Use of Adencosine 5'-Triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mr. 1977, p Copyright 1977 Americn Society for Microbiology Vol. 33, No. 3 Printed in U.S.A. Use of Adencosine 5'-Triphosphte s n Indictor of the Microbiot Biomss in Rumen Contents C. W. FORSBERG* AND K. LAM Deprtment ofmicrobiology, College ofbiologicl Science, University ofguelph, Guelph, Ontrio, NIG 2Wl, Cnd Received for publiction 20 September 1976 A number of techniques were tested for their efficiency in extrcting denosine 5'-triphosphte (ATP) from strined rumen fluid (SRF). Extrction with 0.6 N H2SO4, using modifiction of the procedure described by Lee et l. (1971), ws the most efflcient nd ws better suited for extrcting prticulte smples. Neutrlized extrcts could not be stored frozen before ssying for ATP becuse lrge losses were incurred. The inclusion of internl stndrds ws necessry to correct for incomplete recovery of ATP. The ATP concentrtion in rumen contents from cow receiving rtion of dried roughge (minly lflf hy) rnged from 31 to 56,ug of ATP per g of contents. Approximtely 75% of the ATP ws ssocited with the prticulte mteril. The ATP ws primrily of microbil origin, since only trces of ATP were present in the feed nd none ws found in "cell-free" rumen fluid. Frctiontion of the bcteril nd protozol popultions in SRF resulted in the isoltion of n enriched protozol frction with 10-fold higher ATP concentrtion thn tht of the seprted rumen bcteri. The ATP pool sizes of nine functionlly importnt rumen bcteri during the exponentil phse of growth rnged from 1.1 to 17.6,ug of ATP per mg of dry weight. This informtion indictes tht using ATP s mesure of microbil biomss in rumen contents must be done with cution becuse of possible vritions in the efficiency of extrction of ATP from rumen contents nd differences in the concentrtion of ATP in rumen microbes. Currently used indictors of microbil biomss in rumen contents include the mino cid 2,6-diminopimelic cid for bcteri nd 2-minoethylphosphonic cid for protozo (8). 2,6-Diminopimelic occurs in the bcteril cell wll peptidoglycn nd is bsent from plnt nd niml tissues. 2-Aminoethylphosphonic cid is found only in protozo nd niml tissue (1, 12). In protozo, 2-minoethylphosphonic cid occurs in both lipids nd proteins, wheres in higher nimls it is confined minly to lipids nd is probbly result of digestion nd bsorption of protozol mtter (15). These specific indictors re intended for monitoring the biomss of bcteri nd protozo seprtely. However, for studies where this seprtion is unnecessry or undesirble, more representtive nd rpid ssy would be dvntgeous. Furthermore, n indictor whose concentrtion is intimtely relted to the metbolic ctivity nd vibility of cells would be very useful for the study of fctors ffecting microbil numbers nd ctivity in rumen contents. Adenosine 5'-triphosphte (ATP) is wellccepted indictor of microbil biomss in qutic nd terrestril environments (2, 6, 10). The primry resons for mesuring this cellulr constituent include: (i) ATP is present in ll living cells nd bsent from ded cells (11, 20); (ii) ATP is considered to be present in uniform concentrtion in ll microbes (2, 10); nd (iii) the nlyticl techniques for the extrction nd ssy of ATP re specific, reltively quick, esy to perform, nd inexpensive. Becuse of these unique fetures of ATP mesurement, the fesibility of using this technique s relible indictor of microbil biomss in rumen contents ws exmined. MATERIALS AND METHODS Bcteril cultures, culturl conditions, nd medi. The nerobic rumen bcteri listed in Tble 9 were generously provided by M. P. Brynt, University of Illinois, Urbn, nd K. J. Cheng, Reserch Sttion, Agriculture Cnd, Lethbridge, Albert. The nerobic technique used for culturing the bcteri ws essentilly tht of Hungte (13) s modified by Brynt (5). The rumen bcteri were cultured nerobiclly in the rtificil medium described by Scott nd Dehority (21). The cultures were stored t -65 C on gr slnts. Before use in 528

2 VOL. 33, 1977 growth experiment, ech culture ws subcultured two or three times t 24-h intervls t When the culture reched the lte exponentil phse of growth during the lst subculture, it ws used to inoculte 300 ml of liquid medium to give finl culture density of 20,.g of cells (dry weight)/ml. The cultures were incubted sttionry in wter bth t 370C. At given times of incubtion, the culture ws mixed nd fluid ws removed nerobiclly for opticl density (OD) mesurements, clibrtion of OD redings to dry weight, nd extrction of ATP. The OD mesurements were mde using mtched cuvettes (13 by 100 mm) in Spectronic 20 t 675 nm. The redings were corrected for devitions from Beer's lw using the tbles of Toennies nd Gllnt (22). The OD vlues of ech culture were correlted with the dry weight of cells by mens of seprte clibrtion curve for ech culture relting the OD of cells (hrvested t n OD of 0.5 to 0.7 nd wshed twice in wter) to the dry weight fter drying of n liquot to constnt weight t 1050C. From the curves it ws determined tht n OD reding of 1.0 ws equilvlent to 0.48 mg of Streptococcus bovis, 0.37 mg of Bcteroides ruminicol, 0.36 mg of Selenomons ruminntium, 0.46 mg of Butyrivibrio fibrisolvens, 0.45 mg of Megspher elsdenii, 0.43 mg of Ruminococcus lbus, 0.36 mg ofbcteroides succinogenes, 0.58 mg of Bcteroides mylophilus, nd 0.53 mg of Eubcterium ruminntium per ml. Rumen microbiot. Rumen contents were obtined from two fistulted cows. A Holstein cow ws initilly fed rtion of roughge plus corn-bsed supplement (2:1), nd lter rtion of roughge lone. A Hereford cow used s source of rumen contents for one experiment ws fed rtion of roughge plus rolled ots. The roughge ws mixture of lflf nd brome grss contining primrily lflf. The smples of rumen contents were collected 3 to 4 h fter feeding vi the fistul nd tken to the lbortory in prewrmed (370C) Thermos flsk. Strined rumen fluid (SRF) ws prepred by filtrtion of rumen contents through four lyers of surgicl guze. The plnt mteril retined by the guze ws clled the prticulte mteril. Methods for extrcting ATP. (i) "Boiling" Tris buffer. A 0.5-ml smple of SRF ws trnsferred to screw-cp vil, nd 4.5 ml of 0.04 M tris(hydroxymethyl)minomethne (Tris) buffer, ph 7.8, just off the boil, ws dded. The vil ws quickly seled, nd the contents were mixed nd then plced in boiling wter bth for 5 min with occsionl mixing. After cooling, the contents of the vil were centrifuged t 12,800 x g for 10 min (4C) nd the superntnt ws sved. A portion of the superntnt ws diluted to one-fifth of the originl concentrtion with Tris buffer. (ii) Chloroform ( modifiction of the method of Dhople nd Hnks [9]). A 0.5-ml smple of SRF ws dded to test tube contining 0.15 ml of chloroform. The contents of the tube were mixed for 1 min with Vortex mixer, nd the tube ws plced in boiling wter bth for 5 min. During the lst minute of heting, the tube ws shken to fcilitte vporiztion of the chloroform. The solution ws then ATP AS INDICATOR OF RUMEN MICROBIOTA BIOMASS 529 mde up to 5 ml with 0.1 M NHCO3 (ph 8.5) nd centrifuged t 12,800 x g for 10 min (4 C). A 1-ml smple of the superntnt ws vcuum-evported to remove residul CHCl3 nd then mde up to 10 ml with 0.4 M Tris buffer, ph 7.8. (iii) "Boiling" NHCO3 buffer (6). Liquid smples were hndled s described in (i) except 8 ml of 0.1 M NHCO3 (ph 8.5), just off the boil, ws dded in the plce of Tris buffer. After the centrifugtion step, the superntnt ws decnted nd portion ws diluted to one-tenth of the originl concentrtion with 0.4 M Tris buffer, ph 8.5. In the cse of prticulte mteril (totl rumen contents or strined prticulte mteril), 4- to 7-g smple ws wshed into preheted stinless-steel Wring blender jr with 125 ml of 0.1 M NHCO3 (ph 8.5), just off the boil. The contents were mixed t 10,000 rpm for 4 min. After cooling, portion ws treted s described for liquid smples. (iv) Cold 0.6 N H2S04 ( modifiction of the method described by Lee et l. [171). This is the technique recommended for use. A 1-ml smple of SRF ws dded to 4 ml of ice-cold 0.6 N H2SO4 in centrifuge tube nd mixed intermittently. The tube ws mintined for exctly 5 min in n ice bth nd then centrifuged t 39,000 x g for 10 min (40C). A 2- ml volume of the superntnt nd 1.5 ml of 10% (wt/vol) suspension of N+-sturted ction-exchnge resin (Amberlite IR-120) suspended in wter were immeditely dded to test tube nd mixed for 30 s. Two milliliters of 0.5 M Tris buffer (ph 7.8) ws dded, nd the contents were mixed while 1.0 to 1.2 ml of 1 N NOH ws dded (i.e., sufficient to djust the mixture to ph 7.8 in the bsence of Tris buffer), followed by 10 to 15 ml of wter. Smples were processed to this stge without interruption. The extrct ws seprted from the resin by spirtion, nd the resin ws wshed twice with 2-ml liquots of distilled wter. The combined extrct nd wshes were pooled nd mde to volume of 25 ml with wter. In the cse of prticulte mteril (totl rumen contents or seprted prticulte mteril), 4- to 7-g smple of thoroughly mixed mteril ws dded to centrifuge tube, 25 ml of 0.6 N H2S04 ws dded, nd the contents were mixed vigorously. The subsequent mnipultions were identicl to those for processing smples of SRF. When nonrdioctive internl stndrd of ATP ws dded to liquid smple, 3 ml of 0.6 N H2SO4 ws first mixed with the 1-ml smple; then 1 ml of 0.6 N H2SO4 contining the stndrd (100 nm of nonrdioctive ATP) ws immeditely dded, nd the contents were mixed gin. For prticulte smples, the smple ws mixed with 20 ml of 0.6 N H2SO4, nd then 5 ml of 0.6 N H2SO4 contining the stndrd (750 nm nonrdioctive ATP) ws dded. The H2SO4 solutions of ATP to be used s internl stndrds were prepred immeditely before use. Rdioisotope usge. [U-14C]ATP (mmonium slt, 554 mci/mmol) ws obtined from Amershm/ Serle Corp., Okville, Ontrio. It ws dded without dilution to 0.6 N H2SO4 to give specific ctivities of 0.04 MCi/ml for ddition to liquid smples nd 0.1,uCi/ml for prticulte smples. Neutrlized sm-

3 530 FORSBERG AND LAM ples were suspended in dioxne-bse scintilltion fluid (4) for counting in Beckmn LS-255 scintilltion counter. Seprtion of bcteri nd protozo. Two liters of rumen fluid collected from the cow on hy rtion ws strined through six lyers of surgicl guze into prewrmed (370C) insulted continer nd quickly brought to the lbortory. All pprtus nd glsswre used were prewrmed to 370C. A bcteril frction, free from protozo, ws obtined by first centrifuging SRF t 175 x g for 5 min t 37 C to sediment the protozo nd feed prticles. The superntnt contining bcteri ws spirted off nd smpled for the mesurement of ATP. Thirty milliliters ws dded to ech of four tubes, which were then centrifuged t 39,000 x g for 5 min t 37 C to sediment the bcteri. The superntnt fluid ws poured off, nd two cell pellets were seprtely extrcted with 20 ml of 0.6 N H2SO4; fter mixing, 5 ml of ech ws dded to 100 nmol of ATP (0.1 ml). The extrction procedure ws continued s described bove for both the two 5-ml smples with internl stndrd nd the two 5-ml smples without. The other two cell pellets were wshed twice t 40C with 25-ml volumes of wter, centrifuging ech time t 27,000 x g for 10 min; then ech pellet ws mde up to 10 ml with wter, nd 5 ml ws dried to constnt weight t 1050C. To obtin n enriched frction of protozo, two 750-ml volumes of SRF were dded to 1-liter seprtory funnels, pregssed with CO2. The hed spces of the funnels were gssed with C02, stoppered, nd plced in 370C incubtor for 60 min. The white sediments in the funnels were collected, pooled, nd suspended in 45 ml of freshly prepred "cell-free" rumen fluid (prepred by two successive centrifugtions t 27,000 x g for 30 min, wrmed to 37 C, nd gssed with CO2). The protozol suspension ws divided eqully mong four tubes nd centrifuged t 175 x g for 8 min t 350C to sediment the protozo, nd the culture superntnt ws crefully spirted off ech cell pellet. Two cell pellets were resuspended in 10 ml of 0.6 N H2SO4 nd treted s described for the bcteri. The two remining cell pellets were wshed twice s described for the bcteri, nd the dry weights were determined. Lysis of the bcteril nd protozol frctions during the wter wshes preceding the dry-weight determintions did not pper to occur becuse similr dry weights were obtined when the cells were pretreted with either glutrldehyde or formldehyde. Phse-contrst microscopy reveled tht the protozo were contminted with few bcteri. Further purifiction ws not ttempted to void disturbing the metbolism of the protozo, which would led to chnged ATP content. Estimtion of ATP. ATP ws determined by the firefly bioluminescence technique. The rection mixture contined 0.02 to 0.5 nmol of ATP in 0.3 ml of 0.04 M Tris buffer, ph 7.8, nd 0.2 ml of freshly prepred solution of M sodium rsente-0.05 M mgnesium chloride solution, ph 7.8. The rection ws strted by ddition of 0.1 ml of reconstituted firefly lntern extrct (one vil of FLE-50 homogenized with 5 ml of wter, centrifuged t 12,000 x g APPL. ENVIRON. MICROBIOL. for 15 min t 40C to remove prticulte mteril nd stored on ice; Sigm Chemicl Co., St. Louis, Mo.). The intensity of the bioluminescence ws recorded fter 30 s, using Chem-Glow Photometer (Americn Instruments Co., Silver Spring, Md.). The concentrtion of ATP in experimentl smples ws determined by reference to stndrd curve relting light intensity to ATP concentrtion prepred from disodium ATP. All experimentl smples were ssyed t three or four seprte concentrtions nd in some cses with dded internl stndrds of ATP to check for inhibition of light emission by components present in extrcts. Sodium crbonte inhibited luciferse ctivity, but t the highest concentrtion present in experimentl smples it cused less thn 5% inhibition nd ws disregrded. Extrction nd mesurement of the ATP pool sizes of bcteri. A 1-ml volume of culture ws mixed with 0.2 ml of 3.6 N H2S04 nd set on ice for 20 min. One milliliter of N+-sturted ction-exchnge resin (Amberlite IR-120) suspended in wter ws dded. The contents were shken vigorously nd then centrifuged t 12,000 x g for 15 min t 40C to remove cell debris nd resin. A 1.5-ml volume of the superntnt ws removed, nd the ph ws djusted to 7.8. The solution ws mde 0.04 M with respect to Tris buffer (ph 7.8) in volume of 10 ml. When internl stndrds of ATP (100 nm in 0.1 ml) were dded, they were mixed into the smples immeditely fter ddition of the H2SO4. These extrcts were ssyed for ATP by using minivils in Beckmn model LS-255 scintilltion counter becuse of its incresed sensitivity over the Chem- Glow Photometer. The ssy regents used were the sme s those used with the Chem-Glow Photometer except tht (i) the firefly lntern extrct ws ged for 2 h t 40C, (ii) the volume of smple plus 0.04 M Tris buffer (ph 7.8) ws incresed to 1.0 ml nd the volume of the rsente-mgnesium solution ws incresed to 0.6 ml, nd (iii) 0.2 ml of firefly extrct ws used to strt the enzymtic ssy. Ech smple ws mixed nd counted for three 0.1-min cycles, using gin nd window settings used for counting tritium. The counts per minute recorded for the third 0.1-min cycle ws recorded. A stndrd curve, plotted on log-log grph pper, relting counts per minute to ATP concentrtion ws prepred by ssying stndrd solution of ATP. The recovery of dded internl stndrd usully rnged from 70 to 105% nd ws used to correct the ATP concentrtions to 100% recovery. RESULTS Extrction techniques. The ATP content of rumen fluid ws determined by vriety of techniques with the intention of selecting the best method for the extrction of ATP. The results illustrted in Tble 1, experiment 1, re typicl of those obtined in number of experiments nd show tht hot 0.1 M NHCO3 (ph 8.5) with heting for 5 min gve the gretest recovery of ATP. The efficiency of extrction by this technique ws compred with tht of the

4 VOL. 33, 1977 ATP AS INDICATOR OF RUMEN MICROBIOTA BIOMASS 531 TABLE 1. Effect of extrction technique on the mount ofatp extrcted from strined rumen fluid from cow on rtion of hy plus cerel Expt Extrction technique 1Ag of ATP/ml no. of SRF 1 (i) Boiling 0.04 M Tris 69.5 buffer, ph 7.8 (ii) Chloroform 31.1 (iii) NHCO3-chloroform blending (iv) BoilingNHCO (iv) Boiling NHCO ± 6.9b (v) Cold 0.6 N H2SO Five milliliters of SRF plus 45 ml of 0.1 M NHCO3 (20 C) were dded to blender. Fifteen milliliters of CHCl3 ws dded, nd the mterils were mixed for 5 min. b Men nd the stndrd devition. (iv, four smples; v, five smples). method described by Lee et l. (17) using cold 0.6 N H2SO4. In the ltter technique, fter ddition of 0.6 N H2SO4 to the smple, the mixture ws incubted on ice for 60 min nd the smple ws centrifuged to remove prticulte mteril. A 2-ml volume of the clrified extrct ws treted btchwise with two 0.5-ml mounts of ction-exchnge resin, nd the ph ws djusted to 7.8 by titrtion with 1 N NOH. The extrct ws then mde to volume of 25 ml nd 0.04 M with respect to Tris (ph 7.8). There ws no significnt difference (t the 95% confidence level) in the reltive extrction efficiency between the two methods (Tble 1, experiment 2). The recoveries of ATP (100 nm) dded s internl stndrds to the NHCO3 nd H2SO4 extrctions were 94 nd 105%, respectively. In seprte experiments, the efflciency of the HC104 extrction technique published by Bgnr nd Finch (3) ws compred with the H2S04 extrction technique for mesuring ATP in the prticulte frction of rumen contents. Extrction with HC104 gve only 16% of the ATP obtined using H2S04, nd comprble recovery of internl stndrd ws observed. Becuse it is more convenient, the NHCO3 extrction technique ws used initilly, nd results using this method re reported in Tbles 1, 2, nd 3; however, becuse the H2SO4 extrction technique ws found to give higher recoveries of ATP from prticulr smples, it ws used in the reminder of the study. To determine how the time of incubtion ffected the ATP content of SRF, smple ws extrcted t intervls fter removl of rumen contents from cow. There ws no detectble chnge in the ATP content of the SRF over 160-min period (Tble 2). The use of ATP s relible indictor of microbil biomss in rumen contents depends upon there being low mount of ATP in the feed consumed by the ruminnt niml. The ATP content of number of feed constituents ws determined by extrction with hot 0.1 M NHCO3, using the procedure involving blending. Less thn 0.24 ug of ATP per g ws extrcted from lflf hy, nd no ATP ws detected in either ground corn or the pelleted corn-bsed supplement contining soyben mel, complete vitmin mixture, nd minerls. Similr results were obtined using the H2SO4 extrction technique. Observed distribution of ATP in rumen contents. The distribution of ATP between the SRF nd prticulte frction of rumen contents ws determined for the cow on hy-corn-bsed TABLE 2. Effect ofincubtion t 37 C on the mount ofatp extrcted from strined rumen fluid from cow on rtion of hy plus cerel Time intervl (min) Ag of ATP/ml ± ± ± ± ± ± 5.3 A 300-ml volume of strined rumen fluid contined in 500-ml round bottom flsk ws incubted under n tmosphere of crbon dioxide. The NHCO3 extrction technique ws used to extrct ATP. TABLE 3. Distribution ofatp in rumen contents from cows on rtion of hy plus cerel tlgofatp/gdistri- Expt Smple jug of ATP/g bution no. observed serve 1 Rtion of hy + cornbsed supplement Mixed rumen contents 84.9b 100 SRF Prticulte mteril Rtion of hy + rolled ots Mixed rumen contents c 100 SRF 37.0 ± Prticulte mteril Guzed 2 The NHCO3 extrction technique ws used for experiment 1, nd the H2SO4 extrction technique of Tble 1 ws used for experiment 2. In both cses the extrcts were ssyed for ATP immeditely fter preprtion. b Men vlue of duplictes. c Men nd stndrd devition of smples extrcted nd ssyed in triplicte. d The moist guze with ssocited trces of rumen contents ws immersed in 200 ml of 0.6 N H2SO4, nd the fluid ws processed s described for SRF smples.

5 532 FORSBERG AND LAM supplement rtion nd the cow on hy-rolled ots rtion (Tble 3). In both cses the ATP detected in the two frctions ccounted for greter thn 90% of the ATP in the mixed rumen contents. Very little ATP remined ssocited with the guze used for the filtrtion process. When the seprtion ws repeted with rumen contents tken fter the diet of the first cow hd been chnged to hy, the observed overll recovery of ATP ws 42% when using the NHCO3 extrction procedure nd 55% for the H2SO4 extrction procedure used in Tble 1, thus indicting tht both methods gve incomplete recoveries of ATP. To llow for incomplete recovery of ATP, the experiment ws repeted with internl stndrds, which were dded during the extrction procedure (Tble 4, experiment 1). In this prticulr experiment the neutrlized extrcts were stored frozen t -20 C overnight. There ws very little binding of [14C]ATP to prticulte mterils since little rdioctivity ws lost s result of the centrifugtion step nd resin tretment. However, there were lrge decreses in the mount of nonrdioctive ATP internl stndrd detected in ll smples except the SRF smple. When internl stndrds were dded to the luciferse ssy system, ll the dded ATP ws detected, indicting tht no inhibitors of luciferse were present in the extrcts. In the second experiment (Tble 4, experiment 2), the loss of internl stndrd ws found to be less when the ssys for ATP were conducted immeditely. Nevertheless, the overll recovery of ATP ws low. In both experiments the gretest loss in detectble ATP ws in the prticulte frction. TABLE 4. Fctors ffecting the mount of ATP detected in rumen contents using the H2SO4 extrction technique. Since the extrction of ATP from the prticulte frction ws the lest efficient, this frction ws exmined in the gretest detil. The effect of freezing nd thwing of neutrlized extrcts on the concentrtion of ATP detected ws first exmined (Tble 5). There ws decrese in the concentrtion of ATP detected in treted smples of both SRF nd prticulte mteril s consequence of freezing nd thwing, nd the decreses were greter in extrcts from prticulte smples. These decreses could not be due to the ctivity of n cid-stble enzyme tht ws ctive while the neutrlized smples were thwed, becuse TABLE 5. Effect of freezing nd thwing on the mount ofatp detected in neutrlizing H5S04 extrcts prepred from frctionted rumen contents,ug of ATP/g of contents Smple Un- Frozen Frozen frozen once twice SRF SRF + internl stndrd Prticulte mteril Prticulte mteril + in ternl stndrd Smples of rumen contents, obtined from cow on rtion of hy, were frctionted, nd the SRF nd prticulte frctions were extrcted with 0.6 N H2SO4 for 60 min s described for Tble 1. The extrcts were ssyed for ATP, frozen by storge t - 20 C for 20 h nd then thwed t 22 C, nd ssyed gin for ATP; nd the cycle ws repeted second time. Distribution of ATP in rumen contents from cow on rtion of hy Recovery of internl,ug of ATP/g of contents Distribution (%) Smple - Nonr- ['4C]ATP dioctive Observed Clcultedb Observed Clculted ATP Expt 1 Mixed rumen contents ± 1.4c SRF ± Prticulte mteril ± Guze ± Expt 2 Mixed rumen contents _d ± SRF ± Prticulte mteril ± Guze Smples were extrcted with 0.6 N H2S04 s described for Tble 1. In experiment 1 the neutrlized smples were stored frozen before nlysis, nd in experiment 2 the smples were ssyed immeditely. b The clculted vlues hve been corrected for the verge loss of nonrdioctive internl stndrd (smples contining internl stndrds were lso extrcted in triplicte). c Men nd stndrd devition of smples extrcted nd ssy in triplicte. d _, Not determined. APPL. ENVIRON. MICROBIOL.

6 VOL. 33, 1977 incubtion of extrcts t 30 C for n extended period of time (15.5 h) (Tble 6) resulted in only minor decrese in ATP content. This might be expected since contminting microbes could hve grown nd hydrolyzed some of the ATP in tht time intervl. Freezing nd thwing of neutrlized extrcts of prticulte smples fter heting t 1000C for 5 min sometimes resulted in n increse in the mount of ATP detected. For exmple, fter portion of smple 2 from Tble 6 hd been frozen nd thwed, the concentrtion of ATP detected ws 54.3,tg/g. An identicl smple contining n internl stndrd treted in prllel incresed from 91.3 to 132.0,ug of ATP per g fter the freezing nd thwing procedure. This effect ws not reproducible from one experiment to the next; however, it did increse the mount of ATP detected fter one freezing nd thwing cycle s compred with unheted smples (Fig. 1). Since there ws n incomplete recovery of ATP in the experiment illustrted in Tble 4, experiment 2, even though the smples hd been ssyed immeditely fter neutrliztion, the effect of extrction time with H2SO4 ws exmined. Extrction times longer thn 5 min cused decrese in the mount of ATP detected (Fig. 1). Therefore these experiments showed tht rpid extrction procedure must be used nd the neutrlized smples should not be frozen before being ssyed. Distribution of ATP in rumen contents from cow on rtio of hy. The distribution of ATP in rumen contents from the cow on rtion of hy ws determined by using the modified H2S04 extrction procedure. The recovery TABLE 6. Effect ofincubtion t 30 C on the mount ofatp detected in neutrlized H5S04 extrcts of the prticulte frction of rumen contents from cow on rtion of hy ug of ATP/g Smple Oh 4h 15.5h 1. Extrcted with 0.6 N H2 S04b 2. Neutrlized extrct from ws heted t 100'C for 5 min 3. H2SO4 extrct from 1 fil c tered through UM-10 Amicon ultrfilter nd then neutrlized Aliquots of smples 1, 2, nd 3 were incubted t 30'C immeditely fter preprtion. The ssys for ATP were conducted immeditely fter smpling t ech time intervl ḃ Extrctions were conducted s described for Tble 1. c -, Not determined. ATP AS INDICATOR OF RUMEN MICROBIOTA BIOMASS ' 20O 011 0) Minutes FIG. 1. Effect of extrction time in 0.6 NH5$04 on the mount of ATP detected in untreted (0), untreted nd frozen (0), nd heted nd frozen (v) extrcts from the prticulte frction of rumen contents obtined from cow on rtion of hy. After extrction, ech smple ws rpidly processed s described for Tble 1. Aliquots of ech neutrlized smple were treted s described in Tble 5 without or with n initil heting t 100 C for 5 min. of internl stndrd ws uniformly high in ll frctions, nd the overll recovery ws 80%o bsed on the observed distribution of ATP (Tble 7). Comprison of these results with those in Tble 3 shows tht the observed ATP concentrtion in rumen contents ws pprently greter for n niml receiving cerel in the diet. The proportion of ATP ssocited with the prticulte mteril ws greter when the niml ws on the hy rtion. There ws no ATP present in "cell-free" rumen fluid, probbly becuse of the ctivity of n ATP hydrolse(s) tht ws found to be present (Fig. 2). ATP content of seprted bcteril nd protozol frctions. To determine whether the ATP in rumen contents ws distributed uniformly between bcteri nd protozo (on dryweight bsis), enriched frctions of ech were prepred. The bcteril frction ws prepred by differentil centrifugtion of SRF. The recovery of dded internl stndrd in one experiment (Tble 8, experiment 1) exceeded 100%. This occurred infrequently for SRF smples but mde the inclusion of internl stndrds essentil. A low-speed centrifugtion (175 x g) to sediment the protozo nd feed prticles removed 74% of the ATP (Tble 8, experiment 1). By mesuring the mount of ATP in the super-

7 534 FORSBERG AND LAM TABLE 7. Recovery nd distribution of ATP in rumen contents from cow on rtion of hy using the modified H2S04 extrction procedure Recovery of,ug of ATP/g of contents Distribution (%) Smple internl stndrd (%) Observed Clcultedb Observed Clculted Mixed rumen contents c SRF ± Prticulte mteril ± Guze ± ,000 x g SRF supernte Recovery (%) Extrction procedure is outlined in Mterils nd Methods. b Clculted vlues hve been corrected s described in Tble 4. c Men nd stndrd devition of smples extrcted nd ssyed in triplicte Minutes FIG. 2. ATP hydrolse ctivity in "cell-free" rumen fluid from cow on rtion of hy. Six milliliters of rumen fluid superntnt tken fter two successive centrifugtions t 45,000 x g for 30 min ws mixed with 200 nm ATP (0.2 ml), gssed with C02, nd incubted t 37 C. One-milliliter smples were extrcted with H2SO4. ntnt nd using this to compute the concentrtion of ATP in bcteri, the vlue obtined (2.10,ug of ATP/mg) ws 28% higher thn when the bcteril sediment fter high-speed centrifugtion ws extrcted (1.64,Ag of ATP/mg). The pprent decrese in the concentrtion of ATP in cells fter centrifugtion could be ttributed in prt to the loss of slime-encpsulted bcteri tht formed loose top lyer over the sedimented cells nd s result ws lost when the superntnt ws poured off. The protozol frction prepred by combintion grvity sedimenttion nd subsequent low-speed centrifugtion step, designed to remove residul bcteri, contined 16.9,ug of ATP per mg of dry weight. This concentrtion of ATP ws 10-fold higher thn tht observed in the bcteri. ATP pool sizes of pure cultures of rumen bcteri. To obtin informtion bout the vrition in ATP pool sizes mong rumen bcteri, the ATP concentrtion ws determined for APPL. ENVIRON. MICROBIOL. TABLE 8. ATP pool sizes of bcteril nd protozol frctions from strined rumen fluid from cow on rtion of hy Recovery g Of "g of of in- ATm ATP/mg stndrd lted e clcul- ted Frction ternl clcu- (dry wt) Expt 1 SRF SRF fter centrifug tion t 175 x g for 5 min Bcteri Before centrifugtion Cell pellet Protozo Expt 2 Bcteri Before centrifug tion5 Cell pellet After correction of the vlues to 100% recovery. batp content of SRF fter centrifugtion of 470 x g for 4 min t 37'C to remove protozo; expressed on the bsis of dry weight of wshed bcteri prepred from n liquot of the fluid. All other mnipultions in experiments 1 nd 2 were crried out s described in Mterils nd Methods. number of the functionlly importnt species. Figure 3 illustrtes the vrition of the ATP content of Selenomons ruminntium during growth. The level of ATP ws high during the erly exponentil phse of growth nd decresed by hlf in the lte exponentil phse; it then dropped off precipitously during the sttionry phse. This type of profile for ATP ws typicl for ll the species of bcteri studied (Tble 9) except tht the ATP pools of some species, e.g., Bcteroides mylophilus nd Ruminococcus lbus, remined nerly constnt during the exponentil phse of growh. DISCUSSION A number of methods were tested to determine their efficiency in extrcting ATP from either SRF or its prticulte frction. The two

8 VOL. 33, E 0.6 c 0Q5 0.4 L) O.3 0o 0.2 ATP AS INDICATOR OF RUMEN M[CROBIOTA BIOMASS 535 o U - 6 Xn - 5-7t 3 E I Minutes FIG. 3. ATP pool size of Selenomons ruminntium during growth. most efficient methods for extrcting ATP were those using boiling 0.1 M NHCO3 (modifiction of the method by Christin et l. [6]) nd cold 0.6 N H2SO4, described by Lee et l. (17). Both techniques gve incomplete recoveries of ATP when used to mesure the ATP in prticulte mterils from rumen contents of cow on rtion of hy. The recovery of ATP using the H2SO4 extrction procedure ws the greter, nd, since it ws better suited for prticulte smples, the fctors influencing the recovery of ATP by this technique were exmined. The inclusion of [14C]ATP in the extrction of ATP from whole rumen contents, SRF, nd prticulte mterils reveled tht the low recovery of endogenous ATP ws not cused by binding of ATP to sedimentble mterils. The lowest recovery of dded nonrdioctive ATP occurred when the strined prticulte frction of rumen contents ws extrcted, nd the losses were incresed if smples were frozen before ssy. By decresing the time of extrction with H2SO4 nd ssying smples immeditely fter extrction, the low recovery of mesurble ATP ws lrgely corrected. The pprent loss of ATP from neutrlized extrcts of the prticulte mterils did not occur when they were incubted t 300C. Heting the neutrlized extrcts decresed the mount of ATP lost during freezing nd thwing; in fct, in some cses the concentrtion of ATP ws ctully incresed. These observtions re consistent with the loss in ATP being cused by the extrction of soluble component(s) from plnt mteril, which forms complexes with ATP, either in cid solution or during freezing nd thwing, nd thereby prevents the rection with luciferse. The higher recovery of ATP in neutrlized extrcts prepred 0) from the SRF frction nd the decresed losses resulting from freezing nd thwing suggest tht the inhibitor ws bsent from the SRF. The greter proportion of the ATP in rumen contents of the cow on rtion of hy plus cerel ws present in the SRF frction, wheres the inverse ws observed when the cow ws on hy rtion. Seventy-four percent of the ATP in the SRF from the ltter niml ws sedimented by n extremely low centrifugl force (175 x g for 5 min), indicting tht the bulk of the ATP ws present in microbes ssocited with plnt mteril nd protozo. From n pplied point of view, these observtions suggest tht the proportion of microbil biomss present in SRF my depend upon the niml rtion. Since the types of bcteri in the fluid nd on the prticulte mteril re different (18), the use of SRF in in vitro experiments could be open to criticism becuse the popultion of microbes in the inoculum is chnged from whole rumen contents. To serve s relible indictor of microbil biomss, ATP must be present t uniform concentrtion in vible microbes nd bsent from ded microbes nd other mterils. ATP ws bsent from the niml rtion (dried lflf hy nd corn-bsed supplement) fed in these experiments. The ATP content of fresh grss nd silge hs not been determined. Therefore, no recommendtions cn be mde bout the suitbility of ATP s n indictor of rumen microbil biomss for nimls on rtions of fresh grss nd silge. There presumbly is continul sloughing of epithelil tissue from the rumen wll; however, becuse of the incresed lysozoml ctivity in this tissue (16), it probbly is prtilly utolyzed nd therefore depleted in ATP. We found tht ATP dded to "cell-free" rumen TABLE 9. ATP pool sizes of nine species of rumen bcteri during the exponentil phse ofgrowth in rtificil medium Gener- ug of Bcterium tion time ATP/mg (min) (dry wt) Streptococcus bovis S-b Bcteroides ruminicol subsp. rum inicol 23 Selenomons ruminntium strin D Butyrivibrio fibrisolvens Dl Megspher elsdenii B Ruminococcus lbus B Bcteroides succinogenes S Bcteroides mylophilus Eubcterium ruminntium GA Ech vlue is the verge of three extrctions t culture OD redings of pproximtely 0.20, 0.40, nd 0.70 during exponentil growth.

9 536 FORSBERG AND LAM fluid grdully disppered (t112 = 85 min; Fig. 2). This loss in ATP ws probbly due to the presence of ATP-hydrolyzing enzymes. Since no ttempt ws mde to identify rection products, this proposl cnnot be confirmed. The observed bsence of ATP in the superntnt from centrifuged rumen fluid supports this contention. Thus, under our experimentl conditions, the ATP in the rumen is limited to bcteri nd protozo. The ATP concentrtion in the isolted protozol frction ws 10-fold higher thn in the bcteril frction. Although precutions were tken to mintin nerobiosis nd constnt temperture, the possibility cnnot be excluded tht the vlues obtined, prticulrly for the protozo, were chnged s result of metbolic disturbnces tht my hve occurred during the seprtion procedure. For exmple, it hs been observed tht exposure of Clostridium cetobutylicum to erobic conditions leds to fll in the ATP pool size (19). The ATP pool sizes of functionlly importnt rumen bcteri growing exponentilly in rtificil medi vried by fctor of 16, from 1.1,ug of ATP per mg of dry weight for R. lbus to 17.6,ug of ATP per mg for S. bovis. Most of the vlues were higher thn tht found for the isolted popultion of rumen bcteri. Brring some effect relted to the extrction procedures, this is expected for number of resons. The rumen contins mny gener of bcteri tht undoubtedly hve different ATP pool sizes. The different types of bcteri hve different degrees of ffinity for prticulte mteril (18), wheres the isolted bcteril frction contined only those free in solution. In the rumen, microbil growth my be limited by the vilbility of crbohydrte (14), nd for this reson bcteri, nd prticulrly those free in solution, my be expected to hve decresed ATP pool size. This effect possibly is ccentuted in nimls on roughge diet. In Escherichi coli, glucose-limited growth leds to lower pool levels of ATP (7). Whether ATP reflects the overll metbolic ctivity of the rumen microbiot remins to be determined. The correltion of the concentrtion of ATP in the microbil frction throughout the dy with feed intke nd rtion composition should shed light on this subject. These dt show tht considerble cution must be used in interpreting microbil biomss dt in rumen contents bsed on ATP concentrtions. ATP presumbly cn be used s n indictor of biomss where nimls re receiving uniform rtion free from ATP. However, APPL. ENVIRON. MICROBIOL. mesurement of ATP in frctionted rumen contents cn led to discrepncies. ACKNOWLEDGMENTS We would like to thnk J. Burton (Deprtment of Nutrition) for ccess to his fistulted cows. This investigtion ws supported by grnts to C. W. F. from Agriculture Cnd nd the Ontrio Ministry of Agriculture nd Food. LITERATURE CITED 1. Abou Akkd, A. R., D. A. Messmer, L. R. Fin, nd E. E. Brtley Distribution of 2-minoethylphosphonic cid in some rumen microorgnisms. J. Diry Sci. 51: Ausmus, B. S The use of the ATP ssy in terrestril decomposition studies. Bull. Ecol. Res. Commun. 17: Bgnr, A. S., nd L. R. Finch Quntittive extrction nd estimtion of intrcellulr nucleoside triphosphtes from Escherichi coli. Anl. Biochem. 45: Bry, G. A A simple efficient liquid scintilltor for counting queous solutions in liquid scintilltion counter. Anl. Biochem. 1: Brynt, M. P Commentry on the Hungte technique for culture of nerobic bcteri. Am. J. Clin. Nutr. 25: Christin, R. R., K. Bncroft, nd W. J. Wiebe Distribution of microbil denosine triphosphte in slt mrsh sediments t Spelo Islnd, Georgi. Soil Sci. 119: Cole, H. A., J. W. T. Wimpenny, nd D. E. Hughes The ATP-pool in Escherichi coli. I. Mesurement of the pool using modified luciferse ssy. Biochim. Biophys. Act 143: Czerkwski, J. W Methods for determining 2,6- diminopimelic cid nd 2-minoethyl-phosphonic cid in gut contents. J. Sci. Food Agric. 25: Dhople, A. M., nd J. H. Hnks Quntittive extrction of denosine triphosphte from cultivble nd host-grown microbes: clcultion of denosine triphosphte pools. Appl. Microbiol. 26: Holm-Hnsen, The use of ATP determintions in ecologicl studies. Bull. Ecol. Res. Commun. 17: Holm-Hnsen, O., nd C. R. Booth The mesurement of denosine triphosphte in the ocen nd its ecologicl significnce. Limnol. Ocenogr. 11: Horiguchi, M., nd M. Kndtsu Isoltion of 2- minoethne phosphonic cid from rumen protozo. Nture (London) 184: Hungte, R. E The nerobic mesophilic cellulolytic bcteri. Bcteriol. Rev. 14: Hungte, R. E The rumen microbil ecosystem. Annu. Rev. Ecol. Syst. 6: Kittredge, J. S., nd E. Roberts A crbon-phosphorus bond in nture. Science 164: Lvker, R. M., nd A. G. Mtoltsy Formtion of horny cells: the fte of cell orgnelles nd differentition products in ruminl epithelium. J. Cell Biol. 44: Lee, C. C., R. F. Hrris, J. D. H. Willims, D. E. Armstrong, nd J. K. Syers Adenosine triphosphte in lke sediments: I. Determintion. Soil Sci. Soc. Am. Proc. 35: Minto, H., A. Endo, M. Higuchi, Y. Ootomo, nd T. Uemur Ecologicl tretise on the rumen fermenttion. I. The frctiontion of bcteri ttched

10 VOL. 33, 1977 ATP AS INDICATOR OF RUMEN MICROBIOTA BIOMASS 537 to the rumen digest solids. J. Gen. Appl. Microbiol. 12: O'Brien, R. W., nd J. G. Morris Oxygen nd the growth nd metbolism of Clostridium cetobutylicum. J. Gen. Microbiol. 68: Ptterson, J. W., P. L. Brezonik, nd H. D. Putnm Mesurement nd significnce of denosine triphosphte in ctivted sludge. Environ. Sci. Technol. 4: Scott, H. W., nd B. A. Dehority Vitmin requirements of severl cellulolytic rumen bcteri. J. Bcteriol; 89: Toennies, G., nd D. L. Gllnt The reltion between photometric turbidity nd bcteril concentrtion (Bcterimetric studies. IV). Growth 8:7-20.