The influence of glucose and nitrate concentrations upon denitrification rates in sandy soils

THE INFLUENCE OF GLUCOSE AND NITRATE CONCENTRATIONS liPON DENITRIFICATION RATES IN SANDY SOILS

Summar!--Dentrilic:ltion rates in twso soils were ;~ssesscd separately as a function of NO; concentration while provldinp a constant mitial glucose concent]-atIon. and as a function of glucose concentration while providing a constant initial NO; -N concentration. Of the soils used. a Hanford sandy loam and a Coachella tine sand. the bacteria in the former produced higher rates of den~triticatio~l with a maximum loss of 15OOp~ NO;-!4 ml da>-’ as compared to a loss of 15O;~pNO:-N:ml day-’ from the latter. Rates of loss closeI> approximated Michaelis Mentcn kinetics in the Coachella sand, and K, values for glucoseC and N03--N wcrc 500 pg ml and I70 pg ml. respectively. Rates of loss of NOT-N from the Hanford soil did not approximate Michaehs- Mcntcn kinetics. and this was attributed to failure to saturate enzyme systems in the dcnitrifying bacteria \\,ith glucose and nitrogen when each was held constant. C,!N ratios around 2 appeared to provide the greatest rates of denitrification. High C’N ratios or high glucose concentrations (I.8 per cent) retarded d~llitri~~~~tion. with fungal groxvth and a subsequent drop in pH occuring. ,\ Pvt,itrio~l~itirrls \+as incubated acrohicallq for 24 h followed hp a 72 h anaerobic incubation with nitrate as the sole nitrogen source at 0. 10. SO. 100. 150 and 500 mg N:ml concentrations. Assimilatory nitriitc reduction never cxcccdcd 75 mp N’ml, and it was concluded that this mode of mtrate reduction IS insignificant at hisher nitrate concentrations by comparison to dissimilatory nitrate reduction. i.e. drni-

Despite the long historic awareness of denitrification (Gayon and Dupetit, I XS6). the kinetic properties and magnitude ofdenitrification rates in soils arc not clear. F‘or the most part. dcnitrification rates have been thought to be independent of nitrate concentration (zero order kin&s) over‘ a fairly wide range (Broadbent, 195 1: Wijkr and Del\%i&e. 19%: N~)rnrni~ 1956; Brcmner and Shaw. 195X: Cooper and Smith, 1963). Some investigations (Nommik. 1956: Cooper and Smith, 1963) indicated that reduction of nitrous oxide to molcculnr nitrogen did not occur until the greater part of the nitrate was consumed, and that the rate MX unaffected h\; nitrate concentration. The ~~~~~~il~ibiIit~ of organic matter as a necessary rcductant in the denitrification process has been well rccngni&. Wijler and Dclwichc ( 1954) shelved that dcnitritication rates wcrc increased with increasing concentrations of ahllir. Hrcmncr and Shau (1058) found that denitrilication was directlq correlated tvith ihc type of organic matter. being more rapid with glucose and celiulosc and icss rapid with lignin and sawdust. Simiiarly. Nommik ( 1955) sI10wec1 that glucose increased denitrilication more so than wheat straw. McGarit) (lY61) reported that freezing and thawing of -* Present address: Department of Apronomy. Colorado Stntc I.:niversity. Fort CoIlin\. Colorado X0321.. C:.S.A.

soils stimulated denitri~cation as a consequence of increased availability of organic matter.. Woldcndorp (1962) found that denitrification was higher in the rhizosphere than other areas of soil and attributed the increr~scd activit) to availability of organic root debris and exudates. The question frequently arises as to whether nitrate disappearance can be attributed to immobilization (assimilatorv nitrate reduction) rather than denitrification (dissin&tory nitrate reduction). In a defined medium, Sacks and Barker (1952) showed that Y8 per cent of the added NO; (717 icg!ml N) was converted to N1 and the remainder left intact with growing cells of PWM~JRXJUS ricnitrificar~s. All of the ammonium and cellular nitro_gen appeared to have been derived from glutamic acid, the only other nitrosen so~mr present. Thus. ii would appear that immobilization of nitrate under anaerobic conditions is highly unlikely cm thermodynamic principles if ammomum and amino nitrogen compounds arc present in suf%ienf quantities. The purpose of the current it~vesti~~tion was to determine {a) both the magnitude ofdenltri~cation and the kinetic order of the over-all reaction as functions of nitrate and glucose concentration, (b) whether the maximum rates of denitrification differed significantly between two different soils and (c) whether or not nitrate losses by bacterial immobilization are consequential under anaerobic conditions.

A Hanford

santlq loam and a Coachclla line sand containing 0.75 and 0 1I”,, organic matter. rcspcctively. were used throughout the investigation. Maximal denitrification rates were assessed in two sets of experiments. In the first instance. the energy source (glucose) was added at constant co~lccntr~~tions of 1000 and hOo0 ~cg:ml. rcspcctivcl~. to Hanford and ~‘oachella soils containing varytn! quantities of nitrate as Ca(NO,),. In the second instance. NO, was added at ii constant concentration of 1000 lag, ml for both soils and the concentration of plucosc IV;ISvaried. In all soil incubation cxpcriments. 125 ml flasks containing 50 g soil and SO ml ofsolution were incuhatcd at 3 C in an anacrohic in~Llb~t~~r c~nt~linirl~ ;I N2 atmosphcrc. Two experiments asscssin, (’ the ctrL!ct o(‘ (’ N mtios upon dcnitrification were performed on the Hanford soil only. In one instance. the C”N ratio of 1.8 was maintained in each treatment while the respective concentration of each clement was varied. In the other instance, the NO;-N conc~ntr~iti~~ll was held constant at lo00 pg’rnl, and glucose was added at ct~nc~ntr~ttio~ls to give the respect&c C’;N ratios shown in EGg. 6. An isolate identified as ;I Psc,r,1k)r77o17tr.~ (Xkcrman. 1967) was ohtaincd from soil by sclcctivc cnrichmcnt in a mineral salts medium consisting of I.7 g K,HPO,. 360 mg NaH,f’O,. 131)mg MgSO,. SO0 jcg FcSO, and 3% ,~g CaCIZ. and 36 g KN03 and 5 g glucose pet liter. The culture was inoculated asepticall? into sidenrm-ncphclorlietrv ilasks containing 100 ml of btcrile media with the diffcrcnt concentrations of NO, shown in Table 2. The cultures wcrc grown acrohicall> on a rotary-platform shaker at 30 c‘ and were sampled at the end of 24 h to measure the parameters shown in Table 2. The flasks wcrc then p&cd in an anaerobic incubator at 30 <’ for an additional 72 h. after ~%hich the same paramctcrs mcntioncd abo\~c wcrc mcasurcd. Cell concentrations wcrc measured turbidimctricallq at 525 nm and by plate counts on nitrate apar (Difcol.

NO3 -N was measured by the ~I~enoldisLilfol~ic acid method (Bremner. 1963) and by use of a spucific NO3 electrode (Langmuir and Jacobson, 1970). Ammonium and organic (cellular) nitrogen were analyzed by the Kjeldahl procedure (Bremner, 1965). Organic matter was determined by the Walkley---Black method (Allison. 1965) and soil ~~c~iit~.ifyingbacteria were enumerated by the tn~~st-pl-~~h~~ble number method (Focht and Joseph. 197.3).

Denitrification rates in both soils were dependent on NO; coliccntr~itioll (Figs. I and 3) and glucose conccilti-~~tioil (Figs. ‘7and 4). Rates in the Coachclla sand (Figs. I and 3) closely approximated Michaelis Menten kinetics: they were substrate-dependent at lower concentrations approximating first order kinetics and gradually diminished at higher concentrations to bccomc independent of either suhstratc concentration (zero order kinetics). The respect% li, values for glucose-C and NO< -N were 500 and I70 /~g: ml. The maximal rates (r;,,+,,)observed in both cxperimcnts were almost identical. I:;,,,, obtained when the carbon source was constant (Fig. 1) was 150 /~g N/ml per day (Fig. I ) as opposed to 1,,,,,i= 135 (Fig. 2) when the NO, concentration was held constant. The lower I ,,,_,\ obtained (Fig. 2) was probably due to a less than maGmaI co!iccntr~~tiotl of NO, -N added as seen in Fig. I : the reaction mtc had not quite I-cached l;,,,,at ;I NOj -N concentration of IO00 ,&ml. Dcnitrifcation rates in the Hanford sandy loam did not follow Michaclis Menten kinetics. There appears to bc a sharp transition (Fig. 4). particularly between first order and zero states. Though both experiments (Figs. 3 and 4) vicldcd identical values for the apparent I ;,,,,,(6?0 ,q N;
Dentrification I

I

I

I

I

I

rates in sandy soils

I

COACHELLA

I50

600

-

500

-

0 HANFORD

t::::

0” ? c 400-

/r-1

s d

0 d

75-

2 300 z 110

z ‘!o S

9

5o-l

0

-

200-

IOO-

I

Fig.

2.

centration

I 2000

I

I 4000 GLUCOSE-C.

I

I 6000 rg/ml

I

~,~,,,,~

8000

Denitrilication rates as a function of glucose conma Coachella fine sand with an initially constant NO; -N concentration of 1000 icg/ml.

each respective case was saturating. For example. the NOT-N concentration of 1000 &ml (Fig. 4) was far below the maximum amount needed to attain V”,,,, as shown in Fig. 3. Thus the apparent linearity observed in both casts at lower concentrations would be expected if these points represent only the lower end of the real curve. The kinetics of dual substrate reactions as given by Bray and White (1965. pp. 2855288) can be applied here : C’ = b;,,,, CN,!(C + K,)(N

+ K,,).

where C’ is the denitrificirtion rate. I,,,, is the maximum rate, C is the carbon concentration. N is the NO; concentration, K, is the Michaelis constant for glucose-C and K,, is the Michaelis constant for NO;

4000 GLUCOSE

2000

I

HANFORD

500

0

I500 NO3 - N, pg/ml

IO,000

8000

6000 pg/ml

Fig. 4. Denitrlfication rates as a function of gltlcosc centration in a Hanford sandy loam with an initially stant NO;-N concentration of lOO0,~g ml.

concon-

N. Thus, the rate will not be solely dependent on NO, concentration if the glucose concentration is not high enough to ensure that zero order kinetics rclativc to glucose apply [i.e. C,/(C + K,) 2 I]. The problems involved in selecting suitable concentrations of substrates to saturate both enzyme or cellular systems to achieve maximal rates are compounded in dual substrate reactions. Stoicheometritally the C/N ratio for complete denitrification using glucose as an energy source is 1.25. The quotient of the respective carbon and nitrogen saturation constants (K,‘s) for the Coachella sand was 2.9. This is in agreement with studies by Bremner and Shaw (195X) who obtained maximum denitrification rates at C:‘N ratios I 10,000 -

I

I

I -

-y

II

-C,

HANFORD

I

2500

Fig. 3. Denitrification rates BS a function of NO.; ccntration in a Hanford sandy loam with an initially stant glucose-C concentration of 2000 /cg.ml.

concon-

Fig. 5. Nitrate losses from a Hanford sandy loam with glucose as the oxidizable substrate using a constant CN = I.8 at Initial concentrations of lO.OOOA, 50000. 10000. IO000 and 5001 pg;‘ml.

300

R. A. BOWMAN and D. D. FKHI I

I

I

I

I

I

HANFORD

7

_-_--0

PH

6

5 0=

C/N=2

A= A = 0 = + = 0 = *; 0 =

C/N=O,B

. =C/N

41 _

0

I

/

= 4

C/N = IO Blk (no C; no N) Blk (WC; N) C/N=20 Blk(C; noN) C/N =40 I 2

I

3

I 5

I 4

I 6

DAYS

Fig. 6. The effect of different C:N ratios upon changes in pH in a Hanford sandy loam using an mitially constant NO, concentration of 1000 p&/ml.

of 2: 3. The C/N ratio necessary to effect maximal denitritication rates of course will vary depending on the electron donors per mole of carbon substrate: for example, a mole of hexane will provide twice as many electrons as an equivalent mole of glucose. Holding the C.!N ratio constant in the Hanford soil (Fig. 5). while increasing both elements showed that the observed maximum denitrification rate was greater than 640 /Lg N/ml per day and was closer to 1500 big N/ml per day when the amount of NO;-N lost in 2 days with an initial concentration of 5000 L[g N/ml was taken into account. However. high glucose-C concentrations (1.8per cent) appeared to inhibit denitrification during the incubation period. and fungal growth was visible Table I. Final bacterial

numbers

in this sample. Figure 6 illustrates the decrease in pH that was transitory at moderate glucose concentrations and continuing at higher concentrations for the duration of the incubation. Our results indicate that denitrihcation rates are dependent on NO; concentration according to Michaelis Mcntcn kinetics in contrast to previous investigations (cited earlier) showing that denitrification was a zero order reaction. This discrepancy may be explained by the USCof relatively high concentrations of NO;. thus assuring that zero order kinetics would prevail. and by the expression of concentrations on a soil rather than solution basis. For example. the lowest concentration used by Nommik (1956. p. 206. Table 6) U;LS 3X ;~g NO; -N/g soil. Though no data on soils water relationships were given. he did state that the cxperiments were carried out at the water-holding capacity of the soil. If we assume a water-holding capacity of 30 per cent and a pore space of 65 per cent for a heavy clay. the solution concentration works out to I80 /lg:rnl NOj -N. For coarser-textured soils, the diffcrence between soil and solution concentrations would be greater. Furthermore Nommik’s data showed that the rates (over a IO-day period) increased from 38 pg N to 160 keg N (per g soil) beyond which they remained constant, Throughout this study. nitrogen losses due to immobilization were assumed to be insignificant. The results of the pure culture studies (Table 2) show that this is true for high concentrations of NOT-N since the maximum amount converted to NH: or organic-N did not cxccod 75 mg N/ml. Because the numbers of total and dcnitrifying bacteria were much lower in soil (Table I ) than in pure culture (Table 7). it is apparent that immobilization losses are insignificant at high nitrate concentrations. At lower concentrations. immobilization losses appear to be more significant, although it should be noted that the nitrate immobilized was aerobic conditions for all congreater under centrations.

(per ml) in both soils as a function

of carbon

and nitrogen

concentration

Treatment< Glucose-C Wml)

NO1 -N (&ml)

0 0 2OOOO 20000 ‘0000 70000

0 500 0 500 I I IO 2500

10000

I 0000

Total bacteria

Dcnitrifiers

Dcnitrifiers

(x IV)

I x lOi)

(“,J

Hanford 3.3 ?- I 160 1600 3800

0. IO 0~45 4.0 140 3800

3 14 2 9 I 00

164 1.0

164 I .i

100 78

c’oachclln 0 800

IO00

70

1800

I 000

1400

500

540

6000

0

2.0

0. I

17 1400 6X

3

24 too 13

Dcntrification

rata

301

in sandy soils

Table 2. Bacterial-N. NO.;-N. pH change. and cell densities of a P.srlrt/o,,~o,~rr.s culture incubated aerobically lowed by a 72 h anaerobic incubation with 0.09”,, glucose-C and variable NO,?-N concentrations

Incubation

tlmc

24 h (Aerobic) Optical densit! NO; -N. bcg:ml Assimilated N I&ml PH Bacterial cells IO’ ml 72 h (Cumulative aerobic and anaerobic) Optical density NO, -N 118 ml Asaimilatcd-N jog ‘ml PH Bacterul cclla IO’ ml

~~l~~lo~~/~,~l~~cr,lcrlr --This work Public Health &vice Training

0

0 0

0 6.9

0

0 0 6.9

IO

Initial NOi -N concentration. SO 100

0.05 0 II

6.9 I .4

0.09 0 I2 6.9 3.6

was supported by a U.S. Grant No. ES 000X5-05.

ALL.ISON L. E. (1965) Organic Carbon. In !j,fr,fhotl,\ c!f Soil .A~~trly.vs, Agronomy 9 (c‘. A. Black. Ed.). Part 2. pp. 1367

1378. American Society of Agronomy. Madison, Wisconsin. Bt
0.1 I 3, ;3 6.9 34

0.28 0 40 7.0 so

/@ml 250

0.2 I 32 62 7.3 > 300

0.33 135 74 7.7 > 300

0.50 0

0.5 I 0

72 7.6 > 300

for 14 h fol-

75 7.9 > 300

FOCHT D. D. and JOSLPH H. (1973) An improved

500

0.34 320 60 7.5 > 300

0.50 0 72 79 > 300

method for the enumeration of denitrifying bacteria. Soil Sci. Sw. .~IJZ. Proc.. 37, 69X-699. BAYOU U. and DI.PI ‘TIT G. (1X86) Recherches sur la rcduction dcs nitrates par Ies infnimcnts pctits. Sot,. Sci. P/IX.\. !\;clt. Bwdctrlr.~. %r. 3, 101 307. LAWMLIR D. and JACOICWN R. L. (1970) Specific-ion clectrodc determination of nitrate in some fresh waters and savage eltluents. Enriiou. Sci. Twhwl. 4, X35. MCG.mm J. W. (1961) Denitrification stud& on some South Australian soils. Plarlt trud Soil 14, I 71. Nowal< H. (1956) Investigations on demtrilication in soil. .l<,ttr :k/,‘ic. Sctrd. 6, I95m 228. S4( I(S L. E. and BARECLK H. A. (1952) Suhstrnte oxldatlon and nitrous oxide utilization in dcnitrification. .1. 5trt.r. 64. 247 -. ‘5’_. SKI I