5
Adenosine Triphosphate in Lake Sediments: I. Determination 1 C. C. LEE, R. F. HARRIS, J. D. H. WILLIAMS, D. E. ARMSTRONG, AND J. K. SYERS- ABSTRACT A modification of the luciferin-Iuciferase bioluminescence technique was developed for determining adenosine triphos- phate (ATP) in sediments. The method involves extraction with cold H2SO4, clean up with a cation exchange resin and use of living Aerobacter aerogenes cells as an internal stand- ard to correct for incomplete ATP recovery from the sediments. ATP recovery ranged from 20 to 85%, was a characteristic, reproducible property of a given sediment, but was not related consistently to any other sediment property. The detection limits of the method were about 0.05 ,ug ATP/g oven dry sediment but were dependent on the recovery characteristic of the sediment and the amounts of bioluminescence-inhibitory solutes present in the extract used for final ATP analysis. Precision was low at ATP levels approaching the detection limit, primarily because of the high coefficient of variation shown at low ATP concentrations (about 3 X 10~ 10 M for the instrumentation and luciferin-Iuciferase extracts used in this investigation). Theoretical considerations supported by prelim- inary experimental results indicate that the method should be applicable to soils as well as sediments. The ATP contents of nine sediment samples obtained from different lakes in Wis- consin ranged from 0.34 to 9.5 /j.g ATP/g sediment. Additional Key Words for Indexing: Adenosine triphosphate in soil, microbial biomass, luciferin-Iuciferase method, sediment- water interactions, lake eutrophication. R ECENT evidence (6) supports the high potential of aden- osine triphosphate (ATP) as an index of microbial biomass in aquatic environments. The firefly luciferin- Iuciferase bioluminescence method for ATP analysis has been used extensively for determination of ATP in diverse biological (3, 5, 10), fresh water (9), marine (6), sewage (14), and soil (12) systems. The method is based on mea- surement of the light emitted from the interaction of ATP with luciferin (LH 2 ), luciferase (E), and atmospheric oxygen, the amount of light energy emitted being propor- tional to the concentration of ATP added, as long as the other constituents of the reaction are present in excess (15). A simplified representation of the bioluminescence reaction is as follows (13): Ms ++ ATP + LH 2 + E -=?—>. E-LH 2 - AMP + P-P E-LH 2 -AMP + 0 2 PH E + product + CO 2 + AMP + light . This paper presents a modified luciferin-Iuciferase method for determining ATP in lake sediments and dis- cusses the problems associated with extracting intact ATP from sediments and soils in a state compatible with the requirements of the bioluminescence reaction. MATERIALS AND METHODS Bioluminescence Measurement of ATP Lucijerin-luciferase—The luciferin-Iuciferase used was ob- tained as an arsenate-buffered, powdered firefly lantern extract (Sigma Chemical Co., St. Louis, Mo.) which is stable indefi- nitely in the dessicated state at —fOC. Prior to use, the extract in each vial was reconstituted with 5.0 ml Tris buffer (0.02M, pH 7.8), allowed to stand at room temperature for 3 to 4 hr, or overnight at 4C, to reduce light emission from endogenous ATP and ATP derived from reactions catalyzed by transphos- phorylases (7, 15). The luciferin-Iuciferase suspension was cen- trifuged at 1,000 » for fO min to remove solids. The resultant clear enzyme extract was used within 2 hr of final preparation. ATP Analysis—The standard or unknown ATP solution (1.8 ml) was pipetted into a standard glass liquid scintillation counting vial. At zero time the reconstituted enzyme extract (0.2 ml) was added, the mixture was shaken by hand for ex- actly 10 sec, and then the vial was placed into the counting chamber of a liquid scintillation counter (Packard Tri-Carb, Model 3365) and the light emission integrated over a 5-sec period by activating the repeat cycle (Fig. f). All light measure- ments were made at instrument settings for tritium (gain 50%, discriminator 50-1000 divisions). Blank samples (1.8 ml Tris buffer) were interspersed throughout the ATP samples to enable calculation of net recorded light for each ATP sample. Stock ATP solutions (10~ 3 M) were prepared by dissolving crystalline disodium ATP (Nutritional Biochemical Co., Cleve- land, Ohio) in Tris buffer (0.02M, pH 7.8). The stock ATP solutions were stable for at least 3 months at 10C. Whenever ATP determinations were to be made, a series of standard ATP solutions (10~ 9 M, 10~ 8 M, and 10~ 7 M) was prepared by dilut- ing the stock ATP solution with Tris buffer. The net light response versus ATP concentration in the 3 X 10~ 10 to 10~" M range is approximately linear when plotted on log-log paper (Fig. 1). Regression analysis of the relationship between ATP concentration and light emitted gave a best fit curve of log light emitted = 1.315 log ATP cone. + b. The slope and line- arity of the curve are reproducible but the intercept (b) varies for different vials of firefly extracts, necessitating that ATP standards be run for each set of ATP determinations involving the use of different firefly extracts. Precision declines markedly as the ATP concentration approaches the detection limit (Table 1: 4. 11). ATP Extraction from Aerobacter Aerogenes Cells Stationary phase cells of A. aerogenes (NRRL 199) contain- ing relatively constant amounts of ATP (1 to 2 X 10- 10 /*g ATP/viable cell) were obtained by inoculating a 1% nutrient broth culture solution and shaking overnight at room tempera- ture (8). The cells were harvested by centrifugation and the resultant cell paste resuspended in a small volume (5 ml) of 1 Approved for publication by the Director of the Research Division, College of Agricultural and Life Sciences in coopera- tion with the Engineering Exp. Sta., Univ. of Wis. Supported in part by Office of Water Resources Research Project no. 14-01-0001-1961 (B-022-WIS) and in part by Federal Water Pollution Control Admin. Project no. WP-01470-01, admin- istered through the Univ. of Wis. Water Resources Center. Presented in part before Div. S-3, Soil Science Society of America, Detroit, Mien., Nov. 10, 1969. Received Apr. 17, 1970. Approved Oct. 16, 1970. 2 Research Assistant, Associate Professor and Visiting Assis- tant Professor of Soils, Assistant Professor of Water Chemistry, and Assistant Professor of Soils, respectively, Univ. of Wis., Madison. 82

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Page 1: Adenosine Triphosphate in Lake Sediments: I. Determination1

Adenosine Triphosphate in Lake Sediments: I. Determination1

C. C. LEE, R. F. HARRIS, J. D. H. WILLIAMS, D. E. ARMSTRONG, AND J. K. SYERS-

ABSTRACT

A modification of the luciferin-Iuciferase bioluminescencetechnique was developed for determining adenosine triphos-phate (ATP) in sediments. The method involves extractionwith cold H2SO4, clean up with a cation exchange resin anduse of living Aerobacter aerogenes cells as an internal stand-ard to correct for incomplete ATP recovery from the sediments.ATP recovery ranged from 20 to 85%, was a characteristic,reproducible property of a given sediment, but was not relatedconsistently to any other sediment property. The detectionlimits of the method were about 0.05 ,ug ATP/g oven drysediment but were dependent on the recovery characteristic ofthe sediment and the amounts of bioluminescence-inhibitorysolutes present in the extract used for final ATP analysis.Precision was low at ATP levels approaching the detectionlimit, primarily because of the high coefficient of variationshown at low ATP concentrations (about 3 X 10~10M for theinstrumentation and luciferin-Iuciferase extracts used in thisinvestigation). Theoretical considerations supported by prelim-inary experimental results indicate that the method should beapplicable to soils as well as sediments. The ATP contents ofnine sediment samples obtained from different lakes in Wis-consin ranged from 0.34 to 9.5 /j.g ATP/g sediment.

Additional Key Words for Indexing: Adenosine triphosphatein soil, microbial biomass, luciferin-Iuciferase method, sediment-water interactions, lake eutrophication.

R ECENT evidence (6) supports the high potential of aden-osine triphosphate (ATP) as an index of microbial

biomass in aquatic environments. The firefly luciferin-Iuciferase bioluminescence method for ATP analysis hasbeen used extensively for determination of ATP in diversebiological (3, 5, 10), fresh water (9), marine (6), sewage(14), and soil (12) systems. The method is based on mea-surement of the light emitted from the interaction of ATPwith luciferin (LH2), luciferase (E), and atmosphericoxygen, the amount of light energy emitted being propor-tional to the concentration of ATP added, as long as theother constituents of the reaction are present in excess(15). A simplified representation of the bioluminescencereaction is as follows (13):

Ms++ATP + LH2 + E -=?—>. E-LH2- AMP + P-P

E-LH2-AMP + 02PH E + product

+ CO2 + AMP + light .

This paper presents a modified luciferin-Iuciferasemethod for determining ATP in lake sediments and dis-cusses the problems associated with extracting intact ATPfrom sediments and soils in a state compatible with therequirements of the bioluminescence reaction.

MATERIALS AND METHODS

Bioluminescence Measurement of ATP

Lucijerin-luciferase—The luciferin-Iuciferase used was ob-tained as an arsenate-buffered, powdered firefly lantern extract(Sigma Chemical Co., St. Louis, Mo.) which is stable indefi-nitely in the dessicated state at — fOC. Prior to use, the extractin each vial was reconstituted with 5.0 ml Tris buffer (0.02M,pH 7.8), allowed to stand at room temperature for 3 to 4 hr,or overnight at 4C, to reduce light emission from endogenousATP and ATP derived from reactions catalyzed by transphos-phorylases (7, 15). The luciferin-Iuciferase suspension was cen-trifuged at 1,000 » for fO min to remove solids. The resultantclear enzyme extract was used within 2 hr of final preparation.

ATP Analysis—The standard or unknown ATP solution(1.8 ml) was pipetted into a standard glass liquid scintillationcounting vial. At zero time the reconstituted enzyme extract(0.2 ml) was added, the mixture was shaken by hand for ex-actly 10 sec, and then the vial was placed into the countingchamber of a liquid scintillation counter (Packard Tri-Carb,Model 3365) and the light emission integrated over a 5-secperiod by activating the repeat cycle (Fig. f). All light measure-ments were made at instrument settings for tritium (gain 50%,discriminator 50-1000 divisions). Blank samples (1.8 ml Trisbuffer) were interspersed throughout the ATP samples toenable calculation of net recorded light for each ATP sample.

Stock ATP solutions (10~3 M) were prepared by dissolvingcrystalline disodium ATP (Nutritional Biochemical Co., Cleve-land, Ohio) in Tris buffer (0.02M, pH 7.8). The stock ATPsolutions were stable for at least 3 months at — 10C. WheneverATP determinations were to be made, a series of standard ATPsolutions (10~9 M, 10~8 M, and 10~7 M) was prepared by dilut-ing the stock ATP solution with Tris buffer. The net lightresponse versus ATP concentration in the 3 X 10~10 to 10~" Mrange is approximately linear when plotted on log-log paper(Fig. 1). Regression analysis of the relationship between ATPconcentration and light emitted gave a best fit curve of loglight emitted = 1.315 log ATP cone. + b. The slope and line-arity of the curve are reproducible but the intercept (b) variesfor different vials of firefly extracts, necessitating that ATPstandards be run for each set of ATP determinations involvingthe use of different firefly extracts. Precision declines markedlyas the ATP concentration approaches the detection limit (Table1: 4. 11).

ATP Extraction from Aerobacter Aerogenes CellsStationary phase cells of A. aerogenes (NRRL 199) contain-

ing relatively constant amounts of ATP (1 to 2 X 10-10 /*gATP/viable cell) were obtained by inoculating a 1% nutrientbroth culture solution and shaking overnight at room tempera-ture (8). The cells were harvested by centrifugation and theresultant cell paste resuspended in a small volume (5 ml) of

1 Approved for publication by the Director of the ResearchDivision, College of Agricultural and Life Sciences in coopera-tion with the Engineering Exp. Sta., Univ. of Wis. Supportedin part by Office of Water Resources Research Project no.14-01-0001-1961 (B-022-WIS) and in part by Federal WaterPollution Control Admin. Project no. WP-01470-01, admin-istered through the Univ. of Wis. Water Resources Center.Presented in part before Div. S-3, Soil Science Society ofAmerica, Detroit, Mien., Nov. 10, 1969. Received Apr. 17,1970. Approved Oct. 16, 1970.

2 Research Assistant, Associate Professor and Visiting Assis-tant Professor of Soils, Assistant Professor of Water Chemistry,and Assistant Professor of Soils, respectively, Univ. of Wis.,Madison.

82

Page 2: Adenosine Triphosphate in Lake Sediments: I. Determination1

LEE ET AL.: ATP IN LAKE SEDIMENTS: I. DETERMINATION 83

10'

10'

Table 1—Precision of bioluminescence response

10- 10- 10"ATP, M

Fig. 1—Relationship between ATP concentration and lightemission from the reaction of ATP with the firefly luciferin-luciferase system. Statistical analysis of the data gave aregression curve of y = 1.315 * + b.

distilled water to provide about 109 to 1010 viable cells/ml. Foruse of the cells as an internal standard, ATP extraction involvedmixing the cell suspension (1 ml) with ice-cold 0.6N H2SO4(1 ml) for about 2 min, followed by neutralization with NaOHto pH 7.8. Time of extraction is not critical over a 5-sec to24-hr extraction period, nor is acid concentration, as long asthe final amount of acid exceeds 0.17V. The neutralized ATPextract was adjusted to 10 ml with distilled water and eitheranalyzed or stored at — 10C. Immediately prior to analysis,the extract was diluted 50- to 100-fold with Tris buffer to en-sure complete elimination of inhibition caused by excessiveconcentrations of ions (1, 15). For this reason the amount ofacid used for extraction should be minimized to prevent un-necessary sample dilution.

ATP Extraction from Sediments

Sediment samples were collected with an Eckman dredgeand transported immediately in glass bottles to the laboratoryfor characterization (7, 16); all sediments used in this investiga-tion were stored moist at 5C in sealed glass containers for 3to 6 months. For ATP extraction, duplicate sediment samples(containing about 0.2 g, dry wt basis) were weighed into cen-trifuge tubes; high water content samples were then centrifuged(1,000 g for 10 min) and the supernatant discarded. Extractionwith boiling Tris buffer was accomplished essentially as de-scribed by Holm-Hansen and Booth (7) and with N-bromosuc-cinimide by the method of MacLeod et al. (12). Extractionprocedures using HC104 (0.6/V), neutral DMSO (90% in0.05M Tris buffer), and acid DMSO (90% in 0.1/V H2SO4)were comparable to the H2SO4-extraction procedure describedbelow.

H2SO4-Cation Exchange Resin Method—Extraction withH2SO4 involved addition of ice-cold 0.6N H2SO4 (5 ml) to asediment sample (0.2 g dry wt) and intermittent agitation overa 5-min period using a vortex mixer. The tubes were main-tained ice-cold (usually about 20 min) prior to extract separa-tion from the particulate matter by centrifugation (1,000 g for5 min). The extract volume was recorded and then 2 ml wasshaken with 0.5 ml Na+-saturated cation exchange resin (Am-berlite IR-120) for 3 min. Following extract removal by glasswool filtration, the resin was washed three times with 1-mlaliquots of distilled H2O. The combined washings and extractwere shaken with a fresh 0.5-ml batch of resin and the extract

ATP

Blankf3 x lCTi°t1 x 10-'ti x icr't1 x lO-'J

Grosslight output

StandardMean deviation

310 35636

1.67435,012

623,668

47127475

3, 517

Gross light. 0

11.127.447.611.360.56

Net light

17.809.681.370.56

* Coefficient of variation - Standard deviation/Mean. Net light = Gross light of sample-Gross light of blank,

t Ten determinations. t Five determinations.

removal and washing process repeated except that the resinwas washed two rather than three times with distilled H2O. Thediluted extract (approximately 6-8 ml) was adjusted to pH 7.8with NaOH solution, and the neutralized extract volume madeup to 10 ml with distilled H2O. The final extracts were main-tained ice-cold for ATP assay the same day or stored at — 10Cfor assay within 3 or 4 days. Immediately prior to ATP deter-mination the extracts were diluted 10-fold with Tris buffer(Q.02M, pH 7.8) to dilute solutes interfering with the biolumi-nescence.

ATP Recovery—Determination of ATP recovery from sedi-ments to enable correction of sediment ATP values obtainedby the H2SO4-cation exchange resin and other methods involvedATP extraction from a sediment sample (0.2 g dry wt) andfrom a comparable sample to which an accurately measurableamount of ATP in the form of viable cells of A. acrogencs(1 ml) had been added immediately prior to extraction. Theinternal standard choice of ATP in bacterial cell rather thanin pure chemical form was based on the probability that ATPexists only in association with living cells (8) and on the diffi-culty of assessing possible chemical and enzymatic degradationof unprotected ATP between the time of ATP addition to thesediment and subsequent extraction.

RESULTS AND DISCUSSION

Successful determination of ATP in sediments by thebioluminescence method necessitates fulfillment of manyrequirements (Table 2) that are not major problems insimpler biological systems. Incomplete ATP recovery fromsediments may be real, because of ATP retention or hy-drolysis during extraction, or may be apparent, beingcaused by the presence in the extract of solutes inhibitoryto the bioluminescence reaction. The extent of apparentincomplete recovery can be estimated by diluting the ex-tract to the point where continued dilution causes no in-crease in ATP recovery, but this approach is not feasiblewhen the ratio of inhibitory solutes to ATP in the originalextract is so high that the ATP is diluted beyond the detec-tion range before the solutes are diluted to noninhibitory

Table 2—Requirements for determination of ATP in sedimentsand potential of the H2SO.j-resm method to fulfill

these requirements

Requirements H2SO4-resin methodExtraction;

Complete release of intact ATP fromliving cells; no ATP hydrolysis; noATP adsorption.

Extract separation;Complete removal of ATP in solutionphase.

Extract storage:No ATP hydrolysis.

Extract conditions for analysis:Neutral pH; absence of inhibitingsolutes (anions, cations andchelatlng agents).

Cold 0. 6N H2SO4 ;Cells killed; ATP-ase Inactivated,ATP stable; minimal retention through(un-lonized) phosphate groups butadenlne group protonated.

Centrifugation:Extract clear (sediment flocculated).

Refrigeration (0 C):ATP stable > 24 hr.

Cation exchange clean-up, pHneutralization, dilution: Excess Fe,Al and Ca removed; neutralizedextract clear; Inhibitory solutesdiluted out. _______________

Page 3: Adenosine Triphosphate in Lake Sediments: I. Determination1

84 SOIL SCI. SOC. AMER. PROC., VOL. 35, 1971

Table 3—Recovery of ATP from sediments by differentextraction methods

Recovery of added ATP,in extracts diluted^

Method*

Boiling Tris bufferN-bromosuccinlmlde

DMSO (neutral)(0. IN acid)

HClOj

H,S04

Sediment

Tomahawk 2Geneva 1Trout 1Little John 2

Tomahawk 2Mendota 1Tomahawk 2Mendota 1Little John 2Geneva 1Trout 1

0

3116

4511

17262915

X5

NM21—

_ _

_ _

6533_-_ _35

X10

NM_-49

4545158533857040

X50

NM

32NM

NMNM

88358835847140

All acid extraction methods involved 15% (approx.) ATP removal during cation ex-change resin clean-up; 85% recovery indicates practically complete extraction of in-tact ATP for these methods. Level of internal standard ATP added was 2. 5ftg persediment sample for the Little John and 1. 1 to 1. 3 ^g for the other sediments.NM = ATP concentration of diluted extract beyond measurable limit.

concentrations. An internal standard such as the bacterialcell one used in this investigation compensates for allsources of errors. However, the level of ATP determinationattainable by any method depends on the efficiency ofintact ATP extraction accomplished by the method andon the concentration of inhibitory solutes present in theresultant extract. This becomes particularly important forsystems such as soils and sediments which contain rela-tively low amounts of ATP; even if all the ATP is extractedintact, the ATP concentrations of a > 25-fold dilution ofof the resultant extract approaches the detection limitof the method described in this paper.

Typical ATP recoveries for the different extractionmethods evaluated are presented in Table 3. All acid ex-traction methods involved a cation exchange resin clean-upstep. Experiments with ATP extracted from pure A. aero-genes systems showed that the resin treatment caused a con-sistent 12-18% loss of ATP from the extracts so that 85%ATP recovery is close to the maximum obtainable by acidextraction methods.

Boiling Tris buffer (pH 7.8) extracted ATP efficientlyfrom pure bacterial cell systems but recovered, with poorreproducibility, < 5% of added bacterial ATP from sedi-ment systems. This incomplete recovery was not due to thepresence of inhibitory solutes in the undiluted extract sincedilution caused no increase in ATP recovery. The mostlikely explanation is adsorption of ATP through the ionizedphosphate groups by sediment components such as Fe andAl hydrous oxides at neutral pH. Errors associated with thedifficulty of maintaining boiling conditions to ensure rapidextraction and inactivate phosphatase enzymes (3) mayalso have contributed to low recoveries by the boiling Trisbuffer extractant.

The recently published N-bromosuccinimide method forATP determination in soils (12) was evaluated for itsapplicability to sediments. This method involves soil ex-traction with N-bromosuccinimide, EDTA, and arsenatebuffer, followed by treatment of the resultant extract withborohydride prior to ATP assay. For sediments, the finalextract necessitated dilution to overcome the presence ofinhibitory solutes (Table 3). However, the most seriousdrawback of this method was poor extraction of intact

ATP: comparable recoveries obtained by the N-bromo-succinimide and H2SO4 methods were 32 and 70%, re-spectively, for the calcareous Geneva sediment and 4 and40% for the noncalcareous Trout sediment. The majorreason for incomplete recovery of ATP by the N-bromo-succinimide method was probably the same as that for theboiling Tris buffer, namely strong ATP retention at a neu-tral pH by sediment inorganic components. The EDTAand arsenate components of the extraction system wereapparently not present in sufficient concentration to com-pete effectively with the ATP for the P-adsorption sites.Accordingly an attempt was made to improve the methodby increasing the EDTA concentration. However, theresultant excess EDTA in the extract interfered with thebioluminescence reaction, presumably by chelating theMg2+ needed for catalysis of the ATP-luciferin-luciferaseinteraction.

Poor recovery of ATP by neutral DMSO (Table 3) wasprobably also related to ATP retention at neutral pH, asindicated by the increased recovery obtained with acidic(0.1 N H2SO4) DMSO; recovery by the latter extractantwas still lower than that obtained by Q.6N H2SO4 extrac-tion (Table 3). However, there was little evidence of thepresence of excess inhibitory solutes in the DMSO extractssince ATP recovery was not changed by 10-fold dilutionof the extracts. In contrast, HC1O4, although accomplishingas good an extraction of intact ATP as H2SO4, required10-fold dilution to overcome the strong inhibitory effectof the neutralized extract (5) on the bioluminescencereaction.

Evaluation of the H2SO4-Cation Exchange Method—The advantages of this method in relation to the require-ments for determining ATP in sediments are summarizedin Table 2. The acid kills the cells and inactivates ATP-asewith quantitative extraction of intact ATP. At pH < 1 thephosphate components of the ATP should be fully un-ionized and thus show little tendency for adsorption ontocationic sediment components. In addition, the most reac-tive P-retaining components, amorphous Fe and Al sesqui-oxides and CaCO3, should tend to be dissolved underthese acid conditions, thereby increasing ATP extractabil-ity. This dissolution, however, has negative implicationsfrom other standpoints: apart from the inhibitory effectof multivalent cations on the bioluminescence reaction,co-precipitation of ATP with Fe and Al on neutralization ofthe acid extract results in practically complete loss of ATPfrom the extract. For this reason, multivalent cations wereremoved from the acid extracts with a cation exchangeresin prior to neutralization.

Data obtained using the H2SO4 method (Table 3) indi-cate that it is the best method of those evaluated. Theundiluted extract contains inhibitory solutes but not in suf-ficient concentration to prevent ATP measurement usingthis extract. Five-fold dilution is sufficient to obtain maxi-mum recovery for some sediments and 10-fold dilution hasbeen sufficient for all sediments tested to date. ATP recov-ery from diverse sediments using the H2SO4 method wasnot related consistently to any specific sediment property(Table 4), although the ATP recovery value for a spe-

Page 4: Adenosine Triphosphate in Lake Sediments: I. Determination1

LEE ET AL.: ATP IN LAKE SEDIMENTS: I. DETERMINATION 85

Table 4—ATP recovery and ATP content (determined by theH2SC>4-resin method) as related to sediment properties

Sediment

Wingra 4Geneva 1Mendota 1Monona 1

CaC03

57392733

Fe20,

Ca1111

Property

Org. C ClayP-

sorption*

Recoveryof

added ATPf

Icareous Sediments8687

50—5235

54929878

76702824

Noncalcareous SedimentsTomahawk 2Little John 2Crystal 1Devils 1Trout 1

_ __ _

----

6512

10

1530258

13

5737415537

10099

1009999

8585766745

810

22

64567

6318

392U2

ATPcontent

7 0.8558

757348

0.90.21. 7

0.50.50.20.130.4

Percent of inorganic P removed by 1. 5 g sediment (oven-dry basis) from 50 ml ofKH2PO4 solution containing 25 ^g P/ml.

t ATP removal during resin clean-up was approx. 15%; 85% recovery indicates prac-tically complete extraction of ATP from the sediments.

cific sediment was a reproducible characteristic of thesediment. Taking into consideration that the resin clean-uptreatment was responsible for about 15% of the ATP lossfrom the acid extracts, the H2SO4 achieved over 85%recovery of intact ATP from many of the sediments (Table3). However, for some sediments, namely Trout, Monona,and Mendota, ATP recovery was < 60%. Accordingly,selected methodological variables were evaluated in anattempt to ascertain the reasons for incomplete ATPrecovery from these sediments and also to define moreprecisely the general scope of the H2SO4 method.

The effect of acid extraction time on ATP recovery wasevaluated using the Mendota sediment. The amounts ofATP extracted were comparable for extraction times vary-ing from 5 min to 2 hr, indicating that inadequate exposureto acid conditions was not the reason for incomplete ATPextraction from this sediment. Similarly, acid concentrationin the 0.3./V to 1.ON range was not critical for either theMendota or the Trout sediment as long as there was suffi-cient acidity to ensure a sediment pH of < 1. This pH wasdefinitely attained in all sediments evaluated based on therelative amounts of acid (5 ml of 0.6N H2SO4) andsediment (0.2 g dry wt) used for sediment ATP determi-nation. From another standpoint, ATP recovery valueswere not a function of the level of ATP added, since nochange in ATP recovery occurred when the level of addedATP was increased two-fold (Mendota sediment) or 10-fold (silt loam soil of 45% ATP recovery characteristic).An additional potential source of error, the possibilityof changes in the ATP content or viability of the bacterialcells used as the internal standard occurring immediatelyon contact with the sediment systems, was discounted:viable cell numbers determined using nutrient agar showedno change following mixture with sediments, and ATP/vi-able cell relationships of the supernatant of sediment-watermixtures were similar to those of the initial inocula. Finally,ATP hydrolysis during storage was probably not responsi-ble for incomplete recovery since ATP in acid extractsmaintained at OC was completely stable up to 7 hr andshowed only slight decrease (< 10%) after 30 hr; ATPin resin-treated neutral extracts was stable for at least3 to 4 days at -IOC.

The above data indicate that the most likely explana-tion for incomplete ATP recovery from sediments is related

to retention of ATP by the sediments against acid extrac-tion. The nature of the sediment components and themechanisms involved have yet to be resolved. One possi-bility is sorption through the positive charge possessed bythe adenine (pKa = 4.1) of the ATP molecule; the 15%ATP retention in acid extracts by the cation exchange resinsupports this possibility. Interaction of the ATP phosphategroups with sediment components is also conceivable basedon the known insolubility of Fe salts of organic phosphateesters and sorption of organic phosphates by clays underacid conditions (2).

On the basis of limited experiments with four soils, theH2SO4 method appears applicable to soils as well as sedi-ments, giving ATP contents ranging from 0.2 to 0.6 /*g/gsoil and recoveries ranging from 33 to 60% after correc-tion for ATP retention by the resin used for extract cleanup. The ATP content of the sediments was not related tosediment properties of potential importance in the retentionand stabilization of organic P compounds (Table 4). Theorigin and significance of the ATP in the sediments arediscussed elsewhere (8).

Detection limits of the H2SO4 method using 0.2 g sam-ple and 10-fold dilution of the cleaned-up extract are 0.04to 0.16 ,ug ATP/sample, depending on the recovery char-acteristic of the sediment. The sensitivity of the methodcould probably be increased by treatment of the firefly ex-tract with apyrase to reduce the background light emissioncaused by the presence of endogenous ATP in the extract(11), or by the use of purified luciferin and luciferase (4).Light response measurement with instrumentation speciallydesigned for quantizing light emission immediately follow-ing mixture of the ATP sample and the luciferin-luciferasesystem would increase the sensitivity by one to three ordersof magnitude (4, 12); in this case, it is possible that theexcess multivalent cations in the acid extracts could bediluted out prior to neutralization, thus eliminating theresin clean up step. However, the many potential errorsassociated with ATP determination in sediments and soilsemphasize the necessity of using a reliable internal stan-dard as an integral part of any method for determining theATP content of such systems.

Page 5: Adenosine Triphosphate in Lake Sediments: I. Determination1

SOIL SCI. SOC. AMER. PROC., VOL. 35, 1971