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Ind. Eng. Chem. Res. 1997, 36, 4572-4580
Ethanol Production from Enzymatic Hydrolyzates of Cellulosic Fines and Hemicellulose-Rich Liquors Derived from Aqueous/Steam Fractionation of Forages Khaled Belkacemi,† Ginette Turcotte,*,† Philippe Savoie,‡ and Esteban Chornet§ Department of Food Science and Nutrition, Universite´ Laval, Pavillon Comtois, Sainte-Foy, Quebec, Canada G1K 7P4, Agriculture and Agri-Food Canada, 2650 boul. Hochelaga, Sainte-Foy, Quebec, Canada G1V 2J3, and Department of Chemical Engineering, Universite´ de Sherbrooke, 2500 boul. Universite´ , Sherbrooke, Quebec, Canada J1K 2R1
This study was aimed at evaluating perennial forages (timothy grass, alfalfa, and reed canary grass) as substrates for ethanol production. Two fractions, derived from the aqueous/steam fractionation of these plants, were used as carbon sources for ethanol production: (i) a solution containing water-soluble hemicelluloses and (ii) cellulosic fines recovered after a delignification step. Both fractions were enzymatically hydrolyzed. The hemicellulose-rich fraction was easily saccharified with 90% of theoretical yield. Cellulosic fines were saccharified at 60-70% of theoretical yield. Increasing the delignification of cellulosic fines by alkaline peroxide treatment resulted in higher sugar yields. The glucose-rich hydrolyzate from cellulosic fines was easily fermented to 80-90% of theoretical ethanol yield with Saccharomyces cerevisiae or Pachysolen tannophilus. The pentose-rich hydrolyzate from water-soluble hemicelluloses was fermented to only 20% of theoretical ethanol yield with Pachysolen tannophilus. A lime treatment of the hemicellulose-rich liquors improved cell growth but did not improve ethanol production. Introduction Conversion of renewable bioresources into energy can offer significant benefits for the environment, increase economic activity, and improve independence from fossil fuel utilization. However, in the current economic paradigm, it is difficult for biofuel systems to compete with fossil fuels (Olsson and Hahn-Ha¨gerdal, 1996). Even with recent developments in pentose fermentation to ethanol (Ohta et al., 1991; Barbosa et al., 1992; York and Ingram, 1996), economic viability of the conversion of biomass to ethanol is not currently attainable. At least two technical breakthroughs are required for economic feasibility: a low-cost pentose fermentation process; developments in lignin conversion into valuable byproducts, and perhaps, a link with fiber production. In this context, Keller (1996) overviewed the technical and economic aspects of alternative approaches taken to integrate several unit operations into improved biomass-to-ethanol processes with emphasis on recycle technology and coproduct generation for other improved industries. Many attemps to develop truly integrated, environmentally benign, renewable biomass refineries in which ethanol is one of many coproducts are reported, for example, integrated biomass facility around Stake II technology (Glaser et al., 1992), aromatic hydrocarbons production from biomass (Bushe, 1989; Vasllakos and Barreiros, 1984), protein recovery from herbaceous grasses using AFEX treatment (Dale and de la Rosa, 1982), and synthesis of methanol from CO2 byproduct generated by ethanolic fermentation (Hollberg, 1991). Bioenergy research in eastern Canada is currently considering the use of perennial forages and agricultural * Author to whom inquiries should be addressed. Telephone: (418) 656-2131, ext. 3522. Fax: (418) 656-3353. E-mail:
[email protected]. † Universite ´ Laval. ‡ Agriculture and Agri-Food Canada. § Universite ´ de Sherbrooke. S0888-5885(97)00105-X CCC: $14.00
residues as substrates for ethanol and the production of value-added chemicals (Alvo et al., 1996). One strategy to convert perennial forages and agricultural residues into valuable products consists in the fractionation of the whole plant into major constituents: water extractives, hemicelluloses, lignin, and cellulose. The approach is described in Figure 1, based on research and development work by Kemestrie Inc. (Jollez et al., 1997). The aim of this paper was to evaluate specific fractions generated by aqueous/steam fractionation (hemicellulose-rich fraction and cellulosic fines) as substrates for ethanol production. Experimental Section Feedstock. Mature timothy grass (Phleum pratense, Basho cultivar), alfalfa (Medicago sativa, Apica cv.), and reed canary grass (Phalaris arundinacea, Vantage cv.) were mowed in July 1995 and stored as baled hay. Their average water content was 7-8% w/w. Enzymes. Two enzyme complexes derived from the fermentation of selected strains of Trichoderma longibrachiatum were purchased from Genencor International (Rochester, NY). Multifect Cellulase 300 was a soluble powder with a total declared activity of 180190 Genencor Cellulase Units (GCU)/g of powder. Spezyme CP was a liquid with a declared global activity of 90 GCU/mL. Microorganisms and Inoculation. Saccharomyces cerevisiae ATCC 36859 and Pachysolen tannophilus ATCC 32691 were obtained from the American Type Culture Collection (Rockville, MA). Working stock cultures were grown at 30 °C for 24 h on agar slants containing yeast extract, 3 g/L, malt extract, 3 g/L, peptone, 5 g/L, D-glucose, 20 g/L, D-xylose, 20 g/L, and agar, 20 g/L. A loopful of cells was inoculated in sterile 200 mL of the culture medium described previously but without agar. The pH was adjusted to 5.0. The 250mL Erlenmeyer flasks were agitated at 150 rpm for 48 © 1997 American Chemical Society
Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997 4573
Figure 1. Schematics of the integrated approach for biomass conversion by the aqueous/steam fractionation process (Jollez et al., 1997).
Figure 2. Schematic diagram of the aqueous/steam fractionation process generating two residues potentially transformable into ethanol.
h at 25-27 °C. Cells were harvested by centrifugation at 16 390g for 10 min with an RC5C model centrifuge (Sorvall Instruments, Dupont Canada Inc., Mississauga, Ontario). The resulting pellet was resuspended in sterile distilled water to obtain 10 g/L for S. cerevisiae and 6-7 g/L for P. tannophilus. Aqueous/Steam Fractionation. Five kilograms of each biomass were chopped to an average 5-cm particle size, impregnated with water at 25 °C in a 4:1 water: biomass ratio, and pressure filtered (see Figure 2). The water-saturated material was placed in a 4.5 L MVC (mini-vapo-cracker) batch reactor located in Universite´ de Sherbrooke (Sherbrooke, Canada). The water-
saturated material was steam-treated at 220 °C and 2.4 MPa for 120 s. The contents of the vessel were then suddenly released into a receiving container and the defibrillated material was washed at 80 °C with the impregnation liquor, generating a hemicellulose-rich liquor. About 30 wt % of the forages (dry basis) was solubilized in the hemicellulosic solution with a total dissolved solid content varying between 3.0 and 5.5% w/v for the individual forages. These solutions were used as one type of ethanol substrates. The washed defibrillated materal (about 60 wt % of the biomasses) was delignified with NaOH (1:5 NaOH:dry material) at 160 °C for 90 min. A filtration step generated a black lignin-rich liquor and delignified fibers. Refining these fibers generated long and short (fines) cellulose-rich fibers. Only the cellulosic fines (10% dry weight of the original biomass) were used as the other type of ethanol substrates, as 2.2-2.8% w/v suspensions. Both ethanol substrates were frozen and then shipped to Universite´ Laval (Que´bec, Canada) where they were thawed and prepared for hydrolysis. The cellulosic fines were filtered on a Whatman filter no. 4, washed with water, and resuspended in 0.05 M acetate buffer solution (pH 4.85) to their initial concentration. An aliquot of the first filtrate was stored at -30 °C for subsequent sugar analyses. The hemicellulose-rich liquors were centrifuged (AlfaLaval continuous Model LAPX 202, 160 mL/min, 10 000 rpm) in order to remove small amounts of solid particles formed during the freeze-thaw cycle. Hydrogen Peroxide Treatment of Cellulosic Fines. Some of the cellulosic fines were reacted with hydrogen peroxide to further delignify them prior to filtration, washing, and resuspension. The treatment consisted in reacting the cellulosic fines with a H2O2 commercial solution at a level of 1% v/v and 4 M NaOH solution at a level of 0.5% v/v for 20 h at room temperature. The resulting pH 10.5-11.0 of the solution represented the optimum level for delignification, according to Gould (1984, 1985).
4574 Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997
Lime Treatment of the Hemicellulose-Rich Substrates. In order to remove toxic compounds potentially present in the solutions, part of a noncentrifuged hemicellulose-rich liquor was treated with calcium oxide, until the pH of the solution reached 10-11. It was then heated in a boiling water bath at 100 °C for 1 h. After cooling and decantation at ambient temperature, the precipitate was removed by centrifugation at 16 390g for 10 min. The supernatant was adjusted to pH 5.0 by addition of a few drops of concentrated sulfuric acid. Enzymatic Hydrolysis. In general, hydrolysis was done in 250-mL Erlenmeyer flasks with a working volume of either 150 mL for the cellulosic fines (2.42.8% w/v) or 100 mL for the hemicellulose-rich solutions (3.0-5.5% w/v). After the addition of enzymes, flasks were capped and placed in a rotary incubator (Queue Orbital Shaker, Queue Systems Inc., Parkersburg, WV) at 50 °C and 290 rpm. The media were kept sterile with NaN3 (0.005% w/w). One-milliliter samples were withdrawn at specific time intervals, placed in a boiling water bath for 15 min to deactivate the enzymes, and then passed through a 0.2 µm filter. A portion of 0.2 mL was used for measurement of the total reducing sugars by the dinitrosalicylic acid (DNS) method (Miller, 1959) and compared to a glucose standard. Remaining filtrates were stored at -30 °C for subsequent sugar analyses by GC and HPLC. Hydrolysis of cellulosic fines was carried out by adding Multifect Cellulase 300 at levels of 3, 5, or 10 GCU/g of dry fines (0.017, 0.028, or 0.056 g of cellulase/g of dry fines). Concentration of Spezyme CP was kept constant at a level of 2 µL/g of dry fines (0.18 GCU/g of dry fines). The choice of enzyme concentration for fines was based on previous work with whole-plant forages pretreated by ammonia explosion (Belkacemi et al., 1996). After preliminary trials, hemicellulose-rich liquors were found to be hydrolyzed quickly with Spezyme CP only, without Cellulase 300. Levels of 0.01, 0.02, and 0.03% v/v of Spezyme CP were compared; they represented a range between 0.16 and 0.90 GCU/g of a dry hemicellulose-rich substrate. The potential maximum yield of total sugars (TSmax) was evaluated either by strong acid hydrolysis with H2SO4 24.1 N at 30 °C for 30 min followed by weak acid hydrolysis with H2SO4 0.82 N at 120 °C for 55 min (Belkacemi et al., 1996) for the fines or only by weak acid hydrolysis for the hemicellulosic substrates (Belkacemi, 1990). The efficiency of enzymatic hydrolysis was expressed as a percentage of saccharification, i.e., the ratio of actual sugars released (TSt) over the potential maximum yield of sugars, corrected for soluble sugars initially present (TSi) in the nonhydrolyzed substrates. In the case of fines, TSi was found negligible:
saccharification (%) )
TSt - TSi × 100 TSmax - TSi
(1)
When larger quantities of hydrolyzates were required for subsequent fermentations, the initial solutions of fines or hemicellulose-rich liquors were not supplemented with NaN3. In these cases, the fines solutions were hydrolyzed in 500-mL flasks with a working volume of 300 mL whereas the hemicellulose-rich liquors were hydrolyzed in 12-L 316 SS mechanically agitated bioreactors (New Brunswick, Scientific Co. Inc., Edison, NJ) at 300 rpm. The enzyme loadings were those found optimum with the 250-mL flasks experiments.
Fermentation. The hydrolyzates of cellulosic fines containing up 24 g/L of reducing sugars (mainly glucose) were supplemented with 0.25 g/L of KCl and 0.4 g/L of H3PO4 and then autoclaved at 120 °C for 20 min. After the mixture was cooled to 30 °C, 2 g/L of urea and 1 mL/L of a vitamin solution (1.0 g/L of thiamin HCl, 1.0 g/L of calcium pantothenate, 0.6 g/L of biotin, and HCl 0.05 N) were aseptically added to the fermentation medium which was then inoculated at 0.5 g/L with S. cerevisiae or P. tannophilus. The amount of added nutrients were those found optimum and satisfactory for ethanol production by Beck (1986). Separate 150mL Erlenmeyer flasks were prepared for each sample and filled with a working volume of either 100 mL when P. tannophilus was used or 50 mL when S. cerevisiae was used. Flasks were placed in a Model G-53 rotary shaker (New Brunswick Scientific Co. Inc., Edison, NJ) at 75 rpm with the former microorganism or at 150 rpm with the latter, and at ambient temperature (25 °C). Fermentations were conducted in duplicate. Nutrients (KCl, H3PO4, urea, and vitamins) were added to both types of hemicellulosic hydrolyzates (treated or not with lime) at levels similar to those for the fines and in a similar manner. Fermentations were carried out in duplicate, using 1-L Erlenmeyer flasks, shaken at 75 rpm, and incubated at 25-27 °C. Eight hundred milliliters of hydrolyzates (20-23 g/L of sugars, pH ) 4.3-4.8) were inoculated at 1.0 g/L with P. tannophilus. Fermentations were monitored for 2-4 days by removing 5-mL samples for sugar, ethanol, and cells analyses. Analytical Methods. Cellulosic fines were analyzed for lignin, cellulose, ash, and hemicellulose contents using methods described previously (Belkacemi et al., 1996). Furfural in the hemicellulose-rich substrates was determined by a colorimetric method using orcinol and FeCl3 as coloring reagents as well as by HPLC (Waters Associates Inc., Milford, MA) using a Bio-Rad HPX-87P column at the following conditions: 85 °C, 0.5 mL/min, and a water carrier; results were compared with a standard solution of pure furfural (Sigma-Aldrich Canada Ltd., Oakville, Ontario). The protein content of the hemicellulose-rich substrates was determined using the micro-Lowry method (protein assay kit, procedure no. P 5656, Sigma Diagnostics, Sigma-Aldrich Canada Ltd., Oakville, Ontario) using bovine serum albumin as standard. After hydrolysis with 4% H2SO4 at 120 °C for 55 min, neutralization with a saturated Ba(OH)2 solution and centrifugation, a sugar analysis of the hemicelluloserich substrates was performed by HPLC (WAT-085188 Model, Waters Associates Inc., Milford, MA) using a BioRad column (HPX-87P, 85 °C, 0.6 mL/min, nano-pure water carrier) or as silylated sugars using a HP 5890 GC system equipped with an FID detector. Standard oxime (STOX) and (trimethylsilyl)imidazole (TMSI) (Pierce Chemical Co., Rockford, IL) were used as derivatization reagents, while myo-inositol was used as an internal standard. The total reducing sugars of these solutions was also determined by the DNS method of Miller (1959). The same methods were used for a sugar analysis of the enzymatically hydrolyzed samples. Samples from the fermentation experiments were centrifuged with an RC5C model centrifuge (Sorvall Instruments, Dupont Canada Inc., Mississauga, Ontario) at 16 390g for 10 min at 4 °C. Supernatants were analyzed for ethanol, acetaldehyde, and carbohydrates
Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997 4575 Table 1. Composition of Substrates components
reed canary grass (% dry weight)
timothy (% dry weight)
alfalfa (% dry weight)
R-cellulose hemicelluloses klason lignin ash total potential reducinga sugars from acid hydrolysis concentration of dry matter (g/L)
80.2 5.4 6.1 8.3 100.0 94.0 28.0
82.5 7.5 7.4 1.6 99.0 97.9 22.5
80.3 5.3 13.4 1.0 100.0 94.1 28.0
69.4 10.4 4.5 4.5 1.0 10.1 36.0
58.7 13.5 11.5 1.0 0.6 14.7 44.8
52.8 20.8 5.0 3.7 0.7 15.8 32.7
substrate fines
hemicellulose ash substrate
total sugars proteins soluble lignin furfural unknown concentration of dry matter (g/L)
aNote that the values are near the theoretical potential equal to 1.11 × cellulose + 1.12 × hemicelluloses. The factors 1.11 and 1.12 are introduced in order to include water hydration during hydrolysis.
Table 2. Comparative Analysis of Monomeric Sugars in Hemicellulose-Rich Substrates of Aqueous/Steam Fractionated Forages glucose (g/L)
xylose (g/L)
mannose (g/L)
galactose (g/L)
arabinose (g/L)
fructose (g/L)
total sugars (g/L)
reducing sugars (DNS method) (g/L)
NHa AHb
2.0 7.1
2.9 14.3
1.4 4.0
0.5 0.5
0.3 1.7
0.0 1.3
7.1 28.9
24.5
reed canary grass
NH AH
1.5 4.4
7.9 18.1
0.5 0.6
0.5 0.6
0.6 0.7
0.5 0.6
11.5 25.0
25.5
alfalfa
NH AH
1.5 6.2
1.2 7.3
0.2 0.7
0.0 1.4
0.1 0.9
0.1 0.1
3.1 16.6
3.7 17.2
forages timothy
a
Soluble sugars prior to any hydrolysis. b Sugars from weak acid hydrolysis of the substrates.
while pellets were washed with 8% w/v saline (NaCl) solution and then with distilled water, centrifuged again, and dried at 105 °C until they reached constant weight. Ethanol and acetaldehyde were determined by gas chromatography (GC) equipped with an FID detector, an HP 19395A headspace injector, and auto-sampler system. The column used was an HP DB-WAX, 30-m × 0.25-mm capillary column (J. & W. Scientific Inc., Rancho Cordova, CA). Ethyl formate was used as internal standard. Simple carbohydrates (glucose, xylose, arabinose, mannose, and fructose) were determined by HPLC using the system previously described. The oligosaccharides were determined by the HPLC system (Waters 501) with a differential refractometer detector using a REZEX RSO oligosaccharides column (Phenomenex Inc., Torrana, CA) maintained at 85 °C. The carrier water phase was filtered through 0.45 µm, sonicated, and then supplied isocratically to the system at a flow rate of 0.3 mL/ min and 150 psi. Malto-oligosaccharides (Sigma-Aldrich Canada Ltd., Oakville, Ontario) with a degree of polymerization (DP) from 2-7 were used as standards. Results and Discussion Characterization of Substrates. The composition of the three feedstocks was reported elsewhere (Belkacemi et al., 1997). Table 1 shows the main components of cellulosic fines and hemicellulose-rich liquors obtained after aqueous/steam fractionation of the three feedstocks. Cellulosic fines contained 80-83% cellulose on a dry weight basis. Less than 8% hemicelluloses still remained in the fines which also contained 6-14 wt % lignin. Fines from the three feedstocks were considered relatively homogeneous for ethanol production because cellulose was the main source of sugars.
Composition of the hemicellulose-rich liquors was quite different among the three feedstocks. Alfalfaderived liquor was characterized by a higher protein content, up to 2-fold higher than that of the other two liquors. This is not surprising since alfalfa is known as a rich protein source for human and animal feed. Reed canary grass hemicellulose-rich liquor showed a higher sugar content; it also contained 42% more furfural than that of timothy. Reed canary grass would appear to be more sensitive to degradation than the other forages during aqueous/steam fractionation. A fraction of 10-16 wt % of these hemicellulose-rich liquors was not characterized. This fraction probably contained carboxylic acids such as acetic acid released from the acetyl groups in the polymeric chains of the plant hemicellulose polysaccharides (Christov and Prior, 1993). The monomeric sugar composition of these liquors is depicted in Table 2. Glucose and xylose were the major sugars in all cases, whether as soluble sugars or after a weak acid hydrolysis of the liquors. Reed canary grass and timothy grass were richer in xylose than was alfalfa. The ratio of xylose over total sugars were 72.6, 61.2, and 43.9%, respectively. Analyses of the hemicellulose-rich liquors (Table 3) revealed a mixture of complex carbohydrates in soluble polymeric, oligomeric, and monomeric form. This is one of the main features of a hemicellulosic fraction after aqueous/steam fractionation of lignocellulosic plants. Previous work conducted on agricultural residues such as corn stalks and one graminae Stipa tenacissima (Belkacemi, 1990) showed a similar distribution. Sugars in oligomeric form were quantified as degree of polymerization (DP). The oligomers of DP higher than 7 were estimated by a mass balance as explained in Table 3. Oligosaccharides with a DP of 4, 3, and even
4576 Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997 Table 3. Oligosaccharide Analysis of Selected Hemicellulose-Rich Substrates of Aqueous/Steam Fractionated Forages forages
monomers NHa (g/L)
monomers AHb (g/L)
DP2 (g/L)
DP3 (g/L)
DP4 (g/L)
DP5 (g/L)
DP6 (g/L)
DP7 (g/L)
DP > 7c (g/L)
reed canary grass alfalfa
11.5 3.1
25.0 16.6
0.8 0.0
1.8 2.1
5.3 0.0
0.1 0.0
0.0 0.0
0.0 0.0
4.0 10.0
a Soluble sugars prior to any hydrolysis. b Sugars from weak acid hydrolysis of the substrates. c The amount of oligosaccharides with DP > 7 was estimated by mass balance using the following equation:
DP > 7 )
(monomersAH - monomersNH)
7
1.12
∑DP
i
i)2
Table 4. Extent of Saccharification of Lignocellulosic Substrates with and without Peroxide Treatment after 24 h of Hydrolysis extent of saccharification (% of theoretical saccharification) materials
enzyme loading
without H2O2
with H2O2
alfalfa fines reed canary grass fines timothy fines wheat straw corn stalks corn cobs corn husks foxtail alfalfa hay
Multifect Cellulase 300 at 5 GCU/g (28 mg/g of solids) and 2 L/g of Spezyme CP
52.7 79.0 42.8 27.2 49.8 32.1 62.3 27.0 40.9
79.0 82.7 64.0 93.0 100.0 100.0 99.0 81.7 93.6
a
crude Trichoderma reesei cellulases at 200 mg/g of solids
refs this work Goulda (1984)
The lignocellulosic samples were ground to pass a 2 mm screen. The enzymatic hydrolysis was carried out at 45 °C, pH 4.50.
2 were found in the hemicellulose-rich liquor of reed canary grass while less oligomers with a DP e 7 were found in the alfalfa hemicellulose-rich liquor. Also, 50% of potential total sugars found in the reed canary grass hemicellulose-rich liquor were released in monomeric form during aqueous/steam fractionation against only 20% from alfalfa proving, once again, that reed canary grass was more sensitive to thermomechanical and chemical degradation induced by aqueous/steam fractionation. Enzymatic Hydrolysis. (a) Hydrolysis of Fines. Saccharification of the cellulosic fines (Figure 3) hydrolyzed with Cellulase 300 and 2 µL of Spezyme CP/g of fines reached about 40% of the theoretical yield for alfalfa and timothy substrates after 30 h of reaction, at a Cellulase 300 concentration of 3 GCU/g whereas saccharification was more pronounced (60%) for reed canary grass. An increase to 10 GCU/g accelerated the rate of hydrolysis and the extent of saccharification reached a maximum of 60% for timothy, 70% for alfalfa, and 80% for reed canary grass after 30 h of reaction. Increasing the time of reaction above 30 h led to a leveling-off effect and sometimes even to a decrease in saccharification. This behavior might be partly attributed to end-product inhibition and to the lignin content of the fines, more important for alfalfa fines. All hydrolysis profiles were characterized by a relatively high initial rate of saccharification followed by a gradually slower rate after a few hours of reaction. The initially rapid rate of cellulose hydrolysis has been associated with the easy access of the enzymes to the substrate’s surface as reported by Gregg and Saddler (1996). These authors also suggested that lignin remaining on cellulosic fibers acts as a physical barrier throughout hydrolysis and hinders intimate contact between the substrate and the active sites of enzymes. In order to investigate this hypothesis, lignin was removed by a treatment with hydrogen peroxide. Table 4 reports saccharification yields after 24 h. The greatest effect is shown with alfalfa fines where both the initial rate of enzyme hydrolysis and the maximum sacchari-
Figure 3. Hydrolysis of cellulosic fines with Multifect Cellulase 300 and 2 µL of Spezyme CP/g of fines at 50 °C and pH 4.85: [, 3 GCU; 9, 5 GCU; 10 GCU/g of dry fines.
fication, up to 80% of the potential, were increased. As expected, peroxide had a reduced effect on timothy and reed canary grass since fines of these two feedstocks had lower initial levels of lignin (Table 1). The results of Gould (1984) of highly increased enzymatic hydrolysis of various feedstocks after peroxide treatment are also reported in Table 4. The susceptibility of cellulose to enzymes was enhanced up to 3-fold for milled feedstocks
Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997 4577
Figure 5. Time course of mono- and oligosaccharides during enzymatic hydrolysis of the hemicellulose-rich substrate of reed canary grass with 0.01 % v/v of Spezyme CP: 0, xylose; ], glucose; 9, DP2; [, DP3; 2, DP4; 4, DP5; b, DP6.
Figure 4. Hydrolysis of hemicellulose-rich substrates with Spezyme CP at 50 °C and pH 4.85; ], 0.01 % v/v; 9, 0.02% v/v; 2, 0.03% v/v.
(Gould, 1984) and up to 1.5-fold (Gould, 1985) for fines obtained by aqueous fractionation. Ramos et al. (1993) also confirmed that enzymatic hydrolysis is facilitated for steam-exploded Eucalyptus viminalis chips after alkaline peroxide treatment. (b) Hydrolysis of Hemicellulose-Rich Substrates. Figure 4 shows the saccharification profile for the hemicellulose-rich liquor of the three feedstocks with varying concentrations of Spezyme CP (preliminary studies showed that no Multifect Cellulase 300 was required). Up to 80-90% of saccharification was obtained after only a few hours of reaction. After 10 h, there was no significant difference in saccharification profiles for the three levels of Spezyme CP, indicating that this enzyme was sufficiently active and efficient at 0.01% v/v level. At this level of enzymes, reed canary grass reached 90% saccharification after only 2 h of reaction since 50% of the soluble sugars were already in the form of monomers. Activity assays conducted on Spezyme CP revealed that this commercial enzyme contained several hydrolytic activities, among them xylanolytic and β-D-xylosidic, which act synergistically as demonstrated by Poutanen and Puls (1989) in the hydrolysis of wheat straw arabinoxylan using purified enzymes of Trichoderma reesei. Oligosaccharides released and hydrolyzed by the Spezyme CP enzyme complex showed the transient nature of these sugars (Figure 5). It must be remembered that the final desired products were the monomeric sugars. Fermentation. (a) Fermentation of Cellulosic Fines Hydrolyzates. The fermentation profiles are
Figure 6. Fermentation profiles of cellulosic fines hydrolyzates from aqueous/steam fractionated forages: (a) substrate from alfalfa with S. cerevisiae; (b) substrate from timothy with P. tannophilus; 0, sugars; 9, ethanol; 2, dry cells; b, acetaldehyde.
reported on Figure 6 for hydrolyzates of alfalfa and timothy grass fines using S. cerevisiae and P. tannophilus, respectively. In the case of alfalfa (Figure 6a), fermentation was nearly completed after 10 h where 98% of the total initial reducing sugars were transformed into ethanol and acetaldehyde (10 and 0.25 g/L, respectively). Acetaldehyde reached a maximum of 0.4 g/L after 24 h and disappeared completely after 48 h. S. cerevisiae cells grew rapidly in the first 6 h of fermentation (reaching 1.5 g/L), remained constant during the production phase of acetaldehyde, and underwent a second growth period until a final concentration of almost 2.5 g/L. The constant 10 g/L of ethanol produced represents 85% fermentation efficiency. This efficiency is based on the theoretical yield of 0.51 g of ethanol/gram of total initial sugars. Average ethanol productivity over the first 8-h period was 1.30 g/L‚h. This compares well with those reported by Miyakawa et al. (1986) ranging between 1 to 2 g/L‚h. Productivities for continuous fermentation systems are much higher.
4578 Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997 Table 5. Fermentation by Saccharomyces cerevisiae and Pachysolen tannophilus of Cellulosic Fines Hydrolyzates performance S. cerevisiae forage alfalfa reed canary grass timothy
P. tannophilus
productivityb sugar uptake yielda productivityb sugar uptake yielda (% initial content) (g of EtOH/g of sugar) (g of EtOH/L‚h) (% initial content) (g of EtOH/g of sugar) (g of EtOH/L‚h) 93.4 95.4 97.1
0.4 0.4 0.5
1.3 1.3 1.3
99.4 99.5 99.4
0.4 0.5 0.4
1.0 1.0 1.0
a Ethanol yields reported are based on total sugar uptake after 24 h of reaction. b Productivities are reported after 8 h of reaction for S. cerevisae and 6 h for P. tannophilus.
Values up to 30 g/L‚h were obtained by Miyakawa et al. (1986) and Sola` et al. (1986) working with the same substrates. P. tannophilus also metabolized sugars from a cellulosic substrate of timothy as depicted in Figure 6b. On the basis of an initial sugar concentration of 17 g/L, the ethanol production of 7 g/L represents 82% efficiency. A very weak production of acetaldehyde was detected, and the final concentration of this product was 0.1 g/L. Growth of P. tannophilus showed two distinct periods, similar to those of S. cerevisiae, and reached 2.4 g/L after 30 h of reaction. A further increase in the reaction time led to a decrease in cell growth, probably due to substrate limitation or to some inhibitory effect associated with the continuous presence of acetaldehyde. P. tannophilus took longer times (24 h) to produce an ethanol level above an 80% theoretical yield than did S. cerevisiae (8 h). Ethanol productivities with P. tannophilus averaged 0.7 g/L‚h during the first 8 h and 0.3 g/L‚h over the first 24 h. As a comparison, ethanol production ranging between 70-86% of theoretical yield and productivities between 0.521 and 0.656 g/L‚h were obtained by Doran and Ingram (1993) when fermenting enzymatically-hydrolyzed crystalline cellulose (Sigmacell 50, Sigma Co.) by Klebsiella oxytoca strain P2 containing chromosomally-integrated Zymomonas mobilis genes. Table 5 summarizes results of fermentation of enzymatically-hydrolyzed cellulosic fines from the three forage crops studied. These hydrolyzates were well fermented by both yeasts. (b) Fermentation of the Hemicellulose-Rich Substrates. The ability of P. tannophilus to ferment sugars found in hemicellulose-rich substrates is illustrated in Figure 7a for reed canary grass. Poor fermentation was observed as 78% of the total sugars remained in the broth of the nontreated hydrolyzate even after 50 h of reaction. The substrate was apparently toxic, as shown by a decrease in the yeast concentration. Less than 20% of ethanol’s theoretical yield was obtained and traces of acetaldehyde were detected. When the hydrolyzate was treated with CaO, slight improvements were noticed (Figure 7b). Cell growth was observed and the concentration reached 2.4 g/L after 30 h of reaction. Sugar uptake was more pronounced and about 52% of total sugars were consumed. However, ethanol production was not improved. In the present experiment, the main goal of liming was to neutralize acidic components in the hydrolyzate and to remove some of the inhibitory products such as phenolic and polyphenolic compounds by precipitation with divalent calcium ions. However more than 60% of the furfural initially present remained in the hydrolyzate and might account for the inhibition. These results shed no more light on the subject of liming than those of McMillan (1994) who reported conflicting results concerning the extent to which inhibitory components were removed by liming. According to him,
Figure 7. Fermentation of non-treated and CaO-treated hemicellulose-rich substrate of reed canary grass by P. tannophilus: 0, sugars; 9, ethanol; 2, dry cells; b, acetaldehyde.
liming removed furfural, metal ions, and acetic acid in some cases while in others the concentration of these inhibitory compounds was unchanged. In all hemicellulose-rich hydrolyzates, poor performance of fermentation was obtained with P. tannophilus despite reports to the contrary with pure xylose and glucose as carbone sources (Schneider et al., 1981; Dekker, 1982; Slinninger et al., 1982, 1987; Du Preez and Prior, 1985). However, the hemicellulose-rich liquors obtained after a high-temperature high-pressure treatment are known to contain various compounds such as furfural and its derivatives and phenolic compounds, namely vanilin, that can strongly inhibit fermentation to ethanol (Clark and Mackie, 1984; Tran and Chambers, 1985; Ando et al., 1986; McMillan, 1994; Delgenes et al., 1996; Palmqvist et al., 1996). In order to explain the poor ethanolic conversions from the hemicellulose-rich substrates, synthetic solutions containing a mixture of pure xylose (28 g/L) and glucose (12 g/L) were supplemented (results not shown here) with furfural, vanillin, and m-cresol at 0.001-1 g/L. A P. tannophilus ATCC 32691 strain was shown
Ind. Eng. Chem. Res., Vol. 36, No. 11, 1997 4579
to be very sensitive to these components even at concentrations lower than 0.01 g/L. Since furfural in the presently studied hemicellulose-rich substrates varied between 0.1 and 0.3 g/L, the presence of this component alone could partially explain the poor performance of the yeast. Another possible explanation could be catabolic repression of xylose-fermenting yeasts by glucose (Panchal et al., 1988). The same phenomenon was also reported by Takahashi et al. (1994) for ethanolic fermentation of a mixture of pure arabinose, xylose, and glucose by E. coli ATCC 11303. Conclusion The ethanolic potential of two residues (cellulosic fines and hemicellulose-rich solutions) from perennial forages, fractionated to recover valuable long cellulose fibers for paper-making, was found partially interesting. Cellulosic fines were hydrolyzed at 60-70% of saccharification using 5 GCU (0.028 g) of Multifect Cellulase 300 and 0.18 GCU (2 µL) of Spezyme CP by gram of dry matter. The extent of saccharification increased to more than 80% after treatment with caustic hydrogen peroxide. Fermentation of the resulting hydrolyzates led to 84% of theoretical ethanol yield. On the other hand, the hemicellulose-rich substrates were easily hydrolyzed to monomeric sugars with 80-90% saccharification yield using only 0.01% v/v of Spezyme CP (2 µL/g of dry matter), but fermentation of these solutions was poor and appeared inhibited by compounds released during the aqueous/steam fractionation. According to these results, up to 380 L of ethanol per dry tonne of cellulosic fines and 60 L of ethanol per dry tonne of hemicellulose-rich substrates could be expected from the studied low initial substrate concentrations used in both the hydrolysis and the fermentation steps. Commercial operations, however, would require concentrated residues to attain ethanol concentration around 10%. The accumulation of steam explosion lignin- and sugar-related byproducts prior to the hydrolysis of the hemicellulose-rich substrates would certainly inhibit this step and would require costly processes to neutralize and circumvent their inhibitory effects. Moreover, even if improved, the post-treated substrates may not allow efficient hydrolysis because of end-products inhibition, more apparent in concentrated solutions. Ethanol production from these liquors thus appears problematic, and alternate valorization routes may have to be considered. Ethanol production from cellulosic fines, however, could easily be integrated within the “biorefinery” concept since no inhibitory steam explosion byproducts are recovered with them and high yields of ethanol can be obtained. Acknowledgment This work was supported by a research contract from the Government of Canada within its Green Plan project under the supervision of Agriculture and Agri-Food Canada. The authors thank Dr. Paul Nadeau for his valuable assistance with HPLC analyses and Dr. Real Michaud for advice and supervision of the project. H. Gauvin and F. Gaumond are acknowledged for their contributions to the preparation of the substrates. Nomenclature DP: degree of polymerization TSmax: total sugars released after acid hydrolysis of hemicellulose-rich substrates (g/L)
TSi: total soluble sugars initially present in the hemicellulose-rich substrates (g/L) TSt: total sugars enzymatically released at a given time of hydrolysis (g/L) EtOH: ethanol
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Received for review January 31, 1997 Revised manuscript received June 4, 1997 Accepted June 11, 1997X IE970105J
Abstract published in Advance ACS Abstracts, September 15, 1997. X
