A Balan, N Magalinggam, D Ibrahim, R Rahim
aeration, agitation, bioreactor, geobacillus thermodenitrificans, inoculum concentration, thermostable lipase
A Balan, N Magalinggam, D Ibrahim, R Rahim. Thermostable Lipase Production By Geobacillus Thermodenitrificans In A 5-L Stirred-Tank Bioreactor. The Internet Journal of Microbiology. 2009 Volume 8 Number 2.
Lipases from thermophiles have gained interest in recent years as it has various applications in industries. It plays a significant role in industries as it has high stability and resistant to chemical denaturation. Its extensive application in industries requires its production in a large scale. In this study, thermostable lipase production by
Lipase (triacylglycerol acylhydrolase, EC 22.214.171.124), which acts only on an ester-water interface, is capable of catalyzing the hydrolysis of long-chain triglycerides with the formation of diacylglycerol and carboxylate, as well as the reverse reaction with the synthesis of esters formed from fatty acids and glycerols (1). Lipase is present in diverse organisms including animals, plants, fungi and bacteria; however, only microbial thermostable lipases are of commercial importance (2-3). Extracellular microbial lipases can be produced inexpensively in large quantity by fermentation (4).
The extensive application of thermostable lipases in industries requires its production in a large scale. In this study, production of a thermostable lipase by the thermophilic bacteria
Materials and Methods
Cultivation in Erlenmeyer flasks
Cultivation in a 5-L stirred-tank bioreactor
Lipase production by
Cell growth determination
Cell growth was determined based on the method of (10). Biomass concentration was measured as OD560 with a Spectronic Unicam Genesys10UV. The obtained values were converted to g cell dry wt/L by using a calibration curve.
Lipase activity determination
Lipase activity was determined by using polyvinyl alcohol and olive oil in a 3:1 ratio as the reaction substrate (11). Five milliliters of reaction substrate and 4.0 ml of phosphate buffer 0.2 M (pH 6.8) were incubated for 10 minutes at 65ºC. Then, 1.0 ml of the crude enzyme supernatant of was added to the substrate. The reaction mixture incubated for 30 minutes at 65ºC with shaking at 150 rpm. The enzyme reaction was stopped by adding 20 ml of ethanol-acetone (1:1). The free fatty acid was titrated with 50mM NaOH with 0.1 ml of phenolphthalein as the titration indicator. One unit of lipase activity was defined as the amount of enzyme releasing 1 mole of fatty acid per minute.
Protein Content Determination
Protein content in the sample was determined using a modified method of (12).
Optimization for maximum thermostable lipase production by G. thermodenitrificans
Lipase production was measured at different aeration rates - 1, 2, 3 and 4 L min-1 and agitation speeds -100, 200, 300, 400 and 500 rpm. Inoculum sizes evaluated were 0.5%, 1%, 1.5%, 2% and 2.5% (v/v; 5 x 106 cell/ml) inoculum. Samples were collected every 48 hours, centrifuged (6000g, 15 min, room temperature) and lipase activity, protein content, pH and the amount of cell growth determined.
Results and Discussion
Fermentation in a 5-L stirred tank bioreactor increased the production of thermostable lipase. After four days of cultivation in the bioreactor, lipase production was highest at 48 hours of cultivation with 84 U/ml activity (Figure 1A). Enzyme activity then dropped until 96 hours of cultivation with 31.5 U/ml activity. Protein content followed the same pattern, with the highest value, 35 mg/ml, at 48 hours and then dropping afterwards. Bacterial growth also was highest at 48 hours with 0.5 mg/ml cell dry weight.
Cultivation in a stirred tank bioreactor increased thermostable lipase production 3-fold compared to the shake flask. This increase could be due to the higher dissolved oxygen concentration in the bioreactor.
Total lipase activity in a stirred tank bioreactor by
Effect of agitation rate
Agitation rate of the impellers in a stirred tank bioreactor plays an important role in the production of thermostable lipase enzyme. Agitation showed to be an important parameter to ensure nutrient availability in a growth medium having olive oil (14). An agitation rate of 400 rpm produced the highest lipase activity of ~ 125 U/ml with cell growth of 0.55 mg/ml (Figure 2A). Higher agitation may create condition of higher availability of the carbon sources to microorganisms (15).
Agitation helps maintain uniform conditions within the fermenter and promotes effective mass transfer to the liquid medium in the fermenter. Higher agitation also increases gas dispersion, and increaseed gas dispersion allows more mass transfer (15). The 100 - rpm-air-sparged run was oxygen limited due to the low agitation speed, and had a low enzyme activity as the oxygen transfer coefficients are strongly influenced by the amount of agitation Production of lipase by
Effect of aeration
Under the conditions studied (400 rpm and 55°C), it was found that cell growth and lipase production occurred only when air was supplied continuously to the bioreactor. The aeration rate in the stirred tank bioreactor is important for growth of the bacteria, and also may increase the oxygen exchange rate and help mix the medium (17).
Aeration at 2 lpm produced the highest lipase enzyme activity level 105.5 U/ml (Figure 2B). Lipase production decreased at other aeration rate. The cell growth also was highest, 0.34 mg/ml at 2 lpm. Aeration rate less than 2 lpm might not provide enough oxygen for cell growth. Whereas, an aeration rate > 2 lpm might exceed the oxygen need and result in lower enzyme activity.
Effect of inoculum size
The larger volume of the stirred tank bioreactor means that a higher inoculum concentration also was needed. An inoculum size of 2% (v/v; 5x106cells/ml) produced the highest lipase activity of 115 U/ml (Figure 2C). Cell growth also was highest at that inoculum size with at 0.60 mg/ml. Inoculum size influences the utilization of glucose in the medium (19). Thus, the higher cell concentration at a time, the greater was the production of enzyme that can be expected (20). However, at a lower level of inoculum, enzyme activity was < 115 U/ml. This could be due to the depletion of oxygen on account of the high cell concentration. Studies on the effect of inoculum size on l-leucine amino peptidase production by
Thermophilic lipase production after physical parameters optimization
All the optimized physical parameters were used and the fermentation was run for four days (Figure 1A). The highest lipase activity was detected at 72 hours of cultivation with ~ 180 U/ml of biomass compared to 84 U/ml prior to optimization. After 72 hours of cultivation, lipase production decreased.
Protein content also was highest 3.62 U/mg protein at 72 hours, an increase ~ 53% over the results obtained prior to optimization. The thermostable lipase activity obtained after physical parameter optimization in the bioreactor was ~ 6-fold higher than that attained in shake flask cultures. The highest cell growth (Figure 1B) also occurred after 72 hours of fermentation with 0.64 mg/ml. This amount was also 23% higher than the growth prior to optimization. Thus, both growth and metabolic activity are higher in cultures grown under optimized parameters in a stirred tank bioreactor. A 4-fold enhancement in lipase production and approximately 3-fold increase in specific activity by
In conclusion, the results obtained from the present work, indicate that a thermostable lipase can be produced on a bioreactor scale. The enzyme yield from the stirred tank bioreactor was significantly higher than that obtained in shake flask cultures. Optimization of the physical parameters for the stirred tank bioreactor fermentation also increased production of the thermostable lipase and the biomass concentration. There were no operational problems detected, in spite of the high culture temperature employed. The results obtained in this work suggest the possibility of applying this process to larger-scale systems which will be beneficial for industrial purpose.
The author, A.B. thanks USM Fellowship Scheme for financial assistance.