Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Differential degradation of polycyclic aromatic hydrocarbon mixtures by indigenous microbial assemblages in soil P. Sawulski1, B. Boots1,2, N. Clipson1 and E. Doyle1 1 Environmental Microbiology Group, School of Biology and Environmental Science and Earth Institute, University College Dublin, Dublin, Ireland 2 School of Biosystems Engineering, Agriculture and Food Science Centre, University College Dublin, Dublin, Ireland
Significance and Impact of the Study: Contaminated sites generally contain complex mixtures of pollutants. Development of effective bioremediation strategies for contaminated soils requires knowledge of the response of soil microbial communities to such mixtures. This study provides information on the degradation of different mixtures of three priority pollutants in soil with a history of polycyclic aromatic hydrocarbon contamination and examines the response of soil bacterial and fungal communities to the presence of these pollutants as sole contaminants or as part of a mixture. This is one of few studies todate to compare the effects of single compounds and pollutant mixtures on more than one soil microbial community.
Keywords bacterial communities, biodegradation, diversity, fungal communities, polycyclic aromatic hydrocarbons, soil. Correspondence Evelyn Doyle, Environmental Microbiology Group, School of Biology and Environmental Science and Earth Institute, University College Dublin, Belfield, Dublin, Ireland. E-mail: [email protected] 2015/0618: received 25 March 2015, revised 11 May 2015 and accepted 17 May 2015 doi:10.1111/lam.12446
Abstract Environmental contamination by polycyclic aromatic hydrocarbons (PAHs) typically occurs as mixtures of compounds. In this study, the response of indigenous soil bacterial and fungal communities to mixtures containing phenanthrene, fluoranthene and benzo(a)pyrene in various combinations was examined using molecular fingerprinting techniques and quantification of a key PAH degradative gene. Results were compared to a parallel study by Sawulski et al. (2014) which examined the effect of these PAHs on soil microbial communities when added as single contaminants. The rate of degradation of individual PAHs varied depending on whether the PAH was present as a single contaminant or in a mixture; phenanthrene was degraded most rapidly when present as a sole contaminant, fluoranthene was removed faster in the presence of the lower molecular weight phenanthrene and the rate of benzo(a)pyrene degradation was reduced in the presence of the 4-ring PAH, fluoranthene. Bacterial and fungal assemblages differed significantly between treatments regardless of which PAH was added to soil. Although less abundant than the Gram-negative PAH-RHDa gene, the gene associated with Grampositive bacteria responded to a greater extent to the presence of PAHs, either as single compounds or as mixtures and this increase was significantly correlated with PAH degradation.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are recognized as significant health risks and consequently listed as priority pollutants by environmental protection agencies worldwide. Combustion of fossil fuels is a major contributor to the widespread occurrence of PAHs in the environment, with 90% of emissions ending up in the top
20 cm layer of soil (Maliszewska-Kordybach 1999). PAHs are chemically very stable and the rate of degradation decreases, and toxicity increases as the number of benzene rings of the molecule increases (Cerniglia 1993). Many studies have demonstrated the ability of bacteria and fungi to degrade a wide range of PAHs (Peng et al. 2008). Soils typically harbour a great diversity and abundance of microbes (Daniel 2005) and often contain
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
199
P. Sawulski et al.
Microbial degradation of PAH mixtures in soil
micro-organisms capable of degrading a range of PAHs. Genes involved in degradation of PAHs, such as those encoding ring hydroxylating dioxygenases (RHD) serve as useful biomarkers for the presence of potential PAHdegrading bacteria in soil (Doyle et al. 2008). Recently, Sawulski et al. (2014) showed that soil microbial assemblages responded differently to phenanthrene, fluoranthene and benzo(a)pyrene when these PAHs were added to soil as single contaminants. However, soils contaminated with PAHs typically contain mixtures of compounds differing in structure, susceptibility to degradation and toxicity. Effective bioremediation strategies for such soils require an understanding of how indigenous soil microbial communities respond to the presence of PAH mixtures rather than single compounds. Studies have reported a significant increase in the degradation of high molecular weight PAHs such as fluoranthene, pyrene or benzo(a)pyrene in the presence of lower molecular weight PAHs such as phenanthrene or naphthalene (Juhasz and Naidu 2000; Somtrakoon et al. 2008). However, most studies on PAH mixtures have focussed on pure cultures (Bouchez et al. 1995; van Herwijnen et al. 2003) or soils inoculated with microorganisms (Somtrakoon et al. 2008), with little information concerning the effect of PAH mixtures on indigenous soil communities available. In this study, the response of soil microbial communities to the addition of various combinations of PAHs (3 ring phenanthrene, 4-ring fluoranthene and 5-ring benzo(a)pyrene) was investigated in a microcosm-based system. This work was carried out in parallel to the study by Sawulski et al. (2014) which examined the effect of these PAHs on soil microbial community dynamics when added as single contaminants; results from both studies are compared.
(a)
PAH remaining (%)
100
Results and discussion Degradation of PAH mixtures Phenanthrene and fluoranthene were almost completely removed from the soil microcosms regardless of mixture composition (Fig. 1). Less than 1% of the initial concentration of phenanthrene remained in soil amended with any of the mixtures after 20 days. Although fluoranthene degradation progressed more slowly, 96% of this 4-ring PAH was removed from the mixtures after 20 days. In contrast, less than 20% benzo(a)pyrene was removed from soil from any of the microcosms and when phenanthrene and fluoranthene were both present, even less (6%) benzo(a)pyrene was removed. The rate at which the three PAHs was degraded when present as components of a mixture was then compared with data from the parallel study by Sawulski et al. (2014), where the same PAHs were added to soil as sole contaminants (Table 1). Of the PAHs examined, only fluoranthene was removed faster when present in a mixture. The presence of phenanthrene, or a combination of phenanthrene and benzo(a)pyrene significantly increased (P < 0001) the rate of fluoranthene degradation, reducing its half-life in soil by ~50% (Table 1). In contrast, the lower molecular weight phenanthrene had a significantly greater (P < 0001) half-life in soil when fluoranthene and/or benzo(a)pyrene were also present. Although phenanthrene on its own had no effect on the rate at which benzo(a)pyrene was degraded, the 5-ring PAH was removed three times more slowly in the presence of phenanthrene and fluoranthene, with its estimated half-life increasing from 80 23 to 206 64 days. The more rapid degradation of lower molecular weight PAHs compared to high molecular compounds has been widely
(b)
100
75
75
75
50
50
50
25
25
25
0
5
10 Time (days)
15
20
0
5
10 Time (days)
15
20
(c)
100
0
5
10
15
20
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Figure 1 Degradation of individual polycyclic aromatic hydrocarbons, in different mixtures consisting of (a) phenanthrene and fluoranthene; (b) phenanthrene and benzo(a)pyrene; (c) phenanthrene, fluoranthene and benzo(a)pyrene. Markers represent (●) phenanthrene (200 mg kg1), (▲) fluoranthene and (■) benzo(a)pyrene (each 50 mg kg1). Each data point is a mean of three measurements with error bars representing SEM.
200
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
P. Sawulski et al.
Microbial degradation of PAH mixtures in soil
YðtÞ ¼ a expðb tÞ
ð1Þ
With Y(t) the concentration (in %) of PAH at time t (days), a being the initial concentration and b being the exponential decay constant (day1). From this, half- life values (in days) of each PAH were estimated using: t1=2 ¼
lnð1Þ b
ð2Þ
With k the decay constant obtained from (1). The mean residence times (s) of the PAH in days were computed following: s ¼ lnð2Þ t1=2
ð3Þ
With t1/2 being the half-life obtained from (2). Goodness of fit was assessed by computing adjusted R2 values, acknowledging the limitations to use with nonlinear regressions. Permutation tests were used to assess significances of the fits. All regressions were calculated using SIGMAPLOT ver. 11. Univariate data, including degradation rates, dehydrogenase activity, pah-rhda gene copy numbers from GN and positive bacteria (GP), and species richness of bacteria (Sbac) and fungi (Sfungi) were analysed using a twoway ANOVA with Time (fixed, four levels: 0, 2, 10 and 20 days) and Treatment (fixed, the several levels of mixtures of PAHs) as fixed factors. When significant differences were detected by ANOVA, post-hoc pairwise comparisons (using Tukey) were computed to further explore differences. Significance was considered at a < 005. All univariate statistics were computed using SIGMAPLOT ver. 11 (Systat Software Inc, San Jose, CA). Multivariate methods were used to test whether treatments affected TRFLP and ARISA profiles. Abundance data (represented by relative fluorescent units) were square root transformed and Bray-Curtis dissimilarity matrices were computed among samples. Permutational multivariate analyses of variance (PERMANOVA) were computed to test null hypotheses (at a = 005) of no differences among assemblages across levels within the factors using the same model as for the univariate analyses. Probabilities were calculated based on 9999 permutations of residuals under a reduced model (Type III SS). Results of the PERMANOVA were supported and visualized with 2-dimensisional ordination using canonical analysis of principal coordinates (CAP). This approach fits a set of axes through the multivariate cloud that are best at discriminating among different a priori (hypothesis-based) treatment groups or time points. All multivariate statistics were computed using PRIMER v6 with PERMANOVA add-on (PRIMER-E Ltd. Plymouth, UK). Degradation rates of individual PAHs, dehydrogenase activity, pah-rhda gene copy numbers from GN and
206
Gram-positive bacteria (GP), species richness of bacteria (Sbac), fungi (Sfungi), bacterial and fungal assemblages obtained during this experiment were compared with respective data reported in Sawulski et al. (2014). For this, a two-way ANOVA including Time (fixed, three levels: 2, 10, 20) and Treatment (fixed, two levels: single and mixture) was computed. Post-hoc pairwise comparisons tests (Tukey) were computed to test differences between single and mixed PAH treatments for each time point using SIGMAPLOT ver. 11 (Systat Software Inc). Acknowledgements This work was funded under the Irish Research Council Postgraduate Scholarship Programme (grant no. RS/2005/ 187) and the Irish Environmental Protection Agency STRIVE programme 2007–2013 (grant no. 2008-PhDWRM-1). Conflict of Interest No conflict of interest declared. References Bamforth, S.M. and Singleton, I. (2005) Bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions. J Chem Technol Biotechnol 80, 723–736. Bouchez, M., Blanchet, D. and Vandecasteele, J.P. (1995) Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism. Applied Microbiology and Biotechnology 43, 156–164. Bouchez, M., Blanchet, D., Bardin, V., Haeseler, F. and Vandecasteele, J.P. (1999) Efficiency of defined strains and of soil consortia in the biodegradation of polycyclic aromatic hydrocarbon (PAH) mixtures. Biodegradation 10, 429–435. Cerniglia, C.E. (1993) Biodegradation of polycyclic aromatic hydrocarbons. Curr Opin Biotechnol 4, 331–338. Daniel, R. (2005) The metagenomics of soil. Nat Rev Microbiol 3, 470–478. Dean-Ross, D., Moody, J. and Cerniglia, C.E. (2002) Utilization of mixtures of polycyclic aromatic hydrocarbons by bacteria isolated from contaminated sediment. FEMS Microbiol Ecol 41, 1–7. Doyle, E., Muckian, L., Hickey, A.M. and Clipson, N. (2008) Microbial PAH degradation. Adv Appl Microbiol 65, 27– 66. Griffiths, R.I., Whiteley, A.S., O’Donnell, A.G. and Bailey, M.J. (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA-based microbial community composition. Appl Environ Microbiol 66, 5488–5491.
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
P. Sawulski et al.
TRF (bp) 52 60 61 62 64 73 86 88 92 112 113 127 128 129 137 138 139 144 145 146 147 148 149 150 151 158 159 160 161 189 197 227 265 303 432 485 486 487 488 514 515 540
Day 2
Day 10
Day 20
Relative abundance contribution (%) 1 2 3 4 5
10
20
30
40
50
(b)
ITS (bp) 55 68 73 197 200 201 202 203 204 206 211 213 215 216 218 219 220 221 222 223 224 225 226 227 228 230 231 330 331 340 341 474 482 508 568 572 573 582 583 587 588 598 599 623 624 634 676 677 679 680 681 704 726
no ph P A H e flu ba p ph e+ ph flu e ph +bap e+ no flu+ ph PAH bap e flu ph phe+flu e ph +bap e+ no flu+ ph P A H bap e flu ba p ph e+ ph flu e ph +ba e+ p flu +b ap
(a)
no ph P A H e flu ba p ph e+ ph flu e ph +bap e+ no flu+ ph P A H bap e flu ph e ph +flu e+ ph bap e no +flu+ ph PAH bap e flu ba p ph e+ ph flu e ph +ba e+ p flu +b ap
Microbial degradation of PAH mixtures in soil
Day 2
Day 10
Day 20
Figure 2 Heat maps of (a) bacterial and (b) fungal community profiles obtained from soils amended with single polycyclic aromatic hydrocarbons and their mixtures on days 2, 10 and 20 (phe = phenanthrene, flu = fluoranthene, bap = benzo(a)pyrene). The greyscale indicates relative abundance, with white being absent and black representing the most abundant fragments.
day 2, fungal assemblages in PAH mixtures containing fluoranthene were significantly different from the other treatments (P < 005), including the unamended soil 202
(Fig. 3b, Table S1). In contrast to the bacteria, fungal communities on day 20 remained significantly different (P < 005) from unamended controls suggesting a more
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
P. Sawulski et al.
Microbial degradation of PAH mixtures in soil
Table 2 Pearson product–moment correlation coefficients (r) between polycyclic aromatic hydrocarbon (PAH) degradation rates and changes in copy numbers of the gene encoding the a-subunit of the PAH ring hydroxylating dioxygenase associated with Gram-positive bacteria (PAH-RHDa GP) and a similar gene associated with Gram-negative bacteria (PAH-RHDa GN), species number of bacteria and fungi. Superscripts are P-values (based on separate two-tailed t-tests assuming true correlation is equal to 0) with those in bold being
ORIGINAL ARTICLE
Differential degradation of polycyclic aromatic hydrocarbon mixtures by indigenous microbial assemblages in soil P. Sawulski1, B. Boots1,2, N. Clipson1 and E. Doyle1 1 Environmental Microbiology Group, School of Biology and Environmental Science and Earth Institute, University College Dublin, Dublin, Ireland 2 School of Biosystems Engineering, Agriculture and Food Science Centre, University College Dublin, Dublin, Ireland
Significance and Impact of the Study: Contaminated sites generally contain complex mixtures of pollutants. Development of effective bioremediation strategies for contaminated soils requires knowledge of the response of soil microbial communities to such mixtures. This study provides information on the degradation of different mixtures of three priority pollutants in soil with a history of polycyclic aromatic hydrocarbon contamination and examines the response of soil bacterial and fungal communities to the presence of these pollutants as sole contaminants or as part of a mixture. This is one of few studies todate to compare the effects of single compounds and pollutant mixtures on more than one soil microbial community.
Keywords bacterial communities, biodegradation, diversity, fungal communities, polycyclic aromatic hydrocarbons, soil. Correspondence Evelyn Doyle, Environmental Microbiology Group, School of Biology and Environmental Science and Earth Institute, University College Dublin, Belfield, Dublin, Ireland. E-mail: [email protected] 2015/0618: received 25 March 2015, revised 11 May 2015 and accepted 17 May 2015 doi:10.1111/lam.12446
Abstract Environmental contamination by polycyclic aromatic hydrocarbons (PAHs) typically occurs as mixtures of compounds. In this study, the response of indigenous soil bacterial and fungal communities to mixtures containing phenanthrene, fluoranthene and benzo(a)pyrene in various combinations was examined using molecular fingerprinting techniques and quantification of a key PAH degradative gene. Results were compared to a parallel study by Sawulski et al. (2014) which examined the effect of these PAHs on soil microbial communities when added as single contaminants. The rate of degradation of individual PAHs varied depending on whether the PAH was present as a single contaminant or in a mixture; phenanthrene was degraded most rapidly when present as a sole contaminant, fluoranthene was removed faster in the presence of the lower molecular weight phenanthrene and the rate of benzo(a)pyrene degradation was reduced in the presence of the 4-ring PAH, fluoranthene. Bacterial and fungal assemblages differed significantly between treatments regardless of which PAH was added to soil. Although less abundant than the Gram-negative PAH-RHDa gene, the gene associated with Grampositive bacteria responded to a greater extent to the presence of PAHs, either as single compounds or as mixtures and this increase was significantly correlated with PAH degradation.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are recognized as significant health risks and consequently listed as priority pollutants by environmental protection agencies worldwide. Combustion of fossil fuels is a major contributor to the widespread occurrence of PAHs in the environment, with 90% of emissions ending up in the top
20 cm layer of soil (Maliszewska-Kordybach 1999). PAHs are chemically very stable and the rate of degradation decreases, and toxicity increases as the number of benzene rings of the molecule increases (Cerniglia 1993). Many studies have demonstrated the ability of bacteria and fungi to degrade a wide range of PAHs (Peng et al. 2008). Soils typically harbour a great diversity and abundance of microbes (Daniel 2005) and often contain
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
199
P. Sawulski et al.
Microbial degradation of PAH mixtures in soil
micro-organisms capable of degrading a range of PAHs. Genes involved in degradation of PAHs, such as those encoding ring hydroxylating dioxygenases (RHD) serve as useful biomarkers for the presence of potential PAHdegrading bacteria in soil (Doyle et al. 2008). Recently, Sawulski et al. (2014) showed that soil microbial assemblages responded differently to phenanthrene, fluoranthene and benzo(a)pyrene when these PAHs were added to soil as single contaminants. However, soils contaminated with PAHs typically contain mixtures of compounds differing in structure, susceptibility to degradation and toxicity. Effective bioremediation strategies for such soils require an understanding of how indigenous soil microbial communities respond to the presence of PAH mixtures rather than single compounds. Studies have reported a significant increase in the degradation of high molecular weight PAHs such as fluoranthene, pyrene or benzo(a)pyrene in the presence of lower molecular weight PAHs such as phenanthrene or naphthalene (Juhasz and Naidu 2000; Somtrakoon et al. 2008). However, most studies on PAH mixtures have focussed on pure cultures (Bouchez et al. 1995; van Herwijnen et al. 2003) or soils inoculated with microorganisms (Somtrakoon et al. 2008), with little information concerning the effect of PAH mixtures on indigenous soil communities available. In this study, the response of soil microbial communities to the addition of various combinations of PAHs (3 ring phenanthrene, 4-ring fluoranthene and 5-ring benzo(a)pyrene) was investigated in a microcosm-based system. This work was carried out in parallel to the study by Sawulski et al. (2014) which examined the effect of these PAHs on soil microbial community dynamics when added as single contaminants; results from both studies are compared.
(a)
PAH remaining (%)
100
Results and discussion Degradation of PAH mixtures Phenanthrene and fluoranthene were almost completely removed from the soil microcosms regardless of mixture composition (Fig. 1). Less than 1% of the initial concentration of phenanthrene remained in soil amended with any of the mixtures after 20 days. Although fluoranthene degradation progressed more slowly, 96% of this 4-ring PAH was removed from the mixtures after 20 days. In contrast, less than 20% benzo(a)pyrene was removed from soil from any of the microcosms and when phenanthrene and fluoranthene were both present, even less (6%) benzo(a)pyrene was removed. The rate at which the three PAHs was degraded when present as components of a mixture was then compared with data from the parallel study by Sawulski et al. (2014), where the same PAHs were added to soil as sole contaminants (Table 1). Of the PAHs examined, only fluoranthene was removed faster when present in a mixture. The presence of phenanthrene, or a combination of phenanthrene and benzo(a)pyrene significantly increased (P < 0001) the rate of fluoranthene degradation, reducing its half-life in soil by ~50% (Table 1). In contrast, the lower molecular weight phenanthrene had a significantly greater (P < 0001) half-life in soil when fluoranthene and/or benzo(a)pyrene were also present. Although phenanthrene on its own had no effect on the rate at which benzo(a)pyrene was degraded, the 5-ring PAH was removed three times more slowly in the presence of phenanthrene and fluoranthene, with its estimated half-life increasing from 80 23 to 206 64 days. The more rapid degradation of lower molecular weight PAHs compared to high molecular compounds has been widely
(b)
100
75
75
75
50
50
50
25
25
25
0
5
10 Time (days)
15
20
0
5
10 Time (days)
15
20
(c)
100
0
5
10
15
20
Time (days)
Figure 1 Degradation of individual polycyclic aromatic hydrocarbons, in different mixtures consisting of (a) phenanthrene and fluoranthene; (b) phenanthrene and benzo(a)pyrene; (c) phenanthrene, fluoranthene and benzo(a)pyrene. Markers represent (●) phenanthrene (200 mg kg1), (▲) fluoranthene and (■) benzo(a)pyrene (each 50 mg kg1). Each data point is a mean of three measurements with error bars representing SEM.
200
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
P. Sawulski et al.
Microbial degradation of PAH mixtures in soil
YðtÞ ¼ a expðb tÞ
ð1Þ
With Y(t) the concentration (in %) of PAH at time t (days), a being the initial concentration and b being the exponential decay constant (day1). From this, half- life values (in days) of each PAH were estimated using: t1=2 ¼
lnð1Þ b
ð2Þ
With k the decay constant obtained from (1). The mean residence times (s) of the PAH in days were computed following: s ¼ lnð2Þ t1=2
ð3Þ
With t1/2 being the half-life obtained from (2). Goodness of fit was assessed by computing adjusted R2 values, acknowledging the limitations to use with nonlinear regressions. Permutation tests were used to assess significances of the fits. All regressions were calculated using SIGMAPLOT ver. 11. Univariate data, including degradation rates, dehydrogenase activity, pah-rhda gene copy numbers from GN and positive bacteria (GP), and species richness of bacteria (Sbac) and fungi (Sfungi) were analysed using a twoway ANOVA with Time (fixed, four levels: 0, 2, 10 and 20 days) and Treatment (fixed, the several levels of mixtures of PAHs) as fixed factors. When significant differences were detected by ANOVA, post-hoc pairwise comparisons (using Tukey) were computed to further explore differences. Significance was considered at a < 005. All univariate statistics were computed using SIGMAPLOT ver. 11 (Systat Software Inc, San Jose, CA). Multivariate methods were used to test whether treatments affected TRFLP and ARISA profiles. Abundance data (represented by relative fluorescent units) were square root transformed and Bray-Curtis dissimilarity matrices were computed among samples. Permutational multivariate analyses of variance (PERMANOVA) were computed to test null hypotheses (at a = 005) of no differences among assemblages across levels within the factors using the same model as for the univariate analyses. Probabilities were calculated based on 9999 permutations of residuals under a reduced model (Type III SS). Results of the PERMANOVA were supported and visualized with 2-dimensisional ordination using canonical analysis of principal coordinates (CAP). This approach fits a set of axes through the multivariate cloud that are best at discriminating among different a priori (hypothesis-based) treatment groups or time points. All multivariate statistics were computed using PRIMER v6 with PERMANOVA add-on (PRIMER-E Ltd. Plymouth, UK). Degradation rates of individual PAHs, dehydrogenase activity, pah-rhda gene copy numbers from GN and
206
Gram-positive bacteria (GP), species richness of bacteria (Sbac), fungi (Sfungi), bacterial and fungal assemblages obtained during this experiment were compared with respective data reported in Sawulski et al. (2014). For this, a two-way ANOVA including Time (fixed, three levels: 2, 10, 20) and Treatment (fixed, two levels: single and mixture) was computed. Post-hoc pairwise comparisons tests (Tukey) were computed to test differences between single and mixed PAH treatments for each time point using SIGMAPLOT ver. 11 (Systat Software Inc). Acknowledgements This work was funded under the Irish Research Council Postgraduate Scholarship Programme (grant no. RS/2005/ 187) and the Irish Environmental Protection Agency STRIVE programme 2007–2013 (grant no. 2008-PhDWRM-1). Conflict of Interest No conflict of interest declared. References Bamforth, S.M. and Singleton, I. (2005) Bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions. J Chem Technol Biotechnol 80, 723–736. Bouchez, M., Blanchet, D. and Vandecasteele, J.P. (1995) Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism. Applied Microbiology and Biotechnology 43, 156–164. Bouchez, M., Blanchet, D., Bardin, V., Haeseler, F. and Vandecasteele, J.P. (1999) Efficiency of defined strains and of soil consortia in the biodegradation of polycyclic aromatic hydrocarbon (PAH) mixtures. Biodegradation 10, 429–435. Cerniglia, C.E. (1993) Biodegradation of polycyclic aromatic hydrocarbons. Curr Opin Biotechnol 4, 331–338. Daniel, R. (2005) The metagenomics of soil. Nat Rev Microbiol 3, 470–478. Dean-Ross, D., Moody, J. and Cerniglia, C.E. (2002) Utilization of mixtures of polycyclic aromatic hydrocarbons by bacteria isolated from contaminated sediment. FEMS Microbiol Ecol 41, 1–7. Doyle, E., Muckian, L., Hickey, A.M. and Clipson, N. (2008) Microbial PAH degradation. Adv Appl Microbiol 65, 27– 66. Griffiths, R.I., Whiteley, A.S., O’Donnell, A.G. and Bailey, M.J. (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA-based microbial community composition. Appl Environ Microbiol 66, 5488–5491.
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
P. Sawulski et al.
TRF (bp) 52 60 61 62 64 73 86 88 92 112 113 127 128 129 137 138 139 144 145 146 147 148 149 150 151 158 159 160 161 189 197 227 265 303 432 485 486 487 488 514 515 540
Day 2
Day 10
Day 20
Relative abundance contribution (%) 1 2 3 4 5
10
20
30
40
50
(b)
ITS (bp) 55 68 73 197 200 201 202 203 204 206 211 213 215 216 218 219 220 221 222 223 224 225 226 227 228 230 231 330 331 340 341 474 482 508 568 572 573 582 583 587 588 598 599 623 624 634 676 677 679 680 681 704 726
no ph P A H e flu ba p ph e+ ph flu e ph +bap e+ no flu+ ph PAH bap e flu ph phe+flu e ph +bap e+ no flu+ ph P A H bap e flu ba p ph e+ ph flu e ph +ba e+ p flu +b ap
(a)
no ph P A H e flu ba p ph e+ ph flu e ph +bap e+ no flu+ ph P A H bap e flu ph e ph +flu e+ ph bap e no +flu+ ph PAH bap e flu ba p ph e+ ph flu e ph +ba e+ p flu +b ap
Microbial degradation of PAH mixtures in soil
Day 2
Day 10
Day 20
Figure 2 Heat maps of (a) bacterial and (b) fungal community profiles obtained from soils amended with single polycyclic aromatic hydrocarbons and their mixtures on days 2, 10 and 20 (phe = phenanthrene, flu = fluoranthene, bap = benzo(a)pyrene). The greyscale indicates relative abundance, with white being absent and black representing the most abundant fragments.
day 2, fungal assemblages in PAH mixtures containing fluoranthene were significantly different from the other treatments (P < 005), including the unamended soil 202
(Fig. 3b, Table S1). In contrast to the bacteria, fungal communities on day 20 remained significantly different (P < 005) from unamended controls suggesting a more
Letters in Applied Microbiology 61, 199--207 © 2015 The Society for Applied Microbiology
P. Sawulski et al.
Microbial degradation of PAH mixtures in soil
Table 2 Pearson product–moment correlation coefficients (r) between polycyclic aromatic hydrocarbon (PAH) degradation rates and changes in copy numbers of the gene encoding the a-subunit of the PAH ring hydroxylating dioxygenase associated with Gram-positive bacteria (PAH-RHDa GP) and a similar gene associated with Gram-negative bacteria (PAH-RHDa GN), species number of bacteria and fungi. Superscripts are P-values (based on separate two-tailed t-tests assuming true correlation is equal to 0) with those in bold being
