Original Article

Media optimization studies for enhanced biomass production of two

lignolytic fungi

Parvathy R. Nair1, A.S. Athira3, Saimukund1, J.S. Anoop1, Nived Santhosh1,

K. Shynidevi1, S. Aswin3, Shebin K. Bency3, A. Jayakumaran Nair3,

Ramya R. Prabhu1, O. Veena3

1PG Department of Biotechnology, College of Arts and Science, Thiruvananthapuram,

2Department of Biotechnology, A J College of Science and Technology, Thonnakkal,

3Department of Biotechnology, University of Kerala, Thiruvananthapuram, Kerala, India

Corresponding author: O. Veena, Email: dr.veena@keralauniversity.ac.in

Journal of Experimental Biology and Zoological Studies. 2(2): p 154-9, Jul-Dec 2026.

Received: 05/06/2026; Revised: 16/06/2026; Accepted: 17/06/2026; Published: 05/07/2026

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Abstract

The present study evaluated the effect of 27 different Mineral Salt Medium-Lignin (MSM-L)

formulations on the biomass production and sporulation of two laccase-producing Aspergillus

species, Aspergillus nomiae and Aspergillus niger. Laccase activity was confirmed using the

guaiacol plate assay, demonstrating the ligninolytic potential of both species. Furthermore,

optimization of culture medium composition for enhanced growth and sporulation was carried out.

The findings indicate that these two Aspergillus species may serve as promising candidates for

laccase production and other biotechnological applications involving lignin degradation and

bioremediation.

Keywords: Aspergillus nomiae, Aspergillus niger, fungal growth, laccase, lignin degradation,

mineral salt medium.

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Introduction

Lignolytic enzymes are extracellular enzymes produced by fungi for the degradation of lignin and

other complex aromatic compounds present in plant biomass. Among these enzymes, laccase

[benzenediol:oxygen oxidoreductase (E.C. 1.10.3.2)] is a multicopper-containing enzyme that

catalyses the oxidation of a wide variety of aromatic compounds, accompanied by the concomitant

reduction of molecular oxygen to water.[1] Laccase plays a significant role in lignin degradation

and has gained considerable attention because of its applications in bioremediation, textile dye

decolorization, paper and pulp industries, and wastewater treatment.[2-4] Fungal laccases are widely

preferred for biotechnological applications over bacterial laccases due to their stability and

sustained activity across a broad range of temperatures and pH.[3,5] Fungi are considered efficient

producers of laccase, particularly filamentous fungi such as Aspergillus species.[6-8] The production

of laccase is influenced by several nutritional factors, that include carbon and nitrogen sources,

mineral composition, and incubation period. Optimization of these factors is important for

improving enzyme production as well as fungal biomass formation.[3,9] Recent studies have

focused on optimization of physiological and nutritional parameters to enhance laccase production

in Aspergillus spp. under submerged fermentation conditions.[10] Mineral Salt Medium (MSM) is

commonly used for fungal growth and enzyme production studies because it provides the essential

nutrients required for fungal metabolism while allowing the modification of nutritional

requirements for optimization studies. Different combinations of carbon and nitrogen sources in

MSM can significantly affect fungal growth and laccase production.[8,9] In the present study,

qualitative screening of two characterized species of Aspergillus - Aspergillus nomiae and

Aspergillus niger -was carried out using a guaiacol-containing medium, and their growth and

biomass formation were subsequently evaluated under various MSM formulations.

Materials and Methods

Guaiacol Assay

Characterized strains of A. nomiae and A. niger, obtained from laboratory stock cultures

maintained at the Department of Biotechnology, University of Kerala, were used in this study.

Laccase activity was confirmed by a zone assay on SDA supplemented with 0.01% guaiacol,

followed by incubation at room temperature for 7 days.

Media optimization with different MSM-L formulations.

Mineral salt medium supplemented with lignin (MSM-L) were prepared for media optimization

studies. Lignin (1% w/v) was incorporated as the sole source of carbon. Three crucial components

of the MSM were selected for optimization at three concentrations each: NH4H2PO4 (0.5, 0.75 and

1 g/ L), FeSO4 (0.01, 0.03 and 0.06 g/L), and MgCl2 (0.5, 0.75 and 1 g/L), resulting in a total of 27

formulations.

Table 1: 3 x 3 x 3 MSM-L formulations used in the study and their component concentrations

Concentration of

NH4H2PO4 (g/L)

(A)

Concentration of

FeSO4 (g/L)

(B)

Concentration of

MgCl2 (g/L)

(C)

0.051

0.011

0.51

0.752

0.032

0.51

0.752

0.063

0.51

0.752

0.011

0.51

0.752

0.032

0.51

0.752

0.06l3

0.51

0.752

0.011

0.51

0.752

0.032

0.51

0.752

0.063

0.51

0.752

‘A’ represents NH4H2PO4, ‘B’ represents FeSO4, and ‘C’ represents MgCl2, while the numerical superscripts 1, 2,

and 3 denote the concentration of each component in the medium. The 27 MSM formulations were labelled according

to the concentrations of the individual components used in each formulation. For example, A1B3C2 represents a

medium in which the concentrations of NH4H2PO4, FeSO4, and MgCl2 are 0.05g/L, 0.06 g/L, and 0.75 g/L respectively.

The 3 × 3 × 3 optimization design used for the preparation of the various MSM formulations

evaluated in this study is presented in Table 1. The same experimental design was applied to both

A. nomiae and A. niger to facilitate comparison of their growth under identical culture conditions.

For the preparation of each MSM formulation, autoclaved media components in the respective

proportions outlined in Table 1 were dispensed into 50 mL sterile flasks containing double distilled

water. The final volume of the medium was brought to 25 mL. The pH of the medium was

subsequently adjusted to 6.5 using a 0.1 N HCl solution. The fungal strains were sourced from the

mother culture. A 50 μL aliquot of the inoculum, taken from a vortexed, overnight MSM-L broth

culture of each Aspergillus species, was transferred into the sterile flasks. The cultures were then

incubated at room temperature under controlled humidity without agitation (statically) to prevent

mechanical damage to the developing mycelia. The preparation of the culture medium, inoculation,

and incubation were carried out under aseptic conditions. Following incubation, visible biomass

production and sporulation were monitored at specific intervals over a period of 20 days.

Observations were recorded using a qualitative scoring system. Biomass formation was scored as

follows: no growth was represented by ‘0’, while increasing levels of biomass accumulation were

denoted by ‘+’ and ‘++’, indicating moderate and high biomass production, respectively.

A similar scoring system was used to assess sporulation, where ‘0’, ‘+’, and ‘++’ represented no,

moderate, and high sporulation, respectively.

Heatmap analyses

Heatmap analyses of data derived from culture medium optimization studies were prepared using

Microsoft Excel.

Results

Confirmation of laccase production using guaiacol assay

Qualitative screening for laccase activity using a guaiacol-containing medium confirmed laccase

secretion in both A. nomiae and A. niger (Figure 1). The formation of a reddish-brown colouration

around the fungal colonies confirmed the oxidation of guaiacol by laccase.[8]

Media optimization of nutrient conditions using different MSM-L formulations

Both A. nomiae and A. niger showed variations in biomass formation when grown in different

MSM formulations. Some formulations of MSM showed enhanced biomass formation and

sporulation after a definite period of incubation. Among the 27 MSM formulations evaluated,

only nine supported enhanced fungal growth and were, therefore, selected for further comparative

analysis using the growth-scoring system (Tables 2 and 3).

Figure 1: Guaiacol plate assay showing positive laccase activity. (a) A. nomiae (b) A. niger

Table 2: Qualitative scoring of biomass formation and sporulation in A. nomiae grown on various

MSM formulations

used for culture

Day of observation

DAY 3

DAY 6

DAY 10

DAY 17

DAY 20

A1B1 C1

S+

A1B1C2

S+

A1B1 C3

S+

A1B2 C1

S+

A1 B2 C2

S+

A1 B2 C3

S+

A1 B3 C1

S+

A1 B3 C2

S+

A1 B3 C3

S+

MSM=Mineral salt medium; G=Growth; s=Sporulation; 0, + and ++ indicate no, moderate and high growth or

sporulation, respectively

Table 3: Qualitative scoring of biomass formation and sporulation in A. niger grown on various

MSM formulations

used for culture

Day of observation

DAY 3

DAY 6

DAY 10

DAY 17

DAY 20

A1B1 C1

S+

A1B1C2

A1B1 C3

A1B2 C1

S+

A1 B2 C2

S+

A1 B2 C3

S+

A1 B3 C1

A1 B3 C2

A1 B3 C3

S+

MSM=Mineral salt medium; g=Growth; s=-Sporulation; 0, + and ++ indicate no, moderate and high growth or

sporulation, respectively

MSM=Mineral salt medium; The intensity of colour is proportional to the extent of the observed response, increased

colour intensity indicating a stronger response. For ease of interpretation, colour intensity is also represented

numerically on a scale of 0 - 2, where 0 indicates no response and 2 represents the maximum response

Figure 2: Heatmap analysis of fungal growth and sporulation in A. nomiae (A) and A. niger (B),

from culture medium optimization studies

Heatmap analyses of data

From the heatmap analysis of the quantitative scoring system, it was observed that both A. nomiae

and A. niger exhibited distinct growth and sporulation patterns under different MSM formulations

as may be seen in Figure 2.

Discussion

Aspergillus species can inhabit a wide variety of ecological environments, with soil serving as

their primary habitat.[11] In soil ecosystems, they play a crucial role as saprophytic decomposers of

organic plant materials. However, certain species are also recognized as opportunistic pathogens.

For instance, Aspergillus fumigatus is widely reported to cause severe allergic reactions and life-

threatening systemic infections in humans, collectively known as aspergillosis.[12] Additionally,

some members of this genus act as phytopathogens that target economically important crops.[11]

Despite their pathogenic potential, many Aspergillus species are beneficially utilized in industrial

biotechnology for the large-scale production of enzymes, vitamins, organic acids, and other

bioactive secondary metabolites.[13]

The present study shows that MSM composition significantly influences fungal growth in

Aspergillus spp. which is consistent with previous reports on the effects of nutritional factors on

fungal growth. [3,8] Enhanced growth of fungal isolates in lignin-containing MSM suggests their

ability to utilize lignin through lignolytic enzymes such as laccase. This observation indicates that

Aspergillus spp. can be utilised for lignin degradation.[2]

The quantitative scoring of data from media optimization studies revealed that among the 27 MSM

formulations tested, A1B3C2 and A1B3C3 consistently supported enhanced growth and sporulation

in A. nomiae and A. niger respectively, throughout the period of observation. Therefore, A1B3C2

and A1B3C3 combinations were selected as the optimal media for culturing A. nomiae and A. niger

respectively. A. nomiae reached maximum growth on day 6, while in A. niger this was on day 17,

indicating differences in nutrient utilization and growth response. Heatmap analysis is a powerful

data visualization tool widely used in fungal studies to represent complex datasets using colour

gradients, thereby enabling rapid identification of patterns, clusters, and correlations.[14] In the

present study, the heatmap analysis of growth and sporulation responses of Aspergillus spp.

maintained on different MSM formulations corroborated the results obtained through qualitative

scoring. The approach proved useful in identifying the optimal composition of medium

components for enhanced fungal growth and sporulation.

It is observed in the present study that both fungal species exhibited enhanced growth at a

comparatively lower NH4H2PO4 concentration of 0.5g/L. Fungi are well known for their ability to

degrade lignin. Lignin contains very little or no readily available nitrogen and hence, is not

considered a significant source of nitrogen; but it may serve as a carbon-rich substrate, for fungal

growth.[15,16] Fungi are well known for their ability to degrade lignin. During lignin degradation,

fungi secrete extracellular ligninolytic enzymes such as laccases, lignin peroxidases, and

manganese peroxidases, which break down the complex lignin polymer into smaller aromatic

compounds that can be further metabolized. The production of these enzymes is often stimulated

under conditions of nitrogen limitation, indicating that low nitrogen availability can enhance lignin

degradation and ligninolytic activity.[2] The present study shows that both species of Aspergillus

can survive and grow well in a culture medium with relatively low nitrogen content. This

observation further supports the existence of a nitrogen-limitation response in Aspergillus spp. in

which reduced ammonium availability in the culture medium appears to have caused increased

secretion of laccase for lignin degradation. Thus, under conditions of low nitrogen availability,

lignin may contribute indirectly to fungal growth by providing carbon and energy through its

degradation products.[15,16] In the present study, MSM supplemented with 1% lignin was used for

culturing the fungal isolates based on standardization trials. Thus, with lignin as the sole carbon

source, both fungal species may have relied on ligninolytic enzymes for substrate utilization, for

growth and sporulation.[8] It is further observed that different culture medium formulations

significantly influence ligninolytic enzyme production and fungal growth.[9,17,18]

The successful growth of fungi in MSM indicates their ability to utilize complex carbon sources

under nutrient-limited conditions. MSM provides essential inorganic nutrients while minimizing

the availability of readily metabolizable carbon, thereby promoting the utilization of alternative

substrates such as lignin and lignocellulosic compounds. Several studies have reported that fungi,

particularly white-rot and lignolytic species, exhibit substantial growth and enzymatic activity in

MSM supplemented with lignin or related aromatic compound.[10,19]

Conclusion

The present study demonstrated that both A. nomiae and A. niger exhibited enhanced biomass

production in culture media containing comparatively a lower concentration of NH₄H₂PO₄ (0.5

g/L), with optimum growth observed on the 6th and 17th days of incubation, respectively. Since

lignin served as the sole carbon source, the increased biomass under these conditions suggests

efficient lignin utilization, which is probably associated with enhanced ligninolytic enzyme

activity, including laccase production. The MSM-L formulations designated as A1B3C2 for A.

nomiae and A1B3C3 for A. niger yielded the best results in supporting fungal growth and

sporulation. These formulations may therefore be considered potentially suitable media for laccase

production by Aspergillus species.

References

1. Gianfreda L, Xu F, Bollag JC. Laccases: a useful group of oxidoreductive enzymes. Bioremediation Journal 1999; 3: 1–26.

2. Baldrian P. Fungal laccases – occurrence and properties. FEMS Microbiol Rev 2006; 30(2): 215–42.

3. Madhavi V, Lele SS. Laccase: properties and applications. BioResources 2009; 4(4): 1694–717.

4. Couto SR, Herrera, TJL. Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 2006; 24(5):500–

13.

5. Mate DM, Alcalde M. Laccase engineering: from rational design to directed evolution. Biotechnol Adv 2015; 33(1): 25-40

6. Bagewadi ZK, Mulla SI, Ninnekar HZ. Purification and characterization of endo β-1,4-d-glucanase, xylanase and laccase

from Aspergillus niger. 3 Biotech 2017; 7: 302

7. Shekher SR, Sehgal S, Kamthania M, Kumar A. Laccase: microbial sources, production, purification, and potential

biotechnological applications. Enzyme Res 2011; 2011:217861.

8. Viswanath B, Rajesh B, Janardhan A, Kumar AP, Narasimha G. Fungal laccases and their applications in bioremediation.

Enzyme Res 2014;2014:163242.

9. Papagianni, M. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnology Advances

2004; 22(3): 189–259.

10. Zaidi S, Mirza SA. Optimization of laccase production parameters by Aspergillus niger from different ecological sites of

Lahore. Punjab Univ J Zool 2024;39(2):141-9.

11. Niji QN, Babalola OO, Mwanza M. Soil Aspergillus Species, Pathogenicity and Control Perspectives. J Fungi 2023; 9(7):

766.

12. Frías-De-León GM, García-Salazar E, Acosta-Altamirano G. Virulence attributes in Aspergillus fumigatus. In: Razzaghi-

Abyaneh M, Rai M, Shams-Ghahfarokhi M, Rodrigues-Morales AJ, editors. Aspergillus and Aspergillosis - Advances in

Genomics, Drug Development, Diagnosis and Treatment. London (UK):Intech Open Limited;2023. Available from:

https://www.intechopen.com/chapters/87187 [Last accessed on 2026 May 15].

13. Hou X, Liu L, Xu D, Lai D, Zhou L. Involvement of Lae A and velvet proteins in regulating the production of mycotoxins

and other fungal secondary metabolites. J Fungi (Basel) 2024; 10(8): 561.

14. Feng W, Wang J, Jin Q, Guo Z, Cai W. Analysis of Soil Fungal Community Characteristics of Morchella sextelata Under

Different Rotations and Intercropping Patterns and Influencing Factors. Agriculture 2025;15(8): 823.

15. Kumar V, Prasher IB. Ligninolytic enzymes production by endophytic fungus Diaporthe phaseolorum under the influence of

different carbon and nitrogen sources. Studies in Fungi 2021; 6(1): 531–42.

16. Dashtban M, Schraft H, Syed TA, Qin W. Fungal biodegradation and enzymatic modification of lignin. Int J Biochem Mol

Biol 2010; 1(1): 36–50.

17. Cairns T C, Nai C, Meyer V. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol

Biotechnol 2018; 5: 13.

18. Upadhyay P, Shrivastava R, Agrawal PK. Bioprospecting and biotechnological applications of fungal laccase. 3 Biotech 2016;

6: 15.

19. Elisashvili V, Kachlishvili E, Penninckx M. Effect of growth substrate, method of fermentation, and nitrogen source on

lignocellulose-degrading enzymes production by white-rot basidiomycetes. J Ind Microbiol Biotechnol 2008; 35(11): 1531–

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