Fermentation of Hemicellulosic Sugars and Sugar Mixtures by Candida shehatae

Transcription

1 Fermentation of Hemicellulosic Sugars and Sugar Mixtures by Candida shehatae Thomas W. Jeffries* United States Department of Agriculture, Forest Products Laboratory, Madison, Wisconsin Hassan K. Sreenath* Department of Chemical Engineering, University of Wisconsin, -Madison, Wisconsin Accepted for publication February 10, 1987 D-xylose is a relatively-abundant and easily recovered sugar from the hemicellulose of angiosperms, and it could be used as a feedstock for the production of ethanol. The economics of such a process depend in part on the fermentation rate and ethanol yield achieved. Cundidu shehutue is a yeast capable of relatively rapid fermentation of D-xylose. Volumetric rates of ethanol production reported for this organism are as high as 1.3 g/l/h. Specific rates of ethanol production range from 0.28 to g/g/h, and yields range as high as g ethanol/g xylose.** Even though these values compare well with characteristic volumetric and specific ethanol production rates of 0.22 g/g/h and g/g/h attained with Puchysoten tunnophilus on ~ylose,~,~,~ they are still far short of respective rates of 1 I. 8 and 1.8 g/g/h attained by Succharomyces cerevisiue on glucose. Moreover, the ethanol yields achieved with xylose are generally significantly lower than those obtained with gl~cose.~ The factors limiting xylose and glucose utilization in C. shehatae are still not fully understood. Most of the attention given to fermentation by C. shehutue has focused on its ability to utilize xylose. Its capacity to ferment glucose has been largely assumed. Whereas P. tunnophilus ferments glucose at about five times faster than it does xylose, showing a specific rate of 1.0 with the hexose sugar, C. shehatae utilizes glucose only slightly faster than xylose, and it shows a specific glucose fermentation rate of 0.5. Moreover, with P. tannophilus, the yield of ethanol is higher on glucose than on xylose, and batchwise or periodic feeding of glucose to a xylose fermentation enhances the xylose utilization rate and yield.. In contrast, with C. shehutae, the yield of ethanol from * To whom all correspondence should be addressed. Maintained in cooperation with the University of Wisconsin, Madison Wisconsin. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture of any product or service to the exclusion of others which may be suitable. Present address: Citrus Research and Education Center, University of Florida, Lake Alfred, Florida glucose is not greatly different from that obtained from xylose, and continuous feeding of glucose to a xylose fermentation by C. shehutue increases the ethanol production rate but not the yield. In P. tunnophilus, enzymes essential for the utilization of xylose are known to be inducible. I3,l4 Regulatory processes have not been so well characterized in C. shehutue. It was of interest, therefore, to study how fermentative activity varies with the carbon source used for growth. Xylose is virtually never obtained in a pure form in hydrolysates of hemicellulose, so it was also of interest to characterize the ability of this organism to utilize glucose, xylose and other hemicellulosic sugars alone and in various mixtures. In the experiments described here, batchwise fermentations were employed with cells induced by growth on either glucose or xylose, and fermentation kinetic constants were determined. MATERIALS AND METHODS Microorganism Cundidu shehutue ATCC was obtained from the American Type Culture collection (Bethesda, MD) and maintained on yeast malt peptone glucose agar (YMPG from Difco) at 32 C. Culture Medium The fermentation medium was prepared in 0.05M citrate buffer, ph 3.2, containing filter-sterilized 0.17% yeast base (YB) without ammonium sulfate or amino acids (YB from Difco) but with 0.113% urea along with 0.325% peptone as nitrogen sources. The sugar solutions were separately autoclaved and added aseptically to give the desired concentrations. lnoculum Preparation and Fermentations A loopful of culture from a three-day-old YMPG plate was inoculated into 50 ml of YB containing 90 g/l glu- Biotechnology and Bioengineering, Vol. 31, Pp (1988) John Wiley & Sons, Inc. CCC /88/ $04.00

2 cose or xylose in a 125-mL Erlenmeyer flask, and shaken at 100 rpm at C. The culture broth was centrifuged at 3.4 x lo g for 10 min; the cells were suspended to 10 ml in distilled water and used as the inoculum. Fermentations were conducted for 72 h under the same conditions used for inoculum preparation. For studies of glucose and xylose fermentations, inocula were prepared by growing cells on either the same or the opposite sugars; 72-h-old cells at an initial cell density of 1 g (dry wt)/l were used for the inoculum. For a separate experiment on the fermentation of glucose, xylose, mannose, galactose, and L-arabinose, 24- h-old xylose-grown inocula at an initial cell density of 0.3 g/l were used for the inoculum. For fermentation of acid hydrolysate, 24-h-old xylose-grown inocula were used for the inoculum at a density of 3.4 g/l in the initial fermentation, and 1.2 g/l was used in the second stage. For fermentation studies with individual sugars, each was tested at 60 g/l and at the concentration at which it was present in an acid hydrolysate of wood. Preparation of Acid Hydrolysate An acid hydrolysate of southern red oak (Quercus falcata Michx.) was prepared as described earlier and filter-sterilized. The acid hydrolysate consisted of the following sugars (g/l): xylose, 88.7; glucose, 23.4; mannose, 12.4; galactose, 12.0; and arabinose, 6.1 (total sugars was g/l). The total sugar concentration in the final YB medium was 120 g/l. Eight replicate flasks were inoculated and incubated. Four did not receive further treatment. These flasks were termed acid hydrolysate. The other four were harvested after six days; the supernatant broth was autoclaved and reinoculated after the manner of Hajny. The media in these flasks were termed conditioned hydrolysate. In addition, three flasks of filter-sterilized hydrolysate medium were autoclaved with nutrients prior to inoculation. These were termed autoclaved hydrolysate. These flasks of autoclaved hydrolysate were necessary controls for the con- ditioned hydrolysate because nonenzymatic browning reactions can occur between media components and inhibitors contained in the hydrolysate. Finally, three flasks of medium were prepared using a mixture of pure sugars at the concentrations found in the acid hydrolysate. Analytical Except where noted, each experimental condition was performed in triplicate cultures. Samples (1.O ml) were removed at intervals for analysis. Cell densities were measured in all samples by absorbance at 525 nm. Dry weights were correlated with optical densities (OD) of cell suspensions between 0.05 and 0.5 OD units at 525 nm. An OD of 1.O was equivalent to 0.19 mg dry wt cells/ml. Ethanol was determined by gas chromatography. l6 Xylose, glucose, xylitol, and glycerol were determined by high perfomance liquid chromatography (HPLC). l7 RESULTS The glucose and xylose utilization rates of glucose-grown and xylose-grown inocula were not significantly different on the individual sugars. Xylose alone was taken up at ca. 75% of the rate observed with glucose alone, and the rates were essentially the same regardless of the carbon source used for inoculum preparation (Table I). With the cell densities used in this experiment, growth was slow and limited to a few doublings before sugar was exhausted. Approximately one cell doubling took place in the first 18 h and a total of only two doublings occurred in 72 h. The growth rates did not increase greatly in the presence of glucose, but glucose supplements stimulated fermentations. Fermentations employing xylose-grown inocula were affected more than those employing glucosegrown inocula. The effect of supplemental sugar depended on the history of the cells. Addition of either glucose or xylose reduced Table I. Fermentation of xylose, glucose, and their mixtures by Candida sheharue. Fermentation and growth conditions employed 9% x 6%X + l%gb 6%X + 3%G 9% G 6%G + l%xb 6%G + 3%X Fermentation pararnetef X G X G X G X G X G X G * a All rates were determined in the first 18 h incubation. 1% Glucose or xylose were added at 0, 24, and 48 h. X is xylose-grown inoculum; G is glucose-grown inoculum. means not applicable. Ethanol yield was determined at the maximum concentration attained. COMMUNICATIONS TO THE EDITOR 503

3 the utilization of the opposite sugar to a greater extent with glucose-grown inoculum than with xylose-grown inoculum. Even though either sugar could inhibit utilization of the other, glucose supplements were much more effective in repressing xylose utilization than xylose supplements were in repressing glucose utilization. For example, compare the effect of glucose on the R,X obtained with 9% xylose and sugar mixtures to the effect of xylose on the R,G in analogous controls (Table I). Ethanol production rates were higher with xylose-grown inocula than with glucose-grown inocula. This comparison held true for all of the combinations of glucose and xylose tested. The ethanol production rate was highest for a fermentation of 6% xylose supplemented with 3% glucose by xylose-grown inoculum. The next highest rates were obtained with the fermentation of pure glucose and the glucose-xylose mixture by xylose-grown inoculum. Ethanol yields did not appear to be greatly affected by the sugars tested. Formation of other products varied with the carbon source. Xylitol production was observed only in the presence of xylose, and xylitol was formed to a lesser extent by glucose-grown than by xylose-grown inocula. The yield of xylitol was ca g/g with xylose-grown inocula and 0.11 g/g with glucose-grown inocula. With xylose-grown inocula, glycerol yields were 0.02 g/g on glucose and 0.04 g/g on xylose. With glucose-grown inocula, the corresponding values were 0.03 and 0.05 (experiment of Table I, data not shown). The rates of mannose, glucose, xylose, galactose, and L-arabinose utilization by xylose-grown inocula were determined in a separate experiment (Table 11). Mannose (6%) was used at a higher rate than any of the other sugars tested. Mannose also showed the highest rate of ethanol production. Most of the mannose taken up, however, could not be accounted for in the ethanol produced, so it is possible that significant amounts of mannitol were formed. The D-galactose was fermented at a significantly lower rate than glucose, xylose, or mannose. The L-arabinose was not consumed, as one would expect from taxonomic descriptions of this organism.i8 The ethanol fermentation rate observed was much lower with an autoclaved acid hydrolysate than with the conditioned hydrolysate or a mixture of individual sugars. Con- tacting the hydrolysate with cells for an extended period of time prior to autoclaving greatly improved fermentability, but this strain of C. shehutue was still very susceptible to inhibition by components in the hydrolysate (Table 111). DISCUSSION Du Preez and co-w~rkers'~ previously reported on the fermentation of hexose and pentose sugars by C. shehatue. These authors also studied the fermentation of xylose at concentrations ranging from 10 to 100 g/l, and noted that peak specific ethanol production rate obtained was 0.48 g/ g/h at 50 g/l xylose.*' The maximal volumetric fermentation rate they obtained was ca g/l/h. Whereas their specific rate was about three times higher than what we report here, the volumetric rates were similar given the different strain, media, and fermentation conditions employed in the two studies. Moreover, we have observed that the specific fermentation rate varies greatly with the cell density and age of the culture, being higher in young, vigorously-growing cultures and lower in older cultures. In fact, the volumetric rate can be constant while the cell density increases several-fold, thereby decreasing the specific fermentation rate proportionately. Du Preez and co-workers had noted diauxie in the utilization of glucose and xylose by C. shehatue. l9 They employed 1% each of glucose, xylose, cellobiose, galactose, and mannose with an inoculum of 0.2 g/l. The glucose and mannose were taken up before the xylose but the cellobiose was not utilized. In our study, considerably higher concentrations of xylose were employed, and although it was apparent that inhibition of xylose utilization occurred, Table 111. Fermentation of sugar mixtures and acid hydrolysates by C. shehatae. Fermentation sugar Conditioned Autoclaved parameter mixture hydro1 y sate' hydrolysateb P RPE 1.oo YE Ethanol (g/l) a Acid hydrolysate was preconditioned by inoculation with cells prior to autoclaving with YB and fermentation with a fresh inoculum. Acid hydrolysate was autoclaved with YB and inoculated. Table 11. Fermentation of hemicellulosic sugars by xylose-grown cells of Candida shehatue. Mannose Glucose Xylose Galactose Arabinose Fermentation parameter 6% 1.24% 6% 2.34% 6% 8.87% 6% 1.2% 6% 0.61% P Rs Qs RPE QPE YE Ethanola a Maximum concentration is shown in g/l. 504 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 31, APRIL 1988

4 we did not employ sufficient time points to determine if glucose and xylose utilization were sequential, as in the case of diauxie. Glucose was used more effectively than xylose, but the difference was not related to the growth history of the cells. Rather, cells grown on glucose or xylose used either sugar equally well. Moreover, supplemental xylose inhibited glucose utilization to a small extent in a fashion analogous to glucose inhibiting xylose utilization. The inhibition of glucose utilization by xylose could have resulted from direct competition for the same sugar transport system. Lucas and van Uden2 have shown that C. shehatue possesses a facilitated diffusion sugar transport system in cells exponentially growing on either glucose or xylose (repressed) and a proton symport system in cells starved for 2.5 h (derepressed). Sugar uptake with the two systems is quite different. Whereas a single, facilitated diffusion system is responsible for the transport of either glucose or xylose, two separate proton symport systems are employed. Moreover, with the facilitated diffusion system, the V,,, value for xylose is ca. 10-fold higher than for glucose, but with the proton symport system, the V,,, value for xylose is ca. half that for glucose. Both the facilitated diffusion and proton symport systems exhibit a lower K, for glucose than for xylose-as much as 100-fold in the case of facilitated diffusion. Nonetheless, because of the higher V,,, value observed with facilitated diffusion of xylose, Lucas and van Uden calculated that in the presence of 6% xylose and 3% glucose, C. shehutue would exhibit a theoretical xylose uptake rate of 0.76 g/g/h. The actual xylose utilization rate which we observed was less than half of this theoretical value, thereby suggesting that sugar transport per se does not limit the xylose uptake rate under repressed, fermentative conditions. In fact, the substrate affinities for the facilitated diffusion system used by C. shehutue for taking up glucose and xylose are similar to those observed with S. cerevisiae. 22 Lucas and van Uden also showed that the inhibition constants for transport of glucose and xylose by the facilitated diffusion system were almost identical (Ki = 20.6mM for inhibition of xylose transport by glucose and 22mM for inhibition of glucose by xylose). Yet glucose inhibited xylose utilization to a far greater extent than xylose inhibited glucose. This finding suggests that at least part of the inhibitory effects of glucose results from interactions further down the metabolic pathway. Although the effects of these two sugars on intermediary metabolism are probably profoundly different and incalculable from what we presently know, the first point of direct competition between the two assumed pathways probably would be at phosphofructokinase, and regulatory effects must be examined there, and at D-xylulokinase. The idea that levels of intermediate enzymes might be important in determining the overall fermentative rate is further supported by the observation that xylose-grown inocula actually showed slightly higher initial fermentation rates than glucose-grown inocula. The idea here is that xylose is metabolized slower, hence levels of some intermediate enzymes important in fermentation could be derepressed. It is not obvious why mannose should have been utilized more rapidly than glucose. It is transported by the same facilitated diffusion system as glucose and xylose.21 Mannose utilization presumably proceeds via hexokinase and phosphomannanase to form fructose 6-phosphate, but the pathway has not been described for this organism. Galactose metabolism likewise requires further clarification. The toxicity of various components in acid hydrolysates has been recognized for at least 50 years.23 Furfural and tannin are among the more toxic materials present. Furfural can be removed by yeasts,24 and by employing high cell densities, the toxic effects can be alle~iated.~~ Large yeast inocula along with various other treatments such as activated charcoal, anion exchange, steam distillation, overliming and solvent extraction have been used to improve the fermentability of acid hydrolysates. The method employed here was relatively simple and fairly effective, perhaps because the tannins were bound and removed by the cells. NOMENCLATURE References specific growth rate (h- ) volumetric xylose uptake rate (g/l/h) volumetric glucose uptake rate (g/g/h) specific xylose uptake rate (g/l/h) specific glucose uptake rate (g/g/h) volumetric ethanol production rate (g/l/h) specific ethanol production rate (g/l/h) ethanol yield (g/g) J. C. du Preez, and J. P. van der Walt, Biotechnol. Lett., 5, 357 (1983). H. Dellweg, M. Rizzi, H. Methner, and D. Debus, Biotechnol. Lett., 6, 395 (1984). J. C. du Preez, B. A. Prior and A. M. T. Monteiro, Appl. Microbiol. Biotechnol., 19, 261 (1984). A. Toivola, D. Yarrow, E. van den Bosch, J.P. van Dijken, and W. A. Scheffers, Appl. Environ. Microbiol., 47, 1221 (1984). J. C. du Preez and B. A. Prior, Biotechnol. Left., 7, 241 (1985). P. J. Slininger, R. J. Bothast, J. E. van Cauwenberge, and C. P. Kurtzman, Biotechnol. Bioeng., 24, 371 (1982). T. W. Jeffries, Trends Biotechnol., 3, 208 (1985). B. L. Maiorella, H. W. Blanch, and C. R. Wilkie, Biorechnol. Bioeng., 26, 1003 (1984). T. W. Jeffries, J. H. Fady, and E. N. Lightfoot, Biorechnol. Bioeng., 27, 171 (1985). T. W. Jeffries, in Energy Applications ofbiomoss, M. Z. Lowenstein, Ed. (Elsevier, New York, 1985), p M. J. Beck, Biotechnol. Lett.. 8, 513 (1986). H. K. Sreenath, T. W. Chapman, and T. W. Jeffries, Appl. Microbiol. Biotechnol., 24, 294 (1986). K. L. Srniley and P. L. Bolen, Biotechnol. Lett., 4, 607 (1982). P. L. Bolen and R. W. Detroy, Biotechnol. Bioeng., 27, 302 (1985). G. J. Hajny, Biological Utilization of Wood for Production of Chemicals and Foodstuffs, US. Department of Agriculture, Forest Service Research, Madison, WI. Forest Products Lab, 1980, paper FPL 385. T. W. Jeffries, Biotechnol. Bioeng. Symp., 12, 103 (1982). COMMUNICATIONS TO THE EDITOR 505

5 17. L. A. th. Verhaer and B. E M. Kuster, J. Chromufogr., 210, 279 (1981). 18. S. A. Meyer, D. G. Ahearn, and D. Yarrow, Cundida shehafue, in The Yeusfs, 3rd ed., N. J. W. Kreger-van Rij, Ed. (Elsevier, Amsterdam, 1984). p J. C. du F reez, M. Bosch, and B. A. Prior, Appl. Microbiol. Biotechnol., 23, 228 (1986). 20. J. C. du Preez, M. Bosch, and B. A. Prior, Enzyme Microb. Technol., 8, 361 (1986). 21. C. Lucas and N. van Uden, Appl. Microbiol. Biotechnol., 23, 491 (1986). 22. A. Kotyk and K. Janacek, Cell Membrane Trunsporf (Plenum, New York, 1975). 23. H. Liiers, 2. Spiritusid., 60, 70 (1937). 24. A. F. Azhar, M. K. Bery, A. R. Colcord, R. S. Roberts, and G.V. Corbitt, Biotechnol. Bioeng. Symp., 11, 293 (1981). 25. I. S. Chung and Y. Y. Lee, Biotechnol. Bioeng.. 27, 308 (1985). 26. A. V. Tran and R. P. Chambers, Enzyme Microb. Technol., 8, 439 (1986). 506 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 31, APRIL 1988