Introduction

Lignocelluloses of plant cell walls are composed of cellulose, hemicellulose, pectin and lignin. The major sugars produced after hydrolysis of lignocellulosic biomass are glucose, galactose, mannose, xylose, and arabinose. Efficient conversion of biomass to ethanol requires microorganisms with the ability to ferment the sugars generated after hydrolysis. The yeast, Pichia stipitis, produces ethanol from glucose, galactose, mannose, xylose, and cellobiose with high ethanol yields and low amounts of xylitol (Dellweg et al. 1984; du Preez et al. 1986).

One promising technology for converting lignocellulosic biomass to ethanol is the enzyme-based process, where enzymes are used to hydrolyze the fibers after pretreatment. Dilute sulphuric acid pretreatment at high temperatures extensively hydrolyzes the hemicellulose to soluble sugars (Schell et al. 2003; Fenske et al. 1998). The residual acid after pretreatment is neutralized with alkalis such as Ca(OH)2 (van Zyl et al. 1988; Eken-Saracoglu and Arslan 2000), NH4OH (Alriksson et al. 2005; Persson et al. 2002) and NaOH (Nilvebrant et al. 2005). In some cases, these alkalis are added in excess to reduce inhibitor concentrations in dilute acid pretreated biomass (Tran and Chambers 1986; Nigam 2001a, b).

Although the inhibitors generated after dilute acid pretreatment can decrease ethanol production (Delgenes et al. 1996; Tran and Chambers 1986), Persson et al. (2002) have shown that the improved fermentability after alkali treatment are difficult to explain by the removal of inhibitors only. Therefore, the salts produced after neutralizing the excess H2SO4 with alkalis could play a role in the fermentation. The purpose of this study was to determine how CaSO4, Na2SO4, and (NH4)2SO4 salts affect the cell growth, xylose consumption, ethanol production, ethanol yield, and xylitol production in Pichia stipitis.

Materials and methods

Microorganism

Pichia stipitis CBS 6054 was generously supplied by Dr. Thomas Jeffries of the Forest Products Laboratory, USDA. The cells were grown overnight in a filter-sterilized fermentation medium containing per liter: 1.7 g yeast nitrogen base (without amino acid or ammonium sulfate), 2.27 g urea, 6.56 g peptone, and 20 g xylose. The cells were centrifuged at 3,000g for 5 min and resuspended in 5 ml sterile water to serve as inoculum.

Media and fermentation

Three different salt media were prepared by adding 3.3 ml H2SO4 (7.6 M) to 25 ml Ca(OH)2 (1 M), 50 ml NH4OH (1 M) or 50 ml NaOH (1 M). The salts were diluted with distilled water to a total liquid volume of 200 ml and a final pH of 6.0. The control was 200 ml distilled water at the initial pH of 6.1. Xylose, 12 g, was dissolved in each solution to give 60 g/l. Each sugar solution was filter-sterilized using a 0.2 μm filter. Nutrient solution (50× the concentration used) was prepared by dissolving 1.7 g yeast nitrogen base, 2.27 g urea and 6.56 g peptone in 20 ml water. Fermentations were performed in sterile 125 ml Erlenmeyer flasks (with 0.2 μm vent cap) in at 30°C and shaken at 100 rev/min. Each Erlenmeyer flask contained 50 ml sugar media, 1 ml nutrient solution, and 2 ml inoculum. All these experiments were performed in triplicate at the same initial cell concentration of 1.5 g/l.

Analytical methods

Samples, 1 ml, were periodically removed for analyses. The concentrations of xylose, xylitol, and ethanol were determined using an Agilent HPLC System with an analytical Bio-rad Aminex HPX–87H column and a Bio-rad Cation H refill guard column. The cell concentrations were determined as OD600 values; an OD of 1 = 0.23 g of dry cells/l.

Results and discussion

Effect of the salts on cell growth

The growth of Pichia stipitis on different salt media is shown in Fig. 1 and the pH during fermentation is shown in Fig. 2. Xylose consumption is shown in Fig. 3 and ethanol production in Fig. 4. A synopsis of the key fermentation data is given in Table 1. Pichia stipitis reached its final cell concentration after 90 h (Fig. 1). An initial cell concentration of 1.5 g/l grew to different final cell concentrations on the different salt media after 118 h of fermentation (Fig. 1). Inhibition by (NH4)2SO4 on the growth of Pichia stipitis is in agreement with observations by Guebel et al. (1992). The medium containing (NH4)2SO4 had the lowest cell concentration. Cell concentration on Na2SO4 medium was lower than the control initially (t < 40 h), and became higher towards the end (Fig. 1). The initial pH for all the salts and control was 5.5–6.0 and the final pH was 4.0–4.5. Studies on Saccharomyces cerevisiae suggest that the initial response of cells to saline conditions is the efflux of water, which leads to cell shrinkage (Blomberg 2000). Pichia stipitis might also respond similarly to Saccharomyces cerevisiae on Na2SO4 medium, since the cells did not grow well initially (compare to control). The highest cell growth was on CaSO4 medium. Guebel and Nudel (1994) obtained maximum cell growth rates at low Ca2+ (0.34 mM) concentrations and low growth at high Ca2+ (1 mM) concentrations. The high cell growth of Pichia stipitis in our study might be because of low Ca2+ in solution due to the poor solubility of CaSO4.

Fig. 1
figure 1

The effect of salts on cell growth of Pichia stipitis. The control was fermentation with no salts added. CaSO4, Na2SO4, and (NH4)2SO4 were produced by adding 3.3 ml H2SO4 (7.6 M) to 25 ml Ca(OH)2 (1 M), 50 ml NH4OH (1 M), and 50 ml NaOH (1 M) respectively, and adding distilled water to a final liquid volume of 200 ml. Pichia stipitis fermentations in 60 g xylose/l at an initial cell concentration of 1.5 g/l in a shake flask incubator at 30°C. Cell concentrations were determined from OD600 values using Cary 3C UV-Visible spectrophotometer *Error bars are ± 1 std

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Fig. 2
figure 2

Effect of salts on pH during fermentation using Pichia stipitis. This graph shows pH of samples taken at various time points using Orion portable pH meter *Error bars are ±1 std

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Fig. 3
figure 3

The effect of salts on xylose consumption by Pichia stipitis *Error bars are ±1 std

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Fig. 4
figure 4

The effect of salts on ethanol production by Pichia stipitis*Error bars are ±1 std

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Table 1 Average fermentation parameters on the effect of salts on xylose fermentation by P. stipitis after 140 h of fermentation
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Effect of the salts on xylose consumption and ethanol production

The xylose consumption was initially (t < 60 h) faster on the CaSO4 medium but towards the end, xylose consumption was faster in the (NH4)2SO4 medium (Fig. 3). The fast rate of xylose consumption initially in CaSO4 medium can be attributed the high cell growth in Fig. 1. However, towards the end of the fermentation (t > 90 h), xylose consumption slowed down in all the treatments apart from the (NH4)2SO4 medium (Fig. 3). This makes the xylose consumption of the entire fermentation period (140 h) higher in the (NH4)2SO4 medium compared to the other treatments.

The highest ethanol concentration was produced in the (NH4)2SO4 medium and the lowest ethanol concentration was in the CaSO4 medium (Fig. 4). The low ethanol concentration in CaSO4 medium is due to high cell biomass production, because the xylose was used to produce cell mass instead of ethanol. Although (NH4)2SO4 medium produced the highest ethanol concentration, the cell biomass produced was the lowest. The NH3 produced from the dissociation of NH +4 stimulate ethanol production in Pichia stipitis (Guebel et al. 1992). Xylose consumption and ethanol production in the control and Na2SO4 medium were similar (Figs. 3 and 4).

The highest ethanol yield was in the control 0.39 g/g, which is not significantly different from 0.38 g/g for (NH4)2SO4 medium, whereas the ethanol yield was 0.35 g/g and 0.36 g/g in CaSO4 and Na2SO4 media respectively (Table 1). Ethanol yield per g of cell was higher on (NH4)2SO4 compared to the other treatments because of stimulatory effect of ammonia on ethanol production in Pichia stipitis and the low cell concentration in (NH4)2SO4 medium. Xylitol yield in CaSO4 medium, 0.02 g/g was higher than all the other treatments. The control and (NH4)2SO4 media had a xylitol yield of 0.01 g/g whereas there was no xylitol in Na2SO4 medium. CaSO4 stimulated xylitol production, (NH4)2SO4 had no effect on xylitol production and Na2SO4 inhibited xylitol production in Pichia stipitis.

Conclusion

Xylose consumed after 140 h of fermentation was highest in the (NH4)2SO4 medium compared to the other treatments. The maximum ethanol concentration after 140 h of fermentation was 18.9 g/l in the (NH4)2SO4 medium whilst the lowest ethanol concentration was 15.7 g/l in the CaSO4 medium. The production of ammonia from (NH4)2SO4 enhanced ethanol production by Pichia stipitis, and therefore ethanol yield per g of cells was 3.3 g/g. The salts produced after neutralizing the excess H2SO4 with alkalis such as Ca(OH)2, NaOH and NH4OH have an effect on the cell growth, xylose consumption, ethanol production, ethanol yield, and xylitol production in Pichia stipitis.