Accepted Manuscript

Title: Entrapped elemental selenium nanoparticles affectphysicochemical properties of selenium fed activated sludge

Author: Rohan Jain Marina Seder-Colomina Norbert JordanPaolo Dessi Julie Cosmidis Eric D.van Hullebusch StephanWeiss Francois Farges Piet N.L. Lens

PII: S0304-3894(15)00248-4DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2015.03.043Reference: HAZMAT 16691

To appear in: Journal of Hazardous Materials

Received date: 30-11-2014Revised date: 5-3-2015Accepted date: 21-3-2015

Please cite this article as: Rohan Jain, Marina Seder-Colomina, Norbert Jordan,Paolo Dessi, Julie Cosmidis, Eric D.van Hullebusch, Stephan Weiss, Francois Farges,Piet N.L.Lens, Entrapped elemental selenium nanoparticles affect physicochemicalproperties of selenium fed activated sludge, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2015.03.043

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Entrapped elemental selenium nanoparticles

affect physicochemical properties of selenium

fed activated sludge

Rohan Jaina,b*, Marina Seder-Colominab,d, Norbert Jordanc, Paolo Dessia, Julie

Cosmidisd#, Eric D. van Hullebuschb, Stephan Weissc, François Fargesd, Piet N.L. Lensa

aUNESCO-IHE, Institute for Water Education, Westvest 7, 2611AX Delft, The

Netherlands

b Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM,

77454 Marne la Vallée, France

cInstitute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf e.V.,

Bautzner Landstr. 400, D-01328 Dresden, Germany

dInstitut de Minéralogie, de Physique des Matériaux, et de Cosmochimie (IMPMC).

Sorbonne Universités - UPMC Univ Paris 06, UMR CNRS 7590, Muséum National

d’Histoire Naturelle, IRD UMR 206, Paris, France.

#Present address: Department of Geological Sciences, University of Colorado. UBC

399, 2200 Colorado Avenue. Boulder, CO 80309-0399.

*Corresponding author:

Phone: +31 152151715, fax: +31 152122921, e-mail: rohanjain.iitd@gmail.com, mailto:

UNESCO-IHE, Institute for Water Education, Westvest 7, 2611AX Delft, The

Netherlands

Highlights

• 93% of trapped Se in the Se fed activated sludge is in the elemental form of Se

• Se fed activated sludge has better settleability vis-à-vis control activated sludge

• Se fed activated sludge has more negative surface vis-à-vis control activated

sludge

Graphical abstract

Abstract

Selenite containing wastewaters can be treated in activated sludge systems, where the

total selenium is removed from the wastewater by the formation of elemental selenium

nanoparticles, which are trapped in the biomass. No studies have been carried out so

far on the characterization of selenium fed activated sludge flocs, which is important for

the development of this novel selenium removal process. This study showed that more

than 93% of the trapped selenium in activated sludge flocs is in the form of elemental

selenium, both as amorphous/ monoclinic selenium nanospheres and trigonal selenium

nanorods. The entrapment of the elemental selenium nanoparticles in the selenium fed

activated sludge flocs leads to faster settling rates, higher hydrophilicity and poorer

dewaterability compared to the control activated sludge (i.e. not fed with selenite). The

selenium fed activated sludge showed a less negative surface charge density as

compared to control activated sludge. The presence of trapped elemental selenium

nanoparticles further affected the spatial distribution of Al and Mg in the activated

sludge flocs. This study demonstrated that the formation and subsequent trapping of

elemental selenium nanoparticles in the activated sludge flocs affects their

physicochemical properties.

Keywords : selenium, nanoparticles, activated sludge, physicochemical, settleability,

surface charge

1. Introduction

Selenium is an essential element and is required in low doses for synthesizing

selenoproteins, preventing cardiovascular disease, assisting in sperm mobility and

avoiding miscarriage [1]. However, at higher doses selenium oxyanions (selenate and

selenite) can cause toxicity to humans, but also to animals and aquatic organisms.

Thus, they need to be removed from wastewaters prior to discharge [2–4]. Physico-

chemical remediation such as adsorption of selenite by nanoscale zero-valent iron [5],

magnetic nanoparticle-graphene oxide composite [6], maghemite [7] are successfully

demonstrated However, physico-chemical remediation is expensive and yet sometimes

ineffective in achieving the stringent selenium discharge criteria (less than 50 µg L–1)

[3,8]. Anaerobic microbial reduction of selenium oxyanions to elemental selenium is

often a recommended biological process for selenium oxyanions containing wastewater

treatment [9,10]. However, biological anaerobic reduction of selenium oxyanions leads

to the formation of biogenic elemental selenium nanoparticles (BioSeNPs) in the

discharged wastewaters [11]. These BioSeNPs have to be removed prior to discharge,

which requires an additional treatment step that further increases the remediation cost

[12–14].

Aerobic reduction of selenium oxyanions to either volatilized selenium compounds

followed by gas trapping [15] or to elemental selenium entrapment in microbial biomass

would be a one-step process for the treatment of selenite containing wastewaters. The

entrapment of selenium in the biomass is progressive as the selenium is then in the

solid state and much easier to handle as compared to volatilized selenium trapped in

concentrated HNO3 [15]. Aerobic reduction of selenite by microorganisms has been

described for Bacillus cereus [16], Escherichia coli [17] and activated sludge [18]. The

activated sludge is more suitable as compared to pure cultures for treating selenium rich

wastewater as it can easily handle variation in the influent and the wastewaters need

not be sterilized for continuous operations [19]. However, there are so far no studies on

the characterization of selenium fed activated sludge. This characterization is important

for the development of activated sludge based selenium remediation processes.

Therefore, the present work focused on the characterization of the selenium fed

activated sludge.

In this study, activated sludge loaded with selenium was produced by aerobic reduction

of selenite in batch reactors. The localization, speciation and crystallinity of selenium in

the activated sludge flocs were identified by Scanning Electron Microscopy-Energy

Disperse X-ray Spectroscopy (SEM-EDXS), Transmission Electron Microscopy (TEM),

sequential extraction, X-ray Diffraction (XRD) and Selected Area Electron Diffraction

(SAED). The elemental constituents, morphology, hydrophilicity, sludge volume index

(SVI), capillary suction time (CST) and surface groups density of the selenium fed

activated sludge were compared with a selenium-free control activated sludge.

2. Materials and methods

2.1 Production of selenium fed activated sludge

The activated sludge used in this study was collected from a full scale domestic

wastewater treatment plant in Harnaschpolder (The Netherlands). The synthetic

wastewater was composed of (in mg L–1): Na2SeO3 (173), NH4Cl (600), MgCl2.2H2O

(200) and CaCl2.2H2O (20). Glucose (2000 mg L–1) was used as electron donor and

carbon source. The total suspended solid (TSS) concentration added was 1300 ± 100

mg L–1.

The selenite removal was carried out under continuous aeration by an air flow (15-20 L

air h–1) in a 5L batch reactor. Mixing was carried out with a magnetic stirrer (500 rpm).

The pH was maintained at 7.5-8.0 by manual correction or addition of Na2CO3 when

needed. After the reduction of selenite, selenium fed activated sludge was collected by

settling and the supernatant was discarded. Control activated sludge without the

addition of sodium selenite was produced using the same procedure and collected by

settling as well.

2.2 Elemental composition

Concentrated HNO3 (10 mL) was added to 0.5 g (TSS) of selenium fed activated

sludge. The sludge was destroyed using microwave digestion (Duo Temp, MARS 5)

operated at 1600 W. The program consists of heating first for 10 minutes to 165 oC,

followed by raising the temperature to 175 oC and maintaining this temperature for 8

minutes, followed by 60 minutes cooling. The samples were appropriately diluted and

elemental concentrations were measured by Inductively Coupled Plasma-Mass

Spectrometry (ICP-MS). The experiments were done in triplicate.

2.3 Selenium localization and speciation analysis

SEM-EDXS and TEM were carried out to locate selenium in the microorganisms and

activated sludge flocs. For SEM-EDXS analysis, each sample was diluted in Milli-Q

water (18 MΩ cm) and filtered using 0.22 µm pore-size polycarbonate membrane filters.

Filters were deposited on carbon tape, dried at ambient temperature and finally coated

with a thin carbon film. Samples for TEM were diluted in Milli-Q water, deposited on a

copper TEM grid covered with a lacey formvar film and dried at ambient temperature.

Selenium speciation was determined by carrying out sequential extraction following the

KCl based protocol described by Wright et al. [20], but using a 30 times higher

extractant to solid ratio for complete selenium recovery. Sequential extraction analysis

was carried out in quadruplicates. The crystallinity of the trapped selenium in the

activated sludge flocs was determined by XRD and SAED associated with the TEM.

2.4 Physicochemical activated sludge properties

The SVI and CST of the selenium fed and control activated sludge were determined as

per standard methods [21]. For the SVI, 1.8 g L–1 of TSS was used. The relative

hydrophilicity (RH) was measured following the protocol described in Laurent et al. [22].

Acid-base titration was carried out for determining the pKa of the functional groups using

a Metrohm autotitrator unit. Selenium fed and control activated sludge (0.0266 g dry

weight) was suspended in 30 mL of Milli-Q water with 1 mM of NaCl background

electrolyte. The titration was carried out by automatic addition of 0.1 mL HCl (0.01214

M). The acid-base titration data were fitted using a PROTOFIT software [23] as

described by Laurent et al. [24]. Briefly, a non-electrostatic model with four discrete

acidic sites and an extended Debye-Huckel activity coefficient were used. Prior to

simulation using PROTOFIT, the derivative of the acid-base titration versus moles of

HCl added was plotted to determine the local minima. These local minima represent the

pKa of the functional groups [25]. Taking these pKa as the initial guess, the data fitting

was carried out.

2.5 Analytical methods

Selenium measurements by ICP-MS and XRD analysis were carried out as described

previously [26]. SEM was performed with a Field Emission Gun Scanning Electron

Microscope (GEMINI ZEISS Ultra55) operated at 2 kV. SEM-EDXS analyses were done

on the selected particles at 15 kV. TEM and SAED analyses were performed using a

JEOL 2100F (FEG) operating at 200 kV and equipped with a field emission gun, a high-

resolution UHR pole piece, and a Gatan energy filter GIF 200.

3. Results

3.1 Elemental composition of selenium fed activated sludge

The selenite was removed from aqueous phase in the batch reactor and more than 78%

of the fed selenium was entrapped in the biomass. The concentration of trapped

selenium in the activated sludge is 55 ± 2 mg of Se per g of TSS. The concentrations of

Na, Mg Al, K, Ca and Fe in selenium fed activated sludge are presented in Table 1.

Other elements such as Cu, Mn, Zn, Ba, Cr, V, Ni, Co, Pb and Mo were less than 1.0

mg per g of TSS.

Table 1.

3.2 Characterization of trapped selenium in the activated sludge

Sequential extraction of selenium trapped in the activated sludge suggests that 94 ± 9%

of the trapped selenium is in the form of elemental selenium. The sequential extraction

method was able to account for 95.7% of the total selenium trapped in the activated

sludge. All other fractions, i.e. the soluble/exchangeable fraction, adsorbed fraction and

residual fraction, were insignificant as they constitute only 2 ± 1% of the total trapped

selenium.

SEM images showed the presence of two different morphologies of selenium:

nanospheres and nanorods (Figure 1a). EDXS analysis confirmed that these

morphologies are composed of selenium (Figure 1b1, b2). The presence of selenium

nanorods and nanospheres was further confirmed in SEM images and its corresponding

cartography (Figure 1.e, f). SEM images of the bacterial cells suggest that the selenium

is partly trapped in the biomass and present inside the bacterial cells (Figure 1c). The

white colored spheres and black dots in Figure 1c are selenium nanospheres and the

pores of the filters, respectively. The TEM image showed that the selenium

nanospheres are closely associated with the bacterial cells. Some of the spheres are

indeed present at the surface of the bacteria, while others are possibly found inside the

bacteria, confirming the SEM observations (Figure 1d).

Figure 1.

Elemental selenium exists mainly in trigonal, monoclinic or amorphous forms [11]. In

order to determine the crystallinity of trapped elemental selenium, XRD was carried out

on selenium fed activated sludge. XRD results showed the features at 2-theta values of

23.4 and 30.0, corresponding to, respectively, the 100 and 101 crystallographic planes

of trigonal selenium, while the feature at 2-theta value 26.9 corresponds to 220 planes

of β-monoclinic selenium [27] (Figure 2a). The XRD also showed a hump like structure

between 2-theta values of 15 to 40, suggesting the presence diffuse scattering related

to the presence of aperiodic structures (nanocrystallized and/or “amorphous”). In

contrast to selenium fed activated sludge, the control activated sludge showed no

features in the XRD.

The diffuse SAED pattern of the spherical particles present in the selenium-fed

activated sludge suggests that these nanospheres are amorphous or poorly crystalline

(Figure 2b). Selenium nanospheres are known to be either aperiodic or monoclinic

[26,28]. Thus, the SAED pattern of the Se nanospheres and XRD of selenium fed

activated sludge suggest the presence of a mixture of amorphous and different

crystalline structures of elemental selenium: trigonal (nanorods) and

monoclinic/aperiodic (nanospheres) as also observed in the SEM images (Figure 1a, e).

It is interesting to note that the biological conversion of selenium oxyanions always

results in the formation of aperiodic or monoclinic nanospheres [28,29], which may later

transform to trigonal nanorods [30].

Figure 2.

3.3 Spatial distribution of elements in selenium fed activated sludge flocs

Figure 3a shows the SEM image of a single bacterium from the selenium fed activated

sludge. Figure 3b indicates that the nanospheres observed in the SEM image of Figure

3a are mainly composed of selenium, but Al and Mg are also found to be closely

associated to the selenium particles (Figures 3c, d). Other elements such as Ca, Fe,

Cu, Zn, Pb and Ba were not found to have gradient towards elemental selenium trapped

in the sludge (data not shown).

Figure 3.

3.4 Functional groups in the activated sludges

To evaluate the surface charge present on the selenium fed activated sludge, an acid-

base titration was carried out. The delta pH versus micromoles HCl added is plotted in

Figure 4. The local maxima in Figure 4a represent the maximum shift in pH and hence

the equivalence points. Similarly, the local minima represent the minimum change in pH

and hence the pKa of the functional groups present [25,31] The local minima for

selenium fed activated sludge were observed at pH 7.1, 6.9, 5.2, 3.7, 3.4 and 3.1

(Figure 4a). Similarly, the local minima for control activated sludge are observed at pH

7.2, 5.1 and 3.3. The local minima at 6.9-7.2 correspond to phosphoryl groups [31,32].

Carboxyl groups can be assigned to local minima at pH 5.1-5.2 [31,32]. The local

minima observed at pH of 3.1-3.7 are due to the presence of phosphodiester/ carboxyl

groups [31,33,34].

Figure 4.

The simulation of the acid-base titration data fitted well with the experimental data

(Figure 4b). The surface charge density of the selenium fed activated sludge was twice

more negative than the control activated sludge at neutral pH (Figure 4c). The

simulation of acid-base titration data predicted the pKa of the functional groups at pH

3.2, 5.2, 7.1 and 9.7 for selenium fed activated sludge (Figure 4d). For the control

activated sludge, the predicted pKa values of the functional groups are at pH 3.9, 5.0,

7.2 and 9.5. Assignment of predicted pKa to various functional groups is presented in

Figure 4d. It is important to note that we may not observe the local minima in the

derivate of pH vs moles of acid graph at the start and end of the titration due to a small

change in pH and hence the most acidic and basic groups may be missed out. But as

shown above, these groups can be successfully predicted by the simulation.

3.5 Physical properties of selenium fed and control activated sludge

The SVI of the selenium fed and control activated sludge was 61.1 ± 0.3 and

138.8 ± 0.1 mL g–1, respectively (Figure 5a). The CST of both the selenium fed and

control activated sludge was in the range of 20 seconds at a TSS of 3 g L–1 (Figure 5b).

With the increase in TSS, the CST of the selenium fed activated sludge increased to a

value as high as 67.2 seconds as compared to 19.0 s for control activated sludge at

TSS value of 9 g L–1. The RH of selenium fed activated sludge was 1.6 times higher

than that of the control activated sludge (Figure 5c).

Figure 5.

4. Discussion

4.1 Entrapment of elemental selenium affects the physical properties of activated sludge

flocs

This study demonstrates for the first time the effect on the physicochemical properties of

activated sludge flocs after treating the synthetic selenium contaminated wastewater.

Sequential extraction procedure and XRD provided the experimental proof, for the first

time, about the trapping of elemental selenium nanoparticles in the activated sludge

flocs. The trapping of microbiologically produced elemental selenium nanoparticles lead

to an improved settleability and relative hydrophilicity, but a negative impact on the

dewaterability of the selenium fed activated sludge. Elemental selenium is 4.5 times

denser than the activated sludge. The improved settleability can consequently be

attributed to the dense elemental selenium nanoparticles that increase the density of

activated sludge and thus, lead to better settling. The higher RH of activated sludge with

entrapped elemental selenium as compared to the control activated sludge suggests the

presence of more polar groups such as hydroxyl, carboxyl or phosphodiester groups in

the flocs (Figure 4d and 5c) [22].

The higher CST of selenium fed activated sludge at high TSS values as compared to

the control indicates a poorer dewaterability of the sludge (Figure 5b). The reason for

the poor dewaterability can be due to the blockage of filter pores by elemental selenium

nanoparticles (Figure 1c, d), thus, leading to a higher CST. The blocking of the filter

pores by nanoparticles can be due to the agglomeration of selenium nanoparticles

caused by their interaction with metals such as Al and Mg [26] (Figure 3c, d) or due to

the presence of nanorods that can be micrometers in length. The poor dewaterability of

the activated sludge with entrapped elemental selenium nanoparticles as compared to

control activated sludge can also be attributed to a different extracellular polymeric

substances (EPS) content [35,36]. Selenite removal has been shown to cause stress on

Bacillus sp., leading to the production of larger amounts of EPS, with a different

composition, as compared non stressed microorganisms [37].

4.2 Entrapment of elemental selenium affects the surface properties of the activated

sludge flocs

The less negative surface charge density of the selenium fed activated sludge flocs as

compared to the control activated sludge can be due to the higher concentration of

phosphodiester groups as well as their lower pKa value (3.2 for selenium fed activated

sludge as compared to 3.9 for control activated sludge) (Figure 4d). The difference in

the EPS content can be a one of the reasons for the difference in the surface charge

density of the selenium fed and control activated sludge. The other reason for the less

negative selenium fed activated sludge is the presence of elemental selenium

nanoparticles. Elemental selenium nanoparticles are known to have a negative ζ-

potential with an acidic iso-electric point (pH 3.5) due to the presence of organic

moieties produced by microorganisms on their surface [4,38]. These negative ζ-

potential nanoparticles might contribute to the less negative surface density of the

selenium fed activated sludge.

Due to the negative ζ-potential of these elemental selenium nanoparticles, the spatial

distribution showed a higher concentration of Al and to some extent of Mg near the

elemental selenium nanoparticles trapped in the activated sludge flocs (Figure 3), as

observed in case of Cu [39], Zn [26] as well as other cations such as Ca and Ba [12].

However, it is interesting to see that other than Al and Mg, all the other possible

elements such as Ca and Fe did not show any concentration gradient towards

elemental selenium.

The preference of any cation towards an adsorbent follows 1) a decrease in ionic

radius, 2) an increase in electronegativity of the metal ion or 3) an increase in ratio of

the ionisation potential and ionic radius [40]. In the previous study by the same authors,

the preference of divalent cations at equimolar concentration (Zn, Ca and Mg) towards

BioSeNPs followed ratio of ionization potential and ionic radius [26]. Fe has a higher

ratio of ionization potential to ionic radius when compared to Al, Mg and Ca (–0.68

compared to –3.1, –3.3, 2.9, respectively), higher concentration in the activated sludge

than Mg and Al (Table 1), would be present as a trivalent cation and still no gradient

towards BioSeNPs was observed. This might be due to very low solubility of Fe3+ at the

pH of this study and is precipitating as hematite as predicted by Visual MINTEQ. Hence,

Fe is not available to compete for adsorption sites on the elemental selenium

nanoparticles.

The ratio of ionization potential to ionic radius and the concentration in the selenium fed

activated sludge was slightly higher for Ca when compared to Al, but as a trivalent ion,

the tendency of Al for forming complexes with the protein coatings of EPS present on

elemental selenium nanoparticles [38] would be higher than Ca, thus Al would

outcompete other divalent cations including Ca and Mg. It is interesting to note that

though Mg has lower ionization potential to ionic radius and concentration in the

selenium fed activated sludge than Ca, Mg showed a gradient towards BioSeNPs and

Ca does not (Figure 3d). This might be again due to non availability of Ca which might

be due to the precipitation of Ca as hydroxyapatite as predicted by the Visual MINTEQ.

4.3 Practical implications

The presence of elemental selenium nanoparticles in the activated sludge flocs

improves their settleability. This is important for the activated sludge process as gravity

settling is a cost-effective method for industrial scale solid-liquid separation [41]. The

poorer dewaterability of the selenium fed activated sludge might affect the activated

sludge process, as the entrapped elemental selenium nanoparticles affect the CST at

TSS concentrations (> 3 g L–1). As the selenium is a scarce resource and used as a

fertilizer for selenium deficient soils [42], one of the possible uses of waste selenium fed

activated sludge is a slow release selenium fertilizer [43]. Elemental selenium is known

to adsorb heavy metals including mercury [44–46], copper [39] and zinc [26], thus

selenium fed activated sludge can also be used for decontamination of heavy metal and

specially mercury polluted soils [47]. However, further research is required to realize the

full potential of selenium fed activated sludge as fertilizer or remediation agent.

5. Conclusions

This study showed that most of the trapped selenium in activated sludge flocs is in the

form of elemental selenium. SEM and TEM images suggest that elemental selenium is

present both inside and outside the microbial cell. XRD and SAED suggest the

presence of aperiodic and β-monoclinic nanospheres as well as trigonal nanorods. The

trapping of this elemental selenium in the activated sludge flocs improves the

settleability and hydrophilicity of the sludge, but reduces the dewaterability of the

activated sludge (only at higher TSS concentrations). The selenium fed activated sludge

also showed a less negative surface charge density as compared to control activated

sludge.

Acknowledgements

The authors thank Ferdi Battles (UNESCO-IHE, The Netherlands) for ICP-MS analysis

and Rohit Kacker (TU Delft, The Netherlands) for XRD analysis.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given

approval to the final version of the manuscript.

Funding Sources

This research was supported through the Erasmus Mundus Joint Doctorate

Environmental Technologies for Contaminated Solids, Soils, and Sediments (ETeCoS3)

(FPA n°2010-0009). The purchase of the SEM facility of the Institut de Minéralogie, de

Physique des Matériaux et de Cosmochimie (IMPMC) was supported by Région Ile-de-

France grant SESAME 2006 N°I-07-593/R, INSU-CNRS, INP-CNRS, University Pierre

et Marie Curie – Paris 6, and by the French National Research Agency (ANR) grant no.

ANR-07-BLAN-0124-01. The TEM facility at IMPMC was supported by Région Ile-de-

France grant SESAME 2000 E 1435, INSU-CNRS, INP-CNRS and University Pierre et

Marie Curie (UPMC) – Paris 6.

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Table 1. Metal(loids) presence in the selenium fed activated sludge (As+Se).

Se Na Mg Al K Ca Fe

(mg per g TSS)

AS+Se 55

± 2

27

±4

4.6

± 0.3

3.1

± 0.7

3

± 2

31

± 2

14.0

± 0.3

Table 1.

List of figures

Figure 1. SEM (a) and EDX spectra of nanospheres (b1) and nanorods (b2) present in

the selenium fed activated sludge. Zoomed in SEM image (c), TEM image (d)

suggesting the presence of selenium inside the cells and flocs and (e) SEM images and

(f) corresponding cartography (carbon red, phosphorous green and selenium blue)

clearing showing the presence of selenium nanorods.

Figure 2. (a) XRD of selenium fed () and control (without selenium, ―-―-) activated

sludge and (b) SAED pattern of trapped elemental selenium nanospheres.

Figure 3. (a) SEM image of a single bacterium from selenium fed activated sludge and

EDXS analyses corresponding to the spatial distribution of (b) Se, (c) Al and (d) Mg in

the SEM image. Note that the distortion of one of the nanospheres of selenium

observed in (a) is due to the damage by the electron beam while performing the

measurements.

Figure 4. (a) Derivative of acid-base titration data of selenium fed (―) and control (―

―) activated sludge, (b) Titration data for selenium fed (◊ Raw, ― Simulated) and

control (∆ Raw, ― ― Simulated) activated sludge, (c) Surface charge density of

activated sludge trapping selenium (―) and control activated sludge (― ―) and (d) %

sites with corresponding pKa values of various functional groups for selenium fed and

control activated sludge.

Figure 5. SVI (a), CST (b) and RH (c) of selenium fed (◊) and control (∆) activated

sludge.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.