Silica-carrageenan hybrids used for cell immobilization realizing high-temperature degradation of nitrile substrates

Article abstract of DOI:10.2478/s11532-010-0140-x

In this work the application of hybrid materials, containing TEOS as source of SiO2 and k-carrageenan in different percentage, synthesized by the sol-gel method at room temperature was studied. They were used as matrices for entrapment of whole Bacillus sp. UG-5B cells, producers of thermostable nitrilase. The effect of the surface area and size and quantity of pores in the synthesized materials on the enzyme activity was evaluated. The process of biodegradation of different concentrations of toxic, potentially carcinogenic and mutagenic substrates by the obtained biocatalysts was investigated. The enzyme reaction takes place by the nitrilase pathway, catalysing nitrile hydrolysis directly to the corresponding carboxylic acid, forming ammonia. At batch experiments the influence of the substrate concentration of different nitriles was tested and 20 mM concentration was found most suitable. A two-step biodegradation process in a laboratory-scale column bioreactor of o-, m- and p-tolunitrile as a mixture was followed. After operation of the system for nine hours for the mixture of substrates at a flow rate of 45 mL h^-1 and at 60°C, the overall conversion realized was above 90%, showing a good efficiency of the investigated process. Versita Sp. z o.o.

Full text of DOI:10.2478/s11532-010-0140-x

Cent. Eur. J. Chem. • 9(2) • 2011 • 232-239
DOI: 10.2478/s11532-010-0140-x
Central European Journal of Chemistry
Silica-carrageenan hybrids used for cell
immobilization realizing high-temperature
degradation of nitrile substrates
Research Article
Lyudmila V. Kabaivanova1, Georgi E. Chernev2*,
Isabel M. Miranda Salvado3, Maria H.V. Fernandes3
1Institute of Microbiology, Bulgarian Academy of Science,
1400 Sofia, Bulgaria
2Department of Silicate Technology, University of Chemical Technology
and Metallurgy, 1756 Sofia, Bulgaria
3Department of Ceramic and Glass Technology, University of Aveiro,
CICECO, 3810-193 Aveiro, Portugal
Received 10 September 2010; Accepted 23 November 2010
In this work the application of hybrid materials, containing TEOS as source of SiO2 and k-carrageenan in different percentage,
synthesized by the sol-gel method at room temperature was studied. They were used as matrices for entrapment of whole Bacillus
sp. UG-5B cells, producers of thermostable nitrilase. The effect of the surface area and size and quantity of pores in the synthesized
materialsontheenzymeactivitywasevaluated.Theprocessofbiodegradationofdifferentconcentrationsoftoxic,potentiallycarcinogenic
and mutagenic substrates by the obtained biocatalysts was investigated. The enzyme reaction takes place by the nitrilase pathway,
catalysing nitrile hydrolysis directly to the corresponding carboxylic acid, forming ammonia. At batch experiments the influence of the
substrate concentration of different nitriles was tested and 20 mM concentration was found most suitable. A two-step biodegradation
process in a laboratory-scale column bioreactor of o-, m- and p-tolunitrile as a mixture was followed. After operation of the system for
nine hours for the mixture of substrates at a flow rate of 45 mL h-1 and at 60oC, the overall conversion realized was above 90%, showing
a good efficiency of the investigated process.
Abstract:
Keywords: Sol-gel • Carrageenan • Entrapment • Biodegradation • Cyanides
© Versita Sp. z o.o.
1. Introduction
been tested for immobilization of various types of living
cells to achieve utilization of biochemical functions of
active biomolecules, giving good yield of the developed
processes [6,7].
Creating biocatalysts by immobilized whole
microbial cells finds its application in a wide spectrum
of biotechnological processes, starting from the ethanol
production [8] to phenol degradation [9,10]. Immobilized
cell bioreactors have been used due to their stability
and possibility to achieve a continuous biodegradation
process [11].
Using different biocatalysts is expected to reduce the
energy expenses and their employment could decrease
the distribution of various pollutants in the environment
[11-13]. The wastewater treatment technologies could
be significantly developed by the introduction of new
Cell immobilization technology consists in the
attachment of cells to a certain separated insoluble
phase different from the phase of the free solution but
capable of exchanging substrate and product molecules
and accomplishing of the enzyme substrate reactions
[1-3]. Immobilization methods are mainly chemical and
physical. Chemical methods involve covalent bond
formationbetweenthesurfaceofthecellandcarrier,while
the physical ones are characterized with electrostatic
interactions, ionic bonds, hydrophobic influences [4,5].
According to the purpose of the experiments, adsorption,
chemical binding after activation or entrapment inside
the volume of the carrier can be chosen as to realize an
effective and low cost process. Different carriers have
* E-mail: georgi_chernev@yahoo.com
232

L. V. Kabaivanova et al.
microbial treatment [14,15]. One part of this problem is chemical properties can be controlled by variations
the detoxification of contaminated hot industrial waters in the quantities of the constituents and depend on
before cooling. The application of biological systems for the synergism between them [25]. It is supposed that
conversion of nitrile compounds to the corresponding interaction between phases in the hybrid materials,
acid and ammonia is an attractive alternative to the where the inorganic phase is often covalently bonded
chemical methods due to the good yield, the mild with the organic polymer, will lead to more valuable
reaction conditions and the specific activities of the properties compared to classical composites [26].
enzyme systems [16].
The aim of the present study was sol-gel synthesis
Nitriles are toxic, carcinogenic and mutagenic of hybrid matrices containing k-carrageenan for
organocyanides, which are often found in the obtaining stable and effective biocatalysts for enzyme
environment as a result of different industries and may biodegradation processes of different toxic substances
persist in soil or water for a long period and cause at high temperature.
harmful disturbances to human health. There exists an
urgent need for development of procedures that reduce
2. Experimental Procedure
the nitrile concentrations throughout the environment in
that way reducing the impact of nitrile wastes streams.
Immobilization of nitrile-converting bacteria as means of 2.1. Microorganism and culture conditions
increasing catalyst longevity in chemical synthesis [17] Cells of the moderate thermophilic strain Bacillus sp.
or for their use in detoxification of contaminated sites UG-5B with nitrilase activity, deposited in National Bank
has been of particular interest lately [18].
of Industrial Microorganisms and Cell Cultures-Bulgaria
Combining silicon chemistry with life sciences makes #8021/2001, isolated from polluted industrial waters
possible the application of hybrid biomaterials as carriers near the refinery “Neftochim” Bourgas, were used in the
for different biomolecules, including cells [18,19]. The immobilization procedures. Cultivation was carried out
preparation of bioactive composites could be achieved at 60°C. The medium composition was (g L-1): Reptone-
by the developments in sol-gel techniques [20,21]. For 5.00 (Oxoid); Yeast extract-5.00 (Oxoid); K HPO -5.00;
4
successful implementation of this task the biological MgSO -0.10; NaCl-1.00; FeS0 •7H 0-0.03;2benzonitrile
4
2
reagent should remain intact within the suitable hybrid -20 m4M, pH-7.5. Bacillus sp. UG-5B was grown in
material with high activity and long-term stability, good Erlenmayer flasks (500 mL) for 14 h. The shake-flask
resistance to leaching and desorption [22]. Sol-gel culture was grown in a rotary shaker (Gyrotory water
materials are promising for application since they have bath shaker New Brunswick Scientific). Separation of
demonstrated to be highly compatible with enzymes the cells from the culture medium was performed at the
and cells even stabilizing and protecting them with their beginning of stationary state by centrifugation and after
great mechanical, thermal and biological resistance that they are re-suspended in a phosphate buffer solution
[23,24]. A new field in the sol-gel technology is the (0.06 M, pH 7.2). Cell suspension with a concentration
synthesis of hybrid materials, simultaneously containing of 35 mg mL-1
dry cells and nitrilase activity of 2.8 U mL-1
inorganic and organic components. Their physical and
was used for entrapment.
Figure 1. Schematic presentation of laboratory scale bioreactor used for biodegradation of the mixture of cyanide compounds
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Silica-carrageenan hybrids used for cell immobilization
realizing high-temperature degradation of nitrile substrates
regulated the flow-rate. The bioreactor performances
2.2 Chemicals
were carried out at a flow rate of 45 mL h-1.
The silicon alкoxide precursor: tetraethylortosilicate
(TEOS) purchased by “Merck” was used. H2O; 0.1 N
HCl and phosphate buffer with pH=7.2 ± 0.02 at 20°C
(0.06 M) -“Merck” were also used in the synthesis.
The organic part involved was k-carrageenan (Merck)
in quantity 5 wt% (solubility 0.5 g in 100 mL hot water,
20°C). Benzonitrile and tolunitriles, used as substrates
in the enzyme reactions were purchased by “Fluka”.
2.5. Assay methods
Optical density was measured at 660 nm to estimate
cell quantity in the rinsing waters and leakage of cells.
Enzyme activity was assayed by measurement of
ammonia released due to nitrilase action according to
the phenol-hypochloride method of Fawcett and Scott
[27]. One enzyme unit (U) is defined as the amount of
enzyme, producing 1 µmol ammonia min-1 at pH 7.2,
45°C and 20 mM benzonitrile as a substrate. All runs
were replicated and averaged values are reported.
2.3. Matrix synthesis
Sol-gel transparent silica hybrid matrix with 5, 10 and
20 wt% organic component was synthesized at room
temperature and controlled pH conditions. A poly-step
sol-gel procedure was carried out at strictly controlled
conditions in order to obtain the desired structured
materials. No alcohol was added as a co-solvent. 5 mLof
0.1 N HCl were introduced to reach pH~1.5 of the initial
solution for increasing hydrolysis rate. The inorganic-
organic hybrid materials were prepared by substituting
part of the inorganic precursor with k-carrageenan.
The pH was raised up to pH=7.2±0.02 at 20°C with
phosphate buffer to keep cell vitality. The quantity
of buffer solution varies for the different sol mixtures
depending on the organic component content from 15
to 20 mL to increase pH to 7.0 of the final mixture before
introduction of the cell suspension. Before addition we
shake the cell suspension well, after which, the whole
solution including cell suspension and sol mixture is
slightly stirred to obtain a homogeneous mixture. In all
2.6. Matrix investigations
For studying the structure of the synthesized hybrids
the following methods were used: FT-IR (IR- MATSON
7000–FTIR), XRD (X-ray PW1730/10, the diffracted
intensity of Cu Kα radiation was measured with scan
rate of 0.02° min-1 in 2θ range between 4° and 80°),
BET-Analysis (Gemini 2370 V5), Energy Dispersive
System (EDS) (RONTEC EDS), SEM (Philips-515),
AFM (NanoScope Tapping ModeTM) and roughness
analysis. The influence of the hybrid matrix, synthesized
with TEOS and k-carrageenan on the cells of the
investigated strain was evaluated by scanning electron
microscopy (Scanning device attached to Zeiss electron
microscope-model 10C, at 20 kV accelerating voltage
with electron beam 5-6 nm) on the surface of a piece of
this type of matrix.
cases the ratio precursor/H O was kept constant and
2
equal to 1. No phase separation was observed before
and after the gelation point. The addition of organic
component leads to a faster polymerization. Thin
transparent hybrid flakes were obtained in the hybrid
synthesis. The drying procedure was carried out at room
temperature overnight.
3. Results and Discussion
The FT-IR spectra of synthesized inorganic-organic
materials show the bands at 1080 cm-1, 790 cm-1 and
480 cm-1. They are assigned to νas, νs and δ of Si-O-
Si vibrations, but at the same time these bands can
be related to the presence of Si-O-C, C-O-C and Si-C
bonds. The band at 960 cm-1 is due to a stretching Si-
OH vibration. The bands at 1439 cm-1 are assigned to
C-O-H vibrations. The characteristic bands at around
3450 cm-1 and at 1640 cm-1 assigned to H-O-H vibration
can also be found (Fig. 2). It can be seen that the
transmittance of hybrids decreases with increasing of
carrageenan content.
The results from the XRD-analysis show that all
the studied hybrids were in an amorphous statе. It was
established that the diffraction peaks intensity decreased
with an increase of organic component content. At the
same time the type of the XRD patterns indicates that
some processes of ordering were carried out.
2.4. Immobilization and bioreactor setup
Sol-gel hybrids containing 5 wt% k-carrageenan were
prepared as flakes with the inclusion of 10 mL of the cell
suspension. The living cells were trapped directly within
the gel matrix before gelation. The obtained biocatalysts
were applied for packing of the laboratory column
bioreactor (Fig. 1). It possesses a working volume of
100 mL and glass walls, forming a cylinder. After packing
with the biocatalyst the temperature was controlled and
maintained at 60°C by circulating water through the water
jacket connected to a thermostatic bath. The reactor
contained 45 g of the ready biocatalyst. The substrate
solution (20 mM mixture of o-, m- and p-tolunitrile) was
fed into the bioreactor using a peristaltic pump, which
234

L. V. Kabaivanova et al.
The chemical content in the samples determined by
EDS showed presence of Si, O, N, Na, P, S, Cl and K.
From the data of BET analysis it was established that
the surface area is in the range of 396 to 260 m2 g-1. The
results clearly show that with increasing the percent of
the organiccomponent, the surface area decreases.
From the AFM analysis
a
self-organized
nanostructure was established (Fig. 3). All synthesized
hybrid samples have surfaces with irregularities of quite
small height. In the obtained hybrids the nanobuilding
blocks are made up of nanounits of SiOx groups and that
van der Walls and Hydrogen bonding or electrostatic
interactions between the nano-building blocks exist. In
the synthesized samples the average size of particles is
about 8 - 12 nm and the dimensions of their aggregates
are about 16 - 49 nm. The surface roughness of
Figure 2. FT-IR spectra of synthesized hybrid materials containing
TEOS and different percent k-carrageenan
a
b
c
Figure 3. AFM and roughness analysis of synthesized hybrid materials containing TEOS and different percent k-carrageenan (a) 5 wt%;
(b) 10 wt% and (c) 20 wt%
235
235

Silica-carrageenan hybrids used for cell immobilization
realizing high-temperature degradation of nitrile substrates
enzymatic activity and could then behave just as a ‘‘bag
of enzymes’’ [29].
Our previous investigations showed that the type of
matrix influences to a certain extent the activity reached,
together with other factors that have been changed for
optimization of the process. We have used different type
of organic constituents: agar, PAAG, PEO, alginate,
chitosan and chitin in different percentage [30-33].
Hydrolysing activity of nitrilase from Bacillus sp.
UG-5B in batch experiments was established towards
different concentration of nitriles for free and immobilized
cells (Table 1). The enzyme activity towards benzonitrile
was assumed as 100%. For 20 mM concentration, 100%
corresponded to 2.8 U mL-1; for 30 mM concentration –
2.2 U mL-1 and for 40 mM concentration - 2.1 U mL-1.
In this study the relative enzyme activity of free and
entrapped cells was compared for o-, m- and p-tolunitrile,
representatives of the carboxylic nitriles. From the group
of heterocyclic nitriles, 4-cyanopyridine was tested to
find that with this type of matrix the values of the relative
nitrilase activity for entrapped cells did not exceed these
for the free cells. This is the reason why 4-cyanopyridine
was not subjected to degradation in the reactor with the
immobilized biocatalyst. The catalytic route of nitrile
transformations is of particular interest because the
nitrile-degrading enzymes can convert diverse nitrile
substrates into various amides and carboxylic acids
under relatively moderate process conditions with
excellent chemo-, regio- and stereoselectivities. An
important development in the area of nitrile and amide
hydrolytic bioprocesses has been the use of immobilized
biocatalysts, which confers the benefits of biocatalyst
retention and reuse in continuous process strategies
[34-36]. As it can be seen from Table 1 highest enzyme
activity was shown towards p-tolunitrile, probably due
to a stereo-selectivity, characteristic for the nitrile –
converting microbial enzymes [37] and the type of the
synthesized matrix, so o- and m- tolunitriles are less
susceptible to hydrolysis for both free and immobilized
cells. The final product of enzyme reaction was only
benzoic acid detected by gas chromatography analyses.
Benzamide was not found as an intermediate product.
The influence of the concentration of the substrate was
also shown, confirming that concentration of 20 mM
was most appropriate to carry out the enzyme-substrate
reaction. It is evident that the increase of the substrate
concentration up to 40 mM did not lead to increase in
the nitrilase activity. Higher concentrations of these toxic
substrates probably affect negatively the cells and their
enzyme systems.
2 μm
Figure 4. Image taken by scanning electron microscopy of the
cells on a piece of hybrid matrix with k-carrageenan
450
400
350
300
250
200
150
100
50
3,5
3
Surface area
Enzyme activity
2,5
2
1,5
1
0,5
0
0
5
10
20
Organic component, %
Figure 5. Correlation between surface area and enzyme activity
using three types of matrices – with 5, 10 and 20 wt%
organic constituent
the synthesized hybrids depends on the quantity of
k-carrageenan. With increasing of its percentage, the
surface roughness increases.
The obtained hybrid material appeared to be
compatible with cells. Water solutions of silica precursor
with the purpose to reduce the quantity of alcohol in the
primary mixture were used. The time for homogenization
before the addition of organic part and cell suspension
was increased in order part of the alcohol formed
during hydrolysis to be evaporated. Addition of
phopshate buffer for raising the pH of initial sol in a way
decreases the exposure of cells to acid and high alcohol
concentration [28]. The remaining quantity of alcohol
did not appear deleterious to the cells as the enzyme
systems responsible for nitrilase synthesis function.
All immobilized cells showed nitrile-degrading activity
immediately after the preparation and lasting for a long
period of time. Immobilized cells can be re-used in batch
cycles up to 20 each with fresh substrate medium. Their
cellular organization and enzymatic activity appeared
to be preserved after encapsulation. Bacteria that
are damaged or even dead may still maintain some
The scanning electron micrograph represents part of
the matrix where undamaged cells can be clearly seen
to prove that they keep their integrity and no lysis was
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236

L. V. Kabaivanova et al.
Figure 6. Quantity of ammonia released, corresponding to the quantity of degraded substrate-mixture of tolunitriles in the bioreactor;
- first
step of the process; - second step
Table 1. Influence of the substrate concentration on the relative enzyme activity at batch experiments.
Type of substrate
Substrate
concentration, mM
Relative enzyme activity
of free cells, %
Relative enzyme activity
of entrapped cells,%
20
30
40
20
30
40
20
30
40
20
30
40
14
8
14
30
20
19
80
30
28
39
30
10
48
36
12
95
80
80
o-tolunitrile
m-tolunitrile
p-tolunitrile
benzonitrile
100
100
100
100
100
100
observed (Fig. 4). We know from literature data that the hybrid material with 5 wt% carrageenan, containing the
sol-gel process allows the encapsulation of living cells entrapped bacterial cells. After entrapment, the whole
due to its features, such as low temperature processing, cell quantity used (350 mg) was found within the gel.
high stability, pH regulation and contact surface area, Leaching was 0.003%.
which facilitates permanent interactions between the
microorganisms and the substrate, and does not prevent substrates: o-; m- and p-tolunitrile in two steps at 60°C
cell reproduction [38]. were followed. The remaining quantities of not degraded
The process of enzyme biodegradation of the three
The influence of the surface area and the size of substrates at the first step were treated for a second time
pores on the enzyme activity of entrapped cells were in the reactor filled with the already released solution
also studied (Fig. 5). The obtained results showed that after the first step for another five hours for the mixture
the surface area of the samples with 5% k-carrageenan of tolunitriles.
is the biggest, reaching 396 m2 g-1 and the pore size
In nature the pollutants often occur as mixtures so
is 1.6 nm which promotes highest enzyme activity in o-; m- and p-tolunitrile were treated as a model system
comparison to the sample with 20% k-carrageenan in the bioreactor. To realize the biodegradation of this
where the surface area is 260 m2 g-1 and the pore size mixture of nitriles nine hours were needed to obtain
is 2.3 nm. In the sample with 5 wt% organic part the almost full substrate degradation. At the first step (four
pore size is the least, but enough for the substrate and hours) a degradation of 150 mM was achieved. After the
oxygen to penetrate.
fifth hour a depletion of the substrate was established
The next step of the investigation was to prepare (Fig. 6) to realize a bioconversion of the model system
a laboratory-scale column bioreactor filled with 45 g of of tolunitriles up to 94.9 % for nine hours.
237
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Silica-carrageenan hybrids used for cell immobilization
realizing high-temperature degradation of nitrile substrates
Toobtainbiocatalystswithbroadsubstratespecificity, retaining their bioactivity, we tried to prove this feature.
increased operational stability and thermostability The obtained biocatalysts on the basis of silica sol-gel
by applying new carriers for effective immobilization hybrids with 5 wt% k-carrageenan proved to be suitable
and new methods for biotransformation of complex for this purpose.
molecules, becomes the aim of the investigations of
many researchers. Using biocatalytic processes, nitrile
substrates can be readily hydrolyzed under moderate 4. Conclusions
conditions, compared with the relatively severe
conditions required for analogous chemocatalytic A poly-step sol-gel procedure was used at strictly
processes [39,40].
controlled conditions different from the conventionally
Our results confirm the statement that microbial used, concerning the solvents and acidity in order
cells could be involved by immobilization in processes to keep cell vitality and to obtain the desired hybrid
of remediation of contaminated sites, particularly when materials. The introduction of an organic constituent to
polluted industrial waters come out hot and can be easily produce a hybrid material showed its favourable effect
treated immediately without cooling for a short period, on the biocatalyst formed. Introduction of 5 wt% of this
since with the increase in temperature the reaction organic substance is sufficient and promoting higher
rate increases while energy requirements decrease. enzyme activity reached 3.0 U mL-1, compared to the
The biocatalysts with immobilized cells possess the this obtained when the cells were immobilized in hybrid
advantage of carrying out an economic process of matrices containing 10 wt% (2.4 U mL-1) and 20 wt%
continuous use at stable conditions and control over organic part (2.1 U mL-1).
the specific enzyme reactions. The technique of
The accomplishment of a biodegradation process
immobilization in sol-gel matrices has proved to be in a bioreactor proved no washout, allowing high and
biocompatible and easy to perform [41]. Preservation of stable process efficiency even under variations in
microorganisms is an important feature for biotechnology toxicity, concentration and at high temperature. In
[42]. Together with viability and activity of the enzyme, general, the diffusion of organic pollutants correlates to
a long-term maintenance is required. Here is the role of the solubility, which increases with temperature in two
immobilization. The porous structure of the hybrids is a steps for 9 hours to reach degradation of 94.9% of the
major parameter for the efficiency of the immobilization nitrile mixture. An increase in the reaction temperature
process as the pores of the hybrid materials permit the had its favourable effect on the bioreactor efficiency,
diffusion of the substrates and products of the enzyme probably due to increased mass transfer. The bacterial
reaction.
cells immobilized in the hybrid materials have shown
Certain differences were established between preservation of the enzyme systems, responsible for
free and entrapped cells at the batch experiments, the nitrilase synthesis. In this study the high efficiency of
using different nitriles which fact can be devoted to nitrile degradation by immobilized bacterial cells showed
the formation of a microenvironment around the cell at a high potential as a bioremediation technique for the
entrapment. This is valid in the cases when the enzyme treatment of organocyanides as the strain Bacillus
activity is higher for entrapped than for the free cells. The sp. UG-5B has shown a broad substrate specificity,
same fact was proved by Graham [23] who stated the hydrolysing aliphatic, carbocyclic and heterocyclic
immobilization inside a gel lead to additional stabilization nitirlies. The problem about the biological treatment of
of the cells towards high concentration of organocyanide hot waste waters before cooling has always been of
substrates. For the remaining biocatalysts where importance. Immobilization of such thermophilic cells in
the enzyme activity of free cells is higher it could be sol-gelhybridmatricespermits,performingoftheprocess
suggested that the formation of a barrier may serve as of biodegradation at high temperature with high speed
a hindrance for the penetration of the substrate inside and least contamination. The possibility to carry out this
the cells.
particular biotransformation in a series of reactors with
Since one of the fastest growing research directions immobilized biocatalysts at high temperature, reaching
in the field of sol-gel science and technology is the almost full bioconversion values is feasible.
immobilization of different living cells in sol-gel matrices
238
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L. V. Kabaivanova et al.
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