Other Organic Inhibitors of Soil Nitrification

W o r k w i t h p u r e cultures of nitrifying organisms ( 1 6 1 - 1 6 3 ) a n d t h e soil perfusion technique ( 1 2 8 a - c ) has shown t h a t g u a n i d i n e is a h i g h l y effective inhibitor of nitrification of a m m o n i u m ions. T h e effect of guanidine is to produce a long lag, extending to about 20 days, before nitrification commences. T h e r a t e of nitrification t h e n proceeds nor-m a l l y ( 1 9 6 ) .

T h i o u r e a a n d allylthiourea inhibit soil nitrification at low concentra-tions (e.g., 0.3 m M ) ( 1 9 6 ) . T h i o u r e a is toxic to h i g h e r plants (173) a n d

T A B L E V I I

EFFECTS OF VARIOUS URETHANES ( 3 . 3 mM) ON RATES OF SOIL NITRIFICATION AT 7 0 ° F OF 1 0 mM A M M O N I U M CHLORIDE0

Estimated lag period Urethane used in presence of

1 0 m M ammonium chloride

Time in days to produce 7 0 Mg/ml nitrate nitrogen

before nitrification begins

None 9 0

Isopropyl carbanilate 4 1 3 2

C6H5N H C O O C H ( C H3)2

Ethyl-iV-butyl carbamate 3 8 2 9

C A - N H C O O C A

Ethyl carbamate (urethane) 2 0 1 1 N H2- C O . O C2H6

Ethyl carbanilate 1 5 . 5 6 . 5

e6H5-NH.CO-0-C2H5

Ethyl-iV-isopropyl carbamate 1 5 6 ( C H3)2C H . N H . C O O C2H6

aF r o m Quastel and Scholefield ( 1 9 7 ) .

inhibits Nitrosomonas i n p u r e culture (103, 124). Conceivably its effect m a y be due to combination at low concentrations w i t h metallic ions, e.g., copper, w h i c h m a y p l a y a role in the process of soil nitri-fication (120a, 121, 170). J e n s e n (100) has found t h a t thiourea, w h i c h is n o t a n available source of nitrogen to h i g h e r plants, will sup-port t h e growth of fungi.

I t is of interest to n o t e t h e effects on a m m o n i a oxidation i n soil b y a variety of organicnitrogen compounds. Using t h e W a r b u r g m a n o m e t -ric technique, it is possible to estimate t h e rates of oxygen consumption of soils enriched w i t h nitrifying organisms both i n t h e absence a n d presence of a m m o n i u m ions a n d other substances. I n this w a y Quastel a n d Scholefield (194) w e r e able to demonstrate t h e h i g h l y inhibitory effect of thiourea on t h e process of a m m o n i a oxidation b y nitrifying

710 J. H . Q U A S T E L

cells in soil. Since t h a t t i m e t h e m a n o m e t r i c technique has become in-creasingly used for the study of soil microbial metabolism (30, 39a, b, 72, 112a, b, 196, 198, 2 1 0 ) . Some results w h i c h show qualitatively the ef-fects of a n u m b e r of organic nitrogen compounds on t h e oxygen con-sumption of soils w h i c h are oxidizing a m m o n i u m ions are given in Table V I I I ( 1 9 6 ) .

T A B L E V I I I

T H E EFFECTS OF VARIOUS COMPOUNDS ON THE OXYGEN CONSUMPTION OF SOILS"

Inhibition of respiration of enriched nitrifying soils Substance in presence of NH⁴C1

Methylamine

+

Ethylamine 0

Ethanolamine 0

Ethylurethane

+

iV-Methylurethane

+

Guanidine

+

N- Methylguanidine

+

Arginine, creatine, glycine 0

Methionine sulfoxide

+

Urea, acetamide 0

Thiourea

+

aF r o m Quastel and Scholefield (196).

N o doubt these effects a r e brought about b y t h e inhibition of one or m o r e of t h e processes involved i n t h e nitrification of a m m o n i u m ions, t h e details of w h i c h are still obscure.

F . O X I D A T I O N O F N I T R I T E I N S O I L

Since t h e discovery a n d isolation of the organisms responsible for nitrite oxidation in soil b y W i n o g r a d s k y (275) a n d t h e early studies of Meyerhof ( 1 6 1 - 1 6 3 ) , little was k n o w n about t h e process of nitrite conversion to n i t r a t e in soil u n t i l the advent of the soil perfusion tech-nique. By this method, t h e r a t e of oxidation of nitrite in soil was fol-lowed ( 1 2 8 a - c ) a n d t h e fact t h a t increasing concentrations of nitrite in soil give rise to increasing lag periods before nitrite metabolism com-mences was established. Moreover, it was shown t h a t soils t h a t h a v e been exposed to high concentrations of nitrite acquire t h e ability to oxidize nitrite at higher rates t h a n soils t h a t h a v e been exposed to low concentrations of nitrite ( 1 9 6 ) . A process of adaptation in Nitrobacter seems to occur w h e r e b y it is able not only to oxidize nitrite a t relatively high rates, but also to proliferate in w h a t would n o r m a l l y be toxic

con-6 . M I C R O B I A L A C T I V I T I E S O F S O I L A N D P L A N T N U T R I T I O N 7 1 1

centrations of nitrite. It has been suggested, as a result of studies w i t h suspensions of Nitrobacter ( 1 2 9 ) , t h a t nitrite oxidation involves the participation of cytochrome components. Possibly the cells' content of these components is increased in the adaptation process.

Inhibitors of Nitrite Oxidation in Soil

a. Chlorate. T h e bacteriostatic action of chlorate at low concentrations ( 1 0 ~⁵ to 1 0^{- 6}M ) on t h e development of Nitrobacter was first observed with t h e perfusion technique ( 1 2 7 ) . Such concentrations suppress the proliferation of nitrite-oxidizing organisms, b u t t h a t of the a m m o n i a oxidizers proceeds unchecked. T h e result is t h a t w h e n nitrogenous sub-stances a r e added to soil in t h e presence of small quantities of chlorates, nitrites (but not nitrates) accumulate. Chlorate does not poison or in-terfere w i t h the bacterial oxidation of nitrite to n i t r a t e , except at rela-tively high concentrations, for w i t h a soil enriched w i t h Nitrobacter

cells t h e conversion of nitrite to n i t r a t e proceeds at a constant r a t e u n i n -fluenced b y the low concentrations of chlorate t h a t inhibit the prolifer-ation of the organisms involved ( 1 9 6 ) . H o w e v e r , chlorate a t 1 0 ~³M inhibits oxidation of nitrite b y a n enriched soil. T h e chlorate inhibition of Nitrobacter growth m a y be alleviated b y the presence of n i t r a t e

( 1 2 7 ) . It h a s been pointed out ( 1 2 9 ) t h a t t h e kinetics of t h e inhibition of nitrite oxidation b y chlorate m a y be explained on t h e basis of the destruction, b y chlorate, of a cytochrome component i n Nitrobacter whose concentration determines t h e r a t e of oxidation of nitrite. Chlo-rate, however, does not inhibit nitrite oxidation directly b u t is con-verted, d u r i n g the course of nitrite oxidation, into a substance, presum-ably chlorite, w h i c h is inhibitory. I t is well k n o w n t h a t chlorite is h i g h l y inhibitory to bacterial g r o w t h a n d t h a t chlorite m a y be derived from chlorate b y certain microorganisms ( 2 0 2 ) . T h e alleviating action of n i t r a t e is held to be d u e to its retardation of chlorite formation ( 1 2 9 ) . b. Methionine. T h i s a m i n o acid has a h i g h l y inhibitory action on nitrite oxidation in soil ( 1 9 6 ) , t h e effect being confined to t h e prolifer-ating cells. T h e oxidation of nitrite b y resting Nitrobacter cells, in a n enriched soil, is n o t affected b y m e t h y l - , or ethyl-, m e r c a p t a c e t a t e or mercaptopropionate at concentrations t h a t a r e inhibitory to t h e grow-ing organisms in soil ( 2 8 ) .

c. Other inhibitors of nitrite oxidation in soil. N i t r o u r e a is h i g h l y inhibitory to Nitrobacter oxidation of n i t r i t e in soil ( 1 9 6 ) . Possibly the effect is due to the formation of cyanate, w h i c h is also a n effec-tive inhibitor of nitrite oxidation b y Nitrobacter ( 1 2 9 ) . T h e anti-biotic Chloromycetin is h i g h l y inhibitory to soil oxidation of nitrite

( 1 9 6 ) at concentrations t h a t seem not to affect appreciably t h e

oxida-712 J. H . Q U A S T E L

tion of a m m o n i u m ions. T h i o u r e a is h i g h l y inhibitory. E t h y l u r e t h a n e has no effect on nitrite oxidation i n soil, or Nitrobacter proliferation, at concentrations t h a t m a r k e d l y affect t h e activities of Nitrosomonas.

The Role of Oxidation of Hydroxylamine

K l u y v e r a n d D o n k e r (115) considered t h a t t h e oxidation of a m m o n i a to nitrite in Nitrosomonas takes place w i t h t h e intermediate formation of h y d r o x y l a m i n e hyponitrite. Hof m a n a n d Lees (96) claim t h a t the oxidation of a m m o n i a b y Nitrosomonas can proceed, in t h e presence of h y d r a z i n e as a n inhibitor, w i t h accumulation of h y d r o x y l a m i n e . H y d r o x y l a m i n e is h i g h l y inhibitory to soil nitrification, b u t i n t h e presence of p y r u v a t e full nitrification of t h e nitrogen of h y d r o x y l a m i n e takes place. T h i s is accomplished b y heterotrophic organisms b y the oxidation of p y r u v i c oxime formed as a n intermediate ( 1 2 8 a - c , 200, 2 0 1 ) . It h a s been shown, however, t h a t at low concentrations h y d r o x y l a m i n e m a y itself be oxidized b y washed Nitrosomonas cells as r a p -idly as the a m m o n i u m ion ( 1 2 5 ) .

G . C O N V E R S I O N O F O T H E R N I T R O G E N C O M P O U N D S T O N I T R A T E

1. Oximes

Certain oximes, n o t a b l y p y r u v i c oxime a n d oxalacetic oxime, u n -dergo conversion to nitrite i n soils b y heterotrophic organisms w h i c h h a v e been isolated a n d studied i n p u r e culture (98, 101, 130, 200, 2 0 1 ) . A t least t h r e e species of organisms t h a t attack p y r u v i c oxime are in-volved; two fall into t h e genera Achromobacter a n d a third is identi-fied as a Corynebacterium (200, 2 0 1 ) . A p p a r e n t l y p y r u v i c oxime is oxidized directly to p y r u v i c acid a n d nitrous acid b y a n e n z y m e (py-ruvic oxime oxidase) w h i c h is present in t h e heterotrophic organisms mentioned. T h e oxidase is inhibited b y certain oximes, e.g., those of p h e n y l p y r u v i c acid a n d of α-ketoglutaric acid, w h i c h are not attacked b y t h e e n z y m e i n question. T h i s heterotrophic formation of n i t r i t e in soil from oximes is not affected b y chlorate, nitrourea, thiourea, m e t h -ionine, or ethyl u r e t h a n e (98, 101, 130, 194, 196). It is, however, in-hibited b y sulfadiazine, Chloromycetin, h y d r a z i n e , azide, or cyanide

(98, 101, 130, 200, 2 0 1 ) .

2. Amines (196)

A m i n e s ( m e t h y l a m i n e , e t h y l a m i n e , e t h a n o l a m i n e ) a r e converted to n i t r a t e in soil after p r e l i m i n a r y conversion to a m m o n i u m ions. M e t h y l -a m i n e inhibits nitrific-ation of -a m m o n i u m ions, t h e inhibition being -a logarithmic function of t h e concentration. Organisms t h a t develop in soil i n response to t h e presence of e t h y l a m i n e can oxidize this a m i n e

6 . M I C R O B I A L A C T I V I T I E S O F S O I L A N D P L A N T N U T R I T I O N 7 1 3

but can o n l y feebly oxidize e t h a n o l a m i n e ; organisms t h a t develop in soil in response to t h e presence of e t h a n o l a m i n e can attack this a m i n e but t h e y cannot oxidize e t h y l a m i n e . T h u s organic substances in soil stimulate therein the development, b y adaptation, of organisms (or of enzymes) t h a t m a y specifically attack t h e substances in question.

3 . Amino Acids

A m i n o acids, as already mentioned, are converted to a m m o n i a prior to nitrification and, a p a r t from cysteine a n d methionine, t h e y h a v e little ef-fect on t h e nitrification of a m m o n i u m ions. T h e recovery of a m i n o nitro-gen as n i t r a t e decreases w i t h increase of the C : N ratio of the a m i n o acid

( 1 9 4 ) . T h e a m i n o acids t e n d to r e t a r d the disappearance of admixed nitrite, probably as t h e y themselves give rise to nitrite. Cysteine a n d methionine, as pointed out earlier, h a v e h i g h l y inhibitory effects on nitrite utilization.

A r g i n i n e , w h e n admixed w i t h nitrite, gives rise in soil to nitrite and, only after a l e n g t h y period, to nitrate. T h i s p h e n o m e n o n is due to t h e gradual liberation from a r g i n i n e of u r e a , w h i c h is t h e n hydrolyzed b y soil organisms to a m m o n i u m carbonate, t h u s raising t h e soil p H . T h i s i n t u r n facilitates t h e conversion of a m m o n i u m ions to nitrite, the o p t i m u m p H for w h i c h is 8 . 6 , and, b y m a k i n g available a m m o n i a at a high p H , brings about a n inhibition of nitrite oxidation b y Nitrobacter

( 1 9 6 ) .

Most of t h e c o m m o n a m i n o acids ( w i t h t h e exception of t h r e o n i n e a n d methionine) decompose in soil i n a similar m a n n e r , a rapid deam-ination taking place. Some of t h e carbon a n d nitrogen of t h e a m i n o acids a r e retained i n t h e soil, possibly i n a protein form ( 7 8 , 1 8 1 , 1 9 4 ) . T h r e o n i n e decomposes v e r y slowly ( 7 8 ) , a n d m e t h i o n i n e decomposition depends on the presence of other organic constituents in t h e soil, e.g., glucose ( 2 3 3 ) .

4. Chitin

According to V e l d k a m p ( 2 4 8 ) , t h e addition of chitin to soil results in the development of organisms t h a t attack chitin, w i t h the u l t i m a t e production of nitrate. T h e percentage of chitin nitrogen w h i c h can be recovered as n i t r a t e depends on t h e t y p e of soil a n d on t h e soil con-ditions, b u t as m u c h as 6 0 % of t h e nitrogen originally present in chitin has been recovered as n i t r a t e nitrogen.

H . D E N I T R I F I C A T I O N I N S O I L

T h e problems concerned w i t h n i t r a t e a n d nitrite reduction in micro-organisms h a v e been discussed at length b y Verhoeven; T a n i g u c h i , Sato, and E g a m i ; N a s o n ; M c E l r o y a n d Spencer; D e l w i c h e ; a n d others

714 J. H . Q U A S T E L

in a symposium devoted to t h e subject ( 1 4 9 ) . T h e y will be discussed, here, only as t h e y are relevant to soil problems.

T h e loss of nitrogen, in a gaseous form, from soils is usually referred to as denitrification. T h e conditions u n d e r w h i c h this process occurs are not well understood, but it seems t h a t the loss of nitrogen from the soil is greatly increased b y poor drainage a n d lack of aeration. It m a y be of considerable m a g n i t u d e , however, even in well-managed cropped soils a n d possibly also in lands still in a virgin condition.

G a y o n a n d D u p e t i t ( 6 7 a - c ) showed t h a t nitrous oxide as well as nitro-gen are formed b y denitrifying bacteria a n d pointed out t h a t n i t r a t e undergoes reduction at the expense of organic substrates b y the micro-flora concerned. T h e y reported u p o n t h e culture a n d behavior of some of the microorganisms w h i c h are capable of denitrification, two of w h i c h t h e y called Bacterium denitrificans A a n d B. Strain A produced N²0 a n d N², strain Β formed N², on synthetic media. T h e r e is probably a universal distribution in soil a n d w a t e r of denitrifying organisms.

V e r y m a n y microorganisms are capable of reducing n i t r a t e to nitrite, t h e former molecule acting as a h y d r o g e n acceptor to a variety of facul-tative anaerobes (202) a n d competing w i t h oxygen as a source of e n e r g y for these organisms. Denitrifiers, w h i c h w e r e studied b y such authors as G a y o n a n d Dupetit, W i n o g r a d s k y , Burris a n d Stutzer a n d others, belong to t h e genera Pseudomonas, Micrococcus, a n d Spirillum.

Beijerinck (19) demonstrated denitrification w i t h concomitant oxida-tion of sulfur b y Thiobacillus denitrificans a n d Thiobacillus thioparus.

K l u y v e r a n d Verhoeven (116a, b) found t h a t Micrococcus denitrificans will b r i n g about oxidation of h y d r o g e n at the expense of nitrate.

As pointed out b y Delwiche ( 1 4 9 ) , soils h a v e u n i q u e properties t h a t m a k e t h e m v e r y efficient as denitrifying systems. A m m o n i u m ions, de-rived from organic m a t t e r i n t h e surface layers of the soil, a r e held b y the soil colloids b y base exchange a n d there undergo nitrification by the nitrifying organisms. T h e n i t r a t e formed is no longer held b y base exchange a n d is leached to lower levels of t h e soil w h e r e there are diminished tensions of oxygen. H e r e t h e n i t r a t e is reduced to nitrite a n d finally to nitrogen or is lost b y leaching into lower w a t e r strata. Chap-m a n , Broadbent, a n d others (25, 26, 36, 88, 271) h a v e Chap-m a d e lysiChap-metric studies of losses of salts in this w a y , a n d t h e y h a v e studied the soil nitrogen-loss problem in its various aspects. Using a n electrolytic respirometer to study soil denitrification, M c G a r i t y , Gilmour, a n d Bollen (150) have concluded t h a t t h e criterion for denitrification in well-drained field soils depends, not o n l y on the availability of n i t r a t e i n excess of t h a t needed for assimilation b y t h e microflora, b u t on a critical r a t e of oxygen consumption whose m a g n i t u d e depends on such

6 . M I C R O B I A L A C T I V I T I E S O F S O I L A N D P L A N T N U T R I T I O N 7 1 5

soil characteristics as s t r u c t u r e a n d permeability. It is a l r e a d y k n o w n ( 2 2 1 ) t h a t active denitrification of n i t r a t e occurs o n l y w h e n the oxygen supply is depleted. T h u s t h e oxygen tension in t h e soil will influence the process of denitrification. T h i s tension will depend on a n u m b e r of factors, n a m e l y , t h e microflora present, the organic substrates available, the permeability to air, w h i c h in t u r n depends on soil structure a n d w a t e r content, a n d on t h e depth of t h e soil. W i j l e r a n d Delwiche ( 2 7 1 ) found, in soil systems w h i c h simulated field conditions, t h a t even less t h a n 1 % of oxygen w a s sufficient to suppress denitrification to about 1 2 % of t h a t obtained u n d e r anaerobic conditions.

T h e evidence indicates t h a t oxygen a n d n i t r a t e compete as h y d r o g e n acceptors in the denitrifying cells, t h e affinity for oxygen exceeding t h a t for nitrate. It is of interest t h a t cells w h i c h a r e g r o w n aerobically show a lag period i n t h e utilization of n i t r a t e , t h u s pointing perhaps to a n adaptive m e c h a n i s m operating for n i t r a t e breakdown.

T h e denitrification of n i t r a t e to nitrogen or to nitrous oxide leads to a rise in p H . W i j l e r a n d Delwiche ( 2 7 1 ) found t h a t a t different h y d r o g e n ion concentrations t h e gaseous nitrogenous products differ in their a m o u n t s ; t h e evolution of nitrous oxide is favored at a n e u t r a l or al-kaline condition, t h a t of nitric oxide is favored u n d e r acid conditions a m o u n t i n g to as m u c h as 2 0 % of t h e total nitrogen evolved at p H 5 . T h u s t h e buffering power of a soil m a y m a r k e d l y affect the n a t u r e of the nitrogenous gases evolved in denitrification. A fact of importance is t h a t nitrous oxide m a y itself act as a h y d r o g e n acceptor w i t h m a n y denitrifying organisms, e.g., Pseudomonas denitrificons, P. {Bacterium) stützen ( 3 6 , 2 6 , 2 5 , 4 8 , 8 8 , 2 7 1 ) .

î. Nitrate Reduction to Nitrite

This process, taking place u n d e r biological conditions, involves the operation of a n e n z y m e ( 2 0 2 ) w h i c h is cyanide sensitive ( 5 , 2 0 4 , 2 3 5 ) . T h e e n z y m e , n o w t e r m e d n i t r a t e reductase, is a flavoprotein linked w i t h triphosphopyridine nucleotide ( 1 6 9 , 1 7 2 ) , m o l y b d e n u m also being involved in the activity of the flavin a d e n i n e dinucleotide

(cf. Chapter 4 ) . T h e following general p a t h w a y has been suggested

( 1 6 9 , 1 7 2 ) :

substrate —> triphosphopyridine nucleotide—• flavin adenine nucleotide—» Mo —• N 03~ 2. Nitrite Reduction

This process is not well understood. N a j j a r a n d C h u n g ( 1 4 9 ) con-clude t h a t the reduction of nitrite to nitric oxide requires t h e presence of the p y r i d i n e a n d t h e flavin nucleotides as electron carriers w i t h

716 J. H . Q U A S T E L

possible participation of copper a n d iron. T h e y consider t h a t the cyto-chromes m a y b e involved i n t h e process. Reduction of nitric oxide to nitrogen seems to involve t h e s a m e components. E v a n s a n d McAuliffe

(149) h a v e shown t h a t reduced diphosphopyridine nucleotide a n d ascorbic acid at p H 3 - 6 can r e d u c e nitrite to N O , N²0 , a n d N², nitric oxide being t h e m a i n product. M c E l r o y a n d Spencer (149) conclude t h a t pyridoxine, or some derivative of it, is directly involved in n i t r i t e reduction a n d assimilation.

T h e steps shown i n Eqs. 1-4 occur i n t h e reduction of n i t r a t e to N2 [Delwiche ( 4 7 ) ] . T h i s scheme (Eqs. 1-4), however, does

2H

NOr > N 0²- + H²0 (1)

nitrate reductase 2H

N 02- > NO" (nitroxyl) + H20 (2)

2NO- » N202- - (hyponitrite) (3)

N20 + H20

2H// ?*

N²0²— (4)

4 H

\

N² + 2H²0

not indicate the mode of formation of nitric oxide, unless this results from t h e dissociation of nitrous acid t h u s : 2 H N 02 -> N 02 + N O -f H20 . T h i s suggestion is tentative a n d t h e r e is c u r r e n t l y m u c h discussion as to t h e possible role of nitric oxide, nitroxyl, a n d nitrous oxide as intermediates i n t h e process of denitrification. Sacks a n d Barker

(213) h a v e observed t h a t u n d e r some conditions the utilization of nitrous oxide b y nitrate-adapted cells of Pseudomonas denitrificans show a lag w h i c h indicates t h a t adaptation to n i t r a t e does not neces-sarily include adaptation to nitrous oxide; t h e y h a v e also concluded that, i n this organism, nitrous oxide is not a necessary intermediate.

Allen a n d v a n N i e l (2) suggest t h a t nitrous oxide is not a n inter-mediate in the formation of nitrogen gas from nitrite, b u t t h a t it is reversibly derived from a n intermediate product i n t h e denitrifica-tion process. K l u y v e r a n d Verhoeven (116a, b ) conclude t h a t nitrous oxide is n o r m a l l y a n i n t e r m e d i a t e i n t h e denitrification process, b u t t h e y suggest t h a t t h e r e a r e two possible paths of denitrification a n d t h a t t h e r e occurs hydrogénation of a n intermediate N202H2. Using isotopically labeled n i t r a t e or nitrite, Delwiche (48) h a s observed the conversion of these anions to nitrous oxide (or nitrogen) a n d t h e utiliza-tion of nitrous oxide b y P. denitrificans. W i t h low levels of n i t r a t e these cells quickly adapt to utilization of nitrous oxide, b u t w i t h high levels of nitrate, adaptation to nitrous oxide occurs after a long lag period. Delwiche concludes t h a t w h e n t h e supply of n i t r a t e or n i t r i t e

6. M I C R O B I A L A C T I V I T I E S O F S O I L A N D P L A N T N U T R I T I O N 717

is limited t h e nitrous oxide formed is reabsorbed a n d reduced to nitro-gen gas.

In document Nutrition of As (Pldal 39-47)