Bioresource Technology 193 (2015) 357–362
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Application of ethylene diamine tetra acetic acid degrading bacterium Burkholderia cepacia on biotreatment process Wei-Ting Chen a,⇑, Shu-Min Shen b, Chi-Min Shu b a b
Department of Cosmetic Application & Management, St. Mary’s Junior College of Medicine, Nursing and Management, Yilan 26644, Taiwan, ROC Department of Safety, Health, and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan, ROC
h i g h l i g h t s EDTA can be properly biodegraded by Burkholderia cepacia. Continuous process and batch treatment were used in EDTA wastewater degradation. The strain YL-6 was identified as Burkholderia cepacia. The results showed that batch treatment was more suited for EDTA biodegradation. Removal efficiency of EDTA and COD mostly researched to 100% by batch treatment.
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Article history: Received 29 April 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 25 June 2015 Keywords: Ethylene diamine tetra acetic acid (EDTA) Burkholderia cepacia Batch degradation Nutrients Activated sludge
a b s t r a c t Ethylene diamine tetra acetic acid (EDTA), the effluent of secondary biotreatment units, can be properly biodegraded by Burkholderia cepacia. Through batch degradation of EDTA, the raw wastewater of EDTA was controlled at 50 mg/L, and then nutrients was added in diluted wastewater to cultivate activated sludge, which the ratio of composition is depicted as ‘‘COD:N:P:Fe = 100:5:1:0.5’’. After 27 days, the removal efficiency of Fe-EDTA and COD was 100% and 92.0%, correspondingly. At the continuous process, the raw wastewater of EDTA was dictated at 166 mg/L before adding nutrients to cultivate activated sludge, in which the ratio of composition did also follow with batch process. After 22 days, the removal efficiency of Fe-EDTA and COD for experimental group was 71.46% and 62.58%, correspondingly. The results showed that the batch process was more suited for EDTA biodegradation. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Ethylene diamine tetra acetic acid (EDTA) is extensively used in dyeing industry, textile industry, paper industry, metal manufacturing, printing industry, cosmetic industry, pharmaceutical industry, food industry, and nuclear facilities (Diez et al., 2005; Mansilla et al., 2006; Balaji et al., 2007; Harraz et al., 2010; Lu and Wei, 2011; May et al., 2012; Voglar and Lestan, 2012). In addition, EDTA is a chelating agent which is employed to sequester metal ions, such as Cu2+ and Fe3+. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity. Moreover, the chelating stability constant of EDTA with Cu2+ and Fe3+ usually reaches up 1015, so the highly stable chelator, EDTA, is widely applied in wastewater treatment processes for containments diminishing. In natural waters or industrial wastewater, EDTA exists mainly in the form of Ca-EDTA, Zn-EDTA and Ni-EDTA ⇑ Corresponding author. Tel.: +886 3 989 7396; fax: +886 3 989 7386. E-mail address: [email protected] (W.-T. Chen). http://dx.doi.org/10.1016/j.biortech.2015.06.099 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
(Mansilla et al., 2006). Complexes with high stability constants, such as Fe(III)-EDTA, are not readily removed and are responsible for the EDTA remaining in growth experiments (Chen et al., 2005; Jing et al., 2012; Dong et al., 2012; Chandrashekhar et al., 2013; Kavitha et al., 2013; Zhang et al., 2015). Moreover, EDTA’s low biodegradability is responsible for the presence of several complexes in sewage effluents, freshwater, and ground-water. Although EDTA is a useful chelating agent, it causes an environmental pollution problem because of its high stability. Once metal ions are being bound by EDTA, the chelate cannot degrade readily. Therefore, several attempts about degradation of EDTA have been made, and some explored methods, such as via electrochemical process (Voglar and Lestan, 2012; Zhang et al., 2015), photodegradation (Lauff et al., 1990), activated-sludge adsorption (Chen et al., 2005; Kavitha et al., 2013, 2014; Ginkel et al., 1997; Rodriguez et al., 1999), and biological and UV/O3 degradation (Glass and Popovic, 2005), are developed technologies for EDTA removal. The procedure of EDTA removal was that the raw wastewater was pretreated by chemical coagulation to remove
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heavy metals, and then added the bioaugmentation agent – Burkholderia cepacia to biodegrade the target compound – EDTA. So far many researches are investigated as mentioned in the above descriptions, and there are many cases regarding the biological degradation on wastewater treatment in Taiwan. In practice, the biological degradation is the most economic and environmental-friendly way in aspect of economic benefits and environmental qualities. According to the previous researches, these six EDTA degradation bacteria, such as Agrobacterium sp., activated sludge, bacterial strain DSM 9103, bacterial consortia strain BNC1, soil consortia and agricultural sediment, surface and subsurface soil consortia, are usefully employed in treatment of EDTA degradation. The aim of this study was to apply the screened EDTA degradation bacteria, B. cepacia YL-6, which are screened from activated sludge in a wastewater plant, to undergo the model testing of EDTA degradation. The target of this study is normally on wastewater from a PC board plant in Taiwan. Prior to the treatment of EDTA degradation, the chemical coagulation is a pre-treated method to remove heavy metals of wastewater. After the above process, activated sludge in continuous process was used to eliminate EDTA and COD efficiently. Furthermore, experimental and controlled groups are applied in continuous process to acquire efficient group. Apart from the continuous process, the batch treatment was also employed in this study for degradation of EDTA. The intention of this study was to detect the optimal methodology of EDTA wastewater degradation from biodegradation experiments of continuous process and batch treatment. 2. Methods 2.1. Source of bacteria The attempt of this study was to screen EDTA degradation bacteria, B. cepacia YL-6, from activated sludge in a wastewater plant at Douliou industrial park (Douliou, Yunlin, Taiwan), which were applied to degrade EDTA, and then these bacteria were cultivated with different nutrients, such as Fe-EDTA-based medium, ethylamine-based medium, and Fe-EDTA/ethylamine-based medium, before degradation. 2.2. Component of liquid medium and isolation of bacteria After the target bacteria, B. cepacia YL-6, was scanned, a minimal saline agar medium (Fe-EDTA 1000 mg; ethylamine 1%; ethylenediamine 1%; potassium acetate 250 mg; (NH2CO + (NH4)2SO4) 2000 mg (ratio of 1:1, w/w); KH2PO4 1000 mg; NaCl 100 mg; CaCl2 25 mg; FeSO47H2O 20 mg; MgSO47H2O 20 mg; ZnSO46H2O 10 mg; CuSO46H2O 10 mg; MnSO44H2O 5 mg; CoCl26H2O 0.5 mg; Na2MoO47H2O 0.5 mg) was employed to cultivate the target bacteria (Chen et al., 2005). The components of liquid medium are shown in Table 1 (Chen et al., 2005; Jing et al., 2012). Moreover, Fe-EDTA and urea were the majority of carbon resource and nitrogen resource, respectively. The main phosphate resource are ammonium sulfate and monopotassium phosphate, and potassium acetate (CH3COOK) is regarded as the co-metabolic carbon source. 1.5–2.0% of agar is added in plate before agar melting, sterilization, and agar congealment to be a culture medium. An agar plate is then used to purify bacterial specie and conserve bacterial strain before identification of mycology. 2.3. Analysis methods 2.3.1. Identification of target bacteria There are three methods, including API 20NE, Biolog, and Phoenix ID 100, which are used to determine the target bacteria.
Table 1 Component of liquid medium for bacteria cultivation (Chen et al., 2005; Jing et al., 2012). Composition
Concentration (mg/L)
Percentage (%)
Fe-EDTA Potassium acetate (NH2)CO + (NH4)2SO4 KH2PO4 NaCl CaCl2 FeSO47H2O MgSO47H2O ZnSO46H2O CuSO46H2O MnSO44H2O CoCl46H2O NaMoO47H2O Ethylamine
1000 250 2000 (1:1) 1000 100 25 20 20 10 10 5 0.5 0.5 49
22.27 5.57 44.54 22.27 2.22 0.57 0.45 0.45 0.23 0.23 0.11 0.001 0.001 1.09
Components of the API 20NE strip include microtubes containing dehydrated media and substrates. The conventional tests are described as media microtubes inoculated with a bacterial suspension which reconstitutes the media. After incubation process, indicator systems or the addition of reagents are employed to monitor the metabolic end products. The substrate microtubes consist of assimilation tests which are inoculated with a minimal medium. The bacteria will grow if they are capable of utilizing the corresponding substrate (Glass and Popovic, 2005). A single colony was chosen and emulsified into ‘inoculating fluid A’ (Biolog) for subsequent inoculation on to the MicroPlate test plate (Biolog) for the most cases. According to the manufacturer’s instructions, more particular organisms, especially on capnophilic strains, were cultivated on alternative media, and inocula was prepared to a specified transmittance using a turbidimeter, as specified in the user guide. In each isolate process, according to growth characteristics, 100 lL of the cell suspension was inoculated into each well of the MicroPlate, where a multichannel pipette was used with incubation at 37 °C for 20 h and aerobic condition of 7.5% CO2. MicroPlates were identified by the MicroStation semiautomated reader after 20 h and results were demonstrated by the identification system software (GEN III database, version 5.2.1). If incubation period is over 20 h, the system indicated which isolates could not be identified. Hence, such isolates were re-incubated and re-read between 3 and 6 h later (Holmes et al., 1994; Wragg et al., 2014). The Phoenix 100 ID/AST system is innovated by Becton Dickinson Cooperation (Becton Dickinson Co., Sparks, MD, USA), which is an automated system for identification (ID) and antimicrobial susceptibility testing (AST). The strains were stayed at 70 °C defibrinated sheep blood and were undergone three times on 5% sheep blood agar before testing. All panels were processed according to the manufacturer’s directions. Each panel has to be inoculated within 2 h after its foil pouch is opened, and the panels need to be loaded into the instrument within 30 min of inoculation. Only cotton-tipped swabs or wooden applicators are acceptable for preparation of the suspensions. A suspension of each 24-h-old isolate was made in the Phoenix 100 ID/AST broth to match the turbidity of a 0.5 McFarland standard by using a CrystalSpec nephelometer (Becton Dickinson) (O’Hara, 2006; Giani et al., 2012). 2.3.2. Measurement of bacteria growth concentration The Lowry protein assay method for protein concentration determination is one of the most venerable and extensively-used protein assays. The Lowry method was first described in 1951 by Lowry et al. (1951). The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret
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test) with the oxidation of aromatic protein residues. The Lowry method is best employed with protein concentrations of 0.01– 1.0 mg/mL and is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The concentration of the reduced Folin reagent is measured by absorbance at 660 nm (Lowry et al., 1951). As a result, the total concentration of protein in the sample can be detected by using this method, and the amount of bacteria consequently is the concentration of protein which is divided by 0.6. 2.3.3. Measurement of EDTA concentration According to previous studies (Loyaux-Lawniczak et al., 1999; Cagnasso et al., 2007; Narola et al., 2011), HPLC was broadly used to determine the concentration of EDTA in numerous fields, such as pharmacy, natural water, and drink. As the result, Perkin Elmer Series 200 HPLC is used in quantitative analysis of the EDTA concentration in this study. The analysis conditions are listed in the following sentences: chromatographic column (SUPELCO 516-C18 5 lm, inner radius 250 mm 4.6 mm), solvent (66% of methanol), flow rate (0.8 mL min 1), detecting wavelength (254 nm), and duration time (2.7 min). 2.3.4. Measurement of chemical oxygen demand (COD) test The COD test is widely employed in environmental chemistry to indirectly measure the amount of organic compounds in water, which is adopting the method, NIEA W517.50B, from Environmental Analysis Laboratory, Executive Yuan, Taiwan, ROC. Most applications of COD determine the amount of organic pollutants found in surface water (e.g., lakes and rivers) or wastewater, making COD a useful measure of water quality. It is expressed in milligrams per liter (mg/L) also referred to as ppm (parts per million), which indicates the mass of oxygen consumed per liter of solution (Environmental Analysis Laboratory, 2009). 2.3.5. Analysis of component’s metal ion concentration The metal ion concentration in the aqueous phase and the ores were available to be determined by an inductively coupled plasma atomic emission spectrometer (ICP-AES; Optima, 5100DV). For the partition studies, equal volumes (10 mL) of the aqueous and organic phases were shaken at room temperature (25 ± 3 °C) for 5 min to ensure complete equilibration. The two phases were separated, and a suitable aliquot of the aqueous phase was assayed for the metal ion concentration. Based on three tests, the value of 95% extraction for metal ion was associated with a variation coefficient of ±3% (Skoog et al., 2003). 2.4. Pre-treatment test of wastewater The raw wastewater sample was obtained from a PC board plant in Taiwan industrial park, so it consisted of various kinds of heavy metals. Before EDTA removal procedure, chemical coagulation was pretreated to remove heavy metals in raw wastewater. The chemical coagulation processes are listed in following sentences: 1, 3, 5, 7, or 10 g of ferric trichloride or ferrite powder was added in 1 L raw wastewater, respectively, to coagulate heavy metals. After the addition of ferric trichloride, the mixing liquid was stirred at fast stir (110 rpm, 2 min) and then was stirred at slow stir (22 rpm, 20 min). Subsequently, supernatant liquid and deposit can be separated by centrifugation process. The next step is that supernatant liquid mixed with sodium hydroxide for its pH value adjustment to remove those residual heavy metals which is exited in supernatant liquid after addition of ferric trichloride. As a result, ICP-AES was employed to analyze the concentration of wastewater which was underwent the pre-treatment test.
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2.5. Batch treatment Sequencing batch reactors (SBR) are industrial processing tanks for the treatment of wastewater. SBR treat wastewater such as sewage or output from anaerobic digesters or mechanical biological treatment facilities in batches. Oxygen is bubbled through the wastewater to reduce biochemical oxygen demand (BOD) and COD to make them suitable for discharge into sewers or for use on land (Arnaldo et al., 2007; Piyawadee and Alissara, 2011; Adriana et al., 2012). For sequencing batch reactor activated sludge process, the raw wastewater was controlled at 50 mg/L, and then nutrients were added in diluted wastewater to cultivate activated sludge, in which the ratio of composition is depicted as ‘‘COD:N:P:Fe = 100:5:1:0.5’’. Moreover, cultivation cycle is based on 1 day, 23 h aeration and 1 h sedimentation, and the sludge age is controlled 12 days. In addition, cultivation conditions are controlled as the following sentences during cultivation period: pH value at 7.0 ± 0.2, temperature at 25 °C, dissolved oxygen (DO) at 1–3 mg/L, sludge volume index (SVI) at 80–120 mL/g, and BOD–MLSS loading at 0.2–0.4 kg BOD kg/MLSS. 2.6. Continuous treatment As different from the SBR test, continuous process was employed in activated sludge cultivation to identify the optimal method of cultivation. The advantages of continuous culture include: (1) the growth condition is mainly substrate-limiting so that substrate-abundant uncoupling (Liu et al., 1998) could not occur; (2) maintenance energy requirements are balanced because cell compositions (including enzymes) are in a stabilized state (Russell, 2007). In view of practical applications, information on how continuous flow activated sludge yield is affected by the influent of substrate is also valuable in formulating operational plans, thereby to improve treatment plant performance (Chong et al., 2011). The ratio of nutrients which was employed in a continuous system was the same as the ratio of nutrients in a batch reactor system. Additionally, the speed of nutrient addition was controlled at 1.3 L/h, and hydraulic retention time was maintained at 8 h. Moreover, the cultivation conditions employed in a continuous system was also similar with the cultivation conditions in a batch reactor system. As a result, one of above mentioned systems can be identified as the optimal method to cultivate activated sludge. 2.7. Experimental and control tests The addition of bacterial agent, B. cepacia YL-6, was used in experimental test to certify the efficiency of B. cepacia YL-6. In contrast with experimental test, the test (without addition of bacterial agent) was designed as control test to observe the comparison of experimental and control tests. These two tests could absolutely examine how bacterial agent worked on metal-EDTA pollutants removal. 3. Results and discussion 3.1. Identification of EDTA-degradable bacteria This study adopted three systems, API 20NE, Biolog, and Phoenix ID 100, to identify biological species of the bacteria. Strain YL-6 was rod-shaped and ranged from 1 to 3 lm in length. The Biolog identification system showed a similarity greater than 99%. The identification result of three systems is depicted in Table 2, and precision of each result reaches above 95%. On the basis of these characteristics and its 16S rDNA sequence, strain
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YL-6 was identified as B. cepacia and designated B. cepacia YL-6. After biological species identification, the bacteria were cultivated with various kinds of carbon sources for EDTA degradation.
Table 3 Original concentration of contaminant in wastewater before chemical coagulation processes (Environmental Protection Administration, 2014).
3.2. Effects of ferric trichloride on chemical coagulation processes The original COD and EDTA of the PC board wastewater were approximately 38,000–40,000 mg/L and 850 mg/L, correspondingly. Moreover, wastewater consisted of various kinds of heavy metals, such as cupper (Cu), nickel (Ni), manganese (Mn), lead (Pb), and ferrite (Fe), and the concentration of above metals are 1,895, 150.3, 1050, 588, and 126 mg/L, respectively, as shown in Table 3. Ferrite powder and ferric trichloride are used as coagulation agents with three rations addition, 1, 3, and 5 mg/L, to remove the above mentioned heavy metals before metal-EDTA degradation. In Table 4, increasing the dosage of ferric powder and ferric trichloride (1 g/L) had no effect on the improvement of heavy metals removal at various pH values from the wastewater. Furthermore, tests of heavy metals removal at different pH values are depicted in Table 4, which show better efficiency of heavy metals removal with ferric trichloride addition. Thus, 1 g/L of ferric trichloride in the pH 6 was determined as the appropriate dosage for PC board wastewater pre-treatment. After a variety of tests as shown in Table 4, Table 5 demonstrates that the final COD is 22,233 mg/L, and the removal efficiency is 41.4% at a solution pH of 6 with 1 g/L of ferric trichloride. In addition, Table 5 shows that the removal efficiency of heavy metals, Cu, Fe, Mn, Ni, and Pb, were 79.5%, 53.2%, 99.7%, 96.7%, and 95.1%, respectively. Because of the high stability of EDTA, EDTA cannot degrade easily once metal ions are being bound by EDTA. As the result, the EDTA concentration has no effect during chemical coagulation processes. Therefore, bacterial degradation approaches (batch and continuous treatment with experimental and control tests) were applied to degrade metal-EDTA pollutants after chemical coagulation processes. 3.3. Batch treatment Figs. 1 and 2 show that the degradation efficiency and residual concentration with four diluted concentrations of wastewater (50, 100, 200, and 500 mg/L) during batch treatment. When the initial concentration of EDTA was controlled at 50 mg/L, and then nutrients was added in diluted wastewater to cultivate activated sludge, which the ratio of composition is depicted as ‘‘COD:N:P:Fe = 100:5:1:0.5’’. After 27 days of incubation, the removal efficiency of Fe-EDTA and COD is 100% and 92.0%, correspondingly. In addition, the raw wastewater of EDTA was controlled at higher concentrations, compared with the concentration of 50 mg/L, to cultivate activated sludge, the removal efficiency of Fe-EDTA and COD decreased when the initial concentration was higher than
Items
Con. of pre-treatment (mg/L)
Emission standards (mg/L)
EDTA COD Cu Fe Mn Ni Pb pH
850 38,000 1895 126 1050 150.3 588 9.15–9.75
60.0 100.0 3.0 10.0 10.0 1.0 1.0
*
Con. = concentration.
Table 4 Metal-ions concentration of wastewater after coagulation of ferrite powder and ferric trichloride at pH = 5–10. Element
Con. of post-treatment ferrite powder addition
Con. of post-treatment ferric trichloride addition
1 (mg/L)
3 (mg/L)
5 (mg/L)
1 (mg/L)
3 (mg/L)
5 (mg/L)
(pH = 5) Cu Fe Mn Ni Pb
992 109 4 48 37
1655 107 19 56 42
1401 120 20 114 47
575 474 17 22 25
823 103 14 20 31
807 96 13 16 23
(pH = 6) Cu Fe Mn Ni Pb
1453 28 18 74 42
1453 76 21 59 35
1657 123 29 62 34
388 59 3 5 29
556 88 4 4 22
815 12 12 15 28
(pH = 7) Cu Fe Mn Ni Pb
1601 117 12 63 46
1005 159 13 93 24
1537 114 18 55 25
596 235 6 16 36
575 269 6 59 28
613 211 8 7 29
(pH = 8) Cu Fe Mn Ni Pb
1585 18 8 61 42
1405 81 21 55 38
1274 118 25 63 39
601 380 6 8 25
753 109 9 17 32
666 14 8 8 18
(pH = 9) Cu Fe Mn Ni Pb
1712 16 22 66 39
1671 82 18 54 12
1437 133 13 51 34
778 182 8 21 35
878 148 16 37 21
712 38 7 13 30
(pH = 10) Cu Fe Mn Ni Pb
1816 27 23 73 45
1550 72 18 55 34
1112 68 16 37 28
693 327 5 14 29
790 119 11 16 29
624 376 7 11 27
Table 2 Comparison on identification result of three systems. Identification system
Name of bacteria
Precision (%)
API 20NE
Burkhol cepacia
Precisely matched
Biolog
Alcaligenes calcoaceticus Acinetobacter baumannii Pseudomonas stutzer Photobacterium logei
97 95 Similar to 100 Similar to 100
Pseudomonas aeruginosa Pseudomonas putida Achromobacter baumannii Chromobacterium violaceum Pseudomonas stutzer Sphingobacterium paucimobilis Achromobacter species
Above Above Above Above Above Above Above
Phoenix ID 100
95 95 95 95 95 95 95
Table 5 Removal efficiency of contaminant in wastewater on chemical coagulation processes. Items
Con. of pre-treatment (mg/L)
Con. of post-treatment (mg/L)
Removal efficiency (%)
EDTA COD Cu Fe Mn Ni Pb pH
850 38,000 1,895 126 1,050 150.3 588 9.15–9.75
850 22,233 388 59 3 5 29 6.0 ± 0.2
0 41.1 79.5 53.2 99.7 96.7 95.1
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Fig. 1. Effects of four diluted concentrations of wastewater (50, 100, 200, and 500 mg/L) on the EDTA degradation efficiency by strain YL-6.
Fig. 2. Effects of four diluted concentrations of wastewater (50, 100, 200, and 500 mg/L) on the COD degradation efficiency by strain YL-6.
100 mg/L of EDTA. According to the results of Figs. 1 and 2, the removal efficiency of Fe-EDTA and COD at 100 mg/L is 100% and 45.93%, respectively. With increase of wastewater concentration, the removal efficiency of Fe-EDTA and COD at 200 mg/L is 95.29% and 16.30%, correspondingly. Moreover, the removal efficiency of Fe-EDTA and COD at 500 mg/L is 60.65% and 3.17%, correspondingly. The higher availability of the carbon source may affect enzyme activity to inhibit the growth rate of the strain. A series of similar reports about substrate inhibition of cell growth were also found by Sugimori and Diez’s study which showed the EDTA removal efficiency was ranged from 1% to 9% when the initial concentration of EDTA controlled at 80– 320 mg/L after 7 days of incubation during continuous treatment (Diez et al., 2005). Thus, strain YL-6 has the most efficient EDTA degrading ability of the tested strains during lower concentration of EDTA. 3.4. Continuous treatment Figs. 3 and 4 show that the degradation efficiency and residual concentration with experimental and control groups during continuous treatment. When the initial concentration of EDTA was controlled at 166 mg/L before adding nutrients to cultivate activated sludge, in which the ratio of composition did also follow with batch process, and hydraulic retention time (HRT) was controlled for 8 h. The strain YL-6 grew slowly for first 7 days, the cell grew gradually stabilized after 14 days. After 22 days, the removal efficiency of EDTA and COD for experimental group was 71.5% and 62.6%, correspondingly. According to Diez’s study (Diez et al.,
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Fig. 3. Biodegradation of EDTA during continuous treatment with experimental (with addition of bacterial agent) and control (without addition of bacterial agent) groups by strain YL-6.
Fig. 4. Biodegradation of COD during continuous treatment with experimental (with addition of bacterial agent) and control (without addition of bacterial agent) groups by strain YL-6.
2005), EDTA removal efficiency was lower than 25% after 600 days of incubation during continuous treatment. Comparisons of previous study found by Diez (Diez et al., 2005), the present study found a threefold greater EDTA removal efficiency than Diez’s study. Moreover, the results of experimental and control groups were similar because the concentration of wastewater pulsed simultaneously with addition of raw wastewater, which resulted in lower EDTA removal performance of strain YL-6. Therefore, the batch process was more suited for EDTA biodegradation than continuous process. 4. Conclusions An efficient EDTA degrading strain, strain YL-6, was isolated and characterized. On the basis of these characteristics and its 16SrDNA sequence, strain YL-6 was identified as B. cepacia and designated as B. cepacia YL-6. The original COD for PC board wastewater was approximately 38,000–40,000 mg/L before biodegradation process. The removal efficiency of EDTA and COD are 100% and 92.0% after 27 days of incubation in batch treatment and 71.5% and 62.6% after 22 days in continuous bioreactor, respectively. The results showed that the batch process was more suited for EDTA biodegradation. Acknowledgement This study was guided by the late professor Hung-Yuan Fang and carried out by his team of environmental and microbiological
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researchers. Without his meticulous instruction and positive feedback, this study should not be accomplished.
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Jing, G., Zhou, J., Zhou, Z., Lin, T., 2012. Reduction of Fe(III)-EDTA in a NOx scrubbing solution by magnetic Fe3O4 chitosan microspheres immobilized mixed culture of iron-reducing bacteria. Bioresour. Technol. 108, 169–175. Kavitha, S., Kumar, S.A., Yogalakshmi, K.N., Kaliappan, S., Banu, R.J., 2013. Effect of enzyme secreting bacterial pretreatment on enhancement of aerobic digestion potential of waste activated sludge interceded through EDTA. Bioresour. Technol. 150, 210–219. Kavitha, S., Jayashree, C., Kumar, S.A., Yeom, I.T., Banu, J.R., 2014. The enhancement of anaerobic biodegradability of waste activated sludge by surfactant mediated biological pretreatment. Bioresour. Technol. 168, 159–166. Lauff, J.J., Steele, D.B., Coogan, L.A., Breitfeller, J.M., 1990. Degradation of the ferric chelate of EDTA by a pure culture of an Agrobacterium ap. Appl. Environ. Microbiol. 56, 3346–3353. Liu, Y., Chen, G.H., Paul, E., 1998. Effect of the So/Xo ratio on energy uncoupling in substrate-sufficient nature of activated sludge. Water Res. 32, 2883–2888. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Loyaux-Lawniczak, S., Douch, J., Behra, P., 1999. Optimisation of the analytical detection of EDTA by HPLC in natural waters. Anal. Bional. Chem. 364, 727–731. Lu, M., Wei, X., 2011. Treatment of oilfield wastewater containing polymer by the batch activated sludge reactor combined with a zero valent iron/EDTA/air system. Bioresour. Technol. 102, 2555–2562. Mansilla, H.D., Bravo, C., Ferreyra, R., Litter, M.I., Jardim, W.F., Lizama, C., Freer, J., Fernández, J., 2006. Photocatalytic EDTA degradation on suspended and immobilized TiO2. J. Photochem. Photobiol. 181, 188–194. May, C.C., Young, L., Worsfold, P.J., Heath, S., Bryan, N.D., Keith-Roach, M.J., 2012. The effect of EDTA on the groundwater transport of thorium through sand. Water Res. 39, 4870–4882. Narola, B., Singh, A.S., Mitra, M., Santhakumar, P.R., Chandrashekhar, T.G., 2011. A validated reverse phase HPLC method for the determination of disodium EDTA in meropenem drug substance with UV-detection using precolumn derivatization technique. Anal. Chem. Insights 6, 7–14. O’Hara, M.C., 2006. Evaluation of the Phoenix 100 ID/AST system and NID panel for identification of enterobacteriaceae, vibrionaceae, and commonly isolated nonenteric gram-negative bacilli. J. Clin. Microbiol. 44, 928–933. Piyawadee, S., Alissara, R., 2011. Biological hydrogen production from sweet sorghum syrup by mixed cultures using an anaerobic sequencing batch reactor (ASBR). Int. J. Hydrogen Energy 36, 8765–8773. Rodriguez, J.B., Mutis, A., Yeber, M.C., Freer, J., Baeza, J., Mansilla, H.D., 1999. Chemical degradation of EDTA and DTPA in a totally chlorine free (TCF) effluent. Water Sci. Technol. 40, 267–272. Russell, J.B., 2007. The energy spilling reactions of bacteria and other organisms. J. Mol. Microbiol. Biotechnol. 13, 1–11. Skoog, D.A., West, D.M., Holler, F.J., Crouch, S.R., Kinsel, G.R., 2003. Fundamentals of Analytical Chemistry, 8th ed. Brooks/Cole, Belmont, California, USA. Voglar, D., Lestan, D., 2012. Electrochemical treatment of spent solution after EDTAbased soil washing. Water Res. 46, 1999–2008. Wragg, P., Randall, L., Whatmore, A.M., 2014. Comparison of Biolog GEN III MicroStation semi-automated bacterial identification system with matrixassisted laser desorption ionization-time of flight mass spectrometry and 16S ribosomal RNA gene sequencing for the identification of bacteria of veterinary interest. J. Microbiol. Methods 105, 16–21. Zhang, J., Zhang, Y., Quan, X., Chen, S., 2015. Enhancement of anaerobic acidogenesis by integrating an electrochemical system into an acidogenic reactor: effect of hydraulic retention times (HRT) and role of bacteria and acidophilic methanogenic Archaea. Bioresour. Technol. 179, 43–49.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Application of ethylene diamine tetra acetic acid degrading bacterium Burkholderia cepacia on biotreatment process Wei-Ting Chen a,⇑, Shu-Min Shen b, Chi-Min Shu b a b
Department of Cosmetic Application & Management, St. Mary’s Junior College of Medicine, Nursing and Management, Yilan 26644, Taiwan, ROC Department of Safety, Health, and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan, ROC
h i g h l i g h t s EDTA can be properly biodegraded by Burkholderia cepacia. Continuous process and batch treatment were used in EDTA wastewater degradation. The strain YL-6 was identified as Burkholderia cepacia. The results showed that batch treatment was more suited for EDTA biodegradation. Removal efficiency of EDTA and COD mostly researched to 100% by batch treatment.
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 25 June 2015 Keywords: Ethylene diamine tetra acetic acid (EDTA) Burkholderia cepacia Batch degradation Nutrients Activated sludge
a b s t r a c t Ethylene diamine tetra acetic acid (EDTA), the effluent of secondary biotreatment units, can be properly biodegraded by Burkholderia cepacia. Through batch degradation of EDTA, the raw wastewater of EDTA was controlled at 50 mg/L, and then nutrients was added in diluted wastewater to cultivate activated sludge, which the ratio of composition is depicted as ‘‘COD:N:P:Fe = 100:5:1:0.5’’. After 27 days, the removal efficiency of Fe-EDTA and COD was 100% and 92.0%, correspondingly. At the continuous process, the raw wastewater of EDTA was dictated at 166 mg/L before adding nutrients to cultivate activated sludge, in which the ratio of composition did also follow with batch process. After 22 days, the removal efficiency of Fe-EDTA and COD for experimental group was 71.46% and 62.58%, correspondingly. The results showed that the batch process was more suited for EDTA biodegradation. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Ethylene diamine tetra acetic acid (EDTA) is extensively used in dyeing industry, textile industry, paper industry, metal manufacturing, printing industry, cosmetic industry, pharmaceutical industry, food industry, and nuclear facilities (Diez et al., 2005; Mansilla et al., 2006; Balaji et al., 2007; Harraz et al., 2010; Lu and Wei, 2011; May et al., 2012; Voglar and Lestan, 2012). In addition, EDTA is a chelating agent which is employed to sequester metal ions, such as Cu2+ and Fe3+. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity. Moreover, the chelating stability constant of EDTA with Cu2+ and Fe3+ usually reaches up 1015, so the highly stable chelator, EDTA, is widely applied in wastewater treatment processes for containments diminishing. In natural waters or industrial wastewater, EDTA exists mainly in the form of Ca-EDTA, Zn-EDTA and Ni-EDTA ⇑ Corresponding author. Tel.: +886 3 989 7396; fax: +886 3 989 7386. E-mail address: [email protected] (W.-T. Chen). http://dx.doi.org/10.1016/j.biortech.2015.06.099 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
(Mansilla et al., 2006). Complexes with high stability constants, such as Fe(III)-EDTA, are not readily removed and are responsible for the EDTA remaining in growth experiments (Chen et al., 2005; Jing et al., 2012; Dong et al., 2012; Chandrashekhar et al., 2013; Kavitha et al., 2013; Zhang et al., 2015). Moreover, EDTA’s low biodegradability is responsible for the presence of several complexes in sewage effluents, freshwater, and ground-water. Although EDTA is a useful chelating agent, it causes an environmental pollution problem because of its high stability. Once metal ions are being bound by EDTA, the chelate cannot degrade readily. Therefore, several attempts about degradation of EDTA have been made, and some explored methods, such as via electrochemical process (Voglar and Lestan, 2012; Zhang et al., 2015), photodegradation (Lauff et al., 1990), activated-sludge adsorption (Chen et al., 2005; Kavitha et al., 2013, 2014; Ginkel et al., 1997; Rodriguez et al., 1999), and biological and UV/O3 degradation (Glass and Popovic, 2005), are developed technologies for EDTA removal. The procedure of EDTA removal was that the raw wastewater was pretreated by chemical coagulation to remove
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heavy metals, and then added the bioaugmentation agent – Burkholderia cepacia to biodegrade the target compound – EDTA. So far many researches are investigated as mentioned in the above descriptions, and there are many cases regarding the biological degradation on wastewater treatment in Taiwan. In practice, the biological degradation is the most economic and environmental-friendly way in aspect of economic benefits and environmental qualities. According to the previous researches, these six EDTA degradation bacteria, such as Agrobacterium sp., activated sludge, bacterial strain DSM 9103, bacterial consortia strain BNC1, soil consortia and agricultural sediment, surface and subsurface soil consortia, are usefully employed in treatment of EDTA degradation. The aim of this study was to apply the screened EDTA degradation bacteria, B. cepacia YL-6, which are screened from activated sludge in a wastewater plant, to undergo the model testing of EDTA degradation. The target of this study is normally on wastewater from a PC board plant in Taiwan. Prior to the treatment of EDTA degradation, the chemical coagulation is a pre-treated method to remove heavy metals of wastewater. After the above process, activated sludge in continuous process was used to eliminate EDTA and COD efficiently. Furthermore, experimental and controlled groups are applied in continuous process to acquire efficient group. Apart from the continuous process, the batch treatment was also employed in this study for degradation of EDTA. The intention of this study was to detect the optimal methodology of EDTA wastewater degradation from biodegradation experiments of continuous process and batch treatment. 2. Methods 2.1. Source of bacteria The attempt of this study was to screen EDTA degradation bacteria, B. cepacia YL-6, from activated sludge in a wastewater plant at Douliou industrial park (Douliou, Yunlin, Taiwan), which were applied to degrade EDTA, and then these bacteria were cultivated with different nutrients, such as Fe-EDTA-based medium, ethylamine-based medium, and Fe-EDTA/ethylamine-based medium, before degradation. 2.2. Component of liquid medium and isolation of bacteria After the target bacteria, B. cepacia YL-6, was scanned, a minimal saline agar medium (Fe-EDTA 1000 mg; ethylamine 1%; ethylenediamine 1%; potassium acetate 250 mg; (NH2CO + (NH4)2SO4) 2000 mg (ratio of 1:1, w/w); KH2PO4 1000 mg; NaCl 100 mg; CaCl2 25 mg; FeSO47H2O 20 mg; MgSO47H2O 20 mg; ZnSO46H2O 10 mg; CuSO46H2O 10 mg; MnSO44H2O 5 mg; CoCl26H2O 0.5 mg; Na2MoO47H2O 0.5 mg) was employed to cultivate the target bacteria (Chen et al., 2005). The components of liquid medium are shown in Table 1 (Chen et al., 2005; Jing et al., 2012). Moreover, Fe-EDTA and urea were the majority of carbon resource and nitrogen resource, respectively. The main phosphate resource are ammonium sulfate and monopotassium phosphate, and potassium acetate (CH3COOK) is regarded as the co-metabolic carbon source. 1.5–2.0% of agar is added in plate before agar melting, sterilization, and agar congealment to be a culture medium. An agar plate is then used to purify bacterial specie and conserve bacterial strain before identification of mycology. 2.3. Analysis methods 2.3.1. Identification of target bacteria There are three methods, including API 20NE, Biolog, and Phoenix ID 100, which are used to determine the target bacteria.
Table 1 Component of liquid medium for bacteria cultivation (Chen et al., 2005; Jing et al., 2012). Composition
Concentration (mg/L)
Percentage (%)
Fe-EDTA Potassium acetate (NH2)CO + (NH4)2SO4 KH2PO4 NaCl CaCl2 FeSO47H2O MgSO47H2O ZnSO46H2O CuSO46H2O MnSO44H2O CoCl46H2O NaMoO47H2O Ethylamine
1000 250 2000 (1:1) 1000 100 25 20 20 10 10 5 0.5 0.5 49
22.27 5.57 44.54 22.27 2.22 0.57 0.45 0.45 0.23 0.23 0.11 0.001 0.001 1.09
Components of the API 20NE strip include microtubes containing dehydrated media and substrates. The conventional tests are described as media microtubes inoculated with a bacterial suspension which reconstitutes the media. After incubation process, indicator systems or the addition of reagents are employed to monitor the metabolic end products. The substrate microtubes consist of assimilation tests which are inoculated with a minimal medium. The bacteria will grow if they are capable of utilizing the corresponding substrate (Glass and Popovic, 2005). A single colony was chosen and emulsified into ‘inoculating fluid A’ (Biolog) for subsequent inoculation on to the MicroPlate test plate (Biolog) for the most cases. According to the manufacturer’s instructions, more particular organisms, especially on capnophilic strains, were cultivated on alternative media, and inocula was prepared to a specified transmittance using a turbidimeter, as specified in the user guide. In each isolate process, according to growth characteristics, 100 lL of the cell suspension was inoculated into each well of the MicroPlate, where a multichannel pipette was used with incubation at 37 °C for 20 h and aerobic condition of 7.5% CO2. MicroPlates were identified by the MicroStation semiautomated reader after 20 h and results were demonstrated by the identification system software (GEN III database, version 5.2.1). If incubation period is over 20 h, the system indicated which isolates could not be identified. Hence, such isolates were re-incubated and re-read between 3 and 6 h later (Holmes et al., 1994; Wragg et al., 2014). The Phoenix 100 ID/AST system is innovated by Becton Dickinson Cooperation (Becton Dickinson Co., Sparks, MD, USA), which is an automated system for identification (ID) and antimicrobial susceptibility testing (AST). The strains were stayed at 70 °C defibrinated sheep blood and were undergone three times on 5% sheep blood agar before testing. All panels were processed according to the manufacturer’s directions. Each panel has to be inoculated within 2 h after its foil pouch is opened, and the panels need to be loaded into the instrument within 30 min of inoculation. Only cotton-tipped swabs or wooden applicators are acceptable for preparation of the suspensions. A suspension of each 24-h-old isolate was made in the Phoenix 100 ID/AST broth to match the turbidity of a 0.5 McFarland standard by using a CrystalSpec nephelometer (Becton Dickinson) (O’Hara, 2006; Giani et al., 2012). 2.3.2. Measurement of bacteria growth concentration The Lowry protein assay method for protein concentration determination is one of the most venerable and extensively-used protein assays. The Lowry method was first described in 1951 by Lowry et al. (1951). The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret
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test) with the oxidation of aromatic protein residues. The Lowry method is best employed with protein concentrations of 0.01– 1.0 mg/mL and is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The concentration of the reduced Folin reagent is measured by absorbance at 660 nm (Lowry et al., 1951). As a result, the total concentration of protein in the sample can be detected by using this method, and the amount of bacteria consequently is the concentration of protein which is divided by 0.6. 2.3.3. Measurement of EDTA concentration According to previous studies (Loyaux-Lawniczak et al., 1999; Cagnasso et al., 2007; Narola et al., 2011), HPLC was broadly used to determine the concentration of EDTA in numerous fields, such as pharmacy, natural water, and drink. As the result, Perkin Elmer Series 200 HPLC is used in quantitative analysis of the EDTA concentration in this study. The analysis conditions are listed in the following sentences: chromatographic column (SUPELCO 516-C18 5 lm, inner radius 250 mm 4.6 mm), solvent (66% of methanol), flow rate (0.8 mL min 1), detecting wavelength (254 nm), and duration time (2.7 min). 2.3.4. Measurement of chemical oxygen demand (COD) test The COD test is widely employed in environmental chemistry to indirectly measure the amount of organic compounds in water, which is adopting the method, NIEA W517.50B, from Environmental Analysis Laboratory, Executive Yuan, Taiwan, ROC. Most applications of COD determine the amount of organic pollutants found in surface water (e.g., lakes and rivers) or wastewater, making COD a useful measure of water quality. It is expressed in milligrams per liter (mg/L) also referred to as ppm (parts per million), which indicates the mass of oxygen consumed per liter of solution (Environmental Analysis Laboratory, 2009). 2.3.5. Analysis of component’s metal ion concentration The metal ion concentration in the aqueous phase and the ores were available to be determined by an inductively coupled plasma atomic emission spectrometer (ICP-AES; Optima, 5100DV). For the partition studies, equal volumes (10 mL) of the aqueous and organic phases were shaken at room temperature (25 ± 3 °C) for 5 min to ensure complete equilibration. The two phases were separated, and a suitable aliquot of the aqueous phase was assayed for the metal ion concentration. Based on three tests, the value of 95% extraction for metal ion was associated with a variation coefficient of ±3% (Skoog et al., 2003). 2.4. Pre-treatment test of wastewater The raw wastewater sample was obtained from a PC board plant in Taiwan industrial park, so it consisted of various kinds of heavy metals. Before EDTA removal procedure, chemical coagulation was pretreated to remove heavy metals in raw wastewater. The chemical coagulation processes are listed in following sentences: 1, 3, 5, 7, or 10 g of ferric trichloride or ferrite powder was added in 1 L raw wastewater, respectively, to coagulate heavy metals. After the addition of ferric trichloride, the mixing liquid was stirred at fast stir (110 rpm, 2 min) and then was stirred at slow stir (22 rpm, 20 min). Subsequently, supernatant liquid and deposit can be separated by centrifugation process. The next step is that supernatant liquid mixed with sodium hydroxide for its pH value adjustment to remove those residual heavy metals which is exited in supernatant liquid after addition of ferric trichloride. As a result, ICP-AES was employed to analyze the concentration of wastewater which was underwent the pre-treatment test.
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2.5. Batch treatment Sequencing batch reactors (SBR) are industrial processing tanks for the treatment of wastewater. SBR treat wastewater such as sewage or output from anaerobic digesters or mechanical biological treatment facilities in batches. Oxygen is bubbled through the wastewater to reduce biochemical oxygen demand (BOD) and COD to make them suitable for discharge into sewers or for use on land (Arnaldo et al., 2007; Piyawadee and Alissara, 2011; Adriana et al., 2012). For sequencing batch reactor activated sludge process, the raw wastewater was controlled at 50 mg/L, and then nutrients were added in diluted wastewater to cultivate activated sludge, in which the ratio of composition is depicted as ‘‘COD:N:P:Fe = 100:5:1:0.5’’. Moreover, cultivation cycle is based on 1 day, 23 h aeration and 1 h sedimentation, and the sludge age is controlled 12 days. In addition, cultivation conditions are controlled as the following sentences during cultivation period: pH value at 7.0 ± 0.2, temperature at 25 °C, dissolved oxygen (DO) at 1–3 mg/L, sludge volume index (SVI) at 80–120 mL/g, and BOD–MLSS loading at 0.2–0.4 kg BOD kg/MLSS. 2.6. Continuous treatment As different from the SBR test, continuous process was employed in activated sludge cultivation to identify the optimal method of cultivation. The advantages of continuous culture include: (1) the growth condition is mainly substrate-limiting so that substrate-abundant uncoupling (Liu et al., 1998) could not occur; (2) maintenance energy requirements are balanced because cell compositions (including enzymes) are in a stabilized state (Russell, 2007). In view of practical applications, information on how continuous flow activated sludge yield is affected by the influent of substrate is also valuable in formulating operational plans, thereby to improve treatment plant performance (Chong et al., 2011). The ratio of nutrients which was employed in a continuous system was the same as the ratio of nutrients in a batch reactor system. Additionally, the speed of nutrient addition was controlled at 1.3 L/h, and hydraulic retention time was maintained at 8 h. Moreover, the cultivation conditions employed in a continuous system was also similar with the cultivation conditions in a batch reactor system. As a result, one of above mentioned systems can be identified as the optimal method to cultivate activated sludge. 2.7. Experimental and control tests The addition of bacterial agent, B. cepacia YL-6, was used in experimental test to certify the efficiency of B. cepacia YL-6. In contrast with experimental test, the test (without addition of bacterial agent) was designed as control test to observe the comparison of experimental and control tests. These two tests could absolutely examine how bacterial agent worked on metal-EDTA pollutants removal. 3. Results and discussion 3.1. Identification of EDTA-degradable bacteria This study adopted three systems, API 20NE, Biolog, and Phoenix ID 100, to identify biological species of the bacteria. Strain YL-6 was rod-shaped and ranged from 1 to 3 lm in length. The Biolog identification system showed a similarity greater than 99%. The identification result of three systems is depicted in Table 2, and precision of each result reaches above 95%. On the basis of these characteristics and its 16S rDNA sequence, strain
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YL-6 was identified as B. cepacia and designated B. cepacia YL-6. After biological species identification, the bacteria were cultivated with various kinds of carbon sources for EDTA degradation.
Table 3 Original concentration of contaminant in wastewater before chemical coagulation processes (Environmental Protection Administration, 2014).
3.2. Effects of ferric trichloride on chemical coagulation processes The original COD and EDTA of the PC board wastewater were approximately 38,000–40,000 mg/L and 850 mg/L, correspondingly. Moreover, wastewater consisted of various kinds of heavy metals, such as cupper (Cu), nickel (Ni), manganese (Mn), lead (Pb), and ferrite (Fe), and the concentration of above metals are 1,895, 150.3, 1050, 588, and 126 mg/L, respectively, as shown in Table 3. Ferrite powder and ferric trichloride are used as coagulation agents with three rations addition, 1, 3, and 5 mg/L, to remove the above mentioned heavy metals before metal-EDTA degradation. In Table 4, increasing the dosage of ferric powder and ferric trichloride (1 g/L) had no effect on the improvement of heavy metals removal at various pH values from the wastewater. Furthermore, tests of heavy metals removal at different pH values are depicted in Table 4, which show better efficiency of heavy metals removal with ferric trichloride addition. Thus, 1 g/L of ferric trichloride in the pH 6 was determined as the appropriate dosage for PC board wastewater pre-treatment. After a variety of tests as shown in Table 4, Table 5 demonstrates that the final COD is 22,233 mg/L, and the removal efficiency is 41.4% at a solution pH of 6 with 1 g/L of ferric trichloride. In addition, Table 5 shows that the removal efficiency of heavy metals, Cu, Fe, Mn, Ni, and Pb, were 79.5%, 53.2%, 99.7%, 96.7%, and 95.1%, respectively. Because of the high stability of EDTA, EDTA cannot degrade easily once metal ions are being bound by EDTA. As the result, the EDTA concentration has no effect during chemical coagulation processes. Therefore, bacterial degradation approaches (batch and continuous treatment with experimental and control tests) were applied to degrade metal-EDTA pollutants after chemical coagulation processes. 3.3. Batch treatment Figs. 1 and 2 show that the degradation efficiency and residual concentration with four diluted concentrations of wastewater (50, 100, 200, and 500 mg/L) during batch treatment. When the initial concentration of EDTA was controlled at 50 mg/L, and then nutrients was added in diluted wastewater to cultivate activated sludge, which the ratio of composition is depicted as ‘‘COD:N:P:Fe = 100:5:1:0.5’’. After 27 days of incubation, the removal efficiency of Fe-EDTA and COD is 100% and 92.0%, correspondingly. In addition, the raw wastewater of EDTA was controlled at higher concentrations, compared with the concentration of 50 mg/L, to cultivate activated sludge, the removal efficiency of Fe-EDTA and COD decreased when the initial concentration was higher than
Items
Con. of pre-treatment (mg/L)
Emission standards (mg/L)
EDTA COD Cu Fe Mn Ni Pb pH
850 38,000 1895 126 1050 150.3 588 9.15–9.75
60.0 100.0 3.0 10.0 10.0 1.0 1.0
*
Con. = concentration.
Table 4 Metal-ions concentration of wastewater after coagulation of ferrite powder and ferric trichloride at pH = 5–10. Element
Con. of post-treatment ferrite powder addition
Con. of post-treatment ferric trichloride addition
1 (mg/L)
3 (mg/L)
5 (mg/L)
1 (mg/L)
3 (mg/L)
5 (mg/L)
(pH = 5) Cu Fe Mn Ni Pb
992 109 4 48 37
1655 107 19 56 42
1401 120 20 114 47
575 474 17 22 25
823 103 14 20 31
807 96 13 16 23
(pH = 6) Cu Fe Mn Ni Pb
1453 28 18 74 42
1453 76 21 59 35
1657 123 29 62 34
388 59 3 5 29
556 88 4 4 22
815 12 12 15 28
(pH = 7) Cu Fe Mn Ni Pb
1601 117 12 63 46
1005 159 13 93 24
1537 114 18 55 25
596 235 6 16 36
575 269 6 59 28
613 211 8 7 29
(pH = 8) Cu Fe Mn Ni Pb
1585 18 8 61 42
1405 81 21 55 38
1274 118 25 63 39
601 380 6 8 25
753 109 9 17 32
666 14 8 8 18
(pH = 9) Cu Fe Mn Ni Pb
1712 16 22 66 39
1671 82 18 54 12
1437 133 13 51 34
778 182 8 21 35
878 148 16 37 21
712 38 7 13 30
(pH = 10) Cu Fe Mn Ni Pb
1816 27 23 73 45
1550 72 18 55 34
1112 68 16 37 28
693 327 5 14 29
790 119 11 16 29
624 376 7 11 27
Table 2 Comparison on identification result of three systems. Identification system
Name of bacteria
Precision (%)
API 20NE
Burkhol cepacia
Precisely matched
Biolog
Alcaligenes calcoaceticus Acinetobacter baumannii Pseudomonas stutzer Photobacterium logei
97 95 Similar to 100 Similar to 100
Pseudomonas aeruginosa Pseudomonas putida Achromobacter baumannii Chromobacterium violaceum Pseudomonas stutzer Sphingobacterium paucimobilis Achromobacter species
Above Above Above Above Above Above Above
Phoenix ID 100
95 95 95 95 95 95 95
Table 5 Removal efficiency of contaminant in wastewater on chemical coagulation processes. Items
Con. of pre-treatment (mg/L)
Con. of post-treatment (mg/L)
Removal efficiency (%)
EDTA COD Cu Fe Mn Ni Pb pH
850 38,000 1,895 126 1,050 150.3 588 9.15–9.75
850 22,233 388 59 3 5 29 6.0 ± 0.2
0 41.1 79.5 53.2 99.7 96.7 95.1
W.-T. Chen et al. / Bioresource Technology 193 (2015) 357–362
Fig. 1. Effects of four diluted concentrations of wastewater (50, 100, 200, and 500 mg/L) on the EDTA degradation efficiency by strain YL-6.
Fig. 2. Effects of four diluted concentrations of wastewater (50, 100, 200, and 500 mg/L) on the COD degradation efficiency by strain YL-6.
100 mg/L of EDTA. According to the results of Figs. 1 and 2, the removal efficiency of Fe-EDTA and COD at 100 mg/L is 100% and 45.93%, respectively. With increase of wastewater concentration, the removal efficiency of Fe-EDTA and COD at 200 mg/L is 95.29% and 16.30%, correspondingly. Moreover, the removal efficiency of Fe-EDTA and COD at 500 mg/L is 60.65% and 3.17%, correspondingly. The higher availability of the carbon source may affect enzyme activity to inhibit the growth rate of the strain. A series of similar reports about substrate inhibition of cell growth were also found by Sugimori and Diez’s study which showed the EDTA removal efficiency was ranged from 1% to 9% when the initial concentration of EDTA controlled at 80– 320 mg/L after 7 days of incubation during continuous treatment (Diez et al., 2005). Thus, strain YL-6 has the most efficient EDTA degrading ability of the tested strains during lower concentration of EDTA. 3.4. Continuous treatment Figs. 3 and 4 show that the degradation efficiency and residual concentration with experimental and control groups during continuous treatment. When the initial concentration of EDTA was controlled at 166 mg/L before adding nutrients to cultivate activated sludge, in which the ratio of composition did also follow with batch process, and hydraulic retention time (HRT) was controlled for 8 h. The strain YL-6 grew slowly for first 7 days, the cell grew gradually stabilized after 14 days. After 22 days, the removal efficiency of EDTA and COD for experimental group was 71.5% and 62.6%, correspondingly. According to Diez’s study (Diez et al.,
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Fig. 3. Biodegradation of EDTA during continuous treatment with experimental (with addition of bacterial agent) and control (without addition of bacterial agent) groups by strain YL-6.
Fig. 4. Biodegradation of COD during continuous treatment with experimental (with addition of bacterial agent) and control (without addition of bacterial agent) groups by strain YL-6.
2005), EDTA removal efficiency was lower than 25% after 600 days of incubation during continuous treatment. Comparisons of previous study found by Diez (Diez et al., 2005), the present study found a threefold greater EDTA removal efficiency than Diez’s study. Moreover, the results of experimental and control groups were similar because the concentration of wastewater pulsed simultaneously with addition of raw wastewater, which resulted in lower EDTA removal performance of strain YL-6. Therefore, the batch process was more suited for EDTA biodegradation than continuous process. 4. Conclusions An efficient EDTA degrading strain, strain YL-6, was isolated and characterized. On the basis of these characteristics and its 16SrDNA sequence, strain YL-6 was identified as B. cepacia and designated as B. cepacia YL-6. The original COD for PC board wastewater was approximately 38,000–40,000 mg/L before biodegradation process. The removal efficiency of EDTA and COD are 100% and 92.0% after 27 days of incubation in batch treatment and 71.5% and 62.6% after 22 days in continuous bioreactor, respectively. The results showed that the batch process was more suited for EDTA biodegradation. Acknowledgement This study was guided by the late professor Hung-Yuan Fang and carried out by his team of environmental and microbiological
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researchers. Without his meticulous instruction and positive feedback, this study should not be accomplished.
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