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Influence of nitrogen sources on ethanol fermentation in an integrated ethanol–methane fermentation system
Bioresource Technology 120 (2012) 206–211
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Influence of nitrogen sources on ethanol fermentation in an integrated ethanol–methane fermentation system Ke Wang, Zhonggui Mao ⇑, Chengming Zhang, Jianhua Zhang, Hongjian Zhang, Lei Tang The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
h i g h l i g h t s " Effects of anaerobic effluent and urea on ethanol fermentation were studied. " Anaerobic effluent led to higher growth and ethanol production rates than urea. " Anaerobic effluent can be used as nitrogen source instead of urea.
a r t i c l e
i n f o
Article history: Received 6 December 2011 Received in revised form 17 May 2012 Accepted 14 June 2012 Available online 26 June 2012 Keywords: Ethanol–methane fermentation system Two-stage anaerobic digestion Ammonium Urea
a b s t r a c t An integrated ethanol–methane fermentation system was proposed to resolve wastewater pollution in cassava ethanol production. In the integrated system, wastewater originating from ethanol distillation was treated by two-stage anaerobic digestion and then used in medium for the next batch of ethanol fermentation. Ammonium and other components in the effluent promoted yeast growth and fermentation rate but did not increase the yield of ethanol. Fermentations with the effluent as the nitrogen source showed higher growth and ethanol production rates (0.215 h 1 and 1.276 g/L/h, respectively) than urea that resulted in corresponding rates of 0.176 h 1 and 0.985 g/L/h, respectively. Results indicated that anaerobic digestion effluent can be used as nitrogen source for the ethanol fermentation instead of urea in the ethanol–methane fermentation system. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Distillery waste generation has limited the development of cassava-based ethanol industry. Direct recycling of distillery waste has been studied (Bialas et al., 2010; Ding et al., 2009; Kim et al., 1997), but this approach faces problems. Since the distillery waste contains sand, liquid–solid separation at high temperature and low pH causes serious physical wear and chemical corrosion in the separation equipment and consumes lots of energy. By-products in the fermentation liquor such as low-carbon organic acids, glycerol, ethanol homologues (butanol, amyl alcohol and isoamyl alcohol) and other organic compounds are difficult to remove by distillation and are bound to accumulate when the wastewater is directly recycled. To avoid some of these problems, an ethanol–methane fermentation system was developed for wastewater reutilization (Zhang ⇑ Corresponding author. Address: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Tel./fax: +86 510 85918296. E-mail address: [email protected] (Z. Mao). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.06.032
et al., 2010a,b). In this process (Fig. 1), cassava starch is transformed into ethanol by fermentation while fiber, pectin, and other metabolites of Saccharomyces cerevisiae are converted to biogas by anaerobic digestion. The biogas can be used to produce electricity and the anaerobic digestion effluent reused in ethanol fermentation. The solid materials withdrawn from liquid–solid separation can be used as fertilizer. Zero wastewater discharge and low energy consumption are two major advantages of this process; however, the quality of the anaerobic digestion effluent can influence ethanol fermentation performance (Zhang et al., 2011; Wang et al., 2011) since the constituents of the effluent are very complex, which include the suspended substance, organic substance and inorganic salt. In the ethanol–methane fermentation system, urea is used as an additional nitrogen source for the yeast. Since it is known that the concentration and type of nitrogen source affect yeast growth and metabolite formation (Albers et al., 1996; Ter Schure et al., 2000; Thomas and Ingledew, 1990; Torija et al., 2003), the present study examined the effects of ammonium contained in the effluent and of added urea on ethanol fermentation to further optimize the ethanol–methane fermentation system.
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K. Wang et al. / Bioresource Technology 120 (2012) 206–211
Fig. 1. Flowchart of the proposed ethanol–methane fermentation system.
2. Methods
when the residual sugar concentration was below 2 g/L as determined by high-performance liquid chromatography (HPLC).
2.1. Anaerobic digestion and effluent collection 2.4. Analytical methods Anaerobic digestion was conducted in two-stage anaerobic digestion reactors using thermophilic anaerobic sludge and mesophilic anaerobic granular sludge, respectively. Each reactor had a working volume of 10 L. Distillage wastewater was pumped into the thermophilic anaerobic reactor using a peristaltic pump, where the temperature was maintained at 60 °C by circulation of heated water through a water jacket. The effluent from the thermophilic anaerobic reactor was centrifuged (4000g for 30 min) and the supernatant was pumped into a mesophilic anaerobic reactor, where the temperature was kept at 35 °C in a water bath. One cycle was completed in about 24 h, including 2 h of feeding, 10 h reaction, 12 h settling, and 1 min drainage. The two-stage anaerobic digestion effluent was centrifuged at 4000g for 30 min and the supernatant was stored at 20 °C. 2.2. Magnesium ammonium phosphate (MAP) precipitation experiments MAP precipitation was used to remove the ammonium from the anaerobic digestion effluent. MgCl26H2O and Na2HPO412H2O (Mg2+:NH4+:PO43 molar ratio of 1:1:1.25) were added to the effluent, and stirred for 30 min. The pH was adjusted to 10.5 with NaOH and the mixture was allowed to settle for 1 h. The mixture was centrifuged at10,000g for 10 min and filtered through a 0.45lm cellulose acetate membrane. The ammonium nitrogen in the filtrate was measured by standard method (APHA, 1995). 2.3. Ethanol fermentations Angel ethanol yeast (ADY, a commercial strain of S. cerevisiae for ethanol production) was obtained from Hubei Angel Yeast Co. Ltd., China. The seed medium consisted of: glucose 20 g/L, yeast extract 8.5 g/L, (NH4)2SO4 1.3 g/L, MgSO47H2O 0.1 g/L and CaCl22H2O 0.06 g/L. The fermentation medium consisted of: glucose 120 g/L, MgSO47H2O 0.1 g/L and CaCl22H2O 0.06 g/L. The nitrogen sources in the fermentation water were (1) ammonium sulfate in deionized (DI) water (NH4+–N of 0.47 g/L), (2) urea in DI water (0.1% w/v), (3) anaerobic digestion effluent (NH4+–N of 0.47 g/L), and (4) MAPtreated anaerobic digestion effluent. One loopful of S. cerevisiae was inoculated into a 500-ml Erlenmeyer flask containing seed medium. The flask was incubated on a rotating shaker at 100 rpm, at 30 °C for 19 h. A 10% (v/v) inoculum was transferred into 250-ml bottles with a cotton cap stopper and filled with 150-ml of the fermentation medium. All fermentations were carried out at 30 °C without shaking and the initial pH was adjusted to 5.0 with 300 g/L H2SO4. Samples were taken at appropriate time intervals. Fermentation was considered to be over
The concentrations of glucose, ethanol, glycerol, acetic acid, propionic acid and butyric acid were determined by high-performance liquid chromatography (HPLC). The samples collected were centrifuged (10,000g for 10 min) and the supernatant was filtered (0.20-lm filter) prior to analysis. A 20-ll portion or a standard solution was injected into a Bio-Rad HPX-87H Aminex ion exclusion column. The column was operated at 65 °C and sulfuric acid (0.005 mol/L) was used as mobile phase at a flow rate of 0.6 ml/ min. A refractive index detector (Shodex RI-101, Japan) was used for detection. Data were processed using Chromeleon software (Dionex, USA). Cell growth was determined by absorbance at 600 nm. Ammonium nitrogen, COD, conductivity and true color were determined by using standard methods (APHA, 1995). 3. Results and discussion 3.1. MAP precipitation experiments Characteristics of the anaerobic digestion effluent used are listed in Table 1. Ammonium could be removed at the optimum pH of 10.5 (Fig. 2B). A Mg2+:NH4+:PO43 molar ratio of 1:1:1.25 was optimal to remove ammonium effectively and to avoid excess PO43 and Mg2+ in the effluent (Fig. 2C). A higher ammonium removal ratio was generated by adding MgCl26H2O and Na2HPO412H2O compared to MgO and H3PO4 (Fig. 2A). Under optimized conditions, the ammonium nitrogen concentration of the anaerobic digestion effluent treated by MAP was below 10 mg/L. 3.2. Ethanol fermentations The shortest cultivation time was achieved with effluent as the nitrogen source (Fig. 3). The higher rate of the conversion of glucose (Fig. 3) was also reflected in the maximum specific growth rate (lmax) (Table 2), where the average lmax of 0.215 h 1 was higher than that for growth on effluent treated by MAP precipitaTable 1 Characteristics of the anaerobic digestion effluent.
a
Parameter and unit
Value
Parameter and unit
Value
pH COD (mg/L) Ammonium nitrogen (mg/L) Conductivity (103 ls/cm)
8.10 ± 0.05 2100 ± 100 470 ± 12
Acetic acid (mM) Propionic acid (mM) Butyric acid (mM)
NDa ND ND
True color (TCU)
1800 ± 100
ND: not detectable.
7.3 ± 0.1
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Fig. 2. NH4+-N removal ratio of MAP technique at different factors (A) NH4+–N removal ratio at different chemical combinations of magnesium salt and phosphate (pH = 9.5, Mg2+:NH4+:PO43 = 1:1:1) (B) NH4+–N removal ratio at different pH (MgCl26H2O and Na2HPO412H2O were used, Mg2+:NH4+:PO43 = 1:1:1) (C) NH4+–N removal ratio at different molar ratio of Mg2+:NH4+:PO43 (MgCl26H2O and Na2HPO412H2O were used, pH = 9.5) (Values = average ± SD).
tion (0.185 h 1) and ammonium sulfate (0.165 h 1). The calculated maximal ethanol production rate was higher for the effluent (1.276 g/L/h) than for the effluent treated by MAP precipitation (0.872 g/L/h) and ammonium sulfate (0.834 g/L/h) (Table 2). However the ethanol yields of the three fermentations were almost the same. These results indicated that both ammonium and other components in the effluent increased the conversion rate of glucose and production rate of ethanol, but did not increase ethanol yield. Growth of S. cerevisiae was promoted as well. With urea as the nitrogen source, the lmax (0.176 h 1) and maximal ethanol production rate (0.985 g/L/h) were 7% and 18% higher, respectively, while the ethanol yield was nearly equal compared to ammonium sulfate-grown cultures. This result suggested that urea led to relatively higher growth and ethanol production rates than ammonium sulfate. The choice of nitrogen source also affected the formation of acetic acid and glycerol (Fig. 4, Table 3). For cultures grown on ammonium sulfate as the nitrogen source, the yields of acetic acid and glycerol were higher than those grown
on the other nitrogen sources. The yield of acetic acid was the smallest in the fermentation with effluent treated by MAP precipitation. In addition, the evolution of the pH during the fermentations was monitored during the process (Fig. 5). For all fermentations, the minimum pH was reached after 30 h of fermentation and remained unchanged thereafter. However, when ammonium sulfate and effluent were used as the nitrogen sources, the final pH was lower than that when urea and the effluent treated by MAP precipitation were used. Since fermentation with the effluent as the nitrogen source showed the best performance in terms of the yeast growth and ethanol production rates, anaerobic digestion effluent can be used as nitrogen source for ethanol fermentation instead of urea in the present ethanol–methane fermentation system. When distillery waste is treated by anaerobic digestion, proteins are hydrolyzed to amino acids by extracellular proteases, and these amino acids are taken up by microorganisms for intracellular metabolism. Ammonium is thus produced by deamination of amino
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Fig. 3. Representative profiles of anaerobic batch growth of S. cerevisiae with different nitrogen source: ammonium sulfate (j), urea (d), anaerobic digestion effluent (N), anaerobic digestion effluent treated by MAP precipitation (.) (Values = average ± SD).
Table 2 Growth rates and product yields for anaerobic growth of S. cerevisiae on glucose with different nitrogen source. Parameter and unit
Maximal specific growth rate (h 1) Ethanol yield (mol/mol of glucose) Glycerol yield (mmol/mol of glucose) Acetic acid yield (mmol/mol of glucose) Maximal ethanol production rate (g/L/h)
Value with the following nitrogen source 1
2
3
4
0.165 2.17 80 23 0.834
0.176 2.20 72 17 0.985
0.215 2.23 75 16 1.276
0.185 2.20 75 11 0.872
acids. A small part of the ammonium is used for the growth of anaerobic microorganism while most of which is left in the effluent (He, 1998). When the effluent is recycled for ethanol fermentation, ammonium remained in the fermentation batch. The proportion of nitrogenous compounds in yeast cells is about 50% (by weight), so the synthesis of yeast biomass is clearly dependent on the content of nitrogen in the growth medium. Low levels of yeast assimilable nitrogenous (YAN) compounds have been related to lower fermentation rates and longer fermentative periods. Limiting YAN is thought to affect yeast by reducing yeast cell multiplication and by decreasing the rate of glycolysis (Bely et al., 1990). Therefore, when ammonium was removed from the effluent by MAP precipitation, the yeast growth and ethanol production rates decreased. The results depicted in Table 2 suggest that other components of the effluent could promote the yeast growth. Noguera et al. (1994) and Schiener et al. (1998) have maintained that the majority of the soluble organic matter in effluents from biological treatment processes is actually soluble microbial products (SMP). Some of the SMP have been identified as nucleic acids, organic acids, amino acids, extracellular enzymes, siderophores (Barker and Stuckey, 1999), which could increase the fermentation rate. Amino acids were detected in the effluent (Table 3). The positive effect of amino acids on growth and fermentation rates has previously been noted by Thomas and Ingledew (1990) and Albers et al. (1996). This effect may be explained by the decreased need for amino acid
synthesis in this case. Moreover others have shown that a mixed source (ammonium and amino acids) is more effective for promoting yeast growth and fermentation rate (Ribéreau-Gayon et al., 2000). The type of nitrogen source can also affect the yeast growth rate and formation of metabolites. Ter Schure et al. (2000) reported that growth on good nitrogen sources such as ammonia, glutamine and asparagine seems to yield relatively higher growth rates than on poor ones such as proline and urea; however, in the present research, urea as a nitrogen source led to a relatively higher growth rate and ethanol production rate than ammonium. Actively growing yeasts acidify their medium through a combination of differential ion uptake, proton secretion during nutrient transport, direct secretion of organic acids, and CO2 evolution (Walker, 1998). The pH of all fermentations decreased to values of 2.3–2.8. And the final pH of cultures with ammonium sulfate and the effluent were lower than from those with urea and the effluent treated by MAP precipitation. This result was in accord with those reported by Dartiguenave et al. (2000) and Torija et al. (2003). The differences between the fermentations are mainly due to how the nitrogen sources are transported across the cell membrane. The transport of ammonium is directly driven by the electrochemical plasma membrane potential, generated by the plasma membrane ATPase (Peña et al., 1987; Van der Rest et al., 1995), in which the ion is taken up and the proton is later excreted
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Fig. 4. Representative profiles of metabolism of S. cerevisiae with different nitrogen source: ammonium sulfate (j), urea (d), anaerobic digestion effluent (N), anaerobic digestion effluent treated by MAP precipitation (.) (Values = average ± SD).
Table 3 Amino acids contained in the anaerobic digestion effluent. Type
Concentration (mg/L)
Type
Concentration (mg/L)
Type
Concentration (mg/L)
asp glu ser his gly
28.20 4.51 0.02 0.05 17.00
thr arg ala tyr phe
1.83 0.52 1.69 1.49 74.2
ile leu lys pro Total
0.062 1.18 1.59 12.7 145
to the medium to keep a constant intracellular pH. This process causes acidification of the extracellular medium. Amino acids, on the other hand, cross the membrane using a symporter with one or more protons. This system is also usually coupled with an ATPase pump, but in this case, the protons are extruded to compensate for their entry from the extracellular medium; therefore, acidification outside the membrane is reduced (Torija et al., 2003). 4. Conclusions Both ammonium and other components (especially amino acids) in the effluent promoted yeast growth and fermentation rate. Fermentation with the effluent as the nitrogen source showed the best performance in terms of yeast growth and ethanol production rates when different nitrogen sources were used, although urea led to a
Fig. 5. Evolution of pH in fermentations with different nitrogen source: ammonium sulfate (j), urea (d), anaerobic digestion effluent (N), anaerobic digestion effluent treated by MAP precipitation (.).
relatively higher growth rate and ethanol production rate than ammonium. Therefore, for the present ethanol–methane fermentation system, anaerobic digestion effluent can be used as
K. Wang et al. / Bioresource Technology 120 (2012) 206–211
nitrogen source for the ethanol fermentation and there is no need to add urea to the fermentation culture. Acknowledgements This research was financially supported by the National High Technology Research and Development Program of China (863 Program, No. 2008AA10Z338). References Albers, E., Larsson, C., Liden, G., Niklasson, C., Gustafsson, L., 1996. Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl. Environ. Microbiol. 62, 3187–3195. APHA, 1995. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association (APHA), New York. Barker, D.J., Stuckey, D.C., 1999. A review of soluble microbial products (SMP) in wastewater treatment systems. Water Res. 33, 3063–3082. Bely, M., Sablayrolles, J.M., Barre, P., 1990. Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions. J. Ferment. Bioeng. 70, 246–252. Bialas, W., Szymanowska, D., Grajek, W., 2010. Fuel ethanol production from granular corn starch using Saccharomyces cerevisiae in a long term repeated SSF process with full stillage recycling. Bioresour. Technol. 101, 3126–3131. Dartiguenave, C., Jeandet, P., Maujean, A., 2000. Study of the contribution of the major organic acids of wine to the buffering capacity of wine in model solutions. Am. J. Enol. Viticult. 51, 352–356. Ding, Z.Y., Zhang, L., Fang, Y., Xu, L., Zhang, K., Shi, G.Y., 2009. Application of full permeate recycling to very high gravity ethanol fermentation from corn. Korean J. Chem. Eng. 26, 719–723. He, Y.L., 1998. Anaerobic Biological Treatment for Wastewaters. China Light Industry Press, Beijing. Kim, J.S., Kim, B.G., Lee, C.H., Kim, S.W., Jee, H.S., Koh, J.H., Fane, A.G., 1997. Development of clean technology in alcohol fermentation industry. J. Clean. Prod. 5, 263–267.
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Noguera, D.R., Araki, N., Rittmann, B.E., 1994. Soluble microbial products (SMP) in anaerobic chemostats. Biotechnol. Bioeng. 44, 1040–1047. Peña, A., Pardo, J.P., Ramírez, J., 1987. Early metabolic effects and mechanism of ammonium transport in yeast. Arch. Biochem. Biophys. 253, 431–438. Ribéreau-Gayon, P., Dubourdieu, D., Donèche, B., Lonvaud, A., 2000. Handbook of Enology. Wiley, West Sussex. Schiener, P., Nachaiyasit, S., Stuckey, D.C., 1998. Production of soluble microbial products (SMP) in an anaerobic baffled reactor: composition, biodegradability, and the effect of process parameters. Environ. Technol. 19, 391–399. Ter Schure, E.G., van Riel, N.A.W., Verrips, C.T., 2000. The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 24, 67–83. Thomas, K.C., Ingledew, W., 1990. Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes. Appl. Environ. Microbiol. 56, 2046–2050. Torija, M.J., Beltran, G., Novo, M., Poblet, M., Rozès, N., Mas, A., Guillamón, J.M., 2003. Effect of organic acids and nitrogen source on alcoholic fermentation: study of their buffering capacity. J. Agric. Food Chem. 51, 916–922. Van der Rest, M., Kamminga, A.H., Nakano, A., Anraku, Y., Poolman, B., Konings, W.N., 1995. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis. Microbiol. Mol. Biol. Rev. 59, 304–322. Walker, G.M., 1998. Yeast Physiology and Biotechnology. Wiley, Sussex. Wang, K., Jiang, L., Mao, Z.G., Zhang, C.M., Zhang, J.H., Zhang, H.J., 2011. Effect of recycled two-stage anaerobic digestion effluent on very high gravity cassavabased ethanol fermentation. J. Biobased Mater. Bio. 5, 351–356. Zhang, C.M., Mao, Z.G., Wang, X., Zhang, J.H., Sun, F.B., Tang, L., Zhang, H.J., 2010a. Effective ethanol production by reutilizing waste distillage anaerobic digestion effluent in an integrated fermentation process coupled with both ethanol and methane fermentations. Bioprocess Biosyst. Eng. 33, 1067–1075. Zhang, Q.H., Lu, X., Tang, L., Mao, Z.G., Zhang, J.H., Zhang, H.J., Sun, F.B., 2010b. A novel full recycling process through two-stage anaerobic treatment of distillery wastewater for bioethanol production from cassava. J. Hazard. Mater. 179, 635– 641. Zhang, C.M., Jiang, L., Mao, Z.G., Zhang, J.H., Tang, L., 2011. Effects of propionic acid and pH on ethanol fermentation by Saccharomyces cerevisiae in cassava mash. Appl. Biochem. Biotechnol. 165, 883–891.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Influence of nitrogen sources on ethanol fermentation in an integrated ethanol–methane fermentation system Ke Wang, Zhonggui Mao ⇑, Chengming Zhang, Jianhua Zhang, Hongjian Zhang, Lei Tang The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
h i g h l i g h t s " Effects of anaerobic effluent and urea on ethanol fermentation were studied. " Anaerobic effluent led to higher growth and ethanol production rates than urea. " Anaerobic effluent can be used as nitrogen source instead of urea.
a r t i c l e
i n f o
Article history: Received 6 December 2011 Received in revised form 17 May 2012 Accepted 14 June 2012 Available online 26 June 2012 Keywords: Ethanol–methane fermentation system Two-stage anaerobic digestion Ammonium Urea
a b s t r a c t An integrated ethanol–methane fermentation system was proposed to resolve wastewater pollution in cassava ethanol production. In the integrated system, wastewater originating from ethanol distillation was treated by two-stage anaerobic digestion and then used in medium for the next batch of ethanol fermentation. Ammonium and other components in the effluent promoted yeast growth and fermentation rate but did not increase the yield of ethanol. Fermentations with the effluent as the nitrogen source showed higher growth and ethanol production rates (0.215 h 1 and 1.276 g/L/h, respectively) than urea that resulted in corresponding rates of 0.176 h 1 and 0.985 g/L/h, respectively. Results indicated that anaerobic digestion effluent can be used as nitrogen source for the ethanol fermentation instead of urea in the ethanol–methane fermentation system. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Distillery waste generation has limited the development of cassava-based ethanol industry. Direct recycling of distillery waste has been studied (Bialas et al., 2010; Ding et al., 2009; Kim et al., 1997), but this approach faces problems. Since the distillery waste contains sand, liquid–solid separation at high temperature and low pH causes serious physical wear and chemical corrosion in the separation equipment and consumes lots of energy. By-products in the fermentation liquor such as low-carbon organic acids, glycerol, ethanol homologues (butanol, amyl alcohol and isoamyl alcohol) and other organic compounds are difficult to remove by distillation and are bound to accumulate when the wastewater is directly recycled. To avoid some of these problems, an ethanol–methane fermentation system was developed for wastewater reutilization (Zhang ⇑ Corresponding author. Address: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Tel./fax: +86 510 85918296. E-mail address: [email protected] (Z. Mao). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.06.032
et al., 2010a,b). In this process (Fig. 1), cassava starch is transformed into ethanol by fermentation while fiber, pectin, and other metabolites of Saccharomyces cerevisiae are converted to biogas by anaerobic digestion. The biogas can be used to produce electricity and the anaerobic digestion effluent reused in ethanol fermentation. The solid materials withdrawn from liquid–solid separation can be used as fertilizer. Zero wastewater discharge and low energy consumption are two major advantages of this process; however, the quality of the anaerobic digestion effluent can influence ethanol fermentation performance (Zhang et al., 2011; Wang et al., 2011) since the constituents of the effluent are very complex, which include the suspended substance, organic substance and inorganic salt. In the ethanol–methane fermentation system, urea is used as an additional nitrogen source for the yeast. Since it is known that the concentration and type of nitrogen source affect yeast growth and metabolite formation (Albers et al., 1996; Ter Schure et al., 2000; Thomas and Ingledew, 1990; Torija et al., 2003), the present study examined the effects of ammonium contained in the effluent and of added urea on ethanol fermentation to further optimize the ethanol–methane fermentation system.
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Fig. 1. Flowchart of the proposed ethanol–methane fermentation system.
2. Methods
when the residual sugar concentration was below 2 g/L as determined by high-performance liquid chromatography (HPLC).
2.1. Anaerobic digestion and effluent collection 2.4. Analytical methods Anaerobic digestion was conducted in two-stage anaerobic digestion reactors using thermophilic anaerobic sludge and mesophilic anaerobic granular sludge, respectively. Each reactor had a working volume of 10 L. Distillage wastewater was pumped into the thermophilic anaerobic reactor using a peristaltic pump, where the temperature was maintained at 60 °C by circulation of heated water through a water jacket. The effluent from the thermophilic anaerobic reactor was centrifuged (4000g for 30 min) and the supernatant was pumped into a mesophilic anaerobic reactor, where the temperature was kept at 35 °C in a water bath. One cycle was completed in about 24 h, including 2 h of feeding, 10 h reaction, 12 h settling, and 1 min drainage. The two-stage anaerobic digestion effluent was centrifuged at 4000g for 30 min and the supernatant was stored at 20 °C. 2.2. Magnesium ammonium phosphate (MAP) precipitation experiments MAP precipitation was used to remove the ammonium from the anaerobic digestion effluent. MgCl26H2O and Na2HPO412H2O (Mg2+:NH4+:PO43 molar ratio of 1:1:1.25) were added to the effluent, and stirred for 30 min. The pH was adjusted to 10.5 with NaOH and the mixture was allowed to settle for 1 h. The mixture was centrifuged at10,000g for 10 min and filtered through a 0.45lm cellulose acetate membrane. The ammonium nitrogen in the filtrate was measured by standard method (APHA, 1995). 2.3. Ethanol fermentations Angel ethanol yeast (ADY, a commercial strain of S. cerevisiae for ethanol production) was obtained from Hubei Angel Yeast Co. Ltd., China. The seed medium consisted of: glucose 20 g/L, yeast extract 8.5 g/L, (NH4)2SO4 1.3 g/L, MgSO47H2O 0.1 g/L and CaCl22H2O 0.06 g/L. The fermentation medium consisted of: glucose 120 g/L, MgSO47H2O 0.1 g/L and CaCl22H2O 0.06 g/L. The nitrogen sources in the fermentation water were (1) ammonium sulfate in deionized (DI) water (NH4+–N of 0.47 g/L), (2) urea in DI water (0.1% w/v), (3) anaerobic digestion effluent (NH4+–N of 0.47 g/L), and (4) MAPtreated anaerobic digestion effluent. One loopful of S. cerevisiae was inoculated into a 500-ml Erlenmeyer flask containing seed medium. The flask was incubated on a rotating shaker at 100 rpm, at 30 °C for 19 h. A 10% (v/v) inoculum was transferred into 250-ml bottles with a cotton cap stopper and filled with 150-ml of the fermentation medium. All fermentations were carried out at 30 °C without shaking and the initial pH was adjusted to 5.0 with 300 g/L H2SO4. Samples were taken at appropriate time intervals. Fermentation was considered to be over
The concentrations of glucose, ethanol, glycerol, acetic acid, propionic acid and butyric acid were determined by high-performance liquid chromatography (HPLC). The samples collected were centrifuged (10,000g for 10 min) and the supernatant was filtered (0.20-lm filter) prior to analysis. A 20-ll portion or a standard solution was injected into a Bio-Rad HPX-87H Aminex ion exclusion column. The column was operated at 65 °C and sulfuric acid (0.005 mol/L) was used as mobile phase at a flow rate of 0.6 ml/ min. A refractive index detector (Shodex RI-101, Japan) was used for detection. Data were processed using Chromeleon software (Dionex, USA). Cell growth was determined by absorbance at 600 nm. Ammonium nitrogen, COD, conductivity and true color were determined by using standard methods (APHA, 1995). 3. Results and discussion 3.1. MAP precipitation experiments Characteristics of the anaerobic digestion effluent used are listed in Table 1. Ammonium could be removed at the optimum pH of 10.5 (Fig. 2B). A Mg2+:NH4+:PO43 molar ratio of 1:1:1.25 was optimal to remove ammonium effectively and to avoid excess PO43 and Mg2+ in the effluent (Fig. 2C). A higher ammonium removal ratio was generated by adding MgCl26H2O and Na2HPO412H2O compared to MgO and H3PO4 (Fig. 2A). Under optimized conditions, the ammonium nitrogen concentration of the anaerobic digestion effluent treated by MAP was below 10 mg/L. 3.2. Ethanol fermentations The shortest cultivation time was achieved with effluent as the nitrogen source (Fig. 3). The higher rate of the conversion of glucose (Fig. 3) was also reflected in the maximum specific growth rate (lmax) (Table 2), where the average lmax of 0.215 h 1 was higher than that for growth on effluent treated by MAP precipitaTable 1 Characteristics of the anaerobic digestion effluent.
a
Parameter and unit
Value
Parameter and unit
Value
pH COD (mg/L) Ammonium nitrogen (mg/L) Conductivity (103 ls/cm)
8.10 ± 0.05 2100 ± 100 470 ± 12
Acetic acid (mM) Propionic acid (mM) Butyric acid (mM)
NDa ND ND
True color (TCU)
1800 ± 100
ND: not detectable.
7.3 ± 0.1
208
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Fig. 2. NH4+-N removal ratio of MAP technique at different factors (A) NH4+–N removal ratio at different chemical combinations of magnesium salt and phosphate (pH = 9.5, Mg2+:NH4+:PO43 = 1:1:1) (B) NH4+–N removal ratio at different pH (MgCl26H2O and Na2HPO412H2O were used, Mg2+:NH4+:PO43 = 1:1:1) (C) NH4+–N removal ratio at different molar ratio of Mg2+:NH4+:PO43 (MgCl26H2O and Na2HPO412H2O were used, pH = 9.5) (Values = average ± SD).
tion (0.185 h 1) and ammonium sulfate (0.165 h 1). The calculated maximal ethanol production rate was higher for the effluent (1.276 g/L/h) than for the effluent treated by MAP precipitation (0.872 g/L/h) and ammonium sulfate (0.834 g/L/h) (Table 2). However the ethanol yields of the three fermentations were almost the same. These results indicated that both ammonium and other components in the effluent increased the conversion rate of glucose and production rate of ethanol, but did not increase ethanol yield. Growth of S. cerevisiae was promoted as well. With urea as the nitrogen source, the lmax (0.176 h 1) and maximal ethanol production rate (0.985 g/L/h) were 7% and 18% higher, respectively, while the ethanol yield was nearly equal compared to ammonium sulfate-grown cultures. This result suggested that urea led to relatively higher growth and ethanol production rates than ammonium sulfate. The choice of nitrogen source also affected the formation of acetic acid and glycerol (Fig. 4, Table 3). For cultures grown on ammonium sulfate as the nitrogen source, the yields of acetic acid and glycerol were higher than those grown
on the other nitrogen sources. The yield of acetic acid was the smallest in the fermentation with effluent treated by MAP precipitation. In addition, the evolution of the pH during the fermentations was monitored during the process (Fig. 5). For all fermentations, the minimum pH was reached after 30 h of fermentation and remained unchanged thereafter. However, when ammonium sulfate and effluent were used as the nitrogen sources, the final pH was lower than that when urea and the effluent treated by MAP precipitation were used. Since fermentation with the effluent as the nitrogen source showed the best performance in terms of the yeast growth and ethanol production rates, anaerobic digestion effluent can be used as nitrogen source for ethanol fermentation instead of urea in the present ethanol–methane fermentation system. When distillery waste is treated by anaerobic digestion, proteins are hydrolyzed to amino acids by extracellular proteases, and these amino acids are taken up by microorganisms for intracellular metabolism. Ammonium is thus produced by deamination of amino
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Fig. 3. Representative profiles of anaerobic batch growth of S. cerevisiae with different nitrogen source: ammonium sulfate (j), urea (d), anaerobic digestion effluent (N), anaerobic digestion effluent treated by MAP precipitation (.) (Values = average ± SD).
Table 2 Growth rates and product yields for anaerobic growth of S. cerevisiae on glucose with different nitrogen source. Parameter and unit
Maximal specific growth rate (h 1) Ethanol yield (mol/mol of glucose) Glycerol yield (mmol/mol of glucose) Acetic acid yield (mmol/mol of glucose) Maximal ethanol production rate (g/L/h)
Value with the following nitrogen source 1
2
3
4
0.165 2.17 80 23 0.834
0.176 2.20 72 17 0.985
0.215 2.23 75 16 1.276
0.185 2.20 75 11 0.872
acids. A small part of the ammonium is used for the growth of anaerobic microorganism while most of which is left in the effluent (He, 1998). When the effluent is recycled for ethanol fermentation, ammonium remained in the fermentation batch. The proportion of nitrogenous compounds in yeast cells is about 50% (by weight), so the synthesis of yeast biomass is clearly dependent on the content of nitrogen in the growth medium. Low levels of yeast assimilable nitrogenous (YAN) compounds have been related to lower fermentation rates and longer fermentative periods. Limiting YAN is thought to affect yeast by reducing yeast cell multiplication and by decreasing the rate of glycolysis (Bely et al., 1990). Therefore, when ammonium was removed from the effluent by MAP precipitation, the yeast growth and ethanol production rates decreased. The results depicted in Table 2 suggest that other components of the effluent could promote the yeast growth. Noguera et al. (1994) and Schiener et al. (1998) have maintained that the majority of the soluble organic matter in effluents from biological treatment processes is actually soluble microbial products (SMP). Some of the SMP have been identified as nucleic acids, organic acids, amino acids, extracellular enzymes, siderophores (Barker and Stuckey, 1999), which could increase the fermentation rate. Amino acids were detected in the effluent (Table 3). The positive effect of amino acids on growth and fermentation rates has previously been noted by Thomas and Ingledew (1990) and Albers et al. (1996). This effect may be explained by the decreased need for amino acid
synthesis in this case. Moreover others have shown that a mixed source (ammonium and amino acids) is more effective for promoting yeast growth and fermentation rate (Ribéreau-Gayon et al., 2000). The type of nitrogen source can also affect the yeast growth rate and formation of metabolites. Ter Schure et al. (2000) reported that growth on good nitrogen sources such as ammonia, glutamine and asparagine seems to yield relatively higher growth rates than on poor ones such as proline and urea; however, in the present research, urea as a nitrogen source led to a relatively higher growth rate and ethanol production rate than ammonium. Actively growing yeasts acidify their medium through a combination of differential ion uptake, proton secretion during nutrient transport, direct secretion of organic acids, and CO2 evolution (Walker, 1998). The pH of all fermentations decreased to values of 2.3–2.8. And the final pH of cultures with ammonium sulfate and the effluent were lower than from those with urea and the effluent treated by MAP precipitation. This result was in accord with those reported by Dartiguenave et al. (2000) and Torija et al. (2003). The differences between the fermentations are mainly due to how the nitrogen sources are transported across the cell membrane. The transport of ammonium is directly driven by the electrochemical plasma membrane potential, generated by the plasma membrane ATPase (Peña et al., 1987; Van der Rest et al., 1995), in which the ion is taken up and the proton is later excreted
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Fig. 4. Representative profiles of metabolism of S. cerevisiae with different nitrogen source: ammonium sulfate (j), urea (d), anaerobic digestion effluent (N), anaerobic digestion effluent treated by MAP precipitation (.) (Values = average ± SD).
Table 3 Amino acids contained in the anaerobic digestion effluent. Type
Concentration (mg/L)
Type
Concentration (mg/L)
Type
Concentration (mg/L)
asp glu ser his gly
28.20 4.51 0.02 0.05 17.00
thr arg ala tyr phe
1.83 0.52 1.69 1.49 74.2
ile leu lys pro Total
0.062 1.18 1.59 12.7 145
to the medium to keep a constant intracellular pH. This process causes acidification of the extracellular medium. Amino acids, on the other hand, cross the membrane using a symporter with one or more protons. This system is also usually coupled with an ATPase pump, but in this case, the protons are extruded to compensate for their entry from the extracellular medium; therefore, acidification outside the membrane is reduced (Torija et al., 2003). 4. Conclusions Both ammonium and other components (especially amino acids) in the effluent promoted yeast growth and fermentation rate. Fermentation with the effluent as the nitrogen source showed the best performance in terms of yeast growth and ethanol production rates when different nitrogen sources were used, although urea led to a
Fig. 5. Evolution of pH in fermentations with different nitrogen source: ammonium sulfate (j), urea (d), anaerobic digestion effluent (N), anaerobic digestion effluent treated by MAP precipitation (.).
relatively higher growth rate and ethanol production rate than ammonium. Therefore, for the present ethanol–methane fermentation system, anaerobic digestion effluent can be used as
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