World J Microbiol Biotechnol (2010) 26:153–159
DOI 10.1007/s11274-009-0154-8
ORIGINAL PAPER
Production of mosquitocidal Bacillus sphaericus by solid state
fermentation using agricultural wastes
Magda A. El-Bendary
Received: 15 April 2009 / Accepted: 5 August 2009 / Published online: 23 August 2009
Ó Springer Science+Business Media B.V. 2009
Abstract In this study, Bacillus sphaericus NRC 69 was
grown in culture media, in which 12 agricultural wastes
were tested as the main carbon, nitrogen and energy sources
under solid state fermentation. Of the 12 tested agricultural
by-products, wheat bran was the most efficient substrate for
the production of B. sphaericus mosquitocidal toxins
against larvae of Culex pipiens (LC50 1.2 ppm). Mixtures of
tested agricultural wastes separately with wheat bran
enhanced the produced toxicity several folds and decreased
LC50 between 3.7- and 50-fold in comparison with that of
agricultural wastes without mixing. The toxicity of
B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and
wheat bran/barley straw at 1/4 (g/g) showed the same tox-
icity as that in wheat bran medium (LC50 decreased 17- and
16-fold, in comparison with that in rice hull or barely straw
media, respectively). In wheat bran medium, the maximum
toxicity of the tested organism obtained at 50% moisture
content, inoculum size 84 9 106 CFU/g wheat bran and
incubation for 6 days at 30°C. Addition of cheese whey
permeate at 10% to wheat bran medium enhanced the tox-
icity of B. sphaericus NRC 69 about 46%.
Keywords Bacillus sphaericus
Agricultural wastes

Solid state fermentation
Culex pipiens
Introduction
Over the past couple of years, solid state fermentation
(SSF) involving growth of microbes on moist solid
M. A. El-Bendary (&)
Microbial Chemistry Department, National Research Centre,
El-Behoos Street, Doki, Giza, Egypt
e-mail: tasnim41@yahoo.com
substrate(s) in the absence of free flowing water, has gained
a tremendous momentum owing to certain advantages over
the conventional submerged fermentation like low pro-
duction cost, saving of water and energy, less waste
effluent problem and stability of the product due to less
dilution in the medium (Pandey 2003; Holker and Lenz
2005). Therefore, SSF has found several potential appli-
cations in the industrial production of value added products
such as industrially important microbial enzymes, bioin-
secticides, secondary metabolites and pharmaceuticals
(Robinson et al. 2001; Pandey 2003).
At present, the overall cost of bio-insecticides production
are very high due to the high cost of the substrates and
media used (Prabakaran and Balaraman 2006). The cost of
raw materials used for the production of Bacillus thuringi-
ensis based bio-pesticides as example was about 35–59% of
the production cost as estimated by Stanbury et al. 1995.
Therefore, there is an urgent need to find high yielding, cost
free or low cost and year round available raw materials for
bio-insecticide production (Yezza et al. 2006). This goal
can be achieved by using agricultural wastes as substrates.
In Egypt, there is about 26 million tons of plant
by-products produced annually. The main approach for dis-
posing of these wastes is in vogue incineration and treatment
for animal feed (Economic and statistics institute 2000).
Bacillus sphaericus (B. sphaericus) is a gram positive,
aerobic, spore-former bacterium. Some strains of B. sph-
aericus are toxic to mosquito larvae and used as biocontrol
agent for disease-transmitting mosquitoes (de Barjac
1990). Mosquitocidal strains of B. sphaericus traditionally
produced by submerged fermentation. However, this
technology is constrained by capital costs and requirements
for fermentation expertise. Inexpensive media were used
for production of B. sphaericus under submerged fermen-
tation such as cotton seed meal and soybean meal (Singer
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1981), cow blood and bambara beans (Obeta and Okafor Table 1 Agricultural by-products composition
1983), corn steep liquor (Kuppusamy and Balaraman Agricultural by- Total proteins Total carbohydrates Ash
1991), mustard seed meal (Gangurde and Shethna 1995), products (%) (%) (%)
potato sugar and chick pea (Poopathi et al. 2002), brewery
Wheat bran 16.08 78 5.92
residues (Martins et al. 2006), peanut cake powder (Pra-
bakaran et al. 2007) and feather meal (Poopathi and Abidha Rice hull 2.48 74.18 23.34
2007). Although B. sphaericus production by solid state Wheat straw 2.24 86.96 10.8
fermentation (SSF) requires less capital investment and Rice straw 6.46 79.48 14.06
modest technical skills, few reports have been found. Foda Barely straw 3.28 82.12 14.60
et al. 2003, produced B. sphaericus 14N1 with high level of Corn stover 4.44 85.43 10.13
toxicity against Culex pipiens on cotton seed meal, sesame Corn cobs 6.38 85.01 8.61
seed meal, fodder yeast and linen seed meal in the presence Cotton stakes 5.14 88.52 6.34
of wheat bran as a carrier material using SSF technology. Olive meal 18.83 74.15 7.02
Also, B. sphaericus was efficiently produced by using Date stone 15.84 81.62 2.54
whey permeate under submerged and solid state fermen- Pea peels 7.91 87.28 4.81
tations by El-Bendary et al. 2008. Potato peels 13.11 69.81 17.08
Optimization of B. sphaericus production using the
agricultural by-products under solid state fermentation can
effectively promote the use of this bacterium in disease-
transmitting mosquito management programs, especially in Nutrient broth/agar medium was used for inoculum
developing countries and it also reduces environmental preparation and maintenance of tested culture.
pollution.
The aim of the present study was investigation of the SSF and optimization of process conditions
potential of locally available cost free or inexpensive
agricultural wastes and agro-industrial by-products as For screening the various substrates/supports for mosqui-
substrates/solid supports for production of mosquitocidal tocidal production, initially the same volumes of course
toxins by B. sphaericus under solid state fermentation substrates were taken individually in 250 ml Erlenmeyer
technology. Optimization of the fermentation conditions flasks (to maintain the same aeration conditions) and mixed
such as moisture content of the materials, aeration level with a predetermined quantity of tab water for each sub-
and optimal mixing ratios of promising substrates with strate. Because of these materials, greatly differ in their
wheat bran was investigated. Also, inoculum size, incu- water holding capacity, a suitable moisture content for each
bation period and addition of some additives for the best substrate was roughly determined by adding water to the
substrate were studied. tested substrate until it was moistened. Weights of different
substrates and their final moisture content can be shown in
Table 2. These flasks were autoclaved and inoculated with
Materials and methods one ml of B. sphaericus NRC 69 (about 21.1 9 107 CFU)
and then incubated at 30°C for 6 days under static condi-
Solid agricultural wastes used as substrates/solid tions. Each fermentation test was repeated two times in
supports duplicate.
Effect of moisture content was studied by varying the
A group of dried ground agricultural by-products was moisture content between 20 and 90%.
employed as substrates/supports and the main source of The influence of aeration level on mosquitocidal activity
nutrients under SSF for growth and mosquitocidal toxin of B. sphaericus NRC 69 was investigated for the most
production by B. sphaericus NRC 69 without any pre- promising substrates by transferring different amounts of
treatment. These substrates were from Animal Nutrition substrates into 250 ml Erlenmeyer flasks. The amounts
Department, National Research Center. Table 1 shows the varied between 1.5 and 12 g for wheat straw, rice straw,
agricultural wastes used and their composition according to corn stover, barely straw and cotton stakes, however, for
Economic and statistics institute 2000. wheat bran, rice hull and corn cobs were varied between
2.5 and 20 g.
Microorganism Effect of mixing different substrates separately with
wheat bran at different ratios was tested for improving the
Egyptian isolate, B. sphaericus NRC 69 (El-Bendary produced toxicity by B. sphaericus NRC 69 grown on these
1999), was used in this study. substrates against Culex pipiens larvae.

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Table 2 Effect of agricultural by-products on toxin production by B. sphaericus NRC 69

Substrate Humidity Final pH Viable count Spore count Mortality % after 48 h at (ppm)
(g/250 ml flask) (%) (9109 CFU/g) (9109 CFU/g)
100 10

Wheat bran (5) 50 7.9 120 116 100 100

Rice hull (5) 50 7.5 11 9 100 10
Wheat straw (2.5) 50 7.8 38 25 40 10
Rice straw (2) 67 8.3 19 4 90 0
Barely straw (2.5) 74 7.9 45 38 95 10
Corn stover (3) 73 7.3 9 6.5 70 20
Corn cobs (8.5) 41 7.9 7 5 10 0
Cotton stakes (4) 67 8.5 19 11.5 60 0
Olive meal (12) 33 7.9 6 3.5 0 0
Date stone (10) 44 7.9 2.5 1 0 0
Pea peels (10) 50 7.4 7 1 0 0
Potato peels (8.5) 45 7.4 6.5 0.5 0 0

Effect of inoculum size of the tested culture on toxin
production in wheat bran medium was studied by varying the
inoculum size between 10.5 9 106 and 336 9 106 CFU/g
wheat bran.
Effect of incubation period was studied for extended
incubation periods (2–9 days) on wheat bran medium.
Addition of selected nutritional supplements to wheat
bran medium such as NYSM salts (0.2–1%), dipotassium
hydrogen phosphate (50–200 mM), corn steep liqure (0.2–
1%) and whey permeate (10–100%) was tested.
Total viable cell and spore counts were estimated by
plate count method.
Bioassay of mosquitocidal activity
Bioassay of mosquitocidal activity of B. sphaericus NRC
69 produced under SSF was adopted from Ampofo (1995)
with some modifications. Toxicity was determined with
laboratory reared second instar larvae of Culex pipiens.
One gram of the final product was mixed with dechlori-
nated tap water (100 ml) and shaken for 1 h. Serial dilu-
tions were prepared and the dilutions were placed into
100 ml beakers in triplicate along with 20 second instar
Culex pipiens larvae. About 10 mg of ground fish meal was
added to each cup. The beakers were covered with muslin
and kept at 26 ± 2°C with 10 h light/14 h dark cycle. The
mortality percentage was recorded after 48 h by counting
the number of living larvae and corrected by using
appropriate control and adopting Abbott’s formula (Abbott
1925). Each bioassay was replicated two times.
Statistical analysis
Data obtained from this study were statistically analyzed
according to SPSS system (SPSS 1998) using one-way
analysis and the Duncan’s multiple range test (Duncan
1955) to determine the significance between means. Data
were expressed as mean values ± standard errors.
Prpbit regression analysis (Proban version 1.1, Dr Rys-
zard A. Jedrychowski 1991) was carried out to calculate
LC50 as well as 95% fiducial limits.
Results and discussion
Screening of different agricultural wastes
for mosquitocidal toxin production
The results presented in Table 2 showed that wheat bran
supported the highest growth, sporulation and toxicity of
B. sphaericus NRC 69 against second instar larvae of Culex
pipiens. B. sphaericus cannot utilize carbohydrates as
carbon source due to the lack of the early enzymes of
Embeden Meyerhoff Parnas and hexose monophosphate
pathways (Russel et al. 1989). Instead, it can use amino
acids as carbon and nitrogen sources. It is known that
wheat bran contains 78% carbohydrate, 16% protein, 6%
minerals and vitamins. Therefore, wheat bran medium
almost contains all the requirements for growth and toxin
production by B. sphaericus NRC 69.
Wheat bran has been widely used for production of
entomopathogenic fungi (Jagadeesh and Geeta 1994;
Sangeetha et al. 1993). It was reported that Bacillus thur-
ingiensis israelensis produced using 100 l-fermentor using
wheat bran extract giving a product with higher toxicity
than the growth in NYSM medium (Prabakaran and Bal-
araman 2006). Also, Bacillus thuringiensis produced on
wheat bran medium under SSF by Suyanandona et al.
1996; Hongzhang et al. 2002; Devi et al. 2005.

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The growth of tested organism on rice hull, wheat straw,
rice straw, barely straw, corn stover and cotton stakes
showed promising activities against second instar larvae of
Culex pipiens. On the other hand, corn cobs, olive meal,
date stone, pea peels and potato peels did not support the
growth or toxicity of the tested organism. Lower growth
and lower toxicity of the tested organism grown in these
substrates may be related to lower protein level in this
group or unavailability of the soluble protein for spore and
crystal metabolism or carryover of some inhibitory factors
into the medium. The sporulation of B. sphaericus NRC 69
grown in these substrates is not related to the toxicity
degree with exception of wheat bran. The toxicity of the
tested organism produced on rice straw as example nearly
the same as that produced on barely straw while the
sporulation degree is lower 9.5 fold.
Although, the crystal protein synthesis in B. sphaericus
is dependent on early sporulation (El-Bendary et al. 2005),
the independence of toxicity level and extent of sporulation
in B. sphaericus cultures has been observed by several
authors (Yousten et al. 1984; Yousten and Wallis 1987;
Karim et al. 1993; El-Bendary 1999; Foda et al. 2003; El-
Bendary et al. 2008).
Effect of initial moisture content
Moisture content is a critical factor in SSF. Its importance
for microbial growth and thereby metabolites production
has been well established. As well as, optimum level of
initial moisture content is required for maximum substrate
utilization in SSF.
The maximum toxicity of B. sphaericus NRC 69
(expressed as mortality percent of Culex pipiens larvae)
grown on wheat bran (63.3 ± 2.2 at 5 ppm) and pea peels
(69.5 ± 1.8 at 500 ppm) was at 50% moisture content.
While 70% moisture was the most suitable for rice hull
(73.3 ± 2.1 at 50 ppm), wheat straw (50.4 ± 2.3 at
100 ppm), rice straw (88.8 ± 2.2 at 100 ppm), corn stover
(61.7 ± 1.9 at 100 ppm), corn cobs (61.7 ± 1.8 at
100 ppm) and date stone (50.8 ± 2.6 at 500 ppm). Opti-
mum moisture content was between 60 and 80% for the
highest mosquitocidal activity of tested organism grown on
barely straw (78.8 ± 2.5–84.2 ± 1.4 at 100 ppm). The
toxicity of B. sphaericus NRC 69 grown on potato peels
(48.3 ± 1.7 at 500 ppm) and cotton stakes (57.5 ± 1.6 at
100 ppm) was the maximum at 60% and 80% moisture
content, respectively. Olive meal showed the lowest
moisture content (40%) for growth and toxicity (50 ± 1.6
at 500 ppm) of the tested organism.
The moisture level in SSF has a great impact on the
physical properties of the substrate (Mahanta et al. 2007).
An increase in moisture content causes a decrease in
porosity of the substrate, thereby decreasing the gas
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exchange. A low moisture level leads to sub-optimal
growth and a lower degree of substrate swelling which also
decreases microbial metabolites production (Pandey 2003).
Earlier reports indicated that 50% moisture content was the
best for the maximum mosquitocidal toxin production by
B. sphaericus grown on wheat bran-cotton seed meal and
wheat bran-whey permeate media under SSF (Foda et al.
2003; El-Bendary et al. 2008).
Effect of aeration level
The toxicity of B. sphaericus NRC 69 was the maximum
when grown in ten grams of wheat bran (95.8 ± 1.2 at
5 ppm) or corn cobs (70.4 ± 1.3 at 100 ppm) in 250 ml
flask while the toxicity was stable (about 79.6 ± 2.6 at
50 ppm) within a wide range of increasing the quantity of
rice hull in the flasks (2.5–15 g/flask). The maximum
toxicity of the organism grown in wheat straw (97.5 ± 0.8
at 100 ppm) or cotton stakes (98.8 ± 0.9 at 100 ppm) were
at 1.5–3 g/flask., however, for rice straw (99 ± 0.6 at
100 ppm) or corn stover (89.2 ± 2.3 at 100 ppm) were at
6 g/flask while for barely straw (95.4 ± 1.4 at 100 ppm)
was at 3 g/flask.
Aeration fulfils four main functions in SSF, namely (1)
to maintain aerobic conditions, (2) for carbon dioxide
desorption, (3) to regulate substrate temperature and (4) to
regulate the moisture level (Raimbault 1998).
Effect of mixture of different substrates separately with
wheat bran
Research into suitable agricultural by-products for mass-
production of bio-pesticides can effectively contribute for
promoting use of these by-products in insect pest man-
agement programs especially in less industrialized coun-
tries. Therefore, the effect of mixing of some substrates to
wheat bran for enhancing the toxicity of B. sphaericus
NRC 69 grown on these substrates against second instar
larvae of Culex pipiens is shown in Tables 3, 4 and 5. The
organism grown on wheat bran/rice hull medium at 8/2 (g/
g) and wheat bran/barely straw medium at 1/4 and 2/3 (g/g)
showed high toxicities and they were comparable to that of
the tested organism grown on wheat bran medium. LC50 of
the organism grown on these media were decreased about
17- and 15.7-fold in comparison with that in rice hull or
barley straw media, respectively. The product of wheat
bran/corn cobs medium at 8/2 (g/g) showed lower LC50
(6.9 ppm) than that of corn cobs medium (59.6 ppm) i.e.
LC50 decreased about 8.6 fold. While wheat bran/pea peels
and wheat bran/potato peels media at 6/4 and 4/6 (g/g),
respectively decreased the LC50 of the product about 42.4
and 50.2 fold in comparison with that of pea peels and
potato peels media. LC50 of B. sphaericus NRC 69 grown
World J Microbiol Biotechnol (2010) 26:153–159 157

Table 3 Effect of mixtures of wheat bran with rice hull, potato peels, pea peels or corn cobs on mosquitocidal activity of B. sphaericus NRC 69
grown under SSF
Wheat bran/by- Mortality (%) after 48 h exposure to B. sphaericus NRC 69 grown on
products (w/w)
Wheat bran/rice hull Wheat bran/potato peels Wheat bran/pea peels Wheat bran/corn cobs
At 5 ppm At 10 ppm At 10 ppm At 10 ppm

1/9 40.8 ± 1.7 d 0 5.0 ± 0.6 f 0

2/8 49.2 ± 1.6 c 0 19.6 ± 1.3 e 0
4/6 52.1 ± 2.4 c 50.4 ± 1.6 b 50.0 ± 2.1 d 0
6/4 78.3 ± 2.0 b 19.6 ± 1.9 c 90.0 ± 2.2 b 22.9 ± 1.6 c
8/2 88.3 ± 2.2 a 10.4 ± 1.1 d 58.8 ± 2.2 c 79.2 ± 1.7 b
10/0 90.8 ± 1.5 a 100 ± 0.0 a 100 ± 0.0 a 100 ± 0.0 a
0/10 0 0 0 0
Mortality % is presented as means ± SE. Values for each treatment followed by different letters are significantly different at P = 0.05

Table 4 Effect of mixtures of wheat bran with wheat straw, rice straw, barely straw, corn stover or cotton stakes on mosquitocidal activity of
B. sphaericus NRC 69 grown under SSF
Wheat bran/by- Mortality (%) after 48 h exposure to B. sphaericus NRC 69 grown on
products (w/w)
Wheat bran/wheat Wheat bran/rice Wheat bran/barely Wheat bran/corn Wheat bran/cotton
straw straw straw stover stakes
At 10 ppm At 7.5 ppm At 5 ppm At 5 ppm At 7.5 ppm

0.5/4.5 0 0 29.2 ± 7.3 b 0 0

1/4 0 0 77.5 ± 1.9 a 0 0
2/3 24.2 ± 1.2 d 0 79.6 ± 2.3 a 0 10.4 ± 1.3 d
3/2 44.6 ± 2.3 c 29.6 ± 2.3 c 19.2 ± 1.5 c 19.1 ± 1.5 c 31.7 ± 1.9 c
4/1 57.9 ± 1.8 b 39.6 ± 2.2 b 0 70.8 ± 2.0 b 56.3 ± 1.3 b
5/0 100 ± 0.0 a 85.0 ± 1.8 a 79.2 ± 2.4 a 76.7 ± 1.5 a 86.3 ± 1.1 a
0/5 0 0 0 0 0
Mortality % is presented as means ± SE. Values for each treatment followed by different letters are significantly different at P = 0.05

on wheat bran/wheat straw, wheat bran/rice straw, wheat
bran/corn stover and wheat bran/cotton stakes media at
4/1(g/g) decreased about 3.7, 4.4, 14.2 and 5.2 fold,
respectively in comparison with that of the organism grown
on wheat straw, rice straw, corn stover, and cotton stakes
media.
Although, mixing of these substrates with wheat bran
enhanced the toxicity of the tested organism in comparison
with that produced by growth on the tested substrates
without mixing several times, however, LC50 still higher
than that of wheat bran medium with exception of wheat
bran/rice hull at 8/2 (g/g) medium and wheat bran/barley
straw at 1/4 (g/g) medium. These substrates may have
inhibitory factors or their protein contents are very low or
are not readily available for growth and production of the
tested organism. Perhaps, addition of wheat bran provided
relatively high concentrations of nitrogen, carbon, inor-
ganic compounds, vitamins and amino acids required for
growth and metabolism.
Effect of inoculum size
Inoculum concentration also plays a crucial role in
microbial metabolites production. In the present study,
wheat bran was the best by-product for production of
B. sphaericus under study. Therefore, further tests were
employed on this substrate. As shown in Table 6, there was
an increase in mosquitocidal activity with increasing the
inoculum size up to 4% (84 9 106 CFU/g solid medium)
where 60% mortality was obtained. With further increase
in inoculum size, the mosquitocidal activity decreases. It
was reported that inoculum concentration plays a signifi-
cant role in production of active compounds under SSF
(Pandey 1994; Kashyap et al. 2002).
Effect of incubation period
The toxicity of B. sphaericus NRC 69 was increased with
increasing the incubation period up to 6 days where the

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Table 5 LC50 of B. sphaericus NRC 69 grown on agriculture by- Table 7 Effect of supplementation of wheat bran with additives on
product media mosquitocidal activity of B. sphaericus NRC 69
Substrate LC50 (95% fiducial limits) Supplements/concentration Mortality (%) after
48 h at 1 ppm
Wheat bran 1.2 (1.0–1.5)
Rice hull 28.8 (22.3–34.2) NYSM salts (%)
Wheat straw 35.8 (28.9–41.3) 0.2 60.4 ± 1.1 c
Rice straw 39.2 (31.2–46) 0.5 62.1 ± 2.1 c
Barely straw 42.3 (36.7–48.6) 1 64.6 ± 1.6 bc
Corn stover 48.3 (40.6–56.3) Dipotassium hydrogen phosphate (mM)
Corn cobs 59.6 (49.3–69.9) 50 42.1 ± 1.7 e
Cotton stakes 33.7 (27.9–39.8) 100 46.3 ± 1.6 e
Pea peels 296.6 (230.4–362.5) 200 42.5 ± 1.4 e
Potato peels 507 (444.2–569.5) Corn steep liqure (%)
Wheat bran/rice hull (8/2) 1.7 (1.2–2.2) 0.2 69.2 ± 2.1 b
Wheat bran/wheat straw (4/1) 9.8 (8–11.6) 0.5 65.0 ± 1.5 bc
Wheat bran/rice straw (4/1) 9 (7.8–10.2) 1 15.0 ± 1.2 f
Wheat bran/barely straw (1/4) 2.7 (2.1–3.2) Whey permeate (%)
Wheat bran/corn stover (4/1) 3.4 (2.8–4.1) 10 95.0 ± 1.5 a
Wheat bran/corn cobs (8/2) 6.9 (5.7–8) 25 52.5 ± 1.0 d
Wheat bran/cotton stakes (4/1) 6.5 (5.6–7.3) 50 51.3 ± 2.0 d
Wheat bran/pea peels (6/4) 7.0 (6.2–7.8) 100 44.6 ± 1.3 e
Wheat bran/potato peels (4/6) 10.1 (8.2–12) Control (without additive) 65 ± 1.4 bc
Mortality % is presented as means ± SE. Values for each treatment
followed by different letters are significantly different at P = 0.05
Table 6 Effect of inoculum size of B. sphaericus NRC 69 grown on
wheat bran under SSF on mosquitocidal activity
concentration of whey permeate significantly decreased the
Inoculum size Mortality (%) after
(9106 CFU/g) 48 h at 1 ppm toxicity. NYSM salts had no effect on the activity of the
organism. A decrement in mosquitocidal activity of B.
10.5 4.6 ± 1.0 e sphaericus NRC 69 was noted at all tested concentrations
21 10.4 ± 1.1 d of dipotassium hydrogen phosphate. Addition of corn steep
42 43.3 ± 2.1 b liqure at 0.2–0.5% showed no effect on toxicity, however,
84 60.4 ± 1.8 a increasing its concentration to 1% has a dramatic effect on
210 21.3 ± 0.9 c mosquitocidal activity.
336 11.7 ± 0.7 d It is known that whey permeate contains amino acids
Mortality % is presented as means ± SE. Values for each treatment mainly glutamic acid, aspartic acid, proline and minerals
followed by different letters are significantly different at P = 0.05 (El-Bendary et al. 2008) which promote the growth and
toxin production of B. sphaericus (Klein et al. 1989; Lacey
1984; Shevtsov et al. 1990).
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1 ppm). Further extension of incubation period resulted in
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