Environ. Sci. Technol. 2008, 42, 1938–1943
Assessing the Impact of Nanomaterials on Anaerobic Microbial Communities LEILA NYBERG,† RONALD F. TURCO,‡ A N D L O R I N G N I E S * ,† School of Civil Engineering, Purdue University, West Lafayette, Indiana 47907-2051, and Department of Agronomy, Purdue University, West Lafayette, Indiana 47907-1150
Received August 13, 2007. Revised manuscript received December 12, 2007. Accepted December 19, 2007.
As the technological benefits of nanotechnology begin to rapidly move from laboratory to large-scale industrial application, release of nanomaterials to the environment is inevitable. Little is known about the fate and effects of nanomaterials in nature. Major environmental receptors of nanomaterials will be soil, sediment, and biosolids from wastewater treatment. Analysis of anaerobic microbial activity and communities provides needed information about the effects of nanoparticles in certain environments. In this study, biosolids from anaerobic wastewater treatment sludge were exposed to fullerene (C60) in order to model an environmentally relevant discharge scenario. Activity was assessed by monitoring production of CO2 and CH4. Changes in community structure were monitored by denaturing gradient gel electrophoresis (DGGE), using primer sets targeting the small subunit rRNA genes of Bacteria, Archaea, and Eukarya. Findings suggest that C60 fullerenes have no significant effect on the anaerobic community over an exposure period of a few months. This conclusion is based on the absence of toxicity indicated by no change in methanogenesis relative to untreated reference samples. DGGE results show no evidence of substantial community shifts due to treatment with C60, in any subset of the microbial community.
Introduction The rapid pace of research and development for carbonbased manufactured nanoparticles (CMNP) has raised urgent discussion within the scientific community about potential effects of CMNP release to the environment (1, 2). Wastewater treatment sludge is a receptor and a possible vector to the environment for any new chemicals released during manufacturing or to an industrial waste stream (3). As nanotechnology matures from research discovery to industrial applications to large-volume manufacturing, releases are inevitable. Industrial CMNP releases will almost certainly occur as components of wastewater discharges. Since CMNPs are extremely hydrophobic, they will strongly partition into the biomass in wastewater treatment plants and ultimately anaerobic sludge digesters. Therefore, microbial communities in anaerobic digesters are excellent sentinel communities for evaluation of the effects of CMNPs. Determination of the * Corresponding author phone: (765) 494-8327; fax: (765) 4963145; e-mail:
[email protected]. † School of Civil Engineering. ‡ Department of Agronomy. 1938
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biodegradation potential of a chemical in anaerobic systems is also important for environmental risk assessment (4).The objective of this study was to assess toxicity and potential for anaerobic biodegradation of C60 fullerene, by examining its effect on the structure and function of the microbial community in digester sludge. This engineered system also serves as a model for natural anaerobic systems such as soils and sediments. Currently, no anaerobic toxicity mechanism of C60 has been described. The impact of C60 fullerene on microorganisms has just begun to be explored. Photoinduction of fullerenes has been explored for their use as antibiotics (5). Molecular simulations have shown that C60 is capable of binding to DNA and deforming strands, potentially interfering with DNA repair mechanisms (6). Recent literature shows evidence of toxicity of an aqueous suspension of C60 fullerene to pure cultures of Escherichia coli and Bacillus subtilis growing in minimal media under both aerobic and anaerobic conditions (7, 8). One possible mechanism of aqueous fullerene toxicity to eukaryotic cells has been shown to be lipid peroxidation (9). However, it remains unclear how much of this effect was due to facilitated exposure or experimental artifacts such as solvent effects. It is well-known that fullerenes interact strongly with cell membranes, but this property has no inherent relationship to toxicity (1). Experiments with human cells suggest that C60 is benign in nonaqueous forms, which may increase its usefulness as an antioxidant for biomedical applications (10). It is difficult to extrapolate any of these conclusions to complex microbial ecosystems since only preliminary data exist on the environmental fate and transport of C60. Tong et al. (11) showed that neither solid nor nC60 had any effect on soil microbial respiration, with a corresponding lack of impact on Bacterial 16s rRNA genes or phospholipid profiles. To the authors’ knowledge, this is the first study of the effect of CMNP on the structure and function of anaerobic microbial communities. The effects of several different treatments of C60 were studied in order to model a typical environmental release scenario. The hydrophobic nature of this nanomaterial suggests that the most likely form seen in high concentrations in wastewater treatment systems will be in complexes with various organic solvents and biomass. Fullerene C60 is highly soluble in toluene (12) and o-xylene (13), so these solvents were used to plate C60 on dried sludge biosolids, and this fullerenecoated sludge was then applied to anaerobic microcosms at concentrations up to 50000 mg/(kg of biomass) (dry weight (d/w)). This was the highest concentration obtainable without a significant volume displacement in the microcosms, taking into consideration the highest volume of solvent that could be completely volatilized leaving a crust of C60 on the dried sludge. As previously discussed, it is critical to differentiate between the effect of C60 and any solvents used in its preparation or delivery to living cells. For this experiment, ample time was allowed for the solvents to volatilize after the C60 was plated on the dried sludge. However, trace solvent was certain to remain, so appropriate controls were carefully designed, taking into account the known properties and interactions of C60 with each solvent. The effect of aqueous C60 was also explored, using a suspension of C60 in water at low concentration, as well as C60 dissolved in methanol and ethanol, which were provided along with glucose as substrates for methanogenic gas production. An anaerobic toxicity assay was used to assess community function in response to C60 by measuring methanogenesis for several weeks. Microbial community structure was analyzed using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis 10.1021/es072018g CCC: $40.75
2008 American Chemical Society
Published on Web 02/14/2008
TABLE 1. Fullerene Preparation and Treatment for Anaerobic Toxicity Assay sample treatment ID
C60 concn (mg/(kg of biomass) (d/w))
fullerene prepn
substratea
ref samples
C60 aqueous (estd, mg/L)
A B C D
0.321 8.6 30000 50000
dissolved in MeOH/EtOH aqueous C60 plated on dried sludge (toluene) plated on dried sludge (o-xylene)
+b +/+/+/-
H+ H+/G+ G- EF+/- G+/-
3.26 × 10-6 7.96 × 10-6 7.96 × 10-6 7.96 × 10-6
a Substrate (G/M/E) concentrations in microcosms were 0.6 mM glucose, 0.186 mM methanol, and 0.122 mM ethanol for each time samples were fed with substrate. “+/-“ indicates that sample sets were prepared with and without these substrates. b *No reference sample could be prepared without G/M/E substrate due to the nature of the fullerene treatment for this sample set.
(DGGE). All three domain-level subsets of the microbial community were targeted with primers specific for Bacteria, Archaea, and Eukarya. Representative groups of environmentally relevant microorganisms known to be present either in wastewater sludge or other anaerobic environments are shown in Figure S1 of the Supporting Information. To fully understand the effect of a new chemical in these environments, it is important to consider its impact on microbial groups which comprise a small but ecologically significant subset of the community. Primer sets were chosen in order to detect, for example, ciliate protozoa, with which methanogenic Archaea form endosymbiotic relationships (14). A delicate balance is maintained between methanogens and acetogenic bacteria in a functioning anaerobic digester (15); simultaneous comparison of Archaeal and Bacterial community profiles facilitates discovery of minor changes that could lead to a disruption of this relationship. We believe this is the first study using PCR-DGGE to study the effect of any anthropogenic chemical input on all three branches of the universal phylogenetic tree. Gas production data showed no toxicity due to any fullerene treatment. Nor was biodegradation of C60 indicated by an increase in gas formation. In general, community fingerprints were similar between treated and untreated samples. These data support the hypothesis that C60 release to wastewater treatment would not affect the structure or function of the anaerobic community. Long-term studies of microbial communities will be required to determine the overall environmental impact of fullerenes. The time frame for evolution of biodegradation of a new chemical in anaerobic systems may be particularly long (16), so it is too early to conclude that microbial ecosystems and biogeochemical cycles will be unaffected by C60. For example, partial transformations of PAHs (17) and PCBs (18) yield toxic metabolites, and a similar process could occur with C60. Many questions remain about its fate, transport, and resulting bioavailability. Long-term studies are needed to address the complexity of microbial interactions in environmental receptors such as soil, water, and wastewater treatment sludge.
Experimental Section Sample Collection and Anaerobic Toxicity Assay. Anaerobic digester sludge was collected from the Greater Lafayette Wastewater Treatment Plant. Using a procedure modified from Owen et al. (19), parallel sets of benchtop microcosms were established and maintained anaerobically. Live sludge biosolids were diluted 25% (v/v) in sodium phosphate buffer and aliquoted, 100 mL each, into glass serum bottles (Wheaton, 125 mL). Sample sets were prepared in quadruplicate. A set of untreated reference samples was prepared, as well as a set of reference samples each with 600 µL of glucose (1 M), 75 µL of ethanol, and 75 µL of methanol (henceforth noted as G/M/E) provided as substrates. Fullerenes were added to experimental samples as follows: bulk C60 (Materials and Electrochemical Research (MER) Corp., 99.9%) plated on dried sludge in o-xylene or toluene,
fullerenes dissolved in methanol and ethanol provided as substrate, and an aqueous suspension of C60 prepared according to the method of Cheng et al. (20). Parallel sets of microcosms without glucose, methanol, or ethanol were also treated with each form of C60 applied in the study. Reference samples were also prepared with o-xylene- or toluene-treated sludge without C60 and with untreated dried sludge. Serum bottles were stored in the dark at ambient temperature after being closed with Teflon-coated septa and aluminum crimp seals. One untreated replicate was sacrificed at time zero for community analysis. Gas formation was measured for 89 days (treatment D, Table 1) and 154 days (A, B, C, Table 1) by syringe sampling of microcosm headspace. After gas production reached a plateau, microcosms were fed again with the same concentration of glucose, methanol, and ethanol. Samples treated with C60 dissolved in methanol and ethanol received this preparation each time the microcosms were fed. Cumulative gas production of experimental samples was compared with that of reference samples and theoretical methane yield (by volume). Theoretical gas formation for each substrate was calculated using the Buswell equation: CaHbOc + (a - (b ⁄ 4) - (c ⁄ 2))H2O ⇒ ((a ⁄ 2) - (b ⁄ 8) + (c ⁄ 4))CO2 + ((a ⁄ 2) + (b ⁄ 8) - (c ⁄ 4))CH4 where the subscripts a, b, and c are derived from the molecular formula of the substrate (21). One replicate for each set was sacrificed every two to four weeks for community analysis by PCR-DGGE. Fullerene Preparation and Sample Treatment. Fullerene C60 (99.9%) was purchased from MER Corp. The fullerenes were purified by chromatography and sublimation; traces of C70 and C60 oxide remained as impurities. An 8200 mg/L stock of fullerenes in o-xylene was prepared and plated on airdried sludge. The dried sludge pellets were initially immersed in the fullerene solution. The solvent was allowed to evaporate overnight. During the volatilization step, when a small volume of solvent remained, the mixture was stirred frequently to ensure coating of the C60 over a large surface area of the sludge. One gram of dried sludge plated with 50 mg C60 was added to each microcosm for a final concentration of 50000 mg of C60/(kg of biomass) (d/w) (Table 1). Reference samples with xylene-treated sludge were treated with the same initial volume of xylene as the samples treated with fullerenes (Table 2). Xylene forms solvates with C60, on a 1:2 molar basis (C60: solvent) at 298 K (13), so the same volume of o-xylene was added back to the dried sludge reference sample without C60 as would be expected to remain on the sludge in solvates with C60. Reference samples were also prepared with untreated, dried sludge to account for gas formation from metabolism of the dried biomass. A stock solution of C60 in toluene was also prepared (∼2300 mg/L). Since toluene does not form solvates with C60 at 298 K (13), reference samples were treated with toluene which was allowed to volatilize before setting up the experiment, but no toluene was added VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Reference Samples without Fullerene for Anaerobic Toxicity Assay sample treatment ID E F G Hb
treatment live live live live
sludge + dried sludge/toluene sludge + dried sludge/o-xylene sludge + dried sludge sludge
substratea +/+/+/-
a Substrate (G/M/E) concentrations in microcosms were 0.6 mM glucose, 0.186 mM methanol, and 0.122 mM ethanol for each time samples were fed with substrate. “+/ -“ indicates that sample sets were prepared with and without these substrates. b This reference for treatments A, B, and C is labeled H1 in the figures; H2 is a reference for treatment D.
back to the microcosms. The concentration of C60 in these microcosms was about 30000 mg/(kg of biomass) (d/w). Fullerene C60 dissolved in methanol and ethanol at solubility (0.027 and 1.4 mg/L, respectively (13), was added to a set of microcosms each time reference samples were fed with pure methanol and ethanol as substrates. The concentration of fullerenes added to these samples was 0.107 mg/(kg of biomass) (d/w) per feeding, for a total concentration of 0.321 mg/(kg of biomass) (d/w) after three treatments. A set of microcosms with and without substrate were treated with an aqueous suspension of C60. Concentration of this C60 suspension was determined by spectrophotometry at 330 nm, using a method modified from Fortner et al. (7). The final concentration of aqueous C60 in the microcosms was 8.6 mg/(kg of biomass) (d/w). Equilbrium water-phase concentrations were estimated for C60 on the basis of an aqueous solubility of 7.96 × 10-6 mg/L (22), a log Kow ) 6.67 (22) and a linear sorption isotherm (23). Where estimated values exceed the solubility limit, it was assumed that the water-phase concentration of C60 would reach solubility at equilibrium (Table 1). DNA Extraction and PCR. Genomic DNA was extracted from fresh samples using the Bio101 FastDNA SPIN Kit for soil. A total of 6 mL (three replicates for each sample, 2 mL each) were centrifuged at 14000g. The resulting supernatants were discarded, and DNA was extracted from pelleted biosolids. Before PCR amplification, the DNA recovered from the three replicates extracted for each sample were combined and concentrated by ethanol precipitation. Precipitated DNA was resuspended in Dnase/Rnase-free water (Invitrogen). Fragments (∼200 bp) of the V3 region of the small subunit rRNA genes were amplified using primer sets for Bacteria and Archaea. Bacterial primers were PRBA338f (5′ AC TCC TAC GGG AGG CAG CAG 3′) (24) and PRUN518r (5′ ATT ACC GCG GCT GCT GG 3′) (25). A GC-clamp was added to the forward primer. (5′ CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G 3′) (25). Program parameters were as follows: initial denaturation, 94 °C for 5 min; denaturation, 92 °C for 30 s; annealing, 55 °C for 30 s; elongation, 72 °C, 30 s. Steps 2-5 of this program were repeated for a total of 25 cycles, followed by a final elongation of 72 °C for 15 min. Amplification of the Archaeal fragment was achieved using a nested approach, as previously described (26). A 1072 base-pair fragment was amplified using primers PRA46f and PREA1100r. The inner primer set PARCH340f (with GC clamp) and PARCH519r were used to amplify a small (
