Effect of Plant Extracts on Laboratory Organisms and Biodegrading Agents Found in the Industrial Effluent

Abstract

To determine whether or not the plant extracts affecting biodegrading agents that normalize industrial effluent sample plants known as bionomalizers i.e. Carica papaya Linn., Moringa oliefera Lam. and Musa sapientum L. were taken into consideration through dose mortality against the stored grain pest Tribolium castaneum (Hbst.), cytotoxicity against the brine shrimp Artemia salina nauplii and antibacterial activity against the 9 bacterial isolates from the tannery effluent and 7 certain other bacteria as laboratory test agents were done. Leaf, stem and roots of C. papaya; fruit, leaf, stem bark, stem wood, root bark and root wood of M. oliefera, and leaf, stem and roots of Mu. sapientum were extracted in petroleum ether, chloroform and methanol. For C. papaya extracts against T. castaneum beetles the dose mortality was done through residual film assay to yield the highest and the lowest mortality for the CH3OH extract of roots (LD50 0.114mg cm-2) and CHCl3 extracts of stem (LD50 2.053mg cm-2) after 48h of exposure, while the CHCl3 extracts of leaf and root didn’t offer any mortality to the beetles. According to the intensity of activity observed through dose mortality test against the adult beetles the potentiality of the Pet.E., CHCl3 and CH3OH extracts could be arranged in a descending order: root (CH3OH) > stem (CH3OH) > leaf (CH3OH) > root (Pet.E.) > leaf (Pet.E.) > stem (Pet.E.) > stem (CHCl3) extract. For M. oliefera the highest and the lowest mortality have been observed for the CH3OH extract of root bark (LD50 0.276mg cm-2) and Pet.E. extract of root wood (LD50 0.629mg cm-2) after 48h of exposure. According to the intensity of activity observed through mortality of the adult beetles the potentiality of the Pet.E. and methanol extracts could be arranged in a descending order: root bark (CH3OH) > stem bark (CH3OH) > root bark (Pet.E.) > fruit (CH3OH) > root wood (CH3OH) > stem bark (Pet.E.) > stem wood (CH3OH) > root wood (Pet.E.) extract. For Mu. sapientum the highest and the lowest mortality have been observed for the CH3OH extract of stem (LD50 0.163mg cm-2) and Pet.E. extract of leaf (LD50 1.195mg cm-2) after 48h of exposure. According to the intensity of activity observed through mortality of the adult beetles the potentiality of the extracts could be arranged in a descending order: stem (CH3OH) > root (CH3OH) > leaf (CH3OH) > stem (Pet.E.) > root (Pet.E.) > leaf (Pet.E.) extract. For C. papaya against the A. salina nauplii the highest and the lowest mortality have been observed for the CHCl3 extract of leaf (LC50 1.326ppm) and CH3OH extracts of leaf (LC50 183.443ppm) after 24h of exposure. According to the intensity of activity observed through dose mortality test against the adult beetles the potentiality of the Pet.E., CHCl3 and CH3OH extracts could be arranged in a descending order: leaf (CHCl3) > root (CH3OH) > stem (CHCl3) > stem (Pet.E.) > stem (CH3OH) > root (CHCl3) > root (Pet.E.) > leaf (Pet.E.) > leaf (CH3OH) extract. For M. oliefera the highest and the lowest mortality have been observed from the CHCl3 extract of root bark (LC50 4.197ppm) and CH3OH extracts of fruit (LC50 234.246ppm) after 24h of exposure respectively. According to the intensity of activity observed through dose mortality test against the adult beetles the potentiality of the Pet.E., CHCl3 and CH3OH extracts could be arranged in a descending order: root bark (CHCl3) > root bark (CH3OH) > stem wood (CHCl3) > stem wood (CH3OH) > root wood (CHCl3) > stem wood (Pet.E.) > stem bark (CHCl3) > fruit (Pet.E.) > root bark (Pet.E.) > fruit (CHCl3) > root wood (Pet.E.) > fruit (CH3OH) > stem bark (CH3OH) > leaf (Pet.E.) > root wood (CH3OH) > leaf (CH3OH) extract. For Mu. sapientum the highest and the lowest mortality have been observed from the CH3OH extract of leaf bark (LC50 22.991ppm) and Pet.E. extract of leaf (LC50 127.604ppm) after 24h of exposure. According to the intensity of activity observed through dose mortality test against the adult beetles the potentiality of the Pet.E., CHCl3 and CH3OH extracts could be arranged in a descending order: leaf (CH3OH) > leaf (CHCl3) > stem (CHCl3) > root (Pet.E.) > stem (Pet.E.) > root (CHCl3) > root (CH3OH) > stem (CH3OH) > leaf (Pet.E.) extract. Bacterial strains were isolated from tannery effluent. Fifteen colonies were screened from initial level of effluent on nutrient agar media. Out of fifteen colonies, nine isolates were selected for biochemical test and other studies. Selected isolates were: two isolates from sample 1 (isolate 1 and 2), four from sample 2 (isolate 3, 4, 5 and 6) and three from sample 3 (isolate 7, 8 and 9). The isolates were determined as 1. Bacillus cereus, 2. Klebsiella oxytoca, 3. Staphylococcus aureus, 4. Escherichia coli (I), 5. Escherichia coli (II) 6. Citrobacter freundii, 7. Proteus vulgaris, 8. Bacillus subtilis, 9. Salmonella typhimurium. Physico–chemical characteristics of the tannery effluent were also determined along with the characterization of the found bacterial isolates. The Pet.E., CHCl3 and CH3OH extract of C. papaya (leaf, stem and root),M. oliefera (fruit, leaf, stem bark, stem wood, root bark and root wood) and Mu. sapientum (leaf, stem and root) were tested against 7 selected bacteria (2 Gram positive bacteria Bacillus subtilis, Staphylococccus aureus and 5 Gram negative bacteria Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis, Shigella flexneri and Shigella sonnei) to evaluate their antibacterial potential at a concentration of 200 and 400μg disc-1 along with a standard antibiotic, Ampicillin 10μg disc-1 and 9 isolates (from industrial effluent) to evaluate their antibacterial potential at a concentration of 400μg disc-1 along with a standard antibiotic, Kanamycin30μg disc-1. Among the C. papaya extracts the root extracts showed the highest antibacterial activity. Only the B. subtilis (roots/CHCl3 gave 20mm diam. for 400μg disc-1), K. pneumoniae (stem/CHCl3 gave 09mm in diam. for 400μg disc-1) and St. aureus (leaf/Pet.E. gave 09mm in diam, stem/CHCl3 gave 10mm in diam., roots/Pet.E. and CH3OH gave 15mm and 10mm in diam. respectively, while all the tests were for 400μg disc-1) were responsive among the selected test bacteria. Among the M. oleifera extracts the stem wood extracts showed the highest antibacterial activity. Only the (fruit/Pet.E. and stem bark/CHCl3 both gave 09mm in diam. for 400μg disc- 1), K. pneumoniae (fruit and stem wood/CHCl3 gave 11 and 10mm in diam. both for 400μg disc-1) and St. aureus (fruit/Pet.E. and CHCl3 gave 10mm and 09mm in diam., leaf/ Pet.E. gave 09mm in diam., and stem bark/Pet.E. and CH3OH gave 09 and 10mm in diam. respectively, stem wood/Pet.E. and CH3OH gave 10 and 12mm in diam. respectively, root bark/Pet.E. and CHCl3 gave 11mm and 09mm in diam., and root wood/CHCl3 gave 10mm in diam., while all the tests were for 400μg disc-1) were responsive among the selected test bacteria. Among the Mu. sapientum extracts the root extracts showed the highest antibacterial activity. Only the St. aureus (stem and roots/Pet.E. gave 08mm and 10mm in diam., for 400μg disc-1) were responsive among the selected test bacteria. According to the susceptibility test bacteria could be arranged in a descending order of St. aureus > B. subtilis > K. pneumoniae. The Pet.E., CHCl3 and CH3OH extract of C. papaya (leaf, stem and root),M. oliefera (fruit, leaf, stem bark, stem wood, root bark and root wood) and Mu. sapientum (leaf, stem and root) were tested against 9 isolates viz. 1. Bacillus cereus, 2. Klebsiella oxytoca, 3. Staphylococcus aureus, 4. Escherichia coli (I), 5. Escherichia coli (II), (for the iolates 4 and 5 the strains were different but determination was not done) 6. Citrobacter freundii, 7. Proteus vulgaris, 8. Bacillus subtilis, 9. Salmonella typhimurium. Among the 9 Isolates Isolate 8 B. subtilis was highly responsive to the Pet.E. and CHCl3 extracts of C. papaya stem (15 and 08mm), M. oliefera fruit (14 and 09mm) and root bark (10 and 16mm), and Mu. sapientum root (10 and 10mm) respectively; to the Pet.E. extracts of C. papaya root (11mm), M. oliefera root wood (11mm), Mu. sapientum stem (10mm), and to the CHCl3 extract of M. oliefera stem bark (08mm). Next to the Isolate 8 it was Isolate 2 K. oxytoca responsive to the Pet.E. extract of C. papaya leaf (08mm), M. oliefera fruit (08mm), Mu. sapientum leaf (15mm) and root (10mm); and to the CHCl3 extract of C. papaya leaf (08mm), M. oliefera stem bark (08mm), stem wood (08mm) and root bark (08mm); followed by the Isolate 1 B. cereus which was responsive to the Pet.E. extracts of C. papaya stem (16mm), M. oliefera fruit (10mm) and Mu. sapientum stem (8mm) and root (10mm); this was followed by Isolate 3 St. aureus which was responsive to the Pet.E. extracts of C. papaya stem (10mm), M. oliefera fruit (13mm) and Mu. spaientum root (08mm) and again the CHCl3 extract of M. oliefera stem bark (08mm). This was followed by the Isolate 6 Citrobacter freundii where CHCl3 extract of C. papaya stem (08mm) and Pet.E. extract of Mu. sapientum leaf (10mm) were found responsive. Isolate 4 E. coli (I) and 5 E. coli (II) (but different strains) and 9 Sa. typhimurium show response against Pet.E. extract of C. papaya stem (08mm), M. oliefera fruit (08mm) and CHCl3 extract of M. oliefera stem wood (09mm) respectively. According to the susceptibility test bacteria could be arranged in a descending order of B. subtilis > K. oxytoca > St. aureus = B. cereus > Citrobacter freundii > E. coli (I) = E. coli (II) = Sa. typhimurium. Isolate 7 P. vulgaris was not responsive to any of the 12 extracts of the 3 test plants. For Kanamycin 30g disc-1 the inhibition zones for the Isolate 1, 2,3, 4, 5, 6, 7, 8 and 9 were 50, 35, 35, 40, 36, 42, 42, 55 and 40mm respectively. The differences of the clear zones given by Ampicillin 10μg disc-1(tested against the 7 bacteria) and Kanamycin 30g disc-1 (tested against the 9 isolates) were nearly the same in majority of the test cases. The bacterial isolates found in the industrial effluent, i.e. B. subtilis, St. aureus, one of the E coli strains, another species of Klebsiella, K. oxytoca, another species of Salmonella, Sa. typhimurium were among the list of available 7 test bacteria of the Institute of Environmental Science (IES), R.U. and the response in that separate experiments nearly resembles the results got against the 9 isolates carried out afterwards. However, E coli among the 7 didn’t show any response at all. The activity of the Pet.E., CHCl3 and CH3OH extract of C. papaya (leaf, stem and root), M. oliefera (fruit, leaf, stem bark, stem wood, root bark and root wood) and Mu. sapientum (leaf, stem and root) were mild in activity in general, and P. vulgaris, Sa. typhimurium, both E. coli and Citrobacter freundii didn’t give any considerable clear zone. Thus, it could be mentioned that C. papaya, M. oliefera and Mu. sapientum plant materials are not so much effective against the biodegrading bacteria and obviously they are helpful in biodegradation of industrial effluents causing very insignificant harm to biodegrading bacteria.