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The biomass and activity of bacteria in the sediments of Marion Lake, British Columbia Perry, E. A. 1974

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THE BIOMASS AND ACTIVITY OF BACTERIA IN THE SEDIMENTS OF MARION LAKE, BRITISH COLUMBIA, by EDWARD ALFRED PERRY B. S c . , Yor k U n i v e r s i t y , 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t he department o f Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t , 197^ In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requ i rement s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I ag ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada i i ABSTRACTs THE BIOMASS AND ACTIVITY OF BACTERIA IN THE SEDIMENTS OF MARION LAKE, BRITISH COLUMBIA. Two biomass indicators (direct counts and ATP analysis) and two a c t i v i t y estimators (glucose uptake and dehydrogenase a c t i v i t y ) were used to study the bacteria at 1 m water depth i n Marlon Lake sediments. Direct count-biomass estimates for bacteria averaged 0 . 6 l gC/m^, were high i n summer, declined rapidly i n f a l l , then increased during the winter. Microorganisms less than approximately 30 jd m diameter had a mean biomass of 1.28 gC/m2 as measured by ATP analysis. Seasonal variations i n t h i s figure p a r a l l e l e d changes i n the a l g a l population, although a l g a l contributions to the microbial biomass were less than 50 per cent. ATP analysis was also used to estimate the biomass of the sediment community, excluding animals greater than approximately 5 mn> i n length. The mean community biomass was 4.69 gC/m2. Comparison of ATP data with enumeration data obtained by others, suggests that ATP i s a good biomass indicator, except when c e l l u l a r ATP l e v e l s are changed i n reaction to b i o t i c or ab i o t i c environmental factors. It i s proposed that, i n s i t u a t -ions such as intense grazing or rapid Increases or decreases i n temperature, ATP measurements r e f l e c t not only biomass but also a c t i v i t y . At such times ATP-biomass data may Indicate biomass potential, or the capacity of the population to maintain i t s biomass under abnormally high rates of l o s s . Dehydrogenase a c t i v i t y , or respiratory potential, of the sediment bacteria was assayed using trlphenyl tetrazolium H i chloride. The estimate for annual rate of carbon loss as CO2 from the b a c t e r i a l population (19.3 g C/m 2.year), was almost i d e n t i c a l to previously reported data obtained by measuring oxygen consumption i n normal and a n t i b i o t i c - t r e a t e d sediment cores. Uptake of -^C-glucose was determined i n mixed, d i l u t e d sediments. The maximum uptake rate (9.6 S C/m 2.year), natural uptake rate (5.3 S C/m2.year) and the natural turn-over time (0.31 h) were similar to previous data for these sediments. This suggests that, at least i n terms of function, the b a c t e r i a l population i s quite stable from year to year. Biomass and a c t i v i t y of the Marion Lake sediment bacteria were found to be i n phase i n late spring through early f a l l , but a c t i v i t y remained low i n winter despite Increases i n the microbial biomass, and increased much more r a p i d l y than bio-mass i n early spring. The dynamics of the b a c t e r i a l population are discussed i n terms of these r e l a t i o n s h i p s . The size of the a l g a l and b a c t e r i a l populations and organic matter reservoirs, and the fl u x rates for carbon between these compartments are summarized. i v TABLE OF CONTENTS Abstract i i Table of Contents i v L i s t of Tables v i L i s t of Figures v i i Acknowledgment i x Introduction 1 1 . Objectives and background 1 2 . B a c t e r i a l biomass - b a c t e r i a l counts 5 - ATP analysis 7 3 . A c t i v i t y of microorganisms - r e s p i r a t i o n and dehydro-genase (oxidoreductase) a c t i v i t y 10 - uptake of radiotracers 13 Introduction to the appendices 16 Materials and Methods 17 Sampling 17 Methods 19 Results and Discussion 27 1 . Physical and chemical data 27 2 . Biomass estimates 31 (a) Total sediment biomass estimated by ATP analysis 31 (b) Microbial biomass i n the sediments estimated by ATP analysis 39 (c) B a c t e r i a l biomass i n the sediments estimated by microscopic counts *EL V (d) Relations between biomass estimates 3. A c t i v i t y estimates 50 (a) Dehydrogenase a c t i v i t y measurements 50 (b) Heterotrophic uptake of ^C-glucose 53 (c) Relations between a c t i v i t y estimators 57 Biomass and a c t i v i t y of Marion Lake bacteria 60 (a) Qualitative seasonal description 60 (b) Quantitative description 65 5. Summary and Conclusions 68 References 71 Appendices 79 1. Method for extraction of t o t a l ATP from fresh sediments (based on Lee et a l . . 1971a). 79 2. Method for extraction of t o t a l ATP from lyoph--i l i z e d sediments. 81 3. Method for extraction of microbial ATP from fresh sediments. ' 83 Method for ATP assay. 8U-5. Recovery of ATP added to sediments. 87 6. Relation between ATP concentration and biomass carbon. 89 7. The e f f e c t of incubation time and oxygen on dehydrogenase a c t i v i t y measurements, 91 8. Calculations of heterotrophic uptake parameters. 95 v i LIST OF TABLES I. Carbon to ATP r a t i o s from various sources. 32 I I . S t a t i s t i c a l analysis of biomass data. 35-36 I I I . Sediment ATP - biomass carbon data. 38 IV. S t a t i s t i c a l analysis of dehydrogenase a c t i v i t y data. 52 V. Maximum uptake rates, and natural turnover times and uptake rates for glucose i n sediments and water. 56 A I. Methods tested for extraction of ATP from l y o p h i l i z e d sediments. 82 A l l . E f f i c i e n c y of the various ATP extraction methods. 88 AIII. The r e l a t i o n s h i p between ATP, biomass carbon and c e l l numbers. 90 AIV. The ef f e c t of aerobic versus anaerobic conditions on dehydrogenase a c t i v i t y assayed at d i f f e r e n t depths i n the sediment. 93 v i i LIST OF FIGURES 1. Marion Lake - morphometry and sampling area. 18 2. Scanning electron micrographs of Marion Lake sediment p a r t i c l e s . 29 3. Seasonal changes i n temperature (a), t o t a l organic matter (b), and dissolved glucose (c) i n the sediments. 4. Seasonal changes of t o t a l biomass carbon i n l y o p h i l i z e d (a) and fresh (b) sediments and of microbial biomass carbon ( c ) . 5. Seasonal changes of b a c t e r i a l biomass carbon. ^3 6. Seasonal change i n the proportion of sediment biomass carbon i n b a c t e r i a l compared to t o t a l biomass- b a c t e r i a l compared to microbial biomass; microbial compared to t o t a l biomass, Ur$ 7. Seasonal changes of dehydrogenase a c t i v i t y i n the sediments. 51 8. Seasonal changes i n the sediments of maximum (v"m) and actual (U n) rates of glucose uptake, and the actual turnover time ( T n ) . 5*4-9. Diagrammatic representation of the seasonal r e l a t i o n s between a c t i v i t y and biomass of the sediment bacteria as proportions of t h e i r maxima, 62 10. Structure and function of the Marion Lake sediment ecosystem. 66 v i i i A 1. T y p i c a l standard curve f o r the ATP assay (May 18, 197*0. 86 A 2. E f f e c t of incubation time on dehydrogenase a c t i v i t y measurements. 92 A 3. Graphical representation i l l u s t r a t i n g the modified Lineweaver-Burk p l o t . 97 ACKNOWLEDGMENT This research was conducted as part of the Canadian IBP Marion Lake Project, located at the University of B r i t i s h Columbia, and funded by the National Research Council (NRC) of Canada. I would l i k e to acknowledge f i n a n c i a l support from NRC and from B r i t i s h Columbia Packers Limited and Fisheries Association of B r i t i s h Columbia. A l l the members of the Marion Lake team were h e l p f u l and encouraging throughout the project. In p a r t i c u l a r , I would l i k e to thank Drs. I. E. Efford, P. Kleiber, K, Ha l l and B. K. Burnison for t h e i r ideas and discussions. I would l i k e to acknowledge the constructive comments and c r i t i c i s m of the members of my research committee, including Drs. P. Larkln, H. Blackburn, K. H a l l and I. E. Efford. Dr Blackburn was very generous, allowing me to monopolize his research f a c i l i t i e s . F i n a l l y I would l i k e to thank Corinne Perry, for being the way she i s . INTRODUCTION 1 1. Objectives and background. Many b i o l o g i c a l c h a r a c t e r i s t i c s of Marion Lake, B r i t i s h Columbia, have been studied i n d e t a i l i n an e f f o r t to describe the rate and control of energy flow between the major compartments of the system (Hall and Hyatt, 197-+). The studies since 19&3 were supported by the International B i o l o g i c a l Program as part of i t s commitment to assess productivity i n diverse environments. This emphasis on ecological energy flow stems h i s t o r i c a l l y from the q u a l i t a t i v e d escription of food webs by Forbes i n I887 (quoted i n Odum, 1968) and the energy flow by trophic l e v e l concept i n t r o -duced by Lindeman (19^2). The quantitative approach to aquatic ecosystem des c r i p t i o n using the Lindeman concept has been used In S i l v e r Springs, F l o r i d a (Odum, 1957). a temperate cold spring (Teal, 1957) and a s a l t marsh (Teal, 1962). Marion Lake Is situated 50 km east of Vancouver, B r i t i s h Columbia, at an elevation of 300m. The climate Is t y p i c a l l y coastal being r e l a t i v e l y mild and wet with an annual p r e c i p i t a t i o n of 2^0 cm. The lake i s small (13 hectares), shallow (mean depth 2.^ m) and subject to short turnover time (as short as three days during periods of high water inflow). Planktonlc production i s extremely low due , d i r e c t l y or in d i r e c t l y , to the high f l u s h rate (Efford 1967. 1969). Recent work has therefore focused on determining compartment size and carbon or energy fluxes between the compartments of the benthic environment. 2 Al g a l biomass In the Marlon Lake sediments i s approx-imately 0 . 0 4 3 gC/m2 (Efford, unpublished). B a c t e r i a l numbers were estimated by Fraker, using plate counts, to vary from 5 x 10^ cel l s / m l i n winter to 2 x 10^ ce l l s / m l i n summer (unpublished). Using microscopic counting methods, Ramey (1972) and Burnison (unpublished) estimated numbers of bacteria at the sediment surface to be 10^ cel l s / m l and l O 1 ^ c e l l s / g respectively. Both these workers found a decrease with depth below 2 cm. H a l l and Hyatt (197*0, using Burnis-on' s data and an average c e l l volume of 0.36jjm3 derived a fresh weight of 3.9 mg/g sediment. This i s equivalent to approximately 0.39 gC/m2, or about nine times the a l g a l biomass. Net dissolved and pa r t i c u l a t e allochthonous inputs to the lake are 28 gC/m2/year and 36 gC/m2/year respectively (Geen, unpublished; Odum, unpublished). Autochthonous i n -puts include primary production by phytoplankton (8 gC/m2/ year - Efford, 1967)t epibenthic algae ( 4 0 . 4 - 44.2 gC/m2/ year - Hargrave,: 1969»Greundllng, 1971). Carbon losses as carbon dioxide from the benthic com-munity t o t a l 57 gC/m2/year (Hargrave, 1969). Estimates of b a c t e r i a l r e s p i r a t i o n were obtained by Hargrave by com-paring oxygen uptake i n normal versus a n t i b i o t i c - t r e a t e d cores but t h i s approach has been c r i t i c i z e d (Cameron, 1973; Yetka and Wiebe, 197*0. An estimate of 4.2 - 24 gC/m2/year for b a c t e r i a l r e s p i r a t i o n was obtained by Kleiber (1972) using the data of H a l l et a l ^ (1972). 3 Within the sediment ecosystem, transfers from the chemical environment to microorganisms i n the form of dissolved and particulate material have been studied by Hall et a l ^ (1972) and Hall et a l ^ (1973), using r a d i o -active chemicals and leaf material. Klelber (1972) reported the flux from algae to the chemical environment was 1 . 5 -8 . 8 gC/m2/year. Using the data of Hall et a l ^ (1972) he further estimated the oarbon flux from the environment to bacteria to be 21 - 120 gC/m2/year. The importance of the benthic bacterial population i s evident from these data. Total net inputs to the lake are about 132 gC/m2/year and the use of organic material by bacteria (21 - 120 gC/ m2/year) is equivalent to 16 - 91# of this net input. The purpose of this study was to assess biomass and activity of bacteria i n Marion Lake sediments. As described above some measurements have been obtained but the bacterial biomass was not measured during the course of the hetero-trophic uptake studies (Hall et a l . . 1972) fand bacteria have not been quantified seasonally except by plate count methods. No approach to either biomass or activity measurement is accepted as a "standard method". Many papers report inform-ation on various parameters of microbial populations but methodology i s diverse. A few studies comparing multiple approaches simultaneously have been reported (Witkamp, 1973. i n soils* Hobble et a l . . 1972, In the ocean). Work comparing two methods is more abundant and Is referred to In the discussion. During this study, two biomass estimators (adenosine-4 5'-triphosphate (ATP) and direct microscopic counts) and two activity estimators (dehydrogenase activity and ^C-glucose utilization) were employed. The changes,within and between these methods,seasonally, are presented. 5 2 , Bacterial Biomass Bacterial counts Plate count methods have long been used to estimate the numbers of microorganisms i n water, soils and sediments. Many reports on various experiments ranging from response to f e r t i l i z a t i o n to vertical distribution of bacteria i n soils s t i l l use this approach. There i s no doubt that the technique is useful for looking at specific physiological groups of microorganisms but the use of the aerobic, hetero-trophic plate count as an indicator of total viable bacterial numbers i s invalid (Schmidt, 1973) . Microscopic counts of bacteria with conversion of the numbers to biomass using an average c e l l volume have a number of inherent d i f f i c u l t i e s . Besides being time con-suming, i t i s d i f f i c u l t to differentiate between detrital particles, dead bacteria and viable bacteria. In sediments particularly, many particles are in the size range of bacteria and many of these adsorb chemicals used to stain c e l l s . The irregular shape of many bacteria makes accurate estimation of c e l l volume d i f f i c u l t . The technique was used i n this study to give an upper limit for the bacterial biomass. Direct counting methods have been employed i n diverse environments including water (Sorokin, 1970i Hobble et a l . . 1972) , s o i l (Babiuk and Paul, 1970$ Trolldenier, 1973)# beach-sands (Khiyama and Makemson, 1973) and sediments (Olah, 1972) . Fluorescent stains are helpful in making small bacterial 6 cells on particles more v i s i b l e . Quantitlve work using fluorescent stains such as acridlne orange (Trolldenier, 1973) and fluorescein isothiocyanate (FITC) (Babiuk and Paul, 1970) has been reported. One problem involved i n the method is to obtain a sample uniformly dispersed on a microscope slide so that random sampling yields results with minimal variance. In previous research, samples have been spread in thin layers on slides and allowed to dry fTrolldenier, 1973. Babiuk and Paul, 1970) or thin layers of agar containing s o i l samples were formed (Jones and Mollison, 19^8), Unfortunately materials prepared In this manner may have a variable vertical distribution so repeated focusing &f the objective lens i s required because of Its short depth of f i e l d . In addition the objective of uniform d i s t r i b -ution i s often not achieved. An ideal way to ensure random dispersion of samples would be by f i l t r a t i o n through bacteria-retaining membranes. Previous use of this technique has been limited to light microscopy probably because of interference in fluorescent work from the o i l used to clear the f i l t e r s . A method, devised by B. Kent Burnison (unpublished), which employs f i l t r a t i o n of samples, clearing In acetone vapors and FITC staining has been used i n this study. This technique allows cells to be uniformly distributed with negligible background fluorescence and the cells are bound i n the surface layer of the cleared membrane f i l t e r . 7 ATP Analysis ATP is present i n a l l liv i n g c e l l s (Mahler and Cordes, 1966) but is not associated with dead cells (Holm-Hansen and Booth, 19661 Lee et a l . . 1971asPatterson et a l ^ , 1970 j this study). Presence of ATP i s therefore indicative of l i f e . Knowledge of numerical relations between ATP and organic carbon associated with li v i n g organisms (biomass carbon) permit extrapolation of ATP concentrations to biomass carbon. This relationship has been studied in bacteria (Ausmus, 1973? Ernst, 1970j Hamilton and Holm-Hansen, 1967) , algae (Berland et a l . . 1972; Ausmus, 1973) actinomycetes (Ausmus, 1973)» fungi (Ausmus, 1973) and nematodes (Ernst, 1970) . Consider-able work has been done determining ATP content per bacterial c e l l (for example Chapelle and Levin, 1968) but information on numbers is less relevant to ecosystem studies than i s in^ formation on biomass. The ATP to biomass carbon relation varies between groups of organisms, between species and within species at different times of the l i f e cycle. Despite these d i f f i c u l t i e s a weight relation of carbon to ATP of 250 seems to be meaningful. The ATP assay i s based on the finding of McElroy (1947), that luminescent reactions i n f i r e f l y t a i l extracts require ATP. Since then, light production has been shown to be proportional to ATP i f other reactantes are i n excess (Strehler, 1965)» Mg ATP + l u c i f e r i n ». adenyl-luciferin +PPi luciferase 8 o 2 adenyl-luciferln •» adenyl-oxyluciferln + H2O + light ATP has been measured i n fresh water (Rudd and Hamilton, 1973- Holm-Hansen and Paerl, 1972) , oceans (Hobble et a l . , 1972; Holm-Hansen and Paerl, 1972) , sewage (Patterson et a l . . 1970; Brezonik and Patterson, 1971) , soils (Ausmus, 1973j Conklin and MacGregor, 1972j MacLeod et a l . . 1969) , marine sediments (Ernst,' 1970) and lake sediments (Lee et a l . , 1971 a and b). Only two previous studies on temporal changes of biomass as measured by ATP have been reported, that being the work of Rudd and Hamilton (1973) and Holm-Hansen and Paerl (1972) on lake water columns. Application of the method to estimation of bacterial biomass is d i f f i c u l t . Measurements in water masses are often preceded by f i l t r a t i o n through nets (60 - 150/jm mesh) to remove large gooplankton (Holm-Hansen and Paerl, 1972) , but analyses s t i l l include bacteria, algae, protozoa and many other organisms. These authors further attempted to measure bacterial biomass by subtracting algal biomass,determined by direct counting, from total biomass of organisms less than 60 jm measured by ATP analysis. Even i n this size range there i s the possibility of protozoa biomass being included. Another problem is the uncertainty of the condition of the algae. Direct counts often include dead c e l l s , so overestimatlon of algal biomass and therefore underestimation of bacterial mass could result. Rudd and Hamilton (1973) followed changes i n biomass of various size groups by differential f i l t r a t i o n of water samples but no estimate of bacterial biomass was 9 obtained because of the presence of small algae less than 10jum, particularly during summer. Size fractionation, while not simple nor precise i n aquatic samples, i s even more d i f f i c u l t i n sediments. No previous attempts have been made to categorize biomass of different size groups by ATP analysis in soils or sediments. In this study, an estimate of the microbial biomass (<approximately 30 pm) was ob-tained by physical removal of larger size classes. 3 . Act iv i ty of Microorganisms 10 It i s d i f f i c u l t to assess what microorganisms are doing in s i t u . It i s not possible to "see" what they are eating, assimilating and excreting and as a result chemical and radio-tracer techniques are used. Respiration and dehydrogenase (oxidoreductase) act iv i ty The tradit ional method for determinimg microbial act iv i ty Involves measuring respiration by changes in oxygen and/or carbon dioxide concentrations (Hargrave,19^9> Beyers et a l . . 1963) . The oxygen method was used rigourously in Marion Lake for one year but the va l id i ty of carbon flow information from this data depends on the accuracy of the assumption of a respiratory quotient (RQ)of 0 .85 (Hargrave, I 9 6 9 ) . Unfortunately, RQ values for sediments range from 0 .27 -O.96 (Teal and Kanwisher,1961} Pamatmat, 1968) and for epilithophyton the value of 1 . 1 + 0.9 has been reported (Schindler et a l ^ , 1973) . The measurement of inorganic carbon is d i f f i c u l t in lakes that do not exhibit re lat ive ly high alkal in i ty ,unless sophisticated equipment is available at the s i te (Schindler, 1973) . One conclusion from the above discussion is that reports of carbon exchange based on gas analysis are often relat ive rather than absolute. Another way to estimate respirat ion is by measuring dehydrogenase act i v i t y . In such assays triphenyl tetrazolium chloride (TTC) i s used to intercept electrons flowing through the electron transport system , 11 and competes with oxygen as electron acceptor. This assay Is a relat ive act iv i ty estimator since i t measures respiratory potential rather than respirat ion, but i t i s a convenient method for comparing act iv i ty of samples given different treatments or obtained at different times of the yeais. Dehydrogenase act iv i ty has been used as an act iv i ty index In water (Olah, 1972) , sewage (Lenhard, 1968) , s o i l (Casida et a l . . 196^1 Lenhard, 1956) and sediments (Olah, 1972; Pamatmat and Bhagwat, 1973; Edwards and Rolley, 1965) . The experimental approach suggests there should be a correlation between respirat ion as measured by oxygen up-take or carbon dioxide evolution and respiratory potential as measured by dehydrogenase. Such correlation has been demonstrated in some s o i l studies (Skujins, 1973; Casida et a l t . 196*0 but not others (Howard, 1972) . Stevenson (1959) found oxygen uptake was related to dehydrogenase act iv i ty in 2k so i ls (r = 0.837) but in soi ls which were amended with decomposing plant matter correlation was reduced (r * 0 . 5 H ) . Howard (1972) reported that formazan production was always less than expected from oxygen consumption data. This may be due to a negative response by microorganisms to the presence of TTC since the insoluble reduced formazan i s deposited within the c e l l s . Furthermore,the substitution of TTC in place of oxygen as electron acceptor reduces the amount of ATP (energy) obtained by the c e l l per mole of substrate reduced. Pamatmat and Bhagwat (1973) reported correlation between heat production and dehydrogenase act iv i ty in Lake Washington sediments but oxygen uptake by sediment cores consistently 12 underestimated heat production, presumably because oxygen uptake did not measure anaerobic act iv i ty while the de-hydrogenase assay d id , Edwards and Rolley (19^5) also found no correlation between oxygen consumption and dehydro-genase act iv i ty In sediments. That both anaerobic and aerobic bacter ial act iv i ty could be assayed by TTC reduction was demonstrated by Clah (1972) , He followed ATP levels and dehydrogenase act iv i ty in aerated and anaerobic incubation flasks enriched with powdered Phragmltes. During aerobic incubation changes in dehydrogenase seemed to para l le l changes in ATP, but anaerobic conditions yielded higher ATP concen-trat ion and lower dehydrogenase act iv i ty than was found in the aerated culture. Sediments are a complex environment,and even anaerobic sediments may contain a community with populations other than bacteria (Fenchel, 1969) . Does the dehydrogenase measurement assay the act iv i ty of these other organisms? Pamatmat and Bhagwat (1973) noted the presence of chlronomid larvae in some of their samples but did not know whether or not their act iv i ty was included in dehydrogenase measurements. Packard (1970) and Curl and Sandberg (1961) homogenized animal tissues to assay dehydrogenase act i v i t y , Packard noted the necessity to disrupt not only the outer walls of zooplankton but also the mitochondria before the assay could be performed. Bacteria however can reduce TTC during normal growth (Eidus et a l . . 1959) . On the basis of present know-ledge i t seems probable that the dehydrogenase assay measures act iv i ty of prokaryotlc organisms only, unless 13 samples are homogenized. The enzyme dehydrogenase was assayed during this study to determine aerobic and anaerobic, prokaryotic act iv i ty at different times of the year. Uptake of radiotracers The u t i l i za t ion of radioactive compounds by micro-organisms is another approach to the estimation of microbial ac t i v i t y . The most common experimental design for u t i l i za t ion measurements is to test uptake at different concentrations of the radioactive substrate. These data are treated by one or more of the available l inear transformations (Hall et a l . . 1972) of the Michaelis - Menten enzyme kinetics equation. Information may be obtained on the turnover time of the substrate (T), the maximum rate of substrate uptake (Vm), and a transport constant plus natural substrate concen-t rat ion (Kt + S n ) . If S n can be determined independently the actual rate of uptake (Un) may be calculated. The or ig inal low substrate concentration application of Michaelis - Menten kinetics analysis to study active uptake by heterogeneous bacter ial populations was by Wright and Hobble (1965). The work of Parsons and Strickland (1962) was at substrate concentrations high enough that di f fusion. into algae probably occurred. Wright and Hobble (1966) assumed respiratory losses of carbon as CO2 would be negligible but many studies (Hall et a l . . 1972j Burnlson and Morita, 19731 Crawford et a l . . 1974) have disproved th i s . Most experimenters 14 now use modified techniques which allow measurement of gross uptake, that i s , the sum of assimilated or p a r t i c u l a t e uptake and respiratory losses. There is,unfortunately, no t h e o r e t i c a l j u s t i f i c a t i o n for a p p l i c a t i o n of Michaelis - Menten k i n e t i c s to hetero-geneous populations.The a n a l y t i c a l technique, o r i g i n a l l y r the Langmuir isotherm of Michaelis - Menten equation, was developed to describe r e l a t i v e l y simple reactions of gases or enzymes. In fact,the equation has been found inapplicable i n a number of studies i n natural systems (Vaccaro and Jannasch, 1967; H a l l et aXg., 19721 Kleiber, 1972» Crawford et al^., 1974):. The use of i t to analyze multispecies reactions with v a r i a b l e values of V m and Kt was tested with a computer model by Williams (1973) . He reported that (Kt + S n) and T are sensitive to deviations from the expected r e l a t i o n between uptake rate and substrate concentration, p a r t i c u l a r l y at low concetrations,but V m showed l i t t l e change. Since natural substrate concentrations are often very low ( 1 -50 / i g / l ) , erroneous estimates for T would i n turn e f f e c t U n c a l c u l a t i o n s , Burnison and Morita (1973) tested the occurrence of competitive i n h i b i t i o n for amino a c i d uptake i n Klamath Lake waters. Even at low substrate concentrations, competition was evident i n some cases. This i s i n contrast to the report of Crawford et a l . ( 1 9 7 4 ) ,that competition between amino acids i n estuarine waters seemed to be of l i t t l e consequence. Burnison and Morita found that V m was not effected by com-p e t i t i v e I n h i b i t i o n , while T and (Kt + S n) were both i n -15 creased. From the discussions of Williams (1973) and Burnison and Morita (1973), i t i s apparent that V m i s the most useful parameter for comparing heterotrophic uptake p o t e n t i a l i n d i f f e r e n t water masses because i t i s less sensitive than T and (K^ + S n ) , and therefore U n, to changes i n i n h i b i t o r y i n t e r r a c t i o n s between substrates and to non-kinetic responses of uptake v e l o c i t y to substrate concentrations V m i s a " p o t e n t i a l " uptake estimate though,and U n and T n (the actual rate of uptake and turnover) are the values required for estimates of carbon f l u x i n an ecosystem. The actual rates for glucose have been used i n t h i s study as an index of microbial a c t i v i t y . 16 k. Introduction to the appendices A number of diverse topics have been relegated to the appendices in an attempt to make the methods and results sections as concise as possible. It i s hoped that compre-hension of the work does not require reference to the appendices. Appendices I - III are detai ls of methods used to extract ATP in three different experimental situations. Appendix IV includes preparation of enzymes used to measure ATP concentrations, and an example of a standard curve obtained for the assay. Appendix V describes the approach and results of exper-iments designed to estimate the eff iciency of the three ATP extraction procedures. Appendix VI describes the approach and results of expert-lments designed to study the re lat ion between biomass carbon and ATP. Appendix VII outlines the effect of incubation time and oxygen concentration on the assay for dehydrogenase ac t i v i t y . Appendix VIII gives the mathematical analysis used to calculate V m , T, (K t + S n ) , T n and U n in hetero-trophic uptake studies. MATERIALS AND METHODS 17 Sampling Undisturbed sediment samples (Hargrave, 1969) were obtained along a i m transect at approximately monthly intervals (Figure 1) . From each of four samples, three subsamples were taken with glass corers (12.5 cm long, 5.0 cm diameter). The cores were sectioned at the s i te and the upper 2 cm of sediment were transferred to a s ter i le Jar. The mixed sample was kept on ice during transport, then stored at 2 - 3 ° . A l l analyses requiring fresh sediment (ATP,total organic matter, dehydrogenase act iv i ty and radiotracer experiments) were carried out within 2 - 3 days. Within 2 - 3 hours of sampling, i n t e r s t i t i a l water for o glucose analysis was extracted and stored at -20 and sediment subsamples were lyophil ized for later use (direct bacter ial counts, ATP and carbohydrate analyses). 1.8 Figure 1. Marion Lake - morphometry and sampling area ( ). 18 a Methods Chemical analyses Duplicate fresh samples for t o t a l organic matter were o f i l t e r e d (10)psi) through previously combusted (550 /lh) g l a s s - f i b r e f i l t e r s (Reeve-Angel 93^ AH - 2 A cm d i a . ) . The samples were dried (80 /2kh), weighed, combusted (550 /3h) then reweighed. Carbohydrate concentrations i n the l y o p h i l -ized sediments were measured c o l o r i m e t r i c a l l y with the phenol-sulphuric acid method (Gerchakov et a l . , 1972) . The assay was standardized with ^-D-glucose. Organic carbon content of the sediments was analyzed using a carbon analyzer (Beckman model 9 1 5 ) . Methane production was measured by analysis of the atmos-phere above sealed sediment cores (obtained A p r i l 2 1 , 1974) , incubated at either k or 20 for 3 days, using gas chromato-graphy. To obtain I n t e r s t i t i a l water approximately 100 ml of mixed sediment were g r a v i t y - f i l t e r e d through Whatman no. 1 f i l t e r paper at k . The f i l t r a t e was f i l t e r e d through a M i l l i p o r e GS membrane f i l t e r , pore size 0 .22^111 using a vacuum of 7 p s i . The a water was stored at - 2 0 i n clean test tubes. Glucose concentrations i n the i n t e r s t i t i a l water were determined enzymatically by the method of Hicks and Carey (1968) . A fluorometer (Turner no. 110) , equipped with a green phosphor-escent lamp (GE-F4-T5-G), and a constant temperature door (25 )• was used to measure unknown glucose concentrations r e l a t i v e to standard solutions of B-D-glucose. F i l t e r s used were, on the ex-c i t a t i o n side, a Wratten 58 , a \% neutral density and a polaroid lens , on the emission 20 side, a Wratten 23A. Blanks were identical to standard or i n t e r s t i t i a l water samples except glucose-6-phosphate dehydrogenase was omitted from the reaction mixture. The detection l imit for the assay i s about 1/igglucose/l. Direct counts of bacteria Bacteria were counted in samples prepared from lyoph-l l i z e d sediments using a method developed by B. Kent Burnison (unpublished). A l l solutions were f i l t e r - s t e r i l i z e d . To 2 ml of d i s t i l l e d water plus 0.5 ml of 0.5 N KOH in a tissue homogenizer tube, was added lOmg of lyophil ized sediment. The dry weight was determined by drying para l le l samples (100°/24h). The sample was homogenized for 2 min at which time very few,or no,large part icles remained. The mixture was transferred to a s c i n t i l l a t i o n v i a l using 4 ml of d i s t i l l e d water and sonicated for 15 sec at 50-60 cps with an intensity of 50 (Bronwill Sc ient i f ic Biosonik II). The sample was then adjusted to 100 ml i n a volumetric f lask with d i s t i l l e d water, and mixed. From this suspension 1.00 ml was transferred to a f i l t r a t i o n apparatus and adjusted to 10 ml with water. The sample was f i l te red (10 psi) through a membrane f i l t e r (0.22>um) pre-viously boiled in 0»1% sodium pyrophosphate for 2 min and o rinsed with bacteria-free water. The f i l t e r was dried at 60 . One-half the area of a sl ide was coated with transparent glue. The dried f i l t e r was placed on this f i lm, then cleared in acetone fumes, a i r dried and sealed with Permount. The ce l ls were stained with a solution of FITC using the buffer system suggested by Babiuk and Paul (1970). Cel ls 21 were stained for 30 min with a s o l u t i o n of 1.3 ml of 0.5 M sodium carbonate buffer (pH 9.6), 6.0 ml of 0.01 M potassium phosphate buffer (pH 7.2), 5.7 ml of 0.8# saline and 5.3 mg of FITC. The s l i d e s were washed i n 0.5M sodium carbonate buffer (pH 9.6) for 20 min, and i n 1% sodium pyrophosphate for 2 min. Duplicate s l i d e s were prepared f o r each sample and c e l l s i n 15 ocular g r i d f i e l d s per s l i d e counted under o i l immersion using a Reichert microscope equipped with a HBO 200 mercury vapour lamp. ATP Analysis Determination of unknown ATP concentrations requires extraction of the compound and assay of i t s abundance i n the extract. A correction factor for extraction e f f i c i e n c y must be determined. The methods summarized below are presented i n d e t a i l i n appendices I - VI. (a) Total ATP extraction from fresh sediments The method used was that of Lee et a l . (1971a). Three r e p l i c a t e 3 ml samples of mixed sediment (150-200 mg dry wt.) were extracted for each monthly sample. The main features are extraction of ATP with i c e - c o l d 0.6 N H2SO4 and removal of i n t e r f e r i n g cations with cation exchange r e s i n (for d e t a i l s see appendix I ) . (b) Total ATP extraction from lyophlized sediments In an attempt to check the fresh sediment ATP values obtained each month, l y o p h i l l z e d sediments which had been o stored for up to 1 year at -20, were extracted on one occasion and assayed using single enzyme and standard ATP preparations. Bromosuccinlmide extraction was found to be the most e f f i c i e n t of eight procedures tested (Appendix II) and was used for subsequent experiments (for d e t a i l s see appendix I I ) . (c) Microbial ATP extraction from fresh sediments Sediment samples were cleaned of large organisms (< approximately 30/"") using micropipettes i n an attempt to measure microbial ATP. The separation method and subsequent ATP extraction i s described i n appendix I I I . (d) ATP assay The method used was the l u c i f e r l n - l u c i f e r a s e biolum-inescence assay. Preparation of the enzymes i n appropriate buffer solutions, a d d i t i o n of standard or unknown ATP samples and measurement of l i g h t production are described i n appendix IV. (e) C a l c u l a t i o n of biomass carbon concentration The equation used for conversion of raw assay data to biomass carbon per gram dry weight sediment wast biomass ATP x 1 x d i l u t i o n x 1 x carbon x 10" assayed sample factor extraction ATP dry weight e f f i c i e n c y The assayed ATP had units ng/mlt dry wt. was i n gramsi the d i l u t i o n factor was 5 for experiments on l y o p h i l i z e d s e d i -ments and microbial ATP and (50 x extract volume (ml) x &) for t o t a l ATP i n fresh sediments? extraction e f f i c i e n c y was .125, .106 and .205 for t o t a l fresh, t o t a l l y o p h i l i z e d and microbial fresh ATP extractions respectively (Appendix V)t C/ATP was 250 (Appendix VI)» the factor 10  J converted the 23 data from ng to g. Dehydrogenase act iv i ty Dehydrogenase act iv i ty was measured using a method modified from that of Sorokin and Kadota (1972). The reagent consisted of a solution of 0.1 M t r i s buffer,adjusted to pH 7.5 with 2N HCl,to which was added 0.9 M 2, 3, 5 - t r l -phenyltetrazolium chloride (TTC). This reagent was stored at 2° in the dark. To perform the assay, 5 ml of fresh mixed sediment was pipetted into a 125 ml erlenmeyer f lask . Control samples were routinely steam-killed then treated as normal samples. Blanks of this type were equivalent to adding formalin, or to leaving TTC out of the reagent. To each sample 10 ml of the TTC reagent was added. Rubber stoppers with inlet and outlet tubes were f i t ted and the samples were bubbled with nitrogen for 10 min to remove oxygen. The flasks were sealed, o wrapped in aluminum f o i l and Incubated 4 h and 24 h at 30 with shaking at 100 RPM. Four replicates and one control were used for each incubation period. Extraction of the water-insoluble formazan was with an acetone-methanol solution (9/l» V/V). Twenty-five ml was added to sample f lasks, shaken for 1 h, then f i l te red through cheesecloth under subdued l ight conditions. The f i l t r a t e was adjusted to 50 ml with extractant poured through the sediment part ic les . Absorption was measured at 540 ^ against an acetone-methanol blank. AAstandard curve was obtained by dissolving 2, 3, 5-24 triphenyl formazan In extractant sol u t i o n . The e f f e c t s of incubation time and oxygen on dehydro-genase a c t i v i t y measurements are described i n appendix VII. Heterotrophic uptake of -glucose A l l experiments were performed using fresh mixed sed-iments, d i l u t e d with autoclaved lake water ( f i n a l d i l u t i o n 50»1). Standard solutions of ^ C-glucose (U), 215 mc/mM (New England Nuclear), were stored frozen i n f i l t e r - s t e r i l i z e d d i s t i l l e d water. These solutions were adjusted to 0.1 >ic/ml and l . O y u g glucose/ml p r i o r to use. Two l i v e and one control (formalin-killed) samples were equilibr a t e d for 1 h at lake temperature a f t e r d i l u t i o n . The radiotracer was then added at four solute concentrations (10, 40, 100, 200JJ g / l ) , and incubated under subdued l i g h t conditions for 40 or 60 min with shaking at 100 RPM. The Incubation f l a s k s , ^C-CC^ trapping system and f i l t r a t i o n of the pa r t i c u l a t e f r a c t i o n were described i n d e t a i l by Kleiber (1972). Bray's s c i n t i l l a t i o n s o l u t i o n (Bray, i960) was used for sample r a d i o a c t i v i t y measurements. Quench curves were pre-pared with ^C-toluene (417,000 dpm/ml, New England Nuclear) and chloroform i n Bray*s s o l u t i o n for the l i q u i d s c i n t i l l a t i o n counters used (Nuclear Chicago Mark I and Isocap 300), by the external standard r a t i o (ESR) method. i l l E f f i c i e n c y of C-CO2 counting was determined by com-paring the sample ESR to the quench curve. This value was further corrected f o r ^C-CC^ trapping e f f i c i e n c y (mean of 84# i n 4 samples using lZ*C-Na H CO3). The e f f i c i e n c y of counting p a r t i c u l a t e material sus-25 suspended with Aer - 0 - S i l (Degussa Chemicals) In Bray's so l u t i o n was calculated from an e f f i c i e n c y versus weight -of-sediment curve. The curve was obtained by incubating sediment with ^ C - glucose overnight. Aliquots of d i f f e r e n t volumes were f i l t e r e d through tared membrane f i l t e r s (0.22 JJ m pore s i z e ) . The samples were dried (100°/24 h) and dry wt. determined. One-half of the samples were combusted at 900° In a tufee furnace (Lindberg Hevi-Duty model 55035), The evolved lifC-C02 was c o l l e c t e d i n 8 ml of ethanolamine and ethylene g l y c o l monomethyl ether (1/7, V/V) and counted i n 10 ml of toluene f l u o r (0,5% PPO, 0.03% POPOP). The method has been described by Burnison and Perez (1974). Suitable quench curves were prepared for the toluenesascintillation solution. The remainder of the samples were counted i n the usual way, that i s d i s s o l u t i o n of the f i l t e r s i n Bray*s so l u t i o n and suspension with Aer - 0 - S i l . Comparison of the dpm per g dry wt. obtained by combusting samples, to cpm per g dry wt. by suspending samples, yielded an efficiency-weight curve. Sediment counting e f f i c i e n c y ranged from 54.2 - 58,7#. The methods used to calculate V m, T t, U n and T n are described i n appendix VIII, S t a t i s t i c a l methods Seasonal data obtained from ATP and dehydrogenase experiments and b a c t e r i a l counts were analyzed for d i f f e r -ences between the means by analysis of variance (anova). Logarithmic transformations were required for dehydrogenase data and for the t o t a l fresh ATP data to correct hetero-26 geneous variance within the data. S p e c i f i c means were then compared at the \% p r o b a b i l i t y l e v e l using the new multiple range test of Duncan ( 1 9 5 5 ) . Heterotrophic uptake data were not s t a t i s t i c a l l y analyzed due to the complexity of obtaining information on the errors involved, Kleiber (1972) has proposed a method to handle t h i s problem. 27 RESULTS AND DISCUSSION 1. Physical and chemical data The sediments in Marlon Lake are a deep flocculent ooze except near the springs and the in le t . The sample area was free of macrophytic growth. Macroscopically the sediments contain some large invertebrates, a lgal colonies and mats, chironomld tubes and leaf and twig fragments. The sediment part ic les as seen microscopically are a complex conglomeration of mineral and organic matter with a size range of 20 - bOOpm although most are between 70 -200yUm (Figure 2 ) . The part icles shown were prepared by c r i t i c a l point drying and coated twice with gold, then examined using a Cambridge Stereoscan scanning electron microscope. Temperature during the study was typical of Marlon Lake (Efford, 19&7), although the warming trend usually evident i n May was delayed in 197*+ (Figure 3»). Changes in total organic matter in the sediments were not dramatic, f luctuating around a mean of 414 mg/ g dry wt. except in September (Figure 3b). The maximum deviation within samples was 3*6% of the mean. The Sep-tember peak may be due to a lgal or invertebrate growth within the lake,or allochthonous inputs although the latter do not peak unt i l late f a l l (Odum, unpublished). Organic carbon (dissolved plus particulate) was about 20% of the sediment dry weight. Glucose concentrations in the i n t e r s t i t i a l water were 28 high i n December and January (about 50pg/l) and very low i n A p r i l and May (5 -20j jg/l) (Figure 3 c ) . The low spring concentrations may r e f l e c t heterotrophic a c t i v i t y of bacteria, a p o s s i b i l i t y which i s assessed i n section 4 , T o t a l carbohydrates i n the sediments fluctuated i r r e g u l a r l y with an annual range of 135-184 mg/g dry wt. or 3 2 . 6 - 4 4 . 5 # of the t o t a l organic matter. These carbohydrate concentrations are approximately twice as high as those previously found by H a l l and Doel (1972) and may r e f l e c t v a r i a t i o n s i n methodology. Figure 2. Scanning electron micrographs of Marion Lake sediment p a r t i c l e s . 2 9 * Figure 3. Seasonal changes i n temperature (a), t o t a l organic matter (b) and dissolved . glucose (c) i n the sediment (bars indicate standard deviation of each sample)• 31 2 . Biomass estimates (a) Total sediment biomass estimated by ATP analysis Recovery of ce l lu lar ATP added to sediments was low in this study (12.5% for the fresh sediment extraction method - Appendix V) compared to other work that has been reported. Lee et a l . (1971a) obtained recoveries of ATP added as bacteria of 2^-85% for nine different sediments. Ernst (1970) reported recoveries of ce l lu lar ATP and pure ATP added to marine sediments of 60-97# and 6l-104# respect-ively . Low recovery eff iciency in Marion Lake sediments may be due to high adsorptive capacity, however, pure ATP added to these sediments was recovered at 52-63/6. This indicates the poor recovery is related to extraction from the ce l ls i n addition to subsequent recovery from the extraction mixture. Ce l l carbon to ATP ratios vary considerably between species (Table I). Although ATP per c e l l also varies a great deal within the l i f e cycle of a single species (Ausmus, 1973» Lee et a l . , 1971b) , the ATP to carbon rat io may remain re lat ive ly 'constant unless energy sources are completely exhausted (Harrison and Maitra, 1969? Holms et a l . . 1972) . The conversion of ATP concentrations to c e l l or biomass carbon by a factor of 250 (Hamilton and Holm-Hansen, 19#7) was supported by experiments during this study (Appendix VI). It i s recognized that biomass carbon estimates thus obtained are subject to error but i t is the best estimate avai lable. The tota l biomass carbon at 1 m water depth in Marion 32 Table I. Carbon to ATP r a t i o s from various sources references organism Ausmus (1973) bacteria - 5 s p p . fungi - 8 spp. actinomycetes-6 sppV algae - 6 s p p . Berland et a l . algae - 7 s p p . (1972) Hamilton and V i b r i o spp. Holm-Hansen (1967) bacteria - 7 s p p . Holms et a l , (1972T Appendix VI Escherichia c o l l growth stage exponential stationary 1" exponential exponential and stationary v a r i e t y of growth rates bacteria - 3 s p p . Streptomyces spp,  Anacystls nldulans stationary C/ATP r a t i o range (mean) 357-833(500) 179-313(233) 179- 238(217) 104-278(143) 180- 592(366) 1220 153 91-333(250) 485-808 112-439(281) 376 218 The authors assumed carbon content per c e l l was the same i n stationary and exponential phase. 33 Lake sediments, as measured by ATP analysis, is presented in Figure 4 b . This graph summarizes data obtained by extraction of fresh sediment samples. Two features of these data are the peaks in May each year and the homogenity of the tota l biomass throughout the remainder of the year. A posteriori comparisons among means by the new multiple range test (NMRT) (Table II) indicate that biomass in May, 1973 was s igni f icant ly higher (P<.©1) than on any other sampling date. Biomass estimates in June, 1973 and May, 1974 were not s igni f icant ly different (joined by a l ine), but are s igni f icant ly higher than samples in July to A p r i l . A l l other samples were not s igni f icant ly different from each other (joined by a l i ne ) . Analysis of to ta l ATP In lyophil ized sediments con-firmed the peaks in May each year but introduced much more var iab i l i t y between other samples (Figure 4 a , Table I I ) . The increased var iab i l i t y compared to fresh sediment extract-ion and the high biomass in May, 1974 and December, 1973 may result from the fact that the sample size was only 20 mg dry wt. for the lyophil ized samples compared to 1 5 0 -200 mg dry wt. for the fresh samples. The biomass estimate is generally higher using lyophil ized sediments compared to fresh sediments (Figures 4 a , b) fbut this may be due to an inaccurate estimation of extraction eff ic iency for lyophil ized sediments. The eff ic iency of 10,6% was based on only one experimental organism, compared to fresh extraction eff ic iency which was measured using six organisms (Appendix V). The 34 Figure 4. Seasonal changes of t o t a l biomass carbon i n l y o p h i l i z e d (a) and fresh (b) sediments and of microbial biomass carbon ( c ) . Micro biomass carbon (g/m2) Total biomass carbon -fresh (g/mz) Total biomass carbon - l y o p h i l i z e d (g/m2) 35 Table I I . S t a t i s t i c a l analyses of biomass data. 1. Total fresh sediment ATP analysis Analysis of variance (Anova) F source df. SS ms among samples 11 .6635 .0603 within samples 23 .1849 .0080 t o t a l 34 .8484 F.01(10, 23) = 3.21 s i g n i f i c a n t at P<0.01 New multiple range te s t (NMRT) - Samples are ranked by increasing mean values. Lines j o i n samples not s i g n i f -i c a n t l y d i f f e r e n t at the 1% l e v e l . Nov Feb Dec Oct Apr 1 Sep Jan J u l Apr 21 June May 74 May 73 2. Total l y o p h i l i z e d sediment ATP analysis Anova source df ss ms F among 11 78.6379 7.1489 11.6124 within 13 8.0032 .6156 t o t a l 24 86.6411 F.01(10, 13) s NMHT J u l Apr 1 June Nov Oct Feb Jan Apr 21 Sep Dec May 73 May 74 (continued) 36 3 . Microbial ATP analysis Anova source df among 8 within 9 t o t a l 17 F.01(8, 9) 8 3 5 .4? F . 0 2 5(8, 9) = ^ • 1 0 NMRT Jan Dec Nov Feb June J u l Oct May 74 Sep Two samples i n A p r i l , 1974 were omitted from the s t a t -i s t i c a l analyses because only single values were obtained. 4 . Microscopic b a c t e r i a l counts Anova source among samples among s l i d e s within s l i d e s t o t a l F . 0 1 ( 6 , 12) F . 0 1 ( 1 2,oo) F . 0 5 ( 1 2 , o o ) ss ms F „ 3.8935 .4867 4.5400 0.9648 .1072 4.8583 * s i g n i f i c a n t at P<0.025 df ss ms F 11 44.498 4.045 2 0 . 7 3 3 M Q 12 2.341 .195 . 5 6 7 N S 336 115.576 .344 359 162.416 «= 4.8 e 2 .2 =1.8 N S not s i g n i f i c a n t at P<.05 NMRT Oct Jan Nov Apr 1 May 74 Feb May 73 Apr 21 Sep June Dec J u l 37 r e s u l t s suggest that quick freezing of samples at the s i t e u n t i l l y o p h l l i z a t i o n and ATP analysis could be performed, would be a good method fo r storing samples. Concentrations of ATP i n sediments have been reported by other workers (Table I I I ) , Ernst (1970) converted his data to biomass carbon using a C/ATP r a t i o of 5 0 / 1 . To compare his data and that of Lee et a l . (1971a) and Karl and LaHock (1974) to data obtained during t h i s study, a C/ATP r a t i o of 250 was assumed. The main features of the estimated biomass concentrations are the low values i n marine sediments and the high values i n Marion Lake. Concentrations of biomass carbon of 100>ig/g are equivalent to about 2 x l 0 1 0 bacteria/g (Appendix VI) which i s higher than plate count methods indicate for many sediments (eg. Bianchi, 1973) but much lower than d i r e c t counts indicate (eg. Antipchuk, 1972) . Any sediment with lOOjug biomass would contain v i r t u a l l y no organisms other than bacteria. This would be unusual even i n anaerobic environments (Fenchel, 1969) . Low values i n marine sediments may indicate either very small l i v i n g populations or that the C/ATP r a t i o i s incorrect, although even a 10 f o l d increase i n the r a t i o would not have too great an e f f e c t . I t i s hard to evaluate the data i n Table III since no information was reported on the nature of the sediment communities, but Marlon Lake sediment biomass has been independently estimated by counting organisms of various species (Efford, unpublished). This offers a unique opportunity to compare ATP-blomass carbon estimates to enumeration-blomass carbon estimates. The mean biomass of microorganisms and invertebrates i n Table I I I . Sediment ATP-biomass carbon data. reference Ernst (1970) Lee et a l . (1971a) K a r l and LaRock (1974) This study l o c a t i o n 6 s i t e s North Sea (28 - 345m) 9 Wisconsin lakes beach sand A t l a n t i c ocean (4000 m) Marion Lake (seasonal) reported data 6.6 - 33.4>ug biomass carbon/ml 0.34 - 9 . 5 / f g ATP/g 145 - 228 ng ATP/g 9 - 1 0 . 3 ng ATP/g biomass carbon (yug/g dry w t . ) 1 165 - 835 82 - 2 ,375 36 - 57 2 - 3 2,810 - 12 ,600 Assuming C/ATP i s 250 and, f o r Ernst's (1970) data, that 1 ml = 200 mg dry wt. 39 Marion Lake sediments by enumeration is approximately 4-6g C/m2. ATP-measured biomass carbon in fresh samples ranges from 3.5 - 12 . 8 g C/m2 with an integrated mean of 4.7 g C/m . Discrepancies may be due to inclusion i n the enumeration data of large invertebrates (eg. Slalls ) which were removed prior to ATP extraction, variations i n the benthos from year to year (not a l l organisms were enumerated during the same 12 month period), variations in the depth of sediment sampled (eg. i n this study the top two cm) and variations in different parts of the lake (not a l l counts were obtained at 1 m water depth or along the same transect as were ATP data). Despite a l l these i n -consistencies, the data suggest that ATP analysis does provide a quick approximation of sediment biomass in Marion Lake and that the data presented in Table III are approximately correct. (b) Microbial biomass i n the sediments estimated by ATP analysis Biomass of microorganisms less than approximately 30 JJm diameter was quantified by removing larger organisms from sediment samples and measuring residual ATP. This method is subject fco a l l the assumptions concerning efficiency of extraction and biomass carbon to ATP ratios discussed In the previous section. Seasonal changes i n microbial biomass, including bacteria, fungi, actinomycetes, some algae and a few proto-zoa, do not parallel changes in total biomass. Peaks occurred i n late summer and early spring with a nine-fold 40 difference between maximum and minimum concentrations compared to an approximately four - fo ld var iat ion in to ta l biomass (Figures 4b, c ) . Microbial biomass ranged from o 0,25 - 2.16 g C/m (January - September). S ta t i s t i ca l analysis of the data support the conclusion that warm months have a higher microbial biomass than winter months (Table II). Data for A p r i l 1 and 21, 1974 were excluded from the analyses because replicate samples were los t . Temperatures in July, October, September, 1973 and May, 1974 were higher than 9°• Mean microbial biomass i s s igni f icant ly higher in these months than in January, December and November when the temperature was 4 ° , June biomass data are also higher than in winter months but not at the 1% confidence leve l . Seasonal changes in ATP associated . with plankton In the 0.22 - 250jam size range were reported by Rudd and Hamilton (1973). Their data should represent biomass more equivalent to tota l ATP-blomass than microbial ATP-biomass In this study considering the size range, but in fact resembles the lat ter (Figure 4c). This ref lects the s tab i l i t y of tota l benthlc compared to planktonic communities (Cameron, 1973). Biomass of microorganisms may be calculated for sediment at 1 m water depth using enumeration data. The annual mean biomass of bacteria is 0.61 g C/m (next section), 2 of protozoa is 0.0013 g C/m (Kool and Stachurska, un-published) and of algae is approximately 0.23 g C/m2 (calculated from Greundling, 1971). Actinomycetes are i n -cluded in the bacter ial counts. Fungi have been quantified 41 only by plate count methods and their numbers are low compared to to ta l bacter ial counts (1.8 - 8.2 x 10? fungal propagules/m2 - Chang, unpublished? compared to lO 1 ^ bacteria/m 2). Dick (197D suggested many fungi were present as spores of allochthonous or ig in . Very few healthy fungal filaments were seen during this study except on decaying f i sh in the lake or on sediments in nutrient-enriched microcosms. A rough estimation of fungal biomass may be obtained by assuming a weight of 5 x 10 g C per propagule (Shields et a l . . 1973. reported a mean diameter of 2.5>ini for fungi i n soi l t an average hyphal of spore length of 10^ u m was assumed here to allow the calculation to be made). Cultivable,fungal biomass is then 9-41 x 10"^ g C/m . The sum of the enumeration-microbial biomass is 0,84 g C/m2. The biomass of protozoa and fungi i s negligible in this system. The annual mean ATP-measured microbial b io -mass i s 1.28 g C/m2, indicating the C/ATP rat io may be inaccurate or that the enumeration data underestimate the biomass. The discrepancy is even greater than this indicates because a fract ion (large filaments, colonies and diatoms) of the algae were removed prior to ATP analysis, (c) Bacterial biomass in the sediments estimated by micro-scopic counts Bacterial biomass was estimated by counting ce l l s in known dilutions of sediment and converting the numbers to biomass assuming that ce l l s have a density of lg/cc, are 80$ water and that 50% of the dry wt. i s carbon (Shields et  a l . . 1973). Another requirement for this calculation is the average size per c e l l , but this is d i f f i c u l t to measure in 42 sediment systems. Some microbiologists have measured bacterial ce l l s i n . s o i l . These data were converted here to give an average of 1 . 9 x K T ^ g dry wt./cel l (Bae et a l ^ , 1972) , 5 .7 x IO" 1 4 g/cell (Babiuk and Paul, 1970) and 1 . 6 x lCT^g/ce l l (Zvaginsev, 1973) . Mean sizes for sediment bacteria meas-ured by Antipchuk (1972) are 3 .7 x 10" 1 3 g/rod and 5 .8 x 1 0 ~ 1 / + g/coccus. Burnison (unpublished) suggested a mean volume of 0 .36 y um3 for Marlon Lake sediment bacteria which corres-_-i k. ponds to 7 . 2 x 10 g/cel l . During this study very large spirochaetes (50 - 100/(m length) were seen a few times, but 12 never while counting bacteria. Cel ls up to 5 . 6 x 10 g (4 x 3yU m) were more common while at the other extreme 14 ce l ls weighing 2 . 0 x 10 g (0 .8 x 0 . 4 / 1 m) were noted. Most bacteria were 3 . 9 x l O ' ^ g - 2 .4 x 1 0 ~ 1 3 g (1 - 1 . 5 x . 5 - Ijdm). Not enough ce l ls were measured to determine an accurate mean, but 1 0 " 1 3 g dry wt ./cel l , or 5 x lO"*1^ g C/cell was chosen as a representative value. Bacterial biomass peaked in July and was at a minimum in October (Figure 5 ) . July biomass data are s igni f icant ly higher than only those in October, January, November and early Apr i l (Table II), With the exception of the high biomass in December the seasonal trend was a maximum in early to mid-summer, a rapid decline in early f a l l then a slow recovery during the winter. Plate counts were similar in pattern except that the crash occurred in August in 1969 (Fraker, unpublished). Figure 5» Seasonal changes of b a c t e r i a l biomass carbon. B a c t e r i a l biomass (gC/m2) Most published estimates f o r b a c t e r i a l biomass i n sediments are low because they were based on p l a t e counts. Data are u s u a l l y r e p o r t e d as numbers per g so the conversion f a c t o r of 5 x l C T ^ g C / c e l l was a p p l i e d t o i n f o r m a t i o n r e -ported by the authors c i t e d below. Z o b e l l (1963) reviewed the l i t e r a t u r e and found b a c t e r i a l biomass estimated from 5 x 10"7^j g c - 5°yUg C/ g sediments. Khiyama and Makemson (1973) used l i g h t microscopy and found 1 - IOJJL g C/g i n beach sands. Surface sediments i n f i s h ponds were found by Antipchuk (1972) t o c o n t a i n approximately 1000 - 2000ps C/g.His biomass estimates f o r one sample at each of s i x l o c a t i o n s i n three seasons tend t o peak i n s p r i n g or summer then d e c l i n e i n f a l l , a p a t t e r n not u n l i k e t h a t i n Marion Lake. The biomass of Marion Lake sediment b a c t e r i a ranges from 340 - 650^ug C/g (January - J u l y ) . These estimates are much higher than reported concentrations p r i o r t o the l a s t decade, but tha t they are not overestimates i s suggested by the m i c r o b i a l ATP data, and the s i m i l a r i t y w i t h previous d i r e c t counts i n Marlon Lake sediments (Ramey, 1972 t Bur-n i s o n , unpublished). (d) R e l a t i o n s between biomass estimates The v a r i o u s biomass data may be compared by examinimg p r o p o r t i o n a l changes i n each f r a c t i o n r e l a t i v e t o the others (Figure 6). B a c t e r i a l biomass f l u c t u a t e s between 5 -20/6 of the t o t a l biomass, r e f l e c t i n g changes i n the b a c t e r i a l c o n c e n t r a t i o n except when t o t a l biomass estimates were high i n May. The m i c r o b i a l f r a c t i o n of the community 45 Figure 6, Seaonal changes i n the proportion of sed-iment biomass carbon i n b a c t e r i a l compared to t o t a l biomass ( o o ) , b a c t e r i a l comp-ared to microbial biomass (+ +); microbial compared to t o t a l biomass (• • ) . *5a CO •p c a £ +> - * C d. O 8 ,0 O P o a) O rH as CQ -H CO U o o •H «J .o .o O C c « O rH •H C8 -P -H U fi o o 0 u O O ^ .H a, a 2 - 0 1 1-5-1-0-0. - i r-M J 1 9 7 3 S N J M M 1 9 7 4 46 i s high i n the summer and low i n midwinter, i n c r e a s i n g during l a t e winter t o another peak i n e a r l y s p r i n g . This p a t t e r n resembles t h a t f o r e p l b e n t h i c algae a t 1 m (Greund-l i n g , 1 9 7 D . The p r o p o r t i o n of b a c t e r i a i n the m i c r o b i a l biomass decreases during the summer, r i s e s s h a r ply i n th§ l a t e f a l l , dropping a g a i n i n l a t e w i n t e r . This f i t s a model i n which the b a c t e r i a l c o n t r i b u t i o n i s d i l u t e d by seasonal increases i n the a l g a l biomass. I n December and January, b a c t e r i a were more than 150% of the ATP-microblal biomass. This suggests b a c t e r i a l counts overestimated the b a c t e r i a l p o p u l a t i o n or t h a t the m i c r o b i a l p o p u l a t i o n was a t a p h y s i o l o g i c a l low, probably w i t h a C/ATP rsfcio greater than 250. Fast temperature d e c l i n e s t o 0° were shown by Cole et. a l . (1967) t o cause a sharp r i s e i n C/ATP r a t i o s i n E, c o l l . I f t h i s a p p l i e s to n a t u r a l m i c r o b i a l communities l i v i n g a t 4° f o r s e v e r a l months, then low ATP c o n c e n t r a t i o n s , and t h e r e f o r e under-estimates of the community biomass may be expected. The subsequent Increase i n m i c r o b i a l ATP i n February may r e f l e c t not only the increase i n a l g a l biomass (Greundling, 1971) , but a l s o a c c l i m a t i z a t i o n of the b a c t e r i a . Such a p o s s i b i l i t y i s suggested by the data of Cole et a l . (1967) . Comparison of the A T P - t o t a l biomass data (Figure 4b) t o seasonal v a r i a t i o n of the enumeration data ( E f f o r d , un-published) r e v e a l s another discrepancy. ATP-biomass peaks i n May (12 g C/m2) and i s almost constant f o r the remainder of the year (4 g C/m2). Enumeration data are low i n s p r i n g (about 4 g C/m ) but constant f o r the remainder of the year (about 6 g C/m 2), ATP a n a l y s i s u s i n g a C/ATP of r a t i o of 250 47 appears t o be h y p e r s e n s i t i v e i n May. Cole et a l . (1967) r e p o r t e d periods of over- and under-production of ATP i n E. c o l l . The C/ATP r a t i o decreased d u r i n g h i g h growth but then increased despite c o n t i n u i n g high growth r a t e s . I f t h i s i s g e n e r a l l y true (Holms et a l . . 1971, reported con-f l i c t i n g d a t a ) , and c o n s i d e r i n g t h a t p r i o r t o May the benthos has been r e s t r i c t e d by low temperatures, and t h a t i n May r e s p i r a t i o n i n Marion Lake sediments Increases e x p o n e n t i a l l y (Hargrave, 1969), then over-production of ATP might occur. Using a C/ATP r a t i o of 250 would overestimate the community biomass i n such circumstances. One c o n c l u s i o n from t h i s d i s c u s s i o n i s t h a t ATP i s an adequate i n d i c a t o r f o r biomass a t "average" physio-l o g i c a l c o n d i t i o n s , but under periods of s t r e s s , ATP data may be confounded by r e p r e s e n t i n g both biomass and a c t i v i t y . P ublished data f o r n a t u r a l systems support t h i s . Holm-Hansen and P a e r l (1972) found high primary production, high ATP-biomass and low a l g a l biomass i n surface waters of Lake Tahoe, but a t 80 m, recorded low production, low ATP-biomass and high a l g a l biomass. Rudd and Hamilton (1973) r e p o r t e d a s i m i l a r phenomenon i n Lake 227 of the Experimental Lakes Area, Ontario. ATP may then be thought of, not as measuring biomass at any i n s t a n t , but measuring the p o t e n t i a l biomass through time. An example i s the b a c t e r i a i n Marion Lake sediments. I n January, m i c r o b i a l ATP i s low (Figure 4c) and b a c t e r i a l biomass i s p r o p o r t i o n a l l y high (Figures 5 . 6 ) . At any i n s t a n t i n time more b a c t e r i a l carbon i s a v a i l a b l e 48 t o grazers than ATP data i n d i c a t e . However, i f b a c t e r i a are grazed a t t h i s time, biomass replacement may be slow due t o temperature (or other f a c t o r ) l i m i t a t i o n * Such replacement occurs w i t h i n a few days at warm temperatures (Fenchel, 1970). I n September, ATP-microbial biomass i n the sediment i s t e n -f o l d higher but enumeration-microbial biomass i s only two-f o l d higher than i n January. The suggestion i s that i n September there i s not twice as much p o t e n t i a l m i c r o b i a l biomass a v a i l a b l e f o r consumption, but up t o t e n times as much* Stated another way, t h i s means t h a t i n September there should be 5 times more biomass a v a i l a b l e i f the system i s s t r e s s e d than i s i n d i c a t e d by d i r e c t count data. This p r e d i c t i o n could be t e s t e d by i n c r e a s i n g g r a z i n g pressure on the m i c r o b i a l p o p u l a t i o n t o f i n d t h e i r maximum growth r a t e . Hargrave (1970) d i d t h i s type of experiment u s i n g a range of d e n s i t i e s of H y a l e l l a a z t e c a . Assuming t h a t h i s measurement of b a c t e r i a l r e s p i r a t i o n u s i n g a n t i b i o t i c s approximates b a c t e r i a l growth or production r a t e s , then h i s data may be a p p l i e d t o the problem. Maximum s t i m u l a t e d b a c t e r i a l r e s p i r a t i o n was about 13 times t h a t i n f r e s h , u n a l t e r e d cores. Considering the i n c o n s i s t e n c i e s i n temp-e r a t u r e , time, e t c . between the experiment and the model i t appears t h a t ATP i s i n f a c t a measure of both biomass and a c t i v i t y , or simply biomass p o t e n t i a l . Under average c o n d i t i o n s a C/ATP r a t i o of 250 represents biomass. Under s t r e s s c o n d i t i o n s such as intense g r a z i n g , c o l d temperatures or r a p i d temperature increases the c e l l u l a r ATP balance i s changed and, by d e f i n i t i o n , ATP becomes an a c t i v i t y i n d i c a t o r . Interpretation of ATP-biomass data may therefore be d i f f i c u l t . High values may r e f l e c t large, inactive populations or small, productive populations. In studies concerned with trophic dynamics, however,more concise Information may not be required. 50 3 . A c t i v i t y estimates (a) Dehydrogenase a c t i v i t y measurements Dehydrogenase a c t i v i t y tmithe sediments i s higher i n summer than any other time of the year (Figure 7 . Table IV). Assays incubated for 24 or 4 hours usually showed si m i l a r d i r e c t i o n a l response to changes i n sediment r e s p i r a t i o n , but the absolute rate of response was greater using the short i n -cubation time. This was expected (Appendix VII). The range of data for dehydrogenase a c t i v i t y ( . 0 6 5 5 - . 6 8 2 5 mg formazan/g.h) for the two incubation times over the 12 months i s sim i l a r to the range reported by Pamatmat and Bhagwat (1973) for d i f f e r e n t locations i n Lake Washington ( . 1 3 - .42 mg formazan/g.h, con-verting t h e i r absorption readings to formazan equivalents), although t h e i r methodology was quite d i f f e r e n t . The dehydrogenase data probably r e f l e c t s aerobic and anaerobic p o t e n t i a l a c t i v i t y of prokaryotes i n the sedi-ments since the samples were not homogenized. The suggestive evidence for t h i s was presented i n the Introduction, This observation may explain the lack of c o r r e l a t i o n between oxygen uptake and dehydrogenase a c t i v i t y i n some natural samples, Howard (1972) reported s o i l dehydrogenase a c t i v i t y was always less than that predicted by oxygen uptake data, Pamatwat and Bhagwat (1973) reported that sediment oxygen uptake was le s s than that predicted by dehydrogenase a c t i v i t y . It i s suggested here that, i n aerobic s o i l s , r e s p i r a t i o n due to eukaryotes i s s i g n i f i c a n t , but not measured by the de-hydrogenase assay, Anderson and Domsch (1973) reported that 51 Figure ?. Seasonal changes of dehydrogenase a c t i v i t y i n the sediments (4h incubation o — o ; 24 h incubation . . ) . 52 Table IV. S t a t i s t i c a l analysis of dehydrogenase a c t i v i t y data. 1, Dehydrogenase a c t i v i t y measured with 4 hour incubation Anova source df ss ms F ^ among 10 .7396 .0740 42.6292 within 27 .0468 .0017 t o t a l 37 .7865 F.01(10. 25) = 3 ' 1 3 NMRT Apr 1 Apr 21 Jan May ?4 Dec Feb Oct Nov J u l June Sep 2 . Dehydrogenase a c t i v i t y measured with 24 hour incubation Anova source df ss ms F # # among 11 1.2581 .1144 63.3919 within 29 .0523 .0018 t o t a l 40 1.3104 p . 0 1 ( 1 0 , 25) = 3 * 1 3 NMRT Apr 1 Apr 21 Jan May 74 Dec Feb Oct Nov May 73 Sep J u l June 53 b a c t e r i a were r e s p o n s i b l e f o r only 22% of t o t a l s o i l oxygen consumption i n t h e i r s t u d i e s . I n sediments,many anaerobic processes occur, p a r t i c u l a r l y i n reduced environments, which are not measured by oxygen uptake but do r e a c t w i t h TTC (Pamatmat and Bhagwat, 1973» Olah, 1972). Hargrave (1969) estimated b a c t e r i a l oxygen consumption i n Marion Lake sediments by s u b t r a c t i n g oxygen uptake i n a n t i b i o t i c i s t r e a t e d cores from t h a t i n normal cores. Although the usefulness of a n t i b i o t i c s t o s e l e c t i v e l y e l i m i n a t e s p e c i f i c populations i n n a t u r a l environments i s dubious (Cameron, .1973 * Yetka and Wiebe, 197*0, comparison of h i s r e s u l t s w i t h dehydrogenase a c t i v i t y measurements i s u s e f u l . Hargrave found b a c t e r i a l r e s p i r a t i o n decreased g r a d u a l l y between September and December then increased g r a d u a l l y u n t i l May, When the sediments reached approximately 10°, b a c t e r i a l a c t i v i t y increased r a p i d l y w i t h i n c r e a s i n g temperature. T h i s b a s i c p a t t e r n i s repeated f o r the de-hydrogenase data (Figure 7), although the November sample was i n e x p l i c a b l y h i g h I n a c t i v i t y and maximum a c t i v i t y was recorded i n September (4h incubation) or June (24h incubation) which were not the dates of maximum temperature (19.5° i n J u l y ) , 14 (b) Heterotrophic uptake of C-glucose Gross uptake ( r e s p i r e d plus a s s i m i l a t e d uptake) of C-glucose i n mixed, d i l u t e d sediments f o l l o w e d a dramatic seasonal p a t t e r n (Figure 8), Both p o t e n t i a l uptake r a t e (V m) and n a t u r a l uptake r a t e (Ujj) were maximum I n J u l y when the sediments reached 19.5 • Uptake dur i n g winter was c o n s i s t e n t l y 54 Figure 8. Seasonal changes i n the sediments of maximum (V m) and actual (U n) rates of glucose uptake, and the actual turnover time (T n).(V m o 0 } U n+—+ i T n ). low (<4 mg glucose/m^.h) but s t a r t e d t o increase as the temperature climbed above 4° ( 9 . 5 ° i n May,1974). The A p r i l 21 data were discarded because they d i d not f i t Michaelis-Menten k i n e t i c s . N atural turnover time (T n) di s p l a y e d an inverse r e l a t i o n s h i p t o uptake r a t e (Figure 8). The time r e q u i r e d f o r the b a c t e r i a t o completely use glucose equivalent t o the i n s i t u c o n c e n t r a t i o n ( S n) increased as uptake r a t e d e c l i n e d . H a l l et a l . (1972) r e p o r t e d s i m i l a r values f o r V m (2 . 6 - 38,0/1 g glucose/g.h) and T n ( , 0 6 l - ,400h) as were found here ( V m 2.2 - 3 9 . 3 j u g glucose/g.h, T n .033 - .727h). The p a t t e r n of seasonal v a r i a t i o n was almost i d e n t i c a l , i n d i c a t i n g that the b a c t e r i a l p o p u l a t i o n , w i t h respect t o glucose metabolism, does not change r a d i c a l l y from year t o year (1971 - 72 compared t o 1973 - 7 4 ) . The n a t u r a l uptake r a t e of glucose (U n) was estimated by K l e i b e r (1972) t o be about 1.8 - 10 . 6 >ig glucose/g.h f o l l o w i n g a seasonal t r e n d s i m i l a r t o that of V m. During the present study U n ranged from 0 . 6 - 15 .9;ug glucose/g.h. Wood (1970) found h e t e r o t r o p h i c uptake was maximal i n the s p r i n g and low i n winter i n e s t u a r i n e sediments, but had no data f o r June and J u l y . Some p r e v i o u s l y reported data f o r parameters of hetero-t r o p h i c uptake of glucose i n n a t u r a l environments are summar-i z e d i n Table V. Wood (1970) d i d not determine S n so T n may be low. H a r r i s o n et a l . (1971) used mixed but not d i l u t e d sediments, so T n could be determined d i r e c t l y by e x t r a p o l -a t i o n t o zero added substrate (Appendix V I I I ) . Uptake i n Table V. Maximum uptake rates, and natural turnover times and uptake rates for glucose i n sediments and water. reference Hobble et a l . (1972T Azam and Holm-Hansen (1973) Crawford at a l . (1974) Harrison et a l . (1971) Wood (1970) l o c a t i o n ocean water (10 - 200 m) ocean water (10 - 200 m) estuarine water Klamath Lake sediments estuarine sediments H a l l et a l . (1970) Marion Lake Kleiber T l 9 7 2 ) -mixed Tn Vm V m U n (h) Ojg/g.h) (jug/l.h) (;jg/g.h) 10 -128 days 7.2 2.25 . 0 6 sediments -undisturbed .89-sediments 420 This study Marlon Lake -mixed sediments . 3 1 2.4 299 .26 9.4 9 . 6 .0005 - .006 2 5 . 6 170 627 535 3 . 9 5 . 3 u n (jug/l.h) 260 295 undisturbed cores of Marion Lake sediment occurred w i t h a longer T n than was found i n mixed sediment experiments ( H a l l et a l . . 19721 K l e i b e r , 1972). The f a s t e r uptake i n mixed samples i s probably due t o increased a v a i l a b i l i t y of d i s s o l v e d organics t o the he t e r o t r o p h i c organisms. I n open water compared t o sediments, T n i s much longer and V m i s much lower. This r e f l e c t s d i f f e r e n c e s i n the pop u l a t i o n d e n s i t y of heterotrophs i n the two environments (eg. Marion Lake waters i n March, 1973 contained an equivalent of 74 >*g dry wt. of b a c t e r i a per 1 while the top two cm of the sediment contained approximately 6 0 , 0 0 0/ig dry wt. of b a c t e r i a / 1 ) , and emphasizes the importance of the benthos i n n u t r i e n t c y c l i n g . (c) R e l a t i o n s between a c t i v i t y estimators D i f f e r e n t a c t i v i t y assays need notyneeeasarily,, ~ measure the same parameters of m i c r o b i a l populations due to t h e i r heterogeneous biochemical a b i l i t i e s . Glucose i s not a subs t r a t e f o r a l l b a c t e r i a . The a b i l i t y t o reduce TTC t o formazan i n the dehydrogenase assay may be r e s t r i c t e d ; degrees of the a b i l i t y c e r t a i n l y e x i s t (Eidus et a l . . 1959) . There i s u s u a l l y , however, good c o r r e l a t i o n between the va r i o u s methods. S k u j i n s (1973) reported a high degree of c o r r e l a t i o n between s o i l oxygen consumption, p r o t e o l y t i c a b i l i t y , n i t r i f i c a t i o n p o t e n t i a l and dehydrogenase a c t i v i t y . Hobble et a l . (1972) found a reasonable degree of r e l a t i o n between estimates of marine plankton r e s p i r a t i o n u s i n g oxygen uptake, dehydrogenase a c t i v i t y and ATP. A l l the a c t i v i t y estimators a p p l i e d t o Marion Lake sediments i n d i c a t e b a c t e r i a respond t o increased temperatures 58 i n May, reaching a peak sometime i n l a t e May t o e a r l y Sept-ember, then g r a d u a l l y slow down as the temperature drops. B a c t e r i a l a c t i v i t y remains low a l l w i n t e r . Heterotrophic uptake of glucose ( H a l l et a l . . 1972; F i g u r e 8 ) , g l y c i n e and acetate ( H a l l et a l . , 1972), and oxygen consumption (Hargrave, 1969) demonstrate sharper seasonal maxima than dehydrogenase a c t i v i t y (Figure 7 ) . This may be due t o the f a c t t h a t dehydrogenase i s a measure of p o t e n t i a l , although V m, the p o t e n t i a l uptake r a t e of glucose, has a very sharp peak i n midsummer. Rad i o t r a c e r uptake s t u d i e s , p a r t i c u l a r l y those i n which U n and T n are determined, are perhaps the best way to estimate energy flow from p a r t i c u l a r carbon sources i n t o b a c t e r i a l p opulations. But assumptions o f t e n made,for example, th a t glucose uptake i s r e p r e s e n t a t i v e of uptake of a l l other d i s s o l v e d carbohydrates, may be i n v a l i d . The s o l u t i o n would be t o have a uniformly l a b e l l e d pool of a l l the n a t u r a l substrates i n t h e i r i n s i t u c o n c e n t r a t i o n , but t h i s i s not p r a c t i c a l c o n s i d e r i n g present technology. Dehydrogenase a c t i v i t y may be more i n d i c a t i v e of t o t a l m i c r o b i a l production than e i t h e r h e t e r o t r o p h i c uptake or oxygen consumption as suggested by Pamatmat and Bhagwat (1973) . T h e i r data c o r -r e l a t i n g heat production t o dehydrogenase a c t i v i t y i n sed-iments i s promising because heat production, which i s very d i f f i c u l t t o measure i n n a t u r a l communities, may be the u l t i m a t e t o o l f o r measuring t o t a l m i c r o b i a l a c t i v i t y (Brock, 1967) . 59 The r e l a t i o n between h e t e r o t r o p h i c uptake, oxygen uptake and dehydrogenase a c t i v i t y i n Marion Lake sediments may be examined q u a n t i t a t i v e l y . Hargrave (I969) estimated b a c t e r i a l r e s p i r a t i o n t o be 19.5 g C/m 2.year which i n d i c a t e s t o t a l uptake of 97.5 g C/m 2.year assuming a s s i m i l a t i o n e f f i c i e n c y of 80% ( H a l l e t a l ^ , 1972). K l e i b e r (1972) estimated carbon f l u x i n t o the b a c t e r i a a t 22 - 120 g C/m2. year w i t h r e s p i r a t o r y l o s s e s of 4 - 24 g C/m 2.year. Using a r e g r e s s i o n f o r dehydrogenase a c t i v i t y on heat production (Patmatmat and Bhagwat, 1973). and assuming approximately 100 K c a l heat are produced per mole of carbon o x i d i z e d (Giese, 1968), the p o t e n t i a l b a c t e r i a l r e s p i r a t i o n i s 19 .3 g C/m 2.year using the 4 hour i n c u b a t i o n assay data. This i s equivalent t o gross uptake of 96.5 g C/m 2.year. This agree-ment between p o t e n t i a l and a c t u a l r e s p i r a t o r y data i s un-expected, but, de s p i t e p o s s i b l e e r r o r s i n the v a r i o u s assumptions, I t increases confidence i n the carbon flow estimates. 60 4 . Biomass and a c t i v i t y of Marlon Lake b a c t e r i a (a) Q u a l i t a t i v e seasonal d e s c r i p t i o n There i s evidence t h a t biomass of b a c t e r i a i n n a t u r a l environments does not always c o r r e l a t e w i t h a c t i v i t y measure-ments. Hobble ejt a l . (1972) reported t h a t high h e t e r o t r o p h i c uptake of r a d i o t r a c e r s i n ocean water was a s s o c i a t e d , not n e c e s s a r i l y w i t h high numbers of b a c t e r i a , but a high number of m o t i l e b a c t e r i a . S k u j i n s (1973) showed there was no c o r r e l a t i o n between dehydrogenase a c t i v i t y and numbers of c u l t i v a b l e b a c t e r i a i n s o i l . Wood (1970) however, found c o r r e l a t i o n f o r h e t e r o t r o p h i c uptake of acetate and glucose a g a i n s t d i r e c t counts of b a c t e r i a i n sediments. Uptake of s i x of nine organic a c i d s t e s t e d was shown to be c o r r e l a t e d w i t h numbers of c u l t i v a b l e b a c t e r i a w i t h the a b i l i t y t o u t i l i z e each of the s i x a c i d s as a source of carbon (Robinson et a l . . 1973). Th e i r estimatesof the p o p u l a t i o n s i z e of each biochemical group a r e , however, questionable. Considering the number of c o l o n i e s formed per ml of sample, and the methods they used, most of the s o l e carbon source p l a t e s contained fewer than 15 c o l o n i e s . The normally accepted l i m i t s f o r p l a t e counts are 20 - 200 c o l o n i e s per p l a t e , w i t h numbers l e s s than 20 considered s t a t i s t i c a l l y u n r e l i a b l e (Parkinson et a l . . 1971) . An Important c o n t r i b u t i o n t o understanding n a t u r a l populations of b a c t e r i a was made by Stanley and S t a l e y (1974) . They demonstrated, a p p l y i n g the uptake approach used i n t h i s study and autoradiography, that hetero-t r o p h i c a s s i m i l a t i o n of -'H-acetate i n an a e r a t i o n lagoon was due t o b a c t e r i a , t h a t the uptake per c e l l increased 61 l i n e a r l y over time, and tha t w i t h i n the p o p u l a t i o n there were d i f f e r e n c e s i n net uptake r a t e of at l e a s t t e n - f o l d by d i f f e r e n t s p e c i e s . This work suggests that uptake r a t e may not be p r o p o r t i o n a l t o numbers of b a c t e r i a because of the heterogeneity between b a c t e r i a l metabolic a b i l i t i e s . This d i s c u s s i o n i s i n c o n c l u s i v e . There are r e p o r t s of c o r r e l a t i o n and r e p o r t s of unrelatedness between b i o -mass and a c t i v i t y of b a c t e r i a , A simple e x p l a n a t i o n i s that biomass and a c t i v i t y are sometimes out of phase even i n one p a r t i c u l a r environment, Holm-Hansen and P a e r l (1972) found maximum he t e r o t r o p h i c uptake of acetate lagged behind maximum ATP-biomass of b a c t e r i a i n Lake Tahoe, I n Marion Lake, sediment b a c t e r i a l biomass and a c t i v i t y appear t o be i n phase i n l a t e s p r i n g , summer and e a r l y f a l l (Figure 9 ) , During the remainder of the year a c t i v i t y e i t h e r remains constant while biomass Increases, or increases very r a p i d l y compared t o biomass. The shaded area i n Figure 9 i n d i c a t e s the range of r e l a t i v e a c t i v i t y a t d i f f e r e n t times of the year, c a l c u l a t e d u s i n g the data of Hargrave (1969) on b a c t e r i a l r e s p i r a t i o n and uptake of glucose, acetate and g l y c i n e r e p o r t e d by H a l l et a l . (1972) . S i m i l a r bounds f o r p o t e n t i a l r e s p i r a t i o n are i n d i c a t e d . R e l a t i v e biomass i n d i f f e r e n t seasons was c a l c u l a t e d from d i r e c t count data. These s h i f t s between a c t i v i t y and biomass are i n t i m a t e l y r e l a t e d t o p h y s i c a l and chemical f l u c t u a t i o n s , and r e s u l t a n t changes i n b i o l o g i c a l f a c t o r s such as primary production and gr a z i n g . Considering the r e l a t i o n s found (Figure 9 ) . a 62 Figure 9. Diagrammatic r e p r e s e n t a t i o n of the seasonal r e l a t i o n s between a c t i v i t y and biomass of the sediment b a c t e r i a as pr o p o r t i o n s of t h e i r maxima, ( b a c t e r i a l biomass • • j range f o r h e t e r o t r o p h i c uptake of glucose, g l y c i n e and acetate and f o r oxygen consumption I . I ; range f o r dehydrogenase assay I.- ) A c t i v i t y or biomass as a p r o p o r t i o n of the maximum. ON 63 q u a l i t a t i v e , h y p o t h e t i c a l d e s c r i p t i o n of the b a c t e r i a l p o p u l a t i o n dynamics i s p o s s i b l e . During summer, there are high numbers of b a c t e r i a , probably experiencing r e l a t i v e l y high g r a z i n g pressure, but a t a production l e v e l high enough to maintain t h e i r numbers. I n f a l l , t h i s r eproductive c a p a c i t y d e c l i n e s s h a r p l y , despite f r e s h allochthonous inputs t o the l a k e , due t o decreasing temperature and day length and a l l t h e i r b i o l o g i c a l r a m i f i c a t i o n s . B a c t e r i a l biomass drops s i m u l t -aneously probably as a r e s u l t of g r a z i n g pressure. The b a c t e r i a l biomass then increases g r a d u a l l y during the winter and e a r l y s p r i n g during which p e r i o d growth r a t e , although not a t i t s maximum, appears t o be greater than removal due t o g r a z i n g . When the temperature climbs above 4° b a c t e r i a appear t o respond q u i c k l y . This i s suggested by the sharp d e c l i n e i n glucose concentrations i n the i n t e r s t i t i a l water i n A p r i l and May (Figure 3 c ) . The t o t a l p o p u l a t i o n i s probably s t i l l a t a low a c t i v i t y l e v e l however, due t o a l i m i t e d supply of r e a d i l y - a v a i l a b l e carbon sources. D e t r i t a l m a t e r i a l breakdown i n t o molecules of a s i z e which b a c t e r i a can t r a n s p o r t across t h e i r membranes i s probably the r a t e -l i m i t i n g step a t t h i s time. The consequence of t h i s i s the low a c t i v i t y measurements obtained i n A p r i l and sometimes i n e a r l y May(eg. Figure 8 ) . With s t i l l higher temperatures, primary production by e p l p e l i c algae increases r a p i d l y (Hargrave, 1969; Greundling, 1971) . Carbon and other n u t r i e n t s become a v a i l a b l e t o b a c t e r i a as a l g a l exudates ( K l e i b e r , 1972J. 6k Complex molecules i n the d e t r i t a l material are decomposed more r a p i d l y at higher temperatures. The b a c t e r i a l population should, t h e o r e t i c a l l y , ©xplode at t h i s time of year. And i t probably does. The population i s very "active" i n terms of a l l the a c t i v i t y indicators applied to the system. Biomass, however, does not respond as dramatically, presumably due to increased outputs to higher trophic l e v e l s . This descriptive model could be examined with two b i t s of information. Grazing e f f e c t s on microbial pop-ulations have been studied (eg, Hargrave, 1970; Fenchel, 1970), but data on seasonal v a r i a t i o n i n t h i s factor are lacking. The other requirement Is information on b a c t e r i a l growth rates at d i f f e r e n t times of the year under natural conditions. Attempts to obtain such data i n t h i s study f a i l e d due to heterogeneity between subsamples of the sediments. I f the a c t i v i t y estimators already applied to the sediments do r e f l e c t b a c t e r i a l growth rate (as suggested by the work of Stanley and Staley, 197*0, and i f grazing pressure i s r e l a t i v e l y high i n May to September, and low i n winter, then confidence i n the above de s c r i p t i o n of b a c t e r i a l population dynamics would be increased. 65 (b) Q u a n t i t a t i v e d e s c r i p t i o n The annual mean inputs and outputs of the b a c t e r i a l p o p u l a t i o n ( i n t e g r a t e d mean s i z e of . 6 1 g C/m2 based on d i r e c t counts) are summarized i n Figure 1 0 . The range f o r gross uptake and r e s p i r a t o r y l o s s e s of organic carbon are from K l e i b e r (1972) . The values i n brackets i n d i c a t e the estimates from oxygen uptake(Hargrave, 1969) and r e s p i r a t o r y p o t e n t i a l . The l o s s of carbon as CH^ was estimated from experiments w i t h sediment cores, and appears to be a minor component of the system. Ep i b e n t h i c a l g a l biomass was c a l c u l a t e d from Greundling (1971) and gross p r o d u c t i v i t y from h i s data and tha t of Hargrave (1969) . E x c r e t i o n of a l g a l photosynthate t o the d i s s o l v e d organic carbon (DOC) pool (pool s i z e c a l c u l a t e d from H a l l and Hyatt, 197*0 was determined by K l e i b e r (1972) . A l g a l r e s p i r a t i o n was c a l c u l a t e d from Hargrave's (1969) estimate of r e s p i r a t i o n due t o b a c t e r i a and algae and co r r e c t e d f o r b a c t e r i a l r e s p i r a t i o n . Losses t o g r a z i n g organisms were c a l c u l a t e d by d i f f -erence between inputs and outputs t o the a l g a l and bac-t e r i a l p o p u l a t i o n s . R e c y c l i n g of m a t e r i a l to the d i s s o l v e d and p a r t i c u l a t e organic carbon (POC) pools from higher t r o p h i c l e v e l s i s unknown but may be s i g n i f i c a n t (eg. Hargrave, 1970) . H a l l et a l . (1973) estimated the c o n t r i b u t i o n of organic carbon to the DOC of the water column from a l l o c h -thonous l e a f m a t e r i a l was 1 . 1 - 3 . 1 g C/m 2.year .Net. DOC .inputs — 2 ' from the i n l e t are 28 gC/m .year(Geen,unpublished).Other Figure 10. S t r u c t u r e and f u n c t i o n of the Marlon Lake sediment ecosystem ( a l l compartment s i z e s are expressed i n gC/m2 and a l l f l u x e s are In gC/m .year, unless otherwise s p e c i f i e d - see t e x t f o r e x p l a n a t i o n and sources). 66 a ailochthonous m a t e r i a l b i o l o g i c a l processes — \ ( 1 ) ? / ? DOC 7 *<5 mg/1 water y DOC PO ( sediment .14-.36' 240 * C**4 (1) 29.1-31.1 (2) 42.3 (3) 1.5-8.8 (4) 6.9-26.7(11.5) (5) 22.0-29.3 (6) 21.0-120(97.0) (7) 4.2-24.0(19..4) (8) 76.6 (9) <0.5 CO) 28 67 inputs to t h i s pool come d i r e c t l y or i n d i r e c t l y from phytoplankton and macrophytes and the communities they support. Exchange between water column DOC and sediment DOC has not been quantified, but may be small since d i f f u s i o n processes would be expected to operate i n the opposite d i r e c t i o n . The major unknown component i n the system i s the rate of decomposition of natural sediment POC (including l i v i n g and dead organic matter, but primarily the l a t t e r ) to u t i l i z a b l e DOC, This occurs as a r e s u l t of chemical and b i o l o g i c a l processes, but quantitative assessment of i t s importance w i l l require further development of tech-nology. 68 SUMMARY AND CONCLUSIONS The prime f u n c t i o n a l r o l e s of b a c t e r i a are conversion of d i s s o l v e d and p a r t i c u l a t e organic matter i n t o b a c t e r i a l t i s s u e s which are then a v a i l a b l e t o higher t r o p h i c l e v e l s , and m i n e r a l i z a t i o n of d i s s o l v e d and p a r t i c u l a t e organic n u t r i e n t s , making them a v a i l a b l e t o photosynthetic organisms. The l a t t e r process has not been st u d i e d i n Marion Lake. The major sources of organic matter f o r the het e r o t r o p h i c p o p u l a t i o n appear t o be allochthonous i n p u t s from the watershed and epibent h i c a l g a l production. Much of the l a t t e r i s consumed by higher t r o p h i c l e v e l s and i s not, t h e r e f o r e , d i r e c t l y a v a i l a b l e t o b a c t e r i a . During t h i s study, i t was demonstrated th a t ATP a n a l y s i s o f f e r s r a p i d e s t i m a t i o n of both t o t a l and m i c r o b i a l sediment biomass. B a c t e r i a l biomass was estimated by d i r e c t counts, and was the l a r g e s t component of the m i c r o b i a l approximately 30JJm) standing crop. Changes i n the p r o p o r t i o n of b a c t e r i a l and m i c r o b i a l biomass i n the t o t a l community biomass were a t t r i b u t e d t o changes w i t h i n the b a c t e r i a l and m i c r o b i a l p o p u l a t i o n s . A n a l y s i s of the dis c r e p a n c i e s between ATP- and enumeration-biomass data I n d i c a t e that ATP i s not only a biomass measurement, but, i n s t r e s s s i t u a t i o n s , i s confounded by r e f l e c t i n g a c t i v i t y . I n such s i t u a t i o n s , ATP may r e f l e c t biomass p o t e n t i a l , or the a b i l i t y of a p o p u l a t i o n t o maintain i t s s i z e I f ex-69 posed t o u n n a t u r a l l y h i g h l o s s e s , f o r example, t o higher t r o p h i c l e v e l s . Previous estimates of b a c t e r i a l a c t i v i t y i n Marion Lake sediments were reviewed and compared t o hetero-t r o p h i c uptake and r e s p i r a t o r y p o t e n t i a l measurments obtained during t h i s study. Close c o r r e l a t i o n between he t e r o t r o p h i c uptake of glucose i n 1971-72 and 1973-74 i n d i c a t e d that the b a c t e r i a l p o p u l a t i o n i s comparable, a t l e a s t i n f u n c t i o n , from year t o year. The seasonal responset-of b a c t e r i a l biomass was i n t e r -preted w i t h respect t o s e v e r a l a c t i v i t y measurements, assuming these measurements r e f l e c t e d growth r a t e , and that g r a z i n g i n t e n s i t y was r e l a t i v e l y h i g h i n months when temperatures were above approximately 10 . The q u a n t i t a t i v e r o l e of b a c t e r i a i n the carbon budget of the sediments was described u s i n g acquired data and tha t of s e v e r a l previous students of the Marion Lake benthos. The c o n t r i b u t i o n of allochthonous compared t o autoch-thonous inputs i s u n c l e a r , although estimates of t h e i r c o n t r i b u t i o n s t o the d i s s o l v e d organic matter pools are a v a i l a b l e . 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M i c r o b i a l ecology as studi e d by luminescence microscopy i n i n c i d e n t l i g h t . B u l l . E c o l . Res. Comm. (Stockholm) 17*61 -65 . APPENDICES 79 Appendix 1. Method f o r e x t r a c t i o n of t o t a l ATP from f r e s h sediments (based on Lee et a l . , 1971a). E x t r a c t i o n Add 3 g wet wt. (150-200mg dry wt.) sediment t o a c h i l l e d c e n t r i f u g e tube. Add 5 ml. of i c e - c o l d O.6NH2SO4 t o tube, mix i n t e r m i t t e n t l y f o r 5 min keeping sample c o l d then s t o r e on i c e f o r 20 min. Centrifuge (5000g/5min) and t r a n s f e r the super-natant t o a 10 ml graduated c y l i n d e r . Record the volume (approximately 7 ml). Transfer the p e l l e t t o a t a r e d weighing pan f o r dry wt, determination. P u r i f i c a t i o n T ransfer 2 ml of e x t r a c t t o a t e s t tube keeping i t I c e -c o l d . Add 0.4 mis s e t t l e d volume i n a wide mouth p i p e t t e of c a t i o n exchange r e s i n (Amberlite 3LR-20, converted to Na -form by washing w i t h IN NaOH and d i s t i l l e d water). Mix the contents f o r 3 min. S t r a i n the sample through g l a s s wool u s i n g 3 - 1 ml a l i q u o t s of d i s t i l l e d water t o wash the tube. Repeat the r e s i n treatment, t h i s time u s i n g 2 - 1 ml washes. Adjust the pH t o 7.8 w i t h NaOH (,6N, ,06N, ,006N). The pH changes very r a p i d l y above pH 3. Adjust the volume of the e x t r a c t t o 10 ml w i t h c o l d T r i s b u f f e r (.02 M, pH 7.8). Assay the ATP Immediately or s t o r e a t -20°. P r i o r t o assay i t was found t h a t It5 d i l u t i o n of the e x t r a c t I n T r i s b u f f e r gave optimum r e s u l t s , but t h i s r a t i o v a r i e s w i t h sediment type. The p u r i f i c a t i o n step increased recovery of ATP t e n - f o l d . 81 Appendix I I . Method f o r e x t r a c t i o n of t o t a l ATP from l y o p h l l l z e d sediments. Add 3 ml of I c e - c o l d bromosuccinlmide e x t r a c t i o n reagent (.01 M n-bromosuccinimide, .01 M EDTA, .02 M Na2HAs0jj. ,pH 7.4) t o 20 mg of l y o p h l l l z e d sediment I n a c e n t r i f u g e tube. Keep on Ice f o r 25 min w i t h o c c a s i o n a l mixing, then c e n t r i f u g e (5000g/ 5 min). Add 2 ml of i c e - c o l d water to 1 ml of the supernatant, and a d j u s t pH to 7 . 8 w i t h IN HCl. Adjust f i n a l volume t o 5 ml wi t h T r i s b u f f e r (.02M, pH 7 .8) and assay Immediately or store a t -20°. No d i l u t i o n i s necessary p r i o r t o assay. S e v e r a l reagents were t e s t e d before n-bromosuccinimide was chosen f o r t h i s procedure (Table A 1). H2SO4 and a c i d i c DMSO w i t h EDTA added are almost as e f f i c i e n t as t h i s reagent. The amount of sediment e x t r a c t e d (20 mg) can be a c c u r a t e l y weighed, but does not c o n t a i n high enough concentrations of ca t i o n s t o i n t e r f e r e w i t h the assay as suggested by the f a c t t h a t c a t i o n exchange treatment of ^ SO/j. e x t r a c t s Jaad l i t t l e e f f e c t , and the recovery per u n i t weight of sediment was reduced when a sediment weight of 100 mg was used. Table A l . Methods t e s t e d f o r e x t r a c t i o n of ATP from l y o p h l l i z e d sediments (sample s i z e 20 mg l y o p h l l l z e d wt. of sediment). reagent reference T r i s b u f f e r (.02 M, pH 7.8) Holm-Hansen and Booth (1966) Na HC0 3(.1M) HC10 4 ( .6N) DMSO ( n e u t r a l -90# i n .05 M T r i s b u f f e r ) DMSO(acidic -90# i n O i l N H2SO4) Bromesuccinimide (see t e x t ) H3PO4 (.6N) H 2S0^ (.6N) Bancroft e_t a l ^ (1974) Lee et a l ^ (1971a) Lee et a l . (1971a) Lee et a l ^ . (1971a) MacLeod et a l ^ (1969) B. K. Burnison(unpubllshed) Lee et a l . (1971a) + c a t i o n exchange treatment3 1 Values not c o r r e c t e d f o r e x t r a c t i o n e f f i c i e n c y 2 ND = not detectable 3 t h i s was the only sample r e c e i v i n g c a t i o n exchange treatment EDTA(.01 M) ATPjl(/Jg/g oven dry wt) 0.55 * 0.56 3.75 1.49o + ND 2 6.05 + 3.40 5.34 + 10.24 + 10.70 5 . 0 5 6 . 0 5 7 .54 1.13 8.22 00 83 Appendix I I I . Method f o r e x t r a c t i o n of m i c r o b i a l ATP from f r e s h sediments. D i l u t e 1 ml of f r e s h sediment w i t h 6 ml f i l t e r - s t e r i l -i z e d lake water. A l i q u o t s of t h i s suspension are d r i e d f o r dry wt. determination. Place 0.35 ml i n a spot d i s h and add 0.5 ml lake water. Examine u s i n g l6x and 25x m a g n i f i c a t i o n of a b i n o c u l a r microscope w i t h a bl a c k sample stage. Remove a l l v i s i b l e animals and algae w i t h micropipettes c o n t r o l l e d v i a rubber tubing by the mouth. Tease l a r g e p a r t i c l e s and a l g a l clumps a p a r t . This process takes 20 - 40 min per sample. Transfer the residue t o a c e n t r i f u g e tube i n an i c e bath, a d j u s t i n g the volume t o 1 ml w i t h water. Add 1 ml i c e -c o l d O.6NJH2SO14. and mix. A f t e r 10 min, c e n t r i f u g e (5000g/5min) a d j u s t supernatant t o pH 7.8 w i t h Na OH (.6, .06, .006N) and add T r i s b u f f e r (.02M, pH 7.8) t o 5 ml. Store the e x t r a c t a t -20° or assay immediately. Recovery I s not improved by d i l u t i o n of the sample. Variance between r e p l i c a t e s tends t o be l a r g e , but the sample s i z e i s l i m i t e d . b y the time r e q u i r e d t o run the e x t r a c t i o n . 84 Appendix IV. Method f o r ATP assay. P r e p a r a t i o n of standard s o l u t i o n s Prepare standard s o l u t i o n s of ATP (lmg/ml, Sigma Chemical o Co.) and st o r e i n 0.2 ml a l l q u o t s at -20 . These are s t a b l e f o r a t l e a s t 7 months. For the assay prepare d i l u t i o n s i n T r i s b u f f e r (.5, 1.0, 2.0, 5.0, 10,0 ng/ml). These are s t a b l e f o r s e v e r a l hours i f kept i c e - c o l d . P r e p a r a t i o n of l u c l f e r i n - l u c i f e r a s e enzyme system Homogenize 200 mg of l y o p h i l i z e d enzyme (Sigma Chemical Co., FLE -50) i n 5 ml f r e s h , c o l d .1 M Na2HAs0i|., .04 M MgSO/j,, .03 M mercaptoethanol (pH 7.4) i n a c h i l l e d t i s s u e homogen-i z i n g tube. Rinse the homogenizer w i t h 5 ml of the b u f f e r . Store the e x t r a c t s overnight a t 2°, then c e n t r i f u g e (5000g/10 min). Add 1000 ml f r e s h .01 M Na2HAsOij., .004 M MgSO^, .03 M mercaptoethanol, ,5# bovine serum albumin (Sigma A 4503) (pH7.4). E q u i l i b r a t e a t 2° f o r 1 h p r i o r t o use. The enzyme shows l i t t l e d e t e r i o r a t i o n of response even 6 h a f t e r i n i t i a l use i f kept on Ice. This p r e p a r a t i o n method, devised by Hammerstedt (1973), was not used during the e a r l y stages of t h i s study. T t i s s u p e r i o r to other methods i n which a c t i v i t y decreased over time so samples measured a t d i f f e r e n t times were not d i r e c t l y comparable. Assay procedure Set up the counting apparatus as r e q u i r e d . I n t h i s study a l i q u i d s c i n t i l l a t i o n counter was used (Nuclear Chicago o Mark I or U n i l u x I I ) a t 4 , Windows were wide ppen (00 - 99) w i t h a t t e n u a t i o n a t 3H s e t t i n g s (A 200). Coincidence c i r c u i t r y was e l i m i n a t e d . 85 Add 1 ml of .04 H g l y c y l g l y c l n e , .003 M MgSO^ (pH 7.4) t o a s c i n t i l l a t i o n v i a l (washed i n a c i d and d i s t i l l e d water). Then add 1 ml of the e q u i l i b r a t e d enzyme mixture. Lower the v i a l i n t o the counting chamber and wait 5 sec t o reduce phosphorescence. Count the background f o r 1 min. F i v e seconds a f t e r completion of the background count, add 1 ml of unknown or standard ATP sample (automatic p i p e t t e s g i v i n g quick d e l i v e r y are advantageous), and lower the v i a l i n t o the counting chamber. Twenty seconds a f t e r completion of the back© ground count, i n i t i a t e a 1 minute gross count. Subtract background t o c a l c u l a t e net a c t i v i t y . P l o t net cpm versus ATP c o n c e n t r a t i o n and read unknown concentrations from t h i s curve. The r e l a t i o n s h i p i s l i n e a r over a wide c o n c e n t r a t i o n range (Figure A 1). 86 Figure A I . T y p i c a l standard curve f o r ATP assay (May 18, 1974). Net a c t i v i t y (cpm x 10" ) 87 Appendix V. Recovery of ATP added t o sediments. The e f f i c i e n c i e s of the e x t r a c t i o n procedures were determined i n a s e r i e s of experiments i n which pure c u l t u r e s of organisms were added t o sediment samples. Measurement of ATP i n the pure c u l t u r e s (using a method analogous t o t h a t i n Appendix I I I ) , i n ;the sediment w i t h added organisms and i n c o n t r o l samples permits s o l u t i o n of the f o l l o w i n g r e l a t i o n -s h i p ! e f f i c i e n c y « ATP(sediment + organisms) - ATP sediment ATP organisms The r e s u l t s using a number of species f o r the three e x t r a c t i o n methods are shown i n t a b l e A I I , The i s o l a t e s from Marion Lake were the most abundant organisms obtained on b r a i n heart i n f u s i o n agar p l a t e s In two p l a t e count s e r i e s . I s o l a t e s 73-1 and 74-1 are small Gram negative rods and 74-2 i s a l a r g e Gram negative rod. 88 Table A l l . E f f i c i e n c y of the v a r i o u s ATP e x t r a c t i o n methods. e x t r a c t i o n method organism e f f i c i e n c y HgSO/^  " t o t a l " 1 f r e s h sediment) H2S0i(, "micro" ( f r e s h sediment) I s o l a t e 73-1 .091 7 3 - 1 .129 , 7 4 - 1 .052 | .128 7 4 - 2 .209 B a c i l l u s s u b t l l l s ] l 6 l O s c i l l a t o r l a spp. .102 t l l g A n a c y s t l s nldulans.129 I s o l a t e 73-1 .248 B a c i l l u s s u b t l l l s .161 «-205 .125 Bromosuccinimlde B a c i l l u s s u b t l l l s .106 ( l y o p h i l i z e d sediment1 89 Appendix V I . R e l a t i o n between ATP c o n c e n t r a t i o n and biomass carbon. The r a t i o of organic carbon a s s o c i a t e d w i t h l i v i n g organ-isms t o ATP i s v a r i a b l e , b u t an average of 250il has been suggested (Hamilton and Holm-Hansen, 196?). This r a t i o was determined f o r a number of organisms i n t h i s study (Table A I I I ) . B a c t e r i a were grown i n n u t r i e n t b r o t h + 0.5# yeast e x t r a c t , the actinomycete i n a medium reporte d by Rodina (1972, p. 373). and the blue-green bacterium inBG-11 ( S t a n i e r et a l . . 1971). The b a c t e r i a were i n e a r l y s t a t i o n a r y phase when harvested while the blue-green c u l t u r e was approximately three weeks o l d and appeared healthy. C e l l s were concentrated by c e n t r i f u g a t i o n (3000g/5min) and resuspended a f t e r one wash w i t h water. ATP was e x t r a c t e d from 1 ml of the suspension u s i n g the method described f o r m i c r o b i a l ATP i n f r e s h sediments (Appendix I I I ) . Organic carbon was measured i n a carbon a n a l y z e r (Beckman Model 915)• V i a b l e counts were determined on agar p l a t e s of the same medium as the c u l t u r e s were grown i n . The mean C/ATP r a t i o of 28? (Table A I I I ) was i n t e r -preted as a c o n f i r m a t i o n of the popular value of 250. 90 Table AIII. The relationship between ATP, biomass carbon and c e l l numbers. organism isolate 74-1 74-2 Bacil lus subt l l l s  Streptomyces spp.  Anacystls nldulans ATP/cell (>ug x 10°) 2.35 4.62 14.90 7.33 biomass carbon/ATP 112 292 439 376 218 287 91 Appendix V I I . The e f f e c t of i n c u b a t i o n time and oxygen on dehydrogenase a c t i v i t y measurements. Incu b a t i o n time The i n c u b a t i o n t i m e - a c t i v i t y measurement response i s shown i n f i g u r e A 2. This experiment shows that a c t i v i t y , measured as formazan production, i s r a p i d during the f i r s t few hours, then drops t o a lower r a t e . The r e s u l t s suggest i n c u b a t i o n times l e s s than 6 h would be most s e n s i t i v e t o d i f f e r e n c e s i n r e s p i r a t o r y p o t e n t i a l i n n a t u r a l samples. I n response to previous s t u d i e s ( P a t t e r s o n et a l . . 1970; S o r o k i n and Kadota, 1972) 24 h i n c u b a t i o n was used. Four hour samples were run simultaneously t o t e s t s e n s i t i v i t y changes. The seasonal data (presented i n R e s u l t s ) i n d i c a t e a c t i v i t y measurements at the two i n c u b a t i o n periods are c l o s e -l y c o r r e l a t e * . S e n s i t i v i t y was g reatest using the s h o r t e r i n c u b a t i o n time - the maximum d i f f e r e n c e was 456 mg formazan produced/m 2.h (Sep. 1973 - Apr. 1974) f o r 4 h i n c u b a t i o n com-pared to 168 (June, 1973 - Apr. 1, 1974) f o r 24 h Incubation. Oxygen P r i o r t o Incubation, sample f l a s k s were f l u s h e d w i t h n i t r o g e n f o r 10 minutes. The r a t i o n a l e f o r t h i s i s that oxygen and TTC compete f o r e l e c t r o n s i n the e l e c t r o n t r a n s -port system and v a r i a b l e oxygen concentrations i n samples would reduce the c o m p a r a b i l i t y of TTC-measured dehydrogenase a c t i v i t y i n d i f f e r e n t samples. The m i n i m i z a t i o n of t h i s c ompetition not only makes a c t i v i t y measurements more con-s i s t e n t , but a l s o higher (Table A I V ) . The data from d i f f e r e n t depths i n the sediments f u r t h e r support t h i s p o i n t . The Figure A 2. E f f e c t of Incubation time on dehydrogenase a c t i v i t y measurements. 92 a cd to U o >> •5-0--p o as ^ 2.5-» to 4) A to-a o a> u o •d 3 X O 0) U a a 0 0 4 8 12 16 2 0 2 4 Time (h) 93 Table AIV. The effect of aerobic versus anaerobic conditions on dehydrogenase act iv i ty assayed at different depths in the sediment (March 14, 1973). sediment horizon Incubation condition formazan production (cm) (ps/g dry wt./h) 0 - 1 aerobic 20 anaerobic 103 1 - 2 aerobic 15 anaerobic 108 2 - - 3 aerobic 44 anaerobic 88 94 aerobic value from 2 - 3 cm depth i s probably high r e l a t i v e t o the anaerobic value because of the normal absence of oxygen i n t h i s l a y e r . T h i s i s i n agreement w i t h the f i n d i n g s of Lenhard (1968) t h a t removal of oxygen from anaerobic sludge had l i t t l e e f f e c t on dehydrogenase a c t i v i t y . 95 Appendix V I I I . C a l c u l a t i o n s of h e t e r o t r o p h i c uptake parameters. Theory-Many d e s c r i p t i o n s of the method f o r c a l c u l a t i n g uptake parameters i n water samples have been published since 1965. The f o l l o w i n g i s an o u t l i n e of a procedure developed by K l e i b e r (1972) f o r d i l u t e d sediment samples. Turnover time, T, i s r e l a t e d t o substrate c o n c e n t r a t i o n , S , and uptake r a t e , V, by the formulat T «= S_ E l V I f the n a t u r a l turnover time and substrate c o n c e n t r a t i o n , T n and S N , are known then the r e a l r a t e of uptake, U N , may be c a l c u l a t e d : U N « E2 S N can be measured independently of uptake experiments. The experimental design of the experiments does not permit d i r e c t e s t i m a t i o n of T n because S i n the i n c u b a t i o n f l a s k s i s not S N . I t i s i n s t e a d the added r a d i o a c t i v e substrate c o n c e n t r a t i o n ( S A ) , plus the q u a n t i t y n a t u r a l l y i n the sed-iments c o r r e c t e d f o r the d i l u t i o n f a c t o r , d, such t h a t i s 0 8 s a + S J I E3 d T i s a f u n c t i o n of S , so determination of T n must be performed a t S N . I n n o n - k i n e t i c experiments, f o r Instance u s i n g ^ ^ - b i c a r b o n a t e t o measure primary production i n water samples, t h i s i s not a problem i f S N i s not g r e a t l y changed by the added s u b s t r a t e . The sediment s t u d i e s r e q u i r e d i l u t i o n of the samples,however. E x t e r n a l substrate i s then added a t incremental l e v e l s which h o p e f u l l y encompass the n a t u r a l , u n d i l u t e d substrate l e v e l . I n each experiment, there i s an estimated turnover time, T e, f o r each S a. This i s c a l c u l a t e d byt T p = Rn t E4 where R a i s r a d i o a c t i v i t y (dpm) added t o the sample, R u i s the gross uptake of r a d i o a c t i v i t y and t i s the i n c u b a t i o n time. These values of T e can be measured without i n f o r m a t i o n about S ( i e , S a or S n ) , but are not independent of S. The v a r i e t y of l i n e a r transformations ( H a l l et a l . . 1972) of the h y p e r b o l i c Michaelis-Menten f u n c t i o n assume th a t T e i s a l i n e a r f u n c t i o n of S. The t r a n s f o r m a t i o n used In t h i s study has the formi T e • Kt-. + Sn + 1 S a E5 where Kt i s the transportaconstant and V M i s the maximum uptake r a t e . By p l o t t i n g T e versus S a (modified Lineweaver-Burke p l o t ) estimates of V M , (Kt + SLQ) and T 0, the turnover d time f o r Sa«= o or S«=SW (E3) may be obtained (Figure A3). d The work of Williams (1973) and Burnison and Morita (1973) discussed i n the I n t r o d u c t i o n i s a p p l i c a b l e here. Estimates of V M and (Kt + Sn) are both dependent f o r t h e i r d accuracy on the v a l i d i t y of the assumption of l i n e a r i t y . T 0 i s an e m p i r i c a l l y d e r i v e d q u a n t i t y and Independent of t h i s assumption. Determination of T n, the turnover time a t S n, i s 97 Figure A 3. Graphic r e p r e s e n t a t i o n i l l u s t r a t i n g the modified Lineweaver-Burk p l o t . 9? a 98 accomplished by determining the value of S a which corresponds to S n, This value 1st S a = S n - Sj2 E6 d * From the T e versus S a | > l d t , T e, the turnover time corresponding to S a, may be determined. D i v i s i o n of t h i s q u a n t i t y by d accounts f o r d i l u t i o n of sediment b a c t e r i a i n the experiment so» Tr, = TjL . E7 d T n c a l c u l a t e d i n t h i s manner, and the independent value of S n are plugged i n t o E 2 t o give U n. Data processing The experimental data c o n s i s t e d of the dry weight of sediment per f l a s k , the i n c u b a t i o n time, the added ^ C -glucose (Ra = cpm ijmg), the r e s p i r e d and p a r t i c u l a t e r a d i o -a c t i v i t y (cpm) f o r two sample f l a s k s and one c o n t r o l a t each of four values of S a. I n t e r s t i t i a l glucose concentrations were determined independently but on the same mixed batch of sediment. Gross uptake (R u) was c a l c u l a t e d as the sum of the average, net, l i v i n g , r e s p i r e d and p a r t i c u l a t e uptake, converted t o dpm. T e (h) was c a l c u l a t e d f o r each l e v e l of S a ( j u g / l ) and a l i n e a r r e g r e s s i o n equation determined (E5). By sub-s t i t u t i n g S a • S n - S n/50, where 50 was the d i l u t i o n f a c t o r , i n t o the r e g r e s s i o n equation, T e was obtained. T n was c a l -c u l a t e d by E? and D n by E 2 . The r e g r e s s i o n c o e f f i c i e n t was l / V m . 

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