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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 substrate(s) in the absence of free flowing water, has gained
grown in culture media, in which 12 agricultural wastes a tremendous momentum owing to certain advantages over
were tested as the main carbon, nitrogen and energy sources the conventional submerged fermentation like low pro-
under solid state fermentation. Of the 12 tested agricultural duction cost, saving of water and energy, less waste
by-products, wheat bran was the most efficient substrate for effluent problem and stability of the product due to less
the production of B. sphaericus mosquitocidal toxins dilution in the medium (Pandey 2003; Holker and Lenz
against larvae of Culex pipiens (LC50 1.2 ppm). Mixtures of 2005). Therefore, SSF has found several potential appli-
tested agricultural wastes separately with wheat bran cations in the industrial production of value added products
enhanced the produced toxicity several folds and decreased such as industrially important microbial enzymes, bioin-
LC50 between 3.7- and 50-fold in comparison with that of secticides, secondary metabolites and pharmaceuticals
agricultural wastes without mixing. The toxicity of (Robinson et al. 2001; Pandey 2003).
B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and At present, the overall cost of bio-insecticides production
wheat bran/barley straw at 1/4 (g/g) showed the same tox- are very high due to the high cost of the substrates and
icity as that in wheat bran medium (LC50 decreased 17- and media used (Prabakaran and Balaraman 2006). The cost of
16-fold, in comparison with that in rice hull or barely straw raw materials used for the production of Bacillus thuringi-
media, respectively). In wheat bran medium, the maximum ensis based bio-pesticides as example was about 35–59% of
toxicity of the tested organism obtained at 50% moisture the production cost as estimated by Stanbury et al. 1995.
content, inoculum size 84 9 106 CFU/g wheat bran and Therefore, there is an urgent need to find high yielding, cost
incubation for 6 days at 30°C. Addition of cheese whey free or low cost and year round available raw materials for
permeate at 10% to wheat bran medium enhanced the tox- bio-insecticide production (Yezza et al. 2006). This goal
icity of B. sphaericus NRC 69 about 46%. can be achieved by using agricultural wastes as substrates.
In Egypt, there is about 26 million tons of plant
Keywords Bacillus sphaericus  Agricultural wastes  by-products produced annually. The main approach for dis-
Solid state fermentation  Culex pipiens 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,
Introduction aerobic, spore-former bacterium. Some strains of B. sph-
aericus are toxic to mosquito larvae and used as biocontrol
Over the past couple of years, solid state fermentation agent for disease-transmitting mosquitoes (de Barjac
(SSF) involving growth of microbes on moist solid 1990). Mosquitocidal strains of B. sphaericus traditionally
produced by submerged fermentation. However, this
technology is constrained by capital costs and requirements
M. A. El-Bendary (&)
for fermentation expertise. Inexpensive media were used
Microbial Chemistry Department, National Research Centre,
El-Behoos Street, Doki, Giza, Egypt for production of B. sphaericus under submerged fermen-
e-mail: tasnim41@yahoo.com 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 analysis and the Duncan’s multiple range test (Duncan
production in wheat bran medium was studied by varying the 1955) to determine the significance between means. Data
inoculum size between 10.5 9 106 and 336 9 106 CFU/g were expressed as mean values ± standard errors.
wheat bran. Prpbit regression analysis (Proban version 1.1, Dr Rys-
Effect of incubation period was studied for extended zard A. Jedrychowski 1991) was carried out to calculate
incubation periods (2–9 days) on wheat bran medium. LC50 as well as 95% fiducial limits.
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. Results and discussion
Total viable cell and spore counts were estimated by
plate count method. Screening of different agricultural wastes
for mosquitocidal toxin production
Bioassay of mosquitocidal activity
The results presented in Table 2 showed that wheat bran
Bioassay of mosquitocidal activity of B. sphaericus NRC supported the highest growth, sporulation and toxicity of
69 produced under SSF was adopted from Ampofo (1995) B. sphaericus NRC 69 against second instar larvae of Culex
with some modifications. Toxicity was determined with pipiens. B. sphaericus cannot utilize carbohydrates as
laboratory reared second instar larvae of Culex pipiens. carbon source due to the lack of the early enzymes of
One gram of the final product was mixed with dechlori- Embeden Meyerhoff Parnas and hexose monophosphate
nated tap water (100 ml) and shaken for 1 h. Serial dilu- pathways (Russel et al. 1989). Instead, it can use amino
tions were prepared and the dilutions were placed into acids as carbon and nitrogen sources. It is known that
100 ml beakers in triplicate along with 20 second instar wheat bran contains 78% carbohydrate, 16% protein, 6%
Culex pipiens larvae. About 10 mg of ground fish meal was minerals and vitamins. Therefore, wheat bran medium
added to each cup. The beakers were covered with muslin almost contains all the requirements for growth and toxin
and kept at 26 ± 2°C with 10 h light/14 h dark cycle. The production by B. sphaericus NRC 69.
mortality percentage was recorded after 48 h by counting Wheat bran has been widely used for production of
the number of living larvae and corrected by using entomopathogenic fungi (Jagadeesh and Geeta 1994;
appropriate control and adopting Abbott’s formula (Abbott Sangeetha et al. 1993). It was reported that Bacillus thur-
1925). Each bioassay was replicated two times. ingiensis israelensis produced using 100 l-fermentor using
wheat bran extract giving a product with higher toxicity
Statistical analysis than the growth in NYSM medium (Prabakaran and Bal-
araman 2006). Also, Bacillus thuringiensis produced on
Data obtained from this study were statistically analyzed wheat bran medium under SSF by Suyanandona et al.
according to SPSS system (SPSS 1998) using one-way 1996; Hongzhang et al. 2002; Devi et al. 2005.

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

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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 Effect of inoculum size
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, Inoculum concentration also plays a crucial role in
respectively in comparison with that of the organism grown microbial metabolites production. In the present study,
on wheat straw, rice straw, corn stover, and cotton stakes wheat bran was the best by-product for production of
media. B. sphaericus under study. Therefore, further tests were
Although, mixing of these substrates with wheat bran employed on this substrate. As shown in Table 6, there was
enhanced the toxicity of the tested organism in comparison an increase in mosquitocidal activity with increasing the
with that produced by growth on the tested substrates inoculum size up to 4% (84 9 106 CFU/g solid medium)
without mixing several times, however, LC50 still higher where 60% mortality was obtained. With further increase
than that of wheat bran medium with exception of wheat in inoculum size, the mosquitocidal activity decreases. It
bran/rice hull at 8/2 (g/g) medium and wheat bran/barley was reported that inoculum concentration plays a signifi-
straw at 1/4 (g/g) medium. These substrates may have cant role in production of active compounds under SSF
inhibitory factors or their protein contents are very low or (Pandey 1994; Kashyap et al. 2002).
are not readily available for growth and production of the
tested organism. Perhaps, addition of wheat bran provided Effect of incubation period
relatively high concentrations of nitrogen, carbon, inor-
ganic compounds, vitamins and amino acids required for The toxicity of B. sphaericus NRC 69 was increased with
growth and metabolism. 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).
maximum toxicity was obtained (67.5 ± 2.1% mortality at
1 ppm). Further extension of incubation period resulted in
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