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Biosorption properties of extracellular polymeric substances (EPS) resulting from activated sludge according to their type: Soluble or bound
Process Biochemistry 41 (2006) 815–823 www.elsevier.com/locate/procbio
Biosorption properties of extracellular polymeric substances (EPS) resulting from activated sludge according to their type: Soluble or bound Sophie Comte, Gilles Guibaud *, Michel Baudu Laboratoire des Sciences de l’Eau et de l’Environnement, Faculte´ des Sciences, 123 Avenue Albert Thomas, 87060 LIMOGES Cedex, France Received 15 April 2005; received in revised form 11 October 2005; accepted 12 October 2005
Abstract Extracellular polymeric substances (EPS) are one of the main components of activated sludge. EPS can be found in two forms, soluble or bound depending on their localisation and/or their role in microbial metabolism. In this study, soluble and bound EPS are operationally defined: soluble EPS, which can be extracted by centrifugation alone, and bound EPS in floc biomass which require additional treatments for extraction. The two kinds of EPS extracted were characterized by their organic fractions, their carbon, nitrogen and phosphorus contents, their biochemical composition and their pKa and PEC (protonic exchange capacity) values. Organic carbon content of EPS underlined qualitative differences between soluble and bound EPS, which were better established by polysaccharide content. At pH 7, whatever the EPS considered, the analysis of all biosorption results showed an EPS affinity for metals in descending order: Cu2+ > Pb2+ > Ni2+ Cd2+. The study of EPS biosorption properties showed different behaviour for soluble and bound EPS depending on the metal studied. For Cu2+ (only for EPS A), Pb2+ and Ni2+, soluble EPS showed stronger biosorption properties than bound EPS. For Cd2+ and Cu2+ (case of EPS B), biosorption properties of the two kinds of EPS studied were close due to the weak affinity of Cd2+ for EPS and the different possible binding mechanisms implicated by the speciation of Cu2+ at pH studied. At pH 7, due to the PEC value and pKa, the numbers of binding dissociated sites, could be assumed greater for soluble EPS than for bound EPS and could explain in part the biosorption results obtained. We can guess that soluble EPS play the role of a protective barrier against toxic metals for the microorganisms in the activated sludge flocs. # 2005 Elsevier Ltd. All rights reserved. Keywords: Biosorption; Extracellular polymeric substances; Metal; Sludge
1. Introduction The activated sludge process is commonly used in wastewater treatment. This process is based on organic pollutant degradation by bacteria contained in flocs, under aerobic conditions. The presence of heavy metals in wastewater is of interest because of their known toxic effects on the receiving environment and also on the performance of biological waste treatment processes [1]. The activated sludge flocs are composed of microorganisms, extracellular polymeric substances (EPS), colloids, mineral particles and ionic components such as divalent cations [2]. EPS are biopolymers resulting from active bacterial secretion, shedding of cell surface material, cell lysis materials
* Corresponding author. Tel.: +33 5 55 45 73 67; fax: +33 5 55 45 72 03. E-mail addresses: [email protected] (S. Comte), [email protected] (G. Guibaud), [email protected] (M. Baudu). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.10.014
and from adsorption of organics from the environment [3]. They are composed of a variety of organic substances: carbohydrates and proteins being major constituents with humic substances, uronic acids and nucleic acids in smaller quantities [4,5]. Many authors have defined two kinds of EPS in flocs or biofilms: soluble and bound EPS. Bound EPS include microbially produced bound polymers but also lysis and hydrolysis products as well as absorbed or attached matter [6]. Soluble EPS include microbially produced soluble polymers, hydrolysis products of attached organic matter and organic molecules released by cell lysis. EPS are located at/or outside the cell surface independent of their origin. In biofilms or activated sludges, EPS may be only loosely attached to the cell surface as peripheral capsules (called bound EPS) and can be shed into the surrounding environment as a less organized (amorphous) slime (called soluble EPS) [3]. In flocs or biofilms, bacteria are embedded in EPS (that is to say bound EPS are closely associated with the cells), while soluble EPS do not
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have any direct contact with the cells. In activated sludge, bound EPS on the floc biomass are composed of sheaths, capsular polymers, condensed gel, loosely bound polymers and attached organic material and soluble EPS with soluble macromolecules, colloids and slimes [7,8]. To differentiate the two kinds of EPS, a theory based on metabolism of bacterial cells and electron flux has been suggested [8]. Soluble EPS are defined as Soluble Microbial Products (SMP) divided in two categories: substrate-utilization-associated products (UAP), which are produced directly during substrate metabolism, and biomass-associated products (BAP), which are formed from biomass. If we consider the ‘‘unified theory’’ of [8], soluble EPS are actively secreted by bacteria and are biodegradable and bound EPS are polymers bound to active and inert biomass or are molecules resulting from cell lysis. Ref. [9] discuss this issue and shows that EPS cannot be the only source of BAP. These authors suggest that EPS in nonhydrolyzed form may indeed constitute part of the high molecular weight SMP. However, cell lysis may also result in the release of intracellular high molecular weight SMP, and these compounds are also a source of BAP [9]. Besides EPS, its hydrolysis products, and other intracellular compounds released as a result of cell lysis, SMP may also be comprised of more specific compounds that could be deliberately excreted by microorganisms to play a specific role in metal mitigation, and such organics would be classified as UAPs according to the SMP theory. In conclusion, the origin of EPS is complex and different approaches are thus possible to define soluble and bound EPS. In experiments, EPS are defined rather by the method used to separate/extract the EPS than by a theoretical consideration of the physical state of the polymers [3]. In biofilms or activated sludge systems, EPS can be found in two operationally defined forms: soluble EPS which can be extracted by centrifugation alone and bound EPS, on the floc biomass, which require additional treatment for their extraction [3]. The most important functions of EPS are adhesion to surfaces, aggregation of bacterial cells in flocs, stabilization of the floc structure, formation of a protective barrier that provides resistance to biocides or other harmful effects, retention of water, sorption of exogenous organic compounds for the accumulation of nutrients from the environment, and accumulation of enzymatic activities, such as digestion of exogenous macromolecules for nutrient acquisition, aiding the cells in uptaking metal nutrients [3,10,11]. In fact, the EPS matrix is a medium that allows cooperation and communication between cells resulting in microbial aggregates [8]. Several studies have shown that EPS also play a crucial role in biosorption of heavy metals [12,13,10]. Moreover, in the presence of a heavy metal (chromium), the biomass seemed to have produced more EPS material (principally SMP or soluble EPS), indicating that this might have helped it cope with the metal stress [9]. The aim of this study is to compare biosorption properties of soluble EPS and bound EPS towards metallic cations (Cu2+, Pb2+, Ni2+, Cd2+), taking into account the physico-chemical characteristics of both EPS types.
2. Material and methods 2.1. Activated sludge samples and EPS extraction Extraction was carried out on activated sludges obtained from the aeration tanks of two wastewater treatment plants (WWTP) (called A and B). The WWTPs A and B present different characteristics. The treatment capacity in inhabitant equivalent is 285,000 and 4000, respectively for sludges A and B. The organic loads are between 0.24 and 0.30 and between 0.13 and 0.16 kg BOD5/m3/day respectively for A and B sludge. The volatile suspended solid of sludge A and B were respectively 68% and 72% of the total suspended solid of sludges. The sludge volume index (SVI) of sludge A and B were respectively 90 and 160 mL g1 of TSS. Before the extraction step, sludges were concentrated at 4300 g for 10 min at 4 8C. More over, this step remove the supernatant of sludges which contain minerals ions, the remains of organic matter from effluent and probably very soluble EPS very easily released by sludges. The residues were recovered and suspended in ultra-pure water. Two procedures were used for EPS extraction from each sludge (A and B). The first consisted of a centrifugation at 4000 g for 20 min at 4 8C, allowing soluble EPS to be extracted [14]. The second was a 40 W sonication for 2 min associated with a cationic exchange resin (Dowex 50X8) treatment, followed by two ultracentrifugations (20,000 g for 20 min at 4 8C then 10,000 g for 15 min at 4 8C) [15]. The EPS extracted with the latter protocol were called bound EPS but this procedure in fact extracted both soluble and bound EPS. Extracted EPS from both methods were finally purified in ultra-pure water with a dialysis membrane (3500D, Cellu Sep). The EPS were stored at 18 8C before being used.
2.2. EPS characterization The main characteristics of extracted EPS were determined: dry weight content (DW) (at 105 8C); volatile dry weight content (VDW) (at 550 8C); total organic carbon (TOC) and Nitrogen content (using a Dorhmann Apollo 9000 TOC-meter equipped with a nitrogen analyser); total phosphorus content (using the European standard EN 1189, 2001). The biochemical composition (protein, humic acids, polysaccharide, uronic acid, lipid, nucleic acid content was defined using colorimetric methods as described by [15,16].
2.3. Infra-red (IR) spectrometry One milligram of freeze-dried EPS solution was mixed with 180 mg of KBr and compacted to form a pellet. The IR-spectra were then determined using a spectrum 1000 (Perkin-Elmer).
2.4. pKa determination The determination of the pKa for the EPS solutions was not possible by simple acid–base titration. According to [15], a method previously developed for solids chemistry was modified and applied to EPS in order to determine the pKa of the EPS. The acid–base surface reactions are described only by the law of mass action based on the protonation (under the form: S–OH and S–OH2+) of the surface functional groups (S–O) and determined by analogy with amphoteric compounds. According to Eq. (1), the total number of surface [S]tot sites was ½Stot ¼ ½SOH2 þ þ ½SOH þ ½SO ¼ PEC
(1)
with [S]tot: total number of surface sites, PEC: protonic exchange capacity, Note that the PEC, is the experimental determination by potentiometric titration of the total number of surface [S]tot (see below), that is to say the total number of sites potentially active for metal complexation [17]. SOH2 þ , SOH þ Hþ
(2)
S. Comte et al. / Process Biochemistry 41 (2006) 815–823 SOH , SO þ Hþ with : Ka1 ¼
Ka2 ¼
½Hþ ½SOH ½SOHþ 2
½Hþ ½SO ½SOH
(3)
3. Results
(4)
3.1. EPS characterization
(5)
In this study, the determination of both the EPS surface charge (Q) and the EPS protonic exchange capacity (PEC) were carried out by potentiometric titration. A solution of EPS (25 mL) was placed in a 22 8C thermostated cell. Titration was carried out adding NaOH or HNO3 (0.01 M) with an automatic titrator, Metrohm 716 DMS, coupled to a Metrohm 727 Ti Stand and equipped with a pH electrode (pH 0–14/0; 80 8C; KCl 3 M). Finally, evolution of Q parameter calculated from acidic or alkaline titration curve of EPS and PEC value allowed two Ka of EPS solutions (Ka1 and Ka2 ) to be calculated for the predominant functional groups. Ka1 and Ka2 were expressed under their logarithmic forms that is to say respectively pKa1 and pKa2 .
2.5. Metal biosorption of EPS The determination of the biosorption parameters at 22 2 8C and pH 7 was carried out by measuring the free metal content in the polymer solution after addition of Cu2+, Pb2+, Ni2+ or Cd2+ (under nitrate form at concentration of 102 M). Polarographic methods could be useful tools to investigate metal biosorption by biological materials such as bacteria [18]. According to [19,20], investigations on free metals were carried out by polarography, using the static mercury dropping electrode mode (SMDE). A 626 polarecord polarograph coupled to a 663 VA metrohm stand was used (time of drop fall: 1 s). Operating conditions were as follow: In the measuring cell at 22 2 8C: 10 mL of ultra-pure water; 10 mL of HEPES buffer at pH 7 (Sigma-Aldrich) 101 M. HEPES buffer is recognised as a non-complexant buffer and can be used as a supporting electrolyte [21]. 1 mL of KNO3 (Flucka) 1 M as the electrolyte; addition of quantities of metallic solution (from 20 to 50 mL) at a concentration of 102 M at pH 4; 1 ml of EPS. Potential range scanned for free metal determination Cu2+: 0.10 to 0.20 V; Pb2+: 0.20 to 0.50 V; Cd2+: 0.45 to 0.75 V; Ni2+: 0.65 to 1.15 V. Polarographic titration consists in measuring the evolution of free metal content in the solution of EPS after each addition of metal. The analysis of the curves of the polarographic titration allowed, from Ruzic’s modelling, the different parameters characteristic of the metal binding by EPS to be obtained: the concentration of ligands (or quantity of potentially active sites) and the conditional constants of formation of the complex [22]. The theoretical study of the complexation equilibrium associated to the law of mass action can be described as follows, assuming the formation of a 1:1 complex. M þ L , ML
(6)
½ML ½M½L
(7)
K¼
with M: free metal, L: free ligand (EPS), ML: ligand–metal complex, K: conditional binding constant. The number of sites initially occupied by the metals studied was not taken into account for the determination of the total number of sites in EPS as it was negligible (<1% total complexation determined).
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In order to define soluble (extracted by centrifugation alone) and bound EPS (extracted by additional treatment), the quantity of EPS extracted, the carbon content, the nitrogen content, the phosphorus content and the biochemical composition were compared. The extraction yields of EPS according to the extraction protocol used are presented in Fig. 1. For both sludge samples (A and B), EPS amounts extracted by centrifugation alone (soluble EPS) were less than 1% whereas those extracted by sonication and cationic resin (bound EPS) were about 3%. Due to the extraction method used, EPS obtained from the second procedure were composed of both bound and soluble EPS. We can assume that real bound EPS is about 70% of the bound fraction extracted. This means that real bound EPS are predominant in the bound EPS extraction and justifies the denomination of bound EPS. In biofilms, the proportion of EPS can vary between roughly 50% and 80% of the total organic matter content [6] and the proportion of EPS can be affected by the age of the biofilm [23]. Thus the bound EPS fraction extracted in this study is low (about 3%) when compared to the total amount of EPS present in biofilms. Physical means (ultracentrifugation, cationic exchange resin, sonication, . . .) used to extract EPS are recognized as giving low extraction yields [24] but do not affect the quality of extracted EPS [25]. Information on the general characterization of EPS such as the carbon, nitrogen and phosphorus contents as well as the organic mass fractions of EPS (expressed in % of VDW/DW) is given in Table 1. EPS are mainly composed of an organic fraction (the VDW/DW ratio of EPS varies from 69% to 82%). The nitrogen values varied from 84 to 97 mg N g1 of EPS VDW, and were close for both soluble and bound EPS. EPS N contents came from the organic fraction as it was verified that NO3, NO2 and NH4+ were not present in EPS solution. The organic carbon values varied from 184 to 267 mg C g1 of EPS VDW, the amounts of organic carbon being higher for soluble EPS than for bound EPS and
Fig. 1. Extraction yield of EPS for sludge A and B (% EPS VDW/sludge VSS).
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Table 1 Main characteristics of EPS for sludge A and B according to their origin Soluble
Bound
A
B
A
B
82 5 93 5 241 12 2.6 24 1
74 4 97 5 267 13 2.7 19 1
69 3 84 4 184 9 2.2 28 2
70 4 91 5 228 11 2.5 18 1
Biochemical composition of EPS in mg g1 EPS VDW Proteins 385 15 Polysaccharides 207 17 Humic acids 77 4 Uronic acids 25 1 Lipids <39 Nucleic acids 22 2 Protein/polysaccharide 1.9
334 13 210 17 201 10 80 4 <33 69 7 1.6
365 15 149 12 182 9 69 3 <20 73 7 2.4
381 15 162 13 224 11 71 4 <22 51 5 2.4
underlined qualitative differences between soluble and bound EPS. Moreover, the C/N ratio varied from 2.6 to 2.7 and from 2.2 to 2.5 for soluble and bound EPS, respectively and show a slight qualitative difference between both sorts of EPS. The phosphorus content, did not underline any difference between soluble and bound EPS (values varied from 18 to 28 mg P g1 of EPS DW). The biochemical composition of EPS is also detailed in Table 1. The nucleic acid content is used as an indicator of cellular lysis during the extraction process [24] and its values varied from 22 to 73 mg g1 of EPS VDW (corresponding to 0.2–2.1 mg g1 of sludge VSS (volatile suspended solid)). No contamination of EPS by the intracellular materials was noted when comparing the above results with the literature data [24]. More over, the nucleic acid content of EPS A and B varied according to the sludges origin according to Sponza [5], but also with the EPS location in the sludges (Table 1). Nevertheless, Table 1 shows that the amounts of nucleic acids are higher for soluble EPS than for bound EPS for sludge A, but as to sludge B, that is contrary, but it is hard to explain evolution of these values.
Proteins and polysaccharides formed the biggest fraction: from 334 to 385 mg proteins g1 of EPS VDW, and from 149 to 210 mg polysaccharides g1 of EPS VDW. The protein values were close for all EPS extracted whereas the highest polysaccharide contents referred to soluble EPS solutions. Protein/polysaccharide ratios were 1.9 and 1.6 for soluble EPS and 2.4 for bound EPS. Ref. [24] have shown that the protein:polysaccharide ratio for activated sludge EPS varies over a wide range: from 0.5 to 21.2. The ratios determined in this study (Table 1) are low and are in accordance with the ones referenced by [24] and confirm that the EPS extracted in this study were not contaminated by intracellular materials from bacteria of activated sludge flocs. Biochemical characterization (Table 1) confirmed that there were qualitative differences between soluble EPS and bound EPS. IR spectra of soluble and bound EPS from sludge sample B are presented in Fig. 2. The spectra of both EPS are similar and indicate the presence of the same functional groups mentioned in Table 2. Several intense characteristic bands can be attributed to protein and polysaccharide functional groups. These different
General characterization of EPS VDW/DW (%) EPS VDW (mg N g1) mg C g1 EPS VDW C/N ratio mg P g1 EPS DW
Fig. 2. IR spectra of soluble and bound EPS (sample B).
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Table 2 Main functional groups observed from IR-spectra of soluble and bound EPS Wave number (cm1)
Vibration type
Functional type
3200–3420 2930–2935 1640–1660 1550–1560 1450–1460 1400–1410
Stretching vibration of OH Asymmetric stretching Vibration of CH2 Stretching vibration of C O and C–N (Amide I) Stretching vibration of C–N and deformation vibration of N–H (Amide II) Deformation vibration of CH2 Stretching vibration of C O Deformation vibration of OH Deformation vibration of C O Stretching vibration OH Stretching vibration C–O–C Stretching vibration of OH fingerprint zone Several bands visible
OH in polymeric compounds
1235–1245 1130–1160 1040–1080 <1000
functional groups observed agree with results of [15,16] and are in accordance with the EPS biochemical composition. 3.2. EPS biosorption site characterization The protonic exchange capacities (PEC) and the pKa values are presented in Table 3. No significant difference was noted between the pKa1 and pKa2 for the soluble and bound EPS: pKa1 values varied from 6.1 to 6.6 and pKa2 values varied from 8.4 to 9.2. According to the IR-spectra, the EPS composition and the literature data [4,15,16], pKa1 is characteristic of carboxylic and phosphoric functional groups and pKa2 is attributed to phenolic and amino functional groups. In EPS solution, the pKa of the carboxyl/ phosphoric site was determined at about 6 and that of the amine/ phenolic site about 9.6 [4]. The PEC represents an estimation of EPS total binding sites [17]. The PEC value of EPS was 4000 mmol g1 EPS DW and varied from 2400 to 2500 mmol g1 EPS DW for soluble and bound EPS, respectively. The capacities for exchange of protonic ions are greater for soluble EPS than for bound EPS. The carboxylic and phosphoric groups identified with pKa1 values could be implicated in the binding of metal ions by EPS at pH 7 [26,4]. As a consequence, a different biosorption ability for the two types of EPS could be expected. 3.3. EPS biosorption properties The EPS binding constants (expressed as log K) and the numbers of EPS fixation sites obtained with the polarographic
Table 3 Physico-chemical characterization of EPS from sludge A and B Soluble
pKa1 pKa2 PEC (mmol g1 EPS DW)
Bound
A
B
A
B
6.1 0.1 9.0 0.1 4000 100
6.6 0.1 9.2 0.1 4000 100
6.1 0.1 8.4 0.1 2400 50
6.2 0.1 9.1 0.1 2500 50
Proteins (peptidic bond) Proteins (peptidic bond) Carboxylates Alcohols and phenols Carboxylic acids Phenols Polysaccharides Phosphate or sulphur functional groups
titration curves and by Ruzic’s modelling are presented respectively in Figs. 3 and 4. Whatever the EPS (soluble, bound) and the metal considered (Cu2+, Pb2+, Ni2+, Cd2+), the binding constant in Fig. 3 did not show significant differences in the strength of binding formed between the different EPS and metals by the polarographic method used. The number of fixation sites in Fig. 4 varied for the soluble and bound EPS. This parameter is more sensitive for demonstrating differences in EPS biosorption properties depending on EPS types. The number of fixation sites of both EPS towards Cu2+ varied from 4114 to 5795 mmol g1 of DW EPS. Soluble EPS A showed better biosorption abilities than bound EPS B for Cu2+ instead of the biosorption abilities of both EPS B were close. The binding values of both EPS towards Pb2+ varied from 2051 to 3131 mmol g1 of DW EPS. The results with Pb2+ varied over a smaller range than these with Cu2+. For Pb2+ with EPS A and B, soluble EPS had a higher number of fixation sites than those of bound EPS. The number of fixation sites of soluble EPS towards Ni2+ varied from 2086 to 3120 mmol g1 of DW EPS and those of bound EPS varied from 486 to 917 mmol g1 of DW EPS. Soluble EPS exhibited stronger biosorption properties than bound EPS for Ni2+. The number of fixation sites varied by a factor of four between soluble and bound EPS. As for the biosorption results for Cd2+, the values for soluble EPS varied from 251 to 289 mmol g1 of DW EPS and those for bound EPS varied from 295 to 426 mmol g1 of DW EPS. For Cd2+, bound EPS and soluble EPS exhibited close metal binding properties. The EPS biosorption affinity towards Cd2+ was very weak which made it difficult to find any significant difference between the EPS studied. To summarize, whatever the EPS considered, the analysis of all metal binding sites showed an EPS affinity for metals in the following order: Cu2+ > Pb2+ > Ni2+ Cd2+ which is in accordance with the literature data. Several published values predict the same affinity of metal to EPS for organo-metal complex formation [27,15].
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Fig. 3. Binding constant expressed as log K for soluble and bound EPS according to the metal studied.
4. Discussion The results showed that soluble EPS has a greater capacity to exchange protonic ions than bound EPS. The PEC parameter can be used to evaluate the capacity of EPS to bind ligands [17]. The greater the PEC the more the EPS are able to bind cationic ligands. Due to the pKa1 value and PEC (Table 3), we can assume that the number of dissociated sites of soluble EPS at pH 7, is greater than that of bound EPS. This difference between soluble and bound EPS expressed by the number of dissociated sites could explain the biosorption abilities of the type of EPS toward metals. The numbers of binding sites of EPS towards Cu2+ (only EPS A), Pb2+ and Ni2+ are, in fact, greater for soluble EPS than for bound EPS. Moreover, pKa1 is characteristic of functional groups, carboxylic and phosphoric groups would. pKa2 is attributed
to phenolic and amino functional groups. The carboxylic and phosphoric groups would therefore seem to play a major role in the metal binding at pH 7. Nevertheless, it can be seen that the number of binding sites with Cu2+ is superior to the potential total number of sites (PEC) at pH 7, so different mechanisms could be implicated in Cu2+ biosorption: exchange with protons but also electrostatic interactions and precipitation [28]. The speciation of Cu2+ at pH 7 could influence the evaluation of the EPS biosorption abilities. The study of EPS binding properties showed different behaviour between soluble and bound EPS depending on the metal considered. For Cu2+ (only for EPS A), Pb2+ and Ni2+, soluble EPS exhibited stronger binding properties than bound EPS. The extraction protocol for bound EPS resulted in bound EPS being in fact composed of soluble and bound EPS, so the
S. Comte et al. / Process Biochemistry 41 (2006) 815–823
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Fig. 4. Number of fixation sites (in mmol/g of EPS DW) depending on the type of EPS and the metal studied.
metal binding results for Cu2+ (only for EPS A), Pb2+ and Ni2+ highlight that the contribution of bound EPS to the biosorption affinity was even less important than that of soluble EPS. Soluble EPS are more reactive towards metallic cations, except Cd2+ and Cu2+ (case of EPS B).
The composition of EPS has shown that soluble and bound EPS are mainly composed of proteins and polysaccharides. More precisely, polysaccharide content is greater for soluble EPS than for bound EPS. Moreover, this study showed that biosorption ability of soluble EPS is greater than that of bound
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EPS for Cu2+ (only for EPS A), Pb2+ and Ni2+. Ref. [29] stated that different metal adsorption sites appear to exist on neutral polysaccharides and anionic polysaccharides. Neutral polysaccharides may bind metal cations to the hydroxyl groups of hexose or pentose molecules and anionic polysaccharides may bind metal cations to their carboxyl groups. The biosorption ability of polysaccharides could explain in part why the number of fixation sites is greater for soluble EPS than for bound EPS for Cu2+ (in the case of EPS A), Pb2+ and Ni2+. A function frequently attributed to EPS is their general protective effect against toxic compounds [24]. The sorption of heavy metals is part of a protection strategy against toxic effects [30]. It has been shown by a large number of studies that the type and amount of EPS produced by microorganisms are influenced by environmental conditions [3]. When there are changes in the growth medium, such as the presence or not of Cu, cells of Pseudomonas aeruginosa respond by producing EPS which have large Cu binding capacities [31]. The EPS synthesis by cells resistant to Cu showed elevated Cu binding ability (320 mg g1 EPS) compared to that produced by cells sensitive to Cu presence (270 mg g1 EPS). Moreover, biofilms in the presence of Cu have a high EPS to cell ratio, suggesting that EPS production may provide an important defence strategy against toxic effects, perhaps through sequestration of toxic copper ions [32]. Furthermore, according to [9], the SMP (soluble microbial product or soluble EPS according to the ‘‘unified theory’’ of [8] accumulation in anaerobic sludge from chemostat seems to be proportional to the dose of toxicant (chromium). According to the results of this study and the literature, the chemical nature of soluble EPS seems to be more adapted to protecting bacterial cells against metallic pollution. Qualitative differences such as carbon and polysaccharide contents, C/N ratio and PEC have been underlined between soluble and bound EPS. The two types of EPS could not perform the same functions. Bound EPS are loosely attached to the cell surface whereas soluble EPS are situated in the surrounding environment of bacterial cells. Depending on their location, soluble EPS form a first protective barrier that prevents any harmful effects of heavy metals on floc microorganisms. Moreover, soluble EPS are actively secreted by microorganisms [8]. The bacteria could synthesize soluble EPS to protect themselves against harmful effects of toxic metals. Active secretion of soluble EPS supports the hypothesis that soluble EPS play a major role in the protective strategy of microorganisms in flocs against the effects of heavy metals. However, the potential role of EPS (bound and/or soluble) in aiding the cells in uptaking metal nutrients (such as Cu, Ni, . . .) could be considered. For instance, only a mechanism to scavenge iron has been discovered for aerobic bacteria [11] and involves the excretion of siderophores into the bulk solution. However, as siderophores are deliberately excreted by cells, they are not of extracellular origin, and should be referred to as specific SMP rather than soluble EPS [8,9]. 5. Conclusion Soluble EPS gave highest results for carbon content and to a lesser extend C/N ratio which underlines that there were
qualitative differences between soluble EPS and bound EPS. Both of the EPS studied were comprised mainly of proteins and polysaccharides, the concentration of polysaccharides being higher for soluble EPS than bound EPS. Biochemical characterization results also indicated that there were qualitative differences between the two kinds of EPS. The analysis of the biosorption results of the two types of EPS showed an EPS affinity for metals in the following sequence: Cu2+ > Pb2+ > Ni2+ Cd2+. At the pH of the study, pH 7, carboxylic and phosphoric groups characterized with pKa1 values could have been implicated in the binding of metal ions by EPS through their protonic exchange ability. The numbers of total potential binding sites estimated by the PEC indicated another qualitative difference between soluble and bound EPS and revealed that different mechanisms could be implicated in the metal biosorption of EPS, principally in the case of Cu2+. However, the study of EPS biosorption properties points out the difference in behaviour between soluble and bound EPS. For Cu2+ (only for EPS A), Pb2+ and Ni2+, the biosorption abilities were greater for soluble EPS than those for bound EPS. The ability of polysaccharides to bind metallic cations could be involved in the greater biosorption abilities of soluble EPS in comparison with bound EPS. In this study, soluble EPS were the most reactive EPS towards the metallic cations studied (Pb2+, Ni2+, . . .), therefore, they may play a role as a microbial protective barrier. Acknowledgement This study was supported by a research grant provided by the Conseil Regional du Limousin. References [1] Arican B, Gokcay F, Yetis U. Mechanistics of nickel sorption by activated sludge. Process Biochem 2002;37:1307–15. [2] Urbain V, Block JC, Manem J. Bioflocculation in activated sludge: an analytic approach. Water Res 1993;27:829–38. [3] Wingender J, Neu TR, Flemming HC. Microbial extracellular polymeric substances: characterisation, structure and function. Berlin: Springer, 1999. p. 123. [4] Liu H, Fang HP. Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data. Biotechnol Bioeng 2002;80:806–11. [5] Sponza DT. Extracellular polymer substances and physicochemical properties of flocs in steady and unsteady-state activated sludge systems. Process Biochem 2002;37:983–98. [6] Nielsen PH, Jahn A, Palmgren R. Conceptual model for production of exopolymers in biofilms. Water Sci Technol 1997;36:11–9. [7] Hsieh KM, Murgel GA, Lion LW, Shuler ML. Interactions of microbial biofilms with toxic trace metals: observation and modelling of cell growth, attachment, and production of extracellular polymer. Biotechnol Bioeng 1994;44:219–31. [8] Laspidou CS, Rittmann BE. A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water Res 2002;36:2711–20. [9] Aquino SF, Stuckey DC. Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds. Water Res 2004;38: 255–66.
S. Comte et al. / Process Biochemistry 41 (2006) 815–823 [10] Ledin M. Accumulation of metals by microorganisms-processes and importance for soil systems. Earth-Sci Rev 2000;51:1–31. [11] Andrews SC, Robinson AK, Rodrguez-Quinones F, Rodriguez-Quinone SF. Bacterial iron homeostasis. FEMS Microbiol Rev 2003;27: 215–37. [12] Brown MJ, Lester JN. Metal removal in activated sludge: the role of bacterial extracellular polymers. Water Res 1979;13:817–37. [13] Brown MJ, Lester JN. Role of bacterial extracellular polymers in metal uptake in pure bacterial culture and activated sludge-I. Water Res 1982; 16:1539–48. [14] Higgins MJ, Novak JT. Characterization of exocellular protein and its role in bioflocculation. J Environ Eng Am Soc Civil Eng 1997;123:479– 85. [15] Guibaud G, Tixier N, Bouju A, Baudu M. Relation between extracellular polymers composition an dits ability to complex Cd, Cu and Pb. Chemosphere 2003;52:1701–10. [16] Guibaud G, Comte S, Bordas F, Dupuy S, Baudu M. Comparison of the complexation potential of extracellular polymeric substances (EPS), extracted from activated sludges and produced by pure bacteria strains, for cadmium, lead and nickel. Chemosphere 2005;59:629–38. [17] Nowack B, Behaviour of EDTA in groundwater—a study of the surface reactions of metal–EDTA complexes, Dissertation ETH Zurich No. 11392, 1996. [18] Savvaidis I, Hughes MN, Poole RK. Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions. Van Leeuw J Microbiol 2003;84:99– 107. [19] Perret S, Morly C, Cromer MR, Vittori O. Polarographic study of the removal Cd(II) Pb(II) from dilute aqueous solution by a synthetic flocculant comparison with Cu(II) and Ni(II). Water Res 2000;34: 3614–20. [20] Guibaud G, Tixier N, Bouju A, Baudu M. Use of a polarographic method to determine copper, nickel and zinc constants of complexation by extracellular polymers extracted from activated sludge. Process Biochem 2004;39:833–9.
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[21] Witter AE, Malbury SA, Jones AD. Copper II complexation in northern California rice field waters: an investigation using differential pulse anodic stripping electrode voltammetry. Sci Total Environ 1998;212:21–37. [22] Ruzic I. Trace metal complexation at heterogeneous binding sites in aquatic systems. Mar Chem 1996;53:1–15. [23] Zhang T, Fang HHP. Quantification of extracellular polymeric substances in biofilms by confocal laser scanning microscopy. Biotechnol Lett 2001;23:405–9. [24] Liu Y, Fang HP. Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge. Crit Rev Environ Sci Technol 2003;33:237–73. [25] Comte S, Guibaud G, Baudu M. Relations between extraction protocols of the activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS Part II. Consequences on EPS complexation properties. Enzyme Microb Technol 2006;38:246–52. [26] Singh S, Pradhan S, Rai LC. Metal removal from single and multimetallic systems by different biosorbent materials as evaluated by differential pulse anodic stripping voltammetry. Process Biochem 2000;36:175–82. [27] Jang A, Kim SM, Kim SY, Lee SG, Kim IS. Effect of heavy metals (Cu, Pb, and Ni) on the compositions of EPS in biofilms. Water Sci Technol 2001;43:41–8. [28] Buffle J, Hovai G, In situ monitoring of aquatic systems. In: Wiley Europe (Eds.). Chemical analysis and speciation. 2000. [29] Brown MJ, Lester JN. Role of bacterial extracellular polymers in metal uptake in pure bacterial culture and activated sludge-II. Water Res 1982; 16:1549–60. [30] Decho AW. Microbial biofilms in intertidal systems: an overview. Cont Shelf Res 2000;20:1257. [31] Kazyl SK, Sar P, Singh SP, Sen AK, D’Souza SF. Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis, chemical nature and copper binding. World J Microbiol Biotechnol 2002;18:583–8. [32] Keevil CW. The physico-chemistry of biofilm-mediated pitting corrosion of copper pipe supplying potable water. Water Sci Technol 2004;49:91–8.
Biosorption properties of extracellular polymeric substances (EPS) resulting from activated sludge according to their type: Soluble or bound Sophie Comte, Gilles Guibaud *, Michel Baudu Laboratoire des Sciences de l’Eau et de l’Environnement, Faculte´ des Sciences, 123 Avenue Albert Thomas, 87060 LIMOGES Cedex, France Received 15 April 2005; received in revised form 11 October 2005; accepted 12 October 2005
Abstract Extracellular polymeric substances (EPS) are one of the main components of activated sludge. EPS can be found in two forms, soluble or bound depending on their localisation and/or their role in microbial metabolism. In this study, soluble and bound EPS are operationally defined: soluble EPS, which can be extracted by centrifugation alone, and bound EPS in floc biomass which require additional treatments for extraction. The two kinds of EPS extracted were characterized by their organic fractions, their carbon, nitrogen and phosphorus contents, their biochemical composition and their pKa and PEC (protonic exchange capacity) values. Organic carbon content of EPS underlined qualitative differences between soluble and bound EPS, which were better established by polysaccharide content. At pH 7, whatever the EPS considered, the analysis of all biosorption results showed an EPS affinity for metals in descending order: Cu2+ > Pb2+ > Ni2+ Cd2+. The study of EPS biosorption properties showed different behaviour for soluble and bound EPS depending on the metal studied. For Cu2+ (only for EPS A), Pb2+ and Ni2+, soluble EPS showed stronger biosorption properties than bound EPS. For Cd2+ and Cu2+ (case of EPS B), biosorption properties of the two kinds of EPS studied were close due to the weak affinity of Cd2+ for EPS and the different possible binding mechanisms implicated by the speciation of Cu2+ at pH studied. At pH 7, due to the PEC value and pKa, the numbers of binding dissociated sites, could be assumed greater for soluble EPS than for bound EPS and could explain in part the biosorption results obtained. We can guess that soluble EPS play the role of a protective barrier against toxic metals for the microorganisms in the activated sludge flocs. # 2005 Elsevier Ltd. All rights reserved. Keywords: Biosorption; Extracellular polymeric substances; Metal; Sludge
1. Introduction The activated sludge process is commonly used in wastewater treatment. This process is based on organic pollutant degradation by bacteria contained in flocs, under aerobic conditions. The presence of heavy metals in wastewater is of interest because of their known toxic effects on the receiving environment and also on the performance of biological waste treatment processes [1]. The activated sludge flocs are composed of microorganisms, extracellular polymeric substances (EPS), colloids, mineral particles and ionic components such as divalent cations [2]. EPS are biopolymers resulting from active bacterial secretion, shedding of cell surface material, cell lysis materials
* Corresponding author. Tel.: +33 5 55 45 73 67; fax: +33 5 55 45 72 03. E-mail addresses: [email protected] (S. Comte), [email protected] (G. Guibaud), [email protected] (M. Baudu). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.10.014
and from adsorption of organics from the environment [3]. They are composed of a variety of organic substances: carbohydrates and proteins being major constituents with humic substances, uronic acids and nucleic acids in smaller quantities [4,5]. Many authors have defined two kinds of EPS in flocs or biofilms: soluble and bound EPS. Bound EPS include microbially produced bound polymers but also lysis and hydrolysis products as well as absorbed or attached matter [6]. Soluble EPS include microbially produced soluble polymers, hydrolysis products of attached organic matter and organic molecules released by cell lysis. EPS are located at/or outside the cell surface independent of their origin. In biofilms or activated sludges, EPS may be only loosely attached to the cell surface as peripheral capsules (called bound EPS) and can be shed into the surrounding environment as a less organized (amorphous) slime (called soluble EPS) [3]. In flocs or biofilms, bacteria are embedded in EPS (that is to say bound EPS are closely associated with the cells), while soluble EPS do not
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have any direct contact with the cells. In activated sludge, bound EPS on the floc biomass are composed of sheaths, capsular polymers, condensed gel, loosely bound polymers and attached organic material and soluble EPS with soluble macromolecules, colloids and slimes [7,8]. To differentiate the two kinds of EPS, a theory based on metabolism of bacterial cells and electron flux has been suggested [8]. Soluble EPS are defined as Soluble Microbial Products (SMP) divided in two categories: substrate-utilization-associated products (UAP), which are produced directly during substrate metabolism, and biomass-associated products (BAP), which are formed from biomass. If we consider the ‘‘unified theory’’ of [8], soluble EPS are actively secreted by bacteria and are biodegradable and bound EPS are polymers bound to active and inert biomass or are molecules resulting from cell lysis. Ref. [9] discuss this issue and shows that EPS cannot be the only source of BAP. These authors suggest that EPS in nonhydrolyzed form may indeed constitute part of the high molecular weight SMP. However, cell lysis may also result in the release of intracellular high molecular weight SMP, and these compounds are also a source of BAP [9]. Besides EPS, its hydrolysis products, and other intracellular compounds released as a result of cell lysis, SMP may also be comprised of more specific compounds that could be deliberately excreted by microorganisms to play a specific role in metal mitigation, and such organics would be classified as UAPs according to the SMP theory. In conclusion, the origin of EPS is complex and different approaches are thus possible to define soluble and bound EPS. In experiments, EPS are defined rather by the method used to separate/extract the EPS than by a theoretical consideration of the physical state of the polymers [3]. In biofilms or activated sludge systems, EPS can be found in two operationally defined forms: soluble EPS which can be extracted by centrifugation alone and bound EPS, on the floc biomass, which require additional treatment for their extraction [3]. The most important functions of EPS are adhesion to surfaces, aggregation of bacterial cells in flocs, stabilization of the floc structure, formation of a protective barrier that provides resistance to biocides or other harmful effects, retention of water, sorption of exogenous organic compounds for the accumulation of nutrients from the environment, and accumulation of enzymatic activities, such as digestion of exogenous macromolecules for nutrient acquisition, aiding the cells in uptaking metal nutrients [3,10,11]. In fact, the EPS matrix is a medium that allows cooperation and communication between cells resulting in microbial aggregates [8]. Several studies have shown that EPS also play a crucial role in biosorption of heavy metals [12,13,10]. Moreover, in the presence of a heavy metal (chromium), the biomass seemed to have produced more EPS material (principally SMP or soluble EPS), indicating that this might have helped it cope with the metal stress [9]. The aim of this study is to compare biosorption properties of soluble EPS and bound EPS towards metallic cations (Cu2+, Pb2+, Ni2+, Cd2+), taking into account the physico-chemical characteristics of both EPS types.
2. Material and methods 2.1. Activated sludge samples and EPS extraction Extraction was carried out on activated sludges obtained from the aeration tanks of two wastewater treatment plants (WWTP) (called A and B). The WWTPs A and B present different characteristics. The treatment capacity in inhabitant equivalent is 285,000 and 4000, respectively for sludges A and B. The organic loads are between 0.24 and 0.30 and between 0.13 and 0.16 kg BOD5/m3/day respectively for A and B sludge. The volatile suspended solid of sludge A and B were respectively 68% and 72% of the total suspended solid of sludges. The sludge volume index (SVI) of sludge A and B were respectively 90 and 160 mL g1 of TSS. Before the extraction step, sludges were concentrated at 4300 g for 10 min at 4 8C. More over, this step remove the supernatant of sludges which contain minerals ions, the remains of organic matter from effluent and probably very soluble EPS very easily released by sludges. The residues were recovered and suspended in ultra-pure water. Two procedures were used for EPS extraction from each sludge (A and B). The first consisted of a centrifugation at 4000 g for 20 min at 4 8C, allowing soluble EPS to be extracted [14]. The second was a 40 W sonication for 2 min associated with a cationic exchange resin (Dowex 50X8) treatment, followed by two ultracentrifugations (20,000 g for 20 min at 4 8C then 10,000 g for 15 min at 4 8C) [15]. The EPS extracted with the latter protocol were called bound EPS but this procedure in fact extracted both soluble and bound EPS. Extracted EPS from both methods were finally purified in ultra-pure water with a dialysis membrane (3500D, Cellu Sep). The EPS were stored at 18 8C before being used.
2.2. EPS characterization The main characteristics of extracted EPS were determined: dry weight content (DW) (at 105 8C); volatile dry weight content (VDW) (at 550 8C); total organic carbon (TOC) and Nitrogen content (using a Dorhmann Apollo 9000 TOC-meter equipped with a nitrogen analyser); total phosphorus content (using the European standard EN 1189, 2001). The biochemical composition (protein, humic acids, polysaccharide, uronic acid, lipid, nucleic acid content was defined using colorimetric methods as described by [15,16].
2.3. Infra-red (IR) spectrometry One milligram of freeze-dried EPS solution was mixed with 180 mg of KBr and compacted to form a pellet. The IR-spectra were then determined using a spectrum 1000 (Perkin-Elmer).
2.4. pKa determination The determination of the pKa for the EPS solutions was not possible by simple acid–base titration. According to [15], a method previously developed for solids chemistry was modified and applied to EPS in order to determine the pKa of the EPS. The acid–base surface reactions are described only by the law of mass action based on the protonation (under the form: S–OH and S–OH2+) of the surface functional groups (S–O) and determined by analogy with amphoteric compounds. According to Eq. (1), the total number of surface [S]tot sites was ½Stot ¼ ½SOH2 þ þ ½SOH þ ½SO ¼ PEC
(1)
with [S]tot: total number of surface sites, PEC: protonic exchange capacity, Note that the PEC, is the experimental determination by potentiometric titration of the total number of surface [S]tot (see below), that is to say the total number of sites potentially active for metal complexation [17]. SOH2 þ , SOH þ Hþ
(2)
S. Comte et al. / Process Biochemistry 41 (2006) 815–823 SOH , SO þ Hþ with : Ka1 ¼
Ka2 ¼
½Hþ ½SOH ½SOHþ 2
½Hþ ½SO ½SOH
(3)
3. Results
(4)
3.1. EPS characterization
(5)
In this study, the determination of both the EPS surface charge (Q) and the EPS protonic exchange capacity (PEC) were carried out by potentiometric titration. A solution of EPS (25 mL) was placed in a 22 8C thermostated cell. Titration was carried out adding NaOH or HNO3 (0.01 M) with an automatic titrator, Metrohm 716 DMS, coupled to a Metrohm 727 Ti Stand and equipped with a pH electrode (pH 0–14/0; 80 8C; KCl 3 M). Finally, evolution of Q parameter calculated from acidic or alkaline titration curve of EPS and PEC value allowed two Ka of EPS solutions (Ka1 and Ka2 ) to be calculated for the predominant functional groups. Ka1 and Ka2 were expressed under their logarithmic forms that is to say respectively pKa1 and pKa2 .
2.5. Metal biosorption of EPS The determination of the biosorption parameters at 22 2 8C and pH 7 was carried out by measuring the free metal content in the polymer solution after addition of Cu2+, Pb2+, Ni2+ or Cd2+ (under nitrate form at concentration of 102 M). Polarographic methods could be useful tools to investigate metal biosorption by biological materials such as bacteria [18]. According to [19,20], investigations on free metals were carried out by polarography, using the static mercury dropping electrode mode (SMDE). A 626 polarecord polarograph coupled to a 663 VA metrohm stand was used (time of drop fall: 1 s). Operating conditions were as follow: In the measuring cell at 22 2 8C: 10 mL of ultra-pure water; 10 mL of HEPES buffer at pH 7 (Sigma-Aldrich) 101 M. HEPES buffer is recognised as a non-complexant buffer and can be used as a supporting electrolyte [21]. 1 mL of KNO3 (Flucka) 1 M as the electrolyte; addition of quantities of metallic solution (from 20 to 50 mL) at a concentration of 102 M at pH 4; 1 ml of EPS. Potential range scanned for free metal determination Cu2+: 0.10 to 0.20 V; Pb2+: 0.20 to 0.50 V; Cd2+: 0.45 to 0.75 V; Ni2+: 0.65 to 1.15 V. Polarographic titration consists in measuring the evolution of free metal content in the solution of EPS after each addition of metal. The analysis of the curves of the polarographic titration allowed, from Ruzic’s modelling, the different parameters characteristic of the metal binding by EPS to be obtained: the concentration of ligands (or quantity of potentially active sites) and the conditional constants of formation of the complex [22]. The theoretical study of the complexation equilibrium associated to the law of mass action can be described as follows, assuming the formation of a 1:1 complex. M þ L , ML
(6)
½ML ½M½L
(7)
K¼
with M: free metal, L: free ligand (EPS), ML: ligand–metal complex, K: conditional binding constant. The number of sites initially occupied by the metals studied was not taken into account for the determination of the total number of sites in EPS as it was negligible (<1% total complexation determined).
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In order to define soluble (extracted by centrifugation alone) and bound EPS (extracted by additional treatment), the quantity of EPS extracted, the carbon content, the nitrogen content, the phosphorus content and the biochemical composition were compared. The extraction yields of EPS according to the extraction protocol used are presented in Fig. 1. For both sludge samples (A and B), EPS amounts extracted by centrifugation alone (soluble EPS) were less than 1% whereas those extracted by sonication and cationic resin (bound EPS) were about 3%. Due to the extraction method used, EPS obtained from the second procedure were composed of both bound and soluble EPS. We can assume that real bound EPS is about 70% of the bound fraction extracted. This means that real bound EPS are predominant in the bound EPS extraction and justifies the denomination of bound EPS. In biofilms, the proportion of EPS can vary between roughly 50% and 80% of the total organic matter content [6] and the proportion of EPS can be affected by the age of the biofilm [23]. Thus the bound EPS fraction extracted in this study is low (about 3%) when compared to the total amount of EPS present in biofilms. Physical means (ultracentrifugation, cationic exchange resin, sonication, . . .) used to extract EPS are recognized as giving low extraction yields [24] but do not affect the quality of extracted EPS [25]. Information on the general characterization of EPS such as the carbon, nitrogen and phosphorus contents as well as the organic mass fractions of EPS (expressed in % of VDW/DW) is given in Table 1. EPS are mainly composed of an organic fraction (the VDW/DW ratio of EPS varies from 69% to 82%). The nitrogen values varied from 84 to 97 mg N g1 of EPS VDW, and were close for both soluble and bound EPS. EPS N contents came from the organic fraction as it was verified that NO3, NO2 and NH4+ were not present in EPS solution. The organic carbon values varied from 184 to 267 mg C g1 of EPS VDW, the amounts of organic carbon being higher for soluble EPS than for bound EPS and
Fig. 1. Extraction yield of EPS for sludge A and B (% EPS VDW/sludge VSS).
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Table 1 Main characteristics of EPS for sludge A and B according to their origin Soluble
Bound
A
B
A
B
82 5 93 5 241 12 2.6 24 1
74 4 97 5 267 13 2.7 19 1
69 3 84 4 184 9 2.2 28 2
70 4 91 5 228 11 2.5 18 1
Biochemical composition of EPS in mg g1 EPS VDW Proteins 385 15 Polysaccharides 207 17 Humic acids 77 4 Uronic acids 25 1 Lipids <39 Nucleic acids 22 2 Protein/polysaccharide 1.9
334 13 210 17 201 10 80 4 <33 69 7 1.6
365 15 149 12 182 9 69 3 <20 73 7 2.4
381 15 162 13 224 11 71 4 <22 51 5 2.4
underlined qualitative differences between soluble and bound EPS. Moreover, the C/N ratio varied from 2.6 to 2.7 and from 2.2 to 2.5 for soluble and bound EPS, respectively and show a slight qualitative difference between both sorts of EPS. The phosphorus content, did not underline any difference between soluble and bound EPS (values varied from 18 to 28 mg P g1 of EPS DW). The biochemical composition of EPS is also detailed in Table 1. The nucleic acid content is used as an indicator of cellular lysis during the extraction process [24] and its values varied from 22 to 73 mg g1 of EPS VDW (corresponding to 0.2–2.1 mg g1 of sludge VSS (volatile suspended solid)). No contamination of EPS by the intracellular materials was noted when comparing the above results with the literature data [24]. More over, the nucleic acid content of EPS A and B varied according to the sludges origin according to Sponza [5], but also with the EPS location in the sludges (Table 1). Nevertheless, Table 1 shows that the amounts of nucleic acids are higher for soluble EPS than for bound EPS for sludge A, but as to sludge B, that is contrary, but it is hard to explain evolution of these values.
Proteins and polysaccharides formed the biggest fraction: from 334 to 385 mg proteins g1 of EPS VDW, and from 149 to 210 mg polysaccharides g1 of EPS VDW. The protein values were close for all EPS extracted whereas the highest polysaccharide contents referred to soluble EPS solutions. Protein/polysaccharide ratios were 1.9 and 1.6 for soluble EPS and 2.4 for bound EPS. Ref. [24] have shown that the protein:polysaccharide ratio for activated sludge EPS varies over a wide range: from 0.5 to 21.2. The ratios determined in this study (Table 1) are low and are in accordance with the ones referenced by [24] and confirm that the EPS extracted in this study were not contaminated by intracellular materials from bacteria of activated sludge flocs. Biochemical characterization (Table 1) confirmed that there were qualitative differences between soluble EPS and bound EPS. IR spectra of soluble and bound EPS from sludge sample B are presented in Fig. 2. The spectra of both EPS are similar and indicate the presence of the same functional groups mentioned in Table 2. Several intense characteristic bands can be attributed to protein and polysaccharide functional groups. These different
General characterization of EPS VDW/DW (%) EPS VDW (mg N g1) mg C g1 EPS VDW C/N ratio mg P g1 EPS DW
Fig. 2. IR spectra of soluble and bound EPS (sample B).
S. Comte et al. / Process Biochemistry 41 (2006) 815–823
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Table 2 Main functional groups observed from IR-spectra of soluble and bound EPS Wave number (cm1)
Vibration type
Functional type
3200–3420 2930–2935 1640–1660 1550–1560 1450–1460 1400–1410
Stretching vibration of OH Asymmetric stretching Vibration of CH2 Stretching vibration of C O and C–N (Amide I) Stretching vibration of C–N and deformation vibration of N–H (Amide II) Deformation vibration of CH2 Stretching vibration of C O Deformation vibration of OH Deformation vibration of C O Stretching vibration OH Stretching vibration C–O–C Stretching vibration of OH fingerprint zone Several bands visible
OH in polymeric compounds
1235–1245 1130–1160 1040–1080 <1000
functional groups observed agree with results of [15,16] and are in accordance with the EPS biochemical composition. 3.2. EPS biosorption site characterization The protonic exchange capacities (PEC) and the pKa values are presented in Table 3. No significant difference was noted between the pKa1 and pKa2 for the soluble and bound EPS: pKa1 values varied from 6.1 to 6.6 and pKa2 values varied from 8.4 to 9.2. According to the IR-spectra, the EPS composition and the literature data [4,15,16], pKa1 is characteristic of carboxylic and phosphoric functional groups and pKa2 is attributed to phenolic and amino functional groups. In EPS solution, the pKa of the carboxyl/ phosphoric site was determined at about 6 and that of the amine/ phenolic site about 9.6 [4]. The PEC represents an estimation of EPS total binding sites [17]. The PEC value of EPS was 4000 mmol g1 EPS DW and varied from 2400 to 2500 mmol g1 EPS DW for soluble and bound EPS, respectively. The capacities for exchange of protonic ions are greater for soluble EPS than for bound EPS. The carboxylic and phosphoric groups identified with pKa1 values could be implicated in the binding of metal ions by EPS at pH 7 [26,4]. As a consequence, a different biosorption ability for the two types of EPS could be expected. 3.3. EPS biosorption properties The EPS binding constants (expressed as log K) and the numbers of EPS fixation sites obtained with the polarographic
Table 3 Physico-chemical characterization of EPS from sludge A and B Soluble
pKa1 pKa2 PEC (mmol g1 EPS DW)
Bound
A
B
A
B
6.1 0.1 9.0 0.1 4000 100
6.6 0.1 9.2 0.1 4000 100
6.1 0.1 8.4 0.1 2400 50
6.2 0.1 9.1 0.1 2500 50
Proteins (peptidic bond) Proteins (peptidic bond) Carboxylates Alcohols and phenols Carboxylic acids Phenols Polysaccharides Phosphate or sulphur functional groups
titration curves and by Ruzic’s modelling are presented respectively in Figs. 3 and 4. Whatever the EPS (soluble, bound) and the metal considered (Cu2+, Pb2+, Ni2+, Cd2+), the binding constant in Fig. 3 did not show significant differences in the strength of binding formed between the different EPS and metals by the polarographic method used. The number of fixation sites in Fig. 4 varied for the soluble and bound EPS. This parameter is more sensitive for demonstrating differences in EPS biosorption properties depending on EPS types. The number of fixation sites of both EPS towards Cu2+ varied from 4114 to 5795 mmol g1 of DW EPS. Soluble EPS A showed better biosorption abilities than bound EPS B for Cu2+ instead of the biosorption abilities of both EPS B were close. The binding values of both EPS towards Pb2+ varied from 2051 to 3131 mmol g1 of DW EPS. The results with Pb2+ varied over a smaller range than these with Cu2+. For Pb2+ with EPS A and B, soluble EPS had a higher number of fixation sites than those of bound EPS. The number of fixation sites of soluble EPS towards Ni2+ varied from 2086 to 3120 mmol g1 of DW EPS and those of bound EPS varied from 486 to 917 mmol g1 of DW EPS. Soluble EPS exhibited stronger biosorption properties than bound EPS for Ni2+. The number of fixation sites varied by a factor of four between soluble and bound EPS. As for the biosorption results for Cd2+, the values for soluble EPS varied from 251 to 289 mmol g1 of DW EPS and those for bound EPS varied from 295 to 426 mmol g1 of DW EPS. For Cd2+, bound EPS and soluble EPS exhibited close metal binding properties. The EPS biosorption affinity towards Cd2+ was very weak which made it difficult to find any significant difference between the EPS studied. To summarize, whatever the EPS considered, the analysis of all metal binding sites showed an EPS affinity for metals in the following order: Cu2+ > Pb2+ > Ni2+ Cd2+ which is in accordance with the literature data. Several published values predict the same affinity of metal to EPS for organo-metal complex formation [27,15].
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Fig. 3. Binding constant expressed as log K for soluble and bound EPS according to the metal studied.
4. Discussion The results showed that soluble EPS has a greater capacity to exchange protonic ions than bound EPS. The PEC parameter can be used to evaluate the capacity of EPS to bind ligands [17]. The greater the PEC the more the EPS are able to bind cationic ligands. Due to the pKa1 value and PEC (Table 3), we can assume that the number of dissociated sites of soluble EPS at pH 7, is greater than that of bound EPS. This difference between soluble and bound EPS expressed by the number of dissociated sites could explain the biosorption abilities of the type of EPS toward metals. The numbers of binding sites of EPS towards Cu2+ (only EPS A), Pb2+ and Ni2+ are, in fact, greater for soluble EPS than for bound EPS. Moreover, pKa1 is characteristic of functional groups, carboxylic and phosphoric groups would. pKa2 is attributed
to phenolic and amino functional groups. The carboxylic and phosphoric groups would therefore seem to play a major role in the metal binding at pH 7. Nevertheless, it can be seen that the number of binding sites with Cu2+ is superior to the potential total number of sites (PEC) at pH 7, so different mechanisms could be implicated in Cu2+ biosorption: exchange with protons but also electrostatic interactions and precipitation [28]. The speciation of Cu2+ at pH 7 could influence the evaluation of the EPS biosorption abilities. The study of EPS binding properties showed different behaviour between soluble and bound EPS depending on the metal considered. For Cu2+ (only for EPS A), Pb2+ and Ni2+, soluble EPS exhibited stronger binding properties than bound EPS. The extraction protocol for bound EPS resulted in bound EPS being in fact composed of soluble and bound EPS, so the
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Fig. 4. Number of fixation sites (in mmol/g of EPS DW) depending on the type of EPS and the metal studied.
metal binding results for Cu2+ (only for EPS A), Pb2+ and Ni2+ highlight that the contribution of bound EPS to the biosorption affinity was even less important than that of soluble EPS. Soluble EPS are more reactive towards metallic cations, except Cd2+ and Cu2+ (case of EPS B).
The composition of EPS has shown that soluble and bound EPS are mainly composed of proteins and polysaccharides. More precisely, polysaccharide content is greater for soluble EPS than for bound EPS. Moreover, this study showed that biosorption ability of soluble EPS is greater than that of bound
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EPS for Cu2+ (only for EPS A), Pb2+ and Ni2+. Ref. [29] stated that different metal adsorption sites appear to exist on neutral polysaccharides and anionic polysaccharides. Neutral polysaccharides may bind metal cations to the hydroxyl groups of hexose or pentose molecules and anionic polysaccharides may bind metal cations to their carboxyl groups. The biosorption ability of polysaccharides could explain in part why the number of fixation sites is greater for soluble EPS than for bound EPS for Cu2+ (in the case of EPS A), Pb2+ and Ni2+. A function frequently attributed to EPS is their general protective effect against toxic compounds [24]. The sorption of heavy metals is part of a protection strategy against toxic effects [30]. It has been shown by a large number of studies that the type and amount of EPS produced by microorganisms are influenced by environmental conditions [3]. When there are changes in the growth medium, such as the presence or not of Cu, cells of Pseudomonas aeruginosa respond by producing EPS which have large Cu binding capacities [31]. The EPS synthesis by cells resistant to Cu showed elevated Cu binding ability (320 mg g1 EPS) compared to that produced by cells sensitive to Cu presence (270 mg g1 EPS). Moreover, biofilms in the presence of Cu have a high EPS to cell ratio, suggesting that EPS production may provide an important defence strategy against toxic effects, perhaps through sequestration of toxic copper ions [32]. Furthermore, according to [9], the SMP (soluble microbial product or soluble EPS according to the ‘‘unified theory’’ of [8] accumulation in anaerobic sludge from chemostat seems to be proportional to the dose of toxicant (chromium). According to the results of this study and the literature, the chemical nature of soluble EPS seems to be more adapted to protecting bacterial cells against metallic pollution. Qualitative differences such as carbon and polysaccharide contents, C/N ratio and PEC have been underlined between soluble and bound EPS. The two types of EPS could not perform the same functions. Bound EPS are loosely attached to the cell surface whereas soluble EPS are situated in the surrounding environment of bacterial cells. Depending on their location, soluble EPS form a first protective barrier that prevents any harmful effects of heavy metals on floc microorganisms. Moreover, soluble EPS are actively secreted by microorganisms [8]. The bacteria could synthesize soluble EPS to protect themselves against harmful effects of toxic metals. Active secretion of soluble EPS supports the hypothesis that soluble EPS play a major role in the protective strategy of microorganisms in flocs against the effects of heavy metals. However, the potential role of EPS (bound and/or soluble) in aiding the cells in uptaking metal nutrients (such as Cu, Ni, . . .) could be considered. For instance, only a mechanism to scavenge iron has been discovered for aerobic bacteria [11] and involves the excretion of siderophores into the bulk solution. However, as siderophores are deliberately excreted by cells, they are not of extracellular origin, and should be referred to as specific SMP rather than soluble EPS [8,9]. 5. Conclusion Soluble EPS gave highest results for carbon content and to a lesser extend C/N ratio which underlines that there were
qualitative differences between soluble EPS and bound EPS. Both of the EPS studied were comprised mainly of proteins and polysaccharides, the concentration of polysaccharides being higher for soluble EPS than bound EPS. Biochemical characterization results also indicated that there were qualitative differences between the two kinds of EPS. The analysis of the biosorption results of the two types of EPS showed an EPS affinity for metals in the following sequence: Cu2+ > Pb2+ > Ni2+ Cd2+. At the pH of the study, pH 7, carboxylic and phosphoric groups characterized with pKa1 values could have been implicated in the binding of metal ions by EPS through their protonic exchange ability. The numbers of total potential binding sites estimated by the PEC indicated another qualitative difference between soluble and bound EPS and revealed that different mechanisms could be implicated in the metal biosorption of EPS, principally in the case of Cu2+. However, the study of EPS biosorption properties points out the difference in behaviour between soluble and bound EPS. For Cu2+ (only for EPS A), Pb2+ and Ni2+, the biosorption abilities were greater for soluble EPS than those for bound EPS. The ability of polysaccharides to bind metallic cations could be involved in the greater biosorption abilities of soluble EPS in comparison with bound EPS. In this study, soluble EPS were the most reactive EPS towards the metallic cations studied (Pb2+, Ni2+, . . .), therefore, they may play a role as a microbial protective barrier. Acknowledgement This study was supported by a research grant provided by the Conseil Regional du Limousin. References [1] Arican B, Gokcay F, Yetis U. Mechanistics of nickel sorption by activated sludge. Process Biochem 2002;37:1307–15. [2] Urbain V, Block JC, Manem J. Bioflocculation in activated sludge: an analytic approach. Water Res 1993;27:829–38. [3] Wingender J, Neu TR, Flemming HC. Microbial extracellular polymeric substances: characterisation, structure and function. Berlin: Springer, 1999. p. 123. [4] Liu H, Fang HP. Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data. Biotechnol Bioeng 2002;80:806–11. [5] Sponza DT. Extracellular polymer substances and physicochemical properties of flocs in steady and unsteady-state activated sludge systems. Process Biochem 2002;37:983–98. [6] Nielsen PH, Jahn A, Palmgren R. Conceptual model for production of exopolymers in biofilms. Water Sci Technol 1997;36:11–9. [7] Hsieh KM, Murgel GA, Lion LW, Shuler ML. Interactions of microbial biofilms with toxic trace metals: observation and modelling of cell growth, attachment, and production of extracellular polymer. Biotechnol Bioeng 1994;44:219–31. [8] Laspidou CS, Rittmann BE. A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water Res 2002;36:2711–20. [9] Aquino SF, Stuckey DC. Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds. Water Res 2004;38: 255–66.
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