cost effective medium for the prodution of biopesticide

1. European Journal of Biotechnology and Bioscience
12
European Journal of Biotechnology and Bioscience
Online ISSN: 2321-9122, Impact Factor: RJIF 5.44
www.biosciencejournals.com
Volume 4; Issue 8; August 2016; Page No. 12-16
Cost-effective medium for the production of mosquitocidal toxins from a novel strain Bacillus
thuringiensis subsp. israelensis/tochigiensis (H14/H19)
Shankar K, Prabakaran G, *
Manonmani AM
Vector Control Research Centre (Indian Council of Medical Research) Indira Nagar, Pondicherry, India
Abstract
An indigenous isolate of Bacillus thuringiensis sharing the antigens of israelensis/tochigiensis was for the first time reported to
exhibit mosquito larvicidal activity. In this study a cost effective medium using peanut milk was developed for production of the
mosquitocidial endotoxin for this strain. Shake flask experiments were conducted using 2.5%, 5.0% and 7.5% of peanuts and
compared with the conventional medium for biomass production, spore count and mosquitocidal activity. In stationary phase of
growth, Peanut milk medium 5% gave dry cell mass yield of 3.14 g/L, spore count of 1.5 x 109
CFU/ml and mosquito larvicidal
activity (LC50) of 0.010 µg/ml against late third instar larvae of Culex quinquefasciatus by 18 h as against the dry cell mass yield
of 2.40 g/l, spore count of 1.2 x 109
CFU/ml and mosquito larvicidal activity (LC50) 0.007 µg/ml obtained using 24 h old culture of
the conventional Nutrient yeast Extract Salt Medium (NYSM).
Keywords: Bacillus thuringiensis subsp. israelensis/tochigiensis, Peanut milk, Culex quinquefasciatus, Biomass, Spore count,
Toxicity
1. Introduction
Bacillus thuringiensis subsp. israelensis is a gram positive
spore forming bacteria found ubiquitously in nature. It is well
known for its ability to produce parasporal crystals containing
delta endotoxin concomitantly with sporulation exhibiting
larvicidal upon ingestion by mosquito larvae. B. thuringiensis
subsp. israelensis has drawn much attention due to its specific
activity to mosquito larvae and its lack of effect on mammals
and non-target organisms. B. thuringiensis subsp. israelensis
has been successfully produced, formulated and evaluated in
both laboratory and field conditions [1-4]
. Attempts to isolate
alternate mosquitocidal bacteria from mangrove habitats of
Andaman and Nicobar island of India resulted in a strain of B.
thuringiensis sharing the antigens of israelensis and
tochigiensis, and hence identified as B. thuringiensis subsp
israelensis/tochigiensis (H14/H19). This strain has been
deposited at the culture collection of Vector Control Research
Centre (Indian Council for Medical Research), Pondicherry,
India and given the accession number B-474. Serotypes B.
thuringiensis subsp. tochigiensis (H19) and B. thuringiensis
subsp. israelensis/tochigiensis (H14/H19) are known mosquito
pathogens [5-6]
. This isolate of B. thuringiensis sharing the
antigens of two serotypes could be a potential alternative for
biological control of mosquitoes [7]
.
The conventional laboratory production medium being used
for production of B. thuringiensis subsp. israelensis are Luria
Bertani (LB), Nutrient Yeast Extract Salt Medium (NYSM),
Tryptose broth (TB), Nutrient broth (NB) respectively. These
synthetic media are expensive and a major drawback while up
scaling the production to pilot scale level. Hence, it is an
important requisite to have alternate cheap source of nutrients
for production of biocides which will significantly reduce the
cost of production. Locally available agro industrial wastes
and by products such as coconut cake, neem cake, groundnut
cake, rice bran, coconut water have been used for the
production of B. thuringiensis subsp. Israelensis [8-10]
. Peanut
milk is an extract containing carbohydrates, proteins, fats,
sodium and potassium [11]
. The present study was undertaken
to optimize the process condition for the production of B.
thuringiensis subsp. israelensis/tochigiensis in shake flask
culture using peanut milk as the production media.
2. Material & Methods
2.1 Organism and materials
B. thuringiensis subsp. israelensis/tochigiensis (VCRC B-474)
was obtained from the culture collection of Vector Control
Research Centre (Indian Council of Medical Research),
Pondicherry, India5
. The strain was maintained in the
Modified Nutrient Yeast Salt medium (NYSM), containing 5.0
g glucose, 5.0 g peptone, 5.0 g sodium chloride, 3.0 g beef
extract, 0.5 g yeast extract, 20 g agar, 0.203 g MgCl2, 0.102 g
CaCl2 and 0.01 g Mncl2 (Sigma) per liter of water, pH 7 [12]
.
2.2 Media preparation
Different quantities of the kernel of fresh peanuts (Arachis
hypogaea L.) viz., 2.5 g, 5.0 g and 7.5 g were crushed by
adding small quantity of distilled water using mortar and
pestle. The volume of the extract was than made up to 100 ml
and the resultant slurry was filtered with double layered
cheese cloth to remove all the insoluble solid particles present
in the medium and labeled as PM1, PM2 and PM313
. The
Modified Nutrient Yeast Salt Medium (NYSM) was used as
the reference medium. The pH of each of the media was
adjusted to 7.0. All the media were sterilized by autoclaving at
121°C for 15 min.
2.3 Seed culture preparation
The seed culture was prepared by using shake flasks. First
stage seed was prepared by inoculating 10 ml of NYSM broth
with one loopful culture of B. thuringiensis. subsp.
israelensis/tochigiensis and incubating the tube on a rotary
shaker at 30°C, 250 rpm for 12 hours. Second stage seed was

2. European Journal of Biotechnology and Bioscience
13
prepared by transferring 2.5 ml of first stage seed to 250 ml
flask containing 50 ml of NYSM broth and incubated in a
shaker at 30°C, 250 rpm for 7 h.
2.4 Shake flask experiment
The second stage seed thus prepared was transferred to 100 ml
of the peanut milk media (PM1, PM2 and PM3) and NYSM
medium, each contained in 500 ml Erlenmeyer flask at 2%
level (v/v). The flasks were incubated on a rotary shaker at
30˚C, 250 rpm for a period of 48 h. Cultures were harvested
after 48 h and used for assessing significant differences (P<
0.05) in the cell mass, sporulation and larvicidal activity.
Based on statistical significance the best among the three
peanut milk media comparable with NYSM was selected for
further study. Inoculum at 2% level from actively growing
second stage seed was inoculated to 100 ml of selected peanut
milk and NYSM media contained in 500 ml Erlenmeyer flask
and incubated on a rotary shaker at 250 rpm for a period of 48
hour. Flasks were removed at 6 h interval for up to 48 h and
used for assessing significant differences (P< 0.05) in the cell
mass, sporulation, and larvicidal activity.
2.5 Cell mass
Hundred milliliters of samples collected at different hours
were centrifuged at 15,000 g for 20 min. The supernatant was
discarded and the cell pellet was lyophilized. Dry weight was
calculated and expressed in grams per liter. The lyophilized
sample was used for determination of larvicidal activity.
2.6 Spore count
One milliliter of the culture samples collected from the various
media at different hours was heat treated at 80°C for 15 min,
serially diluted and plated on NYSM agar plates. Plates were
incubated at 30°C for 48 h and the developing colonies were
counted and expressed in CFU (colony forming units) per ml.
2.7 Toxicity test
The spore crystal complex produced in various media at
different hourly intervals was assayed against laboratory
reared late third instar larvae of Culex quinquefasciatus. A
lyophilized powder of 10 milligram was added with 10 ml of
sterile distilled water and mixed using a homogenizer to obtain
a uniform suspension. Range finding bioassays were
conducted using different concentration of suspensions added
to disposable cups, containing 25 larvae in 100ml of chlorine
free tap water. Each experimental bioassay included 5 doses of
four replicates each along with the appropriate control. Larval
mortality was scored after 24 h and corrected for control
mortality, using Abbott’s formula [14]
. The experiment was
done thrice on different days. The results were expressed in
µg/ml.
2.8 Statistical analysis
The significance of difference between the LC50 of the
different studied media with that of the strain B. thuringiensis
subsp. israelensis/tochigiensis was tested by the 95%
confidence limits of lethal concentration ratio (LCR) at the
LC50. Lethal concentration was significantly different (P<
0.05) when the confidence limit did not include the value one
[15]
. All the results are average of three repetitions using
different cultural condition. Experimental data for cell mass
and spore counts was analyzed using the Tukey’s test
performed after analysis of variance (ANOVA) for
comparison of the means. All differences were regarded as
significant (P< 0.05). The statistical analyses were performed
using SPSS v 15.0 Software (SPSS Inc., Chicago, IL, USA).
3. Results
3.1 Biomass production in peanut milk and NYSM
medium
The dry weight of the mean biomass produced by 48 hours in
PM1, PM2, PM3 and NYSM were 1.71 g/l, 2.70 g/l, 1.53 g/l
and 1.97 g/l respectively (Fig 1). The biomass production in
the 4 media showed significant difference (F= (3, 8) =120.07,
p< 0.5). A Tukey post hoc multiple comparison test, (p< 0.05)
indicated that PM2 had significantly higher biomass than the
other three media. All other comparisons were not significant.
Fig 1: Dry cell mass of B. thuringiensis subsp. Israelensis /
tochigiensis grown in PM1, PM2, PM3 and NYSM medium.
3.2 Spore count in Peanut milk and NYSM medium
Highest spore count was obtained in PM2 Medium 12.5 x 108
CFU/ml followed by NYSM 11.7 x 108
CFU/ml (Fig 2). In
PM1 and PM3, 2.6 x 108
CFU/ml and 2.3 x 108
CFU/ml were
obtained. The one-way ANOVA showed a statistically
significant difference across the mean spore counts obtained in
peanut milk media and NYSM media (F=3, 8) =24.46, p<.05).
A Tukey test indicated that PM2 (M=12.5, SD=2.24) and
NYSM (M=11.6, SD=2.35) produced more spores than PM1
(M=2.66, SD=1.52) and PM3 (M=2.33, SD=1.52)
respectively. The Tukey post-hoc multiple comparison test, (p
> 0.05) indicated that there was no significant difference in the
spore count obtained in PM2 and conventional medium
NYSM, whereas the spore count in PM1 and PM3 were
significantly lower (P< 0.05).
Fig 2: Spore count of B. thuringiensis subsp. israelensis/tochigiensis
grown in PM1, PM2, PM3 and NYSM medium (CFU/ml).

3. European Journal of Biotechnology and Bioscience
14
3.3 Toxicity of the lyophilized cell mass obtained from
Peanut milk and NYSM medium
The LC50 of the cell mass obtained from PM1, PM2 and PM3
were 0.021, 0.012 and 0.025 µg/ml respectively whereas that
obtained from NYSM was 0.007 µg/ml. Maximum toxicity
was noticed in PM2 and conventional medium NYSM (Table
1). There was no significant difference between the LC50
values obtained from PM2 and NYSM medium based on
overlap of 95% fidicial limits.
Table 1: Toxicity of B. thuringiensis subsp. israelensis/tochigiensis grown in different production media
against late third instar larvae of C. quinquefasciatus.
Coefficients of probit
Regression equation a
Y=a + b log10 X
Peanut milk and
Nysm medium
a b χ2
LC50 (µg/ml) (95% FL) LC90 (µg/ml) (95% FL)
PM1 -1.490 .690 55.9 0.021(0.018-0.026) 0.039(0.032-0.056)
PM2 -1.153 .092 02.90 0.012(0.009-0.013) 0.020(0.018-0.031)
PM3 -1.408 .733 47.24 0.025(0.022-0.030) 0.046(0.043-0.054)
NYSM -1.312 .274 32.13 0.007(0.005-0.009) 0.015(0.014-0.018)
a
y=probit mortatlity, log 10X = log dose in microgram of spore crystal complex/ml of chlorine free tap water
3.4 Dynamics of biomass, sporulation and toxin synthesis
in PM2 and NYSM Medium
The cultures collected at 6 hourly intervals from NYSM and
PM2 was analyzed for biomass production, sporulation and
toxin synthesis. The biomass produced during the different
hours in there 2 media ranged from 2.2 to 2.7 and 2.44 to 3.48
g/L respectively (Fig 3). The conventional medium did not
show any toxin production up to 12 h. Toxins were observed
from 18 h onwards and reached a maximum level at 24 h.
From 24 to 48 h there was no increase in toxicity. In PM2
medium toxin production started from 12 h onwards and
reached maximum level at 18 h. From 18 to 48 h there was no
increase in toxicity (Tables 2 and 3). The highest spore count
obtained in PM2 and NYSM were 1.5 x 109
(CFU/ml) and 1.2
x 109
(CFU/ml) respectively at 18 and 24 h (Table 4). The
mean difference in the spore count between two media was
not significant (student’s t-test for independent samples, t =
0.923, d.f = 46, p=0.92).
Fig 3: Dynamics of cell mass production of B. thuringiensis subsp.
israelensis/tochigiensis in PM2 and NYSM medium.
Table 2: Toxicity of different hourly cultures of B. thuringiensis subsp. israelensis/tochigiensis grown in NYSM media.
Coefficients of probit
Regression equation a
Y=a + b log10 X
Hours a b χ2
LC50 (µg/ml) (95% FL) LC90 (µg/ml) (95% FL)
6 - - - - -
12 -1.16 .002 12.05 0.057(0.052-0.062) 0.100(0.091-0.111)
18 -2.75 .020 4.60 0.031(0.028-0.036) 0.069(0.066-0.080)
24 -2.04 .028 26.09 0.007(0.005-0.008) 0.011(0.009-0.013)
30 -2.95 .033 18.65 0.008(0.006-0.011) 0.012(0.010-0.015)
36 -1.88 .020 19.03 0.009(0.005-0.013) 0.016(0.013-0.019)
42 -2.00 .019 13.93 0.010(0.006-0.015) 0.017(0.015-0.021)
48 -2.00 .018 14.51 0.011(0.009-0.013) 0.018(0.016-0.023)
a
y=probit mortatlity, log 10X = log dose in microgram of spore crystal complex/ml of chlorine free tap water
Table 3: Toxicity of different hourly cultures of B. thuringiensis subsp. israelensis/tochigiensis grown in PM2 media.
Coefficients of probit
Regression equation a
Y=a + b log10 X
Hours a b χ2
LC50 (µg/ml) (95% FL) LC90 (µg/ml) (95% FL)
6 - - - - -
12 -1.101 .022 25.5 0.049(0.019-0.060) 0.101 (0.092-0.110)
18 -1.392 .253 52.53 0.010 (0.008-0.012) 0.020 (0.017-0.033)
24 -1.376 .252 20.99 0.012 (0.010-0.013) 0.022 (0.019-0.027)
30 -1.339 .252 19.27 0.013 (0.010-0.015) 0.025 (0.017-0.029)
36 -1.399 .252 13.13 0.015 (0.011-0.018) 0.026 (0.020-0.039)

4. European Journal of Biotechnology and Bioscience
15
42 -1.541 .254 10.54 0.014 (0.010-0.019) 0.028 (0.021-0.033)
48 -1.36 .252 24.6 0.015(0.009-0.021) 0.029(0.018-0.040)
a
y=probit mortatlity, log 10X = log dose in microgram of spore crystal complex/ml of chlorine free
Table 4: Spore count of B. thuringiensis subsp. Israelensis /
tochigiensis grown in different time intervals in NYSM and PM2
media.
Hours NYSM (CFU/ml) PM2 (CFU/ml)
6h Nil Nil
12h Nil 7 X 105
18h 3 x105
1.5 x 109
24h 1.2 x 109
1.3 x 109
30h 1.1 x 109
1.5 x 109
36h 1.2 x 109
1.5 x 109
42h 1.0 x 109
1.4 x 109
48h 1.2 x 109
1.5 X 109
4. Discussion
The difference in growth and toxic activities of B.
thuringiensis subsp. israelensis/tochigiensis in different media
may be due to the different growth nutrients available in the
production medium. Peanut milk is the water extract of peanut
containing carbohydrates, proteins, fats and minerals [11]
when
the ratio of peanut extract with water is much diluted, then the
fat content of the peanut milk will be very low too. Since it is
low it will not affect the growth and reported to support good
growth of lactic acid bacteria [16]
. Variability in production
using media derived from various nutrient sources has been
reported by several research workers. Researchers used tofu-
whey as production media for B. thuringiensis subsp.
israelensis and showed toxicity to second instar larvae of A.
aegypti with LC50 value of 0.7µg/ml [17]
. Other raw materials
such as soybean flour, ground nut cake powder and wheat bran
extract have been used for culturing B. thuringiensis subsp.
israelensis in 100L fermentor. The sporulation, toxicity and
biomass yield were satisfactory in all the three media.
However, the cell mass harvested from soya bean culture
medium exhibited maximum toxicity LC50 8.89 ng/ml against
the third instar larvae of C. quinquefasciatus [18]
. A cost
effective medium for B. thuringiensis subsp. israelensis was
developed based on coconut water where in a cell mass of
3.1g/l, spore count of 3.4 x 1011
and mosquito larvicidal
activity (LC50 of 14.85 ng/ml) against fourth instar larvae of
Aedes aegypti were obtained with 30 h old culture which was
similar to that obtained with NYSM [10]
. Other media with less
expensive ingredients like chicken feather and rice bran
required prior boiling, and longer cultivation period up to 120
hours [19]
. In Brazil corn steep liquor, a corn industrial
processing by product and tryptose broth with/without
addition of sugar, were compared. The medium composed of
glucose plus corn steep liquor gave higher sporulation and
higher toxicity value for B. thuringiensis subsp. israelensis
after 20 hours [20]
. In this newly designed peanut milk medium
(PM2), maximum level of toxicity was observed by 18 h,
when the LC50 against late third larvae of C. quinquefasciatus
was 0.010 µg/ml, spore count 1.5 x109
CFU/ml and cell mass
3.14 g/l, compared to NYSM which exhibited maximum
toxicity by 24 h (LC50 - 0.007 µg/ml), gave a spore count of
1.2 x 109
CFU/ml and cell mass yield of 2.40 g/l. There was no
significant difference (P > 0.05) between the LC50 values of
the toxins produced in PM2 and NYSM medium as their 95%
confidence limits overlap. Spore counts are known to be more
accurate than dry mass for yield determinations, because dry
biomass is affected by suspended solids in the media [21]
. This
is very clearly seen in this study as the dry mass obtained in
peanut medium was relatively higher than that obtained in
NYSM medium while the spore count was relatively on par
with NYSM medium. Therefore harvesting the culture at the
end of the stationary phase will allow a maximum recovery of
the spore crystal complex. Once autolysis begins
sedimentation of the spore crystal complex is difficult and
some crystals will go in to the solution. The production cost of
peanut milk medium is US $ 0.050 per liter when compared to
NYSM medium which is US$ 0.50. Hence, the newly
designed medium for production works out to be 10 times
cheaper than the conventional NYSM medium, produces
mosquitocidal toxin 6 hours earlier and the raw material
namely peanut seeds are available throughout the year. Hence
this study has brought out a cheap and easily available raw
material, peanut seeds as a source for the production of
mosquitocidal toxins of B. thuringiensis subsp.
israelensis/tochigiensis (VCRC B-474).
5. Conclusion
The new production medium based on peanuts was found to
be very useful medium in small scale production of
mosquitocidal toxin. Further, the cost of the peanut milk
medium was 10 times cheaper than the conventional medium
(NYSM).
6. Acknowledgment
We are grateful to Dr. P. Jambulingam, Director, VCRC,
Pondicherry, for his critical suggestion. We thank Dr. I.
Geetha and other technical staff of the microbiology division
for their support in this study.
7. References
1. Adams TT, Eiteman MA, Adang MJ. B. thuringiensis
subsp. kurstaki spore production in batch culture using
broiler litter extracts as complex medium. Bioresour.
Technol 1999; 67(1):83-87.
2. Manonmani AM, Prabakaran G, Hoti SL. Retention of
mosquito larvicidial activity of lyophilized cells and WDP
formulation of B. thuringiensis subsp. israelensis on long-
term storage. Acta Trop 2008; 105(2):170-175.
3. Gunasekaran K, Prabakaran G, Balaraman K. Efficacy of
a floating sustained release formulation of B.
thuringiensis subsp. israelensis in controlling Culex
quinquefasciatus larvae in polluted water habits. Acta
Trop 2002; 83(3):241-247.
4. Prabakaran G, Hoti S.L. Immobilization of alginate as a
technique for the separation of B. thuringiensis subsp.
israelensis spore crystal complex. Biol. Control 2012;
61(2):128-133.
5. Geetha I, Prabakaran G, Paily KP, Manonmani AM,
Balaraman K. Characterization of three mosquitocidal
Bacillus strains isolated from mangrove forest. Biol.
Control 2007; 42(1):34-40.
6. Lonc E, Doroszkieewicz W, Klowden MJ, Rydzanicz K,
Galgan A. Entomopathogenic activities of environmental

5. European Journal of Biotechnology and Bioscience
16
isolates of B. thuringiensis against dipteran larvae. J.
Vector. Ecol. 2001; 26(1):15-20.
7. Maeda M, Mizuki E, Hara M, Tanaka R, Akao T,
Yamashita S, Ohba M. Isolation of B. thuringiensis from
intertidal brackish sediments in mangroves. Microbiol.
Res 2001; 156(2):195-198.
8. Poopathi S, Archana B. A Novel cost effective medium
for the production of B. thuringiensis subsp. israelensis
for mosquito control. Trop. Biomed 2012; 29(1):81-91.
9. Poopathi S. Current research technology and education
topics in applied microbiology and microbial
biotechnology. Spain, Edn 2, 2010; 1:349-358.
10. Prabakaran G, Hoti SL, Manonmani AM, Balaraman K.
Cocunut water as a cheap source for the production of
delta endotoxin of B. thuringiensis subsp. israelensis, a
mosquito larvicidal agent. Acta Trop 2008; 105(1):35-38.
11. Isanga J, Zhang G. Production and evaluation of some
physiochemical parameters of peanut milk yoghurt. LWT-
Food Science and Technol 2009; 42(6):1132-1138.
12. Myers PS, Yousten AA. Localization of a mosquito-larval
toxin of B. sphaericus 1593. Applied Environ. Microbiol
1980; 39(6):1205-1211.
13. Utami T, Giyarto, Djaafar TF, Rahayu ES. Growth of
Lactobacillus paracaesai SNP-2 in peanut milk and its
survival in fermented peanut milk drink during storage.
Indonesian Food and Nutrition Progress 2014; 13(1):11-
16.
14. Abbott WS. A Method for computing the effectiveness of
an insecticide. J. Econ. Entomol. 1925; 18(2):265-267.
15. Robertson JL, Priesler HK. Pesticide bioassays with
Arthropods, CRC Press, Boco Raton, Florida, Edn 1,
1992; 1:125.
16. Giyarto, Djaafar TF, Rahaya ES, Utami. Fermentation of
peanut milk by Lactobacillus acidophilus SNP-2 for
production of non-dairy probiotic drink. Proceedings of
3rd
International Conference of Indonesian Society for
Lactic acid bacteria: Better life with LAB: Exploring
Novel functions of LAB, 2012, 9p.
17. Purnawati R, Sunarti TC, Syamsu K, Rahayuningsih M.
Characterization of novel B. thuringiensis isolated from
Attacus atlas and its growth kinetics in the cultivation
media of Tofu whey for bio insecticide production. J.
Biol. Agric. Healthcare. 2014; 4(16):33-39.
18. Prabakaran G, Balaraman K. Development of a cost
effective medium for the large scale production of B.
thuringiensis subsp. israelensis. Biol. Control 2006;
36(3):288-292.
19. Poopathi S, Abidha S. Use of feather based culture
medium for the production of mosquitocidal bacteria. Biol
Control 2007(1); 43:49-55.
20. Ernades S, Del Bianchi VL, Moraes IO. Evaluation of two
different culture media for the development of
bioinsecticides based on B. thuringiensis and their
application in larvae of Aedes aegypti. Acta Sci. Biol
2013; 35(1):11-18.
21. Devidas PC, Pandit BH, Vitthalrao PS. Evaluation of
different culture media for improvement in bioinsecticides
production by indigienous B. thuringiensis and their
application against larvae of Aedes aegypti. Sci World J.
2014(2014):1-6.