Cellular Nucleotide Measurementsand Applications in Microbial · CELLULAR NUCLEOTIDE MEASUREMENTS...

MICROBIOLOGICAL REVIEWS, Dec. 1980, p. 739-796 Vol. 44, No.40146-0749/80/04-0739/58 $02.00/0

Cellular Nucleotide Measurements and Applications inMicrobial Ecology [ i

DAVID M. KARLDepartment of Oceanography, University ofHawaii, Honolulu, Hawaii 96822

INTRODUCTION ....................... .... ............. .......... 740NUCLEOTIDES, NUCLEOSIDES, AND RELATED INTRACELLULAR

COMEPOUNDS ............ ............................. 741Cellular Bioenergetics ....... .......................... ... ...... ..... 741Regulation of Cellular Metabolism ......................................... 742Adenylate energy charge ................................................. 742Phosphorylation state .................................................... 742Oxidation-reduction state.742Indirect energy coupling.743Cyclic adenosine 3',5'-monophosphate, guanosine 5'-triphosphate, and gua-

nosine tetraphosphate.743Analytical Procedures ........................... 743Sampling 744Extraction.745Measurement.750

(i) Firefly bioluminescence .................. ........... ...... 750(ii) Bacterial bioluminescence.751(iii) Renilla biolumnescence.751(iv) Immobilized enzymes .............................................. 751

NUCLEOTIDE LEVELS IN MICROBIAL CELLS: EFFECTS OFGROWTH RATE,CULTURE CONDMONS, NUTRIENT STRESS, AND ENVIRON-MENTAL PERTURBATIONS ...................................... 751

Adenine Nucleotides and Energy Charge Ratios.751Procaryotes ............................. .... ...... ....... .. .. 751Unicellular eucaryotes .............................. 755Metazoa ................... 755

Environmental Perturbations and Nutrient Limitations . .. . 756Transient states. ...756Nutrient limitation and starvation.... 757

(i) Carbon..... 757(ii) Nitrogen, sulfur, and iron.... 758(iii) Phosphorus ......................................... 758

Nonadenine Nucleotide Triphosphates ..760Diel Rhythms and Specific Cell-Cycle-Related Events ..760Nicotinamide and Flavin Nucleotides ..761

NUCLEOTIDE FINGERPRINTS IN NATURE ..761Detection of Life ............................................... .... 761Adenosine 5'-Triphosphate (ATP) asa Biomass Indicator....... .763

Obligate association ofATP with living organisms ..763C/ATP ratios .......................... ................................. 764Correlations of ATP with other measures of biomass and activity .. 766

Selected Environmental ATP Applications ..767Partitioning of bacterial, algal, and metazoan carbon ..767ATP applications in oceanography ..769

(i) Geographical and vertical distributions of ATP in the ocean. 769(ii) Sedimentary ATP distributions.771(iii) ATP-based biochemical indices of physiological state.772(iv) ATP and respiration rates.774(v) ATP and growth rates.774(vi) Downward vertical flux ofATP in the ocean.774

Terrestrial and aquatic decomposition studies ..775Wastewater treatment, disinfection control, and pollution assessment .... 776

Environmental Adenylate Energy Charge.777Guanosine 5'-Triphosphate and Cellular Biosynthesis.780Cyclic Adenosine 3',5'-Monophosphate.782

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Nucleotide Metabolism and Growth ......................................... 782Dissolved nucleotides and microbial heterotrophy ........................ 782Nucleic acid biosynthesis ........................................... 782

CONCLUDING STATEMENT ................................................. 784LITERATURE CITED ......................................................... 785

INTRODUCTION

Ecology is physiology under the worst possibleconditions

T. Brock, 1966

Experimental microbial ecology representsone of the least developed areas of microbiolog-ical research. The organization and dynamics ofmicrobial communities are such that they shouldbe studied as integrated units if an understand-ing of the mechanisms and regulation of vitalbiogeochemical cycles is to be achieved. Theprimary objective of ecosystems research, orsynecology, is to study and ultimately under-stand the exchange of energy and matter be-tween and among the various living and nonliv-ing compartments. A detailed quantitative as-sessment of the total amount of living carbon(i.e., biomass) and of the magnitude of commu-nity metabolic activity and growth is fundamen-tal to our understanding of natural processes.One motivation for measuring biomass and ac-tivity is to estimate the total amount of energystored in the biota and to monitor the rate ofmaterial flux through the living system.Ecology is defined as the integrated study of

biological relationships among organisms shar-ing a common habitat and of the interactionsoccurring between the biotic and abiotic com-ponents of that environment. Although naturalecosystems are of primary concem to the exper-imental microbial ecologist, environments de-signed, constructed, and primarily controlled byhuman activities (e.g., industrial and agriculturalcomplexes and urbanized areas) are also of con-siderable interest in applied microbial ecology.Therefore, the field of microbial ecology extendsbeyond the study of soil and aquatic environ-ments and includes investigations of such areasas microbial disease and infection, contamina-tion of processed foods, wastewater and sewagetreatments, purification of potable water sup-plies, and even the search for extraterrestriallife, to name a few.

In the context of this review, the term micro-bial shall encompass all organisms less than200 ,um in greatest dimension. Accordingly,

bacteria, actinomycetes, unicellular algae, yeastsand fungi, protozoa, and many metazoan taxaare all regarded as components of the naturalmicrobial assemblage. Their small size (i.e., high

surface-to-volume ratio) is the sole unifyingcharacteristic of this heterogeneous group; theresult is an intimate contact and potentiallylarge interaction with their abiotic environment.It is important to remember that microorga-nisms live in microenvironments defined onscales of micrometer and millimeter dimensionsand that the physical and chemical characteris-tics of these microhabitats are generally quitedifferent from those of the ambient macroenvi-ronments.

Classical microbiological methods generallyhave not been suitable for enumerating thesparse populations or for assessing the relativelyslow growth rates that occur in most naturalenvironments. Even more recently developedlaboratory methods for estimating biomass (e.g.,fresh or dry weight determinations, optical den-sity, total protein or cell carbon, and direct mi-croscopy) are generally unsatisfactory when ap-plied to natural populations due to the hetero-geneity and variable size spectrum of the indi-vidual microbial assemblages, the presence ofdead cells, and the varying proportion of nonliv-ing organic materials (i.e., detritus) which areclosely associated with the living cells. The de-velopment of reliable techniques which can beused unequivocally to measure biomass and toestimate the specific growth rates of autochtho-nous microbial assemblages is still considered tobe the most fundamental research objective ofexperimental microbial ecology. Extensive lab-oratory studies have been conducted in an at-tempt to characterize the physiology and bio-chemistry of bacteria isolated from soil, fresh-water, and marine ecosystems; however, it isunknown whether or not these metabolic poten-tials are ever realized in situ among the naturallyoccurring microbial populations. The experi-mental microbial ecologist must ultimately havea "commitment to nature" (38) and sooner orlater must complement laboratory studies withfield observations and measurements.No single approach to the study of microbial

ecology is universally accepted. One option is toinvestigate a single group of organisms or asingle metabolic process through an analyticalbreakdown of the natural environment. The sec-ond, and perhaps more difficult, route is to studythe complex ecosystem in toto. This review em-phasizes the latter approach and specifically isorganized around a presentation and discussion

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of the use (and misuse) of nucleotide measure-ments in experimental microbial ecology. Pre-vious reviews concerning the pathways of nu-cleotide metabolism (129), the role of adeninenucleotides in the regulation of cellular metab-olism (8, 10, 47, 48, 125, 194), and selected topicsin cellular bioenergetics (118) have been mosthelpful in formulating the concept of "nucleotidefingerprinting" of complex microbial communi-ties.

In spite of the large number of papers pub-lished under the general theme of cellular nu-cleotide measurements and applications in mi-crobial ecology, no comprehensive review of thesubject has been written. The present effort isintended to: (i) offer a brief review of the fun-damental physiological principles which providethe motivation for considering nucleotide mea-surements in experimental microbial ecology, (ii)outline the principles and current limitations ofthe analytical procedures used to extract andquantify nucleotides from microbial assemblagesin nature, (iii) appraise the available laboratoryand field data concerning predictable effects ofthe environment on steady-state levels of cellu-lar nucleotides, and (iv) present specific appli-cations and interpretations of the measurementof nucleotide fingerprints in naturally occurringpopulations of microorganisms. These goals aresought by organizing and discussing the ecolog-ical data within an appropriate framework ofmicrobial physiology and nucleotide metabo-lism. It is hoped that this review will stimulateand facilitate the application of nucleotide fin-gerprinting of microbial populations in nature asa means of assessing biomass, growth, and activ-ity and concurrently provide caution for theinterpretation of existing data.

NUCLEOTEDES, NUCLEOSIDES, ANDRELATED INTRACELLULAR

COMPOUNDSCellular Bioenergetics

All living cells contain an identical suite ofmolecules, referred to collectively as nucleosidesand nucleotides, which are essential for viabilityand growth. Although in the strictest sense nu-cleosides and nucleotides are derived from nu-cleic acids, the terms are now used in a muchbroader sense to include structurally relatedcompounds as well (129). More than 100 differ-ent nucleosides and nucleotides have now beenisolated and identified (167); however, the 5'-ribonucleotides of the bases adenine, guanine,uracil, and cytosine compose the bulk (90 to95%) of the free nucleotide derivatives of mostcells. Less abundant and more exotic derivatives,

such as the deoxyribonucleotides, higher-levelpolyphosphates, 3',5' cyclic monophosphates,adenine-sulfuryl nucleotides, conjugated nucleo-tides, naturally occurring nucleoside and nucleo-tide antibiotics, vitamins, and coenzymes alsohave specific functions in cellular metabolism.In addition, the 16S and 23S ribosomal ribonu-cleic acid (RNA) and transfer RNA componentsofmany procaryotes are known to contain struc-turally modified nucleosides as well as "natural"bases. The chemistry and biochemistry of thesemodified nucleotides have been concisely re-viewed by Hall (120).

It is now well established that nucleotideshave at least four functions, including: (i) storageand transport of cellular metabolic energy, (ii)synthesis of deoxyribonucleic acid (DNA) andRNA, (iii) activation and transfer of precursorsfor cellular biosynthesis, and (iv) control andregulation of cellular metabolism. The centralrole of the adenine nucleotides, and especiallyadenosine 5'-triphosphate (ATP), as intermedi-ate carriers of chemical energy linking catabo-lism and biosynthesis has been known since thepublication of Lipmann's classic paper in 1941(221). The production of ATP, however, shouldbe viewed not as a mechanism for the storage ofchemical potential energy, but rather as a sys-tem for rapid and specific mobilization of cellularenergy. Turnover times for the intracellularATP pool of 0.1 to 1.0 s are not uncommon forgrowing bacteria (47, 126, 151, 195, 241). There-fore, the amount of chemical energy representedby the intracellular ATP pool is only a verysmall percentage of the potential energy fluxthrough the cell.Bauchop and Elsden (25) have proposed that

the extent of growth of a microorganism is pro-portional to the amount of ATP available to itfrom the degradation of an energy source. Thisallows for the calculation of a yield coefficient,YATP, defined as the number of grams (dryweight) of cells produced per mole of ATP gen-erated from catabolism (103). Several discus-sions of YATP have since appeared (103, 239),including a challenge to the universality of thisconcept (301, 302). Furthermore, it is not knownwhether yield studies conducted in the labora-tory have predictive value for estimating micro-bial growth yields in nature. Tempest (314) hasraised an interesting question regarding the im-portance of growth yield as a selective force inthe evolution of microorganisms. He has con-cluded that the ability to scavenge low levels ofnutrients probably represents a more importantselection factor than the ability to metabolizethem optimally. Currently, it is unknownwhether ATP production and growth are uncou-

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pled in naturally occurring populations of micro-organisms or whether natural microbial assem-blages are functioning at or near their maximumtheoretical YATP limits.

Regulation of Cellular MetabolismSince the maximum potential rate of energy

production in microorganisms is much greaterthan the requirement for ATP, living systemshave evolved mechanisms to ensure integration,correlation, and control of cellular processes.Bacterial metabolism is regulated by mecha-nisms which function at various biochemicallevels and time scales. These range from coarsegenetic control of enzyme synthesis and activity(i.e., induction, repression, derepression, etc.) tofine metabolic tuning effected through kineticand thermodynamic aspects of the system (163).Several examples of the latter involve cellularnucleotides.Adenylate energy charge. In vitro studies

have revealed that the activities of certain en-zymes are affected by the concentration ofATP,others are affected by adenosine 5'-diphosphate(ADP) or adenosine 5'-monophosphate (AMP),and still others are affected by the ATP/AMPor ATP/ADP ratios. Present usage expressesthe cumulative effects of the adenosine phos-phates on the rate of cellular metabolism interms of the "adenylate energy charge" param-eter (12). The adenylate energy charge (ECA) isequal to one-half of the number of anhydride-bound phosphate groups per adenine moiety, or

ECA = [ATP] + 1/2[ADP][ATP] + [ADP] + [AMP]

and is equivalent to a linear measure of the totalamount of chemical potential energy momentar-ily stored in the adenine nucleotide pool. In vitrorate responses of several "key" enzymes in cel-lular metabolism to variations in ECA have pro-vided the background data for this control hy-pothesis. A discussion of the unique metabolicsignificance of the adenylate system in stoichio-metric coupling of cellular energy and a reviewof the extensive laboratory data supportingthese theoretical considerations have been pre-sented in detail previously (7-11, 47, 48, 125,194).The ECA is unitless, which limits the useful-

ness of this parameter in supplying informationabout intracellular nucleoide concentrations orthe rate of ATP turnover; these can vary signifi-cantly at a fixed ECA (194). Furthermore, Lowryet al. (226) have argued that the ECA is aninsensitive metabolic indicator, since smallchanges in ECA actually disguise much larger

changes in the absolute ATP/ADP or ATP/AMP ratios to which the enzymes are actuallyresponding. The validity of the ECA concept asa mechanism for the control of cellular metabo-lism has also been attacked on more theoreticalgrounds (108, 265). This criticism pertains to thestate of equilibrium of the adenylate kinase re-action and to the intracellular availability ofHP042- and Mg2+. Furthermore, conceptualproblems arise in the interpretation of the ECAcontrol hypothesis as a result ofknown examplesof barrier and kinetic compartmentation of ad-enine nucleotide pools. Failure ofthe ECA theoryin encompassing certain thermodynamic consid-erations of biological energy flow is the basis foradditional criticisms. However, Atkinson (10)has recently emphasized that the componentparts of a cell are far from equilibrium and thatthe kinetic, and not thermodynamic, controlsprovide the stabilizing influence on cellular me-tabolism. Despite these objections, an over-whelming proportion of the published data in-dicate that the relative molar concentrations ofthe adenine nucleotides in actively metabolizingcells are maintained within the stringent limitspredicted by the ECA hypothesis (for compila-tions, see references 47, 48, 125, and 194).Phosphorylation state. Independent of the

ECA hypothesis, the phosphorylation state([ATP]/[ADP][HPO42-]) has been proposed asa fundamental regulatory parameter controllingthe oxidation-reduction state of the cytoplasmicnicotinamide adenine dinucleotide (NAD)-re-duced NAD (NADH) couple and the rates ofrespiration and oxidative phosphorylation inmitochondria (308, 335). More recently, Reed(269) found a positive linear correlation betweenECA and phosphorylation state and postulatedthat the phosphorylation state is regulated bythe ECA.Oxidation-reduction state. Even though

the majority of energy-producing and energy-requiring reactions of a cell are coupled directlyto the formation or hydrolysis of ATP, manyimportant metabolic processes proceed as anindirect result of the adenylate system. Thepyridine nucleotides (NAD, NAD-phosphate[NADP]), and specifically the numerical valuesof the oxidized-reduced pyridine nucleotide cou-ples (i.e., [oxidized NAD]/[NADH] and [oxi-dized NADP]/[NADPH]), affect (and are af-fected by) cellular dehydrogenase activity, theoxidative phosphorylation state, and the energycharge of the adenylate system. The intracellu-lar oxidation-reduction state of the pyridine nu-cleotides may also be an important factor in thenetwork of metabolic control. Andersen and vonMeyenburg (4) have defined the catabolic re-duction charge ([NADH]/[NADH + oxidized

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NAD]) and the anabolic reduction charge([NADPH]/[NADPH + oxidized NADP]) as

important regulatory parameters in cellular me-tabolism.

Indirect energy coupling. In addition todirect utilization ofATP in cellular metabolism,the free energy of hydrolysis of the y-phosphategroup of ATP can be reversibly transferred tothe diphosphates of additional intracellular nu-

cleotides as required for specific biosyntheticreactions. This "phosphate shuttle" is catalyzedby the enzyme nucleosidediphosphate kinase.Since the AGo' values for the hydrolyses of allnucleoside triphosphates (NTPs) are compara-

ble, the nucleosidediphosphate kinase reactionhas a K., of 1.0 and is therefore freely reversible.With a few exceptions, the activity of nucleo-sidediphosphate kinase is absolutely essentialfor the continued supply of the nonadenineNTPs. This indirect coupling ofchemical energy(i.e., use of guanosine 5'-triphosphate (GTP),cytidine 5'-triphosphate, uridine 5'-triphos-phate, etc., rather than ATP directly) is requiredexclusively for the synthesis of new cellular ma-terials. Thompson and Atkinson (317) have spec-

ulated that the use of nonadenine NTPs specif-ically for growth allows the cells to preferentiallysuspend most endergonic biosynthetic reactionsduring periods of nutrient and energy depletion,thereby conserving their scarce metabolic re-

sources for the maintenance of homeostasis.They also determined that the activity of nu-

cleosidediphosphate kinase is directly coupledto the cellular ECA in such a way that the flowof phosphate into the nonadenine NTP pools issharply curtailed when ECA ratios fall below a

critical minimum level (317). Since the nucleo-sidediphosphate kinase reaction represents a sig-nificant potential drain on the available ceilularATP, it may be one of the most importantenzymatic reactions in the regulation of cellularbioenergetics.Cyclic adenosine 3',5'-monophosphate,

guanosine 5'-triphosphate, and guanosinetetraphosphate. Three additional cellular nu-

cleotides which may exert a significant influenceon the control of metabolic processes are cyclic

adenosine 3',5'-monophosphate (cAMP), GTP,and guanosine tetraphosphate. Since the classicwork of Makman and Sutherland in 1965 (232),cAMP has been implicated as an active compo-nent of many important microbial regulatorymechanisms. One of the most important func-tions of cAMP is its effect (in concert with theappropriate inducer molecule) on the synthesisof inducible enzymes. The ability of exogenouscAMP to overcome the effects of cataboliterepression is also noteworthy. The appearance

of cAMP in cells has been regarded by Tomkins

(322) as an important "metabolic symbol" forcarbon starvation and as a biochemical mecha-nism for modulating the growth and develop-ment of both procaryotic and eucaryotic cells.The guanine nucleotides may also play a

unique and important role in the regulation ofRNA and protein syntheses. GTP has beenshown to be an essential factor for the initiationand the aminoacyl-transfer RNA binding pro-cesses and for the polypeptide elongation se-quence of protein biosynthesis. Karl (169, 174)has recently proposed that the intracellularGTP/ATP ratio may serve as a specific nucleo-tide fingerprint which can be used to estimatethe rate of protein synthesis within naturallyoccurring microbial populations. A second im-portant guanine ribonucleotide, guanosine tetra-phosphate, first isolated by Cashel and Gallantin 1969 (44), has since been implicated in thecontrol of ribosomal RNA synthesis (99, 208) aswell as protein synthesis (200).

Undoubtedly, the fine-tuning mechanisms forthe control and integration of cellular energyproduction and utilization are complex and ex-tend well beyond our formulations of chargeratios, nucleotide fingerprints, and metabolicsymbols. Nevertheless, it should be realized thatendeavors to think in functional terms haveprovided a conceptual and experimental frame-work around which to conduct meaningful phys-iological studies on the role of nucleotides inliving cells.

Analytical ProceduresAn overwhelming and justified emphasis on

methodology is a familiar feature in the litera-ture pertaining to environmental nucleotide ap-plications. Numerous investigators have devel-oped methods for extraction of cellular nucleo-tides; however, no single procedure has emergedas the most efficient. Undoubtedly, it would beuseful to have a single or even a few standardtechniques which could be used for analyses ofa wide spectrum of sample materials and micro-bial habitats. Unfortunately, extreme variabilityin the physicochemical and microbiologicalcharacteristics of individual habitats precludesthe existence and mitigates the usefulness of any"universally" applicable technique. Therefore,the procedure that is ultimately selected forroutine nucleotide extraction and analysis willdepend on the nature of the sample material.Innumerable improvements in nucleotide ex-traction and assay procedures have surfaced inrecent years, and the continuing state of flux of"preferred methods" tends to limit the useful-ness of any personal recommendations. The bestadvice that can be given is that several differenttechniques should be evaluated and compared,

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using natural representative sample materials,before selecting the preferred extractionmethod. Most deleterious effects of sampling,extraction, and analysis ofNTPs generally resultin decreased concentrations of NTPs, especiallyATP. A high yield ofNTPs and high ECA valuesshould therefore be taken as primary criteria forevaluating the relative extraction efficiencies ofvarious methods. In comparative studies of thisnature, it is imperative to be aware of, andcorrect for, specific interferences which may beunique to a given chemical extractant, extractionprocedure, or particular environmental substra-tum.

In view of the potential variability in extrac-tion procedures and efficiencies, a brief discus-sion of the following points seems warranted: (i)criteria for efficient nucleotide extraction, (ii)common sources of analytical interference, and(iii) limitations of our "state of the art" tech-niques. In this context, readers should directtheir attention towards various symposia andworkshops (33, 34, 52, 281) which have at-tempted to resolve a few of the outstandinginadequacies of existing techniques and to for-mulate concise, universally acceptable method-ologies.The procedures for measuring environmental

nucleotide concentrations can be convenientlydivided into three equally important analyticalaspects: (i) sampling, (ii) extraction, and (iii)analysis.Sampling. No technique for sampling micro-

organisms in the field is more rapid than re-ported rates of ATP turnover. Consequently,quantitative measurements of intracellular ade-nine nucleotides are accurate only if concentra-tions remain constant throughout the samplingperiod. Biochemists and microbial physiologistshave designed sophisticated sampling devices tominimize the time elapsed between samplingfrom the culture and the extraction. Spring-loaded syringes precharged with the appropriatereagents can now efficiently and reproduciblyremove cells from growth chambers and extractall the intracellular nucleotides in less than 1 s.Unfortunately, microorganisms from naturalecosystems must first be collected, transported,often sorted, and frequently concentrated beforeextraction. To cite an example, marine micro-biologists samplingthe deep-sea environment(depths of 22,000 m) usually wait a minimum of30 min merely for the collected material to reachthe ocean surface, in addition to the time re-quired for shipboard manipulations and for nu-cleotide extraction. In situ growth conditionsshould be maintained as constant as possibleduring this sampling period. If this cannot be

achieved, caution must be exercised in subse-quent interpretation of the data.Romano and Laborde (275) have compared

the ATP and ECA values of water samples ex-tracted in situ with the results obtained by fol-lowing the more conventional sampling andshipboard extraction procedures. Their resultsfrom a single station in the Mediterranean Seaindicated that there were no deleterious sam-pling effects to depths of 30 to 40 m. However,more extensive and systematic studies are nec-essary to further assess the quantitative effectsof sampling, transportation, storage, and addi-tional experimental manipulations of samplematerials on the resultant intracellular nucleo-tide levels. Transitions in light, temperature, pH,pressure, moisture, and dissolved gases may allaffect the physicochemical properties of thesample and thereby directly or indirectly influ-ence the intracellular nucleotide concentrations(and their ratios).Most aqueous environmental samples require

concentration before nucleotide extraction, al-though certain eutrophic ecosystems (and mostlaboratory cell cultures) may be processed bydirect injection of the sample into the extractionmedium (165). The most convenient and prac-tical method for concentrating cell material isvia vacuum filtration; however, reverse filtrationtechniques (149), dialysis plates (160), hollow-fiber concentrator systems (160), and centrifu-gation (61, 87) have also been used. The choiceof membrane filter and pressure differential willbe dictated by the size and fragility of the cells,and all efforts should be taken to minimize filterclogging and cell lysis during the concentrationprocess.

Several investigators have reported a delete-rious effect of vacuum filtration on the finalATP concentrations ofsample extracts (165,180,271, 293, 309). Karl and Holm-Hansen (179, 180)have recently reexamined this "filtration effect"in terms of ATP, total adenine nucleotides (AT= ATP + ADP + AMP), and ECA. Their dataindicated that both the total intracellular ATPconcentration and the ECA decreased, when nor-malized to a standard volume, as the amount ofwater filtered increased. This response was at-tributed to metabolic stress imposed by vacuumfiltration. Since the AT concentration was foundto be conservative (i.e., AT per unit of volumewas independent of volume filtered), these datadiscount cell lysis as an important factor. Theobserved filtration effect was not a linear func-tion ofthe volume filtered, but instead was foundto be hyperbolic, with the asymptote presum-ably corresponding to some maximum level ofmetabolic stress (ECA = 0.5-0.6). It has previ-

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ously been demonstrated that centrifugationalso has a detrimental effect on the ATP levelsof Escherichia coli (61), suggesting a common

physiological mechanism. In addition, Karl andHolm-Hansen (179, 180) have reported that themagnitude of this filtration effect is influencedby the species composition and cell density ofthe microorganisms, as well as by the in situcommunity ECA. Similarly, Sutcliffe et al. (309)have discovered that only certain coastal marinewaters are affected by this phenomenon andthat open-ocean samples (i.e., presumablyslower-growing microbial populations) are rela-tively free from this source of interference. Infreshwater ecosystems, Rudd and Hamilton(276) were unable to demonstrate any effect ofvacuum filtration on ATP levels during an in-vestigation of two Precambrian shield lakes, andRiemann (271) was only able to elicit this re-

sponse in the most eutrophic of four lakes incentral Denmark. At present, the variable re-

sponse of natural populations to the effects ofvacuum filtration, especially with regard to mea-surements and interpretations of ECA data, isone of the most serious, and as yet unresolved,problems in the application of nucleotide deter-minations in aquatic ecosystems. A thoroughunderstanding ofthe precise physiological mech-anism(s) occurring at the cellular level is neededto eliminate the analytical interference causedby vacuum filtration.Sampling artifacts influencing the determina-

tion of environmental ECA values are not re-

stricted to filtration effects. Skjoldal and Bam-stedt (294) have demonstrated a significant"capture stress" associated with net collectionsof planktonic metazoans. The ECA values ofvarious zooplankton species were shown to de-crease to -0.2 immediately upon collection, butrecovered to 0.8 after 24 h in an aquarium.Therefore, in many environmental applicationsthe use of total adenylates may be preferable toATP measurements in order to circumvent thehigher level of variability which may be intro-duced by transient fluctuations in cellular ATPlevels.Extraction. Numerous methods are available

for the extraction of nucleotides from microbialcells. Media for initial extraction may be dividedinto the following categories: (i) boiling aqueousbuffers, (ii) inorganic acids, (iii) organic acids,(iv) organic solvents, and (v) inorganic bases.Under all circumstances, however, the basic cri-teria for a successful extraction procedure are

identical. The most important requisites includerapid cell death and lysis, complete nucleotiderelease, complete and irreversible inactivation ofenzyme activity, and long-term stability of the

extracted nucleotides (i.e., no chemical or enzy-matic hydrolysis). In addition, there may beproblems arising from adsorption (especiallyonto soil and sediment particles), ionic interfer-ences (both organic and inorganic), colorquenching, turbidity, and poor extraction effi-ciencies of microorganisms associated with non-living organic and inorganic materials.Table 1 lists the most commonly used nucleo-

tide extraction procedures, and Table 2 presentsa summary of the numerous comparative studieswhich have been conducted to optimize ATPextraction from various sample materials. Al-though several investigators have recommendedidentical extraction procedures for similar sam-ple materials (e.g., cold H2SO4 for soils and sed-iments), it is apparent that no single extractionmethod can be regarded as universally accepta-ble (Table 2). Nevertheless, a few general com-ments can be made concerning the extraction ofnucleotides from natural microbial assemblages.(i) Boiling buffers have consistently yielded vari-able and inconsistent results when applied tosoils, sediments, and metazoans. One explana-tion which has been offered for these observa-tions is termed the "heat gradient hypothesis,"which is a consideration of thermal extractionefficiencies based on the kinetics of heat flow intwo-phase systems (177, 182, 183). (ii) Stronglyalkaline conditions should be avoided during theextraction process due to the chemical labilityof many polynucleotides, nucleoside diphos-phate esters of sugars, aminoacyl adenylates,and related compounds. This is especially im-portant in the determination of ECA values,where the production of ADP (from the hydrol-ysis of ADP sugars) or AMP (from the break-down ofNAD or nucleic acids) may yield falselylow ECA values. (iii) Internal standards shouldalways be used to correct for losses (or apparentlosses) of nucleotides resulting from hydrolysis,adsorption, coprecipitation, organic and inor-ganic ionic interferences, turbidity, color quench,pH spectral shifts, and other factors. Internalstandards have been added as aqueous ATP-saltsolutions, bacterial cells, and radiolabeled (14C,3H, 32p) ATP. It should be emphasized that thefunction of the internal standard is not to assessthe efficiency of a given extraction medium, butrather to aid in evaluating the "apparent" and"real" losses resulting from various sources ofanalytical interference. Therefore, the most ac-curate method of introducing the internal stand-ard is through the use of aqueous ATP solutions.Transient fluctuations in cellular ATP levelswith environmental perturbations render the useof live microbial cells inadequate as a primaryinternal standard. Radioisotopic tracer tech-

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TABLE 1. Spectrum ofmethods available for extraction of cellular nucleotidesa

Menstruum Conditions of extraction Reference

Aqueous buffersTRISTRIS-EDTATRIS-arsenate-EDTA-butanol

Glycine-EDTASodium bicarbonatePhosphate (Sorensen)Phosphate-citrate (McIlvaine)HEPESWater

Inorganic acidsNitricPerchloricPerchloric-EDTAPhosphoric-EDTASulfuric-charcoal adsorptionSulfuric-EDTASulfuric-ion exchangeSulfuric-ion exchange-ethanolamineTriton X-100-phosphoric-sulfuricSulfuric-oxalic

Organic acidsAceticFormicFormic-EDTATrichloroaceticTrichloroacetic-EDTATrichloroacetic-phosphate-paraquat

Organic solventsAcetoneAcetone-trichloroacetic acid-etherN-Bromosuccinimide-EDTAFornamideDichloromethaneMethanolButanol-EDTAButanol-octanolChloroformChloroform-EDTAChloroform phosphate

Dimethyl sulfoxideDimethyl sulfoxide-sulfuric acidEthanol-EDTA

Inorganic basesPotassium hydroxide-EDTA

pH 7.75, 20 mM, 1000CpH 7.75, 20 mM-2 mM, 100°CpH 7.4, 100 mM-10 mM-10mM-6%, 1000C

pH 10, 10 mM-5 mM, 1000CpH 8.5, 100 mM, 1000CpH 7.7, 65 mM, 1000CpH 7.7, 40 mM-20 mM, 1000CpH 7.5, 25 mM, 1000CpH 5 to 8, 55 M, 1000C

0.1 N, 200C0.4 M, 00C2.3 M-67 mM, 00C1 M-48 mM, 40C0.6 N, 40C0.6 N-48 mM, 40C0.6 N-Na+ resin, 40C0.6 N-Na+ resin-5 N, 250C1.2% (vol/vol)-0.75 M-0.075 M1 to 2 N-62 mM, 40C

1 M, freeze-thawpH 3, 2 M, 00C0.46 M-2 mM, 00C5 to 10% (wt/vol), 00C0.5 M-17 mM, 00C0.5 M-0.25 M-0.1 M, 40C

90% (vol/vol)90%-10%-100%10 mM-10 mM, 25°C10%, 250C90%, 250C100%, 250C25% (vol/vol)-15 mM, 200C50 to 90% (vol/vol)-100%23% (vol/vol), 980C100%-100 mM, 200CpH 11.7, 23% (vol/vol)-10 mM,250C

90% in 50 mM TRIS90% in 0.1 N H2SO4pH 7, 96% (vol/vol)-4 mM,780C

10 mM-2 mM, 1000C

146228, 25450, 228

3212440406519

25819

22817613518221088

Christensen and Devolb65

24419

19219

228161

585823019619619662

83, 28478

228321

210210

300, 228

228a Abbreviations: TRIS, tris(hydroxymethyl)aminomethane hydrochloride; EDTA, ethylenediaminetetraace-

tic acid; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.b R. Christensen and A. Devol, submitted for publication.

niques do not allow for a reliable assessment ofall possible sources of interference, since theinstrumentations and methodologies used fordetecting the labeled ATP and the total ATPextracted from the cells are not the same. Thetheory and application of internal ATP stand-ards have been thoroughly reviewed by Strehler

(304) and will not be further discussed in thisreview.Measurement. Presently, there are a variety

of methods available for the quantitative deter-mination of cellular nucleotides. ATP has beenthe most frequently measured nucleotide be-cause of its central role in cellular energetics and



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TABLE 2. Summary of comparative studies to optimize ATP extraction from microbial cultures andenvironmental samples

MethodsaSample material Comments Reference

Tested Recommended

Activated sludge TRIS Gly-EDTA Selected because it resulted in 321

Activated sludge

Algal cultures

Algal cultures

Algal cultures and naturalwater samples

Aufwuchs communities

Gly-EDTAHC104AcetoneDMSOCHCh3-GlyCHCl3-PO4

BuOHHC104TCATRIS

Acetone-TCA-EAcetoneTRIS

H20TRISGly-EDTAEtOHTCAHC104

DMSOTRISBuOH-OcOH

TRISAcetoneAcetone-TCA-E

TRIS

complete extraction and nohydrolytic loss of ATP

Selected on the basis of simplic-ity, reproducibility, and ATPyield

Acetone-TCA-E Acetone gave higher ATPyields than did TRIS

TCA or HC104 HC104 and TCA yielded thehighest ATP/ADP and ECAratios; however, the variabil-ity between methods was spe-cies dependent

DMSO

Acetone-TCA-E

All three techniques wereequivalent with regard toATP yield, but DMSO wasselected due to convenience

Gave most consistent extractionefficiencies; ATP yields werenot significantly different be-tween TRIS and acetone-TCA-E

219

58

205

286

58

Bacterial cultures

Beach sand

Copepods (2 species)

TRIS-EDTAKOHTRIS-arsenate-EDTA-BuOHEtOHBuOHCHC13HCOOHH2SO4TCAHC104

TRISNaHCO3H2SO4-EDTA

TRISH2SO4-EDTA

TCA Evaluation based upon ATPand total adenylate yield andECA; however, TCA was notuniversally acceptable for allspecies

H2504-EDTA Evaluation based upon a com-parison of ATP, total adenyl-ates, and ECA

H2SO4-EDTA Evaluation based on a compari-son of ATP, total adenylates,and ECA

228

177

177

Escherichia coli cultures

Escherichia coli cultures

HC104TCAHCOOHCH3COOHH20

CH3COOHHC104TCA

HC104

CH3COOH

"Weaker" organic acids requirelonger periods for extractionand result in lowered ATPyields

Highest yield of NTPs

19

244

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MICROBIOL. REV.748 KARL

TABLE 2. ContinuedMethodsa

Sample material Comments ReferenceTested Recommended

Lake sediments HEPES H2S04-Ox Oxalate precipitate removed 65inte2rm O"i k1U

Lake sediments

Lake sediments

Marine and freshwatersediments

Marine sediments

Marine sediments

Marine sediments

NaHCO3H2SO4-IEH2S04-EDTAH2S04-Ox

TRISN-bromoDMSODMSO-acidHC104H2S04-IE

H2S04-IEH2S04-CACHCl3-P04

H2504-IE

CHCl3-P04

interfering La-- ancu numlcsubstances and yielded alower coefficient of variationthan did other methods;H2S04-IE, however, resultedin higher ATP yields

Selected on the basis ofrecovery of added ATP; ATPrecovery not related toCaCO3, Fe2O3, organic C,clay, or P-sorption potentialof the sediment

Provided improved ATPrecovery and long-termstability of extracted ATPand resulted in minimalinterference with enzymaticreaction over other methodstested

McIlvaine H3P04-EDTA Selected on the basis of abilityH2S04-EDTA to repress residual alkalineH3P04-EDTA phosphatase activity which is

commonly encountered inenvironmental nucleotideextracts and on acomparative evaluation ofATP and AT yields, ECA, andGTP/ATP ratios

TRISNaHCO3BuOH-OcOHHC104H2S04

TRISNaHCO3McdvaineSorensen

TRISH2S04-IEH2S04-CA

NaHCO3

Mclvaine

H2S04-CA

H2S04 gave the highest ATPyield, but a high coefficient ofvariation

Gave high recoveries of ATPfrom sediments and seawater;extraction at mildly alkalinepH also avoids problems withhigh cation concentrations inextracts

Efficiency of ATP recovery wassuperior with CA purificationafter H2504 extractionbecause ionic interferencewith the luciferase reaction orATP precipitation uponneutralization of acid extractswas avoided

210

321

176

24

40

135

Marine sediments H2504Triton X-100-H3P04-H2S04

TRISInjectionSonicationHomogenization

H2S04-EDTAInjectionSonicationHomogenization

Triton X-100-H3P04-H2S04

H2S04-EDTA,homogenization

Triton X-100-H3PO4-H2504resulted in higheruncorrected yield of ATP butafter internal standardcorrections, there were nostatistical differences in ATPyield

Evaluation based oncomparison of ATP, totaladenylates, and ECA

Metazoa

Christensenand Devolb

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TABLE 2. ContinuedMethodsa

Sample material Comments ReferenceTested Recommended

Rumen fluids TRIS H2SO, H2SO4 was the most efficient 104CHC13NaHCO3-CHCh3H2SO4

Seawater and bacterial-algal HNO3cultures TRIS

Soil TCAH2SO4-IE-EDMSOHC104TRIS

Soil HCOOHN-bromoTRISBuOH-OcOH

Soil HC104TRISDMSO-H2SO4DMSON-bromoH2SO4BuOH

Soil H2SO4TCATCA-PO4TCA-PO4-paraquat

H2SO4-IE-E

and was better suited forextracting particulatesamples

HNO3 best for bacteria, TRISbest for algae; the twotechniques are comparablefor most seawaters

TRIS ATP was less than 3% ofthe H2SO4-IE-E ATP yield

BuOH-OcOH Selected on basis of highestATP yield

H2504

TCA-PO4-paraquat

BuOH and H2SO4 both yieldedthe highest extractionefficiency, but H2SO4 isroutinely used because it ismore rapid and convenientfor processing large numbersof samples

Paraquat binds to clay andcompetes with ATPadsorption sites

Demingc

88

62

13

161

Spiroplasma citri cultures

Urinary tract pathogens

H2S04NaHCO3NaHCO3-CHCl3

TRISHC104TCA

CHC13MeOHEtOHDMSOFormamideBuOHCH2CI2AcetoneHC104HNO3

NaHCO3-CHCla CHC13 prevented adsorption ofATP to sediment particles

TCA

Acetone or HNO3

TRIS yielded greater ATPvalues but lower ECA thanthe acidic procedures

Acetone and HNO3 wereequivalent with regard toATP yield

255

278

196

a See Table 1. Abbreviations: TRIS, tris(hydroxymethyl)aminomethane hydrochloride; Gly, glycine; EDTA, ethylenedia-minetetraacetic acid; BuOH, butanol; DMSO, dimethyi sulfoxide; TCA, trichloroacetic acid; OcOH, octanol; E, ether, EtOH,ethanol; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; IE, ion exchange; OX, oxalic acid; CA, charcoaladsorption; N-bromo, N-bromosuccinimide; MeOH, methanol.

'R. Christensen and A. Devol, submitted for publication.cJ. Deming, unpublished data.

relatively high intracellular concentrations(compared with the less abundant cellular nu-

cleotides). ATP can be separated and detectedby various chromatographic, radioisotopic, and

enzymatic techniques; however, the firefly bio-luminescence method is the most rapid, sensi-tive, and reproducible assay. As a result of theseattributes, the firefly bioluminescence assay for

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MICROBIOL. REV.

ATP has become the most widely acceptedmethod for ecological studies.

(i) Firefly bioluminescence. The lightemission reaction catalyzed by firefly luciferase(EC 2.7.-),

ATP + reduced luciferin + 02firefly luciferase IAMP

+ inorganic pyrophosphate + C02

+ product + light

has been studied extensively since the pioneer-ing work by McElroy in 1947 (238). Excellentreviews describing the purification of firefly lu-ciferase and its physical and chemical propertiesand reaction mechanism have been publishedrecently (71, 74).Although the essence of the firefly biolumi-

nescence assay ofATP has remained unchangedsince its first description by Strehler and Totter(305), several minor modifications have beendescribed for specific applications. Of particularinterest are the preparation, handling, and useof the luciferin-luciferase reagents. In general,the enzyme preparations can be divided into twocategories: crude and "purified." The term pur-ified as used in this context does not necessarilydenote a crystalline enzyme preparation. Puri-fied reagents can be prepared by ammoniumsulfate precipitation and adsorption onto cal-cium phosphate gels (116), Sephadex G-100 gelchromatography (268), diethylaminoethyl-Sephadex chromatography (29), or isoelectricfocusing (227), or they may be purchased com-mercially from several sources. Purified reagentsare generally more sensitive and specific, but aremuch more expensive to use. Increased sensitiv-ity in the ATP bioluminescence assay is of con-siderable importance in many environmental ap-plications (165, 178); however, the choice of re-agents is generally dictated by the specific ap-plication. Commercially available crude lucifer-ase reagents have been used most extensively inecological studies (>90% of the published data).Additional analytical considerations with re-spect to ATP measurements, including (i) therelative merits of peak height versus integratedlight measurements (169, 174), (ii) luciferase en-zyme reaction specificity (71, 72) and kinetics(73), (iii) computer-assisted data reductionmethods (31, 91, 188, 273), (iv) automated meth-ods of ATP analysis (122, 162, 259), and (v)general limitations and trouble-shooting of themethod (148, 181), have been discussed else-where as referenced and will not be consideredin this review.

In addition to direct measurements of ATP,the firefly luciferin-luciferase bioluminescenceassay can also be used to quantify additionalcellular nucleotides which can be enzymaticallycoupled to the production or hydrolysis of ATP.Many other intracellular metabolites and en-zyme activities which are unique to, or specifi-cally associated with, nucleotide metabolismmay also be assayed with firefly luciferase. ADPand AMP are frequently measured after conver-sion to equimolar concentrations of ATP (48,180, 264). Recently a method has been describedfor the quantitative determination of guanosinenucleotides (guanosine 5'-monophosphate, gua-nosine 5'-diphosphate, and GTP) by the fireflyreaction (170, 172). This technique is based uponselective enzymatic hydrolysis of ATP and uri-dine 5'-triphosphate in cell extracts, followed byquantitative determination of GTP based onATP-generated light emission resulting from anucleosidediphosphate kinase-firefly luciferasecoupled reaction i.e.,

GTPnucleosidediphosphate kinase

ATP firefly luciferase light

Guanosine 5'-diphosphate and guanosine 5'-monophosphate may also be coupled to GTPproduction, and hence to firefly luciferase lightemission, by using enzyme reactions analogousto those described previously for the coupledphosphorylation of ADP and AMP (180). ThisGTP assay procedure may also be used for mea-suring uridine 5'-triphosphate concentrations inmixed nucleotide extracts or for cytidine 5'-tri-phosphate, inosine 5'-triphosphate, and thymi-dine 5'-triphosphate analyses in chromatograph-ically purified fractions, although it was not spe-cifically developed for these applications. Anexample of this latter application has been de-scribed by Manandhar and Van Dyke (232).Cyclic mononucleotides (cAMP and cyclic gua-nosine 3',5'-monophosphate) and the activitiesof cyclic nucleotide phosphodiesterases havebeen measured by using appropriate firefly lu-ciferin-luciferase coupled assay systems (98). Fi-nally, recent work in our laboratory has indi-cated that the firefly bioluminescence reactionmay also be used to assay the concentration ofthe sulfur nucleotide adenosine 5'-phosphosul-fate in sulfate adenyltransferase-firefly lucifer-ase coupled reaction (D. B. Craven and D. M.Karl, unpublished data).

(ii) Bacterial bioluminescence. Bacterialbioluminescence also provides a unique systemfor the quantitative determination of specificmetabolites and enzyme activities that are cou-

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pled to these metabolites. Bacterial luciferaserequires reduced flavin mononucleotide (re-duced FMN) in addition to several other reac-tants. Under suitable reaction conditions, therate of light emission is proportional to the con-centration of reduced FMN (299). Most com-mercial and laboratory preparations of bacterialluciferase also contain NADH:FMN oxidoreduc-tase (dehydrogenase) activity, in addition to lu-ciferase, thereby extending the versatility of theassay to include quantitative determinations ofNADH, NADPH, and FMN (299). The specificprocedures for sample and enzyme preparations,detection of light emission, and general analyti-cal considerations have been described by De-Luca (74). Chappelle and Picciolo (51) have alsodevised a technique for the determination offlavin adenine dinucleotide which requires hy-drolysis to FMN before the bacterial biolumi-nescence reaction.

(iii) Renilla bioluminescence. The discov-ery that 3',5'-diphosphoadenosine is required forlight emission in the bioluminescence system ofthe anthozoan Renilla reniformis has led to thedevelopment of a sensitive and specific quanti-tative assay for 3',5'-diphosphoadenosine andadenosine 3'-phosphate-5'-phosphosulfate. Thedescription and application of these methodshave been published (5).

(iv) Immobilized enzymes. In recent years,the technology required for the immobilizationof soluble enzymes and intact viable bacterialcells onto solid supports has advanced tremen-dously. The immobilization of an enzyme gen-erally results in an increased level of stabilityand yields the advantage of recycling while re-taining its inherent substrate specificity, sensi-tivity, reaction kinetics, and general usefulnessas an analytical tool (158). Methods for theimmobilization of firefly and bacterial luciferasesonto arylamine-glass beads (which can then becemented onto glass rods) have been devisedand applied to detect low concentrations ofATP,NADH, and NADPH (119, 157, 211). The newimmobilized systems offer a rapid and inexpen-sive biochemical assay technique and will un-doubtedly be selected for future ecological ap-plications.

NUCLEOTIDE LEVELS IN MICROBIALCELLS: EFFECTS OF GROWTH RATE,CULTURE CONDITIONS, NUTRIENTSTRESS, AND ENVIRONMENTAL

PERTURBATIONSAdenine Nucleotides and Energy Charge

RatiosWithin the past two decades numerous inves-

tigators have attempted to define pool sizes,turnover rates, and concentration ratios of cel-lular nucleotides (and related compounds) inmicroorganisms. The overwhelming majority ofthe published data have emphasized ATP con-centrations (in terms of nanomoles of ATP permilligram [dry weight] of protein or cell N) andadenine nucleotide concentration ratios (i.e.,ATP/ADP, ATP/AMP, or ECA) in bacteria and,to a lesser extent, in eucaryotes. A discussion ofthe occurrence and probable significance ofcAMP, cyclic guanosine 3',5'-monophosphate,guanosine tetraphosphate, and other less abun-dant nucleotides in microbial cells is beyond thescope of this review; however, excellent currentsummaries of the distribution of guanosine tet-raphsphate (292), cyclic guanosine 3',5'-mono-phosphate (113), and cAMP (270) and their in-volvement in metabolic regulation in microbialcells have appeared recently.Although intracellular concentrations of

NTPs are often referred to as "pools," it isessential to bear in mind that the rapid andcontinuous turnover of these molecules (at leastseveral times per second for ATP in bacteria)precludes the existence of a true energy reserve.Therefore, static measurements of ATP poolsand adenine nucleotide ratios are by themselvesinsufficient to fully characterize the rate of cel-lular metabolism. Additional kinetic parametersof catabolism or growth must be used to corrob-orate ATP pool measurements before an assess-ment of total cellular energy flux or an evalua-tion of the efficiency of metabolic energy cou-pling can be made.Procaryotes. Table 3 presents some repre-

sentative ATP levels, ECA values, and carbon-to-ATP (wt/wt) ratios in growing bacteria. Pre-vious reviews, by Chapman and Atkinson (47),Knowles (194), Chapman et al. (48), and Weber(327), have compiled similar data for ATP andECA in microorganisms and higher plant andanimal tissues. The C/ATP ratios will be dis-cussed subsequently in this review.The similarity in the values reported for intra-

cellular ATP pools and adenine nucleotide ratiosfor various bacterial species growing under dif-ferent culture conditions is noteworthy (Table3) in light ofnumerous potential difficulties withATP analysis and interlaboratory differences inexperimental protocol. In general, the ATP poolin exponentially growing cells appears to be reg-ulated around a mean of 2.0 to 6.0 nmol of ATPper mg (dry weight) of cells, regardless of themode of nutrition. Although the ATP pool ap-pears to be constant during logarithmic growth,the absolute value attained is dependent some-what on the specific composition of the culture

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TABLE 3. Representative ATP levels, ECA values, and C/ATP ratios in exponentially growing procaryoticmicroorganisms

ATP(nmol/mgof C/ATpa

~Ref.Species Medium, growth conditions ATP (nmol/mg of ECA C/ATPA er-~~~~~~~~~~~ence

Acetobacter aceti Ethanol-mineral salts, aerobic, 7.6 0.87 130b 18batch

Inorganic salts, constant light,300C, aerobic, batch

Glucose-mineral salts, aerobic,batch (sphere morphology)

Glucose-mineral salts-casein,aerobic, batch (rod morphol-ogy)

Mannitol-mineral salts (N-free)Aerobic, batchAnaerobic, batch

Aerobic, batch

Nutrient broth, aerobic, batch

2.9 0.97 340" 205

3.8-6.7 0.95 149-259" 215

2.2-5.6 179 448b 216

4.8-6.22.0

7.7 0.87

3.3 0.89

128b

195

109

Bacillus sp.

Beneckea harveyii

Beneckea natriegens


Glycerol-mineral salts, aerobic,batch

Succinate-mineral salts, aero-bic, batch

Glucose-mineral salts, aerobic,batch

2.9-9.4 (x= 5.7)

13-16 0.86

7.0 0.90

6.0 0.90

228

105-340" (x = 173) 219

62-76b 186

141 246

165

High light (2.4 mW/cm2)Medium light (0.4 mW/cm2)Low light (0.2 mW/cm2)

Chemostat culturesGalactose-limitedN-limited (galactose)Glucose-limitedN-limited (glucose)

Defined, anaerobic, batch

Ethanol-acetate-bicarbonate,anaerobic

Crotonate-bicarbonate, anaero-bic



4.2-7.84.9-5.03.34.0

5.1-7.25.5-7.05.0-8.55.2-6.7

3.7

0.76 127-2350.82 197-2010.83 247-299

0.91 137-1930.96 141-179b0.87 116-197b0.90 147-190"

267"

241

81

248

5.48 0.79 180"

7.01 0.76 141"

3.5-7.0 0.85 145-281b

4.0 0.76 246"

medium and on coupling conditions of growth(61, 101, 102, 207; Table 3).Studies on the effect of growth rate on the

intracellular ATP pools have produced conflict-ing results. Several investigators have claimed

that the ATP concentration in bacteria (ex-pressed as nanomoles of ATP per milligram ofdry weight) is independent ofthe cellular growthrate (101, 107, 133, 195, 296), whereas othershave indicated a positive correlation between

Anacystis nidulans

Arthrobacter crystallo-poietes

Azotobacter vinelandii

Bacillus brevis

Bacillus cereus

Chromatium strain D

Citrobacter freundii

Clostridium acetobu-tylicum

Clostridium kluyveri

Escherichia coli

Escherichia coli

70

48

79



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VOL. 44, 1980

Species Medium, growt;

Escherichia coli

Escherichia coli

Escherichia coli

Klebsiella aerogenes(K. pneumoniae)

Klebsiellapneumoniae

Photobacterium fis-cheri

Pseudomonas aerugi-nosa

Rhodospirillum rub-rum

Selenomonas ruminan-tium

Staphylococcus aureus

Zymomonas mobilis

Five species of bacteria

Seven species of marinebacteria

Three species of marinebacteria

CELLUL

TABLE 3. Cong

h conditions

Acetate-mineral salts, aerobic,batch



Succinate-mineral salts, aerobic,batch


Glucose-limited, dissolved 02tension (mmHg) of:420220575.33.0

<0.20


Glycerol-complete, aerobic,batch

Bright strain (MJ-1)Inducer (-) mutant (B-61)B-61 plus inducer


Light-saturated, batch

C-limited, continuous culture


Anaerobic, batch, 30°CComplexDefinedMinimalDefined-limiting panothenateMinimal-limiting panothenate

Not given

Continuous culture

Continuous culture

AR NUCLEOTIDE MEASUREMENTS

tinued

ATP (nmol/mg of ECA C/ATP"

4.1 0.77 235

6.1 0.85 162

3.26 (±9.5%)

3.82 (±9%)

2.6-3.8 (X= 3.2)

8.36.65.96.16.53.73.7

2.6

13.813.712.9

3.6

1.8-2.5

303b

258b

0.80

753

Ref-er-ence

226

235

260-379b (X = 308) 219

126

228

0.87

0.97

186

228

394-547b 331

2.3 133

6.9 0.92

3.04 (±10%)3.06 (±14%)7.0 (± 5%)6.2 (±13%)8.9 (± 5%)

1.97 (±10%)

3.0-10.8b ( = 6.9)

1.9-3.9

143b

324b322b141b159b111b

450-500

91-333 (X= 143)

228

207

13

121

250-510b 146

ATP pools and rate of growth (20, 245). Re-cently, Dolezal and Kapralek (81) reported thatthe ATP pool in Citrobacter freundii was inde-pendent of growth rate up to a critical value of

:0.6 of the maximum growth rate (p.,,,), but theATP concentration increased by -50% nearIpn=-Although the precise molar ATP concentra-

llgb149b167b162b152b267b267b

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TABLE 3. Continued

ATP(rumol/mgof ~~~~Ref-Species Medium, growth conditions dryT t)a EC/ATPf er-

ence

Six species of actino- Not given 4.54b 217 (±13%) 13mycetes

Nine species of rumen Batch, exponential 2.2-34.7 (x = 9.7) 28_448b (x = 102) 104bacteria

Percentages in parentheses represent coefficients of variation.b Assumed: organic carbon = 0.50 x dry weight (229).e1 mmHg - 133.3 Pa.

tion in microbial cells is difficult to determinedue to technical problems associated with esti-mating intracellular volumes, most reported val-ues are within the range of 1 to 5 mM (18, 22, 79,295). It appears that the intracellular concentra-tions of adenine nucleotides are probably at ornear saturating levels for the activities of mostrespiratory and metabolic enzymes.Under steady-state conditions of growth,

there is a tightly regulated balance betweenATP utilization and regeneration. Consequently,the rate of ATP turnover should be positivelycorrelated with growth rate (i.e., ATP utiliza-tion), assuming a constant YATP. As the growthrate decreases, however, the proportion of ATPrequired for maintenance increases, and the ex-pected relationship between ATP turnover rateand growth rate deviates from experimental val-ues. By comparison, the turnover rate of thetotal adenine nucleotide pool is positively cor-related with growth, at both fast and slow rates(47), and this pool appears to "turn over" (i.e., isused up in biosynthesis of macromolecules andis regenerated) approximately 30 to 50 times pergeneration (data reviewed in reference 47).The ECA in growing procaryotic cells appears

to be regulated within the well-defined limits ofthe ECA theory. Figure 1 serves to emphasizethat many of the data collected prior to 1970 areprobably unreliable due to improper considera-tions of turnover times and environimental per-turbations. The ECA values of microbial cellshave not increased since 1948, but rather theaccuracy and precision of the methods used tomeasure cellular adenine nucleotides undoubt-edly have improved. Most chemostat studieshave revealed that there is no correlation be-tween ECA and growth rate (81, 311; D. M. Karl,unpublished data), although certain exceptionshave been reported (131; K. P. Ho and V. Munk,Abstr. Annu. Meet. Am. Soc. Microbiol. 1975,033, p. 197). Nevertheless, both in vitro and invivo studies have indicated that ATP-requiringreactions are curtailed at low ECA values (i.e.,ECA C 0.8). Therefore, a positive correlation

0.8

v 0.6

uj0.4

0.2

50 55 60 65 70

YEAR

FIG. 1. ECA (energy charge) values tabulated byChapman et al. (48), plotted against the years inwhich the analyses were reported. The curve drawnis a 3-year moving average ofECA values. The shadedportion covers one apparent standard deviation oneach side of the line. These data emphasize the factthat only recently have proper considerations beengiven to the analytical procedures required for theextraction and analysis ofcellular nucleotides. FromAtkinson (10).

must exist between the growth potential, orfunctional capacity, of a cell and its ECA.Based on a computer simulation model con-

structed from phosphofructokinase activitydata, Goldbeter (114) has proposed that thecellular ECA is maintained at a constant highvalue (>0.9) for most of the time, but periodi-cally (about every 200 s) exhibits a brief drop toa value of 0.5. This oscillatory behavior wouldallow for a greater flexibility in cellular needs byintermittently activating the energy-yieldingprocesses to their maximum potential rates(114). It is unlikely that these ECA transientscan be detected b our current analytical meth-ods, so we must be satisfied with time-weightedaverage values of cellular ECA. Several thoroughdiscussions of metabolic regulation by ECA con-trol have been published (10, 47, 48, 125, 194).Unicellular eucaryotes. Table 4 summa-

rizes selected data from the literature of intra-cellular ATP levels, ECA values, and C/ATP



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ratios for a variety of unicellular eucaryotes.During exponential growth the mean range inATP concentration is between 2.0 to 6.0 nmol ofATP per mg (dry weight) of cells. These intra-cellular concentrations are essentially indistin-guishable from the levels presented above (Ta-ble 3) for procaryotic microorganisms. This sug-gests the occurrence of a similar, or perhapscommon, physiological driving force behind themaintenance of these steady-state ATP pools.Sakshaug (279) and B. L. Hunter (M.S. thesis,

University of Hawaii, Honolulu, 1979) have in-dependently reported a positive and significantcorrelation between cellular ATP levels (ex-pressed as ATP per cell carbon) and growth ratefor two species of marine diatoms under certainculture conditions (Table 4). Nevertheless, sub-stantial interclass and interspecies differenceswere evident, with greater variations occurringat high growth rates (Hunter, M.S. thesis). Fur-thermore, the specific limiting nutrient was im-portant in determining a correlation betweengrowth rate andATP concentrations. In the caseof Thalassiosira fluviatilis, cellular ATP andgrowth rate were positively correlated duringNO3- and PO4-limited chemostat growth condi-tions, but ATP was independent of growth ratein NH4- and light-limited cultures (Hunter, M.S.thesis).Even though the ECA concept has been ex-

tended to considerations of metabolic regulationin eucaryotes, the theoretical assumptions arenot as easily satisfied in eucaryotes as they arein the structurally simpler procaryotes. The the-ory specifies that adenylate kinase be present atall times and that it responds rapidly to disequi-librium conditions. Noda (247) has pointed outthat adenylate kinase is notably absent fromwithin the cellular mitochondrial complex. Thisrequires that the adenine nucleotide translocasereaction be rapid and immediate so as to mini-mize differences between the intramitochondrialECA and the ECA of the cytosol. Klingenberg(191) has indicated that the translocase reactionis specific for ATP and ADP only and that AMPis not affected. There also appears to be a rapidenzymatic transfer of ATP and ADP in plantsfrom the chloroplast to the cytoplasm whichis catalyzed by the phosphoglyceric acid-di-hydroxyacetone phosphate shuttle (128). TheAMP pools of the mitochondria, chloroplasts,and cytoplasm, however, have been shown to bedistinct (191, 282). Nevertheless, it appears thatthe spatially averaged ECA values in exponen-tially growing unicellular microorganisms fitwithin the prediction of the ECA theory. Severalnotable exceptions have been reported, espe-cially the extended viability of yeast and algal

cells at ECA values as low as 0.1 (22, 222). Thesedifferences may be due, at least in part, to theexistence of separate adenine nucleotide poolswithin the highly compartmentalized eucaryoticcells.Metazoa. Table 5 presents the few available

data for ATP levels in microbial metazoans. Onthe average, the steady-state ATP pools aregreater than those reported in unicellular micro-organisms. The correct explanation for this dif-ference is unknown. Karl et al. (177) have sug-gested that it may be related to differences inthe distributions of structural versus living car-bon or to apparent differences in the ATP/non-adenine NTP concentration ratios for these twoheterogeneous groups of organisms. The pre-sumably slower turnover rate (i.e., "metabolicresponse time") of the ATP pools of metazoamay also contribute to a general requirement forelevated steady-state pool levels.The "whole body" ECA ratios of metazoans

also appear to fit into the framework of the ECAtheory, although it is difficult to rationalize theseresults. The conceptual problems of barrier com-partmentation are magnified by the presence ofindividual tissues and organs. Significant differ-ences undoubtedly exist between different por-tions of a multicellular organism as a result ofspecialized functions of individual groups ofcells. Intertissue differences in ECA have beenshown to be significant for different types ofmuscle in both invertebrates and vertebrates(27). Therefore, it may be more meaningful toisolate a specific tissue or organ and to monitorchanges in its ATP levels and ECA values. Thisapproach has been used for the analysis of tis-sues from higher plants, macroscopic inverte-brates, and mammals, but difficulties in excisingtissue or organ samples have precluded its ap-plication to the microbial metazoans.

Environmental Perturbations andNutrient Limitations

Transient states. It is apparent that thesteady-state ATP pools in exponentially growingunicellular microorganisms (procaryotes andunicellular eucaryotes) are poised at a level ofapproximately 2 to 6 nmol of ATP per mg (dryweight) of cell material (Tables 3 and 4). Tran-sient states can be induced by rapid fluctuationsin environmental conditions and by nutrient lim-itation or starvation. These transient decreasesin ATP are generally accompanied by decreasesin ECA, and by a decrease in the total adeninenucleotide pool (i.e., removal of AMP) if thestress is severe. This latter effect is interpretedas a "strategy" used by the cells to maintain ahigh ECA (10, 47). If the cells are maintained

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TABLE 4. ATP levels, ECA values, and C/ATP ratios in representative unicellular eucaryotesATP

Species Medium, growth conditions (nmof/ ECA C/ATP- Referencemg of A CAP eeecdry wt)

AlnaeN03-limited, chemostat; mean forgrowth rates, 0.048-0.0066/h

2.76b 357 (±24%) Hunterc

Cachonina niei

Chlorella fusca

Ditylum brightwellii

Dunaliella marina

Dunaliella tertiolecta

Gonyaulaxpolyedra

Gonyaulax tamarensis

Pavlova lutheri

Scenedesmus quadri-cauda

Skeletonema costatum



Thalassiosira fluviatilis

NH4-grown, deep tankN03-grown, deep tankNutrient starved, deep tank

Turbidostat culture

NH4-grown, deep tankN03-grown, deep tank

N-deficient, batchP-deficient, batch

N03-limited, chemostat; mean forgrowth rates, 0.035-0.005/h

NH4-grown, deep tank

N-deficient, batchP-deficient, batch

N-deficient, batchP-deficient, batchFe-deficient, batch

Exponential growth

N/P ratio of medium1.2 (N-deficient), batch

20 (Fe-deficient), batch310 (P-deficient), batch830 (P-deficient), batch

Maximum batchDialysis culture in NarragansettBay

% of maximum growth rate:95804020105

NH4-limited, chemostat; mean forgrowth rates, 0.039-0.007/h

P-limited, chemostat; mean forgrowth rates, 0.047-0.007/h

Light-limited, chemostat; mean forgrowth rates, 0.037-0.0022/h

under these new environmental conditions andan energy source is available for ATP regenera-tion, the original steady-state ATP levels are

restored, even though the ATP turnover ratesand rates of biosynthesis may be significantlyaltered. These results again suggest that there is

Amphiprora paludosa

4.44b2.86b2.36b

222345417

2.0

4.83b5.33b

1.81b2.86b

4.63b

2.96b

2.02b2.21b

3.08b1.01"0.55b

1.45

0.640.66

0.740.83

307

220

307

274

Hunter'

307

274

280

127

280

279

279

Hunterc

493b

204185

545345

213 (+20%)

333

489447

3201,0001,800

666

95300250490

96230

250500750

1,0001,5002,000

313 (±10%)

454 (+75%)

189 (±14%)

10.4 b

3.29"3.94b2.01"

10.3 b4.29b

3.94"1.97b1.31b1.Ob0.66"0.5"

3.15"

2.17"

5.22"



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TABLE 4. Continued

ATPSpecies Medium, growth conditions (nmol/ ECA C/ATP" Reference

mg ofdry wt)

Thalassiosira pseudo- N-limited, chemostat, growth ratenana of:

0.041/h0.017/h0.0085/h

5.48b3.19b

139180309

256

6 Species of algae

30 Species of marine algae



Exponential growth, batch



Continuous light, batch

6.9b 143 (±11%) 13

286 (+14%) 140

308 (±45%) Laborded

678 (±85%) 146

14 Species of freshwater al-gae

Batch 313 (±35%) 257

FungiCandida utilis N-limited, chemostat

S-limited, chemostat4.3 0.85 229b4.8 0.85 205b

Dictyostelium discoideum

Neurospora crassa

Saccharomyces cerevisiae

Schizosaccharomycespombe

8 Species of fungi

Exponential growth

Exponential growth

6.5

9.0

3.85 0.87 256b

Exponential growth

295

329

263

233 (±9.3) 13a Values in parentheses represent coefficients of variation.b Assumed: organic carbon = 0.50 x dry weight (229).'B. L. Hunter, M.S. thesis, University of Hawaii, Honolulu, 1979.d P. Laborde, Ph.D. thesis, Universite d'Aix-Marseille, Marseilles, France.

an apparent optimum intracellular ATP poolrequired for exponential growth.

In addition to these short-lived and readilyreversible transient effects on cellular ATP poollevels, more permanent and predictable changesin ATP and ECA may also result from (i) exces-sive nutrient limitation and starvation; (ii) tox-icity induced by heavy metals, organic dyes, orbiocides; (iii) antibiotics, (iv) the addition ofuncouplers ofoxidative phosphorylation, and (v)specific cell-cycle-related events.Nutrient limitation and starvation, (i)

Carbon. The effects of nutrient limitation on

the steady-state ATP pools depend upon thenature of the limiting substance and on thespecific culture conditions. For example, ifchemoorganotrophs are cultured under C-lim-ited batch culture conditions, the intracellular

ATP concentrations and ECA values both de-crease upon exhaustion of the limiting organicsubstrate (48). By comparison, cells grown in C-limited chemostats maintain normal ATP poolsand high ECA values (81; Karl, unpublisheddata). Clearly, the chemostat-grown cells are notnutrient limited in the same metabolic or phys-iological sense as are the batch cultures, sincethe cells continue to grow exponentially andtheir intracellular nucleotide fingerprints are

comparable to those of nutrient saturated batchcultures. One serious problem in conductingstarvation experiments in batch cultures is thatthere is no mechanism for assessing the contri-bution of dead or senescent cells to the totalATP, ECA, organic carbon, or dry weight deter-minations. Continuous culture techniques, on

the other hand, select for growing organisms and

Light, 20°CLight, 4°CDark, 20°CDark, 4°C

316

5.48.17.9

14.4

166

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3.15b

183b

122b125"68b

152"


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TABLE 5. ATP levels, ECA values, and C/ATP ratios in microbial metazoansATP

Species Growth stage, wt (nmol/mg ECA C/ATP Referenceof dry wt)

Adoncholaimus thalassophygas 2-14 ug 26.4a 75a 112(nematode)

Anopolstoma viviparum (nema- 2-14 jig 26.6a 74a 112tode)

Calanus finmarchicus (copepod) 0 Days, starvation 8.56b 115 2123 Days, starvation 8.95b 110

Calanus helgolandicus (copepod) Not given 4.93b 200 141

Calanus sp. (copepod) 130 ug of carbon 23.0b 0.93 43-66 177

Labidocera sp. (copepod) 32,g of carbon 37.2b 0.89 26-27 177

Nippostrongylus brasiliensis (nem- Juvenile 0.78-0.85 23atode)

Eight species of zooplankton Variable 18b 0.70-0.89 43 Skjoldalc

Copepod Adult female 10.4b 95 45Copepodite 7.8b 126

Marine nematodesStation 1 2.19 pg 6.33 150 291Station 2 2.83 pg 11.6 85

AssAumed: dry weight = 0.20 x wet weight; organic carbon = 0.10 x wet weight.b Assumed: organic carbon = 0.50 dry weight.'H. R. Skjoldal, unpublished data.

minimize the potential contribution from deador dying cells.

(ii) Nitrogen, sulfur, and iron. Contrary tothe effects observed with carbon (energy) star-vation, depletion of N from the medium eitherhas no effect on the ATP pools and ECA valuesof bacterial cells or results in a slight increase inthe values (79, 80, 246, 326). This increase ac-companying cessation of growth is presumablydue to a decrease in the ATP demand for bio-synthesis coupled with simultaneous ATP pro-duction from the catabolism of endogenous andexogenous reserves.Thomas and Dawson (316) have investigated

the independent effects of N, S, and Fe limita-tions on cultures of the yeast Candida utilis.Their results indicated that cultures grown un-der either N- or S-limited conditions had similarATP pools (4.3 versus 4.8 nmol of ATP per mgof dry weight) and identical ECA values (-0.85);however, in cells cultured under Fe-limited con-ditions, the ATP pool was reduced by approxi-mately 70% (1.5 nmol of ATP per mg of dryweight) and the ECA decreased to -0.6. A re-duction in the number of cytochromes was of-fered as a possible explanation for these results(316). Despite this low ATP pool and ECA, the

cells were able to grow. This result is in apparentconflict with a strict interpretation of the ECAtheory.

(iii) Phosphorus. The nucleotide data re-garding P starvation and P-dependent microbialgrowth are extremely variable, with many ap-parent interspecies and interlaboratory incon-sistencies. A summary is presented in Table 6.With the exception of a few reports (32, 226; B.Hunter and E. Laws, submitted for publication)P limitation has been found to be accompaniedby a significant decrease in the cellular ATPpool, ranging from -50% of the control steady-state level (209, 256, 324) to an extreme of >90%(45, 139, 280).When microorganisms are limited for inor-

ganic phosphate the synthesis of alkaline phos-phatase is derepressed (induced) and rapidlyaccumulates in the cell. Torriani (323) andGaren and Levinthal (110) indicated that underinorganic phosphate limitation this enzymealone may constitute as much as 6% of the totalsoluble protein. Alkaline phosphatase is a rela-tively nonspecific orthophosphoric-monoesterphosphohydrolase and catalyzes the hydrolysisof many different organic phosphate com-pounds, including adenine nucleotides (AMP,



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TABLE 6. Effects ofphosphate concentrations on the intracellular ATP concentrations of microorganismsATP

Species Medium, growth conditions (nmol/ ECA C/ATP Referencemg ofdy wt)

Azotobacter vinelandii P-rich, 12 h, batch 1.7 0.89 580a 324P-deficient, 12 h, batch

N-starved, 7 days, batchP-starved, 7 days, batchP-starved, 10 days, batch

Aerobic, batchC-starvedN-starvedP-starvedExponential growth

0.5

6.9a1.48a1.0a

0.68 1,9720

143667

1,000

2.6 0.78 37902.0 0.75 493a2.9 0.67 3406.1 0.85 1620

139

226

Monochrysis lutheri

Pavlova lutheri

Peridinium sp.

Scenedesmus quadricauda

Selenastrum capricornutum


Thalassiosira fluviatilis

Thalassiosira pseudonana

N-starved, 7 days, batchP-starved, 7 days, batchP-starved, 13 days, batch

P-saturated, exponential growthP-starvation, batch

P-saturated, batchP-deficient, batch

P-saturated, exponential growthP-deficient, batch

P-rich, 4 days, batchBalanced, 4 days, batchP-deficient, 4 days, batch

P-saturated, exponential growthP-starvation, batch

P-limited, chemostat; growthrate of:

0.047/h0.038/h0.032/h0.027/h0.022/h0.013/h0.011/h0.007/h

x for all growth ratesN-limited, chemostat

P-limited, chemostat; growthrate of:

0.041/h0.017/h

1.18a0.50a0.3a

3.08ao.1a

3.3a

0.42a

1.50a0.3a

3.43.11.4

3.94a0.25a

5.51a3.45a

3.25a1.28a1.87a1.08a0.70a1.33a2.3a3.05a

1.3a0.47a

8332,0003,333

3209,500

2972,374

6673,333

290a318a704a

2504,000

179286303769526909

1,429740

0.84 427323

7802,088

a Assumed: cellular carbon = 0.50 x dry weight (229).b B. L. Hunter, M. S. thesis, University of Hawaii, Honolulu 1979.

ADP, and ATP). Moreover, the enzyme is ex-

tremely heat and acid stable and can easilysurvive most of the standard methods for ex-

tracting cellular ATP. This source of analyticalinterference has been shown recently to be the

cause of at least one set of aberrant ATP data(186). The occurrence of alkaline phosphatase inenvironmental samples and its detrimental ef-fects on measured microbial nucleotide levelshave recently been discussed (176). One labora-

Dunaliella tertiolecta

Escherichia coli

139

280

45

127

209

280

Hunterb

256

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MICROBIOL. REV.

tory investigation conducted in full awareness ofthis potential source of interference (Hunter, M.S. thesis) indicated that the ATP pools in P-deficient algal cells are also growth rate depend-ent (Table 6) and that the intracellular ATPpools ofP-deficient cultures may actually exceedthe pool levels in NO3- and light-deficient cul-tures at high growth rates.

Nonadenine Nucleotide TriphosphatesMany important cellular reactions, notably

those associated with microbial biosynthesis andgrowth, are coupled to energy derived fromNTPs other than ATP. For example, GTP anduridine 5'-triphosphate are both required for theactivation and interconversion of carbohydrateprecursors for polysaccharide biosynthesis;cytidine 5'-triphosphate, GTP, and uridine 5'-triphosphate are required for RNA transcrip-tion; and the deoxyribose derivatives of cytidine5'-triphosphate, GTP, ATP, and thymidine 5'-triphosphate are required for DNA replication.In addition, GTP is an obligate requirement forthe initiation, the aminoacyl-transfer RNA bind-ing and the translocation processes of proteinsynthesis. Previous calculations of the energyrequirement for bacterial growth suggested thata significant proportion of the total energy fluxmay proceed through nonadenine nucleotidepools (214). Until recently, routine quantitativeanalyses of nonadenine NTPs in cell extractshave not been possible due to the lack of a rapid,sensitive, and specific assay comparable to thefirefly bioluminescence assay of ATP. Conse-quently, there is a relative paucity ofinformationconcerning NTP pools in microorganisms.Unlike the ATP pool, which is maintained at

a relatively constant level independent ofgrowthrate, the intracellular concentrations of non-adenine NTPs fluctuate in direct proportion totheir requirements for biosynthesis (107, 169,296, 297). In retrospect, this relationship mighthave been predicted, at least for cellular GTPlevels, since (i) the number of sites for proteinsynthesis (i.e., the ribosomes) increases as a lin-ear function of growth rate and (ii) the rate ofprotein synthesis does not appear to be limitedby GTP concentrations under normal steady-state growth conditions (26, 64, 199). The intra-cellular GTP pool must increase as the growthrate increases in order to maintain saturatingGTP concentrations at the ribosomes.The possibility exists that each specific set of

nucleotide b,ases (i.e., adenine, guanine, uracil,cytosine, etc.) maintains a characteristic EC in-dependent of the others. Furthermore, the gua-nylate EC may regulate "GTP-powered" biosyn-thetic reactions (and likewise the cytidylate EC

may affect "CTP-powered" reactions, etc.) in amanner analogous to the model discussed pre-viously for ECA control of "ATP-powered" re-actions (10). Based on in vitro nucleosidediphos-phate kinase reaction rate data, Thompson andAtkinson (317) have rejected this notion andconcluded that there exists a functional cascaderegulation between the individual charge ratiossuch that the overall cellular ECA is maintainedat the expense of the nonadenine NTPs. Thispredicts that the ECA ultimately regulates theflux ofphosphate into the nonadenine nucleotidepools. Any decrease in the ECA would tend tosharply curtail the regeneration of nonadenineNTPs, and hence the EC values for guanylate,cytidylate, uridylate, etc., would decrease morerapidly than the ECA under adverse conditions.Experimental observations, however, have indi-cated that there is no functional cascade regu-latory step between ECA and guanylate EC inthe rat brain in vivo (76). More recently, Swedeset al. (310) have investigated the regulatorymechanism of protein synthesis during energy-limited growth of the yeast Saccharomyces cer-evisiae. They found that the changes in ECA andguanylate EC values paralleled each other, withno evidence for the existence of a regulatorycascade effect.

Diel Rhythms and Specific Cell-Cycle-Related Events

Most of the nucleotide measurements re-ported in the literature have been obtained withasynchronously dividing batch or continuouscultures, and consequently the majority of theexperimental data represent time-averaged in-tracellular nucleotide pools and concentrationratios. Few studies have investigated transientstates induced by specific cell-cycle-related met-abolic events or controlled by diel metabolicrhythms.When synchronously dividing cultures of mi-

croorganisms are compared with randomly di-viding cells, many presumably cell-cycle-relatedoscillations are evident in the pool sizes ofNTPsand deoxy-NTPs, ECA values, and respirationrates (85, 223, 263, 316). Numerous studies haveindicated a conspicuous correlation between thecellular levels of NTPs (especially the deoxy-NTPs) and the various stages of the mitoticcycle (84, 100, 154, 156, 189). Regardless of themechanism(s) involved, variations in NTP poolsassociated with mitotic cycles are of a sufficientmagnitude (up to fourfold) that they may rep-resent important metabolic signals for the initi-ation of cell division (84).

Diel rhythms may be divided into two majorcategories: (i) those based on a passive response

760 KARL


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to environmental fluctuations during the 24-hcycle that cease when the organism is trans-ferred to a constant environment (exogenousdiel rhythms) and (ii) those that are entrainedby environmental variations but ultimately re-sult from physiological processes which persistunder constant conditions (endogenous, or cir-cadian, rhythms) (330). When microorganismsare exposed to light-dark cycles, diel rhythmsoccur in many physiological and biochemicalprocesses. Plesner (261) and Jones (166) havesuggested that cyclic fluctuations in intracellularATP levels may be the underlying mechanisimfor the control of all biological clock phenomena.Several investigators have noted a diel period-icity in the ATP pool of algal cells entrained ona light-dark cycle (82, 330; Hunter, M.S. thesis).Weiler and Karl (330) have found that the cel-lular ECA in Ceratium furca was maintained ata near-constant and high value (i.e., 0.8 to 0.9)throughout the 24-h period even though intra-cellular ATP and AT decreased by .50% duringthe dark period. Furthermore, when algal cellswere placed in constant light (82) or constantdarkness (330), the diel pattern continued, sug-gesting an endogenous rhythm in the rates ofATP production and utilization. The importanceof these rhythms in environmental nucleotidedeterminations will be discussed in a subsequentsection of this review (ATP and growth rates).

Nicotinamide and Flavin Nucleotides

The nicotinamide dinucleotides (NAD,NADH, NADP, and NADPH) and flavin nu-cleotides (FMN, reduced FMN, and flavin ade-nine dinucleotide) occur in all living cells. Col-lectively, they function as coenzymes for morethan 300 different cellular reactions, and to-gether with the cytochromes they are responsi-ble for catalyzing the sequential oxidoreductionreactions of electron transport. A summary ofour knowledge of NAD biosynthesis and pyri-dine nucleotide cycle metabolism in microorga-nisms has recently appeared (105).

Direct measurements of intracellular nicotin-amide nucleotides reveal that their total concen-tration is -10 mM, roughly equivalent to theinternal pool of adenine nucleotides. During ex-ponential growth in bacteria the pool ofNAD(H) (i.e., [NAD] + [NADH]) is 10- to 40-fold greater than the NADP(H) (i.e., [NADP]+ ([NADPH]) concentration (4, 168, 224, 236).Significant variations exist in the nicotinamidecoenzyme concentrations of bacteria exhibitingdifferences in their tolerance to, or requirementsfor, oxygen (168, 224, 336) and within a givenstrain as a function of medium composition (39,

70, 236) and growth conditions (236, 283). Rep-resentative data are presented in Table 7.Anderson and von Meyenburg (4) have re-

cently investigated the catabolic reductioncharge ratio [i.e., NADH/NAD(H)] and the an-abolic reduction charge ratio [i.e., NADPH/NADP(H)] in E. coli. Both ratios were found tobe constant during exponential growth (cata-bolic charge, 0.05; anabolic charge, 0.45) andwere independent of the growth rate (4).

In comparison with the data on nicotinamidenucleotides in microbial cells, relatively few dataare available on the intracellular pools of flavinnucleotides. Chappelle (49) has reported thatthe "total" flavin [i.e., FMN, reduced FMN, andFAD] pool may range from 0.2 x 10-10 to 65 x100-1,ug per cell in a variety of bacterial species.The highest concentrations were found in Fla-vobacterium arborescens and Bacillus globigii,and the lowest were found in Spirillum serpensand Staphylococcus aureus (49). Robrish et al.(272) found a much smaller range for represent-ative oral microorganisms. Most of the totalcellular flavin was associated with protein (49).To date, neither pyridine nor flavin nucleotide

measurements have been applied to ecologicalstudies despite the ubiquitous occurrence ofthese nucleotides in living cells and their well-recognized and obligatory functions in cellularmetabolism.

NUCLEOTIDE FINGERPRINTS INNATURE

Detection of LifeRapid and reliable detection of sparse popu-

lations of microorganisms is a requirement formany basic and applied investigations. Althoughthe possibility of abiogenic synthesis of ATPcannot be discounted (262, 339), it is generallyagreed that there is an obligate association be-tween ATP and living organisms (152). Levin etal. (217) first described the use of the fireflybioluminescence assay of ATP for detecting thepresence of viable microorganisms. Levin andHeim (218) and MacLoed et al. (230) subse-quently proposed the use ofATP measurementsfor detecting extraterrestrial life.The detection of life in extreme habitats, how-

ever, is not restricted to exobiological investiga-tions. Azam et al. (14) have used ATP measure-ments to detect sparse populations of microor-ganisms living beneath the permanent Ross IceShelf in Antarctica. A recent report by Siegel etal. (290) revealed the presence ofATP and otherlife-associated compounds in Don Juan Pond,Antarctica, a previously suspected "abiotic"habitat. Karl et al. (187) have used ATP mea-

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TABLE 7. Nicotinamide nucleotides in microorganisrnmol/mg of dry wt Reduced/oxidized Ref-

Species Medium, growth conditions NADP- NADH/ NADPH/ er-Species ~~~~~~~~~~NAD(H)(H A APences

Bacilus cereus Dormant spores 0.075 0.004 <0.02 <0.3 2836-min germination 0.198 0.104 0.26 2.2

Minimal, acetate, aerobicMinimal, glucose, aerobic

Minimal, aerobicComplex, aerobic

Dormant sporesLog phaseStationary phase

Dormant spores65-min germination120-min germination

Ethanol-acetate-bicarbonate, anaerobicCrotonate-bicarbonate, anaerobic

Minimal, glucose, aerobicMinimal, glucose, anaerobicComplete, glucose, aerobic

Minimal, glucose, aerobicPlus nicotinic acidPlus tryptophan

Minimal, glucose, aerobicMinimal, succinate, aerobic

Lactate-limited, chemostat; growth rateof:

0.5/h0.25/h0.08/h

NH4-limited, chemostat; growth rate of:0.5/h0.25/h0.08/h

Complex, glucose, aerobicComplex, glucose, anaerobic

Glucose-glycine-glutamate, aerobic

2241.66 1.421.14 0.12

0.56 0.11 2241.52 0.24

0.108 0.018 <0.02 <0.062.31 0.958 0.18 1.20.51 0.515 0.03 1.7

0.072 0.016 <0.04 <0.050.026 0.060 0.36 1.10.043 0.073 0.26 1.2

39.828.2

5.90 0.27 1.424.00 0.32 1.57

283

283

70

2.44 0.24 2242.67 0.581.98 0.48

2.60 0.40 0.53 1.863.20 0.40 0.60 2.083.65 0.55 0.62 2.23

0.19 0.040.27 0.03

1.73.755.6

3.53.63.8

0.830.970.83

0.761.240.82

8.26 0.376.70 1.12

0.89 0.08

39

224

236

224

224

surements to support arguments for the produc-tion of organic carbon at deep-sea hydrothermalvents and have discussed the ecological signifi-cance of this primary production.

Microbial biofouling of introduced surfaces inaquatic ecosystems is a well-known phenome-non. Natural waters are used by many industriesfor cooling and other heat exchange purposes.The presence and succession of fouling micro-organisms significantly reduce the efficiency andthermal exchange capacities of these systems. Ithas been estimated that a 25- to 30-,um-thicklayer of microbial cells may reduce the efficiencyof heat exchange in ocean thermal energy con-

version plants to a point beyond which the gen-

eration of energy by temperature differentials is

no longer economically feasible (63). Aftring andTaylor (2) have recently demonstrated the fea-sibility ofATP measurements for evaluating thepresence and rate of increase of microbial bio-fouling.ATP measurements have also been useful for

detecting bacteria and other microorganisms in

pharmaceuticals and domestic and industrial so-

lutions and for the routine monitoring and qual-ity control ofpresumed sterile products. Piccioloet al. (260) have described a semiautomated in-line flow system for ATP detection in wastewa-ter effluents and potable water supplies. TheATP assay procedure has also been adopted bythe food processing industry for assessing theeffectiveness of various preservation techniques

Bacillus cereus

Bacillus megaterium

Bacillus megaterium

Bacillus subtilis

Clostridium kluyveri

Escherichia coli

Neurospora crassa

Pseudomonas fluorescens

Pseudomoncs ap.

Saccharomyces cerevisiae

Streptomyces griseus



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and storage conditions in addition to the routinemonitoring of product contamination and spoil-age (284).

Clinical detection of bacterial contaminationof blood, urine, and other physiological fluids(75, 117, 318) and quantitative methods for eval-uating the susceptibility of microorganis toselected antibiotics (6, 117, 212) have both beenadvanced through the use of ATP measure-ments. In addition to the extremely high levelsof sensitivity and reproducibility of the ATPassay, its application precludes the necessity fortime-consuming culture techniques (usually 24to 48 h) and therefore provides the means formore rapid diagnosis and treatment. Lee andCrispen (212) have demonstrated an excellentagreement (-100%) between the ATP index ofdrug susceptibility testing and conventionalplate count methods, and Gutekunst et al. (117)have also reported a close agreement (>97%)between ATP concentrations and bacterial col-ony counts of 538 clinical urine specimens. ATPmeasurements have also been used to detect thepresence of debilitated, but viable, coliforn bac-teria (69). The inability of stressed or otherwiseinjured cells to grow on diagnostic culture mediaemphasizes the importance and need for alter-nate methods for detecting the presence ofviableand potentially infectious bacterial cells in clin-ical and applied ecological investigations.

Adenosine 5'-Triphosphate (ATP) as aBiomass Indicator

The necessity for quantitative determinationsof total biomass (i.e., living) carbon is a primaryconcern in nearly all ecological studies. Quanti-tative assessment of the total standing stock ofviable microorganisms in environmental sam-ples, however, has proven to be "surprisinglydifficult" (306). In 1966, Hohm-Hansen andBooth (146) proposed the use of ATP measure-ments as a means of estimating total biomasscarbon. Since that time more than 400 papershave appeared in the scientific literature describ-ing specific ecological applications of the ATP-biomass methodology.Four important assumptions are inherent in

the application of this method: (i) all livingorganisms contain ATP, (ii) ATP is easily ex-tracted from microbial assemblages and can beprecisely measured, (iii) ATP is neither associ-ated with dead cells not adsorbed onto detritalmaterials, and (iv) there exists a-fairly constantratio ofATP to total cell carbon for all microbialtaxa independent of metabolic activity or envi-ronmental conditions. Of these assumptions,only the first is unequivocally acceptable. Thesecond assumption is valid under certain exper-

imental conditions which have been discussedabove (Analytical Procedures). The third andfourth assumptions are discussed below.Obligate association of ATP with living

organisms. Several important analytical con-siderations have surfaced in recent years whichhave questioned the original assumptions of theATP-biomass technique. Chappelle et al. (53,54) have reported that viable cell counts de-creased rapidly in batch cultures of E. coli cellswhich had entered the death phase, whereastotal ATP decreased only gradually. They con-cluded that a portion of the total ATP mustoriginate from "dead" (i.e., either nonviable ordebilitated) cells. Furthermore, they suggestedthat the contribution of ATP from nonlivingcells present in environmental samples will de-pend upon the specific cause of death or celldamage, as well as the chemical and physicalnature of the fluid environment. This potentialanalytical interference of ATP associated withnonliving cellular materials should be consideredas a possible source of error in environmentalnucleotide determinations.

In 1973, Koenings and Hooper (197) examinedthe distribution and chemical nature of dissolvedorganic phosphorus compounds collected fromthe interstitial waters of a northern Michiganboglake. Their studies revealed the presence ofhydrolysis products of RNA, as well as freemono-, di-, and triphosphate nucleotides (197).J. W. Deming, D. A. Nibley, and H.Okrend (Abstr. Annu. Meet. Am. Soc. Microbiol.1976, Q48, p. 198) have also reported the pres-ence of soluble (i.e., cell-free) ATP at severalstations in the GulfofMexico. Azam and Hodson(15) confirmed the ubiquitous occurrence of dis-solved ATP (D-ATP) in the marine environ-ment. From the results of carefully designeddialysis bag experiments, they demonstratedthat D-ATP was not an artifact of sample prep-aration or filtration, but was in fact ATP whichwas freely dissolved in the seawater. B. Riemann(unpublished data) has subsequently reportedthis phenomenon in freshwater ecosystems andhas additionally demonstrated significant sea-sonal variations in the steady-state concentra-tion of D-ATP. Clearly, the original assumptionconcerning the obligate association ofATP withliving organisms must be modified. The concen-tration of D-ATP (i.e., <0.2 ,um) in aquatic en-vironments may constitute up to 75% of the"total" ATP in freshwater lakes (Riemann, un-published data) and over 90% of the total ATPin certain marine ecosystems (15). For thesereasons, Karl (173) has cautioned investigatorsagainst the use of direct injection methods forextracting aqueous environmental samples.

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Although the presence of D-ATP in aquaticenvironments is now well documented, the originof the soluble nucleotides is unknown. Labora-tory studies with batch and continuous culturesof microorganisms indicate that cellular RNA isdegraded to oligonucleotides, nucleotide mono-

phosphates, nucleosides, and free bases understarvation conditions. Most of these degradationproducts are subsequently released by the cells

into the medium; however, intracellular nucleo-tide diphosphates and NTPs are rarely, if ever,excreted (60, 86, 186, 216). Therefore, it is un-

likely that excretion by viable cells is the source

of D-ATP in natural waters. More likely, theintracellular ATP is released by feeding preda-tors and also by the production and dissolutionof feces. Lampert (201) has recently shown thatup to 17% of the algal carbon which is ingestedby the cladoceran Daphnia pulex is lost as dis-solved organic carbon during grazing.Although the pool of D-ATP in many marine

and freshwater environments appears to be sig-nificant, it remains to be demonstrated whetheror not this dissolved pool is adsorbed onto sus-

pended particulate materials. Since the particu-late materials from most environmental samplesmust be concentrated before extraction andanalysis of the cellular nucleotides, the contri-bution from D-ATP should be minimal, even

though it may be the most abundant form ofATP. However, if ATP is adsorbed to, or con-

centrated by, nonliving particulate organic ma-

terials, then the ATP concentrations which arepresumed to be associated with living microbialcells will be grossly overestimated.Holm-Hansen (141) dismissed the possibility

that nonliving particulate materials contributeto the total ATP content of a filtered watersample on the basis of the following experimen-tal data: (i) if suspensions of phytoplankton wereheated to 60°C for a few minutes or frozen at-20°C for a few hours, no ATP could be de-tected in the cellular materials; (ii) after autol-ysis of the flexibacterium Saprospira sp., thetotal particulate ATP decreased to undetectablelevels; and (iii) if ATP is added to a representa-tive seawater sample which has been previouslyfrozen and thawed, no significant amount ofATP is recovered in an extract of the particulatefraction. These results, in particular the datafrom the latter experiment, suggest that, underthe laboratory conditions specified, D-ATP isnot associated with organic detrital materials.Nevertheless, due to the ubiquitous occurrenceand relatively high concentrations of D-ATP inmany aquatic environments, additional experi-ments should be conducted with representativeparticulate materials found in a given habitat.

Surface-active suspended materials (especiallyclay particles, soot, and ash), if present, mayreversibly adsorb D-ATP and other soluble nu-cleotides, yielding overestimates of the concen-trations associated with living cells.C/ATP ratios. The most critical and most

widely criticized assumption of the ATP-bio-mass assay is the validity of the application of asingle C/ATP ratio for the purpose of extrapo-lating ATP measurements to estimates of totalmicrobial biomass carbon. Without a doubt, thecommon practice of converting ATP to biomasscarbon loses all the value of the analytical pre-cision of the ATP measurement. From the datapresented in Tables 3 through 6 it is apparentthat the C/ATP ratios of microbial cells varyconsiderably, and somewhat predictably, be-tween individual taxa and even within certainspecies as a function of culture conditions andgrowth rate. The most commonly used conver-sion factor for ecological studies is ATP x 250to derive an estimate of/ total biomass carbon(146). This relationship has been established asthe mean value from data obtained with sevenstrains of marine bacteria (121, 146) and 30species of unicellular marine algae (139) and, forthe most part, has been systematically appliedto diverse ecosystem analyses without furtherverification or critical consideration of its gen-eral validity. Therefore, it is impossible to ascer-tain the accuracy of biomass estimates extrapo-lated from ATP data.Rather than rely on a mean C/ATP ratio or

even consider the range (or extremes) in possibleC/ATP ratios that might occur in microbialcells, it would seem more logical to examine thecoefficient of variation of the C/ATP ratio of agiven species or group of organisms under adefined range of environmental and physiologi-cal conditions. This coefficient of variation willprovide a more reasonable indication of the ac-curacy and reliability of the biomass carbonextrapolations. Skjoldal and Bamstedt (294)have compiled a table of coefficients of variationfor ATP determinations of individual microbialtaxa. The coefficient of variation ranged from±10% for actinomycetes to ±129% for bacteria.They subsequently developed a model to predictthe required miniimum number of species (n) ina sample to have an accuracy of±x% in the ATPdetermination. The equation used was: n = (to.05x coefficient of variation)/x2, where to.06 is theappropriate value from the t-table (294). Consid-ering the species in a particular sample to berandomly selected, values for n at three differentcoefficients of variation and for various values ofx are presented in Table 8. Assuming a value of60% as the coefficient of variation of a mixed

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community of microorganisms, the populationmust be composed of at least 36 species beforetheir mean ATP concentrations attain an accu-racy of ±20% (294). According to this model, thegreater the number of species present, the moreaccurate the ATP-biomass estimate. In practice,however, biomass estimates will be most accu-rate when the natural microbial assemblage ap-proaches a monoculture of a known species andwhen ancillary data are also available on thegrowth rate and nutrient status of a population.To further emphasize the probable magnitude

and significance ofreported variations in C/ATPratios, let us examine a hypothetical environ-mental situation. Imagine that we wish to knowthe phytoplankton biomass at a particular sta-tion in the euphotic portion of the Pacific Ocean.Let us assume that the dominant microorganismis T. fluviatilis, thereby reducing the possibilityof interspecies variations in the C/ATP ratio.Although the ocean is generally considered to beN limited with respect to phytoplankton growth,we cannot discount the existence of inorganicphosphate or light limitation, the latter occur-ring when the depth of the mixed layer exceedsthe 1% light level. Under the conditions speci-fied, the C/ATP ratio may range from 166 to1,250 (Hunter and Laws, submitted for publica-tion), depending upon the limiting nutrient andspecific growth rate. If the growth-limiting nu-trient was known, then the variability of the C/ATP ratio would be reduced to 257 to 286, 250to 417, 179 to 1,250, or 167 to 238 for ammonium,nitrate, phosphate, or light limitations, respec-tively. Finally, if we further assume that theprecise growth rate of the population is known,then the mean and coefficient of variation forthe C/ATP ratio would be further reduced to313 (±10%), 323 (±23%), 455 (±75%), or 187(±14%) for a given growth rate limited by am-monium, nitrate, phosphate, or light, respec-tively (Hunter, M.S. thesis). The residual vari-

TABLE 8. Minimum number of species constitutinga sample required to have an accuracy of ±x% in

the arithmetic mean ATP concentrationa

Degree of ac- No. of species required for a coefficient ofDegreecofmacn variation of:curacy (mean ____________ _____

+x%) 100% 60% 30%

5 1,600 576 14410 400 144 3620 100 36 930 45 16 440 25 9 350 16 6 270 8 3 1100 4 2 1

a From Skjoldal and Bamstedt (294).

ability has been shown to be dependlent upon adiel periodicity in the C/ATP ratio. Althoughthe intent of this hypothetical example was toemphasize the importance of selecting the mostappropriate C/ATP ratio for a particular envi-ronment, it has actually been an example infutility. If we actually knew the precise speciescomposition, growth-limiting nutrient, andgrowth rate of any microbial assemblage in na-ture, it is unlikely that we would desire an esti-mate of the biomass carbon.The original methods for environmental ATP

determinations have been greatly modified andimproved in recent years. Development of newanalytical procedures has, for the most part,proceeded in parallel with acquisition of data.As refined procedures are adopted for generaluse in microbial ecology, there should be a re-evaluation and, if necessary, a new calibration ofthe C/ATP ratio. Since most environmentalstudies have been reported by independent in-vestigators using different methods of samplecollection, extraction, and analysis, caution mustbe exercised in the comparison of biomass car-bon estimates for different environments.

Despite these significant and predictable var-iations in cellular C/ATP ratios, indirect datafrom several independent field- investigationshave indicated that the proposed ratio of -250is a close approximation of the true in situ C/ATP ratio. This is especially true for phyto-plankton-dominated portions of the marine en-vironment where there is no evidence of inor-ganic nutrient limitation. Under such environ-mental conditions, the algal cells would be grow-ing at or near their m, and the C/ATP ratioswould be expected to approach a value of 250(55, 139; E. Sakshaug, Ph.D. thesis, Universityof Trondheim, Trondheim, Norway, 1978). Sim-ilarly, several field studies have made simulta-neous measurements of ATP and total particu-late organic carbon. For the euphotic zone of theocean, an upper limit of 250 to 300 is derived forthe in situ C/ATP ratio (138, 141, 146, 298).

Cavari (45) has reported a range of 200 to5,000 for the C/ATP ratio of plankton popula-tions from Lake Kinneret over an annual cycle,but for the major part of the year it conforms toa 250:1 ratio. The most prominent seasonal fluc-tuation results from a spring algal bloom domi-nated by the dinoflagellate Peridinium. Basedon field and laboratory data, Cavari (45) sug-gested that the increase in C/ATP ratio is cor-related with a phosphorus deficiency in the lake.He further proposed that measurements of thein situ C/ATP ratio may be useful as quantita-tive indicators of P limitation. This relationship,however, appears questionable in light of recent

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data obtained by Karl and Craven (176). Theirresults indicate that certain (presumed inorganicphosphate-limited) microbial habitats containelevated quantities of heat- and acid-stable al-kaline phosphatase activity which catalyzes thehydrolysis of the extracted ATP. This results inan underestimation of the true ATP concentra-tions, an overestimation of the true C/ATP ra-tios, and additional errors in ECA determinationsand GTP measurements.

Paerl and Williams (250) have examined therelationship between the cellular carbon con-tents of microorganisms as determined by auto-radiography and scanning electron microscopyand ATP contents in several freshwater lakes.Their data indicated a consistent C/ATP ratioof 276 (±12), independent of nutrient conditions,species composition, degree of eutrophication, orwater depth (250). The use of in situ dialysiscultures of representative microbial cells, as sug-gested by Sakshaug (279), may prove useful fordetermining reliable C/ATP ratios of individualcomponents of the microbial community. Itshould be emphasized that microbial assem-blages dominated by metazoans will require theapplication of a lower C/ATP ratio due to theirhigher concentrations ofATP per unit of carbonrelative to unicellular microorganisms (Table 5).Correlations ofATP with other measures

of biomass and activity. Although the accu-racy of the ATP-biomass technique has beenquestioned and numerous difficulties in its ap-plication have been cited, it is still by far themost convenient and reliable method for esti-mating total microbial biomass in most environ-mental samples. Biomass estimates based onchemical analyses of particulate organic carbon,particulate organic nitrogen, protein, or nucleicacids cannot be used in ecological studies due tothe preponderance of nonliving organic mate-rials. Direct microscopic enumeration and sizingof individual microbial taxa can be used to esti-mate total living carbon, provided there exists apreviously determined empirical relationship be-tween microbial cell volume and organic carbon.Particle volume may be determined via elec-tronic particle counters; however, there is nomeans by which to distinguish living cells fromnonliving detrital materials. Biochemical indi-ces, such as chlorophyll a (chl a), lipopolysac-charide, and muramic acid, in addition to culturemethods, exist for estimating individual compo-nents of the total microbial assemblage (i.e.,either bacteria or algae).Mel'nikov (240) has compared biomass esti-

mates based on ATP with values obtained fromdirect microscopic examination of bacteria, phy-toplankton, and microzooplankton at several

stations in the Pacific Ocean. Although similartrends were observed in the spatial distributionsof cellular biomass, there were significant quan-titative differences between the two estimates.With the exception of 3 out of a total of 62determinations, the biomass derived from ATPexceeded that calculated from microscopy, thelatter representing from 3 to 27% of the totalATP biomass. It was concluded, and confirmedby electron microscopy, that the occurrence ofultraplankton not detected by light microscopycontributed to this discrepancy between meth-ods.

Sorokin and Lyutsarev (298) conducted a sim-ilar field comparison of direct microscopic andATP techniques for the estimation of total mi-crobial biomass. An excellent agreement wasobserved between the two methods for euphoticzone samples from the southwest Pacific Ocean.The average ratio of ATP-biomass to directmicroscopy biomass was 1.06 (298). They there-fore concluded that the ATP assay can be usedas a rapid measure of total microbial biomass inseawater samples.

J. E. Hobbie and S. W. Watson (Abstr. Annu.Meet. Am. Soc. Limnol. Oceanogr. 1978 p. 18)have compared three independent methods forestimating bacterial biomass in the deep AtlanticOcean off northwest Africa. At one station 12measurements between 150 and 1,800 m yieldedmean biomass estimates of 2.08, 1.96, and 1.70mg of C per m3 for ATP, lipopolysaccharide, andacridine orange epifluorescence microscopicanalyses, respectively. Although each techniquehas its inherent limitations, the excellent agree-ment among the three methods suggested thatthe majority (75 to 96%) ofthe microbial biomassin the aphotic portions of the world's oceans isbacterial and lends further credibility to theindependent application of these biochemicalindices of biomass in the environment.

Several studies have been conducted whereincomparisons of methods for estimating totalphytoplankton carbon have been achieved. In-vestigations of coastal waters off southern Cali-fornia, as well as other geographical areas, haveled to the conclusion that phytoplankton bio-mass estimates calculated from ATP and chl aconcentrations (136, 141), total particulate or-ganic carbon (140, 146), or cell counts (293) wereall in close agreement for the euphotic zone.Sinclair et al. (293) have recently reported anexcellent agreement between ATP-biomass es-timates and phytoplankton carbon calculatedfrom cell counts and volume measurements.They concluded that in high phytoplankton bi-omass areas (i.e., chl a > 10 ILg/liter), ATPdeterminations can be used to estimate total

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phytoplankton carbon (293). However, Eppleyet al. (90) found that the carbon estimates basedon ATP concentrations were generally greaterthan those based on other indirect methods, andin 11 out of 17 groups of stations the ATP-biomass C estimate exceeded the measuredvalue of total particulate organic carbon.

In other ecological studies, significant corre-

lations have been observed between ATP con-

centrations and evolution of C02 from soils (13,337), total dehydrogenase activity in marine sed-iments (252, 253), DNA synthesis in freshwatersediments (1), electron transport system activityin seawaters and lake sediments (164, 313), totallipid phosphate and rates of DNA synthesis inestuarine sediments (332), alkaline phosphataseand phosphodiesterase activities (242), potentialfor hydrocarbon oxidation in oil-polluted har-bors (43), and biological oxygen demand (3, 66).

Selected Environmental ATP ApplicationsPartitioning of bacterial, algal, and

metazoan carbon. The ubiquitous occurrence

of ATP in all living organisms makes it a uni-versal parameter for detecting life, but, ironi-cally, this ubiquity poses a serious limitation onthe ATP-biomass assay. For certain applica-tions, bacteria may be considered to be the sole,or at least the dominant, microorganisms pres-ent (e.g., acid mine wastes, peat bogs, deep-seahabitats, and permanently anoxic environ-ments); however, most naturally occurring mi-crobial communities are composed of a complexassemblage of numerous taxa.

In many ecological studies, especially thoseconcerned with energy transfer and organic pro-ductivity, it is essential to partition the com-

munity into individual trophic compartments.ATP measurements have proved useful for in-vestigations of marine sediments (111, 291, 341)and microplankton communities (42, 150). TotalATP measurements and direct counts of sedi-ment meiofauna were used by several investi-gators to compare the standing stocks of micro-and meiofauna and to estimate the contributionof meiofauna to the total community biomass.Yingst (341) has determined the numericalabundance of various meiofaunal taxa and hasestimated the carbon contribution of each groupin sediment samples from Long Island Sound.She reported that the top 2 cm of sedimentcontained -70% of all of the meiofauna repre-sentatives,with 41% in the top 1 cm (341). Withinthe topmost layer of sediment the meiofaunaconstitute -100% of the total ATP-biomass;however, the cumulative computational errors

were substantial (+50%). Below this depth, ATPcontributed by microorganisms (bacteria and

protozoa) became more discernible. Ernst andGoerke (92) have likewise concluded thatmeiofauna may contribute a large percentage ofthe total ATP in surface sediments from thedeep Atlantic Ocean (250 to 5,510 m). Sikora etal. (291) have described a method for the selec-tive recovery ofnematodes and other meiofaunalgroups from heterogeneous sediment samples.Their results indicated that the proportion oftotal sedimentary ATP contained within thenematode population of littoral marine sedi-ments can vary seasonally from 68% in the sum-mer to >90% in the winter.

Gerlach (111) has recently constructed atrophic model for a hypothetical subtidal, silty,sand marine sediment ecosystem. He reportedthat the distribution of individual componentsof the total sediment biomass yields macroin-fauna (10 g of carbon per m2), bacteria (5 g ofcarbon per m2), meiofauna (1 g of carbon perIn2), foraminiferans (0.5 g of carbon per m2), andciliates plus flagellates (<0.01 g of carbon perm2). Clearly, the species composition and trophicorganization of individual communities are ex-tremely variable, and it is essential to select themost representative C/ATP ratios to evaluatethe biomass of each biological compartment.

Before the extraction ofATP from water sam-ples, prefiltration is generally used to excludelarger metazoans. However, the retention ofchained or clumped unicells and individual cellsattached to larger particulate materials is notprecluded. Should an effective separation of themetazoa be achieved on the basis of size, onewould still be faced with the task of distinguish-ing between the micrometazoans, protozoans,unicellular algae, and bacteria.Holm-Hansen and Paerl (150) have proposed

a method for estimating the relative contribu-tions of bacteria and algae to the total biomasscarbon in aquatic environments. In their studyan attempt was made to estimate bacterial car-bon by calculating the difference between totalATP-biomass carbon and total phytoplanktoncarbon obtained from direct microscopic identi-fication, cell sizing, and enumeration. Their datafrom Lake Tahoe indicated that during thespring bloom, phytoplankton cells represent ap-proximately 80 to 90% of the total biomass car-bon, but in late summer their contribution haddropped to 20 to 50%.

This method of ecosystem analysis based ona two-component model (i.e., bacteria and algae)is not appropriate for all microbial communitiesdue to the presence of protozoa and other micro-zooplankton. Jassby (159) has criticized the useof total ATP and phytoplankton counts to esti-mate bacterial carbon and has developed a sta-

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tistical model to evaluate the reliability of thisapproach. Disregarding systematic errors asso-ciated with the various analyses, he concludedthat the propagation of errors is significantenough to render this method acceptable in onlya very limited number of natural environments.Sorokin and Lyutsarev (298) have proposed a

method for the determination of bacterioplank-ton biomass in the marine environment. Themethod relied upon an empirically derived cor-

rection factor which must be applied to calculatethe contribution of bacteria to total microbialbiomass. The correction factor was equal to theinverse ratio of total ATP-biomass to the bac-terial biomass determined by direct microscopyand varied with the nature of the habitat. Theaverage values were 1.4 (range, 1 to 1.5) for theeuphotic zone of upwelling waters, 2.3 (range, 2to 3) for the euphotic zone of waters with mod-erate productivity, and 4.6 (range, 4 to 7) fordeep waters below the euphotic zone (298). Theaccuracy and usefulness of this approach forgeneral ecological applications are questionable.It may, however, provide a useful measure ofbacterioplankton biomass in specialized habitatswhich do not experience significant temporalcommunity changes (i.e., deep-sea habitats).

Several attempts have been made at a physi-cal separation of individual microbial compo-nents based either on size, by differential filtra-tion (16, 17, 42, 46, 123), or on density, by differ-ential centrifugation (104). Burnison (42) firstsuggested that bacteria and algae may be sepa-rated by Nuclepore filtration techniques. Thisapproach (i) implies nonoverlapping size spectraof the individual microbial groups, (ii) disregardsthe possible association of small microbial cellswith larger particulate materials, (iii) assumes a

nondestructive filtration procedure, and (iv) re-quires that the intracellular ATP pools remainconstant throughout the experimental manipu-lations. It should be emphasized that none ofthe prerequisites is fully satisfied. For example,Burnison (42) found that -18% of the total bac-teria were retained by a 1-,um Nuclepore filter,whereas Azam and Hodson (16) reported thepresence of microflagellates in the 1-,um Nucle-pore filtrate. Azam and Hodson (16) reasonedthat since -90% of the total heterotrophic activ-ity was associated with the <1-gm Nucleporefraction in seawaters from various habitats, mostbacteria must exist in the "free living" (i.e.,unattached) state. However, other data existwhich indicate that high rates of microhetero-trophic activity may be often associated withparticles -3 ,m in diameter (123, 124). Since theratio of free-living to attached bacteria undoubt-edly varies considerably in samples collected

from different environments, the use of size frac-tionation of ATP to assess "bacterial" biomassis not fully justified. Nevertheless, ATP fraction-ation studies have yielded useful informationconcerning the size distribution of living orga-nisms. These studies, when used in conjunctionwith measures of microbial activity and growth,have provided comparisons of the specific activ-ities of various microbial populations (i.e., activ-ity per unit biomass) (42, 123, 143, 234, 237, 288,325).

Ferguson and Rublee (97) have combined ac-ridine orange direct count estimates of bacterialcell numbers, dimensions, and frequencies withATP biomass carbon measurements to estimatethe contribution of bacteria to the total standingcrop of coastal marine plankton. Their estimatesindicated that the mean standing crop of bacte-ria for 19 coastal seawater samples was 5.5 ± 1.9mg of C per m3 and ranged from 4 to 25% of thetotal plankton carbon (mean, 9.8%). A notewor-thy observation was that -80% of the bacterialcells were free-living cocci but composed only-40% of the total bacterial carbon. Moreover,the numerically less abundant (<15%) bacterialcells found in association with particulate ma-terials contained a disproportionate percentageof larger rod-shaped cells and therefore ac-counted for nearly 45% of the total bacterialcarbon. Palumbo and Ferguson (251) have alsoapplied this approach in a study of the NewportRiver Estuary, N.C. They found that bacterialcarbon represented 20 ± 9.5% (range, 7.5 to41.6%, n = 13) of the total living carbon. Inaddition, total ATP was not correlated withbacterial numbers or to the ratio of bacterial Cto total biomass C (251).

Several successful techniques have been de-veloped to differentiate "bacterial" ATP from"nonbacterial" ATP in physiological fluids, suchas urine (75, 117, 318) and blood (53, 75). How-ever, none of these methods has been adaptedfor use with environmental samples. The mostsuccessful approach involves a selective chemi-cal lysis of nonbacterial cells (i.e., eucaryotes)followed by enzymatic hydrolysis of soluble ATPin the extract and subsequent concentration andextraction of bacterial ATP. The method wasdeveloped for the clinical analysis of fluids con-taining high proportions ofmammalian cells andD-ATP, so it is not known whether this ap-proach would be useful for separating procar-yotic and eucaryotic components of naturallyoccurring microbial assemblages. Although thediversity of eucaryotes present in environmentalsamples may frustrate attempts to devise a se-lective chemical lysing agent, chemical separa-tion procedures for partitioning microbial com-

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ponents in natural aquatic environments deservefurther attention and development.ATP applications in oceanography. (i)

Geographical and vertical distributions ofATP in the ocean. Our understanding of basicbiological oceanographic processes, especiallythose regarding food chain dynamics and spatialand temporal distributions of living organisms,has been advanced substantially fromATP mea-surements. Since its first documentation (146),more than 100 papers have appeared concerningspecific applications of ATP measurements inthe ocean. Table 9 summarizes representativedata from selected geographical locations.A "typical" vertical profile ofATP concentra-

tion (versus depth) in the marine environmentis presented in Fig. 2. Several recurrent andtherefore characteristic features of the distribu-tion ofATP in the ocean have been reported. (i)Elevated ATP concentrations are characteristicof the sea-surface interface (0 to 150 ,um). Thisobserved concentration of biomass may be theresult of physical concentrating mechanisms ormay result from microbial growth within thisspecialized habitat. (ii) High concentrations ofATP are present between 0 and 100 m, corre-sponding to the euphotic zone of the ocean.Frequently, a subsurface peak at or near the chla maximum layer is also apparent. The absoluteconcentration ofATP in the euphotic zone variesregionally and is usually significantly correlatedwith phytoplankton biomass and rates of pri-mary production. Typically, surface ATP con-centrations range from >500 ng ofATP per literfor eutrophic waters to 100 to 500 ng ofATP perliter for regions with moderate productivity to<100 ng of ATP per liter for oligotrophic por-tions of the ocean. (iii) Below the euphotic zone,the concentration of ATP is more variable butusually exhibits a rapid decrease with increasingwater depth to a value of <10% of the surfaceconcentration by a depth of 200 to 400 m. (iv)Below 400 m there is a more gradual decrease inATP concentration to minimum values of -0.5to 2 ng ofATP per liter in the deep-sea habitatsexamined thus far, regardless ofthe productivityof the surface waters (Table 9).Although these typical vertical distributions

of ATP have been observed by many investiga-tors, it should be emphasized that intermediate-depth (400 to 1,000 m) ATP maxima occur (130,146, 175, 180, 184; W. Campbell and W. Weibe,Abstr. Annu. Meet. Am. Soc. Limnol. Oceanogr.1978, p. 10), occasionally with concentrations upto 30% of the surface maximna (184). Karl and co-workers (175, 180, 184) have stressed the impor-tance of oceanic discontinuities (e.g., thermo-clines, pycnoclines, ocean fronts, and water mass

interfaces) for the production and maintenanceof these subsurface zones of elevated biomassand activity. However, the organization and re-search objectives ofmost marine microbiologicalinvestigations conducted to date have been suchas to preclude detailed sampling ofocean discon-tinuities, and therefore the few data that do existhave been serendipitous and incomplete. Futurestudies should concentrate on elucidating theroles of oceanic discontinuities in controlling thedistribution, abundance, and metabolic activitiesof the autochthonous microorganisms.There are several notable exceptions to the

generalized vertical profile ofATP in the aphoticzone as described above. Microbiological inves-tigations of specialized marine habitats in Ant-arctica, within permanently anoxic marine ba-sins and at deep-sea hydrothermal vents, haverevealed unique ATP distribution patterns. Thewaters under the Ross Ice Shelf, Antarctica,constitute a unique marine environment for liv-ing organisms due to the absence of an overlyingeuphotic zone resulting from a 400- to 600-m-thick layer of ice. Extremely low levels of ATPhave been detected at all depths from 20 to 200m beneath the ice cover, with typical concentra-tions ranging from 0.04 to 0.6 ng ofATP per liter(14). By comparison, ATP concentrations of sea-water samples collected from similar depths inthe adjacent ice-free Ross Sea were two to threeorders of magnitude higher (145).

In most deep-sea environments circulatoryprocesses supply dissolved molecular oxygen atrates that greatly exceed the biochemical oxygendemand. However, if these processes are im-paired or if the input of organic matter is unusu-ally high, microbial decomposition will result indepletion of dissolved oxygen. The Black Seaand the Cariaco Trench, Venezuela, are twowell-studied anoxic marine environments. Mea-surements of the vertical distributions of ATPin these habitats have yielded several unusual,but consistent, features, including: (i) a largeincrease in ATP at the oxic-anoxic interface, (ii)elevated ATP concentrations (5- to 10-fold)within the anoxic zone relative to oxygenatedoceanic environments of comparable depth, and(iii) a gradual increase in ATP concentrationwith increasing water depth within the anoxiczone. The factors controlling these atypical ATPdistributions and the significance of elevatedATP levels in anoxic marine environments havebeen discussed by Karl and co-workers (171,185). A similar increase in ATP has also beenreported from the hypersaline, anoxic Orca Ba-sin (Table 9) in the Gulf of Mexico (204).One final exception to the generalized distri-

bution of ATP in the aphotic zone is the recent

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TABLE 9. ATP concentrations in neritic and oceanic seawaters

ATP (ng/liter) at:

Station Coordinates 4m00600 >1,000 Referencem m

Pacific OceanNorth of HawaiiSan Diego TroughSouthern California BightSouthem California BightSouthern California BightSouthern California Bight

North Pacific GyreNorth Pacific GyreEast PacificEast PacificCosta Rica DomePeru coastAleutian TrenchGalipagos RiftGalapagos Rift (hydro-thermal vents)

Cowichan Bay, British Co-lumbia, Canada

Shimoda Bay, JapanGulf of MexicoWest coast of Florida

Orca BasinTampa Bay, Fl.Middle GroundsDestin DomeMobile Bay, Ala.Mississippi Bay

Caribbean SeaCariaco Trench, VenezuelaColumbian Basin

Atlantic OceanNorth Inlet Estuary, N.C.Newport River Estuary,

N.C.Cape Lookout, N.C.Sapelo Island, Ga.North of Cape Hatteras,

N.C.Sargasso SeaSargasso SeaBedford Basin, Nova Sco-

tia, CanadaGuinea DomeAzores PlateauMid-Atlantic RidgeNares Abyssal PlainNamibia Cape BGeorges Bank

Mediterranean SeaNear Toulon, FranceMarseilles Bay, FranceMEDIPROD-I

22010' N, 158°000 W32041' N, 117035' W33018.5' N, 118040' W30040' N, 120002' W32031.6' N, 11800.7' W(Several stations 1-10 km

offshore)310 N, 1550 W280000 N, 1550000 W10032' S, 79058' E12032' S, 101050' E

0.900.4' S, 83037' W

00047.0' N, 86008' W

60-9088-96125-35040-67100-150500-1,500

20-11535-11050-1258-21

55-400100-40075-150

4-835-703-105-8

8-1210-201-56-154-611-154-10

0.2-1.5

0.51-21-2

1.52

0.4-1.51.4-51-2

0.2-0.91-2500

300-1,800

100-600

10 km offshore17 km offshore30 km offshore50 km offshore

-27°40' N, .82040' W-28°40' N, -84420' W-29°30' N, -86° W,30° N, -88° W.30° N, -88°40' W

10032.2' N, 64043.4' W1006.5' N, 77017.0' W

34024' N, 76045' W

36025' N, 74043' W

35000' N, 73000' W32050' N, 62030' W

26018' N, 44044' W26050' N, 60014' W

42055' N, 05054' E43018' N, 05017' E42012' N, 05036' E

1,300750320150

50-300180-42090-230350-2,000

1,500-12,000

80-30010-30

500-2,500500-2,400

125-1821,900500

40-8020-3085-680

25-60020-40400190

100-675120-00

13-40160-80040-120

2.5-15

15015014613618090

231147240

77138147187

287

340

Deminga

204203203203203203

10-15 5-10 1854-6 1 175

57251

97123

16-25 132

2-5 1321.2-7 0.4-1.2 115

312

130288

3-5 0.5-1 18440 0.5-1 184

50-100 2-4 Watson et al.b60 41

3 1-2 6767

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TABLE 9. ContinuedATP (ng/liter) at:

Station Coordinates m 400 >1,000 Referencem m

AntarcticaHeald Island 78013' S, 163054' E 100 145Cape Chocolate 77057' S, 164038' E 200 145New Harbor 77038' S, 163050' E 200-400 145White Island 78001' S, 167021'E 300 145Beneath Ross Ice Shelf 82022.5' S, 168037.5' W 0.04-0.6 14, 144

Station J-9Marginal seasHudson Bay, Canada 90-190 213Wadden Zee, The Nether- 200-1,000 234

landsBlackSea 42050'N, 3300' E 60-250 10-15 5-15 171

'J. Deming, unpublished data.b S. W. Watson, H. L. Quinby, F. W. Valois, J. B. Waterbury, J. E. Hobbie, and T. J. Novitsky, unpublished

data.'P. Laborde, Ph.D. thesis, Universit6 d'Aix-Marseille, Marseilles, France, 1972.

00 r-

m.Ug ATP PER LITER50 100 150

1001

200

500

E

1000

3CILl

4000LFIG. 2. Distribution ofATP (in nanograms [mpg]

per liter) with depth in the eastern Pacific Ocean(31045S N, 120)301 W). Note that different scales areused for the intervals 0 to 200 and 200 to 4,000 m.From Holm-Hansen (140).

discovery of deep-sea production at the Gala-pagos Rift hydrothermal vents. Karl et al. (187)have reported ATP concentrations in excess of500 ng/liter for sulfide-rich vent waters, indicat-ing elevated rates of microbial production in thisspecialized deep-sea habitat. It was suggestedthat energy in the form of geothermically re-

duced sulfur compounds is liberated during oxi-dation and is used for the reduction of CO2 toorganic matter by resident chemosynthetic bac-teria (187).A further application of ATP measurements

in biological oceanography has been the esti-mation of biomass C relative to the total partic-ulate organic carbon present in various marinehabitats. Extensive studies have revealed that50 to 100% ofthe total particulate organic carbonwithin the euphotic zone is living carbon. Withincreasing water depth, the proportion decreasesto -5 to 10% at 200 to 300 m and eventually to<1% at depths exceeding -1,500 m. Represent-ative data from samples collected off the coastof southem California are given in Fig. 3 andTable 10. The intermediate-depth ATP maxi-mum at 386 m (Table 10) is again noteworthy inthat a much higher percentage of living carbonis present relative to the surrounding contiguouswaters. These data provide further evidence ofelevated microbial activity and growth withinisolated portions of the water column.

(ii) Sedimentary ATP distributions. Asummary of selected data relating to the distri-bution of ATP in estuarine and marine sedi-ments is presented in Table 11. Several gener-alized trends are evident, including: (i) ATPconcentrations are highest in organic-richcoastal habitats (e.g., salt-marsh ecosystems andintertidal mud flats), substantially lower insandy beaches and subtidal marine sediments,and even lower with increasing water depth anddistance from shore; (ii) there is generally adecrease in ATP concentration with increasingsediment depth; and (iii) in temperate habitats,there is an annual fluctuation in sedimentaryATP concentrations which is most probably re-

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772 KARL

iug C PER LITER0 5 10 15 20 25 30 35 40 45

0, . . l1 I I I

o00l

E

I-

a-

a

400

6001

FIG. 3. Distribution with depth oftotalparticulateorganic carbon (line A) and organic carbon in livingcells, as estimated byATP x 250 (line B). The stationposition was 32041' N, 117°35' W. From Holm-Han-sen (140).

TABLE 10. Distribution ofATP andparticulateorganic carbon (POC) at various depths at ahydrostation located off southern California

(33018.5 N, 118040 14a

Depth ATP (ng/ POC Biomas Cb Mm,onra(in) liter) ter)li (W/liter) tiPOCa10 126.0 79 33.6 0.4230 357.0 120 95.0 0.79103 17.3 33 4.6 0.14183 6.1 51 1.6 0.03200 10.7 44 2.6 0.06315 6.4 40 1.7 0.04386 34.3 34 9.1 0.27500 6.4 57 1.7 0.03722 4.0 45 1.1 0.02

1,072 0.5 20 0.13 0.006a From Holm-Hansen and Booth (146).b Based on assumption that the C/ATP ratio is 266.

lated to temperature and overall ecosystem pro-ductivity.

(iii) ATP-based biochemical indices ofphysiological state. It is well known that thechemical composition ofan assemblage of micro-organisms is directly affected by the nature ofthe environment. The in situ metabolic state ofindividual cells depends upon the rate of supply

of essential limiting nutrients. Deviations fromexponential, nutrient-saturated, balancedgrowth are often reflected in the chemical com-position of the cells. In theory, the chemicalcomposition of natural microbial assemblagesmay yield information concerning their physio-logical state and nature of the limiting nutrient.Several ATP-based biochemical indices havebeen proposed and used in ecological studies.Physiological ratios based on the relative molarconcentrations of the adenine nucleotides (e.g.,ECA) and on the concentrations of cellular GTPand ATP (i.e., GTP/ATP ratios) will be dis-cussed below.Laboratory and field studies have suggested

that the physiological state of phytoplanktoncells, especially in regard to limiting macronu-trients, could be assessed by measurements ofthe chl a/ATP ratios (35, 256, 279, 280; Sak-shaug, Ph.D. thesis). Perry (256) has reportedthat the chl a/ATP ratio in nutrient-deficientcultures of Thalassiosira pseudonana rangedfrom 3 to 5 during N limitation and from 26 to27 during P limitation. Oceanic field observa-tions have generally yielded ratios of <10, sug-gesting N limitation of phytoplankton growth innature (213,256,279). Nevertheless, any attemptto identify the limiting nutrient from biochemi-cal indices at our present level of knowledgeshould be approached with caution. For exam-ple, Sakshaug (279) has demonstrated a strongcorrelation between chl a/ATP ratios and tem-perature, and several investigators have notedfluctuations in the chl a/ATP ratios due to lightintensity (93; Hunter, M.S. thesis). Moreover,Naiman and Sibert (243) have observed signifi-cant and disproportionate diel variations in bothchl a and ATP in natural phytoplankton popu-lations.Lannergren and Skjoldal (202) have moni-

tored chl a/ATP ratios during a plankton bloomin Lindaspollene Fjord, Norway. chl a/ATP ra-tios increased from <1 before the bloom to >20during bloom conditions. They concluded thatvariations in chl a/ATP ratios were indicativeof physiological changes in the phytoplanktonrelated to the rate of growth. Benon et al. (28)have used chl a/ATP measurements to assessthe mortality of phytoplankton entrained intofreshwater intrusions in the Gulf of Fos, France.Increased chl a/ATP ratios were indicative ofcell death and were found to correlate withdecreases in percent living carbon and totalnumber of phytoplankton cells.

Several studies have also used ATP-basedbiochemical indices to monitor heterotrophicprocesses. During an investigation ofthe decom-position of salt-marsh detritus, R. B. Hanson(unpublished data) used C02/AT and 02/AT ra-

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TABLE 11. ATP in estuarine and marine sedimentsATP ATP(ng/g Ref- (ng/g Ref-

Station location and description of dry er- Station location and description of dry er-sedi- ence sedi- encement) ment)

Sapelo Island, Ga. (short Spartina)(0-1 cm)

AprilJulyOct.Dec.Feb.

Newport River estuary, N.C.0-1 cm

5 cm15cm

Newport River estuary, N.C.Winter (0-1 cm)

(5 cm)(15 cm)

Summer (0-1 cm)(5 cm)

Lindaspollene Fjord, NorwaySoft mud (0-1 cm)

(3-4 cm)Silty sand (0-1 cm)

(3-4 cm)Fine sand (0-1 cm)

(3-4 cm)Coarse sand (0-1 cm)

(3-4 cm)Spanish Sahara

21018' N, 17022' W (200 m) (0-1Cm)

21024' N, 17°54' W (1,000 m) (0-1cm)

Northeastern Atlantic Ocean (0-1 cm,all stations)

36043.5' N, 14016.2' W (252 m)36047.2' N, 14016.7' W (408 m)36042.8' N, 14029.9' W (1,445 m)35057.4' N, 18018.1' W (4,897 m)

Nares Abyssal Plain26050.8' N, 60013.6' W (6,011 m)(0-2 cm)(4-6 cm)

1,000a3,570a2,040a2,720a935a

762algoa57a

640a160"40a

1,180a320a

3,8001,5003,8001,4001,090440900

1,400

550a

56

95

96

252

135

177a

1,32094018084

92

2.4 1840.2

" Values expressed as ATP per cm3 of wet sediment.

Black Sea41021' N, 30014' E (450 m) (5 cm)42059' N, 33059' E (2,200 m) (5 cm)

Long IWland SoundApril (0-1 cm)

(5 cm)(10 cm)(15 cm)

June (0-1 cm)(5 cm)(10 cm)

Oct. (0-1 cm)(10 cm)(20 cm)(50 cm)(150 cm)

Southern California sandy beaches(low tide)

Corona Del Mar(0-1 cm)(5 cm)(10 cm)Catalina Wsland(0-1 cm)(5 cm)(10 cm)Ocean Beach(0-1 cm)(5 cm)(10 cm)Government Point(0-1 cm)(5 cm)(10 cm)

350" 171115a

2,873 3412003322

6,356269239

1,0933499721

11712677

4524712

18018295

174

238348116

tios to provide a broad measure of carbon oxi-dation and oxidative metabolism, respectively.When N was supplied to N-deficient organicdetritus, the C02/AT ratio increased substan-tially. Karl (171) suggested that particulate nu-cleic acid/ATP ratios may be used to comparerelative growth rates of individual microbialcommunities in certain environments. In theanoxic waters of the Black Sea, the particulatenucleic acid/ATP ratios increased from 174 at137 m to a maximum of 933 at 2,045 m, with anattendant and substantial increase in ECA withincreasing water depth (171).

Finally, ATP measurements have been used

frequently in ecological studies as a means tonormalize total production or activity estimatesto a per biomass basis. The resultant "specificactivity" indices are generally more meaningfulmeasures of the metabolic response to in situenvironmental conditions.

(iv) ATP and respiration rates. In severalinvestigations, microbial biomass values esti-mated from ATP concentrations have been fur-ther extrapolated to obtain the total respirationrate (i.e., milliliters of 02 utilized per liter peryear) (132, 141, 142, 147). In ATP-based activityrate estimates the assumptions are made that:(i) the respiration rates of all microorganisms

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are identical when expressed in terms of equiv-alent biomass, (ii) respiration rates have approx-imately the same temperature coefficient as de-termined from laboratory studies, and (iii) noneofthe microorganisms in the sample are nutrientstressed to the extent that respiration is mark-edly affected (141). Hobbie et al. (132) haveapplied a respiration factor of 0.25 pl of 02 per,ug of organic C per day at 220C (and a Qlo[increase in the rate of chemical reaction foreach 100C increase in temperature] of 2.0) toestimate total microbial 02 consumption at ahydrostation in the North Atlantic Ocean. TheATP-derived rates of 02 consumption in theeuphotic zone compared favorably with ratesdetermined by measurements of electron trans-port system activity and by direct 02 respiro-metric methods, but the values were less com-parable in deeper waters (132). Williams andCarlucci (334) have compiled a table of deep-searespiration data estimated by several differentmethods. The data seem to converge on a valueof 1 PI of 02 per liter per year, which is close tothe value derived from ATP measurements.This positive correlation, however, may be for-tuitous, and it should be emphasized that theextrapolation ofrespiration rates from totalATPbiomass data is not justified under most environ-mental conditions. In theory, ATP measure-ments cannot be used to estimate metabolicactivity, since such an application ignores thewell-documented relationships between cell sizeand specific metabolic rate. In addition, severallines of evidence and extensive data have previ-ously been presented to suggest that there is noa priori relationship between biomass and met-abolic activity. Although the turnover rate ofthe ATP pool is positively correlated with met-abolic activity (02 uptake) and growth, it hasalso been shown to be independent ofthe steady-state intracellular ATP concentrations.

(v) ATP and growth rates. Several investi-gators have attempted to estimate in situ growthrates of plankton populations in the sea by mea-suring the rate of increase of particulate ATPwith time (41, 285, 288, 289).Sheldon and Sutcliffe (285) have calculated

growth rates (i.e., generation times) of near-sur-face Sargasso Sea microplankton populations tobe 3.35 (+0.35) h in February and 2.91 (+0.21) hin July as determined by increases in ATP dur-ing a 4- to 7-h incubation period. These rates ofproduction were approximately an order ofmag-nitude greater than the rates measured previ-ously by the standard 14C primary productionmethod. However, no mention was made of dielchanges in ATP per cell, which may have par-tially contributed to their observed ATP

changes with time. Therefore, despite the soundtheoretical basis for using ATP to estimate therate of net accumulation of biomass (i.e., netgrowth rate), the likelihood of temporal fluctu-ations in intracellular ATP concentrations sug-gests that this approach must be used with cau-tion under natural field conditions.

Sieburth and co-workers (41, 288, 289) haveestimated the growth rates of North Atlanticpicoplankton (<3-,nm fraction) by measuringchanges in particulate ATP with time. Repre-sentative water samples were placed into diffu-sion culture chambers and were continuouslynourished with seawaters pumped up from spe-cific depths in the water column (e.g., oxygenminimum, chl a maximum). Their results indi-cated bacterial growth rates approaching twoorders of magnitude greater than estimatesbased on ["4C]glucose and [14C]glycolate uptakemeasurements (288). Periodic decreases in totalparticulate ATP (i.e., difference in ATP concen-tration/difference in time < 0) were concludedto be the result of fluctuations in cellular ECAand not due to actual losses of particulate car-bon. The diurnal pattem of growth was found tobe discontinuous, with the greatest increase intotal particulate ATP occurring in the late after-noon to early evening and in the midmorninghours (41). This diel growth pattem has beencorrelated with variations in the flux of dissolvedcarbohydrates and presumably with phyto-plankton excretion.

(vi) Downward vertical flux of ATP inthe ocean. It must be emphasized that ourcurrent understanding and interpretation of bi-ological oceanographic processes are based pri-marily upon the results of static measurementsof the spatial distributions of biologically impor-tant parameters and indices. Traditionally, theoceanic environment has been sampled usingvarious types of water bottles. However, withinrecent years, the importance of numerically lessabundant large particles has been recognized.These larger particles are not usually collected(in a statistical sense) by current sampling pro-cedures, even though they may represent thedominant class ofsuspended particles in the sea.In situ particle collectors of various dimensionsand configurations are currently being devel-oped and deployed to assess the impact of theseparticles on the structure and trophic organiza-tion of communities in the marine environment.Preliminary measurements of the downwardvertical flux of ATP in the northeast PacificOcean indicate that microbial cells associatedwith the sedimenting particles contribute from1.4 to 15.4 (0 to 400 m) to >100 (1,550 m) timesmore biomass carbon than is measured by static

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sampling techniques (D. Fellows, D. Karl, andG. Knauer, submitted for publication). Further-more, the sedimenting particles are very differ-ent from the "background" materials, both interms of their chemical composition and theratio of living to nonliving carbon. The rapidlysinking particles have lower C/N ratios (G.Knauer and J. Martin, unpublished data) andhigher ratios of living to total carbon (D. Fel-lows, D. Karl, and G. Knauer, unpublished data).The potential metabolic significance of thisdownward vertical flux ofviable microorganismsassociated with large particles may require us toalter our present views concerning the produc-tion and cycling of carbon in the open ocean.Terrestrial and aquatic decomposition

studies. Plant litter in terrestrial ecosystemsand particulate organic detritus in aquatic envi-ronments represent significant sources oforganiccarbon and therefore are important in thetrophic organization and energy flow of the re-spective ecosystems. However, this detrital ma-terial is generally not directly grazed by higherorganism, but rather enters a microbe-basedfood web before becoming available to highertrophic levels (94). The decomposition rate andnutritive changes that occur during the growthand succession of the colonized microbial com-munities on detrital materials have been studiedin attempts to assess the overall trophic role ofdetritus.Most of the biochemical changes that occur

during the decomposition process (e.g., changesin total dry weight, C/N ratios, and total protein)are the direct results of microbial colonization,growth, and succession. Many investigators be-lieve that the "nutritive value" or "food quality"of the detritus is directly related to the quantityof associated microorganisms and their totalcommunity metabolism. Quantitative analysesof ATP associated with organic detritus offer aconvenient measure of the standing stock ofmicrobial cells.

E. B. Haines and R. Hanson (J. Exp. Mar.Biol. Ecol, in press) have examined the decom-position of detritus from three salt-marsh plantsincubated in a P-enriched seawater microcosm.Of the various experimental conditions exam-ined, the greatest rate of decrease in ash-free dryweight and increase in ATP occurred under N-supplemented aerobic conditions. An efficientconversion of detritus to microbial carbon wasobserved, ranging from 19% for decomposingSalicornia to 64% for Spartina detritus.Knauer and Ayers (193) have monitored

changes in C, N, chl a, and ATP during thedecomposition of Thalassia testudinum leavesin a continuous-flow seawater microcosm.

Throughout the 52-day experiment, there was asignificant correlation between organic C andtotal ATP (r = 0.97), indicating that the transferof plant C to microbial C was tightly coupled.They suggested that ATP measurements, whenused in conjunction with C and N data, can yielda simple quantitative estimation of the nutri-tional value of the particle-microbe complex.

K. R. Tenore and R. B. Hanson (Linnol.Oceanogr., in press) have recently studied thequalities of several different sources of detritusat various stages of decomposition as foodsources for the marine polychaete Capitella. Ineach case, both the microbial biomass (based onAT measurements) and the total heterotrophicactivity changed with the age of the detritus.They also reported that the maximum incorpo-ration of detritus into Capitella biomass coin-cided with that stage of decomposition exhibit-ing maximum microbial biomass and metabolicactivity as measured by oxygen consumption.Morrison et al. (242) have followed the colo-

nization, outgrowth, and succession of microbialcommunities associated with allochthonousplant litter in Apalachicola Bay, Fla. The initialcolonization was dominated by bacteria as re-vealed by high muramic acid-to-ATP ratios(242). With increasing incubation time, morecomplex microorganisms became dominant, asevidenced by progressive decreases in the mu-ramic acid/ATP ratios. These biochemical in-dices were confirmned by scanning electron mi-croscopy of the plant litter (242) and by changesin the pattern of biosynthesis of characteristicmicrobial lipids (190).Bobbie et al. (30) have compared the biode-

gradability of slash pine needles with that ofmorphologically similar polyvinyl chlorideChristmas tree needles. Following a 14-week in-cubation in seawater, the slash pine detritusexhibited ATP levels, oxygen uptake, alkalinephosphatase and phosphodiesterase activities,and muramic acid concentration which were 2to 10 times higher than those of the polyvinylchloride controls. Furthermore, the polyvinylchloride needles maintained higher muramicacid/ATP ratios, indicating a paucity of eucar-yotic microorganisms. They concluded thatbiodegradable substrata support microbial com-munities that are three to five times more activethan inert surfaces of comparable surface area.Lopez et al. (225) have examined the assimi-

lation of Spartina litter by adult amphipods ofthe genus Orchestria as a function of total mi-crobial biomass. By assuming a C/ATP ratio of285 and an N/ATP ratio of 63, they were able tocalculate the percentages of C and N in theliving and nonliving portions of the detritus. A

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chemical comparison of decomposing Spartinawith the fecal pellets enabled an estimation ofthe relative degradabilities of the two compo-nents. Their results indicated decreases of 2.1,15.9, and 60.5% for total C, N, and ATP, respec-tively. These data clearly demonstrate a prefer-ential removal ofliving microorganisms from thedetritus. It was also shown that the highestconcentration of ATP per milligram of plantlitter and the highest rates of microbial activityand growth all occurred during conditions ofhighest grazing intensity (225). This result indi-cates that grazing has a significant effect on thelikelihood of microbial colonization and growthand thus on the rate of decomposition and theoverall nutritional quality of the detritus.Wastewater treatment, disinfectant con-

trol, and pollution assessment. The success-ful operation of municipal sewage and industrialwastewater treatment plants, the effective proc-essing of potable water supplies, and the impactof thermal pollution and chemical pollution onnatural ecosystems all require detailed microbi-ological investigations. Controlled treatment ofwastewaters is one of the oldest, largest, andmost applied aspects of environmental microbi-ology. In common with other applications inmicrobial ecology, wastewater treatment re-quires methods for rapid and reliable determi-nations of microbial biomass and activity. ATPmeasurements have recently and successfullybeen used in several activated sludge treatmentplants (36, 155, 198, 219, 254, 277, 328). Kucne-rowicz and Verstraete (198) have observed sig-nificant correlations between ATP concentra-tion and substrate removal rate (r = 0.96, p c1%), both ofwhich are important parameters forregulating the inflow of wastwater and return ofsludge to maintain maximum plant efficiency.

Levin et al. (219) have used ATP measure-ments as the primary criterion for regulating themixed liquor biomass in a 20 x 107-gallon/day(ca. 76-Ml/day)-flow sewage treatment plantand have concluded that "ATP control" pro-vided a rapid and logical method for maintainingan optimum waste-to-microorganism ratio. Hy-sert et al. (155) arrived at a similar conclusionwhen they used ATP measurements to monitorthe operation of a 3.5 x 105-gallon/day (ca. 1.3-MI/day) brewery wastewater treatment plant.Patterson and co-workers (36, 254) have furthersupported the use of ATP measurements in theactivated sludge process by demonstrating thatATP values were constant during normal oper-ational conditions, but that ATP concentrationsresponded rapidly to changes in temperature,dissolved oxygen, substrate loading, pH, andheavy-metal concentrations. Therefore, in addi-

tion to providing an indication of biomass, ATPmeasurements may also be useful for monitoringchanges in the metabolic activity of activatedsludge.The rate of disinfectant addition to wastewa-

ters or potable water supplies is generally deter-mined by specific bacterial culture enumerationtechniques. Tifft and Spiegel (319) have pro-posed the use of ATP measurements for disin-fection control. They observed a decrease intotal particulate ATP with the addition of C12 orC102 which paralleled the results from the moretraditional indices of total and fecal coliformbacteria. After disinfection treatment there wasa significant correlation between ATP and totalcoliforms (r = 0.84) and between ATP and fecalcoliforms (r = 0.76) for total ATP values in therange of 0 to 1.5 ,ug/liter. They concluded thatdecisions regarding sanitary water quality basedon ATP measurements would be as sound asthose based on current coliform enumerationcriteria (319).

Several investigations have attempted to as-sess the impact of urbanization and industriali-zation on the quality of surrounding watermasses (28, 43, 59, 89, 106, 206, 266, 327). ATPmeasurements have served as one of severalcriteria for evaluating the environmental re-sponse to pollutant discharge. ATP bioassayscannot yield absolute measures of water quality,per se, since the quality of the environment willdepend upon species composition, diversity, andmetabolic rates as well as total biomass. Eppleyet al. (89) have found evidence of eutrophicationin waters around the Point Loma and WhitesPoint sewage outfalls in southern California bydiscovering increased concentrations of chl a,ATP, particulate carbon, and rates of primaryproduction. This response was attributed to theincreased supply of inorganic nutrients (espe-cially N) to the otherwise nutrient-depleted wa-ters. However, no increased nutrient levels wereever observed in the outfall area, suggesting thatthe phytoplankton populations were efficientscavengers of sewage-derived N and P. Eppleyet al. (89) found no toxic effects of the sewageeffluent on phytoplankton growth, and, in fact,the sewage-enriched waters were regarded asstimulatory.Laws and Redalje (206) have also evaluated

the effects of sewage enrichment on the phyto-plankton populations of Kaneohe Bay, Hawaii.Their data indicated a nearly eightfold declinein living particulate organic carbon between theoutfall station and the extreme northwest sectorof the bay. However, it was determined that thealgal populations at all stations along the south-east (outfall)-to-northwest Kaneohe Bay tran-

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sect were nutrient saturated. Even at the outfallstation, greater than 80% of the nitrogen re-quired for phytoplankton growth was contrib-uted by in situ recycling. They concluded thatthe decline in living particulate organic carbonalong the transect (towards the northwest sta-tion) was due to dilution ofbay water by offshoreoligotrophic seawaters, and therefore the highconcentrations ofphytoplankton adjacent to theoutfall could not be attributed to sewage enrich-ment. This study emphasized the necessity andimportance of a thorough investigation of theenvironment before a valid assessment of pollu-tants can be achieved.Envfronmental Adenylate Energy ChargeThe well-established correlation between cel-

lular ECA and metabolic or growth potentials ofindividual organisms in theory provides a frame-work for estimating metabolic potentials of nat-urally occurring microbial populations. Sincemost microorganisms studied to date have con-trol systems to maintain the relative molar con-centrations of the adenine nucleotides (ex-pressed as ECA) within narrow limits duringnormal growth, marked decreases in ECA mayreflect environmental stress or other conditionswhich interfere with the normal functioning ofthe cells. In this regard, both the rate of proteinsynthesis and the potential for cellular biosyn-thesis have been shown to be much more closelycorrelated with changes in ECA values than withfluctuations in the absolute concentrations ofintracellular ATP, ADP, or AMP (311). Thepotential applications of ECA measurements inexperimental microbial ecology have been dis-cussed by several investigators (28, 68, 148, 180,333, 338).There are, however, several limitations to the

usefulness of the ECA concept in general, andespecially in its application to environmentalstudies. The first major concern is one of meth-odology. Since each adenine nucleotide hasslightly different adsorption and reaction char-acteristics, three sets of internal standards mustbe included in each extract to assess and correctfor individual nucleotide losses. The occurrenceof soluble (i.e., cell-free) ADP and AMP mustalso be considered, especially when direct injec-tion techniques are used or when sediment orsoil samples are processed. Furthermore, diffi-culties have been reported in the determinationof AMP, especially at concentrations typicallyencountered in most environmental extracts.Karl and Holm-Hansen (180), however, recentlydescribed a method to maximize the enzymaticconversion of AMP to ATP by increasing theATP concentration in the sample extract.

Another concern is the validity of "commu-nity" ECA measurements. Most microbial as-semblages are undoubtedly composed of orga-nisms in a variety of physiological and metabolicstates. When the populations are extracted, amass-weighted mean ECA is measured. Conse-quently, it is extremely difficult, if not impossi-ble, to interpret most community ECA measure-ments. For example, if the measured ECA is 0.6,the strict interpretation of the adenylate meta-bolic control hypothesis would lead one to con-clude that the population was stressed, senes-cent, or incapable of cellular biosynthesis. How-ever, a portion of the population may be growingat p with an intracellular ECA of -0.8, and theremainder may be inactive (ECA -0.5). Un-doubtedly, the most successful (and unequivo-cal) ecological applications of ECA measure-ments are those in which the mean communityECA values are 20.8, indicating that probablymost of the microbial assemblage is potentiallycapable of cellular biosynthesis. Future studiescombining microautoradiography with commu-nity ECA measurements will help to determinethe percentage of active cells and render thistechnique more useful.

Finally it must be reemphasized that the com-munity ECA cannot be used to estimate in situgrowth rates or rates of cellular metabolism,since each parameter may vary signifcantly atfixed ECA values. Attempts to compare the rel-ative metabolic activities of different microbialassemblages will require additional estimates ofrespiration, carbon turnover, and growth.

Despite these shortcomings, environmentalECA measurements have provided useful infor-mation concerning the relative growth potentialsand probable metabolic statuses of several nat-urally occurring microbial populations. Karl andHolm-Hansen (180) have measured the concen-trations of ATP, ADP, and AMP and have cal-culated the corresponding ECA values for watersamples collected in the Southern CalifomiaBight (Fig. 4). The ECA values ranged from 0.5to 0.89, with peak values corresponding to thesubsurface maxima ofATP and chl a concentra-tions. Surface (1 to 10 m) water samples werefound to have depressed ECA values (-0.65 to0.75) relative to the ratios measured at the chla maximum. It has been suggested that thislower growth potential is the result of ultravioletlight stress or inorganic nutrient depletion orboth. A similar effect was also noted in theCaribbean Sea, where reduced rates of stableRNA synthesis corroborated the interpretationof metabolically stressed cells within the surfacelayer of the ocean (175). An additional signifi-cant and recurring trend in seawater ECA deter-

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A ATfP ng /-1 ECA

0 600 1200 0.7 0.8

40-

80

120 I

160

ATfP ng l-l

30

AT, ng 1-1 ECA100 2000 0.5

1600 L

FIG. 4. Vertical distribution ofATP, Ar, chi a, and ECA in waters of the Southern California Bight. Thedata were obtained (A) at a station approximately 5 km offshore in water of200 m (33°15' N, 117°41' W) and(B) at a station approximately 100 km offshore in water of 1,850 m. From Karl and Holm-Hansen (180).

minations is the observed discontinuity in ECAvalues at a depth of .40O to 600 m. This discon-tinuity tends to sepaate microorganisms intocommunities with relatively high ECA ratios(-0.7 to 0.9) above this depth and relatively lowcommunity ratios ('0.5 to 0.7) below this depth(175, 180, 333), suggesting a physiological ormetabolic alteration in populations at depths of>500 m such that the community potential forcellular biosynthesis is reduced. Nonetheless, a

community ECA of 0.5 to 0.7 is indicative ofeither a senescent population or an associationof actively growing cells (ECA > 0.8) and deador dying cells (ECA < 0.5). The observation thatin situ stable RNA synthesis continues in micro-

bial assemblages within a portion of the watercolumn where the ECA is <0.8 (175), however,tends to support the latter hypothesis. Wiebeand Bancroft (333) have reported that environ-mental ECA values and community densitiesvary independently, such that the potential formicrobial growth in the ocean is not necessarilycorrelated with in situ biomass. Karl (175) hasrecently provided additional data by measuringrates of stable RNA synthesis to further supportthis contention.The most extensive ecological investigation

involving the use of ECA measurements is a

recent report by Witzel (338) wherein 356 watersamples from several deep lakes in northern

Chli , P9 ,1

0.6 1.2 1.8

EI3Q.U.'a

B0

0

400

1.0

800EI

0c

1200

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Germany were studied. His results indicatedthat the ECA values ranged from 0.16 to 0.97.The highest ECA values were measured in sam-ples collected in the epilimnion. In deepwatersamples (mostly from anoxic hypolimnia), ECAvalues were generally <0.6. The relative fre-quency distribution showed two preferentidgroups, one with an ECA of -0.55 (0.4 to 0.66)and the other with an ECA of -0.75 (0.7 to 0.85).ECA values of <0.3 or >0.85 were rare (338).

Karl (171) has measured the vertical distri-bution of ATP, AT, and ECA at several stationsin the western basin of the Black Sea. Samplescollected from the 02-H2S interface zone (-125m) revealed a vertically restricted layer (-20 to30 m)of cells with ECA values of >0.8 (Fig. 5).These data suggest that the entire microbialpopulation inhabiting the chemocline is capableof cellular biosynthesis, thereby supporting theEgunoff hypothesis for the existence of a meta-bolically active "bacterial plate" in the BlackSea. Within the anoxic portions of the watercolumn, ATP concentrations were 5 to 10 timesgreater than in oxygenated oceanic environ-ments of comparable depth, and both ATP andECA values increased with increasing waterdepth from -600m (Fig. 5). The deepwater ECAdata suggest that the increase in ATP below-600 m is the result of in situ activity, implyinga vertical gradient in specific organics requiredfor growth and metabolism (171).

ATP, ng liter-'

r,Q0

200

400

600

E 800I 1000

L 1200

X 1400

1600

1800

2000

100 30010 20 30 40 50,1 01

1

_ DEPTH,m Nt. ml liter-I74 2.29

_ 95 OA1102 0.34109 0.34113 0.27

I .1

Measurement of ECA values has also beenapplied to studies of marine sediments. H. R.Skjoldal (unpublsihed data) has reported a meanECA of 0.81 for marine sediments in westernNorway. In general, the ECA values decreasedwith increasing sediment depth, suggesting alower potential for microbial community metab-olism in the deeper sedimentary layers. Thecorrelation between ATP concentration andECA was poor (r = 0.28), again supporting theargument that there is no a priori or obligatoryrelationship between biomass and in situ meta-bolic activity. R. Christensen and A. Devol (sub-mitted for publication) have recently comparedATP and AT concentrations and the correspond-ing ECA values of continental shelf sediments offWashington to those of coastal Puget Sound.Although the magnitudes of the ATP and ATvalues were similar at both locations, the meanECA values of the shelf microbial communitieswere lower than those calculated for communi-ties inhabiting the sound. They concluded thatthe microorganisms of the shelf habitat were ina depressed physiological state relative to thosein Puget Sound, and they have supported theseconclusions with data on benthic respirationrates and excess interstitial alkalinity and NH4'concentrations.The most useful environmental application of

the ECA index may be in assessing the impact ofpollution on selected components of an ecosys-

AT, ng liter-' ENERGY CHARGE200 400

20 40 60 80 1001 Q5 0.6 0.7 0.8 0.9 1.0

I.,IA __

FIG. 5. Vertical distribution ofATP, A'r, and ECA for the water column at a station located in the westernbasin of the Black Sea (42o50' N, 33°00 E). Total water depth 2,160 m. From Karl (171).

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tem. The method currently used for assessingthe biological effects of environmental wastedisposal is to conduct 50% lethal dose bioassayswith key representatives from the natural as-semblage of organisms. It has been suggestedthat a knowledge of the adenylate systems ofkey or indicator species with known response toparticular environmental conditions may pro-vide a convenient measure of the ecological"health" of a given ecosystem (267). Addition-ally, ECA measurements may facilitate the de-tection of subtle sublethal effects of pollution ornatural environmental stress on selected aquaticorganisms, although additional laboratory andfield data are needed before any recommenda-tion of this approach can be made.

Guanosine 5'-Triphosphate and CellularBiosynthesis

The guanine nucleotides, GTP and guanosinetetraphosphate, have been shown to exert a ma-jor influence on the rates of protein and RNAsyntheses, respectively. As mentioned previ-ously, GTP is required for the activation andinterconversion of precursors for bacterial cellwall biosynthesis, for DNA replication (asdeoxy-GTP) and RNA transcription, and for theinitiation, the aminoacyl transfer RNA binding,and the translocation processes of protein syn-thesis. This indirect coupling of cellular energyduring biosynthesis suggests that the total fluxthrough the GTP pool may be correlated withcellular growth rates. Accordingly, it has beenreported that the intracellular GTP pool sizesfluctuate in direct proportion to the demand forbiosynthetic energy (107, 169, 174, 296, 297).This response is presumably a regulatory mech-anism for maintaining saturating levels of GTPat all sites for protein biosynthesis (i.e., theribosomes), with the number of ribosomes in-creasing in proportion to the growth rate (249).Karl (169,174) has indicated that the GTP/ATPratios of microorganisms were positively corre-lated with cellular growth rates. He found thatthe intracellular GTP/ATP ratios were muchhigher in growing bacteria (range, -0.5 to 1.7)than in eucaryotes (range, -0.1 to 0.35), presum-ably due to their innate potential for muchhigher specific rates ofprotein biosynthesis. Karlconcluded that quantitative determinations ofGTP/ATP ratios in environmental sample ex-tracts may be useful for estimating and compar-ing community growth rates in naturally occur-ring microbial assemblages (169, 174).The presence of GTP (and other nonadenine

NTPs) in environmental extracts can be de-tected in one ofseveral ways. Crude preparationsof firefly luciferase will react with GTP, uridine

5'-triphosphate, inosine 5'-triphosphate, and, toa lesser extent, cytidine 5'-triphosphate to pro-duce light. The reaction kinetics with theseNTPs are different from the ATP-dependentkinetics, and therefore a semiquantitative as-sessment can be achieved by simply examininga plot of light emission versus time. In general,most environmental sample extracts that areassayed by integrated light flux determinationyield "ATP" values that are higher than peaklight emission data from the same sample. Thedifference (i.e., difference in [ATP] = integrated[ATP] - peak [ATP]), when normalized to thepeak ATP concentration, may yield overesti-mates from 30 to 250% of the actual ATP con-centrations when using crude luciferase prepa-rations (169, 174). Moreover, it has been dem-onstrated that the difference in ATP concentra-tion is significantly correlated with the GTPconcentration (r = 0.96 for sediments [174], r =0.92 for seawater samples [169]), suggesting thatdifferences in ATP concentration may be usedas relative estimates of the GTP concentrationsin environmental nucleotide extracts. It shouldbe emphasized, however, that the actual extentofNTP reactivity is extremely variable from oneenzyme preparation to the next, which mitigatesthe possibility of a precise quantitative assess-ment of GTP by this approach. More recently,Karl (170) has devised a rapid and sensitivemethod for the measurement of guanine ribo-nucleotides (GTP, guanosine 5'-diphosphate,and guanosine 5'-monophosphate) present inmixed nucleotide extracts. The technique re-quires no special equipment or facilities beyondthose already used for routine adenine nucleo-tide analyses and may be easily integrated intomany ongoing research programs.An inclusive summary of environmental GTP

determinations is presented in Table 12. Thevertical distribution ofGTP at a hydrostation inthe Southern California Bight exhibited verticaltrends similar to those of ATP, with the highestconcentrations at the surface, a rapid decreasewith depth between 50 and 175 m, and a moregradual decrease in concentration with increas-ing water depth (169; Table 12). The GTP/ATPratios ranged from 0.10 to 0.19. Throughout thewater column, organisms of <10 Sn had sub-stantially higher GTP/ATP ratios (range, 0.35to 0.40), indicating greater in situ rates ofproteinbiosynthesis. The GTP/ATP ratios measured inthe surface waters of the Black Sea and at ahydrostation located on the Galipagos Rift wereidentical to those obtained for surface waters offsouthern California (Table 12). However, theanoxic portion of the Black Sea (depths of >125m), and the deep-sea hydrothermal vents on the

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TABLE 12. GTP/ATP ratios in environmental samplesConcna

Sample desciption GTP/ATP ReferenceGTP ATP

Southern California seawater (32043.2' N, 117030.1' W)b10 m, total10 m, <10 pm

100 m, total100 m, <10 ,um500 m, total500 m, <10 um

Southern California seawater (33030.1' N, 119020.0' W)5m50m200m500 m

1,000 m1,550 m

Black Sea seawater (42050.0' N, 33000.0' E)10 m75 m123 m195 m489 m974 m

1,992 mGalapagos Rift seawater (00047.0' N, 86°08' W)

50m500 mHydrothermal vents (2,500 m)

Catalina Island (Calif.) sediments (33021' N, 118030' W),low tide

0-1 cm5 cm10cm

Ocean Beach (Calif.) sediments (32045' N, 117016' W),low tide

0-1 cm5 cm10cm

55.7c21.8c31.2c6.3c2.5c1.9c

82688.55.02.62.5

6812.716.614.63.63.06.4

46<0.6832

6209638

208247142

312582216.511.84.5

55052365262020.8

4628941.435.39.1

12.026.2

2868

968

8929324

0.180.320.140.380.220.42

0.150.130.130.190.130.12

0.150.140.400.410.400.250.24

0.16<0.0750.86

0.701.031.58

355 0.59359 0.69187 0.76

a GTP and ATP concentrations are expressed as picomoles per liter for seawaters, and picomoles per cm3 forsediments.

b Total, seawater filtered onto a Reeve Angel 984-H filter disk; <10 pm, 10-,um Nitex filtrate filtered onto a

Reeve Angel 984-H filter disk.[GTP] = [GTP + uridine 5'-triphosphate] x 0.53 (169).

Galapagos Rift (-2,500 m) exhibited signifi-cantly higher GTP/ATP ratios, implying ele-vated rates of cellular biosynthesis and growth.At the hydrothermal vents, the GTP/ATP valuewas 0.86, indicative of extremely rapid in situgrowth. By comparison, GTP/ATP ratios froma variety of southern California intertidal sedi-ments ranged from 0.40 to 1.87, with an overallmean of 0.69 (187; Table 12).Although GTP/ATP ratios in environmental

extracts may be useful for detecting areas ofelevated microbial growth and for comparingrelative rates of community biosynthesis withinnatural microbial populations, it must be em-

phasized that these measurements reflect meancommunity estimates. After more extensive lab-oratory investigations have been conducted on

the nucleotide levels in a wider range of repre-sentative microorganisms, it may be possible totranslate GTP/ATP ratios directly into absoluterates of protein synthesis.

Cyclic Adenosine 3',5'-MonophosphateThe ubiquity of cAMP in bacteria, phyto-

plankton, and metazoans has led to an extensiveinvestigation of its biological function. The roleof cAMP is regulatory, but not essential (270);the intracellular cAMP concentration varies

169

169

169

187

174

174

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with conditions ofgrowth and with the energeticstate ofthe cells. In bacteria, cAMP accumulateswhen cultures are limited by carbon (322), andin phytoplankton, cAMP levels have been cor-related to inorganic nutrient availability (D.Francko and R. Wetzel, unpublished data).Tomkins (322) has designated cAMP an impor-tant metabolic symbol, and, consequently, mea-surements of cAMP concentrations in naturemay contribute important information regardingthe growth states of naturally occurring micro-bial populations.

J. W. Ammerman and F. Azam (Abstr. Annu.Meet. Am. Soc. Microbiol. 1979, N62, p. 189)have reported the presence of dissolved cAMPin seawater at concentrations of up to 10 pmol/liter, and D. White (personal communication)has detected cAMP in nucleotide extracts fromestuarine sediments. Francko and Wetzel (un-published data) have recently investigated sea-sonal variations in particulate and dissolvedcAMP in two lakes of differing trophic status.The concentrations of both cAMP fractions var-ied during the seasons, between the two lakes,and between the littoral and pelagic zones of agiven lake (Francko and Wetzel, unpublisheddata). Dissolved cAMP levels were similar tothose found in the media of phytoplankton cul-tures, although considerable seasonal variationwas evident. Increased cellular cAMP levels co-incided with increased phytoplankton popula-tion density, and a linear relationship was ap-parent between particulate cAMP and primaryproduction in the hypereutrophic ecosystem.Seasonal changes in dissolved and particulatecAMP were correlated with phytoplankton com-munity succession. Clearly, additional work isnecessary to fully understand the metabolic andecological significance of cAMP, but the prelim-inary data presented by Francko and Wetzelcertainly warrant further investigations.

Nucleotide Metabolism and GrowthDissolved nucleotides and microbial het-

erotrophy. The ubiquitous occurrence of D-ATP (and other nucleotides and nucleotide de-rivatives) in aquatic environments and constantsteady-state production from grazing and autol-ysis pose questions concerning the ultimatefate(s) of these molecules in natural ecosystems.When radiolabeled ATP, AMP, adenosine, oradenine is added to a sample of seawater orfreshwater, there is a rapid disappearance ofradioactivity from solution. Azam and Hodson(15) considered three possible mechanisms foradenine nucleotide turnover in nature: (i) spon-taneous hydrolysis, (ii) enzymatic hydrolysis,and (iii) microbial uptake and assimilation. Hu-lett (153) has calculated the half-life of ATP in

artificial seawater at 210C to be -8 years, so itis unlikely that chemical hydrolysis is a signifi-cant pathway. The presence of extracellularadenosine triphosphatase, nucleotidase, acid andalkaline phosphatases, and additional nonspe-cific orthophosphoric-monoester phosphohydro-lases in aquatic environments provides a mech-anism for enzymatic degradation of soluble nu-cleotides. Finally, it is well known that manydiverse groups of microorganisms can assimilateexogenous adenine supplies, in preference to denovo synthesis, if present in the growth medium.ATP uptake is competitively inhibited by ADP,AMP, adenosine, and adenine, suggesting a com-mon transport system (134). Azam and Hodson(15) found that more than 95% of the radioactiv-ity from [U-14C]ATP was assimilated into cel-lular macromolecules with a negligible amountlost through respiration.When freshwater or oceanic samples are in-

cubated with [3H]ATP (or other radiolabeledadenine derivatives) rapid turnover rates areobserved for the dissolved adenine pools (15,145, 175). Size fractionation studies have indi-cated that -80% of the total uptake is due tomicroorganisms of <0.6 ,um (15), suggesting thatadenine may be an important molecule in themetabolism of aquatic bacteria. Rates ofD-ATPand glucose uptakes and calculated turnovertimes for the steady-state soluble pools havebeen calculated to be of similar magnitude (15;Riemann, unpublished data). S. M. McGrathand C. W. Sullivan (unpublished data) haverecently proposed the use of [3H]AMP as amodel compound to monitor the kinetics of up-take and to identify the compartments involvedin the cycling of total adenylates by marineplanktonic communities.

Nucleic acid biosynthesis. The uptake andincorporation of exogenous radiolabeled nucleo-sides have been used extensively in laboratoryand field investigations to estimate the rates ofnucleic acid biosynthesis (14, 37, 175, 204, 303,315, 320). The observed correlations betweenRNA and DNA synthesis, protein syntheses,and cell growth are so universally acceptablethat they lend themselves to the analysis ofcomplex microbial populations, such as one findsin nature. Brock (37) pioneered the use of thy-midine incorporation as an estimate of DNAsynthesis and cell growth in natural assemblagesof microorganisms. His experimental approachused the marine microbe Leucothrix mucor asa model organism. Data relating the rate ofaccumulation of radioactive cells (via autora-diography) to growth rate were obtained over awide range of growth rates in order to establisha numerical constant (1% of the cells becameradiolabeled in 0.002 generation) for estimating

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the growth rate in nature. In situ doubling timesof L. mucor were then calculated to range from660 to 685 min for a wide range of habitats (37).Other investigators have used thymidine up-

take, or occasionally uptake of uridine, as anindirect measure of microbial activity in soils(315), sediments (320), seawater (14, 204), andaerosol samples (303). Although thymidine up-take and ATP-biomass correlated well (320) andare expected to vary in direct proportion tometabolic activity and growth, the rate of label-ing of cellular macromolecules cannot be as-sumed to be equal to the rate of nucleic acidbiosynthesis. The actual rate will vary accordingto the specific transport mechanisms and dilu-tion of the label by exogenous and endogenouspools before incorporation into nucleic acids.Therefore, quantitative measurements of therates of DNA or RNA synthesis also requiredeterminations of the specific radioactivities ofthe intracellular precursor pools. Even for theestimation of relative rates, failure to measure(and correct for) the intracellular specific ra-dioactivities might lead to serious errors andgross misinterpretations of ecological data.Karl (175) has recently devised a technique

for measuring rates of stable RNA synthesis innaturally occurring populations of microorga-isms. The procedure is based upon the uptakeand incorporation of exogenous radiolabeled ad-enine into cellular RNA (Fig. 6). To calculateabsolute rates of synthesis, measurements of thespecific radioactivities of the intracellular ATPpools (precursor to adenine in RNA) and of thetotal amounts of radioactivity incorporated intoRNA per unit time are required. The theoreticalconsiderations and experimental details of thisnew approach to the study of microbial growth

out n

3 3PRW3H * adenine H*AMPk1

rates in nature have been presented and dis-cussed in detail previously (175).This technique has been successfully used to

demonstrate that the microbial populations as-sociated with the intermediate-depth oxygenminimum layer of the Columbian Basin (-450m) maintain a higher specific rate of RNA syn-thesis (i.e., RNA synthesis per unit of biomass)than the assemblages found elsewhere in theentire water column (175). From production andstanding stock measurements, a mean turnoverrate (generation time) of 2.2 to 6.5 h was derivedfor this intermediate-depth microbial popula-tion, assuming steady-state conditions (i.e.,change in biomass/change in time = 0). Morerecent, and as yet unpublished, data from thislaboratory have expanded the application of thisexperimental approach to provide additional in-formation pertaining to the rates of DNA syn-thesis (and, therefore, DNA/RNA synthesis rateratios), adenine nucleotide pool turnover, pro-duction and interconversion of guanine ribonu-cleotides, and exogenous pool sizes of adenine-containing compounds in natural environments.

CONCLUDING STATEMENTThe growth of microorganisms is ultimately

dependent upon innumerable enzyme reactionsand the successful integration ofmany metaboliccontrol systems. Consequently, the implemen-tation of any unifying principles will undoubt-edly yield numerous exceptions when applied tothe study of microorganisms under a variety ofgrowth regimes. This is especially true for ex-trapolating the results of laboratory studies tocomplex natural ecosystems where the variousmicrobial assemblages have evolved to acceptthe diverse challenges of nature.

3H-ADP

3H-ATP u. RNA/ ( H)

mRNA(3H)

FIG. 6. Schematic representation of the methodpresented by Karl (175) for quantitative determinations ofrates of stabk RNA synthesis in seawater samples. Exogenous [3H]adenine is transported across the cellmembrane at rate k1 and is converted into [3H]AMP, [3H]ADP, and [3HJATP, the latter serving as theimmediate precursor to stable RNA. By measuring total [3H]RNA produced after a set period of incubationand by directly measuring the intracelular specific activity oftheATPpool, absolute rates ofRNA synthesiscan be calculated (i.e., picomoles of adenine incorporated into RNA per liter per hour). mRNA, MessengerRNA. From Karl (175).

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TABLE 13. Environmental nuckotide fingerprintingIndex Rationale Ecological information

ATP concn 1. Present in all cells Estimate of biomass2. C/ATP ratio is fairly constant

ECA 1. ECA is a linear measure of stored Measure of community potentialmetabolic energy (range, 0.0- for growth (i.e., mean commu-1.0) nity metabolic status)

2. ECA appears to regulate cellularmetabolism

3. Biosynthesis and growth are possi-ble only at an ECA of 20.8

GTP/ATP ratio 1. GTP is required for protein syn- Estimate of community growththesis rate

2. GTP/ATP ratio is positively cor-related with growth rate

[3H]adenine assimilation and 1. [3H]adenine -c [3H]ATP -* [3H]- Measure of community growthstable RNA synthesis RNA rate

2. 3H assimilation is proportional torate of RNA synthesis

3. Rate of RNA synthesis is propor-tional to growth rate

DNA/RNA synthesis ratio 1. Intracellular DNA concentration Measure of community growthis independent of growth rate rate

2. Stable RNA concentration is pro-portional to growth rate

3. Ratio of rate of DNA synthesis torate of stable RNA synthesisprovides indication of growthrate

In recent years, investigations in the field ofexperimental microbial ecology have returned tothe fundamental principles of biochemistry,physiology, and cell biology. One area of signifi-cant progress has been in the study of cellularnucleotide concentrations and rates of nucleo-tide metabolism. The development of specificand sensitive techniques for quantitative mea-surements of intracellular nucleotides has en-abled researchers to investigate the distributionof microbial biomass and to estimate the ratesof protein and nucleic acid biosyntheses, nucleo-tide metabolisn, metabolic activity, and growthwithin naturally occurring populations of micro-organisms. This review has attempted to providethe physiological rationale and analytical pro-cedures for considering measurements of cellularnucleotides in microbial ecology and to providethe theoretical framework and data base of lab-oratory results necessary for the proper inter-pretation of environmental nucleotide measure-ments. The approach of nucleotide fingerprint-ing (Table 13) is strongly recommended for fieldinvestigations in order to obtain corroborativedata relating to the in situ physiological statesof naturally occurring microbial assemblages. Ihope that future ecological studies of microor-

ganisms will adopt, refine, and ultimately ex-pand the scope of the fingerprint to includeadditional nucleotide-based metabolic indices.

ACKNOWLEDGMENISI am most grateful to numerous colleagues for their

helpful comments and overwhelming response to myplea for preprints, manuscripts, and unpublished data;without their assistance it would have been impossibleto present an up-to-date account of this subject. I amindebted to D. Craven, R. Enos, C. Winn, and D.Wong for criticism, editorial appraisal, and helpfulsuggestions for the improvement of this manuscript.D. Wong also assisted in the preparation of the tablesand was largely responsible for compiling the lengthyreference list; her enthusiasm and dedication to thisproject are deeply appreciated. I thank the variousauthors and publishers for their permission to repro-duce previously published figures and tables. L.Agawa, J. Katsura, and V. Sen cheerfully and flaw-lessly typed several drafts of this review. Stimulatingdiscussions with F. Azam, A. Carlucci, R. Eppley, 0.Hohm-Hansen, H. Jannasch, G. Knauer, P. LaRock, E.Laws, and K. Nealson have provided new ideas andinsights into possible future applications of nucleotidemeasurements in microbial ecology. Finally, I wish toexpress my most sincere aloha to my former mentors,P. LaRock (Florida State University, Tallasse) and0. Holm-Hansen (Scripps Institution of Oceanogra-



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phy, La Jolla, Calif.) for kindling my interests andsupervising my earlier studies in the area of cellularnucleotides and nucleotide metabolism.The original studies conducted in my laboratory

were supported, in part, by research grants OCE 78-18926, OCE 78-20721, and OCE 78-25446 awardedthrough the Biological Oceanography section of theNational Science Foundation.

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