905 © IWA Publishing 2016 Water Science & Technology: Water Supply | 16.

4 | 2016

Fungi in biofilms of a drinking water network: occurrence,

diversity and mycotoxins approach
S. Hurtado-McCormick, L. Sánchez, J. Martínez, C. Calderón, D. Calvo,
D. Narváez, M. Lemus, H. Groot and M. Rodríguez Susa

ABSTRACT
S. Hurtado-McCormick
Results showed that 75% of the analyzed samples in a drinking water network were positive for
L. Sánchez
fungi, in a range of 1–3,000 CFU/mL. Identification resulted in nine species of fungi and four species J. Martínez
D. Calvo
of yeasts being listed: Bjerkandera, Penicillium, Paraconiothyrium, Paecilomyces, Debaryomyces, M. Lemus
M. Rodríguez Susa (corresponding author)
Rhodotorula and Cryptococcus. Although yeasts showed higher traceability than filamentous fungi, Environmental Engineering Research Center,
Department of Civil and Environmental
the fungal genus Penicillium had relevance by both traceability (six species) and its role in mycotoxin
Engineering,
generation. From volatile organic compound (VOC) mycotoxins and extracts analysis from P. Universidad de Los Andes,
Bogotá D.C.,
ochrochloron and P. purpurogenum water–M9 culture, six groups were identified: phenols, alcohols, Colombia
E-mail: manuel-r@uniandes.edu.co
alkenes, monoterpenes, aldehydes and alkanes, phenols being the predominant group (2,4-bis(1,1-
C. Calderón
dimethyl)phenol 40–88%). P. ochrochloron water culture and M9 culture reported signals of toxicity: Mycology and Plant Disease Laboratory,
the first one as genotoxic for 0.5 y 1 mg/mL mycotoxin extract and the second one as cytotoxic. M9 Universidad de Los Andes,
Bogotá D.C,
media promoted a higher number of compounds in both species and a decrease in phenol Colombia

predominance in P. ochrochloron but not in P. Purpurogenum. The results showed Penicillium and D. Narváez
H. Groot
Debaryomyces as prevalent filamentous fungi and yeast in assessed networks, suggesting that these Human Genetic Laboratory,
Universidad de Los Andes,
could be indicators of fungi and yeast presence in drinking water systems. Bogotá D.C,
Key words | biofilms, drinking water, fungi, yeast Colombia

INTRODUCTION

The occurrence of filamentous fungi and yeasts in biofilms
of drinking water distribution systems has already been
reported in other studies (Hageskal et al. ; Pereira
et al. ). Classic and molecular techniques have been
developed to recover and identify biofilm microorganisms
of drinking water networks. This diversity of available pro-
cedures to identify biofilm microorganisms entails
differences between isolation reports and their associated
inferences (Pereira et al. ).
Some of these microorganisms produce secondary
metabolites that modify organoleptic properties of water;
also some fungal genera such as Penicillium spp., Aspergil-
lus spp., Cladosporium spp. and Phialophora spp., have
been reported as mycotoxin producers (Goncalves et al.
; Hageskal et al. ; Pereira et al. ). Penicillium
doi: 10.2166/ws.2016.024
spp. and Aspergillus spp. have widely been identified in
food and beverages as potential producers of flavoring
secondary metabolites (i.e. aldehydes, alcohols), or undesir-
able metabolites that alter the production process (i.e.
aflatoxins, geosmin) (Moreau ; Pitt & Hocking ;
Hageskal et al. ); geosmin, an earthy-muddy-smelling
metabolite, also produced in drinking water, has principally
been associated with taste-and-odor outbreaks (Hageskal
et al. ).
From in vitro studies, in two different water distribution
systems, Kelley () concluded that fungi in water could
produce mycotoxins and other secondary metabolites.
Although mycotoxins in water would be under detection
limits for dilution effects, these concentrations could be
increased when water is stored during long periods
 906 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network
(i.e. tanks, bottles). For example, Mata et al. () detected
frequently aflatoxin B2, and eventually aflatoxin B1, afla-
toxin G1 and ochratoxin in bottled water, and also fungi
of genders Cladosporium, Fusarium and Penicillium were
isolated. For these cases, daily intake of water affected by
fungi growth becomes a concern for human health (Hages-
kal et al. ).
Fungi as ubiquitous organisms are present in soil, air,
food, and water as well as their metabolites. Fungal volatile
organic compounds (VOC) are derived from both primary
and secondary metabolism pathways. Approximately 250
VOC have been identified from fungi where they occur as
mixtures of simple hydrocarbons, heterocycles, aldehydes,
ketones, alcohols, phenols, thioalcohols, thioesters and
their derivatives, including, among others, benzene deriva-
tives, and cyclohexane (Morath et al. ). The analysis of
fungal VOC in the environment and its relation with
human health has been focused on indoor exposure of
fungi isolated from water-damaged, moldy buildings (Insti-
tute of Medicine of the National Academies ; WHO
). Inamdar et al. () tested VOC produced by three
fungi species in Drosophila melanogaster to assess their tox-
icity; negative effects were observed for eight carbon volatile
compounds exposure. Fungal secondary metabolites, vola-
tile (VOC) and non-volatile, in water are still not well
understood and limits for treated water are rare in drinking
water rules. Additionally, in treated water these organic
compounds react with disinfectants, forming disinfection
by-products (DBP), some controlled by water rules for
their probable health effects (Richardson et al. ).
Taking into account this information, fungal presence and
fungi metabolites production must be assessed in drinking
water distribution systems, more specifically in household
systems and tap water, where stagnation is common.
In order to determine the potential impact of filamen-
tous fungi and yeasts on water quality, and to identify
prevalent species, phenotypic and genotypic features were
used to characterize fungal and yeast populations recovered
from biofilm samples in drinking water distribution systems;
also an identification was performed of mycotoxins extract
from isolated species from the main pipeline and household
plumbing, to make an approximation to principal com-
pounds that could be being generated and their genotoxic
effect in mammalian cells.
Water Science & Technology: Water Supply | 16.4 | 2016
MATERIAL AND METHODS
Fungi isolation
Biofilm sampling
Twelve biofilm samples from two Colombian drinking water
networks, which supply surface water treated by conven-
tional processes (i.e. flocculation, settling, filtration and
hypochlorite sodium chlorination), were analyzed. Distri-
bution systems include both primary and secondary
networks. The range of free chlorine was between 0.5 and
1.5 mg Cl/L. The number of samples was limited by the
scheduled maintenance dates of distribution systems,
according to the specifications of the public service oper-
ator. The samples were taken according to previous
protocol (Zhang et al. ) from the water–surface inter-
faces, when sites were in not flow mode and water was
removed by pumping: biofilms were scraped with sterile spa-
tulas and stored in Ziploc® bags at 4 C until analysis.
W
Sampling details are specified in Table 1.
Samples pre-treatment
In order to remove inorganic material that may inhibit
microbial growth, the biomass of samples was extracted
according to the established protocol (Zhang et al. )
and dissolved to obtain the relation 1:1 mL per cm2, with
an additional 30 minute-shaking period in M9 media
(7.25 g Na2HPO4.2H2O, 3 g KH2PO4, 0.71 g NaCl, 2 g
NH4Cl, 1 mL MgSO4 1 mol/L, 0.1 mL CaCl2 1 mol/L,
1 mL FeCl3 10 mmol/L, 2 g yeast extract), which allows
sufficient sample aeration for fungal demands.
Fungal recovery
Fungal and yeast populations were recovered in enriched
and fungal specific culture media Potato Dextrose Agar
(PDA) (4 g potato infusion, 20 g dextrose, 15 g agar), Rose
Bengal Agar (RBC) (5 g soy peptone, 0.1 g chloramphenicol,
0.05 g Rose Bengal, 10 g dextrose, 1 g monopotassium phos-
phate, 0.5 g magnesium sulphate, 15 g agar) and Malt
Extract Agar (MEA) (1 g peptone, 20 g malt extract, 20 g
 907 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network Water Science & Technology: Water Supply | 16.4 | 2016

Table 1 | Biofilm sampling and associated morphotypes

Species

Samplea Siteb Materialc Networkd Fungi Yeasts

M1 DI pipeline of 0.1 m diameter Ductile iron Primary distribution system A NR Dh

M2 DI pipeline of 0.1 m diameter Ductile iron Po; Pg; Pj NR
M4 Elbow Iron NR Dh
M5 Inner side wall of tank Concrete Ba NR
M6 Output pipeline of tank Concrete NR Rg
M7 Output pipeline DWTP* Concrete Primary distribution system B Pcr; Ba Dh
M8 Manhole k4 600 Concrete Ps NR
M10 Shower Zinc Secondary distribution system B NR Rm; Cd
M12 Zinc Pp; PCh; Pi Dh; Rm
a
Used sample labels.
b
Sample origin in distribution systems network.
c
Raw material of the structures.
d
Distribution system types: primary: main network, and secondary: household network. Counting and traceability of identified species. Ps: P. sporulosum, Pcr: P. crustosum, Ba: B. adusta,
Po: P. ochrochloron, Pg: P. glabrum, Pj: P. janthinellum, Pp: P. purpurogenum, Pch: P. chrysogenum, Pl: P. lilacinus, Rg: R. glutinis, Dh: D. hansenii, Rm: R. mucilaginosa, Cd: Cryptococcus
diffluens * Drinking Water Treatment Plant.

glucose, 20 g agar) from samples with and without pre-
treatment. Each sample was serial diluted (10
1 to 10
3),
and 1 mL of each dilution was plated three times. An incu-
bation period at 26.5 C (Deacon ; Cepero de Garcia
W
et al. ) was adjusted to 8–15 days for fungi and 3–5
days for yeasts. Additionally, direct plating of samples with
the same culture conditions was analyzed.
Fungal counting
Colony forming units per millilitre (CFU/mL) of each described
morphotype were visually counted (partial counts), and the
sum of CFU/mL in all plates from each of the first two dilutions
was calculated (total counts). In each case, the most representa-
tive counting per dilution triplicate was reported.
Fungal isolation
Fungal morphotypes were isolated by puncture culture,
while yeast morphotypes were isolated by streaking culture.
Direct plating of predominant recovered morphotypes was
done per duplicate for each sample, followed by an incu-
bation period of 15–30 days for filamentous fungi and 3–5
W W
days for yeasts, both at 26.5 C.
Species identification
Isolated morphotypes were identified by their phenotypic
(macroscopic and microscopic characterizations by Lactophe-
nol Blue Staining) and genotypic characters. Macroscopic
characters were registered at two different incubation periods:
after the recovery process, and after the isolation procedure.
Microscopic characters were registered only at the post-iso-
lation period. DNA was extracted according to the protocol
for fungal endophytes of the Laboratory of Mycology and
Plant Pathology of Universidad de Los Andes. Polymerase
chain reaction (PCR) reactions were carried on with ITS1
and ITS4 universal primers. Sequencing of PCR products
was performed by the Macrogen Company. The resulting
sequences were edited and assembled using Geneious Pro v
4.8.5 software (Geneious, ). Comparisons between these
reports and results from phenotypic and genotypic identifi-
cation allowed us to determine the final species of
filamentous fungi and yeasts from biofilm samples.
Mycotoxins
Penicillum ochrochloron and P. purpurogenum isolated
from the main pipeline and household plumbing were cul-
tured in M9 media and tap water for 7 days (30 C) in a
 908 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network
glass Erlenmeyer flask. The system was fed daily with 72 mL
of sterilized tap water or M9 until it reached the final
volume of 500 mL and was continuously shaken at 300 rpm.
Mycotoxins extraction
At the end, mycotoxins were extracted by liquid–liquid extrac-
tion with methanol, hexane and chloroform based on the
García et al. () protocol. In an Erlenmeyer flask 50 mL
of media and 150 mL of methanol were mixed for 10 min.
After this, the samples were filtered by Whatman No. 4 filter
paper adding 50 mL of methanol. In a separating funnel,
40 mL of hexane were added to the filtered methanolic
samples; after which they were mixed for 1 min and the
phases allowed to separate. The hexane was discarded.
W
Water under 8 C was added and pH was adjusted to 2 with
HCl. The process was repeated twice with 40 mL of chloro-
form at 2 min shaking. Then 30 mL of water were applied
for washing. Finally, chloroform extracts were evaporated at
W
35 C. The solid extracts were suspended in (deionized) DI
water for total organic carbon (TOC) and VOC.
VOC
The protocol used was ASTM 6520-06/EPA 8260B as
follows: solid-phase microextraction with polydimethyl-
siloxane (PDMS) coated fiber was used. Sodium chloride
(3 g) was added to 10 mL of extract suspension. Samples
W
were shaken and heated at 35 C for 20 min. After, the
fiber was exposed into the head space for 10 min. VOC
were detected by Agilent Technologies 6890 N® GC-MS
(gas chromatography–mass spectrometry).
Genotoxicity
DNA damage was determined by Comet Assay. Vero cells,
cultured as for cytotoxicity assay, were exposed to 0.01,
0.1 and 1 mg TOC/mL of extracts for 3 h. Electrophoresis
was run at 25 V and 290 m for 35 minutes. The sheet was
washed with neutralizing solution, pH 7.5, stored with
®
absolute methanol to be dyed subsequently with GelGreen
(Biotium) and read on a fluorescent microscope Zeiss®
(excitation filter BP 450–496 nm, scan filter at 520 nm and
×100 magnification). One hundred cells were analyzed for
Water Science & Technology: Water Supply | 16.4 | 2016
each sample and DNA damage was determined by %
DNA in the tail, using the CASP ®1.2.3b1 program.
RESULTS
Fungi isolation
Fungal and yeast species were present in 75% of the ana-
lyzed samples in a range of 1–3,000 CFU/mL. Initial
recovery of 12 fungal and nine yeast morphotypes was phe-
notypically characterized, and then was expanded to 27
genetically characterized morphotypes. Additionally, the
results supported higher diversity for secondary distribution
systems (household showers), and higher predominance for
the primary distribution systems (Table 1).
Paraconiothyrium sporulosum (P. sporulosum;
2935 CFU/mL), and Paecilomyces lilacinus (P. lilacinus;
108 CFU/mL) were the most numerically abundant fungi,
according to the highest averaged count. Under the same cri-
teria, the most predominant yeasts were Debaryomyces
hansenii (D. hansenii; 143 CFU/mL), Cryptococcus dif-
fluens (C. diffluens; 95 CFU/mL), and Rhodotorula
mucilaginosa (R. mucilaginosa; 70 CFU/mL). Additionally
and by sample origins, secondary distribution systems evi-
denced a wider variety of species (a total of six species)
while primary water networks showed the highest total
counts in M8 sample (maximum of 2,935 CFU/mL); how-
ever in general, the recoveries at both primary and
secondary samples were similar in range (Figure 1). Differ-
ences in diversity in sample M12 could be associated with
low residual chlorine at extreme points in the network, the
higher surface area/volume ratio of water in household pipe-
lines compared to a mains system or a material incidence
(i.e. zinc vs concrete or iron).
Species traceability was higher for yeasts. This tendency
is consistent with the reported yeast ubiquity (Hageskal
et al. ; Pereira et al. ) as a possible consequence
of more robust mechanisms of adaptability, and also with
the apparent soil preference of fungi, since most fungal
identified species are typically found in these environments
(Pitt ). Debaryomyces hansenii was recovered from both
primary and secondary distribution systems. Similarly, Peni-
cillium spp. were found in both types of networks, but with
 909 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network Water Science & Technology: Water Supply | 16.4 | 2016

Figure 1 | Counting and traceability of identified species. Final identification of 13 species was done by comparing phenotypic, genotypic and reviewing of literature results. Reported CFU
of dilutions from 100 to 10-3 were averaged for each species. Samples with negative recovery were not plotted. Ps: P. sporulosum, Pcr: P. crustosum, Ba: B. adusta, Po:
P. ochrochloron, Pg: P. glabrum, Pj: P. janthinellum, Pp: P. purpurogenum, Pch: P. chrysogenum, Pl: P. lilacinus, Rg: R. glutinis, Dh: D. hansenii, Rm: R. mucilaginosa, Cd:
Cryptococcus diffluens.

values of averaged counts less than those reported for yeast tail) for 0.1 and 1 mg/mL mycotoxin extract of
species. However, the wide variability of this genus supports P. ochrochloron cultured in water. The comet assay did
possible complex mechanisms to repair mutations that not detect DNA damage for the other cultures. The com-
could explain their persistence in distribution systems, pound phenol, 2,4-bis-(1,1-dimethylethyl), principal
even after water treatment. substance detected by VOC analysis in mycotoxins extract,
could be important in toxicity due to genotoxic positive
Mycotoxins sample having the highest relative quantity over the total
of detected compounds (87.9%).
Mycotoxin extracts would contribute TOC of 3.5–6.1 mg/L,
depending on fungal culture medium, being that M9 pro-
moted a higher quantity of organic compounds per litre of DISCUSSION
medium. Even when quantity differences between the two
sources of carbon were relevant (water TOC: 1 ± 0.1 mg/L; Results for fungi in water depend on identification tech-
M9 TOC 0.700 ±0.07 g/L), extracts of organic metabolites niques: i.e. sampling, pretreatment, culturing: media,
were not dissimilar in the same order, according to TOC temperature; as a consequence, the first limitation of the
results. From VOC analysis, six groups were identified: analysis is how to compare with other studies. Having in
phenols, alcohols, alkenes, monoterpenes, aldehydes and mind that the recoveries were obtained from non-specific
alkanes, phenols being the predominant group (40–88%) PDA, RBC and MEA media, which were selected for
in mycotoxins extracts (Table 2). being widely used in identification of cultivable fungi, the
M9 medium promoted a higher number of compounds results and the discussion must be interpreted.
in both species and a decrease in phenol predominance in Filamentous fungi and yeasts were identified in samples
P. ochrochloron but not in P. purpurogenum. The extract of both primary and secondary distribution systems, show-
of P. purpurogenum was constituted by phenols, alkenes ing a relevant role in biofilm networks because of the
and aldehydes but these last compounds were not identified amount of species as well as the probable implications of
in P. ochrochloron mycotoxins. The results of genotoxicity their presence on water quality, possibly affecting human
showed an increase in DNA damage (percentage DNA in health. An assessment of exposure to fungi in the
 910 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network Water Science & Technology: Water Supply | 16.4 | 2016

Table 2 | Results of the assessment of mycotoxins from filamentous fungi isolated from drinking water biofilm: VOC

Filamentous fungi species

P. ochrochloron P. purpurogenum

DI pipeline of 4 inch Galvanized shower

Site of biofilm sampling
Group VOC Water M9 Water M9

Phenols 2,4-Bis(1,1-dimethyl)phenol (%) 87.9 36.7 79.5 80.7

3,5-Bis(1,1-dimethyl)-1,2-benzenediol (%) 2.07
Alcohol 1-Hexadecanol (%) 0.245
Alkenes 1-Tetradecene (%) 0.406 12.6 1.97 1.55
1-Hexadecene (%) 4.41 26.8 4.39 3.19
1-Octadecene (%) 2.63
Monoterpenes Camphene (%) 0.106
Aldehydes Octanal (%) 0.715
Decanal (%) 0.548
Alkanes Tetradecane (%) 0.863
TOC extracts (mg C/L culture) 5.773 6.164 3.565 6.118
Genotoxicity extract: %DNA in tail respect to negative control 0–1 mg/mL TOC 6 NS NS NS

NS: not significant.

environment (i.e. water, air, food) would help understanding
of the relevance of their occurrence in drinking water,
which would be different, for example, in a water-damaged,
moldy buildings scenario (Inamdar et al. ) than in new
buildings. Additionally, mycotoxins, secondary metabolites
and special by-products of disinfection would be dissimilar
or specific in drinking water systems, so the health response
would depend not only on the species but also on the
environment in which it grows.
Six identified species of Penicillium corroborate their
reported ubiquity, even in aquatic environments. Moreover,
four of these species are potential producers of mycotoxins
such as Penitrem A by P. crustosum, Rubratoxin by P. purpur-
ogenum, Verruculogen by P. janthinellum and Citromycetin
by P. glabrum (Pitt ; Bial – Arístegui Pharmaceutical Lab-
oratories ). In addition, the genus Paraconiothyrium that
was identified in the highest count (2,935 CFU/mL) can pro-
duce mycosis infections (Quillet-Dye et al. ). Moreover,
we also identified yeast pathogens for humans. For example,
R. glutinis is another opportunistic pathogen associated with
brain diseases such as meningitis in immunocompromised
patients (Lanzafame et al. ). Cryptococcus diffluens
causes subcutaneous cryptococcosis (Serda Kantarcioglu
et al. ) through skin colonization of patients with atopic
dermatitis (Sugita et al. ), and D. hansenii has been
reported as a participant in diseases such as extrinsic allergic
alveolitis (Yamamoto et al. ). As a conclusion, 10 of 13
species identified by culturing in biofilm samples of analyzed
drinking water distribution systems could have an impact on
consumers’ health. In spite of these results, a total profile of
fungi in water-biofilm by total DNA sequencing is needed to
support the relevance of these organisms and specifically
pathogen species in drinking water systems diversity, because
only a few microorganisms grow in culture media.
Taking into account sample location, an increased risk of
negative effects on consumer health is suggested by the reco-
vering of P. chrysogenum (M12 sample), P. purpurogenum
(M12 sample), and C. diffluens (M10 sample) from samples
of the secondary networks. Some reports are consistent
with these potential negative effects of microbial presence
in distribution systems. For example, bacterial pathogens
such as Mycobacterium avium, M. gordenae and Legionella
pneumophila were identified in shower biofilm samples,
which were partially volatilized by usage processes (Feazel
et al. ). As a consequence, microbial aerosols may consti-
tute a risk of illness because of possible inhalation of
contaminated water droplets (Feazel et al. ).
In addition, the occurrence of the identified species
could be associated in some cases with pipeline material
or aggregates. For example, P. ochrochloron is commonly
 911 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network
isolated from copper environments (Mohapatra ). Its
presence in a ductile iron pipeline (M2 sample) could be
supported by the high affinity of iron with anionic copper
(Moreno ). Nevertheless, this fungus was not recovered
from the other ductile iron pipeline (M1 sample), suggesting
that some spatial differences, related to water–pipeline inter-
actions, are important to determine fungal traceability.
Another example is P. janthinellum, which is able to solubil-
ize phosphorus in terrestrial environments. Its presence in
the same ductile iron pipeline (M2 sample) could be
linked to the high affinity of iron with phosphorus
(Moreno ). Also, the genus Paraconiothyrium was ident-
ified in biofilm from a pipeline (M8 sample) 4 km from
DWTP output, where manganese concentration is high
because KMnO4 is used as the oxidant agent in treatment.
This genus has been associated with Mn(II) oxidation,
lacasse production and it has been found in Mn rich
stream sediments and surface water (Miyata et al. ). Fur-
thermore, as was mentioned, the highest variety of fungal
species in the M12 sample could be explained by the low dis-
infectant concentrations in household systems provisioned
by tanks, such as the secondary networks analyzed in this
study. This condition and other physical factors may
enhance microbial proliferation. Bacteria such as Bacillus
cereus, which were detected in the same kind of network
with similar characteristics (Rueda ), are an example of
potential proliferating microorganisms. Even though some
apparent relations between pipeline material and water
characteristics with fungi diversity were observed, more
samples and also inorganic analysis are needed to point at
specific or relevant dependency in the complex system com-
prising biofilms and drinking water networks.
Although in culturing techniques the number of filamen-
tous fungi is underestimated for their growth in hyphae form,
from filamentous fungi and yeast counts it was possible to
infer an opposite occurrence of these types of microorgan-
isms in primary distribution network B. For example, in the
M7 sample, the yeast D. hansenii had an averaged count of
143 CFU/mL while filamentous fungi P. crustosum and B.
adusta had only 1 CFU/mL. In contrast, in the M8 sample
the fungus P. sporulosum had the most averaged count of
2,935 CFU/mL while no yeasts were recovered (Figure 1).
Based on this relationship between counts, the interspecific
competition could be a determinant factor of microbial
Water Science & Technology: Water Supply | 16.4 | 2016
identification studies, especially those of bacterial species,
which constitutes the 80% of biofilm diversity. Previous
studies of the samples used reported bacterial species such
as Citrobacter freundii (M4 and M3 samples), Klebsiella
oxytoca (M1 and M2 samples), Aeromonas hydrophila (M7
and M8 samples), Micrococcus kristinae (Rueda ), and
Herminiimonas sp. (M6 sample) (Viancha ). These
species could inhibit fungal growth and permanence by com-
petition, even more so in drinking water distribution systems,
being consistent with the same phenomenon in mineral water
environments (Fujikawa et al. ). Inhibition results from
the effects of different factors such as disinfectant concen-
trations, which may influence the proliferation0 s rate of
competitive bacteria. Hence, a higher competition level
between species recovered from the M6 sample could exist
due to low disinfectant concentrations in the storage tank,
located far away from the drinking water treatment plant.
Nevertheless, correlation between fungi/yeasts and bacteria
in biofilm samples and their impact on water quality are
not totally understood because of the scarce knowledge of
additional factors that determine the survival of particular
species. Specifically for D. hansenii, biocontrol mechanisms
could be considered. Transformation of this yeast with plas-
mids encoding toxins has been suggested to regulate the
presence of other bacteria (Hernández-Montiel et al. ).
Despite being unproved, this strategy is conceivable from
average counts of this species (M1, M4, M7 and M12
samples) which were higher than the fungal ones. The bio-
control mechanisms could explain the prevalence of D.
hansenii in primary and secondary networks. Biedunkiewicz
et al. () also found D. hansenii in tap water from two
different cities in Poland and bottled water, Exophiala jean-
selmei and Aspergillus fumigates being the only species, of
36 identified, recorded in the three types of samples. These
tendencies of prevalence could be associated with D. hanse-
nii’s capacity to oxidize a wide range of organic compounds
such as alcohols, carbohydrates, amino acids and others
(Arlyapov et al. ) that are present in treated water, and
also in biofilms as secondary metabolites of other microor-
ganisms such as those found from P. purpurogenum culture
(Table 2). Even though the absence of enough reports of
D. hansenii in biofilms from other distribution systems does
not allow proposing it as a quality indicator, these results
give preliminary information about constant D. hansenii
 912 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network
presence in different points through the studied networks.
More specific analysis in other systems based on temporal
variation are suggested.
Yeast traceability in samples of both primary and second-
ary distribution systems (with an average count of 2 log
CFU/mL; Figure 1) could support resistance mechanisms
of species like D. hansenii which show a wide tolerance to
saline and halogenated environments (Hernández-Montiel
et al. ). Halogenated compounds are frequently detected
in drinking water networks because of the presence of
chlorine and/or disinfection by-products (DBPs) such as
trihalomethanes and haloacetic acids (Rodríguez et al.
). Also, some crystalline and amorphous compounds
of iron–manganese are found (Moreno ). Hence, these
substances may not only be tolerated by yeasts at different
disinfectant concentrations, but also may enhance yeast sur-
vival by selection pressure.
The production of secondary metabolites could meet bac-
terial nutritional demands, facilitating their survival. Volatile
metabolites, phenols, alcohols, alkenes, and alkanes, pro-
duced in both P. ochrochloron and P. purpurogenum
cultured in tap water and M9 medium respectively, and
additionally aldehydes for P. purpurogenum in M9 and ter-
penes for P. ochrochloron in water (Table 1) are normally
produced in fungal fermentation, principally alcohols
(Larsen & Frisvad ). Those volatile compounds have
been related to by-products of enzymatic reactions over poly-
unsaturated fatty acids, linoleic or linolenic acids by
lipoxygenase and hydroperoxide lyase converting to C8 vola-
tile and C10 non-volatile compounds in the case of
Penicillium. sp. (Kermasha et al. ). From specific com-
pounds determined in our experiment, only decanal, octanal
and tetradecane have been also recorded from Penicillium
under other culture conditions (i.e. dry cured ham inoculated
by P. chrysogenum; Martin et al. ); additionally, to our
knowledge, the most relevant 2,4-bis(1,1-dimethyl)phenol
has not been reported as a Penicillium or P. ochrochloron
and P. purpurogenum metabolite. According to Lattanzio
et al. (), phenolic compounds are scarce in bacteria,
fungi, and algae, but as common secondary metabolites in
plants play a role in pigmentation, growth, reproduction and
resistance to pathogens. Similar to plants, fungi metabolize
bioactive substances with medical properties in pathogen con-
trol. Penicillium purpurogenum produced eight phenolic
Water Science & Technology: Water Supply | 16.4 | 2016
compounds: seven antraquinone derivates and one xanthone
that exerted brine shrimp toxicity, moderate inhibition of
G. saubinettii, and moderate phytotoxic activities against
radish seedling growth (Li et al. ). Based on this infor-
mation, the function of 2,4-bis(1,1-dimethyl)phenol could be
linked to survival requirements of fungi in a hostile environ-
ment such as drinking water biofilm, where bacteria could
represent 80% of total microorganisms and could have other
toxicological effects. Even when a direct relationship between
the volatile compounds and toxicity is difficult to make, it was
observed that both extracts of P. ochrochloron water culture
and M9 culture, reported signals of toxicity: the first one as
genotoxic for 0.5 y 1 mg/mL mycotoxin extract and the
second one as cytotoxic, having the highest relative quantity
over the total of detected compounds (87,9%) of phenol, 2,4-
bis-(1,1-dimethyl), also presence of 3,5-bis(1-1 dimethyl)-
1,2benzediol and 1-hexadecanol. Additionally, fungal anti-
biotics production such as penicillin by P. chrysogenum
(Bial – Arístegui Pharmaceutical Laboratories ) could
reduce populations of susceptible bacteria, modifying disinfec-
tant action in the analyzed water networks. In addition, even
low concentrations of antibiotics in drinking water could have
unpredictable effects on consumer health related to bacterial
resistance development.
According to specific parameters such as prevalence, D.
hansenii and Penicillium spp. could be assessed as tracers of
yeast and filamentous fungi presence in water networks,
which compromise water quality by their occurrence (Holt
& Miller ). Parameters that preliminary D. hansenii
and Penicillium spp. accomplish and must be more deeply
assessed in a possible bioindicator selection are: detection,
ubiquity, and secondary metabolites. Other species such as
R. mucilaginosa and P. lilacinus deserve attention in bioin-
dicator studies because of their outstanding presence in
secondary distribution systems.
CONCLUSIONS
From biofilm sampled in drinking water distribution sys-
tems, filamentous fungi and yeast were isolated and
identified as being D. hansenii and Penicillium spp. found
at different points within the network; more analyses are
needed in order to identify if they are also prevalent in
 913 S. Hurtado-McCormick et al. | Fungi in biofilms of drinking water network
other networks in primary and secondary systems. A higher
diversity was detected in household systems and specifically
P. purpurogenum isolated from a shower, which should gen-
erate Rubratoxin, as has been reported before, and also
other metabolites such as phenols, alkenes and aldehydes
that could produce other biological reactions. Similar P.
ochrochloron, under water and M9 culture conditions,
metabolized volatile compounds with unknown effects
in biofilm dynamics and on water quality but signals of tox-
icity were observed on Vero cells as genotoxicity for 0.5 y
1 mg/mL mycotoxin extract of P. ochrochloron cultured in
water. To our knowledge, P. purpurogenum and P. ochro-
chloron have not been assessed as secondary metabolite
producers in drinking water systems either genotoxic
assays of them have been made. It would be the first
approach, so other deeper analyses, in water, biofilm, with
higher number of samples are required to observe the rel-
evance of fungi the presence in water.
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