Environmental Microbiology (2000) 2(6), 703±708

Brief report

Monitoring of Desulfitobacterium frappieri PCP-1in pentachlorophenol-degrading anaerobic soilslurry reactors

M. Lanthier, R. Villemur, F. LeÂpine, J.-G. Bisaillon and

R. Beaudet*

INRS-Institut Armand-Frappier, Centre de Microbiologie

et Biotechnologie, Universite du QueÂbec, Ville de Laval,

Qc, Canada H7V 1B7.

Summary

Anaerobic biodegradation of pentachlorophenol (PCP)

was studied in rotative bioreactors containing 200 g

of PCP-contaminated soil and 250 ml of liquid

medium. Reactors were bioaugmented with cells of

Desulfitobacterium frappieri strain PCP-1, a bacter-

ium able to dehalogenate PCP to 3-chlorophenol.

Cells of strain PCP-1 were detected by quantitative

PCR for at least 21 days in reactors containing

500 mg of PCP per kg of soil but disappeared after

21 days in reactors with 750 mg of PCP per kg of soil.

Generally, PCP was completely removed in less than

9 days in soils contaminated with 189 mg of PCP per

kg of soil. Sorption of PCP to soil organic matter

reduced its toxicity and enhanced the survival of

strain PCP-1. In some non-inoculated reactors, the

indigenous microorganisms of some soils were also

able to degrade PCP. These results suggest that

anaerobic dechlorination of PCP in soils by indigen-

ous PCP-degrading bacteria, or after augmentation

with D. frappieri PCP-1, should be possible in situ and

ex situ when the conditions are favourable for the

survival of the degrading microorganisms.

Introduction

Pentachlorophenol (PCP) is a toxic compound that has

been used since the 1930s as a biocide and a wood-

preserving agent. Utilization of this chemical on a world-

wide scale has led to important soil and groundwater

pollution, principally at wood-treating plants. Aerobic

biotreatments of PCP-contaminated soils are already

available, but as soil is anoxic at a depth of a few

centimetres usage of anaerobic in situ biotreatments

could be more advantageous. Anaerobic ex situ biotreat-

ments are also advantageous because they are less

costly than aerobic treatments and because they do not

need any aeration system, produce less biomass and

methane recuperation can increase their profitability.

However, anaerobic bacteria often work in consortium,

and the lack of knowledge of these systems often limits

their industrial use. Study of these microorganisms

could lead to the development of performing large-scale

anaerobic treatment.

Desulfitobacterium frappieri strain PCP-1 (ATCC

300357) is a strict anaerobic bacterium that has been

isolated from a methanogenic consortium that can

degrade PCP (Bouchard et al., 1996). This microorganism

can dehalogenate PCP to 3-chlorophenol (3-CP) via

reductive dehalogenation. Also, strain PCP-1 can dechlor-

inate at ortho, meta and para positions a large variety of

aromatic molecules with substituted hydroxyl or amino

groups (Dennie et al., 1998). Furthermore, D. frappieri

PCP-1 was able to compete with other microorganisms of

a mixed bacterial community in a continuous anaerobic

reactor, degrading PCP and augmenting with PCP-1 cells

(Tartakovsky et al., 1999).

In the development of PCP-degrading bioprocesses

involving strain PCP-1, its monitoring is important for

understanding its population dynamic, the degradation

mechanisms implicated and also to acquire information

about the reliability of the process. The polymerase chain

reaction (PCR) and competitive PCR (cPCR) have been

used with success to monitor D. frappieri strain PCP-1

and Desulfitobacterium dehalogenans introduced into

non-sterile soil or soil slurry microcosms (El Fantroussi

et al., 1997a, b; LeÂvesque et al., 1997, 1998). A previous

report showed that strain PCP-1 can dechlorinate PCP in

anaerobic soil slurry microcosms and can be monitored by

PCR when introduced in this system (Beaudet et al.,

1998).

The objective of the present work was to measure the

impact of PCP on the population of D. frappieri strain

PCP-1 introduced into anaerobic rotative bioreactors

Q 2000 Blackwell Science Ltd

Received 11 June, 2000; revised 8 July, 2000; accepted 17 August, 2000.*For correspondence. E-mail rejean.beaudet@INRS-iaf.uquebec.ca;Tel. (11) 450 686 5010; Fax (11) 450 686 5501.

containing diverse PCP-contaminated soils. Monitoring of

the PCP-1 population was carried out by PCR and cPCR.

PCP biodegradation and PCR monitoring of

D. frappieri strain PCP-1 in rotative bioreactors

Effect of different concentrations of PCP

The effect of PCP concentrations on the survival of strain

PCP-1 was determined with five bioreactors containing

MS soil (for soil description, see Table 1) contaminated,

respectively, with 0, 100, 300, 500 and 750 mg of PCP

per kg of soil. Each reactor was inoculated with 38.9 ml of

an exponentially growing culture (OD600 � 0.395) of cells

of strain PCP-1. The results obtained by PCR analysis

showed that strain PCP-1 was detected in all samples

(solid and liquid) taken at days 0, 3, 7, 12 and 21 from the

bioreactors contaminated with 0, 100, 300 and 500 mg of

PCP per kg of soil (data not shown). In the reactor with

750 mg of PCP per kg of soil, a PCR signal for cells of

strain PCP-1 was obtained in all samples except at day

21, where no signal was observed in the solid and liquid

phase samples (Fig. 1A). The fluctuations of the popula-

tion of strain PCP-1 in this bioreactor was determined by

quantitative PCR (Table 2). At day 0, the population was

estimated at 1.3 � 109 cells g21 in the soil fraction and at

1.9 � 107 ml21 in the liquid phase, and this population

was mostly stable until day 7. At day 12, an important

decrease was observed as PCP-1 cell concentration was

estimated at 2.7 � 104 cells g21 in the soil fraction and

was not detected in the liquid phase. At day 21, PCP-1

was not detected in both phases.

Effect of agitation

This experiment was carried out to determine whether a

continuous agitation of the bioreactors would have a

greater impact on the PCP biodegradation and on the

survival of cells of strain PCP-1 compared with a

1 h day21 agitation. Three bioreactors containing MS

soil contaminated with PCP were rotated continuously

and three others were rotated for only 1 h day21. Each

group of bioreactors was composed of an abiotic control

(contaminated with 100 mg of PCP per kg of soil) and two

bioreactors inoculated with approximately 106 cfu ml21 of

strain PCP-1 (contaminated, respectively, with 100 or

750 mg of PCP per kg of soil).

Complete degradation of PCP was observed (data not

shown) in less than 7 days in the bioreactors contami-

nated with 100 mg of PCP per kg of soil and inoculated

with PCP-1 cells, independently of the type of agitation. A

PCR signal for PCP-1 cells was generally obtained in

these bioreactors (samples taken at days 0, 3, 7, 13 and

21). Quantitative PCR analysis of samples showed no

important fluctuations of the PCP-1 populations between

the continuously agitated (at day 0, 7.3 � 107 cells g21

and 1.6 � 108 cells ml21; at day 13, 1.4 � 108 cells g21

and 7.5 � 108 cells ml21) or only 1 h day21 agitated

reactors (at day 0, 3 � 107 cells g21 and 7.2 � 107

cells ml21; at day 13, 2.1 � 108 cells g21 and 8.2 � 107

cells ml21). Some biodegradation products such as

trichlorophenols (TCPs) and dichlorophenols (DCPs)

Table 1. Characteristics of the soils used in the biodegradationexperiments in rotative bioreactors.

Soil Pollutant

Organiccarbon(%)

Organicmatter(%)

Watercontent(%) pH

ITB PCP: 180 mg kg21 6.3 12.5 7.6 6.9Creosote: 750 mg of PAH kg21

MSa No 3.1 6.2 7.8 6.8TS No 9.1 18.5 43.4 7.8RIM PCP: 1 mg kg21 1.6 3.2 8.2 ND

PAH, polycyclic aromatic hydrocarbons; ND, not determined.a. 50% sand, 37% silt, 13% clay. All soils were sieved (3±4 mm) andkept at 48C in the dark. The ITB soil (heavily contaminated with PCPand creosote) was obtained from a wood-treating plant in theprovince of Quebec, Canada. These soils have a sandy appearancebut differ in their total organic carbon content.

Fig. 1. Detection of strain PCP-1 in rotative bioreactors by PCR.Total DNA was extracted from samples and amplified by PCR withPCP-1-specific primers (PCP1G/PCP4D). PCR products wereelectrophoresed onto 1.2%21.4% agarose gel.A. MS soil contaminated with 750 mg of PCP per kg of soil andinoculated with 106 PCP-1 cells ml21. Solid and liquid samples weretaken at day 0 (lanes 1 and 2), day 3 (lanes 3 and 4), day 7 (lanes 5and 6), day 12 (lanes 7 and 8) and day 21 (lanes 9 and 10).B. MS soil contaminated with 105 mg of PCP per kg21 of soil.Samples were taken at day 0 (lanes 1±4) and day 7 (lanes 5±8)from the abiotic control (lanes 1 and 5), the biotic control (lanes 2and 6) and both bioreactors inoculated with strain PCP-1 (104 PCP-1 cells ml21 at lanes 3 and 7 and 107 PCP-1 cells ml21 at lanes 4and 8).

Table 2. Estimation of cell concentration of strain PCP-1 in rotativebioreactor containing 750 mg of PCP kg21 of MS soil by cPCR.

Days In soil (cells g21) In liquid phase (cells ml21)

0 1.3 � 109 1.9 � 107

3 2.3 � 108 1.6 � 108

7 1.0 � 108 2.6 � 108

12 2.7 � 104 Not detected21 Not detected Not detected

704 M. Lanthier et al.

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 703±708

were detected in all bioreactors except the abiotic control.

3-CP was also detected, but only in the bioreactors

contaminated with 100 mg of PCP per kg of soil. No PCP

biodegradation and no PCP-1-specific PCR signals were

observed after a 3 day incubation in the bioreactors

contaminated with 750 mg of PCP per kg of soil and

inoculated with PCP-1 cells (data not shown).

Effect of inoculum size

Four bioreactors containing MS soil contaminated with

105 mg of PCP per kg of dry soil were used in an

experiment examining the effect of the inoculum size.

The bioreactors consisted of an abiotic control, a non-

inoculated reactor and two bioreactors inoculated, respec-

tively, with approximately 104 and 107 PCP-1 cells ml21.

The PCP was completely degraded in less than 7 days in

both inoculated bioreactors and the non-inoculated

reactor (data not shown). TCPs, DCPs and 3-CP were

detected in these three bioreactors. No PCP biodegrada-

tion occurred in the abiotic control. At day 0, a specific

PCR signal for strain PCP-1 was detected only in the

bioreactor inoculated with 107 PCP-1 cells ml21 (Fig. 1B).

The PCP-1 concentration in the bioreactor inoculated with

104 cells ml21 was probably below the limit of detection of

the PCR method. However, specific PCR signals for strain

PCP-1 were obtained at day 7 in the two inoculated

reactors but surprisingly also in the non-inoculated

reactor. PCR analysis of samples taken from the non-

inoculated reactor carried out independently, to avoid

cross-contamination, generated the same results.

Assays with TS soil

PCP biodegradation was evaluated in reactors containing

TS soil contaminated with 189 mg of PCP per kg of dry

soil and aged at 48C for 40 days (for soil description, see

Table 1). Two groups of bioreactors were used in this

experiment: the first group was composed of reactors

containing 250 ml of liquid medium and the second group

of reactors with only 25 ml of liquid medium. All the

reactors contained 200 g of PCP-contaminated soil. Each

group was composed of an abiotic control, a non-

inoculated control and a reactor inoculated with approxi-

mately 107 PCP-1 cells per g of soil.

Complete removal of PCP in less than 12 days was

observed in the inoculated reactors but also in non-

inoculated reactors, as observed with the MS soil (Fig. 2).

PCP dechlorination was slightly faster in reactors contain-

ing 25 ml of liquid medium than in those containing 250 ml

of liquid medium. Less chlorinated phenols, TCPs, 3,5-

DCP and 3-CP were detected in the different reactors

except for the abiotic reactor. 3-CP was slowly or

not degraded after 18 days incubation. However, in the

non-inoculated reactor containing 250 ml of liquid medium,

the 3-CP was almost completely degraded. PCP concen-

tration in the liquid phase of all bioreactors never exceeded

5 mg l21 throughout the experiment, suggesting that PCP

was adsorbed to the soil.

The PCR monitoring of PCP-1 cells showed that a

specific PCR signal was obtained in samples taken from

the inoculated bioreactors. No PCP-1-specific PCR signal

was obtained in the samples taken from the non-

inoculated reactors at day 0, but a signal was detected

in samples at days 9, 18 and 25. No PCP-1-specific PCR

signal was obtained in samples taken from the abiotic

reactor.

Assays with RIM soil

The RIM soil is a sandy soil that was slightly contaminated

with PCP (for soil description, see Table 1). It was con-

taminated by the addition of PCP to a final concentration

A150

40

30

20

10

0A2

40

30

20

10

0

PCPTriCPs3,5-DCP3-CP

A340

30

20

10

0 B140

30

20

10

0B2

40

30

20

10

00 4 9 18

Tota

l CP

s (µ

mo

l) in

rea

cto

r

Days

Fig. 2. PCP dechlorination in anaerobic rotative bioreactorscontaining 200 g of PCP-contaminated TS soil and (A) 250 ml or(B) 25 ml of liquid medium. A1, abiotic control; A2 and B1, non-inoculated reactors; A3 and B2, reactors inoculated with 107 PCP-1cells ml21.

Monitoring of D. frappieri PCP-1 705

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 703±708

of 224 mg of PCP per kg of dry soil. Three reactors were

used: an abiotic control, a non-inoculated control and a

reactor inoculated with approximately 107 PCP-1 cells per

g of soil. The bioreactors contained 187.5 ml of liquid

medium and 150 g of RIM soil, except the abiotic control,

which contained 200 g of RIM soil and 250 ml of liquid

medium.

No PCP degradation was observed in any of the

bioreactors used in this experiment. A PCP concentration

of 100 mg l21 and over was observed in the liquid phase

of all bioreactors. No PCP-1-specific PCR signals were

obtained in samples taken from the abiotic and the non-

inoculated bioreactors. A PCP-1-specific PCR signal for

strain PCP-1 was obtained in the inoculated bioreactor at

days 0 and 7, but not at days 14 and 21.

Assays with a mixture of ITB and MS soil

Because the ITB soil was already heavily contaminated

with creosote and PCP (approximately 180 mg of PCP

per kg of dry soil), this soil was diluted with the non-con-

taminated MS soil to reduce its toxicity (for soil descrip-

tion, see Table 1). Each bioreactor contained 250 ml of

liquid medium and 200 g of a mixture of MS and ITB soils.

Five bioreactors were used: an abiotic control, a non-

inoculated reactor and three reactors inoculated with

approximately 107 PCP-1 cells ml21. The abiotic control,

the non-inoculated control and one inoculated bioreactor

contained 83% MS soil and 17% ITB soil. Of the two other

inoculated reactors, one contained a mixture of 50% ITB

and 50% MS soil and the other contained 100% ITB soil.

PCP was added in the reactors containing MS soil to

obtain the same concentration of PCP in all bioreactors.

No PCP biodegradation and no degradation products

were observed in all bioreactors used in this experiment,

even in the reactors containing the most diluted ITB soil. A

PCP-1-specific PCR signal was only observed in the

inoculated bioreactors at days 0, 4 and 8, but not at days

14 or 21.

Discussion

Rapid PCP degradation in less than 9 days was observed

in rotative bioreactors containing MS or TS soil con-

taminated with up to 189 mg of PCP per kg of dry soil.

The addition of D. frappieri PCP-1 to the reactors was not

necessary to obtain the PCP degradation. The indigenous

microorganisms in those soils were able to degrade PCP

as effectively as the reactors bioaugmented with strain

PCP-1. Beaudet et al. (1998) have also described the

PCP-degrading activity of the indigenous microflora in

some anaerobic soil slurry microcosms. They showed that

the inoculation of D. frappieri was necessary to obtain the

PCP degradation when the indigenous microorganisms were

unable to degrade PCP. El Frantroussi et al. (1997) also

observed the anaerobic degradation of chlorinated com-

pounds in soil slurry microcosms inoculated with D.

dehalogenans or Desulfomonile tiedjei. The inoculation

resulted in a shortening of the period required for the

dechlorination of 3-chloro-4-hydroxyphenoxyacetic acid.

The PCP biodegradation was as effective in the reactor

rotated for 1 h per day as in continuously rotated

reactors and in reactors containing only 25 ml of liquid

medium as compared with 250 ml, suggesting that in situ

biodegradation in submerged soils should be possible.

Less chlorinated phenols, principally TCPs, DCPs and

MCPs (3-CP), accumulated in the reactors containing MS

or TS soil, as observed by Mikesell and Boyd (1988) from

anaerobic biodegradation studies of PCP-contaminated

soils. Degradation of 3-CP is the limiting step in the PCP

degradation as it accumulated in the reactors and was

generally slowly degraded in the conditions used. Under

anaerobic conditions, the lesser chlorinated phenols

were degraded more slowly than the higher chlorinated

phenols.

No PCP biodegradation was observed in reactors

containing ITB or RIM soils. As the ITB soil was heavily

contaminated with creosote, the high toxicity found in

those conditions might be responsible for the elimination

of PCP-1 cells in the reactors, as determined by PCR

analysis and for the absence of PCP degradation. With

reactors containing the RIM soil, a high PCP concentra-

tion in the liquid phase (over 100 mg l21) was found,

suggesting that these conditions are toxic for the

degrading microorganisms. This was confirmed by PCR

analysis in which PCP-1 cells were detected after 7 days

but not after 14 days of incubation. As D. frappieri PCP-1

is a spore-forming bacterium, it can probably survive in

soil when the conditions are unfavourable. However,

the resistance of spores and their number make their

detection by PCR analysis difficult. The low total organic

carbon content (1.6%) of the RIM soil has probably limited

the sorption of PCP to soil particles. On the contrary, MS

and TS soils have, respectively, a content of 3.1% and

9.5% of total organic carbon and the PCP concentrations

determined in the liquid phase of reactors containing

these soils were always lower than 5 mg l21. This result

suggests that sorption of PCP to soil organic matter might

reduce its toxicity and enhance the survival of strain PCP-

1 and other degrading microorganisms. The sorption of

PCP to organic matter has been reported previously and a

variation in the PCP extraction yield has been observed

according to the soil used. Indeed, Lagas (1988),

McFarland et al. (1994) and Scheunert et al. (1995)

showed that a fraction of the PCP added in soil is bound

irreversibly and that the proportion of these inextractable

complexes increases with time. This could explain the low

extraction yields (50%) obtained with TS soil as this soil

706 M. Lanthier et al.

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 703±708

was aged for 40 days after the addition of PCP, thus

favouring the formation of inextractable PCP-bound resi-

dues. As suggested by Boyd et al. (1989) and McFarland

et al. (1994), the formation of inextractable compounds

could be mediated by bacteria and microscopic fungus.

The PCP degradation observed in the non-inoculated

reactors suggests that PCP-degrading microorganisms

were present in TS and MS soil. A PCR signal for D.

frappieri was not detected initially in these soils but was

obtained after few days of incubation, suggesting that the

number of cells of D. frappieri in these soils was under the

limit of detection (4 � 104 cells ml21; LeÂvesque et al.,

1997). As soluble PCP is relatively toxic for strain PCP-1

and inhibited its growth at a concentration between 5 and

10 mg l21, the growth of PCP degraders is probably

caused by the optimal culture conditions and the supple-

ment of glucose, formate and yeast extract added to the

medium rather than by PCP addition.

This work suggests that anaerobic dechlorination of

PCP in soil by the indigenous PCP-degrading bacteria, or

after bioaugmentation with D. frappieri, should be possible

in situ and ex situ when the conditions are favourable for

the survival of the degrading microorganisms.

Experimental procedures

Microorganisms and culture conditions

D. frappieri strain PCP-1 (ATCC 700357) was cultivated at378C in 70 ml glass serum bottles containing 35 ml ofanaerobic liquid medium supplemented with 55 mM pyruvateand 0.1% yeast extract (Dennie et al., 1998). A concentrationof 50 mM 2,4,6-trichlorophenol (2,4,6-TCP) was added to themedium to induce the ortho-dechlorinating activity. The cellconcentration of strain PCP-1 in the vial cultures wasdetermined by cfu plating on anaerobic Columbia agarmedium ANA 1121 (Laboratoires Quelab) and/or by theoptical density at a wavelength of 600 nm.

Rotative bioreactors

Bioreactors were 1 l Roller glass bottles (10 � 15 cm)(Fisher Scientific) containing 200 g of soil and 250 ml ofanaerobic liquid medium. The soils were weighed and put inthe reactors. These were kept overnight in an anaerobicchamber under a gas mixture composed of 80% N2/10% H2/10% CO2. An anaerobic liquid mineral salt medium supple-mented with 2.8 mM glucose and 16 mM sodium formate and0.1% yeast extract was added to the anoxic soil (Beaudetet al., 1998). Some bioreactors were inoculated withexponentially growing PCP-1 cells at an initial concentrationof 1062107 cells ml21 of liquid medium, unless stateddifferently. Abiotic controls were made with a reactorcontaining autoclaved soil (1 h, two times at 24 h interval)and 10 g l21 of sodium azide. All bioreactors were rotated atone revolution min21 with a Bello-Corbeil culture systemapparatus (Belco Glass) and kept in the dark at 298C.

Samples of liquid medium (5±15 ml) and wet soil (5±15 g)were taken periodically for chlorophenol and molecularbiology analyses.

Duplicate samples of solid and liquid phases of thebioreactors were analysed periodically. Extraction of chlor-ophenols and analysis by high-performance liquid chromato-graphy (HPLC) were performed by a method alreadydescribed by Beaudet et al. (1998). PCP extraction yield atday 0 varied between 50% and 95% (according to the soilused and its organic matter content) compared with the initialPCP concentration in the bioreactors.

DNA extraction, PCR and competitive PCR

DNA extraction and PCR protocols were described byLeÂvesque et al. (1997), except that, instead of SephadexG-200 columns, polyvinylpolypyrrolidone (PVPP) columnswere used as the last step of DNA purification (Berthelet et al.,1996). The internal standard and the competitive PCRprotocol used in these experiments were described byLeÂvesque et al. (1998).

All PCR reactions contained 0.5 mg ml21 bovine serumalbumine. Universal 16S rDNA eubacterial primers were 5 0-AGAGTTTGATCCTGGCTCAG-3 0 and 5 0-TTACCGCGGC[T/G]GCTGGCAC-3 0 corresponding to positions 8±27 and533±515, respectively, in the 16S rRNA gene of Escherichiacoli (GenBank accession no. J01695). Specific primers for D.frappieri were PCP1G (5 0-CGAACGGTCCAGTGTCTA-3 0)and PCP4D (3 0-AGGTACCGTCATGTAAGTAC-5 0) (LeÂv-esque et al., 1997) and for the genus DesulfitobacteriumDe1 (5 0-GCTATCGTTA[G/A]T[G/A]GATGGAT-3 0) and De2(5 0-CCTAGGTTTTCACACCAGACTT-3 0), corresponding topositions 322±341 and 725±704, respectively, in the 16SrRNA gene of D. frappieri strain PCP-1 (U40078). De1 andDe2 were checked with the PCR PROBE program of theRibosomal Database Project (http://www.cme.msu.edu/RDP/html/index.html) and matched only Desulfitobacterium 16SrRNA gene sequences. PCR amplifications with the De1/De2primers were carried out at 508C instead of 558C. Positivecontrol for PCR amplifications was carried out with theuniversal primers.

Acknowledgements

This work was supported by the Natural Sciences and Engineer-ing Research Council of Canada (NSERC) and by Fonds pour la

Formation de Chercheurs et l'Aide aÁ la Recherche (FCAR).

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