Composition of wheat components in % dry solids.
Ethanol production from agricultural products mainly corn, wheat, sweat potato and residue are gaining importance and requires an industrially viable novel technology namely simultaneous saccharification and fermentation process. This process has an advantage of carrying out saccharification using enzyme and fermentation using yeast in a single fermenter. The investment cost for industrial ethanol production using cheap agricultural residues can be well achieved using SSF process. The success of SSF process greatly depends upon the pretreatment methods using different enzymes to break the complex carbohydrates to simple sugars. Optimization of key process variables is essential to maximize the ethanol yield from suitable substrates. The key process variables affecting the SSF process are pH, temperature, fermentation time, enzyme concentration and substrate concentration. The medium components are to be screened for effective nitrogen, potassium and phosphorous sources to increase the ethanol yield.
- simultaneous saccharification and fermentation
- yeast pH and temperature
The raw materials for ethanol production can be classified based on the type of carbohydrates they contain, i.e., sugar, starch, or cellulose by fermentation. Sucrose, glucose, or fructose for ethanol production for simultaneous saccharification and fermentation process are derived from any of the two classes of raw materials namely, starchy and cellulosic materials .
Ethanol production from simple sugars derived from sugarcane molasses, beet sugar is commercially well established. The yeast or bacterial cells can metabolize the simple sugars directly without the necessity of pretreatment step. The starch and cellulose polymers must be hydrolyzed to simple sugars before they can be fermented by yeast or bacteria [2, 3, 4]. Although cellulosic materials are available in plenty than starchy and sugar-containing raw materials, the process of conversion of it to fermentable sugars is often a very expensive pretreatment step using enzymes [5, 6]. Starch-containing substrates must be hydrolyzed by enzymes or acid to simple sugars and can be used for the production of ethanol. The carbon, hydrogen, and oxygen are normally provided by a complex carbohydrate source such as cane or beet molasses in industries. Vitamins and minerals may be added as additional nutrients. The sources of nitrogen are generally ammonium sulfate and urea, but they require biotin for effective utilization . Other cheaper raw materials such as spent sulfite liquors, and whey also are sources of fermentable sugars. The sugar concentration in the above-mentioned industrial effluents is very much lower than in usual starchy and cellulosic substrates. Spent sulfite liquors contain 20–30 gL−1 of hexose while whey contains 40–50 gL−1 of lactose. Cellulosic raw materials on acid or enzyme hydrolysis give a maximum sugar concentration of around 40–60 gL−1 . Ammonium or potassium phosphate provides the potassium and phosphorous required for growth of yeast. The magnesium sulfate, chloride and biotin can be provided as additional supplements [8, 9]. In a study by Qureshi and Manderson  four renewable agricultural resources were considered, namely wood, molasses, whey permeate, and starch. He reported that molasses sugars were cheaper than sugars derived from the other raw materials.
The simultaneous saccharification and fermentation (SSF) process was conceptualized in the late 1970s by Wright et al., Takagi et al., and Blotkamp et al. [11, 12]. This process employs fermentative microorganisms in combination with amylolytic enzymes in a single fermenter. Sugar accumulation in the fermenter is minimized in this process that favors increased hydrolysis and ethanol yield when compared to separate hydrolysis and fermentation. The main advantage process over separate hydrolysis and fermentation is that high substrate concentration, long residence time and high enzyme concentration can be used in same reactor. Optimization of process variables namely substrate concentration, enzyme concentration, pH and temperature are important to maximize the ethanol yield.
Starches that can be used for ethanol production by fermentation, includes grains, cassava (manioc, tapioca), sweet potato, sweet sorghum, and Jerusalem artichoke, corn, wheat, rice, potatoes, and sugar beets are the mostly used feedstocks in Europe and North America, sugarcane, molasses, cassava, babassu nuts, and sweet potatoes appear to provide the most promising feed for ethanol for countries such as Brazil.
1.1 Substrates for ethanol production using SSF process
According to Miranowski , corn is the most viable feedstock for ethanol production. The main factors are high yield, broad geographical cultivation range and available at cheaper cost. Annual production of corn biomass is about 300 × 106 tons (dry basis), about 40% of which are residues which is suitable for ethanol production. Extremely efficient systems are already in place for corn production from seed at very low cost. In evaluating the potential of corn (and any other food crop) for the production of energy, the moral issue of food vs. fuel must be considered. Approximately 66% of the grain produced consumed as food. The proportion of grain that are unsuitable for food production is about 5% of the annual grain production and it is suitable for alcohol production. In many countries corn is used as a raw material. The suitability of corn for ethanol production using SSF process depends on the contents of starch. A high content of horny endosperm leads to problems in ethanol production using SSF processes. The starch isolated from horny endosperm is difficult to gelatinize, and has low swelling, swelling value, and α-amylase digestibility is very less when compared to floury endosperm. Pre-treatment of horny endosperm is difficult and requires more enzyme concentration.
Wheat is mostly used in distilleries, because it yields a mild and smooth distillate. The starch content of wheat is usually about 60%. Wheat containing more than 13% raw protein causes problems in fermentation. Wheat mashes with high protein forms foam during fermentation and the use of antifoam agent (e.g., silicone anti-foam) is necessary. Table 1 shows the composition of key components in wheat grain and Table 2 shows the average composition of wheat.
|Components||Composition in g/100 g of flour|
|Components||Composition in g/100 g of flour|
1.1.4 Sweet potato
Sweet potato (
1.1.5 Sweet sorghum
Sweet sorghum (
The adaptability to the majority of the world’s agricultural regions, its resistance to draught, and its efficient utilization of nutrients make it as a viable raw material for ethanol production using SSF process .
Barley is mostly used as malting grain in ethanol production. It is also an interesting raw material in ethanol production using SSF process. The disadvantages of barley as a feed stock in distilleries are the husks surrounding the kernels and the content of glucans that leads to high viscosities in mashes. Therefore, special pretreatment step before SSF process is necessary in preparing mashes from barley. Table 4 shows an average analysis of barley. Barley with 55% starch is also a major feedstock for beer production. Potable distillates produced from barley are smooth, but they have a more powerful grain taste.
|Components||Composition in g/100 g of flour|
1.2 Pre-treatment of substrates used in SSF process
1.2.1 Enzymatic liquefaction of starch in SSF process
It is essential to liquefy the starch as a pretreatment step before using the substrate SSF process. Liquefying enzymes are virtually all α-amylases (α-l, 4-glucane 4-glucanohydroase, E.C. 220.127.116.11) that split α-1,4 bonds in amylose and amylopectin that are basically derived from plants, bacteria and fungi. Liquefying enzymes may be classified as endo-acting enzymes and exo-acting enzymes. The α-1,6 glycosidic bonds are not hydrolyzed by alpha amylase since they are endo-acting enzymes. The enzyme activity of α-amylase is majorly dependent on the type of microorganisms or plants from which it is synthesized. α-Amylases rapidly decrease the viscosity due to its endo-acting nature and is used in simultaneous saccharification and fermentation process for pretreatment.
1.2.2 Treatment with α-amylase of
The optimum conditions of pH for enzyme hydrolysis of starch using TBA is between 6 and 7 and the optimum temperature is in the range of 85–90°C . The hydrolysis of corn starch with TBA, mainly produces maltotriose, maltopentaose, and maltohexaose. TBA enzyme is highly unstable and degrade at temperatures above 65°C in absence of Calcium ions and substrate. Senn  established an optimum pH range from 6.2 to 7.5, and pH values below 5.6 lead to a rapid decrease in enzyme activity. Enzyme activity is influenced greatly by the proportion of horny to floury endosperm present in the corn feed stock. Liquefaction of corn mashes using TBA yields mainly starch fragments with a maltotriose as well as maltose and glucose.
1.2.3 Treatment with α-amylase of
BAA synthesized using
1.2.4 Treatment with α-amylase expressed by
BAB, a new technical enzyme produced with a genetically engineered strain of
1.2.5 Treatment with fungal α-amylase of
Fogarty and Kelly , reported that FAA contains only a few amino acid residues and is highly stable in acidic pH. The enzyme activity is maximum in a pH between 5.5–5.9 and at a temperature of 40°C. FAA can hydrolyze starch granules at a pH of 7.2 and temperature of 37°C and only 40% of starch was dextrinized in pretreatment step after 60 hour. The optimum pH ranges from 5.0 to 6.0 while corn is used as a substrate. The optimum temperature is reported between 50 and 57°C. FAA reduces the viscosity which is desirable for saccharification and is more effective in producing dextrins.
1.2.6 Enzymes for starch saccharification in SSF process
Glucoamylase (EC 18.104.22.168) enzyme, hydrolyzes α-1,4, α-1,6, and α-1,3 glycosidic linkages of starch molecules. Hydrolysis rate of starch is based upon the size and structure of the molecules .
1.2.7 Treatment with glucoamylase of
1.2.8 Treatment with glucoamylase of
GAR enzyme shows a maximum activity at temperature of 40°C and a pH value of 4.5–6.3 . Glucoamylase 1 exhibits maximum debranching activity and totally degrades starchy materials to fermentable sugars in SSF process. Saccharification using GAR was carried out in a temperature range of 55–60°C and a pH of 4.4–5.4 ; GAR was also stable in acidic pH while corn is used as substrate.
1.2.9 Enzyme combinations in saccharification process
Single enzymes are rarely used for saccharification process. Enzymes may be combined successfully in mashing processes and fermentation. As reported by , different combinations of technical enzymes may exhibit either complementary or inhibitory effects. “OPTIMALT” is an industrially used enzyme combination off GAR GAA and FAA . The concentration of fermentable sugars in mashes rises rapidly when enzyme combination is used in SSF process.
1.3 Microorganisms for ethanol production using SSF process
The yeast species mainly
Yeasts can utilize a variety of substrates. In general, they are able to grow and efficiently ferment in a pH between 3.5–6.0 and temperature in the range of 28–35°C. The overall productivity of the fermentation was less due to ethanol product inhibition and substrate inhibition . This drawback of substrate inhibition can be overcome in SSF process where simultaneous utilization of substrate by microbes and synthesis of glucose by enzymes at faster rates.
Yeast, under anaerobic conditions, converts glucose to ethanol by the Embden-Meyerhof pathway and is shown in Figure 1. 2 mol of ethanol, CO2, and ATP per mol of glucose fermented were synthesized in this pathway with a yield coefficient of 0.51 g alcohol .
1.4 Simultaneous saccharification and fermentation (SSF) process and key variables
Simultaneous saccharification and fermentation SSF is a process in which sugars from the liquefied substrates are saccharified and fermented in a single fermenter using enzyme and yeast. The drawback of SSF of cellulose using enzymes is feedback inhibition by the product. Separate Hydrolysis and Fermentation uses different temperature for hydrolysis and fermentation but the main disadvantage is the end product inhibition of glucose that accumulates in the hydrolysis step . SSF process overcomes this difficulty of accumulation of sugars inside the fermenter by simultaneous fermentation of sugar by suitable yeast [32, 34]. The flow sheet of the SSF process using corn starch is shown in Figure 2.
Verma et al.  studied the conversion of starch to ethanol in a SSF process using co culture of amylolytic yeast and
Amutha et al.  studied the ethanol from pretreated cassava starch by co-immobilized cells of
Neves et al.  studied the ethanol production from wheat flour by SSF process. SSF process was conducted at 5°C and a controlled pH of 4.5 using glucoamylase 200 U/g of flour and
Davis et al.  studied the production of ethanol using waste starch stream by SSF process using
Nakumara et al 1997  studied the production from raw wheat flour using glucoamylase and
Pavla et al.  had studied the SSF process using wheat bran as substrate. Wheat bran was pre-treated with FAA followed by saccharification using glucoamylase. Pre-treatment temperature for FAA was 55°C and pH 6.0 for 4 hour and saccharification at 55°C for 48 hour to ensure the total hydrolysis of starch. The fermentation of filtrates resulting from pre-treatment using
Reddy et al.  had studied the direct fermentation of potato starch to ethanol by co culture of
SSF process using maize starch as substrate by glucoamylase and
Kadam and Newman  evaluated several industrially available nutrient sources for their effectiveness in the SSF of pretreated starch with
The pH and temperature of the medium plays a vital role in all types of fermentation processes. As temperature increases the rate of biological reactions also increases upto a certain temperature and further increase in temperature may result in lesser product formation. That temperature was always chosen as the optimum temperature for the fermentation. This characteristic is similar to chemical reaction. This increase in rate of biological reaction may be due to more production of required enzymes at the faster rate. The ethanol producing microorganisms such as
|Culture||Source of starch||Process and fermentation conditions||Ethanol concentration gL−1||Ethanol productivity gL−1 h−1||Ethanol yield g/g of starch||Reference|
|Glucoamylase + yeast||cassava||batch fermentation||16.5||0.14||0.49||Ueda et al. |
|Co-immobilized ||Rice||Mini jar fermenter||40||0.18||0.48||Lee et al. |
|Potato||SSF pH—5.5, T—30°C, S—100 gL−1||13.5||0.18||0.135||Reddy et al. |
|Glucoamylase + ||Raw wheat flour||SSF process pH—4.5, T—35°C, S—150 gL−1||60||9.5||0.40||Nakumara et al 1997 |
|Co-immobilized ||liquefied cassava||continuous fermentation pH—6.0, T—30°C, S—150 gL−1||69.6||0.99||0.46||Amutha et al. |
|Cassava||SSF pH—5.5, T—30°C||90||0.5||0.45||Roble et al., (2002)|
|Glucoamylase + ||Raw starch||Fed-batch fermentation pH—5.0, T—30°C||20–30||0.60||0.35||Konda et al. |
|Mutant ||Raw starch||SSF pH—5.5, T—35°C, S—150 gL−1||50||1.42||0.33||Rajoka et al |
|Co-immobilized glucoamylase + ||Sago starch||SSF pH—4.9, T—32°C, S—150 gL−1||55.3||0.98||0.36||Bantaru et al. |
SSF process is found to be a promising technology for industrial ethanol production from cheaper substrates like cellulose and starchy substrates. The success of the SSF process depends mainly on pre-treatment step using suitable enzymes for cellulose hydrolysis and starch hydrolysis. Starchy substrates can be easily liquefied using low cost commercially available alpha amylase enzymes at optimum conditions and can be utilized in SSF process. But the pre-treatment steps in cellulosic materials are more challenging because of the presence of lignin and hemicelluloses. A suitable pre-treatment steps to separate cellulose from naturally occurring lignin and hemicelluloses substrates involves energy intensive process. Furthermore, presence of inhibitory end products from hemicelluloses may hinder the SSF process. SSF process using starch substrates are more promising and also commercial industrial production is feasible in many countries. The advantages of the process are reduction in investment by having single fermenter for both saccharification and fermentation. The feedback inhibition of sugars is greatly reduced. The fermentation time is very less in SSF process.