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Neira-Gladys Rosero*1, Astrid-Lorely Pimienta*1, Fanny Dugarte2 and Fredy-Gonzalo Carvajal3
Ecopetrol S.A. – Instituto Colombiano del Petróleo, A.A. 4185 Bucaramanga, Santander, Colombia 2 Ambar S.A., Venezuela 3 Universidad Autónoma de Bucaramanga, Facultdad de Ingeniería en Energía, Bucaramanga, Santander, Colombia e-mail: email@example.com e-mail: firstname.lastname@example.org (Received 22 May 2002; Accepted 4 November 2003)
his work presents the results obtained from the laboratory-scale experimentation for the optimization of production of rhamnolipid-type biosurfactant in a batch process, through the calculation and analysis of yield parameters. Different carbon/nitrogen ratios were studied, for which the production rates of rhamnolipid under nitrogen limitation was defined. Bacterial growth yield parameters YX/N and YX/C, were also calculated.
Este trabajo presenta resultados de la fase de laboratorio, correspondientes a la optimización del proceso de producción de un biosurfactactante tipo ramnolípido, mediante la obtención y análisis de sus parámetros de rendimiento. Se estudiaron diferentes relaciones carbono/nitrógeno determinando la velocidad de producción de ramnolípido en condiciones de limitación por nitrógeno. También se estimaron los parámetros de rendimiento del crecimiento bacteriano YX/N y YX/C, logrados con los substratos de nitrógeno y carbono.
Keywords: rhamnolipid, biosurfactant, nitrogen limitation, yield parameters.
* To whom correspondence may be addressed
CT&F - Ciencia, Tecnología y Futuro - Vol. 2 Núm. 4
.. Limitation of multivalent cations. In addition. Pimienta et al. biodegradability has currently become the main criterion for the selection of additives. 1996). given the urgent and increasing need of protecting the environment. 4 Dic. other factors that should be considered for cost reduction purposes include bacterial functional stability and reproductiveness. such as iron (+2). a mechanism called quorum sensing is activated whenever nitrogen deficiencies are defined (Pearson et al. Itoh and Suzuki. 1996. energetic cost reductions in stirring and aeration efforts (Rosero et al. Banat. This condition is achieved when the formulation of culture is nitrogen limitation-driven (Venkata and Karanth. This survey is part of the pilot project for the development of a production process for a biosurfactant called Tensobiol-ICP. Sundari and Sandhya. among others.. Fiechter. 2000).. 1991) vegetable oil (Oschner et al. and twice through the production saline medium. 1995. Nitrogen concentration is relevant for the definition of an optimal biomass concentration. 1993). sources such as nitrate. Mulligan and Gibbs. Pimienta et al. oleic acid (Banat et al. 2003 . efforts have focused on the achievement of their financial competitiveness against synthetic surfactants. especially when spillages may not be avoided (Lang and Wullbrandt. 1990) have been used as the main insoluble sources. as based on these results. 1991). 2000). Various soluble and insoluble carbon sources have been assessed for biosurfactant production. Kosaric. 1993).. As a requirement for rhamnolipid accumulation.. In continuous production systems. 1970 in Kosaric. bioremediation.Vol. Ecological and technical advantages provided by biosurfactants are not significant enough to drive a process design at industrial levels. as well as for the definition of the yield parameters. therefore. octadecane (Zhang and Miller. propyleneglycol and glycerol (Oschner et al. Tecnología y Futuro . olive oil (Mercadé et al.... 1993 in Kosaric. 1974. Robert et al. while the concentration of hydrophobic carbon source defines the conversion of available carbon into biosurfactant (Hommel et al. magnesium or calcium.. Application of such substances include tank-bottom cleaning processes. Linhardt et al. 1992). paraffin (Montes de Oca. 1984 in Kosaric et al.. the additives selection criteria focused on their efficiency and cost. 1993). Díaz. 1997) have been used as the main soluble sources.. However. in 24-hour intervals. Bognolo. which was isolated from oily sludge and as included in the strain collection of the Biotechnology Laboratory at the Instituto Colombiano del Petróleo (ICP). 1989. 1987.Ciencia. 1989. 36 CT&F .. the type of nitrogen source has an effect on this process. Inocula preparation For each inoculum. ammonia. supported a significant reduction in related production costs (Rosero et al.. Until a few years ago. Fiechter.. polycyclic aromatic hydrocarbons (Déziel et al. while hexadecane (Montes de Oca. 1989. therefore... MacElwee et al. optimization of carbon sources and micronutrients concentration and nature. Fiechter. 1995). Further experiments. Jain et al.. and. Pimienta et al.. 1989). Excluding genetic enhancements (due to their high costs). 1993). 1992. 1992. 1989. 1995. 1999. 1992. 2 Núm.. Brennan et al. 1995. a vial (cryo-preserved at 193 K) was taken and successively passed twice through a nutritive broth.. 1999.. 1997).. 1992. using Pseudomonas aeruginosa. in order to enhance biosurfactant overproduction (Gruber et al. 1987. 1997). urea and amino acids have been tested.NEIRA-GLADYS ROSERO et al. 1992). nitrogen concentrations are reduced in the feeding medium. 1992. and differently-sourced crude oils (Sundari and Sandhya. and enhanced recovery. 1996. METHODOLOGY Microorganism Pseudomonas aeruginosa ICP70 strain. also increases rhamnolipid production rates (Syldatk et al. Pimienta et al. It includes results for the initial stages of design and kinetic assessment of the culture’s medium. among others (Mulligan and Gibbs. 1984. INTRODUCTION Surface-activity substances (surfactants) are widely used in the oil industry. 1997). 1986). and Guerra-Santos et al. Glucose (Guerra-Santos et al.
variable as per C/N ratio. as well as for the other spectrofotometrical procedures was the spectrophotometer UV-VIS CARY 1E VARIAN. 80 and 100) keeping above mentioned glycerol concentration. Rhamnolipid quantification Rhamnolipid quantification was performed through the measurement of rhamnose via the thioglycolic acid method (Whistler and Wolfrom. For the C/N ratio of 20 it was made a second evaluation. Microbiological follow-up The dry weight technique was used to quantify microbial growth (Madigan et al. through the culture’s absorbance at 540 nm . Tecnología y Futuro . For C/N ratios ranging from 40 to 100. Operating conditions 2 dm3 Erlenmeyer flasks with 3 cm deep baffles were used. 1962). glycerol) dt : Delta value for process hours n : Number of measurements taken during the exponential growth interval Each assay and measure was assessed threefold to guarantee the reproducibility of the results. pH value verifications were performed during the fermentation process. Nitrogen determination.2 and 7.. K2HPO4.5.497) x dilution factor The equipment used for this analytical method. biosurfactant) dS : Delta value for substrate consumption (nitrogen.PARAMETERS EXAMINATION OF A BIOSURFACTANT PRODUCTION Culture conditions The medium consisted of (per dm-3of drinking water): Glycerol. the medium’s pH control was maintained between 6. temperature of 305 K . 1997) as bacterial density. The following conditions were used: work volumes of 800 cm3 . 40. 4 Dic. 2003 37 .dm-3 of K2HPO4 and controlling the pH with a 1N sodium hydroxide solution. as per the method described by Whistler and Wolfrom.Vol.. pH-measurement Measurement of pH was carried out with the potentiometric analyzer ORION Model 720A.6 g. 1. Controls on the media were processed in parallel. 2 Núm. (NH4)2SO4.dm-3 . Different C/N ratios were evaluated (20.5 cm3 . 1. 30.5-7. pH 6. 1992).18 x Absorbance(400-430)-1.1 g . Glycerol quantification was carried out via colorimetry with cupric chloride. MgSO4. using the same samples which had been taken for rhamnolipid and nitrogen determinations. due CT&F . for all the different C/N ratios experimented. except for nitrogen. since growth values were limited by the nitrogenous content. were supplied in quantities enough to support bacterial growth. 0. This implies that all ingredients. in order to eliminate any interference due to the presence of suspended solids. The highest and lowest concentrations of biomass were observed for C/N ratios of 20 and 100.0 . Calculation of the RL concentration was made as follows (Rosero et al. KH2PO4.0 . 60. 0.Ciencia. 3 g ..75 and 0. Corresponding results are stated as ammoniacal nitrogen (mg N-NH3/dm-3). and orbital stirring of 140 rpm. (1962). respectively. Yield factors The following formula was used to calculate nitrogen and glycerol yields for biomass production: Yx/s (dx / dt ) / n (dS / dt ) / n (1) Where: x : Concentration of biomass or generated product (biomass. nessler method Measurement of ammoniacal Nitrogen was performed via a method based on Nessler’s colorimetric technique (APHA. RESULTS AND DISCUSSION Figure 1 shows the biomass concentration variation versus time. while the ammonia sulfate concentrations were respectively: 3.8*(54. using 1 g. Glycerol determination. 2000): [RLppm] =1. 7 g .
pH 7.0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (hours) C/N 20 Ratio C/N 100 Ratio C/N 80 Ratio C/N 40 C/N 20 Ratio C/N 100 Ratio C/N 60 Ratio C/N 40 Ratio C/N 80 Ratio C/N 60 -3 Figure 1. while using pH control with 1N NaOH.dm ) 3. cells are still metabolically active and their performance depends on cell density. from the fourth to the sixteenth hour term. followed by the ratio of 60 (33. however. For C/N ratio of 20 the phosphate contents used in the culture broth was not enough to control a higher quantity of acids released into the medium as a result of the microbial activity of a higher biomass concentration produced.5 0.0 were observed after 20 hours.0 0. it is observed that deficiency of pH control affected the activity of enzymes and therefore the amount of nitrogen consumption. 2 Núm.5 4.5 2. as well as on pH and available carbon content.h-1). the ratio of 80 (25 mg. and values below 5. During the stationary phase. 4 Dic. commercial grade medium to phosphate salts being present (data not displayed). while pH was kept above 38 CT&F .Ciencia. Growth kinetics of Pseudomonas aeruginosa ICP 70 with glycerol as carbon source at different C/N ratios. volume: 800 cm3 . eventhough the net growth rate is zero. Yx/C). Figure 3 indicates that for the same C/N ratio of 20. However.dm3 -1 h (data not displayed). (that is.0 2.21 h-1).Vol.dm-3h-1.5 1. and the ratio of 100 (19.12 mg.dm-3h-1). For these conditions can be expected that cellular metabolic control system is set to achieve maximum rates of biomass production. to quantify in-biomass nitrogen and carbon yield factors (Yx/N.dm-3. For the C/N ratio of 20. after 20 hour related with the medium’s acidity. the data used on bacterial concentrations and nutrients were the ones measured for the exponential growth phase in assessments on C/N ratios of 40 and 20. production rate obtained for the 12 to 20 hour term was of 54 mg.8 mg.0 were observed after 60 hours of processing (Figure 2). temperature 305 K .0 1. for the C/N ratio of 20. It also shows that the biosurfactant is mainly produced during the stationary phase. 4. When the C/N ratio of 20 was tested.The highest specific growth speed was observed for C/N ratios of 40 and 60 (0.dm-3h-1). The highest production rate was observed for the C/N ratio of 40 (45 mg. 2003 . Rhamnolipid production rate was calculated for the stationary phase (16 to 60 hour of processing). Tecnología y Futuro . Process time: 60 hours. a sharp reduction in rhamnolipid productivity was observed.5 [Biomass] (g.dm-3h-1). the exponential growth phase).NEIRA-GLADYS ROSERO et al. Figure 4 shows specific growth rate. the biosurfactant’s productivity for the 24 to 72 hour was of 52 mg. 140 rpm . Furthermore. pH values below 6.0 3. defined for each C/N ratio.
PARAMETERS EXAMINATION OF A BIOSURFACTANT PRODUCTION 1400 1200 7. 140 rpm . Process time: 60 hours. C/N 40 Rl. temperature 305 K . commercial grade medium 700 600 2500 2000 [N . Effects of C/N: 20 of the rhamnolipid concentration and pH’s changes of Pseudomonas aeruginosa ICP 70 with glycerol as carbon source. 140 rpm .dm-3 ) 500 400 300 200 500 100 0 0 10 20 30 Time (hours) N. volume: 800 cm3 . Process time: 60 hours. C/N 80 40 50 60 0 1500 1000 N.dm-3 ) pH CT&F . C/N 80 Rl. commercial grade medium [Rhamnolipid] (mg. pH 7. C/N 60 Rl.5 4 0 10 20 30 Time (hours) RL.5 5 4. temperature 305 K . C/N 40 N.5 7 6.Ciencia. 4 Dic.NH4+ ] (mg. Tecnología y Futuro . C/N 60 N. C/N 20 Rl. C/N 20 N. C/N 20 pH 40 50 60 [Rhamnolipid] (mg. 2003 39 .5 6 5. C/N 100 Figure 3.Vol. 2 Núm. pH 7. C/N 100 Rl. volume: 800 cm3 .dm-3 ) 1000 800 600 400 200 0 Figure 2. Productivity evaluation of rhamnolipid and nitrogen consumption of Pseudomonas aeruginosa ICP 70 with glycerol as carbon source to different C/N ratios.
Growth specific velocity of Pseudomonas aeruginosa ICP 70 with glycerol as carbon source to different C/N ratios. pH control. Related calculations showed a variation coefficient under 12%. 2 Núm. under the fermentation conditions used. the following was established: Yx/N is 6. Process time: 60 hours. stirring patterns.2 Growth Specific Rate (h-1) 0. It was found that the pH and carbon content provided during the stationary phase may improve productivity rhamnolipid rate and keep for a longer period of time the biomass activity.1 0 20 40 60 Ratio C/N Figure 4. pH 7.16 0. level should be taken into consideration parameters such as aeration. volume: 800 cm3 .5 g of biomass per gram Nitrogen Yx/Glyc is 0.22 0. 140 rpm .14 0. temperature 305 K . CONCLUSIONS • Biomass apparent yield factors found for the nitrogen and glycerol substrates. and feeding strategies for the achievement of high and metabolically active biomass to get optimal production of biosurfactant. and that biosurfactant production rate is proportional to the bacterial density achieved at fermentation. and compare efficiency of different sources for these ingredients. Based on these factors.26 g of biomass per gram Glycerol Based on these values is possible to define the medium contents for higher cell densities. it is possible to design culture media that would support higher bacterial concentrations. support the quantification of the nutritional requirements by the microorganism discussed herein.0. 4 Dic. ACKNOWLEDGEMENTS The authors wish to state their gratitude to Sandra Alvarez. commercial grade medium 80 100 120 6.18 0.12 0. keeping in mind the use of feeding strategies to avoid inhibitory concentrations of nutrients. Tecnología y Futuro . for their invaluable cooperation in the development of this survey. 2003 . as a result. 40 CT&F .Vol.NEIRA-GLADYS ROSERO et al. • As part of the process design stage at a pilot scale • The experiments carried out using different carbon/ nitrogen ratios showed that nitrogen depletion induces rhamnolipid-type biosurfactant production.Ciencia. 0.
5: 25-32. M. and Pickenhagen. 4 Dic. Vol. Biochem. H. Horne. Vol. A. J. Prentice Hall. F. 48. Tesis de Maestría Fac.. 1993. P. 1993. L. “Biosurfactant production by a soil pseudomonas strain growing on polycyclic aromatic hydrocarbons”... 1984. Vol. L. Biotechnology Bioeng.. R.). 2003 41 . I.. USA. Käpelli. Stuber. and Trevors.. Universidad Industrial de Santander. Surfactant Science Series. 2 Núm.. Kosaric. and Parker.. Bioresource Technology. Microbiol. R.. 7: 80-88. Díaz.. G. Manresa. Marcel Dekker. “Brock biology of microorganisms”. 152: 41-52... S. 1987. A. Guerra-Santos. Biol. USA. Inc. Production – Properties – Applications”. Appl. T. Inc. D... 483pp. L. and Thomas. W. and Banerjee. (Ed. M. Haferburg. D. New York. Microbiol. 1992. 1993. Bognolo. M. 168 pp. Marcel Dekker. N. In: “Biosurfactants.. 48.). Tecnología y Futuro . “Biosurfactants. J. 68: 645-654. Eng. 1992. P. Biotechnol. CT&F . Lehane. D.. Marcel Dekker. and Fietcher. G. 10: 87-93. 48. Käppelli O. Brennan. 986pp. Cairns.. C. G.. J. Hommel.Ciencia.. 62: 1908-1912. Villemur.. and Trevors. Marcel Dekker. N. 1997. Eur. A: Physicochem. Stuwer. Microbiol. A. Agr. J. L. C.. N.. 4-75 . 1999. Lee. Industri. J. Ingeniería Química. “Effect of addition of pseudomonas aeruginosa UG2 inocula or biosurfactants on biodegradation of selected hydrocarbons in soil”.. “Biosurfactants as emulsifying agents for hydorcarbons”. W. A. K. Déziel. “Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation”. 51: 1-12. Aspects. “Fructose-lipids of arthrobacter. Lang.. Industri. Appl. Elsevier. Biotechnol. G. Production – Properties – Applications”. Vol.. N. and Kleber. “Biosurfactants. “Standard methods for the examination of water and wastewater”. “Biosurfactants and biotechnology”. (Ed. 1995. (Ed. Inc. 483pp. and Fiechter.. “Dependence of pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors”. “Microbially produced rhamnolipid as a source of rhamnose”. “Integrated process for continous rhamnolipid biosynthesis”. Kosaric. M. M. Banat. 13:117. 1970.). Banat. H. Chmiel. Linhardt. Paquette. Hauser. N.. Madigan. Itoh. 48: 301-305. 1992.. Martinko. 1954. 1993. Microbiol. P. 483pp. J. “Estudio preliminar de la aplicación de biosurfactantes en el manejo de crudos pesados”. microbial production and aplication potential”. M... Microbiol. O. 25. “Production of extracellular emulsifying agent by pseudomonas aeruginosa UG1”. Microbiol. Fiechter..).. In: Surfactant Science Series. World J. Jain. 1993. Microbiol & Biotech. O. O. Elsevier. Murad. Bakhit. Guerra-Santos... and Wullbrandt. 26:199. nocardia and mycobacteria grown on fructose”.. 1987. New Jersey... (Eds. WEF. H. New York. W. 1996. Inc. APHA. Marcel Dekker. D. S.. Biotechnol. J.. A. New York. Surfactant Science Series. In: “Biosurfactants. 483pp. “Rhamnose lipids – biosynthesis. 24: 443-448. 43: 1-6.. 33: 365-368. J. R. Chem. Colloids Surfaces.. USA. New York.. Biotechnol.. and Suzuki. C. E. Inc. T. Samarah. Bucaramanga. and Guinea. M. Mercadé. R. Tibtech. 48. Bioresource Technology. (Ed.PARAMETERS EXAMINATION OF A BIOSURFACTANT PRODUCTION BIBLIOGRAPHY APHA. M. USA. H. J. Kosaric. 1989. USA. Mayerl. 1993. USA. L.. Surfactant Science Series. 51: 22-32. “Biosurfactants. 10: 208-217. AWWA... Vol. 1986. 342pp. N. moving toward industrial application”. M.. Daniels.Vol.. 18ª Ed. D.. S.. Surfactant Science Series. Lépine. production – properties – applications”. 1991. Environ. R. F. Lee. Appl. M. Microbiol. Robert. Environ. Espuny. Vol. I. P.. “Olive Oil Mill Effluent (OOME) new substrate for biosurfactant production”. H. 1991... Gruber. USA. Appl. J. C. Kosaric. N. H. Production – Properties – Applications”.. and Bisaillon J.).. 38: 1443-1449. Sticher. 1974.. 1999.4-80.. W. and Karnovski. T. “Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source”. “Biosurfactant production and use in oil tank clean-up”.. 1990. and Gray. New York. Kosaric.. De Andrés. M. “Studies on the production of glycolipide by pseudomonas aeruginosa”. corynebacteria. E. Appl.. and Fietcher. Käppeli.. J. MacElwee. P.
T. J. 25. P. J. H. 24(4): 68-72. Manresa. G. L.. Y. 58: 3276-3282.. and Greenberg. and Karanth. 1 (3): 95-108. 1995.. L.. 92: 1490-1494. 42 CT&F . Cairns. Microbiol.NEIRA-GLADYS ROSERO et al. 342pp. Nieto. Carvajal. R. and Fiechter. 171-182. 2000. C.. Zhang. Espuny. Whistler. Pimienta. A. C. F. 45: 249-257. Biotech. Tecnología & Futuro. L. Parra.... Oschner. B. Acad. Lett. M.. Berlin Heidelberg.. B. P.).. 1: 53. and Miller.. Passador. G. B. and Grosso. Vol. and Gibbs. and Gray. “Factors affecting biosurfactant producing using pseudomonas aeruginosa CFTR-6 under submerged conditions”. 2 Núm.Vol.. M. USA. H. L. and Guinea. Environ. Montes de Oca. 1995. H. “Correlation of nitrogen metabolism with biosurfactant production by pseudomonas aeruginosa”. Syldatk. 55: 3016-3019. N. “Producción de biosurfactantes microbianos”.). In: Advances in Biochemical Engineering Biotechnology. 1962. 53. New York.. CT&F . “Enhanced octadecan dispersion and diodegradation by a pseudomonas rhamnolipid surfactant (Biosurfactant)”. 1987. A.. Proc. L. 1996. G.. Vol. N. and Wolfrom. E. S. M. T. Robert. L. “Methods in Carbohydrate Chemistry”. 1992. Malutovic. “Production of Ramnolipid Biosurfactants”. 1984. 2003 . Hembach. Pearson.Ciencia.. USA.. R.. 1992.. Sci. Carvajal. Tecnología y Futuro . Environ. 11: 871-874... M. Fiechter.. Kosaric. N. Revista del Instituto Mexicano del Petróleo. J. 4 Dic. C. A. Inc. “Extracellular microbial lipids from psedudomonas Sp Ls1 AS bioemulsifiers”. Sundari. C... Bosch. and Sandhya. M. Biotechnol. Pimienta. U. P. A. Díaz. Health. Rosero. “Production of biosurfactants (Rhamnolipids) by pseudomonas aeruginosa isolated from colombian sludges”. 1989. A. N. W.. Iglewski.. F. Mulligan.. (Ed. F. S.Ciencia. “Effect of the carbon source on biosurfactant production by pseudomonas aeruginosa 44T1”.. N. Environ. 1997. Vol. Academic Press. P. Chem. “A second N-acylhomoserine lactone signal produced by pseudomonas aeruginosa”. K. New York. R. Natl. Dugarte. P. Appl. and Alvarez. “Biotech Forum”. Microbiol. 1989.. L. Surfactant Science Series. Marcel Dekker. F. F. (Eds. 1.. “Producción de biosurfactantes”. and Wagner. Appl. Springer.. J. 1989. Tech.. J. In: Biosurfactants and Biotechnology. Informe Técnico de Avance Ecopetrol-ICP. SCI. New York. G. J. Mercadé. J. M. G. Venkata Ramana. A.
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