Removal of ibuprofen, ketoprofen, COD and nitrogen compounds from pharmaceutical wastewater using aerobic suspension-sequencing batch reactor (ASSBR)

Separation and Purification Technology 157 (2016) 215–221

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Removal of ibuprofen, ketoprofen, COD and nitrogen compounds from pharmaceutical wastewater using aerobic suspension-sequencing batch reactor (ASSBR) Hassimi Abu Hasan a,b,⇑, Siti Rozaimah Sheikh Abdullah a, Ali Waheid Nakemish Al-Attabi a, Daniah Ali Hassoon Nash a, Nurina Anuar a, Norliza Abd. Rahman a, Harmin Sulistiyaning Titah c a

Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia Research Centre for Sustainable Process Technology (CESPRO), Faculty of Engineering & Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia c Department of Environmental Engineering, Faculty of Civil Engineering and Planning, Institut Teknologi Sepuluh Nopember (ITS), 60111, Keputih, Sukolilo, Surabaya, Indonesia b

a r t i c l e

i n f o

Article history: Received 3 December 2014 Received in revised form 9 November 2015 Accepted 13 November 2015 Available online 14 November 2015 Keywords: Pharmaceutical wastewater Micropollutants Effective microbes Sequencing batch reactor Nitrogen compounds

a b s t r a c t This study was performed to remove emerging contaminants ibuprofen (IBU) and ketoprofen (KETO), COD and nitrogen compounds from pharmaceutical wastewater. The ASSBR was operated under a constant aeration rate at 1.0 L/min, 24 h HRT and various loadings of IBU (1.71–5.1 mg/m3 day), KETO (0.39–2.1 mg/m3 day), COD (1.2–10 kg/m3 day) and ammonia (NH3–N: 4.3–6.3 g/m3 day). The results showed that IBU, KETO, COD, NH3–N and nitrate (NO3–N) were efficiently removed in the range of 63– 90%, 13–92%, 88.7–89.3%, 77.2–96%, 35.7–92.5%, respectively. Isolation and screening of effective microbes found three isolates identified as Bacillus pseudomycoides, Rhodococcus ruber and Vibrio mediterranei, which had a higher toxicity resistance towards IBU and KETO. Thus, the pharmaceutical wastewater especially IBU and KETO could be biologically removed with the presents of valuable effective microbes in the ASSBR system. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Large quantities of pharmaceutical and personal care products are consumed by humans in different aspects of daily life. Pharmaceutically active compounds (PhACs) such as analgesics (ibuprofen, IBU; ketoprofen, KETO; and propoxyphene), anti-depressants (fluoxetine, paroxetine and sertraline), antibiotics (amoxicillin, penicillin and azithromycin) and hormones are detected existing in municipal and natural water systems via residential or commercial discharges. The scope and magnitude of the challenges posed by the pharmaceutical compounds are not known until now due to a shortage of research data. However, several concerns about PhACs might threaten the physiological and reproductive processes of micro- and macro-aquatic organisms [1]. Pharmaceutical compounds have emerged specifically as a main class of micropollutants, given their extend usage and known bio-

⇑ Corresponding author at: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. E-mail addresses: [email protected], [email protected] (H. Abu Hasan), [email protected] (S.R. Sheikh Abdullah). http://dx.doi.org/10.1016/j.seppur.2015.11.017 1383-5866/Ó 2015 Elsevier B.V. All rights reserved.

logical effects. The ubiquitous occurrence of compounds as environmental micropollution is reported in ground water [2], surface water [3,4], and streams [5], as well as in sludge, soil, and sediment samples [6,7]. The adverse effects of the pharmaceutical compounds are still lacking despite their significantly increased levels in the aquatic environment. Although the contaminants are present in low concentration, they will be accumulated when continuously discharged for long periods of time. Some concerns have been raised that these pharmaceutical compounds are increased cancer patients [8], along with abnormal physiological processes and reproductive deterioration [9], increased toxicity of chemical mixtures, and bacteria that are resistant to antibiotics [5]. The previous investigation reported that analgesic compounds such as IBU or KETO have a very high toxicity to bacteria [10]. The IBU was also reported to have toxic effects on invertebrates and algae [11]. Many treatment technologies have been explored, with the intention of finding a suitable polishing technique to further reduce pharmaceutical contents in water. These technologies include conventional activated sludge [12,13], membrane bioreactor [14,15], activated carbon [16], ultraviolet irradiation [17], ozonation [18] and wetland system [19]. A sequencing batch

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reactor (SBR) is a method of treatment which does not require separate tanks for aeration and sedimentation and there is no sludge return. From the engineering aspect, the SBR system is distinguished by the enforcement of controlled short-term unsteady state conditions leading in the long-term to a stable steady state with respect to composition and metabolic properties of the microbial population growing in the reactor. In this study, an ASSBR system was designed specifically to remove IBU and KETO from pharmaceutical wastewater. This system is a combination of activated sludge with the aeration system, creating an aerobic condition and suspending the microbes throughout the reactor. This allows microbes to have more surface area, by which to obtain sufficient oxygen to degrade IBU and KETO. This system requires less electricity and is more economical compared with the other system, as well as membrane bioreactors, which face clogging problems and are more expensive. Moreover, adsorption technology such activated carbon also faces a problem of saturated adsorption capacity, but ASSBR technology could continuously produce natural adsorption matters of microbes. Therefore, the objective of this study was to remove IBU, KETO, COD and nitrogen compounds from pharmaceutical wastewater using the ASSBR system. The ASSBR was operated under four phases as well as different loading rates at a constant aeration rate and hydraulic retention time (HRT). Moreover, the effective microbes involved in the removal were also characterised and identified through microscopic observations, and biochemical and screening tests.

2. Materials and methods 2.1. Bacteria source and pharmaceutical wastewater The mixed culture used as effective microbes in the ASSBR system was obtained from the agricultural industry located in Bandar Baru Bangi, Malaysia. The synthetic pharmaceutical wastewater containing complexed chemicals was prepared using distilled water. The chemical compositions and concentrations are listed in Table 1. The synthetic wastewater was simulated with carbon, nitrogen and sufficient trace elements for microbial growth. The pharmaceutical contaminants containing IBU and KETO were prepared in a range of 2–40 and 0.2–2.0 lg/L, respectively. These ranges are based on contamination levels in the pharmaceutical wastewater [20,21]. The properties of the IBU and KETO are shown in Table 2. These pharmaceuticals were selected as a model because they are persistently used and detected in the water and wastewater environments [5,25,26]. The evaluation of IBU and KETO levels in the sludge treatment plant effluent and surface water also shown that both micro-contaminants were categorised to possess high environmental risk (risk quotient > 1) [27].

Table 1 Compositions of synthetic pharmaceutical wastewater. Chemicals

Parameters

Concentration

Glucose (Merck Germany)

COD

Magnesium (II) sulphate heptahydrate (Merck, Germany) Sodium bicarbonate (Merck, Germany) Mangan(II) chloride tetrahydrate (Merck, Germany) Potassium dihydrogen phosphate (Merck, Germany) Ammonium chloride (Merck, Germany) Ibuprofen (Sigma–Aldrich, USA) Ketoprofen (Sigma–Aldrich, USA)

Mg

1200– 10,000 mg/L 53 mg/L

HCO3 Mn

500 mg/L 0.3 mg/L

PO43

4 mg/L

NH3–N IBU KETO

4.3–6.3 mg/L 1.7–39 lg/L 0.1–2.1 lg/L

2.2. Setup and operation of laboratory ASSBR system A laboratory-scaled ASSBR system was made of Perspex with a height (H) of 30 cm  diameter (D) of 24 cm. A schematic diagram of ASSBR system is depicted in Fig. 1. The SBR system was equipped with DO (Model 5400 GLI, USA) and ORP probes (Model PD1R1 GLI, USA) connected to respective GLI metres (Model 33, USA) for online monitoring. A computer programme developed in Microsoft Visual Basic 6.0 (Version 8176) was used for the online monitoring. The A/D converter interface and data acquisition card (EX92026, Taiwan) connected the personal computer with the DO and ORP metres. The air diffuser was located at the bottom of the ASSBR system to distribute the aeration supply and to suspend the effective microbes throughout the reactor. The ASSBR system was fed daily with 10 L of synthetic pharmaceutical wastewater and was operated for 24 h HRT. For each cycle, the sequence of the ASSBR operation including Fill (0.5 h), React (22 h), Settle (1 h) and Draw (0.5 h) without eliminating the biomass suspension. The aerobic condition was maintained during the reaction period by supplying an aeration of 1 L/min. After the reaction and settling period, samples were collected during the draw period for analysis of COD, IBU, KETO and nitrogen compounds. As summarised in Table 3, the operation of SBR in the treatment of pharmaceutical wastewater was divided into four phases: Phase I (Day 0–18), II (Day 19–62), III (Day 63–100) and IV (Day 101– 135). In Phase I, the mixed culture was initially acclimatised with the environmental conditions by supplying an approximate carbon and nutrient source for bacterial growth. After the acclimatisation phase, the COD loading was increased to 2160 mg/L day (Phase II). IBU and KETO were only loaded into the SBR system during Phase III with loading in the range of 1.71–5.10 and 0.39–1.47 lg/L day, respectively. After day 100 (Phase IV), the loadings of COD, IBU and KETO were increased to 10,000 mg/L day, 1.98–39.33 and 0.10–2.10 lg/L day, respectively. 2.3. Analytical methods 2.3.1. COD, NH3–N and NO3 –N concentrations A water sample was taken during the draw period at the end of each run and collected in 1-L plastic bottles. The bacteria biomass was filtered using a 0.45-lm nitrate cellulose membrane filter (Whatman, USA) according to the standard method [28]. The concentrations of COD, NH3–N and NO3 –N were measured by the reactor digestion method (Method 8000), Nesslerization method (Method 8038) and Cadmium reduction method (Method 8039), respectively, using a HACH DR/2010 (USA) spectrophotometer. 2.3.2. Solid phase extraction and IBU and KETO determination The solid phase extraction (SPE) (Jones Chromatography, USA) was performed using 200 mg/6 mL Oasis HLB extraction cartridges (Waters Corporation, Massachusetts, USA) with the purpose of concentrating the IBU and KETO levels in the water sample. The HLB cartridges were preconditioned with 3 mL of acetonitrile, 3 mL of methanol and 3 mL of deionised water at pH 2. The sample was then percolated through the cartridge at a controlled flow rate using a vacuum manifold. After percolation, the HLB cartridge was rinsed with 10 mL of deionised water and the eluent was discarded. Finally, the analyte was eluted with 4 mL of acetonitrile and was collected in a vial for HPLC measurement. The eluted sample was measured using HPLC (Agilent 1200 Series, USA) for the IBU and KETO determination through a standard curve calibration using Zorbax C8 column (250 mm  4.6 mm, 5 lm) and a UV wavelength set at 230 nm. The mobile phases used were methanol and water in the ratio of 60:40%, with a flow rate of 1.0 L/min.

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H. Abu Hasan et al. / Separation and Purification Technology 157 (2016) 215–221 Table 2 Properties and structural formula of IBU and KETO. Pharmaceuticals

Chemical formula

Ketoprofen

Ibuprofen

Structural formula

pKa

log Kow

Water solubility (%)

References

C16H14O3

4.45

3.12

51

Domínguez et al. [23]

C13H18O2

4.91

3.97

21

Collado et al. [24]

and incubated overnight at 37 °C. The wells of the Biolog plates were inoculated with 100 lL of the bacterial suspensions adjusted to the transmittance range of 90–98%, as recommended by the manufacturer. After incubation for 18–24 h at 30 °C, the development of colour was automatically recorded using a microreader. The identification was performed using Biolog System software of MicroLog 3 (Version 4.20.04, USA). The scanning electron microscopy (SEM) analysis was performed to determine the effect of pharmaceutical compounds (IBU and KETO) on the appearance of the completion method of bacterial cells. SEM analysis was conducted using FESEM Model Supra55VP (Zeiss, Germany). SEM analysis was conducted after 48 h of exposure. Sample preparation was carried out using the method from Kalab et al. [29]. The specific growth rate of strain was calculated using the equation of ln X/X0 = lt, where l = specific growth rate, X0 = is the cell concentration at initial and X = is the cell concentration at time, t.

Control panel

DO

ORP Air pump

AD converter

DO probe

ORP probe Air flow metre Influent

Effluent

Personal computer

2.5. Statistical analysis

Fig. 1. Schematic diagram of lab scale ASSBR system.

The results were statistically analysed using a one-way analysis of variance (ANOVA) with a significant difference of p < 0.05 to determine the significant effect of different loading rates on the removal of IBU and KETO. Statistical calculations were executed with SPSS software for Windows, version 16.0 (SPSS Inc. USA).

2.4. Characterisation and identification of effective microbes Effective microbes were characterised under two conditions, i.e. original mixed culture (OMC: before addition into the ASSBR) and mixed culture sample in the ASSBR (UMC: after exposure to IBU and KETO). About 50 mL of each mixed culture was sampled and agitated to obtain homogeneous suspensions. Approximately 1 mL of the suspensions was added to 9 mL of sterile saline water to make a 10 mL mixture solution, and was gradually diluted from 10 1 to 10 7. Afterwards, 0.1 mL of each dilution sample was spread on Tryptic Soya Agar (TSA) (Difco, USA) plates and incubated in a growth chamber (Ecocell 55, Germany) at 37 °C for 1– 2 days. When the cell colonies had grown on the TSA, a new streaking was prepared on fresh agar using a sterile loop to obtain pure strains. The isolated colonies were characterised based on the morphology properties under microscopic observation (Leica – DM 1000, Singapore) and biochemical testing. Biolog Micro System GEN III commercialised by Biolog Inc. (USA) was used for strain identification. Before inoculation of Biolog GEN III plates (Biolog, Inc., Hayward, CA, USA) containing 95 different carbon sources, the isolates were streaked on fresh TSA

3. Results and discussion 3.1. COD removal in ASSBR The variation of removal and influent–effluent concentrations of COD are shown in Fig. 2. The average removals of COD for all phases were 89.3% (Phase I), 88.4% (Phase II), 89% (Phase III) and 88.7% (Phase IV). As the COD loading increased from 1.2 to 2.2 kg/m3 day (Phase II), the removal immediately decreased from 95.8% (day 18) to 55.6% (day 25) but increased to 90.5% (after day 30). It is because the microbes inside the ASSBR system could not adapted with a high loading due to a sudden increase of COD. After day 30, the ASSBR showed stable COD removal, even the loading was increased to 4700 (Phase III) and 10,000 mg/L day (Phase IV). At a higher COD initial concentration of 12,000 mg/L, Badawy et al. [30] found that the combination of Fenton-biological treatment (FBT) could remove COD up to 92.1% with effluent concentration of 950 mg/L. Although the system performed well in removing

Table 3 The contaminants loading into ASSBR system. Phases

Run (days)

HRT (h)

NH3–N (mg/L day)

NO3–N (mg/L day)

COD (mg/L day)

IBU (lg/L day)

KETO (lg/L day)

I II III IV

0–18 19–62 63–100 101–135

24 24 24 24

4.3–6.3 4.3–6.3 4.3–6.3 4.3–6.3

0.2–3.4 0.2–3.4 0.2–3.4 0.2–3.4

1200 2200 4700 10,000

– – A: 1.71–5.10 B: 2.0–12.9 C: 16.3–27.4 D: 28.9–39.3

– – A: 0.39–1.47 B: 0.10–0.54 C: 0.66–1.12 D: 1.63–2.01

H. Abu Hasan et al. / Separation and Purification Technology 157 (2016) 215–221

80% 70% I

II

III

IV

60% 50% 40% 30% 20% 10%

0% 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

7 6 5 4 3 2 1 0

100% 90% 80% 70% 60% 50% I II III IV 40% 30% 20% 10% 0% 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Removal (%)

(a)

90%

0

Time (Days)

Time (Days) Effluent

Removal

Fig. 2. The variation of influent, effluent and removal of COD.

COD, there was no significant difference of COD removal performance between FBT and ASSBR. Nevertheless, the ASSBR provided a low setup, operation and maintenance cost compared with the Fenton-biological system. Another study by Zupanc et al. [13] found that the COD removal from pharmaceutical wastewater using three different systems, i.e. activated sludge reactor (ASR), moving bed biofilms reactor filled with Kaldnes (K1 and K2) and Mutag biochips carriers were 94.2%, 83.3% and 85.7%, respectively. 3.2. IBU and KETO removal in ASSBR Fig. 3 shows the variation of influent, effluent and removal of IBU and KETO during operation of the ASSBR in Phases III and IV. The pharmaceutical compounds were not loaded into the ASSBR

Fig. 3. The variation of influents, effluents and removals of (a) IBU and (b) KETO.

(b) NO 3-N concentraon (mg/L)

Influent

Influent

6

4 3 2 1 0

0

Removal

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 10 20 30 40 50 60 70 80 90 100 110 120 130 140 I

5

Effluent

II

III

IV

Removal (%)

100%

NH3-N concentraon (mg/L)

11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0

Removal (%)

COD concentration (mg/L)

218

Time (Days) Influent

Effluent

Removal

Fig. 4. The influent, effluent and removal of nitrogen compounds.

during Phase I and II because the microbes were still in the acclimatisation stage during that period. In Phase III, when 1.7– 5.1 lg/L day of IBU and 0.4–1.5 lg/L day of KETO were loaded into the ASSBR, the removals were observed to apparently fluctuate from day 65 until day 100. The average removals of IBU and KETO during Phase III were recorded as 63% (±11%) and 18% (±13%), respectively. In Phase IV, three different loadings of IBU was loaded into, i.e. B: 2.0–12.9 (day 101–115), C: 16.3–27.4 (day 116–127) and D: 28.9–39.3 (day 128–135) lg/L day. Meanwhile, loadings for KETO during this phase were B: 0.1–0.54 (day 101–115), C: 0.66–1.12 (day 116–127) and D: 1.63–2.01 (day 128–135) lg/L day. As the loading increased, the average removal of IBU and KETO also significantly increased (p < 0.05), where the maximum average removals were recorded as 90 (effluent 3.24 lg/L) and 92% (effluent 0.14 lg/L), respectively. Using conventional ASR operating under 48 h HRT, the removal of IBU and KETO were only 86% and 78%, respectively [13]. Verlicchi et al. [19] found in their study that 94% and 59% of IBU and KETO, respectively, could

Fig. 5. The pH, DO and ORP values prolonged the ASSBR operation.

H. Abu Hasan et al. / Separation and Purification Technology 157 (2016) 215–221

Specific growth rate (μ, h-1)

0.06

Control with IBU & KET

0.05 0.04

219

be removed by using the activated sludge system, followed by a horizontal subsurface flow bed of phytoremediation technology using Phragmites australis. 3.3. NH3–N and NO3–N removal in ASSBR

0.03 0.02 0.01 0 Vibrio mediterranei

Rhodococcus ruber

Bacillus pseudomycoides

Species Fig. 6. The specific growth rate of high resistance B. pseudomycoides, V. mediterranei and R. Ruber exposed with IBU and KETO.

(a)

Control (without IBU and KETO)

The influent, effluent and removal of NH3–N and NO3–N are depicted in Figs. 4 and 5, respectively. From the plots, it can be seen that the influent and effluent of NH3–N and NO3–N fluctuated, prolonging the 135 days of operation. The NH3–N removal was in the range of 77.2–96% with effluent quality of 0.2–1.4 mg/L, while, the achieved removal for NO3–N was in the range of 35.7–92.5% with effluent quality of 0.1–1.7 mg/L, showing that in the presence of dissolved oxygen (aerobic condition), the ASSBR could also simultaneously remove NO3–N. By using different systems, as well as a pilot scale integrated membrane-aerated biofilm reactor (MABR), up to 98% of the NH3–N was removed with the effluent quality below 3 mg/L [31]. Meanwhile, Chen et al. [32] found that a combined system of an up-flow anaerobic sludge blanket (UASB),

Exposed with IBU and KETO

(b)

(c)

Fig. 7. SEM observation of highly resistant effective microbes exposed with IB and KT (a) B. pseudomycoides, (b) R. ruber and (c) V. mediterranei.

220

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a micro-aerobic hydrolysis acidification reactor (NHAR), the twostage aerobic process comprised cyclic activated sludge system (CASS) and the biological contact oxidation tank (BCOT) could remove NH3–N to a final concentration of 9.4 mg/L (93.4% removal).

OMC and UMC in which only three strains could germinate in medium containing IBU and KETO. The three isolations were identified as B. pseudomycoides, R. ruber and V. mediterranei.

Acknowledgements 3.4. The pH, DO and ORP monitoring The long-term monitored parameters of pH, DO and ORP during operation of the ASSBR are shown in Fig. 5. From the plots, the effluent values of the pH, DO and ORP at the end of the 24 h HRT were recorded in the ranges of 6.5–8.2, 5.5–7.6 mg/L and 152– 191 mV, respectively. By monitoring the parameters for 24 h (small plot), the DO and ORP were observed to decrease from the outset to 11 h, but both profiles increased until the end of treatment cycle. Meanwhile, there was no significant change observed in the pH profile. The increasing DO showed the complete oxidation of NH3–N and COD, which was related to deterioration in bacterial respiration. Since the ORP is related with the DO logarithm in a linear relationship [33], it increased similarly, as observed on the DO profile. 3.5. Effective microbes communities The suspended biomass representing effective microbes was monitored to be within the range of 3000–3500 mg/L, which prolonged operating the ASSBR system to degrade COD, IBU, KETO and nitrogen compounds. There were five isolated colonies in each OMC and UMC. Through the biochemical test, about 75% were classified as Gram positive and Oxidase negative, while 100% were categorised as catalase positive, indicating aerobic strains. Comparisons between OMC and UMC in terms of colony similarities and biochemical characteristics resulted in six similar strains labelled R, B, R1, S, P and O. By exposing all of the strains into nutrient broth contaminated with IBU (40 lg/L) and KETO (4 lg/L), it was found that only strains B, R and S grew and had a higher resistance to the contaminants. Based on this observation, the strains B, S and R were identified by Biolog Micro System GEN III technology as Bacillus pseudomycoides (B), Vibrio mediterranei (S) and Rhodococcus ruber (R). From Fig. 6, it can be seen that the specific growth rates of the microbes were higher when inoculated in tryptic soy broth contaminated with IBU and KETO because of the existence of an extra carbon source compared to those grown without contaminants. According to Wen et al. [34], the bacterial strain Paracoccus denitrificans could utilise pyridines in pharmaceutical wastewater as its sole source of carbon and nitrogen. However, based on SEM analysis, the cell structures of B. pseudomycoides, V. mediterranei and R. Ruber were found to be slightly damaged due to the toxic affect (Fig. 7). In the future study, all bacteria will be used to enhance the removal of IBU and KETO in pharmaceutical wastewater using the bio-augmentation technique in biological treatment. Wen et al. [34] reported that the bacterial strain of P. denitrificans was added into a membrane bioreactor (MBR) to enhance the treatment of pharmaceutical wastewater. 4. Conclusions The ASSBR was designed specifically to remove IBU, KETO, COD and nitrogen compounds from the pharmaceutical wastewater. By increasing the IBU and KETO loading, it increased the removal of both micropollutants where the maximum removal achieved up to 90% (effluent 3.24 lg/L) and 92% (effluent 0.14 lg/L), respectively. Meanwhile COD, NH3–N and NO3–N were removed efficiently up to 89%, 96% and 92.5%, respectively, at the end of 24 h HRT. Identifications of effective microbes found five strains in each

This research was financially supported by the Ministry of Science, Technology and Innovation, Malaysia (MOSTI) with Grant Number 02-01-02-SF1045 and the Ministry of Education Malaysia (MOE) with Grant Number FRGS/1/2014/TK05/UKM/02/1.

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