80

CHAPTER 3

MATERIALS AND METHODS

3.1 OVERVIEW

This chapter describes the methodology adopted to enrich the

ANAMMOX bacteria in batch studies, the startup and stabilization of

ANAMMOX process in AnMBR, and the nitrogen removal performance of

AnMBR treating simulated and actual landfill leachate. The enrichment

studies and bench scale AnMBR study were done in the Research Laboratory

at Centre for Environmental Studies (CES), Anna University, Chennai. The

development of CANON process in a laboratory scale MBBR was conducted

at the Department of Earth and Environmental Engineering, Columbia

University, New York. An overview of the methodology is schematically

depicted in Figure 3.1. The scope of the study included

i. enrichment of ANAMMOX bacteria using seeds of aerobic

and anaerobic origin in batch reactor systems.

ii. development and stabilization of ANAMMOX and CANON

process in AnMBR and MBBR and evaluation of nitrogen

removal performance by ANAMMOX and CANON process

in AnMBR and MBBR under varying Nitrogen Loading Rates

(NLRs) and Hydraulic Retention Time (HRT).

iii. identification of the development of AOB, NOB and

ANAMMOX growth, and concentration in MBBR (both

qualitative and quantitative).

The experimental setup, sampling techniques, methodologies and

the analytical techniques used for monitoring are detailed in this chapter.

81

Figure 3.1 Overview of the research methodology

Identification and Analysis of microbial

ecology of AOB, NOB and ANAMMOX

growth and concentration in MBBR

ANAMMOX activity

confirmation by N2H4, NH2OH

and qPCR, DGGE/Sequencing

Enrichment

mode

Operation

Variable

Anaerobic seed from

Vegetable waste digester

Anaerobic seed from

Biosolids digester

Aerobic seed from

Activated Sludge

Seed :

Enrichment

medium ratio

Fed-batch

ANAMMOX activity

confirmation by N2H4,

NH2OH and SEM

Variable

Biological Variable

(NLR)

Physical Variables

( Flow rate, HRT)

Source of seed

Enrichment of

ANAMMOX

populations

ANAMMOX process in

Anaerobic Membrane

Bioreactor (AnMBR)

CANON process in

Moving bed biofilm

reactor (MBBR)

82

3.2 ANAMMOX ENRICHMENT THROUGH BATCH CULTURE

Enrichment of ANAMMOX bacteria from the anaerobic and

aerobic seeds were carried out in batch culture. The anaerobic seed was

collected from the vegetable waste digester and biosolids digester. Aerobic

seed was collected from activated sludge process unit of a sewage treatment

plant (STP). The seeds were collected, brought to laboratory and analyzed

immediately.

3.2.1 Experimental setup

Fourteen ANAMMOX enrichment units (A1, A2, B1, B2, B3, C1,

C2, C3, C4, C5, D1, D2, E1 and E2) were filled with different ratios of seed

and enrichment medium as food, along with NH4Cl / NaNO2 as supplement,

as presented in Table 3.1, were sealed with rubber cork. The C1 – C5 reactors

were covered with butyl rubber stoppers and sealed with aluminium stoppers.

The experimental setup (2.6 L and 5 L capacity reactors) used for A1–A2, B1

– B3, D1 – D2 and E1 – E2, is presented in Figure 3.2 (a) and that of C1–C5

(100 mL capacity reactors) is presented in Figure 3.2 (b). Light interference

was avoided by covering the enrichment units in dark cloth and aluminum foil

as shown in the photographs of the experimental setup in Figure 3.3. Varying

dilutions of seed culture were adopted based on its initial characteristics. The

enrichment medium used (Graaf et al 1996), contained major nutrients and

trace metals, as presented in Table 3.2 and Table 3.3 respectively.

83

Table 3.1 Details of ANAMMOX enrichment units

Sl

No Source of seed culture

NH4+-N in the

seed culture

(mg/L)

Reactor

capacity

Reactor

labels

Food / Seed

ratio (%)

NH4+-N in the

reactor (mg/L)

Study

period

(d)

Sampling

frequency

1.

Anaerobic seed from

Vegetable waste digester

25040

2.6 L

A1 60 / 40 6745 70 Once in 10 d

A2 60 / 40 2015

B1 60 / 40 6750 200

Once in 10 d B2 60 / 40 7100

B3 60 / 40 25000

100 mL

C1 60 / 40 2900

30 Once in a

day

C2 40 / 60 1680

C3 60 / 40 1120

C4 40 / 60 1120

C5 50 / 50 1960

2. Anaerobic seed from

Biosolids digester 315 5 L

D1 40 / 60 796 70 Once in 10 d

D2 60 / 40 424

3.

Aerobic seed from

Activated Sludge process

in STP

280 5 L

E1 50 / 50 199

70 Once in 10 d E2 50 / 50 200

84

(a) 2.6 and 5 L capacity reactors

(b) 100 mL capacity reactors

Figure 3.2 Experimental setup for ANAMMOX enrichment

HEAD SPACE (40% OF

REACTOR VOLUME)

SEED + MEDIUM

PORT FOR MEDIUM

ADDITION

WATER

DISPLACEMENT

JAR

PORT FOR SAMPLING

BATCH REACTOR

HEAD SPACE (40% OF

REACTOR VOLUME)

SEED + MEDIUM

PORT for sampling &

medium addition

HEAD SPACE (40% OF

REACTOR VOLUME)

SEED + MEDIUM

PORT for sampling &

medium addition

85

(a) 2.6 and 5 L capacity reactors

(b) 100 mL capacity reactors

Figure 3.3 Photographs of the experimental setup for ANAMMOX

enrichment

86

Table 3.2 Composition of ANAMMOX enrichment medium

Sl No Compound Concentration (mg/L)

1. Potassium dihydrogen phosphate 25

2. Calcium chloride 300

3. Ferrous sulfate 12

4. EDTA 7

5. Sodium bicarbonate 1050

6. Magnesium chloride 165

7. Trace metal solution as per Table 3.3 1.25 mL/L

Source: Graaf et al (1996)

Table 3.3 Trace metal solution composition for ANAMMOX

enrichment medium

Sl No Compound Concentration (mg/L)

1. EDTA 15000

2. Zinc sulfate 430

3. Cobalt chloride 240

4. Manganese sulphate 990

5. Copper sulphate 250

6. Nickel chloride 190

7. Sodium selenite 320

8. Boric acid 14

Source: Graaf et al (1996)

87

3.2.2 Strategy of operation

The batch cultures were maintained at neutral pH using 1M HCl /

NaOH solution. Mixing was done by manual shaking of the reactors once a

day. During the startup period NaNO3 (10 mg/L) was added along with the

enrichment medium to favor the elimination of denitrifiers and to prevent the

generation of H2S by sulfur reducing bacteria (Wang et al 2009). Periodical

replenishment of the medium was performed once in 2 d. Batch cultures of

B1, B2 and B3, systems were operated for 200 d, A1, A2, D1, D2, E1 and E2

systems were operated for 70 d, in fed batch mode, by varying the Food /

Seed ratio. Anoxic condition was maintained in the reactors and gas

generation was monitored by water displacement method. Whenever NH4+-N

or NO2--N was found consumed, as indicated by reduction in their

corresponding concentrations, external supplements of NH4Cl / NaNO2 along

with the enrichment medium was added to maintain the NH4+-N and NO2

--N

ratio at 1:1. The ANAMMOX enrichment units C1 – C5 of 100 mL capacity

were operated with enrichment medium addition every day, but with no

supplement of NH4+-N and/or NO2

--N. These systems were operated in fed

batch mode for a period of 30 d.

3.2.3 Sampling and analysis

The seed culture obtained from the plants were used in the study for

initial characterization after carrying out double filtration using muslin cloth,

followed by centrifugation at 5000 rpm for 15 min. 100 mL samples were

collected every 10 d, from the sampling port after manual shaking from A1,

A2, B1, B2, B3, D1, D2, E1 and E2, while 1 mL samples were collected

every day from C1, C2, C3, C4 and C5 using syringe through the common

88

sampling/medium addition port. All the samples were filtered through 0.45 µ

filter paper (Whatman). Nitrogen transformations were studied from the

analyses of NO2--N and NO3

--N performed by spectrophotometric method and

NH4+-N by distillation method (APHA 1998). ANAMMOX biomass

development was determined from the metabolites namely N2H4 and NH2OH

(Watt and Chrisp 1952; Frear and Burrell 1955) and indirectly by the TSS,

MLVSS and MLSS estimations, which was carried out as per standard

methods (APHA 1998).

3.3 ANAEROBIC MEMBRANE BIOREACTOR (ANMBR)

The experimental setup is schematically depicted in Figure 3.4. The

AnMBR was composed of hollow fiber membrane module immersed in the

reactor of size 480 mm X 200 mm X 260 mm made of transparent Plexiglas,

with a working volume of 15 L. The AnMBR was filled with a mix of

anaerobic seed (60 %) from biosolids digester and enrichment medium (40 %)

as food, along with NH4Cl / NaNO2 as supplement, to promote the growth of

ANAMMOX bacteria, after excluding the 40 % of headspace in total volume.

The characteristics of the membrane module are presented in Table 3.4. The

feed tank containing enrichment medium was continuously stirred by an

overhead stirrer at 100 rpm to promote homogeneity of the influent, prevent

the entrapment of bubbles and to promote the process stability. The AnMBR

effluent was continuously filtered by the membrane module driven by a

permeation peristaltic pump (Watson Marlow 313) through the solenoid

valve. This operation was controlled by cyclic timer, operating with a

filtration cycle of 10 min and 2 min cut off.

89

Figure 3.4 Schematic of the experimental setup of the Anaerobic Membrane Bioreactor (AnMBR)

SLUDGEWITHDRAWAL

FEED PUMP

PERMEATION

PUMP

MEMBRANE

MODULECOLLECTION

TANK

TIMER

MERCURY

MANOMETER

BALL VALVE

SOLENOID VALVE

STIRRER

WATER LEVEL

SENSOR

MIXED LIQUOR

RECIRCULATION

Amm-N

TANK

NITRITE

TANK

MEDIUM

TANK

SAMPLING

PORT

PERMEATION

PUMP

MEMBRANE

MODULEPERMEATE

COLLECTION

TANK

TIMER

MERCURY

MANOMETER

BALL VALVE

SOLENOID VALVE

STIRRER

WATER LEVEL

SENSORAmm-N

TANK

NITRITE

TANK

MEDIUM

TANK

AMM - N

TANK

NITRITE

TANK

ENRICHMENT

MEDIUM

TANK

SAMPLING

PORT

ANAEROBIC MBR

NH4+-N

NO2--N

90

Table 3.4 Characteristics of membrane module

Sl No Items Details

1. Membrane material Polyethylene

2. Membrane type Hollow fiber

3. Pore size 0.42 µm

4. Surface area 0.2 m2

5. Membrane manufacturer Mitsubhishi rayon, Japan

Source: Sterapore Membrane Manual (2000)

The water level sensor controlled the feed pump to maintain the

liquid level in the bioreactor during the experimental period. Periodical

replenishment of the medium was performed once in 2 d to avoid issues

related to accumulation or lack of nutrients. Anoxic condition was maintained

and the reactor was covered with black cover to prevent the development of

phototrophic algal growth and O2 generation as noticed in the photograph of

the experimental setup in Figure 3.5.

3.3.1 Startup and stabilization of ANAMMOX process in AnMBR

NH4+-N and NO2

--N were supplemented along with mineral

medium as required in the form of NH4Cl and NaNO2 respectively in 3

separate lines. The same composition of the enrichment medium was used

(Graaf et al 1996), with modifications, where concentrations of Calcium

chloride and Magnesium chloride, was halved, with further addition of

1 mg/L of yeast extract (Star et al 2008), favoring the growth of completely

suspended ANAMMOX bacteria as free cells (Star et al 2008). Influent pH

was maintained in the range 6 to 8 without adjustment.

91

Figure 3.5 Photograph of the experimental setup of the Anaerobic Membrane Bioreactor (AnMBR)

Water

Level Sensor

NO2--N

tank

Enrichment

Medium tank

Permeate

Collection Tank

Stirrer

AnMBR

Feed

PumpCyclic

Timer

Solenoid

Valve

Permeation

Pump

NH4+-N

tank

92

The membrane in the AnMBR was immersed in the anaerobic seed

(MLSS 50680 mg/L; MLVSS 23450 mg/L) culture for about 35 d in batch

mode with continuous recirculation of the filtrate during which the biomass

and nitrogen concentrations were monitored every 10 d. NaNO3

(10 mg/L) was also added along with the medium and seed initially to favor

the elimination of degradable biomass by denitrifiers and to prevent the

generation of H2S by sulfur reducing bacteria (Wang et al 2009). This period

is referred as Phase I (1 to 35 d) where ANAMMOX process was initiated.

The reactor operation was then shifted to Phase II (36 to 112 d) which is in

semi continuous mode with nitrogen loading of NH4+-N and NO2

--N (50 mg/L

each) and nutrient feeding every 3 to 4 d, with continuous filtration at 2 d

HRT. During Phase III (113 to 242 d), the reactor was continuously operated

at flow rate of 7.5 L/ d (5 mL/ min) with 2 d HRT and NH4+-N and NO2

--N

(50 mg/L each) feed. During Phase II and III, ANAMMOX process was

stabilized. The system was operated with continuous stirring at 30 rpm. The

resistance of the membrane used in the AnMBR was measured by filtering

pure water at different filtration fluxes and the corresponding transmembrane

pressure (TMP) before the operation of AnMBR. The TMP was regulated in a

range of 0.13 to 0.53 kPa. Regular backwash of the membrane using the

permeate was performed and the membrane was cleaned when it was found

difficult to regulate the TMP in this range due to membrane fouling.

3.3.2 Optimization of Nitrogen loading rate (NLR)

The NH4+-N concentration in the feed was gradually raised from 50

to 10,000 mg/L with 2 d HRT ensuring an NH4+-N removal efficiency of at

least 95 % and/or the effluent NH4+-N concentration less than 10 mg/L. The

NO2--N concentration in the feed was maintained < 150 mg/L, being the toxic

threshold to ANAMMOX process (Strous et al 1999; Egli et al 2001; Dapena-

Mora et al 2007). The performance of the system was evaluated using effluent

quality (pH, ORP, COD, NH4+-N, NO3

--N and NO2

--N) and sludge

characteristics (pH, DO, MLSS and MLVSS).

93

3.3.3 Optimization of Hydraulic retention time (HRT)

The AnMBR was operated at five different HRTs of 1, 1.5, 2, 2.5

and 3 d, with a constant NH4+-N feed concentration of 10,000 mg/L. The

HRT study was conducted in 4 months and the performance was assessed in

terms of nitrogen removal efficacy (NH4+-N, NO3

--N and NO2

--N). The flow

rate was controlled using peristaltic pump. During the course of startup and

stabilization, the dissolved oxygen (DO) concentration was in the range of 0.2

to 0.5 mg/L. The pH of the AnMBR system was in the range of 5.88 to 8.53,

with an ORP range of 50 to – 107 mV. It was expected to be at – 250 mV, to

attain complete anoxic condition, as reported by Sabumon (2009).

3.3.4 Operation of AnMBR treating landfill leachate

Once the AnMBR was optimized for retention time, permeate flow

and NLR, the experiments with actual landfill leachate was undertaken. With

1.5 d HRT as the optimal retention time, nitrogen removal performance of

ANAMMOX process in AnMBR was performed. Upon performing the

leachate characteristics, it was diluted 10 times and spiked with NH4Cl to

make the NH4+-N concentration up to 10,000 mg/L. The performance of the

AnMBR system at optimized condition using the landfill leachate was

evaluated using effluent quality (pH, ORP, COD, NH4+-N, NO3

--N and

NO2--N); sludge characteristics (pH, DO, MLSS and MLVSS); fouling

characteristics (carbohydrate).

3.3.5 Membrane cleaning

During times when the TMP of the membrane increased above 25

kPa, the membrane was cleaned as per the Sterapore membrane manual

(2000). The schematic of the membrane cleaning procedure adopted is

depicted in Figure 3.6. The physical cleaning of the membrane was performed

carefully to remove the thick sludge cake layer deposited on the membrane as

depicted in Figure 3.7, by spraying pressurized water.

94

Figure 3.6 Schematic of the Membrane Cleaning Protocol (Adapted

from Sterapore Membrane Manual 2000)

If the membrane flux was not regained after this cleaning procedure

then the membrane was immersed in chemical cleaning tank containing mixed

solution of NaOCl (effective chlorine concentration of 3000 mg/L) and NaOH

(4 %) for 24 h. After chemical cleaning, the membrane was thoroughly rinsed

with tap water to remove traces of residual chlorine and checked for 80 %

recovery of flux. If the flux was still not recovered, then acid cleaning was

performed with 2 % hydrochloric acid (HCl) solution for 2 to 15 h. Finally the

membrane was rinsed with clean water and flux was measured again before

use.

Physical cleaning using Spray cans

YES

NO

Chemical cleaning (Effective Chlorine 3000 mg/L)

YES

Membrane used for

Operation

NO

Chemical cleaning (2 % HCl)

YES

NO

Change of Membrane

Membrane Flux

Recovery (min 80%)

Membrane Flux

Recovery (min 80%)

Membrane Flux

Recovery (min 80%)

95

(a) Fouled membrane (b) Cleaned membrane

Figure 3.7 Photographs of the Hollow Fibre Membrane module

3.3.6 Analytical techniques

The anaerobic seed from biosolids digester was initially

characterized after its double filtration using muslin cloth, followed by

centrifugation at 5000 rpm for 15 min. Sampling of the influent, effluent and

the MLSS of the AnMBR was performed every 10 d during startup and then

sampling frequency was increased to every day, for which about 100 mL

sample was collected from the sampling/sludge port. The samples were

prepared by filtering through 0.45 µ filter paper (Whatman), prior to analysis.

The Nitrogen transformations were studied from the analyses of NH4+-N,

NO3--N and NO2

--N (APHA 1998) and ANAMMOX biomass development

was determined from the metabolites, N2H4 and NH2OH (Watt and Chrisp

1952; Frear and Burrell 1955) and indirectly by the MLVSS and MLSS

96

estimations (APHA 1998). The various analytical techniques adopted to carry

out the study are presented in Table 3.5.

The phenol – sulphuric acid method of (Dubois et al 1956), was

used for carbohydrate estimation, wherein Glucose was used as the standard.

TMP was monitored using mercury manometer. Free ammonia (NH3) and free

nitrous acid (HNO2) concentrations were calculated by equilibrium equations

(3.1) and (3.2) suggested by Anthonisen et al (1976), Yamamoto et al (2008)

and Furukawa et al (2009)

NH3mgL

1714

Total Ammonia as N mgL

10pH

e(6344 273 T⁄ ) 10pH

(3.1)

HNO2mgL

4614

NO2

mgL

e 2300

273 T⁄ 10pH 3.2

Table 3.5 Analytical Techniques

Sl

No Parameter Method Instrument

Reference

(APHA 1998)

1. pH

Potentiometry

Ecoscan pH/mV/0C meter

(Eutech Instruments,

Singapore)

4500 B 2. ORP

3. COD Dichromate

digestion COD digester 5220 C

4. Alkalinity Titrimetry - 2320 B

5. MLSS Gravimetry

Oven, Balance 2540 B

6. MLVSS Muffle Furnace 2540 E

7. NH4+-N

Colorimetry

Spectrophotometer 4500 NH4

+-N C

8. NO3--N Colorimetry Spectrophotometer 4500 NO3

--N C

9. NO2--N Colorimetry Spectrophotometer 4500 NO2

--N C

10. N2H4 Colorimetry Spectrophotometer Watt and Chrisp 1952

11. NH2OH Colorimetry Spectrophotometer Frear and Burrell 1955

12. Carbohydrates Colorimetry Spectrophotometer Dubois et al 1956

97

3.3.7 Scanning Electron Microscope (SEM) analysis

The physical nature, the foulant constituents and the surface

morphology of the nascent and fouled membrane were determined using SEM

analysis by cutting out a piece of the membrane fiber (1 cm length) from the

membrane module. The biomass suspended in the AnMBR was also analyzed

using SEM. The membrane fiber and the biomass samples were dried,

dehydrated with ethanol (An et al 2009), Gold coated by an Ion sputter

(Model E – 1010, Hitachi) and observed in the Scanning Electron Microscope

(SEM S – 3400N, Hitachi, Germany). The Energy Dispersive X – ray

analyzer (EDX analyzer, Thermo Electron Corporation, Noran System Six

supported by SDS software for interpretation) was also employed to

determine the inorganic components of the cake layer on the membrane. The

ANAMMOX activity was inferred based on Nitrogen stoichiometry and the

variations in COD, alkalinity and biomass concentrations, with supplementary

support from SEM – EDX analysis.

3.4 MOVING BED BIOFILM REACTOR (MBBR)

A 6 L laboratory scale moving bed biofilm reactor (MBBR) was

filled 33 % (v/v) with Kaldnes K1 carriers having an effective surface area of

490 mm2/ carrier piece (Odegaard et al 1994) as depicted in Figure 3.8 and

3.9. The MBBR was operated initially as ANAMMOX reactor (1 to 82 d),

and then changed to CANON reactor (83 to 248 d). Then the CANON MBBR

was optimized for varying NLR and HRT (1 to 290 d).The pH was controlled

at 7.45 to 7.55 using 1M NaHCO3 with the temperature varying between 33.4

to 35°C. The effluent from MBBR was allowed to pass through a settling

flask prior to discharge. The detached biomass from the carrier collected in

the settling flask was stored at 00C, with no intentional biomass wasting.

98

Figure 3.8 Photograph of the experimental setup of the Moving Bed Biofilm Reactor (MBBR)

Buffer

Peristaltic

pump

DO meter

pH and DO probe

Stirrer

Air pumpFlow meter

MBBR

99

Figure 3.9 Photograph of the K1 Kaldnes carriers used in MBBR

exhibiting development of attached phase

3.4.1 ANAMMOX process in MBBR

The MBBR was inoculated with anaerobic seed (NH4+- N of 615 ± 164

mg/L) obtained by centrifuging the supernatant from biosolids digester and

stored at -800C. About 50 mL of the thawed pellet of the seed was added to

the reactor initially. The MBBR was operated in ANAMMOX process with 6

d HRT with an influent flow of 1 L/d for 82 d. Complete anaerobic condition

in MBBR was created by N2/ CO2 sparging to allow buildup of biomass. The

carriers were initially soaked with enrichment medium of composition based

on Graaf et al (1996) prior to seeding with inoculum. NH4+-N was

supplemented with the enrichment medium as required in the form of

(NH4)2SO4 in the feed tank. The NH4+-N concentration in the feed was based

on the NH4+-N conversion, NO2

--N oxidation and NO3

--N production during

the ANAMMOX process. About 24 mg NO2--N/L and 0.083 mg N2H4/L was

100

added to the MBBR to kick start the ANAMMOX process (Third et al 2005).

The NH4+-N concentration in the feed was gradually raised from 360 mg/L to

872 mg /L with 6 d HRT ensuring a consistent NH4+-N removal efficiency >

50 %, as depicted in Table 3.6. Accumulation of NO2--N was avoided by

adjusting the influent NH4+-N loading rate.

3.4.2 Startup of CANON process in MBBR

The MBBR system was shifted from ANAMMOX process to

CANON process on day 83 with intermittent aeration for 5/90 seconds on/off

manner. This practice of switching from ANAMMOX to CANON i.e. from

anaerobic to anoxic condition was adopted to improve the growth of biomass

from suspended to the attached mode, based on the interaction between the

attached phase on the carriers and suspended phase in the system (Park et al

2010a; Park et al 2010b). The aeration time was gradually increased up to

30/60 seconds on/off with an airflow rate of 196 L/ min/ mm2

on day 90, to

maintain 2.5 to 3 mg O2/L of average DO until day 248 of MBBR operation.

The NO2--N concentration in the feed was maintained close to zero thereby

avoiding the inhibition of ANAMMOX bacteria by NO2--N (Third et al 2005).

During this period of CANON initiation, the feed NH4+-N was 375 mg/L. The

feed NH4+-N concentration was then raised to 545 mg/L on day 111 with the

highest influent NH4+-N concentration of 757 mg/L on day 223 as depicted in

Table 3.6. Upsets to the MBBR due to the failure of pumps occurred on day

235, which was eventually rectified. The average influent NH4+-N

concentrations during the period of operation from 83 to 248 d were

363.80 ± 197.27 mg/L, with the CANON stabilization process was performed

with mean nitrogen loading rate of 0.06 ± 0.03 kg N/ m3/ d at 6 d HRT.

101

Table 3.6 Operational Strategy of MBBR

Sl

No

MBBR

operation

mode

Operation

period (d)

Time

(d)

Influent NH4+-N

concentration

(mg/L)

NLR

(kg

NH4+-

N/m3/d)

HRT

(d)

Aeration

time

(seconds

on/off)

1. ANAMMOX

process 1 to 82

1 362 0.06

6 N2/CO2

sparging

3 360 0.06

15 147 0.02

22 184 0.03

50 535 0.09

73 872 0.15

2.

CANON

startup and

stabilization

process

83 to 248

83 375 0.06

6

5/90

90 482 0.08

30/60 111 545 0.09

223 757 0.13

247 551 0.09

3.

CANON

optimization

process

1 to 290

1 615 0.10 6

30/60

8 127 0.04 3

24 112 0.07

1.5

53 264 0.18

75 293 0.20

100 465 0.31 45/60

200 558 0.37 Continuous

290 483 0.32 Continuous

102

3.4.3 Optimization of Nitrogen Loading Rate and Hydraulic

retention time during CANON process in MBBR

The Nitrogen Loading Rate (NLR) of the MBBR was varied by

decreasing hydraulic retention time (HRT) and varying the influent NH4+-N

concentration, with the NLR optimized to 0.33 kg NH4+-N/ m

3/ d. When the

MBBR was operated with 6 d HRT, the feed NH4+-N concentration was in the

range of 147 to 872 mg/L. It was reduced to 3 d on day 9 of CANON

optimization process. Further decrease of HRT from 3 d to 1.5 d was applied

on day 24. NLR was then gradually increased to 0.18 and to 0.20 kg NH4+-N/

m3/ d at day 53 and 75, respectively. On day 100, intermittent aeration was

further increased to 45/60 to provide enough DO. There was a change from

intermittent to continuous aeration of 73.5 L/ min/ mm2 from day 200 by

maintaining 0.7 – 1.5 mg O2/L. At the end of the study, the MBBR was run

with feed NH4+-N concentration of 500 mg/L with 73.5 L/ min/ mm

2

continuous aeration. Upsets occurred during the CANON optimization

process on the MBBR due to the failure of air pumps on days 100, 121 and

207, which were resolved. Aeration was increased momentarily from 147 to

294 L/ min/ mm2

to ward off the effect of nitrogen accumulation on day 207.

The NLR of 0.33 kg NH4+-N/ m

3/ d at 1.5 d HRT and 4 L/ d flow rate was

achieved in MBBR by the end of 290 d.

3.4.4 Analytical techniques

The MBBR reactor performance was monitored three times a week

using NH4+-N (Fisher accumet gas sensing electrode, Waltham,

Massachusetts), NO2--N (colorimetric detection, APHA 1998), NO3

--N

(Fisher accumet ion selective electrode), N2H4 (colorimetric detection, Watt

and Chrisp 1952), and NH2OH (colorimetric detection, Frear and Burrell

103

1955) measurements. The dual channel DO meter (YSI 5300) was interfaced

to a personal computer and the online data acquisition was performed using

virtual instrument codes implemented on LABVIEW, version 8.0 (National

Instruments, Austin, Texas). Gaseous N2O (gas filter correlation, Teledyne

API 320E, San Diego, California) and NO (Chemiluminescence, CLD 64,

Ecophysics, Ann Arbor, Michigan) concentrations were measured twice a

week, each over at least one hour period at a frequency of 1 per second and

time averaged.

3.5 DETERMINATION OF MICROBIAL ECOLOGY OF

BIOMASS IN THE MBBR

The microbial abundance from the suspended and attached phase of

the CANON reactor was determined by quantitative PCR and its molecular

fingerprinting by DGGE/Sequencing.

3.5.1 Quantitative PCR

The samples of biofilm rings and the suspension were regularly

collected and stored at -80C for subsequent DNA extraction process. DNeasy

mini kit (Qiagen, California) was used for DNA extraction following

manufacturer’s instruction. The ensuing bacterial DNA concentration and

quality was measured by UV spectrophotometry (Varian, California).

Bacterial abundance of ANAMMOX, AOB, NOB and total bacteria were

determined from MBBR operation in triplicates via SYBR Green chemistry

quantitative PCR (qPCR). It specifically targeted surrogates such as

ANAMMOX 16S rRNA (AMX 16S), ammonia monooxygenase subunit A

(amoA), Nitrobacter 16S rRNA (Nb 16S), Nitrospira 16S rRNA (Ns 16S),

and total bacterial 16S rRNA (EUB 16S). The bacterial abundance was

quantified using these targeted primers and qPCR assays were conducted on

104

an iQ5 real time PCR thermal cycler (BioRad Laboratories, Hercules,

California). The iQ5 software calculated the cycle at which the fluorescence

intensity goes beyond the threshold value represented as Ct. From the

obtained standard curves, gene copies were calculated with respect to each

PCR reaction. The gene copies/PCR reaction was in turn converted to gene

copies/L depending on the amount of template DNA added to PCR reaction

and the amount of extracted DNA from the source sample. Standard curves

for qPCR were produced via serial decimal dilutions of plasmid DNA that has

specific target gene inserts. From the standard dilution series of plasmid DNA

concentration, the copy numbers of targeted gene inserts was calculated as

represented in Equation (3.3).

Gene copies in Template DNA (Copies L

) 6.022 x 1023 x ( AB x C

) (3.3)

where

A = DNA plasmid concentration (g/µL)

B = sum of PCR amplicon and vector (base pair (bp))

C = average molecular weight / nucleotide base pair (i.e) 660 g/

mole/ bp

Melt curve analysis was performed to confirm the primer

specificity and the lack of primer-dimers. The doubling time (td) of

ANAMMOX bacteria during the CANON process was approximated as per

the Equation (3.4), which was adopted from (Star et al 2007).

Doubling time td ln 2 t t0ln C C0

⁄

105

where

t – t0 = time relative to the initial time, t0 over which td is

calculated (d)

C = concentration of ANAMMOX at t (copies/mL)

C0 = initial concentration of ANAMMOX at t0 (copies/mL)

The maximum specific growth rate of ANAMMOX (µmax,ANAMMOX)

bacteria during the CANON process can be calculated based on the change in

ANAMMOX concentrations from the Equation (3.5), adopted from Star et al

(2007). It was determined by combining the results from molecular assays and

nitrogen removal performance in MBBR.

lnXANAMMOX lnXANAMMOX,0 max,ANAMMOX t (3.5)

where

XANAMMOX, 0 = initial ANAMMOX bacteria concentration

(copies/mL)

XANAMMOX = ANAMMOX bacteria concentration at time t

(copies/mL)

µmax,ANAMMOX = specific growth rate of ANAMMOX bacteria (d-1

)

From the generated µmax, ANAMMOX value for CANON process in

MBBR, along with the reported ANAMMOX specific decay coefficient

(bANAMMOX) of 0.004 d-1

(Star et al 2007) the minimum SRT (θC, min,ANAMMOX)

that is needed to maintain ANAMMOX populations in the reactor was

calculated from Equation (3.6), which is adopted from (Star et al 2007).

(θC,min,ANAMMOX) 1 max,ANAMMOX bANAMMOX

3.6

106

where

µmax,ANAMMOX = specific growth rate of ANAMMOX bacteria

(d-1

)

bANAMMOX = specific decay coefficient of ANAMMOX

bacteria (d-1

)

θC, min,ANAMMOX = minimum SRT needed to maintain

ANAMMOX population in reactor (d)

3.5.2 Molecular fingerprinting

Molecular fingerprinting was performed by using 1055F/1392R-

GC primer set as described in Table 3.7 for Denaturing Gradient Gel

Electrophoresis (DGGE) on a Dcode system (Bio-Rad Laboratories,

California). The DGGE was performed at 600C using 1 x TAE buffer at 75 V

for 13 h on the Dcode system using 8 % polyacrylamide gel with 30 – 60 %

(M/V) gradient urea – formamide denaturant. Post staining of the gel was

carried out using Ethidium bromide and bands were visualized under UV

transilluminator (Fotodyne UV 21). Specific bands observed on the gel were

excised using a sterile scalpel. Once the single bands of the excision were

confirmed by running a secondary DGGE, the bands were reamplified,

purified with QIAEX II (Qiagen, California), and sequenced (ABI3730XL

DNA analyzer, Applied Biosystems, California). Sequences were aligned

using MEGA (Kumar et al 2004) and analyzed using BLAST (Altschul et al

1990). Phylogenetic trees were constructed using the Neighbor – Joining

method with a bootstrap of 1000 replications, and Jukes – Cantor

computational model (Jukes and Cantor 1969).

10

Table 3.7 Primers used in qPCR and PCR – DGGE

Sl

No

Bacterial

community Target

Primers

name Primer sequence (5’ – 3’) Reference

I For qPCR

16S rRNA Pla 46F GGATTAGGCATGCAAGTC Star et al

(2007) 1. ANAMMOX Amx667R ACCAGAAGTTCCACTCTC

2. AOB amoA amoA-1F GGGGTTTCTACTGGTGGT Rotthauwe et al

(1997) amoA-2R CCCCTCKGSAAAGCCTTCTTC

3. NOB

Nitrospira

16S rRNA

NTSPAf CGCAACCCCTGCTTTCAGT Kindaichi et al

(2006) NTSPAr CGTTATCCTGGGCAGTCCTT

Nitrobacter

16S rRNA

Nitro-1198f ACCCCTAGCAAATCTCAAAAAACCG Graham et al

(2007) Nitro-1423r CTTCACCCCAGTCGCTGACC

4. Total bacteria Universal 16S

rRNA

1055F ATGGCTGTCGTCAGCT Ferris et al

(1996)

1392R ACGGGCGGTGTGTAC

II For DGGE 1055F ATGGCTGTCGTCAGCT

5. Total bacteria 1392R - GC [GC-Clamp]ACGGGCGGTGTGTAC [GC–Clamp sequence] = [CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC] (Adapted from Park et al 2010a, Park et al 2010b)