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Chemical Engineering Journal

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Greywater treatment using an oxygen-based membrane biofilm reactor:Formation of dynamic multifunctional biofilm for organics and nitrogenremoval

Yun Zhoua,b, Ran Lia,b, Bing Guob, Lei Zhangb, Xin Zoub, Siqing Xiac, Yang Liua,b,⁎

a College of Petroleum Engineering, Xi’an Shiyou University, Xi’an 710065, Shaanxi Province, ChinabUniversity of Alberta, Department of Civil and Environmental Engineering, Edmonton, Alberta T6G 1H9, Canadac State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China

H I G H L I G H T S

• Highly dynamic multifunctional bio-film formed on fiber surfaces of theO2-MBfR.

• The MBfR achieved high and simulta-neous organics and nitrogen removalat HRT of 7.68 h.

• DO variation induced the biofilm for-mation with aerobic, anoxic, andanaerobic layers.

• Nitrification and aerobic denitrifica-tion in aerobic biofilm achieved or-ganics and nitrogen removal.

• Partial nitrification and anaerobic de-nitrification exist in aerobic-anoxic-anaerobic biofilm.

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:GreywaterOxygen-based membrane biofilm reactor (O2-MBfR)Dynamic multifunctional biofilm (DMB)Partial nitrificationAerobic denitrification

A B S T R A C T

A bench-scale oxygen-based membrane biofilm reactor (O2-MBfR) was used to treat greywater for organics andnitrogen removal. Highly dynamic multifunctional biofilm formed on fiber surfaces of the O2-MBfR. With anorganics loading up to 4.26 g COD/m2-day, the MBfR successfully achieved simultaneous organics and nitrogenreduction, with average removal ratios of 95% for total chemical oxygen demand (TCOD), 98% for linear al-kylbenzene sulfonates (LAS), and 99% for inorganic nitrogen (InON). Increasing feed loading rates led to thegradually decrease of dissolved oxygen (DO) concentration from 1.67 to 0.37 mg/L in the reactor, inducing theformation of complex biofilm containing distinct aerobic, aerobic-anoxic, and aerobic-anoxic-anaerobic layers;these all contributed to the simultaneous removal of both organics and nitrogen in MBfR. Mechanisms of or-ganics and nitrogen removal included nitrification and aerobic denitrification in aerobic biofilm, partial ni-trification in the aerobic-anoxic biofilm, and partial nitrification and anaerobic denitrification in the aerobic-

https://doi.org/10.1016/j.cej.2019.123989Received 29 October 2019; Received in revised form 18 December 2019; Accepted 28 December 2019

Abbreviations: MBfR, membrane biofilm reactor; O2-MBfR, oxygen-based membrane biofilm reactor; AOB, ammonia-oxidizing bacteria; NOB, nitrite-oxidizingbacteria; ADB, aerobic denitrifying bacteria; DO, dissolved oxygen; 2D-DMB, two-dimensional dynamic multifunctional biofilm; TSS, total suspended solids; EEM,fluorescence emission-excitation matrix; TCOD, total chemical oxygen demand; SCOD, soluble chemical oxygen demand; PCOD, particulate chemical oxygen de-mand; LAS, linear alkylbenzene sulfonates; TN, total nitrogen; InON, inorganic nitrogen; ON, organic nitrogen; FIX, fluorescence index; BIX, biological index; HIX,humification index; EPS, extracellular polymeric substances; PM, protein-like matters; HAM, humic acid-like matters; DOM, dissolved organic matter; AnDB,anaerobic denitrifying bacteria

⁎ Corresponding author at: Department of Civil and Environmental Engineering, 7-263 Donadeo Innovation Centre for Engineering, University of Alberta,Edmonton, Alberta T6G 1H9, Canada.

E-mail address: yang.liu@ualberta.ca (Y. Liu).

Chemical Engineering Journal 386 (2020) 123989

Available online 30 December 20191385-8947/ © 2020 Elsevier B.V. All rights reserved.

T

anoxic-anaerobic biofilm due to the co-existence of multifarious functional microorganisms in the O2-MBfR. Thisstudy lays the foundation of process optimization and cost-cutting for the practical application of O2-MBfR forgreywater treatment.

1. Introduction

Greywater (GW), which accounts for 50–80% of urban wastewater,typically includes domestic wastewater from bathrooms, hand basins,laundry, and occasionally dishwashers and kitchen sinks, depending onthe greywater collection system design [1–3]. Greywater quality variesdepending on the collection sources, leading to varying levels of con-taminants. Greywater treatment systems should be designed based onthe greywater characteristics, but often need to consider organics, ni-trogen, suspended solids, and especially high surfactant contents [1].

Various systems containing physical, chemical, and biologicaltechnologies have been applied for greywater treatment. However,physical processes are typically limited to post treatment [1], and theeffluent qualities from chemical processes is often insufficient to meetthe non-potable reuse guidelines, especially for high-strength greywater[2]. For biological treatment processes, aerobic processes have beenshown to achieve excellent removal efficiencies for organics, nitrogen,and turbidity [4–7], however, the poor efficiencies for all of them makeanaerobic biological process unsuitable for greywater treatment andrecycling [1,2]. Various membrane and biofilm-related aerobic treat-ment technologies containing membrane bioreactors (MBR) [8–12],membrane-aerated biofilm reactors (MABR) [13], and moving bedbiofilm reactors (MBBR) [14–16] have been employed for GW treat-ment; Table 1 summarizes the greywater treatment performance forsuch systems. Aerobic biofilms achieve organics biodegradation byheterotrophs as well as the oxidation of ammonia to nitrite and nitrateby ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria(NOB) [17,18]. Recently, aerobic denitrifying bacteria (ADB, e.g.,Pseudomonas, Zobellella) have been found to achieve aerobic deni-trification and organics biodegradation [19,20]. Anoxic biofilm havebeen shown to lead to the partial nitrification of ammonia to nitrite byAOB [17]. Anaerobic biofilm can achieve anaerobic denitrification aswell as organics removal [18]. Together, these processes enable the

simultaneous removal of organics and nitrogen. However, for MBRsystems, serious membrane biofouling, which may occur after longerdurations of operation, could increase the GW treatment cost [1].Further, direct gas bubbling can produce large amounts of bubbles andcause the uncontrolled loss of biomass [21]. Aside from the MBR pro-cess, most other biological processes are typically followed by a filtra-tion and disinfection step to meet the non-potable reuse guidelines [2],increasing overall cost and system complexity.

Nowadays, membrane biofilm reactor (MBfR) technology has beenwidely used for wastewater treatment, especially for the removal oforganics and nitrogen when supplying O2 [22] as well as the reductionof oxidized contaminants when H2 [23] or CH4 are supplied [24]. In theoxygen-based MBfR (O2-MBfR), the pressurized membranes supply O2

to the biofilm present on the exterior of the membrane fiber [25].Compared to traditional biological treatment technologies, O2-MBfRcould achieve a high O2 transfer rate, leading to a reduced reactor sizeand providing significant energy savings [23]. Bubbleless aeration inMBfR prevent the stripping of volatile organics, reduce greenhousegases [25], and help to avoid serious foaming problems that typicallyoccur during similar processes when treating GW containing highamounts of surfactants [1]. Moreover, the gradient distribution of O2

reduces the formation of unique microbial community structures on themembrane surface, allowing the simultaneous removal of organics andnitrogen from greywater [25]. Unstable feed loading causes the varia-tion of O2 gradient distribution in the biofilm layers, leading to achange in microbial community and may affect greywater treatmentperformance. The application of O2-MBfR for greywater treatment hasnot been investigated. Moreover, there has yet to be a study focused onfeed loading and how it may contribute to the formation of multi-functional biofilms responsible for organics and nitrogen removal.

In this study, we evaluated the treatment of synthetic greywater bya bench-scale O2-MBfR. We studied how feed loading affects the re-moval of organics and nitrogen, and impacts to the effluent dissolved

Table 1Membrane and biofilm-related greywater aerobic treatment technologies.

Process HRTa (h) CODb (mg/L) LASc (mg/L) TNd (mg/L) Ref

Ine Rrf

(%)Reg

(g/m3-h)In Rr

(%)Re(g/m3-h)

In Rr(%)

Re(g/m3-h)

MBRh 9 109 85 10.3 0.3 97 0.03 15 63 1.06 [8]10 463 86 39.8 6.45 97 0.63 – – – [9]9.15 356 91 35.4 45.8 71 3.55 – – – [10]14.6 466 87 27.8 37 80 2.03 33 40 0.90 [11]11 675 26 16.0 30.8 99 2.77 25 52 1.18 [12]

MABRi 6–24 253 69 29.1 – – – 2.5 60 0.00 [13]MBBRj 24 240 64 6.40 – – – 6.5 79 0.21 [14]

33.6 396 93 11.0 – – – 5 78 0.12 [15]34 413 94 11.4 – – – 13 63 0.24 [16]

MBfRk 7.68 508 95 62.8 158 98 20.2 13 82 1.39 This study

a HRT is hydraulic retention time.b COD is chemical oxygen demand.c LAS is linear alkylbenzene sulfonates.d TN is total nitrogen.e In is the influent of the reactor.f Rr is the removal ratio.g Re is the removal efficiency.h MBR is the membrane bioreactor.i MABR is the membrane-aerated biofilm reactor.j MBBR moving bed biofilm reactor.k MBfR is the membrane biofilm reactor.

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oxygen (DO) concentration in the MBfR. Further, we assessed the rolefeed loading may play in the dynamics of the microbial community inthe biofilm. Our results document how feed loading reduced the for-mation of two-dimensional dynamic multifunctional biofilm (2D-DMB)responsible for the simultaneous removal of nitrogen and organics inthe O2-MBfR. This research lays the foundation for process optimizationand cost-cutting for the practical application of O2-MBfR’s in greywatertreatment.

2. Methods and materials

2.1. Greywater media and inoculated biomass

Greywater was prepared according to NSF/ANSI 350 (2011) [26]with the minor modification of adding 1.5 mL/L primary sludge tomake the total nitrogen concentration similar with the actual greywater[1], and 1.0 M NaOH was added to greywater to adjust the pH to7.1 ± 0.1. Table S1 shows the components and concentrations addedto the greywater influent formulation. The main parameters of theprimary sludge and synthetic greywater are shown in Tables S2 and S3,respectively. As the determined total phosphorus concentration in thegreywater was lower than 1.0 mg/L, thereby the variation of TP con-centration was now determined in this research. Once prepared, thesynthetic greywater was stored at 4 °C in fridge and used within2 weeks. The MBfR was operated at room temperature (21.5 ± 0.3 °C)and inoculated with 5.0-mL of aerobic activated sludge obtained fromthe aeration tank of a wastewater treatment in the City of Edmonton.The inoculated sludge had a total suspend solids (TSS) and volatilesuspended solids of 2.84 ± 0.12 g/L and 2.07 ± 0.15 g/L, respec-tively.

2.2. O2 supply rate of the MBfR

The O2 transfer experiment was carried out in a 1.0-L glass bottle;Fig. S1 depicts the schematic of the measuring device. Prior to datacollection, DO in the distilled (DI) water was removed using pure N2

until the DO concentration declined to 0 mg/L. Fig. 1(a) shows the DOconcentration in DI water at different lumen pressure. A linear re-lationship between DO concentration and pressure was exhibited forDO concentrations lower than 4 mg/L, and the related slope could beused to calculate the O2 transfer rate of the poly(vinylidene fluoride)(PVDF) hollow fibers. Fig. 1(b) presents the linear relationship betweenO2 transfer rate and lumen pressure; the MBfR had an O2 supply rate of86.6 mg O2/m2 membrane surface⋅hr⋅psi at room temperate(21.5 ± 0.3 °C) and ambient pressure (93.0 KPa).

2.3. MBfR setup and operation

Experiments were carried out in an O2-MBfR; refer to the schematicshown in Fig. 2 for system design details. The building materials usedfor the setup of the MBfR consisted of plastic tubes connected with si-licone tubes, plastic fittings, and three-way polycarbonate stopcocks forsampling ports. Table S4 shows the specifications of the membranemodule. For the MBfR system, one plastic tube with the diameter of 1.0-cm had a set of 20 pieces of 18.6-cm length fibers (main bundle), andboth ends were glued into an air-supply manifold to supply O2. Theother plastic tube with the diameter of 0.5-cm had a set of 8 pieces of18.6-cm length fibers (“coupon” bundle for biofilm sampling) and onlyconnected to the air-supply manifold on top end and knotted at theother. Complete mixing was achieved inside the MBfR by using aperistaltic pump (BT100-2 J, LongerPump®, China) with a recirculationrate of 200 mL/min. Another peristaltic pump provided an influent feedrate at 0.015–0.24 mL/min that achieved the HRT at 6–96 h. The ef-fluent tube was connected with a top-sealed 100-mL glass bottle todetermine the effluent DO and pH values. After inoculation, the MBfRwas maintained in an extended batch mode for 5 days to ensure

formation of biofilm. The air lumen pressure to the MBfR was main-tained at 4.0 psi (0.27 atm) throughout the experiments. Other opera-tional conditions were summarized in Table 2.

2.4. Sampling and analytical assays

Liquid samples were collected from the reactor with 15-mL poly-propylene centrifuge tubes (BD Falcon, VWR, USA) for routine analyses.A portion of the samples were filtered immediately through 0.45 μmfilters (25 mm syringe filter, Fisher Scientific, UK) and were used for themeasurement of fluorescence emission-excitation matrix (EEM) spectra.Unfiltered samples were used for the measurement of linear alkylben-zene sulfonates (LAS), TCOD, SCOD, TN, NH4

+-N, NO2−-N, and NO3

−-N.

TCOD and SCOD concentrations were measured according toStandard Methods [27], and the particulate COD (PCOD) was calculatedas the difference between TCOD and SCOD. The concentrations ofNH4

+-N, NO3−-N, NO2

−-N, and TN were measured using a benchtopspectrophotometer (DR 3900, HACH, USA) with the following HACHkits and reagents: NH4

+-N kits, TNT (2–47 mg/L NH3-N, HACH®, USA);NO3

−-N kits, TNT 835 (0.2–13.5 mg/L NO3-N, HACH®, USA); Ni-triVer®2Nitrite Reagent Powder Pillows (2–250 mg/L NO2

−, HACH®,USA); and Nitrogen (Total) kits, TNT 827 (5–40 mg/L N, HACH®, USA).The organic nitrogen (ON) concentration was calculated as the differ-ence of TN and inorganic nitrogen (InoN, containing NH4

+-N, NO3−-N

and NO2−-N). The LAS concentration was determined using the me-

thylene blue spectrophotometric method according to Zhou et al. [28].

Fig. 1. (a) Oxygen supply rate of the PVDF membrane at different lumenpressure, and (b) the leaner relationship between oxygen transfer rate andlumen pressure.

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The pH of the samples was measured using a pH meter (B40PCID, VWR,SympHony), and the lumen pressure of the membrane was measuredwith a pressure meter (GMH 3100 Greisinger, Germany).

The fluorescence spectra of the filtered samples were measuredusing an EEM fluorescence spectrophotometer (Varian Cary Eclipse,Agilent, Australia). Table S5 shows the detailed information of thefluorescence regions in the EEM spectrum. Three fluorescent para-meters, fluorescence index (FIX), biological index (BIX), and humifi-cation index (HIX), were calculated based on the EEM spectrum [29].FIX is the ratio of fluorescence intensities between emission (Em) 450and 500 nm at Ex 370 nm; BIX is the ratio of Em intensity at 380 nmdivided by the Em intensity maximum in 420–435 nm, obtained atexcitation (Ex) 310 nm; and HIX is the area under the Em 435–480 nmdivided by the peak area under the Em 300–3454+35–480 nm, at Ex254 nm [29].

2.5. Loading and flux calculation

Surface loading and removal flux of TCOD (JTCOD, gTCOD/m2-day)and PCOD (JPCOD, gPCOD/m2-day) were calculated according to thefollowing equation:

=× −J Q C C

A( )0

(1)

where C0 is the influent concentrations of TCOD and PCOD (gCOD/L); Cis the effluent concentrations of TCOD and PCOD (gCOD/L); Q is thereactor influent flow rate (L/day); and A is the total membrane surfacearea (m2).

2.6. DNA extraction and microbial community analysis

Biofilm samples were collected from the MBfR on days 1, 40, 80,120, 134, and 190 (labeled as BiofIni, Biof40, Biof80, Biof120, Biof134, andBiof190). Approximately 0.30 g dry weight of biomass was used forgenomic DNA extraction using a DNeasy PowerSoil Kit (Qiagen Inc.,Toronto, Canada) according to the manufacture’s protocol. The DNAsamples were sent for barcoding and 454 pyrosequencing on anIllumina Miseq PE250 platform at McGill University and the GénomeQuébec Innovation Centre (Montréal, Québec, Canada). Raw data wasprocessed using QIIME2 version 2018.8 [30], as explained in detailpreviously [31]. The taxonomy was assigned with 99% similarity usingthe GreenGenes reference database (version 13_5) according to Werneret al. [32].

2.7. Statistical analysis

TCOD, SCOD, NH4+-N, NO3

−-N, NO2−-N, and TN were measured

in triplicate for each sample. Results are expressed as the mean andstandard deviation of the triplicate measurements (mean ± SD).Filtered samples obtained at the noted times were assayed one time forEEM spectra. Statistical analyses were performed using OriginLab 8.1.5software (OriginLab Corp, Northampton, USA) to identify the strengthof the relationship between two given parameters. The Pearson’s cor-relation coefficient, R2, was used to estimate the linear correlation be-tween two parameters. Correlations were considered statistically sig-nificant at a 95% confidence interval (P < 0.05).

3. Results and discussion

3.1. Removal characteristics of organic matter in the MBfR

Fig. 3 shows the TCOD and PCOD concentrations, surface loading,removal ratio, and flux in the steady state at each stage as well as theratio of PCOD/TCOD in the influent and effluent of the O2-MBfR. Withthe HRT higher than 9 h, the average effluent concentrations of TCODand PCOD remained relatively stable at 29.6 and 7.0 mg/L, with re-moval ratios of 94.5 and 97.5%, respectively. The O2-MBfR systemexhibited higher removal efficiency of organic matter when comparedwith other aerobic biological treatment methods (Table 1). Moreover,as the conversion factor of TSS equivalents to PCOD is 1.32 g PCOD/gTSS in activated sludge related biomass [28,33], the effluent TSS inMBfR was approximately 5.3 mg/L at the steady state, meaning that theeffluent concentrations of COD and TSS from O2-MBfR were in com-pliance with the greywater reuse guidelines and standards in the UnitedStates Environmental Protection Agency (USEPA) [34].

The feed loading was increased to 5.53 g TCOD/m2-day by reducing

Fig. 2. Schematic of the bench-scale oxygen-based membrane biofilm reactor(O2-MBfR) used for greywater treatment.

Table 2Operation conditions and effluent qualities for the O2-based MBfR systems during the whole processing time for greywater treatment.

Stage Time (day) HRTa (hr) LASTotalb (mg/L) TN (mg/L) DOc (mg/L)

Influent Rrd (%) Influent Rr (%) Influent Effluent

S1 0–32 96 159 (2)e 98.6 12.5 (0.2) 81.4 0.92 (0.01) 1.67 (0.08)S2 33–62 48 149 (1) 98.4 12.6 (0.5) 80.5 0.87 (0.02) 1.29 (0.02)S3 63–84 24 148 (1) 98.3 12.4 (0.1) 82.1 0.96 (0.01) 0.86 (0.03)S4 85–108 12 158 (1) 98.9 12.1 (0.3) 80.7 0.91 (0.03) 0.61 (0.01)S5 109–126 9 156 (2) 98.8 12.3 (0.2) 80.8 0.93 (0.01) 0.40 (0.04)S6 127–134 6 158 (1) Unstable 12.6 (0.3) Unstable 0.91 (0.02) 0.32 (0.03)S7 135–172 9 159 (3) 98.2 13.1 (0.4) 80.4 0.88 (0.01) 0.49 (0.01)S8 173–200 7.86 158 (1) 96.3 12.8 (0.1) 81.5 0.93 (0.03) 0.37 (0.02)

a HRT is the hydraulic residence time.b LASTotal is total linear alkylbenzene sulfonates (LAS).c DO is the concentration of dissolved oxygen.d Rr is the removal ratio.e Numbers in the parentheses are the standard deviation of the average parameter values.

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the HRT to 6 h in phage 6 (Ph.6), and the removal ratios for TCOD andPCOD dramatically dropped to 82.7 and 91.5%, respectively, within2 days and further decreased to 57.1 and 81.2%, respectively, afteranother 4 days. Additionally, the increased feed loading resulted in adecreased removal flux of TCOD and PCOD from 4.94 and 2.68 g COD/m2-day, respectively, to 2.53 and 2.32 g COD/m2-day, respectively,after totally 6 days operation. High feed loading seemed to con-tinuously inhibit the biodegradation of organics, perhaps by reducingthe activity of the aerobic bacteria and even causing cell lysis due to thehigh surface loading of LAS in the system [35]. Earlier research foundthat LAS with the dosage of 0–0.1 g/g TSS could cause the solubiliza-tion of extracellular polymeric substances (EPS) from activated sludge,but cell lysis and the release of intracellular organics occur by adding ahigher dosage of LAS [28].

By increasing the HRT to 9 h, the removal ratios for TCOD andPCOD rebounded to 92.5 and 96.7%, respectively, and remained at 88.4and 95.4%, respectively, even with the HRT of 7.86 h. The PCOD/TCODratio was 53% in the influent but reduced and remained stable at 22%in the effluent (Fig. 2[c]), indicating the stable organics removal per-formance in the O2-MBfR system.

3.2. Dynamics of dissolved organic matters

Fig. 4 shows the selected EEM fluorescence spectra of soluble or-ganics in the influent and effluent, as well as the fluorescence intensityand indices of the peaks in the steady state at each stage. The EEMfluorescence spectra for all samples are shown in Fig. S2. Comparedwith the non-peak influent, three peaks were found in the dissolvedorganic matter (DOM) within the effluent: Peak A (Ex/Em 250–300/280–380 nm), which is associated with protein-like matters (PM), andPeaks B (Ex/Em 380–420/420–500 nm) and C (Ex/Em 250–300/420–500 nm), which are associated with humic acid-like matters(HAM) [36]. In phases 1–5 (HRT decreased from 4 days to 9 h), theHAM concentration remained relatively stable and much higher thanthat of PM. LAS could improve the solubilization of EPS from biofilm,leading to the increased concentrations of HAM and PM in DOM [28].Moreover, PM are more readily biodegraded compared to HAM[28,37], leading to the relatively low effluent PM concentration. BothHAM and PM continuously increased when the feed loading was in-creased in phase 6, a trend that mirrors the results of previous studies,which found that high doses of surfactants could cause cell lysis andlead to sharp increases in concentrations for HAM and PM [38–40].When reducing feed loading in phase 7 and even increasing that of inphase 8, the peak intensity of HAM quickly rebounded and recovered tothe initial level.

Fig. 3. Concentrations of (a) TCOD and (b) PCOD, as well as the related surfaceloading and removal flux in the stable phase at each stage, and (c) the ratio ofPCOD/TCOD in the influent and effluent of the O2-based MBfR for treatinggreywater. Ph.1, Ph.2, Ph.3, Ph.4 and Ph.5 ≡ days 28, 56, 80, 104 and 122,respectively; Ph.6-1, Ph.6-2, Ph.6-3 and Ph.6-4 ≡ days 130, 132, 133 and 134,respectively; Ph.7 and Ph.8 ≡ days 166 and 190, respectively.

Fig. 4. (a) EEM fluorescence spectra of filtrated samples for reactor influent andeffluent at Ph.6-2, the variations of (b) peaks fluorescence intensity and (c)fluorescence indices in the stabel phase at each stage. Ph.1, Ph.2, Ph.3, Ph.4 andPh.5 ≡ days 28, 56, 80, 104 and 122, respectively; Ph.6-1, Ph.6-2, Ph.6-3 andPh.6-4 ≡ days 130, 132, 133 and 134, respectively; Ph.7 and Ph.8 ≡ days 166and 190, respectively. FIX, BIX and HIX are the fluorescence, biological andhumification index, respectively; Peak A represents protein-like matters, Peak Band C represent humic acid-like matters.

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Fluorescence indices containing FIX, BIX, and HIX were used tocharacterize the DOM. However, by gradually reducing the HRT to 9 h,the FIX sharply rose to 1.96 within 2 days and further increased to 2.13after another 6 days, indicating DOM may be a dominant microbialsource from extra- and intracellular organics release due to the im-proved EPS solubilization as well as cell lysis from the high LAS dose[28,29]. With the influent value of 1.08, the FIX slightly rose to 1.30and then remained stable in other stages. A low FIX is likely related tothe terrestrial DOM sources [41], indicating the stable performance ofthe MBfR for organics removal. Moreover, the BIX gradually increasedfrom 1.30 to 1.72, but the opposite trend was observed for HIX in stage6, a trend that mirrors the results of Zhou et al, showing that high doseof LAS could sharply improve the release of extra- and intracellularorganics [28], leading to the increased biodegradable organics butdropped humification degree of DOM. In other stages, both of the BIXand the HIX basically remained stable at a relatively low value, showingthat effluent DOM mainly contained the autochthonous or freshly-produced organics [29]. The above results make sense in light of thewith TCOD dynamics (Fig. 2[a]), and the MBfR system can be disin-tegrated under the excessive feeding loadings.

3.3. Removal of nitrogen in the MBfR

Fig. 5 shows the concentrations of various nitrogen species, removal

ratios of organic and inorganic nitrogen, and the distribution of variousnitrogen species in the influent and effluent. The concentrations of ef-fluent InoN contained NH4

+-N, NO2−-N, and NO3

−-N and remainedrelatively stable with an average InoN removal ratio of 98.5%throughout the process, indicating the high efficiency of both ni-trification and denitrification; this enabled the nearly complete removalof InoN in the O2-MBfR. The ON had an influent concentration of–3.5 mg/L; Interestingly, the effluent ON concentration was initiallyhigher than the influent concentration, resulting in a negative ON re-moval ratio. LAS contains a long linear hydrocarbon group and formsmicelles to increase the aqueous solubility of extracellular polymericsubstances (EPS), leading to initially increased concentrations of ni-trogen-containing organic matter (e.g., proteins) [28,39,40]. In thisstudy, nitrogen-containing organic matter concentrations graduallydecreased and reached steady state at ~2.0 mg/L with a removal ratioof 44%. In stages 4 and 5, the concentration of effluent ON increasedinitially because solubilization of nitrogen-containing organics fromEPS by LAS [28], which then rebounded quickly and reached steadystate with and an average ON removal ratio of ~43%. With the HRT of6 h, the effluent ON concentration dramatically increased to 13.4 mg/Lwithin 2 days and further rose to 32.3 mg/L after another 8 days, whichmay have resulted from the release of extra- and even intracellularnitrogen-containing organics due to EPS release and cell lysis caused bythe high loadings of LAS [28]. By increasing the HRT to 9 h, the effluent

Fig. 5. Measures of nitrogen removal in the O2-based MBfR during continuous operation; (a) Influent and effluent of various nitrogen species, (b) removal ratio oforganic nitrogen (ON) and inorganic nitrogen (InoN), and (c) the distribution of various nitrogen species in the influent and effluent. The HRT of stages S1-S8 are4 days, 2 days, 1 day, 12 hrs, 9 hrs, 6 hrs, 9 hrs and 7.86 hrs, respectively.

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ON concentration gradually decreased to ~3.0 mg/L and furtherdropped until it reached a steady concentration of 2.5 mg/L with an ONremoval ratio of 29% with the HRT of 7.86 h. In the influent, totalnitrogen mainly consisted of NH4

+-N (~50%), ON (~26%), and NO3−-

N (~22%). However, ON dominated in the effluent, accounting forbetween 92 and 98% of the total nitrogen (Fig. 5[c]), meaning the InoNwas almost completely removed and the non-biodegraded nitrogen-containing organics were the main source of effluent nitrogen. Further,the effluent concentrations of both InoN and ON from the O2-MBfR metthe reuse guidelines in the United States Environmental ProtectionAgency (USEPA) [34].

3.4. Microbial community structure of the biofilm

3.4.1. Functional bacteria at class and family levelsHigh-throughput sequencing of 16S rRNA gene amplicons was used

to identify the microbial community in the O2-MBfR. Fig. 6 shows themicrobial community distribution at the family level and Fig. S3 re-presents the taxonomic classification at the class level. The main de-tectable bacteria in class level were β-Proteobacteria (11–28%), α-Pro-teobacteria (9–41%), γ-Proteobacteria (7–25%), and δ-Proteobacteria(2–10%) (Fig. S2), all of which belong to Proteobacteria and are func-tional for denitrification and the biodegradation of organic compounds

[42]. [Saprospirae] initially had a proportion of 14–20%, however, itdramatically decreased in proportion to 2–3% under excess loadings.Flavobacteriia (0.6–5.0%), Cytophagia (0.3–4.6%), Actinobacteria(0.9–4.8%), and Planctomycetia (0.2–1.3%), which have been reportedas nitrogen removal related bacteria [43], were also detected in the O2-MBfR.

The predominant families (Fig. 6) included Comamonadaceae(5.6–14.9%) and Rhodocyclaceae (1.2–7.5%), which both belong to β-Proteobacteria, and Haliangiaceae (0–2.4%) which belongs to δ-Proteo-bacteria, all of which gradually decreased by increasing feeding loading.Rhodobacteraceae (1.8–3.9%), Hyphomicrobiaceae (0.6–20.1%), Sphin-gomonadaceae (1.6–6.0%), and Caulobacteraceae (0.3–4.7%) belong toβ-Proteobacteria, and Xanthomonadaceae (3.9–16.4%) belongs to γ-Pro-teobacteria, all of which continuously increased in proportion. The fa-milies belonging to Proteobacteria are functional for denitrification andorganics biodegradation [42]. Other predominant families found in theO2-MBfR were Cytophagaceae (0.9–4.5%), which belongs to Cytophagia;Flavobacteriaceae (0–2.2%) and [Weeksellaceae] (0.1–2.2%), which be-long to Flavobacteriia; and Gordoniaceae (0–3.4%), which belongs toActinobacteria, all of these organisms have been confirmed primarilyresponsible for nitrogen removal [43].

Fig. 6. Distribution of microbial community at family level with the samples taken from MBfR system revealed by high-throughput sequencing of 16S rRNA geneamplicons. Bacteria at family level with the relative abundance of over 1% in all samples are shown. BiofIni, Biof40, Biof80, Biof120, Biof134 and Biof190 represent thebiofilm samples in MBfR taken on days 1, 40, 80, 120, 134 and 190, respectively.

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3.4.2. Functional bacteria at genus levelFig. 7 presents the relative abundances of the most abundant mi-

crobial phylotypes in the biofilm at the genes level. The detectablenitritation bacteria contained Nitrosomonas and Sphingomonas, whichare AOB, and Nitrospira, which is NOB [18,44]. Sphingomonas con-tinuously increased in abundance, whereas both Nitrosomonas and Ni-trospira increased initially then declined and even disappeared at ex-cessive feed loading rates. The nitritation bacteria enabled the highoxidation efficiency of ammonia to nitrite and nitrate in the system.ADB were responsible for denitrification and organics removal underaerobic conditions [19,44]; this includes Pseudomonas, Zoogloea, Aci-netobacter, Aeromonas, Comamonas, and Dechloromonas, which werefound in the system, in descending order of total abundances. Pseudo-monas was found to increase in abundance as the feed loading rateincreased. Moreover, anaerobic denitrifying bacteria (AnDB), con-taining Rhodanobacter, Thermomonas, Enterobacter, Caulobacter, andAcidovorax, were detected in the system. The AnDB genus contributedto denitrification and organics removal under anaerobic conditions[18] and continuously increased with the HRT at 4 days–7.86 h withoutcausing cell lysis. Results indicated that the O2-MBfR could achievedenitrification under aerobic and anaerobic conditions due to the en-richment of related ADB and AnDB in the biofilm, and the entirety ofthe detected functional genus enabled the highly efficient removal ofnitrogen within the system. It should be noted that DNA-based analysisas presented in the current study can only demonstrate the geneticpotential of the microbial community developed in the reactor, furtherRNA (or protein) -based studies are needed to further elucidate andconfirm their activities in the O2-MBfR.

3.5. Formation of the dynamic multifunctional biofilm and the removalorganisms of organics and nitrogen in the O2-MBfR

Fig. 8 describes a proposed schematic diagram showing the feedloading to be dependent on the formation of the dynamic two-dimen-sional biofilm and the proposed nitrogen and organics removal path-ways in the two-dimensional biofilm of the O2-MBfR systems. With airas the target gas in the MBfR, O2 diffuses through the membrane anddirectly enters the biofilm attached on the surface of the membrane[45]. This means that biofilm on the membrane has a high DO con-centration, which however gradually dropped as the biofilm thicknessincreased, due to the utilization of O2 by the functional bacterial toaerobically oxidize the target contaminants [23]. In this study, DO inthe influent was stable at 0.91 ± 0.03 mg/L (Table 1), however, theeffluent DO gradually decreased and reached 0.86 mg/L at stage 3,meaning aerobic biofilm was able to form with the HRT of 12 h(Fig. 8[a]). Because of the complete mixing of the culture, effluent andthe bulk solution in the reactor should have the same DO concentra-tions. When the HRT reduced to 9 h, the effluent DO dropped to0.40 mg/L, indicating that the DO concentration in the biofilm-mem-brane and biofilm liquid phase surface should be higher than otherdepths inside the membrane due to the utilization of O2 by the func-tional microorganisms [23]. The following DO concentrations are idealfor aerobic, anoxic, and anaerobic conditions: > 0.5, 0.2–0.5, and<0.2 mg/L, respectively [46], indicating the formation of aerobic-anoxicbiofilm in the O2-MBfR with the HRT between 9 and 12 h. By furtherreduced the HRT to 6 h, the effluent DO further decreased to 0.32 mg/L,which is much lower than the influent DO concentration. The biofilm-liquid phase surface had the higher DO concentration, which graduallydecreased and could format the anoxic layer inside the biofilm in thisstage. This indicates the formation of aerobic-anoxic-anaerobic biofilmwith HRT lower than 9 h (Fig. 8[a]). Moreover, the O2 pressure insidethe membrane fiber should be uniformly distributed because both endsof the reactor were glued into an air-supply manifold, and membranesurface at different lengths should be attached the homogeneous bio-film; changing the feed loading could foster the formation of two-di-mensional dynamic multifunctional biofilm, which could contribute for

the simultaneous removal of organics and nitrogen in the O2-MBfR.Under low feeding loading conditions, organics could be directly

biodegraded by the heterotrophs in the aerobic biofilm, and ammoniacould be oxidized to nitrite and nitrate by AOB and NOB [17,18]. Be-cause of the existence of ADB in this system, e.g., Pseudomonas, Zoo-gloea, Acinetobacter, etc. (Fig. 7[b]), the produced nitrate could be re-duced to nitrogen gas, and the heterotrophs could biodegrade theorganics during the denitrification process [19,20]. The processes in theaerobic biofilm achieved the simultaneous removal of organics andnitrogen in O2-MBfR. For the aerobic-anoxic biofilm, part of ammoniacould be only oxidized to nitrite by the AOB in the anoxic layer duringthe partial nitrification process [17], and the produced nitrite wasfurther oxidized to nitrate and then reduced to N2 in the aerobic layerduring the aerobic denitrification process. This process was also re-sponsible for the removal of organics, and further studies on detailedaerobic denitrification kinetics are needed. Huang et al. [47] alsoconfirmed that denitrification by ADB can be achieved under aerobic

Fig. 7. Distribution of microbial community at genus lever with the samplestaken from MBfR system revealed by high-throughput sequencing of 16S rRNAgene amplicons: (a) nitritation bacterial, (b) aerobic denitrifying bacterial and(c) anaerobic denitrifying bacterial. BiofIni, Biof40, Biof80, Biof120, Biof134 andBiof190 represent the biofilm samples in MBfR taken on days 1, 40, 80, 120, 134and 190, respectively.

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conditions; enabling the coexistence of nitrification and denitrificationin a single system and help to reduce the costs for wastewater treat-ment., The anaerobic layer, which was present in the aerobic-anoxic-anaerobic biofilm under high feed loadings, was beneficial to the re-actor due to the extremely low inside-O2 concentration; such aerobicconditions enabled the removal of organics accompanied with deni-trification [18]. The aerobic-anoxic-anaerobic biofilm increases theperformance of MBfR as it includes nitritation, partial nitrification, andaerobic and anaerobic denitrification processes, enabling the simulta-neous removal of organics and nitrogen. Moreover, increasing feedloading led to a dramatically reduced DO in the biofilm as well as in theeffluent, enabling the formation of multifunctional aerobic-anoxic-anaerobic biofilm functional for the removal of organics and nitrogen,which could help to reduce the cost for the practical application of O2-MBfR in greywater treatment.

4. Conclusions

Two-dimensional dynamic multifunctional biofilm comprisingmultifarious bacteria functional for organics and nitrogen removalformed on membrane surface of the O2-MBfR. Improving feed loadingled to reduced DO concentration at different depths of the biofilm, in-ducing the growth of two-dimensional dynamic multifunctional bio-film. This biofilm contains aerobic, aerobic-anoxic, and aerobic-anoxic-anaerobic layers, all of which could contribute to the simultaneousremoval of organics and nitrogen. With an average organics loading upto 4.26 g COD/m2-day, the MBfR successfully achieved and maintainedthe removal of TCOD, PCOD, LAS, InON, and TN at removal ratios 95%,

97%, 98%, 99% and 82% at steady state, respectively. Organics andnitrogen removal mechanisms include nitrification and aerobic deni-trification in aerobic biofilm, but also partial nitrification in aerobic-anoxic biofilm and partial nitrification and anaerobic denitrification inthe aerobic-anoxic-anaerobic biofilm due to the coexistence of multi-farious functional microorganisms in the O2-MBfR.

Declaration of Competing Interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgements

The authors acknowledge the financial support for this projectprovided by research grants from a Natural Sciences and EngineeringResearch Council of Canada, Canada (NSERC) collaborative researchand development (CRD) project, Strategic Partnership Grants, anNSERC Industrial Research Chair (IRC) Program in Sustainable UrbanWater Development (Liu, Y.) through the support by EPCOR WaterServices, Canada, EPCOR Drainage Operation, Canada, AlbertaInnovates, Canada, and WaterWerx, Canada, and the Canada ResearchChair (CRC) in Future Community Water Services (Liu, Y.).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cej.2019.123989.

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