Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Kalmi Shak or water spinach (Ipomoea aquatica) is a Bangladeshi indigenous green leafy vegetable
and herbaceous aquatic or semi aquatic perennial plant. A primary study was conducted to elucidate the
multi functionalities of this vegetable. Extract of Kalmi Shak exhibited high antioxidant properties with
hydrophilic-oxygen radical absorbance capacity (H-ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scav-
enging activity being 341.92 ± 1.32 and 37.67 ± 2.63 µmol Trolox equivalent / gram of dry weight (TE/g
DW), respectively. The total polyphenols content was estimated to be 12.56 ± 0.08 mg gallic acid equiva-
lent / gram of dry weight (mg GAE/g DW), and moisture content was found to be 85%. The extract also
showed anti-mutagenic effect on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98, and anti-
tumor activity to mouse myeloma cell line P388. The extract of this vegetable also exhibited anti-bacterial
activities against several spoilage and pathogenic bacteria. The multi functionalities, economic price and
availability during the entire year have made this indigenous Bangladeshi vegetable important from both
medicinal and industrial aspects.
InTRoduCTIon
Leafy vegetables have been extensively investi-
gated as new sources of natural antioxidants as well
as other bioactive compounds of human health ben-
efits (Lakshmi and Vimala, 2000). Epidemiological
studies have shown that consumption of vegetables
is associated with reduced risk of chronic diseases. It
has been reported that leafy vegetable extracts could
Received: September 7, 2010, Accepted: October 21, 2010. Released Online Advance Publication: March 25, 2011. Correspondence: Hossain Uddin Shekhar, [email protected],Tel: - + 81-298-38-8055, Fax: +81-298-38-7996
be used to reduce blood sugar level (Villansennor
et al., 1998) and as an antibiotic against Escherichia
coli, Pseudomonas aeruginosa, Bacillus subtilis and
other microorganisms (Bhakta et al., 2009). Increased
consumption of vegetables containing high levels of
phytochemicals has been recommended to prevent
chronic diseases related to oxidative stress in the
human body (Chu et al., 2002). Natural antioxidants
increase the antioxidant capacity of the plasma and
reduce the risk of certain diseases such as cancer,
heart diseases and stroke (Prior and Cao, 2000). The
secondary metabolites including phenolics and
Multi food functionalities of Kalmi Shak (Ipomoea aquatica) grown in Bangladesh
Hossain Uddin Shekhar1, Masao Goto1, Jun Watanabe1, Ichiho Konishide-Mikami1, Md. Latiful Bari2 and Yuko Takano-Ishikawa1
1National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan2Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh
Keywords: Kalmi Shak, water spinach, antioxidant, anti-mutagenic activity, anti-tumor activity, anti-bacterial activity
Agric. Food Anal. Bacteriol. 1: xx-xx, 2011
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
flavonoids from plants have been reported to be
potent free radical scavengers (Chiang et al., 2004).
They are found in all parts of plants such as leaves,
fruits, seeds, roots and bark (Mathew and Abraham,
2006). There are many synthetic antioxidants in use,
however, it is reported that they have several side ef-
fects, such as risk of liver damage and carcinogen-
esis in laboratory animals (Gao et al., 1999; Ito et al.,
1983; Osawa and Namiki, 1981). Therefore, a search
for natural antioxidants from plant may help to find
safer, more potent, less toxic and cost effective an-
tioxidants.
Kalmi Shak, a semi-aquatic plant water spinach (Ip-
omoea aquatica) belongs to Convolvulaceae family,
not only grows wild but is also cultivated throughout
Southeast Asia, and is one of the widely consumed
vegetable in the region (Huang et al., 2002). It is a ten-
der, trailing or floating perennial aquatic plant, found
in most soils along the margins of fresh water, ditch-
es, marshes and wet rice field. It is usually found year
round and treated as a leafy vegetable unlike other
common vegetables in Bangladesh which are mostly
seasonal. Kalmi Shak represents one of the richest
sources of carotenoids and chlorophylls (Chen and
Chen, 1992). The leaves contain adequate quantities
of most of the essential amino acids in accordance with
the WHO recommendation pattern for an ideal dietary
protein (Prasad et al., 2008). Consequently, when com-
pared with conventional food crops such as soybeans
or whole egg, it has potential for utilization as a food
supplement. Ayurveda, a system of traditional medi-
cine native to the Indian subcontinent, has identified
many medicinal properties of Kalmi Shak, and it is
effectively used against nosebleeds and high blood
pressure (Perry, 1980). However, very limited scientific
studies have been conducted on its functional aspects.
Most of the studies have focused on the inhibition of
prostaglandin synthesis (Tseng et al., 1992), effects on
liver diseases (Badruzzaman and Husain, 1992), con-
stipation (Samuelsson et al., 1992) and hypoglycemic
effects (Malalavidhane et al., 2003). There have been
no reports on the systematic study of the indigenous
Kalmi Shak of Bangladesh to evaluate its potentiality
as a functional food or food supplement. The objective
of this study was to investigate the antioxidant activity,
total phenolic content, anti-tumor, anti-mutagenic, and
antimicrobial properties of the extracts of indigenous
fresh green Kalmi Shak.
MATeRIAlS And MeThodS
Materials
RPMI-1640, penicillin-streptomycin solution (Hy-
bri-Max®), Dulbecco’s phosphate buffered saline
(PBS), DPPH, and 0.4% Trypan Blue solution, 6-hy-
droxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid
(Trolox), and fluorescein sodium salt (FL) were pur-
chased from Sigma Chemical Co. (St. Louis, MO,
USA). Fetal calf serum (FCS) and Folin-Ciocalteau
(F-C) reagent were purchased from JRH Biosciences
(Lenexa, KS, USA) and MP Biomedical, LLC (Illkirch,
France), respectively. S9-mix (rat liver homogenate
containing rat liver microsome S9 fraction) was ob-
tained from Kikkoman Co. Ltd. (Tokyo, Japan). Cell
proliferation reagent WST-1 was purchased from
Takara Bio Inc. (Siga, Japan). Methanol, acetone,
gallic acid, 3-amino-1-methyl-5H-pyrido[4,3-b]in-
dole (Trp-P2), 2,2’-azobis(2-amidinopropane) di-
hydrochloride (AAPH) and dimethyl sulfoxide
(DMSO) and all other chemicals were purchased
from Wako Pure Chemical Co. (Osaka, Japan).
Plant material and sample preparation
Fresh Kalmi Shak was collected within 24 hour of
harvest from the Dhaka (Dhaka is the capital of Ban-
gladesh and one of the major cities of south Asia)
new market during the summer period (mid April to
June onwards). One hundred grams of green leaves
and veins were cleaned with water, and finally freeze-
dried and kept at -20°C until use. One gram of the
freeze-dried sample was sequentially extracted with
hexane: dichloromethane (1:1) (v/v) and with metha-
nol: water: acetic acid (MWA) solvent at the ratio of
90:9.5:0.5 (v/v/v) using an automatic accelerated sol-
vent extraction system (ASE 350; Dionex, Sunnyvale,
CA, USA). Lipohilic fraction was collected (3 times) by
hexane: dichloromethane at 70°C, 5 min stand at 1500
psi. Hydrophilic fraction was collected thrice by MWA
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
solvent at 80°C, 5 minute stand at 1500 psi. The result-
ing MWA extract of the Kalmi Shak which was used
for subsequent experiments was filled up to 50 ml by
MWA. For cell culture and microbiological analyses,
MWA fraction was dried in vacuo and dissolved in
DMSO.
Determination of hydrophilic-oxygen radical absorbance capacity (H-ORAC)
H-ORAC assay was performed according to the
method described by Cao et al. (1993), and Prior
et al. (2003) with slight modifications. In brief, MWA
extracts or Trolox standard solution diluted with 75
mmol/L potassium phosphate buffer (pH 7.4) were
added to a 96-well microplate (#3072, Becton Dickin-
son, NJ, USA). Following the addition of 115 µl of 111
nmol/ LFL to the wells, the plates were incubated at
37°C for 10 min. After the addition of 50 µl of 31.7
mmol/l AAPH to the wells, fluorescence intensities
were measured every two min. for 90 min. by a mi-
croplate reader (Powerscan HT; DS Pharma Biomedi-
cal, Osaka, Japan) with excitation wavelength of 485
nm and emission wave length of 530 nm. H-ORAC
values were expressed as micromole Trolox equiva-
lent per gram of dry sample weight (µmol TE/g DW).
All measurements were done in triplicate.
Measurement of total polyphenols content
Total polyphenols content was measured by the
Folin-Ciocalteu assay according to Sun et al. (2005)
and Velioglu et al. (1998) with slight modifications.
Briefly, three volumes of F-C reagent was diluted by
five volume of water before use. Reaction mixture
containing 80 µl of samples or gallic acid standard
(diluted with MWA) and 56 µl of diluted F-C reagent
was placed in 96 well-microplate (Sumilon, Sumito-
mobakelite, Tokyo, Japan), and incubated for five
min at room temperature. After the addition of 120
µl of 2% (w/v) sodium carbonate, the plate was al-
lowed to stand for 15 min at room temperature. Ab-
sorbance at 750 nm was measured by a microplate
reader (Powerscan HT; DS Pharma Biomedical). To-
tal polyphenols content was expressed as milligram
gallic acid equivalent per gram of dry sample weight
(mg GAE/g DW). All measurements were conducted
in triplicate.
DPPH radical scavenging activity (DPPH-RSA)
DPPH-RSA of MWA extract was examined ac-
cording to the method of Oki et al. (2001) with slight
modifications. Briefly, the same volume of 10% meth-
anol and MWA extract were mixed, and the mixture
was further diluted with 50% methanol. A 50 µl of
diluted MWA extract and 50 µl of 0.2 M morpho-
linoethanesulfonic acid (MES) buffer (pH 6.0) were
subsequently placed in a 96-well microplate (Sum-
ilon, Sumitomobakelite). The reaction was initiated
by adding 50 µl of 800 µM DPPH in ethanol. After
incubation for 20 min. at room temperature, the ab-
sorbance at 520 nm was measured using a micro-
plate reader (Powerscan HT; DS Pharma Biomedical).
DPPH radical scavenging activity was expressed as
micromole Trolox equivalent per gram of dry sample
weight (µmol TE/g DW). All the determinations were
conducted in triplicate.
Determination of moisture content
The moisture content was determined by dry-
ing the samples in a drying oven at 105°C for 24 h
(AOAC, 1984). The leaf and vein (edible portion) of
fresh Kalmi Shak (5.0 g) were cut by dual razor blades
into small pieces, subsequently placed in aluminum
cups and weighed before and after drying. The per-
centages of moisture content were calculated by
subtracting the two values. At least 10 samples per
experiment were replicated, and mean values for
each replicate were calculated.
Determination of the anti-mutagenic effect on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98
The assay was carried out according to the modi-
fied Ames test (Ames et al., 1975) with Salmonella
Typhimurium TA98. In brief, TA98 strain was cultured
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
aerobically in Nutrient broth no. 2 (Oxoid Ltd., Bas-
ingstoke, UK) at 37°C for 12-14h. Trp-P2 was dis-
solved in DMSO to give a working concentration
of 100 ng/ml. The reaction mixture consisted of 0.7
ml of 0.1 mol/l phosphate buffer (pH 7.0), 50 µl of
sample (40 µg/ml in DMSO), 100 µl of S-9 mix, 50 µl
of Trp-P2 and 100 µl of S. Typhimurium TA98. The
positive control contained the same concentration
of perilla leaves extract in DMSO instead of the
sample. Following incubation at 37°C for 20 min in
water bath shaker, two milliliters of soft agar contain-
ing histidine and biotin was added, and the mixture
was immediately plated on a minimal glucose agar.
After incubation at 37°C for two days, the number
of developed revertants was scored. The experi-
ment was performed in triplicate and the mean val-
ues are presented. Anti-mutagenic activities of the
Kalmi Shak extract were calculated according to the
equation described by Hosoda et al. (1992).
Anti-tumor effects to mouse myeloma P388 cells
Anti-tumor activities were measured by the vi-
abilities of myeloma P388 cells using WST-1 cell pro-
liferation reagent (Shinmoto et al., 2001). In brief,
P388 cells (Japan Health Sciences Foundations,
Osaka, Japan) were seeded in 96-well culture plates
(#353072, Falcon) at a density of 5,000 cells (100 µl)
per well in RPMI-1640 medium supplemented with
10% heat-inactivated fetal calf serum (FCS) and 100
units/ml penicillin and 100 µg/ml streptomycin and
incubated at 37°C in a humidified atmosphere with
5% CO2. DMSO solutions of Kalmi Shak with vari-
ous concentrations were added to each well (final
concentrations of 0 (negative control), 50, 100 and
200 µg/ml). Final concentration of DMSO was 0.4%.
Rosemary (Rosmarinus officinalis) extract at varying
concentrations (50 to 200 µg/ml) in DMSO were used
for positive control. After 48 hours incubation, 10 µl
of premixed WST-1 cell proliferation assay reagent
was added to each well. Two hours after the addition
of WST-1, the degree of cell viability was measured
by the absorbance at 450-650 nm of the cell culture
media using microplate reader (Thermomax, Molecu-
lar Devices Co., Tokyo, Japan). Results were reported
as percentage of the inhibition of cell viability, where
the optical density measured from DMSO-treated
control cells was considered to be 100% of viability.
Percentage of inhibition of cell viability was calcu-
lated as follows:
Test organisms
Fifteen strains/species of frequently reported
food borne pathogens or food spoilage bacteria
were used in the study (Table 2). The stock cultures
of the test organisms in 20% glycerol (Sigma) con-
taining medium in cryogenic vials were maintained
at -84°C. Working cultures were kept at 4°C on Tryp-
to Soy Agar (TSB) slants (Nissui Chemical Co. Ltd,
Tokyo, Japan) and were periodically transferred to
fresh slants.
Anti-microbial sensitivity testing
The anti-microbial activity of the Kalmi Shak ex-
tracts was done according to the method of Bauer
et al. (1966). The 8 mm in diameter discs (Toyo Roshi
Kaisha, Ltd. Tokyo, Japan) were impregnated with 50
µl of different concentration of Kalmi Shak extract
before being placed on the inoculated agar plates.
The inocula of the test organisms were prepared by
transferring a loopful of respective bacterial culture
into 9 ml of sterile TSB medium and incubated at
37°C for 5 to 6 h. The bacterial culture was compared
with McFarland (Jorgensen et al., 1999) turbidity
standard (108 CFU/ml) and streaked evenly in three
planes maintaining a 60° angle onto the surface of
the Mueller Hinton agar plate (5 x 40 mm) with ster-
ile cotton swab. Surplus suspensions were removed
from the swabs by rotation against the side of the
tube before the plate was inoculated. After the inoc-
ula dried, the impregnated discs were placed on the
agar using an ethanol dipped and flamed forceps
and were gently pressed down to ensure contact.
Plates were kept at refrigeration temperature (4°C)
1-Aexp group
Acontrolx 100( )
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
for 30 to 60 min for better absorption, during which
microorganisms should not grow but absorption of
extracts should take place. Negative controls were
prepared using the same solvent without the plant
extract. Reference antibiotics (streptomycin, genta-
mycin, and rifampicin) were used as positive control.
The inoculated plates containing the impregnated
discs were incubated in an upright position at 37°C
for overnight and/or 24 to 48 h (depending on the
appearance of colonies). The results were expressed
as positive/negative depending on the zone of inhibition.
Statistical analysis
Statistical analysis was performed using Microsoft
Excel (2007). The data were expressed as means ±
standard deviation (SD) for foods having sample
numbers greater than 2.
ReSulTS And dISCuSSIon
It was observed that Kalmi Shak possessed 341.92
± 1.32 µmol TE/g DW of H-ORAC value (Table 1).
From the moisture content, the H-ORAC value in
fresh weight basis can be calculated as 51.28 µmol
TE/g fresh weight (FW). Wu et al. (2004) reported
that H-ORAC values of common vegetables in USA
were between 0.87 (cucumber) and 145.39 (small
red beans) µmol TE/g FW. The most values were in
a range from 5 to 20 µmol TE/g FW. It is suggested
that H-ORAC value of water spinach is relatively high
when compared with those of common vegetables
and fruits.
Mikami et al. (2009) studied antioxidant activi-
ties of 11 crops from Ibaraki prefecture, Japan, and
found that DPPH-RSA ranged from 0.38 (melon) to
91.0 (ginger) µmol TE/g FW. Pellegrini et al. (2003)
studied 34 vegetables and found that spinach ex-
hibited the highest antioxidant capacity (8.49 µmol
TE/g FW). The DPPH-RSA of water spinach in our
study (Table 1) was nearly equal to that of spinach,
though the methodologies of determination were
slightly different.
It has been reported that the total polyphe-
nols contents of 10 vegetables examined by Cieslik
et al. (2006) were between 0.59 to 2.90 mg GAE/g
FW of samples. Wu et al. (2004) observed that total
polyphenols of 23 vegetables were between 0.24 ±
0.05 (cucumber) and 12.47 (red kidney beans). Water
spinach is available in Bangladesh during the entire
year and our collection period originated from early
onset of summer. It has been reported that leaves
harvested in the spring exhibited much higher levels
of total polyphenols content and ORAC value than
the leaves harvested in the fall (Howard et al., 2002).
Consequently, further study should be undertaken
to see the seasonal variation of antioxidant content
of this leafy vegetables.
MWA extract of Kalmi Shak exhibited anti-muta-
genic effects on Trp-P2 induced mutagenicity to S.
Typhimurium TA98 when tested with perilla as posi-
tive control (Fig. 1). Kanazawa et al. (1995) reported
0
10
20
30
40
50
60
anti
-mut
agen
ic a
ctiv
ity
(%)
Perilla Kalmi shak
Table 1. Anti-oxidative activity, total polyphenols content and moisture content of indigenous Kalmi Shak in Bangladesh
acµmol Trolox equivalent (TE)/g DW ± SD
bdµmol Trolox equivalent (TE)/g FW
emg galic acid equivalent (GAE)/g DW ± SD
fmg galic acid equivalent(GAE)/g FW g
Percentage
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
that flavonoids were very strong anti-mutagens
against Trp-P2. In our study, anti-mutagenic activi-
ties against Trp-P2 were observed to be 52.62% and
50.79% for perilla and Kalmi Shak, respectively (Fig.
1). However, other established mutagens such as
MNNG, AF-2, AB1 etc. were not tested. Therefore,
it is necessary to check the anti-mutagenic activities
of Kalmi Shak extract on the mutagenicity of these
agents in future studies.
MWA extract of Kalmi Shak yielded detectable
anti-tumor activity in the mouse myeloma P388 cell
line. Rosemary extract was used as a positive control
in this experiment, since rosemary leaves exhibit po-
tent anti-tumor and anti-inflammation effects (Peng
et al., 2007). Dose-dependent increase on the anti-
tumor activity was observed in both Kalmi Shak and
rosemary extract (Fig. 2). At a concentration of 50
µg/ml, the corresponding cell viability of rosemary
and Kalmi Shak was 61.36% and 67.56 %, respective-
ly (Fig. 2). At a concentration of 200 µg/ml, cell viabil-
ity of rosemary was 1.66%. On the other hand, that of
Kalmi Shak was 47.59%. Since Kalmi Shak inhibited
the cell viability by more than 50% cell at this con-
centration, and almost 100% cells were not viable in
the case of rosemary. We reported, here, that Kalmi
Shak extract is capable of working against P388 cell
viability.
MWA extract of Kalmi Shak exhibited anti-microbi-
al activities against several spoilage and food borne
pathogenic bacteria within tested fifteen selected
bacteria. The result is presented in Table 2. The extract
of Kalmi Shak exhibited in vitro anti-microbial activi-
ties against spoilage bacteria P. aeroginosa, P. putida,
and pathogenic bacteria such as E. coli O157:H7 and
C. freundii. The result of this study also suggests that
Kalmi Shak extracts include compounds possessing
anti-microbial properties that may be useful to con-
trol food borne pathogens and spoilage organisms.
Further studies need to be done with other food
borne pathogens and spoilage organisms to see the
anti-microbial activities of Kalmi Shak. It would also
be of interest to apply this extract to actual food to
assess the microbiological condition of the particular
food or food products with an extended shelf life.
Figure 2. Anti-tumor activity on mouse myeloma P388 cells
Figure 1. Anti-mutagenic activity on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98
Concentration µg/ml
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
ConCluSIon
In conclusion, the results from in vitro experiments,
including H-ORAC, DPPH-RSA, total polyphenols con-
tent, anti-mutagenic activity, anti-tumor activity, and
anti-bacterial activity demonstrated that Bangladeshi
water spinach variety possessed potent anti-oxidative
and anti-tumor activities. Hence water spinach can be
used as an easy accessible source of natural antioxi-
dants, as a food supplement or in the pharmaceutical
or medical industries. Further work should be per-
formed to isolate and identify the anti-oxidative, anti-
mutagenic, anti-cancer, and anti-bacterial components
of this indigenous vegetable of Bangladesh.
Test Organisms Origin Anti-microbial activity
Spoilage bacteria
Lactobacillus planterum (ATCC 8014) Mexican style cheese -
Perdicoccus pentosaceus(JCM 5890) Dried American beer yeast -
Lactoccus lactis (IFO 12007) Unknown -
Salmonella Enteritidis (SE1) Chicken feces -
Pseudomonas aeroginosa (PA 01) Unknown +
Enterobacter faecalis (NFRI 010618-8) Unknown -
Klebsilla pneumonia (JCM 1662) Trevisan 1887 -
Bacillus subtilis (IFO 13719) Wound -
Pseudomonas putida (KT 2440) Unknown +
Pathogenic bacteria
Escherichia coli (NFRI 080618-8) Celery +
Escherichia coli O157:H7 (CR 3) Bovine feces +
Escherichia coli O157:H7 (MY 29) Bovine feces +
Citrobacter freundi (JCM 1657) Werkman and Gillen 1932 +
Bacillus cereus (IFO 3457) Unknown -
Alcaligenes faecalis (IFO 12669) Unknown -
Table 2. Test organisms used, their source and antibacterial activity of DMSO suspended MWA extract of Kalmi Shak against selected food borne pathogens and spoilage bacteria
+ , - : indicates positive and no positive activity found in preliminary screening, respectively
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
ACknowledgeMenT
This research work was supported by Kirin Holdings
Co., Ltd. (former Kirin Brewery Co., Ltd.) Tokyo, dur-
ing UNU-Kirin fellowship at National Food Research
Institute, Tsukuba, Japan in 2010-11, and its Follow-up
Project in 2011-2013. Authors expressed their sincere
gratitude to the authorities of the NFRI for providing
laboratory facilities and logistic supports to carry out
this investigation.
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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Hydrogen—limited continuous culture was used to isolate autotrophic acetogenic bacteria from rumen
contents of cattle on either a high roughage or a high concentrate diet. Twenty bacterial isolates were ob-
tained and were presumptively identified as acetogenic bacteria. They were able to use H2:CO2 and they
produced acetic acid as their sole end—product. Two isolates were selected for further studies based upon
their low hydrogen threshold values. The acetogenic strain H3HH was a strictly anaerobic gram positive
coccus with a hydrogen threshold of 1390 ppm. The acetogenic strain Al0 was a facultatively anaerobic
gram positive coccus with a hydrogen threshold of 209 ppm. The use of H2 limited continuous culture to
isolate low H2 threshold ruminal acetogens suggests that not only do acetogens with these properties exist
in the rumen but this approach could be used in other ecosystems as well.
Keywords: Methane, greenhouse gasses, methanogens, acetogens, ruminants, rumen, hydrogen,
continuous culture, carbon dioxide
InTRoduCTIon
Ruminants are characterized by having a four com-
partment stomach (Russell and Rychlik, 2001). The
largest compartment, the rumen, has a volume of
nearly 80 liters and is located before the gastric com-
partment (Weimer et al., 2009). The rumen ecosys-
tem is essentially isothermal, there is a constant flux
of feed and H2O and the fermentation of substrates
Received: September 12, 2010, Accepted: November 17, 2010. Released Online Advance Publication: April, 2011. Correspondence: John Patterson, [email protected]: +1 -765-494-4826 Fax: +1-765-494-9347
results in the production of a large amount of acids
(Weimer et al., 2009). Functionally important rumen
microorganisms representing a varied and mixed
population of bacteria, archaea, protozoa, and fungi
hydrolyze complex and soluble feedstuffs primar-
ily to sugars and other hydrolysis products such as
ammonia (Ricke et al., 1996; Stevenson and Weimer,
2007; Uyeno et al., 2007). Glucose is subsequently
fermented in the rumen by rumen microorganisms
to short chain volatile fatty acids (VFA) with the end
products of fermentation including acetate, H2, CO2,
and reduced fermentation products (lactate, butyr-
Using Hydrogen- Limited Anaerobic Continuous Culture to Isolate Low Hydro-gen Threshold Ruminal Acetogenic Bacteria
P. Boccazzi¹,² and J. A. Patterson²
¹ Current address: Massachusetts Institute of Technology, Department of Biology and Health Sciences and Technology, 77 Massachusetts Ave. Room 68-370, Cambridge, MA 02139
² Department of Animal Sciences, Purdue University, 1026 Poultry Building, Room 115, West Lafayette, IN 47907-1026
Agric. Food Anal. Bacteriol. 1: XX-XX, 2011
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
ate, propionate, ethanol) along with microbial cells
(Stevenson and Weimer, 2007; Weimer et al., 2009).
Hydrogen and formate are produced by many mi-
croorganisms in the rumen; however, methanogens
are also present in the rumen and convert H2, and
CO2 to CH4 (Wright et al., 2006). Methanogenesis
represents the primary H2 consumer in the rumen
and energy captured as methane escapes the rumen
via eructation (Boadi et al., 2004, Martin et al., 2010).
Energy lost as methane represents a 2 to 7% loss in
gross energy intake energy of the animal (Branine
and Johnson, 1990) and a loss of 10 to 15% of the
apparently digestible feed energy to the host animal
(Blaxter and Clapperton, 1965). However, direct inhi-
bition of rumen methane production also results in
energy loss in the form of eructated H2 and reduced
microbial protein (Chalupa, 1980).
Chemo-lithoautotrophic acetogens are bacteria
that utilize CO2 as their sole source of carbon and
reduce it to acetate with H2 as the source of energy
(Drake et al., 2008; Ragsdale, 2008). Acetogens are
known to be present in the rumen but they are less
numerous and considered to be less efficient than
methanogens for utilization of hydrogen as a sub-
strate (Martin et al., 2010). Replacement of metha-
nogenesis with acetogenesis could decrease energy
losses and increase the efficiency of ruminant pro-
duction. Consequently, research on acetogenesis in
ruminant animals has been focused toward two re-
lated areas of interest and application. First of all,
since methane formed as a result of ruminal fermen-
tation is subsequently eructated and is lost to the
animal; thus, it would increase energetic efficiency
of the host animal if this loss of feed energy and car-
bon could be minimized (Boadi et al., 2004; Martin
et al., 2010). Secondly, there is increasing interest in
global warming forced by the production of green-
house gasses such as CO2, CH4, and NO2 (Boadi et
al., 2004; Morrison, 2009). Reductive acetogenesis
is a means for developing alternative H2 sinks away
from methanogens that produce CH4 (Joblin, 1999).
Acetogenesis may provide an important model to
find solutions for limiting CH4 emissions from live-
stock and livestock wastes (Morrison, 2009). Efforts
to enhance in vivo acetogenesis in the rumen have
not been as successful as in vitro studies (Fonty et
al., 2007). Methanogens are thought to outcompete
acetogens because methanogens have a lower hy-
drogen threshold (Martin et al., 2010); however, most
acetogens have been isolated in batch culture in the
presence of high hydrogen concentrations and have
not been selected for low hydrogen thresholds. A
key may be a better understanding of hydrogen use
by acetogens. The objective of this study was to use
H2-limited continuous culture to demonstrate that it
could be used to isolate ruminal acetogenic bacte-
ria able to grow on low threshold concentrations of
H2 utilizing CO2 as their sole carbon source.
MATeRIAlS And MeThodS
Source of Organisms
Acetogenic bacterial strains were isolated ei-
ther from rumen contents collected either from
a ruminally fistulated Angus steer fed a diet of al-
falfa and orchard grass hay at maintenance or of a
ruminally fistulated lactating Holstein Friesian dairy
cow consuming a 60:40 percent hay and corn silage:
corn grain diet at 2.6% of her body weight. Rumen
contents were used to inoculate H2-limiting continu-
ous cultures. Individual strains were isolated after at
least 8 turnovers of the continuous culture.
Media and Growth Conditions
All media were prepared by the anaerobic tech-
niques of (Hungate, 1966) as modified by (Balch and
Wolfe, 1976; Bryant, 1972). The basal semidefined
acetogen medium used for growth and nutritional
studies and the methanogen medium are listed in
Table 1. The medium was boiled under a stream of
oxygen-free CO2, sealed, and autoclaved (120°C, 18
Ib/in², 15 min). The pH of the medium was adjusted
to 6.8 with NaOH before boiling. The cooled me-
dium was transferred into an anaerobic glove box
(Coy Laboratories, Ann Arbor, MI) containing 95%
CO2: 5% H2. For all media, the reducing agents, car-
bonate buffer and vitamins were added separately
to the medium in the anaerobic glove box as sterile
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
anaerobic solutions. The medium was subsequently
dispensed into 120 ml serum bottles or into 20 ml se-
rum tubes which were then closed with sterile black
butyul rubber serum stoppers and aluminum crimp
closures (BellCo Inc., Vineland, NJ). Solid medium
for isolation of pure cultures consisted of acetogen
medium with the addition of 2% (w/v) agar (Difco).
Continuous culture medium was the same as the
acetogen medium, but the rumen fluid was previ-
ously incubated at 37°C for 6 days to remove carbo-
hydrates (Greening and Leedle, 1989). For chemo-
lithoautotrophic growth of bacterial cultures, serum
bottles (120 ml) and Erlenmeyer flasks (330 ml) were
flushed for 30 seconds and then pressurized at 2.0
atm with either a H2:CO2 (80:20) or a N2:CO2 (75:25)
gas phase, by insertion of sterile disposable needles
through the black butyl stoppers. In all growth and
nutritional studies cultures were incubated at 39°C.
Isolation Procedures
Hydrogen-limited continuous cultures were uti-
lized in an attempt to isolate acetogenic bacteria
with low H2 thresholds from the bovine rumen. The
isolation medium contained 5 mM 2-bromoeth-
anesulfonic acid (BES) (LeVan et al., 1998) to inhibit
methanogens. The growth vessel (200 ml) was initially
half filled with isolation medium, and BES was added
to give an initial concentration of 40 mM for the full
volume of the growth vessel. The inoculum, 40 ml
of rumen fluid, was collected from either the steer
or the lactating dairy cow prior to morning feeding
and strained through a bilayer of cheesecloth under
a stream of CO2 and was added to the growth vessel.
The medium pump was started immediately after in-
oculation and the medium contained 5 mM BES.
The reservoir and growth vessel of the continu-
ous culture were flushed with a stream of humidified
oxygen-free 100% CO2 gas through a glass diffusion
stone. Although the flow rate was not measured,
humidified oxygen-free 100% H2 gas was bubbled
into the growth vessel at a rate to provide 5 to 10
bubbles/ minute. The dilution rate of the continuous
culture was 0.06 h-1 during isolation of acetogenic
bacteria from the steer and 0.28 h-1 during isolation
of acetogenic bacteria from the lactating dairy cow.
The growth vessels were incubated at 39°C. After
8 fluid volume turnovers, 1 ml of fermentation fluid
from the growth vessel was serially diluted in anaero-
bic dilution solution. Each dilution was plated in trip-
licate on solid acetogen medium containing 5 mM
BES. Plates were subsequently incubated anaero-
bically under 1.5 atm of H2:CO2 (80:20) for 6 days
at 39°C. Ten single colonies from each continuous
culture were selected at random and transferred
a Na2CO3 (8% w/v), Cys·HCl (2.5% w/v) and Na2S·9H2O (2.5% w/v) were added separately as sterile anaerobic solutions, to autoclaved and cooled medium.b Greening and Leedle (1989)c Balch and Wolf (1976)
Table 1. Media Composition
Components Acetogen Methanogen(g/L or ml/L) (g/L or ml/L)
K2HPO4 0.24 0.3KH2PO4 0.24 0.3(NH4)SO4 0.24 0.3NaCl 0.48 0.6MgS04·7H2O 0.1 0.13CaCl2·2H20 0.07 0.008NH4Cl 0.54 1
Na2CO3a 4 5
Cys·HCl a 0.25 0.25
Na2S·9H2O a 0.25 0.25
Yeast Extract 0.5 2Resazurin 0.001 0.001Hemin 0.0001 0.001Trypticase - 2CoM - 0.01FeSO4·7H2O - 0.2
Clarified Rumen Fluid (CRF) 50.0ml 100.0ml
Vitamin Sol.b 10.0ml 10.0mlTrace Min. Sol. 10.0ml 10.0ml
Wolf's Trace Min. Sol. 10.0ml -
KH2PO4 (200nM, pH=7)
- 50.0ml
VFA-Mc - 10.0ml
c
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
anaerobically to 10 ml of acetogen medium in dupli-
cate serum bottles. The bottles were pressurized at
2 atm of either H2:CO2 (80:20) or N2:CO2 (75:25) and
incubated on their sides in a rotatory shaker (New
Brunswick Scientific Co., Inc. Model M52, Edison,
NJ) operating at 200 rpm for 3 days at 39°C.
Volatile Fatty Acid (VFA) Assay
After incubation, the supernatant of each culture
was analyzed by gas chromatography using a Varian
3700 (Varian, Inc., Palo Alto, CA) gas chromatograph,
to determine VFA composition. Bacterial isolates
producing at least a 4 fold increase in acetate in
bottles containing H2:CO2 over that produced in
bottles containing N2:CO2 were retained for further
characterization.
H2 Threshold Assay
In order to isolate acetogenic strains with low H2
thresholds, a series of three experiments were per-
formed. The general protocol was to grow cultures
in a complex medium to increase cell number, then
adapt the cells to H2:CO2 flush excess H2:CO2, and
determine H2 thresholds using lower concentrations
of H2. Culture vessels were incubated at 39°C on their
sides in a rotatory shaker operating at 200 rpm.
Experiment 1
Triplicate cultures of each acetogenic isolate were
grown in acetogen medium containing 27.8 mM
glucose for 60 h. Serum bottles were pressurized to
1.5 atm with H2:CO2 (80:20) and the cultures were
incubated an additional 60 h. Cultures were sub-
sequently flushed and pressurized to 1.5 atm with
N2:CO2 (75:25) and incubated for 36 h to lower re-
sidual H2 concentration. Cultures were subsequently
flushed and pressurized to 1.5 atm with H2:CO2:N2
(1:24:75) and incubated for 60 h. Methanogens were
grown on methanogen medium for 120 h on H2:CO2
(80:20) at 1.5 atm, then were flushed with N2:CO2
(75:25) and incubated with H2:CO2:N2 (1:24:75) at
1.5 atm for 60 h.
Experiment 2
The format was similar to experiment 1 in incuba-
tion times and sequence of gas phases. Differences
were duplicate cultures were used and the initial me-
dium contained 0.2% (w/v) Brain Heart Infusion Broth
(BHI) instead of glucose. Cultures were incubated for
130 h under H2:CO2:N2 (1:24:75) after flushing with
N2:CO2 (75:25).
Experiment 3
The format was similar to experiment 2 where
bacterial cultures were initially grown in BHI and
then flushed with N2:CO2 (75:25), except that 10 ml
fresh acetogen medium (without glucose or BHI) was
added prior to pressurizing with H2:CO2:N2 (1:24:75).
After incubation, the head space of each culture
vessel was analyzed by gas chromatography using
a varian 3700 gas chromatograph, to determine H2
concentration. Bacterial isolates with the lowest H2
thresholds were retained for further characteriza-
tion. Selected strains were further purified on solid
acetogen medium under H2:CO2 (80:20) and stored
as broth cultures in glycerol at -4°C as described by
(Teather, 1982).
Characterization studies
Gram stain, flagella stain, optimum pH, and heat
test for spore determination were performed accord-
ing to (Holdeman et al., 1977). Optimum tempera-
ture of growth was determined by growing cultures
in acetogen medium containing 5.6 mM glucose at
the respective temperatures for 48 h with optimum
temperature being defined as that temperature that
yielded the highest OD measured at 660 nm at 48
hours. Oxygen sensitivity was tested by three meth-
ods: a) degree of growth throughout stab cultures
in acetogen medium containing 0.5% (w/v) glucose
and 0.4% (w/v) agar, in which the topmost layer was
allowed to oxidize; b) zone of growth in PYG mol-
ten agar medium (Holdeman et al, 1977); and c)
growth on non-reduced, aerobically prepared solid
acetogen medium or in aerobic acetogen broth con-
taining 0.5% (w/v) glucose. For colony formation,
isolates were plated on solid acetogen medium con-
taining 5.6 mM glucose and incubated aerobically
or anaerobically at 39° C. Nitrate reduction, cata-
lase, oxidase, esculin hydrolysis and utilization, and
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
starch-hydrolysis tests were performed according to
(Holdeman et al., 1977). GC-fatty acid methyl ester
(FAME) analysis was performed on strains H3HH and
A10 grown in acetogen medium (Table 1) by Mi-
crobe Inotech Laboratories (St. Louis, MO). Similari-
ty and distance coefficients were evaluated between
strains A10 and H3HH and known bacterial species
using the anaerobe database (Moore, ver. 3.7).
Nutritional Studies
The ability of isolates to utilize organic substrates
as energy sources was determined using acetogen
medium containing 0.5% (w/v) of the substrate tested.
Each organic substrate tested was added to the me-
dium as a sterile anaerobic stock solution. Substrate
utilization was assessed by an increase in OD, 660 nm,
after 36 h of growth at 39°C. Determination of cell dry
mass was performed directly on cells washed in saline
solution (NaCl, 0.1% w/v) and harvested from distilled
water. For molar growth yields, cell net dry weight
of 500 ml cultures were compared with the amount
of substrate consumed. Glucose concentration was
measured enzymatically using glucose oxidase re-
agents from Sigma Chemical Co. (St. Louis, MO).
The requirement of isolates for rumen fluid and
yeast extract was determined using Erlenmeyer flasks
(300 ml, BellCo Inc., Vineland, NJ) modified by addi-
tion of a side arm (130 mm x 16 mm) and a serum bot-
tle (20 mm) closure at the top. The bottles were filled
with 20 ml of acetogen medium and then were pres-
surized to 2 atm with either H2:CO2 (80:20) or N2:CO2
(75:25). The inoculum was 0.2 ml (1%, w/v) of a third
transfer of a culture grown under H2:CO2 (80:20). The
flasks were incubated upright at 39°C and agitated
at 200 rpm on a rotatory shaker. The growth of each
organism was followed by measuring the increase in
optical density at 660 nm with time.
Growth Studies
For the assessment of growth and stoichiometry of
acetic acid production and H2 consumption, serum
bottles (120 ml, BellCo Inc.) were filled with either
10 ml of basal acetogen medium or with acetogen
medium containing 5.6 mM glucose and pressurized
to 2 atm with either H2:CO2 (80:20) or N2:CO2 (75:25).
Cultures from each isolate were transferred three
times in medium containing the test substrate and
then a 0.1 ml of the culture was used to inoculate se-
rum bottles for growth determination. Serum bottles
were incubated on their sides and agitated at 200
rpm on a rotatory shaker (New Brunswick Scientific
Co.). Growth of each isolate was measured as an in-
crease in OD. Hydrogen utilization was determined
by measuring reduction in gas volume with a system
similar to that described by (Balch and Wolfe, 1976).
For each sample time, a 4 ml sample of the culture
liquid from each culture was frozen (-4°C) for subse-
quent VFA analysis.
Analytical Methods
Optical density was measured at 660 nm using a
Spectronic 70 spectrophotometer (Bausch & Lomb,
Rochester, NY). Volatile fatty acid production by iso-
lates was measured by gas-liquid chromatography
(GLC) (Holdeman et al., 1977). The frozen samples
were thawed and centrifuged at 15,000 rpm for 5
min, the supernatant was subsequently acidified by
adding 20% (w/v) of methaphopsphoric acid (25%,
w/v) and analyzed. A 3 ft long column, packed with
SP 1220 (Supelco, Bellefonte, PA, USA), was used in
a Varian 3700 GLC with a flame ionization detector.
Oven temperature was 130° C (isothermal), injector
temperature was 170° C, and detector temperature
was 180°C, with carrier gas (N2) flowing at rate of 30
ml per minute.
For the measurement of H2 uptake and CH4 produc-
tion, gas samples were analyzed using a Varian 3700
gas chromatograph equipped with a thermal con-
ductivity detector and a silica gel column (Supelco).
Temperatures of the injector, oven and detector were
room temperature, 130°C, and 120°C respectively,
with carrier gas (N2) flowing at 30 ml per minute.
Microscopy
Determination of cell morphology and presence
of spores and flagella were assessed by phase con-
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
trast microscopy (Carl Zeiss D-7082, Oberkochen,
Germany). Scanning electron micrographs were pre-
pared from cells grown to midlog or early stationary
phase in acetogen medium containing 27.8 mM glu-
cose. For scanning electron microscopy, a poly-D-
lysine coated cover slip was immersed in culture fluid
for one hour. Each coverslip was then fixed for 3 h
with 5% (w/v) glutaraldehyde and 1% (w/v) osmium
tetroxide in 0.1 M phosphate buffer, pH = 6.8. The
material was dehydrated by a series of graded etha-
nol solutions (Lamed et al., 1987). The cells on the
cover slips were critical point dried with liquid CO2.
The samples were sputter coated with gold palladi-
um in a Technics Hummer I and viewed with a JEOL
JSMM840 scanning electron microscope (JEOL Ltd.
Tokyo, Japan).
DNA Base Composition
For determination of mole percent guanine plus
cytosine, chromosomal DNA was extracted from
bacterial cells using the procedure described by
(Marmur, 1961). The mole percentage guanine plus
cytosine was calculated from the inflection point of
the temperature melting profile of isolated DNA with
DNA from Escherichia coli strain K12 as the reference
(Marmur and Doty, 1962). The temperature melting
profiles were analyzed using a Perkin-Elmer Lambda
3A spectrophotometer (Norwalk, CT) equipped with
a thermal cuvette.
ReSulTS And dISCuSSIon
Isolation of Bacteria
Twenty strains of acetogenic bacteria were iso-
lated from rumen contents of either an Angus steer
fed a high forage diet or a lactating dairy cow (Hol-
stein Friesian) fed a 40% concentrate diet. All iso-
lates produced at least five fold more acetate un-
der H2:CO2 than under N2:CO2. Acetate production
ranged from 40 to 75 mM on H2:CO2 and from 2
to 8 mM on N2:CO2. The production of other short
chain VFA was minimal for all strains designated.
Bacterial Characterization
All isolates stained gram positive. Strain A4 and
A9 were short rods while strains A2, A10, H3HH, and
H3HP were oval cocci (Table 3). However, H3HH was
pleomorphic, especially during exponential growth
on a rich carbohydrate medium. No flagella were ob-
On the basis of morphology, growth characteristics,
and H2 threshold values, acetogenic strains H3HH,
H3HP, A10, A2, A4, and A9 were selected for further
characterization. Hydrogen threshold of strain A10
and H3HH were the closest to those of the metha-
nogen strain NI4A (Table 2) and were more com-
pletely characterized. These threshold values are
lower than those reported by (LeVan et al., 1998) for
other ruminal acetogens and are comparable to the
values for non-ruminal acetogens (Cord-Ruwisch et
al., 1998).
Table 2. Hydrogen threshold values of methano-gen strain NI4A and of acetogenic strains A10, A2, A9, A4, and H3HH.
a Acetogenic isolates initially grown in 10 ml acetogen medium containing 27.8 mM glucose then flushed and incubated with 1% H2 for an additional 60 h
b Acetogenic isolates initially grown in 0.2% (w/v) BHI then flushed and incubated with 1% H2 for an ad-ditional 130 h
c Acetogenic isolates initially grown in 0.2% (w/v) BHI with 10 ml fresh medium added prior to incubation with 1% H2 for additional 130 h
d ND=not done
H2 Concentration (ppm)
Culture EXP 1a EXP 2b EXP 3c
Initial 10702 9993 9993NI4A 92 90 NDd
A10 1284 994 208A2 2516 1852 1383A9 5383 66157 1619A4 8007 NDd NDH3HH 1390 ND ND
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
served by either electron microscopy (Figures 1 and
2 for A10 and H2HH) or by standard staining proce-
dures using light microscopy. No spores were ob-
served by phase contrast microscopy and no spores
were produced by heating the cultures at 80° C for 30
min (Holdeman et al., 1976).
All strains when grown in H2:CO2 or glucose were
mesophilic. Strains H3HH, A10 and A2 grown in ace-
togen medium plus 0.1 (w/v) glucose, reached higher
OD at 30° C, however all strains grew faster at 39°C
(Table 4). Thus 39°C was considered the optimum tem-
perature of growth. Cells grew within a temperature
range of 17° to 45°C. strains H3HH and A2 reached
higher OD at 30°C and grew poorly or did not grow at
17°C. Strain A10 grew at almost the same rate at 30° C
and 39°C and was uniquely different from strain H3HH
and A2 in that it was able to grow at 17° C. The opti-
mum pH for growth in acetogen medium plus 0.5%
(w/v) glucose was 7.0 for strains A10 and H3HH and
7.5 for strains A2 and H3HP (Table 3). The pH range for
growth was 5.5 to 8.0 (data not shown). While strains
H3HH, H3HP, and A2 were strict anaerobes, strain A10
was considered to be facultative, because it grew in
all the media and conditions used to determine oxy-
gen sensitivity (Table 3). Most acetogens isolated have
been more strict anaerobes although several can tol-
erate low concentrations of O2 after exhibiting a lag
phase in growth (Karnholz et al., 2002).
Table 3. Morphological characteristics of isolates.
Cat. = catalase Sens. = sensitivity pleom. = pleomorphic
Figure 1. Picture A10: Morphology of strain A10 at late log-phase grown in acetogen medium containing 27.8 mM glucose (Scanning Electron Microscopy, x 11, 000).
Figure 2. Picture H3HH: Morphology of strain H3HH at late log-phase grown in acetogen medium containing 27.8 mM glucose (Scanning Electron Microscopy, x 11, 000).
Culture Gram stain Shape Cell size
µmOxygen
Sens. Cat. Spore MotileOptimum
Temp. (°C)
Optimum pH
G+C Mol%
H3HH + cocci-pleom 0.6-0.8 x 1.0-1.2 anaerobe - No No 39 6.8-7.0 ND
H3HP + oval-cocci 0.6-0.8 x 1.0-1.3 anaerobe - No No 39 7.5 ND
A10 + oval-cocci 0.6-0.8 x 1.0-1.4 facultative + No No 39 6.8-7.5 51.5
A2 + oval-cocci 0.6-0.8 x 1.0-1.5 anaerobe - No No 39 6.8-7.5 ND
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
After 5 days of autotrophic growth on solid ace-
togen medium under H2:CO2, strain H3HH colonies
were 1 mm in diameter, entire, slightly convex with a
regular margin and the colonies were white in color.
Colonies of strain A10 were approximately 0.7 mm
in diameter, the colonies were entire with a regular
margin and were transparent. When incubated aero-
bically on solid acetogen medium containing 27.8
mM glucose, strain A10 colonies were 1.2 mm in di-
ameter, were convex with a regular margin and were
white in color (data not shown). The FAME compo-
sition of strain A10 and H3HH was not found to be
similar to any of the species existing in the anaerobe
database (Moore, ver. 3.7). Species of interest clos-
est to these isolates that were in the FAME database
included Peptostreptococcus productus, Clostridi-
um thermoaceticum, Clostridium thermoautotrophi-
cum, and Eubacterium limosum. The mol% G+C for
strain A10 was 51.5% but was not determined for the
other isolates.
Nutrition Studies, Growth Studies, and Fermentation
Yeast extract, rumen fluid, or both were required
to support initial growth of strains A10 and H3HH
(data not shown). Both strains reached higher op-
tical density when growing on a basal acetogen
medium plus yeast extract, but initially they grew
faster when both yeast extract and rumen fluid were
added to the basal acetogen medium. All strains
utilized a wide range of carbohydrates as an energy
source (Table 4). Cellobiose, lactose, and sucrose
supported the highest growth. No strain was able
to utilize arabinose. Esculin was utilized poorly even
though all strains were able to hydrolyze it. Pectin
and casein were poorly utilized. Strain A10 was the
only strain that did not utilize glycerol. Simple acids
listed in Table 4 were either poorly or not utilized.
Strains H3HH, H3HP, and A2 were all oxidase and
catalase negative (Table 3). Strain A10 was oxidase
negative, but showed a weakly positive response in
the catalase test. Strains A10, A2, H3HH, and H3HP
were able to hydrolyze esculin and both strains A10
and H3HH were able to reduce nitrate (Table 5).
Strain A10, but not strain H3HH, was able to hydro-
lyze starch.
Strain H3HH differed from the ruminal acetogen
Acetitomaculum ruminis, in cell shape, absence of
flagella, and substrate utilization (Greening and
Leedle, 1989). Strain H3HH also differed from the
ruminal acetogen Eubacterium limosum, in sub-
strate utilization (Genthner et al., 1981; Genthner
and Bryant, 1982, 1987) and the ability to reduce
nitrate to nitrite. The species that most closely re-
sembled strain H3HH was Peptostreptococcus pro-
ductus which had been isolated from the calf rumen
(Bryant et al., 1958). However, strain H3HH was not
Table 4. Growtha of selected acetogenic strains on various substrates.
a Absorbance (660 nm) values represent the increase in OD after 36 hours of incubation at 39°C.
b Growth tests were carried out in acetogen medium plus 0.5% (w/v) of the desired substrate. Substrates were added separately as sterile anaerobic solutions to the autoclaved and cooled medium.
Substrateb A10 H3HP H3HH A2
Arabinose 0.13a 0.09 0.12 0.08Cellobiose 3.1 1.71 2.6 2.39Fructose 1.54 0.89 1.29 1.56Galactose 1.38 0.76 1.93 1.35Glucose 1.5 1.1 2.1 1.7Lactose 2.16 1.28 2.48 2.12Maltose 1.97 1.25 2.13 1.56Sucrose 2.58 1.23 2.14 1.99Casein 0.33 0.34 0.37 0.43Esculin 0.59 0.72 0.45 0.23Glycerol 0.22 1.04 0.69 1.2Mannitol 0.38 0.36 1.19 0.21Pectin 0.23 0.25 0.28 0.2Starch 1.5 1.35 0.12 0.15Glutamic acid 0.11 0.05 0.18 0.09
Formic acid 0.17 0.48 0.17 0.1
Fumaric acid 0.08 0.03 0.03 0.05
Lactic acid 0.13 0.17 0.03 0.13Succinic acid 0.05 0.08 0.09 0.05
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
related to P. productus based on GC-FAME analysis.
Strain H3HH differed from Clostridium thermoaceti-
cum and C. thermoautotrophicum in cell shape and
because it did not form spores (Fontaine et al., 1941;
Wiegel et al., 1981). Both strain H3HH and A. kivui
lack flagella and do not produce spores but strain
H3HH differed from A. kivui in cell shape, growth
temperature range and substrate utilized (Klemps et
al., 1987; Leigh et al., 1981; Leigh and Wolfe, 1983).
Strain A10 differed from strain H3HH in substrate
utilization profile (Table 4), and because strain A10
is catalase positive, could also hydrolyze starch, and
is a facultative anaerobe. Strain A10 differed from
the ruminal acetogen Acetitomaculum ruminis, in
cell shape, absence of flagella, substrates utilized,
and DNA base composition (Greening and Leedle,
1989). Strain A10 also differed from the ruminal ace-
togen Eubacterium limosum, in being able to reduce
nitrate and in substrate utilization profile (Genthner
et al., 1981; Genthner and Bryant, 1982, 1987). Strain
A10 was not related to P. productus based on GC-
FAME analysis. Strain A10 differed from Clostridium
thermoaceticum, and C. thermoautotrophicum in
cell shape, lack of flagella and a lower optimum
temperature for growth (Fontaine et al., 1941; Wie-
gel et al., 1981). Strain A10 differed from Acetoge-
nium kivui in cell shape, growth temperature range,
and substrate utilization profile ( Klemps et al., 1987;
Leigh et al., 1981; Leigh and Wolfe, 1983).
Acetate was the major VFA produced when strains
A10 and H3HH were growing in H2:CO2, glucose, or
glucose plus H2:CO2 When strains A10 and H3HH
were grown under H2:CO2 (80:20) strain A10 achieved
a maximum OD of 0.84 and a doubling time of 16 h
(Table 5), strain H3HH achieved a maximum OD of
0.81 and a doubling time of 13 h. These OD values
are lower than those achieved by E. limosum but the
doubling time is shorter (Genthner et al., 1981; Gen-
thner and Bryant, 1982, 1987). The maximum acetate
production of strains A10 and H3HH growing under
H2:CO2 (80:20) was 69 and 35 mM respectively. Both
C. thermoautotrophicum and E. limosum have been
shown to produce slightly more acetate (Genthner
et al., 1981; Genthner and Bryant, 1982, 1987; Wie-
gel et al., 1981). When strains A10 and H3HH were
grown on glucose (5.6 mM) strain A10 achieved a
maximum OD of 0.79 and a doubling time of 1.4 h,
strain H3HH achieved a maximum OD of 0.65 and a
doubling time of 1 h. The maximum acetate produc-
tion of strains A10 and H3HH growing on glucose (5.6
mM) was 14 and 15 mM respectively. When grown on
glucose plus H2:CO2 strain A10 and H3HH achieved
a maximum OD of 1.27 and 1.22 respectively.
When grown on H2:CO2 as an energy source, H2
consumption by strain A10 and H3HH was close to
the theoretical stoichiometry:
4H2 + 2CO2 g 1CH3COOH + 2H2O (Table 6)
Molar growth yields (g dry weight cell/mol sub-
strate consumed) for strain A10 and strain H3HH
were 0.67 and 0.51 g/mole respectively which were
Table 5. Growth and fermentation characteristics of isolates
Doub. = doubling Prod. = production
Culture
O.D. H2:CO2
Doub. Time
H2:CO2
Acetate Prod. (mM)
H2 Threshold
(ppm)
y (H2) (g
DW/mole)
Esculin Hydrol.
Nitrate Reduction
Starch Hydrol.
H3HH 0.81 13h 35 1390 0.51 + + -H3HP 0.73 15h 62 ND ND + ND NDA10 0.84 16h 68.8 209 0.67 + + ND
A2 0.43 24h 30 1383 ND + ND ND
Hydrol. = hydrolysis ND = Not Determined
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
lower than the values reported for C. thermoauto-
trophicum and E. limosum (Genthner et al., 1981;
Genthner and Bryant, 1982, 1987; Wiegel et al.,
1981). When grown on glucose as energy source,
glucose consumption by strain A10 and H3HH
was consistent with the theoretical stoichiometry:
C6H12O6 (glucose)g 3CH3COOH (Table 7)
Molar yields (g dry weight cell/mol substrate con-
sumed) were 36.9 for strain A10 and 47.4 for strain
H3HH.
ConCluSIon
Ruminants are one of the many sources of bio-
genic methane, hence the interest in reducing emis-
sions (Boadi et al, 2004; Fonty et al., 2007; Morrison,
2009). Ruminants may provide an important model
to enhance the animal production efficiency while
at the same time reduce global warming effects. In
addition, assuming that the energy lost as methane
by ruminants represents a loss of potential energy
to the animal (Branine and Johnson, 1990), sig-
nificant savings in feed cost to the producer could
Table 6. Growth yields and stoichiometry of fermentation of strains A10 and H3HH, grown on H2 + CO2(80:20)
aCell dry weights were determined from 500 ml cultures grown in acetogen medium under a H2:CO2 (80:20) gas atmosphere.
b Assimilation of acetate into cell material was calculated by the equation: 17C2H3O2 + 11H20 g 8<C4H7O3> + 2HCO3 + 150H; thus, 20.6 µmol acetate was required for 1.0 mg of cell dry matter (Eichler and Schink, 1984).
cH2 present in fermentation products as percentage of H2 consumed
Table 7. Growth yields and stoichiometry of fermentation of strains A10 and H3HH, grown in acetogen medium containing 5.6 mM glucose.
a Cell dry weights were determined from 500 ml cultures grown in acetogen medium under a H2:CO2 (80:20) gas atmosphere.
b Assimilation of acetate into cell material was calculated by the equation: 17C2H3O2 + 11H20 g 8<C4H7O3> + 2HCO3 + 150H; thus, 20.6 µmol acetate was required for 1.0 mg of cell dry matter (Eichler and Schink, 1984).
Culture
H2 uptake mM
Cell Dry
Weighta mg/ml
Acetate
Assimilatedb
mM
Acetate Produced mM
%H2
Recoveryc
Y(H2) g/mole
A10 421.6 0.28 5.83 68.86 71 0.67
H3HH 128.3 0.07 1.36 35 114 0.51
Culture
Initial Glucose Conc. mM
Cell Dry
Weighta mg/ml
Acetate
Assimilatedb
mM
Acetate Produced
mM
%Carbon Recovery
Y(Glc) g/mole
A10 5.6 0.22 4.54 14.37 113 36.9
H3HH 5.6 0.26 5.42 11.18 99.6 47.4
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
be realized if the potential energy presently lost as
methane is captured as fermentation products. Con-
sequently, methanogenesis in the rumen could be
inhibited and acetogenesis in the rumen enhanced
sufficiently to act as an electron sink and convert en-
ergy in H2 to acetate, which in turn can be utilized
by the animal (Boadi et al., 2004; Morrison, 2009).
Several limitations remain in the study because un-
fortunately these isolates have now been lost. First,
the taxonomy of the isolates was not resolved since
standard nutritional and physiological methods were
used instead of molecular methods. A next step
would have been to use 16S rRNA gene sequence
analysis of these isolates to provide phylogenetic
identification of the isolates. Such information would
have allowed design of FISH probes or PCR primers
for quantifying these acetogens both in vivo and in
vitro, thus expanding greatly the direction of future
research in this area. Unfortunately, the current iso-
lates were lost due to freezer malfunction and further
phylogenetic characterization is not possible. Despite
these limitations, the current study does demonstrate
that H2 limited continuous culture is a possible ap-
proach for isolating low H2 threshold isolates from the
rumen or other anaerobic ecosystems.
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We thank Kenneth Maciorowski, Purdue University,
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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Microbial keratinases have become biotechnologically important since they target the hydrolysis of
highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known
proteolytic enzymes trypsin, pepsin and papain. Keratinases are produced in a medium containing kerati-
nous substrates such as feathers and hair. This paper reports on the optimization of keratinase production
by Bacillus subtilis NCIM 2724. One factor-at-a-time method was used to investigate the effect of carbon
sources, nitrogen sources and pH on keratinase production. An L8 orthogonal array design was adopted to
select the most important fermentation parameters influencing the yield of keratinase. Response surface
methodology (RSM) was used to develop a mathematical model to identify the optimum concentrations of
the key parameters for higher keratinase production, and confirm its validity experimentally. The effect of
various amino acids on the production of keratinase was also studied. The final optimized medium gave a
maximum yield of 12.32 KU ml-1 of keratinase. Keratinases are commercially important among the proteases
that have been studied since they attack the keratin residues and hence find application in developing
cost-effective feather by-products for feeds and fertilizers.
Keywords: Keratinase, Fermentation, Bacillus subtilis, Optimization, Orthogonal Array Design, Response
surface methodology
InTRoduCTIon
Keratin is a fibrous and insoluble structural protein
extensively cross linked with hydrogen, disulphide
and hydrophobic bonds. It forms a major component
of the epidermis and its appendages viz. hair, feath-
ers, nails, horns, hoofs, scales and wool (Anbu et al.,
Received: September 27, 2010, Accepted: October 29, 2010. Released Online Advance Publication: May 6, 2011. Correspondence: Ishwar B. Bajaj, [email protected] Tel: - +91 22 24145616, Fax: +91 22 24145614
2007). Feather keratin exhibits an elevated content of
several amino acids such as glycine, alanine, serine,
cysteine and valine. The intensive cross-linkage in ker-
atins hinders their degradation by commonly known
proteolytic enzymes (Gupta and Ramnani, 2006). Deg-
radation of feathers will not only decrease the envi-
ronmental problem caused due to their accumulation
but could also act as source of some nutritionally im-
portant amino acids.
Currently, some industries have produced feather
Optimization of Fermentative Production of Keratinase From Bacillus Subtilis NCIM 2724
S. M. Harde1, I. B. Bajaj1, R. S. Singhal1
1Food Engineering and Technology Department, Institute of Chemical Technology,
Matunga, Mumbai, Maharashtra, India, 400 019
Agric. Food Anal. Bacteriol. 1: xx-xx, 2011
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
meal by steam pressure cooking. This technique re-
quires high energy input and may degrade amino
acids. The enzymatic hydrolysis of feather may be a
viable alternative to steam pressure cooking (Grazzi-
otin et al., 2006). The use of crude enzymes from Ba-
cillus species particularly Bacillus licheniformis and
Bacillus subtilis have been extensively studied due
to their effectiveness in terms of feather degradation
(Manczinger et al., 2003).
Keratinases [EC.3.4.99.11] belong to the group of
serine proteases capable of degrading keratin. It is
an extracellular enzyme produced in a medium con-
taining keratinous substrates such as feathers and
hair. Keratinases have applications in traditional in-
dustrial sectors including feed, detergent, medicine,
cosmetics and leather manufacturers (Farag and
Hassan, 2004), they also find application in more re-
cent fields such as prion degradation for treatment
of the dreaded mad cow disease (Langeveld et al.,
2003), biodegradable plastic manufacture and feath-
er meal production and thus can be appropriately
called “modern proteases”. The use of keratinases
to enhance drug delivery in some tissues and hydro-
lysis of prion proteins arise as novel potentially high
impact applications for these enzymes (Brandelli,
2007). Although many applications of keratinases are
still in the stage of infancy, a few have found their
way to commercialization, particularly the use of Bio-
resource International’s (BRI) Versazyme for feather
meal production. The crude enzyme can also serve
as a nutraceutical product, leading to significant im-
provement in broiler performance (Odetallah et al.,
2003). The most promising application of keratinase
is in the production of nutritious, cost effective and
environmentally benign feather meal (Gupta and
Ramnani, 2006). Nutritional enhancement can be
achieved by hydrolysis of feather meal/raw feather
using keratinase which significantly increases the lev-
els of essential amino acids methionine, lysine and
arginine (Williams et al., 1991). The present work focuses on trying to produce
keratinase from nonpathogenic microorganisms
and utilization of chicken feathers as a sole carbon
source. Several bacteria produce keratinase as an
extracellular material. Most of these belong to the
genus Bacillus. These bacteria use keratinous sub-
strates such as chicken feathers as carbon sources
for the production of keratinase. Aspergillus fu-
migatus was previously reported to be able to
use chicken feather flour as carbon and nitrogen
source (Santos et al., 1996). Addition of glucose,
sucrose and lactose resulted in strong inhibition
of keratinase production (Brandelli, 2007). The
production of keratinase is usually most notice-
able when chicken feathers are used as a sole car-
bon source (Williams et al., 1990). Farag and Has-
san (2004) used chicken feathers as a sole carbon,
nitrogen and sulphur sources for keratinase pro-
duction and observed 26.69 U/mg of keratinase
activity. Lin et al. (1992) used chicken feathers as a
sole carbon, nitrogen and energy sources for ker-
atinase production. Suntornsuk and Suntornsuk
(2003) reported that keratinase activity increased
upto 0.9 U/ml by using chicken feathers as a sub-
strate and sole carbon source from Bacillus sp. FK
46. They also varied the feather concentration for
production of keratinase and observed that higher
feather concentrations cause substrate inhibition
or repression of keratinase production, resulting
in a low percentage of feather degradation. El-
Refai et al. (2005) used different substrates for ker-
atinase production from Bacillus pumilus FH9 like
feather, muscle protein and wool. They observed
that wool gave the maximum keratinase activity of
647 U/ml. According to Kim et al. (2001), B. cereus
gave the maximum keratinase activity of 117 U/
ml by using feathers as a carbon source. Anbu et
al. (2007) produced keratinase from Scopulariop-
sis brevicaulis by using glucose and feather ascar-
bon sources and observed 1% glucose and 1.5%
feather to achieve a maximum keratinase activity
of 6.2 KU/ml.
Besides carbon sources, factors such as nitro-
gen sources (Thyes et al., 2006) and medium pH
(Suntornsuk and Suntornsuk, 2003) can influence
the productivity of keratinase. B. licheniformis
produced keratinase at neutral pH (Wang and
Shih, 1999). Anbu et al. (2007) studied the effect
of several organic and inorganic nitrogen sources
on keratinase production and found maximum
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
production in the presence of 1.5 to 2% sodium
nitrate (6.2 KU/ml) followed by peptone (6 KU/
ml) and potassium nitrate (5.5 KU/ml). Sodium
nitrate below 1.0 to 1.5% permitted enzyme syn-
thesis, but was inhibitory above 2%. Thyes et al.
(2006) studied the effect of feather meal, soybean,
gelatin, casein, yeast extract, cheese whey and
peptone at 10 g/L for production of protease from
Microbacterium arborescenes. Among the various
nitrogen sources studied, maximum keratinase was
produced in feather meal (96.5 U/ml), followed by
soybean protein (73.8 U/ml) and gelatin (45.8 U/ml).
The initial pH of the medium greatly affects the
bacterial growth, percentage of feather degradation
and keratinase production (Suntornsuk and Suntorn-
suk, 2003). It was observed that Bacillus species is
most active under neutral or basic conditions. The
optimum pH for B. cereus was 7.0 (Kim et al., 2001),
while that for B. pumilus was 8.0 (El-Refai et al., 2005).
For B. subtilis, highest enzyme production was ob-
tained over a broad range of pH 5 to 9.
According to Wang and Shih (1999) maximum
growth rate and keratinase productivity of B. subtilis
occurred at 42°C instead of 37°C, and the fermen-
tation time could also be shortened. However, the
maximum keratinase activity was observed at 37°C.
Elevated temperature increased cell growth, but not
enzyme production. The temperature differential
effect on growth versus keratinase production was
more obvious in B. licheniformis, where cells grew
best at 50°C, but keratinase production was best at
37°C. High temperature may increase the protein
turnover rate. According to El-Refai et al. (2005) the
optimal reaction temperature recorded for B. pumi-
lus FH9 keratinase is higher than those reported for
other B. pumilus strains.
This paper reports on optimization of keratinase
production using a statistical approach. Effects of
pH, carbon source and nitrogen source were inves-
tigated by using one factor at-a-time method. Initial
screening of the medium components was done
by using an L8 orthogonal array design to under-
stand the significance of their effect on the product
formation, and then a few of the more significant
parameters were selected for further optimization
using response surface methodology (RSM).
MATeRIAlS And MeThodS
Materials
Chicken feathers were collected from the Devgiri
poultry farm, Wadegavhaon, Pune, India. Chicken
feathers were ashed three times with distilled water
followed by defattening with chloroform: methanol
(1:1), dried and ground. All chemicals used were of
the AR grade and were purchased from Hi Media
Limited, Mumbai, India.
Bacterial strain and medium
A bacterial strain of Bacillus subtilis NCIM 2724 was
used in the present study. The medium used for the
growth and maintenance contained (g L-1), ammoni-
um chloride, 0.5; magnesium sulphate, 0.1; yeast ex-
tract, 0.1; sodium chloride, 0.5; dipotassium hydrogen
phosphate, 0.3; potassium hydrogen phosphate, 0.3;
feathers, 10 (pH 7.5 ± 0.2). Bacterial cells in agar slants
were incubated at 37°C for 24 h and stored at 4°C.
The medium was sterilized in an autoclave for 15 min
at 121°C.
For the production of keratinase, a medium re-
ported by El-Refai et al. (2005) was used, which con-
tained (g L-1) Feather, 10; Yeast extract, 0.1; MgSO4,
0.1; NH4Cl, 0.5; K2HPO4, 0.3; KH2PO4, 0.3; NaCl, 0.5.
Initial pH of the medium was adjusted to 7.5 ± 0.2 with
Tris–HCl buffer. The medium was sterilized in an auto-
clave for 15 min at 121°C.
Inoculum and fermentation
One ml cell suspension from a slant was trans-
ferred to 20 ml of the seed medium containing (g
L-1) peptone, 5; yeast extract, 1.5; beef extract, 1.5
and sodium chloride, 5; (pH 7 ± 0.2) and incubated
at 37°C and 200 rpm for 24 h. This was used as the
inoculum. Fermentation was carried out in 250 ml Er-
lenmeyer flasks, each containing 50 ml of the sterile
production medium. The medium was inoculated
with 5% (v/v) of 12 h old B. subtilis culture containing
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
approximately 2×106 cells/ml. The flasks were inoculat-
ed on a rotary shaker at 37 ± 2 °C and 200 rpm for 48 h.
All the experiments were carried out at least in triplicate.
Optimization of fermentation medium us-ing one factor-at-a-time method
In order to investigate the effect of initial pH of me-
dium on keratinase production, fermentation runs were
carried out by adjusting initial pH of the medium in the
pH range of 5 to 8, and analyzing the samples for kera-
tinase production after 48 h. To study the effects of dif-
ferent nitrogen sources on keratinase production, yeast
extract in the medium was replaced with different or-
ganic nitrogen sources, such as peptone, malt extract,
and beef extract at 0.1 g L-1, and ammonium chloride
was replaced with different inorganic nitrogen sources,
such as sodium nitrate, potassium nitrate, ammonium
sulphate or ammonium nitrate at 0.5 g L-1 and fermen-
tation was carried out as described in the previous sec-
tion. To check the effect of additional carbon sources
on the production of keratinase, fermentation medium
containing chicken feathers was supplemented with ad-
ditional carbon sources, viz., glycerol, sucrose, soluble
starch, maltose, lactose, fructose, glucose. All carbon
sources were used at 10 g L-1.
Optimization of fermentative production by using Orthogonal Array Design
An L8 orthogonal array method was used for screen-
ing of the most significant fermentation parameters
influencing keratinase production. The design for
the L8 orthogonal array was developed and analyzed
using MINITAB 13.30 software (Pennsylvania State
university, University Park, Pennsylvania). The L8 or-
thogonal array design is shown in Table 1. Seven fac-
tors at two levels were studied viz. chicken feather,
Run Feathers Beef extract MgSO4 KH2PO4 K2HPO4 NaCl NH4Cl Keratinasea
(KU ml-1)
1 1 (5) 1 (0.05) 1 (0.05) 1 (0.1) 1 (0.1) 1(0.15) 1 (0.15) 1.33 ± 0.04
2 1 (5) 1 (0.05) 1 (0.05) 2 (0.5) 2 (0.5) 2 (0.75) 2 (0.75) 2.37 ± 0.1
3 1 (5) 2 (0.25) 2 (0.25) 1 (0.1) 1 (0.1) 2 (0.75) 2 (0.75) 1.1 ± 0.23
4 1 (5) 2 (0.25) 2 (0.25) 2 (0.5) 2 (0.5) 1 (0.15) 1 (0.15) 1.05 ± 0.04
5 2 (25) 1 (0.05) 2 (0.25) 1 (0.1) 2 (0.5) 1 (0.15) 2 (0.75) 3.66 ± 0.07
6 2 (25) 1 (0.05) 2 (0.25) 2 (0.5) 1 (0.1) 2 (0.75) 1 (0.15) 1.90 ± 0.25
7 2 (25) 2 (0.25) 1 (0.05) 1 (0.1) 2 (0.5) 2 (0.75) 1 (0.15) 2.88 ± 0.2
8 2 (25) 2 (0.25) 1 (0.05) 2 (0.5) 1 (0.1) 1 (0.15) 2 (0.75) 4.2 ± 0.08
Table 1. Orthogonal project design for 2 levels of 7 variables used for media optimization for keratinase production.
a Results are mean ± SD of three determinations Values in the parenthesis indicate the real values of variables
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
ammonium chloride, beef extract, potassium dihy-
drogen phosphate, potassium hydrogen phosphate,
magnesium sulphate and sodium chloride for their
significance in production of keratinase by B. subtilis
NCIM 2724.
Optimization of concentrations of the selected medium components using Response Surface Methodology (RSM)
Response surface methodology is an empirical
statistical modeling technique employed for mul-
tiple regression analysis using quantitative data
obtained from properly designed experiments to
solve multivariable equations simultaneously (Puri
et al., 2002). RSM was used to determine the opti-
mum nutrient concentrations for the production of
keratinase. A central composite design (CCD) for
four independent variables was used to obtain the
combination of values that optimizes the response
within the region of three dimensional observation
spaces, which allows one to design a minimal num-
ber of experiments. The experiments were designed
using the software, Design Expert Version 6.0.10 trial
version (State Ease, Minneapolis, MN).
The medium components (independent variables)
selected for the optimization were chicken feather,
ammonium chloride, magnesium sulphate, and di-
potassium hydrogen phosphate. Regression analysis
was performed on the data obtained from the de-
sign experiments. Coding of the variables was done
according to the following equation:
where: xi, dimensionless value of an independent
variable; Xi, real value of an independent variable;
Xcp, real value of an independent variable at the
center point; and ∆Xi, step change of real value of
the variable i corresponding to a variation of a unit
for the dimensionless value of the variable i.
The experiments were carried out at least in tripli-
cate, which was necessary to estimate the variability
of measurements, i.e. the repeatability of the phe-
nomenon. Replicates at the center of the domain in
three blocks permit the checking of absence of bias
between several sets of experiments. The relation-
ship of the independent variables and the response
was calculated by the second order polynomial
equation:
Y is the predicted response; ß0 a constant; ßi the lin-
ear coefficient; ßii the squared coefficient; and ßij the
cross-product coefficient, k is number of factors. The
second order polynomial coefficients were calculated
using the software package Design Expert Version
6.0.10 to estimate the responses of the dependent
variable. Response surface plots were also obtained
using Design Expert Version 6.0.10.
Effect of amino acids on keratinase production by B. subtilis NCIM 2724
To study the effect of amino acids on keratinase
production, various amino acids including L-cysteine,
L-serine, L-valine, L-alanine, L-methionine, L-glutamic
acid, L-threonine, L-histidine, L-arginine and L-lysine
were added individually at 0.05 g L-1, 0.10 g L-1 and
0.50 g L-1 in the RSM optimized medium.
Keratinase assay
Keratinase activity was determined by the method
reported by Yu et al. (1968). Chicken feathers (20 mg)
were suspended in 3.8 ml of 100 mM Tris–HCl buffer
(pH 7.8), to which 0.2 ml of the culture filtrate (enzyme
source) was added. The reaction mixture was incubat-
ed at 37°C for 1 h. After incubation, the assay mixture
was dipped into the ice cold water for 10 min and the
remaining feathers were filtered out by Whatman fil-
ter paper (Whatman® Schleicher and Schuell, Mum-
bai, India). The absorbance of the clear mixture was
measured at 280 nm. The keratinase activity was ex-
pressed as one unit of the enzyme corresponding to
an increase in the absorbance value 0.1 (1KU= 0.100
corrected absorbance).
∆Xi
(Xi -Xcp)xi = i= 1,2,3,... k
i=1 i=1 i<ji<j
kkk k
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
ReSulTS And dISCuSSIon
Optimization using one-factor-at-a-time
An initial pH of 8.0 supported maximum produc-
tion of keratinase of 3.3 KU ml-1. pH is a significant
factor that influences the physiology of a microor-
ganism by affecting nutrient solubility and uptake,
enzyme activity, cell membrane morphology, by-
product formation and oxidative-reductive reac-
tions. During production of keratinase, keratin utili-
zation occurs more rapidly and to a great extent at
pH 7.5 (Suntornsuk and Suntornsuk, 2003). Friedrich
and Antranikian (1996) described maximum kerati-
nase production at alkaline pH. Alkaline pH favors
keratin degradation at higher pH, probably by modi-
fying the cystine residues to lanthionine and making
it accessible for keratinase action. The optimum pH
reported for keratinase production by B. cereus is 7.0
(Kim et al., 2001), chryseobacterium sp. is 9.0 (Casa-
rin et al., 2008), while that by B. pumilus FH9 is 8.0
(El-Refai et al., 2005). For B. subtilis, highest enzyme
production has been reported over a range of pH
of 7 to 9. It was observed that maximum keratinase
production occurs at alkaline pH.
It was found that ammonium chloride and beef
extract supported maximum keratinase activity of
4.05 KU ml-1 and 4.15 KU ml-1 respectively. These re-
sults are in accordance with the results obtained by
El-Refai et al. (2005), where ammonium chloride and
yeast extract supported maximum keratinase pro-
duction in B. pumilus FH9. Some researchers have
considered feather meal as a nitrogen source for
keratinase production (Thyes et al., 2006).
B. subtilis NCIM 2724 produced keratinase in pres-
ence of chicken feathers as the sole carbon source
which supported a maximum production of 4.14 KU
ml-1. Addition of simple carbon sources reduced the
production of keratinase. A decrease in the keratin-
ase production due to the addition of conventional
carbon sources is reported in literature. Addition of
fructose and maltose in medium decreased the kera-
tinase production in Trichophyton rubrum (Meevoo-
tisom and Niederpruem, 1979) and B. licheniformis
(Sen and Satyanarayana, 1993), respectively. These
results may be due to the catabolic repression of
keratinase (Anbu et al., 2007; Ignatova et al., 1999;
Yamamura et al., 2002; Santos et al., 1996). It has
been reported that chicken feathers act as the best
carbon source for keratinase production.
Statistical media optimization Optimization of fermentative production
by using Orthogonal Array Design
Once the best carbon and nitrogen sources were
selected, the medium was subjected to screening
of the most significant parameters for keratinase
production using the L8 orthogonal array. The re-
sponses for means (larger is better) and for signal
to noise ratios obtained using the L8 orthogonal ar-
ray are shown in Table 2. The last two rows in the
tables show delta values and ranks for the system.
Rank and delta values help in assessing which factors
have the greatest effect on the response character-
istic of interest. Delta measures the size of the effect
by taking the difference between the highest and
lowest characteristic average for a factor. A higher
delta value indicates a greater effect of that compo-
nent. Rank orders the factors from the greatest effect
(on the basis of the delta values) to the least effect
on the response characteristic. The order in which
the individual components affected the fermenta-
tion process were feather > ammonium chloride >
magnesium sulphate > dipotassium hydrogen phos-
phate > sodium chloride > beef extract > potassium
dihydrogen phosphate suggesting that feathers had
a major effect, while K2HPO4 had the least effect on
keratinase production by B. subtilis NCIM 2724.
Optimization by RSM
Based on the L8 orthogonal array design, feather
(A), ammonium chloride (B), magnesium sulphate (C)
and dipotassium hydrogen phosphate (D) were se-
lected for further optimization by RSM. To examine
the combined effect of these medium components
(independent variables) on keratinase production,
a central composite factorial design of 24 =16 plus
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
6 center points and (2 × 4 = 8) star points lead-
ing to a total of 30 experiments were performed.
A CCRD matrix of independent variables along
with responses of each experimental trial is given
in Table 3.
The ANOVA of the quadratic regression model
indicated the model to be significant (P < 0.05) (Ta-
ble 4). The P values were used as a tool to check
the significance of each of the coefficients, which,
in turn, are necessary to understand the pattern of
the mutual interactions among the test variables.
The smaller the magnitude of the P, the more sig-
nificant is the corresponding coefficient (Thys et al.,
2006). Among the test variables used in the study,
A (feather), B (ammonium chloride), C (magnesium
sulphate), D (dipotassium hydrogen phosphate), A2
(feather2), B2 (ammonium chloride2) and D2 (dipotas-
sium hydrogen phosphate2) are significant model
terms. Interactions between B (ammonium chlo-
ride) and C (magnesium sulphate); B (ammonium
chloride) and D (dipotassium hydrogen phosphate);
and C (magnesium sulphate) and D (dipotassium
hydrogen phosphate) are also significant. Other in-
teractions were found to be insignificant.
The corresponding second-order response
model found after analysis for the regression was
keratinase (KU ml-1) = 3.66 + 1.09 * feather + 0.51 *
ammonium chloride + 0.72* magnesium sulphate
+ 1.26 * dipotassium hydrogen phosphate + 1.20
* feather2 + 0.36 * ammonium chloride2 + 0.12 *
magnesium sulphate2 + 0.36 * dipotassium hydro-
gen phosphate2 - 0.084 * feather * ammonium chlo-
ride + 0.15 * feather *magnesium sulphate - 0.23 *
feather * dipotassium hydrogen phosphate - 0.24 *
ammonium chloride * magnesium sulphate + 0.68 *
ammonium chloride * dipotassium hydrogen phos-
phate + 0.43 * magnesium sulphate * dipotassium
hydrogen phosphate.
The fit of the model was also expressed by the
coefficient of regression R2, which was found to
be 0.98, indicating that 98.0% of the variability in
keratinase yield could be explained by the model.
Other parameters of ANOVA for response surface
quadratic model were also studied. The ‘Pred R-
Squared’ of 0.92 is in reasonable agreement with
the ‘Adj R-Squared’ of 0.96. ‘Adeq Precision’ mea-
sures the signal to noise ratio.
The special features of the RSM tool, “contour
plot generation” and “point prediction” were also
studied to find optimum value of the combination
of the four media constituents. It was observed
that medium containing (g L-1), feather, 60.0; am-
monium chloride, 1.0; magnesium sulphate, 0.08;
and dipotassium hydrogen phosphate, 0.2 yielded
LevelA
FeathersB
Beef ExtractC
MgSO4
DKH2PO4
EK2HPO4
FNaCl
GNH4CL
S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean S/N Mean
1 2.82 2.82 6.70 2.31 7.71 2.60 5.94 2.24 5.14 2.04 6.48 2.47 4.43 1.79
2 9.42 9.42 5.54 2.21 4.54 1.92 6.31 2.29 7.11 2.49 5.77 2.06 7.81 2.74
Delta 6.60 6.60 1.15 0.09 3.17 0.67 0.37 0.04 1.96 0.44 0.70 0.40 3.38 0.95
Rank 1 6 3 7 4 5 2
Table 2. Response table for means and S/N ratio.
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
Sr. no.
Feather (g L-1)
NH4CL(g L-1)
MgSO4
(g L-1)K2HPO4
(g L-1)Keratinase a
(KU ml-1)
1 -1 (30) -1 (0.5) -1 (0.04) -1 (0.1) 2.77 ± 0.14
2 1 (60) -1 (0.5) -1 (0.04) -1 (0.1) 5.5 ± 0.11
3 -1 (30) 1 (1.0) -1 (0.04) -1 (0.1) 3.1 ± 0.20
4 1 (60) 1 (1.0) -1 (0.04) -1 (0.1) 5.13 ± 0.13
5 -1 (30) -1 (0.5) 1 (0.08) -1 (0.1) 3.65 ± 0.10
6 1 (60) -1 (0.5) 1 (0.08) -1 (0.1) 6.81 ± 0.18
7 -1 (30) 1 (1.0) 1 (0.08) -1 (0.1) 3.41 ± 0.13
8 1 (60) 1 (1.0) 1 (0.08) -1 (0.1) 5.75 ± 0.17
9 -1 (30) -1 (0.5) -1 (0.04) 1 (0.2) 3.45 ± 0.16
10 1 (60) -1 (0.5) -1 (0.04) 1 (0.2) 4.59 ± 0.14
11 -1 (30) 1 (1.0) -1 (0.04) 1 (0.2) 6.36 ± 0.17
12 1 (60) 1 (1.0) -1 (0.04) 1 (0.2) 7.75 ± 0.23
13 -1 (30) -1 (0.5) 1 (0.08) 1 (0.2) 6.14 ± 0.20
14 1 (60) -1 (0.5) 1 (0.08) 1 (0.2) 8.24 ± 0.18
15 -1 (30) 1 (1.0) 1 (0.08) 1 (0.2) 7.91 ± 0.24
16 1 (60) 1 (1.0) 1 (0.08) 1 (0.2) 9.93 ± 0.13
17 -2 (15) 0 (0.75) 0 (0.06) 0 (0.15) 6.18 ± 0.21
18 2 (75) 0 (0.75) 0 (0.06) 0 (0.15) 10.84 ± 0.8
19 0 (45) -2 (0.25) 0 (0.06) 0 (0.15) 4.15 ± 0.04
20 0 (45) 2 (1.25) 0 (0.06) 0 (0.15) 6.12 ± 0.03
21 0 (45) 0 (0.75) -2 (0.02) 0 (0.15) 3.14 ± 0.20
22 0 (45) 0 (0.75) 2 (0.1) 0 (0.15) 5.21 ± 0.20
23 0 (45) 0 (0.75) 0 (0.06) -2 (0.05) 2.13 ± 0.05
24 0 (45) 0 (0.75) 0 (0.06) 2 (0.25) 8.14 ± 0.17
25 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.55 ± 0.11
26 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 2.75 ± 0.25
27 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 4.12 ± 0.04
28 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.86 ± 0.02
29 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.86 ± 0.17
30 0 (45) 0 (0.75) 0 (0.06) 0 (0.15) 3.8 ± 0.20
Table 3. The CCRD matrix of independent variables in coded form and actual values with their corresponding response in terms of production of keratinase by B. subtilis NCIM 2724.
a Results are mean ± SD of three determinations Values in the parenthesis indicate the real values of variables
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
Factor a CoefficientEstimate
Sum of squares
Standard Error DF b F value p
Intercept 3.66 139.52 0.18 1 53.27 < 0.0001
A 1.09 28.67 0.088 1 153.24 < 0.0001
B 0.51 6.13 0.088 1 32.77 < 0.0001
C 0.72 12.51 0.088 1 66.89 < 0.0001
D 1.16 38.18 0.088 1 204.07 < 0.0001
A2 1.20 39.46 0.083 1 210.94 < 0.0001
B2 0.36 3.47 0.083 1 18.55 0.0006
C2 0.12 0.37 0.083 1 1.96 0.1815
D2 0.36 3.47 0.083 1 18.55 0.0006
AB -0.084 0.11 0.11 1 0.61 0.4474
AC 0.15 0.34 0.11 1 1.81 0.1981
AD -0.23 0.81 0.11 1 4.35 0.0544
BC -0.24 0.94 0.11 1 5.0 0.0409
BD 0.68 7.38 0.11 1 39.47 < 0.0001
CD 0.43 3.02 0.11 1 16.14 0.0011
Table 4. Analysis of variance (ANOVA) for the experimental results of the central-composite design (Quadratic Model).
a A = Feathers, B = NH4Cl, C = MgSO4, D =K2HPO4b Degree of freedom
Figure 1. Contour plot for keratinase production (-Effect of MgSO4 and NH4Cl).
Figure 2. Contour plot for keratinase production (Effect of K2HPO4 and NH4Cl).
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
maximum (10.6 KU ml-1) keratinase.
Accordingly, three-dimensional graphs were gener-
ated for the pair-wise combination of the four factors,
while keeping the other two at their center point levels.
Graphs for interactions are given here to highlight the
roles played by these factors (Figure 1 and Figure 2).
From the central point of the contour plot the optimal
process parameters were identified. The keratinase yield
(10.6 KU/ml) in the present study is quite high as com-
pared to the literature reports. The maximum keratinase
production reported till date by using most widely used
strain Bacillus subtilis S1 MTCC 2616 is 4.89 KU/ml and
Scopulariopsis brevicaulis MTCC 2170 is 6.2 KU/ml.
The effect of amino acids on keratinase pro-duction by B. subtilis NCIM 2724
Effect of amino acids on production of keratinase is
shown in Figure 3. All of the amino acids examined sup-
ported keratinase production, but the maximum kerati-
nase activity of 12.32 KU ml-1 was observed with 0.5 g L-1
of L-valine. Further increases in L-valine concentration
did not increase keratinase activity (Data not shown).
Addition of amino acids is of considerable impor-
tance in the protease synthesis in terms of metabolic
driving force. Feather keratin is composed of vari-
ous amino acids including glycine, alanine, serine,
cysteine and valine that are extensively cross linked
with hydrogen, disulphide and hydrophobic bonds.
Degraded feathers may act as source of some nutri-
tionally important amino acids and also serves as an
inducer for keratinase production.
ConCluSIon
Statistical nutrient optimization was done to optimize
keratinase production from B. subtilis NCIM 2724. Tagu-
chi design (L8 orthogonal array) demonstrated the effect
of feather, ammonium chloride, K2HPO4 and MnSO4 to
be significant. Further optimization of the most signifi-
cant factors by RSM revealed complex nutrient interac-
tions among them, and also increased the production
of keratinase by B. subtilis NCIM 2724 from 3.0 KU ml-1
to 10.6 KU ml-1. All amino acids supported keratinase
production and the maximum keratinase activity of
12.32 KU ml-1 was observed with 0.5 g L-1 of L-valine.
Figure 3. Effect of amino acids on keratinase production by B. subtilis NCIM 2724.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
RefeRenCeS
Anbu, P., S. C. B. Gopinath, A. Hilda, T. Lakshmipriya,
G. Annadurai. 2007. Optimization of extracellu-
lar keratinase production by poultry farm isolate
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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
www.afabjournal.comCopyright © 2011
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Listeria monocytogenes adapts to diverse stress conditions including cold, osmotic, heat, acid, and alkali
stresses encountered during food processing and preservation which is a serious food safety threat. In this
review, we have presented the major findings on this bacterium’s stress response proteomes to date along
with the different approaches used for its proteomic analysis. The key proteome findings on cold, heat
shock, salt, acid, alkaline and HHP stresses illustrate that the cellular stress responses in this organism are
a culmination of multiple protein expression changes in response to a particular stress stimuli. Moreover,
a number of key proteins may be involved in conferring the cross protective effects against various stress
environments. As an example, ferritin-like protein (designated as Fri or Flp) is induced during cold, heat,
and HHP stresses. Similarly, general stress protein Ctc is induced in cold and osmotic stresses while mo-
lecular chaperones such as GroEL and DnaK are induced in cold and heat stresses. Furthermore, a number
of stress proteins also contribute towards L. monocytogenes virulence and pathogenicity. Future research
may lead to understanding the stress proteomes of this pathogen induced on various food matrices and
processing environments in which it can persist for long periods of time.
Keywords: Listeria monocytogenes, proteome, cold stress, osmotic stress, heat stress, acid stress, alkali stress.
InTRoduCTIon
Listeria monocytogenes is an important food-
borne pathogen with significant public health threats
and economic impacts on the food industry. It causes
Received: November 22, 2010, Accepted: April 9, 2011. Released Online Advance Publication: April, 2011. Correspondence: Ramakrishna Nannapaneni,
[email protected]: +1 -662-325-7697 Fax: +1-662-325-8728
“listeriosis” in humans, which is associated with a
variety of symptoms ranging from flu-like illness to
severe life threatening meningitis as well as high
mortality (Lennon et al., 1984). Epidemiological stud-
ies estimate that listeriosis to be responsible for ap-
proximately 19 % of food-related deaths in the Unit-
ed States annually (Scallan et al., 2011). Suspected
L. monocytogenes contamination is also among the
leading causes of food recalls resulting in significant
REVIEWAn Overview of Stress Response Proteomes in Listeria monocytogenes
Kamlesh A. Soni1, Ramakrishna Nannapaneni1*, and Taurai Tasara2
1Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, USA
2Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Zurich, Switzerland
Agric. Food Anal. Bacteriol. 1: XX-XX, 2011
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
financial losses to the food industry due to the “zero
tolerance” standard adopted for the ready-to-eat
food products in the USA (Kramer et al., 2005; Mars-
den, 2001; Teratanavat and Hooker, 2004).
The prevalence of L. monocytogenes is mainly
due to its wide-spread distribution and its ability to
withstand adverse environmental conditions. This
includes the ability of this pathogen to survive and
grow at low temperatures, and resistance to high
osmolarity, acidic and alkaline environments. Cold
adaptation of this organism is of growing concern
due to the changing life styles over the years that
have increased the consumption of refrigerated
and minimally processed food products. Besides
cold storage, elevated salt concentrations are an al-
ternative means of food preservation, but L. mono-
cytogenes is also highly salt tolerant and has been
documented to grow in the presence of as high
as 10% NaCl (McClure et al., 1991). Jensen et al.
(2007) recently showed that L. monocytogenes cells
can display increased aggregation and biofilm for-
mation when exposed to NaCl stress. Additionally
this bacterium exhibits acid tolerance responses
(ATR), which significantly increases its resistance to
a subsequent lethal acid (pH 3.0-3.5) stress expo-
sure after an initial encounter with the non-lethal
acidic (pH 5.0-5.5) conditions. As an example, 4-log
higher survival was observed in L. monocytogenes
cells exposed to acid stress at pH 3.5 for 6 h after
an initial 90 minute exposure to a mild acidic condi-
tion at pH 5.5 (Koutsoumanis et al., 2004). Similarly,
L. monocytogenes may also acquire an increased
alkaline stress tolerance subsequent to sublethal al-
kaline stress exposure (Mendonca et al., 1994). Dur-
ing food processing and preservation, L. monocyto-
genes cells may become exposed to multiple forms
of sublethal stresses, leading to “stress hardening”.
Consequently, L. monocytogenes exposure to mild
forms of particular stresses may inadvertently in-
duce cross protection against subsequent expo-
sures to lethal levels of other unrelated stresses. For
example, it has been shown that acid (pH 4.5 for 1
h) or cold (10°C for 4 h) stressed L. monocytogenes
LO28 (serotype 1/2c) cells tend to be more resistant
to high hydrostatic pressure (HHP) in comparison
to the non-stress adapted cells (Wemekamp-Kam-
phuis et al., 2002). Lou and Yousef (1997) reported
that the heat stress of L. monocytogenes results
in cell-hardening and subsequent osmoprotection
and higher resistance of these cells to ethanol treat-
ment. Likewise, L. monocytogenes cells were also
found to be more thermotolerant after a combined
acid and heat shock or after osmotic and heat shock
treatments (Skandamis et al., 2008).
Stress adaptation events in L. monocytogenes, as
in other microorganisms, includes coordinated in-
duction of different stress protection systems within
the affected cells. Proteomics and transcriptomics
are both invaluable tools in delineation of the dif-
ferent mechanisms of stress response in microbes.
Transcriptome analysis technologies while impor-
tant in deciphering the global mRNA expression
changes during stress responses, fail to capture all
aspects of these molecular responses since mRNA
transcripts changes may not directly correlate with
protein expression due to the fact that transcripts
produced in abundance may be rapidly degraded,
translated poorly, or influenced through post-trans-
lational modifications. Therefore complementa-
tion of the transcriptome based analysis of stress
responses with the proteome studies is important
to get a clearer picture as proteins are the key func-
tional units involved in physiological stress respons-
es. As a result of new developments in microbial
cell global protein profiling based on the protein
identification approaches and bioinformatics, re-
searchers are now also able to monitor and deter-
mine the importance of stress induced proteins in
L. monocytogenes during its adaptation to diverse
conditions. A number of proteome profiling studies
performed on this organism so far have already pro-
vided extensive preliminary insights into gene and
protein expression changes that are associated with
the environmental stress adaptation in this bacte-
rium. The purpose of this review is to discuss the
significant developments in proteomic analysis of
the stress-adaptation in L. monocytogenes with fo-
cus on cold, heat, osmotic, acid, alkaline, and HHP
adaptation along with cross linking between stress
proteins and virulence.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
PRoTeomIC TeChnologIeS APPlIed In L. monocytogenes AnAlySIS
The summary of different gel-based and non-gel
based techniques and their assay principles are dis-
cussed in-depth in recent review articles by Haynes
et al. (2007) and Nesatyy and Suter (2007). For L.
monocytogenes, the most commonly applied pro-
tocols to date have used two-dimensional gel elec-
trophoresis (2DE) for protein separation (Folio et al.,
2004; Mujahid et al., 2007; Ramnath et al., 2003; Scha-
umburg et al., 2004). The majority of the L. mono-
cytogenes stress proteome studies utilized soluble
cellular proteins (excluding the extracellular fraction)
that were fractionated using mechanical disruption
alone or protocols combining mechanical disrup-
tion and enzymatic lysis. In the earlier studies most of
the proteins were not identified (; Bayles et al., 1996;
Phan-Thanh and Gormon, 1995), although in later
studies a significant number of the 2DE separated
proteins were identified by mass spectrophotometry
(MS) (Abram et al., 2008b; Dumas et al., 2008; Folio
et al., 2004; Mujahid et al., 2007; 2008; Phan-Thanh
and Jansch, 2006; Schaumburg et al., 2004 ). More re-
cently however, non-gel based approaches that com-
bine liquid chromatography (LC) separation and MS
(LC-MS/MS) are increasingly used. The fractionated
complex bacterial protein mixtures are digested into
peptides, separated by liquid chromatography and
analyzed in MS, taking advantage of the advances in
bioinformatics to identify even larger numbers of the
fractionated proteins (Abram et al., 2008b; Calvo et
al., 2005; Trost et al., 2005).
The majority of studies that have compared pro-
tein expression between normal versus stress ex-
posed L. monocytogenes cells using 2DE gel-based
protein separation with or without subsequent appli-
cation of MS to identify separated proteins (Bayles et
al., 1996; Duche et al., 2002a; Esvan et al., 2000; Phan-
Thanh and Gormon, 1995; 1997; Phan-Thanh and
Mahouin, 1999; Wemekamp-Kamphuis et al., 2004a).
A 2DE reference map covering an estimated 28.8%
of potential gene products was generated from the
soluble subproteome of L. monocytogenes EGDe
serotype 1/2a strain (Folio et al., 2004). Ramnath et
al. (2003) also used this approach and detected two
proteins found in L. monocytogenes EGDe but were
absent in some food isolates. The identification of
these proteins revealed they were involved in glyco-
lytic pathway and metabolism of coenzymes, but the
relevance of their differential expression specifically
in such food isolates remains unknown. The draw-
backs of gel based 2DE proteomics include poor
reproducibility in separation of highly basic or hydro-
phobic proteins, gel-to-gel variations and poor reso-
lution of high molecular weight protein complexes.
Attempts to overcome these drawbacks include the
recent use of 2D-DIGE (Two dimensional-difference
gel electrophoresis) based proteomics analysis. By
using different fluorescent dyes such as Cy2, Cy3
or Cy5 for protein labeling, such approaches allow
protein mixtures of different origins to be analyzed
within the same gel run. Thus these approaches are
more amenable to stress proteome response studies
where protein expression patterns of stress-adapted
cells and control samples can be directly compared
within the same gel run to minimize the influence of
gel-to-gel variations. Folsom and Frank (2007) used
a 2DE-DIGE based proteomics approach to analyze
protein expression changes associated with chlorine
resistance and biofilm formation in a hypochlorous
acid tolerant variant of the L. monocytogenes Scott
A (4b) strain. They found 19 proteins that were dif-
ferentially expressed between planktonic and biofilm
cells of a hypochlorous acid tolerant cultural variant
of this strain (Folsom and Frank, 2007). Six of these
differentially expressed proteins were subsequently
identified by peptide-mass mapping. They included
three ribosomal proteins (L7, L10 and L12), perox-
ide resistance protein (Dpr/Flp/Fri), sugar-binding
protein (Lmo0181), and a putative protein Lmo1888
of yet unknown function. This study also revealed
that peroxide stress resistance proteins Fri that is in-
volved in multitude of other stresses was expressed
2.2-fold times higher in biofilm than in planktonic
cells. At phenotypic level it was observed that L.
monocytogenes cells present in biofilm mass were
more resistant to sanitization treatments compared
to planktonic cells (Pan et al., 2006). Although not yet
widely adapted for L. monocytogenes analysis, LC
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
based techniques seem capable of detecting even
higher numbers of proteins compared to tradition-
al 2DE gel-based techniques. As an example when
the same protein fraction of cell free supernatant
(extracellular) of L. monocytogenes EGDe was ana-
lyzed, 105 proteins were identified using LC-MS/MS
compared to 58 detected by 2DE (Trost et al., 2005).
Forty-five of the detected proteins were found to be
common between the two methods. The analysis of
differential protein expression between L. monocyto-
genes 10403S and its σB null mutant strain using the
LC-MS/MS with iTRAQ (isotope tag for relative and
absolute quantification) identified 35 σB regulated
proteins, whereas the 2DE approach only managed
to detect 13 proteins. Four proteins were common
between the two methods (Abram et al., 2008b). A
combination of SDS-PAGE and LC-MS/MS detected
301 membrane associated proteins of L. monocy-
togenes EGDe (Wehmhoner et al., 2005). This was
greater than 79 proteins detected using the 2DE ap-
proach by Mujahid et al. (2007). One possible reason
for increased protein detection with SDS-PAGE/LC-
MS/MS might be increased protein solubilization in
the SDS-PAGE sample buffer in comparison to the
urea based sample buffer applied in the 2DE-MS ap-
proach (Haynes and Roberts, 2007).
In another example LC-LC-MS/MS combination
also called the multidimensional protein identifica-
tion technique (MudPIT), has also been used for pro-
teome analysis of L. monocytogenes cells. Fifteen
proteins that covalently bound the LPXTG motif were
identified in the subproteome fraction of cell wall as-
sociated proteins of L. monocytogenes strain EGDe
(Calvo et al., 2005). The SrtA and SrtB enzymes an-
chor surface proteins to the cell wall. Surface proteins
recognized by these two sortases were also analyzed
using LC-LC-MS/MS in the EGDe strain. A total of
13 and 2 LPXTG-containing proteins were identi-
fied in srtA and srtB null mutant strains (Pucciarelli
et al., 2005). Recently, MudPIT was used to study the
differences that exist between serotype 1/2a (strain
EGD) and 4b (strain F2365) (Donaldson et al., 2009).
In total, 1754 EGD proteins and 1427 F2365 proteins
were detected representing 50-60% of total Liste-
ria proteome coverage. In total 1077 proteins were
common to both serotypes and of these 413 proteins
displayed significantly differential expression level
between the two serotypes.
PRoTeome AnAlySIS In STReSS-AdAPTed L. monocytogenes CellS
The ability of L. monocytogenes to sense and re-
spond to a particular stress factor has implications
for both survival and virulence properties of this bac-
terium. Stress exposure elicits various fundamental
changes in this organism’s cellular physiology. These
changes are mediated via multiple and specific
changes in gene and protein expression profiles in
cells. Proteins associated with cold, heat, osmotic,
acid, and high hydrostatic pressure stress adaptation
will be discussed in the following sections.
Cold stress adaptation
The growth of L. monocytogenes on cold pre-
served food products is one of its important food
safety challenges. In addition to decreased meta-
bolic capacity, cold stress exposed microorgan-
isms are faced with a wide range of structural and
functional impediments in membrane structures,
nucleic acids (DNA and RNA), and macromolecular
assemblies such as ribosomes (Schumann, 2009).
The putative integral membrane protein PgpH,
whose deletion leads to impaired cold growth, has
been proposed as a possible cold sensing factor in
L. monocytogenes (Liu et al., 2006). Based on the
proposed model, environmental cold stress sensed
through membrane bound PgpH proteins is con-
veyed intracellularly through homeodomain depen-
dent signaling pathways.
Using 2DE gel-based proteome analysis, initial
studies revealed modulation in expression of be-
tween 10 to 38 proteins in association with cold stress
adaptation of this organism (Bayles et al., 1996; He-
braud and Guzzo, 2000; Phan-Thanh and Gormon,
1995). Of these differentially expressed proteins vi-
sualized, the predominating cold shock protein was
subsequently identified through microsequencing as
ferritin (Fri) (designated as Flp or Fri) (Hebraud and
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
Guzzo, 2000). The role of this protein in cold adap-
tation was also later phenotypically confirmed when
Dussurget et al. (2005) created a fri null mutant strain
in L. monocytogenes EGDe, which exhibited a cold
sensitive phenotype. Although physiological and
cold adaptation roles of Fri are not yet well under-
stood, it is hypothesized that it might facilitate alle-
viation of oxidative stress environments developing
in cold stress exposed L. monocytogenes cells (Liu
et al., 2002; Tasara and Stephan, 2006). Wemekamp-
Kamphuis et al. (2002) described four approximately
7 kDa protein that were cold inducible in L. mono-
cytogenes LO28 as determined by using a combi-
nation of 2DE gel electrophoresis and immunoblot-
ting. These proteins designated Csp1-Csp4, were
described as the L. monocytogenes cold shock fam-
ily proteins based on their cross reactivity with anti-B.
subtilis CspB polyclonal antibodies. Although their
identity as such was not confirmed by peptide mass
fingerprinting (PMF) in this work, genomic informa-
tion show that L. monocytogenes harbors three pro-
teins of the cold shock domain protein family (Glaser
et al., 2001). Two of these L. monocytogenes Csp
proteins, CspL (CspA) and CspD have now been
confirmed to be functionally vital for efficient cold
growth in this bacterium (Schmid et al., 2009). CspA
and CspD proteins, based on knowledge from oth-
er microorganisms, are also presumed to facilitate
cold growth possibly through nucleic acid (DNA and
RNA) chaperone-like functions (Horn et al., 2007).
This facilitates DNA replication and gene expression
events that may otherwise be hampered through
secondary structures that tend to form in bacterial
cells at low temperatures.
Meanwhile, a more comprehensive cold adapta-
tion proteome analysis in this bacterium has been
recently described. Cacace et al. (2010) performed
detailed proteome analysis on L. monocytogenes
cells grown for 13 days at 4°C with subsequent MAL-
DI (Matrix-assisted laser desorption/ionization) anal-
ysis. Proteome analysis revealed that 57 proteins in
total were over-expressed and eight were repressed
in cold grown cells compared to cells cultivated at
37°C. Proteome changes detected in this study in-
dicated the increased synthesis of proteins linked to
energy production, oxidative stress resistance, nutri-
ent uptake, lipid synthesis, and protein synthesis and
folding. Cold stress adaptation proteins identified
by this study that are of particular interest include:
OppA, Ctc, GroEL and DnaK. The OppA protein,
which facilitates accumulation of short peptide sub-
strates, is important for efficient cold growth in this
bacterium and at phenotypic level oppA null mutant
of this bacterium was unable to grow at low tem-
perature (5°C) (Borezee et al., 2000). Ctc is a gen-
eral stress protein which has been found to promote
the adaptation of L. monocytogenes cells to high
osmolarity conditions (Gardan et al., 2003b). The
GroEL and DnaK proteins are molecular chaperones
that promote proteins refolding and degradation of
stress damaged proteins that accumulate under dif-
ferent suboptimal conditions including heat stress
(Sokolovic et al., 1990). The cold growth associated
induction of the Ctc, GroEL and DnaK proteins, which
have been previously associated with adaptation to
other stresses (i.e. Ctc for cold and osmotic stress
and GroEL-DnaK for cold and heat stress) conditions
may thus indicate commonality of some stress adap-
tive responses in this bacterium (Cacace et al., 2010;
Duche et al., 2002a,b; Gardan et al., 2003b; Soko-
lovic et al., 1990).
The accumulation of compatible solutes espe-
cially glycine, betaine, and carnitine also promotes
cold growth in various bacteria including L. mono-
cytogenes (Mendum and Smith, 2002; Smith, 1996;
Wemekamp-Kamphuis et al., 2004b). There are no
enzymatic systems for the de novo synthesis of main
cryoprotective compatible solutes glycine, betaine,
and carnitine in L. monocytogenes, but transport
systems (Gbu, BetL and OpuC) that accumulate
them from environmental sources are present, and
deletion of genes coding for these transporters has
confirmed that they facilitate efficient cold growth of
this bacterium (Angelidis et al., 2002; Ko and Smith,
1999; Sleator et al., 1999). Analysis of cold-sensitive
mutants in which Lmo1078 (Chassaing and Auvray,
2007), and LtrC (Chan et al., 2007) proteins are in-
activated also indicates that these proteins func-
tionally contribute to cold adaptation processes in
L. monocytogenes. The Lmo1078 protein is a UDP-
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
glucose pyrophosphorylase proposed to promote
cold adaptation through enhanced UDP-glucose
production at low temperatures. UDP glucose is an
essential substrate in lipoteichoic acid production
and might facilitate maintenance of architectural in-
tegrity in cell wall and membrane structures leading
to protection of bacterial cells from cold stress dam-
age (Chassaing and Auvray, 2007).
Heat stress adaptation
The understanding of heat stress adaptation in
food-borne pathogens is an important issue since
heating constitutes one of the major food process-
ing and preservation methods. The heat shock re-
sponse is one of the most universal and extensively
studied physical stress responses in living organisms.
This process involves increased production of vari-
ous cell protective protein systems, which ultimately
promotes general environmental stress resistance
and enhanced thermal tolerance (Gandhi and Chi-
kindas, 2007; Klinkert and Narberhaus, 2009; Muga
and Moro, 2008; van der Veen et al., 2007). Similar
to other bacteria, L. monocytogenes synthesizes
a highly conserved set of proteins, also defined as
heat shock proteins (Hsps), upon exposure to high
temperatures (>45°C). Hsps include highly con-
served molecular chaperones and proteases that
functionally prevent nonproductive protein aggre-
gations under different stress environments. GroEL
and DnaK are major Hsps that promote refolding
and degradation of damaged proteins through ATP-
dependent mechanisms (Kandror et al., 1994; Sher-
man and Goldberg, 1996; van der Veen et al., 2007).
These two proteins are highly conserved among liv-
ing organisms and also constitute as the main Hsp
chaperones observed in L. monocytogenes (Gahan
et al., 2001; Hanawa et al., 2000)
Using proteome analysis, the induction of up to
15 Hsps in response to heat shock (48°C/30 min) was
observed using SDS-PAGE (Sokolovic et al., 1990). Of
these, two Hsps were identified as GroEL and DnaK
in L. monocytogenes CLIP 54149 (serotype 1/2a)
based on immunological detection. In another study
the induction of as many as 32 Hsps was observed
using preparative 2DE gels of L. monocytogenes
EGD in response to a temperature shock of 49°C/15
min (Phan-Thanh and Gormon, 1995). One identified
predominant protein, Fri, with molecular weight 18
kDa and pI of 5.1 showed 50.6-fold inductions due
to heat shock. This very same protein spot was 10.5-
fold induced in response to cold shock (Phan-Thanh
and Gormon, 1995). Similarly, other researchers have
also observed the transcriptional induction of fri
transcripts in response to heat (Hebraud and Guz-
zo, 2000; van der Veen et al., 2007) and cold stress
(Dussurget et al., 2005). Phenotypically fri gene null L.
monocytogenes EGDe cells also failed to reach the
maximal optical density compared to the wild type
strain during growth under heat at 45°C (Dussur-
get et al., 2005). These findings together suggest
that ferritin-like protein is important for high and
low temperature adaptation in L. monocytogenes.
Recently, Agoston et al. (2009) compared the effect
of mild and prolonged heat treatments on L. mono-
cytogenes cells using 2DE analysis. In line with the
reduced metabolic activity at suboptimal tempera-
ture, large numbers of metabolic proteins were sup-
pressed during heat exposure in this study which is
also consistent with the observation from other stud-
ies (Phan-Thanh and Gormon, 1995; Phan-Thanh and
Jansch, 2006). Importantly, L. monocytogenes stress
protein DnaN, a beta subunit of polymerase III, was
highly induced in response to different heat shock
treatments. Observed induced expression of DnaN,
involved in DNA synthesis process, may indicate its
role in increased synthesis of some HSPs.
Osmotic stress adaptation
The osmotolerance of L. monocytogenes is an-
other property crucial to survival and growth of this
pathogen at high salt levels and low water activity
environments encountered in conserved food prod-
ucts. Osmotic stress adaptation in microorganisms
depends on the modulation of both ionic and or-
ganic solute pools so as to sustain cytoplasmic water
and turgor pressure at levels, which are compatible
with cell viability and growth at low water activity
(Booth and Louis, 1999; Wood, 2007).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
L. monocytogenes cells cope with elevated levels
of osmolarity through appropriate changes in pro-
tein expression levels. Significant modulation (ap-
proximately 32 proteins) in protein expression under
hyper osmotic conditions (3.5% to 6.5% NaCl con-
centration) was first visualized in preparative 2DE
gels (Esvan et al., 2000) and some of the salt stress
adaptation proteins were later identified (Duche
et al., 2002a,b). Identified osmotic stress proteins
include those related to general stress (Ctc and
DnaK), transporters (GbuA and AppA), ribosomal
proteins (RpsF, 30S ribosomal protein S6), as well as
proteins involved in general metabolism processes
(Ald, CcpA, CysK, TufA (EF-Tu), Gap, GuaB, PdhA,
PdhD, and Pgm) (Duche et al., 2002a,b). Among the
salt stress induced proteins, the role of Ctc in osmo-
tolerance was further characterized by Gardan et al.
(2003b), who demonstrated that ctc gene is involved
in L. monocytogenes osmotolerance. They found
that growth of the ctc mutant strain was signifi-
cantly impaired compared to its isogenic wild type
L. monocytogenes LO28 strain in minimal medium
with 3.5% NaCl.
Other than the differential expression of salt stress
proteins, increased uptake of glycine betaine and
carnitine osmolytes via betL, gbu, and opuC en-
coded transporter porters is crucial under hyper-os-
motic conditions. Accumulation of these osmolytes
prevents the intracellular water loss by counteracting
external osmolarity and keeping the macromolecu-
lar structure of the cells intact. Indeed, the induced
expression (>2-fold) of GbuA transporter protein un-
der high osmolarity (3.5% NaCl) has been observed
in 2DE analysis of L. monocytogenes LO28 (Duche
et al., 2002a). Meanwhile the induction of compat-
ible solute transporter encoding genes, betL, gbu,
and opuC in response to higher osmolarity has been
reported at the transcriptional level in L. monocyto-
genes cells (Fraser et al., 2003). Interestingly these
transporter systems expressed under hyper-osmotic
stress conditions are the same as the ones expressed
under cold stress (Mendum and Smith, 2002; Smith,
1996; Wemekamp-Kamphuis et al., 2004b), suggest-
ing that some of the mechanisms counteracting os-
motic and cold stress may be common in L. mono-
cytogenes. Moreover, the cold shock protein CspD
also facilitates both osmotic and cold stress adapta-
tion in L. monocytogenes and a mutant strain lack-
ing cspD gene also display a stress sensitive phe-
notype under NaCl salt stress conditions (Schmid et
al., 2009). Other important proteins in L. monocyto-
genes salt stress adaptation are HtrA (Wonderling
et al., 2004) and Lmo 1078 (Chassaing and Auvray,
2007). The HtrA protein is a general stress response
serine protease that contributes to osmotic stress
adaptation functions through its role in degradation
of salt stress damaged proteins. At the phenotypic
level the L. monocytogenes htrA null mutant displays
diminished growth in presence of NaCl stress. The
Lmo1078 promotes both cold and osmotic tolerance
based on its proposed functional contribution to
maintenance of cell wall and membrane architectur-
al integrity in this bacterium. The CstR transcriptional
repressor protein is also involved in modulation of L.
monocytogenes osmotic stress tolerance functions
since a CstR null mutant of this bacterium displays
improved growth under NaCl salt stress conditions
(Nair et al., 2000b).
Acid stress adaptation
The adaptation of microorganisms to acid stress
environments includes significant gene and protein
expression changes associated with, among other
response, the mobilization of cellular mechanisms
that consume acids and generate basic amines
(Foster, 2004; Merrell and Camilli, 2002). L. monocy-
togenes cells face acid stress conditions in low pH
foods and at various stages during human infection.
L. monocytogenes counteracts acidic stress condi-
tions by production of various acid stress response
proteins (ASPs). ASPs were initially designated based
on their location on the preparative 2DE gels (Davis
et al., 1996; O’Driscoll et al., 1997) and some were
later identified by PMF (Phan-Thanh and Mahouin,
1999; Wemekamp-Kamphuis et al., 2004a). Some
of the identified ASPs include: proteins involved in
respiration (enzyme dehydrogenases and reductas-
es), osmolyte transport (GbuA), protein folding and
repair (Chapronin, GroEL, ClpP), general stress re-
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
sponse (sigma H homologous of B. subtilis), flagella
synthesis (FlaA), and metabolism (Pfk, GalE) (Phan-
Thanh and Mahouin, 1999; Wemekamp-Kamphuis
et al., 2004a).
The acid tolerance response (ATR) is character-
ized by increased microbial cell resistance to le-
thal acid after an exposure to mild acidic condition
(Koutsoumanis et al., 2004). This phenomenon has
been examined by a number of studies in L. mono-
cytogenes cells. When the synthesis of ASPs in L.
monocytogenes LO28 produced under both mild
(pH 5.5 for 2 h) and lethal (pH 3.5 for 15 min) acidic
conditions were compared to a normal pH (~7.2), a
total of 37 proteins were induced under mild acidic
treatment and 47 under lethal acidic treatment, with
23 of the induced proteins being common under
both conditions (Phan-Thanh and Mahouin, 1999).
The different aspects of acid stress adaptive mecha-
nisms in L. monocytogenes are well elucidated from
acid stress adaptation mechanisms studies in this
bacterium (Abram et al., 2008a; Ferreira et al., 2003;
Phan-Thanh and Jansch, 2006; Ryan et al., 2008b ). In
brief, when exposed to a lower external pH, bacte-
rial cells attempt to maintain their cytoplasmic pH by
decreasing the membrane permeability to protons,
buffering their cytoplasm, and by equilibrating the
external pH through catabolism (Phan-Thanh and
Jansch, 2006). One of the ways that limit the bac-
terial permeability to proton is through changes in
the lipid bilayer of cell membrane. Giotis et al. (2007)
suggested that there was an increased concentration
of straight chain fatty acids and decreased concen-
tration of branched chain fatty acids in L. monocy-
togenes 10403S cells grown under acidic conditions
(pH 5.0 to 6.0) compared to neutral pH. Another im-
portant approach that the bacterial cells use for dis-
pelling the protons outside the cells is to accelerate
electron transferring reactions through enhanced
oxidation reduction potential. The ASPs identified
as dehydrogenases (GuaB, PduQ and lmo0560) and
reductases (YcgT) together with respiratory enzymes
are implicated to play an important role in main-
taining pH homeostasis by active proton transport
(Phan-Thanh and Jansch, 2006).
Organic acid salts such as sodium lactate and
sodium diacetate are extensively used in ready-to-
eat (RTE) meat products as antiListerial food preser-
vatives. Recently Mbandi et al. (2007) used 2DE to
evaluate the protein induction in L. monocytogenes
Scott A by these organic salts. Experiments were
conducted in defined medium with either sodium
lactate (2.5%) or sodium diacetate (0.2%) or in com-
bination. Some of the proteins that showed substan-
tial up or down regulation (>10 fold) were identified
using PMF. Oxidoreductase and lipoproteins were
upregulated whereas DNA-binding proteins, alpha
amylase and SecA were repressed during exposure
to these organic acid salts. Identified enzyme protein
oxidoreductase in L. monocytogenes has been pre-
viously suggested to be involved in dispelling proton
molecules to maintain cell homeostasis (Phan-Thanh
and Jansch, 2006).
The glutamate decarboxylase (GAD) and arginine
deiminase (ADI) are well described major acid adap-
tive mechanisms in L. monocytogenes. L. monocyto-
genes LO28 strain with a mutation in genes of GAD
proteins GadA, GadB and GadC displayed higher
acid stress sensitivity in an acidified reconstituted
skim milk background (Cotter et al., 2001b) and gas-
tric fluid (Cotter et al., 2001a). The L. monocytogenes
ADI system includes proteins ArcA, ArcB and ArcC
and ArcD for the conversion and transfer of arginine
into ornithine and deletion in functional genes of
ADI leads impaired growth in mildly acidic condi-
tions (pH 4.8) and survival in lethal pH conditions (pH
3.5) (Ryan et al., 2009).
Alkaline stress adaptation
L. monocytogenes cells are more resistant to
alkaline stress in comparison to other foodborne
pathogens such as Salmonella Enteritidis and E. coli
O157:H7 (Mendonca et al., 1994). At pH 12, L. mono-
cytogenes F5069 (serotype 4b) cell concentrations
decreased by only 1-log in 10 min compared to 8-log
reductions observed for E. coli and S. Enteritidis
within 15 s. Earlier, 2DE analysis of alkaline stressed
(pH 10.0 for 35 min) L. monocytogenes EGDe cells by
Phan-Thanh and Gormon (1997) showed induction
of 16 proteins, synthesis of 11 novel proteins, and
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
repression of nearly half of the total proteins in com-
parison to non-stressed cells. Recently, Giotis et al.
(2008) also reported the repression of a large number
of proteins along with synthesis of 8 novel proteins
in response to alkaline stress of L. monocytogenes
10403S strain. In addition to proteomic analysis, they
also evaluated the alkaline stress adaptive mecha-
nism using microarray transcriptional profiling and
found 390 gene transcripts differentially expressed
(Giotis et al., 2008). Protein identification of four dif-
ferentially expressed proteins by peptide-mass map-
ping revealed induction of heat shock proteins DnaK
and GroEL and repression of DdlA (alanine ligase)
and AtpD (ATP synthase). These identified proteins
spots were also found to be induced or repressed in
microarray analysis. In addition, screening library of
Tn917- lac insertional mutants in L. monocytogenes
LO28 identified 12 mutants sensitive to alkaline con-
ditions, though identification of transposition target
suggest they all carried mutations in only putative
transporter genes (Gardan et al., 2003a).
High hydrostatic pressure (HHP) stress adaptation
L. monocytogenes cells undergo mechanical
stress following HHP treatment. The usual pressure
range employed in HHP is in the range of 200-600
MPa for 5-10 min depending on the food matrices.
Such high pressure damages the cell membrane and
results in leakage of cell content along with disso-
ciation of protein complexes (Gross and Jaenicke,
1994). However, HHP treated L. monocytogenes
cells were found to be sublethally injured with their
metabolic-activity largely maintained and had the
potential for a gradual recovery (Ritz et al., 2006). In
addition although L. monocytogenes cells in HHP
treated cooked ham displayed a lag phase lasting
up to 1.5 months, they subsequently recovered to
grow more than 5-logs over 3 months (Aymerich et
al., 2005).
To characterize the HHP induced proteins en-
abling resistance to mechanical stress, Jofre et al.
(2007) conducted 2DE analysis of L. monocytogenes
CTC1011 (serotype 1/2c) after treatment with 400
MPa for 2 h and observed expression of 23 proteins
being modulated. These high pressure induced pro-
teins were related to ribosomal function (RplJ, RplL,
RpsF, RpsB, IleS, GatA), transcription (GreA), protein
degradation (PepF, PepT), protein folding (GroES),
metabolism (PflB, Pta, Zwf, Ald,), general stress (Fri)
and unknown functions. Of these high pressure in-
duced proteins, chaperone GroES may be necessary
in refolding of dissociated protein complexes follow-
ing HHP treatment, and peptidases (PepF, PepT) may
contribute to degradation of proteins that cannot be
folded by molecular chaperones. Flp has been pre-
viously elucidated to have roles in cold, heat, and
oxidative stress adaptation (Dussurget et al., 2005).
Moreover, L. monocytogenes shows increased resis-
tant to HHP treatment following prior exposure to
cold stress along with induced expression of cold
shock proteins following HHP treatment.
Implications of L. monocytogenes stress adaptation to virulence responses
The stress responses of L. monocytogenes are
not only important in survival of hostile external
and food-associated environments but also dur-
ing host colonization processes. The pathogenicity
of food-borne L. monocytogenes also depends on
their physiological status at infection, which is deter-
mined by, among other factors, the environmental
stress challenges encountered and stress responses
activated prior to interaction with susceptible hosts.
Besides the fact that acid stress adaptation of this
bacterium promotes survival in acidic food environ-
ments, this process has been also shown to modu-
late various aspects of virulence in this pathogen. As
an example, the pathogenic potential of this bacte-
rium can be increased through improved viability in
the gastrointestinal tract, which includes increased
survival of the gastric acid stress challenges. The
increased expression of virulence genes as well as
enhanced cell adhesion and invasion has been re-
ported in association with acid stress adaptation of
L. monocytogenes cells (Conte et al., 2000; Garner et
al., 2006; Olsen et al., 2005; Werbrouck et al., 2009).
Conte et al. (2000) detected enhanced Caco-2 cell
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
invasion ability, in addition to improved survival and
proliferation in activated murine macrophages of L.
monocytogenes cells preadapted by mild organic
acid stress exposure. Werbrouck et al. (2009) also
described increased cellular invasiveness and inlA
mRNA levels in their analysis of acid stress adapted
L. monocytogenes cells. In similar fashion there was
an increased transcription of virulence genes such as
prfA, inlA and inlB, as well as enhanced adhesion and
invasion of Caco-2 cells in two L. monocytogenes
strains adapted to prolonged acid stress (Olesen et
al., 2009). Another stress commonly encountered by
L. monocytogenes cells in food associated environ-
ments considered to potentially influence virulence
of this bacterium is NaCl osmotic stress. NaCl stress
exposure is associated with increased expression of
various general stress resistance and virulence genes
in this bacterium suggesting that osmotic stress ad-
aptation events along the food supply chain may
enhance subsequent pathogenicity (Kazmierczak et
al., 2003; Olesen et al., 2009; Sue et al., 2004). Phe-
notypically increased cell adhesion and invasion in
vitro has been observed in NaCl stress adapted L.
monocytogenes cells (Garner et al., 2006; Olesen
et al., 2009). The significance of these phenotypic
observations however remains to be further exam-
ined. One study, which examined the growth of
some food environment persistent strains and clini-
cal isolates under NaCl osmotic stress, was not able
to detect significant influence of this stress exposure
on pathogenicity of these strains using several viru-
lence models (Jensen et al., 2008). Similarly, Wałecka
et al., (2011) did not find increased expression of in-
ternalins with salt stress and suggested that bacterial
growth phase instead of salt stress was direct deter-
minant of L. monocytogenes invasiveness. Hence
the above mentioned reports determining the in-
volvement of salt stress show conflicting findings
and more work in this direction would be required
to understand the factors that result in such differ-
ing view. The expression of prfA controlled virulence
genes and cell invasion capacity of L. monocyto-
genes cells is temperature dependent and pathoge-
nicity in some meat-processing plant derived strains
of this bacterium was reported to decrease during
long term cold storage at 4°C (Duodu et al., 2010;
Johansson et al., 2002; McGann et al., 2007). Simi-
larly, cold stress exposed wild type and mutants lack-
ing csp genes in the L. monocytogenes EGDe strain
were significantly impaired in cell invasion relative
to corresponding controls grown at 37°C (Loepfe et
al., 2010). Temperature dependent virulence gene
expression repression as well as membrane damage
and cell surface modifications in these organisms ex-
posed at low temperatures might lead to phenotypic
virulence defects observed in cold adapted L. mono-
cytogenes organisms.
Van de Velde et al. (2009) compared proteomes
between L. monocytogenes cells grown in human
THP-1 monocytes versus those growing extracel-
lularly in TSB broth using 2D-DIGE. Down regula-
tions of general stress protein Ctc and oxidative
stress protein Sod was detected suggesting that
compared to extra cellular environment the intra-
cellular uptake by host cells may be more favorable
environment for L. monocytogenes survival and ad-
aptations. Shin et al. (2010) observed the increased
σB activity, as measured by ß-galactosidase lacZ pro-
moter assay, to vancomycin antibiotic stress. While
subsequent proteome analysis of L. monocytogenes
σB wild type and null mutant strains using LC-ESI-
MS/MS also revealed among other proteins the in-
creased production of the virulence protein InlD. Fri
protein is another general stress response protein
with virulence promoting functions in L. monocyto-
genes. It has been shown by using both mice chal-
lenge and macrophage cell virulence models that
fri null strains of L. monocytogenes are significantly
impaired (Dussurget et al., 2005; Mohamed et al.,
2006; Olsen et al., 2005). Proteome analysis of the fri
mutant and wild type strain was compared to reveal
repression in Hly (Listeriolysin O) and stress response
proteins CcpA (Catabolite control protein A) and
OsmC (Dussurget et al., 2005).
The stress induced chaperone proteins ClpB,
ClpC, ClpE, ClpP have all been shown to provide
virulence promoting activities in L. monocytogenes
and thus it is possible that their induction in this
bacterium in response to stress in food associated
environments also increases the capacity of stress
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
adapted organism to survive hostile host envi-
ronments as well as enhance their pathogenicity
(Chastanet et al., 2004; Gaillot et al., 2000; Nair et
al., 1999; 2000a;). Meanwhile Olesen et al. (2009)
in their recent study showed that acid exposed L.
monocytogenes cells displaying increased Caco-
2 cell virulence also displayed increased expres-
sion of genes encoding the ClpC and ClpP. The
RNA binding regulatory protein Hfq, is another
general stress response modulating protein which
has been shown to protects cells from osmotic and
ethanol stress as well as facilitate enhanced patho-
genicity in L. monocytogenes infected mice (Chris-
tiansen et al., 2004). Stack et al. (2005) found that
the HrtA serine protease, which protects L. mono-
cytogenes from various stresses including expo-
sure to acidic conditions also contributes towards
virulence capablities of this bacterium. The gen-
eral stress response protein σB, which facilitates L.
monocytogenes adaptation to multiple stresses
has also been shown to promote virulence and cell
invasiveness in this bacterium (Garner et al., 2006;
Ivy et al., 2010). Recently it was shown that the im-
portance of σB responses in these aspects might
be lineage specific with its activity being important
in lineage I, II, IIIB strains but not in IIIA (Oliver et
al., 2010).
Role of alternative sigma factor (σB ) in L. monocytogenes stress adaptation
In L. monocytogenes, σB is a major stress re-
sponse regulator and mutant strain lacking σB ac-
tivity shows increased sensitivity to a wide range of
stresses including cold (Becker et al., 2000; Chan
et al., 2007; 2008; Moorhead and Dykes, 2004;
Raimann et al., 2009; Wemekamp-Kamphuis et al.,
2004a; ), heat (Hu et al., 2007a,b; van der Veen et
al., 2007), osmotic (Becker et al., 1998, Fraser et al.,
2003; Okada et al 2008; Raimann et al., 2009), acid
(Cotter et al., 2001a,b; Ryan et al., 2008a; Weme-
kamp-Kamphuis et al., 2004a), and HHP (Weme-
kamp-Kamphuis et al., 2004a). The main role of σB
in L. monocytogenes is to regulate the expression
of various stress response associated genes. As an
example, Flp is a general stress protein involved
in cold, oxidative and heat stress adaptation. The
expression of fri gene encoding Flp protein is par-
tially regulated through σB-dependent pathways in
L. monocytogenes 10403S (Chan et al., 2007).
To identify the proteins that show σB dependent
expression in the acidic conditions, 2DE analysis of
acid adapted (pH 4.5) and non-adapted cells (both
wild type and σB mutant) was performed (Weme-
kamp-Kamphuis et al., 2004a). The expression of 9
proteins was dependent on σB during acid stress
and some of these proteins were identified using
PMF. The identified proteins with σB dependent
expression in response to HHP stress included Pfk,
GalE, ClpP, and Lmo1580. The Pfk (6-phospho-
fructokinase) and GalE are enzymes involved in
glycolysis and sugar metabolism, respectively, and
ClpP is the ATP-dependent chaperone protease
that plays a role in preventing the accumulation of
misfolded proteins. The induction of ClpP protein
expression may be necessary in acidic conditions
to help in resolution of protein aggregations that
are likely to occur due to acid stress induced pro-
tein damage.
Recently, the role of σB regulon on L. monocyto-
genes 10403S cells grown to stationary phase in the
presence or absence of 0.5 M NaCl was evaluated
using both 2DE and iTRAQ (Abram et al., 2008b).
Using a combination of these two approaches a
total of 38 proteins (17 induced and 21 repressed)
were identified whose expression was σB depen-
dent. Among these σB controlled proteins, 10
proteins (7 positively regulated and 3 negatively
regulated by σB) were further classified based on
their potential role in stress related functions. Of
these 7 σB positively regulated proteins, two pro-
teins OpuC and HtrA were previously conferred to
have role in L. monocytogenes stress adaptation
(Fraser et al., 2003; Wonderling et al., 2004). OpuC
is involved in osmolyte transfer needed for os-
motic and cold adaptation (Fraser et al., 2003) and
HtrA serves as a protease whose deletion leads to
growth defects under NaCL stress (Wonderling et
al., 2004). Intracellular accumulation of glycine be-
taine and carnitine osmolytes is necessary in cold
xx Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011
as well as osmotic stress. Expressions of the os-
molyte transporter proteins, Gbu and Opu, have
been shown to be at least partially dependent
on the σB activity (Cetin et al., 2004; Fraser et al.,
2003;). Also L. monocytogenes 10403S strain with
a null mutation in the σB gene showed substantial
defects in its ability to accumulate glycine betaine
and carnitine osmolytes (Becker et al., 1998; 2000).
Moreover, σB deletion impairs the ability of L.
monocytogenes 10403S cells to withstand against
heat stress (55°C for 30-60 min) and class II heat
shock genes, which also includes the osmolyte
transporter gene opuC, are positively upregulated
following heat shock (48°C for 3 min) in L. mono-
cytogenes EGDe strain (Hu et al., 2007a; van der
Veen et al., 2007). Using transcriptional analysis,
Ryan et al. (2008a) reported the induction of the σB
in response to sublethal levels of detergent stress.
In addition, following HHP treatment of 300 MPa
of 20 min, the parent strain (EGDe) showed 100-
fold higher survival compared to σB mutant strain
(Wemekamp-Kamphuis et al., 2004a).
Apart from σB, other sigma factors σc, σH, and
σL (RpoN) are also known to play important roles
in stress adaptation of L. monocytogenes. L.
monocytogenes strain lacking σB, σc, σH encoding
proteins have been shown to have significantly
impaired growth compared to wild type strain at
4°C for 12 days (Chan et al., 2008). Raimann et
al. (2009) reported that L. monocytogenes strain
lacking σL has impaired cold growth due to in
part by the repressed transcript production of oli-
gopeptide-binding OppA protein that facilitates
accumulation of short peptide substrates which
are also important for efficient cold growth in this
bacterium (Borezee et al., 2000). Absence of σc in-
creases the L. monocytogenes sensitivity to ther-
mal treatment, thus highlighting the importance
of this regulatory factor in conferring L. monocy-
togenes adaptation to heat stress (Zhang et al.,
2005). σL aids in L. monocytogenes ability to grow
at high salt concentrations (Okada et al., 2006) as
well as control carbohydrate metabolism through
its influence on expression of phosphotransferase
system genes (Arous et al., 2004).
ConCluSIon And FuTuRe PeRSPeCTIveS
The ability of L. monocytogenes cells to survive
adverse physiological conditions is a serious food
safety and public health concern. The physiological
changes in response of environmental stress stimuli’s
reflect key changes instituted by microbial cells at
gene or protein expression levels. In the future an
improved understanding of fundamental changes
occurring at genes or proteins level in L. monocy-
togenes cells in response to adverse environmental
conditions will provides new insights that can be har-
nessed in developing more effective practical food
preservation approaches (Gandhi and Chikindas,
2007; Tasara and Stephan, 2006).
The physiological changes mounted in response
to particular environmental stress stimuli in L. mono-
cytogenes are a consequence of changes at gene
transcription and/or protein expression levels. The
cold adaptive nature of this organism is probably
one of the most important concerns to food produc-
tion due to the ability of this pathogen to grow and
achieve high concentrations in long shelf life ready-
to-eat products preserved by refrigeration. Vari-
ous cold adaptive mechanisms such as synthesis of
conserved cold shock proteins (Schmid et al., 2009),
increased uptake of cryoprotective osmolytes (An-
gelidis and Smith, 2003), increased membrane per-
miablity (Borezee et al., 2000), increased production
of general stress proteins Fri (Dussurget et al., 2005),
etc have been identified that may directly or indi-
rectly confer this bacterium with an ability to multiply
and/or survive at lower temperatures. However, at
this stage it is unclear if these different mechanisms
work in any coordinated manner or if they work on
separate niches leading overall cold stress resistance
of L. monocytogenes cells. Future experiments are
warranted to understand the complex hierarchy be-
tween these different stress response mechanisms.
One way to do this would be to conduct gene knock
out studies where the related genes/proteins of a
particular stress adaptive mechanism (i.e. deletion of
cold shock proteins) is deleted and use these strains
to understand the modulations in genes/proteins of
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 1, Issue 1, 2011 xx
other stress mechanisms. The adaptation of this bac-
terium to osmotic stress also involves complex sets
of cellular responses. Notably some osmotic stress
response mechanisms, such as compatible solute
uptake systems as well as alternative sigma factors
are also involved in cold stress adaptation (Fraser et
al., 2003; Wemekamp-Kamphuis et al., 2004b), which
suggests that some cellular response mechanism to-
wards food related environmental (cold and osmotic)
stresses in this bacterium are common.
The main limitation of current studies is that
large numbers of genes/proteins are tabulated as
being differentially expressed but there is little or
no insight on what the modulations in these gene/
proteins mean. In any event, perturbation in physiol-
ogy of living cells is likely to change the expression
levels of various genes/proteins. Such information is
of limited value without further functional character-
izations of such putative stress adaptation genes or
proteins. While it may not be practical to use such
approach for hundreds of genes/proteins that are
differentially expressed along with each stress, it is
necessary to do follow-up studies on genes/proteins
that exhibit substantially large changes in expression
level. So far only in a few cases of stress proteins has
the follow-up work been done in elucidating their
molecular roles during stress adaptation of L. mono-
cytogenes. Some key examples are: (a) Flp protein,
first identified to be highly induced in cold and heat
stress, and subsequently confirmed through fri mu-
tant strain of L. monocytogenes EGDe, which is im-
paired under both stress conditions (Dussurget et
al., 2005; Hebraud and Guzzo, 2000; Phan-Thanh and
Gormon, 1995); (b) Ctc protein is induced under salt
stress and L. monocytogenes LO28 ctc mutant strain
is found defective in growth under NaCl stress con-
ditions (Duche et al., 2002a; Gardan et al., 2003b);
and (c) GbuA osmolyte transporter protein, induced
under high osmolarity, (at 3.5% NaCl) was confirmed
by gbu mutant strain of L. monocytogenes LTG59
as defective in growth in the absence of osmolyte
uptake activity (Duche et al., 2002a; Mendum and
Smith, 2002). Moreover most of the current stress
adaptation findings are based on laboratory media
and it is crucial that to design new experimental
strategies that detect stress adaption response in L.
monocytogenes cells exposed to different food ma-
trices. The experiments with food substrate may be
designed to see how different food components and
food preservatives modulate the expression of stress
proteins identified using broth media.
ACknowledgemenT
This research was supported in part by Food Safe-
ty Initiative award to RN by the Mississippi Agricul-
tural and Forestry Experiment Station (MAFES), Mis-
sissippi State University.
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