The Incorporation of Chromium in Rice Straw Fermented with Ganoderma Lucidum

The research was designed to evaluate chromium incorporation by Ganoderma lucidum in rice straw substrate supplemented with chromium chloride. Treatments were combination of Cr level (0, 500, 1000, 1500, 2000, 25000, and 3000 ppm) and fermentation time (0, 2, 3, and 4 wk). The treatments were arranged in a factorial 7 x 4 and allocated in completely randomized design with three replications. G. lucidum was grown in potato dextrose agar (PDA) medium for 10 days and was inoculated to the substrate which have been sterilized and mixed with CrCl3.6H2O. The moisture of substrate was maintained at 70%. Fiber and protein components of growth media of G. lucidum was determined and analyzed for their Cr content. The finding result showed that addition of Cr up to 3000 ppm into the substrate stimulated the G. lucidum growth. Chromium was incorporated into the fiber and protein components of the growth substrate of G. lucidum during fermentation. Incorporation of Cr into the protein of substrate containing3000 ppm Cr was highest when fermented for 4 wk. Protein component of substrate contained 9.29% Cr while in NDF and ADF was 27.20% and 10.55% Cr, respectively. It is concluded that Cr was incorporated into the G. lucidum cells during fermentation.


INTRODUCTION
Chromium (Cr) is one of the micro mineral and its essentiality has been proved in many researches. Chromium is important for animals (NRC, 1997) because of their role in the metabolism of carbohydrates, fats, proteins and nucleic acids (Pechova & Pavlata, 2007). Chromium from inorganic sources is poorly absorbed. The conversion of inorganic Cr to an organic complex improves its bioavailability (NRC 1997;Pechova & Pavlata, 2007). Supplementation of mixed Cr in fermented feed and G. lucidum improved immune response of lactating cows (Agustin et al., 2011). Therefore, efforts should be made to convert inorganic Cr to organic form. Effective Cr level for G. lucidum growth in solid state fermentation of oil palm by-product was 3000 ppm with incorporation value of 68.23% (Agustin et al., 2010). Metal ions including Cr can accumulate intracellularly in fungal cells (Sen & Dastidar, 2010). The ability of microorganism cell to bind metal ions is associated with its cell wall (Shugaba et al., 2012).
G. lucidum is able to use sawdust as a substrate for its growth (Chang & Miles, 2004) and it can also use oil palm by product for its growth (Agustin et al., 2010). A source of fiber such as rice straw is available and its availability is abundant. Rice straw may meet the criteria as a source of fiber for G. lucidum growth. Misra et al., (2007) reported that mustard straw fermented by G. lucidum at a temperature of 35 o C for 21 d can increase in vitro digestibility and delignification. G. lucidum can bind and can accumulate metal ions intracellularly. However, information about the use of rice straw as a growth medium of G. lucidum for the purpose of producing organic Cr have not available yet. The experiment was designed to determine the incorporation of Cr by G. lucidum in rice straw as a main component of substrate. The result obtained is an effort to synthesize organic Cr to be used as a mineral supplement for livestock. In this case the whole fermented substrate is used as the product.

Preparation Starter of Ganoderma lucidum
Inoculum stock of G. lucidum was obtained from the collection of Indonesian Biotechnology Research Institute for Estate Crops (IBRI), Bogor. Starter was obtained by rejuvenating inoculums stock at IBRI Microbial Laboratory and Laboratory of Microbiology, Department of Nutrition and Feed Technology, Faculty of Animal Science, Bogor Agricultural University. The rejuvenation of inoculums was carried out by culturing isolates of stock culture G. lucidum into petridish containing PDA medium (potato-dextrose-agar) (Wagner et al., 2003), and was incubated for 10 d or until the mycelium of G. lucidum met petridish. The production of the inoculums was a crucial step affecting significantly the performance of the subsequent fermentation.

Substrate Preparation and Inoculation
The main material used in this research was derived from rice straw obtained from rice fields. Rice straw was chopped to the size of 2-3 cm and dried in the sun to the moisture content of about 10%. Rice straw was mixed with a solution containing CrCl 3 .6H 2 O with varying Cr concentration: 0, 500, 1000, 1500, 2000, 2500, and 3000 mg Cr per kg of dry matter and conditioned at 70% moisture content. The mixed substrate was inserted into the 300 ml bottle, covered with aluminum foil, and sterilized for 30 min at pressure of 1.2 and temperature of 121 o C. After cooling, sterilized bottle were inoculated with starter aseptically with a plug diameter of 0.5 cm in the laminar flow cabinet. The inoculated substrate was incubated at 27-28 o C for 0, 2, 3, or 4 wk. Fermentation products were harvested according to the time of incubation.

Experimental Design
The experiment was carried out in a 7 x 4 factorial design with 3 replications for each treatment. There were seven levels of Cr supplemented into the rice straw, i.e. 0, 500, 1000, 15000, 1500, 2000, 2500, and 3000 ppm, at the time factor. There were four fermentation times as the second factor, i.e. 0, 2, 3, and 4 wk.

Sample and Data Analysis
Percentage of dry matter (DM) and organic matter (OM) loss were calculated. Total Cr in the fermented biomass was measured by using Atomic Absorption Spectrophotometer (AAS) Shimadzu AA680 models (Carry & Allaway, 1971). The amount of Cr bound fiber and protein components of biomass was measured separately. Measurement of total Cr in solid substrate was conducted by using a sample of 5 g. Samples were crushed in a mortal porcelain and put into a test tube, add 5 mL of 10% TCA and was homogenized with a vortex, and then allowed to stand for one hour. The solution was then centrifuged at 3000 rpm for 10 min. Chromium concentration in supernatant was measured. The content of the neutral detergent fiber fraction of fiber (NDF) and acid detergent fiber (ADF) were determined by analysis of Van Soest (Van Soest & Wine 1967). Levels of Cr contained in the NDF and ADF are determined from the sample after the determination of NDF and ADF. Biomass protein content was analyzed (Bradford 1976) and its Cr levels were measured.
The data were analyzed statistically according to two-way Analysis of Variance (ANOVA) and the difference between treatment means was determined using Duncan's Multiple Range Test (DMRT).

RESULTS AND DISCUSSION
G. lucidum grew rapidly on PDA on the fifth until 10 d of incubation. The mycelium reached maximum growth on stable condition when the level of water was 70%. The water content of the rice straw substrate in this study was higher than recommended by Chang & Miles (2004) which was 60%-65%. This is because the substrate for cultivation of G. lucidum was different. Chang & Miles (2004) used saw dust as a substrate, while in this study we used rice straw as a main component of substrate. The growth of G. lucidum varied widely, depending on the kind of substrate (Erkel, 2009). Fermentation with G. lucidum was carried out at temperatures ranged from 26-28 o C and pH of substrate 5.5. The optimum fermentation condition appeared to depend on temperature and pH, and this substrate conditions was suitable for the growth of G. lucidum according to the recommendation of Chang & Miles (2004). The growth of fungi G.
lucidum on rice straw substrate containing up to 3000 ppm Cr began to appear after the age of 1 wk. After 2 wk of fermentation, its white mycelium had covered half of the substrate surface of the media in the bottle. Mycelium growth in 3000 ppm Cr levels up to 4 wk of incubation was excellent but has not formed fruiting bodies.

Fermented Rice Straw
Dry matter content of fermented rice straw was influenced by the fermentation time and Cr levels (P<0.01) but there was no interaction between the two factors (Table 1). Dry matter content declined sharply in the second week of fermentation, from 29.24% to 26.5%, suggesting that the nutrients contained in the substrate has been used by fungi.
Dry matter content of substrate continued to decrease up to 4 wk of incubation. It was suggested that the nutrients in rice straw was used by G. lucidum to increase the number of cells as indicated by the growth of mycelium. There was a linear relationship between the dry matter content of rice straw media with fermentation time according to the equation, Y= -1.11 X + 29.70 (R 2 = 0.8053, P <0.01 where Y= change in the levels of dry matter, %; X= length of fermentation, days). This means that as the fermentation time increased by one unit there was a reduction of dry matter by 1.11%. Ligninolytic enzyme produced by G. lucidum degraded lignin from the rice straw, and this was associated with fermentation time and the reduce of dry matter. Product of degraded lignin was used by G. lucidum to its mycelium growth.
Chromium supplementation appeared to decrease the rate of decline in dry matter content of rice straw substrate. Slowing decline in dry matter content of rice straw occurred at 1500 ppm Cr levels up to 3000 ppm. It is expected that high levels of Cr inhibits the growth of G. lucidum so that decomposition of organic material into H 2 O and CO 2 slowed and ultimately decreased the proportion of dry matter of fermented rice straw. However, mycelium growth was not impaired as it was observed that mycelium growth covered the media even at Cr levels of 3000 ppm.
The rate of change in the content of organic matter of fermented rice straw was similar to that of dry matter, which was somewhat influenced by fermentation time and Cr levels (P<0.01) (Figure 1). A decline in organic matter content was observed with increasing duration of fermentation from 0 to 4 wk. The decline of organic matter content was from 23.95% to 19:55% in fermentation time of 4 wk. Oxidases such as laccase from G. lucidum are enzymes which were involved in oxidative conversions of organic compounds and materials (Mtui, 2012).
This result showed that the proportion of inorganic materials with organic media remains the same for all the different levels of Cr. It is thought that the formation of organic matter occurred in the form of mycelium and was comparable to the decreasing levels of organic matter or dry matter content.

Fermented Rice Straw
The loss of dry matter and organic matter in fer-ry matter and organic matter in fermented rice straw was significantly increased (P<0.01) significantly increased (P<0.01) with increasing length of fermentation time. There was Note: Means in the same row or column with different superscript differ significantly (P<0.01). no interaction between the fermentation periods with Cr levels ( Table 2). Dry matter loss in rice straw increased with increasing duration of fermentation, followed by growth of the mycelium which filled the surface of the substrate and covered the growing medium. Dry matter loss begins in 2 wk (12.49%) and continued to increase up to 4 wk (17.19%). This condition suggests that with increasing dry matter loss, more nutrients were utilized by G. lucidum for the metabolism and growth of its mycelium. There was a linear relationship between the dry matter loss to the fermentation time of fermented rice straw by following equation: Y= 5.356X-2.35 (R 2 = 0.83, P<0.01 where Y= dry matter loss, %, X= length of fermentation, week), but rate of dry matter loss decreases with increasing Cr at a level of more than 2000 ppm Cr. This suggests that the levels of more than 2000 ppm Cr decreased the growth rate of G. lucidum. In our research, the dry matter loss in fermented rice straw have the different profile with the dry matter loss in oil palm byproduct fermented with G. lucidum. In contrast, in case of oil palm by-product, the increase of Cr level in substrate up to 3000 ppm Cr did not affect the growth rate of G.

Cr level
lucidum (Agustin et al., 2010). The loss of dry matter on cultivation of oyster mushroom in wheat straw substrate was 9.4% (Pant et al., 2006), while in this study, the loss of dry matter was 17.19%. This difference in result could be due to G. lucidum being a white rot fungus which is capable of producing ligninolytic enzyme (laccase, manganese peroxidase, lignin peroxidase) (Ke et al., 2011) and able to degrade fiber components of rice straw. The pattern of organic matter loss was the same with the dry matter loss in fermented rice straw in relation to fermentation time and levels of Cr in media. Means of organic matter loss in fermented rice straw was influenced by the fermentation time (P<0.01) and Cr levels (P<0.05). There was no interaction between Cr levels with long fermentation (Figure 2). Organic matter loss begins at week 2 (14.97%) and continued to increase until at week 4 fermentation period (21.88%). Increasing the rate of organic matter loss in rice straw showed an increase in the amount of nutrients utilized by G. lucidum for its metabolic activity and its cell growth.
At the early stage of fermentation, G. lucidum utilized the digestible nutrients contained in rice straw, after which the fungus utilized structural carbohydrates, especially complex compounds that are bound together with lignin. G. lucidum produces laccase enzyme which is capable of hydrolyzing lignin binding with other compounds (Baldrian, 2003;Ke et al., 2011). Increased organic matter loss decreased with increasing fermentation time. These results indicated that G. lucidum was able to hydrolyze and utilize structural carbohydrates and lignin.

Cr Incorporation in Fermented Products
The element Cr bound in media was probability bound with media components and was converted into mycelial cell component of G. lucidum. Chromium incorporation into the cell of G. lucidum (Figure 3) is thought to occur because it grew well in the rice straw substrate. The binding and incorporation of Cr was significantly affected (P<0.01) by the Cr levels of substrate. There was no interaction between Cr levels with fermentation time.
G. lucidum using Cr together with the use of organic matter of rice straw fiber components. This was indicated by the amount of Cr bound and incorporated in fermentation product. Incorporated Cr in fermentation product of G. lucidum was increase along with the higher Cr levels in substrate. The binding and incorporation of Cr in Cr levels of 1000 ppm was only 120 ppm Cr. The amount of incorporated Cr increase continuously until its value 1410.82 ppm Cr at the Cr levels of 3000 ppm based on dry matter. There was a linear relationship between the binding and incorporation of Cr with a Cr content of the substrate following the equation: Y= 296.72X -471.86 (R 2= 0.98, P<0.01 where Y= Cr bound, %; X= Cr content of the media, 500 ppm). This result showed that G. lucidum was more resistance to supplementation of Cr in high concentration because mycelia growth gave the organism a larger surface area, which provides greater protection to sensitive organelles of the fungi. In addition, fungi are eukaryotic cells, which contain more genes that can inhibit toxic metals (Sen & Dastidar, 2010;Shugaba et al., 2012).
The highest Cr level supplemented into rice straw substrate was 3000 ppm. The highest Cr incorporation value was also found on substrate with Cr level of 3000 ppm (Figure 3). The element Cr was needed in the nutrient metabolism of carbohydrates and protein of G.
lucidum cell for 4 weeks of fermentation. This indicates that G. lucidum was able to adapt to the medium with high levels of Cr. G. lucidum was also able to use the media components that are difficult to degrade.
G. lucidum was able to incorporate Cr into its cell proteins with the value of 9.29% (Table 3) when cultured in substrates containing 3000 ppm Cr. Some Cr was deposited in the neutral detergent fibre (NDF) (27.20%) and acid detergent fibre (ADF) (10.55%). These results showed that there has been a conversion of inorganic to organic forms of Cr by G. lucidum. Cr founded in the protein of G. lucidum cell is an organic compound. The highest efficiency of Cr incorporation was 53.58% showed in treatment with 3000 ppm Cr and fermentation time of 4 wk ( Figure 3). It has been suggested that chromium ions can be incorporated into G. lucidum cells by two step, the first step involves the surface binding of Cr (III) ions to the cell wall and extracellular material, which is not depend on metabolism process. The second step is depending on the metabolic activity because uptake Cr (III) into the cell across the cell membrane and at that time is occurred intracellular accumulation (Sen & Dastidar, 2010).

CONCLUSION
The loss of dry matter and organic matter in fermented rice straw was related to the growth of mycelium of G lucidum. The binding of Cr occurs by fiber matrix of substrate. Inorganic Cr conversion into organic forms by fungi G. lucidum on rice straw substrate was evidenced by the presence of Cr in protein of fermented product. Protein component of fermented product contained 9.29% Cr while in NDF and ADF was 27.20% and 10.55% Cr, respectively at level of 3000 ppm Cr. Chromium was incorporated into the G. lucidum cells during fermentation and G. lucidum is tolerant of high Cr levels up to 3000 ppm.