Improvement of biological nitrogen removal with

Bioresource Technology 219 (2016) 624–631

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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Improvement of biological nitrogen removal with nitrate-dependent Fe (II) oxidation bacterium Aquabacterium parvum B6 in an up-flow bioreactor for wastewater treatment Xiaoxin Zhang a,b, Ang Li a, Ulrich Szewzyk b, Fang Ma a,⇑ a b

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China Department of Environmental Microbiology, Technical University of Berlin, Berlin 10587, Germany

h i g h l i g h t s Strain B6 exhibited nitrate-dependent Fe(II) oxidation metabolism. A continuous up-flow bioreactor inoculated with NDFO strain B6 was established. The bioreactor achieved high nitrogen removal rate under low C/N ratio and HRT. Strain B6 was in relatively dominant position in the anoxic section after 50 days. The existence of B6 could enhance the enrichment of denitrifying Bacillus.

a r t i c l e

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Article history: Received 16 June 2016 Received in revised form 7 August 2016 Accepted 10 August 2016 Available online 12 August 2016 Keywords: Nitrate-dependent Fe(II) oxidation Biological nitrogen removal Microbial community Up-flow reactor

a b s t r a c t Aquabacterium parvum strain B6 exhibited efficient nitrate-dependent Fe(II) oxidation ability using nitrate as an electron acceptor. A continuous up-flow bioreactor that included an aerobic and an anoxic section was constructed, and strain B6 was added to the bioreactor as inocula to explore the application of microbial nitrate-dependent Fe(II) oxidizing (NDFO) efficiency in wastewater treatment. The maximum NRE (anoxic section) and TNRE of 46.9% and 79.7%, respectively, could be obtained at a C/N ratio of 5.3:1 in the influent with HRT of 17. Meanwhile, the taxonomy composition of the reactor was assessed, as well. The NDFO metabolism of strain B6 could be expected because of its relatively dominant position in the anoxic section, whereas potential heterotrophic nitrification and aerobic denitrification developed into the prevailing status in the aerobic section after 50 days of continuous operation. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Nitrate contamination in wastewater effluent is a critical concern emerging in recent years due to its negative effects on human health after being discharged with wastewater and transported into groundwater systems (Kim et al., 2005). Currently, biological denitrification for wastewater is considered a reliable nitrate removal process, in which heterotrophic bacteria utilize the available organic substances as carbon sources and nitrate as a terminal electron acceptor (Ghafari et al., 2008) for its relatively lower cost and generation of fewer byproducts (Liu et al., 2012). Previous researchers have indicated that the addition of external carbon source is indispensable to ensure the COD/NO 3 -N mass ratio remains above 4 for sufficient denitrification (Sun et al., 2012; ⇑ Corresponding author. E-mail address: [email protected] (F. Ma). http://dx.doi.org/10.1016/j.biortech.2016.08.041 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Yuan et al., 2015). Although wastewater effluent contains a certain amount of COD, deficiency of biodegradable COD significantly affects nitrate reduction pathway, as well as the carbon utilization patterns. Thus, as a result, the biological removal rate of nitrogen is impeded. (Bortone, 2009; Zhao et al., 2013; Shen et al., 2015). Nitrate-dependent Fe(II) oxidizing (NDFO) bacteria, including both autotrophic and heterotrophic organisms (Chakraborty et al., 2011; Weber et al., 2009) observed in diverse environments, such as freshwater, paddy soils and marine sediments (Kumaraswamy et al., 2006; Edwards et al., 2003), gained considerable scientific interest in recent years as a potential pathway for biological nitrate reduction. Studies have shown that beside molecular oxygen, nitrate can also serve as an electron acceptor under neutrophilic conditions due to the low redox potentials of iron composites. In previous studies, several Aquabacteria species, such as Aquabacterium strain BrG2 (Buchholz-Cleven et al., 1997) and Aquabacterium fontiphilum sp. nov. (Lin et al., 2009), have been

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X. Zhang et al. / Bioresource Technology 219 (2016) 624–631

mentioned as being capable of NDFO. Moreover, isolates from the sediments in the littoral of Lake Constance, which were closely related to the Aquabacterium cluster, were also able to conduct NDFO (Benz et al., 1998). These researchers emphasized the pure bacterial suspension of NDFO bacteria, focusing its utilization in groundwater treatment. On the other hand, notably few reports have described NDFO isolates that were inoculated in a biological reactor for wastewater treatment. Ru et al. (2015) enhanced the NDFO efficiency of the denitrifying sludge (HDS) for wastewater treatment via bio-augmentation process. Working performance and operation conditions of the NDFO granular sludge using anaerobic sludge bed (UASB) reactor were studied as well (Meng et al., 2014). However, no investigation has been conducted on the performance of bioreactor with NDFO strain and activated sludge inoculated separately in different sections while the microbial community of the nitrogen removal reactors as well as the microbial function of the NDFO in the system remains unavailable as well. To compensate for the inefficient nitrogen removal rate of the current wastewater treatment process produced aroused by the deficiency of carbon sources, as well as to facilitate further applications of the NDFO strains in bioreactor systems, the present study was carried out primarily using Aquabacterium parvum strain B6. A continuous up-flow bioreactor was established to investigate the feasibility of strain B6 in biological wastewater treatment process, the operational performance of the bioreactor was evaluated, while the taxonomic composition and the microbial function of strain B6 in the operational process was assessed as well. 2. Materials and methods

4% (v/v) B6 culture grown on the aerobic modified freshwater medium (MFM) mentioned above (approximately 5 103 cells/mL). All cultures were incubated at 28 °C in the dark without shaking. Heat-inactivated biomass (121 °C, 20 min) was used as a control. 2.3. Bio-reactor experiment A polymethyl methacrylate (PMMA) column bioreactor with an inner diameter of 15 cm and a height of 45 cm was established for the experiments, including an anoxic section (height: 15 cm) and an aerobic section (height: 30 cm). The effective capacity of the bioreactor was 7.5 L with a hydraulic retention time (HRT) of 17 h and an inflow quantity of around 0.007 L/min. The schematic of the bioreactor is demonstrated in Fig. 1. The porous plate which connected the two sections was also PMMA with regularly arranged pores (diameter: 3 mm). An aeration plate was settled on the bottom of the aerobic section with an aeration rate of 4 L/ min (0.8 vvm). On the bottom of the reactor, the internal rotor and a magnetic stirrer with a speed of 100 r/min ensured thorough mixing of the anoxic section. The average DO in the aerobic and anoxic sections was kept at approximately 3.0 mg/L and 1.0 mg/L, respectively. Polyurethane carriers (30 mm 30 mm 5 mm) attached with a pre-cultured bacterial suspension of B6 (approximately 4 105 cells/mL) and activated sludge (initialsludge concentration of 3.0 g TSS/L) obtained from aeration tank of Taiping Municipal Wastewater Treatment Plant (Harbin, China) were inoculated into the anoxic section and aerobic section, respectively. The packing rate of Polyurethane carriers was 30%, and specific surface area was1120 m2/m3. 2.4. Synthetic wastewater

2.1. Bacterial strains and cultivation conditions Aquabacterium parvum strain B6 (DSM 11986, GenBank No. AF035052), characterized approximately two decades ago, is a Gram-negative, rod-shaped b-proteobacterium isolated from the Berlin drinking water system (Kalmbach et al., 1999). Strain B6 was grown in modified R2A medium by replacing starch with 0.1% (v/v) Tween 80. The temperature range for the growth of strain B6 was 14–34 °C. Growth occurred between pH 6.5 and 10.0 (Schmidt et al., 2014). 2.2. Cultivation experiment An oxygen-free, modified, non-reduced bicarbonate/CO2buffered freshwater mineral medium under a N2 headspace was used in cultivation studies. Modified freshwater medium (MFM) containing 0.15 g CaCl2, 0.52 g KCl, 0.41 g MgCl2, 0.02 g KH2PO4, 0.27 g NH4Cl, 1 ml of SL9 trace minerals (Table 1), 1 ml of vitamin B12 solution, 0.5 ml of mixed-vitamin solution and 1 g of yeast extract per liter was mixed. Next, 1 mM NO-3 as electron acceptor and 1 mM ferrous iron from an anoxic 1 M Fe(II)-NTA stock solution were added after cooling the basic mineral medium to ensure adequate substrate concentration for NDFO reaction. Growth experiments were performed in serum bottles closed with butyl rubber stoppers. A total of 17 serum bottles (10 mL) were washed with 1 M HCl and distilled water prior to sterilization by autoclaving. 5 mL of filtered medium was added anoxically into each bottle (headspace N2). Bottles were inoculated with a fresh

Synthetic wastewater (SWW) was utilized as the influent for the column reactor experiments in this work. The influent was supplied continuously from the bottom of the reactor by a peristaltic pump. The SWW was prepared based on data of the composition of sewage in Northern China. Unless otherwise specified, the inlet components indicating: CH3COONa and glucose (270 mg/L COD), NH4Cl (40 mg/L NH4+-N), KH2PO4 (10 mg/L PO3 FeCl2 4 -P), (3.0 mg/L Fe2+), CaCl2 (23 mg/L Ca2+), MgSO4 (8 mg/L Mg2+) and 1 ml of SL9 trace elements. The final pH of the SWW was adjusted to 7.2 with NaOH or HCl solution. During the experiment, the reactor was operated in continuous mode with a recirculation ratio of 50%. 2.5. Analytical methods For analysis of total Fe(II) and Fe(III), 1-mL samples were taken at different times under sterile and anoxic conditions (Kappler and Newman, 2004) and analyzed using phenanthroline as the colorimetric reagent (Klueglein et al., 2015). Fe(II) was determined directly after the dissolution of a sample (1 mL) taken from the culture fluid in 1 M HCl (9 mL). Total Fe was determined by reducing an aliquot of the sample with hydroxylamine hydrochloride before the addition of the o-phenanthroline reagent. Fe(III) was calculated by subtracting the amount of Fe(II) from the amount of total Fe. The orange phenanthroline-Fe(II) complex was quantified using a spectrophotometer at 510 nm (Nanodrop 2000c, Thermo Scientific, USA). Three parallel measurements were performed per sample. A calibration line with 0, 10, 25, 50,100, 250, and 500 mM ferrous

Table 1 The SL 9 trace element composition. Composition Concentration (g/L)

FeCl24H2O 2.00

H3BO3 0.01

ZnCl2 0.07

NiCl26H2O 0.02

MnCl42H2O 0.08

CoCl26H2O 0.19

Na2MoO42H2O 0.04

NTA 12.80

626


Pump Effluent

Aerobic Outlet Carrier

Aerobic Section

Pump

Aeration Plate

Air

Recycling Porous Plate

Anoxic Outlet

Anaerobic Section

Carrier Rotor Sludge Discharge

Magnetic Stirrer

Influent Fig. 1. Schematic diagram of up-flow bioreactor.

iron (FeCl24H2O) was used for calculations. For soluble CODCr, NH+4-N, NO 2 -N, and NO3 -N determination, sample water was filtered with 0.45 lm membrane before analysis with standard methods (American Public Health Association (APHA), 1998). For microscopic quantification of cell growth, a hemocytometer and ImageJ software were combined to semi-automatically count fluorescently labeled cells. Next, 1-mL samples were sterilely taken from iron-nitrate grown cultures. After dissolving the particulate Fe(III) using 0.5 mM oxalic acid, cells were stained with 4,6diamidino-2-phenylindole (DAPI) (final concentration of 10 lg/ mL). 10 lL of stained cells were added to Hemocytometer and counted from 10 random large squares of 1 mm 1 mm 0.1 mm (0.1 lL) each using a fluorescence microscope (Axioplan 2, objective 40, Zeiss Germany). According to the protocols of Fero, 2004, the cell density was calculated as

Cell density ðcells=mLÞ ¼ Average Cell Count ðcells=large squareÞ

0.8 lL of each primer (5 lM), 0.4 lL of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s instructions and quantified using QuantiFluorTM –ST (Promega, U.S.). Purified amplicons were pooled in equimolar and paired-end sequenced (2 250) on an Illumina MiSeq platform according to the standard protocols. The sequences were analyzed by the Quantitative Insights into Microbial Ecology (QIIME version 1.17) and Operational Units (OTUs) were clustered with 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier (http:// rdp.cme.msu.edu/) against the silva (SSU115)16S rRNA database using confidence threshold of 70% (Amato et al., 2013).

10; 000 ðlarge squares=mLÞ d:f:ðdilution factor; if anyÞ 3. Results and discussion 2.6. Microbial diversity analysis

3.1. Anaerobic cultivation of strain B6

In order to comprehensively analyze the microbial communities during the operation, activated sludge and Polyurethane carrier samples, collected from aerobic section and anoxic section for Illumina MiSeq sequencing and marked as A2 and An respectively, were taken on Day 50 when the stable removal performance was achieved. Seed sludge sample marked as A1 was taken on the initial day of operation as well. Microbial DNA was extracted from the samples by E.Z.N.A.Ò Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s protocols. The V4–V5 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 °C for 3 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s and a final extension at 72 °C for 10 min) using primers 338F 50 -barcode-AC TCCTACGGGAGGCAGCAG and 806R 50 -GGACTACHVGGGTWTC TAAT-3, where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 lL mixture containing 4 lL of 5 FastPfu Buffer, 2 lL of 2.5 mM dNTPs,

Strain B6 was cultured in anoxic MFM for the evaluation of Fe (II) oxidation and nitrate reduction and organic substrate degradation in batch cultures. Batch culture experiments with strain B6 in anoxic MFM showed that strain B6 was able to oxidize Fe(II) and reduce nitrate simultaneously under anoxic circumstances. As shown in Fig. 2(a), after the initial lag phase of 5 days, a simultaneous decline of Fe(II) and nitrate was observed, while Fe(III) and the number of living cells in the iron-nitrate grown cultures began to gradually accumulate. Nitrate reduction was clearly initiated with the dramatic decrease of Fe(II) from 49.42 mg/L to 39.16 mg/L, and the NO 3 -N consumption from 60.01 mg/L to 48.27 mg/L during the following 10 days (Fig. 2a). Organic substrate in the batch culture was also consumed, decreasing from 247.92 mg/L to 217.98 mg/L. In this process, nitrite accumulation was observed from day 10 to day 20, while the concentration of NO 2 -N was kept at a low level for the remainder of the period, suggesting that the nitrate was converted to N2O or N2. Meanwhile, culture yields began to increase

627


260

300

240

250

55 50 45

220

40 200

35 30 25

180

Fe(II) Fe(III) Nitrate COD Nitrite

20 15 10 5 0

0

5

10

15

20

160

No. of Cells (103/mL)

60

Concentration of COD (mg/L)

Concentration of Fe and Nitrogen (mg/L)

65

200

150

100

Cell 50

140 25

0

0

5

10

15

20

25

Time (d)

Time (d)

(a)

(b)

Fig. 2. Characterization of nitrate-dependent iron-oxidizing bacteria B6. (a)Transformations of NO 3 , NO2 , Fe(II), Fe(III) and organic compounds by Aquabacterium parvum B6 in batch culture using SFM media. (b) Culture growth of Aquabacterium parvum B6 in batch culture using SFM (yeast extract) media. The data are presented as the mean ± the standard deviations (n = 3). When not shown, error bars are smaller than the symbol size.

steadily and rose from 4.22 104cell/mL (around 2.03 108mg/L) to 2.11 105cell/mL (around 1.06 107mg/L) on day 15 (Fig. 2b). Theoretically, the stoichiometric denitrification reaction is

5C þ 2H2 O þ 4NO3 ! 2N2 þ 4OH þ 5CO2

ð1Þ

indicating that besides a certain amount of COD for cell yields, 1 mg NO 3 -N hypothetically demands 2.86 mg biodegradable COD to accomplish the denitrification process. Previous research results noted that the hypothetical, calculated optimal C/N ratio is 3.74, without competition from other heterotrophs (Chiu and Chung, 2003). An increased C/N ratio (higher than 6) could benefit the bacterial growth via ensuring adequate carbon sources, thus increasing nitrogen removal efficiency (Su et al., 2015; Zhou et al., 2015). In the present study, the concentration of NO 3 -N varied from 60.62 mg/L to 49.20 mg/L coupled with the drop in concentration of COD from 247.92 mg/L to 217.98 mg/L during the period from day 5 to day 15. Specifically, the COD/ NO 3 -N ratio was approximately 2.63:1 for the denitrification of strain B6, which is lower than that of the conventional denitrification process. From day 15 to day 25, the nitrate reduction and Fe(II) oxidation in the batch culture were decelerated comparing with the previous 10 days and the cell counts decreased slightly from 2.73 105 to 2.53 105 during the last 5 days of the batch culture due to the occurrence of cell encrustation. Many of the reported NDFO bacteria, such as Acidovorax sp. and Aquabacterium sp., are known as organotrophic nitrate reducers that require the presence of organic substrates, e.g., acetate for efficient Fe(II) oxidation (Picardal, 2012). In addition, Weber et al.(2009) reported that the NDFO strain 2002 is also capable of utilizing a variety of organic substrate as the carbon and energy source with nitrate as the terminal electron acceptor which indicating that both organic compound and Fe(II) can provide energy source for the denitrification process of NDFO strains. Therefore in the present study COD was utilized for heterotrophic biomass growth as well as for the denitrification process of stain B6. 3.2. Bioreduction of nitrogen and Fe(II) oxidation in an up-flow bioreactor An up-flow bio-reactor was established and batch experiments were carried out for the evaluation of nitrogen removal and Fe(II) oxidation in the process. The concentrations of ammonia-

nitrogen, nitrate, nitrite, Fe and organic co-substrate were monitored to verify the occurrence of biological removal of nitrogen and Fe(II) oxidation beginning at the start-up of the bioreactor (Fig. 3). The constant inlet concentration demonstrated in 2.4 was adapted in Fig. 3 to have a better assessment of the overall removal rate of the substrate. Experimental results indicated that an up-flow bioreactor inoculated with strain B6 could achieve nitrate reduction coupled with Fe(II) concentration. The concentration of NH+4-N in the aerobic section effluent (Aerobic Outlet) decreased from 42.90 mg/l and reached approximately 7.41 mg/L by the end of the experiment, with fluctuation during the whole process due to the recirculation, while the NH+4-N concentration in effluent in the anoxic section from Anoxic Outlet was kept approximately around the 40 mg/L. The increase of nitrite concentration from day 1 to day 11 indicated the occur rence of nitrosation, then the concentration of NO 3 -N and NO2 -N in aerobic section (Aerobic Outlet) decreased continuously and stayed at approximately 8.78 mg/L and 3.38 mg/L, respectively, while the NH+4-N decreased to approximately 7.41 mg/L by the end of the experiment (Fig. 3b). However, the concentration of NO 3 -N and NO2 -N in the anoxic section was constantly lower than that in the aerobic section. According to Fig. 3(c), NO 3 -N in the anoxic section (Anoxic Outlet) increased slightly before declining with fluctuations and finally remained at a consistently low level (approximately 2.19 mg/L) during the last 15 days of the experiment due to enzymatic activity of B6. The accumulation of nitrite in the anoxic section was observed during the initial experimental phase before it dropped steadily and was maintained at around 1 mg/L until the end of experiment. This suggests that nitrite might be further converted to N2O, N2 or NH+4. These results are very similar to those obtained in previous studies by Li et al. (2015) of the Fe(II)-oxidizing Citrobacter freundii strain PXL1. The overall ammonia nitrogen removal rate of the bioreactor and the nitrate removal rate of the anoxic section achieved 82.7% and 46.6% (approximately 8.64 g NO /m2support d) 3 -N respectively. The decline of the Fe(II) concentration that occurred in both sections indicates the onset of iron oxidation (Fig. 3c). The concentration of Fe(II) in the anoxic section (Anoxic Outlet) was detected at 3.08 mg/L, initially, and was reduced biologically to approximately 1.50 mg/L on day 49, while in the aerobic section (Aerobic Outlet),

628


55

Nitrate and Nitrite Concentration (mg/L)

35

50

NH+4-N Concentration(mg/L)

45 40 35 30 25 20 15

Aerobic Outlet Anoxic Outlet Influent

10 5 0

0

10

20

30

40

Aerobic Outlet-Nitrite Anoxic Outlet-Nitrite Aerobic Outlet-Nitrate Anoxic Outlet-Nitrate

30 25 20 15 10 5 0 0

50

10

20

(a)

40

50

(b)


4

3

2

1


800

COD Concentration (mg/L)

5

Fe(II) Concentration (mg/L)

30

Time (d)

Time (d)

600

400

200

0

0 0

10

20

30

40

50

Time (d)

(c)

0

10

20

30

40

50

Time (d)

(d)

Fig. 3. Biological removal of nitrogen and iron in an up-flow reactor. (a)Transformations of NH+4, (b) nitrate and nitrite, (C) Fe(II) and (d) organic compounds in the effluent of aerobic section (Outlet 1) and anoxic section (Outlet 2).

the Fe(II) concentration dropped from 2.94 mg/L to 0.74 mg/L due to the aeration. The concentration of COD declined to 64.66 mg/L and 106.96 mg/L in the aerobic and anoxic sections, respectively, by the end of the test. The existence of the heterotrophic microorganisms, such as Bacillus and Enterococcus in anoxic section and Massilia and Sphingobacterium in aerobic section, enhanced the COD degradation of the bioreactor (Fig. 4b). Putting Fig. 3(b and c) together, the NO 3 -N removal was corelated with the Fe2+ removal in anoxic section. During the first 2+ 15 days, both NO decreased with fluctuation while the 3 -N and Fe COD concentration remained at high level (Fig. 3d) indicating that the NDFO process of inoculated strain B6 was predominant during this period of time. After that, the variation of taxonomy composition took place, both the NO 3 -N and Fe (II) concentration increased evidently from Day 15–20 since the NDFO of strain B6 was significantly influenced by microbial community variation. After day 20, the community structure of the bioreactor gradually reached the balance with the enrichment of heterotrophic denitrifying Bacillus microorganisms (Fig. 4). The NO 3 -N and COD concentration decreased synchronously. The relationship between nitrate, Fe(II)

and COD indicated that the extra addition of strain B6 during reactor operation was essential to ensure the NDFO activity of the anoxic section. To assess the biological nitrogen removal efficiency of the upflow bioreactor, the values of C/N ratio, HRT, nitrate removal efficiency (NRE) and total nitrogen removal efficiency (TNRE) attained during the last 10 days of the operation were compared with other related biotechnological systems in Table 2 According to Table 2, the maximum TNRE of 79.7% and NRE of 46.6% were obtained with a C/N ratio of 5.3:1 in the influent which indicating the feasibility as well as the efficiency of the bioreactor inoculated with B6. In comparison with the UASB reactor (No. 2 in Table 2), the up-flow bioreactor was capable of achieving higher treatment efficiency of NDFO. Comparing to previously examined low C/N ratio bioreactors for wastewater treatment (No. 3 and No. 4 in Table 2), the up-flow bioreactor showed a promising nitrogen performance with a relatively lower HRT. This finding can be attributed to the presence of the acclimated biomass of strain B6, which utilize Fe(II) as an electron donor to accomplish the denitrification process. Meanwhile, in contrast with bioreactor systems

629


100

Relative abundance (%)

90 80 70 60 50 40 30 20 10 0

A1

Others SHA-109 Candidate_division_WS6

A2

Sample

An

Candidate Saccharibacteria Acidobacteria Deinococcus-Thermus Chloroflexi Actinobacteria Bacteroidetes Firmicutes

Chlorobi Proteobacteria

(a)Phyla level distribution of the sludge samples

(b) Genus level distribution of the sludge samples Fig. 4. Taxonomic distribution of sludge samples from an up-flow bioreactor. (a) Phyla level distribution of the sludge samples, (b) Genus level distribution of the sludge samples. A1 represents the sample at the initial day from the aerobic section of the bioreactor, whereas A2 and An were samples taken from the aerobic and anoxic sections, respectively, after 50 days of continuous operation.

containing pure bacterial suspension of the NDFO isolates (No. 5 and No. 6 in Table 2), the up-flow bioreactor also demonstrated its capacity of nitrogen removal under a lower C/N ratio and HRT. Interestingly, numerous studies have indicated that cell encrustation could be observed as the C/N ratio increased during

the microbial NDFO process (Dippon et al. 2012). The precipitates, adhering to the surface of cells, might limit nutrient and substrate uptake or constrain cell mobility (Kappler and Straub 2005), inhibiting the denitrification process. Many of the reported NDFO bacteria, such as Acidovorax sp. and Aquabacterium sp., are reported

630


Table 2 Comparison of the performance of up-flow bioreactor in nitrogen removal with the literature. Bioreactor

Carbon Source

C/N ratio

HRT (h)

NRE (%)

TNRE (%)

Reference

Up-flow bioreactor UASB reactor

Synthetic wastewater Synthetic wastewater

5.3

17

46.6

79.7

—

18

45.6

45.6

Sequencing batch reactor

Domestic wastewater

2.1– 5.3

14.6

—

77.0

Sequencing batch reactor

Rural wastewater

3.5

24

50.0

53.8

Anaerobic upflow bioreactor Anaerobic denitrification reactor

Synthetic wastewater Groundwater

13.1

24

66.1

75.5

6.8

48

90.0

97.0

Present study Meng et al. (2014) Wang et al. (2015) Bernat et al. (2003) Zhou et al. (2015) Su et al. (2015)

to grow only mixotrophically. These strains required the presence of organic substrates, e.g., acetate for efficient Fe (II) oxidation (Picardal, 2012). It has also been reported that the concentration of co-substrates influence (and maybe even control) the mineralogy of the Fe (III) minerals produced by Fe (II)-oxidizing microorganisms (Kappler and Straub, 2005). Therefore, C/N ratio can influence cell encrustation by affecting the formation of iron precipitant. In this study, the comparatively low C/N ratio of the bioreactor could also impede cell encrustation; thus, biological the denitrification was ensured. Therefore, the present study clearly indicates that an up-flow bioreactor with the enzymatic activity of B6 present in the anoxic section is an efficient biological improvement for wastewaters containing various compounds of nitrogen.

3.3. Taxonomy composition of the sludge sample in the reactor Fig. 4(a) shows the relative abundance of microbial communities in the samples at the phylum level. In general, the two communities from the aerobic section demonstrate a parallel abundance on the phylum level. Proteobacteria, Firmicutes, Actinobacteria and Chloroflexi exhibited a high similarity in sample A1 and A2 while Candidatus Saccharibacteria, Acidobacteria and Chlorobi only presented in sample A1. The selective enrichment of the phyla in sample A2 might result from the specified operational conditions of the bioreactor. The results are in accordance with a previous study reported by Zhang et al. (2011), who investigated the abundance in active sludge in the municipal WWTP. Compared with the samples from the aerobic section, sample An from the anoxic section of the bio-reactor exhibited a difference in the abundance on a phylum level. Notably, the relative abundances of Proteobacteria and Bacteroidetes were considerably lower in sample An than that in sample A1 and A2. Firmicutes was predominant in the anoxic section, while Actinobacteria and Chloroflexi also showed higher abundance in contrast to the aerobic samples. A smaller fraction of the members of Candidate, Deinococcus-Thermus, Chlorobi, Acidobacteria, and Candidatus Saccharibacteria also presented. This implies that the existence of strain B6 and the anoxic condition could significantly influence the microbial community in the bioreactor. As demonstrated in the genus level distribution (Fig. 4b), Aquabacterium, accounting for 8.06%, was the third most present microbe in sample An (from the anoxic section of the bioreactor), indicating that the microbial function of strain B6 was ensured after an operational period of 50 days. Additionally, potentially facultative anaerobic bacteria, such as Bacillus (22.58%), Enterococcus

(8.24%), Lactococcus (7.41%), and Anaerolineaceae (1.30%), also served as dominant communities due to the absence of DO in the anoxic section. Conversely, comparison with initial sample A1 found that Massilia was predominant in A2, accounting for 23.20% after 50 days of operation. The members of Sphingobacterium, Chryseobacterium, Pseudochrobactrum and Bacillus followed behind with 20.34%, 12.08%, 7.75% and 7.14%, respectively, throughout the sample. According to previous studies by Bailey et al. (2013), Massilia appears to be beneficial for biofilm adhesion and contains a complete dissimilatory nitrate reduction pathway. Recent reports have also indicated that strains from the Sphingobacterium, Chryseobacterium, and Bacillus genera in sample A2 are capable of heterotrophic nitrification and aerobic denitrification (Zhou et al., 2014; Kundu et al., 2014;), while Pseudochrobactrum strains were isolated and characterized for its high metal-reducing ability (Long et al., 2013). Interestingly, the coexistence of Brevundimonas, Acinetobacter and Flavobacterium which account for 3.58%, 1.36% and 1.35%, respectively, of the total in sample A2, was highly similar to the experimental results of Li et al. (2014) who indicated that the system could be recommended for the treatment of groundwater containing high levels of arsenic, iron, nitrogen and sulfur. Remarkably, the presence of Comamonadaceae_unclassified in samples A1, A2 and An, which accounted for 1.18%, 0.71% and 1.33%, respectively, of the total readings, implied the highly possible existence of a nitrate-dependent Fe(II)-oxidizing bacteria in the sludge sample because Comamonadaceae contains various currently known NDFOs such as Acidovorax, Aquabacterium and Ideonella. Therefore, it could be an indication that a certain amount of NDFO bacteria also exists in the system, although their functions of Fe(II) oxidation and nitrate consumption might be diminutive. The up-flow bioreactor ensured the diversity of microbial communities in both sections. The NDFO metabolism by strain B6 could be expected because it has a relatively dominant position in the anoxic section. In addition, the operational condition and the existence of B6 in anoxic section could enhance the enrichment of denitrifying Bacillus and other heterotrophic microorganisms which could also benefit the nitrate removal and COD degradation of the bioreactor. Meanwhile, heterotrophic nitrification and aerobic denitrification metabolisms developed into a dominant status, while the autotrophic bacteria, such as Flavobacterium, suffering less restriction due to comparatively low C/N ratio and DO consumption, were also able to predominate in the aerobic section. Thus, the up-flow reactor was promising for the biological application of nitrogen, Fe (II) oxidation and organic compound removal . 4. Conclusions In this study, the performance and microbial community makeup of an up-flow bioreactor treating synthetic wastewater were investigated. A maximum NRE and TNRE of 46.9% and 79.7%, respectively, could be obtained at a low C/N ratio (5.3:1) and HRT (17 h). The NDFO metabolism by strain B6 could be expected because it occupies a relatively dominant position in the anoxic section, whereas potential heterotrophic nitrification and aerobic denitrification metabolisms developed as the prevailing status in the aerobic section after 50 days of operation. Thus, the up-flow reactor is promising for biological applications of nitrogen, Fe(II) oxidation and organic compound removal. Acknowledgements This work was supported by a doctoral scholarship from China Scholarship Council (CSC) and National Natural Science Foundation of China (NSFC-51478140).


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