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Chen, H., Liu, Y., Kong, F., Lucia, L. A., and Feng, X. (2015). "Application of the protoplast fusion technique to engineer a recombinant microorganism to more efficiently degrade chlorophenols," BioRes. 10(3), 5720-5729.


Recombinant strain Xz6-1 was constructed by the protoplast fusion technique with the goal of endowing it with the ability to efficiently degrade pentachlorophenol (PCP). This compound was considered as a representative of possible compounds that can be obtained during the bleaching of pulp for papermaking. The potential of Xz6-1and Pseudomonas putida to treat PCP synthetic wastewater was explored. The majority of PCP was removed within the first 20 h; two degradation curves were obtained that followed first-order reaction kinetics. The kinetics data revealed that the rate constant for degradation of PCP for Xz6-1 was 0.063 h-1, a value that was over 50% greater than that of Pseudomonas putida (0.040 h-1). Aerobic granular sludge was highly fortified with Xz6-1 and Pseudomonas putida to provide PCP degradability improvements of 180.9% and 98.3%, respectively, relative to the original sludge. All results demonstrate that the protoplast fusion technique is an effective approach to construct a high-activity chlorophenol-degrading strain.

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Application of the Protoplast Fusion Technique to Engineer a Recombinant Microorganism to More Efficiently Degrade Chlorophenols

Honglei Chen,a,b,* Yu Liu,a Fangong Kong,a Lucian A. Lucia,a,c and Xiaojing Feng d

Recombinant strain Xz6-1 was constructed by the protoplast fusion technique with the goal of endowing it with the ability to efficiently degrade pentachlorophenol (PCP). This compound was considered as a representative of possible compounds that can be obtained during the bleaching of pulp for papermaking. The potential of Xz6-1 and Pseudomonas putida to treat PCP synthetic wastewater was explored. The majority of PCP was removed within the first 20 h; two degradation curves were obtained that followed first-order reaction kinetics. The kinetics data revealed that the rate constant for degradation of PCP for Xz6-1 was 0.063 h-1, a value that was over 50% greater than that of Pseudomonas putida (0.040 h-1). Aerobic granular sludge was highly fortified with Xz6-1 and Pseudomonas putida to provide PCP degradability improvements of 180.9% and 98.3%, respectively, relative to the original sludge. All results demonstrate that the protoplast fusion technique is an effective approach to construct a high-activity chlorophenol-degrading strain.

Keywords: Protoplast fusion technique; Pentachlorophenol; Synthetic wastewater; Biodegradability

Contact information: a: Key Lab of Pulp and Paper Science and Technology of the Ministry of Education (Shandong province), Qilu University of Technology, Jinan, 250353, Shandong, China; b: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, 510640, Guangdong, China; c: Laboratory of Soft Materials & Green Chemistry, North Carolina State University, Department of Forest Biomaterials, Raleigh, NC 27695-8005, United States; d: Shandong Academy of Environmental Science, Jinan, 250013, Shandong, China; *Corresponding author:


Chlorophenols are in general very toxic and recalcitrant compounds produced from industrial operations. Such compounds are discharged into receiving waters and may gradually accumulate in the environment (Bum et al. 1999; Takeuchi et al. 2000; Essam et al.2006). They were widely produced in industries such as glue manufacture, pesticides, paint, leather, and in the bleaching of pulp for papermaking (Liu et al. 1991; Wang and Chen 2005). Consequently, it is necessary to take steps for the development and application of economic, simple, and efficient methods to eliminate these contaminants.

Worldwide, there are several existing treatment alternatives available for the removal of chloroaromatics from industrial effluents, such as aerobic, anaerobic, electrochemical, photocatalysis, and Fenton treatment (Xu et al. 2003; Belmonte et al. 2006; Hong et al. 2008). The biological oxidation process is often considered as the common option due to its cost-effectiveness, versatility in handling a wide variety of organic pollutants, and it helps to avoid secondary pollution.

Many researchers have focused on the biodegradation of chloroaromatics with single dominant bacteria and have demonstrated that the Pseudomonas putida strain is capable of effectively degrading chlorophenols as sole substrates (Farrell and Quilty 2002; Fakhruddin and Quilty 2007).

The objective of the present investigation was to construct a high-activity pentachlorophenol (PCP)-degrading strain by using the protoplast fusion technique. The improvement of chlorophenol-degrading ability of the recombinant strain was investigated by comparing the degradation properties of the recombinant strain and its parent strain. An engineered microorganism can be of paramount significance for the treatment of industrial wastewaters rich in chlorophenols.




Pseudomonas putida and Psathyrella candolleana were obtained from the State Key Laboratory of Pulp and Paper Engineering, South China University of Technology.

Preparation of PCP synthetic wastewater

PCP synthetic wastewater was obtained by a previously published procedure (Chen et al. 2013). Three kinds of effluents from ClO2bleaching (D), chelating treatment (Q), and H2O2 bleaching (P) were selected and mixed in accordance with the ratio of 1:1:1 to form DQP bleaching effluent. The mixing effluent was diluted for 5 times, and the COD of diluted effluent was observed to be 212 mg/L. PCP synthetic wastewater were prepared by dissolving a certain amount of PCP in diluted DQP bleaching effluent, and the PCP concentration was 100 mg/L.


Microorganisms cultivation

Pseudomonas putida and recombinant strains were cultivated using the method as follows: One loop of strain from the culture-contained agar was separately transferred to 100 mL of the nutrient medium (beef extract 1.5 g/L; glucose 1.0 g/L; peptone 6.0 g/L; yeast extract 3.0 g/L; pH 7.0.) in a glass flask and activated at 30 °C for 24 h. These activated cells were harvested by centrifugation (5000 rpm for 5 min) as inocula in the late exponential phase, and the cells collected were washed with phosphate buffer solution (PBS, NaCl 8 g/L; KCl 0.2 g/L; KH2PO4 0.2 g/L; K2HPO4 1.15 g/L.).

Psathyrella candolleana was transferred to 100 mL of the potato dextrose broth medium (PDB = 200 g potatoes were peeled and cut into 1 cm3 pieces and boiled in 500 mL of deionized water for 20 min; the extract was collected by filtration through gauze, followed by addition of 20 g glucose and water to 1000 mL in total volume of neutral pH value) from potato dextrose agar medium (PDA, 2% agar in PDB) after scattering with a parallel scrambler and cultivation for 72 h. Mycelium was gathered and then transferred to 25 mL of basal medium (glucose 10 g/L; CaCl2 0.01g/L; KH2PO4 2 g/L; MgSO40.25 g/L; ethylenediamine tartrate 0.5 g/L; NH4NO3 0.5 g/L; and 10 mL of trace elements solution (FeSO4·7H2O 7.5mg/L; MnSO4·H2O 2.5mg/L; ZnSO4·7H2O 2 mg/L; CoCl2 ·6H2O 3 mg/L; CuSO4·5H2O 6 mg/L; Na2BO3 3 mg/L)) after scattering. Inoculated media were incubated for 12 h at 30 °C.

Isolation of protoplasts

Pseudomonas putida cells were harvested in a 5 mL tube by centrifugation at 3000 rpm for 10 min, washed with PBS buffer and re-suspended in 1 mL of NSM buffer (NaCl 0.55 mol/L; sodium succinate 0.2 mol/L; MgCl2·6H2O 0.02 mol/L; pH 6.8) containing 1 mL of lysozyme solution (lysozyme powder was dissolved in 50 mL of NSM solution and filtered with 0.22 μm filter.) and 1 mL EDTA solution (Na2EDTA 0.13 mol/L; NaCl 0.55 mol/L). The cells were shaken at 150 rpm at 37 °C for 60 min to allow the digestion of the peptidoglycan. The protoplasts were obtained and stained with fluorescein 5-isothiocyanate at the concentration of 50 mg/L in PBS for 3 min.

The mycelium of Psathyrella candolleana was harvested in a 5 mL tube by centrifugation at 3000 rpm for 10 min, washed with PBS buffer, and resuspended in 1 mL NSM buffer containing 1 mL mixed enzyme solution (1% cellulase and 0.2% helicase, two enzymes were dissolved in 0.6 mol/L MgSO4 solution) and 1 mL EDTA solution. The mycelium was shaken at 150 rpm at 37 °C for 60 min to begin removal of the cell wall, and the protoplasts were then stained with rhodamine 6B at the concentration of 200 mg/L in PBS for 3 min.

Protoplast fusion

The protoplasts of two parent strains were harvested by centrifugation at 2000 rpm for 10 min at 5 °C, respectively, following by mixing and re-suspended in 1 mL of PEG buffer (30% PEG6000, Sigma Chemicals Co., USA, prepared in STC buffer containing 0.6 M sorbitol; 10 mM Tris–HCl; 10 mM CaCl2, pH 6.5). The fusion mixture was incubated at 37 °C for 5 min, and then diluted with 1 mL of NSM buffer. The hybrid cells with both fluorescent labels were selected using a flow cytometer (Guava easyCyte 8, Merck Millipore Co., USA) and re-suspended in 1.5 mL of NSM buffer.

Immobilization of cells

Pseudomonas putida and Xz 6-1 were immobilized according to the conventional immobilization procedure using Na-alginate (Nagadomiet al. 1999), as follows: The cell suspension in 100 mL deionized water (OD600 = 0.4) was mixed with autoclaved alginate solution (6%, w/v) of 100 mL. The mixture was stirred until stable gelatinous liquor was formed, put into an injector, and extruded from the needle into a 2% (w/v) CaCl2 solution to form microspheres of small size (3 to 5 mm). The microspheres were rinsed for three times with sterile water and kept in PBS buffer.

Intensification of granular sludge

The collected cells were transferred into a 300 mL conical flask which contains 100 mL of fresh LB medium (tryptone, 10.0 g/L; yeast powder, 5 g/L; NaCl,10 g/L) and 0.5 g (dry weight) of granular sludge. Microbial cells and sludge were co-cultured with shaking of 150 rpm in 30 °C for 24 h, in order to fix the cells on the granular sludge. Fortified sludge was collected by centrifugation at a speed of 5000 rpm and stored with PBS buffer after washing.

Biodegradation with free cells

Recombinant strains selected were propagated to the 15th generation and then used to carry out PCP biodegradation experiment, comparing the results with Pseudomonas putida. The cells collected were inoculated into PCP synthetic wastewater to give an initial optical density at 600 nm (OD600) of 0.20±0.01. After inoculation, the conical flasks were capped with cotton plugs and placed in a shaker controlled at 150 rpm and 30 °C. Cell concentration was tested every two hours, and each experiment was stopped when there was no further increase of OD600 (stationary phase). The experiments were performed in triplicate.

Biodegradation with immobilized cells

Immobilized cells were inoculated in a glass flask with 200 mL of PCP synthetic wastewater with a volume ratio of immobilized microspheres and wastewater 3:20, and then the mixture was shaken at 150 rpm at the optimal mesophilic temperature range (30±1°C) for 20 h. The experiments were performed in three replicates.

Biodegradation with granular sludge

The various sludges were added separately into a 500 mL glass flask containing 200 mL of PCP synthetic wastewater to give a sludge concentration of 3 g/L (dry weight), and then cultured with shaking (150 rpm) in 30 °C. 10 mL of wastewater was taken from each conical flask for the analysis of PCP every 8 h, and the experiment was terminated when there was no further reduction of PCP. The experiments were performed in three replicates.

Measurement of UV-visible spectroscopy

All spectrophotometric measurements were made with an Aglient-8453 UV/VIS spectrophotometer (Agilent Technologies, USA), equipped with 1.0 cm quartz cells. Distilled water was used as the blank solution. All spectra were recorded from 190 to 1100 nm with 1.0 nm bandwidth and a scan speed of 1000 nm/min.

Measurement of PCP in wastewater

PCP in synthetic wastewater was quantified based on a UV-vis spectroscopy method, as has been proposed in a previous study (Chen et al. 2013).


Protoplast Fusion

Fusion between Pseudomonas putida and Psathyrella candolleana was performed to construct high-activity chlorophenol-degrading strains. Twelve recombinant strains were isolated based on the morphological characteristics of colonies and an antagonism phenomenon (Table 1), whereas four strains were verified for chlorophenol-degrading capability (Fig. 1).

Figure 1 shows a characteristic UV-Vis peak at 321 nm due to the PCP in wastewater (Chen et al. 2013). The absorbance of original PCP synthetic wastewater was 2.64, and it dropped by varying extents when treated with Xz 6-1Xz 6-3Xz 6-5Xz 8-2, and Pseudomonas putida. The PCP degradation rates were obtained using a PCP determination method proposed in a previous study (Chen et al. 2013), and are shown in Table 2. It was shown that the PCP degradation of Xz8-2 and Xz6-5 were less than the parent strain (Pseudomonas putida), while Xz6-1 and Xz6-3 degraded PCP more effectively than Pseudomonas putida. Xz6-1, especially, showed a remarkable removal rate of 77.1%, a value that improved by 22.9% over Pseudomonas putida, and improved by 14.1% than the encapsulated cells method reported in the literature (Cassidy et al.1997).

Thus, Xz6-1 was further used as a recombinant strain with high-activity chlorophenol degradation capacity.

Table 1. Colonial Morphologies of Recombinant Strains and Parent Strains

Fig. 1. UV-visible spectra of synthetic wastewaters treated by different strains

Table 2. PCP Removal Rates of the Different Degraders

Degradation Kinetics of Non-Immobilized Pseudomonas putidaand Xz6-1 for PCP

The degradation curves for the non-immobilized cells were plotted and are shown in Fig. 2.

Fig. 2. Degradation curves of PCP by Pseudomonas putida and Xz6-1

Fig. 3. Degradation kinetics of PCP by Pseudomonas putida and Xz6-1

The degradation pattern of Xz6-1 is similar to Pseudomonas putida. PCP was degraded within the first 20 h at high rates and was then slowed down significantly to 71.2% and 52.4% within 20 h, respectively. The degradation kinetics curves of the two strains within 20 h are shown in Fig. 3, which showed the variation of the log of (C/C0) as a function of time (where C is the residual PCP concentration at a specific processing time; C0 is the original PCP concentration in synthetic wastewater, and t is time). For both strains, the kinetics showed that there is a good linear relation between ln (C/C0) and t within 20 h indicating that the PCP degrading processes of Pseudomonas putida and Xz6-1 followed first order reaction kinetics models.

The first order reaction kinetics equation can be described as:


Equation (1) was integrated to obtain Eqs. 2 and 3,



where k is the reaction rate constant or the negative slope of the kinetics curves from Eq. (3). Therefore, PCP degradation kinetics models of Pseudomonas putida and Xz6-1 were established (Table 3). The value of k can reflect the PCP biodegradability by the strains. As previously demonstrated, a k value of 0.063 h-1 was obtained for Xz6-1 at a half-life (t1/2) = 17.33, a half-life that is significantly much higher than that of Pseudomonas putida (k = 0.040 h-1and t1/2=10.98).

Table 3. PCP Degradation Kinetics Models

PCP Degradation by Immobilized Cells

The UV-Vis spectra of different wastewater samples treated by various immobilized cells are shown in Fig. 4. Peak absorbance values at λ=321 nm dropped to 1.11 and 1.54 after treatment with immobilized Xz6-1 and immobilized Pseudomonas putida, respectively.

The PCP removal rates of immobilized cells were obtained based on precedent (Chen et al. 2013). For Xz6-1, the removal rate was 82.7%, which is much higher than the 62.1% removal rate attributed to Pseudomonas putida (Table 2), indicating that the ability of immobilized Xz6-1 to degrade PCP is also superior to that of its immobilized parent strain. Meanwhile, a contrasting analysis was performed between immobilized cells and free cells for Xz6-1, which showed that the removal rate improved from 77.1% to 82.7% upon cell immobilization.

Fig. 4. UV-visible spectra of synthetic wastewater treated by immobilized strains

PCP Degraded by Granular Sludges

Figure 5 shows the UV-Vis spectra of synthetic wastewaters treated by different granular sludges, including the original sludge, the sludge fortified with Pseudomonas putida (abbreviated as Spp), and the sludge fortified with Xz6-1 (abbreviated as Sxz). As can be seen in Fig. 5, the PCP degradability of the original sludge was the lowest. A slight reduction of the absorbance value at 321 nm from 2.64 to 2.39 was observed.

Fig. 5. UV-visible spectra of synthetic wastewaters treated by granular sludge.

Bioaugmentation improved PCP degradability by introduction of exogenous bacteria into the original sludge. For synthetic wastewaters treated by Spp and Sxz, the absorbance values (321 nm) dropped to 2.24 and 2.05, respectively. Table 2 summarizes the removal extents of PCP in synthetic wastewater by different granular sludges. The value is just 10.7% for the original sludge, while the removal rates of Spp and Sxz were 21.2% and 30.0%, which means the PCP degradability of two fortified sludges were enhanced by 98.3% and 180.9%, respectively, relative to the original sludge. It also indicated that the fortification by the recombinant strain (Xz6-1) is superior to that of parent strain (Pseudomonas putida). van Limbergen et al. (1998) reported that few reports existed in the literature describing the augmentation of activated sludges with microorganism possessing catabolic activities, resulting in improved degradation of xenobiotics. However, our research have proved the bioaugmentation of activated sludge can result in enhanced degradation of chlorophenols.


1. A new strain (Xz6-1) with the ability to degrade chlorophenols efficiently by protoplast fusion between Pseudomonas putida and Psathyrella candolleana is herein described. During PCP degradation using Xz6-1 and Pseudomonas putida, most of the PCP was removed within the first 20 hours following first order reaction kinetics. The kinetics models of two strains have been established. The kinetic parameter k of Xz6-1 was 0.063 h-1, while it was 0.040 h-1 for Pseudomonas putida.

2. The PCP removal rates reached to 82.7% and 62.1%, when the PCP synthetic wastewaters were treated with immobilized cells of Xz6-1 and Pseudomonas putida, respectively.

3. Granular sludge was fortified with Xz6-1 and Pseudomonas putida, respectively. The PCP removal rate for the original sludge was only 10.7%, while the removal rates were 21.2% and 30.0%, respectively, when Spp and Sxz were used. The PCP degradability of two fortified sludges was improved by 98.3% and 180.9% over the original sludge.


The authors are grateful for the financial support obtained from the Project of Shandong Province Higher Education Science and Technology Program (No. J13LD03), the Open Fund of State Key Laboratory of Pulp and Paper engineering (No. 201405), and the National Science Foundation of China (Grant Nos. 31070525 and 31270627).


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Article submitted: June 11, 2015; Peer review completed: June 29, 2015; Revised version received and accepted: July 14, 2015; Published: July 27, 2015.

DOI: 10.15376/biores.10.3.5720-5729