Author's Accepted Manuscript
Biochemical oxygen demand measurement by mediator method in flow system Ling Liu, Lu Bai, Dengbin Yu, Junfeng Zhai, Shaojun Dong
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(15)00069-7 http://dx.doi.org/10.1016/j.talanta.2015.02.001 TAL15369
To appear in:
Talanta
Received date: 27 December 2014 Revised date: 28 January 2015 Accepted date: 1 February 2015 Cite this article as: Ling Liu, Lu Bai, Dengbin Yu, Junfeng Zhai, Shaojun Dong, Biochemical oxygen demand measurement by mediator method in flow system, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.02.001 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 galley proof before it is published in its final citable 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.
Short communication
Biochemical Oxygen Demand Measurement by Mediator Method in Flow System Ling Liu, Lu Bai, Dengbin Yu, Junfeng Zhai and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. Abstract Using mediator as electron acceptor for biochemical oxygen demand (BOD) measurement was developed in the last decade (BODMed). However, until now, no BODMed in a flow system has been reported. This work for the first time describes a flow system of BODMed method (BODMed-FS) by using potassium ferricyanide as mediator and carbon fiber felt as substrate material for microbial immobilization. The system can determine BOD value within 30 minutes and possesses a wider analytical linear range for measuring glucose-glutamic acid (GGA) standard solution from 2 up to 200 mg L-1 without the need of dilution. The analytical performance of the BODMed-FS is comparable or better than that of the previously reported BODMed method, especially its superior long-term stability up to 2 months under continuous operation. Moreover, the BODMed-FS has same determination accuracy with the conventional BOD5 method by measuring real samples from a local wastewater treatment plant (WWTP). Keywords: rapid BOD; BODMed; flow system; native biofilm. 1. Introduction Biochemical oxygen demand (BOD) is an international regulatory environmental index for monitoring wastewater pollutants [1]. The legislated standard test for BOD monitoring is the widely accepted 5 days method (BOD5). However, it requires complicated procedures, skilled analysts and time-consuming. Over the last decade, several mediated methods have been developed for rapidly measuring BOD (BODMed)
*
Corresponding author phone: +86 431 85262101; e-mail: [email protected]; fax:
+86 431 85689711.
1
and showed great potential as an alternative to the standard BOD5 assay [2-12]. Unlike the oxygen-type BOD biosensors which is limited by the electron acceptor concentration (~ 8.7 mg L-1 at 25 ºC of O2), the mediated-type BOD methods using ferricyanide as electron acceptor, is ~10,000 fold more soluble in water than O2 [13]. Combing a large population of microbes, the BODMed method made the dilution process of sample before testing unnecessary, thus increasing the accuracy and reliability of BOD results. Furthermore, the BODMed strategy offered a simple way to resolve the problem of fluctuations of response signals resulted from the disturbance by aerated operation of oxygen-type BOD biosensors. So, the BODMed method showed remarkable improved abilities for BOD measurement [14-16]. Until now, almost all reported BODMed methods proceed in a quiescent system and no works related to arrange BODMed assay in a flow system (BODMed-FS) has been studied yet. To accelerate the application and commercialization of BODMed approach, developing a facile flow system combined with immobilized microbes and long-term stabilities is highly demanded. Therefore, to build a high performance BODMed-FS, it is very important to find an appropriate means to immobilize the microbes, because the activity of microbes would be affected significantly when exogenous mediator is added in the flow system. More recently, our group reported a phenomenon that the negative effect of mediator toward microbes could be recovered [13] and established a BODMed bioreactor by using carbon fiber felt as the substrate for immobilizing microbes to form native biofilm [17]. Inspired by these achievements, in this work, we try to further develop a BODMed-FS with extraordinarily high biodegradation ability, superior accuracy and reliability. The present approach reported in this work would pave a way for future development of practical BOD determination systems for a wide range of applications. . 2. Experimental 2.1. Materials Broth medium was purchased from Fluka (CASO, Fluka Chemie GmbH CH-9471 Buchs) which contains casein peptone, soybean flour and peptone broth. Phosphate buffer solution (PBS, 0.12 M Na2HPO4/0.08 M K2HPO4/0.1 M KCl, pH 7.0) was used for sample dilution and flow system rinsing unless otherwise stated. To prepare standard BOD solutions with different concentrations, appropriate dilution of the glucose-glutamic acid (GGA) solution according to the American Public Health Association (APHA) method that contained 1.5 g L-1 glucose and 1.5 g L-1 glutamic acid (BOD value of ~1980 mg O L-1, signed as GGA1980) was used [1,18]. The BOD5 values of the real samples were determined by the standard dilution method [1]. All chemicals used in this study were analytical reagent grade and all solutions were prepared with sterile deionized water. 2.2. Flow system 2
The BODMed-FS, according to scheme 1, was composed of a biofilm reactor (BFR) with microbes deposited on a carbon fiber felt and an electrochemical analyzer (CHI 832, CHI Co., Shanghai, China) as the signal collector. The flow system was driven by a pump and a dispensing controller (FK-1C, Baoding Longer, China). Activated sludge collected from a local municipal wastewater treatment plant (WWTP) was used as the microbial seeds for biofilm formation. 3 g of commercial CASO broth medium was pulled into a culture flask with 100 mL supernatant liquid of activated sludge. In order to optimize the measuring conditions, several parameters including incubation periods (under 35 ºC and pH 7), incubation temperatures (under 24 h and pH 7) and pH values of the incubation solution (under 24 h and 35 ºC) were studied. After culture, the flow system was sufficiently rinsed by PBS. 2.3. Measurement procedures The mediator solutions were prepared by appropriate dilution of the 0.33 M potassium ferricyanide. The PBS and sample solutions were both placed in the thermostatic bath to keep consistent temperature while deaerated by nitrogen (N2). Endogenous control solutions were prepared by adding PBS to replace samples. A fixed flow rate of 3.8 mL min-1 was set for the flow system. Before and after wastewater measurements, a calibration procedure (standard GGA solution) must be checked, so that the BODMed-FS can be adjusted and any influence of the sample on the sensor can be detected. After incubation, the samples were pumped out, centrifuged at 10, 000 rpm for 3 min to obtain a supernatant solution and analyzed microbially produced ferrocyanide. The response signals were collected using an electrochemical analyzer and conducted with amperometric mode. The electrode system and detailed operating instructions can be found in our previously reported study [17]. 3. Results and Discussion 3.1. BODMed-FS Scheme 1 shows the established BODMed-FS used in this study and different parts of the flowing system were connected by airtight tubes. The top-left image in Scheme 1 shows an optical microscopy of the immobilized microbes on a carbon fiber felt. As seen from it, the microbes are uniformly and tightly deposited on the carbon fiber felt support which suggests the feasibility of the present flow system for BOD measurement. The mediator reduced by BFR is detected by the signal changed on the electrode. As shown in the top-right image of Scheme 1, a net current ∆I between blank and sample is observed, indicating the analytical result of sample can be quantified by the relationship of ∆I and concentration. Scheme 1 3
3.2. Balance and optimization of BODMed-FS The long-term stability is an important factor for the practical application of BOD biosensor, which was affected by the performance of microbial activity. Generally, an accordant microbial consortium to the microbes contained in the tested water was obtained by a native cultured process. However, the type of species and the amount of microbial consortium could be changed during the measuring and storing processes, especially influenced by the storage conditions. Moreover, the microbes in the flow system could be divided continuously, leading to the reproduction of new bacteria and sacrifice of old bacteria. Therefore, keeping high biodegradability of the BFR which could be affected by different storage conditions is extraordinary important when the BODMed-FS was left in an idle state. Different storage conditions were studied in this work. As shown in Fig. 1, three different storage conditions including pH 7 PBS with GGA200 solution (box a), tap water (box b) and tap water with GGA20 solution (box c) were studied in this work using our established BODMed-FS. A higher but unstable electrochemical signals were obtained in box a whereas a stable signals were observed in box b and c with box b shows the lowest signal behaviour. It should be mentioned here that some strange signals are appeared at the first measurement cycle in box a and box b each day which may be attributed to the measured high blank signals caused by endogenous respiration. We supposed that the bioactivity was high in the initial stage of biofilm formation and the biodegradation was un-balanced. This phenomenon could be prevented by changing the storage conditions which is the case of box 3 where tap water with GGA20 solution was used to maintain the stability of BODMed-FS. The net currents of the electrochemical signals were shown in Fig. S1. Figure 1 The flow system used in this study was established based on our previously published work [17]. Therefore, the same basic measuring conditions are adopted according to the published reports by changing several other conditions in the present work. A tubes-type BFR that successfully applied in previous report by oxygen-type BOD biosensors [19,20] was adopted to compare with the present carbon fiber felt-type BFR for investigating the biodegradability. As shown in Table S1, under the same volumes and incubation time, the electrochemical signal produced by carbon fiber felt-type BFR (90 nA) is about 3 times higher than that by tubes-type (27 nA). This result indicates that in comparison with tubes-type BFR, the carbon fiber felt-type BFR is more suitable for establishing high performance BODMed-FS. The reproducibility of BODMed-FS was examined by GGA20 solution. As shown in Fig. S2, a relative standard deviation of 3.29% was obtained by using ten pieces of carbon fiber felt as substrates for microbial immobilization, which indicated good 4
reproducibility of the presented BODMed-FS approach. Furthermore, in this study, we also found the performance of the BODMed-FS can be influenced when different working electrodes are used. Herein, three different types of working electrodes including 1 mm Pt electrode, Pt microelectrode array 1 (25 µm×4) and array 2 (25 µm×31) were compared. As shown in Table S2, the electrochemical signal obtained at 1 mm Pt electrode was the largest, but it cannot be carried at a short time which is not good for rapid measurement. The sensitivity of Pt microelectrode array 2 was better than that of array 1. Comparing ratio of noise/100 µM ferrocyanide of these two electrodes, Pt microelectrode array 2 was used for establishing BODMed-FS. The influence of incubation time, temperature and solution pH on the response signals of the flow system was evaluated by using GGA20 solution. As shown in Fig. S3, the optimized condition with an incubation temperature of 35 ºC, incubation time of 30 min and pH of 7.0 was selected for BODMed-FS measurement. 3.3. Long-term stability and real polluted wastewater measurement Under the optimized condition, the storage stability of the BODMed-FS over two months was monitored (Fig. 2a), and excellent long-term stability performance suggested a consistent biodegradability of BFR in a long test period. The good long-term stability was benefit from the negative effect of mediator to microbes can be recovered as reported in our previous work [13]. Such great stability of biodegradability would ensure accurate BODMed-FS measurement. Although the BODMed-FS approach validated the applicability for synthetic samples, the ultimate purpose of application to real samples has yet to be confirmed. In this work, the wastewater sample was collected from local WWTPs and analyzed by BODMed-FS method using GGA standard solution calibration. As seen from Fig. 2b, the linear range of the BODMed-FS was further checked by using GGA standard solution. A response range from 2 to 400 mg O L-1 GGA organic substrate and an optimized linear correlation from 2 to 200 mg O L-1 (Fig. 2b) were obtained. All measured signals were obtained by subtracting the background current. The BODMed-FS value 15.9 mg O L-1 of real sample was comparable with BOD5 value 17.9 mg L-1 indicating the BODMed-FS method are promising for real wastewater measurement applications. Figure 2 4. Conclusion remarks In summary, we have reported and experimentally validated a BODMed-FS analytical system for determination of biodegradable organic matters for the first time. The BODMed-FS can measure BOD values within 30 min and possess a long-term stability up to 2 months. It overcomes the problems widely existed in quiescent 5
system of previously reported BODMed method that microorganism suspension and microorganism culture are required before measuring each time. The findings and the strategy used in this work pave a way for future development and application of mediated method for BOD measurement. Acknowledgements This work was supported by National key foundation for exploring scientific instrument (Nos. 2013YQ170585) and the 973 Project (Nos. 2011CB911002). References [1] Standard methods for the examination of water and wastewater, 19th ed. American Public Health Association. Washington, D.C. 1997. [2] N. Pasco, K. Baronian, C. Jeffries, J. Hay, Appl. Microbial. Biotechnol. 53 (2000) 613. [3] N. Yoshida, K. Yano, T. Morita, S. J. McNiven, H. Nakamura, I. Karube, Analyst 125 (2000) 2280. [4] K. Morris, K. Catterall, H. Zhao, N. Pasco, R. John, Anal. Chim. Acta 442 (2001) 129. [5] N. Yoshida, J. Hoashi, T. Morita, S. J. McNiven, K. Yano, A. Yoshida, H. Nakamurab, I. Karube, Analyst 126 (2001) 1751. [6] K. Catterall, K. Morris, C. Gladman, H. Zhao, N. Pasco, R. John, Talanta 55 (2001) 1187. [7] K. Catterall, H. Zhao, N. Pasco, R. John, Anal. Chem. 75 (2003) 2584. [8] A. Tizzard, J. Webber, R. Gooneratne, R. John, J. Hay, N. Pasco, Analytica Chimica Acta 522 (2004) 197. [9] N. Pasco, K. Baronian, C. Jeffries, J. Webber, J. Hay, Biosens. Bioelectron. 20 (2004) 524. [10] K. Morris, H. Zhao, R. John, Aust. J. Chem. 58 (2005) 237. [11] N. Pasco, J. Hay, A. Scott, Webber, J. Aust. J. Chem. 58 (2005) 288. [12] H. Chen, T. Yea, B. Qiu, G. Chen, X. Chen, Anal. Chim. Acta 612 (2008) 75. [13] L. Liu, L. Shang, C. Liu, B. Zhang, S. Dong, Talanta 81 (2010) 1170. [14] M. A. Jordan, D. T. Welsh, P. R. Teasdale, K. Catterall, R. John, Water Res. 44 (2010) 5981. 6
[15] M. A. Jordan, D. T. Welsh, R. John, K. Catterall, P. R. Teasdale, Water Res. 47 (2013) 841. [16] M. A. Jordan, D. T. Welsh, P. R. Teasdale, Talanta 125 (2014) 293. [17] L. Liu, L. Deng, D. Yong, S. Dong, Talanta 84 (2011) 895. [18] L. Liu, S. Zhang, L. Xing, H. Zhao, S. Dong, Talanta 93 (2012) 314. [19] C. Liu, H. Zhao, Z. Ma, T. An, C. Liu, L. Zhao, D. Yong, J. Jia, X. Li, S. Dong, Environ. Sci. Technol. 48 (2014) 1762. [20] C. Liu, H. Zhao, S. Gao, J. Jia, L. Zhao, D. Yong, S. Dong, Biosens. Bioelectron. 45 (2013) 213. Legends
Current
Scheme 1. Schematic diagram of the BODMed-FS Figure 1. Electrochemical signals for measuring GGA20 and black by using BODMed-FS at storing conditions were pH 7 PBS with GGA200 solution (box a), tap water (box b) and tap water with GGA20 solution (box c). Figure 2. (a) The long-term stability of BODMed-FS in 2 months, (b) calibration of GGA standard solution.
∆I Time
microphotograph of the cultivated biofilm optical photograph of the bioreator PBS or sample
Reduced Fe(Ⅱ)
pump Bioreactor
waste
Scheme 1
7
600
a
20
GGA blank
Current/nA
500
b
400 300
c
200 100 0
Apr. 08----May 06 Figure 1
140
a
GGA
20
120 100
∆I/nA
80 60 40 20 0 4.22 4.24 4.25 4.26 4.27 5.05 5.06 6.07 6.08 6.09 6.11 6.12 6.15 6.17 6.19 6.20 6.21 6.23 6.25 6.27 6.29 7.04 7.08 7.14
date
8
800
b
∆I/ nA
600
400
200
Y = 9.21 + 4.17X 2 R = 0.9930
0 0
50
100
150
200
-1
Concentration/mg O L
Figure 2 Highlights: 1.
A flow system of mediator method for BOD measurement (BODMed-FS) was reported at the first time.
2.
The BODMed-FS overcame the problems of previous BODMed method using suspended microorganism.
3.
It can measure BOD values within 30 min and possesses a long-term stability up to 2 months.
4.
A linear range from 2 up to 200 mg L-1 was obtained for measuring GGA without the need of dilution.
9
Current
∆I Time
microphotograph of the cultivated biofilm optical photograph of the bioreator PBS or sample
Reduced Fe(Ⅱ)
pump Bioreactor
waste
Biochemical oxygen demand measurement by mediator method in flow system Ling Liu, Lu Bai, Dengbin Yu, Junfeng Zhai, Shaojun Dong
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(15)00069-7 http://dx.doi.org/10.1016/j.talanta.2015.02.001 TAL15369
To appear in:
Talanta
Received date: 27 December 2014 Revised date: 28 January 2015 Accepted date: 1 February 2015 Cite this article as: Ling Liu, Lu Bai, Dengbin Yu, Junfeng Zhai, Shaojun Dong, Biochemical oxygen demand measurement by mediator method in flow system, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.02.001 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 galley proof before it is published in its final citable 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.
Short communication
Biochemical Oxygen Demand Measurement by Mediator Method in Flow System Ling Liu, Lu Bai, Dengbin Yu, Junfeng Zhai and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. Abstract Using mediator as electron acceptor for biochemical oxygen demand (BOD) measurement was developed in the last decade (BODMed). However, until now, no BODMed in a flow system has been reported. This work for the first time describes a flow system of BODMed method (BODMed-FS) by using potassium ferricyanide as mediator and carbon fiber felt as substrate material for microbial immobilization. The system can determine BOD value within 30 minutes and possesses a wider analytical linear range for measuring glucose-glutamic acid (GGA) standard solution from 2 up to 200 mg L-1 without the need of dilution. The analytical performance of the BODMed-FS is comparable or better than that of the previously reported BODMed method, especially its superior long-term stability up to 2 months under continuous operation. Moreover, the BODMed-FS has same determination accuracy with the conventional BOD5 method by measuring real samples from a local wastewater treatment plant (WWTP). Keywords: rapid BOD; BODMed; flow system; native biofilm. 1. Introduction Biochemical oxygen demand (BOD) is an international regulatory environmental index for monitoring wastewater pollutants [1]. The legislated standard test for BOD monitoring is the widely accepted 5 days method (BOD5). However, it requires complicated procedures, skilled analysts and time-consuming. Over the last decade, several mediated methods have been developed for rapidly measuring BOD (BODMed)
*
Corresponding author phone: +86 431 85262101; e-mail: [email protected]; fax:
+86 431 85689711.
1
and showed great potential as an alternative to the standard BOD5 assay [2-12]. Unlike the oxygen-type BOD biosensors which is limited by the electron acceptor concentration (~ 8.7 mg L-1 at 25 ºC of O2), the mediated-type BOD methods using ferricyanide as electron acceptor, is ~10,000 fold more soluble in water than O2 [13]. Combing a large population of microbes, the BODMed method made the dilution process of sample before testing unnecessary, thus increasing the accuracy and reliability of BOD results. Furthermore, the BODMed strategy offered a simple way to resolve the problem of fluctuations of response signals resulted from the disturbance by aerated operation of oxygen-type BOD biosensors. So, the BODMed method showed remarkable improved abilities for BOD measurement [14-16]. Until now, almost all reported BODMed methods proceed in a quiescent system and no works related to arrange BODMed assay in a flow system (BODMed-FS) has been studied yet. To accelerate the application and commercialization of BODMed approach, developing a facile flow system combined with immobilized microbes and long-term stabilities is highly demanded. Therefore, to build a high performance BODMed-FS, it is very important to find an appropriate means to immobilize the microbes, because the activity of microbes would be affected significantly when exogenous mediator is added in the flow system. More recently, our group reported a phenomenon that the negative effect of mediator toward microbes could be recovered [13] and established a BODMed bioreactor by using carbon fiber felt as the substrate for immobilizing microbes to form native biofilm [17]. Inspired by these achievements, in this work, we try to further develop a BODMed-FS with extraordinarily high biodegradation ability, superior accuracy and reliability. The present approach reported in this work would pave a way for future development of practical BOD determination systems for a wide range of applications. . 2. Experimental 2.1. Materials Broth medium was purchased from Fluka (CASO, Fluka Chemie GmbH CH-9471 Buchs) which contains casein peptone, soybean flour and peptone broth. Phosphate buffer solution (PBS, 0.12 M Na2HPO4/0.08 M K2HPO4/0.1 M KCl, pH 7.0) was used for sample dilution and flow system rinsing unless otherwise stated. To prepare standard BOD solutions with different concentrations, appropriate dilution of the glucose-glutamic acid (GGA) solution according to the American Public Health Association (APHA) method that contained 1.5 g L-1 glucose and 1.5 g L-1 glutamic acid (BOD value of ~1980 mg O L-1, signed as GGA1980) was used [1,18]. The BOD5 values of the real samples were determined by the standard dilution method [1]. All chemicals used in this study were analytical reagent grade and all solutions were prepared with sterile deionized water. 2.2. Flow system 2
The BODMed-FS, according to scheme 1, was composed of a biofilm reactor (BFR) with microbes deposited on a carbon fiber felt and an electrochemical analyzer (CHI 832, CHI Co., Shanghai, China) as the signal collector. The flow system was driven by a pump and a dispensing controller (FK-1C, Baoding Longer, China). Activated sludge collected from a local municipal wastewater treatment plant (WWTP) was used as the microbial seeds for biofilm formation. 3 g of commercial CASO broth medium was pulled into a culture flask with 100 mL supernatant liquid of activated sludge. In order to optimize the measuring conditions, several parameters including incubation periods (under 35 ºC and pH 7), incubation temperatures (under 24 h and pH 7) and pH values of the incubation solution (under 24 h and 35 ºC) were studied. After culture, the flow system was sufficiently rinsed by PBS. 2.3. Measurement procedures The mediator solutions were prepared by appropriate dilution of the 0.33 M potassium ferricyanide. The PBS and sample solutions were both placed in the thermostatic bath to keep consistent temperature while deaerated by nitrogen (N2). Endogenous control solutions were prepared by adding PBS to replace samples. A fixed flow rate of 3.8 mL min-1 was set for the flow system. Before and after wastewater measurements, a calibration procedure (standard GGA solution) must be checked, so that the BODMed-FS can be adjusted and any influence of the sample on the sensor can be detected. After incubation, the samples were pumped out, centrifuged at 10, 000 rpm for 3 min to obtain a supernatant solution and analyzed microbially produced ferrocyanide. The response signals were collected using an electrochemical analyzer and conducted with amperometric mode. The electrode system and detailed operating instructions can be found in our previously reported study [17]. 3. Results and Discussion 3.1. BODMed-FS Scheme 1 shows the established BODMed-FS used in this study and different parts of the flowing system were connected by airtight tubes. The top-left image in Scheme 1 shows an optical microscopy of the immobilized microbes on a carbon fiber felt. As seen from it, the microbes are uniformly and tightly deposited on the carbon fiber felt support which suggests the feasibility of the present flow system for BOD measurement. The mediator reduced by BFR is detected by the signal changed on the electrode. As shown in the top-right image of Scheme 1, a net current ∆I between blank and sample is observed, indicating the analytical result of sample can be quantified by the relationship of ∆I and concentration. Scheme 1 3
3.2. Balance and optimization of BODMed-FS The long-term stability is an important factor for the practical application of BOD biosensor, which was affected by the performance of microbial activity. Generally, an accordant microbial consortium to the microbes contained in the tested water was obtained by a native cultured process. However, the type of species and the amount of microbial consortium could be changed during the measuring and storing processes, especially influenced by the storage conditions. Moreover, the microbes in the flow system could be divided continuously, leading to the reproduction of new bacteria and sacrifice of old bacteria. Therefore, keeping high biodegradability of the BFR which could be affected by different storage conditions is extraordinary important when the BODMed-FS was left in an idle state. Different storage conditions were studied in this work. As shown in Fig. 1, three different storage conditions including pH 7 PBS with GGA200 solution (box a), tap water (box b) and tap water with GGA20 solution (box c) were studied in this work using our established BODMed-FS. A higher but unstable electrochemical signals were obtained in box a whereas a stable signals were observed in box b and c with box b shows the lowest signal behaviour. It should be mentioned here that some strange signals are appeared at the first measurement cycle in box a and box b each day which may be attributed to the measured high blank signals caused by endogenous respiration. We supposed that the bioactivity was high in the initial stage of biofilm formation and the biodegradation was un-balanced. This phenomenon could be prevented by changing the storage conditions which is the case of box 3 where tap water with GGA20 solution was used to maintain the stability of BODMed-FS. The net currents of the electrochemical signals were shown in Fig. S1. Figure 1 The flow system used in this study was established based on our previously published work [17]. Therefore, the same basic measuring conditions are adopted according to the published reports by changing several other conditions in the present work. A tubes-type BFR that successfully applied in previous report by oxygen-type BOD biosensors [19,20] was adopted to compare with the present carbon fiber felt-type BFR for investigating the biodegradability. As shown in Table S1, under the same volumes and incubation time, the electrochemical signal produced by carbon fiber felt-type BFR (90 nA) is about 3 times higher than that by tubes-type (27 nA). This result indicates that in comparison with tubes-type BFR, the carbon fiber felt-type BFR is more suitable for establishing high performance BODMed-FS. The reproducibility of BODMed-FS was examined by GGA20 solution. As shown in Fig. S2, a relative standard deviation of 3.29% was obtained by using ten pieces of carbon fiber felt as substrates for microbial immobilization, which indicated good 4
reproducibility of the presented BODMed-FS approach. Furthermore, in this study, we also found the performance of the BODMed-FS can be influenced when different working electrodes are used. Herein, three different types of working electrodes including 1 mm Pt electrode, Pt microelectrode array 1 (25 µm×4) and array 2 (25 µm×31) were compared. As shown in Table S2, the electrochemical signal obtained at 1 mm Pt electrode was the largest, but it cannot be carried at a short time which is not good for rapid measurement. The sensitivity of Pt microelectrode array 2 was better than that of array 1. Comparing ratio of noise/100 µM ferrocyanide of these two electrodes, Pt microelectrode array 2 was used for establishing BODMed-FS. The influence of incubation time, temperature and solution pH on the response signals of the flow system was evaluated by using GGA20 solution. As shown in Fig. S3, the optimized condition with an incubation temperature of 35 ºC, incubation time of 30 min and pH of 7.0 was selected for BODMed-FS measurement. 3.3. Long-term stability and real polluted wastewater measurement Under the optimized condition, the storage stability of the BODMed-FS over two months was monitored (Fig. 2a), and excellent long-term stability performance suggested a consistent biodegradability of BFR in a long test period. The good long-term stability was benefit from the negative effect of mediator to microbes can be recovered as reported in our previous work [13]. Such great stability of biodegradability would ensure accurate BODMed-FS measurement. Although the BODMed-FS approach validated the applicability for synthetic samples, the ultimate purpose of application to real samples has yet to be confirmed. In this work, the wastewater sample was collected from local WWTPs and analyzed by BODMed-FS method using GGA standard solution calibration. As seen from Fig. 2b, the linear range of the BODMed-FS was further checked by using GGA standard solution. A response range from 2 to 400 mg O L-1 GGA organic substrate and an optimized linear correlation from 2 to 200 mg O L-1 (Fig. 2b) were obtained. All measured signals were obtained by subtracting the background current. The BODMed-FS value 15.9 mg O L-1 of real sample was comparable with BOD5 value 17.9 mg L-1 indicating the BODMed-FS method are promising for real wastewater measurement applications. Figure 2 4. Conclusion remarks In summary, we have reported and experimentally validated a BODMed-FS analytical system for determination of biodegradable organic matters for the first time. The BODMed-FS can measure BOD values within 30 min and possess a long-term stability up to 2 months. It overcomes the problems widely existed in quiescent 5
system of previously reported BODMed method that microorganism suspension and microorganism culture are required before measuring each time. The findings and the strategy used in this work pave a way for future development and application of mediated method for BOD measurement. Acknowledgements This work was supported by National key foundation for exploring scientific instrument (Nos. 2013YQ170585) and the 973 Project (Nos. 2011CB911002). References [1] Standard methods for the examination of water and wastewater, 19th ed. American Public Health Association. Washington, D.C. 1997. [2] N. Pasco, K. Baronian, C. Jeffries, J. Hay, Appl. Microbial. Biotechnol. 53 (2000) 613. [3] N. Yoshida, K. Yano, T. Morita, S. J. McNiven, H. Nakamura, I. Karube, Analyst 125 (2000) 2280. [4] K. Morris, K. Catterall, H. Zhao, N. Pasco, R. John, Anal. Chim. Acta 442 (2001) 129. [5] N. Yoshida, J. Hoashi, T. Morita, S. J. McNiven, K. Yano, A. Yoshida, H. Nakamurab, I. Karube, Analyst 126 (2001) 1751. [6] K. Catterall, K. Morris, C. Gladman, H. Zhao, N. Pasco, R. John, Talanta 55 (2001) 1187. [7] K. Catterall, H. Zhao, N. Pasco, R. John, Anal. Chem. 75 (2003) 2584. [8] A. Tizzard, J. Webber, R. Gooneratne, R. John, J. Hay, N. Pasco, Analytica Chimica Acta 522 (2004) 197. [9] N. Pasco, K. Baronian, C. Jeffries, J. Webber, J. Hay, Biosens. Bioelectron. 20 (2004) 524. [10] K. Morris, H. Zhao, R. John, Aust. J. Chem. 58 (2005) 237. [11] N. Pasco, J. Hay, A. Scott, Webber, J. Aust. J. Chem. 58 (2005) 288. [12] H. Chen, T. Yea, B. Qiu, G. Chen, X. Chen, Anal. Chim. Acta 612 (2008) 75. [13] L. Liu, L. Shang, C. Liu, B. Zhang, S. Dong, Talanta 81 (2010) 1170. [14] M. A. Jordan, D. T. Welsh, P. R. Teasdale, K. Catterall, R. John, Water Res. 44 (2010) 5981. 6
[15] M. A. Jordan, D. T. Welsh, R. John, K. Catterall, P. R. Teasdale, Water Res. 47 (2013) 841. [16] M. A. Jordan, D. T. Welsh, P. R. Teasdale, Talanta 125 (2014) 293. [17] L. Liu, L. Deng, D. Yong, S. Dong, Talanta 84 (2011) 895. [18] L. Liu, S. Zhang, L. Xing, H. Zhao, S. Dong, Talanta 93 (2012) 314. [19] C. Liu, H. Zhao, Z. Ma, T. An, C. Liu, L. Zhao, D. Yong, J. Jia, X. Li, S. Dong, Environ. Sci. Technol. 48 (2014) 1762. [20] C. Liu, H. Zhao, S. Gao, J. Jia, L. Zhao, D. Yong, S. Dong, Biosens. Bioelectron. 45 (2013) 213. Legends
Current
Scheme 1. Schematic diagram of the BODMed-FS Figure 1. Electrochemical signals for measuring GGA20 and black by using BODMed-FS at storing conditions were pH 7 PBS with GGA200 solution (box a), tap water (box b) and tap water with GGA20 solution (box c). Figure 2. (a) The long-term stability of BODMed-FS in 2 months, (b) calibration of GGA standard solution.
∆I Time
microphotograph of the cultivated biofilm optical photograph of the bioreator PBS or sample
Reduced Fe(Ⅱ)
pump Bioreactor
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Scheme 1
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Apr. 08----May 06 Figure 1
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80 60 40 20 0 4.22 4.24 4.25 4.26 4.27 5.05 5.06 6.07 6.08 6.09 6.11 6.12 6.15 6.17 6.19 6.20 6.21 6.23 6.25 6.27 6.29 7.04 7.08 7.14
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Y = 9.21 + 4.17X 2 R = 0.9930
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Figure 2 Highlights: 1.
A flow system of mediator method for BOD measurement (BODMed-FS) was reported at the first time.
2.
The BODMed-FS overcame the problems of previous BODMed method using suspended microorganism.
3.
It can measure BOD values within 30 min and possesses a long-term stability up to 2 months.
4.
A linear range from 2 up to 200 mg L-1 was obtained for measuring GGA without the need of dilution.
9
Current
∆I Time
microphotograph of the cultivated biofilm optical photograph of the bioreator PBS or sample
Reduced Fe(Ⅱ)
pump Bioreactor
waste
