Advances in Biological Sciences Research, volume 13

Proceedings of the International Seminar on Promoting Local Resources for

Sustainable Agriculture and Development (ISPLRSAD 2020)

Selection of Culture Medium and Incubation Time for

Growth and Production of Beauvericin by Local
Beauveria bassiana

Y. P. Roswanjaya1*, N. A. Saryanah1, W. Nawfetrias1, H. Rosdayanti1 and A. L. Putri2

1
Centre of technology for Agricultural Production, Agency for the Assessment and Application of Technology (BPPT),
Serpong, South Tangerang 15314, Indonesia
2
Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Bogor 16911, Indonesia
*
Corresponding author. Email: [email protected]

ABSTRACT
Beauveria bassiana, an entomopathogenic fungus, is a high producer of beauvericin (BEA). BEA is a proven
and useful compound as a mycoinsecticide for plant pest control and a potential antifungal and anticancer agent
for human. BEA produced by Beauveria bassiana fungi, mainly found as an intracellular product, means its
production is dependent on the growth of the fungi in the culture medium. This study investigated four culture
mediums and two incubation times to enhance growth and BEA production by Beauveria bassiana isolated
from the infected insects in Kediri and Mojokerto, East Java, Indonesia. The four culture mediums were Potato
Dextrose Broth (PDB), Yeast and Malt Extract Broth (YMB), Malt Extract Broth (MB), and Fusarium Defined
Medium (FDM), and two incubation times were 6 and 12 days. The biomass and BEA production were studied
in a batch culture without agitation. Our data shows that YMB was the optimum culture medium to produce
high biomass of the fungal mycelium in both strains for 6 and 12 days of incubation. However, instead of in
YMB, the highest BEA production for both strains was obtained from Beauveria bassiana grown in PDB for 6
days and in MB for 12 days. Correlation between biomass and BEA production in every culture medium is then
calculated to see the BEA specific production. The highest BEA specific production has resulted from
Beauveria bassiana grown for 12 days in MB medium with BEA yield was 103,42 mg/L and 237,49 mg/L for
strain Kediri and Mojokerto, respectively.

Keywords: Beauveria bassiana, beauvericin, culture medium, incubation time, beauvericin specific
production
1. INTRODUCTION
The hypocrealean fungus Beauveria bassiana generally 2-3 x 2-2.5 µm. The conidia are formed in
(Bals.) Vuill. (Clavicipitaceae) is a well-known clusters, like snowballs or cotton balls.
entomopathogen because of its ability to infect hundreds of B. bassiana attack their host insects percutaneously,
host species belonging to most insect orders [1]. and the infection pathway of this fungi consist of several
Distribution of this fungi is worldwide, found on infected crucial steps [6]. The following steps are the attachment of
insects both in temperate and tropical areas throughout the the conidia to the cuticle, germination, penetration through
world. Besides its entomopathogenic ability, this fungi has the cuticle, overcoming the host response and immune
also been shown to live saprophytically in the soil, to defense reactions, proliferation within the host by the
colonize the rhizosphere, to have antagonistic activities formation of hyphal bodies/blastospores, and saprophytic
against plant pathogens [2], [3], as well as to grow outgrowth from the dead host and production of new
endophytically inside plant tissues [4]. Based on the conidia. Invasion of the fungi is synergic with the host
description by Rehner & Buckley [5], B. bassiana is response activities, and these interactions are complex
characterized by white, later yellowish, or occasionally processes and comprise many molecular and cellular
reddish colonies. The reverse is uncolored, or yellowish to reactions [7]. The successful infection is marked by hyphal
pinkish. Conidia cells consist of globose to flask-shaped fungi that invade tissues of the host insect by extensive
basal part and up to 20-µm long rachis, mostly forming a vegetative growth and the production of toxins. This
zig-zag. Conidia are hyaline, globose to broadly ellipsoidal, process is followed by the death of the insect and the end of
pathogenesis.

Copyright © 2021 The Authors. Published by Atlantis Press B.V.

This is an open access article distributed under the CC BY-NC 4.0 license -http://creativecommons.org/licenses/by-nc/4.0/. 59
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B. bassiana produces several toxins in vitro and in correlation between biomass and BEA production in every
vivo, but the most important toxin is beauvericin (BEA) [8], culture medium.
[9]. BEA is a cyclic hexadepsipeptide mycotoxin that
belongs to the enniatin antibiotic family. It contains three- 2. MATERIAL AND METHODS
D-hydroxyisovaleryl and three N-methylphenylalanyl
residues in alternating sequence [10], [11]. BEA has also 2.1. Microorganism and culture conditions
already known produce by other fungi such as Isaria B. bassiana was isolated from the infected insects in
fumosorosea and Isaria farinose (formerly Paecilomyces the cocoa plantation in Kediri and Mojokerto, East Java,
fumosoroseus and Paecilomyces farinosus) [12] and by Indonesia. Both fungi were identified as B. bassiana
some Fusarium species [13]–[15]. The difference of BEA according to the morphological criteria described by Lacey
produced by B. bassiana with another fungi species is in the [28]. Stock and working culture of B. bassiana strain Kediri
nature of the N-methylamino acid, causing the difference in and Mojokerto were maintained on solid Potato Dextrose
their bioactivities. Besides has insecticidal activity, BEA Agar (PDA) at 28oC. Fungal biomass and BEA production
reported has antibacterial activity [16], [17], antifungal were studied in a batch culture without agitation at 28oC.
activity [18], [19], antiviral activity [20], and anticancer The inoculum was prepared by taking a full colony of B.
activity [21]–[23]. The broad range function of BEA has bassiana from 6 days old of working culture with a cork-
motivated several research studies on its application as a borer. Three agar plates with 1 cm in diameter fully grown
potential antibiotic and anticancer agent for human health by B. bassiana were put aseptically to each 250 mL
care. Erlenmeyer flask containing 100 mL of different culture
As a fungal metabolite, mycelial fermentation may mediums.
be a feasible and efficient method to scale-up the production
of BEA. The essential factor in developing an efficient
fermentation process is the choice of culture medium for
2.2. Selection of culture medium
fungal growth. Nutrients and physicochemical conditions For the selection of fungal growth and BEA
will be a major key to the successful high production of production, both B. bassiana were cultivated in the
BEA. However, the report studies on the selection of culture following four types of complete culture medium. The
medium for BEA production by B. bassiana is very limited. detailed composition of every medium (amounts were listed
Instead of for B. bassiana, most of the studies were focused per liter) were: 1) Potato Dextrose Broth (PDB): infused
on investigating culture medium for Fusarium species [24]– potato, 200 g; and dextrose, 20 g. 2) Malt Extract Broth
[26]. As shown in those studies, the highest mycelia growth (MB): malt extract base, 6 g; yeast maltose, 1.8 g; dextrose,
and BEA production were reached in Fusarium Defined 6 g; and yeast extract, 1.2 g. 3) Yeast and Malt Extract Broth
Medium with a level of BEA was 0.2 g/L [24]. Furthermore, (YMB): malt extract base, 3 g; yeast extract, 3 g; peptone,
increasing 9 g of glucose and 3 g of peptone from the 5 g; and dextrose, 10 g. 4) Fusarium Defined Medium
Fusarium Basal Media was also increasing BEA level from (FDM): sucrose, 25 g; NaNO3 4.25 g; NaCl, 5 g: MgSO4-
156 mg/L to 198 mg/L [26]. In addition, glucose feeding 7H2O, 2.5 g; KH2PO4, 1.36 g; FeSO4-7H2O, 0.01 g; and
combined with resin to Fusarium Basal Media in the early ZnSO4-7H2O, 0.0029 g.
stationary growth phase (day 7) was increasing BEA yield
from 194 to 265 mg/L [25]. 2.3. Determination of fresh biomass, BEA
During B. bassiana or other fungi fermentation,
BEA can be found mainly as an intracellular product. This
content, and BEA specific production
fact suggests that BEA production is dependent on the B. bassiana mycelial mass was separated from the
fungal growth in the culture medium. However, at a high surface of the liquid medium and put on the filter paper, and
level, the accumulation of BEA within the fungal mycelia then air-dried at room temperature until all liquid medium
can cause growth inhibition due to its antibiotic properties. residues evaporated and mycelial mass became relatively
Because of this feedback inhibition, incubation time also dry. Biomass in the results was to represent the fresh weight
plays an important role during the fermentation process. of fungal biomass.
The maximal BEA concentration produced by Beauveria BEA extraction procedure followed the method
sp. FKI-1366 was achieved after 6 days of fermentation in developed by Moretti et al. [29] with modifications. For
the liquid culture medium [19]. Moreover, in Fusarium, each B. bassiana culture, a 20 g sample was extracted in a
Madry et al. [27] reported that the optimal harvesting time blender with 100 mL of MeOH : 1% aqueous NaCl (55 : 45)
of BEA from mycelial culture was four days of for 3 minutes and filtered through filter paper (Whatman
fermentation. After this period, the production of No.1) using a vacuum pump. The filtrate (50 mL) was
biologically active compounds did not further increase. transferred into a separatory funnel and defatted twice using
Our laboratory has two local isolates of B. bassiana, 50 mL of n-hexane. The upper n-hexane layer was
isolated from the infected insects in cocoa (Theobroma discarded, and the methanol layer was extracted with
cacao) plantation in Kediri and Mojokerto, East Java, chloroform (3 x 30 mL). The CH2Cl2 extracts were
Indonesia. In this present study, we investigated four collected, evaporated to dryness, dissolved in 1 mL of
different culture mediums for the maximal production of methanol, and then analyzed for BEA content.
BEA in two incubation times for both isolates. Furthermore,
BEA specific production was calculated to see the

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BEA was quantified by High Performance Liquid (Figure 1A). However, we could not see this phenomenon
Chromatography (HPLC), as previously described by in strain Mojokerto. Furthermore, PDB, as a general
Logrieco et al. [30] with modifications. HPLC was carried medium for fungal growth, did not show a significant effect
out using a Symmetri® C18 5 µm, 150 x 4.6 mm column, for B. bassiana. This medium was effected fungal growth
and a PDA UV detector, set at 225 nm using acetonitrile and as high as the MB medium after 12days, much lower
water (85:5 v/v) as an eluent, with a flow rate of 1.3 mL/min compared to YMB for both strains.
under a pressure 5.5 MPa. Quantification by HPLC
procedures was done by comparison of the peak height of
BEA against a calibration curve of the peak height obtained
with the BEA standard. BEA reference standard was
purchased from Sigma Chemical Co.
BEA specific production was calculated by divided
BEA content produced by fungi with the initial grams of the
sample used for extraction, then multiple it with fresh
biomass produced in the same medium and at the same time
point. This normalization was conducted to see the real
correlation between biomass and BEA production in every
culture medium.

2.4. Experiment design and statistical analysis

This experiment was constructed by factorial
completely randomized design on all four culture mediums
and two incubation times to examine their effects on fungal
growth for each isolate. The measurement of fresh biomass
in this study was carried out in triplicate. However, the
effects of different growth medium and incubation time on
BEA content were carried out once without replication. We
used R version 3.6.0 to conduct statistical tests. To compare
fungal biomass among treatments, we tested the average of
fresh biomass for significance differences with ANOVA
and a post-hoc Duncan tests for pairwise comparison with a
95% confidence interval.

3. RESULTS
3.1. Growth of B. bassiana in different culture Figure 1. Growth of B. bassiana strain Kediri (A) and
mediums Mojokerto (B) in four different culture
In this experiment, we found that B. bassiana strain mediums for 6 and 12 days of incubation.
Kediri and Mojokerto were well grown in all mediums Different letters above the bars indicate
tested (Figure 1). The pattern of growth in every medium statistical significance (p< 0.05) as
from both strains was similar; fresh biomass was determined by the ANOVA test in
significantly increased from 6to 12 days of incubation. combination with Duncan’s post-hoc test.
However, B. bassiana strain Kediri shows better growth
compared to strain Mojokerto (Figure 1 A & B). The highest 3.2. Profile of BEA produced by B. bassiana in
fresh biomass of both strains was reached when fungi were different culture mediums
grown in YMB for 6 or 12days. Moreover, In strain Kediri,
the highest increasing level of fresh biomass from 6 to BEA can be isolated from both strains of B.
12days was also shown in YMB. In contrast, for strain basianna in all four culture mediums, even though the
Mojokerto, although YMB was the optimum medium to contents were highly variable (Figure 2). The highest BEA
induce growth, the highest increasing fresh biomass level content has resulted from B. bassiana strain Kediri grown
was in the MB medium with a value of 2,29 grams (Figure in PDB for 6days and in MB for 12days with the contents
1B). were 51 and 419 mg/L, respectively (Figure 2A). A similar
The FDM specifically was formulated for the trend was also found in strain Mojokerto; the highest BEA
growth of Fusarium species, but our result shows that FDM content for 6dayshas resulted from B. bassiana grown in
was could also induce growth of B. bassiana, especially for PDB (1402 mg/L), meanwhile for 12days was in MB (1151
strain Kediri. In this medium, the fresh biomass of mycelial mg/L) (Figure 2B). In general, the BEA content increased
was the second highest after YMB in both time points from 6 to 12days, except for the B. bassiana grown in PDB

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for both strains. In the PDB, there was a significant 3.3. BEA specific production
reduction in the BEA content from 6 to 12days, especially We calculated BEA specific production for our two
for strain Mojokerto. Our data also shows that in YMB, the strains in every culture medium and in both time points
BEA contents produced by both strains were relatively (Table 1). Fungal biomass was the crucial factor in this
constant in the two-time points. To our surprise, although calculation as it can be seen that higher BEA specific
YMB was the optimum medium for B. bassiana growth, the production was affected by higher biomass. Our data shows
effect of this medium on BEA content was weaker this clear event in B.bassiana strain Kediri and Mojokerto
compared to other mediums. Furthermore, in our grown in FDM for 6days. Although BEA content for both
experiment, we also found that the production of BEA was strains was similar (13 mg/L), but because their biomass
low in FDM for both strains, which means that Fusarium was different, BEA specific production for strain Kediri was
specific medium can be used to grow B. bassiana, but this higher than strain Mojokerto (2,56 and 1,11 mg/L,
medium was failed to induce a high BEA content. respectively). Generally, the trends for BEA specific
production in this experiment have followed the trends for
BEA content, except for strain Kediri grown in PDB. There
was a reduction of BEA content from day 6th to 12th.
Meanwhile, in their BEA specific production, the levels
were relatively the same, 7,96 and 8,39 mg/L for 6 and
12days of incubation.

Tablel 1. BEA specific production from B. bassiana

strain Kediri and Mojokerto

4. DISCUSSION
In vitro BEA production by fungi B. bassiana is
affected by many environmental factors such as culture
medium, temperature, pH condition, and incubation time.
The precise association among those factors will create an
optimum condition for fungal growth and production of
secondary metabolites, including BEA. To determine the
best environmental factor for growth and BEA production
of B. bassiana, we aimed to select a culture medium and
incubation time trough selection of four different culture
Figure 2. Profile of BEA content produced by B. mediums and two incubation times. We did not investigate
bassiana strain Kediri (A) and Mojokerto the pH condition and temperature in this experiment. As a
(B) in four different culture mediums for 6 consequence, we referred to some reports explaining this
and 12 days of incubation. condition. Fargues et al., and Hallsworth & Magan, have
reported that the optimum temperature for B. bassiana
growth was 28oC [31], [32], whereas the optimum pH
medium was 7 [33]. In this paper, we showed that with a
specific culture medium and with longer incubation time,
fungal biomass and BEA content produced at a high level
for both strains (Figure 1 & 2).

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Components of the nutrient in the medium used for foundafter four days of fermentation, BEA content did not
growing B. bassiana or other fungi as general influence further increase.
biomass yield and production of metabolites. Moreover, the Among the mediums we used, PDB showed an
most important component in a culture medium for this induction to BEA content that differs from another medium.
purpose is carbon and nitrogen. As an imperfect fungi The BEA content was higher on day 6th, then decrease
Deuteromycetes, B. bassiana can be grown on a medium significantly on day 12th, whereas the other mediums show
containing only simple carbon like sucrose, nitrogen, and the opposite results (Figure 2A & B). The BEA content
mineral components [34]. Our result indicates that four reductionin this medium might be caused by the initial BEA
culture mediums with different carbon and nitrogen sources content that wasalready high on day 6th, indicates that after
can still be used to grow B. bassiana. The YMB, which reach a maximal level, BEA content decreases over time.
contains double carbon (dextrose and malt extract) and This finding confirms the results from Xu et al. [25] that
nitrogen (yeast extract and peptone) sources, was the most showed BEA at a high level has negative feedback to the
optimum medium to produce high biomass in both strains. downstream of another BEA production cycle in a longer
However, the biomass from strain Kediri is higher than incubation.
strain Mojokerto. It suggests that the demand for a particular Since the trend of BEA content is similar to BEA
nutrient frequently varies not only within a single species specific production, which was affected by biomass, we
but also for individual strains of a species. The increasing found that although grown in the same medium, the ability
pattern of fungal biomass shows the similarity between our of the two strains used in this experiment is different in
two strains in all mediums. Nevertheless, the differences in producing BEA. Our BEA contents and BEA specific
the biomass yield confirm that both strains were completely production comparison between two strains revealed that
different. strain Mojokerto was stronger in producing BEA in all four
In contrast to fungal biomass, YMB found as the mediums and in both time points than strain Kediri. This
weakest medium to induce BEA content in both strains, significant difference might be affected by strain
indicates that fungal growth was not linearly correlated with specificity, support the hypothesis that BEA content was
the BEA content. The use of different types of carbon and strain-dependent [36]. Even though in the same species, a
nitrogen sources in the four culture mediums are not clear different strain can produce BEA content differently.
to determine their effects on fungal biomass or BEA
content. Our data only reveals that a medium can be the 5. CONCLUSION
most optimum, but which components have a high impact
are hard to specify. The way to know the effect of every The medium needed for optimum growth and BEA
component is by doing optimization on basal medium with production is different. These differences could help us to
various defined carbon and nitrogen sources. Optimization select which medium to be used based on our goals. YMB
of the carbon/nitrogen ratio can be done by response surface is the optimum medium to yield high biomass, whereas MB
methodology that has been proven to select the medium for is the most suitable medium to produce high BEA content.
growth and BEA production from Fusarium oxysporum Additionally, 12 days of incubation is the optimum
KFCC 11363P [24]. However, in this experiment, we used incubation time for both purposes. The selection results
only complete mediums to simplify the selection process. obtained from this study may be useful for the efficient and
Even though without C/N ratio optimization, our result economical production of BEA on batch culture
shows that some medium was suitable for producing BEA fermentation.
with a high content in our two strains.
Observation of incubation time for fungal biomass
yield indicates that prolonged incubation time will increase AUTHORS’ CONTRIBUTIONS
the biomass. This is a common phenomenon for
microorganism growth. As long as still in the exponential YPR and NAS planned and designed the research;
phase, microorganisms will continue to grow until a YPR, NAS, WN, HR, and ALP performed the experiment
particular time [35]. In this experiment, the fresh biomass and analyzed the data; and YPR wrote the manuscript.
of both strains was increased from 6 to 12 days of
incubation, confirm that from 6 to 12 days are still in the
range of the exponential phase. In the case of BEA content, ACKNOWLEDGMENT
our data shows that between time points observed,12 days
was the best incubation time compared to 6 days. This result
The authors like to thank the staff at Nusantara
was opposite to Fukuda et al. [19], which reported that
Plantation Company (PTPN) XII Kediri andthe Plantation
maximal BEA content was reached after 6 days of
office of Mojokerto Regency for help with sample
fermentation. They did not observe BEA content from
collection. The authors are grateful to Lira Hikaru for
Beauveria sp. FK1-1366 after 6 days, so we do not know
guiding in HPLC analysis. This work was supported by the
the pattern of BEA content produced by this Beauveria
Issuance of spending authority (DIPA) BPPT to the Centre
species after 6 days. In contrast, BEA contents in our
of Technology of Agricultural Production under the
observation were also high on day 6th, but after12 days, the
“Optimization of Cocoa Cultivation TechniquesTrough
levels were even higher in almost all culture mediums. This Pest Control Management” Project.
result was also contradictive with Madry [27] that

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