Biotechnol. Prog. 1995, 11, 619-625
619
ARTICLES Conversion of Mixed Waste Office Paper to Ethanol by Genetically Engineered KZebsieZZa oxytoca Strain P2 T.A. Brooks and L. 0. Ingram" Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611
Unsorted, mixed waste office paper (MWOP) is an excellent substrate for conversion into fuel ethanol using a recombinant strain of Klebsiella oxytoca which ferments cellobiose and cellotriose to ethanol at near theoretical yields, eliminating the need for supplemental P-glucosidase. This organism was tested with commercial fungal cellulase in optimized simultaneous saccharification and fermentation experiments (SSF) using MWOP as a substrate (pH 5+5.2, 35 "C). Similar rates and yields were obtained with dilute acid-pulped (hydrolysis of hemicellulose) and water-pulped MWOP on a dry weight basis although viscosity was reduced by the acid pretreatment. In simple batch fermentations, 40 g/L ethanol was produced after 48-72 h with 100 g/L MWOP and 1000 filter paper units (FPU) of cellulaseL, a yield of 550 L of ethanol/ metric ton. Cellulase usage was further reduced by recycling SSF residues containing ch reduced the requirement bound enzymes in m for fungal cellulase tion and high ethanol yield. In our optimal design ethanol were produced e of 80 h (567 FPU of in three successive s fungal cellulaseL; ents a yield of 0.426 g of ton. MWOP contains apethanol/g of subst cy for saccharification and proximately 90% c fermentation to ethanol was 83.3%of the theoretical maximum.
Introduction In the United States, lignocellulose to ethanol plants using concentrated mineral acids for hydrolysis and yeast-based fermentations date back to 1917 (Jacobs, 1950). Improved processes using cellulase enzymes offer the potential to provide a large portion of current automotive fuel needs with many benefits to the economy and environment (Lynd et al., 1991). Lignocellulose is primarily composed of two carbohydrates, cellulose and hemicellulose, integrated into a phenolic matrix of lignin (Eriksson et al., 1990). Both polymers must be solubilized prior to fermentation. Pretreatment such as dilute acid hydrolysis of hemicellulose or extraction with base (Grohmann, 1993;Grohmann et al., 1985)is essential for enzymatic hydrolysis. In 1987 (Ingram et al.), our laboratory developed recombinant bacteria which convert all hexose and pentose sugars in acid hydrolysates of hemicellulose to ethanol at high yield (Barbosa et al., 1992;Beall et al., 1992; Lawford, 1993; Lawford and Rousseau, 1991). These organisms have been improved (Beall et al., 1991; Ingram et al., 1991;Lindsay et al., 1994; Ohta et al., 1991a)while other studies have been initiated to develop alternative bacterial catalysts (Beall and Ingram, 1993; Ohta et al., 1991b;Zhang et al., 1995). Cellulose conversion is more challenging. A yeastbased process for enzymatic conversion of cellulose to
* Corresponding author: L. 0. Ingram, Department of Microbiology and Cell Science, P.O. Box 110700,Gainesville, FL 32611. Phone: 904/392-8176. Fax: 904/846-0969.
ethanol was patented in 1976 (Gauss et al., 1976)which remains, with minor modifications, the best available technology. This process, termed simultaneous saccharification and fermentation (SSF),has been demonstrated at pilot scale (Easely et al., 1989;Emert et al., 1983; Katzen, 1991). Although the production of fungal cellulases is now a mature industry, economic evaluations confirm that the cost-effective use of cellulases remains as a major obstacle (Grohman, 1993;Lee et al., 1995;von Sivers and Zacchi, 1995). The discovery of cellobiose-fermentingyeasts (Barnett, 1975;Freer and Detroy, 1983;Maleszka et al., 1982)can reduce the requirement for ,&glucosidase during SSF (Freer, 1991;Spindler et al., 1992). Our laboratory has genetically engineered a cellobiose-fermentingbacterium using genes encoding the Zynomonas mobilis ethanol pathway, Klebsiella oxytoca strain P2 (Wood and Ingram, 1992). This recombinant contains native enzymes for the uptake and fermentation of cellobiose, cellotriose, xylobiose, and xylotriose in addition to hexose and pentose sugars (Burchhardt and Ingram, 1992;Wood and Ingram, 1992). The performance of strain P2 (Doran and Ingram, 1993)exceeded that of cellobiose-fermentingyeasts (Spindler et al., 1992)when both were tested with the same substrate. Parametric studies optimized SSF conditions for strain P2 (pH 5.0-5.5,35-37 "C) using crystalline cellulose (Doran and Ingram, 1993) and acid-treated bagasse (Doran et al., 1994). A minimum loading of 10 filter paper units (FPU) of fungal cellulase per gram of substrate was required for the production of 40 g/L
8756-7938/95/3011-0619$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers
Biotecbnol. Prog., 1995, Vol. 11, No. 6
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Figure 1. Conversion of MWOP to ethanol by SSF using K.oxytoca strain P2. (A) Comparison of water pulping and acid pulping (120 g L ) . Fermentation contained 1000 FPU of Spezyme CP cellulase/L. (B)Effect of substrate concentration (AP-MWOP)on ethanol production (1000 FPU of cellulaseL). (C)Effect of partial saccharification (12 h, pH 4.8, 48 "C)of AP-MWOP (120 g/L)on ethanol production (1000 FPU of cellulaseL). (D) Effects of cellulase concentration. Fermentations contained 120 g/LAP-MWOP and were subjected to a 12-h partial saccharification (pH 4.8,48 "C), prior to inoculation. An enzyme loading of 8.3 FPUlg of substrate corresponds to 1000 FPU of cellulaseL of fermentation broth.
ethanol from either substrate, although fermentation times were excessive. Previous SSF studies with yeasts have demonstrated that sorted white office paper has the potential for high ethanol yields (Wayman et al., 1992). Sorted white office paper is quite valuable for recycling (Badar, 1993) although mixed waste office paper which typically contains over one-half white paper may represent a more realistic substrate for commercial ethanol production (Kerstetter and Lyons, 1991). Minimal process goals have been proposed for SSF bioconversion (Ingram and Doran, 1995): 40 g/L ethanol, fermentation times under 96 h, and cellulase enzyme loadings of less than 10 FPU/g of substrate. Our current study demonstrates that these goals can be met for mixed waste office paper (MWOP) using K.oxytoca strain P2 using a simple method for cellulase recycling.
Materials and Methods Preparation of Mixed Waste Office Paper (MWOP). MWOP was pulped by heating to 140 "C for 30 min in 1%sulfuric acid to hydrolyze hemicellulose (AP-MWOP)or in tap water (WP-MWOP)using a stirred reactor. AP-MWOP was washed with tap water until the eMuent reached pH 5.0. Both pulps were concentrated by centrifugation. Each contained approximately 90% carbohydrate and 10%lignin plus other nonfermentables (dry weight basis). WP-MWOP contained 10%-12% hemicellulose while only cellulose remained in AP-
mop.
Bacterial Strain. K. ozytoca strain P2 was used in these studies (Ohta et al., 1991b; Wood and Ingram, 1992). Stock cultures were stored in 40% glycerol at -75 "C. Working stocks were maintained at 30 "C on TYE medium (giL: 20 glucose, 10 tryptone, 5 yeast extract, 5
NaC1,15 agar, 0.04 Cm) or TYE broth containing 50 g/L glucose (without agar). Preparation of Inocula. Colonies were inoculated into 500-mL flasks containing 250 mL of TYE broth and incubated for 18 h at 30 "C without agitation. Cells were harvested by centrifugation. SSF fermentations were inoculated to an initial density of 150 mg dry weighs. Fermentation Experiments. SSF experiments were conducted at 35 "C (pH 5.0-5.2) in modified 800-mL beakers (Corning Fleaker) with a 500 mL working volume (Beall et al., 1991). Fermenters were agitated by overhead motors (120 rpm) attached to glass rods with stainless steel paddles. Double-strength"YE and MWOP were sterilized by autoclaving at 121 "C for 15 min. HC1 was used to adjust the initial pH prior to inoculation. Minor pH adjustments were made using 2 N KOH during fermentation. Spezyme CP cellulase was generously provided by Genencor International (South San Francisco, CA). It was sterilized by filtration using polysulfone membranes and contained 100 FPU/ml. Fermenters were either inoculated at the time of cellulase addition or preincubated with cellulase at 48 "C (pH 4.8) for 12 h (partial saccharification). Analyses. Ethanol was measured by gas chromatography (Beall et al., 1991). The composition of MWOP was determined by sequential acid hydrolysis (Doran et al., 1994) using high-performance liquid chromatography (Lindsayet al., 1994). Maximum volumetric productivity was estimated from the initial 24-h period. For calculations of theoretical yield, MWOP is assumed to contain 90% fermentable, polymeric carbohydrate (cellulose + hemicellulose). The maximum theoretical yield is 0.568 g of ethanollg of polymeric carbohydrate or 0.511 g of
Biotechnol. Prog., 1995,Vol. 1 1 , No. 6
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Table 1. Summary of Batch and Enzyme-Recycle SSF Experiments with MWOP and K oxytoca Strain P2 substrate replicates (gL)
FPUL
FPU/gS
partial" vol. prod.b ferm. sacchar. (h) [gE/(L.h)l time (h)
Water-Pulped MWOP and Spezyme CE 0 0.62 144 37.5 f 2.4 0 0.68 144 42.0 f 2.6 0 0.75 96 40.8 Acid-Pulped MWOP and CP Spezyme CP 0 0.82 96 35.8 f 1.4 0 0.83 96 43.5 0 0.87 96 42.0 0 0.77 96 32.0 f 2.6 12 0.88 72 41.4 12 0.49 144 27.3 12 0.67 144 35.1 f 1.9 12 0.76 96 37.1 12 0.88 96 43.7 f 2.0 12 0.87 48 38.4 12 0.81 96 37.5 Recycling AP-MWOP Residue
3 3 2
120 120 120
500 750 1000
4.2 6.3 8.3
4 2 2 4 2 2 8 2 9 2 2
80 100 120 140 100 120 120 120 120 120 120
1000 1000 1000 1000 1000 300 500 750 1000 2000 2500
12.5 10.0 8.3 7.1 10.0 2.5 4.2 6.3 8.3 16.7 20.8
120 100 110
500 500 500
4.2 5.0 4.5
12 12 12
0.64 0.78 0.71
144 120 132
120 100 110
750 250 500
6.3 2.5 4.5
12 12 12
0.76 0.68 0.72
120 100 110
1000 250 625
8.3 2.5 5.7
12 12 12
120 90 80 97
1000 800 800 867
8.3 8.9 10.0 8.9
120 90 80 97
1000 600 600 733
120 90 80 97 100 90 90 93
expt l a stage 1 stage 2 average expt 1b stage 1 stage 2 average expt IC stage 1 stage 2 average expt 2a stage 1 stage 2 stage 3 average expt 2b stage 1 stage 2 stage 3 average expt 2c stage 1 stage 2 stage 3 average expt 3 stage 1 stage 2 stage 3 average
ethanol (gL)
yieldd 2 N KOHc gE/lOOO (ml/L) gE/gS FPU LEA theor 5.5 2.6 4.7
0.313 0.350 0.340
75.0 56.0 40.8
396 61.3% 443 68.5% 430 66.5%
9.7 6.5 7.0 12.3 6.3 18.2 12.6 9.5 8.5 5.3 5.3
0.448 0.435 0.350 0.229 0.414 0.228 0.293 0.309 0.364 0.320 0.313
35.8 43.5 42.0 32.0 41.4 91.0 70.2 49.5 43.7 19.2 15.0
567 550 443 290 524 288 371 391 461 405 396
32.9 38.7 35.8
12.0 7.6 9.8
0.325
71.6
411 63.6%
144 120 132
38.9 33.9 36.4
9.6 9.8 9.7
0.331
72.8
419 64.8%
0.87 0.68 0.78
144 120 132
43.3 35.2 39.3
8.0 8.0 8.0
0.357
62.9
452 69.9%
12 12 12 12
0.95 0.89 0.85 0.87
96 120 96 104
43.8 48.2 42.2 44.7
10.4 12.4 10.4 11.1
0.461
51.6
584 90.2%
8.3 6.7 7.5 7.6
12 12 12 12
0.89 0.83 0.80 0.84
96 120 96 104
45.2 47.3 41.4 44.6
10.8 11.2 10.2 10.7
0.460
60.8
582 90.0%
1000 400 400 600
8.3 4.4 5.0 6.3
12 12 12 12
0.80 0.75 0.76 0.77
96 120 96 104
37.3 44.5 41.3 41.0
8.8 10.4 13.0 10.7
0.423
68.3
535 82.8%
1000 400 300 567
10.0 4.4 3.3 6.1
12 0 0 4
0.88 1.06 0.86 0.93
72 72 96 80
41.4 39.5 37.9 39.6
6.2 7.6 8.0 7.3
0.426
69.8
539 83.3%
87.7% 85.5% 68.5% 44.8% 81.0% 44.6% 57.3% 60.5% 71.2% 62.6% 61.3%
Partial saccharification by preincubation at 48 "C. Volumetric productivity during intial24 h (maximum). Base added to maintain pH 5.0-5.2. Abbreviations: expt, experiment; gE/gS, grams of ethanol produced per gram of substrate; gE/1000 FPU, grams of ethanol produced per 1000 FPU of cellulase; LEA, liters of ethanol per metric ton (dry weight) of substrate (ethanol = 790 gL); theor, percentage of theoretical yield assuming 90% polymeric carbohydrate and 0.568 g of ethanovg of carbohydrate polymer. a
ethanoyg of MWOP (647 L of ethanoymetric ton of MWOP, or 155 gaYU.S. ton of MWOP ethanol density = 0.79 g/mL, 3 kg of ethanol = 1 gal).
Results Effects of Acid Pulping. Figure 1A shows a comparison of SSF with AP-MWOP and WP-MWOP at a substrate concentration of 120 g dry weightL with 8.3 FPU of Spezyme CP/g of substrate. Both produced broths containing similar ethanol concentrations (Table 1 1, although fermentation of AP-MWOP was consistently faster. Ethanol yields were 66.5-68.5% of the theoretical maximum; considerable pulp residue remained undigested in both after 96 h. A marked difference was observed in mixing. WP-MWOP was more difficult to stir and sample, often overloading the small motors during
the initial 12 h of fermentation. Both substrates were well-mixed and easily sampled after 24 h. Effects of MWOP Concentration. The effects of different concentrations of AP-MWOP were examined at a constant level of cellulase, 1000 FPUL (Figure 1A,B; Table 1). Maximum volumetric productivity was not strongly affected by changes in substrate concentration from 80 to 140 g/LAP-MWOP. Differenceswere observed in ethanol yields and final concentrations. The highest yield (87.7% theoretical yield) was observed with 80 g/L AP-MWOP although this broth contained only 35.8 g/L ethanol. At 100 g/Lsubstrate, 43.5 g/L ethanol was produced after 96 h; 40 g/L ethanol was produced after only 48 h with 120 g/L AP-MWOP. The highest substrate concentration tested (140 g/L) did not mix well and appeared to stop fermenting after 36 h. Thus the optimal
Biotechnol. Prog., 1995, Vol. 11, No. 6
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Table 2. ComDarison to SSF Result&! from Other Studies
biocatalyst yeast yeast yeast yeast yeast yeast
K.orytoca P2 K. orytoca P2 K.orytocaP2 K.orytoca P2 K.orytocaP2 K. orytocaP2 (recycleexpt 3)
substratea(per L)
yieldC partial* ferm. cellobiase sacchar. time ethanol gE/1000 FPUL FPU/gS (IUL) (h) (h) gE/gS FPU LEA
(a)
100 g of ST-SWOP 100 g of AT-SWOP 200 g of SWOP 200 g of SWOP 200 g of SWOP 200 g of SWOP 100 g of Sigmacell 50 100 g of AT-bagasse
2649 2649 3440 3440 6880 6880
100gofAP-MWOP 100 g of AP-MWOP 120gofW-MWOP 93gofAP-MWOP
1000
1000 1000 1000
1000 567
26.5 26.5 17.2 17.2 34.4 34.4 10.0 10.0 10.0 10.0 8.3 6.0
274 274 387 387 773 773
6 6 6 6 6 6
none none none none none none
none none none 12
none 4
48 48 72 96 72 96 96 96 96 72 96 80
29.4 35.6 55.8 59.3 63.0 65.8 38.0 22.0 43.5 41.4 40.8 39.6
0.294 0.356 0.279 0.297 0.315 0.329 0.380 0.220 0.43% 0.414 0.340 0.426
11.1 13.4 16.2 17.2 9.2 9.6 38.0 22.0 43.5 41.4 40.8 69.8
372 451 353 376 395 415 481 278 550 524 430 539
ref Capek-Menard et al., 1992 Capek-Menard et al., 1992 Waymanet al., 1992 Waymanet al., 1992 Waymanetal., 1992 Waymanetal., 1992 Doran and Ingram, 1993 Doran et al., 1994 this paper this paper this paper this paper
Substrates: AT-bagasse, residue after removal of hemicellulose with 1%sulfuric acid at 140 "C (60% carbohydrate, 40% ligin); WPMWOP, water-pulped mixed waste office paper; SWOP, shreded white office paper; ST-SWOP, residue after cooking with stream at 210 "C; AT-SWOP, residue after cooking with 0.5%sulfuric acid at 210 "C; Sigma Cell 50, highly crystalline purified cellulose (Sigma Chemical Co., St. Louis, MO). 6 Partial saccharification by preincubation at 45-48 "C. Abbreviations: gE/gS, grams of ethanol produced per gram of substrate; gE/lOOO FPU, grams of ethanol produced per 1000 FPU of cellulase; LEA, liters of ethanol per metric ton (dry weight) of substrate (ethanol = 0.790 kg/L).
conditions for SSF with MWOP are 100-120 g/L MWOP with 1000 FPUL (8.3-10.0 FPUlg of substrate). Effects of Partial Saccharificationprior to SSF. We have investigated the utility of a 12-h preincubation (48 "C, pH 4.8) of AP-MWOP (120 g/L) with cellulase (1000 FPU/L) to provide partial saccharification prior to fermentation (Figure 1C; Table 1). Partial saccharification did not substantially alter the rate of ethanol production or yield, although mixing was improved. Effects of Cellulase Concentration. Figure 1D shows the effects of cellulase loadings on ethanol production at a constant substrate concentration of 120 g/L APMWOP (12-h partial saccharification). Over 40 g/L ethanol was produced in 72 h with a cellulase loading of 8.3 FPU/g of substrate; 96 h was required to reach maximum ethanol (1000 FPU and 120 g/L AP-MWOP). This fermentation produced 0.364 g of ethanoVg of substrate; slightly lower yields were obtained without partial saccharification or acid-pulping (Table 1). Ethanol productivity per 1000 FPU cellulase declined with increased enzyme loading (Figure 2A). At the lowest level of cellulase (300 FPU/L), 91 g of ethanol was produced per 1000 FPU while only 44 g of ethanoVlOO0 FPU were produced in SSF experiments containing 1000 FPUL. The large amount of ethanol produced per 1000 FPU at the lowest cellulase level (300 FPU/L) may result from the preferential digestion of amorphous regions of cellulose rather than higher enzyme efficiency. The rate of ethanol production and ethanol yield from AP-MWOP increased with cellulase concentrations up to 1000 FPU/ L. Cellulase loading was also examined for WP-MWOP (120 g/L) without a partial saccharification step (Table 1). Ethanol levels above 40 g/L were achieved with 8.3 FPU/g of substrate and 6.2 FPU/g of substrate after fermentation times of 96 and 144 h, respectively. Again, ethanol production per 1000 FPU cellulase declined with increasing cellulase although ethanol yield increased and fermentation time decreased. Comparison of SSF with a Second Saccharification Step and Reinoculation. Previous SSF studies with acid-treated sugar cane bagasse and strain P2 (Doran et al., 1994) have shown that a second saccharification after SSF (48 "C, pH 4.8) followed by reinoculation and a second SSF dramatically improved ethanol yield even without additional cellulase. A similar treatment, however, was of no benefit with highly purified
crystalline cellulose (Doran and Ingram, 1993) or with AP-MWOP as the substrate (120 g/L substrate, 8.3 FPU/g of substrate; data not shown). Times from 2-12 h were examined at different stages of fermentation; no conditions were found to increase ethanol production or yield. Recycling of Cellulase. The binding of Trichoderma longibrunchiatum endoglucanases and cellobiohydrolases to cellulose is well established. Although efficient stripping of these enzymes in active form from lignocellulose for reuse is problematic (Hogan and Mes-Hartree, 19901, a significant portion of these enzymes can be recovered bound to SSF residues or to freshly added lignocellulose (Eklund et al., 1992; Lee et al., 1995). We have investigated the feasibility of recycling SSF residues as a means of increasing the effective use of cellulase enzymes. An initial substrate level of 120 g/L AP-MWOP was selected to provide residual cellulose for enzyme binding. Our first experiments explored feasibility by collecting the undigested residue (centrifugation at 5000g, 5 min) and adding this as a supplement to a subsequent SSF fermentation. The first-stage SSF contained either 500, 750, or 1000 FPU/L. Each was allowed to proceed for 144 h, producing broths which contained 32.9, 38.9, and 43.3 g/L ethanol, respectively. As expected, a significant amount of undigested residue remained in all cases (43.3 g of ethanoVl2O g of substrate = 70%of theoretical yield). This residue was resuspended in fresh broth containing either 500 or 250 FPU/L of new cellulase and 100 g/L AP-MWOP, heated for 12 h at 48 "C to provide partial saccharification and promote enzyme redistribution, cooled to 35 "C, and reinoculated to start the second SSF. In all cases, recycling of residues increased productivity and ethanol yield (Table 1;Figure 2B). In experiment la, 18%more ethanol was produced in stage two with recycling than in stage 1 although 500 FPUL had been added to both stages. With the addition of 250 FPU to stage 2, 27% more ethanol was produced in this recycle stage (average experiments lb,c) than in a single-stage SSF containing 300 FPU/L (Table 1). A second series of recycling experiments (Table 1, experiment 2; Figure 2B) were conducted to further optimize yields per FPU cellulase and substrate (APMWOP) and to reduce fermentation times using two recycling steps. Stage 1 contained 120 gfL AP-MWOP and 1000 FPU/L of cellulase. After 96 h, residues were harvested and resuspended in fresh broth containing 90 g/L new substrate and 400-800 FPU of new cellulase,
Biotechnol. Prog., 1995, Vol. 11, No. 6
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300 500 750 1000 2000 2500 ENZYME LOADING (FPU celluiase/liter) Exa2 - r-
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Figure 3. Recycling of residue with bound cellulase during SSF of AP-MWOP using K. oxytoca strain P2 as the biocatalyst. Refer to Table 1,experiment 3, for details.
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cellulase containing approximately 43 g of ethanol&. However, fermentation times remained too long. A third recycling experiment (Table 1, experiment 3) was conducted to test the feasibility of reducing fermentation times and saccharification times and further minimizing the level of cellulase (Table 1; Figures 2B and 3). In the first stage, 41.4 g/L ethanol was produced after 72 h but declined slightly to 39.5 g/L in the second stage (400 FPU of cellulase added). The ethanol concentration in the third stage (300 FPU of cellulase added) was only 35.9 g/L ethanol after 72 h but was allowed to proceed for an additional 24 h, reaching 37.9 g/L ethanol. A fourth stage was terminated prior to completion. This third experimental design represents our most successful combination. During the first three stages, an average of 39.6 g/L ethanol was produced at high yield (83.3%of theoretical yield) in 84 h (4-h saccharification 80-h average fermentation) with a minimum of fungal cellulase (567 FPU/L). Using this approach, approximately 539 L of ethanol would be produced per metric ton of APMWOP and exceed the proposed process goals (Ingram and Doran, 1995). It is likely that the average enzyme loading in this experiment approaches the minimum required to achieve 40 g/L ethanol in less than 96 h even with further optimization or additional recycling steps.
+
" 3440
3440 6880 6880 2649 2649 ENZYME LOADING (FPU cellulase/literl
-
Figure 2. Comparison of ethanol yield. (A) Effect of cellulase concentration (per L of fermentation broth) on ethanol yield during simple batch SSF containing 120 g/L AP-MWOPand K. oxytoca strain P2. (B)Effect of average cellulase concentrations (per L of fermentation broth) on ethanol yield during multistep batch fermentations with enzyme recycling and K. oxytoca strain P2. Average times for partial saccharification (S) and fermentation (F) are listed for each experiment. Refer to Table 1 for further details. (C) Ethanol production from sorted, white office paper (SWOP) using yeast as the biocatalyst (Capek-Menard et al., 1992; Wayman et al., 1992). In addition to the cellulase listed, these fermentations also included commercial cellobiase as a second enzyme supplement (approximately 0.1 IU of cellobiase/l FPU of cellulase). The paper substrate was either autoclaved (unlabeled), steam-treated a t 210 "C (ST-SWOP),or acid-treated at 210 "C (AT-SWOP) prior to fermentation. Fermentation times are listed above each set of bars.
heated for 12 h at 48 "C,cooled, and reinoculated (stage 2). After 120 h of fermentation, harvested residues were resuspended in fresh broth containing 80 g/L new substrate and 400-800 FPU of new enzyme, heated, reinoculated, and incubated for a final 96 h (stage 3). Again recycling was clearly beneficial. Fermentation broths averaged 42.1 g/L ethanol after the first stage (1000 FPU/ L), 46.7 g/L ethanol after the second stage, and 41.6 g/L ethanol after the third stage despite the low levels of new cellulase. Ethanol yields from MWOP were 83-90% of the theoretical maximum. In these experiments, an average of 60 g of ethanol was produced per 1000 FPU
Discussion Although the technology is currently available for the conversion of lignocellulosic biomass to ethanol, the challenge of integrating this conversion into a costeffective process remains. The purchase or production of cellulase and the costs of substrate per liter of ethanol represent the two largest expenses after capital costs (Kerstetter and Lyons, 1991; von Sivers and Zacchi, 1995). Although many studies have focused on cellulase loadings per gram of substrate as an important parameter, improvements in cost effectiveness are best evaluated in terms of ethanol yield per unit of substrate and per unit of cellulase ( z t cellobiase). Overall cost of conversion can be minimized by careful selection of feedstocks which provide high yields. Mixed waste paper is a plentiful form of lignocellulose which may be particularly advantageous for near-term conversion (Kerstetter and Lyons, 1991; Scott et al., 1994). Mixed waste office paper is unusually susceptible to enzymatic digestion even without additional treatments. In SSF fermentations using K. oxytoca strain P2 as the biocatalyst, WP-MWOP and AP-MWOP were more readily fermented than either highly purified crystalline cellulose (Doran and Ingram, 1993) or acid-treated bagasse (Doran et al., 1994) (Table 2).
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Previous SSF studies with (sorted) white office paper (20%solids) have demonstrated excellent rates of ethanol production using conventional yeast as the biocatalyst (Wayman et al., 1992). These fermentations reached 55.8-63.0 g/L ethanol in 72 h and 59.3-65.8 g/L ethanol in 96 h but required high levels of fungal cellulase supplemented with fungal cellobiase (Table 2). Under these conditions, yields after 96 h were 376 L of ethanoU ton using 3440 FPU of cellulase 387 international units (IU) of cellobiaseL and 415 L of ethanoyton using 6880 FPU 773 IU of cellobiase/L. Assuming a specific activity of 0.8 FPU/mg of fungal protein, 57 and 103 g of fungal cellulase protein, respectively, were required to produce 1L of ethanol (0.22-0.39 kg of cellulase proteid gal of ethanol cellobiase). Even at $6/kg of cellulase protein (Ballerini et al., 19941, these large amounts of fungal enzyme would be prohibitively expensive. Using conventional yeasts and lower levels of both cellulase (2649 FPU/L, 274 IU of cellobiase)and paper (100 g/L), 29.4 g/L ethanol was produced from steam-treated white office paper and 35.6 g/L ethanol from acid-treated white office paper (Capek-Menard et al., 1992). Although the ethanol yield per ton was good for the acid-treated paper (451 Uton), again the large amount of fungal cellulase (74 and 89 g of protein/L of ethanol; $0.44-0.53/L) plus cellobiase would be quite expensive. From these examples, it is clear that commercialization of SSF will require either the production of cellulase activity at a fraction of the estimated costs or higher ethanol yields per unit of cellulase activity without sacrificing conversion efficiency. The use of K. oxytoca strain P2 as the biocatalyst in our studies allowed a dramatic reduction in the amount of fungal cellulase required for ethanol production from MWOP and eliminated the need for supplemental cellobiase. Note that our studies used mixed waste office paper rather than the sorted white office paper investigated with yeast (Capek-Menard et al., 1992; Wayman et al., 1992). Although K. oxytoca strain P2 does not have the high ethanol tolerance of yeasts, 41 g/L ethanol was produced in 72 h in batch fermentations. This represents 65-73% of the volumetric productivity of yeast during a 72-h SSF (Wayman et al., 1992) while allowing a 60-80% reduction in cellulase and total elimination of supplemental cellobiase. Figure 2 compares ethanol yields for SSF investigations with K. oxytoca strain P2 and yeast. In singlestage batch fermentations (96 h), ethanol yields per 1000 FPU (100 g/L MWOP) were 2-4-fold higher than reported for yeast (Wayman et al., 1992) with a 28% increase in ethanol yield per ton of substrate (524-550 Uton). Even higher yields were obtained with enzyme recycling reaching a maximum of 72.8 g of ethanoV1000 FPU and 584 L of ethanoyton of MWOP. Experiment 3 (Table 1;Figure 3) represents our most promising approach for the SSF of MWOP to ethanol with K. oxytoca strain P2. After an average of 80 h of fermentation, broths averaged 39.6 g/L ethanol and required only 567 FPU of cellulaseL. The yield under these conditions was 539 IJton of substrate, 69.8 g of ethanol per 1000 FPU of cellulase (14 g of fungal p r o t e i d of ethanol; 0.053 kg of fungal proteidgal of ethanol). Although hemicellulose had been removed by acid hydrolysis, this acid treatment may not be essential since hydrolysis did not significantly alter digestibility in simple batch fermentations. K. oxytoca strain P2 converts all hemicellulose-derived sugars to ethanol in addition to cellooligosaccharides. The 12-h partial saccharification was marginally beneficial when tested alone in simple batch fermentations and also may be unneces-
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sary. However, both hemicellulose hydrolysis with acid and partial saccharificationat 48 "C dramaticallyreduced viscosity and improved the ease of mixing. The dilute acid treatment hydrolyzed 10-12% of the dry weight (hemicellulose),producing soluble sugars which could be used for seed growth (Lawford and Rousseau, 19931, with a corresponding reduction in fungal cellulase per untreated ton of MWOP. Heating of the residues to 48-55 "C during recycling may prove useful as a simple method to control contamination.
Acknowledgment This research was supported by the Florida Agricultural Experimental Station (PublicationNumber R-04405)
and by grants from the Department of Energy, Office of Energy Biosciences (FG05-86ER3574),and the Department of Agriculture (92-37308-7471and 58-3620-2-112).
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243. Accepted August 4, 1995.@
BP9500526 @
Abstract published in Advance ACS Abstracts, October 1,
1995.
