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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)