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Bioconversion of Hg0 into HA-Hg for simultaneous removal of Hg0 and NO in a denitrifying membrane biofilm reactor
Chemosphere 244 (2020) 125544
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Bioconversion of Hg0 into HA-Hg for simultaneous removal of Hg0 and NO in a denitrifying membrane biofilm reactor Z.S. Huang, Z.S. Wei*, X.L. Xiao, B.L. Li, S. Ming, X.L. Cheng, H.Y. Jiao School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou, 510275, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Hg0 was finally bio-oxidized to humic acids bound mercury in living microbes. Non-living microbial matrix performed oxidative Hg0 biosorption. Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification crucially contributed to simultaneous removal of Hg0 and NO.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2019 Received in revised form 1 December 2019 Accepted 3 December 2019 Available online 6 December 2019
Bacterial mercury oxidation coupled to denitrification offers great potential for simultaneous removal of elemental mercury (Hg0) and nitric oxide (NO) in a denitrifying membrane biofilm reactor (MBfR). Four potentially contributory mechanisms tested separately, namely, membrane gas separation, medium absorption, biosorption and biotransformation, which contributed 4.9%/7.2%, 8.1%/8.9%, 38.8%/9.5% and 48.2%/84.9% of overall Hg0/NO removal in MBfR. Herein, Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification played leading roles in simultaneous removal of Hg0 and NO. Living microbes performed simultaneous Hg0 bio-oxidation and denitrification, in which Hg0 as electron donor was biologically oxidized to oxidized mercury (Hg2þ), while NO as the terminal electron acceptor was denitrified to N2. The Hg2þ further complexed with humic acids in extracellular polymeric substances via functional groups (-SH, eOH, -NH- and -COO-) and formed humic acids bound mercury (HA-Hg). Non-living microbial matrix performed oxidative Hg0 biosorption, in which Hg0 may be physically adsorbed by cellular matrix, then non-metabolically oxidized to Hg2þ via oxidative complexation with eSH in humic acids and finally cleavage of SeH bond and surface charge transfer led to formation of HA-Hg. Therefore, bioconversion of Hg0 to HA-Hg by Hg0 bio-oxidation and oxidative Hg0 biosorption coupled with NO denitrification to N2 dynamically cooperated to accomplish simultaneous removal of Hg0 and NO in MBfR. © 2019 Published by Elsevier Ltd.
Handling Editor: Yongmei Li Keywords: Membrane biofilm reactor Hg0 bio-oxidation Hg0 biosorption Denitrification Mechanism
1. Introduction
* Corresponding author. E-mail address: [email protected] (Z.S. Wei). https://doi.org/10.1016/j.chemosphere.2019.125544 0045-6535/© 2019 Published by Elsevier Ltd.
Mercury (Hg) is an extremely toxic metal element and mostly notorious for its long-range mobility in global atmosphere, longterm persistence in ecosystem, neurotoxicity and bio-
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accumulation along food chains (Zhao et al., 2017). Nitrogen oxides (NOx) give rise to acid rain, haze, photochemical smog, and tropospheric ozone depletion (Zheng et al., 2016). Anthropogenic Hg and NO are mainly generated by combustion of fossil fuels in coal-fired power plants, medical waste incinerators, municipal waste combustors and sewage sludge incinerators (Zhao et al., 2017). Elemental mercury (Hg0) and nitric oxide (NO) are frequently present in flue gas, which account for 94% of total Hg and 95% of NOx, (Liu et al., 2014; Niu et al., 2014). These worsening situations stress the need of technologies for simultaneous flue gas demercuration and denitration. Membrane biofilm reactor (MBfR) is a technologically feasible, economically viable and environmentally friendly biotechnology for flue gas decontamination (Sahinkaya et al., 2011; Gu et al., 2018; Wei et al., 2019). A denitrifying MBfR removed 73e86% NO in simulated flue gas with long-term stability (Zhang et al., 2013). Our previous studies also proved the feasibility of MBfR for simultaneous removal of Hg0 and NO (Huang et al., 2019b, 2019c). In principle, the overall removal of Hg0 and NO in the denitrifying MBfR is the integrated result of multiple processes including membrane gas separation, medium absorption, biosorption and biotransformation (Kumar et al., 2008). Hollow fiber membrane offers vast specific gas-liquid interfacial area to enhance mass transfer of poorly soluble gas (Wang et al., 2018). Coupling membrane module to a biofilter can increase styrene removal efficiency by 20.7%, owing to selective membrane gas separation (Li et al., 2012). Chemical absorption can convert poorly soluble Hg0 and NO to water soluble Hg2þ and NO 3 to facilitate their removal. Chemical absorption accounted for 30.5% of NO removal in a bubble column reactor (Khan and Adewuyi, 2010). Chemical oxidation of dissolved elemental mercury (DEM) occurred in the presence of organic thiol groups, which are typical metabolic byproducts (Zheng et al., 2013). Non-living biomass exhibited vast capacity for Hg biosorption by functional groups (-SH, -S-S-, eCOOH, eSO3H, -PO3H2, eNH2, eN2C3H3 and etc.) on cell walls (Siddiquee et al., 2015; Karthik et al., 2017). Heavy metals biosorption by dead cells squez and Dussan, even outcompeted that by living cells (Vela 2009). Biomass of Bacillus cereus achieved 104.1 mg g1 Hg biosorption (Sinha et al., 2012). In our preliminary studies, Hg0 biooxidation was achievable by nitrifying/denitrifying bacteria (Huang et al., 2019b, 2019c). Hg0 bio-oxidation coupled to denitrifying NO reduction played vitally important roles in simultaneous removal of Hg0 and NO in membrane biofilm reactor. Current theories had extensively demonstrated that membrane gas separation, medium absorption, biosorption and biotransformation can all play critical roles in removal of Hg0 and NO. Nevertheless, how these processes jointly or differentially contributed to simultaneous removal of Hg0 and NO in MBfR may be poorly understood and well worth further investigation. To the best of our knowledge, very few studies managed to clarify this. The objective of this work is to clarify contribution of membrane gas separation, medium absorption, biomass adsorption and biotransformation to simultaneous removal of Hg0 and NO in a denitrifying MBfR. Transformation patterns of Hg0 and NO were comparatively analyzed. Mercury speciation in biofilm was determined by sequential extraction processes (SEPs) and by inductively coupled mass spectrometry (ICP-MS). The SEPs extracts were further characterized by excitation-emission matrix spectra (EEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Morphology of biofilm was studied by scanning electron microscopy (SEM). Mechanisms of simultaneous removal of Hg0 and NO jointly or differentially by multiple processes were therefore comprehensively elucidated.
2. Materials and methods 2.1. Experimental set-up The denitrifying MBfR systematically consisted of a polyvinylidene fluoride (PVDF) hollow fiber membrane reactor (Blue cross membrane technology Co., Ltd, Tianjin, China), gas generation-mixing system and nutrient medium recirculation system (Fig. S1). Hg0 vapor was generated by heating a mercury permeation tube (Qingan scientific instruments Co., Ltd, Suzhou, China) at 60e70 C thermostat water bath. NO was generated by a gas cylinder. Airstreams of Hg0 and NO were finely mixed with compressed air in a buffer bottle then pumped into the hollow fiber membrane reactor. The recirculating medium was sprayed over the membrane surface for microbial inoculation using a subaqueous pump. Tail gas was treated by 10% v/v H2SO4-5% m/v KMnO4 solution. The experimental set-up of MBfR had been detailed in our previous work (Huang et al., 2019b). Simultaneous removal of Hg0 and NO in membrane reactor (MR), membrane medium reactor (MMR), membrane biomass reactor (MBmR) and membrane biofilm reactor (MBfR) were comparatively evaluated in a 35-day operation. MBmR, MMR and MR were set up in parallel by modifying configuration of MBfR as following: (1) MR: recirculation system was removed to study contribution of membrane gas separation; (2) MMR: the recirculating mineral medium was sterilized (132 C, 205 kPa) and but not inoculated with bacteria to study overall contribution of membrane gas separation and medium absorption; (3) MBmR: glutaric dialdehyde (2.5% v/v) was added to mineral medium to obtain immobilized non-living biomass and study overall contribution of membrane gas separation, medium absorption and biosorption. MBfR, MBmR, MMR and MR underwent 35 days’ operation under identical operational conditions as following: Hg0 inlet load, 109.9 ± 0.5 mg m3 h1; NO inlet load, 147.8 ± 0.5 g m3 h1; gas residence time (GRT), 5.5 s; pH in the medium, 7.0e7.3; dissolved oxygen (DO) in the medium, 0.08e0.2 mg L1; temperature, 25 C; medium flow rate, 1 60 mL min . Phase I (Days 1e10) was a start-up phase for microbial inoculation in MBfR and MBmR, then the biocide was added to MBmR on Day 10. Afterwards, performance of simultaneous removal of Hg0 and NO in four reactors were comparatively evaluated in Phase II (Days 11e35). Concentration of oxidized mercury (Hg2þ) and nitrate nitrogen (NO 3 -N) in the recirculating medium of MMR, MBmR and MBfR were determined every 2e3 days to clarify transformation patterns of Hg0 and NO. 2.2. Mercury speciation Mercuric species of different chemical forms in the biomass of MBmR and MBfR were qualitatively and quantitatively analyzed by sequential extraction processes (SEPs) followed by inductively coupled plasma mass spectrometry (ICP-MS, iCAP Qc, Thermo Scientific Inc., USA) as per previous methods (Biester and Scholz, 1997). SEPs classified Hg species in biomass into 6 fractions, namely, water soluble mercury (WSeHg), ion exchangeable mercury (IE-Hg), methyl-mercury (MeHg), humic/fulvic acids bound mercury (HA/FA-Hg), organically bound mercury sulfide (Hg-SR) and mercury sulfide (HgS). These fractions were leached sequentially by chemical treatments, oxidized to total mercury (Tot-Hg) by aqua regia digestion and separately quantified by ICP-MS. Detailed procedures were as per following: (1) Total mercury (Tot-Hg): 2.5 g biofilm sample was digested with 16 mL aqua regia at 160 C hotplate for 3 h; (2) WS-Hg: 20 g biofilm was sampled, air-dried overnight and weighted. 50 mL DDI water was added to 20 g
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biofilm sample and the mixture was shaken by vortex at 200 rpm for 2 h then centrifuged at 10,000 rpm for 3 min. Sediment was subject to secondary extraction and the supernatants were collected. 10 mL evenly mixed supernatant was digested with 4 mL aqua regia at 100 C water bath for 1 h; (3) IE-Hg: 50 mL 1 M NH4Ac (aq) was added to the residue in (2) followed by procedures in (2); (4) MeHg: 20 mL 2% HCl-10% ethanol solution was added to residue in (3). The mixture was shaken by vortex at 200 rpm for 20 min, sonicated at 60 ± 2 C for 20 min, centrifuged at 5000 rpm for 10 min and digested as per procedures in (2); (5) HA/FA-Hg: 50 mL 1 M NH3$H2O was added to residue in (4) followed by procedures in (2). 10 mL evenly mixed supernatant was digested with 10 mL concentrated HCl and 10 mL 30% H2O2 at 85 C water bath for 2 h; (6) Hg-SR: The residue (4) was first digested with 10 mL 0.02 M HNO3 at 85 C water bath for 2 h and subsequently with 16 mL 30% H2O2 at 85 C water bath for 1 h 50 mL 1 M NH4Ac-6% HNO3 solution was added to the digest followed by procedures in (2); (7) HgS: residue in (6) was repeatedly rinsed with DDI water for 2 h and air-dried overnight. 2.5 g residue was digested as per procedures in (1). 2.3. Characterization The dominant fractions HA/FA-Hg in mercury speciation were further characterized by EEM, XPS and FTIR. EEM: The fluorescent spectral properties of HA/FA-Hg extracts in MBfR and MBmR were characterized by an excitation emission matrix F-7000 fluorescence spectrophotometer (Hitachi Co., Ltd, Japan). XPS: Chemical form of Hg in HA/FA-Hg extracts were further characterized by an ESCALab250 X-ray photoelectron spectrometer (Thermo Fisher Scientific Inc., USA). HA/FA-Hg extracts were acidified using 1 M HCl (final pH < 1) and stored at 4 C for 1 d to facilitate humic acid precipitation. The precipitates were recovered by centrifugation and airdried overnight. The air-dried HA/FA-Hg precipitates were used for XPS analysis. FTIR: The organic functional groups in HA-Hg precipitates were characterized by a VERTEX 80 Fourier transform infrared spectrometer (Bruker Optics Co., Ltd, Germany). SEM-EDS: The morphology of biofilm samples from MBfR and MBmR on day 10, day 20 and day 30 were studied by a JSM-6330F Field Emission Scanning Electron Microscope (Japan Electron Optics Laboratory Co, Ltd). 2.4. Analytical methods Concentration of Hg0 in flue gas was determined by a mercury vapor indicator (MVI, Ion science company, UK). Concentration of Hg2þ in the recirculating medium was quantified by non-N2purgeable total mercury and determined by aqua regia digestion followed by atomic fluorescence spectrometry (AFS) using a dual channel atomic fluorescence spectrometer (AFS-820, Jilin University Little Swan Instrument Co., Ltd, China). Concentrations of NO 3N in the recirculating medium was determined by ultraviolet spectrophotometry using a UV spectrophotometer (T6, Beijing Persee Instruments Co., Ltd, China). Instrumentation for determinations of DO, pH and flow rate have been specified in our previous work (Huang et al., 2019b). 3. Results and discussion 3.1. Roles of Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification in simultaneous removal of Hg0 and NO Membrane gas separation, medium absorption, biomass adsorption, and biotransformation were the four potentially contributory mechanisms for simultaneous removal of Hg0 and NO
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in the denitrifying MBfR. To understand the role of each process, a 35-day long-term experiment was conducted to evaluate simultaneous removal of Hg0 and NO in MR, MMR, MBmR and MBfR, respectively. As shown in Fig. 1aeb, in start-up phase (Days 1e10), removal efficiency of Hg0 and NO in MBfR gradually increased from 67.5 ± 3.5% and 50.7 ± 3.5% to 89.9 ± 1.5% and 73.5 ± 1.4%, respectively. Likewise, removal efficiency of Hg0 and NO in MBmR increased from 65.2 ± 3.9% and 51.3 ± 3.1% to 88.9 ± 2.0% and 71.6 ± 0.7%, respectively. Afterwards, removal efficiency of Hg0 and NO in MBfR steadily maintained at 90.9 ± 0.9% and 74.8 ± 1.1%, respectively during Days 11e35. After addition of biocide on day 11, Hg0 removal efficiency in MBmR gradually decreased and reached a steady state at 62.4 ± 1.4% during Days 16e22. Subsequently, Hg0 removal efficiency in MBmR further decreased and maintained at 17.1 ± 1.5% during Days 31e35. Meanwhile, NO removal efficiency in MBmR kept decreasing during Days 11e18 then maintained at 13.2 ± 0.6% during Days 19e35. Nevertheless, removal efficiency of Hg0 and NO maintained stayed at low levels in MMR (12.4 ± 1.8%; 10.9 ± 1.0%) and MR (5.8 ± 2.5%; 5.2 ± 1.0%) throughout the operation. The net contribution of membrane gas separation, medium absorption, biosorption and biotransformation to Hg0 removal in MBfR were 4.9%, 8.1%, 38.8% and 48.2%, respectively, while those to NO removal in MBfR were 7.2%, 8.9%, 9.5% and 84.9%, respectively. Biosorption and biotransformation may play crucially important roles in Hg0 removal. However, NO removal was dominantly attributed to biotransformation. Membrane gas separation, medium absorption, biosorption and biotransformation contributed 4.9%/7.2%, 8.1%/8.9%, 38.8%/9.5% and 48.2%/84.9% of overall Hg0/NO removal in MBfR. Simultaneous removal of Hg0 and NO in MBfR showed higher performance in comparison to that in previous studies (Hg0 removal efficiency: 95%, GRT: 6e70 s; NO removal efficiency 75e99%, GRT: 30e132 s) (Wang et al., 2006; Philip and Deshusses, 2008; Jiang et al., 2009; Zhang et al., 2013; Han et al., 2016). As shown in Fig. 1ced, Hg2þ concentration in recirculating medium of MBfR periodically increased from 0 to 11.2e13.2 mg L1 every 10 days. Nevertheless, that in MBmR showed similar increasing trends within narrowing ranges (0e10.6 mg L1; 0e8.4 mg L1; 0e5.3 mg L1; 0e0.8 mg L1) and that in MMR merely reached 0.6 mg L1. Hg0 oxidation took place periodically in both MBfR and MBmR, suggesting that Hg0 removal in MBfR was dominantly attributed to Hg0 bio-oxidation and Hg0 biosorption in MBmR may be an oxidative process. Hg0 oxidation took place steadily in MBfR but progressively weakened in MBmR resulting in lower Hg0 removal. Hg0 oxidation didn’t take place in medium of MMR, suggesting that Hg0 may be dissolved or flushed away by the medium to achieve Hg0 removal in MMR. Meanwhile, NO 3 -N concentration in medium of MMR, MBmR and MBfR irregularly ranged within 0e0.6 mg L1, 0e1.6 mg L1 and 1.5 mg L1, respectively. Average nitrogen removal rate and nitrogen production rate in MMR, MBmR and MBfR were 1.52/1.58 mgN d1, 2.44/1.52 mgN d1and 21.79/ 0.72 mgN d1, respectively. NO removal in MBfR can be due in large part to denitrification (NO/N2), while that in MMR and MBmR may be ascribed to chemical oxidation. Hg0 acted as electron donor and NO acted as terminal electron acceptor in simultaneous Hg0 bio-oxidation and denitrification to form an overall redox in MBfR (Huang et al., 2019c). Hg0 bio-oxidation can be coupled to sulfate bio-reduction, in which Hg0 acted as electron donor, while sulfate acted as terminal electron acceptor (Huang et al., 2019a). A sulfuroxidizing biotrickling filter achieved Hg0 removal by Hg0 biooxidation (Philip and Deshusses, 2008). These combinations indicated that Hg0 bio-oxidation can potentially interact with many microbial metabolic processes. MBmR hardly performed denitrification but had weak ability for Hg0 oxidation, suggesting some
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Fig. 1. Simultaneous removal of Hg0 and NO in MR, MMR, MBfR and MBfR: (a) Hg0 removal performance in MR, MMR, MBfR and MBfR; (b) NO removal performance MR, MMR, MBfR and MBfR; (c) Variation of Hg2þ concentration in recirculating medium of MMR, MBfR and MBfR; (c) Variation of NO 3 -N concentration in recirculating medium of MMR, MBfR and MBfR.
intermediate electron acceptors may oxidize Hg0 to Hg2þ in Hg0 biosorption. Thiol compounds can acted as the electron acceptors for Hg0 by forming self-assembled monolayers (SAMs) in oxidative complexation (Zheng et al., 2013). In summary, simultaneous Hg0 bio-oxidation (48.2% Hg0 removal) and denitrification (84.9% NO removal) aided by oxidative Hg0 biosorption (38.8% Hg0 removal) made major contribution to simultaneous removal of Hg0 and NO in MBfR. 3.2. Mercury speciation: bio-oxidation of Hg0 to humic acids bound mercury As shown in Fig. 2, in Phase I, Hg speciation in biofilms of MBfR (Tot-Hg: 3.55 ± 0.08 mg g1; HA/FA-Hg: 2.85 ± 0.09 mg g1; IE-Hg: 0.51 ± 0.02 mg g1) and MBmR (Tot-Hg: 3.48 ± 0.04 mg g1; HA/FAHg: 2.77 ± 0.03 mg g1; IE-Hg: 0.45 ± 0.04 mg g1) didn’t show significant difference (p > 0.13, student’s t-test). Herein, HA/FA-Hg accounted for 79.5e80.0% of Tot-Hg and dominated Hg speciation in biofilms. In addition, IE-Hg accounted for 12.9e14.2% of Tot-Hg was the subdominant Hg species. In Phase II, Tot-Hg in biofilms of MBfR and MBmR attained 12.99 ± 0.19 mg g1 and 7.71 ± 0.09 mg g1, respectively, suggesting that living biofilms had higher capacity for Hg bioaccumulation than dead biomass. Furthermore, HA/FA-Hg maintained dominance in Hg speciation in biofilms of MBfR (10.30 ± 0.04 mg g1, 79.3% of Tot-Hg) and MBmR (6.09 ± 0.02 mg g1, 79.0% of Tot-Hg). Likewise, IE-Hg was the second dominant Hg species in biofilms of MBfR (1.87 ± 0.07 mg g1, 14.4% of Tot-Hg) and MBmR (1.02 ± 0.05 mg g1, 13.2% of Tot-Hg). Besides, Tot-Hg in Inoculum was almost negligible (<0.2 ng g1). It’s estimated that 73.2% and 71.5% of bioaccumulated Hg0 was oxidized to HA/FA-Hg in MBfR and MBmR. Formation of HA/FA-Hg
can be attributed to Hg2þ complexation with selective binding sites in extracellular polymeric substances (EPS) and cell walls (functional groups: SH, -S-S-, eCOOH, eSO3H, -PO3H2, eNH2, eN2C3H3 and etc.) and even dead biomass exhibited vast capacity for complexation (Siddiquee et al., 2015; Karthik et al., 2017). The microbial metabolites EPS can considerably sequestrate and immobilize bioavailable Hg2þ via complexation to achieve Hg2þ detoxification (Chai et al., 2013; Ouyang et al., 2017). Our previous study also confirmed HA-Hg played critical role in biofilm Hg speciation (Huang et al., 2019a, 2019b, 2019c). Since HA/FA-Hg was the dominant Hg species in both MBfR and MBmR, it can be inferred that both living biofilm and dead biomass can oxidize Hg0 to Hg2þ potentially by means of Hg0 bio-oxidation and oxidative Hg0 biosorption. Hg2þ further reacted with selective binding sites in EPS and cell walls from dead or living cells to dominantly form HA/FAHg. In summary, the removed Hg0 was dominantly oxidized to HA/ FA-Hg potentially by Hg0 bio-oxidation in MBfR and oxidative Hg0 biosorption in MBmR. 3.3. Characterization of humic acids bound mercury As shown in Fig. 3, all EEM spectra of the HA/FA-Hg extracts from MBfR and MBmR showed two prominent peaks at Ex/ Em¼(285e287 nm, 343e346 nm) and Ex/Em¼(345e346 nm, 419e421 nm), respectively, which could be attributed to soluble microbial products-like compounds (Region IV: Ex > 250 nm, Em < 380 nm) and humic acid-like compounds (Region V: Ex > 280 nm, Em > 380 nm) (Chen et al., 2003), providing chemical basis for formation of humic acids bound mercury (HA-Hg) in MBfR and MBmR. Previous researches demonstrated that dissolved organic matter (DOM) especially humic acids can play decisive role
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Fig. 2. Quantitative analysis of mercury speciation: (a) Mercury speciation in biofilms from MBfR; (b) Mercury speciation in biofilms from MBmR.
Fig. 3. Characterization of HA/FA-Hg extract: (a) and (b) EEM spectra of the HA/FA-Hg extract from MBfR; (c) and (d) EEM spectra of the HA/FA-Hg extract from MBmR.
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in Hg speciation, because strong affinity between Hg2þ and functional groups in DOM (-SH, eNH2, eCOOH, eOH) led to formation of highly chemically stable Hg-DOM complexes (logKs ¼ 23.6e38.3) (Haitzer et al., 2002; Ding et al., 2018; Jiang et al., 2018). Moreover, Hg2þ-humic acids complexation considerably abated bioavailability of Hg2þ and acted as a vital mechanism for heavy metal detoxification physiologically (Benoit et al., 2001; Chai et al., 2013; Ouyang et al., 2017). Our previous research also confirmed critical role of HA-Hg in Hg speciation (Huang et al., 2019a). Therefore, gaseous Hg0 was biologically oxidized to Hg2þ and took its final dominant form of HA-Hg in MBfR. As shown in Fig. 4a, FTIR spectra of HA-Hg precipitates from MBfR and MBmR showed prominent peaks corresponding to stretching vibrations of OeH (3139.41 cm1), eSH (2541.47 cm1), eNH (1637.01 cm1), eNHe (1532.14 cm1), -COO- (1402.45 cm1), CeOeC/CeCeO (1232.69 cm1) and CeO/CeC/CeOeC (1042.66 cm1) (Kogelheide et al., 2016; Kamnev et al., 2017). This further elucidated that oxidized mercury may be complexed with humic acids by functional groups eSH, eOH, -NH- and -COO-, which are typical binding sites for ionic mercury (Ding et al., 2018). As shown in Fig. 4b, all XPS spectra of HA-Hg precipitates from MBfR and MBmR showed two distinct groups of peaks ascribed to doublet separation of Hg 4f core level into Hg 4f5/2 (104.5e105.2 eV) and Hg 4f7/2 (100.4e101.1 eV), respectively, potentially attributed to
divalent mercuric species (Hutson et al., 2007). In Phase II, Hg 4f5/2 binding energy of HA-Hg in MBfR (101.1 eV) was higher than that in MBmR (100.4 eV), suggesting that Hg potentially existed in a more oxidized form in HA-Hg from MBfR. Hg0 may be more thoroughly oxidized in Hg0 bio-oxidation in MBfR than oxidative Hg0 biosorption in MBmR. As shown in Fig. 4c, an abundance of rod-shaped bacterial cells in biofilms from MBfR maintained intact throughout the operation. However, destruction of cells (Day 20) and complete autolysis (Day 30) were observed in bacterial cells in biofilms from MBmR. This visually differentiated between living biofilms in MBfR and dead biomass in MBmR. In summary, the final formation of HA-Hg in both MBfR and MBmR was attributed to complexation of oxidized mercury with functional groups eSH, eOH, -NH- and -COO- in humic acids. Hg0 may be more thoroughly oxidized in Hg0 biooxidation by living cells than oxidative biosorption by dead cells. 3.4. Mechanisms of Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification Biotransformation of Hg0 and NO in MBfR was primarily attributed to Hg0 bio-oxidation to HA-Hg and biological denitrification. Moreover, Hg0 biosorption was potentially an oxidative process and achieved oxidation of Hg0 to HA-Hg. Medium
Fig. 4. Characterization of HA/FA -Hg precipitates: (a) FTIR spectra of HA/FA-Hg precipitates; (b) XPS spectra of HA/FA-Hg precipitates (c) SEM images and EDS spectra of the biofilm samples.
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Fig. 5. Mechanisms of contribution of multiple processes to simultaneous removal of Hg0 and NO in MBfR.
absorption served to chemically oxidize NO and physically dissolve Hg0. On the basis of these, the mechanisms of simultaneous removal of Hg0 and NO in MBfR by joint contribution of membrane gas separation, medium absorption, biosorption and biotransformation was hypothesized.
2Hg0 þ 2NO þ 4Hþ /2Hg2þ þ N2 þ H2 O
(1)
Hg2þ þ 2HA SH / ðHA SÞ2 Hg2þ þ 2Hþ
(2)
Hg2þ þ 2HA OH / ðHA OÞ2 Hg2þ þ 2Hþ
(3)
Hg2þ þ 2HA NH2 / ðHA NHÞ2 Hg2þ þ 2Hþ
(4)
2þ
Hg
þ 2HA COOH / ðHA COOÞ2 Hg
2þ
Hg0 þ 2HA SH / ðHA SÞ2 Hg2þ þ 2H
SeH bond and surface charge transfer led to oxidative complexation of Hg0 with eSH and final formation of HA-Hg (Zheng et al., 2013) (Eq. (6)). Besides, NO in oxidized form of NO 3 can also be electrostatically bound to functional groups in biopolymers to achieve biosorption (Sarode et al., 2019). Therefore, molecular diffusion-based membrane gas separation, physical/chemical medium absorption, non-metabolic oxidative biosorption by nonliving microbial matrix and metabolic simultaneous Hg0 biooxidation and denitrification by living microbes differentially or jointly contributed to simultaneous removal of Hg0 and NO in MBfR. 4. Conclusions
þ
þ 2H
(5) (6)
As shown in Fig. 5, influx of Hg0 and NO permeated the membrane wall vertically by molecular diffusion and gradually concentrated in permeant flux, which finally limited the concentration gradient-driven molecular diffusion (Janes et al., 2008; Monsalve-Bravo and Bhatia, 2018). The permeant flux of Hg0 and NO entered the medium phase, in which NO may be chemically 0 oxidized to NO2 by O2 and dissolved in the form of NO 3 , while Hg may be physically absorbed in the form of DEM (Thiemann et al., 2005; Zheng et al., 2013). Subsequently, Hg0 and NO interacted with biofilms over membrane surface. Firstly, living cells actively performed simultaneous Hg0 bio-oxidation and denitrification via enzymatic metabolic processes (Halbach et al., 1988; Smith et al., 1998; Siciliano et al., 2002; Colombo et al., 2014), in which Hg0 acted as electron donor and was biologically oxidized to Hg2þ while NO acted as terminal electron acceptor and was denitrified to N2 (Eq. (1)). The oxidized mercury further complexed with humic acids in EPS via selective binding functional groups (-SH, eOH, -NH- and -COO-) and dominantly formed HA-Hg (Eqs. (2)e(5)). Secondly, non-living microbial matrix (cell walls, EPS and metabolic byproducts) may provide intermediate electron acceptors and passively performed Hg0 oxidation via chemical non-metabolic processes (Vijayaraghavan and Yun, 2008; Chojnacka, 2010). Hg0 was physically adsorbed by eSH in humic acids, then cleavage of
This study comparatively evaluated contribution of multiple processes to simultaneous removal of Hg0 and NO in MBfR, which was dominantly attributed to Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification. Living microbes performed simultaneous Hg0 bio-oxidation and denitrification, in which Hg0 as electron donor was oxidized to Hg2þ while NO as terminal electron acceptor was denitrified to N2. The Hg2þ further complexed with humic acids in EPS and finally formed HA-Hg. Non-living microbial matrix performed oxidative Hg0 oxidation, in which Hg0 was physically adsorbed and finally formed HA-Hg via oxidative complexation with humic acids. Therefore, contribution of multiple processes was clarified. Author contribution statement The authors (Zhenshan Huang, Zaishan Wei*, Xiaoliang Xiao, Bailong Li, Song Ming, Xiangling Cheng, Huaiyong Jiao) finished the paper entitled "Bioconversion of Hg0 into HA-Hg for simultaneous removal of Hg0 and NO in a denitrifying membrane biofilm reactor" together, among which Zhenshan Huang takes charge of Investigation, Data curation, Formal analysis and Writing, Zaishan Wei* of Supervision and review & editing, Xiaoliang Xiao of Formal anlysis, Bailong Li of Formal analysis, Song Ming of Visualization, Xiangling Cheng of Software , Huaiyong Jiao of Investigation. We hereby certify that this paper consists of original, unpublished work which is not under consideration for publication elsewhere. I hope your favorable consideration for publication to Chemosphere.
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Acknowledgements The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (21677178). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125544. References Benoit, J.M., Gilmour, C.C., Mason, R.P., 2001. Aspects of bioavailability of mercury for methylation in pure cultures of desulfobulbus propionicus (1pr3). Appl Environ Microb 67, 51e58. Biester, H., Scholz, C., 1997. Determination of mercury binding forms in contaminated soils: mercury pyrolysis versus sequential extractions. Environ. Sci. Technol. 31, 233e239. Chai, X., Hao, Y., Liu, G., Li, Z., Zhao, Y., 2013. The effect of aerobic conditions on the complexation ability between mercury and humic acid from landfill leachate and its implication for the environment. Chemosphere 92, 458e463. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence ExcitationEmission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701e5710. Chojnacka, K., 2010. Biosorption and bioaccumulation e the prospects for practical applications. Environ. Int. 36, 299e307. Colombo, M.J., Ha, J., Reinfelder, J.R., Barkay, T., Yee, N., 2014. Oxidation of Hg(0) to Hg(II) by diverse anaerobic bacteria. Chem. Geol. 363, 334e340. Ding, L., Zhao, K., Zhang, L., Liang, P., Wu, S., Wong, M.H., Tao, H., 2018. Distribution and speciation of mercury affected by humic acid in mariculture sites at the Pearl River estuary. Environ. Pollut. 240, 623e629. Gu, Y., Huang, J., Zeng, G., Shi, L., Shi, Y., Yi, K., 2018. Fate of pharmaceuticals during membrane bioreactor treatment: status and perspectives. Bioresource Technol 268, 733e748. Haitzer, M., Aiken, G.R., Ryan, J.N., 2002. Binding of mercury(II) to dissolved organic matter: the role of the mercury-to-DOM concentration ratio. Environ. Sci. Technol. 36, 3564e3570. Halbach, S., Ballatori, N., Clarkson, T.W., 1988. Mercury vapor uptake and hydrogen peroxide detoxification in human and mouse red blood cells. Toxicol Appl Pharm 96, 517. Han, L., Shaobin, H., Zhendong, W., Pengfei, C., Yongqing, Z., 2016. Performance of a new suspended filler biofilter for removal of nitrogen oxides under thermophilic conditions and microbial community analysis. Sci. Total Environ. 562. Huang, Z., Wei, Z., Xiao, X., Tang, M., Li, B., Ming, S., Cheng, X., 2019a. Bio-oxidation of elemental mercury into mercury sulfide and humic acid-bound mercury by sulfate reduction for Hg0 removal in flue gas. Environ. Sci. Technol. 53, 12923e12934. Huang, Z., Wei, Z., Xiao, X., Tang, M., Li, B., Zhang, X., 2019b. Nitrification/denitrification shaped the mercury-oxidizing microbial community for simultaneous Hg0 and NO removal. Bioresource Technol 274, 18e24. Huang, Z.S., Wei, Z.S., Xiao, X.L., Tang, M.R., Li, B.L., Zhang, X., 2019c. Simultaneous mercury oxidation and NO reduction in a membrane biofilm reactor. Sci. Total Environ. 658, 1465e1474. Hutson, N.D., Attwood, B.C., Scheckel, K.G., 2007. XAS and XPS characterization of mercury binding on brominated activated carbon. Environ. Sci. Technol. 41, 1747e1752. Janes, D.W., Durning, C.J., van Pel, D.M., Lynch, M.S., Gill, C.G., Krogh, E.T., 2008. Modeling analyte permeation in cylindrical hollow fiber membrane introduction mass spectrometry. J Membrane Sci 325, 81e91. Jiang, R., Huang, S., Chow, A.T., Yang, J., 2009. Nitric oxide removal from flue gas with a biotrickling filter using Pseudomonas putida. J. Hazard. Mater. 164, 432e441. Jiang, P., Liu, G., Cui, W., Cai, Y., 2018. Geochemical modeling of mercury speciation in surface water and implications on mercury cycling in the everglades wetland. Sci. Total Environ. 640e641, 454e465. Kamnev, A.A., Mamchenkova, P.V., Dyatlova, Y.A., Tugarova, A.V., 2017. FTIR spectroscopic studies of selenite reduction by cells of the rhizobacterium Azospirillum brasilense Sp7 and the formation of selenium nanoparticles. J. Mol. Struct. 1140, 106e112.
Karthik, C., Barathi, S., Pugazhendhi, A., Ramkumar, V.S., Thi, N., Arulselvi, P.I., 2017. Evaluation of Cr(VI) reduction mechanism and removal by Cellulosimicrobium funkei strain AR8, a novel haloalkaliphilic bacterium. J. Hazard Mater. 333, 42e53. Khan, N.E., Adewuyi, Y.G., 2010. Absorption and oxidation of nitric oxide (NO) by aqueous solutions of sodium persulfate in a bubble column reactor. Ind. Eng. Chem. Res. 49, 8749e8760. Kogelheide, F., Kartaschew, K., Strack, M., Baldus, S., Metzler-Nolte, N., Havenith, M., Awakowicz, P., Stapelmann, K., Lackmann, J., 2016. FTIR spectroscopy of cysteine as a ready-to-use method for the investigation of plasma- induced chemical modifications of macromolecules. J. Phys. D Appl. Phys. 49, 84004. Kumar, A., Dewulf, J., Van Langenhove, H., 2008. Membrane-based biological waste gas treatment. Chem. Eng. J. 136, 82e91. Li, L., Lian, J., Han, Y., Liu, J., 2012. A biofilter integrated with gas membrane separation unit for the treatment of fluctuating styrene loads. Bioresource Technol 111, 76e83. Liu, Y., Zhang, J., Pan, J., 2014. Photochemical oxidation removal of Hg0 from flue gas containing SO2/NO by an ultraviolet irradiation/hydrogen peroxide (UV/H2O2) process. Energ Fuel 28, 2135e2143. Monsalve-Bravo, G.M., Bhatia, S.K., 2018. Comparison of hollow fiber and flat mixed-matrix membranes: theory and simulation. Chem. Eng. Sci. 187, 174e188. Niu, H., Leung, D.Y., Wong, C., Zhang, T., Chan, M., Leung, F.C., 2014. Nitric oxide removal by wastewater bacteria in a biotrickling filter. J Environ Sci-China 26, 555e565. Ouyang, K., Yu, X., Zhu, Y., Gao, C., Huang, Q., Cai, P., 2017. Effects of humic acid on the interactions between zinc oxide nanoparticles and bacterial biofilms. Environ. Pollut. 231, 1104e1111. Philip, L., Deshusses, M.A., 2008. The control of mercury vapor using biotrickling filters. Chemosphere 70, 411e417. Sahinkaya, E., Hasar, H., Kaksonen, A.H., Rittmann, B.E., 2011. Performance of a sulfide-oxidizing, sulfur-producing membrane biofilm reactor treating sulfidecontaining bioreactor effluent. Environ. Sci. Technol. 45, 4080e4087. Sarode, S., Upadhyay, P., Khosa, M.A., Mak, T., Shakir, A., Song, S., Ullah, A., 2019. Overview of wastewater treatment methods with special focus on biopolymer chitin-chitosan. Int. J. Biol. Macromol. 121, 1086e1100. Siciliano, S.D., O’Driscol, N.J., Lean, D.R.S., 2002. Microbial reduction and oxidation of mercury in freshwater lakes. Environ. Sci. Technol. 36, 3064e3068. Siddiquee, S., Rovina, K., Azad, S.A., 2015. Heavy metal contaminants removal from wastewater using the potential filamentous fungi biomass: a review. J. Microb. Biochem. Technol. 7 (6). Sinha, A., Pant, K.K., Khare, S.K., 2012. Studies on mercury bioremediation by alginate immobilized mercury tolerant Bacillus cereus cells. Int Biodeter Biodegr 71, 1e8. Smith, T., Pitts, K., McGarvey, J.A., Summers, A.O., 1998. Bacterial oxidation of mercury metal vapor, Hg(0). Appl Environ Microb 64, 1328e1332. Thiemann, M., Scheibler, E., Wiegand, K.W., 2005. Nitric Acid, Nitrous Acid, and Nitrogen Oxides. Ullmann’s Encyclopedia of Industrial Chemistry. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. squez, L., Dussan, J., 2009. Biosorption and bioaccumulation of heavy metals on Vela dead and living biomass of Bacillus sphaericus. J. Hazard Mater. 167, 713e716. Vijayaraghavan, K., Yun, Y., 2008. Bacterial biosorbents and biosorption. Biotechnol. Adv. 26, 266e291. Wang, H., Dai, K., Wang, Y., Wang, H., Zhang, F., Zeng, R.J., 2018. Mixed culture fermentation of synthesis gas in the microfiltration and ultrafiltration hollowfiber membrane biofilm reactors. Bioresource Technol 267, 650e656. Wang, J., Wu, C., Chen, J., Zhang, H., 2006. Denitrification removal of nitric oxide in a rotating drum biofilter. Chem. Eng. J. 121, 45e49. Wei, Z., Yu, S., Huang, Z., Xiao, X., Tang, M., Li, B., Zhang, X., 2019. Simultaneous removal of elemental mercury and NO by mercury induced thermophilic community in membrane biofilm reactor. Ecotox Environ Safe 176, 170e177. Zhang, X., Jin, R., Liu, G., Dong, X., Zhou, J., Wang, A., 2013. Removal of nitric oxide from simulated flue gas via denitrification in a hollow-fiber membrane bioreactor. J Environ Sci-China 25, 2239e2246. Zhao, H., Mu, X., Yang, G., George, M., Cao, P., Fanady, B., Rong, S., Gao, X., Wu, T., 2017. Graphene-like MoS2 containing adsorbents for Hg0 capture at coal-fired power plants. Appl Energ 207, 254e264. Zheng, M., Li, C., Liu, S., Gui, M., Ni, J., 2016. Potential application of aerobic denitrifying bacterium Pseudomonas aeruginosa PCN-2 in nitrogen oxides (NOx) removal from flue gas. J. Hazard Mater. 318, 571e578. Zheng, W., Lin, H., Mann, B.F., Liang, L., Gu, B., 2013. Oxidation of dissolved elemental mercury by thiol compounds under anoxic conditions. Environ. Sci. Technol. 47, 12827e12834.
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Bioconversion of Hg0 into HA-Hg for simultaneous removal of Hg0 and NO in a denitrifying membrane biofilm reactor Z.S. Huang, Z.S. Wei*, X.L. Xiao, B.L. Li, S. Ming, X.L. Cheng, H.Y. Jiao School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou, 510275, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Hg0 was finally bio-oxidized to humic acids bound mercury in living microbes. Non-living microbial matrix performed oxidative Hg0 biosorption. Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification crucially contributed to simultaneous removal of Hg0 and NO.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2019 Received in revised form 1 December 2019 Accepted 3 December 2019 Available online 6 December 2019
Bacterial mercury oxidation coupled to denitrification offers great potential for simultaneous removal of elemental mercury (Hg0) and nitric oxide (NO) in a denitrifying membrane biofilm reactor (MBfR). Four potentially contributory mechanisms tested separately, namely, membrane gas separation, medium absorption, biosorption and biotransformation, which contributed 4.9%/7.2%, 8.1%/8.9%, 38.8%/9.5% and 48.2%/84.9% of overall Hg0/NO removal in MBfR. Herein, Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification played leading roles in simultaneous removal of Hg0 and NO. Living microbes performed simultaneous Hg0 bio-oxidation and denitrification, in which Hg0 as electron donor was biologically oxidized to oxidized mercury (Hg2þ), while NO as the terminal electron acceptor was denitrified to N2. The Hg2þ further complexed with humic acids in extracellular polymeric substances via functional groups (-SH, eOH, -NH- and -COO-) and formed humic acids bound mercury (HA-Hg). Non-living microbial matrix performed oxidative Hg0 biosorption, in which Hg0 may be physically adsorbed by cellular matrix, then non-metabolically oxidized to Hg2þ via oxidative complexation with eSH in humic acids and finally cleavage of SeH bond and surface charge transfer led to formation of HA-Hg. Therefore, bioconversion of Hg0 to HA-Hg by Hg0 bio-oxidation and oxidative Hg0 biosorption coupled with NO denitrification to N2 dynamically cooperated to accomplish simultaneous removal of Hg0 and NO in MBfR. © 2019 Published by Elsevier Ltd.
Handling Editor: Yongmei Li Keywords: Membrane biofilm reactor Hg0 bio-oxidation Hg0 biosorption Denitrification Mechanism
1. Introduction
* Corresponding author. E-mail address: [email protected] (Z.S. Wei). https://doi.org/10.1016/j.chemosphere.2019.125544 0045-6535/© 2019 Published by Elsevier Ltd.
Mercury (Hg) is an extremely toxic metal element and mostly notorious for its long-range mobility in global atmosphere, longterm persistence in ecosystem, neurotoxicity and bio-
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accumulation along food chains (Zhao et al., 2017). Nitrogen oxides (NOx) give rise to acid rain, haze, photochemical smog, and tropospheric ozone depletion (Zheng et al., 2016). Anthropogenic Hg and NO are mainly generated by combustion of fossil fuels in coal-fired power plants, medical waste incinerators, municipal waste combustors and sewage sludge incinerators (Zhao et al., 2017). Elemental mercury (Hg0) and nitric oxide (NO) are frequently present in flue gas, which account for 94% of total Hg and 95% of NOx, (Liu et al., 2014; Niu et al., 2014). These worsening situations stress the need of technologies for simultaneous flue gas demercuration and denitration. Membrane biofilm reactor (MBfR) is a technologically feasible, economically viable and environmentally friendly biotechnology for flue gas decontamination (Sahinkaya et al., 2011; Gu et al., 2018; Wei et al., 2019). A denitrifying MBfR removed 73e86% NO in simulated flue gas with long-term stability (Zhang et al., 2013). Our previous studies also proved the feasibility of MBfR for simultaneous removal of Hg0 and NO (Huang et al., 2019b, 2019c). In principle, the overall removal of Hg0 and NO in the denitrifying MBfR is the integrated result of multiple processes including membrane gas separation, medium absorption, biosorption and biotransformation (Kumar et al., 2008). Hollow fiber membrane offers vast specific gas-liquid interfacial area to enhance mass transfer of poorly soluble gas (Wang et al., 2018). Coupling membrane module to a biofilter can increase styrene removal efficiency by 20.7%, owing to selective membrane gas separation (Li et al., 2012). Chemical absorption can convert poorly soluble Hg0 and NO to water soluble Hg2þ and NO 3 to facilitate their removal. Chemical absorption accounted for 30.5% of NO removal in a bubble column reactor (Khan and Adewuyi, 2010). Chemical oxidation of dissolved elemental mercury (DEM) occurred in the presence of organic thiol groups, which are typical metabolic byproducts (Zheng et al., 2013). Non-living biomass exhibited vast capacity for Hg biosorption by functional groups (-SH, -S-S-, eCOOH, eSO3H, -PO3H2, eNH2, eN2C3H3 and etc.) on cell walls (Siddiquee et al., 2015; Karthik et al., 2017). Heavy metals biosorption by dead cells squez and Dussan, even outcompeted that by living cells (Vela 2009). Biomass of Bacillus cereus achieved 104.1 mg g1 Hg biosorption (Sinha et al., 2012). In our preliminary studies, Hg0 biooxidation was achievable by nitrifying/denitrifying bacteria (Huang et al., 2019b, 2019c). Hg0 bio-oxidation coupled to denitrifying NO reduction played vitally important roles in simultaneous removal of Hg0 and NO in membrane biofilm reactor. Current theories had extensively demonstrated that membrane gas separation, medium absorption, biosorption and biotransformation can all play critical roles in removal of Hg0 and NO. Nevertheless, how these processes jointly or differentially contributed to simultaneous removal of Hg0 and NO in MBfR may be poorly understood and well worth further investigation. To the best of our knowledge, very few studies managed to clarify this. The objective of this work is to clarify contribution of membrane gas separation, medium absorption, biomass adsorption and biotransformation to simultaneous removal of Hg0 and NO in a denitrifying MBfR. Transformation patterns of Hg0 and NO were comparatively analyzed. Mercury speciation in biofilm was determined by sequential extraction processes (SEPs) and by inductively coupled mass spectrometry (ICP-MS). The SEPs extracts were further characterized by excitation-emission matrix spectra (EEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Morphology of biofilm was studied by scanning electron microscopy (SEM). Mechanisms of simultaneous removal of Hg0 and NO jointly or differentially by multiple processes were therefore comprehensively elucidated.
2. Materials and methods 2.1. Experimental set-up The denitrifying MBfR systematically consisted of a polyvinylidene fluoride (PVDF) hollow fiber membrane reactor (Blue cross membrane technology Co., Ltd, Tianjin, China), gas generation-mixing system and nutrient medium recirculation system (Fig. S1). Hg0 vapor was generated by heating a mercury permeation tube (Qingan scientific instruments Co., Ltd, Suzhou, China) at 60e70 C thermostat water bath. NO was generated by a gas cylinder. Airstreams of Hg0 and NO were finely mixed with compressed air in a buffer bottle then pumped into the hollow fiber membrane reactor. The recirculating medium was sprayed over the membrane surface for microbial inoculation using a subaqueous pump. Tail gas was treated by 10% v/v H2SO4-5% m/v KMnO4 solution. The experimental set-up of MBfR had been detailed in our previous work (Huang et al., 2019b). Simultaneous removal of Hg0 and NO in membrane reactor (MR), membrane medium reactor (MMR), membrane biomass reactor (MBmR) and membrane biofilm reactor (MBfR) were comparatively evaluated in a 35-day operation. MBmR, MMR and MR were set up in parallel by modifying configuration of MBfR as following: (1) MR: recirculation system was removed to study contribution of membrane gas separation; (2) MMR: the recirculating mineral medium was sterilized (132 C, 205 kPa) and but not inoculated with bacteria to study overall contribution of membrane gas separation and medium absorption; (3) MBmR: glutaric dialdehyde (2.5% v/v) was added to mineral medium to obtain immobilized non-living biomass and study overall contribution of membrane gas separation, medium absorption and biosorption. MBfR, MBmR, MMR and MR underwent 35 days’ operation under identical operational conditions as following: Hg0 inlet load, 109.9 ± 0.5 mg m3 h1; NO inlet load, 147.8 ± 0.5 g m3 h1; gas residence time (GRT), 5.5 s; pH in the medium, 7.0e7.3; dissolved oxygen (DO) in the medium, 0.08e0.2 mg L1; temperature, 25 C; medium flow rate, 1 60 mL min . Phase I (Days 1e10) was a start-up phase for microbial inoculation in MBfR and MBmR, then the biocide was added to MBmR on Day 10. Afterwards, performance of simultaneous removal of Hg0 and NO in four reactors were comparatively evaluated in Phase II (Days 11e35). Concentration of oxidized mercury (Hg2þ) and nitrate nitrogen (NO 3 -N) in the recirculating medium of MMR, MBmR and MBfR were determined every 2e3 days to clarify transformation patterns of Hg0 and NO. 2.2. Mercury speciation Mercuric species of different chemical forms in the biomass of MBmR and MBfR were qualitatively and quantitatively analyzed by sequential extraction processes (SEPs) followed by inductively coupled plasma mass spectrometry (ICP-MS, iCAP Qc, Thermo Scientific Inc., USA) as per previous methods (Biester and Scholz, 1997). SEPs classified Hg species in biomass into 6 fractions, namely, water soluble mercury (WSeHg), ion exchangeable mercury (IE-Hg), methyl-mercury (MeHg), humic/fulvic acids bound mercury (HA/FA-Hg), organically bound mercury sulfide (Hg-SR) and mercury sulfide (HgS). These fractions were leached sequentially by chemical treatments, oxidized to total mercury (Tot-Hg) by aqua regia digestion and separately quantified by ICP-MS. Detailed procedures were as per following: (1) Total mercury (Tot-Hg): 2.5 g biofilm sample was digested with 16 mL aqua regia at 160 C hotplate for 3 h; (2) WS-Hg: 20 g biofilm was sampled, air-dried overnight and weighted. 50 mL DDI water was added to 20 g
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biofilm sample and the mixture was shaken by vortex at 200 rpm for 2 h then centrifuged at 10,000 rpm for 3 min. Sediment was subject to secondary extraction and the supernatants were collected. 10 mL evenly mixed supernatant was digested with 4 mL aqua regia at 100 C water bath for 1 h; (3) IE-Hg: 50 mL 1 M NH4Ac (aq) was added to the residue in (2) followed by procedures in (2); (4) MeHg: 20 mL 2% HCl-10% ethanol solution was added to residue in (3). The mixture was shaken by vortex at 200 rpm for 20 min, sonicated at 60 ± 2 C for 20 min, centrifuged at 5000 rpm for 10 min and digested as per procedures in (2); (5) HA/FA-Hg: 50 mL 1 M NH3$H2O was added to residue in (4) followed by procedures in (2). 10 mL evenly mixed supernatant was digested with 10 mL concentrated HCl and 10 mL 30% H2O2 at 85 C water bath for 2 h; (6) Hg-SR: The residue (4) was first digested with 10 mL 0.02 M HNO3 at 85 C water bath for 2 h and subsequently with 16 mL 30% H2O2 at 85 C water bath for 1 h 50 mL 1 M NH4Ac-6% HNO3 solution was added to the digest followed by procedures in (2); (7) HgS: residue in (6) was repeatedly rinsed with DDI water for 2 h and air-dried overnight. 2.5 g residue was digested as per procedures in (1). 2.3. Characterization The dominant fractions HA/FA-Hg in mercury speciation were further characterized by EEM, XPS and FTIR. EEM: The fluorescent spectral properties of HA/FA-Hg extracts in MBfR and MBmR were characterized by an excitation emission matrix F-7000 fluorescence spectrophotometer (Hitachi Co., Ltd, Japan). XPS: Chemical form of Hg in HA/FA-Hg extracts were further characterized by an ESCALab250 X-ray photoelectron spectrometer (Thermo Fisher Scientific Inc., USA). HA/FA-Hg extracts were acidified using 1 M HCl (final pH < 1) and stored at 4 C for 1 d to facilitate humic acid precipitation. The precipitates were recovered by centrifugation and airdried overnight. The air-dried HA/FA-Hg precipitates were used for XPS analysis. FTIR: The organic functional groups in HA-Hg precipitates were characterized by a VERTEX 80 Fourier transform infrared spectrometer (Bruker Optics Co., Ltd, Germany). SEM-EDS: The morphology of biofilm samples from MBfR and MBmR on day 10, day 20 and day 30 were studied by a JSM-6330F Field Emission Scanning Electron Microscope (Japan Electron Optics Laboratory Co, Ltd). 2.4. Analytical methods Concentration of Hg0 in flue gas was determined by a mercury vapor indicator (MVI, Ion science company, UK). Concentration of Hg2þ in the recirculating medium was quantified by non-N2purgeable total mercury and determined by aqua regia digestion followed by atomic fluorescence spectrometry (AFS) using a dual channel atomic fluorescence spectrometer (AFS-820, Jilin University Little Swan Instrument Co., Ltd, China). Concentrations of NO 3N in the recirculating medium was determined by ultraviolet spectrophotometry using a UV spectrophotometer (T6, Beijing Persee Instruments Co., Ltd, China). Instrumentation for determinations of DO, pH and flow rate have been specified in our previous work (Huang et al., 2019b). 3. Results and discussion 3.1. Roles of Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification in simultaneous removal of Hg0 and NO Membrane gas separation, medium absorption, biomass adsorption, and biotransformation were the four potentially contributory mechanisms for simultaneous removal of Hg0 and NO
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in the denitrifying MBfR. To understand the role of each process, a 35-day long-term experiment was conducted to evaluate simultaneous removal of Hg0 and NO in MR, MMR, MBmR and MBfR, respectively. As shown in Fig. 1aeb, in start-up phase (Days 1e10), removal efficiency of Hg0 and NO in MBfR gradually increased from 67.5 ± 3.5% and 50.7 ± 3.5% to 89.9 ± 1.5% and 73.5 ± 1.4%, respectively. Likewise, removal efficiency of Hg0 and NO in MBmR increased from 65.2 ± 3.9% and 51.3 ± 3.1% to 88.9 ± 2.0% and 71.6 ± 0.7%, respectively. Afterwards, removal efficiency of Hg0 and NO in MBfR steadily maintained at 90.9 ± 0.9% and 74.8 ± 1.1%, respectively during Days 11e35. After addition of biocide on day 11, Hg0 removal efficiency in MBmR gradually decreased and reached a steady state at 62.4 ± 1.4% during Days 16e22. Subsequently, Hg0 removal efficiency in MBmR further decreased and maintained at 17.1 ± 1.5% during Days 31e35. Meanwhile, NO removal efficiency in MBmR kept decreasing during Days 11e18 then maintained at 13.2 ± 0.6% during Days 19e35. Nevertheless, removal efficiency of Hg0 and NO maintained stayed at low levels in MMR (12.4 ± 1.8%; 10.9 ± 1.0%) and MR (5.8 ± 2.5%; 5.2 ± 1.0%) throughout the operation. The net contribution of membrane gas separation, medium absorption, biosorption and biotransformation to Hg0 removal in MBfR were 4.9%, 8.1%, 38.8% and 48.2%, respectively, while those to NO removal in MBfR were 7.2%, 8.9%, 9.5% and 84.9%, respectively. Biosorption and biotransformation may play crucially important roles in Hg0 removal. However, NO removal was dominantly attributed to biotransformation. Membrane gas separation, medium absorption, biosorption and biotransformation contributed 4.9%/7.2%, 8.1%/8.9%, 38.8%/9.5% and 48.2%/84.9% of overall Hg0/NO removal in MBfR. Simultaneous removal of Hg0 and NO in MBfR showed higher performance in comparison to that in previous studies (Hg0 removal efficiency: 95%, GRT: 6e70 s; NO removal efficiency 75e99%, GRT: 30e132 s) (Wang et al., 2006; Philip and Deshusses, 2008; Jiang et al., 2009; Zhang et al., 2013; Han et al., 2016). As shown in Fig. 1ced, Hg2þ concentration in recirculating medium of MBfR periodically increased from 0 to 11.2e13.2 mg L1 every 10 days. Nevertheless, that in MBmR showed similar increasing trends within narrowing ranges (0e10.6 mg L1; 0e8.4 mg L1; 0e5.3 mg L1; 0e0.8 mg L1) and that in MMR merely reached 0.6 mg L1. Hg0 oxidation took place periodically in both MBfR and MBmR, suggesting that Hg0 removal in MBfR was dominantly attributed to Hg0 bio-oxidation and Hg0 biosorption in MBmR may be an oxidative process. Hg0 oxidation took place steadily in MBfR but progressively weakened in MBmR resulting in lower Hg0 removal. Hg0 oxidation didn’t take place in medium of MMR, suggesting that Hg0 may be dissolved or flushed away by the medium to achieve Hg0 removal in MMR. Meanwhile, NO 3 -N concentration in medium of MMR, MBmR and MBfR irregularly ranged within 0e0.6 mg L1, 0e1.6 mg L1 and 1.5 mg L1, respectively. Average nitrogen removal rate and nitrogen production rate in MMR, MBmR and MBfR were 1.52/1.58 mgN d1, 2.44/1.52 mgN d1and 21.79/ 0.72 mgN d1, respectively. NO removal in MBfR can be due in large part to denitrification (NO/N2), while that in MMR and MBmR may be ascribed to chemical oxidation. Hg0 acted as electron donor and NO acted as terminal electron acceptor in simultaneous Hg0 bio-oxidation and denitrification to form an overall redox in MBfR (Huang et al., 2019c). Hg0 bio-oxidation can be coupled to sulfate bio-reduction, in which Hg0 acted as electron donor, while sulfate acted as terminal electron acceptor (Huang et al., 2019a). A sulfuroxidizing biotrickling filter achieved Hg0 removal by Hg0 biooxidation (Philip and Deshusses, 2008). These combinations indicated that Hg0 bio-oxidation can potentially interact with many microbial metabolic processes. MBmR hardly performed denitrification but had weak ability for Hg0 oxidation, suggesting some
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Fig. 1. Simultaneous removal of Hg0 and NO in MR, MMR, MBfR and MBfR: (a) Hg0 removal performance in MR, MMR, MBfR and MBfR; (b) NO removal performance MR, MMR, MBfR and MBfR; (c) Variation of Hg2þ concentration in recirculating medium of MMR, MBfR and MBfR; (c) Variation of NO 3 -N concentration in recirculating medium of MMR, MBfR and MBfR.
intermediate electron acceptors may oxidize Hg0 to Hg2þ in Hg0 biosorption. Thiol compounds can acted as the electron acceptors for Hg0 by forming self-assembled monolayers (SAMs) in oxidative complexation (Zheng et al., 2013). In summary, simultaneous Hg0 bio-oxidation (48.2% Hg0 removal) and denitrification (84.9% NO removal) aided by oxidative Hg0 biosorption (38.8% Hg0 removal) made major contribution to simultaneous removal of Hg0 and NO in MBfR. 3.2. Mercury speciation: bio-oxidation of Hg0 to humic acids bound mercury As shown in Fig. 2, in Phase I, Hg speciation in biofilms of MBfR (Tot-Hg: 3.55 ± 0.08 mg g1; HA/FA-Hg: 2.85 ± 0.09 mg g1; IE-Hg: 0.51 ± 0.02 mg g1) and MBmR (Tot-Hg: 3.48 ± 0.04 mg g1; HA/FAHg: 2.77 ± 0.03 mg g1; IE-Hg: 0.45 ± 0.04 mg g1) didn’t show significant difference (p > 0.13, student’s t-test). Herein, HA/FA-Hg accounted for 79.5e80.0% of Tot-Hg and dominated Hg speciation in biofilms. In addition, IE-Hg accounted for 12.9e14.2% of Tot-Hg was the subdominant Hg species. In Phase II, Tot-Hg in biofilms of MBfR and MBmR attained 12.99 ± 0.19 mg g1 and 7.71 ± 0.09 mg g1, respectively, suggesting that living biofilms had higher capacity for Hg bioaccumulation than dead biomass. Furthermore, HA/FA-Hg maintained dominance in Hg speciation in biofilms of MBfR (10.30 ± 0.04 mg g1, 79.3% of Tot-Hg) and MBmR (6.09 ± 0.02 mg g1, 79.0% of Tot-Hg). Likewise, IE-Hg was the second dominant Hg species in biofilms of MBfR (1.87 ± 0.07 mg g1, 14.4% of Tot-Hg) and MBmR (1.02 ± 0.05 mg g1, 13.2% of Tot-Hg). Besides, Tot-Hg in Inoculum was almost negligible (<0.2 ng g1). It’s estimated that 73.2% and 71.5% of bioaccumulated Hg0 was oxidized to HA/FA-Hg in MBfR and MBmR. Formation of HA/FA-Hg
can be attributed to Hg2þ complexation with selective binding sites in extracellular polymeric substances (EPS) and cell walls (functional groups: SH, -S-S-, eCOOH, eSO3H, -PO3H2, eNH2, eN2C3H3 and etc.) and even dead biomass exhibited vast capacity for complexation (Siddiquee et al., 2015; Karthik et al., 2017). The microbial metabolites EPS can considerably sequestrate and immobilize bioavailable Hg2þ via complexation to achieve Hg2þ detoxification (Chai et al., 2013; Ouyang et al., 2017). Our previous study also confirmed HA-Hg played critical role in biofilm Hg speciation (Huang et al., 2019a, 2019b, 2019c). Since HA/FA-Hg was the dominant Hg species in both MBfR and MBmR, it can be inferred that both living biofilm and dead biomass can oxidize Hg0 to Hg2þ potentially by means of Hg0 bio-oxidation and oxidative Hg0 biosorption. Hg2þ further reacted with selective binding sites in EPS and cell walls from dead or living cells to dominantly form HA/FAHg. In summary, the removed Hg0 was dominantly oxidized to HA/ FA-Hg potentially by Hg0 bio-oxidation in MBfR and oxidative Hg0 biosorption in MBmR. 3.3. Characterization of humic acids bound mercury As shown in Fig. 3, all EEM spectra of the HA/FA-Hg extracts from MBfR and MBmR showed two prominent peaks at Ex/ Em¼(285e287 nm, 343e346 nm) and Ex/Em¼(345e346 nm, 419e421 nm), respectively, which could be attributed to soluble microbial products-like compounds (Region IV: Ex > 250 nm, Em < 380 nm) and humic acid-like compounds (Region V: Ex > 280 nm, Em > 380 nm) (Chen et al., 2003), providing chemical basis for formation of humic acids bound mercury (HA-Hg) in MBfR and MBmR. Previous researches demonstrated that dissolved organic matter (DOM) especially humic acids can play decisive role
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Fig. 2. Quantitative analysis of mercury speciation: (a) Mercury speciation in biofilms from MBfR; (b) Mercury speciation in biofilms from MBmR.
Fig. 3. Characterization of HA/FA-Hg extract: (a) and (b) EEM spectra of the HA/FA-Hg extract from MBfR; (c) and (d) EEM spectra of the HA/FA-Hg extract from MBmR.
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in Hg speciation, because strong affinity between Hg2þ and functional groups in DOM (-SH, eNH2, eCOOH, eOH) led to formation of highly chemically stable Hg-DOM complexes (logKs ¼ 23.6e38.3) (Haitzer et al., 2002; Ding et al., 2018; Jiang et al., 2018). Moreover, Hg2þ-humic acids complexation considerably abated bioavailability of Hg2þ and acted as a vital mechanism for heavy metal detoxification physiologically (Benoit et al., 2001; Chai et al., 2013; Ouyang et al., 2017). Our previous research also confirmed critical role of HA-Hg in Hg speciation (Huang et al., 2019a). Therefore, gaseous Hg0 was biologically oxidized to Hg2þ and took its final dominant form of HA-Hg in MBfR. As shown in Fig. 4a, FTIR spectra of HA-Hg precipitates from MBfR and MBmR showed prominent peaks corresponding to stretching vibrations of OeH (3139.41 cm1), eSH (2541.47 cm1), eNH (1637.01 cm1), eNHe (1532.14 cm1), -COO- (1402.45 cm1), CeOeC/CeCeO (1232.69 cm1) and CeO/CeC/CeOeC (1042.66 cm1) (Kogelheide et al., 2016; Kamnev et al., 2017). This further elucidated that oxidized mercury may be complexed with humic acids by functional groups eSH, eOH, -NH- and -COO-, which are typical binding sites for ionic mercury (Ding et al., 2018). As shown in Fig. 4b, all XPS spectra of HA-Hg precipitates from MBfR and MBmR showed two distinct groups of peaks ascribed to doublet separation of Hg 4f core level into Hg 4f5/2 (104.5e105.2 eV) and Hg 4f7/2 (100.4e101.1 eV), respectively, potentially attributed to
divalent mercuric species (Hutson et al., 2007). In Phase II, Hg 4f5/2 binding energy of HA-Hg in MBfR (101.1 eV) was higher than that in MBmR (100.4 eV), suggesting that Hg potentially existed in a more oxidized form in HA-Hg from MBfR. Hg0 may be more thoroughly oxidized in Hg0 bio-oxidation in MBfR than oxidative Hg0 biosorption in MBmR. As shown in Fig. 4c, an abundance of rod-shaped bacterial cells in biofilms from MBfR maintained intact throughout the operation. However, destruction of cells (Day 20) and complete autolysis (Day 30) were observed in bacterial cells in biofilms from MBmR. This visually differentiated between living biofilms in MBfR and dead biomass in MBmR. In summary, the final formation of HA-Hg in both MBfR and MBmR was attributed to complexation of oxidized mercury with functional groups eSH, eOH, -NH- and -COO- in humic acids. Hg0 may be more thoroughly oxidized in Hg0 biooxidation by living cells than oxidative biosorption by dead cells. 3.4. Mechanisms of Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification Biotransformation of Hg0 and NO in MBfR was primarily attributed to Hg0 bio-oxidation to HA-Hg and biological denitrification. Moreover, Hg0 biosorption was potentially an oxidative process and achieved oxidation of Hg0 to HA-Hg. Medium
Fig. 4. Characterization of HA/FA -Hg precipitates: (a) FTIR spectra of HA/FA-Hg precipitates; (b) XPS spectra of HA/FA-Hg precipitates (c) SEM images and EDS spectra of the biofilm samples.
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Fig. 5. Mechanisms of contribution of multiple processes to simultaneous removal of Hg0 and NO in MBfR.
absorption served to chemically oxidize NO and physically dissolve Hg0. On the basis of these, the mechanisms of simultaneous removal of Hg0 and NO in MBfR by joint contribution of membrane gas separation, medium absorption, biosorption and biotransformation was hypothesized.
2Hg0 þ 2NO þ 4Hþ /2Hg2þ þ N2 þ H2 O
(1)
Hg2þ þ 2HA SH / ðHA SÞ2 Hg2þ þ 2Hþ
(2)
Hg2þ þ 2HA OH / ðHA OÞ2 Hg2þ þ 2Hþ
(3)
Hg2þ þ 2HA NH2 / ðHA NHÞ2 Hg2þ þ 2Hþ
(4)
2þ
Hg
þ 2HA COOH / ðHA COOÞ2 Hg
2þ
Hg0 þ 2HA SH / ðHA SÞ2 Hg2þ þ 2H
SeH bond and surface charge transfer led to oxidative complexation of Hg0 with eSH and final formation of HA-Hg (Zheng et al., 2013) (Eq. (6)). Besides, NO in oxidized form of NO 3 can also be electrostatically bound to functional groups in biopolymers to achieve biosorption (Sarode et al., 2019). Therefore, molecular diffusion-based membrane gas separation, physical/chemical medium absorption, non-metabolic oxidative biosorption by nonliving microbial matrix and metabolic simultaneous Hg0 biooxidation and denitrification by living microbes differentially or jointly contributed to simultaneous removal of Hg0 and NO in MBfR. 4. Conclusions
þ
þ 2H
(5) (6)
As shown in Fig. 5, influx of Hg0 and NO permeated the membrane wall vertically by molecular diffusion and gradually concentrated in permeant flux, which finally limited the concentration gradient-driven molecular diffusion (Janes et al., 2008; Monsalve-Bravo and Bhatia, 2018). The permeant flux of Hg0 and NO entered the medium phase, in which NO may be chemically 0 oxidized to NO2 by O2 and dissolved in the form of NO 3 , while Hg may be physically absorbed in the form of DEM (Thiemann et al., 2005; Zheng et al., 2013). Subsequently, Hg0 and NO interacted with biofilms over membrane surface. Firstly, living cells actively performed simultaneous Hg0 bio-oxidation and denitrification via enzymatic metabolic processes (Halbach et al., 1988; Smith et al., 1998; Siciliano et al., 2002; Colombo et al., 2014), in which Hg0 acted as electron donor and was biologically oxidized to Hg2þ while NO acted as terminal electron acceptor and was denitrified to N2 (Eq. (1)). The oxidized mercury further complexed with humic acids in EPS via selective binding functional groups (-SH, eOH, -NH- and -COO-) and dominantly formed HA-Hg (Eqs. (2)e(5)). Secondly, non-living microbial matrix (cell walls, EPS and metabolic byproducts) may provide intermediate electron acceptors and passively performed Hg0 oxidation via chemical non-metabolic processes (Vijayaraghavan and Yun, 2008; Chojnacka, 2010). Hg0 was physically adsorbed by eSH in humic acids, then cleavage of
This study comparatively evaluated contribution of multiple processes to simultaneous removal of Hg0 and NO in MBfR, which was dominantly attributed to Hg0 bio-oxidation, oxidative Hg0 biosorption and denitrification. Living microbes performed simultaneous Hg0 bio-oxidation and denitrification, in which Hg0 as electron donor was oxidized to Hg2þ while NO as terminal electron acceptor was denitrified to N2. The Hg2þ further complexed with humic acids in EPS and finally formed HA-Hg. Non-living microbial matrix performed oxidative Hg0 oxidation, in which Hg0 was physically adsorbed and finally formed HA-Hg via oxidative complexation with humic acids. Therefore, contribution of multiple processes was clarified. Author contribution statement The authors (Zhenshan Huang, Zaishan Wei*, Xiaoliang Xiao, Bailong Li, Song Ming, Xiangling Cheng, Huaiyong Jiao) finished the paper entitled "Bioconversion of Hg0 into HA-Hg for simultaneous removal of Hg0 and NO in a denitrifying membrane biofilm reactor" together, among which Zhenshan Huang takes charge of Investigation, Data curation, Formal analysis and Writing, Zaishan Wei* of Supervision and review & editing, Xiaoliang Xiao of Formal anlysis, Bailong Li of Formal analysis, Song Ming of Visualization, Xiangling Cheng of Software , Huaiyong Jiao of Investigation. We hereby certify that this paper consists of original, unpublished work which is not under consideration for publication elsewhere. I hope your favorable consideration for publication to Chemosphere.
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