Fermentation Studies on Glucose and Xylose. The fermentation performance of the parent yeast and the genetically engineered yeast was studied on glucose and xylose separately. In both these experiments the substrate concentrations selected were well above the growth limiting concentrations and not inhibitory to growth of the yeasts.

The results of the glucose fermentation study using yeasts 1400 and 1400 (pLNH33) is shown in Figures la and lb, respectively. These experiments were performed using approximately 100 g/L glucose as the substrate with an initial cell density in the range of 0.05-0.10 g/L. In both cases, a maximum cell density in the range of 11-11.5 g/1 was obtained after 14 hours. The specific growth rates of the yeast was calculated from the slope of a semi-log plot of cell dry weight versus time. For the yeast 1400, the specific growth rate under the experimental conditions was calculated to be 0.4910.02 hr’1 while for the recombinant yeast 1400 (pLNH33), the specific growth rate was calculated to be 0.4810.02 hr’1 . Thus the specific growth rates of both yeasts on glucose are identical. The specific glucose utilization rates and ethanol yields in both cases were 2.3 g/g-hr and 0.49, respectively. Based on a theoretical yield of 0.51 g/g, these yields correspond to 96 % of the theoretical yield.

The results of the xylose fermentation study using the above yeasts are shown in Figures 2a and 2b. These experiments were performed using approximately 50 g/L xylose as the substrate with an initial cell density of 0.05-0.10 g/L. As seen in Figure 2a, the yeast 1400 is unable to grow and ferment xylose to ethanol. In comparison, the recombinant yeast 1400 (pLNH33) grows to a cell density of 9.4 g/L, and produces 20.45 g/L ethanol from 52.06 g/L initial xylose after 36 hours. Glycerol and xylitol are the by-products that are produced in minor amounts, to the extent of 1.73 g/L and 2.85 g/L respectively. The specific growth rate of the recombinant yeast was calculated, as explained earlier, to be 0.19±0.02 hr"1. The specific xylose utilization rate was calculated to be 0.30 g/g-hr. Based on the xylose consumed, the ethanol yield was calculated to be 0.40 g/g. This corresponds to 78 % of the theoretical yield (based on a theoretical yield of 0.51 g/g). These results indicate that the specific xylose utilization rate and ethanol yield from xylose are lower than those from glucose.

Fermentation Study on a Glucose-Xylose Mixture. From the above studies on single substrates, it is clear that the fusion product 1400 lacks the ability to ferment xylose. On the other hand, the recombinant yeast 1400 (pLNH33) shows a good fermentation performance on xylose. Typically, both glucose and xylose are present in lignocellulosic hydrolysates. This requires that the fermentative microorganism be able to ferment xylose in presence of glucose. The recombinant yeast 1400 (pLNH33) has been genetically designed to ferment both glucose and xylose present in the same medium (9). To demonstrate this, fermentation of a 1:1 mixture of glucose and xylose was performed. The composition of the sugar mixture was 52.8 g/L glucose and 56.3 g/L xylose and the initial cell density was 2.3 g/L. As shown in Figure 3, the recombinant yeast ferments glucose and xylose simultaneously to ethanol. However, the glucose utilization rate at 8.11 g/L-hr is relatively higher than the xylose utilization rate at 1.77 g/L-hr. A final cell density of 11.5 g/L and an ethanol concentration of 47.9 g/L were achieved after 48 hours. Based on a theoretical yield of 0.51 g/g, the ethanol yield from this experiment was calculated to be 80%.

й І В

Figure 1. Growth and fermentation performance of a) yeast strain 1400 and b) recombinant yeast strain 1400 (pLNH33) on glucose.

Time (hours)

Figure 2. Growth and fermentation performance of a) yeast strain 1400 and b) recombinant yeast strain 1400 (pLNH33) on xylose.

Figure 3. Fermentation of a glucose-xylose mixture by recombinant yeast strain 1400 (pLNH33).

Fermentation Kinetics. The effects of substrate and product inhibition on glucose and xylose fermentation were studied in order to determine the optimal fermentation conditions for achieving highest ethanol yields and productivities.

Substrate Inhibition. These experiments were conducted by using different initial glucose or xylose concentrations in the fermentation media. For the glucose fermentation, significant lag times are observed in experiments with an initial concentration greater than 200 g/L (72). This can be attributed to the effect of glucose inhibition on the yeast growth, as all other conditions such as temperature and availability of nutrients are favorable. Similar studies have also been conducted for the xylose fermentation using recombinant yeast 1400 (pLNH33). These studies indicate that the effect of xylose inhibition for yeast growth is significant above 70 g/L (data on specific growth rates not included).

A classical process methodology for concentrated sugar fermentations is to feed the substrate into the fermentation medium in steps, instead of feeding all of it initially (77,72). To achieve higher ethanol concentrations from glucose fermentation, this modified feeding scheme was used as shown in Figure 4. The fermentation was begun with 106 g/L glucose initially, followed by four successive additions of glucose with nutrients later during the fermentation. The arrows in the figure indicate the times at which glucose with nutrients was added into the fermentation medium. The addition of nutrients along with glucose helped in maintaining a high cell viability throughout the fermentation. At the end of 67 hours, an ethanol concentration of 133 g/L was obtained at a productivity of 1.99 g/L-hr. This reflects the high ethanol tolerance of the Saccharomyces strain 1400. Since ethanol tolerance significantly influences the economics of ethanol recovery, this high ethanol tolerance yeast has potential applications in commercial ethanol production.

This fed-batch experimental methodology was also used for the xylose fermentation using the recombinant yeast 1400 (pLNH33), as shown in Figures 5a and 5b. An ethanol concentration of 50.34 g/L was obtained after 193 hours of fermentation, giving a productivity of 0.26 g/L-hr. Thus in comparison to the glucose fed-batch fermentation, relatively lower ethanol concentrations and productivities were achieved. Ethanol inhibition of the xylose fermentation may be one of the several reasons possible for this observation.

Product Inhibition. In order to determine the effect of ethanol on the cell growth rates and the fermentation rate, experiments with a range of initial ethanol concentrations in the fermentation media were performed. This study was carried using both glucose and xylose as substrates. The sugar concentrations in both experiments were initially 50 g/L, which is well in excess of the saturation constant. This sugar concentration was selected in order to separate ethanol inhibition effects from those of substrate or nutrient limitation. The effects of ethanol concentration as a single independent variable can be clearly discerned using this method as compared to batch studies with produced ethanol.

Figure 6 shows the experimental data of variation in specific growth rate of yeast 1400 on glucose as a function of the initial ethanol concentration. When there is no initial ethanol in the medium, the highest specific growth rate of 0.6 hr’1 is

Time (hours)

Figure 5. a) Xylose and b) ethanol concentration profiles during a xylose fed — batch fermentation using the recombinant yeast strain 1400 (pLNH33).

Figure 7. Effect of ethanol on specific growth rate of recombinant yeast strain 1400 (pLNH33) on xylose.

obtained. There is a decline in the specific growth rate as the initial ethanol increases. The effect of ethanol inhibition becomes significant beyond a concentration of -100 g/L. The cell growth stops completely above an ethanol concentration of 136 g/L.

The above experiment was also performed with the recombinant yeast using xylose as the fermentation substrate. The slower growth rate on xylose is evident from the highest specific growth rate of 0.19 hr’1, which is about 3 times lower than the highest specific growth rate on an equal concentration of glucose. The inhibitory effect of ethanol on the cell growth on xylose is also stronger, in comparison to cell growth on glucose. The cell growth on xylose is inhibited strongly beyond an ethanol concentration of 60 g/L, as seen in Figure 7. However, the genetically engineered yeast has been designed to overcome this ethanol inhibition by being able to use glucose for growth, and then cofermenting the rest of the glucose and xylose to ethanol.

Fermentation Performance of the Recombinant Saccharomyces 1400 (pLNH33) on Lignocellulosic Hydrolyzates. The results which have been discussed above were obtained from studies conducted on pure sugars. These studies are useful, as they provide insight into the fermentation kinetics. In order to study the fermentation performance of this recombinant yeast on “real” substrates, some experiments conducted using com fiber and com cob as model feedstocks are presented.

The pretreatment of the lignocellulosic material is an important process step to achieve higher ethanol yields. Various physical and chemical pretreatment methods have been reported in the literature. These include physical treatments, chemical treatment using strong acids and bases, steam explosion and the low temperature ammonia fiber explosion (AFEX) process (13). Pretreatment studies in our laboratory have resulted in the development of two pretreatment techniques: dilute acid hydrolysis and ammonia steeping followed by dilute acid hydrolysis. Pretreatment of the com fiber was accomplished by acid hydrolysis using 0.5% HC1. The com cob which contains a higher fraction of lignin was pretreated by the ammonia steeping process followed by the dilute acid hydrolysis.

Corn Fiber Studies. Com based starch that is mainly obtained by com wet milling is a predominant feedstock for fuel ethanol production. A low value side stream called “com fiber” is generated in the com wet milling process. This stream contains the hulls, fine fibers and residual starch granules from washing the starch. About 9 to 10% (w/v) of the original dry weight of the com is recovered in the com fiber stream. Thus com fiber is an attractive starting material for ethanol production, in view of its availability, limited use and low cost.

The typical composition of com fiber used in these studies is as follows: 25% starch, 25% hemicellulose and 15% cellulose. Pretreatment of com fiber was accomplished by hydrolysis using 0.5% (w/v) HC1 at 120°C for 45 minutes. This pretreatment by dilute acid hydrolysis readily hydrolyses the starch to glucose, and hemicellulose to primarily xylose. The cellulosic residue is then treated with the cellulase enzyme to hydrolyze it to glucose.

In the simultaneous saccharification and fermentation (SSF) studies, the recombinant yeast 1400 (pLNH33) and the Iogen cellulase are added together to the medium containing the pretreated com fiber. Figure 8 shows the result of a batch SSF process using 10% w/v dry pretreated com fiber with 0.46 FPU/ml (about 6 FPU per gram of cellulose from com fiber) Iogen cellulase enzyme in 50 ml medium. As seen from the substrate profiles, there is a simultaneous utilization of glucose and xylose by the recombinant yeast. An ethanol concentration of 25.1 g/L was obtained at the end of 72 hours of SSF. Based on 15 g/L of glucose released from cellulose, 24.6 g/L glucose and 16 g/L xylose from the pretreated com fiber, the total fermentable sugar is 55.6 g/L. The ethanol yield obtained in this experiment was 0.45 g/g, that corresponds to 88 % of the theoretical yield at 0.51 g/g.

The fed-batch SSF process is an effective process methodology for achieving high ethanol productivities and reducing enzyme costs by lower cellulase loadings. Figure 9a shows a fed-batch SSF process starting with 10% (w/v) pretreated com fiber, followed by an identical feeding to give a total of 20% (w/v) solids. Ethanol concentrations of 38.9 g/L and 41 g/L were obtained after 72 and 96 hours, respectively. Instead of a single feeding, multiple feeds can also be used in the fed — batch SSF process. Figure 9b shows the result of such an experiment, in which the SSF is started with 10% (w/v) pretreated com fiber as before, followed by two identical feedings at 12 hour intervals to give a total of 30% (w/v) solids. Ethanol concentrations of 44.4 g/L and 48.7 g/L were obtained after 72 and 96 hours, respectively. Based on these results, the ethanol productivities during SSF of pretreated com fiber lie in the range of 0.44-0.62 g/L-hr.

Corn Cob Studies. Com cob is a low value agricultural residue having limited use. A typical composition of com cob used in these studies is as follows: 41.1% cellulose, 36% xylan, 6.8% lignin and 3.2% acetate. Compared to com fiber, the percentage of xylose in sugars obtained from com cob will be typically higher. This requires the use of a xylose fermenting microorganism for effectively fermenting xylose to ethanol.

Recently, an effective pretreatment process for lignocellulosic biomass has been developed (Cao, N. J., personal communication). This process involves steeping the raw material in 10% ammonium hydroxide solution at ambient temperatures for 24 hours. It has been determined that this ammonia steeping process efficiently removes lignin, acetate and extractives. Following this treatment, a dilute acid hydrolysis is used to hydrolyze the hemicellulose fraction. The residual fraction is primarily comprised of cellulose, that can be subjected to enzymatic hydrolysis. This process methodology systematically separates lignin, hemicellulose, cellulose and enables separate processing of each fraction. Since the lignin is removed in the initial stages of the process, the adsorption of cellulase on the lignin is minimized. Thus this pretreatment method also allows lower cellulase loadings in the SSF process. By coupling this pretreatment method with the use of the recombinant Saccharomyces 1400 (pLNH33) in the fermentation, promising results have been obtained (Cao, N. J., et al. Biotechnol. Lett., in press).

The ammonia steeping pretreatment removes almost all the acetate from the raw material. This is a key step in the process, as acetic acid has been determined to be inhibitory for xylose fermentation (14). As discussed earlier, a dilute HC1 pretreatment hydrolyses the hemicellulose and a xylose rich hydrolyzate is obtained,

Figure 8. Simultaneous saccharification and fermentation (SSF) of 10% (w/v) pretreated com fiber using the recombinant yeast strain 1400 (pLNH33) and Iogen cellulase.

Glucose Xylose Ethanol —-e—

Figure 9a. Single stage fed-batch SSF process of pretreated corn fiber using the recombinant yeast strain 1400 (pLNH33) and Iogen cellulase.

Figure 9b. Two stage fed-batch SSF process of pretreated com fiber using the recombinant yeast strain 1400 (pLNH33) and Iogen cellulase.

Figure 10. Batch fermentation of cellulose-hemicellulose hydrolyzate from com cob using the recombinant yeast strain 1400 (pLNH33).

that is treated with a weak base anion exchange resin. Iogen cellulase was used to hydrolyze the cellulosic residue, which results in a glucose rich hydrolyzate. Both the hemicellulose and cellulose hydrolyzates were mixed and fermented after adjusting the pH to 5. Figure 10 shows the result of a batch fermentation of this mixed hydrolyzate containing 52.8 g/L glucose and 55.7 g/L xylose using the recombinant yeast. An ethanol concentration of 46.9 g/L was obtained within 36 hours, giving a high yield of 84% (based on a theoretical yield of 0.51 g/g).

Results of the SSF process of cellulosic residue (pretreated by different methods) obtained from 20 g com cob using the Iogen cellulase and the recombinant

□ Ethanol Concentration Ш Ethanol Yield

Figure 11. Effectiveness of different pretreatment methods for the SSF of cellulosic residue from com cob using the recombinant yeast strain 1400 (pLNH33) and Iogen cellulase.

yeast 1400 (pLNH33) at 35°C are shown in Figure 11. The best results were obtained with the pretreatment involving ammonia steeping followed by dilute acid hydrolysis. An ethanol concentration of 40.7 g/L was obtained after 48 hours with a yield of 86% based on dry cellulose from com cob. Application of the pretreatment method allowed a low cellulase loading (8.5 IFPU/g com cob) to be used in the SSF process.

Conclusions

Fermentation studies using the recombinant yeast 1400 (pLNH33) show good results with high ethanol yields achieved on glucose, xylose and their mixtures. The simultaneous sugar utilization pattern exhibited by this yeast is beneficial for the SSF process, as the xylose fermentation time is reduced significantly. Moreover, this recombinant yeast retains the high ethanol tolerance of its parent (the fusion yeast strain 1400). This improves the economics of ethanol recovery greatly, as high final ethanol concentrations can be achieved in the fermentation broth.

Promising results with the yeast 1400 (pLNH33) have also been obtained on lignocellulosic feedstocks like com fiber and com cob. Significant process improvements have also been made in the biomass pretreatment technology. The use of this recombinant yeast coupled with improved pretreatment techniques provides a firm base for developing a mature biomass to ethanol technology.

Acknowledgments

This work was funded by Amoco Corporation and the Consortium for Plant Biotechnology Research. The com cob studies were conducted by M. S. Krishnan with N. J. Cao and C. S. Gong. We acknowledge the assistance of Jean Lu and Xuezhi Yu for the HPLC analysis.