Chemosphere 92 (2013) 633–638

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Chemosphere

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Functional groups characteristics of EPS in biofilm growing on differentcarriers

0045-6535/$ - see front matter Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.01.059

⇑ Corresponding author at: College of Urban Construction and EnvironmentalEngineering, Chongqing University, Chongqing 400045, China. Tel./fax: +86 2365128095.

E-mail address: guo0768@cqu.edu.cn (J.-S. Guo).

You-Peng Chen, Peng Zhang, Jin-Song Guo ⇑, Fang Fang, Xu Gao, Chun LiKey Laboratory of the Three Gorges Reservoir Region’s Eco-Environments of MOE, Chongqing University, Chongqing 400045, ChinaCollege of Urban Construction and Environmental Engineering, Chongqing University, Chongqing 400045, China

h i g h l i g h t s

" Each EPS fraction had a distinctbioflocculation activity.

" The SCP obtained significantlyhigher biomass and EPS comparedwith ACF.

" The TB-EPS fraction of SCP had betterflocculating ability compared withACF.

" Carriers can potentially affect theproduction and characteristics ofEPS.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 August 2012Received in revised form 10 January 2013Accepted 11 January 2013Available online 7 March 2013

Keywords:BiofilmCANONCarrierEPSFT-IRRaman

a b s t r a c t

This study investigated extracellular polymeric substances (EPSs), including soluble EPS, loosely boundEPS and tightly bound EPS (TB-EPS), in biofilms growing on different carriers from a CANON system toelucidate their different compositions and characteristics. The zeta potentials of all EPS fractions of thetwo samples were decreased systematically. The soft combination packing (SCP) was more hydrophilicthan activated carbon fiber (ACF), and it obtained a significantly higher biomass. However, Raman spec-troscopy and Fourier transform infrared spectroscopy revealed the same EPS fraction had similar func-tional groups between two carriers. Especially for the TB-EPS, total amount and the contents ofproteins and polysaccharides in the SCP sample were much higher than those in the ACF sample. More-over, the TB-EPS fraction of the SCP sample had better bioflocculation activity compared with that of theACF sample. Therefore the result demonstrated that the characteristics of EPS varied from carrier tocarrier.

Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Biofilm formation is primarily caused by the adhesion of cellsonto the carrier surface, hence indicating the surface characteris-tics of cells and carriers play an important role in the deposition

of microbes onto surfaces (Renner and Weibel, 2011). Hydrophobicand rough mutant bacterial cells adhere more easily onto carriersurface and form biofilms compared with hydrophilic and smoothspecies (Mazumder et al., 2010). The surface roughness and surfaceenergy of carriers are essential to the early biofilm developmentand the attachment of the organisms (Lakshmi et al., 2012). Manybacterial cells more readily settle on the hydrophobic surfaces ow-ing to hydrophobic interactions, whereas others are prefer to ad-sorb on the hydrophilic surfaces (Gomez-Suarez et al., 2002).Bacterial cell surfaces are always carry a negative charge in

634 Y.-P. Chen et al. / Chemosphere 92 (2013) 633–638

solution under subacid, neutral and alkalescent conditions, andthey are thus more inclined to adhere onto positive surfaces dueto electrostatic attraction (Bernstein et al., 2011). Subsequently,the physicochemical properties of carrier surfaces induce bacterialcells to secrete the extracellular polymeric substances (EPSs) whenthese cells are deposited onto the carriers (Renner and Weibel,2011).

EPS are high-molecular-weight molecules consisting of polysac-charides, DNA, proteins, lipids and humic acids that are released bymicrobes and adhere onto the cell surfaces of activated sludge,granular sludge, and biofilms in wastewater treatment (Flemmingand Wingender, 2010). EPS often divide into two major fractions:soluble EPS (SEPS) and bound EPS (Li and Yang, 2007). The adhe-sion of SEPS to cells is weak, and as a result of which SEPS are oftendissolved in solution. The inner layer of bound EPS consists oftightly bound EPS (TB-EPS), whereas the outer layer consists ofloosely bound EPS (LB-EPS). TB-EPS have certain morphology whileLB-EPS have no a distinct boundary (Sheng et al., 2010). Accordingto previous studies, the production of EPS is influenced by themedium used (e.g. carbon resource, C/N) (Miqueleto et al., 2010),growth conditions, operating conditions (e.g. sludge retentiontime, DO) (Dvorak et al., 2011) and environmental conditions(e.g. metals, drugs, shear forces) (Avella et al., 2010). Furthermore,the high hydrophobicity and charges of bacterial surfaces have agreat propensity to enable the formation of EPS (Harimawanet al., 2011). The adsorption and adhesion of EPS onto carrier andcells alter the physicochemical characteristics of surfaces, conse-quently affecting the cell deposition and biofilm formation. More-over, the attachment and deposition of EPS and bacterial cells ontocarrier surfaces modify the surface characteristics (Tansel et al.,2006; Sheng et al., 2010). Overall, however, few studies focusingon the characteristics of EPS in biofilms growing on different car-rier surfaces have been reported to date (Choi et al., 2001).

The CANON (completely autotrophic nitrogen removal over ni-trite) system can achieve nitrification and anaerobic ammoniumoxidation in a single reactor with oxygen-limiting treatment stepsto remove ammonium from wastewater (Sliekers et al., 2002). How-ever, microbial growth and biofilm formation under this system areslow, resulting in a longer start-up period. We have previouslyinvestigated the CANON process (Li et al., 2012; Chen et al.,2012a), and we have also demonstrated that the hydrophilic-mod-ified activated carbon fiber (ACF) has better biological affinity com-pared with unmodified ACF in CANON systems (Chen et al., 2012b).Therefore, the biofilm growth is also significantly influenced by thesurface properties of carriers, whereas the compositions and charac-teristics of EPS from different carriers require further investigation.

In this work, the EPS including SEPS, LB-EPS and TB-EPS in bio-films growing on two different carriers from a CANON system wereextracted. Biochemical information on the molecular structures ofthe EPS was obtained using Raman and Fourier transform infrared(FT-IR) spectra. In addition, the surface properties of different car-riers and bacterial cells were also measured.

2. Materials and methods

2.1. Biofilm origin

Biofilms were sampled from a sequencing batch biofilm reactor.The reactor had two biomass carriers: ACF, which is polyacryloni-trile-based, and soft combination packing (SCP), whose key compo-sition is polyvinyl formal. Both the two carriers are fibers. Thereactors were fed with synthetic inorganic wastewater which in-cluded ammonium bicarbonate and sodium bicarbonate as carbonsources, as well as ammonium bicarbonate also as the sole nitro-gen source. Details of the synthetic wastewater composition are

as follows (in mg L�1): 1975 NH4HCO3, 328 NaHCO3, 70 KH2PO4,2 mL trace element solution (in g L�1) [5.0 EDTA, 1.6 CoCl2�6H2O,2.2 ZnSO4�7H2O, 5.1 MnCl2�4H2O, 1.6 CuSO4�5H2O, 1.1(NH4)6Mo7O24�4H2O, 5.5 CaCl2�2H2O, 5.0 FeSO4�7H2O]. The reactortemperature (32 ± 2 �C), pH (8.0 ± 0.2), DO (1.8 mg L�1) andhydraulic retention time (2 d) were controlled.

2.2. EPS extraction and chemical analysis

The SEPS, LB-EPS and TB-EPS of biofilm samples were extractedusing the method developed by Liang et al. (2010). Approximately20 mL of fresh biofilm collected from each kind of carrier wereplaced in 50 mL centrifuge tubes, mixed with deionized water toform 45 mL suspensions and then treated by ultrasound at20 kHz and 40 W for 30 s. The suspensions were then centrifugedat 2000g and 4 �C for 15 min. The supernatant fluids were SEPS.The sediments at the bottom of the centrifuge tubes were resus-pended to their initial volume with deionised water. The suspen-sions were horizontally vibrated in a thermostat incubator for1 h and then centrifuged at 5000g and 4 �C for 15 min. The super-natant fluids were thought to be LB-EPS. The sediments wereresuspended to their initial volume with phosphate buffered salineto extract TB-EPS by heating (suspensions were heated to 80 �C for30 min). Subsequently, the suspensions were centrifuged at10,000g and 4 �C for 15 min. All supernatant fluids were filteredthrough a 0.45 lm filter and stored at �20 �C before chemical anal-ysis. The extraction tests were performed twice.

The polysaccharides content was determined by anthrone col-orimetry (Raunkjaer et al., 1994). The humic acid content and theprotein content were determined using a modified Lowry method(Frolund et al., 1995). Lastly, the DNA content was determinedwith the diphenylamine colorimetric method (Sun et al., 1999).The flocculating efficiency of EPS in a kaolin suspension was deter-mined according to Yu et al. (2009).

2.3. Carrier and biofilm property

The compositions of the materials ACF and SCP were comparedusing an FT-IR spectrophotometer (FT-IR 550II Series, Nicolet,USA). The hydrophobicity of both ACF and SCP samples was ob-tained by determining the contact angle of a single fiber as de-scribed by Dumitrascu and Borcia (2006) every 10th time for thesame sample. The volatile suspended solids (VSS) determinationwas according to the method described by Chinese NEPA (2002).The zeta-potential of EPS and biofilm cells was measured using anano-particle and zeta potential analyzer (Zetasizer Nano ZS90,Malvern, UK). The biofilms were prepared as cell suspensions,and their OD546 was adjusted to 0.1 for the zeta-potential measure-ments (Pembrey et al., 1999; Bernstein et al., 2011).

2.4. Raman and IR spectra

EPS extracts were freeze-dried at �50 �C, and the dried powdersof EPS were then analyzed using the FT-IR spectrometer and an FT-Raman spectrometer (RFS 100, Bruker, Germany). The FT-Ramanspectrometer equipped with Nd: YAG laser (k = 1064 nm). The la-ser intensity was 50 mW (sample scan 100 times), and the spectralresolution was 4 cm�1.

3. Results

3.1. Carrier and biofilm

The FT-IR spectra of the ACF and SCP carriers are shown in Fig. 1.The assignments of IR bands were according to the referenced

Fig. 1. The IR spectra of the ACF and SCP carriers.

Table 1Characteristics of biofilms growing on ACF and SCP.

Biofilm Source Zeta potential (mV) Dry weight,g g�1 carrier

Initial After extraction

ACF �14.2 �19.9 2.30 ± 0.18SCP �14.1 �16.4 4.28 ± 0.52

Table 2Zeta potential and flocculating efficiency of EPS extracted from two biofilm samples.

EPS fraction Zeta potential (mV) Flocculating efficiency (%)

ACF SCP ACF SCP

SEPS �27 �23 5.2 ± 2.7 7.3 ± 1.3LB-EPS �24 �21 3.7 ± 0.7 4.9 ± 1.9TB-EPS �14 �13 7.9 ± 1.6 8.3 ± 1.5

Fig. 2. The contents of EPS extracted from biofilms growing on ACF and SCP carrier.

Y.-P. Chen et al. / Chemosphere 92 (2013) 633–638 635

literatures (Maquelin et al., 2002; Liang et al., 2010). The largebands around 3424 and 3450 cm�1 were related to the OAHstretching mode of the hydroxyl functional groups. The bands at2940, 2919 and 2856 cm�1 were assigned to CAH stretching vibra-tions, whereas those at 1430 and 1384 cm�1 were characteristics ofCAH deformation vibrations. The intensities of OAH stretching,CAH stretching and deformation vibrations in SCP were greaterthan those in ACF. The bands at 1635 and 1637 cm�1 were attrib-uted to the C@O stretching vibrations. SCP had more functionalgroups compared with ACF. The bands in the 1016–1173 cm�1 re-gion were correlated to CAO or CAC stretching vibrations in SCP,whereas the band at 1319 cm�1 was assigned to CAOAC deforma-tion vibrations. Unsaturated bonds were found in the regions oflower than 900 cm�1. The water contact angles of ACF and SCPwere 50.7 ± 2.5� and 36.7 ± 3.1�, respectively, which indicated thatthe SCP was more hydrophilic than the ACF.

Table 1 shows the dry weight and surface charges of biofilmsgrowing on ACF and SCP. The biofilm dry weight of the SCP samplewas 4.28 g g�1 carrier, and it was larger than that of the ACF sam-ple (2.30 g g�1 carrier). The zeta potentials (negative) of biofilmcells were changed before and after the EPS extraction. The abso-lute value of zeta potential increased from 14.2 to 19.9 mV forthe ACF sample and that increased from 14.1 to 16.4 mV for theSCP sample after EPS extraction.

3.2. Characteristics and contents of EPS

Table 2 lists the zeta potentials and flocculating efficiency of theEPS fractions from the ACF and SCP biofilm samples, all of which

decreased systematically. However, the zeta potentials of SEPS,LB-EPS and TB-EPS from the SCP sample were slightly lower thanthose from the ACF sample. The flocculating experiments ofTB-EPS were performed in kaolin suspension. The flocculating effi-ciencies varied in each EPS fraction and the type of biofilm carrier.

Fig. 2 shows the contents and constituents of EPS extractedfrom both biofilms samples. The amount of EPS (total content ofSEPS, LB-EPS and TB-EPS) was 119 mg g�1 VSS in the biofilm grow-ing on ACF, with the SEPS, LB-EPS and TB-EPS fraction measuring35%, 19% and 46%, respectively. The amount of EPS from the biofilmgrowing on SCP was 181.7 mg g�1 VSS, and the fractions of SEPS,LB-EPS and TB-EPS were 36%, 14% and 51%, respectively. The re-sults revealed that the EPS extracted from the two samples hadvarious yield but similar fractions of each EPS.

As shown in Fig. 2, the polysaccharide, DNA and humic acid con-tents of SEPS from the SCP sample were higher than those from theACF sample, but the samples did not significantly differ in theirprotein content. For the LB-EPS, the polysaccharide and humic acidcontents from the SCP sample were higher than those from the ACFsample, but the samples did not differ in their protein and DNAcontents. Moreover, for the TB-EPS, the protein, polysaccharideand DNA contents from the SCP sample were higher than thosefrom the ACF sample, whereas the humic acid content from theSCP sample were lower than that from the ACF sample. Thus, thecontents and compositions of EPS were varied significantly be-tween the carriers of adherent microbial aggregates.

3.3. Raman spectra

Fig. 3 shows the Raman spectra of EPS extracted from both sam-ples. Raman bands were assigned as previously described (Chenand Lord, 1976; Tang and Fung, 1997; Maquelin et al., 2002;Schalnat et al., 2011). The bands in the region of

Fig. 3. Raman spectra of SEPS (a), LB-EPS (b) and TB-EPS (c) extracted from biofilmsgrowing on ACF and SCP carriers.

Fig. 4. IR spectra of SEPS (a), LB-EPS (b) and TB-EPS (c) extracted from biofilmsgrowing on ACF and SCP carriers.

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2851–2937 cm�1 were attributed to CAH stretching vibrations,whereas the CAH deformation vibrations were observed in the1433–1464 cm�1 region. Amide I vibrations were appeared in the re-gion of 1655–1670 cm�1, whereas amide III vibrations were detectedin the region of 1246–1267 cm�1. The bands in the 1061–1124 cm�1

region were related to the chain CAC stretching vibrations of lipids orthe CAN stretching vibrations of proteins. The bands near 1049 and715 cm�1 could be attributed to the nitrate group. The CAC stretch-ing vibrations in the region of 820–957 cm�1 were also obtained. Theband near 850 cm�1 was assigned to tyrosine, whereas the bands at619 and 1001 cm�1 were assigned to phenylalanine. A band of G(guanine) was observed at approximately 681 cm�1, whereas thedeoxyribose bands were found at approximately 950 and990 cm�1. The bands in the region of 120–140 cm�1 were attributedto the metal–molecule stretching vibrations.

Fig. 3 shows similar qualitative Raman spectra of SEPS andLB-EPS; however, some differences between them and TB-EPS weredetected. The nitrate group was presented in SEPS and LB-EPS butnot in TB-EPS. The CAC stretching vibrations in SEPS and LB-EPSsamples were assigned to random coils, but those in TB-EPSsamples were assigned to the a-helix. The vibrations of amide IIIof SEPS and LB-EPS were also assigned to a-helix. In TB-EPS, thevibrations of amide III represented the a-helix and random coils,whereas the vibrations of amide I were assigned to the a-helix.Moreover the intensities of the bands of CH2 and CH3 symmetricstretching and deformation vibrations were quite strong. Theresults showed that CH2, CH3 groups and amino structures wereabundant presented in TB-EPS.

The qualitative Raman spectra of the same EPS fraction from thetwo biofilm samples were mostly similar. In the SEPS, the amide IIIvibration at 1267 cm�1 of the a-helix was present in the ACF sam-ple but absent in the SCP sample. The Raman spectra of LB-EPS

from the SCP sample was very similar to those of LB-EPS fromthe ACF sample. For the TB-EPS, the amide III vibration at1246 cm�1 of random coils was present in the ACF sample only.

3.4. FT-IR spectra

The IR spectra of EPS obtained from the two biofilms samplesare shown in Fig. 4. The IR bands were also assigned according tothe existing literatures (Maquelin et al., 2002; Comte et al.,2006). The broad band around 3430 cm�1 was assigned to thestretching vibration of OAH. The visible weak bands at 2850–2959 cm�1 were attributed to the CAH stretching vibrations, whilethe sharp peak at 1384 cm�1 was related to the CAH deformationvibrations. The bands at 1637–1660 cm�1 were assigned to amide Ivibration of proteins, and the weak bands at 1272 and 1288 cm�1

were assigned to amide III vibration. The bands at 1000–1132 cm�1 were the CAO and CAC stretching, CAOAC and CAOAHdeformation vibrations of the polysaccharides structures. The C@Osymmetric stretching vibration of COO� was observed at1400 cm�1. Bands in the regions of lower than 1000 cm�1 couldbe assigned to the phosphate or sulfur functional groups.

Fig. 4 shows that the IR qualitative spectra of each EPS from thetwo samples were highly consistent. However there were somedifferences between SEPS, LB-EPS and TB-EPS in qualitative points.The amide I in SEPS was assigned to the a-helix and b-sheet,whereas that in TB-EPS was assigned to random coils (Tretinnikovand Tamada, 2001). The amide III vibration representing the b-turnwas present in LB-EPS but absent in SEPS and TB-EPS, and theamide I vibration in LB-EPS was assigned to the b-sheet (Cai andSingh, 1999; Tretinnikov and Tamada, 2001). In addition, the func-tional groups in TB-EPSs mainly referred to proteins andpolysaccharides.

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4. Discussion

4.1. Characteristics of EPS

Proteins were the key EPS compositions, and their fractions ran-ged from 51% to 56%, from 47% to 52% and from 43% to 57% in SEPS,LB-EPS and TB-EPS, respectively. The high protein content may beattributing to the low C/N ratio of influent (Bura et al., 1998; Yeet al., 2011). The protein/polysaccharide (PN/PS) ratios of SEPS,LB-EPS and TB-EPS were 2.9, 3.4 and 1.3, respectively, in the ACFsample. The corresponding values in the SCP sample were 2.4,2.3 and 1.9, respectively. The high PN/PS ratios in the present studywere consistent with that reported by Wang et al. (2010), and EPSsecreted by nitrifiers have much higher protein content than car-bohydrates (Liang et al., 2010). The results also suggested thatthe extracellular polysaccharides were mainly presented in theTB-EPS fraction, which were agreement with the finding of Lianget al. (2010). Furthermore, proteins and polysaccharides were ma-jor composition according to the Raman and IR spectra. The proteincontent of TB-EPS was high, and the bioflocculation ability of mi-crobes improved with increasing protein content (Sheng et al.,2010). The presence of a-helix and b-sheet structures in TB-EPS en-hanced their bioflocculation activity of TB-EPS. The zeta potentialsof TB-EPS were in the range of �12.7 to �14.3 mV, which weremuch lower than those of SEPS and LB-EPS. Indeed, flocculatingtest showed the TB-EPS had better bioflocculation activity com-pared of SEPS and LB-EPS. Research has shown that extracellularprotein and DNA played key roles in biofilm formation(Whitchurch et al., 2002; Sheng et al., 2010). The DNA fractionsin LB-EPS were 16.1% and 12.9% for ACF and SCP, respectively,which were much higher than those in SEPS (7.3% and 6.7%) andTB-EPS (1.8% and 1.3%). Liu et al. (2010) found that the LB-EPSplayed a positive role in the sludge aggregations. Similarly, Sunet al. (2009) determined that the LB-EPS had strong binding andflocculating capacity. However, as the LB-EPS content exceeded5.5 mg TOC g�1 SS, its bioflocculation capacity and effluent qualityworsened (Li and Yang, 2007). In the present study, according tothe Raman and IR spectra of LB-EPS, the b-sheet, b-turn and ran-dom coils indicated that the LB-EPS had the flocculation anddeflocculation segments (Badireddy et al., 2010). The Raman andIR spectra also indicated that the EPS contained not only such an-ions as nitrate, phosphate and sulfur functional groups, but alsometal cations. The bivalent cations were presented mainly in theSEPS fraction, whereas trivalent cations were appeared primarilyin the TB-EPS fraction (Yu et al., 2009).

4.2. Difference of EPS on two samples

The data demonstrated that the EPS yields were influenced bythe properties of the carrier to which microbes attached. This indi-cated that the amount of EPS in the SCP sample was much greaterthan that in the ACF sample. The amount of EPS could be correlatedwith the amount of biomass. Choi et al. (2001) reported that thecontents of EPS extracted from biofilm growing on four carrierswere similar, but they increased with biofilm growth. Moreover,Zhang and Bishop (2001) demonstrated that thicker biofilm tendedto produce larger EPS yields. In the present study, the dry weight ofbiofilm growing on ACF was much lower than that of biofilm grow-ing on SCP, which implied that the ACF biofilm was thinner thanthe SCP biofilm. Therefore, the larger EPS yields were detected inthe thicker biofilm growing on SCP carrier. However, the IR spectraof EPS from both samples were very similar, and the structuralinformation obtained by Raman spectroscopy showed that differ-ences between the two samples were not significant. The flocculat-ing ability of EPS enhanced with an increase in the amounts of

proteins, carbohydrates and total EPS (Badireddy et al., 2010). Forthe TB-EPS, either the total amount or the content of proteinsand polysaccharides in the SCP sample was much higher than thatin the ACF sample. The zeta potentials of TB-EPS from the SCP sam-ple was slightly lower than those of TB-EPS from the ACF sample.In addition, the flocculating test also suggested that the TB-EPSfraction of the SCP sample had better flocculating ability comparedwith that of the ACF sample.

Bacterial cells actively recognized the carrier surface and pro-duced EPS after the initial deposition (Lower et al., 2001), whichled to the alterations in surface properties such as zeta potentialand hydrophobicity, and promoted the adsorption of cells (Liuet al., 2010). Therefore, surface properties would affect the cells’initial attachment, EPS production and biofilm formation. Our pre-vious study demonstrated that the hydrophilic surface is more pro-pitious to microbial enrichment in the CANON system, in thepresent study; the hydrophilic surface of SCP also obtained signif-icantly higher biomass and more EPS. Conversely, the higher yieldof EPS would promote the biofilm growth.

5. Conclusions

The finding related to bioflocculation activity revealed byRaman spectroscopy and FT-IR spectroscopy was consistent withthe result of flocculating test. The SCP was more hydrophilic com-pared with the ACF, and it obtained a significantly higher biomass.The same EPS fraction from both carriers had similar functionalgroups. For the TB-EPS, the total amount and content of proteinsand polysaccharides in the SCP sample were much higher thanthose in the ACF sample. Moreover, the TB-EPS fraction of theSCP sample had better bioflocculation activity compared with thatof the ACF sample. These findings indicate that carriers can poten-tially affect the production and characteristics of EPS.

Acknowledgements

The authors gratefully acknowledge the financial support of theNatural Science Foundation of China (51108482 and 51278509)and the Research Fund for the Doctoral Program of Higher Educa-tion of China (2010019120035).

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