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Citation: Hamma, S.; Boucherba, N.;
Azzouz, Z.; Le Roes-Hill, M.; Kernou,
O.-N.; Bettache, A.; Ladjouzi, R.;
Maibeche, R.; Benhoula, M.; Hebal, H.;
et al. Statistical Optimisation of
Streptomyces sp. DZ 06 Keratinase
Production by Submerged
Fermentation of Chicken Feather
Meal. Fermentation 2024,10, 500.
https://doi.org/10.3390/
fermentation10100500
Academic Editor: Demao Li
Received: 18 July 2024
Revised: 20 September 2024
Accepted: 24 September 2024
Published: 28 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
fermentation
Article
Statistical Optimisation of Streptomyces sp. DZ 06 Keratinase
Production by Submerged Fermentation of Chicken Feather Meal
Samir Hamma 1, Nawel Boucherba 1, * , Zahra Azzouz 1, Marilize Le Roes-Hill 2, Ourdia-Nouara Kernou 3,
Azzeddine Bettache 1, Rachid Ladjouzi 1, Rima Maibeche 1, Mohammed Benhoula 1, Hakim Hebal 1,4,
Zahir Amghar 1, Narimane Allaoua 1, Kenza Moussi 1, Patricia Rijo 5, 6, * and Said Benallaoua 1
1Laboratoire de Microbiologie Appliquée, Facultédes Sciences de la Nature et de la Vie, Universitéde Bejaia,
Bejaia 06000, Algeria; samir.hamma@univ-bejaia.dz (S.H.);zahra.azzouz@univ-bejaia.dz (Z.A.);
azzeddine.bettache@univ-bejaia.dz (A.B.); rachid.ladjouzi@univ-bejaia.dz (R.L.);
rima.maibeche@univ-bejaia.dz (R.M.); mohammed.benhoula@univ-bejaia.dz (M.B.);
hakim.hebal@univ-biskra.dz (H.H.); zahir.amghar@univ-bejaia.dz (Z.A.);
narimane.allaoua@univ-bejaia.dz (N.A.); kenza.moussi@univ-bejaia.dz (K.M.);
said.benallaoua@univ-bejaia.dz (S.B.)
2Applied Microbial and Health Biotechnology Institute, Cape Peninsula University of Technology,
P.O. Box 1906, Bellville 7535, South Africa; leroesm@cput.ac.za
3
Laboratoire de Biomathématiques, Biophysique, Biochimie, et Scientométrie (L3BS), Facultédes Sciences de la
Nature et de la Vie, Universitéde Bejaia, Bejaia 06000, Algeria; ourdia.kernou@univ-bejaia.dz
4Faculty of Exact Sciences and Sciences of Nature and Life, Department of Biology, Mohamed Khider
University of Biskra, Biskra 07000, Algeria
5CBIOS-Centro de InvestigaçãoemBiociências e Tecnologias da Saúde, Universida de Lusófona, Campo
Grande 376, 1749-028 Lisbon, Portugal
6
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa,
1649-003 Lisboa, Portugal
*Correspondence: nawal.boucherba@univ-bejaia.dz (N.B.); p1609@ulusofona.pt (P.R.)
Abstract: This study focused on the isolation of actinobacteria capable of producing extracellular
keratinase from keratin-rich residues, which led to the selection of an actinobacterial strain refer-
enced as Streptomyces strain DZ 06 (ES41). The Plackett–Burman screening plan was used for the
statistical optimization of the enzymatic production medium, leading to the identification of five key
parameters that achieved a maximum activity of 180.1 U/mL. Further refinement using response
surface methodology (RSM) with a Box–Behnken design enhanced enzyme production to approxi-
mately 458 U/mL. Model validation, based on the statistical predictions, demonstrated that optimal
keratinase activity of 489.24 U/mL could be attained with 6.13 g/L of chicken feather meal, a pH
of 6.25, incubation at 40.65
◦
C for 4.11 days, and an inoculum size of 3.98
×
10
7
spores/mL. The
optimized culture conditions yielded a 21.67-fold increase in keratinase compared with the initial
non-optimized standard conditions. The results show that this bacterium is an excellent candidate for
industrial applications when optimal conditions are used to minimize the overall costs of the enzyme
production process.
Keywords: keratin residues; chicken feather meal; keratinase activity; actinobacteria; biodegradation;
Plackett–Burman; response surface methodology
1. Introduction
The poultry industry is the main driver of global meat production [
1
]. This sector
continues to grow and industrialize in many parts of the world. The increase in population,
purchasing power, and urbanization have been powerful drivers of growth for this sector.
The latest forecast of the Food and Agriculture Organization of the United Nations (FAO)
predicts that world poultry meat production is expected to reach 146 million tonnes in
2024, increasing by 0.8% year-on-year [
2
]. The Agricultural Outlook 2024–2033 of the
Fermentation 2024,10, 500. https://doi.org/10.3390/fermentation10100500 https://www.mdpi.com/journal/fermentation
Fermentation 2024,10, 500 2 of 28
Organisation for Economic Cooperation and Development (OECD) and the FAO predicted
that worldwide, poultry, pigmeat, beefmeat, and sheepmeat consumption is projected to
grow 16%, 8%, 11%, and 16%, respectively, by 2033. By that year, poultry meat is expected
to account for 43% of the total protein from meat sources, with pig, beef, and sheepmeat
following in consumption [
3
]. This development is unfortunately inseparable from the
increase in accompanying organic products, in the form of viscera, feet, heads, bones,
blood, and feathers [
4
]. Chicken feathers from poultry processing industries are generated
in abundance, as iskeratin-rich waste, which poses a serious ecological concern due to
the large meat quantities produced and their highly resistant characteristics in terms of
degradation [
1
,
4
]. The consumption of poultry meat leads to an annual production of
around 8.5 million tonnes of feather waste worldwide [5].
In nature, keratin represents the third most abundant biomass. It represents almost
90% of the feather’s total weight, thus constituting an important source of amino acids for
various biotechnological applications [
6
,
7
]. The recalcitrant nature of these wastes makes
conventional disposal methods such as incineration and landfills inappropriate, given the
ecological constraints involved, affecting both human health and the whole environment [
8
].
The development of non-polluting methods guaranteeing the production of interesting
products becomes essential. In this regard, the use of microorganisms degrading keratin
materials represents a useful alternative biotechnological solution [
9
]. Keratinolytic microor-
ganisms include fungi such as members of the genera Trichoderma [
10
], Trichosporum [
11
],
Aspergillus, and Fusarium [
12
,
13
], bacteria such as the members of the genera Bacillus [
14
],
Serratia [
15
], Fervidobacterium [
16
], Pseudomonas [
17
], or specifically actinobacteria such
as Streptomyces [
18
], Actinomadura [
19
], Arthrobacter [
20
], and Brevibacterium [
21
]. These
microorganisms can grow on media containing keratin substrates as carbon and nitrogen
sources, mainly due to their aptitude to producekeratinolytic proteases/keratinases that
exhibit high specific hydrolytic activity towards keratin substrates [22].
Keratinases (E.C 3.4.21/24/99.11) consist of a particular class of protein-hydrolyzing
enzymes synthesized by microorganisms with the ability to degrade insoluble keratinous
substrates into value-added products [
6
,
23
,
24
]. These catalysts generally hydrolyze soluble
proteins efficiently compared to insoluble ones, like keratins, and have advantages over
conventional proteolytic enzymes regarding stability over a wide class of environmental
conditions [
24
,
25
]. Most keratinases are classified as serine proteases, while others belong
to the metalloproteases class [
26
]. They are generally inducible, extracellular enzymes
secreted by various microorganisms in a keratin-containing medium and exhibit high
specificity towards the keratin substrate [
5
,
27
,
28
]. Keratinolytic proteases are promising
tools in different biotechnological fields like biotransformation of keratin waste into animal
feed and nitrogen fertilizers, as well as application in the cosmetic and detergent industries,
pharmaceutical fields, textile manufacturing, and leather tanning [29,30].
Among the numerous microorganisms involved in the production of keratinases,
actinobacteria are known as a rich source of bioactive molecules for various industry appli-
cations [
31
]. In nature, this Gram-positive bacterial group represents enormous potential
in terms of biodegradation/bioconversion of animal or plant complex biopolymers. They
are capable of synthesizing varied types of biocatalysts like proteases, keratinases, lipases,
cellulases, and chitinases [32].
On the other hand, the optimal culture conditions for keratinase production vary from
one organism to another; the identification and examination of physicochemical factors
running the production of these enzymes from a given isolate for a better production of
these molecules, is of great importance [
33
]. The limitations of classic optimization methods,
which consist of modifying one factor at a time while maintaining all other factors invariable
(OFAT: One-Factor-at-A-Time), have given rise to alternative statistical approaches using
experimental designs [
33
,
34
]. Thus, studies of factor selection influencing a response given
by linear estimation of the variables main effects can be carried out via a two-level factorial
design with minimal trials, such as the Plackett–Burman design. The Plackett–Burman’s
selected variables can be optimized by response surface methodology (RSM) analysis using
Fermentation 2024,10, 500 3 of 28
Box–Behnken design, which is a statistical and mathematical optimization strategy that
estimate the correlation between the variables and predicts the response in an efficient and
reproducible experimental design [35].
Here we report the isolation, identification, and description of a new thermophilic
actinobacterial strain from the Kabylia region in the north-eastern of Algeria. Isolation and
fermentation media, as well as buffered solutions based on keratinious substrates, were pre-
pared from locally collected chicken feathers. The strain showed rapid growth on a medium
based on chicken feather meal as the only source of carbon and nitrogen. Keratinolytic ac-
tivity was detected under fermentation conditions, and statistical approaches were used to
select and determine the optimal cultivation conditions required for keratinase production.
2. Materials and Methods
2.1. Substrates and Chemicals
Unless specified otherwise, all substrates, chemicals, and reagents were of analyt-
ical grade or the highest available purity and were purchased from Sigma–Aldrich Co.
(St. Louis, MO, USA)
2.2. Culture Medium Substrate Preparation
The culture substrate used was chicken feather meal prepared from locally collected
poultry feathers. The feathers, which had not undergone any physicochemical treatment,
were washed with tap water, then distilled water, air-dried, and then dried in an oven. The
dried feathers were ground and sieved [17].
2.3. Sampling and Sample Pretreatment
Samples of chicken compost manure (CCM), soil amended with chicken feathers
(CFS), and decomposing feathers (DF) were taken from various sites located in the Bejaia
district of Kabylia in north-eastern Algeria. A total of 21 samples were collected from three
locations, including nine samples from the DjebiraBoukhelifa site, seven samples from the
Fenaia Ilmathen site, and five samples from the Ighil Ali site (Figure 1).
Fermentation 2024, 10, x FOR PEER REVIEW 4 of 30
Figure 1. Sampling site maps showing the country and region where sampling took place. Maps
were generated using the Google Maps service.
For all samples, the top five centimeters of decomposing manure, soil, and feathers
were removed, and sufficient quantities of samples were taken sterilely and put into
sterile plastic pouches, then transferred directly to the laboratory and stored at 4 °C until
use.
To facilitate the isolation of actinobacteria, all the samples underwent physical and
chemical pretreatment to minimize the development of non-mycelial bacteria, which
grow faster than actinobacteria, and fungi, which form invasive colonies and inhibit the
development of actinobacteria. The samples were heated to 60 °C for 1 h, followed by the
addition of 1% (w/w) calcium carbonate (CaCO3) (1 g of CaCO3 for every 100 g of sample
in sterile vials) [36]. The presence of calcium carbonate alkalized the pH of treated sam-
ples, which favored the development of actinobacteria [37]. To promote the development
of thermophilic actinobacteria, the containers were incubated at 45 °C for 21 days [19].
2.4. Actinobacterial Isolation and Culture
During the incubation of the samples, 10 g of each sample was taken after 7, 14, and
21 days. Ten-fold dilutions were performed after a 10% (w/v) stock solution was made in
sterile distilled water. The stock solutions and their dilutions were spread-plated (200 μL)
onto feather basal salt medium (FBM), which contained (g/L): 20.0 chicken feather meal,
1.0 dipotassium hydrogen phosphate (K2HPO4), 0.5 magnesium sulfate heptahydrate
(MgSO4·7H2O), 3.0 calcium carbonate (CaCO3), 0.01 iron sulfate heptahydrate
(FeSO4·7H2O), 0.5 sodium chloride (NaCl), and 20.0 agar, with the pH adjusted to 8.2 ±
0.2. After incubating each plate for seven to ten days at 45 °C, the existence of colonies
with the morphological characteristics of actinobacteria with the presence or absence of a
distinct hydrolysis halo surrounding the colony was evaluated [19]. The colonies of in-
terest were subcultured to obtain pure cultures. A selection of strains was maintained at
room temperature (22 ± 3 °C) on solid FBM medium (pH 8.2 ± 0.2) and stored at −20 °C in
a 20% (v/v) glycerol suspension.
Figure 1. Sampling site maps showing the country and region where sampling took place. Maps
were generated using the Google Maps service.
Fermentation 2024,10, 500 4 of 28
For all samples, the top five centimeters of decomposing manure, soil, and feathers
were removed, and sufficient quantities of samples were taken sterilely and put into sterile
plastic pouches, then transferred directly to the laboratory and stored at 4 ◦C until use.
To facilitate the isolation of actinobacteria, all the samples underwent physical and
chemical pretreatment to minimize the development of non-mycelial bacteria, which grow
faster than actinobacteria, and fungi, which form invasive colonies and inhibit the de-
velopment of actinobacteria. The samples were heated to 60
◦
C for 1 h, followed by the
addition of 1% (w/w) calcium carbonate (CaCO
3
) (1 g of CaCO
3
for every 100 g of sample
in sterile vials) [
36
]. The presence of calcium carbonate alkalized the pH of treated samples,
which favored the development of actinobacteria [
37
]. To promote the development of
thermophilic actinobacteria, the containers were incubated at 45 ◦C for 21 days [19].
2.4. Actinobacterial Isolation and Culture
During the incubation of the samples, 10 g of each sample was taken after 7, 14,
and 21 days. Ten-fold dilutions were performed after a 10% (w/v) stock solution was
made in sterile distilled water. The stock solutions and their dilutions were spread-plated
(200
µ
L) onto feather basal salt medium (FBM), which contained (g/L): 20.0 chicken
feather meal, 1.0 dipotassium hydrogen phosphate (K
2
HPO
4
), 0.5 magnesium sulfate
heptahydrate (MgSO
4·
7H
2
O), 3.0 calcium carbonate (CaCO
3
), 0.01 iron sulfate heptahy-
drate (FeSO
4·
7H
2
O), 0.5 sodium chloride (NaCl), and 20.0 agar, with the pH adjusted to
8.2 ±0.2
. After incubating each plate for seven to ten days at 45
◦
C, the existence of colonies
with the morphological characteristics of actinobacteria with the presence or absence of a
distinct hydrolysis halo surrounding the colony was evaluated [
19
]. The colonies of interest
were subcultured to obtain pure cultures. A selection of strains was maintained at room
temperature (22
±
3
◦
C) on solid FBM medium (pH 8.2
±
0.2) and stored at
−
20
◦
C in a
20% (v/v) glycerol suspension.
2.4.1. Solid-State Screening of Keratinase-Producing Strains
Using the same isolation medium containing chicken feather meal (2%w/v) as the sole
source of organic carbon and nitrogen, pH 8, an initial screening for keratinase-producing
actinobacteria was performed. The media were then inoculated with the isolated strains
and incubated at 45
◦
C for 4 days. The strains presenting pronounced growth with or
without clear hydrolysis zones around the colonies were selected for further work [38].
2.4.2. Screening in Liquid Media for Keratinase-Producing Strains
Strains exhibiting pronounced growth with or absence of hydrolysis zones on FBM
agar were inoculated into FBM liquid medium (pH 8) and incubated at 45
◦
C for 2 days.
The pre-cultures were used to inoculate 50 mL FBM broth containing chicken feather meal
(2%, w/v) as substrate, which was supplemented with 1 mL of trace salts solution (g/L:
0.9 ZnSO
4
, 0.7 CaCl
2
, 0.2 MnSO
4·
7H
2
O, 0.3 KCl). The pH was adjusted to 8.2
±
0.2. The
cultures were cultivated for 10 days at 45
◦
C under agitation at 150 rpm. After 3, 5, 7, and
10 day
s, 1 mL samples were taken and centrifuged at 10,000
×
gfor 20 min at 4
◦
C. The cell-
free supernatant obtained was then used for keratinase activity assay (Section 2.6) [
19
,
24
].
All the strains were cultivated in triplicate.
2.5. Bacterial Strain, Growth Conditions, and Preparation of Spore Solutions
A spore suspension was prepared from FBM agar plates inoculated with the strain
that exhibited the highest keratinase activity that was previously incubated for 4 days
at 45
◦
C. The spores were gently harvested to prevent mycelial detachment by mixing
10 mL of sterile distilled water with 1% (v/v) Tween 80, then collecting the mixture in
sterile flasks to serve as an inoculum for the synthesis of keratinase. The suspension was
diluted 1:10 (v/v), and the spores were counted in a counting chamber (Malassez REF 06
106 10 MARIENFELD, Lauda-Königshofen, Germany) [
39
]. For submerged fermentation
experiments, 250-mL flasks containing 50 mL of FBM broth were prepared, adjusted to
Fermentation 2024,10, 500 5 of 28
varying pH levels, autoclaved, inoculated with strain ES41, and then placed in an orbital
shaker at given rotational speeds and temperatures for specified times. After the required
incubation, the culture broths were harvested and centrifuged at 10,000
×
gfor 20 min at
4◦C
. The supernatants were then used for enzyme assays using chicken feather meal as a
substrate [
40
]. For selection and optimization studies of the factors involved in keratinase
production, the culture medium composition and conditions were varied according to the
experimental data.
2.6. Enzyme Activity Assay
The standard operating conditions for measuring keratinolytic activity were followed
as described by Wawrzkiewicz et al. [
41
]. The enzyme solution (0.2 mL) was added to
1.8 mL of 0.5% (w/v) soluble keratin in 0.05 M Tris-HCl buffer (pH 8.2
±
0.2) and then
incubated in a water bath for 10 min at 50
◦
C. After placing the reaction mixture in an ice
bath and adding two milliliters of 0.4 M trichloroacetic acid (TCA), the reaction was halted.
Without adding keratin, the enzyme solution was incubated with 2.0 mL TCA to create
the control. After that, the mixture was centrifuged for 20 min at 4
◦
C and 5000
×
g. At
280 nm, the supernatant’s absorbance was measured in comparison to the control. Under
the previously mentioned experimental conditions, a rise in absorbance at 280 nm with
the blank of 0.01 per minute is defined as one unit per milliliter (U/mL) of keratinolytic
activity. The activity is calculated by the following Equation (1) [38]:
A(U/mL)=V·N·A280 /(0.01 ·T)(1)
where, Ais the keratinase activity; Nis the dilution factor; Vis the final reaction volume
(mL); Tis the incubation time (min); A
280
is the absorbance of the reaction mixture measured
at 280 nm against the control. Each assay in this study was performed in triplicate.
2.7. Morphological Characterization and Molecular Identification of the Keratinase-Producing
Strain, ES41
For better guidance in the identification and classification process of the selected
strain, macromorphological characterization (size, pigmentation, shape, and appearance
of colonies) and micromorphological characterization (Gram staining, morphology, and
arrangement of cells and spores of the isolate studied) were carried out on media recom-
mended for culturing actinobacteria [42].
Molecular identification was carried out using the GF-1 Nucleic Acid Extraction Kit
(Vivantis Technologies Sdn. Bhd, Shah Alam, Selangor DE, Malaysia). The polymerase
chain reaction (PCR) was performed using universal 16S rRNA primers (27: 5
′
–AGA GTT
TGA TCC TGG CTC AG–3
′
, and 1478 R: 5
′
–CCG TCA ATT CCT TTG AGT TT-3
′
) [
43
].
A thermal cycler (Bio-Rad bicycler, Hercules, CA, USA) was used to conduct the PCR,
whereas the measure of the amplicon concentrations was performed using a nanodrop
spectrophotometer (NanoDropTM 2000, Thermo Fisher Scientific, Waltham, MA, USA) [
44
].
The sequencing was performed using the Sanger method by electrophoretic migration
profile analysis of the DNA fragments. The sequences obtained were compared with
similar sequences by submission to BLAST (Basic Local Alignment Search Tool, National
Center for Biotechnology Information) and then identified using the GenBank database.
The phylogeny of the keratinase-producing strain was established using a web application
(Phylogeny.fr) via the neighbor-joining method. [45].
2.8. Determination of Influencing Physicochemical Parameters Using the
Plackett-BurmanApproach (PBD)
Using the Plackett–Burman experimental design, the critical factors influencing the
response, namely the production of keratinases when strain ES41 is cultured in a liquid
medium based on chicken feather meal as the only carbon and nitrogen sources, were
determined [
46
]. Ten two-level independent variables represent the initial requirements
for keratinase synthesis in strain ES41. These include sodium chloride (NaCl): X
1
, in-
Fermentation 2024,10, 500 6 of 28
oculum size: X
2
, incubation time: X
3
, initial pH: X
4
, dipotassium hydrogen-phosphate
(K
2
HPO
4
): X
5
, orbital agitation: X
6
, chicken feather meal: X
7
, calcium carbonate (CaCO
3
):
X
8
, incubation temperature: X
9,
and magnesium sulphate heptahydrate (MgSO
4·
7H
2
O):
X10 (Table 1).
Table 1. Selected independent variables and their level variation in Plackett–Burman design (PBD)
for the optimization of keratinase production by strain ES41.
Study Type Screening
Design Type Plackett-Burman
Design Mode Principal Effect First Order Model No Blocks Runs
Response R Keratinase Activity 20
Factor Name Units Type Level (−1) Level (+1)
X1NaCl g/L Numeric 0 3
X2Inoculum size Spores/mL Numeric 1.5 ×1041.5 ×1010
X3Incubation time Days Numeric 2 14
X4initial pH Numeric 4 12
X5K2HPO4g/L Numeric 0 3
X6Orbital agitation rpm Numeric 0 250
X7Chicken feather meal % (w/v) Numeric 0.2 2
X8CaCO3g/L Numeric 0 4
X9Incubation temperature ◦C Numeric 30 50
X10 MgSO4·7H2O g/L Numeric 0 3
The higher (+1) and lower (
−
1) variation levels of the factors were defined taking into
account the experimental limits of development and keratinase production by strain ES41
when using the OFAT (One-Factor-at-A-Time) method and spaced to effectively determine
the factors affecting significantly the response (Table 1) [40,47].
The Minitab 19 statistical software (trial version) used to elaborate the Plackett–Burman
trials was constructed and organized according to the chosen selection plan. The ten-factor,
two-level PBD method comprised 20 randomized experiments (Table 2).
Table 2. The PBD trial plan layout and associated responses for keratinase activity of strain ES41.
Run Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7 Factor 8 Factor 9 Factor 10 Response
X1X2X3X4X5X6X7X8X9X10 R
Sodium
Chloride
Inoculum
Size
Incubation
Time InitialpH
Dipotassium
Hydrogeno
Phosphate
Orbital
Agitation
Chicken
Feather Meal
Calcium
Carbonate
Incubation
Temperature
Magnesium
Sulphate
Hyptahydrate
Keratinase
Activity
g/L Spores/mL Days g/L rpm %(m/v) g/L ◦C g/L U/mL
1 0 1.5 ×1010 2 12 3 250 2 0 30 3 180.1 ±5.08
2 3 1.5 ×1042 12 3 0 2 4 30 0 142.3 ±4.89
3 0 1.5 ×10414 12 0 250 2 0 30 0 150.6 ±3.91
4 3 1.5 ×1010 14 4 0 250 2 0 50 3 71.3 ±4.47
5 0 1.5 ×1042 12 0 250 0.2 4 50 3 77.2±2.50
6 0 1.5 ×1010 14 4 0 0 0.2 4 30 3 59.7 ±1.63
7 3 1.5 ×1010 2 4 0 0 2 0 50 0 110.8 ±2.72
8 0 1.5 ×1010 14 4 3 250 0.2 0 30 0 62.3 ±1.55
9 3 1.5 ×1042 4 0 250 0.2 4 30 3 69.2±3.36
10 0 1.5 ×1042 4 0 0 0.2 0 30 0 51.4±3.07
11 3 1.5 ×1010 14 12 0 0 2 4 30 3 162.6 ±5.90
12 3 1.5 ×10414 12 3 250 0.2 0 50 3 51.4 ±1.16
13 0 1.5 ×1010 2 12 0 250 2 4 50 0 143.8 ±5.30
14 3 1.5 ×10414 12 3 0 0.2 0 50 0 47.4 ±1.19
15 3 1.5 ×10414 4 3 250 2 4 30 0 92.6 ±3.29
16 3 1.5 ×1010 2 4 3 250 0.2 4 50 0 53.9±5.18
17 0 1.5 ×1042 4 3 0 2 0 50 3 87.1 ±4.09
18 0 1.5 ×1010 14 12 3 0 0.2 4 50 0 59.6 ±3.51
19 3 1.5 ×1010 2 12 3 0 0.2 0 30 3 133.2 ±5.49
20 0 1.5 ×10414 4 3 0 2 4 50 3 43.9 ±4.17
The trials were carried out in three replicates. The experimental response average
obtained wasevaluated by a first-order polynomial model using the following Formula (2):
Fermentation 2024,10, 500 7 of 28
R(U/mL)=β0+
k
∑
i=1
βiXi(2)
where Ris the response;
β0
is the regression coefficient,
βi
is the linear coefficient, and X
i
is
the level of the independent variable.
The set of results was evaluated by analyzing the variance (ANOVA). The factor’s
impact on keratinase production was estimated using the probability value ‘p’ for each
factor. An effect is statistically significant when the p-value indicates a value less than
0.05 [48].
2.9. Determination of Optimal Physicochemical Parameters Using a Response Surface Model Based
on the Box-Behnken Design
Based on the PBD experimental results, keratinase yield in strain ES41 was optimized
by applying the response surface methodology (RSM). In the current investigation, the Box–
Behnken (BBD) model given by the Design Expert 13
®
software (version 13.0.12.0, Statease,
Minneapolis, MN, USA—trial version) was chosen to guide the experimental design with
five variables selected from those tested by the two-stage PBD experimental design. The
selected factors were chicken feather meal: A, initial pH: B, incubation temperature: C,
incubation time: D, and inoculum size: E. The parameters were defined at 3 levels: low
(
−
1), medium (0), and high (+1), while keratinase activity was defined as the response
(Table 3) [49].
Table 3. Operating parameters and their level variations in the Box-Behnken design (BBD).
Study Type Response Surface Subtype Randomized
Design Type Box-Behnken Runs 46
Design Mode Quadratic No Blocks Levels
Factor Name Units Type low (−1) high (+1) Medium (0)
A Chicken feather meal g/L Numeric 2 8 5
B Incubation time Days Numeric 4 8 6
C Initial pH Numeric 5 9 7
DIncubation
temperature
◦C Numeric 35 45 40
E Inoculum size Spores/mL Numeric 1.00 ×1061.00 ×1085.05 ×107
Response Name Unit Observations Analysis
Polynomial
R Keratinase activity U/mL 46
The five-factor, three-level BBD method comprised 46 randomized experiments with
triplicates at the central point (Table 3). The required experimental number (N) was defined
according to Equation (3):
N=2k·(k−1) + C0(3)
where Nrepresents the number of experiments; kand C
0
are the numbers of factors and
central points of the experiments (6), respectively.
The developed and organized 46-trial matrix was executed according to the chosen
optimization plan. The tests were carried out in triplicate (Table 4).
Table 4. The BBD trial plan layout and associated responses for keratinase activity of strain ES41.
Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Response
Run
A: Chicken Feather
Meal
g/L
B: Incubation
Time
Days
C: Initial
pH
D: Incubation
Temperature
◦C
E: Inoculum
Size
Spores/mL
R: Keratinase
Activity
U/mL
1 2 6 9 40 5.05 ×107268.12 ±2.5
2 5 6 7 40 5.05 ×107451.70 ±0.4
Fermentation 2024,10, 500 8 of 28
Table 4. Cont.
Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Response
Run
A: Chicken Feather
Meal
g/L
B: Incubation
Time
Days
C: Initial
pH
D: Incubation
Temperature
◦C
E: Inoculum
Size
Spores/mL
R: Keratinase
Activity
U/mL
3 2 4 7 40 5.05 ×107289.53 ±1.9
4 5 6 7 40 5.05 ×107448.69 ±1.9
5 2 6 7 40 1.00 ×106287.20 ±3.2
6 5 8 7 40 1.00 ×106324.47 ±0.3
7 5 6 9 40 1.00 ×108214.17 ±4.6
8 5 6 7 35 1.00 ×106267.00 ±1.7
9 5 4 7 35 5.05 ×107225.00 ±0.8
10 5 6 5 35 5.05 ×107263.92 ±1.7
11 5 6 7 40 5.05 ×107448.23 ±2.7
12 8 6 7 40 1.00 ×108318.00 ±6.3
13 8 6 7 45 5.05 ×107204.00 ±1.8
14 5 8 7 45 5.05 ×107179.00 ±4.6
15 8 6 9 40 5.05 ×107259.00 ±2.3
16 5 6 5 40 1.00 ×106295.78 ±5.1
17 8 6 5 40 5.05 ×107363.52 ±0.4
18 5 8 7 40 1.00 ×108322.25 ±0.7
19 8 6 7 40 1.00 ×106353.00 ±1.6
20 5 4 9 40 5.05 ×107251.54 ±1.4
21 5 6 7 35 1.00 ×108251.00 ±2.7
22 5 6 7 40 5.05 ×107449.88 ±0.8
23 8 6 7 35 5.05 ×107326.73 ±4.9
24 5 6 7 40 5.05 ×107450.32 ±3.1
25 2 6 7 40 1.00 ×108230.00 ±4.3
26 5 4 7 45 5.05 ×107376.26 ±5.6
27 8 4 7 40 5.05 ×107423.00 ±1.5
28 5 6 7 40 5.05 ×107455.00 ±0.8
29 5 6 9 35 5.05 ×107174.40 ±1.7
30 5 6 5 45 5.05 ×107184.00 ±2.2
31 5 6 9 45 5.05 ×107233.12 ±0.9
32 5 6 7 45 1.00 ×108177.15 ±6.2
33 2 6 5 40 5.05 ×107204.00 ±0.7
34 8 8 7 40 5.05 ×107326.23 ±2.4
35 5 4 7 40 1.00 ×108327.28 ±3.4
36 5 8 5 40 5.05 ×107234.00 ±0.6
37 5 6 9 40 1.00 ×106322.98 ±3.6
38 5 8 7 35 5.05 ×107342.81 ±2.1
39 2 6 7 45 5.05 ×107245.64 ±3.8
40 5 4 5 40 5.05 ×107421.00 ±1.2
41 2 6 7 35 5.05 ×107147.98 ±1.8
42 5 8 9 40 5.05 ×107355.00 ±4.1
43 2 8 7 40 5.05 ×107312.00 ±0.7
44 5 4 7 40 1.00 ×106458.00 ±1.3
45 5 6 7 45 1.00 ×106288.00 ±2.3
46 5 6 5 40 1.00 ×108295.43 ±0.8
The Design Expert 13
®
software, having provided the Box-Behnken experimental
design for optimized keratinase production in strain ES41, was used to analyze the obtained
results, validate the statistical model, and predict the optimal operating parameters for
enzyme production.
Keratinase production was assessed by multiple regression treatment of the data via
examination of the response surfaces and ANOVA. Examination of the response surfaces
traced by varying the values of two factors while maintaining those of the other factors
Fermentation 2024,10, 500 9 of 28
constant at level (0) enabled us to detect, the interactions existing between the various
factors by highlighting linear, quadratic, and interaction effects, while the ANOVA allowed
us to determine the adequacy of the model and thus develop a second-order polynomial
equation (quadratic model) according to the general Equation (4) used for predicting the
ideal requirements for keratinase synthesis.
R=β0+βAA+βBB+βCC+βDD+βEE+βAB AB +βACAC+βAD AD +βAEAE +βBC BC +βBDBD
+βBEBE +βCDCD +βCECE +βDEDE +βAAA2+βBBB2+βCCC2+βDDD2+βEEE2+ε(4)
Rdesignates response surfaces (keratinase activity);
β0
is the steady term (y-intercept),
A, B, C, D, and E represent independent variables;
βA
,
βB
,
βC
,
βD
, and
βE
represent
linearcoefficients;
βAA
,
βBB
,
βCC
,
βDD
, and
βEE
represent quadratic coefficients;
βAB
,
βAC
,
βAD
,
βAE
,
βBC
,
βBD
,
βBE
,
βCD
,
βCE
, and
βDE
represent the interaction coefficients;
and
ε
is a random error component that represents other sources of response variability
notaccounted for in the model, including effects such as measurement error on the response,
inherent system variation such as instrumental background, and the effects of unstudied
variables [
50
,
51
]. To assess the statistical validity of the model used, it is necessary to
evaluate the coefficient of regression (R
2
), which indicates the quality of the fit, and the
adjusted coefficient of regression (R
2
adj), representing the proportion of variance described
by the model. The closer the value of the coefficient of determination (R
2
) is to 1, the better
the model [51].
2.10. Experimental Model Validation
The relevance of the statistical model was verified by performing predicted experi-
ments generated by the Design Expert 13.0.12.0 software by analysis of the quadratic model
under optimal conditions and the determination of the appropriate values of the parameters
involved in the production of keratinase by strain ES41 [
48
]. The predicted experiments
were verified by applying two solutions proposed by the model, comprising predicted
optimal values of the selected variables and predicted responses under these conditions.
3. Results and Discussion
A total number of 35 strains were obtained on a solid medium composed of chicken
feather meal, mainly based on the morphological features of actinobacteria and also the
appearance of hydrolysis zones around the colonies. After the selection of pure colonies,
a second selection in a submerged medium was carried out by quantitative estimation of
keratinase production. Through this selection, five strains were preserved for subsequent
studies, particularly for their ability to produce keratinase in a liquid medium.
3.1. Isolation of Keratinase-Producing Actinobacterial Strains
Selective isolation of actinobacterial strains on solid FBM culture medium yielded
35 distinct bacterial strains with morphological characteristics consistent with actinobacteria.
Figure 2a,b showsthe proportions of isolates obtained on agar medium by sampling site
and type of sample, respectively. Of the strains selected, 15 strains representing 43%
were isolated from the Djebira (Boukhelifa) sampling site, compared with 13 and 7 strains
comprising 38% and 19% from the Fenaia-Ilmathen and Ighil Ali sampling sites, respectively
(Figure 2a). When evaluated by sampling type, chicken feather amended soil (CFS) yielded
the highest number of isolates (68%, 58%, and 53%), followed by chicken compost manure
(CCM) (19%, 27%, and 33%) and decomposing feathers (DF) (13%, 15%, and 14%) for the
three sampling sites, respectively (Figure 2b).
Fermentation 2024,10, 500 10 of 28
Fermentation 2024, 10, x FOR PEER REVIEW 10 of 30
variation such as instrumental background, and the effects of unstudied variables [50,51].
To assess the statistical validity of the model used, it is necessary to evaluate the coeffi-
cient of regression (R2), which indicates the quality of the fit, and the adjusted coefficient
of regression (R2adj), representing the proportion of variance described by the model. The
closer the value of the coefficient of determination (R2) is to 1, the better the model [51].
2.10. Experimental Model Validation
The relevance of the statistical model was verified by performing predicted experi-
ments generated by the Design Expert 13.0.12.0 software by analysis of the quadratic
model under optimal conditions and the determination of the appropriate values of the
parameters involved in the production of keratinase by strain ES41 [48]. The predicted
experiments were verified by applying two solutions proposed by the model, comprising
predicted optimal values of the selected variables and predicted responses under these
conditions.
3. Results and Discussion
A total number of 35 strains were obtained on a solid medium composed of chicken
feather meal, mainly based on the morphological features of actinobacteria and also the
appearance of hydrolysis zones around the colonies. After the selection of pure colonies,
a second selection in a submerged medium was carried out by quantitative estimation of
keratinase production. Through this selection, five strains were preserved for subsequent
studies, particularly for their ability to produce keratinase in a liquid medium.
3.1. Isolation of Keratinase-Producing Actinobacterial Strains
Selective isolation of actinobacterial strains on solid FBM culture medium yielded 35
distinct bacterial strains with morphological characteristics consistent with actinobacte-
ria. Figure 2a,b showsthe proportions of isolates obtained on agar medium by sampling
site and type of sample, respectively. Of the strains selected, 15 strains representing 43%
were isolated from the Djebira (Boukhelifa) sampling site, compared with 13 and 7 strains
comprising 38% and 19% from the Fenaia-Ilmathen and Ighil Ali sampling sites, respec-
tively (Figure 2a). When evaluated by sampling type, chicken feather amended soil (CFS)
yielded the highest number of isolates (68%, 58%, and 53%), followed by chicken com-
post manure (CCM) (19%, 27%, and 33%) and decomposing feathers (DF) (13%, 15%, and
14%) for the three sampling sites, respectively (Figure 2b).
Fermentation 2024, 10, x FOR PEER REVIEW 11 of 30
Figure 2. Proportion of isolates obtained: (a) according to sampling sites; (b) according to the types
of samples collected; CCM = chicken compost manure; CFS = soil amended with chicken feathers;
DF = decomposing feathers.
3.1.1. Pre-Screening on FBM Solid Medium
Based on the existence of pronounced growth of colonies with the appearance or ab-
sence of clear zones of hydrolysis around them due to the secretion and diffusion of
keratinases produced by the isolates identified on the selection medium, the isolation on
solid medium made it possible to select five putative keratinase-producing isolates (Figure
3) [52].
Figure 3. Putative keratinase-producing isolates On feather basal salt medium (FBM) agar plates. (a)
ES41 and (b) EP41 indicate the isolates from Djebira (Boukhelifa) chicken compost manure; (c) ES31
and (d) EP33 show the isolates from Fenaia-Ilmathen chicken compost manure and decomposing
feathers, respectively; and (e) EP22 shows the isolate from Ighil Ali chicken feather amended soil.
Figure 2. Proportion of isolates obtained: (a) according to sampling sites; (b) according to the types
of samples collected; CCM = chicken compost manure; CFS = soil amended with chicken feathers;
DF = decomposing feathers.
3.1.1. Pre-Screening on FBM Solid Medium
Based on the existence of pronounced growth of colonies with the appearance or
absence of clear zones of hydrolysis around them due to the secretion and diffusion of
keratinases produced by the isolates identified on the selection medium, the isolation
on solid medium made it possible to select five putative keratinase-producing isolates
(Figure 3) [52].
Actinobacteria are largely found in nature, especially in soil. They constitute a notable
proportion of the telluric microbial flora [
53
]. The organic-rich soil samples containing
chicken feathers as a potential carbon source promote the growth of actinobacterial strains
with keratinous material’s ability degradation. A large number of actinbacterial gen-
era/species isolated from different soil sites have been reported as keratinase producers,
mainly by the hydrolysis of several keratin materials, citing wool, feathers, and hair. The
Fermentation 2024,10, 500 11 of 28
production ofkeratinolytic enzymes is considered to be inductive; their synthesis takes
place mainly in response to the presence of a keratin substrate [30,54].
Fermentation 2024, 10, x FOR PEER REVIEW 11 of 30
Figure 2. Proportion of isolates obtained: (a) according to sampling sites; (b) according to the types
of samples collected; CCM = chicken compost manure; CFS = soil amended with chicken feathers;
DF = decomposing feathers.
3.1.1. Pre-Screening on FBM Solid Medium
Based on the existence of pronounced growth of colonies with the appearance or ab-
sence of clear zones of hydrolysis around them due to the secretion and diffusion of
keratinases produced by the isolates identified on the selection medium, the isolation on
solid medium made it possible to select five putative keratinase-producing isolates (Figure
3) [52].
Figure 3. Putative keratinase-producing isolates On feather basal salt medium (FBM) agar plates. (a)
ES41 and (b) EP41 indicate the isolates from Djebira (Boukhelifa) chicken compost manure; (c) ES31
and (d) EP33 show the isolates from Fenaia-Ilmathen chicken compost manure and decomposing
feathers, respectively; and (e) EP22 shows the isolate from Ighil Ali chicken feather amended soil.
Figure 3. Putative keratinase-producing isolates On feather basal salt medium (FBM) agar plates.
(a) ES41 and (b) EP41 indicate the isolates from Djebira (Boukhelifa) chicken compost manure; (c) ES31
and (d) EP33 show the isolates from Fenaia-Ilmathen chicken compost manure and decomposing
feathers, respectively; and (e) EP22 shows the isolate from Ighil Ali chicken feather amended soil.
In this perspective, exploring diverse sources of keratinous waste, including by-
products from animal slaughterhouses (such as horns, hooves, and hides), as well as waste
and effluents from textile industries and tanneries, is crucial. This investigation aims to
isolate microorganisms with keratinolytic capabilities and to develop methods for the
treatment and valorization of these wastes.
3.1.2. Screening in FBM Submerged Medium
The results of the submerged screening performed to quantify the keratinase activ-
ity of the five most promising isolates are shown in Figure 4. Strain ES41 (Figure 3a)
isolated from Djebira (Boukhelifa) chicken compost manure showed the highest enzy-
matic activity at 22.4
±
0.5 U/mL. It was followed by strain EP41 (Figure 3b) from the
same site and sample type as the preceding strain, with a recorded keratinase activity of
around
18.3 ±0.2 U/mL
.Then followed strains ES31 and EP33 (Figure 3c,d) isolated from
Fenaia-Ilmathen chicken compost manure and decomposing feathers, respectively, which
successively achieved enzyme yields of 17.7
±
0.2 and 13.8
±
0.3 U/mL. Finally, strain
EP22 (Figure 3e) isolated from Ighil Ali chicken feather amended soil showed a keratinase
production of 10.3 ±0.3 U/mL.
Statisticalanalysis using t-tests indicated significant differences in keratinase activity
among the isolates. For instance, ES41 exhibitedsignificantlyhigher keratinase activity
comparedto: EP41 (p= 0.03), ES31 (p= 0.01), EP33 (p= 0.01), and EP22 (p< 0.001), where-
asstrains EP41 and ES31 show a non-significant difference in enzyme yields (p> 0.05), as
shown in Figure 4.
Bacteria and fungi are regularly cited in the literature as producers of keratinase.
Most fungal producers like Trichophyton and Microsporum and Gram-negative bacteria,
citing Vibrio sp. strain kr2, Citrobacterdiversus, and Pseudomonas aeruginosa 4-3, have limited
applications due to a certain degree of pathogenicity [6,55–58].
Fermentation 2024,10, 500 12 of 28
Fermentation 2024, 10, x FOR PEER REVIEW 13 of 30
Figure 4. Keratinase activity of the five most promising isolates when cultivated under submerged
fermentation conditions in the presence of chicken feather meal as the sole carbon and nitrogen
source; p < 0.05:*; p < 0.01: **; p < 0.001: ***; p > 0.05: not significant.
Gram-positive bacteria, represented mainly by the genus Bacillus and Actinobacte-
ria, are the most recommended for the degradation of keratin materials. Keratinase
producers from the Bacillus genus include Bacillus licheniformis PWD-1, Bacillus subtilis,
Bacillus amyloliticus, Bacillus cereus,and Bacillus thuringiensis, while the main actinobacte-
rial keratinase producers belong to the Streptomyces genus, including Streptomyces albi-
cansand Streptomyces fradiae [59,60]. Compared to a few applications mainly from B. li-
cheniformis strains and based on previous research, actinobacterial species have not been
widely applied for keratin waste bioconversion. Actinobacteria offer advantages when
used in keratinase production, as they present broad physiological tolerances, the capac-
ity and adaptability to grow on different types of biopolymeric substrates, particularly on
a wide range of keratin substrates, and the ability to synthesize a wide range of bioactive
substances such as keratinases. Moreover, the metabolic variability of actinobacteria en-
ables them to be used in both submerged and solid-state fermentation, depending on
their need for water potential. Actinobacteria thus offer potential advantages in terms of
their multiple industrial uses [61]. For this reason, this research aimed to explore the
ability of actinobacteria to produce keratinases and the possibilities of applying them in
several industrial fields.
3.2. Identification and Classification of the Keratinase-Producing Strain ES41
3.2.1. Morphological Characterization of Strain ES41
The macromorphological and micromorphological characteristics of strain ES41
were found to be important for the identification and classification of the microorganism.
Colonies of strain ES41 grown on various agar media at 45 °C for 24 to 48 h were circular
with a powdery and cottony appearance on Williams and International Streptomyces
Project 2 (ISP-2) media (Figure 5a,b) and a powdery and rough appearance on glucose
yeast extract agar (GYEA) medium (Figure 5c), with a diameter ranging from 2 to 10 mm.
The isolate exhibited abundant growth and sporulation on Williams medium.
Growth was relatively average on GYEA medium but weak on the ISP-2 medium. The
Figure 4. Keratinase activity of the five most promising isolates when cultivated under submerged
fermentation conditions in the presence of chicken feather meal as the sole carbon and nitrogen
source; p< 0.05: *; p< 0.01: **; p< 0.001: ***; p> 0.05: not significant.
Gram-positive bacteria, represented mainly by the genus Bacillus and Actinobacteria,
are the most recommended for the degradation of keratin materials. Keratinase producers
from the Bacillus genus include Bacillus licheniformis PWD-1, Bacillus subtilis,Bacillus amy-
loliticus, Bacillus cereus, and Bacillus thuringiensis, while the main actinobacterial keratinase
producers belong to the Streptomyces genus, including Streptomyces albicans and Strepto-
myces fradiae [
59
,
60
]. Compared to a few applications mainly from B. licheniformis strains
and based on previous research, actinobacterial species have not been widely applied for
keratin waste bioconversion. Actinobacteria offer advantages when used in keratinase
production, as they present broad physiological tolerances, the capacity and adaptability
to grow on different types of biopolymeric substrates, particularly on a wide range of
keratin substrates, and the ability to synthesize a wide range of bioactive substances such as
keratinases. Moreover, the metabolic variability of actinobacteria enables them to be used in
both submerged and solid-state fermentation, depending on their need for water potential.
Actinobacteria thus offer potential advantages in terms of their multiple industrial uses [
61
].
For this reason, this research aimed to explore the ability of actinobacteria to produce
keratinases and the possibilities of applying them in several industrial fields.
3.2. Identification and Classification of the Keratinase-Producing Strain ES41
3.2.1. Morphological Characterization of Strain ES41
The macromorphological and micromorphological characteristics of strain ES41 were
found to be important for the identification and classification of the microorganism.
Colonies of strain ES41 grown on various agar media at 45
◦
C for 24 to 48 h were cir-
cular with a powdery and cottony appearance on Williams and International Streptomyces
Project 2 (ISP-2) media (Figure 5a,b) and a powdery and rough appearance on glucose yeast
extract agar (GYEA) medium (Figure 5c), with a diameter ranging from 2 to 10 mm.
Fermentation 2024,10, 500 13 of 28
Fermentation 2024, 10, x FOR PEER REVIEW 14 of 30
aerial mycelium was white, stable, and not fragmented. The substrate mycelium pre-
sented various colors depending on the medium used, ranging from yellow on Williams
medium to dark brown on GYEA medium and light brown on ISP-2. Diffusible pigments
were not produced on any of the test media.
Spore chain arrangements and Gram type determination were observed using a
Euromex optical microscope (at 100× magnification). The aerial mycelium takes the form of
coiled and entangled thick filaments bearing chains of short spores arranged in primary
or secondary whorls. The verticilliums are made up of short sporophores that emerge
from a common point and carry the spore chains (Figure 5d). ES41 strain’s Gram staining
showed that it is Gram-positive (Figure 5e). All these characteristics are in favor of as-
signing the isolate to the genus Streptomyces [42].
Figure 5. Morphology of strain ES41, which was isolated from Djebira (Boukhelifa) poultry com-
post manure: (a) growth on Williams agar medium; (b) growth on ISP2 agar medium; and (c)
growth on GYEA medium; (d) micro-morphology (100× magnification) of spore chain disposition;
and (e) Gram staining.
3.2.2. Molecular Typing of ES41 Strain
To identify and classify the ES41 strain, the 16S rRNA gene was amplified and se-
quenced using PCR and the Sanger method. Alignment of the obtained sequence with
those of the NCBI (National Center for Biotechnology Information) database from the
BLASTn (Basic Local Alignment Search Tool) program showed that ES41 belongs to the
genus Streptomyces (99.66% identity).
The submission of this sequence (1478 bp) to the GenBank database with the name
Streptomyces sp. DZ 06 had been assigned the following accession number: OQ195253.1
[43]. A phylogenetic tree based on partial 16S rRNA sequence is presented in Figure 6,
which illustrates the relationship between Streptomyces sp. strain DZ 06 (ES41) and other
strains within the same as well as related actinobacteria [45]. The 16S rRNA sequences of
Bacillus cabrialesii strain NOK82 (ON 287158.1), Brevibacillusagri strain IHB B 1387
(GU186123.1), and Escherichia coli strain JMC 1649 (LC069032.1) were utilized as
out-groups for neighborhood joining.
Figure 5. Morphology of strain ES41, which was isolated from Djebira (Boukhelifa) poultry compost
manure: (a) growth on Williams agar medium; (b) growth on ISP2 agar medium; and (c) growth on GYEA
medium; (d) micro-morphology (100×magnification) of spore chain disposition; and (e) Gram staining.
The isolate exhibited abundant growth and sporulation on Williams medium. Growth
was relatively average on GYEA medium but weak on the ISP-2 medium. The aerial
mycelium was white, stable, and not fragmented. The substrate mycelium presented
various colors depending on the medium used, ranging from yellow on Williams medium
to dark brown on GYEA medium and light brown on ISP-2. Diffusible pigments were not
produced on any of the test media.
Spore chain arrangements and Gram type determination were observed using a
Euromex optical microscope (at 100×magnification). The aerial mycelium takes the form
of coiled and entangled thick filaments bearing chains of short spores arranged in primary
or secondary whorls. The verticilliums are made up of short sporophores that emerge from
a common point and carry the spore chains (Figure 5d). ES41 strain’s Gram staining showed
that it is Gram-positive (Figure 5e). All these characteristics are in favor of assigning the
isolate to the genus Streptomyces [42].
3.2.2. Molecular Typing of ES41 Strain
To identify and classify the ES41 strain, the 16S rRNA gene was amplified and se-
quenced using PCR and the Sanger method. Alignment of the obtained sequence with
those of the NCBI (National Center for Biotechnology Information) database from the
BLASTn (Basic Local Alignment Search Tool) program showed that ES41 belongs to the
genus Streptomyces (99.66% identity).
The submission of this sequence (1478 bp) to the GenBank database with the name
Streptomyces sp. DZ 06 had been assigned the following accession number: OQ195253.1 [
43
].
A phylogenetic tree based on partial 16S rRNA sequence is presented in Figure 6, which
illustrates the relationship between Streptomyces sp. strain DZ 06 (ES41) and other strains
within the same as well as related actinobacteria [
45
]. The 16S rRNA sequences of Bacillus
cabrialesii strain NOK82 (ON 287158.1), Brevibacillusagri strain IHB B 1387 (GU186123.1),
and Escherichia coli strain JMC 1649 (LC069032.1) were utilized as out-groups for neighbor-
hood joining.
Fermentation 2024,10, 500 14 of 28
Fermentation 2024, 10, x FOR PEER REVIEW 15 of 30
Figure 6. 16S rRNAneighbor-joining phylogenetic tree for Streptomyces sp. Strain DZ 06 (ES41) and
related actinobacterial taxa.
3.3. Screening of Critical Factors Affecting Keratinase Production via the
Plackett-Burman Design
The production of keratinase from bacterial genus/species has often been optimized
using a single-step statistical experimental design and response surface methodology.
Among these investigations were enzymatic bioconversion of feather waste with
keratinases of Bacillus cereus PCM 2849 [1], statistical optimization of keratinase produc-
tion by Bacillus cereus GJBBR [62], enhanced production, purification, and characteriza-
tion of alkaline keratinase from Streptomyces minutiscleroticus DNA38 [63], keratinase
production by Bacillus pumilus GHD in solid-state fermentation using sugar cane bagasse
[64], plus response surface methodology optimization of keratinase production from al-
kali-treated feather waste and horn meal using Bacillus sp. MG-MASC-BT [65]. However,
few studies have been reported using a complete statistical experimental process, in-
cluding statistical methods for screening the various influencing factors and optimizing
their variation levels, as reported in this study.
In the present study, the determination of keratinase production influencing factors
by isolating Streptomyces sp. strain DZ 06 (ES41) was carried out by practical testing of the
PBD plan generated by the statistical software Minitab 19 (trial version). The results of
the obtained PBD experiments are presented in Tables 2 and Figures 7 and 8. Table 2
shows the PBD design matrix provided for screening ten two-level independent variables
representing the initial production conditions and corresponding responses. The col-
lected response data showed variable keratinase activities ranging from 43.99 U/mL
(minimum) to 180.13 U/mL (maximum). The different combinations of high and low
levels of the various influencing factors are at the origin of the variations in response [66].
This wide variation reflected the eminence of optimizing production conditions in order
to reach high levels in terms of enzyme production. The maximum production of
keratinase was obtained in the 1st series (180.13 U/mL), with inoculum size (X2), initial
pH (X4), K2HPO4(X5), orbital agitation (X6), chicken feather meal (X7), and MgSO4·7H2O
(X10) present at high levels. While NaCl (X1), incubation time (X3), CaCO3 (X8), and incu-
bation temperature (X9) were present at low levels (Table 2).
Figure 6. 16S rRNAneighbor-joining phylogenetic tree for Streptomyces sp. Strain DZ 06 (ES41) and
related actinobacterial taxa.
3.3. Screening of Critical Factors Affecting Keratinase Production via the Plackett-Burman Design
The production of keratinase from bacterial genus/species has often been optimized
using a single-step statistical experimental design and response surface methodology.
Among these investigations were enzymatic bioconversion of feather waste with keratinases
of Bacillus cereus PCM 2849 [
1
], statistical optimization of keratinase production by Bacillus
cereus GJBBR [
62
], enhanced production, purification, and characterization of alkaline
keratinase from Streptomyces minutiscleroticus DNA38 [
63
], keratinase production by Bacillus
pumilus GHD in solid-state fermentation using sugar cane bagasse [
64
], plus response
surface methodology optimization of keratinase production from alkali-treated feather
waste and horn meal using Bacillus sp. MG-MASC-BT [
65
]. However, few studies have been
reported using a complete statistical experimental process, including statistical methods for
screening the various influencing factors and optimizing their variation levels, as reported
in this study.
In the present study, the determination of keratinase production influencing factors
by isolating Streptomyces sp. strain DZ 06 (ES41) was carried out by practical testing of the
PBD plan generated by the statistical software Minitab 19 (trial version). The results of the
obtained PBD experiments are presented in Table 2and Figures 7and 8. Table 2shows the
PBD design matrix provided for screening ten two-level independent variables representing
the initial production conditions and corresponding responses. The collected response data
showed variable keratinase activities ranging from 43.99 U/mL (minimum) to 180.13 U/mL
(maximum). The different combinations of high and low levels of the various influencing
factors are at the origin of the variations in response [
66
]. This wide variation reflected
the eminence of optimizing production conditions in order to reach high levels in terms of
enzyme production. The maximum production of keratinase was obtained in the 1st series
(180.13 U/mL), with inoculum size (X
2
), initial pH (X
4
), K
2
HPO
4
(X
5
), orbital agitation (X
6
),
chicken feather meal (X
7
), and MgSO
4·
7H
2
O (X
10
) present at high levels. While NaCl (X
1
),
incubation time (X
3
), CaCO
3
(X
8
), and incubation temperature (X
9
) were present at low
levels (Table 2).
Fermentation 2024,10, 500 15 of 28
Fermentation 2024, 10, x FOR PEER REVIEW 16 of 30
The adequacy of the statistical model was verified by highlighting the effects of the
variables examined via Fisher’s F test and analysis of variance. For p-values < 0.05, factors
were considered to have a significant effect on response [67]. The ANOVA and the
p-value of the model and for each parameter are presented in Table 5.
Table 5. ANOVA for principal effect model used for the identification of key factors influencing
keratinase production by Streptomyces sp. Strain DZ 06 (ES41).
Source DF Adjusted Sum of Squares Adjusted Mean Square F-Value p-Value
Regression 8 28,305.9 3538.23 52.45 0.000
NaCl 1 211.5 211.48 3.13 0.137
Inoculum size 1 2037.9 2037.93 30.21 0.003
Incubation time 1 1141.4 1141.42 16.92 0.009
Initial pH 1 4925.3 4925.29 73.01 0.000
K2HPO4 1 218.2 218.24 3.23 0.132
Orbital agitation 1 327.5 327.5 4.85 0.079
Chicken feather meal 1 6903.8 6903.8 102.34 0.000
Incubation temperature 1 3497.5 3497.52 51.84 0.001
Error 5 337.3 67.46
Total 13 28
,
643.2
R2 98.82%
Adjusted R2 96.94%
Predicted R2 91.12%
Df = the degree of freedom; F Value = the value of F obtained by carrying out an F test. Prob (P) > F =
the value of p, indicating the probability and applying it to significance.
Among the variables studied and in decreasing order of influence, the factors iden-
tified, chicken feather meal (p = 0.000), initial pH (p = 0.000), incubation temperature (p =
0.001), inoculum size (p = 0.003), and incubation time (p = 0.009), showed highly signifi-
cant effects on keratinase production with p values < 0.01. The remaining variables
showing p values > 0.05 are considered non-influential factors. This suggests that low
concentrations of these factors are sufficient for keratinase production within the strain
studied [66].
At the same time, the Pareto chart of the effects of the independent variables re-
vealed the factors influencing the response represented by horizontal bluebars exceeding
the red line representing the significance level, thus confirming the results obtained from
the analysis of the statistical model.(Figure 7) [68].
Figure 7. Pareto plot of the independent factors’ main impacts on keratinase synthesis by the
Streptomyces sp. strain DZ 06 (ES41) according to the PBD results.
Fermentation 2024, 10, x FOR PEER REVIEW 17 of 30
Figure 7. Pareto plot of the independent factors’ main impacts on keratinase synthesis by the
Streptomyces sp. strain DZ 06 (ES41) according to the PBD results.
Figure 8 shows the main plots of the effect of the significant factors on the response
and confirms the results reported in Table 5 and Figure 7. Increases in chicken feather
meal concentrations, initial pH values, and inoculum size greatly influenced keratinase
production, while increases in incubation temperature and incubation time values hurt
keratinase activity detected [69].
Figure 8. Ratio of the principal factors impacts on the keratinase activity by the Streptomyces sp.
strain DZ 06 (ES41).
The regression model “F” value (52.45) was found to be significant. The coefficient of
determination R2provides an explanation of the model fit goodness, which is used to as-
sess the explanatory power of regression models and reflects the variation in response in
the proposed statistical model. [70].
The regression equation acquired showed an R2 value of 0.9882, indicating that
98.82% of the total variation detected for the responses could be interpreted by the model,
demonstrating that the design is highly significant in predicting the factors effects on
keratinase activity by the studied strain. An R2 value > 0.75 indicates fit to the biological
models [71]. The correlation of the predicted obtained responses in the present study is
explained by the closeness of the R2, adjusted R2, and predicted R2values: 98.82%, 96.94%,
and 91.12%, respectively (Table 5). The design performance was valuated by a first-order
model analysis, showing its fitting to the experimental data using the following Equation
(5):
23479
R=52.07+29.29X -18.91X +45.53X +50.69X -37.85X (5)
The PBD results showed that the factors, chicken feather meal (% w/v), initial pH,
incubation temperature (°C), incubation time (days), and inoculum size (spores/v), exert
significant effects on keratinase production, with chicken feather meal concentration
forming a major contributor. This result is identical to those obtained by Abdul Gafar et
Figure 8. Ratio of the principal factors impacts on the keratinase activity by the Streptomyces sp. strain
DZ 06 (ES41).
The adequacy of the statistical model was verified by highlighting the effects of the
variables examined via Fisher’s F test and analysis of variance. For p-values < 0.05, factors
were considered to have a significant effect on response [
67
]. The ANOVA and the p-value
of the model and for each parameter are presented in Table 5.
Among the variables studied and in decreasing order of influence, the factors iden-
tified, chicken feather meal (p= 0.000), initial pH (p= 0.000), incubation temperature
(
p= 0.001
), inoculum size (p= 0.003), and incubation time (p= 0.009), showed highly signifi-
cant effects on keratinase production with pvalues < 0.01. The remaining variables showing
pvalues > 0.05 are considered non-influential factors. This suggests that low concentrations
of these factors are sufficient for keratinase production within the strain studied [66].
Fermentation 2024,10, 500 16 of 28
Table 5. ANOVA for principal effect model used for the identification of key factors influencing
keratinase production by Streptomyces sp. Strain DZ 06 (ES41).
Source DF
Adjusted Sum of Squares
Adjusted Mean Square F-Value p-Value
Regression 8 28,305.9 3538.23 52.45 0.000
NaCl 1 211.5 211.48 3.13 0.137
Inoculum size 1 2037.9 2037.93 30.21 0.003
Incubation time 1 1141.4 1141.42 16.92 0.009
Initial pH 1 4925.3 4925.29 73.01 0.000
K2HPO41 218.2 218.24 3.23 0.132
Orbital agitation 1 327.5 327.5 4.85 0.079
Chicken feather meal 1 6903.8 6903.8 102.34 0.000
Incubation temperature 1 3497.5 3497.52 51.84 0.001
Error 5 337.3 67.46
Total 13 28,643.2
R298.82%
Adjusted R296.94%
Predicted R291.12%
DF = the degree of freedom; F-Value = the value of F obtained by carrying out an F test. Prob (P) > F = the value of
p, indicating the probability and applying it to significance.
At the same time, the Pareto chart of the effects of the independent variables revealed
the factors influencing the response represented by horizontal bluebars exceeding the
red line representing the significance level, thus confirming the results obtained from the
analysis of the statistical model.(Figure 7) [68].
Figure 8shows the main plots of the effect of the significant factors on the response
and confirms the results reported in Table 5and Figure 7. Increases in chicken feather
meal concentrations, initial pH values, and inoculum size greatly influenced keratinase
production, while increases in incubation temperature and incubation time values hurt
keratinase activity detected [69].
The regression model “F” value (52.45) was found to be significant. The coefficient
of determination R
2
provides an explanation of the model fit goodness, which is used to
assess the explanatory power of regression models and reflects the variation in response in
the proposed statistical model. [70].
The regression equation acquired showed an R
2
value of 0.9882, indicating that 98.82%
of the total variation detected for the responses could be interpreted by the model, demon-
strating that the design is highly significant in predicting the factors effects on keratinase
activity by the studied strain. An R
2
value > 0.75 indicates fit to the biological models [
71
].
The correlation of the predicted obtained responses in the present study is explained by
the closeness of the R
2
, adjusted R
2
, and predicted R
2
values: 98.82%, 96.94%, and 91.12%,
respectively (Table 5). The design performance was valuated by a first-order model analysis,
showing its fitting to the experimental data using the following Equation (5):
R=52.07+29.29X2−18.91X3+45.53X4+50.69X7−37.85X9(5)
The PBD results showed that the factors, chicken feather meal (% w/v), initial pH,
incubation temperature (
◦
C), incubation time (days), and inoculum size (spores/v), exert
significant effects on keratinase production, with chicken feather meal concentration form-
ing a major contributor. This result is identical to those obtained by Abdul Gafar et al. and
Laba et al. when PBD was utilized in the screening procedure.It was shown that the con-
centration of chicken feather meal was the most important influencing factor for keratinase
synthesis by Bacillus sp. UPM-AAG1 and the actinobacterium strain Kocuriarhizophila p3-3,
respectively [
67
,
72
]. In work undertaken by Manivasagan et al. and Demir et al., the same
result was obtained where the yield in keratinase production was observed when chicken
feather meal was employed as culture substrate during submerged fermentation using Acti-
noalloteichus sp. MA-32 and Streptomyces sp. 2M21, respectively [
73
,
74
]. The use of chicken
feather meal as an organic material source shows the ability of the strain Streptomyces sp.
Fermentation 2024,10, 500 17 of 28
DZ 06 (ES41) to grow and to obtain its carbon and nitrogen requirements directly from
this substrate. Furthermore, variations in the nutritional needs of each feather-degrading
microorganism as well as the type of the keratinolytic proteases generated by the producer
microorganisms may be the primary causes of the disparities in the role of the substrate
in keratinase production among various feather-degrading bacteria, e.g., which carbon
and/or nitrogen sources would act as inducers [75,76].
Similar outcomes to those obtained in the present research showing the significant
effect of the initial culture pH during optimization work appeared in the literature. Among
these works are those carried out by Fakhfakh–Zouari et al. using Bacillus pumilus A1 and
Abd El-Aziz et al. on Streptomyces swerraensis KN23 [
75
,
77
]. The pH of the medium culture
affects the microbial growth, effectiveness of feather degradation, and keratinase synthesis
via influencing the reaction environment, enzymatic process, and movement of nutrients
across the cell membrane of bacteria [1,20].
Apart from this, the important role of incubation temperature in the production
of keratinases is revealed during this work and in those conducted by Demir et al. on
Streptomyces sp. 2M21 and by Matikeviˇcien
˙
e et al. using Actinomyces fradiae 119 [
74
,
78
].
The importance of incubation temperature is crucial for best enzyme production due to
modifications in the structure and characteristics of microbial proteins with changes in
temperature. Metabolic activities are reduced at temperatures below or above the optimal
temperature, resulting in inhibition of growth and enzyme synthesis [79].
The role of incubation time as a determinant factor for keratinase production obtained
during this work was similar to studies on keratinase production by Streptomyces sp. 2M21
and Streptomyces swerraensis KN23 [
74
,
77
]. Incubation time is an important parameter for
keratinase production. It varies according to the microorganism, nature of the substrate
used, and the production medium conditions [80].
Similarly, it was reported that the inoculum size used had a significant effect on
keratinase production by Amycolatopsis sp. strain MBRL 40, A. fradiae 119, and B. licheniformis
ALW1 [
40
,
78
,
81
]. The inoculum size is the initial bacterial mass required to carry out
a fermentation, hence the need to test the selection of this factor and determining its
variation levels. Keratinolytic activity is often increased with increasing inoculum size.
No discernible increase or even decrease in activity is seen above the necessary initial
rate [
82
].The selected parameters influencing keratinase production in Streptomyces sp. DZ
06 (ES41) are factors usually considered to influence most microorganisms when testing
the production of different types of bioactive substances, in particular keratinases; this
suggests that the strain studied has no particular requirements and that testing these factors
for optimization via response surface methodology using the Box–Behnken design would
enable keratinase production to be better controlled in terms of both quantitative and
qualitative yields.
3.4. Box-Behnken Examination of Keratinase Synthesis-Based on RSM Design
Plackett–Burman Design, as indicated above, made it possible to select the main
parameters influencing keratinase production by Streptomyces sp. strain DZ 06 (ES41). These
independent variables, namely chicken feather meal, initial pH, incubation temperature,
incubation time, and inoculum size, were evaluated at three levels (
−
1, 0, +1) to investigate
their interaction and their effects on keratinase production applying a Box–Behnken design
of experiment. For growth and keratinase production by Streptomyces sp. strain DZ 06
(ES41), the insignificant factors, including mineral salts and orbital agitation, were used at
low levels during the enzyme production process [74].
The matrix experiments and results of the different trials performed by the Box–
Behnken design are shown in Table 4. The highest keratinase production of 458 U/mL
was obtained with 5 g/L of chicken feather meal (A), after 4 days of incubation time
(B), at initial pH 7 (C), at incubation temperature of 40
◦
C (D), and an inoculum size of
1.00 ×106spores/mL
(run 44). In addition, that corresponding to the central points of the
factor values tests (run: 2, 4, 11, 22, 24, and 28) showed the closeness and the repeatability
Fermentation 2024,10, 500 18 of 28
of the responses obtained (448.23
±
1.7 to 450.32
±
0.4) (U/mL) and consequently the
relevance of the statistical model used (Table 4). The performance and adequacy of the
quadratic model were verified through variance analysis (ANOVA) using Design Expert
13®software (Table 6).
Table 6. Determined regression coefficients for the analysis of variance (ANOVA) and the quadratic
polynomial model.
Source Sum of Squares Df * Mean Square F-Value p-Value
Model 3.423 ×10520 17,116.77 10.68 <0.0001 significant
A-Poultry feather meal 22,724.06 1 22,724.06 14.17 0.0009
B-Incubation