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A high yield multi-method extraction protocol for protein quantification in activated sludge
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Bioresource Technology 99 (2008) 7464–7471
A high yield multi-method extraction protocol for protein quantification in activated sludge Ras Monique a, Girbal-Neuhauser Elisabeth a, Paul Etienne b, Lefebvre Dominique a,* a
Laboratoire de Biologie applique´e a` l’Agro-alimentaire et a` l’Environnement, Institut Universitaire de Technologie, Universite´ Toulouse III, 24 Rue d’Embaque`s, 32000 Auch, France b Laboratory of Biosystems and Process Engineering, UMR5504 CNRS/INSA and UMR792 INRA/INSA, 135 avenue de Rangueil, 31077 Toulouse cedex 4, France Received 6 September 2007; received in revised form 14 February 2008; accepted 17 February 2008 Available online 18 April 2008
Abstract A multi-method extraction protocol based on mechanical, ionic and hydrophobic methods was investigated on two types of activated sludge samples. Extraction methods were chosen with regards to optimal protein yield without cell disruption. Sonication, EDTA and Tween extraction methods were selected and combined. The total amount of protein released by the multi-method protocol sums up to 191 and 264 mg equiv. BSA/g VSS for the two different sludge samples. Protocol repetition on the same sample showed that protein yield after each successive protocol fitted an exponential curve model. The total amount of extractable proteins was evaluated by model predictions, 423 and 516 mg equiv. BSA/g VSS for the two sludge samples. The multi-method extraction protocol appears relevant for harvesting a representative quantity of proteins from the original sample (45–49%), moreover the multi-method criterion of the protocol also offers a heterogeneous pool of proteins. Thus, further qualitative studies may not be biased by the extraction protocol. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Exopolymeric protein; Physicochemical treatment; Extraction strategy; Activated sludge
1. Introduction In biological wastewater treatment systems, bacteria aggregate together in order to form suspended floc structures or granules as well as attached biofilms. Several studies have shown that extracellular polymeric substances (EPS) take part in forming a gel-like matrix which acts as cement in bacterial aggregates. EPS are molecules produced by bacteria, brought by the incoming wastewater (Urbain et al., 1993) or are released from cells when lysis occurs. Humic acids (Frohlund et al., 1995), polysaccharides (Rideau and Morfaux, 1976), proteins (Dignac et al., 1998), nucleic acids (Jahn and Nielsen, 1997) and other non determined molecules (Wile´n et al., 2003) have been reported as EPS compounds in the matrix. In spite *
Corresponding author. Tel.: +33 5 62 61 05; fax: +33 5 62 61 63 01. E-mail address: [email protected] (L. Dominique).
0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.02.025
of this wide range of macromolecules, recent studies have shown the relative importance of proteins in wastewater treatment systems from a quantitative aspect (Liao et al., 2001; Sponza, 2002), as well as from a functional point of view (Denecke, 2006). Proteins have been proven to increase biofilm adherence in bioreactors and cause fouling which has considerable consequences on water treatment by membrane filtration (Masse´, 2004; Meng et al., 2006). Enzyme activity measurements showed that enzymes were integrated in the protein EPS pool (Frohlund et al., 1995) and that inherent proteases were the most active enzymes (Cadoret et al., 2002; Jung et al., 2002). Martinez et al. (2004) showed that protein content was linked to sludge processes, and that the types of proteins were rather linked to sludge settleability. Total protein quantification and characterisation could therefore bring relevant information on bacterial aggregation processes as well as on treatment efficiency.
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471
Protein quantification is generally carried out by a prior extraction step. The most popular EPS extraction method is Cation Exchange Resin (CER), (Frohlund et al., 1995). Over many studies using this same method, proteins are showed as the predominant EPS in biofilm (Jahn and Nielsen, 1995) and in aerobic activated sludge extracts (Bura et al., 1998; Frohlund et al., 1995; Liu and Fang, 2002; Wile´n et al., 2003). Sonication and formaldehyde extraction methods have also shown that proteins were the most represented EPS in activated sludge samples (Comte et al., 2006; Liu and Fang, 2002). However other extraction methods such as EDTA applied on the same sludge samples released more polysaccharides and humic substances than proteins (Denecke, 2006; Liu and Fang, 2002). The wide variety of extraction methods used in literature could partly explain the large variation of protein content reported in sludge, between 3.9 and 510 mg/g VSS (Liu and Fang, 2002; Masse´, 2004; Wile´n et al., 2003), without omitting other factors such as sludge origin and process designs which are also described as influencing EPS content (Comte et al., 2006; Sponza, 2002). However, total protein quantification in sludge via extracts can be questionable since only one extraction method is carried out. Moreover, proteins are characterised by ionic, hydrophobic and neutral amino acids. Therefore, specific bonds such as electrostatic, hydrophobic and low energy hydrogen or Van der Waals interactions play a major role in linking proteins to the EPS matrix. Hence, the use of one type of extraction method is likely to promote the study of ‘‘one kind of protein” in quantification or characterisation studies. As shown by Dignac et al. (1998), extracellular proteins released by CER contained a majority of negatively charged amino-acids. General remarks in literature stipulate that quantification and characterisation of EPS compounds in sludge are often biased by extraction procedures which do not take into account the specificity of extraction methods towards certain types of EPS (Comte et al., 2007; Dignac et al., 1998). Thus, in order to assure a homogenous protein pool in extracts, it would be appropriate to apply three different extraction methods which aim on the three major types of bonds linking proteins to the EPS matrix, i.e. low energy, hydrophobic and ionic interactions. Protein quantification in sludge and sludge extracts is frequently carried out by modified Lowry method (Frohlund et al., 1995). However, studies have shown that bicinchoninic acid (BCA) is less sensitive to chemical extraction methods (i.e. EDTA), offers a better protein to protein response (Smith et al., 1985), as well as, a lower quantification limit (Ras et al., in press). This present paper offers a multi-method extraction protocol based on three types of extraction methods (mechanical, ionic and hydrophobic) in order to obtain a diversified protein pool which can stand for the studied sludge. Several extraction methods were chosen to be investigated according to their frequency in literature and care towards cell disruption and protein denaturation. This includes mechanical and ionic extraction methods such as
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sonication (Azeredo et al., 1998; Comte et al., 2006; Dey et al., 2006), cation exchange resin (Cadoret et al., 2002; Comte et al., 2006; Park and Novak, 2007; Sesay et al., 2006), and EDTA (Comte et al., 2006; Liu and Fang, 2002; Sheng et al., 2006). EPS extraction methods which aim hydrophobic interactions in sludge are randomly found in literature. However, among the several non ionic surfactants employed in previous studies, Tween and ethanol have been investigated as hydrophobic extraction methods. Each method was controlled for their possible effect on cell disruption and protein denaturation, as well as, for their interference towards the protein quantification method. The most efficient methods were selected and applied in series according to an original three step extraction strategy. This study will also give more information on the total amount of extractable proteins which can be released from activated sludge. 2. Methods 2.1. Activated sludge samples: origin and handling The extraction methods were undertaken on sludge samples from two different pilots. Organic supply and sludge characteristics are described in Table 1. Samples were collected the previous day, stored at 4 °C and diluted with a Phosphate Buffer Saline (PBS, pH 7) to 5 g VSS/L of sludge before each extraction. Total solids (TS) were measured by desiccating at 105 °C and volatile suspended solids (VSS) were measured by calcinate at 510 °C. 2.2. Extraction instruments and chemicals Extraction tests were undertaken on 50 mL sludge samples. Stirring intensities were all performed at 500 rpm. 2.2.1. Mechanical extractions Ultrasounds were carried out with a vibra cell sonication probe from Bioblock (2.5 mm 49 mm cylinder). Table 1 Pilot and sludge characteristics Pilot 1
Pilot 2
Charge Treated effluent
g DCO/L/d Type
0.2 Domestic wastewater
Sludge residential time (SRT) Sludge concentration Dissolved oxyge`ne Oxygenation time Anoxic time Volatile suspended solids/total solids Temperature (°C)
Days
8
0.06 Synthetic (propionate/ acetate/starch/ ethanol) 8
g TS/L mg/L min min g VSS/g TS
1.5 3 40 40 0.8–0.85
1.5 3 40 40 0.8–0.85
20
20
20
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Sonication was performed at 37 W (0.092 W/mm2), with 10 mm probe tip dipped in 50 mL sludge samples (0.75 W/mL power density). Sonication times were investigated by discontinuous applications with 2 min interval. Time applications varied from 2 min 1 to 2 min 5. Blender treatments were performed on 50 mL of sludge with a waring commercial laboratory blender. Time applications varied from 5 min 1 to 5 min 5 with 5 min interval. For both methods, samples were kept at 4 °C during extraction. 2.2.2. Ionic extractions CER (Dowex, Fluka) was used at 70 g/g VSS (Frohlund et al., 1995) and ethylene diamino tetra acetic acid (EDTA, Acros) was prepared at concentrations ranging from 0.5% to 2% in Tris–HCl buffer 0.3 M pH 8 or in NaOH at concentrations between 0.15 and 2 M. CER was tested for different contact times from 45 min to 3 h 45 min. EDTA tests were carried out for 1 h application. 2.2.3. Hydrophobic extractions Ethanol (Acros) was tested at concentrations between 20% and 50% in PBS buffer and Tween 20 (Acros) was prepared in PBS buffer at concentrations between 0.25% and 1%. Contact time was investigated for both methods, between 1 and 2 h applications. 2.3. EPS quantification Protein measurements were performed on all soluble extracts with the bicinchoninic acid or BCA reagent (Sigma), according to Smith et al. (1985). Bovine serum albumin (BSA) was used as standard. Total nitrogen (TN) measurements by Kjeldahl method was undertaken for indirect protein determination. Polysaccharides were determined using the anthrone method (Dreywood, 1946). Glucose was used as standard. 2.4. Enzyme assays The intracellular glucose-6-phosphate dehydrogenase (G6P-DH) activity measurements were carried out on extracts in order to detect cell lysis. G6P-DH measurements were applied according to Lessie and Vander Wyk (1972). Substrate solutions were prepared with 0.2 M Tris–HCl pH 8.5, 0.2 M 2-mercaptoethanol (Acros), 0.0005 M of nicotine adenine dinucleotide (NAD, Acros) and 0.01 M D-glucose-6-phosphate (Fluka). The supernatant extracts (200 lL) were incubated with 800 lL of substrate solution at room temperature for 30 min. Absorbance values (340 nm) were recorded every 10 s. Results are expressed as units: nmol of nicotine adenine dinucleotide hydrogen (NADH) formed per minute per gram of volatile suspended solids (VSS). An enzymatic screening of 19 different enzymes was performed using the semi quantitative micro method APIZYM (Biome´rieux).
All absorbance measurements were undertaken with a Miltron Roy recordable spectrophotometer (Spectronik 1201).
3. Results and discussion 3.1. Selection of extraction methods In order to offer an efficient protocol for extracting EPS from sludge, physical and chemical treatments were investigated. For each kind of treatment, two methods were compared in order to select the most relevant method in terms of EPS content in extracts and preservation of cell integrity. Since Lowry quantification method is particularly affected by EDTA (Comte et al., 2006; Smith et al., 1985), BCA method was chosen for protein quantification whereas sugars were quantified by the anthrone method. Table 2 presents the amount of proteins and the G6PDH activity obtained in each extract. Mechanical disintegration acts randomly on the different interactions holding the EPS matrix together. Discontinuous sonication during one to five times 2 min treatments was compared to a Blender disruption method for protein release from activated sludge samples. Table 2 shows that sonication releases increasing proportions of proteins with treatment time, from 100 mg equiv. BSA/g VSS for 1 2 min treatment to 160 mg equiv. BSA/g VSS for 5 2 min treatment. The protein/sugar ratio (P/S) decreases from 12 to 7 showing that extended sonication extracts preferentially polysaccharides (Table 3). Blender, on the other hand, releases stable but relatively less proteins compared to sonication (70–80 mg equiv. BSA/g VSS, Table 2). Nevertheless, 25 min blender application discharges spontaneously large proportions of proteins which are comparable to 5 2 min discontinuous sonication. However, ultrasounds reveal a more reliable protein extraction profile with a more convenient and rapid procedure compared to Blender treatments. G6P-DH activity measurement indicated no intracellular leakage in either soluble extracts, whether from sonication or from Blender treatments. It is known that ultrasound power densities affect cell lysis (Zhang et al., 2007). Salhani and Uelker-Deffur (1997) have shown that two minutes sonication under 1.3 W/mL power density was defined as the best mechanical method for floc disruption without reducing cell viability. Zhang et al. (2007) showed that 6 min sonication under 0.5 W/mL extracted 220 mg proteins/g VSS without biomass inactivation. Here, six minutes discontinuous sonication (3 2 min) under an intermediate power density (0.75 W/mL) extracts 140 mg equiv. BSA/g VSS without cell leakage. These results are in agreement with literature. Sonication is a more rapid, efficient and reliable mechanical extraction method for extracellular proteins compared to Blender applications and shall be selected for the multimethod extraction protocol.
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471 Table 2 Comparison of different methods for protein extraction Protein (g equiv. BSA/g VSS)
G6 P-DH Activity (nmol NADH/min/g VSS)
Sonication 2 min 4 min 6 min 8 min 10 min
97 ± 3 121 ± 0 137 ± 1 149 ± 2 162 ± 1
0 0 0 0 0
Blender 5 min 10 min 15 min 20 min 25 min
73 ± 1 78 ± 0 85 ± 2 82 ± 1 157 ± 2
0 0 0 0 0
Tween 0.25% 1 h 0.5% 1 h 0.5% 2 h 1% 1 h 1% 2 h
19 ± 2 36 ± 0 53 ± 0 52 ± 1 60 ± 1
0.05 0.74 0.23 0.39 0.13
Ethanol 20% 1 h 20% 2 h 40% 1 h 40% 2 h 50% 1 h
36 ± 0 44 ± 0 19 ± 0 50 ± 0 24 ± 1
0 0 0 0 0
CER 45 min 1h30 2h15 3h 3h45
39 ± 1 37 ± 1 44 ± 0 47 ± 0 50 ± 1
0 0 0 0 0
EDTA 0.5% Tris 1% Tris 2% Tris 2% NaOH 0.15 mol L1 2% NaOH 1 mol L1 2% NaOH 2 mol L1
64 ± 2 68 ± 5 133 ± 70 3±5 476 ± 16 592 ± 32
0 0 0 0 2.47 10.50
Protein concentration and G6 P-DH activity were measured in each soluble extract. Table 3 Range of protein/sugar (P/S) ratio and tendency Extraction method
P/S Ratio
Tendency
Sonicationa Blenderb Tweenb Ethanolb EDTA in Trisb CERa
12–7 15–8 2–4 2–6 4–10 10–6
Decrease Decrease Increase Variable Increase Decrease
Ratios obtained in each soluble extract from each extraction method in different conditions. a Increasing treatment time. b Increasing concentrations.
The non ionic surfactant Tween 20 and ethanol solvent were both studied for hydrophobic EPS extraction. Tween and ethanol treatments extract less proteins compared to sonication. Table 2 shows that Tween 20 releases increasing
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proportions of proteins, from 20 to 60 mg equiv. BSA/g VSS with increasing Tween concentrations and with increasing extraction time. Proteins are preferentially extracted compared to polysaccharides (P/S increase from 2 to 4, table 3). The effect of increasing Tween concentrations is more pronounced than increasing time applications. Increasing Tween concentration from 0.25% to 1% (1 h treatment) doubles protein content in the extract. Ethanol on the other hand, extracts less protein compared to Tween. Moreover, extracted proteins follow a discontinuous and incoherent profile through increasing ethanol concentrations and time applications (Table 2). Thus, Tween treatment is considered as more reliable compared to the ethanol extraction method. Yet, intracellular G6P-DH activity was recorded in all soluble extracts from 0.25% to 1% Tween treatments (between 0.2 and 0.7 nmol NADH/min/g VSS). Considering the small absorbance/ minute values recorded for these extracts, less than 0.003, little attention should be given to the actual differences of G6P-DH activity for all Tween extraction conditions. Previous studies have shown the effect of Tween treatments on cell membranes: 0.1% of Tween 80 affected cell permeability (Zhang et al., 2003) and disrupted cells after 3 days treatment (Reese and Maguire, 1969). One hour application with 0.25% of Tween 20 is shown as a good compromising procedure for minimum cell disruption and reduction in chemical and time consumption and will therefore be chosen for hydrophobic EPS extraction. CER is the most popular extraction method used for EPS extraction from activated sludge (Frohlund et al., 1995; Jahn and Nielsen, 1995; Sheng et al., 2006; Wile´n et al., 2003). The organic ligand, tetra sodium salt EDTA is also studied as an ion-associated extraction method (Brown and Lester, 1980). Both methods are likely to interact with ionic bonds in the EPS matrix (Park and Novak, 2007) and will be compared towards protein extraction efficiencies. In literature, EDTA is reported as less efficient than CER (Comte et al., 2006; Zhang et al., 1999). Here, the obtained results show, on the contrary, a greater protein release with EDTA in Tris buffer compared to CER treatments (Table 2). Increasing concentrations of EDTA in Tris extract between 70 and 130 mg equiv. BSA/g VSS without cell leakage. EDTA 2% in Tris appears as the most efficient combination. In order to increase the effect of EDTA, NaOH was added at various concentrations (0.15–2 N) and these treatments were compared with the effect of EDTA in Tris buffer alone. The EDTA (2%) extraction efficiency is increased with the presence of NaOH particularly 1 N and 2 N (between 500 and 600 mg equiv. BSA/g VSS). However, a strong cell lysis is measured (up to 10 nmol NADH/min/g VSS), showing that these NaOH–EDTA conditions are inappropriate for extracting only extracellular substances. EDTA 2% in Tris buffer remains the best combination for protein release without cell damage and therefore will be selected. Chemical interferences were controlled towards protein determination by BCA method. Standard curves of BSA
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in PBS, in Tween 0.25% and in EDTA 2% in Tris–HCl showed good linearity (R2 = 1, 0.98 and 0.99, respectively), but with a 7% protein underestimation in Tween and 4% overestimation in EDTA compared to BSA in PBS buffer. Thus, protein contents in Tween and EDTA extracts were corrected accordingly. In conclusion, sonication (3 2 min), Tween (0.25% in PBS) and EDTA (2% in Tris buffer) are three different extraction methods allowing maximal extracellular protein release with minimal contamination by intracellular proteins.
3.2. Protocol extraction strategy The three selected extraction methods will be used to establish a multi-method extraction strategy. The suggested extraction protocol will undertake each method separately with intermediate centrifugation steps, in order to extract three different soluble EPS fractions from the same sample: sonication-extract, EDTA-extract and Tween-extract. This strategy is meant to disrupt, respectively, random, ionic and hydrophobic interactions. Sonication will be applied at first in order to disaggregate the sludge flocs and increase contact between the EPS matrix and chemicals, i.e. EDTA and Tween. Two sequences were tested: sonication, EDTA then Tween and sonication, Tween then EDTA. Exchanging order between EDTA and Tween did not affect the total amount of extracted proteins by the protocol. The multi-method protocol was performed on two different sludge samples, sludge 1 and sludge 2 collected from pilots fed, respectively, with domestic or synthetic effluent. The total amount of extracted proteins obtained by the multimethod protocol sums up to 264 ± 10 and 191 ± 45 mg equiv. BSA/g VSS for sludge 1 and sludge 2, respectively, (Fig. 1). Variation between both sludge samples (32%) is greater than the error brought by the extraction protocol (4 and 23% for sludge 1 and 2, respectively). These results show that protein content in sludge 1 fed on urban waste water is stronger than in sludge 2 fed on a synthetic solution.
Tween
mg eq. BSA / g VSS
300
EDTA
250
Sonication
200 150 100 50 0 Protocol sludge 1
Protocol sludge 2
Sonication x3 EDTA x3 sludge 2 sludge 2
Tween x3 sludge 2
Fig. 1. Protein content in extracts obtained by the multi-method extraction protocol applied on sludge 1 and on sludge 2, and by the stepwise extraction strategy applied on sludge 2 samples.
On the other hand, the total extracted polysaccharide content revealed no difference between both sludge samples with 28 ± 1 and 27 ± 4 mg equiv. BSA/g VSS for sludge 1 and 2, respectively. No significant G6P-DH activity was measured in the obtained extracts (results inferior to 0.1 nmol NADH/min/g VSS). A rapid APIZYM screen over nineteen different enzymatic activities was performed on all the extracts obtained by the multi-method extraction protocol. Enzymes detected in the whole sludge were also detected in all extracts which informs that protein structure was not significantly affected by the extraction protocol. A stepwise extraction strategy is defined as the same extraction method applied three times on the same sample (sludge 2). Stepwise extraction strategies based on sonication (3 2 min), EDTA (2% in Tris) and Tween (0.25%) were investigated in order to determine whether the multi-method extraction protocol is as efficient in protein extraction as repeating one single method. As shown in Fig. 1, when repeating three times EDTA and Tween treatments, total protein content in extracts reached, respectively, 70 and 50 mg equiv. BSA/g VSS, which is less than the extraction protocol (191 ± 45 mg equiv. BSA/g VSS). No G6P-DH activity was found in either extracts. It is therefore clear that a prior mechanical disruption method (sonication) in the extraction protocol increases the protein extraction efficiency of EDTA and Tween methods. Although tripled sonication is as efficient in protein extraction (210 mg equiv. BSA/g VSS, without cell damage) as the multi-method extraction protocol, concern should now be given towards the qualitative aspect. Recent studies have shown the specificity of extraction methods towards certain types of EPS (Comte et al., 2007; Park and Novak, 2007). Single method extraction protocols could be liable in restricting protein content in extracts to one category of proteins. However, this argument should be confirmed with further qualitative analyses of extracts obtained from stepwise and multi-method protocols.
3.3. Validation of protocol extraction efficiency Protocol extraction efficiency was evaluated by adding known proportions of BSA protein in sludge samples, and then by measuring protein release in protocol extracts. For three different concentrations of added BSA (0.5, 1 and 2 g/L), a yield of 78% is obtained for BSA recovery using the extraction protocol. Therefore, 22% of the added BSA is systematically withheld in the sludge after one protocol extraction treatment. This suggests that more proteins in the EPS matrix could still be released with other successive extractions, and was confirmed by repeating the multi-method extraction protocol three times on the same sludge samples (sludge 1 and 2). Both sludge samples offer the same decreasing profile of protein content in all successive extracts (Fig. 2). Extractions as well as protein measurements were done in duplicate for error estimations.
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471 100
Sludge 1 Sludge 2
mg eq. BSA/g VSS
80
60
40
20
0
US-1 Tween-1 Edta-1 US-2 Tween-2 Edta-2 US-3 Tween-3 Edta-3
Protocol 1
Protocol 2
Protocol 3
Fig. 2. Multi-method protocol repetition: protein content in each extract from sludge 1 (j) and sludge 2 ( ), after three successive protocol treatments (Protocol 1, 2 and 3). Error bars represent error on doubled extractions and doubled measurements.
The total amount of proteins extracted by the protocol repeated three times sums up to 460 ± 10 and 348 ± 11 mg equiv. BSA/g VSS for sludge 1 and 2, respectively. Total polysaccharide content in all extracts is lower and sums up to 68 ± 3 and 53 ± 1 mg equiv. glucose/g VSS for sludge 1 and 2, respectively. The large majority of proteins over polysaccharides in these sludge extracts show stronger P/S ratios compared to literature (6.6 and 7 for sludge 1 and 2, respectively). In literature, P/S ratio values obtained by one single extraction method varies between 1.6 and 4.9 depending on the extraction method and sludge
Table 4 Protein content in the soluble organic fraction
Sludge VSS (g/L) Extracted VSS (g/L) mg Proteins/g extracted VSS (BCA method) mg Proteins/g extracted VSS (TNK method)
Sludge 1
Sludge 2
3.22 1.85 803 876
6.07 3.25 650 –
The extracted VSS was determined by the difference of VSS values between the initial sludge and the final pellet obtained after three protocol treatments. Protein determination by the total nitrogen Kjeldahl method (TNK) was evaluated by difference between the initial sludge and the final pellet obtained after three protocol treatments.
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origin (Comte et al., 2006, 2007; Park and Novak, 2007; Sponza, 2002). This could suggest either that our protocol extracts preferentially proteins from sludge samples or that proteins are particularly dominant in the investigated samples. Protein content in the extracted organic soluble fraction (VSSsoluble) was determined by BCA and NTK methods (Table 4). Both methods showed comparable results, between 803 and 876 mg proteins/g VSSsoluble. This reveals that between 80 and 87% of extracted molecules are proteins. Protocol repetition released 264, 134 then 62 mg proteins/g VSS for sludge 1 and 192, 100 then 56 mg proteins/g VSS for sludge 2. Interestingly, each successive extraction protocol releases around 50% of the proteins obtained from the previous extraction protocol (Fig. 2). This trend can describe a predicting model which calculates for each protocol application (n) the total amount of extracted protein (Pn) depending on the amount of proteins extracted by the first protocol application (P1) and the constant decay rate (a): Pn = P1 a (n1). Decay rates (0.48 and 0.55) and P1 values (269 and 192) for sludge 1 and sludge 2, respectively, were solved for an optimal curve fit, i.e. minimum R([Pexperimental][Psimulated])2. Model construction was performed on three data sets for sludge 1 and one data set for sludge 2. Model validation was carried out with one data set (not used for the model construction) for each sludge sample (Table 5). Maximum error between model predictions and experimental values reached 17% which is acceptable for model validation and model reliability (Table 5). By running this model, protein exhaustion is reached after 9 extraction protocols for both sludge samples (Fig 3). Hence, the total amount of soluble proteins should sum up to 516 and 423 mg equiv. BSA/g VSS for sludge 1 and sludge 2, respectively. In general terms, we can estimate that the first extraction protocol applied on a sludge sample extracts between 45% and 49% of proteins in sludge. Therefore, this multimethod extraction protocol is relatively efficient by providing a representative quantity of soluble proteins from sludge. Moreover, by repeating the protocol three times, up to 82–89% of the total soluble proteins can be harvested. The multi-method extraction strategy is also likely to offer different types of proteins which can stand for the initial protein pool found in the sludge.
Table 5 Model validation on sludge 1 and sludge 2 samples n
1 2 3
Sludge 1
Sludge 2
Pexperimental
Psimulated
Error (%)
Pexperimental
Psimulated
Error (%)
253 138 73
269 129 62
6 7 17
192 93 56
192 106 58
0 12 4
Concentrations are indicated as mg equiv. BSA/g VSS. Sludge 1 solved parameters: a = 0.48 and P1: 268, sludge 2 solved parameters: a = 0.55 and P1 = 192.
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P : [protein] mg eq. BSA/g VSS
300
Sludge 1 (− model) Sludge 2 (− model)
250 200 150 100 50 0
1
2
3
4 5 6 X : number of protocols
7
8
9
Fig. 3. Exponential curve model (line) fitted to experimental data from sludge 1 (j) and sludge 2 ( ): protein content in extracts vs. number of protocols. Black curve line (—) for sludge 1 and grey curve line ( ) for sludge 2.
4. Conclusion An extraction protocol was developed to improve protein extraction from sludge. The experimental design was established by selecting different extraction methods which act on the various chemical interactions found in activated sludge aggregates. Regarding the extracellular protein extraction efficiency (maximal protein release with minimal cell lysis), discontinuous sonication (3 2 min), Tween (0.25% 1 h) and EDTA (2% in Tris buffer, 1 h) were selected. These three methods did not counteract cell lysis measurements nor did they interfere in protein determination by BCA method. Extraction methods applied in series as described in the multi-method protocol are more efficient in protein release than EDTA and Tween methods applied alone. Protocol repetition showed that proteins can still be released from the sludge aggregates. Protein release vs. number of protocols progressively decreased and fitted a specific exponential model. Model predictions show that protein exhaustion is reached after 9 protocol applications. This suggested multi-method extraction protocol applied once releases between 45% and 49% of the total amount of extractable proteins from a sludge sample, which is quantitatively significant for sludge representativeness. Considering the specificity of extraction methods towards certain types of EPS, we can consider that the obtained soluble protein pool is also qualitatively characteristic of the studied sludge samples. Further qualitative studies are to be undertaken for fingerprint diagnosis. References Azeredo, J., Oliveira, R., Lazarova, V., 1998. A new method for extraction of exopolymers from activated sludges. Water Sci. Technol. 37, 367– 370. Brown, M.J., Lester, J.N., 1980. Comparison of bacterial extracellular polymer extraction methods. Appl. Environ. Microbiol. 40, 179–185. Bura, R., Cheung, M., Liao, B., Finlayson, J., Lee, B.C., Droppo, I.G., Leppard, G.G., Liss, S.N., 1998. Composition of extracellular poly-
meric substances in the activated sludge matrix. Water Sci. Technol. 37, 325–333. Cadoret, A., Conrad, A., Block, J.-C., 2002. Availability of low and high molecular weight substrates to extracellular enzymes in whole and dispersed activated sludges. Enzyme Microbiol. Technol. 31, 179–186. Comte, S., Guibaud, G., Baudu, M., 2006. Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexion properties. Part I. Comparison of the efficiency of eight EPS extraction methods. Enzyme Microb. Technol. 38, 237–245. Comte, S., Guibaud, G., Baudu, M., 2007. Effect of extraction method on EPS from activated sludge: an HPSEC investigation. J. Hazard. Mater. 140, 129–137. Dey, E.S., Szewczky, E., Wawrzymczyk, J., Norrlow, O., 2006. A novel approach for characterization of exopolymeric material in sewage sludge. J. Res. Sci. Technol. 3, 97–103. Denecke, M., 2006. Protein extraction from activated sludge. Water Sci. Technol. 54, 175–181. Dignac, M.-F., Urbain, V., Rybacki, D., Bruchet, A., Snidaro, D., Scribe, P., 1998. Chemical description of extracellular polymers: implication on activated sludge floc structure. Water Sci. Technol. 38, 45–53. Dreywood, R., 1946. Qualitative test for carbohydrate material. Ind. Eng. Chem. 18, 499. Frohlund, B., Palmgren, R., Keiding, K., Nielsen, P.H., 1995. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 30, 1749–1758. Jahn, A., Nielsen, P.H., 1995. Extraction of extracellular polymeric substances (EPS) from biofilms using cation exchange resin. Water Sci. Technol. 32, 157–164. Jahn, A., Nielsen, P.H., 1997. Cell biomass and exopolymer composition in sewer biofilms. Water Sci. Technol. 37, 17–24. Jung, J., Xing, X.H., Matsumoto, K., 2002. Recoverability of protease released from disrupted excess sludge and its potential application to enhance hydrolysis of proteins in wastewater. Biochem. Eng. J. 10, 67– 72. Lessie, T.G., Vander Wyk, J.C., 1972. Multiple forms of Pseudomonas multivarans glucose-6-phosphate and 6-phosphogluconate deshydrogenase: differences in size, pyridine nucleotide specificity and susceptibility to inhibition by adenosine 50 -triphosphate. J. Bacteriol. 110, 1107–1117. Liao, B.Q., Allen, D.G., Droppo, I.G., Leppard, G.G., Liss, S.N., 2001. Surface properties of sludge and their role in bioflocculation and settleability. Water Res. 35, 339–350. Liu, H., Fang, H.P., 2002. Extraction of extracellular polymeric substances (EPS) of sludges. J. Biotechnol. 95, 249–256. Martinez, F.O., Lema, J., Me´ndez, R., Cuervo-Lopez, F., 2004. Role of exopolymeric protein on the settleability of nitrifying sludges. Bioresour. Technol. 94, 43–48. Masse´, A., 2004. Biore´acteur a` membranes immerge´es pour le traitement des eaux re´siduaires urbaines: spe´cificite´s physico–chimiques du milieu biologique et colmatage, Doctorat Sciences des proce´de´s. Institut National des Sciences Applique´es, Toulouse. Meng, F., Zhang, H., Yang, F., Zahng, S., Li, Y., Zhang, X., 2006. Identification of activated sludge properties affecting membrane fouling in submerged membrane bioreactors. Sep. Purif. Technol. 51, 95–103. Park, C., Novak, J.T., 2007. Characterization of activated sludge exocellular polymers using several cation-associated extraction methods. Water Res. 41, 1679–1688. Ras, M., Girbal-Neuhauser, E., Paul, E., Spe´randio, M., Lefebvre, D., in press. Protein extraction from activated sludge: an analytical approach. Water Res. Reese, E.T., Maguire, A., 1969. Surfactants as stimulants of enzyme production by microorganisms. Appl. Microb. 17, 242–245. Rideau, J.P., Morfaux, J.N., 1976. Etude des polisides du mucilage des boues actives. Polisides study on activated sludge mucilage. Water Res. 10, 999–1003. Salhani, N., Uelker-Deffur, A., 1997. Improved quantification of aggregated bacteria by combined enzymatic and mechanical treatment of
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471 flocs and biofilms from a rotating drum bioreactor. Water Res. 32, 1287–1295. Sesay, M.L., Ozcengiz, G., Sanin, F.D., 2006. Enzymatic extraction of activated sludge extracellular polymers and implications on bioflocculation. Water Res. 40, 1359–1366. Sheng, G.P., Yu, H.Q., Li, X.Y., 2006. Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS). Biotechnol. Bioeng. 93, 1095–1102. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using Bicinchoninic Acid. Anal. Biochem. 150, 76–85. Sponza, D.T., 2002. Extracellular polymer substances and physicochemical properties of floc in steady and unsteady-state activated sludge systems. Proc. Biochem. 37, 983–998.
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Urbain, V., Block, J.C., Manem, J., 1993. Bioflocculation in activated sludge: an analytic approach. Water Res. 27, 829–838. Wile´n, B.M., Jin, B., Lant, P., 2003. The influence of key chemical constituents in activated sludge on surface and flocculating properties. Water Res. 37, 2127–2139. Zhang, H., Yao, M., Morrison, R.A., Chong, S., 2003. Commonly used surfactant, tween 80, improves absorption of P-glycoprotein substrate, digoxin, in rats. Arch. Pharm. Res. 26, 768–772. Zhang, P., Zhang, G., Wang, W., 2007. Ultrasonic treatment of biological sludge: floc disintegration, cell lysis and inactivation. Bioresour. Technol. 98, 207–210. Zhang, X., Bishop, P.L., Kinkle, B.K., 1999. Comparison of extraction methods for quantifying extracellular polymers in biofilms. Water Sci. Technol. 39, 211–218.
Bioresource Technology 99 (2008) 7464–7471
A high yield multi-method extraction protocol for protein quantification in activated sludge Ras Monique a, Girbal-Neuhauser Elisabeth a, Paul Etienne b, Lefebvre Dominique a,* a
Laboratoire de Biologie applique´e a` l’Agro-alimentaire et a` l’Environnement, Institut Universitaire de Technologie, Universite´ Toulouse III, 24 Rue d’Embaque`s, 32000 Auch, France b Laboratory of Biosystems and Process Engineering, UMR5504 CNRS/INSA and UMR792 INRA/INSA, 135 avenue de Rangueil, 31077 Toulouse cedex 4, France Received 6 September 2007; received in revised form 14 February 2008; accepted 17 February 2008 Available online 18 April 2008
Abstract A multi-method extraction protocol based on mechanical, ionic and hydrophobic methods was investigated on two types of activated sludge samples. Extraction methods were chosen with regards to optimal protein yield without cell disruption. Sonication, EDTA and Tween extraction methods were selected and combined. The total amount of protein released by the multi-method protocol sums up to 191 and 264 mg equiv. BSA/g VSS for the two different sludge samples. Protocol repetition on the same sample showed that protein yield after each successive protocol fitted an exponential curve model. The total amount of extractable proteins was evaluated by model predictions, 423 and 516 mg equiv. BSA/g VSS for the two sludge samples. The multi-method extraction protocol appears relevant for harvesting a representative quantity of proteins from the original sample (45–49%), moreover the multi-method criterion of the protocol also offers a heterogeneous pool of proteins. Thus, further qualitative studies may not be biased by the extraction protocol. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Exopolymeric protein; Physicochemical treatment; Extraction strategy; Activated sludge
1. Introduction In biological wastewater treatment systems, bacteria aggregate together in order to form suspended floc structures or granules as well as attached biofilms. Several studies have shown that extracellular polymeric substances (EPS) take part in forming a gel-like matrix which acts as cement in bacterial aggregates. EPS are molecules produced by bacteria, brought by the incoming wastewater (Urbain et al., 1993) or are released from cells when lysis occurs. Humic acids (Frohlund et al., 1995), polysaccharides (Rideau and Morfaux, 1976), proteins (Dignac et al., 1998), nucleic acids (Jahn and Nielsen, 1997) and other non determined molecules (Wile´n et al., 2003) have been reported as EPS compounds in the matrix. In spite *
Corresponding author. Tel.: +33 5 62 61 05; fax: +33 5 62 61 63 01. E-mail address: [email protected] (L. Dominique).
0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.02.025
of this wide range of macromolecules, recent studies have shown the relative importance of proteins in wastewater treatment systems from a quantitative aspect (Liao et al., 2001; Sponza, 2002), as well as from a functional point of view (Denecke, 2006). Proteins have been proven to increase biofilm adherence in bioreactors and cause fouling which has considerable consequences on water treatment by membrane filtration (Masse´, 2004; Meng et al., 2006). Enzyme activity measurements showed that enzymes were integrated in the protein EPS pool (Frohlund et al., 1995) and that inherent proteases were the most active enzymes (Cadoret et al., 2002; Jung et al., 2002). Martinez et al. (2004) showed that protein content was linked to sludge processes, and that the types of proteins were rather linked to sludge settleability. Total protein quantification and characterisation could therefore bring relevant information on bacterial aggregation processes as well as on treatment efficiency.
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471
Protein quantification is generally carried out by a prior extraction step. The most popular EPS extraction method is Cation Exchange Resin (CER), (Frohlund et al., 1995). Over many studies using this same method, proteins are showed as the predominant EPS in biofilm (Jahn and Nielsen, 1995) and in aerobic activated sludge extracts (Bura et al., 1998; Frohlund et al., 1995; Liu and Fang, 2002; Wile´n et al., 2003). Sonication and formaldehyde extraction methods have also shown that proteins were the most represented EPS in activated sludge samples (Comte et al., 2006; Liu and Fang, 2002). However other extraction methods such as EDTA applied on the same sludge samples released more polysaccharides and humic substances than proteins (Denecke, 2006; Liu and Fang, 2002). The wide variety of extraction methods used in literature could partly explain the large variation of protein content reported in sludge, between 3.9 and 510 mg/g VSS (Liu and Fang, 2002; Masse´, 2004; Wile´n et al., 2003), without omitting other factors such as sludge origin and process designs which are also described as influencing EPS content (Comte et al., 2006; Sponza, 2002). However, total protein quantification in sludge via extracts can be questionable since only one extraction method is carried out. Moreover, proteins are characterised by ionic, hydrophobic and neutral amino acids. Therefore, specific bonds such as electrostatic, hydrophobic and low energy hydrogen or Van der Waals interactions play a major role in linking proteins to the EPS matrix. Hence, the use of one type of extraction method is likely to promote the study of ‘‘one kind of protein” in quantification or characterisation studies. As shown by Dignac et al. (1998), extracellular proteins released by CER contained a majority of negatively charged amino-acids. General remarks in literature stipulate that quantification and characterisation of EPS compounds in sludge are often biased by extraction procedures which do not take into account the specificity of extraction methods towards certain types of EPS (Comte et al., 2007; Dignac et al., 1998). Thus, in order to assure a homogenous protein pool in extracts, it would be appropriate to apply three different extraction methods which aim on the three major types of bonds linking proteins to the EPS matrix, i.e. low energy, hydrophobic and ionic interactions. Protein quantification in sludge and sludge extracts is frequently carried out by modified Lowry method (Frohlund et al., 1995). However, studies have shown that bicinchoninic acid (BCA) is less sensitive to chemical extraction methods (i.e. EDTA), offers a better protein to protein response (Smith et al., 1985), as well as, a lower quantification limit (Ras et al., in press). This present paper offers a multi-method extraction protocol based on three types of extraction methods (mechanical, ionic and hydrophobic) in order to obtain a diversified protein pool which can stand for the studied sludge. Several extraction methods were chosen to be investigated according to their frequency in literature and care towards cell disruption and protein denaturation. This includes mechanical and ionic extraction methods such as
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sonication (Azeredo et al., 1998; Comte et al., 2006; Dey et al., 2006), cation exchange resin (Cadoret et al., 2002; Comte et al., 2006; Park and Novak, 2007; Sesay et al., 2006), and EDTA (Comte et al., 2006; Liu and Fang, 2002; Sheng et al., 2006). EPS extraction methods which aim hydrophobic interactions in sludge are randomly found in literature. However, among the several non ionic surfactants employed in previous studies, Tween and ethanol have been investigated as hydrophobic extraction methods. Each method was controlled for their possible effect on cell disruption and protein denaturation, as well as, for their interference towards the protein quantification method. The most efficient methods were selected and applied in series according to an original three step extraction strategy. This study will also give more information on the total amount of extractable proteins which can be released from activated sludge. 2. Methods 2.1. Activated sludge samples: origin and handling The extraction methods were undertaken on sludge samples from two different pilots. Organic supply and sludge characteristics are described in Table 1. Samples were collected the previous day, stored at 4 °C and diluted with a Phosphate Buffer Saline (PBS, pH 7) to 5 g VSS/L of sludge before each extraction. Total solids (TS) were measured by desiccating at 105 °C and volatile suspended solids (VSS) were measured by calcinate at 510 °C. 2.2. Extraction instruments and chemicals Extraction tests were undertaken on 50 mL sludge samples. Stirring intensities were all performed at 500 rpm. 2.2.1. Mechanical extractions Ultrasounds were carried out with a vibra cell sonication probe from Bioblock (2.5 mm 49 mm cylinder). Table 1 Pilot and sludge characteristics Pilot 1
Pilot 2
Charge Treated effluent
g DCO/L/d Type
0.2 Domestic wastewater
Sludge residential time (SRT) Sludge concentration Dissolved oxyge`ne Oxygenation time Anoxic time Volatile suspended solids/total solids Temperature (°C)
Days
8
0.06 Synthetic (propionate/ acetate/starch/ ethanol) 8
g TS/L mg/L min min g VSS/g TS
1.5 3 40 40 0.8–0.85
1.5 3 40 40 0.8–0.85
20
20
20
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Sonication was performed at 37 W (0.092 W/mm2), with 10 mm probe tip dipped in 50 mL sludge samples (0.75 W/mL power density). Sonication times were investigated by discontinuous applications with 2 min interval. Time applications varied from 2 min 1 to 2 min 5. Blender treatments were performed on 50 mL of sludge with a waring commercial laboratory blender. Time applications varied from 5 min 1 to 5 min 5 with 5 min interval. For both methods, samples were kept at 4 °C during extraction. 2.2.2. Ionic extractions CER (Dowex, Fluka) was used at 70 g/g VSS (Frohlund et al., 1995) and ethylene diamino tetra acetic acid (EDTA, Acros) was prepared at concentrations ranging from 0.5% to 2% in Tris–HCl buffer 0.3 M pH 8 or in NaOH at concentrations between 0.15 and 2 M. CER was tested for different contact times from 45 min to 3 h 45 min. EDTA tests were carried out for 1 h application. 2.2.3. Hydrophobic extractions Ethanol (Acros) was tested at concentrations between 20% and 50% in PBS buffer and Tween 20 (Acros) was prepared in PBS buffer at concentrations between 0.25% and 1%. Contact time was investigated for both methods, between 1 and 2 h applications. 2.3. EPS quantification Protein measurements were performed on all soluble extracts with the bicinchoninic acid or BCA reagent (Sigma), according to Smith et al. (1985). Bovine serum albumin (BSA) was used as standard. Total nitrogen (TN) measurements by Kjeldahl method was undertaken for indirect protein determination. Polysaccharides were determined using the anthrone method (Dreywood, 1946). Glucose was used as standard. 2.4. Enzyme assays The intracellular glucose-6-phosphate dehydrogenase (G6P-DH) activity measurements were carried out on extracts in order to detect cell lysis. G6P-DH measurements were applied according to Lessie and Vander Wyk (1972). Substrate solutions were prepared with 0.2 M Tris–HCl pH 8.5, 0.2 M 2-mercaptoethanol (Acros), 0.0005 M of nicotine adenine dinucleotide (NAD, Acros) and 0.01 M D-glucose-6-phosphate (Fluka). The supernatant extracts (200 lL) were incubated with 800 lL of substrate solution at room temperature for 30 min. Absorbance values (340 nm) were recorded every 10 s. Results are expressed as units: nmol of nicotine adenine dinucleotide hydrogen (NADH) formed per minute per gram of volatile suspended solids (VSS). An enzymatic screening of 19 different enzymes was performed using the semi quantitative micro method APIZYM (Biome´rieux).
All absorbance measurements were undertaken with a Miltron Roy recordable spectrophotometer (Spectronik 1201).
3. Results and discussion 3.1. Selection of extraction methods In order to offer an efficient protocol for extracting EPS from sludge, physical and chemical treatments were investigated. For each kind of treatment, two methods were compared in order to select the most relevant method in terms of EPS content in extracts and preservation of cell integrity. Since Lowry quantification method is particularly affected by EDTA (Comte et al., 2006; Smith et al., 1985), BCA method was chosen for protein quantification whereas sugars were quantified by the anthrone method. Table 2 presents the amount of proteins and the G6PDH activity obtained in each extract. Mechanical disintegration acts randomly on the different interactions holding the EPS matrix together. Discontinuous sonication during one to five times 2 min treatments was compared to a Blender disruption method for protein release from activated sludge samples. Table 2 shows that sonication releases increasing proportions of proteins with treatment time, from 100 mg equiv. BSA/g VSS for 1 2 min treatment to 160 mg equiv. BSA/g VSS for 5 2 min treatment. The protein/sugar ratio (P/S) decreases from 12 to 7 showing that extended sonication extracts preferentially polysaccharides (Table 3). Blender, on the other hand, releases stable but relatively less proteins compared to sonication (70–80 mg equiv. BSA/g VSS, Table 2). Nevertheless, 25 min blender application discharges spontaneously large proportions of proteins which are comparable to 5 2 min discontinuous sonication. However, ultrasounds reveal a more reliable protein extraction profile with a more convenient and rapid procedure compared to Blender treatments. G6P-DH activity measurement indicated no intracellular leakage in either soluble extracts, whether from sonication or from Blender treatments. It is known that ultrasound power densities affect cell lysis (Zhang et al., 2007). Salhani and Uelker-Deffur (1997) have shown that two minutes sonication under 1.3 W/mL power density was defined as the best mechanical method for floc disruption without reducing cell viability. Zhang et al. (2007) showed that 6 min sonication under 0.5 W/mL extracted 220 mg proteins/g VSS without biomass inactivation. Here, six minutes discontinuous sonication (3 2 min) under an intermediate power density (0.75 W/mL) extracts 140 mg equiv. BSA/g VSS without cell leakage. These results are in agreement with literature. Sonication is a more rapid, efficient and reliable mechanical extraction method for extracellular proteins compared to Blender applications and shall be selected for the multimethod extraction protocol.
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471 Table 2 Comparison of different methods for protein extraction Protein (g equiv. BSA/g VSS)
G6 P-DH Activity (nmol NADH/min/g VSS)
Sonication 2 min 4 min 6 min 8 min 10 min
97 ± 3 121 ± 0 137 ± 1 149 ± 2 162 ± 1
0 0 0 0 0
Blender 5 min 10 min 15 min 20 min 25 min
73 ± 1 78 ± 0 85 ± 2 82 ± 1 157 ± 2
0 0 0 0 0
Tween 0.25% 1 h 0.5% 1 h 0.5% 2 h 1% 1 h 1% 2 h
19 ± 2 36 ± 0 53 ± 0 52 ± 1 60 ± 1
0.05 0.74 0.23 0.39 0.13
Ethanol 20% 1 h 20% 2 h 40% 1 h 40% 2 h 50% 1 h
36 ± 0 44 ± 0 19 ± 0 50 ± 0 24 ± 1
0 0 0 0 0
CER 45 min 1h30 2h15 3h 3h45
39 ± 1 37 ± 1 44 ± 0 47 ± 0 50 ± 1
0 0 0 0 0
EDTA 0.5% Tris 1% Tris 2% Tris 2% NaOH 0.15 mol L1 2% NaOH 1 mol L1 2% NaOH 2 mol L1
64 ± 2 68 ± 5 133 ± 70 3±5 476 ± 16 592 ± 32
0 0 0 0 2.47 10.50
Protein concentration and G6 P-DH activity were measured in each soluble extract. Table 3 Range of protein/sugar (P/S) ratio and tendency Extraction method
P/S Ratio
Tendency
Sonicationa Blenderb Tweenb Ethanolb EDTA in Trisb CERa
12–7 15–8 2–4 2–6 4–10 10–6
Decrease Decrease Increase Variable Increase Decrease
Ratios obtained in each soluble extract from each extraction method in different conditions. a Increasing treatment time. b Increasing concentrations.
The non ionic surfactant Tween 20 and ethanol solvent were both studied for hydrophobic EPS extraction. Tween and ethanol treatments extract less proteins compared to sonication. Table 2 shows that Tween 20 releases increasing
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proportions of proteins, from 20 to 60 mg equiv. BSA/g VSS with increasing Tween concentrations and with increasing extraction time. Proteins are preferentially extracted compared to polysaccharides (P/S increase from 2 to 4, table 3). The effect of increasing Tween concentrations is more pronounced than increasing time applications. Increasing Tween concentration from 0.25% to 1% (1 h treatment) doubles protein content in the extract. Ethanol on the other hand, extracts less protein compared to Tween. Moreover, extracted proteins follow a discontinuous and incoherent profile through increasing ethanol concentrations and time applications (Table 2). Thus, Tween treatment is considered as more reliable compared to the ethanol extraction method. Yet, intracellular G6P-DH activity was recorded in all soluble extracts from 0.25% to 1% Tween treatments (between 0.2 and 0.7 nmol NADH/min/g VSS). Considering the small absorbance/ minute values recorded for these extracts, less than 0.003, little attention should be given to the actual differences of G6P-DH activity for all Tween extraction conditions. Previous studies have shown the effect of Tween treatments on cell membranes: 0.1% of Tween 80 affected cell permeability (Zhang et al., 2003) and disrupted cells after 3 days treatment (Reese and Maguire, 1969). One hour application with 0.25% of Tween 20 is shown as a good compromising procedure for minimum cell disruption and reduction in chemical and time consumption and will therefore be chosen for hydrophobic EPS extraction. CER is the most popular extraction method used for EPS extraction from activated sludge (Frohlund et al., 1995; Jahn and Nielsen, 1995; Sheng et al., 2006; Wile´n et al., 2003). The organic ligand, tetra sodium salt EDTA is also studied as an ion-associated extraction method (Brown and Lester, 1980). Both methods are likely to interact with ionic bonds in the EPS matrix (Park and Novak, 2007) and will be compared towards protein extraction efficiencies. In literature, EDTA is reported as less efficient than CER (Comte et al., 2006; Zhang et al., 1999). Here, the obtained results show, on the contrary, a greater protein release with EDTA in Tris buffer compared to CER treatments (Table 2). Increasing concentrations of EDTA in Tris extract between 70 and 130 mg equiv. BSA/g VSS without cell leakage. EDTA 2% in Tris appears as the most efficient combination. In order to increase the effect of EDTA, NaOH was added at various concentrations (0.15–2 N) and these treatments were compared with the effect of EDTA in Tris buffer alone. The EDTA (2%) extraction efficiency is increased with the presence of NaOH particularly 1 N and 2 N (between 500 and 600 mg equiv. BSA/g VSS). However, a strong cell lysis is measured (up to 10 nmol NADH/min/g VSS), showing that these NaOH–EDTA conditions are inappropriate for extracting only extracellular substances. EDTA 2% in Tris buffer remains the best combination for protein release without cell damage and therefore will be selected. Chemical interferences were controlled towards protein determination by BCA method. Standard curves of BSA
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R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471
in PBS, in Tween 0.25% and in EDTA 2% in Tris–HCl showed good linearity (R2 = 1, 0.98 and 0.99, respectively), but with a 7% protein underestimation in Tween and 4% overestimation in EDTA compared to BSA in PBS buffer. Thus, protein contents in Tween and EDTA extracts were corrected accordingly. In conclusion, sonication (3 2 min), Tween (0.25% in PBS) and EDTA (2% in Tris buffer) are three different extraction methods allowing maximal extracellular protein release with minimal contamination by intracellular proteins.
3.2. Protocol extraction strategy The three selected extraction methods will be used to establish a multi-method extraction strategy. The suggested extraction protocol will undertake each method separately with intermediate centrifugation steps, in order to extract three different soluble EPS fractions from the same sample: sonication-extract, EDTA-extract and Tween-extract. This strategy is meant to disrupt, respectively, random, ionic and hydrophobic interactions. Sonication will be applied at first in order to disaggregate the sludge flocs and increase contact between the EPS matrix and chemicals, i.e. EDTA and Tween. Two sequences were tested: sonication, EDTA then Tween and sonication, Tween then EDTA. Exchanging order between EDTA and Tween did not affect the total amount of extracted proteins by the protocol. The multi-method protocol was performed on two different sludge samples, sludge 1 and sludge 2 collected from pilots fed, respectively, with domestic or synthetic effluent. The total amount of extracted proteins obtained by the multimethod protocol sums up to 264 ± 10 and 191 ± 45 mg equiv. BSA/g VSS for sludge 1 and sludge 2, respectively, (Fig. 1). Variation between both sludge samples (32%) is greater than the error brought by the extraction protocol (4 and 23% for sludge 1 and 2, respectively). These results show that protein content in sludge 1 fed on urban waste water is stronger than in sludge 2 fed on a synthetic solution.
Tween
mg eq. BSA / g VSS
300
EDTA
250
Sonication
200 150 100 50 0 Protocol sludge 1
Protocol sludge 2
Sonication x3 EDTA x3 sludge 2 sludge 2
Tween x3 sludge 2
Fig. 1. Protein content in extracts obtained by the multi-method extraction protocol applied on sludge 1 and on sludge 2, and by the stepwise extraction strategy applied on sludge 2 samples.
On the other hand, the total extracted polysaccharide content revealed no difference between both sludge samples with 28 ± 1 and 27 ± 4 mg equiv. BSA/g VSS for sludge 1 and 2, respectively. No significant G6P-DH activity was measured in the obtained extracts (results inferior to 0.1 nmol NADH/min/g VSS). A rapid APIZYM screen over nineteen different enzymatic activities was performed on all the extracts obtained by the multi-method extraction protocol. Enzymes detected in the whole sludge were also detected in all extracts which informs that protein structure was not significantly affected by the extraction protocol. A stepwise extraction strategy is defined as the same extraction method applied three times on the same sample (sludge 2). Stepwise extraction strategies based on sonication (3 2 min), EDTA (2% in Tris) and Tween (0.25%) were investigated in order to determine whether the multi-method extraction protocol is as efficient in protein extraction as repeating one single method. As shown in Fig. 1, when repeating three times EDTA and Tween treatments, total protein content in extracts reached, respectively, 70 and 50 mg equiv. BSA/g VSS, which is less than the extraction protocol (191 ± 45 mg equiv. BSA/g VSS). No G6P-DH activity was found in either extracts. It is therefore clear that a prior mechanical disruption method (sonication) in the extraction protocol increases the protein extraction efficiency of EDTA and Tween methods. Although tripled sonication is as efficient in protein extraction (210 mg equiv. BSA/g VSS, without cell damage) as the multi-method extraction protocol, concern should now be given towards the qualitative aspect. Recent studies have shown the specificity of extraction methods towards certain types of EPS (Comte et al., 2007; Park and Novak, 2007). Single method extraction protocols could be liable in restricting protein content in extracts to one category of proteins. However, this argument should be confirmed with further qualitative analyses of extracts obtained from stepwise and multi-method protocols.
3.3. Validation of protocol extraction efficiency Protocol extraction efficiency was evaluated by adding known proportions of BSA protein in sludge samples, and then by measuring protein release in protocol extracts. For three different concentrations of added BSA (0.5, 1 and 2 g/L), a yield of 78% is obtained for BSA recovery using the extraction protocol. Therefore, 22% of the added BSA is systematically withheld in the sludge after one protocol extraction treatment. This suggests that more proteins in the EPS matrix could still be released with other successive extractions, and was confirmed by repeating the multi-method extraction protocol three times on the same sludge samples (sludge 1 and 2). Both sludge samples offer the same decreasing profile of protein content in all successive extracts (Fig. 2). Extractions as well as protein measurements were done in duplicate for error estimations.
R. Monique et al. / Bioresource Technology 99 (2008) 7464–7471 100
Sludge 1 Sludge 2
mg eq. BSA/g VSS
80
60
40
20
0
US-1 Tween-1 Edta-1 US-2 Tween-2 Edta-2 US-3 Tween-3 Edta-3
Protocol 1
Protocol 2
Protocol 3
Fig. 2. Multi-method protocol repetition: protein content in each extract from sludge 1 (j) and sludge 2 ( ), after three successive protocol treatments (Protocol 1, 2 and 3). Error bars represent error on doubled extractions and doubled measurements.
The total amount of proteins extracted by the protocol repeated three times sums up to 460 ± 10 and 348 ± 11 mg equiv. BSA/g VSS for sludge 1 and 2, respectively. Total polysaccharide content in all extracts is lower and sums up to 68 ± 3 and 53 ± 1 mg equiv. glucose/g VSS for sludge 1 and 2, respectively. The large majority of proteins over polysaccharides in these sludge extracts show stronger P/S ratios compared to literature (6.6 and 7 for sludge 1 and 2, respectively). In literature, P/S ratio values obtained by one single extraction method varies between 1.6 and 4.9 depending on the extraction method and sludge
Table 4 Protein content in the soluble organic fraction
Sludge VSS (g/L) Extracted VSS (g/L) mg Proteins/g extracted VSS (BCA method) mg Proteins/g extracted VSS (TNK method)
Sludge 1
Sludge 2
3.22 1.85 803 876
6.07 3.25 650 –
The extracted VSS was determined by the difference of VSS values between the initial sludge and the final pellet obtained after three protocol treatments. Protein determination by the total nitrogen Kjeldahl method (TNK) was evaluated by difference between the initial sludge and the final pellet obtained after three protocol treatments.
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origin (Comte et al., 2006, 2007; Park and Novak, 2007; Sponza, 2002). This could suggest either that our protocol extracts preferentially proteins from sludge samples or that proteins are particularly dominant in the investigated samples. Protein content in the extracted organic soluble fraction (VSSsoluble) was determined by BCA and NTK methods (Table 4). Both methods showed comparable results, between 803 and 876 mg proteins/g VSSsoluble. This reveals that between 80 and 87% of extracted molecules are proteins. Protocol repetition released 264, 134 then 62 mg proteins/g VSS for sludge 1 and 192, 100 then 56 mg proteins/g VSS for sludge 2. Interestingly, each successive extraction protocol releases around 50% of the proteins obtained from the previous extraction protocol (Fig. 2). This trend can describe a predicting model which calculates for each protocol application (n) the total amount of extracted protein (Pn) depending on the amount of proteins extracted by the first protocol application (P1) and the constant decay rate (a): Pn = P1 a (n1). Decay rates (0.48 and 0.55) and P1 values (269 and 192) for sludge 1 and sludge 2, respectively, were solved for an optimal curve fit, i.e. minimum R([Pexperimental][Psimulated])2. Model construction was performed on three data sets for sludge 1 and one data set for sludge 2. Model validation was carried out with one data set (not used for the model construction) for each sludge sample (Table 5). Maximum error between model predictions and experimental values reached 17% which is acceptable for model validation and model reliability (Table 5). By running this model, protein exhaustion is reached after 9 extraction protocols for both sludge samples (Fig 3). Hence, the total amount of soluble proteins should sum up to 516 and 423 mg equiv. BSA/g VSS for sludge 1 and sludge 2, respectively. In general terms, we can estimate that the first extraction protocol applied on a sludge sample extracts between 45% and 49% of proteins in sludge. Therefore, this multimethod extraction protocol is relatively efficient by providing a representative quantity of soluble proteins from sludge. Moreover, by repeating the protocol three times, up to 82–89% of the total soluble proteins can be harvested. The multi-method extraction strategy is also likely to offer different types of proteins which can stand for the initial protein pool found in the sludge.
Table 5 Model validation on sludge 1 and sludge 2 samples n
1 2 3
Sludge 1
Sludge 2
Pexperimental
Psimulated
Error (%)
Pexperimental
Psimulated
Error (%)
253 138 73
269 129 62
6 7 17
192 93 56
192 106 58
0 12 4
Concentrations are indicated as mg equiv. BSA/g VSS. Sludge 1 solved parameters: a = 0.48 and P1: 268, sludge 2 solved parameters: a = 0.55 and P1 = 192.
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P : [protein] mg eq. BSA/g VSS
300
Sludge 1 (− model) Sludge 2 (− model)
250 200 150 100 50 0
1
2
3
4 5 6 X : number of protocols
7
8
9
Fig. 3. Exponential curve model (line) fitted to experimental data from sludge 1 (j) and sludge 2 ( ): protein content in extracts vs. number of protocols. Black curve line (—) for sludge 1 and grey curve line ( ) for sludge 2.
4. Conclusion An extraction protocol was developed to improve protein extraction from sludge. The experimental design was established by selecting different extraction methods which act on the various chemical interactions found in activated sludge aggregates. Regarding the extracellular protein extraction efficiency (maximal protein release with minimal cell lysis), discontinuous sonication (3 2 min), Tween (0.25% 1 h) and EDTA (2% in Tris buffer, 1 h) were selected. These three methods did not counteract cell lysis measurements nor did they interfere in protein determination by BCA method. Extraction methods applied in series as described in the multi-method protocol are more efficient in protein release than EDTA and Tween methods applied alone. Protocol repetition showed that proteins can still be released from the sludge aggregates. Protein release vs. number of protocols progressively decreased and fitted a specific exponential model. Model predictions show that protein exhaustion is reached after 9 protocol applications. This suggested multi-method extraction protocol applied once releases between 45% and 49% of the total amount of extractable proteins from a sludge sample, which is quantitatively significant for sludge representativeness. Considering the specificity of extraction methods towards certain types of EPS, we can consider that the obtained soluble protein pool is also qualitatively characteristic of the studied sludge samples. Further qualitative studies are to be undertaken for fingerprint diagnosis. References Azeredo, J., Oliveira, R., Lazarova, V., 1998. A new method for extraction of exopolymers from activated sludges. Water Sci. Technol. 37, 367– 370. Brown, M.J., Lester, J.N., 1980. Comparison of bacterial extracellular polymer extraction methods. Appl. Environ. Microbiol. 40, 179–185. Bura, R., Cheung, M., Liao, B., Finlayson, J., Lee, B.C., Droppo, I.G., Leppard, G.G., Liss, S.N., 1998. Composition of extracellular poly-
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