WikiJournal Preprints/Screening of potential microorganisms from pharmaceutical effluence capable of degrading environmental pollutants

Introduction
Pharma effluent refers to the wastewater that is generated from pharmaceutical manufacturing processes, research laboratories, and other related activities in the pharmaceutical industry. This type of effluent can contain various types of pollutants, including organic and inorganic compounds, heavy metals, pharmaceutical residues, and other substances.

The composition of pharma effluent can vary widely depending on the specific processes and products involved. Some common contaminants found in pharma effluent include solvents, detergents, acids, alkalis, antibiotics, hormones, and other chemicals used in drug production. Due to the potential environmental and health impacts of pharma effluent, regulatory bodies around the world have established guidelines and standards for its treatment and disposal. Effective treatment of pharma effluent is essential to prevent the release of harmful pollutants into the environment and to protect public health.

Pharma effluent refers to the wastewater that is generated from pharmaceutical manufacturing processes, research laboratories, and other related activities in the pharmaceutical industry. This type of effluent can contain various types of pollutants, including organic and inorganic compounds, heavy metals, pharmaceutical residues, and other substances.

The composition of pharma effluent can vary widely depending on the specific processes and products involved. Some common contaminants found in pharma effluent include solvents, detergents, acids, alkalis, antibiotics, hormones, and other chemicals used in drug production. Due to the potential environmental and health impacts of pharma effluent, regulatory bodies around the world have established guidelines and standards for its treatment and disposal. Effective treatment of pharma effluent is essential to prevent the release of harmful pollutants into the environment and to protect public health.

Treatment of pharma effluent is necessary to remove or reduce the concentration of harmful pollutants and contaminants before it is discharged into the environment. The treatment process may vary depending on the specific characteristics of the effluent, but generally, it involves the following steps:

The treatment process for pharma effluent can be complex and may require specialized equipment and expertise. It is important to comply with local regulations and standards to ensure the effective treatment and safe disposal of pharma effluent.
 * 1) Preliminary Treatment: The first step in the treatment process is the removal of large particles, such as grit, sand, and other debris, using physical processes such as screening or sedimentation.
 * 2) Primary Treatment: This step involves the removal of suspended solids and organic matter using physical and chemical processes such as coagulation, flocculation, and sedimentation.
 * 3) Secondary Treatment: The effluent is then subjected to biological treatment to remove dissolved organic matter using activated sludge or other biological treatment processes.
 * 4) Tertiary Treatment: In some cases, additional treatment may be required to remove specific contaminants such as heavy metals, pharmaceutical residues, or other persistent pollutants. This may involve chemical, physical, or biological processes such as reverse osmosis, adsorption, or advanced oxidation.
 * 5) Disinfection: Finally, the treated effluent is disinfected using chemical or physical processes to kill any remaining pathogens before it is discharged into the environment.

Microbial degradation is a process in which microorganisms, such as bacteria or fungi, break down complex organic compounds into simpler, less harmful substances. This process can be used to treat pharma effluent by utilizing the natural ability of microorganisms to degrade and remove pollutants.

There are different types of microbial degradation processes that can be used to treat pharma effluent, including aerobic and anaerobic processes.
 * 1) Aerobic degradation: This process involves the use of oxygen to break down organic compounds in the presence of aerobic microorganisms. In this process, microorganisms oxidize the organic compounds, converting them into carbon dioxide and water.
 * 2) Anaerobic degradation: This process involves the use of microorganisms that do not require oxygen to break down organic compounds. In this process, microorganisms convert organic compounds into methane, carbon dioxide, and other simpler compounds.

Microbial degradation can be used as a standalone treatment or as a complementary treatment with other physicochemical processes. The effectiveness of microbial degradation depends on various factors, including the type and concentration of pollutants in the effluent, the type of microorganisms used, and the operating conditions.

Overall, microbial degradation is a promising technology for the treatment of pharma effluent due to its potential to reduce the concentration of pollutants and its compatibility with sustainable and environmentally friendly approaches.

Microbial methods have been developed and utilized for the removal of pharma waste. Microbial degradation is an effective process that uses microorganisms to break down complex organic compounds into simpler, less harmful substances. There are different approaches to using microbial methods for the removal of pharma waste, including the following:


 * 1) Bioremediation: Bioremediation is the use of microorganisms to degrade pollutants in the environment. In the case of pharma waste, bioremediation can be used to treat contaminated soil and water by utilizing microorganisms that are capable of breaking down organic compounds present in the waste.
 * 2) Biodegradation: Biodegradation is the process in which microorganisms break down organic compounds present in pharma waste. The microorganisms use the organic compounds as a source of energy and carbon, which leads to the degradation of the waste.
 * 3) Composting: Composting is a process that uses microorganisms to decompose organic waste, including pharma waste. Composting involves the controlled degradation of the waste material, leading to the production of a nutrient-rich soil amendment that can be used in agriculture.
 * 4) Microbial fuel cells: Microbial fuel cells (MFCs) are devices that use microorganisms to generate electricity. In MFCs, microorganisms break down organic compounds in the waste, producing electrons that are harvested as electrical energy.

The use of microbial methods for the removal of pharma waste is a promising approach that can help reduce the environmental impact of the waste. However, the effectiveness of these methods depends on various factors, including the type and concentration of pollutants in the waste, the type of microorganisms used, and the operating conditions. Therefore, careful selection and optimization of microbial methods are necessary for the successful removal of pharma waste.

There have been several studies on the treatment of pharmaceutical effluent, which is a type of industrial wastewater that contains various chemicals, drugs, and pollutants. Some of the previous studies on the treatment of pharmaceutical effluent include: Advanced Oxidation Processes (AOPs) are a group of chemical treatment processes that involve the generation of highly reactive oxidizing species, such as hydroxyl radicals, to degrade organic pollutants. Studies have shown that AOPs can effectively treat pharmaceutical effluent by reducing the concentration of pollutants and increasing the biodegradability of the effluent. Membrane filtration is a physical separation process that uses a semi-permeable membrane to separate suspended solids and pollutants from the effluent. Studies have shown that membrane filtration can effectively remove pharmaceuticals and other contaminants from the effluent, and can be used in combination with other treatment processes to achieve higher levels of purification. Biological treatment involves the use of microorganisms to degrade organic pollutants in the effluent. Studies have shown that biological treatment can be effective in treating pharmaceutical effluent, especially when combined with other treatment processes such as AOPs or membrane filtration. Adsorption is a process that involves the attachment of pollutants to a solid surface. Studies have shown that various materials such as activated carbon, zeolites, and biochar can be used as adsorbents to remove pharmaceuticals and other contaminants from the effluent. Electrochemical treatment involves the use of an electric current to degrade pollutants in the effluent. Studies have shown that electrochemical treatment can be effective in removing pharmaceuticals and other contaminants from the effluent, and can be used in combination with other treatment processes for enhanced performance. Overall, there are several treatment options available for pharmaceutical effluent, and the choice of treatment process will depend on the specific characteristics of the effluent and the desired level of purification.

Isolation of Microorganisms
For isolation of potential microorganisms nutrient agar was selected as primary media. Samples from aeration tanks of pharmaceutical effluents of the various industires located in Ahmedabad districts were collected in sterile containers and immediately transferred to laboratory under cold conditions. After serial dilution, 50 µl samples from each diluted tubes were spreaded on nutrient agar plates and incubated for 24 hrs at 37ºC. After the incubation, colonies were screened based on colony morphology. Based on colony morphology 18 different microorganisms were selected for further study. For determination of pollutant degradation efficiency, active culture (100µl) of all the selected species were inoculated in Bushnell haas media containing 1% pharmaceutical effluent waste and incubated for 24 hrs at 37 ºC. After incubation, based on the visual observation, best 4 species were selected for identification through 16S rRNA sequencing. The efficacy of degradation of pollutants was analyzed by FTIR and HPLC. FTIR analysis was carried out from 400nm to 4000nm on Shimadzu IR affinity –I with ATR. For HPLC analysis samples were prepared by solid phase extraction. Cartridges for extraction were conditioned with 3 mL of methanol and 3 mL of distilled water. 20 µL of 0.5 M Na2EDTA solution and 100 µL of acetic acid were added to 200 mL of sample just before loading of samples. Samples were loaded at flow rate of 5 mL/min on columns and then dried for 30 min under vacuum. Elution was carried out by 6 mL of methanol. The elutes were allowed to dry and in water bath at 40 ºC temperature and obtained samples were reconstituted in 100 µL of water/methanol (80:20 ratio).

HPLC carried out using a Shimadzhu 2010 with UV/Vis detector. C18 column (100 × 2.1 mm, 2.6 µm) was used for separation of compounds. The mobile phase consisting of 0.1% formic acid in water and methanol, delivered at the flow rate of 0.2 mL/min. A gradient program of 20% of mobile phase (B) used from 0 to 1.0 min, 20% (B) to 95% (B) from 1.0 to 5.0 min, maintained at 95% (B) from 5.0 to 7.0 min, then decreased back to 20% (B) from 7.0 to 7.1 min and finally the column was re-equilibrated with 20% (B) from 7.0 to 10 min.

Results and Discussion
Results of isolation have shown presence of vast diversity of microorganisms. Based on the morphology presence of rods and cocci were observed. Primary screening based on the colony morphology total 18 different species were isolated and used for further study. When these 18 isolated were analyzed for their degradation efficiency, only four species have shown significant degradation visually. These species were designated as A, E, HG and L. The selected four isolates were further identified using 16s rRNA sequencing. Out of the four, two species labelled as A and E were found to be Bacillus licheniformis and remaining two belongs to Bacillus amyloliquefaciens.

FTIR Analysis
FTIR analysis is carried out the find the presence of particular function groups of the compound. It also helps in identification of compound. However, certain studies have shown potential use of FTIR to determine the degradation of particular compounds. A comparative study with the undigested sample may help in interpretation of degradation. Figure 1 shows the digestion of sample collected from pharmaceutical samples.



Based on the comparative analysis of FTIR results with the digested effluent, it was found that many compounds were destroyed by the microorganisms resulting into less number of peaks as compare to untreated samples. It was also seen that microorganism labeled with A and E are more efficient in digestion of alkenes and alkynes molecules which generally shown peaks at 2200 nm. Not only this, digestion of N-H, O-H and =C-H and amide compounds were also observed by this species (frequency area between 3000nm to 3400nm) The other two species have also shown similar pattern of digestion but still presence of molecules with C-O and C-N were seen. Overall it was found to have digestion of many compounds by the microorganisms, earlier present in the undigested samples. Many earlier studies have used FTIR as a primary and effective tool for analysis of digestion efficiency of microorganisms. It enables to determine the type of compound digested by the microorganisms.

HPLC Analysis
Results of HPLC analysis have shown that the numbers of small molecules are higher as compare to the undigested samples (Figure 2). This indicates conversion of larger molecules into the smaller molecules with the help of microorganisms. The total absorption areas by the molecules were found half the total area of undigested samples. This means a significant digestion of molecules is possible using microorganisms. Based on the graph pattern it was found that microorganisms labeled as Sample HG and E have shown maximum peaks indicating production of more smaller molecules. Least number of peaks was obtained in sample L however it was more than blank. HPLC coupled with MS can be used for accurate identification of compound produced upon digestion of pharmaceutical waste. Here the research was focused on the digestion of waste material only, so the study was restricted to HPLC only instead of MS study.

There are various approaches which can be applied to treat the wastewater generated by pharmaceutical industries. Chemical methods are among one of the earlier methods to be used but leads to unnecessary contamination of water and land. It may also be an expensive approach. Another approach is to use the biomolecules including enzymes; however it has limited efficiency and can digest only selected molecules. The stability of these molecules is also an issue. Many times combination physical and chemical methods are applied which give better results as compare to either of them. Use of potential microorganisms is considered as one of the best approach to digest waster material. Microorganisms used to digest such material to fulfill their growth requirement and may produce other important by products. Here also the similar approach was used and got good results. Isolation of potential microorganisms from the effluent tank has enable to screening adapted microorganisms with better efficiency.

Conclusion
In conclusion, the research underscores the pivotal role of microbial degradation, particularly by Bacillus species, in efficiently treating pharmaceutical effluent. The robust analyses using FTIR and HPLC reveal the substantial degradation of a diverse range of compounds. This eco-friendly strategy not only provides an effective solution for pharmaceutical waste management but also emphasizes the imperative need for customized microbial methods in comprehensive wastewater treatment. The study encourages further exploration and optimization of such sustainable approaches for broader industrial applications.

Conflict of Interest
Authors declares no conflict of interest.

Authors Contribution
Jagruti Patel has designed and executed the entire study and has written the manuscript. She has interpreted the results of the various analysis. Rita N Kumar has guided for the entire study and planning of execution of work. Not limited to this, vital support in data analysis and review of the manuscript was also done by her.

Funding Sources
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.