Soil Biol. Biochem.Vol. 19. No. 6, pp. 66S-6-672.

1987 0038-0717/8753.00 + 0.00

Pvintcdin Great Brimin. All rights-cd Copyright c 1987 Pergamon Journals Ltd

INTERRELATIONSHIP BETWEEN pH AND SURFACE

GROWTH OF NITROBACTER
G. A. KEEN and J. I. PROSSER
Department of Genetics and Microbiology, Marischal College, University of Aberdeen,
Aberdeen AB9 IAS, Scotland

(Accepted 10 March 1987)

Stunmary-The effect of pH on surface growth and activity of Nitrobacter was studied in batch and
continuous flow systems and on glass coverslips and anion-exchange resin beads. In batch culture with
an initial nitrite concentration of 50 pg NO?-N ml-’ freely-suspended cells had a pH optimum of 7.5 and
a pH minimum of 6, which was reduced to 5.5 at lower initial nitrite concentrations. Free cells grew in
continuous culture at pH 53 and nitrite oxidation rate per cell decreased with decreasing pH. Attachment
of Nitrobacter to anion-exchange resin beads increased with pH over the range 5.5-8.0. No such increase
was observed on glass coverslips but attached cells grew approximately 20% faster than free cells.
Attachment to glass did not affect the pH optimum or minimum in batch culture. In continuous flow
culture, the nitrite-oxidizing activity of attached ceils was less than that of free cells and responses to
transient changes in pH were reduced. Established attached cells were covered in extracellular slime
material and significant nitrite oxidation occurred at pH 4.5. Surface growth was the major factor in the
response of Nitrobacter to pH and allows for the possibility of autotrophic nitrification in acid soils.

lNTRODUCTlON
The conversion of ammonium to nitrate, via nitrite,
by autotrophic nitrifying bacteria is generally be-
lieved to occur in the soil at neutral to alkaline pH
values, while nitrilication in acid soils is thought to
result from the activity of heterotrophic organisms,
oxidizing either ammonium or nitrite through sec-
ondary metabolic processes (Focht and Verstraete,
1977). This belief arises mainly from observations of
growth of nitrifying bacteria in liquid batch culture,
where reported optima for Nitrosomonas and Nitro-
butter lie in the ranges 7.5-9.0 and 7.0-9.3 re-
spectively (Painter, 1986). Nitrite oxidation is re-
duced at alkaline pH through competitive inhibition
between NO; and OH-, while inhibition at low pH
is due to formation of free nitrous acid.
There are three major differences of particular
relevance between the soil, other natural environ-
ments and the liquid batch culture systems used for
determination of pH profiles. Firstly. concentrations
of nitrite in the soil are usually less than 1 pg
NOT-N ml-’ and are much lower than those used in
laboratory culture media, usually SO-200 c(g NO;-N
ml-‘. Inhibition by nitrous acid at low pH may
therefore be reduced in the soil. Secondly, the physio-
logical state of the organisms may be different as, in
the soil, cells will exist in a variety of conditions
ranging from feast and famine type situations to
prolonged growth at sub-optimal rates, with a con-
tinuous supply of substrate at growth-limiting con-
centrations. Thirdly, microbial cells in the soil will
exist at a solid-liquid interface and there is strong
evidence that soil nitrification is associated with the
surface of soil particles (Lees and Quastel, 1946). The
effects of surface attachment on microbial growth
and activity are many and appear to vary with the
665
organisms, the nature of the surface and other factors
(Fletcher, 1985). Surface attachment may stimulate
or inhibit growth and activity, but there are few
reports on the effects of surfaces on growth at
different pH and on growth of nitrite-oxidizing bac-
teria and pH may also influence the degree of revers-
ible attachment of microorganisms by altering the cell
surface charge. In turn, the pH-activity response of
attached cells has been reported to differ from that of
free cells, as demonstrated with Escherichia coli, and
Micrococcus luteus attached to anion-exchange resin
(Hattori and Furusaka, 1960; Hattori and Hattori,
1963) and Nitrobacter agilis attached to cation-
exchange resins and soil particles (McLaren and
Skujins, 1963). The effect of pH on microbial growth
and activity in homogeneous liquid culture may
therefore be very different to that in the soil.
It is impossible to study these factors in soil and we
have attempted to isolate them using model labora-
tory systems and a pure culture of Nitrobacter.
Growth at low nitrite concentrations was studied
in batch and continuous culture and the latter was
also used to study cells growing at sub-optimal
growth rates. Attached growth was studied on glass
coverslips and on anion-exchange resins, providing
surfaces of opposite charge and of defined chemical
composition. Anion-exchange resins provide ideal
surfaces for adsorption and growth of Nitrobacter
(Keen and Prosser, 1987) and have been used here to
study growth in an air-lift column fermenter in which
resin beads were circulated such that the liquid phase
was homogeneous with respect to substrate and
product concentrations. This provides advantages
over continuous flow fixed columns (Prosser and
Bazin, 1987) where the establishment of gradients in
biomass, substrate and product concentrations com-
plicates analysis of data on activity of attached cells.
666 G. A. KEZN and
MATERIALS AND METHODS
A Nitrobaczer strain isolated from soil by Dr R. M.
Macdonald, Rothamsted Experimental Station,
Harpenden, was maintained and subcultured as de-
scribed by Keen and Ptosser (1987).
Batch culture
Growth in batch culture was investigated by inocu-
lating 5 ml of a late exponential phase culture of
Nitrobacter into a 250 ml Erlenmeyer flask containing
IOOml of inorganic growth medium (Skinner and
Walker, 1961) containing 5Opg NOT-N ml-’ as
sodium nitrite. Following autociaving at 120°C for
15 min, the pH was adjusted in the range 5.&-8.0 by
addition of either 0. I N HCl or 1.5% sodium carbon-
ate. Four replicates were used at each pH and flasks
were incubated at 30°C shaking at 200 revmin-i.
Samples (1 ml) were removed periodically for esti-
mation of nitrite and nitrate concentrations using a
Technicon Autoanalyzer II System. Attached cell
inocula were used to investigate growth at pH 6, 7
and 8 and were obtained by colonization of glass
microscope coverslips in a fed-batch system. Glass
microscope coverslips (Chance Propper Limited,
22 x 22 mm) were soaked overnight in nitric acid and
rinsed with the growth medium described above.
Three coverslips were then suspended by means of
nylon thread in 250 ml Erienmeyer flasks containing
150 ml growth medium at the required pH. Following
inoculation with IOml of an exponentially growing
culture of Nitrobacter, flasks were incubated at
I50 rev min-‘, 30°C. until nitrite was completely
oxidized. Spent growth medium was then syphoned
off and replaced with the same volume of fresh
growth medium. This procedure was repeated three
times and the final number of attached cells was
determined by ultrasonication followed by micro-
scopic enumeration using a counting chamber (Keen
and Prosser, 1988). The mean numbers of attached
cells per glass coverslip were 6.12 x 10’ (SE
0.21 x IO’), 5.76 x lO’(SE 0.13 x 10’) and 6.17 x 10’
(SE 0.23 x 10’) at pH 6, 7 and 8 respectively. Co-
lonized coverslips were then transferred aseptically to
Erlenmeyer flasks containing I50 ml growth medium
at the appropriate pH. Control flasks were inoculated
with the same number of free cells subcultured twice
in medium at the pH to be investigated. Samples were
removed and analyzed for nitrite and nitrate.
The effect of pH on the extent of colonization to
glass coverslips and anion-exchange resins was also
investigated in batch culture. Flasks were prepared
containing 150 ml medium and either one glass
coverslip, suspended as described above, or 0.2 g
anion~x~hange resin (Amberlite resin IRA-400,
chloride form. analytical grade, BDH Chemicals
Limited), which provided an equivalent surface area.
Resin beads were washed by boiling alternately in
0.01 N HCl and 0.1 N NaOH for 30 min followed by
washing in several changes of deionized water and
equilibration in growth medium of the appropriate
pH. Phenol red was excluded from the growth me-
dium as it concentrated at resin surfaces. Following
autoclaving, the pH of the medium was adjusted in
the range X5-8.0 with three replicates for each at-
tachment surface at each pH. Flasks were inoculated
J. 1. FROSER
with 10 ml of an ex~nentiaily-growing culture of
Nitrobacter that had been grown in medium of the
same pH and Rasks were then incubated. When
nitrite was completely utilized attached cell number
for each surface was determined by ultrasonication
and microscopic enumeration as described by Keen
and Prosser (1988).
Continuous culture
Continuous culture in the absence of particulate
material was investigated in an LH Engineering
Modular Fermenter System (Series II) modified to
reduce wall growth (Keen and Prosser, 1987). Culture
volume was 800ml and Skinner and Walker’s
medium containing 50 pg NO; -N ml-’ was supplied
continuously to give a dilution rate of 0.016 h-t. The
pH of the growth medium was maintained by addi-
tion of 5% sodium carbonate to the medium reser-
voir. Effluent was sampled continuously using an
LKB Ultrorac Fraction Collector every 1 or 2 h in
test tubes containing potassium ethyl xanthate which
prevented further oxidation. Samples were ana-
lyzed for nitrite and nitrate concentrations and total
cell concentration was determined by microscopic
enumeration. Establishment of steady-states was
assessed using Williams’ coefficient of sequential
variation (Keen and Presser, 1987).
Continuous culture in the presence of anion-
exchange resin beads was studied in an air-lift column
fermenter (Keen and Presser, 1988). Washed anion
exchange resin beads (5 g) were autoclaved within the
column fermenter containing 600 ml of Skinner and
Walker’s medium which was inoculated with IO ml of
an exponentially-growing culture of Nitrobacter. Fol-
lowing complete utilization of nitrite, fresh medium
was supplied to give a dilution rate of 0.085 h-r.
Effluent samples were collected and analyzed. In
addition, ion-exchange resin beads were sampled and
attached cell concentration determined. Beads were
also prepared for scanning electron microscopy by
critical point drying and were observed using a
Stereoscan electron microscope (Cambridge Scientific
Instruments). Changes in pH were imposed by alter-
ing the pH of the supplied medium.
RESULTS
Batch culture in the absence of surfaces
Growth in liquid batch culture was monitored by
changes in nitrate concentration which increased
exponentially following an initial lag phase. The
duration of the lag phase was pH dependent, in-
creasing as pH decreased below the optimum for
growth. Specific growth rate was calculated as the
slope of a semi-log plot of nitrate con~ntration vs
time during exponential production (Keen and Pros-
ser, 1987). A maximum specific growth of 0.0048 h-’
occurred at pH 7.5. There was a sharp cut-off at sub-
optimal pH, and although the specific growth rate at
pH 6 was 58.7% of the maximum, there was no
nitrite oxidation at pH 5.5. Inocula for this experi-
ment had been cultured at pH 8.0. A possible require-
ment for adaptation of inoculated cells to low pH was
tested for by inoculation of growth medium (5Opg
NO;-N ml-‘) with cells cultured at pH 6 and with
 pH and surface growth of Mrrobucfer 667

6
7

5 4.0
3.5
40 3.0
2.5
2.0
30
15

I I I

750 1000 1250 1500

Time (h)
Fig. I. Changes in nitrite concentration (O), Nifrobacrer cell concentration (0) and pH (0) in a
chemostat operated at a constant dilution rate of 0.016 h-l. Changes in pH were achieved by altering the
pH of the supplied growth medium at times indicated by arrows. Nitrite concentrations were determined
every l-l.5 h and data have been smoothed, to improve presentation, by plotting the means of groups
of 30 successive values.

cells taken from chemostat cultures growing at pH
5.5 (see below). Both inocula gave growth at pH 6 but
no nitrite was oxidized at pH 5.5. In order to test
whether the inability of Nitrobacter to grow in batch
culture at pH 5.5 was related to the initial nitrite
concentration as proposed by Anthonisen et al.
(1976), duplicate flasks containing growth medium at
pH 5.5 and nitrite concentrations in the range
S-50 pg NO;-N ml-‘, were inoculated with Nitro-
batter. Flasks were incubated in the normal manner
and nitrite was completely oxidized at ail nitrite
concentrations up to 40 pg NO;-N ml-’ but not at
50 pg NOT-N ml.
Continuous culture in the absence of surfaces
Nitrobacter was grown in homogeneous con-
tinuous culture at a dilution rate of 0.016 h-l, equiv-
alent to a specific growth rate 33% of the maximum
observed in batch culture and 22% of that reported
for this strain in continuous culture (Keen and Pros-
ser, 1987). Growth was followed by changes in total
cell concentration and nitrite concentration (Fig. I).
A steady state was first established at pH 8, and
subsequent changes in pH were achieved by adjust-
ment of the pH of the medium contained in the
reservoir. The pH of the medium in the culture vessel
did not, therefore, change in a step-wise fashion but
approached new values asymptotically.
Steady-states in nitrate concentration were estab-
lished at each pH studied down to and including pH
5.5 and steady-state data are presented in Table 1,
.with coefficients of sequential variation. These
coefficients were used to determine establishment of
steady states and steady-state nitrite concentrations.
Nitrite concentrations for each steady state were
significantly different and multiple steady states were
therefore obtained at pH 8. Nitrite oxidation rate per
cell decreased with pH but the occurrence of multiple
steady states prevents quantitative analysis of the
relationship. All changes in pH result&d in under-
shoots before establishment of new steady-state
nitrite concentrations, but total cell concentration
varied little between steady states, even at pH 5.5.
The transient response of nitrite concentration to
pH may be explained with reference to the pH profile
for Nitrobacter in batch culture. During both in-

Table I. Changes in steady-state nitrite concentration, coefficient of sequential variation and nitrite
oxidation rate per ccl1 for Nifrobucrer growing in a chcmostal and in an air-lift column fermenter containing
anionsxchanne resin beads at several sleadv slates and DH values
Sleady-state Coefficient Nitrite
Dilution nitrite of oxidation rate
rate concentration sequential per cell. per h
PH (h-l) bgNO;-N ml-‘) variation (pg NO,-N cell-’ h-l)

Chcmostat 8.0 0.016 19.2 0.017 0.122

6.0 0.016 21.2 0.012 0.119
8.0 0.016 14.5 0.013 0.140
5.5 0.016 30.5 0.017 0.102

Air-lift column 8.0 0.085 4.5 0.024 0.052

fermenter 6.0 0.085 1.7 0.021 0.086
8.0 0.085 0.4 0.019 0.075
4.5 0.042 5.7 0.271 0.046
8.0 0.016 5.5 0.017 0.041
5.5 0.016 21.3 0.022 0.034
 668 G. A. KEEN
creases and decreases in pH between values of 6 and
8, pH crossed the optimum for growth of 7.5. The
undershoots may be attributed to an increase in
growth rate as the pH moves towards the optimum
with a subsequent decrease as the pH moves beyond
this value. An undershoot may therefore occur fol-
lowing an increase or decrease in pH as long as the
optimum pH is within the range imposed. However,
the shape of undershoots varied with the direction of
the pH change. This appears to reflect the time at
which the pH crossed the optimum value, in com-
parison with the time taken for the total pH change.
The trough of the nitrite undershoot occcurred earlier
following the step decrease in pH from 8 to 6 than
that fo~Iowing the step increase in pH from 6 to 8.
Undershoots in nitrite concentration therefore reflect
the asymptotic nature of the pH change rather
than adjustments in metabolic activity of the cells. A
final pH reduction to 5 was imposed at 1600 h but
nitrite concentration increased and complete washout
occurred.
Batch culture in the presence of surfaces
The effect of pH on attachment to anion exchange
resin beads and glass microscope coverslips following
oxidation of 50 pg NO;-N ml-’ is illustrated in Fig.
2. The number of ceils attached to resin beads
increased signifi~ntly with pH in contrast to the
relatively smalt effect of pH on colonization of glass
coversfips. However, attachment to glass surfaces was
greater at low pH with no significant difference in
attachment to either surface at pH 6 and 6.5. At
neutral and alkaline pH values, therefore, anion-
exchange resins provided a much better surface for
attachment of Nitrobacter. Despite the small effect of
pH on colonization of glass coverslips, cells attached
to glass exhibited higher specific growth rates than
those of freely-suspended cells (Fig. 3) with specific
growth rates of attached cells 24.8, 24.6 and 20.4%
higher at pH values of 6, 7 and 8 respectively than
those of free cells (values were significantly different
at the 5, I and 5% levels of signifi~n~). Althou~
specific growth rates were higher, the pH profiles of
attached and freely-suspended cells Were similar, the
pH minima were unchanged and attached cells did
not grow in media of pH 5.5 and 5.
PH
Fig. 2. The effect of pH on attachment of Nirrobacrer to
glass coverslips (0) and anion-exchange resin beads (0).
Error bars represent standard errors.
and J. I. PROSER
PH
Fig. 3. Variation in the specific growth rate with pH of
Mrrobocrer with pH in suspended culture (0) and attached
to glass covenlips (0). Each value is the mean of three
replicates and error bars represent standard errors.
Continuous culture in the presence of surfaces
The effect on growth of Nitrobacter cells attached
to anion exchange resins was investigated in two
experiments involving air-lift column fermenters.
Steady-state values for nitrite concentration and
attached and free cefl con~nt~tions at each pH
investigated are given in Table 1.
Figure 4 illustrates changes in nitrite concentration
and in attached and free cell concentration following
changes in pH during the first of these experiments,
in which the system was operated at a constant
dilution rate of 0.085 h-l. This value is in excess of
the critical dilution rate at which freely-suspended
cells are washed out. The column had been operated
for 2600 h at pH 8 prior to data presented in Fig. 4.
The pH change from 8 to 6 at 100 h resulted in an
undershoot in nitrite concentration, which then fell to
a lower steady-state value of 1.7 fig NO;-N ml-‘.
Attached cell con~t~tions fell during the period of
the pH change, with a cor~~nding transient in-
crease in free cell concentration. On returning the pH
to 8 at 600 h, nitrite was completely oxidized for 16 h
and nitrite concentration then rose to the steady-state
concentration of 0.4 c(g NOT-N ml-‘. Nitrite concen-
tration showed little variation during the period of
the pH change from 8 to 5.5, fluctuating in the range
O-l fig NOT-N ml-‘. Following a reduction in pH to
3.5, nitrite concentration fell, but this was due to
chemical instability of nitrite below pH 4 rather than
to biological activity. The column was flushed with
three vofumes of fresh growth medium at pH 8 and
continuous flow was halted. Despite the low pH
encountered, reinoculation of the column was not
necessary and 5Opg NO;-N ml-’ was completely
oxidized within 14 days, after which the system was
again operated continuously at the lower dilution
rate of 0.042 h-l. On commencing continuous flow,
the pH was adjusted to 5.0. Apart from the small
initial transient increase in nitrite concentration,
complete nitrite oxidation was maintained as the pH
fell to 5, free cell concentration showed a slight but
not significant increase and attached cell concen-
tration did not change. A steady state was subse-
 pH and surface growth of Nitrobacter 669

Time Ih)

Fig. 4. Changes in nitrite ~n~nt~tion (CJ), free (tf) and attached (8) cell ~on~ntration and pH (@)
in an air-lift column fermenter inoculated with Nilrobafrer and containing anion-exchange resin beads.
Changes in pH were achieved by altering the pH of the supplied growth medium at times indicated by
arrows. Nitrite concentration is plotted as deskbed in Fig. 1. The dilution rate was 0.085 h-l until I200 h
after which washout occurred. The system was then operated in batch for 14 days followed by con!inuous
flow at a dilution rate of 0.042 h-‘.

quently established following a pH reduction to 4.5,
with no further change in free or attached cell
concentration and a steady-state nitrite concentration
of 5.7 pg NOT-N ml-’ was obtained.
To determine whether attached cells had become
adapted to or selected for growth at low pH, a sample
of resin beads was removed from the column at
1180 h and used to inoculate into flasks containing
IOOml growth medium (50~gNO;- ml-‘) at pH
values in the range 5-8. There was again no growth
in batch culture below pH 6.
The response to low pH was further investigated in
a second experiment at a dilution rate of 0.016 h-i.
A steady state was initially established at pH 8 and
a subsequent reduction in pH to 5.5 resulted in an
undershoot in nitrite concentration which fell to
complete oxidation before a steady state of
21.5 pg NOT-N ml-’ became established. Reduction
in pH was accompanied by a slight decrease in
attached cell concentration but free cell concentration
did not change significantly.
Changes in pH were frequently followed by under-
shoots in nitrite concentration before new steady
states became established, but these were sometimes
obscured by complete oxidation of nitrite. In some
cases monotonic changes in nitrite concentration
occurred but overshoots were never observed. As
discussed above, undershoots may be due to
changes in maximum specific growth rate and other
factors as pH optima are approached and passed.
Reduction in pH was, in one case, also associated
with a decrease in attached cell concentration and an
increase in free ceil concentration, indicating desorp.
tion of attached cells. This may correspond with
colonization of resin beads in batch culture, which
decreased with decreasing pH, but was not always
observed.
Table I gives values for steady-state data for both
air-lift column fermenter experiments including rates
of nitrite oxidation per cell. In both experiments,
most of the free cells would have originated from
attached cells through desorption, detachment and
shearing from surfaces and the ratio of attached-to-
free cells was high. Nitrate oxidation rate per cell was
therefore calculated by dividing the total nitrite oxi-
dixed (i.e. the product of dilution rate and steady-
state nitrate con~ntration) by the total number of
cells. Attached cell concentration was estimated from
the final attached cell count made during each steady
state and attached cell activity was based on the
assumption that all attached cells were viable and
oxidized nitrite. This assumption may not be valid
however, particularly for cells attached on the inside
of the biofilm or cells covered in slime material to
which the diffusion of substrates and oxygen may be
limited.
Attached cell activity increased from 0.052 pg N
cell-‘h-’ to 0.086pg N cell-‘h-i following the
reduction in pH from 8 to 6 in the first column
experiment. This contrasts with the effect of pH on
cell activity in homogen~~ continuous culture,
where cell activity decreased with decreasing pH and
in the second air-lift column fermenter experiment,
where attached cell activity fell with the decrease in
pH from 8 to 5.5. The increase in attached cell
activity in the first experiment may be related to the
release of attached cells following the change in pH
from 8 to 6. Attached cell concentration was high at
 G. A. m and 3. I. PRoSa

sved
Fig. 5. Scanning electron micrograph of an anion-exchange resin bead colonized by Nifrobacfer remo
from the air-lift column fermenter described in Fig. 4 at 980 h and prepared by critical point drying: (a)
3lar
a sparsely colonized region, (b) a heavily colonized area showing polysaccharide slime material and p’
orientation of some cells. Scale bars represent 2pm.
 pH and surface growth of
pH 8 and the surface coverage of 73% was estimated
assuming growth as a monolayer. Therefore, the
reduction in attached cell concentration may have
alleviated limitation due to diffusion of substrates,
enhancing the activity of the remaining attached
population. However, attached cell concentration
rose again following the increase in pH to 8 and cell
activity remained high. The effect of pH on activity
of attached cells appears to be complicated by other
factors such as the extent of colonization and the
history of the population, but in all cases was lower
than the activity of free cells at the same pH.
Attached cell concentrations were in the range
0.13-1.5 x lo8 cells cmm2 which, assuming growth as
a monolayer, would provide a 6575% coverage of
the surface. However, biomass was not distributed
evenly, with some areas sparsely populated while
others supported colonies several cells deep. Figure 5
shows examples of two such areas, with heavily-
colonized areas associated with extracellular slime
material and cells apparently attached in polar orien-
tation. Figure 5 also shows preferential development
of colonies in pitted regions at the resin surface which
may offer protection from abrasion and shearing
forces.
DISCUSSION
Optimum reported pH values for nitrification in
liquid culture are usually on the alkaline side of
neutrality. Direct comparison with other studies is
difficult due to the use of mixed inocula and different
growth media and incubation temperatures, but our
results are similar to published data.
The effect of pH on attachment to glass and
anion-exchange surfaces may be.explained in terms of
the variation in cell surface charge with pH. Most
reported isoelectric points for bacterial cells lie in the
acidic range and, although the isoelectric point for
Nitrobacter is not known, it is considered unlikely to
be above pH 6. Surface charge was unlikely to have
been reversed over the pH range investigated and it
is therefore proposed that the cell surface was nega-
tively charged and that enhanced attachment to
positively charged resin surfaces at alkaline pH values
was due to an increase in the magnitude of this
charge. Conversely electrostatic repulsion between
negatively charged cells and glass surfaces would
occur over the entire pH range investigated, resulting
in a smaller number of attached cells. Enhanced
attachment to glass surfaces at pH 6 and 6.5 may
result from a reduction in the net cell surface negative
charge at pH values closer to the isoelectric point. In
continuous culture studies, reductions in pH resulted,
in one case, in desorption of a proportion of the
attached population. This phenomenon was not
always observed and the effect of pH on established
attached populations is thought to be complicated by
irreversible attachment processes, such as extra-
cellular slime production. This has been shown (Keen
and Prosser. 1987) to provide long-term permanent
adhesion of Nitrobacter cells on anion-exchange resin
beads (Cox et al., 1980).
In addition to electrostatic interactions, other
forces may be important in the association between
Nitrobacter cells and anion-exchange resins. In par-
Mrrobucrer 671
ticular the attraction and repulsion of charged
substrates has been shown to be important in
colonization of anion- and cation-exchange resins
beads by Nitrosomonus and Nitrobucter (Underhill
and Prosser, 1987). Despite the relatively minor effect
of pH on numbers of cells attached to glass surfaces
compared to anion-exchange resins, those attached to
glass exhibited a specific growth rate between 20 and
25% greater than that of free cells. In common with
free cells, however, attached cells were incapable of
growth at pH values below 6 in batch culture. An
altered pH response of cells attached to various
surfaces, compared to that of free cells, has been
reported for several bacteria. Hattori and Furusaka
(1960) reported similar pH-activity curves for the
oxidation of a number of growth substrates by cells
of Escherichiu coli attached to anion-exchange resins
and free cells. However, the activity curves were
shifted to the alkaline side for attached cells and
maximum oxidation occurred at approximately I pH
unit higher for attached cells compared to free cells.
This was explained in terms of a cationic layer formed
outside an anionic layer on the surface of the resin,
such that attached ceils were exposed to a higher H+
concentration than free cells. Conversely, Hattori and
Hattori (1963) reported a pH optimum for succinate
oxidation by Pseudomonas jluorescens attached to
cation-exchange resins to be 1 pH unit lower than for
free cells. This was interpreted as the exposure of
attached cells to a lower H+ concentration than free
ceils. McLaren and Skujins (1963) reported a 0.5 pH
unit increase in pH optimum for nitrite oxidation by
Nitrobucter in soil, compared to liquid culture. In all
of these examples the altered activity of attached cells
was interpreted in terms of a pH difference of the
attachment surface compared to that of the bulk
phase. However, the enhanced specific growth rate of
Nitrobucter cells in our study was independent of pH
and both attached and free. cells exhibited the same
pH optimum. In addition the similar net charge of
glass surface and growth substrate, nitrite ions, does
not suggest that the enhanced rate is due to substrate
accumulation at the attachment surface. Therefore,
two major factors which are frequently quoted to
explain the alteration of microbial activity at solid
surface, concentration of nutrients and an altered
surface pH, do not appear to apply here for Nitro-
butter cells attached to glass surfaces.
Colonization of anion-exchange resins in batch
culture was reduced at lower pH values. This corre-
sponds with the release of attached cells in the first
column experiment following the pH reduction from
8 to 6. In both cases colonization was at an early
stage and a significant proportion of the cells may
have been only reversibly attached. Other reductions
in pH did not result in a decrease in attached cell
concentration or an increase in free cell concen-
tration. Attached cells removed from the first column
experiment at 980 h were observed by scanning
electron microscopy to be covered in slime material
and such cells were unlikely to be susceptible to pH
induced detachment.
The transient response of attached cells in con-
tinuous culture following changes in pH was more
variable than that of free cells. In both cases over-
shoots were never observed following an increase or
672 G. A. KaN and J. I.
decrease in pH but, while freely suspended cells
always exhibited undershoots, attached cells often
gave monotonic changes to new steady-state values.
For example the reduction in pH from 8 to 6 imposed
in the first column experiment resulted in an under-
shoot in nitrite concentration, although a similar pH
change from 8 to 5.5 imposed 800 h later did not
result in an undershoot. The largest undershoot was
observed when a reduction in pH was accompanied
by desorption of attached cells. The attached popu-
lation was therefore more robust to changes in en-
vironmental conditions, particularly when cells were
reversibly attached, and similar behaviour was ob-
served by Keen and Prosser (1987) and Bazin et al.
(1982) following step changes in dilution rate.
The three factors atTecting the influence of pH on
growth of Nitrobacter which were specifically in-
vestigated in our study were nitrite concentration, the
physiological state of the cells and surface growth.
Data from both batch and continuous culture
demonstrated that the pH minimum for growth could
be. reduced from 6 to 5.5 by reducing nitrite concen-
tration to 4Opg NO; ml-’ or below, but growth at
pH 5 was never observed in batch or continuous
culture in the absence of surfaces. This applied even
when freely suspended cells or cells attached to
anion-exchange resins taken from continuous culture
systems at pH 5.5 were used as inocula. Low nitrite
concentrations found in soil may therefore be
important in reducing pH minima but would not
explain nitrification in soils of pH less than 5.
The physiological state of the cells did not appear
to affect response to pH for freely-suspended cells but
was important for attached cells. Growth in batch
culture occurred down to pH 6 (initial nitrite concen-
tration 50 pg NOT-N ml-‘) or pH 5.5 (initial nitrite
concentration less than 40 pg NO, -N ml-‘), regard-
less of the source of inoculum. In continuous culture
freely-suspended cells grew at pH 5.5, because of
reduced nitrite concentration, but would not grow at
pH 5. Ceils attached to anion-exchange resins were
active at pH 5 and below, but only in continuous
culture. When used as inocula for batch culture,
growth did not occur below pH 6 (initial nitrite
concentration 50 pg NO, -N ml-‘).
The major factor affecting the response to pH
was surface growth, which allowed nitrite oxidation
in continuous culture down to a pH of 4.5. Our
experiments did not distinguish between growth and
activity and nitrite oxidation at pH 4.5 may be
possible without growth. However, the implications
for nitrite oxidation in soil still apply. Established
populations of Nitrobacter irreversibly adsorbed to
soil particles and provided with a continuous supply
of low concentrations of nitrite may be capable of
high rates of nitrite oxidation. There are many other
PROSER
factors which may influence activity in acid soils, e.g.
microenvironments of neutral or alkaline pH, selec-
tion of acidophilic populations, but our results indi-
cate that these considerations are not necessary to
explain activity of autotrophic nitrite oxidizers in
such soils.
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