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Enhanced sweet sorghum stalk to ethanol by fungus Mucor indicus using solid state fermentation followed by simultaneous saccharification and fermentation
Industrial Crops and Products 49 (2013) 580–585
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Enhanced sweet sorghum stalk to ethanol by fungus Mucor indicus using solid state fermentation followed by simultaneous saccharification and fermentation Maryam Molaverdi a , Keikhosro Karimi a,b,∗ , Morteza Khanahmadi c , Amir Goshadrou a a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran c Agricultural Engineering Department, Isfahan Agriculture and Natural Resources Research Center, Isfahan 81785-199, Iran b
a r t i c l e
i n f o
Article history: Received 13 March 2013 Received in revised form 15 June 2013 Accepted 17 June 2013 Keywords: Sweet sorghum Mucor indicus Fungal solid state fermentation Simultaneous saccharification and fermentation Ethanol
a b s t r a c t Sweet sorghum is one of the most promising energy crops for ethanol production. Fungal solid state fermentation (FSSF) of dry sweet sorghum stalk particles (DSSSPs) for ethanol production was conducted using fungus Mucor indicus and followed by simultaneous saccharification and fermentation of the residual solid without any pretreatment and addition of fresh microorganism cells. The effects of important variables including temperature (28, 32, and 36 ◦ C), moisture level (65, 75, 80, and 85%), initial fungal biomass concentration (0.001, 1, and 5 g/L), and particle size (<80, 20–80, and >20 mesh) on the yield of ethanol production by FSSF were investigated. The results showed that M. indicus was able to utilize almost all the glucose and fructose within 48 h, whereas the maximum ethanol yield (0.48 g produced ethanol/g consumed sugars) was achieved by FSSF at 32 ◦ C, 80% moisture, and particle size of 20–80 mesh with 5 g/L fungal biomass concentration. Moreover, simultaneous saccharification and fermentation of the stalk glucan (10, 25, and 50 g/L) was performed at 32, 35, and 37 ◦ C with different cellulase and glucosidase enzymes loading for 48 h. In the best case, 85.6% of ethanol yield was achieved when 50 g glucan/L was saccharified using 15 FPU cellulase and 30 IU -glucosidase per gram glucan and simultaneously fermented to ethanol at 37 ◦ C for 48 h. The results indicated that the FSSF acted as a pretreatment stage and assisted the subsequent simultaneous saccharification and fermentation process of the residual solid, resulted in up to 4.3 times improvement in the ethanol production yield. © 2013 Elsevier B.V. All rights reserved.
1. Introduction About 28% of the world energy consumption is utilized for transportation which is directly related to greenhouse gas (GHG) emissions to the atmosphere (Antoni et al., 2007). Using alcoholfueled engines is an attractive way to prevent harmful GHG emissions and global warming effects. Amongst alcoholic fuels, ethanol is the best alternative liquid fuel since it is less toxic and can be produced from abundant renewable resources all around the world (Vaidya and Rodrigues, 2006). Ethanol is nowadays produced biologically from different feedstocks such as sugary (e.g., molasses), starchy (e.g., corn), and lignocellulosic materials (e.g., agricultural and forestry residues) (Taherzadeh and Karimi, 2008). For multiple reasons, such as environmental impacts and
∗ Corresponding author at: Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran. Tel.: +98 3113915623; fax: +98 3113912677. E-mail addresses: [email protected], [email protected] (K. Karimi). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.06.024
increasing the food prices, conversion of food crops to fuel has drawn a lot of opposition in recent years. On the other hand, complexity and production cost of the process depend on the feedstock and go from the simple conversion of sugars by fermentation to the multi-stage conversion of lignocellulosic biomass into ethanol. Nevertheless, cultivation of low-cost and high output energy crop plants for making ethanol could be among the promising alternatives for fuel ethanol production. Sweet sorghum (Sorghum bicolor L. Moench) is one of the most promising energy crops characterized by its high sugar and biomass yield. It is also considered as a drought-resistant plant and can be cultivated in temperate, subtropical, and tropical climates (Goshadrou et al., 2011). Sweet sorghum stalk is rich in soluble sugars (i.e., sucrose, glucose, and fructose) and insoluble carbohydrates (i.e., cellulose and hemicelluloses), which both can be utilized as raw materials for fermentative ethanol production (Liu et al., 2008). Despite the potential of sweet sorghum, there are two major drawbacks attached to the practical use of this plant for industrial applications. First, the plant should be harvested in a few days after ripening while long-term storage of the fresh stalks are
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accompanied by sugars deterioration. Second, the lignocellulosic stalk cannot be easily hydrolyzed enzymatically to fermentable sugars due to the presence of free sugars inhibiting the hydrolytic enzymes action (Taherzadeh and Karimi, 2007); thus, the sugars in the stalk should be removed before the enzymatic hydrolysis of the cellulosic part of the plant. As the sweet sorghum stalk is harvested, the juice can be extracted and fermented by submerge fermentation. Meanwhile, avoiding the sugar loss, the juice has to be concentrated. However, the juice is very dilute (typically contains 15% sugar) and its concentration (e.g., up to 80%) has a high energy demand. Drying the fresh stalk is suggested as an alternative to the sugar extraction. The sugars in the stalk can be then converted to ethanol by solid state fermentation. The solid state fermentation has a number of advantages such as lower energy and water requirements, less capital investment and operating costs, and less pollution problems compared with the submerge fermentation. Moreover, it has been generally claimed that product yields are mostly higher by solid state fermentation in comparison to the submerge fermentation (Kargi et al., 1985; Pandey, 2003; Yu et al., 2008). After any of the mentioned methods, the residual lignocellulosic waste, i.e., sorghum bagasse, can be further proceed to fermentable sugars by enzymatic hydrolysis (Bvochora et al., 2000; RongHou et al., 2005). The sweet sorghum bagasse is a lignocellulosic material containing cellulose, hemicellulose, and lignin. Typically, bioconversion of a native lignocellulose is inefficient due to the compact structure of lignocellulosic biomass and high crystallinity of cellulose, therefore pretreatment is an essential stage for efficient conversion of the cellulose to fermentable sugars. However, developing an effective pretreatment is a technically challenging issue, and this step is the most expensive processing part of the ethanol production accounting for almost 20% of the total ethanol production cost (Agbor et al., 2011; Demirbas, 2011; Yang and Wyman, 2008). After a pretreatment, the carbohydrates within the biomass can be enzymatically hydrolyzed to fermentable sugars followed by conversion to ethanol. The fermentation process can be performed using a separated hydrolysis and fermentation or simultaneous saccharification and fermentation process. Simultaneous saccharification and fermentation has several advantages such as minimized end-product inhibition for the enzymes, lower risk of microbial contaminations, lower capital cost of the process, lower enzyme requirement, and higher ethanol yields from cellulose (Taherzadeh and Karimi, 2007). Selection of a suitable microorganism is one of the key factors in solid state fermentation processes. The microorganism should be able to utilize the solid, moist substrate, and grow under reduced moisture conditions. Recently, zygomycetes fungi such as Mucor indicus and Mucor hiemalis have been introduced for ethanol production with some advantages compared to the industrial ethanol-producing microorganisms (i.e., Saccharomyces cerevisiae). They are able to utilize both hexoses and pentoses and grow at slightly higher optimum temperature compared to the baker’s yeast leading to lower contamination risk and increased performance through the simultaneous saccharification and fermentation. Besides, valuable biomass of the fungi is suitable for various applications in water purification, drug, and food industries (Ab et al., 2011; Asachi et al., 2011; Heidary Vinche et al., 2012a, b; Millati et al., 2008; Pandey, 2003). However, to our knowledge, no previous work has been reported on application of zygomycetes fungi in FSSF for ethanol production. The present work aimed at FSSF of dry sweet sorghum stalk particles (DSSSPs) for ethanol production by the fungus M. indicus. Moreover, the effects of important variables, such as initial fungal biomass concentration, temperature, moisture level, and particle size on the yield of ethanol production through the solid state fermentation process were investigated. Subsequently, the solid
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state fermentation residue (i.e., sweet sorghum bagasse and the microorganism) was subjected to simultaneous saccharification and fermentation without any pretreatment or addition of fresh microorganism. Likewise, the effects of initial glucan and enzymes loading, as well as temperature on the yield of ethanol production were investigated. 2. Materials and methods 2.1. Raw material The sweet sorghum cultivar Sofra (Italy) was utilized as substrate in all experiments. It was cultivated in Isfahan University research farm (Isfahan, Iran) according to Almodares et al. (2008). The stems were separated from the other plant parts, dried at room temperature, and milled and screened to different particle sizes of <80 (less than 0.178 mm), 20–80 (between 0.178 and 0.853 mm), and >20 (larger than 0.853) meshes. The DSSSPs were stored in plastic bags. The moisture content after drying was about 1.7%, measured by drying at 105 ◦ C until a constant weight. 2.2. Microorganism and medium The zygomycetes fungus M. indicus CCUG 22424 was supplied from Culture Collection University of Gothenburg (Sweden) and used in all fermentation experiments. It was cultivated at 32 ◦ C for 5 days on an agar medium contained (g/L): glucose, 40; agar, 20; and peptone; 10 (Asachi et al., 2011) and stored at 4 ◦ C before use. The fungus has different morphologies (e.g., yeast-like and filamentous) (Sharifia et al., 2008). The yeast-like morphology is used for the solid state fermentation in this work, since it can form a homogeneous mixture in the solid medium. 2.3. Biomass production The required fungal biomass for the solid state fermentation was produced in a 250 mL Erlenmeyer flask with 100 mL pre-culture medium containing (g/L): glucose, 50; yeast extract, 5; (NH4 )SO4 , 7.5; MgSO4 ·7H2 O, 0.75; K2 HPO4 , 3.5; and CaCl2 , 1. The medium was autoclaved (121 ◦ C, 20 min) and cooled down to room temperature followed by addition of 25 mL inoculum suspension containing 6 × 106 spores/mL of M. indicus. The medium was incubated at 32 ◦ C and 110 rpm for 24 h. The produced biomass was then aseptically separated by centrifugation (4000 rpm, 20 min). 2.4. Ethanol production by FSSF Fungal solid state fermentation (FSSF) of DSSSPs was performed in 250 mL Erlenmeyer flasks. The solid medium contained (w/w): DSSSPs, 35%; (NH4 )2 SO4 , 0.3%; KH2 PO4 , 0.15%; and MgSO4 .7H2 O; 0.06% at pH 5.5 (Wang and Liu, 2009). The medium was thoroughly mixed and sterilized by autoclaving at 121 ◦ C for 15 min. It was then cooled to room temperature, inoculated with different fungal biomass concentrations (0.001, 1, and 5 g/L) with moisture level of 65, 75, 80, and 85%, and anaerobically fermented at different temperatures (28, 32, and 36 ◦ C) for 96 h. 2.5. Ethanol extraction after FSSF Considering the inhibitory effects of residual sugars and produced ethanol on the hydrolytic enzymatic activities (Taherzadeh and Karimi, 2007), the fermentation medium was washed aseptically with distilled water. The extract was then vacuum-filtered (Whatman No. 44), and the clear filtrate was subjected to ethanol
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and water-soluble sugars analyses. The yield of ethanol production was calculated according to the following equation: Ethanol yield (g/g) =
Produced ethanol (g/L) Total consumed sugars (g/L)
(1)
The filter cake was then subjected to subsequent simultaneous saccharification and fermentation.
Table 1 Chemical composition (% wt) of the sweet sorghum stalk. Sweet sorghum fraction
Components (%)
Free sugars in stalk
Glucose 8.0
Bagasse
Glucan 47.8
a b
2.6. Simultaneous saccharification and fermentation The residual material after the solid state fermentation and ethanol extraction was subjected to simultaneous saccharification and fermentation without any pretreatment. The experiments were performed in 50 mM sodium citrate buffer solution (pH 5.5) contained (g/L): glucan loading, 50; yeast extract, 5; CaCl2 ·2H2 O, 1; (NH4 )2 SO4 , 7.5; MgSO4 ·7H2 O, 0.75; and K2 HPO4 , 3.5. The fermentation medium was supplemented with different amounts of cellulase (Celluclast 1.5L, Novozyme, Denmark) and -glucosidase (Novozyme 188, Novozyme, Denmark). The enzymes activities were 51.5 FPU/mL and 240 IU/mL for cellulase and -glucosidase, respectively, measured according to the previously reported methods (Adney and Baker, 2008; Ximenes et al., 1996). The produced fungus biomass during solid state fermentation was used as fermenting microorganism, and thus no sterilization was done before simultaneous saccharification and fermentation. The mixture was incubated at 120 rpm and different temperatures (32, 35, and 37 ◦ C) for 48 h, and the liquid samples were periodically taken. Besides, simultaneous saccharification and fermentation of DSSSPs with 1 g/L fungal biomass was also conducted as a control in parallel. The yield of ethanol production was calculated based on maximum theoretical ethanol yield as follows: Ethanol yield (%) =
Produced ethanol (g/L) × 100 Initial bagasse concentration (g/L) × F × 1.111 × 0.51
(2)
where F is the bagasse glucan fraction (Table 1). 2.7. Analytical methods The carbohydrates and lignin content of the sweet sorghum bagasse were determined according to the standard protocol presented by National Renewable Energy Laboratory (NREL) (Sluiter et al., 2008). The acid-soluble lignin was determined using spectroscopy at 320 nm. The sugars concentration were determined using HPLC. For determination of free sugars (glucose, sucrose, and fructose) concentration in the sweet sorghum stalks, 10 g of DSSSPs was added to 100 mL sterile distilled water and thoroughly mixed at 100 ◦ C for 20 min to dissolve the free sugars. The sugar concentrations were measured by HPLC. The sugars and metabolites concentration in the liquid samples from fermentation experiments were analyzed by high performance liquid chromatography (HPLC, Jasco International Co., Tokyo, Japan) equipped with UV and RI detectors. Ethanol concentration was analyzed using an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 60 ◦ C with 0.6 mL/min of 5 mM sulfuric acid as a mobile phase and measured from UV chromatograms. The sugars were separated using an ion-exchange Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) at 85 ◦ C with 0.6 mL/min ultra-pure water as an eluent and measured from RI chromatograms. All the experiments and analyses were performed at least in duplicate and the average values of the two replications were reported.
Xylan 16.8
Fructose 5.0
Sucrose 16.1
ASLa 1.4
AILb 14.4
Ash 1.2
Acid-soluble lignin. Acid-insoluble lignin.
3. Results and discussion Sweet sorghum, as an energy crop, has a high potential for production of ethanol and M. indicus is a promising strain for fermentation of carbohydrates in the sorghum. The fungus is nonpathogenic dimorphic microorganism that is able to utilize a wide range of hexoses, pentoses, and disaccharides (e.g., cellobiose) and efficiently produce ethanol. The yield and productivity of ethanol by this microorganism are the same as that by S. cerevisiae and Pichia stipitis. Another advantage is the highly valuable biomass of this fungus, since it contains relatively high concentrations of chitosan that received a number of industrial applications, e.g. in chemical, biotechnology, medical, veterinary, dentistry, agriculture, food processing, environmental protection, water purification, cosmetic and textile industries (Karimi and Zamani, 2013). In this study, the free sugars available in its stalk was converted to ethanol by fungal solid state fermentation at different moisture levels, temperatures, fungal biomass concentrations, and substrate particle sizes. After the free sugars fermentation, the residual solid was subjected to simultaneous saccharification and fermentation for ethanol production from the stalk cellulose without any pretreatment and use of fresh fungal biomass supplementation. 3.1. Compositional analysis The sugar analysis showed that the DSSSPs mainly contained sucrose (16.1%), glucose (8.0%), and (5.0%) fructose (Table 1). The sweet sorghum bagasse was rich in glucan (47.8%), while xylan was the main constituent of hemicellulose (16.8%). It had also less than 15% acid-insoluble lignin and low amounts of acid-soluble lignin (Table 1). Similar composition for sweet sorghum bagasse has been previously reported by Goshadrou et al. (2011). 3.2. Solid state fermentation 3.2.1. Effect of temperature Effect of temperature on the yield of ethanol production was investigated by solid state fermentation of DSSSPs with 80% moisture using 5 g/L of fungal biomass concentration at different growth temperatures of 28, 32, and 36 ◦ C. As shown in Fig. 1a, the maximum ethanol production (0.48 g/g) was obtained at 32 ◦ C after 48 h. Temperature is one of the most important physical parameters, which is directly related to the growth of microorganism and product formation (Krishna, 2005). The fungus M. indicus was able to withstand fermentation temperatures within 25–38 ◦ C, and increasing growth temperature up to 36 ◦ C did not significantly affect the ethanol production yield (Fig. 1a). Heat transfer problem is one of the main difficulties in handling of solid state fermentation. Fermentation is an exothermic process and therefore heat is generated in the solid medium. The wet solid has poor thermal conductivity; thus, heat removal from the medium is usually a slow process leading to consequent heat accumulation and temperature elevation (Pandey, 2003). Therefore, the ability of M. indicus to resist wide temperature ranges is technically favorable in solid state fermentation.
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0.6
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0.6
(b)
Ethanol yield (g/g)
(a) 0.5
0.5
0.4
0.4
0.3 0.3
0.2
0.2
0.1
0.1
0 28
32
66
36
Temperature ( C)
80
85
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(c)
0.5 Ethanol yield (g/g)
75
Moisture level (%)
0.4
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0
0 0.001
1
(d)
0.5
< 80
5
20-80
> 20
Parcle size (mesh)
Fungal biomass concentraon (g/L)
Fig. 1. Effects of temperature (a), moisture level (b), biomass loading (c), and particle size (d) on ethanol production yield during FSSF.
3.2.2. Effect of moisture content The DSSSPs were inoculated with 5 g/L of fungal biomass and fermented to ethanol at 32 ◦ C and different moisture levels ranged from 66 to 85%. The moisture level was adjusted by addition of sterile distilled water. The results (Fig. 1b) indicated that the ethanol yield was enhanced by increasing solid moisture level from 66 to 80%, and maximum production yield of 0.48 g/g was obtained when the initial moisture level was 80%. It is acknowledged that the water activity (aw ) of the substrate is a key factor affecting microbial activity, and an optimum moisture level has to be maintained during the solid state fermentation. Low moisture level of the solid material reduces the chance of contamination during fermentation
M. indicus Nutrients
process. However, it hampers nutrient diffusion and therefore reduces the fungal growth, enzyme stability, and substrate swelling. On the other hand, higher moisture levels lead to particle agglomeration, competition of contaminating bacteria, and gas transfer limitation between air and the solid surface (Gowthaman et al., 2001; Pandey, 2003; Roukas, 1994; Shen and Liu, 2008; Yu et al., 2008). No significant change in the ethanol production yield was observed by further increase of the moisture level from 80 to 85%. The results were well supported by Yu et al. (2008) who reported that the moisture level of 75–80% was optimal for solid state fermentation of sweet sorghum using a thermotolerant yeast strain.
Ethanol Water
(from free sugars)
Nutrients Enzymes (cellulase &β-glucosidase)
Liquid
Solid state fermentaon
Washing
Separaon
(a)
Residual solid & microorganism
Simultaneous saccharificaon and fermentaon
Ethanol (from cellulose)
Sweet sorghum stalk (b)
Simultaneous saccharificaon and fermentaon
M.indicus
Ethanol (from free sugars and cellulose)
Nutrients
Enzymes (cellulase & β-glucosidase) Fig. 2. Ethanol production from DSSSPs by (a) solid state fermentation followed by simultaneous saccharification and fermentation and (b) simultaneous saccharification and fermentation.
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3.2.4. Effect of particle size Size of particles plays key role in the product formation by solid state fermentation since the microorganisms need to have an access to the inner parts of the moist solids. To investigate the effect of particle sizes on ethanol production yield from sweet sorghum stalks, DSSSPs with different particle sizes including <80, 20–80, and >20 meshes were subjected to solid state fermentation at 32 ◦ C and 80% humidity using 5 g/L fungal biomass. As can be seen from the Fig. 1d, fermentation of the fine (<80 mesh) and the large DSSSPs (>20 mesh) resulted in ethanol yields of 0.34 and 0.29 g/g, respectively, which were lower than the yield (0.48 g/g) obtained from the 20–80 meshes particles. It is known that the size of particle is directly related to the specific surface area and porosity of the solid medium. In general, big particles have less surface area leading to poor accessibility to the microorganism, whilst small particles having larger surface area are preferred for high production yields. Furthermore, small particles provide a higher solid medium density and decrease the void fraction, which are both favorable for anaerobic fermentation. However, too small particles lead to medium compaction which may diminish the heat transfer and carbon dioxide exchange rates. Therefore, an optimum particle size of 0.178–0.853 mm which is corresponding to 20–80 meshes has to be considered for fermentation of DSSSPs. 3.3. Simultaneous saccharification and fermentation In the FSSF process, the free sugars were initially converted to ethanol, and the cellulosic part of the residual materials was then subjected to simultaneous saccharification and fermentation. 3.3.1. Effect of FSSF on the following simultaneous saccharification and fermentation The solid residue after the FSSF was identically subjected to simultaneous saccharification and fermentation, and the results were compared to that of fresh DSSSPs (Fig. 2). While the simultaneous saccharification and fermentation of DSSSPs resulted in only 20.0% ethanol yield, the FSSF significantly improved (4.3 fold) the subsequent ethanol production yield. The limiting stage in simultaneous saccharification and fermentation is typically hydrolysis. Free sugars are well known inhibitors for the hydrolytic enzymes
Ethanol yield (% Theorical yield)
100
24 h, 5:10 (FPU:IU) 24 h, 15:30 (FPU:IU)
80
(a)
60 40 20 0 32 100
Ethanol yield (% Theorical yield)
3.2.3. Effect of fungal biomass concentration M. indicus is among the dimorphic fungi and it is possible to change and control its morphology. Generally, using yeast-like morphology of the fungus for fermentation of the solid medium has several technical advantages over the mycelial biomass, e.g., the biomass can be distributed uniformly on the solid surface (Gowthaman et al., 2001). Fermentation of 80% moist DSSSPs was conducted at 32 ◦ C using different fungal biomass concentrations and the most important results are illustrated in Fig. 1c. The ethanol production yield was only 0.22 g/g as the substrate was inoculated with 0.001 g/L fungus (3 × 104 spores/ml). Interestingly, the ethanol yield was markedly increased (1.6 times) using 1 g/L of fungus biomass as inocula and reached the maximum value of 0.48 g/g during fermentation by 5 g/L of the fungal biomass. The results were in contrast to the previous studies, which reported lower ethanol yield by solid state fermentation with higher initial yeast cell concentration, e.g., yeast inoculation rate higher than 0.25 (Shen and Liu, 2008). However, it has been mentioned that the lower ethanol yield could be due to the overuse of fermentable sugars for growth and maintenance at high cell concentrations. Besides, slow heat removal along with too fast fermentation results in heat accumulation and hot spots formation which may consequently decrease the yeast activity. None of the aforementioned effects was observed in the case of fermentation by fungus M. indicus. (Karimi and Zamani, 2013).
35
37
24 h, 5:10 (FPU:IU) 24 h, 15:30 (FPU:IU)
80
(b)
60 40 20 0 32
35
37
100
Ethanol yield (% Theorical yield)
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24 h, 5:10 (FPU:IU) 24 h, 15:30 (FPU:IU)
80
(c)
60 40 20 0 32
35
37
Temperature (C) Fig. 3. Ethanol production by simultaneous saccharification and fermentation from 10 (a), 25 (b), and 50 g/L (c) glucan.
(Taherzadeh and Karimi, 2007); thus, the lower hydrolysis and consequently ethanol yield by simultaneous saccharification and fermentation of fresh DSSSPs is most likely attributed to the presence of free soluble sugars in the DSSSPs. On the other hand, the fungus can produced variety of enzymes including xylanase during the aerobic cultivations. Thus, at aerobic conditions, cultivation of the fungus could act as a biological pretreatment for the lignocellulosic part of DSSSPs and make it more digestible for subsequent enzymatic hydrolysis in simultaneous saccharification and fermentation. However, under anaerobic cultivation, the fungus has no effects on lignin and hemicellulosic polymers (Karimi and Zamani, 2013). The yield of ethanol production from untreated sweet sorghum bagasse of the same culture was reported to be less than 50% of theoretical yield, thus a pretreatment stage is necessary to reach higher production yields (Goshadrou et al., 2011). It has been shown that the sodium hydroxide and phosphoric acid pretreatments improved the ethanol yield to about 80%. However, with no pretreatment stage in this work, more than 85% ethanol yield was observed in simultaneous saccharification and fermentation of the solid residue after FSSF using M. indicus.
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3.3.2. Optimization of simultaneous saccharification and fermentation The effects of process parameters including initial glucan concentration, temperature, and enzyme loading on the yield of ethanol production by simultaneous saccharification and fermentation from the bagasse were investigated. The experiments were performed at 32, 35, and 37 ◦ C, with 10, 25, and 50 g glucan/L using 5 and 15 FPU cellulase and 10 and 30 IU -glucosidase per gram glucan. The ethanol yields at 32, 35, and 37 ◦ C were respectively 31.0, 46.3, and 47.0% after 24 h with 10 g/L initial glucan using 15 FPU cellulase and 30 IU -glucosidase per gram of glucan (Fig. 3a). As shown, the yields were correspondingly increased to 48.4, 77.1 and 79.5% by continuation of the process up to 48 h. However, the yields were much less by loading of 5 FPU cellulase and 10 IU -glucosidase per gram of glucan. Likewise, effects of temperature, fermentation time, and enzymes loading in the experiments with 10, 25, and 50 g/L glucan were the same (Fig. 3b and c). The maximum ethanol yield from 10 g/L substrate (79.5%) was almost unaffected as the glucan loading increased to 25 g/L, whilst it was significantly improved to 85.6% for 50 g/L initial glucan concentration. Consequently, the maximum ethanol yield was achieved when 50 g/L glucan was saccharified using 15 FPU cellulase and 30 IU glucosidase per gram of glucan and simultaneously fermented to ethanol at 37 ◦ C for 48 h. The results are in agreement with a previous study on ethanol production from sweet sorghum bagasse by simultaneous saccharification and fermentation using S. cerevisiae (Wang et al., 2013). 4. Conclusions The present study was undertaken to assess the ethanol production from dry sweet sorghum stalk by FSSF using M. indicus followed by simultaneous saccharification and fermentation of the residual solid. Optimal values for FSSF parameters were found to be 5 g/L fungal biomass, 20–80 mesh particle size, 80% substrate moisture, and 32 ◦ C, while the best results for simultaneous saccharification and fermentation were obtained by 50 g/L glucan loading with particle sizes of 20–80 mesh and addition of 15 FPU cellulase together with 30 IU -glucosidase per g glucan at 37 ◦ C for 48 h. Moreover, the FSSF prior to simultaneous saccharification and fermentation significantly enhanced the ethanol production yield from about 20% to more than 85% without any pretreatment and addition of fresh microorganism. Acknowledgment The authors would like to thank Dr. Almodares (Department of Biological Science, University of Isfahan, Iran) for providing the sweet sorghum stalks. References Ab, S., Darah, I., Omar, I.C., 2011. Utilization of palm kernel cake for the production of mannanase by an indigenous filamentous fungus, Aspergillus niger USM F4 under solid substrate fermentation. Internet J. Microbiol. 9 (1), http://dx.doi.org/10.5580/2779. Adney, B., Baker, J., 2008. Measurement of cellulase activities. National Renewable Energy Laboratory, NREL/TP-510-42628.
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Agbor, V.B., Cicek, N., Sparling, R., Berlin, A., Levin, D.B., 2011. Biomass pretreatment. Fundamentals toward application. Biotechnol. Adv. 29, 675–685. Almodares, A., Hadi, M.R., Ahmadpour, H., 2008. Sorghum stem yield and soluble carbohydrates under different salinity levels. Afr. J. Biotechnol. 7, 4051–4055. Antoni, D., Zverlov, V.V., Schwarz, W.H., 2007. Biofuels from microbes. Appl. Microbiol. Biotechnol. 77, 23–35. Asachi, R., Karimi, K., Taherzadeh, M.J., 2011. Fungal autolysate as a nutrient supplement for ethanol and chitosan production by Mucor indicus. Biotechnol. Lett. 33, 2405–2409. Bvochora, J., Read, J., Zvauya, R., 2000. Application of very high gravity technology to the cofermentation of sweet stem sorghum juice and sorghum grain. Ind. Crops Prod. 11, 11–17. Demirbas, A., 2011. Competitive liquid biofuels from biomass. Appl. Energy 88, 17–28. Goshadrou, A., Karimi, K., Taherzadeh, M.J., 2011. Bioethanol production from sweet sorghum bagasse by Mucor hiemalis. Ind. Crops Prod. 34, 1219–1225. Gowthaman, M.K., Krishna, C., Moo-Young, M., 2001. Fungal solid state fermentation—an overview. Appl. Mycol. Biotechnol. 1, 305–352. Heidary Vinche, M., Asachi, R., Zamani, A., Karimi, K., 2012a. Ethanol and chitosan production from wheat hydrolysate by Mucor hiemalis. J. Chem. Technol. Biotechnol. 88 (2), 255–260. Heidary Vinche, M., Karimi, K., Zamani, A., Asachi, R., 2012b. Chitosan: a valuable byproduct of ethanolic fermentation by Rhizopus oryzae. J. Biobased Mater. Bioenergy 6, 552–557. Kargi, F., Curme, J.A., Sheehan, J.J., 1985. Solid state fermentation of sweet sorghum to ethanol. Biothecnol. Bioeng. 27, 34–40. Karimi, K., Zamani, A., 2013. Mucor indicus: biology and industrial applications perspectives: a review. Biotechnol. Adv., http://dx.doi.org/10.1016/j.biotechadv. 2013.01.009. Krishna, C., 2005. Solid-state fermentation systems—an overview. Crit. Rev. Biotechnol. 25, 1–30. Liu, R., Li, J., Shen, F., 2008. Refining bioethanol from stalk juice of sweet sorghum by immobilized yeast fermentation. Renew. Energy 33, 1130–1135. Millati, R., Karimi, K., Edebo, L., Niklasson, C., Taherzadeh, M.J., 2008. Ethanol production from xylose and wood hydrolyzate by Mucor indicus at different aeration rates. Bioresources 3, 1020–1029. Pandey, A., 2003. Solid-state fermentation. Biochem. Eng. J. 13, 81–84. RongHou, L., JinXia, L., Fei, S., Qing, S., 2005. Ethanol fermentation of sweet sorghum stalk juice by immobilized yeast. Trans. Chin. Soc. Agric. Eng. 21, 137–140. Roukas, T., 1994. Solid-state fermentation of carob pods for ethanol production. Appl. Microbiol. Biotechnol. 41, 296–301. Sharifia, M., Karimi, K., Taherzadeh, M.J., 2008. Production of ethanol by filamentous and yeast-like forms of Mucor indicus from fructose, glucose, sucrose, and molasses. J. Ind. Microbiol. Biotechnol. 35, 1253–1259. Shen, F., Liu, R., 2008. Research on solid-state ethanol fermentation using dry sweet sorghum stalk particles with active dry yeast. Energy Fuel 23, 519–525. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory, NREL/TP-510-42618. Taherzadeh, M.J., Karimi, K., 2008. Bioethanol: market and production processes. In: Nag, A. (Ed.), Biofuels Refining and Performance. McGraw-Hill, New York, pp. 69–106. Taherzadeh, M.J., Karimi, K., 2007. Enzyme-based hydrolysis processes for ethanol from lignocellulosic materials: a review. Bioresources 2 (4), 707–738. Vaidya, P.D., Rodrigues, A.E., 2006. Insight into steam reforming of ethanol to produce hydrogen for fuel cells. Chem. Eng. J. 117, 39–49. Wang, F., Liu, C.Z., 2009. Development of an economic refining strategy of sweet sorghum in the inner Mongolia region of China. Energy Fuel 23, 4137–4142. Wang, L., Luo, Z., Shahbazi, A., 2013. Optimization of simultaneous saccharification and fermentation for the production of ethanol from sweet sorghum (Sorghum bicolor) bagasse using response surface methodology. Ind. Crops Prod. 42, 280–291. Ximenes, E.A., Felix, C.R., Ulhoa, C.J., 1996. Production of cellulases by Aspergillus fumigatus and characterization of one b-glucosidase. Curr. Microbiol. 32, 119–123. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod. Bioref. 2, 26–40. Yu, J., Zhang, X., Tan, T., 2008. Ethanol production by solid state fermentation of sweet sorghum using thermotolerant yeast strain. Fuel Process. Technol. 89, 1056–1059.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Enhanced sweet sorghum stalk to ethanol by fungus Mucor indicus using solid state fermentation followed by simultaneous saccharification and fermentation Maryam Molaverdi a , Keikhosro Karimi a,b,∗ , Morteza Khanahmadi c , Amir Goshadrou a a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran c Agricultural Engineering Department, Isfahan Agriculture and Natural Resources Research Center, Isfahan 81785-199, Iran b
a r t i c l e
i n f o
Article history: Received 13 March 2013 Received in revised form 15 June 2013 Accepted 17 June 2013 Keywords: Sweet sorghum Mucor indicus Fungal solid state fermentation Simultaneous saccharification and fermentation Ethanol
a b s t r a c t Sweet sorghum is one of the most promising energy crops for ethanol production. Fungal solid state fermentation (FSSF) of dry sweet sorghum stalk particles (DSSSPs) for ethanol production was conducted using fungus Mucor indicus and followed by simultaneous saccharification and fermentation of the residual solid without any pretreatment and addition of fresh microorganism cells. The effects of important variables including temperature (28, 32, and 36 ◦ C), moisture level (65, 75, 80, and 85%), initial fungal biomass concentration (0.001, 1, and 5 g/L), and particle size (<80, 20–80, and >20 mesh) on the yield of ethanol production by FSSF were investigated. The results showed that M. indicus was able to utilize almost all the glucose and fructose within 48 h, whereas the maximum ethanol yield (0.48 g produced ethanol/g consumed sugars) was achieved by FSSF at 32 ◦ C, 80% moisture, and particle size of 20–80 mesh with 5 g/L fungal biomass concentration. Moreover, simultaneous saccharification and fermentation of the stalk glucan (10, 25, and 50 g/L) was performed at 32, 35, and 37 ◦ C with different cellulase and glucosidase enzymes loading for 48 h. In the best case, 85.6% of ethanol yield was achieved when 50 g glucan/L was saccharified using 15 FPU cellulase and 30 IU -glucosidase per gram glucan and simultaneously fermented to ethanol at 37 ◦ C for 48 h. The results indicated that the FSSF acted as a pretreatment stage and assisted the subsequent simultaneous saccharification and fermentation process of the residual solid, resulted in up to 4.3 times improvement in the ethanol production yield. © 2013 Elsevier B.V. All rights reserved.
1. Introduction About 28% of the world energy consumption is utilized for transportation which is directly related to greenhouse gas (GHG) emissions to the atmosphere (Antoni et al., 2007). Using alcoholfueled engines is an attractive way to prevent harmful GHG emissions and global warming effects. Amongst alcoholic fuels, ethanol is the best alternative liquid fuel since it is less toxic and can be produced from abundant renewable resources all around the world (Vaidya and Rodrigues, 2006). Ethanol is nowadays produced biologically from different feedstocks such as sugary (e.g., molasses), starchy (e.g., corn), and lignocellulosic materials (e.g., agricultural and forestry residues) (Taherzadeh and Karimi, 2008). For multiple reasons, such as environmental impacts and
∗ Corresponding author at: Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran. Tel.: +98 3113915623; fax: +98 3113912677. E-mail addresses: [email protected], [email protected] (K. Karimi). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.06.024
increasing the food prices, conversion of food crops to fuel has drawn a lot of opposition in recent years. On the other hand, complexity and production cost of the process depend on the feedstock and go from the simple conversion of sugars by fermentation to the multi-stage conversion of lignocellulosic biomass into ethanol. Nevertheless, cultivation of low-cost and high output energy crop plants for making ethanol could be among the promising alternatives for fuel ethanol production. Sweet sorghum (Sorghum bicolor L. Moench) is one of the most promising energy crops characterized by its high sugar and biomass yield. It is also considered as a drought-resistant plant and can be cultivated in temperate, subtropical, and tropical climates (Goshadrou et al., 2011). Sweet sorghum stalk is rich in soluble sugars (i.e., sucrose, glucose, and fructose) and insoluble carbohydrates (i.e., cellulose and hemicelluloses), which both can be utilized as raw materials for fermentative ethanol production (Liu et al., 2008). Despite the potential of sweet sorghum, there are two major drawbacks attached to the practical use of this plant for industrial applications. First, the plant should be harvested in a few days after ripening while long-term storage of the fresh stalks are
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accompanied by sugars deterioration. Second, the lignocellulosic stalk cannot be easily hydrolyzed enzymatically to fermentable sugars due to the presence of free sugars inhibiting the hydrolytic enzymes action (Taherzadeh and Karimi, 2007); thus, the sugars in the stalk should be removed before the enzymatic hydrolysis of the cellulosic part of the plant. As the sweet sorghum stalk is harvested, the juice can be extracted and fermented by submerge fermentation. Meanwhile, avoiding the sugar loss, the juice has to be concentrated. However, the juice is very dilute (typically contains 15% sugar) and its concentration (e.g., up to 80%) has a high energy demand. Drying the fresh stalk is suggested as an alternative to the sugar extraction. The sugars in the stalk can be then converted to ethanol by solid state fermentation. The solid state fermentation has a number of advantages such as lower energy and water requirements, less capital investment and operating costs, and less pollution problems compared with the submerge fermentation. Moreover, it has been generally claimed that product yields are mostly higher by solid state fermentation in comparison to the submerge fermentation (Kargi et al., 1985; Pandey, 2003; Yu et al., 2008). After any of the mentioned methods, the residual lignocellulosic waste, i.e., sorghum bagasse, can be further proceed to fermentable sugars by enzymatic hydrolysis (Bvochora et al., 2000; RongHou et al., 2005). The sweet sorghum bagasse is a lignocellulosic material containing cellulose, hemicellulose, and lignin. Typically, bioconversion of a native lignocellulose is inefficient due to the compact structure of lignocellulosic biomass and high crystallinity of cellulose, therefore pretreatment is an essential stage for efficient conversion of the cellulose to fermentable sugars. However, developing an effective pretreatment is a technically challenging issue, and this step is the most expensive processing part of the ethanol production accounting for almost 20% of the total ethanol production cost (Agbor et al., 2011; Demirbas, 2011; Yang and Wyman, 2008). After a pretreatment, the carbohydrates within the biomass can be enzymatically hydrolyzed to fermentable sugars followed by conversion to ethanol. The fermentation process can be performed using a separated hydrolysis and fermentation or simultaneous saccharification and fermentation process. Simultaneous saccharification and fermentation has several advantages such as minimized end-product inhibition for the enzymes, lower risk of microbial contaminations, lower capital cost of the process, lower enzyme requirement, and higher ethanol yields from cellulose (Taherzadeh and Karimi, 2007). Selection of a suitable microorganism is one of the key factors in solid state fermentation processes. The microorganism should be able to utilize the solid, moist substrate, and grow under reduced moisture conditions. Recently, zygomycetes fungi such as Mucor indicus and Mucor hiemalis have been introduced for ethanol production with some advantages compared to the industrial ethanol-producing microorganisms (i.e., Saccharomyces cerevisiae). They are able to utilize both hexoses and pentoses and grow at slightly higher optimum temperature compared to the baker’s yeast leading to lower contamination risk and increased performance through the simultaneous saccharification and fermentation. Besides, valuable biomass of the fungi is suitable for various applications in water purification, drug, and food industries (Ab et al., 2011; Asachi et al., 2011; Heidary Vinche et al., 2012a, b; Millati et al., 2008; Pandey, 2003). However, to our knowledge, no previous work has been reported on application of zygomycetes fungi in FSSF for ethanol production. The present work aimed at FSSF of dry sweet sorghum stalk particles (DSSSPs) for ethanol production by the fungus M. indicus. Moreover, the effects of important variables, such as initial fungal biomass concentration, temperature, moisture level, and particle size on the yield of ethanol production through the solid state fermentation process were investigated. Subsequently, the solid
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state fermentation residue (i.e., sweet sorghum bagasse and the microorganism) was subjected to simultaneous saccharification and fermentation without any pretreatment or addition of fresh microorganism. Likewise, the effects of initial glucan and enzymes loading, as well as temperature on the yield of ethanol production were investigated. 2. Materials and methods 2.1. Raw material The sweet sorghum cultivar Sofra (Italy) was utilized as substrate in all experiments. It was cultivated in Isfahan University research farm (Isfahan, Iran) according to Almodares et al. (2008). The stems were separated from the other plant parts, dried at room temperature, and milled and screened to different particle sizes of <80 (less than 0.178 mm), 20–80 (between 0.178 and 0.853 mm), and >20 (larger than 0.853) meshes. The DSSSPs were stored in plastic bags. The moisture content after drying was about 1.7%, measured by drying at 105 ◦ C until a constant weight. 2.2. Microorganism and medium The zygomycetes fungus M. indicus CCUG 22424 was supplied from Culture Collection University of Gothenburg (Sweden) and used in all fermentation experiments. It was cultivated at 32 ◦ C for 5 days on an agar medium contained (g/L): glucose, 40; agar, 20; and peptone; 10 (Asachi et al., 2011) and stored at 4 ◦ C before use. The fungus has different morphologies (e.g., yeast-like and filamentous) (Sharifia et al., 2008). The yeast-like morphology is used for the solid state fermentation in this work, since it can form a homogeneous mixture in the solid medium. 2.3. Biomass production The required fungal biomass for the solid state fermentation was produced in a 250 mL Erlenmeyer flask with 100 mL pre-culture medium containing (g/L): glucose, 50; yeast extract, 5; (NH4 )SO4 , 7.5; MgSO4 ·7H2 O, 0.75; K2 HPO4 , 3.5; and CaCl2 , 1. The medium was autoclaved (121 ◦ C, 20 min) and cooled down to room temperature followed by addition of 25 mL inoculum suspension containing 6 × 106 spores/mL of M. indicus. The medium was incubated at 32 ◦ C and 110 rpm for 24 h. The produced biomass was then aseptically separated by centrifugation (4000 rpm, 20 min). 2.4. Ethanol production by FSSF Fungal solid state fermentation (FSSF) of DSSSPs was performed in 250 mL Erlenmeyer flasks. The solid medium contained (w/w): DSSSPs, 35%; (NH4 )2 SO4 , 0.3%; KH2 PO4 , 0.15%; and MgSO4 .7H2 O; 0.06% at pH 5.5 (Wang and Liu, 2009). The medium was thoroughly mixed and sterilized by autoclaving at 121 ◦ C for 15 min. It was then cooled to room temperature, inoculated with different fungal biomass concentrations (0.001, 1, and 5 g/L) with moisture level of 65, 75, 80, and 85%, and anaerobically fermented at different temperatures (28, 32, and 36 ◦ C) for 96 h. 2.5. Ethanol extraction after FSSF Considering the inhibitory effects of residual sugars and produced ethanol on the hydrolytic enzymatic activities (Taherzadeh and Karimi, 2007), the fermentation medium was washed aseptically with distilled water. The extract was then vacuum-filtered (Whatman No. 44), and the clear filtrate was subjected to ethanol
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and water-soluble sugars analyses. The yield of ethanol production was calculated according to the following equation: Ethanol yield (g/g) =
Produced ethanol (g/L) Total consumed sugars (g/L)
(1)
The filter cake was then subjected to subsequent simultaneous saccharification and fermentation.
Table 1 Chemical composition (% wt) of the sweet sorghum stalk. Sweet sorghum fraction
Components (%)
Free sugars in stalk
Glucose 8.0
Bagasse
Glucan 47.8
a b
2.6. Simultaneous saccharification and fermentation The residual material after the solid state fermentation and ethanol extraction was subjected to simultaneous saccharification and fermentation without any pretreatment. The experiments were performed in 50 mM sodium citrate buffer solution (pH 5.5) contained (g/L): glucan loading, 50; yeast extract, 5; CaCl2 ·2H2 O, 1; (NH4 )2 SO4 , 7.5; MgSO4 ·7H2 O, 0.75; and K2 HPO4 , 3.5. The fermentation medium was supplemented with different amounts of cellulase (Celluclast 1.5L, Novozyme, Denmark) and -glucosidase (Novozyme 188, Novozyme, Denmark). The enzymes activities were 51.5 FPU/mL and 240 IU/mL for cellulase and -glucosidase, respectively, measured according to the previously reported methods (Adney and Baker, 2008; Ximenes et al., 1996). The produced fungus biomass during solid state fermentation was used as fermenting microorganism, and thus no sterilization was done before simultaneous saccharification and fermentation. The mixture was incubated at 120 rpm and different temperatures (32, 35, and 37 ◦ C) for 48 h, and the liquid samples were periodically taken. Besides, simultaneous saccharification and fermentation of DSSSPs with 1 g/L fungal biomass was also conducted as a control in parallel. The yield of ethanol production was calculated based on maximum theoretical ethanol yield as follows: Ethanol yield (%) =
Produced ethanol (g/L) × 100 Initial bagasse concentration (g/L) × F × 1.111 × 0.51
(2)
where F is the bagasse glucan fraction (Table 1). 2.7. Analytical methods The carbohydrates and lignin content of the sweet sorghum bagasse were determined according to the standard protocol presented by National Renewable Energy Laboratory (NREL) (Sluiter et al., 2008). The acid-soluble lignin was determined using spectroscopy at 320 nm. The sugars concentration were determined using HPLC. For determination of free sugars (glucose, sucrose, and fructose) concentration in the sweet sorghum stalks, 10 g of DSSSPs was added to 100 mL sterile distilled water and thoroughly mixed at 100 ◦ C for 20 min to dissolve the free sugars. The sugar concentrations were measured by HPLC. The sugars and metabolites concentration in the liquid samples from fermentation experiments were analyzed by high performance liquid chromatography (HPLC, Jasco International Co., Tokyo, Japan) equipped with UV and RI detectors. Ethanol concentration was analyzed using an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 60 ◦ C with 0.6 mL/min of 5 mM sulfuric acid as a mobile phase and measured from UV chromatograms. The sugars were separated using an ion-exchange Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) at 85 ◦ C with 0.6 mL/min ultra-pure water as an eluent and measured from RI chromatograms. All the experiments and analyses were performed at least in duplicate and the average values of the two replications were reported.
Xylan 16.8
Fructose 5.0
Sucrose 16.1
ASLa 1.4
AILb 14.4
Ash 1.2
Acid-soluble lignin. Acid-insoluble lignin.
3. Results and discussion Sweet sorghum, as an energy crop, has a high potential for production of ethanol and M. indicus is a promising strain for fermentation of carbohydrates in the sorghum. The fungus is nonpathogenic dimorphic microorganism that is able to utilize a wide range of hexoses, pentoses, and disaccharides (e.g., cellobiose) and efficiently produce ethanol. The yield and productivity of ethanol by this microorganism are the same as that by S. cerevisiae and Pichia stipitis. Another advantage is the highly valuable biomass of this fungus, since it contains relatively high concentrations of chitosan that received a number of industrial applications, e.g. in chemical, biotechnology, medical, veterinary, dentistry, agriculture, food processing, environmental protection, water purification, cosmetic and textile industries (Karimi and Zamani, 2013). In this study, the free sugars available in its stalk was converted to ethanol by fungal solid state fermentation at different moisture levels, temperatures, fungal biomass concentrations, and substrate particle sizes. After the free sugars fermentation, the residual solid was subjected to simultaneous saccharification and fermentation for ethanol production from the stalk cellulose without any pretreatment and use of fresh fungal biomass supplementation. 3.1. Compositional analysis The sugar analysis showed that the DSSSPs mainly contained sucrose (16.1%), glucose (8.0%), and (5.0%) fructose (Table 1). The sweet sorghum bagasse was rich in glucan (47.8%), while xylan was the main constituent of hemicellulose (16.8%). It had also less than 15% acid-insoluble lignin and low amounts of acid-soluble lignin (Table 1). Similar composition for sweet sorghum bagasse has been previously reported by Goshadrou et al. (2011). 3.2. Solid state fermentation 3.2.1. Effect of temperature Effect of temperature on the yield of ethanol production was investigated by solid state fermentation of DSSSPs with 80% moisture using 5 g/L of fungal biomass concentration at different growth temperatures of 28, 32, and 36 ◦ C. As shown in Fig. 1a, the maximum ethanol production (0.48 g/g) was obtained at 32 ◦ C after 48 h. Temperature is one of the most important physical parameters, which is directly related to the growth of microorganism and product formation (Krishna, 2005). The fungus M. indicus was able to withstand fermentation temperatures within 25–38 ◦ C, and increasing growth temperature up to 36 ◦ C did not significantly affect the ethanol production yield (Fig. 1a). Heat transfer problem is one of the main difficulties in handling of solid state fermentation. Fermentation is an exothermic process and therefore heat is generated in the solid medium. The wet solid has poor thermal conductivity; thus, heat removal from the medium is usually a slow process leading to consequent heat accumulation and temperature elevation (Pandey, 2003). Therefore, the ability of M. indicus to resist wide temperature ranges is technically favorable in solid state fermentation.
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0.6
583
0.6
(b)
Ethanol yield (g/g)
(a) 0.5
0.5
0.4
0.4
0.3 0.3
0.2
0.2
0.1
0.1
0 28
32
66
36
Temperature ( C)
80
85
0.6
0.6
(c)
0.5 Ethanol yield (g/g)
75
Moisture level (%)
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0
0 0.001
1
(d)
0.5
< 80
5
20-80
> 20
Parcle size (mesh)
Fungal biomass concentraon (g/L)
Fig. 1. Effects of temperature (a), moisture level (b), biomass loading (c), and particle size (d) on ethanol production yield during FSSF.
3.2.2. Effect of moisture content The DSSSPs were inoculated with 5 g/L of fungal biomass and fermented to ethanol at 32 ◦ C and different moisture levels ranged from 66 to 85%. The moisture level was adjusted by addition of sterile distilled water. The results (Fig. 1b) indicated that the ethanol yield was enhanced by increasing solid moisture level from 66 to 80%, and maximum production yield of 0.48 g/g was obtained when the initial moisture level was 80%. It is acknowledged that the water activity (aw ) of the substrate is a key factor affecting microbial activity, and an optimum moisture level has to be maintained during the solid state fermentation. Low moisture level of the solid material reduces the chance of contamination during fermentation
M. indicus Nutrients
process. However, it hampers nutrient diffusion and therefore reduces the fungal growth, enzyme stability, and substrate swelling. On the other hand, higher moisture levels lead to particle agglomeration, competition of contaminating bacteria, and gas transfer limitation between air and the solid surface (Gowthaman et al., 2001; Pandey, 2003; Roukas, 1994; Shen and Liu, 2008; Yu et al., 2008). No significant change in the ethanol production yield was observed by further increase of the moisture level from 80 to 85%. The results were well supported by Yu et al. (2008) who reported that the moisture level of 75–80% was optimal for solid state fermentation of sweet sorghum using a thermotolerant yeast strain.
Ethanol Water
(from free sugars)
Nutrients Enzymes (cellulase &β-glucosidase)
Liquid
Solid state fermentaon
Washing
Separaon
(a)
Residual solid & microorganism
Simultaneous saccharificaon and fermentaon
Ethanol (from cellulose)
Sweet sorghum stalk (b)
Simultaneous saccharificaon and fermentaon
M.indicus
Ethanol (from free sugars and cellulose)
Nutrients
Enzymes (cellulase & β-glucosidase) Fig. 2. Ethanol production from DSSSPs by (a) solid state fermentation followed by simultaneous saccharification and fermentation and (b) simultaneous saccharification and fermentation.
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3.2.4. Effect of particle size Size of particles plays key role in the product formation by solid state fermentation since the microorganisms need to have an access to the inner parts of the moist solids. To investigate the effect of particle sizes on ethanol production yield from sweet sorghum stalks, DSSSPs with different particle sizes including <80, 20–80, and >20 meshes were subjected to solid state fermentation at 32 ◦ C and 80% humidity using 5 g/L fungal biomass. As can be seen from the Fig. 1d, fermentation of the fine (<80 mesh) and the large DSSSPs (>20 mesh) resulted in ethanol yields of 0.34 and 0.29 g/g, respectively, which were lower than the yield (0.48 g/g) obtained from the 20–80 meshes particles. It is known that the size of particle is directly related to the specific surface area and porosity of the solid medium. In general, big particles have less surface area leading to poor accessibility to the microorganism, whilst small particles having larger surface area are preferred for high production yields. Furthermore, small particles provide a higher solid medium density and decrease the void fraction, which are both favorable for anaerobic fermentation. However, too small particles lead to medium compaction which may diminish the heat transfer and carbon dioxide exchange rates. Therefore, an optimum particle size of 0.178–0.853 mm which is corresponding to 20–80 meshes has to be considered for fermentation of DSSSPs. 3.3. Simultaneous saccharification and fermentation In the FSSF process, the free sugars were initially converted to ethanol, and the cellulosic part of the residual materials was then subjected to simultaneous saccharification and fermentation. 3.3.1. Effect of FSSF on the following simultaneous saccharification and fermentation The solid residue after the FSSF was identically subjected to simultaneous saccharification and fermentation, and the results were compared to that of fresh DSSSPs (Fig. 2). While the simultaneous saccharification and fermentation of DSSSPs resulted in only 20.0% ethanol yield, the FSSF significantly improved (4.3 fold) the subsequent ethanol production yield. The limiting stage in simultaneous saccharification and fermentation is typically hydrolysis. Free sugars are well known inhibitors for the hydrolytic enzymes
Ethanol yield (% Theorical yield)
100
24 h, 5:10 (FPU:IU) 24 h, 15:30 (FPU:IU)
80
(a)
60 40 20 0 32 100
Ethanol yield (% Theorical yield)
3.2.3. Effect of fungal biomass concentration M. indicus is among the dimorphic fungi and it is possible to change and control its morphology. Generally, using yeast-like morphology of the fungus for fermentation of the solid medium has several technical advantages over the mycelial biomass, e.g., the biomass can be distributed uniformly on the solid surface (Gowthaman et al., 2001). Fermentation of 80% moist DSSSPs was conducted at 32 ◦ C using different fungal biomass concentrations and the most important results are illustrated in Fig. 1c. The ethanol production yield was only 0.22 g/g as the substrate was inoculated with 0.001 g/L fungus (3 × 104 spores/ml). Interestingly, the ethanol yield was markedly increased (1.6 times) using 1 g/L of fungus biomass as inocula and reached the maximum value of 0.48 g/g during fermentation by 5 g/L of the fungal biomass. The results were in contrast to the previous studies, which reported lower ethanol yield by solid state fermentation with higher initial yeast cell concentration, e.g., yeast inoculation rate higher than 0.25 (Shen and Liu, 2008). However, it has been mentioned that the lower ethanol yield could be due to the overuse of fermentable sugars for growth and maintenance at high cell concentrations. Besides, slow heat removal along with too fast fermentation results in heat accumulation and hot spots formation which may consequently decrease the yeast activity. None of the aforementioned effects was observed in the case of fermentation by fungus M. indicus. (Karimi and Zamani, 2013).
35
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24 h, 5:10 (FPU:IU) 24 h, 15:30 (FPU:IU)
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(b)
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37
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Ethanol yield (% Theorical yield)
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24 h, 5:10 (FPU:IU) 24 h, 15:30 (FPU:IU)
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(c)
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Temperature (C) Fig. 3. Ethanol production by simultaneous saccharification and fermentation from 10 (a), 25 (b), and 50 g/L (c) glucan.
(Taherzadeh and Karimi, 2007); thus, the lower hydrolysis and consequently ethanol yield by simultaneous saccharification and fermentation of fresh DSSSPs is most likely attributed to the presence of free soluble sugars in the DSSSPs. On the other hand, the fungus can produced variety of enzymes including xylanase during the aerobic cultivations. Thus, at aerobic conditions, cultivation of the fungus could act as a biological pretreatment for the lignocellulosic part of DSSSPs and make it more digestible for subsequent enzymatic hydrolysis in simultaneous saccharification and fermentation. However, under anaerobic cultivation, the fungus has no effects on lignin and hemicellulosic polymers (Karimi and Zamani, 2013). The yield of ethanol production from untreated sweet sorghum bagasse of the same culture was reported to be less than 50% of theoretical yield, thus a pretreatment stage is necessary to reach higher production yields (Goshadrou et al., 2011). It has been shown that the sodium hydroxide and phosphoric acid pretreatments improved the ethanol yield to about 80%. However, with no pretreatment stage in this work, more than 85% ethanol yield was observed in simultaneous saccharification and fermentation of the solid residue after FSSF using M. indicus.
M. Molaverdi et al. / Industrial Crops and Products 49 (2013) 580–585
3.3.2. Optimization of simultaneous saccharification and fermentation The effects of process parameters including initial glucan concentration, temperature, and enzyme loading on the yield of ethanol production by simultaneous saccharification and fermentation from the bagasse were investigated. The experiments were performed at 32, 35, and 37 ◦ C, with 10, 25, and 50 g glucan/L using 5 and 15 FPU cellulase and 10 and 30 IU -glucosidase per gram glucan. The ethanol yields at 32, 35, and 37 ◦ C were respectively 31.0, 46.3, and 47.0% after 24 h with 10 g/L initial glucan using 15 FPU cellulase and 30 IU -glucosidase per gram of glucan (Fig. 3a). As shown, the yields were correspondingly increased to 48.4, 77.1 and 79.5% by continuation of the process up to 48 h. However, the yields were much less by loading of 5 FPU cellulase and 10 IU -glucosidase per gram of glucan. Likewise, effects of temperature, fermentation time, and enzymes loading in the experiments with 10, 25, and 50 g/L glucan were the same (Fig. 3b and c). The maximum ethanol yield from 10 g/L substrate (79.5%) was almost unaffected as the glucan loading increased to 25 g/L, whilst it was significantly improved to 85.6% for 50 g/L initial glucan concentration. Consequently, the maximum ethanol yield was achieved when 50 g/L glucan was saccharified using 15 FPU cellulase and 30 IU glucosidase per gram of glucan and simultaneously fermented to ethanol at 37 ◦ C for 48 h. The results are in agreement with a previous study on ethanol production from sweet sorghum bagasse by simultaneous saccharification and fermentation using S. cerevisiae (Wang et al., 2013). 4. Conclusions The present study was undertaken to assess the ethanol production from dry sweet sorghum stalk by FSSF using M. indicus followed by simultaneous saccharification and fermentation of the residual solid. Optimal values for FSSF parameters were found to be 5 g/L fungal biomass, 20–80 mesh particle size, 80% substrate moisture, and 32 ◦ C, while the best results for simultaneous saccharification and fermentation were obtained by 50 g/L glucan loading with particle sizes of 20–80 mesh and addition of 15 FPU cellulase together with 30 IU -glucosidase per g glucan at 37 ◦ C for 48 h. Moreover, the FSSF prior to simultaneous saccharification and fermentation significantly enhanced the ethanol production yield from about 20% to more than 85% without any pretreatment and addition of fresh microorganism. Acknowledgment The authors would like to thank Dr. Almodares (Department of Biological Science, University of Isfahan, Iran) for providing the sweet sorghum stalks. References Ab, S., Darah, I., Omar, I.C., 2011. Utilization of palm kernel cake for the production of mannanase by an indigenous filamentous fungus, Aspergillus niger USM F4 under solid substrate fermentation. Internet J. Microbiol. 9 (1), http://dx.doi.org/10.5580/2779. Adney, B., Baker, J., 2008. Measurement of cellulase activities. National Renewable Energy Laboratory, NREL/TP-510-42628.
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