Biotechnology Letters 23: 1597–1602, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Nitrification rates in a saline medium at different dissolved oxygenconcentrations

Omar Sanchez1, Marıa C. Martı2, Estrella Aspe1 & Marlene Roeckel1,∗1Departamento de Ingenierıa Quımica, 2Departamento de Farmacologıa, Casilla 160-C, Universidad deConcepcion, Concepcion, Chile

Received 14 May 2001; Revisions requested 30 May 2001; Revisions received 23 July 2001; Accepted 23 July 2001

Key words: kinetics, nitrification, oxygen

Abstract

A kinetic study was carried out in a saline medium to assess the effect of O2 on the two-step nitrification process:for the first nitritation step, 2–26 mg dissolved O2 (DO) l−1 was used and for the second nitratation step, 0.5–24 mg DO l−1 was used. Nitritation rate was measured in the presence of sodium azide so as to inhibit nitratation.Ammonia-oxidizing (AOB) and nitrite-oxidizing (NOB) bacterial in the mixed culture were determined by 16 SrRNA hybridization. The affinity constants for oxygen O2 of the AOB and the NOB were 1.66 mg O2 l−1 and 3 mgO2 l−1 respectively. The larger than the previously reported values of these constants might be due to the high saltcontent in the medium. High O2 concentrations did inhibit the nitrification rate.

Introduction

Ammonia can be produced by the anaerobic treatmentof proteins in fishery effluents (Vidal et al. 1997).In this particular case, the effluent of the anaerobicreactor not only contains a high total ammonia con-centration, 1–1.5 g l−1 (Aspé et al. 1997), but also ishighly saline (24 g NaCl l−1; 1–3 g Na2SO4 l−1) asthe water fraction of the effluent being treated is sea-water. The disposal of this effluent to the coastal watercontributes to environmental unbalances, such as O2decrease, generation of anaerobic and highly reductiveenvironments and eutrophication.

A combined nitrifying and denitrifying biologicalprocess has been used for the ammonia removal ofeffluents from different sources. A sequential treat-ment that includes these processes is being studied atour laboratory to treat liquid residues from the fishprocessing industry.

According to the following sequence, and omit-ting intermediates such as NH2OH, NO2, N2O, thenet result is the ammonia transformation to elementalnitrogen:

NH+4 → NO−

2 → NO−3 → NO−

2 → N2. (1)

Nitrification is the biological conversion of ammo-nia to nitrate; nitrification involves two steps: nitrita-tion (transformation of ammonia to nitrite) and nitrata-tion (transformation of nitrite to nitrate). According tostudies carried out with molecular biology techniques,the predominant ammonia-oxidizing bacteria (AOB)in nitrifying reactors belong to the Nitrosomonas andNitrosospira genera whereas the nitrite-oxidizing bac-teria (NOB) belong to the Nitrospira and Nitrobactergenera (Schramm et al. 1998, Juretschko et al. 1998).

The nitrification rate at dissolved O2 (DO) concen-trations higher than the ones obtained by saturating asalt-free medium with air (7.54 mg l−1 at 30 ◦C) andthe variation in the nitrification rate at different O2concentrations in the presence of salt concentrationsnear the seawater content (24 g NaCl l−1) have beenscarcely addressed (Sharma & Ahlert 1977). Sten-strom & Poduska (1980) reviewed the literature on theeffect of the DO concentration on the nitrification rate.They concluded that the high variability of the resultscould be due to the effect of mass transfer limita-tion, as the experiments were performed with biomassflocs, and to the exclusion of the kinetic effect of theelectron donor in the analyses. Although later workshave addressed the effect of the O2 concentration on

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the process conversion, they did not focus on the de-termination of the kinetic parameters (Hanaki et al.1990, Stenstrom & Song 1991, Beccari et al. 1992,Lazarova et al. 1998). On the other hand, Jayamohanet al. (1988) and Laanbroeck et al. (1994) studiedthe affinity constants for O2 of the AOB and NOB,in mixed and pure cultures of Nitrosomonas europeaand Nitrobacter hamburgensis, respectively, but theirwork was performed in non-saline medium.

Thus, the goal of this work was to carry out a ki-netic study in a saline medium (24 mg NaCl l−1) of theeffect of DO concentration on the rates of the two stepsof the nitrification process. Based on these results, akinetic model is proposed that includes the effect ofthe DO concentration on the nitrification process rate.

Materials and methods

Nitrifying culture

Kinetic studies were carried out with cells collectedin cold (T = 5–8 ◦C) at the exit of a stable continu-ous reactor without sludge recycle (at least 8 monthsin steady-state operation), so as to ensure a constantspecific growth rate and a similar culture history forall the kinetic experimentation (Grady et al. 1996).This reactor had a useful volume of 10 l, 30 ◦C (con-trolled temperature), pH 7.5 (controlled), hydraulicretention time time of 55.5 h, and a 90% ammo-nia removal. The composition of the synthetic sub-strate, prepared with distilled water, was: 1.885 g(NH4)2SO4 l−1 (400 mg NH+

4 l−1 at pH = 7.5), 24 gNaCl l−1, 4.75 g Na2HCO3 l−1; microelements con-centrations are specified in Table 1. Measurementsof ammonium, nitrite and nitrate were performed us-ing a flow injection analysis (FIA) equipment (FossTecator, Sweden). In this equipment, ammonium isdetermined after its conversion in an alkaline mediumto gaseous ammonia, diffusion of the gas through amembrane, mixing with an acid-base indicator so-lution and spectrophotometric reading at 590 nm;nitrite is measured at 540 nm as the coloured prod-uct after reaction with sulfanilamide diazotized withN-(1-naphtyl)-ethylenediamine dihydrochloride; andnitrate is reduced to nitrite in a cadmium column andmeasured as described for nitrite (APHA 1985).

These analyses were carried out during the wholeexperimenting period so as to determine the nitrify-ing continuous reactor performance; the average outletconcentrations were: 8.5 mg N–NH+

4 l−1, 43.07 mg

Table 1. Concentration of the mi-croelements in the substrate.

Compound Concentration

(mg l−1)

MgSO4 · 7H2O 40

CaCl2 20

KH2PO4 100

FeSO4 · 7H2O 2.7

MnCl2 · 4H2O 0.1

CoCl2 · 6 H2O 0.024

NiCl2 · 6H2O 0.024

CuCl2 · 2H2O 0.017

ZnCl2 0.068

Na2MoO4 0.024

N-NO−2 l−1 and 313.5 mg N-NO−

3 l−1. The latterindicated that 90% of the fed nitrogen was being trans-formed to nitrite and nitrate indicating a stable reactoroperation and that nitrification was the predominantprocess taking place in the reactor.

Ammonia-oxidizing bacteria (AOB) andnitrite-oxidizing bacteria (NOB) consortium

The AOB and NOB consortium in the mixed cultureexiting from the continuous reactor was determined bythe dot blot hybridization technique with probes for16S-rRNA. RNA was extracted and purified accord-ing to Summers (1970) and hybridization was carriedout with 6 probes (Interactiva, Germany): EUB338,specific for all eubacteria (Schramm et al. 1998),NSO190 and NSO1225, specifics for AOB of theβ-Proteobacteria subclass (Schramm et al. 1998, Ju-retschko et al. 1998), NIT3, especific for the Nitrobac-ter genius (Juretschko et al. 1998) and NSR826 andNTSPA, specific for the Nitrospira genius (Hovanecet al. 1998). Hybridization was performed accordingto Sthal (1997). Probes intensities were determined byimage analyses. Probe EUB338 intensity was consid-ered as the total intensity, probe NSO190 intensity asthat of the AOB and the addition of probes NIT3 andNSR826 intensities as that of the NOB. To determineconsortium of AOB and NOB in the mixed culture,the intensity for each group was divided by the totalintensity.

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Stoichiometry

The stoichiometric coefficients relating O2 with am-monia and nitrite were determined by assays wherethe substrate, ammonia or nitrite was completely con-sumed. The stoichiometric coefficients for ammoniaand nitrite were determined by dividing O2 consump-tion by the amount of injected substrate.

Respirometry assays

The experimental procedure was based on the methoddescribed by Cech et al. (1984). The respirometriccell had a volume of 85 ml; temperature and pH werekept at 30 ◦C and 7.5 (buffer Tris/HCl), respectively.The initial biomass concentration was in the 1.4–1.6 gvolatile suspended solids (VSS) l−1 range and mea-sured by standard methods (APHA 1985). Assayswere started by filling the cell with a medium (Ta-ble 1) containing the mixed culture; the medium wasdevoid of either ammonia or nitrite. The DO concen-tration was raised up to the saturation point (34 mgl−1 at 30 ◦C) by injecting pure O2; the O2 supplywas stopped and the DO concentration measured asa function of time by means of a Biological OxygenMeter (Model 53, Yellows Springs Instruments, USA)on line with a PC. After 15 min had elapsed, 0.2 ml(NH4)2SO4 or NaNO2 solution was injected to givean initial concentration of 5 mg N-NH+

4 l−1 or 15 mgN-NO−

2 l−1 (T = 30 ◦C and pH = 7.5); the lattervalues were used to ensure substrate limitation and noinhibition of the ammonia or nitrite oxidation. At thesubstrate injection point the DO concentration variedbetween 0.5–24 mg DO l−1 for nitritation and 2–26 mg DO l−1 for nitratation. Sodium azide (1.56 mgl−1) was used as a nitratation inhibitor (Ginestet et al.1998) in experiments performed to measure ammoniaconsumption rate.

Substrate consumption rate

Figure 1 shows a typical respirometric assay and itsexperimental curve. From this curve and the oneobtained by applying the following equations the sub-strate consumption rate is:

rT = rC + rE , (2)

rC = rT − RE , (3)

dCC

dt= dCT

dt− rE , (4)

Fig. 1. Respirometric assay. The arrow indicates the point at whichthe substrate was injected to the system.

CC = CT − rEt , (5)

rN = rC

YNO

, (6)

where rc is the O2 consumption rate associated to thesubstrate consumption [mg O2 g−1 VSS min], rE isthe rate of endogenous respiration [mg O2 g−1 VSSmin], rN is the ammonia or nitrite consumption rate[mg DO g−1 VSS min], rT is the total rate of O2consumption [mg O2 g−1 VSS min], CC is the O2 con-centration corrected for endogenous respiration [mgDO l−1], CT is the O2 concentration before substrateinjection [mg DO l−1], YNO is the stoichiometriccoefficient [mg O2 mg−1 N] and t is the time [min].

Results and discussion

Stoichiometric coefficients (YNO )

Figures 2A and 2B show typical experiments for am-monia oxidation and for nitrite oxidation, respectively,from which the stoichiometric coefficients were de-termined. Values for ammonia oxidation and nitriteoxidation were YNO = 3.37 mg O2 mg−1 N-NH+

4and YNO = 0.95 mg O2 mg−1 N-NO−

2 , respectively.These values are in agreement with those reportedelsewhere: 3.22 mg O2 mg−1 N-NH+

4 (Drtil et al.1993), 3.16 mg O2 mg−1 N-NO−

2 (Wiesmann 1994),1.1 mg O2 mg−1 N-NO−

2 (Wiesmann 1994).

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Fig. 2. Respirometric assay to determine YNO . (A) Ammonia ox-idation (2.7 mg NH+

4 -N l−1, 1.5 g VSS l−1); (B) nitrite oxidation

(3.8 mg NO−2 -N l−1, 1.6 g VSS l−1). OC representas the oxygen

consumption.

Biomass for respirometric assays

Figure 3 shows a film with the spot correspondingto each probe used in this work. Quantification ofthese spots, by image analysis, showed that 92% ofthe EUB338 probe intensity was due to AOB andNOB. This means that a 16S-rRNA fraction does nothybridize with the used probes and, hence, a bacter-ial fraction that cannot be quantified by this method.The latter can be either AOB or NOB that do not be-long to the groups to which the probes are directed orheterotrophic bacteria. Quantification by image analy-ses showed that in the continuous reactor the AOBfraction was 0.57 while the NOB fraction was 0.35;furthermore, the latter fraction was made up of 10%Nitrobacter and 25% Nistrospira. No genus could be

Fig. 3. Spots for each probe in the hybridization assay. Num-bers identify probes: (1) EUB338; (2) EUB338; (3) NSO190;(4) NSO1225; (5) NIT3; (6) NSR826; (7) NTSPA. Spot (1) cor-responds to a E. coli standard while the remaining spots correspondto a sample from the continuous reactor.

specified for the AOB as the probes are not intendedfor discerning different strains but for AOB of the Ni-trosomonas, Nitrosospira, Nitrosovibrio, Nitrosolobusand Nitrosococus genera (Schramm et al. 1998).

Effect of the oxygen concentration on the kinetics ofnitrification

Figures 4 and 5 showed the nitritation and nitratationrate at different DO concentrations, respectively. The

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Fig. 4. Effect of the dissolved oxygen concentration on the nitrita-tion kinetics, pH = 7.5, T = 30 ◦C. (•), Experimental data; (–),values predicted by the model.

curves show a characteristic hyperbolic equation and,hence, data was modeled with the Monod equation:

rN = rNOCO

KO + CO

(7)

The term rNO includes the effect of the ammoniaconcentration (5 mg NH+

4 -N l−1) or the nitrite con-centration (15 mg NO−

2 N l−1) on the nitritation andnitratation rate, respectively. As this method measuresinitial rates, this term remains constant since all theexperiments were carried out at the same ammonia ornitrite concentration.

Ammonia and nitrite exert substrate inhibitionupon the AOB and NOB, respectively; therefore, theAndrews equation (Wiesmann 1994) has been used tomodel the relationship between the kinetics and theconsumption rate. In contrast to the Monod equation,the substrate consumption rate in the Andrews equa-tion is a function of the ammonia concentration inthe whole range and therefore, the RNO correspondsto the ammonia or nitrite consumption rate at the setconcentrations and do not reflect the maximal con-sumption rate. The ammonia and nitrite concentrationsused in the experiments were smaller than the experi-mentally determined concentrations for maximal rate,32.6 mg NH+

4 -N l−1 and 26.7 NO−2 -N l−1 (Sánchez

et al. 2000). This ensured that the assays were car-ried out in the substrate limitation zone and not inthe substrate inhibition zone. The rNO for ammoniaoxidation was 0.73 mg NH+

4 -N g−1 VSSAO min andKO = 1.66 mg O2 l−1 while for nitrite oxidation

Fig. 5. Effect of the dissolved oxygen concentration on the nitrata-tion kinetics, pH = 7.5, T = 30 ◦C. (•), Experimental data; (–),values predicted by the model.

the rNO was 1.75 mg NO−2 g−1 VSSNO min and

KO = 3 mg O2 l−1.The value of the affinity constant, KO , is larger

for the NOB than for the AOB, in agreement withresults reported in the literature (Table 2). However,the KO values obtained in this work for the AOB andNOB are larger than the ones reported in the literature;this might be explained by the different media used(24 g NaCl l−1) as compared to the works cited inTable 2, that were carried out in a salt-free medium.It has been reported that NaCl affects the nitrificationrate (Hunik et al. 1992, 1993); an increase in this saltconcentration usually diminishes the nitrification rate.The large KO value could reflect a decrease of affinitydue to the presence of high salt concentration in themedium and, hence, smaller nitrification rates at lowO2 concentrations. Furthermore, it has been reportedthat KO increases at increasing temperatures (Wies-mann 1994). KO values in Table 2 were reported fora temperature of 20 ◦C, while our data for estimatingKO were obtained at 30 ◦C.

The higher KO value for the NOB than the AOBimplies that upon an O2 decreasing, the nitrite oxida-tion is more affected than the ammonia oxidation and,hence, the nitrite concentration increases although themaximal consumption rate is larger for nitrite thanfor ammonia. This effect has been proposed as away to obtain a large nitrite concentration in nitri-fying reactors (Garrido et al. 1997); thus, a 50%conversion of nitrite and a total ammonia conversionhas been obtained. This favors a combined nitrifying-denitrifying treatment, as denitrification from nitrite

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Table 2. Affinity constant (KO ) values for oxygen of the am-monia-oxidizing bacteria (AOB) and nitrite oxidizing bacteria(NOB).

Consortium KO Referencesa

(mg O2 l−1)

AOB 0.3 Stenstrom & Poduska (1980)

NOB 0.83–2.46 Stenstrom & Poduska (1980)

AOB 0.6 Wiesmann (1994)

NOB 1.3 Wiesmann (1994)

AOB 0.3 Jayamohan et al. (1988)

NOB 1.1 Jayamohan et al. (1988)

AOB 1.66 This work

NOB 3 This work

aAt 20 ◦C, except this work.

is advantageous as compared to denitrification fromnitrate.

According to our results, for the two steps involvedin nitrification, high DO concentrations do not elicitsubstrate inhibition on the bacteria and its effect on thekinetics can be represented by the Monod equation.

Conclusions

The effect of DO in the kinetics of the two steps in-volved in the nitrification process may be representedby the Monod model, even at larger concentrationsthan the air saturation O2 concentration. This indicatesthat DO, at the studied concentration range, does notelicit substrate inhibition effects. The affinity constantfor the AOB is smaller than the affinity constant valuefor the NOB.

Acknowledgements

This work was possible through grants provided bythe Fondo de Desarrollo Científico y Tecnológico(Fondecyt 1980537 and Fondecyt 2990062).

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