PAKISSAN.com; Environmental Biotechnology Division

Environmental Biotechnology Division

Searched out, for the environmental pollution especially caused by the industrial waste water. Research on a particular problem is carried out in collaboration with different industries and a tailor made process is developed and handed over. In this connection, microorganism based process of biosorption and biodegradation has been developed for treating the effluent from "Dyeing and Printing sections" of Crescent Textile Mills, Faisalabad. Similarly microorganisms required and process details for treating effluents from two pharmaceutical industries have been developed and passed on to the end users. Details of this work have been provided in NIBGE Activity Report of 1996 and 1998. Different biosorbents have been used for removal of chromium from leather tanning industries effluent have been discussed previously. Similarly work on petroleum degradation and establishment of a battery of tests for genotoxicity testing have been reported earlier. Further studies in this reg!
ards have been continued and some of the findings are given below.

1. TEXTILE INDUSTRY

Effluent from Dyeing and Printing sections have been found to contain hazardous chemicals. Results of laboratory scale, fermentor level and pilot plant scale studies, for detoxification of effluent, have been reported earlier.

1.1. Analysis of Samples for Pollution Level

For designing and fabrication of treatment plant for industrial waste water, level and type of pollution load is a prerequisite. Forty samples, representative from different sections at various time period were analysed for COD, BOD, TSS, TDS, Fats, oil and greases, chloride and phenolics. Wide variation was observed in samples from different sections and collected at varied time. This analytical data will be utilized for designing of the treatment plant for the industry.

1.2. Removal of Dyes

1.2.1. Preparation of Cheap and Economical Biosorbents:

Biosorbents were prepared after several process steps (washing, drying, grinding, sizing and activation etc) from different sources: fungal waste biomass (FW) from a pharmaceutical industry; dried biomass of Azolla pinnata (AB) from Azolla nursery at NIBGE, sesame seed cake (SS) and mustard seed cake (MS) from local oil mill.

1.2.2. Removal of Dyes From Their Aqueous Solutions

These prepared biosorbents were tested for their biosorption capacities for congo red and rhodamine-B. After optimization of pH, adsorbent dosage and initial dye concentration, adsorption capacities of AB, MS, SS and FW were found to be 48, 15, 2.0 and 2.5 mg/g respectively for rhodamine-B and 20, 10, 7.5 and 4.0 mg/g respectively for congo red in shake flask studies. Removal of these dyes was also successful in the columns packed with these biosorbents with no significant loss in their adsorption capacities. All the loaded adsorbents were successfully desorbed and regenerated with 0.5N NaOH and 0.5N H2SO4 respectively Although these dye removal capacities especially those of Azolla and mustard seed cake seems to be very promising, but still these capacities can be further improved by optimizing the adsorption conditions.

2.LEATHER TANNING INDUSTRY

2.1. Pollution Load in Effluents

Effluent samples from various sections like soaking, liming, deliming, pickling, chrome tanning and mixed effluent released out of tanneries of Lyallpur Tannery (L.T.) Faisalabad and National Tanneries (N.T.) Muredke were collected and chemically analysed for various pollutant parameters. Both these samples were analysed for pH, Ec, Cr, chemical oxygen demand (COD) and biological oxygen demand (BOD), nutrients (N, P, K), anions and cations Na, Mg, Ca, K, Cl, SO4. The results revealed that some of these samples were highly polluted as indicated by the Tables 1a, 1b, 2a, and 2b.

Total organic carbon in samples collected from L.T. was higher than from N.T. The values from different sections ranged from 200 to 15,000 with a value of 283 and 2,900 in mixer from N.T. and L.T. respectively. Biological analysis of samples from N.T. has revealed that effluent from soaking, liming, deliming and mixture contained 8600, 700, 3300 and 7810 CFU (Colony forming Units)/ml of bacteria.

Samples from dyeing and chrome tanning sections were found toxic (based on growth inhibition of Photobacterium phosphoreum) and mutagenic (SOS Spot test, an indicative of induction of sfiA gene which switches on only in response to DNA damaging agents. This is judged by the induction of -galactosidase on indicator plates containing a substrate (IPTG) which on hydrolysis with the enzyme releases blue colour as a ring around the zone of inhibition.) in N.T. while these were weakly toxic and weakly mutagenic in case of L.T. Samples from dyeing sections were also found toxic and mutagenic. Samples of liming section and mixture from N.T. were found toxic at high concentration while non toxic at lower concentration. This suggests a separate process for treatment of chrome tanning section.

2.2. Microbial Detoxification of Effluent from Leather Tanning Industry

Enriched culture obtained through chromium tolerance studies and from sludge have been employed for reduction in COD, using mixed effluent from Lyallpur Tannery, Faisalabad (8125 mg/l COD). During incubation, different level of reduction in COD have been achieved by different sources used for inoculation of bacteria. Mixed effluent from L.T. showed maximum percent reduction of 56.5, 59.3 and 32.97 using above mentioned cultures respectively. Mixed effluent from National Tannery Mureedke was also biologically treated and almost 76 % reduction in COD could be obtained within 144 hours of incubation

2.3. Biosorption of Metal (Cr) From Industrial Effluent

Above mentioned biosorbents (AB, MS, SS and FW) were also used to remove Cr (VI) from its aqueous solution. After optimization of pH, adsorbent dosage and initial chromium concentration, absorption capacities of AB, MS, SS and FW were found to be 125, 110, 23 and 25 mg/g respectively at pH 2.

2.4.Effect of Tannery Effluents on Growth of Brassica campestris and Uptake of Chromium

Considering the hazardousness of effluents released by the leather tanning industries and its use for irrigation, samples of different crops growing in Kasur area were analysed for toxic heavy metals especially chromium, a dangerous chemical used for rendering colour to the hides. It was found that the level of chromium in wheat (13 ppm), oat (63 ppm), mustard (31 ppm), sugar cane (13 ppm) and grass (363 ppm) was much higher than standard permissible level. To look into the role of industrial effluents in irrigation, effluent released by different sections of tannery industries was collected and analysed for various pollutant parameters especially the heavy metal chromium. All the sections were found to contain high pollution load and chrome tanning section effluent contained high chromium. Mixed effluent also contained appreciable amount of chromium. Thus, two main effluents (effluent from chrome tanning section and a mixture of various sections effluent) were studied f!
or their effect on growth parameters of a very popular, in both villages and cities, vegetable Brassica campestris (Sarsoon) used as a pot herb and for cooking oil. These effluents were tested independently, in pot experiment, and were mixed with irrigation water in different ratios (1:1, 1:3, 3:1). Irrigation water only was used as control and these industrial waste water samples without dilution were also included to see their severe effect. Irrigating once at second irrigation and twice (second dose at fourth irrigation) was tested and the samples were analysed for different "pollutants" including chromium.

Mixed effluent when mixed with irrigation water (at the minimum level i.e., one fourth), increased the vegetative growth of the plant, a positive influence. On the other hand, these did contain chromium in amount not permissible for human consumption. Highest dose applied resulted in chromium accumulation more than 1200 ppm (dry weight basis) in roots and almost 700 ppm (dry weight basis) in shoots. In appearance there was no sign of accumulation of chromium and the plant shoots enter into human food and animal feed and may cause serious health problem like cancer.

2.5. Characterization of Metal Resistant Bacterial Strains Against Cr (VI)

Chromium resistant bacteria have been isolated from these and sludge samples. These have shown resistance to chromium, indicated by their growth in mineral medium containing up to 500 ppm chromium in the solution. This is a very high level of tolerance shown by the enriched culture. Individual bacteria present in this mixture have been isolated and their level of tolerance to chromium has also been studied.

Bacterial strains MF 'K' and MF 'L' already checked for being resistant to different heavy metals like Cd, Zn, Co, Ni were checked for Cr resistance. Effect of Cr on their growth was also studied. The two bacterial strains were found resistant to 1000 ppm of Cr+6. During growth on minimal media containing 100 and 1000ppm of Cr+6 , these bacterial strains can remove 60% Cr+6 from the solution.

2.7. Scaled Up of Biosorption Studies.

Biosorption process for the removal of Cr (VI) from its aqueous solution has been scaled up to column level. Dried biomass of an aquatic fern, Azolla, was packed into columns (10 mm internal diameter, 13 cm bed, flow rate 1ml/min.) and was found to adsorb chromium (VI) with an adsorption capacity of more than 100mg/g at pH 2.0 at 60% saturation of biomass. The saturated biomass was treated with 0.5M HCl with a regeneration efficiency of about 50%.

3.PHARMACEUTICAL INDUSTRIES

3.1. Biodegradation of Phenol

Several bacterial isolates capable of degrading phenol were enriched and isolated from pharmaceutical and tanneries effluents and soil. Enrichments were carried out using shake flask method with increasing phenol concentration periodically. Five promising isolates designated as HQ1, HQ2, HQ3, HQ4 and HQ5 were selected for their ability to degrade higher than 1500mg/L of phenol utilizing phenol as a sole source of carbon and energy. The strains HQ3 and HQ5 were able to degrade even higher than 2000mg/L of phenol. A significant decrease (80-99%) in COD and BOD was also observed following degradation by the tested strains. Biodegradation of phenol was observed to be growth associated.

3.2. Bacterial Detoxification of Phenols in Effluent Containing Inorganic Chemicals

Eight phenol degrading bacteria were isolated from different sources like effluent and soil contaminated by phenols collected from surroundings of Pharmaceutical industry. These were compared for their growth and phenol degrading potential and the best two; strain MB and strain 3B were selected for further studies.

Effect of higher concentration of inorganic chemicals was studied on their growth and phenol degrading abilities. Effect of addition of Na2SO4, Na2CO3, NaCl, K2SO4, K2CO3 and KCl on degradation of phenol supplied at 1000 ppm was studied. Strain MB was found better to tolerate these chemicals. Mineralization of phenol in presence of these chemical could be achieved within 48 hours of incubation. Selected bacterial strains can be applied for degradation of phenol in effluents containing phenol and high concentrations of these chemicals.

3.3.Adsorption of Phenol onto Activated Carbon Cloth.

Adsorption of phenol onto activated carbon cloth, was very promising. Activated carbon cloth adsorbed appreciable amount of phenol (146 mg/g ) under optimized conditions and adsorption mechanism was found to obey both Langmuir and Freundlich isotherms. When phenol adsorbed on activated carbon cloth was subjected to biodegradation by mixed culture of HQ3 and HQ5 (bacterial strains mentioned above) in the presence/absence of glucose, more than 80% of phenol (3000 ppm) was consumed by the mixed culture after 6 weeks in presence of glucose.

3.4. Adsorption of Phenol on Different Adsorbents.

Adsorption of phenol was checked on calcium alginate beads, diatomaceous earth, diatomaceous earth treated with cetyl trimethyl ammonium bromide (CTAB) and denatured biomass of Rhizopus arrhizus. No significant adsorption of phenol was observed.

3.5. Studies with Enterobacter cloacae QW3.

A chromium resistant isolate Enterobacter cloacae QW3 was grown on phenol (500mg/L) in the presence of 0.5mM chromate Cr(VI). This culture not only utilized the total phenol but also lowered the Cr(VI) concentration to 0.1mM in the solution within 24 hours. Cr(VI) was determined by DPC method. Further studies are in progress to see if the Cr(VI) was reduced to Cr(III) or it was accumulated by the bacterial cells.

3.6.Removal of Phenolics by Enzymatic Precipitation.

Horseradish peroxidase (HRP) catalyses the oxidation of aromatic compounds (especially phenolics) in the presence of H2O2 and the oxidized products polymerize to form insoluble precipitates which readily separate out from the aqueous solution. Reaction conditions were optimized to precipitate phenol from its aqueous solution by using HRP in the presence of H2O2. Initially it was observed that the process is adversely affected by the presence of organic solvents. In order to overcome this problem two iso-enzymes of HRP were isolated and purified on mono-Q anion exchange resin using FPLC. One of these isoenzymes was found to precipitate and thus remove phenol in the presence organic solvent with no significant loss in its activity.

After optimization of conditions for the removal of phenol from its aqueous solution, effect of various additives i.e., Polyethylene glycol (PEG), Carboxymethyl cellulose (CMC), Pectin and CTAB on the phenol removal efficiency of peroxidase enzyme was examined. Of these PEG and CMC were found to be the best which improved phenol removing efficiency of peroxidase by more than 100%. CTAB and pectin improved that efficiency by 70 and 30 % respectively. Thus PEG and CMC are found to play an important role in commercializing this technology.

Thermodynamic and kinetic studies of phenol removal by HRP has also been completed and the process is now being upscaled up to a 5 litre bio-reactor to treat the synthetic/real phenolic effluents.

3.7. Effect of Various Chemicals on Enzymatic Precipitation of Phenolics.

Since industrial effluents also contain variable amount of salts, effect of various salts i.e., Na2SO4, MgSO4, Na2SO3, Na2S2O3 and CaCl2 was also examined. Na2SO4 improved phenol removing efficiency as its concentration increased from 0 ‑ 30,000 ppm. MgSO4 improved this efficiency upto its concentration of 6000 ppm, while there was no significant effect of it on phenol removal from 6000 ‑ 30,000 ppm. Na2SO3 improved phenol removal efficiency up to 1600 ppm while further increase in its concentration decreased this efficiency significantly. Na2S2O3 was inhibiting even at its lower concentrations 100 ppm and there was no significant phenol removal at its concentration of 1000 ppm. Similar was the case for CaCl2 which severely affected phenol removal efficiency by peroxidase enzyme even at its concentration of 80 ppm and no significant phenol removal was observed when its concentration increased up to 1000 ppm.

3.8.Plasmid Isolation of Phenol Degrading Strains.

Various methods were employed to isolate plasmid DNA of selected strains but the yield of plasmid DNA was very low, suggesting that either plasmid DNA in these strains was of high molecular weight or these strains are plasmid less. However, a strain, Enterobacter cloacae QW3 yielded a plasmid of ~23 kb.

4. PETROLEUM INDUSTRY

4.1.Production of Biosurfactant by Pseudomonas aeruginosa Strain S8 Under

Nitrogen and Phosphorus Limiting Condition.

Pseudomonas aeruginosa strain S8 was previously isolated and characterized as biosurfactant producing bacteria on the basis of tensiometric, emulsification and hemolytic activities. Three minimal media having hexadecane and two complex media with glucose were evaluated as possible substrates for biosurfactant production in shake flasks at 30oC and 100 rpm.

On hexadecane, when nitrogen and phosphate became limited, growth of bacteria stopped and with the presence of available carbon, the metabolism shifted towards glycolipid production. Phosphate deficient proteose peptone glucose salt (PPGAS) medium yielded higher biosurfactant (surface tension <30 mN/m) as compared to the same medium with normal phosphate levels (48mN/m) after 72 hrs incubation. Thus De novo synthesis of rhamnose by cultures grown on hexadecane pass through the process of gluconeogenesis and the formation of lipid moiety produced in glycolipids in glucose grown culture is a result of lipogenesis.

4.2Enhanced Biosurfactant Production by P. aeruginosa Mutant Strain EBN8

Deficient in Rhamnose Utilization.

Enhanced biosurfactant production and hydrocarbon utilization by a gamma ray induced mutant was observed during growth on hexadecane in minimal medium. Decrease in surface tension (ST) of cell free culture broth (CFCB) and reciprocal of critical micelle concentration (CMC-1) was 2.8 times higher in case of mutant. Time course for the growth of both the parent and the mutant (CFU/ml), pH changes and metabolite production were compared using hexadecane as carbon and energy source. These studies were carried out for 15 days at 37oC and 32oC. Growth of EBN8 was much more faster as compared to the parent as well as it was 2-3 times hyperproductive for biosurfactant.

4.3.Bioremediation of Oil Contaminated Soil and Water by Different Indigenous

Bacterial Strains

Implementation of bioremediation is to improve the useful material (like soil) in contaminated environment by accelerating the natural biodegradation process. The main objective was to study the parameters for enhanced oil degradation in contaminated soil on site, to make it fit for growth of grass and trees.

Oil biodegradation in soil was studied by using 4 locally isolated bacterial strains (27, 33, 6B and EBN-8) individually and in mixture. Three of these were isolated from oil contaminated soil from Kot Addu Power Plant and EBN-8 was a mutant of previously isolated bacterial strain. Soil microcosms were established to study the oil (30g/kg soil) in soil and to see the contribution of supplementation of nitrogen and phosphorus. In case where soil was provided with nitrogen and phosphorus, 75% of oil was degraded in 6-7 weeks time when 25% of nitrogen and 83% of phosphorus was found unavailable in soluble form.

Similar experiment related to bioremediation and biodegradation of furnace oil was carried out in contaminated soil under field conditions at high temperature ranging from 25-45oC. Evaporation and photo-oxidation at higher temperature resulted in 6.6% losses. Native bacteria degraded 13% oil. While in case of inoculated with these bacteria, the observed degradation was 33% over and above the control (with native bacteria) in 7 weeks. However, the biodegradation under field conditions was lower than that recorded in Lab experiment.

Furnace oil biodegradation in aqueous phase was found to be 61, 55 and 37 % by bacterial strains 6B, 33 and 27 respectively. To check the release of oil from soil by biosurfactant produced by bacterial strains and its further utilization, oil contaminated soil was suspended in liquid mineral medium. It was observed that 50, 48 and 56 % of oil was degraded by strains 33, EBN-8 and mixed culture respectively. Bacterial population increased in first 6 weeks.

For quick and increased growth of oil degrading bacteria, these were provided with molasses (a liquid waste from cane sugar industry) as carbon and energy source, in liquid mineral medium containing oil. During incubation for 4 weeks, 43-46 % of molasses were used and 37-48 % oil was utilized by different bacteria. Presence of molasses increased growth of these bacteria but was inhibitory for oil utilization.

4.4. Molecular Biology of Biosurfactant Producing Strains.

4.4.1 Cloning of Rhamnolipid (rhl) Genes From P. aeruginosa Strain K3.

To identify the rhamnolipid (rhl) encoding genes in wild type biosurfactant producing local isolate, chromosomal DNA of P. aeruginosa strain K3 was digested with SalI and resolved on the gel. The gel was southern hybridized to pUO58 (containing the 5.8 Kb insert of rhlABR gene). DNA digested with SalI hybridized to the rhl gene probe at two positions on the gel (2.0 and 5.0 Kb respectively).The above sized fragments were cut out from the native gel, eluted and cloned in the pBluescript vector. The ligation mixture was transformed in DH5 cells. The recombinant plasmids (white colonies) were screened on LB plates having ampicillin and X-gal. To check for the desired inserts, plasmid DNA was isolated from several recombinants, digested with XbaI and XhoI and resolved on the 1% agarose gel. The transformants having insert size in the range of 1.5-2.0 Kb and 4.5-5.0 Kb were screened for the presence of rhl locus genes. More than 400 clones were analyzed by restriction dige!
stion analysis for the desired inserts. About 160 were found to contain inserts in the range of 1.5 to 2.5 Kb and 35 clones contain inserts in the range of 4.5-5.5 Kb (as shown by hybridization with pUO58 with K3 genomic DNA) All these clones were dot blotted and hybridized to pUO58 to eliminate the recombinants for the undesirable inserts. This screening yielded 34 clones, which were further confirmed by southern hybridizing with pUO58 to identify positive clones for rhl genes.

Seven clones (pMF7, pMF8, pMF18, pMF19, pMF53, pMF60, pMF66) hybridized with the probe suggesting that these clones contain gene of interest. The size of the inserts of different clones vary between 1.5 Kb to 3.0 Kb suggesting that these clones contain rhl related DNA fragments and were showing homology to the pUO58 probe (containing full length rhamnolipid rhlABR gene). The presence of structural and regulatory sequences in these clones and their homology with the probe have to be confirmed by DNA sequencing.

4.4.2. Subcloning in Shuttle Vector pUCP19

In order to study the effect of cloned rhl gene in hexadecane utilization and biosurfactant production in K3 strain, Xba1 and Xho1 2.0 Kb insert in pMF19 was subcloned in shuttle vector pUCP19. Several clones were screened for the desired inserts and the selected clone (pMF36) having the desired 2.0 Kb Xba1and Xho1 insert was selected for further studies.

4.4.3. Effect of Different Carbon Sources on Growth and Biosurfactant

Production by pMF36 (clone carrying the 2.0 kb rhl gene of K3 ).

4.4.3.1. Effect of Different Concentration of Glucose.

The effect of cloned rhl gene of K3 strain on growth, hexadecane utilization and biosurfactant production in pMF36 (clone carrying 2.0 Kb XbaI and XhoI related rhl segment of strain K3 ) was studied using different carbon sources. Different parameters like O.D, ST reduction, pH and rhamnose concentration was studied. Effect of different concentration of glucose (5g/l, 10g/l, 15g/l) on biosurfactant production was checked. It was found that pMF36 showed maximum growth (O.D) at 10 mg/l glucose concentration after seven days of incubation, whereas growth at 5 and 15 g/l glucose concentration was same for both the strains i.e K3 (Control) and pMF36 respectively. However, pMF36 showed two times higher rhamnolipid production at 5 and 10 g/l glucose concentration. At 15 g/l glucose concentration, rhamnolipid concentration was equal for both the strains. Similarly pMF36 was able to lower the surface tension of the culture broth at low glucose concentration (5g/l) as compared to!
K3 suggesting higher rhamnose production by pMF36.

4.4.3.2. Effect of Different Concentration of Heptadecane

Different concentration of heptadecane (1%, 3%) was used to study rhamnolipid production by pMF36 and K3 respectively. It was found that pMF36 produced two times higher rhamnolipids as compared to K3 when grown in the presence of 1% heptadecane. Production of rhamnolipids was three times higher by pMF36 as compared to control (K3) when grown in the presence of 3% heptadecane suggesting that increase number of rhl related genes in pMF36 resulted in enhanced biosurfactant production at both concentration tested and utilizes heptadecane efficiently for biosurfactant production as compared to control (K3). Optimization of substrate concentration is still required to achieve maximum biosurfactant production.

4.4.3.3. Effect of Different Concentration of Vegetable Oil on Biosurfactant

Production.

Effect of vegetable oil (1%) on growth, hexadecane utilization and biosurfactant production, was studied on pMF36 (clone carrying 2.0 Kb XbaI and XhoI related rhl segment of strain K3 ) and K3 respectively. Similar results were obtained when vegetable oil was used as carbon and energy source. Production of rhamnolipids was three times higher by pMF36 as compared to control (K3) when grown in the presence of 1% vegetable oil as carbon and energy source. It was observed that in all cases, use of different carbon sources while studying their effect on growth, hexadecane utilization and biosurfactant production on pMF36, carrying cloned rhl gene of K3 strain, resulted in two to three times enhanced biosurfactant production.

5. GENOTOXICITY TESTING

Genotoxicity assays are used specifically to evaluate genotoxic potential of environmental and industrial effluent samples, medicinal plants, consumer products, pharmaceutical and veterinary drugs and agricultural chemicals. The selected test systems can also used to screen products and chemicals for anti‑mutagenic properties. NIBGE is the only institute in Pakistan having expertise in genetic toxicology and operating in compliance with the international guidelines. Our services are particularly useful to the environmental agencies, pharmaceutical companies as well as manufacturers of medical devices, cosmetics, and agricultural and industrial chemicals, who are required to comply with the government legislation. In order to complement genotoxicity test "battery", following new methods are being developed and applied for environmental monitoring.

5.1. Comet Assay ‑ New Biomarker for Detecting Genotoxicity Induced by

Chemicals and Radiation.

The Comet assay is one of the most sensitive methods to measure direct DNA damage to individual cells by different genotoxic agents. This technique is extremely sensitive in detecting DNA damage by radiation. The comet assay is rapid, requires only small cell samples, and is also reasonably economical. The comet assay also known as single cell gel electrophoresis (SCGE) assay is a new sensitive and simple method for the detection of DNA damage in human and other organisms. The relevance of SCGE lies in its requirement for very small cell sample, and in its ability to evaluate DNA damage in proliferating as well as non proliferating cells. This technique is preferred because results can be obtained in a single day and the cost of performing the assay is also reasonably economical.

5.1.1. Genotoxicity of Air Particulate Matter.

The comet assay can provide an important indication on the genotoxic potentials of air borne particulate. The DNA damaging activities associated with inhaleable particulate matter (PM10) collected from Faisalabad (an industrial city of Pakistan) have been determined using comet assay. Air samples from seven different locations of the city were collected on fibre glass filters using high flow air sampler. To study the induction of comets by airborne particulate matter, MCL-5 cells were exposed for 30 minutes at 37C to both inorganic (50% nitric acid) and organic (methylene chloride) extracts of the air particulate. These cells were then subjected to alkaline single cell gel electrophoresis under optimised conditions. Out of 7 organic extracts, 3 induced significant DNA damage to MCL-5 cells, comets with tail length greater than 50µm were observed. While inorganic extracts of same samples were also positive. The longer exposure (18 hours) of MCL-5 cells to air particulat!
e samples resulted in higher cytotoxicity and longer mean tail length of comets. To determine the effect of DNA repair inhibitors on comets, additional studies were set up to perform comet assay with and with out repair inhibitor compounds (HU/araC). Relatively higher number of comets with greater tail lengths were induced with repair inhibitor than without inhibitors. The results of the present study indicated, that there is a need to monitor airborne particulate on regular basis for their potential risk to damage genetic material of living organisms.

5.1.2.Comet Assay to Detect

Genotoxicity of Tamoxifen in Rat Tissues.

This was a part of a long term study related to toxicity testing of tamoxifen drug. Albino rats were administered with tamoxifen. This drug induced comets in the different tissues (liver, spleen, kidney, lungs, stomach and intestine etc) of treated rats.

5.1.3. Detection of DNA Damage in Irradiated Meat Samples by Comet Assay.

DNA damage in irradiated (1,3 &5 KGy) meat samples of chicken, beef, mutton and fish was detected by comet assay. An increase in DNA damage with increase in the radiated dose was observed in all the meat samples. DNA damage in the form of comets was visualized by silver staining method under microscope.

5.1.4.Detection of Effect of UV Radiation on DNA of Chicken RBC by Comet

Assay.

Using comet assay a preliminary study was carried out to observe the effect of UV radiation on DNA of chicken red blood cells. The DNA was stained with ethidium bromide and comets were observed under fluorescent microscope. The UV radiation caused DNA damage to chicken RBC which was indicated by induction of comets.

5.2.Setting-up of SOS Chromo Test.

SOS chromotest, is a colorimetric bacterial assay for detection of genotoxins, and is based on the induction by DNA damaging agents of the SOS function SfiA, whose level of expression is monitored by means of SfiA::lacZ operon fusion. The E. coli tester strain PQ37 provided by Dr. Phillips Quillardet (France) was used for this assay. The purity of the tester strain & -galactosidase activity was checked by SOS spot test. Induction of -galactosidase activity with known genotoxins was studied at different concentrations (0.1,.0.2,0.3,0.4,0.5 µg/assay) of known mutagens, mitomycin and K2Cr2O7.

The results were expressed in enzyme units. The response was found to be dose dependent i.e., increase in concentration of mutagen resulted in increase in -galactosidase activity (enzyme units). Optimization of conditions like O.D of test culture, time of incubation for color development & mutagen (tested compound) concentration are in progress.

5.2.1. SOS Assay of the Air Pariticulate Matter Samples.

Air samples from different areas of Faisalabad city was collected through the air sampler. Using SOS spot test, the organic extracts were found to be more genotoxic to the E. coli PQ37 test strain as determined by the zone of growth inhibition followed by induction of blue ring around this zone indicating that the spotted samples can induce SfiA::lacZ operon. Hence they can damage DNA. Negative control (distilled water) did not give any induction of SfiA::lacZ fusion. Both the organic and inorganic fractions of the air particulate samples were found cytotoxic as well as genotoxic to the test strain as indicated by the presence of blue ring around zone of inhibition.

5.2.2.Genotoxicity of Tanneries Effluent by SOS Chromotest.

Ten samples collected from different sections of tannery industry i.e soaking, chrome tanning, deliming, pickling were subjected to SOS spot test. Only one sample from chrome tanning section was found toxic and was capable of inducing SfiA::lacZ induction as indicated by the presence of blue ring around zone of inhibition.

5.3.Postlabelling Assay.

The 32P-Postlabelling technique most sensitive method for the detection of a wide range of compounds bound to DNA. For hydrophobic, aromatic DNA adducts, such as polycyclic aromatic hydrocarbon PAH-DNA adducts, this method can detect 1 adduct in 109-1010 bases by selective removal of unmodified nucletides by enzymic methods. The DNA adducts are then phophorylated via enzyme catalysed transfer of 32P-phosphate to the deoxyribose of the adduct. The high sensitivity of the assay has led to broad use of 32P-postlabelling assay in studies with human and other animals for assessing exposure to environmental genotoxins.

5.3.1.Study on Effect of Tamoxifen® (Anti Breast Cancer Drug) on Rat Liver.

There was a dose dependent increase in adduct formation (DNA covalent linkages indicating DNA damage) was noted. There were 220-350 adducts/108 nucleotides with 45mg/kg dosage indicating a high level of DNA damaging potential of the tamoxifen®.

5.3.2.Monitoring Human DNA Samples Collected From Faisalabad for DNA

Adducts.

Blood samples were collected from different normal individuals. From blood samples WBC layer was separated and DNA was isolated following standard techniques. The DNA samples were subjected to post labelling studies. Out of 20 samples only 5 samples indicated some adduct formation possibly related to smoking. However some unidentified adduct were indicated in 3 samples.

5.4.Microtox Test.

Microtox test, an acute toxicity test which is based on the measurement bioluminescence of luminous bacteria has been commercialized by some companies. These commercial toxicity analysers and test cultures are very expensive. Efforts are being carried out to use spectrofluorometer (available in the lab) and (ATCC-11040), Photobacterium phosphorium culture to develop toxicity testing system for industrial waste and other pollutants. This alternative test system would eliminate dependence on commercial supply of culture and equipment. Growth conditions for Photobacterium phosphorium have been optimized. The bioluminescence of the strain was confirmed by appearance of dark spots on x-ray film which was exposed to the bacterial colonies in the dark. The emission spectrum of the Photobacterium culture gave maximum fluorescence intensity at 460 nm.

5.5.Ames Test to Monitor Mutagenicity of Phenolics..

Mutagenicity of effluent samples and treated phenolic samples was tested by Ames test using Salmonella typhimurium strains TA98 and TA100. The non mutagenicity of biodegraded phenolic samples indicated that biodegradation of phenol has resulted into complete removal of toxicity of phenols. Where as all the effluent samples collected from AW Pharmaceutical were toxic to these strains.

5.5.1. Ames Assay of the Air Samples.

Air samples collected onto fibre glass filters from seven different locations of Faisalabad city by a high volume air sampler were separated into inorganic fraction (extraction with 50% Nitric acid) and organic fraction (extraction with Methylene Chloride). These two fractions were then subjected to Standard Ames Mutation assay to check their mutagenicity. 4 out of 8 samples were found to be toxic and potentially mutagenic.

5.5.2. Genotoxicity of Chlorpyrofos Pesticide of Organophosphate Group.

Ames Salmonella reversion assay was performed with TA98 strain to check the genotoxicity of the pesticide by spot test. Different dilutions (2, 4, 6, 8, 10 ppm) were prepared and filter sterilized. Their mutagenicity was determined. Spot test showed that 100 ppm of pesticide was quite toxic to the test strain as indicated by the absence of background lawn. Revertant colonies were observed around the filter paper disc. Spot test of different dilutions were also found toxic and mutagenic to the test strain.

5.6. Antimicrobial Activity of Azadiracta indica.

Culture sensitivity of Azadiracta indica against gram positive (Bacillus) and gram negative (E. coli) was checked by preparing different concentration each (mg/ml: 4, 2, 0.4, 0.2, 0.1) of two extracts i.e, 50/50 (ethanol/aqueous) and 80/20 (ethanol/aqueous) by agar disc It was found that different concentration of aqueous extract of medicinal plant (Azadiracta indica) was non‑mutagenic as the mutagenicity index was less than 2.0.

It was found that different concentration (mg/ml: 4, 2, 0.4, 0.2, 0.1) of each extract of Azadiracta indica showed no antimicrobial activity against gram positive and negative strains after 24 hours of incubation. However, culture sensitivity was observed with Bacillus strain with increasing concentration of ampicillin i.e ,4, 2, 0.4, 0.2, 0.1 mg/ml respectively. In case of E. coli some activity was observed at concentration of 4 mg/ml ampicillin.

5.7. Setting-up ELISA and Molecular Based Diagnostic Technologies for

Rinderpest.

Rinderpest is a most fatal viral disease of animals. It can cause heavy losses to livestock within a short time. Although it has been eradicated from most of the countries of the world. But perhaps the Pakistan is the only country where out breaks of this disease still occur. In an effort to eradicate rinderpest from Pakistan work on a recently awarded TC-IAEA project has been initiated. The main aim of this project is to set up ELISA and PCR based diagnostic and differential diagnostic methodologies for rinderpest.

5.8. Estimation of Formaldehyde from Milk Samples.

Twenty four milk samples were collected from milkmen of different localities of Faisalabad. These samples were then analysed for formaldehyde after distilling it out in phosphate buffer. Distillate was then subjected to formaldehyde estimation by Nash reagent method. No formaldehyde was observed in 6 samples while its concentration in 9 samples was between 0-1 ppm, that of 8 samples between 1-4 ppm and one sample contained exceptionally higher concentration of formaldehyde i.e., 16 ppm.

5.9.Phytoremediation.

Potential use of living Azolla pinnata to remove was examined. In vivo study showed that it is able to grow in the presence of up to 6 ppm Cr (VI) and 12 ppm Cr (III) without any apparent negative effect on its growth rate. It was also able to remove about 50 % of the chromium within one month. Higher concentration of chromium slowed down its growth ultimately leading to its death. Similar studies were conducted using another beautiful floating plant Pistia stratiotes this plant was also able to tolerate 4 ppm Cr (VI) and 8 ppm Cr (III) without any apparent negative effect on its growth rate. It was also able to remove about 50 % of the chromium within one month. Potential of Azolla pinnata to grow in the presence of toxic phenol was also examined and was found to grow in the presence of up to 250 ppm phenol with out any significant affect on its growth rate.

Source: Dr. Khalid Shouq

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