Decomposition of rice chaff using a cocultivation system of Thermobifida fusca and Ureibacillus thermosphaericus

: Lignocellulosic biomass comprises cellulose, hemicellulose, and lignin and is a potential source of fuels and chemicals. Although this complex biomass is persistent, it can be cooperatively decomposed by a microbial consortium in nature. In this study, a coculture of the moderately thermophilic bacteria Thermobifida fusca and Ureibacillus thermosphaericus was used for biodegradation of rice chaff. The bacterial strains were incubated in modified Brock’s basal salt medium (pH 8.0) supplemented with yeast extract and rice chaff at 50°C for 7 days. The concentration of reducing sugars and the enzymatic activities of laccase, lignin peroxidase, cellulase, and xylanase in the supernatant of the culture medium were measured every day. The concentrations of reducing sugars in solo cultures of T. fusca and U. thermosphaericus and a mixed culture of the two strains after 7 days of incubation were 0.047 mg/mL, 0.040 mg/mL, and 0.195 mg/mL, respectively, indicating that the decomposition of rice chaff was enhanced in the coculture. Based on the results, it is thought that the lignin surrounding the cellulose was decomposed by laccase and lignin peroxidase secreted from U. thermosphaericus , resulting in cellulose and hemicellulose in the rice chaff being easily decomposed by enzymes from T. fusca .


Introduction
Lignocellulosic biomass constitutes the cell wall of plants, and this decomposition product has been attracting attention as a raw material for bioethanol and biodegradable plastics.However, this biomass is not easily decomposed, as it has a strong structure in which polymers such as cellulose, hemicellulose, and lignin are linked by hydrogen bonds or covalent bonds.For this reason, decomposition treatment with an acid has been attempted, but this method has the problem that aromatic compounds are generated and mix with the decomposition products.On the other hand, enzymatic decomposition is less efficient than chemical decomposition.In nature, lignocellulose is degraded through a complex multistep process by the cooperation of various microorganisms that produce synergistic cellulolytic enzymes, hemicellulytic enzymes and lignin-degrading enzymes [1].In addition, coculture of microorganisms may induce higher enzyme production than single cultures due to synergistic effects [2,3].It is considered very difficult to maintain a wide variety of microbial communities in a reactor and perform specific biomass decomposition, but it may be possible to construct a coculture system for a few species of microorganisms.In fact, Shiotani et al. [4] reported that the decomposition of aquatic plants was promoted under the coculture of the cellulose/hemicellulose-degrading bacterium Thermobifida fusca and the lignin-degrading bacterium Ureibacillus thermosphaericus.These bacteria are moderately thermophilic and have similar optimal growth temperatures and pH values.In this study, a coculture of T. fusca and U. thermosphaericus was used for the biodegradation of rice chaff.

Materials and Methods
Thermobifida fusca DSM43792 and Ureibacillus thermosphaericus DSM10633 were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).The bacterial strains were incubated in modified Brock's basal salt medium [5] (pH 8.0) supplemented with yeast extract (0.5%) and rice chaff (0.5%) at 50°C for 7 days.The concentration of reducing sugars was determined using the dinitrosalicylic acid method [6].The enzymatic activities of laccase, lignin peroxidase, cellulase, and xylanase in the supernatant of the culture medium were measured every day according to standard methods [7][8][9].The viable cells in each culture were counted as colony-forming units.To investigate the effect of the mixing ratio of the two strains on rice chaff decomposition, a culture experiment was conducted in which the mixing ratio of the strains was varied.

Cell density
The cell density of T. fusca gradually increased, reaching 2.3 × 10 6 cells/mL on the 7th day.On the other hand, the cell density of U. thermosphaericus showed a maximum value of 5.3 × 10 9 cells/mL on the second day and then gradually decreased.The cell density of the coculture reached 4.6 × 108 CFU/mL on the second day and was maintained at >3.3 × 10 7 CFU/mL throughout the study period (Fig. 1a).

Concentration of reducing sugar
The concentrations of the reducing sugars in the single cultures of T. fusca and U. thermosphaericus and the mixed culture of the two strains showed maximum values of 0.111 mg/mL, 0.040 mg/mL, and 0.225 mg/mL, respectively, indicating that the decomposition of rice chaff was enhanced in the coculture (Fig. 1b).

Enzymatic activities
The activities of lignin peroxidase, cellulase, and xylanase were higher in the coculture than in the single culture.The laccase activity was higher in the culture of U. thermosphaericus alone than in the coculture (Fig. 2).

Influence of the mixture ratio of the coculture
The enzymatic activities were higher in the coculture than in the single cultures except when the inoculation amount of T. fusca was very small.It was possible to start a coculture with a relatively wide range of inoculum mixture ratios.

Conclusions
A cocultivation system of T. fusca and U. thermosphaericus was applied for the decomposition of rice chaff, a model compound of lignocellulosic biomass.Both the concentration of reducing sugars and the enzymatic activities were higher in the coculture than in the single cultures.Based on the results, it is thought that the lignin surrounding the cellulose was decomposed by laccase and lignin peroxidase secreted from U. thermosphaericus, resulting in the cellulose and hemicellulose in the rice chaff being easily decomposed by the enzymes from T. fusca.

Figure 1 .
Figure 1.Cell densities (a) and reducing sugar production (b) in the cultures of T. fusca, U. thermosphaericus and both strains.

Figure 3 .
Figure 3. Influence of the mixture ratio of T. fusca and U. thermosphaericus inoculums on each enzyme activity after 7 days of incubation.Laccase and lignin peroxidase activities are expressed in ratios based on the U. thermosphaericus monoculture.The cellulase and xylanase activities are expressed in ratios based on the T. fusca monoculture.