Effect of an acid hydrolyzate of southern pine softwood on the growth and fermentation ability of yeast Saccharomyces cerevisiae.

An alternative energy source could come from microbial fermentation of the sugars present in acid hydrolyzates derived from agricultural biomass. The fermenting microbes must be tolerant of acidic conditions and able to ferment sugars present in the hydrolyzates. Saccharomyces cerevisiae has been shown as an ideal microorganism that can ferment hexose sugars present in lignocellulose-derived hydrolyzates. The objective of this study was to assess the effect of the acid hydrolyzate of a southern pine softwood sawdust on the viability and fermentation capability of S. cerevisiae. Acid hydrolyzate of the sawdust (pH -0.63) was diluted with fermentation media at 100, 300, 400, and 800 fold. Four-day old cultures of S. cerevisiae (ATCC #765) were then added. During the 96-hour incubation (30 [degrees]C), turbidity of the medium, colony count and ethanol production were monitored. Viable number of cells and ethanol production in 300X- and 400X-diluted groups were significantly higher than other groups in the first 24 hr and reached the maximum after 24 hr and 48 hr of incubation, respectively. In a separate experiment pH of the fermentation medium was adjusted with [H.sub.2]S[O.sub.4] (SA) to simulate those of the 300X and 400X diluted acid hydrolyzate (AH) groups. The results indicated that the colony counts of both AH300 and SA300 were significantly higher than the other groups after 48 hours, with AH300 being the highest among all groups. However, this pattern was not paralleled by ethanol production activity. Thus ethanol fermentability of S. cerevisiae exists in the extreme environment such as diluted acid hydrolyzate.

Keywords: acid hydrolyzate, ethanol, fermentation, Saccharomyces cerevisiae

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Fossil fuels have been the major resource to meet the demanding need for energy. With the constant increase of energy consumption and the constant use of nonrenewable fuel chemicals, there is a need to find a more economical, environmentally friendly and renewable energy source (Sun and Cheng, 2002). Research has proven that ethanol meets these qualifications. The fact that ethanol is a byproduct of microbial fermentation makes it a renewable energy source. As an oxygenated fuel, ethanol is environmentally friendly. The major challenge is how to produce ethanol economically. Ethanol production costs have been the limiting factor in the ability of gasohol and ethanol blended fuels to compete with current gasoline cost. Processing methods and feedstock are the major factors driving ethanol cost and of the two, feedstock contributes over 50% of the total cost. Production costs can significantly be reduced with use of low cost feedstock (Mani et al., 2002; Zerbe, 1992). Lignocellulosic biomass, such as sawdust and wood chips, is of low commercial value but can provide inexpensive sources for ethanol production. Lignocellulosic biomass is usually composed of three primary constituents including cellulose, hemicellulose and lignin (Reshamwala et al., 1995; Cheung and Anderson, 1997; Boopathy, 1998; Dewes and Hunsche, 1998). Although wood residues such as sawdust and woodchips are readily available, the sugars suitable for fermentation processes and alcohol yield are only found in cellulose and hemicellulose constituents. Cellulose contains hexose sugars (mainly glucose) and hemicellulose contains pentose sugars (mainly xylose). In order to obtain the sugars needed for fermentation pre-treatment of lignocellulosic biomass is necessary.

The most widely used pre-treatment method is acid hydrolysis. Concentrated acid hydrolysis, one of the acid hydrolysis methods, involves use of concentrated acid (sulfuric acid, [H.sub.2]S[O.sub.4] or hydrochloric acid, HCl) in the presence of high temperature (190 [degrees]C-215 [degrees]C) to split cellulose and hemicellulose into simple sugars (Springfield and Hester, 1999). The concentration of sugars tends to be higher if the lignocellulosic biomass is treated with extreme acidity and high temperature; however, the products of acid hydrolysis (acid hydrolyzate) become very acidic and may contain a variety of inhibitory compounds such as furfural, 5-hydroxymethyl-furfural (5-HMF), acetate and other phenolic compounds (Luo et al. 2002; Palmqvist and Hahn-Hagerdal 2000). In order to maximize recycling of the acid present in the hydrolyzate, it is desirable to use acid-tolerant microorganisms to conduct ethanol fermentation process. Saccharomyces cerevisiae has been shown as the yeast that can best ferment hexose sugars present in lignocellulose-derived hydrolyzates due to its ethanol-producing capacity and high inhibitor to lerance (Hahn-Hagerdal et al., 1991; Olsson and Hahn-Hagerdal, 1993). Even though S. cerevisiae grows optimally around pH 5 (ATCC 2001 Product Information Sheet for ATCC #765), one study showed that the yeast is able to grow at a pH as low as 2.5 (Taherzadeh et al., 1997b). The objective of this study was to assess the effect of the acid hydrolyzate of a southern pine softwood sawdust on the viability and potential fermentation capability of S. cerevisiae. The ultimate goal is to develop a specific acid hydrolysis procedure to minimize the amount of inhibitory hydrolyzate constituent for S. cerevisiae and to maximize ethanol fermentation within the most tolerable acidity level. Consequently, the cost for recycling the sulfuric acid and the associated impact on environmental health would be reduced.

MATERIALS AND METHODS

A 4 day old culture of Saccharomyces cerevisiae (ATCC#765) was used in the experiment. The 4 day old culture was prepared by rehydrating the freeze-dried culture in 5 mL sterile distilled water overnight and then transferred to potato dextrose broth (24 g/L, pH 4.2) for enrichment (ATCC 2001 Product Information Sheet for ATCC #765). According to chemical analysis with the method developed by National Renewable Energy Laboratory (NREL) at Golden, Colorado (Ruiz and Ehrman, 1996), pre-treatment of southern pine softwood sawdust by acid hydrolysis ([H.sub.2]S[O.sub.4]) yielded an acid hydrolyzate (pH -0.63) that contained the following sugars and other organic compounds: 26.65 mg/mL glucose, 6.7 mg/mL mannose, 4.48 mg/mL xylose, 2.78 mg/mL galactose, 1.4 mg/mL arabinose, and 1.64 mg/mL acetic acid. Xylitol, lactic acid, glycerol, ethanol, HMF and furfurals were not detected. In Pyrex flasks containing 90 mL fermentation media (Atlas, 1995) (1% trypticase, 0.1% beef extract, 0.5% dextrose and 0.5% NaCl, pH 6.4), 10 mL of S. cerevisiae (in PDB, cell density 15X[10.sup.5]/mL) was added. A different amount of acid hydrolyzate was added to the flasks to reach a final concentration of 100, 300, 400, and 800 fold dilution of the acid hydrolyzate. The flask containing only fermentation media and the cells served as the control. Fermentation flasks were incubated at 30 [degrees]C and sampled at time points 0, 24, 48, 72, and 96 hours. The following assays were conducted: (1) turbidity of the growth medium (Benson, 1994); (2) colony count on potato dextrose agar plate (Benson, 1994); and (3) ethanol production. Briefly, turbidity (Klett unit) was measured with the photoelectric colorimeter at spectral range 520-580 nm (Tube Model 800-3, Scienceware, Bel-Art Products, Pequannock, NJ). Concurrently pH and colony count (CFU/mL) on potato dextrose agar were recorded. The samples were then filtered through a 0.2 [micro]m HT Tufftyn[R] membrane filter (Acrodisc syringe filter, Pall Corp., Ann Arbor, MI) and alcohol production was measured by gas chromatograph (Shimadzu GC-14A, Shimadzu Corp., Kyoto, Japan) with a flame ionization detector (FID). Samples were separated on a 30 m X 0.53 mm fused-silica capillary column with 1.0 [micro]m thickness film. The oven temperature was programmed as follows: 40 [degrees]C (0.3 min), 40-85 [degrees]C (20 [degrees]C/min), and 85-125 [degrees]C (40 [degrees]C/min). Injector temperature was 230 [degrees]C. Injection volume was 0.1 mL.

In a separate experiment the pH values of the 300- and 400-fold diluted acid hydrolyzate were simulated by adding 0.6 and 1 mL of 2N [H.sub.2]S[O.sub.4] to the Pyrex flasks containing the mixture of 90 mL fermentation media and 10 mL S. cerevisiae, respectively. Various sugars present in the diluted acid hydrolyzate were added at the corresponding concentrations. The same assays were conducted following the aforementioned procedures. Difference in the experimental data between the treatment groups or between a treatment group and its relevant control group is determined with either SAS or Student's t-test (p < 0.05).

[FIGURE 1 OMITTED]

RESULTS AND DISCUSSION

One of the requisites for efficient growth and ethanol production is the tolerance of the fermenting microbe (S. cerevisiae) to the wood hydrolyzate. However, our previous experiments showed that the undiluted wood hydrolyzate (pH -0.63) inhibited the growth and the fermentation ability of S. cerevisiae in the media containing undiluted hydrolyzate (data not shown). Due to the toxicity of the undiluted acid hydrolyzate, the hydrolyzate was diluted with fermentation medium up to 800 fold. After 1, 2, 3, and 4 days of incubation, the mean pH, growth in terms of Klett unit and number of colony forming units per mL of medium, and percent ethanol production for each treatment (or dilution) and for the control were determined. As evidenced by the pH data of the control group (yeast in fermentation medium without acid hydrolyzate), fermentation process decreased pH from the initial value of 5.8 to 4.6 and stabilized it around 4.6 within the 4-day incubation period. Consistent pH values were also observed for the other groups, with the 100X group maintaining the lowest pH number (Figure 1a). Concurrent measurements of viability, biomass and ethanol production levels are indicated in units of Klett turbidity unit (Figure 1b), cfu X [10.sup.5]/mL (Figure 1c) and percent ethanol production (Figure 1d), respectively. All numbers of the 100X group remained low throughout the incubation period, indicating that cellular growth and fermentation activity of the yeast culture were inhibited by the 100 X-diluted acid hydrolyzate (pH 1.8-2.0). At extracellular pH values below the pKa value of acetic acid (4.75), the undissociated acid can diffuse through the cellular membrane and dissociate intracellularly. ATP consumption rate will increase and hence the growth rate will decrease when the proton pump process is activated to maintain a constant intracellular pH (Taherzadeh et al., 1997a).

[FIGURE 2 OMITTED]

Viable number of cells (expressed as colony forming units X [10.sup.5]/mL of medium) and ethanol production of the test yeast in 300X- and 400X-diluted groups were significantly higher than other groups in the first 24 hr and reached the maximum after 24 hr and 48 hr of incubation, respectively (Figure 1c & 1d). Figure 1b indicates that Klett units of the 300X and 400X-diluted groups are the highest among all groups; however, the turbidity units of all groups continued to increase during the incubation period with the exception of 100X group. Although clinically Klett colorimetry has been extensively used to monitor the growth curve of microorganisms, the turbidity scale does not necessarily correlate with active or living biomass of the test microorganisms, except during the exponential growth phase. The experiment was repeated several times and the yeast populations consistently grew and fermented better in the 300-400X groups. Yeasts exhibit a considerable tolerance to extremes of pH. They are capable of maintaining active fermentation in glucose solution between a pH range of 2.4 to 7.4 (http://www.home.earthlink.net, August, 2004). For optimum production of ethanol, routine practice dictates that the pH of the fermenting medium is maintained within the range of 4.0 to 6.0. The explanation to the test yeast's ability to grow and ferment in 300X, 400X- and 800X-diluted groups (pH 3.0-4.2; Figure 1a & 1d) is that the intracellular pH of the yeast cells remains quite constant at about pH 5.8, regardless of the wide pH variations in the surrounding environment. The enzymes involved in fermentation thus operate in an optimum pH environment within the yeast cells. Addition of acetate to a growth medium during anaerobic continuous and batch cultivations was shown to lower the biomass yield while enhancing the ethanol yield by S. cerevisiae (Verduyn et al., 1990; Taherzadeh et al., 1997a). The increase in ethanol yield was speculated to occur due to the uncoupling effect of acetic acid on ATP reductions, provided that byproduct formation does not increase during the sugar to ethanol conversion process (Taherzadeh et al. 1997b). Our results partially contradict those findings. In the case of 300X and 400X-diluted groups in our batch study, both biomass and ethanol production rates were significantly enhanced in 24 hours (SAS; p < 0.05). The net effect of acetate on biomass yield or ethanol formation rate may vary, depending on the growth conditions or whether the specific growth rate is affected (Taherzadeh et al., 1997a). Further studies are needed to confirm the effect of acetic acid on biomass yield and ethanol production.

In a separate experiment the pH of the fermentation medium was adjusted with [H.sub.2]S[O.sub.4] (SA) to simulate those of the 300X- and 400X-diluted acid hydrolyzate (AH) groups (Figure 2a). Colony count and ethanol production of the SA and AH groups were compared (Figures 2b and 2c). Colony counts of both AH300 and SA300 were significantly higher than the other groups at 48 hours (Figure 2b), with AH300 being the highest among all groups (p < 0.05; Student's t-test). However, this pattern was not paralleled by the ethanol production (Figure 2c) in which AH300 and SA300 were indistinguishable from other treatments in the first 48 hr. Except for acetic acid, all sugars in the diluted acid hydrolyzate were added to the 300X and 400X SA groups. Since various sugars and acetic acid were the only prevalent organic species detected in the acid hydrolyzate, the significant increase in the number of viable cells of the 300X groups could not be attributed to the presence of acetic acid. Apparently more study is needed to understand the complicated growth energetics of S. cerevisiae in the presence of acid hydrolyzate. Results of this study indicate that the ethanol fermentation capability of the yeast exists in the extreme environment such as diluted acid hydrolyzate. However, low sugar concentrations in the diluted acid hydrolyzate could not support the test yeast's fermentation activity. Other pre-treatment methods, such as overliming and ion exchange, were reported to result in higher ethanol productivity (Palmqvist and Hahn-Hagerdal 2000). Comparison studies on the effects of the aforementioned pre-treatment methods on sugar concentrations and ethanol production will be conducted in the near future.

ACKNOWLEDGEMENTS

This research was supported by: (1) the U.S. Department of Energy #DE-FG02-00ER45830 with subcontract to JSU through Mississippi State University and (2) U.S. Department of the Army # DAAD 19-01-1-0733 (to JSU). We thank Dr. Roger Hester of the Department of Polymer Science, University of Southern Mississippi for providing acid hydrolyzate and Dr. Bonnie R. Hames of the National Renewable Energy Lab, Golden, Colorado for chemical analysis of acid hydrolyzate. We also thank Ms. Ana L. Balarezo for providing technical assistance in this study.

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Yi Zhang (1), Huey-Min Hwang (1,2), Maria F.T. Begonia (1), Ken Lee (3), and Kui Zeng (1)

(1) Department of Biology, Jackson State University, 1400 Lynch Street, Jackson, MS 39217; and (3) Department of Chemistry, Jackson State University, 1400 Lynch Street, Jackson, MS 39217

(2) Author for correspondence: TEL: [phone]; E-mail: [e-mail]