10.6092/issn.2531-7342/18345
Penicillium species research has progressed far beyond their ability to produce secondary metabolites with potential biological applications, particularly as antimicrobial agents. In this work, Penicillium bilaiae EWB-3 was isolated from electronic waste and identified using morphological and molecular (ITS and β-tubulin regions) methods. For 15 days, Penicillium bilaiae EWB-3 was grown into Czapek Yeast Broth using an orbital shaker. Finally, the secondary metabolites in this strain’s filtrates were extracted using ethyl acetate. The agar well diffusion method tested this crude extract for antimicrobial activity. The Penicillium bilaiae EWB-3 extract exhibited strong antimicrobial potential against all tested microorganisms, including Pseudomonas aeruginosa, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans and Aspergillus niger. With diameters of 31, 26, and 25 mm, the largest inhibition zones were observed against C. albicans, S. aureus, and E. faecalis, respectively. The presence of 24 active compounds was revealed by gas chromatography-mass spectrometry (GC-MS) analysis of the crude extract of Penicillium bilaiae EWB-3. The findings suggest that the secondary metabolite extract of Penicillium bilaiae EWB-3 could be a promising active pharmaceutical component.
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Secondary metabolites
Metabolic profiling of antimicrobial secondary metabolites produced by <em>Penicillium bilaiae</em> EWB-3 isolated from electronic wastes in Algeria
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Zhao X, Liu X, Zhao H, Ni Y, Lian Q, Qian H, He B, Liu H, Ma Q (2021) Biological control of Fusarium wilt of sesame by Penicillium bilaiae 47M-1. BioControl 158:104601. https://doi.org/10.1016/j.biocontrol.2021.104601
Metabolic profiling of antimicrobial secondary metabolites produced by <em>Penicillium bilaiae</em> EWB-3 isolated from electronic wastes in Algeria
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                     IJM - Italian Journal of Mycology
                     ISSN 2531-7342 - Vol. 53 (2024): 13-28
                     Journal homepage: https://italianmycology.unibo.it/

Research article


      Metabolic profiling of antimicrobial secondary metabolites
        produced by Penicillium bilaiae EWB-3 isolated from
                     electronic wastes in Algeria
      Dounya Achwak Chemmam1, Ghania Bourzama2, Nouari Sadrati3, Moussa Houhamdi4

1
  Molecular and Cellular Biology Laboratory, Faculty of Nature and Life Sciences, University of Jijel, Jijel, 18000,
Algeria.
2
  Biochemistry and Environmental Toxicology Laboratory, Faculty of Sciences, Badji Mokhtar University, Annaba,
23000, Algeria
3
  Characterization and Valorization of Natural Resources Laboratory, Faculty of Nature and Life and Earth Sciences and
the Universes, University Mohamed El Bachir El Ibrahimi of Bordj Bou Arreridj, 34000, Algeria.
4
  Biology, Water and Environment Laboratory, Faculty of Sciences of Nature and Life and Sciences of the Earth and the
Universe, University 8 Mai 1945, Guelma, 24000, Algeria.

Corresponding author e-mail: chemmam.da@univ-jijel.dz
______________________________________________________________________________

ARTICLE INFO
Received 29/10/2023; accepted 14/12/2023
https://doi.org/10.6092/issn.2531-7342/18345

Abstract
Penicillium species research has progressed far beyond their ability to produce secondary metabolites with potential
biological applications, particularly as antimicrobial agents. In this work, Penicillium bilaiae EWB-3 was isolated from
electronic waste and identified using morphological and molecular (ITS and β-tubulin regions) methods. For 15 days,
Penicillium bilaiae EWB-3 was grown into Czapek Yeast Broth using an orbital shaker. Finally, the secondary
metabolites in this strain’s filtrates were extracted using ethyl acetate. The agar well diffusion method tested this crude
extract for antimicrobial activity. The Penicillium bilaiae EWB-3 extract exhibited strong antimicrobial potential against
all tested microorganisms, including Pseudomonas aeruginosa, Bacillus cereus, Enterococcus faecalis, Escherichia coli,
Salmonella typhimurium, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae,
Candida albicans and Aspergillus niger. With diameters of 31, 26, and 25 mm, the largest inhibition zones were observed
against C. albicans, S. aureus, and E. faecalis, respectively. The presence of 24 active compounds was revealed by gas
chromatography-mass spectrometry (GC-MS) analysis of the crude extract of Penicillium bilaiae EWB-3. The findings
suggest that the secondary metabolite extract of Penicillium bilaiae EWB-3 could be a promising active pharmaceutical
component.

Keywords
Antimicrobial activity, electronic waste, GC-MS, Penicillium bilaiae, secondary metabolites.

________________________________________________________________________________



                                                      ______________
    Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
    The journal is hosted and maintained by AlmaDL. © 2024 The Authors. This work is licensed under CC BY 4.0
Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
https://doi.org/10.6092/issn.2531-7342/181345
___________________________________________________________________________________________________________________________________________________

Introduction

Penicillium is one of the largest and most well-known fungal genera (Ali Shah et al., 2022).
Secondary metabolites identified in this genus with various structural properties have piqued the
interest of the pharmaceutical industry and mycologists, beginning with the discovery of penicillin
and progressing to all of the different synthetically derived drugs currently in use (Omeike et al.,
2019). Therefore, the role of Penicillium species as suppliers of bioactive compounds for medical use
is crucial (Ashtekar et al., 2022). Studies have reported that industrial waste from current production
processes can produce bioactive metabolites (Stierle and Stierle, 2014). It is possible to find novel
antibiotics by exploring environments that have not yet been explored (Velasco-Rodríguez et al.,
2021). The discovery of various commercially available drugs derived from extreme habitats such as
salt mines, marine habitats and compost has elevated the importance of microbial research (Yadav et
al., 2018; Kour et al., 2019; Abdel-Razek et al., 2020). Nature offers a broad spectrum of structurally
distinct secondary metabolites. These structural differences reflect many biological activities (Yadav
et al., 2020). Natural products can be arranged into multiple classes, terpenoids and steroids, fatty-
acid-derived substances and polyketides, alkaloids, and nonribosomal polypeptides (Vemireddy et
al., 2020). Developing microbial resistance against common antibacterial, antifungal, and antiviral
medications is a major threat to human health (Hashem et al., 2021). Therefore, research of new
bioactive compounds with antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral,
cytotoxic, antitumoral and immunomodulatory activity through several pathogen-killing ways is
necessary (Toghueo and Boyom, 2020). The screening procedure for producing secondary
metabolites from filamentous fungi, which have already shown interesting biological activity, has
been the subject of a few studies in Algeria. Penicillium bilaiae Chalab. is frequently used as a seed
inoculant to improve phosphorus efficiency in various crops (Raymond et al., 2018). Thus,
discovering new natural compounds from this filamentous fungus piqued our interest as a research
topic. This study aims to isolate Penicillium spp., and in particular P. bilaiae, from new resources as
electronic wastes that can produce the bioactive secondary metabolites and identify them by gas
chromatography-mass spectrophotometry (GC-MS), evaluating their antimicrobial activity against
human pathogens.


Materials and Methods

Sampling
In 2021, electronic samples were aseptically collected from a mixture of batteries and printed circuit
boards waste at a depth of 10 cm below the surface of waste storage tanks in the Bordj-Bou Arreridj
region (Algeria). These samples were immediately transferred to the laboratory for analysis.

Isolation of fungal strain
The fungal strain was isolated from samples electronic waste under aseptic conditions on Potato
Dextrose Agar (PDA) plates using a standard serial dilution plating technique. Ten g of a mixture of
batteries and printed circuit boards waste were washed using sterile distilled water, then stirred in 90
ml of sterile physiological water for 45 min. Serial dilutions from the supernatant were prepared until
103. One hundred µl were put on Petri plates containing PDA. In 2021, electronic samples were
aseptically collected from a mixture of batteries and printed circuit boards waste at a depth of 10 cm

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 below the surface of waste storage tanks in the Bordj-Bou Arreridj region (Algeria). These samples
were immediately transferred to the laboratory for analysis.
        The plates were incubated at 28 °C for 7 days during repeated sub-culturing, and distinct
isolates were purified. The isolated strains were then transferred in plates filled with five different
culture media: Malt Extract Agar (MEA), Sabouraud Dextrose Agar (SDA), Czapek Yeast Agar
(CYA), and Oxytetracyclin-Glucose-Agar (OGA). After three days, the morphological characteristics
of filamentous fungal colonies, including spore morphology, colony colors, and hyphal patterns, were
observed. Based on their characteristics, macroscopic and microscopic slides were used to identify
and select Penicillium isolates. Pure fungal isolates were stored at 4 °C for further examination
(Yadav et al., 2018).

Molecular analyses
DNA extraction, PCR amplification and sequencing
Genomic DNA extraction from a selected fungal culture growing on PDA was performed using a
NucleoSpin Plant II DNA isolation kit (Macherey-Nagel, Germany), following the manufacturer’s
instructions. The PCR amplification of Internal Transcribed Spacer (ITS) region of the fungal genome
and 5.8S ribosomal genes was performed using the universal primers ITS1F (5’ CTTGGTCATTTA
GAGGAAGTAA 3’) and ITS4 (5’ TCCTCCGCTTATTGATATGC 3’) and the primers BT2A
(5’GGTAACCAAATCGGTGCTGCTTTC 3’), BT2B (5’ACCCTCAGTGTAGTGACCCTTGGC
3’) for the β-tubulin gene. In total, 25 µl of the reaction mixture was used to amplify all fragments,
including 5 µl of Taq Buffer (Solis Biodyne). One and a half µl of 25 mM MgCl2, 0.2 µl of 25 mM
dNTP, 1 µl of each primer (ITS1F and ITS4), 2 µl of genomic DNA, 0.2 µl of 5 U µl-1 Taq polymerase
(Solis Biodyne) and ultra-pure water (Kumar et al., 2016).
   The thermal cycle program starts at 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 55
°C for 30 s, 72 °C for 45 s and a final extension at 72 °C for 7 min. The PCR conditions for the β-
tubulin region are 5 min at 95 °C for pre-denaturation, followed by 35 cycles at 95 °C for 30 s, 30 s
at 60 °C, 72 °C for 45 s and a final extension at 72 °C for 7 min (Guo et al., 2020). The amplified
PCR products were separated by electrophoresis and then purified by NucleoSpin® Gel and PCR
Clean-up from Macherey-Nagel (Germany) Mini kit. The Sanger technique sequenced the purified
PCR products (Sanger et al., 1977) using the BigDye v3.1 kit from Applied Biosystems and the PCR
primers used to amplify fragments of interest. The obtained sequences were analyzed and cleaned
using the CHROMAS PRO software. Then the final sequences were compared with those of the
GenBank database by BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgiBlast) for the identification
of studied isolates based on the percentage homology with reference strains.

Phylogenetic analysis
The ITS and β-tubulin sequences of EWB-3 isolate and those of phylogenetically related species
obtained from NCBI were aligned using a muscle algorithm in MEGA7 software to generate the
phylogenetic trees. The obtained individual sequences, including ITS and β-tubulin, and the
concatenated sequences were subjected to maximum likelihood analyses. The analyses were
conducted in MEGA7 software with 1,000 replicates of the Tamura-Nei model. The trees were rooted
with Penicillium levitum CBS: 345.48.




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Fermentation and extraction of secondary metabolites
The cultures in shake flasks were performed in 500 ml Erlenmeyer flasks containing 200 ml Czapek’s
liquid medium by inoculating agar blocs of actively growing pure culture (6 mm in diameter) of the
selected fungal strain. The cultures were incubated at 28 °C at 150 rpm on a rotary shaker for 15 days
for the highest production. The Czapek medium composed of (g L-1): NaNO3, 0.3 g; K2HPO4, 0.1 g;
KCl, 0.05 g; MgSO4, 7H2O, 0.05 g; FeSO4 7H2O, 0.001 g; sucrose, 3.0 g; Yeast Extract, 5.0 g (Pitt,
1979). After incubation, the cultures were filtered through Whatman paper N°1 to separate the culture
filtrate and mycelial material. The filtrate was extracted three times with an equal volume of ethyl
acetate (EtOAc) using a separating funnel. Combined ethyl acetate extract evaporated at 40 °C by
rotary evaporator (BUCHI R-100). The crude extracts were then dissolved in Dimethyl Sulfoxide
(DMSO) and stored at at 4 °C for further analysis (Lykholat et al., 2021; Nischitha and Shivanna,
2021).

In vitro antimicrobial activity
Agar well diffusion method
Antimicrobial activity of crude ethyl acetate extract was performed by agar well diffusion method
described by Fatima et al. (2016) against ten pathogenic bacterial and fungal strains Klebsiella
pneumoniae ATCC 70063, Enterococcus faecalis ATCC 29212, Methicillin-Resistant
Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 29522, Salmonella
typhimurium ATCC 13311, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC
43306, Bacillus cereus ATCC, Aspergillus niger CTM 10099 and Candida albicans ATCC 10231.
Bacterial and fungal suspensions were adjusted to a final concentration of 108 CFU ml-1 and 106 CFU
ml-1, respectively. One hundred μl of each bacterial and fungal suspension were spread on Mueller-
Hinton agar and Sabouraud chloramphenicol agar plates, respectively. Then, wells with a diameter
of 6 mm were created, and 20 μl of the metabolic extract was added. Dimethyl sulfoxide (DMSO)
was used as a negative control. Subsequently, the plates were incubated for 24 h at 37 °C for bacteria
and 48 h at 28 °C for fungi. All the assays were conducted in triplicate. The antibacterial and
antifungal activities were evaluated by measuring the diameter of inhibition zones (mm).

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicide Concentration
(MFC)
The minimal inhibitory concentration was established using the 96-well broth microdilution
technique (Pierce et al., 2008). The final concentrations of tested ethyl acetate extract varied between
(90 - 45 - 22.5 - 11.2 - 5.6 - 2.8 - 1.4 - 0.7 - 0.3 µg ml-1), and the bacterial and fungal suspension was
adjusted to 0.5 McFarland. The Gentamycin (40 µg ml-1) and Fluconazole (50 µg ml-1) antimicrobial
agents were used as controls, and the indicator strains were used for antimicrobial assays. Each well
was inoculated with 90 µl of different ethyl acetate crude extract concentrations and 10 µl of bacterial
and fungal suspensions. After incubation at 37 °C for 24 h for bacterial strains and 48 h for fungal
strains, the presence or absence of growth was observed with the resazurin fluorometric/colorimetric
assay by addition of 20 µl of resazurin to each well (Eloff, 2019). The MIC was defined as the lowest
concentration of ethyl acetate crude extract without any microbial growth.




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Characterization of compounds
Gas chromatography-mass spectrometry (GC-MS) analysis of bioactive metabolites
The protocol described by Lykholat et al. (2021), with some modifications, was used to search for
volatile metabolites. GC-MS (Thermo Fisher Scientific) was used to analyze the biologically active
fungal crude ethyl acetate extract, and the sample was derivatized to impart volatility to the
compounds for maximum detection. The derivatization procedure was realized by adding 50 μl of a
sample extract (20% in ethyl acetate) was placed into a 2 ml glass vial and treated sequentially with
100 μl of acetonitrile and 100 μl of NO-Bis (trimethylsilyl)trifluoroacetamide (BSTFA). The sample
was shaken at room temperature for 1 min then the vial was heated at 60 °C for 30 min. The resulting
solution is analyzed directly by capillary gas chromatography connected to a mass-spectrometry
assay. GC-MS analysis of the extracts was performed using a Thermo Fisher ZB-5 MS column
(length 30 m × 0.25 mm, film thickness 0.25 μm) containing 5% diphenyl/ 95% dimethyl polysiloxane
as a fixed liquid phase. The column temperature was maintained at 50 °C for 5 min. After that, the
temperature gradient was programmed to rise by 10 °C each minute, achieving 300 °C and
maintaining there for 10 min. The carrier gas helium passed at a 20 ml/min flow rate. Injection volume
1 µl was employed (split ratio 20%); the injector temperature was 280 °C.

Identification of compounds
The National Institute of Standards and Technology (NIST) database was used to interpret the GC-
MS mass spectrum. The obtained spectrum was compared to the spectrum of the database’s standard
component. Each compound’s name, retention time, and chemical structure were also determined.

Statistical analysis
The data were analyzed using Microsoft Excel 2013 and were expressed as mean ± SD value.

Results

Isolation and identification of the fungal strain
Based on morphological characteristics, one of the isolated fungal colonies was identified as
Penicillium sp. from preliminary screening of electronic waste. After 10 days at 28 °C, this isolate
grew on various growth media, including PDA, OGA, CYA, MEA, and SDA, as shown in Figure 1.
On most culture media, this isolate grew at a similar rate, with colony diameters ranging from 22 to
30 mm. However, its colonial morphology was significantly different, with colonies on PDA
appearing green with a white outline, mycelium on CYA, OGA, and MEA becoming green with
yellow centers, covered with yellow exudates for MEA with age, and mycelium on SDA appearing
green with a white center. A diffusion of yellow pigment around colonies was observed in all culture
media.
After sequencing and correction, the blast of the obtained sequences from ITS and β-tubulin regions
against the previously archived sequences in the NCBI databases revealed that both sequences had a
similarity percentage of 98% with those of the P. bilaiae strain NRRL 3391. A total of 34 ITS and β-
tubulin sequences of the closely related species to the sequences of EWB-3 isolate downloaded from
NCBI, one sequence from this study and another sequence as an outgroup, were used for the
paleogenetic analyses and to construct the trees. The Maximum likelihood phylogenetic analysis
results indicate that fungal isolate EWB-3 is classified within the Penicillium genus, specifically in

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section Sclerotiorum, series Adametziorum. Phylogenetic trees created using ITS sequences, β-
tubulin, and concatenated data generally exhibit identical topologies. The EWB-3 isolate is positioned
among the other species in the same section and has the closest phylogenetic affinity to P. bilaiae,
supported by very high bootstrap values of 100% (ITS tree), 99% (β-tubulin tree) and 98%
(concatenated tree) (Figs. 2-4). The internal transcribed spacer (ITS) and β-tubulin sequences of the
isolate EWB-3, was deposited to the GenBank with accessions numbers: ITS: OR225221, β-tubulin:
OR243415.




                     Fig. 1 – Morphological characteristics of EWB-3 isolate on different growth media
                     after 10 days at 28°C: conidiophores (a-c), conidia (d-e). Scale bars: a, c, = 250 µm;
                     b = 500 µm; d, e = 10 µm.

                                                     91             Penicillium restingae 43M6
                                                            Penicillium adametzii CBS 209.28
                                                    99 Penicillium amaliae CBS 134209
                                                         Penicillium lilacinoechinulatum CBS 454.93
                                                                      Penicillium jugoslavicum CBS 192.87
                                                           72                      EWB-3                                  ser. Adametziorum
                                                                         100 Penicillium bilaiae NRRL 3391

                                                                                    Penicillium arianeae CBS 134559
                                                   51                           Penicillium brocae CBS 116113
                                                                    Penicillium angulare CBS 130293
                                                        94                   Penicillium adametzioides CBS 313.59
                                                                                                           Penicillium mellis CBS 142499
                                                   58     Penicillium choerospondiatis HMAS248813
                                                       Penicillium sanshaense HMAS248820
                                                                                                                                              section Sclerotiorum




                                                 57
                                                      Penicillium verrucisporum HMAS248819                            ser. Herqueorum
                                                99
                                                      Penicillium herquei CBS 336.48
                                                    Penicillium malachiteum CBS 647.95
                                                              Penicillium hirayamae CBS 229.60
                                                        55 Penicillium acidum EML-DLW4-1
                                                                Penicillium johnkrugii DAOM 239943
                                                           Penicillium exsudans HMAS248735
                                                           Penicillium mallochii DAOM 239917
                                                            Penicillium vanoranjei DTO99H6
                                                       90
                                                             Penicillium vanoranjei CBS 134406
                                                                   Penicillium sclerotiorum NRRL 2074                  ser. Sclerotiorum
                                                                     Penicillium cainii DAOM 239914
                                                                 64
                                                                           Penicillium daejeonium KACC 46609
                                                                       Penicillium jacksonii DAOM 239937
                                                                  Penicillium guanacastense DAOM 239912
                                                                 Penicillium viticola FKI-4410
                                                                        Penicillium maximae NRRL2060
                                                                     99 Penicillium austrosinicum HMAS248734

                                                         Penicillium alexiae CBS H-21142
                                                    Penicillium levitum CBS:345.48
                                                                                                                           ser. Herqueorum
                                         0.02


                          Fig. 2 – Maximum-likelihood phylogenetic tree inferred from the data set of
                          ITS locus for Penicillium section Sclerotiora. The Bootstrap values are greater
                          than 50%. The phylogram is rooted with Penicillium levitum CBS: 345.48.

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                                                               84               Penicillium restingae 43M6
                                                                             Penicillium adametzii CBS 209.28
                                                                              Penicillium amaliae CBS 134209
                                                                  96      Penicillium lilacinoechinulatum CBS 454.93
                                                                          Penicillium arianeae CBS 134559
                                                                                Penicillium brocae CBS 116113
                                                                         98        Penicillium adametzioides CBS 313.59
                                                                                                                              ser. Adametziorum
                                                                                 Penicillium angulare CBS 130293
                                                                 51                  EWB-3
                                                 52                        99 Penicillium bilaiae NRRL 3391

                                                                             Penicillium jugoslavicum CBS 192.87
                                                                                         Penicillium alexiae CBS H-21142
                                     51
                                                                Penicillium reconvexovelosoi CCDCA 11500
                                                         96             Penicillium mellis CBS 142499




                                                                                                                                                  Section Sclerotiorum
                                                          Penicillium sanshaense HMAS248820
                                                   92
                                                                    Penicillium   verrucisporum HMAS248819
                                                       68
                                                                   Penicillium choerospondiatis HMAS248813                    ser. Herqueorum
                                                                       Penicillium herquei CBS 336.48
                                                                    Penicillium malachiteum CBS 647.95
                                                        Penicillium hirayamae CBS 229.60
                                                               98 Penicillium austrosinicum HMAS248734
                                                                        Penicillium maximae NRRL2060
                                          72                      Penicillium circulare CNUFC-GEU220-1
                                                                       Penicillium cainii DAOM 239914
                                                              Penicillium jacksonii DAOM 239937
                                                    64
                                                                   Penicillium guanacastense DAOM 239912
                                                               Penicillium viticola FKI-4410
                                                                                                                              ser. Sclerotiorum
                                                              Penicillium daejeonium KACC 46609
                                                       58           Penicillium sclerotiorum NRRL 2074
                                                                99 Penicillium vanoranjei CBS 134406
                                                          76        Penicillium vanoranjei DTO99H6
                                                                   92       Penicillium mallochii DAOM 239917
                                                             94
                                                                        Penicillium johnkrugii DAOM 239943
                                                                 99       Penicillium acidum EML-DLW4-1
                                                                     98 Penicillium exsudans HMAS248735

                                                                                             Penicillium levitum CBS:345.48

                                          0.05


                          Fig. 3 – Maximum-likelihood phylogenetic tree inferred from the data set of
                          BenA locus for Penicillium section Sclerotiora. The Bootstrap values are
                          greater than 50%. The phylogram is rooted with Penicillium levitum
                          CBS:345.48.


                                                               84               Penicillium restingae 43M6
                                                                             Penicillium adametzii CBS 209.28
                                                                              Penicillium amaliae CBS 134209
                                                                  96      Penicillium lilacinoechinulatum CBS 454.93
                                                                          Penicillium arianeae CBS 134559
                                                                                Penicillium brocae CBS 116113
                                                                         98        Penicillium adametzioides CBS 313.59
                                                                                                                              ser. Adametziorum
                                                                                 Penicillium angulare CBS 130293
                                                                 51                  EWB-3
                                                 52                        99 Penicillium bilaiae NRRL 3391

                                                                             Penicillium jugoslavicum CBS 192.87
                                                                                         Penicillium alexiae CBS H-21142
                                     51
                                                                Penicillium reconvexovelosoi CCDCA 11500
                                                         96             Penicillium mellis CBS 142499
                                                                                                                                                  Section Sclerotiorum




                                                          Penicillium sanshaense HMAS248820
                                                   92
                                                                    Penicillium verrucisporum HMAS248819
                                                       68
                                                                   Penicillium choerospondiatis HMAS248813                    ser. Herqueorum
                                                                       Penicillium herquei CBS 336.48
                                                                    Penicillium malachiteum CBS 647.95
                                                        Penicillium hirayamae CBS 229.60
                                                               98 Penicillium austrosinicum HMAS248734
                                                                        Penicillium maximae NRRL2060
                                          72                      Penicillium circulare CNUFC-GEU220-1
                                                                       Penicillium cainii DAOM 239914
                                                              Penicillium jacksonii DAOM 239937
                                                    64
                                                                   Penicillium guanacastense DAOM 239912
                                                               Penicillium viticola FKI-4410
                                                                                                                              ser. Sclerotiorum
                                                              Penicillium daejeonium KACC 46609
                                                       58           Penicillium sclerotiorum NRRL 2074
                                                                99 Penicillium vanoranjei CBS 134406
                                                          76        Penicillium vanoranjei DTO99H6
                                                                   92       Penicillium mallochii DAOM 239917
                                                             94
                                                                        Penicillium johnkrugii DAOM 239943
                                                                 99       Penicillium acidum EML-DLW4-1
                                                                     98 Penicillium exsudans HMAS248735

                                                                                             Penicillium levitum CBS:345.48

                                          0.05


                          Fig. 4 – Maximum-likelihood phylogenetic tree inferred from the ITS and
                          BenA loci combined data set for Penicillium section Sclerotiora. The
                          Bootstrap values are greater than 50%. The phylogram is rooted with
                          Penicillium levitum CBS:345.48.

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Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
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In vitro antimicrobial activity (well diffusion method)

The crude ethyl acetate extract exhibited high antibacterial and antifungal activities by inhibiting
many pathogens (Table 1). A strong antimicrobial activity against B. cereus ATCC 14579, E. faecalis
ATCC 29212, P. aeruginosa ATCC 27853, S. aureus ATCC 43306, C. albicans ATCC 10231 can
be noted, as well as a strong activity against E. coli ATCC 29522, K. pneumoniae ATCC 70063,
MRSA ATCC 43300 and A. niger CTM 10099. On the other hand, the fungal extract displayed a
weak zone for S. typhimurium ATCC 13311of only 11 mm. The inhibition zones varied from 11 to
31 mm in diameter, as shown in Figure 5. The largest inhibition zone was observed against C.
albicans, B.cereus and S. aureus, with 31, 26, and 25 mm, respectively.




        Fig. 5 – Antimicrobial activity of ethyl acetate extract against Escherichia coli (a), Pseudomonas
        aeruginosa (b), Klebsiella pneumoniae (c), Enterococcus faecalis (d), Staphylococcus aureus (e),
        Methicillin-Resistant Staphylococcus aureus (f), Salmonella typhimurium (g), Bacillus cereus (h), Candida
        albicans (i), Aspergillus niger (j).

  Table 1 - Inhibition diameters of antimicrobial activity of ethyl acetate extract against the tested microorganisms.
   Microbial strains                                         Zones of inhibition (mm) ± SD          Negative control
                                                                                                    DMSO
   Escherichia coli ATCC 29522                               17 ± 2.52                              00 ± 00

   Pseudomonas aeruginosa ATCC 27853                                     23 ± 2.08                                    00 ± 00

   Klebsiella pneumoniae ATCC 70063                                      18 ± 2.08                                    00 ± 00

   Salmonella typhimurium ATCC 13311                                     11 ± 1.15                                    00 ± 00

   Enterococcus faecalis ATCC 29212                                      24 ± 5.13                                    00 ± 00

   Bacillus cereus ATCC 14579                                            26 ± 1.11                                    00 ± 00

   Staphylococcus aureus ATCC 43306                                      25 ± 3.46                                    00 ± 00

   Methicillin-Resistant Staphylococcus aureus                           19 ± 1.15                                    00 ± 00
   (MRSA) ATCC 43300
   Candida albicans ATCC 10231                                           31 ± 3.00                                    00 ± 00

   Aspergillus niger CTM 10099                                           13 ± 1.53                                    00 ± 00




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Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
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Table 2 - Minimum Inhibitory Concentration (µg ml-1) of ethyl acetate crude extract.

                                                                    Human bacterial pathogens

                         E.           P.                  K.                    S.                 E.            B.           S.
   Crude ethyl                                                                                                                           MRSA
                        coli      aeruginosa          pneumoniae           typhimurium          faecalis       cereus       aureus
  acetate extract
                        5.62            90                 22.5                  90                 45          1.40         1.40         0.35

MRSA: Methicillin-Resistant Staphylococcus aureus.

   Table 3 - Minimum Fungicide Concentration (MFC µg/ml) of ethyl acetate crude extract.
                                                                     Human Fungal pathogens

       Crude ethyl                               C. albicans                                                  A. niger
      acetate extract
                                                     11.25                                                      5.62


Characterization using Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS analysis provides a representative spectrum of each metabolite in the tested sample. The GC-
MS chromatograms (Supplementary Fig. S1) show that the fungal isolate’s crude ethyl acetate extract
contains the compounds listed in Table 4, their name, retention time (RT), probability %, chemical
structure and formula. In total, 24 different metabolites were identified.

  Table 4 - The detected compounds through GC-MS in a crude extract of Penicillium bilaiae EWB-3.
                                  Retention
    No      Compound name                      Probability %            Chemical structure          Formula
                                 Time (min)
             Ethylene glycol,                                                                     C8H22O2Si2
     1                                9.29          25.39
             2TMS derivative
             Propylene glycol,                                                                     C9H24O2Si2
     2                                9.58          53.88
             2TMS derivative
                                                                                                  C10H26O2Si2
              2.3-Butanediol,
     3                               10.40          51.51
             2TMS derivative

                                                                                                                                C9H22O3Si2
               Lactic acid, 2TMS
      4                                         10.78                  87.10
                   derivative

                                                                                                                                 C8H12O3Si
               3-Furoic acid, TMS
      5                                         12.13                  20.71
                   derivative

                Hydracrylic acid,                                                                                               C9H22O3Si2
      6                                         12.28                  89.33
                2TMS derivative
                                                                                                                                 C10H16OSi
             Benzyl alcohol, TMS
      7                                         12.45                  22.00
                 derivative
                                                                                                                               C10H24O3Si2
                 Propanoic acid,
      8                                         12.54                  92.38
                2TMS derivative
              3-Hydroxyisovaleric                                                                                              C11H26O3Si2
      9          acid, 2TMS                     13.32                  80.67
                  derivative

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Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
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                                                                                                                                C10H22O4Si2
                Butanedioic acid,
     10                                         14.90                  88.93
                2TMS derivative

                                                                                                                               C10H20O4Si2
               2-Butanedoic acid,
     11                                         15.40                  88.49
             (E)-, 2TMS derivative
               2.3-Dihydroxy-2-                                                                                                 C14H34O4Si3
     12       methylbutanoic acid,              15.71                  97.70
                3TMS derivative
              ®-2.3-Dihydroxy-3-                                                                                                C14H34O4Si3
     13       methylbutyric acid,               15.94                  65.12
                3TMS derivative
             Butanoic acid, 2,4-                                                                                               C13H32O4Si3
             bis[(trimethylsilyl)ox
     14      y]-,     trimethylsilyl            16.20                  44.43
             ester

                                                                                                                                C13H24O2Si2
                 Orcinol, 2TMS
     15                                         16.68                  77.25
                   derivative

                                                                                                                                C15H36O4Si3
              2.3-Dihydroxy-3-
     16      methylpentanoic acid,              16.95                  83.24
               3TMS derivative
                                                                                                                                C13H30O5Si3
               Malic acid, 3TMS
     17                                         17.19                  96.91
                  derivative

                                                                                                                                C16H42O4Si4
                Erythritol, 4TMS
     18                                         17.40                  45.18
                   derivative

                                                                                                                               C14H32O5Si3
             Pentanedioic acid, 2-
     19      [(trimethylsilyl)oxy]-,            18.27                  93.34
             bis(trimethylsilyl)
             ester
                                                                                                                                C15H30O6Si3
              Aconitic acid, 3TMS
     20                                         20.28                  54.10
                  derivative

             D-(-)-Fructofuranose,                                                                                              C21H52O6Si5
     21        pentakis, 5TMS                   20.92                  18.92
                   derivative
                                                                                                                                C18H40O7Si5
                Citric acid, 4TMS
     22                                         21.00                  36.02
                    derivative
                                                                                                                                C19H38O5Si4
               Gallic acid, 4TMS
     23                                         22.43                  90.50
                   derivative

               Palmitic acid, TMS                                                                                               C19H40O2Si
     24                                         23.37                  64.62
                   derivative




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Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
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Discussion

Penicillium bilaiae EWB-3 was isolated from electronic waste for the first time in Algeria. The ability
of this EWB-3 isolate to produce bioactive compounds was evaluated. Natural antimicrobial
compounds derived from extremophile fungi are safer for human health and food preservation than
synthetic antimicrobials (Akbar et al., 2019). Penicillium bilaiae EWB-3 showed a very important
antimicrobial activity against all tested pathogens. The findings from previous studies support the
genus Penicillium’s ability to produce a wide variety of secondary metabolites (Toghueo and Boyom,
2020). Earlier studies have employed P. bilaiae for the bioremediation of metal-contaminated soil
(Arwidsson et al., 2010). Moreover, the phosphorus-solubilizing fungus has been used as an inoculant
to promote plant growth (Hansen et al., 2020). Meng et al. (2014) investigated some metabolic
compounds of P. bilaiae MA-267 isolated from the rhizospheric soil of a mangrove plant with high
activity against the plant pathogenic fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.
The current study is considered a first report where P. bilaiae EWB-3 was isolated from an unusual
source, electronic waste, and its characterized metabolites exhibited a high activity against all tested
microorganisms known as human pathogens.
       The media composition influences secondary metabolite profiles. The yeast extract containing
czapek yeast medium is regarded as a secondary metabolite-producing medium (Frisvad, 2012). The
most common organic solvent employed to extract fungal secondary metabolites is ethyl acetate
(EtOAc) with medium polarity extraction (Nawaz et al., 2020). Data from the literature confirm that
the antibacterial effects of fungal extracts are related to the extraction solvent (Ben Mefteh et al.,
2018). Further, Al-Saleem et al. (2022) reported an antimicrobial activity of ethyl acetate extract from
P. chrysogenum against S. aureus with a MIC value of 250 µg ml-1. The authors also observed
fungicidal activity against C. albicans and Cryptococcus neoformans. According to Kumari et al.
(2021), the antibacterial activity of Penicillium citrinum Thom of ethyl acetate extract displayed a
strong activity against human pathogenic bacteria, including S. aureus, Bacillus subtilis, E. coli,
Shigella boydii, Vibrio cholera.
       Some other works affirmed the antibacterial activity of the crude extract of Penicillium
griseofulvum Dierckx (Zerroug et al., 2018) and Penicillium brevicompactum Dierckx ANT13
(Sadrati et al., 2023) against pathogenic bacteria with the maximal activity against E. coli (45.5 mm)
and S. aureus ATCC 25923 (45.5 mm) respectively. Omeike et al. (2019) also reported a potent
activity of P. citrinum ethyl acetate extract with 16 mm of inhibition zone against Klebsiella
peneumoniae. Nischitha and Shivanna (2021) studied the antimicrobial activity of the ethyl acetate
extract of Penicillium pinophilum Hedgc. The researchers obtained good activity for all tested bacteria
except S. aureus and P. aeruginosa. Pan et al. (2017) indicated the potent antibacterial activity of
Penicillium sp. ethyl acetate extract against S. aureus and B. subtilis. Comparatively with our work,
the results revealed that ethyl acetate extract of P. bilaiae EWB-3 inhibited both Gram-positive and
Gram-negative bacteria tested, such as S. aureus, B. cereus and P. aeruginosa with an important
inhibition zone 25 mm and 23 mm, respectively. The antimicrobial activity of these Penicillium spp.
may be explained by the presence of chemical compounds that can inhibit microbial growth.
       Penicillium bilaiae EWB-3 produces a wide range of chemical compounds belonging to
different classes, organic acids, esters, fatty acids and alcohols that appeared during GC-MS analysis.
The most abundant compounds in the fungal extract are 2,3-butanidiol, lactic acid, 3-furoic acid,
propanoic acid, 2-butanedioic acid, malic acid, pentanedioic acid, D-(-)-fructofuranose, 3-

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Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
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___________________________________________________________________________________________________________________________________________________

hydroxyphenylacetic acid, gallic acid, palmitic acid. These compounds have different biological
properties, such as antifungal and antibacterial activities. This study is the first in Algeria to target
the biomolecules produced by this isolate. Research carried out about P. bilaiae has reported the
crucial role of this fungus in dissolving insoluble forms of inorganic phosphorus (Hansen et al., 2020;
Zhao et al., 2021) and highlighted the utility of using P. bilaiae as an inoculant to promote plant
growth or phosphorus uptake in various crops. This process may release organic acids for phosphorus
mobilization and promote plant growth.
        Arwidsson et al. (2010) reported that P. bilaiae could produce organic acids such as citric acid,
oxalic acid, and acetic and formic acid for bioremediation in metal-contaminated soils. In this study,
we characterized unusual and interesting organics compounds isolated for the first time from this
fungus and having potent activity against all tested pathogenic human microorganisms, such as 3-
hydroxyisovaleric acid, pinosylvin, 3-furoic acid, palmitic acid and 2,3-dihydroxy-2-methylbutanoic
acid. On the other hand, work on P. bilaiae in co-culture with P. chermesinum carried out by Meng
et al. (2020) detected new bioactive meroterpenoids in chermebilaenes A and B. The obtained
compounds were tested against the human and aqua-pathogenic bacteria. Only 9, 12-octadecadienoic
acid showed moderate activity against the tested pathogens. Nakahara et al. (2004) isolated a new
acetylenic with nematicidal activity from P. bilaiae against Pratylenchus penetrans. Our results are
according to Lykholat et al. (2021), who analyzed different compounds from ethyl acetate extract
from Penicillium sp., the same compounds using GC-MS analysis were noticed, propanoic acid, 2-
butanoic acid, and pentanedioic acid also known as glutaric acid. In addition, different studies confirm
what we found about the antimicrobial activity of malic acid (Agoramoorthy et al., 2007), 3-furoic
acid (Liu et al., 2012), ethylene glycol (Gurtler and Mai, 2014) and palmitic acid (Zhang et al., 2020).
        Furthermore, 3-hydroxyisolvaleric acid, present in our work, was also identified by Menezes et
al. (2022) as a bioactive secondary metabolite exhibiting antimicrobial and antiparasitic activity. Song
et al. (2020) reported propanoic acid as the antibacterial metabolite against Acinetobacter baumannii,
P. aeruginosa, E.coli and S. aureus. Gong et al. (2014) found that the P. oxalicum synthesis pathway
for generating acids is directly influenced by the presence of a nitrogen source. This strain largely
secreted malic acid, acetic acid, propionic acid and citric acid in this condition. These findings suggest
that fatty acids could enhance the penetration into bacterial cell walls, plasma membranes, protein
synthesis, and nucleic acid metabolism, subsequently increasing the antimicrobial activity against
tested pathogenic microorganisms (Li et al., 2015; Watanabe et al., 2019). Therefore, it can be noted
that P. bilaiae EWB-3 can produce a potential number of secondary metabolites with therapeutic
benefits. These include phenolic acids, esters, and fatty acids.

Conclusion

Penicillium bilaiae EWB-3 was identified morphologically and genetically from electronic waste in
this study. This is the first report of this strain being isolated in Algeria. This filamentous fungus is a
rich source of biologically active natural compounds. Penicillium bilaiae EWB-3 crude extract was
tested for antimicrobial activity against Gram-positive and Gram-negative human pathogenic bacteria
and fungi. As a result, the high potential of this fungal strain suggests that it could be a promising
source of bioactive substances with potential applications in the pharmaceutical industry.



                                                                        24
Chemmam et al.                                                                             Italian Journal of Mycology 53 (2024): 13-28
https://doi.org/10.6092/issn.2531-7342/181345
___________________________________________________________________________________________________________________________________________________

Acknowledgements
The authors acknowledge Ministry of Higher Education and Scientific Research in Algeria for the ﬁnancial support and
the Department of Biochemistry (Badji Mokhtar University) for all facilities provided.



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