Enhanced Mineralization of p-Fluoronitrobenzene in a Methane-based Hollow-fiber

Liu Chunshuang; Yu Haitong;Qu Changyong; Li Wei ;Wang Yannan; Guo Yadong; HanFenglei

(1. College of Chemical Engineering, China University of Petroleum, Qingdao 266580;2. SINOPEC Jinan Refning & Chemical Company, Jinan 250014)

Abstract: In this study, the enhanced mineralization of p-fluoronitrobenzene (p-FNB) was achieved in a methane-based hollow-fiber membrane biofilm reactor (CH4-MBfR). A TOC removal of 73.2% was gained in CH4-MBfR with an influent p-FNB loading of 42―84 mg/L·d, while only 40.6% of TOC was removed in the corresponding control biological reactor(BR). Moreover, the reaction rate constant for TOC removal in CH4-MBfR was 2.48 times that of the BR. VFAs was only detected in CH4-MBfR, although oxidation elimination was the first step for p-FNB removal in both systems. Methylococcus and Methylononas were dominant strains in the CH4-MBfR, which may play an important role in converting CH4 to VFAs and then further enhancing the mineralization efficiency of p-FNB.

Key words: p-fluoronitrobenzene; biodegradation; CH4-MBfR; mineralization

1 Introduction

p-Fluoronitrobenzene (p-FNB), a typical fluorinecontaining intermediate in chemical engineering, has been used extensively in the manufacture of fluorine-based plastics, chemicals, medicines and surfactants[1]. Because of electron-withdrawing nature of the nitro group[2-3]and the specific strength of the carbon-fluorine bond[4], fluoronitrobenzene derivatives have high chemical stability and high ecological toxicity. Therefore,p-FNB-containing wastewaters need to be treated properly.

Numerous studies have suggested that aerobic microbial degradation plays an important role in wastewater purification. Efficientp-FNB removal was achieved in a biological system and defluorination was the first step according to the study of Feng, et al[5]. Microorganisms such asFlavobateria, Clostridia,andIgnavibacteriawere enriched and were used for directp-FNB biodegradation[5]. However, little is known about the mineralization ofp-FNB in biological system.

Inorganic oxidized substrates, such as nitrate[6],chromate[7]and perchlorate[8], were efficiently removed by using CH4-based hollow-fiber membrane biofilm reactors(CH4-MBfR). VFAs usually accumulated, which could further benefit from the reduction of oxidized substrates.

However, little is known aboutp-FNB removal in CH4-MBfR up to now.

Herein, a CH4-MBfR was adopted to treat thep-FNB containing wastewater in this study. Interestingly, higher mineralization efficiency was observed in CH4-MBfR than in controlled biological reactor (BR). Therefore, the differences in the VFAs production,p-FNB mineralization kinetic and pathways during the degradation ofp-FNB in both systems were revealed.

2 Experimental

2.1 Reactor set-up

A CH4-MBfR with a working volume of 80 mL was adopted forp-FNB removal in this study[9]. The CH4-MBfR was equipped with gas-permeable hollow fiber membranes (nonporous polypropylene fiber, 200 μm in OD, 100―110 μm in ID, which was produced by Teijin,Ltd., Japan). One end of the fibers was open and the other was sealed as a dead end in order to well deliver CH4gas. Mixed gas (95% of CH4and 5% of CO2) was sent to the lumen of the fibers from the open end at 1.1 atm controlled by the regulator connected to a gas cylinder.The reactor was mixed continuously by recirculating the liquid with a peristaltic pump at a rate of 10.5 mL/min. An 100-mL overflow bottle with a 75 mL headspace was set up for liquid sampling, pH monitoring, and final effluent discharge. BR was operated without applying the mixed gas.

2.2 Microbial inocula and operation

The composition of the synthetic influent was (per L):KH2PO40.075 g/L, CaCl2·2H2O 0.3 g/L, MgCl2·7H2O 0.23 g/L, MgSO40.05 g/L, NH4Cl 0.09 g/L, 0.5 mL/L of an acidic trace element solution, and 0.2 mL/L of an alkaline trace element solution.p-FNB was added as needed into the medium. All reactors were inoculated with 20 mL of anaerobic sludge from Nibuwan municipal wastewater treatment plant (Qingdao, China) and were operated at ambient temperature (22±2 °C). The hydraulic retention time (HRT) was about 24 h. The pH value of the reactor was maintained at 7―7.5 by manual dosing of a 1 mol/L HCl or 1 mol/L NaOH solution. At the end of the continuous tests, the kinetic characteristics of CH4-MBfR and BR were investigated. And two reactors were operated in batch mode after the medium was added in the form of pulse.

2.3 Analytical methods

Effluent samples taken from the reactors once in two days were immediately filtered through a 0.22 µm membrane.The concentration ofp-FNB was analyzed using high performance liquid chromatography (HPLC) system equipped with a Bio-Rad HPLC column (300 mm×7.8 mm) and a RID detector. Fluoride ions were measured by ion chromatography. Intermediates produced duringp-FNB degradation in the CH4-MBfR and BS were identified using a gas chromatograph-mass spectrometer(GC-MS) according to Zhou, et al[10]. The GC/MS analysis was performed using an Agilent 6890 N GC/5975BSD(Agilent, Corp., USA) equipped with a HP5 capillary column (30 m×0.25 mm, i.d.×0.25 um). The intermediates were compared with available authentic compounds in a mass spectral library to identify the metabolites.

Total genomic DNA of sludge samples was extracted from 0.15―0.20 g of dried sludge using the PowerSoil DNA Isolation Kit (MoBio, Carlsbad, CA, USA)following the manufacturer’s instructions. The 16S rRNA gene of the extracted DNA was amplified using the 515F (5'-GTGCCAGCMGCCGCCC-3') and 907R(5'-CCGTCAATTCMTTTRAGTTT-3') primer set. The bacterial communities were investigated by Illumina high-throughput sequencing, which was conducted by the Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai,China).

3 Results and Discussion

3.1 Performance of CH4-MBfR

The performance of CH4-MBfR was characterized in terms ofp-FNB removal, fluorination, and mineralization.After start-up,p-FNB removal in CH4-MBfR reached 100% the next day and onwards (Figure1). However, the time needed for 100%p-FNB removal was 10 d in the control test (BR). The increasing concentration of influentp-FNB from 0.3 mmol/L to 0.45 mmol/L, and then to 0.6 mmol/L had little effect onp-FNB removal in both CH4-MBfR and BR. Defluorination efficiency reached about 85.1% in both reactors after 86 days of operation and kept stable in the following periods.

The TOC removal efficiency in CH4-MBfR was as high as 75.2%, 74.8% and 70.2%, respectively, at an influentp-FNB concentration of 0.3 mmol/L, 0.45 mmol/L and 0.6 mmol/L. By contrast, the TOC removal efficiency in BR was only about 42.3% when the influentp-FNB concentration was lower than 0.45 mmol/L, and further decreased to 38.6% with the influentp-FNB concentration increasing to 0.6 mmol/L.

To investigate thep-FNB mineralization rate, the kinetic characteristics of CH4-MBfR and BR were compared.Thep-FNB mineralization kinetics was characterized by fitting TOC concentration measured in batch experiments as a function of time (Figure 2). The TOC degradation in both systems could conform to a first-order model (Table 1). The TOC removal rate constant (k) in the CH4-MBfR was 0.0261 h-1which was 2.48 times that in the BR(0.0105-1). In contrast, thep-FNB mineralization rates,p-FNB removal rates and defluorination rates were nearly the same in the two systems after 86 days of operation(Table 1).

Table 1 Kinetics of p-FNB removal, defluorination and TOC removal in CH4-MBfR and BR after 86 days of operation

Figure 1 p-FNB removal (a), fluoride ion release (b) and TOC removal (c) by CH4-MBfR and BR

Figure 2 p-FNB removal, fluoride ion release, p-FA accumulation and TOC removal at batch test in CH4-MBfR (a) and BR (b)

3.2 Primary characteristics of metabolites and pathway

To characterize the metabolites in the CH4-MBfR and BR, GC-MS was utilized. Nop-FA was detected in both systems andp-hydroxybenzoic acid was the central metabolite. In addition, phthalic acid accumulated in both CH4-MBfR and BR systems (Figure 3). This phenomenon indicated that oxidative elimination of the nitro group was the principal degradation step in both systems.

Furthermore, VFAs were also monitored as possible metabolic intermediates. The concentration of residual VFAs was 13.44±2.86 mg/L for CH4-MBfR, which was comparable with data obtained from methane-mediated biological bromate reduction system[11]. By contrast,little was accumulated in the BR system (0.83±0.56 mg/L) (Figure 4). Specifically, VFAs accumulationmay have hastened the mineralization efficiency ofp-FNB, resulting in faster TOC removal with methane meditation than in BR. Besides, the findings denoting thatp-hydroxybenzoic acid and phthalic acid accumulated in both CH4-MBfR and BR, respectively, indicated that defluorination reactions occurred before cleavage of the benzene ring and defluorination might be the first step for biodegradation ofp-FNB in both systems.

Figure 3 GC-MS analysis and mass spectral profiles of the characteristic metabolites in CH4-MBfR (a) and BR (b)

Figure 4 Average concentration of residual VFAs in CH4-MBfR and BR

3.3 Microbial community structures

In order to analyze the microbial community structures,16S rRNA gene sequencing was performed for both the inoculum and two biomass samples (CH4-MBfR and BR). Compared to the inoculum, microbial richness was decreased in both CH4-MBfR and BR systems (Table 2). In particular, the BR harbored less diverse microbial communities in terms of OTU numbers, Simpson and Shannon indexes, suggesting that microbial communities tended to be highly selected. By contrast, microbial diversity slightly increased in CH4-MBfR, implying that more species existed due to CH4mediation.

Table 2 The bacterial richness and diversity of inoculated sludge and bioreactors.

As shown in Figure 5, the percentage of phylum Flavobacteriia increased greatly to 28.7% in CH4-MBfR and 18.2% in BR, but its relative abundance in the inocula was nearly undetected. Additionally, the dominant phyla γ-Proteobacteria (24.2%), Anaerolineae (16.2%),Clostridia (8.5%), and Bacteroidia (5.3%) were mainly abundant in CH4-MBfR, while only some γ-Proteobacteria(6.6%), Anaerolineae (7.5%), Clostridia (4.7%), and Bacteroidia (2.3%) were present in BR. The dominant phyla in BR were affiliated with Ignavibacteria (29.2%)and Sphingobacteriia (20.2%), which were present in very low amounts in CH4-MBfR (4.2% and 3.1%,respectively).

Figure 5 Relative abundance of microbial communities revealed by high-throughput sequences for the inoculum,biomass in CH4-MBfR and BR after 86 days of cultivation at levels of phylum (a) and genus (b)

At genus level,Fluviicola, Bacteroidetes, Caldilineaceae,Anaerolineaceae,Clostridiumsp. andIgnavibacteriumsp. presented in both CH4-MBfR and BR systems.These strains had been shown to be able to degrade aromatic hydrocarbons[12-16]. The degradation process ofp-FNB involved several steps, like transformation of the nitro group, defluorination, and mineralization,etc., which required various functional communities.Thus,Fluviicola,Bacteroidetes,Caldilineaceae,Anaerolineaceae,Clostridiumsp., andIgnavibacteriumsp. might play a role inp-FNB removal. Besides,MethylococcusandMethylononasmainly existed in CH4-MBfR.MethylococcusandMethylononascould convert CH4to VFAs[10,17], which might further enhance the mineralization efficiency ofp-FNB.

4 Conclusions

It was feasible to achieve enhanced mineralization ofp-FNB in CH4-MBfR. High TOC removal efficiency(73.2%) was attained in CH4-MBfR and the reaction rate constant for mineralization ofp-FNB by sludge in CH4-MBfR was 2.48 times that achieved in the control reactor.VFAs were detected andMethylococcusandMethylononaswere dominant strains in the CH4-MBfR, which might play an important role in converting CH4to VFAs to further enhance the mineralization efficiency ofp-FNB.

Acknowledgements:This research was supported by the National Natural Science Foundation of China (No. 21307160),the Natural Science Foundation of Shandong Province(ZR2019MEE038), and the Fundamental Research Funds for the Central Universities (19CX02038A).