E3S Web of Conferences 355, 02017 (2022) https://doi.org/10.

1051/e3sconf/202235502017
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Investigation of low-cost media for Bacillus thuringiensis

subspecies israelensis
Phuwana Mo-on1, Siriwan Panprivech1 and Viyada Kunathigan2*
1Department of Agro-Industry, Faculty of Biotechnology, Assumption University, Bangkok, Thailand, 10240
2Department of Food Biotechnology, Faculty of Biotechnology, Assumption University, Bangkok, Thailand, 10240

Abstract. Bacillus thuringiensis subspecies israelensis (Bti) has been used as a mosquito control agent in agricultural
field and residences. The production of Bti by conventional medium was expensive. The objective of this study was to
investigate low-cost media for Bti’s production with a cost analysis of the medium formulated. Soybean Casein Digest
Medium (TSB) was used as a conventional medium to compare with Saberi medium at room temperature (approximately
21.5˚C) on static and agitation (by obituary shaker at 100 rpm). Saberi medium was also modified to vary the percentage
of nitrogen and carbon source to observe the growth rate and sporulation, compared to the Saberi medium. The growth of
Bti in TSB with agitation for 24 hours yielded the highest growth rate. Sporulation of Bti in TSB with agitation at room
temperature was 6 days which was faster than Bti in the Saberi medium. Modified Saberi nitrogen source and carbon
source medium could not support the growth of Bti due to an imbalance in the percentage of nitrogen source and carbon
source. Determination of sugar utilization showed sugar in the medium was utilized mostly in the exponential phase of
Bti, from 3 hours to 6 hours. The price of TSB was 76.80 Thai baht per liter while Saberi medium’s cost was 20.43 Thai
baht per liter. In this study, the Saberi medium may not be able to replace TSB in terms of growth rate and period of
sporulation. Further study will be needed to optimize the media that use agro-industrial wastes as ingredients following
the concept of circular economy and sustainability.

Keyword. Bti, Bioinsecticide, Low-cost medium

1 Introduction phospholipids [5]. The function of the gut is disrupted,

causing larval death from starvation [4]
Mosquitoes impact human health and good living. They The Bti production for mosquito control using
are the vector of transmitting diseases, such as Malaria laboratory media is expensive. Previous work by Saberi
and Dengue fever. Thailand is a country with tropical (2020) [6] optimized the culture media for the
rainforest, Asian tiger mosquito (Aedes albopictus) is production of B. thuringiensis var. tenebrionis-BN1
commonly found as a carrier of Chikungunya fever [1]. using molasses from sugar beet and sugar cane as a
The solution to mosquito elimination may end up using carbon source while using corn steep liquor as a nitrogen
insecticide, which may contain volatile chemicals that source. The maximum CFU of 107 spore/ml was
have adverse effects on human health and the achieved with the amount of carbon source above 11%
environment. An example of a dangerous insecticide is and nitrogen source less than 2% [6]. For Bt, the media
DDT, dichloro-diphenyl-trichloroethane, which was that support the growth and sporulation consists of:
banned in many countries. Other classes of chemical glucose, 5 g/L; glycerol, 1 g/L; sodium acetate, 0 g/L;
insecticides, such as organophosphates and carbamate (NH4)2SO4, 1 g/L; corn steep liquor, 2 mL/L; yeast
were used instead of using DDT [2]. Environmental and extract, 1 g/L; hydrolyzed casein, 1 g/L; CaCl2, 20 mg/L;
health concerns have led to the alternative MgSO4, 50 mg/L; MnSO4 100 mg/L provided the
environmentally friendly bioinsecticide growth 6.4 x 10-7 cell/mL and 99% of sporulation [10].
Bacillus thuringiensis subspecies israelensis (Bti) has In this study, the use of sugar cane molasses and corn
been used in many countries to control the population of steep liquor which are agro-industrial waste were
mosquitoes [3]. Many studies demonstrated that Bti is explored. These wastes are used in many industries, such
environmentally safe and target-specific bioinsecticide. as supplements of livestock’s feed, to reduce the
Bti targeted three main insect larvae: mosquitoes, black production cost [11]. The compositions of sugar cane
flies, and fungus gnats [3]. Protoxins of Bti is produced molasses and corn steep liquor provide carbon and
in the stationary phase when endospore formation takes nitrogen source which can be used to promote bacterial
place. After ingestion of toxins, protoxin in form of growth. Hence, the use of both sugar cane molasses and
crystals, including Cry (crystal) and Cyt (cytolytic) corn steep liquor to formulate media for the production
toxins that are solubilized in the alkaline condition in the of BTi will enhance the circle of material usage
larva’s gut, which is activated by midgut protease. Cry following the concept of circular economy and
toxins bind to a protein receptor on the outer membrane sustainability.
of gut cells [4], while Cyt toxins bind to the

*
Corresponding author: [email protected]

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
E3S Web of Conferences 355, 02017 (2022) https://doi.org/10.1051/e3sconf/202235502017
RI 2C 2022

2 Materials and methods steep liquor. Inoculum medium, Saberi medium and
modified Saberi media were autoclaved for 10 minutes at
115˚C. The media were prepared in batches of 350 ml.
2.1 Determination of kinetic growth of Bacillus
thuringiensis subspecies israelensis in Table 1: Compositions of Saberi medium and modified Saberi
conventional medium media.

Soyabean Casein Digest medium TSB (HIMEDIA) was Media Compositions of media
Sugar cane Corn steep MgSO4 KH2PO2 K2HPO4
prepared for 300 mL per batch in triplicate. The Bti was molasses liquor (w/v) (w/v) (w/v)
subcultured in modified TSB [6] overnight as inoculum. (v/v) (v/v)
Three percent (v/v) of Bti inoculum was added to the Saberi 12.5% 1.5% 0.3% 0.3% 0.4%
TSB and incubated at room temperature (approximately medium (5.33%w/v (0.73%w/v
21.5˚C) under shaking (100 rpm) and static conditions (SM) [6] sugar) protein)
Modified 8% 1.5% 0.3% 0.3% 0.4%
(Figure 1). The optical density was measured using Saberi (3.41%w/v (0.73%w/v
UNICO 1200 spectrophotometer at wavelength 600 nm carbon sugar) protein)
in triplicate to observed growth. The development of the source
endospore in Bti was observed by the standard endospore (MSC)
Modified 12.5% 3% 0.3% 0.3% 0.4%
staining technique. Saberi (5.33%w/v (1.45%w/v
nitrogen sugar) protein)
source
2.2 Preparation of Saberi medium and modified (MSN)
Saberi media

The production medium was prepared according to the 2.3 Culturing in Saberi medium and modified
formula from Saberi et. al. (2020) [6] and called as Saberi media
Saberi medium (Table 1). Corn steep liquor, NQ. QCA
3051/21, was sponsored by Friendship Corn Starch Bti was cultured overnight on the orbital shaker at 30˚C
Company Limited. The solid content in the corn steep 100 rpm in the modified TSB [6] as inoculum culture.
liquor was 38.05%. The protein content in the corn steep The 3% (v/v) of overnight Bti was inoculated in each
liquor was 48.46% analyzed by the Kjeldahl method. 350 mL of the media (Figure 1). After the inoculum
Sugar cane molasses was purchased from New Friend culture was added to the batch, 1 mL of the media was
Farm Company Limited. Saberi medium was modified collected for growth measurement using OD at 600 nm
into 2 formulas: Modified Saberi Carbon Source (MSC) and 9 mL from each treatment were collected from each
contains 8% (v/v) of sugarcane molasses and Modified time point (0, 1, 2, 3, 4, 5, 6 and 24 hour) for further
Saberi Nitrogen Source (MSN) contains 3% (v/v) of corn analysis.

Figure 1 Schematic diagram of this experiment related to sections 2.1 and 2.2

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2.4 Determination of growth kinetic of Bacillus 3). The amount of reducing sugar in SM, MSC and MSN
thuringiensis subspecies israelensis in Saberi media however showed a constant level throughout the
medium and modified Saberi media course of growth. Total sugar as inverted sugar was
determined by AOAC Official Method 968.28/2012 in
For the growth of the bacteria, 1 mL of sample collected the SM, MSC and MSN during the growth of Bti. After
in 2.3 from each timepoint was centrifugated at 8000 24 hours total sugar in SM and MSC reduced from
rpm for 2 minutes. The supernatant was discarded; the 12.13% of sugar as inverted sugar to 5.68%. While in
pellet was washed with distilled water and vortexed until MSN, the total sugar was not reduced (Figure 4).
the mixtures became homogenized. The washing step
was repeated before the mixtures were observed using a
spectrometer at 600 nm. The growth curve was
constructed and used to compare doubling time and
specific growth rate using the following equation (eq 1-
2).

Doubling time:
tb = ln (2)/B (1)
Specific growth rate:
µ = [2.303(log OD2 – log OD1)/(t1 – t2)] (2)

2.5 Determination of sugar utilization of Bacillus

Figure 2. Growth of B. thuringiensis subspecies israelensis in
thuringiensis subspecies israelensis in Saberi TSB without agitation, TSB, Saberi medium (SM), modified
medium and modified Saberi media Saberi carbon source (MSC) media, and modified Saberi
nitrogen source (MSN) media with agitation (100 rpm) at room
The sample was collected and centrifuged to remove temperature (approximately 21.5˚C), for 24 hours.
solid particles and bacteria cells at 4,000 rpm for 5
minutes. The supernatant was kept at -20˚C for the sugar
analysis. For reducing sugar, samples were analyzed
using the dinitrosalicylic colorimetric method (DNS) in
duplicate. 1 mL of the sample was diluted in 100 mL of
distilled water. 3 mL of DNS reagent were added to 3
mL of supernatant in a lightly capped test tube. The
mixture was heated at 90˚C for 15 minutes until the red
brown color was developed. 1 mL of 40% (w/v)
potassium sodium tartrate solution was added to stabilize
the color. After cooling to room temperature in a cold-
water bath, the absorbance was observed at 575 nm.
For non-reducing sugar, the Association of Official
Agricultural Chemists (AOAC) Official method
923.09/2012 Invert Sugar in Sugar and Syrups was used Figure 3. Reducing sugar during growth of B. thuringiensis
for the preparation of reagents. AOAC Official Method subspecies israelensis in TSB without agitation, TSB, Saberi
968.28/2012 [7]. Total Sugar in Molasses as Invert Sugar medium (SM), modified Saberi carbon source (MSC) media,
was used to analyze total sugar content as invert in the and modified Saberi nitrogen source (MSN) media with
sample at each time point (0, 1, 2, 3, 4, 5, 6 and 24 agitation (100 rpm) at room temperature, for 24 hours.
hours). 8 mL of centrifugated sample was used for the
total invert sugar analysis. Sugarcane molasses was also
analyzed.

3 Results
The growth of Bti in TSB with agitation (100 rpm), at
room temperature for 24 hours yielded the highest
growth of Bti compared to other conditions and media
(Figure 2). Growth of Bti in Saberi medium showed a
higher growth rate compared to TSB without agitation at
room temperature for 24 hours. When the percentage of
nitrogen and carbon source from the Saberi medium was
changed in the modified Saberi media, both MSC and
MSN, the growth rate of Bti were lowered. Figure 4. Non-reducing Sugar in Saberi medium (SM) and
Reduction of reducing sugar was observed during the modified Saberi media (MSN, MSC) with agitation (100 rpm)
growth of Bti in TSB with and without agitation (Figure at 21˚C, room temperature, for 24 hours after additional B.
thuringiensis subspecies israelensis.

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4 Discussion demonstrated in the growth with Saberi medium. The

total sugar as inverted sugar was reduced from 12.13%
Growth of Bacillus thuringiensis subspecies israelensis to 7.83% in the MSC medium. However, the MSC
was observed in the TSB medium with and without medium could not sustain the growth of Bti as the
agitation. The growth rate and reducing sugar utilization growth rate was reduced after 6 hours (Figure 2).
were significantly faster when agitation was provided. Modified Saberi nitrogen source (MSN) medium
This result confirmed the requirement of agitation as consisting of 3% (v/v) of corn steep liquor was
previously described [6]. The sporulation was also formulated and able to support the growth of Bti for a
observed in the population of Bti that was grown in TSB short period. The highest growth was reached within 6
and found that sporulation was faster in the TSB with hours. The increased percentage of nitrogen source in the
agitation (6 days) (Table 2). As the sporulation was modified Saberi nitrogen source medium could not
associated with the production of the Bti protoxin sustained the growth of Bti which was corresponded to
[4,5,6], the media that stimulate the sporulation in a previous study in B. thuringiensis var. tenebrionis-BN1
shorter time would be more economically feasible. [6]. In addition, the sporulation could not be observed in
Hence, the agitation condition was further used to the Bti that grew in MSC or MSN medium within 14
examine the growth and sporulation of Bti in the Saberi days. The effect on the growth demonstrated in MSC and
medium. MSN medium may be caused by imbalanced carbon and
nitrogen ratios. A carbon per nitrogen ratio of 7:1 is
Table 2. Kinetic growth of B. thuringiensis subspecies effective to support the growth of Bti and yields a high
israelensis in different types of media and conditions. rate of Cry production about 150 g/L [9]. The
development of the endospore on Bti and the doubling
Types of Agitation Doubling Specific Sporulation
media (rpm) time (td) growth (day) time is related. If the media are optimum for Bti, Bti will
(hour) rate (μ) reach the stationary faster and have a shorter doubling
(hour-1) time and shorter development of endospore.
TSB - 3.04 0.419 16 DNS method was used to analyze reducing sugar to
TSB 100 1.46 0.440 8 address sugar utilization of Bti in different media. The
Saberi 100 2.20 0.336 14 reducing sugar in TSB was utilized by the growth
medium observed (Figure 2 and 3). Bti in TSB with agitation
Modified 100 3.25 0.214 > 14 utilized reducing sugar from 35.61 mg/mL to 16.96
Saberi mg/mL while Bti in static TSB utilized from 37.80
nitrogen mg/mL to 24.09 mg/mL with 24 hours. The reducing
source sugar observed from the growth of Bti in SM, MSC and
Modified 100 - - > 14 MSN medium did not change over time, while the
Saberi growth was observed hence indicating utilization of the
carbon
other carbon source. Total sugar in SM, MSC and MSN
source
was analyzed to monitor the utilization of non-reducing
In previous work, different types of carbon sources sugar (Figure 4). As Bti was able to utilize sucrose [8],
and nitrogen sources were tested to formulate production the utilization of total sugar correlated with the growth
mediums that have lower costs [6, 8]. These substrates of the Bti.
include sugar beet and sugar cane molasses, sucrose, and The cost of the medium was analyzed in which the
potato. The sugar cane molasses and corn steep liquor TSB medium cost 76.80 Thai baht per liter, while the
were chosen to be used in this experiment due to the cost Saberi medium cost 20.42 Thai baht per liter. However,
and availability of the raw materials in Thailand. when considering the growth rate and the sporulation
The Saberi media was chosen as described previously time, TSB despite the higher cost still has advantage of
[6], and the amount of carbon source (sugar cane shorter operation time. Further optimization is needed to
molasses) and nitrogen source (corn steep liquor) were formulate a medium that is more feasible economically.
varied to investigate the effect of these nutritional Different carbon and nitrogen sources may be explored.
sources on the growth and sporulation of Bti. In addition, other parameter like temperature, pH and
Growth of Bti in TSB with agitation at room agitation will also need to be optimized.
temperature for 24 hours shows the highest growth rate
compared to all media tested. The doubling time of Bti 5 Conclusion
on TSB with agitation (1.46 hours), was faster than the
doubling time of Bti on TSB without agitation (3.04 The Saberi medium (SM) formulated with 12.5% (v/v)
hours) (Table 1). Growth of Bti in Saberi medium sugar cane molasses and 1.5% corn steep liquor with
showed the doubling time of 2.20 hours, faster than Bti supplementary was able to use for the production of
on TSB in static conditions. The Saberi medium consists Bacillus thuringiensis subspecies israelensis (Bti) when
of 12-14% (w/v) of sucrose, the bacteria can use this grown at room temperature (21.5˚C) with agitation (100
non-reducing sugar for growth. The sporulation of Bti rpm on rotary shaker) with 14 days to sporulate. Despite
that grew in the Saberi medium took place after 14 days. the cheaper cost of 20.42 Thai baht per liter of SM
Modified Saberi carbon source (MSC) medium was compared to 76.80 Thai baht per liter of TSB, further
formulated based on the utilization of total sugar optimization is needed to formulate a more feasible

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utilization of agro-industrial wastes as the main O. Tavakoli, Optimization of culture media to
ingredients of the production media would increase enhance the ability of local Bacillus thuringiensis
materials recycling ability that meets the concept of var. tenebrionis. Journal of the Saudi Society of
circular economy and sustainability. Agricultural Sciences, 19, 7, (2020): 468-475
7. AOAC 968.28-1969, Invert sugar in sugars and
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*
Corresponding author: [email protected]