Download Chapter II Isolation identification and characterization

Chapter II
Isolation identification and
characterization of alkaliphilic
bacteria from different habitats
65
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
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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
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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
VSG-1
Rod
+
7-11
Cell
Morphology
Irregular
Colony
Colour
Light
orange
White
VSG-2
cocci
+
6-10
Circular
VSG-3
Rod
-
6-9
Cream
VSG-4
-
7-12
VSG-5
Pleomorphic
rod
Rod
Irregular
spreading
Circular
+
7-12
Circular
Yellow
VSG-6
Cocci
-
6-9
Irregular
Pale pink
VSG-7
Cocci
-
5-8
White
VSG-8
+
6-10
Cream
VSG-9
Pleomorphic
rod
Rod
Irregular
spreading
Irregular
+
7-10
Circular
Pink
VSG-10
Cocci
-
6-10
circular
Orange
Pink
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Table 2.2: Some characteristics of the isolated strains of alkaliphilic bacteria (continued).
VSG-1
Growth at
37 °C
+++
Growth at
pH 5
-
Growth at
7-12
++
Reduction
of Nitrate
-
VSG-2
++
-
+
-
+
VSG-3
+
+
+
+
+
VSG-4
+
-
+
-
+
VSG-5
+
-
++
-
+
VSG-6
++
+
+
+
+
VSG-7
++
+
+
+
+
VSG-8
+
-
++
-
+
VSG-9
+++
-
+
-
+
VSG-10
++
-
+
-
+
Strain
Catalase
+
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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%)
VSG-1
++
+
+
Yeast
Extract
(1%)
+
VSG-2
+
-
-
+
+
VSG-3
-
-
-
+
+
VSG-4
+
+
+
+
++
VSG-5
++
+
+
++
++
VSG-6
-
-
-
+
+
VSG-7
+
+
+
+
+
VSG-8
-
-
-
+
+
VSG-9
-
-
-
-
+
VSG-10
+
+
+
+
++
Peptone
(1%)
++
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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-
Micrococcus luteus
1
VSG-5
Gram’s staining
+
+
Sporulation
-
+
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
+
-
Glucose
+
+
Fructose
+
+
Maltose
+
+
Arabinose
+
+
Sucrose
+
+
Size
Acid production from
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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 sporeforming 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.
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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 realtime 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.jgipsf.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
87
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
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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
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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
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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 indepth characterization of these alkaliphiles.
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