A sensitive crude oil bioassay indicates that oil spills ...

Environmental Pollution 164 (2012) 42e45

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A sensitive crude oil bioassay indicates that oil spills potentially induce a change of major nitrifying prokaryotes from the Archaea to the Bacteria Hidetoshi Urakawa a, *, Juan C. Garcia a, Patricia D. Barreto c, Gabriela A. Molina b, c, Jose C. Barreto b, c a

Department of Marine and Ecological Sciences, Florida Gulf Coast University, 10501 FGCU Boulevard S. Fort Myers, FL 33965-6565, USA Department of Chemistry and Mathematics, Florida Gulf Coast University, FL, USA c Green Technology Research Group, Florida Gulf Coast University, FL, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 November 2011 Received in revised form 13 January 2012 Accepted 14 January 2012

The sensitivity of nitrifiers to crude oil released by the BP Deepwater Horizon oil spill in Gulf of Mexico was examined using characterized ammonia-oxidizing bacteria and archaea to develop a bioassay and to gain further insight into the ecological response of these two groups of microorganisms to marine oil spills. Inhibition of nitrite production was observed among all the tested ammonia-oxidizing organisms at 100 ppb crude oil. Nitrosopumilus maritimus, a cultured representative of the abundant Marine Group I Archaea, showed 20% inhibition at 1 ppb, a much greater degree of sensitivity to petroleum than the tested ammonia-oxidizing and heterotrophic bacteria. The differing susceptibility may have ecological significance since a shift to bacterial dominance in response to an oil spill could potentially persist and alter trophic interactions influenced by availability of different nitrogen species. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Oil spill Nitrification Ammonia-oxidizing bacteria Ammonia-oxidizing archaea Nitrosopumilus maritimus

1. Introduction Major oil spills present some of the most vivid images of ecological damage resulting from human commerce, with released oil coating beaches and marine animals, and entire foods webs impacted by toxic petroleum components. The toxicity of hydrocarbon is different for each marine organism and depends on the body size and mechanisms of toxicity (Echeveste et al., 2010). To investigate the toxicity of crude oil and susceptibility of marine organisms for oil spills, laboratory scale bioassays are an essential tool with a long and successful history of use (Reid and MacFarlane, 2003; Fuller et al., 2004; Mercurio et al., 2004). In contrast to the macrobiota, marine microorganisms are generally viewed not so much as impacted species but as providing service to biodegradation, since many marine bacteria have been shown to actively degrade different petroleum components (Head et al., 2006). Therefore, the major focus on microbial responses to oil contamination has been primarily focused on the characterization of dominant hydrocarbon degrading bacteria (Aldrett et al., 1997; Head et al., 2006). However, since microorganisms serve multiple biogeochemical functions, some functionally significant groups may also be particularly vulnerable to oil spills. Identification of negatively impacted microbial populations,

* Corresponding author. E-mail address: [email protected] (H. Urakawa). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2012.01.009

in addition to providing insight into the biogeochemical significance of oil spills, may also provide good targets for developing new bioassays (Alnafisi et al., 2007; Röling et al., 2004). Although knowledge is limited, we hypothesized that groups of chemolitotrophic microorganisms might be very sensitive to petroleum because these microorganisms cannot directly and indirectly consume crude oil as a carbon source. Specifically, it has been reported that nitrification is sensitive to environmental stress and contaminants (Brandt et al., 2001; Juliette et al., 1993; Stephen et al., 1999; Suwa et al., 1994; Urakawa et al., 2008). To our knowledge very little is known about the sensitivity of nitrifying bacteria, or the recently discovered nitrifying archaea to petroleum contamination. In the present study, the toxicity of crude oil was tested for ammonia-oxidizing bacteria (AOB) and archaea (AOA) to gain insight into ecological significance of the variable response of these two groups to oil contamination. 2. Methods 2.1. Panama City Beach tarball sample The BP Deepwater Horizon oilspill became the largest marine oil spill in history after releasing over 780,000 m3 of oil into the Gulf of Mexico from April 20 to July 15, 2010, a period of 87 days. Panama City Beach, Florida experienced some damage from drifting oil and small numbers of tarballs drifted ashore and were collected on June 25, 2010. A tarball sample was shipped to the Florida Gulf Coast University on ice for bioassay testing.

H. Urakawa et al. / Environmental Pollution 164 (2012) 42e45

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2.2. Oil sample preparation

3. Results and discussion

A Panama City Beach tarball sample was placed in a 20 ml scintillation vial with 10 ml of hexane (final concentration of oil, 10,000 ppm). The scintillation vial was vortexed until the tarball disintegrated. This solution was placed in a microfuge tube and spun for 2 min at 12,100g. Supernatant was then placed in a clean scintillation vial. The vial was rolled to coat the bottom and sides with a thin film and left open in the hood to evaporate the hexane. Ten milliliters of artificial seawater (Instant Ocean, Marineland Labs) were added to the vial and vortexed several times. Hydrogen peroxide (final conc. 0.3%) was added to the scintillation vial to sterilize the solution. A control consisting of artificial seawater only with hydrogen peroxide was also prepared; no microbial contamination was confirmed by adding 100 ml of solution into 2 ml of Marine Broth 2216 (BD).

Many investigators use a diminution in V. fischeri luminescence to determine toxicity in environmental samples; indeed commercial instruments are available for this purpose (Fuller et al., 2004; van Gestel et al., 2001). Most of the assays are acute, meaning that they utilize short exposure times for the toxin; they therefore measure immediate changes in metabolic activity instead of measuring growth, or inhibition of growth (van Gestel et al., 2001). We assumed that petroleum toxicity from a tarball might be detectable with the Microtox assay and used both a commercial Microtox bioluminescence assay and an automated assay we developed in our laboratory. Unexpectedly, no toxicity was detected using either commercial assay (data not shown) and in house assays in which the sample was challenged in the range of 62.5e500 ppm oil (p < 0.05) (Fig. 1). It seems that the V. fischeri tolerates tarball toxins well, and/or, the short acute exposure time (5 min) is insufficient to reveal toxicity when using V. fischeri. We also examined the sensitivity of N. oceani and N. maritimus to petroleum toxicity. The nitrite production of both was completely inhibited in the range of concentration 0.1e10 ppm (data not shown). Thus, the sample was diluted to ppb levels and re-tested (Fig. 2). The low concentration series experiment showed no toxicity in the range of concentration 1e10 ppb but a longer lag phase was observed when the 100 ppb of dose was used to challenge N. oceani (Fig. 2A). For comparison between AOB and AOA, N. maritimus was tested using the same method. These studies showed that N. maritimus is much more sensitive than N. oceani to petroleum (Fig. 2B). Ammonia oxidation by N. maritimus was significantly inhibited at 100 ppb (p < 0.05), showed an inhibitory response at longer incubation times at 10 ppb and a measurable response at concentrations as low as 1 ppb. In general, rapid bioassays are preferable. However, since the growth of ammonia-oxidizing organisms is slower than most heterotrophic bacteria, the sensitivity experiments took more than a week (Fig. 2). As an alternative, we developed an endpoint toxicity assay (Fig. 3). Inhibition of nitrite production was observed among all tested microorganisms at 100 ppb. Especially, N. maritimus showed very high sensitivity to crude oil, with 20% of inhibition at 1 ppb. The sensitivity of N. maritimus was significantly higher than the other AOB (p < 0.01). Although the same trend was observed, the sensitivity of nitrifying bacteria to crude oil varied with species. The

2.3. Bioluminescence assay The Microtox bioassay, which monitors bioluminescence of Vibrio (Allivibrio) fischeri, was used to test for the toxicity of tarball samples. Ten micrograms of lyophilized cultures (Microtox; Aquatox Research) were added to 50 ml of Photobacterium broth (Carolina Biological Supply) and incubated overnight at 26 C (z16 h). The culture was diluted z1:4 with Photobacterium broth (initial read of 10e15 million relative luminescence units [RLU]). One milliliter of diluted culture was placed in each well of a 24-well microplate and the relative luminescence was read before treatment. Control and treatment samples were prepared by adding 20 ml of artificial seawater or oil samples (62.5, 125, 250 and 500 ppm), respectively. Microplates were incubated for 5 min (3 min at room temperature, agitated for 10 s followed by 2 min incubation) then the luminescence was determined by using a TECAN Genios Pro 96 Multifunction Microplate Reader (MTX Lab Systems). 2.4. Toxic effect of an oil sample on the nitrite production of ammonia-oxidizing microorganisms To assess the effect of tarball toxicity, Nitrosococcus oceani and Nitrosopumilus maritimus were grown and used for oil sensitivity experiments. Cells were grown in 10 ml volume of SCM medium containing 1 mM of NH4Cl (Martens-Habbena et al., 2009) in 100 ml medium bottles. The concentration of the challenged sample was adjusted to ppm (0.1, 1, 10 mg l1) and ppb (1, 10, 100 mg l1) doses. The cultures were incubated at 25 C in the dark without agitation. The growth of the cultures was compared between control and challenged samples using nitrite levels to monitor ammonia oxidation. At each time point, subsamples were transferred to cuvettes and used for nitrite assays as described previously (Stickland and Parsons, 1972). 2.5. Endpoint toxicity assays for ammonia-oxidizing microorganisms According to the results of growth curve experiments, 72 h were selected as an end point of toxicity assay based on the nitrite production. The endpoint toxicity assay was conducted for four marine ammonia-oxidizing microorganisms (Table 1). Percent inhibition was calculated based on the net nitrite concentration (subtraction from the end time point [72 h] to the initial time point) between control and challenged samples and normalized to a 100% scale; 100% was the maximum response and 0% was set equal to the no challenge control.

1.2

Four strains, including two Gram-negative bacteria (Serratia marcescens ATCC13880 and Klebsiella pneumoniae WS1680) and two Gram-positive bacteria (Bacillus megaterium WS1625 and Staphylococcus aureus ATCC6538), were used for endpoint toxicity assays. Cells were grown for 24 h with shaking at 25 C in 2 ml volume of nutrient broth medium (BD) in 15 ml plastic tubes. The concentrations of challenged samples were adjusted to be 0.1e10 ppm and compared with no challenge control samples. The cell growth was monitored spectrophotometrically at 600 nm. 2.7. Statistical analysis

Ratio of remaining RLU

2.6. Endpoint toxicity assays for heterotrophic bacteria 1.0

0.8

0.6

The statistical analysis of the experimental data was carried out with SigmaPlot, version 10.1 (Systat Software). A one-way analysis of variance (ANOVA) and a Tukey posthoc test were used for the assessment of toxicity experiments. 0.4 0

Table 1 Ammonia-oxidizing microorganisms used in this study.

62.5

125

250

500

Oil concentration (ppm)

Ammonia-oxidizing microorganism

Strain

Phylogenetic group

Nitrosopumilus maritimus Nitrosococcus oceani Nitrosomonas marina Nitrosomonas cryotolerans

SCM1 ATCC19707 C-113a ATCC49181

Thaumarchaeota Gammaproteobacteria Betaproteobacteria Betaproteobacteria

Fig. 1. The ratio of remaining relative luminescence (RLU) for Vibrio fischeri upon oil treatment. Data are the ratio of before and after 5 min treatments. Controls (0 ppm) should result in a ratio of 1.0 (dashed line). Gray dotted lines indicate 95 percent confidence interval. Toxicity in this assay is indicated by the decrease of the ratio of remaining RLU values. Data are mean SD (n ¼ 6).

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1200

Nitrite (µM)

1000

1200

A

0 ppb 1 ppb 10 ppb 100 ppb

1000

800

800

600

600

400

400

200

200

0

0

2

4

6

8

10

12

14

16

0

B

0 ppb 1 ppb 10 ppb 100 ppb

0

5

10

15

20

25

30

Day

Day

Fig. 2. Toxic effects of oil on the nitrite production of Nitrosococcus oceani (A) and Nitrosopumilus maritimus (B). Data are means SEs for triplicates.

betaproteobacterial ammonia oxidizers, especially N. cryotolerans, were more sensitive to petroleum toxicity than the gammaproteobacterial ammonia oxidizer. It has been reported that the toxicity of hydrocarbon was size dependent for phytoplankton species (Echeveste et al., 2010). Ammonia-oxidizing archaea are among the smallest free living microorganisms (Könneke et al., 2005). Conversely, N. oceani and other Nitrosococcus species have the largest cell size among ammonia-oxidizing bacteria (Campbell et al., 2011). Thus, the variation in petroleum toxicity in ammonia-oxidizing microorganisms might be explained in part by the different surface/volume ratios determined by cell size. Although rapid bioassays are often preferable, evaluation of only an acute response will generally limit sensitivity to the assay. Chronic testing, with growth occurring while the toxin is present, is more likely to yield a higher detection limit and was therefore used to examine the petroleum toxicity of selected heterotrophic bacteria (Fig. 4). The growth response of two heterotrophs, S. marcescens and S. aureus, was confirmed with challenges of 0.1e10 ppm oil. The growth of K. pneumoniae and B. megaterium was strongly inhibited by the challenge of 10 ppm dose but both cultures grew at 1 ppm.

Overall, no obvious petroleum toxicity was detected at 0.1 to 1 ppm doses, suggesting that nitrifiers are more sensitive to petroleum toxicity than heterotrophic bacteria. Therefore, our data indicate that ammonia-oxidizing prokaryotes could be used as an excellent biomonitoring tool for detecting oil and possibly other marine contaminants. Especially, N. maritimus showed very high sensitivity for crude oil and may be the best microorganism now available in culture for developing a petroleum spill bioassay. Literature evidence suggests that oil contamination in the marine environment can induce a shift of microbial communities (Head et al., 2006). A few researchers have compared population changes between archaea and bacteria; their work implied that the archaea would be more sensitive than the bacteria for hydrocarbons (Röling et al., 2004). Until 2005, the function of mesophilic Marine Group I Archaea, which are one of the most common bacterioplankton members in the ocean, was not well understood (Könneke et al., 2005). Although the major physiology of this abundant marine group of non-extreme Archaea is now generally thought to be

Percent inhibition

N. oceani

20 N. marina

40 60

N. maritimus N. cryotolerans

80

Absorbance (600 nm)

1 0

0.1

0.01

Staphylococcus aureus Klebsiella pneumoniae Serratia marcescens Bacillus megaterium

0.001

100 1

10

100

Oil concentration (ppb) Fig. 3. Endpoint toxicity assays using ammonia-oxidizing microorganisms after 72 h. Data are means SEs for 9 replicates (three measurements and three cross-combinations with control samples). Some error bars are smaller than symbol sizes.

0

0.1

1

10

Oil concentration (ppm) Fig. 4. Endpoint toxicity assays using heterotrophic bacteria after 24 h. Bacterial growth was monitored as optical density at 600 nm wavelength. Data are means SDs for triplicate measurements. Most of error bars are smaller than symbol sizes.


ammonia oxidation, no attempt has been done to test this observation directly. In the present study it was demonstrated that Marine Group I archaeon N. maritimus has a much greater sensitivity to oil than the any other bacteria tested. Thus in addition to providing a basis for the development of a novel toxicity bioassay, our demonstration of differing susceptibility of AOA and AOB to petroleum contamination may have ecological significance, since a shift to bacterial dominance in response to an oil spill could potentially persist and alter trophic interactions. These results now provide a foundation to further evaluate key ecosystemlevel questions concerning short and long term response of marine systems to petroleum contamination. Acknowledgements The authors thank David Stahl at University of Washington for providing N. maritimus SCM1 and critical reading of the manuscript. We are grateful to Martin Klotz at University of North Carolina at Charlotte for providing a culture of N. marina C-113a. We would thank Paula Scott of the Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute and Darren Rumbold at Florida Gulf Coast University for providing a Pensacola oil sample. Helpful discussions with Willm Martens-Habbena at University of Washington are acknowledged. We thank Jaffar Ali at Florida Gulf Coast University for helpful suggestion regarding to statistical analysis. Bioluminescence assays developed with funding from the U.S. Department of Defense to J.E.B. contributed to this work. This research was supported by the Florida Gulf Coast University Office of Research and Sponsored Programs Internal Grant Program for H.U. References Aldrett, S., Bonner, J.S., Mills, M.A., Autenrieth, R.L., Stephens, F.L., 1997. Microbial degradation of crude oil in marine environments tested in a flask experiment. Water Research 31, 2840e2848. Alnafisi, A., Hughes, J., Wang, G., Miller 3rd, C.A., 2007. Evaluating polycyclic aromatic hydrocarbons using a yeast bioassay. Environmental Toxicology and Chemistry 26, 1333e1339. Brandt, K.K., Hesselsoe, M., Roslev, P., Henriksen, K., Sorensen, J., 2001. Toxic effects of linear alkylbenzenesulfonate on metabolic activity, growth rate, and microcolony

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