- Email: [email protected]
Microbial communities variation analysis of denitrifying bacteria immobilized particles
Process Biochemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Microbial communities variation analysis of denitrifying bacteria immobilized particles ⁎
Liangang Hou, Jun Li , Yang Liu College of Architecture and Civil Engineering, Beijing University of Technology, Beijing 100124, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microbial immobilized technology Denitrification Microbial community High-throughput sequencing Wastewater treatment
The aim of this study was to investigate the denitrification performance and analyze the variation of microbial communities in immobilized particles which used in real municipal wastewater treatment. The bioreactor has achieved good denitrification effect on real municipal wastewater treatment, the NO3−-N and TN removal efficiencies reached 77.4% and 68.3%, respectively. High-throughput sequencing shows that the community structure and relative abundance of bacteria changed greatly, especially at the phyla and genes levels in immobilized particles. Through the investigation of this paper, we think the influence of microflora variation should be considered when using microbial immobilized technology for wastewater treatment, and we suggest that immobilized particles cannot be used indefinitely and it needs to be replaced periodically.
1. Introduction
2. Materials and methods
Activated sludge are a form of collective life with emergent properties that confer many advantages on their inhabitants [1], and they represent a much higher level of organization than single cells do [2], so it has been widely used in wastewater treatment plants (WWTPs) [3,4]. However, microbial communities will change under different living conditions [5–8] affecting the wastewater treatment effect. Therefore, it is necessary to maintain the stability of the microbial communities during wastewater treatment for WWTPs [9,10]. Microbial immobilization technology (MIT) is a technique that uses chemical or physical methods to limit or localize free cells or enzymes in a specific spatial region and maintain biological activity [11–14]. Due to the high activity and stability [15], MIT has been widely concerned for wastewater treatment [13,16,17]. Although the immobilized carrier has a protective effect on microorganism [18–20], whether the bacteria in the immobilized particles have changed during the operation has not been reported so far. The aim of this study was to investigate the denitrification performance and analyze the variation of microbial communities in immobilized particles which used in real municipal wastewater treatment. This work would add some new insights into microbial immobilization technology and further solve some problems of its application in wastewater treatment.
2.1. Experimental setup and operation
⁎
A certain amount of cultured sludge which contains denitrifying bacteria was mixed with an equal amount of waterborne polyurethane and a suitable amount of water, added 1% (w/v) potassium persulfate solution and 0.5% (w/v) N,N,N,N-tetramethylethylenediamine solution to induced polymerization [21].The mixture was solidified for 5∼10 min at 27 ± 2℃ and formed a solid jelly-bean-shaped block of hydrosol, then cut it into 3 × 3 x 3 mm cubes, cultured in a solution containing sodium nitrate and glucose for later use. The feeding medium used in this experiment was real municipal wastewater, which was taken from effluent of aeration tank, a WWTPs in Liaoning province, China. The major characteristics of the municipal wastewater included: nitrate (NO3−-N) 25 ± 2 mg/L, ammoniacal nitrogen (NH4+-N) < 0.1 mg/L, nitrite (NO2−-N) 0.2 ± 0.1 mg/L, total nitrogen (TN) 26 ± 3 mg/L, chemical oxygen demand (COD) 50 ± 5 mg/L. The pH value of the municipal wastewater fluctuated in the range of 7.6∼7.7 and dissolved oxygen (DO) < 0.5 mg/L. Glucose was added into the bioreactor to provide sufficient carbon source for the denitrification process. The schematic of the laboratory-scale denitrification bioreactor is shown in Fig. 1. The bioreactor was made of a plexiglas cylinder and with an operating volume of 80.0 L. Added 12.0 L(15%, v/V) immobilized particles (Sampled and marked Sample A) into the reactor,
Corresponding author. E-mail address: [email protected] (J. Li).
https://doi.org/10.1016/j.procbio.2019.09.001 Received 12 June 2019; Received in revised form 28 August 2019; Accepted 3 September 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Liangang Hou, Jun Li and Yang Liu, Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.001
Process Biochemistry xxx (xxxx) xxx–xxx
L. Hou, et al.
Fig. 1. Schematic diagram of the denitrification bioreactor.
set the hydraulic retention time (HRT) was 12 h, the temperature maintained around 27∼30℃. Sampling after running about 58 days and marked Sample C. Samples A and C were 30–50 immobilized particles randomly collected in the bioreactor to ensure representative samples. 2.2. Analytical methods Samples for water quality measurements were collected regularly and analyzed every day. The concentrations of NO3−-N,TN, NO2−N,NH4+-N and COD in influent and effluent were analyzed according to Standard Methods for the Examination of Water and Wastewater [22]. Before analysis of the above parameters in liquid, samples were membrane filtered (0.45 μm), and all tests were repeated twice in a temperature-controlled room at 27 ± 1℃.The temperature, pH and DO were measured near the midway of the bioreactor by using a WTW analyzer (Multi 3620IDS,Germany). 2.3. Microbiological analysis High-throughput sequencing of 16S rRNA gene fragments was used to analyze the microbial communities [23] in immobilized particles samples. Bacterial genomic DNA was extracted using an E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Inc.) after the samples (A and C) pretreated, following manufacturer's instructions. The PCR amplification used the universal bacteria primers (for 16S rRNA all bacteria) which incorporated Miseq platformic the V3-V4 hypervariable regions. The final sequences of the primers were 341 F (C C T A C G G G N G G C W G C A G), 805R (G A C T A C H V G G G T A T C T A A T C C).The PCR products were sequenced on the Miseq 2 × 300bp pyrosequencing platform by Sangon Biotech, shanghai, China. Finally, to identify and compare core taxa, OTUs (Operational taxonomic unit) were summarized and normalized at the genus level [24,25]. For analyzing the dataset further, the OTUs table along with associated metadata and representative sequences, were imported, stored, and subset, OTUs and Alpha diversity indices (Ace, Chao 1, Shannon, Simpson and Coverage) were determined by Mothur software (ver.1.30.1), and the taxonomy-based analysis used RDP Classifier(ver.2.12) of the Ribosomal Database Project (RDP) and the National Center for Biotechnology Information
Fig. 2. Changes of concentration and removal efficiency of (Fig. 2a)NO3−-N and(Fig. 2b) TN in the bioreactor.
2
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Table 1 Community richness and diversity indices of Samples A and C. Sample
OTUs Number
Coverage (%)
Chao1
Ace
Shannon
Simpson
A C
3794 4023
0.94 0.94
47308.32 42369.57
150619.59 114729.54
3.36 4.50
0.20 0.05
of Dong et al. [28], which reported that waterborne polyurethane immobilized activated sludge has a high-efficiency denitrification effect for the treatment of acrylonitrile wastewater. 3.2. Bacterial diversity analysis The number of OTUs and the richness as well as diversity of two samples were compared (Table 1). More OTUs were detected in the Samples C compared with A, this implies that the microbial richness increased significantly due to the presence of nitrogen compounds in municipal wastewater, which was also reflected by the Chao1 and Ace indexes. The higher Shannon index and lower Simpson index suggested higher diversity in Sample C, indicating that the microbes were highly selected and community diversity has enlarged due to the special life conditions of immobilized particles. 3.3. Microbial community identification The diversity of the microbial communities in immobilized particles is an important property for determining wastewater treatment quality. The microbial community of immobilized particles was characterized based on 16S rRNA analysis. The result shows a diverse community of immobilized particles during different operation stages, and there are different functional groups involved in the nitrogen cycle, respectively. It can be seen from Fig. 4 that the total of identified phyla for Samples A were 18 and Samples C were 19. Different bacteria were identified inside the immobilized particles, Sample A was mainly composed of Proteobacteria (83.2%), Bacteroidetes (7.7%), Firmicutes (4.4%), Actinobacteria (1.2%) (4 species with relative abundance > 1%). Sample C was mainly represented by Proteobacteria (77.8%), Bacteroidetes (8.6%), Candidatus Saccharibacteria (5.4%), Firmicutes (2.8%), Actinobacteria (1.4%), Ignavibacteriae (1.1%) (6 species with relative abundance > 1%). It was known that the major phyla and relative abundances among the two samples were different, Sample C (19 phyla) compared to Sample A (18 phyla), the relative abundance decreased with 5 phyla, 11 phyla increased and one remained unchanged (Hydrogenedentes (0.01%)), simultaneity, one disappears (Nitrospirae) and two new ones appeared (Euryarchaeota (0.01%) and candidate division WPS−1(0.01%)). The relative abundance of phylum Proteobacteria and Firmicutes in Sample C decreased by 5.3% and 1.5%, respectively. These two bacterial phyla were widely distributed in various wastewater treatment plants [29]. Proteobacteria is the largest phylum in bacteria [30], and many of the denitrification microorganisms belong to this phylum [31].Therefore, phylum Proteobacteria plays an important role in the denitrification process of immobilized particles, meaning that the bioreactor exhibited excellent nitrate removal performance. The relative abundance of phyla Candidatus Saccharibacteria and Bacteroidetes in sample C increased 4.7% and 0.83%, respectively. The phylum Candidatus Saccharibacteria [32] was recently assigned the name Saccharibacteria, owing to their sugar metabolisms, and has been frequently detected in natural environments and activated sludge [33].Within the phylum, Filamentous bacteria are the main contributors to the bulking issues in wastewater treatment as their excessive growth. In this study, Filamentous was found in immobilized particles, but they did not cause bulking issues, it indicated that the microbial immobilization technology was beneficial for maintaining the stability of
Fig. 3. Changes of concentration and removal efficiency of (Fig. 3a) COD, (Fig. 3b) NO2−-N and NH4+-N in the bioreactor.
(NCBI) BLAST.
3. Results and discussion 3.1. NO3−-N and COD removal performances Fig. 2 shows the concentration changes of NO3−-N and TN from municipal wastewater through the treatment of immobilized denitrifying bacteria particles. As shown in Fig. 2, The NO3−-N removal efficiency was 5.5–77.4% while TN removal efficiency was 5.2–68.3% during the experimental operation. Those results meaning that most of the nitrate converted to nitrogen gas by denitrifying bacteria in the immobilized particles. The COD removal rate was 18.9–70.2% in the operating process. It can be seen that when the concentration of TN was 25 mg/L in water, maintaining COD/TN = 4.0 can meet the requirement of denitrification, this result was in agreement with that of previous studies [26,27]. Fig. 3 shows the concentration of NO2−-N in effluent, and the accumulation rate of NO2−-N was only 0.3–3.9 mg/L, the low accumulation rate of nitrite indicated that the denitrification process of immobilized particles was relatively complete. In this study, the content of NH4+-N was relatively low, and its concentration vary was not obvious during the operation, indicating that the bacteria in the immobilized particles were mainly participated in denitrification process. The changes of nitrogen and COD in the bioreactor indicated that the immobilized particles had a good treatment efficiency on the real municipal wastewater. This result was in accordance with the research 3
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Fig. 4. 16sRNA analysis of the immobilized particles, phyla identified based on sequencing are shown.
C), Simplicispira (4.1% of Sample A changed to 22.3% of Sample C), Saccharibacteria genera_incertae_sedis (0.8% of Sample A changed to 5.4% of Sample C), Thauera (0 in Sample A and 4.5% in Sample C), Brevundimonas (0 in Sample A and 3.3% in Sample C). At the genus level, Comamonas has reduced by 36.8%, while Simplicispira, Saccharibacteria_genera_incertae_sedis, Thauera and Brevundimonas have increased by 18.2%,4.7%,4.5% and 3.3%, respectively. The genus Comamonas has been reported as one of the major members of microbial communities in various natural and engineered environment, and some of this genus were facultative anaerobes capable of using nitrate as an alternative electron acceptor [35]. Genus
denitrification. Bacteroidetes played an important role in starch and papermaking wastewater treatment plants containing various aromatic pollutants [34], so we guessed that Bacteroidetes also played an important role in the degradation process of nitrate in this paper. As can be seen from Fig. 5, the vary of genus in immobilized particles was obvious. Compared with Sample A, the relative abundance of some genus varies by more than 0.5% in Sample C, and there are six kinds increased and six kinds decreased. In addition, eight of new genus have emerged and seven have disappeared in Sample C compared with A. The most obvious changes in relative abundance of genus were as follows: Comamonas (47.8% of Sample A changed to 11.0% of Sample 4
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Fig. 5. 16sRNA analysis of the immobilized particles, genus identified based on sequencing are shown.
Results of high-throughput sequencing showed a large variation in the microbial community composition at phylum and genus levels. Our investigation demonstrated that microbial community will change in immobilized particles during operation process. Therefore, the influence of microflora variation should be considered when using microbial immobilized technology for wastewater treatment. We suggest that immobilized particles cannot be used indefinitely and it need to be replaced periodically, because considering the changes of microbial communities will affect the wastewater treatment effect.
Simplicispira belonged to the phylum Proteobacteria, and the members of this genus were defined as Gram-staining-negative, aerobic, rod-shaped bacteria [36] and distributed in water, soil and activated sludge [37]. Thauera was an important denitrifying bacteria, which can convert NO3−-N into N2 [38,39], and its increase of relative abundance indicated that Thauera played an important role in the denitrification process of immobilized particles. Genus Brevundimonas belonged to the phylum Proteobacteria, and its members have been isolated from a wide variety of habitats, species of the genus Brevundimonas played an important role in environment as bioremediation tool, in agriculture as growth promoter, and in medical field as opportunistic pathogens [40]. 5
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4. Conclusions
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Li, Effects of carbon sources on sludge performance and microbial community for 4-chlorophenol wastewater treatment in sequencing batch reactors, Bioresour. Technol. 255 (2018) 22–28. [30] X. Zhang, X. Miao, J. Li, Z. Li, Evaluation of electricity production from fenton oxidation pretreated sludge using a two-chamber microbial fuel cell, Chem. Eng. J. 361 (2019) 599–608. [31] I. Naz, D. Hodgson, A. Smith, J. Marchesi, S. Ahmed, C. Avignone-Rossa, D.P. Saroj, Effect of the chemical composition of filter media on the microbial community in wastewater biofilms at different temperatures, RSC Adv. 6 (2016) 104345–104353. [32] B. Ferrari, T. Winsley, M. Ji, B. Neilan, Insights into the distribution and abundance of the ubiquitous Candidatus Saccharibacteria phylum following tag pyrosequencing, Sci. Rep. 4 (2014) 3957–3966. [33] R. Takenaka, Y. Aoi, N. Ozaki, A. Ohashi, T. Kindaichi, Specificities and efficiencies of primers targeting candidatus phylum saccharibacteria in activated sludge, Materials 11 (2018) 1129–1140. [34] D. Shu, Y. He, H. Yue, Q. Wang, Microbial structures and community functions of anaerobic sludge in six full-scale wastewater treatment plants as revealed by 454 high-throughput pyrosequencing, Bioresour. Technol. 186 (2015) 163–172. [35] Y. Wu, N. Zaiden, B. Cao, The Core- and pan-genomic analyses of the genus comamonas: from environmental adaptation to potential virulence, Front. Microbiol. 9 (2018) 3096–3108. [36] D.W. Hyun, S.J. Oh, M.S. Kim, T.W. Whon, M.J. Jung, N.R. Shin, P.S. Kim, H.S. Kim, J.Y. Lee, W. Kang, J.W. Bae, Simplicispira piscis sp nov., isolated from the gut of a Korean rockfish, Sebastes schlegelii, Int. J. Syst. Evol. Microbiol. 65 (2015) 4689–4694. [37] R.H. Dahal, J. Kim, Simplicispira soli sp nov., a betaproteobacterium isolated from stream bank soil, Int. J. Syst. Evol. Microbiol. 68 (2018) 951–956. [38] N. Yang, G. Zhan, D. Li, X. Wang, X. He, H. Liu, Complete nitrogen removal and electricity production in Thauera-dominated air-cathode single chambered microbial fuel cell, Chem. Eng. J. 356 (2019) 506–515. [39] R. Pishgar, J.A. Dominic, Z. Sheng, J.H. Tay, Denitrification performance and microbial versatility in response to different selection pressures, Bioresour. Technol. 281 (2019) 72–83. [40] D.K. Chaudhary, J. Kim, Brevundimonas mongoliensis sp nov., A novel psychrotolerant bacterium isolated from oil-contaminated soil, Curr. Microbiol. 75 (2018) 1530–1536.
The key finding of this paper is that the bacteria inside the immobilized particles will change in the operation process. In this paper, immobilized denitrifying bacteria particles could remove NO3−-N and TN effectively from real municipal wastewater, and the community structure and relative abundance of bacteria changed greatly, especially at the phyla and genes levels. Therefore, the influence of microflora variation should be considered and we suggest that immobilized particles cannot be used indefinitely, it needs to be replaced periodically when using microbial immobilized technology for wastewater treatment. Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgement This work was supported by the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07103-001). References [1] A.M. Saunders, M. Albertsen, J. Vollertsen, P.H. Nielsen, The activated sludge ecosystem contains a core community of abundant organisms, ISME J. 10 (2016) 11–20. [2] H.C. Flemming, S. Wuertz, Bacteria and archaea on earth and their abundance in biofilms, Nat. Rev. Microbiol. 17 (2019) 247–260. [3] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marinas, A.M. Mayes, Science and technology for water purification in the coming decades, Nature 452 (2008) 301–310. [4] S. Ding, P. Bao, B. Wang, Q. Zhang, Y. Peng, Long-term stable simultaneous partial nitrification, anammox and denitrification (SNAD) process treating real domestic sewage using suspended activated sludge, Chem. Eng. J. 339 (2018) 180–188. [5] R.J. Seviour, T. Mino, M. Onuki, The microbiology of biological phosphorus removal in activated sludge systems, FEMS Microbiol. Rev. 27 (2003) 99–127. [6] T. Zhang, M.F. Shao, L. Ye, 454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants, ISME J. 6 (2012) 1137–1147. [7] J.S. Griffin, G.F. Wells, Regional synchrony in full-scale activated sludge bioreactors due to deterministic microbial community assembly, ISME J. 11 (2017) 500–511. [8] M. Sposob, A. Cydzik-Kwiatkowska, R. Bakke, C. Dinamarca, Temperature-induced changes in a microbial community under autotrophic denitrification with sulfide, Process. Biochem. 69 (2018) 161–168. [9] Y.H. Ahn, Sustainable nitrogen elimination biotechnologies: a review, Process. Biochem. 41 (2006) 1709–1721. [10] C. Yu, D.R. Harrold, J.T. Claypool, B.A. Simmons, S.W. Singer, C.W. Simmons, J.S. VanderGheynst, Nitrogen amendment of green waste impacts microbial community, enzyme secretion and potential for lignocellulose decomposition, Process. Biochem. 52 (2017) 214–222. [11] Y. Kourkoutas, A. Bekatorou, I.M. Banat, R. Marchant, A.A. Koutinas, Immobilization technologies and support materials suitable in alcohol beverages production: a review, Food Microbiol. 21 (2004) 377–397. [12] O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia, R.C. Rodrigues, R. FernandezLafuente, Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties, Biomacromolecules 14 (2013) 2433–2462. [13] O.F. Sarioglu, N.O.S. Keskin, A. Celebioglu, T. Tekinay, T. Uyar, Bacteria immobilized electrospun polycaprolactone and polylactic acid fibrous webs for remediation of textile dyes in water, Chemosphere 184 (2017) 393–399. [14] B. Ranjan, S. Pillai, K. Permaul, S. Singh, Simultaneous removal of heavy metals and cyanate in a wastewater sample using immobilized cyanate hydratase on magneticmultiwall carbon nanotubes, J. Hazard. Mater. 363 (2019) 73–80. [15] H.A. Akdogan, N.K. Pazarlioglu, Fluorene biodegradation by P. osteratus- Part II:biodegradation by immobilized cells in a recycled packed bed reactor, Process. Biochem. 46 (2011) 840–846. [16] S.A. Covarrubias, L.E. de-Bashan, M. Moreno, Y. Bashan, Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae, Appl. Microbiol. Biotechnol. 93 (2012)
6
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Microbial communities variation analysis of denitrifying bacteria immobilized particles ⁎
Liangang Hou, Jun Li , Yang Liu College of Architecture and Civil Engineering, Beijing University of Technology, Beijing 100124, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microbial immobilized technology Denitrification Microbial community High-throughput sequencing Wastewater treatment
The aim of this study was to investigate the denitrification performance and analyze the variation of microbial communities in immobilized particles which used in real municipal wastewater treatment. The bioreactor has achieved good denitrification effect on real municipal wastewater treatment, the NO3−-N and TN removal efficiencies reached 77.4% and 68.3%, respectively. High-throughput sequencing shows that the community structure and relative abundance of bacteria changed greatly, especially at the phyla and genes levels in immobilized particles. Through the investigation of this paper, we think the influence of microflora variation should be considered when using microbial immobilized technology for wastewater treatment, and we suggest that immobilized particles cannot be used indefinitely and it needs to be replaced periodically.
1. Introduction
2. Materials and methods
Activated sludge are a form of collective life with emergent properties that confer many advantages on their inhabitants [1], and they represent a much higher level of organization than single cells do [2], so it has been widely used in wastewater treatment plants (WWTPs) [3,4]. However, microbial communities will change under different living conditions [5–8] affecting the wastewater treatment effect. Therefore, it is necessary to maintain the stability of the microbial communities during wastewater treatment for WWTPs [9,10]. Microbial immobilization technology (MIT) is a technique that uses chemical or physical methods to limit or localize free cells or enzymes in a specific spatial region and maintain biological activity [11–14]. Due to the high activity and stability [15], MIT has been widely concerned for wastewater treatment [13,16,17]. Although the immobilized carrier has a protective effect on microorganism [18–20], whether the bacteria in the immobilized particles have changed during the operation has not been reported so far. The aim of this study was to investigate the denitrification performance and analyze the variation of microbial communities in immobilized particles which used in real municipal wastewater treatment. This work would add some new insights into microbial immobilization technology and further solve some problems of its application in wastewater treatment.
2.1. Experimental setup and operation
⁎
A certain amount of cultured sludge which contains denitrifying bacteria was mixed with an equal amount of waterborne polyurethane and a suitable amount of water, added 1% (w/v) potassium persulfate solution and 0.5% (w/v) N,N,N,N-tetramethylethylenediamine solution to induced polymerization [21].The mixture was solidified for 5∼10 min at 27 ± 2℃ and formed a solid jelly-bean-shaped block of hydrosol, then cut it into 3 × 3 x 3 mm cubes, cultured in a solution containing sodium nitrate and glucose for later use. The feeding medium used in this experiment was real municipal wastewater, which was taken from effluent of aeration tank, a WWTPs in Liaoning province, China. The major characteristics of the municipal wastewater included: nitrate (NO3−-N) 25 ± 2 mg/L, ammoniacal nitrogen (NH4+-N) < 0.1 mg/L, nitrite (NO2−-N) 0.2 ± 0.1 mg/L, total nitrogen (TN) 26 ± 3 mg/L, chemical oxygen demand (COD) 50 ± 5 mg/L. The pH value of the municipal wastewater fluctuated in the range of 7.6∼7.7 and dissolved oxygen (DO) < 0.5 mg/L. Glucose was added into the bioreactor to provide sufficient carbon source for the denitrification process. The schematic of the laboratory-scale denitrification bioreactor is shown in Fig. 1. The bioreactor was made of a plexiglas cylinder and with an operating volume of 80.0 L. Added 12.0 L(15%, v/V) immobilized particles (Sampled and marked Sample A) into the reactor,
Corresponding author. E-mail address: [email protected] (J. Li).
https://doi.org/10.1016/j.procbio.2019.09.001 Received 12 June 2019; Received in revised form 28 August 2019; Accepted 3 September 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Liangang Hou, Jun Li and Yang Liu, Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.001
Process Biochemistry xxx (xxxx) xxx–xxx
L. Hou, et al.
Fig. 1. Schematic diagram of the denitrification bioreactor.
set the hydraulic retention time (HRT) was 12 h, the temperature maintained around 27∼30℃. Sampling after running about 58 days and marked Sample C. Samples A and C were 30–50 immobilized particles randomly collected in the bioreactor to ensure representative samples. 2.2. Analytical methods Samples for water quality measurements were collected regularly and analyzed every day. The concentrations of NO3−-N,TN, NO2−N,NH4+-N and COD in influent and effluent were analyzed according to Standard Methods for the Examination of Water and Wastewater [22]. Before analysis of the above parameters in liquid, samples were membrane filtered (0.45 μm), and all tests were repeated twice in a temperature-controlled room at 27 ± 1℃.The temperature, pH and DO were measured near the midway of the bioreactor by using a WTW analyzer (Multi 3620IDS,Germany). 2.3. Microbiological analysis High-throughput sequencing of 16S rRNA gene fragments was used to analyze the microbial communities [23] in immobilized particles samples. Bacterial genomic DNA was extracted using an E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Inc.) after the samples (A and C) pretreated, following manufacturer's instructions. The PCR amplification used the universal bacteria primers (for 16S rRNA all bacteria) which incorporated Miseq platformic the V3-V4 hypervariable regions. The final sequences of the primers were 341 F (C C T A C G G G N G G C W G C A G), 805R (G A C T A C H V G G G T A T C T A A T C C).The PCR products were sequenced on the Miseq 2 × 300bp pyrosequencing platform by Sangon Biotech, shanghai, China. Finally, to identify and compare core taxa, OTUs (Operational taxonomic unit) were summarized and normalized at the genus level [24,25]. For analyzing the dataset further, the OTUs table along with associated metadata and representative sequences, were imported, stored, and subset, OTUs and Alpha diversity indices (Ace, Chao 1, Shannon, Simpson and Coverage) were determined by Mothur software (ver.1.30.1), and the taxonomy-based analysis used RDP Classifier(ver.2.12) of the Ribosomal Database Project (RDP) and the National Center for Biotechnology Information
Fig. 2. Changes of concentration and removal efficiency of (Fig. 2a)NO3−-N and(Fig. 2b) TN in the bioreactor.
2
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Table 1 Community richness and diversity indices of Samples A and C. Sample
OTUs Number
Coverage (%)
Chao1
Ace
Shannon
Simpson
A C
3794 4023
0.94 0.94
47308.32 42369.57
150619.59 114729.54
3.36 4.50
0.20 0.05
of Dong et al. [28], which reported that waterborne polyurethane immobilized activated sludge has a high-efficiency denitrification effect for the treatment of acrylonitrile wastewater. 3.2. Bacterial diversity analysis The number of OTUs and the richness as well as diversity of two samples were compared (Table 1). More OTUs were detected in the Samples C compared with A, this implies that the microbial richness increased significantly due to the presence of nitrogen compounds in municipal wastewater, which was also reflected by the Chao1 and Ace indexes. The higher Shannon index and lower Simpson index suggested higher diversity in Sample C, indicating that the microbes were highly selected and community diversity has enlarged due to the special life conditions of immobilized particles. 3.3. Microbial community identification The diversity of the microbial communities in immobilized particles is an important property for determining wastewater treatment quality. The microbial community of immobilized particles was characterized based on 16S rRNA analysis. The result shows a diverse community of immobilized particles during different operation stages, and there are different functional groups involved in the nitrogen cycle, respectively. It can be seen from Fig. 4 that the total of identified phyla for Samples A were 18 and Samples C were 19. Different bacteria were identified inside the immobilized particles, Sample A was mainly composed of Proteobacteria (83.2%), Bacteroidetes (7.7%), Firmicutes (4.4%), Actinobacteria (1.2%) (4 species with relative abundance > 1%). Sample C was mainly represented by Proteobacteria (77.8%), Bacteroidetes (8.6%), Candidatus Saccharibacteria (5.4%), Firmicutes (2.8%), Actinobacteria (1.4%), Ignavibacteriae (1.1%) (6 species with relative abundance > 1%). It was known that the major phyla and relative abundances among the two samples were different, Sample C (19 phyla) compared to Sample A (18 phyla), the relative abundance decreased with 5 phyla, 11 phyla increased and one remained unchanged (Hydrogenedentes (0.01%)), simultaneity, one disappears (Nitrospirae) and two new ones appeared (Euryarchaeota (0.01%) and candidate division WPS−1(0.01%)). The relative abundance of phylum Proteobacteria and Firmicutes in Sample C decreased by 5.3% and 1.5%, respectively. These two bacterial phyla were widely distributed in various wastewater treatment plants [29]. Proteobacteria is the largest phylum in bacteria [30], and many of the denitrification microorganisms belong to this phylum [31].Therefore, phylum Proteobacteria plays an important role in the denitrification process of immobilized particles, meaning that the bioreactor exhibited excellent nitrate removal performance. The relative abundance of phyla Candidatus Saccharibacteria and Bacteroidetes in sample C increased 4.7% and 0.83%, respectively. The phylum Candidatus Saccharibacteria [32] was recently assigned the name Saccharibacteria, owing to their sugar metabolisms, and has been frequently detected in natural environments and activated sludge [33].Within the phylum, Filamentous bacteria are the main contributors to the bulking issues in wastewater treatment as their excessive growth. In this study, Filamentous was found in immobilized particles, but they did not cause bulking issues, it indicated that the microbial immobilization technology was beneficial for maintaining the stability of
Fig. 3. Changes of concentration and removal efficiency of (Fig. 3a) COD, (Fig. 3b) NO2−-N and NH4+-N in the bioreactor.
(NCBI) BLAST.
3. Results and discussion 3.1. NO3−-N and COD removal performances Fig. 2 shows the concentration changes of NO3−-N and TN from municipal wastewater through the treatment of immobilized denitrifying bacteria particles. As shown in Fig. 2, The NO3−-N removal efficiency was 5.5–77.4% while TN removal efficiency was 5.2–68.3% during the experimental operation. Those results meaning that most of the nitrate converted to nitrogen gas by denitrifying bacteria in the immobilized particles. The COD removal rate was 18.9–70.2% in the operating process. It can be seen that when the concentration of TN was 25 mg/L in water, maintaining COD/TN = 4.0 can meet the requirement of denitrification, this result was in agreement with that of previous studies [26,27]. Fig. 3 shows the concentration of NO2−-N in effluent, and the accumulation rate of NO2−-N was only 0.3–3.9 mg/L, the low accumulation rate of nitrite indicated that the denitrification process of immobilized particles was relatively complete. In this study, the content of NH4+-N was relatively low, and its concentration vary was not obvious during the operation, indicating that the bacteria in the immobilized particles were mainly participated in denitrification process. The changes of nitrogen and COD in the bioreactor indicated that the immobilized particles had a good treatment efficiency on the real municipal wastewater. This result was in accordance with the research 3
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Fig. 4. 16sRNA analysis of the immobilized particles, phyla identified based on sequencing are shown.
C), Simplicispira (4.1% of Sample A changed to 22.3% of Sample C), Saccharibacteria genera_incertae_sedis (0.8% of Sample A changed to 5.4% of Sample C), Thauera (0 in Sample A and 4.5% in Sample C), Brevundimonas (0 in Sample A and 3.3% in Sample C). At the genus level, Comamonas has reduced by 36.8%, while Simplicispira, Saccharibacteria_genera_incertae_sedis, Thauera and Brevundimonas have increased by 18.2%,4.7%,4.5% and 3.3%, respectively. The genus Comamonas has been reported as one of the major members of microbial communities in various natural and engineered environment, and some of this genus were facultative anaerobes capable of using nitrate as an alternative electron acceptor [35]. Genus
denitrification. Bacteroidetes played an important role in starch and papermaking wastewater treatment plants containing various aromatic pollutants [34], so we guessed that Bacteroidetes also played an important role in the degradation process of nitrate in this paper. As can be seen from Fig. 5, the vary of genus in immobilized particles was obvious. Compared with Sample A, the relative abundance of some genus varies by more than 0.5% in Sample C, and there are six kinds increased and six kinds decreased. In addition, eight of new genus have emerged and seven have disappeared in Sample C compared with A. The most obvious changes in relative abundance of genus were as follows: Comamonas (47.8% of Sample A changed to 11.0% of Sample 4
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Fig. 5. 16sRNA analysis of the immobilized particles, genus identified based on sequencing are shown.
Results of high-throughput sequencing showed a large variation in the microbial community composition at phylum and genus levels. Our investigation demonstrated that microbial community will change in immobilized particles during operation process. Therefore, the influence of microflora variation should be considered when using microbial immobilized technology for wastewater treatment. We suggest that immobilized particles cannot be used indefinitely and it need to be replaced periodically, because considering the changes of microbial communities will affect the wastewater treatment effect.
Simplicispira belonged to the phylum Proteobacteria, and the members of this genus were defined as Gram-staining-negative, aerobic, rod-shaped bacteria [36] and distributed in water, soil and activated sludge [37]. Thauera was an important denitrifying bacteria, which can convert NO3−-N into N2 [38,39], and its increase of relative abundance indicated that Thauera played an important role in the denitrification process of immobilized particles. Genus Brevundimonas belonged to the phylum Proteobacteria, and its members have been isolated from a wide variety of habitats, species of the genus Brevundimonas played an important role in environment as bioremediation tool, in agriculture as growth promoter, and in medical field as opportunistic pathogens [40]. 5
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4. Conclusions
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