Microb Ecol (1987) 13:13-29

MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1987

Bacterial Growth on Dissolved Organic Carbon from a Blackwater River Judy L. Meyer, Richard T. Edwards, and Rebecca Risley Department of Zoologyand Institute of Ecology,University of Georgia, Athens, Georgia 30602, USA

Abstract. Different nominal molecular weight (nMW) fractions of D O C from a southeastern blackwater river were concentrated by ultrafiltration and added to sieved river water to assess each fraction's ability to stimulate bacterial growth. Bacterial growth was measured using change in bacterial biomass from direct counts and using 3H-thymidine incorporated into DNA. Bacterial growth and amount of DOC used was greatest in the low MW enrichment (< 1,000 nMW) and least in the intermediate MW enrichment (1,000-10,000 nMW). The high MW fraction (> 10,000 nMW) supported more growth than did the intermediate MW fraction, apparently because o f lower MW compounds complexed with a high MW refractory core. The low M W fraction of DOC from a clearwater mountain stream, a boreal blackwater river, and leachate from water oak and willow leaves also stimulated more bacterial growth than did other fractions. However, the high MW DOC from these other sources was not as biologically available as high MW DOC from a blackwater river. Bacteria converted blackwater river DOC to bacterial biomass with an efficiency o f 31%. Bacteria produced at the expense o f abundant riverine DOC provide a t r o p h i c resource for protozoa and higher levels of the microbial food web of a blackwater river.

Introduction Dissolved organic carbon (DOC) is an abundant organic carbon source in natural waters (e.g., Ref. 47), yet it is incompletely characterized either with respect to its chemical composition (e.g., Ref. 44) or its availability to bacteria. Although there are numerous measures o f bacterial growth on specific compounds (e.g., Ref. 3), these measures are difficult to relate to bacterial growth in nature because single compounds are such a small fraction of total DOC present (e.g., Ref. 24). There have been few attempts to quantify bacterial growth on the heterogeneous mixture o f organics that comprise DOC in natural waters [9, 25]. The question o f which fractions o f the naturally occurring DOC are available to bacteria is of critical importance in evaluating the potential role o f microbial food webs in natural waters. In marine planktonic systems, the link between

14

J.L. Meyeret al.

algal-produced DOC and bacterial growth is clear (e.g., Ref. 4). In rivers such as the blackwater river we are studying, where primary productivity is low JR. T. Edwards (1985) The role ofseston bacteria in the metabolism and secondary production dynamics of southeastern blackwater rivers. PhD Thesis, University of Georgia] compared with allochthonous inputs [T. F. Cuffney (1984) Characteristics of riparian flooding and its impact upon the processing and exchange of organic matter in coastal plain streams of Georgia. PhD Thesis, University of Georgia], algal exudates probably support only a small fraction of observed bacterial production [18]. In these systems bacterial growth would more likely be at the expense of DOC leached from the surrounding watershed. DOC leached from freshly fallen leaves is removed from the water by biotic processes in streams [11], and the lower molecular weight fractions (< 10,000 nominal molecular weight) appear the most available to bacteria [23]. However, it is inappropriate to base predictions about bacterial uptake of riverine DOC from studies of fresh leaf leachate. Much of the labile DOC in sources such as leachate appears rapidly removed, leaving the refractory compounds to be transported downstream [48]. Hence, in the sixth-order blackwater river we are studying, one might expect that much of the labile DOC had already been removed. Blackwater rivers characteristically have tea-colored waters due to the relatively high concentrations of humic substances leached from the sandy soils of their watershed [39]. In our studies of the Ogeechee River, a sixth-order blackwater fiver system in the southeastern United States, we have found high DOC concentrations (mean = 12.7 mg C/liter in the mainstream and 30.8 mg C/liter in a fourth-order tributary [28]. Although DOC concentration is commonly high in blackwater rivers, much of the DOC is humic substances that have been considered refractory to bacterial attack [6]. However, there is evidence that structurally complex aquatic humic substances of high molecular weight may stimulate microbial growth by increasing trace metal availability (e.g., Ref. 34) or by cometabolism [12, 13]. Humic substances in the soil, from which the aquatic material originates, have been demonstrated to stimulate bacterial growth (e.g., Ref. 45). Bacteria are abundant in the water colum of the Ogeechee River and Black Creek, with an annual average density of 1.5 x 10 ~o cells/liter [R. T. Edwards (1985) PhD Thesis, University of Georgia]. We are currently investigating possible sources of bacteria including suspension from the shifting sandy substrate [18], input from the extensive floodplain swamps, and water column production JR. T. Edwards (1985) PhD Thesis, University of Georgia]. Bacterial production in the Ogeechee River is high when water temperature is high. [R.T. Edwards (1985) PhD Thesis, University of Georgia]. Studies of this and other southeastern blackwater rivers have noted high secondary production rates for filter-feeding invertebrates [8, 38]. Since bacterial biomass produced at the expense of the abundant DOC is one possible energy source for the invertebrate filter-feeders in this river, we considered it important to examine the availability to bacteria of different apparent molecular weight fractions of DOC from the Ogeechee River and Black Creek, one of its tributaries. The results of those experiments are presented here.

Bacterial Growth on Blackwater DOC

15

Methods We analyzed the ability of DOC from two southeastern blackwater rivers to stimulate bacterial growth in ten enrichment experiments. In these experiments, the DOC from 1 to 1.5 liter of Ogeechee River or Black Creek water was separated into three different nominal molecular weight (nMW) fractions and concentrated to 50 ml by ultraflltration. We collected water from two similar fourth-order sites on Black Creek: just above its confluence with the Ogeechee River and several km upstream where Georgia Route 280 crosses it. Water samples were collected in March, May, July, and August 1983; February, April, May, June and July 1984; and January 1985. In May 1983 water from the sixth-order Ogeechee River was collected upstream of the point where Interstate 16 crosses the river, which is about 63 km from its mouth. These sites are more fully described elsewhere [28, 46]. To concentrate DOC, river water was first sterile-filtered twice through 0.22-#m Millipore filters (prerinsed with sterile water) to remove bacteria. It was then placed in a 200-ml stirred cell and filtered through an Amicon PM 10 membrane (62-mm diameter at < 10 psi of nitrogen) until 50 ml remained (> 10,000 nMW fraction). The filtrate was then filtered through an Amicon YM2 membrane ( 10,000 nMW

1,000-10,000 nMW

Ogeechee River or Black Creek Matamek River Hugh White Creek Water oak ieachate Willow leachate

1.38 + 0.59 (9) 0.19 (1) 8.03 (1) 3.58 (1) 0.78 (1)

5.85 + 2.12 (9) 0.19 (1) 13.6 (1) 2.47 (1) 1.07 (1)

2.81 + 0.65 (8) 0.58 (1) 23.3 (1) 3.69 (1) 1.28 (1)

< 1,000 nMW 14.54 _+ 4.69 2.22 35.3 16.4 2.43

(7) (1) (1) (1) (1)

"Values are mean + standard error (number of experiments) in units of nM thymidine/liter. Thymidine uptake was measured in water collected on all dates except April 1984

3000 25001 2000

A

0 / %'..

-'"

500[

~ I Z %

i~-.I ......

-.,1~."

"" "'..

0

B

,o z o

30 40

50 60

ro Bb

Fig. 1. Time course of thymidine uptake of bacteria in an experiment in which 4 #m-sieved (A) or GFC glass fiber-filtered (B) blackwater river water was enriched with high MW DOC ( . . . . . O . . . . . ), intermediate MW DOC ( - - - I I - - - ) , low MW DOC ( [] ), or unenriched ( - - ' - - 0 - - ' - - ) . Large populations of protozoa were present in A but not B. Values are the means of three determinations of DPM of ~H-TdR into DNA per hour per #g DOC initially present.

TIME(h)

w e i g h t f r a c t i o n s , it is m o r e a p p r o p r i a t e t o c o m p a r e b a c t e r i a l g r o w t h (in t h i s case thymidine incorporation) per unit DOC present. The time course of thymidine incorporation per unit DOC present over the p e r i o d o f t w o e x p e r i m e n t s is p l o t t e d in Fig. 1. T h e p l o t f o r m o s t e x p e r i m e n t s l o o k e d like t h a t in Fig. 1A , w i t h p e a k i n c o r p o r a t i o n o c c u r r i n g p r i o r t o t h e e n d o f t h e i n c u b a t i o n p e r i o d . W h e n p r o t o z o a n p r e d a t o r s w e r e e x c l u d e d (Fig. 1B), incorporation rates peaked at the end of the incubation period. Despite differe n c e s in p a t t e r n a n d a b s o l u t e a m o u n t o f t h y m i d i n e i n c o r p o r a t e d in t h e s e t w o e x p e r i m e n t s , b o t h s h o w e d g r e a t e s t i n c o r p o r a t i o n in t h e l o w M W e n r i c h m e n t , i n t e r m e d i a t e i n c o r p o r a t i o n in t h e h i g h M W e n r i c h m e n t , a n d l e a s t in t h e i n t e r m e d i a t e M W e n r i c h m e n t . I n o n e e x p e r i m e n t (Fig. 1B) t h e r e w a s l i t t l e d i f f e r e n c e b e t w e e n c o n t r o l a n d i n t e r m e d i a t e M W e n r i c h m e n t s , w h e r e a s in t h e o t h e r e x p e r i m e n t (Fig. 1A) t h e r e w a s less i n c o r p o r a t i o n in t h e i n t e r m e d i a t e MW enrichment. D a t a o n t h y m i d i n e i n c o r p o r a t i o n p e r u n i t D O C p r e s e n t is s u m m a r i z e d f o r all e x p e r i m e n t s in T a b l e 4. T h i s m e a s u r e o f g r o w t h w a s s i g n i f i c a n t l y g r e a t e r i n t h e l o w a n d h i g h M W e n r i c h m e n t s t h a n in t h e u n e n r i c h e d flasks. T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n u n e n r i c h e d flasks a n d t h o s e e n r i c h e d w i t h

20

J.L. Meyer et al.

Table 4. The influence of different DOC enrichments on bacterial growth measured as thymidine incorporated into DNA per g DOC present" Enriched

1,000-10,000

DOC source

Unenriched

> 10,000 nMW

nMW

< 1,000 nMW

Ogeechee River or Black Creek Matamek River Hugh White Creek Water oak leachate Willow leachate

82 - 29 (9) 26 (1) 1,830 (1) 112 (1) 27 (1)

210 + 65 (9) 24 (1) 2,280 (1) 42 (1) 32 (1)

68 + 24 (8) 30 (1) 2,020 (1) 76 (1) 34 (i)

316 + 98 (6) 235 (1) 5,090 (1) 447 (1) 79 (1)

Values are mean _4-standard error (number of experiments) in units ofnM TdR/g C. Thymidine uptake was measured in water collected on all dates except April 1984

-

2O

88

9

16

Iz

9

.~

g~

-6

-4

-2

UNLABELLED

2

4

6

8

I0

Fig. 2. Isotope dilution plot for bacterial incorporation of aH-TdR into DNA in flasks enriched for 29 hours with high MW DOC ( . . . . . 9 . . . . . ) (r 2 = 0.467), intermediate MW DOC ( - - - I I - - - ) (r 2 = 0.573), low MW DOC ( [] ) (r 2 = 0.585), or unenriched ( - - . - - e - - ---) (r= = 0.401). Each point represents one measurement, and the lines are leastsquares regression fit to the 16 points. The intersection of the regression line with the horizontal axis gives the effective pool size.

THYMIDINE ADDED (nM)

i n t e r m e d i a t e M W D O C . T h i s is the s a m e r e s u l t o b t a i n e d w i t h the o t h e r m e a sures o f b a c t e r i a l g r o w t h d e s c r i b e d a b o v e . T h e s e k i n d s o f c o m p a r i s o n s o f t h y m i d i n e i n c o r p o r a t i o n b e t w e e n flasks are a I : p r o p r i a t e o n l y i f i s o t o p e d i l u t i o n is the s a m e i n different flasks. I f the specific a c t i v i t y o f t h e t h y m i d i n e differed greatly b e t w e e n flasks b e c a u s e o f differences in a m o u n t s of unlabeled t h y m i d i n e present either internally or in the m e d i u m , t h e n o n e c o u l d n o t a c c u r a t e l y c o m p a r e rates o f l a b e l e d t h y m i d i n e i n c o r p o r a t i o n . W e c h e c k e d this a s s u m p t i o n o f e q u a l d i l u t i o n i n all flasks i n o n e e x p e r i m e n t (Fig. 2). A l t h o u g h t h e r e was s o m e v a r i a t i o n b e t w e e n flasks i n the d i l u t i o n plots, the differences i n e s t i m a t e s o f the size o f the u n l a b e l e d p o o l s were w i t h i n the e r r o r l i m i t s o f t h e m e a s u r e m e n t s . H e n c e we feel c o n f i d e n t i n c o m p a r i n g thym i d i n e i n c o r p o r a t i o n b e t w e e n flasks.

Changes in DOC. N o t o n l y d i d the a m o u n t o f b a c t e r i a l g r o w t h o b s e r v e d differ b e t w e e n e n r i c h m e n t s , the a m o u n t o f D O C u s e d also differed ( T a b l e 5). T o m a k e d a t a c o m p a r a b l e b e t w e e n e x p e r i m e n t s w i t h different a m o u n t s o f D O C a d d e d , we h a v e e x p r e s s e d D O C use as the p e r c e n t o f a d d e d DOC t h a t was r e m o v e d d u r i n g t h e c o u r s e o f the e x p e r i m e n t , c o r r e c t e d for D O C use i n t h e u n e n r i c h e d flasks. T h i s was c a l c u l a t e d as ( c h a n g e i n D O C i n e n r i c h e d flask -

21

Bacterial G r o w t h on Blackwater D O C T a b l e 5. Percentage o f a d d e d D O C u s e d d u r i n g the course o f a 72hour enrichment experiment ~ T y p e o f D O C used (%) D O C source Ogeechee R i v e r or Black Creek W a t e r oak leachate Willow leachate Matamek River H u g h W h i t e Creek

> 10,000 nMW

1,000-10,000 nMW

21 • 9 (6) 26 (l) 43 (1) 9 (1) 0 (l)

2 + 2 (6) 30 (1) 31 (1) i (l) 31 (l)

< 1,000 nMW 54 ___ 16 168 ~ 100 37 86

(4) (1) (1) (1) (1)

o Values are m e a n _+ s t a n d a r d error ( n u m b e r o f experiments) a n d were calculated as (&DOC enriched - A D O C unenriehed)/(initial D O C enriched - initial D O C unenriched). W a t e r for these experim e n t s c a m e from the Ogeechee in M a y 1983 a n d from Black Creek in M a r c h , July, a n d A u g u s t 1983, F e b r u a r y 1984 a n d J a n u a r y 1985 ~' T h i s is n o t an artifact. It merely m e a n s that s o m e o f the D O C present prior to e n r i c h m e n t was utilized w h e n the low M W fraction was a d d e d b u t n o t used in the u n e n r i c h e d flask. C o m e t a b o l i s m [ 13] would be one possible explanation for this o b s e r v a t i o n

T a b l e 6 Nitrogen c o n t e n t o f flasks enriched with different types o f D O C D a t a are presented as ~ M total N present a n d as m o l a r N / C ratios a Enriched D O C source

Unenriched

>10,000 nMW

1,000-10,000 n M W

_-1 mg C/liter ~'Data from 4 unenriched flasks, and 4, 2, and 5 flasks enriched with high, intermediate, and low MW DOC, respectively " Data from $ unenriched flasks, and 5, 2, and 4 flasks enriched with high, intermediate, and low MW DOC, respectively d Data from one iatermediate and one low MW enrichment , Data from one high, intermediate, and low MW enrichment

DOC from Other Sources The experiments using D O C fractions isolated from sources other than southe a s t e r n b l a c k w a t e r r i v e r s were n o t r e p l i c a t e d . H e n c e a d e t a i l e d c o m p a r i s o n o f b a c t e r i a l g r o w t h i n each size f r a c t i o n is n o t a p p r o p r i a t e s i n c e a statistical p r o b a b i l i t y c a n n o t be a s s i g n e d to differences b e t w e e n t r e a t m e n t s . H o w e v e r , t w o g e n e r a l t r e n d s are a p p a r e n t i n t h e s e d a t a . T h e first is t h a t D O C u p t a k e ( T a b l e 5) a n d b a c t e r i a l g r o w t h ( T a b l e s 1 - 4 ) were greatest i n the l o w M W e n r i c h m e n t . T h i s is the s a m e p a t t e r n o b s e r v e d w i t h Black C r e e k a n d O g e e c h e e R i v e r water. T h e s e c o n d t r e n d c o n t r a s t s w i t h the r e s u l t s o b t a i n e d w i t h s o u t h e a s t e r n b l a c k w a t e r r i v e r D O C : high M W D O C f r o m o t h e r s o u r c e s d i d n o t s t i m u l a t e b a c t e r i a l g r o w t h ( T a b l e 1-4), a n d i n s o m e cases (e.g., M a t a m e k R i v e r , T a b l e s 1-2; w a t e r o a k leachate, T a b l e s 2 - 4 ) a p p e a r e d to r e t a r d it.

Bacterial Conversion Efficiency T h e s e e x p e r i m e n t s were n o t expressly d e s i g n e d to m e a s u r e the efficiency w i t h w h i c h b a c t e r i a c o n v e r t e d n a t u r a l l y o c c u r r i n g D O C to b a c t e r i a l b i o m a s s ; h o w ever, we h a v e m e a s u r e d b o t h b a c t e r i a l g r o w t h a n d D O C use so we c a n c a l c u l a t e c o n v e r s i o n efficiency. T h e o n e diff• i n this is t h a t o u r m e a s u r e o f D O C use was n o t p a r t i c u l a r l y s e n s i t i v e . B e c a u s e we w e r e u s i n g w a t e r f r o m b l a c k w a t e r rivers, i n i t i a l D O C c o n t e n t was q u i t e high ( m e a n = 26.5 m g C / l i t e r i n u n e n r i c h e d flasks). H e n c e , we were t r y i n g to d e t e c t s m a l l c h a n g e s i n D O C a g a i n s t a r e l a t i v e l y h i g h b a c k g r o u n d c o n c e n t r a t i o n . T o try to m i n i m i z e e r r o r s d u e to i n a c c u r a c i e s i n D O C m e a s u r e m e n t , w e h a v e o n l y u s e d d a t a f r o m flasks w h e r e the c h a n g e i n D O C was at least 1 m g C / l i t e r for c a l c u l a t i n g c o n v e r s i o n effi-

Bacterial Growth on BlackwaterDOC

25

ciencies. We calculated efficiency based on both biomass changes and on thymidine incorporation (Table 8). Both measures of efficiency are subject to error and are sensitive to the conversion factors used. They give us rather different measures o f conversion efficiency. The efficiencies calculated from measured thymidine incorporation are probably most accurate since efficiencies calculated from changes in cell num be r are low because o f extensive protozoan grazing.

Discussion These experiments had one consistent result regardless of the bacterial growth measure used: low MW D O C from southeastern blackwater rivers was most available to bacteria, intermediate MW DOC was least available, and high MW DOC was intermediate in availability. When DOC was concentrated from other rivers or leaf leachate, the low M W fraction was also most available; however, the high MW fraction was generally less available than the intermediate MW fraction. Therefore, we conclude that high MW DOC from these blackwater rivers was more available to bacteria than was high MW DOC from a cIearwater mountain stream, a boreal blackwater river, or fresh leachate of oak or willow leaves. High MW DOC leached from leaves o f riparian bushes was also found to be unavailable to bacteria in a piedmont stream [23]. The molecular weight separations that form the basis o f these studies of differential availability were obtained by ultrafiltration. Although this is a commonly used procedure (e.g., Ref. 43), the molecular weights obtained are relative and are influenced by the nature o f the substances being filtered, particularly the charges they bear [ 1]. Although the PM 10 membrane we used to concentrate high MW DOC performed appropriately when used with an aquatic fulvic acid from a southeastern blackwater river [ 1], it is important to recognize that these membranes are not absolute filters. Complex molecules of the same molecular weight may be either retained or may pass through the membrane, depending on a variety o f factors such as their charge distribution [1]. Hence, the low MW concentrates used in these experiments may have been contaminated by higher MW compounds that passed through the membranes, and the intermediate MW concentrates may have had some contamination from lower MW compounds that were retained. However, this does not compromise our major conclusions: if high MW contaminants had not been present, bacterial growth on low MW compounds would have been even higher; if low MW contaminants had not been present, bacterial growth on intermediate MW DOC would have been even less. The results o f the hydrolysis experiments suggest that bacterial growth on the high MW DOC was at the expense of lower MW compounds complexed with a more refractory high M W core. In humic-stained waters of an Oregon river, 60% o f sugars [42] and 96% o f amino acids [26] were associated with the high MW humic acid fraction. Some of the high MW DOC in our experiments may also have been low M W D O C that had formed complexes with metals; the metal complexes would then be separated as high M W DOC by our ultrafiltration procedure [29]. Another possibility is that during the con-

26

J.L. Meyer et al.

centration procedure aggregates were f o r m e d that were too large to pass through the ultrafiltration m e m b r a n e [ 1]. Regardless o f the m e c h a n i s m by which complexes were f o r m e d in D O C f r o m southeastern blackwater rivers, bacterial exoenzymes were able to cleave t h e m and to use the low M W c o m p o u n d s released. T h e same p h e n o m e n o n was not observed in D O C from a boreal blackwater river, a clearwater m o u n t a i n stream, or fresh leaf leachate. Either the complexes were not f o r m e d or they were m o r e resistant to bacterial attack. Others have observed evidence o f bacterial growth at the expense o f D O C from humic-stained waters [13, 36, 37, 40, 41], and in some cases bacterial biomass was correlated with h u m i c or fulvic acid content o f the water [ 13, 21 ]. In most e n v i r o n m e n t s low M W D O C appears the most susceptible to bacterial attack (e.g., Refs. 23 and 33; but see Ref. 20), as we observed in these blackwater rivers. Intermediate M W D O C from a softwater lake was considered to be m o r e refractory than high or low M W D O C [2], although the criterion used was degree o f p h o t o l y s i s when exposed to U V light rather than a direct measure o f its availability to bacteria as we have used here. We are interpreting the observed pattern o f growth stimulation to be a consequence o f differential availability o f the organic m a t t e r as a growth substrate, since clear differences in D O C r e m o v a l were observed between flasks. H o w e v e r , we cannot rule out the possibility that increased growth was a consequence o f trace metal chelation by the added organics, although this has been observed previously with algae rather than bacteria [34]. T h e work with algae also showed that the lowest molecular weight fraction was m o s t stimulatory [34]. It is less likely that the pattern was a consequence o f differential binding o f toxic metals by the added organic m a t t e r because D O C concentrations prior to e n r i c h m e n t were already high (mean _ standard deviation = 26.9 __+ 12.7 mg C/liter, n = 12). At these D O C concentrations there would be a few free ions o f potentially toxic metals such as copper or a l u m i n u m , and adding m o r e organic m a t t e r would have a m i n o r effect on their toxicity. Although the M W fractions had different concentrations o f cations associated with them, the observed differences in bacterial activity could not be explained by variation in cation content. O n the other hand, differences in nitrogen and especially nitrogen/carbon ratios between treatments were related to observed differences in bacterial growth (Table 6). T h e low M W fraction clearly had the most nitrogen-rich carbon and supported the greatest bacterial growth. The nitrogen content o f the high M W D O C was s o m e w h a t greater than that o f the intermediate M W fraction, and the high M W D O C supported m o r e bacterial growth. D O C from a sixth-order sampling site on the Ogeechee R i v e r is primarily o f intermediate MW: 25% is high MW, 61% is intermediate M W , and 17% is low M W [28]. Differential bacterial uptake is one o f several possible mechanisms causing this distribution. Data presented here have shown greatest uptake o f low M W D O C and least uptake o f the intermediate M W fraction, and hence that intermediate M W material could accumulate in the water. Bacterial activity has been implicated as the cause o f observed molecular weight distributions in other aquatic ecosystems [13, 19]. O v e r a 72-hour period about half the low M W D O C was utilized by bacteria (Table 5). H a d bacterial populations not been so heavily grazed by flagellates,

Bacterial Growth on Blackwater DOC

27

D O C use m i g h t h a v e been greater. Although low M W D O C is a small fraction o f total D O C in this river, its average concentration is 2 m g C/liter in the Ogeechee R i v e r and 4 m g C/liter in Black Creek; those c o n c e n t r a t i o n s are greater t h a n total D O C in m a n y clearwater s t r e a m s (e.g., Ref. 27). T h e r e is, then, a sizeable stock o f D O C available for c o n v e r s i o n to bacterial b i o m a s s . This c o n v e r s i o n is not particularly efficient. T h e 30% efficiency r e p o r t e d here for riverine D O C is considerably less t h a n the 5 0 - 8 0 % efficiencies generally recorded for bacteria growing on n o n r e f r a c t o r y dissolved substrates [49], although 2 0 - 3 0 % efficiency for m a r i n e bacteria growing on p h y t o p l a n k t o n detritus has also been o b s e r v e d [5]. It is also less t h a n the 53% efficiency n o t e d for bacterial growth on leachate f r o m Ogeechee R i v e r m a c r o p h y t e s [17], but far greater t h a n the 0.02% e s t i m a t e d for benthic bacteria m e t a b o l i z i n g D O C f r o m a clearwater p i e d m o n t s t r e a m [9]. F u r t h e r m e a s u r e s o f c o n v e r s i o n efficiencies for bacteria growing on c o m p l e x natural substrates are needed before we will be able to reliably m o d e l the c o n v e r s i o n f r o m D O C to bacteria in riverine ecosystems. T h e bacterial b i o m a s s p r o d u c e d f r o m D O C in our e x p e r i m e n t s was cons u m e d by flagellated protozoa, the next step in the " m i c r o b i a l l o o p " (sensu 4). Significant p r o t o z o a n growth has b e e n o b s e r v e d in o t h e r e x p e r i m e n t s where bacteria were grown on natural D O C [ 17, 35]. Similar grazing p r o b a b l y h a p p e n s in the Ogeechee R i v e r where p r o t o z o a n densities are high: annual average a b u n d a n c e is 1.6 x 103 flagellates/ml a n d 21 ciliates/ml (Leslie Carlough, U n i versity o f Georgia, personal c o m m u n i c a t i o n ) . In the river, bacteria m a y also be c o n s u m e d by a b u n d a n t filter-feeding invertebrates such as the net-spinning caddisfly Macronema a n d black f i e s (Sirnutium spp.), which are capable o f capturing bacteria-sized particles [50] as well as protozoa. T h e links between these benthic invertebrates a n d higher trophic levels such as fish are well docu m e n t e d (e.g., Ref. 7). W h e t h e r bacterial b i o m a s s is p r o d u c e d at the expense o f riverine D O C as we o b s e r v e d in these e x p e r i m e n t s or is washed in f r o m the floodplain JR. T. Edwards (1985) P h D Thesis, U n i v e r s i t y o f Georgia], the microbial loop a p p e a r s an i m p o r t a n t c o m p o n e n t o f the trophic d y n a m i c s o f the water c o l u m n in this blackwater river.

Acknowledgments. We thank Drs. R. J. Naiman and J. B. Wallace for providing water from the Matamek River for our experiment. This research was supported by NSF grants DEB-8104427 and BSR-8406631.

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