Bioremediation of pharmaceutical effluent with the use of effective microfloral consortia
Smriti Singh1*, Hari Pathak2, Abhinav Mishra3, Awadesh Gaur4, Utkarsh Pandey5
1,2,3,4,5Department of Biotechnology Engineering, HCST, Farah, Mathura
* Corresponding Author Email: firstname.lastname@example.org | Tel: +918979348826
The enormous amount of the pollutants being dumped constantly from the various industries, including pharmaceutical causes severe damages to the natural resources e.g. water, soil. As an alternative method, bioremediation is used to treat the contaminants present in the pharmaceutical effluent with more potential degradation at very low operational cost. Many microbial species have been identified, which are found to be efficient in the degradation of organic compounds present in the effluent. In the present work, our aim was to find out effective microbial consortia for remedy of an environmental problem generating from the pharmaceutical effluent. In this work, effluent samples were collected from Pharmaceutical industry, Dholpur, Rajasthan, each from Non-ETP(Effluent Treatment Plant) treated and ETP treated effluent. BOD and COD of both samples were determined after sample collection and readings were 2352 mg/L and 15237 mg/l for Non-ETP treated sample and 1124 mg/L and 5327 mg/ml for ETP sample. For bioremediation of effluent; microorganisms were isolated from various sources by crowd plate method. Fifteen isolates, on basis of their different phenetic characters, were purified. These isolates were inoculated in minimum basal medium, supplemented with Non-ETP treated pharmaceutical effluent. During incubation, microbial growth was observed in the medium. However, only seven isolates showed significant growth during incubation with Non-ETP treated effluent. BOD and COD were again estimated and valued as 258 mg/L and 595 mg/L respectively. The results show a significant reduction in BOD and COD of Non-ETP treated effluent, and it was also better than BOD and COD of ETP treated effluent.
Bioremediation, Pharmacuetical, Effluent, Microflora, BOD, COD
Pharmaceuticals include any substance or mixture of substances for use in the diagnosis, treatment, mitigation or prevention of a disease, disorder or abnormal physical state, or its symptoms in human beings or animals (Enick & Moore 2007). Thus, these products include a wide range of structures, functions, behaviors, and activity. The presence of pharmaceutical residues in the environment was reported for the first time in the late 1970s(Jones et al. 2005).Most of the pharmaceuticals produced ultimately make their way directly or indirectly into the environment polluting the flora and fauna to different extents. It has been shown that pharmaceuticals are introduced into the environment by a diverse range of pathways. The main one is human excretion after consumption. In this case, pharmacokinetic studies have shown that a significant proportion, up to 50%, of the original compound may be excreted unchanged (Ternes 1998).When a medication has expired, people dispose of it in a toilet or in the garbage (Ternes et al. 2002). Leaching from landfill sites to groundwater has been demonstrated in some studies (Jones et al. 2004). Often the pharmaceuticals pass into sewage and end up in water bodies without any treatment. For instance, Canadian cities were reported to have an average discharge of 3.25 billion liters per day of essentially untreated sewage into surface water or ocean (Daughton & Ternes 1999). In recent years, there have been many proposals to remediate the situation and to include major changes in the typical wastewater treatment process. Most of these utilize an oxidation process or a combination of these called advanced oxidation processes (Benítez et al. 2008). These techniques are based on the production of reactive and oxidizing radicals, which degrade pharmaceuticals. Ozonation is one technique that can successfully remove many pharmaceuticals, especially compounds with an activated ring or carbon-carbon double bond (Nakada et al. 2007). Unfortunately, the cost related to this kind of advanced technologies is significant and they pose maintenance problems (Jones et al. 2007), which make them economically unfeasible for many municipalities. Modifications, such as increasing the solid retention time for a typical wastewater plant that uses biological treatment has been proposed (Oppenheimer et al. 2007), as it promotes the growth of a more diverse biological community able to degrade xenobiotic compounds. However, studies have shown that this solution did not significantly increase the removal of all drugs tested (Clara et al. 2005).
The failure to remove pharmaceuticals from wastewater raises concerns for researchers due to their potential accumulation in the environment. Although pharmaceuticals represent a small fraction of all the chemicals that are released in the environment, special care must be taken regarding their accumulation for four reasons (Enick & Moore, 2007):
1) Pharmaceuticals are ubiquitous and disseminate well.
2) Pharmaceuticals are specifically designed to act on biological systems.
3) Pharmaceuticals are known to cause a wide range of side effects in non-target organisms.
4) Pharmaceuticals can cause chronic toxicity at low concentrations.
Since concerns regarding the accumulation of drugs have been raised, many studies have reported the presence of pharmaceuticals in different countries and water plants throughout the world (Jones et al. 2005). The usual concentrations stated are in the magnitude of ng/L, and rarely exceed the drinking-water-guidelines (Kolpin et al. 2002). However, still many compounds have not been regulated yet. The adaptation of the biomass consists of an acclimatized period in which the microorganisms are exposed to the target chemical to degrade. The microorganisms then have time to develop enzymes to degrade the compound more effectively, which is usually not a readily used carbon or nitrogen source (Jones et al. 2007). A study by Zhou et al. revealed that during the treatment of high-strength pharmaceutical wastewater and removal of antibiotics in anaerobic and aerobic biological treatment processes (Zhou et al. 2006) a lot of microorganisms are reported with biodegradability for salts present in pharmaceutical product e.g. carbamazepine, desmedipham, phenmedipham and promecarb (Gieg et al. 1996; Andreozzi et al. 2003).
In the present work, we have explored the isolation of microorganisms from effluent and sewage water sample and their efficiency in biodegrading pharmaceutical effluents.
Materials and Methods
2.1. Effluent Sample collection
Pharmaceutical effluent samples were collected from the final drainage effluent of pharmaceutical industry from Dholpur, Rajasthan in sterile bottles. Wastewater samples were subjected to physicochemical as well as microbiological characterization to define their pollution strength and selecting the most effective treatment technology. Later on, the quantitative analysis (DO, BOD, COD) of the water sample collected from pharmaceutical were analyzed.
For isolation of desired microorganisms for bioremediation, soil samples were collected in the sterile bag from the water canal of Bichpuri, Agra.
2.2. Isolation of Microbial Consortia from Pharmaceutical Effluent and Sewage Soil
1 gm of soil sample was added into 50 ml distilled water and kept for shaking on a rotary shaker at 150 rpm on room temperature for 30 minutes then serial dilution was prepared up to 10-6. 100 µl of a serially diluted water sample from 10-5 and 10-6 were poured onto the NB agar and LB agar plate. Plates were incubated at 37ºC for overnight in an incubator for growth. After overnight incubation plates were observed. Colonies of different morphologies were purified on NA and LB agar plates by re-streaking until single colony obtained. Purified colonies were stored in NB agar slants and stored in a refrigerator for short term use as well as glycerol stocks were prepared for long term storage.
2.3.Bioremediation of pharmaceutical effluent
Isolated microbial colonies were inoculated in 150 ml of basal medium supplemented with pharmaceutical effluent water and incubated at 37 ºC for 48 hours. Isolates were inoculated in basal medium as consortia and as single (Pure Culture), in order to determine which type of isolates is actively participated in biodegradation of organic matter present in effluent water. During this incubation, 1 ml of samples was withdrawn for determination of growth kinetics after subsequent 1 hour. Growth was measured at 600 nm with the help of spectrophotometer.
2.4.Determination of DO, COD and BOD
Water quality parameters were again determined after biological treatment with microbial consortia.
Results and Discussions
Our study was aimed at exploring an effective low-cost bioremediation solution for treatment of pharmaceutical effluent by use of microflora. Out of 15 microbial purified isolates utilized in our study, only seven isolates were showing significant results in bioremediation of pharmaceutical effluents.
3.1.Isolation, Culturing & Purification of microbes
After overnight incubation, microbial colonies with different morphology such as branched, round shape, colored; discrete and pinpoint were observed on NA plate and LB agar plate. On the basis of their morphology and appearance fifteen isolates were purified by re-streaking and used for bioremediation in this work. Purified isolates were inoculated in nutrient broth at 4 °C for short time storage and glycerol for long term, storage at
3.2.Estimation of BOD and COD with microbial consortia
After sample collection from the sites of contamination, the biochemical test was performed within 2 hours. BOD and COD of non-treated samples were 2352 and 15237 mg/l initially. After five days of incubation with microbial consortia, BOD was 258 mg/l and COD was 595 mg/l which indicated that microbial consortia were able to reduce BOD and COD of non-treated water at a significant level.
In recent times very little attention was given to the environmental problem arising by pharmaceutical effluents so there was a constant need to find an alternative low cost solution for treatment of pharmaceutical effluent. To address this concern 15 microbial isolates were evaluated for their bioremediation efficiency against pharmaceutical effluent taken from different pharmaceutical industries. However, only seven isolates showed significant growth during incubation with non ETP treated effluent. BOD and COD were again estimated and valued as 258 mg/L and 595 mg/L respectively. The results show a significant reduction in BOD and COD of non ETP treated effluent, and it was also better than BOD and COD of ETP treated effluent.
We are thankful to our Director Prof R. K. Upadhayay, Hindustan College of Science & Technology, Farah, Mathura for his administrative and financial support for this research work and especially thanks to Resource person, Sonotek Pharmaceutical (P) Ltd, Dholpur, Rajasthan for their support in this research work.
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