Xueping Liu,Ping Xue,Feng Jia,Dongya Qiu,Keren Shi,Weiwei Zhang

State Key Laboratory of High-efficiency Coal Utilization and Green Chemical Engineering,College of Chemistry and Chemical Engineering,Ningxia University,Yinchuan 750021,China

Keywords:Polymeric composite Immobilization Biocatalysis Phenolic compounds Degradation Reusability

ABSTRACT Phenol and its derivatives are highly toxic pollutants in industrial wastewater for the ecological environments,so there is essential attention to develop effective means of removing these harmful substances from water.In this work,the microorganism was immobilized into polymeric composite gel beads prepared by the effective recombination of natural abundant chitosan (CS) and industrial polyvinyl alcohol(PVA) for treating phenolic compounds.The degradation rate of 99.5% can be achieved to treat 100 mg﹒L-1 of phenol at 30°C using the fresh resultant immobilized microorganism,where only 21.1%degradation rate was obtained by the free microorganism under the identical conditions.The recycling experiments of repeated 90 times to treat 100 mg﹒L-1 of phenol displayed that the degradation rate of phenol was stable to 99% with the appearance of beads unchanged significantly,indicating the immobilized microorganism possessed excellent operating stability.Moreover,while the phenol derivatives of 100 mg﹒L-1 were treated catalytically including p-methylphenol,catechol,and o-aminophenol for 24 h by the immobilized microorganism,the degradation rates were all above 95%.The immobilized microorganism into PVA-CS polymeric composite with excellent operating stability and degradation activity would provide a feasible solution for treating phenolic compounds in water in industrial applications.?2020 The Chemical Industry and Engineering Society of China,and Chemical Industry Press Co.,Ltd.All rights reserved.

1.Introduction

Phenol and its derivatives including p-methylphenol,catechol and o-aminophenol are highly toxic pollutants,mainly source in industrial wastewater such as papermaking,coking,oil refining,plastics,textiles,and so on,which are common organic pollution in industrial wastewater [1–3].Due to its wide source,hard-todegrade,easy-to-rich and toxic as well as persistent,phenol typically has been listed as a priority pollutant by the Chinese Environmental Protection Agency and the US Environmental Protection Agency [4,5].However,the traditional physical method [6,7] and chemical technology [8] do not address these issues well because the technical,economic and environmental issues associated with each method hinder the development of acceptable strategies for removing these compounds.There are significant disadvantages in physical removal,e.g.high running cost,low processing efficiency and difficult regeneration of adsorbents.A large number of chemical reagents in chemical treatment could be required because of the high cost and secondary pollution.Dissimilarly,the biodegradation using biocatalyst enzyme or microorganism is an effective,non-secondary pollutant,environmentally friendly and sustainable water treatment technology.However,the enzymatic catalysis of biodegradation will be limited substantially by the high price of oxidoreductase in large scale industrial water treatment [9–11].In contrast,the utilizing microorganisms at low cost in biodegradation to treat phenolic water should be the development potential for industry application [12].

The immobilization of microorganisms has always been a quite challenging task due to low operational stability,difficult recycle and reuse of free microorganisms in water treatment applications.Some researchers had reported the immobilized microorganism used as a biocatalyst to remove phenolic compounds.Compared with the traditional biotechnology,immobilized microorganisms technology possesses many superior characteristics:(i) improving the stability and tolerance of microorganisms to thermal and pH;(ii) reducing the running cost and achieving green,security and economic development;(iii) using repeatedly with high treating efficiency in unit processing [13,14].For example,Jiang et al.[15]studied the immobilized Acinetobacter on polyvinyl alcohol to degrade phenol.The results manifested that the degradation rate of phenol(800 mg﹒L-1)treated by immobilized microorganism was 75.8%in 24 h,while that of phenol by free microorganism was only 30.6% under the same conditions,indicating that the biodegradability of Acinetobacter for phenol could be improved by the immobilization.Bacillus subtilis was embed in polyethylene oxide by Satchanska et al.[16] to degrade phenolic aqueous solution with different concentrations (300–600 mg﹒L-1),and the results displayed that the degradation rate of phenol with different concentration could be close to 100% with the extension of reaction time.When the concentration of phenol was 300 mg﹒L-1,400 mg﹒L-1and 600 mg﹒L-1,the required degradation time was 7 d,12 d and 16 d,respectively.Increasing the concentration of phenol to 1000 mg﹒L-1,the degradation rate was less than 10%even if treating time was extended to 28 days,which indicated that phenol solution with high concentration may inhibit the activity of immobilized microorganism.At present,the immobilized microorganism has been applied in phenolic wastewater treatment,but there are still some practical problems to be solved in terms of improving the reusability of biocatalysts and raising treatment efficiency as well as reducing running cost.The expedient choice of immobilizing materials and preparation techniques is critical to solve the above problems[17,18].Polymer microspheres are stable in chemical and mechanical performance and its stabilities are even unaffected by microbial,acid and base.Hence,polymer materials employed as immobilizing supports could improve the stability and reusability of biocatalyst[19–21].The main purpose of this study is to prepare the effective and reusable immobilized microorganisms for treating hazardous phenolic compounds in water,where free microorganisms are difficult to be recycled and reused in current water treatment.The industrial polyvinyl alcohol and bio-friendly natural polymer chitosan were used as raw materials to obtain polymeric composite gel beads encapsulated microorganisms with porous channels by polymeric recombination.The biodegradation ability and reusability of the encapsulated microorganism beads for phenol and its derivatives were investigated systemically.

2.Materials and Methods

2.1.Materials

The commercial microorganism (MO,code Q500206.G86.2987-2006) and its activator were purchased from General Environmental Protection Technology Company and Anxinda Environmental Protection Technology (Ningbo,China) Co.,Ltd.The MO was mainly composed of Pseudomonas (33.7%),Delftia (11.31%),Elizabethkingia (8.25%),Cellulomonas (3.65%) and Bacillus (3.14%),and the activator for microorganism was mainly composed of glucose,starch,protein,dipotassium hydrogen phosphate,magnesium sulfate,ammonium chloride and other required nutrients.Chitosan (CS) was purchased from Jinan Haidebei Bioengineering Limited Corporation(Jinan,China;Degree of deacetylation ≥90%).Industrial polyvinyl alcohol (PVA) was from Zibo Xiechuang Limited Corporation (Zibo,China) PVA.4-aminoantipyrine (purity 98%,Alfa Corporation),N,O-bis(trimethylsilyl)trifluoroacetamide(99:1,Tic Corporation),Phenol,p-Methylphenol,Catechol,Oaminophenol,Boric Acid,Ammonium Chloride and Potassium Ferricyanide(Analytical Pure)were purchased from Sinopharm Chemical Reagent Co.,Ltd.All reagents were of analytical grade without further treatment.

2.2.Preparation and characterization of immobilized microorganism

A facile method for the preparation of immobilized microorganisms was developed by using the biocompatible complex of PVA and CS.The solution was obtained by taking 9.0 g of PVA powder in 40 ml distilled water under the mechanically stirring rate of 800 r﹒min-1at 80–90°C,and it was then cooled down to ambient temperature after complete dissolution.Then a certain amount of chitosan flakes (0.1 g,0.5 g,1.0 g and 1.5 g) was dissolved in 40 ml 1% (v/v) acetic acid solution under the constant magnetic stirring rate of 300 r﹒min-1at room temperature.After the complete dissolution of chitosan flakes,chitosan solution was slowly added to the previously prepared PVA solution,and the gel mixture was kept under constant stirring for another 30 min.The commercial stock solution (20 ml) of the microorganisms was added into complex gel with mechanical stirring at 20°C,and the mixture was dropped into 100 ml saturated boric acid solution with vigorous stirring to form hydrogel beads by using an electronic peristaltic pump with a 23-gauge needle.The beads were incubated for 12 h in 0.5 mol﹒L-1sodium sulfate solution and washed 3 times with 0.15 mol﹒L-1sodium chloride to remove sodium sulfate and free microorganisms.The immobilized microorganisms encapsulated into PVA-CS polymeric composite gel beads were obtained and named as MO/PVA-CS.In addition,to compare the adsorption capacity of phenol with catalyzed degradation of phenol by MO/PVA-CS,the PVA-CS beads without MO were prepared according to the same procedure above.

The morphological analysis of MO/PVA-CS beads was performed by scanning electron microscopy (SEM,JEOL JSM-6360LV)with an acceleration voltage (Acc.V) of 10.0 kV.Prior to being observed by SEM,the samples were freeze-dried and then coated with a thin layer of gold.

2.3.Degradation of phenolic compounds by MO/PVA-CS

The phenol,p-methylphenol,catechol and o-aminophenol simulated aqueous solution were prepared according to the procedure reported in the Standard Methods [22].The degradation experiments of phenol and its derivatives were all performed in a 250 ml flask containing 100 ml phenolic compounds aqueous solution at 30°C and 150 r﹒min-1.The degradation of phenol was carried out by adding 20 ml of free MO solution or MO/PVA-CS (MO/PVA-CS prepared using 20 ml free MO solution),and the acclimated MO/PVA-CS beads were used in other experiments including the degradation of p-methylphenol,catechol and oaminophenol,in other words,the MO/PVA-CS was used 4 times in treating 100 mg﹒L-1of phenol.The MO/PVA-CS beads were separated and the liquid samples were taken out for testing the residual concentration of substrates at the end of the degradation.

2.4.Test of MO/PVA-CS operational stability

The operational stability of MO/PVA-CS beads for phenol degradation was tested as follows:fresh immobilized microorganism of 60 g (wet weight) was added in 250 ml conical flasks for treating 100 ml 100 mg﹒L-1phenol simulated wastewater,then the conical flask was shaken in an air shaker at a speed of 150 r·min-1at 30°C.At the end of each reaction,the MO/PVA-CS beads were separated and washed with 0.85% NaCl solution,and then put into the flask and used repeatedly for treating 100 ml phenol simulated wastewater with the concentration of 100 mg﹒L-1.In the first four repeated tests,the time required for the reaction was recorded while the degradation rate reached 99%.The degradation rate of phenol in each subsequent experiment was recorded in the treatment of 5 h from the fifth to the ninetieth cycling running.

2.5.Analytical method

Fig.1.The image of MO/PVA-CS beads.(a) photo of beads;(b) SEM image of bead surface;(c) SEM image of bead cross profile.

The concentration of phenol,o-aminophenol and catechol in the degradation was determined by Spectrophotometer(L5S;Shanghai Yidian Analytical Instrument Co.,Ltd).This was done with 4-aminoantipyrine in the presence of ammonium hydroxide and potassium ferricyanide which produced a colored antipyrine dye that was measured spectrophotometrically at 510 nm (phenol,oaminophenol) or 270 nm (catechol).The p-methylphenol was monitored by HPLC (Agilent 1100 series,Agilent,Waldbronn,Germany)and it was thoroughly filtered through 0.45 μm filter before injecting(10 μl)into ZORBAX SBC-18(Applied Biosystems)column and eluted using 60% acetonitrile at the flow rate of 1 ml﹒min-1.The eluate was monitored at 280 nm with an ultraviolet detector,and the degradation rate (D) was calculated as follows:

where P1is the initial concentration of phenol (mg﹒L-1),and P2is the concentration of phenol (mg﹒L-1) after treated by MO/PVA-CS beads.

The dissolved oxygen(DO)concentration was measured to analyze the oxygen uptake rate (OUR) and relative biological activity of the MO/PVA-CS beads as follows:Taking 200 ml phenol simulated aqueous solution of 100 mg﹒L-1in the reactor with the dissolved oxygen concentration saturated by aeration pump,and then 5.0 g (wet weight) of MO/PVA-CS beads or the same amount of free MO was added.The changes of dissolved oxygen concentration in the reactor were monitored by MP516 dissolved oxygen analyzer (Shanghai Sidelius precision instrument Co.,Ltd).OUR was calculated according to formula (2):

Where DO1is the dissolved oxygen concentration (mg﹒L-1) at the beginning of test,DO2is the dissolved oxygen concentration(mg﹒L-1) at the end of test,and X is the amount (g﹒L-1) of MO/PVA-CS or free MO,and t is the test time(min).The relative biological activity refers to the ratio of OUR of MO/PVA-CS beads to that of free MO under the same amount.

3.Results and Discussion

3.1.Morphology and property of MO/PVA-CS beads

As shown in Fig.1(a)that the MO/PVA-CS beads are milky white regular spheroid with around diameter of 4 mm and non-adhesive to each other,which is beneficial to separate from the reaction system in the process of use.There are abundant pores on the outer surface of MO/PVA-CS beads and as shown in Fig.1(b).Furthermore,it can be seen from Fig.1(c) that a large number of cavities existed in the interior of beads and the cavity size was significantly larger than that of pores existed on the outer surface.Notably,the size of microorganisms is typically micron-size and the diameter of phenol molecule is 0.69 nm,and the pores orifice on the external surface is smaller than the microorganism size but larger than the size of phenol molecule,thus the phenol molecules can freely enter the interior of MO/PVA-CS beads close to the microorganism and were biodegraded by the catalytic action of microorganisms encapsulated in the cavities.The internal cavity structure can provide enough space for microbial reproduction and survival in MO/PVA-CS beads,which is beneficial for the microorganism preventing leakage,free movement,proliferation and rapid intake of nutrients and dissolved oxygen.At the same time,the cavity structure of MO/PVA-CS beads formed by the polymeric recombination of PVA-CS can effectively prevent the contact between the biological macromolecules in water and microorganisms in the cavity,which is helpful to obtain the highly active and tolerant immobilized microorganism.Besides,the strong hydrogen bonding interactions can be formed by the reaction between plenty of -OH and -NH2groups of chitosan and-OH group of polyvinyl alcohol in the liquid state to reduce the self-agglomeration of polyvinyl alcohol[23].As shown in Fig.2,the polymeric composite gel beads with numerous cavities were constituted by the intermolecular hydrogen bonding between synthetic polyvinyl alcohol and natural chitosan molecules.The microorganisms were encapsulated effectively in the cavities of the polymeric composite gel beads with a large number of pores capable of transporting the substrate molecules.

The relative biological activity and OUR of MO/PVA-CS beads with different mass ratio of PVA to CS were shown in Fig.3.The dissolved oxygen in phenol simulated aqueous solution of the open-system vibrating reactor was determined to be 0.76 mg﹒L-1and the OUR on free MO was 1.04 mg﹒g-1﹒min-1.The OUR on MO/PVA-CS beads varied significantly with the mass ratio of PVA to CS,and it reached the maximum value of 0.73 when the mass ratio of PVA to CS was 9:0.5.Accordingly,the relative biological activity of MO/PVA-CS beads displayed the maximum value of 70% at mass ratio 9:0.5 of PVA to CS.In the following studies,it found that the catalytic degradation of phenol by MO/PVA-CS beads possesses consistent change trend with the relative biological activity with different mass ratio of PVA to CS,that is,the high relative biological activity leads to the high catalytic degradation at mass ratio 9:0.5 of PVA to CS.

3.2.Biodegradation of phenol by MO/PVA-CS

Fig.2.The internal structure schematic of MO/PVA-CS beads.

Fig.3.Biological activity of MO/PVA-CS beads with different mass ratio of PVA to CS.

Fig.4.Reaction time curves of phenol degradation.

The treating results of fresh MO/PVA-CS beads(9:0.5 mass ratio of PVA to CS)and the free MO as well PVA-CS beads to 100 mg﹒L-1of phenol were shown in Fig.4.As can be seen that the degradation rate of phenol by MO/PVA-CS beads reached 99.5% in 120 h,and the degradation rate by free MO was only 21.1%under the identical treatment conditions,indicating that the degradation rate of phenol by immobilized microorganism was more efficiently than that of free microorganism.It suggested that the PVA-CS polymeric composite gel could provide a biocompatible micro-environment for the growth and reproduction of microorganisms,leading to the enhancement of degradation activity.What’s more,it also can be seen in Fig.4 that the adsorption of phenol reached saturation by PVA-CS beads in 70 h with only 17.8% removal of phenol,which was obviously lower than that of phenol by catalytic degradation using MO/PVA-CS beads.From another point of view,the degradation rate remained 99%after 90 cycles repeating use,which objectively reflected that the adsorption of phenol by MO/PVA-CS beads was negligible compared with the catalytic degradation of phenol.

3.3.Effect of temperature and pH on phenol degradation by MO/PVACS

The fresh MO/PVA-CS beads were used to treat phenol solution of 100 mg﹒L-1for 120 h at different temperature and pH values.The phenol solution with different pH values was obtained by preparing phosphate buffer solution with different pH containing 100 mg﹒L-1of phenol.As seen in Fig.5 that the degradation rate of phenol catalyzed by MO/PVA-CS beads was remarkably affected by the reaction temperature and pH of phenol solution,and the degradation rate of 99.5% could be achieved by MO/PVA-CS beads at appropriate temperature of 30°C and pH 7.

3.4.Operational stability of MO/PVA-CS

The reusability of immobilized microorganisms was one of the key qualities in view of economically viable industrial processes.It can be seen in Fig.6 that the MO/PVA-CS beads used in 90 successive phenol-degradation operations display the excellent recycling performance.The catalytic activity of the immobilized microorganism has been greatly improved after first used so that the time required shortened from 120 h to 60 h with phenol degradation rate up to 99%.The activity of the immobilized microorganism catalytically degrading phenol increased by 2–3 times,and the degradation rate of phenol reached 99% in only 5 h at fourth use,indicating the microorganism immobilized into PVA-CS could adapt to the degradation environment and be acclimated gradually.The above results manifested that the immobilized microorganism MO/PVA-CS beads showed a gradual increase trend in degradation efficiency with the increasing number in the first 4 times of use.The acclimatization of immobilized microorganisms in use was also observed in the case of immobilized activated sludge in sodium alginate and photo-crosslinked resin (ENTG-3800) for treating 500 mg﹒L-1phenol wastewater.During the repeated use of immobilized activated sludge,the degradation rate increased from 10.4 mg﹒L-1﹒h-1for the first use to 83.0 mg﹒L-1﹒h-1for the seventh use.This should be attributed to the formation of microbial membrane on the immobilized microorganism surface which could enhance the ability to adapt to the phenolcontaining environment,thus resulting in the improvement of its activity [24,25].The degradation rate of about 99% at 5 h in 90 repeated uses was achieved by microorganism encapsulated into PVA-CS polymer composite gel beads,declaring that the MO/PVA-CS beads possessed excellent degradation efficiency and operational stability.After using 90 times,the beads in appearance shape were significantly unchanged,which may be attributed to the powerful intermolecular hydrogen bonding between polyvinyl alcohol and chitosan molecules in the polymeric composite MO/PVA-CS beads.The MO/PVA-CS beads possessing superior degradation efficiency for phenolic compounds will display potential practical prospects as a biocatalyst in industrial phenol-containing water treatment.

Fig.5.Effect of temperature and pH on phenol degradation.

3.5.Effect of phenol concentration on degradation rate by MO/PVA-CS

Fig.7.Effect of phenol concentrations on degradation rate by acclimated MO/PVACS.

The degradation of phenol solution with different concentrations by the acclimated MO/PVA-CS beads (used 4 times for the degradation of 100 mg﹒L-1phenol) were shown in Fig.7.With the increase of phenol concentration (200 mg﹒L-1–1600 mg﹒L-1),the time required was gradually prolonged as the degradation rate of phenol reached above 99%.When the phenol concentration increased to 1000 mg﹒L-1,the complete degradation of phenol needed 112 h,and a longer degradation time was required for the complete degradation of 1600 mg﹒L-1phenol.Nevertheless,while treating 2000 mg﹒L-1phenolic aqueous solution,only 48.5%of phenol was degraded by the acclimated MO/PVA-CS beads after 182 h,manifesting that the substrate inhibition was significant from the high phenol concentration of 2000 mg﹒L-1.The similar results were also observed by Jiang et al.[15],when the phenol concentrations were over 800 mg﹒L-1,the removal efficiency of phenol decreased obviously.In addition,Satchanska et al.[16]found that the phenol degradation rate could be close 100% by immobilized Bacillus subtilis at phenol concentration of 300 mg﹒L-1,400 mg﹒L-1and 600 mg﹒L-1,while the phenol removal efficiency decreased rapidly when the concentration of phenol was 1000 mg﹒L-1.

3.6.Degradation of phenol derivatives by MO/PVA-CS

The degradation for o-aminophenol,p-methylphenol and catechol simulated wastewater with the same concentration of 100 mg﹒L-1were investigated by the acclimated MO/PVA-CS beads.As shown in Fig.8,the concentrations of catechol,p-methylphenol,and o-aminophenol decreased from 100 mg﹒L-1to 0.4 mg﹒L-1,0.6 mg﹒L-1and 4.7 mg﹒L-1after 24 h treating,and the degradation rate of the immobilized microorganism to all three phenolic compounds reached more than 95%.It concluded that catechol,pmethylphenol and o-aminophenol in water could be effectively degraded by the immobilized microorganism.

Fig.6.Operational stability of MO/PVA-CS beads.

Fig.8.Degradation of phenol derivatives by acclimated MO/PVA-CS.

4.Conclusions

The immobilized microorganism MO/PVA-CS beads with favorable biological compatibility,outer pores and inner cavities structures were prepared by using polyvinyl alcohol and chitosan as raw materials.The degradation rate of 99.0% has been achieved by using fresh MO/PVA-CS beads for treating 100 mg﹒L-1phenol,which is much higher than that of free microorganisms.The MO/PVA-CS beads can be recycled and reused with 99.5% degradation rate in 90-cycle.Furthermore,the degradation rates are more than 95%by the acclimated MO/PVA-CS beads at 1600 mg﹒L-1of phenol and 100 mg﹒L-1of p-methylphenol,catechol or o-aminophenol.The biocatalyst MO/PVA-CS beads with low raw material cost,simple preparation process,excellent degradation performance and high operational stability will provide a feasible solution for treating phenol-containing wastewater.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No.21961028) and the Science and Technology Support Project of Ningxia Province (NX015076).