Bioprocess Engineering 22 (2000) 9599 Springer-Verlag 2000

Scale up studies for the production of biosurfactant in packed column bioreactor

N. K. Veenanadig, M. K. Gowthaman, N. G. K. Karanth

Abstract Biosurfactants capable of emulsifying pesticides have great potential to assist in microbial degradation of the pesticides. Solid State Fermentation (SSF) due to several advantages, is one of the efcient ways of producing these surfactants and seldom receives attention for commercial exploitation. In this study, a packed column bioreactor with wheat bran as the raw material and Bacillus subtilis has been used to produce a biosurfactant specic to disperse Fenthion, an organophosphrous pesticide. The emulsier activity (EA) and surface tension from the packed column bioreactor were compared with ask fermentation experiments, which served as control. Airow rate in the packed column bioreactor was varied from 1020 l/min. Maximum EA and minimum surface tension occurred at airow rate of 20 l/min. Peak EA in the control was 1.2 at 29 h while it was 1.9 in the bioreactor. The least surface tension of 24 dynes/cm was noticed at 54 h in the bioreactor, which was 33% better than the control at the same time period. The results indicate that the packed column bioreactor can become a more acceptable solid state fermentation system for commercial exploitation of Fenthion specic biosurfactant production.

1 Introduction Surfactants are amphiphilic molecules that tend to partition preferentially at the interface between phases of different polarity and water bonding. Considerable amount of surfactants are used in detergents for household and industrial purposes [1]. Currently surfactants are produced through chemical routes [2]. However, many microbes are capable of synthesizing different types of biosurfactants [36]. Hydrocarbons and other insoluble substrates induce
Received: 22 February 1999

biosurfactant production. By virtue of biodegradability, substrate specicity, rapid/controlled inactivation, chemical and functional diversity, biosurfactants are gaining importance in various industries like agriculture, food, textiles, petrochemicals etc. The biodegradative property of biosurfactants makes them ideal for environmental applications, especially pesticides removal. Literature reveals that application of biosurfactants in the eld of pesticides is still in its infancy when compared to the well-developed eld of hydrocarbon research. Also, there are postulations on the possible replacement of synthetic surfactants with biosurfactants in pesticide formulations and residue clean up [79]. Most of the earlier studies on production of biosurfactants have been performed in submerged fermentation (SmF). However, solid state fermentation (SSF) is a simple and efcient means of producing several important chemicals using inexpensive substrates. This study involves the production of a pesticide (Fenthion) dispersing biosurfactant by SSF systems, in a Packed Column Bioreactor (PCBR) using various ow rates. A ask experiment was carried out as control. This surfactant not only reduced the surface tension but also had the property of emulsifying Fenthion an organophosphorous pesticide and a pollutant. This property is very important especially in the cleaning operations for pesticides containers, containing Fenthion and other similar operations where Fenthion may have to be removed.

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2 Materials and methods 2.1 Organism and inoculum preparation Bacillus subtilis FE-2 strain deployed in studies were deployed in the study. Bacillus subtilis obtained from Indian Institute of Sciences, Bangalore, India, was routinely maintained on mineral medium supplemented with Fenthion and subcultured every month. Five ml of the organism from the stock culture was transferred into the 100 ml Luria broth (10 g tryptone, 5 g yeast extract, 5 g NaCl in 1 l of distilled water). A teon coated bar magnet sterilized with 70% ethyl alcohol was placed inside the inoculated culture ask. The culture was incubated at 30 C on a magnetic stirrer for 48 h (nal OD of 2.8) and used as inoculum whenever mentioned. 2.2 Solid state fermentation Coarse wheat bran (solid medium) procured from the Milling and Baking Technology Department, CFTRI,

N. K. Veenanadig, N. G. K. Karanth (&) Food Protectants and Infestation Control Department, Central Food Technological Research Institute, Mysore-570 013, India e-mail: v_nadig@mailciy.com M. K. Gowthaman Fermentation and Bioengineering Department, Central Food Technological Research Institute, Mysore-570 013, India Authors thank Director, CFTRI, Mysore, India for his interest in this study and also for providing the facility. Ms. Veenanadig is grateful to CSIR for a Senior Research Fellowship.

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Mysore was dried in a cross current hot air drier at 45 2.2.3 50 C and stored till use. All the experiments were carried Bioreactor media out at ambient temperature. Five hundred grams of wheat bran was weighed into a tray, mixed with 550 ml potassium phosphate buffer (0.1 M, 2.2.1 pH 7) to get 50% initial moisture. It was transferred to a Flask experiments 3 l ask, autoclaved at 121 C for 60 min and cooled to Ten grams of bran was weighed into 250 ml capacity Er- 30 C before use. The mix was retransferred to a sterile tray lenmeyer ask and mixed with sufcient potassium in the inoculation chamber. To this 250 ml of spore inphosphate buffer (0.1 M pH 7) to get 50% initial moisture. oculum was added as a suspension, followed by 0.5% The moist wheat bran medium was autoclaved at 121 C Fenthion (10 ml of 250 mg/ml as acetone) and handfor 60 min and then cooled to 30 C before use. Bacillus mixed thoroughly to get uniform mixing. Thereafter, the subtilis inoculum (5 ml suspension) was transferred fol- media was packed into the column. lowed by 0.5% Fenthion (200 ll of 250 mg/ml as acetone). Inoculation was performed under aseptic conditions. The 2.3 contents of the asks were mixed thoroughly by gently Moisture determination tapping the ask with hand. Moisture (%w/w) present in the samples was measured in a moisture meter (ARICO-Model M-30 A) tted with an 2.2.2 infra red source for rapid drying of samples.

Packed column bioreactor and experimental arrangement A schematic diagram of the experimental set up consisting of a humidier and a packed column bioreactor (PCBR) is shown in Fig. 1. The humidier consisted of 30 l capacity jacketed stainless steel column lled to 0.75 m height with distilled water. Through a perforated plate (size of the hole 1 mm) located at the bottom of the humidier, ltered air from the compressor was sparged. The humidied air was rst passed through a water trap to remove the excess (condensed) water. The PCBR consisted of a stainless steel column of diameter 150 mm and height 345 mm; sieves placed at the bottom and top of the column served to support the substrate and to prevent the escape of bran particles into the atmosphere, respectively, during the course of fermentation. Samples of fermented bran were drawn for analysis from a hole (25-mm dia) located at a height of 170 mm from the bottom of the column.

2.4 Extraction of the crude biosurfactant The biosurfactant in the ask was extracted from the fermented bran by adding 100 ml phosphate buffer (0.1 M) pH 7 and keeping on a rotary shaker for 30 min at room temperature. Similarly 10 g of fermented bran from the bioreactor was transferred into 250 ml ask and 100 ml phosphate buffer (0.1 M) pH 7 was added to it and kept on a rotary shaker for 30 min at room temperature. The suspension was allowed to separate by keeping it stationary for 10 min. The supernatant was decanted and centrifuged at 3820 g for 20 min using a Sorval RC 5B refrigerated centrifuge. The cell free supernatant was collected and used as the crude biosurfactant. 2.5 Determination of surface tension A clean and dry stalagnometer was xed to a stand in a vertical position. Distilled water was taken in a beaker and placed below it. A piece of rubber tubing was attached to the upper end of the stalagnometer, which carried a pinchcock. Water was sucked into the stalagnometer through the rubber tubing and the pinchcock was applied. The pinchcock was then released slowly and carefully to regulate water ow so that well-dened spherical drops were formed at a rate of 1520 drops per minute. Following this, water was again sucked into the stalagnometer without changing the adjustment of the pinchcock until the water level in the stalagnometer was above the upper mark ``A''. Water in the stalagnometer was then allowed to drop. The number of drops formed between the upper mark ``A'' and the lower mark ``B'' which was below the bulb was noted and counted. The process was repeated 23 times for water and the average number of drops formed was noted. The stalagnometer was drained and dried with acetone. The SSF extract was drawn into it and the experiment repeated as above. The number of drops formed between the marks A and B was counted. This procedure was repeated 23 times and the average number of drops calculated.

Fig. 1.

N. K. Veenanadig et al.: Production of pesticide specic biosurfactant in packed column bioreactor

Density of the liquid = weight of liquid density of water/weight of water.

Surface tension of liquid (dynes/cm) Density of liquid drops of water Surface tension of water Density of water drops of liquid

2.6 Determination of emulsifier activity The ability of an emulsier to aid the formation of an emulsion is related to its ability to absorb and to stabilize the pesticide-water interface. The surfactant activity was assayed by the standard method (1216) i.e., measuring the turbidity due to the formation of pesticide emulsion by the biosurfactant. To 5 ml of the culture supernatant taken in a test tube (25 ml capacity), 20 mg of Fenthion in acetone (0.2 ml) was added; the solution was then vortexed for 60 s, and left undisturbed at room temperature for 24 h. The optical density (O.D) of the emulsion was read in a Shimadzu UV-160A recording spectrophotometer at 660 nm [15]. Similarly, a blank was prepared without Fenthion. The difference in absorbancy between the sample and blank was taken as the Fenthion-specic surfactant activity.

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Fig. 2.

3 Results and discussion The bioemulsier was evaluated in terms of emulsier activity and the biosurfactant by surface tension. The biosurfactant of our system had a low surface tension accompanied by the emulsication of the insoluble substrate. Fenthion in the aqueous phase. A similar phenomenon was 3.2 Surface tension profile in flask and bioreactor observed by a couple of other workers [17, 18]. The proles of surface tension of bioreactor and ask samples are plotted in Fig. 3. The ow rate of 20 l/min, 3.1 which was shown earlier to yield the lowest surface tenSurface tension profile of the bioreactor system sion, has been selected for this purpose. Results show that at different airflow rates Since the bioreactor had been earlier found to be a better system with increase in air ow rates [19] there was a need to test the various air ow rates for the production of the minimum surface tension. The bioreactor was supplied with various airow rates viz., 10, 15, 17.5 and 20 l/min. The results are shown in Fig. 2, which reveals that the maximum airow rate of 20 l/min supported the best production of the surfactant. The least airow rate of 10 l/min hardly reduced the surface tension, whereas, the airow rates of 15, 17.5 and 20 l/min did support good surfactant production in a gradually increasing order. The surface tension of the simple collected from the bioreactor, which was supplied with airow of 20 l/min, reduced the surface tension to the lowest value of 24 dynes/cm. Although, there is a reduction in the surface tension in the experiments with other ow rates, 20 l/min seems to be the best airow to be provided for maximum reduction in surface tension. Oxygen transfer is enhanced at larger ow rates resulting in efcient fermentation, enhanced surfactant production and hence, further reduction in surface tension. Still higher airow rates were not tried due to some technical difculty. Higher airow rates have been found to reduce the optimum moisture content in the solid substrate bed in a packed column reactor [20]. In SSF, as Fig. 3.

the fermentation proceeds the void space or porosity of the bed decreases due to bacterial multiplication which tends to decrease the bed voidage, thereby decreasing the oxygen transfer into the interior of the bed. In addition, the airow rate of 10 l/min itself may not be sufcient to overcome transport resistance and supply the required oxygen to the biomass. These conditions probably have resulted in sub-optimal growth and surfactant production hence, insignicant reduction in surface tension at 10 l/min.

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there was a reduction in surface tension in both the samples collected from the bioreactor as well as the asks. In the ask system reduction in the surface tension took place in the rst 30 h and was reduced from 54 dynes/cm to 34 dynes/cm. Further incubation did not decrease the surface tension. In the contrary in the bioreactor sample, the rate of reduction of the surface tension was similar to the ask system until 30 h. However, the decrease in surface tension continued even after 30 h and decreased to 24 dynes/cm. This amounts to 33% increased efciency in the PCBR. No reduction was observed beyond 56 h. The results indicate the scale up efciency of the PCBR.

3.3 Emulsifier activity (EA) Emulsier activity of the crude surfactant present in the bioreactor as well ask samples (as given in Materials and methods) was estimated in order to compare and evaluate their bioemulsier properties. The results given in Fig. 4 show the EA as an index of emulsier production in these two systems. The emulsier production in the ask samples started around 10 hrs and reached a maximum activity of 1.2 units at the 29th hour and dropped thereafter. In the bioreactor although the activity started around 10 h it increased very shapely to attain a value of 1.9 in 31 h. References Beyond 31 h the reduction was slow up to 46 h. It is in1. Fiechter, A.: Biosurfactants: moving towards industrial application. TIBTECH 10 (1992) 208217 teresting to note that even at 50 h, the surfactant activity in 2. Greek, B.F.: Chem Eng News 69 (1991) 2552 the bioreactor was more (1.4) than the peak of the ask system (1.2). This has a distinct advantage that even by a 3. Cooper, D.G.; Zajic, J.E.; Denis, C.: Surface active properties of a biosurfactant from Corynebacterium lepus. JAOCS 58(1) slight error in the harvesting time, activity will not be af(1981) 7780 fected much. As compared to the ask fermentation, the 4. Cooper, D.G.: Biosurfactant, Microbiol. Sci. 3(5) (1986) 145 activity of the surfactant was almost double. In the case of 149 ask fermentation, to attain the peak activity (1.2) it took 5. Singer, M.E.: In: Zajic, J.E.; Donaldson E.C.: (eds) Microbes and oil recovery (Bioresource Publications, E1 Paso, Texas) 29 h, where as in bioreactor the activity of 1.2 was attained 1985, pp. 1938 in 20 h, thereby again indicating that the emulsier production production was faster in the case of the bioreactor 6. Haferberger, D.; Hommel, R.; Claus, R.; Kleber, H.P.: Extracellular microbial lipids as biosurfactants. Adv. Biochem. system. Thus, the results indicate that the PCBR system Eng/Biotechnol. 33 (1986) 5393 enhanced biosurfactant production and its rate hence, is 7. Banerjee, S.; Duttagupta, S.; Chakrabarty, A.M.: Production an efcient system for scaling up. of emulsifying agent during growth of Pseudomonas cepacia
8. 9. 10. 11. 12. 13. 14. 15. Fig. 4.

4 Conclusions Solid state fermentation is a simple and efcient technique to produce several interesting metabolites, one of which is biosurfactant production reported in this paper. This biosurfactant is specic to Fenthion and has the dual properties of surfactant and emulsier. This surfactant was produced by the addition of Fenthion to the culture medium. Experiments carried out in a packed column bioreactor have shown that the surfactant and emulsier activities are more efcient in the system and strongly dependent on the airow rate. The maximum surface tension reduction (33%) was obtained at an airow rate of 20 l/min. In addition, the bioreactor offered the advantage of achieving high emulsier activity in a short period of time over the ask fermentation system. A much-detailed experimentation will be required to clearly understand the interaction of oxygen transfer and surfactant production on which further work is going on. However, this is probably the rst report for pesticide dispersing biosurfactant production by SSF in a packed column fermentor, which shows promise for scale up application.

with 2,4,5-trichlorophenoxyacetic acid. Arch. Microbiol. 135 (1983) 110114 Patel, M.N.; Gopinathan, K.P.: Lysozyme-senstive bioemulsier for immiscible organophosphrous pesticides. Appl. Environ. Microbiol. 52 (5) (1986) 12241226. Appaiah, A.; Karanth, N.G.K.: Insecticide specic emulsier production by hexachlorocyclohexane utilizing Pseudomonas tralucida Ptm + strain. Biotechnol. Lett., 13:5 (1991) 371374 Veenanadig, N.K.; Karanth, N.G.K.: Proceedings of Annual Conference of Association of Microbiology held at IIT Chennai, Dec 46, (1996)107. Veenanadig, N.K.; Karanth, N.G.K.: Proceedings of Annual Meetings of Society of Biochemist (India) held at Andra University Dec 2224 (1997) 177 Rosenberg, M.: Microbial surfactants. Crit. Rev. Biotechnol. 3:2 (1986) 109132 Singh, M.; Desai J.D.: Hydrycarbon emulsication by Candida tropicalis and Debaryomyces polymorhus. Ind. J. Exp. Biol. 27 (1989) 224226 Patel, M.N.: Microbial emulsication and metabolism of organophosphrous pesticides, Ph.D thesis, Indian Institute of Science, Bangalore, India (1988) Appaiah, A.; Karanth, N.G.K.: Production of extracellular lipase by hexachlorocyclohexane utilising Pseudomonas strain. Lett. Appl. Microbiol. 21 (1995) 6567

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biochemical reactions in packed bed solid state fermentors: 16. Ramesha, N.: Biosurfactant production and biodegradation of Effect of temperature gradients. Enzyme Microb, Technol. 16 alpha HCH by Pseudomonas Ptm + strain. Ph.D thesis, (1994) 253257. CFTRI, Mysore. India (1996) 17. Reisfeld, A.; Rosenberg, E.; Gutnick, D.L.: Microbial degra- 19. Gowthaman, M.K.; Raghava Rao, K.S.M.S.; Gildhyal, N.P.; Karanth, N.G.K.: Gas concentration and temperature gradidation of crude oil: Factors affecting the dispersion in sea ents in a packed bed solid-state fermentor. Biotech. Adv. 11 water by mixed and pure culture. Appl. Environ. Microbiol. (1993) 611620 24 (1972) 363368 18. Ghildyal, N.P.; Gowthaman, M.K.; Raghava Rao, K.S.M.S.; Karanth, N.G.K.: Interaction of transport resistances with

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