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Feasibility of converting lactic acid to ethanol in food waste fermentation by immobilized lactate oxidase
Applied Energy 129 (2014) 89–93
Contents lists available at ScienceDirect
Applied Energy journal homepage: www.elsevier.com/locate/apenergy
Feasibility of converting lactic acid to ethanol in food waste fermentation by immobilized lactate oxidase Hong-zhi Ma ⇑, Yi Xing, Miao Yu, Qunhui Wang Key Laboratory of Educational Ministry for High Efficient Mining and Safety in Metal Mine, University of Science and Technology Beijing, Beijing 100083, China Department of Environmental Engineering, University of Science and Technology, Beijing 100083, China
h i g h l i g h t s Residue lactic acid in food waste could be converted to pyruvic acid. Calcium alginate immobilized the lactate oxidase with high pH and thermal stability. Immobilized enzyme could convert 70% lactic acid to pyruvic acid. Ethanol yield could be increased by 20% with lactate oxidase added.
a r t i c l e
i n f o
Article history: Received 17 February 2014 Received in revised form 24 April 2014 Accepted 28 April 2014
Keywords: Lactate oxidase Lactic acid Food waste Pyruvate Ethanol fermentation
a b s t r a c t Adoption of lactic acid bacteria (LAB) into ethanol fermentation from food waste can replace the sterilization process. However, LAB inoculation will convert part of the substrate into lactic acid (LA), not ethanol. This study adopted lactate oxidase to convert the produced LA to pyruvate, and then ethanol fermentation was carried out. The immobilization enzyme was utilized, and corresponding optimum conditions were determined. Results showed that calcium alginate could successfully immobilize the enzyme and improve pH and thermal stability. The optimum pH and temperature were 6.2 and 55 °C, respectively. The utilization of immobilized enzyme with catalytic time of 5 h could convert 70% LA to pyruvate, and the addition of enzyme increased the ethanol yield by 20% more than that of the control. The process could be applied in food waste storage and can help in reducing carbon source consumption. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The universal concern on energy and environmental preservation has generated increasing research focus on biomass energy worldwide. Finding proper raw materials for biomass energy production is important. Food waste is a type of biomass waste with high organic quantity and moisture content; it is easily perishable and harmful to the environment if not treated properly [1]. High carbon source and abundance of nutrients make food waste an ideal raw material for value-added products, such as methane, lactic acid (LA) fermentation and hydrogen production [2–4]. Ethanol production from food waste can reduce pollution and serve as a resource alternative [5,6]. Fuel ethanol is an important biomass energy because it is simple and has the ability to be mixed with gasoline. Ethanol production usually undergoes cooking process, ⇑ Corresponding author at: Department of Environmental Engineering, University of Science and Technology, Beijing 100083, China. Tel.: +86 13426135537; fax: +86 1062332778. E-mail addresses: [email protected] (H.-z. Ma), [email protected] (Y. Xing). http://dx.doi.org/10.1016/j.apenergy.2014.04.098 0306-2619/Ó 2014 Elsevier Ltd. All rights reserved.
saccharification, fermentation, and distillation. The traditional cooking process consumes 30–40% of the total energy investment and a large quantity of cooling water, which both increase the cost of ethanol production [7–9]. Production cost is one of the barriers for ethanol production application. Uncooked fermentation cancels the cooking and cooling processes, decreases steam and water use, and simplifies the technological route and equipment. The successful utilization of this process for food waste ethanol fermentation will benefit the environment and reduce the ethanol fermentation cost [9]. Another disadvantage of ethanol from food waste is storage for the raw materials. Preventing the occurrence of corruption during storage and deterioration caused by microorganisms is important in this process [10,11]. High organic content in food waste is useful for microorganism growth. LA is accumulated under methophilic condition without lactic acid bacteria (LAB) inoculation [1,12,13]. LAB is the dominant microorganism. No pathogenic microorganisms are observed in this process. The accumulated LA is used to prevent corruption in ethanol production from food waste. A few studies on this utilization are available, and the mutual effect
90
H.-z. Ma et al. / Applied Energy 129 (2014) 89–93
during this process has been studied [14]. Previous studies have focused on removing the corruption effect during the process. No investigation on the effect caused by LAB on the carbon source is available. Ethanol and LA production share the same metabolic pathway, which begin from pyruvic acid. LAB utilization will compete with ethanol production microorganisms for carbon resource. Adopting a proper biochemical method to reduce carbon consumption and prevent corruption results in a double-win effect, which is important for ethanol fermentation from food waste. A possible approach for this goal is to utilize the produced LA during the anti-corruption process to produce pyruvic acid and then yeast in citric acid cycle metabolism for ethanol. The utilization of lactate oxidase can change LA into pyruvic acid [15,16], which is a possible approach to enhance ethanol yield. In fact the pyruvic acid could be produced by fermentation and enzyme-catalyzed reactions [17]. Industrial pyruvate is produced by the dehydration and decarboxylation of tartaric acid [18]. This process has the merit of simple process, while had the disadvantages of expensive cost and low yield. When it came to fermentation method, complex technology route as well as the difficulty in separation had made it hard for industrial application. Enzyme conversion thus became the proper choice [19], there existed several kinds of enzymes for pyruvic production. Lactic dehydrogenase could be used for pyruvic production from lactic acid, since the reaction was a balanced one and corresponding conversion yield was low, and the cost was high [20]. There existed research on pyruvate from lactate by using whole-cell gylcolate oxidase as the enzyme [21]. While among them lactate oxidase was often adopted due to its high yield and low cost [22]. But free enzyme is easily to lose its ability and difficult to be recycled. This would cause difficulty for its separation. If not recycled, the cost for the enzyme would be great. Thus the immobilized enzyme would be easily for recovery of enzyme and could reuse [23]. Take the research for lactate oxidase for example, the utilization for the immobilized enzyme had the following merits, the procedure for cell broken and enzyme extraction could be omit, the stability for the enzyme would be increased, the cost for immobilization had reduced to a large extent, the product could reused for a long time, and it would be easy for separation from product. All these advantages had showed that immobilized enzyme would be a suitable technology. Furthermore, our study utilized food waste as the substrate, which caused a high concentration of suspended solid, which would cause bad effect for the free enzyme, the immobilized enzyme had high tolerance for the impurities inside the food waste ethanol fermentation broth. Which would in turn maintained a high and stable conversion efficiency. Thus the immobilized lactic oxidase should be utilized in this process. Currently more researches were carried out in immobilized enzyme in the field of enzyme electrodes and biosensors [24]. Few research was carried out for its utilization of converting lactic acid inside food waste to ethanol, in this study, sodium alginate will be used for enzyme immobilization, the characteristic for the immobilized enzyme as well as its utilization in food waste were studied in this process, also the relationship between lactic acid and ethanol were studied as well. 2. Materials and methods 2.1. Materials Food waste was collected from the dining room of the University of Science and Technology Beijing, China. LAB, designated as TD175, was obtained from our lab. Escherichia coli [BL21 (DE3)/ pET-LOD] was also obtained from the University of Science and Technology Beijing.
2.1.1. Culture media Luria broth (LB): Peptone (1 g/100 mL), yeast powder (0.5 g/100 mL), NaCl (1.0 g/100 mL), and kanamycin (50 lg/L) were dissolved in water, and pH was adjusted to 7. The solution was sterilized at 121 °C for 15 min. SB3 media: Peptone (44 g/L), yeast powder (30 g/L), NaCl (10 g/L), and kanamycin (50 lg/L) were dissolved in water. The solution was adjusted to pH 7 and sterilized at 121 °C for 15 min. 2.2. Methods 2.2.1. Preparation of lactic oxidase and detection of enzyme ability E. coli was initially cultured in LB media for 12 h and then transferred to SB3 media for 48 h. The fermentation broth was centrifuged at 12,000 r/min for 3 min. Then, the supernatant was discarded and the broth was washed twice with phosphate buffer. The broth was dissolved in 20 mL of PBS, and ultrasonic treatment was carried out for 5 min (minimum power, 200 W; probe amplitude, 40%). After a 20 min centrifugation (n = 10,000 r/min, 4 °C), the supernatant was poured into the tube to collect the lactic oxidase crude enzyme solution. The enzyme was determined based on Ref. [16]. One unit of enzyme was determined as the amount of enzyme that catalyzed LA to produce 1 nmol pyruvate in 1 min. The relative activity was determined as the ratio of the enzyme detected and the maximum enzyme under the condition.
Relative enzyme activity ¼ enzyme activity detected=maximum enzyme activity 2.2.2. Lactate oxidase immobilization Lactic oxidase enzyme solution (20 mL) was mixed with 20 mL of 4% sodium alginate solution. This solution was slowly added into 400 mL of 0.2 mol/L CaCl2 solution with a syringe. The solution was then filtered, washed, dried to obtain the particulate immobilized lactate oxidase, and solidified at 25 °C for 2 h. 2.2.3. Measurement methods for LA, pyruvic acid, ethanol, and reducing sugar content Lactic acid, ethanol concentration was measured by SBA 40 C biosensor [14]. Pyruvate acid was determined as followed, 1 ml of fermentation supernatant was put into a test tube, added 2 mL 8% trichloroacetic acid plus 1.0 mL of 0.1% 2,4-dinitrophenylhydrazine solution. Shaked properly then added 5.0 mL 1.5 mol/L, NaOH solution, detect the absorbance at wavelength of 520 nm, then referred to the standard solution. The reducing sugar was determined as DNS method [25]. All the detection was performed for three times and the average was used for discussion. 2.2.4. Optimization of catalytic conditions for immobilized lactate oxidase 2.2.4.1. Optimum temperature. Phosphate buffer (0.5 mL) was added to each test tube with 0.2 mL of 20 mmol/L LA and 0.1 g of immobilized enzyme. Each tube was placed at 25, 37, 45, 55, 65, and 75 °C, respectively. The water bath was maintained for 10 min, and then 1 mL of 1 mol/L 2,4-dinitrophenylhydrazine was added. The reaction was performed at 55 °C for 20 min, and then 5 mL of 0.04 mol/L NaOH was added to the solution. Finally, the absorbance values were determined at 520 nm wavelength. For comparison, the free enzyme was used as control. 2.2.4.2. Optimum pH. Phosphate buffer with different pH levels (pH 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, and 8.0) was prepared in advance. Then, 0.5 mL of phosphate buffer, 0.2 mL of 20 mmol/L LA, and 0.1 g of immobilized enzyme were placed into each tube. The solution
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2.2.5. Utilization of immobilized enzyme for pyruvate acid production with LA produced during storage of food waste The food waste in this study was obtained from the dining room of the University of Science and Technology Beijing. Corresponding treatment and LA fermentation for food waste storage were based on Ref. [14]. The inoculum size for LAB was determined to be 1% (V/W). During the storage, pH and LA were determined every 12 h. After 48 h of storage, saccharification of food waste was performed using glucoamylase with the addition of 100 U/g. Saccharification of fermentation broth was carried out at 45 °C for 5 h. Then, the broth was separated into solid and liquid parts. The liquid part was used for ethanol fermentation. To utilize the LA residue, the immobilized enzyme was inoculated with a dosage of 20 g/100 mL, and the reaction was catalyzed at 55 °C for 5 h. The fermentation broth was inoculated with yeast to produce ethanol. A corresponding fermentation procedure was mentioned in our previous study [5,6]. One group, which served as control treatment, underwent ethanol fermentation without lactic oxidase added. 3. Result and discussion 3.1. Thermal characteristics of immobilized enzyme For enzyme utilization, thermal stability is important in comparing the temperature tolerance ability between immobilized and free enzymes. These enzymes were both cultured at 65 °C for 1 h. The result in Fig. 1 demonstrates that the free enzyme almost lost its ability, whereas the immobilized enzyme could maintain 79% ability. This result indicates that immobilization could improve thermal stability, which may be attributed to the connection of the enzyme and its carrier. After the immobilization, the enzyme protein was fixed in the gel and its movement was restricted. Thus, the reverse factor for the enzyme also decreased, and the stability for the enzyme structure was enhanced. The thermal stability increase of immobilized enzyme resulted not only in the increase in the reaction speed, but also prevented the pollution caused by microorganisms, which was suitable for its industrial application. 3.2. Optimum catalytic conditions for immobilized and free enzymes To evaluate the optimum temperature for immobilized and free enzymes, the experiment was carried out as mentioned in Section 2.2.4. The result shown in Fig. 2 demonstrates that the optimum temperature for the immobilized enzyme was 55 °C,
100
Relative activity/ %
was placed in a water bath at 55 °C for 10 min, and final absorbance was measured at 520 nm wavelength value, as mentioned in Section 2.2.4. For comparison, free enzyme was used as control.
40
immobilization enzyme free enzyme
20
40
60
80
t/ Fig. 2. The optimum temperature for immobilization and solution enzyme.
and the relative activity could reach almost 100%. For the free enzyme, the optimum temperature was 37 °C. A sharp decrease and a total loss of enzyme ability were observed at temperatures over 37 and 65 °C, respectively. This result is in agreement with those obtained for immobilized enzyme utilization. Such treatment could fix the protein properly, increasing its tolerance in severe condition. The saccharification process usually requires a temperature as high as 50–60 °C. This process was carried out in this study. If the enzyme is affected at a high temperature, no cooling process would be needed, thus reducing the energy consumption in the process. Fig. 3 shows the optimum pH for enzyme. For immobilized enzyme, the highest relative enzyme appeared at 6.2, whereas for free enzyme, the optimum condition was 7.4. When the pH was over 7, the ability of the immobilized enzyme significantly decreased. This result may be due to the calcium alginate inside the immobilization enzyme, which dissolved in alkaline condition. Given that ethanol fermentation generally occurs in an acidic condition, the pH-tolerant enzyme can be used directly in fermentation broth without pH adjustment. The utilization of immobilized enzyme would simplify the process at a greater degree compared with the use of free enzyme. 3.3. Use of enzyme in food waste ethanol production 3.3.1. Effect of inoculums of LAB on food waste storage The details for the utilization of LA in food waste ethanol fermentation were presented in [14]. This study utilized 1% inoculum size, and the storage time was 48 h during the whole process. Fig. 4 shows the pH and LA yield. The LA concentration increased to more than 4 mg/L, and pH decreased from more than 6 to nearly 4. A previous study showed that acidic condition can prevent pollution in the fermentation system caused by microorganisms. Ethanol fermentation could not be significantly affected by the interaction
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0 20
100 80 60 40
immobilization enzyme 20 0
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free enzyme 25
35
100 90 80 70 60 50 immobilization enzyme free enzyme
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30 5.5
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7
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5
7
6 Lactic acid (g/L)
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pH
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1 0
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Time (h) Fig. 4. pH and lactic acid during food waste storage.
with the produced LA. This study aims to utilize LAB for food waste storage. The produced LA would further be converted to pyruvate and then changed to ethanol during fermentation.
3.3.2. Utilization of immobilized enzyme for LA produced during food waste storage The produced LA can prevent fermentation from contamination, but it will occupy some carbon sources and reduce ethanol concentration. To utilize the produced LA, this study adopted immobilized enzyme to convert LA to pyruvate and then to ethanol. Corresponding LA utilized and pyruvate produced in the fermentation process were showed in Fig. 5. The result demonstrates that LA concentration decreased and pyruvate increased as time progressed. When the reaction time was more than 5 h, LA concentration ended. The conversion rate for LA was over 70%. This result shows that the utilization of immobilized enzyme could successfully change the remaining LA to pyruvate. The conversion ratio should be related to the reaction time as well as the substrate concentration. The research carried out by Gu showed that 116 mmo l/DL-lactic acid could convert 76.6 mmol/L pyruvate with the conversion rate of 66%, if the time could prolong to 48 h, the conversion rate could enhance to 82% [26]. The research by S. Gough demonstrated that conversion of lactate to pyruvate was optimal at a concentration of 0.5 M with reaction time of 24 h, resulting in 100% conversion. When it came to 1 M, conversion rate decreased to 60%. From this point of view, since only 5 h was used in our study, the conversion rate was good. In consideration of the residue lactic acid in our study was not high, it would be useless to prolong the time to achieve the higher conversion rate.
3 2.5 3.0
2 lactic acid
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2.0
pyruvate acid
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Pyruvate (g/L)
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Fig. 6. Reducing sugar for food waste ethanol fermentation with and without enzyme added.
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3.3.3. Ethanol fermentation from food waste for groups with and without enzyme addition To evaluate the addition of enzyme for ethanol production, the broth was inoculated with yeast to produce ethanol after the treatment in Section 3.3.2. The corresponding procedure was mentioned in Section 2.2.5. For comparison, the group without enzyme was used as control. Ethanol and reducing sugar for the two groups were detected, and corresponding results are shown in Figs. 6 and 7. Fig. 6 demonstrates that the reduced sugar barely changed with and without the addition of enzyme. In both groups, nearly no sugar could be observed after 60 h; 24–36 h could be regarded as the end of fermentation because only a small amount of sugar was left. Thus, a comparative short time period could be obtained in industrial application. This result is different from previous studies [5,6]. A previous study selected a mixture broth of solid and liquid food wastes as substrate, whereas the present study used saccharification broth. The solid parts may have required additional time to release the sugar inside, so the fermentation time was prolonged. Given this reason, liquid fermentation may be the proper choice. Fig. 7 demonstrates that ethanol concentration in the group with enzyme could be 20% higher than those of the control. Similar to reducing sugar, the maximum ethanol concentration was observed within 24–36 h. The result showed that the enzyme could convert LA to pyruvate and then to ethanol. The conversion rate of LA to pyruvate was 70%. LAB was used to store the food waste, and the produced LA could kill the pathogenic microorganisms. Immobilized enzyme was introduced to convert the produced LA to pyruvate and then to ethanol. This whole process
Reducing sugar (g/L)
92
no enzyme added enzyme added
Time (h) Time (h) Fig. 5. Lactic acid and pyruvate acid during food waste ethanol fermentation with enzyme added.
Fig. 7. Ethanol fermentation with food waste with and without enzyme added.
H.-z. Ma et al. / Applied Energy 129 (2014) 89–93
could prevent corruption and improve ethanol, which is important for food waste ethanol fermentation. The utilization of LAB in ethanol fermentation has been investigated using different approaches. Zhang reported that the adoption of LAB can be used successfully for food waste storage and ethanol fermentation [13]. Solem et al. attempted to use the gene method to allow ethanol fermentation of Lactococcus lactis. The advantage in their study is that the microorganism can tolerate high acid condition, and no other by-product could be produced [27]. Ethanol production from engineered LAB is a short and efficient approach. The difference between their study and the present study is that they used LAB as a platform organism for engineering bacteria to produce ethanol. The present study aims to use LAB to determine the anti-corruption effect. Glucose fermentation was used in their research, and the engineered strain was suitable in obtaining ethanol as its major product with minor by-products. Our materials were food waste that did not undergo sterilization treatment. Their study had a good research topic, but warranted further investigation for industrial application. The present study attempted to utilize LAB in a double-win approach, which not only realized anti-corruption, but also increased ethanol yield from a carbon source utilization pathway. The economical analysis should be carried out with a large-scale utilization. Accurate analysis is required for a true evaluation of the process and its further application. 4. Conclusion The utilization of calcium alginate could successfully immobilize lactate oxidase. The corresponding optimum pH and temperature were 55 °C and 6.2, respectively. The immobilized enzyme preferred high thermal stability. The utilization of the enzyme could convert LA produced during food waste storage to pyruvate with an efficiency of nearly 70%. The corresponding ethanol fermentation was 20% higher than that of the control. This technology could tentatively achieve anti-corruption and reduction of carbon source consumption. Acknowledgements The work was supported by International Science & Technology Cooperation Program of China (2013DFG92600) and National Scientific Funding of China (51008020, 51378003), Beijing Higher Education Young Elite Teacher Project, Beijing Nova Program (NO. Z111106054511043). References [1] Naomichi N, Yutaka N. Recent development of anaerobic digestion processes for energy recovery from wastes. J Biosci Bioeng 2007;103(2):105–12. [2] Moon HC, Song IS. Enzymatic hydrolysis of food waste and methane production using UASB bioreactor. Int J Green Energy 2011;8(3):361–71.
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[3] Sun KH, Hang SS. Biohydrogen production by anaerobic fermentation of food waste. Int J Hydrogen Energy 2004;29(6):569–77. [4] Wang QH, Ma HZ, Xu WL, Gong LJ, Zhang WY, Zou DX. Ethanol production from kitchen garbage using response surface methodology. J Biochem Eng 2008;39(3):604–10. [5] Ma HZ, Wang QH, Zhang WY, et al. Optimization of the medium and process parameters for ethanol production from kitchen garbage by Zymomonasmobilis. Int J Green Energy 2008;5:480–90. [6] Ma HZ, Wang QH, Qian DY, et al. The utilization of acid-tolerant bacteria on ethanol production from kitchen garbage. Renewable Energy 2009;34: 1466–70. [7] Arunachalam M, Ricke Steven C. Current perspectives on detection of microbial contamination in bioethanol fermentors. Bioresour Technol 2010;101: 5033–42. [8] Antonio G, Hikmate A, Rosario LL, Nabil BO. Bacteriocin-based strategies for food biopreservation. Int J Food Microbiol 2007;120:51–70. [9] Ma HZ, Wang QH, Gong LJ, Wang XM, Wang XQ, Yin W. Ethanol production from kitchen garbage by Zymomonas mobilis. Chem Biochem Eng Q 2008;22(3):369–75. [10] Igor M, Michiel K. 10 years of the nisin-controlled gene expression system in Lacetoceoccus lactis. Appl Microbiol Biotechnol 2005;68(6):705–17. [11] Sakai K, Fujii N, Chukeatirote E. Racemization of L-lactic acid in pH-swing open fermentation of kitchen refuse by selective proliferation of Lactobacillus plantarum. J Biosci Bioeng 2006;102(3):227–32. [12] Liu DW, Liu DP, Zeng RJ, et al. Hydrogen and methane production from household solid waste in the two-stage fermentation process. Water Res 2006;40:2230–6. [13] Zhanga Wenyu, Ma Hongzhi. Qunhui Wang research on the adoption of lactic acid bacteria in food waste storage and ethanol production. Int J Green Energy 2012;9(5):456–66. [14] Xu P, Yano T, Yamamoto K, Suzuki H, Kumagai H. Screening for several strains of gram negative bacteria producing the lactate oxidase. J Ferment Bioeng 1996;81:357–9. [15] Xu P, Yano T, Yamamoto K, Suzuki H, Kumagai H. Characterization of a lactate oxidase from a strain of gram negative bacterium. Appl Biochem Biotechnol 1996;56:277–88. [16] Patel NG, Erlenkotter A, Cammann K, et al. Fabrication and characterization of disposable type lactate oxidase sensors for dairy products and clinical analysis. Sens. Actuators, B 2000;67:134–41. [17] Tsujino T, Ohigashi S, Sugiyama S, Kawashiro K, Hayashi H. Oxidation of propylene glycol and lactic acid to pyruvic acid in aqueous phase catalyzed by lead-modified palladium-on-carbon and related systems. J Mol Catal 1992;71:25–35. [18] Howard JW, Fraser WA. Preparation of pyruvic acid. New York: Wiley; 1948. p. 475–480. [19] Ogawa J, Soong CL, Masashi I, Shimizu S. Enzymatic production of pyruvate from fumarate—an application of microbial cyclic-imide transforming pathway. J Mol Catal 2001;11:355–9. [20] Blanchard M, Green DE, Nocito Carroll V, et al. L–H y-droxy acid oxidase [J]. Biol Chem 1946;162:137. [21] Gavagan JE, Fager SK, Seip JE. Glyoxylic acid production using microbial transformant catalysts. J Org Chem 1995;60:3957–63. [22] Gough S, Dostal L. A. Howe Production of pyruvate from lactate using recombinant Pichia pastoris cells as catalyst. Process Biochem 2005;40:2597–601. [23] Schmid A, Dordick JS, Hauer B, et al. Industrial biocatalys-is today and tomorrow [J]. Nature 2001;409:258–68. [24] Suman S, Singhal Rahul, Sharma Amit L, et al. Development of a lactate biosensor based on conducting copolymer bound lactate oxidase. Sens. Actuators, B 2005;107:768–72. [25] Miller GL. Use of DNS reagent for determination of reducing sugars. Anal Chem 1959;31:426–8. [26] Gu J, Xu P, Qu Y. A biocatalyst for pyruvate preparation from DL-lactate: lactate oxidase in a Pseudomonas sp. [J]. J Mol Catal B Enzym 2002;18:299. [27] Solem C, Dehli T, Jensen PR. Rewiring Lactococcus lactis for ethanol production. Appl Environ Microbiol 2013;79:2512–8.
Contents lists available at ScienceDirect
Applied Energy journal homepage: www.elsevier.com/locate/apenergy
Feasibility of converting lactic acid to ethanol in food waste fermentation by immobilized lactate oxidase Hong-zhi Ma ⇑, Yi Xing, Miao Yu, Qunhui Wang Key Laboratory of Educational Ministry for High Efficient Mining and Safety in Metal Mine, University of Science and Technology Beijing, Beijing 100083, China Department of Environmental Engineering, University of Science and Technology, Beijing 100083, China
h i g h l i g h t s Residue lactic acid in food waste could be converted to pyruvic acid. Calcium alginate immobilized the lactate oxidase with high pH and thermal stability. Immobilized enzyme could convert 70% lactic acid to pyruvic acid. Ethanol yield could be increased by 20% with lactate oxidase added.
a r t i c l e
i n f o
Article history: Received 17 February 2014 Received in revised form 24 April 2014 Accepted 28 April 2014
Keywords: Lactate oxidase Lactic acid Food waste Pyruvate Ethanol fermentation
a b s t r a c t Adoption of lactic acid bacteria (LAB) into ethanol fermentation from food waste can replace the sterilization process. However, LAB inoculation will convert part of the substrate into lactic acid (LA), not ethanol. This study adopted lactate oxidase to convert the produced LA to pyruvate, and then ethanol fermentation was carried out. The immobilization enzyme was utilized, and corresponding optimum conditions were determined. Results showed that calcium alginate could successfully immobilize the enzyme and improve pH and thermal stability. The optimum pH and temperature were 6.2 and 55 °C, respectively. The utilization of immobilized enzyme with catalytic time of 5 h could convert 70% LA to pyruvate, and the addition of enzyme increased the ethanol yield by 20% more than that of the control. The process could be applied in food waste storage and can help in reducing carbon source consumption. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The universal concern on energy and environmental preservation has generated increasing research focus on biomass energy worldwide. Finding proper raw materials for biomass energy production is important. Food waste is a type of biomass waste with high organic quantity and moisture content; it is easily perishable and harmful to the environment if not treated properly [1]. High carbon source and abundance of nutrients make food waste an ideal raw material for value-added products, such as methane, lactic acid (LA) fermentation and hydrogen production [2–4]. Ethanol production from food waste can reduce pollution and serve as a resource alternative [5,6]. Fuel ethanol is an important biomass energy because it is simple and has the ability to be mixed with gasoline. Ethanol production usually undergoes cooking process, ⇑ Corresponding author at: Department of Environmental Engineering, University of Science and Technology, Beijing 100083, China. Tel.: +86 13426135537; fax: +86 1062332778. E-mail addresses: [email protected] (H.-z. Ma), [email protected] (Y. Xing). http://dx.doi.org/10.1016/j.apenergy.2014.04.098 0306-2619/Ó 2014 Elsevier Ltd. All rights reserved.
saccharification, fermentation, and distillation. The traditional cooking process consumes 30–40% of the total energy investment and a large quantity of cooling water, which both increase the cost of ethanol production [7–9]. Production cost is one of the barriers for ethanol production application. Uncooked fermentation cancels the cooking and cooling processes, decreases steam and water use, and simplifies the technological route and equipment. The successful utilization of this process for food waste ethanol fermentation will benefit the environment and reduce the ethanol fermentation cost [9]. Another disadvantage of ethanol from food waste is storage for the raw materials. Preventing the occurrence of corruption during storage and deterioration caused by microorganisms is important in this process [10,11]. High organic content in food waste is useful for microorganism growth. LA is accumulated under methophilic condition without lactic acid bacteria (LAB) inoculation [1,12,13]. LAB is the dominant microorganism. No pathogenic microorganisms are observed in this process. The accumulated LA is used to prevent corruption in ethanol production from food waste. A few studies on this utilization are available, and the mutual effect
90
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during this process has been studied [14]. Previous studies have focused on removing the corruption effect during the process. No investigation on the effect caused by LAB on the carbon source is available. Ethanol and LA production share the same metabolic pathway, which begin from pyruvic acid. LAB utilization will compete with ethanol production microorganisms for carbon resource. Adopting a proper biochemical method to reduce carbon consumption and prevent corruption results in a double-win effect, which is important for ethanol fermentation from food waste. A possible approach for this goal is to utilize the produced LA during the anti-corruption process to produce pyruvic acid and then yeast in citric acid cycle metabolism for ethanol. The utilization of lactate oxidase can change LA into pyruvic acid [15,16], which is a possible approach to enhance ethanol yield. In fact the pyruvic acid could be produced by fermentation and enzyme-catalyzed reactions [17]. Industrial pyruvate is produced by the dehydration and decarboxylation of tartaric acid [18]. This process has the merit of simple process, while had the disadvantages of expensive cost and low yield. When it came to fermentation method, complex technology route as well as the difficulty in separation had made it hard for industrial application. Enzyme conversion thus became the proper choice [19], there existed several kinds of enzymes for pyruvic production. Lactic dehydrogenase could be used for pyruvic production from lactic acid, since the reaction was a balanced one and corresponding conversion yield was low, and the cost was high [20]. There existed research on pyruvate from lactate by using whole-cell gylcolate oxidase as the enzyme [21]. While among them lactate oxidase was often adopted due to its high yield and low cost [22]. But free enzyme is easily to lose its ability and difficult to be recycled. This would cause difficulty for its separation. If not recycled, the cost for the enzyme would be great. Thus the immobilized enzyme would be easily for recovery of enzyme and could reuse [23]. Take the research for lactate oxidase for example, the utilization for the immobilized enzyme had the following merits, the procedure for cell broken and enzyme extraction could be omit, the stability for the enzyme would be increased, the cost for immobilization had reduced to a large extent, the product could reused for a long time, and it would be easy for separation from product. All these advantages had showed that immobilized enzyme would be a suitable technology. Furthermore, our study utilized food waste as the substrate, which caused a high concentration of suspended solid, which would cause bad effect for the free enzyme, the immobilized enzyme had high tolerance for the impurities inside the food waste ethanol fermentation broth. Which would in turn maintained a high and stable conversion efficiency. Thus the immobilized lactic oxidase should be utilized in this process. Currently more researches were carried out in immobilized enzyme in the field of enzyme electrodes and biosensors [24]. Few research was carried out for its utilization of converting lactic acid inside food waste to ethanol, in this study, sodium alginate will be used for enzyme immobilization, the characteristic for the immobilized enzyme as well as its utilization in food waste were studied in this process, also the relationship between lactic acid and ethanol were studied as well. 2. Materials and methods 2.1. Materials Food waste was collected from the dining room of the University of Science and Technology Beijing, China. LAB, designated as TD175, was obtained from our lab. Escherichia coli [BL21 (DE3)/ pET-LOD] was also obtained from the University of Science and Technology Beijing.
2.1.1. Culture media Luria broth (LB): Peptone (1 g/100 mL), yeast powder (0.5 g/100 mL), NaCl (1.0 g/100 mL), and kanamycin (50 lg/L) were dissolved in water, and pH was adjusted to 7. The solution was sterilized at 121 °C for 15 min. SB3 media: Peptone (44 g/L), yeast powder (30 g/L), NaCl (10 g/L), and kanamycin (50 lg/L) were dissolved in water. The solution was adjusted to pH 7 and sterilized at 121 °C for 15 min. 2.2. Methods 2.2.1. Preparation of lactic oxidase and detection of enzyme ability E. coli was initially cultured in LB media for 12 h and then transferred to SB3 media for 48 h. The fermentation broth was centrifuged at 12,000 r/min for 3 min. Then, the supernatant was discarded and the broth was washed twice with phosphate buffer. The broth was dissolved in 20 mL of PBS, and ultrasonic treatment was carried out for 5 min (minimum power, 200 W; probe amplitude, 40%). After a 20 min centrifugation (n = 10,000 r/min, 4 °C), the supernatant was poured into the tube to collect the lactic oxidase crude enzyme solution. The enzyme was determined based on Ref. [16]. One unit of enzyme was determined as the amount of enzyme that catalyzed LA to produce 1 nmol pyruvate in 1 min. The relative activity was determined as the ratio of the enzyme detected and the maximum enzyme under the condition.
Relative enzyme activity ¼ enzyme activity detected=maximum enzyme activity 2.2.2. Lactate oxidase immobilization Lactic oxidase enzyme solution (20 mL) was mixed with 20 mL of 4% sodium alginate solution. This solution was slowly added into 400 mL of 0.2 mol/L CaCl2 solution with a syringe. The solution was then filtered, washed, dried to obtain the particulate immobilized lactate oxidase, and solidified at 25 °C for 2 h. 2.2.3. Measurement methods for LA, pyruvic acid, ethanol, and reducing sugar content Lactic acid, ethanol concentration was measured by SBA 40 C biosensor [14]. Pyruvate acid was determined as followed, 1 ml of fermentation supernatant was put into a test tube, added 2 mL 8% trichloroacetic acid plus 1.0 mL of 0.1% 2,4-dinitrophenylhydrazine solution. Shaked properly then added 5.0 mL 1.5 mol/L, NaOH solution, detect the absorbance at wavelength of 520 nm, then referred to the standard solution. The reducing sugar was determined as DNS method [25]. All the detection was performed for three times and the average was used for discussion. 2.2.4. Optimization of catalytic conditions for immobilized lactate oxidase 2.2.4.1. Optimum temperature. Phosphate buffer (0.5 mL) was added to each test tube with 0.2 mL of 20 mmol/L LA and 0.1 g of immobilized enzyme. Each tube was placed at 25, 37, 45, 55, 65, and 75 °C, respectively. The water bath was maintained for 10 min, and then 1 mL of 1 mol/L 2,4-dinitrophenylhydrazine was added. The reaction was performed at 55 °C for 20 min, and then 5 mL of 0.04 mol/L NaOH was added to the solution. Finally, the absorbance values were determined at 520 nm wavelength. For comparison, the free enzyme was used as control. 2.2.4.2. Optimum pH. Phosphate buffer with different pH levels (pH 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, and 8.0) was prepared in advance. Then, 0.5 mL of phosphate buffer, 0.2 mL of 20 mmol/L LA, and 0.1 g of immobilized enzyme were placed into each tube. The solution
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2.2.5. Utilization of immobilized enzyme for pyruvate acid production with LA produced during storage of food waste The food waste in this study was obtained from the dining room of the University of Science and Technology Beijing. Corresponding treatment and LA fermentation for food waste storage were based on Ref. [14]. The inoculum size for LAB was determined to be 1% (V/W). During the storage, pH and LA were determined every 12 h. After 48 h of storage, saccharification of food waste was performed using glucoamylase with the addition of 100 U/g. Saccharification of fermentation broth was carried out at 45 °C for 5 h. Then, the broth was separated into solid and liquid parts. The liquid part was used for ethanol fermentation. To utilize the LA residue, the immobilized enzyme was inoculated with a dosage of 20 g/100 mL, and the reaction was catalyzed at 55 °C for 5 h. The fermentation broth was inoculated with yeast to produce ethanol. A corresponding fermentation procedure was mentioned in our previous study [5,6]. One group, which served as control treatment, underwent ethanol fermentation without lactic oxidase added. 3. Result and discussion 3.1. Thermal characteristics of immobilized enzyme For enzyme utilization, thermal stability is important in comparing the temperature tolerance ability between immobilized and free enzymes. These enzymes were both cultured at 65 °C for 1 h. The result in Fig. 1 demonstrates that the free enzyme almost lost its ability, whereas the immobilized enzyme could maintain 79% ability. This result indicates that immobilization could improve thermal stability, which may be attributed to the connection of the enzyme and its carrier. After the immobilization, the enzyme protein was fixed in the gel and its movement was restricted. Thus, the reverse factor for the enzyme also decreased, and the stability for the enzyme structure was enhanced. The thermal stability increase of immobilized enzyme resulted not only in the increase in the reaction speed, but also prevented the pollution caused by microorganisms, which was suitable for its industrial application. 3.2. Optimum catalytic conditions for immobilized and free enzymes To evaluate the optimum temperature for immobilized and free enzymes, the experiment was carried out as mentioned in Section 2.2.4. The result shown in Fig. 2 demonstrates that the optimum temperature for the immobilized enzyme was 55 °C,
100
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was placed in a water bath at 55 °C for 10 min, and final absorbance was measured at 520 nm wavelength value, as mentioned in Section 2.2.4. For comparison, free enzyme was used as control.
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and the relative activity could reach almost 100%. For the free enzyme, the optimum temperature was 37 °C. A sharp decrease and a total loss of enzyme ability were observed at temperatures over 37 and 65 °C, respectively. This result is in agreement with those obtained for immobilized enzyme utilization. Such treatment could fix the protein properly, increasing its tolerance in severe condition. The saccharification process usually requires a temperature as high as 50–60 °C. This process was carried out in this study. If the enzyme is affected at a high temperature, no cooling process would be needed, thus reducing the energy consumption in the process. Fig. 3 shows the optimum pH for enzyme. For immobilized enzyme, the highest relative enzyme appeared at 6.2, whereas for free enzyme, the optimum condition was 7.4. When the pH was over 7, the ability of the immobilized enzyme significantly decreased. This result may be due to the calcium alginate inside the immobilization enzyme, which dissolved in alkaline condition. Given that ethanol fermentation generally occurs in an acidic condition, the pH-tolerant enzyme can be used directly in fermentation broth without pH adjustment. The utilization of immobilized enzyme would simplify the process at a greater degree compared with the use of free enzyme. 3.3. Use of enzyme in food waste ethanol production 3.3.1. Effect of inoculums of LAB on food waste storage The details for the utilization of LA in food waste ethanol fermentation were presented in [14]. This study utilized 1% inoculum size, and the storage time was 48 h during the whole process. Fig. 4 shows the pH and LA yield. The LA concentration increased to more than 4 mg/L, and pH decreased from more than 6 to nearly 4. A previous study showed that acidic condition can prevent pollution in the fermentation system caused by microorganisms. Ethanol fermentation could not be significantly affected by the interaction
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5
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Time (h) Fig. 4. pH and lactic acid during food waste storage.
with the produced LA. This study aims to utilize LAB for food waste storage. The produced LA would further be converted to pyruvate and then changed to ethanol during fermentation.
3.3.2. Utilization of immobilized enzyme for LA produced during food waste storage The produced LA can prevent fermentation from contamination, but it will occupy some carbon sources and reduce ethanol concentration. To utilize the produced LA, this study adopted immobilized enzyme to convert LA to pyruvate and then to ethanol. Corresponding LA utilized and pyruvate produced in the fermentation process were showed in Fig. 5. The result demonstrates that LA concentration decreased and pyruvate increased as time progressed. When the reaction time was more than 5 h, LA concentration ended. The conversion rate for LA was over 70%. This result shows that the utilization of immobilized enzyme could successfully change the remaining LA to pyruvate. The conversion ratio should be related to the reaction time as well as the substrate concentration. The research carried out by Gu showed that 116 mmo l/DL-lactic acid could convert 76.6 mmol/L pyruvate with the conversion rate of 66%, if the time could prolong to 48 h, the conversion rate could enhance to 82% [26]. The research by S. Gough demonstrated that conversion of lactate to pyruvate was optimal at a concentration of 0.5 M with reaction time of 24 h, resulting in 100% conversion. When it came to 1 M, conversion rate decreased to 60%. From this point of view, since only 5 h was used in our study, the conversion rate was good. In consideration of the residue lactic acid in our study was not high, it would be useless to prolong the time to achieve the higher conversion rate.
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Fig. 6. Reducing sugar for food waste ethanol fermentation with and without enzyme added.
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3.3.3. Ethanol fermentation from food waste for groups with and without enzyme addition To evaluate the addition of enzyme for ethanol production, the broth was inoculated with yeast to produce ethanol after the treatment in Section 3.3.2. The corresponding procedure was mentioned in Section 2.2.5. For comparison, the group without enzyme was used as control. Ethanol and reducing sugar for the two groups were detected, and corresponding results are shown in Figs. 6 and 7. Fig. 6 demonstrates that the reduced sugar barely changed with and without the addition of enzyme. In both groups, nearly no sugar could be observed after 60 h; 24–36 h could be regarded as the end of fermentation because only a small amount of sugar was left. Thus, a comparative short time period could be obtained in industrial application. This result is different from previous studies [5,6]. A previous study selected a mixture broth of solid and liquid food wastes as substrate, whereas the present study used saccharification broth. The solid parts may have required additional time to release the sugar inside, so the fermentation time was prolonged. Given this reason, liquid fermentation may be the proper choice. Fig. 7 demonstrates that ethanol concentration in the group with enzyme could be 20% higher than those of the control. Similar to reducing sugar, the maximum ethanol concentration was observed within 24–36 h. The result showed that the enzyme could convert LA to pyruvate and then to ethanol. The conversion rate of LA to pyruvate was 70%. LAB was used to store the food waste, and the produced LA could kill the pathogenic microorganisms. Immobilized enzyme was introduced to convert the produced LA to pyruvate and then to ethanol. This whole process
Reducing sugar (g/L)
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Time (h) Time (h) Fig. 5. Lactic acid and pyruvate acid during food waste ethanol fermentation with enzyme added.
Fig. 7. Ethanol fermentation with food waste with and without enzyme added.
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could prevent corruption and improve ethanol, which is important for food waste ethanol fermentation. The utilization of LAB in ethanol fermentation has been investigated using different approaches. Zhang reported that the adoption of LAB can be used successfully for food waste storage and ethanol fermentation [13]. Solem et al. attempted to use the gene method to allow ethanol fermentation of Lactococcus lactis. The advantage in their study is that the microorganism can tolerate high acid condition, and no other by-product could be produced [27]. Ethanol production from engineered LAB is a short and efficient approach. The difference between their study and the present study is that they used LAB as a platform organism for engineering bacteria to produce ethanol. The present study aims to use LAB to determine the anti-corruption effect. Glucose fermentation was used in their research, and the engineered strain was suitable in obtaining ethanol as its major product with minor by-products. Our materials were food waste that did not undergo sterilization treatment. Their study had a good research topic, but warranted further investigation for industrial application. The present study attempted to utilize LAB in a double-win approach, which not only realized anti-corruption, but also increased ethanol yield from a carbon source utilization pathway. The economical analysis should be carried out with a large-scale utilization. Accurate analysis is required for a true evaluation of the process and its further application. 4. Conclusion The utilization of calcium alginate could successfully immobilize lactate oxidase. The corresponding optimum pH and temperature were 55 °C and 6.2, respectively. The immobilized enzyme preferred high thermal stability. The utilization of the enzyme could convert LA produced during food waste storage to pyruvate with an efficiency of nearly 70%. The corresponding ethanol fermentation was 20% higher than that of the control. This technology could tentatively achieve anti-corruption and reduction of carbon source consumption. Acknowledgements The work was supported by International Science & Technology Cooperation Program of China (2013DFG92600) and National Scientific Funding of China (51008020, 51378003), Beijing Higher Education Young Elite Teacher Project, Beijing Nova Program (NO. Z111106054511043). References [1] Naomichi N, Yutaka N. Recent development of anaerobic digestion processes for energy recovery from wastes. J Biosci Bioeng 2007;103(2):105–12. [2] Moon HC, Song IS. Enzymatic hydrolysis of food waste and methane production using UASB bioreactor. Int J Green Energy 2011;8(3):361–71.
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