Production of Laccase by Marasmius sp. Grown in Rice Straw using a Packed Bed Bioreactor

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1 PROCEEDING 19 th Regional Symposium ISBN: Production of Laccase by Marasmius sp. Grown in Rice Straw using a Packed Bed Bioreactor Hendro Risdianto a,b*, Maya Fitriyanti a, Sri Harjati Suhardi c, Yogi W. Budhi a and Tjandra Setiadi a a Department of Chemical Engineering Faculty of Industrial Technology, Institut Teknologi Bandung, Bandung Indonesia b Center for Pulp and Paper Ministry of Industry, Bandung Indonesia c School of Life Sciences and Technology Institut Teknologi Bandung, Bandung Indonesia * Corresponding Author s hendrorisdianto@yahoo.com Abstract An effort to produce laccase in a continous mode has been performed. Marasmius sp grown on rice straw have been employed in a packed bed which had height and inner diameter of 24 cm and 2.5 cm, respectively. The effect of the liquid nutrient (Kirk medium) rate and the aeration rate were investigated. Both of them flew countinously by counter current along the. The Kirk medium was pumped into the top of at the rates of 0.5 and 1.0 ml/minute, while the forced air flew from the bottom at the rates of 0.05 and 1.5 vvm. The results demonstrated that increasing medium and aeration rate gave an increase of laccase activity and productivity. The highest activity and productivity were U/L and U/L.day, respectively. Keywords: laccase; Marasmius sp. ; rice straw; packed bed ;. 1. Introduction Solid State Fermentation (SSF) is a fermentation process carried out in conditions of little or no free water, using an inert substrate or natural substrate as the solid support (growth media) [Mitchell et. al., 2006]. This fermentation is an alternative to the Submerged Fermentation culture immersion because it is possible to use substrates derived from products/agricultural waste that's cheap and available in abundance. SSF also very suitable for fermentation using fungi. Metabolites produced from the SSF has a high concentration that can be more easily and cost-effective in the purification phase [Pandey et al., 2008]. Laccase is one of the enzymes that are being developed using Solid State Fermentation. Production of laccase by Solid State Fermentation in small scale and batch system has been widely studied [Galhaup and Haltrich, 2001; Couto et al., 2003; Fenice et al., 2003; Rancano et al., 2003; Dominguez et al., 2005]. Laccase has many benefits in the areas of biotechnology, among others, to delignification, color removal of textile waste water and detoxification. In the foreseeable future, the need of laccase is predicted to increase so it requires a strategy of large-scale and continuous production. However, until recently, the development of Solid Fermentation culture on a commercial scale is still limited because of the lack of an established knowledge regarding how to design and operate large-scale. Initiation of use of some of the with a continuous mode has been performed [Feijoo et al., 1994]. Tray, packed bed, expanded bed, and the rotating drum is used for several types of enzyme production by solid fermentation culture. The use of these s are expected to increase the yield of enzyme activity and productivity. The process of enzyme production by SSF method on a scale is still hampered by a lack of data and explanation of the criteria for scale-up, especially for the production process which utilizes a fast-growing fungus on natural growth media. The simplest design of s for SSF is packed-bed. On this type of, static bed condition of the substrate is suitable for the SSF for mixing and agitation can interfere with the growth of fungi. The A-24-1

2 results from Fillis (2001) and Niladevi et al. (2008) showed that the type of packed-bed capable to produce ligninolitic enzymes (LiP, MnP, laccase) from the white rot fungus with a better activity than in the batch culture when grown on solid synthetic and natural media. The previous study on batch culture showed that Marasmius sp grown on rice straw was the most promising fungus and growth media to produce laccase. Based on these results, this study was focused on the laccase production in continuous system using a packed bed. Thus, this study has the objective to study the effect of medium and aeration rates on laccase production processes in a packed-bed and to compare the productivity of continuous and batch system. 2. Experimental A. Microorganisms Marasmius sp., a collection of Laboratory of Microbiology and Bioprocess Technology ITB, was grown on Potato Dextrose Agar (PDA) plates at room temperature (±28 C) for five days. The culture were then kept at 4 C until used. B. Bioreactor Configuration Marasmius sp. was grown as an immobilised culture in a packed bed (Fig. 1). The was made from glass which has height and inner diameter of 24 and 2.5 cm, respectively C. Cultivations Rice straw as a growth media for Marasmius sp was chopped in size of cm length. A 15 grams of rice straw cuttings was soaked in 75 ml of Kirk medium and sterilised. Compositions of Kirk medium are glucose 10 g/l, KH 2 PO g/l, MgSO 4.7H 2 O 0.4 g/l, CaCl g/l, sodium acetate 2.3 g/l, diammonium tartrate 0.4 g/l, MnCl g/l, yeast extract 0.3 g/l, CuSO 4.7H 2 O 0.01 g/l, H 2 MoO g/l, MnSO 4.4H 2 O 0.01 g/l, ZnSO 4. 7H 2 O g/l, and Fe 2 (SO4) 3 0,007 g/l. Sterilised soaked rice straw were transferred into the sterilised. Two pieces inoculums from agar plate in size of 1.5 x 1.5 cm were then transferred aseptically into the top and the middle part of. To investigate the effect of medium and aeration rates, two level of medium and aeration rates were employed as shown in Table 1. The sterilised Kirk medium was flowed into the top of by peristaltic pump while the air was supplied from the bottom of. All of run was carried out for 21 days.and sample was collected daily from the bottom of to analyse the laccase activity and glucose concentration.. A-24-2

3 Run Figure 1. Configuration of a Packed Bed Bioreactor Table 1. Medium and aeration rates Medium rates (F m ) (ml/menit) Aeration rates (F u ) (vvm) D. Laccase and Glucose Assay Laccase activity was determined with 2,2 -azinobis (3-ethylbenzthiazoline -6-sulphonic acid) (ABTS) in 0.4 mm sodium acetate buffer (ph 4.5). Oxidation of ABTS was determined by the increase in A 420 (ε 420 = 36 (mm cm) -1 ) using Spectrophotometer. One unit of enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute. Glucose concentration was analysed using Somogy-Nelson method. E. Productivity The overall activity (U o ) during a period of fermentation was defined as in eq. 1 and the productivity (P r ) was calculated using eq. 2.. (1). (2) 3. Results and Discussion Figure 2 shows the results of laccase production in a continuaous packed bed for 21 days with the variation of medium and aeration rates. The dynamics of production of laccase, glucose A-24-3

4 concentration and ph that comes out of the bottom of the appeared to have a similar pattern. At the beginning of cultivation, the inoculum Marasmius sp. which were inoculated at the top of the bed began to adapt to new environments and will begin to consume glucose for growth and produce laccase. Along with the increased rate of glucose consumption, laccase activity increased sharply until reaching a maximum activity and then decreased. Glucose is supplied in constant rate at any time and at the end of fermentation were almost entirely consumed by Marasmius sp. In the medium flow rate (F m ) 0.5 ml/min and aeration rate (F u ) of 0.05 vvm, laccase activity was observed starting on the second day, i.e 3.88 U/L. Laccase activity increased slightly until the fourth day. This indicates Marasmius sp. still adapting to the environment in the, it is also indicated by the consumption of glucose that still at low rate. Glucose concentration of 11.7 g/l at the beginning of fermentation, while on the fourth day was observed at 10.9 g/l. Laccase activity increased significantly from the fifth day and reached a maximum activity of U/L on day 15. Glucose concentration also experienced a sharp decline of 10.9 g/l on the fourth day to 3.2 g/l on day 15 and continued to decline until it becomes 0.1 g/l on day 21. After the 15 th day, the activity decreases up to U/L on day 21. In the second run, when the aeration rate (F u ) increased to be 1.5 vvm with a medium flow rate as the first run (F m ) 0.5 ml/min, the maximum activity obtained appear higher, which is U/L on day 13. While the consumption of glucose is faster than the first run as it is seen from the concentration of glucose was observed on the fourth day to 8.7 g/l. As with the first run, laccase activity continued to decline until the end of fermentation after reaching the maximum activity. The second run shows that the air flow rate has a positive influence on the activity obtained due to much more oxygen supplied for Marasmius sp. F m = 0.5 ml/min F u = 0.05 vvm F m = 0.5 ml/min F u = 1.5 vvm A-24-4

5 F m = 1.0 ml/min F u = 0.05 vvm F m = 1.0 ml/min F u = 1.5 vvm Figure 2 The dynamics of laccase production in a packed bed ( : laccase activity, ο: residual of glucose, : glucose consumption) The increase in medium flow rate (F m ) to be 1.5 ml/min at aeration rate (F u ) of 0.05 vvm also have a positive influence to obtain the maximum activity of U/L on day 16. Obtaining the maximum activity at the medium flow rate is still lower than the increase in air flow rate. Medium flow rate and air flow rate has an influence on the maximum activity obtained. When both of them are increased to be F m = 1 ml/min and F u = 1.5 vvm, the maximum activity obtained was the highest ( U/L). The ph parameter at all run showed a relatively constant value in the range of 5.5 to 6.0. This profile is in agree with several studies on laccase production in packed bed [Rosales 2007; Guerra, 2008]. Glucose consumption is one of the factors that influence the growth of fungi and enzymes produced. Changes in glucose concentration values in all four experimental variation associated with a given aeration rate. At run 2 and 4 with aeration rate 1.5 vvm, decreased glucose concentration occurred more rapidly than run 1 and 3 at a smaller aeration rate of 0.5 vvm. In addition, the final glucose concentration at run 2 and 4 is smaller than run 1 and 3. This shows that the growth of fungal biomass and laccase production occurs more rapidly in the by providing a greater concentration of oxygen. This is also supported by measurements of fungal growth along the column of. In run 2 and 4, the fungus has grown to fill the entire column bed (24 cm) on day 8 and day 7, respectively. Whereas in run 1 and 3, it was occured on day 9 and 10, respectively. This study showed that the flow rate of medium (Kirk medium) affects the growth of fungus and product formation.. This is because it deals with the water content of wet substrate in the. Naturally, rice straw tends to dry [Niladevi et al., 2008] causing the substrate should be wetted to obtain adequate moisture during the fermentation process. It should be paid attention because of the metabolic heat generated during fermentation could cause a decrease in water content of the substrate. Adequate water levels in SSF will encourage the formation of products due to reducing barriers for fungal hyphae to consume nutrients while decreasing water content will cause a decrease in solubility and availability of nutrients for fungal cultures. In addition, the increase in flow A-24-5

6 rate of medium (liquid) will also improve the distribution medium throughout the and reduce the presence of dead zones [Feijoo et al., 1995]. The liquid medium contained glucose as a simple carbon source that could be directly utilized by fungus. A smaller medium flow rate might cause a certain parts of the substrate is not wetted by the medium so that growth of fungus will be inhibited. However, if the water content is too high (above the optimum value), it would be reduced the laccase production due to reducing space and the interparticle porosity for the growth [Niladevi et al., 2008]. Solid State Fermentation usually involve the growth of aerobic organisms [Mitchell et al., 2006], including the white rot fungus Marasmius sp. Oxygen requirement must be met as an important factor that will affect the growth of fungi. In this study, the oxygen supplied in the form of forced air flow from the bottom of bed so that it can pass through the column bed. The sufficient of aeration for the fungus in system will allow the diffusion of oxygen and a more even spread into the fungal biomass particles that grow attached to a solid substrate. In this study, increasing the aeration rate of laccase activity obtained also increased. At the aeration rate 1.5 vvm, oxygen from the air has met to the minimum dissolved oxygen requirement, therefor a higher activity was obtained than that of aeration rate 0.05 vvm. The level of aeration rate above 1.5 vvm was not studied yet, but possibly by increasing aeration rate would decrease laccase activity due to the decreasing of water content of the fermented material that has negative effect to the growth of microorganisms [Anisha et al., 2008]. The results were in line with Niladevi et al. (2008) who has studied the influence of aeration rate of laccase activity from Streptomyces psammoticus on straw substrate in a packed bed and obtained the optimum aeration rate was 1.5 vvm. In this study, laccase activity and glucose consumption in a packed bed has not reached steady state. In fact one of the intended use of these s is continuously laccase production. Enzyme production was discontinued on day 21 for all variations of the run due to a blockage of the flow due to the overgrowth of Marasmius sp. on the rice straw in the. Therefore, the stability of laccase production in a packed bed needs to be improved. Feijoo et al. (1995) explains a method to increase the stability of the enzyme production by regulating the concentration of residual glucose in the output stream at a certain level when the fungus has reached the stationary phase. The maximum laccase activity obtained in this study was U / L and it is lower than the batch culture (1564 U/L). Another important parameter in the enzyme production is the productivity. Productivity is total unit of enzyme that could be obtained per working volume of per total fermentation time. Table 2 shows the results for each variation of laccase productivity and compared to batch systems, which showed that the highest productivity ( U/L/day) obtained at medium flow rate of 1 ml/min and aeration rate 1.5 vvm. Productivity of a continuous production is higher than that of batch systems (175.81U/L /day). Laccase activity obtained from this study was relatively high when compared to other studies (Table 3). Table 2 Laccase productivity System Medium rate (ml/min) Aeration rate (vvm) Productivity (U/L.day) Continuous Batch Table 3 Comparison to other studies Bioreactor type Organism Substrate/growth media Activity Reference A-24-6

7 Continuous packed bed-biorector Phanerochaete chrysosporium Corn cob 30 U/L Couto et al., 2000 Continuous packed bed-biorector Phanerochaete chrysosporium PUF (polyurethane foam) 30 U/L Couto et al., 2000 Immersion versicolor Nylon sponge (induser Tween 80) 229 U/L Couto et al., 2003 Tray versicolor Nylon sponge (inducer Tween 80) 343U/L Couto et al., 2003 Tray versicolor Barley bran (inducer Tween 80) 3500 U/L Couto et al., 2003 Expanded bed versicolor Barley bran (inducer Tween 80) 600 U/L Couto et al., 2003 Packed bed Phanerochaete chrysosporium PUF (polyurethane foam) 570 U/L Fillis, 2001 Packed bed Streptomyces psammoticus Rice straw U/g fermented substrate Niladevi et al., 2008 Tray hirsuta Grape seed 18,715 U/L Brijwani et al., 2010 Packed bed Pleoratus ostreatus PUF (polyurethane foam) Lakase U/mg protein Prasad et al., 2005 Packed bed Marasmius sp. Rice straw U/L (= U/g protein) This study 4. Conclusion In conclusion, a. medium flow rate and aeration affect laccase activity produced by Marasmius sp. in a packed bed. Highest laccase activity obtained on the condition of medium flow rate of 1.0 ml / min and aeration rate 1.5 VVM of U / L. Activity of this is lower than the batch system but produces higher productivity, Nomenclature F m flow of Kirk Medium (ml/min) F u aeration rate (vvm) U o overall activity (U) t prod fermentation time (day) V R working volume of (L) References 1. Anisha, G.S., John, R.P., Prema, P., Pandey, A. (2008). Investigation on α-galactosidase production by Streptomyces griseoloalbus in a forcefully aerated packed-bed operating in solid-state fermentation condition. Appl. Biochem. Biotechnol., 160, A-24-7

8 2. Brijwani, K., A. Rigdon, and P.V. Vadlani. (2010), Fungal Laccases: Production, Function, and Applications in Food Processing, Enzyme Research, Couto, S., Moldes, D., Liébanas, A., and Sanromán, M. (2003): Investigation of several configurations for laccase production by versicolor operating in solid-state conditions. Biochemical Engineering Journal, 15: Couto, S.R., I. Rivela, M. R. Munoz, A. Sanroman, (2000): Ligninolytic enzyme production and the ability of decoloration of Poly R-478 in packed bed s by Phanerochaete chrysosporium, Bioprocess Engineering, 23, Feijoo, G., Dosoretz, C., dan Lema, M. (1994). Production of lignin peroxidase from Phanerochaete chrysosporium in a packed bed with recycling. Biotechnology Techniques, Vol. 8(5), Feijoo, G., Dosoretz, C., dan Lema, M. (1995). Production of lignin peroxidase from Phanerochaete chrysosporium in a packed bed operated in semi-continuaous mode, Journal of Biotechnology. 42, Fenice, M., Sermanni, G.G., Federici, F. and D Annibale, A. (2003): Submerged and solid-state production of laccase and Mn-peroxidase by Panus tigrinus on olive mill wastewater-based media, J Biotechnol 100, Fillis, V.W. (2001). Design of a Packed-Bed Fungal Bioreactor: The application of enzymes in the bioremediation of organopollutants present in soils and industrial effluent. Theses and Disertation. Faculty of Science. Department of Physical Sciences (Chemical Engineering), Cape Peninsula University of Technology. 9. Galhaup, C. and Haltrich, D. (2001): Enhanced formation of laccase activity by the white-rot fungus pubescens in the presence of copper, Appl Microbiol Biotechnol 56, Guerra, G., Domínguez, O., Ramos-Leal, M., Manzano, M.A., Sánchez, M.I., Hernández, I., Palacios, J., dan arguelles, J., (2008): Production of laccase and manganese peroxidase by white-rot fungi from sugarcane bagasse in solid bed: Use for dyes decolourisation, Sugar Tech., 10(3), Mitchell, D.A., Krieger, N., Berovič, M. (2006). Solid-state fermentation s: fundamental of design and operation. Berlin: Springer, Niladevi, K. N., Sheejadevi P. S. and Prema P. (2008). Strategies for Enhancing Laccase Yield from Streptomyces psammoticus and Its Role in Mediator-based Decolorization of Azo Dyes. Applied Biochemistry and Biotechnology, 151(1), Pandey, A., Soccol, C., dan Laroche, C. (2008). Current development in solid state fermentation. New Delhi: Springer Asiatech Publisher Inc. 14. Prasad, K., Mohan, S., Bhaskar, Y., Ramanaiah, S., Babu, V., Pati, B., et al. (2005): Laccase production using Pleurotus ostreatus 1804 immobilized on PUF cubes in batch and packed bed reactors : influence of culture condition. The Journal of Microbiology, 43: Rancano, G., Lorenzo, M., Molares, N., Rodríguez Couto, S.and Sanromán, A. (2003): Production of laccase by versicolor in an airlift fermentor. Process Biochem 39, Rosales, E., Couto, S.R., dan Sanromán, M.A., (2007): Increased laccase production by hirsuta grown on ground orange peelings, Enzyme and Microbial Technology, 40, A-24-8

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Optimisation of Laccase Production using White Rot Fungi and Agriculture Wastes in Solid State Fermentation

ITB J. Eng. Sci., Vol. 44, No. 2, 2012, 93-105 93 Optimisation of Laccase Production using White Rot Fungi and Agriculture Wastes in Solid State Fermentation Hendro Risdianto 1,2, Elis Sofianti 1, Sri