Chapter II Isolation identification and characterization of alkaliphilic bacteria from different habitats

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2.1. Introduction Extremophilic microorganisms exhibit the ability to grow at the limits of environmental

factors-pH, temperature, salinity, and pressure-which critically influence growth. Among

these organisms, the immense potential of alkaliphiles (syn. alkalophile) has been

realized since the 1960s, primarily due to the pioneering work of Horikoshi (1999).

Products of industrial importance from alkaliphiles have been commercialized, the most

successful of which have been in the detergent and food industries. It is noteworthy that

industrial production of products from alkaliphiles is so far insufficient to meet the

demands.

An industrial study document shows that the enzyme industry worldwide is valued at

$5.1 billion and is predicted to show an annual increase in demand of 6.3 %. Specialty

enzymes with process-specific characteristics and those used for animal feed processing

and ethanol production are envisaged to have increased demand. The study also forecasts

that while developed countries are likely to show increased market share, developing

countries will show the best growth. Alkaline enzymes have a dominant position in the

global enzyme market as constituents of detergents. So, it is pertinent to examine the role

of alkaliphiles from which most of the commercial enzymes are obtained. This review

focuses on the commercialized enzymes and other interesting products from alkaliphilic

bacteria, which could be produced on an industrial scale.

Alkaliphiles consist of two main physiological groups of microorganisms; alkaliphiles

and haloalkaliphiles. Alkaliphiles require pH of 9 or more for their growth and have an

optimal growth pH of around 10, whereas haloalkaliphies require both an alkaline pH

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(>pH 9) and high salinity (up to 33 % NaCl). Alkaliphiles have been isolated mainly

from neutral environments, sometimes even from acidic soil samples and feces.

Haloalkaliphiles have been mainly found in extremely alkaline saline environments such

as Rift Valley lakes of East Africa and the western soda lakes of the United States.

Alkaliphilic microorganisms are not only found in areas having neutral or high pH but

have also been isolated from acidic soil (Horikoshi 1999). The neutral and acidic sites

probably have some alkaline pockets where the alkaliphiles thrive. While these organisms

can be facultative or obligate alkaliphiles, sub-groups can include psychro, meso, thermo,

and haloalkaliphiles. The true alkaliphiles, by and large, grow at and above pH of 9.0 and

show optimal growth pH of 10.0. Alkaline environments can be those with high or low

Ca++. The thermoalkaliphiles (growing optimally at alkaline pH ranges in addition to

temperatures above 50°C) and haloalkaliphiles (requiring high salinity and alkaline pH)

are promising in terms of production of biomolecules suited for industrial applications.

Enzymes from these microorganisms have found major commercial applications such as

in laundry detergents, for efficient food processing, in finishing of fabrics, and in pulp

and paper industries. The major products obtained are described in the following sections.

2.2. Materials and methods 2.2.1. Chemicals

Carboxymethylcellulose (CMC, medium viscosity, 400-800 cP), cellulose powder

(Sigma cell Cellulose, Type 20; particle size-20 μm), oat spelt xylan (OSX), locust bean

gum (LBG), starch, pectin, gelatin and tween-80 were purchased from Sigma Chemical

67

Company (St. Louis, MO, U.S.A). Pectin, tannic acid, starch and casein were purchased

from Himedia Chemicals, Mumbai, India. All other reagents were of analytical grade.

2.2.2. Sample collection and Isolation of alkaliphilic bacteria

Nearly 50 samples were collected from different habitats employing enrichment culture

technique from grain mill effluents of Gulbarga City, Karnataka, India and were streaked

on 1.5% agar plates containing a basal medium containing 0.5 % peptone, 0.2 % yeast

extract, 1.0 % glucose, 0.1 % K2HPO4, 0.5 % NaCl, 0.02 % MgSO4.7H2O. The pH of the

medium was adjusted to around 9 and 10. After 48-72 hours of incubation white, creamy,

yellowish, yellow, orangish, orange and transparent colonies of the alkaliphiles appeared

on these agar plates. Different colonies were picked and re-streaked several times to

obtain pure cultures. They are isolated as pure cultures and further grown in 250 ml

Erlenmeyer flasks containing 50 ml of above fermentation medium. Flasks were

inoculated with 1.0 ml of old culture and incubated at 37°C in a rotary shaker at 180 rpm

for 48 h. the flasks were removed at regular intervals, the contents centrifuged and the

supernatant was used as enzyme source.

2.2.3. Morphological, cultural and physiological characteristics

Isolated strains were examined for colony, cell morphologies and cell motility. Colonial

morphologies were described by using standard microbiological criteria with special

emphasis on pigmentation, diameter, colonial elevation, consistency and opacity (Oren et

al. 1997). These characters were described for cultures grown at optimum temperature,

pH and salt concentration. Isolated strains were examined for motility and morphological

features in wet mounts. Cell morphology was examined by light microscopy of the

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exponentially growing liquid cultures. Gram staining was performed by using acetic acid

fixed samples as described by Dussault (1955).

2.2.4. Antibiotic tests

The antibiotic sensitivity of alkaliphilic strains were examined by spreading bacterial

suspension on agar plates containing above mentioned mineral salt medium and applying

antibiotic discs of bacitracin (10 µg), amphicillin (10 µg), gentamycin (10 µg),

tetracycline (10 µg), cefadroxil (10 µg), cephataxime (10 µg), and oflaxacin (10 µg). The

results were recorded in terms of resistance or sensitivity after 5 days of incubation at 37

°C with sensitivity being defined as the appearance of a zone of inhibition extending at

least 2 mm beyond the antibiotic disc.

2.2.5. Extracellular cellulase activity

Cellulolytic activity of the cultures was screened qualitatively in a saline medium

containing 0.5% carboxymethylcellulose and 10-20 % total mineral salts (Ventosa et al.

1982) in 50 mM phosphate buffer of pH 9.0 sterilized at 121°C for 15 min. about 15 ml

of the medium was poured in a petridish under aseptic conditions and inoculated with the

isolated microorganisms and incubated at 37°C for 48 h. then the plates were flooded

with 2% KI in 0.2 g of iodine solution. The brown color developed in the petriplates and

clear zones were seen around the colonies indicating the hydrolysis of

carboxymethylcellulose by the enzyme.

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2.2.6. Assay of xylanase activity

Xylanase activity of the isolates was detected by screening for zones of hydrolysis around

colonies growing on above mentioned salt medium containing 1% oat spelt xylan (OSX),

after incubation for 48-60 hours.

2.2.7. Extracellular protease activity

Proteolytic activity of the culture was screened qualitatively in a saline medium

containing milk (50%) plus 10-20% total salts (Ventosa et al. 1982) supplemented with

0.5% (w/v) yeast extract and 1% peptone. The medium was solidified by adding 20g/l of

agar. Zones of precipitation of para-casein around the colonies appearing over the next

48-60 hours were taken as evidence of proteolytic activity.

2.2.8. Assay of gelatinolytic activity

The medium contained 2 % (w/v) agar, 1 % (w/v) gelatin in 50 mM glycin NaOH buffer

pH 11.0 sterilized at 120° C for 15 min. About 15ml of the medium was poured in a

petridish under aseptic conditions. Using a sterilized cork borer, two 6mm diameter cups

were made in each of the agar plate. The culture filtrate of the isolated alkaliphilic

bacterium was added carefully into each well. The petridishes were incubated at 37°C

for 48h. After incubation plates were developed with 15% (w/v) mercuric chloride. After

10 min a clear transparent zone, indicated hydrolysis of the gelatin by extracellular

proteases whereas the rest of the plates became opaque due to the coagulation of gelatin

by HgCl2. The diameter of the clear zones was used as a measure of protease activity.

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2.2.9. Extracellular amylase activity

1 % starch and 2 % agar were taken and mixed with 100 ml of above mentioned media

and autoclaved. After solidification of agar the plates were inoculated. These plates were

incubated for 48-60 hours and plates were developed with 2 % KI in 0.2g iodine solution.

The blue color developed in the petri-plates and clear zones were seen around the

colonies indicating the hydrolysis of the starch by the enzyme.

2.2.10. Extracellular lipolytic activity

Lipolytic activity of the isolates was detected by screening of zones of hydrolysis around

colonies growing on above mentioned salt medium containing 1 % Tween -80, after

incubation for 48-60 hours.

2.2.11. Extracellular xylanase activity

Microorganisms were grown in plates containing substrate 0.2 % xylan and incubated for

48-60 hours at 37°C. The xylanolytic activity was detected by flooding with 1 % congo

red solution; a clear zone of hydrolysis indicated the xylanolytic activity.

2.2.12. Biochemical tests

Inoculants for the various biochemical tests were prepared by growing cells of strain

VSG-1 and VSG-5 were aerobically cultured at 37°C in a basal salt medium containing

0.5 % peptone, 0.2 % yeast extract, 0.5 % glucose, 0.1 % K2HPO4, 0.5 % NaCl, 0.02 %

MgSO4.7H2O. The pH of the medium was adjusted to 9.0 and 10.0 using phosphate

buffer respectively. Gelatin, cellulase, xylanase, mannanase, pectinase, amylase, tannase

activities, tween 80 hydrolysis, indole production, methyl red and Voges-Proskauer tests

71

were performed according the procedure mentioned by Birbir and Sesal (2003). The

results were recorded at 48 h of incubation at 37°C.

2.2.13. 16S r DNA sequencing

The cultures were allowed to grow for 48 h. Single colony was re-suspended in 20 µl of

50 mM Tris-HCl-EDTA saline (pH 7.2). The bacterial suspensions were incubated for 10

min at 95°C and centrifuged at 18,500 X g for 2 min. The supernatants were amplified

from the total genomic DNA samples. The bacterial 16 S rRNA genes were amplified

from the genomic DNA using universal eubacteria specific primers which yielded a

product of 1029 and 1024 base pairs. The PCR conditions were 35 cycles of 95°C,

denaturation for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min, in

addition one cycle of extension at 72°C for 10 min. The PCR products were purified by

PEG-NaCl precipitation as described by Sambrook et al. (1989). Briefly, the PCR

products were mixed with 0.6 volumes of PEG-NaCl solution (20 % PEG 6000, 2.5 M

NaCl) and incubated for 0 min. The pellet was washed twice with 70 % ethanol and dried

under vaccum which were then re-suspended in glass distilled water at concentration of

>0.1pmol/ml. The purified products were sequenced by Ocimum Biosolutions,

Hyderabad. The nucleotide sequence analyses were of the sequences was done at

BLAST-n site at NCBI server (www.ncbi.nlm.nih.gov/BLAST). The sequences were

refined manually after cross-checking with the raw data to remove ambiguities and were

submitted to Genbank with the accession numbers, JQ312121 and JQ272845

respectively. The phylogenetic tree was constructed using the aligned sequences by the

neighbor-joining method using MEGA 5.1 software.

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2.3. Results

Screening of bacteria from different environments in south India (Karnataka) led to the

isolation of a total 18 extreme alkaliphilic bacteria and few with bacteria able to produce

different hydrolases (cellulases, mannanases, pectinases, amylase, xylanases and

proteases). The plating efficiency of native populations was not determined because it is

well established that no one medium composition or set of growth conditions can provide

the growth requirements of the entire “viable” bacterial flora.

2.3.1. Cell and colony morphology

Morphologies of all isolated strains were extremely pleomorphic, appearing as irregular,

short, long swollen and bent rods, spheres and triangles. Approximately cell dimensions

were in length 1.0-4.5 µm and width 0.5-1.0 µm. Colonies of all strain on medium were

1-2mm in size, circular, convex, opaque with entire margin. Moreover, all of them were

gram positive and non-motile. Phenotypic characteristics of strains isolated from various

samples are presented in Table 2.1.

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Table 2.1: Some characteristics of the isolated strains of alkaliphilic bacteria.

Strain Morphology Motility pH Cell Morphology

Colony Colour

VSG-1 Rod + 7-11 Irregular Light orange

VSG-2

cocci + 6-10 Circular White

VSG-3 Rod - 6-9 Irregular spreading

Cream

VSG-4 Pleomorphic rod

- 7-12 Circular Pink

VSG-5

Rod + 7-12 Circular Yellow

VSG-6

Cocci - 6-9 Irregular Pale pink

VSG-7 Cocci - 5-8 Irregular spreading

White

VSG-8 Pleomorphic rod

+ 6-10 Irregular Cream

VSG-9

Rod + 7-10 Circular Pink

VSG-10

Cocci - 6-10 circular Orange

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Table 2.2: Some characteristics of the isolated strains of alkaliphilic bacteria (continued).

Strain Growth at 37 °C

Growth at pH 5

Growth at 7-12

Reduction of Nitrate Catalase

VSG-1

+++ - ++ - +

VSG-2

++ - + - +

VSG-3

+ + + + +

VSG-4

+ - + - +

VSG-5

+ - ++ - +

VSG-6

++ + + + +

VSG-7

++ + + + +

VSG-8

+ - ++ - +

VSG-9

+++ - + - +

VSG-10

++ - + - +

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Table 2.3: Antibiotic sensitivity of the isolated strains of alkaliphilic bacteria.

Strain

Amphicillin

Tetracycline

Gentamycin

Erythromycin

Oflaxacin

VSG-1

R R R R R

VSG-2

S R R S R

VSG-3

S R S S R

VSG-4

R S S R R

VSG-5

R R R R R

VSG-6

S R R S S

VSG-7

S R R S R

VSG-8

R S S R R

VSG-9

S R R S R

VSG-10

S R R S S

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Table 2.4: Carbon and nitrogen sources utilized by isolated strains of alkaliphilic bacteria.

Strain Glucose (1%)

Fructose (1%)

Maltose (1%)

Yeast Extract

(1%)

Peptone (1%)

VSG-1

++ + + + ++

VSG-2

+ - - + +

VSG-3

- - - + +

VSG-4

+ + + + ++

VSG-5

++ + + ++ ++

VSG-6

- - - + +

VSG-7

+ + + + +

VSG-8

- - - + +

VSG-9

- - - - +

VSG-10

+ + + + ++

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2.3.2. Salt and pH tolerance

Biochemical characteristics of 18 strains isolated from various samples are presented in

Table 2.2. Optimum growth for all strains occurred at 00-16 % NaCl at 37°C except 4

strains were able to grow at 00-16% NaCl. Therefore, all the 18 strains were defined as

mesophilic extreme halotolerent alkaliphiles. The pH values above 6 were suitable for all

strains, however growth at pH 11 was found to be optimal. Nitrate reduction was not

observed in 11 strains for isolated alkaliphilic bacteria.

2.3.3. Antibiotic sensitivity test

Eighteen strains were tested by the disc diffusion method for their sensitivity to 5

different antibiotics (Table 2.3). Four strains were sensitive to amphicillin (10 µg). Six

strains were resistant to erythromycin (10 µg) and 5 strains were sensitive to gentamycin

(10 µg). Only 4 strains were sensitive to oflaxacin (10 µg) and 7 were resistant to

tetracycline. Strains VSG-1 is resistant to all the antibiotics tested.

2.3.4. Effect of carbon and nitrogen sources

Growth tests on carbohydrates and complex medium demonstrated that strains of

alkaliphilic bacteria grew on most of the substrate tested. The study demonstrated that

the alkaliphilic bacteria have diverse metabolic requirements. Table 2.4 summarizes the

carbon and nitrogen sources utilized by alkaliphilic strains VSG-1, VSG-2, VSG-8 and

VSG-10 were able to utilize the glucose and maltose. Most of the strains had better

growth in peptone than yeast extract. Excellent growth was observed for strains VSG-1,

VSG-2, VSG-8 and VSG-10 in glucose, fructose maltose (1 % w/v) containing medium.

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2.3.5. Extracellular hydrolytic enzymes by isolated alkaliphilic bacteria

Table 2.5 shows the production of extracellular hydrolytic enzymes by the 10 alkaliphilic

newly isolated strains. Pectinase hydrolyzing enzyme activity was observed in 9 strains.

Cellulase activity was not observed in 4 of the isolated alkaliphilic bacteria. Nearly 6

strains were that showed amylase activity. Mannanolytic activity was observed in 8

strains. The cellulolytic activity was observed in only 4 strains.

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Table 2.5: Extra cellular enzymes of the isolated strains of alkaliphilic bacteria.

Strain

Cellulase Xylanase Amylase Mannanase Pectinase

VSG-1

+++ + ++ + ++

VSG-2

- - + ++ +

VSG-3

- + + + ++

VSG-4

- + + + ++

VSG-5

++ + ++ + +

VSG-6

- + - - +

VSG-7

- - - + -

VSG-8

+ + - - +

VSG-9

- + - + ++

VSG-10

++ + + + +

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Table 2.6. Biochemical characterization of isolated bacteria. Tests Exiguobacterium sp. VSG-

1

Micrococcus luteus

VSG-5

Gram’s staining + +

Sporulation - +

Size 0.5–5.0 mm 0.5-3.0 mm

MR test + +

VP test - -

Starch hydrolysis + +

Casein hydrolysis + +

Citrate utilization + +

Indole production - -

H2S production - -

Arginine utilization + +

Nitrate reduction - -

Catalase + +

Urease + -

Oxidase + -

Acid production from

Glucose + +

Fructose + +

Maltose + +

Arabinose + +

Sucrose + +

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Fig. 2.1. Neighbour-joining phylogenetic dendrogram based on 16S rRNA gene sequence

data indicating the position of strain VSG-1 among members of the genus

Exiguobacterium. Accession numbers of 16S rRNA gene sequences of reference

organisms are indicated. Bootstrap values from 1000 replications are shown at branching

points; only values above 60 are shown. Bar, 0.01 substitutions per 100 nt.

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Fig. 2.2. Neighbour-joining phylogenetic dendrogram based on 16S rRNA gene sequence

data indicating the position of strain VSG-5 among members of the genus Micrococcus.

Accession numbers of 16S rRNA gene sequences of reference organisms are indicated.

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(a) Exiguobacterium sp. VSG-1

(b) Micrococcus luteus VSG-5

Fig. 2.3. Petriplates showing the colonies of (a) Exiguobacterium sp. VSG-1 and (b)

Micrococcus luteus VSG-5.

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2.3.6. Biochemical tests

Strain VSG-1 and VSG-5 are Gram positive bacteria. Strain VSG-1 is rod shaped

whereas strain VSG-5 is cocci. VSG-1 is non- spore-forming but VSG-5 is of spore-

forming bacteria with catalase positive, MR positive and VP negative (Table 2.6). Both

are positive for starch hydrolysis, casein hydrolysis, citrate utilization and arginine

utilization and negative for indole utilization, H2S production and nitrate reduction. Both

strains VSG-1 and VSG-5 produce acid from glucose, fructose, arabinose and maltose.

Optimum growth was observed at pH 9.0 and temperature 37°C. The 16 S rRNA

sequence was compared with other known bacteria and constructed the dendrogram.

They were aligned 100 % to Exiguobacterium sp. and Micrococcus luteus respectively as

shown in Fig. 2.1 and Fig. 2.2. The plates showing colonies of both the bacteria (Fig.

2.3). The NCBI accession numbers of 16 S rRNA gene sequences of stains VSG-1 and

strain VSG-5 were determined in this study as JQ312121 and JQ272845 respectively.

Description of Exiguobacterium sp. Nov

Exiguobacterium sp. is one of the group of rod shaped, Gram positive, aerobic (under

some conditions) or anaerobic bacteria widely found in soil. The genus Exiguobacterium

was first described in 1983 by Collins et al. (1983) with characterization of the type

species Exiguobacterium aurantiacum. In 1994, Farrow et al. included the species

formerly identified as Brevibacterium acetylicum incertae sedis into the genus

Exiguobacterium, as E. acetylicum (Farrow et al. 1994). Since then, 11 new species have

been added to the genus (Chaturvedi et al. 2008; Chaturvedi and Shivaji 2006; Crapart et

al. 2007; Fruhling et al. 2002; Kim et al. 2005; Lopez-Cortes et al. 2006; Rodrigues et al.

85

2006; Yumoto et al. 2004). In addition to the type strains, Exiguobacterium spp. have

been isolated from, or molecularly detected in, a wide range of habitats including cold

and hot environments with temperature range from -12 to 55°C. Exiguobacterium spp.

has been detected in Siberian permafrost, temperate and tropical soils by multilocus real-

time PCR (Rodrigues and Tiedje 2007). The Exiguobacterium genus comprises

psychrotrophic, mesophilic, and moderate thermophilic species and strains

(Vishnivetskaya et al. 2005), with pronounced morphological diversity (ovoid, rods,

double rods, and chains) depending on species, strain, and environmental conditions

(Vishnivetskaya et al. 2007).

Several Exiguobacterium strains possess unique properties of interest for applications in

biotechnology, bioremediation, industry and agriculture. Exiguobacterium strain Z8 was

capable of neutralizing highly alkaline textile industry wastewater (Kumar et al. 2006);

strain 2Sz showed high potential for pesticide removal (Lopez et al. 2005); strain WK6

was capable of reducing arsenate to arsenite (Anderson and Cook, 2004); other

Exiguobacterium strains could rapidly reduce Cr[VI] over a broad range of temperature,

pH and salt concentrations (Okeke et al. 2007; Pattanapipitpaisal et al. 2002). A panel of

mercury resistant Exiguobacterium strains harbor determinants homologous to

meroperons (Petrova et al. 2002) or mercury-resistance transposons (Bogdanova et al.

2001). Furthermore, several enzymes (alkaline protease, EKTA catalase, guanosine

kinase, ATPases, dehydrogenase, esterase) with stability at a broad range of temperatures

were purified from different Exiguobacterium strains (Hara et al. 2007; Hwang et al.

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2005; Kasana and Yadav 2007; Suga and Koyama 2000; Usuda et al. 1998; Wada et al.

2004).

While reports about isolation of new Exiguobacterium strains continue to appear,

information on genomic diversity of strains already isolated from different habitats

remains quite limited. On the basis of small-subunit ribosomal RNA sequences, the

species of the genus Exiguobacterium were clustered in proximity to Bacillus

benzoevorans, B. circulans, and B. siralis in the order Bacillales, phylum Firmicutes

(Yarza et al. 2008). The genome of E. sibiricum 255-15 has been sequenced in the

context of the Joint Genome Institute Microbial Sequencing program (http://genome.jgi-

psf.org/draft_microbes/ exigu/exigu.home.html). This strain was chosen for genome

sequencing on the basis of excellent survival potential after exposure to a long-term

freezing at -20°C in trypticase soy broth without addition of cryoprotectants (Ponder et

al. 2005), rapid growth at temperatures as low as -6°C (Vishnivetskaya et al. 2007), and

the age (2-3 million years) of the permafrost sediment from which it was derived

(Vishnivetskaya et al. 2006). The genome of E. sibiricum 255-15 contains a 3.0 Mbp

chromosome and two small plasmids of 4.9 and 1.8 kbp, respectively, with a total of

3,015 predicted protein-encoding genes and G+C content of 47.7 % (Rodrigues et al.

2008). Genome sequence analysis of E. sibiricum 255-15 revealed that it shared 829 and

544 orthologous genes (50 % similarities over 90 % lengths) with B. halodurans and B.

subtilis, respectively (Vishnivetskaya et al. 2008). Recently, the genome sequencing of a

thermophilic Exiguobacterium isolate, strain AT1b from a Yellowstone hot spring has

also been undertaken. The draft sequence of Exiguobacterium sp. AT1b revealed a 2.8

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Mbp genome with a G+C content of 48.3% and 3,046 candidate protein-encoding genes

(http://genome.ornl.gov/microbial/exig_AT1b/).

The fact that certain strains (e.g., those from ancient permafrost) can grow at

temperatures as low as -6°C whereas others (e.g., those from hot pools) have optimum

growth temperatures above 45°C confers substantial interest to Exiguobacterium as a

potential model system for the investigation of evolutionary mechanisms and genomic

attributes that may correlate with adaptations of organisms to diverse thermal regimes.

The strain VSG-1 is classified as shown below.

Kingdom : Bacteria

Phylum : Firmicutes

Class : Bacilli

Order : Bacillales

Family : Bacillales Incertae Sedis

Genus : Exiguobacterium

Species : Exiguobacterium sp.

Strain : Exiguobacterium sp. VSG-1

Description of Micrococcus luteus sp. Nov

Micrococcus luteus was originally isolated by Alexander Fleming in 1929 as

Micrococcus lysodeikticus. It was the primary experimental microbe used in Fleming’s

discovery of lysozyme. The microbe can be found in a variety of environments including

soil, water, animals, and some dairy products. Micrococcus is generally thought to be a

88

saprotrophic or commensal organism, though it can be an opportunistic pathogen. This is

particularly true in hosts with compromised immune systems.

Micrococci, like many other representatives of the Actinobacteria, can be catabolically

versatile. It has the ability to utilize a wide range of potentially toxic substrates, such as

carbon-based pyridine, pesticides, crude oil and petroleum by-products. As a species,

they are likely involved in detoxification or biodegradation of many other environmental

pollutants. Other Micrococcus isolates can synthesize various useful products, such as

long-chain (C21-C34) aliphatic hydrocarbons for lubricating oils and the biosynthesis of

terpenes1. Thus the full sequencing of Micrococcus luteus has been supported due to its

potential as a bio-remediator of contaminated water and soil as well as in current and

future biotechnology applications.

Micrococcus luteus is able to survive in the environment for long periods. It is very

capable of survival under stress conditions, such as low temperature and starvation.

However, M. luteus does not form spores as survival structures, as is common in other

bacterium. Instead M. luteus undergoes dormancy without spore formation.

More recently, a non-spore forming cocci, identified as Micrococcus Luteus, was isolated

from a 120 million year old block of amber. Although comparison of rRNA sequences

from other isolates is unable to confirm the precise age of the bacteria, it is estimated that

Micrococcus luteus has survived for at least 34,000 to 170,000 years on the basis of 16S

rRNA analysis. It seems that M.luteus and other related modern members of the genus

have numerous genetic adaptations for survival. This includes extreme, nutrient-poor

conditions. These phenotypes have assisted the microbe in persistent and prevalent

89

dispersal within the environment. This species has an ability to utilize succinate and

terpine related compounds (which themselves are major components of natural amber) to

enhance and ensure its survival in oligotrophic environments (Greenblatt, et al. 2004).

Micrococcus luteus is an organism that is capable of growth on pyridine. Pyridine is a

natural byproduct of coal and oil gasification. It is also mobile in soil and is considered

an environmental teratogen. M. luteus contains a gene that codes for the enzyme

succinate-semialdehyde dehydrogenase. Although the mechanism is not completely

understood, the enzyme is actually induced by pyridine. It permits the oxidation of

pyridine as a metabolic carbon source and thereby provides cellular energy. In the

process it releases the nitrogen contained in the pyridine ring as ammonium (NH3). M.

luteus, like species of Bacillus and Corynebacterium, require the -amino acids arginine,

valine, leucine and methionine for enhanced growth on pyridine (Sims et al. 1986).

Miccrococus luteus contains two structural genes (hex-a, hex-b) that encode two essential

components of Hexaprenyl disphosphate synthase (HexPS). When these two components

are combined, they mechanize prenyl transferase activity. This enzyme complex will

produce the precursor of the prenyl side chain of menaquinon-6 (HexPP; C30).

Terpenoid-Menaquinon biosynthesis in prokaryotes function as electron carriers within

the cytoplasmic membrane, and each is required for respiration using different, although

overlapping subsets of terminal electron acceptors. Menaquinone is also known as the

essential Vitamin K-2, because it is a nutrient that cannot be synthesized by mammals

(Shimizu et al. 1998).

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The strain VSG-5 is classified as shown below. Kingdom : Bacteria Phylum : Actinobacteria Order : Micrococcales Family : Micrococcaceae Genus : Micrococcus Species : Micrococcus luteus Strain : Micrococcus luteus VSG-5

2.4. Discussion

Our ecological studies in hyperalkaline environments reveal a wide extent of diversity of

alkaliphilic bacteria endowed with the potential to hydrolyze a rather range of structurally

non-related polymers and nucleic acids. Enzymes from alkaliphilic are expected to show

optimal activities in extreme conditions; thus, the possibility to have a wide variety of

alkaliphiles producing extremozymes will be of invaluable help for biotechnological

applications.

There are no precise definitions of what characterizes an alkaliphilic or alkali-tolerant

organism. Several micro-organisms exhibit more than one pH optimum for growth

depending on the growth conditions, particularly nutrients, metal ions, and temperature.

Therefore, the term “alkaliphile” is used for micro-organisms that grow optimally or very

well at pH values above 9, often between 10 and 12, but cannot grow or grow only

slowly at the near-neutral pH value of 6.5 (Horikoshi, 1999). Most of them require 0-10

salt to grow and the entire bacterial community could grow with or without salt. They

can be considered to be extremely halo-tolerant alkaliphilic. Cells face many challenges

in an alkaline environment; they must make their cytoplasm more acidic to buffer the

alkalinity. In addition, enzymes both excreted and surface located must be resistant to the

91

effects of extreme pH. Finally, the pH gradient must be reversed to carry out ATP

synthesis.

The antibiotic resistant studies showed that most of the bacteria were resistant to the 5

antibiotic tested. Strains VSG-1, VSG-2, VSG-8 and VSG-10 were resistant to all

antibiotic tested, which is characteristic of extreme haloalkaliphiles. All 10 strains grew

between pH 6 and 12 but none exhibited growth at acidic pH 5. Because no one medium

or culture condition is known to support the growth of all alkaliphilic bacteria, the

general-purpose, peptone based medium was employed for both enrichments and direct

plate streaking. All strains showed catalase activity. Finally, strain VSG-1 exhibited

high cellulase and xylanase activity. Among 10 strains only 6 strains produced

extracellular cellulase. The VSG-1 and VSG-5 strains also secreted xylanase. The strain

VSG-1 and VSG-5 having maximum cellulase and xylanase activity has been selected for

detailed studies. The next chapter describes the identification and characterization of

these bacteria. The optimization of culture conditions for extracellular enzymes

production and biochemical characterization from alkaliphilic strain VSG-1 and VSG-5

were studied.

Alkaliphiles are the most likely sources of enzymes showing optimal activities at

different pH concentrations and temperature, because not only are their enzymes are

alkaliphilic but many are thermophilic. Hence these enzymes from the above isolated

may possess commercial importance. Further studies are described in order to select the

best producers of cellulase and xylanase, the investigation were directed towards the in-

depth characterization of these alkaliphiles.

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