Organic Acids Production of Rice Straw Fermented with Several Types of Microorganism at Different Temperatures

The experiment was carried out to examine the organic acids production of rice straw fermentedwith some types of microorganisms at different temperatures. The experiment was designed as SplitPlot-Completely Randomized Design. The main plot was temperatures treatments (25, 35, 45°C) and thesub plot were microorganisms (Control, Control+Mollases, Lactobacillus fermentum, Bacillus subtilis,Bacillus coagulant, Saccharomyces cerevisiae, Aspergillus Niger). The highest lactic acid productionswas in B. coagulans treatment at 35°C (53.79 g/kg DM). The highest acetic acid productions was in L.fermentum at 35°C (13.20 g/kg DM), while the highest propionic acid productions were in Controltreatment at 35°C (0.37 g/kg DM).


INTRODUCTION
Rice straw (Oryza sativa) is a large portion of the world crop by-products that is not used efficiently. It has been well recognized as an alternative feed for ruminant because of abundant in stock and cheap in price so it can give more economic advantages due to the lower production cost. Rice straw has also been used as a roughage in the diet of beef and dairy cattle when drought limits the availability of other conserved forages in small farms. However, rice straw has limited nutritive value as ruminant feed because of low protein (2-7% on a dry matter basis), low digestibility and high fiber contents (Drake et al., 2002).
Numerous methods of physical, chemical, biological treatments have been widely studied and developed in order to improve the utilization of rice straw for ruminant feed (Trach, 1998). Microbial fermentation was studied in the past and many studies determined to improve the fibrous feed qualities (Han and Anderson, 1974). Fermentation usually implies that the action of the Organic Acids Production of Fermented Rice Straw (Y. Yanti et al.) microorganisms is desirable. Cellulase (a complex multienzyme system) acts collectively to hydrolyze cellulose from agricultural waste to produce simple glucose units (John, 1996). Cellulases are synthesized by cellulolytic fungi such as Aspergillus spesies (Milala et al., 2005;Vijaya and Singaracharya, 2005) and Sacharomices species (Lila et al., 2006;Callaway et al., 1997), and Bacillus species (Eun et al., 2006), whereas Lactobacillus species were used widely as additives in silage fermentation of forage to improve preservation efficiency. The epiphytic lactic acid bacteria ferment the watersoluble carbohydrates in the crop to lactic acid (Weinberg and Muck, 1996).
Fungal growth resulted from the complex interaction of several factors, such as temperature, water availability and incubation time (Pardo et al., 2005). The optimum-growth temperature corresponds to the usual temperature of the natural habitat of the species, and to the temperature at which the essential bacterial enzymes function best (Burdon and William, 1965). The effects of fungal growth on the chemical composition and hence nutritive value of fibrous residues is depend on the type of fungus, the nature of the residue and the conditions of growth.
Although many reports suggest fermentation method with microbes could improve quality of fibrous feed but selections of the type microbes in suitable temperature of fermentation have not been determined yet. The aim of this research was to determine the nutritive value of rice straw, especially organic acids, fermented with some types of microorganisms at different temperatures. Hypothesis of this research was the different type of microbes need different temperatures may result in differ nutritive value of fermented rice straw.

This research was conducted in Tropical
Grassland Chemical and Utility Laboratory, Faculty of Agriculture University of the Ryukyus, Okinawa, Japan. Rice straw was obtained from rice fields in Ishigaki District, Okinawa, Japan. The types of microbes used were Lactobacillus fermentum, Bacillus subtilis, Bacillus coagulanst Saccharomyces cerevisiae, and Aspergillus niger. Water was added to rice straw prior to fermentation. Molasses were added to rice straw 5% dry matter basis. Split plot completely randomized design was used in this experiment to study the effect of rice straw fermented with microbes in different temperatures on organic acids content. Treatments were assigned as mainplot and subplot. The mainplot was temperature (25, 35, 45°C)  The methods used in the fermentation process were as follow: 1) Rice straws were chopped into small pieces at 1-2 cm length. 2) Water was added to the rice straws at ratio water:rice straw = 2:3. 3) All types of microbes were cultivated in culture media agar specific for each types in plate and the occurred cell then were transferred to increase the cell numbers in culture media broth specific for each types in a 100 ml flasks to make microbes juice. The cell was then incubated for 3 days of each types. 4) The 5% microbe juice and 5% molasses were mixed with 1 kg of the rice straw based on dry matter. The control treatments were not mixed with molasses and the other one were mixed with molasses. 5) Approximately 150 g of mixture were transferred into a plastic pouches. 6) Fermentation method: Ferment bag by plastic pouchs were sealed by vacuum sealer in groups of control, L. fermentum, B. subtilis and B. coagulanst. Exception in group fermented with S. cerevisiae and A. niger the ferment plastic pouches were not sealed but were loosely closed. 7) Rice straw bags were stored at 25, 35 and 45°C according to the treatment for 3 weeks.
After 3 weeks fermentation, a representative sample of rice straw (20 g fresh matter) was macerated with 70 ml of distillated water and stored at 4°C for 12 hours. The extract was filtered through No. 5A filter paper (Toyo Roshi Co. Ltd., Tokyo, Japan). The filtrate was collected to analyze the concentration of Volatile Fatty Acids (VFA). VFA content was measured by HPLC (Shim-pack SCR-102H, 300 mm x 8.0 mm i.d; column temperature, 40°C; flow rate, 0.8 ml/min, Shimadzu Co. Ltd., Kyoto, Japan). After 3 weeks fermentation, the fermented materials were dried in a 60-70°C drying oven for 48 hours to determine water content.
Effect of treatment was analyzed using the analysis of variance (ANOVA). The Duncan's New Multiple Range Test was performed to determine the significant differences among treatments. The Least Square Different (LSD) was used when interaction between temperature and microorganisms were found (Sastrosupadi, 2000).

RESULTS AND DISCUSSION
The lactic acids, acetic acid and propionic acid production of fermented rice straw are presented in Table 1. There were temperatures, microorganisms and its interaction effects (P<0.01) on lactic, acetic and propionic acid production of fermented rice straw. Interaction between temperatures and microorganisms on lactic, acetic and propionic acid are shown in Figure 1.
The lactic acid production of Control+molasses and L. fermentum treatments at temperature 25°C showed higher (P<0.01) than those in others microorganism treatments. It could be explained that 25°C temperature may give a better environment for Control+mollases and L. fermentum to produce lactic acid. The high content of lactic acid in Control+molasses treatment might be caused by lactic acid bacteria that grow and ferment molasses to produce lactic acid. This result confirmed to the report of Gao et al. (2008), who isolated microbial community from fermented rice straw identified as L. fermentum, L. plantarum and L. paracasei, these species were lactic acid bacteria producing lactic acid as well as acetic acid. This result was coincided with the report of McDonald et al. (1991), lactic acid bacteria grow within the range 5-50°C, while for most strain grow optimally around 30°C.
B. coagulans at temperature 35°C and 45°C gave lactic acid production higher (P<0.01) than othr treatments. The high content of lactic acid in B. coagulans treatment was similar to the report of Woolford (1977) citated by McDonald et al. (1991). Woolford examined the activities of B. coagulans in grass with low content of water soluble carbohydrates (WSC) and found that organism fermented sugar largely produced lactic and acetic acids. B. coagulans at 35°C produced high lactic acid, and this confirmed the report of Davey et al. (1966) that B. coagulans optimally grow at temperature 35-55°C, except at 45°C the growth rate decreased.
S. cerevisiae and A. niger treatments gave lower lactic acid and acetic acid production (P<0.01) than those in other treatments in all temperatures. This might be the effect of loosely sealing which caused the lactic acid evaporation during fermentation. This is also agreed to McDonald et al. (1991) who reported that yeast and mold produced only small amount of lactic and acetic acids.
Among the treatments, the highest (P<0.01) acetic acid production was found in L. fermentum treatment in all temperatures (Table 1). It was because L. fermentum is a heterofermentative   (Gao et al., 2008) that ferments WSC into lactic, acetic acids and CO 2 (Cai et al., 1998). The propionic acids production at 25°C showed that L. fermentum gave higher (P<0.01) than that in other treatments. While, propionic acids production at 35°C showed that Control and B. subtilis gave higher (P<0.01) than that in other treatments. The propionic acids production at 45°C showed that Control+mollases gave higher (P<0.01) among the treatments. The unexpected result was found in this B. subtilis treatment. There were no previous reports explained that B. subtilis produced propionic acid. Propionic acids were sometimes detected in silage in small amount (McDonald et al., 1991). The only microorganisms presented in silage which were likely responsible for propionic acid formation were clostridia and propionic acid bacteria.
In present study, the butyric acid was not detected in all treatments. This condition could be considered as a prove of high quality fermentation. This result was in agreement with Manabu et al. (2006) and Cai et al. (2003), that high fermentation quality was indicated by high lactic acid production and lower butyric acid production. Butyric acid was a degradation product of lactic acid by clostridia (McDonald et al., 1991), the undesirable microorganism in feed fermentation, especially in silage, because clostridia compete with the LAB for the available sugars, and degrade protein, causing problems in reduction of feeding value and the production of biogenic amines.
The LSD tests for organic acids are shown in Figure 1. This figure figured the effect of interaction between temperatures and microorganisms. The LSD test showed that control+mollases and L. fermentum at 25°C and B. coagulans at 35°C produced the highest (P<0.01) of lactic acid among the treatments. L. fermentum at 35°C also showed the highest (P<0.01) production of acetic acid, while control, control+mollases and B. subtilis at 35°C produced the highest of propionic acid (P<0.01) among the treatments. Rice straw fermented with L. fermentum at 35°C showed the highest (P<0.01) production of acetic acid among the treatments (Figure 1). This result was similar to the report of Mikelsaar et al. (2007) that the optimal temperature for L. fermentum growth was 37°C, eventhough this bacteria still grow at 45°C, but it does not grow at 15°C. This result was in contrast with McDonald et al. (1991) and Sneath et al. (1986) who reported that L. fermentum was exceptionally grows optimum at 45°C. Control, control+molasses and B. subtilis at 35°C produced higher (P<0.01) propionic acid than other treatments. It had explained previously that propionic acids were sometimes detected in silage in small amount (McDonald et al., 1991). The only microorganisms present which were likely to be responsible for its formation were clostridia and propionic acid bacteria.

CONCLUSIONS
The highest lactic acid production was in B. coagulans treatment at 35°C (53.79 g/kg DM). The highest acetic acid productions were in L. fermentum at 35°C (13.20 g/kg DM), while the highest propionic acid productions was in Control treatment at 35°C (0.37 g/kg DM).