Isolamento di funghi da ambiente marino mediante esche legnose per selezionare specie potenzialmente utilizzabili nella mycoremediation
10.6092/issn.2531-7342/10769
I funghi, grazie alle loro attività enzimatiche, vengono impiegati in molti studi e applicazioni per il biorisanamento di contaminanti organici come, per esempio, gli idrocarburi. In particolare, i funghi ligninolitici isolati in ambiente marino possono essere sfruttati per tale proposito, in quanto svolgono già un ruolo attivo nei cicli biogeochimici su substrati legnosi in ambiente marino.Al fine di selezionare specie potenzialmente utilizzabili in protocolli  di mycoremediation, è stata verificata la presenza di funghi ligninolitici di origine marina nel bacino portuale di Genova (Italia nord-occidentale). A tale scopo sono state immerse diverse tipologie di esche legnose nella colonna d'acqua. In totale, sono stati individuati 437 ceppi di microfunghi appartenenti a 12 generi e 23 specie. Sono emerse differenze tra le specie trovate alle varie profondità indagate, ma sono state anche individuate specie più comuni rispetto ad altre, in particolare, Penicillium solitum e Galactomyces geotrichum.
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Mirca Zotti
Italian Journal of Mycology
marine port environment
Trapping of marine-derived fungi on wooden baits to select species potentially usable in mycoremediation
Text.Serial.Journal
https://italianmycology.unibo.it/
49
Articles
2020-09-28
2020-04-14
2020-02-10
2020-09-28
101-115
Ital J Mycol
Mirca Zotti
2020/09/28
10.6092/issn.2531-7342/10769
101
DISTAV - University of Genoa, 26 Corso Europa, I-16132, Genoa, Italy
2531-7342
marine port environment
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Zafra G, Cortés-Espinosa DV (2015) Biodegradation of polycyclic aromatic hydrocarbons by Trichoderma species: a mini review. Environmental Science and Pollution Research 22: 19426-19433. https://dx.doi.org/10.1007/s11356-015-5602-4
Trapping of marine-derived fungi on wooden baits to select species potentially usable in mycoremediation
https://italianmycology.unibo.it/article/download/10769/11618
49
Greco et al.                                                            Italian Journal of Mycology 49: 101-115 (2020)

DOI: 10.6092/issn.2531-7342/10769

Original paper

Trapping of marine-derived fungi on wooden baits to select species
             potentially usable in mycoremediation
               Giuseppe Greco, Laura Cutroneo, Simone Di Piazza, Marco Capello, Mirca Zotti

DISTAV - University of Genoa, 26 Corso Europa, I-16132, Genoa, Italy

Corresponding author e-mail: simone.dipiazza@unige.it



Article info
Received 14/04/2020; accepted 14/09/2020
DOI: 10.6092/issn.2531-7342/10769

Abstract
Thanks to their enzymatic activities fungi are used in many studies and applications for remediation purposes in order
to degrade organic contaminants such as hydrocarbons. In particular, ligninolytic marine-derived fungi can be applied
to biodegradation, since they already play an active role in the biogeochemical cycles of marine substrates. To select
potentially usable species in mycoremediation, the occurrence of ligninolytic marine-derived fungi was investigated in
the water mass of a commercial port (Port of Genoa, north-western Italy) by exposing baits of different wood-types in
the water column. In total, 437 microfungal strains were found belonging to 12 genera and 23 species; the most common
fungal species were Penicillium solitum and Galactomyces geotrichum, and differences in the investigated water column
were highlighted.

Keywords
wooden baits; marine-derived fungi; fungal diversity; marine port environment




Introduction

Organic pollution is one of the major ecological problems in the world (Revenga et al., 2000;
Latimer and Zheng, 2003). Several studies have shown how the organic waste products are extremely
persistent in marine environment and can be harmful to both animal and human health (Walker and
Livingstone, 1992; Naso et al., 2005; Nfon et al., 2008; Farrington and Takada, 2014; Seltenrich,
2015). Among the organic pollutants, hydrocarbons are well known to alter the immune system,
to determine carcinogenic action and to induce genetic mutations (Man et al., 2013). In marine
environment, hydrocarbon pollution is mostly due to accidental or intentional spills, or to the fallout
and consequent deposition of airborne polluting substances (Singh, 2006; Davis et al., 2018; Davis
et al., 2019). Moreover, pollution from polycyclic aromatic hydrocarbons (PAHs) is very common
in ports due to industrial activities, ship traffic, street run-off water and accidental spills from fuel
distributors.
       To solve this ecological problem, bioremediation techniques (such as phytoremediation and
microbial remediation) have received during the last two decades wide acclaim becoming promising
alternatives to the traditional techniques because they are eco-friendly, they require little energy and
may detoxify persistent organic compounds (Singh, 2006). In this context, degradation of PAHs can
be induced by the action of extremophile organisms, such as bacteria and fungi (Boonchan et al.,

ISSN 2531-7342 Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2020 The Authors. This work is licensed under CC BY 4.0
Greco et al.                                                     Italian Journal of Mycology 49: 101-115 (2020)

DOI: 10.6092/issn.2531-7342/10769


2000; Cerniglia and Sutherland, 2010; Kostka et al., 2011). In fact, several studies have shown how
some fungal species can adapt to adverse marine environmental conditions (for both abiotic factors -
e.g. high salinity and hydrostatic pressure - and nutrient deficiency) which would be limiting for most
living organisms (Gunde-Cimerman et al., 2005; Gadd, 2007; Gonçalves et al., 2013).
        Fungi from marine environments, in particular the ligninolytic marine-derived fungi, play a
central and active role in the biogeochemical cycles of hydrocarbon pollutants. These fungal strains
were originally terrestrial that have been overtime adapted to the marine environment, developing
different genetic makeup and gene expression (Pang et al., 2016). In recent years, many mycological
researches have investigated the microfungal communities of environments contaminated by organic
substances (Rydin et al., 1997; Schmit and Mueller, 2007; Pautasso and Zotti, 2009), but few studies in
marine environment on ligninolytic marine-derived microfungal communities have been carried out.
Among the first scientists who dealt with this topic, Montemartini Corte in 1979 laid wooden panels
of Fraxinus ornus L., Abies alba Mill., Ochroma pyramidale (Cav. Ex Lam), Fagus sylvatica L.,
Larix decidua Mill., Pinus pinaster Aiton, Populus alba L. and Olea europaea L. to catch cellulolytic
fungi and investigate the mycodiversity of the Portofino bay (north-western Italy). In the following
years, lignicolous marine-derived fungal species have been isolated from driftwood, pilings, wooden
boats, mangrove roots and tree trunks (Shearer et al., 2007; D’Souza-Ticlo et al., 2009; Liu et al.,
2011; Rämä et al., 2014). These researches ranged from the surface to the depth of 1200 m (Shearer
et al., 2007; Dupont et al., 2009) and from the poles to the equator (Booth, 1983; Rämä et al., 2014).
        Moreover, wood-inhabited marine-derived fungi produce extracellular enzymes such as
peroxidases and laccases capable of degrading or modifying lignin and complex molecules with
similar chemical structure, such as hydrocarbon products and aromatic persistent environmental
pollutants (Hammel, 1995; Passarini et al., 2011). Hence, thanks to their ecological plasticity and
the ability to produce cellulolytic extracellular enzymes, marine-derived fungi may be used in real
biotechnological processes (Kumar et al., 2011). With regards to this Cecchi et al. (2019) reported that
the exploitation of fungi can prove to be as potential tool to be able to restore port sediments deriving
from dredging operations (ex situ) or to remediate limited marine areas affected by hydrocarbon spills
(in situ).
        Therefore, starting from the previous studies (Montemartini Corte, 1979; Greco et al., 2018),
our study aims i) to deepen the fungal characterisation of the marine environment of the Port of
Genoa (north-western Italy), ii) to investigate the lignicolous microfungal component in the water
column and iii) to isolate (from wooden panels) lignicolous marine-derived fungi potentially usable
for bioremediation purposes.

Materials and methods

Study area
The study area, the Port of Genoa (Fig. 1), is the most important commercial port of the north-western
Mediterranean Sea, characterised by several commercial activities such as shipyards, ferry terminals,
marinas, industries (e.g. a steel mill). Several terminals are dedicated to receive containers or different
materials, such as dry or liquid bulk coal and crude oil transported by oil tankers.
      Many different water types finish into the port basin which includes several city streams and all
the wastewater from Genova city itself (population of 600,000 units) which is directly overlooking
the basin. In addition, the basin is affected daily by freshwater inflows from the mouths of the Bisagno
and Polcevera torrents (http://www.porto.genova.it). Due to these water and discharge inputs, marine
waters inside the port are often rich in nutrients, faecal coliform, chlorophyll α, metals and hydrocarbon
compounds (Ruggieri et al., 2011; Cutroneo et al., 2015, 2017).



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Fig. 1 - Map of the Port of Genoa and position of the four sampling stations (red dots) where ropes with wood baits were
moored. Station 1: on the breakwater in the Sampierdarena channel; station 2: in the municipal dock in the inner part of
the port; stations 3 and 4: at the western port entrance.




Preliminary test
A preliminary test aimed to assess the presence of marine-derived fungi in the water column was
carried out. For this purpose, were employed six panels (12 x 4 x 2 cm) of fir wood (A. alba) as an
organic substrate, to allow the isolation of lignicolous marine-derived fungi. Abies alba panels were
sterilised and mounted on a rope, using plastic ties, arranged at 1 m from each other, and moored
in the water column at station 1 (Fig. 1) starting from the sea surface for a period of 1 month. The
different wooden baits placed in several sampling stations highlight differences in the distribution of
marine-derived fungi at varied depth in different areas inside the port. Later was carried out a detailed
fungal characterisation once the effectiveness of the method was proven and the presence of the
marine-derived fungi along the water column was confirmed.

Sampling strategy
The marine-derived fungi were trapped using natural (A. alba) and artificial wooden baits (Medium
Density Fibreboard) in order to evaluate possible differences in number and type of isolated fungi. The
Medium Density Fibreboard (hereinafter MDF) in an engineered wood typically produced starting
from wood fibres (82%) combined with urea-formaldehyde resin (9%) and paraffin wax (1%) and
treated with high temperature and pressure to produce panels.
       Six wooden baits (12 x 4 x 2 cm) were sterilised and then mounted on a rope, using plastic
ties, arranged at 1 m from each other forming a bait chain (Fig. 2). Bait ropes were positioned in four
different sampling stations in the Port of Genoa (Fig. 1): one rope of fir wood and one rope of MDF
were positioned at stations 1 and 2; one rope of fir wood baits was positioned at station 3; one rope of
MDF was positioned at station 4. Each station was characterised by different depth, hydrodynamics
and rate of potential water contamination due to its position in the inner or external parts of the port.
Ropes were tied to the piers starting from the water surface and kept vertical with a weight on the
sea bottom. Ropes at stations 1 and 2 were recovered after 269 and 399 days, respectively, whereas,
ropes at stations 3 and 4 were not found at the recovery time maybe because of the bad sea conditions.
Characteristics of sampling stations and sampling time for each bait chain are reported in Table 1.



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Table 1 - Sampling stations positions and sampling time for each bait chain. MDF: Medium Density Fibreboard.


  Sampling                                          Chain of      Chain of      Sampling period       Number of
               Position in the port basin
   station                                        A. alba baits   MDF baits      (dd/mm/yyyy)       sampling days
                                                                                   16/11/2016
               Channel affected by industrial         YES            YES                                  76
                                                                                   31/01/2017
      1        activities and passage of
               merchant vessels and tugs                                           31/01/2017
                                                      YES            YES                                 269
                                                                                   27/11/2017
               Inner part of the port impacted
                                                                                   16/12/2016
      2        by stream waters and city run-         YES            NO                                  399
                                                                                   19/01/2018
               off, trawler and tugs traffic




                                 Fig. 2 - A) Wood panels of A. alba; B) Experimental
                                 design of wood bait chain; C) Wood panel at the
                                 recovery time.

Laboratory treatment
Wooden panels were recovered and transported in refrigerated bags to the mycological laboratory
where they were immediately processed. Later, the panels were scratched and washed with sterile
autoclaved seawater to remove the fouling organisms. To isolate marine-derived fungi, 1 mL of
homogenized washing water was pipetted in Petri dishes (9 cm diameter) containing a modified
culture medium Rose Bengale Agar seawater (RBs; Fig. 3). The plates were incubated at 24 ± 1°C,
in the dark, for 7 days.
       To enable the sporulation of the latent fungi, were incubated wooden panels at room temperature
for a total of 21 days in Petri dishes in contact with sterilised filter paper (Fig. 3). The panels were
checked weekly to be able to isolate marine-derived latent fungal species from wooden substrate.
       Later, all the vital fungal strains were isolated in axenic cultures using test tubes containing
different media, such as RBs, Malt Extract Agar seawater (MEAs), Czapek Yeast Agar medium (CYA),


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Potato Dextrose Agar medium (PDA) and Oatmeal Agar medium (OA), specifically usable based on
founded fungal species. The isolated fungal strains were deposited at the Mycological Laboratory of
the Department of Earth, Environment and Life Sciences of the University of Genoa.




                    Fig. 3 - The six MDF (Medium Density Fibreboard) and A. alba wood
                    panels in Petri dishes with sterilised filter paper (above) and respective
                    Petri dishes containing RBs (below) used for the fungal characterisation
                    in the water column of the station 2.


      Strains were identified with an integrated morphological and molecular approach. At first, the
micro- and macro-morphological characteristics were evaluated with specific taxonomical keys (e.g.,
Raper and Fennel, 1977; Pitt, 1979; Klich, 2002; Domsch et al., 2007; Samson and Frisvad, 2004).
For this purpose, was employed an optical microscope (10×/0.30 and 40×/0.75). Genomic DNA
was extracted from 5-days old cultures by a modified CTAB method (Doyle and Doyle, 1987). The
morphological identifications were confirmed by amplification of the ITS region using the universal
primers ITS1F/ITS4 (Gardes and Bruns, 1993) and the β-tubulin gene using the primers Bt2a and
Bt2b (Glass and Donaldson, 1995). The PCR reaction contained 24 μl mix (15.875 μl Milli-Q water,
1.5 μl 50 mM MgCl2, 5 μl 5× Green GoTaq® Buffer, 0.5 μl 10 mM dNTPs, 0.5 μl 10 µM of each
primer, 0.125 GoTaq® 5 U µl-1) and 1 μl of DNA template. The PCR program was: 2 min 95 °C, 35
× (45 sec 95 °C, 45 sec 55 °C, 2 min 72 °C), 5 min 72 °C. PCR products were purified and sequenced
using MACROGEN Inc. (Seoul, Republic of Korea). The sequences obtained were compared with the
Gen Bank database using the BLASTN algorithm using 97% as threshold for species identification.

Statistical analysis
The biodiversity levels of each sampling series (on MDF and A. alba at stations 1 and 2) were evaluated
with Shannon’s biodiversity index (H′) that was calculated in log2 (Shannon, 1948) considering the
number of fungal strains found for each species in the whole water column.

Results

Preliminary test results
In the preliminary test carried out with A. alba panels, a total of 50 microfungal strains were found
(Table 2); 5 genera and 9 species have been identified. The most recurrent genera were Aspergillus


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and Penicillium, while among the isolates, the most common fungal species were Penicillium solitum
Westling (from 2 to 4 m depth), and Trichoderma longibrachiatum Rifai (from 2 to 5 m depth; Fig. 4).
      No fungal presence was found on wooden panels placed on the surface layer (0 m depth), while
the highest fungal abundance was observed at 3 m depth.




                      Fig. 4 - Fungal abundance on A. alba panels in the water column in the
                      preliminary test.



    Table 2 - Fungal distribution in the water columns in the preliminary test. Zero values for the species were not
    shown to the table.

     Fungal species                                                            Preliminary test on Abies alba
                                                                       0m        1m     2m      3m      4m      5m
     Aspergillus tubingensis Mosseray                                             1
     Aspergillus westerdijkiae Frisvad & Samson                                           2
     Cladosporium cladosporioides (Fresen.) G.A. de Vries                                        5               1
     Eurotium amstelodami L. Mangin                                               2
     Galactomyces geotrichum (Butl & Peter.) Redhead &
                                                                                                         1
     Malloch
     Penicillium solitum Westling                                                         5      8       6
     Penicillium sizovae Baghd.                                                           1
     Trichoderma harzianum Rifai                                                                 4               4
     Trichoderma longibrachiatum Rifai                                                    3      1       2       4
     Total fungal strains                                               0         3      11      18      9       9
     Total fungal species                                               0         2       4      4       3       3




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Mycodiversity results
A total of 437 microfungal strains were isolated from the wooden panels, 187 in station 1 (Table 3)
and 250 in station 2 (Table 4). All the isolates are well-known marine-derived fungi and some of the
microfungal strains belonged to Ascomycota. Twelve genera and 23 species have been identified;
among these, 11 genera and 18 species have been isolated from A. alba panels, whereas 9 genera
and 16 species from MDF. The sequences obtained were deposited in GenBank under the following
accession numbers: MT791370-MT791380 for ITS, and MT820427-MT820429, MT820431-
MT820435, MT876625 for β-tubulin.

 Table 3 - Fungal distribution at different depth (0-5 m) in the water column at station 1 on A. alba and MDF panels.

 Fungal species                           STATION 1 – Abies alba                          STATION 1 – MDF
                                   0m     1m      2m     3m     4m     5m     0m     1m      2m    3m     4m      5m
 Acrostalagmus luteoalbus
 (Link) Zare, W. Gams &                                   1      2      1
 Schroers
 Aspergillus europaeus Hubka,
 A. Nováková, Samson,
                                                                        3
 Houbraken, Frisvad, M.
 Kolařík
 Aspergillus flavus Link                                         1                                   3
 Aspergillus tubingensis
                                                          8             1                                   2
 Mosseray
 Aspergillus terreus Thom                                 2      3      4             3
 Aspergillus westerdijkiae
                                                                               2
 Frisvad & Samson
 Amphichorda felina (DC.) Fr.                                                         1

 Cladosporium sp.                                                3      3             1       1      3      2

 Epicoccum nigrum Link                      1      2             3                    1

 Fusarium sp.                               1                    4
 Galactomyces geotrichum (Butl
                                                  17      3      2                    2       2      1
 & Peter.) Redhead & Malloch
 Geotrichum candidum Link                  20

 Mucor hiemalis Wehmer                      2      2                                  2

 Penicillium solitum Westling               1      6      6      11     2             5       5     20      8      6

 Rhizopus arrhizus A. Fish.                                                                                 1

 Trichoderma harzianum Rifai                                            1
 Total fungal strains                0     25     27     20     29      15     2      15      8     27     13      6
 Total fungal species                0      5      4      5      8      7      1      7       3      4      4      1




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   Table 4 - Fungal distribution at different depth (0-5 m) in the water columns at station 2 on A. alba and MDf
   panels.

     Fungal species                   STATION 2 – Abies alba                      STATION 2 – MDF
                               0m     1m     2m     3m     4m      5m    0m     1m     2m     3m     4m     5m
     Acrostalagmus
     luteoalbus (Link) Zare,                                       2
     W. Gams & Schroers

     Aspergillus flavus Link
                                                     1
     Eurotium amstelodami L.
                                                                                                             1
     Mangin
     Fusarium sp.
     Galactomyces
     geotrichum (Butl &
                                              6      3         2                        5      2      2
     Peter.) Redhead &
     Malloch
     Mucor hiemalis Wehmer                                                 8            2      1      2
     Mucor racemosus Fresen      1                                                                           2
     Mucor sp.                                                                                               5
     Penicillium camponoti
     Visagie, David Clark &                                        1
     Seifert
     Penicillium solitum
                                 7     10     34     24        2   2      12     23     41     9      7      5
     Westling
     Rhizopus stolonifer
                                 2     1                           1      19
     (Ehrenb.) Vuill.
     Trichoderma koningii
                                                                   1
     Oudem
     Trichoderma
                                                               3           1
     longibrachiatum Rifai
     Total fungal strains       10     11     40     28        7   7      40     23     48     12     11    13
     Total fungal species        3     2      2      3         3   5       4     1      3      3      3      4

      The recurrent genera were Aspergillus and Penicillium, while the most common fungal species
were P. solitum and Galactomyces geotrichum (Butl & Peter.) Redhead & Malloch (Fig. 5); among
the most common fungal species, P. solitum was isolated in both sampling stations at different depths
in the water column and on both A. alba and MDF wooden panels; while G. geotrichum was not
found on the surface and bottom layers.
      Penicillium solitum (from 1 to 5 m depth) and G. geotrichum (from 2 to 4 m depth) were the
most common isolates in the water column at station 1 on A. alba (Fig. 6), while only P. solitum was
highlighted as the most abundant (from 0 to 5 m depth) on MDF at station 1 (Fig. 6). Instead, in
the whole water column (from 0 to 6 m depth) at station 2, P. solitum was the most common fungal
species both on A. alba and MFD (Fig. 6).

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  Fig. 5 - Pure culture and micromorphological features of G. geotrichum (A and B) and P. solitum (C and D).




        Fig. 6 - Fungal abundance in the water column at stations 1 (above) and 2 (below) on A. alba (left) and
        MDF (right) panels.




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      On the surface layer (0 m depth) at station 1, there was not fungal presence on A. alba, while
only one species (Aspergillus westerdijkiae Frisvad & Samson) was isolated from the MDF panels.
At this station, on both A. alba and MDF, the highest fungal abundance was observed from 1 to 4 m
depth. Otherwise, at station 2, the highest abundance was found from 0 to 3 m depth (from 0 to 3 m
depth on A. alba, and from 0 to 2 m depth on MDF). At 5 m depth, fungal abundance on A. alba and
MDF was significantly reduced at both station 1 and station 2. The highest H′ values, as shown in
table 5, were observed at station 1 on both the wooden panels, confirming the higher fungal diversity
present in this station than in station 2.


                                    Table 5 - Shannon’ index (H’) results.


                                      Station    Wood panel        H’
                                         1          A. alba       3.18
                                         1           MDF           2.11
                                         2          A. alba       1.34
                                         2           MDF          1.68



Discussion

All the fungal colonies isolated belong to well-known marine-derived Taxa; this information support
the hypothesis that fungal spores/conidia may be transported in seawater by the inputs from the
waterways and/or by the wastewater discharge from treatment plants surrounding the site. Moreover,
results highlight a significant presence of microfungal communities in the water columns at stations 1
and 2 on both A. alba and MDF panels which confirm a high level of adaptability to the environmental
contamination. In this case, wax and resin binders composing MDF do not influence the fungal
presence.
       Fungal absence was observed in the surface layer in the preliminary test, while only one species
(A. westerdijkiae) was isolated in the surface layer at station 1. These results are probably due to the
high hydrodynamics and wave-effect that characterised this port section, which is exposed to strong
winds from the North and to the traffic of merchant vessels and tugboats (Cutroneo et al., 2017), these
conditions prevent the microfungal colonization of the wooden substrates.
       At 5-m depth at stations 1 and 2, on both A. alba and MDF, fungal abundance was significantly
reduced; these results are due to the variations of physical parameters in relation to depth (e.g. light,
temperature and dissolved oxygen) that often strongly impact on the composition of microfungal
communities, as reported by Li et al. (2016).
       Penicillium solitum has been commonly observed on A. abies and MDF panels in the whole
water column at both station 1 (except in the surface layer) and station 2, showing high levels of
adaptability to the environmental pollution that characterised the area. At both sampling stations with
P. solitum, also G. geotrichum and some species belonged to Trichoderma genera (except in MDF
panels of station 1) were frequently isolated from the two different panel types. P. solitum was reported
as the most recurrent fungal species also in the previous survey carried out by Greco et al. (2018).
Penicillium solitum has a wide geographical spread and in fact, it was also isolated from glacial ice
in the extremely cold Polar Regions, and it is also known to grow at low temperatures and at high
salinity levels (Sonjak et al., 2006). It was considered an important pan-global contaminant of foods

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such as refrigerated dry meat, cheese, apples, pears or nuts (Pitt, 1979; Samson and Frisvad, 2004;
Domsch et al., 2007). Moreover, P. solitum can degrade various polymeric substrates (Gonçalves
et al., 2013) and it is a well-known degrader of cellulose, which has a structure very similar to
many hydrocarbon compounds; that’s why it could be used effectively in marine environments for
bioremediation purposes. Furthermore, its hydrocarbon-degradation capacity is not well-known
because no preliminary tests have been carried out.
       Along with P. solitum, also G. geotrichum is known for its ecological plasticity; it grows at a
range of temperatures between 25-30 °C and at a pH between 3 and 11 with an optimum around 5-7
(Grygier et al., 2017). It was isolated from different environments including freshwater (Nagahama,
2006) and marine sediments (Sutani et al., 2015). As indicated by Grygier et al. (2017), G. geotrichum
is very important in the dairy industry and has been isolated from milk and derivatives and alcoholic
beverages. This species plays an important role in ecology because it can be used to improve
environmental quality being often used in the flocculation processes of wastewater treatment.
Moreover, Grygier et al. (2017) indicates G. geotrichum as able to degrade hazardous substances
such as DDT (Dichlorodiphenyltrichloroethane), and Khan et al. (2015) showed that it is an effective
petroleum-degrading fungus.
       As highlighted also by H’ values, the highest level of mycodiversity and fungal strains was
reached on the wooden panels placed at station 1 (Fig. 1), in front of the area dedicated to receiving
coal and crude oil transported by oil tankers. In this station the water column is affected by PAH
contamination, as reported by Cutroneo et al. (2015). The area is also affected by strong hydrodynamics
due to its exposure to strong winds from N (Cutroneo et al., 2017). Hence, achieved data suggest that
the high hydrocarbon concentration can produce a considerable increase of fungal species adapted to
organic polluted environments. Station 2 presents a lower level of mycodiversity this may be due to
the high level of organic contamination of the water column (ammonia–nitrogen and fecal coliform),
low value of oxygenation (40-50%) and low hydrodynamics, with strong parameter variation due to
the direct input of a city stream especially during rainy autumn days (Ruggieri et al., 2011).
       Trichoderma longibrachiatum and T. harzianum, even if found in very small numbers, are
cosmopolitan filamentous fungi, known as successful colonizers of common habitats (Zafra and Cortés
Espinosa, 2015). Their enzymatic activity due also to cellulases, chitinases, glucanases, and proteases
production, enable the decomposition and utilization of substrates present in soils, but in particular, the
degradation of lignocellulosic material (Jaklitsch, 2009). These cosmopolitan species were studied in
relation to diverse biotechnological applications and they are associated with the ability to metabolise
a variety of both high and low molecular weight polycyclic aromatic hydrocarbons (PAHs) such as
naphthalene, phenanthrene, chrysene, pyrene, and benzo[a]pyrene (Zafra and Cortés Espinosa, 2015).
       Trichoderma longibrachiatum and T. harzianum, together with A. flavus, A. tubingensis, A.
terreus, Fusarium sp., G. geotrichum, M. racemosus, P. solitum, R. arrhizus, and R. stolonifer were
also found in the previous study of Greco et al. (2018) in the Port of Genoa, that reported them as
species adapted to contaminated marine environment.
       Even though the TRL (Technology Readiness Level) of these techniques has yet to rise,
bioremediation with fungi is undoubtedly a cost-effective technology. Microfungi may grow easily
and fast in bioreactors, so they can be directly used in situ during sediment dredging, to remediate
hydrocarbon-polluted waters. Another option is to inoculate solutions containing conidia of the
selected microfungi and periodically turning the soil to enable oxygenation and therefore ease the
colonization of soil by selected strains that make the sediments ready for the colonization of other
less tolerant organisms.




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Greco et al.                                                  Italian Journal of Mycology 49: 101-115 (2020)

DOI: 10.6092/issn.2531-7342/10769



Conclusions

This research shows that a quite diverse microfungal community is present in the water of the Port of
Genoa and that the isolated lignicolous marine-derived fungi are perfectly adapted to the surrounding
organic-polluted environments.
      Among the isolated, P. solitum, G. geotrichum, and the species belonged to Trichoderma genus
are the most common microfungi founded in the investigated port. Thanks to their ability to adapt
to extreme environments, such as those of the port in question, these marine-derived fungi may be
employed in in situ and ex situ sustainable remediation techniques with the aim to degrade persistent
organic contaminants (e.g. hydrocarbon products). However, the degradative potential of the above-
mentioned fungal species, not yet fully investigated, should be studied thorougly, in order to draw up
protocols aimed to allow the mycoremediation of marine polluted port matrices.

Acknowledgements

The authors wish to thank Dr. G. Maurici for the laboratory activities. The research was supported
with internal funds.

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