Microbes-Heavy Metal Reduction Bacillus cereus B66- Deliming Effluent

Abstract

The present study aims to isolate, and identify bacteria that could reduce heavy metals (chromium, nickel cadmium copper, lead) from tannery effluent. Screening of isolates on various heavy metals chromium, cadmium, nickel, lead and copper on MIC at 10,000 ppm were determined. The reduction potentiality a concentration of 100 ppm was assessed using Atomic Absorption Spectrophotometer by various pH (5,7,9), Temperature (37℃,45℃, 55℃) incubation time(24,48,72hrs) were optimized. Strong resistance isolate was identified by 16S rRNA sequencing as Bacillus cereus B66. The degrading potentiality was assessed using Atomic Absorption Spectrophotometer. Bacillus cereus B66 showed maximum degradation at 37℃ pH 9 Cr 90%, Pb 92%, Ni 91%, Cu 92% and Cd 85% 48 hrs incubation at 45℃, pH 9 Cr 92%, Pb 94%, Ni 91%, Cu 93%, Cd 81%. But at 55℃ pH 9 Cr 93%, Pb 95%, Ni 94%, Cu 95%, Cd 93% for 48 hrs of incubation. The most significant factors (p < 0.0001) were incubation time, pH and temperature.

Specification

Description:FIELD OF INVENTION
The present invention generally relates to industrial waste management. More specifically the invention describes the application of a specific bacterial strain in addressing heavy metal pollution through biological means.
BACKGROUND OF THE INVENTION
Heavy metals are metallic elements that have a greater density than water. Heavy metals also include metalloids, such as arsenic, which can be dangerous at low levels of exposure, based on the idea that heaviness and toxicity are connected. Because of their physical and chemical properties, arsenic (As), cobalt (Co), iron (Fe), and manganese (Mn) are less common heavy metals (Mn). In recent years, there has been a growing worry about the environmental damage caused by these metals.
Furthermore, human exposure has increased considerably as a result of their widespread usage in a variety of industrial, agricultural, residential, and technical applications. Mining, foundries, and smelters, as well as other metal-based industrial operations, are major sources of environmental pollution.
Heavy metal poisoning (toxicity) is caused by exposure to heavy metals such as lead, mercury, and arsenic. Heavy metals bond to sections of your cells, preventing your organs from functioning normally. Heavy metal poisoning symptoms can be life-threatening and irreversible.
Lead poisoning occurs when lead accumulates in the body, often over months or years. Even little levels of lead can have major health consequences. Children under the age of six are particularly sensitive to lead poisoning, which can have serious consequences for their mental and physical development. Lead poisoning can be lethal in excessive doses.
Cadmium is used for a variety of applications, including electroplating, storage batteries, vapor lamps, and some solders. Overexposure might result in fatigue, headaches, nausea, vomiting, stomach cramps, diarrhea, and fever. Furthermore, increasing loss of lung function, abnormal fluid buildup within the lungs, and shortness of breath may occur.
Chromium is used in the making of automobiles, glass, ceramics, and linoleum. Overexposure to chromium can lead to lung and respiratory tract cancer, as well as renal problems. Furthermore, chromium overexposure can result in gastrointestinal symptoms such as diarrhea and vomiting, which is commonly accompanied by blood. Mercury can harm the lungs, kidneys, brain, and skin.
Traditional methods for treating heavy metal contamination include:
Chemical precipitation in water and wastewater treatment is the transformation of elements dissolved in water into solid particles that can be removed. However, this approach is pricey and produces dangerous sludge.
Resins have a finite exchange capacity. Resins can be used individually or in combination to remove ionic pollutants from water.
Ion exchange can also be employed to remove arsenic, as well as other metalloids and metals. Other potential alternatives, such as membrane separation, may be more efficient, but may also cost more.
A scientific literature titled "Heavy Metal Tolerance and Removal Efficiencies by Soil Bacterial Strains: Effects of Carbon and Nitrogen Sources", [M Y Asunmo et al., Nov 2023] the aim of this study was to evaluate the effects of carbon and nitrogen sources on tolerance to lead, nickel and cadmium by soil bacterial strains. The effects of carbon, nitrogen sources and carbon-nitrogen ratio on the bacteria strains were also explored.
Another scientific literature titled "Degradation of Hydrocarbons and Heavy Metal Reduction by Marine Bacteria in Highly Contaminated Sediments", [F Dell'Anno et al.,2020] in this study, five bacterial strains, Halomonas sp. SZN1, Alcanivorax sp. SZN2,Pseudo alteromonas sp. SZN3, Epibacterium sp. SZN4, and Virgibacillus sp. SZN7, were isolated from polluted sediments from an abandoned industrial site, and tested for their bioremediation efficiency on sediment samples collected from the same site. These bacteria were added as consortia or as individual cultures into polluted sediments to assess biodegradation efficiency of polycyclic aromatic hydrocarbons and heavy metal immobilisation capacity.
Therefore, there exists a need to develop an advanced method that should overcome the shortcomings of the currently existing methods
SUMMARY OF THE INVENTION
The following summary is provided to facilitate a clear understanding of the new features in the disclosed embodiment and it is not intended to be a full, detailed description. A detailed description of all the aspects of the disclosed invention can be understood by reviewing the full specification, the drawing and the claims and the abstract.
The objective of the present invention is to isolate heavy metal tolerant bacteria from tannery effluent and its characterization.
The aforementioned aspects along with the objectives and the advantages can be achieved as described herein.
Another objective of the present invention is to analyse Heavy metal reduction efficiency of isolated bacterial species.
According to the embodiment of the present invention, Bacillus cereus B66 have substantial reduction potential and it is optimum at various temp and pH without affecting its physical and chemical conditions
In an aspect of the present invention, bioremediation is a sustainable and economical substitute for traditional waste management because microbes can effectively break down and convert such contaminants into less toxic and simpler compounds.
Further in accordance with the present invention, microbes can be treated to reduce or remove heavy toxins and clean up the pollutants in an environment.
BRIEF DESCRIPTION OF FIGURES
Other features and advantages of the present invention will become apparent from the detailed description of the invention which follows, when considered in light of the accompanying drawings in which:
Fig. 1 illustrates the process flow diagram to isolate heavy metal reducing bacteria from tannery effluent and its characterization.
Fig. 2 illustrates the process flow diagram to analyse heavy metal reduction efficiency.
Fig. 3(a) illustrates the screening of D1 isolate and control plate on Nickel resistant at 10,000 ppm.
Fig. 3(b) illustrates the screening of D1 isolate and control plate on Chromium resistant at 10,000 ppm.
Fig. 3(c) illustrates the screening of D1 isolate and control plate on Copper resistant at 10,000 ppm.
Fig. 3(d) illustrates the screening of D1 isolate and control plate on Cadmium resistant at 10,000 ppm.
Fig. 3(e) illustrates the screening of D1 isolate and control plates on lead resistant at 10,000 ppm.
Fig. 4 illustrates the gram staining (a) D1- Gram Positive, (b) Catalase test.
Fig. 5 illustrates the phylogenetic tree of Bacillus cereus and other strains on 16Sr RNA sequences.
Fig. 6 illustrates the growth optimization (a) Control (b) Temp 37 oC (c) Temp 45 oC (d) Temp 55 oC
Fig. 7(a) illustrates the growth curve of Bacillus cereus B66 at pH 5 and temp 37 oC
Fig. 7(b) illustrates the growth curve of Bacillus cereus B66 at pH 7 and temp 37 oC
Fig. 7(c) illustrates the growth curve of Bacillus cereus B66 at pH 9 and temp 37 oC
Fig. 7(d) illustrates the growth curve of Bacillus cereus B66 at pH 5 and temp 45 oC
Fig. 7(e) illustrates the growth curve of Bacillus cereus B66 at pH 7 and temp 45 oC
Fig. 7(f) illustrates the growth curve of Bacillus cereus B66 at pH 9 and temp 45 oC
Fig. 7(g) illustrates the growth curve of Bacillus cereus B66 at pH 5 and temp 55 oC
Fig. 7(h) illustrates the growth curve of Bacillus cereus B66 at pH 7 and temp 55 oC
Fig. 7(i) illustrates the growth curve of Bacillus cereus B66 at pH 9 and temp 55 oC
Fig. 8(a) illustrates the comparison of Incubation time a*** - control vs 24 hrs, b***- control vs 48 hrs, c***- control vs 72 h, ***p < 0.001, ****p < 0.0001 and ns - non-significant.
Fig. 8(b) illustrates the comparison of pH 5, pH 7, pH 9 at 37°C for 48 hours a ****- control vs pH 5, b****- control vs pH 7, c****- control vs pH 9, ****p < 0.0001, ***p < 0.001 and ns - non-significant.
Fig. 8(c) illustrates the omparison of pH 5, pH 7, pH 9 45 °C for 48 hours a****- control vs pH 5, b**** - control vs pH 7, c**** - control vs pH 9, ***p < 0.001, ****p < 0.0001 and ns - non-significant.
Fig. 8(d) illustrates the comparison of pH 5, pH 7, pH 9 (incubation time of 48 hours) a**** - control vs pH 5, b**** - control vs pH 7, c**** - control vs pH 9, ****p < 0.0001, ***p < 0.001 and ns - non significant.
DETAILED DESCRIPTION
The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.
The embodiments will be described in detail with corresponding marked references to the drawings, in which the illustrative components of the invention are outlined. The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are construed such that it provides a complete and a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.
It must also be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred, systems and methods are now described.
The objective of the present invention is to isolate heavy metal tolerant bacteria from tannery effluent and its characterization. Another objective of the present invention is to analyse Heavy metal reduction efficiency of isolated bacterial species.
Heavy metal salts of Chromium, Copper, Lead, Nickel, Cadmium (CrO3, Cu(NO3)2, Pb (NO3)2, Nicl2, Cd(NO3)2) were dissolved in distilled water and its solutions were sterilized using filter membrane with a pore size of 0.22µm. Heavy metals were prepared for stock solutions and carried out for further experiments.
Sample collection and physiochemical properties
For bioremediation studies, deliming effluent samples were collected from a tannery sector. Samples were collected in sterilized bottles and was immediately transported to the laboratory and maintained at 4℃ for further analysis. Physicochemical parameters including BOD, COD, pH, electrical conductivity, total dissolved solids, calcium, magnesium, sodium, manganese and heavy metals was also determined.
Isolation of Bacteria
The nutrient agar was prepared and sterilized at 121°C for 15 mins. Serial dilution was separately spread for spread and pour plates and incubated at 37°C for 24 hrs. After incubation, the plates were observed for predominant colonies.


Assessing Minimum Inhibitory Concentration (MIC)
For selective screening, isolates were streaked on Nutrient agar plates supplemented with various metals Cr, Cu, Cd, Ni, Pb with increasing concentrations ranging from 50 ppm to 10,000 ppm. The isolates were incubated at 37°C for 72hrs (3 days). After incubation, heavy metal resistance capacity of each isolate was assessed.
Biochemical and Molecular characterization of bacterial isolate
The bacterial strains were characterized based on their cultural, morphological and biochemical characteristics for identification of bacteria. Activities of oxidase, catalase isolates were biochemically characterized. A pure bacterial culture was used to extract the genomic DNA of the isolate using DNA extraction kit.
The kit manual's recommendations for the extraction process were followed, and the sample was frozen at -4℃ until a PCR reaction was conducted. The amplification process involved 35 cycles: 5 minutes of initial denaturation at 95°C,1 minute of denaturation at 95°C, 1 minute of annealing, 2 minutes of primer extension at 72°C, and 10 minutes of final extension at 72°C.
The size of the DNA was ascertained by running the PCR result on a 1% agarose gel that was pre-stained with a side marker and ethidium bromide. After dipping a 14-well comb into the gel, it was given about 30 minutes to settle. After the gel had solidified, the comb was taken out and the gel tray was put inside the buffer tank. The gel was able to descend to a depth of roughly 2 to 5 mm by adding 0.5 × TBE to the tank. After purification, the amplified 16S rRNA material was sent for sequencing
Phylogenetic Analysis
The sequences were identified using BLAST tool on NCBI database. Based on maximum identity score, first ten sequences were selected and aligned using multiple sequence alignment software program Clustal W. Distance matrix and phylogenetic tree was constructed using MEGA 10 software. The Accession numbers were requested from NCBI by sending the sequences in GenBank.
Experimental set up for bacterial Growth with Heavy Metals
The bacterial growth in the presence of various heavy metals like Chromium, Copper, Cadmium, Nickel, Lead were assessed in laboratory. Briefly, conical flask with only NB media and heavy metal was considered as control, whereas experimental conical flask was inoculated with isolated bacteria along with media and heavy metal with 100 ppm concentration. This was repeated for other heavy metals with same conditions. Growth pattern of the bacterial isolates was recorded under various physical parameters like pH and temperatures as follows.
In brief, bacterial culture while reaching 0.6 OD @600nm, 100 ppm of sterilized heavy metal from stock were added. The isolates were grown in rotatory shaker at 150 rpm with various pH 5, 7, 9 at various temperatures 37℃, 45℃, 55℃ for 24 hrs. The optical density was measured at 600 nm using UV spectrophotometer for 24 hrs. Experiments were performed in triplicates.
Bioreduction Assay
Bacterial isolate was cultured into flask in NB broth medium in a rotatory shaker at 150 rpm. After optical density reached at 0.6, sterilized heavy metals (Cr, Cu, Cd, Ni, Pb)100 ppm was added to each conical flask and incubated at 37℃,45℃, 55℃ for 24,48,72 hrs. After incubation, culture was centrifuged at 12000 rpm for 12 mins.
The supernatants were separated and mixed to the double volume of concentrated HNO3.Mixtures were heated to 100℃ on hotplate stirrer for acid digestion till volume decreases to initial supernatant volume. Through Whatman filter paper extract was filtered and collected into flask and then diluted. This extract was analyzed for Atomic Absorption Spectrometry and results were compared to control for reduction capacity %. This experiment was performed for all parameters.
Statistical Analysis
The statistical analysis was performed based on the degradation percentage of various temp, pH, incubation period of isolate. The analysis of all the data was conducted by using Microsoft Excel and GraphPad Prism. Between-group comparisons Two-way ANOVA method were used and combined with Tukey's Test analysis. The data showed significance at P < 0.0001.
Physiochemical parameters, Screening of Spread and pour plates, Minimum Inhibitory Concentration (heavy metals concentration), Multiple resistance capacity, Biochemical characterization, DNA isolation, PCR Technique, construct phylogenetic tree, Growth optimization set up for bacterial Growth with Heavy Metals various pH, temperature Bio-reduction Assay- pH, Temperature and incubation time. The result is Colony forming unit: 2218CFU/ml.
Deliming effluent
Analyzation of Physiochemical Parameters
Parameters Unit Results Test Method
Color -
pH@25℃ - 6.71 IS 3025 (Part 11): 1983/ APHA 23 rd Edition; 4500-H+B: 2017
Electrical Conductivity@25℃ µs/cm 6800 IS 3025 (Part 14): 1984/ APHA 23 rd Edition; 2510 B: 2017
Potassium mg/L 69 APHA 23 rd Edition; 3111 B : 2017
Calcium mg/L 994 IS 3025 (Part 40/Sec-6): 1991/ APHA 23 rd Edition, 3111 B: 2017
Magnesium mg/L 184 IS 3025 (Part 46/Sec-6): 1994/ APHA 23 rd Edition, 3111 B: 2017
Sodium mg/L 401 APHA 23 rd Edition; 3111 B: 2017
Total Dissolved Salts mg/L 4352 IS 3025 (Part-16): 1984 (By Calculation)
Manganese mg/L <DL (0.01) APHA 23 rd Edition; 3111 B: 2017
Biochemical Oxygen Demand (BOD) 3 days @27 ℃ mg/L 6830 5210-B APHA 23rd Edition. 2017
Chemical Oxygen Method (COD) mg/L 40995 5220 -B APHA 23rd Edition. 2017
Analyzation of Heavy Metals
Iron mg/L 1.95 APHA 23 rd Edition; 3111 B : 2017
Zinc mg/L 0.39
Copper mg/L 0.12
Lead mg/L <DL (0.1)
Cadmium mg/L <DL (0.1)
Chromium mg/L <DL (0.1)
Nickel mg/L <DL (1.0)

Physiochemical characteristics of Deliming effluent
Reduction Assay
Effect of Incubation time
Bacillus cereus B66
Hrs Pb 100 ppm Cu 100 ppm Cd 100 ppm
24 59.00% 55.00% 43.00%
48 76% 79% 60%
72 95% 93% 83%

Values are expressed in mean ± SD (n = 3), statistically significant test for comparison was done by TWO WAY ANOVA followed by Turkey Test. Comparison of Incubation time a*** - control vs 24 hrs, b***- control vs 48 hrs, c***- control vs 72 h, ***p < 0.001, ****p < 0.0001 and ns - non-significant.



Effect of temperature and pH
37℃
pH Cr Pb Ni Cu Cd
5 54.00% 64% 69% 71% 47%
7 60% 76% 72% 79% 60%
9 90% 92% 91.00% 92% 85%

45℃
pH Cr Pb Ni Cu Cd
5 81.12% 85% 82% 89% 72%
7 90.30% 90% 90.00% 91% 79%
9 92% 94% 91.00% 93% 81%

55℃
pH Cr Pb Ni Cu Cd
5 89.00% 90% 88% 87% 70%
7 91.30% 93% 91.00% 93% 77%
9 93% 95% 94.00% 95% 83%

Future Enhancements or Advancements
Microbes can be used in Agricultural and tannery Industries for bioremediation, phytoremediation, bioventing, bio-stimulation, landfarming, bio attenuation. Other remediations are also possible thermal desorption, bio leaching and soil washing.
Microbe can be genetically developed or enhanced or mutated genes can be obtained for genetics purposes. To enhance microbe can be infused with nanoparticle for nanotechnology. This productive nature of isolate in the case of biodegradation could efficiently be increased by altering their metabolism when grown in different carbon sources. When using biological products or living sources, bioremediation techniques have a negligible environmental impact.
The best-preferred method for bioremediation is created by the above kind's economical and ecologically beneficial qualities. Additionally, multiple bacterial strains can be co-cultured to achieve effective results in biodegradation under challenging environmental conditions, and competent bacterial strains can be genetically improved. Current invention can be treated to clean up the contaminated environment.
Application Areas
 Treating wastewater
 Environmental management
 Agricultural industries
 Leather Industries
 Mining Industries
It is emphatise that the Abstract of the Disclosure is provided to allow a reader to quickly as certain the nature of the technical Disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in a single embodiment for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the Following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separate embodiment. In the appended claims, the terms "including" and "in which" are used as the plain-English equivalents of the respective terms "comprising" and "wherein," respectively. Moreover, the terms "first", "second," "third," and so forth, are used merely as labels, and are not intended to impose numerical requirements on their objects.
What has been described above includes examples of the disclosed architecture. It is, of course, not possible to describe every conceivable combination of components and/or methodologies, but one of ordinary skill in the art may recognize that many further combinations and permutations are possible. Accordingly, the novel architecture is intended to embrace all such alterations, modifications and variations that fall within the spirit and scope of the appended claims.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the illustrative examples, make and utilize the present invention and practice the claimed methods. It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It will be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention.
, Claims:We Claim,
1. A method for isolating heavy metal-tolerant bacteria from tannery effluent characterized by the steps of:
a) Dissolving heavy metal salts, including Chromium (CrO₃), Copper (Cu(NO₃)₂), Lead (Pb(NO₃)₂), Nickel (NiCl₂), and Cadmium (Cd(NO₃)₂), in distilled water and sterilizing the solutions using a filter membrane with a pore size of 0.22 µm;
b) Preparing nutrient agar and sterilizing it at 121°C for 15 minutes;
c) Performing serial dilution of tannery effluent and spreading the dilutions on nutrient agar plates for spread and pour plate methods;
d) Incubating the plates at 37°C for 24 hours and observing the plates for the presence of predominant bacterial colonies.
2. The method of claim 1, wherein the isolated bacterial colonies are tested for heavy metal tolerance by inoculating them into nutrient broth (NB) media containing 100 ppm of the heavy metal salts dissolved in sterilized water, with a control flask containing only the NB media and heavy metal but no bacterial inoculation.
3. The method of claim 1, wherein the isolated bacteria are further grown in nutrient broth with 100 ppm concentration of the heavy metals under varying physical parameters, including:
a) pH values of 5, 7, and 9;
b)Temperatures of 37°C, 45°C, and 55°C;
c) A shaking speed of 150 rpm in a rotatory shaker for 24 hours.
4. The method of claim 1, wherein the bacterial growth pattern is measured by monitoring the optical density (OD) at 600 nm using a UV spectrophotometer after the cultures reach 0.6 OD at 600 nm, with heavy metal solutions added at 100 ppm concentration.
5. The method of claim 1, wherein the experiment is performed in triplicate to ensure the accuracy of results for the heavy metal tolerance and growth conditions of the bacterial isolates.

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