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Bioformulations of Bacillus Spores for using as Biofertilizer



A maximum spore percentage of Bacillus megatherium (B. megatherium) (89 %) was recorded after 96 hours of inoculation into a modified nutrient medium containing a mixture of 500 ppm of MnSo4 , CaCl2, ZnSo4 and KCL. These spores were incorporated into 21 different talc, cellulose and clay based formulations and their viability were assessed over 6 months at room temperature. Of these bioformulations, Talc -glucose, Talc -yeast and Cellulose -clay based powder formulations were selected for additional in vivo testing because of their highest levels of viability . Field experiment was conducted to evaluate the efficiency of the treatment of bean seeds with selected powder bioformulations on the growth , yield parameters and root colonization ability of B. megatherium . The powder bioformulations as well as the free spore suspension effectively enhanced plant biomass ,increased the yield and accelerate the rhizosphere colonization by the bacterium under field condition . So, the commercially acceptable powder bioformulations of the B. megatherium which have a long storage life , aid product delivery, and promote the plant growth parameters were prepared to be used instead of the traditionally used free spore suspension. [Amal. M. Omer. Bioformulations of Bacillus Spores for using as Biofertilizer. Life Science Journal 2010;7(4):124-131]. (ISSN: 1097-8135).
Life Science Journal, Volume 7, Issue 4, 2010
Bioformulations of Bacillus Spores for using as Biofertilizer
Amal. M. Omer
Soil Microbiology Unit, Desert Research Center, Cairo, Egypt
Abstract: A maximum spore percentage of Bacillus megatherium (B. megatherium) (89 %) was recorded after 96
hours of inoculation into a modified nutrient medium containing a mixture of 500 ppm of MnSo4 , CaCl2, ZnSo4
and KCL. These spores were incorporated into 21 different talc, cellulose and clay based formulations and their
viability were assessed over 6 months at room temperature. Of these bioformulations, Talc - glucose, Talc - yeast
and Cellulose - clay based powder formulations were selected for additional in vivo testing because of their highest
levels of viability . Field experiment was conducted to evaluate the efficiency of the treatment of bean seeds with
selected powder bioformulations on the growth , yield parameters and root colonization ability of B. megatherium .
The powder bioformulations as well as the free spore suspension effectively enhanced plant biomass ,increased the
yield and accelerate the rhizosphere colonization by the bacterium under field condition . So, the commercially
acceptable powder bioformulations of the B. megatherium which have a long storage life , aid product delivery, and
promote the plant growth parameters were prepared to be used instead of the traditionally used free spore
[Amal. M. Omer. Bioformulations of Bacillus Spores for using as Biofertilizer. Life Science Journal
2010;7(4):124-131]. (ISSN: 1097-8135).
Key words: Formulations, Sporulation, Bacillus megatherium, Talc, Cellulose, Clay
1. Introduction:
Formulations generally composed of the active
material which must be preserved or maintained in
viable condition to produce its biological effect, the
carrier material may or may not include the
incorporation of enrichment materials or additives.
Generally, amendments can be grouped as either
carriers (fillers, extenders) or amendments that
improve the chemical, physical, or nutritional
properties of the formulated biomass (Schisler et al.,
2004). The active material is mixed with carrier
materials such as water, clay, talc, oil or others to
make the formulation safer to handle, easier to apply
and better suited for storage. In some formulations,
enrichment materials comprising of nutrient-rich
medium such as, molasses, trehalose, maltose and
sucrose are incorporated to further enhance the
viability of core (active) materials (Brar et al., 2006;
Tu and Randall, 2005).
The commercial use of plant growth-
promoting rhizobacteria requires inoculum that
retains a high cell viability and easily be transported
and applied to seed. The aims of formulating viable
cells are to ensure that adequate cell viability is
sustained to increase the efficacy of the cells and to
facilitate the delivery and handling processes (Filho
et al., 2001). For commercialization, a long shelf-life
is an advantage for any inoculant (Fages 1990, 1992).
This can be achieved by producing granular
formulations, powder or dust formulations,
microcapsules, or oil-emulsion formulations (Brar et
al., 2006).
A formulated microbial product, for purpose of
this paper, is defined as a powder product composed
of biomass of a phosphate dissolving bacteria and
ingredients to improve the survival and efficient of
the product.
Most often, dry formulations are generally
preferred over wet formulations because they provide
extended shelf life and are easier to store and
transport (Lumsden et al., 1995).
In powder formulation, the active material is
preferred to be in spore form to increase the shelf life
and efficiency of active material. Gram-positive
microorganism that produce heat- and desiccation-
resistant spores that can be formulated into stable,
dry-powder products offer a biological solution to the
problem of biofertilizer agent formulation (Caesart
and Burr 1991)
A crucial initial step toward preserving biomass
viability during formulation is to optimize
fermentation protocols for not only maximal total
biomass but also for maximal spores production.
When producing biomass of Bacillus spp., in most
instances fermentation protocols should be designed
to maximize the production of efficacious spores
rather than vegetative cells (Driks, 2004).
Different factors can enhance the
sporulation process of Bacillus spp.It is well known
that endospore formation in B. subtilis can be
promoted by a high cell density (Grossman and
Life Science Journal, Volume 7, Issue 4, 2010
Losick, 1988), nutrient limitation (Schaeffer et al.
1965), high mineral composition and transition
metals, especially Zn and Cu ( Kihm, et al., 1988 ).
Several investigators have shown that there is
considerable variation in spore production depending
on the mineral composition of the medium (Krueger
and Kolodziej, 1977, Mallidis and Scholefield,
The spore forming bacteria B. megatherium
serve as phosphate dissolving bacteria for
solubilizing inorganic phosphatic compounds into
soluble forms which is available for plant. It is well
known that seed inoculation of phosphorus
solubilzing microbes enhance P uptake and yield of
economic parts (Gharib, et al., 2004). There is
increasing evidence that phosphorus solubilizing
bacteria improve plant growth due to biosynthesis of
plant growth substances rather than their action in
releasing available phosphorous and the
solubilization effect is generally due to the production
of organic acids by these organisms. They are also
known to produce amino acids, vitamins and growth
promoting substances like indole acetic acid (IAA)
and gibberellic acid (GA3) which help in better
growth of plants. (Ponmurugan and Gopi 2006 ) .
The inert carriers used in the formulations
were talc, clay and cellulose. For each carrier type,
1% carboximethylcellulose (CMC) as binder, traces
of sodium benzoate as stabilizer ,15% CaCo3 as
buffer and 0.25% of different enrichment materials
were incorporated .The enrichment materials
incorporated to be tested were : glucose , sucrose,
mannitol , yeast and peptone . Also, combinations of
the three carriers (talc-cellulose, talc-clay, and
cellulose-clay) were developed to be evaluated. In this study, heat-resistant endospores of B.
megatherium were formulated with various
combinations of organic carriers with and without the
incorporation of enrichment and additive materials to
develop a formulation which has potential for
large-scale applications.
2. Materials and Methods:
Bacillus megatherium (Phosphate dissolving
bacteria) was grown on nutrient, Pikovskaya
(Pikovskaya, 1948) .and modified Bunt and Rovira
media (Abd El- Hafez, 1966) for 24h, 48 h, 72 h, and
96 h of cultivation on an orbital shaker at 150 rpm at
300C. Both viable and heat - resistant spore counts
were determined. For heat - resistant spore counts,
cultures were heated at 80°C for 15 min to kill any
vegetative cells present. Spores were then
subsequently enumerated by plating aliquots of serial
dilutions onto nutrient agar media which were
incubated for 3 days at 30°C.
Enhancement of sporulation by addition of different
Nutrient broth medium was used as the basal
medium to which different metals at a concentration
of 500 ppm were added, total viable count, heat -
resistant spore count and the spore percentage were
determined after 72 h of cultivation as described
before. Duplicate flasks were set up for each
The metals tested were: KCL , MgSo4, MgCl2,
FeSo4, CaCl2, Na2So4, MnSo4, CuSo4 and K2So4 .
Preparation of B. megatherium spore yield:
B. megatherium were grown on a modified nutrient
medium supplemeted with a mixture of MnSo4,
CaCl2, ZnSo4 and KCL at a concentration of 500 ppm
for 3 days on an orbital shaker at 150 rpm at 300C till
the maximum spore yield was produced, these were
harvested and subsequently washed by repeated
centrifugation at 5,000 × g for 20 min at 4°C
/resuspension in sterile distilled water (Warriner and
Waites, 1999). Finaly, the spore pellet was re-
suspended in sterile distilled water and used as active
material in different formulations .The final spore
titer was 108 CFU/ml .
Formulation of B. megatherium :
The inert carriers, enrichment and additive
materials were mixed and sterilized by autoclaving.
Twenty ml of spore suspension were added into them,
mixed well under aseptic conditions, then the
mixtures were air dried in a laminar flow chamber for
48 hours. After drying, a 1-g sample was removed for
initial population counts. Powder formulations were
then placed in plastic petri plates, sealed with
parafilm, stored at room temperature, and sampled for
viability assessment.
Viability assessment:
In the viability assessment, population counts
of bacteria among various formulations were
determined by serial dilutions from formulations and
plated in triplicate on nutrient agar, and the CFU per
gram of formulation were enumerated at intervals of
1 to 6 months.
Seed coating with bioformulation:
One gram of the selected bacterial formulation
(Talc-glucose, Talc-yeast and Clay-cellulose) was
added to 100 g bean seeds wetted with 1 ml sterile
distilled water in a sterile plastic bag. The mixture
was shaken until the seeds were thoroughly coated
with the formulation. Also, 100 g bean seeds were
mixed with free-spore suspension of Bacillus
megatherium (108spore/ml) using CMC (1%) as
sticker (Amran, 2006). Three corn seeds coated with
the formulation were taken randomly and placed
Life Science Journal, Volume 7, Issue 4, 2010
separately into test tubes containing 10 ml sterile
distilled water. With a sterile pipette, 0.1 ml from a
10-2 dilution was placed onto nutrient plates and
spread using a sterile glass rod hockey. The plates
were sealed and incubated at 300C. After 48 hours,
bacterial colonies were counted.
Field evaluation of selected bioformulation on growth
and yield parameters of bean plant:
A field experiment was conducted in a complete
randomized design with three replicates at Maryut
Experimental Station of the Desert Research Center
(DRC), Alexandria. A standard plot size of 5 x 4 m2
was maintained for all treatments. Soil in all
treatments was amended with recommended dose of
super phosphate (15.5% P2O5) at a rate of 250 kg/fed,
ammonium nitrate (33.3% N ) at a rate of 300 kg/fed
and K-sulphate (48% K2O) at a rate of 200 kg/fed .
Seeds of faba bean (Vicia faba cv Giza 40)
purchased from agriculture ministry, Giza, Egypt.
were treated with selected bacterial formulations
(Talc-Glucose , Talc- yeast and Clay –Cellulose ) at
the rate of 1% ( powder formulation : seeds ) to give
a bacterial population of 107CFU/seed of
formulation , For the free-spore suspension treatment,
seeds were moistened in CMC solution (1%) before
application of inoculum to get a thin, uniform coating
of inoculum on seeds. Inoculated seeds were dried in
shade before sowing (Samasegaran et al., 1982), an
untreated control was maintained. .
Plant height, weight of 100seeds, seed and straw
yield (kg/fed.) of bean plants were recorded at the
time of harvest for all treatments
Chemical analysis of bean seeds and straw was
carried out after harvest to determine total
phosphorus, nitrogen and protein in both straw and
The plant materials were dried in an oven at 70
°C until a constant mass was reached and then they
were grounded for chemical analysis. Total nitrogen
was determined according to (Bremner and
Mulvaney, 1982). and phosphorus was determined
spectrophotometrically according to by the ascorbic
acid method at 650 nm according to Watanabe and
Olsen (1965).
The degree of rhizosphere colonization was
estimated as following: After 30 days from sowing,
roots and attached soil were divided into primary
roots by cutting just below the seed. Each primary
root was cut into thirds according to length, and the
top section of each root was placed into sterile
distilled water. Bacterial colonization was
significantly higher in the uppermost section of the
root compared to that of lower sections (Oliver, et al,
2004( Root sections and attached soil were sonicated
for 5 min. The resulting bacterial suspensions were
vortexed , serially diluted and grown on selective
modified Bunt and Rovira agar plates for counting of
phosphate dissolving bacteria and on nutrient media
for total count . Colonies were counted after 48 h of
incubation at 30°C, and the CFU per cm of the root
system was estimated.
Statistical analysis:
Data were subjected to statistical analysis using
the method described by (Snedecor, 1966). The least
significant difference (L.S.D) was used to
differentiate means according to (Waller and Duncan,
3. Results and Discussion:
From table (1), the highest values of TVC and
HRSC detected were 400×10 8
CFU/ ml and 100
×10 8 spore/ml after 72 h of cultivation,
respectively. Whilst the highest spore percentage (35
%) was recorded after 96 h of incubation on nutrient
media although both total viable count and heat-
resistant spore count decreased.
The nutrient media increased both the total
viable count and spore yield over 1 and 2.5 folds
compared to Bunt and Rovira medium and over 2.5
and 3.3 folds compared to Pikovskaya medium.
As the nutrient media enhanced the sporulation
process and increased the sporulating percentage of
B. megatherium, so it was selected to be a basal
medium for the production of spore yield.
Table (1): Effect of different media used on total viable cell counts, spore count and spore percentage;
Media used
Nutrient Bunt and Roveira Pikovskaya
% TVC×108
% TVC×108
24 h 70 5 7 20 2 10 10 1 10
48 h 180 18 10 45 6 13 60 8 13
72 h 400 100 25 200 40 20 160 30 19
96 h 80 28 35 120 30 25 20 5 25
TVC: total viable count HRSC: Heat-resistant spore count
Life Science Journal, Volume 7, Issue 4, 2010
As shown from table ( 2 ), incorporation of
metals to the basal sporulating media generally
increase the sporulation process except for Cu, and
this may be due to increasing of osmotic pressure of
media which have appositive effect on sporulation
process , this is supported by the fact that certain
transition metals including iron (Fe) and manganese
(Mn) in a complex sporulation medium stimulated
spore formation in certain strains of Clostridium
botulinum, but sporulation was drastically decreased
by the addition of copper (Cu) to the medium (David,
et al., 1990).
Medium containing 500 ppm of MgSo4 ,MnSo4,
K2So4 or ZnSo4 showed the highest values of both
total viable count and heat-resistant spore count
compared with other concentrations tested and this is
agree with a fact that there is a correlation between
growth rate and spore yield. (Osadchaya, et al., 997).
Addition of a mixture composed of 500 ppm
of MnSo4 , CaCl2 , ZnSo4 and KCL (the metals gave
the spore percentage over 75% ) to the basal nutrient
medium enhanced the sporulation process
,increased the spore yield to about 160 ×108CFU ml-
1and increased the spore percentage to 89% (over
64% compared to control) .
The specific functions of metal ions in
sporulation are probably that they act as activators of
the various enzyme systems necessary for sporulation
(Bruno and Ralph, 1964).
Varying the metal concentration in the
sporulation media is known to influence the thermal-
resistance spores due to inducuction of genes coding
for the two small acid soluble proteins earlier during
sporulation in the media that contained higher metal
concentrations (Oomes and Brul, 2004).
Table (2): Effect of different metals on the enhancement of sporulation process (total viable cell counts, spore
count and spore percentage):
Metals added
at 500 ppm Total viable count
×108CFU ml-1 Heat-resistant spore
×108 spore ml-1
Spore percentage
Control 80 20 25
KCL 96 72 75
MgSo4 160 88 55
MgCl2, 80 32 40
MnSo4 120 96 80
CaCl2 90 74 82
FeSo4 55 30 55
Na2So4 34 22 65
K2So4 190 80 42
CuSo4 0, 002 0.0004 20
ZnSo4 110 86 78
*Mixture 180 160 89
*Amixture composed of each of the following: 500 ppm of MnSo4, CaCl2, ZnSo4 and KCL.
In all formulations, bacterial populations
declined steadily over time, the bacteria survived
even up to 180 days of storage with different
percentage although the population declined from 30
days of formulation as shown from table (3) .
The type of carriers used influenced the viability of
bacterial cells, the bacterial populations for both
cellulose and talc--based formulations were
generally higher than that of clay-based formulation..
All cellulose-based formulations with and without
enrichments were able to maintain the highest viable
cell count throughout the storage period, with a mean
cell count of 81.7 x 108CFU/g of formulation
compared to talc -based formulations and clay-based
formulations, with 80 and 9.3 x 108CFU/g ,
respectively after 180days of storage . Recent studies
on beneficial rhizobacteria have investigated the
efficacy of powder formulations in combination with
methylcellulose and xanthan gum (Kloepper and
Schroth, 1981, Suslow and Schroth , 1982).
Among the formulations, enrichment materials
proved to be the most useful as highest number of
viable cells were recovered.
These are in agreement with previous research
which showed that high-molecular-weight (C6 to
C12) compounds such as sucrose and trehalose
enhanced survival of bacteria in dried biopolymers
(Ilyina et al., 2000). Among these enrichment
materials, glucose and yeast were the most efficient
ones in preserving bacterial populations at different
Also, the formula composed of clay- cellulose
was the most effective one among other formulations
in survival of bacterial populations. This is may be
due to the advantages of both clay and cellulose as
Life Science Journal, Volume 7, Issue 4, 2010
This showed that clay materials benefited the
cells by providing large surface areas which act as an
effective survival unit for nutrient absorption and
protection (Lunsdorf et al., 2000, Ting et al., 2010).
In addition to the catalytic and shielding properties,
clay also has good cation exchange capacity which
enhances the bacterial metabolic activity (Adamis et
al., 2005). High cation exchange capacity enhances
bacterial metabolic activity leading to higher viability
(Beveridge, 1988),
The great value of porosity and capacity of
cellulose to absorbe the hydrophobic and
hydrophilic liquids may be useful for application as
carrier for microorganisms for agriculture objectives.
These properties are important for distribution of
microorganisms in the granules and absorption of
substrates limiting the growth of microbial population
(Ilyina et al., 2000).
This indicates the possibility of developing
simple powder formulations with the capacity to
provide long-term survival of beneficial
rhizobacterial strains at high populations.
Among these formulations, five formulations (T-
glucose, T-yeast, Ce -glucose, Cellulose - yeast and
Cellulose-clay) were selected for additional testing
because they had higher levels of viability amongst
bacterial cell populations.
Table (3): Effect of different carriers and amendments on survival of bacteria in powder formulations
x108CFU/g of formulation Formulation
0day 30day 60day 90day 120day 150day 180day
Ta lc 200 190 80 60 500 40 40 20
T-glucose 220 180 180 160 160 145 150 68
T-sucrose 190 190 160 90 70 50 50 26
T-.manitol 210 200 90 90 60 60 45 21
T-yeast 200 200 200 190 180 170 150 75
T- pe pt on 180 180 120 80 70 45 45 25
Clay 200 110 30 10 6 5 2 1
C-glucose 190 180 90 50 40 20 20 11
C-sucrose 180 150 40 15 12 4 3 1.6
C-manitol 210 200 170 35 20 10 7 3
C-yeast 210 170 150 70 60 30 18 8.5
C-pepton 200 140 35 30 20 10 6 3
Cellulose 210 210 200 160 150 140 70 33
Ce-glucose 230 230 180 140 140 120 120 58
Ce-sucrose 180 160 160 140 100 100 70 40
Ce-.manitol 190 160 130 120 120 100 70 37
Ce-yeast 200 200 190 190 160 140 110 55
Ce-pepton 200 180 160 90 90 80 50 40
Talc-Cellulose 190 180 145 100 74 60 57 30
Talc-Clay 210 200 175 140 130 105 105 50
Cellulose-Clay 200 200 200 190 170 170 160 80
Percent survival of each formulation was determined as follows: [(CFU/g of formulation at sampling)/CFU/g of
formulation at beginning of experiment)] x 100
Results from table (4) showed that all seeds were
successfully coated with bacterial spores of B.
megatherium with different ranges , the highest
bacterial population (2.3x 107CFU /seed) was on seed
coated with the cellulose - clay based-formulation
followed by talc based-formulations and the lowest
was on seed with the cellulose - based formulations
(0.8 x 107 CFU /seed). It has been proposed that a
uniform coating of approximately 107 CFU of
bacteria per seed is necessary for successful
bacterization (Suslow, 1982).
Generally, bacterial populations on seeds treated
with free cell suspension and CMC was higher than
that of powder based-formulations except for
cellulose - clay based-formulation which gave the
highest value.
The ability of any PGPB to colonize its target
plant roots and to produce growth effects is an
ultimate test (Bashanand, 1997; Glick and Bashan
The obtained data from the table (6) generally
showed that application of biofertilizer in the form of
free-spore suspension or powder formulations
considerably stimulates both total microbial and
phosphate dissolvers counts in the rhizosphere of
bean plants. Maximum numbers of inoculated
Life Science Journal, Volume 7, Issue 4, 2010
bacteria were recovered from the rhizosphere of free-
spore suspension and cellulose - clay formulation
treated plants after 4 weeks of growth.
These indicate that the colonization capacity of
B. megatherium in free-spore suspension was
superior to that of other powder formulations except
for cellulose -clay formulation which gave the same
colonization capacity.
Table ( 4 ): Bacterial populations on seed surface treated with different formulations :
Formulation x107CFU/seed Formulation x107CFU/seed
FSS 2 Ce-glucose 0.8
T- glucose 1.5 Ce - yeast 0.9
T- yeast 1.3 Ce - clay 2.3
FSS: free spore suspension
Table ( 5 ): The effect of selected formulations on the degree of rhizosphere colonization:
Treatments Total count
x106CFU/cm PDB count
Control 9 8
Fss 20 16
T-glucose 18 10
T-yeast 17 11
Ce-clay 21 16
Generally, data represented in table (6) indicated
that seed inoculation with all bioformulations were
found to enhance the plant height, weight of 100
seeds ,seed and straw yield of the faba bean plant
over the control . no significant differences among
bioformulations treatments were detected .
For plant height, the highest remarkable increase
was recorded with half free spore suspension
application followed by Cellulose-clay based
formulation relative to control.
Free spore suspension recorded the significance
increase over the control and other powder
formulations for straw yield,, while Cellulose-clay
based formulation record the heights significance
value for seed yield .
Biofertilization treatments caused an effective
action on N and P uptake by different parts of faba
bean plants (seed and straw) relative to the control.
Concerned to N % , there is no significant differences
among formulations of B. megatherium in case of
straw although the free spore suspension gave the
maximum N % in seeds followed by other powder
However, the overall mean of both seeds and
straw P % as affected by B. megatherium inoculation
indicated a relative increase by about 18 % and 25 %,
respectively over the untreated one.
Higher accumulation of P in seeds were
recorded with inoculation of free spore suspension
followed by other powder formulations where there is
no significant differences among them .For P % in
straw, there is no significant differences among
different biotreatments. Of these treatments, the free
spore suspension and Cellulose-clay based powder
formulation were more effective than the other
formulations and the spore application without a
carrier as compared with other treatments.
Table (6): Evaluation of selected formulations on the bean growth and yield parameters:
Yield kg/fed Nitrogen % Phosphorus %
Weight of
seeds(gm) Total
Control 48 c 80 b 2592c 1160c 1432b 2.9 b 1.5 a 0.66 b 0.5 b
Fss 62 a 95 a 3100a 1288b 1812a 3.5a 1.9 a 0.88 a 0.79 a
T-glucose 52 bc 90 ab 2760bc 1300b 1460b 3.2ab 1.9a 0.81ab 0.73 a
T-yeast 55abc 90 ab 2800b 1364b 1436b 3.2ab 1.7 a 0.8 ab 0.73 a
Ce-clay 60ab 93 a 2800b 1500a 1300b 3.4ab 1.8 a 0.85ab 0.76 a
LSD(0.5%) 7.6 11.1 174.5 89.04 183.7 0.56 0.77 0.139 0.173
- FS: Free spore suspension
- Values with same letter are not significantly different (P = 0.05).
Life Science Journal, Volume 7, Issue 4, 2010
4. Conclusion
This study reports the results of investigations
on the different formulation combinations in
maintaining the efficacy and viability of the B.
megatherium endospores.
When heat-resistant endospores of B. megatherium
produced from modified nutrient medium were
formulated with different inorganic carriers, the
dormancy of the endospores was maintained for 6
months at room temperature depending on the carrier
type, also the enrichment materials were effective in
enhancing the cell viability especially glucose and
yeast .Of these bioformulations, cellulose-clay and
talc based powder formulations were more effective
than other formulations tested.
From the field application, it is indicated that
seed treatment with powder formulations especially
Cellulose –clay based formulation is an effective
delivery system that can provide a conducive
environment for B. megatherium to solubilize
phosphate ,enhance growth and yield parameters of
plants and has the potential for utilization in
commercial field application .The application of
powder formulation of phosphate solubilizer B.
megatherium spores undoubtedly shows their
advantages over traditionally used free spore
inoculation as they increase the efficacy of the spore
, enhance cell viability , facilitate the delivery and
handling processes and promote the growth
parameters of the plant.
Finally, the powder bioformulations have a
potential for large-scale applications instead of the
traditionally used free spore inoculation.
Corresponding author:
Amal. M. Omer
Soil Microbiology Unit, Desert Research Center,
Cairo, Egypt
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... When producing Bacillus spp. biomass, fermentation protocols should be designed to maximize the production of spores rather than vegetative cells (Driks, 2004;Omer, 2010). ...
... Generally, biofertilizers have been developed with 10 6 -10 9 CFU g −1 of PGPBs in carriers [16,44,53,54]. In our study, the three rice varieties responded to 10 5 and 10 7 CFU mL −1 of B. pumilus TUAT1 (Table S3), whereas 10 5 did not solely show the enhancement of each variety (Figure 1). ...
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Bacillus pumilus strain TUAT1 is a plant growth-promoting bacterium (PGPB) applied as a biofertilizer, containing its spores, for rice. In this study, we analyzed the short-term effects of biofertilization on plant growth in the nursery and long-term effects on plant vegetative growth, yield, and lodging resistance in paddy fields using animal feed rice (‘Fukuhibiki’ and line LTAT-29 which was recently officially registered as a cultivar ‘Monster Nokodai 1′) and fodder rice (line TAT-26). The effects of the biofertilization were analyzed under two nitrogen treatments and at two transplanting distances in the field. The application of 107 colony forming units (CFU) mL−1 bacterial spore solution to seeds on plant box significantly improved the initial growth of rice. The biofertilizer treatment with this strain at 107 CFU g−1 onto seeds in nursery boxes increased the nitrogen uptake at the early growth of rice in the field, resulting in higher growth at the late vegetative growth stage (e.g., tiller number and plant height). Furthermore, the improvement of growth led to increases of not only yield components such as the total panicle number (TPN) and the number of spikelets in a panicle (NSP) in LTAT-29 but also the straw yield and quality of TAT-26. The lodging resistances of these forage rice plants were also improved due to the increased root development and photosynthesis creating tougher culms.
... Presently, many forms of bio-formulation are available in the market in liquid and solid states with their own pros and cons. For instance, dry formulations have longer shelf lives and are easy to store and transport, while liquid formulations are faster to approach the plant system [1,[4][5][6][7][8][9]. ...
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The present study was an attempt to evaluate the bio-formulations of phosphate-solubilizing fungus Aspergillus awamori S29 using two economically viable carriers (calcium alginate and agar) in repeated batch fermentation. Further, the viable cell count under storage and response of these stored bio-formulations on the growth of wheat plants were studied at the end of 2, 4, and 6 months of incubation. Also, the response of these formulations in next season on pearl millet (bajra) was studied without further inoculation. In repeated batch fermentation assay, immobilized form performed significantly better than free form. The viability of fungal inoculant was 88.2% in calcium alginate-based bio-formulation after six months of storage. These bio-formulations showed not only a statistically significant increase in the growth of wheat crop in first season but also of pearl millet in next season. This work strengthens the re-usability potential of immobilized bio-formulations for next season crop.
... To improve the isolation of microorganisms in soil, a particular culture medium (SIA) allowing the isolation of viable but non-culturable strains within 1-3 months of incubation at 28 • C could help develop different cultural methods for the diagnosis of unculturable fungi (Sia et al., 2018). However, different co-formulants could behave differently on the survival of bacteria in formulations (Omer, 2010;. As alternatives, or in combination, to the classical cultivation methods, the MicroResp (Creamer et al., 2016), and communitylevel physiological profiles (Frąc et al., 2017) methods can be used for the identification of bacteria species. ...
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Microorganisms promised to lead the bio-based revolution for a more sustainable agriculture. Beneficial microorganisms could be a valid alternative to the use of chemical fertilizers or pesticides. However, the increasing use of microbial inoculants is also raising several questions about their efficacy and their effects on the autochthonous soil microorganisms. There are two major issues on the application of bioinoculants to soil: (i) their detection in soil, and the analysis of their persistence and fate; (ii) the monitoring of the impact of the introduced bioinoculant on native soil microbial communities. This review explores the strategies and methods that can be applied to the detection of microbial inoculants and to soil monitoring. The discussion includes a comprehensive critical assessment of the available tools, based on morpho-phenological, molecular, and microscopic analyses. The prospects for future development of protocols for regulatory or commercial purposes are also discussed, underlining the need for a multimethod (polyphasic) approach to ensure the necessary level of discrimination required to track and monitor bioinoculants in soil.
... Currently, plant-based antifungal formulations are used to manage the diseases of the plants. These antifungal formulations comprise effective compounds accompanied by specific inert carrier material (Omer, 2010). Anis et al. (2012) established a Naalginate based bioformulation to control the charcoal rot disease of sunflower. ...
Leaf spot disease caused by Curvularia lunata is one of the major constraints affecting the cultivation of maize in India. Recently, it has been reported that the severity of curvularia leaf spot was prevalent in moderate to severe intensities and cause extensive damage to the crop thus lowering the yields. In the present study, in vivo preventive effect of Lawsonia inermis Linn. bioformulation on curvularia leaf spot disease of maize has been studied. All the experiments were conducted in pots. Percent disease index (PDI), percent efficacy of disease control (PEDC), chlorophyll contents, total carotenoids, and others various growth characteristics like plant height, number of leaves per plant, total carbohydrate and protein content were recorded. Percent seed germination was also observed for seeds treated with all formulations. A significant control of leaf spot disease was recorded with bioformulations treatment T3 [seeds were treated with partially purified acetone extract (4 ml): 100% clove bud oil cake (4 ml): 100% cow dung (2 ml)], T4 [seeds were treated with partially purified acetone extract (3 ml): 100% clove bud oil cake (4 ml): 100% cow dung (3 ml)], T2 [seeds were treated with 100% alcoholic crude extract (2 ml): 100% clove bud oil cake (6 ml): 100% cow dung (2 ml)] and T1 [seeds were treated with 100% alcoholic crude extract (4 ml): 100% clove bud oil cake (4 ml): 100% cow dung (2 ml)] as compared to other bioformulation treatments and its PDI and PEDC were recorded 9.10%, 10.00%, 17.50%, 19.00% and 89.47%, 88.43%, 79.76%, 78.03%, respectively. Results showed that treatment with bioformulation treatment T3, notably increased plant height, number of leaves per plant, chlorophyll, carotenoids, and total soluble sugar content followed by formulations number T4, T2, and T1. However, total soluble protein content was observed to be less in T3 as compared to control. This study suggests that these bioformulations could be essential towards sustainable agricultural science deprived of harming the ecosystem. The synthesized bioformulations have enormous potential to be commercially explored for agriculture use.
The rhizosphere is a thin film of soil that surrounds plant roots and the primary location of nutrient uptake, and is where important physiological, chemical, and biological activities are occurring. Many microbes invade the rhizosphere and have the capacity to promote plant growth and health. Bacillus spp. is the most prominent plant growth promoting rhizobacteria due to its ability to form long-lived, stress-tolerant spores. Bacillus-plant interactions are driven by chemical languages constructed by a wide spectrum of metabolites and lead to enhanced plant growth and defenses. Thus, this review is a synthesis and a critical assessment of the current literature on the application of Bacillus spp. in agriculture, highlighting gaps that remain to be explored to improve and expand on the Bacillus-based biostimulants. Furthermore, we suggest that omics sciences, with a focus on metabolomics, offer unique opportunities to illuminate the chemical intercommunications between Bacillus and plants, to elucidate biochemical and molecular details on modes of action of Bacillus-based formulations, to generate more actionable insights on cellular and molecular events that explain the Bacillus-induced growth promotion and stress resilience in plants.
The growing global demand for animal products and processed meat has created a challenge for the livestock sector to enhance animal productivity without compromising product quality. The restriction of antibiotics in animal feeds as growth promoters makes the use of probiotics a natural and safe alternative to obtain functional foods that provide animal health and quality and to maintain food safety for consumers. To incorporate these additives into the diet, detailed studies are required, in which in vitro and in vivo assays are used to prove the efficacy and to ensure the safety of probiotic candidate strains. Studies on the use of Bacillus subtilis natto as a spore-forming probiotic bacterium in animal nutrition have shown no hazardous effects and have demonstrated the effectiveness of its use as a probiotic, mainly due to its proven antimicrobial, anti-inflammatory, antioxidant, enzymatic, and immunomodulatory activity. This review summarizes the recent scientific background on the probiotic effects of B. subtilis natto in animal nutrition. It focuses on its safety assessment, host-associated efficacy, and industrial requirements.
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Bioaugmentation is commonly employed as a remediation technology. However, numerous studies indicate that introduced microorganisms often do not survive in the environment and thus do not increase contaminant remediation. This review details several new approaches that may increase the persistence and activity of exogenous microorganisms and/or genes following introduction into the environment. These techniques include: (1) bioaugmentation with cells encapsulated in a carrier such as alginate; (2) gene bioaugmentation where the goal is for the added inoculant to transfer remediation genes to indigenous microorganisms; (3) rhizosphere bioaugmentation where the microbial inoculant is added to the site along with a plant that serves as a niche for the inoculant's growth; and (4) phytoaugmentation where the remediation genes are engineered directly into a plant for use in remediation without a microbial inoculant. Additionally, the review discusses the generation of genetically engineered microorganisms for use in bioaugmentation along with methods for the control of the engineered microorganisms in the environment, and the potential effects of the release on indigenous organisms. Various methods for the detection of introduced microorganisms such as real-time polymerase chain reaction (PCR) and reporter genes are also addressed. Ultimately, these new approaches may broaden the application of bioaugmentation as a remediation technology.
Bacillus subtilis is a food spoilage spore-forming bacterium. The spores can be very heat-resistant and may cause problems in the production of foods. Varying the metal concentration in the sporulation media is known to influence the heat resistance of the spores. The effect of changing the metal composition of the sporulation media was monitored by means of transcriptome analysis. Heat resistance was measured based on the method described by Kooiman and the transcriptome analysis was performed with micro-arrays of B. subtilis wt168 DNA. The micro-array analysis showed that genes coding for the two small acid soluble proteins (SASPs a and b) were induced earlier during sporulation in the media that contained higher metal concentrations and produced thermal-resistant spores. The results also showed that the spores of a product isolate (strain B. subtilis A163) were much more heat-resistant than the spores produced under the same environmental conditions by strain B. subtilis PS832. In addition to other genes, SASP a was also significantly induced in cells of strain B. subtilis A163 at early to middle stages of sporulation.
The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to =98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.
The relation between heat resistance characteristics and the composition of the bacterial spores was studied in five strains of Bacillus stearothermophilus. A relationship was found to exist between the dipicolinic acid (Dpa) and calcium content of the spore and the heat resistance characteristics. The stability of the Dpa chelates was also significantly related to heat resistance.
From recycling/de-inking pulp residues of paper industry, ThermoFiberGen Inc. (Bed-ford, Massachusetts) makes absorbent granules currently marketed under the trade name BIODAC. In this work it was demonstrated the possibility to use the granules of BIODAC 12/20 as the carrier to microorganisms that can be useful for bioremediation applications. As studying models the six different types of microorganisms (bacteria and fungi) isolated from petroleum hydrocarbon-contaminated soils and their consortium, were used. It was demonstrated that BIODAC did not inhibit the microbial growth in the liquid culture me-dia as well as in the solid phase of agar-agar. The microorganisms were alive after desicca-tion with granules and their storage during 2 months at least. The major number of CFU (approximately at 6 times) was detected in the petroleum hydrocarbon-contaminated soil inoculated with preparation of consortium-BIODAC as compared with the soil inoculated with the same quantity of consortium only. The presence of BIODAC affected the soil capacity to retain the moisture. It was observed the buffer property of this product and that BIODAC using (at reasonable limits) did not affect the growth of plants (canary grass and kidney bean). Therefore, it may be applied as carrier to microbial inoculation of soils.
Spore crops of Bacillus subtilis PS 346 were increased by the addition of a combination of glucose and ribose to the sporulating medium. The increase in spore yield was over 100% higher in glucose:ribose-containing cultures, compared with cells grown on glucose as a sole carbon and energy source. Spore crops obtained from cultures grown on glucose:ribose had similar thermal resistance and hydrodynamic mean radius to those obtained when cultivated solely on glucose- or ribose-containing media. When other combinations of dual carbohydrates were tested it was apparent that those substrates primarily channelled through the pentose phosphate pathway gave enhanced sporulation. This effect was also observed by supplementing glucose-containing media with pyruvate but not citrate or malate. Those substrates that are channelled through the glycolytic pathway were also less effective in this respect. From the results obtained it appears that the enhancement of sporulation could be attributed to alteration in carbon and electron flow through metabolic pathways leading, directly or indirectly, to metabolites that play a role in sporogenesis regulation.