Effects of Iron Salts on Rhamnolipid Biosurfactant Production

Transcription

1 BIOLOGIA (PAKISTAN) 211, 57 (1&2), PK ISSN Effects of Iron Salts on Rhamnolipid Biosurfactant Production * MUHAMMAD IRFAN MAQSOOD 1, ASIF JAMAL 2, & HAFIZ ABDUL AZEEM 3 1 Department of Molecular Biology and Biotechnology, University of Lahore, Lahore, 2 Department of Microbiology, Quaid-e-Azam University, Islamabad 3 IEER, University of Engineering and Technology, Lahore ABSTRACT In this research, previously identified Pseudomonas aeruginosa (IMBB Strain) was re-streaked onto a fresh nutrient agar slants and stored at 4 C for further use. Iron limited mineral salt medium with the prescribed composition was used for the production of biosurfactants. Three different salts, namely ferrus chloride, ferrus sulfate and ferrous ammonium sulfate, were selected to analyze their effect on rhamnolipid production. The experiments were designed in three batches containing varying concentrations of ferrous chloride, ferrous sulfate and ferrous ammonium sulfate with one control flask. The produced rhamnolipid was detected physically and chemically by surface tension measurement and Orcinol assay, respectively. There was highest yield of Rhamnolipid of 3.81 g/l when the medium of Manitol was varied with.8 g/l of ferrous sulfate. The Rhamnolipid production using.4 g/l ferrous chloride was estimated as 1.85 g/l. INTRODUCTION Biosurfactans are structurally diverse group of surfactants (surface active compounds) produced by living organisms (microorganisms) and contain both hydrophobic and hydrophilic moieties that reduce surface tension and interfacial tension between individual molecules at the surface and at interfaces respectively (Lin et al., 1998). Biosurfactants have applications in a wide variety of industrial processes involving emulsification, foaming, detergency, wetting, dispersing or solubilization. There is almost no modern industrial operation where properties of surfaces and surface active agents are not exploited (Fiechter, 1992). Biosurfactants attracted attention as hydrocarbon dissolution agents in the late 196s and their applications have been greatly extended in the past five decades as an improved alternative to chemical surfactants (carboxylates, sulphonates and sulphate acid esters) especially in food, pharmaceuticals and oil industry (Banat et al., 2.). The reason for their popularity as high value microbial products is primarily because of their specific action, low toxicity, higher biodegradability, effectiveness at extreme temperature, ph, salinity, widespread applicability, and their unique structures which provides new properties that classical surfactants may lack (Kosaric, 1992). Biosurfactants possess the characteristic property of reducing the surface and interfacial tension using the same mechanism like chemical surfactants. Unlike chemical surfactants which are mostly derived from the petroleum and feedstock, these molecules can be prepared by the microbial fermentation processes using cheaper agro-based substrates and waste materials. During the past few years biosurfactant production by various *Corresponding author: muhammadirfanmaqsood@gmail.com

2 122 M. I. MAQSOOD ET AL BIOLOGIA (PAKISTAN) microorganisms has been studied extensively. Also various aspects of biosurfactant such as their biomedical and therapeutic properties, natural roles, production on cheap alternative substrates, and commercial potential have been reviewed by Gautam & Tyagi (25). Unlike chemically synthesized surfactants, which are classified according to the nature of their polar grouping, biosurfactants are characterized mainly by their chemical composition and their microbial origin. In general their structure includes a hydrophilic moiety consisting of amino acid or peptides anions or cations; mono, di, or polysaccharides; and a hydrophobic moiety consisting of unsaturated or saturated fatty acids (Desai & Banat, 1997). Rhamnolipid, biosurfactants were first isolated from Pseudomonas aeruginosa and described by Jarvis & Johnson (1949). These compounds are predominantly constructed from the union of one or two rhamnose sugar molecules and one or two β-hydroxy (3-hydroxy) fatty acids (Banat et al., 2). Rhamnolipid with one sugar molecule are referred to as mono-rhamnolipid, while those with two sugar molecules are di Rhamnolipid. The length of the carbon chains found on the β-hydroxyacyl portion of the Rhamnolipid can vary significantly. However in the case of Pseudomonas aeruginosa 1-C molecule chain is predominantly formed (Singh & Cameorta, 24). Pseudomonas aeruginosa is a facultative anaerobe and non-spore forming bacterium. Pseudomonas aeruginosa involves secretion of Pyoverdin (florescence- a flourish yellow-green siderophores under iron limited conditions, such other siderophores are Pyocyanin (blue-green), Pyorubin (Red-brown) pigments. Pseudomonas aeruginosa produces exopolysacharides (Slime Layers). Secretion of exopolysacharide makes it difficult for Pseudomonas aeruginosa to be phagocytozed by human white blood cells. Rhamnolipid from Pseudomonas aeruginosa decrease the surface tension of the water to 26 mn/m and the interfacial tension of water / hexadecane to < 1mN/m (Hisatsuka et al., 1971). Some biosurfactants are stable even after autoclave (121 C /2 min ) and after 6 months at -18 C, the surface activity did not change from ph 5 to 11 (Nitschke & Pastore, 199). Microbial products like surfactants are easily degradable and particularly suited for environmental applications such as bioremediation and dispersion of oil spills (Mulligan, 25). MATERIALS AND METHODS Microorganism and MSM Previously identified Pseudomonas aeruginosa (IMBB Strain) was kindly provided by the Institute of Molecular Biology and Biotechnology. The organism was re-streaked onto a fresh nutrient agar slants and stored at 4 C for further use. Manitol was used as carbon source. Iron limited mineral salt medium was used for the production of biosurfactants. The composition of MSM was Na2HPO4 (4 g/l), KH2PO4 (1 g/l), NaNO3 (2 g/l), MgSO4.7H2O; (.8 g/l), CaCl2.2H2O; (.1 g/l), FeSO4.7H2O (.1 g/l). Inoculum preparation From the fresh growth of Pseudomonas aeruginosa-imbb, a loop full of the bacteria was taken and transferred into 1 ml sterilized nutrient broth media. The flask was then placed into an orbital shaker at 37 C and 12 rpm for 48 hours. After 48 hours the turbidity of the cultured broth was measured using

3 Vol. 57 (1&2) Effects of Iron Salts on Rhamnolipid 123 Unicam spectrophotometer as Nutrient Broth + Agar (1.5 %) was used as solidifying agent Production of Biosurfactants After the preparation of inoculum, 1 ml of inoculum was taken for each flask, containing mineral salt medium for the production of biosurfactants with different types and composition of iron containing compounds like ferrous sulfate, ferrous chloride and ferrous ammonium sulfate. The fermenting flasks were placed into an orbital shaker at 37 C for 24 hours and after every 24 hours, 1 ml sample was taken for the detection and quantification of Rhamnolipid. The supernatant was separated after the interval of every 24 hours upto 12 hours and was centrifuged at 1, rpm for 2 minutes. After every 24 hours, growth was monitored by spectrophotometer at 6nm and supernatants were preserved for the estimation of Rhamnolipid and surface tension measurement. Studying the effect of various iron containing salts on biosurfactant production Three different salts containing iron were selected to understand the effect of these compound on Rhamnolipid production namely ferrus chloride, ferrus sulfate and ferrous ammonium sulfate. The experiments were designed in batch modes. Three batches were made. In the first batch, varying concentrations of the ferrous chloride were used (.1,.4,.8 and.12 g/l). The composition of the media remained the same as described previously and one control flask with no salt concentration(any) and named as control. All the flasks with media containing salts and the control were autoclaved at 12 C for 2 minutes at 15 lbs pressure. After autoclaving, the flasks were kept in the laminar flow hood and cooled and inoculated with 1ml prepared inoculum and then these flasks were kept in orbital shaker at 37 C for 24 hours. The bacterial growth was monitored by spectrophotometer after every 24 hours by taking 1 ml from each flask and the remaining were centrifuged at 1, rpm for 2 minutes with Ultracentrifuge machine. The supernatants were separated after every twenty four hours and preserved at the cool temperature for the characterization of the produced biosurfactants. In the second batch, varying concentrations of the ferrous sulfate were used (.1,.4,.8 and.12 g/l) and one control flask with no salt concentration and named as control. The procedure was repeated and supernatants were obtained after monitoring the bacterial growth. The supernatants were preserved at the cool temperature for the characterization of the produced biosurfactants. In the third batch, varying concentrations of the ferrous ammonium sulfate were used (.1,.4,.8 and.12 g/l) and one control flask. The same batch procedure of the first and second batch repeated with the flasks of the third batch and bacterial growth was monitored and supernatants were preserved for the estimation and characterization of the produced biosurfactant. All the fermentation experiments were done twice and the most probable reading was considered. Detection of biosurfactants The produced Rhamnolipid was detected physically and chemically by the Surface tension measurement and Orcinol assay, respectively.

4 124 M. I. MAQSOOD ET AL BIOLOGIA (PAKISTAN) Surface tension measurement The Rhamnolipid production was detected by the measurement of surface tension by the Tensiometer model Kruss K-1 in the bioremediation lab, environmental biotechnology division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. The Tensiometer was optimized and corrected at zero; the ring of the Tensiometer was sterilized by fire and suspended on the hook. The cuvette was washed by the distilled water and filled with distilled water for the determination of the surface tension of the water and then another cuvette was filled with the supernatant separated from the flasks of daily fermentation for the detection of the Rhamnolipid biosurfactant. The same experiment was repeated with each of the samples collected after fermentation and the surface tension was measured by the readings of the Tensiometer as numeric digits. Orcinol Assay The Rhamnolipid production was determined by the Orcinol assay in which 35 μl of supernatants of each sample and 1 ml of diethylether were added. Then it was vortexed for two minutes by vortex machine and kept undisturbed for 1 hour. After one hour organic phase was separated and pooled for evaporation at 37 C for 24 hours. After 24 hour of evaporations, the resultant powder was dissolved in.5 ml distilled water. Then 1 μl was obtained and mixed with the 9 μl of 97% Orcinol reagent and incubated at 8 C for 2 minutes and then it was cooled for 15 minutes and assayed using UV/VIS spectrophotometer. The obtained Optical density Y was applied for each sample into following formula and Rhamnolipid was measured as X from the following formula. X(g/L) = Y RESULTS & DISCUSSION Bacterial Growth Monitoring Bacterial grown is an important phenomenon in fermentation especially when biosurfactant production is the main goal. Different concentrations of iron salts have been used indicated as conc. 1 is for.1 g/l, conc. 2 is for.4 g/l, conc. 3 is for.8 g/l and conc. 4 is for.12 g/l. The growth was measured in optical density after each 24 hours upto 12 hours for all concentrations of different salts used in batch experiments as shown in Table 1, 2 and 3. The growth patterns are expressed in graphs in Fig., 1, 2 and 3 for different salt concentrations used in medium.

5 Vol. 57 (1&2) Effects of Iron Salts on Rhamnolipid 125 Table 1: Bacterial Growth Monitoring after 12 hours of incubation using different concentration of ferrous sulfate salt and measured as Optical Density Time (hours) Control Conc. 1 Conc. 2 Conc. 3 Conc. 4.1 g/l.4 g/l.8 g/l.12 g/l hours hours hours hours hours Table 2: Bacterial Growth Monitoring after 12 hours of incubation using different concentration of ferrous chloride salt measured as optical density Time (hours) Control Conc. 1 Conc. 2 Conc. 3 Conc. 4.1 g/l.4 g/l.8 g/l.12 g/l hours hours hours hours hours Table 3: Bacterial Growth Monitoring after 12 hours of incubation using different concentration of ferrous ammonium sulfate salt measured as Optical Density Time (hours) Control Conc. 1 Conc. 2 Conc. 3 Conc. 4.1 g/l.4 g/l.8 g/l.12 g/l hours hours hours hours hours

6 Optical 6 nm Optical 6 nm 126 M. I. MAQSOOD ET AL BIOLOGIA (PAKISTAN) Bacterial Growth using different conc. of FeSo g/l.4 g/l.8 g/l.12 g/l.5 hr 24 hr 48 hr 72 hr 12 hr Time (hr) Fig., 1: Bacterial growth using different conc. of FeSO 4 Bacterial Growth using different conc. of FeCl g/l.4 g/l.8 g/l.12 g/l hr 24 hr 48 hr 72 hr 12 hr Time (hr) Fig., 2: Bacterial growth using different conc. of FeCl 2

7 Optical 6 nm Vol. 57 (1&2) Effects of Iron Salts on Rhamnolipid 127 Bacterial Growth using different conc. of Fe(NH4)2(SO4) g/l.4 g/l.8 g/l.12 g/l.5 hr 24 hr 48 hr 72 hr 12 hr Time (hr) Fig., 3: Bacterial growth using different conc. of Fe(NH 4 ) 2 (SO 4 ) 2 Measurement of Surface Tension Reduction Surface tension reduction is the major property of biosurfactants. Measurement of surface tension reduction helps us to measure the efficiency of a biosurfactant. Table 4, 5 and 6 demonstrates the surface tension reduction of the supernatants achieved after every 24 hours of fermentation experiment with different concentrations of ferrous sulphate, ferrous chloride and ferrous ammonium sulphate, respectively. The surface tension was measured as digit by digital Tensiometer of all the supernatants separated after every 24 hours. The reduction patterns are articulated as graphs in Fig., 4, 5 and 6 for all three kind of above mentioned salts. Table 4: Surface Tension (mn/m) Reduction during 12 hours of incubation using different concentration of FeSO 4 Time (hours) Conc.1 Conc. 2 Conc. 3 Conc. 4.1 g/l.4 g/l.8 g/l.12 g/l hr hr hr hr hr

8 Surface Tension 128 M. I. MAQSOOD ET AL BIOLOGIA (PAKISTAN) Table 5: Surface Tension (mn/m) Reduction during 12 hours of incubation using different concentration of FeCl 2 Conc.1 Conc. 2 Conc. 3 Conc. 4 Time (hours).1 g/l.4 g/l.8 g/l.12 g/l hr hr hr hr hr Table 6: Surface Tension (mn/m) Reduction during 12 hours of incubation using different concentration of ferrous ammonium sulfate Control Conc. 1 Conc. 2 Conc. 3 Conc. 4 Time (hours).1 g/l.4 g/l.8 g/l.12 g/l hr hr hr hr hr Surface Tension Reduction using different conc. of FeSo g/l.4 g/l.8 g/l.12 g/l 2 1 hr 24 hr 48 hr 72 hr 12 hr Time (hr) Fig., 4: Surface Tension Reduction using different conc. of FeSO 4

9 Surface Tension Surface Tension Vol. 57 (1&2) Effects of Iron Salts on Rhamnolipid 129 Surface Tension Reduction using different conc. of FeCl g/l.4 h/l.8 g/l.12 g/l 2 1 hr 24 hr 48 hr 72 hr 12 hr Time (hr) Fig., Surface 5: Surface Tension Tension Reduction Reduction using different using conc. different of Fe(NH4)2(SO4)2 conc. of FeCl g/l.4 g/l.8 g/l.12 g/l 2 1 hr 24 hr 48 hr 72 hr 12 hr Time (hr) Fig., 6: Surface Tension Reduction using different conc. of Fe(NH 4 ) 2 (SO 4 ) 2

10 Rhamnolipid (g/l) 13 M. I. MAQSOOD ET AL BIOLOGIA (PAKISTAN) Estimation of Rhamnolipid Production Table 7 explains the production of rhamnolipid after every 24 hours upto 12 hours of the fermentation duration. The Rhamnolipid produced after 12 hours was measured as 1.62 g/l in the control medium, 1.85 g/l in the medium of ferrous chloride with concentration 2 (.4 g/l), 3.71 g/l in the medium of ferrous sulfate with concentration 3 (.8 g/l) and 2.7 g/l in the medium of ferrous ammonium sulfate with concentration 2 (.4 g/l) (Fig., 7). Table 7: Estimation of rhamnolipid production in control and different medium Medium containing ferrous ammonium sulfate (Conc..4 g/l) X= Rh. (g/l) Y=OD Medium containing ferrous sulfate (Conc..8 g/l) X= Rh. Y=OD (g/l) Medium containing ferrous chloride (Conc..4 g/l) X= Rh. (g/l) Y=OD Control Medium (without salt) X= Rh. (g/l) Y=OD Time (hours) hr hr hr hr Estimation of rhamnolipid production Control Medium Medium containing FeCl2 Medium containing FeSO4 Medium containing (NH4)2Fe(SO4) hr 48 hr 72 hr 12 hr Time (hours) Fig., 7: Estimation of Rhamnolipid production different medium

11 Vol. 57 (1&2) Effects of Iron Salts on Rhamnolipid 131 After every 24 hours, there is continuous production of rhamnolipid from hours to 12 hours of the fermentation (Fig. 7) as it has been discussed in table 7. The highest production was observed in the batch of medium containing ferrous sulphate with.8 g/l concentration. Rhamnolipid produced by Pseudomonas aeruginosa are the subject of interest due to their diverse application area. Literature reveals that there is a few studies have been conducted to explore the effect of iron on rhamnolipid production. The role of iron is imperative and should be cleared to design an appropriate fermentation process. Less attention towards this direction left some conceptual errors hence present investigation is aimed to explore the effect of iron containing compounds on the bacterial physiology and synthetic capabilities. The best property to characterize the rhamnolipid biosurfactant is its surface tension reduction ability, as it was described by Hisatsuka, et al., in Researches show that rhamnolipid reduced the surface tension of water from 72 to 27 mn/m. The biosurfactant with surface tension reduction activity range from 27-3 mn/m are considered as excellent Biosurfactants. The pure form of the Rhamnolipid shows the surface tension reduction of 27 mn/m (Lang & Wagner, 1987). Rhamnolipid produced by Pseudomonas aeruginosa IMBB strain shows 33.4 mn/m of surface tension reduction in controlled medium as it is shown in table 6. The measurement of surface tension reduction in the medium using various concentrations of different salts (ferrous sulfate, ferrous chloride and ferrous ammonium sulphate) is also shown in table 4, 5 and 6 respectively. The fermentation of Manitol with the medium free from iron salts yields 1.62 g/l rhamnolipid after the incubation for 12 hours as it is shown in Table 7. Table 7 also explains the rhamnolipid production when the medium was changed with different concentrations of iron containing compounds. There was highest yield of rhamnolipid of 3.81 g/l when the medium of Manitol was varied with.8 g/l of ferrous sulfate. The Rhamnolipid production using.4 g/l ferrous chloride was estimated as 1.85 g/l. Acknowledgements We would like to thank to the administration of the University of Lahore and National Institute for Biotechnology and Genetic Engineering (NIBGE) for allowing to conduct this research in the research laboratories of the Institute of Molecular Biology and Biotechnology (IMBB) and Environmental biotechnology division. REFERENCES Banat, I.M., Makkar, R.S. & Cameotra, S.S., 2. Potential commercial application of biosurfactants. Appl. Microbiol. Biotechnol., 53: Desai, J.D. & Banat, I.M., Microbial production and their commercial potential. Micr. Mol. Biol. Rev., pp:47-64 Fiechter, A., Biosurfactants moving towards industrial application. Trends Biotechnol., 1: Gautam, K.K. & Tyagi, V.K., 25. Microbial surfactants: A review. J. Oleo. Sci., 55:

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