A . T H E P E R F U S I O N T E C H N I Q U E
T h e perfusion t e c h n i q u e (7, 120b, 122, 126, 1 2 8 a - c ) w a s p r i m a r i l y designed for t h e s t u d y of metabolic p a t t e r n s i n soils. It has t h e ad-v a n t a g e oad-ver techniques w h i c h inad-volad-ve t h e u s e of p u r e cultures of organisms i n t h a t it enables studies to be m a d e of soil metabolic processes, a n d of t h e factors w h i c h influence t h e proliferation of t h e responsible organisms, u n d e r conditions w h i c h a p p r o x i m a t e to those in t h e field. Investigations a r e confined to studies of organisms proliferat-ing i n contact w i t h t h e m a n y species of microorganisms n o r m a l l y present i n soil. T h u s , it m a y be shown t h a t a c o m p o u n d (e.g., m e t h i -onine) w h i c h n o r m a l l y has b u t little effect on a n o r g a n i s m g r o w n
734 J. H . Q U A S T E L
in a p u r e culture, is h i g h l y inhibitory to t h e development of the organism in soil. T h i s result is due to t h e compound giving rise in soil to other substances t h a t are h i g h l y inhibitory to t h e organism u n d e r investigation. It also h a p p e n s t h a t a substance h i g h l y inhibitory to a n organism g r o w n in p u r e culture (e.g., certain antibiotics) has b u t little effect on t h e same organisms as t h e y develop in soil. T h i s results from t h e rapid decomposition of t h e substance in soil or from the de-velopment of resistant or adapted strains of t h e organisms u n d e r study.
Problems of adaptation of m u t a t i o n m a y be conveniently investigated w i t h the soil perfusion technique.
1. Perfusion Apparatus
T h e a p p a r a t u s used at present [ A u d u s ( 7 ) ] is a modification of t h a t used b y Lees a n d Quastel ( 1 2 6 ) , as the latter is somewhat complicated a n d involves the use of a continuous s t r e a m of w a t e r to produce t h e i n t e r m i t t e n t perfusion. T h e modification (7) is shown i n Fig. 1; the technique is described below in some detail.
T h e soil, 30 g m of air-dried sieved crumbs ( 2 - 4 m m in d i a m e t e r ) , is contained in a glass tube between pads of glass wool. T h e perfusing solution is in t h e separating funnel, F. A constant small suction is applied at A. T h i s suction is transmitted back t h r o u g h t h e lengths of t h e r m o m e t e r tubing, R± a n d R2 (or other suitable resistances), a n d the soil column in Ρ to t h e perfusing solution in tube T. T h i s causes air to be d r a w n in at t h e bottom of the side tube, 5 , t h e r e b y detaching a column of solution which is d r a w n u p tube Τ a n d falls on t h e top of t h e soil column. T h i s discharge releases the partial v a c u u m , a n d the solution again rises above t h e base of S u n t i l it reaches t h e level of t h e solution in F. M o r e air is t h e n d r a w n in, t h r o u g h S, a n d the whole process is repeated. T h e liquid discharged from t u b e Τ perfuses t h r o u g h t h e column of soil, w h i c h t h u s becomes saturated w i t h fluid a n d is i n t e r m i t t e n t l y aerated. T h e a p p a r a t u s m a y be insulated against aerial organisms b y a cotton plug, b u t this has not been found to be necessary. It m a y be linked w i t h a n y source of gas (oxygen, nitrogen, helium, carbon dioxide, or mixtures of these gases) b y connecting w i t h a n appropriate gas cylinder. F o r analysis, t h e suction is stopped a n d fluid is allowed to rise in S, w h e n c e aliquots are removed. After such removal, the suction is restored a n d perfusion recommences. This m a y proceed indefinitely. T h e soil is not h a n d l e d u n t i l the end of t h e ex-periment, so as not to interfere w i t h t h e equilibria established i n t h e soil. T h e soil m a y of course be removed at a n y time for analysis of adsorbed constitutents. E x p e r i m e n t s are r u n in a thermostatically con-trolled room (21 °C) in t h e dark. As the a p p a r a t u s is inexpensive a n d
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 735
FIG. 1. Apparatus for intermittent perfusion of soil. See text for description of the technique. From Audus (7).
easy to h a n d l e , experiments m a y be r u n in duplicate or triplicate and m a n y such perfusion u n i t s m a y be assembled into a bank.
A substance whose metabolism in t h e soil is being investigated is dissolved in t h e perfusion fluid or mixed w i t h t h e column of soil. T h e volume of perfusate is kept constant b y periodic additions of w a t e r to replace the a m o u n t lost b y evaporation.
T h i s technique for investigating soil metabolism has a variety of advantages, including the following:
736 J. H . Q U A S T E L
1. T h e w a t e r content of t h e soil is kept constant a n d it is homo-geneously distributed in the soil t h r o u g h o u t t h e experiment.
2. M a x i m a l aeration of t h e soil is effected.
3. Gases entering the a p p a r a t u s can be controlled, a n d metabolic events in a n y defined atmosphere m a y be studied.
4. Substances such as biological poisons or inhibitors m a y be added to t h e soil solution (or perfusate) d u r i n g t h e course of t h e experi-m e n t , at a n y period corresponding to a k n o w n experi-metabolic activity of the soil.
5. T h e soil solution can be replaced at a n y time b y a solution of a n y metabolite whose transformations b y a soil w i t h k n o w n metabolic activity a r e t h e subject of study.
6. Ionic equilibria between soil a n d solution are quickly established a n d a r e not further affected except insofar as t h e equilibria a r e dis-turbed b y the products of metabolism in soil.
7. T h e soil is not h a n d l e d i n a n y w a y d u r i n g the experiment, analysis being confined to the constituents of t h e perfusate. T h e soil m a y be sampled after a n y a r b i t r a r y t i m e for analysis of ions a n d other substances adsorbed onto t h e soil.
8. Gases leaving t h e a p p a r a t u s m a y be analyzed for C 02, etc.
9. T h e a p p a r a t u s minimizes biological variation between one sample of soil a n d another a n d lends itself to quantitative kinetic studies w h i c h m a y be reproduced w i t h considerable accuracy.
T h e perfusion u n i t has been modified b y Lees (78, 120b, 122), b y T e m p l e ( 2 4 2 ) , a n d b y Collins a n d Sims ( 4 2 ) .
2. The Soil and the Perfusate
Experience has shown t h a t t h e ideal soil for t h e perfusion a p p a r a t u s is one t h a t has been air dried for a week at room t e m p e r a t u r e a n d w h i c h consists of particles 2 - 4 m m in diameter. A garden soil or agri-cultural soil of good structure is to be preferred. Details of the h a n d l i n g of soils from various sources a r e given b y Quastel a n d Scholefield ( 1 9 9 ) . I n most experiments in w h i c h the soil perfusion technique is utilized, no n u t r i e n t s other t h a n t h e substrate u n d e r consideration are added.
Soils do not seem to require the addition of minerals, vitamins, essential amino acids, or a n y accessory growth factors for metabolism to take place. F o r example, to obtain good proliferation of t h e nitrifying organisms in soil it is necessary only to perfuse a m m o n i u m chloride solution.
T h e concentration of a substrate m a y b e varied w i t h i n w i d e limits, while ensuring good rates of metabolism. H i g h concentrations produce only a n increase in t h e lag period preceding proliferation, a n d low
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 737
concentrations suffer o n l y from t h e drawback t h a t most of t h e substrate is oxidized or metabolized before t h e proliferating stage is reached. I n general, a concentration of 10~2M substrate has been found convenient.
3. Enriched Soils
W h e n a metabolizable substrate, e.g., a m m o n i u m ions, nitrite or thiosulfate ions, m a n g a n e s e or arsenite ions, is perfused t h r o u g h soil, t h e r e occurs a n initial lag period before metabolism commences. Its duration varies w i t h t h e t y p e of soil, concentration of substrate, etc.
It diminishes w i t h subsequent perfusions. Eventually, after repeated perfusions a constant r a t e of b r e a k d o w n of substrate or other metabolite occurs. W i t h each perfusion a n increased e n r i c h m e n t of the soil w i t h those microorganisms t h a t attack t h e substrate takes place, a n d finally a state of saturation of t h e soil occurs after w h i c h no further increase i n t h e velocity of b r e a k d o w n of t h e metabolite takes place. Such a soil is often referred to as a n enriched or bacterially saturated soil. W i t h m a n y forms of metabolism in soil, a n d especially those involving the autotrophs, t h e entire metabolism seems to occur at the surfaces of the soil particles. W h e n the soil is removed, no further changes take place in t h e soil perfusate, w h i c h is u s u a l l y w a t e r clear a n d shows re-m a r k a b l y few organisre-ms u n d e r t h e re-microscope. T h e organisre-ms are adsorbed by, a n d probably proliferate at, the soil c r u m b surfaces.
Such a n enriched, or saturated, soil m a y be used for a variety of experimental purposes ( 1 9 6 ) . It m a y be used for the metabolic events u n d e r study, or it m a y be used directly for kinetic a n d stoichiometric studies. E x p e r i m e n t s show t h a t such a n enriched soil acts in every w a y as though it w e r e a preparation of resting cells of t h e responsible organisms. T h e fact t h a t t h e soil organisms are not proliferating u n d e r these conditions is shown b y t h e absence of a n y inhibitory effect of such a growth inhibitor as sulfanilamide. M a n y other properties of t h e resting organisms in soil m a y be studied in a similar m a n n e r .
E n r i c h e d soil c r u m b s m a y be used to inoculate fresh samples of soil a n d so diminish, or avoid, lag periods.
It is k n o w n n o w t h a t a soil m a y become enriched at t h e same t i m e w i t h at least two different sets of organisms ( 1 9 8 ) , it being a p p a r e n t t h a t specific organisms a d h e r e to, or proliferate at, specific sites on the soil c r u m b surfaces.
W h e n w e t " e n r i c h e d " soils (e.g., those capable of oxidizing thio-sulfate) a r e quickly dried i n a c u r r e n t of cold air, t h e y m a y retain their high oxidizing activities for several m o n t h s if t h e y a r e stored between 0° a n d 4°C. H o w e v e r , if t h e soils are kept at room tem-perature, this r a p i d oxidizing power m a y be lost in a few days. T h e
738 J . H . Q U A S T E L
retention of oxidizing ability of stored " e n r i c h e d " soils depends on t h e efficiency, a n d t e m p e r a t u r e , of d r y i n g a n d on t h e n a t u r e of t h e organisms involved.
B. M E T A B O L I C S T U D I E S A N D M A N O M E T R I C S T U D I E S O F S O I L
1. Metabolic Studies
T h e following a r e some of t h e metabolic studies in soil, m a n y of w h i c h h a v e a bearing on p l a n t nutrition, t h a t h a v e been carried out b y the perfusion technique:
1. Nitrification i n soil a n d n i t r a t e formation from organic nitrogen compounds (37, 38, 78, 128a-c, 194, 1 9 6 ) .
2. Effects of chlorates on soil nitrification ( 1 2 7 ) .
3. Effects of alkylthio compounds on soil nitrification ( 2 8 ) .
4. Effects of u r e t h a n e s on soil nitrification a n d metabolism of u r e t h a n e s ( 1 9 7 ) .
5. Conversion of oximes to nitrites (200, 2 0 1 ) . 6. M a n g a n e s e metabolism ( 1 5 1 ) .
7. Thiosulfate a n d tetrathionate metabolism ( 7 5 ) . 8. Arsenite conversion to arsenate ( 1 9 8 ) .
9. Iron metabolism ( 7 3 ) .
10. Breakdown of indoleacetic acid, coumarin, a n d herbicides such as 2,4-dichlorophenoxyacetic acid (8a,b, 9 ) .
11. Metabolism of bile acids in soil ( 8 4 ) . 12. T h i o c y a n a t e oxidation in soil ( 7 4 ) .
2. Rocking Percolation Technique
A rocking percolation, or perfusion, technique for soil studies has been devised b y Greenwood a n d Lees ( 7 8 ) ; this technique allows for m e a s u r e m e n t s of oxygen consumption. It h a s been modified (79, 238, 270) in such a w a y t h a t automatically m e t e r e d a n d electrolytic generated oxygen w i t h i n t h e a p p a r a t u s replaces the oxygen consumed b y metabolic processes. T h i s electrolytic, rocking, percolation u n i t re-tains t h e advantages of t h e original m a n o m e t r i c , rocking, percolating a p p a r a t u s a n d allows m e a s u r e m e n t s to be m a d e of oxygen u p t a k e d u r i n g t h e breakdown of a n y substance in a soil at a given partial pressure of oxygen.
3. Manometric Studies of Soils
Samples of soil taken either directly from the field, or from the perfusion a p p a r a t u s after soil has been enriched w i t h organisms t h a t
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 739
attack a particular substrate, m a y b e placed i n a conventional W a r b u r g m a n o m e t r i c a p p a r a t u s a n d t h e i r rates of oxygen consumption i n the presence or absence of t h e substrates or of other substances m a y be m e a s u r e d (75, 194, 196). I n this w a y , for example, it is easily shown t h a t for every molecule of thiosulfate added to a suitably enriched soil four atoms of oxygen corresponding to t h e complete oxidation of thio-sulfate to thio-sulfate a r e taken u p ( 7 5 ) . T h e process is affected b y respir-atory inhibitors a n d b y substances w h i c h u n c o u p l e phosphorylation a n d oxidation, so t h a t it becomes possible to m a k e observations on t h e mech-anisms involved i n t h e respiratory process b y investigations only on the enriched soils. A g a i n m a n o m e t r i c studies of suitably enriched soils show t h a t the theoretical oxygen consumption occurs in the oxidation in soil of arsenite to arsenate (198) a n d t h a t certain oxides of arsenic a n d a n t i m o n y can inhibit this process. A n o t h e r p e r t i n e n t e x a m p l e is the m a n o m e t r i c study (197) of soils enriched w i t h organisms t h a t de-compose ethyl u r e t h a n e . T h e oxygen consumed b y such soils in the oxidation of ethyl u r e t h a n e is i n accordance w i t h t h e conclusion t h a t acetic acid is t h e end product. Acetic acid is, a p p a r e n t l y , not further oxidized or is oxidized o n l y v e r y slowly. T h e kinetic a n d stoichiometric evidence leads to t h e conclusion t h a t e t h y l u r e t h a n e is first broken d o w n b y soil organisms to ethanol, a m m o n i a , a n d carbon dioxide w i t h t h e subsequent formation of acetic a n d nitric acids [see also ( 2 3 4 ) ] .
Katznelson a n d Rouatt (111) h a v e pointed out, on t h e basis of m a n o m e t r i c studies, t h a t t h e oxygen consumption of t h e soil in the rhizosphere is greater t h a n t h a t of nonrhizosphere soils, both w i t h a n d without added substrates. M a n o m e t r i c studies of soils h a v e been carried out to s t u d y t h e effects of t h e a d m i x t u r e of soils w i t h straw a n d straw extracts, w i t h nitrates a n d a m m o n i u m salts on soil respira-tion ( 7 2 ) . Various studies of microbial metabolism i n soil, using the m a n o m e t r i c method, h a v e been m a d e (30, 39a,b, 5 1 , 112a,b, 210) a n d these indicate the potentialities of this technique for soil studies in relation to p l a n t nutrition. T h e use of t h e m a n o m e t r i c m e t h o d for studies of soil aeration h a s long been advocated (54, 203) a n d w a s of considerable service i n the study of synthetic soil conditioners.
C. R O L E O F M A N G A N E S E A N D O F I R O N
It is k n o w n (cf. C h a p t e r 4 ) t h a t for t h e h e a l t h y growth of plants traces of a variety of elements such as iron, copper, boron, zinc, m a n -ganese, a n d m o l y b d e n u m a r e necessary, a n d probably also of cobalt, v a n a d i u m a n d sodium. T h e a m o u n t s of some of these elements w h i c h are required for t h e h e a l t h y development of a given p l a n t m a y be exceedingly small. Deficiencies of these substances, however, lead to a
740 J. H . Q U A S T E L
great v a r i e t y of p l a n t diseases. It does not follow that, if a n essential element is present i n t h e soil, it is necessarily available to t h e plant.
1. Manganese Metabolism
A deficiency of m a n g a n e s e i n soil—and soils rich i n organic m a t t e r a n d lime are prone to this deficiency—leads to p l a n t diseases, such as g r a y speck of oats or m a r s h spot of peas, a n d to a substantial reduction i n t h e yield of a potato (Solanum tuberosum) crop or to t h e complete failure of a n oat crop. But m a n y of these deficient soils—as diagnosed b y inspection a n d analysis of t h e crop—often contain relatively large quantities of manganese. T h u s it is a p p a r e n t t h a t m a n g a n e s e exists i n t h e soil in at least two forms, of w h i c h only one is available for the plant. So far as is k n o w n it is only the base-exchangeable form of m a n g a n e s e (probably the divalent m a n g a n o u s ions) w h i c h is available for t h e plant. T h e question n o w arises w h y certain soils, w h i c h con-tain a m p l e quantities of m a n g a n e s e , a r e " m a n g a n e s e deficient" and w h y other soils, w h i c h m a y contain m u c h less m a n g a n e s e , a r e " m a n -ganese available." T h i s problem is i n t i m a t e l y connected w i t h the metabolic transformations to w h i c h m a n g a n e s e is subjected in soil.
I t is n o w k n o w n (151) t h a t w h e n m a n g a n e s e sulfate is perfused t h r o u g h soil, oxidation of t h e m a n g a n e s e takes place. I n n e u t r a l or slightly alkaline soil this oxidation is almost entirely accomplished b y t h e microorganisms w h i c h a r e present. T h i s is so because t h e r a t e of oxidation of m a n g a n e s e in soil at 21 °C follows the logarithmic course expected if proliferating organisms are responsible for the oxidation, and b y t h e fact t h a t sterilization of a soil, or its t r e a t m e n t w i t h cell poisons a n d narcotics, m a y result i n a complete cessation of m a n g a n e s e oxida-tion. It was a l r e a d y k n o w n (69, 70a, 188) t h a t soil contains organisms w h i c h are capable of oxidizing bivalent m a n g a n e s e , b u t quantitative studies w i t h t h e perfusion a p p a r a t u s h a v e established h o w m u c h of the oxidation which takes place in t h e soil is in fact d u e to microorganisms.
Nonbiological autoxidation of m a n g a n e s e i n soil h a d been t h o u g h t to be a d o m i n a n t process. N o t only is bivalent m a n g a n e s e oxidized to states of higher valency b y soil microorganisms, b u t similar agencies are re-sponsible for t h e reduction of tervalent a n d q u a d r i v a l e n t m a n g a n e s e to the bivalent form. M a n y substances in soil will reduce m a n g a n e s e dioxide, e.g., polyphenols, thiol compounds, a n d ferrous ions, but bacterial suspensions will also accomplish the reduction so long as there are present traces of such h y d r o g e n carriers as bacterial pigments or hematins. N o r m a l l y , biological reduction of t h e higher valency forms of m a n g a n e s e takes place in t h e soil at t h e expense of t h e organic substances present w h i c h a r e themselves oxidized. T h e presence
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 4 1
of glucose, a n d other carbohydrates, i n a soil containing m a n g a n e s e dioxide brings about a n increased production of m a n g a n e s e ions, for t h e glucose stimulates t h e g r o w t h of organisms w h i c h accomplish t h e reduction of the h i g h e r v a l e n c y states of m a n g a n e s e . T h i s p h e n o m e n o n is seen i n t h e curves shown i n Fig. 2.
3 0 0
ε
CL
2 0 0 *
100 i σ
ο» c 2 σ
0
0 2 4 6 8
Time,days
FIG. 2. Effects of perfusing glucose at 21 °C on exchangeable (bivalent) manganese of Rothamsted soil. From Mann and Quastel (151).
Autoxidation at high pH Taken up by
the plant
( M n203)
FIG. 3. Manganese cycle in soil under aerobic conditions. From Mann and Quastel (151).
T h e facts point to t h e existence in soil of a cycle of biological changes w h i c h involve m a n g a n e s e oxidation a n d reduction, such as t h a t shown in Fig. 3. T h e kinetics of t h e processes in this cycle d e t e r m i n e t h e a m o u n t of bivalent m a n g a n e s e available i n soil a t a n y m o m e n t . T o prevent m a n g a n e s e deficiency i n t h e field, so long as some form of m a n g a n e s e is present, it is necessary to b r i n g about a suitable shift
S a Glucose j
\
Manganese Je \
t i l l
742 J. H . Q U A S T E L
in the equilibrium. This m a y be accomplished b y sulfur [ a n d possibly thiosulfate ( 1 9 3 ) ] t r e a t m e n t , b u t it is u s u a l l y accomplished b y spray-ing w i t h m a n g a n e s e sulfate (for fruits, peas, a n d oats). D i o n a n d M a n n
(49) h a v e shown t h a t t h e first product of biological oxidation of divalent m a n g a n e s e i n soil is t h e trivalent ion w h i c h , u n d e r acid condi-tions, gives rise to a m i x t u r e of divalent m a n g a n e s e ion a n d m a n g a n e s e dioxide. T h i s dismutation a p p a r e n t l y goes on i n soil so t h a t it is clear t h a t t h e biological oxidation of bivalent m a n g a n e s e i n n e u t r a l or slightly acid soils eventually leads w h o l l y to m a n g a n e s e dioxide.
U n d e r anaerobic conditions, or i n t h e presence of respiratory poisons such as sodium azide, t h e m a n g a n e s e equilibrium shifts m a r k e d l y to-w a r d divalent m a n g a n e s e , a n d u l t i m a t e l y all t h e m a n g a n e s e to-w o u l d a p p e a r in t h a t form if oxidative conditions w e r e completely suppressed.
M a n g a n e s e dioxide can act as a n oxidant, or t e r m i n a l h y d r o g e n acceptor, i n a v a r i e t y of biological systems ( 9 5 ) , a n d its biological oxidizing properties m a y h a v e i m p o r t a n t consequences i n soil chem-istry. It is well k n o w n t h a t thiol compounds act i n a h a r m f u l m a n n e r in t h e soil either b y depriving soil microflora of oxygen, or b y en-couraging t h e development of anaerobes, or b y accomplishing specific toxic effects. T h e presence of m a n g a n e s e dioxide i n a soil will, so long as it is i n some excess, r e n d e r t h e soil relatively free from thiol com-pounds. W h e n t h e anaerobic conditions a r e replaced b y t h e aerobic, t h e m a n g a n e s e ions a r e reconverted to m a n g a n e s e dioxide a n d t h e soil store of this substance is replenished. T h u s t h e m a n g a n e s e dioxide-m a n g a n e s e cycle acts as a n oxidant buffer systedioxide-m protecting the soil organisms, including plants, against deleterious agents, such as a variety of sulfur compounds a n d other reducing bodies.
2. Ferrous Oxidation
Just as biological oxidation of divalent m a n g a n e s e takes place i n soil so does a biological oxidation of ferrous ions occur ( 7 3 ) . Specific iron-oxidizing organisms are a b u n d a n t i n soil. A n y ferric ions, t h a t are formed, can also u n d e r g o biological reduction, so it is a p p a r e n t that, as w i t h m a n g a n e s e , a cycle of oxidation-reduction takes place w i t h iron.
Such cyclic processes involve t h e proliferation both of autotrophic organisms, w h i c h accomplish, for example, t h e oxidation of divalent iron, a n d of heterotrophic organisms t h a t b r i n g about t h e reduction of these substances to lower v a l e n c y states.