Effects of selected by-products of an acid hydrolyzate on cell growth and ethanol fermentation by Saccharomyces cerevisiae.

ABSTRACT

Acid hydrolysis of lignocellulosic biomass results in the release of various sugars as well as inhibitory compounds such as furfural, 5-hydroxymethylfurfural (HMF), vanillin, and vanillic acid that could inhibit microbial growth and ethanol fermentation. During the process of acid hydrolysis of pinewood sawdust conducted in this study, heavy metals such as chromium (Cr) were released from the extruder of the processing reactor. The purpose of this research was to study the effect of selected by-products in an acid hydrolysate on cell growth and ethanol fermentation of Saccharomyces cerevisiae. Furfural, HMF, vanillin, vanillic acid, and Cr were added at different concentrations to fermentation media. S. cerevisiae growth and ethanol yield were measured after 18 hours of incubation. The results showed that individual presence of furfural, HMF, vanillin, and vanillic acid at concentrations as low as 1.0, 2.0, 2.0, and 0.5 mg/ml, respectively, significantly inhibited ethanol production, while Cr did not show any inhibitory effect at concentrations up to 20 ppm. Effects of the mixture of furfural, HMF, vanillin, vanillic acid, and Cr on ethanol yield and cell growth were also studied. Results showed that at low concentration such as 0.5 mg/ml, the by-products stimulated not only the S. cerevisiae growth but also the ethanol production. At 1.0 mg/ml, ethanol production and cell growth were significantly inhibited. Co-existence of Cr did neither enhance nor reduce the inhibitory/stimulating effect.

INTRODUCTION

There is a growing demand for the already depleting energy resources for fuel. One of the alternative solutions to the problem is ethanol production, which is a cost effective and environmentally friendly process of producing fuel (Luo et al., 2002). Processing method and feedstock are the determining factors in ethanol production costs. Currently in the U.S., corn is the primary feedstock for ethanol production and accounts for 60% of the total production costs (Kim and Dale, 2002). The use of feedstock, such as lignocellulosic biomass that has no or low commercial value, could significantly reduce ethanol production costs. The processing of lignocellulosic biomass to obtain the monomeric sugars needed for fermentation is one of the major factors contributing to the non-competitive costs of lignocellulose derived ethanol. The conversion of the polysaccharides in lignocellulose to monosaccharides can be achieved by dilute or concentrated acid hydrolysis. The drawback of acid hydrolysis is that it produces a product (acid hydrolysate) that contains not only the sugars needed for fermentation but also furan and phenolic compounds as well as weak acids that could inhibit fermentation and microbial growth.

The known by-product compounds found in acid hydrolysate include furfural and 5-hydroxymethyl furfural (HMF) formed from high temperature degradation of pentose and hexose sugars respectively (Larsson et al., 1999; Palmqvist et al., 1999; Taherzadeh et al., 1997, 2000). Others include phenolics formed from the partial breakdown of lignin (Ando et al., 1986; Jonsson et al., 1998; Larsson et al., 1999) and weak organic acids such as acetic acid as well as formic and levulinic acids that are formed from the breakdown of HMF and furfural (Larsson et al., 1999; Martin and Jonsson, 2002; Martinez et al., 2001). The presence of furfural at 1 mg/ml was reported to decrease the C[O.sub.2] evolution (Sanchez and Bautista, 1988), the cell multiplication (Azhar et al., 1981), and the total viable cell count (Chung and Lee, 1985) in the early phase of fermentation by yeast. HMF was an inhibitor to the growth of S. cerevisiae, but not as strong as furfural (Sanchez and Bautista, 1988). An early study showed that HMF at the concentration of 1 mg/ml had an inhibitory effect on baker's yeast (Ingram et al., 1955). A more recent study reported that only at higher concentration (2 mg/ml), HMF decreased bacterial biomass yield by 23% (Boopathy et al., 1993). Vanillin was one of the strongest inhibitors among the hydrolysis by-products (Delgenes et al., 1996). It significantly inhibited ethanol production by Saccharomyces carlsbergensis W34 at 5 mg/ml (Pfeifer et al., 1984). When the concentration was < 1 mg/ml, vanillic acid had no effect on ethanol fermentation by S. cerevisiae (Ando et al., 1986).

To extract sugars from lignocellulose of pinewood sawdust in this study, a twin-screw extruder reactor was used for continuous impregnation with sulfuric acid. In our previous study (Zhang et al., 2005), we reported that the growth and fermentation ability of S. cerevisiae were severely inhibited by undiluted and slightly diluted (eg., 100 X) acid hydrolyzates. Extreme acidity was speculated to be a major reason for the inhibition. Recently we found out that besides various organic by-products, certain heavy metals such as chromium (Cr) were also released into the hydrolyzate due to acid corrosion of stainless steel of the extruder. Indeed, we found that there was up to 16 ppm of Cr present in the hydrolyzate.

Although studying the inhibitory effect of individual by-products is important for formulating hydrolyzate production, synergism could occur due to the presence of multiple by-products in most hydrolyzates. Therefore, it is also important to study the effect of the presence of multiple by-products on cell growth and ethanol fermentation by S. cerevisiae. The objectives of this study were: (1) to determine the effect of individual organic by-products and chromium on cell growth and ethanol fermentation of S. cerevisiae; and (2) to determine the effect of the by-products mixture on cell growth and ethanol fermentation of S. cerevisiae.

MATERIALS AND METHODS

Chemicals and Microorganisms. 2-furaldehyde (furfural), 5-hydroxymethylfurfural (HMF), vanillin, vanillic acid, and water (HPLC grade) were purchased from Sigma Chemical Co. (St. Louis, MO). Chromium nitrate [Cr(N[O.sub.3])[.sub.3]9[H.sub.2]O] was purchased from Fisher Scientific (Houston, TX). The fermentation medium (Atlas, 1995) was prepared and consisted of 1% trypticase, 0.1% beef extract, 0.5% dextrose and 0.5% NaCl (pH 6.4). To prepare the inoculum, S. cerevisiae (ATCC # 765) cells were incubated in potato dextrose broth for 18 h at 28 [degrees]C, on a rotary shaker at 200 rpm.

Effects of the by-products on growth and ethanol production Furfural, HMF, vanillin, vanillic acid, and chromium were detected in our hydrozylates (unpublished data). To study the effects of these individual organic by-products on the growth and ethanol fermentation, 10 ml of S. cerevisiae cell suspension containing about [10.sup.7] cells were transferred to a 250-ml flask containing 100 ml fermentation medium. Furfural, HMF, vanillin, and vanillic acid were added separately to achieve the final furfural concentrations of 0, 1, 2, and 4 mg/ml, plus final HMF, vanillin, and vanillic acid concentrations of 0, 0.5, 1, and 2 mg/ml. Chromium nitrate was added to achieve the final chromium concentrations of 0, 5, 10, and 20 ppm. The flasks were incubated at 30 [degrees]C for 18 hours before samples were withdrawn for cell count and ethanol concentration analysis. To study the effects of the organic by-products mixture on the growth and ethanol fermentation, different amounts of furfural, HMF, vanillin, and vanillic acid were added together to achieve the final concentrations of 0, 0.5, or 1 mg/ml each of the organic by-products in the mixture solution. The chromium nitrate was also added to achieve the final concentrations of 0, 8, and 16 ppm. In this mixture, S. cerevisiae was inoculated and samples were incubated as described in the aforementioned procedure.

The colony count was carried out on potato dextrose agar plates (Benson, 1994). The ethanol concentration was determined by gas chromatography (Shimadzu GC-14A, Shimadzu Corp., Kyoto, Japan) equipped with a flame ionization detector. The oven temperature was 125 [degrees]C and injector temperature was 230 [degrees]C.

Chromium Analysis The paste from the extruder was treated with water in a 1:1 weight ratio and filtered using a glass Buchner funnel having 10-20 [micro]m porocity. The chromium concentration in the filtrate was analyzed by atomic absorption spectroscopy (Shimadzu AA-6701F Atomic Absorption Flame Emission Spectrophotometer). Several standard solutions of various concentrations were prepared for quantitative measurement of chromium concentrations.

Statistical Analysis Samples were run in triplicate. All results are expressed as mean [+ or -] SD. Differences between treatment groups were tested by one-way analysis of variance (ANOVA) using the SAS System for Windows (V8, SAS Institute Inc., Gray, NC). Differences at p< 0.05 are considered significant.

RESULTS AND DISCUSSION

To maximize ethanol production from fermenting sugars in the acid hydrolyzate by S. cerevisiae, the by-products from acid hydrolysis of lignocellulose must be characterized to assure that they are not toxic to microbial growth and ethanol production. In this study, the effects of the selected by-products which were commonly present in our acid hydrolyzate were tested.

The results indicated that furfural at concentrations as low as 1 mg/ml resulted in a significant reduction of ethanol production (p<0.05) (Fig. 1) compared to the control (0 mg/ml). Ethanol production decreased from 0.273% (control) to 0.188% (1 mg/ml furfural), indicating that ethanol production was inhibited 31%. This result was in agreement with the report by Modig et al. (2002) who showed that furfural could inhibit glycolytic enzymes at > 1 g/1. At 2 mg/ml, furfural also inhibited the growth of S. cerevisiae. For example, with the addition of 2 mg/ml furfural, only 18% of the yeast cells survived. It is also interesting that at lower concentrations such as 1 mg/ml, furfural stimulated the growth of S. cerevisiae. Specifically, cell numbers increased significantly from 8.4 x [10.sup.6] CFU/ml (control) to 1.17x [10.sup.7] CFU/ml (1 mg/ml group; p< 0.05). We speculated that at a lower concentration furfural could be used as a growth substrate for S. cerevisiae.

[FIGURE 1 OMITTED]

The HMF was less inhibitory than the furfural. At 2 mg/ml, it significantly inhibited the ethanol production (p< 0.05), while there was no significant effect on cell growth at the test concentration (p> 0.05) (Fig. 2). The results also showed that vanillin and vanillic acid at 2 and 0.5 mg/ml, respectively, significantly inhibited ethanol production (p< 0.05) (Fig. 3 and 4). The inhibitory concentrations were lower in our experiment as compared to some other studies which showed that vanillin inhibited ethanol production by Saccharomyces carlsbergensis W34 at 5 mg/ml (Pfeiter et al., 1984) and vanillic acid had no effect on ethanol fermentation by S. cerevisiae when the concentration was < 1 mg/ml (Ando et al., 1986). It was also shown that at lower vanillic acid concentrations such as 0.5 mg/ml and 1 mg/ml, vanillic acid stimulated the growth of S. cerevisiae. Such stimulating effect was not detected with lower concentrations of vanillin. We speculated that lower concentrations of vanillic acid could also be used as a growth substrate for S. cerevisiae.

[FIGURE 2 OMITTED]

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[FIGURE 4 OMITTED]

It was reported that some microelements including Zn, Cu, Cr, and Fe might influence the stability of cell membranes, as well as the synthesis of nucleic acids and stability of the double helix of DNA while forming hydrogen bonds (Beran et al., 1995; Davis and Vincent, 1997; Zetic et al., 2001). It was also reported that the addition of 200 mg/1 Cr[Cl.sub.3] (66 ppm chromium) into the medium stimulated both the yeast growth and the ethanol production (Zetic et al., 2001). In this study, the results showed that chromium at a concentration as high as 20 ppm had no effect on either ethanol production or microbial growth (Table 1). Chromium found in our acid hydrolyzate sample was under 20 ppm. Therefore, chromium released from the extruder reactor did not have any adverse effect on ethanol fermentation activity and cell growth or S. cerevisiae.

Effect of the by-products mixture including chromium on ethanol production and colony-forming units was also performed in this study. Results showed that at the concentration of 1.0 mg/ml, ethanol production and cell growth were significantly inhibited (p< 0.05) (Fig. 5). Ethanol production decreased from 0.277% (control) to 0.01% (1.0 mg/ml by-products mixture), reflecting a 96.4% inhibition on ethanol production. At the same time, with the addition of the 1.0 mg/ml inhibitors mixture, only 23.4% cells survived the inhibition. While at lower concentrations such as 0.5 mg/ml, the by-products stimulated not only the S. cerevisiae growth but also the ethanol production. Cells increased from 4.67x[10.sup.6] CFU/ml (control) to 1.37x[10.sup.7] CFU/ml (0.5 mg/ml by-products mixture). Ethanol production increased from 0.277% (control) to 0.294% (0.5 mg/ml by-products mixture), reflecting a 6.5% enhancement of ethanol production. Although the addition of 0.5 mg/ml and 1.0 mg/ml of the by-products decreased the pH of the medium from 6.4 to 5.0 and 4.5, respectively, the pH change should not have contributed to the difference in ethanol production and cell growth. The result of a separate experiment indicated that growth and ethanol production were the same in the control medium with pH 4.5, 5.0 and 6.4 (data not shown). Therefore, the changes of ethanol production and cell growth were caused by the by-products instead of pH. Results also showed that no effect on ethanol production and cell growth was caused by the co-existence of chromium. With the final chromium concentration of 0, 8, and 16 ppm, no change in the stimulating/inhibition pattern in ethanol production and cell growth was observed (Fig. 5). This indicates that chromium did not enhance nor reduce the inhibitory effect under the test condition.

[FIGURE 5 OMITTED]

Processing method and feedstock are considered as the determining factors in ethanol production costs. Use of sawdust, a lignocellulosic biomass of no or low commercial value, could significantly reduce ethanol production costs and provide an environmentally friendly renewable energy source. However, production of various inhibitors by acid hydrolysis indicates the need for treatment before ethanol fermentation. The costs for the treatment must be reasonably low to make this route of ethanol production price-competitive. Treatment with resins is being studied in our laboratory and will be reported in the near future.

CONCLUSIONS

Our results indicated that individual presence of furfural, HMF, vanillin, and vanillic acid at as low as 1.0, 2.0, 2.0, and 0.5 mg/ml, respectively, significantly inhibited ethanol production, while Cr did not show any inhibitory effect at concentrations up to 20 ppm. At low by-products concentrations such as 0.5 mg/ml, the mixture of furfural, HMF, vanillin, and vanillic acid stimulated not only the S. cerevisiae growth but also the ethanol production, while at the concentration of 1.0 mg/ml, the ethanol production and cell growth were significantly inhibited. Co-existence of chromium did neither enhance nor reduce the inhibitory/stimulating effect of the organic by-products.

ACKNOWLEDGEMENTS

This research was supported by: (1) the U.S. Department of Energy #DE-FG02-00ER45830 with subcontract to JSU through Mississippi State University; (2) NIH-Rise # GM 067122 (to JSU); (3) NIH-SCORE # S06GM 08047 (to JSU); and (4) NSF-HBCU-UP # HRD-0411559.

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Jun Gao (1), Yi Zhang, Jennifer Ntoni (1), Maria F.T. Begonia (1), Ken S. Lee (2), Lenore Hicks (3), Wayne W. Hwang (3), and Huey-Min Hwang (1*)

(1) Department of Biology and (2) Department of Chemistry Jackson State University, Jackson, MS 39217 (3) Jackson Preparatory School, 3100 Lakeland Dr., Flowood, MS 39232

*Author for correspondence: Department of Biology, Box 18540, 1400 Lynch Street, Jackson State University, Jackson, MS 39217. E-mail: [e-mail] Tel: [phone] Fax: [phone]

Table 1. Effect of chromium on ethanol production and colony-forming
units (CFU) of S. cerevisiae grown in potato dextrose broth amended with
different amounts of chromium. Samples were triplicated.

Chromium (ppm)  CFU(x [10.sup.6]/ml)  Ethanol (%)

 0              21.2[+ or -]1.7       0.36[+ or -]0.006
 5              20.9[+ or -]5.7       0.35[+ or -]0
10              22.8[+ or -]2.8       0.35[+ or -]0
20              26.6[+ or -]4.8       0.34[+ or -]0