Effect of Ethanol and Lactic Acid Pre-fermentation on Putrefactive

Mar 17, 2016 - †Department of Environmental Engineering, School of Civil and Environmental Engineering, and ‡Beijing Key Laboratory on Resource-Or...
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Effect of Ethanol and Lactic Acid Pre-fermentation on Putrefactive Bacteria Suppression, Hydrolysis, and Methanogenesis of Food Waste Nana Zhao,† Miao Yu,† Qunhui Wang,*,†,‡ Na Song,† Shun Che,† Chunfu Wu,†,‡ and Xiaohong Sun*,§ †

Department of Environmental Engineering, School of Civil and Environmental Engineering, and ‡Beijing Key Laboratory on Resource-Oriented Treatment of Industrial Pollutants, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083, People’s Republic of China § Beijing Reseaching Center of Agricutural Biotechnology, Beijing 100089, People’s Republic of China ABSTRACT: Lactic acid pre-fermentation is widely used as an effective method for the bacteriostatic preservation of food waste, and ethanol pre-fermentation can alleviate acidification and maintain system stability of methane fermentation. This study investigated the effects of methods of ethanol and lactic acid pre-fermentation on bacteriostatic preservation and anaerobic digestion. Results showed that high levels of lactic acid production reduced the pH and decreased the populations of Staphylococcus aureus and Escherichia coli by 99.9 and 99.8%, respectively. Meanwhile, serious acidification during methane production caused the gas production to halt. Ethanol pre-fermentation did not lower the pH of the system but still decreased the S. aureus and E. coli populations by 98.5 and 96.5%, respectively, compared to the control group. This trend was attributed to the presence of some metabolites in the pre-fermentation broth as proven by the Oxford cup method and the selective-medium method. In addition, the total volatile fatty acid content as well as the propionic and acetic acid concentrations were the lowest with ethanol pre-fermentation. However, methane production was 43.9 and 49.6% higher in the ethanol pre-fermentation group than lactic acid pre-fermentation and control groups, respectively. Therefore, ethanol pre-fermentation is an effective treatment that can benefit the bacteriostasis of food waste while promoting hydrolysis, alleviating acidification, and stabilizing the fermentation system to improve the methane production rate.

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INTRODUCTION According to the Food and Agriculture Organization of the United Nations (FAO) survey, nearly 1.3 billion metric tons of food, including fresh vegetables, fruits, meat, bread, and dairy products, is missing in the food supply chain each year;1 the food waste mainly includes carbohydrate polymers (starch, cellulose, and hemicelluloses), lignin, proteins, lipids, organic acids, and inorganic fraction. The conventional food waste treatments have been landfill, incineration, composting, etc.2 However, these conventional treatments can cause secondary pollution easily, and energy recovery efficiency is very low.3 Moreover, food waste treatment has become a critical issue for the Chinese government since the “illegal cooking oil” and “hogwash pig” issues were disclosed by the media. In 2010− 2015, a total of 100 Chinese cities were chosen as model cities of “legitimate treatment and resource utilization of kitchen waste” and anaerobic digestion technology was adopted by more than 68 cities. Anaerobic digestion can transform the organic substance in food waste to a high-calorific-value substance, such as methane, so that it can mitigate the energy crisis.4−6 Thus, anaerobic digestion has become the mainstream. Besides, the biogas market is growing, and to replace the expensive maize silage, more and more industrial organic waste is used in biogas plants.7 Therefore, the study of anaerobic fermentation for food waste is becoming more and more widespread, such as anaerobic co-digestion with animal manures, the improvement of the fermentation conditions, etc.8−10 Large-scale food waste recycling is a current major issue because the collection of food waste is dispersed. © XXXX American Chemical Society

Consequently, the collection and transport of food waste takes time. However, food waste easily promotes bacterial growth because it is rich in water and organic substrates. The bacteriostatic preservation of food waste is a key problem in food waste recycling. Our group found that the addition of lactic and acetic acids in food waste could inhibit bacterial reproduction and maintain the concentrations of useful compounds for subsequent resource utilization (such as the production of ethanol, lactic acid, etc.).11,12 Lactic acid bacteria and their metabolites could inhibit the growth of pathogenic bacteria that can lead to food spoilage and food poisoning.13 However, the effect of lactic acid on the subsequent methanogenesis is unknown. Ethanol prefermentation has been proven to be optimal for the acidification phase of two-phase anaerobic digestion.14 Ethanol prefermentation could also promote hydrolysis−acidification, increase the alkalinity of the system, and maintain the stable pH, thereby increasing the methane production. However, whether ethanol pre-fermentation inhibits bacterial reproduction has not been reported to date. On the basis of these questions, this paper designed ethanol and lactic acid pre-fermentation systems, with a corresponding control group. During pre-fermentation, we counted the number of Staphylococcus aureus and Escherichia coli to confirm that ethanol pre-fermentation can cause bacteriostatic preserReceived: November 25, 2015 Revised: February 25, 2016

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DOI: 10.1021/acs.energyfuels.5b02779 Energy Fuels XXXX, XXX, XXX−XXX

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Energy & Fuels

Pre-fermentation Methods. A total of 1.5 g of activated yeast, lactic acid, or water was first added to three 250 mL conical flasks (i.e., ethanol pre-fermentation, lactic acid pre-fermentation, and control group, respectively), each with 100 g of FW. According to the preexperiment (data not shown), the original concentration of the yeast and lactic bacteria in ethanol pre-fermentation and lactic acid prefermentation group should be higher than 4 × 107 individuals/g of FW. The three conical flasks were numbered and placed in a table concentrator at 37 °C and 60 rpm. The pre-fermentation time was 2.5 days. Samples were collected every 12 h. Fermentation Methods. Three parallel experiments were carried: after pre-fermentation, the FW was placed into the 500 mL methane fermentation tank before DG and AS were put into the tank. The additive amounts of FW, DG, and AS were 40, 30, and 300 g, respectively. To release the remaining air, we purged the reactor with nitrogen (5 min) before the fermentation tank was placed in a THZ82 digital temperature air bath oscillator (60 rpm at 35 ± 1 °C). Biogas production was measured daily. The total volatile fatty acid (TVFA), composition of the organic acid ratio, ethanol concentration, and pH were measured every 3 days. Analytical Techniques. The populations of S. aureus and E. coli were determined by the plate count method, and S. aureus and E. coli used mannitol salt agar medium and deoxycholate agar medium, respectively. The weight loss method was used to measure total solid (TS) and volatile solid (VS). The pH value was measured using PHS-3C type digital acidity. TVFA was measured by colorimetry (725N spectrophotometer). Ethanol, acetic acid, propionic acid, butyric acid, and pentanoic acid were measured by gas chromatography (Shimadzu GC-2010 Japan).

vation before we analyzed its bacteriostatic mechanism via the Oxford cup method and the selective-medium method. Simultaneously, we investigated the effect of the two prefermentation systems on the hydrolysis−acidification and methane production of food waste. We attempted to identify an effective pre-fermentation method that could facilitate bacteriostatic preservation, promote hydrolysis, alleviate acidification, and stabilize the fermentation system to improve the methane production rate.

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MATERIALS AND METHODS

Feedstocks and Inoculums. Food waste (FW) was obtained from a student canteen in the University of Science and Technology in Beijing. The waste was shredded and then stored at −20 °C. Considering their rich lignocellulose content and hard-degraded properties, feedstock distiller’s grain (DG) was added to adjust the C/N ratio and alleviate the acidification of the original digestion substrate. It was obtained from a liquor distillery in Beijing. The DG was ground and then stored at −20 °C. The activated sludge (AS) was allowed to acclimatize for 2 months to the degrading ability of FW and DG before the experiments. The chemical characteristics of the wastes and sludge are shown in Table 1.

Table 1. Composition of Anaerobic Digestion Substrates

a

materiala

TS (%)

VS (%)

VS/TS (%)

pH

DG FW AS

36.83 16.67 15.48

33.67 16.13 9.72

92.32 96.75 62.81

4.35 5.50 7.50

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DG, distiller’s grain; FW, food waste; and AS, activated sludge.

RESULTS AND DISCUSSION Effects of Pre-fermentation on Bacteriostasis. E. coli was reported as one of the causes linked to hemolytic uremic syndrome, a leading cause of kidney failure in children.15 S. aureus is an etiological agent of human and animal diseases.16 S. aureus and E. coli are the two most common and important food-borne pathogens.17 Therefore, this paper examined the populations of both pathogens during pre-fermentation compared to the control group. The results are shown in Figure 1. Figure 1 shows that bacteriostasis was observable in the two pre-fermentation systems compared to the control group. S. aureus and E. coli in food waste were reduced by 2 orders of magnitude or more. Although the inhibitory effect of ethanol pre-fermentation was lower than that of lactic acid prefermentation, relatively strong bacteriostasis was still observed

Ethanol pre-fermentation uses Angel instant dry yeast (produced by Angel Yeast Co., Ltd.). Lactic acid bacteria is Lactobacillus casei (bought in the China Center of Industrial Culture Collection). Experimental Methods. Activation and Cultivation of Yeast and Lactic Acid Bacteria. Yeast. For preparation of the yeast inoculum, 10 g of dry yeast, with the yeast concentration of 3.15 × 1010 individuals/g and 84.48% survival rate, was suspended in 100 mL of 5% glucose solution. The yeast inoculum was activated at 35 °C for 6 h before use. Lactic Acid Bacteria. The strains were grown on agar slant culture medium [agar was placed in the de Man, Rogosa, and Sharpe (MRS) liquid culture medium] and used to inoculate 250 mL conical flasks filled with 100 mL of MRS liquid culture. The conical flasks were placed in a constant temperature box at 35 °C for 12 h. Subsequently, 5 mL of the activated bacteria was transferred to the MRS liquid culture and cultivated at the same conditions for 12 h.

Figure 1. Bacteriostasis of the two pre-fermentation systems: (a) S. aureus and (b) E. coli. B

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fermentation. We conducted further experiments to determine the bacteriostatic mechanism of ethanol pre-fermentation, which has not been previously reported to the best of our knowledge. After literature review and analysis, we initially speculated three possible reasons for the bacteriostatic mechanism of ethanol pre-fermentation. First, yeast and E. coli and S. aureus bacteria may have antagonistic effects; yeast can compete for nutrients and inhibit the growth of pathogenic bacteria. Second, the ethanol produced during pre-fermentation may lead to pathogen dehydration and death.19 However, the maximum ethanol concentration of the pre-fermentation system is only 10 g/L (Figure 2c), and the common medical sterilization concentration of ethanol is 75%. Third, some metabolites (not just ethanol) produced during the pre-fermentation process may inhibit pathogen growth. Given these three possibilities, we performed the Oxford cup method. E. coli and S. aureus were inoculated on the respective selective media before we placed three Oxford cups, in which were 10 g/L ethanol solution (simulating the ethanol concentration in fermentation broth, marked “E”), 5‰ of yeast culture (simulating the inoculation of pre-fermentation, marked “J”), and the supernatant after pre-fermentation (marked “S”). Both cultures were incubated at 37 °C for 24 h. Finally, we observed the changes in the culture media, and the results are shown in Figure 3.

in comparison to the control group. For example, the number of S. aureus and E. coli in the first 2.5 days of ethanol prefermentation were lower by 98.5 and 96.5%, respectively, than the control group. Analysis of the Mechanism of Pre-fermentation to Bacteriostasis. The resulting changes in the pH and the concentrations of ethanol and lactic acid are shown in Figure 2.

Figure 3. Antibiotic sensitivity via the Oxford cup method: (a) S. aureus and (b) E. coli.

Figure 3 shows that no zones of inhibition were formed around Oxford cup “E” and Oxford cup “J”, thereby indicating that 10 g/L ethanol and yeast had no significant inhibitory effects for the two pathogens. Meanwhile, an obvious zone of inhibition appeared around Oxford cup “S”. The yeast supernatant may be an important reason for the inhibition of the growth and reproduction of pathogenic microorganisms. To further verify the above-mentioned results, we smeared E. coli bacteria on three types of culture media, which contained 10 g/L ethanol, the fermentation supernatant, and water, respectively. The three plates were incubated at 37 °C for 24 h. Figure 4 shows that E. coli quickly proliferated on the first two medium plates, but the proliferation of E. coli on the supernatant medium was greatly inhibited. This result was consistent with results of the Oxford Cup method. These results further illustrated that ethanol pre-fermentation broth contains an inhibitor of E. coli proliferation. In fact, many studies on food preservation have discussed inhibitors in the metabolites of yeast. Cavalero and Cooper20 reported that Candida bombicola could produce extracellular glycolipids, which have an inhibitory effect on Candida albicans

Figure 2. Effect of pre-fermentation on the pH and concentration of ethanol and lactic acid.

With an increased lactic acid concentration, the pH of the lactic acid pre-fermentation group obviously decreased, whereas the pH value of the ethanol pre-fermentation group gradually dropped. In increasing order, the pH of the pre-fermentation process was lactic acid group < control group < ethanol group (Figure 2a). Given the known pH suitable for the growth of pathogenic microorganisms (E. coli is pH-neutral, and S. aureus is pH 5−9), the bacteriostatic mechanisms of lactic acid prefermentation were the reduced pH of the system. This result is consistent with those reported in most papers.18 However, the pH of the ethanol pre-fermentation group was higher than that of the control group. Therefore, the pH was not the reason for the bacteriostasis during ethanol preC

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Figure 4. E. coli growth on different media: (a) water, (b) ethanol, and (c) pre-fermentation supernatant.

and S. aureus. Zhao21 found that Saccharomycodes and Brettanomyces exhibited antibacterial effect on E. coli and that the antibacterial substances were almost in the fermentation supernatant of yeast. Chen22 demonstrated that the metabolites of Kluyveromyces marxianus and Saccharomyces cerevisiae could inhibit the growth of E. coli. The main inhibitors were toxin protein and organic acid. The antibacterial substances of the used yeast produced in food waste may be extracellular glycolipids, toxin protein, organic acids, or other antibiotic factors. However, the exact substance has not yet been determined. Thus, a further study on the identification of the antibacterial substances and bacteriostasis mechanism is necessary. Effect of Pre-fermentation on the Hydrolysis of Food Waste and Carbon Flow. Gas chromatography and liquid chromatography showed that the major products of the prefermentation are ethanol, lactic acid, and volatile fatty acids (VFAs), such as acetic, propionic, and butyric acids, among others. This study selected the total main hydrolyzate (TVFA + LA + EtOH) concentration to measure the degree of hydrolysis. Results of the analysis are shown in Figure 5.

higher than that of the control group. This result is similar to the result of He et al.;23 their work disposed perishable organic waste by two-phase anaerobic digestion. When the pH value was kept at 7.0, the hydrolysis−acidification efficiency was the highest. However, when the pH value was not kept constant, hydrolysis−acidification was severely inhibited. Although the pH value of the present study was not maintained at 7.0, the pH value was higher in the ethanol pre-fermentation group than in the lactic acid pre-fermentation and control groups. This trend may explain why the degree of hydrolysis was higher with ethanol pre-fermentation than in the other groups. The degree of substrate hydrolysis, which was represented by the concentration of the hydrolysis product (TVFA + LA + EtOH), was largest in the ethanol pre-fermentation group compared to the lactic acid pre-fermentation and control groups (Figure 5). The pH of the ethanol pre-fermentation group was also the highest among the three groups. This trend can be explained by the change in the carbon source distribution. The carbon source distribution of each group is shown in Table 2. Table 2. Effect of Different Pre-fermentation Methods on the Carbon Distributiona treatmentb

acetic acidc (%)

propionic acidd (%)

lactic acide (%)

ethanolf (%)

control EP LAP

28.56 17.49 20.77

3.91 2.58 4.59

49.75 38.96 64.98

17.79 40.97 9.66

All of the test data were from the first 2.5 days of pre-fermentation. TVFA, total volatile fatty acid; LA, lactic acid; EtOH, ethanol; EP, ethanol pre-fermentation; and LAP, lactic acid pre-fermentation. cAs defined in terms of acetic acid/(TVFA + LA + EtOH). dAs defined in terms of propionic acid/(TVFA + LA + EtOH). eAs defined in terms of lactic acid/(TVFA + LA + EtOH). fAs defined in terms of ethanol/ (TVFA + LA + EtOH). a b

Figure 5. Total main hydrolyzate (TVFA + LA + EtOH) concentration.

The hydrolysis products of the pre-fermentation process were mostly ethanol, lactic acid, and TVFA. After 2.5 days, the ratio of the ethanol concentration to the total concentration of hydrolysis products was the highest in the ethanol prefermentation group, with an increase by 23.2% relative to the control group. Meanwhile, the ratios of the acetic acid concentration, the propionic acid concentration, and the lactic acid concentration to the total concentration of hydrolysis products were all the lowest, with a decrease by 11.1, 1.3, and 10.8%, respectively, compared to the control group (see Table 2). These results implied that most of the carbon source in the ethanol pre-fermentation group transformed into the neutral ethanol. Consequently, the production of acetic acid, propionic acid, and lactic acid decreased. Therefore, the pH of the ethanol

As shown in Figure 5, on the first day of fermentation, the degree of hydrolysis of food waste in descending order was ethanol group ≈ lactic acid group > control group. The pH of the lactic acid pre-fermentation group decreased with an increased hydrolyzate concentration (Figure 2a); the pH of the system was too low to inhibit bacterial activity. Consequently, the hydrolyzate did not increase after the first day. Finally, the hydrolyzate concentration of the lactic acid pre-fermentation and control groups was similar. In contrast, the degree of hydrolysis continued to increase during ethanol pre-fermentation; thus, the concentration of the hydrolyzate was 30.3% D

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Figure 6. Daily production and cumulative production of methane by different groups.

pre-fermentation group was higher than the other two groups, thereby preventing acidification. The lactic acid pre-fermentation group had the highest ratio of the lactic acid concentration to the total concentration of hydrolysis products, which was 15.23% more than the control group. In the lactic acid pre-fermentation group, the carbon source was converted into lactic acid in advance. Lactic acid is easily converted to propionic acid; thus, the ratio of the propionic acid concentration to the total concentration of hydrolysis products was 0.68% higher than that in the control group, thereby easily leading to acidification in the subsequent methanogenesis. Effect of Pre-fermentation to Methanogenesis. In addition to antibacterial preservation, promotion of substrate hydrolysis, and remission of acidification, the most important purpose of ethanol pre-fermentation is to improve the methane production. Consequently, we mixed the food waste without pretreatment or after ethanol or lactic acid pre-fermentation with the distiller’s grain and seed sludge in three respective tanks. The tanks were placed in the same environment to start methane fermentation. The daily methane production was recorded. In this study, the daily methane production and cumulative production of each group are shown in Figure 6. Figure 6a shows that the ethanol pre-fermentation group had the highest daily gas production; gas production was maintained for 16 days. The lactic acid pre-fermentation group rapidly produced biomass, but gas production was strongly inhibited after the first 7 days and stopped until the fermentation end. As seen in Figure 6b, the accumulated methane yield in ascending order was ethanol group (2988 ± 133.6 mL) > lactic acid group (1675 ± 123 mL) > control group (1507 ± 96.5 mL). The methane production of the ethanol pre-fermentation group was twice as large as that of the control group. Therefore, ethanol pre-fermentation favors the subsequent fermentation process. This paper analyzed the effect of different pre-fermentation systems on the pH value and TVFA in the methane fermentation system; the results are shown in Figure 7. As shown in Figure 7, at the beginning of methanogenesis, the pH of the mixed materials ranged from 7.2 to 7.7, whereas the TVFA concentration was 2.0−2.7 g/L. By the third day of fermentation, the pH value significantly decreased as the TVFA concentration continued to rise. This phenomenon indicated that the acidification stage still existed during the early process of methanogenesis. The acidification degree of the ethanol prefermentation group was significantly lower than that of the

Figure 7. Effect of pre-fermentation on pH and TVFA of methanogenesis.

control group because of the high ethanol concentration. The acidification degree of the lactic acid pre-fermentation group was higher than that of the control group because of the high concentration of lactic acid. This finding indicated that the system of the ethanol pre-fermentation group was the most stable. After 3 days, the pH value increased rapidly and the concentration of TVFA decreased quickly. After 9 days, the pH value and the concentration of TVFA were stable. The pH value and TVFA of the ethanol pre-fermentation group changed with the smallest margin compared to those of the other two groups. The changes in the concentrations of ethanol, acetic acid, and propionic acid as well as the ratio of propionic acid/acetic acid during methanogenesis in the three experimental groups are shown in Figure 8. The ethanol concentration of the ethanol pre-fermentation group during methanogenesis was higher than that of other groups (Figure 8a), but the acetic acid and propionic acid concentrations were lower than other groups (panels b and c of Figure 8). This result is consistent with the carbon flow in the hydrolysis stage (Figure 5 and Table 2). The trend can be attributed to the fact that, during the process of methanogenesis, hydrolysis acidification also occurred through the glycolytic pathway [Embden−Meyerhof−Parnas (EMP)]. The sugar in organic matter produced pyruvate, which is converted by different metabolic pathways into different products. Under the same amount of pyruvate, ethanol pre-fermentation was equal to the strength of metabolic pathways during ethanol E

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Figure 8. Effect of pre-fermentation on the main hydrolyzate and the ratio of propionic acid/acetic acid during methanogenesis.

Besides TVFA, the composition of VFAs and the ratio of propionic acid/acetic acid are also an important factor for CH4 fermentation. In a previous study, this ratio was always used to predict the stability of the digestion system and generally the ratios of ≤0.5 resulted in a faster methane production and VFA decomposition compared to ratios of ≥1.27,28 The increase in this ratio always indicated that the system will breakdown in the near future.29 The ratio of propionic acid/acetic acid in the current study was as high as 0.43 and lasted for a long duration, thereby causing irreversible variation in the system. Consequently, the gas production was stopped after 6 days. However, the ratio of propionic acid/acetic acid of ethanol prefermentation was the lowest, and the system was the most stable.

production, which converted more of the carbon source into ethanol. In comparison to methane-producing bacteria (MPB) that use hydrogen, MPB that use acetic acid have a dominant role in the anaerobic digestion system. That is, MPB can directly use acetic acid but not ethanol. Although the acetic acid concentration of the control group during the pre-fermentation was higher than that of other groups, the CH4 fermentation was inhibited by the high TVFA concentration and significant decrease in pH. In contrast, the acetic acid concentration was initially not high. However, produced ethanol could be continuously transformed into acetic acid by ethanol-oxidizing bacteria in the system. This trend could be caused by the Gibbs energy, which was relatively lower when ethanol was transformed into acetic acid.24 In addition, the suitable pH for ethanol-oxidizing bacteria was similar to that of the MPB; a synergetic effect may exist in both bacterial systems. In the combined system, MPB can use the acetic acid, which was directly produced from ethanol by ethanol-oxidizing bacteria, to generate CH4. That is, the ethanol-oxidizing bacteria can supply the “slow release acetic acid matter” for the MPB.25 Therefore, the ethanol pre-fermentation can create sufficient nutrition and a stable pH environment for MPB. The ethanol and acetic acid concentrations of the lactic acid pre-fermentation group were similar to those of the control group. However, the propionic acid concentration was significantly higher than that of the other two groups. Propionic acid is difficult to be used by methanogens, and its toxic effects on methanogenic bacteria are potent even at a low concentration.26 Thus, lactic acid pre-fermentation is not conducive to the methane fermentation of food waste.

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CONCLUSION

(1) During the food waste pre-fermentation stage, bacteriostasis was observable during ethanol and lactic acid prefermentation compared to the control group, such that the numbers of S. aureus and E. coli in food waste were reduced by 2 orders of magnitude or more. Ethanol pre-fermentation can inhibit the growth and reproduction of pathogenic microorganisms, which is caused by some metabolites during prefermentation. In addition, the concentration of the hydrolyzate (TVFA + LA + EtOH) was the highest for the ethanol prefermentation, such that it was 30.30 and 34.34% higher than the control and lactic acid pre-fermentation groups, respectively. More carbon was converted to ethanol (accounting for 40.97% of the total) with ethanol pre-fermentation, whereas carbon was F

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(10) Yang, L.; Huang, Y.; Zhao, M.; Huang, Z.; Miao, H.; Xu, Z.; Ruan, W. Enhancing biogas generation performance from food wastes by high-solids thermophilic anaerobic digestion: Effect of pH adjustment. Int. Biodeterior. Biodegrad. 2015, 105, 153−159. (11) Wang, Q.; Yamabe, K.; Narita, J.; Morishita, M.; Ohsumi, Y.; Kusano, K.; Shirai, Y.; Ogawa, H. I. Suppression of growth of putrefactive and food poisoning bacteria by lactic acid fermentation of kitchen waste. Process Biochem. 2001, 37 (4), 351−357. (12) Wang, Q.; Narita, J.; Ren, N.; Fukushima, T.; Ohsumi, Y.; Kusano, K.; Shirai, Y.; Ogawa, H. Water, Air, Soil Pollut. 2003, 144, 405−418. (13) Gutiérrez, S.; Martínez-Blanco, H.; Rodríguez-Aparicio, L. B.; Ferrero, M. A. Effect of fermented broth from lactic acid bacteria on pathogenic bacteria proliferation. J. Dairy Sci. 2016, DOI: 10.3168/ jds.2015-10439. (14) Wu, C.; Wang, Q.; Yu, M.; Zhang, X.; Song, N.; Chang, Q.; Gao, M.; Sonomoto, K. Effect of ethanol pre-fermentation and inoculum-tosubstrate ratio on methane yield from food waste and distillers’ grains. Appl. Energy 2015, 155, 846−853. (15) Karmali, M.; Petric, M.; Steele, B.; Lim, C. Lancet 1983, 321, 619−620. (16) Alva-Murillo, N.; Medina-Estrada, I.; Báez-Magaña, M.; OchoaZarzosa, A.; López-Meza, J. E. The activation of the TLR2/p38 pathway by sodium butyrate in bovine mammary epithelial cells is involved in the reduction of Staphylococcus aureus internalization. Mol. Immunol. 2015, 68 (2), 445−455. (17) Zhang, Y.; Liu, X.; Jiang, P.; Li, W.; Wang, Y. Mechanism and antibacterial activity of cinnamaldehyde against Escherichia coli and Staphylococcus aureus. Mod. Food Sci. Technol. 2015, 05, 31−35 (in Chinese). (18) Li, X.; Shao, W.; Diao, E.; Zhang, H. Inhibition study on E. coli by temperature, pH and natural drug with microcalorimetric method. Food Sci. 2007, 06, 252−255 (in Chinese). (19) Wei, F.; Liu, X.; Cao, H.; Qiu, B.; Liu, J.; Ma, S. Effects of different alcohol concentrations on the biofilms of two common bacteria. Chin. J. Microecol. 2013, 25 (8), 907−910 (in Chinese). (20) Cavalero, D. A.; Cooper, D. G. The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214. J. Biotechnol. 2003, 103 (1), 31−41. (21) Zhao, M. A research on the isolation, identification and antibacterial property of yeasts in the Koumiss. Master’s Thesis, Inner Mongolia University, Huhhot, Inner Mongolia, China, 2002 (in Chinese). (22) Chen, Y. Study of antibacterial mechanism of yeasts metabolites from Koumiss on pathogenic Escherichia coli. Master’s Thesis, Inner Mongolia University, Huhhot, Inner Mongolia, China, 2015 (in Chinese). (23) He, P.; Pan, X.; Lv, F.; Shao, L. The influence of pH value on anaerobic hydrolysis and acidogenesis rates of biodegradable organic waste. China Environ. 2006, 01, 57−61 (in Chinese). (24) Refai, S.; Wassmann, K.; Deppenmeier, U. Short-term effect of acetate and ethanol on methane formation in biogas sludge. Appl. Microbiol. Biotechnol. 2014, 98 (16), 7271−7280. (25) Lim, J. W.; Chen, C. L.; Ho, I. J. R.; Wang, J. Y. Study of microbial community and biodegradation efficiency for single- and two-phase anaerobic co-digestion of brown water and food waste. Bioresour. Technol. 2013, 147, 193−201. (26) Wang, Y.; Zhang, Y.; Wang, J.; Meng, L. Effects of volatile fatty acid concentrations on methane yield and methanogenic bacteria. Biomass Bioenergy 2009, 33 (5), 848−853. (27) Wang, L.; Wang, Q.; Cai, W.; Sun, X. Influence of mixing proportion on the solid-state anaerobic co-digestion of distiller’s grains and food waste. Biosystems Engineering. 2012, 112 (2), 130−137. (28) Wagner, A. O.; Reitschuler, C.; Illmer, P. Effect of different acetate:propionate ratios on the methanogenic community during thermophilic anaerobic digestion in batch experiments. Biochem. Eng. J. 2014, 90, 154−161.

mostly converted to lactic acid (accounting for 64.98% of the total) and propionic acid with lactic acid pre-fermentation. (2) During the methanogenesis stage, the ethanol prefermentation system had the largest pH value, the minimum amount of VFAs, and the lowest ratio of propionic acid/acetic acid; that said, the methane fermentation system was stable. However, the lactic acid pre-fermentation system was the opposite, and acidification appeared at 7 days. The accumulated methane yield after 21 days was in the following order: ethanol group (2988 ± 133.6 mL) > lactic acid group (1675 ± 123 mL) > control group (1507 ± 96.5 mL). In conclusion, lactic acid pre-fermentation has good bacteriostatic and hydrolytic acidification effects but caused acid inhibition of the subsequent methanogenesis. In contrast, ethanol pre-fermentation realized antimicrobial preservation of food waste, promoted hydrolysis, alleviated acidification, kept the system stable, and improved methane production. Therefore, ethanol pre-fermentation is a simple, economic, and effective pretreatment.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone/Fax: +86-010-6233-2778. E-mail: wangqh59@ sina.com. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Key Technology R&D Program (2014BAC24B01) and the National Natural Science Foundation of China (Grant 51278050).

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REFERENCES

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DOI: 10.1021/acs.energyfuels.5b02779 Energy Fuels XXXX, XXX, XXX−XXX

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Energy & Fuels (29) Wang, Q.; Noguchi, C. K.; Kuninobu, M.; Hara, Y.; Kakimoto, K.; Ogawa, H. I.; Kato, Y. Influence of hydraulic retention time on anaerobic digestion of pretreated sludge. Biotechnol. Tech. 1997, 11 (2), 105−108.

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DOI: 10.1021/acs.energyfuels.5b02779 Energy Fuels XXXX, XXX, XXX−XXX