A biological solution that bypasses the severe practical difficulties posed by growing ethanologens in concentrated solutions of potentially toxic hydrolysates of lignocel — lulosic materials is to replace physicochemical methods of biomass substrate hydro­lysis with enzymic breakdown (cellulase, hemicellulase, etc.) under milder conditions — especially if enzyme-catalyzed hydrolysis can be performed immediately before the uptake and utilization of the released sugars in a combined hydrolysis/fermenta — tion bioprocess. Extrapolating back up the process stream and considering a totally enzyme-based hydrolysis of polysaccharides, an “ideal” ethanol process has been defined to include193

• Lignin removal during pretreatment to minimize unwanted solids in the substrate

• Simultaneous conversion of cellulose and hemicellulose to soluble sugars

• Ethanol recovery during the fermentation to high concentrations

• Immobilized cells with enhanced fermentation productivity

An even closer approach to the ideal would use enzymes to degrade lignin suffi­ciently without resort to extremes of pH to fully expose cellulose and hemicellulose before their degradation to sugars by a battery of cellulases, hemicellulases, and ancillary enzymes (esterases, etc.) in a totally enzymic process with only a minimal biomass pretreatment, that is, size reduction. No such process has been devised, but because pretreatment methods could solubilize much of the hemicellulose, two dif­ferent approaches were suggested in 1978 and 1988 with either cellulolytic microbes (whole cell catalysis) or the addition of fungal cellulase and hemicellulase to the fermentation medium.194 195 These two options have become known as direct micro­bial conversion (DMC) and SSF, respectively.

DMC suffers from the biological problems of low ethanol tolerance by the (usually) clostridial ethanologens and poor ethanol selectivity of the fermentation (see section 3.3.2.5).196 Commercialization has been slow, few studies progressing beyond the laboratory stage. The phytopathogenic[41] fungus Fusarium oxysporium is the sole nonbacterial wild-type microbe actively considered for DMC; the ability of the organism to ferment xylose as well as hexose sugars to ethanol was recognized in the early 1980s, and several strains can secrete cellulose-degrading enzymes.197198 Hemicellulose sugars can also be utilized in acid hydrolysates, although with low conversion efficiencies (0.22 g ethanol per gram of sugar consumed).199 Extensive metabolic engineering of F. oxysporium is, therefore, likely to be required for an efficient ethanologen, and detailed analysis of the intracellular biochemical networks have begun to reveal potential sites for intervention.200-202

Metabolic engineering of S. cerevisiae to degrade macromolecular cellulose has been actively pursued by research groups in South Africa, the United States, Can­ada, Sweden, and Japan; fungal genes encoding various components of the cellulase complex have successfully been expressed in ethanologenic S. cerevisiae, yielding strains capable of utilizing and fermenting either cellobiose or cellulose.203-206 Cal­culations show that, based on the growth kinetics and enzyme secretion by cellulose degraders such as H. jecorina, approximately 1% of the total cell protein of a recom­binant cellulase-secreting S. cerevisiae would be required, perhaps up to 120-fold more than has been achieved to date.207,208

In contrast, SSF technologies were installed in North America in the early 1990s in production plants generating between 10 million and 64 million gallons of ethanol/year from starch feedstocks.122 In addition to starch breakdown and sugar fermentation, the technology can also include the stage of yeast propagation in a cas­caded multifermentor design (figure 4.11). Extensive research worldwide has defined some factors for successful process development:

• If yeasts are to act as the ethanologens, thermotolerant strains would per­form more in harmony with the elevated temperatures at which cellulases work efficiently.209,210

• Bacteria are more readily operated in high-temperature bioprocesses, and recombinant Klebsiella oxytoca produced ethanol more rapidly under SSF conditions than did cellobiose-utilizing yeasts; coculturing K. oxytoca and S. pastorianus, K. marxianus, or Z. mobilis resulted in increased ethanol production in both isothermal and temperature-profiled SSF to increase the cellulase activity.211

• Both K. oxytoca and Erwinia species have the innate abilities to trans­port and metabolize cellobiose, thus reducing the need to add exogenous P-glucosidase to the cellulase complex; moreover, chromosomally integrat­ing the E. chrysanthemi gene for endoglucanase and expressing the gene at a high level results in high enzyme activities sufficient to hydrolyze cellulose and even produce small amounts of ethanol in the absence of added fungal

cellulase.212,213

• Although high ethanol concentrations strongly inhibit fermentations with recombinant E. coli and glucose or xylose as the carbon substrate, SSF with cellulose and added cellulase showed a high ethanol yield, 84% of the theoretical maximum.214

• Simulations of the SSF process to identify the effects of varying the operat­ing conditions, pretreatment, and enzyme activity highlight the importance of achieving an efficient cellulose digestion and the urgent need for contin­ued R&D efforts to develop more active cellulase preparations.215

• Reducing the quantity of cellulase added to ensure efficient cellulose diges­tion would also be beneficial for the economics of the SSF concept; add­ing nonionic surfactants, polyethylene glycol, and a “sacrificial” protein to decrease nonproductive absorption of cellulase to lignin binding sites have also been demonstrated to increase cellulase action so that cellulose diges­tion efficiency can be maintained at lower enzyme:substrate ratios.8216

The importance of the quantity of cellulase added was underlined by a Swedish study that showed that reducing the enzyme loading by 50% actually increased the production cost of ethanol in SSF by 5% because a less efficient cellulose hydro­lysis reduced the ethanol yield.217 At low enzyme loading, there are considerable advantages by growing the yeast inoculum on the pretreated biomass material (bar­ley straw); the conditioned cells can be used at a reduced concentration (2 g/l, down from 5 g/l), and with an increased solids content in the SSF stage.218

The cost of commercially used fungal cellulase has decreased by over an order of magnitude because of the efforts of enzyme manufacturers after 1995.219 Multiple efforts have been made to increase the specific activity (catalytic efficiency) of cel — lulases from established and promising novel microbial sources (see section 2.4.1), and recently, the National Center for Agricultural Utilization Research, Peoria, Illi­nois, has focused on the cellulase and xylanase activities from the anaerobic fungus Orpinomyces, developing a robotic sampling and assay system to improve desirable gene mutations for enzymic activity.220-222 Inserting genes for components of the cellulase complex into efficient recombinant ethanol producers has also continued as part of a strategy to reduce the need to add exogenous enzymes; such cellulases can be secreted at levels that represent significant fractions of the total cell protein and increase ethanol production capabilities.223-227 This is of particular importance for the accumulation of high concentrations of ethanol because ethanol at more than 65 g/l inhibits the fungal (H. jecorina) cellulase commonly used in SSF studies.228

SSF has been shown to be superior to independent stages of enzymic hydrolysis and fermentation with sugarcane bagasse, utilizing more of both the cellulose and hemicelluloses.229 A continued industry-wide commitment to SSF is evident in the numbers of publications on SSF technologies applied to ethanol production with a wide variety of lignocellulosic feedstocks (table 4.3).32, 230-238 Issues of process eco­nomics are discussed in chapter 5. Prominent in the list of lignocellulosic feedstocks in table 4.3 is corn stover, a material that has the unique distinction of having a specific biocatalyst designed for its utilization.239 This fusion of the biochemical abilities of Geotrichum candidium and Phanerochaete chrysosporium points toward a long-term option for both SSF and DMC, that of harnessing the proven hypercapabilities of some known microbes to degrade lignocellulose (see section 2.4.1) and converting them to ethanologens by retroengineering into them the ethanol biochemistry of Z. mobilis (see section 3.3.2). Before then, attempts will without doubt continue to introduce fungal genes for starch degradative enzymes into candidate industrial ethanologens and explore the possible advantages from combining genetic backgrounds from two microbes into a single hybrid designed for high amylase secretion.240-242 On a paral­lel track, commercial use of food wastes such as cheese whey, a lactose-rich effluent stream, has prompted the construction of strains with P-galactosidase to hydrolyze lactose extracellularly and use both the released glucose and galactose simultaneously for ethanol production under anaerobic conditions.243

As a final option — and one that mimics the evolution of natural microbial com­munities in soils, forest leaf litter, water-logged areas, and stagnant pools — coculti­vation of a good ethanologen together with an efficient secretor of enzymes to degrade polymeric carbohydrates and/or lignocelluloses is a route avoiding introducing genetically manipulated (GM) organisms and could be adapted to continuous tech­nologies if a close control of relative growth rates and cell viabilities can be achieved. One or more of the microbial partners can be immobilized; table 4.4 includes two examples of this approach together with the cocultivation of different ethanologens to ferment glucose/xylose mixtures and pretreated lignocellulosics.244-249

Simultaneous Saccharification and Fermentation Applied to Fuel Ethanol Production from Lignocellulosic Feedstocks

TABLE 4.4

Cocultivations of Ethanologenic and Ethanologenic Plus Enzyme-Secreting Microbes for DMC/SSF Processes