Abstract

Background: Metals are required in minor amounts in the human body to perform vital functions, but beyond that level, they become toxic and cause many harmful effects. At present, polluted water is a large source of these heavy metals inside our body. Bioremediation is one of best treatments for the removal of these heavy metals from water. Objectives: The main objective of the current study was to isolate metal-reducing bacteria from soil and wastewater samples from different industries. Furthermore, the metal reducing potential of the bacteria was also evaluated under various environmental conditions (pH, temperature, incubation time, and UV exposure).


Methodology: Different bacterial strains were isolated that were resistant to different concentrations of zinc (Zn) and chromium (Cr) from wastewater and soil samples from four different industries (Riaz textile mills, Sitara chemical industry, Mandiali paper mills and Siddique leather works). Morphological characterization was carried out with the help of Gram staining, spore staining and motility tests. Biochemical tests were performed, such as catalase, oxidase, and H2S production tests, starch hydrolysis tests and Simon citrate tests. Chromium reduction after UV exposure was calculated to check mutation effects on chromium reduction.


Results: A total of 14 bacterial strains were isolated from the soil and wastewater samples. Six strains were Zn resistant, while eight strains were chromium resistant. A total of five strains were isolated from wastewater out of fourteen strains, while the remaining nine strains were isolated from soil samples of these industries. The bacterial strains were rod-shaped cocci and coccobacillus. The growth of bacterial strains under different environmental conditions, such as temperature, pH and incubation time, was observed, and the best growth was found at 37◦C, pH 7.0 and after 48 hrs. Maximum reduction at different concentrations was observed at pH 7.0 and 37◦C and after 48 hrs.


Conclusion: In conclusion, the bacterial strains isolated from industrial wastes and soils showed significant resistivity against various concentrations of Zn and Cr and reduced it efficiently under different conditions (pH, incubation time, temperature and UV light). The use of microorganisms for bioremediation is an environmentally friendly and cost-effective approach to reduce heavy metals present in our water and soil.


INTRODUCTION

Heavy metals are defined as metals with a particular density greater than 5 g/cm3 that have a negative impact on the environment and living beings. When present in extremely low quantities, these metals are important for maintaining different biochemical and physiological processes in living organisms; nevertheless, when concentrations surpass specific thresholds, they become toxic 1, 2. Heavy metals are emitted by natural and anthropogenic activities into the environment. Most heavy metals reach the environment from mining operations3. Heavy metals also persist in the environment even when mining activities are stopped. This was due to anthropogenic activities such as different foods, cosmetics and other chemicals that we used in our routines contain heavy metals 4. Heavy metals are present in the waste of many industries, such as the tanning, textile and electroplating industries5. When added to other water resources, including drinking water, their wastewater becomes a cause of toxicity 6. Water contaminated with these heavy metals when applied to agricultural areas enters into food crops through the water uptake of plants7. Due to their nonbiodegradable nature, heavy metals enter the food chain and affect living species8. When we consume food with heavy metals, they enter the human body and affect bodily functions, causing disease depending upon the metal. The most frequent heavy metals detected in waste water are arsenic, cadmium, chromium, copper, lead, nickel, and zinc, all of which pose health and environmental hazards9.

Chromium pollution of the environment, specifically hexavalent chromium pollution, has been a major problem in recent years10. Cr(III) is oxidized to Cr(VI) in the presence of sufficient oxygen in the environment, which is incredibly poisonous and readily soluble in water11. Excess chromium in the environment is harmful to plants because it alters the biological components of the plant and enters the food chain via the ingestion of these plant materials7, 12. Reduced root development, leaf chlorosis, germination rate inhibition, and low biomass are all common symptoms of Cr phytotoxicity13, 14, 15. Chromium (VI) has corrosive properties and can induce allergic responses in the body. As a result, inhaling excessive quantities of chromium (VI) can irritate the nasal lining and lead to nose ulcers16. It can also harm sperm and the male reproductive system17, causing anemia18, irritations, and ulcers in the small intestine and stomach19. Chromium also induces allergic responses, including significant skin redness and edema20. Humans may have serious cardiovascular, pulmonary, hematological, gastrointestinal, renal, hepatic, and neurological consequences, as well as mortality, when exposed to excessively high concentrations of chromium(VI) compounds16, 21, 22.

Several technologies have been developed for the treatment of water containing heavy metals and other waste. Different synthetic compounds are used to treat such waste types, but they are too expensive. One and only effective and cost-effective method is the biodegradation of heavy metals from bacteria. The main objective of the current study was to isolate these bacteria from soil and wastewater samples from different industries, including the leather industry, paper mill, chemical industry and textile industry, which have the potential to reduce and degrade chromium (Cr) at different temperatures, pH values and incubation times. For bacterial characterization, different morphological, biochemical and physiological tests were also performed. The effect of DNA mutation on chromium reduction was also observed after exposure to UV.

METHODOLOGY

Sample collection

Samples were collected from different industries, including Siddique leather works, Mandiali paper mills, Sitara chemical industry and Riaz textile mills. Waste water and soil samples of these four industries were collected by using a bottle following safety precautions. These samples were stored in properly labeled bottles. These samples were stored under proper conditions before laboratory experiments to avoid reactions (Table 1).

Table 1.

Soil and wastewater samples from different industries

Sr.no Samples Industries
1 Sample no 1 (L1) Siddique leather works
2 Sample no 2 (L2) Mandiali paper mills
3 Sample no 3 (L3) Sitara chemical industries
4 Sample no 4 (L4) Riaz textile mills

Table 2.

Zinc and Chromium resistant bacterial strains isolated from different industrial samples

Sr.no Strains Samples
1 S 1 ZnL 1 Soil sample of Siddique leather works
2 S 2 ZnL 1 Soil sample of Siddique leather works
3 S 3 ZnL 1 Soil sample of Siddique leather works
4 S 4 ZnL 1 Soil sample of Siddique leather works
5 S 5 ZnL 3 Soil sample of Sitara chemical industries
6 W 1 ZnL 1 Water sample of Siddique leather works
7 S 2 CrL 2 (A) Soil sample of Mandiali paper mills
8 S 2 CrL 2 (B) Soil sample of Mandiali paper mills
9 W 1 CrL 3 (A) Water sample of sitara chemical industries
10 W 1 CrL 3 (B) Water sample of sitara chemical industries
11 S 4 CrL 4 Soil sample of Riaz textile mills
12 W 4 CrL 4 Water sample of Riaz textile mills
13 W 3 CrL 2 Water sample of Mandiali paper mills
14 S 5 CrL 1 Soil sample of Siddique textile mills

Table 3.

Colony morphology of zinc (Zn)- and chromium (Cr)-resistant strains

Sr.no Strains Colony morphology
Size Form Pigment Margin Elevation Texture Opacity
1 S 1 ZnL 1 Large Circular Cream Entire Convex Mucoid Non- opaque
2 S 2 ZnL 1 Medium Irregular Off white Entire Umbonate Shiny Opaque
3 S 3 ZnL 1 Medium Circular Off white Entire Flat Shiny Non- opaque
4 S 4 ZnL 1 Medium Circular Yellow Entire Flat Shiny Non- opaque
5 S 5 ZnL 3 Large Circular Cream Entire Raised Mucoid Non- opaque
6 W 1 ZnL 1 Large Circular Off white Entire Flat Shiny Non- opaque
7 S 2 CrL 2 (A) Medium Circular Cream Entire Raised Mucoid Non- opaque
8 S 2 CrL 2 (B) Small Irregular White Lobate Umbonate Rough Non- opaque
9 W 1 CrL 3 (A) Medium Circular White Entire Flat Mucoid Non- opaque
10 W 1 CrL 3 (B) Medium Circular Off white Entire Flat Mucoid Non- opaque
11 S 4 CrL 4 Small Circular Cream Entire Convex Mucoid Non- opaque
12 W 4 CrL 4 Small Circular Yellow Entire Umbonate Shiny Opaque
13 W 3 CrL 2 Medium Circular Off white Entire Flat Mucoid Non- opaque
14 S 5 CrL 1 Large Rhizoid White Filifom crateriform Rough Non- opaque

Table 4.

Cell morphology of zinc (Zn)- and chromium (Cr)-resistant strains

Sr.no Strains Cell shape Gram Staining Spore formation Motility
1 S 1 ZnL 1 Coccus + + -
2 S 2 ZnL 1 Rod + + -
3 S 3 ZnL 1 Rod - + -
4 S 4 ZnL 1 Coccus + + -
5 S 5 ZnL 3 Coccobacilli + + -
6 W 1 ZnL 1 Rod + + -
7 S 2 CrL 2 (A) Rod + + -
8 S 2 CrL 2 (B) Coccus + + -
9 W 1 CrL 3 (A) Coccus + + -
10 W 1 CrL 3 (B) Coccus + + -
11 S 4 CrL 4 Coccobacilli + + -
12 W 4 CrL 4 Rod + + -
13 W 3 CrL 2 Rod + + -
14 S 5 CrL 1 Rod - + -

Table 5.

Biochemical characterization of isolated bacterial strains

Sr.no Strains Catalase Oxidase Indole test H2S production test Starch Hydrolysis test
1 S 1 ZnL 1 + + - - -
2 S 2 ZnL 1 + + - - -
3 S 3 ZnL 1 + + - - -
4 S 4 ZnL 1 + + - - +
5 S 5 ZnL3 + + - - +
6 W 1 ZnL 1 + + - - +
7 S 2 CrL 2 (A) + + - - -
8 S 2 CrL 2 (B) + + - - +
9 W 1 CrL 3 (A) + + - - +
10 W 1 CrL 3 (B) + + - - -
11 S 4 CrL 4 + + - - +
12 W 4 CrL 4 + + - - +
13 W 3 CrL 2 + + - - -
14 S 5 CrL 1 + + - - -

Table 6.

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 100 µl concentration

Sr no Strains Cross Metal resistance at 100µl concentration
Zinc (Zn) Chromium (Cr) Cobalt (Co) Manganese (Mn) Nickle (Ni) Silicon (Si) Selenium (Se)
1 S 1 ZnL 1 o o o o o × o
2 S 2 ZnL 1 o o o o o × o
3 S 3 ZnL 1 o × o o o × o
4 S 4 ZnL 1 o o o o o o o
5 S 5 ZnL 3 o o o o o × o
6 W 1 ZnL 1 o o o o o o o
7 S 2 CrL 2 (A) o o o o o × o
8 S 2 CrL 2 (B) × o o o o o o
9 W 1 CrL 3 (A) o o × o o × o
10 W 1 CrL 3 (B) o o × o o o o
11 S 4 CrL 4 o o o o o × o
12 W 4 CrL 4 o o o o o × o
13 W 3 CrL 2 o o o o o × o
14 S 5 CrL 1 o o × o o o o

Table 7.

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 150 µl

Sr no Strains Cross Metal resistance at 150µl concentration
Zinc (Zn) Chromium (Cr) Cobalt (Co) Manganese (Mn) Nickle (Ni) Silicon (Si) Selenium (Se)
1 S 1 ZnL 1 o o o o o × o
2 S 2 ZnL 1 o o o o o × o
3 S 3 ZnL 1 o × o o o × o
4 S 4 ZnL 1 o o o o o o o
5 S 5 ZnL 3 o o o o o × o
6 W 1 ZnL 1 o × o o o o o
7 S 2 CrL 2 (A) o o o o o × o
8 S 2 CrL 2 (B) × o o o o × o
9 W 1 CrL 3 (A) o o × o o × o
10 W 1 CrL 3 (B) o o × o o × o
11 S 4 CrL 4 o o × o o × o
12 W 4 CrL 4 o o o o o × o
13 W 3 CrL 2 o o o o o × o
14 S 5 CrL 1 × o × o o o o

Table 8.

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 200 µl

Sr.n o Strains Cross Metal resistance at 200µl concentration
Zinc (Zn) Chromium (Cr) Cobalt (Co) Manganese (Mn) Nickle (Ni) Silicon (Si) Selenium(Se)
1 S1ZnL1 o o o o o × o
2 S2ZnL1 o o o o o × o
3 S3ZnL1 o × o o × × o
4 S4ZnL1 o o o o o o o
5 S5ZnL3 o o × o o × o
6 W1ZnL1 o × o o o o o
7 S2CrL2 (A) o o o o o × o
8 S2CrL2 (B) × o o o o × o
9 W1CrL3 (A) o o × o o × o
10 W1CrL3 (B) × o × o × × o
11 S4CrL4 × o × o o × o
12 W4CrL4 o o o o o × o
13 W3CrL2 × o o o o × o
14 S5CrL1 × o × o × o o

Table 9.

Heavy metal resistance of zinc (Zn)- and chromium (Cr)-resistant strains at 300 µl

Sr no Strains Cross Metal resistance at 300µl concentration
Zinc (Zn) Chromium (Cr) Cobalt (Co) Manganese (Mn) Nickle (Ni) Silicon (Si) Selenium (Se)
1 S 1 ZnL 1 o o o o × × o
2 S 2 ZnL 1 o o o o × × o
3 S 3 ZnL 1 o × o o × × o
4 S 4 ZnL 1 o o o o × o o
5 S 5 ZnL 3 o o × o × × o
6 W 1 ZnL 1 o × o × × o o
7 S 2 CrL 2 (A) o o o o × × o
8 S 2 CrL 2 (B) × o o o × × o
9 W 1 CrL 3 (A) × o × o × × o
10 W 1 CrL 3 (B) × o × o × × o
11 S 4 CrL 4 × o × o o × o
12 W 4 CrL 4 o o o o × × o
13 W 3 CrL 2 × o o o × × o
14 S 5 CrL 1 × o × o × o o

Table 10.

Growth of zinc (Zn)- and chromium (Cr)-resistant strains after different time intervals

Sr. no Strains Time (hours)
2 4 6 18 24 48
1 S 1 ZnL 1 0.041 0.472 0.561 1.89 1.989 2.000
2 S 2 ZnL 1 0.058 0.530 0.676 1.213 1.24 0.949
3 S 3 ZnL 1 0.031 0.452 0.613 0.921 1.924 1.543
4 S 4 ZnL 1 0.059 0.563 0.721 0.986 1.014 1.986
5 S 5 ZnL 3 0.050 0.439 0.689 0.890 0.909 1.893
6 W 1 ZnL 1 0.030 0.621 0.798 0.940 0.953 1.923
7 S 2 CrL 2 (A) 0.073 0.534 0.890 1.231 1.240 2.100
8 S 2 CrL 2 (B) 0.045 0.567 0.888 0.987 0.990 1.712
9 W 1 CrL 3 (A) 0.035 0.478 0.650 0.989 1.001 1.754
10 W 1 CrL 3 (B) 0.023 0.211 0.567 0.811 0.813 1.432
11 S 4 CrL 4 0.041 0.314 0.659 0.989 0.990 1.976
12 W 4 CrL 4 0.121 0.362 0.789 0.912 0.930 2.213
13 W 3 CrL 2 0.090 0.450 0.889 0.995 0.100 2.221
14 S 5 CrL 1 0.061 0.313 0.540 0.777 0.780 1.653

Table 11.

Effect of pH on the growth of zinc (Zn)- and chromium (Cr)-resistant strains

Sr. no Strains Optical Density at Different Ph (O.D)
3 5 9
1 S 1 ZnL 1 0.412 1.226 0.728
2 S 2 ZnL 1 0.451 1.162 0.577
3 S 3 ZnL 1 0.460 0.944 0.633
4 S 4 ZnL 1 0.409 1.200 0.409
5 S 5 ZnL 3 0.375 1.052 0.721
6 W 1 ZnL 1 0.286 0.796 1.100
7 S 2 CrL