Substrate and Product Inhibition on Yeast ... - ACS Publications

Jan 27, 2015 - Program in Environment and Ecology, Faculty of Science and Engineering, Meisei University, 2-1-1, Hodokubo, Hino, Tokyo. 1918506, Japan...
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Substrate and Product Inhibition on Yeast Performance in Ethanol Fermentation Qi Zhang,† Deyi Wu,† Yan Lin,*,† Xinze Wang,† Hainan Kong,† and Shuzo Tanaka‡ †

School of Environmental Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Program in Environment and Ecology, Faculty of Science and Engineering, Meisei University, 2-1-1, Hodokubo, Hino, Tokyo 1918506, Japan

‡

S Supporting Information *

ABSTRACT: A batch fermentation utilizing Saccharomyces cerevisiae BY4742 was conducted to determine the inhibitory effects of highly concentrated substrate and product levels on yeast. Experiments were performed to determine the largest dosage of substrate and the largest product concentration that the yeast could tolerate in a very high gravity fermentation process. The yeast’s growth and fermentation activities were characterized by changes in the biomass and ethanol yield under different substrate and product concentrations during fermentation. All of the experiments were performed at a pH of 5.0 and a temperature of 35 °C with a stirring rate of 180 r/min and a fermentation time of 96 h. Furthermore, five cycles of acclimatization were conducted to improve the yeast’s tolerance to ethanol. Ethanol yield was maximized at 95% with a product concentration of 39 g/L and substrate dosage of 80 g/L. The system exhibited an obvious increase in cell growth and ethanol production with increasing substrate dosage up to a critical point of 160 g/L glucose (53 g/L ethanol fermented and an ethanol yield of 65%). Above this point, cell growth and ethanol production were inhibited with the final product concentration increasing only slightly with an increase in the initial substrate concentration. The end product (ethanol) was shown to be the primary factor inhibiting yeast growth and fermentation activity because the yeast would completely stop growing and fermenting when the initial exogenous ethanol concentration exceeded 70 g/L. The endogenous ethanol exerted a greater impact on yeast performance during anaerobic fermentation than exogenous ethanol. Five cycles of acclimatization significantly improved the yeast density, cell morphology, and ethanol production during very high gravity fermentation. The ethanol yield increased from 6% to 30% under an initial exogenous ethanol concentration of 60 g/L.

1. INTRODUCTION Lignocellulosic ethanol, a typical second-generation biofuel, is gaining increased public attention for several reasons: it can be directly used as a liquid fuel, it can be produced from inexpensive and abundant sources, it does not compete with food production as some other biofuels may, and it may potentially decrease dependence on fossil fuels, aiding international energy security.1,2 Ethanol fermentation, which converts the products of cellulose hydrolysis (glucose and other residual sugars) into ethanol, is the key procedure in the process of producing ethanol fuel from lignocellulose and determines the ultimate yield.3 Yeast such as Saccharomyces cerevisiae (S. cerevisiae), the main microorganism of anaerobic ethanol fermentation, plays an important role in determining the ultimate yield and financial benefit of lignocellulosic ethanol. Of all the fermentation technologies, very high gravity fermentation (VHGF) is expected to possess the most benefits due to associated water and energy savings that allows greatly increased plant capacities. As a result, VHGF may decrease costs and improve the economic benefits of ethanol fermentation.4,5 Nevertheless, the high concentrations of the substrate and end products present in VHGF severely inhibit the performance of yeast, limiting the production of ethanol and restricting the promotion of industrialized production of ethanol from lignocellulose.4,6 Therefore, it is necessary to study the inhibitory effects of the product and substrate on the yeast © XXXX American Chemical Society

during anaerobic ethanol fermentation to discover the maximum allowable dose of substrate while decreasing the influence of critical inhibitory factors. This knowledge will improve the ultimate ethanol production potential of VHGF and accelerate the industrialization of lignocellulosic ethanol.

2. MATERIALS AND METHODS 2.1. Materials. 2.1.1. Yeast Strain. S. cerevisiae BY4742, the yeast used in this study, was preserved in the Biology Laboratory of School of Environmental Science and Engineering (SESE), Shanghai Jiao Tong University (SJTU) at −80 °C. This strain was originally from EUROSCARF, Germany and offered by the Department of Chemistry, Meisei University, Japan. 2.1.2. Culture Medium.7,8 Yeast extract peptone dextrose (YPD) liquid medium, which consists of 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose, was used. The solid culture medium used for plate screening contained 10 g/L agar in addition to the YPD medium. According to the experimental design, certain concentrations of ethanol were added to the YPD medium for acclimatization. All of the culture media were maintained at a pH in the range of 4.0 to 4.5 and sterilized at 121 °C for 25 min before use. 2.1.3. Fermentation Medium.9,10 Glucose was selected as the fermentation substrate because it accounts for more than 70% of the hydrolysate-reducing sugar in the hydrolysis process, the step prior to fermentation.11 The other fermentation medium was added into the Received: October 17, 2014 Revised: January 27, 2015

A

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Energy & Fuels liquor at the beginning of each fermentation experiment to ensure the high activity of yeast cells. This medium contained the following: 3.00 g/L NH4Cl, 0.70 g/L KH2PO4, 0.35 g/L MgSO4·7H2O, 0.10 g/L CaCl2, 0.10 g/L NaCl, 0.11 g/L MnSO4·4H2O, 1.0 mg/L CuSO4· 5H2O, 21.0 mg/L ZnSO4·7H2O, 4.0 mg/L CoSO4·7H2O, 40.0 mg/L H3BO3, 0.2 mg/L Na2MoO4·2H2O, and 0.14 g/L FeSO4. 2.2. Experimental Design. The goal of the experiment was to investigate key factors involved in inhibiting the efficiency of bioethanol production from lignocellulose during VHGF such as very high concentrations of substrate and product. The research focused on studying the influence of these inhibitory factors on yeast growth and fermentation activity and attempted to identify the optimal substrate dosage and maximum product concentration that the yeast could tolerate. Further efforts were made to improve the yeast’s viability and activity in the associated harsh environment through acclimatization. The cell growth, glucose consumption, and ethanol production analyses were performed under different initial substrate and/or ethanol concentrations during fermentation with all other parameters controlled. The glucose concentrations used in the substrate inhibition experiments were the following: 40, 80, 120, 160, 200, and 280 g/L. These experiments were conducted at pH 4.0 and a temperature of 35 °C with an agitation rate of 180 r/min and an initial yeast cell concentration of 2 g/L in the fermentation medium. The total fermentation time for each batch was 96 h, and the samples were removed from the reactor every 24 h. All of the fermentations were performed in a 1 L automatic anaerobic reactor. Before the experiment, the recovered yeast was cultured in YPD medium at 37 °C, shaking at 180 r/min for 16 to 18 h until a sufficient number of yeast cells for the fermentation tests were produced. The initial ethanol concentrations used in the product inhibition experiments were as follows: 0, 10, 20, 30, 40, 50, 60, 70, and 80 g/L. In these experiments, the substrate concentration was 80 g/L, and the other parameters were the same as in the substrate inhibition experiments. Additionally, the average yeast-specific growth and ethanol fermentation rates during the first 12 h of fermentation under the same endogenous ethanol (produced by yeast itself) and exogenous ethanol (added at the beginning of the fermentation) concentrations were compared to identify the differences between their inhibitory effects. Furthermore, the yeasts were subjected to five cycles of acclimatization to achieve higher tolerance to ethanol. In each cycle, 1 mL of 50 g/L yeast was inoculated into 200 mL of culture medium that contained a certain amount of ethanol. After 24 h of cultivation at 35 °C and 180 r/min, 10 mL of the broth was transferred to 200 mL of culture medium containing a higher concentration of ethanol. After three passages in one acclimatization cycle, plate spreading and streaking were performed to screen, separate, and purify the acclimatized yeast. The purified yeast was cultivated for a contrast test and the next round of acclimatization.12,13 The ethanol concentrations in each culture medium were the following: cycle one, 20, 25, and 30 g/L; cycle two, 30, 35, and 40 g/L; cycle three, 35, 40, and 45 g/L; cycle four, 40, 45, and 50 g/L; and cycle five, 45, 50, and 55 g/L. 2.3. Analytical Methods. Cell samples from the fermentation were diluted 1:5 before the test. The yeast cell growth was determined by measuring optical density (OD) at 600 nm using a spectrophotometer (UV2600, SHUNYUHENGPING, Shanghai, China).14 The cell concentration of the samples was obtained using a calibration curve, as shown in eq 1

A = 0.14804 + 1.19501CY

Japan). An Aminex HPX-87P column (Bio-Rad, USA) with a safe guard column was used at 80 °C, and pure-grade water was used as the mobile phase for the separation at a flow rate of 0.6 mL/min. Ten microliters of each of the samples was injected after being filtered through a 0.45 μm membrane.16 All of the HPLC tests were performed at the Instrument Analysis Center of SESE, SJTU. Cell morphology was observed through an inverted microscope (X71, OYMPUS, Kyoto, Japan). 2.4. Calculation. The maximum theoretical ethanol concentration from cellulose was calculated according to the stoichiometric relationship represented by eq 2, i.e., 100 g of glucose could theoretically produce 51.1 g of ethanol. The ethanol yield (defined as the practical ethanol concentration in the fermentation experiments versus the theoretical concentration based on the total initial glucose concentration) was calculated according to eq 3 as follows C6H12O6 → 2CH3CH 2OH + 2CO2

(2)

YE = CE /(CG × 0.511) × 100%

(3)

where YE is the ethanol yield, CE is the ethanol concentration of the fermentation liquid (g/L), and CG is the concentration of glucose added at the beginning of fermentation (g/L). The yeast-specific growth rate (defined as the increase of cell mass per unit cell mass per unit time) and the ethanol fermentation rate (defined as the increase of ethanol per unit cell mass per unit time) were calculated according to eq 4 and eq 5, respectively

μ = (1/CY ) × (dCY /dt )

(4)

RE = (1/CY ) × (dCE /dt )

(5)

where μ is the yeast-specific growth rate (1/h), RE is the ethanol fermentation rate (gethanol/(gyeast·h)), CY is the yeast concentration (g/ L), dCY is the change in the yeast concentration (g/L), dCE is the change in the ethanol concentration (g/L), and dt is the reaction time (h).

3. RESULTS AND DISCUSSION 3.1. Substrate Inhibition of Ethanol Fermentation. The hypertonic environment caused by excessive levels of substrate (glucose) could weaken the viability and fermentation-ability of yeast. During VHGF, if the substrate concentration is higher than a certain value, the product (ethanol) and yeast concentrations will not increase with additional substrate, wasting resources, and energy.6,17 Figure 1 shows the changes in the yeast growth in response to different initial substrate concentrations. Under a low glucose concentration, the final yeast concentration grew considerably with the increase of initial substrate concentration

(1)

where A is the sample absorbance at 600 nm, and CY is the cell concentration (g/L). The glucose and ethanol concentrations were determined by highperformance liquid chromatography (HPLC), utilizing the protocol introduced in Pessani’s and Zhang’s research.15,16 The fermentation liquid was diluted 1:10 and analyzed by HPLC (LC-10AD, SHIMADZU, Kyoto, Japan). The HPLC instrument was equipped with a refractive index detector (RID-10A, SHIMADZU, Kyoto,

Figure 1. Changes in the yeast concentration under different initial substrate concentrations. B

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Figure 3 describes the changes in the ethanol yield throughout the fermentation process for different initial

up to 160 g/L. This growth was observed because glucose is not only the most important carbon source for yeast cells but is also an important signal molecule for yeast. Yeast can change their own enzyme expression and activity according to their environment in order to achieve the ideal metabolism for growth and the optimal density based on the substrate concentration.18 This essentially means that the yeast grew better as a result of increased availability of a carbon source in their environment. When the initial concentration of glucose was greater than 160 g/L, a very high concentration of glucose, the yeast rapidly increased their population density to accelerate their substrate consumption rate in order to improve the severe fermentation environment. Therefore, the cell concentration grew significantly during the first 24 h. However, the yeast concentration ultimately stopped increasing and even slightly decreased as the glucose concentration increased. This is most likely due to a significantly slowed yeast growth rate during the later period as a result of damage induced by the hypertonic solution on membrane fluidity and enzyme activity. These phenomena illustrate the suppression of yeast growth and reproduction induced by the hypertonic solution containing very highly concentrated substrate.6 As shown in Figure 2, YX/S, yield of biomass on substrate (cell growth per unit mass of substrate consumption, gyeast/gglucose)

Figure 3. Comparison of the ethanol yield under different initial substrate concentrations.

substrate concentrations. As shown in Figure 3, it could be inferred that 80 g/L is the optimal substrate concentration, because at this concentration, the ethanol yield reached 95% within 72 h and produced ethanol to a concentration of 39 g/L. In this experiment, high fermentation temperatures (around 35 °C) facilitated an increased rate of the fermentation process that, when scaled up, could mean decreased costs associated with cooling systems. As seen in Figures 1 and 3, the cell and product concentrations continued to increase with increasing substrate concentration. The maximum yield of 80% obtained over the whole fermentation time with substrate concentrations greater than 80 g/L was found with a substrate concentration of 120 g/L at 72 h. However, this value is still acceptable for industrial production, because the most important factor in industrial production is the total production per unit of time, corresponding to the concept and advantages of VHGF.4 Although the yield was slightly lower, 20% more product could be produced compared to initial substrate concentration of 80 g/L within the same time period without experiencing a waste of materials. The study of the substrate inhibition of yeast was carried out to determine the most appropriate substrate dosage with which optimal production of ethanol and high substrate utilization could still be obtained. As seen in Figures 3 and 4, when the substrate concentration was 160 g/L, the product concentration was 53 g/L, and the yield at 96 h decreased from 95% to 65%. As shown in Figure 4,

Figure 2. Fitting of the average YX/S for different initial substrate concentrations.

decreased with an increase in the initial glucose concentration, C0, following eq 6: YX / S = −0.0074ln(C0) + 0.052

(6)

Equation 6 also demonstrates the inhibition of anaerobic ethanol fermentation induced by a high substrate concentration. YX/S decreased with an increase in the substrate concentration because, under the severe fermentation conditions produced by the high substrate concentration, additional energy and carbon sources were consumed by the yeast not for growth or fermentation but instead for survival in the overly hypertonic environment.19 Long-term exposure to a hypertonic solution can lead to a severe decrease in cell membrane fluidity, making it more difficult for the substrate to enter and for the product to exit the cell.17 Therefore, additional carbon sources were consumed by the yeast to maintain the activity of the transport system of essential materials instead of being fermented to the final product ethanol.

Figure 4. Changes in the maximum ethanol concentration under different initial substrate concentrations. C

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Energy & Fuels the final ethanol concentration increased with an increase in the initial substrate concentration during the fermentation. However, the final product concentration almost stopped increasing when the substrate concentration was higher than 160 g/L. This indicates that, of all the substrate concentrations used in the experiment, 160 g/L is the critical point where concentrations beyond this value did not improve the fermentation, only resulting in a waste of resources. Because the yeast produced more ethanol during the later period of the VHGF process than the beginning, the long-term accumulation of endogenous ethanol and the high substrate concentration may concurrently inhibit the yeast fermentation capacity. According to Figure 3, when the substrate concentration was greater than 160 g/L, the ethanol yield decreased markedly, and the final biomass concentration did not increase. This resulted in a waste of both resources and time. The ethanol yield decreased to less than 40% when the initial substrate concentration was 280 g/L. Additionally, the maximum ethanol concentration obtained was 55 g/L, only 2 g/L higher than that obtained with an initial substrate concentration of 160 g/L indicating that the yeast fermentation activity was seriously restrained. The ethanol yield for a substrate concentration of 280 g/L was only 40%, 55% lower than the yield obtained at a substrate concentration of 80 g/L. The results were similar to those of previous studies, which showed that the yeast fermentation ability is significantly inhibited by substrate concentrations greater than 150 g/L.19 The following reasons may explain the inhibition of yeast growth and fermentation ability by a high substrate concentration. As discussed above, the decrease in membrane fluidity caused by hypertonic solutions may lead to the accumulation of ethanol and other toxic metabolites in the cells, causing biological damage to the yeast cell including damage to its transport and metabolism systems. Additionally, the excessive loss of water from the cell might result in cell atrophy and organelle dehydration.6,17 However, the high substrate concentration may alter the activity of the intracellular enzyme activities, reducing substrate utilization and ethanol fermentation as well as influencing yeast growth and reproduction. For instance, hexokinase (HK) and pyruvate kinase (PK) are the key enzymes of the Embden-MeyerhofParnas (EMP) pathway. HK converts glucose into glucose-6monophosphate in the first step of the EMP pathway. This step is incremental in determining the speed of the subsequent reactions. PK catalyzes the conversion of phosphoenolpyruvate to pyruvic acid. Therefore, the energy supply of the cell is mainly determined by the activities of PK and HK. In this study, the highly concentrated substrate greatly restricted the normal expression of HK and PK and thus directly reduced the flux intensity of the EMP pathway. The metabolic disorder of glucose is also expected to inhibit downstream ethanol fermentation and yeast growth.20−22 Moreover, because fermentation is a comprehensive system that is affected by various factors, the overaccumulation of the product during the later period of VHGF also synergistically inhibits the yeast’s growth and growth activity. This may happen in response to excess product interfering with key enzymes of the glucose transport system and damaging the cell structure, particularly the membrane components and permeability.23 Further exploration of the inhibitory effect of the product alone is required. Based on the discussion above, it could be concluded that, of all the experimental concentrations used, 160 g/L was the

highest substrate concentration that could be adopted in industrial ethanol fermentation because the yeast growth and fermentation capacity would be tremendously inhibited at initial substrate concentrations above this level. 3.2. Product Inhibition. As a toxic metabolite, ethanol strongly inhibits yeast cell growth and ethanol production, which limits the production of products.24,25 During the late stage of VHGF, synergistic and coinhibitory effects involving the substrate and product concentrations on the yeast’s performance exist. To measure the inhibition induced by ethanol alone, an artificial fermentation system with a low concentration of substrate but a high product concentration was simulated. Figure 5 shows the impacts of the initial concentration of ethanol on yeast growth. A significant decrease in the cell

Figure 5. Changes in the yeast concentration under different initial ethanol concentrations.

growth was observed with an increase in the exogenous ethanol concentration. For ethanol concentrations lower than 30 g/L, the maximum yeast concentration still increased by more than 70% of its initial concentration, and the yeast completed growth after 24 h. This indicates that the ethanol did not strongly suppress cell growth. However, at a higher ethanol concentration of 30−50 g/L, the suppression was markedly more severe. The cell growth decreased with an increase in the exogenous ethanol concentration within this range. Compared with the control group, the increase in the yeast density decreased from 110% to 30%, and the adaptable phase of the cells correspondingly increased from less than 24 to 48 h. This indicates a significant ethanol-induced inhibition of the yeast cells. For ethanol concentrations greater than 50 g/L throughout the fermentation period, the cells experienced a slight increase in growth, i.e., less than 10% after 96 h. The yeast stopped growing and reproducing when the ethanol concentration was greater than 70 g/L. Large exogenous ethanol concentrations inhibited the fermentation capacity much more seriously than the yeast growth. As illustrated in Figure 6, the glucose conversion rate (defined as the proportion of glucose utilized by yeast) decreased markedly with an increase in the exogenous ethanol concentration. In the control group, all of the substrate was consumed within 96 h. The substrate utilization rate decreased from 100% to 60% when 10 g/L of ethanol was initially added, and the glucose utilization rate decreased to 30% when the initial ethanol concentration was 20 g/L. The yeast failed to metabolize any glucose when the initial ethanol concentration was greater than 70 g/L. D

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concentration reached 40 g/L, its inhibitory effect was so severe that the ethanol yield maintained a very low value and almost did not respond to the change of substrate concentration. Figure 7 further shows that while both the product and highly concentrated substrate could inhibit the yeast fermentation activity, the product was the key inhibitory factor. In the artificial fermentation system, ethanol severely inhibited the yeast growth and fermentation capability and eventually limited the benefits of producing bioethanol from lignocellulose. The following reasons may account for this phenomenon:27,28 a) ethanol significantly restricted the activity of the glucose transport and metabolism pathways in the cell, b) ethanol reduced the adenosine triphosphatase (ATPase) activity in the cell membrane leading to dissolution of the proton gradient concentration and a loss of nutrition in the cell, thereby inhibiting the yeast metabolic activity and reducing its viability, c) ethanol changed the proportion of phospholipids and ergosterols in the cell membrane, thereby increasing the membrane’s nonspecific permeability and weakening its protection of the cell, and d) ethanol may have caused oxidative damage to the mitochondria and induced the production of reactive oxygen in the cells, which eventually interferes with the anaerobic environment of the ethanol fermentation system. Due to these damages to the cell structure and function, the viability and activity of the yeast were greatly reduced, and the ethanol production was therefore decreased. From the results and discussion above, it can be concluded that end-product ethanol was the primary inhibitory factor during the fermentation. Yeast cells are very sensitive to ethanol, and the ethanol concentration should be maintained at less than 30 g/L to ensure high yeast growth and fermentation activity. The yeast would completely stop growing, reproducing, and fermenting if the exogenous ethanol concentration was greater than 70 g/L. Therefore, weakening the negative effects of ethanol on the yeast cells is the first step to improving the efficiency of ethanol production and accelerating the industrialization of fermentation. Several basic conclusions regarding ethanol-induced inhibition of yeast growth can be drawn from the results obtained from the artificial fermentation environment described above. Nevertheless, there are a number of differences between the artificial and the actual fermentation processes. For instance, the maximum ethanol concentration produced in the natural fermentation processes is only 55 g/L (shown in Figure 4), whereas the yeast was found to tolerate up to 70 g/L of ethanol in the artificial experiment. In addition to the synergistic inhibitory effects of high substrate concentration and high product concentration, the difference between endogenous and exogenous ethanol may also be significant. Therefore, it was necessary to perform another series of experiments. The initial exogenous ethanol concentration was set from 10 g/L to 80 g/ L at 10 g/L intervals. The samples for the analyses of glucose, ethanol, and yeast concentrations were collected during the first 12 h to remove the interference of endogenous ethanol.29 To observe the contrast with the artificial fermentation environment, another fermentation experiment was concurrently conducted to show the inhibition induced by endogenous ethanol using yeast at the same stage without added ethanol. The samples were collected every 12 h from 0 to 96 h. All of the other test conditions were the same as above. Figure 8 illustrates the difference of the endogenous and exogenous ethanol’s inhibitory effect on yeast growth and fermentation activity. As shown in Figure 8 (A), when the

Figure 6. Comparison of the glucose conversion rate and ethanol yield under different initial ethanol concentrations.

Also shown in Figure 6, the changes in the glucose conversion rate and the ethanol yield in response to exogenous ethanol were similar, indicating that nearly all of the glucose consumed by the yeast was successfully converted into ethanol. Therefore, it can be inferred that glucose transport and metabolism are the main restrictive factors throughout the ethanol fermentation process. The low activity of the glucose transport and metabolic systems in yeast greatly limit the downstream metabolism of ethanol.26,27 Thus, priority should be given to the enhancement of the cellular enzyme activity in the transport and glycolytic pathways to improve the fermentation capability of yeast in the presence of high product concentrations. The correlation among substrate (glucose) concentration, product (ethanol) concentration, and ethanol yield is shown in Figure 7, which illustrates the interactions of highly

Figure 7. Interactions of substrate and product concentration on ethanol yield.

concentrated glucose and ethanol on the final ethanol yield. This figure pointed out the ethanol and the highly concentrated glucose (over 80 g/L) both had inhibitory effects on ethanol yield. By contrast, the product had a far more obvious influence on the ethanol yield. As shown in Figure 7, at comparatively low substrate concentrations, the increase of initial ethanol concentration in the system led to the inhibition of the fermentation activity of yeast, greatly reducing the ethanol yield. Similarly, at low product concentration values, the ethanol yield also descended with increased substrate concentration, although the displayed downward trend was not as sharp as the former. Particularly, when the product E

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As inferred from Figure 8 (B), endogenous ethanol exhibited a greater inhibitory effect on the fermentation rate than the same amount of exogenous ethanol. The speed of yeast fermentation was determined by two factors: the yeast density and the fermentation-ability of each cell. Based on the discussion above, the cell density varied greatly with ethanol concentration. Therefore, the fermentation rate was defined as the mass of ethanol that was produced by one gram of yeast in 1 h to study the fermentative capacity of each cell alone and eliminate the interference of cell density. The yeast fermentation rate decreased to 0.005 h−1 with 38 g/L endogenous ethanol and remained greater than 0.2 h−1 with the same concentration of exogenous ethanol. The rate decreased linearly with an increase in the concentration of exogenous ethanol and decreased through a power function with an increase in the concentration of endogenous ethanol. The differences between the exogenous or endogenous ethanol were most likely caused by the long-term accumulation and penetration of the high intracellular ethanol concentration, which led to irreversible damage of the cell structure and function. This damage was markedly more serious than that obtained from the sudden subjection to highly concentrated exogenous ethanol.30 Additionally, the protection of the membrane also reduced the toxicity of exogenous ethanol on the interior parts of the cell. Therefore, in practical VHGF, the yeast suffered a far more severe inhibitory effect from selfproduced ethanol, with a maximum tolerable concentration of ethanol of less than 70 g/L. These results further confirmed that the improvement of yeast growth and fermentation activity is crucial for increasing the final production of bioethanol through VHGF. 3.3. Acclimatization of Yeast Tolerance to Ethanol. Oriented acclimatization is an effective method for the improvement of the specific fermentation performance of yeast, such as its ability to utilize pentose and tolerate high temperatures or high concentrations of product and substrate.31 This research was dedicated to enhancing the yeast tolerance to the product, i.e., improving the yeast growth and fermentation activity under high ethanol concentrations. In total, five cycles of acclimatization were conducted following the procedure described in section 2.2. Figure 9 shows the microscopic views of the yeast cell morphology. After fermentation with 30 g/L exogenous ethanol for 96 h, the yeast cells were observed through microscopy. Under the same conditions, the yeast density and cell morphology were improved after five cycles of acclimatization. The microscopic views shown in Figure 9 (A) demonstrate that

Figure 8. Comparison of the 12-h average yeast-specific growth rate (A) and ethanol fermentation rate (B) under endogenous and exogenous ethanol.

ethanol concentration was less than 15 g/L, the endogenous and the exogenous ethanol showed no obvious difference in yeast growth inhibition. However, when the concentration increased, the yeast-specific growth rate with endogenous ethanol decreased much faster than that observed with exogenous ethanol. For instance, when the exogenous ethanol concentration was 60 g/L, the yeast maintained a low specific growth rate of 4.30 × 10−3 h−1, and the yeast could tolerate up to 70 g/L exogenous ethanol.

Figure 9. Micro-observations of unacclimatized yeast (A) and five-cycle-acclimatized yeast (B) after 96 h of fermentation. F

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6% V/V (47 g/L) ethanol at 30 °C. The higher fermentation temperature meant less cooling costs and a better coupling with hydrolysis. The tolerance the yeast developed to the higher product concentration also led to a higher-gravity fermentation. It can be expected that a longer acclimatization period will result in a continued improvement in the yeast growth and fermentation activity, even in a higher-gravity fermentation. The effect of acclimatization on the yeast tolerance to ethanol represents an amazingly bright prospect for improving the production and accelerating the industrialization of lignocellulosic ethanol with additional research.31 Moreover, this procedure of yeast acclimatization could also be applied in industrial-scaled production of lignocellulosic ethanol, as shown in Scheme 1. The significant inhibition of highly concentrated ethanol on yeast performance during VHGF limited the final production yield and restricted the promotion of the industrialized production of lignocellulosic ethanol. The lab-scale acclimatization process was shown effective at improving the yeast’s fermentation activity under high concentrations of ethanol. Since the results of the lab scale acclimatization study were successful, the process in Scheme 1 is expected to work at the industrial-scale. The yeast collected from the fermentation liquor, which survived the high concentration of substrate and product present, was partially proliferated and readded into the fermentation reactor. It was also partially used as the source yeast to further acclimatization for higher tolerance to ethanol. Then, the acclimatized yeast with enhanced tolerance was adopted in the industrial VHGF together with the previous yeast to raise the final ethanol yield. From the successful experience of increasing the ethanol yield by four times under the high ethanol concentration of 60 g/L, it could be expected that the final yield of industrial VHGF production should be significantly improved after certain cycles of acclimatization. To realize this protocol in industrial-scale ethanol production, there is still a lot of work to do in future research such as identifying the dosage of acclimatized yeast and the recycle fraction of yeast to optimize the ethanol fermentation process.

the unacclimatized yeast cells were of different shapes and sizes and the cellular autolysis of a small proportion of the yeasts could be observed. This indicates that the yeast cells failed to remain at a high activity under VHGF conditions. In contrast, according to Figure 9 (B), the acclimatized yeast cells had a regular round or oval shape and were approximately the same size indicating that the cell structures remained highly active. A cell concentration analysis performed with a spectrophotometer also demonstrated improvement in yeast growth after acclimatization with cell concentration of yeast subjected to five cycles of acclimatization increasing 16% after 96 h of fermentation with an initial ethanol concentration of 30 g/L. Figure 10 demonstrates the improvement in the yeast activity in terms of growth and fermentation after the five cycles of

4. CONCLUSIONS The effects of substrate and product inhibition on performance of ethanol fermentation using the yeast S. cerevisiae BY4742 was systematically studied in this research. Within the range of the tested substrate concentrations, 80 g/L was found to be the optimal substrate concentration for this yeast strain. At this substrate concentration, the ethanol yield reached 95% after 72 h at 35 °C. In contrast, the critical substrate concentration was found to be 160 g/L. The high concentration of substrate decreased the membrane fluidity and caused cell atrophy as well as organelle dehydration. It could also be concluded that in conditions of excessive levels of substrate, the consequent metabolic disorder of glucose led to the decrease of yeast fermentation and growth activity. The highly concentrated end-product ethanol severely inhibited the glucose transport and metabolism system and thus limited the downstream metabolism pathway. This made ethanol the primary inhibitory factor during the fermentation. The yeast could not tolerate an ethanol concentration greater than 70 g/L. Moreover, the self-designed contrast experiment indicated that due to the long-term accumulation and penetration of the highly concentrated intracellular ethanol, endogenous ethanol exerted a more severe inhibitory effect on

Figure 10. Comparison of the final yeast concentration (A) and the final ethanol yield (B) after each cycle of acclimatization.

acclimatization. The yeast concentration at the end of the fermentation increased by 18% with 60 g/L ethanol, which is similar to the increase that was found with 30 g/L ethanol (16%). This result indicates that the growth and reproductive activities of the yeast were successfully improved to a certain extent. As a whole, the final yeast concentration increased with acclimatization. As the acclimatization continued, the increase in the ethanol yield was more substantial under a fermentation environment with a higher product concentration. With an initial ethanol concentration of 60 g/L, the yield increased from 6% to 30%, whereas under an initial ethanol concentration of 30 g/L, the yield increased from 21% to 40%. The acclimated yeast could tolerate 60 g/L ethanol at 35 °C and maintained their fermentation and growth activity. The final ethanol concentration achieved 73 g/L, higher than that of traditional VHGF using S. cerevisiae, which was commonly 7−8% (V/V) 55−63 g/L, reported by Bai et al.7 Voorst et al.14 used mutant yeast from the same origin strain, BY4742, which could tolerate G

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Energy & Fuels Scheme 1. Flowchart of Yeast Acclimatization in Industrial Production of Lignocellulosic Ethanol

the yeast performance than exogenous ethanol during anaerobic fermentation. The five cycles of yeast acclimatization to ethanol tolerance improved the yeast density, cell morphology, and ethanol production parameters under high concentrations of ethanol. Particularly, the ethanol yield increased from 6% to 30% with an initial exogenous ethanol concentration of 60 g/L. This acclimatization procedure could also be adopted into the industrial-scale VHGF as the increased final product yield could promote the industrialization of lignocellulosic ethanol.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

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HPLC = high-performance liquid chromatography HK = hexokinase PK = pyruvate kinase EMP = Embden-Meyerhof-Parnas ATPase = adenosine triphosphatase

REFERENCES

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* Supporting Information S

Figure titled “interaction among the glucose, ethanol and yeast cellular activity” and the Scheme titled “flow chart of the experimental procedure”. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Phone: 86-21-54744540. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research project was sponsored by the Major Science and Technology Program for Water Pollution Control and Treatment (2012ZX07105-003) and the Shanghai Natural Science Foundation (No. 11ZR1417200). The authors would also like to thank Dr. Hui Lin and Dr. Tyler Barzee at University of California, Davis for their discussions and help on this work.

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ABBREVIATIONS S. cerevisiae = Saccharomyces cerevisiae VHGF = very high gravity fermentation SESE = School of Environmental Science and Engineering SJTU = Shanghai Jiao Tong University YPD = yeast extract peptone dextrose OD = optical density H

DOI: 10.1021/ef502349v Energy Fuels XXXX, XXX, XXX−XXX

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DOI: 10.1021/ef502349v Energy Fuels XXXX, XXX, XXX−XXX