Geobacillus thermodenitrificans was cultured on nutrient agar and maintained at 65ºC. It was previously isolated from a hot spring in Labok, Machang, Kelantan, Malaysia.

G. thermodenitrificans was grown in medium containing 1% (w/v) glucose, 0.45% (w/v) NaCl, 1.25% (w/v) yeast extract and 0.1% (v/v) olive oil. Seven percent (v/v, 5 x 10⁶ cell/ml) of culture suspension, which was prepared from a 24 hour old culture, was inoculated in 100 ml of medium, pH 6.8. Fermentation was done in a 250 ml Erlenmeyer flask and incubated at 65ºC on an orbital shaker at 200 rpm for 48 hours. Contents of the flasks were collected after 48 hours and centrifuged (6000g, 15 min, room temperature). The supernatant was used for lipase assays and protein content determination.

Lipase production by G. thermodenitrificans was carried out in a 5-L stirred-tank bioreactor (INFORS AG, Switzerland) in 2L of medium. The medium (pH 6.8) was sterilized at 121ºC for 20 minutes in an autoclave. The medium was inoculated with 2.0% of (v/v; 5 x 10⁶ cell/ml) inoculum and the fermentation was carried out at 55ºC. SPAN 20 10% was used as the antifoam. The impeller speed was initially adjusted to 100 rpm and compressed sterile air was sparged into the medium at the rate of 1 L min^-1. Samples (15 ml) were withdrawn every 6 hours and analyzed for lipase activity, protein concentration, pH and cell growth.

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 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 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.

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 Candida rugosa in a triple impeller system was highest at 600 rpm (16).

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.

G. thermodentrificans was isolated from high temperature environment where the dissolved oxygen concentration was quite low. Its adaptation to this low oxygen environment could be the reason for lower lipase production and cell growth at higher aeration rates. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate rather than dissolved oxygen concentration. Improvement in oxygen transfer rate by aeration resulted in an increase in lipase yield by Acinetobacter radioresistens and/or a reduction in fermentation time (18).

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 Streptomyces gedanensis IFO13427, showed that LAP production progressively increased up to 2% (v/v) (1.2 × 10⁹ CFU/ml) inoculum and decreased thereafter (21).

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 B.cepacia was obtained in a 14 l bioreactor within 15 h after statistical optimization following shake flask culture (22).

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.

Figure 1

Figure 1: Profile of lipase production and growth of before and after physical parameters optimization, in a 5-L stirred tank bioreactor (A) lipase activity and protein content, (B) growth. All the experiments were carried out independently in triplicates. 1A closed triangles -lipase activity before optimization, closed diamond – lipase activity after optimization, closed circles – protein content before optimization and closed square – protein content after optimization. 1B closed triangle – cell dry weight before optimization and closed square – cell dry weight after optimization

Figure 2

Figure 2: Effects of agitation rate (A), aeration levels (B) and inoculum sizes (C) on lipase production and cell growth. Bar chart – lipase activity and line – cell dry weight.