EFFECTS OF RESIDUAL COD ON MICROBIAL GROWTH KINETICS IN A NITRIFYING UCBR

Three reactor set-ups were investigated, namely nitrification UCBR prior to coupling, coupled UCBR-packed bed system for nitrogen removal, and de-coupled recovering UCBR. Batch studies were conducted on the biofilm particles taken from the UCBR. A mathematical model was developed to estimate the growth kinetics of the biofilm autotrophic and heterotrophic bacteria. Heterotrophs was noted to accumulate on the nitrifying biofilm due to the effect of residual COD from the packed bed column during the coupled reactor phase. The growth of heterotrophs had affected the substrate removal rates, biofilm morphology, and growth kinetics of nitrifying bacteria. For example, μm of ammonium oxidizing bacteria decreased from 0.55 to 0.19 d-1, while Ks increased from 1.42 to 3.34 mg N/L. This study demonstrated that μm and Ks of the nitrify bacteria changed with the type of substrate. Hence, growth kinetics would not be the same for nitrifying bacteria that is exposed to residual carbon in a coupled UCBR-packed bed system and a single UCBR that is fed on organic-carbon free substrate.

EFFECTS OF RESIDUAL COD ON MICROBIAL GROWTH KINETICS IN A NITRIFYING UCBR

L.F Song, S.L Ong, J.Y Hu, K.B Chia, L.Y Lee and W.J Ng

Wastewater Biotreatment Group, Department of Civil Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260

ABSTRACT Three reactor set-ups were investigated, namely nitrification UCBR prior to coupling, coupled UCBR-packed bed

system for nitrogen removal, and de-coupled recovering UCBR Batch studies were conducted on the biofilm

particles taken from the UCBR A mathematical model was developed to estimate the growth kinetics of the

biofilm autotrophic and heterotrophic bacteria Heterotrophs was noted to accumulate on the nitrifying biofilm due

to the effect of residual COD from the packed bed column during the coupled reactor phase The growth of

heterotrophs had affected the substrate removal rates, biofilm morphology, and growth kinetics of nitrifying

bacteria For example, µ m of ammonium oxidizing bacteria decreased from 0.55 to 0.19 d -1 , while Ks increased

from 1.42 to 3.34 mg N/L This study demonstrated that µm and Ks of the nitrify bacteria changed with the type of

substrate Hence, growth kinetics would not be the same for nitrifying bacteria that is exposed to residual carbon in

a coupled UCBR-packed bed system and a single UCBR that is fed on organic-carbon free substrate

KEYWORDS Biofilm; half saturation constant; heterotroph; maximum specific growth rate; nitrifying bacteria; UCBR

INTRODUCTION

The Ultra Compact Biofilm Reactor (UCBR) has been shown to be efficient for nitrification (Yu, 1998) The design of a biofilm system is usually governed by the intrinsic process parameters such as growth and decay rates of microorganisms The reported growth kinetics by various researchers have been different for different

bioreactor configurations such as fluidized bed, chemostat and activated sludge (Stevens et al., 1989; Hanaki et

age, bacteria genus, degree of turbulence and mixing in the bioreactor This study aimed to determine the values

of growth kinetics, µm and Ks, of a nitrifying UCBR and the effects different system set-ups, through single UCBR, reactors coupled and de-coupled reactors, have on these growth parameters

MATERIALS AND METHODS This study was carried out in three phases

1 Phase 1 investigated and optimized UCBR nitrification performance at a NH4+- N loading rate of 2.8 kg NH4+- N/m3.d

2 Phase 2 studied biofilm nitrification activity in the nitrification UCBR column coupled with a denitrifying packed bed column under the influence of residual COD remaining in the effluent of denitrifying column The NH4+-N and COD loading rates for the coupled reactors system were 2.8 kg NH4+- N/m3.d and 10.2 kg COD/m3.d, respectively

3 Phase 3 studied biofilm recovery in a single UCBR after de-coupling the reactors The NH4+-N loading was decreased to 0.5 kg NH4+-N/m3.d and then gradually increased to 2.8 kg NH4+-N/m3.d over a period

of 42 days

Experimental set-up

UCBR The UCBR had two concentric draught tubes which divided the reactor column into two zones, the riser

and downcomer A three-phase separator was mounted at the top of the reactor Compressed air was introduced

at 2.0 cm/s into the riser column via a metallic sparger located at the bottom of the UCBR The air supplied provided mixing and dissolved oxygen to the UCBR

(Schotts Pte Ltd, Germany) with a dimension of 25mm x 25mm (diameter x height) Backwashing to prevent clogging was performed once every two days using an industrial grade N2 gas

Coupled UCBR- Packed Bed System During the coupled reactor phase, a recycle ratio of 4.0 was used This

recirculation ratio was chosen to avoid short circuit in the flow of the wastewater Recirculation between the two columns was achieved using a peristaltic pump to connect the flow from the bottom of the packed bed column to the bottom of the UCBR column The geometry of the UCBR and packed bed column are given in Table 1

Table 1 Geometry of UCBR and Packed Bed column

Total reactor volume (L) 4.35 3.30

was conducted in a 1 L beaker Compressed air was supplied through a diffuser and the reactor was adequately mixed using a floating magnetic stirrer Dissolved oxygen was maintained at above 4.0 mg/L throughout the experiment pH of the reactor was maintained at 7.5 + 0.3 with manual addition of acid (HCl) and base (NaHCO3) Approximately 15 ml of biofilm particles was taken from the UCBR to inoculate 1 L of synthetic feed for each batch test The synthetic feed consisted of 382.0 mg/L NH4Cl, 294.0 mg/L CH3COONa, 600.0 mg/L NaHCO3, 22.5 mg/L K2HPO4 and 0.2 ml/L trace elements Each liter of trace elements solution contains

10 g CaCl2⋅H2O, 8 g FeCl3⋅6H2O, 5 g MgSO4⋅7H2O, 2 g CoCl2⋅6H2O, 2 g Thiamine-HCl, 1 g NaSiO3⋅9H2O,

550 mg Al2(SO4)3⋅16H2O, 50 mg MnCl2⋅2H2O, 1 mg (NH4)6Mo7O24⋅6H2O, 1 mg CuSO4⋅5H2O, 1 mg ZnSO4⋅7H2O and 1 mg H3BO4 Samples were collected at 20 to 30 minutes intervals All the samples were

filtered through 0.45 µm pore size filter papers The filtrate was tested for NH4+-N, NO2--N, NO3--N and COD

in accordance with Standard Methods for Water and Wastewater (APHA, 1995) DO and pH were recorded at each sample collection interval MLSS concentration, particle size and biofilm density were determined at the end of each experiment The biofilm particle size was determined using an Image Analyzing System (Image-Pro Plus version 3.0 for Windows from Media Cybernatics, U.S.A) The batch tests were terminated when ammonium has been completely converted to nitrate

Modeling with AQUASIM AQUASIM 2.0 (Reichert, 1998) was used to estimate the kinetic parameters for

autotrophs and heterotrophs as shown in Table 2 Their values would be based on data obtained from the batch tests

Table 2 Kinetic coefficients obtained with AQUASIM

growth rate (d-1)

Mass Fraction Half saturation

constant (mg/l)

nA (XA = nA X) KS,NO2

(XH = nH X) KS,H

A simple mathematic model based on Monod’s kinetics for substrate ultilization within the batch reactor was derived and used to estimate values of the parameters shown in Table 3 The mass fraction parameters, nA and

nH have been introduced to differentiate the mixed-population biofilm into functions of ammonium oxidizing and COD oxidizing fractions Convergence tolerance was set to 0.0005 and the secant minimization algorithm was selected as the optimization routine for parameter estimation

Table 3 Process matrix for parameter estimation

Stoichiometric coefficient

COD SNH4-N SNO2-N SNO3-N Heterotrophs















 +















 +













=

DO K

DO S

K

S Y

X n dt

dS

H O COD H S COD H

H H m COD

, ,

, µ

-1 Nitrosomonas











 +













+









=

−

−

−

−

DO K

DO S

K

S Y

X n dt

dS

A O N NH N NH S

N NH A

A NH m N NH

, 4

4 , 4 4

,

-1 +1

Nitrobacter







 +









+









=

−

−

−

−

DO K

DO S

K

S Y

X n dt

dS

A O N NO N NO S

N NO A

A NO m N NO

, 2

2 , 2 2

,

-1 +1

RESULTS & DISCUSSION

In Phase 1, nitrification efficiency in the UCBR was virtually 100% During Phase 2, 80% nitrogen removal efficiency was achieved initially Over a coupled reactors period of 16 days, the nitrogen removal efficiency decreased to 55% The corresponding conversion rate of NH4+-N to NO3--N deteriorated from 100% to 55% over the same period The deterioration in the specific ammonium oxidation rate was due to the rapid growth of heterotrophs over the slower growing nitrifiers The decrease in nitrification activities could be due to 3 reasons:

1 Competition between heterotrophs and nitrifiers for common substrate such as dissolved oxygen and ammonium

2 Increase in diffusional resistance of substrate into the biofilm due to the growth of the outer heterotrophic layer over the slower growing nitrifers During Phase 2, the diameter of biofilm particles increased from around 600 to 850 µm At the same time, density of particle decreased from 153 to 97 g/L Heterotrophic bacteria growth had contributed to a less dense biofilm during Phase 2 According to

Tijhuis et al (1995), biofilm density of the heterotrophs was about 7 times lower than the biofilm

formed by the predominantly nitrifying bacteria

3 Reduction in gas-liquid mass transfer due to decreased turbulence in the UCBR caused by a higher solids holdup This reduction in turbulence could have reduced the oxygen transfer necessary for nitrification

As shown in Fig 1, the specific activities of heterotrophs increased from 0.29 g COD/g VSS.d to 0.68 gCOD/gVSS.d while the ammonia oxidation specific activity decreased from 0.33 g NH4+-N/g VSS.d to 0.04 gNH4+-N/gVSS.d during transition from Phase 1 to Phase 2 Tijhuis et al (1995) reported that biofilm specific

ammonium oxidation activity was approximately 1.4 g NH4+-N /g VSS.d in a Biofilm Airlift Suspension Reactor (BAS) In comparison, the value obtained in this study was rather low However, it must be noted that the value reported in this study was an average specific rate for a batch system The maximum value of specific ammonium oxidation rate obtained in the batch test during Phase 1 was approximately 0.97 g NH4+-N/g VSS.d

This value was closer to the value reported by Tijhuis et al (1995) The different operating conditions between

the two studies could have led to the difference The NH4+-N loading rate used in Tijhuis et al (1995) study was

5 kg N/m3.d which is higher than the 2.8 kg N/m3.d used in this study As specific substrate utilization rate will increase with substrate loading, one would therefore expect that a higher NH4+-N loading rate would lead a higher specific ammonium oxidation rate

Fig 1 Specific activity vs time

The microbial growth kinetics was obtained in this study by fitting the experimental batch test results to the

mathematical model A comparison between the nitrifying and heterotrophs growth kinetics obtained in this

study with those reported in literature is summarized in Table 4

Table 4 Kinetic coefficients for nitrifiers Nitrosomonas Nitrobacter Heterotrophs Type of reactor µm,NH4

(1/d)

Ks,NH4 (mgN/l) µm,NO2

(1/d)

Ks,NO2 (mgN/l) µm,H

(1/d)

Ks,H (mgCOD/l) UCBR1

Phase 1 0.55 + 0.08 1.42 + 0.09 0.03 + 0.01 3.68 + 0.32 0.95 + 0.04 4.45 + 0.17

Phase 2 0.19 + 0.12 3.34 + 2.05 0.06 + 0.04 4.14 + 0.29 1.26 + 0.13 5.18 + 0.30

Phase 3 0.58 + 0.25 1.57 + 0.22 0.05 + 0.08 3.58 + 0.13 1.04 + 0.38 4.40 + 0.32

Biofilm Air-lift Suspension

1This study

2 Hanaki et al., (1990)

3 Harald and Dietmar (1997)

4 Metcalf and Eddy (1991)

0.00 0.20 0.40 0.60 0.80 1.00

Day of operation

Specific NH4-N rate Specific COD rate Phase 1 Phase 2 Phase 3

A sensitivity analysis of the model parameters using the absolute-relative sensitivity function showed that maximum specific growth rate, µm, and mass fraction, n, had high sensitivity, while half saturation concentration, Ks, had a low sensitivity The purpose of a sensitivity analysis is to check if the model parameters can be adequately determined with the aid of the available data and to estimate the uncertainty of the parameter estimated

The µm obtained in this study was lower for both heterotrophs and nitrifiers as compared to the activated sludge process (Metcalf & Eddy, 1991) This could be due to a lower specific surface loading rate in the UCBR The

KS on the other hand was higher, which could be due to a higher diffusional resistance within the immobilized biofilms as compared to the suspended flocs in an activated sludge process As noted from Fig 2 and Fig 3, the increase in µm,H due to the influx of COD into the nitrification UCBR was rapid (from 0.99 to 1.36 d-1 in 3 days), whereas the reduction in µm,NH4 was relatively gentle (from 0.55 to 0.10 d-1 in 21 days) On the other hand, the change in µm,NO2 due to the coupled reactors arrangement was less compared to µm,NH4 It was also noted that in the recovery phase, µm,H reached its original values faster than µm,NH4 As shown in Fig 4 and Fig 5, KS for both heterotrophs and autotrophs increased due to the coupled reactors arrangement Ks for heterotrophs increased from 4.8 to 5.3 mgCOD/L over a period of 13 days, while KS for ammonium oxidizers and nitrite oxidizers increased from 1.42 to 3.34 mgN/L and from 3.68 to 4.41 mgN/L, respectively This could be due to the growth of the outer heterotrophic layer leading to a higher diffusional resistance of substrate to the microorganisms in the inner layer of the biofilm

0

0.5

1

1.5

2

Days of Operation

0.2 0.4 0.6 0.8

Days of Operation

Maximum specific growth rate (1/d)

Nitrosomonas Nitrobacter Fig 3 Maximum specific growth of nitrifiers

0.00

1.00

2.00

3.00

4.00

5.00

Days of operation Half saturation constant (mgN/l)

nitrosomonas nitrobacter

0 2 4 6

Days of Operation

Fig 4 Half saturation concentration Ks of nitrifiers Fig 5 Half saturation concentration Ks of heterotrophs.

Mass fraction of the microorganism also indicated that percentage of heterotrophs increased, while nitrifying bacteria population decreased during coupled reactor period (Fig 6) The mass fraction, nH, for heterotrophic bacteria indicated the presence of heterotrophic bacteria in the biofilm even in the absence of COD in Phases 1

and 3 This observation is similar to the findings of Rittmann et.al (1994) After the reactors have been

de-coupled in Phase 3, the fraction of nitrifiers was restored to its Phase 1 values within a few days However performance of the reactor and specific removal rates were still below the values obtained in Phase 1 During Phase 1, the average particle diameter was around 600 µm, while experimental results showed that the diameter

of particles in Phase 3 was smaller, approximately 400µm This suggested that sloughing of outer biofilm layers

Phase 1 Phase 2 Phase 3

Fig 2 Maximum specific growth of heterotrophs

Phase 1 Phase 2 Phase 3

may have taken place after de-coupling This could have resulted in nitrifying bacteria in inner layers being sheared off during the sloughing process and washed out from the reactor

0 0.2 0.4 0.6 0.8 1

5 12 15 19 22 26 33 40 47 57 62 69

Days of operation

Nitrifiers Heterotrophs

CONCLUSIONS This study showed that the growth kinetics of nitrifiers, µm and Ks, changed in accordance to the prevailing type

of substrate and other operational parameters, such as system configuration Due to the effect of residual COD,

µm,NH4 decreased from 0.55 to 0.19 d-1 over a period of 21 days, whereas KS,NH4 increased from 1.42 to 3.34 mgN/L over a period of 13 days Heterotrophic activity had also increased due to residual COD µm,H increased from 0.99 to 1.36 d-1 in 3 days, while KS,H increased slightly from 4.8 to 5.3 mgCOD/L Experimental results showed that the kinetic parameters, µm and KS, for UCBR nitrification would differ between a coupled UCBR-packed bed system and a single UCBR Hence appropriate choice of kinetic values would have to be made when applying kinetic model for reactor design

REFERENCES

1 Hanaki K., Wantawin C., and Ohgaki S (1990) Effects of the activity of heterotrophs on nitrification in

suspended growth reactor Wat Res., 24 (3), 289-296

2 Harald H and Dietmar C.H (1997) Growth and decay in an auto/heterotrophic biofilm Wat Res., 31(9),

2243-2252

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in biological treatment Wat Sci Tech., 30 (6),1-8

6 Sheintuch M., Tartakovsky B., Narkis N and Rebhun M (1995) Substrate inhibition and multiple states

in continuous nitrification process Wat Res., 29(3), 953-963

7 Standard Methods for the Examination of Water and Wastewater (1995) 19th ed., American Public

Health Association/ American Water Works Association/ Water Environment Federation, Washington

DC, USA

8 Stevens D.K., Berthouex P.M and Chapman T.W (1989) Dynamic model of nitrification in fluidized

bed J Envir Eng., 32 (1), 71-80

9 Tijhuis L., Huisman J.L., Hekkelman H.D., van Loosdrecht M.C.M and Heijnen J.J (1995) Formation

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Fig 6 Fraction of auto/heterotrophs by mass