Industrial pollution is one of the leading causes of pollution worldwide. Because of its size and scope, industrial pollution is a serious problem for the entire planet. They are creating menace and futile efforts are made to reduce this pollution. Industries are causing air pollution by emitting smokes, gases, and fumes, land pollution by dumping their wastes and water pollution by releasing effluents into water bodies. Insufficient management of wastes release from industries is more common custom of developing countries. In these countries economic instability prevent the industrialist to spend their money on managing environmental issues.
Pollution is giant monster of 21st century and we the human being are the creator and victim of this monster and it is mounting bigger every day. Environment is degrading, climate is changing, intensity and rate of natural disaster has increased, new diseases, food shortage; all these miseries due to this monster. Industries are necessary evil of today's life. Industrialization is vital to a nation's socioeconomic development as well as its political stature in the international committee of nations. It provides ready employment opportunities for good percentage of the population in highly developed economics. Industries differ according to sizes, process technology, nature of products, characteristics and intricacy of wastes discharged (Amuda, 2006).
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In this research bacterial diversity of effluents of Scottman pharmaceutical industry will be determined. The bacteria will be isolated and identified from the effluents of pharmaceutical industry. The bacterial diversity will be helpful in studying the effects of effluent on the environment. The findings of this research work can be helpful to ensure how much this industrial effluent is playing its role in antibiotic resistance and environmental degradation.
Due to Industrialization and man's activities our environment has turned into dumping sites for waste materials. This makes many water resources detrimental and perilous to man and other living systems (Bakare et al., 2003). The lethal substances discharged into water bodies get accumulated through the food chain (Odiete, 1999).
In Pakistan, very few industries are equipped with satisfactory operating treatment facility set up, so there is common trend that industries dispose off untreated effluents via open and covered routes into the water ways which degrade water quality (Farid, 2003). Hence these industrial effluents are the most potential source of water and soil pollution. These effluents contain heavy metals as well as nutrients, which affect plant and soil in variety of ways (Dhevagi & Oblasam, 2002). Heavy metals are accumulated in the living cells causing a reduction of cell activities, inhibition of growth and various deficiencies/ diseases in plants (Shafiq & Iqbal, 2005; Kabir et al., 2008; Farooqi et al., 2009). Effluents discharged from various industries have variable characteristics and are potential threat to underground water contamination (Tariq et al.,2006)
Pharmaceutical industry is imperative industry in today's world as diseases are becoming very common and unique in nature. The primary charter of the pharmaceutical industry is to produce substances that have therapeutic value for humans and animals. Pharmaceutical effluents are waste engendered by pharmaceutical industry during the process of drugs manufacturing. The steps involved in the compounding of drugs (which may include extraction, processing, purification and packaging) generate air emission, liquid wastes and solid wastes (Duon, 1993).
Pharmaceutical should be considered as pollutant and they can pose threat to living beings when they enter in environment. One of the major problems related to pharmaceutical can be making bacteria resistant to antibiotics. High levels of antibiotics in the water are a cause for alarm as there is an increased risk of selecting resistant bacteria, an issue of global concern. This can lead to some highly effective antibiotics that are becoming ineffective. The term "eco-shadow" has been introduced to describe the ecological impact of antibiotics. Antibiotics with a wide spectrum that are also stable will have a greater impact on the bacterial flora (a long eco-shadow) than those with a narrow antibacterial spectrum which disintegrates more rapidly (a short eco-shadow) (Wennmalm et al., 2010)
Wastewater pollution is the main issue of this sector. In pharmaceutical industries wastewater is mainly generated through the washing activities of the equipment. Though the wastewater discharged is small in volume; but it is highly polluted, because of presence of substantial amounts of organic pollutants (Overcash, 1986). Solid waste usually comprises of expired or rejected medicines, spent solvents, packaging material and damaged bottles. Level of wastewater pollution varies from industry to industry depending on the type of process and the size of the industry (Garcia et al., 1995).
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Liquid effluents resulting from equipment cleaning after batch operation contain toxic organic residues. Their composition varies depending on the product manufactured, the materials used in the process and other process details ( Pollution Prevention and Abatement Handbook of World Bank Group, 1998). The biochemical oxygen demand (BOD), chemical oxygen demand (COD), suspended solids as well as phenol and pH of pharmaceutical effluent is however not consistent, depending on the product manufactured, materials used and the processing details. Pharmaceutical effluents changes the some chemical properties of soil (Osaigbovo. et al., 2006) as the chemical properties changes the micro fauna of soil also get effected. Most substances found in a pharmaceutical industrial wastewater are structurally complex organic chemicals that are resistant to biological degradation (Cokgor et al., 2004 Ren et al., 2008).
The growing use of Pharmaceutical products is becoming a new environmental challenge. For several years now the use of Pharmaceutical products has been on a steady rise, mainly due to a rise in the aging population in most nations. In the United States, for example, the pharmaceutical industry has been growing twice as fast as the rest of the US economy. The same happens in much other country to varying degrees. (Farre, et al.,2001). This rise in the use of pharmaceutical products results also in the proportional rise in the possibility of these compounds being present in the environmental samples. Pharmaceutical chemicals have potentials to produce long term effects on the environmental species (Velagaleti et al 2002)
The high level of pharmaceuticals in treated effluents from pharmaceutical industries show very high chronic toxicity to bacteria. The bacteria may acquire high degree of resistance and their resistance gene can spread to human pathogens. (Larsson et al., 2007 Martinez 2008).
Bacteria occupy a critical place in the web of life. Bacteria are the simplest life form and are the most numerous organisms with respect to number of species and total biomass. They are small, unicellular procaryotic organisms . Bacteria are classiï¬ed by structure (morphology), response to chemical stains, nutrition, and metabolism. Except for ï¬lamentous forms, cyanobacteria, and spirochetes, the range in sizes of most bacteria is from 0.3 to 3 Âµm. Filamentous bacteria such as Sphaerotilus natans usually are >100 Âµm in length.The unicellular cyanobacteria have photosynthetic pigments in cell membranes and range in size from 5 to 50 Âµm. Freeliving spirochetes commonly are found in wastewater treatment plants and may be up to 50 Âµm in length. Although the size range for bacteria is described as a diameter or largest side of the bacterial cell, there are many bacteria that are not spherical. The size of these organisms may be described according to their length and width. For example Escherichia coli,a common bacterium found in human feces and wastewater treatment plants, is approximately 2 Âµm in length and 0.5 Âµm in width.(Michael, 2006)
Over the billions of years of their existence, they have evolved to occupy conceivable niche on earth, indeed they influence almost everything we experience. Bacteria generally occur in variations of three major forms: rod-shaped bacteria (bacilli), spherical bacteria (cocci) and long spiral form bacteria (spirochetes if rigid or spirilla if flexible).
Bacteria are best known to the public as agents of human disease. Antibiotics are commonly used against bacteria but now bacteria are developing antibiotic resistance making it difficult to cure. The possible increase of antibiotic-resistant bacteria in sewage associated with the discharge of wastewater from a hospital and a pharmaceutical plant was investigated by usingÂ AcinetobacterÂ species as environmental bacterial indicators. The discharge of wastewater from the pharmaceutical plant was associated with an increase in the prevalence of both single- and multiple-antibiotic resistance among AcinetobacterÂ species in the sewers. (Guardabassi, et al., 1998) Antibiotics and antimicrobial agents (e.g. triclosan) are of concern because they could have direct effects over microbial populations, altering the community structure and increasing the resistance of human pathogens in the environment (Daughton & Ternes, 1999; Gobel et
al., 2004; Hirsch et al., 1999).
The Pakistan Pharmaceutical Industry is a high technology, essential and even strategically important industry that represents the single largest collective multinational financial investment in Pakistan. It is an industry which manufactures a wide range of research based life saving and life-enhancing medicines. It is a major contributor to the health of the Pakistani People, and to the economic well being of the country. The pharmaceutical industry in Pakistan started its development almost immediately after the country was created in 1947. All along, the research based multinational pharmaceutical companies played a key and ever-increasing role in making an enormous financial investment, transferring technology, and providing the latest innovative excellent quality medicines to the deserving consumers throughout the country (Zafar, 1995).
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In Pakistan, only the formulation of pharmaceutical products is carried out and wide variety of chemical and pharmaceutical products are produced which includes anesthetics, disinfectants, water soluble salt, muscle relaxants, anti clotting agents, analgesic, anti hypertensives, antibiotics, diuretics, anti-infective, cardiovascular, central nervous system and vitamin in the form of capsules, tablets, ampoules, syrups, creams, etc. Pharmaceutical sector can be classified as one of the most organized sector with respect to its institutional arrangements (Imran 2005).
In the effluents of pharmaceutical industry bacterial count was 2.15x105 c.f.c. the organism present in the effluent was Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Serratia marcescens and Pseudomonas aeruginosa. 25 bacterial strains was isolated from effluents which show resistance to antibiotics.(Lateef, 2003)
Pharmaceuticals within the environment, although currently at an amount considered to be negligible, gives rise to multiple concerns. These compounds are specifically designed to create a specific response within those it is administered to (humans, animals). Therefore, the amounts within the environment and the limited amount of information regarding the breakdown and effects are areas in need of development. Some concerns that have been voiced include "abnormal physiological processes and reproductive impairment, increased incidences of cancer, development of antibiotic-resistant bacteria, and the potential increased toxicity of chemical mixtures" (Kolpin, et al., 2002)
In research on a final effluent of hospital effluent identification confirmed that the bacterial population is composed of 27 species belonging to 17 genera; Escherichia coli 1, Klebsiella pneumoniae, K. ornithinolytica, Providencia alcalifaciens, Salmonella arizonae, Enterobacter cloacae, E. saburiae, E. gergoviae, Yersinia pestis, Citrobacter frundii, Serratia marcens, Pseudomonas aeruginosa, P. putida, Flvimonas oryzihabitans, Chryseomona luteda, Stenotrophomonas maltophila, Shewanella putrifaciens,Aeromonas hydrophila, A. salmonicida, Chryseobacterium meningiosepticum, Ch. Indologenes, Pasteurella multocida, Pas. pneumotropica and Moraxella catarrhalis, affiliated to 5 families; Enterbacteriaceae, Pseudomonadaceae, Vibrionaceae, Pasteurellaceae and Moraxellaceae. Gram negative bacterial strains dominated specially those of family Enterobacteriaceae and Pseudomonadaceae. The higher distribution patterns amongst the isolated strains, from almost all samples were for E. coli, followed by Enterobacter cloacae and Chryseobacterium meningiosepticum.. Antibiotic assay (9 antibiotics from different families) on 153 representative strains using antibiotic serial dilutions from 10 up to 100 Âµg/ml- to determine MIC (s), MBC (s) and the MIC/MBC indexes. All the studied strains exhibited resistance to at least 3 of the 9 tested antibiotics. Many bacterial isolates resist the whole 9 antibiotics and to a concentration of more than 100 Âµg/ml. The majority of the resistance strains were gram negative and the mechanism of action proved to be inhibitory.(Diab, et.al. 2008) .
Wastewaters from two pharmaceutical production processes, cotrimoxazole B wastewater (BWW) and Piriton wastewater (PWW), were examined microbiologically and for physico-chemical parameters. Furthermore, the wastewaters were also screened for genotoxicity using Allium cepa assay to assess the risk associated with the discharge of untreated pharmaceutical wastewaters into the environment. The effluents induced various types of chromosomal aberrations, namely, disturbed spindle, vagrant and chromosome bridge, and also showed a dose-dependent reduction in the number of dividing cells. The mitotic inhibition ranged from 38.6 to 67.2%. The mean root length at 20% of BWW and all concentrations except 1% of PWW were significantly different from the control values (p < 0.05). The EC50Â of the root growth inhibition was 4.17 and 12.45% for PWW and BWW, respectively. The wastewater physico-chemical analysis revealed that most parameters were within the allowable limits. The wastewaters had similar microbial load index of 107Â cfu mlâˆ’1, indicating dense populations of bacteria, which may be due to the richness of the wastewaters in nutrients particularly sulphate, nitrate and phosphate. Coliform bacteria concentrations in the PWW and BWW wastewaters were 50MPN/100 ml and 550MPN/100 ml, respectively. The identified bacterial isolates included Staphylococcus aureus, Escherichia coli, Serratia marcescens, Klebsiella sp, Streptococcus pyogenes, Bacillus licheniformis, Yersinia sp, Proteus vulgaris and Bacillus subtilis. The resistance of the bacterial isolates ranged from 10% for gentamicin to 100% for augmentin, amoxycillin, cloxacillin and nalidixic acid. PWW isolates were more resistant. Seven patterns of multiple drug resistance ranging from 5 to 11 antibiotics were obtained amongst the isolates. (Lateef et al., 2007).
OBJECTIVE OF THE RESEARCH PROJECT:
Isolation and identification of bacteria from the effluents of pharmaceutical industry Rawalpindi.
SCOTTMAN PHARMACEUTICALS (PVT) LTD
5D, I-10/3, Industrial Area, I-9, Islamabad
MATERIALS AND METHODS
Effluent sample for the research purpose was obtained from the Scottmann Pharmaceutical industry Islamabad, on the issuance of permission letter from university. Effluent samples were collected in dry sterilized containers.
Isolation of Bacterial Strains:
Nutrient agar media was prepared by mixing 20g of nutrient agar in 1 litre of distilled water. Media was autoclaved at 121oC for 15 minutes, and then pouring was done. After sometime nutrient agar get solidifies. Nutrient agar is a general purpose media manufactured by Merck and its pH was 7.2 at 25oC. The pH and electrical conductivity of pharmaceutical effluent was measured. Bacteria were isolated by serial dilution, spread plate and bacterial colonies are purified with streak plate method.
For serial dilution fill test tubes with 9 ml of distilled water and autoclave them. After autoclaving add 1 ml of sample in first test tube. This test tube is direct dilution. For second test tube take 1 ml from the first test tube, for third test tube take 1 ml from the second tube and so on. Make ten dilutions in this way for each sample.
Pour plate method:
In this method take 100Âµl(0.1 ml) with the help of micropipette and a sterile tips from first, third, fifth, seventh and ninth test tube and spread each on nutrient agar plate and spread it with the help of sterile L- shaped spreader while rotating the plate in order to distribute the bacteria evenly. After place the plates in incubators for 24 hours at 37oC, as this temperature is optimum fro bacterial growth.
Streak Plate Method:
In streak plate method, colonies are picked from the incubated nutrient agar plates and streaked on the new agar plates. All colonies with different morphology are streaked on separate plate. Streaking is done by picking colony with the help of sterilized inoculating loop and streak on the plate on three sides at right angle to each other and the last streak is a zigzag line. After each streak inoculating loop is sterilized. It is expected that on zigzag line one can get single cell colony. Single cell colony is that colony of bacteria which is derived from a single cell. By doing 3-4 rounds of streaking one can get pure cultures of bacterial strains. The pure cultures were kept at 4oC and were time to time refreshed to avoid contamination.
For the sake of convenience and future reference the isolated strains were named as SF1, SF2, SF3, SF4, SF5, SF6, SF7, SF8, SF9, SF10, SF11, SF12, SF13, SF14, SF15, SF16, SF17, SF18, SF19, SF20, SF21, SF22, SF23 and SF24.
IDENTIFICATION OF BACTERIAL STRAINS:
Isolated pure strains are identified on the basis of morphological characteristics and biochemical tests.
Determining the morphology of a single colony growing on the surface of a plate culture can be an important tool in the description and identification of microorganisms Morphological characteristics of all isolated strains grown on nutrient agar plates were observed to identify and classify the strains. Following are the morphological characteristics observed:
Table 1: Common description of colony morphology
Punctiform, small, medium, large
Entire, Undulate, filiform, curled, lobate
Circular, irregular, filamentous, rhizoid
Raised, convex, flat, umbonate
Yellow, orange, pale yellow, off-white
The test was originally developed by Christian Gram in 1884, but was modified by Hucker in 1921. Gram stain permits the separation of all bacteria into two large groups, those which retain the primary dye (gram-positive) and those that take the color of the counterstain (gram-negative). The primary dye is crystal violet and the secondary dye is usually either safranin O or basic fuchsin.
Both Gram-positive (Gm+) and Gram-negative (Gm) organisms form a complex of crystal violet and iodine within the bacterial cell during the Gram-staining procedure. Gm+ organisms are thought to resist decolorization by alcohol or acetone because cell wall permeability is markedly decreased when it is dehydrated by these solvents. Thus, the dye complex is entrapped within the cell, resist being washed out by the solvents, and Gm+ bacteria remain purple following this differential stain.
In contrast, cell wall permeability of Gm- organisms isÂ increasedÂ by ethyl alcohol washing because it removes the outer membrane from the Gram-negative cell wall. This allows the removal of the crystal violet-iodine complex from within the cell. The decolorized Gm- cell can then be rendered visible with a suitable counterstain, in this case Safranin O, which stains them pink. Pink which adheres to the Gm+ bacteria is masked by the purple of the crystal violet.Â
Table 2: composition of gram stains
Under sterile conditions a smear of each isolate colony was prepared. For it put a small drop of water on clean slide. Pick a colony with sterilized inoculating loop and spread it on the slide. After spreading, let it dry. When slide get dried heat fix it by passing through flame 3 to 4 times.
Cover the smear with crystal violet (primary stain). Left it for 1 min. After that rinse off the stain with distilled water. Then flood the slide with gram iodine (mordant) and left it for 1 min. After 1 min, rinse off iodine with distilled water. Then add few drops of 75% alcohol and rinse off immediately. Then add safranine (counter stain) on slide and left it for 40 sec. After 40 sec, wash the stain with distilled water and left for air drying.
OBSERVATION UNDER MICROSCOPE:
When slide get air dried add a drop of oil emulsion and cover it with cover slip. After doing this observe the slide under microscope at 100X resolution.
The isolated were biochemically characterized according to the bergey's manual of determinative bacteriology (bergey et al.,1994)
CITRATE UTILIZATION TEST:
This test is for the ability of bacteria to convert citrate (an intermediate of the Kreb's cycle) into oxaloacetate (another intermediate of the Kreb's cycle). In this media, citrate is the only carbon source available to the bacteria. If it cannot use citrate then it will not grow. If it can use citrate, then the bacteria will grow and the media will turn a bright blue as a result of an increase in the pH of the media.Â
Media is prepared by dissolving 22.5 g of Simmon's citrate agar in 1 litre of water. Mix well and gently heat the mixture so that agar gets complete dissolve in water. Then Simmon's citrate agar is autoclave at 121oC for 15 minutes. The pH of the media is 6.8. Simmon's citrate agar plates were prepared and inoculated with 24 hour fresh culture. After inoculation agar plates are placed in incubator for 24hours at 37oC. Citrate positive strain will give growth on agar transforming green agar into blue.
GEL LIQUEFACTION TEST:
This media is used to test if bacteria can digest the protein gelatin. To digest gelatin, the bacteria must make an enzyme called gelatinase. After digestion, gelatine is converted into smaller components.
Media prepared for Gel Liquefaction test has following composition:
Table 3 Composition of media for Gelatine liquefaction test
Media is prepared and suspended in test tubes. Autoclave the test tubes at 121oC for 15 min. Inoculate with 24 hour fresh culture and place it in incubator at 37oC for 24 hours. Before observing the results kept the test tubes in refrigerator at 4oC for 1 hour. If the suspended media remain liquid then results are positive.
This test is used to detect the enzyme amylase, which breaks down starch. After incubation the plate is treated with Gram's iodine. If starch has been hydrolyzed (broken down) then there is a reddish color or a clear zone around the bacterial growth; if it has not been hydrolyzed then there is a black/blue area indicating the presence of starch.
For starch hydrolysis, media has following composition:
Table 4 Composition of Starch Agar Media
Starch agar media
Star agar media plates were made and inoculated with 24-hour culture and incubate it at 37oC for 24 hours. To check starch hydrolysis adds iodine in plates after 24 hour incubation. Clear zones around the colony will indicate positive result for starch hydrolysis.
TRIPLE SUGAR IRON TEST:
Triple Sugar Iron Agar is used for the differentiation of microorganisms on the basis of dextrose, lactose, and sucrose fermentation and hydrogen sulfide production.
Peptone mixture and the Beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group. TSI contains three carbohydrates (Dextrose, Sucrose and Lactose) as sources of carbon and energy. When these are fermented the acid production is indicated by the Phenol red indicator, being the color changes yellow for acid production and red for alkalinization. Sodium thiosulfate is reduced to Hydrogen sulfide, which reacts with the iron salt to give the black iron sulfide. Ferric ammonium citrate is a H2S indicator. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Bacteriological agar is the solidifying agent
Suspend 64.5 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes and sterilize in autoclave at 121°C for 15 minutes. Allow to cool in a slanted position in order to obtain butts of 1.5 - 2.0 cm. depth. The color is red.
Inoculate the butt by stabbing the butt and inoculate the slant my moving inoculating loop to and fro on the slant. An alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only. An acid slant-acid butt (yellow/yellow) indicates fermentation of dextrose, lactose and/or sucrose. An alkaline slant-alkaline butt (red/red) indicates dextrose or lactose were not fermented (non-fermenter). Cracks, splits, or bubbles in medium indicate gas production. A black precipitate in butt indicates hydrogen sulfide production.
Some bacteria can produce indole from amino acid tryptophan using the enzyme typtophanase. Production of indole is detected using Kovac's reagent. Indole reacts with the aldehyde in the reagent to give a red color. An alcoholic layer concentrates the red color as a ring at the top.
Bacterium to be tested is inoculated in peptone water, which contains amino acid tryptophan and incubated overnight at 37oC. Following incubation few drops of Kovac's reagent are added. Kovac's reagent consists of para-dimethyl aminobenzaldehyde, isoamyl alcohol and con. HCl.. Formation of a red or pink coloured ring at the top is taken as positive.
indole + pyruvic acid+ NH3
Table 5 Peptone water
Table 6 Kovac's Reagent
METHYL RED-VOGAS PROSKEUR (MR-VP) TEST:
MR-VP media are designed to test the method by which an organism ferments glucose on the basis of the by-products that are produced in the fermentation process.
METHYL RED TEST (MRVP BROTH)
Mixed acid fermentation - Many gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids produced lowers the pH of the medium - Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas.
Inoculate bacteria into MRVP broth. Incubate it for 48 hours. The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1 ml of broth and place into a sterile tube before performing the methyl red test if you are going to use the same broth for the VP test. Add 3-4 drops of methyl red to the original broth. Do not shake the tube. A positive result has a distinct red layer at the top of the broth. A negative result has a yellow layer.
Table 7 Methyl-Red indicator
: Organisms that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.
Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days. Read the VP test when you have good turbidity. A clear broth indicates that your organism did not grow and cannot be tested. Barritt's reagent A (VP A) contains Î±-naphthol and Barritt's B (VP B) contains KOH.
Table 8 Barritt's Reagents (Solution A)
Table 9 Barritt's reagent (Solution B)
Test 1 ml of your culture from the MRVP broth. If you have already conducted the methyl red test, you should have already placed 1 ml of untested broth in a sterile tube. Add 0.6 ml of barritt reagent A and 0.2 ml of Barriet reagent B. Shake well. This reaction will take a few minutes before you will see a color change. Shake the tube every few minutes for best results. With a positive reaction the medium will change to pink or red indicating that acetoin is present. With a negative reaction the broth will not change color or will be copper colored. Wait at least 15 minutes for color to develop before calling the test negative.