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