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Effect of gaseous mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor
Accepted Manuscript Short communication Effect of gaseous mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor Z.S. Wei, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L. Xiao PII: DOI: Reference:
S1385-8947(17)30096-7 http://dx.doi.org/10.1016/j.cej.2017.01.085 CEJ 16387
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
7 November 2016 20 January 2017 21 January 2017
Please cite this article as: Z.S. Wei, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L. Xiao, Effect of gaseous mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.01.085
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Effect of gaseous mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor
Z.S. Wei*, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L.Xiao School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou 510275, China
*
Corresponding author Tel: +86 20 84037096; fax: +86 20 39332690
E-mail address: [email protected] (Z.S.Wei)
Abstract The effect of gaseous elemental mercury on nitric oxide removal performance, microbial community and microbial nitrogen metabolism of a hybrid catalytic membrane biofilm reactor (HCMBR) were evaluated. A gaseous Hg0 concentration of 25±5 µg·m-3 reduced the NO removal efficiency of HCMBR from 86.4% to 41.9%. In 80 days of operation, NO removal efficiency was up to 68.8%, and Hg0 removal efficiency achieved 81.7%, respectively. The addition of gaseous elemental mercury affected the microbial community structure and changed microbial nitrogen metabolism, as shown by metagenomics sequencing method. Some phyla, such as Azospirillum, and Delftia, increased in abundance, whereas others, such as Weeksella , decreased. Hg0 could combine with merP and be transported by merT, and then be oxidized into Hg2+ by KatG or KatE. The nitrogen metabolism related processes such as catalysis, nitrification and denitrification, ammoniation and biological nitrogen fixation processes, exists in HCMBR and HCMBRHg. Keywords: Gaseous elemental mercury; Nitric oxide; Microbial community; Nitrogen metabolism; Mercury metabolism
1. Introduction Nitrogen oxides (NOx) are major air pollutants that trigger serious environmental problems, including acid rain, photochemical smog, and haze [1]. Mercury is considered one of the most toxic heavy metals with harmful effects on humans and other terrestrial and aquatic organisms due to its toxicity, volatility, persistence, and bioaccumulation [2]. The main sources of NOx and mercury pollution are power plants, coal–fired boiler, municipal waste combustors, medical waste incinerators, chlor–alkali plants and cement plants [3,4]. The microbial remediation of nitrogen oxides may offer a greener and cheaper technology that removes of NOx from contaminated point sources. Biofiltration of nitric oxide has been undertaken using perlite, compost, biofoam, soil compost, carbon foam and lava as packing materials [5-7].
A rotating drum biofilter was
applied to denitrifying removal of nitric oxide (NO) using glucose as electron donor [8] due to the lower mass transfer resistance and higher effective utility of packing materials [9]. A biotrickling filter using P. mendocina was fabricated for treating NO in a simulated exhaust gas [10]. Denitrification removal of NO by a biotrickling filter inoculated P. putida SB1 was an effective protocol for eliminating NOx from flue gas [11]. The suspended biofilter inoculating the thermophilic denitrifying bacterium Chelatococcus daeguensis TAD1 could remove NO efficiently with great elimination capacity [12]. A bench scale biotrickling filter using Chelatococcus daeguensis was capable of converting nitric oxide NOx under thermophilic condition [13, 14]. NO removal in an anoxic biofilter packed with four compost types at temperatures of 55–60°C was investigated [15]. An integrated process of Fe(II)EDTA absorption coupled with two-stage microbial regeneration using immobilized microorganisms was used to removal of NOx[16]. A mixed absorbent had been proposed to enhance
the chemical absorption–biological reduction process for NOx removal from flue gas [17]. A newly isolated thermophilic Anoxybacillus sp. HA, identified by 16S rRNA sequence analysis, could simultaneously reduce Fe(II)EDTA-NO and Fe(III)EDTA [18]. The nitrifying hollow fiber membrane bioreactor (MBR) is a promising technology for NOx control because it provides high surface area for mass transfer of nitrogen oxides to nitrifying bacteria at temperatures between 20 and 55°C [19]. A hallow-fiber membrane bioreactor was stable and highly efficient for denitrifying removal of NO from simulated flue gas, NO removal was inversely proportional to the inlet oxygen concentration, low-concentration sulfur dioxide had no great influence on denitrification removal of NO[20]. The thermophilic membrane biofilm reactor using the synergy of nitrifying and aerobic denitrifying microorganisms could be used for NO removal from flue gas at 60 °C [21]. A hybrid catalytic membrane biofilm reactor offered potential for flue gas denitrification, membrane photocatalysis, and nitrification/denitrification could contribute to the removal of NO [22]. Elemental mercury emission from coal-fired flue gas is often contained in the flue gas even after the electrostatic precipitator or baghouse and wet flue gas desulfurization (WFGD) treatment, which might have toxic effects on the nitrification and denitrification microorganism in the biotreatment of nitrogen oxides system. Fewer studies have been conducted to the effect of gaseous elemental mercury on nitric oxide removal in hybrid catalytic membrane biofilm reactor. Three of the strains (Enterobacter helveticus, Citrobacter amalonaticus, and Cronobacter muytjensii) can detoxify the environment from mercury due to wastes of florescent lamps[23]. Biogenic palladium nanoparticles (Bio-Pd NPs) do not exert toxicity towards the bioluminescent marine bacterium V. fischeri and may have limited inhibitory effects towards some respiratory metabolisms [24]. The deduced amino acid sequence of
merA gene indicated a sequence homology with different organisms from the alpha proteobacteria group [25]. The objective of this work is to study the effect of gaseous elemental mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor (HCMBR). The study analyzes bacterial community composition assessed by metagenomics sequencing method, and evaluates nitrogen metabolism, mercury metabolism. The mechanism for simultaneous denitration and demercuration in HCMBRHg is elicited, which is believed to promote the application of the MBR.
2. Methods 2.1. Experimental procedure The experimental flow loop effect of mercury on nitric oxide removal in hybrid catalytic membrane biofilm reactor used in the study was shown schematically in Fig.1. The HCMBR included an N-TiO2/PP hybrid catalytic membrane biofilm reactor, a waste gas mixture (NO, Hg and clean air) generation system, and a nutrient supply system. A N-TiO2-coated polypropylene (PP) hollow fiber membrane consisted of 2400 hydrophobic, microporous membrane polysulfone fiber bundles with the inner diameter of 0.38 mm, outer diameter of 0.52 mm and effective length of 300 mm. Nitrification and denitrification bacterials adhered to the surface of the surface of N-TiO2/PP to form the biofilm. The NO supplied from the gas cylinders, was first diluted with the compressed air, passed through an air mixture bottle, then flowed upwards the bottom of the hybrid catalytic membrane bioreactor. Batch tests on Hg0, Hg0 vapor equipment unit consists of an elemental mercury permeation tube, a bottle of gas mixture, a water bath. The elemental mercury permeation tube was
placed in a water bath with a temperature of 328 K. Hg0 vapor supplied from the mercury permeation unit, was first diluted with the compressed air, passed through an air mixture bottle; simulated NO and Hg0 flue gas were flowed upwards through the HCMBR. 2.2. Metagenomics sequencing data analysis Bacterial community compositions and the gene function of bacterial in the hybrid catalytic membrane bioreactor were assessed by 16S rDNA and metagenomics method, and identify the colonies of the predominant microorganisms by the procedures of total DNA extraction, polymerase chain reaction (PCR) amplification of 16S rDNA, cloning and sequencing, calculation of similarity and diversity indexes, analysis of the successional route of the community. 2.2.1 Extraction of genome DNA Total genome DNA from samples was extracted using CTAB method. DNA concentration and purity was monitored on 1% agarose gels. To obtain a specific concentration, DNA was diluted to 1ng/µl using sterile water. The samples were collected from the outface of the membrane in HCMBR and were stored at -80ºC before DNA extraction. The samples were added into a Eppendorf
tube ， which is mixed with 1000ul CTAB and certain amount of
Lysozyme. The tube was heated in 65 ºC water bath. Afterwards，the samples were centrifuged at 3000rpm for 5min before supernatant was removed. And then， combined with Phenol，Chloroform and Isoamyl alcohol at a ratio of 25：24：1(v/v/v) ， the samples were centrifuged at 12000 rpm for 10min before supernatant was collected. Take 1.5 mL of supernatant into a new centrifuge tube containing Isoamyl alcohol, shake the tube upwards and downwards for several times, then place the tube for precipitation at -20 ºC. After the new tube was centrifuged at 12000 rpm for
10min, keep the sediment instead of the supernatant. The sediment was rinsed by 1ml 75% alcohol twice and then air-dried in clean benches. Used ddH2O to dissolve the DNA sample and if necessary，the sample can be put into 55-60℃ environment for10min for solubilization. At last，the RNA in the sample was dispelled by 1ul RNase and then placed in static state in 37℃ for 15min. 2.2. 2 Amplicon Generations 16S rRNA genes were amplified by the specific primer (515F-806R) with the barcode. All PCR reactions were carried out in 30µL reactions with 15µL of Phusion® High-Fidelity PCR MasterMix (New England Biolabs); 0.2μM forward and reverse primers, and about 10 ng templates DNA. Thermal cycling consisted of 1-minute initial denaturation at 98 ºC, 10-second 30 cycles of denaturation at 98 ºC, 30-second annealing at 50 ºC, and 30-second elongation at 72 ºC. Finally the sample is preserved at 72 ºC for 5 min. 2.2. 3 PCR Products quantification and qualification Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis in 2% agarose gel for detection. Samples with bright main strip between 400-450bp were chosen for further experiments. 2.2. 4 PCR Products Mixing and Purification PCR products were mixed in accordance with equidensity ratios. Then, mixture PCR products were purified by GeneJET Gel Extraction Kit (Thermo Scientific). 2.2. 5 Library preparation and sequencing Sequencing libraries were generated by NEB Next® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer
(Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina HiSeq platform and 250 bp/300bp paired-end reads were generated. 2.2.6 Data analysis Paired-end reads from the original DNA fragments are merged by using FLASH -a very fast and accurate analysis tool which is designed to merge paired-end reads when overlaps occur between reads1 and reads2. Paired-end reads were assigned to each sample according to the unique barcodes. Sequences were analyzed by using QIIME software package (Quantitative Insights Into Microbial Ecology), and in-house Perl scripts were used to analyze alpha- (within samples) and beta- (among samples) diversity. Sequences with ≥97% similarity were assigned to the same OTUs. A representative sequences for each OTU was picked and the RDP classifier to annotate taxonomic information for each representative sequence was used. 2.3. Analytical methods Nitric oxide concentration was measured by a Testo350 flue gas analysis device (Testo AG, Germany). The inlet and outlet of the bubbler were sampled for gaseous mercury inaccordance with Ontario Hydro Method of U.S. EPA Method 23 and U.S.DOE. The concentrations of mercury were quantified by atomic fluorescence spectrophotometry (AFS). Gas flow rates and Liquid flow rates were measured using Model LZB-1 flow meters. Illumination intensity were measured using SIGMA AR823 Separation of illuminance analysis device (SIGMA, Hong Kong).The pH values were measured by a Model pHB-3 pH Tester ( Sanxin Instrument Company, Shanghai, China).
3. Results and discussion
3.1. The effect of elemental mercury on nitric oxide removal performance Fig.2 showed the effect of elemental mercury on nitric oxide removal performance of the HCMBR during 130-d continuous running test under the conditions of pH of 6.8~7.2, sprinkling amount of 60 ml·min-1, NO inlet load of 135~145 g·m-3·h-1, gas residence time (GRT) of 9 s at normal temperature and visible Light. As shown in Fig.1, two distinct phases were observed: phase I under no gaseous elemental mercury, lasted from days 1 to 50; in phase II the addition of gaseous Hg0 concentration of 25 µg·m-3, from days 51 to 130, effect of elemental mercury on total performance of the HCMBR was investigated. In phase I, NO removal efficiency (RE) changed from 47.8 to 61.6% before 8 d, then increased from 64% at 9thd to 73.6% at 14th d. RE had a slight fluctuation around 74% from days 15 to 40. NO removal efficiency drastically increased from 72.1% at 40thd to 83.8% at 41th d. RE changed small from 86.1% at 42th d to 86.3% at 50th d. The increase in the removal efficiency was attributed to the acclimatization phase, nitrification and denitrification bacteria were attached to the surface of N-TiO2/PP to form the biofim. In phase II, NO removal efficiency sharply fell from 86.3 to 41.9 % after 24 h of the supply of gaseous Hg0 concentration of 25µg·m-3. The possible reason for the fall in NO removal efficiency could be that gaseous elemental mercury was toxic to microorganism, and inhibited NO removal. Subsequently, NO removal efficiency can ascend to about 60% surprisingly after a few days. After the gradual ascent, NO removal efficiency maintained at about 63% for almost 70 days. Hg0 removal efficiency was a striking upward trend from 25% at 51thd to 80% at 60th d, after that, the Hg removal efficiency experienced an obvious fluctuation and decreased from 81.7% at 61th d to 63.5% at 130th d. This illustrated that HCMBR showed a fast
response, could efficiently handle this applied influence, and had good NO and mercury removal performance. NO and mercury are bioremediation by the nitrification and denitrification microorganism in the biofilm attached to the surface of N-TiO2/PP. 3.2. The bacterial community in the HCMBR before and after mercury addition The addition of gaseous elemental mercury affect the microbial community structure, as shown by denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA genes. 94.75% of the sequences were assigned to bacteria and only few sequences belonged to archaea in total. At phylum level, the relative abundances of dominant bacteria Proteobacteria, Bacteroidetes and Firmicutes of HCMBR and HCMBRHg were in the change of 35.7 to 45.78%, 24.61 to 15.22% and 0.75 to 1.06%, respectively (Fig.3a). Some phyla, such as Azospirillum, Delftia, Firmicutes and Proteobacteria, increased in abundance, whereas others, such as Weeksella, and Bacteroidetes, decreased. For comparison, in the presence of HCMBR, with/without gaseous elemental mercury in flue gas treatment process, the relevant abundance of Azospirillum and Delftia in HCMBRHg was 13 times and 6 times of that in HCMBR, respectively. However, the relevant abundance of Weeksella in HCMBR was 794 times of that in HCMBRHg. Proteobacteria has the functions of nitrification, denitrification and nitrogen fixation using ammonia, methane and some kind of volatile fatty acids as electron donor [27-29]. Bacteroidetes can make use of nitrate or nitrite as the ultimate electron acceptor at hypoxia or anaerobic condition [30]. At the class level, comparison of HCMBR and HCMBRHg, the microbial community structure changed from 8 to 13 classes (Fig.3b). Seven classes that included
Enterobacteriaceae,
Burkholderiales,
Rhizobiales,
Flavobacteriales,
Bacteroidetes, Bacillales and Desulfobacterales, were unchanged. Six classes that include
Pseudomonadales,
Xanthomonadales,
Clostridiales,
Actinomycetales,
Sphingobacteriales and Caulobacterales, were appeared. One classe, Chlorobi, disappeared due to its inability to survive the mercury environment. At the genus level, the dominant bacteria genus in the HCMBRHg, and HCMBR, such as Burkholderia, Delftia, Azospirillum, Elizabethkingia, Acinetobacter, Weeksella, Proteiniphilum, Bacteroides , Sphingo bacterium were identified. All two communities were dominated by the Burkholderia, with the relative abundances of 11.99%, and 9.61% in HCMBRHg, and HCMBR, respectively (Fig.4). Burkholderia is was a genus of ß-proteobacteria and has the function of nitrification and denitrification. Burkholderia cenocepacia and Achromo bacterxylosoxidans were more resistant to the recombinant enzyme produced by a synthetic gene encoding dispersin B of Aggregatibacter actinomycetemcomitans [31]. Delftia could make use of aniline, organic nitrogen as the carbon source and the nitrogen source [32,33]. Sphingobacterium, Bacteroides and Acinetobacter possen the function of nitrification and denitrification[34-35]. Azospirillum could utilize the nitrogen in air or dissolved in the water, or even the nitrogen generated by denitrifying bacteria to conduct nitrogen fixation [36]. 3.5 Microbial mercury metabolism As shown in Fig. 5a, eight classes mercury resistant bacteria group with function gene of mercury metabolism in HCMBRHg that included Burkholderiales, Enterobacteriales,
Rhizobiales,
Xanthomonadales,
Bacillales,
Clostridiales,
Selenomonadales and Actinomycetales. Burkholderiales had seven function genes of KatE, KatG, merA, merR, merT, merP and Amerl with mercury metabolism. Enterobacteriales had four function genes of KatE, KatG, merT and merP. As Fig. 5a
shown, the relative abundances of Hg resistant gene KatE, KatG, merA, merR, merT, merP and Amerl were 1.71, 0.67, 0.56, 0.38, 0.35, 0.29 and 0.07%, respectively. Hg0 could combine with merP and be transported by merT and then，oxidized into Hg2+ by KatG or KatE. Mercury could be oxidized to Hg(II) by catalase peroxidase (KatG, KatE). Mercuric oxidase enzyme mediated Hg(0) oxidation was a complex process that includes diffusion through outer membrane, active transport (by MerT and MerP) across the periplasm and the inner membrane. Mer operon was involved in the mercuric resistant mechanism, MerR of Mer operon en codes the proteins related to the regulation, transport and reduction of mercury ion, respectively.
Hg(II) was
microbial methylated to methyl mercury. Small amount of MeHg could be deoxygenized by Amerl and merA or radicals. Burkholderiales, Actinomycetales with alkylmercury lyase (Amerl) degrade methyl mercury to inorganic mercury through de-methylation. Burkholderiales, Actinomycetales with mercuric reductase (MerA) enzyme reduced Hg(II) to Hg(0). MerA was part of an elaborate resistance mechanism that is mediated by the mercury resistance (mer) operon. Methylation of mercury in HCMBRHg was extremely weak. Small amount of MeHg could be deoxygenized by Amerl and merA or radicals. Mercury resistant bacteria could reduce Hg2+ to Hg0 by merA [36]. MeHg could be deoxygenized by microbial reductase (merA), organic mercury lyase (merB) to release CH4 and Hg0 [37]. MerRTPA participated in control, transport, and reduction of mercury resistance [38]. Eukaryotic fungi. merA encoded enzyme mercuric reductase activity was evident in the crude protein of the isolate in mercury bioremediation [25]. 3.6 The effect of elemental mercury on microbial nitrogen metabolism pathway As shown in Fig. 5b, the relative abundances of ammonification related genes (Gln, Gdh, AadA, Nit, Gls ) was the highest(4.52%), followed by denitrification
(Naright, Nas, Nir, NosB, NosZ, Nap)(3.35%), nitrification (PmoA, HAO,NRT, Nod)(1.39%) and nitrogen fixation (Nif) (0.61%) in the HCMBR. The relative abundances of ammonification related genes (Gln, Gdh, AadA, Nit, Gls) was the highest (5.57%), followed by denitrification (Naright, Nas, Nir, NosZ,NosB, Nap)(4.52%), nitrification (PmoA, HAO,NRT, Nod)(1.23%) and nitrogen fixation (Nif) (0.58%) in the HCMBRHg. The relative abundances of Gln and NarIGH increased from 1.68% to 1.93%, from 1.36% to 1.83% after the addition of gaseous elemental mercury, respectively, this suggests that the ammoniation and denitrification are enhanced. For comparison, in the presence of hybrid catalytic membrane biofilm reactor, with/without gaseous elemental mercury in flue gas treatment process, the relative abundances of ammonification, denitrification related genes increased, while the relative abundances of nitrification, nitrogen fixation related genes small decreased, thus the addition of gaseous elemental mercury affected the microbial community structure, did not changed microbial nitrogen metabolism. GDH gene was differently affected by Cd, the repressive effect of Cd on nitrogen metabolism at the level of the enzyme and gene was significantly alleviated by cysteine [39]. As Figure 6a shows, there was a microbial nitrification and denitrification process, ammoniation and a biological nitrogen fixation process and organic nitrogen metabolism process in the HCMBR. Nitric oxide can be oxidized to nitrite by Nod(Enterobacteriaceae), and then was further oxidized into nitrate by NarIGH (Enterobacteriales, Bacteroidetes, Chlorobi). Nitrite was assimilated into ammonium by Nrf (Bacteroidetes). Ammonium was oxidized to hydroxylamine by PmoA (Burkholderiales), and then was further oxidized into nitrate by HAO (Burkholderiales). Nitrite was converted to nitric oxide by NirKS (Enterobacteriales,
Bacillales, Flavobacteriales, Bacteria). Nitric oxide could be reduced to nitrous oxide by NorB(Flavobacteriales), and then was further reduced into nitrogen gas by NorZ(Bacteroidetes, Chlorobi). Nitrogen fixation was that nitrogen was reduced to ammonium by NifDHK(Enterobacteriaceae, Bacteroidetes). As shown in Fig.6b, there were also nitrification and denitrification, ammoniation, and biological nitrogen fixation process in the HCMBRHg. Nitrification was the process in which NO was oxidized to nitrite by Nod (Enterobacteriaceae), and then was further oxidized into nitrate by NarIGH (Enterobacteriales, Burkholderiales, Pseudomonadales). Nitrite was assimilated into ammonium by Nrf(Bacteroidates). Ammonium was oxidized to hydroxylamine by PmoA (Burkholderiales), and then was further oxidized into nitrate by HAO (Burkholderiales). Nitrite was converted to nitric oxide by NirKS (Enterobacteriales, Bacillales). Nitric oxide could be reduced to nitrous oxide by NorB(Caulobacterales, Burkholderiales, Flavobacteriales), and then was further reduced into nitrogen gas by NorZ (Flavobacteriales, Rhizobiales, Sphingobacteriales). Nitrogen fixation was that nitrogen was reduced to ammonium by Nif (Clostridiales, Burkholderiales). These results confirmed that nitrification, denitrification, nitrogen fixation and ammonification existed in both samples HCMBRHg and HCMBR and the pathways of which are consistent with the results of microorganism identification, but different bacteria in nitrogen metabolism pathways. These results also demonstrated that mercury biooxidation, methylation, deoxygenized, and mercuric reductase existed in HCMBRHg.
4. Conclusions The paper revealed that the addition of gaseous elemental mercury affected the
microbial community structure, and not changed microbial nitrogen metabolism within the HCMBR. Gaseous elemental mercury decreased the NO removal efficiency of the hybrid catalytic membrane biofilm reactor by half. In 80 days of operation, NO removal efficiency was up to 68.8%, and Hg0 removal efficiency achieved 81.7%, respectively. Hg0 could combine with merP, and be transported by merT, and then be oxidized into Hg2+ by KatG or KatE. The nitrogen metabolism related processes such as nitrification/denitrification, ammoniation and biological nitrogen fixation processes, existed in HCMBR and HCMBRHg. There were microbial mercury metabolic processes in HCMBRHg.
Acknowledgements The authors gratefully acknowledge the financial support from the Nation Nature Scientific Research Foundation of China (21377171, 21677178).
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“Cyclobacteriaceae” with nitrate reducing activity, Systematic Appl. Microbiol. 35(2012) 320-325. [30] O.Y. Dobrynina, Disruption of bacterial biofilms using recombinant dispersin B. Microbiol. 84(2015) 498-501. [31] C.Xiao, J. Ning, H.Yan, X. Sun, J. Hu, Biodegradation of Aniline by a Newly Isolated Delftia sp. XYJ6, Chin. J. Chem. Eng. 17(2009) 500-505. [32] J.Mayer, N-Acetyltaurine dissimilated via taurine by Delftia acidovorans NAT, Arch. Microbiol. 186(2006), 61-67. [33] F. Carvalho, R.Souza, F.Barcellos, M. Hungria, A.Vasconcelos, Genomic and evolutionary comparisons of diazotrophic and pathogenic bacteria of the order Rhizobiales, BMC Microbiol. 10(2010) 37. [34] V.E.Vallejo, M.M.Gómez, A.M.Cubillos, F.Roldán, Effect of land use on the density of nitrifying and denitrifying bacteria in the Colombian Coffee Region, Agronomía Colombiana, 29(2011) 455-464. [35] O. Steenhoudt, J. Vanderleyden, Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects, FEMS Microbiol. Rev. 24(2000) 487-506. [36] Z Freedman, C Zhu, T Barkay, Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic aquificae, Appl. Environ. Microbiol. 78(2012) 6568-6575. [37] M C Marvin-DiPasquale, R S Oremland, Bacterial methylmercury degradation in florida everglades peat sediment, Environ. Sci. Technol.32(1998) 2556-256. [38] Hirak R. Dash, Surajit Das, Bioremediation of mercury and the importance of bacterial mer genes, Int. Biodeterior. Biodegrad. 75(2012) 207–213. [39] S.Erdala, H.Turk, Cysteine-induced upregulation of nitrogen metabolism-related genes and enzyme activities enhance tolerance of maize seedlings to cadmium stress, Environ. Exp. Bot. 132(2016) 92-99.
Figure captions and Table
Fig.1 Schematic diagram effect of mercury on nitric oxide removal in hybrid catalytic membrane biofilm reactor (HCMBR): (1)air pump;(2)NO container; (3)gas buffer bottle;
(4)gas
flowmeter;
(5)hollow
composite
fiber
membrane
biofilm
reactor(HCMBR) ; (6)mixed gas entrance; (7)reacted gas exit; (8)light source; (9)circular pool; (10)submersible pump; (11)Hg vapor generator; (G) sampling port Fig.2 The effect of elemental mercury on nitric oxide removal performance of the HCMBR during 130-d continuous running test. Fig. 3. Relative abundance of bacterial community at phylum and class levels in HCMBR and HCMBRHg. Fig. 4. Relative abundance of bacterial community at genus levels in HCMBR and HCMBRHg. Fig.5. Types and relative abundances of genes (a) nitrogen metabolism, (b) mercury metabolism. Fig. 6. Mechanism of denitration, demercuration in HCMBR and HCMBHg
7
5
4
8
11 1
4
6
G
4
1
2
3
1 G 10 9
Fig.1
90
300
80 250
60
200
50 150 40 30
100
20
NO removal efficiency
10
NO elimination capacity NO inlet load
0 0
Fig.2.
50
Hg removal efficiency
10
20
30
40
50
60 70 days
80
90
0 100
110
120
130
NO Load，g·m-3· h -1
Removal Efficiency，%
70
Relative Abundance，%
50 40 30 20
(a) phylum HCMBR HCMBRHg
10 0
Relative Abundance.%
35 30
(b) class
25 20
HCMBR
15
HCMBRHg
10 5 0
Fig.3.
Acidiphilium Mesorhizobium Tannerella Salmonella Methylobacterium Castellaniella Pseudomonas Clostridium Riemerella Brevundimonas Niabella Cupriavidus Achromobacter Pedobacter Comamonas Lachnoclostridium Stenotrophomonas Niastella Klebsiella Flavobacterium Enterococcus Chryseobacterium Brevibacillus Paenibacillus Sphingobacterium Bacteroides Proteiniphilum Weeksella Acinetobacter Elizabethkingia Azospirillum Delftia
HCMBRHg HCMBR
0
1
2
3
Relative Abundance，‰
Fig.4.
4
5
2.0
Relative abundance，%
(a) nitrogen metabolism
1.5
HCMBR HCMBRHg
1.0
0.5
0.0
Functional Gene 2.0
Relative abundance，%
(b) mercury metabolism 1.6 1.2 0.8 0.4 0.0 KatE
Fig.5
KatG
merA
merR
merT
merP
Amerl
(a) HCMBR
(b) HCMBRHg
Fig.6
Highlights Effect of gaseous elemental mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor Z.S. Wei*, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L.Xiao
School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou 510275, China *Corresponding author. tel.: +86-20- 84037096; fax: +86-20-39332690 E-mail address: [email protected]
Highlights
► NO and Hg0 removal efficiency achieved 68.8 and 81.7%, respectively. ►Mercury affected microbial structure, but not changed nitrogen metabolic
pathways. ► Hg0 could be transported by merT, and then be oxidized into Hg2+ by KatG or
KatE. ► Mechanism of simultaneous denitration and demercuration in HCMBRHg was
proposed.
S1385-8947(17)30096-7 http://dx.doi.org/10.1016/j.cej.2017.01.085 CEJ 16387
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
7 November 2016 20 January 2017 21 January 2017
Please cite this article as: Z.S. Wei, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L. Xiao, Effect of gaseous mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.01.085
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Effect of gaseous mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor
Z.S. Wei*, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L.Xiao School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou 510275, China
*
Corresponding author Tel: +86 20 84037096; fax: +86 20 39332690
E-mail address: [email protected] (Z.S.Wei)
Abstract The effect of gaseous elemental mercury on nitric oxide removal performance, microbial community and microbial nitrogen metabolism of a hybrid catalytic membrane biofilm reactor (HCMBR) were evaluated. A gaseous Hg0 concentration of 25±5 µg·m-3 reduced the NO removal efficiency of HCMBR from 86.4% to 41.9%. In 80 days of operation, NO removal efficiency was up to 68.8%, and Hg0 removal efficiency achieved 81.7%, respectively. The addition of gaseous elemental mercury affected the microbial community structure and changed microbial nitrogen metabolism, as shown by metagenomics sequencing method. Some phyla, such as Azospirillum, and Delftia, increased in abundance, whereas others, such as Weeksella , decreased. Hg0 could combine with merP and be transported by merT, and then be oxidized into Hg2+ by KatG or KatE. The nitrogen metabolism related processes such as catalysis, nitrification and denitrification, ammoniation and biological nitrogen fixation processes, exists in HCMBR and HCMBRHg. Keywords: Gaseous elemental mercury; Nitric oxide; Microbial community; Nitrogen metabolism; Mercury metabolism
1. Introduction Nitrogen oxides (NOx) are major air pollutants that trigger serious environmental problems, including acid rain, photochemical smog, and haze [1]. Mercury is considered one of the most toxic heavy metals with harmful effects on humans and other terrestrial and aquatic organisms due to its toxicity, volatility, persistence, and bioaccumulation [2]. The main sources of NOx and mercury pollution are power plants, coal–fired boiler, municipal waste combustors, medical waste incinerators, chlor–alkali plants and cement plants [3,4]. The microbial remediation of nitrogen oxides may offer a greener and cheaper technology that removes of NOx from contaminated point sources. Biofiltration of nitric oxide has been undertaken using perlite, compost, biofoam, soil compost, carbon foam and lava as packing materials [5-7].
A rotating drum biofilter was
applied to denitrifying removal of nitric oxide (NO) using glucose as electron donor [8] due to the lower mass transfer resistance and higher effective utility of packing materials [9]. A biotrickling filter using P. mendocina was fabricated for treating NO in a simulated exhaust gas [10]. Denitrification removal of NO by a biotrickling filter inoculated P. putida SB1 was an effective protocol for eliminating NOx from flue gas [11]. The suspended biofilter inoculating the thermophilic denitrifying bacterium Chelatococcus daeguensis TAD1 could remove NO efficiently with great elimination capacity [12]. A bench scale biotrickling filter using Chelatococcus daeguensis was capable of converting nitric oxide NOx under thermophilic condition [13, 14]. NO removal in an anoxic biofilter packed with four compost types at temperatures of 55–60°C was investigated [15]. An integrated process of Fe(II)EDTA absorption coupled with two-stage microbial regeneration using immobilized microorganisms was used to removal of NOx[16]. A mixed absorbent had been proposed to enhance
the chemical absorption–biological reduction process for NOx removal from flue gas [17]. A newly isolated thermophilic Anoxybacillus sp. HA, identified by 16S rRNA sequence analysis, could simultaneously reduce Fe(II)EDTA-NO and Fe(III)EDTA [18]. The nitrifying hollow fiber membrane bioreactor (MBR) is a promising technology for NOx control because it provides high surface area for mass transfer of nitrogen oxides to nitrifying bacteria at temperatures between 20 and 55°C [19]. A hallow-fiber membrane bioreactor was stable and highly efficient for denitrifying removal of NO from simulated flue gas, NO removal was inversely proportional to the inlet oxygen concentration, low-concentration sulfur dioxide had no great influence on denitrification removal of NO[20]. The thermophilic membrane biofilm reactor using the synergy of nitrifying and aerobic denitrifying microorganisms could be used for NO removal from flue gas at 60 °C [21]. A hybrid catalytic membrane biofilm reactor offered potential for flue gas denitrification, membrane photocatalysis, and nitrification/denitrification could contribute to the removal of NO [22]. Elemental mercury emission from coal-fired flue gas is often contained in the flue gas even after the electrostatic precipitator or baghouse and wet flue gas desulfurization (WFGD) treatment, which might have toxic effects on the nitrification and denitrification microorganism in the biotreatment of nitrogen oxides system. Fewer studies have been conducted to the effect of gaseous elemental mercury on nitric oxide removal in hybrid catalytic membrane biofilm reactor. Three of the strains (Enterobacter helveticus, Citrobacter amalonaticus, and Cronobacter muytjensii) can detoxify the environment from mercury due to wastes of florescent lamps[23]. Biogenic palladium nanoparticles (Bio-Pd NPs) do not exert toxicity towards the bioluminescent marine bacterium V. fischeri and may have limited inhibitory effects towards some respiratory metabolisms [24]. The deduced amino acid sequence of
merA gene indicated a sequence homology with different organisms from the alpha proteobacteria group [25]. The objective of this work is to study the effect of gaseous elemental mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor (HCMBR). The study analyzes bacterial community composition assessed by metagenomics sequencing method, and evaluates nitrogen metabolism, mercury metabolism. The mechanism for simultaneous denitration and demercuration in HCMBRHg is elicited, which is believed to promote the application of the MBR.
2. Methods 2.1. Experimental procedure The experimental flow loop effect of mercury on nitric oxide removal in hybrid catalytic membrane biofilm reactor used in the study was shown schematically in Fig.1. The HCMBR included an N-TiO2/PP hybrid catalytic membrane biofilm reactor, a waste gas mixture (NO, Hg and clean air) generation system, and a nutrient supply system. A N-TiO2-coated polypropylene (PP) hollow fiber membrane consisted of 2400 hydrophobic, microporous membrane polysulfone fiber bundles with the inner diameter of 0.38 mm, outer diameter of 0.52 mm and effective length of 300 mm. Nitrification and denitrification bacterials adhered to the surface of the surface of N-TiO2/PP to form the biofilm. The NO supplied from the gas cylinders, was first diluted with the compressed air, passed through an air mixture bottle, then flowed upwards the bottom of the hybrid catalytic membrane bioreactor. Batch tests on Hg0, Hg0 vapor equipment unit consists of an elemental mercury permeation tube, a bottle of gas mixture, a water bath. The elemental mercury permeation tube was
placed in a water bath with a temperature of 328 K. Hg0 vapor supplied from the mercury permeation unit, was first diluted with the compressed air, passed through an air mixture bottle; simulated NO and Hg0 flue gas were flowed upwards through the HCMBR. 2.2. Metagenomics sequencing data analysis Bacterial community compositions and the gene function of bacterial in the hybrid catalytic membrane bioreactor were assessed by 16S rDNA and metagenomics method, and identify the colonies of the predominant microorganisms by the procedures of total DNA extraction, polymerase chain reaction (PCR) amplification of 16S rDNA, cloning and sequencing, calculation of similarity and diversity indexes, analysis of the successional route of the community. 2.2.1 Extraction of genome DNA Total genome DNA from samples was extracted using CTAB method. DNA concentration and purity was monitored on 1% agarose gels. To obtain a specific concentration, DNA was diluted to 1ng/µl using sterile water. The samples were collected from the outface of the membrane in HCMBR and were stored at -80ºC before DNA extraction. The samples were added into a Eppendorf
tube ， which is mixed with 1000ul CTAB and certain amount of
Lysozyme. The tube was heated in 65 ºC water bath. Afterwards，the samples were centrifuged at 3000rpm for 5min before supernatant was removed. And then， combined with Phenol，Chloroform and Isoamyl alcohol at a ratio of 25：24：1(v/v/v) ， the samples were centrifuged at 12000 rpm for 10min before supernatant was collected. Take 1.5 mL of supernatant into a new centrifuge tube containing Isoamyl alcohol, shake the tube upwards and downwards for several times, then place the tube for precipitation at -20 ºC. After the new tube was centrifuged at 12000 rpm for
10min, keep the sediment instead of the supernatant. The sediment was rinsed by 1ml 75% alcohol twice and then air-dried in clean benches. Used ddH2O to dissolve the DNA sample and if necessary，the sample can be put into 55-60℃ environment for10min for solubilization. At last，the RNA in the sample was dispelled by 1ul RNase and then placed in static state in 37℃ for 15min. 2.2. 2 Amplicon Generations 16S rRNA genes were amplified by the specific primer (515F-806R) with the barcode. All PCR reactions were carried out in 30µL reactions with 15µL of Phusion® High-Fidelity PCR MasterMix (New England Biolabs); 0.2μM forward and reverse primers, and about 10 ng templates DNA. Thermal cycling consisted of 1-minute initial denaturation at 98 ºC, 10-second 30 cycles of denaturation at 98 ºC, 30-second annealing at 50 ºC, and 30-second elongation at 72 ºC. Finally the sample is preserved at 72 ºC for 5 min. 2.2. 3 PCR Products quantification and qualification Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis in 2% agarose gel for detection. Samples with bright main strip between 400-450bp were chosen for further experiments. 2.2. 4 PCR Products Mixing and Purification PCR products were mixed in accordance with equidensity ratios. Then, mixture PCR products were purified by GeneJET Gel Extraction Kit (Thermo Scientific). 2.2. 5 Library preparation and sequencing Sequencing libraries were generated by NEB Next® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer
(Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina HiSeq platform and 250 bp/300bp paired-end reads were generated. 2.2.6 Data analysis Paired-end reads from the original DNA fragments are merged by using FLASH -a very fast and accurate analysis tool which is designed to merge paired-end reads when overlaps occur between reads1 and reads2. Paired-end reads were assigned to each sample according to the unique barcodes. Sequences were analyzed by using QIIME software package (Quantitative Insights Into Microbial Ecology), and in-house Perl scripts were used to analyze alpha- (within samples) and beta- (among samples) diversity. Sequences with ≥97% similarity were assigned to the same OTUs. A representative sequences for each OTU was picked and the RDP classifier to annotate taxonomic information for each representative sequence was used. 2.3. Analytical methods Nitric oxide concentration was measured by a Testo350 flue gas analysis device (Testo AG, Germany). The inlet and outlet of the bubbler were sampled for gaseous mercury inaccordance with Ontario Hydro Method of U.S. EPA Method 23 and U.S.DOE. The concentrations of mercury were quantified by atomic fluorescence spectrophotometry (AFS). Gas flow rates and Liquid flow rates were measured using Model LZB-1 flow meters. Illumination intensity were measured using SIGMA AR823 Separation of illuminance analysis device (SIGMA, Hong Kong).The pH values were measured by a Model pHB-3 pH Tester ( Sanxin Instrument Company, Shanghai, China).
3. Results and discussion
3.1. The effect of elemental mercury on nitric oxide removal performance Fig.2 showed the effect of elemental mercury on nitric oxide removal performance of the HCMBR during 130-d continuous running test under the conditions of pH of 6.8~7.2, sprinkling amount of 60 ml·min-1, NO inlet load of 135~145 g·m-3·h-1, gas residence time (GRT) of 9 s at normal temperature and visible Light. As shown in Fig.1, two distinct phases were observed: phase I under no gaseous elemental mercury, lasted from days 1 to 50; in phase II the addition of gaseous Hg0 concentration of 25 µg·m-3, from days 51 to 130, effect of elemental mercury on total performance of the HCMBR was investigated. In phase I, NO removal efficiency (RE) changed from 47.8 to 61.6% before 8 d, then increased from 64% at 9thd to 73.6% at 14th d. RE had a slight fluctuation around 74% from days 15 to 40. NO removal efficiency drastically increased from 72.1% at 40thd to 83.8% at 41th d. RE changed small from 86.1% at 42th d to 86.3% at 50th d. The increase in the removal efficiency was attributed to the acclimatization phase, nitrification and denitrification bacteria were attached to the surface of N-TiO2/PP to form the biofim. In phase II, NO removal efficiency sharply fell from 86.3 to 41.9 % after 24 h of the supply of gaseous Hg0 concentration of 25µg·m-3. The possible reason for the fall in NO removal efficiency could be that gaseous elemental mercury was toxic to microorganism, and inhibited NO removal. Subsequently, NO removal efficiency can ascend to about 60% surprisingly after a few days. After the gradual ascent, NO removal efficiency maintained at about 63% for almost 70 days. Hg0 removal efficiency was a striking upward trend from 25% at 51thd to 80% at 60th d, after that, the Hg removal efficiency experienced an obvious fluctuation and decreased from 81.7% at 61th d to 63.5% at 130th d. This illustrated that HCMBR showed a fast
response, could efficiently handle this applied influence, and had good NO and mercury removal performance. NO and mercury are bioremediation by the nitrification and denitrification microorganism in the biofilm attached to the surface of N-TiO2/PP. 3.2. The bacterial community in the HCMBR before and after mercury addition The addition of gaseous elemental mercury affect the microbial community structure, as shown by denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA genes. 94.75% of the sequences were assigned to bacteria and only few sequences belonged to archaea in total. At phylum level, the relative abundances of dominant bacteria Proteobacteria, Bacteroidetes and Firmicutes of HCMBR and HCMBRHg were in the change of 35.7 to 45.78%, 24.61 to 15.22% and 0.75 to 1.06%, respectively (Fig.3a). Some phyla, such as Azospirillum, Delftia, Firmicutes and Proteobacteria, increased in abundance, whereas others, such as Weeksella, and Bacteroidetes, decreased. For comparison, in the presence of HCMBR, with/without gaseous elemental mercury in flue gas treatment process, the relevant abundance of Azospirillum and Delftia in HCMBRHg was 13 times and 6 times of that in HCMBR, respectively. However, the relevant abundance of Weeksella in HCMBR was 794 times of that in HCMBRHg. Proteobacteria has the functions of nitrification, denitrification and nitrogen fixation using ammonia, methane and some kind of volatile fatty acids as electron donor [27-29]. Bacteroidetes can make use of nitrate or nitrite as the ultimate electron acceptor at hypoxia or anaerobic condition [30]. At the class level, comparison of HCMBR and HCMBRHg, the microbial community structure changed from 8 to 13 classes (Fig.3b). Seven classes that included
Enterobacteriaceae,
Burkholderiales,
Rhizobiales,
Flavobacteriales,
Bacteroidetes, Bacillales and Desulfobacterales, were unchanged. Six classes that include
Pseudomonadales,
Xanthomonadales,
Clostridiales,
Actinomycetales,
Sphingobacteriales and Caulobacterales, were appeared. One classe, Chlorobi, disappeared due to its inability to survive the mercury environment. At the genus level, the dominant bacteria genus in the HCMBRHg, and HCMBR, such as Burkholderia, Delftia, Azospirillum, Elizabethkingia, Acinetobacter, Weeksella, Proteiniphilum, Bacteroides , Sphingo bacterium were identified. All two communities were dominated by the Burkholderia, with the relative abundances of 11.99%, and 9.61% in HCMBRHg, and HCMBR, respectively (Fig.4). Burkholderia is was a genus of ß-proteobacteria and has the function of nitrification and denitrification. Burkholderia cenocepacia and Achromo bacterxylosoxidans were more resistant to the recombinant enzyme produced by a synthetic gene encoding dispersin B of Aggregatibacter actinomycetemcomitans [31]. Delftia could make use of aniline, organic nitrogen as the carbon source and the nitrogen source [32,33]. Sphingobacterium, Bacteroides and Acinetobacter possen the function of nitrification and denitrification[34-35]. Azospirillum could utilize the nitrogen in air or dissolved in the water, or even the nitrogen generated by denitrifying bacteria to conduct nitrogen fixation [36]. 3.5 Microbial mercury metabolism As shown in Fig. 5a, eight classes mercury resistant bacteria group with function gene of mercury metabolism in HCMBRHg that included Burkholderiales, Enterobacteriales,
Rhizobiales,
Xanthomonadales,
Bacillales,
Clostridiales,
Selenomonadales and Actinomycetales. Burkholderiales had seven function genes of KatE, KatG, merA, merR, merT, merP and Amerl with mercury metabolism. Enterobacteriales had four function genes of KatE, KatG, merT and merP. As Fig. 5a
shown, the relative abundances of Hg resistant gene KatE, KatG, merA, merR, merT, merP and Amerl were 1.71, 0.67, 0.56, 0.38, 0.35, 0.29 and 0.07%, respectively. Hg0 could combine with merP and be transported by merT and then，oxidized into Hg2+ by KatG or KatE. Mercury could be oxidized to Hg(II) by catalase peroxidase (KatG, KatE). Mercuric oxidase enzyme mediated Hg(0) oxidation was a complex process that includes diffusion through outer membrane, active transport (by MerT and MerP) across the periplasm and the inner membrane. Mer operon was involved in the mercuric resistant mechanism, MerR of Mer operon en codes the proteins related to the regulation, transport and reduction of mercury ion, respectively.
Hg(II) was
microbial methylated to methyl mercury. Small amount of MeHg could be deoxygenized by Amerl and merA or radicals. Burkholderiales, Actinomycetales with alkylmercury lyase (Amerl) degrade methyl mercury to inorganic mercury through de-methylation. Burkholderiales, Actinomycetales with mercuric reductase (MerA) enzyme reduced Hg(II) to Hg(0). MerA was part of an elaborate resistance mechanism that is mediated by the mercury resistance (mer) operon. Methylation of mercury in HCMBRHg was extremely weak. Small amount of MeHg could be deoxygenized by Amerl and merA or radicals. Mercury resistant bacteria could reduce Hg2+ to Hg0 by merA [36]. MeHg could be deoxygenized by microbial reductase (merA), organic mercury lyase (merB) to release CH4 and Hg0 [37]. MerRTPA participated in control, transport, and reduction of mercury resistance [38]. Eukaryotic fungi. merA encoded enzyme mercuric reductase activity was evident in the crude protein of the isolate in mercury bioremediation [25]. 3.6 The effect of elemental mercury on microbial nitrogen metabolism pathway As shown in Fig. 5b, the relative abundances of ammonification related genes (Gln, Gdh, AadA, Nit, Gls ) was the highest(4.52%), followed by denitrification
(Naright, Nas, Nir, NosB, NosZ, Nap)(3.35%), nitrification (PmoA, HAO,NRT, Nod)(1.39%) and nitrogen fixation (Nif) (0.61%) in the HCMBR. The relative abundances of ammonification related genes (Gln, Gdh, AadA, Nit, Gls) was the highest (5.57%), followed by denitrification (Naright, Nas, Nir, NosZ,NosB, Nap)(4.52%), nitrification (PmoA, HAO,NRT, Nod)(1.23%) and nitrogen fixation (Nif) (0.58%) in the HCMBRHg. The relative abundances of Gln and NarIGH increased from 1.68% to 1.93%, from 1.36% to 1.83% after the addition of gaseous elemental mercury, respectively, this suggests that the ammoniation and denitrification are enhanced. For comparison, in the presence of hybrid catalytic membrane biofilm reactor, with/without gaseous elemental mercury in flue gas treatment process, the relative abundances of ammonification, denitrification related genes increased, while the relative abundances of nitrification, nitrogen fixation related genes small decreased, thus the addition of gaseous elemental mercury affected the microbial community structure, did not changed microbial nitrogen metabolism. GDH gene was differently affected by Cd, the repressive effect of Cd on nitrogen metabolism at the level of the enzyme and gene was significantly alleviated by cysteine [39]. As Figure 6a shows, there was a microbial nitrification and denitrification process, ammoniation and a biological nitrogen fixation process and organic nitrogen metabolism process in the HCMBR. Nitric oxide can be oxidized to nitrite by Nod(Enterobacteriaceae), and then was further oxidized into nitrate by NarIGH (Enterobacteriales, Bacteroidetes, Chlorobi). Nitrite was assimilated into ammonium by Nrf (Bacteroidetes). Ammonium was oxidized to hydroxylamine by PmoA (Burkholderiales), and then was further oxidized into nitrate by HAO (Burkholderiales). Nitrite was converted to nitric oxide by NirKS (Enterobacteriales,
Bacillales, Flavobacteriales, Bacteria). Nitric oxide could be reduced to nitrous oxide by NorB(Flavobacteriales), and then was further reduced into nitrogen gas by NorZ(Bacteroidetes, Chlorobi). Nitrogen fixation was that nitrogen was reduced to ammonium by NifDHK(Enterobacteriaceae, Bacteroidetes). As shown in Fig.6b, there were also nitrification and denitrification, ammoniation, and biological nitrogen fixation process in the HCMBRHg. Nitrification was the process in which NO was oxidized to nitrite by Nod (Enterobacteriaceae), and then was further oxidized into nitrate by NarIGH (Enterobacteriales, Burkholderiales, Pseudomonadales). Nitrite was assimilated into ammonium by Nrf(Bacteroidates). Ammonium was oxidized to hydroxylamine by PmoA (Burkholderiales), and then was further oxidized into nitrate by HAO (Burkholderiales). Nitrite was converted to nitric oxide by NirKS (Enterobacteriales, Bacillales). Nitric oxide could be reduced to nitrous oxide by NorB(Caulobacterales, Burkholderiales, Flavobacteriales), and then was further reduced into nitrogen gas by NorZ (Flavobacteriales, Rhizobiales, Sphingobacteriales). Nitrogen fixation was that nitrogen was reduced to ammonium by Nif (Clostridiales, Burkholderiales). These results confirmed that nitrification, denitrification, nitrogen fixation and ammonification existed in both samples HCMBRHg and HCMBR and the pathways of which are consistent with the results of microorganism identification, but different bacteria in nitrogen metabolism pathways. These results also demonstrated that mercury biooxidation, methylation, deoxygenized, and mercuric reductase existed in HCMBRHg.
4. Conclusions The paper revealed that the addition of gaseous elemental mercury affected the
microbial community structure, and not changed microbial nitrogen metabolism within the HCMBR. Gaseous elemental mercury decreased the NO removal efficiency of the hybrid catalytic membrane biofilm reactor by half. In 80 days of operation, NO removal efficiency was up to 68.8%, and Hg0 removal efficiency achieved 81.7%, respectively. Hg0 could combine with merP, and be transported by merT, and then be oxidized into Hg2+ by KatG or KatE. The nitrogen metabolism related processes such as nitrification/denitrification, ammoniation and biological nitrogen fixation processes, existed in HCMBR and HCMBRHg. There were microbial mercury metabolic processes in HCMBRHg.
Acknowledgements The authors gratefully acknowledge the financial support from the Nation Nature Scientific Research Foundation of China (21377171, 21677178).
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Figure captions and Table
Fig.1 Schematic diagram effect of mercury on nitric oxide removal in hybrid catalytic membrane biofilm reactor (HCMBR): (1)air pump;(2)NO container; (3)gas buffer bottle;
(4)gas
flowmeter;
(5)hollow
composite
fiber
membrane
biofilm
reactor(HCMBR) ; (6)mixed gas entrance; (7)reacted gas exit; (8)light source; (9)circular pool; (10)submersible pump; (11)Hg vapor generator; (G) sampling port Fig.2 The effect of elemental mercury on nitric oxide removal performance of the HCMBR during 130-d continuous running test. Fig. 3. Relative abundance of bacterial community at phylum and class levels in HCMBR and HCMBRHg. Fig. 4. Relative abundance of bacterial community at genus levels in HCMBR and HCMBRHg. Fig.5. Types and relative abundances of genes (a) nitrogen metabolism, (b) mercury metabolism. Fig. 6. Mechanism of denitration, demercuration in HCMBR and HCMBHg
7
5
4
8
11 1
4
6
G
4
1
2
3
1 G 10 9
Fig.1
90
300
80 250
60
200
50 150 40 30
100
20
NO removal efficiency
10
NO elimination capacity NO inlet load
0 0
Fig.2.
50
Hg removal efficiency
10
20
30
40
50
60 70 days
80
90
0 100
110
120
130
NO Load，g·m-3· h -1
Removal Efficiency，%
70
Relative Abundance，%
50 40 30 20
(a) phylum HCMBR HCMBRHg
10 0
Relative Abundance.%
35 30
(b) class
25 20
HCMBR
15
HCMBRHg
10 5 0
Fig.3.
Acidiphilium Mesorhizobium Tannerella Salmonella Methylobacterium Castellaniella Pseudomonas Clostridium Riemerella Brevundimonas Niabella Cupriavidus Achromobacter Pedobacter Comamonas Lachnoclostridium Stenotrophomonas Niastella Klebsiella Flavobacterium Enterococcus Chryseobacterium Brevibacillus Paenibacillus Sphingobacterium Bacteroides Proteiniphilum Weeksella Acinetobacter Elizabethkingia Azospirillum Delftia
HCMBRHg HCMBR
0
1
2
3
Relative Abundance，‰
Fig.4.
4
5
2.0
Relative abundance，%
(a) nitrogen metabolism
1.5
HCMBR HCMBRHg
1.0
0.5
0.0
Functional Gene 2.0
Relative abundance，%
(b) mercury metabolism 1.6 1.2 0.8 0.4 0.0 KatE
Fig.5
KatG
merA
merR
merT
merP
Amerl
(a) HCMBR
(b) HCMBRHg
Fig.6
Highlights Effect of gaseous elemental mercury on nitric oxide removal performance and microbial community of a hybrid catalytic membrane biofilm reactor Z.S. Wei*, J.B. Wang, Z.S. Huang, Y.M. He, J.L. Pei, X.L.Xiao
School of Environmental Science and Engineering, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou 510275, China *Corresponding author. tel.: +86-20- 84037096; fax: +86-20-39332690 E-mail address: [email protected]
Highlights
► NO and Hg0 removal efficiency achieved 68.8 and 81.7%, respectively. ►Mercury affected microbial structure, but not changed nitrogen metabolic
pathways. ► Hg0 could be transported by merT, and then be oxidized into Hg2+ by KatG or
KatE. ► Mechanism of simultaneous denitration and demercuration in HCMBRHg was
proposed.