Bioresource Technology 102 (2011) 2387–2393

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Bioresource Technology
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Biodegradation of chicken feathers waste directed by Bacillus subtilis

recombinant cells: Scaling up in a laboratory scale fermentor
Taha I. Zaghloul a,⇑, Amira M. Embaby a, Ahmed R. Elmahdy b
a
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Egypt
b
Department of Food Science and Technology, Faculty of Agriculture, Alexandria University, Egypt

a r t i c l e i n f o a b s t r a c t

Article history:
Received 19 August 2010
Received in revised form 12 October 2010
Accepted 23 October 2010
Available online 28 October 2010
Keywords:
Bio Flo 110 laboratory scale fermentor
Chicken feathers waste
Bacillus subtilis recombinant cells
Feathers hydrolysis end products
Keratinolytic alkaline protease
Biodegradation of chicken feathers waste directed by Bacillus subtilis DB 100 (p5.2) cells was successfully
carried out in 14 L Bio Flo 110 laboratory scale fermentor. Seven liters of feathers-based modified basal
medium II, feathers-based tap water and feathers-based distilled water separately in the fermentor were
inoculated with activated bacterial cells. The fermentation processes were conducted at 37 °C, 700 rpm
agitation speed and 0.7 vvm air flow rate in the absence of kanamycin. Highest net levels of released
feathers hydrolysis end products [soluble proteins and NH2–free amino groups] and keratinolytic alkaline
protease activity in the fermentor were greatly comparable to those of shake flasks. Interestingly, the
plasmid (p5.2) inside the recombinant B. subtilis cells growing in the fermentor displayed 100% stability
till the fifth day of incubation and this presents a great challenge. Data certainly would encourage the
transfer to larger scale fermentors to carry out feathers biodegradation process.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction essential amino acids such as methionine and histidine (Papadopo-

The incremental increase in poultry industry allover the world
resulted in the generation of millions of tones of chicken feathers
waste (Williams et al., 1990; Zaghloul et al., 1998; Vasileva-Tonkova
et al., 2007). Keratin, a hard to degrade insoluble animal protein,
represents 90% of this keratinous waste (Böckle et al., 1995). It dis-
plays high mechanical stability and resistance to hydrolysis by
common proteolytic enzymes. Complexity in keratin structure is
due to high cross linking between the polypeptide chains as a result
of S–S bonds, hydrogen bonds and hydrophobic interactions (Böckle
et al., 1995). The keratinous waste along with the inefficient utiliza-
tion imposed some environmental pollution problems (Onifade
et al., 1998; Zaghloul et al., 1998). Although, researchers have paid
a great attention to this keratinous waste based on its high protein
content, the high recalcitrant nature of this waste greatly hinders
its utilization in the native state as an animal feedstuff unless it
had been undergone physicochemical treatments. Currently, the
production of commercial feather meals demands the use of these
physicochemical methods. High cost and intensive energy are two
prerequisites for the completion of this process. However, the
resulting product has a low nutritional value and is poor in some
⇑ Corresponding author. Address: Department of Biotechnology, Institute of
Graduate Studies and Research, 163 El Horreya Aven., EL Chatby, P.O. Box 830,
Alexandria, Egypt. Tel.: +20 16 42 55 327.
E-mail addresses: tahazaghloul@yahoo.com (T.I. Zaghloul), amira_mohamed_
2000@yahoo.com (A.M. Embaby), elmahdy200966@yahoo.com (A.R. Elmahdy).
ulos et al., 1986).
Much attention has been turned toward the valorization of this
waste through alternative biotechnological methods (Shih, 1993;
Sangali and Brandelli, 2000; Suzuki et al., 2006). Isolation and puri-
fication of keratinolytic enzymes from different bacteria, actino-
mycetes and a lesser from fungi have been reported (Williams
et al., 1990; Gradisar and Friedrich, 2000; Ionata et al., 2008; Cao
et al., 2009; Brandelli et al., 2010; Mazotto et al., 2010; Xie et al.,
2010). These enzymes display a great capability to hydrolyze kerat-
inous wastes efficiently and this is reported under biotechnological
valorization (Onifade et al., 1998; Deivasigamani and Alagappan,
2008). Some reports highlighted certain trails to clone and express
genes encoding keratinolytic enzymes in a variety of expression
systems (such as Bacillus subtilis, Escherichia coli, Pichia pastoris,
and others) (Lin et al., 1995, 2009). However, in most of these trials
plasmid instability and low gene expression were the two obsta-
cles to maximize the amount of keratinase produced by genetically
modified host cells (Gupta and Rammani, 2006). On the other
hand, high expression levels of the keratinolytic serine protease
gene (aprE) in the recombinant B. subtilis DB 100 (pS1) and B. sub-
tilis DB 100 (p5.2) have been reported (Zaghloul et al., 1994, 2004;
Oulad Haddar et al., 2009). Moreover, biodegradation of keratin in
the form of chicken feathers by the recombinant B. subtilis DB 100
(p5.2) cells was accompanied by the production of considerable
levels of alkaline protease, soluble proteins and NH2-free amino
groups on a bench scale (Oulad Haddar et al., 2009). This alkaline
protease (aprE) enzyme, a key enzyme in the keratin degradation

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.10.106
2388 T.I. Zaghloul et al. / Bioresource Technology 102 (2011) 2387–2393
process, was reported to be a dual enzyme. It works as a protease
and as a keratinase at the same time (Zaghloul, 1998).
The biodegradation of chicken feathers waste by a myriad of
wild type keratinase-producing bacteria was reported on a flask
scale. Only one report highlighted production of keratinase from
Bacillus licheniformis PWD-1 strain and three recombinant strains
of B. subtilis carrying the keratinase gene (KerA) of B. licheniformis
PWD-1 in a bioreactor (Wang and Shih, 1999). As a matter of fact,
scaling up of any fermentation process is usually carried out step
wise. Steps involve the use of laboratory scale up fermentors fol-
lowed by using pilot scale fermentors and finally the use of indus-
trial scale fermentors. Moreover, transferring from shake flasks to
fermentors is a great challenge due to the geometric differences
between the two systems. However, transferring from small scale
fermentors to large scale fermentors is much more predictable.
To the best of our knowledge, this is the first report highlighting
biodegradation of chicken feathers waste directed by a keratinase–
producing recombinant bacterial strain in a bioreactor. The objec-
tive of the present study is to test the possibility of scaling up
feathers biodegradation process directed by B. subtilis DB 100
(p5.2) recombinant cells in a laboratory scale fermentor as a first
step on the way to the industrial scaling strategy. The aim is
extending to propose a low cost feathers-based medium to be used
in the course of feathers biodegradation.
2. Methods
2.1. Chemicals and reagents
Yeast extract, peptone and bacteriological agar were Oxoid type.
Ninhydrin, hydrindantin, Coomassiae Brilliant Blue G250 (CBB
G250), methylcellosolve, ethyl alcohol leucine and Hide Powder
Azure (HPA) were purchased from Sigma. Bovine serum albumin
(BSA) was purchased from WinLab.
2.2. Bacterial strain and plasmid
B. subtilis DB100 his- met-(p5.2) was used in this study to direct
the biodegradation of chicken feathers waste. The strain harbors a
multicopy recombinant plasmid (p5.2) (4.7 kbp) that carries the
complete alkaline protease (aprE) gene (Oulad Haddar et al., 2009).
2.3. Media
Peptone yeast extract (PY) medium (Bernhardt et al., 1978)
(10 g bactopeptone, 5 g yeast extract, and 5 g NaCl per liter) was
used to activate the bacterial strain. PA medium is PY supple-
mented with 1.5% agar. PA km medium is PA medium supple-
mented with kanamycin (km) at a final concentration of 10 lg/
mL. PA milk km medium is PA km medium supplemented with
1% (w/v) skimmed milk. Basal medium II (Williams et al., 1990)
(0.5 g NH4Cl, 0.5 g NaCl, 0.3 g K2HPO4, 0.4 g KH2PO4, 0.1 g MgCl2
and 0.1 g yeast extract per liter), the core medium after some mod-
ification (omission of yeast extract and NH4Cl) was used in the pro-
cess of feathers biodegradation. Additionally, tap water and
distilled water instead of modified basal medium II were used in
the process of feathers biodegradation. All feathers–based media
were supplemented with 2% (w/v) chicken feathers waste. Kana-
mycin (km) was added to the growth media at a final concentra-
tion of 10 lg/mL unless otherwise stated.
2.4. Chicken feathers waste
Chicken feathers waste was collected from white hens from
slaughtering houses after slaughtering processes in Alexandria
City, Egypt. White male and female hens were the source of this
waste. The waste was washed with tap water then with distilled
water. After that, the waste was allowed to air dry. Chicken feath-
ers waste was used as such without defatting.
2.5. Bio Flo 110 laboratory scale fermentor
Bio Flo 110 laboratory fermentor (New Brunswick, Bio Flo 110
fermentor, USA) was used in the study. The fermentor is equipped
with a Control Processing Unit (CPU) capable of controlling the fer-
mentation parameters, including temperature and agitation. The
glass vessel has a total volume of 14 L with minimum and maxi-
mum working volumes of 4.0 and 10.0 L, respectively. Foaming
was controlled by addition of 1% antifoam solution as required.
The Bio Flo 110 fermentor containing certain medium was steril-
ized in 80 L semi automatic autoclave (ALP, Japan) for 1 h at 121 °C.
2.6. Activation of the recombinant B. subtilis cells
Single fresh colonies of B. subtilis recombinant cells were used
to inoculate 50 mL of PY km medium in 250 mL Erlenmeyer flasks.
The culture was grown at 37 °C with agitation at 200 rpm for 2.5 h.
Five milliliters of this growing culture were used to inoculate
100 mL of feathers-based media in shake flasks while 350 mL of
the same culture were used to inoculate 7L of feathers-based med-
ia in the Bio Flo 110 fermentor.
2.7. Shake flask experiments
All bench scale experiments were carried out in 250 mL Erlen-
meyer flasks. One hundred milliliters of modified basal medium
II, tap water and distilled water separately, each supplemented
with 2% (w/v) chicken feathers waste, was inoculated as above.
The fermentation process was conducted at 37 °C with agitation
at 150 rpm (New Brunswick incubator shaker, USA) for four days.
2.8. Laboratory fermentor experiments
All laboratory scale up experiments were carried out in the Bio
Flo 110 laboratory fermentor. Seven liters of modified basal med-
ium II, tap water and distilled water, separately each supple-
mented with 2% (w/v) chicken feathers waste were inoculated as
above. Feathers biodegradation processes were conducted at
37 °C, 700 rpm agitation speed of two six-bladed impellers,
0.7 vvm (volume of air per volume of medium per minute) air flow
rate unless otherwise stated in the absence of kanamycin for four
days.
2.9. Determination of viable cells, NH2-free amino groups, and soluble
proteins
Viable bacterial cells were determined according to the method
described earlier (Pelczar and Chan, 1977). NH2-free amino groups
resulted from feathers biodegradation were determined according
to the method described before (Pearce et al., 1988). A standard
curve using the amino acid leucine was established. Soluble pro-
teins resulted from feathers biodegradation were determined as
described by Bradford, 1976. A standard curve using bovine serum
albumin (BSA) was established.
2.10. Proteolytic activity
Proteolytic activity was determined as described earlier by Cliff
and Law (1982) using Hide Powder Azure as a substrate. One unit
of enzyme activity is defined as the amount of the enzyme that
 T.I. Zaghloul et al. / Bioresource Technology 102 (2011) 2387–2393 2389

causes a change (1) in absorbance at 595 nm over that of the con- cells on the above three media after 72, 24 and 48 h of incubation,
trol after 30 min at 37 °C. respectively (Fig. 1A, B and C).
On the other hand, the highest net levels of released NH2–free
2.11. Keratinolytic activity amino groups (106.37–118.6 lmole leucine/mL) were similar to a
great extent after 72 h of incubation in the three present cultures.
The keratinolytic activity of the alkaline protease enzyme was Generally, the rate of feathers biodegradation was greatly
determined as described earlier (Oulad Haddar et al., 2009;
Zaghloul et al., 2010) based on the NH2-free amino groups that
were released as a result of keratin biodegradation (here chicken
feathers waste) by the bacterial cells. NH2-free amino groups were
determined using ninhydrin as described by Pearce et al., 1988. A
standard curve for leucine was established.

2.12. Monitoring NH2-free amino groups, soluble proteins and alkaline

protease activity

The performance of the biodegradation process in both shake

flasks and Bio Flo 110 laboratory fermentor was evaluated by mon-
itoring the levels of released keratin hydrolysis end products (sol-
uble proteins and NH2-free amino groups) and alkaline protease
enzyme as well. One mL samples were withdrawn daily from
either shake flasks or Bio Flo 110 laboratory fermentor, centrifuged Fig. 1A. Levels of feather hydrolysis end products upon growing B. subtilis
at 7000 rpm in a microcentrifuge for 3 min and the supernatant recombinant cells on feathers–based modified basal medium II in shake flasks
was used to determine the levels of released soluble proteins, and Bio Flo 110 fermentor. Symbols h & j represent soluble proteins in shake flasks
and Bio Flo 110 feather hydrolysates, respectively. Symbols D & N represent levels
NH2-free amino groups and alkaline protease activity. of NH2–free amino groups in shake flasks and Bio Flo 110 feather hydrolysates,
respectively.
2.13. Plasmid stability

Segregational and structural stability of the recombinant plas-

mid (p5.2) in B. subtilis cells grown in the Bio Flo 110 fermentor
was tested. Seven liters of modified basal medium II supplemented
with 2% (w/v) chicken feathers waste were inoculated with acti-
vated bacterial cells and the process was conducted as described
above. Plasmid stability was monitored throughout five days of
the biodegradation process. Each day, a portion of the culture
was plated on PA plates, incubated at 37 °C, for 24 h. The resulted
colonies were screened for their proteolytic activity and resistance
to kanamycin (km) by tooth picking them on PA milk km plates.

2.14. Analysis of amino acids

Free amino acids, except tryptophan, resulted from feathers bio-
degradation were determined using Beckman 119 CL amino acid
analyzer (Speckman et al., 1958).
Fig. 1B. Levels of feather hydrolysis end products upon growing B. subtilis
recombinant cells on feathers–based tap water medium in shake flasks and Bio
Flo 110 fermentor. Symbols h & j represent soluble proteins in shake flasks and Bio
Flo 110 feather hydrolysates, respectively. Symbols D & N represent levels of NH2-
free amino groups in shake flasks and Bio Flo 110 feather hydrolysates, respectively.

3. Results and discussion

3.1. Feathers degradation: the shake flasks level

Previous data from our group greatly encouraged the biodegra-

dation of feathers directed by B. subtilis DB 100 (p5.2) cells upon
growing on feathers–based basal medium II. Considerable levels
of soluble proteins, NH2–free amino groups and alkaline protease
were produced in shake flasks (Oulad Haddar et al., 2009). Consid-
ering cost effectiveness and achieving higher levels of released
feathers hydrolysis end products, modified basal medium II (basal
medium II without NH4Cl and yeast extract), tap water and dis-
tilled water were tested instead of basal medium II. Three feath-
ers-based media, feathers–based modified basal medium II,
feathers-based tap water and feathers-based distilled water were Fig. 1C. Levels of feather hydrolysis end products upon growing B. subtilis
inoculated with activated bacterial cells in shake flasks (100 mL recombinant cells on feathers–based distilled water medium in shake flasks and
Bio Flo 110 fermentor. Symbols h & j represent soluble proteins in shake flasks and
each), separately. The processes were conducted as described in Bio Flo 110 feather hydrolysates, respectively. Symbols D & N represent levels of
Materials and Methods. The highest net levels of released soluble NH2-free amino groups in shake flasks and Bio Flo 110 feather hydrolysates,
proteins were 0.53, 0.7 and 0.63 mg/mL upon growing bacterial respectively.
2390 T.I. Zaghloul et al. / Bioresource Technology 102 (2011) 2387–2393
accelerated and enhanced upon using the above three feathers–
based media described above when compared to that obtained
upon using feathers–based basal medium II (Oulad Haddar et al.,
2009). Highest levels of released soluble proteins and NH2–free
amino groups obtained upon using the present three media are
achieved after 24–72 and 72 h of incubation, respectively. How-
ever, highest levels of released soluble proteins and NH2–free ami-
no groups obtained upon using feathers-based basal medium II
were achieved after 96 and 144 h of incubation, respectively
(Oulad Haddar et al., 2009). Fold increases of 2.12, 2.8 and 2.52
in the levels of released soluble proteins were achieved upon using
the three feathers–based media; feathers–based modified basal
medium II, feathers–based tap water medium and feathers–based
distilled water medium, respectively when compared to those lev-
els obtained upon using feathers–based basal medium II (Oulad
Haddar et al., 2009). On the other hand, the levels of released
NH2-free amino groups obtained upon using the present three
feathers-based media described above were somewhat compara-
ble to that obtained upon using feathers-based basal medium II
(Oulad Haddar et al., 2009). Complete solubilization of 2% (w/v)
feathers was obtained after 48 h of incubation in the shake flask
system upon using the three above feathers-based media. Con-
versely, intact feathers were completely degraded by a feather-
degrading B. subtilis strain isolated from forest soil after cultivation
for five days in an improved basal medium II containing 0.1% (w/v)
feathers and 0.1% sorbitol (Jeong et al., 2010).
Moreover, this recombinant B. subtilis strain displays superior
feathers degradation capability when compared to that of a wild
type bacterium, Meiotherums ruber since feathers hydrolysates of
the latter bacterium contains 37.5 lmole leucine/mL after six days
released from 3% (w/v) chicken feathers waste (Matsui et al., 2009).
The present finding confers the ability of the above recombinant
bacterial cells to utilize chicken feathers waste as a sole source of
carbon and nitrogen. Moreover, the present finding greatly recom-
mends the use of the three above feathers-based media in the
course of feathers biodegradation. Although, present data indicate
that one can use tap water instead of modified basal medium II and
this would be of a great value from the standpoint of cost effective-
ness, the quality of tap water is undergoing seasonal variation con-
cerning water treatments.
3.2. Feathers degradation: the Bio Flo 110 fermentor level
Transferring a fermentation process from shake flaks to fermen-
tors is considered to be a great challenge due to substantial differ-
ences in the mode of agitation, aeration, pH control, temperature
control and dissolved oxygen as well as the geometric differences
in both systems. On the other hand, scaling up from small scale
to large scale fermentors is often much more predictable than di-
rect up scaling from flasks to fermentors. Feathers biodegradation
through a laboratory scale fermentor was carried out as a first step
attempt on the way to scale up at industrial level. Seven liters of
the three aforementioned feathers-based media in 14 L Bio Flo
110 laboratory scale fermentor were inoculated with activated re-
combinant bacterial cells. The processes were conducted as de-
scribed in Materials and Methods and the performance of all
processes was evaluated by measuring the levels of released end
products. The highest net levels of released soluble proteins in
the Bio Flo 110 fermentor were 1.0, 1.0 and 1.4 mg/mL in cultures
containing these feathers-based media; feathers–based modified
basal medium II, feathers–based tap water medium and feath-
ers–based distilled water medium after 24, 26 and 32 h of incuba-
tion, respectively (Fig. 1A, B and C). Regarding the levels of released
NH2-free amino groups in the Bio Flo 110 fermentor, the highest
net levels 120, 82.1 and 96.5 lmole leucine/mL were achieved in
cultures containing the above three feathers–based media after
48–72 h of incubation, respectively (Fig. 1A, B and C).
Accelerated rate of feathers biodegradation and enhanced levels
of released end products were noticed in the Bio Flo 110 fermentor
upon using feathers-based modified basal medium II when com-
pared to the situation in shake flasks using the same medium
(Fig. 1A). A fold increase of 5.88 in the level of released soluble pro-
teins was noticed in the Bio Flo 110 fermentor after 24 h of incuba-
tion. However, the highest levels of released NH2-free amino
groups were noticed 24 h earlier than that obtained in shake flasks
(Fig. 1A).
The situation was different to some extent upon using feathers–
based tap water medium where quite similarity in the rate of re-
leased soluble proteins was noticed in both Bio Flo 110 fermentor
and shake flasks. A fold increase of 1.4 in the net levels of released
soluble proteins was obtained after 26 h of incubation when com-
pared to those of shake flasks (Fig. 1B). However, the levels of re-
leased NH2-free amino groups were slighter higher in shake
flasks when compared to those of Bio Flo 110 fermentor (Fig. 1B).
Upon using feathers-based distilled water medium a fold in-
crease of 2.25 in the net levels of released soluble proteins was no-
ticed in the Bio Flo 110 fermentor after 48 h of incubation when
compared to those of shake flasks (Fig. 1C). Conversely, the levels
of NH2-free amino groups were comparable in both shake flasks
and Bio Flo 110 fermentor.
Generally, physical appearance of feathers revealed its complete
solubilization after 24 h of incubation in the Bio Flo 110 fermentor
(Fig. 1D), while, complete solubilization of feathers was obtained
after 48 h of incubation in the shake flask system upon using the
three above feathers-based media. Present finding is in partially
agreement with that of other report stating that E. coli HB101 har-
boring pEZZ18 ker BL2 from B. licheniformis strain ER-15 degraded
feathers completely within 24 h at 37 °C in shake flasks (Tiwary
and Gupta, 2010).
Conclusively, data indicate that, the fermentation process that
applies the three feathers-based media had been scaled up suc-
cessfully upon using the Bio Flo 110 laboratory scale fermentor.
If one wants to obtain more soluble proteins, it is better to direct
the process using feathers–based distilled water medium. On the
Fig. 1D. Physical appearance of feathers in the Bio Flo 110 fermentor (7 L working
volume, 700 rpm and 0.7 vvm) after 24 h of incubation with B. subtilis recombinant
cells. A: Bio Flo 110 fermentor containing 7 liters of recombinant B. subtilis feathers
hydrolysate. B: Physical appearance of feathers in the Bio Flo 110 fermentor at day
zero and after one day of incubation.
 T.I. Zaghloul et al. / Bioresource Technology 102 (2011) 2387–2393
other hand, if more NH2-free amino groups are needed, it is better
to direct the process using feathers–based modified basal medium
II. However, moderate levels of end products along with a lowest
cost, can be achieved by using feathers–based tap water medium
dependent system.
3.3. Feathers biodegradation: implementing two sets of agitation speed
and air flow rate
It is known that, mixing is a very crucial aspect to get the max-
imum productivity in microbial fermentations. It could be achieved
by means of aeration and agitation. It is important to provide opti-
mum combination of aeration and agitation in free-cell batch bio-
reactor operation to avoid shear stress and cell disruption. In an
attempt to enhance the levels of released end products resulting
from the biodegradation of feathers in the Bio Flo 110 fermentor,
the process was conducted at a two different combination sets of
agitation speed along with airflow rate. The first combination set
was 500 rpm agitation speed of the impeller with 1.0 vvm air flow
rate while the second set was 700 rpm agitation speed with
0.7 vvm air flow rate. Implementing the second set resulted in an
accelerated rate of feathers biodegradation and obtaining 1.92-fold
enhanced levels of released soluble proteins (1.0 mg/mL) in feath-
ers hydrolysates after 24 h of incubation (Fig. 1E). On the other
hand, the highest net levels of NH2-free amino groups were 120
and 150 lmole leucine/mL present in feathers hydrolysates when
the process was conducted at 700 rpm with 0.7 vvm and 500 rpm
with 1.0 vvm after 48 h and 72 h of incubation, respectively. It
seems that, increasing the agitation speed from 500 rpm to
700 rpm has no adverse effect on the growing cells since almost
quite similar colony forming units in both cases were obtained
(data not shown). Moreover, the levels of alkaline protease enzyme
produced under the two stated combination sets were almost quite
similar (data not shown). Although, agitation speed and high air
flow rates are generally prerequisites during the log phase of the
growth to accelerate the rate of growth and consequently to in-
crease the number of cells, increasing the air flow rate from 0.7
to 1.0 vvm in the present case had no positive impact on the
growth of cells. It could be that by reaching the stationary phase
bacterial cells start to lower their growth rate step wisely and there
will be no need to utilize higher air flow rates at this phase. As a
matter of fact, expression of the cloned keratinolytic alkaline pro-
tease enzyme produced by B. subtilis recombinant cells starts at
late log phase, the great bulk of this enzyme, a key enzyme in
the biodegradation process, is mainly produced at the stationary
phase (Oulad Haddar et al., 2009). This aspect highlights the issue
Fig. 1E. Levels of feathers hydrolysis end products in the Bio Flo 110 fermentor
upon using two different combination sets of agitation speed and air flow rate.
Symbols h & j represent soluble proteins upon using 500 rpm with 1.0 vvm and
700 rpm with 0.7 vvm, respectively. Symbols D & N represent levels of NH2–free
amino groups upon using 500 rpm with 1.0 vvm and 700 rpm with 0.7 vvm,
respectively.
2391
of non-growth related products formation observed in many
microbial fermentation processes (Genckal and Tari, 2006).
The enhanced levels of soluble proteins obtained upon using
higher agitation speed (700 rpm) could be attributed to the good
enzyme/substrate mixing allover the bioreactor. In other words, a
satisfactory contact between the keratinolytic alkaline protease
enzyme and feathers had been achieved. A proper choice of which
combination set of agitation speed and air flow rate to be imple-
mented will depend on some issues such as the purpose of the pro-
cess (production of soluble proteins or NH2-free amino groups) and
the power required for impeller and motor driving.
3.4. Monitoring bacterial growth and levels of alkaline protease in
shake flasks and Bio Flo 110 fermentor
It is reported that, shear stress, which is a common phenome-
non in stirred tank reactors, could impose adverse effects on the
growth of bacterial cells and the produced enzymes as well. To
examine this issue, bacterial growth in the Bio Flo 110 fermentor
as well as the alkaline protease activity was monitored in this
study. Generally, bacterial cells exhibited quite similar pattern of
growth in terms of colony forming units (CFU) in both shake flasks
and Bio Flo 110 fermentor upon growing of the recombinant cells
on the three aforementioned feathers-based media. Moreover, data
revealed that, bacterial cells in both shake flasks and Bio Flo 110
fermentor showed an exponential phase extending to 5–6 h with-
out lag phase. It seems that, there is no shear stress falling that
could affect cells upon using the above stated conditions in the
Bio Flo 110 fermentor. Additionally, the levels of the produced ker-
atinolytic alkaline protease in the three above cultures in the Bio
Flo 110 fermentor were greatly comparable to those of shake flasks
(Fig. 2). The maximal levels of alkaline protease enzyme (6.13–7.6
Units/log CFU) were achieved at the third day of incubation in the
three feathers–based cultures using the Bio Flo 110 fermentor
(Fig. 2). Similarly, the maximal levels of alkaline protease activity
(5.6–6.7 Units/log CFU) were achieved during the third day of incu-
bation in the three above cultures using the shake flasks system. It
seems that, there is no shear stress that could affect the enzyme
upon applying the above stated conditions in the Bio Flo 110 fer-
mentor. It is clear that, the maximal levels of the produced alkaline
protease are achieved during the stationary phase of growth in
Fig. 2. Levels of alkaline protease produced by B. subtilis recombinant cells upon
growing on three feathers-based media in shake flasks and Bio Flo 110 fermentor.
Symbols h, D and s represent levels of alkaline protease in shake flasks upon
growing bacterial cells on three feathers–based media; feathers–based modified
basal medium II, feathers–based tap water medium and feathers-based distilled
water medium, respectively. Symbols j, N and d represent levels of alkaline
protease in the Bio Flo 110 fermentor upon growing bacterial cells on three
feathers–based media; feathers-based modified basal medium II, feathers-based tap
water medium and feathers-based distilled water medium, respectively.
2392 T.I. Zaghloul et al. / Bioresource Technology 102 (2011) 2387–2393

both systems and this in agreement with the onset of the alkaline
protease gene expression.
3.5. Plasmid stability
Plasmid instability is a common phenomenon when recombi-
nant cells are used specifically in stirred tank reactors. This phe-
nomenon greatly addresses the indispensable need for examining
the segregational and structural stability of the recombinant plas-
mid (p5.2) upon growing B. subtilis cells in the Bio Flo 110 fermen-
tor. The plasmid (p5.2) displays considerable segregational and
structural stability in shake flasks upon growing B. subtilis recom-
binant cells on feathers-based medium (Oulad Haddar, 2006). The
plasmid stability was examined in cultures grown in the Bio Flo
110 fermentor as described previously in Section 2. Our results re-
veal that, this plasmid exhibits an excellent segregational and
structural stability (100%) till the fifth day of cells cultivation on
feathers-based medium in the Bio Flo 110 fermentor (Fig. 3). Sur-
prisingly, stability of the plasmid (5.2) in the Bio Flo 110 fermentor
is greatly superior to that obtained in shake flasks. Plasmid stabil-
ity in shake flasks was found to be 100% till the second day only.
However, after two days of incubation in shake flasks, plasmid sta-
bility has dropped to reach 85% (Oulad Haddar, 2006). Data confer
that, the present recombinant system [B. subtilis cells and the re-
combinant plasmid (p5.2)] is a promising system in scaled up pro-
Fig. 3. Stability the recombinant plasmid (p5.2) upon growing B. subtilis
recombinant cells on feathers-based medium in the Bio Flo 110 fermentor. Symbols
D and h represent plasmid and gene stability, respectively.
cesses. The present finding would greatly reduce the cost of the
process upon shifting to larger scale up fermentors since there is
no need to add kanamycin in the fermentors during the course of
feathers biodegradation. The obtained results greatly encourage
shifting up to other consecutive scales (pilot scales then industrial
scales).
3.6. Amino acids profiles in feathers hydrolysates
The amino acids resulting from the biodegradation of keratin-
containing materials are one important product that can promote
several new industries. There has been an increased demand for
amino acids to be used in many areas such as food, feed additives,
drug and pharmaceutical manufacturing. Since, keratin is the main
component of chicken feathers, representing nearly 90% of feathers
weight, biotechnological applications greatly consider the use of
keratin-degrading microorganisms or keratinolytic enzymes in
the production of amino acids and peptides (Oulad Haddar et al.,
2009).
The amino acids resulted from chicken feathers biodegradation
in shake flasks and Bio Flo 110 fermentors are shown in Table 1.
Generally, feathers hydrolysates of Bio Flo 110 fermentor upon
growing the recombinant cells on the three aforementioned feath-
ers–based media were rich in some amino acids such as phenylal-
anine, tyrosine, valine, leucine, isoleucine, serine, alanine, glycine
and threonine. Moreover, the levels of some amino acids in the
Bio Flo 110 feathers hydrolysates were greatly enhanced than that
of shake flasks. For example, a fold increase of 8.8, 3.16, 2.36, 2.0
and 1.8 in the level of alanine, serine, valine, threonine and threo-
nine, respectively was noticed in the Bio Flo 110 feathers hydroly-
sate upon growing of cells on feathers–based modified basal
medium II when compared to those of shake flasks under the same
conditions. On the other hand, the amino acids isoleucine, glutamic
and tyrosine showed a fold increase of 3.8, 2.6 and 1.97, respec-
tively in the Bio Flo 110 feathers hydrolysate upon growing of cells
on feathers–based tap water medium when compared to those of
shake flasks under the same conditions. Moreover, isoleucine, leu-
cine, proline, threonine and glycine were increased by 6.22, 5.43,
2.1, 1.8 and 1.57, respectively in the Bio Flo 110 feathers hydroly-
sate upon growing of cells on feathers–based distilled water med-
ium when compared to that of shake flasks under the same
conditions. The present finding is partially in disagreement with
that of shake flask feathers hydrolysate of Vibrio sp. (Betrsh and

Table 1
Amino acids profiles of feathers hydrolysates produced in shake flasks and Bio Flo 110 fermentor.

Amino acid Net mg amino acids/100 gm chicken feathers

Feathers-based modified basal medium II Feathers-based tap water medium Feathers-based distilled water medium
Shake flasks Bio Flo 110 fermentor Shake Flasks Bio Flo 110 fermentor Shake Flasks Bio Flo 110 fermentor
Asparatic acid 532.5 341.5 351.0 493.0 106.5 278.5
Threonine 330.5 666.0 134.5 98.5 231.0 428.0
Serine 366.0 1157.0 203.5 41.0 72.0 319.5
Glutamic acid 536.0 217.0 300.0 786.5 279.5 364.5
Proline 437.5 N.D 66.0 N.D 42.0 87.5
Glycine 181.5 N.D 173.5 142.0 437.5 686.5
Alanine 95.0 837.0 38.0 4.0 0.00 4.5
Cystine 245.0 281.0 41.0 120.5 86.5 56.0
Valine 821.5 1941.0 828.0 431.0 907.5 1101.5
Methionine 591.5 52.5 274.0 41.0 172.0 246.0
Isoleucine 343.0 431.0 168.0 648.0 346.0 2153.5
Leucine 376.0 205.5 370.0 180.0 366.0 1987.0
Tyrosine 1441.5 1472.0 738.5 1458.0 798.0 737.0
Phenylalanine 2038 2491.5 1260.5 2719.5 1873.0 1811.5
Histidine 17.50 156.0 61.5 N.D 0.00 10.5
Lysine 206.0 126.0 527.5 418.0 443.5 320.5
Arginine 464.5 837.0 118.8 78.5 61.0 14.0
 T.I. Zaghloul et al. / Bioresource Technology 102 (2011) 2387–2393
Coello, 2005) and that of a feather-degrading B. subtilis isolated
from forest soil (Jeong et al., 2010). This difference could be mainly
attributed to a strain difference that imposes different characteris-
tics on the specificity of the produced keratinase enzyme.
Present data would greatly recommend the use of recombinant
B. subtilis feathers hydrolysates as feed additives since it is very
rich in free amino acids. Moreover, high content of soluble proteins
and free amino acids of the B. subtilis DB 100 (p5.2) feathers
hydrolysates would greatly recommend its possible use in microbi-
ological practices as a low cost alternative instead of the more
expensive bacteriological peptone (Vasileva-Tonkova et al., 2007).
4. Conclusions
Present study highlights a successful process of feathers biodeg-
radation directed by B. subtilis recombinant cells in a laboratory
scale fermentor. Four challenges are highlighted upon transferring
to larger fermentors. The first challenge entitles the cost effective-
ness when feathers-based tap water medium is used. The second
challenge entitles the no need to add kanamycin (a stress factor)
during the course of feathers biodegradation. The third one focuses
on the feasibility of growing recombinant bacterial strain in a fer-
mentor. The last one addresses the superior yield of feathers
hydrolysis end products obtained in the fermentor compared to
that of shake flasks. Data encourage carrying out stiochiometric
kinetic study in the future.
Acknowledgements
The authors are very grateful to the Egyptian Scientific Acad-
emy for Research and Technology for its financial support through
a project under the national strategy of genetic engineering and
biotechnology programme.
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