introduction methods discussion and results€¦ · scan electron microscopy flow cytometry...

INTRODUCTION

B.R. Oliveira1,2, M. T. Barreto Crespo1,2, V.J. Pereira1,2

1 iBET - Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal

Limited attention has been given to the presence of fungi in the aquatic environment compared to other microorganisms such as bacteria and virus. Our previous research showed that fungi occur widely in drinking water

sources [1,2] and described many fungi species that have not been previously reported in the aquatic environment. Moreover, many filamentous fungi species present in water were found to be able to grow at high

temperatures and have conidia measurements lower than 5 µm, being therefore considered as potential pathogenic species to humans and animals [2].

Chlorine is the most widely used disinfectant in water treatment. However, Penicillium and Aspergillus species showed a higher resistance to free and combined chlorine disinfection than certain Cladosporium and Phoma

species tested [3,4] and so may resist the conventional treatment. Further research is therefore needed to address the efficiency of different disinfectants for the inactivation of fungi. The use of UV for water treatment has

increased along the years since it is extremely effective to achieve inactivation of protozoans, virus and bacteria and does not require chemical addition. This will also decrease the chlorine dose needed as a final disinfectant

in the distribution system and consequently, decrease the formation of chlorination disinfection by products.

Light-emitting diodes (LED) recently emerged as a promising treatment technology due to their advantages: mercury free lamps, no stabilization time, long lifetimes, and diversity of wavelengths available. LEDs have

already been used for several purposes including the inactivation of several microorganisms in real water sources but, to the best of our knowledge have not been tested in terms of their ability to inactivate filamentous fungi

in water. The aim of this study is therefore to evaluate: (i) the inactivation efficiencies of LEDs with different wavelengths (255 nm and 265 nm) on three Aspergillus’ species (A. fumigatus, A. niger and A. terreus) that were

isolated from drinking water sources and; (ii) the effect of those light sources on their morphology, membrane permeability and enzymatic activity.

METHODS DISCUSSION and RESULTS

Initial After 3 weeks

A. fu

mig

atu

s

A. fumigatus A. niger A. terreus

LE

D 2

55

nm

UV Fluence

Fu

ngal

in

acti

vat

ion

Growth Studies

10 µm10 µmA. fu

mig

atu

s

10 µm10 µm

A. n

iger

10 µm10 µm

A. te

rreu

s

Spores and Mycelium grown for 7 days at 27 °C

1x108 spores/mL spiked into surface water matrix

LED 255 nm and 265 nm

0, 0.5, 1, 5, 10, 15, 30, 45 and 60 min

LE

D 2

55

nm

LE

D 2

65

nm

A. fumigatus A. niger A. terreus

Mem

bra

ne

per

mea

bil

ity a

nd

enzy

mat

ic a

ctiv

ity Size and Granulation

LiveDormant

Dead Damage

FL3 – Propidium Iodide

FL1 - Fluorescein Diacetate

The LED that emits at 265 nm is more efficient to inactivate the fungi species;

A. niger is the most resistant species.

The LED that emits at 265 nm has a higher effect on the fungal spores

morphology, regardless the fungi species

A. fumigatus A. niger A. terreus

No

Ex

po

sure

LE

D 2

55

nm

LE

D 2

65

nm

Fu

ngal

in

acti

vat

ion

Ph

eno

typ

ic e

ffec

t

Karnovsky’s Fixative (overnight):

- Glutaraldehyde (2.5 % v/v)

- Paraformaldehyde (2.0 % v/v)

- Phosphate saline solution (0.1 M)

Osmium tetroxide (1.0 % v/v) for 2 hours

Dehydration with 30, 50, 70, 80, 90, 95 %, 5 min

and 100 %, 10 min, of ethanol

Freeze dried for 30 min

Mounting samples on carbon conductive tape

Cover samples with gold and palladium

Scan Electron Microscopy

Flow Cytometry

CONCLUSIONS

Acknowledgement: Financial support from Fundação para a Ciência e a Tecnologia through the fellowship SFRH/BD/111150/2015 is gratefully acknowledged. iNOVA4Health - UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e

Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is gratefully acknowledged. The authors also thank Dr. David Bastien from SYSMEX for helping with the flow cytometry

analysis and SYSMEX that has kindly provided the flow cytometer used in these experiments.

References: [1] Pereira, V.J., Basílio, M.C., Fernandes, D., Domingues, M., Paiva, J.M., Benoliel, M.J., Crespo, M.T. and Romão, M.V.S. (2009) Occurrence of filamentous fungi and yeasts in three different drinking water sources. Water Research 43(15), 3813-3819.

[2] Oliveira, B.R., Barreto Crespo, M.T., San Romão, M.V., Benoliel, M.J., Samson, R.A. and Pereira, V.J. (2013) New insights concerning the occurrence of fungi in water sources and their potential pathogenicity. Water Research 47(16), 6338-6347. [3] Pereira, V.J.,

Marques, R., Marques, M., Benoliel, M.J., Barreto Crespo, M.T. (2013) Free chlorine inactivation of fungi in drinking water sources. Water Research, 47, 517-523. [4] Pereira, V.J., Marques, M., Marques, R., Benoliel, M.J., Barreto Crespo, M.T. (2017) Inactivation of

fungi in treated surface water by chloramination. J Am Water Works Assoc, 109, 19-23.

Mem

bra

ne

per

mea

bil

ity a

nd

enzy

mat

ic a

ctiv

ity

The LED that emits at 265 nm has a higher effect on the membrane

permeability and the enzymatic activity of the fungal spores

A. niger is the most resistant species followed by A. fumigatus and A. terreus

The LED that emits at 265 nm is more efficient than the LED that emits

at 255 nm which may be due to the DNA peak absorption at 264 nm

A. niger A. fumigatus A. terreus

Spores’ size: 3.5-4.5 µm Spores’ size: 2.5-3.0 µm Spores’ size: 1.5-2.5 µm

Aspergillus niger was the most resistance species which may also be

due to its higher spores’ size and/or due to the presence of pigments

LiveDormant

Dead Damage

SEM and Flow Cytometry proved to be suitable techniques to evaluate

spores’ morphology, membrane integrity and enzymatic activity

1 mm 1 mm 1 mm

250 µm

250 µm