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Riksfardini Annisa Ermawar, Dede Heri Yuli Yanto, Fitria, and Euis Hermiati

Research and Development Unit for Biomaterials – Indonesian Institute of Sciences

ABSTRACT

Rice straw probably is the most abundant biomass in Indonesia. Rice straw can potentially be converted into various different value added products. However, many recent researches on biomass have focused only on the productions of a few high-value products, such as ethanol and pulp/paper. For these particular purposes, lignin contained in the biomass should be removed, so that the cellulose and hemicellulose can be further hydrolyzed either chemically or enzymatically. Therefore, the biomass should be pretreated to remove the lignin as much as possible. One of pretreatment methods of biomass is using white-rot fungi. White-rot fungi are the most effective basidiomycetes for biological pretreatment of lignocellulosic materials. These fungi produce a set of enzymes which are directly involved in lignin decay. The aims of this research were to study the effects of white-rot fungi and incubation time that applied for pretreatment of rice straw on its biodegradation. Rice straw meal used in this study was prepared from the IR-type rice plants. The rice straw meal was steamed before inoculated by Pleurotus ostreatus, Ceriporiopsis subvermispora, Coriolus versicolor, Pycnoporus sanguineus, and Schizophyllum commune. The cultivation was performed stationery at 27ºC for 8 weeks. Sampling was carried out at 2, 4, 6 and 8 weeks. Lignin and holocellulose content at each sample were determined using Wise Methods. Results of this study showed that the five fungi can be used for biodegradation of lignin in rice straw. However, for particular purposes, such as ethanol or pulp production, C. versicolor is the most suitable species among the five fungi studied, while optimum incubation time achieved after 4 weeks of incubation.

Keywords : biomass, rice straw, white-rot fungi, pretreatment, lignin.

INTRODUCTION

Biomass has always been a major source of energy for mankind and it is presently estimated to contribute of the order 10-14% of the world’s energy supply. Biomass, as large amounts of lignocellulosic waste, is generated through forestry and agricultural practices (Howard, et al., 2003). Since Indonesia is the country which produce rice extensively, rice straw becomes the most abundant biomass. Indonesia produced 41 million tons of rice straw each year and only 31-38 % has been using for feedstock (Utomo, et al., 2005).

It has also been recognized that lignocellulosic materials can potentially be converted into various different value added products. However, many recent researches on biomass have focused only on the productions of a few high-value products, such as productions of ethanol and pulp/paper (Howard et al, 2003). Moreover, productions of ethanol and other alternative fuels from lignocellulosic biomass, which treated by biological methods, can reduce urban air pollution, decrease the release of carbon dioxide in the atmosphere, and provide new markets for agricultural wastes. In addition, pretreatment of agricultural wastes with ligninolytic fungi enables their use as raw material for pulp and paper manufacturing (Perez, et al., 2002).

The efficiency and effectiveness in producing ethanol depends on the proportion of the material in the biomass and the conversion techniques used. The proportion of cellulose and lignin in biomass are important in biochemical conversion processes. The biodegradability of cellulose is greater than that of lignin. Thus, for the production of ethanol, biomass with high cellulose/hemi cellulose content is more preferred to provide high, liter/tons yield to the ones with higher proportion of lignin (McKendry, 2002).

The pulp and paper industry utilized mechanical and chemical pulping processes or a combination of these to produce pulps with desired characteristics. These processes have their own disadvantages. Mechanical processes are electrical energy-intensive and produce paper with higher color reversion rate (tendency to turn yellow with time) as a result of high lignin content in raw materials. Chemical pulping involves the use of excessive chemicals to degrade and dissolved lignin and releasing high-cellulose fibers from wood cell walls (Akhtar, et al., 2002). Thus, it is important to apply or combine several methods in order to reduce lignin content in biomass more economically and produce better quality pulp/paper.

The effect of lignin on bioavailability of other cell wall components is thought to be largely a physical restriction, with lignin molecules reducing the surface area available to enzymatic penetration and activity (Haug, 1993 in Richard, 2000). Meanwhile, the structural complexity of lignin, its high molecular weight and its insolubility make its degradation very difficult (Perez, et al., 2002). Due to its resistance to enzymatic attack, naturally occurring cellulosic biomass must be pretreated before it can be enzymatically hydrolyzed. Pretreatment of lignocelluloses materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose (Sun & Cheng, 2002).

White-rot fungi are defined as the microorganisms that most efficiently degrade lignin from wood (Perez, et al., 2002). Another reference describes that white-rot fungi are the most effective basidiomycetes for biological pretreatment of lignocelluloses materials (Fan et al., 1987 in Sun & Cheng, 2002). The term white rot fungi have been applied to certain ligninolytic basidomycetes with a relatively high selectivity to degrade lignin in wood. These fungi produce a set of enzymes which are directly involved in lignin decay. Two major families of enzymes are involved in ligninolysis by white-rot fungi, those are peroxidases and phenol-oxidase termed laccase. Peroxidases divided into lignin peroxidase (LiP) and manganese peroxidase (MnP). Each species secretes a particular assortment of this enzymatic machinery to the medium in which it is growing. Thus, some strains produce all of the major families of enzymes, others only two of them, or even one (Lobos et al., 2001; Perez, et al., 2002). Therefore, it is interesting and important to know the activity of these fungi in degrading lignin of rice straw.

The objectives of this research were to study the effects of using white-rot fungi during pretreatment and the incubation time on biodegradation of lignin in rice straw. Moreover, results this study hopefully can become valuable information

regarding the feasibility of using biological pretreatments in lignocellulosic materials in further industrial processes.

MATERIALS AND METHODS

Rice Straw

The rice straw was originated from IR type rice and was taken from Bogor. The rice straw was air-dried to approximately 5 % of moisture content, then milled and screened into 40-60 mesh size.

Fungus

Five strains of the white-rot fungi were used based on their abilities to decay wood. Ceriporiopsis subvermispora and Coriolus versicolor were obtained from Research Center for Chemistry, Indonesian Institute of Sciences, Serpong. Pleurotus ostretus No. 226 and Pycnoporus sanguineus No. 915 were obtained from Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences, Bogor and Schizophylum commune was originaly isolated from decaying-wood at Research and Development Unit for Biomaterials, Indonesian Institute of Sciences, Cibinong. Cultures were continuously maintained in potato dextrose agar slants. Working cultures were prepared from the stock cultures as needed and refrigerated until used. In preparing the inocula, the strains were cultivated on sterile Potato Dextrose Agar (PDA) plates for 7 days.

Rice Straw Meal Preparation and Inoculation

Prior to treatment by white-rot fungi, the rice straw meal was steamed at 100ºC for one hour. Forty grams (40 g) of steamed rice straw was added into 250 ml bottles, and then sterilized in 121ºC for 30 minutes. The rice straw meal was then ready to be inoculated by the white rot fungi after cooled at room temperature (± 27–29 ºC). Ten plugs cut (each plug has 1 cm diameter size) of 7 day-old potato dextrose agar plate of the fungal culture, from the previous pre-cultures, were inoculated on the rice straw meal. The cultivation was performed stationery at 27ºC for 8 weeks.

Klason Lignin and Holocellulose Content

After 2, 4, 6, and 8 weeks of cultivation, lignin was analyzed by Klason lignin and holocellulose content was determined using Wise methods.

Experimental Design and Statistical Analyses

Factorial design was used in this study. There were two factors studied, species of fungi (Ceriporiopsis subvermispora, Coriolus versicolor, Pleurotus ostretus, Pycnoporus sanguineus, Schizophylum commune) and incubation time (2, 4, 6, and 8 weeks). Data resulted from this study were statistically analyzed by Analysis of Variance (ANOVA) and further by Duncan Multiple Range Test.

RESULTS AND DISCUSSIONS

Results of this study showed that species of fungi and incubation time used in pretreatment of rice straw as well as their interactions have significant effects on weight loss, lignin loss and holocellulose loss of rice straw. Weight loss indicates the degree of degradation of components in rice straw, therefore the higher the weight loss means the more active the fungi degrading all components in rice straw. However, fungi that give the highest weight loss did not always mean that they are the best ones, since for this purpose we expect fungi that degrade lignin extensively but did not degrade holocelluloce very much.

Weight loss in rice straw pretreated by white-rot fungi at each incubation time can be seen in Figure 1. The highest average of weight loss was found in rice straw pretreated by P. ostreatus, followed by those treated by C. versicolor, P. sanguineus and S. commune, while the lowest was found in rice straw pretreated by C. subvermispora (Table 1). Further statistical analyses using Duncan Multiple Range Test showed that weight loss caused by P. ostreatus was sigficantly different from those caused by the other four fungi. The difference was probably due to the ability of those fungi in degrading the components of lignocellulosic materials in rice straw. Previous study using Scanning Electron Microscope (SEM) demonstrated that P. ostreatus degraded cell walls and middle lamella of beech wood during 8 weeks, meanwhile C. subvermispora preferentially attacked middle lamella without intensive morphological damage of the cell walls (Syafwina, et al., 2002). Unfortunately, scientific studies on the ability of C. versicolor, P. sanguineus and S. commune using SEM were not reported yet.

Figure 2 shows lignin loss in rice straw during pretreatment by white-rot fungi, while Figure 3 shows the holocellulose loss. Lignin loss in rice straw pretreated by P. sanguineus, C. versicolor and P. ostreatus was significantly higher than those pretreated by S. commune and C. subvermispora, while holocellulose loss in rice straw pretreated by P. ostreatus was significantly higher than those pretreated by the other four fungi (Table 1). One of previous studies reported that C.subvermispora has performed better as a high-selectively lignin degarading fungi among basidiomycetes (Lobos et al, 2001). However, in this study degradation ability of C. subvermispora was the lowest. This was probably due to unsuitable incubation and cultivation conditions for the fungus. The high lignin loss in rice straw pretreated by P. ostraetus and the low lignin loss in that pretreated by C. subvermispora was probably due to ligninolytic enzymes secreted by the fungi. It was reported that these fungi produce a set of enzymes which are directly involved in lignin decay (Perez, 2002). It has been known that P. ostreatus produced three kinds of ligninolytic enzymes: laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP), while C. subvermispora produced two kinds of ligninolytic enzymes: laccase and manganese peroxidase. (Kofujita, 1991; Rutimann, et al., 1992 in Lobos et al., 2001). Moreover, there was a suggestion that an indirect oxidation by LiP of low-molecular-weight diffusible compounds capable of penetrating the cell wall and oxidizing the polymer. Microscopic studies of selective lignin biodegradation reveal that white-rot fungi remove the polymer from inside the cell wall. So far, it is the most effective peroxidase and can oxidize phenolic and non-phenolic compounds, amines, aromatic ethers and polycyclic aromatics with appropriate ionization potential. (Perez, et al., 2002). Therefore, it is important to know the kinds of enzymes produce by C. versicolor, P. sanguineus and S. commune which probably involved in lignin decay, but unfortunately scientific studies on those had not reported yet.

Since we have to choose fungi that could degrade as much as possible lignin and as little as possible holocellulose in rice straw, it seems that P. ostreatus is not suitable due to its highest capability in degrading holocellulose. The relatively lower

capability in degrading holocellulose was found in C. versicolor and P. sanguineus; therefore, it seems that the two fungi were the most suitable fungi among the three fungi that have the highest lignin degrading ability.

Table 1. The effects of species of fungi on weight loss, lignin loss and holocelluolose loss average in rice straw during pretreatment.

Data on the same column that have the same letter do not significantly different

Table 2 showed that the longer the incubation time, the higher the weight loss. The highest weight loss average was found in rice straw after incubated for 6 and 8 weeks, which reached 29.47% and 30.93%, respectively. There was not significant difference of weight loss between six weeks and eight weeks of incubation, but there was significant difference of weight loss between two and four weeks of incubation. The highest lignin loss average was also found in rice straw incubated for 8 weeks (34.24%). However, this did not differ from the one that incubated for 4 weeks (31.06%). Hollocellulose loss average reached as high as 39.47% after 8 weeks of incubation, which is also the highest loss, and this did not differ from that of 6 weeks of incubation (38.53%). Since more lignin loss and less holocellulose loss were expected, it seems that 4 weeks of incubation was the optimum incubation time to get high lignin loss and not too much loss of holocellulose. In Figure 2 it can also be seen that the lignin loss caused by C. versicolor and P. sanguineus, which have highest lignin degrading ability, reached optimum after 4 weeks of incubation.

Table 2. The effects of incubation time on weight loss, lignin loss and holocelluolose loss average of rice straw during pretreatment by white-rot fungi.

Data on the same column that have the same letter do not significantly different

Figure 1. Weight loss in rice straw pretreated by white-rot fungi

Figure 2. Lignin loss in rice straw pretreated by white-rot fungi

Figure 3. Holocellulose loss in rice straw pretreated by white rot fungi

CONCLUSIONS

Species of fungi and incubation time used in pretreatment of rice straw as well as their interactions have significant effects on weight loss, lignin loss and holocellulose loss of rice straw. Of the five white-rot fungi studied, P. ostreatus, C. versicolor and P. sanguineus were the most active fungi in degrading lignin as well as holocellulose in rice straw. Considering the average of lignin loss and holocellulose loss in rice straw pretreated by the fungi, it seems that C. versicolor is the most suitable fungus. In general, the longer the incubation time, the higher the weight loss, lignin loss and holocellulose loss. However, it was found that 4 weeks of incubation reached optimum in degrading lignin in rice straw. Lignin loss caused by C. versicolor, which has the highest lignin degrading ability, reached optimum after 4 weeks of incubation as well.

ACKNOWLEDGEMENT

The authors would like to gratefully acknowledge Ms. Tami Idiyanti from Research Center for Chemistry, Indonesian Institute of Sciences, Serpong and Ms. Suciatmih from Research Center for Biology, Indonesian Institute of Sciences, Bogor for helping in providing cultures of fungi.

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