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Exploring the potential of applying proteomics for tracking bisphenol A and nonylphenol degradation in activated sludge
Chemosphere 90 (2013) 2309–2314
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Technical Note
Exploring the potential of applying proteomics for tracking bisphenol A and nonylphenol degradation in activated sludge Neus Collado a,b, Gianluigi Buttiglieri b,⇑, Boris A. Kolvenbach c, Joaquim Comas a, Philippe F.-X. Corvini c, Ignasi Rodríguez-Roda a,b a b c
LEQUIA, Institute of the Environment, University of Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain ICRA, Catalan Institute for Water Research, Carrer Emili Grahit, 101, Parc Científic i Tecnològic de la Universitat de Girona, 17003 Girona, Spain Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland
h i g h l i g h t s " Proteomics applied to a pure culture of Sphingomonas TTNP3 and its proteome analysed. " The pure strain hydroquinone dioxygenase enzyme used as a proteomic marker. " Dilutions of the pure strain with activated sludge were done to validate the method. " Diluted samples protein patterns were compared with the pure strain proteome. " The protocol detection limit is noteworthy for Sphingomonas TTNP3 in activated sludge.
a r t i c l e
i n f o
Article history: Received 5 August 2012 Received in revised form 4 October 2012 Accepted 5 October 2012 Available online 31 October 2012 Keywords: Enzymes Pure culture Phenolic substances Micropollutants Proteomics Activated sludge
a b s t r a c t A significant percentage of bisphenol A and nonylphenol removal in municipal wastewater treatment plants relies on biodegradation. Nonetheless, incomplete information is available concerning their degradation pathways performed by microbial communities in activated sludge systems. Hydroquinone dioxygenase (HQDO) is a specific degradation marker enzyme, involved in bisphenol A and nonylphenol biodegradation, and it can be produced by axenic cultures of the bacterium Sphingomonas sp. strain TTNP3. Proteomics, a technique based on the analysis of microbial community proteins, was applied to this strain. The bacterium proteome map was obtained and a HQDO subunit was successfully identified. Additionally, the reliability of the applied proteomics protocol was evaluated in activated sludge samples. Proteins belonging to Sphingomonas were searched at decreasing biomass ratios, i.e. serially diluting the bacterium in activated sludge. The protein patterns were compared and Sphingomonas proteins were discriminated against the ones from sludge itself on 2D-gels. The detection limit of the applied protocol was defined as 10 3 g TTNP3 g 1 total suspended solids (TSSs). The results proved that proteomics can be a promising methodology to assess the presence of specific enzymes in activated sludge samples, however improvements of its sensitivity are still needed. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Currently, there is an increasing awareness of the presence of micropollutants (e.g. surfactants, plasticizers, pharmaceuticals, pesticides and so on) in domestic wastewater (Carballa et al., 2004; Barceló and Petrovic, 2007; Buttiglieri and Knepper, 2008). Nonylphenol (NP) and bisphenol A (BPA) are phenolic substances used in high amounts by the industry (Greiner et al., 2007; Soares et al., 2008). Both can act as hormone disrupters and affect the reproductive system in animals causing, for example, feminization ⇑ Corresponding author. E-mail address: [email protected] (G. Buttiglieri). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.10.002
in various aquatic organisms or inhibition of algal populations (Wang et al., 2011; Li et al., 2012; Gyllenhammar et al., 2012). Some studies indicate that negative health effects occur even at very low doses (vom Saal and Hughes, 2005). Effluents from wastewater treatment plants (WWTPs) are a major source of environmental BPA and NP (Vethaak et al., 2005; Crain et al., 2007). Influent WWTP concentrations are generally at low lg L 1 level but concentrations up to 59 lg L 1 are reported for BPA (Fukazawa et al., 2002) and even higher for NP (Klecka et al., 2010). The percentage of BPA removal in WWTPs varies considerably per location from 0% to 96% (Vethaak et al., 2005). WWTP removals of NP are also highly variable (9–94%) depending on the region and type of unit treatment processes employed
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(Soares et al., 2008). NP, in addition, can be produced during the wastewater treatment processes (e.g. the surfactants NPethoxylates can easily degrade back to NP, Soares et al., 2008) and it is often found at higher concentrations in the effluents than in the influents (Farre et al., 2002; Soares et al., 2008). These results are of concern since they indicate that WWTPs are only partially efficient in removing such compounds being released into the aquatic environment. Low micropollutant concentrations, as it is the case of NP and BPA, are not likely to sustain growth of microorganisms, and conventional microbiological techniques based on cultivation of microorganisms will remain ineffective in monitoring biodegradation mechanisms. More information may be achieved using molecular microbiological techniques, e.g. qPCR and FISH. Proteomics is a relatively new approach in environmental microbiology based on the identification and expression levels of proteins. Its central assumption is that fingerprints of expressed proteins are specific to the conditions experienced by the organisms. The goal of the technique is to observe differences and changes in protein expression levels and, hence, to identify key proteins directly involved in the different biological processes (Lopez, 1999). Since the introduction of the metaproteomic approach (meaning proteomics applied to mixed cultures; Wilmes and Bond, 2004) only a few studies have been carried out on marine ecosystems (Banfield et al., 2005; Kan et al., 2005), groundwater and soil (Benndorf et al., 2007). The high potential of metaproteomics has been also successfully demonstrated on activated sludge and specifically on phosphorous removal in conventional WWTPs (Wilmes et al., 2008) but never for tracking proteins of a strain able to degrade micropollutants in activated sludge mixtures. In order to produce a proof-of-concept on the applicability of proteomics in wastewater samples an axenic culture as well as its mixture with activated sludge was used in the present work. A specific strain, Sphingomonas sp. strain TTNP3, responsible for the degradation of the micropollutants BPA and NP was chosen. Experiments were first performed with it and more precisely focusing on the hydroquinone dioxygenase (HQDO). This enzyme has been well characterised (Kolvenbach et al., 2011) but has not been used as a marker for proteomics application before. The subsequent objective was to check if proteins, in our case, belonging to Sphingomonas proteome were still detectable in the overall mixture with activated sludge, and until which dilution, in order to define the detection limit of the applied protocol and to follow the protein match set within the diluted gels. 2. Materials and methods 2.1. Pure strain, purified HQDO Cultures of Sphingomonas (deposited at BCCM under accession number LMG 21268) were cultivated and harvested as previously described including 16 h of incubation with 0.5 mM technical grade NP to induce HQDO expression (Kolvenbach et al., 2011, 2012). HQDO catalyses the ring opening of the latter to produce 4-hydroxymuconic semialdehyde (Fig. 1; Kolvenbach et al., 2007; Porter et al., 2012). Purified HQDO was obtained as described previously by ammonium sulphate precipitation and subsequent hydrophobic interaction and ion exchange chromatography (Kolvenbach et al., 2011). 2.2. Dilution of Sphingomonas sp. strain TTNP3 in activated sludge A set of activated sludge samples were spiked with different amounts of Sphingomonas biomass. Experimental replicates were considered of minor importance in this case, since the objective
was to find out the detection limit of the protocol and to follow not target identified proteins but the general protein patterns. Moreover to be noted that activated sludge itself acts as a pool with already an intrinsic variability of microorganisms and enzymes. The activated sludge was sampled at ARA-BIRS WWTP (Basel, Switzerland), from the aeration tank of the biological treatment with a total suspended solids (TSSs) concentration of 3 g L 1 (100% domestic wastewater). Sphingomonas stock solution had a concentration of dry weight of 3 g TTNP3 L 1. Three logarithmic serial dilutions in the above described activated sludge were prepared from 1:10 (v/v) to 1:103. Thus, the first diluted sample corresponded to a concentration of 3 10 1 g TTNP3 L 1 and the last one of 3 10 3 g TTNP3 L 1. Finally, the cells from these activated sludge samples were harvested by centrifugation at 5000 rpm for 20 min. The supernatant was discarded, and the collected pellet was frozen at 80 °C till analysis. 2.3. Protein extraction, purification and resuspension of the samples All analysed pellets were frozen in liquid nitrogen and lyophilised. The pulverised samples were resuspended by pulsevortexing in 5 mL of lysis buffer consisting of 40 mM Tris-HCl pH 8, 2 mM EDTA, 25 mM NaCl, 25 mM MgCl2, 0.1% SDS (Sodium Dodecyl Sulphate), 2 mM Pefabloc SC, as a protease inhibitor, and 2 mM Pefabloc SC protector reagent (Roche). Cells were then placed on ice and lysed by sonication (four cycles of three min each at pulse 40 and amplitude 21%; Sonics Vibra-Cell). The suspension was then centrifuged at 13,000 rpm (approximately 16,000g) for 30 min to remove cell debris, and the supernatants were stored at 80 °C. Protein concentrations were determined using a BioRad RC DC kit with bovine serum albumin as a standard following the manufacturer’s protocol. For each procedure, protein standards were prepared in the same buffer as the protein samples. Finally, proteins were precipitated in 10% (w/v) trichloroacetic acid, incubated on ice for 30 min and centrifuged at 13,000 rpm for 15 min. The protein pellet was then washed in 80% (v/v) ice-cold acetone overnight at 20 °C. The dried protein pellet was resuspended in 500 lL of 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM DTT (DL-Dithiothreitol) and a trace of bromophenol. 2.4. 2D-page (polyacrylamid gel electrophoresis) The protein fingerprints are obtained by protein separation in two dimensions, i.e. first by isoelectric point (pI) and subsequently by molecular weight (MW), in 2D-gel electrophoresis (Seung et al., 2003). 30 lg of the purified protein sample, 400 lg in the case of the TTNP3 lysate and 250 lg for activated sludge samples were dissolved in 480 lL of rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT and a trace of bromophenol), 1% of IPG Pharmalites 3-10, and were used to impregnate 24 cm pH 4– 7 IPG strips (Immobiline DryStrips, Amersham Biosciences–GE Healthcare) in an Immobiline DryStrip Reswelling Tray (Amersham Biosciences–GE Healthcare) for 16 h. For first-dimension separation, the strips were placed in an IPGphor ceramic manifold, covered with Plusone DryStrip cover fluid and focused for 95,000 V h in an Ettan IPGphor3 isoelectric focusing system (Amersham Biosciences–GE Healthcare). Gels strips were subsequently equilibrated for 20 min in equilibration buffer A (6 M urea, 75 mM Tris-HCl pH 8.8, Glycerol 29.3%, SDS 2%, a trace of bromophenol, milli-Q water, 10 mg mL 1 DTT), followed by 20 min in equilibration buffer B (as buffer A but containing 25 mg iodoacetamide per mL instead of DTT). Second dimension separation was carried out with 12% v/v acrylamide, SDS-PAGE gels, at 100 W in an Ettan DALTsixelectrophoresis system (Amersham Biosciences-GE Healthcare). The gels were stained overnight with Colloidal Coomassie
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OH
R
R
2
R
1
R
4
R
1
R
R
2
+H2 O/ -H
1
HO
(BPA) 2
R
R
+
-H
+H
OH
2
R
OH
O
1
2
2
6
5 +
1
1
R
+
2
C R
3
HqdA HqdB
COOH
COOH
+
COOH COOH
COOH
CHO OH
OH
O
O
7
8
9
10
beta-ketoadipate pathway
Fig. 1. Main degradation pathway for BPA and NP in Sphingomonas sp. strain TTNP3: 1 – BPA (R1 = methyl; R2 = p-phenol) or NP (R1 = ethyl; R2 = 2-pentyl, for the 4-(1-ethyl1,2-dimethylpentyl)phenol) isomer; 2 – quinol intermediate; 3 – carbocationic intermediate; 4 – 4-(2-hydroxypropan-2-yl)phenol with BPA as substrate and 3,5-dimethyl-3heptanol with NP as substrate, respectively); 5 – 4-isopropenylphenol; 6 – 4-isopropylphenol; 7 – hydroquinone; 8 – 4-hydroxymuconic acid semialdehyde; 9 – maleylacetic acid; 10 – 3-oxoadipic acid; rectangles mark the enzymes attributed to the metabolism step (adapted from Corvini et al., 2006; Kolvenbach et al., 2012).
Fig. 2. On the left side, 2D gel from the purified HQDO enzyme with both subunits (HdqA and HdqB). On the right side, the proteome of the Sphingomonas sp. strain TTNP3 with all the spots in blue, the candidate target enzymes (for HqdA, HqdB) in orange and the identified HdqB highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Proteins identification on excised spots candidates to be HQDO subunits. Spot ID
GI
Score
Name
pI
MW
Organism
1 2 3 4 5 6
82702350 254776561 148553363 148554247 148555246 335346123
70 64 90 98 112 88
Glyoxalasa family protein Transcriptional regulator, GntR family protein Transcription elongation factor GreA DNA-directed RNA ploymerase subunit alpha Hydrogenobyrinic acid a,c-diamide cobaltochelatase Hydroquinone subunit B
9.69 7.08 4.73 4.89 5.02 4.94
6719 25,014 17,403 38,091 37,465 36,933
Nitrosospira multiformis ATCC 25196 Mycobacterium avium subsp. Avium ATCC 25291 Sphingomonas wittichii RW1 Sphingomonas wittichii RW1 Sphingomonas wittichii RW1 Sphingomonas TTNP3
Blue (Bio-Rad) and scanned using a (Amersham Biosciences-GE Healthcare).
Labscan
instrument
subunits (HqdA and HqdB). The highly expressed ones in these areas were chosen for further quantification and identification after excision.
2.5. Image analysis of 2D-PAGE gels The acquired gel images were processed and analysed using Image Master Platinum v.7 (Amersham Biosciences-GE Healthcare). The gels were placed within the same match set. Automated and manual spot detection and matching was performed, as well as spot densities determined. As a secondary analysis, and for the gels corresponding to the pure strain dilutions in activated sludge, a match set was performed among the different gel images based on their pI and the MW. 2.6. Spot excision Protein spots, from the Sphingomonas lysate 2D gel, were ranked according to their proximity to the HQDO subunits (HqdA and HqdB) taking into account their theoretical pI (4.53 and 4.94 respectively) and MW (19 and 38 kDa respectively) of the HQDO
2.7. Matrix assisted laser desorption ionisation time-of-flight (MALDIToF) mass spectrometry (MS) and protein identification The excised spots were processed by in gel tryptic digestion, prior to mass spectrometric analyses. Each excised spot was detained by acetonitrile (ACN) treatment then dried using a Speedvac centrifuge. Approximately, 200 ng of trypsin in 25 mM ammonium bicarbonate was added in a volume enough to rehydrate the gel pieces. Digestion was performed overnight at 37 °C. The tryptic peptides were eluted from the gel with 50% ACN/0.1% trifuoracetic acid. 1 lL of eluant was mixed with an equal volume of saturated solution of a-cyano 4-hydroxycinnamic acid in 50% ACN and 0.1% trifluoracetic acid and spotted on a MALDI plate by dried droplet method. The samples were analysed on a Voyager DE-PRO, MALDI-TOF analyser (Applied Biosystems). A 50 Hz nitrogen laser was used to desorb/ionise the matrix/analyte material, the acceleration voltage was 20 kV and the ions were detected in positive ion
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Table 2 Matched peptides in bold and underlined for the identification of HqdB. 1
MAMSEALEII
DFGDSKARTD
51
LTWPGGSHII
PIDAFLRAMM
AGRFNDAYRN LPWGIKNGNN
AGRDHEERFK DEAISRQRVT
QEQPVVEAEP EFILPVEHGN ADIRHQGYST
101 151 201 251 301
TEHLAINNET RDVAWGFFYG
GYRSFRAGGF VVNFDHVFGT
TFTRDEYFAR INHYGEVTMF
SSALMAVFKD
ILSDWTVEGY
DPFAAPMETG
GFEAEVSAYN DRCEWFLQLS
ARRMVGLPGD LFGYLSRSDV DEIVWDVKDK
TPVRTDANGF TWNPSVCSVV ESGKPRARVT
PVNRQFADVP GDSLFCPTSE ARAGDICCMP
KRSMLLVWEN
GSPKIPQMIA
DGTAPVVPVT
F
Table 3 Spots number, percent match and matched spots number between Sphingomonas sp. strain TTNP3 2D gel and the ones serially diluting the pure strain in activated sludge. Gel
Spots number
Matched spots (%)
Matched spot number
Pure strain Dilution 1.10 Dilution 1:100 Dilution 1:1000
709 588 673 357
n.a. 11 4 2
n.a. 78 30 8
reflectron mode. The samples were externally calibrated against a standard mixture of peptides from Bruker, ranging in mass from 1 to 3.2 kDa. Resulting spectra were analysed directly using Data Explorer version 4.0.0.0. MS software (Applied Biosystems). For each MALDI-TOF spectrum, a MASCOT PMF search of the National Centre for Biotechnology Information non-redundant protein database was first undertaken. Searches were performed allowing at least 1 trypsin miscleavage and without restriction on protein mass or pI. Carbamidomethylation of cysteine are a fixed modification and variable modifications included acetylation of N-terminal protein and oxidation of methionine. Peptide mass tolerance was set to ±60 ppm. A MASCOT PMF search of all data was made against the Bacteria (Eubacteria) Taxonomy protein dataset. Significance was established according to expectancy value transformed into a MOWSE score (with significance at p-value < 0.05 at scores over 85), minimum percentage coverage of 18% and theoretical pI and MW compared to the approximate experimental values observed on 2-DE gels. 3. Results and discussion Experiments focused first on Sphingomonas, on the HQDO enzyme and both its subunits (HqdA and HqdB). Purified HQDO was at first studied separately by running the 2D gel. Then, the overall proteome of Sphingomonas was obtained and HqdA and HqdB were searched in it. As a next step, Sphingomonas was spiked at different biomass ratios into an activated sludge mixture (i.e. decreasing the ratio of TTNP3 on activated sludge) to validate the method in such a complex matrix. The patterns of proteins observed in the different diluted samples were compared with the pure strain one. 3.1. Purified HQDO and identification of Sphingomonas sp. strain TTNP3 proteins in 2D gels The 2D gel of the purified HQDO enzyme with the visualisation of the obtained spots is represented in Fig. 2 on the left side while the right one shows the overall Sphingomonas proteome. The two subunits (HqdA and HqdB) were clearly distinguished as separated spots in the 2D gel (Fig. 2, left). The pIs were 4.54 and 4.73 and the molecular weights 18 and 36 kDa for HqdA and HqdB respectively.
The Sphingomonas proteome was also run in a 2D gel to attempt to identify the target enzymes in the overall protein complex (obtained result in Fig. 2, right). As shown, the HqdB was positively identified in the complex resolved proteome of the pure strain (Table 1) in the 2D gel, based on the translated amino acid sequence (GenBank accession number JF440299). A positive match with 42% of protein coverage and a match of 19 out of 103 peptides was observed (Table 2). In contrast, HqdA could not be identified even though several potential candidate spots were analysed (as visible in Fig. 2, marked with orange numbers) possibly for mass detection difficulties (in particular for HqdA due to its smaller MW). 3.2. Dilutions of Sphingomonas sp. strain TTNP3 into activated sludge samples The pure strain was spiked at decreasing ratio with activated sludge matrix and the 2D gels were run for the three corresponding applied dilutions and for the un-spiked control (i.e. activated sludge without Sphingomonas). The acquired gel images were processed, compared and placed within the same match set to prove the reliability of the gels and of the protocol itself. It was confirmed that the gels were correctly run as an average of 38% match within all of them was observed. This value can be considered acceptable taking into account single gel expression variability and the different and decreasing influence of the inoculum of Sphingomonas in the different mixtures. In the spiked diluted samples with activated sludge, none of the subunits of HQDO could be detected. This enzyme is apparently not as abundant as other proteins from the same strain and possibly masked by proteins from other microorganisms present in the activated sludge itself, leading to even less intense spots in the gels from the diluted samples. The detection limit of the method was tested looking for proteins belonging to Sphingomonas proteome among the diluted samples. The 2D gels, corresponding to the un-spiked control and to the dilutions from 1:10 to 1:103, were matched separately with the pure strain 2D gel (Table 3). A promising result was obtained as it was possible to distinguish proteins belonging to Sphingomonas on the 2D gels among the other proteins from activated sludge. Concretely, a match percentage of Sphingomonas proteins in the gel dilution 1:10 was done and 11% of the pure strain proteins were visible in this first dilution (Fig. 3). This operation was repeated for other dilutions and 11 proteins of the pure strain in the dilution 1:103 gel were still found even though with lower intensity (Fig. 4). The detection limit for proteins specific for Sphingomonas calculated in this case study can be considered 10 3 g TTNP3 g 1 TSS according to the obtained results. It is important to note at this point that Sphingomonas lysate gel was loaded with a greater quantity (400 lg) compared to the other gels (250 lg for sample limitation issue) resulting in higher intensities of the TTNP3 lysate gel proteins. Therefore the comparison of spot intensity and the match within the diluted samples was more difficult to assess. Moreover, not all the enzymes may be present,
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pH 4…………………………………………....7 4 ....7
pH 4…………………………………………....7
pH 4…………………………………………....7
Dilution 1:10
(a)
(b)
Dilution 1:10
(c)
Fig. 3. Detected spots are circled in blue and the spots belonging to Sphingomonas sp. strain TTNP3 are circled in red. (a) 2D gel of the un-spiked activated sludge control; (b) 2D gel of the pure strain matched with the 2D gel of the 1:10 diluted sample; (c) 2D gel of the 1:10 diluted sample matched with the 2D gel of the pure strain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
pH 4…………………………………………....7
pH 4…………………………………………....7
Dilution 1:100
Dilution 1:1000
Fig. 4. Detected spots are circled in blue and the spots belonging to Sphingomonas sp. strain TTNP3 are circled in red. On the left 2D gel of the pure strain matched with the 2D gel of the 1:100 diluted sample; on the right 2D gel of the pure strain matched with the 2D gel of the 1:1000 diluted sample. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
active or expressed at similar level. Nonetheless a certain number of proteins belonging to specific bacteria, even if not necessarily connected to the studied biodegradation mechanism, can be identified even in such a complex and heterogeneous media (activated sludge). In the current study using the presented protocol, it is possible to affirm that proteomic techniques had a detection limit of 10 3 g TTNP3 g 1 TSS for the Sphingomonas proteins (limit being pertinent to the chosen pure strain, tested activated sludge and conditions). This detection limit may vary among different proteins and may be too high when applied to activated sludge samples as it may, in some cases, only allow to assess dominant proteins of dominant species of microorganisms in the sludge matrix. Improving the protocol in subsequent studies could allow for better target protein detection and identification, which may eventually give valuable information on surfactants removal in activated sludge and the underlying degradation pathways. Nonetheless the obtained detection limit can be already considered noteworthy even when dealing with activated sludge systems.
4. Conclusions Reliability and detection limit of proteomics applied to specific proteins in a pure strain lysate and serially diluting it into activated sludge samples were investigated. Subunit B of the target enzyme (HQDO) was positively identified in the Sphingomonas proteome but not in the spiked diluted samples with activated sludge.
Afterwards, a significant percentage of target strain proteins were distinguished from other ones of activated sludge lysate, with a protocol detection limit of 10 3 g TTNP3 g 1 TSS. Protocol improvements are needed but proteomics was proven useful in this study not just with pure strain samples but also to find and follow target proteins in complex matrixes as activated sludge. Acknowledgments This study has been co-financed by the Spanish Ministry of Economy and Competitiveness and the European Union through the European Regional Development Fund and through a national Research Project (CTM2009-14742-C02-01). The authors would further like to thank the Servei de Recursos Científics i Tècnics de la Universitat Rovira i Virgili de Tarragona and in particular Dr. Núria Canela Canela. References Banfield, J.F., Ram, R.J., VerBerkmoes, N.C., Thelen, M.P., Tyson, G.W., Baker, B.J., Blake, R.C., Shan, M., Hettich, R.L., 2005. Community proteomics of a natural microbial biofilm. Science 308, 1915–1920. Barceló, D., Petrovic, M., 2007. Pharmaceuticals and personal care products (PPCPs) in the environment. Anal. Bioanal. Chem. 387, 1141–1142. Benndorf, D., Balke, G.U., Harms, H., von Bergen, M., 2007. Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. ISME J. 1, 224–234. Buttiglieri, G., Knepper, T.P., 2008. Removal of emerging contaminants in wastewater treatment: conventional activated sludge treatment. In: Barceló, D., Petrovic, M., (Eds.), Handbook of Environmental Chemistry, Emerging Contaminants From Industrial And Municipal Wastewaters, vol. 5. Springer, Berlin, pp. 1–35 (5 S2).
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Carballa, M., Omil, F., Lema, J.M., Llompart, M., Garcia, C., Rodriguez, I., Gomez, M., Temes, T., 2004. Behavior of pharmaceuticals, cosmetics and hormones in a sewage treatment plant. Water Res. 38, 2918–2926. Corvini, P.F.X., Hollender, J., Ji, R., Schumacher, S., Prell, J., Hommes, G., Priefer, U., Vinken, R., Schaffer, A., 2006. The degradation of alpha-quaternary. Nonylphenol isomers by sphingomonas sp strain TTNP3 involves a type II ipso-substitution mechanism. Appl. Microbiol. Biotechnol. 70, 114–122. Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc, G.A., Guillette, L.J., 2007. An ecological assessment of bisphenol-A: evidence from comparative biology. Reprod. Toxicol. 24, 225–239. Farre, M., Kloter, G., Petrovic, M., Alonso, M.C., de Alda, M.J.L., Barcelò, D., 2002. Identification of toxic compounds in wastewater treatment plants during a field experiment. Anal. Chim. Acta 456, 19–30. Fukazawa, H., Watanabe, M., Shiraishi, F., Shiraishi, H., Shiozawa, T., Matsuhita, H., Terao, Y., 2002. Formation of chlorinated derivatives of bisphenol A in waste paper recycling plants and their estrogenic activities. J. Heal. Sci. 48, 242–249. Greiner, E., Kaelin, T., Nakamura, K., 2007. Bisphenol A. CEH Report By SRI Consulting.
downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3. Appl. Microbiol. Biotechnol. 93, 1315–1324. Li, Z., Zhang, H., Gibson, M., Liu, P., 2012. An evaluation of the combined effects of phenolic endocrine disruptors on vitellogenin induction in goldfish Carassius auratus. Ecotoxicol. 1–9. http://dx.doi.org/10.1007/s10646-012-0925-0. Lopez, M.F., 1999. Proteome analysis I. ‘‘Gene products are where the biological action is’’. J. Chromatogr. 722, 191–202. Porter, A.W., Campbell, B.R., Kolvenbach, B.A., Corvini, P.F.X., Benndorf, D., RiveraCancel, G., Hay, G., 2012. Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram. Appl. Microbiol. Biotechnol. 94, 261–272. Seung, Il.K., Seung-Youl, S., Kyung-Wook, K., Eun-Mi, H., Kye-Heon, O., 2003. Proteomic analysis of the benzoate degradation pathway in Acinetobacter Sp. KS-1. Res. Microbiol. 154, 697–703. Soares, A., Guieysse, B., Jefferson, B., Cartmell, E., Lester, J.N., 2008. Nonylphenol in the environment: a critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ. Int. 34, 1033–1049. Vethaak, A.D., Lahr, J., Schrap, S.M., Belfroid, A.C., Rijs, G.B.J., Gerritsen, A., de Boer, J., Bulder, A.S., Grinwis, G.C.M., Kuiper, R.V., Legler, J., Murk, T.A.J., Peijnenburg, W., Verhaar, H.J.M., de Voogt, P., 2005. An integrated assessment of estrogenic contamination and biological effects in the aquatic environment of The Netherlands. Chemosphere 59, 511–524. Vom Saal, F.S., Hughes, C., 2005. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ. Health Perspect. 113, 926–933. Wang, Z., Hu, F., Song, W., Guo, J., He, W., Ding, F., 2011. Chronic toxic effect of three estrogens to algae (Scenedesmus obliquus). In: Proceedings 2011 International Conference on Human Health and Biomedical Engineering, HHBE 2011, pp. 877–880, doi: 10.1109/HHBE.2011.6028962. Wilmes, P., Bond, P.L., 2004. The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. Environ. Microbiol. 6, 911–920. Wilmes, P., Wexler, M., Bond, P.L., 2008. Metaproteomics provides functional insight into activated sludge wastewater treatment. PLoS One. 3, Article number e1778.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Technical Note
Exploring the potential of applying proteomics for tracking bisphenol A and nonylphenol degradation in activated sludge Neus Collado a,b, Gianluigi Buttiglieri b,⇑, Boris A. Kolvenbach c, Joaquim Comas a, Philippe F.-X. Corvini c, Ignasi Rodríguez-Roda a,b a b c
LEQUIA, Institute of the Environment, University of Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain ICRA, Catalan Institute for Water Research, Carrer Emili Grahit, 101, Parc Científic i Tecnològic de la Universitat de Girona, 17003 Girona, Spain Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland
h i g h l i g h t s " Proteomics applied to a pure culture of Sphingomonas TTNP3 and its proteome analysed. " The pure strain hydroquinone dioxygenase enzyme used as a proteomic marker. " Dilutions of the pure strain with activated sludge were done to validate the method. " Diluted samples protein patterns were compared with the pure strain proteome. " The protocol detection limit is noteworthy for Sphingomonas TTNP3 in activated sludge.
a r t i c l e
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Article history: Received 5 August 2012 Received in revised form 4 October 2012 Accepted 5 October 2012 Available online 31 October 2012 Keywords: Enzymes Pure culture Phenolic substances Micropollutants Proteomics Activated sludge
a b s t r a c t A significant percentage of bisphenol A and nonylphenol removal in municipal wastewater treatment plants relies on biodegradation. Nonetheless, incomplete information is available concerning their degradation pathways performed by microbial communities in activated sludge systems. Hydroquinone dioxygenase (HQDO) is a specific degradation marker enzyme, involved in bisphenol A and nonylphenol biodegradation, and it can be produced by axenic cultures of the bacterium Sphingomonas sp. strain TTNP3. Proteomics, a technique based on the analysis of microbial community proteins, was applied to this strain. The bacterium proteome map was obtained and a HQDO subunit was successfully identified. Additionally, the reliability of the applied proteomics protocol was evaluated in activated sludge samples. Proteins belonging to Sphingomonas were searched at decreasing biomass ratios, i.e. serially diluting the bacterium in activated sludge. The protein patterns were compared and Sphingomonas proteins were discriminated against the ones from sludge itself on 2D-gels. The detection limit of the applied protocol was defined as 10 3 g TTNP3 g 1 total suspended solids (TSSs). The results proved that proteomics can be a promising methodology to assess the presence of specific enzymes in activated sludge samples, however improvements of its sensitivity are still needed. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Currently, there is an increasing awareness of the presence of micropollutants (e.g. surfactants, plasticizers, pharmaceuticals, pesticides and so on) in domestic wastewater (Carballa et al., 2004; Barceló and Petrovic, 2007; Buttiglieri and Knepper, 2008). Nonylphenol (NP) and bisphenol A (BPA) are phenolic substances used in high amounts by the industry (Greiner et al., 2007; Soares et al., 2008). Both can act as hormone disrupters and affect the reproductive system in animals causing, for example, feminization ⇑ Corresponding author. E-mail address: [email protected] (G. Buttiglieri). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.10.002
in various aquatic organisms or inhibition of algal populations (Wang et al., 2011; Li et al., 2012; Gyllenhammar et al., 2012). Some studies indicate that negative health effects occur even at very low doses (vom Saal and Hughes, 2005). Effluents from wastewater treatment plants (WWTPs) are a major source of environmental BPA and NP (Vethaak et al., 2005; Crain et al., 2007). Influent WWTP concentrations are generally at low lg L 1 level but concentrations up to 59 lg L 1 are reported for BPA (Fukazawa et al., 2002) and even higher for NP (Klecka et al., 2010). The percentage of BPA removal in WWTPs varies considerably per location from 0% to 96% (Vethaak et al., 2005). WWTP removals of NP are also highly variable (9–94%) depending on the region and type of unit treatment processes employed
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(Soares et al., 2008). NP, in addition, can be produced during the wastewater treatment processes (e.g. the surfactants NPethoxylates can easily degrade back to NP, Soares et al., 2008) and it is often found at higher concentrations in the effluents than in the influents (Farre et al., 2002; Soares et al., 2008). These results are of concern since they indicate that WWTPs are only partially efficient in removing such compounds being released into the aquatic environment. Low micropollutant concentrations, as it is the case of NP and BPA, are not likely to sustain growth of microorganisms, and conventional microbiological techniques based on cultivation of microorganisms will remain ineffective in monitoring biodegradation mechanisms. More information may be achieved using molecular microbiological techniques, e.g. qPCR and FISH. Proteomics is a relatively new approach in environmental microbiology based on the identification and expression levels of proteins. Its central assumption is that fingerprints of expressed proteins are specific to the conditions experienced by the organisms. The goal of the technique is to observe differences and changes in protein expression levels and, hence, to identify key proteins directly involved in the different biological processes (Lopez, 1999). Since the introduction of the metaproteomic approach (meaning proteomics applied to mixed cultures; Wilmes and Bond, 2004) only a few studies have been carried out on marine ecosystems (Banfield et al., 2005; Kan et al., 2005), groundwater and soil (Benndorf et al., 2007). The high potential of metaproteomics has been also successfully demonstrated on activated sludge and specifically on phosphorous removal in conventional WWTPs (Wilmes et al., 2008) but never for tracking proteins of a strain able to degrade micropollutants in activated sludge mixtures. In order to produce a proof-of-concept on the applicability of proteomics in wastewater samples an axenic culture as well as its mixture with activated sludge was used in the present work. A specific strain, Sphingomonas sp. strain TTNP3, responsible for the degradation of the micropollutants BPA and NP was chosen. Experiments were first performed with it and more precisely focusing on the hydroquinone dioxygenase (HQDO). This enzyme has been well characterised (Kolvenbach et al., 2011) but has not been used as a marker for proteomics application before. The subsequent objective was to check if proteins, in our case, belonging to Sphingomonas proteome were still detectable in the overall mixture with activated sludge, and until which dilution, in order to define the detection limit of the applied protocol and to follow the protein match set within the diluted gels. 2. Materials and methods 2.1. Pure strain, purified HQDO Cultures of Sphingomonas (deposited at BCCM under accession number LMG 21268) were cultivated and harvested as previously described including 16 h of incubation with 0.5 mM technical grade NP to induce HQDO expression (Kolvenbach et al., 2011, 2012). HQDO catalyses the ring opening of the latter to produce 4-hydroxymuconic semialdehyde (Fig. 1; Kolvenbach et al., 2007; Porter et al., 2012). Purified HQDO was obtained as described previously by ammonium sulphate precipitation and subsequent hydrophobic interaction and ion exchange chromatography (Kolvenbach et al., 2011). 2.2. Dilution of Sphingomonas sp. strain TTNP3 in activated sludge A set of activated sludge samples were spiked with different amounts of Sphingomonas biomass. Experimental replicates were considered of minor importance in this case, since the objective
was to find out the detection limit of the protocol and to follow not target identified proteins but the general protein patterns. Moreover to be noted that activated sludge itself acts as a pool with already an intrinsic variability of microorganisms and enzymes. The activated sludge was sampled at ARA-BIRS WWTP (Basel, Switzerland), from the aeration tank of the biological treatment with a total suspended solids (TSSs) concentration of 3 g L 1 (100% domestic wastewater). Sphingomonas stock solution had a concentration of dry weight of 3 g TTNP3 L 1. Three logarithmic serial dilutions in the above described activated sludge were prepared from 1:10 (v/v) to 1:103. Thus, the first diluted sample corresponded to a concentration of 3 10 1 g TTNP3 L 1 and the last one of 3 10 3 g TTNP3 L 1. Finally, the cells from these activated sludge samples were harvested by centrifugation at 5000 rpm for 20 min. The supernatant was discarded, and the collected pellet was frozen at 80 °C till analysis. 2.3. Protein extraction, purification and resuspension of the samples All analysed pellets were frozen in liquid nitrogen and lyophilised. The pulverised samples were resuspended by pulsevortexing in 5 mL of lysis buffer consisting of 40 mM Tris-HCl pH 8, 2 mM EDTA, 25 mM NaCl, 25 mM MgCl2, 0.1% SDS (Sodium Dodecyl Sulphate), 2 mM Pefabloc SC, as a protease inhibitor, and 2 mM Pefabloc SC protector reagent (Roche). Cells were then placed on ice and lysed by sonication (four cycles of three min each at pulse 40 and amplitude 21%; Sonics Vibra-Cell). The suspension was then centrifuged at 13,000 rpm (approximately 16,000g) for 30 min to remove cell debris, and the supernatants were stored at 80 °C. Protein concentrations were determined using a BioRad RC DC kit with bovine serum albumin as a standard following the manufacturer’s protocol. For each procedure, protein standards were prepared in the same buffer as the protein samples. Finally, proteins were precipitated in 10% (w/v) trichloroacetic acid, incubated on ice for 30 min and centrifuged at 13,000 rpm for 15 min. The protein pellet was then washed in 80% (v/v) ice-cold acetone overnight at 20 °C. The dried protein pellet was resuspended in 500 lL of 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM DTT (DL-Dithiothreitol) and a trace of bromophenol. 2.4. 2D-page (polyacrylamid gel electrophoresis) The protein fingerprints are obtained by protein separation in two dimensions, i.e. first by isoelectric point (pI) and subsequently by molecular weight (MW), in 2D-gel electrophoresis (Seung et al., 2003). 30 lg of the purified protein sample, 400 lg in the case of the TTNP3 lysate and 250 lg for activated sludge samples were dissolved in 480 lL of rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT and a trace of bromophenol), 1% of IPG Pharmalites 3-10, and were used to impregnate 24 cm pH 4– 7 IPG strips (Immobiline DryStrips, Amersham Biosciences–GE Healthcare) in an Immobiline DryStrip Reswelling Tray (Amersham Biosciences–GE Healthcare) for 16 h. For first-dimension separation, the strips were placed in an IPGphor ceramic manifold, covered with Plusone DryStrip cover fluid and focused for 95,000 V h in an Ettan IPGphor3 isoelectric focusing system (Amersham Biosciences–GE Healthcare). Gels strips were subsequently equilibrated for 20 min in equilibration buffer A (6 M urea, 75 mM Tris-HCl pH 8.8, Glycerol 29.3%, SDS 2%, a trace of bromophenol, milli-Q water, 10 mg mL 1 DTT), followed by 20 min in equilibration buffer B (as buffer A but containing 25 mg iodoacetamide per mL instead of DTT). Second dimension separation was carried out with 12% v/v acrylamide, SDS-PAGE gels, at 100 W in an Ettan DALTsixelectrophoresis system (Amersham Biosciences-GE Healthcare). The gels were stained overnight with Colloidal Coomassie
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OH
R
R
2
R
1
R
4
R
1
R
R
2
+H2 O/ -H
1
HO
(BPA) 2
R
R
+
-H
+H
OH
2
R
OH
O
1
2
2
6
5 +
1
1
R
+
2
C R
3
HqdA HqdB
COOH
COOH
+
COOH COOH
COOH
CHO OH
OH
O
O
7
8
9
10
beta-ketoadipate pathway
Fig. 1. Main degradation pathway for BPA and NP in Sphingomonas sp. strain TTNP3: 1 – BPA (R1 = methyl; R2 = p-phenol) or NP (R1 = ethyl; R2 = 2-pentyl, for the 4-(1-ethyl1,2-dimethylpentyl)phenol) isomer; 2 – quinol intermediate; 3 – carbocationic intermediate; 4 – 4-(2-hydroxypropan-2-yl)phenol with BPA as substrate and 3,5-dimethyl-3heptanol with NP as substrate, respectively); 5 – 4-isopropenylphenol; 6 – 4-isopropylphenol; 7 – hydroquinone; 8 – 4-hydroxymuconic acid semialdehyde; 9 – maleylacetic acid; 10 – 3-oxoadipic acid; rectangles mark the enzymes attributed to the metabolism step (adapted from Corvini et al., 2006; Kolvenbach et al., 2012).
Fig. 2. On the left side, 2D gel from the purified HQDO enzyme with both subunits (HdqA and HdqB). On the right side, the proteome of the Sphingomonas sp. strain TTNP3 with all the spots in blue, the candidate target enzymes (for HqdA, HqdB) in orange and the identified HdqB highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Proteins identification on excised spots candidates to be HQDO subunits. Spot ID
GI
Score
Name
pI
MW
Organism
1 2 3 4 5 6
82702350 254776561 148553363 148554247 148555246 335346123
70 64 90 98 112 88
Glyoxalasa family protein Transcriptional regulator, GntR family protein Transcription elongation factor GreA DNA-directed RNA ploymerase subunit alpha Hydrogenobyrinic acid a,c-diamide cobaltochelatase Hydroquinone subunit B
9.69 7.08 4.73 4.89 5.02 4.94
6719 25,014 17,403 38,091 37,465 36,933
Nitrosospira multiformis ATCC 25196 Mycobacterium avium subsp. Avium ATCC 25291 Sphingomonas wittichii RW1 Sphingomonas wittichii RW1 Sphingomonas wittichii RW1 Sphingomonas TTNP3
Blue (Bio-Rad) and scanned using a (Amersham Biosciences-GE Healthcare).
Labscan
instrument
subunits (HqdA and HqdB). The highly expressed ones in these areas were chosen for further quantification and identification after excision.
2.5. Image analysis of 2D-PAGE gels The acquired gel images were processed and analysed using Image Master Platinum v.7 (Amersham Biosciences-GE Healthcare). The gels were placed within the same match set. Automated and manual spot detection and matching was performed, as well as spot densities determined. As a secondary analysis, and for the gels corresponding to the pure strain dilutions in activated sludge, a match set was performed among the different gel images based on their pI and the MW. 2.6. Spot excision Protein spots, from the Sphingomonas lysate 2D gel, were ranked according to their proximity to the HQDO subunits (HqdA and HqdB) taking into account their theoretical pI (4.53 and 4.94 respectively) and MW (19 and 38 kDa respectively) of the HQDO
2.7. Matrix assisted laser desorption ionisation time-of-flight (MALDIToF) mass spectrometry (MS) and protein identification The excised spots were processed by in gel tryptic digestion, prior to mass spectrometric analyses. Each excised spot was detained by acetonitrile (ACN) treatment then dried using a Speedvac centrifuge. Approximately, 200 ng of trypsin in 25 mM ammonium bicarbonate was added in a volume enough to rehydrate the gel pieces. Digestion was performed overnight at 37 °C. The tryptic peptides were eluted from the gel with 50% ACN/0.1% trifuoracetic acid. 1 lL of eluant was mixed with an equal volume of saturated solution of a-cyano 4-hydroxycinnamic acid in 50% ACN and 0.1% trifluoracetic acid and spotted on a MALDI plate by dried droplet method. The samples were analysed on a Voyager DE-PRO, MALDI-TOF analyser (Applied Biosystems). A 50 Hz nitrogen laser was used to desorb/ionise the matrix/analyte material, the acceleration voltage was 20 kV and the ions were detected in positive ion
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Table 2 Matched peptides in bold and underlined for the identification of HqdB. 1
MAMSEALEII
DFGDSKARTD
51
LTWPGGSHII
PIDAFLRAMM
AGRFNDAYRN LPWGIKNGNN
AGRDHEERFK DEAISRQRVT
QEQPVVEAEP EFILPVEHGN ADIRHQGYST
101 151 201 251 301
TEHLAINNET RDVAWGFFYG
GYRSFRAGGF VVNFDHVFGT
TFTRDEYFAR INHYGEVTMF
SSALMAVFKD
ILSDWTVEGY
DPFAAPMETG
GFEAEVSAYN DRCEWFLQLS
ARRMVGLPGD LFGYLSRSDV DEIVWDVKDK
TPVRTDANGF TWNPSVCSVV ESGKPRARVT
PVNRQFADVP GDSLFCPTSE ARAGDICCMP
KRSMLLVWEN
GSPKIPQMIA
DGTAPVVPVT
F
Table 3 Spots number, percent match and matched spots number between Sphingomonas sp. strain TTNP3 2D gel and the ones serially diluting the pure strain in activated sludge. Gel
Spots number
Matched spots (%)
Matched spot number
Pure strain Dilution 1.10 Dilution 1:100 Dilution 1:1000
709 588 673 357
n.a. 11 4 2
n.a. 78 30 8
reflectron mode. The samples were externally calibrated against a standard mixture of peptides from Bruker, ranging in mass from 1 to 3.2 kDa. Resulting spectra were analysed directly using Data Explorer version 4.0.0.0. MS software (Applied Biosystems). For each MALDI-TOF spectrum, a MASCOT PMF search of the National Centre for Biotechnology Information non-redundant protein database was first undertaken. Searches were performed allowing at least 1 trypsin miscleavage and without restriction on protein mass or pI. Carbamidomethylation of cysteine are a fixed modification and variable modifications included acetylation of N-terminal protein and oxidation of methionine. Peptide mass tolerance was set to ±60 ppm. A MASCOT PMF search of all data was made against the Bacteria (Eubacteria) Taxonomy protein dataset. Significance was established according to expectancy value transformed into a MOWSE score (with significance at p-value < 0.05 at scores over 85), minimum percentage coverage of 18% and theoretical pI and MW compared to the approximate experimental values observed on 2-DE gels. 3. Results and discussion Experiments focused first on Sphingomonas, on the HQDO enzyme and both its subunits (HqdA and HqdB). Purified HQDO was at first studied separately by running the 2D gel. Then, the overall proteome of Sphingomonas was obtained and HqdA and HqdB were searched in it. As a next step, Sphingomonas was spiked at different biomass ratios into an activated sludge mixture (i.e. decreasing the ratio of TTNP3 on activated sludge) to validate the method in such a complex matrix. The patterns of proteins observed in the different diluted samples were compared with the pure strain one. 3.1. Purified HQDO and identification of Sphingomonas sp. strain TTNP3 proteins in 2D gels The 2D gel of the purified HQDO enzyme with the visualisation of the obtained spots is represented in Fig. 2 on the left side while the right one shows the overall Sphingomonas proteome. The two subunits (HqdA and HqdB) were clearly distinguished as separated spots in the 2D gel (Fig. 2, left). The pIs were 4.54 and 4.73 and the molecular weights 18 and 36 kDa for HqdA and HqdB respectively.
The Sphingomonas proteome was also run in a 2D gel to attempt to identify the target enzymes in the overall protein complex (obtained result in Fig. 2, right). As shown, the HqdB was positively identified in the complex resolved proteome of the pure strain (Table 1) in the 2D gel, based on the translated amino acid sequence (GenBank accession number JF440299). A positive match with 42% of protein coverage and a match of 19 out of 103 peptides was observed (Table 2). In contrast, HqdA could not be identified even though several potential candidate spots were analysed (as visible in Fig. 2, marked with orange numbers) possibly for mass detection difficulties (in particular for HqdA due to its smaller MW). 3.2. Dilutions of Sphingomonas sp. strain TTNP3 into activated sludge samples The pure strain was spiked at decreasing ratio with activated sludge matrix and the 2D gels were run for the three corresponding applied dilutions and for the un-spiked control (i.e. activated sludge without Sphingomonas). The acquired gel images were processed, compared and placed within the same match set to prove the reliability of the gels and of the protocol itself. It was confirmed that the gels were correctly run as an average of 38% match within all of them was observed. This value can be considered acceptable taking into account single gel expression variability and the different and decreasing influence of the inoculum of Sphingomonas in the different mixtures. In the spiked diluted samples with activated sludge, none of the subunits of HQDO could be detected. This enzyme is apparently not as abundant as other proteins from the same strain and possibly masked by proteins from other microorganisms present in the activated sludge itself, leading to even less intense spots in the gels from the diluted samples. The detection limit of the method was tested looking for proteins belonging to Sphingomonas proteome among the diluted samples. The 2D gels, corresponding to the un-spiked control and to the dilutions from 1:10 to 1:103, were matched separately with the pure strain 2D gel (Table 3). A promising result was obtained as it was possible to distinguish proteins belonging to Sphingomonas on the 2D gels among the other proteins from activated sludge. Concretely, a match percentage of Sphingomonas proteins in the gel dilution 1:10 was done and 11% of the pure strain proteins were visible in this first dilution (Fig. 3). This operation was repeated for other dilutions and 11 proteins of the pure strain in the dilution 1:103 gel were still found even though with lower intensity (Fig. 4). The detection limit for proteins specific for Sphingomonas calculated in this case study can be considered 10 3 g TTNP3 g 1 TSS according to the obtained results. It is important to note at this point that Sphingomonas lysate gel was loaded with a greater quantity (400 lg) compared to the other gels (250 lg for sample limitation issue) resulting in higher intensities of the TTNP3 lysate gel proteins. Therefore the comparison of spot intensity and the match within the diluted samples was more difficult to assess. Moreover, not all the enzymes may be present,
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pH 4…………………………………………....7 4 ....7
pH 4…………………………………………....7
pH 4…………………………………………....7
Dilution 1:10
(a)
(b)
Dilution 1:10
(c)
Fig. 3. Detected spots are circled in blue and the spots belonging to Sphingomonas sp. strain TTNP3 are circled in red. (a) 2D gel of the un-spiked activated sludge control; (b) 2D gel of the pure strain matched with the 2D gel of the 1:10 diluted sample; (c) 2D gel of the 1:10 diluted sample matched with the 2D gel of the pure strain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
pH 4…………………………………………....7
pH 4…………………………………………....7
Dilution 1:100
Dilution 1:1000
Fig. 4. Detected spots are circled in blue and the spots belonging to Sphingomonas sp. strain TTNP3 are circled in red. On the left 2D gel of the pure strain matched with the 2D gel of the 1:100 diluted sample; on the right 2D gel of the pure strain matched with the 2D gel of the 1:1000 diluted sample. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
active or expressed at similar level. Nonetheless a certain number of proteins belonging to specific bacteria, even if not necessarily connected to the studied biodegradation mechanism, can be identified even in such a complex and heterogeneous media (activated sludge). In the current study using the presented protocol, it is possible to affirm that proteomic techniques had a detection limit of 10 3 g TTNP3 g 1 TSS for the Sphingomonas proteins (limit being pertinent to the chosen pure strain, tested activated sludge and conditions). This detection limit may vary among different proteins and may be too high when applied to activated sludge samples as it may, in some cases, only allow to assess dominant proteins of dominant species of microorganisms in the sludge matrix. Improving the protocol in subsequent studies could allow for better target protein detection and identification, which may eventually give valuable information on surfactants removal in activated sludge and the underlying degradation pathways. Nonetheless the obtained detection limit can be already considered noteworthy even when dealing with activated sludge systems.
4. Conclusions Reliability and detection limit of proteomics applied to specific proteins in a pure strain lysate and serially diluting it into activated sludge samples were investigated. Subunit B of the target enzyme (HQDO) was positively identified in the Sphingomonas proteome but not in the spiked diluted samples with activated sludge.
Afterwards, a significant percentage of target strain proteins were distinguished from other ones of activated sludge lysate, with a protocol detection limit of 10 3 g TTNP3 g 1 TSS. Protocol improvements are needed but proteomics was proven useful in this study not just with pure strain samples but also to find and follow target proteins in complex matrixes as activated sludge. Acknowledgments This study has been co-financed by the Spanish Ministry of Economy and Competitiveness and the European Union through the European Regional Development Fund and through a national Research Project (CTM2009-14742-C02-01). The authors would further like to thank the Servei de Recursos Científics i Tècnics de la Universitat Rovira i Virgili de Tarragona and in particular Dr. Núria Canela Canela. References Banfield, J.F., Ram, R.J., VerBerkmoes, N.C., Thelen, M.P., Tyson, G.W., Baker, B.J., Blake, R.C., Shan, M., Hettich, R.L., 2005. Community proteomics of a natural microbial biofilm. Science 308, 1915–1920. Barceló, D., Petrovic, M., 2007. Pharmaceuticals and personal care products (PPCPs) in the environment. Anal. Bioanal. Chem. 387, 1141–1142. Benndorf, D., Balke, G.U., Harms, H., von Bergen, M., 2007. Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. ISME J. 1, 224–234. Buttiglieri, G., Knepper, T.P., 2008. Removal of emerging contaminants in wastewater treatment: conventional activated sludge treatment. In: Barceló, D., Petrovic, M., (Eds.), Handbook of Environmental Chemistry, Emerging Contaminants From Industrial And Municipal Wastewaters, vol. 5. Springer, Berlin, pp. 1–35 (5 S2).
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Carballa, M., Omil, F., Lema, J.M., Llompart, M., Garcia, C., Rodriguez, I., Gomez, M., Temes, T., 2004. Behavior of pharmaceuticals, cosmetics and hormones in a sewage treatment plant. Water Res. 38, 2918–2926. Corvini, P.F.X., Hollender, J., Ji, R., Schumacher, S., Prell, J., Hommes, G., Priefer, U., Vinken, R., Schaffer, A., 2006. The degradation of alpha-quaternary. Nonylphenol isomers by sphingomonas sp strain TTNP3 involves a type II ipso-substitution mechanism. Appl. Microbiol. Biotechnol. 70, 114–122. Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc, G.A., Guillette, L.J., 2007. An ecological assessment of bisphenol-A: evidence from comparative biology. Reprod. Toxicol. 24, 225–239. Farre, M., Kloter, G., Petrovic, M., Alonso, M.C., de Alda, M.J.L., Barcelò, D., 2002. Identification of toxic compounds in wastewater treatment plants during a field experiment. Anal. Chim. Acta 456, 19–30. Fukazawa, H., Watanabe, M., Shiraishi, F., Shiraishi, H., Shiozawa, T., Matsuhita, H., Terao, Y., 2002. Formation of chlorinated derivatives of bisphenol A in waste paper recycling plants and their estrogenic activities. J. Heal. Sci. 48, 242–249. Greiner, E., Kaelin, T., Nakamura, K., 2007. Bisphenol A. CEH Report By SRI Consulting.
downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3. Appl. Microbiol. Biotechnol. 93, 1315–1324. Li, Z., Zhang, H., Gibson, M., Liu, P., 2012. An evaluation of the combined effects of phenolic endocrine disruptors on vitellogenin induction in goldfish Carassius auratus. Ecotoxicol. 1–9. http://dx.doi.org/10.1007/s10646-012-0925-0. Lopez, M.F., 1999. Proteome analysis I. ‘‘Gene products are where the biological action is’’. J. Chromatogr. 722, 191–202. Porter, A.W., Campbell, B.R., Kolvenbach, B.A., Corvini, P.F.X., Benndorf, D., RiveraCancel, G., Hay, G., 2012. Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram. Appl. Microbiol. Biotechnol. 94, 261–272. Seung, Il.K., Seung-Youl, S., Kyung-Wook, K., Eun-Mi, H., Kye-Heon, O., 2003. Proteomic analysis of the benzoate degradation pathway in Acinetobacter Sp. KS-1. Res. Microbiol. 154, 697–703. Soares, A., Guieysse, B., Jefferson, B., Cartmell, E., Lester, J.N., 2008. Nonylphenol in the environment: a critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ. Int. 34, 1033–1049. Vethaak, A.D., Lahr, J., Schrap, S.M., Belfroid, A.C., Rijs, G.B.J., Gerritsen, A., de Boer, J., Bulder, A.S., Grinwis, G.C.M., Kuiper, R.V., Legler, J., Murk, T.A.J., Peijnenburg, W., Verhaar, H.J.M., de Voogt, P., 2005. An integrated assessment of estrogenic contamination and biological effects in the aquatic environment of The Netherlands. Chemosphere 59, 511–524. Vom Saal, F.S., Hughes, C., 2005. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ. Health Perspect. 113, 926–933. Wang, Z., Hu, F., Song, W., Guo, J., He, W., Ding, F., 2011. Chronic toxic effect of three estrogens to algae (Scenedesmus obliquus). In: Proceedings 2011 International Conference on Human Health and Biomedical Engineering, HHBE 2011, pp. 877–880, doi: 10.1109/HHBE.2011.6028962. Wilmes, P., Bond, P.L., 2004. The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. Environ. Microbiol. 6, 911–920. Wilmes, P., Wexler, M., Bond, P.L., 2008. Metaproteomics provides functional insight into activated sludge wastewater treatment. PLoS One. 3, Article number e1778.