Microb Ecol (1983) 9:41-55

IYIICROBI,4I ECOLOGY 9 1983 Springer-Verlag

Heterotrophic Bacterial Guild Structure: Relationship to Biodegradative Populations Lawrence M. Mallory* and Gary S. Sayler Department of Microbiologyand the Graduate Program in Ecology,The Universityof Tennessee, Knoxville, Tennessee 37916 USA Abstract. Numerical taxonomic analysis o f a freshwater bacterial guild demonstrated that the bacteria capable of growth on phenanthrene and polychlorinated biphenyl media were representative o f the taxa obtained from low nutrient oligotrophic media. The diversity of heterotrophic bacteria and members o f new taxa recovered from the guild followed a poisson distribution relative to the number of isolation media used. Moderately high nutrient, yeast extract peptone and glucose agar was found to be the most selective isolation medium relative to the total number of taxa recovered whereas low nutrient, lake water agar was the least selective medium used. Carbon source utilization patterns of the isolated taxa indicated that taxa within the guild had broad niche ranges and could potentially occupy many niches within a dynamic environment. The structure of the bacterial guild was dominated by mesophilic oligotrophs. The results o f this investigation demonstrate that potential biodegradative populations are representative o f the diverse taxa found in uncontaminated freshwater environments.

Introduction An aquatic microbial community is composed of an assemblage of highly diverse populations of eucaryotic and procaryotic microorganisms. The elucidation and definition of the structure o f the bacterial component of communities within estuarine and oceanic ecosystems has been the focus of numerous investigations [5, 15, 22, 25]. Such studies have demonstrated a wide diversity ofphototrophic, chemolithotrophic, and heterotrophic microbial populations in the water column of aquatic ecosystems. A fundamental role of the heterotrophic bacterial populations within an aquatic ecosystem is the transformation o f energy and the cycling of matter. The efficiency of these transformations relates directly to niche specialization by the microorganisms. In recent years, considerable interest has arisen concerning the role of these microbial populations in the transformation (biodegradation) of environmental contaminants, the impact of environmental contaminants on microbial processes, and the utility of microbiological assessment in examining the occur* P r e s e n t address:

Department of Agronomy, Cornell University, Ithaca, NY. 0095-3628/83/0009-0041502.80

42

L.M. Mallory and G. S. Sayler

r e n c e a n d fate o f e n v i r o n m e n t a l c o n t a m i n a n t s . A s a result, t e c h n i q u e s for t h e elucidation of microbial populations capable of transforming environmental contaminants are abundant. However, little attention has been given to the significance of the technique, or the microbial populations enumerated with r e s p e c t to t h e i r r e l a t i o n s h i p to t h e m i c r o b i a l c o m m u n i t y a n d t h e n i c h e s t h o s e organisms occupy. Numerical taxonomic techniques have been used to examine the relations h i p s a m o n g specific g r o u p s o f a q u a t i c b a c t e r i a [1, 4, 8, 28], as w e l l a s h i g h l y d i v e r s e b a c t e r i a l p o p u l a t i o n s [2, 18, 19, 24, 35]. I t w a s h y p o t h e s i z e d t h a t numerical taxonomy could aid in describing the niches of natural bacterial p o p u l a t i o n s a n d m i c r o b i a l r e s p o n s e to e n v i r o n m e n t a l c o n t a m i n a n t s . T h e p r i n cipal goal of the investigation was to characterize the aerobic and facultative anaerobic portions of the heterotrophic aquatic bacterial guild structure in the photic zone of an oligotrophic freshwater environment. This community would b e e x p e c t e d to b e r e s p o n s i v e to e n v i r o n m e n t a l c o n t a m i n a n t s as j u d g e d f r o m previous studies reporting biodegradative and resistant bacteria isolated from a q u a t i c e n v i r o n m e n t s . T h e specific o b j e c t i v e s o f t h i s s t u d y w e r e 3 - f o l d a n d i n c l u d e d (1) e s t a b l i s h i n g t h e c o m p a r a t i v e r e l a t i o n s h i p a m o n g b i o d e g r a d a t i v e / resistant bacterial populations recovered from selective media to total cultura b l e h e t e r o t r o p h s e n u m e r a t e d o n l o w a n d h i g h n u t r i e n t m e d i a ; (2) d e f i n i n g phenetic groups and estimating bacterial niche structure based on the physiological and biochemical diversity of taxonomic characteristics of the bacterial g u i l d w i t h i n t h e a q u a t i c c o m m u n i t y ; a n d (3) e x a m i n i n g t h e l i m i t a t i o n s a s s o ciated with both isolation and characterization of aquatic bacterial populations within the framework of numerical taxonomy.

Materials and Methods Reservoir samples were collected from a single site located on Center Hill Reservoir in central Tennessee in January 1977. This oligotrophic reservoir has been previously described [29] and studies have been conducted on the biodegradation of polychlorinated biphenyls (PCB) [33] and polyaromatic hydrocarbons [32], as well as the effects of these contaminants on heterotrophic microbial populations and processes [29]. Physical and chemical sample characteristics at the time of sampling are given in Table 1. The reservoir was sampled using a Niskin Sterile Bag Sampler (General Oceanics, Miami, FL). A 3 liter pooled sample was maintained at ambient water temperature during transport and was.processed at the field station facilities within 1 h of initial sampling. Microbial populations were enumerated and isolated using the spread plate technique following a 10-fold dilution series in quarter strength Ringers buffer. Four media were inoculated in triplicate over the dilution range using a 0.1 ml sample inoculum. The media used included a moderately high nutrient yeast extract-peptone-glucoseagar (YEPGA) [29]; low nutrient, lake water agar (LWA) formulated with 1.8% (w/v) purified agar in aged Center Hill Reservoir water, phenanthrene agar (PHA) [29] consisting of basal salts, yeast extract, 1.8% purified agar, and 1.0 g 1-1 certified phenanthrene (Eastman Organic Chemicals, Rochester, NY) in distilled water; and polychlorinated biphenyl agar (PCBA) [29] identical to PHA except that phenanthrene was replaced with 1.0 g 1-~ Aroclor 1254 (Monsanto Chemical Co., St. Louis, MO), a mixture of polychlorinated biphenyls, 54% chlorine by weight. Inoculated plates were incubated 4 weeks at 25~ in the dark. Following incubation, 240 well isolated colonies were randomly chosen for study. Sixty colonies

Heterotrophic Bacterial Guild Structure

43

Table 1. Physical and chemical parameters of site 4 at the time of sampling Variable Water temperature Transparency Dissolved organic carbon Phosphate Nitrate Dissolved oxygen Conductivity pH Suspended sediments

Measured value 4.5~2 4.0 meter 50 mg/l 18 ug/l 920 ~g/l 12.5 mg/l 100 ~tmohs 7.75 1.3 mg/l

were picked from each medium. Each isolated strain, the operational taxonomic unit (OTU), was subcultured. Three successive isolation streaks were performed for each OTU. Purity was determined from uniform colonial morphology and from microscopic examination. OTUs were maintained on YEPGA slants. Fresh slants were inoculated every 8 weeks. A total of 28 OTUs were lost upon initial subculturing or shortly thereafter. In addition to the unknown strains described, 13 reference cultures also were examined. These were Alcaligenes dispar (American Type Culture Collection (ATCC) 3121), Bacillus cereus var. mycoides (ATC 6462), Bacillus subtilis (ATCC 6051), Erwinia carotovora (ATCC 8061), Erwinia herbicola (ATCC 12287), Erwinia carnegieana (ATCC 13452), Escherichia coli (ATCC I 1775), Klebsiella aerogenes (National Collection of Type Cultures 8172), Micrococcus luteus (ATCC 4698), Moraxella phenylpyruvica (ATCC 23333), Nocardia cariarum (ATCC 14629), Nocardia corallina (ATCC 4273) and Streptomyces griseus (ATCC 23345). The OTUs were examined for 158 morphological, physiological, and biochemical characters. Test media were inoculated and incubated at 25~ for 4 weeks unless otherwise noted. Tests were carried out once and were repeated only when inconclusive results were obtained. Colonial morphology data were recorded following 7 days incubation on YEPGA. Selected characters from Colwell and Wiebe [9] were used. Fluorescent pigment production was determined using medium B of King et ai. [21 ]. Motility and cellular morphology were determined from wet mounts, prepared from 2 to 3-day-old broth cultures incubated at 25~ and examined by phase microscopy. Gram staining reactions of 2 to 3-day-old broth cultures were determined using Hucker's modification of the Gram stain [16]. Acid fastness was tested using 5-day-old cultures with the method described by Cowen and Steel [1 l]. A decolorizer of 5% (w/v) H2SO4 was used. lntraceUular granulation was detected in 2 to 3-day-old cultures using a methylene blue stain [3]. Gliding motility was determined using the method of Colwell and Wiebe [9]. Growth in the presence of NaC1 (2%, 5%, 7.5%, 10%, 12.5%, and 15%, all w/v) was tested in NaCl-amended YEPGA. Temperature growth range was determined using YEPGA medium. Plates were lightly inoculated and incubated at 4~ 15~ 370C, and 42~ for 4 weeks, 2 weeks, 5 days, and 3 days, respectively. Visible growth was scored as a positive result. Resistance to the following dyes and other inhibitors was determined by using appropriately amended YEPGA: 0.00001% (w/v) each of brilliant green, crystal violet, malachite green, methylene blue, neutral red, and pyronin B; 0.02% (w/v) of both bismuth citrate and potassium tellurite; and 0.04% of sodium azide. Any growth was scored as a positive test. The production of arginine dehydrolase and lysine and ornithine decarboxylase was determined using the method of Moeller [26]. The arginine dehydrolase and lysine and ornithine decarboxylase tests were read at 2 and 4 days incubation and 2 and 4 weeks incubation. The methyl red test, indol production, and acetoin production were determined following 5 days incubation at 25~ [3]. The presence of oxidase [3] and catalase [3] was tested using fresh, newly visible growth from YEPGA. Nitrate and nitrite reduction were determined using the method described by Bailey and Scott [3]. The production of H2S was determined in SIM medium (Difco). Levan production was tested using YEPGA amended with 5% (w/v) sucrose. Copious slime production was scored as a positive

44

L . M . Mallory and G. S. Sayler

Table 2.

Percent positive frequency for characters used in the computer analysis

Character Colonial morphology elevation convex fiat lumpy peaked raised umbonate edge crenate irregular smooth surface matt shiny waxy transparency opaque translucent transparent color white off-white light yellow yellow orange pink red purple diffusible pigment fluorescent pigment Micromorphology length to width ratio coccoid 1.5-3:1 3-6:1 7-15:1 15:1 cell characteristics branching round ends tapered ends stalked

Percent

1 3 3 10 73 10 11 13 76 1 69 30 60 32 8 18 29 7 17 18 7 2 2 4

4 38 72 29 18 4 99 5 3

Character

Percent

curved pleomorphic fragmenting chaining rosette formation motility gliding wet mount Staining reactions gram-positive gram-variable gram negative acid fast granulation Physiology growth on media containing NaC1 2% (w/v) NaCI 5% (w/v) NaCI 7.5% (w/v) NaC1 10% (w/v) NaCI 12.5% (w/v) NaC1 initiation of growth at pH pH 5 pH 6 pH 9 growth at temperature 4~ 15~ 37~ 42~ growth in the presence of brilliant green bismuth citrate crystal violet malachite green methylene blue potassium tellurite pyronin B sodium azide Biochemistry acetoin production arginine dehydrolase catalase

12 8 5 8 3 3 33 25 15 60 1 10

50 28 8 3 1 63 94 95 32 78 40 14 67 27 68 63 60 11 64 1 5 13 60

test. The fermentation of glucose was tested using the method of Hugh and Liefson [ 17]. Under aerobic conditions, acid production from the following carbohydrates was examined: L+ arabinose, cellobiose, dextrin, glucose, inositol, lactose, maltose, melezitose, alpha methyl glucoside, raffinose,

Heterotrophic Bacterial Guild Structure Table 2.

45

Continued Character

Percent

Character

indol production levan production lysine decarboxylase methyl red test nitrate reduction nitrite reduction ornithine decarboxylase oxidase glucose metabolism

7 7 9 3 44 23 3 35

1+ arabinose arginine betaine butanol cellobiose cystine dulcitol D fructose D galactose D glucose

39 35 34 30 46 i2 17 63 56 67

glycerol glycogen inositol inulin

56 46 32 18

lactose lysine malonic acid maltose mannitol mannose melezitose melibiose octanoic acid ornithine proline propanol

43 34 23 58 64 59 29 40 18 42 52 32

rhamnose ribose saliein

37 46 43

salicylic acid sodium acetate sodium aspartate sodium benzoate sodium butyrate sodium citrate sodium formate sodium glutamate sodium lactate sodium oxalate sodium pyruvate sodium succinate serine sorbitol sucrose trehalose tryptophan uracil xylose

1 42 59 15 16 48 18 62 58 13 60 62 23 32 45 55 14 6 50

alkaline reaction fermentative oxidative

15 35 35

degradation o f agar casein DNA egg yolk hypoxanthine starch Tween 20 Tween 40 Tween 60 Tween 80 tyrosine urea

3 40 36 15 20 24 43 45 39 34 45 16

acid production from adonitol arabinose cellobiose

5 28 29

dextrin dulcitol glucose inositol lactose maltose mannitol melezitose melibiose alpha methyl glucoside raftinose salicin sucrose xylose

19 0 44 9 14 33 25 9 19 6 9 8 26 23

utilization as sole carbon source adonitol alanine

24 46

Percent

salicin, surcrose, and xylose. Where possible, the carbohydrates were sterilized by filtration; otherwise sterilization was performed by successive heating to 100~ for 1 h on 3 consecutive days.

46

L . M . Mallory and G. S. Sayler

The sterilized basal medium of Hugh and Liefson [17] was mixed with a carbohydrate and aseptically dispensed into sterile multidishes. The media were inoculated with a multipoint inoculator [23], and were read at 1,2, 4, 7, and 14 days. The degradation ofhypoxanthine, starch, and tyrosine was tested using YEPGA amended with 0.5% (w/v), 1.5% (w/v), and 0.5% (w/v) amounts, respectively. Starch degradation was confirmed by flooding the plate with a dilute iodine solution. Clear zones around colonies were scored as positive. For the insoluble compounds, clearing of the medium around colonies was scored as a positive result. Agar degradation was determined using the method of Colwell and Wiebe [9]. Casine degradation was tested by incorporating 5% (w/v) skim milk into YEPGA. Clearing of the medium was scored as a positive test. The degradation of DNA and urea was carried out using the methods of Bailey and Scott [3]; Tween 20, Tween 40, Tween 60, and Tween 80 degradation was tested using the method of Sierra [34]. The precipitation of calcium salts around areas of bacterial growth was taken to be a positive result. Egg yolk agar was prepared using 5% (v/v) egg yolk in YEPGA. Clearing of the medium around areas of growth was scored as positive. Utilization of sole carbon sources was tested using the method of Stevenson [38]. Carbon sources were formulated in 20% (w/v) solutions and sterilized as previously described. The solutions were added to the support medium to give a final concentration of 0.2% (w/v). The complete media were aseptically dispensed into sterile replidishes and inoculated with multipoint inoculators [23]. Fifty carbon sources were tested (Table 2). Both positive and negative controls were included. Results were recorded after 2 and 4 weeks incubation. The characters were coded into 3 states: "'1" for a positive, " 0 " for a negative test, and " 9 " for a noncomparable or not applicable test. Quantitative multistate characters, such as NaCI tolerance, were coded in a nonadditive fashion. The final n • t matrix contained 225 OTUs and 158 coded characters. The data were examined using both the Jaccard coefficient (SJ) [31], which considers only positive matches, and the simple matching coefficient (SSM) [35], which takes into account both positive and negative matches. Clustering was achieved using the unweighted average linkage (UPGMA) technique [37]. An inverted matrix (t • n) was examined using the SSM and SJ similarity coefficients. Clustering was achieved using the U P G M A technique. Character correlation was assessed by this method. From the inverted matrix, a smaller matrix was generated for carbon compounds, which were used in both the sole carbon source tests and the acid production tests. This smaller matrix was examined in an identical manner as the larger inverted matrix. The programs used included the T A X A N 5 and IGPS 3 program packages obtained from Dr. R. R. ColweU at the University of Maryland. The programs were rewritten for the University of Tennessee, Dec System 10 computer.

Results A t o t a l o f 153 O T U s a n d 1 r e f e r e n c e s t r a i n , c o m p r i s i n g 7 2 % o f t h e t o t a l O T U s , w e r e c l u s t e r e d in 41 p h e n e t i c g r o u p s d e f i n e d at t h e 8 0 - 8 5 % s i m i l a r i t y l e v e l , using the SSM coefficient. Examination of the OTUs using the SJ coefficient produced clusters essentially identical in membership to those obtained using the SSM coefficient. Certain strains were shown to cluster above 80% similarity but were not defined in any groups. When examined using the SJ coefficient, these strains were not recovered in clusters. The high level of similarity observed w i t h t h e S S M c o e f f i c i e n t f o r t h e s e O T U s w a s f o u n d to b e a t t r i b u t a b l e t o a h i g h degree of negative matching. A s i m p l i f i e d d e n d r o g r a m (Fig. I) s h o w i n g d e f i n e d c l u s t e r s w h i l e o m i t t i n g s i n g l e s t r a i n s e x h i b i t s e a c h d u s t e r ' s d e f i n e d s i m i l a r i t y l e v e l a n d its r e l a t i v e size. T h r e e c l u s t e r s (2, 5, a n d 4 0 ) c o n t a i n e d 8 o r m o r e s t r a i n s a n d a c c o u n t e d f o r

Heterotrophic Bacterial Guild Structure

PHENON

NO. OF STRAINS

1.......

3

2 .........

19

3 ..........

2

4 ...............

2-

5. . . . . . . . . .

10

6 .............. ? .......................

2 3

8

6

9 10 11 12 13 14 15 16 17" 18 . 19' 20

47

% SIMILARITY SlSM] 100

90

80

70

60

I

I

I

I

I

I

I

SO

Vmm.

B

3 2 3

....... --2 --2

.

.

.

.

2 6 --2 3 2 2 4

21

5

22

7

23 24 25

3 2 --2

26 ........................ 27 ..................... 28 ....................

4 2 4

29 ....................... 30 ............................ 31 . . . . . . . . . . . . . . . . . . . . . . . . . . 32 .......

2 4 2 6

33 34 35 36 37 38 39

4 2 3 3 2 2 5

40

8

41

2

r

i

simplified dendrogram of grouped Center Hill Reservoir OTUs constructed using the SSM similarity coefficient.

Fig. 1.

A

23% o f the clustered O T U s . There were 27 small clusters with 2 - 3 m e m b e r s each. These O T U s accounted for 41% o f grouped strains. T h e remaining 11 clusters, with 4 - 7 members, contained 36% o f the clustered O T U s . The percent positive responses to the c o d e d characters used in this study are

48

L.M. Malloryand G. S. Sayler

given in Table 2. The majority of positive responses were associated with one character within each category of colonial morphology, with the exception of color. These characters were raised (73%), smooth edge (76%), shiny surface (69%) and opaque transparency (60%). Color tended to fall into 2 broad categories with approximately equal frequency. White and off-white accounted for 47% o f the OTUs and light yellow, yellow, and orange accounted for 42%. The predominant micromorphological form observed was a rod with a length to width ratio o f 3:1-6:1 (72%). Motility was observed in 36% o f the OTUs. Only 3% exhibited gliding motility. Most OTUs (60%) were gram-negative, 25% were gram-positive, and 15% were gram-variable. Tolerance o f salt was low with 50% of the strains growing in the presence of 2% (w/v) NaC1. Only 28% grew in the presence of 5% NaCI. In marked contrast to this, the pH growth range was quite wide. When inoculated onto YEPGA pH 9, 95% o f the O T U s initiated growth. At pH 6 and pH 5, the percent positive responses were 94 and 63%, respectively. As one group, the OTUs could be characterized as low temperature mesophiles with 78% of the strains growing at 15~ Nearly one-third (32%) of the strains grew well at 4~ In the higher temperature range, 40% grew at 37~ and only 14% grew at 42~ A surprising number of strains grew in the presence of dye. Growth in the presence o f brilliant green, crystal violet, malachite green, methylene blue, and pyronin B was 67%, 68%, 63%, 60%, and 64% positive, respectively. Although most strains were capable of growth in the oxidation fermentation test medium, only 35% gave a fermentative reaction and 65% gave either no reaction or an oxidative reaction. Many o f the O T U s that were scored as oxidative exhibited a very weak reaction when compared with an uninoculated control. All other biochemical characters, with the exception of catalase and certain sole carbon source characters, had a positive response frequency of less than 50%. The OTUs examined in this study were found to have a moderately diverse range o f possible carbon sources. O f the 50 carbon compounds tested for utilization, 15 were used by 50% or more of the strains. The 15 carbon compounds included (1) 9 sugars: D fructose (utilized by 63% of the strains), glucose (67%), glycerol (56%), D galactose (56%), maltose (58%), mannitol (64%), mannose (59%), trehalose (55%), and xylose (50%); (2) 3 amino acids: proline (52%), sodium aspartate (59%), and sodium glutamate (62%); and (3) 3 organic acids: sodium lactate (58%), sodium pyruvate (60%), and sodium succinate (62%). Thirty-five sole carbon sources were used by 30% or more of the OTUs examined. Presumptive identifications were made for 24 groups. Although group 2 contained the marker strain Erwinia carnegieana, no identification was made due to the highly atypical results obtained from that strain. The presumptive identifications are given in Table 3. Only 1 group, group 2, contained OTUs isolated from all 4 isolation media. Seven groups (17% o f all groups) contained members from 3 isolation media. Eight groups contained members from only 1 medium. The remaining groups, having strains isolated from 2 media, accounted for 61% of the defined groups. If each unclustered strain represents a distinct taxonomic group, then a total

Heterotrophic Bacterial Guild Structure

49

Table 3. Presumptive group identifications Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Identification Acinetobacter sp, unidentified unidentified unidentified Alcaligenes sp. unidentified Caulobacter sp. Streptothrix sp. Janthinobacterium sp, unidentified Pseudomonas sp. unidentified unidentified unidentified Flexibacter sp. Flexibacter sp. Corynebacterium sp. coryneform Aerornonas hydrophila unidentified unidentified Pseudomonas sp. Aeromonas hydrophila Pseudomonas sp. Kurthia sp. coryneform coryneform unidentified unidentified coryneform unidentified Pseudomonas sp. Pseudomonas sp. coryneform Nocardia sp. unidentified unidentified unidentified Pseudomonas fluorescens Pseudomonas fluorescens unidentified

o f 100 taxa were r e c o v e r e d i n this study. O T U s i s o l a t e d f r o m Y E P G A , P H A , a n d P C B A were r e c o v e r e d as single s t r a i n s o n l y 25% o f the t i m e . S t r a i n s i s o l a t e d f r o m L W A were r e c o v e r e d as single s t r a i n s 4 1 % o f the t i m e . T a b l e 4 s u m m a r i z e s the d i s t r i b u t i o n o f O T U s i n p h e n e t i c g r o u p s b y t h e i r i s o l a t i o n m e d i a . It is i n t e r e s t i n g t h a t Y E P G A r e p r e s e n t e d the m o s t selective m e d i u m i n t h a t 33% o f all O T U s r e c o v e r e d f r o m t h a t m e d i u m w i t h i n phe-

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L_ M. Mallory and G. S_ Sayler

Table 4. Comparative selectivity and commonality of OTU group membership among different isolation media

Isolation media YEPGA LWA PHA PCBA

Selectivity~ (%)

YEPGA

C~176 LWA

h (%) PHA

PCBA

33 5 18 9

100 32 22 42

27 100 41 42

27 56 I00 54

45 50 55 100

Percent of OTUs with phenotypic group's membership unique to the isolation medium Percent of OTUs with group membership shared in common with OTUs from the alternative isolation media

Table 5.

Total viable counts, OTU recovery, and taxa defined from Center Hill Reservoir Isolation medium

Total viable count ~ OTUs recovered Cumulative OTUs Taxa characterized per medium Cumulative new taxa

YEPGA

LWA

PHA

PCBA

3,600 60 60 33 33

2,400 46 106 35 63

180 47 l 53 34 85

370 59 212 35 100

a Expressed as colony forming units per ml lake water

notypic groups were unique to that m e d i u m . Lake water agar represented the least selective m e d i u m with only 6% o f the group O T U s being unique; P H A and P C B A were f o u n d to be slightly m o r e selective-- 18% and 9%, respectively. T h e selectivity o f Y E P G A is also a p p a r e n t in that the c o m m o n a l i t y a m o n g groups containing an O T U isolated f r o m Y E P G A is 27% for groups r e c o v e r e d f r o m L W A a n d P H A . T h e converse also holds true for groups containing an O T U isolated f r o m either L W A or P H A . Forty-five percent o f the groups containing an O T U recovered f r o m Y E P G A are also r e c o v e r e d as an O T U f r o m PCBA. C o m m o n a l i t y a m o n g O T U recovered in groups f r o m LWA, P H A , a n d P C B A was 40% a n d P H A a n d P C B A shared c o m m o n a l i t y o f 50%. T h e n u m b e r o f O T U s taken f r o m each isolation m e d i u m a n d the total n u m ber o f t a x a characterized, including single strains, f r o m each m e d i u m are given in T a b l e 5. It is interesting that the O T U s isolated f r o m L W A had the greatest a p p a r e n t species richness ( n u m b e r o f taxa per total O T U s characterized). By using the degradative characters and the sole carbon characters, 4 large groups can be defined f r o m those O T U s c o n t a i n e d in groups. These groups are (1) 1-11, which average 4.6 c a r b o n sources utilized; (2) 12-20, which average 15.8 c a r b o n sources utilized; (3) 21-24, which average 7.5; and (4) 25-41, which average 21 c a r b o n sources utilized. T h e r e was no significant difference between groups 1 a n d 3 when c a r b o n source characters, which are 9 0 - 1 0 0 % positive, were c o m p a r e d . H o w e v e r , when carbon source characters, which are 10-90% positive within a group, were included it was found that g r o u p 3

Heterotrophic Bacterial Guild Structure

51

averaged 28 carbon sources totally to partially utilized within a group whereas group 1 averaged 21 carbon sources. When partial and total positive degradative characters were examined, group 1 averaged 4.5 characters whereas group 3 averaged 9 characters. With the exception o f group 4 (25--41), the groups do not correspond to any observed higher order grouping, as delineated by the simplified d e n d r o g r a m (Fig. 1). When the characters were examined using the simple matching coefficient in the inverted matrix, it was observed that 43 o f the 50 carbon source characters were clustered in 9 groups at the 50-60% similarity level using the SSM coefficient. Significant intragroup structure was observed in m a n y o f the groups. This intragroup structure was defined at or above the 70% similarity level. When e x a m i n e d using the SJ coefficient, similarity was lower but group structure and intragroup structure were largely maintained. Most groups could be characterized according to the chemical nature o f the carbon sources. G r o u p s fell into 5 broad classes: simple hexoses, disaccharides and polysaccharides, amino acids, sugar alcohols, and organic acids. N o significant correlation was observed between acid production characters and sole carbon source characters. With such a wide spectrum o f O T U s , a higher correlation was observed within acid production characters.

Discussion

The use o f numerical t a x o n o m y to classify bacteria is limited by the n u m b e r of O T U s examined, the n u m b e r and appropriateness o f characters tested, and the similarity coefficients and clustering techniques used [36]. However, bias introduced into this study through the use o f numerical t a x o n o m i c techniques most likely is overshadowed by problems associated with the isolation, relative recovery, and culture o f bacteria from their native aquatic environment. Although the m e t h o d o f choice for isolation appears to be the dilution spread plate technique [7], there is no good choice for a plating m e d i u m capable o f supporting the growth o f all native aquatic bacteria, colonizing diverse habitats, or existing in various physiologically suppressed or d o r m a n t states [12, 39]. Bacterial populations that are most fit in terms o f growth potential in the aquatic e n v i r o n m e n t , are best adapted to the isolation m e d i u m , and are present in relatively high numbers should be recovered on a variety o f isolation media in greatest frequency. When the n u m b e r o f O T U isolation media is increased, the n u m b e r o f observed taxa should also increase. Although "fit" taxa will be recovered on m o r e than one isolation m e d i u m , d o r m a n t or stressed populations having -estricted growth ranges will be recovered on fewer isolation media; hence, fewer new taxa per isolation medium. Both hypotheses are supported by the results o f the investigation (Table 4). N o single taxon within the bacterial guild was found to be d o m i n a n t in this investigation. T h e largest group, phenon 2 (Fig. 1), contained only 9% o f the total isolates, and the second largest phenon contained only 5%. Bacterial diversity (examined using the Shannon diversity index H, where H = c/n (N log 10N - 10g lOni), N is the total n u m b e r o f O T U s examined, ni is the total

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n u m b e r o f individuals in the ith group, and c is a constant equal to 3.3219) was found to be 3.21, indicating high diversity. This level o f diversity is comparable to oligotrophic marine habitats [18, 25]. The use o f multiple isolation media, ranging from low nutrient to high nutrient, resulted in increased H values in this study. The goodness o f fit o f the classification reported was d e m o n s t r a t e d by the m i n o r group rearrangement after controlling for group f o r m a t i o n due to negative matching. H o w e v e r , as reported in other investigations [2, 5, 20, 35], unequivocal identification o f all defined groups was impossible. Only 58% o f the groups could be identified and only 79% o f these to the genus or species level. This problem is most likely due to the nature or definition o f a bacterial species, which can be considered to consist o f a spectrum or series o f integrating forms or phenotypes that give rise to populations that c a n n o t unequivocally be identified using the phenetically defined clusters [10, 14]. The bacterial populations recovered from each o f the 4 media suggest that each m e d i u m selects for different populations (Table 4). Yeast extract-peptoneglucose agar was found to be the most selective; 25% o f the O T U s isolated from Y E P G A were recovered as single strain taxa and 33% o f the groups containing O T U s from Y E P G A were exclusive to that m e d i u m . O v e r 50% o f the taxa isolated from Y E P G A were exclusive to that m e d i u m . The degree o f selectivity found in Y E P G A was not observed a m o n g the other 3 media. However, their patterns o f O T U isolation are by no means identical. All 3 media can be selective for oligotrophic bacteria. Although 2 media, P H A and PCBA, contain high concentrations o f carbon (i.e., phenanthrene and polychlorinated biphenyls, respectively), these compounds cannot be used for growth by m a n y groups o f bacteria. Thus, to initiate growth, an organism would have to be resistant to the toxicant and an oligotroph, or be able to utilize the toxicant. This form o f selection has been d e m o n s t r a t e d and discussed by D i G e r o n i m o et al. [13]. Species richness was lower for P H A and PCBA when c o m p a r e d with LWA. Polychlorinated biphenyl agar was found to be the least selective m e d i u m in this study. Operational t a x o n o m i c units isolated using PCBA exhibited the lowest percent single strain taxa and the lowest percent o f groups recovered as exclusive PCBA isolates (Table 4). This finding is not surprising. Sayler et al. [29], Shiaris and Sayler [33], and Blakemore and Carey [6] have d e m o n s t r a t e d that highly recalcitrant polychlorinated biphenyls appear to have only moderate to no biological effects on heterotrophic bacterial activity. T h e 2 media, P H A and PCBA, were found to select for taxa similar to those recovered from LWA. Similar conclusions were drawn from results o f studies on estuarine bacteria linked to PCB biodegradation [30]. It is appropriate to view taxa recovered from toxicant media as biodegradative or resistant and/ or oligotrophic in nature although linking them specifically to biodegradation is speculative. What is apparent is that such media do recover taxa that are representative o f the heterotrophic guild that is responsible for the biotransformation o f polychlorinated biphenyls and polyaromatic h y d r o c a r b o n in the e n v i r o n m e n t e x a m i n e d [31-33]. If we assume that carbon source utilization represents a c o m p o n e n t o f the fundamental heterotrophic role, it is possible to examine the hypothetical niche

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these o r g a n i s m s o c c u p y . It is s i g n i f i c a n t t h a t 9 d e f i n a b l e g r o u p s o f u t i l i z a b l e c a r b o n s o u r c e s w e r e r e c o v e r e d in t h e i n v e r t e d m a t r i x d e s p i t e t h e p e r m i s s i v e n a t u r e o f c a r b o n s o u r c e testing. T h i s r e s u l t e d in t h e e l u c i d a t i o n o f 5 m a j o r , o v e r l a p p i n g classes o f c a r b o n s o u r c e u t i l i z a t i o n p a t t e r n s w i t h i n t h e m a j o r g r o u p s o f c a r b o n s o u r c e s . I f H u t c h i n s o n ' s p o r t r a y a l o f t h e a q u a t i c e n v i r o n m e n t as d y n a m i c s y s t e m is a c c e p t e d , it f o l l o w s t h a t p o t e n t i a l n i c h e s for h e t e r o t r o p h i c b a c t e r i a , as m e a s u r e d b y a v a i l a b l e c a r b o n s o u r c e s , a r e a l s o in a d y n a m i c state. Consequently the physiological and biochemical potential of the individual defines t h e p o s s i b l e o c c u p i a b l e n i c h e . I f t h e n i c h e is sufficiently s t a b l e o v e r t i m e , c o m p e t i t i o n a m o n g c o e x i s t i n g p o p u l a t i o n s w o u l d r e s u l t in a n a r r o w i n g o f d e f i n a b l e t a x a . C o n v e r s e l y , i f t h e p o t e n t i a l n i c h e s t r u c t u r e is r a p i d l y c h a n g i n g within a community, phenetically similar but genetically distinct taxa could o c c u p y t h e s a m e n i c h e . C o n s e q u e n t l y , p h y s i c a l t r a n s p o r t o r s u r v i v a l m a y be more important than unique adaptations in the ability of an organism to occupy a given niche. These relationships are distorted by the artificiality of the labo r a t o r y e n v i r o n m e n t ; h o w e v e r , t h e y d o p r o v i d e a c o n c e p t u a l f r a m e w o r k in w h i c h to v i e w t h e 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 a c t e r i a l g u i l d w i t h i n t h e a q u a t i c community. Acknowledgments. This investigation was supported by Union Carbide subcontracts, 7182 and 7685, Oak Ridge National Laboratory, and by National Institute for Environmental Health Sciences grant ESO 1521, US Public Health Service. Gary S. Sayler is supported by an NIEHS Research Career Development Award.

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