STUDY ON THE PRODUCTION AND ANTIBACTERIAL ACTIVITIES OF BIOSURFACTANT PRODUCED FROM SOME BACTERIAL SPECIES

* Chioma Okore 1 , Linda Nwaehiri 1 , Ogechukwu Mbanefo 1 , Toochukwu Ogbulie 2 , Assumpta Ugenyi 2 ,Ifeanyi Ogbuka 1 ,Agunna Ejele 3 and Ikechukwu Okwujiako 2 . 1. Environmental Biology Dept, Federal Polytechnic NekedeOwerri, Imo-State. 2. Biotechnology Dept, Federal University of Technology Owerri, Imo-State. 3. Chemistry Dept, Federal University of Technology Owerri, Imo-State. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Biosurfactant are produced by some micro organisms. Kerosene was used as substrate to enhance the production of biosurfactant by Staphylococcus aureus, Bacillus sp., Pseudomonas aeruginosa, Proteus sp. and Corynebacteriumsp. Staphylococcus aureus produced the highest 0.5 g, Bacillus sp., Pseudomonas aeruginosa and Proteus sp. 0.2 g, Corynebacterium sp. the least 0.1 g. The biosurfactant demonstrated antibacterial activity against the test bacteria (Staphylococcus sp. and Pseudomonas aeruginosa). The biosurfactant produced by Pseudomonas aeruginosa gave the highest zone of inhibition against Staphylococcus aureus, Bacillus sp., Corynebacteriumsp., (25 mm). The biosurfactant produced by Staphylococcus aureus gave the highest zone of inhibition against Pseudomonas aeruginosa (39 mm), Corynebacteriumsp., and Proteus sp. (30 mm), Bacillus sp. the least zone (25 mm). The production of biosurfactantand antibacterial efficacy can thus be promising for use in medical, therapeutics, pharmaceuticals, cosmetcs, food and beverages for treatment and control of diseases caused by micro organisms.
The microbially produced surfactants are alternatives to chemical surfactants whose effects have been reported variously by authorities. The effects of surfactants on the human body are divided into effects on the skin and in the body. The main ingredients of modern life detergents are surfactants, long-term use cause skin irritation effect and lead to some degree of damage. After the surfactants enter into the human body, they damage the enzyme activity and thus disrupt the body's normal physiological function. Surfactants have some toxicity and may accumulate in the human body, so it is difficult to degrade (Venhuis and Mehrva, 2004). In general, nonionic surfactants are not electrically charged, not combined with protein. They have minimal irritation to the skin. The toxicity of cationic surfactants is the biggest, and the toxicity of anionic surfactants is between that of non-ionic surfactants and cationic surfactants. Prolonged exposure of skin to surfactants can cause chafing because surfactants (e.g. soap) disrupt the liquid coating that protects skin and other cells. There have been the reports that SDBS (sodium dodecyl benzene sulfonate) is absorbed through the skin, they damage the liver and cause narrowing and other chronic symptoms, as well as teratogenic and carcinogenic (Toll et al., 2000). It is based on these effects that this study was carried out to determine the biosurfactant producing ability of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus sp., Corynebacteriumsp., Proteus sp. and ascertain their antimicrobial prorperties for applications in medical, therapeutics, pharmaceuticals, cosmetcs, food and beverages for treatment and control of diseases caused by micro organisms.

Production of biosurfactant:-
Biosurfactant was produced using stock cultures of Staphylococcus aureus, Bacillus sp., Corynebacterium sp, Pseudomonas aeruginosa, and Proteus sp. A loopful of each of the isolates was placed in 5 ml of sterile peptone water in a test tube and 0.1 ml of hydrocarbon (kerosene) was added to enhance the growth of the bacterial species. The suspensions were then allowed to stand for 48 h. After the 48 h incubation at room temperature, the biosurfactant produced by each microorganism was extracted as described by Anandaraj and Thivakaran, (2010); Okore et al., (2017a; 2017b).

Extraction of biosurfactant:-
The biosurfactant produced by each of the isolates was extracted by centrifuging at 5000 rpm for 20 min to obtain a cell-free supernatant of each of the test organisms. Then l ml supernatant of each of the suspension was taken and placed in a sterile glass Petri-dish and acidified with 1 ml of 2 M H 2 SO 4 to obtain a pH of 2.0. Thereafter, the 583 biosurfactant produced was extracted using a mixture of chloroform and methanol in the ratio of 1:2 (1 ml of chloroform: 0.5 ml of methanol). The mixture of biosurfactant and the extracting solvents (chloroform and methanol) was allowed for 24 h to evaporate at room temperature. Then the biomass of biosurfactant produced was determined by subtracting the total weight of the Petri-dish with the biosurfactant from the initial weight before the experiment (Okore et al., 2017a; 2017b).

Antibacterial activity of the biosurfactant produced:-
The disc technique as described by Osadebe and Ukwueze, (2004) was adopted for this study to evaluate the antibacterial activity of the biosurfactants. About 0.2 ml aliquot of the biosurfactants were dropped on sterile filter paper disc of about 6 mm in diameter and allowed to get absorbed before they were placed into nutrient agar plates inoculated with each of the test organisms Staphylococcus aureus and Pseudomoas aeruginosa and appropriately labelled, discs impregnated with water and ethanol were used as control in each case. The plates were then incubated at 37 o C for 24 h and the zones of inhibition obtained by each of the biosurfactant were measured.

Results:-
The results of the production of biosurfactant by the stock culture of Staphylococcus aureus, Bacillus sp., Corynebacterium sp., Pseudomonas aeruginosa, and Proteus sp. are presented in Table 1 and result for the antimicrobial activities of the biosurfactant produced on Table 2 and Table 3.

Discussion:-
The results of the mass of biosurfactant produced by the different bacterial species as presented in Table 1   The Staphylococcus aureus and Proteus sp. grown on kerosene as the carbon source yielded 0.5 g and 0.2 g of biosurfactant. These two organisms have scarcely been reported in literature as biosurfactant producers, as emphases have been on the use of non pathogenic strains for biosurfactant production with specificity to the area of application.
The above Table 2 (Rodrigues et al., 2006a;2006b;2006c). These biosurfactants can be used in the production of antibiotics that are specific to the target bacteria since the biosurfactants produced will likely be specific to certain genes or genomic composition of the target bacteria.

Conclusion and recommendation:-
Biosurfactant can be produced from bacterial broth cultures supplemented with hydrocarbons (e.g. kerosene) and extracted by acidification followed by liquid liquid extraction with chloroform-methanol mixture in the ratio of 2:1. The biosurfactants also have pronounced antibacterial activities against Staphylococcus aureus and Pseudomonas aeruginosa. The Staphylococcus aureus is well implicated in causing various infections including wound infections and other superficial infections. This thus validates the reported medical importance of biosurfactant. It is therefore recommended that biosurfactant be massively produced as well as purified and used in the production of pharmaceutical products due to their proven antimicrobial activities.