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CATALYTIC FILAMENTOUS CARBONS (CFC)

AND CFC–COATED CERAMICS FOR
IMMOBILIZATION OF BIOLOGICALLY ACTIVE
SUBSTANCES
G.A. KOVALENKO, D.G. KUVSHINOV,
O.V.KOMOVA, A.V. SIMAKOV, N.A. RUDINA
Boreskov Institute of Catalysis, Novosibirsk 630090, Russia

Abstract: Adsorption properties of granulated catalytic filamentous

carbons (CFC) and CFC-coated macrostructured ceramics (honeycomb
monoliths, foams) have been studied for adsorptive immobilization of
biologically active substances including enzyme glucoamylase, invertase-
active yeast membranes and non-growing cells of bacteria. The effect of
morphology of the surface carbon layer on the adsorption efficiency of the
supports and on the biocatalytic properties (stability and activity) of
immobilized enzymes has been investigated.

Key words: catalytic filamentous carbon; carbonized ceramics; adsorptive

immobilization.

1. INTRODUCTION

The problem of developing efficient supports for adsorptive

immobilization of biologically active substances and microorganisms is
still relevant because of its importance for various applications including
medicine, biotechnology and pharmaceutical industry. Obviously, such
supports-adsorbents must meet certain criteria. Firstly, they should have
sufficient adsorption capacity with respect to such substances and
microorganisms and have to strongly bind them to the surface. Secondly,
they should retain and stabilize the biological activity of the immobilized
substances at a relatively high level. Thirdly, they should possess high
mechanical strength and resistance to biological and chemical
degradation. Finally, their cost should be relatively low. Apparently,
carbon-containing inorganic supports satisfy these requirements [2,3,5].
The present investigation has been carried out to study the granulated
catalytic filamentous carbons (CFC) and CFC-coated macrostructured
ceramics as supports – adsorbents for immobilization of enzyme
glucoamylase, invertase-active yeast membranes and the non-growing
bacterial cells. The effect of morphology of surface carbon layer on the
adsorption efficiency of the supports and on the biocatalytic properties
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(stability and activity) of immobilized enzymatic active substances has
been investigated.

2. EXPERIMENTAL
Granulated catalytic filamentous carbons (bulk CFC) with diameter up
to 5 mm were prepared by catalytic pyrolysis of methane on Ni / Al2O3
and Ni, Cu / Al2O3 catalysts [1,2]. The carbon yield (Y) expressed in g of
carbon deposit per 1 g of the catalyst was varied from 1 to 180 g/g.
Macrostructured ceramics were used as honeycomb monoliths and
foams described in [3,4]. These supports were coated by a carbon layer of
different morphology, such as i) graphite-like (G) layer (Fig.1 a), ii) fur-
like layer (F) formed by thick and short carbon filaments (Fig.1 b), and iii)
hair-like layer (H) formed by thin and long carbon filaments (Fig.1 c),
which were synthesized by methods described in [3]. Thus, the graphite-
like carbon layer was synthesized by thermal decomposition of sucrose
impregnated into the support pores at 500°C in H2 flow for 2 hours. The
catalytic filamentous carbon layers were synthesized by catalytic pyrolysis
of a propane-butane mixture over Ni / Al/Si catalyst at 500°C.

Fig.1. Scanning electron micrographs of carbon layers synthesized on ceramic surface.

a) graphite-like carbon layer (G) on foam-like ceramics,
b) fur-like carbon layer (F) on foam-like ceramics and Al/Si honeycomb monoliths,
c) hair-like carbon layer (H) on cordierite honeycomb monolith.

The adsorptive immobilization of the biologically active substances on

the carbon-containing supports was performed under conditions described
in [2,5]. The amount of adsorbate was calculated from the difference of its
contents in solution before and after adsorption and expressed in mg/g of
the support.
Scanning electron microscopy (SEM) studies of the support surfaces
and the adsorbed bacteria were performed with REM-100Y microscope.
The samples for microscopy were prepared as described in [2,5]. The
labels in the micrographs correspond to distance in micrometers (µ.)
3. RESULTS AND DISCUSSION

Comparative investigation of adsorption properties of granulated

bulk CFCs with respect to various biological substances were carried out
in [2]. The main conclusion was that the value of accessible surface area
is of crucial importance in the adsorption of adsorbates of biological
origin [2]. As for microorganisms, they were adsorbed exclusively on the
external surface of CFC granules; inner surface was not accessible for
large bacterial cells (1-2 µ in size) [2,5]. An additional key factor affecting
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the adsorption of bacterial cells was roughness of the surface determined
by the carbon yield (Y) [2,5]. It was shown that the adsorption capacity
for the non-growing bacterial cells (E.coli, Bacillus sp. and Rhodococcus
sp.) was lower for CFCs with dense and smooth surface (at high Y) and
higher for CFCs with rough “carbon fur coating” (at low Y) whereas the
surface area accessible to bacteria did not depend on Y [2]. The cross-
linking of bulk CFC granules by the similar carbon filaments increased
significantly both the inner area accessible for bacteria adsorption due to
channel formation (Fig.2a) and the roughness of the surface via additional
synthesis of “carbon fur coat” (Fig.2b). The adsorptive immobilization of
bacteria on the cross-linked CFC granules was found to be highly
efficient. In the SEM micrograph the adsorbed bacterial cells of
Rhodococcus ruber were packed closely together (Fig.3).
As a conclusion, granulated bulk CFC supports with «carbon fur
coating» were found to be efficient adsorbents for biologically active
substances.

Fig.2. Scanning electron micrographs of the surface of cross-linked CFC granules

a) channels on the external surface of granules;
b) the “carbon fur coating”

Fig.3. Scanning electron micrograph of the Rhodococcus ruber immobilized on cross-linked CFC
granules.

To increase the accessible surface area and the mechanical strength of

the supports and at the same time to retain the morphology of filamentous
carbons, the macrostructured ceramics, in particular foams were used and
methods of synthesis of CFC layers on the alumina-silica surfaces were
developed. To study the effect of carbon layer morphology on stability
and biocatalytic activity of immobilized enzymatic active substances, the
ceramics coated by graphite (G)-, fur (F)- and hair-like (H) carbon layers
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were synthesized. When enzyme glucoamylase was adsorbed on the
supports with graphite-like carbon layer, the stability of biocatalyst was
relatively low [3]. The stability of glucoamylase increased by an order of
magnitude via immobilization of enzyme on ceramics coated by
filamentous carbon layer [3]. Also, filamentous carbon layer was found to
provide the highest operational stability of immobilized glucoamylase. In
the continuous hydrolysis of corn dextrinized starch the activity of the
biocatalyst remained constant for several hundred hours at 50-55oC.
The effect of carbon layer morphology on the stability and biocatalytic
activity of immobilized invertase-active cell membranes of autolyzed
baker yeast was studied. Supports coated by catalytic filamentous carbon
ensured the highest stabilization. The best result on stability was that the
biocatalysts retained over 50% of the initial activity for 0,5-year storage at
ambient temperature. Unlike glucoamylase, activity of immobilized
invertase was found to depend strongly on the morphology of the carbon
layer (Table 1). Invertase-active yeast membranes adsorbed on the
graphite-like carbon layer synthesized on foam-like ceramics (Fig.1, a)
showed a ∼10-fold decrease of biocatalytic activity in comparison with
fur-like carbon (Fig.1, b) perhaps due to hydrophobicity of graphite.
Moreover, invertase adsorbed on hydrophobic carbon supports specially
modified to increase the surface hydrophobicity has lost the biocatalytic
activity completely. The activity of invertase-active yeast membranes
immobilized on hair-like carbon layer synthesized on cordierite monolith
was shown to be comparatively low because of diffusion limitations
(Table 1).
Table 1.The effect of macrostructure of supports and morphology of carbon layer on activity of
biocatalysts prepared via adsorption of yeast cell membrane.

Macrostructure Sspecific, Carbon Sspecific, m2/g Morphology Activity, U/g

of support m2/g of content, of of carbon of biocatalyst
non- w/w% carbonized layer*
carbonized support
support
Foam 0.5 0.6 2.7 G 2.2
Foam 0.5 1.0 6.5 F 18.4
Monolith 24 1.4 27 G 12.5
Monolith 24 5.6 60 F 15.6
Monolith 0.15 0.5 0.8 H 7.2

The following designations are used: G – graphite-like carbon lay er (Fig.1a), F – fur-like carbon
layer (Fig.1 b), H – hair-like carbon layer (Fig.1c)

4. CONCLUSIONS
Comprehensive investigations of granulated CFC supports and CFC-
coated ceramics demonstrate their high adsorption efficiency with respect
to biologically active substances (enzymes, yeast membranes and non-
growing bacteria) due to filamentous morphology of the surface carbon
layer. Cross-linked CFC granules possess the highest adsorption capacity
for the non-growing bacterial cells.
Filamentous carbon provides the highest long-term and operational
stability of the heterogeneous biocatalysts based on immobilized enzyme
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glucoamylase. In the continuous hydrolysis of corn dextrinized starch
their activity remains constant for several hundred hours at 50-55oC.
The activity of immobilized invertase-active yeast membranes depends
strongly on the morphology of carbon layer. Invertase adsorbed on
graphite-like carbon layer has low biocatalytic activity. The significant
(up to 10-fold) increase of invertase activity is observed when yeast
membranes are immobilized on foam-like ceramics with fur-like carbon
layer.
The highly efficient CFC-based adsorbents have potential for
applications in biotechnology and medicine.
Acknowledgement: The work was partly funded by INTAS grant 01-
2151.
References
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substances and non-growing bacterial cells, Carbon 39, 1133 – 1143.
3 Kovalenko GA, Komova OV, Simakov AV, Khomov VV, Rudina NA. (2002)
Macrostructured carbonized ceramics as adsorbent for immobilization of glucoamylase,
J.Mol.Catal.А: Chemical 182-183, 73-80.
4 Pat.RU 2167197 (2001).
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Carbonaceous materials as adsorbents for biologically active substances and bacterial cells,
Colloid J. 61, 729-736.
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