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Author(s): Nagendra Kumar Chandrawanshi*1, KL Tiwari2, SK Jadhav3

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    1School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    2School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    3School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    *Corresponding Author Email-

Published In:   Volume - 2,      Issue - 1,     Year - 2020

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Nagendra Kumar Chandrawanshi, KL Tiwari, SK Jadhav (2020) Antibiotic Efficacy Assessment of Certain Species of Penicillium fungi. NewBioWorld A Journal of Alumni Association of Biotechnology, 2(1):33-37.

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 NewBioWorld A Journal of Alumni Association of Biotechnology (2020) 2(1):33-37            


Antibiotic Efficacy Assessment of Certain Species of Penicillium fungi

Nagendra Kumar Chandrawanshi*, KL Tiwari, SK Jadhav

School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India

*Corresponding Author Email-




Article history:


05 November 2019

Received in revised form

12 December 2019


03 January 2020


Penicillium species; Antimicrobial activity; Broth dilution assay; Bacteria and Inhibition


This study aimed to evaluate the antimicrobial activity of Penicillium species on soil-isolated gram-positive coccus bacteria. The antimicrobial efficacy of Penicillium species filtrate extracts were determined via disc diffusion and broth dilution assay. A kill curve study was conducted with treated and untreated bacterial populations. There were P. oxalicum, and P. purpurogenum showed the maximum zone in solid surface culture methods, followed by other spices with fewer antibiotic properties. The kill curve study has shown an exposure-dependent inhibition for bacterial populations. With the increase in the exposure periods, microbial growth was significantly reduced. In the broth dilution assay methods, among all treated Penicillium species, only P. oxalicum and P. funiculosum species were shown potent antibiotic properties; other species had given the very least activity. The study interpreted that screening of the suitable strain of microbes for very essentially for industrially important bioactive compounds scale up at industrial set up.



Fungi play a significant role in soil ecosystems, along with bacteria, protists, small invertebrates, and plants, through complex trophic interactions. Most soil fungi are regarded as saprobes, decomposing organic matter and contributing to nutrient cycling, while several species form mycorrhizal associations with plants or are plant pathogens. Also recognized as prolific secondary metabolite producers, fungi have provided several bioactive compounds and chemical models currently used as pharmaceuticals, and soils are traditionally the primary source of fungal genetic resources for bioprospection programs (Adrio & Demain, 2003).  Penicillium is a large anamorphic (asexual state) ascomycetous fungal genus with widespread occurrence in most terrestrial environments. This genus comprises more than 200 described species, and many are common soil inhabitants, as well as food-borne contaminants or food ingredients used in the preparation of cheese and sausages.  Penicillium species produce a much-diversified array of active secondary metabolites, including antibacterial, antifungal substances, immunosuppressants, cholesterol-lowering agents, and potent mycotoxins (Lucas et al., 2007). Thousands of Penicillium isolates have probably been screened in bioprospecting programs since the discovery of penicillin (Petit et al., 2009). The synthesis of large numbers of antibiotics over the past three decades caused complacency about the threat of antibiotic resistance. Drug resistance is frequently encountered in hospital-acquired pathogens, usually critically ill or immune-suppressed patients. The resistance of microorganisms to low-cost antibiotics also significantly increases the country's healthcare spending (Friedman et al., 2016). Thus, the pharmaceutical industry and academic institutions invest vast resources to produce safe and effective antimicrobial drugs (Strobel & Daisy, 2003). The members of the genus Penicillium are well known for the production of antibiotics.  P. purpurogenum has been isolated from various substrates, particularly soil, and wood. They are known for producing polyketide red pigments, natural colorants in the food industry (Padmapriya et al., 2015). However, their antimicrobial activity needs to be better characterized. The reports on endophytic strains of P. purpurogenum are also only few available. However, previous penicillin production investigators have yet to develop a highly effective medium successfully. For decades, penicillin yields have been increased through the development of better production strains by classical mutagenesis procedures and optimization of the growth conditions. Penicillin biosynthesis is regulated by environmental factors such as the medium's phosphate, carbon, nitrogen, and oxygen content (Feng et al., 1994). Thus, this study aimed to evaluate the antimicrobial activity of various species of Penicillium against soil-isolated cocci bacteria (cocci morphology). The fungal bioactive constituents of mycelium filtrate of antibacterial activity were characterized via gel diffusion and broth assay analysis.

Materials and Methods

Microorganisms and maintenance of culture 

DOI: 10.52228/NBW-JAAB.2020-2-1-7

The identified microorganisms were obtained from the School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur. The microorganisms used in this study were ten fungi (Penicillium citrinum, P. notatum, P. oxalicum, P. rubrum, P. frequent, P. rugulosum,                        P. chrysogenum, P. variabile, P. purpurogenum, and P. funiculosum. The soil isolated gram-positive cocci, biochemically characterized bacteria used for the antimicrobial assay. The test microorganisms were sub-cultured on nutrient agar before use every two weeks to maintain their viability. The microbial inoculums were prepared by transferring a loopful of microbial colonies into a universal bottle of sterile distilled water. The turbidity of the suspension was adjusted to approximately 108 colony-forming units (CFU)/mL (Yenn et al., 2017).

Culture medium

Potato dextrose agar (PDA) [200g/L potato extract, 20 g/L Dextrose, 15 g/L Agar, (pH 5.6)] was used to cultivate the fungus. Similarly, Nutrient Agar Medium (NAM) [5.0 g/L Peptone, 3.0 g/L Beef extract, 5.0 g/L Nacl, 15 g/L Agar, (pH 7.0)] was prepared for cultivated the bacterium culture. The pH of the medium was adjusted as standard before autoclaving at 121°C for 15 minutes.


The liquid medium was prepared without adding agar (PDB or NBM); this prepared medium is used to cultivate microbes. In this manner, inoculums were prepared by introducing separately all individual fungal plugs of 1.0 cm in diameter into 250 ml Erlenmeyer flasks containing 100 ml of PDB medium. The inoculated flasks were incubated in the dark at 25°C in static condition for 15 days.


According to Tiwari et al. (2010) and Yenn et al. (2017), the procedure was carried out with some minor modifications. After the incubation period, After two weeks of cultivation, the fermented broth and fungal biomass were separated using Whatman No.1 filter paper. The cultures of mycelium separated, and the filtrate was harvested. The collected crude filtrates used potential bioactive compounds. The obtained extract was dried for the solid plate, and crude extract paste was obtained. A control was also prepared using a sterile medium following the same procedure used for fermentative broth. 

Antimicrobial Assay: 

Disc diffusion assay

The antimicrobial efficacy of the extract was evaluated according to standards created by the Clinical Laboratory Standards Institute (CLSI) and Yenn et al. (2017) with some modifications. The assay was performed by transferring the fungal inoculums to the surface of nutrient agar medium after 3 to 5 days of appropriate incubation, inoculated with previously grown bacterial suspension, allowing all plates to be incubated at 37oC for 24 hrs.; furthermore, measured the inhibitory zone by antibiotic zone scale. In the control NAM plate, they have only transferred bacterial suspension. The experiment is consecutive and was done twice times.

Broth Dilution assay

The potential inhibitory concentration was determined by using the broth dilution assay method in different time intervals, according to followed Yenn et al. (2017) and Chandrawanshi & Shukla (2019). Then, previously fully grown bacteria containing NBM medium were added to Penicillium mycelium filtrate extract in equal amounts. These tested tubes were incubated for the next 24 hrs and absorbed recorded at 600 nm for the following treated six days. The bacteria containing NBM with an equal amount of mycelium extracts were used as blank. The minimal lethality concentration was recorded as the lowest extract concentration to kill the test microorganisms, with dose exposure assayed. 


Disc diffusion assay 

The finding of the best suitable Penicillium species was characterized on a solid medium, and significant antimicrobial activity was investigated. The present study found that the highest level of antimicrobial activity was produced by P. oxalicum and P. purpurogenum, as observed from the results obtained by the agar diffusion method; the zone diameter has revealed in Table 1. The activity was considered for inhibition zones wider than 10 mm into the agar surface. The activity against gram-positive (coccus) soil-isolated bacteria. Followed by P. chrysogenum recorded only 8 mm diameter, and the minor zone was observed for P. notatum and P. funiculosum (5 mm); it showed that in the plate assayed, a minute quantity of penicillin substance secreted, which was seen that antibiotic inhibitory zone in the plate. The experiment has taken other species that responded very ineffective, thus considered non detective (nd).

Table 1: Antibiotic sensitivity test, measured of the zone of inhibition



Diameter (in mm)































nd-Not detected


Figure 2: Antibacterial activity by Penicillium filtrates against Gram positive bacteria


Broth Dilution assay

The test microorganisms exhibited susceptibility to fungal extract from fermented Penicillium culture. The initially optimized inhibitory activity on a solid medium was further tested on broth dilution. The assay was performed in a sterile test tube, according to Yenn et al. (2017). The nutrient broth was used for testing bacteria, and mycelium filtrate-containing potato broth was used to examine antibiotic properties. The study showed that P.notatum and P. citrinum showed very little effectiveness on broth assayed because optical density observed data showed in increasing order, indicating that the mycelium filtrate has not potent effective against bacterial culture. P.oxalicum and P. funiculosum showed the hard capacity of antibacterial properties against gram-positive bacteria, has observed that the decreasing order and particular day after the reading was obtained in zero values. It is potent in both assay system broth and solid surface medium. Then other tested fungi species, including P. rubrum, P. frequent, P. variabile, P. rugulosum, P. chrysogenum and P. purpurogenum showed less effective or insignificant antibiotic properties (figure1), thus not observed proper growth inhibition activity, similar to the data were expressed on solid plate medium. Minimal lethality concentration (MLC) was determined based on the exposure of extract to kill the test microorganisms.

Discussion and Interpretation

The present study used potato dextrose medium for crude antibacterial compounds extract successively obtained. The growth media and growth incubation conditions strongly affect secondary metabolite production, and there is no compromise on which culture media are optimal for specific metabolite production. Several culture media like Czapek-Dox broth, Sabourod broth, potato dextrose broth, malt extract broth, and nutrient broth were used to investigate previously. However, potato dextrose broth was a potent medium for producing bioactive antimicrobial metabolites (Mathan et al., 2013). Singh et al. (2012) and Toghueo et al. (2018) reported the potato dextrose agar is the best medium for the maximum growth and production of metabolites. Talia et al. (2011) investigated in vitro synergism between several chalcones substituted in combination with oxacillin, an antibiotic used conventionally against S. aureus ATCC 43 300 that was resistant to meticillin, using the kinetic turbidimetric method developed earlier. The results were satisfactory for all assayed combinations and followed the mechanism of bacteriostatic inhibition. Even so, the size of inhibition zones varied among all microbes, indicating different susceptibility of the test microorganism to the extract. Among the microbial species, the largest clear zone was shown by Streptococcus sp., a gram-positive bacterium. The results obtained supported previous studies that most of the fungal extracts generally exhibit lower antimicrobial activity against gram-negative bacteria than gram-positive bacteria (Mahadevamurthy et al., 2016). Sadrati et al. (2013) reported that the antibacterial activity by plug agar method for the PLR9 isolate might be due to the secretion of diffusible extracellular metabolites in the agar medium, these compounds having a specific effect against bacteria. This technique was easy and widely used to detect non-volatile compounds produced by microorganisms. The significant antibacterial activity obtained in the present study may be due to the presence one or more these molecules in the crude filtrate extracts especially penicillin acid, which is active against bacteria (Zainuddin et al., 2010; Varga et al., 2015). Penicillin is secondary metabolites, its production varying by cultivated factors. Thus, the expression of secondary metabolites might depend on the culture conditions and the strains. P. corylophilum was often isolated from temperate climates (Malmstro¨m et al., 2000). However, in work, they investigated and reported that the strain was isolated from a Brazilian soil sample, a tropical country. According to Gaden-Junior (2000), metabolite production is influenced by medium composition, nutrients availability and others aspects. Microorganisms can use a wide variety of carbon and nitrogen sources. Neverthe less, many secondary metabolic pathways are negatively affected by these sources favorable for growth (Cutler et al., 1989; Sanchez & Demain, 2002). Thus might be the reason for various antibacterial activity expressions among Penicillium species.

Conflict of Interest

The authors declare that there is no conflict of interest.


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