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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 A C T I V I T Y OF BACTERIA  IN THE SEDIMENTS  OF MARION L A K E , B R I T I S H COLUMBIA, by EDWARD ALFRED PERRY B. S c . , Y o r k U n i v e r s i t y ,  1971  A T H E S I S SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENTS  FOR THE DEGREE OF  MASTER OF SCIENCE  in  the department of Zoology  We a c c e p t t h i s  thesis  required  as conforming  to the  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA August,  197^  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  f u r t h e r agree  for  scholarly  by h i s of  this  written  thesis at  the U n i v e r s i t y  make  that  it  purposes  for  freely  permission may  representatives. thesis  in p a r t i a l  financial  is  of  Columbia,  British for  for extensive by  gain  Depa r t m e n t  Columbia  shall  the  requirements I  agree  r e f e r e n c e and copying  of  this  that  not  copying  or  for  that  study. thesis  t h e Head o f my D e p a r t m e n t  understood  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  available  be g r a n t e d  It  fulfilment  or  publication  be a l l o w e d w i t h o u t  my  ii  ABSTRACTs THE BIOMASS AND ACTIVITY OF BACTERIA IN THE SEDIMENTS OF MARION LAKE, BRITISH COLUMBIA. Two biomass i n d i c a t o r s  ( d i r e c t counts and ATP a n a l y s i s )  and two a c t i v i t y e s t i m a t o r s (glucose uptake and a c t i v i t y ) were used t o study the b a c t e r i a a t 1 m i n Marlon Lake sediments. D i r e c t count-biomass  dehydrogenase water  depth  estimates f o r  b a c t e r i a averaged 0 . 6 l gC/m^, were h i g h i n summer, d e c l i n e d rapidly i n f a l l ,  then i n c r e a s e d d u r i n g the w i n t e r . Microorganisms  l e s s than approximately 30 jd m diameter had a mean biomass of 1.28  gC/m as measured by ATP a n a l y s i s . Seasonal v a r i a t i o n s 2  i n t h i s f i g u r e p a r a l l e l e d changes i n the a l g a l p o p u l a t i o n , although a l g a l c o n t r i b u t i o n s t o the m i c r o b i a l biomass were l e s s than 50 per c e n t . ATP a n a l y s i s was a l s o used t o estimate the biomass o f the sediment  community, e x c l u d i n g animals g r e a t e r  than approximately 5 mn> i n l e n g t h . The mean community biomass was 4 . 6 9 gC/m . 2  Comparison  of ATP data w i t h enumeration data obtained by  o t h e r s , suggests that ATP i s a good biomass i n d i c a t o r , except when c e l l u l a r ATP l e v e l s are changed i n r e a c t i o n t o b i o t i c or a b i o t i c environmental f a c t o r s . I t i s proposed t h a t , i n s i t u a t ions such as i n t e n s e g r a z i n g or r a p i d Increases or decreases i n temperature, ATP measurements r e f l e c t not only biomass but a l s o a c t i v i t y . At such times ATP-biomass data may I n d i c a t e biomass p o t e n t i a l , or the c a p a c i t y of the p o p u l a t i o n t o maintain i t s biomass under abnormally h i g h r a t e s of l o s s . Dehydrogenase a c t i v i t y , or r e s p i r a t o r y p o t e n t i a l , of the sediment b a c t e r i a was assayed u s i n g t r l p h e n y l  tetrazolium  Hi  c h l o r i d e . The estimate f o r annual r a t e of carbon l o s s as CO2 from the b a c t e r i a l p o p u l a t i o n (19.3 g C/m .year), was almost 2  i d e n t i c a l t o p r e v i o u s l y r e p o r t e d 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 r a t e n a t u r a l uptake r a t e  (9.6 S C/m .year), 2  (5.3 S C/m .year) and the n a t u r a l t u r n 2  over time (0.31 h) were s i m i l a r t o p r e v i o u s d a t a f o r these sediments. T h i s suggests t h a t , a t l e a s t i n terms of f u n c t i o n , the b a c t e r i a l p o p u l a t i o n i s q u i t e s t a b l e from year t o y e a r . Biomass and a c t i v i t y of the Marion Lake sediment  bacteria  were found t o be i n phase i n l a t e s p r i n g through e a r l y but a c t i v i t y remained  fall,  low i n winter d e s p i t e Increases i n the  m i c r o b i a l biomass, and i n c r e a s e d much more r a p i d l y than b i o mass i n e a r l y s p r i n g . The dynamics of the b a c t e r i a l p o p u l a t i o n are d i s c u s s e d i n terms of these r e l a t i o n s h i p s . The  s i z e of the a l g a l and b a c t e r i a l p o p u l a t i o n s and o r g a n i c  matter r e s e r v o i r s , and the f l u x r a t e s f o r carbon between these compartments a r e summarized.  iv  TABLE OF CONTENTS  Abstract  i i  Table of Contents  iv  L i s t of Tables  vi  L i s t of F i g u r e s  vii  Acknowledgment  ix  1  Introduction 1.  O b j e c t i v e s and background  1  2.  B a c t e r i a l biomass - b a c t e r i a l counts 7  - ATP a n a l y s i s 3 . A c t i v i t y of microorganisms  5  - r e s p i r a t i o n and dehydrogenase (oxidoreductase) activity 10 - uptake of r a d i o t r a c e r s  Introduction  Sampling Methods  16  t o the appendices  M a t e r i a l s and Methods  13  17  17 19  R e s u l t s and D i s c u s s i o n  27  1.  P h y s i c a l and chemical data  2.  Biomass estimates  27  31  (a) T o t a l sediment biomass estimated by ATP a n a l y s i s (b) M i c r o b i a l biomass i n the sediments estimated by ATP a n a l y s i s 39 (c) B a c t e r i a l biomass i n the sediments estimated by m i c r o s c o p i c counts *EL  31  V  (d) R e l a t i o n s 3. A c t i v i t y  between biomass estimates  estimates  50  (a) Dehydrogenase a c t i v i t y measurements  50  (b) H e t e r o t r o p h i c uptake of ^ C - g l u c o s e  53  (c) R e l a t i o n s Biomass and  between a c t i v i t y e s t i m a t o r s a c t i v i t y of Marion 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 (b) Q u a n t i t a t i v e 5. Summary and References  71  Appendices  79  1.  description  Conclusions  60  68  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).  5. Recovery of ATP  assay.  from f r e s h  8U-  added t o sediments.  6. R e l a t i o n between ATP  79  from lyoph--  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 sediments. ' 83 Method f o r ATP  60  65  2. Method f o r e x t r a c t i o n of t o t a l ATP i l i z e d sediments. 81 3.  57  concentration  87 and  biomass carbon.  7. 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, 91 8.  Calculations  of h e t e r o t r o p h i c  uptake parameters.  95  89  vi LIST OF TABLES I. Carbon t o ATP r a t i o s from v a r i o u s s o u r c e s . I I . S t a t i s t i c a l a n a l y s i s of biomass d a t a . I I I . Sediment ATP - biomass carbon d a t a .  32 35-36  38  IV. S t a t i s t i c a l a n a l y s i s of dehydrogenase a c t i v i t y  data.  52  V. Maximum uptake r a t e s , and n a t u r a l t u r n o v e r times and uptake r a t e s f o r glucose i n sediments and water.  56  A I . 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 i l i z e d sediments.  82  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. A I I I . The r e l a t i o n s h i p between ATP, c e l l numbers.  biomass carbon and  90  AIV. The e f f e c t of a e r o b i c v e r s u s a n a e r o b i c c o n d i t i o n s on dehydrogenase  activity  i n the sediment.  93  assayed a t d i f f e r e n t  depths  88  vii LIST OF FIGURES  1. Marion Lake - morphometry and sampling a r e a .  18  2. Scanning e l e c t r o n micrographs of Marion Lake  sediment  29  particles.  3. S e a s o n a l changes  i n temperature  (a), t o t a l organic  matter ( b ) , and d i s s o l v e d glucose (c) i n the sediments. 4. S e a s o n a l changes o f t o t a l biomass carbon i n l y o p h i l i z e d (a) and f r e s h (b) sediments and o f m i c r o b i a l  biomass  carbon ( c ) . 5. S e a s o n a l changes o f b a c t e r i a l biomass carbon. 6. S e a s o n a l change i n the p r o p o r t i o n o f sediment  ^3 biomass  carbon i n b a c t e r i a l compared t o t o t a l biomass-  bacterial  compared t o m i c r o b i a l biomass; m i c r o b i a l compared t o Ur$  t o t a l biomass,  7. S e a s o n a l changes o f dehydrogenase sediments.  a c t i v i t y i n the  51  8. Seasonal changes i n t h e sediments o f maximum (v" ) and m  a c t u a l ( U ) r a t e s o f g l u c o s e uptake, and the a c t u a l n  t u r n o v e r time ( T ) . n  9. Diagrammatic  5*4-  r e p r e s e n t a t i o n of the s e a s o n a l r e l a t i o n s  between a c t i v i t y and biomass o f the sediment as p r o p o r t i o n s o f t h e i r maxima,  62  10. S t r u c t u r e and f u n c t i o n o f the Marion Lake ecosystem.  66  bacteria  sediment  viii  A 1. T y p i c a l standard 197*0.  curve f o r the ATP  assay (May  18,  86  A 2. E f f e c t of i n c u b a t i o n time on dehydrogenase measurements.  activity  92  A 3. G r a p h i c a l 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 Lineweaver-Burk p l o t .  97  modified  ACKNOWLEDGMENT  T h i s r e s e a r c h was conducted as p a r t o f the Canadian IBP Marion Lake P r o j e c t , l o c a t e d a t the U n i v e r s i t y of B r i t i s h Columbia, and funded by t h e N a t i o n a l Research C o u n c i l (NRC) of Canada. I would l i k e t o acknowledge f i n a n c i a l support from NRC and from B r i t i s h  Columbia  Packers L i m i t e d and F i s h e r i e s A s s o c i a t i o n o f 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 p r o j e c t . I n p a r t i c u l a r , I would l i k e t o thank Drs. I . E. E f f o r d , P. K l e i b e r , K, H a l l and B. K. B u r n i s o n f o r t h e i r ideas and d i s c u s s i o n s . I would l i k e t o acknowledge  t h e c o n s t r u c t i v e comments  and c r i t i c i s m of the members of my r e s e a r c h  committee,  i n c l u d i n g Drs. P. L a r k l n , H. Blackburn, K. H a l l and I . E. E f f o r d . Dr B l a c k b u r n was v e r y generous, a l l o w i n g me t o monopolize h i s r e s e a r c h  facilities.  F i n a l l y I would l i k e t o thank Corinne P e r r y , f o r b e i n g the  way she i s .  1 INTRODUCTION  1. O b j e c t i v e s 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 s t u d i e d i n d e t a i l i n a n e f f o r t t o d e s c r i b e the r a t e and c o n t r o l of energy f l o w between the major compartments of the system ( H a l l and H y a t t , 197-+). The s t u d i e s s i n c e 19&3 were supported by t h e I n t e r n a t i o n a l B i o l o g i c a l Program a s p a r t o f i t s commitment t o a s s e s s p r o d u c t i v i t y i n d i v e r s e environments. T h i s emphasis on e c o l o g i c a l 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 e s c r i p t i o n o f food webs by Forbes i n I887 (quoted i n Odum, 1968)  and the energy f l o w by t r o p h i c l e v e l concept i n t r o -  duced by Lindeman (19^2). The q u a n t i t a t i v e approach t o a q u a t i c ecosystem d e s c r i p t i o n u s i n g the Lindeman concept has been used I n S i l v e r S p r i n g s , F l o r i d a (Odum, 1957). a temperate c o l d s p r i n g ( T e a l , 1957)  and a s a l t marsh ( T e a l ,  1962). Marion Lake Is s i t u a t e d 50 km east o f Vancouver, B r i t i s h Columbia, a t a n e l e v a t i o n o f 300m. The c l i m a t e Is t y p i c a l l y c o a s t a l b e i n g r e l a t i v e l y m i l d and wet w i t h a n annual p r e c i p i t a t i o n o f 2^0 cm. The lake i s s m a l l  (13  h e c t a r e s ) , s h a l l o w (mean depth 2.^ m) and s u b j e c t t o s h o r t t u r n o v e r time (as s h o r t as t h r e e days d u r i n g p e r i o d s of h i g h water i n f l o w ) . P l a n k t o n l c p r o d u c t i o n i s extremely low due ,  d i r e c t l y o r i n d i r e c t l y , t o the h i g h f l u s h r a t e  ( E f f o r d 1967. 1969). Recent work has t h e r e f o r e f o c u s e d on d e t e r m i n i n g compartment s i z e and carbon or energy f l u x e s between the compartments o f the b e n t h i c  environment.  2 A l g a l biomass I n the Marlon Lake sediments i m a t e l y 0 . 0 4 3 gC/m  i s approx-  (Efford, unpublished). B a c t e r i a l  2  numbers were estimated by F r a k e r , u s i n g p l a t e counts, t o v a r y from 5 x 10^ c e l l s / m l i n w i n t e r t o 2 x 10^ c e l l s / m l i n summer ( u n p u b l i s h e d ) . U s i n g m i c r o s c o p i c c o u n t i n g methods, Ramey (1972) and B u r n i s o n (unpublished) e s t i m a t e d numbers o f b a c t e r i a a t the sediment cells/g respectively.  s u r f a c e t o be 10^ c e l l s / m l and l O ^ 1  Both these workers found a decrease  w i t h depth below 2 cm. H a l l and Hyatt (197*0, u s i n g B u r n i s on' s data and a n average f r e s h weight  c e l l volume o f 0.36jjm3 d e r i v e d a  o f 3.9 mg/g sediment. T h i s i s e q u i v a l e n t t o  approximately  0.39 gC/m , or about 2  nine times the a l g a l  biomass. Net d i s s o l v e d and p a r t i c u l a t e a l l o c h t h o n o u s i n p u t s t o the l a k e a r e 28 gC/m /year and 36 gC/m /year 2  2  respectively  (Geen, unpublished; Odum, u n p u b l i s h e d ) . Autochthonous i n ( 8 gC/m /  puts i n c l u d e primary p r o d u c t i o n by phytoplankton year - E f f o r d ,  2  1967)t e p i b e n t h i c a l g a e ( 4 0 . 4 - 44.2 gC/m / 2  year - Hargrave, 1969»Greundllng, 1971). :  Carbon l o s s e s as carbon d i o x i d e from the b e n t h i c community t o t a l 57 gC/m /year (Hargrave, 1969). Estimates o f 2  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-  p a r i n g oxygen uptake i n normal v e r s u s 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 o f 4.2 -  24 gC/m /year  f o r b a c t e r i a l r e s p i r a t i o n was o b t a i n e d by K l e i b e r u s i n g t h e data of H a l l e t a l ^ (1972).  2  (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 H a l l et a l ^ (1972) and H a l l 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/m /year. Using the data of H a l l et a l ^ (1972) he 2  further estimated the oarbon f l u x from the environment to bacteria to be 21 - 120 gC/m /year. The importance of the 2  benthic b a c t e r i a l population i s evident from these data. Total net inputs to the lake are about 132 gC/m /year 2  and the use of organic material by bacteria (21 - 120 gC/ m /year) i s equivalent to 16 - 91# of t h i s net input. 2  The purpose of t h i s study was to assess biomass and a c t i v i t y of bacteria i n Marion Lake sediments. As described above some measurements have been obtained but the b a c t e r i a l biomass was not measured during the course of the heterotrophic uptake studies (Hall et a l . . 1972) and bacteria f  have not been quantified seasonally except by plate count methods. No approach to either biomass or a c t i v i t y measurement i s accepted as a "standard method". Many papers report informa t i o n on various parameters of microbial populations but methodology i s diverse. A few studies comparing multiple approaches simultaneously have been reported (Witkamp, 1 9 7 3 . i n s o i l s * Hobble et a l . . 1 9 7 2 , In the ocean). Work comparing two methods i s more abundant and Is referred to In the discussion. During t h i s study, two biomass estimators (adenosine-  4  5'-triphosphate (ATP) and direct microscopic counts) and two a c t i v i t y estimators (dehydrogenase a c t i v i t y and  ^C-  glucose u t i l i z a t i o n ) were employed. The changes,within and between these methods,seasonally, are presented.  5  2 , B a c t e r i a l Biomass  B a c t e r i a l counts Plate count methods have long been used to estimate the numbers of microorganisms i n water, s o i l s 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 v e r t i c a l d i s t r i b u t i o n of bacteria i n s o i l s s t i l l use t h i s approach. There i s no doubt that the technique i s useful f o r looking at s p e c i f i c physiological groups of microorganisms but the use of the aerobic, heterotrophic plate count as an indicator of t o t a l viable b a c t e r i a l numbers i s i n v a l i d (Schmidt, 1 9 7 3 ) . 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 consuming, i t i s d i f f i c u l t to d i f f e r e n t i a t e between d e t r i t a l p a r t i c l e s , dead bacteria and viable b a c t e r i a . In sediments p a r t i c u l a r l y , many p a r t i c l e s are i n the size range of bacteria and many of these adsorb chemicals used to s t a i n c e l l s . The i r r e g u l a r 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 t h i s study to give an upper l i m i t f o r the b a c t e r i a l biomass. Direct counting methods have been employed i n diverse environments including water (Sorokin,  1970i Hobble et a l . .  1 9 7 2 ) , s o i l (Babiuk and Paul, 1970$ T r o l l d e n i e r , 1973)# beachsands (Khiyama and Makemson, 1973) and sediments (Olah, 1 9 7 2 ) . Fluorescent stains are h e l p f u l i n making small b a c t e r i a l  6  c e l l s on p a r t i c l e s more v i s i b l e . Quantitlve work using fluorescent stains such as acridlne orange ( T r o l l d e n i e r , 1973)  and f l u o r e s c e i n isothiocyanate (FITC) (Babiuk and  Paul, 1970)  has been reported. One problem involved i n  the method i s to obtain a sample uniformly dispersed on a microscope s l i d e so that random sampling y i e l d s r e s u l t s with minimal variance. In previous research, samples have been spread i n t h i n layers on s l i d e s and allowed to dry f T r o l l d e n i e r , 1973. Babiuk and Paul, 1970)  or t h i n layers  of agar containing s o i l samples were formed (Jones and Mollison, 19^8), Unfortunately materials prepared In t h i s manner may  have a variable v e r t i c a l d i s t r i b u t i o n 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 u t i o n i s often not An i d e a l way  distrib-  achieved. 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 t h i s technique has been limited to l i g h t microscopy probably because of interference i n fluorescent work from the o i l used to c l e a r 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, c l e a r i n g In acetone vapors and FITC staining has been used i n t h i s study. This technique allows c e l l s to be uniformly d i s t r i b u t e d with n e g l i g i b l e background fluorescence and the c e l l s are bound i n the surface layer of the cleared membrane f i l t e r .  7 ATP Analysis ATP i s present i n a l l l i v i n g c e l l s (Mahler and Cordes, 1966) but i s not associated with dead c e l l s (Holm-Hansen and Booth, 19661 Lee et a l . . 1971asPatterson et a l ^ , 1970 j t h i s study). Presence of ATP i s therefore i n d i c a t i v e of l i f e . Knowledge of numerical r e l a t i o n s between ATP and organic carbon associated with l i v i n g organisms (biomass carbon) permit extrapolation of ATP concentrations to biomass carbon. This relationship has been studied i n bacteria (Ausmus, 1973? Ernst, 1970j Hamilton and Holm-Hansen, 1 9 6 7 ) , algae (Berland et a l . . 1972; Ausmus, 1973) actinomycetes (Ausmus, 1973)» fungi (Ausmus, 1973) and nematodes (Ernst, 1 9 7 0 ) . Considerable work has been done determining ATP content per b a c t e r i a l c e l l (for example Chapelle and Levin, 1968) but information on numbers i s less relevant to ecosystem studies than i s i n ^ formation on biomass. The ATP to biomass carbon r e l a t i o n varies between groups of organisms, between species and within species at d i f f e r e n t times of the l i f e cycle. Despite these d i f f i c u l t i e s a weight r e l a t i o n 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, l i g h t 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  ». a d e n y l - l u c i f e r i n +PPi luciferase  8  o adenyl-luciferln  2  •» adenyl-oxyluciferln + H2O + l i g h t  ATP has been measured i n fresh water (Rudd and Hamilton, 1973- Holm-Hansen and Paerl, 1 9 7 2 ) , oceans (Hobble et a l . , 1972; Holm-Hansen and Paerl, 1 9 7 2 ) , sewage (Patterson et a l . . 1970; Brezonik and Patterson, 1 9 7 1 ) , s o i l s (Ausmus, 1973j Conklin and MacGregor, 1972j MacLeod et a l . . 1 9 6 9 ) , 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 b a c t e r i a l biomass i s d i f f i c u l t . Measurements i n 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, 1 9 7 2 ) , but analyses s t i l l include bacteria, algae, protozoa and many other organisms. These authors further attempted to measure b a c t e r i a l biomass by subtracting a l g a l biomass,determined by d i r e c t counting, from t o t a l biomass of organisms less than 60  jm measured by ATP a n a l y s i s . Even i n t h i s size range there i s the p o s s i b i l i t y of protozoa biomass being included. Another problem i s the uncertainty of the condition of the algae. Direct counts often include dead c e l l s , so overestimatlon of a l g a l biomass and therefore underestimation of b a c t e r i a l mass could r e s u l t . Rudd and Hamilton (1973) followed changes i n biomass of various size groups by d i f f e r e n t i a l f i l t r a t i o n of water samples but no estimate of b a c t e r i a l biomass was  9  obtained because of the presence of small algae less than 10jum,  p a r t i c u l a r l y 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 d i f f e r e n t size groups by ATP analysis i n s o i l s or sediments. In t h i s study, an estimate of the microbial biomass (<approximately 30 pm) was obtained by physical removal of larger size classes.  10  3 . A c t i v i t y of Microorganisms  It  is difficult  i n s i t u . It  to assess what microorganisms are doing  i s not possible to "see" what they are eating,  a s s i m i l a t i n g and excreting and as a r e s u l t chemical and r a d i o tracer techniques are used.  Respiration and dehydrogenase (oxidoreductase)  activity  The t r a d i t i o n a l method for determinimg microbial a c t i v i t y Involves measuring r e s p i r a t i o n by changes i n oxygen and/or carbon dioxide concentrations (Hargrave,19^9> Beyers et a l . . 1 9 6 3 ) . The oxygen method was used rigourously  i n Marion  Lake for one year but the v a l i d i t y of carbon flow information from t h i s data depends on the accuracy of the assumption of a respiratory quotient  (RQ)of 0 . 8 5 (Hargrave,  I969).  Unfortunately, RQ values for sediments range from 0 . 2 7 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 ^ , 1 9 7 3 ) . The measurement of inorganic carbon i s d i f f i c u l t lakes that do not exhibit r e l a t i v e l y  in  high a l k a l i n i t y , u n l e s s  sophisticated equipment i s available at the s i t e  (Schindler,  1973). One conclusion from the above discussion i s that reports of carbon exchange based on gas analysis are often r e l a t i v e rather than absolute. Another way to estimate r e s p i r a t i o n i s by measuring dehydrogenase a c t 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 r e l a t i v e a c t i v i t y estimator since i t measures respiratory p o t e n t i a l rather than r e s p i r a t i o n , but i t i s a convenient method for comparing a c t i v i t y of samples given d i f f e r e n t treatments or obtained at d i f f e r e n t times of the yeais. Dehydrogenase a c t i v i t y has been used as an a c t i v i t y index In water (Olah, 1 9 7 2 ) , sewage (Lenhard, 1 9 6 8 ) ,  soil  (Casida et a l . . 196^1 Lenhard, 1956) and sediments (Olah, 1972; Pamatmat and Bhagwat, 1 9 7 3 ; Edwards and R o l l e y ,  1965).  The experimental approach suggests there should be a c o r r e l a t i o n between r e s p i r a t i o n as measured by oxygen uptake or carbon dioxide evolution and respiratory  potential  as measured by dehydrogenase. Such c o r r e l a t i o n has been demonstrated i n some s o i l studies (Skujins, et a l . 196*0 but not others (Howard, t  (1959)  1 9 7 3 ; Casida  1972).  Stevenson  found oxygen uptake was related to dehydrogenase  a c t i v i t y i n 2k s o i l s  (r = 0.837) but i n  s o i l s which were  amended with decomposing plant matter c o r r e l a t i o n 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 i n 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 c o r r e l a t i o n between  heat production and dehydrogenase a c t i v i t y i n Lake Washington sediments but oxygen uptake by sediment cores  consistently  12  underestimated heat production, presumably because oxygen uptake did not measure anaerobic a c t i v i t y while the dehydrogenase assay d i d , Edwards and Rolley (19^5) also found no c o r r e l a t i o n between oxygen consumption and dehydrogenase a c t i v i t y  In sediments. That both anaerobic and aerobic  bacterial activity  could be assayed by TTC reduction was  demonstrated by Clah ( 1 9 7 2 ) , He followed ATP l e v e l s and dehydrogenase a c t i v i t y  i n aerated and anaerobic incubation  flasks enriched with powdered Phragmltes. During aerobic incubation changes i n dehydrogenase seemed to p a r a l l e l changes i n ATP, but anaerobic conditions yielded higher ATP concent r a t i o n and lower dehydrogenase a c t i v i t y  than was found i n  the aerated c u l t u r e . Sediments are a complex environment,and even anaerobic sediments may contain a community with populations other than bacteria (Fenchel, 1 9 6 9 ) . Does the dehydrogenase measurement assay the a c t i v i t y  of these other organisms?  Pamatmat and Bhagwat (1973) noted the presence of chlronomid larvae i n some of t h e i r samples but did not know whether or not t h e i r a c t i v i t y was included i n dehydrogenase measurements. Packard (1970) and Curl and Sandberg (1961) homogenized animal tissues to assay dehydrogenase a c t 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 . . 1 9 5 9 ) . On the basis of present knowledge i t seems probable that the dehydrogenase assay measures a c t i v i t y  of prokaryotlc organisms only, unless  13 samples are homogenized. The enzyme dehydrogenase was assayed during t h i s study to determine aerobic and anaerobic, prokaryotic  activity  at d i f f e r e n t times of the year.  Uptake of radiotracers The u t i l i z a t i o n of radioactive compounds by microorganisms i s another approach to the estimation of microbial a c t i v i t y . The most common experimental design for measurements i s to test uptake at different  utilization  concentrations  of the radioactive substrate. These data are treated by one or more of the available l i n e a r transformations (Hall et a l . . 1972) of the Michaelis - Menten enzyme k i n e t i c s equation. Information may be obtained on the turnover time of the substrate (T),  the maximum rate of substrate uptake  (V ), m  and a transport constant plus natural substrate concent r a t i o n (Kt + S ) . If S n  can be determined independently the  n  actual rate of uptake (U ) n  may be c a l c u l a t e d .  The o r i g i n a l low substrate concentration a p p l i c a t i o n of Michaelis - Menten k i n e t i c s analysis to study active uptake by heterogeneous b a c t e r i a l populations was by Wright and Hobble (1965). The work of Parsons and Strickland (1962) was at substrate concentrations high enough that  diffusion.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 t h i s . Most experimenters  14 now  use modified techniques  uptake, t h a t i s , the sum  which a l l o w measurement o f  gross  of a s s i m i l a t e d or p a r t i c u l a t e uptake  and r e s p i r a t o r y l o s s e s . There i s , u n f o r t u n a t e l y , 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 M i c h a e l i s - Menten k i n e t i c s t o  geneous populations.The  a n a l y t i c a l technique,  hetero-  originally  r  the Langmuir isotherm of M i c h a e l i s - Menten equation,  was  developed t o d e s c r i b e r e l a t i v e l y simple r e a c t i o n s of gases or enzymes. In f a c t , t h e equation has been found i n a p p l i c a b l e i n a number of s t u d i e s i n n a t u r a l systems (Vaccaro and 1967;  H a l l et aXg.,  19721  K l e i b e r , 1972» Crawford et al^.,  The use of i t t o analyze v a r i a b l e v a l u e s of V model by W i l l i a m s  m  Jannasch, 1974):.  multispecies r e a c t i o n s with  and Kt was  t e s t e d w i t h a computer  ( 1 9 7 3 ) . He r e p o r t e d t h a t (Kt + S )  and  n  T  are s e n s i t i v e t o d e v i a t i o n s from the expected r e l a t i o n between uptake r a t e and at  substrate concentration,  low c o n c e t r a t i o n s , b u t V  m  showed l i t t l e  particularly  change. S i n c e  n a t u r a l s u b s t r a t e c o n c e n t r a t i o n s are o f t e n v e r y low / i g / l ) , erroneous estimates  (  f o r T would i n t u r n e f f e c t  1-50 U  n  calculations, Burnison and competitive  M o r i t a (1973) t e s t e d the occurrence  of  i n h i b i t i o n f o r amino a c i d uptake i n Klamath  Lake waters. Even a t low s u b s t r a t e c o n c e n t r a t i o n s , c o m p e t i t i o n was  evident i n some c a s e s . T h i s i s i n c o n t r a s t t o the r e p o r t  of Crawford et a l . ( 1 9 7 4 ) , t h a t c o m p e t i t i o n between amino a c i d s i n e s t u a r i n e waters seemed t o be of l i t t l e B u r n i s o n and  M o r i t a found t h a t V  p e t i t i v e I n h i b i t i o n , while T and  m  was  consequence.  not e f f e c t e d by com-  (Kt + S ) n  were both i n -  15  creased. From the d i s c u s s i o n s of W i l l i a m s and  Morita  (1973),  (1973)  i t i s apparent t h a t V  and  Burnison  i s the most u s e f u l  m  parameter f o r comparing h e t e r o t r o p h i c 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 l e s s s e n s i t i v e t h a n T and  (K^ + S ) , and t h e r e f o r e U , n  n  t o 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 s u b s t r a t e s and t o n o n - k i n e t i c of uptake v e l o c i t y t o s u b s t r a t e c o n c e n t r a t i o n s " p o t e n t i a l " uptake estimate r a t e of uptake and turnover) estimates glucose  though,and U  n  and  V T  n  m  responses  is a (the a c t u a l  are the v a l u e s r e q u i r e d f o r  of carbon f l u x i n an ecosystem. The  actual rates for  have been used i n t h i s study as an index of m i c r o b i a l  activity.  16  k. Introduction to the appendices A number of diverse topics have been relegated to the appendices i n an attempt to make the methods and r e s u l t s sections as concise as p o s s i b l e . It  i s hoped that compre-  hension of the work does not require reference to the appendices. Appendices I - III  are d e t a i l s of methods used to extract  ATP i n three d i f f e r e n t experimental s i t u a t i o n s . 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 r e s u l t s of experiments designed to estimate the e f f i c i e n c y of the three ATP extraction procedures. Appendix VI describes the approach and r e s u l t s of expertlments designed to study the r e l a t i o n between biomass carbon and ATP. Appendix VII  outlines the e f f e c t of incubation time and  oxygen concentration on the assay for dehydrogenase activity. Appendix VIII gives the mathematical analysis used to calculate V , T, m  (K  t  + S ), T  trophic uptake studies.  n  n  and U  n  i n hetero-  17  MATERIALS AND METHODS 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 t e and the upper 2 cm of sediment were transferred to a s t e r i l e 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 a c t i v i t y and radiotracer experiments) were c a r r i e d 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 l y o p h i l i z e d for l a t e r use (direct b a c t e r i a l counts, ATP and carbohydrate analyses).  1.8  Figure 1. Marion Lake - morphometry and sampling area ( ).  18 a  Methods Chemical a n a l y s e s D u p l i c a t e f r e s h samples f o r t o t a l organic matter were o  filtered  (10)psi) through p r e v i o u s l y combusted  glass-fibre f i l t e r s  (Reeve-Angel 93^ AH - 2 A  samples were d r i e d (80 /2kh),  (550 / l h ) cm d i a . ) . The  weighed, combusted  (550  /3h)  then reweighed. Carbohydrate c o n c e n t r a t i o n s i n the l y o p h i l i z e d sediments were measured c o l o r i m e t r i c a l l y w i t h the phenols u l p h u r i c a c i d method (Gerchakov et a l . ,  1 9 7 2 ) . The assay was  s t a n d a r d i z e d w i t h ^-D-glucose. Organic carbon content of the sediments was a n a l y z e d u s i n g a carbon a n a l y z e r  (Beckman model  915). Methane p r o d u c t i o n was measured by a n a l y s i s of the atmosphere above s e a l e d sediment cores (obtained A p r i l 2 1 , incubated a t e i t h e r k  or 20  f o r 3 days, u s i n g gas  1974),  chromato-  graphy. To o b t a i n I n t e r s t i t i a l water approximately 100 ml o f mixed sediment were g r a v i t y - f i l t e r e d through Whatman no. 1 paper a t k  filter  . 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  membrane f i l t e r ,  pore s i z e  0.22^111  GS  u s i n g a vacuum of 7 p s i . The  a  water was  stored at -20  i n c l e a n t e s t tubes.  Glucose c o n c e n t r a t i o n s i n the i n t e r s t i t i a l water were determined e n z y m a t i c a l l y by the method of H i c k s and Carey A f l u o r o m e t e r (Turner no. 1 1 0 ) ,  (1968).  equipped with a green phosphor-  escent lamp (GE-F4-T5-G), and a constant temperature door (25 )• was used t o measure unknown glucose c o n c e n t r a t i o n s r e l a t i v e t o standard s o l u t i o n s of B-D-glucose. F i l t e r s used were, on the exc i t a t i o n s i d e , a Wratten 5 8 , a \% n e u t r a l d e n s i t y and a p o l a r o i d l e n s , on the e m i s s i o n  20  s i d e , a Wratten 23A. Blanks were i d e n t i c a l 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 i m i t for the assay i s about 1/igglucose/l. Direct counts of bacteria Bacteria were counted i n samples prepared from lyophl 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 i n a tissue homogenizer tube, was added lOmg of l y o p h i l i z e d sediment. The dry weight was determined by drying p a r a l l e l samples (100°/24h). The sample was homogenized for 2 min at which time very few,or no,large p a r t i c l e s 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 S c i e n t i f i c Biosonik II).  The sample was then adjusted to  100 ml i n a volumetric f l a s k with d i s t i l l e d water, and mixed. From t h i s suspension 1.00 ml was transferred to a  filtration  apparatus and adjusted to 10 ml with water. The sample was f i l t e r e d (10 psi) through a membrane f i l t e r viously boiled i n 0»1%  (0.22>um) p r e -  sodium pyrophosphate for 2 min and o  rinsed with b a c t e r i a - f r e e water. The f i l t e r was dried at 60 . One-half the area of a s l i d e was coated with transparent glue. The dried f i l t e r was placed on t h i s f i l m , then cleared i n acetone fumes, a i r dried and sealed with Permount. The c e l l s were stained with a solution of FITC using the buffer system suggested by Babiuk and Paul (1970). C e l l s  21 were s t a i n e d f o r 30 min w i t h a s o l u t i o n of 1.3 sodium carbonate b u f f e r phosphate b u f f e r  (pH 7.2),  (pH 9.6), 5.7  6.0  ml of 0.01  ml of 0.8#  of FITC. The s l i d e s were washed i n 0.5M buffer for  2  (pH 9.6)  ml o f 0.5  M  M potassium  s a l i n e and 5.3  mg  sodium carbonate  f o r 20 min, and i n 1% sodium pyrophosphate  min.  D u p l i c a t e s l i d e s were prepared f o r each sample  and  c e l l s i n 15 o c u l a r g r i d f i e l d s per s l i d e counted under o i l immersion u s i n g a R e i c h e r t microscope equipped w i t h a 200  HBO  mercury vapour lamp.  ATP A n a l y s i s D e t e r m i n a t i o n o f unknown ATP c o n c e n t r a t i o n s r e q u i r e s e x t r a c t i o n of the compound and assay of i t s abundance i n the e x t r a c t . A c o r r e c t i o n f a c t o r f o r e x t r a c t i o n e f f i c i e n c y must be determined. The methods summarized  below are p r e s e n t e d  i n d e t a i l i n appendices I - V I . (a) T o t a l ATP e x t r a c t i o n from f r e s h sediments  (1971a). (150-200 mg  The method used was t h a t of Lee e t a l . replicate  3 ml  samples of mixed sediment  Three dry wt.)  were e x t r a c t e d f o r each monthly sample. The main f e a t u r e s a r e e x t r a c t i o n of ATP w i t h 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 c a t i o n s w i t h c a t i o n exchange r e s i n ( f o r d e t a i l s see appendix I ) . (b) T o t a l ATP e x t r a c t i o n from l y o p h l i z e d  sediments  I n an attempt t o check the f r e s h sediment ATP v a l u e s o b t a i n e d each month, l y o p h i l l z e d sediments which had been o  s t o r e d f o r up t o 1 year a t -20,  were e x t r a c t e d on one  o c c a s i o n and assayed u s i n g s i n g l e enzyme and standard ATP  preparations. Bromosuccinlmide e x t r a c t i o n was found t o be t h e most e f f i c i e n t of e i g h t procedures t e s t e d  (Appendix I I ) and was  used f o r subsequent experiments ( f o r d e t a i l s see appendix II). (c) M i c r o b i a l ATP e x t r a c t i o n from f r e s h sediments Sediment  samples were c l e a n e d of l a r g e  (< approximately  30/"")  organisms  using micropipettes  measure m i c r o b i a l ATP. The s e p a r a t i o n ATP e x t r a c t i o n i s d e s c r i b e d  i n an attempt t o  method and subsequent  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 bioluminescence a s s a y . P r e p a r a t i o n  of the enzymes i n a p p r o p r i a t e  b u f f e r s o l u t i o n s , a d d i t i o n of standard o r unknown ATP samples and measurement o f l i g h t p r o d u c t i o n a r e d e s c r i b e d i n appendix IV. (e) C a l c u l a t i o n of biomass carbon  concentration  The e q u a t i o n used f o r c o n v e r s i o n of raw assay d a t a t o biomass carbon per gram d r y weight sediment wast biomass ATP assayed  x  1 x dilution sample factor dry weight  x  1 extraction efficiency x  carbon ATP  x  10"  The assayed ATP had u n i t s ng/mlt dry wt. was i n gramsi the d i l u t i o n f a c t o r was 5 for  experiments on l y o p h i l i z e d s e d i -  ments and m i c r o b i a l ATP and (50 x e x t r a c t volume (ml) x &) f o r t o t a l ATP i n f r e s h sediments? e x t r a c t i o n e f f i c i e n c y was .125,  .106  and .205  f o r t o t a l f r e s h , t o t a l l y o p h i l i z e d and  m i c r o b i a l f r e s h ATP e x t r a c t i o n s r e s p e c t i v e l y  (Appendix V ) t  C/ATP was 250  converted the  (Appendix VI)» the f a c t o r 10  J  23 data from ng to  g.  Dehydrogenase a c t i v i t y Dehydrogenase a c t i v i t y 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° i n the dark. To perform the assay, 5 ml of fresh mixed sediment was pipetted into a 125 ml erlenmeyer f l a s k . Control samples were routinely steam-killed then treated as normal samples. Blanks of t h i s 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 i n l e t and outlet tubes were f i t t e d and the samples were bubbled with nitrogen for 10 min to remove oxygen. The f l a s k s were sealed, o  wrapped i n aluminum f o i l and Incubated 4 h and 24 h at 30 with shaking at 100 RPM. Four r e p l i c a t e s 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 l a s k s , shaken for 1 h, then f i l t e r e d through cheesecloth under subdued l i g h t conditions. The f i l t r a t e was adjusted to 50 ml with extractant poured through the sediment p a r t i c l e s . Absorption was measured at 540  ^  against an acetone-methanol blank. AAstandard curve was obtained by dissolving 2, 3, 5-  24 t r i p h e n y l formazan I n e x t r a c t a n t s o l u t i o n . The e f f e c t s 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 a r e d e s c r i b e d i n appendix V I I .  H e t e r o t r o p h i c uptake o f A l l experiments  -glucose  were performed  u s i n g f r e s h mixed sed-  iments, d i l u t e d with a u t o c l a v e d lake water ( f i n a l  50»1). (New  Standard  s o l u t i o n s of ^ C - g l u c o s e  (U),  dilution  215  mc/mM  England Nuclear), were s t o r e d f r o z e n 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 s o l u t i o n s were a d j u s t e d t o 0.1 >ic/ml and l . O y u g glucose/ml p r i o r t o use. Two l i v e and one c o n t r o l ( f o r m a l i n - k i l l e d ) samples were e q u i l i b r a t e d f o r 1 h a t l a k e temperature  a f t e r d i l u t i o n . The r a d i o t r a c e r was then added  a t f o u r s o l u t e c o n c e n t r a t i o n s (10,  40, 100, 200JJ g / l ) , and  incubated under subdued l i g h t c o n d i t i o n s f o r 40 o r 60 min w i t h shaking a t 100 RPM. The I n c u b a t i o n f l a s k s , ^ C - C C ^ t r a p p i n g system and f i l t r a t i o n o f the p a r t i c u l a t e were d e s c r i b e d i n d e t a i l by K l e i b e r Bray's  fraction  (1972).  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 f o r  sample r a d i o a c t i v i t y measurements. Quench curves were p r e -  (417,000  pared with ^ C - t o l u e n e  dpm/ml, New England  and c h l o r o f o r m i n Bray*s s o l u t i o n f o r the l i q u i d counters used  Nuclear)  scintillation  (Nuclear Chicago Mark I and Isocap 300), by  the e x t e r n a l standard r a t i o (ESR)  method.  ill  E f f i c i e n c y of  C-CO2 c o u n t i n g was determined  by com-  p a r i n g the sample ESR t o the quench curve. T h i s v a l u e was f u r t h e r c o r r e c t e d f o r ^C-CC^ t r a p p i n g e f f i c i e n c y (mean o f 84# i n 4 samples u s i n g *C-Na H CO3). The e f f i c i e n c y o f c o u n t i n g p a r t i c u l a t e m a t e r i a l suslZ  25  suspended w i t h Aer - 0 - S i l (Degussa Chemicals) I n Bray's s o l u t i o n was c a l c u l a t e d from an e f f i c i e n c y v e r s u s weight of-sediment c u r v e . The curve was o b t a i n e d by i n c u b a t i n g sediment w i t h ^ C  - glucose o v e r n i g h t . A l i q u o t s o f d i f f e r e n t (0.22 JJ m  volumes were f i l t e r e d through t a r e d membrane f i l t e r s  pore s i z e ) . The samples were d r i e d (100°/24 h) and d r y wt. determined. One-half of the samples were combusted a t 900° In a tufee f u r n a c e (Lindberg Hevi-Duty model 55035), The evolved  lif  C-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 e t h e r (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 d e s c r i b e d by B u r n i s o n and Perez (1974). S u i t a b l e quench curves were prepared f o r the t o l u e n e s a s c i n t i l l a t i o n s o l u t i o n . The remainder of the samples were counted i n the u s u a l way, t h a t 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  s o l u t i o n and suspension w i t h Aer - 0 - S i l . Comparison  of the dpm per g dry wt. o b t a i n e d by  combusting samples, t o cpm per g dry wt. by suspending samples, y i e l d e d a n e f f i c i e n c y - w e i g h t c u r v e . Sediment c o u n t i n g e f f i c i e n c y ranged from 54.2 - 58,7#. The methods used t o c a l c u l a t e V , T , U m  are  t  n  and T  n  d e s c r i b e d i n appendix V I I I ,  S t a t i s t i c a l methods Seasonal d a t a o b t a i n e d from ATP and  dehydrogenase  experiments and b a c t e r i a l counts were a n a l y z e d f o r d i f f e r ences between the means by a n a l y s i s of v a r i a n c e (anova). L o g a r i t h m i c t r a n s f o r m a t i o n s were r e q u i r e d f o r dehydrogenase data and f o r the t o t a l f r e s h ATP d a t a t o c o r r e c t h e t e r o -  26 geneous v a r i a n c e w i t h i n the d a t a . S p e c i f i c means were then compared a t the \% p r o b a b i l i t y l e v e l u s i n g the new m u l t i p l e range t e s t of Duncan ( 1 9 5 5 ) . H e t e r o t r o p h i c uptake data were not s t a t i s t i c a l l y a n a l y z e d due t o the complexity  of o b t a i n i n g i n f o r m a t i o n on  the e r r o r s i n v o l v e d , K l e i b e r ( 1 9 7 2 ) has proposed a method to handle t h i s problem.  27  RESULTS AND DISCUSSION  1. Physical and chemical data The sediments i n Marlon Lake are a deep flocculent ooze except near the springs and the i n l e t . The sample area was free of macrophytic growth. Macroscopically the sediments contain some large invertebrates, a l g a l colonies and mats, chironomld tubes and l e a f and twig fragments. The sediment p a r t i c l e s 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 p a r t i c l e s 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 t y p i c a l of Marlon Lake (Efford,  19&7),  although the warming trend usually  evident i n May was delayed i n  197*+  (Figure 3»).  Changes i n t o t a l organic matter i n the sediments were not dramatic, f l u c t u a t i n g around a mean of 414 mg/ g dry wt. except i n September (Figure 3b). The maximum deviation within samples was 3*6% of the mean. The September peak may be due to a l g a l or invertebrate growth within the lake,or allochthonous inputs although the l a t t e r do  not peak u n t i l late f a l l (Odum, unpublished). Organic  carbon (dissolved plus particulate) was about 20% of the sediment dry weight. Glucose concentrations i n 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 v e r y low i n A p r i l and May  (5-20jjg/l) (Figure 3 c ) .  c o n c e n t r a t i o n s may  The low s p r i n g  r e f l e c t h e t e r o t r o p h i c a c t i v i t y of  b a c t e r i a , a p o s s i b i l i t y which i s a s s e s s e d i n s e c t i o n 4 , T o t a l carbohydrates i n the sediments f l u c t u a t e d w i t h an annual range of 1 3 5 - 1 8 4 mg/g  irregularly  dry wt. or 3 2 . 6 - 4 4 . 5 #  of the t o t a l o r g a n i c matter. These carbohydrate c o n c e n t r a t i o n s are  approximately twice as h i g h as those p r e v i o u s l y 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.  F i g u r e 2.  Scanning e l e c t r o n micrographs of Marion Lake sediment  particles.  29 *  F i g u r e 3. Seasonal changes i n temperature ( a ) , t o t a l o r g a n i c matter (b) and d i s s o l v e d . glucose  (c) i n the sediment  (bars  i n d i c a t e standard d e v i a t i o n o f each sample)•  31 2 . Biomass estimates (a) Total sediment biomass estimated by ATP analysis Recovery of c e l l u l a r ATP added to sediments was low i n t h i s 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 d i f f e r e n t sediments. Ernst (1970) reported recoveries of c e l l u l a r ATP and pure ATP added to marine sediments of 60-97# and 6l-104# respecti v e l y . Low recovery e f f i c i e n c y i n 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 i s related to extraction from the c e l l s i n a d d i t i o n to subsequent recovery from the extraction mixture. C e l l carbon to ATP r a t i o s 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 r a t i o may remain  r e l a t i v e l y '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 t h i s study (Appendix It  VI).  i s recognized that biomass carbon estimates thus obtained  are subject to error but i t i s the best estimate a v a i l a b l e . The t o t a l biomass carbon at 1 m water depth i n Marion  32  Table  I . Carbon t o ATP r a t i o s from v a r i o u s  references  organism  Ausmus (1973) b a c t e r i a - 5 s p p . f u n g i - 8 spp. actinomycetes6 sppV algae-6spp.  growth  sources  stage  exponential  180- 592(366)  V i b r i o spp.  stationary " exponential  bacteria-7spp.  e x p o n e n t i a l and stationary  Holms e t a l , E s c h e r i c h i a coll (1972T Appendix VI  357-833(500) 179-313(233) 1 7 9 - 238(217) 104-278(143)  Berland et a l . a l g a e - 7 s p p . (1972) Hamilton and Holm-Hansen (1967)  C/ATP r a t i o range (mean)  1  v a r i e t y of growth r a t e s  bacteria-3spp. stationary Streptomyces spp, Anacystls nldulans  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 s t a t i o n a r y and e x p o n e n t i a l phase.  33 Lake sediments, as measured by ATP a n a l y s i s , i s presented i n Figure 4 b . This graph summarizes data obtained by extraction of fresh sediment samples. Two features of these data are the peaks i n May each year and the homogenity of the t o t a l biomass throughout the remainder of the year. A p o s t e r i o r i comparisons among means by the new multiple range test (NMRT) (Table I I ) indicate that biomass i n May, 1973 was s i g n i f i c a n t l y higher ( P < . © 1 ) than on any other sampling date. Biomass estimates i n June, 1973 and May, 1974 were not s i g n i f i c a n t l y d i f f e r e n t (joined by a l i n e ) , but are s i g n i f i c a n t l y higher than samples i n July to A p r i l . A l l other samples were not s i g n i f i c a n t l y d i f f e r e n t from each other (joined by a l i n e ) . Analysis of t o t a l ATP In l y o p h i l i z e d sediments confirmed the peaks i n May each year but introduced much more v a r i a b i l i t y between other samples (Figure 4 a , Table I I ) . The increased v a r i a b i l i t y compared to fresh sediment extraction and the high biomass i n May, 1974 and December, 1973 may r e s u l t from the fact that the sample size was only 20 mg dry wt. for the l y o p h i l i z e d samples compared to 1 5 0 200 mg dry wt. for the fresh samples. The biomass estimate i s generally higher using l y o p h i l i z e d sediments compared to fresh sediments (Figures 4 a , b ) b u t t h i s may be due to an f  inaccurate estimation of extraction e f f i c i e n c y for l y o p h i l i z e d sediments. The e f f i c i e n c y of 10,6% was based on only one experimental organism, compared to fresh extraction e f f i c i e n c y which was measured using six organisms (Appendix V). The  34  F i g u r e 4. Seasonal changes of t o t a l biomass carbon i n lyophilized  (a) and f r e s h (b) sediments and  of m i c r o b i a l biomass carbon ( c ) .  Micro biomass carbon (g/m ) 2  T o t a l biomass carbon - f r e s h (g/m ) z  T o t a l biomass carbon - l y o p h i l i z e d (g/m ) 2  35 Table I I . S t a t i s t i c a l a n a l y s e s o f biomass d a t a . 1. T o t a l f r e s h sediment ATP a n a l y s i s A n a l y s i s of v a r i a n c e (Anova) source among samples w i t h i n samples total F  df. 11 23 34  . 0 1 ( 1 0 , 23)  SS .6635 .1849 .8484 3.21  =  ms .0603 .0080  F  significant  a t P<0.01  New m u l t i p l e range t e s t (NMRT) - Samples a r e ranked by i n c r e a s i n g mean v a l u e s . L i n e s 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 a t the 1% l e v e l .  Nov Feb Dec Oct Apr 1 Sep J a n J u l Apr 21 June May 74 May 73  2. T o t a l l y o p h i l i z e d  sediment ATP a n a l y s i s  Anova source among within total  df 11 13 24 F  . 0 1 ( 1 0 , 13)  ss 78.6379 8.0032 86.6411  ms 7.1489 .6156  F 11.6124  s  NMHT J u l Apr 1 June Nov Oct Feb J a n Apr 21 Sep Dec May 73 May 74  (continued)  36  3.  M i c r o b i a l ATP a n a l y s i s Anova source among within total F  F  .01(8, 9)  df 8 9 17  ms .4867 .1072  F „ 4.5400  5.4?  83  . 0 2 5 ( 8 , 9)  ss 3.8935 0.9648 4.8583  =  ^•  * s i g n i f i c a n t a t P<0.025  1 0  NMRT J a n 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 a n a l y s e s because only s i n g l e v a l u e s were o b t a i n e d . 4.  Microscopic b a c t e r i a l  counts  Anova source among samples among s l i d e s within slides total F  df 11 12 336 359  ss 44.498 2.341 115.576 162.416  ms 4.045 .195 .344  F 20.733 .567  M Q N S  . 0 1 ( 6 , 12) «= 4.8 2.2  F  .01(12,oo)  F  . 0 5 ( 1 2 , o o ) =1.8  e  N  S  not s i g n i f i c a n t a t P<.05  NMRT Oct J a n 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 t h a t q u i c k f r e e z i n g of samples a t the  site  u n t i l l y o p h l l i z a t i o n and ATP a n a l y s i s c o u l d be performed, would be a good method f o r s t o r i n g samples. C o n c e n t r a t i o n s of ATP  i n sediments have been reported  by other workers (Table I I I ) , E r n s t (1970) converted h i s data t o biomass carbon u s i n g a C/ATP r a t i o of 5 0 / 1 .  To  compare h i s data and t h a t of Lee et a l . (1971a) and  Karl  and LaHock (1974) t o d a t a obtained d u r i n g t h i s study, a C/ATP r a t i o of 250 was  assumed. The  main f e a t u r e s of the  e s t i m a t e d biomass c o n c e n t r a t i o n s a r e the low v a l u e s i n marine sediments and the high v a l u e s i n Marion Lake. C o n c e n t r a t i o n s of biomass carbon of 100>ig/g a r e e q u i v a l e n t t o about 2 x l 0  1 0  b a c t e r i a / g (Appendix  VI) which i s h i g h e r  than  p l a t e count methods i n d i c a t e f o r many sediments (eg. B i a n c h i , 1973)  but much lower than d i r e c t counts i n d i c a t e  Antipchuk,  1 9 7 2 ) . Any  (eg.  sediment w i t h lOOjug biomass would  c o n t a i n v i r t u a l l y no organisms other than b a c t e r i a . T h i s would be unusual even i n anaerobic environments (Fenchel, 1 9 6 9 ) . Low v a l u e s i n marine sediments may  indicate  either  v e r y s m a l l l i v i n g p o p u l a t i o n s or t h a t the C/ATP r a t i o i s i n c o r r e c t , although even a 10 f o l d i n c r e a s e 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 t o e v a l u a t e the data i n Table I I I s i n c e no i n f o r m a t i o n was  r e p o r t e d on  the nature of the sediment communities, but Marlon Lake sediment biomass has been independently estimated by c o u n t i n g organisms of v a r i o u s s p e c i e s ( E f f o r d , u n p u b l i s h e d ) . T h i s o f f e r s a unique o p p o r t u n i t y t o compare ATP-blomass carbon estimates t o enumeration-blomass carbon The mean biomass of microorganisms and  estimates.  invertebrates i n  T a b l e I I I . Sediment ATP-biomass c a r b o n d a t a . reference  location  reported data  biomass c a r b o n (yug/g d r y w t . )  1  E r n s t (1970)  6 s i t e s N o r t h Sea (28 - 345m)  6 . 6 - 33.4>ug biomass carbon/ml  Lee e t a l . (1971a)  9 Wisconsin lakes  0.34 - 9 . 5 / f g ATP/g  82 - 2,375  K a r l and LaRock (1974)  beach sand  145 - 228 ng ATP/g  36 -  A t l a n t i c ocean (4000 m)  9 - 1 0 . 3 ng ATP/g  This study  M a r i o n Lake (seasonal)  165 -  835  57  2 - 3 2,810 - 12,600  Assuming C/ATP i s 250 and, f o r E r n s t ' s (1970) d a t a , t h a t 1 ml = 200 mg d r y wt.  39 Marion Lake sediments by enumeration i s approximately 4-6g C/m . ATP-measured biomass carbon i n fresh samples 2  ranges from 3.5 - 1 2 . 8 g C/m  2  with an integrated mean of  4.7 g C/m . Discrepancies may be due to i n c l u s i o n i n the enumeration data of large invertebrates (eg. S l a l l s ) 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 1 2 month period), variations i n the depth of sediment sampled (eg. i n t h i s study the top two cm) and variations i n d i f f e r e n t 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 i n Marion Lake and that the data presented i n Table I I I 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 r e s i d u a l ATP. This method i s subject fco a l l the assumptions concerning e f f i c i e n c y of extraction and biomass carbon to ATP r a t i o s discussed In the previous section. Seasonal changes i n microbial biomass, including bacteria, fungi, actinomycetes, some algae and a few protozoa, do not p a r a l l e l changes i n t o t a l 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 f o u r - f o l d v a r i a t i o n i n t o t a l biomass (Figures 4b, c ) . Microbial biomass ranged from o  0,25 - 2.16 g C/m  (January - September). S t a t i s t i c a 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 r e p l i c a t e samples were l o s t . Temperatures i n J u l y , October, September, 1973 and May, 1974 were higher than 9°• Mean microbial biomass i s  significantly  higher i n these months than i n January, December and November when the temperature was 4 ° , June biomass data are also higher than i n winter months but not at the 1% confidence level. Seasonal changes i n 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 t o t a l ATP-blomass than microbial ATP-biomass In t h i s study considering the size range, but i n fact resembles the l a t t e r  (Figure 4 c ) . This r e f l e c t s the s t a b i l i t y  of  t o t a 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 i s 0.61 g C/m 2 of protozoa i s 0.0013 g C/m  (next s e c t i o n ) ,  (Kool and Stachurska, un-  published) and of algae i s approximately 0.23 g C/m  2  (calculated from Greundling, 1971). Actinomycetes are i n cluded i n the b a c t e r i a l counts. Fungi have been quantified  41 only by plate count methods and t h e i r numbers are low compared to t o t a l b a c t e r i a l counts (1.8 - 8.2 x 10? fungal propagules/m - Chang, unpublished? compared to 2  l O ^ bacteria/m ). Dick (197D 1  2  suggested many fungi were  present as spores of allochthonous o r i g i n . Very few healthy fungal filaments were seen during t h i s study except on decaying f i s h i n the lake or on sediments i n  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 s o i l t an average hyphal of spore length of 10^u m was assumed here to allow the c a l c u l a t i o n to be made). Cultivable,fungal biomass i s then 9-41 x 10"^ g C/m . The sum of the enumeration-microbial biomass i s 0,84 g C/m . The biomass of protozoa and fungi i s 2  negligible  i n t h i s system. The annual mean ATP-measured microbial b i o mass i s 1.28 g C/m , indicating the C/ATP r a t i o may be 2  inaccurate or that the enumeration data underestimate the biomass. The discrepancy i s even greater than t h i s  indicates  because a f r a c t i o n (large filaments, colonies and diatoms) of the algae were removed p r i o r to ATP a n a l y s i s , (c) B a c t e r i a l biomass i n the sediments estimated by microscopic counts B a c t e r i a l biomass was estimated by counting c e l l s  in  known d i l u t i o n s of sediment and converting the numbers to biomass assuming that c e l l s have a density of lg/cc, are 80$ water and that 50% of the dry wt. i s carbon (Shields et al..  1973). Another requirement for t h i s c a l c u l a t i o n i s the  average size per c e l l , but t h i s i s d i f f i c u l t  to measure i n  42  sediment systems. Some microbiologists have measured b a c t e r i a l c e 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 w t . / c e l l  (Bae et a l ^ , 1 9 7 2 ) , 5 . 7 x  g/cell (Babiuk and Paul, 1970) and 1 . 6 x (Zvaginsev,  IO"  14  lCT^g/cell  1 9 7 3 ) . Mean sizes for sediment bacteria meas-  ured by Antipchuk (1972) are 3 . 7 x 1 0 " g / r o d and 5 . 8 x 1 0 ~ 13  g/coccus. Burnison (unpublished)  1 / +  suggested a mean volume  of 0.36 um3 for Marlon Lake sediment bacteria which corresy  _-i k.  ponds to 7 . 2 x 10 g / c e l l . During t h i s study very large spirochaetes (50 - 100/(m length) were seen a few times, but 12  never while counting b a c t e r i a . C e l l s up to 5 . 6 x 10 g (4 x 3yU m) were more common while at the other extreme 14  c e l l s 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 ~ g  (1 - 1 . 5  1 3  x  .5 -  Ijdm).  Not enough c e l l s were measured to determine an accurate mean, but 1 0 " g dry w t . / c e l l , or 5 x lO"* ^ g C/cell was 1 3  1  chosen as a representative value. B a c t e r i a l biomass peaked i n July and was at a minimum i n October (Figure 5 ) . July biomass data are  significantly  higher than only those i n October, January, November and early A p r i l (Table II),  With the exception of the high  biomass i n December the seasonal trend was a maximum i n early to mid-summer, a r a p i d decline i n early f a l l then a slow recovery during the winter. Plate counts were similar i n pattern except that the crash occurred i n August i n 1969 (Fraker,  unpublished).  F i g u r e 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/m ) 2  Most p u b l i s h e d e s t i m a t e s f o r b a c t e r i a l biomass i n sediments a r e l o w because t h e y were based on p l a t e c o u n t s . Data a r e u s u a l l y r e p o r t e d a s numbers p e r g so t h e c o n v e r s i o n f a c t o r o f 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 p o r t e d by t h e a u t h o r s c i t e d below. Z o b e l l (1963) r e v i e w e d the l i t e r a t u r e and found b a c t e r i a l biomass e s t i m a t e d  from  5 x 1 0 " 7 ^ j g c - 5°yUg C/ g sediments. Khiyama and Makemson  (1973) u s e d l i g h t microscopy a n d found 1 beach sands. S u r f a c e sediments Antipchuk  IOJJL  g C/g i n  i n f i s h ponds were found by  (1972) t o c o n t a i n a p p r o x i m a t e l y 1000 - 2000ps  C/g.His biomass e s t i m a t e s f o r one sample a t each o f s i x l o c a t i o n s i n t h r e e seasons t e n d t o peak i n s p r i n g o r summer t h e n 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 M a r i o n Lake. The biomass o f M a r i o n Lake sediment b a c t e r i a ranges from 340 - 650^ug C/g (January - J u l y ) . These e s t i m a t e s a r e much h i g h e r t h a n r e p o r t e d c o n c e n t r a t i o n s p r i o r t o t h e l a s t decade, b u t t h a t t h e y a r e n o t o v e r e s t i m a t e s i s suggested by the m i c r o b i a l ATP d a t a , and t h e s i m i l a r i t y w i t h p r e v i o u s d i r e c t counts i n M a r l o n Lake sediments  (Ramey, 1 9 7 2 t Bur-  nison, unpublished). (d) R e l a t i o n s between biomass e s t i m a t e s The v a r i o u s biomass d a t a 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 t h e o t h e r s ( F i g u r e 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 o f t h e t o t a l biomass, r e f l e c t i n g changes i n t h e 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 e s t i m a t e s were h i g h i n May. The m i c r o b i a l f r a c t i o n o f t h e community  45  F i g u r e 6, Seaonal changes i n the p r o p o r t i o n of sediment biomass carbon i n b a c t e r i a l t o t o t a l biomass ( o  compared  o ) , bacterial  comp-  a r e d t o m i c r o b i a l biomass (+  +); m i c r o b i a l  compared t o t o t a l biomass (•  •).  *5a  CO  •p c  2-01  a £ +>  -*  C d. O 8 ,0 O P o a)  1-5-  O rH  as  CQ -H CO U o o •H «J .o .o O  1-0-  C  c«  O rH  •H C8 -P -H U  fi  O  O  o o 0u  ^ .H a, a  0.  -i  r-  J  M 1973  S  N  J  M  M 1974  46 i s h i g h i n t h e summer and low i n m i d w i n t e r , i n c r e a s i n g d u r i n g l a t e w i n t e r t o a n o t h e r peak i n e a r l y s p r i n g . T h i s 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 a l g a e a t 1 m l i n g , 197D.  (Greund-  The p r o p o r t i o n o f b a c t e r i a i n t h e m i c r o b i a l  biomass d e c r e a s e s d u r i n g t h e summer, r i s e s s h a r p l y i n th§ l a t e f a l l , dropping again i n l a t e w i n t e r . This f i t s  a  model i n which t h e 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 i n c r e a s e s i n the a l g a l  biomass.  I n December and January, b a c t e r i a were more t h a n of  150%  t h e A T P - m i c r o b l a l biomass. T h i s suggests b a c t e r i a l  counts o v e r e s t i m a t e d t h e b a c t e r i a l p o p u l a t i o n o r t h a t t h e 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, p r o b a b l y w i t h a C/ATP r s f c i o g r e a t e r t h a n 250.  F a s t temperature  d e c l i n e s t o 0° were shown by C o l e et. a l .  (1967) t o cause  a s h a r p 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, t h e n 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 undere s t i m a t e s o f t h e community biomass may be e x p e c t e d . subsequent  I n c r e a s e i n m i c r o b i a l ATP  The  i n F e b r u a r y may  reflect  not o n l y t h e i n c r e a s e i n a l g a l biomass ( G r e u n d l i n g , 1 9 7 1 ) , but a l s o a c c l i m a t i z a t i o n o f t h e 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 d a t a o f C o l e e t a l . ( 1 9 6 7 ) . Comparison to  o f the A T P - t o t a l biomass d a t a ( F i g u r e  4b)  s e a s o n a l v a r i a t i o n o f t h e enumeration d a t a ( E f f o r d , un-  p u b l i s h e d ) r e v e a l s a n o t h e r d i s c r e p a n c y . ATP-biomass peaks i n May  (12 g C/m ) 2  and i s a l m o s t c o n s t a n t f o r t h e remainder  the y e a r (4 g C/m ). Enumeration d a t a a r e low i n s p r i n g 2  4 g C/m  ) but c o n s t a n t f o r t h e remainder of t h e y e a r  6 g C/m ), 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 2  of (about  (about  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 p e r i o d s of o v e r - and u n d e r - p r o d u c t i o n of ATP i n E. c o l l . The C/ATP r a t i o d e c r e a s e d d u r i n g h i g h growth b u t t h e n i n c r e a s e d d e s p i t e c o n t i n u i n g h i g h growth r a t e s . I f t h i s i s g e n e r a l l y t r u e (Holms et a l . . 1971,  r e p o r t e d 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 t e m p e r a t u r e s , and t h a t i n May r e s p i r a t i o n i n M a r i o n Lake sediments I n c r e a s e s e x p o n e n t i a l l y (Hargrave, 1969), t h e n o v e r - p r o d u c t i o n o f ATP might o c c u r . U s i n g a C/ATP r a t i o o f 250 would o v e r e s t i m a t e the community biomass i n such c i r c u m s t a n c e s . 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" p h y s i o -  l o g i c a l c o n d i t i o n s , but under p e r i o d s o f s t r e s s , ATP  data  may be confounded by r e p r e s e n t i n g b o t h biomass and a c t i v i t y . P u b l i s h e d d a t a f o r n a t u r a l systems support t h i s . HolmHansen and P a e r l (1972) found h i g h p r i m a r y p r o d u c t i o n , h i g h ATP-biomass and low a l g a l biomass i n s u r f a c e waters of Lake Tahoe, but a t 80 m, r e c o r d e d low p r o d u c t i o n , low ATP-biomass and h i g h a l g a l biomass. Rudd and H a m i l t o n (1973) r e p o r t e d a s i m i l a r phenomenon i n Lake 227 of t h e E x p e r i m e n t a l Lakes A r e a , O n t a r i o . ATP may t h e n be thought o f , not as measuring biomass a t any i n s t a n t , but measuring t h e p o t e n t i a l biomass t h r o u g h t i m e . An example i s t h e b a c t e r i a i n M a r i o n Lake s e d i m e n t s . I n J a n u a r y , m i c r o b i a l ATP i s low ( F i g u r e 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 h i g h ( F i g u r e s  4c)  5.6).  A t any i n s t a n t i n time more b a c t e r i a l c a r b o n i s a v a i l a b l e  48  t o g r a z e r s t h a n ATP d a t a i n d i c a t e . However, i f b a c t e r i a a r e g r a z e d a t t h i s t i m e , biomass r e p l a c e m e n t may be slow due t o temperature ( o r o t h e r f a c t o r ) l i m i t a t i o n * Such r e p l a c e m e n t o c c u r s w i t h i n a few days a t warm t e m p e r a t u r e s ( F e n c h e l , 1970). I n September, A T P - m i c r o b i a l biomass i n t h e sediment i s t e n f o l d higher but enumeration-microbial  biomass i s o n l y two-  f o l d h i g h e r t h a n i n J a n u a r y . The s u g g e s t i o n i s t h a t i n September t h e r e i s not t w i c e 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, b u t up t o t e n t i m e s a s much* S t a t e d a n o t h e r way, t h i s means t h a t i n September t h e r e s h o u l d be 5 t i m e s more biomass a v a i l a b l e i f t h e system i s stressed  t h a n i s i n d i c a t e d by d i r e c t count d a t a .  T h i s p r e d i c t i o n c o u l d 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 p r e s s u r e on t h e 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 o f experiment u s i n g a range o f d e n s i t i e s o f H y a l e l l a a z t e c a . Assuming t h a t h i s measurement o f 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 a p p r o x i m a t e s b a c t e r i a l growth o r p r o d u c t i o n r a t e s , t h e n h i s d a t a may be a p p l i e d t o t h e 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 t i m e s t h a t i n f r e s h , u n a l t e r e d c o r e s . C o n s i d e r i n g t h e i n c o n s i s t e n c i e s i n tempe r a t u r e , t i m e , e t c . between t h e experiment a n d t h e model i t appears t h a t ATP i s i n f a c t a measure o f b o t h biomass and a c t i v i t y , o r s i m p l y 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 o f 250 r e p r e s e n t s biomass. Under s t r e s s c o n d i t i o n s such a s i n t e n s e g r a z i n g , c o l d t e m p e r a t u r e s or r a p i d temperature i n c r e a s e s t h e c e l l u l a r ATP b a l a n c e i s changed and, by d e f i n i t i o n , ATP becomes a n a c t i v i t y  indicator.  I n t e r p r e t a t i o n of ATP-biomass data may d i f f i c u l t . High v a l u e s may  t h e r e f o r e be  r e f l e c t large, inactive  p o p u l a t i o n s or s m a l l , p r o d u c t i v e p o p u l a t i o n s . In s t u d i e s concerned with t r o p h i c dynamics, however,more c o n c i s e I n f o r m a t i o n may  not be r e q u i r e d .  50 3. Activity  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 h i g h e r i n summer t h a n any other time of the year ( F i g u r e 7 .  Table I V ) .  Assays incubated f o r 24 or 4 hours u s u a l l y showed s i m i l a r d i r e c t i o n a l response t o changes i n sediment r e s p i r a t i o n , but the a b s o l u t e r a t e of response was g r e a t e r u s i n g the s h o r t i n c u b a t i o n time. T h i s was expected (Appendix V I I ) . The range of data f o r dehydrogenase a c t i v i t y  ( . 0 6 5 5 - . 6 8 2 5 mg  formazan/g.h)  f o r the two i n c u b a t i o n times over the 12 months i s s i m i l a r t o the range r e p o r t e d by Pamatmat and Bhagwat ( 1 9 7 3 ) f o r d i f f e r e n t l o c a t i o n s i n Lake Washington ( . 1 3 - . 4 2 mg formazan/g.h, conv e r t i n g t h e i r a b s o r p t i o n r e a d i n g s t o formazan e q u i v a l e n t s ) , a l t h o u g h t h e i r methodology was q u i t e  different.  The dehydrogenase data probably r e f l e c t s a e r o b i c and a n a e r o b i c p o t e n t i a l a c t i v i t y of p r o k a r y o t e s i n the s e d i ments s i n c e the samples were not homogenized. The s u g g e s t i v e evidence f o r t h i s was presented i n the I n t r o d u c t i o n , T h i s o b s e r v a t i o n may e x p l a i n the l a c k 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 n a t u r a l samples, Howard ( 1 9 7 2 ) r e p o r t e d s o i l dehydrogenase a c t i v i t y was always l e s s than t h a t p r e d i c t e d by oxygen uptake data, Pamatwat and Bhagwat ( 1 9 7 3 ) r e p o r t e d t h a t sediment oxygen uptake was l e s s than t h a t p r e d i c t e d by dehydrogenase a c t i v i t y . I t i s suggested here t h a t , i n a e r o b i c 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 dehydrogenase assay, Anderson and Domsch ( 1 9 7 3 ) r e p o r t e d t h a t  51  F i g u r e ?. Seasonal changes of dehydrogenase a c t i v i t y i n the sediments (4h i n c u b a t i o n o — o ; 24 h i n c u b a t i o n .  .).  52  Table IV. S t a t i s t i c a l a n a l y s i s o f dehydrogenase a c t i v i t y data. 1, Dehydrogenase a c t i v i t y measured w i t h 4 hour i n c u b a t i o n Anova source among within total  df 10 27 37  F  ss .7396 .0468 .7865  . 0 1 ( 1 0 . 25)  = 3  '  1  ms .0740 .0017  F ^ 42.6292  3  NMRT Apr 1 Apr 21 J a n May ?4 Dec Feb Oct Nov J u l June Sep  2 . Dehydrogenase a c t i v i t y measured w i t h 24 hour i n c u b a t i o n Anova source among within total  df 11 29 40  p  .01(10,  25)  ss 1.2581 .0523 1.3104  =  3  *  ms .1144 .0018  F 63.3919  #  #  1 3  NMRT Apr 1 Apr 21 J a n 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 o n l y 22% o f t o t a l s o i l consumption i n t h e i r s t u d i e s . I n sediments,many  oxygen  anaerobic  processes occur, p a r t i c u l a r l y i n reduced environments, which a r e not measured by oxygen u p t a k e b u t do r e a c t w i t h TTC  (Pamatmat and Bhagwat, 1973» O l a h , 1972). Hargrave  (1969)  e s t i m a t e d b a c t e r i a l oxygen  consumption  i n M a r i o n 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 c o r e s from t h a t i n normal c o r e s . A l t h o u g h the u s e f u l n e s s 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  eliminate  s p e c i f i c p o p u l a t i o n s i n n a t u r a l environments i s dubious (Cameron,  .1973  * Y e t k a and Wiebe, 197*0, comparison  o f 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 d e c r e a s e d g r a d u a l l y between September and December t h e n i n c r e a s e d g r a d u a l l y u n t i l May, When t h e sediments r e a c h e d a p p r o x i m a t e l y 10°, b a c t e r i a l a c t i v i t y i n c r e a s e d 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 t h e dehydrogenase d a t a ( F i g u r e 7), a l t h o u g h t h e 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 a n d maximum a c t i v i t y was r e c o r d e d i n September ( 4 h i n c u b a t i o n ) o r June (24h i n c u b a t i o n ) w h i c h were n o t t h e d a t e s o f maximum t e m p e r a t u r e (19.5° i n J u l y ) , 14 (b) H e t e r o t r o p h i c uptake o f  C-glucose  Gross uptake ( r e s p i r e d p l u s a s s i m i l a t e d uptake) o f C-glucose i n mixed, d i l u t e d sediments f o l l o w e d a d r a m a t i c s e a s o n a l p a t t e r n ( F i g u r e 8 ) , B o t h 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 t h e sediments r e a c h e d 19.5 • Uptake d u r i n g w i n t e r 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 ) and a c t u a l m  ( U ) r a t e s of g l u c o s e uptake, n  and the a c t u a l t u r n o v e r time ( T ) . ( V n  U +—+ i T n  n  ).  m  o  0  }  low (<4 mg glucose/m^.h) b u t s t a r t e d t o i n c r e a s e as t h e temperature c l i m b e d above 4° ( 9 . 5 ° i n May,1974). The A p r i l 21 d a t a were d i s c a r d e d because t h e y d i d not f i t Michaelis-Menten k i n e t i c s . N a t u r a l turnover time ( T ) n  d i s p l a y e d a n i n v e r s e r e l a t i o n s h i p t o uptake r a t e  (Figure  8 ) . The t i m e r e q u i r e d f o r t h e b a c t e r i a t o c o m p l e t e l y use glucose equivalent t o the i n s i t u  concentration (S ) n  i n c r e a s e d a s uptake r a t e d e c l i n e d . H a l l e t a l . (1972) r e p o r t e d s i m i l a r v a l u e s f o r V ( 2 . 6 - 38,0/1 g g l u c o s e / g . h ) and T  n  m  ( , 0 6 l - ,400h) a s were  found here ( V 2.2 - 3 9 . 3 j u g g l u c o s e / g . h , T m  n  The p a t t e r n o f s e a s o n a l v a r i a t i o n was almost  .033 - .727h). identical,  i n d i c a t i n g that the b a c t e r i a l population, with respect t o g l u c o s e metabolism, does n o t change r a d i c a l l y from y e a r t o y e a r (1971 - 72 compared t o 1973 - 7 4 ) . The n a t u r a l uptake r a t e o f g l u c o s e ( U ) was e s t i m a t e d n  by K l e i b e r (1972) t o be about 1.8 - 1 0 . 6 >ig g l u c o s e / g . h f o l l o w i n g a seasonal trend s i m i l a r t o that of V . During m  the p r e s e n t s t u d y U  n  ranged from 0 . 6 - 15.9;ug g l u c o s e / g . h .  Wood (1970) found h e t e r o t r o p h i c uptake was maximal i n t h e s p r i n g and low i n w i n t e r i n e s t u a r i n e s e d i m e n t s , b u t had no d a t a f o r June and J u l y . Some p r e v i o u s l y r e p o r t e d d a t a f o r parameters o f h e t e r o t r o p h i c uptake o f g l u c o s e i n n a t u r a l environments a r e summari z e d i n T a b l e V. Wood (1970) d i d not determine S  n  so T  n  may  be l o w . H a r r i s o n e t a l . (1971) used mixed b u t not d i l u t e d sediments, so T  n  c o u l d 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 z e r o added s u b s t r a t e (Appendix V I I I ) . Uptake i n  Table V. Maximum uptake r a t e s , and n a t u r a l turnover times and uptake r a t e s f o r glucose i n sediments and water. reference  location  Hobble e t a l .  ocean water (10 - 200 m)  (1972T  Azam and HolmHansen (1973)  Crawford at a l . (1974)  Harrison et a l . (1971)  ocean water (10 - 200 m)  Tn (h)  Vm Ojg/g.h)  V (jug/l.h) m  U (;jg/g.h) n  un (jug/l.h)  .0005 - . 0 0 6  10 128 days  estuarine water  7.2  Klamath Lake sediments  2.25  Wood (1970)  estuarine sediments  H a l l e t a l . (1970) Kleiber Tl972)  Marion Lake -mixed .26 sediments - u n d i s t u r b e d .89sediments 420  T h i s study  Marlon Lake -mixed sediments  .06  .31  25.6  2.4  170  299  9.4  627  3.9  260  9.6  535  5.3  295  u n d i s t u r b e d c o r e s o f M a r i o n Lake sediment o c c u r r e d w i t h a longer T  n  t h a n was found i n mixed sediment e x p e r i m e n t s  ( H a l l e t 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 p r o b a b l y due t o i n c r e a s e d a v a i l a b i l i t y o f d i s s o l v e d o r g a n i c s t o t h e h e 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 l o n g e r and V  m  i s much l o w e r . T h i s r e f l e c t s d i f f e r e n c e s i n t h e p o p u l a t i o n d e n s i t y o f h e t e r o t r o p h s i n t h e two environments ( e g . M a r i o n Lake w a t e r s i n March, 1973 c o n t a i n e d a n e q u i v a l e n t o f 74 >*g d r y wt. o f b a c t e r i a p e r 1 w h i l e t h e t o p two cm o f t h e sediment c o n t a i n e d a p p r o x i m a t e l y 6 0 , 0 0 0 / i g d r y wt. o f b a c t e r i a / 1 ) , and emphasizes t h e importance o f t h e benthos i n nutrient cycling. (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 a s s a y s need n o t y n e e e a s a r i l y , ,  ~  measure t h e same parameters o f m i c r o b i a l p o p u l a t i o n s due t o t h e i r heterogeneous b i o c h e m i c a l a b i l i t i e s . G l u c o s e i s not a s u b s 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 r e d u c e TTC t o formazan i n t h e dehydrogenase a s s a y may be r e s t r i c t e d ; degrees o f t h e a b i l i t y c e r t a i n l y e x i s t ( E i d u s e t 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 t h e v a r i o u s methods. S k u j i n s (1973) r e p o r t e d a h i g h degree o f 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 a n d dehydrogenase Hobble e t a l .  activity.  (1972) found a r e a s o n a b l e degree o f r e l a t i o n  between e s t i m a t e s o f marine p l a n k t o n r e s p i r a t i o n u s i n g oxygen u p t a k e , dehydrogenase a c t i v i t y and ATP. A l l t h e a c t i v i t y e s t i m a t o r s a p p l i e d t o M a r i o n Lake sediments i n d i c a t e b a c t e r i a r e s p o n d t o i n c r e a s e d t e m p e r a t u r e s  58 i n May, r e a c h i n g a peak sometime i n l a t e May t o e a r l y S e p t ember, t h e n g r a d u a l l y slow down as t h e t e m p e r a t u r e d r o p s . 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 . H e t e r o t r o p h i c uptake of g l u c o s e ( H a l l e t a l . . 1972;  Figure 8), glycine  and a c e t a t e ( H a l l e t a l . , 1972), and oxygen ( H a r g r a v e , 1969)  demonstrate s h a r p e r s e a s o n a l maxima t h a n  dehydrogenase a c t i v i t y ( F i g u r e 7 ) . f a c t t h a t dehydrogenase V , m  consumption  T h i s may be due t o t h e  i s a measure o f p o t e n t i a l , a l t h o u g h  the p o t e n t i a l uptake r a t e of g l u c o s e , has a v e r y sharp  peak i n midsummer. R a d 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 t h o s e i n which U  n  and T  n  a r e d e t e r m i n e d , a r e perhaps the b e s t way  t o e s t i m a t e energy f l o w from p a r t i c u l a r c a r b o n s o u r c e s i n t o b a c t e r i a l p o p u l a t i o n s . But assumptions o f t e n made,for  example,  t h a t g l u c o s e uptake i s r e p r e s e n t a t i v e o f uptake o f a l l o t h e r d i s s o l v e d c a r b o h y d r a t e s , may be i n v a l i d . The s o l u t i o n would be t o have a u n i f o r m l y l a b e l l e d p o o l o f a l l t h e n a t u r a l s u b s t r a t e s 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 considering present technology.  Dehydrogenase  a c t i v i t y may be more i n d i c a t i v e o f t o t a l m i c r o b i a l p r o d u c t i o n t h a n e i t h e r h e t e r o t r o p h i c uptake o r oxygen consumption as suggested by Pamatmat and Bhagwat ( 1 9 7 3 ) . T h e i r d a t a c o r r e l a t i n g heat p r o d u c t i o n t o dehydrogenase  a c t i v i t y i n sed-  iments i s p r o m i s i n g because heat p r o d u c t i o n , which i s v e r y 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 ( B r o c k , 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 u p t a k e , oxygen uptake and dehydrogenase  a c t i v i t y i n M a r i o n Lake sediments  may be examined q u a n t i t a t i v e l y . Hargrave (I969) e s t i m a t e d 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 .year which i n d i c a t e s 2  t o t a l uptake o f 97.5 g C/m .year assuming 2  assimilation  e f f i c i e n c y o f 80% ( H a l l e t a l ^ , 1972). K l e i b e r (1972) e s t i m a t e d c a r b o n f l u x i n t o t h e b a c t e r i a a t 22 - 120 g  C/m . 2  y e a r w i t h r e s p i r a t o r y l o s s e s o f 4 - 24 g C/m .year. U s i n g a 2  r e g r e s s i o n f o r dehydrogenase  a c t i v i t y on heat p r o d u c t i o n  (Patmatmat and Bhagwat, 1973). and assuming a p p r o x i m a t e l y 100 K c a l heat a r e produced p e r mole o f c a r b o n o x i d i z e d ( G i e s e , 1968), t h e 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 .year u s i n g t h e 4 hour i n c u b a t i o n a s s a y d a t a . T h i s i s 2  e q u i v a l e n t t o g r o s s uptake o f 96.5 g C/m .year. T h i s a g r e e 2  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 d a t a i s unexpected, b u t , d e 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 M a r l o n Lake b a c t e r i a  (a) Q u a l i t a t i v e s e a s o n a l d e s c r i p t i o n There i s e v i d e n c e t h a t biomass o f b a c t e r i a i n n a t u r a l environments does not a l w a y s c o r r e l a t e w i t h a c t i v i t y measurements. Hobble ejt a l . (1972) r e p o r t e d t h a t h i g h 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 h i g h numbers of b a c t e r i a , but a h i g h 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 t h e r e 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 o f a c e t a t e and  glucose  a g a i n s t d i r e c t c o u n t s of b a c t e r i a i n sediments. Uptake of s i x of n i n e o r g a n i c a c i d s t e s t e d was  shown t o 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 c a r b o n ( R o b i n s o n e t a l . . 1973). T h e i r e s t i m a t e s o f t h e p o p u l a t i o n s i z e of each b i o c h e m i c a l group a r e , however, q u e s t i o n a b l e .  Considering  the number of c o l o n i e s formed per ml of sample, and methods t h e y used, most o f the s o l e c a r b o n source c o n t a i n e d fewer t h a n 15 c o l o n i e s . The f o r p l a t e counts a r e 20 -  the  plates  normally accepted  limits  200 c o l o n i e s per p l a t e , w i t h numbers  l e s s t h a n 20 c o n s i d e r e d s t a t i s t i c a l l y u n r e l i a b l e ( P a r k i n s o n e t a l . . 1 9 7 1 ) . An Important c o n t r i b u t i o n t o n a t u r a l p o p u l a t i o n s of b a c t e r i a was  understanding  made by S t a n l e y  and  S t a l e y ( 1 9 7 4 ) . They demonstrated, a p p l y i n g the uptake approach used i n t h i s study and a u t o r a d i o g r a p h y ,  that hetero-  t r o p h i c a s s i m i l a t i o n o f -'H-acetate i n a n a e r a t i o n l a g o o n due t o b a c t e r i a , t h a t t h e uptake p e r c e l l i n c r e a s e d  was  61 l i n e a r l y over t i m e , and t h a t w i t h i n t h e 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 o f a t 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 . T h i s work s u g g e s t s t h a t uptake r a t e may not be p r o p o r t i o n a l t o numbers o f b a c t e r i a because o f the h e t e r o g e n e i t y between b a c t e r i a l m e t a b o l i c a b i l i t i e s . T h i s 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 a r e 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 o f u n r e l a t e d n e s s between b i o mass and a c t i v i t y o f b a c t e r i a , A s i m p l e e x p l a n a t i o n i s t h a t biomass and a c t i v i t y a r e sometimes out o f phase even i n one p a r t i c u l a r environment, Holm-Hansen and P a e r l found maximum h e t e r o t r o p h i c  (1972)  uptake o f a c e t a t e l a g g e d b e h i n d  maximum ATP-biomass o f b a c t e r i a i n Lake Tahoe, I n M a r i o n 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  9),  (Figure  D u r i n g t h e remainder o f t h e y e a r a c t i v i t y e i t h e r remains c o n s t a n t w h i l e biomass I n c r e a s e s , o r i n c r e a s e s  very r a p i d l y  compared t o biomass. The shaded a r e a i n F i g u r e  9 indicates  the range o f 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 t i m e s o f t h e y e a r , c a l c u l a t e d u s i n g t h e d a t a o f 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 o f g l u c o s e , a c e t a t e and glycine reported  by H a l l e t a l . ( 1 9 7 2 ) . 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 a r e 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 d a t a . These s h i f t s between a c t i v i t y and biomass a r e 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 c h e m i c a l 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 a s p r i m a r y p r o d u c t i o n and g r a z i n g . C o n s i d e r i n g t h e r e l a t i o n s found ( F i g u r e 9 ) .  a  62  F i g u r e 9. Diagrammatic r e p r e s e n t a t i o n of t h e  seasonal  r e l a t i o n s between a c t i v i t y and biomass of t h e sediment b a c t e r i a as p r o p o r t i o n s o f 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 o f g l u c o s e , g l y c i n e and a c e t a t e and f o r oxygen consumption I . I ; range f o r dehydrogenase a s s a y  I.-  )  A c t i v i t y o r biomass as a p r o p o r t i o n o f 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 . D u r i n g summer, t h e r e a r e h i g h numbers of b a c t e r i a , probably experiencing r e l a t i v e l y high grazing pressure, but a t a p r o d u c t i o n l e v e l h i g h enough t o m a i n t a i n t h e i r numbers. I n f a l l , t h i s r e p r o d u c t i v e c a p a c i t y d e c l i n e s s h a r p l y , d e s p i t e f r e s h allochthonous inputs t o the l a k e , due t o d e c r e a s i n g temperature  and day l e n g t h 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 a n e o u s l y p r o b a b l y as a r e s u l t o f g r a z i n g p r e s s u r e .  The  b a c t e r i a l biomass t h e n i n c r e a s e s g r a d u a l l y d u r i n g the w i n t e r and e a r l y s p r i n g d u r i n g w h i c h p e r i o d growth r a t e ,  although  not a t i t s maximum, appears t o be g r e a t e r t h a n r e m o v a l due t o g r a z i n g . When the temperature appear t o respond  c l i m b s above 4° b a c t e r i a  q u i c k l y . T h i s i s suggested by the  sharp  d e c l i n e i n g l u c o s e c o n c e n t r a t i o n s i n t h e i n t e r s t i t i a l water i n A p r i l and May  (Figure 3c).  The t o t a l p o p u l a t i o n i s  p r o b a b l y 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 s u p p l y o f r e a d i l y - a v a i l a b l e carbon s o u r c e s . D e t r i t a l m a t e r i a l breakdown i n t o m o l e c u l e s o f a s i z e w h i c h b a c t e r i a can t r a n s p o r t a c r o s s t h e i r membranes i s p r o b a b l y the r a t e l i m i t i n g s t e p a t t h i s t i m e . The consequence o f t h i s i s the low a c t i v i t y measurements o b t a i n e d i n A p r i l and i n e a r l y May(eg. F i g u r e 8 ) .  With s t i l l higher  sometimes temperatures,  p r i m a r y p r o d u c t i o n by e p l p e l i c a l g a e i n c r e a s e s r a p i d l y (Hargrave, 1969;  G r e u n d l i n g , 1 9 7 1 ) . Carbon and o t h e r 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 m a t e r i a l are decomposed  more r a p i d l y at higher temperatures.  The b a c t e r i a l p o p u l a t i o n  should, t h e o r e t i c a l l y , ©xplode a t t h i s time of year. And i t probably does. The p o p u l a t i o n i s v e r y " a c t i v e " i n terms of a l l the a c t i v i t y i n d i c a t o r s a p p l i e d t o the system. Biomass, however, does not respond as d r a m a t i c a l l y , presumably  due  t o i n c r e a s e d outputs t o higher t r o p h i c l e v e l s . T h i s d e s c r i p t i v e model c o u l d be examined with  two  b i t s of i n f o r m a t i o n . G r a z i n g e f f e c t s on m i c r o b i a l popu l a t i o n s have been s t u d i e d (eg, Hargrave,  1970;  Fenchel,  1970), but d a t a on seasonal v a r i a t i o n i n t h i s f a c t o r are l a c k i n g . The other requirement  Is i n f o r m a t i o n on b a c t e r i a l  growth r a t e s a t d i f f e r e n t times of the year under n a t u r a l c o n d i t i o n s . Attempts t o o b t a i n such data i n t h i s  study  f a i l e d due t o h e t e r o g e n e i t y between subsamples of the sediments.  I f the a c t i v i t y e s t i m a t o r s a l r e a d y a p p l i e d t o the  sediments do r e f l e c t b a c t e r i a l growth r a t e (as suggested the work of S t a n l e y and S t a l e y ,  by  197*0, and i f g r a z i n g  pressure i s r e l a t i v e l y h i g h i n May  t o September, and low i n  w i n t e r , then confidence i n the above d e s c r i p t i o n of b a c t e r i a l p o p u l a t i o n dynamics would be i n c r e a s e d .  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 a n n u a l mean i n p u t s a n d o u t p u t s o f t h e 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 o f . 6 1 g C/m  2  based on  d i r e c t c o u n t s ) a r e summarized i n F i g u r e 1 0 . The range for  g r o s s uptake and r e s p i r a t o r y l o s s e s o f o r g a n i c c a r b o n  are  from K l e i b e r ( 1 9 7 2 ) . The v a l u e s i n b r a c k e t s i n d i c a t e  the  e s t i m a t e s from oxygen u p t a k e ( H a r g r a v e , 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 o f c a r b o n a s CH^ was e s t i m a t e d from e x p e r i m e n t s w i t h sediment c o r e s , and appears t o be a minor component o f t h e system. E p 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 G r e u n d l i n g (1971) and g r o s s p r o d u c t i v i t y from h i s d a t a and t h a t o f Hargrave ( 1 9 6 9 ) . E x c r e t i o n o f a l g a l p h o t o s y n t h a t e t o t h e d i s s o l v e d o r g a n i c c a r b o n (DOC) p o o l ( p o o l s i z e  calculated  from H a l l and H y a t t , 197*0 was d e t e r m i n e d by K l e i b e r 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  (1972).  (1969)  e s t i m a t e o f r e s p i r a t i o n due t o b a c t e r i a and a l g a e and corrected f o r b a c t e r i a l respiration. L o s s e s 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 i n p u t s and o u t p u t s t o t h e a l g a l and bact 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 t o the d i s s o l v e d and p a r t i c u l a t e o r g a n i c c a r b o n (POC) p o o l s from h i g h e r t r o p h i c l e v e l s i s unknown b u t may be s i g n i f i c a n t ( e g . Hargrave, 1 9 7 0 ) . H a l l e t a l . (1973) e s t i m a t e d t h e c o n t r i b u t i o n o f o r g a n i c c a r b o n t o t h e DOC o f t h e 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 .year .Net. 2  DOC .inputs  — 2 ' f r o m t h e i n l e t a r e 28 gC/m . y e a r ( G e e n , u n p u b l i s h e d ) . O t h e r  F i g u r e 10. S t r u c t u r e and f u n c t i o n o f t h e M a r l o n Lake sediment ecosystem ( a l l compartment s i z e s a r e e x p r e s s e d i n gC/m and a l l f l u x e s a r e I n 2  gC/m  .year, u n l e s s o t h e r w i s e 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 s o u r c e s ) .  66 a  ailochthonous material  biological  —  processes ?  ? /  \(1) DOC  7  *<5  mg/1  y  water sediment  (  PO 240  DOC .14-.36'  * **4 C  (1)  29.1-31.1  (4) 6.9-26.7(11.5)  (7) 4.2-24.0(19..4)  (2) 42.3  (5) 22.0-29.3  (8) 76.6  (3) 1.5-8.8  (6) 21.0-120(97.0)  (9)  <0.5  CO)  28  67  i n p u t s t o t h i s p o o l 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 q u a n t i f i e d , but may be s m a l l s i n c e d i f f u s i o n processes would be expected t o operate i n the opposite  direction.  The major unknown component i n the system i s the r a t e of decomposition of n a t u r a l sediment POC  (including  l i v i n g and dead o r g a n i c matter, but p r i m a r i l y the l a t t e r ) to u t i l i z a b l e DOC, T h i s occurs as a r e s u l t of chemical and b i o l o g i c a l p r o c e s s e s , but q u a n t i t a t i v e assessment of i t s importance w i l l r e q u i r e f u r t h e r development o f t e c h nology.  68  SUMMARY AND CONCLUSIONS  The  prime f u n c t i o n a l r o l e s o f b a c t e r i a a r e c o n v e r s 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 o r g a n i c 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 o f 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 p h o t o s y n t h e t i c organisms. The  l a t t e r p r o c e s s has n o t been s t u d i e d  i n M a r i o n Lake. The  major s o u r c e s o f o r g a n i c matter f o r t h e h e t e r o t r o p h i c p o p u l a t i o n appear t o be a l l o c h t h o n o u s i n p u t s from t h e watershed and e p i b e n t h i c  a l g a l p r o d u c t i o n . Much o f t h e l a t t e r  i s consumed by h i g h e r t r o p h i c l e v e l s and i s not,  therefore,  directly available to bacteria. D u r i n g t h i s s t u d y , i t was demonstrated t h a t ATP a n a l y s i s offers rapid estimation  o f b o t h 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 e s t i m a t e d by d i r e c t c o u n t s , a n d was t h e l a r g e s t component o f t h e m i c r o b i a l a p p r o x i m a t e l y 30JJm) s t a n d i n g c r o p . Changes i n t h e proportion  o f b a c t e r i a l and m i c r o b i a l biomass i n t h e  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 . discrepancies Indicate  Analysis  of the  between ATP- and enumeration-biomass d a t a  t h a t ATP i s n o t o n l y a biomass measurement, b u t ,  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 , o r the a b i l i t y o f a p o p u l a t i o n t o m a i n t a i n i t s s i z e I f e x -  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 h i g h e r trophic  levels.  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 r e v i e w e d and compared t o h e t e r o 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 o b t a i n e d d u r i n g t h i s s t u d y . C l o s e c o r r e l a t i o n between h e t e r o t r o p h i c u p t a k e o f g l u c o s e i n 1971-72 and 1973-74 i n d i c a t e d t h a t t h e 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 y e a r . The  s e a s o n a l responset-of b a c t e r i a l biomass was i n t e r -  p r e t e d w i t h r e s p e c t t o s e v e r a l a c t i v i t y measurements, assuming t h e s e measurements r e f l e c t e d growth r a t e , and t h a t 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 a p p r o x i m a t e l y 10 . The  q u a n t i t a t i v e r o l e o f b a c t e r i a i n the c a r b o n budget o f the sediments was d e s c r i b e d u s i n g a c q u i r e d d a t a and t h a t of s e v e r a l p r e v i o u s s t u d e n t s o f t h e M a r i o n Lake  benthos.  The c o n t r i b u t i o n o f a l l o c h t h o n o u s compared t o a u t o c h thonous i n p u t s i s u n c l e a r , a l t h o u g h e s t i m a t e s o f t h e i r c o n t r i b u t i o n s t o t h e d i s s o l v e d o r g a n i c matter p o o l s a r e a v a i l a b l e . I t was n o t e d t h a t a l l t h e e s t i m a t e d t o t a l upt a k e o f o r g a n i c c a r b o n by b a c t e r i a need n o t come d i r e c t l y from e i t h e r o f t h e s e p r i m a r y c a r b o n s o u r c e s , s i n c e t h e r e i s a n unknown q u a n t i t y from r e c y c l i n g w i t h i n the community. Temperature, which l i m i t s the a v a i l a b i l i t y o f d e t r i t u s and d i s s o l v e d o r g a n i c matter t o b a c t e r i a and, p a r t i c u l a r l y i n t h e May t o September p e r i o d , g r a z i n g , a p p e a r  t o be t h e key  factors c o n t r o l l i n g b a c t e r i a l product!  71  REFERENCES Anderson,J.P.E., and K. H. Domsch. 1973. S e l e c t i v e i n h i b i t i o n as a method f o r e s t i m a t i o n o f the r e l a t i v e a c t i v i t i e s of m i c r o b i a l populations i n s o i l s . B u l l . E c o l . Res. Comm. (Stockholm) 1 7 * 4 7 - 5 2 .  Antipchuk, A. F . 1972. Abundance and s i z e o f microorganisms i n bottom sediments o f f i s h ponds. H y d r o b i o l o g i c a l J o u r n a l 2:46-50. Ausmus, B. S. 1973. The use o f the ATP assay i n t e r r e s t r i a l decomposition s t u d i e s . B u l l . E c o l . Res. Comm. (Stockholm)  17s223-234.  Azam, F., and 0 . Holm-Hansen. 1973. Use o f t r i t i a t e d subs t r a t e s i n the study o f heterotrophy i n seawater. Mar. Biol.23»191-196. Babiuk, L. A., and E. A. P a u l . 1970. The use o f f l u o r e s c e i n i s o t h i o c y a n a t e i n the d e t e r m i n a t i o n o f the b a c t e r i a l b i o mass o f g r a s s l a n d s o i l . Can. J . M i c r o b i o l . 1 6 1 5 7 - 6 2 . Bae, H. C., E . H. Cota-Robles, and L. E. C a s i d a , J r . 1972. M i c r o f l o r a o f s o i l as viewed by t r a n s m i s s i o n e l e c t r o n microscopy. A p p l . M i c r o b i o l . 23*637-648. B a n c r o f t , K., E. A. P a u l , and W. J . Wiebe. 1974. E x t r a c t i o n of adenosine t r i p h o s p h a t e from marine sediments w i t h b o i l i n g sodium b i c a r b o n a t e . A b s t r a c t G - 2 0 3 , Ann. Meeting Am. Soc. M i c r o b i o l . , Chicago. B e r l a n d , B. R., D. J . Bonin, P. L. Laborde, S. Y. M a e s t r i n i . 1972. V a r i a t i o n s de quelques f a c t e u r s e s t i m a t i f s de l a biomasse, e t en p a r t i c u l i e r de l'ATP chez p l u s i e r s algues marines p l a n c t o n i q u e s . Mar. B i o l . 1 3 1 3 3 8 - 3 4 5 . Beyers, R. J . , J , Larimer, H. T. Odum, R. B. Parker, and N. E . Armstrong. 1963. D i r e c t i o n s f o r the d e t e r m i n a t i o n of changes i n carbon d i o x i d e c o n c e n t r a t i o n from changes i n pH. P u b l . I n s t . Mar. S c i . Univ. Texas 9 : 4 5 4 - 4 8 9 . B i a n c h i , A. J . M. 1973. V a r i a t i o n s de l a c o n c e n t r a t i o n b a c t e r i e n n e dans l e s eaux e t l e s sediments l i t t o r a u x . Mar. B i o l . 2 2 : 2 3 - 2 9 . Bray, G. A. i 9 6 0 . A simple e f f i c i e n t l i q u i d s c i n t i l l a t o r f o r c o u n t i n g aqueous s o l u t i o n s i n a l i q u i d s c i n t i l l a t i o n counter. A n a l . Biochem. 1 : 2 7 9 - 2 8 5 . Brezonik, P. L., and J . W, P a t t e r s o n . 1 9 7 1 . A c t i v a t e d sludge ATP: e f f e c t s o f environmental s t r e s s . J . S a n i t a r y Eng. Dlv., Proc. Am. Soc. C i v i l Engineers. Brock, T. D. 1967. The ecosystem and the steady Bioscience 17*166-167.  state.  72  B u r n i s o n , B. K., and R. Y. M o r i t a . 1973. Competitive i n h i b i t i o n f o r amino a c i d uptake by the i n d i g e n o u s m i c r o f l o r a o f Upper K l a m a t h L a k e . A p p l . M i c r o b i o l . 25*103-106.  B u r n i s o n , B. K., and K. T. P e r e z . 1974. A s i m p l e method f o r t h e d r y combusion o f ^ C - l a b e l l e d m a t e r i a l . (Accepted f o r p u b l i c a t i o n i n Ecology). Cameron, R. L. 1973. A c o m p a r i s o n of the r e s p o n s e s o f b e n t h i c and p l a n k t o n l c communities t o t h e enrichment of i n o r g a n i c f e r t i l i z e r s . M. S. T h e s i s . U n i v . o f B. C. C a s i d a , L. E., J r . , D. A. K l e i n and T. S a n t o r o . 1964. dehydrogenase a c t i v i t y . S o i l S c i . 98:371-376.  Soil  C h a p e l l e , E. W., and G. V. L e v i n . 1968. Use of the f i r e f l y b i o l u m l n e s c e n t r e a c t i o n f o r r a p i d d e t e c t i o n and c o u n t i n g of b a c t e r i a . Biochem. M e d i c i n e 2:41-52. C o l e , H. A., J . W. T. Wimpenny, and D. W. Hughes. I967. The ATP p o o l i n E s c h e r i c h i a c o l l . 1. Measurement o f the p o o l u s i n g a modified l u c i f e r a s e assay. Biochim. b i o p h y s . A c t a (Amst.) 143I445-453. C o n k l i n , A. R., J r . , and A. N. MacGregor. 1972. S o i l adenosine t r i p h o s p h a t e : e x t r a c t i o n , r e c o v e r y and h a l f l i f e . B u l l . Env. C o n t a m i n a t i o n and T o x i c o l o g y 7:296-300. C r a w f o r d , C. C., J . E. Hobble, and K. L. Webb. 1974. The u t i l i z a t i o n o f d i s s o l v e d f r e e amino a c i d s by e s t u a r i n e m i c r o o r g a n i s m s . E c o l o g y 55*551-563. C u r l , H., J r . , and J . Sandberg. I 9 6 I . The measurement of dehydrogenase a c t i v i t y i n marine organisms. J , Mar. Res. 19*123-138.  D a v l e s , G. S. 1970. P r o d u c t i v i t y of macrophytes i n M a r i o n L a k e , B r i t i s h C o l u m b i a . J . F i s h . Res. Bd. Canada 27:71-81. D i c k , M. W. 1971. The e c o l o g y o f S a p r o l e g n i a c e a e i n l e n t i c and l i t t o r a l muds w i t h a g e n e r a l t h e o r y of f u n g i i n the l a k e ecosystem. J . Gen. M i c r o b i o l . 65*325-337. Duncan, D. B. 1955. M u l t i p l e range t e s t s and m u l t i p l e F t e s t s . B i o m e t r i c s 11i1-42. Edwards, R. W., and H. L. J . R o l l e y . 1965. Oxygen consumption of r i v e r muds. J . E c o l . 53*1-19. E f f o r d , I . E. I967. Temporal and s p a t i a l d i f f e r e n c e s i n p h y t o p l a n k t o n p r o d u c t i v i t y i n M a r i o n Lake, B r i t i s h C o l u m b i a . J . F i s h . Res. Bd. Canada 24:2283-2307.  73  . 1969. Energy t r a n s f e r i n M a r i o n Lake, B r i t i s h Columbia, w i t h p a r t i c u l a r r e f e r e n c e s t o f i s h f e e d i n g , V e r h . I n t e r n a t , V e r e i n , L l m n o l . 17*104-108, E i d u s , L., B, B, D i e n a , and L, Greenberg. 1 9 5 9 . O b s e r v a t i o n s on t h e use o f t a t r a z o l i u m s a l t s i n t h e v i t a l s t a i n i n g o f b a c t e r i a . Can. J . M i c r o b i o l . 5*245-250. E r n s t , W. 1970. ATP a l s I n d i k a t o r f u r d i e Biomasse Sedimente. O e c o l o g i a ( B e r l i n ) 5 * 5 6 - 6 0 .  mariner  F e n c h e l , T. 1 9 6 9 . The e c o l o g y o f marine m i c r o b e n t h o s . I V . S t r u c t u r e a n d f u n c t i o n o f t h e b e n t h i c ecosystem, i t s c h e m i c a l and p h y s i c a l f a c t o r s and t h e m i c r o f a u n a communities with s p e c i a l references t o the c i l i a t e d protozoa, O p h e l i a 611-182. F e n c h e l , T. 1 9 7 0 . S t u d i e s on t h e d e c o m p o s i t i o n o f o r g a n i c d e t r i t u s d e r i v e d from t h e t u r t l e g r a s s T h a l a s s l a t e s t udlnum. L i m n o l . Oceanogr. 1 5 « 1 4 - 2 0 . Gerchakov, S. M., and P. G. H a t c h e r . 1972. Improved t e c h nique f o r a n a l y s i s of carbohydrates i n sediments. L i m n o l . Oceanogr. 17*931-938. G i e s e , A. C. 1 9 6 8 . C e l l P h y s i o l o g y . W. B. Saunders, Co., P h i l a d e l p h i a , pp 1 - 6 7 9 . G r e u n d l i n g , G, K. 1 9 7 1 . E c o l o g y o f t h e e p i p e l i c a l g a l communities i n M a r i o n Lake, B r i t i s h Columbia. J . P h y c o l . 7*239-249. H a l l , K, J , , P, M. K l e i b e r , and I . Y e s a k i . 1 9 7 2 . H e t e r o t r o p h i c uptake o f o r g a n i c s o l u t e s by microorganisms i n t h e sediment. Mem. 1st. I t a l . I d r o b i o l . 29 Suppl.«441-471. H a l l , K. J . , and S, D o e l , 1972. A n a l y s i s o f o r g a n i c c o n s t i t u e n t s i n sediment. M a r i o n Lake P r o j e c t R e p o r t 1 9 7 1 - 7 2 . H a l l , K. J . , I . Y e s a k i , and S, D o e l . 1 9 7 3 . D e c o m p o s i t i o n o f l e a f l i t t e r i n sediment microcosms. 36th M e e t i n g Am. S o c . L i m n o l , Oceanogr., U n i v . o f U t a h , U t a h . H a l l K. J . , and K. H y a t t . 1974. M a r i o n Lake (IBP) - from b a c t e r i a t o f i s h . j . F i s h . R e s . B d . Canada 31:893-91 1. S p e c i a l Issue  for  the  XIX  Congress,  S.  I.  L.  Hammerstedt, R. H. 1 9 7 3 . An automated method f o r ATP a n a l y s i s u t i l i z i n g t h e l u c i f erin-luc I f e r a s e r e a c t i o n . A n a l , Biochem. 52*449-455. H a m i l t o n , R. D., and 0 . Holm-Hansen. 1 9 6 7 . Adenosine t r i phosphate c o n t e n t o f marine b a c t e r i a . L i m n o l . Oceanog. 12i319-324.  74  H a r g r a v e , B. T., 1969. E p l b e n t h i c a l g a l p r o d u c t i o n and community r e s p i r a t i o n i n t h e sediments of M a r i o n Lake. J . F i s h . Res. Bd. Canada 2612003-2026. . 1970. The e f f e c t of a d e p o s i t - f e e d i n g amphlpod on t h e m e t a b o l i s m o f b e n t h i c m i c r o f l o r a . L i m n o l . Oceanogr. 15*21-30. H a r r i s o n , D. E. F., and P, M a i t r a . 1969. C o n t r o l of r e s p i r a t i o n and metabolism i n g r o w i n g K l e b s i e l l a aerogenes. The r o l e o f adenine n u c l e o t i d e s . Biochem. J . 112:647-652. H a r r i s o n , M. J . , R. T. W r i g h t , and R. Y. M o r i t a . 1971. Method f o r measuring m i n e r a l i z a t i o n i n l a k e s e d i m e n t s . A p p l . M i c r o b i o l . 211698-702. H i c k s , S. E., and F, G. Carey. 1968, G l u c o s e d e t e r m i n a t i o n i n n a t u r a l w a t e r s . L i m n o l . Oceanogr. 1 3 * 3 6 1 - 3 6 3 . Hobble, J . E., 0 . Holm-Hansen, T. T. P a c k a r d , L. R. Pomeroy, R. W. S h e l d o n , J . P. Thomas, and W, J , Wiebe. 1972. A s t u d y of t h e d i s t r i b u t i o n and a c t i v i t y of m i c r o o r g a n i s m s i n ocean w a t e r . L i m n o l . Oceanogr. 17*544-555. Holm-Hansen, 0 . , and C. R. Booth. 1966. The measurement of a d e n o s i n e t r i p h o s p h a t e i n t h e oceans and i t s e c o l o g i c a l s i g n i f i c a n c e . L i m n o l . Oceanogr. l i s 5 1 0 - 5 1 9 . Holm-Hansen, 0 . , and H. W. P a e r l . 1972. The a p p l i c a b i l i t y of ATP d e t e r m i n a t i o n f o r e s t i m a t i o n of m i c r o b i a l biomass and m e t a b o l i c a c t i v i t y , Mem. Iafc. I t a l . I d r o b i o l . 29 Suppl.«149-168. Holms, W. H., I . D. H. H a m i l t o n , and A. G. R o b e r t s o n . 1972. The r a t e of t u r n o v e r of the a d e n o s i n e t r i p h o s p h a t e p o o l of E s c h e r i c h i a c o l l growing a e r o b i c a l l y i n s i m p l e d e f i n e d media. A r c h . M i k r o b i o l . 8 3 : 9 5 - 1 0 9 . Howard, P. J . A. 1972, Problems i n t h e e s t i m a t i o n o f o g i c a l a c t i v i t y i n s o i l . O i k o s 23*235-240.  biol-  J o n e s , P. C. T., and J . E. M o l l i s o n . 1948. A t e c h n i q u e f o r t the q u a n t i t a t i v e e s t i m a t i o n o f s o i l m i c r o o r g a n i s m s . J . Gen. Microbiol. 2J54-69. K a r l , D. M. and P. A. LaRock. 1974. E x t r a c t i o n and measurement of a d e n o s i n e t r i p h o s p h a t e i n sediments and s o i l . A b s t r a c t G - 2 0 2 , Ann. M e e t i n g Am. Soc. M i c r o b i o l . , C h i c a g o . Khiyama, H. M., and J . C. Makemson. 1973. Sand beach b a c t e r i a : e n u m e r a t i o n and c h a r a c t e r i z a t i o n . A p p l . M i c r o b i o l . 2 6 : 2 9 3 - 2 9 7 . K l e i b e r , P. M. 1972. The dynamics of e x t r a c e l l u l a r , d i s s o l v e d o r g a n i c m a t e r i a l i n t h e sediments of M a r i o n Lake, B r i t i s h Columbia. Ph. D. T h e s i s . U n i v . o f C a l i f o r n i a , D a v i s ,  75  Lee, C. G., R. F. H a r r i s , J . D. H. W i l l i a m s , D. E. Arms t r o n g , a n d J . K, S u e r s . 1971a. Adenosine t r i p h o s p h a t e i n lake sediments. 1 , Determination. S o i l S c i . Soc, Amer. P r o c . 3 5 * 8 2 - 8 6 . . 1971b. Adenosine t r i p h o s p h a t e i n l a k e s e d i m e n t s , 2 . O r i g i n and s i g n i f i c a n c e . S o i l S c i . Soc. Amer. P r o c . 35*86-91. Lenhard, G. 1 9 5 6 . D i e d e h y d r o g e n a s e a k t i v i t a t des Bodens a l s Mass f u r d i e M i k r o o r g a n i s m e n t a t i g k e i t im Boden. Z. P f l E r n a h r . Dung. 7 3 * 1 - 1 1 . . 1968. A s t a n d a r d i z e d procedure f o r t h e d e t e r m i n a t i o n o f dehydrogenase a c t i v i t y i n samples from a n a e r o b i c t r e a t m e n t systems. Water Res. 2 i l 6 l - l 6 7 . Llndeman, R. L. 1 9 4 2 . The t r o p h i c - d y n a m i c a s p e c t s o f ecology. E c o l . 23t399-418. MacLeod, N. H., E. W. C h a p e l l e , and A. M. C r a w f o r d . I 9 6 9 . ATP a s s a y o f t e r r e s t r i a l s o i l s * a t e s t o f a n e x o b i o l o g i c a l e x p e r i m e n t . Nature 223*267-268, M a h l e r , H, R,, and E, H, Cordes. 1 9 6 6 , B i o l o g i c a l C h e m i s t r y . Harper a n d Row, New Y o r k , pp. I - 8 7 2 . Mc E l r o y , W. D. 1 9 4 7 . The energy s o u r c e f o r b i o l u m i n e s c e n c e i n a n i s o l a t e d system. P r o c . N a t l . A c a d . S c i . U. S, 33*342-345. Odum, H. T. 1 9 5 7 . T r o p h i c s t r u c t u r e and p r o d u c t i v i t y o f S i l v e r S p r i n g s , F l o r i d a , E c o l , Monogr, 2 7 * 5 5 - 1 1 2 . Odum, E. P. 1 9 6 8 . Energy f l o w i n ecosystems* r e v i e w . Amer. Z o o l o g i s t 8 * 1 1 - 1 8 ,  a historical  O l a h , J , 1 9 7 2 , L e a c h i n g , c o l o n i z a t i o n and s t a b i l i z a t i o n d u r i n g d e t r i t u s f o r m a t i o n , Mem. 1 s t . I t a l . I d r o b l o l . 29 Suppl.* 105-127. P a c k a r d , T. T. 1 9 7 0 , The e s t i m a t i o n o f t h e oxygen u t i l i z a t i o n r a t e i n seawater from t h e a c t i v i t y o f t h e r e s p i r a t o r y e l e c t r o n t r a n s p o r t system i n p l a n k t o n . Ph. D. T h e s i s , U n i v . of Washington. Pamatmat, M. M. 1 9 6 8 . E c o l o g y and m e t a b o l i s m o f a b e n t h l c community on a n I n t e r t i d a l s a n d f l a t . I n t e r n . Rev. Ges. H y d r o b i o l . 53*211-298. Pamatmat, M. M., and A. M. Bhagwat. 1 9 7 3 . A n a e r o b i c metabolism i n Lake Washington s e d i m e n t s . L i m n o l . Oceanogr. 1 8 * 6 1 1 - 6 2 7 . P a r k i n s o n , D., T. R. G. Gray, a n d S. T. W i l l i a m s . 1 9 7 1 .  76  E c o l o g y o f S o i l M i c r o o r g a n i s m s . IBP Handbook No. 1 9 . B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , O x f o r d , pp. 1 - 1 1 6 . P a r s o n s , T. R., and J . D. H. S t r i c k l a n d . 1 9 6 2 . On t h e p r o d u c t i o n o f p a r t i c u l a t e o r g a n i c c a r b o n by h e t e r o t r o p h i c p r o c e s s e s i n sea w a t e r . Deep-Sea Res. 8 1 2 1 1 - 2 2 2 . P a t t e r s o n , J . W., P. L. B r e z o n i k a n d H. D. Putnam. 1 9 7 0 . Measurement a n d s i g n i f i c a n c e o f adenosine t r i p h o s p h a t e i n a c t i v a t e d s l u d g e . E n v i r o n m e n t a l S c i . and T o x i c o l o g y 4s569-575.  Ramey, W. D. 1972. M i c r o s c o p i c e x a m i n a t i o n o f b a c t e r i a l p o p u l a t i o n w i t h i n t h e d e t r i t u s o f M a r i o n Lake. B. S. T h e s i s . U n i v . o f B. C. R o b i n s o n , G. G. C., L. L. Hendzel a n d D. C. G i l l e s p i e . 1 9 7 3 . A r e l a t i o n s h i p between h e t e r o t r o p h i c u t i l i z a t i o n of o r g a n i c a c i d s and b a c t e r i a l p o p u l a t i o n s i n West B l u e Lake, Manitoba. L i m n o l . Oceanogr. 1 8 1 2 6 4 - 2 6 9 . R o d i n a , A. G. 1 9 7 2 . Methods i n A q u a t i c M i c r o b i o l o g y . U n i v e r s i t y P a r k P r e s s , B a l t i m o r e , pp 1 - 4 6 1 , Rudd, J . W, M., and R. D. H a m i l t o n . 1 9 7 3 . Measurement of adenosine t r i p h o s p h a t e (ATP) i n two p r e c a m b r i a n -:• s h i e l d l a k e s o f n o r t h w e s t e r n O n t a r i o . J . F i s h . Res. Bd. Canada 3 0 s 1 5 3 7 - 1 5 4 6 . S c h i n d l e r , D. W. 1 9 7 3 . E x p e r i m e n t a l approaches t o l i m n o l o g y - a n o v e r v i e w . J . F i s h . Res. Bd. Canada 3 0 i l 4 0 9 S c h i n d l e r , D. W., V. E. F r o s t , a n d R. V. Schmidt. 1973. P r o d u c t i o n o f e p i l i t h o p h y t o n I n two l a k e s o f the E x p e r i m e n t a l Lakes A r e a , n o r t h w e s t e r n O n t a r i o . J . F i s h . R e s . - Bd. Canada 30s 1511-1524. Schmidt, E. L. 1 9 7 3 . The t r a d i t i o n a l p l a t e count t e c h n i q u e among modern methods. Chairman's summary. B u l l . E c o l . Res. Comm. (Stockholm) 17s453-454. S h i e l d s , J . A., E, A, P a u l , W. E. Lowe, a n d D. P a r k i n s o n . 1973. Turnover o f m i c r o b i a l t i s s u e i n s o i l under f i e l d c o n d i t i o n s . S o i l B i o l . Biochem. 5s753-764. S k u j i n s , J . 1 9 7 3 . Dehydrogenases a n i n d i c a t o r o f b i o l o g i c a l a c t i v i t i e s i n a r i d s o i l s . B u l l . E c o l . Res. Comm. ( S t o c k holm) 17s235-241. S o r o k i n , Y. I . 1970. I n t e r r e l a t i o n s between s u l f u r a n d carbon turnover i n meromictic l a k e s . Arch, H y d r o b i o l . 66s391-446.  77  S o r o k i n , Y. I . , and H. Kadota ( e d i t o r s ) . 1 9 7 2 . Techniques f o r t h e Assessment o f M i c r o b i a l P r o d u c t i o n and Decomposi t i o n I n F r e s h Waters. I B P Handbook No. 2 3 . B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , Oxford. S t a n i e r , R. Y., R. Kunisawa, M. Mandel, and G, Cohen B a z i r e , 1 9 7 1 . P u r i f i c a t i o n and p r o p e r t i e s o f u n i c e l l u l a r blue-green algae (order Chlorococcales). B a c t e r i o l . Rev. 3 5 : 1 7 1 - 2 0 5 . S t a n l e y , P. M. and J . T. S t a l e y . 1974, A c e t a t e u p t a k e by a q u a t i c b a c t e r i a l communities measured by a u t o r a d i o graphy and f i l t e r a b l e r a d i o a c t i v i t y . A b s t r a c t G - 1 8 3 . Ann. M e e t i n g Am. Soc. M i c r o b i o l . , C h i c a g o . S t e v e n s o n , I . L. 1 9 5 9 . Dehydrogenase Can. J . M i c r o b i o l . 5 * 2 2 9 - 2 3 5 .  activity i n soils.  S t r e h l e r , B. L. I 9 6 5 . Adenosine - 5 ' - t r i p h o s p h a t e and c r e a t i n e phosphate. D e t e r m i n a t i o n w i t h l u c i f e r a s e . (IN) Methods o f E n z y m a t i c A n a l y s i s , pp. 5 5 9 - 5 7 2 . H. U. Bergmeyer ( e d i t o r ) , Academic P r e s s , New Y o r k . T e a l , J . M. 1 9 5 7 . Community m e t a b o l i s m i n a temperate c o l d s p r i n g . E c o l . Monogr. 2 7 * 2 8 3 - 3 0 3 . . 1 9 0 2 . Energy f l o w i n t h e s a l t marsh ecosystems of G e o r g i a . E c o l o g y 4 3 : 6 1 4 - 6 2 4 . , a n d J . W. K a n w i s h e r . 1961. Gas exchange i n a G e o r g i a s a l t marsh. L i m n o l . Oceanogr. 6 : 3 8 8 - 3 9 9 . T r o l l d e n l e r , G. 1 9 7 3 . The use o f f l u o r e s c e n c e m i c r o s c o p y f o r counting s o i l microorganisms. B u l l . E c o l . Res. Comm. (Stockholm) 1 7 * 5 3 - 5 9 . V a c c a r o , R, F., and H. W. J a n n a s c h . 1967. V a r i a t i o n s i n uptake k i n e t i c s f o r g l u c o s e by n a t u r a l p o p u l a t i o n s i n seawater. L i m n o l . Oceanogr. 1 2 : 5 4 0 - 5 4 2 . W i l l i a m s , P. J . LeB. 1973. The v a l i d i t y o f t h e a p p l i c a t i o n of s i m p l e k i n e t i c a n a l y s i s t o heterogeneous m i c r o b i a l p o p u l a t i o n s . L i m n o l . Oceanogr. 1 8 : 1 5 9 - 1 6 5 . Witkamp, M. 1973. C o m p a t i b i l i t y o f m i c r o b i a l measurements. B u l l . E c o l . Res. Comm. (Stockholm) 1 7 * 1 7 9 - 1 8 8 . Wood, L. W. 1970. The r o l e o f e s t u a r i n e sediment m i c r o organisms i n t h e uptake o f o r g a n i c s o l u t e s under a e r o b i c c o n d i t i o n s . Ph. D. T h e s i s , N o r t h C a r o l i n a S t a t e U n i v .  78  W r i g h t , R. T., and J . E. Hobble. 1 9 ^ 5 . The uptake o f o r g a n i c s o l u t e s I n l a k e w a t e r . L i m n o l . Oceanogr. 9« 163-178. . 1966. Use o f g l u c o s e and a c e t a t e by b a c t e r i a and a l g a e i n a q u a t i c ecosystems. E c o l o g y 47»447-464. Y e t k a , J . E., and W. J . Wiebe. 1974. E f f e c t s o f antife b i o t i c s on r e s p i r a t i o n o f b a c t e r i a l p o p u l a t i o n s . Abs t r a c t G - I 8 7 , Ann. Meeting Am. Soc. M i c r o b i o l . , C h i c a g o . Z o b e l l , C. E. 1 9 6 3 . Domain o f t h e marine m i c r o b i o l o g i s t . (IN) C. H. Oppenheimer ( e d i t o r ) . Symposium on Marine M i c r o b i o l o g y , pp 3-24, C. C. Thomas, S p r i n g f i e l d . Z v a g i n s e v , D. G. 1 9 7 3 . M i c r o b i a l e c o l o g y a s s t u d i e d by luminescence m i c r o s c o p y 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) 1 7 * 6 1 - 6 5 .  79 APPENDICES  Appendix 1.  Method f o r e x t r a c t i o n o f t o t a l ATP sediments  (based on Lee e t a l . ,  from f r e s h  1971a).  Extraction Add  3  g wet wt.  (150-200mg  d r y wt.) sediment t o a c h i l l e d  c e n t r i f u g e t u b e . Add 5 ml. of i c e - c o l d O.6NH2SO4 t o t u b e , mix i n t e r m i t t e n t l y f o r 5 min k e e p i n g sample c o l d t h e n s t o r e on i c e for  20  min. C e n t r i f u g e  (5000g/5min)  and t r a n s f e r the s u p e r -  n a t a n t t o a 10 ml g r a d u a t e d c y l i n d e r . Record t h e volume (approximately 7 ml). T r a n s f e r the p e l l e t t o a t a r e d weighing pan f o r d r y wt, d e t e r m i n a t i o n .  Purification T r a n s f e r 2 ml o f e x t r a c t t o a t e s t tube k e e p i n g 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 o f  c a t i o n exchange r e s i n ( A m b e r l i t e 3LR-20, c o n v e r t e d t o Na form by washing w i t h IN NaOH and d i s t i l l e d w a t e r ) . Mix t h e c o n t e n t s f o r 3 min. S t r a i n t h e sample t h r o u g h g l a s s wool using 3 -  1 ml a l i q u o t s o f d i s t i l l e d water t o wash t h e t u b e .  Repeat t h e r e s i n t r e a t m e n t , t h i s t i m e u s i n g 2 A d j u s t t h e pH t o 7.8  w i t h NaOH (,6N,  ,06N,  1 ml washes.  ,006N). The  pH  changes v e r y r a p i d l y above pH 3. A d j u s t t h e volume o f t h e 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 Assay t h e ATP  Immediately or s t o r e a t -20°.  M, pH  7.8).  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 o f t h e 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 t y p e . The p u r i f i c a t i o n s t e p i n c r e a s e d r e c o v e r y o f ATP  ten-fold.  81 A p p e n d i x I I . Method f o r e x t r a c t i o n o f t o t a l ATP from l y o p h l l l z e d sediments. Add 3 ml o f I c e - c o l d b r o m o s u c c i n l m i d e e x t r a c t i o n r e a g e n t (.01  M n-bromosuccinimide,  .01 M EDTA, .02 M Na2HAs0jj. ,pH 7.4)  t o 20 mg o f 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 t u b e . Keep on I c e f o r 25 min w i t h o c c a s i o n a l m i x i n g , t h e n c e n t r i f u g e  (5000g/  5 m i n ) . Add 2 ml o f i c e - c o l d water t o 1 ml o f t h e s u p e r n a t a n t , and a d j u s t pH t o 7 . 8 w i t h I N H C l . A d j u s t f i n a l volume t o 5 ml w i t h T r i s b u f f e r (.02M, pH 7 . 8 ) and a s s a y Immediately o r s t o r e a t -20°. No d i l u t i o n i s n e c e s s a r y p r i o r t o a s s a y . S e v e r a l r e a g e n t s were t e s t e d b e f o r e  n-bromosuccinimide  was chosen f o r t h i s p r o c e d u r e ( T a b l e A 1). H2SO4 and a c i d i c DMSO w i t h EDTA added a r e a l m o s t a s e f f i c i e n t a s t h i s r e a g e n t . The amount o f sediment e x t r a c t e d (20 mg) c a n be a c c u r a t e l y weighed, b u t does not c o n t a i n h i g h enough c o n c e n t r a t i o n s o f c a t i o n s t o i n t e r f e r e w i t h t h e a s s a y a s s u g g e s t e d by t h e f a c t t h a t c a t i o n exchange t r e a t m e n t o f ^SO/j. e x t r a c t s Jaad l i t t l e e f f e c t , and t h e r e c o v e r y p e r u n i t w e i g h t o f sediment was r e d u c e d when a sediment weight o f 100 mg was used.  T a b l e A l . Methods t e s t e d f o r e x t r a c t i o n o f 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. o f s e d i m e n t ) . reagent  reference  Tris buffer  (.02 M, pH 7.8)  Holm-Hansen and Booth  Na HC0 (.1M)  B a n c r o f t e_t a l ^ (1974)  HC10 (.6N)  Lee e t a l ^ (1971a)  3  4  DMSO ( n e u t r a l -90# i n .05 M T r i s buffer)  Lee e t a l . (1971a)  DMSO(acidic  Lee e t a l ^ . (1971a)  -90# i n O i l N H2SO4)  EDTA(.01 M) (1966)  MacLeod e t a l ^ (1969)  H3PO4 (.6N)  B. K. B u r n i s o n ( u n p u b l l s h e d )  H S 0 ^ (.6N)  Lee e t a l . (1971a)  + c a t i o n exchange t r e a t m e n t 3 1 Values not corrected 2  jl  1.49 +  ND  o  2  6.05  Bromesuccinimide (see t e x t )  2  *  ATP (/Jg/g oven dry wt) 0.55 0.56 3.75  +  3.40 5.34  +  10.24  +  5.05 10.70 6.05 7.54 1.13 8.22  for extraction efficiency  ND = n o t d e t e c t a b l e  3 t h i s was t h e o n l y sample r e c e i v i n g c a t i o n exchange t r e a t m e n t  00  83 Appendix I I I . Method f o r e x t r a c t i o n o f 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 o f f r e s h sediment w i t h 6 ml f i l t e r -  steril-  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. d e t e r m i n a t i o n . P l a c e 0.35 ml i n a spot d i s h and add 0.5 ml l a k e w a t e r . 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 b l a c k sample s t a g e . Remove a l l v i s i b l e a n i m a l s and a l g a e w i t h m i c r o p i p e t t e s c o n t r o l l e d via  r u b b e r t u b i n g by t h e 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 . T h i s p r o c e s s t a k e s 20 - 40 min p e r sample. T r a n s f e r t h e r e s i d u e t o a c e n t r i f u g e tube i n a n i c e b a t h , a d j u s t i n g t h e volume t o 1 ml w i t h w a t e r . Add 1 ml i c e c o l d O.6NJH2SO14. and mix. A f t e r a d j u s t supernatant  10 min, c e n t r i f u g e (5000g/5min)  t o pH 7.8 w i t h Na OH (.6,  add T r i s b u f f e r (.02M, pH 7.8) -20° o r a s s a y i m m e d i a t e l y .  .06,  .006N) and  t o 5 ml. Store the extract a t  Recovery I s n o t improved by d i l u t i o n  of t h e sample. V a r i a n c e between r e p l i c a t e s t e n d s t o be l a r g e , b u t t h e sample s i z e i s l i m i t e d . b y t h e time r e q u i r e d t o r u n t h e extraction.  Appendix I V . Method f o r ATP  84  assay.  P r e p a r a t i o n of s t a n d a r d s o l u t i o n s P r e p a r e s t a n d a r d s o l u t i o n s of ATP  (lmg/ml, Sigma C h e m i c a l o  Co.) and s t o r e i n 0.2  ml a l l q u o t s a t -20  . These a r e s t a b l e  f o r a t l e a s t 7 months. F o r t h e a s s a y p r e p a r e 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 a r e 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 Co., FLE -50) .03  mg o f l y o p h i l i z e d enzyme (Sigma  i n 5 ml f r e s h , c o l d .1 M Na HAs0i|., 2  M mercaptoethanol  (pH 7.4)  Chemical  .04 M MgSO/j,,  i n a c h i l l e d t i s s u e homogen-  i z i n g t u b e . R i n s e t h e homogenizer w i t h 5 ml o f t h e b u f f e r .  2°,  S t o r e the e x t r a c t s o v e r n i g h t a t m i n ) . Add 1000  ml f r e s h .01  mercaptoethanol,  ,5#  (5000g/10  then centrifuge  M Na HAsOij., .004 M MgSO^, .03 2  b o v i n e serum a l b u m i n (Sigma A  (pH7.4). E q u i l i b r a t e a t 2° f o r  M  4503)  1 h p r i o r t o u s e . The  enzyme  shows l i t t l e d e t e r i o r a t i o n of r e s p o n s e even 6 h a f t e r  initial  use i f k e p t on I c e . T h i s p r e p a r a t i o n method, d e v i s e d by Hammerstedt  (1973),  was n o t u s e d d u r i n g t h e e a r l y s t a g e s o f t h i s s t u d y . T t i s s u p e r i o r t o o t h e r methods i n which a c t i v i t y d e c r e a s e d over time so samples measured a t d i f f e r e n t t i m e s were not d i r e c t l y comparable. Assay  procedure S e t up the c o u n t i n g a p p a r a t u s as r e q u i r e d . I n t h i s s t u d y  a l i q u i d s c i n t i l l a t i o n c o u n t e r was used ( N u c l e a r Chicago o  Mark I or U n i l u x I I ) a t 4 , Windows were wide ppen (00 with attenuation at H 3  was  eliminated.  s e t t i n g s (A 200).  -  99)  Coincidence c i r c u i t r y  85 Add  1 ml o f .04  H glycylglyclne,  to a s c i n t i l l a t i o n v i a l  .003  M MgSO^ (pH  7.4)  (washed i n a c i d and d i s t i l l e d w a t e r ) .  Then add 1 ml o f the e q u i l i b r a t e d enzyme m i x t u r e . Lower the v i a l i n t o the c o u n t i n g chamber and w a i t 5 sec t o reduce phosphorescence. Count the background f o r 1 min. F i v e seconds a f t e r c o m p l e t i o n of the background c o u n t , add 1 ml o f unknown or s t a n d a r d ATP  sample ( a u t o m a t i c p i p e t t e s g i v i n g q u i c k  d e l i v e r y a r e advantageous), and lower the v i a l i n t o the c o u n t i n g chamber. Twenty seconds a f t e r c o m p l e t i o n o f the back© ground count, i n i t i a t e a 1 minute g r o s s c o u n t .  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 v e r s u s ATP  c o n c e n t r a t i o n and r e a d unknown  c o n c e n t r a t i o n s from t h i s c u r v e . 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 ( F i g u r e A  1).  86  F i g u r e A I . T y p i c a l s t a n d a r d curve f o r ATP (May 18,  1974).  assay  Net a c t i v i t y  (cpm x 10" )  87  A p p e n d i x V. Recovery o f ATP added t o s e d i m e n t s . The e f f i c i e n c i e s o f the e x t r a c t i o n p r o c e d u r e s were d e t e r m i n e d i n a s e r i e s o f e x p e r i m e n t s i n w h i c h pure c u l t u r e s of organisms were added t o sediment samples. Measurement o f ATP i n t h e pure c u l t u r e s ( u s i n g 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 p e r m i t s s o l u t i o n o f the f o l l o w i n g r e l a t i o n ship! efficiency «  ATP(sediment + o r g a n i s m s ) - ATP ATP organisms  sediment  The r e s u l t s u s i n g a number o f s p e c i e s f o r t h e t h r e e e x t r a c t i o n methods a r e shown i n t a b l e A I I , The  isolates  from M a r i o n Lake were t h e most abundant organisms o b t a i n e d on b r a i n h e a r t i n f u s i o n agar p l a t e s I n two p l a t e count s e r i e s . I s o l a t e s 73-1 and 74-2  and 74-1  a r e s m a l l Gram n e g a t i v e r o d s  i s a l a r g e Gram n e g a t i v e r o d .  88  T a b l e A l l . E f f i c i e n c y o f t h e 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  1  HgSO/^ " t o t a l " f r e s h sediment)  organism  efficiency  I s o l a t e 73-1 .091 73- 1 .129 , 74- 1 .052 | .128 74-2 .209 Bacillus subtllls ]l6l O s c i l l a t o r l a spp. .102 g Anacystls nldulans.129 t l l  H2S0i(, " m i c r o " ( f r e s h sediment)  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  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  «-205  .125  89 A p p e n d i x 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 o f o r g a n i c c a r b o n a s s o c i a t e d w i t h l i v i n g organisms t o ATP i s v a r i a b l e , b u t a n average o f s u g g e s t e d ( H a m i l t o n and Holm-Hansen,  250il  has been  196?). T h i s r a t i o was  d e t e r m i n e d f o r a number o f organisms i n t h i s s t u d y ( T a b l e 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 , t h e a c t i n o m y c e t e i n a medium r e p o r t e d by R o d i n a  (1972,  p.  373).  and t h e b l u e - g r e e n b a c t e r i u m inBG-11  (Stanier  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 h a r v e s t e d w h i l e t h e b l u e - g r e e n c u l t u r e was a p p r o x i m a t e l y t h r e e weeks o l d and appeared h e a l t h y . C e l l s were c o n c e n t r a t e d 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  with water. ATP was e x t r a c t e d from 1 ml o f t h e s u s p e n s i o n u s i n g t h e method d e s c r i b e d 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 ) . O r g a n i c c a r b o n was measured i n a c a r b o n a n a l y z e r (Beckman Model 915)•  V i a b l e c o u n t s were d e t e r m i n e d  on a g a r p l a t e s o f t h e same medium as t h e c u l t u r e s were grown in. The mean C/ATP r a t i o o f 28? ( T a b l e A I I I ) was preted as a c o n f i r m a t i o n of the popular value of  inter250.  90  Table AIII. The relationship between ATP, biomass carbon and c e l l numbers. ATP/cell (>ug x 10°)  organism i s o l a t e 74-1 74-2 Bacillus  subtllls  2.35 4.62  112 292  14.90  439  376  Streptomyces spp. Anacystls nldulans  biomass carbon/ATP  7.33  218  287  91  Appendix V I I . The  e f f e c t o f i n c u b a t i o n time and oxygen on  dehydrogenase a c t i v i t y measurements. I n c u 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 r e s p o n s e i s  shown i n f i g u r e A 2. T h i s experiment shows t h a t a c t i v i t y , measured as formazan p r o d u c t i o n , i s r a p i d d u r i n g the  first  few h o u r s , t h e n 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 t h a n 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 t o p r e v i o u s s t u d i e s ( P a t t e r s o n e t a l . . 1970;  S o r o k i n and K a d o t a , 1972)  24 h i n c u b a t i o n was  Four hour samples were r u n s i m u l t a n e o u s l y t o t e s t changes. The  used. sensitivity  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 a t t h e two i n c u b a t i o n p e r i o d s a r e 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 r e a t e s t u s i n g the s h o r t e r  i n c u b a t i o n time - t h e maximum d i f f e r e n c e was 456 produced/m .h (Sep. 1973  - A p r . 1974)  pared t o 168  - A p r . 1, 1974)  2  (June, 1973  mg  formazan  f o r 4 h i n c u b a t i o n comf o r 24 h I n c u b a t i o n .  Oxygen P r i o r t o I n c u b a t i o n , 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 m i n u t e s . The r a t i o n a l e f o r t h i s i s t h a t 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 -  p o r t system and v a r i a b l e oxygen c o n c e n t r a t i o n s 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 o m p e t i t i o n not o n l y makes a c t i v i t y measurements more cons i s t e n t , but a l s o h i g h e r (Table A I V ) . The  d a t a 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  F i g u r e A 2. E f f e c t o f I n c u b a t i o n time on dehydrogenase activity  measurements.  92  a  •5-0-  cd to U  o >>  -p  o as  » ^to 4)  2.5-  A  to-a  o a> uo •d 3 X O 0) U a a  0 0  4  8  12 Time (h)  16  20  24  93  Table AIV.  The effect of aerobic versus anaerobic conditions on dehydrogenase a c t i v i t y assayed at  different  depths i n the sediment (March 14, 1973). sediment horizon (cm) 0-  1-  1  2  2 --3  Incubation condition  formazan production (ps/g dry wt./h)  aerobic  20  anaerobic  103  aerobic  15  anaerobic  108  aerobic  44  anaerobic  88  94 a e r o b i c v a l u e from 2 - 3 cm d e p t h i s p r o b a b l y h i g h r e l a t i v e t o the a n a e r o b i c v a l u e because o f t h e normal absence o f 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 o f Lenhard ( 1 9 6 8 ) t h a t r e m o v a l o f oxygen from a n a e r o b i c s l u d g e had l i t t l e e f f e c t on dehydrogenase  activity.  95  A p p e n d i x 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 p a r a m e t e r s . TheoryMany 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 1965.  parameters i n water samples have been p u b l i s h e d s i n c e The  f o l l o w i n g i s a n o u t l i n e of a procedure d e v e l o p e d by  K l e i b e r (1972) f o r d i l u t e d sediment samples. Turnover t i m e , T, i s r e l a t e d t o s u b s t r a t e S,  and u p t a k e r a t e , V, by t h e T «=  formulat El  S_  V  I f t h e n a t u r a l t u r n o v e r time and T  n  concentration,  substrate  concentration,  and S , a r e known t h e n the r e a l r a t e of u p t a k e , U , N  may  N  be c a l c u l a t e d : U  S The  N  E2  «  N  can be measured i n d e p e n d e n t l y  experimental  of u p t a k e e x p e r i m e n t s .  d e s i g n of the e x p e r i m e n t s does not p e r m i t  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 . I t i s i n s t e a d t h e added r a d i o a c t i v e s u b s t r a t e N  c o n c e n t r a t i o n ( S ) , p l u s the q u a n t i t y n a t u r a l l y i n the A  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  08  s  a +  E3  SJI  d  T i s a f u n c t i o n of S , so d e t e r m i n a t i o n  of T  performed a t S . I n n o n - k i n e t i c e x p e r i m e n t s , f o r N  using ^^-bicarbonate  n  must be Instance  t o measure p r i m a r y p r o d u c t i o n i n water  samples, t h i s i s not a problem i f S by t h e added s u b s t r a t e . The  N  i s not g r e a t l y changed  sediment s t u d i e s r e q u i r e d i l u t i o n  o f the samples,however. E x t e r n a l s u b s t r a t e i s t h e n added a t i n c r e m e n t a l 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 , undiluted substrate l e v e l . I n each experiment, time, T , e  f o r each S .  where R  a  turnover  T h i s i s c a l c u l a t e d byt  a  T  there i s an estimated  = Rn  p  t  E4  i s r a d i o a c t i v i t y (dpm)  added t o t h e sample,  R  u  i s t h e g r o s s uptake of r a d i o a c t i v i t y and t i s t h e i n c u b a t i o n time. These v a l u e s o f T about S ( i e , S  can be measured w i t h o u t i n f o r m a t i o n  e  o r S ) , but a r e not independent o f S.  a  n  v a r i e t y of l i n e a r t r a n s f o r m a t i o n s ( H a l l e t a l . . 1972) hyperbolic Michaelis-Menten l i n e a r f u n c t i o n o f S. The  f u n c t i o n assume t h a t T  e  The o f the  is a  t r a n s f o r m a t i o n used I n t h i s  study  has the formi T  e  • Kt-. + Sn  + 1  S  E5  a  where Kt i s t h e t r a n s p o r t a c o n s t a n t and V uptake r a t e . By p l o t t i n g T  e  Burke p l o t ) e s t i m a t e s of V ,  versus S  a  M  i s t h e maximum  (modified Lineweaver-  (Kt + SLQ) and T , t h e t u r n o v e r d time f o r S «= o o r S«=S (E3) may be o b t a i n e d ( F i g u r e A3). d M  a  0  W  The work o f W i l l i a m s (1973) and B u r n i s o n and  Morita  (1973) d i s c u s s e d 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 h e r e . Estimates of V accuracy T  0  M  and  ( K t + Sn) a r e b o t h dependent f o r t h e i r d  on the v a l i d i t y o f the a s s u m p t i o n o f  linearity.  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. D e t e r m i n a t i o n of T , n  the t u r n o v e r time a t S , n  is  97  F i g u r e A 3. G r a p h i c 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 m o d i f i e d Lineweaver-Burk p l o t .  the  9?  a  98  a c c o m p l i s h e d by d e t e r m i n i n g t h e v a l u e o f S  a  which  corresponds  t o S , T h i s v a l u e 1st n  S  = S  a  n  E6  - Sj2 d *  From t h e T  versus S | > l d t , T , t h e turnover time corresponding  e  a  e  t o S , may be d e t e r m i n e d . D i v i s i o n o f t h i s q u a n t i t y by d a  a c c o u n t s f o r d i l u t i o n o f sediment b a c t e r i a i n t h e experiment so» Tr, = TjL  d  E7  .  T  n  c a l c u l a t e d i n t h i s manner, and t h e independent  S  n  a r e plugged i n t o E 2 t o g i v e U .  value of  n  Data p r o c e s s i n g The e x p e r i m e n t a l d a t a c o n s i s t e d o f t h e d r y weight o f sediment p e r f l a s k , t h e i n c u b a t i o n t i m e , t h e added ^ C g l u c o s e (Ra = cpm ijmg),  t h e 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 f o u r values of S . I n t e r s t i t i a l glucose c o n c e n t r a t i o n s a  were determined of  i n d e p e n d e n t l y b u t on t h e same mixed b a t c h  sediment. Gross uptake  ( R ) was c a l c u l a t e d a s t h e sum o f t h e u  a v e r a g e , n e t , 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, c o n v e r t e d 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 o f S  and a l i n e a r r e g r e s s i o n e q u a t i o n determined stituting S  a  • S  n  S /50,  -  n  where  into the regression equation, T c u l a t e d by E? and D  n  e  50 was  a  (jug/l)  (E5). By sub-  the d i l u t i o n factor,  was o b t a i n e d . T  n  was c a l -  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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