Microelectrode measurements of the activity distribution
in nitrifying bacterial aggregates
Beer, de, D.; Heuvel, van den, J.C.; Ottengraf, S.P.P.
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Applied and Environmental Microbiology
Published: 01/01/1993
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Citation for published version (APA):
Beer, de, D., Heuvel, van den, J. C., & Ottengraf, S. P. P. (1993). Microelectrode measurements of the activity
distribution in nitrifying bacterial aggregates. Applied and Environmental Microbiology, 59(2), 573-579.
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Vol. 59, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1993, p. 573-579
0099-2240/93/020573-07$02.00/0
Microelectrode Measurements of the Activity Distribution in
Nitrifying Bacterial Aggregates
D. DE BEER,* J. C. VAN DEN HEUVEL, AND S. P. P. OTTENGRAF
Department of Chemical Engineering & Biotechnological Center, Nieuwe Achtergracht 166,
1018 WVAmsterdam, The Netherlands
Received 29 July 1992/Accepted 20 November 1992
Microelectrodes for ammonium, oxygen, nitrate, and pH were used to study nitrifying aggregates grown in
fluidized-bed reactor. Local reactant fluxes and distribution of microbial activity could be determined from
the microprofiles. The interfacial fluxes of the reactants closely reflected the stoichiometry of bacterial
nitrification. Both ammonium consumption and nitrate production were localized in the outer shells, with a
thickness of approximately 100 to 120 ,m, of the aggregates. Under conditions in which ammonium and oxygen
penetrated the whole aggregate, nitrification was restricted to this zone; oxygen was consumed in the central
parts of the aggregates as well, probably because of oxidation of dead biomass. A sudden increase of the oxygen
concentration to saturation (pure oxygen) was inhibitory to nitrification. The pH profiles showed acidification
in the aggregates, but not to an inhibitory level. The distribution of activity was determined by the penetration
depth of oxygen during aggregate development in the reactor. Mass transfer was significantly limited by the
boundary layer surrounding the aggregates. Microelectrode measurements showed that the thickness of this
layer was correlated with the diffusion coefficient of the species. Determination of the distribution of nitrifying
activity required the use of ammonium or nitrate microelectrodes, whereas the use of oxygen microelectrodes
alone would lead to erroneous results.
a
In response to environmental problems caused by strongly
increased ammonium emission by intensive agricultural and
industrial activities, wastewater (19) and waste gas (8) purification plants have been designed and applied to reduce
emission of nitrogen compounds. The ultimate removal of
nitrogen compounds can be established by dissimilative
reduction of nitrate to molecular nitrogen. Therefore, ammonium has to be oxidized to nitrate first, which can be
achieved by autotrophic nitrification. The reaction for the
oxidation of ammonium to nitrate is given by: NH4' + 202
+ H20 -+ N03- + 2H30+, thus leading to a stoichiometry
for NH4+-O2-NO3- of 1:2:1. During biological nitrification,
however, nitrogen is also incorporated in biomass (Nbio) and
additional oxidizing equivalents are available from CO2
fixation and reduction. For nitrification, including biomass
synthesis, a stoichiometry for NH4+-02-NO3 -NbiO of
1:1.83:0.98:0.021 has been suggested (10).
Since the growth rates and biomass yields of nitrifying
organisms are low, their application in continuous-flow
processes requires efficient retention of biomass, and development of bacterial aggregates with good settling properties
is needed. The design, scale-up, and operation of such
bioreactors require a profound understanding of biofilm
behavior. Therefore, information about the processes involved and the location of the processes in aggregates is
required. Up to now, most efforts have been directed to
mathematical modelling of nitrification by immobilized biocatalysts (11, 12, 22, 32). According to these studies, ammonium is rate limiting at bulk concentrations lower than 0.2
mM, for biofilms incubated in air saturated medium, because
of diffusive resistances. At higher ammonium concentrations, oxygen is the limiting substrate. Evidence for the
theoretical models has been provided by pilot studies and
correlation of literature data (29). Microprofiles of the reac*
tants inside the biofilm, however, give better insight into the
factors governing the process. Therefore, measurements
were done with ammonium (6) and nitrate (5) microelectrodes, enabling a spatial resolution in micrometers. With
oxygen microelectrode measurements, microprofiles of all
reactants and concomitant fluxes of the biological nitrification can be obtained. Moreover, direct measurement of the
thickness of the active part of the biofilm is possible.
In a reactor with immobilized bacteria, substrate transport
to the cells involves convective transport from the bulk
liquid to the vicinity of the biofilm, convective and diffusive
transport through a boundary layer, and diffusion through
the biofilm to the cells; products follow the same route in
opposite direction. The mass boundary layer 8 of a compound is the zone near the biofilm, where the local concentration gradually changes from the bulk liquid level to the
concentration at the surface of the particle. Because of this
layer, the substrate and product concentrations at the surface of the biofilm may differ considerably from the bulk
concentrations. The thickness of the mass boundary layer is
dependent on the hydrodynamics of the liquid and the
diffusion coefficient of the compound. The local substrate
concentration in the biofilm is determined by the reaction
rate and diffusion resistance. Therefore, transport through
the mass boundary layer must be taken into account if it
significantly contributes to the total diffusional resistance.
With microelectrodes, 8 can be measured directly.
The biological fluidized-bed process is a relatively new
technique in wastewater treatment. Applications have been
developed for anaerobic and aerobic processes, e.g., methanogenesis, acidification (14, 19), and oxidation of sulfide to
sulfate and of ammonium to nitrate (21). For the development of the nitrifying bacterial aggregates used in this study,
a two-phase (liquid-solid) fluid-bed nitrification reactor with
an external aerator was operated successfully. Due to the
relatively low shear forces, as compared with those in
three-phase systems, aggregates developed without addition
Corresponding author.
573
574
DE
APPL. ENVIRON. MICROBIOL.
BEER ET AL.
0
'
(9©
11
FIG. 1. Scheme of the nitrifying reactor setup: 1, fluidized bed
reactor; 2, medium inlet; 3, sink for washed-out biomass; 4, conditioning vessel for aeration and pH adjustment; 5, air inlet; 6,
recirculation loop; 7, effluent outlet.
of carrier material, thus facilitating measurements with fragile microelectrodes.
MATERIALS AND METHODS
Reactor operation. The reactor setup is presented schematically in Fig. 1. A conical 360-ml continuous-flow reactor
with a height of 0.8 m and internal diameters of 1 cm at the
bottom and 3 cm at the top was used. The reactor influent
contained NH4Cl (5.6 mM), Na2CO3 (30 mM), Na2SO4 (1
mM), K2HPO4 (5.7 x 10-1 mM), MgCl2 (8.36 x 10-2 mM),
FeCl3 (1.85 x 10-1 mM), CaCl2 (2 x 10-1 mM), MnCl2 (1 x
10-2 mM), and NaMoO4 (4 x 10' mM). The influent flow
rate was 2.14 x 10-2 ml/s. The liquid phase of the reactor
was recirculated at a rate of 1 ml/s via a 700-ml aeration tank.
The temperature was maintained at 30°C, and the pH was
kept at 8. The reactor was inoculated with activated sludge
from a municipal wastewater treatment plant (R.W.Z.I.oost, Amsterdam). The reactor was covered with black
paper to prevent growth of algae.
Microelectrode preparation and microprofile measurements. Ammonium, nitrate, and pH microelectrodes with tip
diameters of 1 ,um were prepared as described previously
(5-7). Oxygen electrodes with internal reference and a tip
diameter of 10 ,um were prepared (25). Measurements were
performed in a flow cell as described previously (6) at 30°C
with a flow rate of 5 x 10-2 ml/s, resulting in an average
liquid velocity of 0.5 mm/s. The medium used for profile
measurements consisted of NH4Cl (0.3 mM), NaNO3 (0.1
mM), Na2HPO4 (5.7 x 10-1 mM), MgCl2 (8.36 x 10-2 mM),
FeCl3 (1.85 x 10-1 mM), CaCl2 (2 x 10-1 mM), MnCl2 (1 x
10-2 mM), NaMoO4 (4 x 10-3 mM), and EDTA (2.7 x 10-1
mM) adjusted to pH 8. Potassium ions were not added to
avoid interference with the ammonium electrode. The electrodes penetrated the aggregates 90 to 1100 from the front
stagnation point of the flow on the particle; the direction was
almost perpendicular to the flow. Aggregates were sampled
from approximately 0.25 m of the reactor base. All microprofiles were recorded in different aggregates. The biofilmliquid interface was determined by using a dissection microscope, with a small depth of field, focused on the edge of the
aggregate. The microelectrode was brought into the focal
plane and advanced axially until it contacted the aggregate
edge.
Analytical methods. Nitrate and nitrite concentrations
were determined by high-pressure liquid chromatography
(Chrompack Ionospher tm A column; RI detector; ERC 7510
ERMA). Ammonium was determined colorimetrically
(Spectroquant 14752; Merck). Nitrogen incorporated in biomass was estimated from the dry weight washed out from the
reactor by using a microbial element analysis (27).
Calculations. Fluxes (Js) through the interface of aggregates with radius r were calculated by using Fick's first law:
Js = - Ds(dS/dr)r I R
(1)
where Ds is the molecular diffusion coefficient of compound
S in the liquid phase, r is the distance from the center, and
(dS/dr)rlR is the concentration gradient in the boundary
layer at the biofilm-liquid interface. The diffusion coefficients
of oxygen, ammonium, and nitrate at 30°C were taken at 2.72
x 10- , 1.8 x 10-9, and 1.6 x 10-9 m2/s, respectively (1).
Mass transfer between flowing medium and a solid surface
is determined by the thickness of the mass boundary layer
and therefore is dependent on both the diffusion coefficient
of the compound and the hydrodynamics. The hypothetical
boundary layer ah, the zone in the near vicinity of the biofilm
where mass transfer is governed by diffusion, is defined by
the slope of its concentration profile at the surface of the
biofilm (16):
ah
= (Sb - Si)l(dS/dr)r I R
(2)
where Sb is the bulk concentration at an infinite distance and
Si is the concentration at the biofilm-liquid interface. When
the relationship between mass transfer to both hydrodynamics and diffusivity is expressed as the Sherwood number
(Sh), the empirical equation for spheres in flowing liquid is
(24):
Sh = 2 + 0.59 Re'12 Sc113 = kdp/Ds
(3)
where Re is the Reynolds number (pudpl/', dimensionless
number defining the hydraulic regimen), Sc is Schmidt
number (-q/pD,, dimensionless number relating the hydrodynamic and the mass boundary layer), p is the liquid density,
,- is the dynamic liquid viscosity, k is the mass transfer
coefficient, and dp is the diameter of the particle. ah iS
calculated from:
k = DJSh
(4)
An analytical approximation was used for calculation of the
local mass boundary layer thickness 8, which is the distance
from the surface where the concentration equals 90% of the
bulk concentration (18):
8 = 1.266(DR2/u)13(0 - (sin29)/2)1/3/sinO
(5)
where 0 is the angle in radians from the front stagnation point
of the flow on the particle. This equation is only valid for
spherical particles under creeping-flow conditions (Re < 1),
i.e., when the streamlines on both sides of the equator
perpendicular to the flow direction are completely symmetrical.
RESULTS
Reactor performance. During start-up, the original sludge
used as the inoculum disappeared and disc-shaped, smooth
aggregates with a diameter of 1 to 3 mm developed (Fig. 2).
VOL. 59, 1993
ACTIVITY DISTRIBUTION OF NITRIFYING AGGREGATES
575
FIG. 2. Photograph of nitrifying aggregates.
Initially, the nitrite concentration was low, whereas conversion of ammonium to nitrate was optimal after 2 months and
remained stable for at least 9 months. The influent contained
5.6 mM ammonium; in the steady state, 10.26 mol m-3
oxygen was consumed, as calculated from the concentration
difference over the bed and the recirculation time. The
effluent contained approximately 0.1 to 0.3 mM ammonium,
c 0.05 mM nitrite, 0.24 mM biomass nitrogen, and 5.2 mM
nitrate. Thus, the conversion of ammonium to nitrate
amounted to 93%, and approximately 4% of the ammonium
was incorporated in biomass. The amount of ammonium
stripped out of the reactor was negligible. With an oxygen
microelectrode lowered carefully in the reactor, the oxygen
concentration at the sampling height was determined to be
approximately 0.1 mM. Occasionally, we observed protozoa, which were probably predating on the nitrifying population; these protozoa were killed by an anaerobic treatment
of the reactor for 1 h. After resumption of aeration, nitrification proceeded as before.
Microprofiles. The smooth surface of the aggregates facilitated accurate determination of the biofilm-liquid interface.
Simultaneous measurement of ammonium, oxygen, and nitrate profiles in one aggregate is difficult for technical reasons; therefore, three to five profiles of each reactant were
measured in separate aggregates. Some of these microprofiles, measured in air-saturated medium with an oxygen
concentration of 0.23 mM, are presented in Fig. 3. Clearly,
the ammonium and oxygen concentrations in the aggregates
decreased gradually going from the bulk liquid to the center
of the aggregates, whereas the nitrate concentration increased on the same route. Ammonium and nitrate profiles
indicated that under these conditions the nitrifying activity
was located mainly on the outside of the aggregate as a shell
approximately 100 to 120 1±m thick. The oxygen concentration at the 100-,um depth was low (0.04 mM). Therefore, the
oxygen supply seemed the limiting factor for nitrification,
although oxygen was present at a depth of 300 ,um. From
these profiles, the fluxes of the various compounds through
the interface were calculated; their mean values are presented in Table 1. The oxygen fluxes from the nitrifying
outer shell to the deeper non-nitrifying zone were calculated
by assuming that the intraparticle diffusion coefficient was
equal to the molecular diffusion coefficient. Oxygen fluxes
through the interface were corrected by subtracting the
fluxes from the active zone to the center, resulting in the
oxygen consumption in the nitrifying zone (Table 1). The
ratio of the uncorrected fluxes for NH4 -O2-NO3 was
0.40
NNH4+
4-
NO 3
0.30
0.20
[mol/m3]
0.10
0.00L
-500
-250
0
250
500
depth [umi
FIG. 3. Microprofiles of ammonium (+), oxygen (O), and nitrate
(E) in nitrifying bacterial aggregates with a diameter of 2 mm in
air-saturated medium. The dotted line indicates the 100-p.m depth in
aggregates.
576
APPL. ENVIRON. MICROBIOL.
DE BEER ET AL.
TABLE 1. Fluxes of ammonium, oxygen, and nitrate through the
aggregate-bulk liquid interface
Flux rate (10-7 mounM2 s) for:
Medium
Air saturated
Oxygen saturated
NH4+
02
N03
13.8 (3.4)a
28.8 (7.1)
24.8 (7.8)b
25.1 (6.1)
-11.5 (1.6)
8.9 (1.8)
1.15
02
NH44
NO3- 0.30
1.05
[morl/m3]
[mol/m']
0.95
-8.8 (2.8)
15.9 (5.9)"
0.85
a Numbers within parentheses indicate 90% probability intervals (t test of
significance).
b Values corrected for oxygen flux to non-nitrifying center.
0.00I
-250
-500
250
0
0.75
500
depth [um]
1:2.09:0.83, whereas the ratio for the corrected fluxes was
1:1.80:0.83.
Mass boundary layers were observed outside the aggregate mass, and the ammonium and oxygen concentrations at
the surface of the aggregate were considerably lower than
the bulk concentrations. Mean values of measured ah for
oxygen, nitrate, and ammonium and values calculated by
using equations 3 and 4 are summarized in Table 2. Values
for 8, measured as the distance from the aggregate surface
where the local concentration was 90% of the concentration
difference between the bulk and the interface (Sb - Sj) are
presented in Table 2. The Reynolds numbers in the flow cell
were calculated to be 5 for hydraulic flow and 2 for the
submerged aggregate placed in the flow cell. Therefore, the
flow regime in the cell and around the aggregate was laminar.
This was confirmed by observation of the pattern of ink
leaking out of ink-saturated spherical agar beads with a
diameter of 2.5 mm.
To investigate whether oxygen is indeed the limiting factor
for the ammonium oxidation, experiments were performed
in medium conditioned with pure oxygen at concentrations
of up to 1.15 mM. Some of the microprofiles of the reactants
measured under high-oxygen conditions and after an incubation period of at least 1 h are presented in Fig. 4. Oxygen
penetrated the whole aggregate; however, the nitrification
zone was still restricted to the outer 100 p.m of the aggregate,
as shown by the ammonium and nitrate profiles. The concomitant mean fluxes of the reactants through the interface
and those of oxygen corrected for the outgoing fluxes to the
non-nitrifying center (Table 1) did not increase when the
oxygen concentration in the bulk liquid was maintained at
the maximal level for 2 h. On the contrary, the activities of
the aggregates and the ammonium and nitrate fluxes were
significantly lower than those of aggregates under atmospheric oxygen conditions, with a probability of more than
0.95 (t test of significance). The oxygen flux through the
TABLE 2. Thickness of mass boundary layers around nitrifying
aggregates for ammonium, oxygen, and nitrate,a bh being the
hypothetical boundary layer and 8 the distance from the
aggregate surface where S = 0.9(Sb - Si)
Compound
8h (pLm) measured with:
'Tr/2 Equations 3 and 4
80 (6.4)
220
100 (21)
262
0
f
0
=
NH4+
02
213
88 (3.9)
N03
a,h is the hypothetical boundary layer, and
(pm) measured with:
Equation 5
rr/2
interface did not decrease significantly, according to this
test, because of the increased flux to the non-nitrifying
center. However, the net consumption in the active zone,
obtained after correction for the efflux to the non-nitrifying
center, did decrease significantly. The ratio of NH4+-O2NO3- for the uncorrected fluxes was 1:2.8:0.99, whereas
that for the corrected fluxes was 1:1.79:0.99.
Microprofiles of the pH in aggregates incubated under
low- and high-oxygen conditions are presented in Fig. 5. In
air-saturated medium containing 0.23 mM oxygen, the pH in
the biofilm decreased to 6.7 within the outer layer of 100 p.m.
In oxygen-saturated medium containing 1.15 mM oxygen,
the pH decrease in the biofilm was less pronounced, because
of decreased nitrification activity and levelled off at a depth
of 200 p.m to a value of 6.85.
DISCUSSION
for nitrification and biostoichiometry
The steady-state
mass synthesis in the fluid-bed reactor (NH4+-02-NOf3-Nbio)
was 1:1.84:0.94:0.04. The measured biomass yield was
higher than expected; however, within the limits of experimental accuracy the values found are close to those sug8.50
pH
8.00
7.50
-
7.00
=
180 (18)
213 (41)
150 (23)
f
FIG. 4. Microprofiles of ammonium (+), oxygen (O), and nitrate
(El) in nitrifying bacterial aggregates with a diameter of 2 mm in
oxygen-saturated medium. The dotted line indicates the 100-p.m
depth in aggregates.
225
258
216
is the distance from the
aggregate surface where S = 0.9(Sb - Si). Numbers within parentheses
indicate 90% probability intervals (t test of significance).
6.50
-500
-250
0
250
500
depth [um]
FIG. 5. pH microprofiles measured in nitrifying bacterial aggregates incubated in air-saturated (+) and oxygen-saturated (El) medium. The dotted line indicates the 100-p.m depth in aggregates.
VOL. 59, 1993
ACTIVITY DISTRIBUTION OF NITRIFYING AGGREGATES
gested previously (10). The ammonium removal was almost
complete and amounted to 98.2 to 99.5%. The nitrification
capacity of the reactor amounted to 3 x 10' mol/m3 s,
which is lower than the reported performance in a gas-lift
reactor (1.5 x 10'3 mol/m s) (19). The high recirculation/
influent ratio of 47, needed to supply sufficient oxygen for
complete ammonium oxidation, makes this reactor type
unattractive for industrial applications, since a comparable
system with pure oxygen and a recirculation ratio of 10 was
also rejected (19). Moreover, the possibilities for upscaling
the reactor are limited by the oxygen depletion at a reactor
height of less than 1 m. For industrial purposes, three-phase
fluidized bed reactors, with direct aeration of the reactor
content, have been designed and applied (19). However, the
reactor is highly suitable as a laboratory device because the
low shear forces enable the development of large aggregates.
Also the absence of carrier material that may damage the
fragile electrode tips is of considerable advantage for microelectrode studies.
From the microprofiles measured in air-saturated medium
(Fig. 3), it can be observed that the active nitrifying zone was
limited to the outer 100 to 120 p.m of the aggregates. Oxygen
was not depleted completely at 100 p.m depth but penetrated
into the non-nitrifying center of the aggregate, where it was
consumed by a process other than nitrification. As a result,
the oxygen flux through the aggregate interface was 15%
higher than that expected from the stoichiometry of the
nitrification reaction and the fluxes of ammonium and nitrate. Oxygen may have been limiting for the nitrification
rate, since the oxygen concentration at 100 pum depth came
close to its reported Ks (2 x 10-2 mM [29]). In that case, an
increase of the oxygen bulk concentration should result in an
increase of nitrifying activity. Microprofiles measured in
medium saturated with pure oxygen did not show an increase of ammonium and nitrate fluxes or an increase in the
thickness of the active zone. On the contrary, a decrease of
the fluxes of the reactants was observed. Nitrifying activity
was still limited to the outer 100-p.m zone of the aggregates,
although oxygen and ammonium concentrations in the whole
aggregate were high. The centers of the aggregates were
obviously inactive, whereas the active nitrifying bacteria
were located exclusively in the outer 100 p.m.
The distribution of activity might be explained by the
history of the aggregates in the reactor. The thickness of the
active zone of the sampled aggregates can be approximated
from the local reactor circumstances by assuming zero-order
kinetics for oxygen as the limiting substrate. Then the
relation between the interface concentration Si and penetration depth A is given by (23):
A = (2DeffSi/VO)0°5
(6)
where Deff is the effective diffusion coefficient in the biofilm
and V0 is the volumetric reaction rate. Aggregates were
sampled from the lower part of the reactor (0.25 m), where
the mean liquid velocity was 5 x 10-3 m/s. According to
equation 3, Sh was 16 and the resulting mass transfer
coefficient k for oxygen amounted to 2.16 x 10-5 mol/m2 s.
Since the active zone is small compared with the radius of
the particle, a flat film approximation can be used to calculate VO for which it can be deduced that
J= Vox
(7)
In the aggregates investigated, J was approximately 25 x
10- mol/M2 s (Table 1) and X was 10-4 m; therefore, VO for
oxygen consumption is 2.5 x 10-2 mM/s. At steady state the
577
flux through the interface equals the consumption in the
aggregate:
k-Trdp2(Sb - Si) = Voirdp2X
This, in combination with equation 6, results in
k(Sb
-
Si)
(2DeffVo)0 5(5i)05
(8)
(9)
At the relevant reactor height the liquid bulk oxygen concentration Sb was approximately 0.1 mM; therefore, from
equation 9 it follows that Si is approximately 2.5 x 10-2 mM.
According to equation 6, the penetration depth X for oxygen
is 73 p.m under reactor conditions. This value might be a
slight underestimation, because the assumption of zeroorder kinetics; however, it is close to the observed thickness
of the active nitrifying zone. Under the assumption that
ammonium is the limiting substrate, the same calculation
results in an ammonium penetration depth of 103 pum for the
lowest observed ammonium concentration in the reactor
bulk liquid of 0.1 mM. A comparison of the calculated
penetration depths of oxygen and ammonium shows good
evidence that oxygen is the limiting substrate. The thickness
of the active nitrifying zone is determined by the oxygen
supply to the aggregate, and the center of the aggregate
consisted of biomass inactivated by oxygen depletion. The
center probably consists of lysed cells, since it is relatively
flaccid compared with the outer shell and is easily washed
away from broken aggregates (Fig. 2).
For the flow cell, it is concluded that nitrification is limited
by the oxygen transport rate at ammonium concentrations
higher than 0.1 mM. When the dissolved oxygen concentration is lower than the air-saturated value, as occurs under
reactor conditions, it can be expected that the bulk liquid
concentration at which ammonium becomes limiting for
nitrification will even be lower than 0.1 mM. With the
calculated oxygen penetration depth of 73 p.m described
above, ammonium becomes limiting below a bulk liquid
concentration of 0.09 mM, when Si is 0.034 mM. The
observation of a significant mass boundary layer during the
microprofile measurements complicates the translation of
the presented results to reactor circumstances. In the reactor, liquid velocities are higher than those applied in the flow
cell, and consequently the mass boundary layer is smaller. In
the reactor, therefore, the concentrations of reactants at the
aggregate surface are closer to the bulk concentrations. It is
clear that the mass boundary layer may influence the conversion rate of nitrifying aggregates strongly. This phenomenon should be taken into account in the design of experiments and reactors.
There is no general consensus in the definition of 8, which
has been defined as the distance from the surface where the
concentration equals 100% (17, 28), 99% (26), and 90% (18)
of the bulk concentration. For the last case, an analytical
approximation for the local 8 was found for spherical particles under creeping-flow conditions, when Re < 1 (equation
5). The thus calculated values for 8 (Table 2) exceed the
measured values. This could be explained by the fact that
equation 5 is derived for creeping-flow conditions, whereas
the Re of aggregates in the flow cell was 2. An additional
explanation for the differences may be found in the geometry
of the aggregates. Calculations are based on the assumption
of perfect spheres, whereas the aggregates were irregularly
shaped and thus the flow pattern was influenced. Nevertheless, calculations lead to estimations that are correct within
1 order of magnitude. Furthermore, the prerequisite of
creeping flow may be to rigid, and equation 5 may be a
=
578
DE BEER ET AL.
sufficient approximation for flow conditions at which the
flow does not detach from the sphere; namely, for Re < 20
(4).
A different interpretation is given by the definition of the
hypothetical boundary layer ah: a zone in the vicinity of the
biofilm in which mass transfer is governed by diffusion only
(equation 2). This definition leads to a considerably smaller
thickness of 8 and was relevant for the stagnant liquid
conditions prevailing, as apparent from oxygen microelectrode measurements (16). In this study, the estimation of ah
from equations 3 and 4 was higher than the observed values.
It should be stressed that ah thus calculated for aggregates in
flowing media represents a mean value for the whole aggregate, whereas the actual local ah depends on the distance
from the front stagnation point of the liquid on the aggregate.
The slopes of the microprofiles in the boundary layer and
in the outer zone of the aggregate were approximately the
same, indicating that the effective diffusion coefficients in
nitrifying aggregates were close to the molecular diffusion
coefficients. The values in the aggregates may have been
slightly lower than the molecular diffusion coefficients.
Therefore, the oxygen fluxes to the non-nitrifying center
might be overestimated by 10 to 20%.
Microprofiles measured in medium saturated with pure
oxygen did not show the expected increase of ammonium
and nitrate fluxes or increase of the thickness of the active
zone. On the contrary, a decrease of the fluxes of the
reactants was observed. A reduced nitrifying activity was
found in the outer 100-,um zone of the aggregates, whereas
the aggregate center obviously consisted of inactive bacteria
that may have died by starvation. Moreover, high oxygen
concentrations appeared to inhibit nitrification. This is contrary to the results obtained with a Warburg respirometer in
a study in which the rates of ammonium oxidation by biofilm
material were measured under oxygen and normal air (13).
However, since the contents of the Warburg vessel were not
agitated, a considerable drop in the oxygen concentration in
the mass boundary layer might have protected the cells from
inhibition by oxygen. Inhibition of growth of Nitrobacter
cultures was observed under elevated oxygen concentrations (9). Rapid ammonium accumulation upon a sudden
increase of the oxygen concentration was observed during
mixed culture experiments; however, acclimatization of
nitrifiers to high oxygen concentrations was observed (3, 15).
The mechanism of the observed inhibition is unknown, but it
might be speculated that accumulation of free radicals plays
a role. Therefore, adaption of biofilms to high oxygen levels
may involve development of protective mechanisms such as
superoxide dismutase and catalase. The applied incubation
time in our studies (1 to 2 h) clearly is not sufficient for these
adaptions.
In air-saturated medium, the ratio of the reactant fluxes
approximated the stoichiometry of the nitrification reaction,
whereas in oxygen-saturated medium the oxygen flux was
relatively high. Approximately 30% of the oxygen flux in
aggregates incubated in oxygen-saturated medium can be
attributed to processes in the center of the aggregates, most
probably oxidation of dead biomass. Therefore, the thickness of the active nitrifying zone cannot be determined from
oxygen microprofiles under increased oxygen tensions.
Moreover, because of this phenomenon the results obtained
with biological oxygen monitors and Warburg vessels may
lead to erronous conclusions.
The reaction equation for nitrification indicates that H30O
is one of the products. Previous measurements in which a
pH macroelectrode was mounted to the back of a nitrifying
APPL. ENvIRON. MICROBIOL.
biofilm showed that the pH decreased as a result nitrifying
activity (30). Indeed, the pH microprofiles presented in this
report show a substantial decrease of the pH in the aggregates. The thickness of the active zone, as determined from
the pH microprofile measured with air-saturated medium,
also approximated 100 ,um. The profiles measured in oxygensaturated medium were less pronounced than those measured in air-saturated medium. It can be concluded that the
extent of the pH profiles reflected the nitrifying activity. The
unknown oxidation processes in the center of the aggregates
occurring under high oxygen tension had little influence on
the local pH. The decrease of the pH is not sufficient for
inhibition of nitrification, since nitrification will proceed
uninhibited in the pH range of 5.5 to 7.3 (2). Therefore, the
explanation for the restriction of activity to the outer 100-,m
layer must be the absence of nitrifying organisms in the
centers of the aggregates.
In some other studies indications of heterogeneous activity distribution in nitrifying aggregates were found. Development of a heterogeneous biomass distribution was found
in an artificial model system, namely, 2-mm spherical gels
containing immobilized Nitrobacter agilis cells with an initially homogeneous distribution. After a steady state was
reached, 90% of the immobilized cells were localized in the
outer shell of 140 ,um, as shown by image analysis (31).
Another study showed that the specific nitrifying activity of
biofilms decreased during biofilm growth; this decrease was
attributed to development of heterotrophic organisms, diffusion limitation, or segregation of microbial communities (20).
The critical biofilm thickness was reported to be 0.1 mm,
however, without supporting data (19). These observations
are confirmed, however, by the direct measurements presented in this study, which underlines the value of the
microelectrode technique.
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