Letters in Applied Microbiology 2003, 36, 315320

Poly-b-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37

lez3 and M.A. Mart nez1 F.A. Godoy1, M. Bunster2, V. Matus3, C. Aranda1, B. Gonza
1

gicas, Universidad de Departamento de Microbiologa and 2Departamento de Biologa Molecular, Facultad de Ciencias Biolo tica Molecular y Microbiologa, Facultad de Ciencias Biolo gicas, n, Casilla, Concepcio n, Chile, and 3Departamento de Gene Concepcio lica de Chile, Alameda, Santiago, Chile Ponticia Universidad Cato

2002/309: received 17 October 2002, revised 23 January 2003 and accepted 31 January 2003

ABSTRACT
LEZ AND M.A. MART F.A. GODOY, M. BUNSTER, V. MATUS, C. ARANDA, B. GONZA I N E Z . 2003.

Aims: To analyse the possible effect of poly-b-hydroxyalkanoate (PHA) consumption on 2,4,6-trichloro phenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP. Methods and Results: The strain was inoculated in saline solution supplemented with 2,4,6-TCP (25400 lM). Chlorophenol degradation was followed both spectrophotometrically and by chlorine released; viable bacterial counts were also determined. Cells starved for 24, 48 or 72 h were incubated with 25 lM of 2,4,6-TCP and PHA in cells investigated by spectrouorimetric and ow cytometry. Results demonstrated that starvation decreased the ability to degrade 2,4,6-TCP. After 72 h of starvation, degradation of 2,4,6-TCP decreased to less than 10% and the relative PHA content diminished to ca 50% during the rst 24 h. Conclusion: Utilization of PHA may be an important factor for the degradation of toxic compounds, such as 2,4,6-TCP, in bacterial strains unable to use this toxic compound as carbon and energy source. Signicance and Impact of the Study: This is the rst study describing a relationship between intracellular PHA consumption and 2,4,6-TCP degradation. Therefore, PHAs provides an endogenous carbon and energy source under starvation and can play a signicant role in the degradation of toxic compounds. Keywords: poly-b-hydroxyalkanoate, Sphingopyxis chilensis, starvation, 2,4,6-trichlorophenol degradation.

INTRODUCTION Various chlorophenols, 2,4,6-trichlorophenol (2,4,6-TCP) included, are produced secondarily by the cellulose industry and are severe pollutants because they are toxic, persistent and bioaccumulated (Andreoni et al. 1998). Several heterotrophic bacteria have been isolated because of their ability to use a chlorophenol as sole carbon and energy source, and bacterial strains that can degrade but not grow on chlorophenols have also been reported (Chaudhry and Chapalamadugu 1991; Lee et al. 1998). The presence of easily
Correspondence to: M.A. Martnez, Departamento de Microbiologa, Facultad de gicas, Universidad de Concepcio n, Casilla, Concepcio n, Chile (e-mail: Ciencias Biolo mimartin@udec.cl).

metabolizable substrates (glucose, glutamate or rhamnose) lez enhances bacterial degradation by chlorophenols (Gonza and Hu 1991; Aranda et al. 1999). Therefore, chlorophenolinduced bacterial degradation in the environment should be affected by nutrient availability, although environmental bacteria are frequently in a starved state (Morita 1988). Under inanition, bacteria may remain viable for a long time if appropriate intracellular endogenous reserves are available (Kolter et al. 1993; Ruiz et al. 2001). Poly-b-hydroxyalkanoate (PHA) is an intracellular storage form of carbon and energy in bacterial cells (Lee 1996) and provides carbon and energy during starvation (James et al. 1999). PHA might be used as an energy source to transform trichloroethylene by methanotrophic bacteria (Henrysson and McCarty 1993). The aim of this work was to analyse the

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possible effect of PHA consumption on 2,4,6-TCP degradation during starvation by Sphingopyis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP.

Table 1 PCR primers used in this work Primer MON-F MON-R HQ-F HQ-R MAR-F MAR-R Sequence Source

M A T E R I A LS A N D M E T H O D S Bacterial strain The S. chilensis S37 strain was used (Godoy et al. 2002). Some of its degradation properties have been reported previously (Aranda et al. 1999). Degradation studies Cells of strain S37 were grown in R2A broth (72 h, 25C, orbital shaking 150 rev min)1), collected by centrifugation, washed thrice with saline solution and suspended at 1 107 cfu ml)1 in 50 ml of saline solution plus 2,4,6-TCP at various concentrations (25400 lM). For degradation tests, cells were incubated at 25C (150 rev min)1) for 5 days, and sampled every 24 h. Flasks without carbon source were used as viability control and asks without cells for abiotic control. Samples (1 ml) were centrifuged (14 000 g) and the supernatant stored at )80C. Degradation of 2,4,6-TCP was determined spectrophotometrically at 310 nm (A310 nm 456 103). Chloride release was measured by a chromogenic reaction (Aranda et al., 1999). Viable bacteria were determined by the Miles and Misra method in R2A agar (Herbert 1990). To study the effect of starvation on degradation of 2,4,6-TCP, cells (1 107 CFU ml)1) were inoculated in 50 ml of mineral salts medium. After 0, 24, 48 and 72 h, 2,4,6-TCP (25 lM) was added and the cells were further incubated for 24, 48 and 168 h. Samples were obtained every 24 h. All the assays were done in triplicate. Detection of tcp gene sequences Detection of gene sequence for 2,4,6-TCP-4-monooxygenase (tcpA), hydroxyquinol-1,2-dioxygenase (tcpC) and maleylacetate reductase (tcpD) was carried out by PCR analysis using primer pairs based on such sequences from Ralstonia pickettii and R. eutropha (Table 1) (Takizawa et al. nchez, M., Ma rtinez, M. and Gonza lez, 1995; Matus,V., Sa B., unpublished results). Southern DNA analysis was performed using a 1200-bp fragment containing a sequence of R. eutropha JMP134, which is 89% homologous to the tcpA gene reported in R. pickettii (Takizawa et al. 1995), and the 450-bp fragment from the tcpD gene of R. eutropha JMP134, which is 67% similar to the gene of R. eutropha 335 nchez, M., Ma rtinez, M. and Gonza lez, (Matus V., Sa B.,unpublished results). The probes were obtained from the PCR amplication products cloned in pGem-T easy

CGC AAT GTC TGG GTC GGC AA hadA CAC GCG GCC GAT CTT CAC hadA ACC GCC ACC GGC CAC AAG TG hadC AGC GAT TCG CGC ACG CCG AAC ACA G hadC AAA TGA CGC CGA TCT ATG G macA AGC ACA TGG CAG AGC TTG TG tfdFII

had genes from Ralstonia pickettii DTP0602; macA from R. eutropha 335; tfd genes from R. eutropha JMP134 pJP4 plasmid; GenBank accession numbers are: hadA and hadC, D86544; tfdII U16782; macA, AF130250.

(Promega, Madison, WI, USA). Southern blots of DNA digested with ClaI, or BamHI were made using Hybond N+ membranes (Amersham Pharm. Biotech., Piscataway, NJ, USA). Probes were labelled using the KPL labelling system and hybridization detected by KPL nucleic acids detection system (Gibco BRL, Rockville, MD, USA), as recommended by the supplier. Metabolic activity Metabolic activity of cells was determined according to Fry (1990). Between 400 and 500 cells were scored for each condition. Detection and visualization of PHA PHA detection was performed by staining granules with Sudan black and by spectrophometric detection of crotonic acid (Law and Slepecky 1961). PHA granules were visualized by transmission electron microscopy (Lageveen et al. 1988). To detect accumulation of PHA in bacterial colonies, 05 lg ml)1 Nile red was incorporated in R2A agar plates (Spiekermann et al. 1999). For starved cells, PHA was detected spectrouorometrically using Nile red and ow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson) operated with a Macintosh Real-time data acquisition system controlled by CellQuest v. 33 software, while FACSComp software was used for daily set-up and quality control (Degelau et al. 1995). Results for each sample were based on the analysis of 10 000 events. PHA polyester qualitative analysis was performed according to Brandl et al. (1988). The resulting methyl esters of hydroxyalkanoic acids were analysed by gas chromatography (Brandl et al. 1988) with a Hewlett Packard 5890 series II gas chromatograph equipped with a DurabondCarbowax-M15 with megabore capillary column (15 m; 054 mm).

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RESULTS AND DISCUSSION Degradation of 2,4,6-TCP by Sphingopyxis chilensis S37 The effect of concentration of 2,4,6-TCP and the ability of S. chilensis S37 to degrade 2,4,6-TCP was studied in cells previously grown on R2A broth. Biotic and abiotic controls did not show signicant changes during the experiment (data not shown). Although 2,4,6-TCP degradation occurred at 25 and 50 lM, these cells were almost unable to degrade higher chlorophenol concentrations (Figure 1a). Degradation of 2,4,6-TCP was conrmed because over 75% of chlorine was released after 48 h of incubation with 25 and 50 lM 2,4,6-TCP (Figure 1b), in a stoichiometric ratio (3 : 1) and no intermediary metabolite was detected by HPLC (Aranda, C., Godoy, F. and nez, M., unpublished results). Further, with 25 lM Mart of 2,4,6-TCP chlorine release was more than expected. These results are not clear, but suggest that different enzymatic activities might exist in the 2,4,6-TCP degradation metabolic pathway. No biomass increase was detected when 2,4,6-TCP was degraded (Figure 1c), suggesting that trichlorophenol was not used as carbon source. Previous studies have highlighted two types of 2,4,6-TCP degraders. The rst group (mainly Ralstonia strains) degraded high concentrations of chlorophenol (054 mM), using it as sole carbon and energy source ment et al. 1995). Other bacteria were able to degrade (Cle low concentration 2,4,6-TCP but unable to use it as nchez, M., Ma rtinez, M. and carbon source (Matus,V., Sa lez, B.,unpublished results). A common degradative Gonza pathway has been reported for the Ralstonia group, involving 2,4,6-TCP enzymatic conversion to maleylacetate by 2,4,6-TCP-4-monooxygenase, hydroxyquinol-1,2-dioxygenase and maleylacetate reductase (genes tcpA, tcpC ment et al. 1995; Latus et al. and tcpD, respectively) (Cle 1995; Wieser et al. 1997). Genes tcpA, tcpC and tcpD were not detected by PCR in S. chilensis S37. Southern analyses with probes for tcpA and tcpD sequences also did not give positive signals, suggesting that S. chilensis S37 2,4,6-TCP degradation occurs by a different catabolic pathway compared to that reported for the Ralstonia group. It also indicates that S. chilensis S37 is a good model to study bacteria able to degrade but not to grow on 2,4,6-TCP as sole carbon source. Bacterial viability decreases when exposed to 100400 lM 2,4,6-TCP (Figure 1c). Thus, the inability of S. chilensis degrade higher concentrations of 2,4,6-TCP may result from substrate toxicity phenomena that could be a consequence of 2,4,6-TCP producing higher energy cell demand for maintenance (Rittmann 1992).

(a)

80 70

Degradation of 2,4,6-TCP (%)

60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 8
25 M 50 M 100 M 200 M 300 M 400 M

(b)
100

Chloride released (%)

80

60

40

20

0 0 1 2 3 4 5 6 7

(c)

9 8 7

Log (CFU ml )

6 5 4 3 2 1 0 1 2 3 4 5 6 7 8

Ti m e (d ay s)
Fig. 1 Chlorophenol degradation (a), chloride release (b) and bacterial viability (c) after incubation of Sphingopyxis chilensis S37 with different concentrations of 2,4,6-trichlorophenol. Values are means of three replicates. 2,4,6-TCP concentration: d 25 lM; j 50 lM; m 100 lM; 200 lM; . 300 lM

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Effect of starvation on degradation of 2,4,6-TCP by Sphingopyxis chilensis S37 Concentration of 2550 lM 2,4,6-TCP did not detectably sustain S. chilensis S37 growth. To explain this result, the possibility of the strain using PHA as an endogenous carbon and energy source for degradation of 2,4,6-TCP was analysed. S. chilensis S37, harvested in stationary growth, showed abundant inclusion bodies, strong UV uorescence of colonies grown on R2A agar plus Nile-red and detection of crotonic acid (data not shown), indicating the presence of PHA. The constituent of PHA was methyl esters of 3-hydroxybutanoic acid, as detected by gas chromatography. Degradation of 2,4,6-TCP by S. chilensis S37 was affected by the starvation period (Figure 2). After 24 h, degradation of 25 lM 2,4,6-TCP was less than half of that observed in nonstarved cells. In addition, degradation after longer starvation times was almost negligible (Figure 3), probably due to a decrease in bacterial viability, metabolic activity or both. However, viability remained constant after 72 h of starvation (Table 2), and a slight decrease in metabolic activity was observed at 48 and 72 h under starvation (Table 2), but the
Degradation of 2,4,6-TCP (%)

70 60 50 40 30 20 10 0 0 24 48 72 96 120 144 168 Time (h)

0 h of starvation 24 h of starvation 48 h of starvation 72 h of starvation

Fig. 2 Effect of initial starvation time on the degradative activity of Sphingopyxis chilensis S37, after addition of 25 lM 2,4,6-TCP. Starvation time: s 0 h; h 24 h; , 48 h and n 72 h. Values correspond to the mean of three independent experiments

(a)
9000 Relative fluorescence (600 nm)

8000

7000

6000

5000 0 24 48 Time (h) 72

(b)
100 80 60 40 20 0 100 101 102 103

Events

101 104 100 Fluorescence intensity

102

103

104

Fig. 3 Change in PHA content after 0 or 3 days of starvation of Sphingopyxis chilensis S37 determined by (a) spectrouorimetry and (b) ow cytometry of Nile red stained cells, 0 (left) or 3 days (right) of starvation. Spectrouorometry values correspond to the mean of three independent experiments

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Table 2 Metabolic activity and bacterial viability during starvation of Sphingopyxis chilensis S37 Starvation time 0 24 48 72 Metabolically active cells (%) 8 9 8 3 7 1 6 2 2 4 2 1 1 4 2 1 Viable cells (log CFU ml)1) 86 84 84 84 003 009 014 003

Each value corresponds to the mean of three independent experiments.

2,4,6-TCP degradation ability observed in 24-h-starved cells was almost lost. PHA content decreases in S. chilensis S37 during starvation together with 2,4,6-TCP degradation capacity. In fact, the spectrouorimetric analysis showed a decrease of uorescence during the rst 24 h of starvation (Figure 3a), and ow cytometry conrmed this decrease during starvation (Figure 3b). Initially, 85% of the cells showed relative uorescence intensities above 100; however, at 72 h starvation, a signicant proportion of cells had uorescence intensities similar to the control. These results support the idea that energy generated by consumption of intracellular PHA during the rst 24 h would provide enough energy to maintain viability and metabolic activity during starvation in the presence of chlorophenol. This suggests that PHA consumption is associated, time-dependently, with 2,4,6-TCP degradation. The nding described is not a cometabolic process, as complete mineralization of 2,4,6-TCP was observed (Wang and Loh 1999). PHA granules have been also observed in other chlorophenols degrading bacteria (Radeahus and Schmidt 1992). Hence, PHAs might be responsible for providing the energy necessary for chlorophenol degradation, especially when these toxic compounds are not carbon and energy sources. Therefore, PHAs and probably other storage substances provide endogenous carbon and energy sources under starvation and perhaps bacteria that are able to accumulate them play a signicant role in biodegradation of toxic compounds in the environment, with scarce and changing nutrient bioavailability. ACKNOWLEDGEMENTS n Research supported by grant no. 200.036.020-1.0, Direccio n, Universidad de Concepcio n, Chile, and de Investigacio FONDECYT 8990004 and 2980041. F. Godoy and V. Matus are CONICYT-Chile PhD fellows. REFERENCES
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2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 315320