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Interrelationship between pH and surface growth of Nitrobacter
Soil Biol. Biochem.Vol. 19. No. 6, pp. 66S-6-672. 1987 Pvintcdin Great Brimin. All rights-cd
INTERRELATIONSHIP GROWTH
0038-0717/8753.00 + 0.00 Copyright c 1987 Pergamon Journals
Ltd
BETWEEN pH AND SURFACE 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 believed 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 secondary 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 Nitrobutter lie in the ranges 7.5-9.0 and 7.0-9.3 respectively (Painter, 1986). Nitrite oxidation is reduced 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 environments 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 physiological 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 continuous supply of substrate at growth-limiting concentrations. 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 bacteria and pH may also influence the degree of reversible 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 cationexchange 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 laboratory 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 complicates analysis of data on activity of attached cells.
G. A.
666
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 described by Keen and Ptosser (1987). Batch culture
Growth in batch culture was investigated by inoculating 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 carbonate. 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 estimation 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 microscopic 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. Colonized 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 medium 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 attachment 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 addition of 5% sodium carbonate to the medium reservoir. 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 analyzed 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 anionexchange 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. Following 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 altering 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, increasing 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 Prosser, 1987). A maximum specific growth of 0.0048 h-’ occurred at pH 7.5. There was a sharp cut-off at suboptimal 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 experiment had been cultured at pH 8.0. A possible requirement 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
667
pH and surface growth of Mrrobucfer
6 7 6 5
4.0 3.5
40
3.0 2.5 2.0
30
15
I
1000
750 Time
I
I
1250
1500
(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 Nitrobatter. 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 continuous culture at a dilution rate of 0.016 h-l, equivalent 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 Prosser, 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 adjustment 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 established 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 undershoots 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 nitrite concentration bgNO;-N ml-‘)
Coefficient of sequential variation
Nitrite oxidation rate per cell. per h (pg NO,-N cell-’ h-l)
PH
Dilution rate (h-l)
Chcmostat
8.0 6.0 8.0 5.5
0.016 0.016 0.016 0.016
19.2 21.2 14.5 30.5
0.017 0.012 0.013 0.017
0.122 0.119 0.140 0.102
Air-lift column fermenter
8.0 6.0 8.0 4.5 8.0 5.5
0.085 0.085 0.085 0.042 0.016 0.016
4.5 1.7 0.4 5.7 5.5 21.3
0.024 0.021 0.019 0.271 0.017 0.022
0.052 0.086 0.075 0.046 0.041 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 following 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 comparison 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.
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.
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, anionexchange 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.
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 increase 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 concentration 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 concentration did not change. A steady state was subse-
pH and surface growth of Nitrobacter
Time
669
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 undershoots 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-tofree cells was high. Nitrate oxidation rate per cell was therefore calculated by dividing the total nitrite oxidixed (i.e. the product of dilution rate and steadystate 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 heavilycolonized areas associated with extracellular slime material and cells apparently attached in polar orientation. 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 negatively 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 extracellular 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 Nitrobutter cells attached to glass surfaces. Colonization of anion-exchange resins in batch culture was reduced at lower pH values. This corresponds 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 concentration. 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 continuous culture following changes in pH was more variable than that of free cells. In both cases overshoots 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 undershoot 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 population was therefore more robust to changes in environmental conditions, particularly when cells were reversibly attached, and similar behaviour was observed 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 investigated 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 concentration 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 concentration 50 pg NOT-N ml-‘) or pH 5.5 (initial nitrite concentration less than 40 pg NO, -N ml-‘), regardless 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, selection of acidophilic populations, but our results indicate that these considerations are not necessary to explain activity of autotrophic nitrite oxidizers in such soils.
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Bazin M. J., Cox D. J. and Scott R. 1. (1982) Nitrilication in a column reactor: limitations, transient behaviour and effect of growth on a solid substrate. Soil Biology & Biochemistry 14, 477-487. Cox D. J., Bazin M. J. and Gull K. (1980) Distribution of
bacteria in a continuous-flow
nitrification column. Soil
Biology & Biochemistry 12, 241-246.
Fletcher M. M. (1985)Effectof solid surfaces on the activity of attached bacteria. In Bacterial Adhesion: Mechanisms and Significance (D. C. Savage and M. M. Fletcher, Eds),
pp. 339-361. Plenum Pm&New York. Focht 0. 0. and Vestraete W. (1977) Biochemical ecology of nitrification and denitrification. Adrances in Microbial Ecology 1, 135-214. Hattoti T. and Furusaka C. (1960) Chemical activities of Lcherichia coli adsorbed on a.resin. Journol of Biochemistry (Tokyo) 48, 831-837. Hattori R. and Hattori T. (1963)The erect of a liauid-solid
interface on the life of micr&organisms. Ecoldgiccrl Reoiews 16, 63-70. Keen G. A. and Presser J. I. (1987) Steady state and transient growth of autotrophic nitrifying bacteria. Archives oi Microbioiogy 147, 73-79.
Keen G. A. and Presser J. 1. (1988) The surface growth and activity of Nilrobacrer. Mi&obiil Ecology lS;(In press). Lees H. and Quastel J. H. (1946) Biochemistry of nitrification in soil. 2. Site of soil nitrification. Biochemical Journal 40, 8 I S-823. McLaren A. D. and Skujins J. J. (1963) Nit&cation by Nitrobacter ugilis on surfaces and in soil with respect to hydrogen ion concentration. Canadian Journal of Microbiology 9, 729-73 I. Painter H. A. (1986) Nitrification in the treatment of sewage and waste-waters. In Nirrl$cation (J. I. Presser. Ed.), pp. 185-21 I. IRL, Oxford. Prosser J. 1. and Bazin M. J. (1987) The use of packed column reactors to study microbial nitrogen transformations in the soil. In- A Handbook of bborarory Svsrems for Microbial Ecosvsfem Research (J. kimpenny, Ed.), CRC Press; Florida (In press).
W. T.
Skinner F. A. and Walker N. (1961) Growth of Nirrosomonas europaea in batch and continuous culture. Archiu fur Mikrobiologie -38, 339-349.
Underhill S. E. and Presser J. I. (1987) Surface attachment of nitrifying bacteria and their inhibition by potassium ethyl _ xanthate. Mrcrobial Ecology 14, (In press).
INTERRELATIONSHIP GROWTH
0038-0717/8753.00 + 0.00 Copyright c 1987 Pergamon Journals
Ltd
BETWEEN pH AND SURFACE 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 believed 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 secondary 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 Nitrobutter lie in the ranges 7.5-9.0 and 7.0-9.3 respectively (Painter, 1986). Nitrite oxidation is reduced 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 environments 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 physiological 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 continuous supply of substrate at growth-limiting concentrations. 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 bacteria and pH may also influence the degree of reversible 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 cationexchange 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 laboratory 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 complicates analysis of data on activity of attached cells.
G. A.
666
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 described by Keen and Ptosser (1987). Batch culture
Growth in batch culture was investigated by inoculating 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 carbonate. 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 estimation 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 microscopic 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. Colonized 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 medium 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 attachment 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 addition of 5% sodium carbonate to the medium reservoir. 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 analyzed 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 anionexchange 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. Following 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 altering 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, increasing 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 Prosser, 1987). A maximum specific growth of 0.0048 h-’ occurred at pH 7.5. There was a sharp cut-off at suboptimal 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 experiment had been cultured at pH 8.0. A possible requirement 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
667
pH and surface growth of Mrrobucfer
6 7 6 5
4.0 3.5
40
3.0 2.5 2.0
30
15
I
1000
750 Time
I
I
1250
1500
(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 Nitrobatter. 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 continuous culture at a dilution rate of 0.016 h-l, equivalent 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 Prosser, 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 adjustment 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 established 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 undershoots 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 nitrite concentration bgNO;-N ml-‘)
Coefficient of sequential variation
Nitrite oxidation rate per cell. per h (pg NO,-N cell-’ h-l)
PH
Dilution rate (h-l)
Chcmostat
8.0 6.0 8.0 5.5
0.016 0.016 0.016 0.016
19.2 21.2 14.5 30.5
0.017 0.012 0.013 0.017
0.122 0.119 0.140 0.102
Air-lift column fermenter
8.0 6.0 8.0 4.5 8.0 5.5
0.085 0.085 0.085 0.042 0.016 0.016
4.5 1.7 0.4 5.7 5.5 21.3
0.024 0.021 0.019 0.271 0.017 0.022
0.052 0.086 0.075 0.046 0.041 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 following 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 comparison 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.
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.
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, anionexchange 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.
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 increase 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 concentration 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 concentration did not change. A steady state was subse-
pH and surface growth of Nitrobacter
Time
669
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 undershoots 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-tofree cells was high. Nitrate oxidation rate per cell was therefore calculated by dividing the total nitrite oxidixed (i.e. the product of dilution rate and steadystate 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 heavilycolonized areas associated with extracellular slime material and cells apparently attached in polar orientation. 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 negatively 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 extracellular 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 Nitrobutter cells attached to glass surfaces. Colonization of anion-exchange resins in batch culture was reduced at lower pH values. This corresponds 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 concentration. 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 continuous culture following changes in pH was more variable than that of free cells. In both cases overshoots 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 undershoot 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 population was therefore more robust to changes in environmental conditions, particularly when cells were reversibly attached, and similar behaviour was observed 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 investigated 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 concentration 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 concentration 50 pg NOT-N ml-‘) or pH 5.5 (initial nitrite concentration less than 40 pg NO, -N ml-‘), regardless 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, selection of acidophilic populations, but our results indicate that these considerations are not necessary to explain activity of autotrophic nitrite oxidizers in such soils.
REFERENCES
Anthonisen A. C., Loehr R. C.. Prakasam T. B. S. and Srinath E. G. (1976) Inhibition of nitritication by ammonia and nitrous acid. Journo[ of rhe Wafer Pollution Control Federation 48, 835-852.
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