Pergamon

Chemosphere, Vol. 34, No. 3, pp. 655-669, 1997

PII: S0045-6535(96)00360-8

Copyright 1997 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0045-6535197 $17.00+0.00

STIMULATION AND ENRICHMENT OF T W O MICROBIAL POLYCHLORINATED BIPHENYL REDUCTIVE DECHLORINATION ACTIVITIES

William A. Williams

Environmental Laboratory, GE Research and Development Center, P.O. Box 8, Schenectady, New York 12301-0008
(Received in USA 22 June 1996; accepted 16 September 1996)

ABSTRACT Isolation and characterization of the bacterium or bacterial consortium responsible for microbial polychlorinated biphenyl (PCB) reductive dechlorinataon would allow a better understanding of the mechanism of this activity in the environment. Stimulation and enrichment of a PCB dechlorinatlon activity in a culture is necessary for isolation of the microorganisms that cause PCB dechlorination. Two distinct microbial PCB dechlorination activities were independently stimulated in slurries of PCB-contaminated upper Hudson River sediment by adding specific PCB congeners. The addition of 2,4,6-trichlorobiphenyl stimulated a specific para dechlorination activity on Aroclor 1242 (designated a Process Q). In contrast, 2,3,6-trichlorobiphenyl addition stimulated a specific meta dechlorination activity (designated a Process M). Each dechlorination activity was enriched by transferring 10 percent by volume of the sediment slurry containing the dechlorination activity to an autoclaved medium composed of an anaerobic mineral solution, humic acids, and PCBs This is the first report of an enrichment culture of the microbial para dechlorination activity (the Process Q) Both microbial PCB dechlorination activities were inhibited by vancomycin (5 ppm) or nisin (500 ppm). These data suggest that a population of gram-positive bacteria is essential for these PCB dechlorination activities. 1997 Elsevier Science Ltd. All rights reserved

Key words: polychlorinated biphenyls, PCBs, reductive dechlorination, anaerobic microbiology

INTRODUCTION Polychlorinated biphenyls (PCBs) are a group of compounds that were banned from production in the United States in 1977. These compounds were manufactured as mixtures of PCB congeners that differed by the number and configuration of chlorines on the biphenyl nucleus (Figure 1); the mixtures were given trade names such as Aroclor 1242. Widespread use of PCBs resulted in their accumulation in the organic portions of soils,

655

656 sediments, and biota. Aerobic biodegradation of PCBs is well characterized [for a review, see reference 1] but is generally limited to the lightly chlorinated biphenyls. Unlike aerobic PCB biodegradation, anaerobic microbial reductive dechlorination of nearly all the PCB homologs can occur.

para ortho~J ortho metameta metal ~,] meta ortho ~ ortho

,, ,

4 2

2'
3'

3~ 6S
6'
5'

para

4'

Figure I. The Greek and numerical nomenclatures describing the configuration of chlorines in a PCB congener.

Brown and co-workers [2-5] proposed that the distinctive pattems of PCB transformation seen m PCBcontaminated sediments resulted from several different microbial dechlorination activities that are distinguished by congener selectivity. Bedard and Quensen [6] provided a detailed review of the microbial PCB reductive dechlorination activities (also termed, dechlorination processes) and the dechlorination patterns, which have been given letter designations. These investigators noted that chlorine removal (ie., a dechlorination process) is not determined strictly by the position (ortho, meta, or para) of the chlorine relative to the opposite phenyl ring; but also by the surrounding chlorine configuration, the chlorine configuration on the opposite ring, and the total number of chlorines on both rings [4, 6]. Microbial PCB dechlorination pattems M and Q have been observed in sediment slurries or microcosms containing microorganisms from PCB-contaminated upper Hudson River sediments [7-14]. Pattern M

dechlorination activity is characterized as meta dechlorination of 2,3,4-; 2,3,6-; 2,3-; 2,5-; and 3,4-chlorophenyl groups; and possibly meta dechlorination of 3-chlorophenyl groups [6, 15] Pattern Q dechlorination activity is characterized as p a r a dechlorination of 2,4,5-; 2,4-; 3,4-; and 4-chlorophenyl groups; and possibly para dechlorination of 2,3,4-chlorophenyl groups [6, 15]. Pattern Q dechlorination activity is also characterized as
meta dechlorination of 2,3-chlorophenyl groups; and possibly m e t a dechlorination of 2,3,4- and 2,3,6-

chlorophenyl groups [6, 15]. A better understanding of microbial PCB dechlorination could be obtained by isolating and characterizing the bacterium or bacterial consortium responsible for these activities. Enrichment of a

dechlorination activity is one step in isolating microorganisms that cause PCB dechlorination There have been several reports of microbial pattern M dechlorinauon activity enrichment cultures [8, 10, 14, 16], but no report of a microbial pattem Q dechlorination activity enrichment culture. Selective stimulation of a particular dechlorination activity is one method of obtaining an enrichment culture. Some halogenated aromatic compounds, such as halogenated biphenyls or benzoic acids, have been

657 used to stimulate and enrich specific microbial PCB dechlorination activities [17, 18]; but patterns M and Q activities have not been stimulated and enriched in this manner. In a previous study [19], I noted meta and
para, but not ortho dechlonnation of 2,3,6-trichlorobiphenyl (2,3,6-CB) and 2,4,6-trichlorobiphenyl (2,4,6-CB)

by microorganisms in upper Hudson River sediment slurries. These results [17-19] led to the hypothesis and subsequent investigation (described herein) that 2,3,6-CB and 2,4,6-CB could be used to stimulate and then enrich patterns M and Q dechlorination activities in a PCB-contaminated upper Hudson River sediment

MATERIALS AND METHODS Sediment Collection Site. Hudson River sediment was collected in the river near the east bank at Stillwater,
NY (river mile 169) and stored at 4C. The sediment contained -25 [tg/g (dry wt of sediment) meta- and paradechlorinated Aroclor 1242; -60% of the PCBs were 2-chlorobiphenyl (2-CB); 2,2'-CB; 2,6-CB; and 2,6,2'CB.

Sediment Slurry Preparation. The slurry samples were prepared in an anaerobic chamber (Coy Laboratory
Products, Ann Arbor, MI) containing an atmosphere of_>98% N 2 and <2% H2. A slurry of wet sediment and revised anaerobic mineral medium reduced with 0.1,4 L-cysteine hydrochloride (reduced RAMM [20]) was prepared; the total volume of the slurry was 240 mL. The ratio of wet sediment to reduced RAMM was 2:3 (vol:vol); the slurry contained - 3 0 % solids. Aroclor 1242 (neat) was added to the slurry to make a final concentration o f - 1 0 0 lag/g (dry wt of sediment), and the slurry was magnetically stirred for five minutes before it was divided into three parts to which the PCB congeners 2,3,6-trichlorobiphenyl; 2,4,6-trichlorobiphenyl; or both were added as 70 mM stock solutions in acetone. In slurries amended with one trichlorobiphenyl, the concentration of 2,3,6-CB or 2,4,6-CB was 350/aM (-.440 pg/g dry wt of sediment). In slurries amended with both trichlorobiphenyls, the concentration of each congener was 175 /aM With continuous stirring, 15-mL aliquots were transferred from the slurries to 30-mL serum vials; there were five vials for each of the three trichlorobiphenyl treatments All vials were crimp-sealed with Teflon-lined butyl rubber septa (Wheaton,

Millville, NJ) and removed from the chamber. Three of the five vials from each group were pasteurLzed by shaking in a 74C + 4C water bath for 20 min and setting in ice water for 10 man; one of the three pasteurized vials from each group was then autoclaved for three hours. All vials, including the pasteurized and autoclaved vials, were incubated stationary at room temperature (23-26C) in the dark.

Sampling and PCB Analysis. The headspace gas in each sample vial was analyzed for methane before each
sampling for PCB analysis [13]. The sediment slurries were sampled (025 mL) in the anaerobic chamber at 14, 25, 36, 41, 69, and 92 days of room temperature incubation. The sediment slurry vials were crimp-sealed immediately after sampling and returned for further incubation. The PCBs were extracted from each sample by mechanical shaking at room temperature for _>14 hours with 6 mL of diethyl ether and -0.3 mL mercury Mercury was added to precipitate the elemental sulfur in the sediment A 90% extraction efficiency of the

658 PCBs in a sediment slurry was obtained using this extraction procedure [13, 19]. The enrichment cultures were sampled and the PCBs extracted in the same fashion as the sediment slurries at 4, 9, 14, 18, 26, and 36 days of room temperature incubation. The ether extracts were analyzed in a Varian 3500 gas chromatograph (GC) using a fused silica capillary column (30 m by 0.25 mm inner diameter) coated with DB-1 (polydimethylsiloxane, J&W Scientific, Folsom, CA) and an electron capture detector (ECD) at 300C. The concentrations of selected PCB congeners in a sample (listed in Table 1) were determined from a linear, three-point calibration curve of known concentrations of Aroclor 1242 using GC-ECD with the DB-1 column following the procedures previously described [21]. The peak numbering in Table 1 and Figures 2 and 3 is the 118 peak PCB profile from GC-ECD using a DB-1 column described by Frame et al. [21 ]. The concentrations of 2,3,6-CB; 2,4,6-CB; and 2,6-CB in the samples were also determined from a linear, three-point calibration curve of known concentrataons of these congeners. The PCB congeners 2,3,6-CB; 2,4,6-CB; and 2,6-CB (each at >99% purity); and Aroclor 1242 were purchased from Accustandard, Inc. (New Haven, CT). DB-1 peak number 5 containing 2,2'-CB and 2,6-CB was not included in the data of Figures 2 and 3, because both the added trichlorobiphenyls (added in large excess compared to the other PCB congeners) were dechlorinated to 2,6-CB. Including DB-1 peak number 5 in the data would significantly obscure the mole percent PCB congener calculations. Unfortunately, 2,2'-CB and 2,6-CB were the major dechlorination

products of Aroclor 1242. Exclusion of these congeners from the data calculations resulted in skewed mole percent difference histograms. For PCB congeners that were dechlorinated, the actual amounts were more than the calculated values displayed in Figures 2 and 3. For PCB congeners that were dechlorination products, the actual amounts were less than the calculated values displayed in Figures 2 and 3. Any calculations of rate and extent of PCB dechlorination based on this data would underestimate the true values. Nevertheless, the

microbial PCB dechlorination patterns are not obscured by the data calculations and they can be identified in the histograms in Figures 2 and 3. Extraction of Humic Acids. A humic acid extract was prepared using upper Hudson River sediment that did not contain PCBs (PCB-free sediment). The extraction procedure was modified from the method of Stevenson [22]. The sediment was collected upstream from the PCB-contaminated sediment at river mile 205. The wet sediment was extracted with an equal volume of 0.5 M NaOH by mechanical stirring for 14 hours at - 2 5 C The liquid extract was separated from the solids (sand and clay fractions) by gravity and centrifugation The pH of the extract (-11.5) was adjusted to - 6 by the drop-wise addition of concentrated HCI while magnetically stirring the solution. The pH adjustment resulted in precipitation of the humic acids, which were collected by centrifugation. The humic acid extract was washed four times with eight to ten volumes of distilled water and stored as a gel (9% solids by wt) at 4C The humic acid extract was 6% by weight of the sediment Preparation of Enrichment Cultures. Enrichment cultures of the two microbial PCB dechlorination activities were prepared in a medium containing the humic acid extract of PCB-free sediment. The humic acid extract

659 stock was diluted to 3% solids by weight with reduced RAMM. Aroclor 1242, and 2,3,6-CB or 2,4,6-CB were added to make final concentrations of 45 ppm (vol:vol) and 350 BM, respectively. Each sample was autoclaved for 30 minutes and allowed to cool to room temperature, before addition of an unheated, methanogenic sediment slurry (10% by vol) in which one or the other microbial PCB dechlorination activity had occurred. Two bacterial antibiotics, vancomycin and nisin, were added as stock solutions (5000 ppm each in reduced RAMM) to separate enrichment cultures; the final concentrations of either antibiotic were 5, 50, or 500 ppm For each of the two microbial PCB dechlorination activities, a set of duplicate enrichment cultures was treated with vancomycin or nisin at one of three concentrations, or untreated; there were 14 enrichment cultures for each activity. Both antibiotics were purchased from Sigma Chemical Company (St. Louis, MO); the

vancomycin was purchased as the hydrochloride salt and the nisin was purchased as a 2.5% solution in denatured milk solids. The 5000 ppm stock solution of nisin in reduced RAMM was prepared directly with the purchased material; thus enrichment cultures treated with nisin contained denatured milk solids RESULTS

PCB Dechlorination Activity Resulting from Trichlorobiphenyl Treatment.

The microbial PCB

dechlorination activities caused significant changes in the mole percent of specific PCB congeners in the sediment slurries and the enrichment cultures. The histograms in Figures 2 and 3 show the mole percent differences of PCBs in the sediment slurries (i.e., the mole percent of PCB congeners at selected time points minus the mole percent of PCB congeners at time zero). Dechlorination patterns M and Q were identified by decreases in specific congeners (the dechlorination substrates) and increases in the dechlorination products (6, 15) Both patterns M and Q dechlorination activities occurred in slurries amended with both 2,3,6-CB and 2,4,6-CB (top histogram in Figure 2). Pattern M activity occurred first in these slurries; it was evidenced by
meta

dechlorination of PCBs containing 2,5-dichlorophenyl, some 2,3-dichlorophenyl, and 2,3,6-

trichlorophenyl groups. By the next sampling time, pattem Q activity had occurred with p a r a dechlortnation of 2,4-dichlorophenyl and 3,4-dichlorophenyl groups, and p a r a dechlorination of some 2,4,5- and 2,4,6trichlorophenyl groups. At the last sampling time, only 10% of PCBs containing m e t a o r p a r a chlorines

remained. In a separate experiment, pattern M activity but not pattern Q activity was observed in slurries of the same sediment to which Aroclor 1242 but neither 2,3,6-CB nor 2,4,6-CB was added (unpublished data) In slurries amended with only 2,4,6-CB, only pattern Q dechlorination activity occurred An example of the changes in the mole percent of specific PCB congeners indicative of a pattern Q dechlorination is shown in the top histogram in Figure 3. This seemed to be the only PCB dechlorination activity that occurred in these slurries, as evidenced by the loss of the p a r a chlorine from 2,4'-CB and several PCBs containing 2,4- and 3,4dichlorophenyl, and 2,4,5-trichlorophenyl groups; as well as loss of the m e t a chlorine from PCBs containing

660

2
-5

il(llIllIE

11111rl[ItIilFIIil[Iltl!ll

10

13

16

19

22

25

28

31

34

37

40

43

46

49

52

5S

58

61

64

67

70

73

DB-1 Peak N u m b e r

9
8

= Q)7 o co s m

~-3 Q)
-62 ~Z
I
4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 SS 5B 61 64 67 70 73

DB-I Peak N u m b e r

Figure 2. Microbial reductive dechlorination of Aroclor 1242 in unheated, methanogenic Hudson River sediment slurries amended w~th 2,3,6-CB and 2,4,6-CB. The top histogram is the average of duplicate samples of the mole percent differences of PCB congeners between the selected time points and time zero The bottom histogram is a typical mole percent PCB congener distribution in the sediment slurries at time zero A listing of the congeners in each peak is shown in Table 1 2,3-dichlorophenyl, and 2,3,4- and 2,3,6-trichlorophenyl groups. By the last sampling, nearly all the p a r a chlorine-containing congeners were dechlorinated Both patterns M and Q dechlorination activities also occurred in slurries supplemented with only 2,3,6CB (bottom histogram in Figure 3). The pattern M dechlorination in these slurries was similar to the pattern M dechlorination in slurries amended with both trichlorobiphenyls (top histogram in Figure 2). This pattern was

661

2,4,6-CB Addition

s,]i 311 2Jl ,II

0

I i 1 I lllllll l ILIIIIIlIIIIIIIilII

III

lllll illil llill l llI

III I]ilITTIII III il III ]lilll I11 II Ill llllllll]l [[I II[II i1[ [liil[[ll[ Iiili [I
n

~ -1

~3

I IIIliillillll[ ,ll[ll o ! I~ lillllil IIIIItllitl - t! ' , ' " ; " ; ' ; 0 ;] ;~ ,9 2;'2~ 29 ;; ,, ~, ~4 ,, ,6':,~ s2 'd s~ 6, [ 6, ~o 7~
DB-1 Peak Number I FA2SDAYS 141 DAYS 92DAYSi 2,3,6-CB Addition

8
cO

~3 o~ ID -5

~JNIIIIIllllilllllllillllill o I|IIIILIIIIIIIIIII~IIIt[III.UII[LJII[IILLLI[t,

lllNlllllll lllllll'll"

[[! Illliillll[I

s IIIIIlllllllllllllllllllrlllltllllllllllllllllllli
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 ss

IIIIllllllI]l
58 61 64 67 70

[
73

DB-1 Peak Number m2S DAYS 4 1 DAYS 192 DAYS1

Figure 3. Microbial reductive dechlorination of Aroclor 1242 in unheated, methanogenic Hudson River sediment slurries amended with 2,3,6-CB or 2,4,6-CB Each histogram is the average of duplicate samples of the mole percent differences of PCB congeners between the selected time points and time zero A listing of the congeners in each peak is shown in Table 1 first observed as m e t a dechlorinataon of PCBs containing 2,5- and 2,3-dichlorophenyl, and 2,3,5- and 2,3,6trichlorophenyl groups. Additional m e t a dechlorination of 2,3- and 3,4-dicblorophenyl, and 2,3,4-

tnchlorophenyl groups occurred by the next sampling time. Pattern Q activity occurred after pattern M activity; it was identified by the p a r a dechlorination of several PCB congeners, including 2,4,2'-CB (peak 15). By the last sampling period, m e t a and p a r a dechlorination of nearly all the PCB congeners occurred.

662 TABLE 1. A listing of the primary PCB congener(s) quantified for each ~eak ~shown in Figures 2 and 3 DB-1 peak number a 2 6 7 congener(s) in peak 22,4-;':2,5DB-1 peak numbera 34 37 38 39 42 46 47 48 49 50 51 53 54 57 58 59 61 63 69 74 congener(s) in peak 2,4,5,2'-; 2,4,6,4'2,3,2',5'2,3,2',4'-; 3,4,4'-; 2,3,6,3'2,3,4,2'-; 2,3.6,4'2,3,2',3'2,4,5,4'2,5,3',4'2,4,3',4'-; 2,3,6,2',5'2,3,6,2',4'2,3,3 ',4'-; 2,3,4,4'2,3,6,2',3 '-; 2,3,5,2',5'2,3,5,2',4'-; 2,4,5,2',5'2,4,5,2',4'2,4,5,2',3'2,3,4,2",5'-; 2,3,4,6,4'2,3,4,2',4'2,3,6,3',4'2,3,4,2',3 '2,4,5,3',4'-; 2,3,6,2',4',5'2,3,4,3 ',4"-; 2,3,4,2',3',6"-

2,3'2,4'-; 2,32,6,2'2,5,2'-; 4,4'2,4,2'2,6,3'-; 2,3,62,3,2'-; 2,6,4'2,5,3"2,4,3 '2,5,4'2,4,4'2,3',4'; 2,3,3'-; 2,5,2',6'2,3,4'-; 2,4,2',6'2,3,6,2'2,3,2',6'2,5,2',5'2,4,2',5'2,4,2',4'-

8 10
14 15 16b 17 21 22 23 24 25 26 27 29 31 32 33

a This list includes only the peaks shown in Figures and 3. The peak numbers were assigned according to the sequence of 118 PCB peaks detected by DB-1 GC-ECD" the complete table was described by Frame el a1.[21]. b Because 2,6,3"-CB co-migrates with 2.3,6-CB, peak number 16 was not included in the data of slurries to which 2,3,6-CB was added (Figure 2 and the bottom chart of Figure 3).

Acclimation Times for PCB Dechlorination.

The acclimation time for a particular microbial PCB

dechlorination activity is an important indicator of stimulation and enrichment of the activity. An acclimation time was recorded as the first sampling time when the mole percent of a specific PCB congener had decreased (Table 2). Since PCB sampling was done at specific time points, the time at which dechlorination occurred between two testing intervals could not be determined. The acclimation time for pattem M dechlorination activity was the sampling time at which m e t a dechlorination of 2,5,2'-CB (peak 14) was first observed [6, 15]; at that time >60% of the congener was dechlorinated The sampling time at which p a r a dechlorination of 2,4,2'-CB (peak 15) was first observed was the acclimation time for pattern Q dechlorination activity [6, 15]; at

663 that time _>45% of the congener was dechlorinated Both 2,3,6-CB and 2,4,6-CB were _>80% dechlorinated to 2,6-CB at their acclimation times. TABLE 2. Observed acclimation times for PCB dechlorination in the Hudson River sediment slurries PCB dechlorinationa congener addition
CI

pasteurizationb

pattem M c and 2,3,6-CB-->2,6-CB

pattem Qc and 2,4,6-CB---)2,6-CB 41, 69 days d 69 days d

Yes Cl Cl 2,3,6-CB e l Cl ~ Cl 2,4,6-CB 2,3,6-CB & 2,4,6-CB Yes No Yes No No

14 days 25 days

not observed not observed 14 days 25 days

25 days 41 days 25 days 36 days

a No PCB dechlorinationwas observedin the autoclavedsedimentslurries. b Pasteurizationinhibitedmethanogenesis,whichwas observedin the unheated slurries. c Patternsdescribedby Brown [15], and Bedard and Quensen [6]. d Pattem Q was observedin one sampleat 41 days and in the other samplesat 69 days. N~ 2,4,6-CB was addedto these slurries. Some of the acclimation times for the dechlorination activities were the same within a sediment slurry, and between the differently treated slurries (Table 2). Meta dechlorination of 2,3,6-CB and meta dechlorination activity on Aroclor 1242 (pattern M) had the same acclimation time within a sediment slurry, irrespective of whether or not the slurries were treated with both trichlorobiphenyls or only 2,3,6-CB. Likewise, para

dechlorination o f 2,4,6-CB and p a r a dechlorination activity on Aroclor 1242 (pattern Q) had the same acclimation time within a sediment slurry; these dechlorinations also bad the same acclimation time when comparing pasteurized slurries amended with 2,4,6-CB, with and without 2,3,6-CB addition. In slurries of the same sediment to which Aroclor 1242 but neither 2,3,6-CB nor 2,4,6-CB was added, pattern M activity was observed by 36 days and pattern Q was not observed after 120 days (separate experiment, unpublished data) The acclimation times for patterns M and Q dechlorination activities differed relative to each other, and relative to the pasteurization or the trichlorobiphenyl treatments (Table 2). Pattern M activity occurred sooner than pattern Q activity within the same slurry and between the differently treated slurries. Pasteurization of the slurries caused shorter acclimation times for all the dechlorination activities relative to their acclimation times in the unheated slurries. The pattern Q activity occurred sooner in the unheated slurries to which both

664 trichlorobiphenyls were added, than in the unheated slurries to which only 2,4,6-CB was added. Pattern Q activity also occurred in slurries supplemented with only 2,3,6-CB (bottom histogram in Figure 3), but later than in slurries containing 2,4,6-CB. The significance of these acclimation time differences and similarities is discussed below. Enrichment of Microbial PCB Dechlorination Activities. Enrichment cultures of each dechlorination

activity were prepared by inoculating a culture medium with the 2,3,6-CB-amended or 2,4,6-CB-amended, methanogenic sediment slurries at day 43 of the experiment (when M or Q was the only dechlorination pattern seen in each slurry). After 9 days, meta dechlorination of 2,3,6-CB and pattern M dechlorination of the Aroclor 1242 was observed in the 2,3,6-CB-amended enrichment cultures that were inoculated with the sediment slurry in which pattem M was observed. After 14 days, para dechlorination of 2,4,6-CB and pattern Q dechlorination of the Aroclor 1242 was observed in the 2,4,6-CB-amended enrichment cultures that were inoculated vath the sediment slurry in which pattern Q was observed. By the last sampling time (36 days), only the microbial dechlorination activity that was transferred occurred in each enrichment culture. Pattern M activity was maintained through five serial transfers. However, pattern Q activity was not obtained after the third serial transfer. The enrichment culture media differed from the sediment slurry media, in that complete Hudson River sediment was used in the slurry media, whereas a humic acid extract of PCB-free upper Hudson River sediment was used in the enrichment culture media. Antibiotic treatment of a microbial microcosm can be used to identify or select specific microbial populations. The enrichment cultures of the microbial PCB dechlorination activities were treated with two antibiotics, vancomycin and nisin, that are known to inhibit gram-positive microorganisms. Vancomycin is a glycopeptide that inhibits cross linking of the peptidoglycan subunits on the bacterial cell membrane; the large size of the vancomycin molecule (1488 daltons) severely impairs its effectiveness against most gram-negative bacteria, which have several outer layers surrounding the cellular membrane [23]. Nisin is a small polypeptide that inhibits growth of a broad spectrum of gram-positive bacteria and the outgrowth of germinated Clostridia spores [24, 25]. No PCB dechlorination was observed in any enrichment culture treated with 5, 50, or 500 ppm vancomycin; or 500 ppm nisin. PCB dechlorination was observed in the enrichment cultures treated with 5 and 50 ppm nisin. These results show that a gram-positive microbial population is essential for both microbial PCB dechlorination activities. DISCUSSION Trichlorobiphenyl Stimulation of PCB Dechlorination Activities. The additions of 2,3,6-CB and 2,4,6-CB to PCB-contaminated upper Hudson River sediment slurries stimulate the production of reductive dechlorination patterns M and Q, respectively. These conclusions are supported by the following evidence.

665 First, pattems M and Q were observed at the same sampling time as m e t a and para dechlorinatlon of 2,3,6-CB and 2,4,6-CB, respectively (Table 2). Exact acclimation times and dechlorination rates in the heterogeneous sediment slurries could not be measured. However, the dechlorination patterns were identified from the PCB congener distribution in the sediment slurries. For example, in slurries supplemented with both

trichlorobiphenyls, pattern M dechlorination and m e t a dechlorination of 2,3,6-CB were clearly observed before pattern Q dechlorination and p a r a dechlorination of 2,4,6-CB. Second, pattern Q occurred significantly sooner in the 2,4,6-CB-amended slurries than in the 2,3,6-CB-amended slurries. Third, pattern Q but not pattern M was observed in slurries amended with only 2,4,6-CB. Fourth, the acclimation time for pattern M was longer in slurries with no trichlorobiphenyl treatment than in slurries treated with 2,3,6-CB (36 days compared to 25 days); pattern Q was not observed in slurries with no trichlorobiphenyl addition. Fifth, separate enrichment cultures of each microbial dechlorination activity were prepared with amendments of 2,3,6-CB or 2,4,6-CB These data demonstrate that each trichlorobiphenyl amendment is associated with a distinct microbial PCB dechlorination pattern, implying a stimulatory role of the two congeners in the sediment slurries A pattern M and then pattern Q dechlorination sequence was observed in sediment slurries treated with both trichlorobiphenyls (Figure 2) and in sediment slurries amended with only 2,3,6-CB (bottom histogram in Figure 3). In the latter case, pattern Q was observed significantly later than pattern M and also later than pattern Q in the 2,4,6-CB-amended slurries; it is unclear if pattern Q was stimulated by the 2,3,6-CB addition, the pattern M dechlorination products, or another factor(s) within the 2,3,6-CB-amended slurries (discussed further below). There have been several reports that the sequential pattern M and then pattern Q dechlonnation activities occurred in PCB-contaminated upper Hudson River sediment slurries or microcosms [6, 9, 11, 13-15] It was proposed [6, 15] that together these activities cause a pattern C dechlorination, which is an environmental PCB dechlorination pattern found in upper Hudson River sediments [2-5] It is possible that the successive, microbial patterns M and then Q dechlorination activmes do not function independently This possibility is suggested m two ways. First, in slurries to which only 2,3,6-CB was added, pattern M was stimulated by the trichlorobiphenyl addition but pattern Q also occurred, albeit significantly later. Second, in the unheated slurries to which 2,4,6-CB was added there was a shorter

acclimation time for pattern Q activity if pattern M activity had occurred; the acclimation time was 36 days in slurries amended with both trichlorobiphenyls compared to 41 days in slurries amended with only 2,4,6-CB These data suggest that the pattern M activity might have affected stimulation of pattern Q activity The

difference in the acclimation times for pattern Q activity in unheated slurries amended with 2,4,6-CB was not observed in pasteurized slurries, implying that pasteurization eliminates some interaction between the two PCB dechlorination activities. These data could indicate that pattern C dechlorination (the result of combined patterns M and Q) is caused by a microbial consortium. Shelton and Tiedje [26] isolated an anaerobic, bacterial consortium that could dechlorinate and mineralize 3-chlorobenzoic acid

666 The current research shows that pasteurization can be used in conjunction with 2,3,6-CB or 2,4,6-CB to stimulate microbial patterns M and Q dechlorination activities in PCB-contaminated upper Hudson River sediment. This conclusion is supported by the observation that pasteurization caused a decrease in the

acclimation times for both dechlorination activities when compared to their acclimation times in the unheated samples (Table 2). Stimulation and enrichment of two other para dechlorination activities, processes P and H, have recently been reported [17, 27] The congener specificity of process Q is distinctly different from the other

para dechlorination activities. Pattern Q dechlorination results in removal of all para chlorines from mono-, di-,
and trichlorophenyl groups and is not limited to removal of flanked para chlorines like processes P and H [6]. Bedard and co-workers [17] reported stimulation of pattem P dechlorination on PCBs in Housatonic River sediment slurries by the addition of 2,5,3 ',4'-CB. Ye et al. [27] provided evidence that pattem H dechlorination is caused by methanogenic bacteria. The results of the pasteurization and antibiotic treatments in the curren! report, and o f Y e et al. [27] suggest that patterns H and Q are caused by two different microbial populations. Enrichment of Microbial PCB Dechlorination Activities. A 10% by volume inoculation of a culture medium with a sediment slurry containing either PCB dechlorination activity resulted in a decrease in the acclimation time for dechlorination relative to what was observed in the primary cultures (the sediment slurries). These results demonstrate enrichment of each microbial activity For example, the pattern Q activity had a 41-day acclimation time in the unheated sediment slurries, but only a 14-day acclimation time in the enrichment cultures. This is the first report of a microbial pattern Q dechlorination activity enrichment culture. Both Morris et al. [16] and Ye et al. [14] reported loss of pattern Q activity in enrichment cultures prepared with a method similar to the current experiments. Previous laboratory investigations of microbial PCB dechlorination have used sediment both as a source of microorganisms and a growth matrix [6, 8, 10, 16, 14]. While isolation of a bacterium capable of PCB dechlorination has not been successful, May and co-workers [10] were successful in subculturing a PCBdechlorinating, anaerobic enrichment on solid media However, their bacterial colony could only cause

dechlorination in a sediment slurry. A defined culture medium is needed for isolation of the microorganism or consortium that causes PCB dechlorination and for understanding how PCB dechlorination occurs. To this end, the current experiments indicate that the humic acid fraction of river sediment, separate from the sand and the clay fractions, supports microbial PCB dechlorination. The physical or chemical mechanisms by which humic acids support PCB dechlorination are not known. Very little is known about what genera of bacteria might cause PCB reductive dechlorination. Pasteurization experiments have shown that spore-formers cause patterns M and Q dechlorination [13, 14, and the current study]. Bedard and Quensen [6] proposed that pattern M dechlorination could be caused by a sulfate-reducing, spore-forming bacterium such as a species of Desul/btomaculum, which is the only known genus of sulfate-reducing spore-formers Desul~tomac~dllm are gram-negative bacteria; hence they are likely

667 to be resistant to vancomycin or nisin treatments. On the other hand, Clostridia are gram-positive, anaerobic, spore-forming bacteria; but sulfate reduction is uncommon in species of this genus. Further study of the enrichment' cultures of each microbial PCB dechlorination activity should provide insights about the type of bacteria that cause PCB reductive dechlorination.
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