- Email: [email protected]
PAH-degrading microbial consortium and its pyrene-degrading plasmids from mangrove sediment samples in Huian, China
Marine Pollution Bulletin 57 (2008) 703–706
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
PAH-degrading microbial consortium and its pyrene-degrading plasmids from mangrove sediment samples in Huian, China Yi Lin *, Li-Xi Cai Department of Bioengineering and Biotechnology, HuaQiao University, Quanzhou 362021, China
a r t i c l e
i n f o
Keywords: Polycyclic aromatic hydrocarbons (PAHs) Mangrove sediment Microbial consortium Bacillus megaterium Pyrene-degrading plasmids
a b s t r a c t PAH-degrading microbial consortium and its pyrene-degrading plasmids were enriched from the sediment samples of Huian mangroves. The consortium YL showed degrading abilities of 92.1%, 87.6%, 92.3%, and 95.8% for pyrene, fluoranthene, phenanthrene, and fluoene at 50 mg l 1 after 21 days incubation, respectively. The dynamics of pH changes in the cultures was consistent with that of PAH concentration change. Bacillus cereus Py5 and Bacillus megaterium Py6 were isolated from the consortium and observed consuming 65.8% and 33.7% of pyrene (50 mg l 1) within three weeks, respectively. The enriched Escherichia coli DH5a cells containing the plasmids of YL were demonstrated to degrade 85.7% of the original pyrene concentration at the 21st day. Ó 2008 Published by Elsevier Ltd.
1. Introduction
2. Materials and methods
Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic and readily adsorbed onto particulate matter, thus, coastal and marine sediments become the ultimate sinks for PAHs (Hughes et al., 1997; Tam et al., 2001). Generally, low-molecular-weight PAHs (two or three condensed aromatic rings) are readily degradable in nature, but high-molecular-weight PAHs (four or more condensed aromatic rings) are regarded as recalcitrant and often genotoxic (Mortelmans et al., 1986). Microorganisms are considered to be the major degrader for natural PAH contaminants and are of great practical interest to bioremediation. The field of biodegradation of high-molecular-weight PAHs in marine environment especially mangrove sediments is now witnessing some interesting developments such as the isolation of microorganisms capable of decomposing the four-ring-aromatics fluoranthene and pyrene and the five-ring-aromatics benzo[a]pyrene (Yu et al., 2005; Guo et al., 2005; Luo et al., 2005). However, the metabolism pathways of high-molecularweight PAHs degraded by marine microorganisms remained unclear, and the practical bioremediation in the marine sites polluted by high-molecular-weight PAHs still leaved lots of problems to be solved. The present study therefore, aims to obtain high-molecularweight PAH-degrading microbial consortium, bacterial strains, and pyrene-degrading plasmids from mangrove sediments for the isolation of genes or gene clusters responsible for pyrene degradation, and for PAH bioremediation on the spot.
2.1. Sediment sampling and enrichment of PAH-degrading microbial consortium
* Corresponding author. Tel.: +86 595 22692031; fax: +86 595 22691560. E-mail address: [email protected] (Y. Lin). 0025-326X/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2008.03.025
The sediment sampling and enrichment of PAH-degrading microbial consortium were performed mainly according to the methods described by Luo et al. (2005), Yu et al. (2005), and Guo et al. (2005). Ten random surface sediment samples (0–3 cm) were collected at Huian mangroves in Fujian, China during low tides. Each sediment sample, around 2–3 kg fresh weight, was actually a composite sample made up by thoroughly mixing sediments collected from several sampling points. The final sample was made up by thoroughly mixing 200 g fresh sediments from each of the ten samples. Fresh sediments (10 g) from the final sample were immediately used for microbial enrichment. Pyrene (a 4-ring PAH, purchased from Sigma Chemicals, USA, purity >96%) was used as the sole carbon and energy source to enrich PAH-degrading consortium from mangrove sediments. The 10 g fresh sediment was transferred to a flask containing 95 ml 0.85% NaCl solution. An aliquot of 5 ml supernatant was then transferred into a flask containing 45 ml sterilized MSM (mineral salt medium with pH adjusted to 7.2 and salinity at 2%) with the addition of pyrene to a final concentration of 100 mg l 1. The flask was shaken on an orbital shaker at 25 °C at a speed of 150 rpm. After one month incubation, an aliquot of 5 ml enriched culture was transferred into another flask containing 45 ml fresh MSM with the addition of pyrene to a final concentration of 200 mg l 1. Totally, five enrichments with the addition of pyrene to a final concentration of 50, 100, 150, 200, and 250 mg l 1, respectively, were carried out to reach a PAH-degrading microbial consortium.
Y. Lin, L.-X. Cai / Marine Pollution Bulletin 57 (2008) 703–706
At the end of the enrichment, bacterial strains in the consortium were isolated by spreading the 10-fold serial diluted consortium on agar plates coated with a layer of pyrene on the surface. Bacterial colonies producing clear zones were picked up from the plates, further purified by repetitive streaking on nutrient agar plates. The pure isolates were then identified by 16S rDNA sequencing after amplification of the gene by PCR using the set of primers 27F (E. coli position 8-27, 5-AGA GTT TGA TCC TGG CTC AG-3) and 1492R (E. coli position 1510–1492, 5-GGC TAC CTT GTT ACG ACT T-3). The PCR conditions (37 cycles of 3 min at 94 °C, 1 min at 55 °C, 1 min at 72 °C) were performed in a PCR thermal cycler (PTC100TM DNA Engine, MJ Research) with Taq DNA polymerase (Sangon). The 16S rDNA sequences resulted from the purified PCR product sequencing were compared using BLASTN. 2.3. Enrichment of pyrene-degrading E. coli cells transformed with plasmids of the consortium The plasmids of the enriched microbial consortium were extracted according to normal methods and were used to transform E. coli DH5a cells. After cultured for 3 h, 30 ll culture containing the transformed cells were transferred into a flask containing 20 ml sterilized LB medium with the addition of pyrene to a final concentration of 0.1 mg l 1. The flask was shaken on an orbital shaker at 37 °C at a speed of 150 rpm in dark for 24 h. An aliquot of 2 ml culture was then transferred into a flask containing 18 ml sterilized MSM (mineral salt medium with pH adjusted to 7.2 and salinity at 2%) with the addition of pyrene to a final concentration of 50 mg l 1. The flask was shaken on an orbital shaker at 25 °C at a speed of 150 rpm. After about one month incubation, an aliquot of 2 ml enriched culture was transferred into another flask containing 18 ml fresh pyrene-MSM. Totally, three enrichments with the addition of pyrene to a final concentration of 50 mg l 1 in MSM were carried out to reach a pyrene-degrading transformed E. coli. 2.4. Biodegradation of PAHs by enriched consortium, bacterial isolates, and E. coli transformant To start the biodegradation experiment, 1 ml aliquot of the enriched consortium, single bacterial isolate, or the transformed E. coli cells reaching the late exponential growth phase was inoculated into 49 ml MSM containing one of the four PAHs pyrene (4ring), fluoranthene (4-ring), phenanthrene (3-ring), and fluoene (3-ring) (purchased from Sigma Chemicals, USA, purity >96%) at an initial concentration of 50 mg l 1. MSM containing the same amount of PAHs but without any microbial inoculum was used as the control to determine abiotic losses of PAHs. The flasks were shaken in the same way as described above. At regular time intervals during the 21 days incubation, triplicate flasks from each treatment were retrieved and the residual PAH concentrations were extracted two times with dichloromethane and determined by HPLC. The biodegradation percentage of PAHs was calculated as the difference in residual PAH concentrations between the control and the flasks with inoculum.
different PAH at the concentration of 50 mg l 1 to examine the potential of PAH degradation. The remaining PAH concentrations were periodically determined during 21 incubation days, using HPLC. The assays demonstrated that the consortium YL was capable of degrading a number of PAHs, including the 4-ring PAHs pyrene and fluoranthene and the 3-ring PAHs phenanthrene and fluoene. Small but significant degradation rates were observed at the 3rd day with fluoene, at the 6th day with phenanthrene and pyrene, and at the 9th day with fluoranthene. After 21 days of incubation, the biodegradation percentages of pyrene, fluoranthene, phenanthrene, and fluoene were 92.1%, 87.6%, 92.3%, and 95.8%, respectively (Fig. 1A), while the total abiotic losses of all the four PAHs were negligible. The dynamics of pH change in the cultures was consistent with that of PAH concentration change (Fig. 1B), indicating that the PAH biodegradation by the consortium YL can be monitored by determining the pH change. At the end of the assays, the pH value of pyrene-MSM decreased from 6.9 to 2.6, from 7.0 to 2.8 of fluoranthene-MSM, from 6.9 to 3.4 of phenanthrene-MSM, and from 7.1 to 4.2 of fluoene-MSM (Fig. 1B). These results indicated that a number of acidic intermediate products such as succinic acid, acetic acid, fumaric acid and pyruvic acid be produced in the degradation process of PAH. The pH decline resulted from the accumulation of those acid intermediate products. And this may
A 60 Concentration of PAHs(mg/L)
2.2. Isolation and identification of pyrene-degrading bacterial strains
YL+Fluoene
50
YL+Phenanthrene
40
YL+Fluoranthene YL+Pyrene
30 20 10 0 0
B
3
6
9 12 Culture time (d)
15
8
18
21
Fluoene Pheanthrene Fluornanthene
7 6
Pyrene
5
pH
704
4 3 2
3. Results and discussion 3.1. PAH biodegradation in liquid medium by the enriched microbial consortium A microbial consortium named as YL was obtained from the sediment samples enriched with the addition of an increasing concentration of pyrene for five months. YL was cultured in MSM with
1 0 0
3
6
9 12 Culture time (d)
15
18
21
Fig. 1. The degradation curve of fluoene, phenanthrene, fluoranthene and pyrene by the consortium YL (A), and the pH change during the degradation process (B).
705
Y. Lin, L.-X. Cai / Marine Pollution Bulletin 57 (2008) 703–706
be the reason that the change in pH corresponded to the change in PAH concentration. 3.2. Isolation, identification, and biodegradation potencial of bacterial strains from the enriched microbial consortium After the enriched microbial consortium was plated, a number of colonies showed good growth on the plate with pyrene as the sole carbon and energy source. Pure cultures of the pyrene degraders were obtained after four transfers. Five isolates were chosen for further identification by 16S rDNA sequencing. PCR amplification of 16S rDNA from those five isolates resulted in sequences of about 1500 bp. Based on a BLASTN search of GenBank, the closest matches to Py2, Py5, Py7, and Py9 of the five isolates were Bacillus cereus (nucleotide identities all above 99%), and the rest one Py6 isolate was B. megaterium (nucleotide identity above 99%). B. cereus Py5 and B. megaterium Py6 were separately cultured in MSM with pyrene at 50 mg l 1 to examine the potential of pyrene degradation. Very little pyrene biodegradation took place in the first 12 days of incubation, 14.8% with Py5 and 6.8% with Py6 (Fig. 2A), indicating that the initial degradation of pyrene by the two pure bacterial strains were very difficult. At the end of the 21 incubation days, 65.8 and 33.7% of the initial concentration was consumed, respectively (Fig. 2A). Very little pH changes in the cultures were detected for both strains during the whole assay, quite different from that of consortium YL (Figs. 2B and 1B), indicating that the type and quantity of acidic intermediate products depends on the types of strains.
A
60
The degradation capability and single bacterial species of the consortium YL enriched from Huian mangrove sediments were quite different from those of PAH-degrading consortia enriched from other mangrove sediments by Yu et al. (2005), Guo et al. (2005), and Luo et al. (2005), suggesting that mangrove sediments worldwide are huge banks for PAH biodegradors. 3.3. Enrichment for pyrene-degrading E. coli cells transformed by the plasmids of consortium YL The enrichment for pyrene-degrading E. coli cells transformed by the plasmids of consortium YL was carried out according to the method described above. During the enrichment, the culture colour changed and cell growth was observed, while no colour change and cell growth occurred with the inoculum of normal E. coli cultures, indicating that the enriched E. coli cells containing the plasmids of consortium YL can use with pyrene as the sole carbon and energy source. The enriched E. coli cells containing the plasmids of consortium YL were determined to show 85.7% degradation of pyrene at 50 mg l 1 after 21 days incubation, while only 2.0% degradation was observed by the normal E. coli cells (Fig. 3A). The pH value decreased from 7.1 to 3.1 at the end of the assay, similar to that of consortium YL (Figs. 3B and 2B). Pyrene degradation occurred with different kinetics between the consortium YL and the enriched E. coli cells containing the plasmids of consortium YL (Figs. 1A and 3A). The initial adaption period of the transformed E. coli cells was almost the half of that of consortium YL, the degradation rate reached 41.2% after only 6 days of incubation while small degradation was observed with the consortium YL at the 6th day (Figs. 1A and 3A).
A
40 30 20 Py5 Py6
10 0
B
Concentration of pyrene (mg/L)
Concentration (mg/L)
50
0
3
6
9 12 Culture time (d)
15
18
21
60 50 40 30 20 10 0 0
3
6
9 12 Culuture time (d)
15
18
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0
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6
9 12 Culture time (d)
15
18
21
8
B
7
8
6 6
4
PH
pH
5 4
3 Py5 Py6
2
2
1 0 0
3
6
9 12 Culture time (d)
15
18
21
Fig. 2. The degradation curve of pyrene by B. cereus Py5 and B. megaterium Py6 (A), and the corresponding pH change (B).
0
Fig. 3. The degradation curve of pyrene by the enriched E. coli cells containing the plasmids of consortium YL (A), and the corresponding pH change (B).
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Y. Lin, L.-X. Cai / Marine Pollution Bulletin 57 (2008) 703–706
The PAH-degrading genes or gene clusters have been demonstrated to be located in the chromosome or plamids (Menn et al., 1993; Kiyohara et al., 1994; Cho and Kim, 2001; Zhang et al., 2003). A pyrene-degrading plasmid has been identified from a PAH-degrading bacterial strain ZL5, which was isolated from oilcontaminated soil of Liaohe Oil Field, China (Zhang et al., 2003). In this paper, the pyrene-degrading plasmids were obtained from the PAH-degrading consortium YL, which laid a solid foundation for the isolation of related genes or gene clusters to uncover the metabolism pathways of high-molecular-weight PAHs degraded by marine microorganisms. Acknowledgements The work described in this paper was supported by a grant from HuaQiao University. The authors would like to thank Prof. Zheng TL of Xiamen University for invaluable help from his lab. References Cho, J.C., Kim, S.J., 2001. Detection of mega plasmid from polycyclic aromatic hydrocarbon – degrading Sphingomonas sp. strain 14. J. Mol. Microbiol. Biotechnol. 3, 503–506.
Guo, C.L., Zhou, H.W., Wong, Y.S., Tam, N.F.Y., 2005. Isolation of PAH-degrading bacteria from mangrove sediments and their biodegradation potential. Mar. Pollut. Bull. 51, 1054–1061. Hughes, J.B., Beckles, D.M., Chandra, S.D., Ward, C.H., 1997. Utilization of bioremediation processes for the treatment of PAH-contaminated sediments. J. Ind. Microbiol. Biotechnol. 18, 152–160. Kiyohara, H., Torigoe, S., Kaida, N., 1994. Cloning and characterization of a chromosomal gene cluster, pah, that encodes the upper pathway for phenanthrene and naphthalene utilization by Pseudomonas putida OUS82. J. Bacteriol. 176, 2439–2443. Luo, Y.R., Hu, Z., Zheng, T.L., Huang, X., 2005. Biodegradation of Benzo[a]pyrene by Mangrove Microbial Consortium. J Xiamen University (Natural Science) (Suppl.), 75–79. Menn, F.M., Applegate, B.M., Sayler, G.S., 1993. NAH plasmid-mediated catabolism of anthracene and phenanthrenen to naphthoic acids. Appl. Environ. Microbiol. 59, 1938–1942. Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., Zeiger, E., 1986. Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ. Mutagen. 8 (Suppl. 7), 1–119. Tam, N.F.Y., Ke, L., Wang, X.H., Wong, Y.S., 2001. Contamination of polycyclic aromatic hydrocarbons in surface sediments of mangrove swamps. Environ. Pollut. 114, 255–263. Yu, S.H., Ke, L., Wong, Y.S., Tam, N.F.Y., 2005. Degradation of polycyclic aromatic hydrocarbons (PAHS) by a bacterial consortium enriched from mangrove sediments. Environ. Int. 31, 149–154. Zhang, J., Liu, Y.S., Feng, J.X., Bai, X.L., Zhang, Z.Z., 2003. Isolation and identification of PAHs-degrading strain ZL5 and its degradative plasmid. Chin. J. Appl. Environ. Biol. 9, 433–435.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
PAH-degrading microbial consortium and its pyrene-degrading plasmids from mangrove sediment samples in Huian, China Yi Lin *, Li-Xi Cai Department of Bioengineering and Biotechnology, HuaQiao University, Quanzhou 362021, China
a r t i c l e
i n f o
Keywords: Polycyclic aromatic hydrocarbons (PAHs) Mangrove sediment Microbial consortium Bacillus megaterium Pyrene-degrading plasmids
a b s t r a c t PAH-degrading microbial consortium and its pyrene-degrading plasmids were enriched from the sediment samples of Huian mangroves. The consortium YL showed degrading abilities of 92.1%, 87.6%, 92.3%, and 95.8% for pyrene, fluoranthene, phenanthrene, and fluoene at 50 mg l 1 after 21 days incubation, respectively. The dynamics of pH changes in the cultures was consistent with that of PAH concentration change. Bacillus cereus Py5 and Bacillus megaterium Py6 were isolated from the consortium and observed consuming 65.8% and 33.7% of pyrene (50 mg l 1) within three weeks, respectively. The enriched Escherichia coli DH5a cells containing the plasmids of YL were demonstrated to degrade 85.7% of the original pyrene concentration at the 21st day. Ó 2008 Published by Elsevier Ltd.
1. Introduction
2. Materials and methods
Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic and readily adsorbed onto particulate matter, thus, coastal and marine sediments become the ultimate sinks for PAHs (Hughes et al., 1997; Tam et al., 2001). Generally, low-molecular-weight PAHs (two or three condensed aromatic rings) are readily degradable in nature, but high-molecular-weight PAHs (four or more condensed aromatic rings) are regarded as recalcitrant and often genotoxic (Mortelmans et al., 1986). Microorganisms are considered to be the major degrader for natural PAH contaminants and are of great practical interest to bioremediation. The field of biodegradation of high-molecular-weight PAHs in marine environment especially mangrove sediments is now witnessing some interesting developments such as the isolation of microorganisms capable of decomposing the four-ring-aromatics fluoranthene and pyrene and the five-ring-aromatics benzo[a]pyrene (Yu et al., 2005; Guo et al., 2005; Luo et al., 2005). However, the metabolism pathways of high-molecularweight PAHs degraded by marine microorganisms remained unclear, and the practical bioremediation in the marine sites polluted by high-molecular-weight PAHs still leaved lots of problems to be solved. The present study therefore, aims to obtain high-molecularweight PAH-degrading microbial consortium, bacterial strains, and pyrene-degrading plasmids from mangrove sediments for the isolation of genes or gene clusters responsible for pyrene degradation, and for PAH bioremediation on the spot.
2.1. Sediment sampling and enrichment of PAH-degrading microbial consortium
* Corresponding author. Tel.: +86 595 22692031; fax: +86 595 22691560. E-mail address: [email protected] (Y. Lin). 0025-326X/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2008.03.025
The sediment sampling and enrichment of PAH-degrading microbial consortium were performed mainly according to the methods described by Luo et al. (2005), Yu et al. (2005), and Guo et al. (2005). Ten random surface sediment samples (0–3 cm) were collected at Huian mangroves in Fujian, China during low tides. Each sediment sample, around 2–3 kg fresh weight, was actually a composite sample made up by thoroughly mixing sediments collected from several sampling points. The final sample was made up by thoroughly mixing 200 g fresh sediments from each of the ten samples. Fresh sediments (10 g) from the final sample were immediately used for microbial enrichment. Pyrene (a 4-ring PAH, purchased from Sigma Chemicals, USA, purity >96%) was used as the sole carbon and energy source to enrich PAH-degrading consortium from mangrove sediments. The 10 g fresh sediment was transferred to a flask containing 95 ml 0.85% NaCl solution. An aliquot of 5 ml supernatant was then transferred into a flask containing 45 ml sterilized MSM (mineral salt medium with pH adjusted to 7.2 and salinity at 2%) with the addition of pyrene to a final concentration of 100 mg l 1. The flask was shaken on an orbital shaker at 25 °C at a speed of 150 rpm. After one month incubation, an aliquot of 5 ml enriched culture was transferred into another flask containing 45 ml fresh MSM with the addition of pyrene to a final concentration of 200 mg l 1. Totally, five enrichments with the addition of pyrene to a final concentration of 50, 100, 150, 200, and 250 mg l 1, respectively, were carried out to reach a PAH-degrading microbial consortium.
Y. Lin, L.-X. Cai / Marine Pollution Bulletin 57 (2008) 703–706
At the end of the enrichment, bacterial strains in the consortium were isolated by spreading the 10-fold serial diluted consortium on agar plates coated with a layer of pyrene on the surface. Bacterial colonies producing clear zones were picked up from the plates, further purified by repetitive streaking on nutrient agar plates. The pure isolates were then identified by 16S rDNA sequencing after amplification of the gene by PCR using the set of primers 27F (E. coli position 8-27, 5-AGA GTT TGA TCC TGG CTC AG-3) and 1492R (E. coli position 1510–1492, 5-GGC TAC CTT GTT ACG ACT T-3). The PCR conditions (37 cycles of 3 min at 94 °C, 1 min at 55 °C, 1 min at 72 °C) were performed in a PCR thermal cycler (PTC100TM DNA Engine, MJ Research) with Taq DNA polymerase (Sangon). The 16S rDNA sequences resulted from the purified PCR product sequencing were compared using BLASTN. 2.3. Enrichment of pyrene-degrading E. coli cells transformed with plasmids of the consortium The plasmids of the enriched microbial consortium were extracted according to normal methods and were used to transform E. coli DH5a cells. After cultured for 3 h, 30 ll culture containing the transformed cells were transferred into a flask containing 20 ml sterilized LB medium with the addition of pyrene to a final concentration of 0.1 mg l 1. The flask was shaken on an orbital shaker at 37 °C at a speed of 150 rpm in dark for 24 h. An aliquot of 2 ml culture was then transferred into a flask containing 18 ml sterilized MSM (mineral salt medium with pH adjusted to 7.2 and salinity at 2%) with the addition of pyrene to a final concentration of 50 mg l 1. The flask was shaken on an orbital shaker at 25 °C at a speed of 150 rpm. After about one month incubation, an aliquot of 2 ml enriched culture was transferred into another flask containing 18 ml fresh pyrene-MSM. Totally, three enrichments with the addition of pyrene to a final concentration of 50 mg l 1 in MSM were carried out to reach a pyrene-degrading transformed E. coli. 2.4. Biodegradation of PAHs by enriched consortium, bacterial isolates, and E. coli transformant To start the biodegradation experiment, 1 ml aliquot of the enriched consortium, single bacterial isolate, or the transformed E. coli cells reaching the late exponential growth phase was inoculated into 49 ml MSM containing one of the four PAHs pyrene (4ring), fluoranthene (4-ring), phenanthrene (3-ring), and fluoene (3-ring) (purchased from Sigma Chemicals, USA, purity >96%) at an initial concentration of 50 mg l 1. MSM containing the same amount of PAHs but without any microbial inoculum was used as the control to determine abiotic losses of PAHs. The flasks were shaken in the same way as described above. At regular time intervals during the 21 days incubation, triplicate flasks from each treatment were retrieved and the residual PAH concentrations were extracted two times with dichloromethane and determined by HPLC. The biodegradation percentage of PAHs was calculated as the difference in residual PAH concentrations between the control and the flasks with inoculum.
different PAH at the concentration of 50 mg l 1 to examine the potential of PAH degradation. The remaining PAH concentrations were periodically determined during 21 incubation days, using HPLC. The assays demonstrated that the consortium YL was capable of degrading a number of PAHs, including the 4-ring PAHs pyrene and fluoranthene and the 3-ring PAHs phenanthrene and fluoene. Small but significant degradation rates were observed at the 3rd day with fluoene, at the 6th day with phenanthrene and pyrene, and at the 9th day with fluoranthene. After 21 days of incubation, the biodegradation percentages of pyrene, fluoranthene, phenanthrene, and fluoene were 92.1%, 87.6%, 92.3%, and 95.8%, respectively (Fig. 1A), while the total abiotic losses of all the four PAHs were negligible. The dynamics of pH change in the cultures was consistent with that of PAH concentration change (Fig. 1B), indicating that the PAH biodegradation by the consortium YL can be monitored by determining the pH change. At the end of the assays, the pH value of pyrene-MSM decreased from 6.9 to 2.6, from 7.0 to 2.8 of fluoranthene-MSM, from 6.9 to 3.4 of phenanthrene-MSM, and from 7.1 to 4.2 of fluoene-MSM (Fig. 1B). These results indicated that a number of acidic intermediate products such as succinic acid, acetic acid, fumaric acid and pyruvic acid be produced in the degradation process of PAH. The pH decline resulted from the accumulation of those acid intermediate products. And this may
A 60 Concentration of PAHs(mg/L)
2.2. Isolation and identification of pyrene-degrading bacterial strains
YL+Fluoene
50
YL+Phenanthrene
40
YL+Fluoranthene YL+Pyrene
30 20 10 0 0
B
3
6
9 12 Culture time (d)
15
8
18
21
Fluoene Pheanthrene Fluornanthene
7 6
Pyrene
5
pH
704
4 3 2
3. Results and discussion 3.1. PAH biodegradation in liquid medium by the enriched microbial consortium A microbial consortium named as YL was obtained from the sediment samples enriched with the addition of an increasing concentration of pyrene for five months. YL was cultured in MSM with
1 0 0
3
6
9 12 Culture time (d)
15
18
21
Fig. 1. The degradation curve of fluoene, phenanthrene, fluoranthene and pyrene by the consortium YL (A), and the pH change during the degradation process (B).
705
Y. Lin, L.-X. Cai / Marine Pollution Bulletin 57 (2008) 703–706
be the reason that the change in pH corresponded to the change in PAH concentration. 3.2. Isolation, identification, and biodegradation potencial of bacterial strains from the enriched microbial consortium After the enriched microbial consortium was plated, a number of colonies showed good growth on the plate with pyrene as the sole carbon and energy source. Pure cultures of the pyrene degraders were obtained after four transfers. Five isolates were chosen for further identification by 16S rDNA sequencing. PCR amplification of 16S rDNA from those five isolates resulted in sequences of about 1500 bp. Based on a BLASTN search of GenBank, the closest matches to Py2, Py5, Py7, and Py9 of the five isolates were Bacillus cereus (nucleotide identities all above 99%), and the rest one Py6 isolate was B. megaterium (nucleotide identity above 99%). B. cereus Py5 and B. megaterium Py6 were separately cultured in MSM with pyrene at 50 mg l 1 to examine the potential of pyrene degradation. Very little pyrene biodegradation took place in the first 12 days of incubation, 14.8% with Py5 and 6.8% with Py6 (Fig. 2A), indicating that the initial degradation of pyrene by the two pure bacterial strains were very difficult. At the end of the 21 incubation days, 65.8 and 33.7% of the initial concentration was consumed, respectively (Fig. 2A). Very little pH changes in the cultures were detected for both strains during the whole assay, quite different from that of consortium YL (Figs. 2B and 1B), indicating that the type and quantity of acidic intermediate products depends on the types of strains.
A
60
The degradation capability and single bacterial species of the consortium YL enriched from Huian mangrove sediments were quite different from those of PAH-degrading consortia enriched from other mangrove sediments by Yu et al. (2005), Guo et al. (2005), and Luo et al. (2005), suggesting that mangrove sediments worldwide are huge banks for PAH biodegradors. 3.3. Enrichment for pyrene-degrading E. coli cells transformed by the plasmids of consortium YL The enrichment for pyrene-degrading E. coli cells transformed by the plasmids of consortium YL was carried out according to the method described above. During the enrichment, the culture colour changed and cell growth was observed, while no colour change and cell growth occurred with the inoculum of normal E. coli cultures, indicating that the enriched E. coli cells containing the plasmids of consortium YL can use with pyrene as the sole carbon and energy source. The enriched E. coli cells containing the plasmids of consortium YL were determined to show 85.7% degradation of pyrene at 50 mg l 1 after 21 days incubation, while only 2.0% degradation was observed by the normal E. coli cells (Fig. 3A). The pH value decreased from 7.1 to 3.1 at the end of the assay, similar to that of consortium YL (Figs. 3B and 2B). Pyrene degradation occurred with different kinetics between the consortium YL and the enriched E. coli cells containing the plasmids of consortium YL (Figs. 1A and 3A). The initial adaption period of the transformed E. coli cells was almost the half of that of consortium YL, the degradation rate reached 41.2% after only 6 days of incubation while small degradation was observed with the consortium YL at the 6th day (Figs. 1A and 3A).
A
40 30 20 Py5 Py6
10 0
B
Concentration of pyrene (mg/L)
Concentration (mg/L)
50
0
3
6
9 12 Culture time (d)
15
18
21
60 50 40 30 20 10 0 0
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6
9 12 Culuture time (d)
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8
6 6
4
PH
pH
5 4
3 Py5 Py6
2
2
1 0 0
3
6
9 12 Culture time (d)
15
18
21
Fig. 2. The degradation curve of pyrene by B. cereus Py5 and B. megaterium Py6 (A), and the corresponding pH change (B).
0
Fig. 3. The degradation curve of pyrene by the enriched E. coli cells containing the plasmids of consortium YL (A), and the corresponding pH change (B).
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The PAH-degrading genes or gene clusters have been demonstrated to be located in the chromosome or plamids (Menn et al., 1993; Kiyohara et al., 1994; Cho and Kim, 2001; Zhang et al., 2003). A pyrene-degrading plasmid has been identified from a PAH-degrading bacterial strain ZL5, which was isolated from oilcontaminated soil of Liaohe Oil Field, China (Zhang et al., 2003). In this paper, the pyrene-degrading plasmids were obtained from the PAH-degrading consortium YL, which laid a solid foundation for the isolation of related genes or gene clusters to uncover the metabolism pathways of high-molecular-weight PAHs degraded by marine microorganisms. Acknowledgements The work described in this paper was supported by a grant from HuaQiao University. The authors would like to thank Prof. Zheng TL of Xiamen University for invaluable help from his lab. References Cho, J.C., Kim, S.J., 2001. Detection of mega plasmid from polycyclic aromatic hydrocarbon – degrading Sphingomonas sp. strain 14. J. Mol. Microbiol. Biotechnol. 3, 503–506.
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