determination of ammonia oxidation bacteria kinetics ...

Journal of Science and Technology 52 (3A) (2014)

DETERMINATION OF AMMONIA OXIDATION BACTERIA KINETICS IN PARTIAL NITRITATION PROCESS USING RESPIROMETRIC METHOD Nguyen Le Hoang Trung1*, Vo Thuy Khanh Nghi1, Phan The Nhat1 and Nguyen Phuoc Dan1* 1

Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology, Building B9, 268 Ly Thuong Kiet street, district 10, Ho Chi Minh City, Viet Nam. *Email: [email protected], [email protected] Received: 20 June 2014; Accepted for publication: 12 July 2014 ABSTRACT

This study used the respirometric method to determine kinetic parameters of Ammonia Oxidation Bacteria (AOB) sludge in the Partial Nitritation Sequencing Batch Reactor (PNSBR) which was running stably with old landfill leachate at nitrogen loading rate of 1.2 kgN/m3/day. Two different media used in this study were landfill leachate and synthetic wastewater containing ammonia and salts. Respirometric tests were conducted at pH of 8.0 ± 0.2 and temperature of 30 ± 1oC. The determined kinetics of AOB in two media were presented as follow. Maximum specific oxygen uptake rate, oxygen half-saturated constant, and ammonia half-saturated constant of AOB in synthetic wastewater were 0.143 mgO2/mgVSS.h-1, 1.55 mgO2/L, and 154 mgN-NH4+/L respectively, whereas the corresponding values in landfill leachate were 0.054 mgO2/mgVSS.h-1, 1.07 mgO2/L, and 66 mgN-NH4+/L respectively. Maximum growth rate µmax and yield coefficient YX/N of AOB in landfill leachate were found to be 0.12 day-1 and 0.26 mgVSS/mgN-NH4+. Keywords: respirometric, kinetics, AOB, landfill leachate. 1. INTRODUCTION Old landfill leachates are known as wastewater whose treatment is very complicated, and costly due to high loads of refractory compounds, low BOD5/COD ratio, high salinity and high strength of ammonia [1]. The concentration of ammonia in leachate gradually increases according to the age landfill leachate, which can become a great environmental concern in waste management. Partial nitritation (PN) followed by Anammox process may be an option instead of the conventional nitrification–denitrification process for nitrogen removal [2]. To obtain theoretically 1:1.32 (NH4+:NO2-) ratio of Anammox, the PN process require promotion of ammonia oxidizing bacteria (AOB) growth and inhibition of nitrite oxidizing bacteria (NOB) growth by running at high free ammonia (FA) concentration and/or low dissolved oxygen (DO) [3]. The chemistry behind nitritation and nitratation is given in following equations: 1

Nguyen Le Hoang Trung, Vo Thuy Khanh Nghi, Phan The Nhat and Nguyen Phuoc Dan

AOB NH4  1.5O2   NO2  H2O  2H+

(1)

NOB NO2  0.5O2   NO3

(2)

Respirometric techniques have been intensively used for the determination of BOD, toxicity and kinetic parameters of toxic and nontoxic wastewater. Oxygen Uptake Rates (OURs) can be used as a measure of the activity of the nitrifying populations present in the activated sludge. Some studies use inhibitors which suppress AOB and NOB activity, such as allylthiourea or sodium chlorate, respectively [4]. Due to the toxic effect on nitrifying bacteria and their low specific growth rate, it is difficult to apply these compounds to a kinetics study. The present study attempts, therefore, to contribute to the development of a method which will enable kinetic monitoring for nitritation only. The kinetic parameters of AOB such as maximum specific oxygen uptake rate (SOURmax), oxygen and ammonia half-saturated constant (KO, KS) were determined using respirometric method; maximum growth rate µmax and yield coefficient YX/N were calculated based on stoichiometric links between nitrogen removal, oxygen consumption, and activated sludge growth during nitritation process [5]. 2. MATERIALS AND METHODS 2.1. Respirometric reactor Fig. 1 illustrates a 2 L completely sealed mixed batch reactor (3). The reactor is a cylindrical closed vessel with 20o beveled flange which eliminate transferring of oxygen from atmosphere to the solution during experiments. The solution is kept homogenized by a magnetic stirrer (1) at 200 rpm (5). The reactor has 4 ports at the top which allow oxygen supply (6), sampling and injecting substrate (8), residual gas exhaust (7), inserting of DO electrode (Oxy 701, WTW) for monitoring DO levels. The probe is connected to a personal computer, which is used for storing and monitoring DO and temperature data transmitted by the probe. Compressed oxygen (2) is provided through fish aerators. The DO concentrations were controlled by adjusting valve opening.

Figure 1. Schematics of respirometric reactor

2.2. Materials For synthetic wastewater(SW), chlorine-free tap water (using sodium sulfite as a dechlorination agent) was used to produce enrichment media with constant ratio of N:P:Mg = 340:10:1, a micronutrient solution was also added according to Ciudad et al. [6]. Landfill leachate (LC) was taken from Go Cat municipal landfill in Ho Chi Minh City. To remove ammonia completely, air stripping was used by raising pH of the leachate to 12 and aerating for 2

Determination of ammonia oxidation bacteria kinetics in partial nitritation process using respirometric method

3 days. The pH of ammonia-free leachate then was neutralized using hydrochloric acid (HCl). Ammonia concentration in leachate medium, whose conductivity value in the range of 32.4 33.5 mS/cm, was below detection limit. Ammonium chloride (NH4Cl) and sodium nitrite (NaNO2) were also prepared as substrate solutions. Activated sludge (AS) in this study was obtained from a lab-scale PNSBR which was running stably with Go Cat landfill leachate at nitrogen loading rate of 1.2 kgN/m3/day and concentration of 3790 mgN-NH4+/L. MLSS of liquid in the respirometric reactor was approximately 1,000 mg/L. 2.3. Experiments set-up 2.3.1. Operation of the reactor Prior to each experiment, the AS was washed with tap water in order to eliminate remaining oxygen consumable matters such as biodegradable organics and nitrogen compounds that could affect the final result. Subsequently, the AS was aerated and kept free of substrate for 20 minutes to reach the endogenous respiration phase. In each experiment, initial and final samples were taken. Substrate injection and sample taking were performed through a special entrance designed with flexible rubber by using the syringe, as seen in Fig. 1. SS, alkalinity, ammonia, nitrite and nitrate were analyzed according to APHA [7]. Each respirometric experiment was divided into five stages, as shown in Fig. 2, (A) inoculation of the reactor with the washed AS, (B) aeration until reaching the maximum DO level, (C) end of aeration and initiation of the endogenous respiration phase, (D) substrate injection and initiation of the reaction phase and (E) application of intermittent aeration, measurement and evaluation of oxygen consumption over time [8]. Intermittent aeration allowed five respirometric assays per experiment.

Figure 2. Five stages of a respirometric experiment.

2.3.2. Oxygen consumption partition This assay was conducted in stage C of the respirometric experiment. The respirometric reactor was operated with the samples prepared in three following different phases: 1) AS without substrates (endogenous phase) in 20 minutes; 2) AS after nitrite injection (nitratation phase) to investigate NOB activity in 10 minutes; 3) AS after injection of both nitrite and ammonia (nitritation phase). OUR monitoring of all phases was conducted over 4 hours at 30 ± 1oC. Table 1 shows schematically the terms of OURs for the individual conditions. 3


Table 1. Expected oxygen uptake by phases in respirometric reactor Synthetic water

Landfill leachate

Endogenous phase

OURe

OURe + OURb

Nitratation phase

OURe + OURNOB

OURe + OURb + OURNOB

Nitritation phase OURe + OURNOB + OURAOB OURe: Endogenous oxygen uptake rate OURb: Biological oxidation oxygen uptake rate OURAOB: Ammonia oxidation oxygen uptake rate OURNOB: Nitrite oxidation oxygen uptake rate

OURe + OURb + OURNOB + OURAOB

2.3.3. Respirometric assays The kinetic of nitrification process is dependent mainly on substrate and DO concentrations. It is normally described by a Double Monod expression (Eq.(3)). For each experiment, the data were fitted to Eq.(3) using a nonlinear regression module in SIGMAPLOT software to determine maximum SOUR and half saturation coefficients of AOB.

 [DO]  [S]  SOUR  SOUR max     [DO]  K O  [S]  KS 

(3)

Where SOUR is specific oxygen uptake rate for nitritation (mg O2/mgVSS/h), SOURmax is maximum SOUR for nitritation, [S] is substrate concentration (mg N-NH4+/L), [DO] is DO concentration (mg O2/L), KS is half saturation coefficient for substrate (mg N-NH4+/L), and KO is half saturation coefficient for oxygen (mg O2/L). To investigate the effect of substrate concentrations, two tests, using synthetic wastewater and landfill leachate, were conducted in stage (E). Five initial ammonia concentrations of 10, 200, 800, 1600 and 3200 mg N-NH4/L were used to study the influence of ammonia on nitritation rate. To avoid the occurrence of DO limitation in the vessel, the experiment was terminated at 4.0 mg O2/L. The initial DO was at 8.0 mg O2/L. Experiments to examine the effect of bulk DO concentration were conducted in the same manner as described above. An initial ammonia concentration of 1600 mg N-NH4/L was chosen for both synthetic wastewater and landfill leachate tests. 3. RESULTS AND DICUSSION 3.1. Oxygen consumption partition Partitioning the oxygen consumption in the respirometric reactor, it was found that, in experiments with landfill leachate, total 88.86% of the oxygen was consumed for nitrification, including the oxidation of ammonia (88.74%) and nitrite (0.12%). In addition, 8.75 and 2.39% of the total oxygen consumption was used for endogenous oxidation and BOD, respectively (Figure 3). The result of respirometric tests presented that insignificant shares of total oxygen amount were used for NOB in both leachate (0.05%) and synthetic wastewater (0.12%). This points out that there is no significant activity of NOB in the partial nitritation bioreactor. In addition, 2.39% of the total oxygen consumption was used for COD oxidation in tests with 4


landfill leachate. Thus, it showed that heterotrophic micro-organisms existed unremarkably in the partial nitritation bioreactor.

Figure 3. Partitioning of oxygen consumption with synthetic wastewater (a) and landfill leachate (b)

0.16

0.16

0.14

0.14

0.12 0.10 0.08 0.06 0.04

Landfill leachate Synthetic water Synthetic water - Monod fit Landfill leachate - Monod fit

0.02

SOUR (mgO2/mgVSS/h)

SOUR (mgO2/mgVSS/h)

3.2. Effects of ammonia and DO concentration on AOB activity

Landfill leachate Synthetic water Synthetic water - Monod fit Landfill leachate - Monod fit

0.12 0.10 0.08 0.06 0.04 0.02 0.00

0.00 0

1000

2000

0

3000

2

4

6

8

DO (mg/L)

N-NH 4 (mg/L)

Figure 4. Determination of KS, SOURmax,S (a) and KO, SOURmax,DO (b) for synthetic wastewater and landfill leachate

Fig. 4a shows the average SOUR for SW and LC of AOB as a function of substrate concentration. The error bars represent the minimum and maximum values. The SOUR of both medium followed a Monod type equation with respect to substrate concentration. At substrate concentrations of 0 - 150 mg N-NH4/L for SW and 0 - 60 mg N-NH4/L for LC, the AOB growth was limited by substrate and SOUR depended on the substrate concentration. At higher substrate concentrations (>150mg N-NH4/L for SW and >60 mg N-NH4/L for LC), their growth and SOUR reached the maximum and were independent from substrate concentration. The Monod fitting parameters of the data are summarized in Table 2. Effects of DO concentration on SOUR for nitritation are presented in Fig. 4b. Note that there are no error bars in this graph because the measurement of DO concentration could not be controlled to be at the same values 5


between the duplicate experiments. As shown in Fig. 4b, the nitritation activity was affected differently by DO between SW and LC. For LC, at DO < 2 mg/L, the SOUR for nitritation were limited by the DO concentration. At DO > 2 mg/L, the SOUR was maximum and remained constantly. The SOURmax values of SW were higher than those of LC. Table 2. Summary of Monod kinetic coefficients Parameter

Unit

Synthetic water (R2)

Landfill leachate (R2)

Salinity

ppt

3.5

20.4

KO

mg O2/L

1.67 ± 0.36 (0.89)

1.35 ± 0.24 (0.85)

KS

mg N-NH4+/L

SOURmax,DO SOURmax,S

165 ± 29 (0.96)

53 ± 6 (0.98)

mg O2/mg VSS.h

-1

0.141 ± 0.011 (0.89)

0.066 ± 0.004 (0.85)

mg O2/mg VSS.h

-1

0.144 ± 0.005 (0.96)

0.059 ± 0.001 (0.98)

3.3. Calculation of kinetic coefficients of AOB In respirometric assay with landfill leachate, the solution in reactor was sampled twice to analyze pH values, ammonia, nitrite and nitrate nitrogen. The result showed that DO, ammonia nitrogen, alkalinity were decreased by metabolism activities of AOB which contribute to the accumulation of nitrite while nitrate was almost unchanged. Alkaline Yield coefficient Yalk/N (6.3 mgCaCO3/mgN-NH4+) was matched with the current operating conditions of the PNSBR and nearly equal to the theoretical requirement of full nitrification process (7 mgCaCO3/mgNNH4+). The oxygen demand of a reduced nitrogen species in this study was 2.9 mgO2/mgNNH4+ which was different from those in theory (3.43 mg O2/ mg N-NH4+) while previous studies lies between 3.0 - 3.2 mgO2/mgN-NH4+ [2,6]. This happened since total amount ammonia uptake consists of oxidized ammonia to nitrite and assimilated ammonia into cells without oxygen consumption. Chadran and Smets using electron flow of the ammonia oxidation process to present a mechanistic approach estimating bacteria growth rate [5]. Due to numerical correlation between µmax and fS in Monod-type functions, only the parameter combination representing maximum specific oxygen uptake rate (SOURmax) is uniquely identifiable from batch respirograms. When the change in AS concentration is negligible during a batch respirometric assay, the maximum specific growth rates for nitritation, are stoichiometrically related to the maximum SOUR as max 

fS SOUR max   0.12 day1 1  fS 1.42

(4)

The COD per unit mass of VSS and for Nitrosomonas sp. C5H7O2N was 1.42 mg-XCOD/mg VSS [9]. The AS yield coefficient can be calculated as follow: YX/ N  YO/ N 

max  0.26 mg VSS/mg N-NH4 SOUR max

The obtained µmax was considerably smaller compared to Blackburne et al. study [10] which was 0.54 day-1 while YX/N was higher than the reported value in the same study (0.14 mgVSS/mgN-NH4+). 6

(5)


4. CONCLUSIONS The respirometric assays proved to be a quick and reproducible method for calculating the kinetic coefficients of nitritation, without the need for an analytical measurement of concentrations of various nitrogen compounds. Furthermore, through stoichiometric links between nitrogen removal and oxygen consumption during nitritation, the maximum growth rate of AOB can be determined, making this method a more economical alternative compared to the on-line measurement tools available on the market. The method was successfully demonstrated with two different media whose values were summarized as follow. Maximum specific oxygen uptake rate, oxygen half-saturated constant, and ammonia half-saturated constant of AOB in synthetic wastewater were 0.143 mgO2/mgVSS.h-1, 1.55 mgO2/L, and 154 mgN-NH4+/L respectively, whereas the corresponding values in landfill leachate were 0.054 mgO2/mgVSS.h-1, 1.07 mgO2/L, and 66 mgN-NH4+/L respectively. Maximum growth rate µmax and yield coefficient YX/N of AOB in landfill leachate were found to be 0.12 day-1 and 0.26 mgVSS/mgN-NH4+. REFERENCES 1. Renou S., Givaudan J.G., Poulain S., Dirassouyan F., Moulin P. Landfill leachate treatment: Review and opportunity. J. Hazardous Materials, 150 (2008) 468–493. 2. Schmidt I. Sliekers O., Schmid M., Bock E., Fuerst J., Kuenen J., Jetten M., Strous M. New concepts of microbial treatment processes for the nitrogen removal in wastewater. FEMS Microbio, 27 (2003) 481-492. 3. Sri Shalini S., Joseph K. Nitrogen management in landfill leachate: Application of SHARON, ANAMMOX and combined SHARON–ANAMMOX process. Waste Management, 32 (12) (2012) 2385–2400. 4. Ficara E., Musumeci A., Rozzi A. Comparison and combination of titrimetric and respirometric techniques to estimate nitrification kinetics parameters. Water SA, 26 (2) (2000) 217-224. 5. Chandran K., Smets B. Estimating biomass yield coefficients for autotrophic ammonia and nitrite oxidation from batch respirograms. Wat Res, 35 (13) (2001) 3153–3156. 6. Ciudad G., Rubilara O., Munoza P., Ruiz G., Chamy R., Vergara C., Jeison D. Partial nitriﬁcation of high ammonia concentration wastewater as a part of a shortcut biological nitrogen removal process. Process Biochemistry, 40 (2004) 1715–1719. 7. APHA. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association American Water Works Association, Water Environment Federation, Washington, 1999. 8. Ciudad G., Werner A., Bornhardt C., Munoz C., Antileo C. Differential kinetics of ammonia- and nitrite-oxidizing bacteria: A simple kinetic study based on oxygen affinity and proton release during nitrification. Process Biochem, 41 (2006) 1764–1772. 9. Contreras E., Ruiz F., Bertola N. A modified method to determine biomass concentration as COD in pure cultures and in activated sludge systems. Water SA, 28 (4) (2002) 463-468. 10. Blackburne R., Vadivelu V.M., Zhiguo Y., Keller J. Determination of Growth Rate and Yield of Nitrifying Bacteria by Measuring Carbon Dioxide Uptake Rate. Water Environ Res, 79 (2007) 2437-45. 7