Abstract: Dissolved oxygen tension and shear stress as two very important factors in fungal fermentation were studied in the batch cultures of Aspergillus niger. The intention was to maximize the total activity of glucose oxidase produced in a 5-1 bench-top bioreactor. 300 rpm found to be optimum for enzyme production, however in higher mixing rates higher growth was achieved. The maximum activity of glucose oxidase was obtained in 1.5 vvm while the best aeration rate for growth was 2 vvm. Glucose oxidase with the activity of 548 U mL-1 was produced in 1.5 vvm and 300 rpm as the optimum conditions.
INTRODUCTION
Aspergillus niger is well known to produce a lot of organic acids, enzymes, plant growth regulators, mycotoxins and antibiotics. The industrial importance of A. niger is due to its capacity for synthesis more than 35 native products. During the past few years numerous studies have been presented on this most important fungus for production and secretion of protein (Jeenes et al., 1991). The employment of A. niger as a host organism for production and secretion of homologous and heterologous proteins demonstrates many advantages. A. niger is a prodigious exporter species of homologous proteins and is able to produce certain enzymes in quantities of kilograms per cubic meter under the right conditions (Finkelstein, 1987). A. niger has a long history of usage within the fermentation industry and is generally regarded as safe (GRAS). The fermentation industries are very familiar with the conditions required to maximize production of homologous proteins in Aspergillus. Its species are effective secretors of proteins, often in a native, correctly folded form. They tend not to accumulate large quantities of the protein intracellularly, in form of inclusion bodies, as some bacteria and yeast do. Glucose oxidase (β-D-glucose: O2 1-oxidoreductase, E.C. 1.1.3.4) is one of the most important enzyme produced by this fungi (Witteveen et al., 1992). Glucose oxidase catalyses the oxidation of β-D-glucose to D-glucono-δ-lactone and hydrogen peroxide and finally to gluconic acid using molecular oxygen as electron acceptor (Leskovac et al., 2005). It is widely used in the removal of traces of oxygen or glucose from different foods such as dried egg, beer, wine and fruit juices, as a source of hydrogen peroxide in food preservation and in gluconic acid production (Kapata et al., 1998). Glucose oxidase was also found to be antagonistic against different food-borne pathogens such as Salmonella infantis, Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, Campylobacter jejuni and Listeria monocytogens (Kapata et al., 1998). It has been also used as an ingredient of toothpaste (Petrucoili et al., 1999). It is also a key enzyme which is being exploited commercially in biosensors for monitoring the glucose level in blood, as well as in fermentation broth for on-line estimation of residual glucose (Petrucoili et al., 1999; Sierra et al., 1997).
In the present study, the effect of agitation and aeration on glucose oxidase production by Aspergillus niger in 5-l bench-top-fermenter was investigated to increase glucose oxidase production. Aeration and agitation are basic problems of aerobic fermentation processes. Aeration of growing microbial culture is undertaken primarily to supply its oxygen requirements and at the same time to remove waste products. The necessary oxygen for the growth and production of fungal culture can be ensured by agitation and aeration of the culture. The efficiency of aeration can be improved by agitation, resulting in an increased interface between gas and liquid. It is known that the intensive flow of liquid, caused by agitation, forces the air bubbles to disintegrate into a large number of small bubbles. An additional beneficial effect of agitation is to diminish the size of mycelial aggregates, making oxygen more easily accessible to the cells. The enhancement of the production of GOD with increase of oxygen transfer rates in wild-type and recombinant Aspergillus niger was reported (Hellmuth et al., 1995). According to Visser (1991), GOD is induced by high levels of oxygen and by high glucose concentration. The most extensively studied physical factors which have a profound effect on oxygen transfer in a particular reactor, are the speed of agitation and aeration rate (Nienow et al., 1997; Manferidini and Cavallera, 1983). It has also been found that glucose oxidase is formed at a high concentration of dissolved oxygen (Witteveen et al., 1992). Traeger et al. (1991) report that enhanced endocellular glucose oxidase activity was achieved by aerating the reactor with a mixture of pure oxygen and air. This mixture corresponded to a dissolved oxygen concentration of about 100% related to saturation with air at 1 bar total pressure. Studies by Witteveen et al. (1992) reveal that no enzyme activity whatsoever could be detected in experiments performed at 7% dissolved oxygen concentration. It is appropriate combination of impeller speed and aeration rate which is more important for the enhancement of specific glucose oxidase production (Kapat et al., 2001). When designing industrial production processes these different behaviors must be defined in the laboratory in order to achieve optimal production conditions.
The major goal of this research was to enhance the production of glucose oxidase by different optimization techniques. In the first phase, a study was carried out to examine the effect of oxygen transfer conditions on the production level of glucose oxidase. In this report, the influences of aeration and agitation on glucose oxidase production are described.
MATERIALS AND METHODS
Microorganism: Aspergillus niger PTTC 5012 strain from IROST collection in Iran was used, the slant prepared on PDA for vegetation of spores and maintained at 4°C.
Growth media and culture conditions: The strain was grown in 500 mL Erlenmeyer flasks containing 100 mL inoculum medium with the composition (in grams per liter): (NH4)2HPO4, 0.4; KH2PO4, 0.2; MgSO4.7H2O, 0.2; Peptone, 10; Sucrose, 70. The pH was adjusted to 5.5, with 1 N HCL, prior to sterilization. After inoculation, the preculture was incubated for 24 h on a rotary shaker operating at 225 rpm and 30°C (Hatzinikolaou and Maeris, 1994).
Fermentation was carried out in the 5-l stirred glass vessel bioreactor Chemap AG (Switzerland) with a working volume of 3 L. The bioreactor was equipped with two rushton turbine blades impellers with 80 mm diameter. The following probes were installed on the top plate: Mettler Toledo sterilizable Inpro 6000, pH-electrode, pt-100-temperature sensor and a conductive foam sensor. The fermentation parameters were controlled by a digital measurement and control system. The fermentation medium consisted of (g L-1): (NH4)2HPO4, 0.4; KH2PO4, 0.2; MgSO4.7H2O, 0.2; Calcium carbonate, 40; beet molasses, 5%(w/v); corn steep liquor (CSL), 10%(v/v). All media were sterilized at 121°C for 30 min. The fermentation conditions were as follows: the agitation and aeration had been set up as the requirement of the experiment; temperature, 30°C; liquid silicone was used as antifoam agent. The fermentation was inoculated with 10% (v/v) from the 24 h inoculum culture. The pH of culture was kept at value of 5.5 and maintained with pH adjusting from controller.
Biomass measurement: Samples were withdrawn in different intervals of time. Biomass was measured by conventional method of filtration as cell dry weight. Before filtration, the CaCO3 was converted to soluble form of CaCl2 by HCL 4N and reduce pH to 2.5 (Hatziniokolaou and Macris, 1995). The fungal biomass was separated from the culture fluid by filtration and washed with distilled water several times and then dried in 80°C. Concentration of biomass was determined by weighing dried mycelia.
Crude extract of intracellular and extracellular enzyme preparation: Mycelia from the culture liquid were collected, washed two times with distillated water on a sieve (whatman paper No. 42) and suspended in 0.1 M citrate-phosphate buffer, pH 4. The mycelia were incubated into a freezer in -20°C for 24 h. before disruption, 1 mmol L-1 of PMSF and 3 mmol L-1 EDTA reagents were added to samples in order to inactivate intracellular protease enzyme and chelating the metallic ions respectively. Then mycelia were disrupted with glass beads using mechanical force. After disruption the Aliquots of culture fluid were clarified by centrifugation at 6000 rpm for 15 min at room temperature and enzyme activities were measured in the clear supernatant. The obtained data for intracellular activity showed that the concentration of intracellular glucose oxidase was less than half of its extracellular enzyme and could be ignored after 20 h of culture. So that, the enzyme assays was limited to activity of the extracellular source of glucose oxidase. For extracellular enzyme determination, aliquots of culture fluid were clarified by centrifugation at 6000 rpm for 15 min at room temperature and The clear yellow supernatant was used as the source of glucose oxidase.
Analytical methods: All chemicals used were of analytical grade and obtained through Merck Chemical Co. The reduced sugars concentration was measured by the DNS (3, 5 dinitro salicylic acid) method using glucose as the standard (Miller, 1959). The same method was used for measuring enzyme activity, measuring residual reducing sugars. The reducing sugars were treated with DNS which is reduced to 3-amino-5-nitro-salicylic acid. The later was quantified by measuring absorbance at 540 nm using a spectrophotometer (Taktorn model 163 Varian). The DNS reagent consisted of a 1 g DNS dissolved in 20 mL 2 N NaOH and 50 mL distilled water. Thirty grams of potassium sodium tartrate tetrahydrate was added and the volume brought up to 100 mL with distilled water. The reducing sugars were measured as follows: 0.2 mL reducing sugar solution, 1.8 mL distilled water and 2 mL DNS reagent were boiled for 5 min followed by cooling to room temperature and diluting to 24 mL a standard calibration curve was prepared using known concentration of glucose (0.5-5 g L-1). From the standard curve the concentration of reducing sugar was determined (Kona et al., 2001).
The enzyme assay mixture consisted of 0.2 mL reducing sugar solution, 0.2 mL crude enzyme preparation, 1 mL citrate phosphate buffer (pH 5) and 0.6 mL distilled water. The citrate phosphate buffer contained 0.02 g L-1 sodium nitrate to inhibit catalase activity without affecting glucose oxidase activity. The reaction mixture was incubated at 30°C for 30 min. the reaction was stopped by keeping the tube in boiling water. The residual sugar then was measured by the mentioned method. Activity unit was defined as amount of enzyme that convert 1 μmol glucose (β-D-glucose) to gluconic acid and H2O2 in one minute under the above-described conditions.
RESULTS AND DISCUSSION
In the first step the relations between cell growth and enzyme production as a function of different agitation speeds were studied. Therefore the aeration rate was adjusted at 1.5 vvm in witch the presence of sufficient oxygen will assured and the agitation speed was changed from 150 rpm till 400 by 50 rpm interval during different batch runs.
As shown in Fig. 1 the cell growth increased with the time in all cultures studied with more or less the same time of maximum value at about 48 h of cultivation. In any case no further increase in biomass was observed after this time and it diminished for the rest of the cultivation.
| Fig. 1: | Effect of different agitation speeds on A. niger growth in 5-L bioreactor. The aeration rate fixed at 1.5 vvm. (CDW: Cell Dry Weight) |
| Fig. 2: | Effect of different agitation speeds on GOx production by A. niger in 5-L bioreactor. The aeration rate fixed at 1.5 vvm |
The maximum cell dry weight reached 11.7 g L-1 after 48 h in the culture with an agitation speed of 300 rpm. However, increasing the agitation speed till 400 rpm, resulted in lower cell mass due to a higher shear stress. Regarding Markl and Bronnenmeier (1985), a rapid pressure fluctuation around the blade and of shear stress created by the blade tips of rushton turbine at high stirrer speed may damage microorganisms and decrease the biomass concentration.
From Fig. 2 it can be seen increasing the stirrer speed from 150 to 300 rpm resulted in a significant increase in enzyme activity (from 284 to 548 U mL-1). In agreement with Petrucoili et al. (1995), in all cases, GOx activity reached its maximum about 72 h of fermentation and remained almost constant thereafter. It is interesting to note that the glucose oxidase continued to be produced until the last few hours of the run. Several attempts were made to make the production of enzyme continue for longer period of time, but the success was not significant (Rothberg et al., 1999).
| Fig. 3: | The maximum values of cell concentration and GOx production by A. niger obtained at different agitation speeds in a 5-L bioreactor. The aeration rate fixed at 1.5 vvm. (CDW: Cell Dry Weight) |
| Fig. 4: | Effect of different aeration rates on A. niger growth at 300 rpm in 5-L bioreactor (CDW: Cell Dry Weight) |
As shown in Fig. 3, neither high nor low agitation speeds were suitable for A. niger growth and GOx production. The cultivation at a moderate agitation speed was better for production of GOx. This result is in agreement with Zetlaki and Vas (1968). The influence of stirrer speed on GOx production was more significant than its influence on cell growth of A. niger which was also reported by other authors (El-Enshasy et al., 1999). As well, Petrucoili et al. (1995) studied the influence of stirring speed ranged from 300 to 900 rpm on GOx production by Penicillium variabile. Similar result was obtained for this microorganism. They observed a maximal enzyme production in 400 rpm agitated culture. Other study done on the different strains of A. niger also showed that there is an optimum narrow range of stirrer speeds for producing a metabolite like citric acid (Ujcova et al., 1980).
| Fig. 5: | Effect of different aeration rates on GOx activity produced by A. niger at 300 rpm in 5-L bioreactor |
| Fig. 6: | The maximum values of cell concentration and GOx production by A. niger obtained at different aeration rates in a 5-L bioreactor. (CDW: Cell Dry Weight) |
In the other hand Traeger et al. (1991) reported that enhanced endocellular GOx activity was achieved by aerating the reactor with a mixture of pure oxygen and air. This mixture corresponded to a dissolved oxygen concentration of about 100% related to saturation with air at 1 bar total pressure. In fact, the oxygen transfer rate will play a decisive role in the production of GOx by Aspergillus niger (Hellmuth et al., 1995). It will be affected not only by agitation speed but also by aeration rate. Thus, in the second step of this work, we studied the effect of different aeration rates at the optimal agitation speed (300 rpm).
The effect of increasing the aeration rate from 0.5 to 2 vvm on the growth of A. niger is shown in Fig. 4.
Increasing the aeration resulted in an increase in biomass levels of 8.3 to 11.9 g L-1, dry weight after 48 h of fermentation. The maximum biomass concentration was obtained at 2 vvm.
Varying the aeration rate had no particular effect on the enzyme activity, which reached 506 to about 550 U mL-1 after 72 h of fermentation (Fig. 5). From this Fig. it can be seen that the maximal enzyme level obtained at 1.5 vvm. However the effect of aeration rate in the selected range of variation was not very significant and aeration was not very beneficial for GOx production by Aspergillus niger. In Fig. 6 the maximal value of biomass concentration and enzyme activity obtained at different aeration rates were compared.
CONCLUSIONS
Several experiments performed in a laboratory reactor revealed that the influence of stirrer speed on GOx production was more significant than its influence on cell growth of A. niger. This study showed also that neither high nor low agitation speeds were suitable for GOx production. The maximal enzyme production was obtained using an intermediate agitation of 300 rpm which also gave a better cell growth. In contrast, the cell growth of A. niger was affected more than GOx production by variation of aeration rate. The optimal aeration rate for enzyme production was 1.5 vvm. It should be added that the differences in enzyme production in these cultures were mainly due to not only the difference in oxygen transfer but also the morphological feature of fungal growth witch need to be investigated in a separate study.
ACKNOWLEDGMENTS
The authors express their thanks to Mr. Gharib-Ali Farzi for his technical assistance in fermentation.