Category Archives: BIOETHANOL

Basic structure

A hybrid cybernetic model can be given in a general form as follows:

Подпись:dx = SxZrMc + V(хш — x)

Подпись: '■ FTN — FndV


where x is the vector of nx concentrations of extracellular components in the reactor (such as substrates, products and biomass), Sx is the (nxxnr) stoichiometric matrix, and Z is the (nrxnz) EM matrix, rM is the vector of nz fluxes through EMs, Fin and Fout are volumetric feed rates at the inlet and outlet, V is the culture volume, xIN is the vector of nx concentrations of extracellular components in the feed. Eq. (1) can also represent batch operation by setting Fin = FnUT = 0 (i. e., V is constant), and fed-batch systems by setting FnUT = 0. In chemostat operations, Fin = FnUT = F, and F/V is often given as dilution rate D. With Z normalized with respect to a reference substrate, rM implies uptake fluxes through EMs. Fluxes through EMs are given as below:

image084 image085 image086 image087

where the subscript j denotes the index of EM, vM, j is the cybernetic variable controlling enzyme activity, eM, j and eMj are the enzyme level and its maximum value, respectively, and rMnj is the kinetic term. Enzyme level eM, j is obtained from the following dynamic equation, i. e.,

where the first and second terms of the right-hand side denote constitutive and inducible rates of enzyme synthesis, and the last two terms represent the decrease of enzyme levels by degradation and dilution, respectively. In the second term of the right-hand side, uM, j is the cybernetic variable regulating the induction of enzyme synthesis, b is the fraction of internal resources (such as DNA, RNA, protein, lipid and other components) involved in the enzyme synthesis process, and rME j is the kinetic part of inducible enzyme synthesis rate. In the third and fourth terms, pMj and ji are the degradation and specific growth rates, respectively.

Подпись: “M , j Подпись: :_PL_ . Z Pk' k Подпись: JM, j image091 Подпись: (4)

The cybernetic control variables, uMj and vM, j are computed from the following the "Matching Law" and the "Proportional Law"(Kompala et al., 1986; Young & Ramkrishna, 2007), respectively:

where the return-on-investment Pj denotes the carbon uptake flux through the jth EM.

The structure of HCMs is illustrated using Fig. 2.1. In this tutorial example, we get three EMs from the network. The uptake flux is split into three individual fluxes thorough EMs, which are catalyzed by enzymes Ej, E2 and E3, respectively. HCMs view that the uptake fluxes are optimally distributed (by the cybernetic variables u and v) among three EMs for maximizing a metabolic objective function (such as the carbon uptake flux or growth rate). The uptake and excretion rates are represented by nonnegative combinations of individual fluxes through EMs.

“Biomass to ethanol” process and review of improvements

The general scheme of "Biomass to ethanol" process is presented elsewhere in this book. Our purpose in this section is to highlight the numerous and various ways to optimize the whole process from biomass to ethanol at different steps: choice of the biomass, pretreatment, enzyme productions, enzymatic hydrolysis, and ethanol fermentation. First of all, as discussed earlier, in the second generation of biofuels, biomass collection should not compete with food plants. Biomass should be abundant and cultural practices as

sustainable as possible. Interest was recently focused on plants providing good yields of biomass for a given surface as the tall Miscanthus. Reduction of lignin cell wall content is another interesting approach to enhance sugar recovery from biomass, lignin being an abundant and resistant polymer limiting the digestion of biomass in biofuels processes. With anti-sense technology, tobacco plants lines were obtained with 20% lower lignin content (Kavousi et al., 2010). The modified lines displayed a threefold increase of saccharification efficiency compared to wild type. Of course, the application of such studies in larger scales depends on the acceptance of transgenic plants by the society. Decision to use these plants has to be supported by studies of environmental risks and potential benefits (Talukder, 2006). Literature about pretreatment is very abundant, describing various methods: physical, chemical or combination of both (Soccol et al. 2010). Fine optimization of conditions should be performed individually depending on biomass. Among innovative method proposed, dry wheat straw has been treated successfully with supercritical CO2. After treatment, 1kg biomass yields to 149 g sugars (Alinia et al., 2010). Another currently emerging feature for bioethanol process amelioration is protein engineering. For instance, a cellulase from the filamentous bacterium Thermobifida fusca has been modified both in its catalytic domain and in its carbohydrate binding module (Li et al., 2010). A mutant enzyme displays a two fold increase activity, and a better synergy with other enzymes, leading it to be very useful for biomass digestion. At the next step, i. e. sugar fermentation to ethanol, many efforts have been run to allow yeast to perform both hexoses and pentoses fermentations. Industrial yeast Saccharomyces cerevisiae strains, fermenting only hexoses have been modified by addition of xylose degradation enzymes (Hector et al., 2010). Finally the outcome of engineering could be the use of synthetic biology, which is creating cell systems able to convert biomass to sugars and also to ferment them to ethanol. This strategy needs better fundamental knowledge to be developed (Elkins et al., 2010).

As discussed above, the step following the pretreatment of the biomass could be performed via the enzymatic hydrolysis of the cell wall polysaccharides into fermentescible, monomeric sugars. Unfortunately, it is well known that recalcitrance of plant cell wall to enzymatic digestion impairs the process. The behavior and the efficiency of the cell wall degrading enzymes (CWDE) in situ and in vitro with isolated polysaccharides are completely different. The properties of the CWDE, as conformation, hydrophobicity, capacity of adsorption onto the cell wall, interaction with the lignins, and catalytic efficiency in heterogeneous catalysis, are major parameters which should be considered and studied.

This chapter focuses on biomass degradation enzymes. What is the best strategy to produce the most efficient enzymes? What is the best choice depending of up — and downstream steps: commercial enzyme cocktail, enzymes produced by a given microorganism or heterologous production of individual enzymes? Efficiencies and cost of enzymes, two bottlenecks in the process, will be discussed. For some authors, the improvements of the conversion of biomass to sugar offer larger cost-saving potential than those concerning the step from sugar to biofuels (Lynd et al., 2008). These authors evaluated two scenarios; the first based on current technology and the second one including advanced nonbiological steps. In both cases, conversion of polysaccharides from biomass could be improved by increasing polysaccharides hydrolysis yields combined by lowered enzyme inputs. On-site enzyme production was also identified as beneficial for cost of the whole ethanol production process.

Lesson learned from Thai cassava bioethanol industry

The ethanol industry in Thailand has been active since 1961 as one of the Royal Project of His Majesty the King. Later, as an oil-importing country, Thailand has lost economic growth opportunity and energy security due to limited oil supply and price fluctuation. The seek for alternative energy for liquid fuel uses for transportation sector has been developed as a part of National Energy Policy and ethanol was then upgraded as national policy in 1995, initially in order to replace a toxic Octane Booster, i. e. Methyl tert-butyl ether (MTBE) in gasoline. By that time, the consumption rate of gasoline was 20 million liters per day which required 10% Octane Booster or 2 million liter per day; this formula is equivalent to Gasohol E10 (for octane 91 and 95), a blend of unleaded gasoline with 10% v/v anhydrous ethanol. With a rising concern of Global Warming and Clean Development Mechanism (CDM), gasohol containing higher ethanol components has been currently developed; E20 & E85. Presently, there are 47 factories legally licensed to produce biofuel ethanol with a total capacity of 12.295 million liters/day or 3,688.5 million liters per annum (at 300 working days). Two feedstocks, namely sugar cane molasses and cassava are their primary raw materials. A total of 40 factories use only a single feedstock; 14 factories using molasses with a total production capacity of 2.485 million liters/day, 25 factories using cassava with a total production capacity of 8.590 million liters/ day and 1 factory using sugar cane with a total production capacity of 0.2 million liters/ day. A multi-feedstock process using both molasses and cassava is, however, preferred in some factories (7 factories with a total production capacity of 1.020 million liters/day) to avoid feedstock shortage (Department of Alternative Energy Development and Efficiency, DEDE, 2009). A complication of Thai bioethanol industry is generated due to the fact that there are two feedstock types being used in other industries and also other alternative energy for transportation, i. e. LPG (Liquefied petroleum gas) and CNG (Compressed natural gas), being promoted by the government.

Alkali pretreatment

Unlike other pretreatments, the use of strong alkali delignifies biomass by disrupting the ester bonds of cross-linked lignin and xylans, resulting in cellulose and hemicellulose enriched fraction. Alkali pretreatment processes generally utilize lower temperatures, pressures and residence times compared to other technologies (McIntosh & Vancov, 2010).

The main compounds used as pretreatment agents in alkali processes are: sodium hydroxide, ammonia and lime, because of their comparatively lower cost and the possibility of chemical and water recycling (McIntosh & Vancov, 2010). Usually two temperature conditions are used for hydrolysis: mild (60°C) or high (121°C).

Hydrolysis of LCB polysaccharides for the ethanol production

Bioethanol production from LCB includes basically the following steps: (1) hydrolysis of cellulose and hemicelluloses; (2) separation of released sugars from lignin residue (3) fermentation of sugars; (4) recovery and purification of ethanol to meet final specifications. The hydrolysis (saccharification) is one of the most important steps and is technically difficult to perform due to the poor accessibility of cellulose caused by many physical, chemical and structural factors mentioned above. It is an energy consuming task, contributing substantially to the economic costs of the process and is a subject of many research works (Mussatto et al. 2004; Sanchez et al. 2008; Alvira et al. 2010; Sannigrahi et al. 2010). Hydrolysis can be carried out using organic or strong inorganic acids or enzymes as cellulases and hemicellulases. Some characteristics of different conventional and prospective hydrolytic processes are summarised in Table 1.


Hydrolytic processes

Dilute acid

Concentrated acid


Yield of sugars, %

ca 50


ca 50

Acid consumption



Reactivity of hydrolysis lignin




Technological status

Commercial in former USSR

Pilot scale

Pilot scale

Table 1. Process conditions and properties for different hydrolytic processes

Organic acids, mainly acetic and formic acids, are normally used in the autohydrolysis process and arisen upon hydrothermal treatment of LCB at high temperatures (170-220 °С) as the result of partial degradation of macromolecular components (acetylated xylan/ mannan and lignin). These relatively weak organic acids at low concentration are more effective in the hydrolysis of hemicelluloses to a significant extent than of cellulose. Consequently a pre-hydrolysis step is widely used in the production of dissolving pulps by kraft cooking when wood chips are processed prior to pulping by hydrothermal treatment to eliminate significant part of hemicelluloses (Sjostrom 1993). The pre-hydrolysis is also a part of pretreatment strategies aiming to hydrolyse selectively the hemicelluloses in LCB to obtain fermentable sugars and/or to improve cellulose accessibility towards hydrolytic enzymes. In this process, the monomeric sugars from hemicelluloses (xylose, galactose, glucose, mannose, and arabinose) and acetic acid are released in the medium (Lawford et al. 1993; Sanchez et al. 2008). Additionally, degradation of lignin/tannins and sugars originate biologically toxic compounds: gallic acid, syringic acid, pyrogallol, vanillic acid, furfural, 5- hydroxymethylfurfural, among others (Marques et al. 2009). Significant efforts were done to minimize the production of such highly toxic compounds, as well as acetic acid, for ethanol — producing microorganisms. The pretreatment should improve recovery of sugars from hemicelluloses, facilitate the cellulose hydrolysis step (when the main objective is the complete saccharafication of all polysccharides from LCB), and avoid the formation of inhibitors for subsequent fermentation processes (Mussatto et al. 2004; Alvira et al. 2010; Sannigrahi et al. 2010).

Inorganic acids (mainly H2SO4 and HCl) are effective hydrolysis catalysts and allow complete saccharification of LCB polysaccharides. There are some differences between the use of diluted (1-5%) and concentrated acids in the hydrolysis step. In the first case the complete saccharification takes place at high temperatures (160-180 °C) and leads to the formation of residual hydrolysis lignin (cellolignin) as a massive by-product (Sanchez et al. 2008). Due to drastic reaction conditions, sugars are readily degraded via intramolecular dehydration resulting in furfural from pentoses and 5-hydroxymethylfurfural from hexoses. All of these secondary products have a high inhibitory effect on the metabolism of microorganisms. In order to avoid sugars degradation, these compounds should be continuously removed from the reactor by continuous pumping of "fresh" acidic solution through the biomass bed (percolation hydrolysis). This process is used industrially since 1930th in former USSR and nowadays may be considered outdated due to its poor efficiency: low sugars recovery and production of high amounts of chemically inert hydrolysed lignin. The hydrolysis with concentrated acids (50-70% of H2SO4 or 30% HCl) allows for effective saccharification of LCB at moderate temperatures (30-80 °С) for short reaction time with high sugars yield. However, due to the technical difficulties and high consumption of acid, this hydrolysis method is not commercialized yet and is implemented only on pilot scale.

The hydrolysis of polysaccharides by hydrolytic enzymes (cellulases and hemicellulases) is one of the most promising tools for the saccharification of LCB. Hydrolytic enzymes permit highly selective hydrolysis of polysaccharides at relatively low temperatures (30-60 °С), practically without emission of products from sugars degradation. Endo-cellulases break internal bonds to disrupt both the amorphous and the crystalline structures of cellulose, exposing its polysaccharide chains. Exocellulase cleaves two to four units from the ends of the exposed chains produced by endocellulase, while |3-glucosidase hydrolyses the exocellulase product into individual monosaccharides. Since no degradation of glucose occurs, more sugars could be available for a subsequent fermentation, which is the main advantage of this process. However, this process is slower when compared with acidic hydrolysis and hydrolytic enzymes have poor accessibility to polysaccharides of cell wall, especially cellulose. For these reasons this process is time consuming and results in low sugar yields. LCB enzymatic hydrolysis needs a preliminary treatment step to improve the accessibility of enzymatic attack. This preliminary step includes the application of physical methods (mechanical, hydrothermal, etc.) to disintegrate plant tissues and chemical/biochemical treatments to eliminate concomitant biopolymers, mainly lignin and hemicelluloses, hindering the cellulose accessibility. However, the enzymatic efficiency of cellulose conversion still needs to be improved.

The poor efficiency of mild acidic hydrolysis and, particularly, enzymatic biotreatment for direct saccharification of LCB, represents an obstacle for a successful production of second — generation biofuels. For this reason, the development of pretreatment techniques to improve cellulose accessibility and saccharification efficiency is a permanent challenge (Sanchez et al. 2008). A general perspective scheme for LCB conversion into ethanol is presented in Figure 6. The first step presumes LCB pretreatment invoked to degrade strong woody biomass matrix and thus blows away the integral tissues. Different lignocellulosic materials have different physic and chemical characteristics and consequently it is necessary to adopt a specific pretreatment suitable for each raw material. The selected pretreatment will have a determinant effect in the subsequent steps. The amount and type of simple sugars released, toxic compounds formed and their concentration, as well as the overall energy demand and wastewater required in the treatments, depend directly on the specific pretreatment applied (Mussatto et al. 2004; Alvira et al. 2010). Several methodologies for biomass pretreatments have been developed during the last decades. They can be classified into biological (using brown, white and soft-rot fungus or their lignolytic and cellulolytic enzymes to degrade lignin and hemicelluloses), physical (mechanical milling and extrusion), chemical (alkali or
acid pretreatments, ozonolysis, organosolv and pretreatment with ionic liquids) and physicochemical (steam explosion, hydrothermal treatment, ammonia fibre explosion, wet oxidation, microwaves, ultrasound and CO2 explosion) (Balat et al. 2008; Alvira et al. 2010; Sannigrahi et al. 2010). LCB pretreatment leads to partial or major removal of hemicelluloses in the form of mono — or oligosaccharides. Then, cellulose is prepared for the hydrolysis step, if the objective is fermentation of glucose from cellulose, or for further processing to obtain pulps for textile and paper products (Fig. 6). This extra step (dashed) can be catalysed by dilute or concentrated mineral acids or enzymes (cellulases).

Подпись: Bioethanolimage056Biomass

image057 Подпись: Cellulose Подпись: Detoxification image060 image061

Fig. 6. Schematic steps for production of bioethanol from lignocellulosic biomass

Until now, fuel ethanol from LCB is not yet considered a viable alternative, mainly due to the high complexity involved on this process, compared with the cheaper oil derived fuels. However, in the last years, with the oil crisis, environmental concerns and the increased need for energy and fuels, bioethanol has become a realistic option in the energy market (Cardona et al. 2007; Sanchez et al. 2008). New research has been developed in order to overcome cellulosic to ethanol bioconversion problems and to make this process a cost — effective technology, with a process integration that combines different steps into one single unit (Lawford et al. 1993; Cardona et al. 2007). Furthermore, the process integration in other industrial plants, namely large scale industries, can be a good solution for reducing costs of bioethanol production, such as in pulp and paper mill industries, with the advantage of reduced release of subproducts.

Comparison of batch and continuous reactors

Ethanol productivity curves at standard conditions in batch and continuous systems are collected together in Fig. 4.2 for clear comparison. In general, the productivity of growth — associated products in a chemostat is far higher than in a batch reactor. This is not the case with ethanol production because it is suppressed by growth (Shuler & Kargi, 2002). The ethanol productivity from mixed sugars in batch culture is about two to three times higher than in continuous culture (Fig. 4.2(a)). Meanwhile, the foregoing considerations show that chemostats outperform batch fermenters in ethanol production from glucose alone as cells grow relatively fast (Fig. 4.2(b)). Choice of preculture medium affects ethanol productivity of batch fermentation and its effect is more clearly shown for mixed sugars (Fig. 4.2(a)) than a single substrate (Fig. 4.2(b)).

Fig. 4.2. Performance comparison between batch and continuous systems: (a) fermentation of mixed sugars, (b) fermentation of glucose only. Adapted from Song and Ramkrishna (2010).

Native cellulolytic organisms

The quest for cellulolytic organisms has recently gained increased interest because of the potential to circumvent the cost of enzymes used for cellulose hydrolysis. An ideal host for cellulosic ethanol production should possess certain traits, such as a broad substrate range (utilizing both pentoses and hexoses), high productivity, and tolerance to both ethanol and toxic compounds of lignin (Fischer et al. 2008). In order to identify desirable organisms for cellulosic ethanol production, naturally evolved cellulose-degrading microbes have been characterized from several sources, including the rumen of cattle and the gut of insects, and even from marine environments (Hess et al. 2011). However, most of these microbes cannot be cultivated with synthetic media in the laboratory. Hence, DNA isolates were directly sequenced and putative carbohydrate-hydrolyzing genes were identified (Hess et al. 2011). With this metagenomic approach, identification of microbes suitable for cellulosic fuel production has not been possible, because our current knowledge of the genes is limited.

Well-characterized native cellulolytic organisms include Cellulomonas fimi, Fibrobacter succinogenes, Ruminococcus albus, and C. thermocellum. Among these, C. thermocellum is of considerable importance, because it is recognized as a "cellulose-using specialist" (Zhang and Lynd 2005). Cellulolytic organisms produce many isoforms of the three different cellulases. T. reesei, for example, can secrete five endoglucanases, two cellobiohydrolases, and two ^-glucosidases. Apart from cellulases, these organisms also secrete adhesion proteins like glycocalyx, which enables strong adhesion of the cellulolytic organisms to cellulose (Lynd et al. 2002).

Despite the diversity of cellulolytic organisms, none of these organisms are known to produce ethanol efficiently (Xu et al. 2009). Even as the search for a cellulolytic organism with the ability to produce ethanol continues, another strategy would be to engineer efficient ethanol production into cellulolytic organisms such as Clostridium spp. (Lynd et al. 2005). However, a lack of proper genetic tools for manipulating these uncommon laboratory strains and very limited knowledge of their genotypes have resulted in a need to engineer the cellulolytic ability into efficient ethanol producers such as S. cerevisiae, E. coli and Z. mobilis.

image153 Подпись: Heterologous Expression and Extracellular Secretion of Cellulases in Recombinant Microbes 243










о Oo°e




— Crude Cellulases

— Cellulose

— Ethanologenic Microbes




Direct starch fermentation without enzyme

In the industry whereby ethanol is produced from starch, temperature around 140°C-180°C is applied to cook the starch during hydrolysis using a-amylase prior to liquefaction. This high-temperature completely sterilizes harmful microorganisms and increases the efficiency of saccharification for high ethanol yield (Shigechi et al, 2004a, b). Consequently, this resulted in high energy consumption and added cost to amylolitic enzymes used in the process which ultimately increased the overall production cost. Several methods have been developed to reduce the energy consumption by applying milder liquefaction and/or saccharafication temperatures (Kolusheva and Marinova, 2007; Majovic et al., 2006; Montesinos and Navarro, 2000; Paolucci-Jeanjean et al, 2000) and also by exercising non­cooking fermentation (Shigechi et al., 2004b; Zhang et al, 2010). However, these types of fermentation usually required longer process time and sometimes may demand for additional volume of enzyme to maintain same productivity. The cost of enzyme will upset the total process cost.

To overcome this shortcoming, an alternative method of direct fermentation from starch may be employed to reduce the cost of enzyme. However, there are relatively few fermentation microorganisms that are capable of converting starch directly to ethanol since they do not produce starch-decomposing enzymes. One of the attempts to resolve this problem is by constructing recombinant microbes to coproduce a-amylase and glucoamylase with incorporating low temperature cooking of starch prior to fermentation by many research teams as shown in Table 3.

Several investigators reported that direct fermentation of starch using amylolytic microorganism offers a better alternative to the conventional multistage using commercial amylases for liquefaction and saccharification followed by fermentation with yeast (Abouzied and Reddy, 1986; Verma et al., 2000; Knox et al., 2004). By using this amylolytic microorganism in direct fermentation, the ethanol production cost can be reduced via recycling some of the microorganism back to fermentors, thereby maintaining a high cell density, which facilitates rapid conversion of substrate into ethanol. Furthermore by using cell exhibiting amylolytic activities, unlike using liquid enzyme that needs to be replenished or recycled unless if it is in immobilized system, the cell can multiply and reproduce with the enzymes. Fermentation using recombinant microbes, the starch medium can be prepared at low temperature cooking or uncook as a raw starch.

Another attempt of the direct fermentation without utilising any enzyme is by using co­culture microbes in the process. Instead of having enzyme separated and purified in different processes and subsequently to be used for hydrolysis in another separated process





Source of a — amylase

Source of glucoamylase

Type of starch




Max. ethanol concentration


Altinta§ et

al (2002)



Bacillus subtilis



Pure starch in 2.5 L fedbatch



Ulgen et al. (2002)



Bacillus subtilis




5- 80

47.5 (fed- batch culture)

15.6(batch culture)

Knox et al. (2004)







Pure starch




Shigechi et al. (2004a)






Rhizopus oryzae

Corn starch cook at 80° C





Shigechi et al. (2004b)





Rhizopus oryzae

Raw corn starch in shake flask

200 g/L total sugar


Oner et al. (2005)

Respiration — Deficient Recombinant S.


Bacillus subtilis




5% starch + 0.4% (wt/vol) glucose


Khaw et al. (2007)

S. cerevisiae (non — and flocculent)

Not stated

Not stated

Raw corn starch



Kotaka et al. (2008)

S. cerevisiae (Sake yeast strain)

Not required

Aspergillus oryzae Rhizopus oryzae

Corn starch



He et al. (2009a)



Not required



Raw Sweet potato





Table 3. Recombinant microbes for direct fermentation at low cooking temperature.

which contribute to higher expense, co-culture fermentation is worth to be considered as it might reduce the cost by omitting the unnecessary steps. While recombinant microorganism is constructed to provide the amylase activities, co-culture is simply selecting the microorganisms that naturally possess these amylase activities and combine them to work together to produce ethanol from starch.

Not many research works dedicated and related to co-culture fermentation for direct bioconversion of starch to ethanol. From just a few, same conclusions were drawn on the fermentation yield of the co-culture was better compared to mono-culture with improvement in the ethanol fermentation process. For instance study done by Verma et al. (2000), the co-culture fermentation of liquefied starch to ethanol can be carried out effectively with fermentation efficiency up to 93% compared to 78% and 85% when two-step bioconversion process using a-amylase and glucoamylase were applied to hydrolyze starch. Abuzied and Reddy (1986) observed that higher cell mass was produced in monoculture than in co-cultures which suggesting that substantially more carbon is used for cell production in monoculture, whereas in the co-culture most of the substrate carbon is utilized for ethanol production. Studies on co-culture microorganisms and systems are summarized in Table 4. The co-culture fermentation can either be simultaneous or subsequent mode for direct fermentation of low-temperature-cooking starch.

Strains for co-culture fermentation can also be obtained inexpensively from dry starter such as Ragi Tapai or Ragi Tape. This is similar to other oriental starter such as Ragi in Malaysia and Indonesia, Bubod in Philipine, Loog-pang in Thailand, Nurok in Korea, Koji in Japan, Banh Men in Vietnam, Chinese yeast in Taiwan and Hamei and Marcha in India. It is a dry — starter culture prepared from a mixture of rice flour and water or sugar cane juice/extract (Merican and Yeoh, 2004, Tamang et al., 2007). Clean rice flour is mixed with water or sugar cane juice to form thick paste. Sometime spices such as chilies, pepper, ginger and garlic which are assumed to carry desirable microorganism or may inhibit the development of undesirable microorganism are added to the paste (Basuki et al., 1996; Merican and Yeoh, 2004). Then the thick paste is shaped into hemispherical balls. Ragi from previous batch is inoculated either on thick paste before or after it is shaped into hemispherical balls. Hesseltine et al. (1988) reported that at least one yeast and one Mucoraceous mold (Mucor, Rhizopus, and Amylomyces) were present with one or two of cocci bacteria in every sample of the dry starter. Apart from the Rhizopus sp. which capable of producing lactic acid besides fermentable sugar and ethanol (Soccol et al., 1994), lactic acid bacteria are among the integral of the dry starter such as Pediococcus pentosaceus, Lactobacillus curvatus, Lactobacillus plantarum and Lactobacillus brevis (Sujaya et al., 2002; Tamang et al., 2007).

The traditional fermented food of tapai or tape’ usually contains ethanol at concentration of 1.58% with high sugar content at concentration of 32.06%. Microaerophilic condition is required for the fermentation condition since fungi are unable to grow under anaerobic conditions and will result in unhydrolyzed starch. At lower temperature of 25°C, higher alcohol content will be produced after 144 h whereas at temperature of 37°C the tapai produces higher sugar content and becomes sweeter. (Merican and Yeoh, 2004). Tapai may contain up to 5% (v/v) of ethanol concentration (Basuki et al., 1996).

The benefit of using strains from dry starter such as ragi is that its application to produce fermented food such as tapai, is proven edible. Moreover, with addition of S. cerevisiae into the medium, the residue from ethanol recovery will contain yeast extract which can be processed as animal feed since it is edible and contain valuable nutrient that suitable for animal consumption as compared to fermentation using microbe such as Escherichia coli. Direct fermentation has several advantages. First, to have multistage processes carried out in one reactor in which the glucose is produced during saccharification and simultaneously is fermented to ethanol can reduce contaminations and process handling cost. Second, direct fermentation reduces energy consumption. The starch medium can be prepared either at low-cooking temperature or by using the raw starch (uncooked starch). Even though some aseptic chemical or method may be required especially in raw starch fermentation, the cost incurred is still lower than the cost of energy consumption used in conventional fermentation.

Third, by applying direct fermentation, it is able to reduce inhibition of reducing sugar on fermenting yeast. In conventional fermentation, when starch is hydrolyzed using enzyme or mineral acid, certain amount of reducing sugar will be produced depending on the starch concentration. High level of reducing sugar in the fermentation medium (above 25% (w/ v)) exerts osmotic pressure to the cells and limits their fermenting activity. This value may vary with different fermenting yeasts. However in direct fermentation, the osmotic pressure can be reduced by simultaneous converting starch to sugar and sugar to ethanol. This is particularly true in the recombinant clone which can co-express both the degrading enzymes. In the case of co-culture fermentation, the suitable inoculation time for the second microorganism needs to be determined. This is to avoid high yield of reducing sugar in


1st microorganism







Type of starch and





Hyun and Zeikus (1985)





14 L


5 % Starch with TYE medium (contains


solution, ammonium chloride, magnesium chloride and trace mineral)

>120 mM

Abouzied and Reddy (1986)

Aspergillus niger



Simultaneous co-culture (500 mL shake flask)

Potato starch recovered from waste water of a potato chip manufacturing plant. (5% (w/v) starch)


Abouzied and Reddy (1987)





Co-Culture fermentation (500 ml shake flask)

Similar to Abouzied and Ready (1986)


Reddy and Basappa (1996)

Endomycopsis fibuligera NRRL 76

Zymomonas mobilis ZM4

Shake flask

22.5% (w/v) cassava starch

10.5% (v/v)

Jeon et al. (2007)

Aspergillus niger



Separate fermentation in serial

bioreactors (1.5 — 3.0 L).

Potato starch 55 g/L/day

22 g/L/day

He et al. (2009b)

Paenibacillus sp.




vs. subsequently co-cultured at 48 h of

fermentation time. (100 mL shake flask)

50.0 g/L raw sweet potato starch (5% w/v starch)

6.6 g/L (120 h

fermentation, pH 6.0) From






Rhizopus sp.



Subsequently co-culture at 24, 48 and 72 h.


14.36 g/L at 24 h



Table 4. The co-culture microorganisms in direct fermentations without enzyme addition.

medium before the second inoculation. When reducing sugar inhibition is avoided, fermentation of high starch concentration can be achieved for high ethanol yield and thus it reduces the water use. Subsequently this will reduce energy consumption in ethanol-water separation.

Direct fermentation is not limited to starch as it had been reported that different sugars from lignocellulosic hydrolysates such as mixture of glucose and pentose sugar for instance; xylose (Murray and Asther, 1984; Kordowska and Targonski, 2001; Qian et al., 2006) were fermented by glucose and pentose-fermenting microorganisms.

Material distribution

At present, sugar is produced following the three stage boiling technology or the three and a half stage boiling technology. It takes a long time and high energy consumption to boil the B sugar and C sugar. The value the by-product is low. There are high costs and weak adaptability to the market.

Generally, it is advantage to regulate sugar production and ethanol production according to market demand the flexibility while applying the "Simultaneous production of sugar and
ethanol" mode. It is necessary to distribute the raw material fluxes rationally. However, less literature is related to juice and syrup distribution for simultaneous production of sugar and ethanol. In this paper, material fluxes balance calculation is carried out according to Brazil experience and the parameters of three and a half stage boiling process. The feed syrup is 60 Bx, the purity is 87%, and the feed syrup fluxes are 100 tons. The sugar combined fuel ethanol process is showed as Sugarcane for simultaneous production of sugar and ethanol

In China, biotechnology research and genetic improvement have led to the development of strains which are more resistant to disease, bacteria, and pests, and also have the capacity to respond to different environments, thus allowing the expansion of sugarcane cultivation. The leading sugar enterprise in charge for applied research on agriculture, together with research developed by state institutes and universities. Efforts have been concentrated in taking advantage of its genetic diversity and high photosynthetic efficiency characteristic, high separation sugarcane population was generated via distant hybridization technology. To obtain the new material of sugarcane for ethanol, we took total biomass, total fermentable sugars as targets and adopted advanced photosynthetic efficiency living early — generation determination technology, molecular markers and cell engineering technology combined with conventional breeding. Then, in order to optimize the selection of energy sugarcane, we took a series of pilot test and technical and economic indexes of evaluation. By 2010, more than 10 sugarcane varieties for simultaneous production of sugar and ethanol are cultivated in China, such as "00-236", "FN91-4710","FN94-0403", FN95-1702","G94-116",
"Y93-159", "Y94-128", "G-22" et al.. Although potential benefits are high, there is still a lack of understanding of the potential impacts of genetically modified organisms on environmental parameters.


Fig. 5. Sugarcane for simultaneous production of sugar and ethanol

Use of residues for energy supply

The stillage from distillation can be separated in a liquid-solid separation step into two fractions. The solid fraction is usually used for solid fuel production. The liquid fraction is either fed to an anaerobic digestion process, generating biogas with a methane concentration of about 60% (Prakash et al., 1998) or is used for solid fuel production together with the solid fraction after evaporation of most of the water. In this case the concentrated liquid fraction is mixed with the solid fraction before drying and pelletizing.

Biogas is used for heat generation or combined heat and power generation for the bioethanol process, whereas solid biofuels can also be sold on the market.