Economics and environmental impact of bioethanol
production technologies: an appraisal (Part -2)

Anuj Kumar Chandel1,2, Chan ES3, Ravinder Rudravaram1, M. Lakshmi Narasu2, L.Venkateswar Rao1 and Pogaku Ravindra3*
Simultaneous saccharification and fermentation (SSF)
The most important process improvement made for the enzymatic hydrolysis of biomass is the introduction of simultaneous saccharification and fermentation (SSF), which has been improved to include the co-fermentation of multiple sugar substrates (Figure 4) (Sreenath et al., 2001; Wingren et al., 2003). This approach combined the cellulase enzymes and fermenting microbes in one vessel. This enabled a one-step process of sugar production and fermentation into ethanol. Simultaneous saccharification of both carbon polymer, cellulose to glucose; and hemicellulose to xylose and arabinose; and, fermentation will be carried out by recombinant yeast or the organism which has the ability to utilize both C5 and C6 sugars. According to Alkasrawi et al. (2006) the mode of preparation of yeast must be carefully considered in SSF designing. A more robust strain will give substantial process advantages in terms of higher solid loading and possibility to recirculate the process stream, which results in increased energy demand and reduced fresh water utilization demand in process. Adaptation of yeast to the inhibitors present in the medium is an important factor for consideration in the design of SSF process. Morerecently, Kroumov et al. (2006) demonstrated an unstructured model of SSF of starch to ethanol by genetically modified strain Saccharomyces cereviseae YPB-G, using two hierarchic levels of concept. In first concept, a mechanism of enzymatic hydrolysis of starch to glucose by combined action of two enzymes (alphaamylase and glucoamylase) secreted by recombinant yeast and the second concept was the enzymatic degradation of starch to glucose and simultaneous utilization of glucose to ethanol by microorganisms. SSF combines enzymatic hydrolysis with ethanol fermentation to keep the concentration of glucose low. The accumulation of ethanol in the fermenter does not inhibit cellulase action as much as high concentration of glucose; so, SSF is good strategy for increasing the overall rate of cellulose to ethanol conversion (Lin and Tanaka, 2006). SSF gives higher ethanol yield while requiring lower amounts of enzyme because end-product inhibition from cellobiose and glucose formed during enzymatic hydrolysis is relieved by the yeast fermentation (Banat et al., 1998). However, it is not feasible for SSF to 020 Biotechnol. Mol. Biol. Rev.

Figure 2. Concentrated acid hydrolysis and separate pentose and hexose sugars fermentation.

meet all the challenges at industrial level due to its low rate of cellulose hydrolysis and most microorganisms employed for ethanol fermentation can not utilize all sugars derived after hydrolysis. To overcome of this problem, the cellulolytic enzyme cocktail should be more stable in wide range of pH and temperature. Also the fermenting microorganisms (yeasts or bacteria) should be able to ferment a wide range of C5 and C6 sugars. Recently Matthew et al. (2005) has found some promising ethanol producing bacteria viz. recombinant E. coli K011, Klebsiella oxytoca, and Zymomonas mobilis for industrial exploitation. SSF process has now improved after including the co-fermentation of multiple sugar substrates present in the hydrolysate. This new variant of SSF is known as simultaneous saccharification and co- fermentation (SSCF) (Wilke et al., 1976; Patel et al., 2005; Wyman et al., 2005). SSF and SSCF are preferred over SHF, since both operations can be performed in the same tank resulting in lower cost, higher ethanol yield and shorter processing time (Wright et al., 1988). The most upgraded form of biomass to ethanol conversion is consolidated bioprocessing (CBP) - featuring cellulose production, cellulose hydrolysis and fermentation in one step-is a highly integrated approach with outstanding potential (Lynd et al., 2005). It has the potential to provide the lowest cost route for biological conversion of cellulolosic biomass to ethanol with high rate and desired yields.

Figure 3. SHF with separate pentose and hexose sugars and combined sugar fermentation.

Figure 4. SSF with combined sugars (pentoses and hexoses) fermentation.

Direct microbial conversion (DMC)
DMC is a method of converting cellulosic biomass to ethanol in which both ethanol and all required enzymes are produced by a single microorganism. The potential advantage of DMC is that a dedicated process step for the production of cellulase enzyme is not necessary. Cellulase enzyme production (or procurement) contributes significantly to the cost involved in enzymatic hydrolysis process. However, DMC is not considered the leading process alternative. This is because there is no robust organism available that can produce cellulases or other cell wall degrading enzymes in conjunction with ethanol with a high yield. Singh and Kumar (1991) found that several strains of Fusarium oxysporum have the potential for converting not only D-xylose, but also cellulose to ethanol in a one-step process. Distinguishing features of F. oxysporum for ethanol production in comparison to other organisms are identified. These include the advantage of in situ cellulase production and cellulose fermentation, pentose fermentation, and the tolerance of sugars and ethanol. The main disadvantage of F. oxysporum is its slow conversion rate of sugars to ethanol as compared to yeast.
Fermentation
Bioconversion of various biomass sources into ethanol by different microorganisms has been summarized in Table 2. The sugar syrup obtained after cellulosic hydrolysis is used for ethanol fermentation. The ability to ferment pentoses along with hexoses is not widespread among microorganisms (Toivolla et al., 1984), S. cereviseae is capable of converting only hexose sugars to ethanol. The most promising yeasts that have the ability to use both C5 and C6 sugars are Pichia stipitis, Candida shehatae and Pachysolan tannophilus. However, ethanol production from sugars derived from starch and sucrose has been commercially dominated by the yeast S. cereviseae (Lin and Tanaka, 2006). Thermotolerant yeast could be more suitable for ethanol production at industrial level. In high temperature process energy savings can be achieved through a reduction in cooling costs. Considering this approach, Sree et al. (1999) developed solid state fermentation system for ethanol production from sweet sorghum and potato employing a thermotolerant S. cereviseae strain (VS3).
Researches are now focusing on developing recombinant yeast, which can greatly improve the ethanol production yield by metabolizing all form of sugars, and reduce the cost of operation. In this contention the researchers have made efforts by following two approaches. The first approach has been to genetically modify the yeast and other natural ethanologens additional pentose metabolic pathways. The second approach is to improve ethanol yields by genetic engineering in microorganisms that have the ability to ferment both hexoses and pentoses (Jeffries and Jin, 2000; Dien et al., 2003; Katharia et al., 2006). Jeffries and Jin (2004) compiled the recent developments happened towards the genetic engineering of yeast metabolism and concluded that strain selectionthrough mutagenesis, adaptive evolution using quantitative metabolism models may help to further improve their ethanol production rates with increased productivities. Piskur et al., (2006) showed the recent developments in comparative genomics and bioinformatics to elucidate the high ethanol production mechanism from Saccharomyces sp.
Though new technologies have greatly improved bioethanol production yet there are still a lot of problems that have to be solved. The major problems include maintaining a stable performance of genetically engineered yeast in commercial scale fermentation operation (Ho et al., 1998, 1999), developing more efficient pre-treatment technologies for lignocellulosic biomass, and integrating optimal component into economic ethanol production system (Dien et al., 2000). Sridhar and co-workers (2002) made an effort to improve the thermo tolerance of yeast isolates by treating them with UV radiation. Fermentation can be performed as a batch, fed batch or continuous process. The choice of most suitable process will depend upon the kinetic properties of microorganisms and type of lignocellulosic hydrolysate in addition to process economics aspects.
Batch fermentation
Traditionally, ethanol has been produced batch wise. At present, nearly, all of the fermentation ethanol industry uses the batch mode. In batch fermentation, the microorganism works in high substrate concentration initially and a high product concentration finally (Olsson and Han- Hagerdal, 1996). The batch process is a multi-vessel process, allows flexible operation and easy control over the process. Generally batch fermentation is characterized by low productivity with an intensive labour (Shama, 1988). For batch fermentation, elaborate preparatory procedures are needed; and because of the discontinuous start up and shut down operations, high labour costs are incurred. This inherent disadvantage and the low productivity offered by the batch process have led many commercial operators to consider the other fermentation methods.
Fed batch fermentation
In fed batch fermentation the microorganism works at low substrate concentration with an increasing ethanol concentration during the course of fermentation process. Fed batch cultures often provide better yield and productivities than batch cultures for the production of microbial metabolites. For practical reasons, therefore, some continuous operations have been replaced by fed batch process(Schugerl, 1987).

Table 2. Various raw materials for ethanol production.

Keeping the low feed rate of substrate solution containing high concentration of fermentation inhibitors such as furfural, hydroxymethyl furfural and phenolics, the inhibitory effect of these compounds to yeast can be reduced. Complete fermentation of an acid hydrolysate of spruce, which was strongly inhibiting in batch fermentation, has been achieved without any detoxification treatment (Taherzadeh, 1999). The productivity 024 Biotechnol. Mol. Biol. Rev. in fed batch fermentation is limited by the feed rate which, in turn, is limited by the cell mass concentration. The specific ethanol productivity has also been reported to decrease with increasing cell mass concentration (Lee and Chang, 1987; Palmqvist et al., 1996). Ideally, the cell density should be kept at a level providing maximum ethanol productivity and yield.

Continuous fermentation
Continuous fermentation can be performed in different kind of bioreactors – stirred tank reactors (single or series) or plug flow reactors. Continuous fermentation often gives a higher productivity than batch fermentation, but at low dilution rates which offers the highest productivities. Alexender et al., (1989) studied the effect of shift in temperature and aeration in steady state continuous culture of C. shehatae to determine the effects of ethanol on xylose metabolism. The accu-mulation of ethanol exerted a delayed inhibitory effect on the specific rate of substrate utilization. Continuous operation offers ease of control and is less labor intensive than batch operation. However contamination is more serious in this operation. Since the process must be interrupted, all the equipments must be cleaned, and the operation started again with the growth of new inoculum. The continuous process eliminates much of the unproductive time associated with cleaning, recharging, adjustment of media and sterilization. A high cell density of microbes in the continuous fermenter is locked in the exponential phase, which allows high productivity and overall short processing of 4 - 6 h as compared to the conventional batch fermentation (24 - 60 h). This results in substantial savings in labour and minimizes investment costs by achieving a given production level with a much smaller plant.
Immobilized cells
A limitation to continuous fermentation is the difficulty of maintaining high cell concentration in the fermenter. The use of immobilized cells circumvents this difficulty. Immobilization by adhesion to a surface (electrostatic or covalent), entrapment in polymeric matrices or retention by membranes has been successful for ethanol production from hexoses (Godia et al., 1987). The applications of immobilized cells have made a significant advance in fuel ethanol production technology. Immobilized cells offer rapid fermentation rates with high productivity – that is, large fermenter volumes of mash put through per day, without risk of cell washout. In continuous fermentation, the direct immobilization of intact cells helps to retain cells during transfer of broth into collecting vessel. Moreover, the loss of intracellular enzyme activity can be kept to a minimum level by avoiding the removal of cells from downstream products (Najafpour, 1990). Immobilization of microbial cells for fermentation has been developed to eliminate inhibition caused by high concentration of substrate and product and also to enhance ethanol productivity and yield. Abbi et al., (1996) observed that the rate of sugar consumption by immobilized cells of C. shehatae NCL-3501 was slightly lower than that of free cells, thus leading to higher ethanol production. When microorganisms are attached to solid supports, fluid viscosity is lower which contributes to better mixing and mass transfer in the system. The work on ethanol production in an immobilized cell reactor (ICR) showed that ethanol production using Z. mobilis was doubled. Amutha and Gunasekaran (2001) reported ethanol production, 46.7 g/l from 150 g/l liquefied cassava starch from co-immobilized cells of Saccharomyces diastaticus and Z. mobilis. Yamada et al. (2002) successfully used recombinant Z. mobilis with high sugar concentration (12-15%) and further observed the significant role of increased biomass concentration in bioreactor performance for the improved ethanol production. A repeated batch fermentation system was used to produce ethanol using an immobilized osmotolerant S. cereviseae, in which ethanol concentration as high as 93 g/l was recorded at 200 g/l glucose concentration (Sree et al., 2000). Nigam (2000) has reported that the ethanol production rate as high as 42.8 g/l/h was achieved from the fermentation of pineapple canary derived sugars by S. cereviseae ATCC 24553.

Recycling of process stream
In an environmentally sustainable process, the use of fresh water, the amount of wastewater and the energy consumption must be minimized. The water consumption is decreased by recirculating process streams for use in the washing and hydrolysis steps (Palmqvist and Hahn- Hagerdal, 2000). Recirculating part of the dilute ethanol stream from the fermenter can increase the ethanol concentration in the feed to the distillation stage. However, computer simulations have shown that recirculation of streams leads to the accumulation of nonvolatile inhibitory compounds (Galbe and Zacchi, 1992; Palmqvist et al., 1996).
To increase the ethanol productivity, cell recycling has been employed by several workers (Fein et al., 1984; Maleszka et al., 1981), while retaining the simplicity of thebatch process. Cell recycling generally does not increase the sugar consumption or ethanol production but the time required for the fermentation can be reduced by 60 - 70%. Schneider (1989) observed a reduction in ethanol production after third cell cycle and suggested the decrease in ethanol production was due to the limitations of oxygen and sugar as a result of an increase in cell density.

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