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Production of propionic acid-enriched volatile fatty acids from co-fermentation liquid of sewage sludge and food waste using Propionibacterium acidipropionici Xiang Li, Hui Mu, Yinguang Chen, Xiong Zheng, Jingyang Luo and Shu Zhao
ABSTRACT Volatile fatty acids (VFA), derived from sludge fermentation, have been used as one effective carbon source for biological nutrient removal, especially favorable with VFA containing with high levels of propionic acid. In this paper, a new fermentation method was employed to significantly produce the propionic acid-enriched VFA from the co-fermentation liquid of sewage sludge and food waste: including (1) mixing food waste with sludge in the anaerobic digester (the first stage) and (2) separating the mixture, sterilizing the first stage liquid and fermenting it after inoculation with Propionibacterium acidipropionici (the second stage). The effect of the key parameters including pH, the mixing ratio of the food waste and sludge, fermentation time and temperature of the first stage
Xiang Li Hui Mu Yinguang Chen (corresponding author) Xiong Zheng Jingyang Luo Shu Zhao State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China E-mail: [email protected]
on the propionic acid-enriched VFA production (the second stage) was individually discussed. By the molecular weight distribution analysis, the comparison of the solubilisation and hydrolysis process in difference parameters was fully elaborated. The optimal combination of the parameters was then obtained. Finally, the propionic acid-enriched VFA fermentation was successfully conducted in a semi-continuous reactor using the first stage liquid from the optimal condition. Key words
| food waste, Propionibacterium acidipropionici, propionic acid, two stage fermentation
INTRODUCTION It has been demonstrated that volatile fatty acids (VFA) can be used as the additional carbon source for biological nutrient removal (BNR), which has been widely used to remove nitrogen and phosphorus from wastewater (Arun et al. ; Abu-ghararah & Randall ; Guisasola et al. ). Normally, 1.07–1.82 and 1.87–3 mg VFA-C (carbon based) was required to remove 1 mg N and 1 mg P, respectively (Daigger & Bowen ; Elefsiniotis & Li ). Since the VFA in most of the municipal wastewater is insufficient, it is therefore necessary to provide VFA to the BNR system. Although chemically synthesized VFA can be used as the supplementary carbon source of municipal wastewater, it will consume the limited organic resources. Therefore, many efforts have been made to convert waste activated sludge (WAS), a by-product of wastewater treatment plants, to acetic acid-containing VFA (Yuan et al. ; Li et al. ; Tan et al. ). However, it was reported that the increase of propionic acid percentage in doi: 10.2166/wst.2013.463
VFA would facilitate the performance of BNR (Chen et al. ; Ji & Chen ). An important reason is that the increase of wastewater propionic/acetic acid ratio is beneficial to the competition of phosphorus-accumulating organisms over glycogen-accumulating organisms (Zhang et al. ). Thus, high propionic acid-containing VFA instead of acetic acid-dominated VFA is preferable for BNR. But until now, the strategy to improve the content of propionic acid in VFA has seldom been reported (Feng et al. ). Recently, a novel two-stage fermentation method was documented for the first time to significantly improve the propionic acid fraction in VFA derived from WAS by using food waste and Propionibacterium acidipropionici (Chen et al. ). First, the food waste, enriching the carbohydrate available, and sewage sludge were fermented for several days. Then, the liquid phase of the above mixture was sterilized, inoculated by P. acidipropionici, and fermented. Finally, propionic acid-enriched VFA was
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obtained and further utilized as a superior carbon source of BNR. In the last study, the first stage fermentation was demonstrated to be the crucial step for the second stage fermentation. Thus, the parameters involved in the first stage fermentation are worth considering. In the literature, pH value and fermentation time were the important factors to affect the anaerobic process (Inanc et al. ; Yuan et al. ; Zhao et al. ), while the mixing ratio of the food waste and sludge determined the organic loading rate to the anaerobic system (Forbes et al. ). Meanwhile, the temperature determined the anaerobic efficiency (Zhang et al. ). However, few studies revealed the effect of those factors from the first stage on the second stage fermentation. In the current study, the effect of key parameters of the first stage, including pH, the mixing ratio of the food waste and sludge (named VSf/VSs), fermentation time and temperature, on the propionic acid-enriched VFA fermentation (i.e., the second stage) were respectively elaborated. Additionally, the molecular weight distribution analysis further revealed the influence of these parameters on the solubilisation and hydrolysis process in the first stage. Furthermore, the second stage fermentation in a semicontinuous reactor was testified feasible using the first stage fermentation liquid obtained from the optimal condition.
Table 1
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METHODS Optimization of the first stage parameters on the second stage fermentation in batch experiments Since the pH value, mixing ratio of food waste and sludge (i.e., volatile solids ratio VSf/VSs), fermentation time and temperature played the important roles in the first stage fermentation, it is necessary to investigate the individual effect of these parameters on the second stage fermentation. The characteristic of the food waste and sludge are shown in Supporting Information (available online at http://www. iwaponline.com/wst/068/463.pdf). A series of identical reactors, with working volume of 1.0 L each, was used in the trials. The details of the arrangement for the trials are shown in Table 1. For each test, certain amounts of sludge and food waste were mixed in the anaerobic reactor, and then tap water was added to achieve the final volume of 1 L and total chemical oxygen demand (TCOD) of 25.0 ± 1.5 g/L. Each reactor was mechanically stirred at 120 rpm and maintained at a certain temperature. After a certain period of fermentation time, the mixture was centrifuged (4,000 rpm for 10 min) and the suspension liquid was obtained. Then 5 mL of each liquid sample was used for the molecular weight distribution analysis. Fifty millilitres of the suspension liquid was transferred to the serum
Experimental arrangement of the variables for the parameters investigated in the first stage fermentation Variable ranges Temperature ( C)
2.5 ± 0.1b
20 ± 1b
0.7/1 ± 0.1/1 2.5/1 ± 0.2/1 9.5/1 ± 0.2/1 17.5/1 ± 0.3/1
2.5 ± 0.1
20 ± 1
8.0 ± 0.3
6.0/1 ± 0.2/1
1.0 ± 0.1 2.0 ± 0.1 3.0 ± 0.1 4.0 ± 0.1
20 ± 1
8.0 ± 0.3
6.0/1 ± 0.2/1
2.5 ± 0.1
5±1 35 ± 1 50 ± 1
pH
Effect of pH on the second stage fermentation
Blanka 6.0 ± 0.3 7.0 ± 0.3 8.0 ± 0.3b 9.0 ± 0.3 10.0 ± 0.3
Effect of VSf/VSs ratio on the second stage fermentation
8.0 ± 0.3
Effect of fermentation time on the second stage fermentation
Effect of temperature on the second stage fermentation a
VSf/VSs ratio
6.0/1 ± 0.2/1b
Blank referred to the test without pH control.
b
W
Time (d)
Parameter investigated
This combination was employed for all the batch tests of the parameters, but only shown in the effect of pH test.
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bottle (volume 100 mL) and adjusted to pH 7. After autoclaving in 121 C and 15 psig for 20 min, each serum bottle was inoculated with 5 mL P. acidipropionici inoculum, flushed with nitrogen gas and sealed with a rubber cap. Then, each bottle was fermented in an air-bath shaker (120 rpm) at 30 ± 1 C and pH 7. After 4 d of second fermentation, the stable propionic acid concentration was observed and the VFA was assayed. The optimal combination of the parameters was obtained from the best performance of the second stage fermentation. W
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phase (flow rate of 0.5 mL/min). The samples were diluted and filtered through a 0.45 μm hydrophilic filtration membrane before being assayed by a refractive index detector (RID-10A). The Mw was calculated with Class VP 5.03 software (Shimadzu Co., Japan).
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Operation of the semi-continuous fermentation for high propionic acid-enriched VFA production The semi-continuous setup is shown in Figure S1 (Supporting Information, available online at http://www.iwaponline. com/wst/068/463.pdf), which included: (I) first stage reactor (working volume of 3.0 L), (II) centrifuge and autoclave equipment, and (III) second stage reactor (working volume of 2.0 L). The setup was operated for 40 d. In the first stage reactor, the mixture of food waste and WAS (VSf/VSs ratio: 6.0/1) was added with tap water to make the TCOD of 25.0 ± 1.5 g/L. The first stage reactor (mechanically stirred at 100 rpm) was maintained at pH 8.0 ± 0.3 using 5 M NaOH or 5 M HCl. The temperature was controlled at 20 ± 1 C by water bath equipment. After 2.5 d of first stage fermentation, 1 L of the suspension liquid was obtained via centrifuge, which was autoclaved in 121 C and 15 psig for 20 min. The suspension liquid was stored for the second stage fermentation. In the beginning of the startup for the second stage reactor, 2 L of the first stage liquid was inoculated with 200 mL P. acidipropionici inoculum. Then every 4 days, 1 L of the second stage mixture was discharged out from the second stage reactor, and the same volume of the first stage liquid obtained above was added to the second stage reactor followed by nitrogen flush. The second stage fermentation was then operated at 30 ± 1 C and pH 7.0 ± 0.3. After Day 28, the second stage reactor stopped refreshing the first stage liquid and fermented in batch for another 12 d. During the semi-continuous fermentation, VFA was measured every day. W
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Analytical methods Molecular weight (Mw) distributions of the first stage fermentation liquid were determined by a gel-filtration chromatography analyzer (Shimadzu Co., Japan). The gel column (TSK G4000SW type Tosoh Co., Japan) was utilized in the current study. Milli-Q water was used as the mobile
RESULTS AND DISCUSSION Effect of the parameters in the first stage on the second stage fermentation Figure 1(a) shows the effect of pH in the first stage on the propionic acid concentration and its percentage in total VFA of the second stage. Obviously, the highest propionic acid concentration, 7.12 g COD/L, was observed when the first stage fermentation was conducted at pH 8. With the decrease of pH from 8, propionic acid concentration dropped rapidly to less than 4.72 g COD/L. Increasing pH to 10 resulted in a propionic acid concentration of only 1.02 g COD/L, which shows a severe inhibition to propionic acid production. From Figure 1(a), the percentage of propionic acid in total VFA was 66.5, 66.6, 69.8 and 69.1% with no significant difference (P value >0.05) from pH 6 to 9. However, the percentage fell to only 32.5% at pH 10. Therefore, pH 8 resulted in the highest propionic acid production. In Figure 1(b), the VSf/VSs ratio of 0.7/1, 2.5/1 and 6.0/1 exhibited similar propionic acid concentration of 6.11, 6.39 and 7.12 g COD/L, respectively. However, with the increase of VSf/VSs ratio from 9.5/1 to 17.5/1, the concentration of propionic acid decreased from 5.29 to 2.81 g COD/L. Similarly, the VSf/VSs ratio from 2.5/1 to 9.5/1 resulted in the same percentage of propionic acid in total VFA (approx. 69.8%). However, further increasing VSf/VSs ratio to 17.5/1 led to significant decrease of propionic acid percentage (to 45.2%). Thus, VSf/VSs ratio of 6.0/1 was the optimal ratio for the second stage fermentation. Figure 1(c) denotes that the optimal fermentation time in the first stage was 2.5 d, which led to the highest propionic acid production. Meanwhile, propionic acid concentration and percentage derived from 2 and 3 d were 6.52 and 6.75 g COD/L, and 67.9 and 68.5% respectively with no significant difference (P value >0.05). However, fermentation for 4 d resulted in a decrease of both propionic acid concentration and percentage, while fermentation for 1 d resulted in propionic acid concentration of 4.43 g COD/L. Figure 1(d) explains the effect of temperature on the propionic acid concentration and percentage.
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Effect of pH, VSf/VSs ratio, fermentation time and temperature in the first stage on the propionic acid-enriched VFA fermentation.
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Fermentation at 50 C, notwithstanding the high propionic acid concentration of 6.49 g COD/L, led to a reduction in the percentage of propionic acid in the VFA to 61.5%. Although, low temperature at 5 C exhibited high percentage of 68.8%, the concentration of propionic acid was the lowest (only 2.58 g COD/L). Despite anaerobic fermentation being favourable at 35 C (Nakasaki et al. ), the propionic acid concentration fell to only 5.45 g COD/L, with the percentage of 56.4%. The mechanisms of the parameters affecting the bio-conversion process during the first stage fermentation will be discussed in the following text. Thus, the optimal temperature should be set at 20 C. W
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Mechanisms study of the parameters affecting the process of solubilisation and hydrolysis in the first stage As shown in Figure S2 (Supporting Information, available online at http://www.iwaponline.com/wst/068/463.pdf), the mixture of sludge and food waste was first dissolved to colloid and polymer organics, which further hydrolyzed to protein and polysaccharide in the first stage fermentation. Afterwards, the second stage fermentation was driven by the first stage liquid containing a variety of metabolic products. Different parameters in the first stage determined the organic content through the solubilisation and hydrolysis process, which could be expressed
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via Mw distribution analysis in Figure S3 (Supporting Information). In Figure S3(a), extreme alkaline fermentation at pH 10 probably caused some toxic macro-matter release from the sludge, which inhibited the growth of P. acidipropionici in the second stage. Similar findings were reported whereby the addition of monovalent ion (such as Naþ) to sludge deteriorated the dewatering capability of sludge, which might result from the release of some unfavourable chemicals (Higgins & Novak ). Low pH value (pH < 8) in the first stage mainly weakened the strength of the solubilisation and hydrolysis process, which reduced the potential substrate for the propionic acid production in the second stage. This was in good agreement with the findings in the literature (Yuan et al. ; Cokgor et al. ). In Figure S3(b), the VSf/VSs ratio from 6.0/1 to 9.5/1 exhibited higher concentration (the area of the peaks) of low Mw hydrolysate than that of other ratios. A previous study demonstrated that the activity of sludge hydrolysis enzyme was significantly improved by the addition of carbohydrate to the sludge hydrolysis system (Feng et al. ). Therefore, the proper VSf/VSs ratio resulted in the highest solubilisation efficiency (Lim et al. ). The effect of first stage fermentation time on the Mw distribution is shown in Figure S3(c). Fermentation time between 2 and 3 d exhibited similar Mw distribution, which was consistent with Figure 1(c). The Mw distribution at different temperature is denoted in Figure S3(d). It is well known that the speed of solubilisation and hydrolysis will be improved with the increase of temperature (Cokgor et al. ; Yuan et al. ). Therefore, the organics were hydrolyzed to smaller molecular matter in the range of 100,000 to 20, 000 g/mol Mw using 20, 35 and 50 C. However, it was over-hydrolyzed at 35 C, which led to depletion of the potential substance for the second stage. Nevertheless, isobutyric acid was accumulated at 50 C in the first stage liquid (data not shown), which weakened the percentage of propionic acid in the second stage. Therefore, the optimal parameters in the first stage fermentation are pH 8, VSf/VSs ratio 6.0/1, fermentation for 2.5 d at 20 C. W
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Figure 2
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Performance of the semi-continuous fermentation under the optimal parameters.
inhibitory effect of the high propionic acid was reduced extensively and P. acidipropionici started to produce propionic acid. Therefore, after the adaptation period (Day 0 to Day 10), the propionic acid concentration improved to 8.19 ± 0.17 g COD/L and total VFA increased to 13.08 ± 0.33 g COD/L on Day 20. The maximal concentration of the VFA was 15.20 ± 0.38 g COD/L on Day 28, and the corresponding propionic acid was 9.65 ± 0.23 g COD/L, which was much higher than the previous studies (Feng et al. ; Chen et al. ). During the last 12 d of batch fermentation (Day 28–40), the concentration of propionic acid became steady at 8.57 ± 0.21 g COD/L after 4 d (i.e., Day 32). Therefore, the retention time of 4 d for the second stage was sufficient to produce propionic acid-enriched VFA. Therefore, the two stage fermentation method by semi-continuous operation was feasible and had high efficiency.
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CONCLUSION It was observed that the optimal parameters in the first stage for the second stage fermentation of propionic acid-enriched VFA production were: pH 8, VSf/VSs ratio 6.0/1, fermentation for 2.5 d and at 20 C. The semi-continuous operation of the second stage fermentation was successfully conducted with 4 d retention time, which could achieve VFA of 15.20 g COD/L with propionic acid of 9.65 g COD/L. W
Performance of the semi-continuous fermentation The performance of the semi-continuous fermentation is explained in Figure 2. During the first 10 d (Day 0–Day 10), the VFA only reached 6.69 ± 0.16 g COD/L with propionic acid of 4.09 ± 0.10 g COD/L. It was reported that high concentration of propionic acid would inhibit the growth of P. acidipropionici (Suwannakham & Yang ). However, by replacing the second stage mixture with fresh liquid, the
ACKNOWLEDGEMENTS This work was supported by the National Hi-Tech Research and Development Program of China (863)
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(2011AA060903), the National Science Foundation of China (51178324), the Fundamental Research Funds for the Central Universities, the Foundation of State Key Laboratory of Pollution Control and Resource Reuse and Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation (STGEF).
REFERENCES Abu-ghararah, Z. H. & Randall, C. W. A proposed model for the anaerobic metabolism of short chain fatty acids in enhanced biological phosphorus removal systems. J. Water Pollut. Control Fed. 61, 1729–1730. Arun, V., Mino, T. & Matsuo, T. Biological mechanism of acetate uptake mediated by carbohydrate consumption in excess phosphorus removal system. Water Res. 22, 565–570. Chen, Y., Randall, A. A. & McCue, T. The efficiency of enhanced biological phosphorus removal from real wastewater affected by different ratios of acetic to propionic acid. Water Res. 38, 27–36. Chen, Y., Li, X., Zheng, X. & Wang, D. Enhancement of propionic acid fraction in volatile fatty acids produced from sludge fermentation by the use of food waste and Propionibacterium acidipropionici. Water Res. 47, 615–622. Cokgor, E. U., Oktay, S., Tas, D. O., Zengin, G. E. & Orhon, D. Influence of pH and temperature on soluble substrate generation with primary sludge fermentation. Bioresour. Technol. 100, 380–386. Daigger, G. T. & Bowen, P. T. Economic considerations on the use of fermenters in biological nutrient removal systems. Use of fermentation to enhance biological nutrient removal, Proceedings of the conference seminar, 67th Annual Water Environment Federation Conference and Exposition. Elefsiniotis, P. & Li, D. The effect of temperature and carbon source on denitrification using volatile fatty acids. Biochem. Eng. J. 28, 148–155. Feng, L., Chen, Y. & Zheng, X. Enhancement of waste activated sludge protein conversion and volatile fatty acids accumulation during waste activated sludge anaerobic fermentation by carbohydrate substrate addition: the effect of pH. Environ. Sci. Technol. 43, 4373–4380. Forbes, C., OReilly, C., McLaughlin, L., Gilleran, G., Tuohy, M. & Colleran, E. Application of high rate, high temperature anaerobic digestion to fungal thermozyme hydrolysates from carbohydrate wastes. Water Res. 43 (9), 2531–2539. Guisasola, A., Pijuan, M., Baeza, J. A., Carrera, J., Casas, C. & Lafuente, J. Aerobic phosphorus release linked to acetate uptake in bio-P sludge: Process modeling using oxygen uptake rate. Biotechnol. Bioeng. 85, 722–733.
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Higgins, M. J. & Novak, J. T. Dewatering and settling of activated sludges: The case for using cation analysis. Water Environ. Res. 69, 225–232. Inanc, B., Matsui, S. & Ide, S. Propionic acid accumulation and controlling factors in anaerobic treatment of carbohydrate: Effects of H2 and pH. Water Sci. Technol. 34, 317–325. Ji, Z. & Chen, Y. Using sludge fermentation liquid to improve wastewater short-cut nitrification-denitrification and denitrifying phosphorus removal via nitrite. Environ. Sci. Technol. 44, 8957–8963. Li, X., Chen, H., Hu, L., Yu, L., Chen, Y. & Gu, G. Pilot-scale waste activated sludge alkaline fermentation, fermentation liquid separation, and application of fermentation liquid to improve biological nutrient removal. Environ. Sci. Technol. 45, 1834–1839. Lim, S., Kim, B. J., Jeong, C. M., Choi, J., Ahn, Y. H. & Chang, H. N. Anaerobic organic acid production of food waste in once-a-day feeding and drawing-off bioreactor. Bioresour. Technol. 99, 7866–7874. Nakasaki, K., Akakura, N., Adachi, T. & Akiyama, T. Use of wastewater sludge as a raw material for production of L-lactic acid. Environ. Sci. Technol. 33, 198–200. Suwannakham, S. & Yang, S. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol. Bioeng. 91, 325–337. Tan, R., Miyanaga, K., Uy, D. & Tanji, Y. Effect of heatalkaline treatment as a pretreatment method on volatile fatty acid production and protein degradation in excess sludge, pure proteins and pure cultures. Bioresour. Technol. 118, 390–398. Yuan, H., Chen, Y., Zhang, H., Jiang, S., Zhou, Q. & Gu, G. Improved bioproduction of short-chain fatty acids from excess sludge under alkaline conditions. Environ. Sci. Technol. 40, 2025–2029. Yuan, Q., Sparling, R. & Oleszkiewicz, J. A. Waste activated sludge fermentation: effect of solids retention time and biomass concentration. Water Res. 43, 5180–5186. Yuan, Q., Sparling, R. & Oleszkiewicz, J. A. VFA generation from waste activated sludge: effect of temperature and mixing. Chemosph. 82, 603–607. Zhang, C., Chen, Y., Randall, A. A. & Gu, G. Anaerobic metabolic models for phosphorus- and glycogenaccumulating organisms with mixed acetic and propionic acids as carbon sources. Water Res. 42, 3745–3756. Zhang, P., Chen, Y. & Zhou, Q. Waste activated sludge hydrolysis and short-chain fatty acids accumulation under mesophilic and thermophilic conditions: effect of pH. Water Res 43, 3735–3742. Zhao, Y., Chen, Y., Zhang, D. & Zhu, X. Waste activated sludge fermentation for hydrogen production enhanced by anaerobic process improvement and acetobacteria inhibition: the role of fermentation pH. Environ. Sci. Technol. 44, 3317–3323.
First received 27 March 2013; accepted in revised form 8 July 2013. Available online 19 October 2013
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© IWA Publishing 2013 Water Science & Technology
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68.9
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Production of propionic acid-enriched volatile fatty acids from co-fermentation liquid of sewage sludge and food waste using Propionibacterium acidipropionici Xiang Li, Hui Mu, Yinguang Chen, Xiong Zheng, Jingyang Luo and Shu Zhao
ABSTRACT Volatile fatty acids (VFA), derived from sludge fermentation, have been used as one effective carbon source for biological nutrient removal, especially favorable with VFA containing with high levels of propionic acid. In this paper, a new fermentation method was employed to significantly produce the propionic acid-enriched VFA from the co-fermentation liquid of sewage sludge and food waste: including (1) mixing food waste with sludge in the anaerobic digester (the first stage) and (2) separating the mixture, sterilizing the first stage liquid and fermenting it after inoculation with Propionibacterium acidipropionici (the second stage). The effect of the key parameters including pH, the mixing ratio of the food waste and sludge, fermentation time and temperature of the first stage
Xiang Li Hui Mu Yinguang Chen (corresponding author) Xiong Zheng Jingyang Luo Shu Zhao State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China E-mail: [email protected]
on the propionic acid-enriched VFA production (the second stage) was individually discussed. By the molecular weight distribution analysis, the comparison of the solubilisation and hydrolysis process in difference parameters was fully elaborated. The optimal combination of the parameters was then obtained. Finally, the propionic acid-enriched VFA fermentation was successfully conducted in a semi-continuous reactor using the first stage liquid from the optimal condition. Key words
| food waste, Propionibacterium acidipropionici, propionic acid, two stage fermentation
INTRODUCTION It has been demonstrated that volatile fatty acids (VFA) can be used as the additional carbon source for biological nutrient removal (BNR), which has been widely used to remove nitrogen and phosphorus from wastewater (Arun et al. ; Abu-ghararah & Randall ; Guisasola et al. ). Normally, 1.07–1.82 and 1.87–3 mg VFA-C (carbon based) was required to remove 1 mg N and 1 mg P, respectively (Daigger & Bowen ; Elefsiniotis & Li ). Since the VFA in most of the municipal wastewater is insufficient, it is therefore necessary to provide VFA to the BNR system. Although chemically synthesized VFA can be used as the supplementary carbon source of municipal wastewater, it will consume the limited organic resources. Therefore, many efforts have been made to convert waste activated sludge (WAS), a by-product of wastewater treatment plants, to acetic acid-containing VFA (Yuan et al. ; Li et al. ; Tan et al. ). However, it was reported that the increase of propionic acid percentage in doi: 10.2166/wst.2013.463
VFA would facilitate the performance of BNR (Chen et al. ; Ji & Chen ). An important reason is that the increase of wastewater propionic/acetic acid ratio is beneficial to the competition of phosphorus-accumulating organisms over glycogen-accumulating organisms (Zhang et al. ). Thus, high propionic acid-containing VFA instead of acetic acid-dominated VFA is preferable for BNR. But until now, the strategy to improve the content of propionic acid in VFA has seldom been reported (Feng et al. ). Recently, a novel two-stage fermentation method was documented for the first time to significantly improve the propionic acid fraction in VFA derived from WAS by using food waste and Propionibacterium acidipropionici (Chen et al. ). First, the food waste, enriching the carbohydrate available, and sewage sludge were fermented for several days. Then, the liquid phase of the above mixture was sterilized, inoculated by P. acidipropionici, and fermented. Finally, propionic acid-enriched VFA was
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obtained and further utilized as a superior carbon source of BNR. In the last study, the first stage fermentation was demonstrated to be the crucial step for the second stage fermentation. Thus, the parameters involved in the first stage fermentation are worth considering. In the literature, pH value and fermentation time were the important factors to affect the anaerobic process (Inanc et al. ; Yuan et al. ; Zhao et al. ), while the mixing ratio of the food waste and sludge determined the organic loading rate to the anaerobic system (Forbes et al. ). Meanwhile, the temperature determined the anaerobic efficiency (Zhang et al. ). However, few studies revealed the effect of those factors from the first stage on the second stage fermentation. In the current study, the effect of key parameters of the first stage, including pH, the mixing ratio of the food waste and sludge (named VSf/VSs), fermentation time and temperature, on the propionic acid-enriched VFA fermentation (i.e., the second stage) were respectively elaborated. Additionally, the molecular weight distribution analysis further revealed the influence of these parameters on the solubilisation and hydrolysis process in the first stage. Furthermore, the second stage fermentation in a semicontinuous reactor was testified feasible using the first stage fermentation liquid obtained from the optimal condition.
Table 1
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METHODS Optimization of the first stage parameters on the second stage fermentation in batch experiments Since the pH value, mixing ratio of food waste and sludge (i.e., volatile solids ratio VSf/VSs), fermentation time and temperature played the important roles in the first stage fermentation, it is necessary to investigate the individual effect of these parameters on the second stage fermentation. The characteristic of the food waste and sludge are shown in Supporting Information (available online at http://www. iwaponline.com/wst/068/463.pdf). A series of identical reactors, with working volume of 1.0 L each, was used in the trials. The details of the arrangement for the trials are shown in Table 1. For each test, certain amounts of sludge and food waste were mixed in the anaerobic reactor, and then tap water was added to achieve the final volume of 1 L and total chemical oxygen demand (TCOD) of 25.0 ± 1.5 g/L. Each reactor was mechanically stirred at 120 rpm and maintained at a certain temperature. After a certain period of fermentation time, the mixture was centrifuged (4,000 rpm for 10 min) and the suspension liquid was obtained. Then 5 mL of each liquid sample was used for the molecular weight distribution analysis. Fifty millilitres of the suspension liquid was transferred to the serum
Experimental arrangement of the variables for the parameters investigated in the first stage fermentation Variable ranges Temperature ( C)
2.5 ± 0.1b
20 ± 1b
0.7/1 ± 0.1/1 2.5/1 ± 0.2/1 9.5/1 ± 0.2/1 17.5/1 ± 0.3/1
2.5 ± 0.1
20 ± 1
8.0 ± 0.3
6.0/1 ± 0.2/1
1.0 ± 0.1 2.0 ± 0.1 3.0 ± 0.1 4.0 ± 0.1
20 ± 1
8.0 ± 0.3
6.0/1 ± 0.2/1
2.5 ± 0.1
5±1 35 ± 1 50 ± 1
pH
Effect of pH on the second stage fermentation
Blanka 6.0 ± 0.3 7.0 ± 0.3 8.0 ± 0.3b 9.0 ± 0.3 10.0 ± 0.3
Effect of VSf/VSs ratio on the second stage fermentation
8.0 ± 0.3
Effect of fermentation time on the second stage fermentation
Effect of temperature on the second stage fermentation a
VSf/VSs ratio
6.0/1 ± 0.2/1b
Blank referred to the test without pH control.
b
W
Time (d)
Parameter investigated
This combination was employed for all the batch tests of the parameters, but only shown in the effect of pH test.
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bottle (volume 100 mL) and adjusted to pH 7. After autoclaving in 121 C and 15 psig for 20 min, each serum bottle was inoculated with 5 mL P. acidipropionici inoculum, flushed with nitrogen gas and sealed with a rubber cap. Then, each bottle was fermented in an air-bath shaker (120 rpm) at 30 ± 1 C and pH 7. After 4 d of second fermentation, the stable propionic acid concentration was observed and the VFA was assayed. The optimal combination of the parameters was obtained from the best performance of the second stage fermentation. W
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phase (flow rate of 0.5 mL/min). The samples were diluted and filtered through a 0.45 μm hydrophilic filtration membrane before being assayed by a refractive index detector (RID-10A). The Mw was calculated with Class VP 5.03 software (Shimadzu Co., Japan).
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Operation of the semi-continuous fermentation for high propionic acid-enriched VFA production The semi-continuous setup is shown in Figure S1 (Supporting Information, available online at http://www.iwaponline. com/wst/068/463.pdf), which included: (I) first stage reactor (working volume of 3.0 L), (II) centrifuge and autoclave equipment, and (III) second stage reactor (working volume of 2.0 L). The setup was operated for 40 d. In the first stage reactor, the mixture of food waste and WAS (VSf/VSs ratio: 6.0/1) was added with tap water to make the TCOD of 25.0 ± 1.5 g/L. The first stage reactor (mechanically stirred at 100 rpm) was maintained at pH 8.0 ± 0.3 using 5 M NaOH or 5 M HCl. The temperature was controlled at 20 ± 1 C by water bath equipment. After 2.5 d of first stage fermentation, 1 L of the suspension liquid was obtained via centrifuge, which was autoclaved in 121 C and 15 psig for 20 min. The suspension liquid was stored for the second stage fermentation. In the beginning of the startup for the second stage reactor, 2 L of the first stage liquid was inoculated with 200 mL P. acidipropionici inoculum. Then every 4 days, 1 L of the second stage mixture was discharged out from the second stage reactor, and the same volume of the first stage liquid obtained above was added to the second stage reactor followed by nitrogen flush. The second stage fermentation was then operated at 30 ± 1 C and pH 7.0 ± 0.3. After Day 28, the second stage reactor stopped refreshing the first stage liquid and fermented in batch for another 12 d. During the semi-continuous fermentation, VFA was measured every day. W
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Analytical methods Molecular weight (Mw) distributions of the first stage fermentation liquid were determined by a gel-filtration chromatography analyzer (Shimadzu Co., Japan). The gel column (TSK G4000SW type Tosoh Co., Japan) was utilized in the current study. Milli-Q water was used as the mobile
RESULTS AND DISCUSSION Effect of the parameters in the first stage on the second stage fermentation Figure 1(a) shows the effect of pH in the first stage on the propionic acid concentration and its percentage in total VFA of the second stage. Obviously, the highest propionic acid concentration, 7.12 g COD/L, was observed when the first stage fermentation was conducted at pH 8. With the decrease of pH from 8, propionic acid concentration dropped rapidly to less than 4.72 g COD/L. Increasing pH to 10 resulted in a propionic acid concentration of only 1.02 g COD/L, which shows a severe inhibition to propionic acid production. From Figure 1(a), the percentage of propionic acid in total VFA was 66.5, 66.6, 69.8 and 69.1% with no significant difference (P value >0.05) from pH 6 to 9. However, the percentage fell to only 32.5% at pH 10. Therefore, pH 8 resulted in the highest propionic acid production. In Figure 1(b), the VSf/VSs ratio of 0.7/1, 2.5/1 and 6.0/1 exhibited similar propionic acid concentration of 6.11, 6.39 and 7.12 g COD/L, respectively. However, with the increase of VSf/VSs ratio from 9.5/1 to 17.5/1, the concentration of propionic acid decreased from 5.29 to 2.81 g COD/L. Similarly, the VSf/VSs ratio from 2.5/1 to 9.5/1 resulted in the same percentage of propionic acid in total VFA (approx. 69.8%). However, further increasing VSf/VSs ratio to 17.5/1 led to significant decrease of propionic acid percentage (to 45.2%). Thus, VSf/VSs ratio of 6.0/1 was the optimal ratio for the second stage fermentation. Figure 1(c) denotes that the optimal fermentation time in the first stage was 2.5 d, which led to the highest propionic acid production. Meanwhile, propionic acid concentration and percentage derived from 2 and 3 d were 6.52 and 6.75 g COD/L, and 67.9 and 68.5% respectively with no significant difference (P value >0.05). However, fermentation for 4 d resulted in a decrease of both propionic acid concentration and percentage, while fermentation for 1 d resulted in propionic acid concentration of 4.43 g COD/L. Figure 1(d) explains the effect of temperature on the propionic acid concentration and percentage.
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Effect of pH, VSf/VSs ratio, fermentation time and temperature in the first stage on the propionic acid-enriched VFA fermentation.
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Fermentation at 50 C, notwithstanding the high propionic acid concentration of 6.49 g COD/L, led to a reduction in the percentage of propionic acid in the VFA to 61.5%. Although, low temperature at 5 C exhibited high percentage of 68.8%, the concentration of propionic acid was the lowest (only 2.58 g COD/L). Despite anaerobic fermentation being favourable at 35 C (Nakasaki et al. ), the propionic acid concentration fell to only 5.45 g COD/L, with the percentage of 56.4%. The mechanisms of the parameters affecting the bio-conversion process during the first stage fermentation will be discussed in the following text. Thus, the optimal temperature should be set at 20 C. W
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Mechanisms study of the parameters affecting the process of solubilisation and hydrolysis in the first stage As shown in Figure S2 (Supporting Information, available online at http://www.iwaponline.com/wst/068/463.pdf), the mixture of sludge and food waste was first dissolved to colloid and polymer organics, which further hydrolyzed to protein and polysaccharide in the first stage fermentation. Afterwards, the second stage fermentation was driven by the first stage liquid containing a variety of metabolic products. Different parameters in the first stage determined the organic content through the solubilisation and hydrolysis process, which could be expressed
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via Mw distribution analysis in Figure S3 (Supporting Information). In Figure S3(a), extreme alkaline fermentation at pH 10 probably caused some toxic macro-matter release from the sludge, which inhibited the growth of P. acidipropionici in the second stage. Similar findings were reported whereby the addition of monovalent ion (such as Naþ) to sludge deteriorated the dewatering capability of sludge, which might result from the release of some unfavourable chemicals (Higgins & Novak ). Low pH value (pH < 8) in the first stage mainly weakened the strength of the solubilisation and hydrolysis process, which reduced the potential substrate for the propionic acid production in the second stage. This was in good agreement with the findings in the literature (Yuan et al. ; Cokgor et al. ). In Figure S3(b), the VSf/VSs ratio from 6.0/1 to 9.5/1 exhibited higher concentration (the area of the peaks) of low Mw hydrolysate than that of other ratios. A previous study demonstrated that the activity of sludge hydrolysis enzyme was significantly improved by the addition of carbohydrate to the sludge hydrolysis system (Feng et al. ). Therefore, the proper VSf/VSs ratio resulted in the highest solubilisation efficiency (Lim et al. ). The effect of first stage fermentation time on the Mw distribution is shown in Figure S3(c). Fermentation time between 2 and 3 d exhibited similar Mw distribution, which was consistent with Figure 1(c). The Mw distribution at different temperature is denoted in Figure S3(d). It is well known that the speed of solubilisation and hydrolysis will be improved with the increase of temperature (Cokgor et al. ; Yuan et al. ). Therefore, the organics were hydrolyzed to smaller molecular matter in the range of 100,000 to 20, 000 g/mol Mw using 20, 35 and 50 C. However, it was over-hydrolyzed at 35 C, which led to depletion of the potential substance for the second stage. Nevertheless, isobutyric acid was accumulated at 50 C in the first stage liquid (data not shown), which weakened the percentage of propionic acid in the second stage. Therefore, the optimal parameters in the first stage fermentation are pH 8, VSf/VSs ratio 6.0/1, fermentation for 2.5 d at 20 C. W
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Performance of the semi-continuous fermentation under the optimal parameters.
inhibitory effect of the high propionic acid was reduced extensively and P. acidipropionici started to produce propionic acid. Therefore, after the adaptation period (Day 0 to Day 10), the propionic acid concentration improved to 8.19 ± 0.17 g COD/L and total VFA increased to 13.08 ± 0.33 g COD/L on Day 20. The maximal concentration of the VFA was 15.20 ± 0.38 g COD/L on Day 28, and the corresponding propionic acid was 9.65 ± 0.23 g COD/L, which was much higher than the previous studies (Feng et al. ; Chen et al. ). During the last 12 d of batch fermentation (Day 28–40), the concentration of propionic acid became steady at 8.57 ± 0.21 g COD/L after 4 d (i.e., Day 32). Therefore, the retention time of 4 d for the second stage was sufficient to produce propionic acid-enriched VFA. Therefore, the two stage fermentation method by semi-continuous operation was feasible and had high efficiency.
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CONCLUSION It was observed that the optimal parameters in the first stage for the second stage fermentation of propionic acid-enriched VFA production were: pH 8, VSf/VSs ratio 6.0/1, fermentation for 2.5 d and at 20 C. The semi-continuous operation of the second stage fermentation was successfully conducted with 4 d retention time, which could achieve VFA of 15.20 g COD/L with propionic acid of 9.65 g COD/L. W
Performance of the semi-continuous fermentation The performance of the semi-continuous fermentation is explained in Figure 2. During the first 10 d (Day 0–Day 10), the VFA only reached 6.69 ± 0.16 g COD/L with propionic acid of 4.09 ± 0.10 g COD/L. It was reported that high concentration of propionic acid would inhibit the growth of P. acidipropionici (Suwannakham & Yang ). However, by replacing the second stage mixture with fresh liquid, the
ACKNOWLEDGEMENTS This work was supported by the National Hi-Tech Research and Development Program of China (863)
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(2011AA060903), the National Science Foundation of China (51178324), the Fundamental Research Funds for the Central Universities, the Foundation of State Key Laboratory of Pollution Control and Resource Reuse and Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation (STGEF).
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First received 27 March 2013; accepted in revised form 8 July 2013. Available online 19 October 2013
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