Zinc tolerance and accumulation capability by <em>Penicillium</em> strains isolated from Neutral Mine Drainage (NDM) colloidal precipitates
10.6092/issn.2531-7342/8651
Lo zinco, in basse concentrazioni, è uno degli oligoelementi essenziali per la vita. Così come molti altri metalli, alte concentrazioni di Zn nell’ambiente risultano tossiche per diversi organismi, compromettendone talvolta il ciclo vitale. Il drenaggio Neutro (o alcalino) di miniera (NMD), caratterizzato da alte concentrazioni di metalli e valori di pH prossimi alla neutralità, è un particolare processo generato dall'alterazione di minerali, spesso di solfuri, (in molti casi Zn e Pb), in ambiente di miniera. Questo fenomeno favorisce la dispersione dei metalli e contribuisce all'aumento della concentrazione di questi elementi tossici nelle acque circolanti minacciando l'ambiente circostante. Oggi è ormai assodata l'efficacia dei funghi (in particolare di ceppi autoctoni) nei processi di biorimedio di metalli ecotossici. Nel nostro lavoro abbiamo studiato la capacità di tolleranza e accumulo di Zn in quattro ceppi di Penicillium (due P. janthinellum, un P. olsonii e un P. waksmanii) isolati da un ambiente soggetto a NMD. I risultati ottenuti hanno mostrato una risposta di crescita positiva di tutti i ceppi in presenza di 750 ppm di Zn, mentre solo due ceppi (P. olsonii e P. waksmanii) sono stati in grado di crescere a concentrazioni pari a 1500 ppm di Zn. Inoltre, ulteriori prove hanno dimostrato una capacità di bioaccumulo del ceppo di P. olsonii fino a 8600 ppm. Questi dati, evidenziando la capacità di bioaccumulo di specie poco studiate in precedenza, sono un'ulteriore conferma della potenziale utilità dei microfunghi autoctoni nel biorisanamento di terreni contenenti metalli pesanti.
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Mirca Zotti
Italian Journal of Mycology
Penicillium olsonii
Zinc tolerance and accumulation capability by <em>Penicillium</em> strains isolated from Neutral Mine Drainage (NDM) colloidal precipitates
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2018-10-25
2018-10-24
2018-01-29
2020-01-20
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Ital J Mycol
Mirca Zotti
2018/10/25
10.6092/issn.2531-7342/8651
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University of Genova
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Penicillium olsonii
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Zinc tolerance and accumulation capability by <em>Penicillium</em> strains isolated from Neutral Mine Drainage (NDM) colloidal precipitates
https://italianmycology.unibo.it/article/download/8651/8401
47
S. Di Piazza, S. Consani, G. Cecchi, C. Carbone, M. Zotti    Italian Journal of Mycology vol. 47 (2018) ISSN 2531-7342
DOI: https://doi.org/10.6092/issn.2531-7342/8651




  Zinc tolerance and accumulation capability by
  Penicillium strains isolated from Neutral Mine
  Drainage (NDM) colloidal precipitates


     Simone Di Piazza, Sirio Consani, Grazia Cecchi, Cristina Carbone, Mirca
                                                        Zotti

  Dipartimento di Scienze della Terra dell’Ambiente e della Vita (DISTAV), Università di Genova, Corso
  Europa 26, 16126 Genova, Italy.

  Correspondig Author e-mail: simone.dipiazza@unige.it

  Abstract

    Zinc, at low concentration, is one of the oligo elements essential for life. As well as many others metals, high
    environmental Zn concentrations are toxic for several organisms and can compromise their lifecycle. Neutral
    (or alkaline) Mine Drainage (NMD) are metal-rich solutions with near-neutral pH values, often generated by
    weathering of sulphide minerals (mainly Zn and Pb sulphides), circulating in mining areas. As this
    phenomenon favours the dispersion of metals and contributes to the rising of concentration of toxic elements in
    the groundwaters, NMD threatens the surrounding environment. The effectiveness of autochthonous fungal
    strains in bioremediation process of ecotoxic metal is today well founded. We investigated the Zn tolerance and
    accumulation capability of four Penicillium strains (two P. janthinellum, one P. olsonii, and one P. waksmanii)
    isolated from an NMD environment. The experimental results showed a positive growth response of all strains
    at 750 ppm of Zn, while only two strains (P. olsonii and P. waksmanii) were able to grow at 1500 ppm of Zn.
    Further tests showed a bioaccumulation capability by P. olsonii strains up to 8600 ppm. These data, reporting
    the bioaccumulation capabilities of fungal species that have not been studied previously, are a further
    confirmation of the potential usefulness of autochthonous microfungi in bioremediation of heavy metals, and
    highlight the need of increase the number of tested strains.

    Keywords: extreme environments; woodwardite; microfungi; mycoextraction; Penicillium olsonii


  Riassunto

    Lo zinco, in basse concentrazioni, è uno degli oligoelementi essenziali per la vita. Così come molti altri metalli,
    alte concentrazioni di Zn nell’ambiente risultano tossiche per diversi organismi, compromettendone talvolta il
    ciclo vitale. Il drenaggio Neutro (o alcalino) di miniera (NMD), caratterizzato da alte concentrazioni di metalli
    e valori di pH prossimi alla neutralità, è un particolare processo generato dall'alterazione di minerali, spesso di
    solfuri, (in molti casi Zn e Pb), in ambiente di miniera. Questo fenomeno favorisce la dispersione dei metalli e
    contribuisce all'aumento della concentrazione di questi elementi tossici nelle acque circolanti minacciando
    l'ambiente circostante. Oggi è ormai assodata l'efficacia dei funghi (in particolare di ceppi autoctoni) nei
    processi di biorimedio di metalli ecotossici. Nel nostro lavoro abbiamo studiato la capacità di tolleranza e
                                                                                                                    55
S. Di Piazza, S. Consani, G. Cecchi, C. Carbone, M. Zotti       Italian Journal of Mycology vol. 47 (2018) ISSN 2531-7342
DOI: https://doi.org/10.6092/issn.2531-7342/8651



       accumulo di Zn in quattro ceppi di Penicillium (due P. janthinellum, un P. olsonii e un P. waksmanii) isolati da
       un ambiente soggetto a NMD. I risultati ottenuti hanno mostrato una risposta di crescita positiva di tutti i ceppi
       in presenza di 750 ppm di Zn, mentre solo due ceppi (P. olsonii e P. waksmanii) sono stati in grado di crescere
       a concentrazioni pari a 1500 ppm di Zn. Inoltre, ulteriori prove hanno dimostrato una capacità di bioaccumulo
       del ceppo di P. olsonii fino a 8600 ppm. Questi dati, evidenziando la capacità di bioaccumulo di specie poco
       studiate in precedenza, sono un'ulteriore conferma della potenziale utilità dei microfunghi autoctoni nel
       biorisanamento di terreni contenenti metalli pesanti.

       Parole chiave: ambienti estremi; woodwardite; microfunghi; micoestrazione; Penicillium olsonii

  1. Introduction

  The weathering of sulphide minerals can generate solutions with low pH values, and a high amount of
  dissolved sulphates and metals of environmental concern. The formation of these acid solutions is
  typical of sulphide ore mining environments, and it is known as Acid Mine Drainage (AMD) (Blowes et
  al., 2003). When mine waters have near neutral pH, the definition of Neutral (or alkaline) Mine
  Drainage (NMD) is applied (Scharer et al., 2000). Although high concentrations of dissolved metals,
  especially Zn and Pb, can be reached in these waters, this phenomenon has received less attention
  than AMD. Nonetheless, these waters represent a threat to the quality of the surrounding aquatic
  system (Iribar, 2004; Frau et al., 2015). In NMD from Cu mines one of the most important secondary
  mineral in the precipitates is woodwardite, (Cu1-xAlx)(OH)2(SO4)x/2·nH2O, a member of the
  hydrotalcites group (Dinelli et al., 1998; Tumiati et al., 2008; Carbone et al., 2013). This mineral forms
  a complete isomorphic series with zincowoodwardite (Witzke and Raade, 2000), thus Cu can be
  substituted by Zn, making woodwardite a good sink for this element. The precipitation of colloidal
  particles generally produces a positive effect on the water quality (Sracek et al., 2011), although
  colloids can be transported for long distances (Schemel et al., 2000), or become a secondary source of
  contaminants (Consani et al., 2017). For these reasons, mine waters and related precipitates have a
  major role in increasing the concentrations of ecotoxic elements in surrounding freshwaters and soils,
  and therefore are a potential risk for the environment and consequently for human health.
  Investigating the capability of some organisms to survive in these inhospitable conditions is an
  intriguing task, since the same organisms could constitute possible agents able to remediate the
  environment. Recently, among the organisms that can survive in polluted areas, fungi have shown a
  huge potential in bioremediation of organic and inorganic pollutants (Gadd, 2010; Aytar et al., 2014;
  Jakubiak et al., 2014; Zotti et al., 2014; Pozdnyakova et al., 2006). In particular, as concern heavy-metal
  mycoremediation (bioremediation using fungi), several fungal strains were tested with various metals,
  such as V (Ceci et al., 2012), Cu (Yap et al., 2011; Wang and Wang, 2013), Ni (Cecchi et al., 2017a), Ag
  (Cecchi et al., 2017b), Pb (Zucconi et al., 2003), and Zn (Fan et al., 2008). Surely, among fungi,
  Penicillium is one of the main and more common genera. This genus includes more than 300
  widespread species, characterised by a wide ecological range. Penicillium species have been isolated
  from various environments and substrata such as soil, freshwater and seawater, food stocks, etc
  (Domsch et al., 2007; Pitt and Hocking, 2009). Owing to this ecophysiological flexibility, Penicillium
  species have been often exploited for industrial and biotechnological purposes (Adrio and Demain,
  2003). For the same reasons, as shown by Leitão (2009), several studies in the last few years have
  highlighted how Penicillium species could be fruitfully exploited for mycoremediation purposes.
  In this context, several studies were carried out in order to assess the microbial flora occurring in AMD
  environments (Colmer and Hinkle, 1947; Das et al., 2009; Bond et al., 2000; Zotti et al., 2014);

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DOI: https://doi.org/10.6092/issn.2531-7342/8651



  conversely, only recently few authors started to study the microbial communities (mainly bacterial) in
  NMD environments (Lindsday et al., 2009; Majzlan et al., 2011; Dockrey et al., 2014).
  Our work investigated the Zn tolerance and accumulation capability of four Penicillium strains
  (belonging to three different species) isolated from a NMD environment. These strains were mostly
  recurrently isolated during a survey (unpublished data) carried out in the Libiola Cu-Fe sulphide mine
  located near the Sestri Levante town (Liguria, North-Western Italy). In this precipitate, Zn is one of the
  major constituents (Carbone et al., 2013), and reaches high concentrations between 0.5% to 2%: for
  this reason we focused our study on this element.
  More specifically, the goal of our work was to verify the in vitro tolerance and bioaccumulation
  capability of native Penicillium strains on two different substrata: greenish-blue colloidal precipitate
  and ZnSO4·7H2O enriched media (MUD-MEA and Zn-MEA). The achieved results propel us to deepen
  our research in order to find the best conditions for exploiting the capability of these strains.

  2. Materials and methods

  2.1. Study area
  The study area was located at the Libiola mine site (lat. 44°17’18’’ N, long. 9° 26’42’’ E), located about 8
  km NE of Sestri Levante (Eastern Liguria, Italy). The mine area extends over an area of about 4 km2
  within the basin of the Gromolo torrent (Fig. 1).




  Fig. 1. Geographical location of the mining area in Italy (bottom right); position of mining area (red dot), nearest
  streams (blue lines), area subject to NMD (dotted area), pictures of two out flow streams and related parameters
  (pH, Eh, Ec).
  Fig. 1. Localizzazione geografica dell’area di miniera rispetto al territorio italiano (in basso a destra); posizione
  dell’area di miniera (punto rosso), dei corsi d’acqua circostanti e dell’area soggetta NMD (area tratteggiata).
  Nelle immagini sono presenti due tratti dei corsi d’acqua con i relativi parametri (pH, Eh, Ec).

  The climate of the area is classified as Mediterranean, is characterised by an average temperature
  during the year of 15 °C (seasonal average 5 °C in Winter, 10.5 °C in Spring, 19 °C in Summer, 13 °C in
  Autumn) and rainfall during the year range between 1100 and 1600 mm/year. The mineralisation is a
  Volcanogenic Massive Sulphide (VMS) deposit occurring, as massive lenses and stockwork mineralised
  veins, within the basalts associated with the Internal Ligurian Ophiolites. Pyrite and chalcopyrite are
  the dominant sulphide minerals in the site, with minor amounts of sphalerite and pyrrhotite, and
  gangue minerals are primarily composed by quartz and chlorite with minor amounts of calcite
  (Zaccarini and Garuti, 2008). The host rocks are mainly represented by basalts, serpentinites, and to a
  lesser extent by ophiolitic breccias (Ferrario and Garuti, 1980). At one site inside the mining area, the
  Margherita adit, the precipitation of greenish-blue precipitates takes place (Dinelli et al., 1998;
  Carbone et al., 2013). Seasonal variations led to pH oscillations from 6.3 to 7.7, and do not affect the
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DOI: https://doi.org/10.6092/issn.2531-7342/8651



  chemical composition of the precipitates, which is always composed mainly by Al, Si, and Cu. The
  amount of Zn varies from 0.5% to 2% (Carbone et al., 2013).
  2.2 Colloidal precipitates sampling
  On the whole five samples of colloidal precipitates by NMD were collected at the Margherita mine adit
  in April 2015. More precisely one sample was used for mineralogical characterization, and the
  remaining four were processed for microfungal isolation. The sampling was performed with a plastic
  syringe, then the solid + liquid suspension was stored in polypropylene bottles, filtered through 16 μm
  filters and air-dried. The pH, Eh and Electrical Conductivity (EC) of related waters were measured in
  the field using a portable pH-metre “PH330i” (WTW, Germany), equipped with “SenTix41” and “SenTix
  ORP” electrodes and a “Eutech XS COND6+” conductivity metre.
  2.3 Isolation of native fungal strains
  Mycological analysis was performed in August 2013. Four samples were collected sucking 50 ml of
  precipitates with a sterile syringe, the samples were put in falcon tubes and stored in a thermal bag at
  6°C until the arrival to the laboratory; then the samples were stored in a refrigerator at 6°C.
  In order to select vial strains, the mycological analysis was performed following a dilution plate
  method modified from Gams (1984), as follows, within 2 days from the sampling.
  The isolation of the strains was performed plating 1 ml of the precipitates both pure and diluted (1:10,
  1:10-2, 1:10-3) on two different media (Rose Bengal Sigma-Aldrich® and Malt Extract Agar added with
  chloramphenicol Sigma-Aldrich®). A total of 16 Petri dishes (9 cm Ø) were inoculated for each sample.
  The dishes were incubated at 24°C for 7 days. Given the high level of toxicity of the matrix, the dishes
  were monitored daily for three more weeks, in order to isolate also the strains whose growth was
  slowed down by the high heavy metal concentrations.
  The strains isolated were identified at genus level by conventional mycological methods by observing
  their micro- and macromorphological features and considering the different trophic and physiological
  requirements. In order to identify species belonging to genus Penicillium, molecular analyses ß-tubulin
  gene was amplified using primers Bt2a and Bt2b (Glass & Donaldson, 1995). The isolated strains were
  conserved in the culture collection of the Mycological Laboratory of DISTAV (University of Genoa,
  Italy).
  2.4 Tolerance and bioaccumulation tests
  Firstly, the ability of the strains to survive and germinate at high Zn concentration was tested on 750
  and 1500 ppm Zn enriched Malt Extract Agar (Zn-MEA750 and Zn-MEA1500). The media were
  prepared by adding 3.706 and 7.412 g of ZnSO4·7H2O, respectively, to a standard MEA (1 L of distilled
  water, 20 g glucose, 20 g malt extract, 1 g peptone, 20 g agar), and autoclaving for 10 minutes at 120°C.
  In order to separate mycelial biomass (essential for subsequent analysis) from the Zn-MEA, a 9 cm
  diam. Cellophane Membrane Backing Biorad® disc was placed in each Petri dish. A conidial suspension
  (5x108 conidia/mL) was obtained by adding 20 ml of deionised water + 10 µL of tween 80 to 14-d-old
  cultures and by scraping the surface, The conidial suspension was then inoculated on Zn-MEA 9 cm
  diam. Petri dishes, and incubated at 24°C for 21 days. The colonies were then separated from the
  media, and analysed with a Tescan Vega scanning electron microscope equipped with an EDS (Energy
  Dispersive Spectrometry) probe (EDAX manufacturer; see paragraph 2.5) to obtain a first evaluation
  of the presence and distribution of Zn in the mycelial biomass.
  The bioaccumulation capability of the strain that showed the higher Zn signal during the EDS analysis
  was tested in vitro. The bioaccumulation test was performed on Zn-MEA750 (see above) and MUD-

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DOI: https://doi.org/10.6092/issn.2531-7342/8651



  MEA (1 L greenish-blue precipitates and water suspension, 20 g glucose, 20 g malt extract, 1 g peptone
  20 agar). As done during the tolerance test, a 9 cm Cellophane Membrane Backing Biorad disc was
  placed in each Petri dish. A 5x108 per ml conidia suspension was prepared as described above. The
  conidial suspension was inoculated on MEA (control), MEA enriched with ZnSO4·7H2O and MEA-Mud
  dishes. The mycelium of 14-days culture was analysed by Tescan Vega scanning electron microscope
  equipped with an EDS (Energy Dispersive Spectrometry) probe in order to verify the heavy metal
  uptake capacity of the mycelium.
  2.5 Instrumental analyses
  The mineralogical characterisation of the natural colloidal precipitate samples was determined by X-
  Ray Diffraction (XRD) with Co Kα radiation (current 20 mA, voltage 40 kV). The sample of colloidal
  precipitates was ground with an agate mortar and pestle, and mounted on zero-background silicon
  plates. The analysis was performed scanning the sample between 5 and 80° 2θ at a scan rate of
  5°/min.
  The major element composition was investigated with Inductively Coupled Plasma-Emission
  Spectrometry ICP-ES on 1 g of sample digested in hot Aqua Regia, while minor and trace elements
  were determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) analysis of a 0.5 g
  samples after modified Aqua Regia digestion at Bureau Veritas Mineral Laboratories (Canada).

  3. Results and discussion

  3.1 Mineralogical characterisation
  The XRD results (Fig. 2) show that the greenish-blue precipitates consist mainly of allophane
  [(Al2O3)(SiO2)1.3-2·2.5-3H2O], an amorphous hydrated Si-Al aluminosilicate, and woodwardite
  [(Cu1-xAlx)(OH)2(SO4)x/2·nH2O] or hydrowodwardite, which differs from woodwardite only for its
  higher water content (Witzke, 1999).




  Fig. 2. XRD diagram of the sample (h-wd= hydrowoodwardite; All= allophane).
  Fig. 2. Diagramma XRD del campione (h-wd= hydrowoodwardite; All= allofane).

  From the bulk chemistry of the sample (Fig. 3) it is evident that the major constituents of the
  precipitates are Al and Si (11.84% and 9.57%, respectively), followed by Cu (8.14%), S (1.37%), and Fe
  (1.16%). The concentration of Zn in the sample is 5.729 mg kg-1.



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DOI: https://doi.org/10.6092/issn.2531-7342/8651



  The colloidal precipitate composition shows a high concentration of several toxic metals (see Fig. 3),
  which are detrimental to living organisms; therefore, this colloidal precipitate can be considered an
  extreme environment.




  Fig. 3. Main chemical compounds of the sample.
  Fig. 3. Principali componenti chimici presenti nel campione.


  3.2 Tolerant/bioaccumulant strains
  As previously reported, we opted to use the native strains to test the Zn tolerance and accumulation
  capability, since these strains are more likely to survive to the high metal concentrations. In previous
  works we displayed that autochthonous microfungi evolved adaptation strategies against limiting
  factors and/or pollutants (Zotti et al., 2014; Cecchi et al., 2017 a,b), and for this reasons are the best
  candidate to show a positive growth response on contaminated media in laboratory test. However, in
  our case, as shown in Table 1, the growth response test (G) performed using the 4 most frequent
  strains present in this NMD area highlight different threshold for different species. Observing the
  Table 1, it is clear that all strains are able to germinate at 750 ppm of Zn, suggesting that the
  propagules of these two species have surely adapted to this environment without completely loosing
  the germination capability. However, only two strains (P. olsonii and P. waksmanii) are able to
  germinate with Zn concentration of 1500 ppm.

  Tab. 1. Positive (+) or negative (-) growth response (G) of the selected strains at 750 and 1500 ppm.
  Tab. 1. Risposta di crescita (G) positiva (+) o negativa (-) dei ceppi saggiati a concentrazioni di 750 e 1500 ppm.


                                                                                                 G         G
         Strain                    Closest match (accession)                     Identities
                                                                                                750       1500
           1       Penicillium janthinellum Biourge (KJ608359.1)                    99%          +         -
           2       Penicillium janthinellum Biourge (DQ486649.1)                    99%          +         -
           3       Penicillium olsonii Bainier & Sartory (AY674443.1)               99%          +         +
           4       Penicillium waksmanii K.M. Zalessky (JQ965078.1)                 99%          +         +



  In spite of the fact that 4 tolerant strains were positive at the 750 ppm G test, and 2 at the 1500 ppm G
  test, the following accumulation screening performed by SEM and EDS probe on the fungal biomass
  samples shows that only P. olsonii biomass grown on Zn-MEA750 is able to accumulate Zn (see Fig. 4
  60
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DOI: https://doi.org/10.6092/issn.2531-7342/8651



  and 5). The SEM image in figure 4 shows a well developped conidiophores of the P. olsonii strain on
  Zn-MEA750; while the results of EDS analysis on the mycelium of the same strain are reported in the
  figure 5 showing a peak of Zn in the right part of the graphic. We must take into account that this EDS
  analysis is a semi quantitative measure, therefore it confirms the presence of at least 1% of Zn in the
  fungal biomass, but cannot provide the exact amount.




  Fig. 4. Scanning Electron Microscope image of a conidiophore of P. olsonii grown on Zn-MEA750.
  Fig. 4. Immagine al microscopio elettronico a scansione di un conidioforo di P. olsonii cresciuto su Zn-MEA750.




  Fig. 5. EDS analysis of conidiophores of P. olsonii grown on Zn-MEA750, in the frame is present the enlargment of
  the Zn peaks.
  Fig. 5. Immagine relativa all’analisi EDS dei conidiofori di P. olsonii cresciti su Zn-MEA750, nel riquadro è
  presente un ingrandimento relativo ai picchi di rilevazione di Zn.

  For this reason the following bioaccumulation tests with a P. olsonii strain on Zn-MEA750 and MUD-
  MEA were carried out to quantify the exact amount of Zn bioabsorbed in the biomass. After the
  incubation period, the ICP-MS analysis on mycelial biomass performed at the ASL Scandinavia AB Lab
  (Luleå, Sweden) confirms, as shown in table 2, the Zn uptake capability of this P. olsonii strain, with a
  concentration of 1520 ppm (from MEA-Mud) and 8630 ppm (from Zn-MEA750).

  Tab. 2. ICP-MS analysis on P. olsonii biomass and related medium on MEA, MEA+ZnSO4·7H2O, and MEA+Mud
  Tab. 2. Analisi ICP-MS del contenuto in Zn nella biomassa di P. olsonii crescito sui differenti terreni (MEA,
  MEA+ZnSO4·7H2O, and MEA+Mud).


                                                                                  Zn
                                Medium                      Sample
                                                                                (mg/kg)
                                MEA               P. olsonii biomass             b.d.l.
                             Zn-MEA1500           P. olsonii biomass             8630
                              MEA-Mud             P. olsonii biomass             1520
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DOI: https://doi.org/10.6092/issn.2531-7342/8651




  Penicillium olsonii is well known as agent of food spoilage, and in the past it has been tested as a
  potential antagonist of the plant pathogen Rhizoctonia solani (Demirci et al., 2011), but till now has not
  been reported in literature to accumulate heavy metals. Our results represent the first report of P.
  olsonii metal tolerance and bioaccumulation capability, and demonstrate that harsh environmental
  conditions make a selection which can favour the evolution of strain-specific strategies in fungi.
  These results invite us to deepen this investigation, in order to increase ì) the number of heavy metal
  tolerant strains ìì) the knowledge of the heavy metals uptake patterns of native filamentous fungi.

  Acknowledgement

  The authors would like to thank Roberto Badano for assistence in XRD analyses, Laura Negretti for
  assistance during XRD analyses and SEM observations, and Carmela Sgrò for assistence during
  mycological analysis.

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