Bioresource Technology 101 (2010) 24012404

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Pretreatment of poultry litter improves Bacillus thuringiensis-based

biopesticides production
Orhan Ozcan a, Bulent Icgen b, Gulay Ozcengiz a,*
a
b

Middle East Technical University, Department of Biological Sciences, 06531 Ankara, Turkey
Krkkale University, Department of Biology, Krkkale, Turkey

a r t i c l e

i n f o

Article history:
Received 26 August 2009
Received in revised form 6 November 2009
Accepted 9 November 2009
Available online 14 December 2009
Keywords:
Bacillus thuringiensis
Bt kurstaki
Bt israelensis
Bt tenebrionis
Poultry litter

a b s t r a c t
Pretreated poultry litter was used in batch cultures for the production of Bacillus thuringiensis (Bt)-based
biopesticide of lepidoptera- and diptera-specic Cry1 and Cry2, diptera-specic Cry4Ba and Cry11Aa and
coleoptera-specic Cry3Aa toxins by Bt subsp. kurstaki 81, subsp. israelensis HD500 and subsp. tenebrionis
3203, respectively. Bt kurstaki 81 showed improved growth and produced more toxin in this medium as
compared to other subspecies. Base and acid hydrolysis were tested as the methods of substrate pretreatment. The use of poultry litter pretreated with 2 N HCl yielded 94% more bioinsecticidal protein than 2 N
NaOH-pretreated poultry litter when Bt kurstaki 81 was cultured. With appropriate pretreatment, poultry
litter demonstrated potential as a valuable raw material for a low-cost complex medium to produce
Bt-based biopesticides.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
The parasporal crystalline inclusions along with the spores of
Bacillus thuringiensis (Bt) have a great potential to control a number
of pest insects belonging to the order Lepidoptera, Diptera and Coleoptera, since these insects have a tendency to develop resistance
towards chemical pesticides (Rowe and Margaritis, 1987). However, commercial application of these biopesticides depends on
the feasibility and economical viability of the production process
and technology. These, in turn, depend mainly on the cost of raw
materials, strain efciency, fermentation cycle, maintenance of
process parameters, bioprocessing of fermentation uid, and formulation of the nished product (Salama et al., 1983; Sachdeva
et al., 2000). The raw materials used for the production of Bt-based
biopesticides represent a substantial part of the overall production
cost. Stanbury et al. (1995) estimated that 3559% of the production cost was related to the fermentation medium. Therefore, for
commercial purposes, there is an urgent need to nd high yielding,
low cost and year round available raw materials for Bt production.
Various reports have dealt with cost reduction of the Bt production process through substitution of high-cost medium ingredients
of soy our and sh meal with complex agro-industrial wastes
(cassava starch, maize starch, rice straw, wheat bran, corn steep
liquor, sugarcane molasses, cheese whey and coconut waste)
(Abdel-Hameed, 2001; Khuzhamshukurov et al., 2001; Adams
* Corresponding author. Tel.: +90 312 210 5170; fax: +90 312 210 7976.
E-mail address: ozcengiz@metu.edu.tr (G. Ozcengiz).
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.11.048

et al., 1999; Vora and Sethna, 1999; Vega, 1999) and wastewater
sludge (Sachdeva et al., 2000; Tirado-Montiel et al., 2001). These
ingredients contain necessary nutritional elements to sustain
growth, sporulation and crystal formation by Bt. This approach of
bioconversion of residues to value added products can substantially reduce the production cost of Bt as well as lead to sustainable
utilization of residues, which would be socially useful and environmentally benign. Effects of low-cost media supplements such as
fodder yeast and agro-industrial by-products (Salama et al.,
1983), legume seeds and dried cow blood (Obeta and Okafor,
1983), gruel and shmeal (Zouari et al., 2002), wheat bran (Vimala
Devi and Rao, 2005), wastewater sludge (Lachhab et al., 2001;
Vidyarthi et al., 2002) and re-use of culture supernatant (Luna
et al., 2004) were studied for anti-lepidopteran and anti-dipteran
toxin production. Adams et al. (1999) proposed that with proper
pretreatment, poultry litter extract can have the potential to be
an excellent medium for the growth, sporulation and protoxin production for Bt subsp. kurstaki but it was also recognized that some
compounds in litter inhibited growth of some microorganisms
(Adams et al., 2002).
Poultry litter is a mixture of excreted manure mixed with
bedding material. As a raw and low-cost substrate that provides
a good source of protein, energy and minerals, poultry litter offers
many potential uses including fertilizing crops, cow feed, biomass
and biogas production. The current study was undertaken to
develop and compare various poultry litter pretreatments with
the aim of developing an economical complex medium for
Bt-based biopesticides production.

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O. Ozcan et al. / Bioresource Technology 101 (2010) 24012404

2. Methods

Three different Bt subspecies; kurstaki (strain 81 locally isolated),

israelensis (strain HD500 supplied by Bacillus Genetics and Stock
Centre) and tenebrionis (strain 3203 supplied by Prof. Feruccio
Gadoni, Philip Morris Europa S.A. Neuchatel, Switzerland), producing lepidoptera- and diptera-specic Cry1 and Cry2, diptera-specic
Cry4Ba and Cry11Aa and coleoptera-specic Cry3Aa toxins were
used in this study. The strains were streaked onto LB agar, subcultured monthly and stored at +4 C. For long term maintenance of
Bacillus strains, stock solutions were stored at 80 C in Luria broth
covered with 20% glycerol. Seed culture of Bt strains were prepared
according to the procedure adopted from Stahly et al. (1992).
Ten kilograms of poultry litter from a local broiler farm
(Ylmazer Tavukuluk ve Yumurta Ltd. Co. Ktahya, Turkey) was
stored in a sealed, triple lined, polyethylene bag at 4 C. The poultry
farm meets the basic requirements for organic poultry including
the use of certied organic feed free of antibiotics, drugs or synthetic parasiticides. After sieving in a #6 ASTM Standard Sieve,
the sample was dried at 80 C till no wet particles remained,
ground to a ne homogenous powder for use as unprocessed litter.
The chemical composition of both processed and unprocessed
poultry litter was analyzed at Middle East Technical University
Central Laboratory, Ankara, Turkey. An elemental analyzer (LECO,
CHNS-932) was used for C and N determination. Trace metal composition was analyzed using an inductively coupled plasma optical
emission spectrometer (Perkin Elmer Optima 4300DV).
The processing of poultry litter by base hydrolysis was adopted
from Caldwell (2006) who treated cellulosic raw materials with base
for a bioethanol process. A 20-g sample of dried poultry litter was
soaked in 25 ml of 2 N NaOH and rapidly stirred constantly at
200 C for 1 h. The resulting poultry litter suspension was cooled
and distilled water was added to increase the volume to 1 L. The
pH was adjusted to 7.2 with 2.6 N NaOH. The resulting poultry-based
media were ready to use after autoclaving at 121 C for 15 min. The
processing of poultry litter in acid was adopted from McMillan
(1994) and Esteghlalian et al. (1997) who used acid treatment method for conversion of lignocellulosic materials into fermentable sugars. Acid hydrolysis was performed in ve different ways: (I) Direct
hydrolysis in 2 N HCl at 50 C for 1 h: 20 g of dried poultry litter
was suspended in 1 L of 2 N HCl which was then stirred at 50 C
for 1 h. After cooling, the pH was adjusted to 7.2. The resulting poultry-based media were ready to use after autoclaving at 121 C for
15 min. (II) Direct hydrolysis in 2 N HCl at room temperature, overnight: as in (I), but hydrolysis was performed overnight at room temperature. (III) Direct hydrolysis in 1 N HCl at 100 C for 1 h: as in (I),
but hydrolysis was performed in 1 N HCl at 100 C for 1 h. (IV) Suspension in distilled water, solid/liquid separation by ltration followed by hydrolysis of solids in 2 N HCl at 50 C for 1 h: 20 g of
dried poultry litter was suspended in 100 mL of distilled water. The
solids were collected and exposed to 5 ml of 2 N HCl at 50 C for
1 h. After cooling, the total volume of liquid collected was made up
to 1 L with distilled water, the pH was adjusted to 7.2 and autoclaved.
(V) Hydrolysis in 2% sulfuric acid at 50 C for 2 h: as in (I), but 2% sulfuric acid instead of HCl was employed for hydrolysis.
For inoculum preparation, the glycerol stock of each Bt strain
was rst inoculated (0.1%) into 50 mL of Difco Sporulation Medium
(DSM) in a 250 mL ask and cultured overnight by shaking at 30 C
at 200 rpm. Aliquots of 1% were used as inocula to start the cultivation in 50 mL of the poultry litter media with 2% (w/v) of solids.
Samples were taken at 2 h intervals and bacterial growth was measured spectrophotometrically as absorbance 600 nm. Viable and
spore counts were determined as described by Icgen et al.
(2002a,b) and cell concentrations were determined as Colony
Forming Units/mL (CFU/mL).

A slightly modied procedure of Armelle (1991) was used for

protein extraction. The modication involved washing of harvested
spores and crystals twice with 10 mM Tris1 mM EDTA in place of
1 mM phenylmethylsulfonyl uoride10 mM EDTA. Protein concentrations were measured by the Bradford Quantication Method
(1976). Proteins were separated by using a vertical polyacrylamide
gel apparatus. The SDSpolyacrylamide gels were prepared as described by Laemmli (1970). Proteins were visualized by Coomassie
Blue R-250 staining of the gels. Gels were photographed by Vilber
Lourmat Gel Imaging System. Coomassie-stained gels were digitized and protein bands were compared using the 2-D image analysis software Delta2D version 3.3 (Decodon, Germany).
3. Results and discussion
3.1. Growth, sporulation and crystal toxin production on poultry litter
Production of each toxin was observed after 24, 48 and 72 h
incubation, but the highest cell and spore counts were obtained
at 72 h for all the strains. Bt kurstaki 81 yielded more cells
(7.3  109 CFU/mL) and spores (6.4  109 spores/mL) in poultry litter-based medium than DSM where the cell and spore counts were
3.8  108 CFU/mL and 3.4  108 spore/mL at 72nd hour. The counts
obtained from Bt israelensis HD500 (8.0  108 CFU/mL and
5.6  108 spore/mL) and Bt tenebrionis 3203 (4.5  108 CFU/mL
and 4.4  108 spore/mL) were not as high as those of Bt kurstaki
81, but were approaching to those obtained from their DSM cultures. Bt kurstaki 81 did not only grow much better in poultry litter
medium, but it consistently produced 3.54.0-fold more toxin as
compared to Bt subsp. tenebrionis 3203 and generally produced
much better than Bt subsp. israelensis HD500 although the latter
exhibited a great variation in toxin yield from experiment to experiment in this medium.
The above mentioned results demonstrated that poultry litter
medium can support high spore concentration which is a prerequisite, even if it might not always be sufcient, for high yields of crystal protein.
3.2. Effect of pretreatment of poultry litter
Further experiments which involved the pretreatment of poultry litter and its use for delta-endotoxin production was performed
by using Bt kurstaki 81. Base and acid hydrolysis were the techniques used for substrate pretreatment. The results showed that
base hydrolysis was more efcient than direct acid hydrolysis (pretreatments I, II, III and V). However, ltration of poultry litter suspension followed by acid hydrolyses of solids (pretreatment IV)
resulted in highest toxin yields (Fig. 1). Therefore, pretreatment
IV was used for poultry litter-based media preparation in the rest

Fig. 1. Cry1 and Cry2 biosynthesis by Bt 81 on pretreated poultry litter-based medium.

M: molecular weight markers. Lane 1: alkali-hydrolyzed; Lane 2: acid-hydrolyzed,
treatment IV; Lane 3: acid-hydrolyzed, treatment I; Lane 4: acid-hydrolyzed, treatment
II; Lane 5: acid-hydrolyzed, treatment III; Lane 6: acid-hydrolyzed, treatment V; Lane 7:
DSM control. The position of Cry1 and Cry2 were identied as in Icgen et al. (2002a,b).

O. Ozcan et al. / Bioresource Technology 101 (2010) 24012404

Table 1
Mineral content of pretreated and untreated poultry litter.
Broiler litter

Pretreated
Untreated

Minerals (mg/L)
C

Fe

Ca

Mg

Mn

770
720

450
1140

1.03
1.19

77.52
85.13

16.10
14.95

0.68
0.67

of the study. It is known that during acid hydrolysis, ammonia and

amino acids from hydrolyzed protein can react with mono sugars
in the hydrolyzed solution under the high temperature acidic conditions and can ultimately inuence the nal sugar yield (Fennema, 1996). Because of this reason, Liao et al. (2004) pretreated dairy
manure by washing it three times with water and separating out
the solids using a centrifuge. These washings were reported to be
enough to cut the nitrogen content of the manure by half, and
the resulting manure was much more qualied as a substrate for
various industrial fermentations. In our study, the pretreatment
IV which involved separation of solids from poultry litter suspension before acid hydrolysis possibly had a benecial effect by
ensuring removal of unwanted ammonia and amino acids from
hydrolysis mixture.

2403

well as the trace elements contents of pretreated and untreated

poultry litter are tabulated in Table 1. There were only small differences between the trace element contents of pretreated and untreated poultry litter. C to N ratios formed the only striking
difference between them. The ratio in untreated poultry litter
was considerably lower than that of pretreated one, indicating that
pretreatment IV has ensured a signicant reduction in nitrogen
content of the litter, at least by half. Concerning the effect of C to
N ratio on delta-endotoxin formation, the study conducted by Farrera et al. (1998) showed that a ratio of 7:1 improved the production of crystal protein Cry1Ac by Bt spp. kurstaki HD73. Poultry
litter medium with a C to N ratio of 8.5 gave much higher sporulation frequency when compared to C to N ratios of 9.5, 12.7 and 13.5
(Adams et al., 2002). In order to nd the optimal C to N ratio and
further improve toxin yields, processed poultry litter-based media
with different C to N ratios (2, 4, 6, 8 and 10) were prepared by
adding appropriate amounts of glucose into each. After 24 h fermentation, a C to N of 4 was ideal for Cry1 and Cry2 production
by Bt 81 (Fig. 2a). Cry4Ba and Cry11Aa biosynthesis by Bt HD500
was not much affected by C to N ratio within a range of 2 to 6
(Fig. 2b). For Cry3Aa toxin biosynthesis, on the other hand, C to
N ratio was found to be a very important parameter as a C to N ratio of 2 was the best, but those higher than 4 decreased delta-endotoxin yield remarkably (Fig. 2c).

3.3. Elemental analysis and effect of C to N ratio

4. Conclusions
Elemental analysis was done to compare the nutritional value of
processed and unprocessed litter. Carbon and nitrogen contents as

A high yield of bioinsecticidal proteins was obtained with a

medium prepared from acid hydrolyzed pretreated poultry litter,
but this pretreatment may not be necessary for all Bt strains, as revealed by the Cry4Ba, Cry11Aa producers. The Bt strains showed
different responses to changing C to N ratios in the litter medium
in that the optimal C to N ratio of poultry litter was 4 and 2 for
subsp. kurstaki 81 and subsp. tenebrionis 3203, respectively while
subsp. israelensis HD500 did not respond to this parameter. Our results indicated that poultry litter which constitutes a valuable,
inexpensive and readily available medium has potential to be used
for the commercial production of Bt-based biopesticides.
Acknowledgements
The authors acknowledge the assistance of Ms. Aslihan Kurt.
The nancial support of Middle East Technical University Scientic
Research Foundation is also gratefully acknowledged.
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Fig. 2. Effect of C to N ratio on Cry1 and Cry2 biosynthesis by Bt 81 (a), Cry4Ba and
Cry11Aa biosynthesis by Bt HD500 (b) and Cry3Aa biosynthesis by Bt 3203 (c) after
24 h incubation. M: molecular weight markers. Lane 1: DSM control; Lane 2:
untreated poultry litter; Lane 3: pretreated poultry litter (C to N ratio 2); Lane 4:
pretreated poultry litter (C to N ratio 4); Lane 5: pretreated poultry litter (C to N
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