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The influence of the filtration membrane air-cathode biofilm on wastewater treatment
Accepted Manuscript The influence of the filtration membrane air-cathode biofilm on wastewater treatment Peng Zhang, Youpeng Qu, Yujie Feng, Jia Liu PII: DOI: Reference:
S0960-8524(18)30146-9 https://doi.org/10.1016/j.biortech.2018.01.124 BITE 19481
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
15 November 2017 19 January 2018 20 January 2018
Please cite this article as: Zhang, P., Qu, Y., Feng, Y., Liu, J., The influence of the filtration membrane air-cathode biofilm on wastewater treatment, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech. 2018.01.124
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The influence of the filtration membrane air-cathode biofilm on wastewater treatment Peng Zhanga, Youpeng Qub, Yujie Fenga, Jia Liua* a
State Key Laboratory of Urban Water Resource and Environment, School of Environment,
Harbin Institute of Technology. No 73 Huanghe Road, Nangang District, Harbin 150090, China b
School of Life Science and Technology, Harbin Institute of Technology. No. 2 Yikuang Street,
Nangang District, Harbin 150080, China
Abstract The aim of this work was to evaluate the influence of FMA biofilm on nutrient removal through the filtration membrane air-cathode (FMA) replacement test. The result showed that the biofilm accounted for only 29.9% of the COD removal, while 82.9% of the TN removal can be due to the contribution of the FMA biofilm. The microbial community analysis showed that most of the primary genus in the FMA biofilm were TN removal related bacteria. This quantitative determination of the FMA biofilm influence on COD and TN removal would promote the further optimization of the MFC reactor with FMA for higher wastewater treatment efficiency. Keywords: Microbial fuel cell; COD; TN; Biofilm; Filtration air-cathode
1. Introduction During last decades, the theme of sustainability has become more and more important due to the global issues, such as fossil-fuel depletion, environmental pollution, water resource crisis and shortage . Microbial fuel cell (MFC) is a microbial electrochemical technology that utilizes *Corresponding Author: E-mail: [email protected];
1
bacteria to simultaneously degrade organic wastewater and produce electricity (Logan & Regan, 2006; Mashkour et al., 2016; Rahimnejad et al., 2015; Rahimnejad et al., 2012). However, this technology is not efficient in achieving high-quality effluent and needs a further treatment process (Ge et al., 2013). Membrane bioreactor (MBR), which combines biological reactions for organic removal with membranes for solid and liquid separation to produce higher quality effluent, has been integrated with MFC to improve the effluent quality (Malaeb et al., 2013). As one of the MFC-MBR integration patterns, a dual-function module, defined as filtration membrane air-cathode (FMA), can serve both as the membrane module of MBR and the air-cathode module of MFC (Katuri et al., 2014; Malaeb et al., 2013). In our recent study (Zhang et al., 2016), for the simple fabrication process and low cost, activated carbon FMA showed promising application potential and achieved the goal of simultaneous energy generation and high wastewater treatment efficiency. During the operation of the reactor with FMA, the biofilm can gradually form on the inner side of FMA and hold back the bacteria and sludge flocs by the formation of a three-dimensional biofilm structure (Ghasemi et al., 2013). However, the influence of the FMA biofilm on the wastewater treatment has not been explored before. In this study, the influence of FMA biofilm on COD and TN removal was investigated quantificationally in an MFC reactor.
2. Experiments 2.1 Fabrication of activated carbon filtration membrane air-cathode FMA consisted of stainless steel mesh and the activated carbon layer, was fabricated by the rolling-press method described in the previous study (Zhang et al., 2016). The detailed information was provided in supporting information (SI). The scanning electron microscope (SEM) image of activated carbon layer showed that micrometer scale pores scattered on the 2
surface, indicating that FMA can also be used for the filtration process as a membrane. 2.2 Bioreactor construction and operation The bioreactor is a cylindrical reactor, which consists of two chambers, the upper chamber, and the nether chamber. The FMA is placed between the two chambers and the effective area of the FMA is 7 cm2. The anode is a graphite brush (3 cm in length and 3 cm in diameter) and is fixed on the top of the upper chamber. The external resistance of 1000 Ω was connected between anode and cathode via Ti wire. The detailed information of the reactor operation was provided in the SI. 2.3 Measurements and analysis Linear sweep voltammetry (LSV) was performed to investigate the catalytic performance of the FMA (detailed information in SI). The chemical oxygen demand (COD) and total nitrogen (TN) were measured in duplication using test kit by a spectrophotometer (HACH Company). To explore the anodic and cathodic microbial communities, the biofilm attached to anode and cathode were sampled at the end of the experiment. Total DNA extraction, PCR-DGGE and 16S rRNA analysis were done as previously described (Xiao et al., 2017). If it is assumed that no biofilm exists on the FMA in cycle 9 (Figure 1b), the COD consumption by the reactor consists of two parts: the COD adsorption by the new FMA (CODadsorption) and the COD degraded by the anode biofilm (CODanode). Thus, the COD consumption by the anode biofilm can be calculated by the following equation (equation 1):
For the COD removal in cycle 8 consisted of the COD degradation by the cathode biofilm (CODcathode) and the CODanode, the rate of CODcathode on the total COD degradation (RateCOD) could be calculated by the following equation (equation 2): 3
Based on the similar calculation method of the COD, the TN degraded by the cathode biofilm (TNcathode) can also be calculated by the date of cycle 8 and 9 (Figure 2b). In cycle 9, the TN consumed by anode biofilm (TNanode) could be calculated by the following equation (equation 3):
And in cycle 8, the rate of the TN degraded by the cathode biofilm (TNcathode) on the total TN removal (RateTN) can be calculated by the following equation (equation 4):
3. Results and discussion 3.1 Effect of FMA biofilm on COD removal The domestic wastewater (COD, 166 mg/L) was used as the raw inoculation influent in the reactor. As showed in Figure. 1a, the variation of the effluent COD and the COD removal rate could be divided into two stages. During the stageⅠ, the effluent COD was greatly decreased from 63.7 ± 5.1 mg/L to 13.0 ± 2.1 mg/L, with the COD removal rate increasing from 60.9 ± 3.2% to 88.9 ± 1.9%. Even the phenomenon of the COD removal rate fluctuation was observed in the stage Ⅱ, which may be caused by the influent quality disturbance, stable effluent quality (11.0 ± 3.5 mg/L) and COD removal (93.2 ± 2.2%) can be finally achieved, indicating the efficient and stable biofilm for COD removal formed in the reactor. After the successful inoculation with domestic wastewater, the synthetic wastewater was used as the influent to test the cathode biofilm effect on COD removal. The synthetic wastewater contained sodium acetate (1.0 g/L; COD, 780.0 mg/L) in 50 mM phosphate buffer solution. As showed in Figure 1b, with the synthetic wastewater, the COD removal rate was just 89.6% in the 4
first cycle, slightly lower than that with domestic wastewater, which may be caused by the COD increase from the domestic wastewater to the synthetic wastewater. From cycle 1 to 8, the effluent COD concentration and COD removal reached about 35.0 mg/L and 95.4%, which was comparable with other membrane air-cathode bioreactors (Malaeb et al., 2013; Zuo et al., 2015). Meanwhile, a layer of biofilm cake formed on the solution-facing side of FMA. To investigate the influence of FMA biofilm on COD removal, a new FMA with similar electrochemical behavior, was used in the bioreactor in cycle 8. In cycle 9, the effluent COD increased to 90.0 mg/L, with the COD removal rate decreasing to 88.5%, indicating that the biofilm on the FMA can influence the COD removal. The COD removal rate recovered to 95.3% again in cycle 10. This phenomenon was supported by the previous result that most of the COD was removed by the exoelectrogens on the anode of single chamber MFC and only a few of the influent COD had been oxidized by heterotrophs in the system (Zhang et al., 2015). The FMA adsorption experiment was conducted in the bioreactor without anode to determine the adsorption COD by this FMA. 169.3 mg/L COD was removed in the first filtration cycle and the adsorption also becomes saturated in the third filtration process, with the COD concentration in the effluent reaching the influent COD concentration. According to the calculation of equation 1 and 2, the biofilm on cathode only accounted for 29.9% (RateCOD) of COD removed by the reactor. Thus, most of the COD degradation would be due to the bacteria on the anode and only a small portion of COD removal could be attributed to the function of the cathode biofilm. 3.2 Effect of FMA biofilm on TN removal The TN removal showed a similar variation trend as the COD, finally achieving the stable TN removal efficiency of about 70.0% and the effluent TN concentration of about 20.0 mg/L 5
(influent TN, 67.0 mg/L). Two apparent stages can be identified during the 10 cycles’ operation (Figure 2a). In stage Ⅰ, from cycle 1 to cycle 8, the TN removal rate gradually increased from 22.3 ± 4.2% to about 70.1 ± 1.2%. During stage Ⅱ, the stable TN removal rate was achieved, indicating the mature and stabilization of the TN removal system in the reactor. The TN removal needed apparent longer time to achieve stable removal rate than COD, indicating that the nitrifying and denitrifying bacteria may need more time for acclimation in the reactor. Similarly, to test the effect of FMA biofilm on TN removal, the synthetic wastewater with stable TN concentration was used. As showed in Figure. 2b, the TN removal rate of the bioreactor gradually reached to 69.6%, with the TN concentration in the effluent just 25.0 mg/L (Influent NH4+-N concentration, 81.2 mg/L). After the new FMA was installed in cycle 9, the sharp decrease of TN removal rate to 21.2% indicated that the biofilm on cathode greatly influenced the TN removal. The TN removal became stable again at cycle 12 with the removal efficiency around 68.5%. The recovery time of TN removal ability was about 4 operation cycles, much longer than that of the COD (1 cycle), which may need the FMA biofilm formation. A similar FMA adsorption experiment for the TN adsorption showed that 7.6 mg/L NH4+-N was adsorbed by the FMA in the first filtration process. The adsorption by the FMA becomes saturated in the second cycle. According to the calculation of equation 3 and 4, the rate of cathode biofilm on the TN removal was calculated from the data of cycle 8 and 9 (Figure 2b): FMA biofilm accounted for 82.9% of the TN degradation in the reactor. This result showed that most of the TN removal by the reactor should be due to the function of FMA biofilm, including nitrifying and denitrifying bacteria (Sotres et al., 2016). In this system, the ammonia is first oxidized to nitrite and nitrate by the nitrification process; then, the oxidized forms of nitrogen can be removed by the denitrification process with electron donor oxidation or cathode 6
oxidation (Yan et al., 2012). 3.3 Microbial community of FMA biofilm The microbial communities on both anode and cathode were analyzed to investigate the primary bacteria in the biofilm. As showed in Figure 3a, there was significant distribution difference in the community structures on anode and FMA. The genus Pseudomonas, as the main genus in the anode community (48.0%), was reported to be able to produce compounds like phenazine pyocyanin that function as electron shuttles between the bacterium and electrode (Newman, 2001). Pseudomonas was also found in the cathode biofilm with a relative abundance of 3.7%. Bacillus, with a relative abundance of 20.8%, was reported to be an electroactive microorganism, whose electron transfer mechanism was also mainly due to the secreted redox compounds (Ghangrekar & Shinde, 2007). The genus Ignavibacteriales is a member of the phylum Chlorobi and represented by Ignavibacterium album, a non-phototrophic fermentative bacterium (Iino et al., 2010), which was also reported before (Yoshizawa et al., 2014; Zhou et al., 2015). Acinetobacter, which had been reported to be an important microorganism accepting electrons from biocathode (Erable et al., 2010), reached a relative abundance of 6.9% on the anode, indicating that this kind of bacterium may also transfer electrons to the anode. These results showed that the bacteria on anode were mainly about electricity generating bacterium utilizing the nutrient in the solution. The first abundant genus on the cathode, Flavobacterium (21.3%), played a key role as denitrifying microorganisms (Chen et al., 2008), but was barely found on the anode (0.02%). Azospira (5.1%) and Nitratireductor (2.3%) on the cathode, members of phylum Proteobacteria, had also been determined to be denitrifying bacteria (Heylen et al., 2006). While the relative abundance of Azospira and Nitratireductor on anode was only 0.16% and 0.07%. The second 7
abundant strain, Chitinophagaceae (13.2%), was proficient in decomposing cellulose and chitin in the domestic sewage (Zhiwei Wang, 2013). The ammonia-oxidizing bacterium Nitrosomonas (0.4%) was mainly found on the cathode electrode, proving the biological nitrification on the FMA. The bacteria distribution of FMA biofilm in Figure 3b showed that TN removal related primary bacteria genus accounted for almost 30.0% of the microbial community; while COD removal related primary bacteria genus accounted for only 16.6%. This result indicated that the major function of FMA biofilm was the TN removal, corresponding to the result that 82.9% of the TN removal by the reactor was due to the biofilm on the FMA.
Conclusion The bioreactor with FMA achieved COD of 94.0% and TN removal efficiency of 70.0%. The new FMA test proved that only 29.9% of the COD removal was due to cathode biofilm, while 82.9% of the TN removal accounted for the cathode biofilm. The microbial community analysis showed that both nitrifying and denitrifying bacteria were found on the FMA, while these nitrogens degraded bacterium were barely found on the anode. This quantificationally analysis of FMA biofilm on COD and TN removal can promote the further optimization of the bioreactor with FMA and deepen the understanding of the corresponding nutrient removal mechanism.
Associated content The supporting information contains the detailed information of the FMA fabrication process, the FMA reactor operation process, and FMA electrochemical test, the Photograph, schematic picture, and SEM image of the FMA (Figure S1), schematic picture of the bioreactor with activated carbon FMA (Figure S2), SEM image of the biofilm on the solution-facing side of FMA (Figure S3), the LSV results of the new and used FMA (Figure S4), the effluent COD 8
concentration and NH4+-N concentration in the new FMA adsorption test (Figure S5), the electricity generation performance of bioreactor with FMA (Figure S6).
Acknowledgements This work was supported by the National Natural Science Fund for Distinguished Young Scholars (Grant No. 51125033), National Natural Science Fund of China (Grant No. 51209061 and 51408156), the Fundamental Research Funds for the Central Universities"(Grant No.HIT.MKSTISP.2016 14)， the Fundamental Research Funds for the Central Universities (HIT. NSRIF. 2015090) and the International Cooperating Project between China and European Union (Grant No. 2014DFE90110).
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Logan, B.E., Regan, J.M. 2006. Electricity-producing bacterial communities in microbial fuel cells. Trends in Microbiology, 14(12), 512-518.
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12. Newman, M.E.H.a.D.K. 2001. Extracellular electron transfer. Cellular and Molecular Life Sciences, 58, 1562-1571. 13. Rahimnejad, M., Adhami, A., Darvari, S., Zirepour, A., Oh, S.-E. 2015. Microbial fuel cell as new technology for bioelectricity generation: A review. Alexandria Engineering Journal, 54(3), 745-756. 14. Rahimnejad, M., Ghasemi, M., Najafpour, G.D., Ismail, M., Mohammad, A.W., Ghoreyshi, A.A., Hassan, S.H.A. 2012. Synthesis, characterization and application studies of self-made Fe3O4/PES nanocomposite membranes in microbial fuel cell. Electrochimica Acta, 85(Supplement C), 700-706. 15. Sotres, A., Cerrillo, M., Vinas, M., Bonmati, A. 2016. Nitrogen removal in a two-chambered microbial fuel cell: Establishment of a nitrifying-denitrifying microbial community on an intermittent aerated cathode. Chemical Engineering Journal, 284, 905-916. 16. Xiao, L., Xie, B., Liu, J., Zhang, H., Han, G., Wang, O., Liu, F. 2017. Stimulation of long-term ammonium nitrogen deposition on methanogenesis by Methanocellaceae in a coastal wetland. Science of The Total Environment, 595, 337-343. 17. Yan, H.J., Saito, T., Regan, J.M. 2012. Nitrogen removal in a single-chamber microbial fuel cell with nitrifying biofilm enriched at the air cathode. Water Research, 46(7), 2215-2224. 18. Yoshizawa, T., Miyahara, M., Kouzuma, A., Watanabe, K. 2014. Conversion of activated-sludge reactors to microbial fuel cells for wastewater treatment coupled to electricity generation. J Biosci Bioeng, 118(5), 533-9. 19. Zhang, P., Qu, Y., Liu, J., Feng, Y. 2016. A new design of activated carbon membrane air-cathode for wastewater treatment and energy recovery. RSC Adv., 6(6), 4587-4592. 20. Zhang, X., He, W., Ren, L., Stager, J., Evans, P.J., Logan, B.E. 2015. COD removal 11
characteristics in air-cathode microbial fuel cells. Bioresource Technology, 176(Supplement C), 23-31. 21. Zhiwei Wang, J.M., Yinlun Xu, Hongguang Yu, Zhichao Wu. 2013. Power production from different types of sewage sludge using microbial fuel cells_ A comparative study with energetic and microbiological perspectives. Journal of Power Sources, 235, 280-288. 22. Zhou, X., Qu, Y., Kim, B.H., Li, H., Liu, J., Du, Y., Li, D., Dong, Y., Ren, N., Feng, Y. 2015. Effects of azide on current generation and microbial community in air-cathode MFCs. RSC Adv., 5(19), 14235-14241. 23. Zuo, K., Liang, S., Liang, P., Zhou, X., Sun, D., Zhang, X., Huang, X. 2015. Carbon filtration cathode in microbial fuel cell to enhance wastewater treatment. Bioresour Technol, 185, 426-30. Figure Captions Figure 1. The variation of domestic wastewater effluent COD concentration and COD removal rate by the reactor along the treatment cycle (a); the variation of synthetic wastewater effluent COD concentration and COD removal rate by the reactor before and after the replacement of a new FMA along the treatment cycle (b). Figure 2. The variation of domestic wastewater effluent TN concentration and TN removal rate by the reactor along the treatment cycle (a); the variation of synthetic wastewater effluent TN concentration and TN removal rate by the reactor before and after the replacement of a new FMA along the treatment cycle (b). Figure 3. Distributions of bacterial populations enriched on the anode and cathode at the genus level (a); the cathode bacteria distribution at genus level associated with COD and TN removal respectively (b). 12
Figure 1
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Figure 2
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Figure 3
15
Highlights
The FMA biofilm accounted for only 29.9% of the COD removal
82.9% of the TN removal can be due to the contribution of the FMA biofilm
Most of the primary genus in the FMA biofilm were TN removal related bacteria
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17
S0960-8524(18)30146-9 https://doi.org/10.1016/j.biortech.2018.01.124 BITE 19481
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
15 November 2017 19 January 2018 20 January 2018
Please cite this article as: Zhang, P., Qu, Y., Feng, Y., Liu, J., The influence of the filtration membrane air-cathode biofilm on wastewater treatment, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech. 2018.01.124
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The influence of the filtration membrane air-cathode biofilm on wastewater treatment Peng Zhanga, Youpeng Qub, Yujie Fenga, Jia Liua* a
State Key Laboratory of Urban Water Resource and Environment, School of Environment,
Harbin Institute of Technology. No 73 Huanghe Road, Nangang District, Harbin 150090, China b
School of Life Science and Technology, Harbin Institute of Technology. No. 2 Yikuang Street,
Nangang District, Harbin 150080, China
Abstract The aim of this work was to evaluate the influence of FMA biofilm on nutrient removal through the filtration membrane air-cathode (FMA) replacement test. The result showed that the biofilm accounted for only 29.9% of the COD removal, while 82.9% of the TN removal can be due to the contribution of the FMA biofilm. The microbial community analysis showed that most of the primary genus in the FMA biofilm were TN removal related bacteria. This quantitative determination of the FMA biofilm influence on COD and TN removal would promote the further optimization of the MFC reactor with FMA for higher wastewater treatment efficiency. Keywords: Microbial fuel cell; COD; TN; Biofilm; Filtration air-cathode
1. Introduction During last decades, the theme of sustainability has become more and more important due to the global issues, such as fossil-fuel depletion, environmental pollution, water resource crisis and shortage . Microbial fuel cell (MFC) is a microbial electrochemical technology that utilizes *Corresponding Author: E-mail: [email protected];
1
bacteria to simultaneously degrade organic wastewater and produce electricity (Logan & Regan, 2006; Mashkour et al., 2016; Rahimnejad et al., 2015; Rahimnejad et al., 2012). However, this technology is not efficient in achieving high-quality effluent and needs a further treatment process (Ge et al., 2013). Membrane bioreactor (MBR), which combines biological reactions for organic removal with membranes for solid and liquid separation to produce higher quality effluent, has been integrated with MFC to improve the effluent quality (Malaeb et al., 2013). As one of the MFC-MBR integration patterns, a dual-function module, defined as filtration membrane air-cathode (FMA), can serve both as the membrane module of MBR and the air-cathode module of MFC (Katuri et al., 2014; Malaeb et al., 2013). In our recent study (Zhang et al., 2016), for the simple fabrication process and low cost, activated carbon FMA showed promising application potential and achieved the goal of simultaneous energy generation and high wastewater treatment efficiency. During the operation of the reactor with FMA, the biofilm can gradually form on the inner side of FMA and hold back the bacteria and sludge flocs by the formation of a three-dimensional biofilm structure (Ghasemi et al., 2013). However, the influence of the FMA biofilm on the wastewater treatment has not been explored before. In this study, the influence of FMA biofilm on COD and TN removal was investigated quantificationally in an MFC reactor.
2. Experiments 2.1 Fabrication of activated carbon filtration membrane air-cathode FMA consisted of stainless steel mesh and the activated carbon layer, was fabricated by the rolling-press method described in the previous study (Zhang et al., 2016). The detailed information was provided in supporting information (SI). The scanning electron microscope (SEM) image of activated carbon layer showed that micrometer scale pores scattered on the 2
surface, indicating that FMA can also be used for the filtration process as a membrane. 2.2 Bioreactor construction and operation The bioreactor is a cylindrical reactor, which consists of two chambers, the upper chamber, and the nether chamber. The FMA is placed between the two chambers and the effective area of the FMA is 7 cm2. The anode is a graphite brush (3 cm in length and 3 cm in diameter) and is fixed on the top of the upper chamber. The external resistance of 1000 Ω was connected between anode and cathode via Ti wire. The detailed information of the reactor operation was provided in the SI. 2.3 Measurements and analysis Linear sweep voltammetry (LSV) was performed to investigate the catalytic performance of the FMA (detailed information in SI). The chemical oxygen demand (COD) and total nitrogen (TN) were measured in duplication using test kit by a spectrophotometer (HACH Company). To explore the anodic and cathodic microbial communities, the biofilm attached to anode and cathode were sampled at the end of the experiment. Total DNA extraction, PCR-DGGE and 16S rRNA analysis were done as previously described (Xiao et al., 2017). If it is assumed that no biofilm exists on the FMA in cycle 9 (Figure 1b), the COD consumption by the reactor consists of two parts: the COD adsorption by the new FMA (CODadsorption) and the COD degraded by the anode biofilm (CODanode). Thus, the COD consumption by the anode biofilm can be calculated by the following equation (equation 1):
For the COD removal in cycle 8 consisted of the COD degradation by the cathode biofilm (CODcathode) and the CODanode, the rate of CODcathode on the total COD degradation (RateCOD) could be calculated by the following equation (equation 2): 3
Based on the similar calculation method of the COD, the TN degraded by the cathode biofilm (TNcathode) can also be calculated by the date of cycle 8 and 9 (Figure 2b). In cycle 9, the TN consumed by anode biofilm (TNanode) could be calculated by the following equation (equation 3):
And in cycle 8, the rate of the TN degraded by the cathode biofilm (TNcathode) on the total TN removal (RateTN) can be calculated by the following equation (equation 4):
3. Results and discussion 3.1 Effect of FMA biofilm on COD removal The domestic wastewater (COD, 166 mg/L) was used as the raw inoculation influent in the reactor. As showed in Figure. 1a, the variation of the effluent COD and the COD removal rate could be divided into two stages. During the stageⅠ, the effluent COD was greatly decreased from 63.7 ± 5.1 mg/L to 13.0 ± 2.1 mg/L, with the COD removal rate increasing from 60.9 ± 3.2% to 88.9 ± 1.9%. Even the phenomenon of the COD removal rate fluctuation was observed in the stage Ⅱ, which may be caused by the influent quality disturbance, stable effluent quality (11.0 ± 3.5 mg/L) and COD removal (93.2 ± 2.2%) can be finally achieved, indicating the efficient and stable biofilm for COD removal formed in the reactor. After the successful inoculation with domestic wastewater, the synthetic wastewater was used as the influent to test the cathode biofilm effect on COD removal. The synthetic wastewater contained sodium acetate (1.0 g/L; COD, 780.0 mg/L) in 50 mM phosphate buffer solution. As showed in Figure 1b, with the synthetic wastewater, the COD removal rate was just 89.6% in the 4
first cycle, slightly lower than that with domestic wastewater, which may be caused by the COD increase from the domestic wastewater to the synthetic wastewater. From cycle 1 to 8, the effluent COD concentration and COD removal reached about 35.0 mg/L and 95.4%, which was comparable with other membrane air-cathode bioreactors (Malaeb et al., 2013; Zuo et al., 2015). Meanwhile, a layer of biofilm cake formed on the solution-facing side of FMA. To investigate the influence of FMA biofilm on COD removal, a new FMA with similar electrochemical behavior, was used in the bioreactor in cycle 8. In cycle 9, the effluent COD increased to 90.0 mg/L, with the COD removal rate decreasing to 88.5%, indicating that the biofilm on the FMA can influence the COD removal. The COD removal rate recovered to 95.3% again in cycle 10. This phenomenon was supported by the previous result that most of the COD was removed by the exoelectrogens on the anode of single chamber MFC and only a few of the influent COD had been oxidized by heterotrophs in the system (Zhang et al., 2015). The FMA adsorption experiment was conducted in the bioreactor without anode to determine the adsorption COD by this FMA. 169.3 mg/L COD was removed in the first filtration cycle and the adsorption also becomes saturated in the third filtration process, with the COD concentration in the effluent reaching the influent COD concentration. According to the calculation of equation 1 and 2, the biofilm on cathode only accounted for 29.9% (RateCOD) of COD removed by the reactor. Thus, most of the COD degradation would be due to the bacteria on the anode and only a small portion of COD removal could be attributed to the function of the cathode biofilm. 3.2 Effect of FMA biofilm on TN removal The TN removal showed a similar variation trend as the COD, finally achieving the stable TN removal efficiency of about 70.0% and the effluent TN concentration of about 20.0 mg/L 5
(influent TN, 67.0 mg/L). Two apparent stages can be identified during the 10 cycles’ operation (Figure 2a). In stage Ⅰ, from cycle 1 to cycle 8, the TN removal rate gradually increased from 22.3 ± 4.2% to about 70.1 ± 1.2%. During stage Ⅱ, the stable TN removal rate was achieved, indicating the mature and stabilization of the TN removal system in the reactor. The TN removal needed apparent longer time to achieve stable removal rate than COD, indicating that the nitrifying and denitrifying bacteria may need more time for acclimation in the reactor. Similarly, to test the effect of FMA biofilm on TN removal, the synthetic wastewater with stable TN concentration was used. As showed in Figure. 2b, the TN removal rate of the bioreactor gradually reached to 69.6%, with the TN concentration in the effluent just 25.0 mg/L (Influent NH4+-N concentration, 81.2 mg/L). After the new FMA was installed in cycle 9, the sharp decrease of TN removal rate to 21.2% indicated that the biofilm on cathode greatly influenced the TN removal. The TN removal became stable again at cycle 12 with the removal efficiency around 68.5%. The recovery time of TN removal ability was about 4 operation cycles, much longer than that of the COD (1 cycle), which may need the FMA biofilm formation. A similar FMA adsorption experiment for the TN adsorption showed that 7.6 mg/L NH4+-N was adsorbed by the FMA in the first filtration process. The adsorption by the FMA becomes saturated in the second cycle. According to the calculation of equation 3 and 4, the rate of cathode biofilm on the TN removal was calculated from the data of cycle 8 and 9 (Figure 2b): FMA biofilm accounted for 82.9% of the TN degradation in the reactor. This result showed that most of the TN removal by the reactor should be due to the function of FMA biofilm, including nitrifying and denitrifying bacteria (Sotres et al., 2016). In this system, the ammonia is first oxidized to nitrite and nitrate by the nitrification process; then, the oxidized forms of nitrogen can be removed by the denitrification process with electron donor oxidation or cathode 6
oxidation (Yan et al., 2012). 3.3 Microbial community of FMA biofilm The microbial communities on both anode and cathode were analyzed to investigate the primary bacteria in the biofilm. As showed in Figure 3a, there was significant distribution difference in the community structures on anode and FMA. The genus Pseudomonas, as the main genus in the anode community (48.0%), was reported to be able to produce compounds like phenazine pyocyanin that function as electron shuttles between the bacterium and electrode (Newman, 2001). Pseudomonas was also found in the cathode biofilm with a relative abundance of 3.7%. Bacillus, with a relative abundance of 20.8%, was reported to be an electroactive microorganism, whose electron transfer mechanism was also mainly due to the secreted redox compounds (Ghangrekar & Shinde, 2007). The genus Ignavibacteriales is a member of the phylum Chlorobi and represented by Ignavibacterium album, a non-phototrophic fermentative bacterium (Iino et al., 2010), which was also reported before (Yoshizawa et al., 2014; Zhou et al., 2015). Acinetobacter, which had been reported to be an important microorganism accepting electrons from biocathode (Erable et al., 2010), reached a relative abundance of 6.9% on the anode, indicating that this kind of bacterium may also transfer electrons to the anode. These results showed that the bacteria on anode were mainly about electricity generating bacterium utilizing the nutrient in the solution. The first abundant genus on the cathode, Flavobacterium (21.3%), played a key role as denitrifying microorganisms (Chen et al., 2008), but was barely found on the anode (0.02%). Azospira (5.1%) and Nitratireductor (2.3%) on the cathode, members of phylum Proteobacteria, had also been determined to be denitrifying bacteria (Heylen et al., 2006). While the relative abundance of Azospira and Nitratireductor on anode was only 0.16% and 0.07%. The second 7
abundant strain, Chitinophagaceae (13.2%), was proficient in decomposing cellulose and chitin in the domestic sewage (Zhiwei Wang, 2013). The ammonia-oxidizing bacterium Nitrosomonas (0.4%) was mainly found on the cathode electrode, proving the biological nitrification on the FMA. The bacteria distribution of FMA biofilm in Figure 3b showed that TN removal related primary bacteria genus accounted for almost 30.0% of the microbial community; while COD removal related primary bacteria genus accounted for only 16.6%. This result indicated that the major function of FMA biofilm was the TN removal, corresponding to the result that 82.9% of the TN removal by the reactor was due to the biofilm on the FMA.
Conclusion The bioreactor with FMA achieved COD of 94.0% and TN removal efficiency of 70.0%. The new FMA test proved that only 29.9% of the COD removal was due to cathode biofilm, while 82.9% of the TN removal accounted for the cathode biofilm. The microbial community analysis showed that both nitrifying and denitrifying bacteria were found on the FMA, while these nitrogens degraded bacterium were barely found on the anode. This quantificationally analysis of FMA biofilm on COD and TN removal can promote the further optimization of the bioreactor with FMA and deepen the understanding of the corresponding nutrient removal mechanism.
Associated content The supporting information contains the detailed information of the FMA fabrication process, the FMA reactor operation process, and FMA electrochemical test, the Photograph, schematic picture, and SEM image of the FMA (Figure S1), schematic picture of the bioreactor with activated carbon FMA (Figure S2), SEM image of the biofilm on the solution-facing side of FMA (Figure S3), the LSV results of the new and used FMA (Figure S4), the effluent COD 8
concentration and NH4+-N concentration in the new FMA adsorption test (Figure S5), the electricity generation performance of bioreactor with FMA (Figure S6).
Acknowledgements This work was supported by the National Natural Science Fund for Distinguished Young Scholars (Grant No. 51125033), National Natural Science Fund of China (Grant No. 51209061 and 51408156), the Fundamental Research Funds for the Central Universities"(Grant No.HIT.MKSTISP.2016 14)， the Fundamental Research Funds for the Central Universities (HIT. NSRIF. 2015090) and the International Cooperating Project between China and European Union (Grant No. 2014DFE90110).
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Figure 1
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Figure 2
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Figure 3
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Highlights
The FMA biofilm accounted for only 29.9% of the COD removal
82.9% of the TN removal can be due to the contribution of the FMA biofilm
Most of the primary genus in the FMA biofilm were TN removal related bacteria
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