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Summary

Methods used for measuring s o i l and l i t t e r r e sp ixa t ion in the laboratory are o u t l i n e d w i t h speci- - reference to manometric methods. The part icular advantages of Dixon and Gilson respiroveters, which are similar in theory, are discussed and t h e th-:7ry of t h e Dixon respirometer is given in deta l l .

In correcting t h e readings of these t w o xesp~zometers to Normal Temperature and P ~ e s s u x e (N.:: .P. ) , t h e bath temperature is o f t e n used, but from t h e t h e o r y of t h e instrument it is clear t h a t t h e ambient temperature should be used, otherwise an error is introduced.

fnt reduction

Many ecologists and s o i l b io log i s t s are interested in measuring t h e nletabolic z c t i v i t y of soil organisms, either ind3vidually or as a general s o i l population, Numbevs of organisms are: not reliable guides to a c t i v i t y c st otzky 1965, p. 1550). However, it is a general b~ochemical property of living matter that energy- req&ing reactions such as protein synthes is are linked via :::iochemical pathways to reactions in w h i c h energy is released, and w h i l e xany micro-organisms can obtain chemical energy from anaerobic processes, aerobic organisms obtain t h e i r energy from the oxidation of food materials by molecu . la r oxygen, carbon dioxid'le being evolved. One or both conF9nent s of this gas exchange (respiration) can be measured to give an index o f the metabolic activity of t h e aexobes.

G e n e r a l Methods

For the study of respira t ion o f soil and l i t t e r animals, z a o l o g i s t s have evolved var ious methods, many of which invo lve the zeplacement of consumed oxygen by t h a t genera ted by electrolysis of a s o l u t i o n , oxygen consumed being registered in a variety of ways and carbon dioxide evolved being absorbed in a lka l i ( e . g . Helvey, 1952; Macfadyen, 1961; Phillipsan, 19621, Similar t y p e s sf equipment have been used in general s o i l sPudies ( e . g , Greenavood and Lees, 1959; McGarity ,, et .,f a1 1958; Swaby and Passey , 1953; Wagcr and Poxter , 1961; Wieringa and Mogot , 1957).

However, electrical methods o f t e n give pxact ica l difficulties and many .-3rk~xs use t he simpler manometric methods, part icular ly the classical Warburg method (e,g. Bernicr , 1960; Chase and Gray, 1957; Drobnik, 196Oa, l 9 4 O b , 1 9 6 0 ~ ; Gilnour et af 1958; Katznelson and - -3 ~ou;.Tt, 1957; K a t znelson and S t e v ~ n s o n , 1956 ; Parkinson

and Coups, 1963 ; Rovira , 1953; Stevenson, 1956; Webley, 1947). In t h i s respirometer the experimental f l a sk is attached to one a r m of a manometer the other a r m of which is open to the a i r . Small changes in bath temperature or atmospheric pressure during t h e experiment have a considerable effect on the readings. T t is necessaryto have at least one extra respirometer w i t h only water in the flask, t h e readings of t h i s xespirometer ( t h e thexmo- barometer) being subtracted from t h o s e of the other respirometers .

O t h e r workers have used compensated respirometers such as t h a t of Itlaldane (Lees, 1949, 1950) or of Barcroft (Stout, 1963; Johns ton , 1953; Drobnik, 1958). These respixometers have t h e experimental f l a s k on one side of the manometer and a compensation f lask of similar dimensions on t h e other s ide . The system, being t o t a l l y enclosed, is independent of changes in atmospheric pressure, and changes in bath temperature r-ffect both flask and cancel out.

Special f lasks have been used where t h e conventional ones w e r e unsu i t ab le (Drobnik, 1958, 1960a - Parkinson and Coups, 1963; Johnston 1953; Stotzky, 1956).

Howevex, Warburg and Barcroft respirometers have a number of practical disadvantages which make them t i m e - consuming and tedious to use, For example, each flask and manometer must be matched, calibrated, and constants must be calculated for t h e volumes of substances in the flask (KOH, water, sample) in each series of measuxements. The oxygen uptake is calculated f r o m the change in h e i g h t of the manometer fluid using t h e s e c o n s t a n t s . If f lasks are broken, we-matching and re-calibration axe required. A direct-reading i n s t rurnent whi & does not require calibration is therefore more convcni,r?t to use and consumes less time. T h i s is p a r t i c u l a r l y t r u e if one w i s h e s to examine a number o f samples of different sizes and moisture c o n t e n t s at different temperatures. Usually in c a l c u l a t i n g t h e flask constants one does not know precisely t h e volume of s o i l , water, or other materials in the sample, particularly if t h e soil sample is fresh and i n t a c t . Webley (1947) gave a method far calcula t ing the f l a sk cons t an t f o r oxygen when using dry soil to which a known volume of water is added. He assumed that there is no i n t e r a c t i o n of oxygen w i t h the s o i l during t h e one hour experimental period w h i c h he used. He pointed out t h a t the method cannot be used f o r carbon dioxide. Webleyts method has been used by Chase and Gray (1957).

The Dixon Respiroraeter

During t h e past four years we have used Dixun respirom~tzrs (Dixon 1952, g , 6 ) for measuring oxygen uptake of decom~osing plant material ( H o w a r d , 19671, of soil, and also of millipedes, (Bocock and Hursman in prep, ) .

T h i s direct reading r~spirometer is very sui table f o r such studies and h a s a number of advantages over Warburg and Barcroft respirameters.

Dixon (1952) did not describe in deta i l t h e f u n c t i o n i n g of his respirometcr, and t h e desc r ip t ion , in U m b r e i t et a1 - - 9 (1957, p. 111) contains an crror which is discussed below. Although the const ruc t ion of t h e Dixon respirorneter is similar to t h a t of t h e Barcroft apparatus, its theory and use are d i f f erent .

In the Dixon respriometex, the pressure is kept constant and the change in gas volume is measured di rec t ly . The theory is as follows: before gas exchange occurs ( F i g , 1) v is the volume in pl of gas in t h e react ion flask, P, is normal atmosphexic pressure (760 mm of mercury), P is the pressure in t h e respriometer, p is t h e vapour pxessure of water in the reaction flask, p 1 is the vapour pressure of w a t e r in t h e burette, X02 represents quantity of oxygen taken up as PI of dry gas at N.T.P., hC is t h e solubility coefficient of oxygen in t h e liquid in t h e reaction flask, v' is t h e i n i t i a l volume of gas i n the burette , V F is the volume of l iqu id in t h e reaction f l a sk . The reaction and compensating flasks are at bath temperature T, but the burette and manometer are at the ambient temperature Tt . T h e tubes connecting the flasks to the nanometer are of equal and constant volume and can be ignored in the following equation. As t h e compensation flask is there merely to isolate t h e apparatus f r o m t h e outside atmosphere and to eliminate the effects of small changes in bath temperatuxe, it does not enter into the calculations,

For mathematical simplicity t h e apparatus is assumed to bc f i l l e d i n i t i a l l y with pure oxygen, and t h i s gas is assumed to be evolved in t h e reaction flask, The evolved gas displaces t h e manometer fluid (Fig. 2). Gas evolution takes place continuously but readings are taken at fixed t i m e s . When a reading is t aken , the level of mercury in the burette is adjusted to b r i n g the manometer f l u i d back to the i n i t i a l level. The new volume in the burette ( v t f ) and t h u s the volume change in the burette ( v t I - v ' ) are read d irec t ly . As t h e burette and manometer are equifi- brated at Tr, it is t h i s temperature at which the change in gas volume is measured, no t the bath temperature T as s ta ted by Umbreit et a1 (1957, p, Ill), This is an -- irnpoxtant point , as the error involved may be large if t h e bath and ambient temperatuxes are greatly d i f ferent , t h i s is i l lus trated in Table 1.

The amount of oxygen present initially is:

and t ha t present finally is:

Table 1

0 0 Oxygen uptake a f decomposing h c s e l leaves measured at 0 C, 5 C, ~ O ' C

. . . . . . . . . . . . . . . , _ . _ _ .I....A... . . . . . . . . . . . . . . . . . . . ,,.. .....-- ...... ! incorrect correct . . . . . - ........ - - . . j - , .* - - . - .

Barometric bath ambient I e r ror if incorrect p:.~,/gU~,h / A.T .Po f ac to r N.T.P. factor f i 0 g 0 ~ / h factorused pressure temperature Oc

uncorrected bath temp.

i ambienttemp, at

mm Hg t % I

The difference between these is obviously X02 and by subtracting (1) from ( 2 ) we get :

Thus, the volume of oxygen evolved is measured d i rec t ly and t h e o n l y calculation needed is conversion of t h e reading t o N . T . P . It is customary to adjust gas volumes to Normal Temperature and Pressure (N.T.P. ) . In doing so, the gas in t h e respirorneter is assumed to be saturated w i t h water vapour. To a d j u s t t h e volume to that of dry gas :;t 760 rnm of mercury and O W , the observed volume is multiplied by a figure ( t h e N . T . P . f ac to r ) which is obtained from standard tables or nomograms.

On s e t t i n g up t h e Dixon respirometer, t h e compensating flask must have the same volume as the experimental f lask so that the effects of small fluctuations in bath temperature cancel o u t . However, as m o s t respirorneter w a t e r baths maintain t h e temperature to w i t h i n 2 0.1 degree C, a certain amount of lat i tude is possible. The compensating flask is set up in t h e same way as the expeximental s lask, but w i t h inac t ive material in place of the sample. For this purpose we use f i l t e r paper when dealing with leaves, small glass beads w i t h loose s o i l , and tubes of glass wool w i t h s o i l cores. Experience soon shows how accurately the volumes must be adjusted for adequate compensation, but c a l c u l a t i o n s h o w s t ha t if the flasks di f f ex in volume by 1 cc, a temperature rise of 0.2 deg. C will result in a movement in manometer fluid p o s i t i o n ind ica t ing a change i n volume of 0.8 pl, the direction of t h e change depending on which f lask is t h e 1 : . The magnitude of t h e change depends upon the difference in volume between t h c flasks and is independent of t h e size of the flasks.

The Gil son Respiromet er

T h e Gilson d i f f e r e n t i a l respirometer (Umbriet et a1 - ' 1964, pp. 104-105), which is one of several recent modifications of the Dixon respirometer, has become available in B r i t a i n only during t h e past four years and its use i s increasing. In t h i s respirometer, instead of each manometer having a compensating f lask , a l l t h e manometers are linked via a mani fo ld to a s i n g l e compensating f lask. The apparatus has three valves: (a) t h e manifold valve, which connects the manifold to the external atmosphere, It can a l so be used f o r introducting special gas mixtures, (b) the disconnect valve, which can be used to disconnect from t h e manifold any manometer not required, (c) t h e operational valve,

which connects the t w o arms of each manometer above t h e f l u i d level. T h i s valve is in t h e open p o s i t ion when setting up and equilibrating and is closed to commence readings. A 1 1 of t h e operat ional valves can be operated simultaneously by a master lever.

T h i s respirometer is fully compensated only if the t o t a l volume of compensation flask plus manifold is equal to the t o t a l volume of t h e experimental f l a s k s plus connectors. The apparatus is provided w i t h a 250 Cc compensating flask designed for use w i t h standard Warburg type respira t ion flasks, These arc too small f o r many soil and l i t t e r s tud ies and we use f l a s k s of 70 to 100 cc volume.* With fauxteen f lasks having a t o t a l volume of 1400 cc and a compensating flask of o n l y 500 cc ( t h e largest possible size) the effect of the imbalance is merely a small rhythmic r i s i n g and falling of the manometex fluid as the bath thermostat switches on and off . In practice we f ind it quite easy to ignore this movement when reading t h e i n s t r umen t . With smaller experimental flasks t h i s effect disappears. Because the temperature control of the Gilson water bath is + 0.03 degrees C, a small volume imbalance prcduces a neglrgible effect.

The Gilson respirorneter has certain advantages over t h e Dixon respirometer: (a) the former is much more compact, (b) t h e f l a s k s axe connected to t h e manometer by plas t i c tubing and ground glass C14 connectors, the l a t t e r being mounted on s ta in less steel clips which f i t into - - brackets on t h e bath. This makes setting up quick and easy fox one operator and minimises t h e xisks of breakage, (c) because t h e Gilsan xespirometer is available w i t h refrigeration (working temperature -5OC to SO°C) and recording facilities, it is very useful f o r s o i l biology studies, Whereas in the Dixon respirometer t h e change in volume is read on a graduated tube of mercury, in t h e Gilson respirometer the mercury column is replaced by a piston operated by a micrometer screw w i t h a d i g i t a l readout i n p l . The micrometers are calibxated to 0.2 pl and w i t h practice a good operator can read t h e i n s t r u m e n t to t h e nearest 1 pl. Greater accuracy is d i f f i c u l t to a t t a i n because nf the di f f i cu l ty in l o c a t i n g accurately t h e manometer fluid meniscus. No doubt t h i s could be overcome by means of a l e n s . Some diffusion of gases thxough t h e p las t ic tubing occurs, but it only becomes a pxoblem if t h e gases in the apparatus d i f f e r great ly in composition from t h e external atmosphexe. In such cases t h e apparatus has to be madifieri.

The theory of t h i s instrument is t h e s a m e as t h a t of t h e Dixon respirometer, and cont rary to the s t a t e m e n t made in t h e ear l ier Gilson handbooks, t h e ambient temperature not the bath temperature should be used in c a l c u l a t i n g the N.T .P . factox.

* See page 7

General Discussion

Biochemists and micxobiolagists have been measuring t h e respiration of bacter ial suspensions and t i s s u e s for many years, but t h e application of t h e i r techniques to s o i l presents cer ta in difficulties. Thus, w i t h bacter ia l suspensions or tissues slices the chemical composition of the m e d i u m can be accurately controlled and one variable ht a t i m e can be altered. In such cases resp ira t ion may be measured as oxygen uptake, carbon dioxide evolut ion or both. and the xespiratory q u o t i e n t (RQ = vol C02 evolved/vol O2 t aken up) calculated. However, w i t h s o i l s , especially those in t h e f i e l d condi t ion , it is not so simple. Not a l l of t h e carbon dioxide released from s o i l s comes from respira t ion. Whilst oxidative decarboxylation is an important source of respiratory carbon dioxide, "st raightU decarboxylations also occur, of t w o main types: (a) reactions catalyzed by carboxylase and co-carboxylase, (b) reactions catalyzed by t h e amino-acid decarboxylases, Reactions of t h e latter type are common in bacteria, but they axe rare and probably small-scale processes among animals (Baldwin, 1952, p, 418). Furthermore, there m a y be inorganic reactions involving exchange of carbon dioxide in s o i l , and these may vary w i t h horizon and s o i l type (e.g. seeRusse11, 1961, p, 106 et s). Methods for determining RQ values of soils have been described (e.g. Stotzky, 1960; Rixon and Bridge 19681, but these must be regarded as suspect. With soif, measurement of oxygen uptake is obviously preferable.

The way in which t h e results of laboratory measurements of soil resp i ra t ion are expressed depends largely on the purpose of the measurements, Results can be calculated on t h e basis of so i l volume, dry weight or carbon content .

With s o i l , a l l laboratoxy xespiration measurements are to some extent a r t i f i c i a l because the so i l has been removed from i t s normal s u r r o u n d i n g s and its aeration and othex factors are altered. These effects become greater if the material is kept in t h e respirorneter fox long periods. Some of these problems are discussed by Stotzky (1965), Drobnikova and Dsobnik ( I N S ) , and Dornsch (1962) discusses methods and results.

With decomposing leaf l i t t e r , our experience has shown t h a t careful handling of t h e sample has no appreciable effect on its respiration,

Flasks f o r use w i t h Dixon and Gilson respirometers

Conventional. flasks are rarely suitable fo r use w i t h s o i l , p lant material, or small animals. We use flasks like t h a t of Parkinson and Coups (19631, but having a B40 base, or those shown i n Fig. 3 , which were designed in our laboratories.

In flask A t h e KOH is introduced i n t o t h e annu la r well v i a t h e C14 connector at the t o p , an automatic pipette w i t h a narrow plas t i c tube is use fu l for t h i s . The bottom ground joint is a R40 cup, and the sample is placed in a base made from a B40 cone. These can be made any desired depth .

If t h e rate of respiration is low, t h e large volume of f l a s k A can be a disadvantage. We therefore designed flask B, also us ing the B40 base, With our shallowest base, flask A has a volume of 135 cc and flask B 80 cc. Flask B has no alkali well, but to ovexcome t h i s t h e sample is placed in a specimen tube and t h e alkali is placed in t h e base. Advantages in using specimen tubes are t h a t handling of t h e sample is easier and fewer bases are needed.

Flask C has been used by Bocock and Horsrnan (in prep.) in studies an millipede xespirat ion. Here again, the sample is placed in a specimen tube which fits into the centre well, An annu la r space is provided fox alkali. This flask has a B29 neck and connects to the respirometer via a B29-C14 adaptor. The total volume of f l a s k plus adaptor is 6 5 cc, but if necessary t h i s can be reduced by f i l l i n g the adaptor with an iner t material which will n o t impede gas exchange ( e . g . glass or plastic beads),

For t h i s type of work it is essential for all ground glass jo ints to be of the highest standard if l eaks are to be avoided. Serious f a u l t s in interchangeable ground glass j o i n t s are: (a) the cup or cone is not pexfectly round (the joint "bindsH in places when xotated); (b) t h e taper of one or both components is incorrect ( t h e joint w i l l rock about i t s long axis).

The large number of f lasks types A and B, and bases, made f o r us by Gallenkamp (Widnes) Ltd., have proved e n t i r e l y sat isfactory. I shall be glad to supply further informat ion W anyone interested.

With s o i l , litter and small animals, one does no t need to shake t h e f l a s k s , as is necessary when l i q u i d media are used , so t h a t the size of t h e f l a s k is not a disadvantage.

Acknowledgements

1 am grateful to Professor M. Dixon f o r h i s comments on t h e f u n c t i o n i n g of t h e Dixon respirometer.

References

BALDWIN, E , ( 1952 ) . Dynamic aspects of biochemistry . Cambridge Univ. Press. 2nd Ed.

BERNIER, B. (1960). The respiratory metabolism of some samples of forest humus. C o n t r . Fond. Rech. For. Univ. Laval. No. 5 . pp, 44,

CHASE, F. E. and GRAY, P , H. H. (1957). Application of the Warburg respixometer in studying respiratory ac t iv i ty in soil. Canad. J. Microbiol., 3, 335-349.

DIXON, M. (1952). Manometric Methods. Cambridge Univ. Press, -

IDOMSCH, K, H. (1962). Bodenatmung: Sammelbericht h e r Methoden und Ergebnisse, +l. Bakt . , 116, 33-78.

DRQBNIK, J. (1958). An apparatus for studying respira- t i o n in a soil sample, Fol. Biol., 5, 188-192.

DROBNIK, J, (1960 a). A Warburg vessel for so i l samples. Nature, Lond,, 188, 686.

DROBNIK, J. (1960 b). Primary oxidation of organic matter in t h e soil: I . The form of respiration curves w i t h glucose as the substrate, Plant and Soil 12 199-211, - 9 '

DROBNIK, J. (1960 c ) , Primary oxidation of organic matter in t h e zoil: T I Inf luence of various kinds of pre-incubations. Plant and Soil, 12, 212-222. -

DROBMIKOVA, V, and DROBNIK, J, (1965). S o i l Respiration. Z b l . B a k t , , 119, 714-729.

GILMOUR, C . M: , DAMSKY, L. and BOLLEN, W. B. (1958). Manometric gas a n a l y s i s as an index of microbial oxidations and reductions in soil. Canad. J. Microbiol. , 4, 287-293.

GREENWOOD, D. J. and LEES, H. (1959). An electrolyt ic rocking percolator. Plan t and S o i l , ll, 87-92.

HELVEY, T. C. (1951). Microrespisometer with automatic e lectr ic cant x o l of constant oxygen p a r t i a l pxessure. J. Sci. I n s t r . , 28 , 354.

HOWARD, P . J . A . (1967)- A method fox studying the respiration and decomposition of l i t t e r .- 1n Progress in Soil Biology, Vieweg and Sohn, Braunschweig, 464-472.

JOHNSTON, D. R . (1953). A laboratory s t u d y of t h e decomposition of vegetable debris in r e l a t i o n to the formation of raw humus, Plant and " o i l , 5, 345-369. .-

KATZNELSON, H. and ROUATT, J. W, (1957) . Manometric studies with rhizosphere,and non-rhizosphere soil. Canad. 3 . Microbioh., 3, 673-678.

KATZNELSON, W. and STEVENSON, 1, L. (1956 ) . observations -on t h e metabolic ac t iv i ty of t h e soil microflora. Canad.. J. Microbial,, - 2 , 611-622.

LEES J (1949). A simple apparatus for measuring t h e oxygen uptake of s o i l s , P l a n t and Soil, 2, 123-128.

LEES, H. (1950), A percolat ing respirometer . Nature, Lond 166, 118. * 9 -

MACFADYEN, A. (1961). A new system for continuous respiromet r y of s m a l l a i r - b r e a t h i n g invertebrates under near-natural conditions. J. exp. B i o l . , 38, 323-341.

McGARITY, J. W., GILMOUR, C, M. and BOLLEN, W. B, (1958). U s e of an electrolytic respirometer to study denitr i f i c a t i o n in soil, Canad, J . Microbiol, , 5, 303-316 +

PARKINSON, D, and COUPS, E. (1963). Microbiol a c t i v i t y in a podsol. In Soil Orqanisms. Eds. J. Qoeksen and J. V a n d e x D r i f t , North Holland P u b l i a i n q C o . , A m s t e r d a m , 167-175.

PHILLIPSON, 5. (1962). Respirometry and t h e study of enexqy turnover in natural systems with particular refeience to harvest spiders- (Phalangiida) . Oikos -9 13, 311-322.

RIXON, A. 3 . and BRIDGE, B. J. (1968). Respiratory quotient a r i s i n g from microbial -1. tivity in re la t ion to matric s u c t i o n and air-filled pore space of soil, Nature, Lond., 218, 961-962.

ROVIRA, A. D. (1953). Use of t h e Warburg apparatus in s o i l metabolism studies. Nature, Lond., 172, 29-30.

RUSSELL, E, W. (1961). S o i l conditions and p lant growth. 9th Ed. Longmans Green & Co .

STEVENSON, I. L. (1956). Some observations an the microbiol a c t i v i t y in remoistened air-dried s o i l s . P l an t and Soil, 8, 170-182.

STOTZKY, G. (1960). A s i m ~ l e method fo r t h e determination of-the respiratory quotient of so i l s . Canad. J. Microbiol., 2, 439-452.

STOTZKY, G. (1965). Microbial resp i ra t ion . In "Methods of Soil A n a l y s i s Vol. 2," No. 9 in the series t'Aqronomy't, published by the American society of Agronomy. pp. 1550-1572,

STOUT, J. D. (1963). Some observations on t h e protozoa 6f some beechwood s o i l s on the C h i l t e x n hills. - J. h i m . Ecol . , 32, 281-287.

SWABY, R . J. and PASSEY, 3. I. (1953). A u s t r a l . J. aqric. R e s 4, 334-339. -- 3 -

UMBREIT, W. W:, BURRIS, R . H. and STAUFFER, J. F. (1957). Manornetclc Techniques, Bursess Pub. Co., U . S . A .

UMBREIT, W. W . , BURRIS, R, H. and STAUFFER, 3. F. (1964). ~anometric ~ e c h n i q u c s . 4th Edition. ~ u r ~ e s s Publishing Co., U . S . A ,

WAGER, H, G. and PORTER, F. A . E. (1961). An apparatus for t h e automatic measurement of oxygen uptake by electrolytic replacement of the oxygen consumed, Biochern. J., -, 81 614-618.

WEBLEY, D. M. (1947). A technique f o r the study of oxygen availability to micro-organisms in s o i l and i t s possible u s e as an index of s o i l &-rat ion. J. agric. s&. , 37, 249-255, -

W I E R I N C A , K. T, and M-, M. K. F. (1957). An apparatus f o x t h e determination of the respiration process in s o i l samples, Plant and Soil, 8, 395-396.

Figure I. Conditions before gas evolution

Candf t ion s after gas evalut ion