International Biodeterioration & Biodegradation 150 (2020) 104940

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International Biodeterioration & Biodegradation

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Molecular identification and evaluation of the impact of red heat damage T

causing halophilic microbes on salted hide and skin
Solomon Enquahonea, Guido van Marleb, Amare Gessessea,c,d, Addis Simachewa,∗
a
Institute of Biotechnology, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia
b
Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, T2N 4N1, Alberta, Canada
c
Department of Microbial, Cellular, and Molecular Biology, Addis Ababa University, P O Box 1176, Addis Ababa, Ethiopia
d
Department of Biological Sciences and Biotechnology, Botswana International University of Science and Technology, Private Bag 16, Palapye, Botswana

ARTICLE INFO ABSTRACT

Keywords: Salt is widely used as preservative to protect animal skin from microbial attack. However, addition of salt creates
Skin defect a favorable condition for halophilic microorganisms associated with biodeterioration termed red heat damage.
Collagen Knowledge about microbial diversity associated with red heat damage is expected to help develop appropriate
Skin degradation control strategies. The objectives of this study were to isolate and characterize halophiles from salted sheepskin
Salt preservation
and evaluate their ability to degrade sheepskin. A total of 85 halophiles were isolated from salted sheepskins
Skin and hide
after enrichment in a medium containing 10%, 20% and 25% (w/v) NaCl and chopped sheepskin. In all the
enrichment cultures the sheepskin was completely dissolved by the microbial consortia. Pure culture isolates
belonging to the genera Halomonas, Bacillus, Salimicrobium, Nesterenkonia, and Marinococcus within Domain
Bacteria and genus Halococcus from Domain Archaea were isolated. When tested independently, the isolates
showed variation in their efficiency to hydrolyze sheepskin collagen. Isolate Halococcus sp. s2535 was the fastest
in hydrolyzing the skin followed by Nesterenkonia sp. s2011, Marinococcus sp. s2526 and Halococcus sp. s2528.
The results of this study demonstrate that halophilic microbes associated with red heat damage of salted
sheepskin are highly diverse and preventive measures should target a diverse group of halophilic microbes.

1. Introduction
The process of leather tanning is as old as human civilization and
allows production of different leather goods from the byproducts of the
meat industry (van Driel-Murray, 2012; Wu et al., 2017). The tech-
nology of leather tanning has gradually evolved and reached its current
level of sophistication employing relatively complex machinery and
process to produce various types of leather products (Berber and Birbir,
2019). Today, trade on leather goods is a significant economic activity
throughout the world. About 19.6 billion square feet of leather with an
estimated value of US$ 82 billion is produced annually (FAO, 2016). In
Ethiopia leather tanning is an important industrial activity employing
thousands of workers and helping the country earn substantial amount
of foreign currency (Abtew, 2015).
About 60–70% of the weight of fresh skin and hide is water and 25%
is protein (or more than 90% dry weight basis) (Gudro et al., 2014;
Vreeland et al., 1999). This makes fresh skins and hides suitable sub-
strates for the growth of different groups of microorganisms causing
significant damage through putrefaction. To avoid such damages fresh
skin needs to be preserved soon after flaying (Berber et al., 2010;
Nigatu and Ayalew, 2003). The methods used for animal skin pre-
servation include cooling, drying, salting and the use of other anti-
microbial agents that prevent microbial growth and thus putrefaction
(Caglayan et al., 2015). Of these, salt curing is the most preferred and
widely used method for the preservation of hide and skin (Aslan and
Birbir, 2011; Birbir, 1997; Kanagaraj et al., 2005; Kannan et al., 2010;
Yilmaz and Birbir, 2019). Fresh skin or hide is treated with salt (almost
always sodium chloride) in the form of dry powder (added about
40–50% of the wet weight of the hide or skin) or as a concentrated salt
brine (at 33% saturation) (Kannan et al., 2010). The salt added to fresh
(green) skin or hide penetrates the skin and also helps to draw water
out of fresh skin or hide making the skin or hide unfavorable for mi-
crobial growth (Bailey, 2003; Vreeland et al., 1999). Hence, in the
absence of microbial growth, there will not be production of protein
degrading enzymes and thus no damage to the skin or hide structure
(Woods et al., 1972).
However, some groups of microorganisms, collectively known as
halophiles, optimally grow in the presence of high salt concentrations

∗
Corresponding author. Institute of Biotechnology, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia. Tel.: +251 911696435.
E-mail addresses: Addissimachew@gmail.com, Addis.simachew@aau.edu.et (A. Simachew).

https://doi.org/10.1016/j.ibiod.2020.104940
Received 10 December 2019; Received in revised form 15 March 2020; Accepted 15 March 2020
0964-8305/ © 2020 Elsevier Ltd. All rights reserved.
S. Enquahone, et al.
(Oren, 2002, 2011). In nature, these microorganisms exist in hypersa-
line environments, such as salt lakes, marine environments, and salt
concentration ponds and remain viable on the dry salt (Grant, 2004;
Oren, 2014). When salt obtained from these sources is used for skin or
hide curing, the halophilic microorganisms present on the dry salt start
to proliferate (Bailey & Birbir, 1993, 1996; Bailey, 2003). Since animal
skin is mainly composed of protein (Gudro et al., 2014), it serves as a
source of nutrient supporting growth of halophilic microbes on salt
cured animal skins and hides. Growth of halophiles on salt cured skin or
hide, therefore, involves production of proteolytic enzymes that hy-
drolyze the major protein component, collagen (Vreeland et al., 1999;
Woods et al., 1972).
Appearance of red spots on the flesh side of salt cured skins and
hides is a result of growth of halophilic microorganisms and the color is
due to a red pigment produced by some of the halophilic microorgan-
isms (Anderson, 1954; Bailey & Birbir, 1993, 1996; Caglayan et al.,
2015; Stuart et al., 1933). The skin or hide with such red spots is da-
maged because these halophilic microorganisms secret enzymes that
hydrolyze the collagen fibers, a condition known as red heat damage
(Bailey & Birbir, 1993, 1996; Vreeland et al., 1999). Growth of such
microorganisms is faster and the damage caused more severe at a
higher storage temperature making the problem more severe in tropical
regions. Therefore, to avoid the impact of red heat damage, salted skin
and hide need to be processed faster (Vreeland et al., 1999). If condi-
tions dictate that long term storage is inevitable, then appropriate
measures aimed at retarding or inhibiting the growth of halophilic
microbes must be put in place.
Red heat damage of salt cured skins and hides has been reported
from all over the world, such as in the USA, France, Russia and Turkey
(Akpolat et al., 2015; Bailey and Birbir, 1993; Birbir, 1997; Bitlisli et al.,
2004; Caglayan et al., 2018). Different halophilic microbes causing red
heat damage on salted skin and hide have been isolated and char-
acterized through culture dependent and molecular techniques
(Akpolat et al., 2015; Bailey and Birbir, 1993; Birbir, 1997; Bitlisli et al.,
2004; Caglayan et al., 2018; Stuart et al., 1933). Depending on the
Fig. 1. Red heat damaged sheepskin showing colonies of red pigmented halophilic microbes
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International Biodeterioration & Biodegradation 150 (2020) 104940
source of salt used for curing, the type of halophilic microorganism
could differ from one location to another (Caglayan et al., 2015, 2017).
Knowledge about the types of microorganisms could therefore help in
the development of appropriate control strategies.
Having the largest number of livestock in Africa and with an esti-
mated annual off take rate of 30–35% for sheep and goats and 7% for
cattle (Mahmud, 2000), the leather tanning industry in Ethiopia gets a
steady supply of skins and hides. As a result, the leather tanning in-
dustry is an important sector in the country. However, in recent years
the Ethiopian leather tanning industry is facing serious challenges due
to mainly quality deterioration of the raw materials, skins and hides.
Some reports indicate that only 10% of processed skins and hides
qualify for top grade designation (Grades 1 to 3), with the rest graded to
a lower grade or rejected (Amde, 2017; Coppeaux et al., 2016).
Previous studies on factors responsible for the damage to skins
and hides in Ethiopia showed that scratches, skin diseases and flay
cuts as major causes of damage on skins and hides (Amde, 2017;
Kahsay et al., 2015; Tesfaye et al., 2012). Based on these findings
several attempts were made to improve the quality of hides and skins
by applying different interventions by addressing the above causes of
skin and hide damage. However, the quality of hides and skins kept
on decreasing indicating that other factors might also be responsible
for quality deterioration (Coppeaux et al., 2016). The objective of this
study was to evaluate the potential impacts of halophilic microbes on
sheepskin and identify the organisms to a genus level using molecular
methods.
2. Materials and methods
2.1. Sample collection and preparation of enrichment culture
Five red heat damaged salt cured sheepskins with visible red pat-
ches were collected from the local collector's stores in Addis Ababa,
Ethiopia (Fig. 1) and used as sources of inoculum for isolation of ha-
lophilic microbes after enrichment.
S. Enquahone, et al.
The basal medium used for enrichment of halophilic microbes
contained (g/l): MgSO4.7H2O, 10; yeast extract, 1; K2HPO4, 0.2;
CaCl2.2H2O, 0.2; KCl, 2; and the required concentration of NaCl
(Schneegurt, 2012). Three different concentration of NaCl, viz. 10%,
20% or 25% (w/v), were used for the enrichment. In addition, 2 g of
chopped sheepskin was added per 100 ml of basal medium. The
chopped sheepskin was prepared from fresh skin collected within 2 h
after flaying. After de-hairing, the skin was cut into smaller pieces of
about 2 cm2 and cleaned of dirt and other attached substances through
repeated washing using distilled water. After sterilization by auto-
claving at 121 °C for 15 min and cooling to a temperature of 50–60 °C,
10 ml/l filter-sterilized trace mineral solution was added from a stock
solution. The stock trace mineral solution was composed of (mg/l):
H3BO4, 30; ZnSO4.7H2O, 10; Na2MoO4.2H2O, 3; MnCl2.4H2O, 3;
NiCl2.6H2O, 2; CoCl2.6H2O, 10; and CuCl2.2H2O, 1 (Atlas, 2010). Each
flask was then inoculated with a loopful of visible red patches scraped
from the red heat damaged sheepskins (Fig. 1) and incubated at 30 °C
with rotary shaking at 121 rpm for up to 15 days. Growth of micro-
organisms was measured spectrophotometrically at 600 nm and hy-
drolysis of the chopped skin was monitored visually by comparing with
a control that was not inoculated.
2.2. Isolation of pure cultures
For isolation of pure cultures on solid media, the same medium
composition as the enrichment media was used except that the chopped
sheepskin was replaced by 0.5% peptone and agar was added as a so-
lidifying agent to a final concentration of 2% (w/v). Trace mineral
solution (1 ml per 100 ml medium) and glucose (0.5%, w/v) were
sterilized separately and added to the rest of the medium after cooling.
The medium was poured into petri-dishes and left at room temperature
overnight before inoculation.
A sample from the enrichment cultures was serially diluted using
sterile NaCl solution in water (10%, w/v). A 100 μl sample was then
spread onto each agar plate and incubated at 30 °C wrapped in a plastic
bag until distinct colonies emerged. A total of 85 colonies were picked
based on their appearance, shape, or color and purified through re-
peated streaking. The purified colonies were stored at 4 °C for further
study.
2.3. Genomic DNA extraction, PCR amplification and restriction
endonuclease digestion of 16S rDNA genes
Genomic DNA was extracted from each pure isolate by following the
freeze thaw method with the modification of a rapid boiling method
designed for the isolation of bacterial plasmid DNA (Moore et al.,
1999). The integrity of the extracted genomic DNA was checked by
agarose gel (0.8%, w/v) electrophoresis at 80 V for 40 min. Gels were
stained with ethidium bromide and bands were visualized using a gel
documentation system (Gel DOC™ XR, Bio-Rad, USA) using Quantity
one software (Bio-Rad, USA). A 1 Kb+ DNA ladder (Fermentas) was
used as size marker.
For bacterial isolates, fragments of 16S rRNA gene positions
8–1542, E. coli numbering, were amplified via PCR from the genomic
DNA using primer set A8f-H1542r (Giovannoni, 1991; Øvreås et al.,
1997). For archaeal isolates fragments of 16S rRNA gene were ampli-
fied using primer set Arch21F-Arch1542r (Delong, 1992; Giovannoni,
1991). PCR amplification of the 16S RNA gene was carried out as de-
scribed previously (Misganaw et al., 2019). The quality of the PCR
product was checked by running 5 μl of each PCR reaction on a 0.9%
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International Biodeterioration & Biodegradation 150 (2020) 104940
agarose gel for 1 h at 80 V using 1 Kb+ DNA ladder (Fermentas) as
molecular marker. Enzymatic digestion of PCR products was carried out
using restriction enzyme, TaqI (5′-T^CGA-3′) (Himedia, India) according
the manufacturer's instruction. Fragments were subjected to ARDRA
(Amplified Ribosomal DNA Restriction Analysis) patterns to determine
OTUs (Operational Taxonomic Units) before sequencing. PCR frag-
ments of one isolate from each OTU (as determined by ARDRA) were
selected for sequencing.
2.4. Purification of PCR products, sequencing and phylogenetic analysis
PCR products were purified using GenElute PCR Clean-Up Kit
(Himedia, India) according to the manufacture's instructions. Purified
PCR products were sequenced at University of Calgary by the University
Core DNA Services, Canada. Nearly full-length 16S rRNA gene was
sequenced using primer sets A8f-H1542f for the bacterial isolates and
A21f-H1542r for the archaeal isolates, from both directions.
Sequences were edited manually upon inspection of the chromato-
gram using the Bioedit (Hall, 1999) software. The sequences were then
assembled by PRABI-Doua CAP 3 software (Huang and Madan, 1999)
and further analyzed in MEGA X (Kumar et al., 2018). Sequence were
used for database search with BLAST (Basic Local Alignment Search
Tool) analysis to provide the identity of the sequence (Altschul et al.,
1990). Reference 16S rRNA sequences were retrieved from NCBI Gen-
Bank database available at http://www.ncbi.nlm.nih.gov based on the
best matching organisms found in the BLAST search.
Phylogenetic relationship of sequences to closest matches in public
database based on 16S rRNA gene sequences was constructed by using
Maximum likelihood method (Olsen et al., 1994) with MEGA X (Kumar
et al., 2018) using distances calculated with Jukes-Cantor correction
(Jukes and Cantor, 1969). The stability and reliability of the relation-
ships of lineages on the inferred trees was tested by bootstrap analysis
(Felsenstein, 1985) for 1000 replicates. Sequences with sufficient length
were included in the alignment; the sequence alignments were then
corrected manually and sequences with ambiguous alignment positions
were removed from the analysis.
2.5. Sequence deposition
The 16S rRNA gene sequences were submitted to the NCBI nu-
cleotide database with accession numbers MK256716-MK256728,
MN560066-MN560068 and MK256207.
2.6. Evaluation of the degree of hydrolysis of sheepskin by pure culture
strains
To check ability to hydrolyze skin collagen, each isolate was grown
in liquid medium having the same composition used for enrichment
(section 2.1 above). A fresh de-haired sheepskin chopped to approxi-
mately 2 cm2 was added to serve as the sole source of carbon and ni-
trogen. After autoclaving and cooling, a loopful of fresh pure culture of
each strain was added into 50 ml medium in a 250 ml Erlenmeyer flask
and incubated at 30 °C with rotary shaking at 121 rpm for up to 15
days. The degree of hydrolysis of the chopped sheepskins was mon-
itored visually and microbial growth was measured spectro-
photometrically at 600 nm.
2.7. Biochemical tests of the representative isolates
Lipase activity was tested on nutrient agar plates containing 1% (v/
S. Enquahone, et al. International Biodeterioration & Biodegradation 150 (2020) 104940

v) of Tween 80 (Sánchez-Porro et al., 2003) and supplemented with
10%, 20% and 25% (w/v) NaCl. Appearance of clear zones around the
colonies was taken as an indicator of lipase production. Casein hydro-
lysis was tested in the same way as for lipase by changing the substrate
to 1% (w/v) casein (Rohban et al., 2009). Gelatinase activity was tested
following the nutrient gelatin stab method of Cruz and Torres (2012)
with slight modification. A 1.5% gelatin in the same medium used
under section 2.1 was prepared, inoculated with each isolate, and in-
cubated for 12 days. Gelatin liquefaction was tested in an ice bath. To
check if the gelatinase is extracellular or cell-bound, cell free culture
supernatant from a 10 day culture was used for the liquefaction of 2%
gelatin after 2 h of incubation at 30 °C. Gram staining was carried out
according to a method described by Pacarynuk (2005). Catalase test
was carried out using 3% H2O2 following the method described by
Reiner (2010).
3. Results
3.1. Hydrolysis of chopped sheepskin by a mixed culture of halophiles
Inoculation of liquid medium containing chopped sheepskin with
samples taken from red coloured patches on salted sheepskin (Fig. 1)
resulted in prolific growth of halophiles and resulted in complete de-
gradation of the sheepskin. Complete degradation occurred faster in the
culture containing 10% NaCl (within 13 days) while it took 15 days and
18 days for cultures containing 20% and 25% salt, respectively. To
check if the degradation of the sheepskin is due to interaction with
medium components due to prolonged incubation, a control without
any inoculum added was prepared for all the three salt concentrations.
In all the three controls the chopped sheepskin remained intact
throughout the entire incubation period showing the hydrolysis was a
result of microbial action.
Compared with the high salt concentrations (20 and 25% salt),
growth of the mixed halophilic culture in the presence of 10% salt was
faster (picking up 24 h after inoculation) and more pronounced. In the
presence of 20% and 25% salt, growth picked up slowly (after the 3rd
day of inoculation) and reached to a maximum after about a week
(Fig. 2). This probably indicates either there are more groups of halo-
philes able to grow at the lowest salt concentration or the high salt
concentrations might retard growth rate of most organisms.
3.2. Identification and phylogenetic analysis of halophiles isolated from
salted sheepskin
Based on colony morphology, color, and other cultural character-
istics, a total of 85 distinct colonies were picked from the three different
enrichment cultures carried out using three salt concentrations. Of
these, 28 were from culture enriched with 10% salt, 27 from culture
enriched with 20% salt, and 30 were from the culture enriched with
25% salt.
Based on TaqI ARDRA profile, the 85 isolates were grouped into a
total of 17 possible OTUs. Seven of the OTUs were from the enrichment
culture carried out using 10% salt and the remaining 10 OTUs were
from the 20% and 25% salt concentration enrichments (5 OTUs from
each of the two enrichments).
3.2.1. Domain and phylum level community abundance and composition
Out of a total of 85 isolates obtained, 89% (a total of 76 isolates)
belong to the Domain Bacteria while 11% (9 isolates) belong to the
Domain Archaea (Supplementary Fig. 1a). The 76 bacterial isolates
belonged to 3 bacterial phyla: Actinobacteria, Firmicutes and Proteo-
bacteria (Supplementary Fig. 1b). Members of Firmicutes and Actino-
bacteria had a similar abundance, accounting for 45% (34 isolates) and
43% (33 isolates) of all bacterial isolates, respectively.
3.2.2. Genus level community abundance and composition
Based on the 16S rRNA gene sequences of representative isolates,
the isolates were grouped into six microbial genera (Table 1). The six
genera include Halomonas, Bacillus, Salimicrobium, Nesterenkonia, Mar-
inococcus and Halococcus. Members of the genus Nesterenkonia were the
most abundant accounting for 39% of the total (33 isolates) followed by
Bacillus accounting for 21% (18 isolates) and Marinococcus accounting
for 15% (13 isolates). Members of the genera Halomonas and Bacillus
were isolated only from the 10% salt enrichment culture while mem-
bers of Halococcus and Marinococcus were restricted to 25% salt culture
conditions. Only members of the genera Salimicrobium and Nester-
enkonia were isolated from enrichment cultures of two different salt
concentrations.
3.2.3. Phylogenetic analysis of isolates
The phylogenetic relationship of the different isolates is presented in
Fig. 3. The bacterial isolates belonging to phylum Firmicutes were di-
verse and consisted of 3 genera, namely Bacillus, Salimicrobium and
Marinococcus. The representative isolate sequences of Bacillus sp. s1081
and Bacillus sp. s1021 representing 10 and 2 isolates, respectively of

Table 1
Genus level abundance of halophilic microbial isolates.
Domain Genus Enrichment salt conc. Total abundance
(% NaCl (w/v))

10 20 25 Number %

Bacteria Halomonas 9 – – 9 11
Bacillus 18 – – 18 21
Salimicrobium 1 – 2 3 3
Nesterenkonia – 27 6 33 39
Marinococcus – – 13 13 15
Archaea Halococcus – – 9 9 11
Total number of 28 27 30 85 100
isolates
Fig. 2. Growth curve of the enrichment microbial community at different salt
concentrations.

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S. Enquahone, et al. International Biodeterioration & Biodegradation 150 (2020) 104940

Fig. 3. Phylogenic tree showing the evolutionary relation of enrichment microbial community. 16S rRNA gene sequence-based phylogenetic tree generated by using
the Maximum like hood method based on the Jukes-Cantor model (Jukes and Cantor, 1969) showing the relationships between the strains studied and close matches.
Numbers at nodes indicate percentages of occurrence in 1000 bootstrapped trees; only values greater than 50% are shown. Bar 0.05 number of substitutions per
nucleotide position. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). Archeoglobus fulgidus (vc-16) (y00275) was used as out-group. a:
Enrichment bacterial community; b: Enrichment archaeal community.

their ARDRA groups, showed > 99% 16S rRNA gene sequence simi-
larity to its closest relatives, Bacillus sp. M10 isolated from banana in
Brazil (Souza et al., 2014) and Bacillus velezensis strain AP183 isolated
from a cotton plant rhizosphere in the USA (Nasrin et al., 2015)
(Fig. 3a). Bacillus sp. s1021 and Bacillus sp. s10112 representing 2 and 1
isolates, respectively showed > 99% 16S rRNA gene sequence simi-
larity to Bacillus subtilis strain CAB 1111 isolated from a polluted river
in South Africa (Benadé et al., 2016), Bacillus amyloliquefaciens
UMAF6614, a plant biocontrol agent against fungal and bacterial pa-
thogens (Magno-Perez-Bryan et al., 2015) and Bacillus sp. RAB14R
isolated from rice (Shrestha et al., 2016).
Salimicrobium sp. s1026, which represent 3 isolates, showed > 98%
16S rRNA gene sequence similarity with Salimicrobium jeotgalis strain
MJ3 isolated from salted, fermented Korean traditional seafood, with
an optimal growth at 10% salt concentration (Choi et al., 2014) and
Salimicrobium salexigens strain 29CMI isolated from salted hides in
Spain (De la Haba et al., 2011).
Marinococcus sp. s2526 representing 13 isolates showed > 99% 16S
rRNA gene sequence similarity to Marinococcus luteus strain YIM 9109
isolated from saline soil from saline lake in China (Wang et al., 2007)
and a Marinococcus halobia strain HB68 isolated from Salicornia, a plant
growing in Tunisian hypersaline soils (Mapelli et al., 2013).
Bacterial isolates affiliated with phylum Actinobacteria belong to a
single genus, Nesterenkonia. The representative isolates Nesterenkonia
sp. s2015, Nesterenkonia sp. s2011, Nesterenkonia sp. s209 and
Nesterenkonia sp. s203 (representing 22, 2, 8 and 1 isolates each,

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respectively) showed > 98% 16S rRNA gene sequence similarity to its
closest matches, Nesterenkonia aethiopica strain DSM 17733 which was
isolated from the shore of Lake Abijata in Ethiopia (Delgado et al.,
2006) and Nesterenkonia halophila strain YIM 70179 isolated from saline
soil in China (Li et al., 2008). Nesterenkonia sp. s2525 (representing 6
isolates) is the only representative of the genus Nesterenkonia which was
isolated from enrichment culture containing 25% salt concentration.
This isolate showed > 98% similarity of 16S rRNA gene sequence with
Nesterenkonia xinjiangensis strain YIM70097 isolated from saline soils in
China (Li et al., 2004) and Nesterenkonia suensis strain Sua-BAC020 also
isolated from high salt environments (salt pans) in South Africa
(Govender et al., 2013).
The bacterial isolates belonging to the phylum Proteobacteria all
belong to one single genus, Halomonas. Isolates Halomonas sp. s106 and
Halomonas sp. s1017 (representing 8 and 1 isolates, respectively)
showed > 98.5% 16S rRNA gene sequence similarity with Halomonas
halodenitrificans strain DSM 735 (Miller et al., 1994), Halomonas sp.
CO12 isolated from marine sediments in France (Alain et al., 2012),
Halomonas halodenitrificans strain L2 isolated from salted sheepskin in
Turkey (Caglayan et al., 2016), and Halomonas alimentaria strain YKJ
isolated from jeotgal, a traditional Korean fermented seafood (Yoon
et al., 2002).
The phylogenetic relationship of the enriched archaeal isolates is
presented in Fig. 3b. The 2 representative isolates sequenced belonged
to the extremely halophilic archaeal genera Halococcus, under the
phylum Euryarchaeota. Halococcus sp. s2528 (representing 4 isolates)
showed > 99% 16S rRNA gene sequence similarity to Halococcus
dombrowskii strain H4 isolated from a Permian alpine salt deposit in
Austria (Stan-Lotter et al., 2002), Halococcus dombrowskii strain 519
isolated from salted hide in Turkey (Bilgi et al., 2012), Halococcus
morrhua (Leffers and Garrett, 1984) and Halococcus thailandensis strain
HDB5-2 isolated from fish sauce in Thailand (Namwong et al., 2007).
Halococcus sp. s2535 (representing 5 archaeal isolates) showed >
99% 16S rRNA sequence similarity with Halococcus qingdaonensis strain
CM5 isolated from a crude sea salt sample in China (Wang et al., 2007),
and Halococcus sp. CBA1201 isolated from plumage of captive fla-
mingoes in Korea (Yim et al., 2015).
3.3. Collagen hydrolysis by pure culture isolates
All isolates representing the 17 OTUs were tested for their ability to
degrade collagen of sheepskin which was provided as sole carbon and
nitrogen source for growth. In a period of about two weeks incubation,
all the 17 isolates tested dissolved the added sheepskin and showed
appreciable growth (Table 2). Isolate Halococcus sp. s 2535 showed the
fastest and more efficient hydrolysis of the sheepskin collagen (Fig. 4)
followed by isolates Nesterenkonia sp. s2011, Marinococcus sp. s2526
and isolate Halococcus sp. s2528. All the other isolates were less ef-
fective in hydrolyzing collagen. It is interesting to note that the most
efficient collagen degraders were all isolated from culture enriched
with 25% salt while most isolates obtained from the enrichment culture
containing 10% salt were less efficient in collagen degradation.

Table 2
Qualitative test for sheepskin degradation by the different halophilic OTUs.
Enrichment Salt concentration (%) OTU Representative isolate/s sequenced Accession number Pigmentation Degree of collagen degradation

10 1 Halomonas sp. s106 MK256716 Yellow **

2 Bacillus sp. s1081 MK256207 Yellow *
3 Bacillus sp. s10111 MN560068 White **
4 Bacillus sp. s10112 MK256725 White **
5 Halomonas sp. s1017 MK256717 Yellow *
6 Bacillus sp. s1021 MK256719 Yellow *
7 Salimicrobium sp. s1026 MN560066 Yellow **
20 8 Nesterenkonia sp. s203 MN560067 White **
9 Nesterenkonia sp. s209 MK256718 White *
10 Nesterenkonia sp. s2011 MK256720 White ***
11 Nesterenkonia sp. s2015 MK256721 White *
12 Nesterenkonia sp. s2020 MK256722 White **
13 Nesterenkonia sp. s2525 MK256723 White *
25 14 Marinococcus sp. s2526 MK256726 Yellow ***
15 Salimicrobium sp. s2527 MK256724 White *
16 Halococcus sp. s2528 MK256727 Pink red ***
17 Halococcus sp. s2535 MK256728 Pink red ****

Key: *: least degrader; **: moderate degrader; ***: very good degrader; ****: excellent degrader.

Fig. 4. Hydrolysis of sheepskin by selected

pure culture isolates. Each isolate was
grown in liquid medium containing
chopped sheepskin as sole carbon and ni-
trogen source. Control refers to a medium
that was incubated same way as the culture
without being inoculated. The pictures
show appearance of the culture for each
isolate after the 15th day growth. a:
Halococcus sp. s2535; b: Halococcus sp.
s2528; and c: Bacillus sp. s1026.

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Table 3 4. Discussion
Growth of isolates at different salt concentration.
Original enrichment Representatives isolate Growth in different salt Salt curing is the most widely used method of preservation of animal
salt conc. for isolation sequenced concentrations (%) skin, a valuable raw material for the leather tanning industry. At high
concentration salt inhibits or significantly retards growth of most mi-
10 20 25 croorganisms (Gudro et al., 2014). However, addition of salt creates a
10% NaCl Halomonas sp. s106 + – – favorable condition for the growth of halophilic microorganisms which
Bacillus sp. s1081 + – – causes damage to salted skins and hides, a condition known as red heat
Bacillus sp. s10111 + – – damage. Damage on salted skin and hide caused by halophilic microbes
Bacillus sp. s10112 + – – is a worldwide problem reported by different authors (Akpolat et al.,
Halomonas sp. s1017 + – –
2015; Bailey and Birbir, 1993; Birbir, 1997; Bitlisli et al., 2004;
Bacillus sp. s1021 + – –
Salimicrobium sp. s1026 + + – Caglayan et al., 2018). In this study halophiles were isolated from red
20% NaCl Nesterenkonia sp. s203 + + – heat damaged sheepskin after enrichment in liquid enrichment medium
Nesterenkonia sp. s209 + + – containing chopped sheepskin and sodium chloride at a concentration
Nesterenkonia sp. s2011 + + –
of 10%, 20%, or 25% (w/v). In the enrichment cultures microbial
Nesterenkonia sp. s2015 + + –
Nesterenkonia sp. s2020 + + – growth was observed in all the salt concentrations tested only after few
25% NaCl Nesterenkonia sp. s2525 + + + days of cultivation and the sheepskin was completely dissolved. Since
Marinococcus sp. s2526 + + + chopped sheepskin was the only nitrogen source, growth indicates
Salimicrobium sp. s2527 + + + ability of these microorganisms to degrade the skin collagen. Therefore,
Halococcus sp. s2528 + + +
the presence of halophiles capable of growing at such high salt con-
Halococcus sp. s2535 + + +
centration (10%–25% or about 1.71 M–4.27 M) and their ability to
Key: + showed growth at the salt concentration tested; - didn't grow at the salt degrade skin collagen indicates the danger these microbes could pose
concentration tested. on the quality of salted hide and skin and its impact on the leather
tanning industry in Ethiopia.
3.4. Growth of pure culture isolates at different salt concentration The 85 halophilic isolates obtained from the three enrichment cul-
ture were grouped into 17 distinct OTUs using restriction fragment
Table 3 shows growth of the 17 OTUs in media containing three analysis of amplified 16S rRNA which belong to six genera, four phyla
different salt concentrations. All the isolates, including those originally and two domains. This shows that halophiles associated with red heat
isolated from enrichment cultures containing 20% and 25% salt con- damage of salted skin belong to diverse taxonomic groups. The six
centration, were able to grow in the presence of 10% salt. On the other genera isolated in this study include Halomonas, Bacillus, Salimicrobium,
hand all isolates originally isolated from 10% salt, except for one iso- Nesterenkonia, Marinococcus and Halococcus. Of these, those belonging
late, were unable to grow at higher salt concentrations. The only ex- to the genera Nesterenkonia were the most abundant accounting for 39%
ception was Salimicrobium sp. s1026 that was able to grow in the pre- of all the isolates. The other five genera accounted between 3 and 21%
sence of 20% salt. All isolates originally isolated from a 20% salt of the isolates. Out of the six genera, five of them were previously
enrichment culture were also unable to grow in the presence of 25% isolated from salted sheepskin in different parts of the world (Akpolat
salt. et al., 2015; Caglayan et al., 2015; De la Haba et al., 2011). However,
the number of genera isolated and the relative abundance of each
genera reported in previous studies differ from the findings of this
3.5. Biochemical and morphological characteristics of the representative study. Since the salt is considered the source of halophilic micro-
isolates organisms on salted skin and hide (Birbir et al., 2002), the observed
difference in occurrence as well as abundance may reflect differences in
The results obtained from biochemical tests and morphological the microbial composition of the salt used for salting in different
analysis are listed in Supplementary Table 1. Most of the representative countries. For example, Akpolat et al. (2015) isolated 179 halophilic
isolates (13 isolates) tested were found to be gram positive and all the isolates from salted sheepskin in Spain that belong to eleven genera, six
17 isolates were catalase positive. Nine of the isolates showed gelati- of them in Domain Bacteria and the remaining five genera belonging to
nase activity. Of these three isolates, Bacillus sp. s1081, Bacillus sp. Domain Archaea. The six bacterial genera they isolated included Alka-
10112, Nesternkonia sp. s203 and Halococcus sp. s2535, produced ex- libacillus, Pseudomonas, Acinetobacter, Salimicrobium, Marinococcus, and
tracellular gelatinase (Fig. 5). None of the isolates were positive for Staphylococcus while the five archaeal genera included Halorubrum,
lipase activity while two isolates, Bacillus sp. s1111 and Nesternkonia sp. Natrinema, Halococcus, Halostagenicola and Haloterrigena. Other authors
s2020 were able to hydrolyzed casein. have also reported isolation of the genera Halomonas and Salimicrobium

Fig. 5. Gelatin liquefaction by A: Halococcus sp. s2535 whole culture; and B: culture filtrate of Halococcus sp. s2535.

7
S. Enquahone, et al.
from salted hides in Turkey and Australia (Caglayan et al., 2015; De la
Haba et al., 2011). To the best of our knowledge Nesterenkonia, the most
abundant halophile isolated in this study, is the only genus, which was
not previously reported from salted skin and hide in other countries.
The halophilic OTUs isolated in this study showed sequence simi-
larities of > 98–99% with other strains previously isolated from natu-
rally occurring saline habitats (Stan-Lotter et al., 2002; Edouard et al.,
2014; Kocur and Hodgkiss, 1973; Stan-Lotter et al., 2002; Wang et al.,
2009; Yilmaz, 2010; Yim et al., 2015). For example the two archaea
isolates, Halococcus sp. s2528 and Halococcus sp. s2535 showed > 99%
16S rRNA gene sequence similarity with Halococcus dombrowskii strains
isolated from Permian alpine salt deposit in Austria (Stan-Lotter et al.,
2002) while Marinococcus sp. s2526 showed > 99% sequence similarity
with Marinococcus luteus strain YIM9109 isolated from soil of a saline
lake in China (Wang et al., 2007). Similarly all the isolates belonging to
genus Nesterenkonia showed from > 98 to > 99 sequence similarity
with other strains of the genus isolated from a soda lake in Ethiopia
(Delgado et al., 2006) saline soil in China (Li et al., 2004, 2008) or salt
pans in South Africa (Govender et al., 2013).
In all the three enrichment cultures (10%, 20%, and 25% NaCl) the
sheepskin added as the sole nitrogen source was completely dissolved.
However, the rate of growth of the culture and the rate of degradation
of the sheepskin was faster at 10% and it gets slower with increasing
salt concentration. When pure culture strains isolated from each en-
richment culture was tested for their ability to grow at the different salt
concentrations, all the 17 OTUs were able to grow in the presence of
10% salt. None of the OTUs, except one, were able to grow at higher
salt concentration than they were isolated from. The only exception was
Salimicrobium sp. s1026 which was isolated from a 10% salt enrichment
but was able to grow in the presence of 20% salt. Therefore, in the
enrichment culture containing 10% salt, all the 17 OTUs were able to
grow while at salt concentrations of 20% only 11 OTUs grew and at
25% salt concentration only 5 OTUs were able to grow. Therefore, this
might justify the faster growth and faster hydrolysis of sheepskin in the
presence of 10% salt and vice versa for the higher salt concentrations.
Each of the 17 OTUs were also independently tested for their ability
to degrade collagen by growing them in a liquid medium containing
chopped sheepskin as the sole source of carbon and nitrogen. All of
them grew in this medium indicating that they were able to degrade the
collagen. However, differences were observed among the 17 isolates.
Thus, seven isolates were rated to have low degradation efficiency, six
isolates were rated to have good degradation efficiency, three isolates
to have very good degradation efficiency, and one isolate was rated to
have excellent collagen degradation efficiency. The isolate that showed
excellent collagen degradation efficiency was Halococcus sp. s2535 and
the three isolates with very good efficiency were Nesterenkonia sp.
s2011, Marinococcus sp. s2526, and Halococcus sp. s2528. It is inter-
esting to note that all these four isolates were isolated from enrichment
cultures containing 20% and 25% salt. As stated earlier, salt is normally
added to fresh animal hide and skin as a preservative at a concentration
of about 30–40% based on the flesh weight of the skin (Gudro et al.,
2014). The reason it is used is to inhibit or retard growth of most
spoilage microorganisms. However, addition of salt creates a favorable
condition for the growth of halophilic microorganisms, some of them
highly efficient collagen degraders, indicating the potential negative
impact this will have on the leather tanning industry.
What is interesting is that the more salt added the more favorable it
becomes for the growth of efficient collagen degraders, such as isolate
Halococcus sp. s2528, that optimally grow at a salt concentration of up
to 25%. These organisms are aerobic and optimally grow at 25–35 °C,
the ambient temperature in tropical countries. This shows that red heat
damage of skin and hide could pose serious challenges in the leather
tanning industry of tropical countries. For example, in Ethiopia, be-
cause of the abundance of skin and hide, the leather tanning industry is
8
International Biodeterioration & Biodegradation 150 (2020) 104940
a major industrial activity creating tens of thousands of jobs (Coppeaux
et al., 2016). Skin and hide are collected throughout the country by
collectors and delivered to processing sites. Because more animals are
slaughtered during festivals, availability of skin and hide is not uniform
through the seasons. Therefore, fresh skin and hide is collected from
each district, preserved using salt bought from local markets, and stored
in warehouses until delivery to processors. Because processors can only
handle a certain amount of skin and hide per day, the skin and hide
could be further stored in a factory warehouse. Similar experiences of
storage for longer periods before processing is also practiced in other
countries as well (Caglayan et al., 2018). The warehouses where skin
and hide are stored are almost always well ventilated. The presence of
high salt concentration, aeration of the warehouse, and the high am-
bient temperature could create an ideal condition for the growth of
halophiles, some of them highly efficient collagen degraders.
The fact that all these isolates degrade sheepskin in liquid media
indicates their ability to produce extracellular enzymes. Test using cell
free culture supernatant of Halococcus sp. s2535 grown on sheepskin as
sole carbon and nitrogen source showed that it hydrolyzes gelatin
within 1 h incubation indicating the presence of extracellular gelatinase
activity. Previously strains belonging to the genera Halomonas,
Salimicrobium, Marinococcus, and Halococcus that were isolated from
salted skin at different places were reported to produce hydrolytic en-
zymes such as proteases and lipases (Akpolat et al., 2015; Caglayan
et al., 2015; De la Haba et al., 2011; Stan-Lotter et al., 2002).
Because some halophiles produce a red pigment (Bailey and Birbir,
1996; Vreeland et al., 1999), appearance of red spots on the flesh sides
of salted skins and hides is used as a sign of damage by red heat.
Therefore, tanners depend on visual inspection for the growth of ha-
lophilic microbes to decide whether to accept or reject skin and hide
from suppliers for further processing. Thus presence of visible red color
on salted animal skin lead to rejection because it indicates growth of
halophilic microbes that might have caused damages to the collagen
fiber (Akpolat et al., 2015). Appearance of red color is a result of a red
pigment produced by some halophilic archaea when grown both under
natural and artificial conditions (Stan-Lotter et al., 2002; Wang et al.,
2007; Yim et al., 2015). In this study, out of the 17 OTUs tested, only
two archaeal isolates (representing 11.2% of the total) produced red
pigments. Even for these pigmented isolates, within the first one week
of growth, no detectable pigment production was observed. In general
pigment production by halophilic archaea in culture is known to start
late in the growth phase, in some cases commencing after two weeks
(Akpolat et al., 2015; Anderson, 1954). The remaining 88.2% of the
OUTs tested appeared yellow or white. This shows that detection of red
heat damage solely based on the appearance of red pigment may not be
a reliable method to decide whether a given salted skin or hide should
be further processed. On the other hand, all the 15 OTUs that did not
produce a red pigment were able to degrade collagen, including two
that were rated as very efficient collagen degraders. Anderson (1954)
also reported both pigmented and non-pigmented halophiles from
salted hide and skin all of them capable of degrading collagen. In the
absence of visible red pigment on salted skin tanners could continue the
tanning process only to discover that the skin or hide is already da-
maged after they reach the final production stage. This, in addition to
adding cost, could lead to the generation of waste for a product that will
have no practical use.
5. Conclusions
Halophilic microorganisms belonging to both Domain Bacteria and
Archaea were isolated from salted sheepskin. All isolates were capable
of degrading sheepskin showing that these microbes can damage the
collagen fiber of salted skin and hide and thus affect the quality of
leather products.
S. Enquahone, et al. International Biodeterioration & Biodegradation 150 (2020) 104940

Declaration of competing interest 11–22.

Cruz, T., Torres, J.M., 2012. Gelatin Hydrolysis Test Protocol. Microbial Library,
American Society for Microbiology, Washington.
The authors declare that they have no known competing financial De la Haba, R.R., Yilmaz, P., Sánchez-Porro, C., Birbir, M., Ventosa, A., 2011.
interests or personal relationships that could have appeared to influ- Salimicrobium salexigens sp. nov., a moderately halophilic bacterium from salted
hides. Syst. Appl. Microbiol. 34, 435–439.
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2006. Nesterenkonia aethiopica sp. nov., an alkaliphilic, moderate halophile isolated
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The study was supported by The Ethiopian Biotechnology Institute Edouard, S., Sankar, S., Dangui, N.P.M., Lagier, J.-C., Michelle, C., Raoult, D., Fournier,
(EBTi) and Institute of Biotechnology (IoB), Addis Ababa University and P.-E., 2014. Genome sequence and description of Nesterenkonia massiliensis sp. nov.
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