1.1 Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes

The ethanol fermentation abilities of basidiomycetes have not been well characterized, we evaluated the ability of F. velutipes in CBP. Preliminary fermentation experiments indicate that F. velutipes convert sugars to ethanol much more under the high concentration of biomass which close to solid state cultivation than liquid cultivation condition. Therefore, we employed solid state cultivation which usually performed in artificial cultivation of mushrooms for the coversion of biomass to produce bioethanol. Sorghum is selected as a possible raw material to produce bioethanol by CBP using F. velutipes. Sorghum is a C4 crop of the grass family belonging to the genus Sorghum bicolor L. It is well adapted to temperate climates and can be cultured from Kyushu to Tohoku area in Japan. The plant grows to a height from about 120 to above 400 cm, depending on the variety and growing conditions, and can be an annual or a short perennial crop. Sorghum is considered to be one of the most drought resistant agricultural crops, as it is able to remain dormant during the driest periods (Xu et al., 2000). These properties of sorghum are suitable as raw material for the ethanol production. We evaluated the ability of F. velutipes in CBP using sorghum strains as a raw material, and solid-state CBP of ground sorghum strains (SIL-05 and Kyushukou No. 4) using F. velutipes was investigated. The possibility of sorghum strains as a raw material in the CBP ethanol production by F. velutipes is also discussed below.

We selected grinding for the pretreatment of sorghum strains. This can be used on both dry and wet materials, and the cost of grinding is one of the cheapest compared to other methods used for milling biomass. The grinding of sorghum was carried out with an ultra­fine friction grinder. Grinding was performed at room temperature, and was repeated twice. To examine the efficiency of grinding as a pretreatment, the degree of saccharification was tested using commercially available enzymes Celluclast 1.5L (Sigma, St. Louis, MO), Novozyme 188 (Sigma) and Multifect xylanase (Genencor Kyowa, Tokyo).

The saccharification yields of SIL-05 and Kyushukou No. 4 by the enzymes were 30.1% and 51.7% respectively (Fig. 5A). Kyushukou No. 4 is one of the sorghum brown mid-rib (bmr) mutants in which cafferic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, activity is reduced as compared to the wild type (Bout & Vermerris, 2003). This property of bmr significantly affected the hydrolysis of polysaccharides in the biomass, but there were no significant differences in the proportions of hydrolysis of the components such as cellulose and hemicellulose (Fig. 5). When the saccharification yields of cellulose and hemicelluloses were compared, degradation of hemicelluloses was slightly higher than for cellulose in both types of sorghum.

Next, solid-state ethanol fermentation by F. velutipes was performed for both sorghum strains. Solid-state fermentation is advantageous because it carries a low ethanol production cost. Generally, sorghums contain 70-80% v/ v water, corresponding to 43-25% w/ v. These concentrations are necessary to obtain relatively high final ethanol concentrations. Furthermore, it is possible to reduce the costs of many procedures, such as amount of water, concentration of biomass, treatment of waste water, and so forth, if the water concentration of the raw materials in the all ethanol production procedures is retained. The Fv-1 strain was selected for further study because it not only produces high levels of cellulases, but also because its ability to ferment ethanol is superior to the other strains. Mycelia of Fv-1 were harvested in the late exponential growth phase by centrifugation at 3,000 x g and washed with sterile water. The prepared wet mycelia (20 mg of dry weight) were mixed with 100 mg of ground sorghum for solid-state fermentation.

A larger amount of ethanol was produced from SIL-05 than from Kyushukou No. 4 (Fig. 6). Because SIL-05 contained a larger amount of soluble sugars than Kyushukou No. 4 (Table 1), it should be advantageous for total ethanol fermentation. The ethanol conversion rates for the soluble sugars contained in SIL-05 and Kyushukou No. 4 were 57.2% and 38.9% respectively. The addition of saccharification enzymes was not effective for SIL-05 (Fig. 6A). This corresponded with the results of enzymatic hydrolysis (SIL-05 just hydrolyzed almost 30%) (Fig. 6). However, the ethanol conversion rate for the degraded cellulose was 85.6%, significantly higher than that for soluble sugars. In contrast, although total ethanol production was not high, ethanol production from Kyushukou No. 4 significantly increased when saccharification enzymes were added to the culture (Fig. 6B). Because the cellulose and hemicellulose in Kyushukou No. 4 were more easily hydrolyzed than SIL-05 by cellulases, significantly more ethanol was produced by the addition of the saccharification enzymes. The ethanol conversion rate for the degraded cellulose of Kyushukou No. 4 (98.3%) was much higher than that of SIL-05 (85.6%). Thus, the bmr mutation appears to be useful for CBP because it gives a high yield of glucose from biomass without acid or alkali pretreatment. However, the results indicate that the production of cellulases by F. velutipes is not sufficient for CBP, or that the saccharification enzymes are suppressed by carbon

catabolites due to the existence of soluble sugars. Therefore, an effective saccharification enzyme inducing method for F. velutipes in the CBP is required.

In this work, we demonstrated CBP ethanol fermentation of sorghum strains by F. velutipes Fv-1. The procedure is quite simple and cost effective, and can reduce energy consumption, because the raw material is simply ground and then mixed with mycelia. We demonstrated the merit of high concentrations of soluble sugars and the bmr mutation in sorghums. Both sorghum strains can be used in CBP. The bmr mutation is only found in sorghums, corn, and pearl millet, giving sorghum an advantage over many other crops for ethanol production. Future studies should focus on the improvement of CBP using F. velutipes and the selective breeding of novel types of sorghums with high concentrations of soluble sugars and the bmr mutation.