Scientific
Publications - Work Done by Microbiology Reader Bioscreen C
Fig. 6 Effect
of growth medium flow rate on the time course of
the carrier surface colonization
by C. maltosa (3 ml min-1 [
];
9 ml min-1 [
]; 30 ml min-1 [ ]; 90 ml min-1 [
])
BIOFILM FORMATION: A TOOL
INCREASING
BIODEGRADATON ACTIVITY
Jan Masak*,Alena Cejkova, Martina Siglova, David
Kotrba,Vladimir Jirku, Petr Hron
Department of Fermentation Chemistry and Bioengineering,
Institute of Chemical Technology,
Prague, Czech Republic. *e-mail: Jan.Masak@vscht.cz
Microbial biofilms are frequently
found 3D constructs in most natural and man-made environments where microbial
cells are associated predominantly with surfaces rather than in a free-
floating state. General
expectations among biofilm researches are that these elaborate
structures have meaning as well as abilities to control own
architecture and environment. In this
connection, a spectrum of
structurally heterogeneous biofilms is observed ranging from dense, amorphous
biofilms to biofilms demonstrating robust, well developed structures
(Lewandowski,
2000). Biofilms are of great importance in industries
such as food processing and water industry / treatment; biofilms
reduce heat transfer, increase resistance and act as a reservoir for
potential pathogens, among others
(Lappin-Scott and Costerton, 1989). Biofilm forms most commonly at water-solid
interfaces although it can appear at an interface between two immiscible
liquids, at air-water interfaces
and at gas-solid surfaces. The complex regulation of surface attachment,
irreversible surface binding, biofilm maturation and ultimately biofilm
detachment is
always affected to at least some degree by the physiology of
microflora involved as well as by physico-chemical parameters of solid surface
and environment (Davis, 2000). Moreover, biofilm
structure requires cells to
contact physically not only the extracellular matrix but also the surface of
neighboring cells. An essential question is whether attached cells use this
physical contact
solely for a structural, nutritional or protective purpose,
or whether these intercellular interactions are also perceived as
mechano-physiological signals, inducing a programmed response, that
may be important for expression
of a differentiated state or the development of a resistance (Jirku et al.,
2001). This
paper investigates the effect of extracellular factors and surface
hydrophobicity on biofilm formation and growth, using two fungal
biodegraders utilizing,
respectively, phenol, acetone, acetonitrile and CN- ions.
Material and methods
Candida maltosa (a
phenol degrader) and Fusarium
proliferatum (an
acetone, acetonitrile and CN- degrader)
were obtained via the
aerobic enrichment of soil
samples microflora and consequent lower fungi
(single
species) isolations (Masak et al., 1997). Degradative
function of yeast and mycelial
biofilms was investigated
using jacketed, tubular reactor (30 cm inner diameter;
70 cm length), enabling
temperature (18 °C) control and
a countercurrent, continual circulation of media (pH 4.7)
and air. Degradative
function of suspended cells was
tested using a bioreactor
(Braun Biotech International,
Germany) with an operating volume of 2.0
l, and
temperature (18 °C), pH (4.7) and rotation speed
(120 rpm) control. Marker
pollutant was used as a sole
source of carbon. Both fungal populations were
grown
aerobically under the effect of stressors (pH 3, pH 5,
pH 8, 0.5 M NaCl, 1 M NaCl, 0.01
M CaCl2, 0.1 g l-1 Cd2+,
0.1 g l-1 Zn2+,
temperature 8 °C and 38 °C, nutrient
starvation) using the
Olson-Johnson (1949) medium.
Growth activity was monitored using a
Bioscreen C
analyzer (Labsystem Oy, Helsinky, Finland). The
cell
surface hydrophobicity was assayed using a procedure
according
to
Rosenberg
(1980)
-
BATH
test.
Ultrasonically cleaned
(Sonorex, Bandelin, Germany)
Na-silica glass (EcoGlas Ginzel, Czech
Republic) was
used as a standard carrier material for the cell adhesion
(attachment) studies; glass
modification was performed,
respectively, through 3h exposure to 6M HCl at 100
°C and
by
silanization
through
gama-aminopropyl-
triethoxysilan hydrolysis.
Glass hydrophobicity was
assayed as contact angle of water drop on
glass surface
(Mozes and Rouxhet, 1987).The 90 x 8 x 4 mm (l x w x
h) flow cell of effective
volume 2.88 ml was made of
stainless steel and polymethylmethacrylate. The
biofilm
carrier used throughout here is a sandwich-like foil with
40 µm polypropylene core
covered with 12 µm
polyester outer layer (Steriking, WIPAK
MEDICAL,
Findland). Microbial attachment to the carrier was
performed under standard
conditions of the carrier-cell
population (statically/flow) contact. The
assessment of
the carrier surface colonization was based on the image
analysis of the carrier, using
LUCIA for Windows NT for
light and fluorescence (microscopy) image processing,
and bound cell proteins
determination (Bradford, 1972).
Cells were imaging via SYTO 13 (Molecular Probes, USA)
DNA detection. A standard
rinsing of the carriers was
used to remove loosely attached cells.A cell
detachment
prior to the bound biomass determination was
performed ultrasonically
(Sonorex TK 100, Bandelin,
Germany). Conclusions made are based on the
amount of
bound (biofilm) biomass determined after 7 days.
Results and discussion
The maximal rates of
phenol, p-cresol and CN- ions
respective biodegradations reveals that the removal rate is
significantly increased if an
attached fungal population, particle-based biofilm (column) reactor, is used
(Fig. 1).The
hydrophobicity data obtained (Table 1) show that
eukaryotic microorganism can exhibit both higher and lower
hydrophobicity of the cell
surface.The differences in this parameter were used to test the capacity of both
strains to
colonize the silica-glass treated to give as well differently hydrophobic
surface (Fig. 2). The results of this study
suggest that a higher extent
of hydrophobicity of the interacting entities (cells / carrier) always
promotes the
adhesion (attachment) of cells and consequent biofilm
formation, and on the contrary. In order to investigate the
effect of sub-physiological
conditions of extracellular environment as an additive tool that potentially
affects the
process of a glass carrier colonization, some combinations of the glass
modification, cell type and physiologically
acting factors were set up and
employed (Figs. 3 and 4).The results obtained indicate that, for materials and
cells that
are mutually repellent, the effect of some stressors can enhance the
adhesion capacity of microbial cell. In
view of the fact that the process
of biofilm formation experiences the condition determined by outer environment
the effect flow rate of growth
medium was investigated in parallel monitoring the formation of C.
maltosa biofilm
under the effect of different (standard medium) flow
rates in a flow cell system. Quantitative data obtained for
yeast biofilm formation under the
flow rates within the range from 3.0 ml.min-1 to
90.0 ml.min-1 (Figs. 5 and 6)
suggest that the intensity of
biofilm formation can be expected to increase with increasing flow rate.
References
Bradford M. A. (1972). A
rapid and sensitive method for the quantitation of
microgram quantities of
protein using the principle of dye binding. Anal.
Biochem. 72, 248-252.
Davis D. G. (2000).
Physiological events in biofilm formation. In: Community
structure and co-operation in
biofilms, D. G. Allison, P. Gilbert, H. M. Lappin-Scott
and M.Wilson (eds.), Cambridge
University Press, Cambridge, pp. 37-52.
Jirku V., Masak J. and
Cejkova A. (2001). Significance of physical attachment of
fungi for bio-treatmet of
water. Microbiol. Res. 156, 1- 4.
Lappin-Scott H.M. and Costerton J.W. (1989).
Bacterial biofilms and surface
fouling. Biofouling 1, 323-342.
Lewandowski Z. (2000).
Structure and function of biofilms. In: Biofilms: Recent
advances in their study
and control, L. V. Evans (ed.), Harwood Academic
Publishers,Amsterdam, pp. 1-18.
Masak J., Cejkova A. and
Jirku V. (1997). Isolation of acetone / ethylene glycol
utilizing and biofilm forming
strains of bacteria. J. Microbiol. Methods 30, 133-139.
Mozes N. and Rouxhet P. G.
(1987). Methods for measuring hydrophobicity of
microorganisms. J. Microbiol.
Methods 6, 99-112.
Olson B. H. and Johnson M. J. (1949). Factors producing
high yeast in synthetic
media. J. Bacteriol. 57, 235-246.
Rosenberg, M. (1984) Bacterial
adherence to hydrocarbons: a useful technique for
studying cell surface
hydrophobicity. FEMS Microbiol. Lett. 22,289-295.
Conclusion
Surface hydrophobicity,
effect of stressors and
fluid dynamics in laminar flow provide a
complex of variables that
affect the evolution of
fungal biofilm (community) structure. However,
the range of possible
interactions among them
requires a more detailed evaluation of this
complex tool that must be
performed in the
relationship to the fungal taxon as well as to the
carrier. In practical terms,
these results point to
new direction of research for the design of more
adhesive materials and their
(optimized) usage
as carriers for fungal biodegraders.
Fig. 1 Maximal
biodegradation rates achieved in suspended [
] and
attached [
] fungal populations
Hydrophobicity
Glass
Contact angle [°]
non-modified
leached by HCl
3.0 ± 1
64.0 ± 2
silanized
69
24
Microorganism
BATH test [%]
Candida maltosa
Fusarium proliferatum
Tab. 1 Carrier
and fugal cell hydrophobicity
Fig. 2 Carrier colonization by C.
maltosa and F.
proliferatum (glass: non-
modified [ ]; leached by HCl
[ ]; silanized [ ]).
Fig. 3 Effect of stressors on the
adhesion of Candida maltosa to glass
leached by HCl [
] and silanized [
]
Fig. 4 Effect
of stressors on the adhesion of Fusarium
proliferatum to
glass leached by HCl [ ] and
silanized [ ]
Fig. 5 Effect of growth medium flow
rate on the number of
attached C. maltosa cells after
24 hours of cultivation.
150
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.20
0.15
0.10
0.05
0.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
100
50
phenol
Candida maltosa
Candida maltosa
Strssors
Fusarium proliferatum
Fusarium proliferatum
p-cresol
KCN
0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
3.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
10.0
15.0
20.0
25.0
5.0
9.0
30.0
90.0
Flow rate [ml min-1]
Time[h]
(Full Text online - PDF)
