Vol. 43, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1982, p. 542-5470099-2240/82/030542-06$02.00/0

Apparent and Measured Rates of Nitrification in theHypolimnion of a Mesotrophic Lake

GRAHAME H. HALLFreshwater Biological Association, Far Sawrey, Ambleside, Cumbria, U.K.

Received 22 June 1981/Accepted 7 November 1981

Three distinct phases were observed in the change of dissolved inorganicnitrogen concentrations in the hypolimnion of Grasmere. The second phase ofdecreasing ammonia and increasing nitrate concentrations was typical of thenitrification process. Observations on nitrate concentration gradients betweensurface sediments and the water column and experiments using the nitrificationinhibitor N-Serve indicated the in situ activity of chemolithotrophic nitrifyingorganisms. Nitrification rates were estimated throughout the period of stratifica-tion by using the N-Serve and [14C]bicarbonate uptake method. Comparison ofthe field nitrate concentrations with the predicted nitrate concentrations (fromestimates of the nitrification rate) indicated that the method underestimated thetrue rate of nitrification. Possible reasons for this are discussed.

Oxidized surface muds have been implicatedas the most important site of nitrification in lakes(13, 28, 39; N. Christofi, Ph.D. thesis, DundeeUniversity, Dundee, Scotland, 1978). Changesin nitrate concentration in the water columns ofcertain lakes, however, suggest that planktonicnitrification in aerobic hypolimnetic water canalso be important in affecting changes in waterchemistry and in deoxygenation of the hypolim-nion (9, 14, 21, 24, 35; P. L. Brezonik, Ph.D.thesis, University of Wisconsin, Madison,1968). Yet increases in nitrate concentrationsalone cannot be taken as a posteriori evidence ofin situ nitrifying activity. Seepage of groundwa-ter into a lake, for example, can add substantialquantities of nitrate; moreover, these sourcesare difficult to quantify (34). Simultaneous de-creases in ammonia concentration provide addi-tional evidence for bacterial involvement (24;Brezonik, Ph.D. thesis, 1968), but actual demon-stration of activity or measured rates of nitrifica-tion are required to provide unequivocable evi-dence. Nitrification in situ in the water columnhas been demonstrated by using inhibitors oflithotrophic nitrifying organisms (Christofi,Ph.D. thesis, 1978), but quantitative estimates ofnitrification rates are limited (14, 35; Brezonik,Ph.D. thesis, 1968). Other estimates of plankton-ic nitrification can be regarded as only qualita-tive since experimental conditions differed indegree of nutrient supplementation and length ofincubation time from those measurements madein situ (11, 15, 42; Christofi, Ph.D. thesis, 1978).

Considerable attention has been drawn re-cently to the use of dark 14CO2 uptake in thepresence and absence of N-Serve [2-chloro-6-(trichloromethyl)pyridine], a known inhibitor of

chemolithotrophic nitrifying organisms (23), forthe estimation of rates of nitrification (14, 19;Christofi, Ph.D. thesis, 1978). This paper ex-pands the chemical evidence for planktonic nitri-fication in the hypolimnion of lakes and furthertests the 14CO2-inhibitor method for measuringrates of nitrification.

MATERIALS AND METHODSSampling. Descriptions of Grasmere have already

been presented (16, 24); in the present study, samplingwas restricted to the deepest point of the lake (21.5 m).Water samples were taken weekly with a 5-liter Frie-dinger bottle at 1-m intervals between the depths of 10and 20 m between May and September 1976 and 1977.Water for chemical analysis or N-Serve sensitivitystudies was transferred directly into 0.5-liter screw-capped glass bottles; that used for the estimation ofrates of nitrification was subsampled into seven 135-mlglass bottles, each being flushed with approximatelytwice its own volume of water. Six of these bottleswere immediately sealed with Suba-seals (Gallen-kamp), and one was sealed with a ground-glass stopperand subsequently used for the determination ofpH andalkalinity.Chemical analysis. The inorganic nitrogen species

ammonia, nitrite, and nitrate were determined onpooled equal portions of individual samples betweenthe depths of 10 to 15 m and 15 to 20 m. Ammonia wasdetermined by the indophenol blue method (12), andnitrate was determined after reduction to nitrite byspongy cadmium (17), the nitrite being subsequentlydetermined by diozatization (5). Nitrite was deter-mined in a similar manner, but the cadmium reductionstage was omitted.pH was determined on individual samples, which

had been allowed to equilibrate to room temperature inthe completely filled glass-stoppered bottle, using aGK 2401 C combination electrode and PHM 62 milli-voltmeter (Radiometer). Alkalinity was determined by

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titratiorn with 0.01 N HCl (36). The total CO2 concen-tration in the samples was calculated from the alkalin-ity, pH values, and bicarbonate equilibrium dissocia-tion constants corrected to in situ temperature values(47).

N-Serve sensitivity studies. Water samples (0.5 liter)were treated with a filter-sterilized (0.2-,jm; Nucle-pore) solution of N-Serve in ethanol to obtain finalconcentrations of 5 1Lg of N-Serve and 53 ,ug of ethanolper ml in the sample. A replicate sample was treatedwith ethanol to a final concentration of 53 ,ug/ml as acontrol (untreated sample). Incubation was at 10°C(hypolimnetic water temperature range during thesampling period was 6.5 to 8.5°C) for 3 to 7 days beforenitrate concentrations in the samples were deter-mined.Measurement of rates of nitrification. Two of the

sealed glass bottles were immediately treated withfilter-sterilized N-Serve in ethanol solution to obtainfinal concentrations of 5 ,g of N-Serve and 53 ,ug ofethanol per ml in the sample. Another two bottlesreceived an equivalent volume of ethanol (untreatedsamples); the remaining two bottles were treated withneutralized Formalin to a final concentration of 2%(vol/vol). Samples were transported back to the labo-ratory in the dark and equilibrated in a water bath at10°C. After equilibration, 5 ,uCi of NaH 14CO3 (59mCi/mmol., Radiochemical Centre, Amersham) wasadded to each bottle, and incubation was continued for19 to 20 h. Duplicate 50-ml samples from each bottlewere filtered though 0.22-p.m membrane filters (Milli-pore Corp.), and the filters were rinsed with approxi-mately 2 ml of 2% (vol/vol) HCI. The filters weredissolved in a fluor containing 1,4-dioxan (900 ml),napthalene (100 g), butyl-PBD [2-(4-tert-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole] (7 g), bis-MSB [1,4-di(2-methylstyryl)-benzene] (0.5 g), and water (100ml). Samples were counted on a Packard 6332 scintilla-tion counter and corrected for quench by the samplechannels ratio method.By using the mean value of the duplicate counts

from each bottle, the dark CO2 uptake was calculated(22). These values were corrected for nonbiologicaluptake, and differences between the means of N-Serve-treated and untreated samples were tested byparametric statistical methods. When significant dif-ferences were found, the N-Serve-sensitive CO2 up-take was converted to a value for nitrogen oxidized,using a factor of 9.01 (6; Christofi, Ph.D. thesis, 1978).This represents the reciprocal of an average carbon-fixed-to-nitrogen oxidized ratio for ammonia- and ni-trite-oxidizing bacteria. The means of the measuredrates of nitrification between the depths of 10 to 15 mand 15 to 20 m were calculated for comparison withchanges in the inorganic nitrogen concentrations of thewater column.

Prediction of nitrate concentrations. The regressionline, on time, of the measured field nitrate concentra-tion was calculated. The intercept of this line wastaken as the nitrate concentration at zero time. Assum-ing that the nitrification rate measured on one sam-pling date prevailed until the date of the next measure-ment, the predicted nitrate concentration wascalculated as a cumulative total. The regression line,on time, of this predicted nitrate concentration wascalculated and compared with the regression for themeasured nitrate concentration.

T6 j J 2 o X40- -~~~~~~~~, 40

m A M J J A N A M J J A

FIG. 1. Variation in ammonia (-- -) and nitrate(-) concentrations in the depth layers of 10 to 15 m(a) and 15 to 20 m (b) in Grasmere (1976). (Arrowsindicate the three phases observed.)

MPN counts of ammonia-oxidizing Uthotrophic bacte-ria. The basal medium of Skerman (43) was diluted to1% (vol/vol) with double-distilled water. The sub-strate, (NH4)2SO4, was added to a concentration of 4mg of N/liter, and the pH was adjusted to 7.6, usingsterile sodium bicarbonate solution; 10-ml portionswere dispensed into sterile test tubes (G. H. Hall,Ph.D. thesis, Brunel University, Uxbridge, Middle-sex, England). Sample dilution intervals of 1:10 wereprepared in filter-sterilized lake water (0.22 ,um; Milli-pore Corp.), and five replicate tubes per dilution wereinoculated. Incubation was prolonged for 100 days at20°C, with fresh substrate being added 2 weeks beforefinal most probable number (MPN) analysis. Positivetubes were detected with both nitrite and nitrate spottests, using the methods described above.

RESULTSEvidence for nitrification in the hypolimnion.

Figure 1 shows the change in ammonia andnitrate concentrations between March and Au-gust 1976 for the layers of depth 10 to 15 m and15 to 20 m, respectively. Nitrite was not detect-ed in concentrations greater than 10 ,ug of N/literin any samples. Thermal stratification becameestablished in mid-April, and the increases inammonia concentrations before this time wereobserved throughout the water column. Threedistinct phases were observed in the change ofdissolved inorganic nitrogen concentrations. Aninitial increase in ammonia concentration wasfollowed by decreasing ammonia and increasingnitrate concentrations (Fig. lb). The final phasewas characterized by decreasing nitrate andincreasing ammonia concentrations (Fig. la).The second phase is typical of the nitrificationprocess. However, changes in inorganic nitro-gen concentrations observed in the water col-umn may not have been a result of in situmicrobial activity since production in and diffu-sion from sediments can affect the observedconcentrations, particularly in the lower hypo-limnion (15 to 20 m). Nitrate concentrations inthe interstitial water of profundal surface sedi-ments during June and July ranged between 20

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TABLE 1. Effect of N-Serve (5 jig/ml) on nitrateproduction in laboratory-incubated water samplestaken during the period of nitrate accumulation

Nitrate production (,zg of N03-N/liter ± 95% CLUDepth Final nitrate(m) Initial nitrate

Untreated N-Serve treated

10 371 ± 12 (11)b 422 ± 19 (14) 370 9 (7)18 505 ± 20 (4) 605 ± 7 (4) 525 8 (4)17 591 ± 22 (4) 659 ± 7 (4) 550 6(4)16 606 ± 7 (4) 745 ± 10 (4) 644 8(4)a CL, Confidence limit.b Numbers in parentheses are degrees of freedom.

and 150 ,ug of N/liter (Hall, Ph.D. thesis, 1981),and therefore diffusion gradients would be intoand not out of the sediments during the periodwhen nitrate was accumulating in the hypolimni-on.The nitrate concentration increased in the two

depth layers simultaneously; nitrate productionat discrete depths and turbulent mixing with thewhole depth layer could be eliminated due to thestability of oxygen gradients in the hypolimnionat this time (Hall, Ph.D. thesis, 1981). Furtherevidence for in situ nitrate production was pro-vided by N-Serve sensitivity studies (Table 1).Water samples, taken from various depths in thehypolimnion and incubated at in situ tempera-tures, continued to accumulate nitrate; more-over, this accumulation was prevented by theaddition of 5 ,ug of N-Serve per ml, a knowninhibitor of chemolithotrophic nitrifying organ-isms (23). However, the observed rates of ni-trate production in these samples could not becompared with the measured rates in the hypo-limnion due to the long incubation times andsubsequent changes in bacterial populations.During the lake nitrification phase, the ratio of

NH3-N lost to N03-N gained was less than unity(Fig. 1). Having established that sediment-de-rived nitrate does not contribute to water col-umn concentrations, there was some evidence ofactive nitrogen mineralization in the water col-umn. However, sources of inorganic ammonia

FIG. 2. Mean measured rates of nitrification (0)for the depth layers of 10 to 15 m (a) and 15 to 20 m (b)in Grasmere (1977). Nitrate concentrations (0) arealso shown. (Arrows indicate the period of nitrateaccumulation.)

TABLE 2. Log MPN counts of lithotrophicammonia-oxidizing bacteria at 18-m depth in

GrasmereDate Log MPN/litera

30 April 1980 3.2311 June 1980 2.9723 June 1980 2.6530 June 1980 3.527 July 1980 3.6914 July 1980 2.8920 July 1980 3.8525 July 1980 4.23

a Standard error of log MPN = 0.259; 95% confi-dence limits = log MPN ± 0.52. Results are signifi-cantly different when log MPN differs by 0.719.

associated with sedimenting particulate materialcannot be discounted. Table 2 shows the MPNcounts of ammonia-oxidizing lithotrophic bacte-ria at 18-m depth in Grasmere. Although notdirectly comparable, the significant increase inthe MPN at the end of June coincided with thenitrate concentration increases in the two depthlayers of the hypolimnion.

Nitrification rates. Figure 2 shows the meanvalues of the measured rates of nitrificationduring 1977 between the depths of 15 to 20 m and10 to 15 m. The corresponding nitrate concentra-tions observed in the field are also shown withthe lake nitrification phase indicated. Two peaksof activity were found, the first in May and thesecond in July. The July peak in the 15- to 20-mdepth layer preceded the one in the 10- to 15-mdepth layer by approximately 2 weeks. Thedetection of significant rates of nitrification be-fore the lake nitrification phase indicated thatthe nitrifying population was already estab-lished. Although not directly comparable, theobservation was further supported by the MPNcounts of ammonia-oxidizing lithotrophic bacte-ria at 18-m depth over this period (Table 2).

Prediction of nitrate concentrations. Knowingthe nitrification rates as a function of time fromthe laboratory measurements, nitrate concentra-tions could be predicted in the hypolimnion. InFig. 3, the predicted and actual concentrationsof nitrate in the hypolimnion are given alongwith regression analysis of the data. It should benoted that the actual nitrate concentrations rep-resent only net changes, as the nitrogen cycle isa dynamic phenomenon. Gross concentrationchanges could be equal to or greater (but neverless) than the net change in concentrations mea-sured. From Fig. 3 it becomes apparent that thepredicted nitrate concentrations were signifi-cantly lower (P < 0.05) than the actual concen-trations measured. However, considerable fluc-tuation in the actual nitrate concentrationoccurred in May and early June before the lake

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_ 2.15±0.5 30c1.15±0o33 300-

MAY JUNE JULY AUG.

FIG. 3. Regression lines of the actual (0) and pre-

dicted (0) nitrate concentrations in the depth layers of10 to 15 m (a) and 15 to 20 m (b) in Grasmere. Slopeand 95% confidence limits are also shown.

nitrification phase. These fluctuations can affectthe slope of the regression line, and a more validcomparison of actual and predicted nitrate con-centrations would be during the period of nitrateaccumulation, when nitrification is the dominantprocess. Figure 4 compares the predicted andactual nitrate concentrations during the lakenitrification phase. Again it is apparent that thepredicted nitrate concentrations were signifi-cantly lower than the actual concentrations.

DISCUSSIONThe observation that nitrate concentrations

increased in aerobic hypolimnetic water is inkeeping with results from other lakes both in thisarea (14, 21) and in other temperate areas of theworld (35; Brezonik, Ph.D. thesis, 1968). In themore eutrophic Blelham tarn, a moving front ofnitrification was observed associated with theboundary between oxic and anoxic conditions asdeoxygenation of the hypolimnion progressedfrom sediments up into the water column (14).This type of effect was noted in Grasmere butover extended time and greater depth intervals.Blelham tarn has been classified as being moreeutrophic than Grasmere (29), and the spatialdifferences noted for the moving front of nitrifi-cation could be a function of the rate of oxygenconsumption in the hypolimnion. The demon-stration that nitrate was produced by planktonicchemolithotrophic nitrifying organisms corre-sponds to similar observations in Blelham tarn(Christofi, Ph.D. thesis, 1978). Why nitrificationbecomes the dominant process is not clear.There is not consistency in the ammonia concen-trations at which nitrate accumulation com-mences in either the upper or the lower hypolim-nion, nor between years (Hall, Ph.D. thesis,1981). The laboratory-measured rates of nitrifi-cation do not increase at this particular time, andthere are insufficient data available to suggest a

decrease in the rate of nitrate assimilation. Suchan effect has been reported to be responsible formidwater nitrate concentration maxima in someGuatemalan lakes (8).

I.-600

500~~~~~~

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4 4.04±0.7 300_ - 3.3±b0.80'--¢ 1.32 ±0.19 o---o 1.21t0.S

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FIG. 4. Regression lines of the actual (0) and pre-dicted (0) nitrate concentration during the lake nitrifi-cation phase in the depth layers of 10 to 15 m (a) and 15to 20 m (b) in Grasmere. Slope and 95% confidencelimits are also shown.

Others who have used the 14CO2-inhibitormethod report good agreement between mea-

sured nitrification rates and other independentlymeasured data (6, 14, 45). The present experi-mental results are within an order of magnitudeof the measured rate of nitrate accumulation inthe field which is the accuracy claimed for themethod (14). However, as the measured rate inGrasmere consistently underestimated the ap-parent rate, it is worthwhile discussing bothmethodological and ecological factors whichcould affect the use of N-Serve-sensitive CO2uptake as an index of nitrogen oxidation.N-Serve is relatively insoluble in water (23),

and although aqueous solutions have been usedto inhibit nitrification in soils (7), the use of suchsolutions on water samples is not possible due tothe degree of dilution required to obtain inhibi-tory concentrations of N-Serve. Organic sol-vents have therefore to be used, but this hasbeen criticized on the grounds that the solventitself could inhibit the nitrifying organisms (7).Investigations have indicated (Hall, Ph.D. the-sis, 1981) that ethanol (53 ,ug/ml), used in thepresent work, has no such effect. Few authorspresent sufficient information to calculate thefinal concentration present in the samples (6,27), although ethanol concentrations of 226 (45)and 6,300 ,ug/ml (Christofi, Ph.D. thesis, 1978)have been used. It is impossible to speculatewhether such concentrations are inhibitory tochemolithotrophic nitrifiers, but it is likely thatthe natural dark CO2 uptake of samples will bestimulated (Hall, Ph.D. thesis, 1981). The use ofN-Serve has also been criticized because itinhibits only ammonia-oxidizing bacteria, andtherefore nitrite-oxidizing bacteria are still ac-tive (25). With ammonia oxidation inhibited, theonly other possible source of nitrite in hypolim-netic water is by nitrate reduction. Bacteriacapable of this reduction have been isolatedfrom Grasmere (26), and the reaction in soils (2)and marine (38) systems is known. However, theability of N-Serve to inhibit completely nitrateproduction in Grasmere (Table 1) indicates that

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nitrite production from nitrate is unimportant.The application of statistical analysis to obtain

a more rigorous assessment of differences be-tween N-Serve-treated and control samples re-duced the number of positive results obtained.Such tests are, however, necessary due to thewithin- and between-sample variations noted formicrobiological data and the effect of samplingtechnique on such results (30). As the methodattempts to detect differences between sampletreatments, zero-time controls may be omitted,and more replicate samples with and without N-Serve may be included. Sensitivity of the meth-od can also be increased by the use of longerincubation times or increasing the 14C concen-tration.The use of autotrophic CO2 uptake as a mea-

sure of lithotrophic nitrification may be subjectto several errors which require further discus-sion. Oxidation of ammonia and nitrite andautotrophic CO2 fixation have been reported tobe closely coupled (1), but other reports statethat they can be separated in time due to theaccumulation of storage products (18). Little isknown of the physiological state of nitrifyingorganisms in situ and, of course, this state couldvary with time. The method also assumes that aconstant carbon-fixed-to-nitrogen oxidized (C/N) ratio is maintained. The C/N ratio for theseorganisms is a measure of the growth yield,which is known to change with conditions in theenvironment (temperature, nutrients, etc.) aswell as the biomass of the organisms (4). It istherefore unlikely that one ratio could possiblycover the ranges of environmental conditionslikely to be encountered. Also, the C/N ratio isusually determined on enrichment cultures ofnitrifying organisms in which population num-bers, nutrient concentrations, and subsequentlyrates of activity are greater than would be foundin natural environments. This procedure couldlead to underestimation of the conversion factorand therefore underestimation of the apparentrate of nitrification. The results could be pre-sented as N-Serve-sensitive CO2 uptake to beused as an index of nitrifying activity (27), butfor ecological measurements a value for nitrogenoxidized is preferred. In addition to this, apopulation of nitrifying bacteria will only in-crease as long as the rate of substrate supply isin excess of that needed to meet the mainte-nance energy requirements of that population(3). Under substrate-limited conditions, it isunlikely that CO2 uptake would be as large asunder conditions of active growth.

In the presence of their specific lithotrophicenergy sources, nitrifying bacteria are known toassimilate low-molecular-weight organic com-pounds (49). Complete heterotrophic growth(biosynthesis and energy production) has been

demonstrated for both Nitrosomonas spp. (40)and Nitrobacter spp. (32, 33, 44, 46). Specializedcultural procedures were, however, required,for Nitrosomonas and Nitrobacter yields werelow in comparison with yields obtained in litho-trophic culture (37). In the presence of organiccompounds and their lithotrophic substrates,growth yields of Nitrobacter and activites percell of Nitromonas have been increased (20, 31)and, moreover, a decrease in the activity ofC02-assimilating enzymes in Nitrobacter hasbeen observed (46). The success of CO2 uptakeas a measure of nitrogen oxidation will dependon the contribution of heterotrophic uptake tothe biosynthetic metabolism of the lithotrophicnitrifiers. Culture studies estimate this figure at10% (37), but the degree of mixotrophy in situ isunknown.

Finally, the method would not detect syner-gistic relationships between Nitrobacter and ni-trite-producing heterotrophic bacteria (41, 48).Although the ecological significance of hetero-trophic nitrification has yet to be resolved, sucha relationship has been demonstrated in culture(10).The absence of reliable data relating to the

factors discussed above must cast doubt on thegeneral applicability of the 14CO2-N-Servemethod as a quantitative measure of nitrifica-tion.

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3. Belser, L. W. 1979. Population ecology of nitrifying bacte-ria. Annu. Rev. Microbiol. 33:309-335.

4. Belser, L. W., and E. L. Schmidt. 1980. Growth andoxidation kinetics of three genera of ammonia oxidizingnitrifiers. FEMS Microbiol. Lett. 7:213-216.

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