ISOLATION AND CHARACTERIZATION OF HEAVY METAL-REDUCING MICROBES FROM TANNERY EFFLUENT AND ITS ACTIVITY ON IMMOBILIZED BEADS

ISOLATION AND CHARACTERIZATION OF HEAVY METAL-REDUCING MICROBES FROM TANNERY EFFLUENT AND ITS ACTIVITY ON IMMOBILIZED BEADS

J. Raveena Jayam and C. Sivaraj *

ARMATS Biotek Training and Research Institute, Chennai, Tamil Nadu, India.

ABSTRACT: Heavy metal pollutants in tannery effluent can be removed using the bioremediation technique. This study intends to extract bacteria that may reduce heavy metals and molecularly characterize them using 16S rRNA PCR in samples taken from tannery effluent. It also studies their cell-free immobilized enzymatic activity. Thirteen bacterial isolates were identified based on morphological, cultural, and biochemical characteristics. The top four isolates, A, B, sp5, and sp3, were identified as Brevundimonas sp, Proteus mirabilis, and Alcaligenes faecalis by morphological, cultural, biochemical, MALDI-TOF Analysis, and molecular characteristics using 16S rRNA. These organisms showed good tolerance to zinc, nickel, and chromium. When heavy metals like zinc and chromium have reduction properties, by ICP-OES to assess the concentration of heavy metals. Further investigation, beads were immobilized, and its activity was analyzed.

Keywords: Heavy metal tolerance, Tannery effluent, Reduction, Bioremediation, inductively coupled plasma-optical emission spectrometry, Immobilization

INTRODUCTION: Bioremediation ¹ refers to biological systems that remove ecological pollutants from the air, gas, and industrial effluent states. The ability of microorganisms to captivate, assemble, and reduce common pollutants has enchanted contamination ¹. Bioremediation decreases the impact of products created from environmental change activities, such as urbanization and agricultural processes. Organic pollutants are generally more susceptible than heavy metals, including free radicals.

Oxidation increases the solubility level in water in vulnerability degraded by the addition of water. Biodegradation converts the elements of hydrogen and carbon to carbon dioxide and water. Bioremediation provides the best solutions for heavy metals ⁷. Heavy metal can be removed by various bioremediation techniques ¹⁶.

The process of treating animal skins and hides to generate leather is known as tanning. A tannery ⁷ is a facility that preserves skins. Sulphites, ammonium salts, and calcium salts are found in the effluents from tannery procedures and de-liming bating. These hairs are plucked and immersed in water for two days. Sulphuric acid, chrome, chlorides, sodium bicarbonate, and sulfates are found in the pickling and chrome tanning effluents. High hazardous pollutants like sulphides in an animal's skin tanning are used to identify tannery effluents. Numerous research investigations on the treatment of tannery effluent have recorded a variety of procedures, including flotation, electrochemical treatment, sedimentation, coagulation, filtration, ultra-filtration and reverse osmosis process. Because so many chemicals are employed in turning animal hides into leather, the tanning industry is the dirtiest. Chromium salts, phenolics, tannins, and organic debris are among the substances that are routinely emitted into the environment by tanneries.

Tannery wastewater ^{7, 9} is defined by the following parameters: suspended solids (SS), chromium, sulphides, biochemical oxygen demand (BOD), and chemical oxygen demand (COD) ¹². Total dissolved salts (TDS) @125°C, Carbonate as Caco3, Bicarbonate as Caco3, Residual Sodium Carbonate, Sodium Adsorption Ratio, Sulphate, Chloride, Nitrate, iron, potassium, Calcium, Magnesium, Sodium, Total dissolved salts (TDS) @125°C, Carbonate as Caco3, Bicarbonate as Caco3, Residual Sodium Carbonate, Sodium Adsorption Ratio Gelatin and glue derived from tannery leftovers. Limited fleshings, trimmings, and shavings are used to make foam-generating compounds. The fleshings can be used to make manure, animal feed, amino acids, grease, and other products.

Heavy metals contain harmful or unpleasant substances. Zinc (Zn), chromium (Cr), and nickel are examples of heavy metals (Ni). The tannery effluent ²⁰ contains 5.50 and 11.0 mg/L of chromium ³, respectively. Zinc concentrations in tannery effluent range between 6.00 and 7.00 mg/L, below WHO. Allowed limits. Numerous factors, including the exposure route, chemical species, dose, and age, gender, genetics, and nutritional status of the exposed individuals, all affect how dangerous they are. Due to their high toxicity, arsenic, cadmium, chromium, lead, and mercury are among the priority metals that concern public health. These metals were considered toxicants known to cause multiple organ injuries even at low exposure levels ⁹. The US Environmental ²³ Protection Agency and the International Agency for Research on Cancer classify them as human carcinogens (known or probable). This study looks at their manufacture, use, and prevalence in the environment, the possibility of human exposure, and the cellular mechanisms underlying their toxicity, genotoxicity, and carcinogenicity. Due to their low concentrations (ppb to 10ppm) in many environmental matrices, heavy metals were also categorized as trace elements. Bioavailability was impacted by physical elements such temperature, phase association, adsorption and sequestration. Chemical factors affect lipid solubility, complexation kinetics, octanol/water partition coefficients, and thermodynamic equilibrium. Additionally, biological aspects including species characteristics, trophic relationships, and physiological and biochemical flexibility have a big impact.

With oxidation ¹⁵ states varying from chromium (II) to chromium (VI), chromium (Cr) is an element that is naturally present in the crust of the planet ⁸. The trivalent [Cr (III)] form of chromium elements, which is stable, is found in ores like ferrochrome. The hexavalent form of [Cr (VI)] is the second stable state. Nature does not include the chromium element [Cr (0)]. There are several natural and phylogenetic sources of chromium that end up in the environment (air, water, and soil), with industry having a substantial impact ¹⁵. Among the sectors with greater chromium subscriptions are those that process metal, have tannery facilities, create chromate, weld stainless steel, produce ferrochrome, and produce chrome pigment. Metallurgical, refractory and other industrial operations, in particular, are the main sources of chromium-releasing air and wastewater.

The most prevalent chromium ¹⁴ assembly in the environment is the hexavalent [Cr (VI)]. Numerous executive and non-executive authorities have identified the hazardous adulterant hexavalent chromium [Cr (VI)] (US Department of Health and Human Services) as a human carcinogen. Depending on its level of oxidation, chromium ¹⁷ poses a variety of health concerns that can range from mild metal lethality to severe toxicity. Because only Cr (III) was present in the air, water, soil, and biological materials, it was once thought that anything containing Cr (VI) was artificial. The World Health Organization's drinking water standard for Cr (VI) is 50 g per litre, yet it has been discovered that Cr (VI) levels in surface and ground waters are greater. Metal is a crucial process in surface treatment and metal deposition for increased product life and ornamentation, despite nickel contamination in the environment. Depending on the particular needs of the objects, metals including nickel, copper, zinc, and chromium can be used for electroplating. Significant volumes of metal ions are discharged into the effluent during the washing of the electroplating tanks ⁵.

Businesses that manufacture storage batteries, zinc base casting and silver refineries, for instance, all have Ni (II) in their effluents. Higher nickel concentrations cause lung, nasal, and bone cancer. Nickel compounds enter the food chain in both humans and animals. One of the negative effects of nickel exposure is dermatitis (the Ni itch) and another is acute Ni toxicity ⁵.

Due to its resistance to corrosion from the environment, zinc is used to galvanise iron in order to prevent it from rusting. Anode materials for galvanic cells, the cyanide method for the preparation of gold and silver, the parks process for the desilverization of lead, and the manufacturing of zinc white and a variety of precious alloys like brass, German silver, and delta metal all use zinc ¹⁰.

Zinc salts are used to make ceramics, textiles, fertilizers, pigments, photographic paper, batteries, wood preservatives, catalysts, and accelerators for rubber vulcanization. It is uncommon, but excessive zinc ingestion might cause stomach distress and diarrhoea.

Using a simple and quick method, developmental conditions for getting maximal intensity in ultrasonic baths are given ¹³. Bath sonication is an indirect sonication technique that uses a water bath. Ultrasonic energy is delivered to a water bath and subsequently into a vessel or several tubes in bath sonication. This approach works best with extremely little samples ²⁴.

It is often not guaranteed that all the polymers will pass through this region when using probe sonicators for sonication and mixing because the power is concentrated into a very small volume with weak forces acting. Sound energy is applied to a liquid containing particles using an ultrasonic bath or probe. It is claimed that high-speed mixing combined with probe sonication is effective ¹⁶. The confinement of a cell or enzyme in a matrix is known as Immobilization. Natural, synthetic, and inorganic materials all benefit from immobilization procedures. Making enzymes more stable, active, and reusable can be accomplished by enzyme immobilization ^{4, 6}, which is frequently utilized. Enzymes or cells can be immobilized using various techniques, including copolymerization, trapping, adsorption, and covalent bonding. Aspergillus oryzae's aminoacylase ⁴ was the first enzyme to be immobilized using immobilization cells.

An enzyme becomes insoluble and inactive when calcium chloride is combined with a sodium alginate solution and enzyme solution mixture. Conditions like pH and temperature can now be changed more easily as a result.

Increasing industries require advanced solutions to rectify the effluent generated by the beverage and leather industries. Leather industries are one of the major wastewater generating hubs, which generate a lot of heavy metals like chromium, nickel, zinc, etc. These heavy metals are difficult to remove from the water before it lets out into the freshwater lake after treatment. Effluents generally contain heavy metals but also more bacteria that can thrive in such an environment. Bioremediation is one such solution to remove heavy metals.

For example, Bacillus spp ¹⁰ can convert hexavalent chromium (VI) into chromium (III). Our study aims to isolate heavy metals reduction and molecular characterization of the bacteria using 16S ¹⁹ rRNA PCR in samples collected from the tannery industry and to study its cell-free immobilized enzymatic activity. These microbes may produce enzymes that can reduce some of the heavy metals. These can be extracted and immobilized into sodium alginate beads, which can do an enzymatic activity, so the beads can be used at industrial and household levels to eliminate heavy metal content.

MATERIALS AND METHODS: 

Sample Processing: Effluent samples were collected from Sugan Leather Enterprises, Nagalkeni-Chrompet. The sample involved effluent and was collected in falcon tubes and preserved at approximately 4℃ to prevent contamination and allow the sample to stay longer.

Measurement of Physicochemical Parameters: Physicochemical parameters like chromium, nickel, zinc, pH@25℃, Sulphate, Chloride, Nitrate, iron, potassium, Calcium, Magnesium, Sodium, TDS@125℃, Carbonate as Caco3, Bicarbonate as Caco3, Residual Sodium Carbonate, Sodium Adsorption Ratio of the effluent was determined ¹².

Screening and Isolation of Bacteria: The nutrient agar was sterilized at 121°C for 15 min and allowed to cool at 40-45℃. The effluent was serially diluted in 9 ml of saline water, and then 1 ml of sample was added to the first test tube to have 10^-1 to 10^-9 and then 0.1ml was screened by standard spread and pour plate method and observed at 37℃ for 24 h. After 24 h of incubation, plates were observed for different morphological appearances ¹².

Multiple Metal Tolerance Capacity: Thirteen isolates (A, B, sp5, sp3, 10^-3, 10^-5, pp3, pp6, pp4C, pp4D, sp3A, sp3B, sp5A) were picked from the spread and pour plates and streaked on heavy metal containing media supplemented with 50 mg/L, 100 mg/L, 200 mg/L of Zinc, Nickel and Chromium and kept for incubation for 24 hrs for heavy metal tolerance ¹¹.

Characterization of Bacterial Isolates: The bacterial isolates were characterized based on their cultural, morphological and biochemical characteristics as described in Bergey's manual for the identification of bacteria. For the activities of gram staining, catalase, oxidase, lecithinase and lipid hydrolysis were analyzed. The four isolates (A, B, sp5, sp3) were characterized using MALDI-TOF and 16s Rrna ^{2, 11} sequencings and were analyzed with the database of NCBI (http://www.ncbi.nlm.nih.gov/BLAST/).

Determination of Heavy Metals: 3.125g of Luria Broth (LB) with 40 ppm of Zn, Cr was mixed with each 125 ml of distilled water and autoclaved at 121°C for 15 minutes before being put into a conical flask and incubated for 24 h at 37℃. A heavy metal test was performed by Inductively coupled plasma-optical emission spectrometry and compared with standard and control ²⁸.

Sonication and Protein Estimation: For sonication ²¹, heavy metal and non-heavy metal-induced samples were sonicated at 20-40 kHz for 30 minutes. The samples were centrifuged at 5000 rpm for 15 min, and the supernatant was collected and stored.

The samples were prepared in different concentrations from S1 to S6, and different volumes of BSA were added with distilled water along with 200 μl of bradford. Triplicates were kept for the samples and analyzed at 595 nm for brad ford assay for protein estimation ²⁷.

Immobilization: 1% sodium alginate and calcium chloride was prepared in each 100 ml of distilled water in two different beakers. Sodium alginate was mixed well with heavy metal-inducing and non-heavy metal-induced samples in the beaker separately. Those samples were induced in calcium chloride through the filler, and immobilization beads were formed and analyzed ²².

RESULTS:

Physicochemical Characteristics of Tannery Effluent: Standard techniques were used to measure the physicochemical characteristics. The effluent was green in color, chromium, nickel, zinc, pH@25℃, Sulphate, Chloride, Nitrate, iron, potassium, Calcium, Magnesium, Sodium, TDS@125℃, Carbonate as Caco3, Bicarbonate as Caco3, Residual Sodium Carbonate, Sodium Adsorption Ratio of the effluent was also analyzed. Zinc 0.21 mg/L, Nickel 0.24 mg/L, 2,156 mg/L, 63 mg/L, and 528 mg/L are among the heavy metals found in effluent. Carbonate as Caco3 is nil, while bicarbonate as Caco3 is 147.25 mg/L.

Other researchers have produced similar findings. This sample has an electrical conductivity of 5400 S/cm, and a total dissolved salts test of 3521 mg/L.

Other characteristics evaluated in the effluent sample included sodium, chloride, nitrate, iron, potassium, calcium, magnesium and sodium was determined to be 897.3 mg/L, 1300.51 mg/L, 3.2 mg/L, 0.24 mg/L, 2,156 mg/L, 63 mg/L, and 528 mg/L. Carbonate as Caco3 is nil, but the Bicarbonate Caco3 test results are 147.25mg/ L. Other researchers have observed similar findings in Table 1.

TABLE 1: PHYSICOCHEMICAL PARAMETERS OF EFFLUENT

Screening and Isolation of Bacteria from Tannery Effluent: More colonies were grown in the spread and pour plates at the same sample location. Totally thirteen single bacterial colonies were isolated and were able to tolerate 50 mg/L, 100 mg/L, 200 mg/L of Zn, Ni, Cr in nutrient agar after 24 hrs of incubation. This collected sample consists of heavy metal tolerance bacteria Fig. 1 and 2.

FIG. 1: SPREAD PLATE

FIG. 2: POUR PLATE

Comparative Analysis of Multiple Heavy Metal Tolerance Capacity: Total of thirteen bacterial isolates, among them four isolates (A, B, sp5, sp3) were selected for further study.

In this experiment, four isolates showed good tolerance in 50 mg/L, 100 mg/L, 200 mg/L of Zn, Ni, Cr.

Characterization and Identification: Thirteen heavy metal tolerance isolates (A, B, sp5, sp3, 10^-3, 10^-5, pp3, pp6, pp4C , pp4D, sp3A, sp3B, sp5A) were characterized based on their morphological and biochemical characteristics as described in Bergey's manual for the identification of bacteria Table 2.

TABLE 2: BIOCHEMICAL TEST

Matrix-assisted Laser Desorption/ Ionization-time of Flight (MALDI-TOF) Mass Spectro-metry (MS): Bacterial isolates A depicts Alcaligenes faecalis which has a 2.05 log score. Isolate B shows Thauera terpenica, which has a log score of 1.275; isolate sp5 shows Alcaligenes faecalis which has a log score of 1.804; isolate sp3 shows Proteus mirabilis, which has a 1.706 log score, as shown in Fig. 3, 4, 5, 6.

FIG. 3: BACTERIAL ISOLATE A - ALCALIGENES FAECALIS- 2.05 

FIG. 4: BACTERIAL ISOLATE B- THAUERA TERPENICA- 1.275

FIG. 5: BACTERIAL ISOLATE SP3-PROTEUS MIRABILIS-1.706

FIG. 6: BACTERIAL ISOLATE SP5-ALCALIGENES FAECALIS- 1.804

Molecular Characterisation: Species were identified using the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/), 100bp ladder, 16S rRNA forward and reverse primer were used to analyze the plasmid and sequencing were done. Brevundimonas sp, Proteus mirabilis, Alcaligenes faecalis were identified Fig. 7.

FIG. 7: LANE 1-100BP LADDER, LANE 2 SHOWS BREVUNDIMONAS SP, LANE 3- SHOWS PROTEUS MIRABILIS, LANE 4 AND 5 SHOWS ALCALIGENES FAECALIS

Determination of Heavy Metals: To measure heavy metals, (Cr and Zn) to determine reduction capacity, the samples were analyzed by inductively coupled plasma-optical emission spectrometry and compared with the control. All four isolates A, B, sp5, sp3 were reduced in concentration mg/L shown in Fig. 8 & 9.

FIG. 8:  CONCENTRATION OF CHROMIUM HEAVY METAL

FIG. 9: CONCENTRATION OF ZINC HEAVY METAL

Sonication and Protein Concentration: Samples prepared in triplicates in various concentrations were done by BSA, distilled water and brad ford 200µl analyzed using optical density at 595 nm. Standard Graph were plotted and concentration of heavy metal-induced and non-heavy metal-induced isolates analyzed, shown in Table 3, Fig. 10, 11.

TABLE 3: BRADFORD CONCENTRATION

FIG. 10: STANDARD GRAPH

FIG. 11: CONCENTRATION OF HEAVY METAL AND NON-HEAVY METAL INDUCED ISOLATES

Immobilization: 1 % of sodium alginate and calcium chloride was prepared in each 100 ml of distilled water and then heavy metal-induced (Zinc sp5, Chromium B) and non-heavy induced (sp5, B) were prepared and analyzed by ICP-OES. Heavy metal concentration (mg/L) in Zinc and Chromium, when compared with control, concentration has been reduced in heavy metal-induced and non-heavy metal-induced beads in effluent Fig. 12, 13.

FIG. 12: IMMOBILIZED BEADS

FIG. 13: CONCENTRATION OF HEAVY METAL-INDUCED AND NON-INDUCED ON IMMOBILIZED BEADS

DISCUSSION: Chennai, as an industrial city, is plagued by risks. Heavy metals are released by the leather and other sectors, endangering humans, wildlife, and agricultural settings. Even though large levels of heavy metals in our ecological system have a negative impact. Bioremediation is a safe, environmentally favourable process that can be stored in its original state.

The main goal is to isolate heavy metal-reducing microbes from tannery effluent and test their activity on immobilized beads, as well as to compare heavy metal remediation concentration and capacity for selective isolates so that they can be used to recover future hazards from tannery effluent containing toxic heavy metals.

Totally thirteen samples were identified A, B, pp4D, sp3B, sp5A, pp3, pp6,sp3A, pp4C, sp5, sp3, 10^-3,10^-5 based on their morphological, cultural, biochemical characterization, and heavy metal tolerance activity in all three heavy metals zinc, nickel and chromium. The best four isolates A, B, sp5, sp3 were taken compared to all three multiple metal tolerance activities in 50 mg/L. Morphological, cultural, biochemical, and molecular analyses were used to characterize these four bacterial isolates. Bergey's manual of bacteriology was used to examine biochemical characterizations. By detecting the peptidoglycan layer, which is present in thick layers of bacteria, all isolates were determined to be gram-negative bacteria. Isolate sp5, sp3, 10^-3, A, B, pp4D, sp5A, sp3 shows catalase positive may be staphylococcus sp. Some isolates that were oxidase negative may be Enterobacteriaceae. In lipid hydrolysis sp5A, sp3B, pp6, pp3, and A bacterial isolates form a zone of precipitation that hydrolyses these isolates and were able to degrade. In the lecithinase test, sp5A, sp3B, pp3, A these isolates form zones of precipitation in which lecithinase in egg yolk is able to degrade. MALDI-TOF analyses were characterized and identified and carried out by DNA sequencing in which four species were analyzed: Brevundimonas sp, Proteus mirabilis, Alcaligenes faecalis. Two isolates were found to be the same species, respectively. The isolates were measured for detection of heavy metals and analyzed through inductively coupled plasma-optical emission spectrometry Cr and Zn ; when compared with reference value all four isolates A, B, sp5, sp3, were reduced in concentration mg/L.  The Sonication process was analyzed for cell lysis so that it may be an intracellular or extracellular protein. In Bradford assay, when compared with standard graph y=0.02x+0068, R²=0.9663, the concentration, and heavy metal-induced and non-heavy metal-induced isolates were compared, i.e., Zn sp5, B, Cr B, sp5. Then for further study, immobilization was processed by forming beads through sodium alginate and calcium chloride. Here, heavy metal and non-heavy metal-induced beads were formed separately, and control was kept. When analyzed through ICP-OES, the concentration level of heavy metal in mg/L, Zinc and Chromium, compared with control concentration, has been reduced in heavy metal-induced and non-induced beads in the effluent. Here beads were analyzed for activity and longevity so they can be revived for further purposes.

CONCLUSION: One sample was taken from the tannery sector, and thirteen isolates were tested in this investigation. Four isolates were found to have multiple heavy metal tolerance capacities. Isolates were identified as Brevundimonas sp, Proteus mirabilis, and Alcaligenes faecalis based on their morphological, biochemical, and molecular characterization. All of the study's findings are consistent with the idea that these three microorganisms possess valuable traits that can be used to develop bioremediation techniques that will detoxify tannery effluent at both the industrial and household levels while removing heavy metals.

ACKNOWLEDGEMENT: I express my sincere gratitude to my project mentor, who has always provided me with moral support and valuable advice. I also want to thank SAIF at IIT Madras for their assistance.

CONFLICTS OF INTEREST: No conflict of interest in this research

REFERENCES:

1. Ayele, Abate and Yakob Godebo Godeto: “Bioremediation of Chromium by Microorganisms and Its Mechanisms Related to Functional Groups.” Journal of Chemistry and Chemical Engineering 2021 (July). https://doi.org/10.1155/2021/7694157.
2. Clarridge Jill E: 3rd. “Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases.” Clinical Microbiology Reviews Table of Contents 2004; 17(4): 840–62.
3. Cohen MD, Kargacin B, Klein CB and Costa M: “Mechanisms of Chromium Carcinogenicity and Toxicity.” Critical Reviews in Toxicology 1993; 23(3): 255–81.
4. Dai, Xiaoli, Jing Lv, Guangxu Yan, Chunmao Chen, Shaohui Guo and Pengcheng Fu: “Bioremediation of Intertidal Zones Polluted by Heavy Oil Spilling Using Immobilized Laccase-Bacteria Consortium.” Bioresource Technology 2020; 309(8): 123305.
5. Es-sahbany H, Berradi M, Nkhili S, Hsissou R, Allaoui M, Loutfi M, Bassir D, Belfaquir M and El Youbi MS: “Removal of Heavy Metals (nickel) Contained in Wastewater-Models by the Adsorption Technique on Natural Clay.” Materials Today: Proceedings 2019; 13(1): 866–75.
6. Homaei, Ahmad Abolpour, Reyhaneh Sariri, Fabio Vianello and Roberto Stevanato: “Enzyme Immobilization: An Update.” Journal of Chemical Biology 2013; 6(4): 185–205.
7. Igiri, Bernard E, Stanley IR Okoduwa, Grace O. Idoko, Ebere P. Akabuogu, Abraham O. Adeyi and Ibe K. Ejiogu: “Toxicity and Bioremediation of Heavy Metals Contaminated Ecosystem from Tannery Wastewater: A Review.” Journal of Toxicology 2018; (9): 2568038.
8. Jacobs JA and Testa SM: 2005. “Overview of Chromium (VI) in the Environment: Background and History.” Chromium (VI) Handbook.https://books.google.com/books?hl=en&lr=&id=DVdCqL9NbOcC&oi=fnd&pg=PA1&dq=Jacobs+JA+Testa+SM+Overview+of+chromium(VI)+in+the+environment+background+and+history+In+Guertin+J+Jacobs&ots=ejB-OPgp05&sig=p2a_XmBIzgFSH23fcCRtlC-4YgI.
9. Jadhav JP, Kalyani DC, Telke AA, Phugare SS and Govindwar SP: “Evaluation of the Efficacy of a Bacterial Consortium for the Removal of Color, Reduction of Heavy Metals, and Toxicity from Textile Dye Effluent.” Bioresource Technology 2010; 101(1): 165–73.
10. Liu, Jianghong, Jian Xue, Xiaohang Wei, Huimin Su and Ruidan Xu: “Optimization of Cr6+ Removal by Bacillus Subtilis Strain SZMC 6179J from Chromium-Containing Soil.” Indian Journal of Microbiology 2020; 60(4): 430–35.
11. Marzan, Lolo Wal, Mehjabeen Hossain, Sohana Akter Mina, Yasmin Akter and A. Masudul Azad Chowdhury: “Isolation and Biochemical Characterization of Heavy-Metal Resistant Bacteria from Tannery Effluent in Chittagong City, Bangladesh: Bioremediation Viewpoint.” The Egyptian Journal of Aquatic Research. https://doi.org/10.1016/j.ejar.2016.11.002.
12. Mustapha, Mohammed Umar and Normala Halimoon: 2015. “Screening and Isolation of Heavy Metal Tolerant Bacteria in Industrial Effluent.” Procedia Environmental Sciences. https://doi.org/10.1016/j.proenv.2015.10.006.
13. Nascentes, Clésia C, Mauro Korn, Clarivaldo S. Sousa, and Marco Arruda AZ: “Use of Ultrasonic Baths for Analytical Applications: A New Approach for Optimisation Conditions.” Journal of the Brazilian Chemical Society 2001; 12(1): 57–63.
14. Nur-E-Alam, Md, Md Abu Sayid Mia, Farid Ahmad and Md Mafizur Rahman: 2020. “An Overview of Chromium Removal Techniques from Tannery Effluent.” Applied Water Science. https://doi.org/10.1007/s13201-020-01286-0.
15. Patlolla, Anita K, Constance Barnes, Clement Yedjou VR, Velma and Paul B. Tchounwou: “Oxidative Stress, DNA Damage, and Antioxidant Enzyme Activity Induced by Hexavalent Chromium in Sprague-Dawley Rats.” Environmental Toxicology 2009; 24(1): 66–73.
16. Ultrasound in chemistry. Analytical applications José-Luis Capelo-Martínez (Ed.) Wiley-VCH ISBN: 978-3-527-31934-3, 171.
17. Pushkar, Bhupendra, Pooja Sevak, Sejal Parab and Nikita Nilkanth: “Chromium Pollution and Its Bioremediation Mechanisms in Bacteria: A Review.” Journal of Environmental Management 2021; 287(6): 112279.
18. Sambo, Francesco, Francesca Finotello, Enrico Lavezzo, Giacomo Baruzzo, Giulia Masi, Elektra Peta, Marco Falda, Stefano Toppo, Luisa Barzon and Barbara Di Camillo:  Optimizing PCR Primers Targeting the Bacterial 16S Ribosomal RNA Gene.” BMC Bioinformatics 2018; 19(1): 343.
19. Saranya D and Shanthakumar S: “An Integrated Approach for Tannery Effluent Treatment with Ozonation and Phycoremediation: A Feasibility Study.” Environmental Research 2020; 183(4): 109163.
20. Shah, Tayyab Raza, Hasan Koten and Hafiz Muhammad Ali: “Chapter 5 - Performance Affecting Parameters of Hybrid Nanofluids.” In Hybrid Nanofluids for Convection Heat Transfer, edited by Hafiz Muhammad Ali Academic Press 2020; 179–213.
21. Shakerian, Farid, Jing Zhao and Shao-Ping Li: “Recent Development in the Application of Immobilized Oxidative Enzymes for Bioremediation of Hazardous Micropollutants - A Review.” Chemosphere 2020; 239(1): 124716.
22. Stedeford, Todd, Q. Jay Zhao, Michael L. Dourson, Marek Banasik and Ching-Hung Hsu: “The Application of Non-Default Uncertainty Factors in the U.S. EPA’s Integrated Risk Information System (IRIS). Part I: UF(L), UF(S), and ‘Other Uncertainty Factors.” Journal of Environmental Science and Health. Part C, Environmental Carcinogenesis & Ecotoxicology Reviews 2007; 25(3): 245–79.
23. Taylor AC: “7 - Advances in Nanoparticle Reinforcement in Structural Adhesives.” In Advances in Structural Adhesive Bonding, edited by David A. Dillard Woodhead Publishing 2010; 151–82.
24. Velma, Venkatramreddy Vutukuru SS and Paul B: Tchounwou. “Ecotoxicology of Hexavalent Chromium in Freshwater Fish: A Critical Review.” Reviews on Environmental Health 2009; 24(2): 129–45.
25. Woese CR and Fox GE: “Phylogenetic Structure of the Prokaryotic Domain: The Primary Kingdoms.” Proceedings of the National Academy of Sciences of the United States of America 1977; 74(11): 5088–90.
26. Kocis and Figura: “Ultrasonic Measurements and Technologies.” (Sensor Physics and Technology Series) Springer 2012; 1996.
27. Balasubramanian S and Pugalenthi V: “Determination of total chromium in tannery waste water by inductively coupled plasma-atomic emission spectrometry, flame atomic absorption spectrometry and UV-visible spectrophotometric methods” 1999.
    

    ---

    How to cite this article:

    Jayam JR and Sivaraj C: Isolation and characterization of heavy metal reducing microbes from tannery effluent and its activity on immobilized beads. Int J Pharm Sci & Res 2023; 14(7): 3392-03. doi: 10.13040/IJPSR.0975-8232.14(7).3392-03.

    ---

    All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.