Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

邮发代号 80-969

2019 Impact Factor: 3.552

Direct ethanol production from rice straw by coculture with two high-performing fungi
Direct ethanol production from rice straw by coculture with two high-performing fungi
Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan

To develop efficient and economical direct ethanol production from fine rice straw crashed mechanically, two high-performing fungi, which can secret hyperactive cellulases and/or ferment effectively various sugars, were selected from some strains belong toMucor circinelloides preserved in our laboratory. The simultaneous saccharification and fermentation (SSF) by coculture with these fungi was investigated. The screening of high-performing fungi resulted in the selection of NBRC 4572 as an ethanol-producing fungus and NBRC 5398 as a cellulase-secreting fungus. The strain 4572 produced ethanol aerobically from glucose and xylose in high yields of 0.420 g/g at 36 h and 0.478 g/g at 60 h, respectively, but secreted fairly low cellulases. On the other hand, the strain 5398 also produced ethanol from glucose in yield of 0.340 g/g though it had a little growth in xylose culture. However, it secreted hyperactive cellulases that are essential for hydrolysis of rice straw in culture and the maximum activities of endo-β-glucanase and β-glucosidase were 2.11 U/L and 1.47 U/L, respectively. In SSF of rice straw by coculture with two fungi selected, the ethanol production reached 1.28 g/L after 96 h when the inoculation ratio of the strain 5398 to the strain 4572 was 9.

Keyword: Mucor circinelloides; ethanol production; cellulase secretion; SSF; coculture; rice straw

Bioethanol production from renewable resources has been received attention because it is expected as clean safe alternative fuel due to the diminishing fossil in recent decades. In fact, bioethanol is industrially produced mainly from sugarcane in Brazil and maize starch in USA. However, since the production of ethanol from these food grains causes the sudden short supply of sugar and fodder, it is urged to produce bioethanol using nonfood crops or agricultural waste materials as a new biomass resource. Rice straw generated from rice farm is an attractive resource for the bioethanol production. The annual rice production was about 689 million tons (FAO, 2008) and concomitant rice straw was estimated to be about 689 - 1034 million tons per year in the world but a goodly portion of rice straw is wildly discarded [ 1]. Rice straw is a lignocellulosic material containing cellulose, hemicellulose, lignin, and ash. The major fraction is cellulose composed of glucose and hemicellulose composed of xylose. To produce ethanol effectively and economically from such cellulosic materials, the enzymatic hydrolysis of biomass by hyperactive cellulases and fermentation by high ethanol-producing microorganism are essentially necessary. Therefore, the cellulase possessed high digesting-activity has been developed [ 2, 3]. In addition, the bioprocesses for ethanol production, separation hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), by combining the commercial cellulase reagents and glucose/xylose fermenting-microorganism has been developed [ 4, 5]. Those processes have not been practically used because the enzyme is now very expensive.

Mucor sp. is one of non-pathogenic filamentous fungi belong to Zygomaycete which has no dissepiment. It is commonly found in soil and plants etc., and capable to assimilated grow aerobically and anaerobically a lot of sugars. Although this fungus is known as a microorganism secreted milk-clotting enzyme (Rennin) widely applied in the cheese-making industries [ 6] and produced γ-linolenic acid, an important fatty acid for human health and nutrition [ 7]. The genus are recently been received attention as an ethanol-producing microorganism, which is capable of ethanol fermentation from cellulosic materials hydrolysate contained not only hexoses such as glucose, fructose, and mannose but also pentose such as xylose. For example, the production of ethanol by M. indicus from hydrolysate of rice straw, wood, and other lignocellulosic materials are investigated [ 8, 9]. The yield and productivity from dilute-acid spruce hydrolysate by M.indicus were 0.45 g/g and 0.83 g/(L·h)-1, respectively [ 10]. Other species of Mucor ( M. corticolous, M. hiemaris, M. miehei, and M. cirsinelloidesetc.) has also been demonstrated on capacity of ethanol production [ 11 14]. Moreover, Mucor sp. has also been known as microorganism which has cellulases secretion ability [ 15 19]. Especially, M. circinelloides has attracted attention because it has a high ability for producing ethanol from hexose and pentose and further was secreted cellulases such as endo-β-glucanase (EBG), cellobiohydrolase (CBH), β-glucosidase (BG), xylanase (X), and β-xylosidase (BX) which are able to convert various cellulosic and hemicellulosic materials to glucose and xylose etc. [ 20 23]. Therefore, M. circinelloides has been selected as a good candidate for direct ethanol production from lignocellulosic materials. Though the fungus is expected achieving at the same time the cellulase secretion and the ethanol fermentation of glucose and xylose, there have been no report on such innovative wild fungus. Therefore, it is desirable under the present situation strategy to construct SSF by coculture with two kinds of fungi that possess different functions. In general though the operation of SSF by coculture with two microorganisms is like pulling teeth because it is difficult to match the culture environment of each microorganism [ 24 30]. Based on this viewpoint, it is expected that the best culture environment is able to achieve easily if the congeneric species are able to use in coculture. However, no research has been reported on bioconversion from lignocellulosic materials to ethanol by coculture with congeneric fungi to the present.

In this study, two high-performing fungi on cellulase secretion and ethanol fermentation were selected from some strains belong to M. circinelloides and further direct conversion of lignocellulosic biomass, fine rice straw crashed mechanically, to bioethanol by using SSF system with coculture of two fungi selected was investigated.

Materials and methods
Microorganisms and cultivation

M. circinelloides was used through this study for screening of cellulase-secreting fungus and ethanol-producing fungus. M. circinelloides f. circinelloides NBRC 4554, 4569, 4570, 4572, 4574, 5382, 5774, and 30470, M. circinelloides f. griseo-cyanus NBRC 4563, and M. circinelloides f. janssenii NBRC 5398 and 6746 were purchased from the NBRC (NITE Biologic Resource Center, Chiba, Japan). It was maintained on potato dextrose agar Petri plate (9 cm diameter) and incubated at 28°C for 3 days as a preculture. The mycelia on a plate was milled by using blender (Milser LM-Plus, Osaka Chemical Co., Osaka, Japan) in 100 mL saline per one plate and it was used as inoculums. The fungi were cultivated in 100 mL flasks with liquid media 25 mL containing (g/L): yeast extract (Oriental Yeast Co., Tokyo), 5; (NH4)2SO4, 7.5; KH2PO4, 3.5; MgSO4·7H2O, 0.75; CaCl2·2H2O, 1.0; and carbon source, 50. Glucose, xylose, and rice straw pretreated were used as carbon sources. All reagents used were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The media including rice straw etc. was adjusted to pH 5.5 and previously autoclaved at 121°C for 15 min before use. After cooled it down, 1 mL suspension of each fungus was inoculated in the sterility and cultivated on shaking at 120 rpm at 28°C.

The high-performing fungi were selected on the basis of the analysis of cellulase activity and ethanol concentration in the broth obtained after the fungi were cultured for 72 h on glucose, 120 h on xylose, and 120 h on rice. The direct fermentation of rice straw by the coculture using two kinds of fungi was performed under the same condition as described above. In coculture, each fungus selected was milled by the blender with saline by method described above and then these were inoculated to medium at appropriate ratios in 1 mL total amount. After cultivation, secreted enzymes, residual sugars, and ethanol in broth were analyzed. Moreover, these fungal forms were confirmed by microscopic visualization in each sampling.

Biomass and pretreatment

High-yielding rice straw named “Kinmaze” was used as cellulosic biomass in this study. The rice straw was harvested in Manto-City (Toyama, Japan) in September 2008, air-dried, and cut into about 1 cm length prior to pretreatment into a fine powder about 40 μm in diameter by a dry-mechanical crusher (Dry Burst, Sugino Machine Ltd., Toyama). The rice straw contained 27.4% cellulose (glucose), 14.4% hemicelluloses (12.6% xylose and 1.8% arabinose), 47.1% lignin, and 10.8% ash, which were measured according to Sun et al. [ 31].

Cellulases activity secreted by M. circinelloides

Activities of enzymes secreted in broth by fungi were analyzed by colorimetric method. Substrates for cellulosic biomass degrading-enzymes involved hemicellulase were Azo-Avicel, Azo-CM-Cellulose, and Azo-Wheat-Arabinoxylan (Megazyme Co., Wicklow, Ireland) for cellobiohydrolase, endo-β-glucanase, and xylanase, respectively, which was prepared by dyeing highly purified and partially depolymerised with Remazolbrilliant Blue dye [ 32]. The reactions were followed by the protocols. One unit of enzymes activity was determined by reference to the standard curve for Meicelase (Meiji Seika Kaisha Co., Tokyo, Japan) to convert absorbance to units of activity per assay on each substrate. Moreover, BG and BX activity were determined using p-nitrophenyl-β-D-glucopyranoside and p-nitrophenyl-β-D-xylopyranoside (Sigma, St Louis, MO) as the substrates by colorimetric method of absorbance at 415 nm. One unit of enzymes activity was defined as the amount of enzymes required to release one micromole of glucose or xylose equivalent from each substrate in one minute.

Analysis methods

After cultivation, fungi were separated from media by filtration (Filter paper No. 131, Advantec Co., Tokyo, Japan) and dry cell weight was measured after 24 h at 90°C. On the other hand, the broth was analyzed for ethanol, residual sugars, by-products, and cellulase and hemicellulase activities. Ethanol produced was measured by gas chromatography (GC) equipped with a flame ionization detector (GC-2010, Shimadzu Co., Kyoto, Japan) using the column of Iert-Cap WAX (GL Sciences Inc., Tokyo, Japan) and helium as the carrier gas with a flow rate of 30 mL/min. Residual sugars and by-products in broth were measured on high performance liquid chromatography (HPLC, LC-10AD, Shimadzu Co.) with a refractive index detector (RID-10A, Shimadzu Co.), an ICSep WA-1 wine analysis column (Transgenomic, Ltd., NE) at 40°C, and mobile phase of 1.25 mmol·L-1 sulfuric acid at flow rate of 0.6 mL/min.

Results and discussion
Screening of high-performing fungi

Ethanol production abilities of fungus, M. circinelloides, based on the fermentation of glucose and xylose were first investigated and the fungus which produced the highest amount of ethanol from both sugars was selected. These fungi were cultured aerobically in media containing 50 g/L glucose or xylose as a carbon source at 28°C and the broths were collected by filtering after 72 h on glucose or 120 h on xylose. Then, the concentration of residual sugars, ethanol, and by-products in the broths were analyzed. Figure 1 shows the relation between ethanol production from glucose and xylose in order to compare the ethanol production ability among 11 species of M. circinelloides. The point in upper right in the figure means high ethanol production ability. It was found that all strains can produce ethanol from both glucose and xylose. Though lignocellulosic biomass contains not only cellulose consisting of glucose but also hemicelluloses consisting of xylose and arabinose, most fermentative microorganisms is not able to ferment xylose from the hydrolysis of hemicellulosic moiety. From the point of this view, M. circinelloides NBRC 4572 was selected because it has the highest fermentation ability and produced 18.3 g/L and 8.45 g/L of ethanol from glucose and xylose, respectively.

Fig.1 Ethanol production from glucose and xylose by several species of M. circinelloides

On the other hand, cellulase and hemicellulase-secreting ability of these fungi was estimated by measuring the enzymatic activity in broth obtained by culture under the same cultivation conditions as shown in Fig. 2. These fungi were cultured aerobically in media containing 50 g/L each sugars at 28°C for 120 h. After the cultivation, broths were separated by filtering and CBH, EBG, X, BG, and BX activities were analyzed. The fungus showing the highest cellulase activity in broth was selected; especially the relation between EBG and BG was paid attention because cellulose is the carbohydrate mainly containing in rice straw as described in Sect. 2.2. Though CBH activity, which is able to hydrolyze microcrystal cellulose moiety, was not observed in the all broths of fungi (data not shown), it was found that the fungus belong to M. circinelloides secreted various lignocellulosic material-digesting enzymes. Unfortunately, the amounts of EBG and BG secreted by high ethanol-producing fungus, strain 4572, were not too high comparing with those of other strains. However, M. circinelloides NBRC 5398 showed obviously higher activity of both EBG and BG at the culture of xylose compared with other strains, and these activities were 2.46 U/L and 2.94 U/L, respectively, though the fermentative ability to xylose by the strain is low. Because the strain 4572 can ferment effectively not only glucose and but also xylose, and the strain 5398 can secret higher BG or EBG than other strains at the glucose culture, it is probable to produce ethanol from rice straw by co-culturing with two high-performing fungi to utilize each advantage.

Fig.2 Cellulases secreted in broth by several species of M. circinelloides

Batch cultivation of two high-performing M. circinelloides

To examine the behavior of mycelia growth and the cellulase secretion of both strains in detail, the high ethanol-producing fungus, the strain 4572, and high cellulase-secreting fungus, the strain 5398, was cultivated aerobically in media containing 50 g/L glucose or xylose at 28°C. Figure 3 shows the result of time course as each strain was cultured with both sugars. For the strain 4572 grown with glucose, glucose was consumed sharply, and the dry cell weight reached 5.51 g/L when glucose was completely consumed after 36 h (Fig. 3(a)). Simultaneously, high ethanol was produced from glucose and the maximum concentration was 21.0 g/L at 36 h, which corresponds to the ethanol productivity of 0.583 g/(L·h)-1 and the ethanol yield of 0.420 g/g. After depletion of glucose, the ethanol concentration slowly decreased to 11.0 g/L after 96 h, which was caused by consuming ethanol as a carbon source to acquire energy by the strain. In the case of 50 g/L xylose, the growth rate was fast as well as glucose cultivation and the dry cell weight was 5.07 g/L at 24 h though xylose was consumed slower than glucose and was consumed completely after 96 h (Fig. 3(b)). It is noted that ethanol produced from xylose reached 17.8 g/L after 60 h (before depletion of xylose) and the productivity and the yield was 0.296 g/(L·h)-1 and 0.478 g/g, respectively. However, the ethanol production stopped despite the presence of residual xylose, and then the simultaneous consumption of xylose and ethanol was advanced for cell growth and maintenance. Moreover, the fungus was able to assimilate other monosaccharide, disaccharide, polysaccharide, and sugar alcohol (mannose, galactose, fructose, maltose, arabinose, cellobiose, lactose, sucrose, maltose, soluble starch, rice starch, Avicel, CMC, xylan, sorbitol, mannitol, xylitol etc.) and also ferment most sugars except a part of sugars (data not shown).

Fig.3 Batch culture of M. circinelloides NBRC 4572 and 5398 by using glucose (A, C) and xylose (B, D) as carbon sources

On the other hand, while the growth rate of the strain 5398 was slower than that of the strain 4572, the mycelium grew with the consumption of glucose and the maximum cell concentration reached 3.90 g/L after 72 h (Fig. 3(C)). However, ethanol was also produced, and its concentration, productivity, and yield reached 19.5 g/L, 0.340 g/g, and 0.202 g/(L·h)-1 after 96 h, respectively. In contrast, the strain 5398 consumed only a little of xylose, and its cell concentration was only 0.748 g/L after 96 h (Fig. 3(D)).

Behavior for cellulase secretion of M. circinelloides

The performance of cellulase secretion by two kinds of M .circinelloides were investigated because the strain 4572 was able to assimilate polysaccharides such as soluble starch, rice starch, CMC, Avicel, xylan, and rice straw. Two high-performing fungi were cultured aerobically in 25 mL media containing rice straw in order to induce the secretion of cellulases and then the broths were separated by filtration and its EBG, X, BG, and BX activities were analyzed, respectively, as shown in Fig. 4. It was found that a few cellulases by the strain 4572 were produced (Fig. 4(A)). The activities of EBG, X, and BG increased slowly, and were 0.578 U/L, 0.546 U/L, and 0.0231 U/L after 96 h, respectively.

Fig.4 Cellulase and hemicellulase secreted in broth by M . circinelloides NBRC 4572 and 5398 as batch cultures was performed with glucose and xylose

On the other hand, these activities of cellulases secreted from the strain 5398 were higher than those of the strain 4572 (Fig. 4(B)). EBG activity increased to 2.11 U/L after 24 h and decreased to 0.584 U/L at 48 h, and then increased again. BG activity began to increase at 36 h and reached 1.47 U/L at 72 h. These results indicate that the strain 5398 has higher secreting ability (4-fold for EBG and 20-fold for BG) than the strain 4572.

Direct ethanol production by SSF with coculture of two high-performing fungi from rice straw

First, the possibility of direct fermentation of rice straw by the strain 4572 or the strain 5398 only from rice straw was examined. The experiments was carried out by using basic medium with 100 g/L fine mechanically crushed rice straw (contained worth 50 g/L sugars) as a carbon source. Each SSF was started by inoculating milled fungus suspension to the medium and aerobically shaking at 28°C. After cultivation, the broths were separated by filtration and residual sugars, ethanol, and by-products were analyzed. For two cultivations after 96 h, not only ethanol but also other organic alcohols and acids were hardly detected (data not shown). Therefore, the ethanol production from rice straw powder by SSF with coculture of two kinds of high-performing fungus was examined. SSF was carried out under the same conditions as previous solo SSF except the inoculation ratio of two fungi. As a result, when the coculture using two selected fungi was carried out at the inoculation ratio of the strain 5398 to 4572 of 1 or 3, no ethanol was directly produced from fine mechanically crushed rice straw (data not shown). This is probably attributed to the fast growth rate and the slow secretion of cellulases of the strain 4572, i.e., once sugars produced from rice straw they were consumed immediately by the strain 4572 and therefore this seems to inhibit the growth of the strain 5398. Based on these results, the coculture was performed at the inoculation ratio of 9. As shown in Fig. 5, glucose and xylose eluted from rice straw were consumed completely after about 24 h and subsequently these sugars were not observed. However, ethanol concentration increased slowly from 12 h to 96 h and reached 1.28 g/L at 96 h. From the microscope observation of the broth obtained through the cultivation, the contamination from some species such as yeast and bacteria was not able to be confirmed.

Fig.5 Direct ethanol production from mechanical crashed rice straw by SSF with a coculture combined with cellulase-secreting fungus (strain 5398) and ethanol-producing fungus (strain 4572)

Meanwhile, though the fungi grew and ethanol was produced by SSF with the coculture system, there were little cellulase activities in broth as shown in Fig. 6. Only EBG activity was observed at about 0.5-0.8 U/L between 12 h and 96 h. The reason why other cellulases activities were little is that EG, X, and XG was adsorbed on the surface of rice straw or mycelia. Moreover, about 25 g/L ethanol is able to obtain theoretically from initial concentration of 100 g/L rice straw ( ca.50% total sugar content). However, in the SSF with coculture system as showed in Fig. 5, the ethanol was produced only 1.28 g/L. Because the difference of growth rates of two selected high-performing fungi was caused by the imbalance of the formation rate of monosaccharides (only glucose and xylose) by cellulases secreted from both fungi and the fermentation rate of these sugars, the improvement of secretion amount of various cellulases at the initial stage of SSF is especially necessary. A further high cellulases-secreting fungus is required to achieve the development of effective SSF with coculture system due to the direct ethanol production from rice straw. It is expected that the development of this system would be successful in more economical ethanol production than that by SSF combined with expensive commercial cellulases and ethanol-producing microorganisms.

Fig.6 Enzymatic activities existed in broth as direct ethanol production from fine mechanically crashed rice straw by SSF with coculture


The development of the direct production process of ethanol is recently demanded from rice straw abandoned voluminously all over the world every year. Therefore, we attempted the development of simultaneous saccharification and fermentation by using only fungi belong to Zygomycetes, especially Mucor circinelloides. M. circinelloides NBRC 5398 that can secrete a large amount of cellulases to obtain fermentable sugars from rice straw and M. circinelloides NBRC 4572 that can ferment glucose and xylose in high yield were selected from among our fungal library. In direct SSF from fine mechanically crushed rice straw by coculture system with two high-performing fungi, about 1.28 g/L ethanol was produced after 96 h when the inoculation ratio of the strain 5398 to the strain 4572 was 9. To increase the yield of ethanol using our proposed SSF with coculture system, the secretion of cellulases by M. circinelloides NBRC 5398 or other strains must be further improved.

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