Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Silver nanoparticle inhibition of polycyclic aromatic hydrocarbons degradation by Mycobacterium species RJGII-135 S.R. Mueller-Spitz1 and K.D. Crawford2 1 Department of Biology and Microbiology, University of Wisconsin Oshkosh, Oshkosh, WI, USA 2 Department of Chemistry, University of Wisconsin Oshkosh, Oshkosh, WI, USA
Significance and Impact of the Study: Silver nanoparticle (AgNP) pollution threatens bacterial-mediated processes due to their antibacterial properties. With the widespread commercial use of AgNP, continued environmental release is inevitable and we are just beginning to understand the potential environmental ramifications of nanoparticle pollution. This study examined AgNP inhibition of carbon metabolism through the polycyclic aromatic hydrocarbon degradation by Mycobacterium species RJGII-135. Sublethal doses altered PAH metabolism, which is dependent upon cell membrane properties and intracellular proteins. The changed carbon metabolism when exposed to sublethal doses of AgNP suggests broad impacts of this pollution on bacterial carbon cycling in diverse environments.
Keywords biodegradation, Mycobacterium, polycyclic aromatic hydrocarbons, silver nanoparticles. Correspondence Sabrina R. Mueller-Spitz, Department of Biology and Microbiology, University of Wisconsin Oshkosh, 800 Algoma Blvd, Oshkosh, WI 54901, USA. E-mail: [email protected] 2013/1262: received 24 June 2013, revised 30 October 2013 and accepted 7 November 2013 doi:10.1111/lam.12205
Abstract Polycyclic aromatic hydrocarbons (PAH) are a common environmental contaminant originating from both anthropogenic and natural sources. Mycobacterium species are highly adapted to utilizing a variety of PAH. Silver nanoparticles (AgNP) are an emerging contaminant that possess bactericidal properties, interferes with the bacterial membrane and alters function. Mycobacterium sp. strain RJGII-135 provided a model bacterium to assess changes in carbon metabolism by focusing on PAH degradation, which is dependent upon passive uptake of hydrophobic molecules into the cell membrane. A mixture of 18 PAH served as a complex mixture of carbon sources for assessing carbon metabolism. At environmentally relevant PAH concentrations, RJGII-135 degraded two-, three-, and four-ring PAH within 72 h, but preferentially attacked phenanthrene and fluorene. Total cell growth and PAH degradation were successively reduced when exposed to 005– 05 mg 1 1 AgNP. However, 005 mg l 1 AgNP inhibited degradation of naphthalene, acenaphthylene and acenaphthalene. RJGII-135 retained the ability to degrade the methylated naphthalenes regardless of AgNP concentration suggesting that proteins involved in dihydrodiol formation were inhibited. The reduced PAH metabolism of RJGII-135 when exposed to sublethal concentrations of AgNP provides evidence that nanoparticle pollution could alter carbon cycling in soils, sediment and aquatic environments.
Introduction Nanoparticle pollution is an emerging concern for aquatic and soil environments. Nanoparticles can enter the environment after release from a commercial product through disposal, weathering, application of sewage sludge or with sewage effluent waters (Kiser et al. 2009; Nowack et al. 330
2012). Both aquatic and soil environments are likely to receive increasing amounts of nanoparticle pollution. A concern is how nanoparticle pollution will affect bacterial-mediated processes because of their antibacterial activities. Silver nanoparticles (AgNP) are commonly used for these antibacterial properties. Key factors controlling nanoparticle toxicity to bacteria are size, shape, number
Letters in Applied Microbiology 58, 330--337 © 2013 The Society for Applied Microbiology
S.R. Mueller-Spitz and K.D. Crawford
of nanoparticles per cell, aggregation, elemental composition, capping agents and interaction with UV light (reviewed in Fabrega et al. 2011; Hajipour et al. 2012). Numerous modes of action have been proposed for the antibacterial activities including membrane destabilization, generation of damaging oxygen radical species, intracellular nanoparticle-DNA or protein interactions and release of metal ions (Hajipour et al. 2012; Schacht et al. 2013). Delayed growth has been commonly reported for various bacterial species on nanoparticle exposure (Sondi and Salopek-Sondi 2004; Li et al. 2010; Dimkpa et al. 2011), which may relate to decreased energy generation because of changes in electron transport (Anas et al. 2013). The ability to tolerate nanoparticles varies among different strains of bacteria based upon cell envelope composition or the production of extracellular polysaccharides (Dimkpa et al. 2011; Jin et al. 2013). Less is known about the physiological response stimulated by sublethal concentrations of nanoparticles. The potential for nanoparticle pollution to kill susceptible bacterial taxa and alter metabolism could have great impact upon bacterial processes such as carbon and nitrogen cycling. Decreased total bacterial counts and reduced diversity following nanoparticle exposure correspond to changes in bacterial community composition (Bradford et al. 2009; Doiron et al. 2012; Rodrigues et al. 2013). Subsequently, these composition changes altered bacterial community function following nanoparticle exposure (Das et al. 2012; Kumar et al. 2012). Choi and Hu (2009) illustrated that 1 mg l 1 AgNP inhibited bacterial nitrification. Similar effects have been seen in soils exposed to carbon nanotubes, where both soil biomass and extracellular enzymes essential for complex carbon utilization were reduced (Chung et al. 2011). These functional changes could reduce bacterial degradation of various recalcitrant organic pollutants. An important area of bacterial metabolism in certain polluted environments is the transformation of organic compounds to less toxic substances. Polycyclic aromatic hydrocarbons (PAH) are a major chemical class of concern because of their continual releases from a myriad of sources, detection in aquatic and terrestrial environments, and their negative impacts upon human health (Tobiszewski and Namiesnik 2012). These molecules are diverse in chemical structure and are classified as both low molecular weight (≤three-rings, LMW) and high molecular weight (≥four-rings, HMW). Regardless the source, the PAH are released as mixtures of LMW and HMW forms (Achten and Hofmann 2009; Tobiszewski and Namiesnik 2012). Environmental persistence is driven by low water solubility and hydrophobicity that increases with number of rings (Sikkema et al. 1995; Kanaly and Harayama 2010). Numerous soil and aquatic
AgNP inhibit PAH degradation
bacterial species can degrade PAH as a sole carbon and energy source, including Pseudomonas, Rhodococcus, Burkholderia, Arthrobacter, Polaromonas and Sphingomonas species (Kanaly and Harayama 2000, 2010). It has been suggested that the ability to degrade these recalcitrant molecules is a common metabolic trait of many bacteria taxa because the enzymes that carry out oxidation of the PAH to a dihydrodiol function against many natural aromatics (Kanaly and Harayama 2010). Yet, the ability to degrade both LWM-PAH and HMW-PAH commonly resides with Mycobacterium species (Cerniglia and Heitkamp 1990; Kanaly and Harayama 2010). Various Mycobacterium species isolated from sediments, and soils can breakdown pyrene, benzo(a)pyrene, fluoranthene, benzo(a)anthracene, naphthalene and anthracene, illustrating the wide metabolic activity of this genera (Cerniglia and Heitkamp 1990; Grosser et al. 1991; Rehmann et al. 1998, 2001; Kanaly and Harayama 2010). Mycobacterium species have mycolic acids that are very hydrophobic forming an impermeable cell wall (Niederweis et al. 2010), which aids in the uptake of hydrophobic PAH (Sikkema et al. 1995). Prior work has focused on characterizing breakdown pathway(s) for an individual PAH when provided as the sole carbon source allowing complex metabolic pathways to be generated. M. vanbaalenii PYR-1 degrades numerous aromatics as demonstrated by the presence of numerous ring-hydroxylating oxygenases, cytochrome P450 monooxygenases and other monooxygenases that are involved in the beginning step of aromatic degradation (Kweon et al. 2011). M. vanbaalenii PYR-1 activates complicated multiprotein degradation pathways to breakdown PAH with different metabolic networks for both LMW and HMW molecules (Kim et al. 2007, 2008, 2009; Kweon et al. 2007, 2011). These metabolic pathways have been detected in various aromatic degraders providing support that Mycobacterium species are highly evolved for PAH degradation (Kweon et al. 2011). The co-occurrence of organic and metal pollutants has long been a major concern for the feasibility of bacterialmediated biodegradation as metal ions reduce bacterial diversity and function. In this study, we examined how sublethal AgNP concentrations would interfere with PAH degradation. Mycobacterium species strain RJGII-135 was used as a model PAH degrader (Grosser et al. 1991, 1995; Schneider et al. 1996; McLellan et al. 2002). RJGII135 was grown in a mixture of PAH provided at low part per billion concentration in a low-carbon media to mimic natural systems in that carbon sources are somewhat limited but diverse (Arp et al. 2011). AgNP interactions with the cell could alter up-take or oxidation of the various PAH providing evidence for altered carbon utilization.
Letters in Applied Microbiology 58, 330--337 © 2013 The Society for Applied Microbiology
331
AgNP inhibit PAH degradation
S.R. Mueller-Spitz and K.D. Crawford
Results and discussion In contaminated environments, PAH occur as complex mixtures, but little is known about RJGII-135 ability to utilize both LMW-PAH and HMW-PAH. The uninduced cells in the presence of alternative carbon sources degraded ten of the 18 PAH, which included all of the two- and three-ring molecules as well as the four-ring molecules pyrene and fluoranthene (Table 1). The HMW-PAH, specifically five- and six-ring molecules, were not degraded over the 72-h incubation period. RJGII-135 preferentially attacked 1-methylnaphthalene, 2-methylnaphthalene, fluorene and phenanthrene with less than 1% of the parent compounds remaining (Table 1). PAH degradation has been shown to be more effective in the presence of other carbon sources (Boldrin et al. 1993), which may account for the rapid loss of ten PAHs from an uninduced culture. However, if given a longer time, there may have been a decrease in the HMW-PAH. RJGII-135 is capable of degrading benzo[a]pyrene (BaP) and benz[a]anthracene (BaA; Schneider et al. 1996), although the bacterium more efficiently mineralizes pyrene and phenanthrene over BaP and BaA (McLellan et al. 2002). The related organism M. vanbaalenii PYR-1 also preferentially degrades phenanthrene over fluorene and pyrene (Kim et al. 2012). Pyrene can inhibit the degradation of benzo[a]pyrene (McLellan et al. 2002), which may account for why RJGII-135 was unable to degrade any of the HMW-PAH with LMW-PAH remaining. The degradation of HMW-PAH may only begin once the preferred PAH are removed due to activation of degradative pathways (Kweon et al. 2011), which relates to the substrate specificity of the rieske nonheme iron proteins (Kweon et al. 2010).
RJGII-135 tolerated double the concentrations of silver ion (Ag+; 1 mg l 1) as compared to AgNP (05 mg l 1) when grown in R2B media. It has been commonly reported that AgNP are more toxic than Ag+, although AgNP toxicity can be driven by the capping agent (Fabrega et al. 2011; Arnaout and Gunsch 2012). For AgNP capped with PVA, similar tolerance levels have been seen in other bacteria (Fabrega et al. 2011). The hydrophobic cell envelope of this fast-growing Mycobacterium species appeared not to increase its tolerance to AgNP. Tolerance to both forms of silver was reduced in 10% strength R2B, which may relate to the higher salt concentrations allowing the AgNP to aggregate reducing their toxicity in R2B. RJGII-135 tolerated up to 025 mg l 1 silver nanoparticles in 01 9 R2B media, which reduced total viable cells from 699 9 109 colony-forming units ml 1 in the control to 319 9 107 colony-forming units ml 1 in the exposed samples after 72 h (data not shown). The 025 mg l 1 AgNP was therefore chosen as upper limit to maintain some viable cells with 05 mg l 1 as the concentration of AgNP that should be lethal to RJGII-135. The presence of AgNP in the PAH mixture reduced total cell growth and PAH degradation. There was a lag in growth for the 005 mg l 1 treatment at 24 h with lower final cell densities (Fig. 1). This indicates there was inhibition despite the presence of simple carbon sources such as pyruvate and glucose. Limited growth was detected at both of the higher AgNP concentrations indicating some cells remained viable. Reduced growth rates have been seen in Escherichia coli, Pseudomonas sp., Bacillus subtilis and Cupriadvius necator on exposure to various NP (Sondi and Salopek-Sondi 2004; Li et al. 2010; Dimkpa et al. 2011; Schacht et al. 2013). These reductions
Table 1 Concentration of parent polycyclic aromatic hydrocarbons (PAH) (average lg l (019 R2B) with and without silver nanoparticles Control PAH
0h
NAPT 1-MeNAPT 2-MeNAPT ACNY ACEN FLUR PHEN ANTH FLTH PYR
25 9 17 185 16 37 15 22 10 10
005 mg l
2 3 2 09 1 4 2 3 2 2
72 h
0h
5 1* ND* ND* 3 2* 4 2* ND* ND* 7 7* 2 2* 2 2*
27 11 15 17 14 36 13 20 10 10
1
and standard deviation) in low strength media
025 mg l 72 h
3 2 2 2 1 4 2 3 2 1
1
11 ND* 23 8 9 07 ND* 6 14 12
0h 4* 07* 3* 4* 07* 1* 03* 03*
29 9 14 18 15 37 13 22 12 11
1
05 mg l 72 h
4 3 3 1 2 5 3 10 3 4
45 17 11 19 13 26 ND* 16 15 18
0h 19† 04* 2† 3 2† 6* 6 3† 2*
25 7 12 17 14 35 12 19 15 13
1
72 h 3 3 1 1 2 3 3 5 2 3
32 4 10 20 13 34 14 19 19 21
15 1* 1* 2* 2 4 2† 4 2* 2*
NAPT: naphthalene, 1-MeNAPT: 1-methylnaphthalene, 2-MeNAPT: 2-methylnaphthalene, ACEN: acenaphthene, ACNY: acenaphthylene, FLUR: fluorene, PHEN: phenanthrene, ANTH: anthracene, FLTH: fluoranthene, PYR: pyrene. *P-values from paired t-test
ORIGINAL ARTICLE
Silver nanoparticle inhibition of polycyclic aromatic hydrocarbons degradation by Mycobacterium species RJGII-135 S.R. Mueller-Spitz1 and K.D. Crawford2 1 Department of Biology and Microbiology, University of Wisconsin Oshkosh, Oshkosh, WI, USA 2 Department of Chemistry, University of Wisconsin Oshkosh, Oshkosh, WI, USA
Significance and Impact of the Study: Silver nanoparticle (AgNP) pollution threatens bacterial-mediated processes due to their antibacterial properties. With the widespread commercial use of AgNP, continued environmental release is inevitable and we are just beginning to understand the potential environmental ramifications of nanoparticle pollution. This study examined AgNP inhibition of carbon metabolism through the polycyclic aromatic hydrocarbon degradation by Mycobacterium species RJGII-135. Sublethal doses altered PAH metabolism, which is dependent upon cell membrane properties and intracellular proteins. The changed carbon metabolism when exposed to sublethal doses of AgNP suggests broad impacts of this pollution on bacterial carbon cycling in diverse environments.
Keywords biodegradation, Mycobacterium, polycyclic aromatic hydrocarbons, silver nanoparticles. Correspondence Sabrina R. Mueller-Spitz, Department of Biology and Microbiology, University of Wisconsin Oshkosh, 800 Algoma Blvd, Oshkosh, WI 54901, USA. E-mail: [email protected] 2013/1262: received 24 June 2013, revised 30 October 2013 and accepted 7 November 2013 doi:10.1111/lam.12205
Abstract Polycyclic aromatic hydrocarbons (PAH) are a common environmental contaminant originating from both anthropogenic and natural sources. Mycobacterium species are highly adapted to utilizing a variety of PAH. Silver nanoparticles (AgNP) are an emerging contaminant that possess bactericidal properties, interferes with the bacterial membrane and alters function. Mycobacterium sp. strain RJGII-135 provided a model bacterium to assess changes in carbon metabolism by focusing on PAH degradation, which is dependent upon passive uptake of hydrophobic molecules into the cell membrane. A mixture of 18 PAH served as a complex mixture of carbon sources for assessing carbon metabolism. At environmentally relevant PAH concentrations, RJGII-135 degraded two-, three-, and four-ring PAH within 72 h, but preferentially attacked phenanthrene and fluorene. Total cell growth and PAH degradation were successively reduced when exposed to 005– 05 mg 1 1 AgNP. However, 005 mg l 1 AgNP inhibited degradation of naphthalene, acenaphthylene and acenaphthalene. RJGII-135 retained the ability to degrade the methylated naphthalenes regardless of AgNP concentration suggesting that proteins involved in dihydrodiol formation were inhibited. The reduced PAH metabolism of RJGII-135 when exposed to sublethal concentrations of AgNP provides evidence that nanoparticle pollution could alter carbon cycling in soils, sediment and aquatic environments.
Introduction Nanoparticle pollution is an emerging concern for aquatic and soil environments. Nanoparticles can enter the environment after release from a commercial product through disposal, weathering, application of sewage sludge or with sewage effluent waters (Kiser et al. 2009; Nowack et al. 330
2012). Both aquatic and soil environments are likely to receive increasing amounts of nanoparticle pollution. A concern is how nanoparticle pollution will affect bacterial-mediated processes because of their antibacterial activities. Silver nanoparticles (AgNP) are commonly used for these antibacterial properties. Key factors controlling nanoparticle toxicity to bacteria are size, shape, number
Letters in Applied Microbiology 58, 330--337 © 2013 The Society for Applied Microbiology
S.R. Mueller-Spitz and K.D. Crawford
of nanoparticles per cell, aggregation, elemental composition, capping agents and interaction with UV light (reviewed in Fabrega et al. 2011; Hajipour et al. 2012). Numerous modes of action have been proposed for the antibacterial activities including membrane destabilization, generation of damaging oxygen radical species, intracellular nanoparticle-DNA or protein interactions and release of metal ions (Hajipour et al. 2012; Schacht et al. 2013). Delayed growth has been commonly reported for various bacterial species on nanoparticle exposure (Sondi and Salopek-Sondi 2004; Li et al. 2010; Dimkpa et al. 2011), which may relate to decreased energy generation because of changes in electron transport (Anas et al. 2013). The ability to tolerate nanoparticles varies among different strains of bacteria based upon cell envelope composition or the production of extracellular polysaccharides (Dimkpa et al. 2011; Jin et al. 2013). Less is known about the physiological response stimulated by sublethal concentrations of nanoparticles. The potential for nanoparticle pollution to kill susceptible bacterial taxa and alter metabolism could have great impact upon bacterial processes such as carbon and nitrogen cycling. Decreased total bacterial counts and reduced diversity following nanoparticle exposure correspond to changes in bacterial community composition (Bradford et al. 2009; Doiron et al. 2012; Rodrigues et al. 2013). Subsequently, these composition changes altered bacterial community function following nanoparticle exposure (Das et al. 2012; Kumar et al. 2012). Choi and Hu (2009) illustrated that 1 mg l 1 AgNP inhibited bacterial nitrification. Similar effects have been seen in soils exposed to carbon nanotubes, where both soil biomass and extracellular enzymes essential for complex carbon utilization were reduced (Chung et al. 2011). These functional changes could reduce bacterial degradation of various recalcitrant organic pollutants. An important area of bacterial metabolism in certain polluted environments is the transformation of organic compounds to less toxic substances. Polycyclic aromatic hydrocarbons (PAH) are a major chemical class of concern because of their continual releases from a myriad of sources, detection in aquatic and terrestrial environments, and their negative impacts upon human health (Tobiszewski and Namiesnik 2012). These molecules are diverse in chemical structure and are classified as both low molecular weight (≤three-rings, LMW) and high molecular weight (≥four-rings, HMW). Regardless the source, the PAH are released as mixtures of LMW and HMW forms (Achten and Hofmann 2009; Tobiszewski and Namiesnik 2012). Environmental persistence is driven by low water solubility and hydrophobicity that increases with number of rings (Sikkema et al. 1995; Kanaly and Harayama 2010). Numerous soil and aquatic
AgNP inhibit PAH degradation
bacterial species can degrade PAH as a sole carbon and energy source, including Pseudomonas, Rhodococcus, Burkholderia, Arthrobacter, Polaromonas and Sphingomonas species (Kanaly and Harayama 2000, 2010). It has been suggested that the ability to degrade these recalcitrant molecules is a common metabolic trait of many bacteria taxa because the enzymes that carry out oxidation of the PAH to a dihydrodiol function against many natural aromatics (Kanaly and Harayama 2010). Yet, the ability to degrade both LWM-PAH and HMW-PAH commonly resides with Mycobacterium species (Cerniglia and Heitkamp 1990; Kanaly and Harayama 2010). Various Mycobacterium species isolated from sediments, and soils can breakdown pyrene, benzo(a)pyrene, fluoranthene, benzo(a)anthracene, naphthalene and anthracene, illustrating the wide metabolic activity of this genera (Cerniglia and Heitkamp 1990; Grosser et al. 1991; Rehmann et al. 1998, 2001; Kanaly and Harayama 2010). Mycobacterium species have mycolic acids that are very hydrophobic forming an impermeable cell wall (Niederweis et al. 2010), which aids in the uptake of hydrophobic PAH (Sikkema et al. 1995). Prior work has focused on characterizing breakdown pathway(s) for an individual PAH when provided as the sole carbon source allowing complex metabolic pathways to be generated. M. vanbaalenii PYR-1 degrades numerous aromatics as demonstrated by the presence of numerous ring-hydroxylating oxygenases, cytochrome P450 monooxygenases and other monooxygenases that are involved in the beginning step of aromatic degradation (Kweon et al. 2011). M. vanbaalenii PYR-1 activates complicated multiprotein degradation pathways to breakdown PAH with different metabolic networks for both LMW and HMW molecules (Kim et al. 2007, 2008, 2009; Kweon et al. 2007, 2011). These metabolic pathways have been detected in various aromatic degraders providing support that Mycobacterium species are highly evolved for PAH degradation (Kweon et al. 2011). The co-occurrence of organic and metal pollutants has long been a major concern for the feasibility of bacterialmediated biodegradation as metal ions reduce bacterial diversity and function. In this study, we examined how sublethal AgNP concentrations would interfere with PAH degradation. Mycobacterium species strain RJGII-135 was used as a model PAH degrader (Grosser et al. 1991, 1995; Schneider et al. 1996; McLellan et al. 2002). RJGII135 was grown in a mixture of PAH provided at low part per billion concentration in a low-carbon media to mimic natural systems in that carbon sources are somewhat limited but diverse (Arp et al. 2011). AgNP interactions with the cell could alter up-take or oxidation of the various PAH providing evidence for altered carbon utilization.
Letters in Applied Microbiology 58, 330--337 © 2013 The Society for Applied Microbiology
331
AgNP inhibit PAH degradation
S.R. Mueller-Spitz and K.D. Crawford
Results and discussion In contaminated environments, PAH occur as complex mixtures, but little is known about RJGII-135 ability to utilize both LMW-PAH and HMW-PAH. The uninduced cells in the presence of alternative carbon sources degraded ten of the 18 PAH, which included all of the two- and three-ring molecules as well as the four-ring molecules pyrene and fluoranthene (Table 1). The HMW-PAH, specifically five- and six-ring molecules, were not degraded over the 72-h incubation period. RJGII-135 preferentially attacked 1-methylnaphthalene, 2-methylnaphthalene, fluorene and phenanthrene with less than 1% of the parent compounds remaining (Table 1). PAH degradation has been shown to be more effective in the presence of other carbon sources (Boldrin et al. 1993), which may account for the rapid loss of ten PAHs from an uninduced culture. However, if given a longer time, there may have been a decrease in the HMW-PAH. RJGII-135 is capable of degrading benzo[a]pyrene (BaP) and benz[a]anthracene (BaA; Schneider et al. 1996), although the bacterium more efficiently mineralizes pyrene and phenanthrene over BaP and BaA (McLellan et al. 2002). The related organism M. vanbaalenii PYR-1 also preferentially degrades phenanthrene over fluorene and pyrene (Kim et al. 2012). Pyrene can inhibit the degradation of benzo[a]pyrene (McLellan et al. 2002), which may account for why RJGII-135 was unable to degrade any of the HMW-PAH with LMW-PAH remaining. The degradation of HMW-PAH may only begin once the preferred PAH are removed due to activation of degradative pathways (Kweon et al. 2011), which relates to the substrate specificity of the rieske nonheme iron proteins (Kweon et al. 2010).
RJGII-135 tolerated double the concentrations of silver ion (Ag+; 1 mg l 1) as compared to AgNP (05 mg l 1) when grown in R2B media. It has been commonly reported that AgNP are more toxic than Ag+, although AgNP toxicity can be driven by the capping agent (Fabrega et al. 2011; Arnaout and Gunsch 2012). For AgNP capped with PVA, similar tolerance levels have been seen in other bacteria (Fabrega et al. 2011). The hydrophobic cell envelope of this fast-growing Mycobacterium species appeared not to increase its tolerance to AgNP. Tolerance to both forms of silver was reduced in 10% strength R2B, which may relate to the higher salt concentrations allowing the AgNP to aggregate reducing their toxicity in R2B. RJGII-135 tolerated up to 025 mg l 1 silver nanoparticles in 01 9 R2B media, which reduced total viable cells from 699 9 109 colony-forming units ml 1 in the control to 319 9 107 colony-forming units ml 1 in the exposed samples after 72 h (data not shown). The 025 mg l 1 AgNP was therefore chosen as upper limit to maintain some viable cells with 05 mg l 1 as the concentration of AgNP that should be lethal to RJGII-135. The presence of AgNP in the PAH mixture reduced total cell growth and PAH degradation. There was a lag in growth for the 005 mg l 1 treatment at 24 h with lower final cell densities (Fig. 1). This indicates there was inhibition despite the presence of simple carbon sources such as pyruvate and glucose. Limited growth was detected at both of the higher AgNP concentrations indicating some cells remained viable. Reduced growth rates have been seen in Escherichia coli, Pseudomonas sp., Bacillus subtilis and Cupriadvius necator on exposure to various NP (Sondi and Salopek-Sondi 2004; Li et al. 2010; Dimkpa et al. 2011; Schacht et al. 2013). These reductions
Table 1 Concentration of parent polycyclic aromatic hydrocarbons (PAH) (average lg l (019 R2B) with and without silver nanoparticles Control PAH
0h
NAPT 1-MeNAPT 2-MeNAPT ACNY ACEN FLUR PHEN ANTH FLTH PYR
25 9 17 185 16 37 15 22 10 10
005 mg l
2 3 2 09 1 4 2 3 2 2
72 h
0h
5 1* ND* ND* 3 2* 4 2* ND* ND* 7 7* 2 2* 2 2*
27 11 15 17 14 36 13 20 10 10
1
and standard deviation) in low strength media
025 mg l 72 h
3 2 2 2 1 4 2 3 2 1
1
11 ND* 23 8 9 07 ND* 6 14 12
0h 4* 07* 3* 4* 07* 1* 03* 03*
29 9 14 18 15 37 13 22 12 11
1
05 mg l 72 h
4 3 3 1 2 5 3 10 3 4
45 17 11 19 13 26 ND* 16 15 18
0h 19† 04* 2† 3 2† 6* 6 3† 2*
25 7 12 17 14 35 12 19 15 13
1
72 h 3 3 1 1 2 3 3 5 2 3
32 4 10 20 13 34 14 19 19 21
15 1* 1* 2* 2 4 2† 4 2* 2*
NAPT: naphthalene, 1-MeNAPT: 1-methylnaphthalene, 2-MeNAPT: 2-methylnaphthalene, ACEN: acenaphthene, ACNY: acenaphthylene, FLUR: fluorene, PHEN: phenanthrene, ANTH: anthracene, FLTH: fluoranthene, PYR: pyrene. *P-values from paired t-test
