1. Summary
Summary
The adherence of microbial cells onto surfaces often results
in a build up of aggregates and formation of what is known as
"biofilm". Biofilm formation is the oldest and most powerful
forms of life; its strength arises from the microbial cell’s ability to
produce layers of extracellular polymeric substance which offers
protection against biocides and toxins. Problems arising from
biofilm formation are due to the cost associated with losses it
causes: the deterioration in plant performance, decrease in the
quality and quantity of the product, the damage of the
constructing material and the cost for cleaning processes, cost of
addition of biocides or labor used to replace or clean the tanks.
There are chemical methods for preventing cell adhesion, such as
biosurfactant and antibiotics. There are also physical methods
which include brushing, scraping and flushing. The attachment
process between fungal spores and/or hyphae and substrates is
considered a very complex process; it depends mainly on the

2. Summary
physicochemical surface interaction, specific molecular factors
such as glycoproteins, hydrophobins, carbohydrates and lipids.
The aim of the present work is to avoid biofilm formation for an
oxidase producing fungus using an eco-friendly method. Twelve
fungal isolates were screened, four of which produced
intracellular phenol oxidase, the highest producing was the fungus
Penicillium purpurogenum, and this fungus produced fungal rings
on the container after incubation. Employing types of abiotic
stress affected the stress response by the fungus, the changes were
observed at pH 5, 30o
C and the addition of tryptone to the
cultivating media. On the other hand, the use of gamma radiation
didn’t result in a consistent change.Therefore, an agro-industrial
by-product which is utilized by microorganism as a carbon
source, was used as a type of stress to control biofilm formation.
Matter of fact, the presence of ethanol increased catalase and lipid
peroxidation, while the surface bound protein decreased at the
tested ethanol concentration. No changes took place in the surface

3. Summary
bound exopolysaccharide (EPS). As for the red pigment secreted
by the fungus, the presence of ethanol exhausted the pigment,
probably by consuming it for protecting the cells against stress.
Combining all types of stress resulted in low biomass; therefore
there were no obvious morphological changes in the fungus. The
addition of different ethanol concentrations to the growth media
showed that 2.5% v/v was un-harming to the fungal growth,
concentrations above which, lower fungal growth and/or decrease
in spore formation. Following the same trend, 2.5%v/v was also
the optimal ethanol concentration which prevented biofilm
formation and adhesion on the walls of the container. It also
induced the phenol oxidase enzyme.
Scanning electron microscope showed that the fungal
growth was loose fungal network in the presence of ethanol as
compared to a tightly formed fungal network in control cultures.
When we tested ethanol as a signaling compound, the
results showed that it was not signaling compound controlling the

4. Summary
adhesion process as we compared to wortmannin which is an
inhibitor of a PI3 signaling pathway and positive control cultures.
The addition of fetal bovine serum as a mode of increasing
germination by enhancing the germ tube formation in fungi in
contradiction to adding ethanol proved that ethanol does not
trigger germination, on the contrary, spores remained unchanged,
and this means that ethanol did not contribute in signaling via
germ tube formation. Therefore, there is another mode for fungal
adhesion. The gene regulator Yap1p was detected in the fungus
under study suggesting that the fungus has the ability to
reprogram itself in the presence of ethanol. It is involved in the
transcription of glutathione gene (GSH1), this was confirmed by
the presence of glutathione as detected by Fourier Transfer Infra
Red scpectroscopy (FTIR). Glutathione is present as stress
response taking place in fungi.
We tested the cell surface charges in the presence of
ethanol, the results showed an increase in cell surface charge.

5. Summary
Performing the Random Amplified Polymorphic DNA (RAPD)
on Penicillium purpurogenum grown in media amended with
different ethanol concentrations showed that the ethanol affected
the DNA polymorphic profile of DNA rendering the fungus
genetically variable. SDS-polyacrylamide protein profile showed
polymorphism in surface bound proteins for cultures amended
with ethanol as compared to control cultures.
Adhesion of the fungus on polystyrene, glass and tin foil
showed that less adhesion for all tested samples in the presence
of ethanol as compared to control cultures when using light
microscopy, While the use of scanning electron microscopy
showed exactly which substrate attached more fungal cells, the
results showed that glass sheets exhibited the lowest growth in the
presence of 2.5% v/v ethanol. This is attributed to the wettability
of each substrate, we tested if we can vary the wettability of a
substrate such as polystyrene using gamma radiation, and the
results showed that the fungal cells were loosely attached to the

6. Summary
polystyrene surface suggesting that we can manipulate the surface
to control biofilm formation, however, the attachment is more
than that observed when we used ethanol to inhibit the adhesion.
The use of ethanol did not affect the degradation of olive mill
waste water, 2.5% v/v also inhibited the biofilm formation on the
container.
Conclusion
fungal adhesion could be manipulated by the addition of
ethanol which could affect the adhesion of both cell-to-cell and
cell-to-substrate. This low-cost by-product will offer a safe
alternative to existing biofilm and biofouling control agents and
also will not exert any toxic effects on the environment as it is
metabolized by the fungus. The addition of ethanol did not affect
the fungus in terms of application. The conclusion drawn from the
results obtained in this study are:
1. There were some biological parameters changed in
Penicillium purpurogenum under stress condition

7. Summary
that affect the biofilm formation as surface bound
protein.
2. Addition of 2.5%v/v ethanol to Penicillium
purpurogenum could affect the cell-cell attachment
and was used as carbon source with no cell loss and
maintaining phenol oxidase activity.
3. The addition of 2.5% ethanol resulted in control of
biofilm formation via the surface bound protein and
not a signaling process.
4. Penicillium purpurogenum contained Yap1p gene
which reprograms the cell to counter base ethanol
stress. The presence of glutathione was on additional
protection for the cells, it was present in the oxidized
form (GSSG).
5. The DNA profile and surface bound protein were
showed polymorphism affected in Penicillium

8. Summary
purpurogenum amended with 2.5 and 5%v/v ethanol
concentrations .
6. The addition of ethanol has an effect on the cell
surface properites such as wettability and surface
charge. Therefore, the adhesion had been affected.
7. Ethanol did not affect the fungus in terms of
application. The efficiency of Olive Mill Wast Water
bioremediation was enhanced, probably due to the
increase in phenol oxidase activity observed after
ethanol addition.