ARTICLE IN PRESS

0043-1354/$ - se

doi:10.1016/j.w

�Correspondfax: +39030 37

E-mail addr

Water Research 39 (2005) 2056–2064

www.elsevier.com/locate/watres

Bacteria enclosure between silica-coated membranes for thedegradation of organic compounds in contaminated water

Roberta Pedrazzania, Giorgio Bertanzab,�, Carlo Maffezzonia, Marina Gelmic,Nino Mancac, Laura E. Deperoa

aDepartment of Mechanical Engineering, Faculty of Engineering, INSTM, University of Brescia, Via Branze, 38, 25123 Brescia, ItalybDepartment of Civil Engineering, Faculty of Engineering University of Brescia, Via Branze, 38, 25123 Brescia, Italy

cInstitute of Microbiology, Faculty of Medicine and Surgery, University of Brescia, P. le Spedali Civili, 1, 25100 Brescia, Italy

Received 22 April 2004; received in revised form 8 September 2004

Available online 10 May 2005

Abstract

A non-conventional technique is proposed for the enclosure of either pure bacterial cultures or entire biocoenoses, for

a possible utilization in the treatment of contaminated water. Biological components have been enclosed between

polyester membranes coated by silica films consisting of: (a) SiO2 and nitrocellulose, (b) SiO2, ZnS crystals and

nitrocellulose, (c) SiO2, TiO2 crystals and nitrocellulose, (d) SiO2, ZnS and TiO2 crystals and nitrocellulose.

Morphological, structural and mechanical features of membranes were investigated by means of optical and electron

microscopy, mercury porosimetry and wear resistance tests. Degradation kinetics have been finally studied by dipping

the entrapped biomass into aqueous solutions of three different model organic compounds (a-D-glucose, ethyl alcoholand peptone). Results are very promising: in fact, no metabolic inhibition mechanisms of microorganisms have been

evidenced. The porosity of the system allows mass transfer through the membranes, hence bacteria can grow and

degrade pollutants. Besides, by this system, cells are constrained, avoiding they to spread across the retainment scaffold.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Biodegradation kinetics; Entrapped biomass; Membranes; Silica coating; Water treatment

1. Introduction

The removal of organic pollutants from surface and

groundwater is a demanding task (CLARINET, 2000;

US EPA, 2004) since human activities have caused wide

environment contamination phenomena which compro-

mise the water use for human consumption. Pollution

problems are correlated with the presence of both

inorganic (e.g., ammonia, nitrates, heavy metals, etc.)

and organic (e.g., chlorinated solvents, pesticides, PAHs,

e front matter r 2005 Elsevier Ltd. All rights reserve

atres.2005.03.013

ing author. Tel.: +39030 3715522;

15503.

ess: giorgio.bertanza@unibs.it (G. Bertanza).

etc.) substances. Common methods for water treatment,

and especially for contaminated groundwater remedia-

tion (like pump-and-treat, air sparging/bio-sparging,

chemical oxidation, etc.), are generally expensive and

often lead to the formation of harmful by-products.

The technique proposed in this paper is based on

biological degradation of pollutants by physically

encapsulated microorganisms. Due to physical features

of the tested system (see description below) its potential

application should concern the treatment of water

characterized by contaminants which are mainly

present in the dissolved form, such as, for instance,

groundwater and chemically physically pre-treated

surface water.

d.

ARTICLE IN PRESS

Fig. 1. Schematic representation of the experimental scaffold

fabricated for biomass enclosure.

R. Pedrazzani et al. / Water Research 39 (2005) 2056–2064 2057

Over the last three decades, the technology of

immobilized biologicals has greatly expanded in several

fields, ranging from synthetic and analytical chemistry,

to environmental monitoring and technology, medical

diagnostics and proteomics (Wijffels and Tramper, 1995;

Leenen et al., 1996; Tartakovsky et al., 1996; Gill and

Ballesteros, 2000a). Bioimmobilizates often require

stringent constrains in order to allow their application.

The major issues are the physico-chemical stability of

retaining matrix and immobilized biologicals, especially

in relation to performances in gaseous and liquid media

under biologically, chemically, and physically aggressive

conditions. Encapsulation is the most widely used

technique for immobilizing biological components

(O’Reilly and Scott, 1995; Nassif et al., 2002): synthetic

polymers can be obtained via sol–gel or non-sol–gel

processes (Gill and Ballesteros, 2000b; Viitala et al.,

2002). Sol–gel processes are generally based on hydro-

lysis, condensation, and polymerization of metal-alk-

oxides and yield matrices of defined porosity and

thickness, optically transparent, which are chemically,

thermally and dimensionally stable (Hench and West,

1990; Dunn et al., 1998; Chia et al., 2002). Sol–gel synthesis

of inorganic glasses is performed at room temperature,

thus enabling metabolic functioning of incorporated

biologicals (Avnir et al., 1994; Dunn et al., 1998). Proteins,

bacterial, algal, fungal and yeast whole cells are not

inhibited by the entrapment in the silica matrix, since its

porosity allows small molecules to diffuse into the matrix

itself (or to be spread out of the glass coating) (Dunn et al.,

1998; Carturan et al., 2000a; Carturan et al., 2000b).

Finally, as stated in literature (Tartakovsky et al., 1996;

Gill and Ballesteros, 2000a; Cohen, 2001; Viitala et al.,

2002), sol–gel-derived silica biomaterials are characterized

by mechanical stiffness and long-term stability.

The aim of the present study was to enclose biomass

between polyester membranes whose physical character-

istics were implemented by SiO2, SiO2/ZnS, SiO2/TiO2,

and SiO2/ZnS/TiO2 coatings (subsequently: S, SZ, ST,

SZT). A colloidal silica precursor was simply evaporated

to form an amorphous unstructured silica film. The

utilized method, indeed, differs substantially from

sol–gel chemistry (which is restricted to polymers

derived from the controlled reaction of silicon alkoxide

precursors) and was proposed because of silica biocom-

patibility and chemical stability, simplicity of the

manufacturing procedure, easy availability of employed

materials on the market and low costs (preliminary

investigations have shown that there should be an

appreciable saving, with respect to the use of conven-

tional synthetic membranes or other conventional

groundwater treatment systems commonly employed in

environmental applications) (US EPA, 2001; Van der

Roest et al., 2002).

A further nitrocellulose coating was also used in order

to prevent the crumbling of silica structure (this is very

important in view of the supposed applications), despite

the possible increase of the diffusion limitations.

Experimental tests focussed on the evaluation of

bioactivity and viability maintenance of entrapped

biomass.

2. Materials and methods

2.1. Experimental scaffolds for bacteria retention

Biomass (in the form of slurry: see below) was

retained between two polyester membranes (mesh:

71.55, expressed in cm, thread: 55 mm), which were

tightened by a stainless steel frame (30 cm in length,

15 cm in width) and completed with four different

coatings: S, SZ, ST or SZT, respectively (see Fig. 1).

Polyester membranes provide a robust physical

support for the silica-based layer; the latter controls

the porosity of the inert barrier in order to avoid the

biomass to spread across.

Silica medium consisted of a colloidal suspension of

SiO2 (Du Pont) in which TiO2 and/or ZnS crystals

(average particle diameter: 27.7mm for ZnS and 0.25mmfor TiO2) were dispersed. SiO2 and ZnS crystals were

mixed in a 6–4 w/w ratio; colloidal SiO2 and TiO2

crystals were mixed in a 9–1 w/w ratio. The four

different dispersions (S, ST, SZ, SZT) were deposed on

the polyester membrane obtaining a homogeneous layer.

Samples were then heated at 80 1C for 10min for drying.

Since the silica layer is fragile and easily crumbles, as

stated above, an additional nitrocellulose coating was

deposed so as to improve the physical features of the

membranes. Cellulose nitrate (Aldrich) was dissolved in

amyl acetate (Aldrich) in a 12.5–87.5 w/w ratio. The

nitrocellulose amyl acetate solution was then sprayed

onto both sides of the membrane. Then, the amyl acetate

was evaporated by heating at 80 1C for 5min.

Biomass was taken from the oxidation tank of a

municipal wastewater activated sludge treatment plant,

since biodegradation kinetics, biotic structures,

ARTICLE IN PRESSR. Pedrazzani et al. / Water Research 39 (2005) 2056–20642058

physiological and metabolic patterns are well known

(Henze et al., 1997). Activated sludge was centrifuged

(5000 rpm, 5min) so as to obtain slurry with high

biomass concentration (about 7 g/L of volatile dry

matter). The slurry (50mL) was finally placed between

two membranes, sealed with silicone (see Fig. 1).

2.2. Characterization of preformed membranes

Microscopy investigations: Membrane structure was

investigated by means of optical and electron micro-

scopy techniques: a Reichart-Jung MeF3 optical micro-

scope (for metallography) and a Cambridge Stereoscan

260 SEM microscope were, respectively, used.

Porosity: Coatings porosity and pore size distribution

were monitored by a PASCAL 240 mercury porosi-

meter, which allows to determine pore size and volume,

in the range of 3.7 and 7500 nm radius and with a

volume resolution of 0.1mm3, by means of mercury

intrusion at high pressure (up to 200MPa). Porosity

analyses were performed on membrane samples of about

700 cm2 (5 g weight).

Membrane retention efficiency and biomass viability: A

preliminary experiment was aimed at verifying both the

biocompatibility of scaffolds (biomass viability) and

their biomass retention effectiveness. This test was

performed preparing a scaffold in which only Escher-

ichia coli was introduced. This apparatus was dipped

into 3L of sterile buffered peptone water (Biolife) and

maintained at a temperature of 37 1C with sterile air

blowing for 2 weeks. Samples of the contact solutions

(10mL, 4 replicates) were taken after 1, 2, 7, 14, 28 days

and submitted to E. coli determination after incubation

(at 44.570.2 1C, for 24 h) in agar plates with Tryptone

Bile X-Glucuronide Medium (Oxoid). After 28 days,

surface sampling was also carried out (4 cm2 surface, 4

replicates), in order to exclude the presence of E. coli

attached onto the external side of membranes.

Dehydrogenase activity was monitored at the begin-

ning and at the end of the test, taking bacteria samples

(1mL sample, 2 replicates) from the apparatus, in order

to assess biomass viability (Andreottola et al., 2002).

Wear resistance: Mechanical tests were carried out to

verify coatings wear resistance under turbulent water

conditions. Membranes were maintained in water for 1

month. This period of time was defined considering that,

in a supposed practical application, membranes should

be substituted every month, due to biomass metabolism

constrains. Turbulence was obtained by rotational

movement of the scaffolds or air blowing. Water

samples were taken at fixed time intervals and Si, Zn

and Ti concentrations were measured in order to check

possible coating failures and consequent metal

release.

The apparatus was fixed to a rotating shaft and

dipped into 3L of distilled water. It was continuously

rotated (40 rpm) for a week. After 64 and 167 h contact

water was replaced and 500mL samples were taken and

submitted to Si, Zn and Ti analyses.

Afterwards, the same apparatus was dipped into 2.7L

of distilled water and air diffusers were placed under the

scaffold. After 64 h, contact water was replaced and a

500mL sample was taken. The same operations were

repeated after 232 and 569 h.

In all cases, contact water samples were filtered using

polycarbonate membranes (Nuclepores Track Etch

membranes, Whatman); both filtered water and mem-

branes were digested with fluoridric acid and then

submitted to ICP analyses which were performed by

means of ICP-AES Jobin Yvon Sequential Spectrometer

JY24.

Besides, coatings surface was periodically monitored

with optical microscope.

2.3. Pollutants degradation efficiency

The evaluation of the organic pollutants degradation

capability was assessed by means of laboratory tests

carried out in batch conditions by comparing metabolic

activity of free and encapsulated cells.

Five parallel experimental runs were performed, for

testing free biomass (as a reference) and biomass

enclosed between S, SZ, ST, SZT preformed coated

membranes, respectively. Microorganisms were dipped

into 6L distilled water tanks; at different time intervals,

nutrients (K2HPO4, KH2PO4 � 3H2O, NH4NO3, NaCl,

Carlo Erba Reagenti) and different carbon sources

(respectively, (a) a-D-glucose, Carlo Erba Reagenti; (b)

ethyl alcohol—anhydrous, denatured with 5% isopropyl

alcohol and 5% methyl alcohol-, Aldrich and (c)

buffered peptone water, Biolife) were dosed. Carbon

compounds were dosed so as to reach a COD

concentration of about 150–200mg/L, while nitrogen

and phosphorous were added in such amounts to avoid

nutrients to be a limiting factor for bacterial activity.

Table 1 shows the composition of ethyl alcohol and

glucose stock solutions and peptone powder employed

for substrates dosage.

The test baths were agitated by means of magnetic

stirrers and dissolved oxygen was supplied by an air

blower throughout the test. Microorganisms degrada-

tion activity was evaluated by measuring the COD and

TOC residual concentration, at fixed time intervals.

Inorganic nitrogen compounds concentration was also

determined. Temperature, pH (WTW Inolab pH meter)

and dissolved oxygen concentration (WTW oxymeter)

were on-line monitored. pH and dissolved oxygen were

adjusted respectively at 8.0 and 6.0mg/L. COD, TOC

and nitrogen concentration measurements were per-

formed accordingly with IRSA-CNR methods (IRSA-

CNR, 1994).

ARTICLE IN PRESS

Table 1

Composition of stock solutions and peptone powder employed during degradation bench tests

Ethyl alcohol stock solution (g/L) Ethyl alcohol NH4NO3 KH2PO4 K2HPO4 � 3H2O

5 3 0.086 2

Glucose stock solution (g/L) Glucose NH4NO3 KH2PO4 K2HPO4 � 3H2O

10 3 0.086 2.56

Biolife peptone powder (%w/w) Peptone NaCl Na2HPO4 NaH2PO4

50 25 17.5 7.5

R. Pedrazzani et al. / Water Research 39 (2005) 2056–2064 2059

2.4. Mass balances

For a more accurate analysis of degradation processes

during bench tests, and for an approximate mass balance

determination, the following phenomena were considered:

�

organic substrate degradation (both under aerobic

and anoxic conditions);

�

cellular lysis and consequent nitrogen release;

�

nitrification and denitrification.

Process simplified modelling was developed through

the following steps:

(1)

(Removed N)/(removed COD) ratio determination

Nitrogen, in the form of ammonia, is assimilated

during organic substrate degradation both under

aerobic and anoxic conditions (biomass growth).

Besides, nitrogen in the form of nitrate is denitrified

under anoxic conditions.

The amount of ammonia required for biomass

production depends on the organic substrate being

degraded and can be calculated if the yield constant

Yobs is known. This can be approximately estimated

for every batch test from experimental data calcu-

lating the actual biomass growth as the difference

between maximum growth [ ¼ Ymax� (degraded

organic substrate)] and decay [ ¼ (decay rate) �

(amount of biomass) � (duration of the test)],

assuming that maximum yield constant (Ymax) and

decay rate are the same for aerobic and anoxic

conditions. Based on this simplified approach,

calculated Yobs values range from 0.26 to 0.45 and

from 0.39 to 0.65 (g biomass COD/g substrate

COD), respectively, for ethyl alcohol and glucose

(depending on several factors such as duration of

the test, F/M ratio, temperature, etc.), where

assumed values for Ymax are 0.55 (g biomass

COD/g substrate COD) for ethyl alcohol and 0.75

(g biomass COD/g substrate COD) for glucose

(Dircks et al., 1999) and assumed decay rate is

0.08–0.10 d�1 as a function of temperature (Henze

et al., 1997). For instance, assuming for Yobs the

values 0.454 (for ethyl alcohol) and 0.647 (for

glucose), obtained during one of the experiments

carried out, the biomass growth equations are the

following:

C2H5OHþ 0:272NH3 þ 1:640O2

! 0:272C5H7NO2 þ 0:640CO2 þ 2:456H2O; ð1Þ

C6H12O6 þ 0:777NH3 þ 2:115O2 ! 0:777C5H7NO2

þ 2:115CO2 þ 4:446H2O; ð2Þ

where C5H7NO2 is the generic composition of

biomass (Henze et al., 1997).

From the above equations, the mass ratio nAER

between assimilated N and COD degraded under

aerobic conditions can be easily calculated, respec-

tively, for ethyl alcohol and glucose:

nAER; C2H5OH ¼ 0:0396g N

g COD,

nAER; C6H12O6¼ 0:0565

g N

g COD.

Similarly, ethyl alcohol and glucose degradation

under anoxic conditions can be described as follows

(provided that the organism assimilates ammonium):

C2H5OHþ 1:312 NO�

3 þ 0:272 NHþ

4 þ 1:040 Hþ

! 0:272 C5H7NO2 þ 0:656 N2

þ 0:640 CO2 þ 3:112 H2O; ð3Þ

C6H12O6 þ 1:692 NO�

3 þ 0:777 NHþ

4 þ 0:915 Hþ

! 0:777 C5H7NO2 þ 0:846 N2

þ 2:115 CO2 þ 5:292 H2O; ð4Þ

leading to the same mass ratios nANOX (assimilated

nitrogen/degraded COD) as calculated for aerobic

conditions:

nANOX; C2H5OH ¼ nAER; C2H5OH ¼ nC2H5OH ¼ 0:0396g N

g COD,

nANOX; C6H12O6¼ nAER;C6H12O6

¼ nC6H12O6¼ 0:0565

g N

g COD.

From Eqs (3) and (4) the mass ratio n0 between

denitrified nitric nitrogen and degraded COD can

ARTICLE IN PRESSR. Pedrazzani et al. / Water Research 39 (2005) 2056–20642060

also be calculated by observing that denitrification

of 1mole of NO3� requires 1mole of C2H5OH or

1mole of C6H12O6. Resulting n0 coefficients are the

following:

n0

C2H5OH ¼ 0:1911g N2NO�

3

g COD,

n0

C6H12O6¼ 0:1230

g N2NO�

3

g COD.

(2)

Determination of N–NH4+ deriving from cellular

lysis

Biomass decay and consequent cellular lysis increase

the ammonia nitrogen content in the bulk liquid

(10%, being the typical nitrogen content in the

biomass expressed as volatile solids) (Metcalf and

Eddy Inc., 1991).

Biomass concentration was known; thus considering

a decay rate of 0.08–0.10 g VSS/g VSSd (as a

function of temperature) it was possible to obtain

the amount of nitrogen release due to biomass decay

and cellular lysis.

(3)

Evaluation of actually removed N–NH4+

Ammonia nitrogen amount was calculated (based

on the measured concentrations and the water

volume) at the beginning ([N–NH4+]0) and at the

end of the test ([N–NH4+]END); by calculating

N–NH4+ produced from cellular lysis ([N–NH4

+]L)

as described in step (2), the actually removed

nitrogen ([N–NH4+]REM) could be evaluated as

follows:

½N2NHþ

4 �REM ¼

½N2NHþ

4 �0 � ½N2NHþ

4 �END þ ½N2NHþ

4 �L.

(4)

Evaluation of assimilated nitrogen

COD concentration was measured at the beginning

and at the end of the test, allowing the calculation of

the amount of degraded COD ([COD]DEG). Assimi-

lated nitrogen ([N]ASS) was calculated by the

following equation:

½N�ASS ¼ n½COD�DEG,

n being calculated in step (1).

(5)

Evaluation of nitrified nitrogen:

On the basis of the amount of removed ammonia

nitrogen ([N–NH4+]REM) and assimilated nitrogen

([N]ASS), nitrified nitrogen ([N–NH4+]NIT) was

calculated:

½N2NHþ

4 �NIT ¼ ½N2NHþ

4 �REM � ½N�ASS.

(6)

Evaluation of denitrified nitrogen

The denitrified nitrogen ([N]DEN) was obtained based

on the amount of oxidized nitrogen forms at the

beginning and at the end of the test ([N–NOx�]0 ,

[N–NOx�]END) and of nitrified nitrogen

([N–NH4+]NIT), as follows:

½N�DEN ¼ ½N�NO�

X �0 � ½N�NO�

X �END þ ½N�NHþ

4 �NIT,

where

½N�NO�

X � ¼ ½N�NO�

2 � þ ½N�NO�

3 �.

(7)

Determination of COD degraded under anoxic

conditions

COD degraded under anoxic conditions ([COD]ANOX)

was calculated, depending on the specific organic

substrate, based on the amount of denitrified

nitrogen, as follows:

½COD�ANOX ¼½N�DEN

n0.

(8)

Determination of COD degraded under aerobic

conditions

Finally, COD degraded under aerobic conditions

([COD]AER) was calculated as a difference be-

tween COD degraded during the overall process

([COD]DEG) and the amount of COD degraded

under anoxic conditions ([COD]ANOX):

½COD�AER ¼ ½COD�DEG � ½COD�ANOX.

3. Results and discussion

3.1. Characterization of preformed membranes

Microscopy investigations: Micrographs (Fig. 2) show

that: SiO2 homogeneously covers the polyester structure

(A) presenting irregular fissures; ZnS crystals (B) seems

to reduce the fissure size while TiO2 (C) tends to

decrease the overall cracking.

Porosity: As a result of porosity tests, the cumulative

distribution of pore volume with respect to pore

diameter was obtained. Experimental data were pro-

cessed by means of a Savitzky–Golay algorithm. Fig. 3

shows a significant difference in coatings pore size

distribution, depending on ZnS and TiO2 crystals

presence. S and ST coatings are characterized by two

main pore diameter domains: 3–5 and 7–9 mm range,

respectively. On the other hand, SZ and SZT coatings

are monodispersed with a pore diameter of 4–5 mm.

Whichever is the cause of two distribution functions of

membrane S (we have to consider that dead volumes are

also measured with the employed technique, so surface

defects can be revealed), the presence of TiO2 (average

particle diameter: 0.25 mm) does not modify significantly

the distribution of pore diameters, although the amount

of larger pores is decreased. In membranes containing

ARTICLE IN PRESS

Fig. 2. Micrographs (SEM) of S (A), SZ (B), ST (C) membranes.

0

0.0005

0.001

0.0015

0.002

0.0025

100 1000 10000 100000

dV

/dD

[m

m3/

nm

]

SSTSZSZT

Pore Diameter [nm]

Fig. 3. Plot of (dV/dr) vs. log (D) for S, SZ, ST, SZT coatings.

R. Pedrazzani et al. / Water Research 39 (2005) 2056–2064 2061

ZnS, these crystals (average particle diameter: 27.7 mm)

reduce the porosity (large cavities and defects are

avoided due to ‘‘filling’’ effect of ZnS) hence a

monodispersed pore distribution resulted. In this case,

the further addition of TiO2 leads only to a broader pore

size distribution.

Membrane retention efficiency and biomass viability:

All E.coli determinations (water and surface samples)

led to absence of bacterial growth on agar plates, thus

showing that no biomass leakage occurred under the

tested conditions.

Dehydrogenase activity tests after 28 days showed the

maintenance of respiratory activity, the average mea-

sured concentration of monoformazans being equal to

11.5mg TPF/mg VSS (free biomass, at the beginning of

the test) and 10.8mg TPF/mg VSS (entrapped biomass,

at the end of the test).

Wear resistance: Optical microscope observations (not

shown) performed during the test did not highlight any

significant structural change on membrane’s coatings.

Besides, Zn, Si and Ti were never detected in contact

water (concentration o5mg/L for Zn and concentration

o9mg/L for Si and Ti). These data suggest a good wear

resistance of all types of silica coatings.

3.2. Pollutants degradation efficiency and mass balances

As far as bioactivity is concerned, Fig. 4 displays, as

an example, only COD concentration time profile

measured in case of S and SZ membranes. As a first

point, it can be seen that different behaviours occurred

with the same substrate (see dosage 1 and dosage 3 for

ethyl alcohol). This was probably due to the lack of

bacteria acclimation in the case of dosage 1 (for ethyl

alcohol) and dosage 2 (for glucose). As a second point,

the degradation rate was higher for glucose and ethanol

and lower for peptone (see dosages 3, 4 and 5); this was

likely caused by molecular complexity of peptone in

comparison with glucose and ethanol.

Considering the nitric nitrogen time profile (Fig. 5), it

appears as the difference between free and enclosed

biomass. Enclosed bacteria were able to remove nitrates

through denitrification. It can be supposed that deni-

trification occurred within the inner layer of biomass,

where anoxic conditions could be established.

Fig. 6 displays the results of mass balance calcula-

tions; the example of glucose as organic substrate is

shown. Fig. 6 summarizes all results obtained with

different types of membrane coatings. Only data

referring to acclimated biomass are reported (for

instance, in the case of S and SZ membranes, only data

concerning dosage 5 are plotted, while dosage 2 is not

considered). It can be observed that no appreciable

differences in COD removal were obtained with

different apparatuses but S and SZ membranes showed

higher removal rates (COD was degraded in 28 h) even if

temperature was relatively low (14 1C). It can be

supposed that in TiO2-coated structures mass transfer

through the membranes was delayed, for instance, due

to a lower global porosity.

In general, there was a high N removal, also thanks to

denitrification, which, actually, did not always occur at

the same extent. In fact, if tests carried out with ST and

SZT membranes (in which glucose was added twice at

different times) are considered, it can be seen that in one

case denitrification was less efficient and a lower COD

degradation under anoxic conditions was obtained. It is

worth noting that aerobic COD degradation was faster

ARTICLE IN PRESS

0

50

100

150

200

250

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

time [h]

SiO2 (S) SiO2+ZnS (SZ)

Dosage 5Glucose +nutrients

Dosage 2Glucose +nutrients

Dosage 3Ethyl alcohol +nutrients

Dosage 4Peptone

413.25

Dosage 1Ethyl alcohol +nutrients

CO

D [

mg

/L]

Fig. 4. COD time profiles obtained during substrate degradation tests (S and SZ membranes).

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

N-N

O3-

[mg

/L]

SiO2 (S) SiO2+ZnS (SZ) free biomass

Dosage 1Ethyl alcohol +nutrients

Dosage 3Ethyl alcohol +nutrients

Dosage 2Glucose +nutrients

Dosage 4Peptone

413.25

Dosage 5Glucose +nutrients

time [h]

Fig. 5. N–NO3� time profiles obtained during substrate degradation tests (S and SZ membranes in comparison with free biomass).

R. Pedrazzani et al. / Water Research 39 (2005) 2056–20642062

than aerobic/anoxic removal. This phenomenon (which

was observed also in the case of ethyl alcohol) can be

explained by considering that denitrification can be

affected by the COD concentration gradient through the

membranes. When the diffusional velocity of organic

substrate allows organic matter to reach the anoxic

zone, denitrification can occur. But, if the diffusional

resistance is increased (for example, due to the

accumulation of degradation by-products on the inner

surface of the membrane which, as a consequence, are

clogged), COD is mainly degraded in the aerobic zone

and partial denitrification occurs. Then, the membranes

porosity and consequently their behaviour are also

influenced by the working time.

ARTICLE IN PRESS

Fig. 6. Results of mass balance calculations (glucose): substrates (expressed in mg substrate/L contact solution) removed during

degradation tests. Dt ¼ time required for substrate degradation.

R. Pedrazzani et al. / Water Research 39 (2005) 2056–2064 2063

4. Conclusions

A novel approach for the realization of a simple and

cheap system for biodegradation of dissolved organics in

contaminated water was proposed: biomass was en-

closed between polyester membranes coated with silica

films consisting of: (a) SiO2 and nitrocellulose; (b) SiO2,

ZnS crystals and nitrocellulose; (c) SiO2, TiO2 crystals

and nitrocellulose; (d) SiO2, ZnS and TiO2 crystals and

nitrocellulose. The system was submitted to several tests;

the following considerations can be drawn:

�

Membrane physical features are affected by the type

of coating: in particular, ZnS crystals reduce the pore

size (a pore diameter domain of 4–5mm was

measured) and leads to a monodispersed pore

distribution curve, while a further TiO2 addition

slightly increases the dispersion around the mean

value. The final nitrocellulose coating avoids that

silica-based layers crumble under turbulent condi-

tions.

�

Obtained features allow the diffusion of substrates

(organics, nutrients, oxygen) from the bulk liquid

through the membranes and the enclosed biomass

avoiding its metabolic inhibition; on the contrary, the

biomass cannot spread across the retainment scaf-

fold, so uncontrolled bacteria diffusion into the

surrounding environment is prevented. Indeed, since

bacteria size (E. coli was used in this test) is of the

same order of magnitude of the measured pore

diameters, it can also be supposed that the nitrocel-

lulose layer plays an important role in this issue.

�

Substrate degradation tests (carried out by entrap-

ping activated sludge from a municipal wastewater

treatment plant) showed that TiO2 coating delays the

mass transfer of substrates through the membranes,

since with ST and SZT membranes COD removal

ARTICLE IN PRESSR. Pedrazzani et al. / Water Research 39 (2005) 2056–20642064

rate was lower with respect to the rate obtained with

S and SZ membranes. Global porosity was supposed

to be reduced by TiO2 presence.

�

Both aerobic and anoxic processes (with N–NO3-

denitrification) could take place at a different extent

within the membranes depending on the substrate

diffusional velocities with respect to the degradation

rates.

In conclusion, even if further investigations are

required for the optimization of material synthesis and

for its testing under different environment conditions

(characteristics of ‘‘target’’ compounds and conse-

quently selected bacteria, flow regime, temperature,

pH, dissolved oxygen concentration, working time,

etc.), the first series of tests has shown the feasibility of

this approach. ZnS crystals are important in homo-

genizing the porosity, while TiO2 seems to decrease the

system performance. Finally, nitrocellulose plays an

important role for both material stability and bacterial

escape prevention.

There is a patent pending on this technique.

References

Andreottola, G., Baldassarre, L., Collivignarelli, C., Pedrazza-

ni, R., Principi, P., Sorlini, C., Ziglio, G., 2002. A

comparison among different methods for evaluating the

biomass activity in activated sludge systems: preliminary

results. Water Sci. Technol. 46 (1–2), 413–417.

Avnir, D., Braun, S., Lev, O., Ottolenghi, M., 1994. Enzymes

and other proteins entrapped in sol–gel materials. Chem.

Mater. 6, 1605–1614.

Carturan, G., Campostrini, R., Dal Monte, R., 2000b.

Secondary metabolites from cells immobilized by a SiO2

sol–gel layer. In: Tundo, P., Anastas, P. (Eds.), Green

Chemistry. Challenging Perspectives. Oxford University

Press, New York, pp. 61–78.

Carturan, G., Dal Monte, R., Muraca, M., 2000a. SiO2

entrapment cells for hybrid bioartificial organs. Mater.

Res. Soc. Symp. Proc. 628 CC10.1-15.

Chia, S.M., Wan, A.C.A., Quek, C.H., Mao, H.Q., Xu, X.,

Shen, Lu., Ng, M.L., Leong, K.W., Yu, H., 2002. Multi-

layered microcapsules for cell encapsulation. Biomaterials

23, 849–856.

CLARINET, 2000. Remediation of contaminated land tech-

nology implementation in Europe: a state of the art of

CLARINET WG 7. Eighth Draft Document 2000

December.

Cohen, Y., 2001. Biofiltration—the treatment of fluids by

microorganisms immobilized into the filter bedding materi-

al: a review. Bioresourc. Technol. 77, 257–274.

Dircks, K., Pind, P.F., Mosbaek, H., Henze, M., 1999. Yield

determination by respirometry—the possible influence of

storage under aerobic conditions in activated sludge. Water

SA 25 (1), 69–74.

Dunn, B., Miller, J.M., Dave, B.C., Valentine, J.S., Zink, J.I.,

1998. Strategies for encapsulating biomolecules in sol–gel

matrices. Acta Mater. 3, 737–741.

Gill, I., Ballesteros, A., 2000a. Bioencapsulation within

synthetic polymers (Part 1): sol–gel encapsulated biologi-

cals. Trends Biotechnol. July, 282–296.

Gill, I., Ballesteros, A., 2000b. Bioencapsulation within

synthetic polymers (Part 2): non-sol–gel protein polymer

biocomposites. Trends Biotechnol. November, 469–479.

Hench, L.L., West, J.K., 1990. The sol–gel process. Chem. Rev.

90, 33–79.

Henze, M., Harremoes, P., Jansen, J.l., Arvin, E., 1997.

Wastewater Treatment, Biological and Chemical Processes.

Springer, Berlin.

IRSA-CNR, 1994. Standard Methods for Water Analysis

(Metodi analitici per le acque). Quaderno n. 100. Istituto

Poligrafico e Zecca dello Stato, Roma (Italy).

Leenen, E.J.T.M., Dos Santos, V.A.P., Grolle, K.C., Tramper,

J., Wijffels, R.H., 1996. Characteristics of and selection

criteria for support materials for cell immobilization in

wastewater treatment. Water Res. 12, 2985–2996.

Metcalf and Eddy Inc., 1991. Wastewater Engineering: Treat-

ment, Disposal and Reuse, third ed, revised by G.

Ichobanoglous and F.L. Burton McGraw-Hill, New York

Nassif, N., Bouvet, O., Rager, M.N., Roux, C., Coradin, T.,

Livage, J., 2002. Living bacteria in silica gel. Nat. Mater. 1,

42–44.

O’Reilly, A.M., Scott, J.A., 1995. Defined coimmobilization of

mixed microorganism cultures. Enzyme Microb. Technol.

17, 636–646.

Tartakovsky, B., Kotlar, E., Sheintuch, M., 1996. Coupled

nitrification–denitrification processes in a mixed culture of

coimmobilized cells: analysis and experiment. Chem. Eng.

Sci. 51 (10), 2327–2336.

US EPA, 2001. Cost analyses for selected groundwater cleanup

projects: pump and treat systems and permeable reactive

barriers. EPA Report 542-R-00-013.

US EPA, 2004. Federal Remediation Technologies Roundtable.

Remediation Technologies: Screening Matrix and Reference

Guide. Version 3.0. http://www.frtr.gov/matrix2/top_pa-

ge.html.

Van der Roest, H.F., Lawrence, D.P., Van Bentem, A.G.N.,

2002. Membrane Bioreactors for Municipal Wastewater

Treatment. IWA Publishing, Colchester, UK.

Viitala, R., Jokinen, M., Peltola, T., Gunnelius, K., Rosen-

holm, J.B., 2002. Surface properties of in vitro bioactive and

non-bioactive sol–gel derived materials. Biomaterials 23,

3073–3086.

Wijffels, R.H., Tramper, J., 1995. Nitrification by immobilized

cells. Enzyme Microb. Technol. 17, 482–492.