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N range and its potential application in phenolic and coking wastewater
Accepted Manuscript Title: Characterization of a microbial consortium capable of heterotrophic nitrifying under wide C/N range and its potential application in phenolic and coking wastewater Author: Ya Yang Yuxiang Liu Ting Yang Yongkang Lv PII: DOI: Reference:
S1369-703X(16)30344-8 http://dx.doi.org/doi:10.1016/j.bej.2016.12.008 BEJ 6615
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
31-8-2016 24-11-2016 9-12-2016
Please cite this article as: Ya Yang, Yuxiang Liu, Ting Yang, Yongkang Lv, Characterization of a microbial consortium capable of heterotrophic nitrifying under wide C/N range and its potential application in phenolic and coking wastewater, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2016.12.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of a microbial consortium capable of heterotrophic nitrifying under wide C/N range and its potential application in phenolic and coking wastewater Ya Yang a, Yuxiang Liu a,*, Ting Yang a, Yongkang Lv b a
College of Environmental Science and Engineering, Taiyuan University of Technology, Shanxi
030024, China b Key
Laboratory of Coal Science and Technology of Shanxi Province and Ministry of Education,
Taiyuan University of Technology, Shanxi 030024, China
Yuxiang Liu: * Corresponding author at: Taiyuan University of Technology, #79 Yingze West Street, Taiyuan 030024, Shanxi, China. Tel/Fax: +86 0351 6010014. E-mail address: [email protected].
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Highlights ●A microbial consortium named FG-06 has excellent heterotrophic nitrifying-aerobic denitrifying ability. ●It had good heterotrophic nitrifying ability under a wide C/N range. ●Simultaneous removal of ammonia nitrogen and COD in phenolic wastewater and coking wastewater were achieved. ●The predominant bacterial groups were identified as Acinetobacter spp. (54.37%) and Pseudomonas spp. (26.52%).
Abstract: Nitrogen pollution has been a serious problem in environmental water particularly in industrial wastewater. A microbial consortium named FG-06 was found to exhibit efficient heterotrophic nitrification and aerobic denitrification ability, with the maximum NH4+-N, NO2--N and NO3--N removal rate of 7.33, 6.53 and 4.53 mg/L/h, respectively. High-throughput sequencing analysis showed that it was mainly made up of Acinetobacter spp. (54.37%) and Pseudomonas spp. (26.52%). The consortium could grow under a wide range of C/N ratio and the NH4+-N removal efficiencies at C/N ratio 4 and 32 were 90.40% and 93.84% separately. Furthermore, simultaneous biodegradation of NH4+-N and COD were achieved by inoculation with enriched consortium in synthetic phenolic wastewater and real coking wastewater samples. Results demonstrated that the consortium had high potential for further practical applications. Keywords: heterotrophic nitrification-aerobic denitrification; C/N; simultaneous removal of NH4+-N and COD; phenolic wastewater; coking wastewater
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1. Introduction Coking wastewater is a kind of refractory and toxic wastewater. Major pollutants of the wastewater have been reported as ammonia, phenols, nitrogen heterocyclic compounds, and polycyclic aromatic hydrocarbons etc. [1]. Usually phenols are the most abundant toxic compounds and comprise most of the chemical oxygen demand (COD) of the wastewater. What’s more, ammonia nitrogen from insufficiently treated effluent of the industrial wastewater is claimed to be the chief culprit that cause human diseases and eutrophication problems in water bodies. Accordingly, it is an urgency to find an effective way which can reduce the ammonia nitrogen and COD in the wastewater [2]. Biological treatment processes are the most used technology to treat the wastewater because they are more cost effective and environmental friendly than their chemical and/or physical counterparts [3]. According to the traditional ammonium removal theory, the biological processes include nitrification, which oxidizes ammonium to nitrite or nitrate by nitrifiers under aerobic condition, and denitrification, which converts nitrite and nitrate to N2 gas by denitrifiers under anaerobic condition [4, 5]. However, the low rate of nitrification and the different requirements for nitrifiers and denitrifiers make it cost-intensive and time-consuming [6]. Recently, more studies have highlighted the existence of bacteria that are capable of performing heterotrophic nitrification and have a marked ability to denitrify their nitrification products under aerobic conditions. Bacteria of this capability have been isolated from soils and
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wastewater treatment systems, such as Pseudomonas, Bacillus, Klebsiella, Alcaligenes, Acinetobacter and Paracoccus [7-12]. These special heterotrophic bacteria with higher growth rates than autotrophs can not only nitrify and denitrify simultaneously but also remove nitrogen and carbon pollutants in a single reactor [13]. In addition, the alkalinity generated during denitrification can partly compensate for the acidification caused by nitrification which could lower the cost of pH-adjusting [11]. However, most of the studies were conducted with only one or two bacterial strains, and no attention has been paid to the degradation of ammonium and organics by a microbial consortium. A microbial consortium has more advantages than a single strain in the industrial application, both from the treatment time and the stability of the treatment results. In recent years, several reports have studied the characteristics of a consortium and offered a basis for further application [14, 15]. However, there is currently few reports about a microbial consortium capable of heterotrophic nitrifying. What’s more, the application of heterotrophic nitrification bacteria was mostly for domestic wastewater or micro-polluted water [4, 7, 16], sterile synthetic wastewater was usually used instead of real industrial wastewater [17, 18]. Lower or higher C/N ratios of actual wastewaters may lead to poor treatment efficiencies. Up to now, limited reports are available on a bacterium which could conduct heterotrophic nitrification aerobically under a wide range of C/N ratio. Although some bacteria could survive under lower or higher C/N ratios, the removal efficiencies of ammonia nitrogen were relatively low [11, 17]. In this study, a microbial consortium named FG-06, capable of heterotrophic
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nitrification and aerobic denitrification under wide C/N range was enriched from coking activated sludge. Ammonium, nitrate and nitrite degradation experiments were conducted under aerobic conditions. The application of the consortium in synthetic phenolic wastewater and real coking wastewater was conducted in order to investigate its potential for use in bioaugmented treatment. The results may provide useful information about heterotrophic nitrification-aerobic denitrification bacteria for bioaugmentation in industrial wastewater treatment or in situ bioremediation at contaminated sites.
2. Materials and Methods 2.1. Chemicals and culture media All of the chemicals used in the experiments were of analytical grade. The consortium was enriched in Luria–Bertani medium (LB) containing (per liter): 10 g tryptone, 10 g beef extract, and 5 g NaCl. Basal medium (BM) used for heterotrophic nitrifying study consisted of the following components (per liter): sodium succinate 3.473 g, (NH4)2SO4 0.472 g, MgSO4·7H2O 0.05 g, K2HPO4 0.2 g, NaCl 0.12 g, MnSO4·4H2O 0.01 g, FeSO4·7H2O 0.01 g. The denitrification medium (DM-1&2) used for nitrate and nitrite reduction studies contained (per liter): 0.72 g KNO3 (0.49 g NaNO2), 4.7 g of sodium succinate, 0.05 g MgSO4·7H2O, 0.2 g K2HPO4, 0.12 g NaCl, 0.01 g MnSO4·4H2O, 0.01 g FeSO4·7H2O. The consortium was inoculated in ammonia-nitrate medium (SNDM-1) and 5
ammonia-nitrite medium (SNDM-2) to study simultaneous nitrification
and
denitrification efficiency contained (per liter): 0.236 g (NH4)2SO4, 0.361 g KNO3 (0.247 g NaNO2), 3.473 g of sodium succinate, 0.05 g MgSO4·7H2O, 0.2 g K2HPO4, 0.12 g NaCl, 0.01 g MnSO4·4H2O, 0.01 g FeSO4·7H2O. Initial pH of all culture media was set at 7.0 and all the culture media were autoclaved for sterility at 121 °C for 20 minutes before use. Conical flasks (250 mL capacity) containing 100 mL medium sealed with sterile breathable membranes were used in the experiments. The other experiment conditions were as follows: culturing temperature 30 °C, shaking speed 120 r/min. 2.2. Enrichment of the consortium The activated sludge was collected from coking wastewater treatment plant in Taiyuan City, Shanxi Province, China in November 2015 and used to enrich the consortium. The activated sludge (20 mL) was inoculated into autoclaved LB medium (200 mL) in an Erlenmeyer flask. The flask was then sealed with sterile breathable membranes and shaken in a rotary shaker at 30 °C and 120 r/min. After a cultivation of 2 days, cell suspension (10 mL) was transferred to 90 mL of sterile LB medium and incubated under the conditions described above for a second enrichment. Thereafter, bacteria suspension (1 mL) was sub-inoculated into another sterile flask containing fresh BM (100 mL) for selective enrichment of ammonium oxidation bacteria. Sodium succinate and (NH4)2SO4 were provided as the sole carbon and nitrogen source. After 20 h of aerobic incubation, the cultures (1 mL) were transferred to another fresh BM (100 6
mL) containing sodium succinate and (NH4)2SO4. This process was repeated three times. The concentration of NH4+-N was measured periodically in each cycle to identify its ability to degrade ammonium nitrogen. Afterwards, a consortium named FG-06 with excellent nitrification ability was picked and individually tested for detailed nitrogen removal capability through shake flask experiments. The consortium was spread in inclined solid medium of coking wastewater for short-term storage and suspended in 25% glycerol solution at -80 °C for long-term storage. 2.3. Microbial consortium analysis 2.3.1. DNA extraction and PCR amplification For genomic DNA extract, the consortium were cultivated overnight and harvested at high bacteria concentration. PCR for high-throughput sequencing was performed with barcode
primers
(Nobar341F
CCTACGGGNGGCWGCAG,
Nobar805R
GACTACHVGGGTATCTAATCC), which targeted the 16S rDNA V3–V4 regions [19]. Barcodes were added at the 5ˊ terminus of forward primers to allow samples multiplexing during sequencing. The total reaction mixture (50 µL) of PCR contained 5 μL of 10× PCR buffer, 2 µL of 10 µM forward and reverse primers, 0.5 μL of 10 mM dNTP mixture, 1.5 U of Taq™ polymerase (Takara Biotech, Dalian, China), 10 ng of genomic DNA, and water (RT-PCR grade, USA) [20]. PCR was performed under the following conditions: denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 30 7
s; and a final extension step at 72 °C for 5 min. Triplicate PCR products were pooled and purified with DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China). Then the purified amplicons were paired-end sequenced on a Miseq platform according to the standard protocols. The two reads were optimized by trimming off the tag sequences and removing the sequences less than 50 bp or with a quality score lower than 20 [21]. During this process, the sequences which contained any errors in barcodes or primers were removed. The number of selected bacterial sequence in the sample was 35,233 after the filtration. 2.3.2. Sequence analysis Operational taxonomic unit (OTU)-based method was employed for describing the enriched consortium. 20,000 sequences from each sample were clustered with 97% similarity and the representative sequence for each OTU was the most abundant one. A total of 13 OTUs were selected and the 16S rDNA sequences of the isolated strains have been deposited in NCBI GenBank under accession number KY054998–KY055010. Then the representative sequences of the most abundant 8 OTUs were compared with available sequences in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) [22]. Phylogenetic reconstructions of the 16S rDNA sequences obtained in this study were performed in MEGA 5.1 software using neighbor-joining (NJ) method with a bootstrap of 1000 [23]. 2.4. Studies on the heterotrophic nitrification characteristics under different conditions
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Single-factor experiments were conducted for studying the heterotrophic nitrification characteristics of the consortium under different culturing conditions, including C/N ratio, initial inoculant dosage and ammonia nitrogen concentration. In C/N ratio experiments, the initial inoculant dosage was kept in 1% and the content of carbon source was changed in order to adjust C/N ratio to 4, 8, 12, 16, 20, 24, 28 and 32 respectively, by fixing nitrogen concentration at 100 mg/L. The inoculant dosage was setted to 1%, 3%, 5%, 7% and 9% in initial inoculant dosage experiments and the later experiments were all conducted under the optimum conditions from C/N test and initial inoculant dosage test. In ammonia nitrogen experiments, initial ammonia nitrogen concentration was adjusted to 100, 200, 400, 600, 800 mg/L, and sodium succinate content varied accordingly to keep the optimum C/N. To evaluate the influence of refractory contaminants on heterotrophic nitrification of the consortium, ammonia nitrogen degradation in mixed carbon sources was also investigated in batch with 100 mL BM containing 100 mg/L of NH4+-N and 50 mg/L of quinoline or nitrobenzene. 2.5. Assessment of aerobic denitrification and simultaneous nitrification and denitrification ability After 18 h cultivation in BM, cell suspension was inoculated into 100 mL sterile DM-1&2 and SNDM-1&2 in 250 mL Erlenmeyer flasks, and was incubated aerobically about 48 h. During incubation, samples were withdrawn and measured periodically to determine the optical density at 600 nm (OD600) and NH4+-N, NO2--N, NO3--N, NH2OH, total nitrogen (TN) and COD.
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2.6. Evaluation of the treatment of phenolic and coking wastewater by the consortium In this study, raw coking wastewater was collected from the same coking wastewater treatment plant. Synthetic phenolic wastewater was used as the experimental wastewater which contained phenol, 1000 mg/L; (NH4)2SO4, 100 mg/L; sodium succinate, 2.05 g/L; MgSO4·7H2O, 50 mg/L; K2HPO4, 200 mg/L; NaCl, 120 mg/L; MnSO4·4H2O, 10 mg/L; FeSO4·7H2O, 10 mg/L. The concentrations of NH4+-N and COD in the wastewater were given in Table 1. For degradation in synthetic phenolic wastewater, the harvested consortium was inoculated into the wastewater previously autoclaved at a dosage of 1%. For degradation in real coking wastewater, the consortium was inoculated into the wastewater previously autoclaved and filtered through a 0.45 μm membrane at a dosage of 3%. The wastewater without the consortium inoculated was as a control. Samples were collected periodically to determine the concentration of NH4+-N and COD. 2.7. Analytical methods and calculations All aqueous samples were centrifuged for 10 min at 10000 rpm before being subjected to subsequent analysis. The growth of the consortium was determined by spectrophotometry at 600nm (OD600). Levels of NH4+-N, NO2--N and NO3--N were measured according to the standard methods [24]. NH4+-N was determined by the method of Nessler’s reagent spectrophotometry at a wavelength of 420 nm; NO2--N was determined
using
spectrophotometry;
N-(1-naphthyl)-1, NO3--N
was
2-diaminoethane
determined 10
by
phenol
dihydrochloride disulphonic
acid
spectrophotometry. NH2OH was measured colorimetrically according to Frear and Burrell [25]. TN was detected by the alkaline persulfate oxidation with a UV spectrophotometric method. COD and pH were measured using a COD analyzer (DR 1010, HACH, USA) and a pH meter (PB-10, Sartorious, Germany) separately. Nitrogen, COD and phenol removal efficiency and removal rate were calculated as: per (%) = (C0 – Cn)/C0 × 100 and (C0 – Cn)/t, respectively, where C0 was the initial concentration (mg/L), Cn was the final concentration at the time when sample was collected for analysis and t was the total period of time for the consortium treatment. Each treatment was performed in triplicate and results were presented as means ± SD (standard deviation of means).
3. Results and discussion 3.1. Composition of the microbial consortium To understand the contribution of biological degradation in the treatment of consortium from wastewater, microbial species in the consortium were analyzed by high-throughput sequencing. As shown in Fig. 1, Acinetobacter spp. and Pseudomonas spp. were the most dominant bacterial groups in the consortium. Phylogenetic relationship of the most abundant genera based on 16S rDNA sequences were depicted in Fig. 2. Within the Acinetobacter cluster (54.37% of the OTUs in FG-06 culture), clone FG-066, clone FG-067 and clone FG-068 were affiliated with Acinetobacter spp.. Furthermore, clone FG-061 (26.52% of the OTUs in FG-06 culture) was part of Pseudomonas spp.. Several lineages of the genus Acinetobacter and Pseudomonas have 11
been reported capable of heterotrophic nitrification and aerobic denitrification, including Acinetobacter sp. SYF26 [26], Acinetobacter sp. Y16 [27], Acinetobacter junii YB [18], Pseudomonas alcaligenes AS-1 [28], Pseudomonas tolaasii Y-11 [29], and Pseudomonas stutzeri [4, 8]. Therefore Acinetobacter spp. and Pseudomonas spp., as the most abundant species in the enriched consortium, potentially played the largest role in biological degradation under aerobic condition. Other species were likewise detected in this microbial consortium. Although their percentages were quite low, certain species, such as Aeromonas and Alcaligenes, have been reported to have heterotrophic nitrification and aerobic denitrification ability [10, 30]. 3.2. Heterotrophic nitrification performance under wide C/N range Fig. 3 showed the effect of C/N ratios on utilization of NH4+-N under aerobic condition in batch culture. The NH4+-N removal percentage was not significantly different among C/N 4-32, all of which could reach a level of 90% in 48 h. The highest removal percentage was 99.94% at C/N ratio 16 and the corresponding maximum removal rate of NH4+-N was 7.33 mg/L/h in 12 h. At a C/N ratio of 4, up to 90.40% of the NH4+-N in the medium was removed by the consortium. With the C/N ratios varying from 8 to 32, the removal efficiencies of NH4+-N were remained higher than 93%. The final OD600 was consistent with the removal of NH4+-N, and the final OD600 under C/N ratio 16 was 1.208 which was the highest among them. Most literatures reported the optimal C/N of heterotrophic nitrification-aerobic denitrification bacteria was 6-20 [17, 31]. However, the microbial consortium FG-06 12
could grow under C/N ratio 4 which was relatively lower for several strains [11, 32]. Even if the C/N ratio was as high as 32, which was too high for the growth of autotrophic nitrifying bacteria, the consortium still exhibited satisfying nitrification ability with a NH4+-N removal percentage of 93.84%. The tolerance of the consortium to a wide C/N range expanded its application scope, regardless of the treatment of piggery waste with C/N of 4-7 or other wastewater with high C/N such as municipal landfill leachate and coking wastewater. Take cost effectiveness into consideration, C/N 16 was used in following experiments. 3.3. Ammonium removal under different conditions 3.3.1. Effect of initial inoculant dosage As shown in Fig. 4a, the NH4+-N removal efficiencies from 1% to 9% of the inoculation in 48 h were 99.98%, 99.95%, 99.12%, 97.56% and 96.58% separately, with the highest one at 1%. The final OD600 after batch tests decreased slightly with increased inoculant dosage. High inoculant dosage may affect the degradation characteristics of bacteria due to the excessive metabolic wastes in the culture medium. Taking into account of the stability of reaction and other factors, inoculant dosage 1% was used in following experiments. 3.3.2. Effect of ammonia concentration The effect of initial ammonia concentration on the heterotrophic nitrification capability of the consortium under 48 h was studied over the initial concentration range
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100-800 mg/L (Fig. 4b). The NH4+-N removal efficiency could reach more than 99% when initial ammonia nitrogen concentration was 100, 200 and 400 mg/L while it was 89.93% with 600 mg/L of initial NH4+-N. It is worth mentioning that the removal efficiency was still about 82.36% under the condition of ammonia nitrogen concentration up to 800 mg/L. Pseudomonas stutzeri YG-24 could survive up to 200 mg/L of NH4+-N but the removal efficiency was only 55.98% [8]. The NH4+-N removal efficiency of Pseudomonas stutzeri T13 was 39.56% when initial NH4+-N was 224.68 mg/L [33]. Above all, the consortium could effectively remove NH4+-N at concentrations ≤ 400 mg/L. 3.3.3. Effect of quinoline and nitrobenzene According to the experimental results obtained using mixed carbon sources containing sodium succinate and quinoline or nitrobenzene, the maximum removal efficiencies of NH4+-N were 98.57% and 99.5% within 48 h, respectively, at an initial NH4+-N concentration of 100 mg/L (Fig. 4c&d). The consortium grew well and the maximum OD600 were 1.186 and 1.225 separately. Compared with NH4+-N degradation in a single carbon source under the optimum C/N condition (Fig. 3a&b), the degradation rate slightly decreased because of the presence of quinoline or nitrobenzene in the mixed carbon sources. In spite of this, the consortium maintained high-efficiency NH4+-N degradation and was tolerant of quinoline and nitrobenzene. The results indicated that the consortium was a promising candidate in engineering practice of coking wastewater treatment.
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3.4. Aerobic denitrification capability of the consortium To evaluate the aerobic denitrification ability of the consortium, two nitrification intermediates, nitrate nitrogen (DM-1) and nitrite nitrogen (DM-2) were used as denitrifying substrates respectively. Fig. 5 showed the changes of various components in a 250-mL flask during the period of nitrogen removal under an aerobic condition. When nitrate was added as the nitrogen source (Fig. 5a), the nitrate concentration decreased significantly with the consortium growing rapidly, as did the TN and COD concentrations. Approximately 50% of the NO3--N was removed in 12 h, which correlated well with the rate of cell growth. Cell growth reached stationary phase in 24 h, as the OD600 increased from 0 to 1.305, 98.2% of the NO3--N and 94.4% of the TN were removed. Between 36 and 48 h, the rate of decrease in NO3--N concentration declined. The same trend was observed for the TN and COD concentration. The accumulation of NO2--N in 24 h reached the maximum amount of 7.53 mg/L. However, the NO2--N concentration decreased subsequently and completely reduced by 48 h. The maximum removal efficiencies of NO3--N, TN and COD by 48 h were 99.64%, 98.53% and 93.60%, separately. The calculated maximum nitrate removal rate in 12 h was as high as 4.53 mg/L/h, which was higher than that of Klebsiella pneumoniae CF-S9 with nitrate removal rate of 2.2 mg/L/h [9] and Rhodococcus sp. CPZ24 with nitrate removal rate of 0.93 mg/L/h [31]. In DM-2 (Fig. 5b), the OD600 increased to 1.32 at 24 h, the maximum removal efficiencies of NO2--N, TN and COD were 98.25%, 98.02% and 95.73%, separately. Meanwhile, no obvious accumulation of nitrate was detected in the medium
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with nitrite as a nitrogen source. The maximum removal rate of NO2--N in 12 h was 6.53 mg/L/h with the fastest removal rate occurring during exponential phase, which was much higher than that of Pseudomonas sp. yy7 (18.20 mg/L/d) [34] and Bacillus methylotrophicus strain L7 (5.81 mg/L/d) [11]. It was clearly demonstrated that both NO3--N and NO2--N could be utilized by the consortium under aerobic conditions, which suggested the existence of aerobic denitrification capacity in FG-06. The consortium was particularly different from other heterotrophic nitrifying bacteria of A. faecalis NR [35] and A. calcoaceticus HNR [13], in which nitrite and nitrate were both not utilized. 3.5. Heterotrophic nitrification and aerobic denitrification abilities in mixed N-source To further study the nitrogen removal by the consortium under aerobic conditions, simultaneous nitrification and denitrification in mixed N-sources were investigated (Fig. 6). When (NH4)2SO4, KNO3 (SNDM-1) or (NH4)2SO4, NaNO2 (SNDM-2) were used as the nitrogen sources of the consortium, the growth of the consortium was very well and the bacterial biomass peaked at 24 h. The growth trend was similar to the conditions when nitrite nitrogen or nitrate nitrogen was used as the sole N-source. In SNDM-1, NH4+-N concentration decreased dramatically from initial 52.85 mg/L to 1.46 mg/L in 24 h. Approximately 97.23% of NH4+-N was removed, and the NO3--N removal efficiency was 96.84% under 24 h. The maximum removal rate in 12 h was 2.91 NO3--N mg/L/h, which was lower than that of the nitrate as sole nitrogen source. The formation of intermediate product, nitrite started from 0 h and the concentration gradually 16
increased to 18.19 mg/L at 12 h and decreased to zero level by 36 h. In SNDM-2, the consortium was able to remove almost all the NH4+-N by 24 h, and the maximum removal efficiency of NO2--N was 99.49% under 48 h. During the inoculation period, a little NO3--N was detected at 12 h and decreased to zero by 48 h. The patterns of total nitrogen removal were consistent with the removal of nitrogen sources, and the removal efficiencies could reach 97.14% and 96.03% respectively. In addition, the removal efficiencies of COD were all above 97%. In this experiment, the NH4+-N removal rate was high with highest removal rate of 2.66 and 3.15 mg/L/h in SNDM-1&2, respectively. Compared to the maximum removal rate under wide C/N range of heterotrophic nitrification, the ammonium removal rate was significantly decreased. Some studies drew a conclusion that nitrification efficiency would be declined in the presence of nitrite [36, 37], which was similar to the findings in this study that the addition of nitrite and nitrate in BM medium inhibited the removal of ammonium. In addition, NH4+-N and NO3--N, NO2--N were degraded by the consortium simultaneously. This was similar to the phenomenon occurred in Paracoccus versutus LYM [38], but differed from Acinetobacter junii YB [18], whereby NO3--N and NO2--N started to decline gradually after the complete removal of NH4+-N. 3.6. The treatment of phenolic and coking wastewater by the consortium Bioaugmentation is a promising improvement to the biological treatment of wastewater, where the employment of microbial strains enhances the removal of certain organic pollutants. The characteristics of nitrogen removal by heterotrophic 17
nitrification-aerobic denitrification bacteria in shaking flask medium culture conditions have been widely reported [26, 29, 35, 39]. However, limited studies concern the removal of nitrogen and organics by the bacteria in phenolic wastewater and coking wastewater. In the present study, the potential application of the enriched consortium was investigated. The results of NH4+-N degradation and COD removal by the consortium in phenolic wastewater and coking wastewater were shown in Fig. 7. Compared with NH4+-N degradation in the BM, NH4+-N degradation in the phenolic wastewater was delayed. However, more than 99% of NH4+-N and 97% of COD were degraded simultaneously within 60 h, the maximum removal rates of COD and NH4+-N were 47.08 and 5.57 mg/L/h, respectively. The removal of COD in synthetic phenolic wastewater indicated that the consortium could also use phenol as the carbon source and achieve the simultaneous removal of NH4+-N and COD. The consortium was inoculated into real coking wastewater having initial concentration about 83.29 mg/L of NH4+-N and 2800 mg/L of COD. At 24 h there was no obvious change in the concentration of NH4+-N. However, the average concentration was reduced to 64.75 mg/L by 48 h, 33.53 mg/L by 120 h and almost completely removed by 168 h. The degradation rate was much lower than that in the BM, and degradation need more time to complete. This indicated that other refractory contaminants present in the coking wastewater were inhibited to the enriched consortium. Even so, the experiments demonstrated that NH4+-N could still be degraded completely by the consortium, indicating that it preserved its ability for ammonia nitrogen degradation
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under unfavorable circumstances. In addition, more than 80% of COD was removed within 168 h which suggested that the consortium could degrade most organic compounds in the coking wastewater. Therefore, the tolerance to high concentration of organics and the simultaneous removal of NH4+-N and COD demonstrated high potential of the consortium for application in real wastewater treatment. But subsequent experiment should be conducted to discuss the strategies of bioaugmentation and the survival capability of the consortium in activated sludge systems [18].
4. Conclusions A newly enriched consortium named FG-06 has both heterotrophic nitrification and aerobic denitrification abilities. High-throughput sequencing result showed that it was mainly made up of Acinetobacter spp. and Pseudomonas spp.. The tolerance of a wide C/N range made the consortium a wider scope of potential application. It could remove NH4+-N and COD in synthetic phenolic wastewater and real coking wastewater which was a valuable feature for reliable sewage treatment. Therefore, application of the consortium may lead to bioaugmented treatment of industrial wastewater containing high concentrations of NH4+-N and COD.
Acknowledgements This work was supported by the Key research and Development Program of Shanxi Province (grant number 201603D321010), International Cooperation Projects of
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Shanxi Province (grant number 201603D421040), National Key research and Development Program (grant number 2016YFB0600502) and Science and Technology Development of Shanxi Province (grant number 20140321013-04).
References [1] Z. Min, J.H. Tay, Q. Yi, S.G. Xia, Coke plant wastewater treatment by fixed biofilm system for COD and NH3-N removal, Water. Res. 32 (1998) 519-527. [2] J. Wang, X. Quan, L. Wu, Y. Qian, W. Hegemann, Bioaugmentation as a tool to enhance the removal of refractory compound in coke plant wastewater, Process. Biochem. 38 (2002) 777-781. [3] H. Li, H. Cao, Y. Li, Y. Zhang, H. Liu, Innovative biological process for treatment of coking wastewater, Environ. Eng. Sci. 27 (2010) 313-322. [4] J. Zhang, P. Wu, B. Hao, Z. Yu, Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001, Bioresour. Technol. 102 (2011) 9866-9869. [5] Z.Y. Ji, Y.G. Chen, Using sludge fermentation liquid to improve wastewater short-cut nitrification-denitrification and denitrifying phosphorus removal via nitrite, Environ. Sci. Tecnol. 44 (2010) 8957-8963. [6] T. Khin, A.P. Annachhatre, Novel microbial nitrogen removal processes, Biotechnol. Adv. 22 (2004) 519-532. [7] T.L. Huang, S.L. Zhou, H.H. Zhang, N. Zhou, L. Guo, S.Y. Di, Z.Z. Zhou, Nitrogen removal from micro-polluted reservoir water by indigenous aerobic denitrifiers, Int. J. Mol. Sci. 16 (2015) 8008-8026. [8] C. Li, J. Yang, X. Wang, E. Wang, B. Li, R. He, H. Yuan, Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a phosphate accumulating bacterium Pseudomonas stutzeri YG-24, Bioresour. Technol. 182 (2015) 18-25. [9] S.K. Padhi, S. Tripathy, R. Sen, A.S. Mahapatra, S. Mohanty, N.K. Maiti, Characterisation of heterotrophic nitrifying and aerobic denitrifying Klebsiella pneumoniae CF-S9 strain for bioremediation of wastewater, Int. Biodeter. Biodegr. 78 (2013) 67-73. [10] M. Shoda, Y. Ishikawa, Heterotrophic nitrification and aerobic denitrification of high-strength ammonium in anaerobically digested sludge by Alcaligenes faecalis strain No. 4, J. Biosci. Bioeng. 117 (2014) 737-741. [11] Q.L. Zhang, Y. Liu, G.M. Ai, L.L. Miao, H.Y. Zheng, Z.P. Liu, The characteristics of a novel heterotrophic nitrification–aerobic denitrification bacterium, Bacillus methylotrophicus strain L7, Bioresour. Technol. 108 (2012) 35-44. [12] Z. Yu, S. Zhuang, M. Chen, X. Dong, J. Zhou, Evaluation of simultaneous nitrification and denitrification under controlled conditions by an aerobic denitrifier culture, Bioresour. Technol. 175c (2014) 602-605. [13] B. Zhao, Y.L. He, J. Hughes, X.F. Zhang, Heterotrophic nitrogen removal by a newly isolated Acinetobacter calcoaceticus HNR, Bioresour. Technol. 101 (2010) 5194-5200. [14] C. Pan, G.Y.A. Tan, L. Ge, C.L. Chen, J.Y. Wang, Microbial removal of carboxylic acids from
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1,3-propanediol in glycerol anaerobic digestion effluent by PHAs-producing consortium, Biochem. Eng. J. 112 (2016) 269-276. [15] A. Salmerón-Alcocer, N. Ruiz-Ordaz, C. Juárez-Ramírez, J. Galíndez-Mayer, Continuous biodegradation of single and mixed chlorophenols by a mixed microbial culture constituted by Burkholderia sp., Microbacterium phyllosphaerae, and Candida tropicalis, Biochem. Eng. J. 37 (2007) 201-211. [16] C. Qian, J. Ni, M. Tao, L. Tang, M. Zheng, Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR, Bioresour. Technol. 183 (2015) 25-32. [17] J.M. Duan, H.D. Fang, B. Su, J.F. Chen, J.M. Lin, Characterization of a halophilic heterotrophic nitrification–aerobic denitrification bacterium and its application on treatment of saline wastewater, Bioresour. Technol. 179 (2015) 421-428. [18] L. Yang, Y.-X. Ren, X. Liang, S.-Q. Zhao, J.-p. Wang, Z.-H. Xia, Nitrogen removal characteristics of a heterotrophic nitrifier Acinetobacter junii YB and its potential application for the treatment of high-strength nitrogenous wastewater, Bioresour. Technol. 193 (2015) 227-233. [19] S.E. Dowd, T.R. Callaway, R.D. Wolcott, Y. Sun, T. Mckeehan, R.G. Hagevoort, T.S. Edrington, Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), BMC Microbiol. 8 (2008) 208-213. [20] Y.P. Mao, X. Yu, Z. Tong, Characterization of Thauera -dominated hydrogen-oxidizing autotrophic denitrifying microbial communities by using high-throughput sequencing, Bioresour. Technol. 128C (2012) 703-710. [21] S. Li, D. Li, W. Yan, Cometabolism of methyl tert-butyl ether by a new microbial consortium ERS, Environ. Sci. Pollut. R. 22 (2015) 10196-10205. [22] S. Kumar, M. Nei, J. Dudley, K. Tamura, MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences, Brief. Bioinform. 9 (2008) 299-306. [23] L. MA, B. G, B. NP, C. R, M. PA, M. H, V. F, W. IM, W. A, L. R, T. JD, G. TJ, H. DG, Clustal W and Clustal X version 2.0, Bioinformatics, 23 (2007) 2947-2948. [24] A. Apha, WEF (1998) Standard methods for the examination of water and wastewater, American Public Health Association, Washington, DC, 1998. [25] D. Frear, R. Burrell, Spectrophotometric method for determining hydroxylamine reductase activity in higher plants, Anal. Chem. 27 (1955) 1664-1665. [26] J.F. Su, K. Zhang, T.L. Huang, G. Wen, L. Guo, S.F. Yang, Heterotrophic nitrification and aerobic denitrification at low nutrient conditions by a newly isolated bacterium, Acinetobacter sp. SYF26, Microbiol. 161 (2015). [27] X. Huang, W. Li, D. Zhang, W. Qin, Ammonium removal by a novel oligotrophic Acinetobacter sp. Y16 capable of heterotrophic nitrification–aerobic denitrification at low temperature, Bioresour. Technol. 146 (2013) 44-50. [28] J.J. Su，K.S. Yeh，P.W. Tseng, A strain of Pseudomonas sp. isolated from piggery wastewater treatment systems with heterotrophic nitrification capability in Taiwan, Current Microbiology, 53 (2006) 77-81. [29] T. He, Z. Li, S. Quan, X. Yi, Q. Ye, Heterotrophic nitrification and aerobic denitrification by Pseudomonas tolaasii Y-11 without nitrite accumulation during nitrogen conversion, Bioresour. Technol. 200 (2016) 493-499.
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[30] M. Chen, W. Wang, F. Ye, X. Zhu, H. Zhou, Z. Tan, X. Li, Impact resistance of different factors on ammonia removal by heterotrophic nitrification–aerobic denitrification bacterium Aeromonas sp. HN-02, Bioresour. Technol. 167C (2014) 456-461. [31] P. Chen, L. Ji, Q.X. Li, Y. Wang, S. Li, T. Ren, L. Wang, Simultaneous heterotrophic nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24, Bioresour. Technol. 116 (2012) 266–270. [32] L. Yu, Y. Wang, H. Liu, C. Xi, L. Song, A novel heterotrophic nitrifying and aerobic denitrifying bacterium, Zobellella taiwanensis DN-7, can remove high-strength ammonium, Appl. Microbiol. Biotechnol. (2016) 1-11. [33] Y. Sun, A. Li, X. Zhang, F. Ma, Regulation of dissolved oxygen from accumulated nitrite during the heterotrophic nitrification and aerobic denitrification of Pseudomonas stutzeri T13, Appl. Microbiol. Biotechnol. 99 (2015) 3243-3248. [34] C. Wan，X. Yang，D.J. Lee，M. Du，F. Wan, C. Chen, Aerobic denitrification by novel isolated strain using NO2--N as nitrogen source, Bioresour. Technol. 102 (2011) 7244-7248. [35] B. Zhao, Q. An, Y.L. He, J.S. Guo, N2O and N2 production during heterotrophic nitrification by Alcaligenes faecalis strain NR, Bioresour. Technol. 116 (2012) 379-385. [36] S. Kai, W. Zhou, H. Zhao, Y. Zhang, Performance of halophilic marine bacteria inocula on nutrient removal from hypersaline wastewater in an intermittently aerated biological filter, Bioresour. Technol. 113 (2012) 280-287. [37] E.W.J.V. Niel, K.J. Braber, L.A. Robertson, J.G. Kuenen, Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis strain TUD, Anton. Leeuw. Int. J. G. 62 (1992) 231-237. [38] Z. Shi, Y. Zhang, J. Zhou, M. Chen, X. Wang, Biological removal of nitrate and ammonium under aerobic atmosphere by Paracoccus versutus LYM, Bioresour. Technol. 148 (2013) 144-148. [39] D. Zhang, X. Huang, W. Li, W. Qin, P. Wang, Characteristics of heterotrophic nitrifying bacterium strain SFA13 isolated from the Songhua River, Ann. Microbiol. (2015) 1-8.
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Fig. 1. Percent relative abundance of abundant groups by high throughput-sequencing.
23
Fig. 2. Phylogenetic tree of microorganisms isolated from FG-06 culture based on 16S rDNA sequences.
24
Fig. 3. Changes in NH4+-N (a) and OD600 (b) under wide C/N range.
25
Fig. 4. Effect of factors on the growth and ammonium removal of the consortium. Initial inoculant dosage (a), ammonia concentration (b), quinoline (c) and nitrobenzene (d). Column symbols indicate percentage of ammonia nitrogen removal; ●, OD600.
26
Fig. 5. Aerobic denitrification ability of the consortium [a- nitrate nitrogen, b- nitrite nitrogen used as sole nitrogen source].
27
Fig. 6. Changes in nitrogen compounds and OD600, COD when ammonia and nitrate nitrogen (a) and ammonia and nitrite nitrogen (b) were added as nitrogen sources under the aerobic condition.
28
Fig. 7. Degradation performance of the consortium in synthetic phenolic wastewater (a) and real coking wastewater (b).
29
Table 1 Composition of the synthetic phenolic wastewater and raw coking wastewater
Phenolic wastewater
Coking wastewater
Items
Levels
Average value
Total COD (mg/L)
2010-2465
2236
NH4+-N (mg/L)
99.55-102.78
101.55
pH
5.90-6.50
6.20
Total COD (mg/L)
2610-2940
2800
NH4+-N (mg/L)
75.78-88.91
83.29
pH
6.10-7.70
6.75
30
S1369-703X(16)30344-8 http://dx.doi.org/doi:10.1016/j.bej.2016.12.008 BEJ 6615
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
31-8-2016 24-11-2016 9-12-2016
Please cite this article as: Ya Yang, Yuxiang Liu, Ting Yang, Yongkang Lv, Characterization of a microbial consortium capable of heterotrophic nitrifying under wide C/N range and its potential application in phenolic and coking wastewater, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2016.12.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of a microbial consortium capable of heterotrophic nitrifying under wide C/N range and its potential application in phenolic and coking wastewater Ya Yang a, Yuxiang Liu a,*, Ting Yang a, Yongkang Lv b a
College of Environmental Science and Engineering, Taiyuan University of Technology, Shanxi
030024, China b Key
Laboratory of Coal Science and Technology of Shanxi Province and Ministry of Education,
Taiyuan University of Technology, Shanxi 030024, China
Yuxiang Liu: * Corresponding author at: Taiyuan University of Technology, #79 Yingze West Street, Taiyuan 030024, Shanxi, China. Tel/Fax: +86 0351 6010014. E-mail address: [email protected].
1
Highlights ●A microbial consortium named FG-06 has excellent heterotrophic nitrifying-aerobic denitrifying ability. ●It had good heterotrophic nitrifying ability under a wide C/N range. ●Simultaneous removal of ammonia nitrogen and COD in phenolic wastewater and coking wastewater were achieved. ●The predominant bacterial groups were identified as Acinetobacter spp. (54.37%) and Pseudomonas spp. (26.52%).
Abstract: Nitrogen pollution has been a serious problem in environmental water particularly in industrial wastewater. A microbial consortium named FG-06 was found to exhibit efficient heterotrophic nitrification and aerobic denitrification ability, with the maximum NH4+-N, NO2--N and NO3--N removal rate of 7.33, 6.53 and 4.53 mg/L/h, respectively. High-throughput sequencing analysis showed that it was mainly made up of Acinetobacter spp. (54.37%) and Pseudomonas spp. (26.52%). The consortium could grow under a wide range of C/N ratio and the NH4+-N removal efficiencies at C/N ratio 4 and 32 were 90.40% and 93.84% separately. Furthermore, simultaneous biodegradation of NH4+-N and COD were achieved by inoculation with enriched consortium in synthetic phenolic wastewater and real coking wastewater samples. Results demonstrated that the consortium had high potential for further practical applications. Keywords: heterotrophic nitrification-aerobic denitrification; C/N; simultaneous removal of NH4+-N and COD; phenolic wastewater; coking wastewater
2
1. Introduction Coking wastewater is a kind of refractory and toxic wastewater. Major pollutants of the wastewater have been reported as ammonia, phenols, nitrogen heterocyclic compounds, and polycyclic aromatic hydrocarbons etc. [1]. Usually phenols are the most abundant toxic compounds and comprise most of the chemical oxygen demand (COD) of the wastewater. What’s more, ammonia nitrogen from insufficiently treated effluent of the industrial wastewater is claimed to be the chief culprit that cause human diseases and eutrophication problems in water bodies. Accordingly, it is an urgency to find an effective way which can reduce the ammonia nitrogen and COD in the wastewater [2]. Biological treatment processes are the most used technology to treat the wastewater because they are more cost effective and environmental friendly than their chemical and/or physical counterparts [3]. According to the traditional ammonium removal theory, the biological processes include nitrification, which oxidizes ammonium to nitrite or nitrate by nitrifiers under aerobic condition, and denitrification, which converts nitrite and nitrate to N2 gas by denitrifiers under anaerobic condition [4, 5]. However, the low rate of nitrification and the different requirements for nitrifiers and denitrifiers make it cost-intensive and time-consuming [6]. Recently, more studies have highlighted the existence of bacteria that are capable of performing heterotrophic nitrification and have a marked ability to denitrify their nitrification products under aerobic conditions. Bacteria of this capability have been isolated from soils and
3
wastewater treatment systems, such as Pseudomonas, Bacillus, Klebsiella, Alcaligenes, Acinetobacter and Paracoccus [7-12]. These special heterotrophic bacteria with higher growth rates than autotrophs can not only nitrify and denitrify simultaneously but also remove nitrogen and carbon pollutants in a single reactor [13]. In addition, the alkalinity generated during denitrification can partly compensate for the acidification caused by nitrification which could lower the cost of pH-adjusting [11]. However, most of the studies were conducted with only one or two bacterial strains, and no attention has been paid to the degradation of ammonium and organics by a microbial consortium. A microbial consortium has more advantages than a single strain in the industrial application, both from the treatment time and the stability of the treatment results. In recent years, several reports have studied the characteristics of a consortium and offered a basis for further application [14, 15]. However, there is currently few reports about a microbial consortium capable of heterotrophic nitrifying. What’s more, the application of heterotrophic nitrification bacteria was mostly for domestic wastewater or micro-polluted water [4, 7, 16], sterile synthetic wastewater was usually used instead of real industrial wastewater [17, 18]. Lower or higher C/N ratios of actual wastewaters may lead to poor treatment efficiencies. Up to now, limited reports are available on a bacterium which could conduct heterotrophic nitrification aerobically under a wide range of C/N ratio. Although some bacteria could survive under lower or higher C/N ratios, the removal efficiencies of ammonia nitrogen were relatively low [11, 17]. In this study, a microbial consortium named FG-06, capable of heterotrophic
4
nitrification and aerobic denitrification under wide C/N range was enriched from coking activated sludge. Ammonium, nitrate and nitrite degradation experiments were conducted under aerobic conditions. The application of the consortium in synthetic phenolic wastewater and real coking wastewater was conducted in order to investigate its potential for use in bioaugmented treatment. The results may provide useful information about heterotrophic nitrification-aerobic denitrification bacteria for bioaugmentation in industrial wastewater treatment or in situ bioremediation at contaminated sites.
2. Materials and Methods 2.1. Chemicals and culture media All of the chemicals used in the experiments were of analytical grade. The consortium was enriched in Luria–Bertani medium (LB) containing (per liter): 10 g tryptone, 10 g beef extract, and 5 g NaCl. Basal medium (BM) used for heterotrophic nitrifying study consisted of the following components (per liter): sodium succinate 3.473 g, (NH4)2SO4 0.472 g, MgSO4·7H2O 0.05 g, K2HPO4 0.2 g, NaCl 0.12 g, MnSO4·4H2O 0.01 g, FeSO4·7H2O 0.01 g. The denitrification medium (DM-1&2) used for nitrate and nitrite reduction studies contained (per liter): 0.72 g KNO3 (0.49 g NaNO2), 4.7 g of sodium succinate, 0.05 g MgSO4·7H2O, 0.2 g K2HPO4, 0.12 g NaCl, 0.01 g MnSO4·4H2O, 0.01 g FeSO4·7H2O. The consortium was inoculated in ammonia-nitrate medium (SNDM-1) and 5
ammonia-nitrite medium (SNDM-2) to study simultaneous nitrification
and
denitrification efficiency contained (per liter): 0.236 g (NH4)2SO4, 0.361 g KNO3 (0.247 g NaNO2), 3.473 g of sodium succinate, 0.05 g MgSO4·7H2O, 0.2 g K2HPO4, 0.12 g NaCl, 0.01 g MnSO4·4H2O, 0.01 g FeSO4·7H2O. Initial pH of all culture media was set at 7.0 and all the culture media were autoclaved for sterility at 121 °C for 20 minutes before use. Conical flasks (250 mL capacity) containing 100 mL medium sealed with sterile breathable membranes were used in the experiments. The other experiment conditions were as follows: culturing temperature 30 °C, shaking speed 120 r/min. 2.2. Enrichment of the consortium The activated sludge was collected from coking wastewater treatment plant in Taiyuan City, Shanxi Province, China in November 2015 and used to enrich the consortium. The activated sludge (20 mL) was inoculated into autoclaved LB medium (200 mL) in an Erlenmeyer flask. The flask was then sealed with sterile breathable membranes and shaken in a rotary shaker at 30 °C and 120 r/min. After a cultivation of 2 days, cell suspension (10 mL) was transferred to 90 mL of sterile LB medium and incubated under the conditions described above for a second enrichment. Thereafter, bacteria suspension (1 mL) was sub-inoculated into another sterile flask containing fresh BM (100 mL) for selective enrichment of ammonium oxidation bacteria. Sodium succinate and (NH4)2SO4 were provided as the sole carbon and nitrogen source. After 20 h of aerobic incubation, the cultures (1 mL) were transferred to another fresh BM (100 6
mL) containing sodium succinate and (NH4)2SO4. This process was repeated three times. The concentration of NH4+-N was measured periodically in each cycle to identify its ability to degrade ammonium nitrogen. Afterwards, a consortium named FG-06 with excellent nitrification ability was picked and individually tested for detailed nitrogen removal capability through shake flask experiments. The consortium was spread in inclined solid medium of coking wastewater for short-term storage and suspended in 25% glycerol solution at -80 °C for long-term storage. 2.3. Microbial consortium analysis 2.3.1. DNA extraction and PCR amplification For genomic DNA extract, the consortium were cultivated overnight and harvested at high bacteria concentration. PCR for high-throughput sequencing was performed with barcode
primers
(Nobar341F
CCTACGGGNGGCWGCAG,
Nobar805R
GACTACHVGGGTATCTAATCC), which targeted the 16S rDNA V3–V4 regions [19]. Barcodes were added at the 5ˊ terminus of forward primers to allow samples multiplexing during sequencing. The total reaction mixture (50 µL) of PCR contained 5 μL of 10× PCR buffer, 2 µL of 10 µM forward and reverse primers, 0.5 μL of 10 mM dNTP mixture, 1.5 U of Taq™ polymerase (Takara Biotech, Dalian, China), 10 ng of genomic DNA, and water (RT-PCR grade, USA) [20]. PCR was performed under the following conditions: denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 30 7
s; and a final extension step at 72 °C for 5 min. Triplicate PCR products were pooled and purified with DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China). Then the purified amplicons were paired-end sequenced on a Miseq platform according to the standard protocols. The two reads were optimized by trimming off the tag sequences and removing the sequences less than 50 bp or with a quality score lower than 20 [21]. During this process, the sequences which contained any errors in barcodes or primers were removed. The number of selected bacterial sequence in the sample was 35,233 after the filtration. 2.3.2. Sequence analysis Operational taxonomic unit (OTU)-based method was employed for describing the enriched consortium. 20,000 sequences from each sample were clustered with 97% similarity and the representative sequence for each OTU was the most abundant one. A total of 13 OTUs were selected and the 16S rDNA sequences of the isolated strains have been deposited in NCBI GenBank under accession number KY054998–KY055010. Then the representative sequences of the most abundant 8 OTUs were compared with available sequences in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) [22]. Phylogenetic reconstructions of the 16S rDNA sequences obtained in this study were performed in MEGA 5.1 software using neighbor-joining (NJ) method with a bootstrap of 1000 [23]. 2.4. Studies on the heterotrophic nitrification characteristics under different conditions
8
Single-factor experiments were conducted for studying the heterotrophic nitrification characteristics of the consortium under different culturing conditions, including C/N ratio, initial inoculant dosage and ammonia nitrogen concentration. In C/N ratio experiments, the initial inoculant dosage was kept in 1% and the content of carbon source was changed in order to adjust C/N ratio to 4, 8, 12, 16, 20, 24, 28 and 32 respectively, by fixing nitrogen concentration at 100 mg/L. The inoculant dosage was setted to 1%, 3%, 5%, 7% and 9% in initial inoculant dosage experiments and the later experiments were all conducted under the optimum conditions from C/N test and initial inoculant dosage test. In ammonia nitrogen experiments, initial ammonia nitrogen concentration was adjusted to 100, 200, 400, 600, 800 mg/L, and sodium succinate content varied accordingly to keep the optimum C/N. To evaluate the influence of refractory contaminants on heterotrophic nitrification of the consortium, ammonia nitrogen degradation in mixed carbon sources was also investigated in batch with 100 mL BM containing 100 mg/L of NH4+-N and 50 mg/L of quinoline or nitrobenzene. 2.5. Assessment of aerobic denitrification and simultaneous nitrification and denitrification ability After 18 h cultivation in BM, cell suspension was inoculated into 100 mL sterile DM-1&2 and SNDM-1&2 in 250 mL Erlenmeyer flasks, and was incubated aerobically about 48 h. During incubation, samples were withdrawn and measured periodically to determine the optical density at 600 nm (OD600) and NH4+-N, NO2--N, NO3--N, NH2OH, total nitrogen (TN) and COD.
9
2.6. Evaluation of the treatment of phenolic and coking wastewater by the consortium In this study, raw coking wastewater was collected from the same coking wastewater treatment plant. Synthetic phenolic wastewater was used as the experimental wastewater which contained phenol, 1000 mg/L; (NH4)2SO4, 100 mg/L; sodium succinate, 2.05 g/L; MgSO4·7H2O, 50 mg/L; K2HPO4, 200 mg/L; NaCl, 120 mg/L; MnSO4·4H2O, 10 mg/L; FeSO4·7H2O, 10 mg/L. The concentrations of NH4+-N and COD in the wastewater were given in Table 1. For degradation in synthetic phenolic wastewater, the harvested consortium was inoculated into the wastewater previously autoclaved at a dosage of 1%. For degradation in real coking wastewater, the consortium was inoculated into the wastewater previously autoclaved and filtered through a 0.45 μm membrane at a dosage of 3%. The wastewater without the consortium inoculated was as a control. Samples were collected periodically to determine the concentration of NH4+-N and COD. 2.7. Analytical methods and calculations All aqueous samples were centrifuged for 10 min at 10000 rpm before being subjected to subsequent analysis. The growth of the consortium was determined by spectrophotometry at 600nm (OD600). Levels of NH4+-N, NO2--N and NO3--N were measured according to the standard methods [24]. NH4+-N was determined by the method of Nessler’s reagent spectrophotometry at a wavelength of 420 nm; NO2--N was determined
using
spectrophotometry;
N-(1-naphthyl)-1, NO3--N
was
2-diaminoethane
determined 10
by
phenol
dihydrochloride disulphonic
acid
spectrophotometry. NH2OH was measured colorimetrically according to Frear and Burrell [25]. TN was detected by the alkaline persulfate oxidation with a UV spectrophotometric method. COD and pH were measured using a COD analyzer (DR 1010, HACH, USA) and a pH meter (PB-10, Sartorious, Germany) separately. Nitrogen, COD and phenol removal efficiency and removal rate were calculated as: per (%) = (C0 – Cn)/C0 × 100 and (C0 – Cn)/t, respectively, where C0 was the initial concentration (mg/L), Cn was the final concentration at the time when sample was collected for analysis and t was the total period of time for the consortium treatment. Each treatment was performed in triplicate and results were presented as means ± SD (standard deviation of means).
3. Results and discussion 3.1. Composition of the microbial consortium To understand the contribution of biological degradation in the treatment of consortium from wastewater, microbial species in the consortium were analyzed by high-throughput sequencing. As shown in Fig. 1, Acinetobacter spp. and Pseudomonas spp. were the most dominant bacterial groups in the consortium. Phylogenetic relationship of the most abundant genera based on 16S rDNA sequences were depicted in Fig. 2. Within the Acinetobacter cluster (54.37% of the OTUs in FG-06 culture), clone FG-066, clone FG-067 and clone FG-068 were affiliated with Acinetobacter spp.. Furthermore, clone FG-061 (26.52% of the OTUs in FG-06 culture) was part of Pseudomonas spp.. Several lineages of the genus Acinetobacter and Pseudomonas have 11
been reported capable of heterotrophic nitrification and aerobic denitrification, including Acinetobacter sp. SYF26 [26], Acinetobacter sp. Y16 [27], Acinetobacter junii YB [18], Pseudomonas alcaligenes AS-1 [28], Pseudomonas tolaasii Y-11 [29], and Pseudomonas stutzeri [4, 8]. Therefore Acinetobacter spp. and Pseudomonas spp., as the most abundant species in the enriched consortium, potentially played the largest role in biological degradation under aerobic condition. Other species were likewise detected in this microbial consortium. Although their percentages were quite low, certain species, such as Aeromonas and Alcaligenes, have been reported to have heterotrophic nitrification and aerobic denitrification ability [10, 30]. 3.2. Heterotrophic nitrification performance under wide C/N range Fig. 3 showed the effect of C/N ratios on utilization of NH4+-N under aerobic condition in batch culture. The NH4+-N removal percentage was not significantly different among C/N 4-32, all of which could reach a level of 90% in 48 h. The highest removal percentage was 99.94% at C/N ratio 16 and the corresponding maximum removal rate of NH4+-N was 7.33 mg/L/h in 12 h. At a C/N ratio of 4, up to 90.40% of the NH4+-N in the medium was removed by the consortium. With the C/N ratios varying from 8 to 32, the removal efficiencies of NH4+-N were remained higher than 93%. The final OD600 was consistent with the removal of NH4+-N, and the final OD600 under C/N ratio 16 was 1.208 which was the highest among them. Most literatures reported the optimal C/N of heterotrophic nitrification-aerobic denitrification bacteria was 6-20 [17, 31]. However, the microbial consortium FG-06 12
could grow under C/N ratio 4 which was relatively lower for several strains [11, 32]. Even if the C/N ratio was as high as 32, which was too high for the growth of autotrophic nitrifying bacteria, the consortium still exhibited satisfying nitrification ability with a NH4+-N removal percentage of 93.84%. The tolerance of the consortium to a wide C/N range expanded its application scope, regardless of the treatment of piggery waste with C/N of 4-7 or other wastewater with high C/N such as municipal landfill leachate and coking wastewater. Take cost effectiveness into consideration, C/N 16 was used in following experiments. 3.3. Ammonium removal under different conditions 3.3.1. Effect of initial inoculant dosage As shown in Fig. 4a, the NH4+-N removal efficiencies from 1% to 9% of the inoculation in 48 h were 99.98%, 99.95%, 99.12%, 97.56% and 96.58% separately, with the highest one at 1%. The final OD600 after batch tests decreased slightly with increased inoculant dosage. High inoculant dosage may affect the degradation characteristics of bacteria due to the excessive metabolic wastes in the culture medium. Taking into account of the stability of reaction and other factors, inoculant dosage 1% was used in following experiments. 3.3.2. Effect of ammonia concentration The effect of initial ammonia concentration on the heterotrophic nitrification capability of the consortium under 48 h was studied over the initial concentration range
13
100-800 mg/L (Fig. 4b). The NH4+-N removal efficiency could reach more than 99% when initial ammonia nitrogen concentration was 100, 200 and 400 mg/L while it was 89.93% with 600 mg/L of initial NH4+-N. It is worth mentioning that the removal efficiency was still about 82.36% under the condition of ammonia nitrogen concentration up to 800 mg/L. Pseudomonas stutzeri YG-24 could survive up to 200 mg/L of NH4+-N but the removal efficiency was only 55.98% [8]. The NH4+-N removal efficiency of Pseudomonas stutzeri T13 was 39.56% when initial NH4+-N was 224.68 mg/L [33]. Above all, the consortium could effectively remove NH4+-N at concentrations ≤ 400 mg/L. 3.3.3. Effect of quinoline and nitrobenzene According to the experimental results obtained using mixed carbon sources containing sodium succinate and quinoline or nitrobenzene, the maximum removal efficiencies of NH4+-N were 98.57% and 99.5% within 48 h, respectively, at an initial NH4+-N concentration of 100 mg/L (Fig. 4c&d). The consortium grew well and the maximum OD600 were 1.186 and 1.225 separately. Compared with NH4+-N degradation in a single carbon source under the optimum C/N condition (Fig. 3a&b), the degradation rate slightly decreased because of the presence of quinoline or nitrobenzene in the mixed carbon sources. In spite of this, the consortium maintained high-efficiency NH4+-N degradation and was tolerant of quinoline and nitrobenzene. The results indicated that the consortium was a promising candidate in engineering practice of coking wastewater treatment.
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3.4. Aerobic denitrification capability of the consortium To evaluate the aerobic denitrification ability of the consortium, two nitrification intermediates, nitrate nitrogen (DM-1) and nitrite nitrogen (DM-2) were used as denitrifying substrates respectively. Fig. 5 showed the changes of various components in a 250-mL flask during the period of nitrogen removal under an aerobic condition. When nitrate was added as the nitrogen source (Fig. 5a), the nitrate concentration decreased significantly with the consortium growing rapidly, as did the TN and COD concentrations. Approximately 50% of the NO3--N was removed in 12 h, which correlated well with the rate of cell growth. Cell growth reached stationary phase in 24 h, as the OD600 increased from 0 to 1.305, 98.2% of the NO3--N and 94.4% of the TN were removed. Between 36 and 48 h, the rate of decrease in NO3--N concentration declined. The same trend was observed for the TN and COD concentration. The accumulation of NO2--N in 24 h reached the maximum amount of 7.53 mg/L. However, the NO2--N concentration decreased subsequently and completely reduced by 48 h. The maximum removal efficiencies of NO3--N, TN and COD by 48 h were 99.64%, 98.53% and 93.60%, separately. The calculated maximum nitrate removal rate in 12 h was as high as 4.53 mg/L/h, which was higher than that of Klebsiella pneumoniae CF-S9 with nitrate removal rate of 2.2 mg/L/h [9] and Rhodococcus sp. CPZ24 with nitrate removal rate of 0.93 mg/L/h [31]. In DM-2 (Fig. 5b), the OD600 increased to 1.32 at 24 h, the maximum removal efficiencies of NO2--N, TN and COD were 98.25%, 98.02% and 95.73%, separately. Meanwhile, no obvious accumulation of nitrate was detected in the medium
15
with nitrite as a nitrogen source. The maximum removal rate of NO2--N in 12 h was 6.53 mg/L/h with the fastest removal rate occurring during exponential phase, which was much higher than that of Pseudomonas sp. yy7 (18.20 mg/L/d) [34] and Bacillus methylotrophicus strain L7 (5.81 mg/L/d) [11]. It was clearly demonstrated that both NO3--N and NO2--N could be utilized by the consortium under aerobic conditions, which suggested the existence of aerobic denitrification capacity in FG-06. The consortium was particularly different from other heterotrophic nitrifying bacteria of A. faecalis NR [35] and A. calcoaceticus HNR [13], in which nitrite and nitrate were both not utilized. 3.5. Heterotrophic nitrification and aerobic denitrification abilities in mixed N-source To further study the nitrogen removal by the consortium under aerobic conditions, simultaneous nitrification and denitrification in mixed N-sources were investigated (Fig. 6). When (NH4)2SO4, KNO3 (SNDM-1) or (NH4)2SO4, NaNO2 (SNDM-2) were used as the nitrogen sources of the consortium, the growth of the consortium was very well and the bacterial biomass peaked at 24 h. The growth trend was similar to the conditions when nitrite nitrogen or nitrate nitrogen was used as the sole N-source. In SNDM-1, NH4+-N concentration decreased dramatically from initial 52.85 mg/L to 1.46 mg/L in 24 h. Approximately 97.23% of NH4+-N was removed, and the NO3--N removal efficiency was 96.84% under 24 h. The maximum removal rate in 12 h was 2.91 NO3--N mg/L/h, which was lower than that of the nitrate as sole nitrogen source. The formation of intermediate product, nitrite started from 0 h and the concentration gradually 16
increased to 18.19 mg/L at 12 h and decreased to zero level by 36 h. In SNDM-2, the consortium was able to remove almost all the NH4+-N by 24 h, and the maximum removal efficiency of NO2--N was 99.49% under 48 h. During the inoculation period, a little NO3--N was detected at 12 h and decreased to zero by 48 h. The patterns of total nitrogen removal were consistent with the removal of nitrogen sources, and the removal efficiencies could reach 97.14% and 96.03% respectively. In addition, the removal efficiencies of COD were all above 97%. In this experiment, the NH4+-N removal rate was high with highest removal rate of 2.66 and 3.15 mg/L/h in SNDM-1&2, respectively. Compared to the maximum removal rate under wide C/N range of heterotrophic nitrification, the ammonium removal rate was significantly decreased. Some studies drew a conclusion that nitrification efficiency would be declined in the presence of nitrite [36, 37], which was similar to the findings in this study that the addition of nitrite and nitrate in BM medium inhibited the removal of ammonium. In addition, NH4+-N and NO3--N, NO2--N were degraded by the consortium simultaneously. This was similar to the phenomenon occurred in Paracoccus versutus LYM [38], but differed from Acinetobacter junii YB [18], whereby NO3--N and NO2--N started to decline gradually after the complete removal of NH4+-N. 3.6. The treatment of phenolic and coking wastewater by the consortium Bioaugmentation is a promising improvement to the biological treatment of wastewater, where the employment of microbial strains enhances the removal of certain organic pollutants. The characteristics of nitrogen removal by heterotrophic 17
nitrification-aerobic denitrification bacteria in shaking flask medium culture conditions have been widely reported [26, 29, 35, 39]. However, limited studies concern the removal of nitrogen and organics by the bacteria in phenolic wastewater and coking wastewater. In the present study, the potential application of the enriched consortium was investigated. The results of NH4+-N degradation and COD removal by the consortium in phenolic wastewater and coking wastewater were shown in Fig. 7. Compared with NH4+-N degradation in the BM, NH4+-N degradation in the phenolic wastewater was delayed. However, more than 99% of NH4+-N and 97% of COD were degraded simultaneously within 60 h, the maximum removal rates of COD and NH4+-N were 47.08 and 5.57 mg/L/h, respectively. The removal of COD in synthetic phenolic wastewater indicated that the consortium could also use phenol as the carbon source and achieve the simultaneous removal of NH4+-N and COD. The consortium was inoculated into real coking wastewater having initial concentration about 83.29 mg/L of NH4+-N and 2800 mg/L of COD. At 24 h there was no obvious change in the concentration of NH4+-N. However, the average concentration was reduced to 64.75 mg/L by 48 h, 33.53 mg/L by 120 h and almost completely removed by 168 h. The degradation rate was much lower than that in the BM, and degradation need more time to complete. This indicated that other refractory contaminants present in the coking wastewater were inhibited to the enriched consortium. Even so, the experiments demonstrated that NH4+-N could still be degraded completely by the consortium, indicating that it preserved its ability for ammonia nitrogen degradation
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under unfavorable circumstances. In addition, more than 80% of COD was removed within 168 h which suggested that the consortium could degrade most organic compounds in the coking wastewater. Therefore, the tolerance to high concentration of organics and the simultaneous removal of NH4+-N and COD demonstrated high potential of the consortium for application in real wastewater treatment. But subsequent experiment should be conducted to discuss the strategies of bioaugmentation and the survival capability of the consortium in activated sludge systems [18].
4. Conclusions A newly enriched consortium named FG-06 has both heterotrophic nitrification and aerobic denitrification abilities. High-throughput sequencing result showed that it was mainly made up of Acinetobacter spp. and Pseudomonas spp.. The tolerance of a wide C/N range made the consortium a wider scope of potential application. It could remove NH4+-N and COD in synthetic phenolic wastewater and real coking wastewater which was a valuable feature for reliable sewage treatment. Therefore, application of the consortium may lead to bioaugmented treatment of industrial wastewater containing high concentrations of NH4+-N and COD.
Acknowledgements This work was supported by the Key research and Development Program of Shanxi Province (grant number 201603D321010), International Cooperation Projects of
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Shanxi Province (grant number 201603D421040), National Key research and Development Program (grant number 2016YFB0600502) and Science and Technology Development of Shanxi Province (grant number 20140321013-04).
References [1] Z. Min, J.H. Tay, Q. Yi, S.G. Xia, Coke plant wastewater treatment by fixed biofilm system for COD and NH3-N removal, Water. Res. 32 (1998) 519-527. [2] J. Wang, X. Quan, L. Wu, Y. Qian, W. Hegemann, Bioaugmentation as a tool to enhance the removal of refractory compound in coke plant wastewater, Process. Biochem. 38 (2002) 777-781. [3] H. Li, H. Cao, Y. Li, Y. Zhang, H. Liu, Innovative biological process for treatment of coking wastewater, Environ. Eng. Sci. 27 (2010) 313-322. [4] J. Zhang, P. Wu, B. Hao, Z. Yu, Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001, Bioresour. Technol. 102 (2011) 9866-9869. [5] Z.Y. Ji, Y.G. Chen, Using sludge fermentation liquid to improve wastewater short-cut nitrification-denitrification and denitrifying phosphorus removal via nitrite, Environ. Sci. Tecnol. 44 (2010) 8957-8963. [6] T. Khin, A.P. Annachhatre, Novel microbial nitrogen removal processes, Biotechnol. Adv. 22 (2004) 519-532. [7] T.L. Huang, S.L. Zhou, H.H. Zhang, N. Zhou, L. Guo, S.Y. Di, Z.Z. Zhou, Nitrogen removal from micro-polluted reservoir water by indigenous aerobic denitrifiers, Int. J. Mol. Sci. 16 (2015) 8008-8026. [8] C. Li, J. Yang, X. Wang, E. Wang, B. Li, R. He, H. Yuan, Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a phosphate accumulating bacterium Pseudomonas stutzeri YG-24, Bioresour. Technol. 182 (2015) 18-25. [9] S.K. Padhi, S. Tripathy, R. Sen, A.S. Mahapatra, S. Mohanty, N.K. Maiti, Characterisation of heterotrophic nitrifying and aerobic denitrifying Klebsiella pneumoniae CF-S9 strain for bioremediation of wastewater, Int. Biodeter. Biodegr. 78 (2013) 67-73. [10] M. Shoda, Y. Ishikawa, Heterotrophic nitrification and aerobic denitrification of high-strength ammonium in anaerobically digested sludge by Alcaligenes faecalis strain No. 4, J. Biosci. Bioeng. 117 (2014) 737-741. [11] Q.L. Zhang, Y. Liu, G.M. Ai, L.L. Miao, H.Y. Zheng, Z.P. Liu, The characteristics of a novel heterotrophic nitrification–aerobic denitrification bacterium, Bacillus methylotrophicus strain L7, Bioresour. Technol. 108 (2012) 35-44. [12] Z. Yu, S. Zhuang, M. Chen, X. Dong, J. Zhou, Evaluation of simultaneous nitrification and denitrification under controlled conditions by an aerobic denitrifier culture, Bioresour. Technol. 175c (2014) 602-605. [13] B. Zhao, Y.L. He, J. Hughes, X.F. Zhang, Heterotrophic nitrogen removal by a newly isolated Acinetobacter calcoaceticus HNR, Bioresour. Technol. 101 (2010) 5194-5200. [14] C. Pan, G.Y.A. Tan, L. Ge, C.L. Chen, J.Y. Wang, Microbial removal of carboxylic acids from
20
1,3-propanediol in glycerol anaerobic digestion effluent by PHAs-producing consortium, Biochem. Eng. J. 112 (2016) 269-276. [15] A. Salmerón-Alcocer, N. Ruiz-Ordaz, C. Juárez-Ramírez, J. Galíndez-Mayer, Continuous biodegradation of single and mixed chlorophenols by a mixed microbial culture constituted by Burkholderia sp., Microbacterium phyllosphaerae, and Candida tropicalis, Biochem. Eng. J. 37 (2007) 201-211. [16] C. Qian, J. Ni, M. Tao, L. Tang, M. Zheng, Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR, Bioresour. Technol. 183 (2015) 25-32. [17] J.M. Duan, H.D. Fang, B. Su, J.F. Chen, J.M. Lin, Characterization of a halophilic heterotrophic nitrification–aerobic denitrification bacterium and its application on treatment of saline wastewater, Bioresour. Technol. 179 (2015) 421-428. [18] L. Yang, Y.-X. Ren, X. Liang, S.-Q. Zhao, J.-p. Wang, Z.-H. Xia, Nitrogen removal characteristics of a heterotrophic nitrifier Acinetobacter junii YB and its potential application for the treatment of high-strength nitrogenous wastewater, Bioresour. Technol. 193 (2015) 227-233. [19] S.E. Dowd, T.R. Callaway, R.D. Wolcott, Y. Sun, T. Mckeehan, R.G. Hagevoort, T.S. Edrington, Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), BMC Microbiol. 8 (2008) 208-213. [20] Y.P. Mao, X. Yu, Z. Tong, Characterization of Thauera -dominated hydrogen-oxidizing autotrophic denitrifying microbial communities by using high-throughput sequencing, Bioresour. Technol. 128C (2012) 703-710. [21] S. Li, D. Li, W. Yan, Cometabolism of methyl tert-butyl ether by a new microbial consortium ERS, Environ. Sci. Pollut. R. 22 (2015) 10196-10205. [22] S. Kumar, M. Nei, J. Dudley, K. Tamura, MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences, Brief. Bioinform. 9 (2008) 299-306. [23] L. MA, B. G, B. NP, C. R, M. PA, M. H, V. F, W. IM, W. A, L. R, T. JD, G. TJ, H. DG, Clustal W and Clustal X version 2.0, Bioinformatics, 23 (2007) 2947-2948. [24] A. Apha, WEF (1998) Standard methods for the examination of water and wastewater, American Public Health Association, Washington, DC, 1998. [25] D. Frear, R. Burrell, Spectrophotometric method for determining hydroxylamine reductase activity in higher plants, Anal. Chem. 27 (1955) 1664-1665. [26] J.F. Su, K. Zhang, T.L. Huang, G. Wen, L. Guo, S.F. Yang, Heterotrophic nitrification and aerobic denitrification at low nutrient conditions by a newly isolated bacterium, Acinetobacter sp. SYF26, Microbiol. 161 (2015). [27] X. Huang, W. Li, D. Zhang, W. Qin, Ammonium removal by a novel oligotrophic Acinetobacter sp. Y16 capable of heterotrophic nitrification–aerobic denitrification at low temperature, Bioresour. Technol. 146 (2013) 44-50. [28] J.J. Su，K.S. Yeh，P.W. Tseng, A strain of Pseudomonas sp. isolated from piggery wastewater treatment systems with heterotrophic nitrification capability in Taiwan, Current Microbiology, 53 (2006) 77-81. [29] T. He, Z. Li, S. Quan, X. Yi, Q. Ye, Heterotrophic nitrification and aerobic denitrification by Pseudomonas tolaasii Y-11 without nitrite accumulation during nitrogen conversion, Bioresour. Technol. 200 (2016) 493-499.
21
[30] M. Chen, W. Wang, F. Ye, X. Zhu, H. Zhou, Z. Tan, X. Li, Impact resistance of different factors on ammonia removal by heterotrophic nitrification–aerobic denitrification bacterium Aeromonas sp. HN-02, Bioresour. Technol. 167C (2014) 456-461. [31] P. Chen, L. Ji, Q.X. Li, Y. Wang, S. Li, T. Ren, L. Wang, Simultaneous heterotrophic nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24, Bioresour. Technol. 116 (2012) 266–270. [32] L. Yu, Y. Wang, H. Liu, C. Xi, L. Song, A novel heterotrophic nitrifying and aerobic denitrifying bacterium, Zobellella taiwanensis DN-7, can remove high-strength ammonium, Appl. Microbiol. Biotechnol. (2016) 1-11. [33] Y. Sun, A. Li, X. Zhang, F. Ma, Regulation of dissolved oxygen from accumulated nitrite during the heterotrophic nitrification and aerobic denitrification of Pseudomonas stutzeri T13, Appl. Microbiol. Biotechnol. 99 (2015) 3243-3248. [34] C. Wan，X. Yang，D.J. Lee，M. Du，F. Wan, C. Chen, Aerobic denitrification by novel isolated strain using NO2--N as nitrogen source, Bioresour. Technol. 102 (2011) 7244-7248. [35] B. Zhao, Q. An, Y.L. He, J.S. Guo, N2O and N2 production during heterotrophic nitrification by Alcaligenes faecalis strain NR, Bioresour. Technol. 116 (2012) 379-385. [36] S. Kai, W. Zhou, H. Zhao, Y. Zhang, Performance of halophilic marine bacteria inocula on nutrient removal from hypersaline wastewater in an intermittently aerated biological filter, Bioresour. Technol. 113 (2012) 280-287. [37] E.W.J.V. Niel, K.J. Braber, L.A. Robertson, J.G. Kuenen, Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis strain TUD, Anton. Leeuw. Int. J. G. 62 (1992) 231-237. [38] Z. Shi, Y. Zhang, J. Zhou, M. Chen, X. Wang, Biological removal of nitrate and ammonium under aerobic atmosphere by Paracoccus versutus LYM, Bioresour. Technol. 148 (2013) 144-148. [39] D. Zhang, X. Huang, W. Li, W. Qin, P. Wang, Characteristics of heterotrophic nitrifying bacterium strain SFA13 isolated from the Songhua River, Ann. Microbiol. (2015) 1-8.
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Fig. 1. Percent relative abundance of abundant groups by high throughput-sequencing.
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Fig. 2. Phylogenetic tree of microorganisms isolated from FG-06 culture based on 16S rDNA sequences.
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Fig. 3. Changes in NH4+-N (a) and OD600 (b) under wide C/N range.
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Fig. 4. Effect of factors on the growth and ammonium removal of the consortium. Initial inoculant dosage (a), ammonia concentration (b), quinoline (c) and nitrobenzene (d). Column symbols indicate percentage of ammonia nitrogen removal; ●, OD600.
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Fig. 5. Aerobic denitrification ability of the consortium [a- nitrate nitrogen, b- nitrite nitrogen used as sole nitrogen source].
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Fig. 6. Changes in nitrogen compounds and OD600, COD when ammonia and nitrate nitrogen (a) and ammonia and nitrite nitrogen (b) were added as nitrogen sources under the aerobic condition.
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Fig. 7. Degradation performance of the consortium in synthetic phenolic wastewater (a) and real coking wastewater (b).
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Table 1 Composition of the synthetic phenolic wastewater and raw coking wastewater
Phenolic wastewater
Coking wastewater
Items
Levels
Average value
Total COD (mg/L)
2010-2465
2236
NH4+-N (mg/L)
99.55-102.78
101.55
pH
5.90-6.50
6.20
Total COD (mg/L)
2610-2940
2800
NH4+-N (mg/L)
75.78-88.91
83.29
pH
6.10-7.70
6.75
30