Chelating properties and molecular weight distribution of soluble microbial products from an aerobic membrane bioreactor

ARTICLE IN PRESS WAT E R R E S E A R C H

40 (2006) 1531 – 1538

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Chelating properties and molecular weight distribution of soluble microbial products from an aerobic membrane bioreactor Ladan Holakooa, George Nakhlaa,, Ernest K. Yanfulb, Amarjeet S. Bassia a

Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ont, Canada N6A 5B9 Department of Civil and Environmental Engineering, University of Western Ontario, London, Ont, Canada N6A 5B9

b

art i cle info

A B S T R A C T

Article history:

This paper presents a detailed study on soluble microbial products (SMPs) in an aerobic

Received 21 July 2005

membrane bioreactor (MBR) treating synthetic wastewater simulating municipal waste-

Received in revised form

water. The concentration of SMP in the reactor conformed to a cyclical pattern of

7 December 2005

accumulation and reduction in relation to SRT. The molecular weight (MW) distribution of

Accepted 1 February 2006

accumulated SMP was determined to vary from o1 kD to 4100 kD. Copper chelating

Available online 29 March 2006

properties of various SMP fractions in the MBR were compared before and after copper

Keywords:

addition to the feed. The conditional stability constant (Log cK), complexation capacity (Cc),

Chelating properties

and SMP-ligand concentration (CL) were evaluated to determine the impact of copper on

Complexation capacity

the chelating properties. The results indicated that accumulated SMP in the aerobic MBRs

Membrane bioreactor (MBR)

without copper addition are moderate chelators with Log cK values of 7.6–8.3 mol
1 for the

Molecular weight (MW) distribution

moderate ligands and 6.3–6.8 mol
1 for the relatively weaker ligands. SMPs with MW of

Soluble microbial products (SMP)

1–10 kD were found to have the highest complexation capacity among all SMP fractions.

Stability constant

The complexation capacity of accumulated SMP after feeding copper was 0.11 mmol/mg of SMP, almost half of its value prior to feeding copper. The reduction of Cc after feeding copper was a result of an increase in large molecular weight SMP (4100 kD). & 2006 Elsevier Ltd. All rights reserved.

1.

Introduction

Soluble microbial product (SMP) are broadly classified as: utilization-associated products (UAP), related to substrate metabolism and biomass-associated products (BAP), generated from biomass as part of cell decay (Namkung and Rittmann, 1986; Laspidou and Rittmann, 2002). SMP comprise a wide range of high and low molecular weight compounds including proteins, polysaccharides, humic and fulvic acids, nucleic acids, enzymes and structural compounds (Rittmann et al., 1987; Parkin and McCarty, 1981; Chudoba et al., 1980) and are therefore difficult to measure. In spite of the ambiguity related to SMP, interest in this area has been

growing over the years due to the progressively stringent discharge criteria being implemented world wide. Membrane bioreactor (MBR) technology is a steadily growing wastewater treatment solution capable of generating high-quality effluent by retaining solids and SMP (Urbain et al., 1998; Gao et al., 2004). The accumulation of SMP has been shown to adversely affect membrane flux, metabolic activity of activated sludge, and nitrification (Chudoba, 1985, Huang et al., 2000). Few studies have examined accumulation, molecular weight (MW) distributions and fate of SMP in aerobic MBRs over a period of only 300 days or less (Huang et al., 2000; Shin and Kang, 2003). Both studies reported an accumulation in SMP followed by a drop in aerobic MBR at an

Corresponding author. Tel.: +1 519 661 2111x85470; fax: +1 519 850 2921.

E-mail address: [email protected] (G. Nakhla). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.02.002

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SRT of 20 days when operating at total suspended solids (TSS) concentrations of 3–8.5 g L
1. Huang et al., (2000) observed that high MW compounds accumulated and dropped, while Shin and Kang (2003) observed a shift from high MW (4100 kD) to low MW (o3 kD) over time. The present study builds upon existing literature, by monitoring the concentration, fate and MW distribution of SMP operating at high TSS concentrations and an extended operational period (600 days). SMP may play a role in metal toxicity and bioavailability. The chelating properties of SMP are acquired through the various functional groups, i.e. carboxylates, hydroxyls, sulfydryls, phenols and amines (Kuo and Parkin, 1996). Chelating properties i.e. the ligand–metal conditional stability constant (cK) and the complexation capacity (Cc) of the natural organic ligands in the environment have been widely studied (van den Berg et al., 1979; Rudd et al., 1984; Hansen et al., 1990; Kunz and Jardim, 2000; Chang and Gray, 2003). These ligands include organics present in surface waters, soil, sewage, microbial and algal excretions, and extracellular polymeric substances (EPS) from various microbial species. However, limited data are available on the chelating properties of SMP in wastewater treatment processes. The study presented here attempts to bridge the gap in information on the chelating properties of SMP in wastewater treatment processes. The objectives of this research were to (1) study the fate and MW distribution of accumulated SMP in an aerobic MBR treating synthetic medium strength municipal wastewater at SRT of 40 days and TSS concentration of 14 g L
1 and over a long period of time (15 SRT turnovers), (2) determine the copper chelating properties of the accumulated SMP with respect to different MW fractions, i.e. 4100, 10–100, 1–10 and o1 kD, in the absence and presence of copper in the feed. The chelating properties were determined through the measurement of conditional stability constant (Log cK), complexation capacity (Cc), and equivalent concentrations of SMP as ligands in the reactor.

2. 2.1.

Material and methods

The laboratory scale aerobic MBR (Fig. 1), operated at 1771 1C, consisted of a 6.6 L Plexiglas unit with two submersible membrane modules, ZeeWeed-1 (ZW-1, Zenon Environmental

Feed pump

Effluent pump

Air flowmeter

Fig. 1 – MBR schematic.

2.2. Composition of synthetic wastewater in mineral medium The synthetic feed was prepared with glucose to facilitate SMP measurements. The selection of glucose was based on prior studies conducted by Kuo et al. (1996) proving higher SMP production relative to acetate as substrate. The feed was prepared to simulate medium strength municipal wastewater (glucose 280 mg L
1) in the following mineral medium: (NH4)2SO4: 126.0 mg L
1; MgSO4  7H2O: 70 mg L
1; CaCl2  2H2O: 22.5 mg L
1; KH2PO4: 31 mg L
1; FeCl3: 11 mg L
1; CuSO4  5H2O: NaMoO4  2H2O: 0.15 mg L
1; MnSO4  H2O: 0.013 mg L
1;
1
1 0.13 mg L ; ZnCl2: 0.23 mg L ; CoCl2  6H2O: 0.42 mg L
1; Na2CO3: 216 mg L
1 and NaHCO3: 169 mg L
1. Sulfuric acid was used to maintain a pH of 7.3.

2.3.

Analytical methods

TSS and volatile suspended solids (VSS) were measured using standard methods (APHA, 1998). Chemical oxygen demand (COD) was measured with COD digestion vials using HACH method 8000. pH was measured using an Orion pH meter model 410A and a pH probe (VWR model SympHony).

2.3.1.

System description

Pressure gauge

Inc., Burlington, ON, Canada) and a mixer. The hollow fiber membrane had a nominal pore size of 0.04 mm and surface area of 0.047 m2, respectively. Aeration was provided through the membranes so as to control membrane fouling and to maintain DO of 42 mg L
1 in the bioreactor. The reactor was seeded with returned activated sludge from an established municipal wastewater treatment plant in London, ON, Canada. The system was operated at a 4 h HRT and 40 day SRT in two phases: (1) 6th–15th turnover of SRTs without influent copper addition (2) 16th–17th with feed copper concentration of 2 mg L
1. The phase separation enabled a controlled study the effect of copper on the chelating properties of accumulated SMP.

SMP measurement and fractionation

The soluble samples were obtained by centrifuging reactor contents at 1250G for 10 min, and then filtering through 0.45 mm. Soluble samples were used for fractionation using stirred cell ultrafiltration (UF) membranes (OMEGACELL from PALL Life Sciences, Ontario, Canada). Three UF membranes with pore size of 100, 10, and 1 kD were used in series with the highest MW cutoff at first and the lowest at last. MW distribution was categorized as follows: 4100, 10–100, 1–10, and o1 kD. The membranes had low protein binding to maximize SMP recovery. Gentle turbulence was created at the membrane surface using a magnetic stirrer to minimize the build-up of a dense macromolecular layer at the membrane surface. Air regulated at 50 psi pressure was used as a driving force for filtration. Based on the degradation pathway for glucose to pyruvate (glycolysis), followed by the citric acid cycle (Kreb cycle) which is the aerobic pathway for the breakdown of pyruvate to water and carbon dioxide (Lehninger et al., 1993), SMPs were determined using eq. (1) after accounting for the COD of glucose (Glu) and all the intermediates, i.e. pyruvic acid (HP),

ARTICLE IN PRESS

citric acid (HC), succinic acid (HS), fumaric acid (HF), and malic acid (HM). SMP ¼ soluble COD21:07ðGluÞ20:91ðHPÞ20:75ðHCÞ21:08ðHSÞ 20:83ðHFÞ20:72ðHMÞ:

(1)

Glucose was measured using Glucose Trinder Assay kit # 220-32, from Diagnostic Chemicals (Charlottetown, PEI, Canada). Pyruvate was measured employing a modified method for the determination with dinitrophenylhydrazine (DNPH) (Anthon and Barrett, 2003). Accordingly, a 25 mL sample was added to 1 mL distilled water and 1 mL 250 mg L
1 2, 4- DNPH solution in 1 M HCl. The mixture was placed in a water bath at 37 1C for 10 min. Then 1.0 mL of 1.5 M NaOH was added to the mixture and absorbance promptly read after the addition of NaOH at wavelength of 420 nm on a Varian UV spectrophotometer model Cary50. Proper blanks and a five standard (0–10 mg L
1) calibration system using sodium pyruvate was adopted. The concentrations of citric acid, succinic acid, fumaric acid and malic acid were analyzed by a HPLC (Agilent model 1100) with an ultraviolet-diode array detector (UV-DAD) and with a ZORBAX SB-Aq column (4.6 mm  150 mm  5 mm). The detection wavelength and the reference wavelength were 210/8 and 360/80 nm, respectively. The mobile phase was 20 mM aqueous phosphate buffer at pH ¼ 2 and acetonitrile 99:1 (V/V) at a flowrate of 1 mL. Column temperature was set at 25 1C.

2.3.2.

SMP titration

Prior to the titration of SMP, SMPs were treated with a chelating cation exchange resin (Amberlite IRC748, SigmaAldrich) to remove the previously complexed copper from the SMP. A Cu-selective electrode, Thermo-Orion model 9629 combination electrode, and a Thermo-Orion pH/ISE meter model 710Aplus were used for the titration of SMP. A 25 mL of SMP sample was titrated with 0.8 mM Cu as CuSO4. As recommended by Orion for low-level measurements of copper, samples were titrated at a low ionic strength to maintain the background ionic strength high and constant relative to variable copper concentrations. An ionic strength adjuster (NaNO3) was added at a concentration of 0.01 M. The pH of the sample was maintained at 6 by addition of HNO3 and NaOH during the course of titration to avoid precipitation of Cu(OH)2. Though the chelating properties of the SMP at MWo1 kD could not be measured accurately due to the presence of salts and inorganic ligands (devoid in other fractions), which could interfere with the response of the ISE, they have been measured for comparison.

2.3.3.

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4 0 (200 6) 153 1 – 153 8

Quality assurance

To minimize errors while using the ion-selective electrode (ISE), precautions were observed in handling all glassware and probes to prevent cross contamination. As recommended by Orion for low-level measurements of copper samples were titrated in plastic containers. The performance of the probe was verified by checking the electrode slope, the change in electrode potential per 10-fold change in concentration. The slope was between 29.2 and 29.6 mv per decade of free Cu concentration which is within the accepted range for copper. During titration, insulation (styrofoam) was provided between the stirrer and the sample beaker to avoid heat

transfer from the magnetic stirrer. Consistency in mixing speed and temperature was maintained. Calibration and verification of probe performance were continued throughout the titrations. Since the total concentration of copper added during titration was low (o0.6 mg L
1), a low- level calibration procedure was followed as recommended by Orion. The calibration was done using non-linear regression analysis since the response of the ISE probe is not linear at low free metal concentrations. The r2 was always 498%. The initial total copper concentration of each sample was measured using atomic absorption spectrophotometry (Philips, model PU9100X) and the values were used to correct the background concentration for titration data analysis.

2.3.4.

Data analysis using Scatchard model

Complexation parameters (cK: conditional stability constant and L: ligand concentration) were determined by graphical analysis of Cu2+ titration data using the Scatchard plot (Scatchard, 1949). Based on the assumption of 1:1 complex formation between SMP and Cu2+, Eqs. (2)–(6) show the derivation of the linear model (Eq (6)). The Scatchard plot of [Cu–L]/[Cu2+] vs. [Cu–L] gives the ligand-metal stability constant K (the slope) and the total ligand concentration L (the xintercept) in the titrated sample as shown in Fig. 2. The slope of the steeper line, K1, is the stability constant of the higher affinity sites and the x-intercept, L1, is the concentration of the high-affinity sites. The slope of the second line, K2, is the stability constant of the relatively lower affinity sites and the x-intercept is the total ligand concentration (LT). The concentration of the lower affinity sites, L2, is the difference between LT and L1. During titration, total copper (CuT) is known at each increment of addition and free copper concentration (Cu2+) is measured by ISE. The concentration of complexed copper (Cu–L) is the difference between CuT and Cu2+. The complexation capacity (Cc) of SMP was defined as mol Cu per mg of SMP and was derived as LT/SMP (mol mg
1). L þ Cu2þ 2Cu
L, c

K¼

½Cu2L ½L½Cu2þ 

(2)

,

(3)

120 100 80 Cu-L/Cu

WAT E R R E S E A R C H

K1 60 40 20 0 0.5

K2

1

2 Cu-L, x10-6 mol

L1 1.5

2.5

LT

3

Fig. 2 – Scatchard plot of SMP titration with copper.

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½LT  ¼ ½L þ ½Cu
L.

40 (2006) 1531– 1538

(4)

soluble COD). Thus, SMP constituted the major fraction of effluent COD. TSS and VSS in the MBR averaged at 13.972.6 and 11.672.2 g L
1, respectively.

By combining Eqs. (3) and (4), a linear relationship is obtained: ½Cu2L ¼ c K½L½Cu2þ  ¼ c Kð½LT 
½Cu
LÞ½Cu2þ , ½Cu2L ½Cu2þ 

(5)

¼ c K½LT 
c K½Cu
L.

(6)

In order to validate the measurements, L and Cc were theoretically calculated for total SMP using the measured L and Cc values of individual SMP fractions (Eqs (7)–(11)). The calculated values for total SMP were then compared to the measured values L1 ¼

n X

L1;i ,

(7)

n X

L2;i ,

(8)

LT;i ,

(9)

i

LT ¼

n X i

Cc ¼

n
 X Xi Cci ;

SMP measurement

3.2.1.

SMP concentrations

Accumulated SMP in the system were monitored from the 6th to 15th SRT turnover prior to feeding copper. The temporal variation of SMP is presented in Fig. 3. It is evident from Fig. 3 that the accumulation of SMP in the system followed a cyclical pattern, an initial buildup followed by a decline. SMP concentrations varied within the range of 8–68 mg L
1 prior to copper addition, averaging at 31715 mg L
1. The system had undergone eight cycles of accumulation and reduction with a cycle period of 1-1.5 SRT (40–60 days) before experiments with copper were initiated. The cyclic trend observed here is consistent with the findings of Shin and Kang (2003) and Huang et al. (2000) in aerobic MBRs treating glucose. Both studies documented an increase in the accumulated SMP in the MBR followed by a decline.

i

L2 ¼

3.2.

(10)

i

, Xi ¼ Ci

n X

3.2.2. Ci ,

i

Where LT, L1 and L2 (mol L
1) are the total, moderate and weak ligand concentrations of SMP, respectively. Li, L1,i and L2,i are the ligand concentration of various SMP fractions. Xi is the weight fraction of various SMP fractions, and Ci is the concentration of various SMP fraction. Cc and Cci (mol mg
1) are the complexation capacities of total SMP and various SMP fractions, respectively.

3.

Results and discussion

3.1.

System performance

SMP molecular weight fractionation

The MW fractionation of accumulated SMP in the MBR from the 6th–14th SRT turnover is depicted in Fig. 4. Despite the 0.04 mm pore size of the membrane and the MW cutoff of 300 kD, SMP with MW less than the membrane cutoff accumulated in the MBR. The percentage of macromolecules (MW4100 kD) accumulating in the MBR steadily increased from 27.9% to 91.4% from the 6th–11th SRT turnover, then dropped to 29.7% by the 12.3 SRT turnover as the MW shifted from high (4100 kD) to lower MW compounds. Meanwhile, the concentrations of the other fractions, i.e. 10–100, 1–10 and o1 kD, increased from 2.4% to 39.4%, 5% to 21%, and 1.3% to 9.7%, respectively. The build up of macromolecules recurred, increasing to 49.5% by the 14th SRT turnover. Fig. 5 shows the temporal concentration variation of various SMP concentrations. It is interesting to note that despite an increase in the percentage of SMP with MW4100 kD from the 6th to the 9th SRT turnover, the concentration of this fraction actually decreased from 8.7 to 4.4 mg L
1. Concentration of other fractions also dropped: 10–100 kD from 6.9 to 1.3 mg L
1; 1–10 kD from 9.0 to

(11)

Detailed performance of the MBR is reported elsewhere (Holakoo et al., 2005). The influent and effluent COD concentrations averaged, respectively, at 291734 and 576 mg L
1 (90 samples). The COD removal efficiency was 98% with effluent glucose concentrations and intermediate products measuring below 0.5 mg L
1 (o10% of the effluent

100

SMP, mg/L

210 190 170 150 130 110 90 70 50

Before addition of Cu After addition of Cu

80 60 40 20 0 6

7

8

9

10

11

12

13

14

15

1

2

time,SRT Fig. 3 – SMP concentration at different turnovers of SRT.

3

ARTICLE IN PRESS

% SMP fraction

WAT E R R E S E A R C H

100 90 80 70 60 50 40 30 20 10 0

1535

4 0 (200 6) 153 1 – 153 8

>100KD 10-100 KD 1-10 KD < 1KD

6*SRT

7*SRT

9*SRT 11*SRT time, SRT turnovers

12.3*SRT

14*SRT

Fig. 4 – %MW fraction of SMP before feeding copper.

60

SMP, mg/L

50 40 30

>100KD 10-100KD 1-10 KD <1KD

20 10 0 6*SRT

7*SRT

9*SRT 11*SRT time, SRT turnovers

12.3*SRT

14*SRT

Fig. 5 – Concentration of SMP fractions before feeding copper.

Table 1 – Chelating properties of different fractions of SMP before feeding copper Faction 4100 kD 10–100 kD 1–10 kD o1 kD SMP41 kD Total SMP

Log K1

Log K2

Cc (mol mg
1)

LT,MBR (mol L
1)

%L1

7.670.1(4) 7.770.3(4) 8.070.3(4) 8.370.1(3) 7.970.1(5) 8.070.2(3)

6.6(4) 6.470.2(4) 6.570.2(4) 6.870.1(3) 6.570.1(5) 6.370.1(3)

(0.1270.06)
10
6(4) (0.2770.04)  10
6(4) (0.4170.06)  10
6(4) (0.1670.01)  10
6(3) (0.2070.05)  10
6(5) (0.2070.02)  10
6(3)

(0.4770.1)  10
6(4) (0.3270.04)  10
6(4) (1.070.16)  10
6(4) (0.3570.03)  10
6(3) (1.870.42)  10
6(5) (1.770.18)  10
6(3)

47.8 25.5 43.8 43.5 37.1 45.5

Average values7standard deviation, numbers in parenthesis are number of samples.

2.3 mg L
1; and o1 kD from 6.4 to 0.9 mg L
1. This clearly illustrates that not only does the fractional concentration of SMP changes over time but also the accumulation of various fractions is cyclical emphasizing the pattern of Fig. 3 discussed earlier. The cyclical pattern of SMP in MBRs is consistent with the findings of Shin and Kang (2003) and Huang et al. (2000) at SRTs of 20d and biomass concentration of 4-6.3 g L
1 and 3-4 g L
1, respectively. Compared to the above cited studies, a higher overall contribution of SMP with MW 4100 kD was observed here possibly due to the relatively longer SRT. At higher biomass concentrations and lower F:M ratios, the specific growth rate (m) is much lower than the maximum growth rate (mmax) and this may cause decay associated SMP to prevail over growth-associated SMP, hence

resulting in accumulation of higher molecular fractions which are products of cell lysis.

3.3.

SMP chelating properties

3.3.1.

Stability constant (Log cK)

Table 1 shows the logarithms of the stability constant of SMP fractions prior to feeding copper. All fractions of SMP were found to be heterogeneous with relatively moderate (Log cK1) and weak (Log cK2) binding sites. The values of Log cK were found to be 7.6–8.3 mol
1 for the moderate binding sites and 6.3–6.8 mol
1 for the relatively weaker binding sites. The results suggest that accumulated SMP in the system were moderate chelators, consistent with the Log cK of 7.25 and

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40 (2006) 1531– 1538

5.86 for the moderate and weak ligand sites observed for copper in secondary sewage effluents (Jardim and Allen, 1984).

3.3.2.

Complexation capacity (Cc)

Table 1 compares the complexation capacities (Cc) of the various SMP fractions before feeding copper. SMPs with MW of 1-10 kD were found to have the highest Cc among all fractions with a complexation capacity of twice the weighted average for total SMP41 kD. Higher MW compounds (4100 kD), which are more likely biomass-associated products and products of cell lysis had the lowest complexation capacity with a capacity of 0.12 m mol mg
1. Table 2 compares the calculated and measured ligand concentration (L) and complexation capacity (Cc) of total SMP41 kD in accordance with Eqs. (7)–(11) using SMP weight fractions (4100 kD: 54.1%, 10–100 kD: 16.1% and 1–10 kD: 29.8) and their respective Cc (Table 1). The theoretical L and Cc values were within 15.2% of the measured values.

3.3.3.

Ligand concentration

The total ligand concentrations of the various SMP fractions along with the percentage of total ligand that is contributed by the relatively strong ligands (L1) are presented in Table 1. In all fractions, L1 comprised less than half of the total ligand concentration. Table 2 shows the comparability of the theoretical and measured ligand concentrations of total SMP41 kD in accordance with Eqs. (7)–(11). The low relative discrepancies between the theoretical and measured values attest to the accuracy of measurements.

3.4.

Effect of copper

3.4.1.

Effect of copper on SMP

Fig. 3 shows the trend of net SMP concentrations in the MBR before and after addition of copper. After adding copper to the feed at a concentration of 2 mg L
1, the accumulated SMP in the MBR increased to 131 mg L
1 within the first SRT, dropped and increased again to 200 mg L
1 within the second turnover of SRT. The cyclic trend of SMP continued after addition of copper with an increase in average SMP concentration to 121737 mg L
1. The increase in net SMP is a consequence of enhanced SMP production and increased cell lysis in the presence of copper. The production of SMP is known to increase in response to environmental stress and toxic substances (Aquino and Stuckey, 2004; Boero et al., 1996) due to enhanced cell decay rate or as a defensive response. Fig. 6 shows the MW distribution of SMP after copper addition, while the actual concentrations are shown in Fig. 7. The MW trend shows that the percentage MW4100 kD increased from 49.5% to 76% within two SRT turnovers. SMP with MW 10–100 kD dropped from 32.3% to 12.4%, 1–10 kD remain relatively constant at 8%—10.6%, and o1 kD dropped from 10.2% to 1%. The trend clearly shows an increase in MW4100 kD after feeding copper. The concentration of MW4100 kD sharply increased from 19 to 91 mg L
1 within one SRT after feeding copper and remained as high as 80 mg L
1 by the second SRT (Fig. 7).

3.4.2.

Effect of copper on chelating properties

Table 3 shows the stability constants (Log cK), complexation capacity (Cc), ligand concentrations (LT and L1) of SMP after addition of copper. The various SMP fractions were found to be heterogeneous with moderate (K1) and relatively weak (K2)

Table 2 – Comparison of combined chelating parameters of SMP with measured values

Combined values (1–10 kD+10–100 kD+4100 kD) Measured values of SMP41 kD % Deviation

L1

L2

LT

Cc

0.76  10
6 0.66  10
6 15.2

1.08  10
6 1.13  10
6 4.4

1.84  10
6 1.79  10
6 2.8

0.23  10
6 0.20  10
6 15.0

L1, L2, and LT: ligand concentrations (mol L
1); Cc: Cu complexation capacity of SMP (mol mg
1).

% SMP fraction

80 70

>100KD 10-100 KD 1-10 KD < 1KD

60 50 40 30 20 10 0 0

1*SRT, CU time, SRT turnovers

2*SRT, CU

Fig. 6 – %MW fraction of SMP after feeding copper at 2 mg L-1.

ARTICLE IN PRESS WAT E R R E S E A R C H

1537

4 0 (200 6) 153 1 – 153 8

100

SMP, mg/L

80 60

>100KD 10-100KD 1-10 KD <1KD

40 20 0 0

1

2

time, SRT turnovers Fig. 7 – Concentration of SMP fractions after feeding copper.

Table 3 – Chelating properties of different fractions of SMP after feeding copper at 2 mg L
1 Faction

Log K1

Log K2

Cc mol mg
1

LT mol L
1

%L1

4100 kD 10–100 kD 1–10 kD o1 kD SMP41 kD Total SMP

7.870.1(4) 8.870.3(4) 8.170.2(4) 8.0(3) 7.870.1(4) 7.970.1(3)

6.370.1(4) 6.770.2(4) 6.670.1(4) 6.870.1(3) 6.870.1(4) 6.3(3)

(0.0570.02)  10
6(4) (0.1870.04)  10
6(4) (0.3670.05)  10
6(4) (0.1870.04)  10
6(3) (0.1170.03)  10
6(4) (0.1070.01)  10
6(3)

(4.370.3)  10
6(4) (2.770.9)  10
6(4) (5.871.1)  10
6(4) (0.770.03)  10
6(3) (13.673.20)  10
6(4) (13.373.33)  10
6(3)

39.2 31.4 46.3 44.2 33.7 38.4

Average values7standard deviation; numbers in parenthesis are number of samples.

ligand sites. The Log K1 was found to be in the range 7.8–8.8 and 6.3–6.8 mol
1 for the weaker ligand (K2) sites. The addition of copper had no significant effect on the stability constants. By comparison of Tables 2 and 3, it is apparent that the complexation capacities of SMP4100 kD and total SMP41 kD after copper addition decreased to 0.05 and 0.11 mmol mg
1, almost half of their original values. This can be attributed to the high amount of large MW compounds (4100 kD) after feeding copper. Similar to the pattern prior to copper addition, SMP with MW of 1–10 kD were found to have the highest Cc among all fractions with a capacity of 0.36 mmol mg
1. The theoretical complexation capacities and ligand concentrations of SMP41 kD, calculated by using Eqs. (7)–(11), were within 10% of the measured values. As apparent from Table 3, after feeding copper, not only did the concentration of the weaker binding sites (L2) dominate the total ligand concentration, but the overall contribution of the moderate and weak ligands sites of SMP were similar to prior copper addition and no distinct difference was observed.

4.

Conclusions

The study presented here focused on the characteristics of SMP and their ability to chelate copper in aerobic MBRs. The following conclusions are drawn: 1. Longer SRT and lower F:M ratio may have led to the accumulation of higher MW fractions (4100 kD) of biomass decay associated SMP compared to previous literature studies.

2. The concentration of SMP in the MBR followed a cyclical pattern of accumulation and reduction over a period of 1–1.5 SRTs, even after copper addition. Concentration of various SMP fractions also followed a cyclical profile. 3. Copper addition increased the accumulation of SMP by four-fold, with high MW SMP contributing to 76% of total SMP. 4. The accumulated SMP were moderate chelators of copper as indicated by Log cK of 7.6–8.8 and 6.3–6.8 for moderate and weaker ligands, respectively. 5. In all fractions, the weaker ligands contributed to more than half of the total ligands concentration. The trend was similar after addition of copper. 6. SMP with MW of 1–10 kD were found to have the highest complexation capacity among all the SMP fractions. 7. Copper addition significantly altered the complexation capacity of the accumulated SMP by reducing it from 0.2 to 0.11 mol mg
1, due to the increase in the MW4100 kD SMP.

Acknowledgment This work was fully funded by Natural Sciences and Engineering Research Council of Canada (NSERC). R E F E R E N C E S

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