Genetic Engineering of Klebsiella axytoca and other Bacteria for the SSF Process.

Many different bacteria have the native ability for cellobiose uptake and metabolism. However, none of these organisms produce ethanol efficiently without genetic modification. Research at the University of Florida has focussed on the genetic engineering of improved organisms for die SSF process using the portable ethanol pathway derived from Z. mobilis. Three organisms have been initially targeted: 1) Klebsiella oxytoca (35), an abundant organism in pulp and paper waste; 2) Erwinia which cause soft-rot of plant tissue (39); and 3) Bacillus (40). All three organisms utilize cellobiose and thus do not require supplemental B-glucosidase. The latter two organisms also secrete endoglucanases which can assist cellulose hydrolysis. Most of our published studies have focussed on K. oxytoca P2 in which the ethanol pathway genes have been chromosomally integrated (41). In this organism and in plasmid­bearing derivatives of Erwinia, ethanol is produced from solubilized sugars at greater than 90% of theoretical yield. Other studies indicate that no major barriers exist to prevent the transfer of the ethanol pathway to Bacillus, although optimal strains have not been reported (40).

Optimization of SSF using K. axytoca P2. Commercial fungal cellulases produced by Trichoderma are most active under conditions too extreme (45-50°C, pH 4.0-5.0) for the growth of strain P2. A series of experiments was conducted with Sigmacell 50 (crystalline cellulose; Sigma Scientific Company, St. Louis, MO) to identify the optimal conditions for an SSF process with this biocatalyst (37). Spezyme CE and Spezyme CP cellulases were generously provided by Genencor International (South San Francisco, CA). Temperature was varied from 30°C-40°C at pH 5.0-pH 6.0. Surprisingly, ethanol yields exceeded 70% of the theoretical maximum (0.568 g ethanol/g cellulose) in most cases. The highest yield and rates of ethanol production were obtained at pH 5.2 and 35°C. Under these conditions, cellulase enzymes from Trichoderma are very stable and continue to be active for many days. Further studies were conducted to examine the dose dependence of cellulase enzymes. With 100 g cellulose/L, a loading of 1000 filter paper units of cellulase (FPU)/L (ie., 10 FPU/g cellulose) approached saturation. Under these conditions, the overall efficiency for fermentation plus saccharification was 72% of the theoretical maximum (Figure 4).

Comparison of SSF with Different Substrates

Time (h) Figure 4. Production of ethanol from different substrates by SSF using K. oxytoca strain P2. Fermentations contained 100 g substrate/L and 1,000 FPU cellulase/L.

Further studies were conducted using sugar cane bagasse which had been treated with dilute acid to remove hemicellulose (Figure 4) (42). Pretreatment was essential for saccharification and fermentation. However, this material was much less digestible than Sigmacell 50 and required approximately twice as much cellulase enzyme to achieve high yields. Partial saccharification of acid-treated bagasse (pH 4.8 and 48°C) for 12 h with enzymes alone (without biocatalysts) improved mixing and fermentability with a modest benefit to yield. In many cases, SSF with acid treated bagasse stopped after less than 50% of the cellulose had been digested. A brief heat treatment (followed by re-inoculation) was found to rejuvenate the saccharification process. Although the basis for this effect is not understood, it is possible that brief heating to around 60°C allows cellulases which are bound at nonproductive sites to be released. Subsequent binding to at new sites may allow saccharification to resume.

Some of our most successful studies have been conducted with mixed office waste paper (38). This substrate is highly digestible with commercial cellulase (Figure 4). Enzyme loadings of around 8.3 FPU/g cellulose approach saturation with Spezyme CP. Over 40 g ethanol/L was produced after 96 h. As with bagasse, partial saccharification prior to inoculation improved mixing but was of little benefit to ethanol yield. Dilute acid pretreatment of mixed waste office paper solubilized approximately 10% of the dry weight and improved mixing in SSF experiments. However, this pretreatment does not appear essential. Yields of around 80% of the theoretical maximum were achieved in batch fermentations with 1000 FPU/L and 100 g MWOP/L (approximately 530 L ethanol/metric ton of mixed office waste paper) (Table III).

The re-utilization of cellulase enzymes in consecutive SSF processes with MWOP allowed a dramatic reduction in the requirement for fungal cellulase (Figure 5). Fungal endoglucanase and cellobiohydrolase have specific cellulose-binding domains which facilitate recycling (38). Cellulosic residue at the end of fermentation contains bound cellulase. By adding this residue to subsequent fermentations, both product yield and enzyme effectiveness were improved. With three consecutive recycles, 40 g ethanol was produced after each 80-h fermentation with 83% of theoretical yield using an average of only 570 FPU/L of fermentation broth. For this substrate, the estimated cost of cellulase enzyme produced on site is $0,085 per liter of ethanol, $0.32 per gallon of ethanol. Approximately 539 liters of ethanol per metric ton are projected using this approach (Figure 6).

Figure 6 shows a comparison of results from SSF fermentations with K. oxytoca P2 and yeasts. Further detail is provided in Table III. Two important parameters are highlighted in this comparison, ethanol production per 1000 FPU commercial cellulase and ethanol yield per metric ton of feedstock. Both represent major cost factors for a commercial process. This comparison illustrates the benefit of utilizing

K. oxytoca P2 containing a cellobiose uptake system for batch fermentations for recycling. SSF processes with P2 allow the maintenance of high ethanol yields with a fraction of the cellulase enzyme needed with yeast as a biocatalysts. However, the cost of fungal cellulase remains substantial and further efforts should be made to reduce the levels of these enzymes needed in bioconversion.