COMBINATION OF SAWDUST, “FILTER CAKE” AND CALCIUM CARBONATE AS GROWTH MEDIUM FOR THE PRODUCTION OF WHITE OYSTER MUSHROOM (PLEUROTUS OSTREATUS)

This study was aimed to investigate the effect of combining sawdust (SD), filter cake (FC) and calcium carbonate as growth medium for the production of white oyster mushroom. Isolate F2 oyster mushroom was cultured on malt extract agar (MEA) and used in the experiment. The culture medium consisted of two treatments: first, A treatment: combined medium (A0=100% SD; A1=100% FC; A2=70% FC and 30% SD; A3=50% FC and 50% SD; A4=30% FC and 70% SD) and second, K treatment: addition of calcium carbonate (K1=2%; K2=3%; K3=4%; K4=5% weight of medium). A Randomized design was used to analyze certain parameters, such as mycelial growth, presence of fruiting body, the number of fruiting bodies and the fresh weight of fruiting body at harvest. The results showed that the highest mycelial growth and fruiting body formation occurred on A0 treatment. However, a high number of fruiting bodies and a high fresh weight of fruiting body at harvest were obtained on A4 treatment. Interaction between the combined medium (A) and addition of calcium carbonate (K) showed that the highest mycelial growth occurred on A0K2 treatment 30 days after incubation. The composition of A4 treatment (30% FC and 70% SD) was found to be the optimal medium for the production of fruiting body. This finding shows that FC with additional nutrition could as a substitute of SD medium for cultivating white oyster


INTRODUCTION
Mushroom is a horticultural commodity and is used as food or nutraceutical to prevent and treat human diseases. Preclinical and clinical studies have shown that direct and indirect consumption of certain mushrooms reduce the occurrence of cancer and cause weight loss (8). Indonesia is a country that has the potential to become an edible mushroom producer. As a tropical country, Indonesia has high mushroom diversity, which makes it possible to grow them in every season. Edible mushrooms with high nutritional value can be used as medicine (19). The high demand for mushroom as a food additive in the society and the increasing mushroom production indicate business opportunities in mushroom cultivation. Setyawati (23) stated that the presence of mushroom farming in some areas increases the value of the commodity. As the tenth largest producer of sugar-cane in the world, Indonesia produces a large amount of waste product in this sector. According to Kuswurj (14), the biomass of sugar-cane waste was 1.1 million ton/hectar/year. Currently, the waste is used primarily as fertilizer, alternative fuel source or abandoned, which causes pollution (9). The composition of filter cake was investigated by Senthil and Das (22), Kumar et al. (13), and Sørensen et al. (12). Boonyuen et al.
(3) reported that filter cake and sugar-cane stem waste consist of high lignocellulose and can be used for fungal growth. A previous research by Suka et al. (6) stated that the addition of various amounts of filter cake to sawdust medium increases the production of Pleurotus ostreatus. They also reported impressive results with respect to mycelial growth, pinhead, fruiting body and harvest period for the medium with 60 g of filter cake. Addition of calcium carbonate (CaCO 3 ) to filter cake is required to increase pH. Mardiana et al. (15) reported a difference in mycelial growth of Pleurotus ostreatus on a medium with CaCO 3 . Therefore, the aim of the current research is to investigate the use of the combined medium of saw dust, filter cake and CaCO 3 as growth medium for the production of white oyster mushroom (Pleurotus ostreatus).

MATERIALS AND METHODS
Isolate F 2 white oyster mushroom was cultured on malt extract agar (MEA) obtained from F 1 Agrotechnology laboratory, Agricultural Faculty, Medan Area University and was used in this experiment. The treatments consist of A treatment, which is a combined medium of filter cake and sawdust (A 0 =100% SD; A 1 =100% FC; A 2 =70% FC and 30% SD; A 3 =50% FC and 50% SD; A 4 =30 FC and 70% SD), and K treatment: addition of calcium carbonate include K 1 =2%; K 2 =3%; K 3 =4%; K 4 =5% weight of medium, which involves the addition of CaCO 3 . A Randomized design was used to analyze certain parameters, such as mycelial growth, the presence of fruiting body, the number of fruiting bodies and the fresh weight of fruiting body at harvest. The experiment using randomized complete design, with three replication analysis of variance (ANOVA) and Duncan multiple range test (DMRT) was formulated as follow: Preparation, Isolation and Incubation Isolate F 2 oyster mushroom was obtained from F 1 culture collection Agrotechnology Laboratory, Agriculture Faculty, Medan Area University. The isolate was cultured on baglog with a volume of 375 cm 3 containing powdered filter cake, sawdust, wheat bran, 10 % CaCO 3. and 0.5% maize flour. Eighty baglogs were made in this experiment. Each baglog was arranged and incubated (25-28 o C) for 35-40 days in the dark on a rack in a musroom house, 7 x5 x 6 m (length, wide, height). Each treatment was made in triplicate. Inoculated eight baglogs with spawn were made for each treatment. The humidity inside the mushroom house (80-90 %) was controlled daily using a hygrometer. To control pest and diseases in each treatment, fly trap was used.

Parameter Observation
Each baglog was observed after 3 days of incubation. Certain parameters, such as mycelial growth, were observed after 5 days of incubation. The size of the fruiting body was observed after 35-40 days, while the number of fruiting bodies and the fresh weight were observed at harvest. Harvesting was conducted two times between 40 and 120 days of incubation using cutter (11). The first harvest took place on the 50th day and the second on the 75th day. The freshly harvested mushroom was weighed using analytical balance type Gs/Vibra, Merk Shinko, Japan.

RESULTS AND DISCUSSION
The medium composition of each baglog was covered by mycelial growth after 35 days. The combined medium with CaCO 3 supports fungal growth. Previous results showed that sawdust and filter cake contain cellulose, which is required by white oyster mushroom for growth (2). Garg and Gupta (9) stated that agro industrial wastes and their derivatives were used as medium for cultivating oyster mushroom. The high lignocellulose produced by mushroom gives the species advantage in growth and development on such environment. In addition to light intensity, the temperature, acidity/alkalinity of the medium (6-7) and relative humidity (60-70%) in the mushroom house affect their growth. Soenanto (18) reported that oyster mushrooms require an area that is 400-800 m above sea level for optimum growth. However, some strains of the species are able to grow on lower than 400 m above sea level as long as the area has low light intesity and cool weather. Our results are in accordance with Chazali and Pratiwi (4), who stated that a temperature range of 23-28 0 C was optimal for mycelial growth of oyster mushroom. High light intensity inhibits growth and metabolism. Suriawiria (2) stated that fungal growth was affected by the nutrients on the medium, particularly phosphorus, potassium, nitrogen, sulphur, calcium and carbon. Previous research by Soenanto (18) and Hanifah (10) showed that the nutrient and moisture contents of the medium determine fungal growth. The result of this study was showed that the presence of nutrients on sawdust and filter cake, the moisture content of both media (60-65%) and their pH (6-7) determine the growth of oyster mushroom.

Mycelial growth covered the Substrat/baglog (cm)
The statistical analysis indicate that the combined medium (sawdust, filter cake and CaCO 3 ) had a significant effect (P< 0.05) on mycelial growth. We assumed that the starting point of mycelial formation was determined by the presence of nutrients in the medium in the baglog. Optimum mycelial growth occurred after 30 days. A 0 , A 2 , and A 3 had no significant effect on mycelial growth. The result indicates that filter cake can be used as a substitute medium of sawdust. The optimum growth of mycelium with respect to the medium containing CaCO 3 was observed on K 2 ; the performance of K 2 is significantly different from those of others. Interaction A 0 K 2 showed optimum mycelial growth. Sawdust and filter cake contain nutrients required for optimum growth of white oyster mushroom. Similar results were also reported by Dewi (5). Agus et al.

-35 days incubation Formation of Fruiting Body
The formation of fruiting body at the first and second harvests on 100% sawdust (SD) medium (A 0 ) did not significantly different compared with the combined medium of 50% SD and 50% FC (A 3 ). The table shows yield increase in the first harvest significantly with A 4 60.88 aA and second harvest significantly with A 1 and A 2. The beginning of fruiting body formation is indicated by the presence of small white buttons surrounding the mycelia. The first apperance of fruiting body during the first and second harvests is shown in Table 2. The availability of nutrients, such as C, N, P, and K, on FC + SD medium promotes the growth of mycelia to the extent of covering the medium and enhances fruiting body formation. Maize flour and wheat bran added to the medium has no effect on fungal production.  The number of fruiting bodies at the first harvest was 3.5-9.81 and that of the second harvest was 3.25-9.16 (Table 3 and Figure 5). The highest number of fruiting bodies was found on combined medium of 30% FC + 70% SD (A 4 ), and did not significantly different from the number of fruiting bodies on the 100% SD medium (A 0 ), 70% FC + 30% SD medium (A 2 ) and 50% FC + 50% SD medium (A 3 ). Regarding the production of fruiting body, filter cake was used as a substitute of sawdust. The nutrients and compounds required for growing oyster mushroom include calcium, potassium, phosphorus, nitrogen, carbon, protein and chitin. Mkhize et al. (17) used nitrogen and carbohydrate for the producton of fruiting body. However, Suriawiria (2) and Hanifah (10) reported that wheat bran and filter cake enriched the medium in terms of nutrients required for the growth of oyster mushroom, and the addition of calcium carbonate controlled the pH of the medium.

Figure 5. Production of fruiting body at the first and second harvests Fresh Harvest Weight
The fresh weight of fruiting body (g/baglog) at harvest is show in Table 4 and Figure 6. High harvest fresh weight was observed on A 4 in first and second harvest. With higher concentration of filter cake in the culture medium, the fresh weight increases. Filter cake consists of crude protein, sugar, cellulose, chlorine, phosphate and fibre (5). Silveira et al. (21) reported that the presence of cellulose, hemicellulose and lignin in culture medium promotes mycelial growth, which increases fruiting body formation. 129.83 Aa 106.75 a A Numbers followed by the same letters in the same column have significant difference at 95% (small letter) and 99% (capital letters)

Figure 6. Fresh weight of fruiting body at first and second harvests
The results showed that the combined medium of sawdust and filter cake increases the productivity and growth of white oyster mushroom. The nutrients in sawdust were complemented by the addition of filter cake to promote fungal growth (16). As a sapotroph, the nutrients required by fungi are provided by other organisms. Environmental factors such as light intensity, temperature, acidity/ alkalinity of medium and humidity also affect fungal growth.

Conclusion
As waste agricultural products, filter cake and sawdust used as a combined medium is suitable for cultivating white oyster mushroom. The combination of both waste products has a beneficial effect on mycelial growth and fruiting body formation.