iron with calcium sulfide and its structure were carefully investigated. Scanning electron microscopy (SEM) with EDS analysis demonstrated that sulfur was incorporated into the zero valent iron core and homogeneously distributed within the nanoparticles. S-nZVI had an optimal Cr (VI) removal capacity of 200mg/L, which was >100% higher than for pristine nZVI. Different molar ratio of polycalcium sulfide and zero-valent iron, initial zero-valent iron addition amount, initial pollutant concentration and initial pH value have different effects on the removal effect. While the S/Fe=0.2, 200mg/L initial Cr (VI) concentration, 2g/L S-nZVI additive amount, pH<5, have the optimum removal rate. Contrast to pristine nZVI, S-nZVI can efficiently sequester high concentration of hexavalent chromium from different contaminated groundwater matrices.</p>

Removal of Cr(VI) from Aqueous Solution Using Activated Cow Dung Carbon

In the present study, response surface methodology (RSM) was employed to investigate the effects of different operating conditions on the removal of hexavalent chromium (Cr (VI)) onto mixed cultures of Pseudomonas aeruginosa and Bacillus subtilis (MCOPAABS) using biosorption processes. Box‐Behnken design (BBD) was used for optimization of the biosorption process and to evaluate the effects and interactions of the process variables, i. e., biomass concentration, pH, temperature and contact time on the removal of Cr (VI). A synthetic aqueous solution with a Cr (VI) concentration of 10 mg/L was used in the experimental study as a fixed input variable. The optimum conditions for maximum uptake (1.44 mg/g) of Cr (VI) onto the biosorbent were established as 0.5 g/L biosorbent dosage, pH 2 for the aqueous solution, 32°C temperature and 23 min contact time.

Bioremediation is an environmentally friendly technology for the treatment of chromium-contaminated sites. Here, a hexavalent chromium [Cr(VI)]-resistant strain was isolated from oil-contaminated soil and designated as Bacillus sp. Y2-7 based on 16S rDNA sequence characterization. The effects of various factors including inoculation dose, pH value, glucose concentration, and temperature on Cr(VI) removal rates were then evaluated. Based on the response surface methodology, optimal Cr(VI) removal efficiency (above 90%) could be achieved at an initial Cr(VI) concentration of 155.0 mg·L−1, glucose concentration of 11.479 g·L−1, and pH of 7.1. The potential removal mechanisms of Cr(VI) by strain Y2-7 were also supposed. The contents of polysaccharide and protein in extracellular polymer (EPS) of strain Y2-7 decreased slowly after cultured with Cr(VI) of 15 mg·L−1 from 1 to 7 days. We thus inferred that EPS bonded with Cr(VI) and underwent morphological changes in water. Molecular operating environment (MOE) analysis suggested that macromolecular protein complexes in Bacillus sp. Y2-7 and hexavalent chromium could establish hydrogen bonds. Collectively, our findings indicate that Bacillus sp. Y2-7 is an excellent bacterial candidate for chromium bioremediation.

Tannery effluent is known to contain large amount of dissolved salt (such as NaCl) used in the preliminary preservation of hides and skin, which is known to be highly soluble and stable in nature, difficult to eliminate and toxic in nature, thereby being a burden to the environment (ecosystem) and human health. The aim of this study is to isolate halophilic organisms from Nigerian Institute of Leather and Science Technology, Zaria tannery dumpsite using selective media. The isolated microbes were characterized microscopically and biochemically using standard methods. The microbes isolated include; Staphylococcus aureus, Bacillus sp, Pseudomonas aeruginosa, and Saccharomyces cerevisiae. The isolates were used for the bioremediation of total soluble salt in the tannery effluent. Before the tannery effluent treatment, physicochemical parameters such as temperature, pH, colour, Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), chloride content, conductivity, total dissolved solid (TDS) and salinity were determined using standard procedures. The bioremediation of tannery effluent was carried out using the isolated organisms individually and in combination. The highest remediation was observed in the combination of microbial consortium, followed by Saccharomyces cerevisiae, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus sp. The temperature of the effluent ranges between 27.4°C to 27.6°C before the bioremediation, which increased to between 28.4°C and 32.3°C after remediation. Majority of the physicochemical parameters analyzed recorded drastic decrease especially the salinity (7.10 ppt to 1011 ppm), conductivity (1788 µS to 1407 mS), colour (faint ash to ash), BOD (942 mg/l to 1156 mg/l), COD (1239 mg/l to 508 mg/l), chloride content (181.9 mg/l to 579.8 mg/l), pH (6.3 to 9.1) and TDS (8.20 ppt to 1322 ppm) respectively. Most of the physicochemical parameters are above the Federal Environmental Protection Agency (FEPA) and Ethiopian Environmental Protection Authority (EEPA) standard safe limit viz; pH (6.9), Conductivity (2500 µScm−¹), BOD5 (50 mg/l and 200 mg/l), COD (500 mg/l). However, temperature (<40 and 40°C) and chloride content (1000 mg/l and 600 mg/l) are within the acceptable limits. Also, there is significant difference (P= 0.000 at 0.005 level of significance) between the chloride content of the tannery effluent before the bioremediation and the chloride content after ten days bioremediation. Thus, it is recommended to use these organisms in combination to remedy total dissolved salt efficiently than in single. Halophiles can be used in bioremediation of total soluble salt in tannery effluent. It is recommended that natural halophilic microbes should be used in the remediation of total soluble salts in preference to chemicals, which may contain heavy metals that cause toxicity and threat to the environment (ecosystem) and human health.

Low-density polyethylene (LDPE) is the predominant single-use plastic and rarely decomposes after disposal. The primary objective of this study was to identify potential bacteria capable of degrading LDPE plastic and investigating the biochemical pathways of this process. Bacteria were isolated from soil samples collected from a local garbage dumping site in Thailand and tested on their capability to degrade LDPE plastic. Two of the bacteria isolated from the dumping site, Bacillus sp. AS3 and Sphingobacterium sp. AS8, demonstrated 3.06% and 2.01% (w/w) LDPE plastic weight loss over four weeks, respectively. Analysis by FTIR showed that both bacterial strains degraded the LDPE in the region of 3200–3400 cm−1, which represents the OH group in a commercial LDPE polymer. Bacillus sp. AS3 caused the formation of a new range in the carbonyl group (C=O stretch) and the alcohol, carboxylic acid, esters, and ethers group (–C–O stretch). GC–MS analysis revealed various depolymerized compounds, such as alkane, alcohol, and carboxylic compounds, during LDPE degradation by Bacillus sp. AS3. Bacillus sp. AS3 illustrated esterase activity as 0.608 ± 0.004 U/mL after incubation. The proposed schematic of the LDPE biodegrading pathway by Bacillus sp. AS3 relies on the identification of depolymerized molecules as evidence. This suggests that Bacillus sp. AS3 possesses extracellular enzymes that break down LDPE into smaller molecules through depolymerization. Moreover, the surface of LDPE degraded by Bacillus sp. AS3 and Spingobacterium sp. AS8 was marked by cavities and a rough texture when observed under SEM analysis. This study provides microbial applications to reduce plastic pollution by utilising microorganisms to assimilate plastic waste as a carbon source.

Aim This study aims to determine the biological activity and explore the antimicrobial compounds producedbya halophilic bacterium, as well as the hemolytic, antioxidant, and antimicrobial properties of extracellular metabolites from Bacillus sp. Methodology The bacterial strain was obtained from the Department of Microbiology at Saveetha Medical College and Hospital, Chennai, India, specifically from the bio-control and microbial product laboratory (BCMPL). The genotype and phenotype of the isolate were characterized while the cultures were maintained in a nutrient broth medium supplemented with 8% sodium chloride (NaCl). The secondary metabolites were extracted using ethyl acetate and concentrated through open evaporation techniques after nine days of growth in the culture medium. Biological compatibility studies were conducted concurrently with the screening of the antimicrobial and antioxidant properties of the secondary metabolites. The chemical composition of the crude metabolites was analyzed using the gas chromatography (GC)-mass spectrometry (MS) technique. Results After phenotypic and genotypic analysis, the obtained potential halophilic bacterium from BCMPL was determined to be Bacillus sp. After the dark brown crude metabolites were extracted, the extracellular metabolites' antimicrobial activity against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Candida albicans (C. albicans) extracellular metabolites was moderately inhibited. Furthermore, the metabolites exhibited a moderate level of hemolytic and antioxidant activity. The GC-MS method depicted the presence of 12 distinct metabolites, each with a distinct retention time. Conclusion To sum up, the halophilic bacteria that were obtained and identified asBacillus species and their crude metabolites demonstrated noteworthy antioxidant and antimicrobial properties. Further investigation may be helpful in identifying possible compounds that Bacillus sp. produces.

This study aims to establish a bacterial consortium to deconstruct the coir, areca, banana, and cotton fibers recalcitrant structure. Hence, lignocellulase secreting bacterial strains were isolated from cow rumen, dung garbage, and vermicompost samples. After selecting desired strains, a mixed-batch bacterial consortium (MBBC) was made by mixing of cellulase batch (Bacillus sp. HSTU-2, Bacillus sp. HSTU-3, Citrobacter sp. HSTU-AAJ4), pectinase batch (Acinetobacter sp. HSTU-6, Bacillus sp. HSTU-7, Enterobacter sp. HSTU-AAH8), and amylase batch (Bacillus sp. HSTU-9, and Bacillus sp. HSTU-10) strains. Separately, each batch and MBBC strains were largely grown in the culture medium enriched with coir, areca, banana, and cotton fibers; as a consequence, the hardy lignocellulosic fibers were degraded. FTIR study indicated that the peak intensities for lignin was diminished, but sharpened for cellulose in the MBBC than that of the single batch pretreatment. The MBBC pretreatment could remove 64–73% lignin from banana, areca, and coir fibers, resulting in increasing cellulose amounts. The crystallinity index was observed 31.5%, 21.71%, 35%, and 29.26% for the untreated and 13.69%, 18.27%, 17.72%, and 25.20% for the MBBC pretreated cotton, areca, coir, and banana fibers, respectively. The MBBC pretreated banana, areca, coir, and cotton fibers could generate reducing sugars that scaled up to 5.3-, 3.9-, 3.68-, and 2.68-fold greater than the untreated samples. Hence, it is radically feasible to produce bioethanol precursors from the 4-days long MBBC pretreated banana, areca, and coir lignocelluloses. This research revealed the shortest lignocellulose pretreatment duration with a bacterial consortium holding minimal community members.

Alkaliphilic bacterial strain termed KSUCr9a was isolated from soil and water samples collected from various soda lakes located in northern Egypt. KSUCr9a was tolerance up to 75 mM Cr (VI), with minimum inhibition concentration (MIC) value of 80 mM, in alkaline medium (pH 10.5) containing 10% NaCl. Analysis of 16S rDNA of strain KSUCr9a identified this bacterial strain as Bacillus sp., with sequence similarity of 99%, and was referred to as Bacillus sp. strain KSUCr9a. In addition to its tolerance to Cr(VI), Bacillus sp. KSUCr9a showed high resistance to other heavy metals including Cd 2+ (50 mM), Mo 2+ (75 mM), Mn 2+ (100 mM), Cu 2+ (2 mM), Ni 2+ (100 mM), Pb 2+ (75 mM), Co 2+ (5 mM) and Zn 2+ (2 mM). Bacillus sp. KSUCr9a demonstrated good chromate bio-reduction ability, as it could rapidly reduce up to 100 μM within 24 h. In addition, at initial Cr(VI) concentration of 200 μM, complete chromate reduction was achieved within 48 h. Furthermore, at initial Cr(VI) concentration of 300, 400 and 500 μM, 92.8, 75.5 and 39.8% of chromate reduction was achieved within 72 h. Bacillus sp. KSUCr9a was able to reduce Cr(VI) in a wide range of NaCl (0 to 20%), indicating the halotolerance nature of this alkaliphilic bacterial strain. Addition of glucose as an electron donor to the culture medium led to significant increase of both growth and chromate reduction by Bacillus sp. KSUCr9a. Maximum Cr(VI) reduction was exhibited in alkaline medium (pH 9) containing 0.8% glucose at incubation temperature of 35°C and under static culture condition. Under optimum Cr (VI) bioreduction conditions, 169.2 μM of Cr(VI) was completely reduced within 24 h, indicating a good ability of Bacillus sp. KSUCr9a of Cr(VI) detoxification under alkaline condition. Furthermore, Cr(VI)-reduction by Bacillus sp. KSUCr9a was slightly induced in the presence of other heavy metals, such as Mn 2+ , Co 2+ , Mo 2+ and Cu 2+ at concentration of 50 mg/L along with Cr(VI) in the culture medium. Moreover, Bacillus sp. KSUCr9a showed the ability of repeated bioreduction of chromate without any addition of exogenous nutrients, indicating its possible application in chromate detoxification. Key words : Chromate reduction, bioremediation, heavy metals, Bacillus sp., soda lakes.