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Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofurancontaminated water ab
S. Laocharoen , P. Plangklang
cd
& A. Reungsang
bce
a
International Postgraduate Programs in Environmental Management, Chulalongkorn University, Bangkok, Thailand b
National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok, Thailand c
Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand
d
Groundwater Research Center, Khon Kaen University, Khon Kaen, Thailand
e
Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand Accepted author version posted online: 01 Mar 2013.Published online: 25 Mar 2013.
To cite this article: S. Laocharoen, P. Plangklang & A. Reungsang (2013) Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofuran-contaminated water, Environmental Technology, 34:18, 2587-2597, DOI: 10.1080/09593330.2013.781226 To link to this article: http://dx.doi.org/10.1080/09593330.2013.781226
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Environmental Technology, 2013 Vol. 34, No. 18, 2587–2597, http://dx.doi.org/10.1080/09593330.2013.781226
Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofuran-contaminated water S. Laocharoena,b , P. Plangklangc,d and A. Reungsangb,c,e∗ a International
Postgraduate Programs in Environmental Management, Chulalongkorn University, Bangkok, Thailand; b National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok, Thailand; c Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand; d Groundwater Research Center, Khon Kaen University, Khon Kaen, Thailand; e Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand
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(Received 11 January 2012; final version received 18 February 2013 ) This study investigated the utilization of agricultural matrices as the support materials for cell immobilization to improve the technique of bioremediation. Coir, bulrush, banana stem and water hyacinth stem in both delignified and undelignified forms were used to immobilize Burkholderia cepacia PCL3 in bioremediation of carbofuran at 5 mg l−1 in synthetic wastewater. Undelignified coir was found to be the most suitable support material for cell immobilization, giving the short half-life of carbofuran of 3.40 d (2.8 times shorter than the treatments with free cells). In addition, it could be reused three times without a loss in ability to degrade carbofuran. The growth and degradation ability of free cells were completely inhibited at the initial carbofuran concentrations of 250 mg l−1 , while there was no inhibitory effect of carbofuran on the immobilized cells. The results indicated a great potential for using the agricultural matrices as support material for cell immobilization to improve the overall efficiency of carbofuran bioremediation in contaminated water by B. cepacia PCL3. Keywords: agricultural residues; bioaugmentation; Burkholderia cepacia PCL3; carbofuran; immobilization
Introduction Carbofuran (2,3-dihydro-2,2 dimethylbenzofuran-7-yl methylcarbamate) is a broad-spectrum insecticide widely used in agriculture to control insects and nematodes on contact or after ingestion. [1] It is of environmental concern because it is soluble in water and highly mobile in soil, resulting in a high potential for groundwater contamination. [2,3] Carbofuran has a chronic affect on aquatic organisms through cholinesterase inhibition, neurotoxicity and reproductive effects. [4] Acute uptake of carbofuran through accidental exposure can cause acute toxicities and fatalities in humans and other organisms. [5] Therefore, the removal of carbofuran from contaminated environments is needed. A variety of pretreatment methods have been reported for carbofuran degradation. In particular, bioremediation has gained recent attention due to the advantages of ease of operation and low operating cost, which make this technique technically and economically feasible in large-scale operations. One of the effective bioremediation techniques used to remediate pesticide contamination is bioaugmentation, which is the addition of microorganisms to aid dissipation of the pesticide. Immobilization of the microorganisms on suitable support materials before augmentation of the contaminated matrices has been reported to improve bioaugmentation efficiency. [6,7] ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
The advantages of immobilized cells over free cells include the allowance of high cell loading, more tolerance against toxic compounds, [8] the removal of the biomassliquid separation step, an increase of the degradation rate and reusability. However, the structure and material of the support material directly influence microbial adhesion and degradation performance; therefore selection of an appropriate support material is an important step for an efficient immobilization system. [9] The selected support materials should be able to maintain their physical, chemical and thermal stability under the treatment conditions and throughout the process. In addition, they should be of low cost, abundant and readily available, and should not hinder the mass transfer between microorganisms and target contaminants. [10] Agricultural matrices are alternative support materials for cell immobilization because they are environmentally friendly, locally available and cheaper than synthetic polymers. Adsorption is the major process for cell immobilization on agricultural support materials. This provides advantages for microbial immobilization because adsorption is easy to handle, inexpensive and there are no constraints due to chemical changes to the support materials and microorganisms. [11] The additional advantage of using agricultural residues as support materials for cell
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immobilization is that the spent materials can be sent back to the land as an organic amendment, hence minimizing waste generation and disposal problems. Various agricultural residues such as papaya stem, [11] pine sawdust, [12] sugarcane bagasse and corncob [13] have been investigated for cell immobilization in contaminant degradation. Different types of agricultural residue differ in structure, physical and chemical properties, including fibre length and width and cell wall architecture. The major physical differences are water sorption, free volume, permeability and strength. Therefore, selection of an appropriate agricultural residue as a support material for cell immobilization is required for efficient contaminant removal. Chemically, agricultural residues differ in lignin, cellulose and hemicellulose content. [14] Lignin is a highly crosslinked molecular complex with an amorphous structure and can act as a wax and encrusting substance on fibre surfaces. The presence of encrusting substances causes the fibres to have an irregular appearance. The removal of surface waxes and encrusting substances makes the fibre surface rough and improves the adhesion of fibres and the polymer matrix. [15,16] The present study aimed to search for an effective local agricultural residue to immobilize the carbofuran degrader Burkholderia cepacia PCL3, for remediating carbofuran in synthetic wastewater. The isolate PCL3 was isolated from carbofuran-phytoremediated rhizosphere soil of rice. [17] It is capable of utilizing carbofuran as its sole carbon source. PCL3 was immobilized by various local agricultural materials: coir, banana stem, bulrush and water hyacinth stem in both delignified and undelignified forms. The degradation abilities, stability and reusability of the immobilized PCL3 cells as well as the effect of carbofuran concentrations on the immobilized PCL3 cells on the suitable support material were then investigated in order to determine its effectiveness.
Materials and methods Chemicals and reagents Carbofuran (98% purity) and carbofuran phenol (99% purity) were purchased from Sigma-Aldrich, USA. 3-keto carbofuran (98.5% purity) was purchased from Ehrenstorfer Quality, Germany. Methanol (high-performance liquid chromatography (HPLC) and analytical grades) was purchased from Merck, Germany. All other chemicals were analytical grade and purchased from BDH, England.
Synthetic wastewater Basal salt medium (BSM) [18] was used as a synthetic wastewater in this study. It consisted of Na2 HPO4 (5.57 g l−1 ), KH2 PO4 (2.44 g l−1 ), NH4 Cl (2.00 g l−1 ), MgCl2 .6H2 O (0.20 g l−1 ), MnCl2 .4H2 O (0.0004 g l−1 ), FeCl3 .6H2 O (0.001 g l−1 ) and CaCl2 (0.001 g l−1 ). The pH
of the synthetic wastewater was adjusted to 7 before being autoclaved at 121◦ C for 15 min.
Support materials preparation Four local agricultural wastes, i.e. coir, banana stem, bulrush and water hyacinth stem, were collected from Khon Kaen Province, Thailand. These materials were chosen for cell immobilization because of their high matrix porosity and pore size, which could enhance the cell adsorption capability, and their local abundance. Each material was cut into 1.0 × 1.0 × 1.0 cm pieces using a knife, then 300 g of each support material was boiled in 3 l of 1% NaOH for 3 h to remove lignin and fibres that might react with the cells. [19] Delignified and undelignified support materials were autoclaved at 121◦ C for 15 min before use. Both delignified and undelignified support materials were evaluated for their immobilization capacity.
Sorption of carbofuran to support materials An adsorption isotherm was determined by conducting batch equilibrium experiments. Support materials were airdried overnight and milled into small pieces using a food blender and passed through a 2-mm sieve. Then, 0.125 g of support material powder was put into 100-ml glass tubes. Then, 25 ml of 0.01 M CaCl2 solution containing carbofuran at concentrations of 0.1, 1.0, 5.0, 10.0 and 20.0 mg l−1 was then added to the tubes. The tubes were shaken horizontally at a constant speed of 100 rpm for 48 h at an average room temperature of 29 ± 2◦ C. After 48 h, the solution was passed through a Whatmann filter paper No. 1 and the filtrate was extracted by the liquid-liquid partitioning method and quantified for carbofuran concentration by HPLC. The data were fitted to the Freundlich equation [20] to describe the kinetics of carbofuran sorption to the support materials.
Microorganism preparation and cell immobilization B. cepacia PCL3 (GenBank accession No. EF990634) [17] was used as the carbofuran degrader. This was grown in 100 ml of nutrient broth (NB) containing 5 mg l−1 of carbofuran at 30◦ C and shaken at 150 rpm for 36 h, before being harvested by centrifugation at 5000 rpm for 10 min at 4◦ C. The cell pellets were washed and re-suspended in BSM and were used as seed inoculums. The immobilization of B. cepacia PCL3 on each support material was conducted following the procedures previously described by Plangklang and Reungsang (2009). [13]
Stability of immobilized cells attached to support materials The stability of the immobilized cell on the support materials was investigated according to the method of Wang
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Environmental Technology et al. (2001). [21] Briefly, 10 g of each sample of immobilized cells was rinsed with sterile BSM and transferred to a 250-ml flask containing 100 ml sterile BSM. The flask was shaken at 150 rpm at 30◦ C for 30 min. The amounts of PCL3 on the support materials and washed out from the support materials to BSM were counted by the drop-plate technique. The moisture content of the immobilized cells on undelignified coir, bulrush, banana stem and water hyacinth stem were 64, 84, 76 and 82%, respectively, while the moisture content of the immobilized cells on delignified coir, bulrush and banana stem were 69, 86 and 82%, respectively. The amount of cells immobilized or washed out was calculated on a dry basis and expressed as cfu per g dry support material. The cell stability (%) was calculated according to Equation (1). IMC Stability (%) = × 100 (1) WC + IMC where IMC is the number of cells observed in the support material (cfu g−1 dry support material) and WC is the number of cells observed in BSM (cfu g−1 dry support material). Carbofuran degradation test with free and immobilized cells Degradation of carbofuran by the immobilized B. cepacia PCL3 cells was conducted in a 250-ml shake-flask in a batch experiment. One hundred ml of synthetic wastewater containing 5 mg l−1 of carbofuran was added to the flask before inoculation with the immobilized PCL3 at a final concentration of 106 cfu ml−1 . The flask was incubated at room temperature and shaken at 150 rpm. The liquid part was sampled at days 0, 3, 6, 10 and 15 to extract carbofuran and its metabolites (i.e. 3-ketocarbofuran and carbofuran phenol) by the liquid-liquid partitioning method and analysed for their concentrations by HPLC. The amount of PCL3 in the support material and in the synthetic wastewater was determined by the drop-plate technique. Degradation of carbofuran by B. cepacia PCL3 in freecell form was conducted in a 500-ml shake flask in a batch experiment as a control. Two hundred ml of synthetic wastewater containing 5 mg l−1 of carbofuran as the sole carbon source was added to the flask before inoculation with 10% inoculum of PCL3 (final cell concentration of 106 cfu ml−1 ). In order to determine the effect of the abiotic degradation process on the removal of carbofuran, sterile support materials were used instead of immobilized cells in the abiotic control treatments. Flasks were incubated and analysed for the concentration of carbofuran and its metabolites and the amount of PCL3 as previously described. After 15 days of incubation, the immobilized cells on each support material were harvested and re-inoculated into fresh synthetic wastewater containing 5 mg l−1 of carbofuran. Carbofuran degradation in synthetic wastewater, cell
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leakage and cell survival were determined at days 0, 3, 6, 10 and 15. This step was repeated three times to investigate reusability of the immobilized cells. Effect of carbofuran concentration This experiment was conducted to determine the maximum carbofuran concentration at which the PCL3 could survive and degrade carbofuran effectively. The biodegradation batch experiments were conducted in the same manner as above using free or suitable immobilized cells of PCL3 as the inoculum, except that the concentration of carbofuran was varied in the range 0–280 mg l−1 and the liquid part was sampled every 3 d until the carbofuran degradation reached steady state. Analysis methods Carbofuran and its metabolites, i.e. carbofuran phenol and 3-ketocarbofuran, were extracted from synthetic wastewater by the liquid-liquid partitioning method [13] with the recovery percentages of 98%, 96% and 95%, respectively. The extracts were analysed using a Shimadzu 10-A HPLC equipped with a 4.6 × 150 mm Lunar 0.5 μm C-18 column (Phenomenex, USA) with an ultraviolet (UV) detector operating at 220 nm. The HPLC operating parameters followed Plangklang and Reungsang (2009) [13] excepting that the column oven temperature was changed from room temperature to 40◦ C. The HPLC retention times of carbofuran, carbofuran phenol and 3-ketocarbofuran were 3.99, 3.34 and 4.56 min, respectively. The half-life (t1/2 ) of carbofuran in the synthetic wastewater was calculated by fitting the data to a modified first-order kinetic model using the SAS software program (SAS Institute Inc., Cary, NC). [22] Data were analysed by SPSS software program Version 10.0 (SPSS Inc., Chicago, IL). The significance of treatments was set at a p-value of less than or equal to 0.05 by the one-way ANOVA with Duncan’s post hoc test. The microstructure of the support materials and immobilized cells were investigated by scanning electron microscopy (SEM). [23] The number of carbofuran degraders in the synthetic wastewater and in the support material was determined by the drop-plate technique. [24] For growth and degradation kinetics analysis, the amount of PCL3 in the free-cell experiment was directly converted to the cell concentration by a linear relationship. For the immobilization experiment, the amount of PCL3 on the support material and leaking cells were added together before being converted to the cell concentration. Growth and degradation kinetic analysis Kinetic analysis for growth of PCL3 and its carbofuran degradation were evaluated by fitting the data to the bacterial growth and substrate degradation model using the Sigmaplot 9.0 software program (SPSS Inc., Chicago, IL).
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The Luong model proposed by Luong (1987) [25] was used to explain the bacterial growth and carbofuran degradation of PCL3 in free-cell form while the Monod (1949) model was used to explain the kinetic behaviour of the immobilized cells. [26] Results and discussions Sorption kinetic of carbofuran All sorption data fitted well to the Freundlich isotherm with the regression coefficient ranging between 0.91 and 0.97. Results indicated that the sorption isotherms were not linear (1/n = 1). The 1/n values were less than 1.0, indicating that concentration change had little effect on the adsorptive capacity. [27] The Kf values were used to describe the extent of sorption between carbofuran and the support materials and found to be correlated to the organic carbon content of the support materials (Table 1). The Kf values of carbofuran on undelignified support materials was less than that found on delignified support materials, which might be due to less organic carbon content in undelignified support materials. Undelignified and delignified coir had higher Kf values (20.88–24.23 l kg−1 ) than the other support materials (8.13–17.25 l kg−1 ). The sorption mechanism is believed to reduce diffusion limitation and facilitate the mass transfer between the contaminant and catalyst (microbial cells or enzyme) on the support materials. [28] Thus, the results implied that undelignified and delignified coir were good candidates as support materials for cell immobilization in bioaugmentation of carbofuran. Quantification and stability of cell attachment on the support The amount of PCL3 on the support materials was in the range of 1.46 × 108 –6.90 × 108 cfu g−1 dry support. The amount of PCL3 on delignified coir (2.90 × 108 cfu g−1 dry support) was approximately two times greater than the number of cells on undelignified coir (1.46 × 108 cfu g−1 dry support). This might be due to the fact that the delignification process removed the microcrystalline structure of lignin on the surface of coir but did not remove its complex layers (Figure 1(a) and (e)). This could result in a better Table 1.
sorption of the cells onto the surface of delignified coir than onto undelignified coir. In the case of bulrush, the amount of B. cepacia PCL3 on its undelignified form (6.87 × 108 cfu g−1 dry support) was greater than its delignified form (1.73 × 108 cfu g−1 dry support), which might be due to the loss of complex structure and pores from the surface of bulrush after delignification (Figure 1(b) and (f)). The amount of PCL3 on undelignified (2.17 × 108 cfu g−1 dry support) and delignified banana stem (1.96 × 108 cfu g−1 dry support) was not significantly different, which might be due to the physical appearance, texture and microstructure of banana stem, which were not changed after delignification (Figure 1(c) and (g). The amount of PCL3 on undelignified water hyacinth stem was relatively high at 6.00 × 108 cfu ml−1 , which could be a result of the clumping of cells on the surface (Figure 1(d)). The capability for high cell density retention is a good characteristic of a supporting material for cell immobilization because it allows high cell density loading to the contaminated area. However, the stability of the cells attached to the support materials should also be taken into account as an important prerequisite. [10] The stability of the immobilized cells attached on each support material is shown in Table 2. Results indicated that the immobilized cells on all support materials, except for delignified bulrush and undelignified water hyacinth stem, had a stability percentage greater than 75% (Table 2) which indicated a good characteristic of support materials for cell immobilization. Delignified water hyacinth stem is not considered to be a good support material since the stability of the immobilized cells was only 57.36%. SEM analysis of the support materials and immobilized B. cepacia PCL3 The microstructures of the support materials were determined by SEM and shown in Figure 1. From SEM, it could be seen that the support materials had complex layers and pores that were suitable for cell immobilization. Coir (Figure 1(a)) has more complex layers than bulrush (Figure 1(b)), banana stem (Figure 1(c)) and water hyacinth stem (Figure 1(d)). The microstructure of delignified coir (Figure 1(e)) remained the same as in the undelignified form (Figure 1(a)), while the complex layer of bulrush
Organic matter content, Freundlich sorption coefficient and sorption constant (1/n).
Type of support materials Undelignified coir Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem a Coefficients
Organic matter content (%)
Freundlich sorption coefficient (Kf ) (l kg−1 )
Sorption constant (1/n)
r 2a
89.11 97.41 80.99 85.76 78.52 79.73 73.63
20.88 24.23 11.36 17.25 11.05 16.84 8.13
0.76 0.55 0.88 0.54 0.44 0.43 0.72
0.91 0.94 0.96 0.92 0.92 0.91 0.97
of determination for linear plots of Freundlich isotherm regression.
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Figure 1. Scanning electron microscopic images of (a) undelignified coir, (b) undelignified bulrush, (c) undelignified banana stem, (d) undelignified water hyacinth stem, (e) delignified coir, (f) delignified bulrush, (g) delignified banana stem; immobilized B. cepacia PCL3 on (h) undelignified coir, (i) undelignified bulrush, (j) undelignified banana stem, (k) undelignified water hyacinth stem, (l) delignified coir, (m) delignified bulrush and (n) delignified banana stem.
(Figure 1(f)) and banana stem (Figure 1(g)) had been removed after delignification. Delignified water hyacinth stem was not observed by SEM and would not be used as a support material for cell immobilization because its structure was broken after delignification. The cell characteristic, attachment patterns and arrangement of B. cepacia PCL3 on the support materials were studied under SEM. As shown in Figure 1(h)–(n), cells were rod-shaped, approximately 1.6 μm in length and 0.7 μm in diameter when they were immobilized on coir, bulrush and banana stem in both undelignified and delignified forms (Figure 1(h)–(j) and (l)–(n)). When cells were immobilized on water hyacinth stem, the sizes of the cells were approximately 2.2 μm in length and 0.7 μm in diameter (Figure 1(k)). It could be observed that cells were arranged singly on all supports, except for water hyacinth stem in
which the cells were arranged as a cluster on its surface. Differences in cell shape and arrangement could be the result of nutrient acquisition and different attachment mechanisms to different surfaces. [29] SEM photographs indicated that B. cepacia PCL3 was able to access into the pores as well as reside on the surface area of the complex layers for all support materials excepting undelignified water hyacinth stem. The possible mechanism of cell attachment on these support materials is initially cell adhesion onto the surface area of support materials by the van der Waals force (an electrostatic force caused by different surface charges of bacterial cells and supports). When the time for cell adhesion is extended, there is a polymeric binding between the cells and the support materials that prevents reversible sorption and makes adhesion between cells and the surface of the support
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Table 2. Stability of the immobilized cells and first-order kinetic parameters including degradation rate constants and half-lives of carbofuran in synthetic wastewater by free cells, immobilized cells and reuse of immobilized cells on each support material.
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Treatment PCL3 in free-cell form Immobilized PCL3 cells on Undelignified coir Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem Reuse immobilized cells on undelignified coir First reuse Second reuse Third reuse Delignified coir First reuse Second reuse Third reuse Undelignified bulrush First reuse Second reuse Undelignified banana stem First reuse Second reuse Delignified banana stem First reuse
Stabilitya (%)
Degradation rate constants (k1 ) (d−1 )
Half-life (t1/2 ) (d)a
r 2b
0.0749 ± 0.0085
9.31c ± 1.05
0.98
0.2102 ± 0.0523 0.2175 ± 0.0030 0.2229 ± 0.0844 0.1012 ± 0.0016 0.1686 ± 0.0536 0.2429 ± 0.0064 0.1598 ± 0.0337
3.40a ± 0.85 3.19a ± 0.04 3.35a ± 1.27 6.85b ± 0.11 4.33a ± 1.38 2.85a ± 0.07 4.44a ± 0.94
0.99 0.99 0.98 0.99 0.99 0.99 0.99
0.277 ± 0.065 0.158 ± 0.017 0.169 ± 0.028
2.58a ± 0.61 4.41a ± 0.48 4.15a ± 0.67
0.98 0.91 0.98
0.211 ± 0.047 0.165 ± 0.007 0.112 ± 0.013
3.36a ± 0.75 4.21a ± 0.19 4.43a ± 0.52
0.99 0.96 0.99
0.232 ± 0.003 0.166 ± 0.017
2.99a ± 0.04 4.19a ± 0.43
0.98 0.92
0.209 ± 0.052 0.135 ± 0.022
3.42a ± 0.84 5.21a ± 0.84
0.99 0.93
0.270 ± 0.010
2.57a ± 0.10
0.98
78.72ab ± 1.84 77.75ab ± 2.41 82.36a ± 1.37 69.71c ± 2.42 77.12b ± 0.92 75.96b ± 1.36 57.36d ± 3.74
a Comparison between treatment in column are significantly different (p ≤ 0.05) if marked in different lowercase letters. b Coefficients of determination for non-linear regressions.
materials firmer and more stable. [11,30] In the case of undelignified water hyacinth stem, where the cells only clustered on the surface, the cell attachment mechanism is initially cell adhesion followed by polymeric linkage between cells with no polymeric binding between cells and supports. This can be the reason for the lower stability of immobilized cells on undelignified water hyacinth stem when compared with other support materials. Degradation of carbofuran by free and immobilized PCL3 cells Degradation of carbofuran in synthetic wastewater was described by a modified first-order kinetic model. The free PCL3 cells could degrade carbofuran in synthetic wastewater with a half-life of 9.31 d while the shorter half-lives in the range 2.85–6.85 d were obtained when immobilized cells were used (Table 2). The results suggested that the degradation ability of B. cepacia PCL3 could be increased by the immobilization technique. The high matrix porosity and porous nature of the support materials might be effective for physical adsorption of carbofuran to the support material and transference of carbofuran to the cells resulting in an enhanced carbofuran degradation efficiency. [31,32] The delignification process did not significantly affect the carbofuran degradation efficiency of the immobilized
cells on coir. This was suggested by the nonsignificant difference (p > 0.05) between the t1/2 of carbofuran in synthetic wastewater augmented with immobilized PCL3 on delignified coir compared to undelignified coir (3.19– 3.40 d) (Table 2). A significant longer t1/2 of carbofuran in synthetic wastewater (6.85 d) was observed when the immobilized PCL3 on delignified bulrush was used in comparison to the immobilized cell on undelignified bulrush (t1/2 of 3.35 d) (Table 2). The delignification process could improve the carbofuran degradation efficiency of immobilized cells on banana stem. The t1/2 of carbofuran in synthetic wastewater augmented with the immobilized PCL3 on delignified banana stem (2.85 d) was significantly shorter than that augmented with undelignified banana stem (t1/2 of 4.33 d). Delignification, in fact, could improve carbofuran sorption efficiency of the support materials (Table 1), which could facilitate the transfer of carbofuran to the cell inside the support materials, thus enhancing the carbofuran degradation efficiency. In addition, after lignin was removed, the fibres of the support material were softened, which could be used easily by the PCL3 as an additional energy source for its growth. The presence of additional energy sources in the pesticide bioremediation system might result in an increase in cell number as well as pesticide degradation activities. [33] However, in some cases, the additional energy sources could worsen the efficiency of pesticide remediation if the
0.065 nd
0.114 0.082 0.107 nd 0.417 0.643 0.408
0.051 0.032 0.053 0.124 0.071 0.028 0.022 nd nd nd 0.063 0.022
1.754 0.449 0.844 0.730 0.764 0.018 0.965
0.061 0.473
0.063 0.221
10 0.036 1.747
nd nd nd nd nd nd nd nd: not detectable.
Carbofuran phenol Immobilized PCL3 cells on Undelignified coir Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem
3-Ketocarbofuran
nd nd nd nd nd nd
0.013 0.116 nd nd 3-Ketocarbofuran Carbofuran phenol PCL3 in free-cell form
Immobilized PCL3 cells on Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem
9 6
nd nd nd nd nd nd nd
0.076 0.034 0.052 0.162 0.082 0.031
0.060 nd
35 15
18
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5 4 2 0 Metabolite Treatment
Incubation time (d)
Metabolites observed during biodegradation of carbofuran in synthetic wastewater by PCL3 in free and immobilized cell forms.
degrader prefers to use the additional energy sources rather than the target pesticide. [24] Carbofuran phenol and 3-keto carbofuran were the metabolites observed in free and immobilized cells experiments (Table 3) while only 3-keto carbofuran (0.051– 0.170 mg l−1 ) (data not shown) was observed in abiotic controls. The results suggested that carbofuran phenol was the metabolite from carbofuran biodegradation by PCL3. The concentration of carbofuran phenol tended to increase to its maximum value (0.449–1.754 mg l−1 ) after a certain time; it then decreased and was not detected at the end of incubation in all treatments (Table 3). This implied that carbofuran phenol could be utilized by B. cepacia PCL3, which is in agreement with previous published reports. [13,17] 3-Keto carbofuran concentration increased over time, and it accumulated until the end of incubation in all treatments. The maximum concentrations of 3-keto carbofuran ranged between 0.031 and 0.162 mg l−1 . Since the concentrations of 3-keto carbofuran observed in biodegradation treatments were not markedly different from the abiotic control, and there had been evidence that PCL3 degraded carbofuran to carbofuran phenol but not 3-keto carbofuran, [13] we suggested that 3-keto carbofuran was the metabolite from the abiotic degradation process. The survival and growth of free and immobilized cells were determined by the amounts of B. cepacia PCL3 in synthetic wastewater (Figure 2(a)) and on the support materials (Figure 2(b)). Results indicated that free cells and the immobilized cells on all materials could survive 15 d of incubation at cell numbers of approximately 106 cfu ml−1 (Figure 2(a)) and 108 to 109 cfu g−1 dry support material, respectively (Figure 2(b)). Cell leakage from the support materials was determined from the cell concentration observed in synthetic wastewater during incubation (Figure 2(c)). At day 0, the number of PCL3 cells in the liquid phase of synthetic wastewater was negligible. After day 3, the amount of PCL3 in the synthetic wastewater was in the range 105 –107 cfu ml−1 (Figure 2(c)). Since the number of cells on support materials was not decreased, we suggested that the leakage of the cells from the support materials might have resulted from the overgrowth of cells within the limited space of the support materials. In addition, there is no barrier between the cell and the synthetic wastewater, which led to the possibility of cell detachment and relocation with the establishment of cell equilibrium inside the support material and synthetic wastewater. [10] The dissipation of carbofuran could be observed in synthetic wastewater augmented with sterile support materials (abiotic control). After 15 d of incubation, approximately 18% of the carbofuran was dissipated in synthetic wastewater with sterile undelignified coir, delignified and undelignified banana stem, undelignified bulrush and undelignified water hyacinth stem; while approximately 30% carbofuran removal was observed with sterile delignified coir and delignified bulrush. The dissipation of carbofuran might
Table 3.
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Figure 2. Amount of B. cepacia PCL3 in (a) free and (b) immobilized cell forms grown in synthetic wastewater and (c) number of leaking cells from the support materials.
be caused by the sorption of carbofuran onto the surface and pores of the support materials during incubation. The greater carbofuran degradation rates observed in the treatments with immobilized cells (t1/2 ranged between 2.85 and 6.85 d (Table 2) than in the abiotic controls suggested that carbofuran dissipation in synthetic wastewater mainly resulted from the biodegradation process.
carbofuran concentration on growth patterns and degradation kinetics of immobilized PCL3 on coir were studied in the following experiment.
Reusability of immobilized B. cepacia PCL3 on each support material
The correlation between specific growth rate of PCL3 in free and immobilized cell forms and the initial concentration of carbofuran is depicted in Figure 3. The decrease in specific growth rate with the increase in initial carbofuran concentration in the free-cell experiment implied that carbofuran acts as an inhibitor on PCL3. Therefore, the substrate inhibition model (Luong model) was used to explain the growth kinetics of PCL3 in free-cell form. The regression coefficients, r 2 , of 0.99 indicate a good fit of the data to the models. The estimated kinetic parameters for free PCL3 cells from the Luong model were maximum specific growth rate (μmax ) = 1.70 d −1 and substrate saturation constant (Ks ) = 33.12 mg l−1 . The empirical constants n (1.22) estimated by the Luong model were greater than 1, suggesting that the non-linear relationship between specific growth rate (μ) and initial carbofuran concentration (S) existed during the inhibition process. The maximum substrate concentration above which carbofuran degradation was completely inhibited (Sm ) was 247.83 mg l−1 . The inhibitory effect of carbofuran on the immobilized PCL3 was not observed at concentrations up to 280 mg l−1 , which confirmed that the immobilization technique could protect the cells of PCL3 from substrate inhibition. The Monod model was used to explain the growth kinetics of the
Investigation into the reusability of PCL3 on undelignified water hyacinth stem and delignified bulrush was not conducted due to the degradation of the support materials after 15 d of incubation. Undelignified coir could be reused three times, undelignified bulrush and undelignified banana stem could be reused two times and delignified banana stem could be reused once. The corresponding kinetic data fitted to a modified first-order kinetic model are tabulated in Table 2. The results revealed that the reused immobilized cells on the tested support materials could effectively degrade carbofuran in synthetic wastewater with a short t1/2 of 2.57–5.21 d (Table 2), which was not significantly different from the t1/2 of carbofuran degraded by the first-time usage immobilized cells (2.85–6.85 d) (Table 2). From the results obtained, it could be concluded that undelignified coir is the most suitable support material to immobilize PCL3 for remediating carbofurancontaminated water. The immobilized PCL3 on undelignified coir possessed a high stability of 78.72%, a high carbofuran degradation ability with a short t1/2 of 3.40 d, and could be reused four times with carbufuran degradation ability still intact (t1/2 of 2.58–4.15 d). The effects of
Effect of initial carbofuran concentration in synthetic wastewater on the growth and carbofuran degradation kinetics of B. cepacia PCL3 Growth kinetics
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Figure 3. (a) Specific growth rate and (b) specific degradation rate of PCL3 in free and immobilized cells forms at various initial carbofuran concentrations.
immobilized PCL3 with an r 2 of 0.98. The estimated growth kinetic parameters for the immobilized PCL3 obtained from the Monod model were μmax = 0.85 d −1 and Ks = 26.77 mg l−1 . The lower μmax value was obtained when the immobilized cells were used in comparison to free cells, which might be because the immobilized cells could not grow freely under the conditions with limited space inside the support material. The lower Ks value obtained indicated that at the low carbofuran concentration the immobilized PCL3 was able to access carbofuran in synthetic wastewater better than free cells. This result confirmed that sorption of carbofuran to the support material could enhance the mass transference between carbofuran and the cells inside the support. Degradation kinetic The degradation rate coefficient of carbofuran was calculated by fitting to a modified first-order kinetic model in order to examine the t1/2 of carbofuran in synthetic
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wastewater (Table 4). Results indicated that immobilized cells were more efficient in degrading carbofuran than free cells at any range of carbofuran concentration used in this study. In the free-cell experiment, the significantly longer t1/2 of carbofuran (p < 0.05) was obtained when the initial carbofuran concentration was increased (Table 4). The t1/2 of carbofuran at the initial carbofuran concentration of 250 mg l−1 was 8.2 times longer than the carbofuran t1/2 at the initial carbofuran concentration of 5 mg l−1 . In the immobilized cell experiments, t1/2 of carbofuran (3.4–6.3 d) was not significantly different at the initial concentrations of 5 to 50 mg l−1 (Table 4). The slightly longer t1/2 of carbofuran t1/2 of 15 d could be found when the concentration of carbofuran was increased from 50 mg l−1 to 280 mg l−1 . These results indicated that the inhibitory effect on carbofuran degradation by PCL3 at a high carbofuran concentration could be reduced by the immobilization technique. The specific carbofuran degradation rate from free and immobilized PCL3 at different initial carbofuran concentrations are shown in Figure 3. The carbofuran degradation kinetic parameters of PCL3 in free-cell form estimated from the Luong model (r 2 = 0.94) were maximum specific carbofuran degradation rate (qmax ) = 1.38 d −1 Ks = 34.78 mg l−1 and n = 1.3. The degradation kinetic parameters of immobilized PCL3 were estimated from the Monod model (r 2 = 0.96) where qmax and Ks were 1.92 d−1 and 30.32 mg l−1 , respectively. The inhibitory effect of carbofuran was observed in the free-cell experiment in which the maximum substrate concentration above which carbofuran degradation was completely inhibited (Sm ) was 252.80 mg l−1 , while an inhibitory effect of carbofuran on the immobilized cells was not observed. The results confirmed that the immobilization technique could protect the PCL3 cell from substrate inhibition, hence enhancing carbofuran degradation efficiency. The enhancement of contaminant degradation activity by the immobilization technique has also been reported for different mechanisms. The immobilization of viable cells could alter their physiological
Table 4. Degradation rate constants (k1 ) and half-lives (t1/2 ) of carbofuran in PCL3-inoculated synthetic wastewater with various initial carbofuran concentrations. Initial carbofuran concentration (mg l−1 ) 5 10 50 100 150 200 250
Free cells of PCL3 k1 (d−1 )
t1/2 (d)a
r 2b
0.074 ± 0.008 0.069 ± 0.028 0.060 ± 0.005 0.046 ± 0.012 0.020 ± 0.003 0.012 ± 0.002 0.009 ± 0.001
9.34a ± 0.95 10.80ab ± 4.33 11.56b ± 0.99 15.68b ± 4.03 35.23c ± 5.87 59.86d ± 8.68 76.30e ± 4.74
0.98 0.99 0.98 0.99 0.99 0.98 0.99
Initial carbofuran concentration (mg l−1 )
Immobilized PCL3 k1 (d−1 )
t1/2 (d)a
r 2b
5 10 50 120 200 280
0.210 ± 0.052 0.163 ± 0.020 0.119 ± 0.045 0.093 ± 0.023 0.071 ± 0.012 0.047 ± 0.008
3.40a ± 0.85 4.30a ± 0.54 6.29ab ± 2.39 7.69b ± 1.90 9.90b ± 1.73 15.00c ± 2.58
0.99 0.99 0.99 0.99 0.97 0.98
between treatments in the column are significantly different (p ≤ 0.05) by Duncan’s Multiple Range test if marked in different small letters. b Coefficients of determination for non-linear regressions.
a Comparison
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features of metabolism such as enhanced induction of the enzyme responsible for contaminant degradation. [34] The support materials could act as a protective shelter against the toxicity of the contaminant and adverse environmental conditions, hence the survival of the cell could be improved, resulting in an increase in degradation efficiency. [13,35] In addition, the immobilized cells could be used as the source for continuously providing the cells of the degraders in the bioremediation system. [36,37]
Conclusions Undelignified coir was the most suitable support material for cell immobilization giving the short t1/2 of carbofuran of 3.40 d (2.8 times shorter than the treatments with free cells). The immobilized cells on undelignified coir could be reused three times without a loss of the ability to degrade carbofuran. The kinetic characterization of free and immobilized PCL3 revealed that the growth and degradation ability of free cells were completely inhibited at the initial carbofuran concentrations of 250 mg l−1 . The immobilization technique was able to reduce the inhibitory effect of carbofuran on PCL3; the growth and carbofuran degradation ability of the immobilized PCL3 on coir did not worsen at concentrations up to 280 mg l−1 . The results from this study indicated the great potential of using immobilization techniques to improve the overall efficiency of carbofuran bioremediation in contaminated water by B. cepacia PCL3. Acknowledgements This work was supported by grants from the Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand and the National Center of Excellence for Environmental and Hazardous Waste Management, Bangkok, Thailand.
References [1] EPA. Interim Reregistration Eligibility Decision (IRED) Document for Carbofuran: Special review and reregistration division, (2006), pp. 44, Available from: http://www. epa.gov/oppsrrd1/reregistration/REDs/carbofuran_ired.pdf. [2] Campbell S, David MD, Woodward LA, Li QX. Persistence of carbofuran in marine sand and water. Chemosphere. 2004;54:1155–1161. [3] Farahani GHN, Sahid IB, Zakaria Z, Kuntom A, Omar D. Study on the downward movement of carbofuran in two Malaysian soils. Bull Environ Contam Toxicol. 2008;81:294–298. [4] Plese LPM, Paraiba LC, Foloni LL, Trevizan LRP. Kinetics of carbosulfan hydrolysis to carbofuran and the subsequent degradation of this last compound in irrigated rice fields. Chemosphere. 2005;60:149–156. [5] Satar S, Sebe A, Yesilagac H. Carbofuran poisoning among farm workers. Toxicol Lett. 2005;S1:135–136. [6] Cho YG, Rhee SK, Lee ST. Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardiodes sp. NSP41. Biodegradation. 2000;11:21–28.
[7] Parameswarappa S, Karigar C, Nagenahalli M. Degradation of ethylbenzene by free and immobilized Pseudomonas fluorescens-CS2. Biodegradation. 2008;19:137–144. [8] Chen F, Cui M, Fu J, Sheng G, Sun G, Xu M. Biodegradation of quinoline by freely suspended and immobilized cells of Comamonas sp. strain Q10. J Gen Appl Microbiol. 2003;49:123–128. [9] Pattanasupong A, Nagase H, Sugimoto E, Hori Y, Hirata K, Tani K, Nasu M, Miyamoto K. Degradation of carbendazim and 2,4-dichlorophenoxyacetic acid by immobilized consortium on loofa sponge. J Biosci Bioeng. 2004;98: 28–33. [10] Kourkoutas Y, Bekatorou A, Banat IM, Marchant R, Koutinas AA. Immobilization technologies and support materials suitable in alcohol beverages production, a review. Food Microbiol. 2004;21:377–397. [11] Lakshmi MB, Muthukumar K, Velan M. Immobilization of Mycoplana sp. MVMB2 isolated from petroleum contaminated soil onto papaya stem (Carica papaya L.) and its application on degradation of phenanthrene. Clean–Soil, Air, Water. 2012;40:870–877. [12] Podorozhko EA, Lozinsky VI, Ivshina IB, Kuyukina MS, Krivorutchko AB, Philp JC, Cunningham CJ. Hydrophobised sawdust as a carrier for immobilisation of the hydrocarbon-oxidizing bacterium Rhodococcus rubber. Bioresour Technol. 2008;99:2001–2008. [13] Plangklang P, Reungsang A. Bioaugmentation of carbofuran residues in soil using Burkholderia cepacia PCL3 adsorbed on agricultural residues. Int Biodeterior Biodegradation. 2009;63:515–522. [14] Kelley SS, Rowell RM, Davis M, Jurich CK, Ibachb R. Rapid analysis of the chemical composition of agricultural fibers using near infrared spectroscopy and pyrolysis molecular beam mass spectrometry. Biomass Bioenerg. 2004;27: 77–88. [15] Mohanty AK, Misra M, Hinrichsen G. Biofibres, biodegradable polymers and biocomposites, an overview. Macromol Mater Eng. 2000;276/277:1–24. [16] Saha SC, Das BK, Ray PK, Pandey SN, Goswami K. SEM studies of the surface and fracture morphology of pineapple leaf fibers. Text Res J. 1990;10:726–731. [17] Plangklang P, Reungsang A. Isolation and characterization of carbofuran degrading Burkholderia sp. PCL3 from carbofuran phytoremediated rhizosphere soil. Chem Ecol. 2012;28:253–266. [18] Mo K, Lora CO, Wanken AE, Javanmardian M, Yang X, Kulpa CF. Biodegradation of methyl tert-butyl ether by pure bacterial cultures. Appl Microbiol Biotechnol. 1997;47: 69–72. [19] Bardi EP, Koutinas AA. Immobilization of yeast on delignified cellulosic material for room temperature and lowtemperature wine making. J Agr Food Chem. 1994;42: 221–226. [20] Sposito G. Derivation of the Freundlich equation for ion exchange reactions in soils. Soil Sci Soc Am J. 1980;44: 652–654. [21] Wang AA, Mulchandani A, Chen W. Whole-cell immobilization using cell surface-exposed cellulose-binding domain. Biotechnol Prog. 2001;17:407–411. [22] SAS Institute. SAS User’s guide. Statistics. 5th ed. Cary, NC: SAS Institue; 1985. [23] Rachman MA, Nakashimada Y, Kakizono T, Nishio N. Hydrogen production with high yield and high evolution rate by self-focculated cells of Enterobacter aerogenes in a packed-bed reactor. Appl Microbiol Biotechnol. 1998;49:450–454.
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[32] Ma YL, Yang BL, Zhao JL. Removal of H2 S by Thiobacillus denitrificans immobilized on different matrices. Bioresour Technol. 2006;97:2041–2046. [33] Robles-Gonzalez IV, Fava F, Poggi-Varaldo HM. A review on slurry bioreactors for bioremediation of soils and sediments. Microb Cell Fact. 2008;7:1–16. [34] Chung TP, Tseng HY, Juang RS. Mass transfer effect and intermediate detection for phenol degradation in immobilized Pseudomonas putida systems. Process Biochem. 2003;38:1497–1507. [35] Wang SG, Liu XW, Zhang HY, Gong WX, Sun XF, Gao BY. Aerobic granulation for 2,4-dichlorophenol biodegradation in a sequencing batch reactor. Chemosphere. 2007;69: 769–775. [36] Mertens B, Boon N, Verstraete W. Slow-release inoculation allows sustained biodegradation of c-hexachlorocyclohexane. Appl Environ Microbiol. 2006;72:622–627. [37] Boon N, de Gelder L, Lievens H, Siciliano S, Top E, Verstraete W. Bioaugmenting bioreactors for the continuous removal of 3-chloroaniline by a slow release approach. Environ Sci Technol. 2002;36:4698– 4704.
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofurancontaminated water ab
S. Laocharoen , P. Plangklang
cd
& A. Reungsang
bce
a
International Postgraduate Programs in Environmental Management, Chulalongkorn University, Bangkok, Thailand b
National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok, Thailand c
Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand
d
Groundwater Research Center, Khon Kaen University, Khon Kaen, Thailand
e
Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand Accepted author version posted online: 01 Mar 2013.Published online: 25 Mar 2013.
To cite this article: S. Laocharoen, P. Plangklang & A. Reungsang (2013) Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofuran-contaminated water, Environmental Technology, 34:18, 2587-2597, DOI: 10.1080/09593330.2013.781226 To link to this article: http://dx.doi.org/10.1080/09593330.2013.781226
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Environmental Technology, 2013 Vol. 34, No. 18, 2587–2597, http://dx.doi.org/10.1080/09593330.2013.781226
Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofuran-contaminated water S. Laocharoena,b , P. Plangklangc,d and A. Reungsangb,c,e∗ a International
Postgraduate Programs in Environmental Management, Chulalongkorn University, Bangkok, Thailand; b National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok, Thailand; c Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand; d Groundwater Research Center, Khon Kaen University, Khon Kaen, Thailand; e Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand
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(Received 11 January 2012; final version received 18 February 2013 ) This study investigated the utilization of agricultural matrices as the support materials for cell immobilization to improve the technique of bioremediation. Coir, bulrush, banana stem and water hyacinth stem in both delignified and undelignified forms were used to immobilize Burkholderia cepacia PCL3 in bioremediation of carbofuran at 5 mg l−1 in synthetic wastewater. Undelignified coir was found to be the most suitable support material for cell immobilization, giving the short half-life of carbofuran of 3.40 d (2.8 times shorter than the treatments with free cells). In addition, it could be reused three times without a loss in ability to degrade carbofuran. The growth and degradation ability of free cells were completely inhibited at the initial carbofuran concentrations of 250 mg l−1 , while there was no inhibitory effect of carbofuran on the immobilized cells. The results indicated a great potential for using the agricultural matrices as support material for cell immobilization to improve the overall efficiency of carbofuran bioremediation in contaminated water by B. cepacia PCL3. Keywords: agricultural residues; bioaugmentation; Burkholderia cepacia PCL3; carbofuran; immobilization
Introduction Carbofuran (2,3-dihydro-2,2 dimethylbenzofuran-7-yl methylcarbamate) is a broad-spectrum insecticide widely used in agriculture to control insects and nematodes on contact or after ingestion. [1] It is of environmental concern because it is soluble in water and highly mobile in soil, resulting in a high potential for groundwater contamination. [2,3] Carbofuran has a chronic affect on aquatic organisms through cholinesterase inhibition, neurotoxicity and reproductive effects. [4] Acute uptake of carbofuran through accidental exposure can cause acute toxicities and fatalities in humans and other organisms. [5] Therefore, the removal of carbofuran from contaminated environments is needed. A variety of pretreatment methods have been reported for carbofuran degradation. In particular, bioremediation has gained recent attention due to the advantages of ease of operation and low operating cost, which make this technique technically and economically feasible in large-scale operations. One of the effective bioremediation techniques used to remediate pesticide contamination is bioaugmentation, which is the addition of microorganisms to aid dissipation of the pesticide. Immobilization of the microorganisms on suitable support materials before augmentation of the contaminated matrices has been reported to improve bioaugmentation efficiency. [6,7] ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
The advantages of immobilized cells over free cells include the allowance of high cell loading, more tolerance against toxic compounds, [8] the removal of the biomassliquid separation step, an increase of the degradation rate and reusability. However, the structure and material of the support material directly influence microbial adhesion and degradation performance; therefore selection of an appropriate support material is an important step for an efficient immobilization system. [9] The selected support materials should be able to maintain their physical, chemical and thermal stability under the treatment conditions and throughout the process. In addition, they should be of low cost, abundant and readily available, and should not hinder the mass transfer between microorganisms and target contaminants. [10] Agricultural matrices are alternative support materials for cell immobilization because they are environmentally friendly, locally available and cheaper than synthetic polymers. Adsorption is the major process for cell immobilization on agricultural support materials. This provides advantages for microbial immobilization because adsorption is easy to handle, inexpensive and there are no constraints due to chemical changes to the support materials and microorganisms. [11] The additional advantage of using agricultural residues as support materials for cell
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immobilization is that the spent materials can be sent back to the land as an organic amendment, hence minimizing waste generation and disposal problems. Various agricultural residues such as papaya stem, [11] pine sawdust, [12] sugarcane bagasse and corncob [13] have been investigated for cell immobilization in contaminant degradation. Different types of agricultural residue differ in structure, physical and chemical properties, including fibre length and width and cell wall architecture. The major physical differences are water sorption, free volume, permeability and strength. Therefore, selection of an appropriate agricultural residue as a support material for cell immobilization is required for efficient contaminant removal. Chemically, agricultural residues differ in lignin, cellulose and hemicellulose content. [14] Lignin is a highly crosslinked molecular complex with an amorphous structure and can act as a wax and encrusting substance on fibre surfaces. The presence of encrusting substances causes the fibres to have an irregular appearance. The removal of surface waxes and encrusting substances makes the fibre surface rough and improves the adhesion of fibres and the polymer matrix. [15,16] The present study aimed to search for an effective local agricultural residue to immobilize the carbofuran degrader Burkholderia cepacia PCL3, for remediating carbofuran in synthetic wastewater. The isolate PCL3 was isolated from carbofuran-phytoremediated rhizosphere soil of rice. [17] It is capable of utilizing carbofuran as its sole carbon source. PCL3 was immobilized by various local agricultural materials: coir, banana stem, bulrush and water hyacinth stem in both delignified and undelignified forms. The degradation abilities, stability and reusability of the immobilized PCL3 cells as well as the effect of carbofuran concentrations on the immobilized PCL3 cells on the suitable support material were then investigated in order to determine its effectiveness.
Materials and methods Chemicals and reagents Carbofuran (98% purity) and carbofuran phenol (99% purity) were purchased from Sigma-Aldrich, USA. 3-keto carbofuran (98.5% purity) was purchased from Ehrenstorfer Quality, Germany. Methanol (high-performance liquid chromatography (HPLC) and analytical grades) was purchased from Merck, Germany. All other chemicals were analytical grade and purchased from BDH, England.
Synthetic wastewater Basal salt medium (BSM) [18] was used as a synthetic wastewater in this study. It consisted of Na2 HPO4 (5.57 g l−1 ), KH2 PO4 (2.44 g l−1 ), NH4 Cl (2.00 g l−1 ), MgCl2 .6H2 O (0.20 g l−1 ), MnCl2 .4H2 O (0.0004 g l−1 ), FeCl3 .6H2 O (0.001 g l−1 ) and CaCl2 (0.001 g l−1 ). The pH
of the synthetic wastewater was adjusted to 7 before being autoclaved at 121◦ C for 15 min.
Support materials preparation Four local agricultural wastes, i.e. coir, banana stem, bulrush and water hyacinth stem, were collected from Khon Kaen Province, Thailand. These materials were chosen for cell immobilization because of their high matrix porosity and pore size, which could enhance the cell adsorption capability, and their local abundance. Each material was cut into 1.0 × 1.0 × 1.0 cm pieces using a knife, then 300 g of each support material was boiled in 3 l of 1% NaOH for 3 h to remove lignin and fibres that might react with the cells. [19] Delignified and undelignified support materials were autoclaved at 121◦ C for 15 min before use. Both delignified and undelignified support materials were evaluated for their immobilization capacity.
Sorption of carbofuran to support materials An adsorption isotherm was determined by conducting batch equilibrium experiments. Support materials were airdried overnight and milled into small pieces using a food blender and passed through a 2-mm sieve. Then, 0.125 g of support material powder was put into 100-ml glass tubes. Then, 25 ml of 0.01 M CaCl2 solution containing carbofuran at concentrations of 0.1, 1.0, 5.0, 10.0 and 20.0 mg l−1 was then added to the tubes. The tubes were shaken horizontally at a constant speed of 100 rpm for 48 h at an average room temperature of 29 ± 2◦ C. After 48 h, the solution was passed through a Whatmann filter paper No. 1 and the filtrate was extracted by the liquid-liquid partitioning method and quantified for carbofuran concentration by HPLC. The data were fitted to the Freundlich equation [20] to describe the kinetics of carbofuran sorption to the support materials.
Microorganism preparation and cell immobilization B. cepacia PCL3 (GenBank accession No. EF990634) [17] was used as the carbofuran degrader. This was grown in 100 ml of nutrient broth (NB) containing 5 mg l−1 of carbofuran at 30◦ C and shaken at 150 rpm for 36 h, before being harvested by centrifugation at 5000 rpm for 10 min at 4◦ C. The cell pellets were washed and re-suspended in BSM and were used as seed inoculums. The immobilization of B. cepacia PCL3 on each support material was conducted following the procedures previously described by Plangklang and Reungsang (2009). [13]
Stability of immobilized cells attached to support materials The stability of the immobilized cell on the support materials was investigated according to the method of Wang
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Environmental Technology et al. (2001). [21] Briefly, 10 g of each sample of immobilized cells was rinsed with sterile BSM and transferred to a 250-ml flask containing 100 ml sterile BSM. The flask was shaken at 150 rpm at 30◦ C for 30 min. The amounts of PCL3 on the support materials and washed out from the support materials to BSM were counted by the drop-plate technique. The moisture content of the immobilized cells on undelignified coir, bulrush, banana stem and water hyacinth stem were 64, 84, 76 and 82%, respectively, while the moisture content of the immobilized cells on delignified coir, bulrush and banana stem were 69, 86 and 82%, respectively. The amount of cells immobilized or washed out was calculated on a dry basis and expressed as cfu per g dry support material. The cell stability (%) was calculated according to Equation (1). IMC Stability (%) = × 100 (1) WC + IMC where IMC is the number of cells observed in the support material (cfu g−1 dry support material) and WC is the number of cells observed in BSM (cfu g−1 dry support material). Carbofuran degradation test with free and immobilized cells Degradation of carbofuran by the immobilized B. cepacia PCL3 cells was conducted in a 250-ml shake-flask in a batch experiment. One hundred ml of synthetic wastewater containing 5 mg l−1 of carbofuran was added to the flask before inoculation with the immobilized PCL3 at a final concentration of 106 cfu ml−1 . The flask was incubated at room temperature and shaken at 150 rpm. The liquid part was sampled at days 0, 3, 6, 10 and 15 to extract carbofuran and its metabolites (i.e. 3-ketocarbofuran and carbofuran phenol) by the liquid-liquid partitioning method and analysed for their concentrations by HPLC. The amount of PCL3 in the support material and in the synthetic wastewater was determined by the drop-plate technique. Degradation of carbofuran by B. cepacia PCL3 in freecell form was conducted in a 500-ml shake flask in a batch experiment as a control. Two hundred ml of synthetic wastewater containing 5 mg l−1 of carbofuran as the sole carbon source was added to the flask before inoculation with 10% inoculum of PCL3 (final cell concentration of 106 cfu ml−1 ). In order to determine the effect of the abiotic degradation process on the removal of carbofuran, sterile support materials were used instead of immobilized cells in the abiotic control treatments. Flasks were incubated and analysed for the concentration of carbofuran and its metabolites and the amount of PCL3 as previously described. After 15 days of incubation, the immobilized cells on each support material were harvested and re-inoculated into fresh synthetic wastewater containing 5 mg l−1 of carbofuran. Carbofuran degradation in synthetic wastewater, cell
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leakage and cell survival were determined at days 0, 3, 6, 10 and 15. This step was repeated three times to investigate reusability of the immobilized cells. Effect of carbofuran concentration This experiment was conducted to determine the maximum carbofuran concentration at which the PCL3 could survive and degrade carbofuran effectively. The biodegradation batch experiments were conducted in the same manner as above using free or suitable immobilized cells of PCL3 as the inoculum, except that the concentration of carbofuran was varied in the range 0–280 mg l−1 and the liquid part was sampled every 3 d until the carbofuran degradation reached steady state. Analysis methods Carbofuran and its metabolites, i.e. carbofuran phenol and 3-ketocarbofuran, were extracted from synthetic wastewater by the liquid-liquid partitioning method [13] with the recovery percentages of 98%, 96% and 95%, respectively. The extracts were analysed using a Shimadzu 10-A HPLC equipped with a 4.6 × 150 mm Lunar 0.5 μm C-18 column (Phenomenex, USA) with an ultraviolet (UV) detector operating at 220 nm. The HPLC operating parameters followed Plangklang and Reungsang (2009) [13] excepting that the column oven temperature was changed from room temperature to 40◦ C. The HPLC retention times of carbofuran, carbofuran phenol and 3-ketocarbofuran were 3.99, 3.34 and 4.56 min, respectively. The half-life (t1/2 ) of carbofuran in the synthetic wastewater was calculated by fitting the data to a modified first-order kinetic model using the SAS software program (SAS Institute Inc., Cary, NC). [22] Data were analysed by SPSS software program Version 10.0 (SPSS Inc., Chicago, IL). The significance of treatments was set at a p-value of less than or equal to 0.05 by the one-way ANOVA with Duncan’s post hoc test. The microstructure of the support materials and immobilized cells were investigated by scanning electron microscopy (SEM). [23] The number of carbofuran degraders in the synthetic wastewater and in the support material was determined by the drop-plate technique. [24] For growth and degradation kinetics analysis, the amount of PCL3 in the free-cell experiment was directly converted to the cell concentration by a linear relationship. For the immobilization experiment, the amount of PCL3 on the support material and leaking cells were added together before being converted to the cell concentration. Growth and degradation kinetic analysis Kinetic analysis for growth of PCL3 and its carbofuran degradation were evaluated by fitting the data to the bacterial growth and substrate degradation model using the Sigmaplot 9.0 software program (SPSS Inc., Chicago, IL).
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The Luong model proposed by Luong (1987) [25] was used to explain the bacterial growth and carbofuran degradation of PCL3 in free-cell form while the Monod (1949) model was used to explain the kinetic behaviour of the immobilized cells. [26] Results and discussions Sorption kinetic of carbofuran All sorption data fitted well to the Freundlich isotherm with the regression coefficient ranging between 0.91 and 0.97. Results indicated that the sorption isotherms were not linear (1/n = 1). The 1/n values were less than 1.0, indicating that concentration change had little effect on the adsorptive capacity. [27] The Kf values were used to describe the extent of sorption between carbofuran and the support materials and found to be correlated to the organic carbon content of the support materials (Table 1). The Kf values of carbofuran on undelignified support materials was less than that found on delignified support materials, which might be due to less organic carbon content in undelignified support materials. Undelignified and delignified coir had higher Kf values (20.88–24.23 l kg−1 ) than the other support materials (8.13–17.25 l kg−1 ). The sorption mechanism is believed to reduce diffusion limitation and facilitate the mass transfer between the contaminant and catalyst (microbial cells or enzyme) on the support materials. [28] Thus, the results implied that undelignified and delignified coir were good candidates as support materials for cell immobilization in bioaugmentation of carbofuran. Quantification and stability of cell attachment on the support The amount of PCL3 on the support materials was in the range of 1.46 × 108 –6.90 × 108 cfu g−1 dry support. The amount of PCL3 on delignified coir (2.90 × 108 cfu g−1 dry support) was approximately two times greater than the number of cells on undelignified coir (1.46 × 108 cfu g−1 dry support). This might be due to the fact that the delignification process removed the microcrystalline structure of lignin on the surface of coir but did not remove its complex layers (Figure 1(a) and (e)). This could result in a better Table 1.
sorption of the cells onto the surface of delignified coir than onto undelignified coir. In the case of bulrush, the amount of B. cepacia PCL3 on its undelignified form (6.87 × 108 cfu g−1 dry support) was greater than its delignified form (1.73 × 108 cfu g−1 dry support), which might be due to the loss of complex structure and pores from the surface of bulrush after delignification (Figure 1(b) and (f)). The amount of PCL3 on undelignified (2.17 × 108 cfu g−1 dry support) and delignified banana stem (1.96 × 108 cfu g−1 dry support) was not significantly different, which might be due to the physical appearance, texture and microstructure of banana stem, which were not changed after delignification (Figure 1(c) and (g). The amount of PCL3 on undelignified water hyacinth stem was relatively high at 6.00 × 108 cfu ml−1 , which could be a result of the clumping of cells on the surface (Figure 1(d)). The capability for high cell density retention is a good characteristic of a supporting material for cell immobilization because it allows high cell density loading to the contaminated area. However, the stability of the cells attached to the support materials should also be taken into account as an important prerequisite. [10] The stability of the immobilized cells attached on each support material is shown in Table 2. Results indicated that the immobilized cells on all support materials, except for delignified bulrush and undelignified water hyacinth stem, had a stability percentage greater than 75% (Table 2) which indicated a good characteristic of support materials for cell immobilization. Delignified water hyacinth stem is not considered to be a good support material since the stability of the immobilized cells was only 57.36%. SEM analysis of the support materials and immobilized B. cepacia PCL3 The microstructures of the support materials were determined by SEM and shown in Figure 1. From SEM, it could be seen that the support materials had complex layers and pores that were suitable for cell immobilization. Coir (Figure 1(a)) has more complex layers than bulrush (Figure 1(b)), banana stem (Figure 1(c)) and water hyacinth stem (Figure 1(d)). The microstructure of delignified coir (Figure 1(e)) remained the same as in the undelignified form (Figure 1(a)), while the complex layer of bulrush
Organic matter content, Freundlich sorption coefficient and sorption constant (1/n).
Type of support materials Undelignified coir Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem a Coefficients
Organic matter content (%)
Freundlich sorption coefficient (Kf ) (l kg−1 )
Sorption constant (1/n)
r 2a
89.11 97.41 80.99 85.76 78.52 79.73 73.63
20.88 24.23 11.36 17.25 11.05 16.84 8.13
0.76 0.55 0.88 0.54 0.44 0.43 0.72
0.91 0.94 0.96 0.92 0.92 0.91 0.97
of determination for linear plots of Freundlich isotherm regression.
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Figure 1. Scanning electron microscopic images of (a) undelignified coir, (b) undelignified bulrush, (c) undelignified banana stem, (d) undelignified water hyacinth stem, (e) delignified coir, (f) delignified bulrush, (g) delignified banana stem; immobilized B. cepacia PCL3 on (h) undelignified coir, (i) undelignified bulrush, (j) undelignified banana stem, (k) undelignified water hyacinth stem, (l) delignified coir, (m) delignified bulrush and (n) delignified banana stem.
(Figure 1(f)) and banana stem (Figure 1(g)) had been removed after delignification. Delignified water hyacinth stem was not observed by SEM and would not be used as a support material for cell immobilization because its structure was broken after delignification. The cell characteristic, attachment patterns and arrangement of B. cepacia PCL3 on the support materials were studied under SEM. As shown in Figure 1(h)–(n), cells were rod-shaped, approximately 1.6 μm in length and 0.7 μm in diameter when they were immobilized on coir, bulrush and banana stem in both undelignified and delignified forms (Figure 1(h)–(j) and (l)–(n)). When cells were immobilized on water hyacinth stem, the sizes of the cells were approximately 2.2 μm in length and 0.7 μm in diameter (Figure 1(k)). It could be observed that cells were arranged singly on all supports, except for water hyacinth stem in
which the cells were arranged as a cluster on its surface. Differences in cell shape and arrangement could be the result of nutrient acquisition and different attachment mechanisms to different surfaces. [29] SEM photographs indicated that B. cepacia PCL3 was able to access into the pores as well as reside on the surface area of the complex layers for all support materials excepting undelignified water hyacinth stem. The possible mechanism of cell attachment on these support materials is initially cell adhesion onto the surface area of support materials by the van der Waals force (an electrostatic force caused by different surface charges of bacterial cells and supports). When the time for cell adhesion is extended, there is a polymeric binding between the cells and the support materials that prevents reversible sorption and makes adhesion between cells and the surface of the support
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Table 2. Stability of the immobilized cells and first-order kinetic parameters including degradation rate constants and half-lives of carbofuran in synthetic wastewater by free cells, immobilized cells and reuse of immobilized cells on each support material.
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Treatment PCL3 in free-cell form Immobilized PCL3 cells on Undelignified coir Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem Reuse immobilized cells on undelignified coir First reuse Second reuse Third reuse Delignified coir First reuse Second reuse Third reuse Undelignified bulrush First reuse Second reuse Undelignified banana stem First reuse Second reuse Delignified banana stem First reuse
Stabilitya (%)
Degradation rate constants (k1 ) (d−1 )
Half-life (t1/2 ) (d)a
r 2b
0.0749 ± 0.0085
9.31c ± 1.05
0.98
0.2102 ± 0.0523 0.2175 ± 0.0030 0.2229 ± 0.0844 0.1012 ± 0.0016 0.1686 ± 0.0536 0.2429 ± 0.0064 0.1598 ± 0.0337
3.40a ± 0.85 3.19a ± 0.04 3.35a ± 1.27 6.85b ± 0.11 4.33a ± 1.38 2.85a ± 0.07 4.44a ± 0.94
0.99 0.99 0.98 0.99 0.99 0.99 0.99
0.277 ± 0.065 0.158 ± 0.017 0.169 ± 0.028
2.58a ± 0.61 4.41a ± 0.48 4.15a ± 0.67
0.98 0.91 0.98
0.211 ± 0.047 0.165 ± 0.007 0.112 ± 0.013
3.36a ± 0.75 4.21a ± 0.19 4.43a ± 0.52
0.99 0.96 0.99
0.232 ± 0.003 0.166 ± 0.017
2.99a ± 0.04 4.19a ± 0.43
0.98 0.92
0.209 ± 0.052 0.135 ± 0.022
3.42a ± 0.84 5.21a ± 0.84
0.99 0.93
0.270 ± 0.010
2.57a ± 0.10
0.98
78.72ab ± 1.84 77.75ab ± 2.41 82.36a ± 1.37 69.71c ± 2.42 77.12b ± 0.92 75.96b ± 1.36 57.36d ± 3.74
a Comparison between treatment in column are significantly different (p ≤ 0.05) if marked in different lowercase letters. b Coefficients of determination for non-linear regressions.
materials firmer and more stable. [11,30] In the case of undelignified water hyacinth stem, where the cells only clustered on the surface, the cell attachment mechanism is initially cell adhesion followed by polymeric linkage between cells with no polymeric binding between cells and supports. This can be the reason for the lower stability of immobilized cells on undelignified water hyacinth stem when compared with other support materials. Degradation of carbofuran by free and immobilized PCL3 cells Degradation of carbofuran in synthetic wastewater was described by a modified first-order kinetic model. The free PCL3 cells could degrade carbofuran in synthetic wastewater with a half-life of 9.31 d while the shorter half-lives in the range 2.85–6.85 d were obtained when immobilized cells were used (Table 2). The results suggested that the degradation ability of B. cepacia PCL3 could be increased by the immobilization technique. The high matrix porosity and porous nature of the support materials might be effective for physical adsorption of carbofuran to the support material and transference of carbofuran to the cells resulting in an enhanced carbofuran degradation efficiency. [31,32] The delignification process did not significantly affect the carbofuran degradation efficiency of the immobilized
cells on coir. This was suggested by the nonsignificant difference (p > 0.05) between the t1/2 of carbofuran in synthetic wastewater augmented with immobilized PCL3 on delignified coir compared to undelignified coir (3.19– 3.40 d) (Table 2). A significant longer t1/2 of carbofuran in synthetic wastewater (6.85 d) was observed when the immobilized PCL3 on delignified bulrush was used in comparison to the immobilized cell on undelignified bulrush (t1/2 of 3.35 d) (Table 2). The delignification process could improve the carbofuran degradation efficiency of immobilized cells on banana stem. The t1/2 of carbofuran in synthetic wastewater augmented with the immobilized PCL3 on delignified banana stem (2.85 d) was significantly shorter than that augmented with undelignified banana stem (t1/2 of 4.33 d). Delignification, in fact, could improve carbofuran sorption efficiency of the support materials (Table 1), which could facilitate the transfer of carbofuran to the cell inside the support materials, thus enhancing the carbofuran degradation efficiency. In addition, after lignin was removed, the fibres of the support material were softened, which could be used easily by the PCL3 as an additional energy source for its growth. The presence of additional energy sources in the pesticide bioremediation system might result in an increase in cell number as well as pesticide degradation activities. [33] However, in some cases, the additional energy sources could worsen the efficiency of pesticide remediation if the
0.065 nd
0.114 0.082 0.107 nd 0.417 0.643 0.408
0.051 0.032 0.053 0.124 0.071 0.028 0.022 nd nd nd 0.063 0.022
1.754 0.449 0.844 0.730 0.764 0.018 0.965
0.061 0.473
0.063 0.221
10 0.036 1.747
nd nd nd nd nd nd nd nd: not detectable.
Carbofuran phenol Immobilized PCL3 cells on Undelignified coir Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem
3-Ketocarbofuran
nd nd nd nd nd nd
0.013 0.116 nd nd 3-Ketocarbofuran Carbofuran phenol PCL3 in free-cell form
Immobilized PCL3 cells on Delignified coir Undelignified bulrush Delignified bulrush Undelignified banana stem Delignified banana stem Undelignified water hyacinth stem
9 6
nd nd nd nd nd nd nd
0.076 0.034 0.052 0.162 0.082 0.031
0.060 nd
35 15
18
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5 4 2 0 Metabolite Treatment
Incubation time (d)
Metabolites observed during biodegradation of carbofuran in synthetic wastewater by PCL3 in free and immobilized cell forms.
degrader prefers to use the additional energy sources rather than the target pesticide. [24] Carbofuran phenol and 3-keto carbofuran were the metabolites observed in free and immobilized cells experiments (Table 3) while only 3-keto carbofuran (0.051– 0.170 mg l−1 ) (data not shown) was observed in abiotic controls. The results suggested that carbofuran phenol was the metabolite from carbofuran biodegradation by PCL3. The concentration of carbofuran phenol tended to increase to its maximum value (0.449–1.754 mg l−1 ) after a certain time; it then decreased and was not detected at the end of incubation in all treatments (Table 3). This implied that carbofuran phenol could be utilized by B. cepacia PCL3, which is in agreement with previous published reports. [13,17] 3-Keto carbofuran concentration increased over time, and it accumulated until the end of incubation in all treatments. The maximum concentrations of 3-keto carbofuran ranged between 0.031 and 0.162 mg l−1 . Since the concentrations of 3-keto carbofuran observed in biodegradation treatments were not markedly different from the abiotic control, and there had been evidence that PCL3 degraded carbofuran to carbofuran phenol but not 3-keto carbofuran, [13] we suggested that 3-keto carbofuran was the metabolite from the abiotic degradation process. The survival and growth of free and immobilized cells were determined by the amounts of B. cepacia PCL3 in synthetic wastewater (Figure 2(a)) and on the support materials (Figure 2(b)). Results indicated that free cells and the immobilized cells on all materials could survive 15 d of incubation at cell numbers of approximately 106 cfu ml−1 (Figure 2(a)) and 108 to 109 cfu g−1 dry support material, respectively (Figure 2(b)). Cell leakage from the support materials was determined from the cell concentration observed in synthetic wastewater during incubation (Figure 2(c)). At day 0, the number of PCL3 cells in the liquid phase of synthetic wastewater was negligible. After day 3, the amount of PCL3 in the synthetic wastewater was in the range 105 –107 cfu ml−1 (Figure 2(c)). Since the number of cells on support materials was not decreased, we suggested that the leakage of the cells from the support materials might have resulted from the overgrowth of cells within the limited space of the support materials. In addition, there is no barrier between the cell and the synthetic wastewater, which led to the possibility of cell detachment and relocation with the establishment of cell equilibrium inside the support material and synthetic wastewater. [10] The dissipation of carbofuran could be observed in synthetic wastewater augmented with sterile support materials (abiotic control). After 15 d of incubation, approximately 18% of the carbofuran was dissipated in synthetic wastewater with sterile undelignified coir, delignified and undelignified banana stem, undelignified bulrush and undelignified water hyacinth stem; while approximately 30% carbofuran removal was observed with sterile delignified coir and delignified bulrush. The dissipation of carbofuran might
Table 3.
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Figure 2. Amount of B. cepacia PCL3 in (a) free and (b) immobilized cell forms grown in synthetic wastewater and (c) number of leaking cells from the support materials.
be caused by the sorption of carbofuran onto the surface and pores of the support materials during incubation. The greater carbofuran degradation rates observed in the treatments with immobilized cells (t1/2 ranged between 2.85 and 6.85 d (Table 2) than in the abiotic controls suggested that carbofuran dissipation in synthetic wastewater mainly resulted from the biodegradation process.
carbofuran concentration on growth patterns and degradation kinetics of immobilized PCL3 on coir were studied in the following experiment.
Reusability of immobilized B. cepacia PCL3 on each support material
The correlation between specific growth rate of PCL3 in free and immobilized cell forms and the initial concentration of carbofuran is depicted in Figure 3. The decrease in specific growth rate with the increase in initial carbofuran concentration in the free-cell experiment implied that carbofuran acts as an inhibitor on PCL3. Therefore, the substrate inhibition model (Luong model) was used to explain the growth kinetics of PCL3 in free-cell form. The regression coefficients, r 2 , of 0.99 indicate a good fit of the data to the models. The estimated kinetic parameters for free PCL3 cells from the Luong model were maximum specific growth rate (μmax ) = 1.70 d −1 and substrate saturation constant (Ks ) = 33.12 mg l−1 . The empirical constants n (1.22) estimated by the Luong model were greater than 1, suggesting that the non-linear relationship between specific growth rate (μ) and initial carbofuran concentration (S) existed during the inhibition process. The maximum substrate concentration above which carbofuran degradation was completely inhibited (Sm ) was 247.83 mg l−1 . The inhibitory effect of carbofuran on the immobilized PCL3 was not observed at concentrations up to 280 mg l−1 , which confirmed that the immobilization technique could protect the cells of PCL3 from substrate inhibition. The Monod model was used to explain the growth kinetics of the
Investigation into the reusability of PCL3 on undelignified water hyacinth stem and delignified bulrush was not conducted due to the degradation of the support materials after 15 d of incubation. Undelignified coir could be reused three times, undelignified bulrush and undelignified banana stem could be reused two times and delignified banana stem could be reused once. The corresponding kinetic data fitted to a modified first-order kinetic model are tabulated in Table 2. The results revealed that the reused immobilized cells on the tested support materials could effectively degrade carbofuran in synthetic wastewater with a short t1/2 of 2.57–5.21 d (Table 2), which was not significantly different from the t1/2 of carbofuran degraded by the first-time usage immobilized cells (2.85–6.85 d) (Table 2). From the results obtained, it could be concluded that undelignified coir is the most suitable support material to immobilize PCL3 for remediating carbofurancontaminated water. The immobilized PCL3 on undelignified coir possessed a high stability of 78.72%, a high carbofuran degradation ability with a short t1/2 of 3.40 d, and could be reused four times with carbufuran degradation ability still intact (t1/2 of 2.58–4.15 d). The effects of
Effect of initial carbofuran concentration in synthetic wastewater on the growth and carbofuran degradation kinetics of B. cepacia PCL3 Growth kinetics
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Environmental Technology
Figure 3. (a) Specific growth rate and (b) specific degradation rate of PCL3 in free and immobilized cells forms at various initial carbofuran concentrations.
immobilized PCL3 with an r 2 of 0.98. The estimated growth kinetic parameters for the immobilized PCL3 obtained from the Monod model were μmax = 0.85 d −1 and Ks = 26.77 mg l−1 . The lower μmax value was obtained when the immobilized cells were used in comparison to free cells, which might be because the immobilized cells could not grow freely under the conditions with limited space inside the support material. The lower Ks value obtained indicated that at the low carbofuran concentration the immobilized PCL3 was able to access carbofuran in synthetic wastewater better than free cells. This result confirmed that sorption of carbofuran to the support material could enhance the mass transference between carbofuran and the cells inside the support. Degradation kinetic The degradation rate coefficient of carbofuran was calculated by fitting to a modified first-order kinetic model in order to examine the t1/2 of carbofuran in synthetic
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wastewater (Table 4). Results indicated that immobilized cells were more efficient in degrading carbofuran than free cells at any range of carbofuran concentration used in this study. In the free-cell experiment, the significantly longer t1/2 of carbofuran (p < 0.05) was obtained when the initial carbofuran concentration was increased (Table 4). The t1/2 of carbofuran at the initial carbofuran concentration of 250 mg l−1 was 8.2 times longer than the carbofuran t1/2 at the initial carbofuran concentration of 5 mg l−1 . In the immobilized cell experiments, t1/2 of carbofuran (3.4–6.3 d) was not significantly different at the initial concentrations of 5 to 50 mg l−1 (Table 4). The slightly longer t1/2 of carbofuran t1/2 of 15 d could be found when the concentration of carbofuran was increased from 50 mg l−1 to 280 mg l−1 . These results indicated that the inhibitory effect on carbofuran degradation by PCL3 at a high carbofuran concentration could be reduced by the immobilization technique. The specific carbofuran degradation rate from free and immobilized PCL3 at different initial carbofuran concentrations are shown in Figure 3. The carbofuran degradation kinetic parameters of PCL3 in free-cell form estimated from the Luong model (r 2 = 0.94) were maximum specific carbofuran degradation rate (qmax ) = 1.38 d −1 Ks = 34.78 mg l−1 and n = 1.3. The degradation kinetic parameters of immobilized PCL3 were estimated from the Monod model (r 2 = 0.96) where qmax and Ks were 1.92 d−1 and 30.32 mg l−1 , respectively. The inhibitory effect of carbofuran was observed in the free-cell experiment in which the maximum substrate concentration above which carbofuran degradation was completely inhibited (Sm ) was 252.80 mg l−1 , while an inhibitory effect of carbofuran on the immobilized cells was not observed. The results confirmed that the immobilization technique could protect the PCL3 cell from substrate inhibition, hence enhancing carbofuran degradation efficiency. The enhancement of contaminant degradation activity by the immobilization technique has also been reported for different mechanisms. The immobilization of viable cells could alter their physiological
Table 4. Degradation rate constants (k1 ) and half-lives (t1/2 ) of carbofuran in PCL3-inoculated synthetic wastewater with various initial carbofuran concentrations. Initial carbofuran concentration (mg l−1 ) 5 10 50 100 150 200 250
Free cells of PCL3 k1 (d−1 )
t1/2 (d)a
r 2b
0.074 ± 0.008 0.069 ± 0.028 0.060 ± 0.005 0.046 ± 0.012 0.020 ± 0.003 0.012 ± 0.002 0.009 ± 0.001
9.34a ± 0.95 10.80ab ± 4.33 11.56b ± 0.99 15.68b ± 4.03 35.23c ± 5.87 59.86d ± 8.68 76.30e ± 4.74
0.98 0.99 0.98 0.99 0.99 0.98 0.99
Initial carbofuran concentration (mg l−1 )
Immobilized PCL3 k1 (d−1 )
t1/2 (d)a
r 2b
5 10 50 120 200 280
0.210 ± 0.052 0.163 ± 0.020 0.119 ± 0.045 0.093 ± 0.023 0.071 ± 0.012 0.047 ± 0.008
3.40a ± 0.85 4.30a ± 0.54 6.29ab ± 2.39 7.69b ± 1.90 9.90b ± 1.73 15.00c ± 2.58
0.99 0.99 0.99 0.99 0.97 0.98
between treatments in the column are significantly different (p ≤ 0.05) by Duncan’s Multiple Range test if marked in different small letters. b Coefficients of determination for non-linear regressions.
a Comparison
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features of metabolism such as enhanced induction of the enzyme responsible for contaminant degradation. [34] The support materials could act as a protective shelter against the toxicity of the contaminant and adverse environmental conditions, hence the survival of the cell could be improved, resulting in an increase in degradation efficiency. [13,35] In addition, the immobilized cells could be used as the source for continuously providing the cells of the degraders in the bioremediation system. [36,37]
Conclusions Undelignified coir was the most suitable support material for cell immobilization giving the short t1/2 of carbofuran of 3.40 d (2.8 times shorter than the treatments with free cells). The immobilized cells on undelignified coir could be reused three times without a loss of the ability to degrade carbofuran. The kinetic characterization of free and immobilized PCL3 revealed that the growth and degradation ability of free cells were completely inhibited at the initial carbofuran concentrations of 250 mg l−1 . The immobilization technique was able to reduce the inhibitory effect of carbofuran on PCL3; the growth and carbofuran degradation ability of the immobilized PCL3 on coir did not worsen at concentrations up to 280 mg l−1 . The results from this study indicated the great potential of using immobilization techniques to improve the overall efficiency of carbofuran bioremediation in contaminated water by B. cepacia PCL3. Acknowledgements This work was supported by grants from the Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand and the National Center of Excellence for Environmental and Hazardous Waste Management, Bangkok, Thailand.
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