World Journal of Microbiology and Biotechnology (2021) 37:154

https://doi.org/10.1007/s11274-021-03122-2

ORIGINAL PAPER

Polyurethane foam as an inert support using concentrated media

improves quality and spore production from Bacillus thuringiensis
Briseida Flores‑Tufiño1   · Francisco Figueroa‑Martínez2   · Gustavo Viniegra‑González1   · Octavio Loera1 

Received: 22 February 2021 / Accepted: 7 August 2021

© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a gram-positive bacterium that produces spores, several virulence
factors and insecticidal toxins, making this microorganism the most used biopesticide worldwide. The use of inert supports
such as polyurethane foam (PUF) in solid cultures has been a great alternative to produce various metabolites, including
those produced by Bt. In this study we compared the yields, productivity and quality of the spores by two wild strains of Bt,
(Y15 and EA3), grown in media with high substrate concentration in both culture systems: liquid and solid (PUF as solid
inert support). Both strains showed 2.5- to 30-fold increases in spore production and productivity in solid culture, which
showed an even greater increase when considering the spores retained in the PUF observed by scanning electron microscopy.
Moreover, spore produced in solid culture showed up to sevenfold higher survival after a heat-shock treatment, relative to
spores from liquid culture. The infectivity against larvae of Galleria mellonella (Lepidoptera:Pyralidae) improved also in
spores from solid cultures. This comparison showed that the culture of Bt on solid support has clear advantages over liquid
culture in terms of the production and quality of spores, and that those advantages can be attributed only to the culture system,
as the same media composition was used in both systems.

Keywords  Bacillus thuringiensis · Infectivity · Liquid culture · Quality · Solid culture · Spores

Introduction this bacterium, including Vip, Sip, Cyt and Cry toxins. Vip
and Sip proteins are normally produced and secreted to the
Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a medium during the exponential growth phase; these proteins
Gram-positive bacterium belonging to the Bacillus cereus have insecticide action towards the insect orders Lepidop-
group that can produce spores, a wide variety of toxins and tera, Hemiptera and Coleoptera (Estruch et al. 1996; Warren
several enzymes of high industrial interest such as proteases et al. 1998; Sattar and Maiti 2011; Palma et al. 2014; Savini
and amylases (Brar et al. 2007; Smitha et al. 2013; Palma and Fazii 2016; Domínguez-Arrizabalaga et al. 2020). Simi-
et al. 2014; Martínez-Zavala et al. 2020). Also, B. thur- larly, Cyt and Cry δ-endotoxins, belonging to the class of
ingiensis is extensively used for pest control since it has a pore-forming toxins (Bravo et al. 2007), accumulate during
formidable arsenal against insects, including lethal insecti- the stationary growth phase and are effective against Lepi-
cide toxins and several virulence factors (phospholipases, doptera, Diptera, Coleoptera and Hymenoptera species (Dul-
enterotoxins, chitinases, and a β-exotoxin) that allow Bt to mage and Aizawa 1982; Feitelson et al. 1992; Lambert and
thrive inside the insect body (Sauka and Benintende 2008; Peferoen 1992; Savini and Fazii 2016; Domínguez-Arriza-
Palma et al. 2014). The broad insecticide spectrum of Bt balaga et al. 2020). Cyt and Cry toxins appear as crystalline
relies on the collection of different toxins associated with inclusions that represent up to 25% of the dry weight of the
sporulated cell (Agaisse and Lereclus 1995).
* Octavio Loera Due to the high diversity of Cry toxins (more than 308
loera@xanum.uam.mx Cry toxins according to Crickmore et al. 2020) as well as
their broad insecticidal spectrum, Cry toxins and Bt spores
1
Departamento de Biotecnología, Universidad Autónoma (spore/crystal complexes) have been used in the formula-
Metropolitana-Iztapalapa, 09340 Mexico City, Mexico
tion of commercial bioinsecticides. The production of the Bt
2
Catedrático CONACyT, Universidad Autónoma spore/crystal complex is carried out in liquid cultures (LCs)
Metropolitana-Iztapalapa, 09340 Mexico City, Mexico

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with carefully balanced carbon/nitrogen ratio, as it affects at the maximum production time. Spore retention in PUF
both Cry protein production and Bt sporulation; a C/N ratio was determined from three experimental units by washing
close to seven favours the production of Cry protein; while a the solid support, after initial spore harvest, using NaCl or
C/N ratio around four favours the production of spores (Far- Tween solution. Viability, bioassay, and total protein analy-
rera et al. 1998; Anderson and Jayaraman 2003; López and ses were also carried out using spore samples obtained from
Torre 2005). In general, glucose is the main carbon source, three independent experimental units.
while the main nitrogen sources for Bt spore/crystal produc-
tion are fishmeal, corn seed, cotton seed, peptone or yeast
Bacterial strain and media
extract.
Recently, the production of spores and Cry protein of
Two B. thuringiensis strains were used, identified as Y15
B. thuringiensis has increased using solid cultures (SCs)
and EA3; both strains were isolated from sugarcane planta-
(Zhuang et al. 2011; Jisha et al. 2015; Ballardo et al. 2016;
tions in Morelos state, Mexico, and stored in the collection
El-Bendary et al. 2016; Lima-Pérez et al. 2019; Rodríguez
of the Laboratory of Plant Parasitology at the Center for Bio-
et al. 2019; Mejias et al. 2020). Generally, SC represents
logical Research of the Universidad Autónoma del Estado de
a cheaper alternative to LC, with lower operating cost and
Morelos, Mexico (Alquisira-Ramírez et al. 2014).
also agro-industrial residues can be used as solid supports
or even substrates. Higher productions of enzymes, spores
and other secondary metabolites have been reported in SC Inoculant preparation
than in LC (Mitchell et al. 2000; Pandey et al. 2000); this
has been associated with higher oxygen diffusion, higher Pre-inoculum was obtained by placing a filter paper strip
contact surface area, and less catabolic repression. However, impregnated with Bt spores in a flask containing 25 mL of
product yield comparisons between solid and liquid cultures Luria–Bertani broth. The flasks were incubated for 12 h
do not consider the use of substrates that differ in chemi- at 150 rpm and 30 °C. After this time, the inoculum was
cal composition. For example, LCs use defined carbon and obtained by transferring 5% v/v of the pre-inoculum to
nitrogen sources, whereas SCs use mainly agro-industrial 50 mL of GYS medium, which comprised glucose (30 g L ­ –1),
–1 –1
residues of variable composition. In addition, the physico- yeast extract (12 g L
­ ), ­(NH4)2SO4 (3 g ­L ); ­CaCl2·2H2O
chemical properties of the substrate/support change over the (0.12 g ­L–1), ­MgSO4·7H2O (1.5 g ­L–1), ­MnSO4·H2O (0.09 g
time course of the culture, making it difficult to quantify ­L–1), ­K2HPO4 (1.5 g L
­ –1), and K ­ –1) (Berbert-
­ H2PO4 (1.5 g L
the actual advantages of the solid over the liquid culture. Molina et al. 2008), followed by 6 h incubation at 30 °C
Therefore, the aim of this work was to improve spore pro- under agitation at 150 rpm.
duction by B. thuringiensis, comparing the quality of spores,
including viability and virulence, and protein yields in solid
Solid and liquid cultures
culture, using polyurethane foam as an inert solid support;
this allows the use of the same media composition as the
The modified medium GYS 4× was used in both systems
liquid cultures. This analysis comprised glucose and media
for comparisons between liquid and solid cultures. Modi-
concentrations higher than those previously reported.
fied GYS 4× medium comprised glucose (120 g ­L–1), yeast
extract (48  g ­L–1), ­(NH4)2SO4 (12  g ­L–1), ­CaCl2·2H2O
(0.48 g ­L–1), ­MgSO4·7H2O (6 g L­ –1), ­MnSO4·H2O (0.36 g
Materials and methods ­L ), ­K2HPO4 (6 g ­L ), and ­KH2PO4 (6 g ­L–1). Under these
–1 –1

conditions the culture medium has a C/N ration of 5.98. The

Experimental design
pH of the medium was adjusted to 7.5 before the sterilisa-
tion (Berbert-Molina et al. 2008). In both culture systems,
Spore production, spore quality and total Cry protein were
the inoculation was performed by adding a volume of the
determined for two strains of B. thuringiensis in solid and
inoculum equivalent to 5% of the medium volume.
liquid culture at high substrate concentration, using the fol-
lowing experimental design. Each experimental unit con-
sisted of 250 mL Erlenmeyer flasks with 14.6 mL of GYSx4 Liquid cultures
medium for liquid culture or 14.6 mL of the same medium
embedded in 1 g of polyurethane foam (PUF) as support Liquid cultures were prepared in 250 mL flasks containing
for solid culture. The maximum spore production time was 14.6 mL of GYS 4× medium. The medium was inoculated
determined for each system by measuring spore production with 0.7 mL of inoculum (5% v/v) of Bt strains Y15 or EA3,
at 21, 28, 35, 42, 48 and 72 h, from at least three experimen- then incubated at 30 °C with constant agitation at 200 rpm.
tal units at each time. All posterior analyses were carried out Samples were taken at different times for 72 h.

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Solid cultures with 1 mL of a suspension of 1 × ­108 spores m ­ L–1. For the
negative control, 10 larvae were placed in Petri dishes using
Solid cultures were also prepared in 250 mL flasks, using triplicates and to each set of larvae was added 0.1 g of cotton
1 g of polyurethane foam cubes of 7 mm (PUF cubes) with impregnated with 1 mL of sterile 1% Tween 80 solution. The
a density of 17 kg ­m–3 as sterile solid support. The support larvae were incubated at 26 °C for 6 days with photoperiods
was impregnated with 14.6 mL of GYS  4× medium inocu- of 12 h/12 h (light/darkness). After the incubation time, the
lated with 0.7 mL of inoculum (5% v/v) of Bt strains Y15 number of dead larvae in each treatment was determined.
or EA3 and homogenised with a sterile spatula. The flasks
were incubated with no agitation at 30 °C (72 h). Spores Protein estimation
and Cry protein harvesting was done by squeezing the PUF
cubes with 60 mL syringes (López et al. 2010). After the Total protein obtained in solid and liquid cultures were
extraction process, the PUF cubes was washed with 35 mL determined by Bradford's method in a microplate, follow-
of 1% Tween 80 solution or 0.85% NaCl to measure spores ing the manufacturer’s instructions (Bio-Rad Protein Assay).
retained in the PUF cubes. Samples were taken at different
times for 72 h. Statistical analysis

Scanning electron microscopy The data are presented in the graphs as mean ± SD. The
effect of time in spore production was determined by analy-
Bt strains grown in the PUF cubes at the time of maximum sis of variance (ANOVA, α = 0.005, P values ˂ 0.05 were
production (48  h) were observed by scanning electron considered significant) and significant differences by Tuk-
microscopy (SEM). Selected samples were subjected to a ey’s test as mean comparison criterion (α = 0.005, P values
glutaraldehyde 3.5% fixation process for 48 h. Then, the PUF ˂ 0.05 were considered significant). Significant differences
cubes samples were washed several times with phosphate between liquid and solid culture at the time of maximum
buffer (0.1 M, pH 7.4). The samples were subjected to a production was determined by Student's t-test (P values of ˂
second fixation with 1% osmium tetroxide for 2 h; after this 0.05 were considered significant), for the quantity of spores,
process, the samples were washed with ethanol solutions quantity of spores recovered by washing with 0.85% NaCl
of increasing concentration (30–100%). The samples were or 1% Tween 80, total protein in the two culture systems,
dried to a critical point and fixed on carbon and graphite survival of G. mellonella larvae at the spore suspension from
tapes. Once fixed, the samples were covered with gold for the two culture systems and resistance of the spores from
observation under the microscope (JEOL JSM-5900LV). both culture systems to heat shock.

Quantification of spores
Results
The quantification of free spores was carried out using a
Neubauer chamber, making serial dilutions when necessary Spore production
with 1% Tween 80 solution (Madigan et al. 2004).
To determine the effect of the culture system on spore pro-
Viability of spores duction of the two Bt strains, liquid and solid cultures were
compared using GYS  4× media in both systems (Fig. 1a
The viability of the obtained spores was determined by col- and b); these represented more than twice the highest con-
ony forming units (CFU). Spore suspensions were adjusted centration reported previously (Lima-Pérez et al. 2019).
to 1 × ­104 spores m
­ L–1, followed by a heat shock at 80 °C Spore production reached the maximum peak at 48  h
for 10 min using a Thermomixer and then cooled on ice for in both culture systems for strain Y15 (Fig. 1a); never-
5 min. Then, 30 μL of each dilution (approx. 300 spores) theless, production was 2.4-fold higher in SC (5.7 × ­109
were placed on Petri dishes containing LB agar and incu- spores ­mL–1) than in LC (2.7 × ­109 spores ­mL–1); in terms
bated at 30 °C for 12–18 h (Zhuang et al. 2011). of productivity, in solid culture 1.19 × ­108 spores m
­ L−1 ­h−1
are produced, while in liquid culture less quantity of
Bioassay spores is produced during the same period (5.69 × ­1 0 7
spores ­mL−1 ­h−1). Differences were more noticeable for
The insecticidal efficiency of both strains of Bt was deter- EA3 strain, with 30 times more spores produced under
mined using Galleria mellonella larvae. For each treatment, SC (3.37 × ­1010 spores ­mL–1) than in LC (1.1 × ­109 spores
10 larvae were placed in Petri dishes using triplicates. To ­m L –1) after 48 h, in fact, spore production for Bt EA3
each set of larvae 0.1 g of cotton was added impregnated in LC remained constant over the sampling time; on the

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Fig. 1  Spore production in liquid and solid culture for B. thuring- (gray bars); Spores recovered after washing the PUF (empty bars)
iensis (Bt) strains Y15 (a) and EA3 (b). LC: liquid culture (broken with 1% Tween 80 and 0.85% NaCl for Bt Y15 (c) and Bt EA3 (d). (a
lines), SC: solid culture (solid lines). Spores at the initial harvest and b with n = 6, c and d with n = 3)

contrary, in SC the spore production reached a maximum production of biofilm was observed, which relates to the
value at 48 h. Similarly, productivity in SC by Bt Y15 poor recovery of spores after washing, in contrast to that
strain (7.07 × ­108 spores m
­ L−1 ­h−1) was higher than that observed for strain Bt Y15 (Fig. 2k and l).
obtained in the same period in LC (2.62 × ­1 0 7 spores After harvesting, several spores were still retained in
­mL−1 ­h−1). the PUF; therefore, the PUF was washed using Tween 80
The growth of B. thuringiensis Y15 and EA3 on PUF 1% or NaCl 0.85% solutions. This strategy allowed the
cubes was observed by SEM at different stages (Fig. 2). recovery of 2.55 × ­109 spores m­ L–1 for Bt Y15 strain, an
Both strains formed cellular aggregates on the walls of the additional 35% of the initial harvest (Fig. 1c, empty bars),
PUF (Fig. 2a–d), often attached to a thread-like structure and 1.13 × ­109 spores ­mL–1 for Bt EA3, representing only
which may have a role in biofilm formation. The SEM also 3.5% of the initial yield for this strain (Fig.  1d, empty
showed that several spores and vegetative cells remained bars). There was no significant difference in spore recov-
attached to the solid support after the harvesting process, ery between washing the PUF cubes either with Tween 80
possibly due to biofilm formation (Fig. 2e–h). In addition, or NaCl (P > 0.05). Vegetative cells and spores retained
pyramidal structures resembling Cry protein crystals were to a less extent in the PUF even after washing the solid
observed (Fig. 2f). In the case of strain Bt EA3, a greater support with Tween 80 or NaCl (Fig. 2i–l).

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Fig. 2  SEM of Bt Y15 and Bt EA3 growing inside polyurethane foam by washing the PUF with 1% Tween 80 solution or 0.85% NaCl (i–l,
before the harvesting process (a–d). Cellular debris, spores, bacilli respectively). Arrows indicate vegetative cells (VC), spores (S), Cry
and cry crystals can still be observed in the PUF after the harvest- crystal (C) and biofilm (B)
ing process (e–h). Most of the PUF-retained spores can be recovered

Y15 and EA3 after a heat shock; for the strain Y15, spores
harvested from SC showed an average viability of 81%, more
than five-fold higher that the viability of spores from LC
subjected to the same treatment. In the case of strain EA3, a
greater viability was observed in spores from SC (63%) rela-
tive to those produced in LC (9%). This result also exhibited
sensitivity differences to heat shock inherent to each strain.
Final survival (%) of larvae of G. mellonella after 6 days
of infection is shown in Fig. 4. For both strains the infectiv-
ity of the spores produced in LC showed very low mortality
under the conditions assayed. Nonetheless for the strain Y15,
spores from SC reached a greater infectivity (75% mortality)
compared to spores produced in LC (< 20%). In contrast,
spores of the EA3 strain, either harvested form SC or LC,
did not display significant difference in the larval survival.

Fig. 3  Percentage of viability of B. thuringiensis spores after thermal Protein estimation

shock (10 min to 80 °C and 5 min on ice): liquid culture (solid gray
bars); Solid culture (empty bars) (n = 3)
Total protein produced by both strains in SC was about twice
that produced when the same strain grew in liquid media
Viability and bioassay (Fig. 5); however, Bt Y15 protein production was higher
than EA3 strain in both systems. These results are consistent
One important aspect to consider in the production of spores with the higher spore production observed in SC, and there-
of B. thuringiensis is the resistance to different variables. fore the higher biomass production. In this experiment we
Figure 3 presents the viability of spores of B. thuringiensis measured the amount of protein after centrifuging an aliquot

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Fig. 4  Survival percentage of
larvae of G. mellonella after the
infection process: liquid culture
(solid gray bars); Solid culture
(empty bars); Control (solid
light gray bars) (n = 3)

Spore yields (5.7 × ­109 spores ­mL–1 and 3.37 × ­1010 spores

­mL–1 by Y15 and EA3, respectively) obtained using SC are
comparable to those obtained in other works (Zhuang et al.
2011; Jisha et al. 2015; Ballardo et al. 2016; El-Bendary
et al. 2016; Lima-Pérez et al. 2019; Rodríguez et al. 2019;
Mejias et al. 2020), all experiments were performed using a
C/N ratio of 5.98. Farrera et al. (1998), mention that a C/N
ratio close to 4 is optimal for spore production whereas a
C/N close to 7 is ideal for production of the insecticidal Cry
protein, therefore, further media optimization could signifi-
cantly increase spore production in SC. In addition, the use
of PUF cubes as solid support allows easy harvesting of
most of the spore-crystal complex, without contaminants
derived from the agro-industrial residues commonly used
in solid-state fermentation (Zhuang et al. 2011; Jisha et al.
2015; Ballardo et al. 2016; El-Bendary et al. 2016; Rod-
Fig. 5  Total protein produced in liquid (solid gray bars) and solid ríguez et al. 2019; Mejias et al. 2020).
culture (empty bars) by Bt strains Y15 and EA3 in GYS  4× medium Bt spore/crystal complexes have been produced com-
(n = 3) monly in LC; however, one disadvantage in LC is growth
inhibition when using high concentrations of substrate.
of the media, thus the higher total protein measured in SC Berbert-Molina et al. (2008) reported a decrease in specific
could be an addition effect of both the biomass production growth rate and yield in Bt cultures in liquid media with
and the Cry protein forming insoluble crystals, which are 75 g ­L–1 of glucose as substrate, whereas 10 to 30 g L ­ –1 of
released into the media after sporulation. glucose were the best conditions for Bt biomass production.
Recently, these inhibition limitations were corroborated at
even higher glucose concentrations, up to 50 g L ­ –1 (Lima-
Discussion Pérez et al. 2019). Moreover, the inhibition caused by high
substrate concentrations in LC could be attributed to the
In this work, the comparison of spore production, productiv- higher oxygen demand required to metabolise the substrate,
ity, and quality in two Bt strains confirmed a better perfor- making oxygen a limiting factor for growth. In fact, Dulmage
mance in SC compared to LC, regardless the strain evaluated (1981) proposed that the growth and sporulation of Bt can be
(Fig. 1a and b). The cultures were carried out using an inert optimised by using high rates of aeration, increasing oxygen
support (PUF cubes) and the same media in both systems, diffusion, but it would increase production costs due to the
avoiding the interference of media with different compo- required mechanical agitation. In addition, the use of solid
sition, as happens when agro-industrial residues are used; culture for spore production could be a cheaper alternative
therefore, the higher spore production in SC can be attrib- to liquid culture because of the higher productivity in CS
uted just to the system culture, as suggested by Lima-Pérez and the possibility to reduce cost by using agro-industrial
et al. (2019) for Bt, and in agreement to reports using other waste such as molasses or reuse the same PUF in several
microorganisms (John et al. 2007; Baños et al. 2009; López production batches. Some authors have reported between
et al. 2010; Lima-Pérez et al. 2018; Selvaraj and Vytla 2018). 32 and 36% decrease in operating cost using CS compared

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to CL (Castilho et al. 2000; Jovanović et al. 2020), although heat shock treatment. In addition to the chaperone proteins,
the economic advantages of each culture system must be Setlow and Setlow (1994) showed the formation of abasic
evaluated on a case-by-case basis. sites in DNA of spores lacking SASP α- and β- after inacti-
The increase in spore production observed by both Bt vating spores by heat, this suggests that SASPs have a role
strains, cultured on PUF cubes as solid support, could be on DNA protection against high temperatures. Considering
related to the fact that the solid support alleviates the two these findings, possibly in our system the spores produced
limitations associated with LC as stated above. On one hand, in liquid culture were not mature enough to resist a thermal
there is no growth inhibition induced by high substrate con- shock, requiring more time for the complete maturation, rel-
centrations, even though the media have high glucose con- ative to the spores produced in solid culture. Furthermore,
centration (120 g ­L–1), probably because the media embed- the culture system could induce a differential expression of
ded into the PUF cubes form substrate micro-gradients in specific proteins for the infection of the insect (Cry, Vip,
the water layers attached to the PUF walls; SC also relieves etc.). Proteomic or transcriptomic analysis could verify that
the osmotic stress due to direct contact with the liquid–air the culture system imposes a metabolic adjustment in the
interface, which cells encounter when growing in the limits microorganism, improving the production of metabolites of
of the water layer. On the other hand, the structure of the interest.
polyurethane foam cubes results in an advantage over the Solid culture favoured the formation of cellular aggre-
liquid culture in relation to the specific area of the system, gates (Fig. 2a–d) whose cross interaction in a small area of
defined as the relation of the total area of the system where the solid support is greater than in LC. Processes such as
the gas exchange takes place and the total volume used in biofilm formation and sporulation are triggered by quorum
the culture. Due to the number of pores present in the polyu- sensing, then the local substrate gradients in the solid sup-
rethane foam, this characteristic favours the formation of port, the non-homogeneous diffusion of molecules in the
extremely thin liquid layers, improving the gas exchange liquid interface, and the high number of cells aggregates
for a greater diffusion of oxygen in solid culture. According could trigger pathways associated to the quorum sensing
to Viniegra-González et al. (2003), the specific area of the leading either to biofilm formation, Cry protein production
polyurethane foam cubes is up to 400 times higher compared or sporulation (Keller and Surette 2006; Rocha et al. 2010;
to that of a liquid culture; this allows complete substrate Verplaetse et al. 2015). Although there is clear advantage
oxidation, and more efficient production of the metabolites of the formation of cellular aggregates, a high production
such as toxins, or even spores. of biofilm can become a disadvantage when recovering the
Solid cultures also improved the quality of B. thuring- spores retained in the PUF cubes, a possible reason why the
iensis spores. In this work, two important quality param- percentage of spore recovery, with Tween or NaCl solutions,
eters were determined: the viability of the spores after a heat of the EA3 strain is very low compared to strain Y15, as seen
shock and the ability of the spores to infect and kill G. mel- in the micrographs of Fig. 2, strain EA3 appears to produce
lonella larvae, probably assisted by other metabolites pro- a high quantity of biofilm compared to strain Y15.
duced. Both strains under SC produced spores with greater In conclusion, this study showed that solid culture, using
tolerance to sudden changes in temperature, compared to the polyurethane foam cubes as solid support with high concen-
spores produced in LC. Several factors have been associated trations of substrate, is a viable option to produce B. thur-
to the resistance of the spores of the Bacillus genus to heat, ingiensis spores, increasing production and productivity
these include the presence and quantity of minerals, water by up to 30 times compared to liquid culture, even without
content and the maturation of the spores. Sanchez-Salas considering the spores retained in the foam that are further
et al. (2011) shows that immature spores (early spores) are recovered. In addition, spores produced on solid media
less resistant to temperature shocks, these authors mention showed higher tolerance to heat-shock treatment and, in the
that the heat resistance of mature spores is due to the com- case of one of the strains, the spores from the solid culture
plete release of spore from the sporangium, related to the showed even greater infectivity. Also, the culture system had
complete maturation in the layers that make up the structure a direct effect on total protein profiles. Taken all together, the
of the spore, or even additional modifications in the structure observed changes are attributable only to the culture system
of the spore cortex exerting full resistance to heat in spores. employed, as the same media composition was used in both
Other works have proved that the higher the mineral content, systems.
the greater the heat resistance (Bender and Marquis 1985;
Ghosh et al. 2011). Some authors have sought the possibility Acknowledgements  We are grateful for the financial support of the
Universidad Autónoma Metropolitana for this research. We also thank
of explaining heat resistance in the expression of different the Consejo Nacional de Ciencia y Tecnología (CONACYT) for the
proteins; Movahedi and Waites (2000) developed proteomic fellowship granted to B. Flores-Tufiño (CVU 613243). We also thank
studies in sporulating cells of Bacillus subtilis where the Dr. Facundo Muñiz-Paredes and Dr. Héctor Escalona-Buendía for the
expression of chaperone proteins increased after applying a critical reviews on this document.

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Author contributions  BFT designed and performed all experiments bacteria-derived pesticidal proteins. J Invertebr Pathol. https://​
and drafted the manuscript. GVG contribute to the discussion and doi.​org/​10.​1016/j.​jip.​2020.​107438
analysis of the data. FFM and OL contribute to the design of the experi- Domínguez-Arrizabalaga M, Villanueva M, Escriche B et al (2020)
ments, analysis of the data and final version of the manuscript. Insecticidal activity of Bacillus thuringiensis proteins against
coleopteran pests. Toxins (basel) 12:430
Funding  The authors received financial support from the Universidad Dulmage HT (1981) Production of bacteria for biological control
Autónoma Metropolitana. of insects. In: Papavizas G (ed) Biological control of crop pro-
duction, Beltsville symposia in agricultural research. Allanhed,
Osmun and Co, Totowa, pp 129–139
Data availability  The datasets generated during and/or analysed dur-
Dulmage HT, Aizawa K (1982) Distribution of B. thuringiensis in
ing the current study are available from the corresponding author on
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Conflict of interest The authors declare that they do not have any
Bacillus thuringiensis vegetative insecticidal protein with a
known competing financial interests or personal relationships that
wide spectrum of activities against lepidopteran insects (lepi-
could have appeared to influence the work reported in this paper.
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Sci USA 93:5389–5394
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