See

discussions, stats, and author profiles for this publication at:

https://www.researchgate.net/publication/7180588

Bacillus thuringiensis andBacillus

sphaericus biopesticides production

Article in Journal of Basic Microbiology April 2006

DOI: 10.1002/jobm.200510585 Source: PubMed

CITATIONS READS

59 763

1 author:

Magda El-bendary
National Research Center, Giza, Egypt
41 PUBLICATIONS 369 CITATIONS

SEE PROFILE

All content following this page was uploaded by Magda El-bendary on 09 February 2017.

The user has requested enhancement of the downloaded file.

J. Basic Microbiol. 46 (2006) 2, 158 170 DOI: 10.1002/jobm.200510585

Review
(Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt)

Bacillus thuringiensis and Bacillus sphaericus biopesticides

production
MAGDA A. EL-BENDARY*

(Received 17 February 2005/Returned for modification 04 June 2005/Accepted 04 July 2005)

The long residual action and toxicity of the chemical insecticides have brought about serious
environmental problems such as the emergence and spread of insecticide resistance in many species of
vectors, mammalian toxicity, and accumulation of pesticide residues in the food chain. All these
problems have highlighted the need for alternative biological control agents.
Entomo-pathogenic Bacillus thuringiensis (Bt) and Bacillus sphaericus (Bs) are two safe biological
control agents. They have attracted considerable interest as possible replacements for the chemical
insecticides. Although microbial insecticides based on Bt and Bs are available for use, their high cost
makes large-scale application impracticable in developing countries. This review focuses on the
economic production of these two microorganisms by submerged fermentation and solid state
fermentation using agro-industrial by-products and other wastes.

Bacillus thuringiensis (Bt)

Bt is a facultative anaerobic gram-positive bacterium present in soil, water and on plant
surfaces. It differentiates into ellipsoidal spore and characteristic protein parasporal crystal
(-endotoxin). This crystal is toxic to a variety of insects, nematodes and protozoan (FEITEL-
SON 1993).
According to ROWE and MARGARITIS (1987) and WHO (1999), there have been nine dif-
ferent toxins described in Bt strains. These toxins are -exotoxin (phospholipase C), -exo-
toxin (thermostable exotoxin), -exotoxin (toxic to sawflies), -endotoxin (protein paraspo-
ral crystal), louse factor exotoxin (active only against lice), mouse factor exotoxin (toxic to
mice and Lepidoptera), water-soluble toxin, Vip3A (Bt vegetative insecticidal protein) and
enterotoxin (produced by vegetative cells). Out of these several toxins produced by Bt
strains, -endotoxin received much attention and have been exploited commercially for
production of bioinsecticides. Bt crystals have various forms (bipyramidal, cuboidal, flat
rhomboid, or a composite with two or more crystal types). The crystal toxins (-endotoxin)
are belonging to two structurally different groups:
1. Cry family, with specific cytolytic activity as Cry1Aa1, Cry1Ba1, Cry2Aa1, etc.
2. Cyt family, which is a nonspecific cytolytic and hemolytic as Cyt1Aa1, Cyt2Aa1, etc.
(WHO 1999 and DELECLUSE et al. 2000).

Bacillus sphaericus (Bs)

Bs is an aerobic rod shaped endospore forming bacterium with the endospore in a swollen
terminal or subterminal position (GORDON et al. 1973). It is widely distributed in soil and
water habitats. Some strains of Bs form crystal protein during sporulation and they are
pathogenic to mosquito larvae.

* Corresponding author: Dr. M. A. EL-BENDARY; e-mail: tasnim41@yahoo.com

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/06/0204-0158

Biopesticides production 159

Intensive studies on larvicidal toxins from various pathogenic strains of Bs revealed the
presence of two types of toxins, the crystal protein or binary toxin (Btx) and mosquitocidal
toxins (Mtx). Bs produces Btx during the sporulation and comprises 41.9- (Bin A) and 51.4-
KDa (Bin B) (BAUMANN et al. 1991, CHARLES et al. 1996, HUMPHREYS and BERRY 1998).
Mtx toxins are produced during the vegetative growth and they are associated with the cell
membrane of Bs (LIU et al. 1996). Mtx toxins are three types, Mtx1, Mtx2 and Mtx3, with
molecular masses of 100-, 31.8- and 35.8-kDa, respectively. Most of highly toxic strains
synthesize Btx toxin and may contain one or more of Mtx toxins. However, low toxic
strains synthesize only Mtx toxins.

Safety of Bt and Bs insecticides

Entomopathogens like chemical insecticides must be evaluated for their safety to both ani-
mals and humans. Microbial safety tests concentrate on acute toxicity and vertebrate infec-
tivity, while chemical safety tests focus on acute toxicity, neurotoxicity and carcinogenicity.
According to DE BARJAC (1990), PRIEST (1992), PORTER (1996), WHO (1999), SIEGEL
(2001), ABDULLAH (2002) and MITTAL (2003), Bt and Bs are completely safe to other non-
target organisms, human, animals, wildlife and environment and they are suitable for com-
munity use.

Mode of action of Bt and Bs

The pathogenicity of these bacteria has been attributed to the expression of toxic proteins
(10 30% dry weight of the bacteria, REGIS et al. 2001) that are produced in crystalline
form. Ingestion of these toxic proteins causes destruction of the larval gut epithelial cells
causing the death of the larvae.
The detailed mode of action of Bt and Bs toxins is still unknown. A number of studies
(BAUMANN et al. 1985 and 1991, KNOWLES and ELLAR 1987, CHARLES 1987, SINGH and
GILL 1988, DAVIDSON 1988, BROADWELL et al. 1990, DE BARJAC 1990, BAUMANN and
BAUMANN 1991, OEI et al. 1992, DAVIDSON 1995, CHARLES et al. 1996 and 1997, REGIS
et al. 2001, MANCEVA et al. 2004 and SMITH et al. 2005) revealed that the action of the
crystal toxin on susceptible larvae involved the following series of steps:
1. The larvae of the target insect ingest crystal proteins from water.
2. The crystal proteins are solubilized and activated under the combination of alkaline pH
and proteinase of the larval midgut.
3. Active toxins bind to apical microvilli of midgut cells. They bind to specific membrane
receptors (Phospholipids, phosphatidyl choline and sphingomyelin, for Bt toxins and a
60 kDa -glucosidase anchored in the apical midgut membrane via a glycosyl-phos-
phatidyl inositol anchor, for Bs toxins).
4. After binding of toxin to the receptor site, a part of the toxin inserts into the membrane
lipid bilayer forming ionic-selective channel or pore, which lead to entry of water into
the cell and exit of ions and other larger components, leading to swelling and lysis of the
cell by a colloid-osmotic lysis mechanism.
5. There is another mechanism for pore formation called theory of oligomeric pore forma-
tion, which proposed that the crystal toxins (Cry produced by Bt or Btx produced by Bs)
form oligomers, which are necessary for pore formation.
6. The most drastic cytological changes caused by Bt toxin consist of swelling of midgut
epithelial cells and lysis, while Bs toxin causes large vacuoles in the midgut cells and
mitochondrial swelling. Late damage of neural tissues and skeletal muscles have also
been reported.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

160 MAGDA A. EL-BENDARY

Factors affecting growth and toxin production by Bt and Bs

Bt and Bs grow in a culture medium containing sources of carbon and nitrogen as well as
mineral salts. The growth of Bt and Bs can be described by three phases: Vegetative growth
(exponential phase), transition phase and sporulation phase. During the sporulation phase,
each cell liberates one spore and a protein toxin crystal. The factors affecting growth, sporu-
lation and toxin formation by Bt and Bs as follows:

1. Carbon source: Bt uses sugars, usually glucose, fructose, maltose, ribose, molasses,
starch, dextrin, wheat flour and inulin, producing acid during the fermentation (NICKERSON
and BULLA 1974, SALAMA et al. 1981, ARCAS et al. 1984, ZAMOLA et al. 1981, FODA et al.
1985, EL-BENDARY 1994, SADEK 2000, ICGEN et al. 2002a and OZKAN et al. 2003).
OZKAN et al. (2003) studied various nutritional and cultural parameters influencing dip-
tera-specific -endotoxin synthesis by Bacillus thuringiensis israelensis (Bti) HD500. They
reported that inulin, dextrin, maltose, lactose, sucrose, whey and glycerol were simulatory,
while glucose, starch and molasses were suppressive.
In contrast to Bt, Bs can not use carbohydrates for growth due to the lack of many of the
enzymes of EMBDEN MEYERHOFF and hexose monophosphate pathways (RUSSELL et al.
1989). Numerous strains of Bs grew with acetate, pyruvate, lactate, glutamate, succinate,
histidine, arginine, as sole major carbon and energy source (GORDON et al. 1973, DE BAR-
JAC et al. 1980, KLEIN et al. 1989, WIDJAYA et al. 1992, AHMED et al. 1993 and 1996).

2. Nitrogen source: With respect to the nitrogen sources suitable for Bt production, the
overwhelming majority of literatures revealed the inability of most of Bt varieties to utilize
inorganic nitrogen source as a sole nitrogen source in the growth medium. Instead, at least
one amino acid particularly glutamate, aspartate, valine, leucine, serine or threonine has to
be added in order to allow growth of the organism in a minimal medium (NICKERSON and
BULLA 1974, NORMANSELL et al. 1980, EL-BENDARY 1994, AVIGNONE-ROSSA and MI-
GNONE 1995 and SADEK 2000). However, cysteine and cystine amino acids showed clear
inhibitory effect on growth, sporulation and toxin formation by Bt (RAJALAKSHMI and
SHETHNA 1980, EL-BENDARY 1994 and SADEK 2000). ICGEN et al. (2002a) found that pep-
tone was the best organic nitrogen source supporting sporulation and toxin production by Bt.
Several studies were reported in the literature regarding nitrogen sources and amino acids
required for growth and sporulation of Bs strains. YOUSTEN (1984) reported that the mos-
quitocidal Bs strains were able to grow in defined broth containing (NH4)2SO4, biotin, thia-
mine, mineral salts and one of a variety of carbon sources. WHITE and LOTY (1980) and
KLEIN et al. (1989) reported that many of Bs strains grew and sporulated on a single amino
acid medium or a mixture of several amino acids as source of both nitrogen and carbon.
YOUSTEN et al. (1984a) found that amino acid analysis of NYSM broth after growth for
seven hours by strain 1593 revealed that glutamic acid, proline, arginine, histidine, aspartic
acid and glycine were readily catabolized. They proposed that these amino acids probably
serve as primary carbon sources for growth in proteinaceous media. The same conclusion
was reported by LACEY (1984) and KLEIN et al. (1989) when they found glutamic acid,
lysine, glycine and valine promoted growth and toxin production of Bs 2362 and 1593.
Moreover, addition of L-proline to glutamic acid enhanced the toxin formation by Bs 2362.
In a later study, SHEVTSOV et al. (1990) concluded that the majority of Bs strains require
arginine, glutamic acid, methionine, thereonine, serine, alanine, and lysine for growth and
spore germination but can not utilize phenylalanine or proline. They also reported that the
most effective germination inducers are arginine, methionine and glutamic acid.

3. Potassium ion: Potassium ion is essential for toxin production by Bt and Bs (WAKISAKA
et al. 1982 and EL-BENDARY 1999). OZKAN et al. (2003) found that an effective synthesis
of Cry4Ba by Bti HD500 required high concentrations of inorganic phosphate (50 to

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Biopesticides production 161

100 mM K2HPO4). The same conclusion was reported by BHATNAGAR (1999) for production
of 135-kDa protoxin protein by Bti HD522.
The effect of phosphate ion concentration on sporulation and toxin formation by Bs was
studied by EL-BENDARY (1999). She reported that the sporulation of Bs 2362 and Egyptian
Bs isolate 69 in the minimal medium without phosphate was lower than that supplemented
with different concentrations of phosphate. Moreover, the toxicity of both cultures against
second instar Anopheles stephensi larvae increased with the increase of phosphate concen-
tration.

4. Metal ions: Metal ions such as Ca2+, Mg2+, Mn2+, Zn2+, Cu2+ and Fe2+ are essential for
production of the highest sporulation and -endotoxin formation by Bt (FALOCI et al. 1986,
SADEK 2000 and ICGEN et al. 2002b).
OZKAN et al. (2003) stated that Mn2+ was the most critical element for the biosynthesis of
Cry4Ba and Cry11Aa by Bti HD500 at 106 M concentration. However, Mg2+ and Ca2+ fa-
vored toxin production when provided at 8 103 M and 5.5 104 M concentrations, respec-
tively, while Fe2+, Zn2+ and Cu2+ negatively influenced toxin biosynthesis. In contrast, SIK-
DAR et al. (1991) have recommended the addition of Fe2+ and Cu2+ for stimulation of Cry-
toxin production by the same subspecies (Bti HD500).
For Bs, It was reported that Mn2+, Mg2+, Ca2+, Zn2+, Fe2+, Ni2+ and Cu2+ in combination
significantly enhanced the toxin production by Bs (AHMED 1994 and EL-BENDARY 1999).

5. pH: The growth of Bt occurs in the pH range of 5.5 8.5 (ROWE and MARGARITIs 1987,
ICGEN et al. 2002b and OZKAN et al. 2003). The usual initial pH is 6.8 7.2; decreasing to
5.8 as acetate is released, then rising to 7.5 8 as it is consumed.
YOUSTEN et al. (1984a) found that the toxic activity of Bs 1593 was increased about ten
fold, by controlling the pH near neutrality in the fermentor. On the other hand, in a later
publication, YOUSTEN and WALLIS (1987) reported that controlling the pH near neutrality
was detrimental to toxin synthesis by Bs 2362. FODA et al. (2000) stated that the maximum
sporulation and toxicity (against second instar Anopheles stephensi larvae) by Bs were ob-
tained in a medium buffered at initial pH 7.

6. Temperature: The normal temperature for growth and toxin production of Bt is 30 C.

OZKAN et al. (2003) found that Cry4Ba synthesis by Bti HD500 was the best when the or-
ganism was grown at 25 C, whereas Cry11Aa synthesis was optimal at 30 C.
YOUSTEN et al. (1984) and LACEY (1984) found that Bs 1593 grew at incubation tempera-
ture between 25 C and 40 C, whereas both spore and toxin development were inhibited at
35 C. In comparable to these finding, DEPIERI and LUDLOW (1992) found the maximum
sporulation yield as a percentage of viable counts of Bs 9602 was <10% at 10 C and 12 C,
while it was >95% at 15 C, 20 C and 30 C, however, at 40 C Bs grew only vegetatively.

7. Effect of aeration: Aeration is very important for Bt fermentation. FODA et al. (1985)
noted the failure of the organism to survive or sporulate under low aeration levels. Most
submerged fermentation of Bt is done using aeration rates approximately one air vol-
ume/volume of medium/minute. Recent studies on metabolism of Bt during growth and spo-
rulation have employed higher aeration level e.g. 1.4 air volume/volume of medium/minute
(ROWE 1990).
Some studies were published in the literature concerning the aeration and toxin formation
of Bs. It has been reported that Bs 1593 was unable to sporulate in the presence of high
levels of oxygen, while toxin production was normal (YOUSTEN et al. 1984). In subsequent
study YOUSTEN and WALLIS (1987) reported that oxygen was required for toxin formation
by strain 2362. However, using of pure oxygen to increase levels of dissolved oxygen dur-
ing fermentation decrease the toxicity although the sporulation is normal. In comparable

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

162 MAGDA A. EL-BENDARY

study KARIM et al. (1993) found that dissolved oxygen (DO) levels of 20%, 50% and 100%
resulted in a rapid formation of free spores, whilst 5% DO slow the formation of free spores.
In addition, the toxicity of the preparations from cells grown at 5%, 20% and 50% DO gave
comparable levels of mosquito-larvicidal activity, whilst that from the cells grown at 100%
DO was significantly lower.

Mass production of Bt and Bs

Because of the economic importance of Bt and Bs as powerful biological control agents
against harmful insect pests, special attention was paid to elucidate and optimize growth
conditions of Bt and Bs that leading to the highest yields of their toxins. SALAMA et al.
(1983) and SACHDEVA et al. (1999) reported that the commercial application of both organ-
isms depends on the cost of raw materials, strain efficiency, fermentation cycle, mainte-
nance of process parameters, bioprocessing of fermentation fluid, and formulation of the
final product. The cost of raw materials is one of the principal costs involved in overall Bt or
Bs production. In the conventional Bt production process, the cost of raw materials varied
between 30 and 40% of the total cost depending on the plant production capacity (EJIOFOR
1991 and LISANSKY et al. 1993). Therefore, local production of this insecticide in develop-
ing countries should depend on the use of production media made of cheap, locally avail-
able sources including agro-industrial by-products (AMPOFO 1995). For large scale produc-
tion of Bt and Bs, different approaches were investigated to construct media that could
support good production of spores and toxins at reasonable costs. Various agricultural and
industrial by-products used as raw material in Bt and Bs production were citrus peels, wheat
bran, corn meal, seeds of dates, beef blood, silkworm pupal skin, ground nut cake, cane
molasses, fish meal, cotton seed meal, soybean meal, residues from chicken slaughter
house, fodder yeast, cheese whey and corn steep liquor (SALAMA et al. 1983, OBETA and
OKAFOR 1984, MUMMIGATTI and RAGHUNATHAN 1990, ABDEL-HAMEED et al. 1990 LEE
and SELEENA 1991, EL-BENDARY 1994 and 1999, SACHDEVA et al. 1999, FODA et al. 2002
and 2003).
Recently, other wastes such as sludge and broiler poultry litter were utilized for biopesti-
cides production (ADAMS et al. 2002 and VIDYARTHI et al. 2002).
In general, two methods of fermentations are used for production of microbial products,
submerged fermentation and solid state fermentation.

1. Submerged fermentation of Bt and Bs

Fermentation media for Bt production

The search for suitable media for industrial production of Bt has been the objective of
several studies reported in the literatures. An early submerged fermentation medium for
Bt production was reported by MEGNA (1963). He used seed medium contained beet molas-
ses (1%), corn steep solids (0.85%) and calcium carbonate (0.1%). While the production
medium contained beet molasses (1.86 %), corn steep solids (1.7%), cotton seed flour
(1.4%) and calcium carbonate (0.1%). The yield was 2.5 109 colony forming units
(CFU)/ml.
DULMAGE (1970) devised a fermentation medium based on defatted cotton seed flour,
which supported the production of large yield of -endotoxin by the tested Bt strains. The
same author (DULMAGE 1971) constructed three fermentation media including a novel me-
dium with defatted soybean meal flour as the major component for production of Bt sero-
type 3. In later study, DULMAGE and DE BARJAC (1973) reported fermentation media for Bt
-endotoxin production based upon cotton seed flour and corn steep liquor.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Biopesticides production 163

SALAMA et al. (1983) investigated several agro-industrial by-products for Bt -endo-

toxin production. They found that fodder yeast, beef blood and chicken slaughter residues
were among the byproducts that produced high sporulation and potent -endotoxin prepa-
rations.
PEARSON and WARD (1988) developed a fermentation process involved two inoculum
stages and a production stage for production of Bti. The inoculum culture was grown in
1S medium (15 g/l, tryptone water; 7.1 g/l, Na2HPO4; 0.2 g/l, MgSO4 7 H2O; 0.001 g/l,
FeSO4 7 H2O; 0.005 g/l, ZnSO4 7 H2O; 0.005 g/l, CuSO4 5 H2O in distilled water; pH 7)
to seed a laboratory fermentor containing 10-l of 2S medium (20 g/l casein, 5 g/l corn steep
solids, 2.7 g/l yeast extract, 5 g/l cane molasses, 5 g/l Na2HPO4,, pH 7). A 16 hr culture pro-
duced in laboratory fermentor was used to inoculate a pilot scale 40-l fermentor containing
P3 (a) medium (15 g/l soybean meal, 10 g/l molasses, 10 g/l starch, 1 g/l CaCO3). The vi-
able count at the end of the 48 h fermentation was 6.5 109 CFU/ml with greater than 95%
sporulation.
WIDJAYA et al. (1992) reported a defined medium containing 1.5% yeast extract for
growing Bti. A final Bti spore yield in this medium was 2.9 109 CFU/ ml. Replacement of
yeast extract with fish meal extended growth phase with similar final values for cell and
spore counts.
KANG et al. (1992) carried out fed-batch culture to increase cell mass followed by batch
culture for spore production of Bt in GYS medium, which contains glucose, yeast extract
and some mineral salts. They found that high cell mass obtained by increasing the feeding
of glucose in constant fed-batch culture did not proceed to spore formation. However, in-
termittent fed-batch culture supported fast cell growth and resulted in good sporulation
during subsequent batch culture (1.25 1010 CFU/ml).
LUI and TZENG (1998) reported that the optimum medium composition for production of
high level of spores by Bti (8.56 108 CFU/ml) was 5.01% tapioca; 5.86% fish meal and
0.06% (NH4)2SO4.
MONTIEL et al. (2001) used sludge as a raw material for the production of Bt based bioin-
secticides using Bt kurstaki. The sludge samples were used under three different prepara-
tions: without pre-treatment, with acid treatment (hydrolyzed sludge) and the supernatant
obtained after centrifugation of the hydrolyzed sludge. The highest viable cell, spore counts
and -endotoxin production were when the organism was grown in hydrolyzed sludge,
while the liquid phase (supernatant) showed the lowest sporulation and toxicity. LACHHAB
et al. (2001) conducted a detailed study on the effect of inoculum and sludge solid concen-
trations on the production of Bt kurstaki in shake flasks and 15-l fermentor. They concluded
that preparation of inoculum in the same sludge as that used for the production medium
resulted in higher sporulation and toxicity values. They also reported that the optimum
sludge solid concentration was 26 g/l, which resulted in an improved potency and high
spore count. In a later study, VIDYARTHI et al. (2002) compared the growth and -endotoxin
production by Bt kurstaki in tryptic soy yeast extract (TSY) medium; soybean based com-
mercial medium and wastewater sludge medium. They found that the highest toxicity was
obtained in a sludge medium and was comparable to that of the concentrated commercial Bt
formulation available in the market (FORAY 48B). They also found that the optimum value
of C: N ratio in combined sludge for Bt production was 7.9 9.9.
ZOUARI et al. (2002) investigated the production of several Bt strains active against Lepi-
doptera and Diptera in gruel (a cheap and abundant byproduct of semolina factories) and
fish meal media. They observed that Ddiptera-specific strains produced less -endotoxin
(1246 1998 mg/l) than Lepidoptera-specific ones (3060 3301 mg/l). However, addition of
10 g/l sodium acetate increased 38 79% -endotoxin production by Diptera-specific strains
in shake flask cultures. Similar -endotoxin production was obtained with or without 10 g/l
sodium acetate with an excess of aeration in a 2-l fermentor.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

164 MAGDA A. EL-BENDARY

Fermentation media for Bs production

DULMAGE et al. (1970) cultured Bs 1593 and Bs 2362 separately in a fermentor on pep-
tonized milk medium with yeast extract and mineral supplements. The fermentor beer was
centrifuged and then resuspended in lactose solution and precipitated with acetone. These
powders were highly insecticidal to Culex quinquifasciatus larvae producing LC50 values in
the range of 102 g/ml.
OBETA and OKAFOR (1983) formulated five media from dried cow blood, mineral salts
and seeds from four species of legumes (ground nut cake, cowpea, mambara beans and
soybean) for production of Bs 1593. Good growth, sporulation and toxin activity of Bs 1593
were obtained with all tested media.
KLEIN et al. (1989) used hydrolyzed industrial peptones (waste product of industry) for
constructing seven media for production of Bs larvicides. These media contained 5 g/l in-
dustrial peptone in 50 mM phosphate buffer (pH 7.0) in combination with other carbon and
nitrogen sources. Industrial peptone medium supplemented with glycerol was the most
efficient medium for growth and larvicides production by Bs 2362.
AMPOFO (1995) used some local row-materials for production of Bs insecticide in Ghana.
He tested anchovy, spent grain from breweries, bambara beans and sprout maize as media
for production of Bs IAB 881. He reported that larvicidal activity of Bs IAB 881 grown in
anchovy, spent grain, bambara beans and sprout maize, was similar to that obtained in a
synthetic medium with LC50 ranging from 0.3 105 to 0.68 106 (dilution). Cell counts
were in the range of 11 108 36 108 CFU/ml and spore counts were between 29 107
and 61 107 CFU/ml.
SASAKI et al. (1998) used corn steep liquor (CSL) as a replacement of yeast extract and
they reported that CSL enhanced the sporulation of Bs 2362. A combinations of yeast ex-
tract and CSL with acetate as carbon source in fed-batch culture resulted in a cell mass of
13 g/l, a high spore count of 16.4 109 CFU/ml and a high toxin production.
EL-BENDARY (1999) used ground agro-industrial by-products and leguminous seeds at
2% final concentration as media for production of Bs in distilled water with or without addi-
tion of NYSM salts. The obtained results indicated that the most of the tested substances
supported the production of appreciably high sporulation yield and toxicity. She also re-
ported that the most efficient media for Bs toxin production were soy flour, cotton seed
flour, corn steep solids and offals meal. Furthermore, it was observed that addition of
NYSM salts to these substances increased the Bs toxicity. Moreover, the toxicity of Bs in-
creased about 1.5 4.5 times when these agro-industrial by-products partially hydrolysed by
nuclease or alkalase before using as media.

Fermentation media for production of both Bt and Bs

In 1985, DHARMSTHITI et al. constructed two media using HDL, a by-product from a mono-
sodium glutamate factory. These media were found to be highly suitable for culturing Bti
and Bs. These media contained 0.05% K2HPO4 and 4% HDL (H4 medium) or 0.05%
K2HPO4 and 7% HDL (H7 medium). The sporulation and LC50 values of Bti grown in H4
medium at 48 hr were 2.5 108 CFU/ml and 107 (dilution), respectively. While those of Bs
grown in H7 medium were 1.4 109 CFU/ml and 108, respectively.
KUPPUSAMY and BALARAMAN (1991) studied the effect of corn steep liquor as a growth
medium for production of three strains of Bti and two strains of Bs as compared with a me-
dium based on peptone and yeast extract using a laboratory fermentor. They concluded that
corn steep liquor can effectively replace both peptone and yeast extract in the media for
large scale production of the two larvicidal bacilli.
DESAI and SHETHNA (1991) formulated three fermentation media for growth of Bti and Bs
1593 using defatted ground nut cake as the first nitrogen source and gram flour, soy bean
and defatted milk powder as the second nitrogen source. They reported that medium con-

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Biopesticides production 165

taining gram flour showed the highest toxicity in case of Bti, whereas medium containing
defatted milk powder enhanced toxicity of Bs 1593.
GANGURDE and SHETHNA (1995) studied the growth of Bti and Bs in media based on
defatted mustard seed meal. They obtained appreciable growth and larvicidal activity of the
tested Bti and Bs strains. Moreover, the potencies of Bs in medium with 4% defatted mus-
tard seed meal were comparable with those of international reference standards.

2. Solid state fermentation (SSF)

Under the circumstances of the developing countries, the use of submerged fermentation for
Bt and Bs production may not be economically feasible due to the high cost of submerged
fermentation equipments such as the cost of the well-equipped deep-tank fermentor, high-
speed cooling centrifuge as well as drying facilities e.g. spray dryer. Accordingly, the SSF
methodology offers an alternative approach. Advantages of solid state fermentations are:
(1) low cost methodology (2) low wastewater output, (3) low capital investments, (4) some
spore-forming microorganisms only sporulate when grown on a solid substrate (MUDGETT
1984, WALTER and PAAU 1992 and CAPALBO 1995).
Although the extensive application of SSF technology in production of different micro-
bial products, little information have so far been published on the possible use of SSF me-
thodology in the production of Bt and other microbial control agents.
The earliest report on possible application of SSF in production of Bt appeared in a form
of U.S. patent by MECHALAS (1963) followed by reports by DULMAGE and RHODES (1971)
and SITTIG (1977).
In China, WANG (1988) reported that stable high quality Bt products were easily obtained
through certain simple and economic SSF process. The medium used was wheat bran, husk
of rice and lime powder. He claimed that this process proved to be power saving with low
cost and popular in provinces of China.
CAPALBO and MORAES (1988) carried out a study on the production of Bt by SSF me-
thodology. They used a group of available agro-industrial by-products as growth media
including wastes from pulp and paper industry, residual fermented malt from beer industry,
meal from residual cookies and biscuits from bakery industry as well as meal from chicken
slaughter house residues. They reported that the successful production of Bt formulations
with high sporulation titers occurred by using paper pulp and fermented malt. However, no
detailed information on the experimental design and fermentation conditions used were
given.
YANG et al. (1994) optimized a method advocated for the production of Bt by SSF proc-
ess. They praised the advantages of low cost, high insecticidal activity and convenience of
storage of the products. In this method, several agricultural wastes were used as solid cul-
ture media for production of -endotoxin by Bt. They investigated the effects of several
culture conditions e.g. inoculum size, pH, seed age, initial moisture content, amount of plant
ash used, and fermentation temperature. Bioassays against fourth instars larvae of Seaio-
thisa cinereasra and Pieris rapes proved the high potency of the product.
CAPALBO et al. (1994) devised two column bioreactors namely an aerated fixed bed and a
fluidized bed fermentors for SSF of Bt. They claimed that these two column bioreactors
could be used to solve the questions addressed and encountered in SSF methodology includ-
ing heat and mass transfer, aeration extent, sterility level as well as productivity of this ap-
proach. CAPALBO (1995) reviewed the aspects of the fermentation process and risk assess-
ment of Bt production in developing countries. She concluded that the local production of
bioinsecticides is highly appropriate for pest control in developing countries. She also re-
ported that Bt could be cheaply produced on a wide variety of low cost organic substrates
under SSF conditions.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

166 MAGDA A. EL-BENDARY

More recently, FODA et al. (2002) produced Bt through SSF technology using ground soy-
bean seeds as a substrate in the presence of talcum powder and wheat bran as carrier materials.
The highest growth and sporulation were obtained at 10% (w/w) of ground soybean.
ADAMS et al. (2002) used several varieties of heat-sterilized broiler litter as substrates in
solid state fermentations to produce biocontrol agents. They studied litter produced by one
flock of broilers from medicated and non medicated controlled rations and litters produced
by two flocks and four flocks on a single application of bedding material from medicated
commercial sources for production of Bt japonensis, a pathogen of Japanese beetle larvae.
Bt japonensis could not grow in unextracted 1-flock litter nor in water extracted litter, but
grew in methanol extracted litter to 5 1010 CFU/g litter and a spore count of 1 1010 CFU/g
litter. It also grew in unprocessed 2-flock and 4-flock litters, achieving cell counts of 3 109
and 1 109 CFU/g litter, respectively and spore counts of 1 109 CFU/g litter. Bioassays of
soil containing over 0.5% (db) litter fermented with Bt resulted in over 90% mortality in
21 days for first instars of Japanese beetle. They concluded that the Bt produced via solid
state fermentation using broiler poultry litter have potential in biocontrol applications in soil
environment.
On the other hand, no published information on possible use of SSF in production of Bs.
Thus, FODA et al. (2003) produced Bs 14N1 (an Egyptian isolate exhibited mosquitocidal
activity higher than that of Bs 2362) using solid state fermentation technology. They re-
ported that the highest levels of toxin production were obtained using cotton seed meal,
sesame seed meal, fodder yeast and linen seed meal in the presence of wheat bran as a car-
rier material. They found that a final spore count and LC50 for Bs 14N1 grown in cotton seed
meal under SSF were 25.2 1010 CFU/g product and 1 ppm, respectively. Supplementation
of this medium with mineral salt solution increased the toxin level several folds.

References

ABDEL-HAMEED, A., CARLBERG, G. and EL-TAYEB, O. M., 1990. Studies on Bacillus thuringiensis
H-14 strain isolated in Egypt. III. Selection of media for -endotoxin production. World J. Micro-
biol. Biotechnol., 6, 313 317.
ABDULLAH, M. A. F., 2002. Characterization of toxicity determinants in Bacillus thuringiensis mos-
quitocidal delta-endotoxins. PhD thesis, Ohio State University, USA.
ADAMS, T. T., EITEMAN, M. A. and HANEL, B. M., 2002. Solid state fermentation of broiler litter for
production of biocontrol agents. Bioresource Technol., 82, 33 41.
AHMED, H. K, 1994. Physiology and toxin gene expression of Bacillus sphaericus: a mosquito patho-
gen for biocontrol. PhD thesis, Heriot Watt University, UK.
AHMED, H. K., MITCHELL, W. J. and PRIEST, F. G., 1993. Catabolic repression of histidase biosynthe-
sis in Bacillus sphaericus by acetate. FEMS. Microbiol. Lett., 106, 71 76.
AHMED, H. K. MITCHELL, W. J. and PRIEST, F. G., 1996. Optimization of mosquitocidal toxin synthe-
sis from Bacillus sphaericus using gene fusion. World J. Microbiol. Biotechnol., 12, 7 11.
AMPOFO, J. A., 1995. Use of local row materials for the production of Bacillus sphaericus insecticide
in Ghana. Biocontrol Sci. Technol., 5, 417 423.
ARCAS, J., YANTORNO, O., ARRARAS, E. and ERTOLA, R., 1984. A new medium for growth and delta-
endotoxin production by Bacillus thuringiensis var kurstaki. Biotechnol. Lett., 6, 495.
AVIGNONE-ROSSA, C. and MIGNONE, C. F., 1995. Bacillus thuringiensis growth and toxicity; Basic
and applied considerations. Molecul. Biotechnol., 4, 55 71.
BAUMANN, L., BROADWELL, A. H. and BAUMANN, P., 1988. Sequence analysis of the mosquitocidal
toxin genes encoding 51.4- and 41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297. J.
Bacteriol., 170, 2045 2050.
BAUMANN, P. and BAUMANN, L., 1991. Effects of components of the Bacillus sphaericus toxin on
mosquito larvae and mosquitocidal-derived tissue culture grown cells. Curr. Microbiol., 23, 51 57.
BAUMANN, P., CLARK, M. A., BAUMANN, L. and BROADWELL, A. H., 1991. Bacillus sphaericus as a
mosquito pathogen: Properties of the organism and its toxins. Microbiol. Rev., 55, 425 436.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Biopesticides production 167
BAUMANN, P., UNTERMAN, B. M., BAUMANN, L., BROADWELL, A. H., ABBENE, S. J. and BOWDITCH,
R. D., 1985. Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-
molecular-weight precursors. J. Bacteriol., 163, 738 747.
BHATNAGAR, N. B., 1999. Inorganic phosphate regulates Cry4A protoxin expression in Bacillus thur-
ingiensis israelensis. Biochem. Bioph. Res. Co., 262, 359 364.
BROADWELL, A. H., CLARK, M. A., BAUMANN, L. and BAUMANN, P., 1990. Construction by site-
directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva processed toxin
of Bacillus sphaericus 2362. J. Bacteriol., 172, 4032 4036.
CAPALBO, D. M. F., 1995. Fermentation process and risk assessment. Mem. Ins. Oswaldo Cruz., 90,
135-138.
CAPALBO, D. M. F. and MORAES, I. O., 1988. Production of protein protoxin by Bacillus thuringiensis
by semi-solid fermentation. In: 12th Simposio Annual da Academia de Ciencias do Fstado de Sao
Paulo, Campinas, 1988. vol. II, pp. 46 55.
CAPALBO, D. M. F., MORAES, I. O. and MORAES, R. O., 1994. Development of a bioreactor for semi-
solid fermentation purposes: Bacterial insecticide fermentation. In: Development in Food Engineer-
ing. Proc. Int. Congress Eng. Food 6th 1993 (pub. 1994) (YANO, T., NATANNO, R. and NAKAMURA,
K., eds.). Blackie, Glasgow, UK.
CHARLES, J.-F., 1987. Ulrastructural midgut events in Culicidae larvae fed with Bacillus sphaericus
2297 spores/crystal complex. Ann. Inst. Past. Microbiol., 138, 471 484.
CHARLES, J.-F., FILHA, M. H. S. and NIELSEN-LEROUX, C., 1997. Binding of the 51- and 42-kDa
individual components from the Bacillus sphaericus crystal toxin to mosquito larval midgut mem-
branes from Culex and Anopheles (Diptera:Culicidae). FEMS Microbiol., 156, 153 159.
CHARLES, J.-F., NIELSEN-LEROUX, C. and DELECLUSE, A., 1996. Bacillus sphaericus toxins: Molecu-
lar biology and mode of action. Ann. Rev. Entomol., 41, 451 472.
DAVIDSON, E. W., 1988. Binding of Bacillus sphaericus (Eubacteriales: Bacillaceae) toxin to midgut
cells of mosquito (Diptera: Culicidae) larvae: relationship to host range. J. Med. Entomol., 25,
151 157.
DAVIDSON, E. W., 1995. Biochemistry and mode of action of the Bacillus sphaericus toxins. Mem.
Inst. Oswaldo Cruz, 90, 81 86.
DE BARJAC, H., 1990. Classification of Bacillus sphaericus strains and comparative toxicity to mos-
quito larvae. In: Bacterial Control of Mosquitoes and Black Flies (DE BARJAC, H. and SOUTHER-
LAND, D., eds.), pp. 228 236. Rutgers University Press, New Jeresy.
DE BARJAC, H., VERON, M. and COSMAO-DUMANNOIR, V., 1980. Characterization biochemique et
serologique de souches de Bacillus sphaericus pathogens ou non pour les mosquites. Ann. Inst.
Past. Microbiol., 131B, 191 201.
DELECLUSE, A., JUAREZ-PEREZ, V. and BERRY, C., 2000. Vector-active toxins: structure and diversity.
In: Entomopathogenic Bacteria: from Laboratory to Field Application (CHARLES, J.-F., DELECLUSE, A.
and NICLSEN-LEROUX, C., eds.), pp. 101 125. Kluwer Academic Publishers, Dordeeht, Netherlands.
DEPIERI, L. A. and LUDLOW, I. K., 1992. Relationship between Bacillus sphaericus spore heat resist-
ance and sporulation temperature. Lett. Appl. Microbiol., 14, 121 124.
DESAI, S. Y. and SHETHNA, Y. I., 1991. Production and formulation of Bacillus thuringiensis var.
israelenesis and Bacillus sphaericus strain 1593. Indian J. Med. Res. Section A Infectious Dis-
eases, 93, 318 323.
DHARMSTHITI, S. C., PANTUWATANA, S. and BHUMIRATANA, A., 1985. Production of Bacillus thur-
ingiensis subsp. israelenesis and Bacillus sphaericus strain 1593 on media using a by-product from
a monosodium glutamate factory. J. Invert. Pathol., 46, 231 238.
DULMAGE, H. T., 1970. Insecticidal activity of HD-1, a new isolate of Bacillus thuringiensis var.
alesti. J. Invert. Pathol., 15, 232 239.
DULMAGE, H. T., 1971. Production of delta-endotoxin by 18 isolates of Bacillus thuringiensis, sero-
type 3, in 3 fermentation media. J. Invert. Pathol., 18, 353 358.
DULMAGE, H. T. and DE BARJAC, H., 1973. HD-187, a new isolate of Bacillus thuringiensis that pro-
duces high yield of delta-endotoxin. J. Invert. Pathol., 22, 273 277.
DULMAGE, H. T. and RHODES, R. A., 1971. Production of pathogen in artificial media. In: Microbial
Control of Insects and Mites (BURGES, H. D. and HUSSEY N. W., eds.), pp. 507. Academic Press,
London.
DULMAGE, H. T., BOENINIG, O. P., REHNBORG, C. S. and HASSEN, G. D., 1971. A proposed standardi-
zation bioassay for formulation of Bacillus thuringiensis based on the international unit. J. Invert.
Pathol., 18, 240 245.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

168 MAGDA A. EL-BENDARY

DULMAGE, H. T., CORREA, J. A. and MARTINEZ, A. J., 1970. Coprecipitation with lactose as a means
of recovering the spore-crystal complex of Bacillus thuringiensis. J. Invert. Pathol., 15, 15 20.
EJIOFORE, A. O., 1991. Production of Bacillus thuringiensis serotype H-14 as bioinsecticides using a
mixture of spent brewers yeast and waste cassava starch as the fermentation medium. Disc Innova-
tion, 3, 85 88.
EL-BENDARY, MAGDA A., 1994. Studies on the production and stability of Bacillus thuringiensis
endotoxin. M.Sc thesis, Faculty of Science, Ain-Shams University, Egypt.
EL-BENDARY, MAGDA A., 1999. Growth physiology and production of mosquitocidal toxins from
Bacillus sphaericus. Ph.D. thesis, Faculty of Science, Ain-Shams University, Egypt.
FALOCI, M. M, ARCAS, J. A. and YANTORNO, O. M., 1986. Influence of certain ions on the sporulation
and formation of delta-endotoxin in Bacillus thuringiensis cultures. Revista Argentina De Microbi-
ologia, 18, 53 62.
FEITELSON, J., 1993. The Bacillus thuringiensis family tree. In: Advanced Engineered Pesticides
(KIM, L., ed.), pp. 63 67. Dekker, New York.
FODA, M. S. ABU-SHADY, M. R., PRIEST, F. G. and EL-BENDARY, M. A., 2000. Physiological studies
on pathogenic strains of Bacillus sphaericus. In: 10th Microbial conference, 12 14 November 2000,
Cairo, Egypt.
FODA, M. S., EL-BENDARY, M. A. and MOHARAM, M. E., 2003. Salient parameters involved in mos-
quitocidal toxins production from Bacillus sphaericus by semi-solid substrate fermentation. Egypt.
Microbiol., 38, 229 246.
FODA, M. S., ISMAIL, I. M. K., MOHARAM, M. E. and SADEK, KH. H. A., 2002. A novel approach for
production of Bacillus thuringiensis by solid state fermentation. Egypt. Microbiol., 37, 135 156.
FODA, M. S., SALAMA, H. S. and SELIM, M., 1985. Factors affecting growth and physiology of Bacillus
thuringiensis. Appl. Microbiol. Biotechnol., 22, 50 52.
GANGURDE, R. P. and SHETHNA, Y. I., 1995. Growth, sporulation and toxin production by Bacillus
thuringiensis subsp. israelenesis and Bacillus sphaericus in media based on mustard-seed meal.
World. J. Microbiol. Biotechnol., 11, 202 205.
GORDON, R. E., HAYNES, W. C. and PANG, C. H.-N., 1973. The genus Bacillus. In: Agricultural Hand-
book No. 427. Washington, DC: United states Department of Agriculture.
HUMPHREYS, M. J. and BERRY, C., 1998. Variants of the Bacillus sphaericus binary toxins: implica-
tions for differential toxicity of strains. J. Invert. Pathol., 71, 184 185.
ICGEN, Y., ICGEN, B. and OZCENGIZ, G., 2002a. Regulation of crystal protein biosynthesis by Bacillus
thuringiensis: II. Effects of carbon and nitrogen sources. Res. Microbiol., 153, 605 609.
ICGEN, Y., ICGEN, B. and OZCENGIZ, G., 2002b. Regulation of crystal protein biosynthesis by Bacillus
thuringiensis: I. Effects of mineral elements and pH. Res. Microbiol., 153, 599 604.
KANG, B. C., LEE, S. Y. and CHANG, H. N., 1992. Enhanced spore production of Bacillus thuringiensis
by fed-batch culture. Biotechnol. Lett., 14, 721 726.
KARIM, M. I., LUCAS, R. J., OSBORNE, K. J. and ROGERS, P. L., 1993. The effect of oxygen on the
sporulation and toxicity of Bacillus sphaericus 2362. Biotechnol. Lett., 15, 47 50.
KLEIN, D., YANAL P., HOFSTEIN, R., FRIDLENDER, B. and BRAUNS, S., 1989. Production of Bacillus
sphaericus larvicide on industrial peptones. Appl. Microbiol. Biotechnol., 30, 580 584.
KNOWLES, B. H. and ELLER, D. J., 1987. Colloid-osmotic lysis as a general feature of the mech-
anism of action of Bacillus thuringiensis -endotoxin. Biochimica et Biophysica Acta, 924, 509
578.
KUPPUSAMY, M. and BALARAMAN, K., 1991. Effect of corn steep liquor on growth and mosquito lar-
vicidal activity of Bacillus thuringiensis var. israelensis DE BARJAC 1978 and Bacillus sphaericus
Neide 1904. Indian Experimen. Biol., 29, 187 189.
LACEY, L. A., 1984. Production and formulation of Bacillus sphaericus. Mosq. News, 44, 153 159.
LACHHAB, K., TYAGI, R. D. and VALERO, J. R., 2001. Production of Bacillus thuringiensis biopesti-
cides using wastewater sludge as a raw material: effect of inoculum and sludge solids concentra-
tion. Process Biochem., 37, 197 208.
LEE, H. L. and SELEENA, P., 1991. Fermentation of a Malaysian Bacillus thuringiensis serotype H-14
isolate, a mosquito microbial control agent utilizing local wastes. Southeast Asian J. Trop. Med.
Public Health, 22, 108 112.
LISANSKY, S. G., QUINLAN, R. and TASSONI, G., 1993. The Bacillus thuringiensis Production Hand-
book. Newbury, UK: CPL Perss.
LIU, B. L. and TZENG, Y. M., 1998. Optimization of growth medium for the production of spores from
Bacillus thuringiensis using response surface methodology. Bioprocess Engineering, 18, 413 418.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Biopesticides production 169
LIU, J. W., PORTER, A. G., WEE. B. Y. and THANABALU, T., 1996. New gene from nine Bacillus
sphaericus strains encoding highly conserved 35.8-kilodalton mosquitocidal toxin. Appl. Environ.
Microbiol., 62, 2174 2176.
MANCEVA, S. D., PUSZTAI-CAREY, M. and PETER, BUTKO, 2004. Effect of pH and ionic strength on the
cytolytic toxin Cyt1A: a fluorescence spectroscopy study. Biochem. Bioph. Acta, 1699, 123 130.
MECHALAS, B. J., 1963. Method for the production of microbial insecticides. U.S. Patent 3086922.
MEGNA, J. C., 1963. Preparation of microbial insecticide. U.S. Patent 3073749.
MITTAL, P. K., 2003. Biolarvicides in vector control: challenges and prospects. J. Vect. Borne. Dis.,
40, 20 32.
MONTIEL, M. D. L. T., TYAGI, R. D. and VALERO, J. R., 2001. Wastewater treatment sludge as a raw
material for the production of Bacillus thuringiensis based biopesticides. Wat. Res., 35, 3807
3816.
MUDGETT, R. E., 1984. Solid state fermentations. In: Manual of Industrial Microbiology (DEMAIN,
A. L., SOLOMON, N. A., eds.), pp. 66 83. American Society for Microbiology).
MUMMIGATTI, S. G. and RAGHUNATHAN, N., 1990. Influence of media composition on the production
of -endotoxin by Bacillus thuringiensis var. thuringiensis. J. Invert. Pathol., 55, 147 151.
NICKERSON, K. W. and BULLA, L. A., 1974. Physiology of sporeforming bacteria associated with
insects: Minimal nutritional requirements for growth, sporulation and parasporal crystal formation
of Bacillus thuringiensis. Appl. Microbiol., 28, 124.
NORMANSELL, P. J. M., BURGES, H. D. and JARRETT, P., 1980. Genetics of Bacillus thuringiensis.
Newsletter of the International Cooperative Program on Bacillus thuringiensis (BURGES, H. D., ed.).
OBETA, J. A. N. and OKAFOR, N., 1983. Production of Bacillus sphaericus 1593 primary powder on
media made from locally obtainable Nigerian agriculture products. Can J. Microbiol., 29, 704 709.
OEI, C., HINDLEY, J. and BERRY, C., 1992. Binding of purified Bacillus sphaericus binary toxin and its
deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains.
J. Gen. Microbiol., 138, 1515 1526.
OZKAN, M., DILEK, F. B., YETIS, U. and OZCENGIZ, G., 2003. Nutritional and cultural parameters influ-
encing antidipteran delta-endotoxin production. Res. Microbiol., 154, 49 53.
PEARSON, D. and WARD, O. P., 1988. Effect of culture conditions on growth and sporulation of Bacil-
lus thuringiensis subsp. israelensis and development of media for production of the protein crystal
endotoxin. Biotechnol. Lett., 10, 451 456.
PORTER, A. G., 1996. Mosquitocidal toxins, genes and bacteria: The hit squad. Parasitol. Today, 12,
175 179.
PRIEST F. G., 1992. Biological control of mosquitoes and other biting flies by Bacillus sphaericus and
Bacillus thuringiensis. J. Appl. Bacteriol., 72, 357 369.
RAJALAKSHMI, S. and SHETHNA, Y. I., 1980. Effect of L-cyctine on macromolecular changes during
spore and parasporal crystal formation in Bacillus thuringiensis var. thuringiensis. J. Biosci., 2,
311 319.
REGIS, L., SILVA-FILHA, M. H., NIELSEN-LEROUX, C. and CHARLES J.-F., 2001. Bacteriological lar-
vicides of dipteran disease vectors. Trends in Paracitol., 17, 377 380.
ROWE, G. E., 1990. Central metabolism of Bacillus thuringiensis during growth and sporulation. Ph.D
thesis, The University of Western Ontario, London, Ontario, Canada.
ROWE, G. E. and MARGARITIS, A., 1987. Bioprocess developments in the production of bio-
insecticides by Bacillus thuringiensis. CRC Critical Rev. Biotechnol., 6, 87 127.
RUSSEL, B. L., JELLEY, S. A. and YOUSTEN, A. A., 1989. Carbohydrate metabolism in the mosquito
pathogen Bacillus sphaericus 2362. Appl. Environ. Microbiol., 55, 294 297.
SACHDEVA, V., TYAGI, R. D. and VALERO, J. R., 1999. Factors affecting the production of Bacillus
thuringiensis bioinsecticides. Rec. Res. Dev. Microbiol., 3, 363 375.
SADEK, KH. H. A., 2000. Studies on some factors affecting growth and sporulation of Bacillus thur-
ingiensis. M.Sc thesis, Faculty of Science, Cairo University, Egypt.
SALAMA, H. S., FODA, M. S. and EL-SHARABY, A., 1981. Potency of spore delta-endotoxin complex of
Bacillus thuringiensis against some cotton pests. Z. Angew. Entomol., 91, 388 398.
SALAMA, H. S., FODA, M. S., SELIM, M. H. and EL-SHARABY, A., 1983. Utilization of fodder yeast and
agro-industrial by-products in production of spores and biologically active endotoxins from Bacil-
lus thuringiensis. Zbl. Mikrobiol., 138, 553 563.
SASAKI, K., JIAVIRIYABOONYA, S. and ROGERS, P. L., 1998. Enhancement of sporulation and crystal
toxin production by corn steep liquor feeding during intermittent fed-batch culture of Bacillus
sphaericus 2362. Biotech. Lett., 20, 165 168.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

 170 MAGDA A. EL-BENDARY

SHEVTSOV, V. V., POTROVA, T. M., KHOVRYCHEV, M. P. LETUNOVA, E. V. and VADIM, V. S., 1990.
Growth and spore germination factors in different strains of Bacillus sphaericus. Microbiol., 59,
304 309.
SIEGEL, J. P., 2001. The mammalian safety of Bacillus thuringiensis based insecticides. J. Invert.
Pathol., 77, 13 21.
SIKDAR, D. P., MAJUMDAR, M. K. and MAJUMDAR, S. K., 1991. Effect of minerals on the production
of the delta-endotoxin by Bacillus thuringiensis subsp. israelensis. Biotechnol. Lett., 13, 511
514.
SINGH, G. J. P. and GILL, S. S., 1988. An electron microscope study of the toxic action of Bacillus
sphaericus in Culex quinquifasciatus larvae. J. Invert. Pathol., 52, 237 247.
SITTING, M., 1977. Pesticides process encyclopedia. NOVO Data Corp., Park Ridge, N.J.
SMITH, A. W., CAMARA-ARTIGAS, A., BRUNE, D. C. and ALLEN, J. P., 2005. Implications of high-
molecular-weight oligomers of the binary toxin from Bacillus sphaericus. J. Invert. Pathol. (In
Press)
VIDYARTHI, A. S., TYAGI, R. D., VALERO, J. R. and SURAMPALLI, R. Y., 2002. Studies on the produc-
tion of Bacillus thuringiensis based bioinsecticides using wastewater sludge as a raw material. Wat.
Res., 36, 4850 4860.
WAKISAKA, Y., MASAKI, E. and NISHIMOTO, Y., 1982. Formation of crystalline -endotoxin or poly-B-
hydroxy-butyric acid granules by asporogenous mutants of Bacillus thuringiensis. Appl. Environ.
Microbiol., 43, 1473.
WALTER, J. F. and OAAU, A. S., 1992. Microbial inoculant production and formulation. In: Soil
Microbial Ecology, Applications in Agricultural and Environmental Management, pp. 579 594.
Marcel Dekker, New York.
WANG, T., 1988. A simple and economic semi-solid fermentation process for Bacillus thuringiensis.
International Symposium on Insecticide of Bacillus thuringiensis. Hubei Academy of Agricultural
Science, Wuhan. October, 1988.
WHITE, P. J. and LOTAY, H. K., 1980. Minimal nutritional requirements of Bacillus sphaericus NCTC
9602 and 26 other strains of this species: The majority grow and sporulate with acetate as sole
major source of carbon. J. Gen. Microbiol., 118, 13 19.
WHO, 1999. Microbial pest control agent Bacillus thuringiensis. Report of UNEP/ILO/WHO (EHC,
217). WHO, Geneva.
WIDJAYA, T. S., OSBORNE, K. J. and ROGERS, P. L., 1992. The effect of acetate on growth and sporula-
tion of the mosquito pathogen Bacillus sphaericus 2362. Proc. 10th Australian Biotechnol. Conf.,
pp. 239 242.
YANG, S., YI, Z., LIANG, S. and LI, Z., 1994. Production of microbial pesticides by solid-state fermen-
tation. In: Sel. Pap. Eng. Chem. Metall. China, 1993 (pub. 1994) (MAA, M. and XIA, G., eds.),
pp. 160 165. Sci. Press, Beijing, Peop. China.
YOUSTEN, A. A., 1984. Bacillus sphaericus: microbiological factors related to its potential as a mos-
quito larvicide. Advan. Biotechnol. Proc., 3, 315 343.
YOUSTEN, A. A. and WALLIS, D. A., 1987. Batch and continuous culture production of the mosquito
larval toxin of Bacillus sphaericus 2362. J. Indust. Microbiol., 2, 227 283.
YOUSTEN, A. A., MADHEKAR, N. and WALLIS, D. A., 1984a. Fermentation conditions affecting growth,
sporulation and mosquito larval toxin formation by Bacillus sphaericus. Dev. Indust. Microbiol.,
25, 757 762.
YOUSTEN, A. A., WALLIS, D. A. and SINGER, S., 1984. Effect of Oxygen on growth, sporulation and
mosquito larval toxin formation by Bacillus sphaericus 1593. Curr. Microbiol., 11, 175 178.
ZAMOLA, B., VALLES, P., MELI, G., MICCOLI, P. and KAJFEZ, F., 1981. Use of the centrifugal separation
technique in manufacturing a bioinsecticides based on Bacillus thuringiensis. Biotechnol. Bioeng.,
23, 1079.
ZOUARI, N., ALI, S. B. S. and JAOUA, S., 2002. Production of Delta-endotoxins by Bacillus thuringien-
sis strains exhibiting various insecticidal activities toward Lepidoptera and Diptera in gruel and fish
meal media. Enzyme and Microbial Technol., 31, 411 418.

Mailing address: Dr. MAGDA A. EL-BENDARY, Microbial Chemistry Department, National Research
Center, Dokki, Cairo, Egypt
E-mail: tasnim41@yahoo.com

View publication stats