DSpace at VNU: Nitrogen removal from old landfill leachate with SNAP technology using biofix as a biomass carrier

Nitrogen removal from old land ﬁll leachate with SNAPFaculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho C

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Nitrogen removal from old land ﬁll leachate with SNAP

Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, VietNam

Received 17 June 2015; accepted 4 January 2016

Available online xxx

Single-stage nitrogen removal using Anammox and partial nitritation (SNAP) is a novel technology developed in

recent years for removing nitrogen To evaluate the ability of SNAP technology to remove nitrogen in old landﬁll

leachate under the conditions in Vietnam, we conducted a survey with 7 different nitrogen loading rates of 0.2, 0.4, 0.6,

0.8, 1.0, 1.2, 1.4 kg-N/m3day and a concentration from 100 to 700 mg-N/L The operating conditions were as follows: DO at

1.0e5.3 mg/L, HRT at 12 h, and pH at 7.5e7.8 The biomass carrier was a bioﬁx made from acrylic ﬁber The maximum

ammonium conversion and nitrogen removal efﬁciency were approximately 98% and 85%, respectively, at 1.2 kg-N/

m3day In general, the nitrogen removal efﬁciency increased and stabilized at the end of each loading rate The ﬁrst step

showed that SNAP could potentially be applied in real life for removing nitrogen from old landﬁll leachate

[Key words: SNAP; Anammox; Nitritation; Old landﬁll leachate; Bioﬁx]

Leachate is generated from landﬁlls of municipal solid waste and

is a major concern for the surrounding environment Its negative

impacts include damaging the receiving sources if it has not been

thoroughly treated In particular, the nitrogen concentration in

leachate is relatively high Therefore, the problem of treating

ni-trogen in the leachate is a subject concerning scientists in particular

and each nation in general Conventional nitri

fication/denitrifica-tion technologies are not suited for treating old landfill leachate

with high ammonium nitrogen concentrations and low

biode-gradable organic matter content (1) Traditional technologies

require additional external carbon sources and large energy

con-sumption and so on

Thus, applied research has been conducted on individual or

combined partial nitritation and anammox processes to reduce

operating costs and ensure the nitrogen processing efﬁciency with

a high nitrogen loading rate, such as single reactor system for high

ammonium removal over nitrite (SHARON-Anammox) (2),

completely autotrophic nitrogen removal over nitrite (CANON)(3),

oxygen-limited autotrophic nitriﬁcation-denitriﬁcation (OLAND)

(4), and single-stage nitrogen removal using Anammox and

par-tial nitritation (SNAP)(5) Compared with conventional nitri

ﬁca-tion/denitriﬁcation technologies, partial nitritation/anammox

reduced 85% of the oxygen requirement, 100% of the carbon

requirement and 83% of the bio-solid production(6)

The SNAP process is based on two biotechnologies In theﬁrst,

partial nitritation, ammonium is partly nitriﬁed to nitrite by

ammonia-oxidizing bacteria (AOB) (Eq 1) (5), and then the

resulting nitrite is denitriﬁed with the residual ammonium in the Anammox (Eq.2)(7)

2NHþ4þ1:5O2/1NHþ4þ1NO2þ1H2Oþ 2Hþ (1) 1NHþ4þ1:146NO2/0:986N2þ0:161NO3þ2H2O (2) The overall reaction is described in Eqs.3and5:

1NHþ4 þ 0:85O2/0:44N2þ 0:11NO3 þ 1:43H2Oþ 1:14Hþ (3)

In a few recent studies on SNAP, Lieu et al.(8)performed more research on SNAP technology with a landfill leachate with low ammonium nitrogen Takekawa et al.(9)studied the effects of the operational conditions of the SNAP process Hien et al.(10) evalu-ated the nitrogen removal efficiency from synthesized wastewater with a high nitrogen concentration using SNAP technology Further research is required on SNAP technology for the treatment of real landfill leachate with high ammonium nitrogen concentrations to create a precondition for the application of this technology in practice under conditions in Vietnam

MATERIALS AND METHODS Experimental set-up and operational conditions The SNAP reactor design features a cylinder with a conical bottom made of acrylic resin and a working volume

of 6.5 L Its internal diameter and useful height tank are 150 mm and 420 mm, respectively The schematic diagram of the experiment is shown in Fig 1 The biomass carrier used in this research is bioﬁx, which is made from a hydrophilic net-type acryl resin ﬁber material (NET Co., Ltd., Hyogo, Japan) The characteristics

of this biomass carrier are shown in Table 1 and Fig 2 The bioﬁx used had a weight of 41.2 g and was ﬁxed by cylindrical frames with an external diameter of

92 mm, inner diameter of 62 mm and height of 300 mm.

The reactor operated with uncontrolled temperature and depended entirely on the ambient temperature, which ranged from 27C to 35C Air was fed into the

* Corresponding author Tel./fax: þ44 8 38639682.

E-mail address: ntphong@hcmut.edu.vn (T.P Nguyen).

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reactor through the bottom of the center tube and controlled by an air-controlling

valve such that the dissolved oxygen (DO) did not exceed 1 mg/L The DO

increased gradually with increasing nitrogen loading rate The pH and hydraulic

retention time (HRT) were suitable for SNAP, pH 7.5e7.8 and 12 h, respectively (5)

NaHCO 3 solution (0.5 N) was used to control the pH value through a pH controller.

In addition, to achieve partial nitritation and avoid the production of nitrate

ðNO

3 NÞ, the operation required certain conditions, such as a low dissolved

oxy-gen concentration (DO) (11e13) , high temperature (14) , pH (15) and free ammonia

concentration (FA) (16) The DO was strictly controlled, as mentioned above, with a

measurement frequency of 2 times/day FA was controlled through the adjustment

of the ammonium concentration and pH in the tank During the Tet holiday, the

research was maintained with the operating condition of an NLR of 0.6 kg-N/m 3 day.

Seed sludge and inﬂuent wastewater The anammox sludge and the

acti-vated sludge seeding in the reactor were 6 g/L and 4 g/L, respectively (5) They were

mixed into the SNAP sludge The total seed sludge was 5 g/L (MLSS) The anammox

sludge and activated sludge were obtained from Kumamoto University, Japan, and

the Tan Binh wastewater treatment plant, Vietnam, respectively.

In this study, a synthetic landﬁll leachate simulating pretreated leachate was

used as the inﬂuent for the start-up phase for the SNAP sludge Then, the synthetic

leachate and old leachate were mixed together at different ratios (the ratios of

synthetic leachate and old leachate were two weeks each of 8:2, 6:4, 4:6, and 2:8)

until the old landﬁll leachate was completely replaced at 0.4 kg-N/m 3

day When real landﬁll leachate was used in the experiment, the nitrogen concentration in the

inﬂuent wastewater increased to achieve nitritation and the SNAP process The

composition of the synthetic and old landﬁll leachate are shown in Tables 2 and 3 ,

respectively.

Chemical analysis Standard Methods for the Examination of Water and

Wastewater (USA) were used to analyze ammonium-nitrogen ðNH þ

4 NÞ, nitrite-nitrogen ðNO

2 NÞ and nitrate-nitrogen ðNO

3 NÞ, including NH þ

4 N:

4500-NH 3 -B Preliminary Distillation Step, NO2 N : 4500NO

2 B Colorimetric Method, and NO3 N : 4500NO

3 E Cadmium Reduction Method (17) The total nitrogen was deﬁned by TCVN 6638:2000 (National Technical Regulation Vietnam), Catalytic digestion-devarda alloy (18) The pH was controlled by pH controller BL 931700, HannaeEngland DO was measured using EXTECH 407510eTaiwan.

Polymerase chain reactionePCR Polymerase chain reaction (PCR) analysis was conducted to conﬁrm the existence and identiﬁcation of nitrifying and anam-mox bacteria in the SNAP sludge The entire analytical process was conducted by the

1 Influent

2 NaHCO

3 Influent

4 NaHCO

5 pH cont

6 Air pum

tank

3 solution pump

3 pump roller p

7 Air-co

8 Reac

9 Biom

10 Dis

11 Cen

ntrolling v tor ass carriers charge valve ter resin tub

alve

sludge e

FIG 1 Schematic diagram of experimental apparatus.

TABLE 1 Properties of acryl-ﬁber biomass carrier.

a Provided by the manufacturer FIG 2 Photos of biomass carriers and biomass carrier fixed on steel frames. Please cite this article in press as: Vo, T T., and Nguyen, T P., Nitrogen removal from old landfill leachate with SNAP technology using biofix as a

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Institute of Tropical Biology Analytical procedures including DNA separation, DNA

ampliﬁcation with special oligonucleotide primers, and 16S rDNA sequencing of

bacteria by the Macrogen Company, Korea, and then the retrieved gene sequences

were compared with homologous genes using BLAST with NCBI databases The

special oligonucleotide primers for anammox are Ana-5 0 (5 0

-TAGAGGGGTTTTGAT-TAT-30) and Ana-30(50-GGACTGGATACCGATCGT-30) (19)

RESULTS AND DISCUSSION Variations in pH, DO, and temperature As shown inFig 3, the pH value was controlled in the appropriate range for the SNAP process by an automatic pH controller with the starting pH value of 7.7 (5) The pH greatly affected two species of AOB and anammox bacteria in SNAP because it was the essential factor for determining the free ammonia (FA) and free nitric acid (FNA) concentration in the tank FA and FNA were two inhibitors for the nitriﬁcation process in general and the partial nitritation process

in particular The concentration of FNA was generally lower than the nitriﬁer-inhibited values of 0.2e2.8 mg/L(16) Hence, FA was consulted more because it was also a contributing factor to the inhibition of unwanted nitrite oxidizing bacteria (NOB) in SNAP with an inhibitive range of FA for NOB and AOB were reported to

be 0.08e0.82 mg/L and 10e150 mg/L, respectively (16) The concentrations of FA and FNA corresponding to the nitrogen loading rate are shown inTable 4

DO was the most important parameter of the SNAP process If the DO was too low or too high, it would directly affect the activity

of AOB and anammox bacteria Therefore, the DO must be adjusted

to optimize partial nitritation reaction and not to inhibit anammox reaction simultaneously DO was controlled based on the analytical results of the effluent nitrite and nitrate concentrations The DO increased from 1.0 to 4.8 mg/L with increase in NLR when the mi-croorganisms stabilized and increased the biomass The DO ob-tained in this study was slightly lower than the reported values by Takekawa et al.(9)and Qiao et al.(20) This DO value is higher than those in the studies by Lieu et al.(8)and Hien et al.(10)because the SNAP reactor was operated under a high loading rate using real landfill leachate as influent and used real landfill leachate There-fore, a higher DO supply was necessary to oxidize a number of other components in the landfill leachate

In addition, temperature was also a notable condition during the experiment because it partly contributed to selecting AOB and eliminating NOB However, the temperature was not controlled and

TABLE 2 Composition of synthetic landﬁll leachate.

a Potassium hydrogen phthalate (C 8 H 5 O 4 K).

b Controlling the DO in inﬂuent of synthetic wastewater.

TABLE 3 Eater quality of old leachate from the Go Cat landﬁll.

Adopted from Biec (30)

25 27 29 31 33 35 37

0 1 2 3 4 5 6 7 8 9

0C)

Time (days)

I - 0.2 kg-N/m3.day IV - 0.8 kg-N/m3.day

II - 0.4 kg-N/m3.day V - 1.0 kg-N/m3.day III - 0.6 kg-N/m3.day VI - 1.2 kg-N/m3.day

FIG 3 Changes in pH, DO, and temperature in the reactor.

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entirely depended on the ambient temperature As shown inFig 3,

it was recorded in the range between 27 and 35C during this

experiment This temperature range was very suitable for the

growth of AOB and anammox bacteria(21,22) In addition, it also

restricted NOB bacterial growth because AOB has a higher speciﬁc

growth rate than NOB under temperatures above 25C(23)

Ammonium conversion, nitrogen removal efﬁciency and

efﬂuent nitrate nitrogen As shown in Fig 4, the ammonium

conversion efﬁciency (ACE) increased and stabilized at the end of

each loading rate In the loading rate of 0.2 kg-N/m3day (run I),

ACE rapidly achieved a high efﬁciency of approximately 88.6% on

day 55 In the second loading (run II), ACE slightly decreased and

had the highest value of approximately 71.4% Then, it also

increased in the following loading rate (run III) This could be

because the bacteria gradually stabilized and grew well In the

last three loading rates (run IVeVI), ACE exceeded 90% regularly

at the end of each run The highest ACE was recorded at the end

of the 1.0 kg-N/m3day loading rate, 98.2% on the 250th day

Furthermore, ACE highly decreased at the beginning of each

loading rate because the ammonium nitrogen concentration

increased abruptly and the higher FA concentration inhibited AOB

bacterial activity(15) The inhibition of AOB indicated that partial nitritation had not occurred as a result and the anammox bacteria could not participate in the reaction of SNAP reactor Similar to the ACE results, the nitrogen removal efﬁciency (NRE) decreased strongly at the beginning of each loading rate Consequently, the

FA concentration must be controlled at an appropriate value in the tank via pH The AOB activity recovered in a timely manner and converted ammonium well again Also, high NRE of 83.1%, 83.7% and 85.2% were obtained on the last day in the last three runs Highest and stable NRE with a standard deviation of6.03% was obtained under NLR of 1.2 kg-N/m3 day This suggested favorable and stable microbial growth was obtained in SNAP reactor

Fig 5shows that the average values of ACE and NRE gradually increased stably over the loading rates The average ACEs for six runs were 55.6%, 58.0%, 60.4%, 76.6%, 79.1% and 83.8% Similar to ACE, NRE also gradually increased to 46.4%, 51.8%, 53.6%, 69.6%, 69.1% and 73.5% under the six loading rates At 1.0 kg-N/m3day, the NRE value did not increase and was close to the value of the pre-vious loading rate The reason for that was the withdrawal of sediment which reduced the probability of water circulation and obstructed exposure of the substrates and microorganisms in the tank at 1.0 kg-N/m3day Additionally, under this loading rate ACE increased only slightly compared with the other loading rates This was consistent with the theory that the AOB growth rate was faster than the anammox growth rate; therefore, ACE increased and NRE did not change compared with the 0.8 kg-N/m3day loading rate On the other hand, the standard deviation of ACE and NRE also decreased signiﬁcantly over the loadings This deviation decreased from19.1% and 17.5% to 7.7% and 6.0%, respectively, with ACE and NRE at 1.2 kg-N/m3day This indicates that the SNAP reactor experienced relatively good activity and stability over the time of operation The changes in the average values and standard de-viations of ACE and NRE over the loading rates proved that an

TABLE 4 FA and FNA over loading rates.

Run NLR (kg-N/m 3 day) FA (mg/L) FNA (103mg/L)

0 20 40 60 80 100

0 1 2 3 4 5 6 7

Time (days)

FIG 4 Variations in ammonium conversion and nitrogen removal efﬁciency and efﬂuent nitrate concentration.

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adequate bacterial density and status existed to perform the

con-version process in the tank

Additionally, according toFig 5, the efﬂuent NO3 N

concen-trations increased for theﬁrst three runs and tended to decrease for

the subsequent loading rates In run I, the SNAP reactor used

syn-thetic wastewater with a little organic matter according to the

autotrophic bacteria activity of the nitriﬁer and anammox

Furthermore, the operating conditions were maintained with a DO

content of 1 mg/L This limited the amount of denitriﬁer; thus,

increasing the NO3 N concentration during ﬁrst three run I to III

may have caused (i) the byproduct of the anammox process or (ii)

NOB partially converting nitrite to nitrate

In run I, NO3 N=NHþ

4 removal N ¼ 0:117, which was nearly equal to the reported reaction ratio of SNAP process Then, this

ratio decreased in the subsequent loading rates to 0.09, 0.08, 0.04,

0.02 and 0.01 As mentioned above, the denitrifier was limited in the early period, but the viability of the denitrifier was still possible in the tank During thickening, the biofilm created anoxic conditions for denitrifier activity Therefore, at the three last loading rates, the NO3 N=NHþ4 removal N decreased significantly and showed a reduction of the effluent NO

3 N concentration This observation was similar to the previous SNAP research of Lieu

et al.(5)and Hien et al.(10) Referring to the study of the partial nitritation and anammox process, the nitrate reduction was also noted by Ganigue et al.(24), Wang et al.(25), and Yamagiwa et al

(26) This contributes to increasing the treatment efﬁciency of the SNAP process

Effect of hydraulic retention time After surveying the treatment capabilities of SNAP through 6 different loading rates under the conditions examined in previous studies of SNAP(5,9,10),

we found that the loading changes were based on increasing the

influent ammonium nitrogen concentration The results showed that the nitrogen removal efficiency of the SNAP model was very high Thus, SNAP technology may be applied in practice The research continued to study the more efficient removal of nitrogen with SNAP with higher loading rates through changes in hydraulic retention time (HRT) During the period of the HRT survey, the experimental conditions were maintained at

NHþ4 ¼ 700 3.6 mg/L, reducing HRT from 12 h to 8 h as shown

in Fig 6 The operational time for each HRT stage was 30 days, similar to the time of the previous stages Observations showed that the ammonium conversion and nitrogen removal efﬁciency tended to decrease Speciﬁcally, at HRT of 12 h corresponding to 1.4 kg-N/m3day, ACE and NRE increased and stabilized similarly

to the 6 stages of surveying treatment efﬁciency of the previous SNAP They ranged from 79 to 98% and 70e85%, respectively The

DO was recorded at approximately 5.3 0.2 mg/L, and this value was maintained throughout the HRT survey At the end of this stage, the biomass in the SNAP reactor was measured, requiring a treatment efﬁciency stabilization period of approximately 10 days Then, other HRTs were surveyed

However, after HRT was reduced by 10 h, ACE and NCE changed strongly, from 98% to 60% and from 89% to 52%, respectively

0

10

20

30

40

50

60

70

80

90

100

Stage

II - 0.4 kg-N/m3.day V - 1.0 kg-N/m3.day

III - 0.6 kg-N/m3.day VI - 1.2 kg-N/m3.day

Value (%)

FIG 5 Average ammonium conversion and nitrogen removal efﬁciency and efﬂuent

nitrate concentration.

0 0.5 1 1.5 2 2.5

0 20 40 60 80 100

3.day

Time(days)

HRT = 12 h

FIG 6 Effect of HRT on SNAP treatment performance.

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Afterward, they increased gradually and stabilized in the range of

70 5% and 57 3%, respectively In the last stage of HRT ¼ 10 h, the

processing efﬁciency was partly reduced by the FA concentration

always exceeding the AOB inhibition of bacteria; thus, ACE was low

during this period At HRT¼ 8 h, ACE and NRE clearly decreased

through the dates of operation By the 15th day under the HRT of 8 h,

the ammonium conversion efﬁciency and nitrogen removal

efﬁ-ciency were 44% and 38%, respectively This result is similar to

pre-vious studies by Lieu et al.(5) The concentration of suspended

biomass increased This suggests that the AOB and anammox

mi-croorganisms were seriously inhibited and that the SNAP treatment

capacity was decreased This was because HRT was not sufﬁcient for

the nitritation process to occur completely Thus, residual ammonium

concentration increased highly and FA exceeded the AOB inhibition

threshold constantly Moreover, the pH of landﬁll leachate was

slightly high in the range of 8e8.5, and the ambient temperature was

approximately 31e33C Hence, FA was three times higher than the

normal value and inhibited the nitriﬁer Through this study, the

au-thors noted that the period in which the microorganism system in the

SNAP model was completely inhibited was over 45 days

The treatment capacity of SNAP required over 30 days for

recovering and achieving a stable NRE in the range of 70%e75%

under HRT of 12 h The NRE values were slightly lower than before HRT was changed This was understandable because the anammox biomass was lost in the stage of complete inhibition; it needed more time to grow again In general, the research approach of surveying the capacity of the nitrogen treatment with high loading rates was suitable for increasing the inﬂuent nitrogen concentration On the other hand, the optimal hydraulic retention time for the SNAP pro-cess was still 12 h as the recommendation by Lieu et al.(5) Observation of morphologic sludge Observed through naked eyes as shown inFig 7, the authors found that the SNAP sludge changed gradually from brown-yellow to venetian red over operational stages This indicated that anammox bacteria grew well in the sludge attached on the biomass carrier because

it is the characteristic color of anammox bacteria As mentioned above, the change in nitrogen loading rate depends on the increase in inﬂuent ammonium nitrogen concentration Thus, the leachate was diluted to the proper concentration for the study At initial loadings, the biomass color was observed relatively easily because of the high dilution ratio During the last loadings, color observation of biomass was low and the liquid color was more fuscous in the reactor

FIG 7 Changes in SNAP sludge color over the experimental period (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article).

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In particular, the amount of biomass was relatively excessive and

covered over the wall tank at loading of 1.4 kg-N/m3day Therefore,

could not observe the sludge color or any productive gas bubbles on

the biomass carrier However, at the previous loading of 1.2 kg-N/

m3day, the sludge nuance partly showed stability and the sludge

attachment was active They exhibited good symbiosis together For

example, ACE and NRE were 97% and 85%, respectively, at the NLR

of 1.2 kg-N/m3day Moreover, microbial consortia analyses were

carried out on day 232 with stable MRE under NLR of 1.0 kg-N/

m3day

The result of the PCR shows clear bands approximately 200 base

pairs (bp), and the special primers for anammox are Ana-50 (50

-TAGAGGGGTTTTGATTAT-30) and Ana-30 (50

-GGACTGGA-TACCGATCGT-30) (Fig 8) The result of the sequence analysis

con-ﬁrms the primers of many well-known anammox species, as shown

inTable 5 In further studies, we will progress towards the

identi-fication of AOB, denitrifiers and other autotrophic denitrifiers in the

SNAP reactor

Conclusion The SNAP reactor required a rather long time for

adaptation and stabilization SNAP technology was capable of

removing ammonium nitrogen under high NLR with closely

controlled condition, such as pH 7.5e7.8, an optimal HRT of 12 h,

and increasing DO with increasing nitrogen loading corresponding

to 5.3 mg/L at 1.4 kg-N/m3day The temperature conditions in the

study area were suitable for the efﬁcient performance of SNAP

The maximum ammonium conversion and nitrogen removal

ef-ﬁciency were approximately 98% and 85%, respectively, at 1.2

kg-N/m3 day This study shows that the denitriﬁcation process may

still occur along with the anammox process It contributes to

increasing the processing performance of SNAP In summary, the

ammonium nitrogen treatment ability of SNAP technology has

been veriﬁed, and it is completely applicable to the real treatment

of old landﬁll leachate in practice under the conditions in

Vietnam

ACKNOWLEDGMENT The authors wish to thank the laboratory staff of the Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, for their support throughout this study

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FIG 8 Electrophoresis proﬁle of PCR-ampliﬁed DNA fragments.

TABLE 5 Result of sequence analysis.

Species identiﬁed Similarity (%) NCBI No Reference

Anaerobic ammonium oxidizing

planctomycete KOLL2a

Uncultured anoxic sludge

bacterium KU2

Candidatus Kuenenia stuttgartiensis

(Anoxic bioﬁlm clone Pla1-1)

Uncultured anoxic sludge bacteria

KU1

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