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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


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


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( )


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


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


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


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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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


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.


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


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


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


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-


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.


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


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


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


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


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


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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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


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


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

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


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


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



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


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.


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


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).


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.


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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


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.


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


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


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-


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).


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-


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


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


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


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


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


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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