{"id":1283,"date":"2015-03-21T08:00:31","date_gmt":"2015-03-21T08:00:31","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=1283"},"modified":"2020-04-25T11:10:36","modified_gmt":"2020-04-25T11:10:36","slug":"screening-optimization-and-production-of-biosurfactants-from-bacillus-and-pseudomonas-sp","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol3no1\/screening-optimization-and-production-of-biosurfactants-from-bacillus-and-pseudomonas-sp\/","title":{"rendered":"Screening Optimization and Production of Biosurfactants from Bacillus and Pseudomonas sp"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Biosurfactants are amphiphilic compounds produced on living surfaces, mostly microbial cell surfaces or excreted extracellularly and contain hydrophobic and hydrophilic moieties that reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively.(Karanth et al.,<\/em> 1999) biosurfactant producing microorganisms\u00a0 were naturally present in oil contaminated soil. Oil contaminated environment contain large amount of hydrocarbons. i.e., aliphatic and aromatic hydrocarbons. Microorganisms exhibit emulsifying activity by producing biosurfactants and utilize\u00a0 the hydrocarbons as substrate often mineralizing them or converting them into harmless products.<\/p>\n

Biosurfactants have gained more importance in the fields of enhanced oil recovery, environmental bioremediation, food processing, and pharmaceuticals owing to their unique properties- higher biodegradability, lower toxicity, and effectiveness at extremes of temperature, pH and salinity. Biosurfactants can be produced by microbial fermentation processes using cheaper agrobased substrates and waste materials.In various industrial processes, they are potencially useful surface-active agents for emulsion polymerization, wetting, foaming, phase dispersionl, emulsification and de-emulsification.(Desai et al<\/em>., 1997)<\/p>\n

Biosurfactants are mainly categorized mainly by their chemical composition and microbial origin. Generally their structures include a hydrophilic moiety consisting of aminoacids or peptides, mono-di or polysaccharides and hydrophobic moiety comprising unsaturated or saturate fatty acids.Accordingly, the major classes of biosurfactants include glycolipids, lipopeptides, lipoproteins, phospholipid, fatty acids, polymeric biosurfactant and particulate biosurfactants.(Manneerat et al.,<\/em> 2005)<\/p>\n

Among the different classes of biosurfactants rhamnolipid and surfactin are best studied. Rhamnolipid is produced by Pseudomonas aeruginosa, a gram-negative, motile, non spore forming bacteria. Surfactin is cyclic lipopeptide commonly used as an antibiotic. Surfactin\u2019s structure consists of a peptide loop of seven aminoacids(L-asparagine, glycine, two L-leucine, L-valine, and two D-Leucins) and an hydrophobic fatty acid chain thirteen to fifteen carbon long.(Peypoux et al.,<\/em> 1999)<\/p>\n

Materials and Methods<\/strong><\/p>\n

The soil samples were collected from polluted sites and isolate the bacteria Bacillus<\/em> and Pseudomonas<\/em> from oil contaminated soil by gram staining and by various biochemical test. Screening of biosurfactants using the following methods.<\/p>\n

Screening for biosurfactant activity<\/strong><\/p>\n

The supernatant was subsequently subjected to the preliminary screenig methods by using 3 different oil namly petrol, Diesel and kerosene.<\/p>\n

Oil spreading techniques<\/strong><\/p>\n

The 50ml of distilled water was added to a large petri dish (15cm diameter) followed by the addition of 20ml of Oil to the surface of water, 10ml of supernatant of culture broth (Rodrigues et al.,<\/em> 2006)<\/p>\n

Emulsification cabacity(E24) test<\/strong><\/p>\n

E24 of culture\u00a0 samples was determined by adding 2ml of oil to the same amount of culture, mixing with a vortex for a 2min and leaving to stand for 24 hours. The E24 index is given as percentage of height of emulsified layer(cm) divided by total height of the liquid column(cm) (Sarubbo 2006)<\/p>\n

Table 1: (A)\u00a0Emulsification Index In\u00a0 Petrol<\/strong><\/p>\n\n\n\n\n
Organisms<\/strong><\/td>\nEmulsifiedLayer (cm)<\/strong><\/td>\nTotal Liquid Column(cm)<\/strong><\/td>\n% of E24<\/strong><\/td>\n<\/tr>\n
\u00a0<\/em>Bacillus subtilis<\/em>Pseudomonas aeruginosa<\/em><\/td>\n0.60.8<\/td>\n2.92.7<\/td>\n2130<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Drop collapse method<\/strong><\/p>\n

For drop collapse method 2ml of oil was added to each well of a 96 well containing microtiter plate lid. The lid was equilibrated for 1 h at room temperature. MSM cultures of bacterial isolates was centrifuged and then 5ml of the drop on the surface of oil was evaluated after 1 min.<\/strong>(Jain et al.,<\/em> 1991)<\/p>\n

Table 1: (B)<\/strong>Emulsification Index In Diesel<\/strong><\/p>\n\n\n\n\n
 <\/p>\n

Organisms<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Emulsified<\/strong><\/p>\n

Layer (cm)<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Total Liquid Column(cm)<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

% of E24<\/strong><\/td>\n<\/tr>\n

\u00a0<\/em><\/p>\n

Bacillus subtilis<\/em><\/p>\n

Pseudomonas aeruginosa<\/em><\/td>\n

 <\/p>\n

0.3<\/p>\n

0.5<\/td>\n

 <\/p>\n

3.2<\/p>\n

3.0<\/td>\n

 <\/p>\n

9<\/p>\n

17<\/p>\n

 <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Haemolytic activity <\/strong><\/p>\n

Isolates were screened on blood agar plates containing 5% sheep blood and incubated at 37\u00b0C for 48 h. Hemolytic activity was detected as the presence of clear zone around bacterial isolates (Plaza et<\/em> al ., 2006)<\/p>\n

\u00a0<\/strong>Table 1: (C)<\/strong>Emulsification Index In Kerosine<\/strong><\/p>\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Organisms<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Emulsified<\/strong><\/p>\n

Layer (cm)<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Total Liquid Column(cm)<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

% of E24<\/strong><\/td>\n<\/tr>\n

\u00a0<\/em><\/p>\n

Bacillus subtilis<\/em><\/p>\n

Pseudomonas aeruginosa<\/em><\/td>\n

 <\/p>\n

0.3<\/p>\n

0.4<\/td>\n

 <\/p>\n

3.2<\/p>\n

3.1<\/td>\n

 <\/p>\n

9<\/p>\n

13<\/p>\n

 <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Surface tension measurement<\/strong><\/p>\n

Surface tension of biosurfactant was measured by drop weight method . A burette is held\u00a0 vertically and liquid drops are allowed to from its lower end at a very solw rate\u00a0 about 8 drops per minute. A clean dry beaker is weighted and placed under flow of burette. 50 drops of the liquid are collected and weighted the beaker. Again repeat the same for 3 times.the average mass of one drop is calculated and then surface tension of liquid containg biosurfactant calculated by ( surface tension(T) = Mg\/3.8r )<\/p>\n

Process optimization and biosurfactant production<\/strong><\/p>\n

The\u00a0 pH ranges from 6,7,and 8, temperature ranges from 35\u00b0C, 37\u00b0C and 39\u00b0C and kerosene concentration from 1%, 3% and 5% for optimization of biosurfactants production in bacillus<\/em> and pseudomona sp<\/em>. Both\u00a0 the organisms have maximum growth rate observed\u00a0 at pH 7, temperature of 37\u00b0C in 0.3% kerosene concentration. Effective biosurfactants production from Bacillus <\/em>and Pseudomonas sp<\/em> can be achieved using Mineral salt broth.<\/p>\n

Result and Discussion<\/strong><\/p>\n

Four isolates of bacillus<\/em> and pseudomonas<\/em> have highest E24 value was observed in diesel (Priya and usharani 2009). <\/strong>In case our study \u00a0Bacillus<\/em> and Pseudomonas <\/em>have the ability to emulsifying oils. The highest E24 value was observed in petrol and Pseudomonas<\/em> showed the better E24 value than bacillus.<\/em> (table \u20131). In oil spreading techniques bacillus<\/em> shows higher zone formation of 4.1mm, 3.6mm, 3.2mm in petrol , diesel, and kerosene respectively. Similarly our study focused Pseudomonas<\/em> showed 3.9mm , 3.3mm, 2.8mm in petrol, diesel, and kerosene.<\/p>\n

Surface tension reduction was measured by Kruss Hamburg Nr2215 Tensio meter. Results were compared to medium composition as negative control\u00a0( Pavitran et<\/em> al.,<\/em> 2004) Since our study of surface tension\u00a0 reduction was measured by drop weight method and Pseudomonas<\/em> shows 0.007 surface tension reduction per drop.<\/p>\n

Blood agar method is often used for a preliminary screening of microorganisms for the ability to produce biosurfactants on hydrophilic media. (Schulz et al .,<\/em> 1991). From our results both the isolates will cause lysis of the blood cells and exhibit a colorless, transparent ring around the colonies.<\/p>\n

Optimization of biosurfactants was carried out by using different pH , temperature and concentration of carbon source ( kerosene) (chart-1)<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 1<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Conclusion<\/strong><\/p>\n

From the above observation, it was concluded that both bacterial isolates of bacillus <\/em>and pseudomonas<\/em>\u00a0 have the ability to secrete surface active agents it is gain more importane in future for industrial and environmental applications.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Desai, J.D., and Banat, I.M., 1997. Microbial production of\u00a0 surfactants and their commercial potencial. Microbiol. Mol. Biol., 61: 47-64.<\/li>\n
  2. Jain, D.K., Collins Thommpson, D.L., Lee, H. 1991. A drop collapsing test for Screening biosurfactant producing microorganisms. J. Microbiol Methods.,<\/em> 13: 271-279.<\/li>\n
  3. Karanth, N.G.K., Deo, P.G.\u00a0 and Veenanadig, N.K. 1999. Microbial Production of biosurfactants and their importance. Curr.Sci.,<\/em> 77: 116-123.<\/li>\n
  4. Maneerat, S. 2005. Biosurfactants from marine microorganisms. Songklanakarin J<\/em> Sci Technol.,<\/em> 27: 1263-1272<\/li>\n
  5. Peypoux, F., Bonmatin, J.M., and Wallach, J. 1999. Recent trends in Biochemistry Of\u00a0 Surfactin. Applied Microbiol Biotechnol.,<\/em> 51: 553-563.<\/li>\n
  6. Plaza, G.A., Zjawiony, I., Banat, I.M. 2006. Use of different methods for detection of thermophilic biosurfactant producing bacteria from hydrocarbon-contaminated and bioremediated soils. J. Petroleum Sci Engg., <\/em>50: 71-77.<\/li>\n
  7. Priya, T., Usharani, G. 2009. Comparative Study for Biosurfactant Production by Using Bacillus subtilis<\/em> and Pseudomonas aeruginosa.<\/em> Bot. Res. Intl., 2(4): 284-287.<\/li>\n
  8. Rodrigues, L.R., Teixeira, J.A., Mei, H.C., and Oliveira, R. 2006. Physiochemical and functional characterisation of a Biosurfactant produced by Lactococcus lactis 53, Colloids and surfaces. B: Biointerfaces.,<\/em> 49: 79-86<\/li>\n
  9. Schulz, D., Passeri, A., Schmidt, M. 1991. Screening for biosurfactants among crude-oil degrading marine microorganisms from the North-sea. z Naturforsch<\/em> ( C )., 46(4): 197-203.<\/li>\n
  10. Saruboo, L.A. 2006. Production and Stability studies of the Bioemulsifier obtained from a strain of Candida glabrata UCP1002. Journal of Biotechnology.,<\/em> 9(4): 400-401.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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