JOURNAL OFBIOSCIENCE ANDBIOENGINEERING Vol. 88, No. 5, 586-589. 1999
Batch Decolorization of Molasses by Suspended and Immobilized Fungus of Geotrichum candidum Dee 1 SEONG JUN KIM AND MAKOTO SHODA* Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8503, Japan Received21 June 1999/Accepted11 August 1999 Geotrichum candidum Dee 1, which exhibits a broad dye-decolorizing spectrum, was used to decolorize molassesduring semi-batch cultivation. Au eighty % decolorizatiou of molassesand a stable peroxidase activity were maintained for approximately four weeks, after which, both activities deteriorated signi&antly. Subsequently, repeated batch cultivation of fungal cells immobilized on polyurethane foam was employed to solve the aforementioned problems and stable decolorization of molassesas well as stable peroxidase activity was realized for more than eight weeks. [Key words: decolorization, molasses,Geotrichum candidum, peroxidase, immobilization]
We isolated a new fungus, Geotrichum candidum Dee 1 (Dee I), which is a dye-decolorizing microorganism that exhibits decolorizing ability for 21 kinds of azoand anthraquinone-dyes tested (1). This fungus requires carbon sources for decolorization in the presence of oxygen, and molasses is one of the easily available carbon sources. However, molasses itself contains highly colored and complex polymerized substances. Therefore, decolorization of molasses as well as dye decolorization is a prerequisite for the application of Dee 1. The characteristics of several strains of dye-degrading microorganisms have been reviewed (2). Among them, Phanerochaete chrysosporium has been extensively studied not only from a biological viewpoint but also from an engineering viewpoint (3-6). However, Dee 1 has several advantages over P. chrysosporium such as a higher growth rate and less sensitivity to shear stress (7) and to high dye concentrations (1). In a previous study, the decolorization of crude molasses by Dee 1 in a batch cultivation was demonstrated (7). In this study, a longterm and stable method for the decolorization of molasses was attempted in a jar fermentor using suspended and immobilized cells of Dee 1. Peroxidase activity, which is responsible for the decolorization of dyes (l), was also measured separately to determine the possibility of simultaneous decolorization of molasses and dyes. G. candidum Dee 1 was cultivated in potato-dextrose agar (PDA) medium for 6 d at 30°C. The spores formed on the surface of PDA were suspended in sterile water and the spore suspension was used as the inoculum for further experiments. The preparation of the molasses medium used was similar to that in a previous study (8). To obtain consistent data, 50 1 of molasses was stored in a reservoir at 4”C, with a supply of air to homogenize the molasses. An aliquot of the homogenized molasses was used in a series of experiments. Adoption of this procedure limited the fluctuation of data to less than 10%. The molasses obtained from the reservoir was diluted to 40-50 g/l with water. A 5 ml spore suspension containing approximately 10’ colony forming units (cfu)/ml was inoculated into 150 ml of fresh molasses medium in
500 ml flasks and agitated at 120 rpm at 30°C for 3 d. This 150 ml culture was added to 5 I fresh molasses medium in a 7 I jar fermentor for semi-batch cultivation. A conventional jar fermentor (nominal volume=7 1, working volume=5 I), which was simplified by removing bafRe plates and water circulating pipes inside the fermentor to avoid adhesion of the fungus to the solid parts, was used for semi-batch cultivation. The structural details of the jar fermentor have been previously described (7). Batch cultivation was initially conducted by agitation at 180 rpm until 80% decolorization of the molasses was observed. The culture broth was then decanted, leaving the fungal cells at the bottom. Aseptic removal of 1.5 I of broth from the jar fermentor using a pump was followed by the addition of the same volume of fresh molasses medium by the pump and the cultivation proceeded until 80% decolorization of the molasses was attained. This procedure was repeated for 41 d. A 60% oxygen-fortified air, which was prepared using a pressure swing adsorption oxygen generator (PSAOG) (RELIANT, Airsep Co., USA) was supplied at a flow rate of 2 I/min (0.4vvm) (7) and the dissolved oxygen (DO) concentration was monitored using a DO sensor. The pH was also monitored continuously with a sensor. Among the various solid materials used for the immobilization of P. chrysosporium, polyurethane foam was preferred because of its lightness and porosity (913). Polyurethane foam (proprietary name: the color foam EBR-1, Inoack Co. Ltd., Nagoya) was selected as the best carrier for the immobilization of Dee 1 among various polyurethane carriers, because the decolorization degree of molasses and the peroxidase activity of the fungus immobilized by EBR-1 were the highest in preliminary shake culture tests (data not shown). The physical properties of EBR-1 are as follows: bulk density, 35 kg/ m3, indentation load deflection, 12.5 kg, tensile strength, 0.7 kg/cm2, elongation, lSO%, and compression set, 5%. A 0.7 1 jar fermentor was used for repeated batch experiments with immobilized cells of Dee 1. Baflle plates were removed to avoid fungal attachment. A two-blade screw impeller was fixed and agitated at 50-60 rpm. Six hundred ml of fresh molasses medium, 200ml of intact EBR-1 which was cut into 5 X 5 x 5 mm cubes, and 15 ml of spore suspension of Dee 1 were mixed in the fer-
* Correspondingauthor. 586
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mentor and 60% oxygen-fortified air was supplied by PSAOG at a rate of 0.5 Nmin. When the decolorization degree of molasses reached approximately 80%, the culture supernatant was decanted and 550ml of fresh molasses medium was then mixed with the residual immobilized polyurethane foam. This operation was repeated for about eight weeks. The decolorization degree of molasses (%) was determined from the difference in absorbance at 475 nm of the culture broth (14, 15), after making the appropriate dilution, between the initial value and the sampling value at each sampling time. The ability to decolorize the dye by the culture broth was monitored by measuring peroxidase activity, because the direct determination of dye decolorization is not possible due to the dark-colored molasses. The dye used was Reactive blue 5 (abbreviated as RB5) (Nippon Kayaku Co. Ltd., Tokyo) (l), an anthraquinone dye. The culture broth was periodically sampled from jar fermentors and centrifuged at 10,000 x g for 5 min. RB5 was added to the supernatant to a final concentration of 500 ppm and pH was adjusted to an optimal value of 3.2 (1, 8). A 0.7 ml aliquot of the sample was taken immediately after the addition of the dye and also 1 h later and then centrifuged at 10,000 xg for 10 min. The obtained supernatants were diluted 5-fold with distilled water and 0.4mM H202 was added. Disappearance of the dye in the supernatant was spectrophotometrically determined by measuring the absorbance at 600nm of absorbance, which is the wavelength for maximum absorbance of the dye (1). The decolorization rate of the dye, which was determined from the difference in absorbance at 6OOnm, was converted into a concentration difference of the dye in the original culture broth per unit time, and expressed as ppm of dye/min (1, 8). The supernatant obtained from the procedure described above was filtered through a 0.2~pm polytetrafluoroethylene (PTFE) membrane (Advantec, Tokyo) and the filtrate was used for HPLC (RID-3000, Nihon Bunko Co. Ltd., Tokyo) on a column (Shodex NH2P-50 4E, 4.6 mm in diameter x 250 mm in length) at 4O”C, to measure the concentrations of glucose and fructose. The eluent of acetonitrile and water (3 : l(v/v)) was supplied at a flow rate of 1 ml/min. Figure 1 shows changes in the decolorization degree of the molasses and the peroxidase activity in semi-batch cultivation in the 7 I jar fermentor. After an initial batch was cultivated for IOd, semi-batch operations started. Every 2 or 3 d, the decolorization degree of molasses reached 80% while the peroxidase activity reached a maximum at day 18. However, the decline in the decolorization degree of molasses and the peroxidase activity became significant after operation for 22 d. During the experimental period, glucose concentration remained at 5-10 g/Z and a sufficient DO concentration of more than 5 mg/f was confirmed. Therefore, to determine the reason for the deterioration of the decolorization activity, an aliquot of the culture broth was taken at 41 d of culture, centrifuged at 8OOOXg for 20min and the precipitated cells and the supernatant were separated. The collected cells were spread on PDA medium and cultivated for 7 d at 3O”C, SO that a spore suspension of the cells was obtained. The spore suspension (3 ml) was then inoculated into 120 ml of fresh molasses medium in a 500 ml flask and cultivated with shaking for 8 d, after which the molasses decolorization and peroxidase activi-
FIG. 1. Changesin decolorizationdegreeof molasses( l ), and decolorizationrate of RB5 (0) of the culturebroth when G. candidurn Dee 1 was grown by semi-batchcultivation.
ties were measured. The cells after cultivation for 41 d showed one-fourth of the molasses decolorization activity of the original cells and exhibited no peroxidase activity. When the cell-free culture broth after cultivation for 41 d was used to grow the original Dee 1 cells, the molasses decolorization and peroxidase activities were also significantly low. This indicates that some inhibitory components had accumulated in the supernatant during the long-term cultivation and these affected cell activity. Although we have not identified any enzymes that are specifically involved in the decolorization of molasses, some enzymes related to melanoidin degradation have been reported, which are unstable at high concentrations of molasses (16, 17). In order to avoid this kind of adverse effect of the molasses medium on cell activity and to realize a stable decolorization activity by Dee 1, repeated-batch cultivation with immobilized Dee 1 on a polyurethane foam carrier was conducted, as shown in Fig. 2. Stable decolorization degree of molasses of 80% and peroxidase activity of about 15 ppm/min were repeatedly obtained, which lasted for 61 d. The pH fluctuations observed approximately every 2 d corresponded with the times when the fresh medium was added, and when the decolorization degree reached 80%. The cell mass on the polyurethane carrier was estimated to be approximately 3 g-cell/g-carrier, which was approximately 3-fold higher than the data reported previously (18). The suspended cell mass was estimated to be less than 5% of the attached cell mass. A fairly stable cell mass was reflected by the constant change in the rate of sugar consumption. Under nutrient-limiting conditions, fungal cells generally cannot remain active during a long-term cultivation. Therefore, the continuous-culture method is not practical and the semi-batch or repeated-batch method can be an alternative for long-term cultivation. During the operation of suspended fungus cultivation, agitation was increased so that the cells were dispersed homogeneously and prevented from adhering to impellers, probes and tubes. However, the enhanced agitation induced the formation of larger pellets of Dee 1 and such large pellets became oxygen-limited (7, 19). To solve this problem, the supply of oxygen-fortified air was found to be effective in this experiment. However, the control of pellet size by controlling agitation or aeration rate is difficult, and thus the immobilization of the fungus on a solid support is an appropriate means for
KIM AND SHODA
J. BIOSCI. BIOENG.,
FIG. 2. Changes in decolorization degree of molasses (0) (a), decolorization rate of RBS (0) (b), consumption rate of sugar (glucose + fructose) ( o ) (c), and pH (A) (d) when immobilized cells of G. candidurn Dee 1 on polyurethane foam were grown by repeated batch cultivation.
controlling the thickness of the biofilm. The immobilization of the fungus offers advantages such as short retention time, easy recovery of the cells and increased activity. Furthermore, in the presence of the foam matrix, pellet size was restricted by the size and the physical properties of the foam. In spite of such advantages of immobilization, the enhanced activity of the enzyme did not last long and a decrease in the maximum level of activity was often observed within a couple of days of operation (20-24). For practical operation, the selection of the carbon source is one of the most important factors. Molasses is the most popular carbon source but color removal after the use of molasses is a matter of concern. We demonstrated that the utilization of molasses as a carbon source and its decolorization were efficiently realized by Dee 1 and stable peroxidase activity, which is involved in dye decolorization, was possible by the immobilization of Dee 1 cells for a fairly long period of operation. The results obtained here may be applied to different systems for dye decolorization, such as one-stage-semi-batch systems or two-stage-continuous systems. The optimal conditions for these methods are under investigation. We thank Oriental Yeast Co. Ltd., and Inoack Co. Ltd., for providing the crude molasses solution and the polyurethane foam samples, respectively. REFERENCES 1. Kim, S. J., Ishikawa, K., Hirai, M., and Shoda, M.: Characteristics of a newly isolated fungus, Geotrichum candidurn Dee 1, which decolorizes various dyes. .I. Ferment. Bioeng., 79, 601-607 (1995). 2. Banat, I. M., Nigam, P., [email protected]
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