CHAPTER 2

SCREENING OF EXOPOLYSACCHARIDE PRODUCING

CULTURE/S
 37

CHAPTER SUMMARY

A developmental programme for microbial products hitherto unexplored usually

begins with screening of cultures producing the desired product in large amounts and
in an economical maimer with the selected strain itself exhibiting good stability
during storage and production alike. Hence, in the present chapter, towards finding a
suitable uronic acid rich EPS as a substitute for sodium alginate from the microbial
sources, isolation and characterization of a bacterial strain producing highly mucoid
i
colonies on a medium having high C:N ratio has been described.

Of more than 150 bacterial cultures capable of producing copious amount of

exopolysaccharide (EPS) isolated from different ecological niches, eight different
isolates were selected for further study on the basis of (i) viscosity of the respective
EPS solutions (1% w/v), (ii) anionic nature, (iii) ability to grow on cheaper substrates,
and (iv) ease of recovery. The selected exopolysaccharides could be precipitated by
cetyl pyridinium chloride (CPC), a cationic surfactant,, suggesting that these EPS were
of anionic nature. Alginate of commerce is also an anionic polysaccharide.

One of the above cultures designated here as BE1 was selected for further studies on
the basis of its anionic nature, high viscosity at low concentration, high consistency
index, pseudoplasticity, thixotrophic nature and better performance as a thickener in
reactive textile printing in terms of the colour yield.

Compared to other isolates obtained, the exopolysaccharide produced by the

bacterial strain BE1 formed a highly viscous solution (6010 cP at a shear rate of
2.9 sec"1) at a concentration of 1% (w/v) and gave high colour strength when used

as thickener (at 10 times less concentration than sodium alginate) in reactive

textile printing. The culture was identified as Rhizobitm radiobacter through 16S
rRNA gene sequence analysis and conventional microbiological methods. When the
whole cell protein profile of the Rhizobium radiobacter BE1 was compared with that
obtained for the reference strain Rhizobium radiobacter ATCC 19358, few
differences in the protein band pattern were observed. The strain was found to be non-
pathogenic to plants as tested by carrot disc infectivity assay.
 38

2.1 INTRODUCTION
There exists a tremendous structural and thereby property diversity in microbial
exopolysaccharides. Even within the “sphingan” family of exopolysaccharides,
existence of minor structural differences has given rise to gellan, welan and rhamsan
kind of exopolysaccharides, which are distinctly different in their properties (Yalpani,
1998). Although xanthan is being produced commercially since 1960, only due to
existence of above referred diversity, a novel EPS having superior rheological
properties than that of xanthan has been reported from Sphingomonas paucimobilis
GS1 (Ashtaputre and Shah, 1995). Hence there is always a scope of getting a novel
culture from natural resources through a well planned or directed screening efforts
(Bueno and Garcia-Cruz, 2006).

2.1.1 Isolation of exopolysaccharide producing microorganisms

A development program for microbial exopolysaccharides essentially involves (i)
obtaining material from various sources for screening of suitable microbial species,
(ii) identification of mucoid (slime- or capsule producing) microorganisms, and (iii)
selection of a number of strains based upon a qualitative assessment of the chemical
and physicochemical properties of the polymers produced by them. Subsequently,
background knowledge of the growth characteristics of the selected organism(s), the
conditions essential for polysaccharide production and a detailed knowledge of the
physical and chemical characteristics of the polysaccharide are obtained (Lawson and
Sutherland, 1977).

2.1.1.1 Sources bf EPS producing microorganisms

Although EPS producing microorganisms occur in a wide variety of environments,
typically they are found in soil, fresh water, sea water, in association with plants or
with food, biofilms on a variety of surfaces and in extreme environments such as high
salt, high temperature or alkaline environments. Also, habitats containing large
amounts of carbohydrate; waste materials from industrial processes with relatively
small amounts of nitrogenous substrates favour the presence of polysaccharide
producing microorganisms. Many plant and animal pathogens produce
exopolysaccharides (Lawson and Sutherland, 1977; Sutherland, 1983). Organisms
producing EP$ have also been isolated from diverse sources such as polluted ground
 39

water (Fusconi and Godinho, 2002), cheese (Kojic et al., 1992), hydrothermal vents
(Geuezennec, 1994; Rougeaux, 1996) and activated sludge (Farrah and Unz, 1976).

2.1.1.2 Strategies to screen EPS producing organisms

The studies toward development of EPS for industrial applications is complicated by
the fact that even today, it has not been possible to correlate structure of a
polysaccharide with either its viscosity, or its functioning in nature or for any specific
application. Although the ability of an organism to produce EPS might be a response
to selective pressures in the natural environment, there is no direct evidence that the
strategies used to screen for EPS-producing microorganisms have been based on the
natural role of the exopolysaccharides (Dudman, 1977). The selection of EPS
producing isolates based on development of mucoid aspect of the strains on solid
media seems to be the only practically feasible method for screening of EPS
producers (Fusconi and Godinho, 2002).

Few rational strategies such as the use of polysaccharide binding dyes e.g. alcian blue,
screening of strains on the basis of bacteriophage resistance, uncoupling of the EPS
biosynthesis by use of antibiotics such as tetracycline, streptomycin and
chloramphenicol have been reported for isolation of EPS producing microorganisms
(Morin, 1998). Ramirez et al. (1988) observed that higher yields of xanthan were
obtained in case of virulent strains of Xanthomonas campestris and proposed that the
degree of virulence could be used as a criteria for selecting and isolating strains
producing high quality xanthan gum. When used for production of milk-based
products, the EPS of lactic acid bacteria (LAB) from ropy strains conferred smoother
consistency and high viscosity along with lower susceptibility to syneresis than EPS
obtained from non-ropy strains. When stained with ruthenium red (which stains
bacterial cell wall), the presence of EPS prevented the uptake of the stain by ropy
strains and hence their colonies appeared white, while the colonies of non-ropy strains
appeared pink (Gancel et al., 1989; Ruas-Modiedo and de Los Reyes-Gavilan, 2005).

2.1.1.3 Guidelines for selection and preliminary studies of EPS producing

organisms for further development

It has been suggested previously that those strains that produce high amount of EPS
and grow luxuriantly in simple media should only be selected for further studies. In
 40

fact, in terms of personnel and equipment, this part of the developmental program is
the most demanding, as it will probably be necessary to test strains for numerous
characteristics as well as the productivity and subsequent characterization of the
exopolysaccharides produced. Using a basic growth medium, the effect of physical
and chemical variables influencing the growth and the EPS production ability of the
organism such as concentration of carbon source, concentration of organic and/or
inorganic nitrogen sources, pH, buffering of the me<iia, concentration of mineral salts
and aeration are studied. Of potential importance from the economic aspect of
polysaccharide production are (i) the use of waste carbohydrate products as carbon
and energy sources, and (ii) the extent of conversion of substrate to polymer. It is
unlikely that organisms producing polysaccharide yields below 40% and in batch
times longer than 90 h, will have a great chance of commercial success (Lawson and
Sutherland, 1977; Sutherland, 1983).

In terms of physical characteristics, the flow behaviour of aqueous solutions and gel
forming ability are tested. Due to potential danger to the operatives and ethical
requirements for the final product, human or animal pathogens must be excluded from
consideration. Such procedures outlined for selection of strains for EPS production
have been routinely followed as exemplified by a study on! EPS production by
Erwinia sp. (Flatt et al., 1992). More importantly, the selected strains should hot
possess degradative enzymes for the EPS that they synthesize. It is well known that
Streptococcus species form both hyaluronic acid and hyaluronidase; Azotobacter
vinelandii synthesizes alginate and also small quantities of alginase and Streptococcus
mutants produce both insoluble dextran-type polymers and ‘mutanase’ which
degrades them (Lawson and Sutherland, 1977).

2.1.1.4 Media and incubation conditions for isolation of EPS producing

organisms
Although there are no selective media which can be used specifically for the isolation
of polysaccharide producing microbial species, most media used in the laboratory for
polysaccharide production are based on high carbon substrate: limiting nutrient ratios,
where nitrogen is usually the favoured component to induce growth limitation and
stimulate exopolysaccharide formation. Such media have been shown to favour
polymer production in Enterobacteriaceae, XanthoLlonas campestris, Pseuodomonas
 41

spp, and many other bacteria and fungi (Lawson and Sutherland, 1977; Sutherland,
1983; Margaritis and Pace, 1985). Glucose or sucrose at concentrations of 2 to 5 %
(w/v) are usually the preferred carbon substrates for EPS production because, (i) these
sugars are utilized by a wide range of microbial species, and (ii) are also widely
available. Most polysaccharide producing microorganisms are incubated at or near
30°C, although incubation at suboptimal temperatures frequently favours
exopolysaccharide production (Sutherland, 1983).

2.2 THE PRESENT STUDY

Most of the previous studies on exopolysaccharides were mainly focused on either the
structural elucidation of the exopolysaccharides, or on nutritional requirements of the
producing organism or the environmental influences on EPS production or the role of
exopolysaccharides in biofilm formation. Previously, Rougeaux et al. (1996) reported
(as part of a systematic screening program) exopolysaccharides of Alteromonas
macloeodi subsp. fijiensis, Pseudoalteromonas species and Vibrio species isolated
from hydrothermal vents. However, they only suggested the potential applications of
the selected exopolysaccharides, while the actual performance of the same in
proposed applications is not known. Similarly, although EPS producing isolates have
been obtained from different hypersaline habitats, the aim was mainly to establish the
relationship between different EPS producers in diverse hypersaline habitats and not
development of an EPS for any applications (Martinez-Canova et al., 2004).

Significantly, functions for the observed diversity in EPS have been ascribed to
pathogens and not for environmental strains (Weiner et al., 1995), even though the
exopolysaccharides of environmental strains are of actual value for commercialization
if found worthy of so. Also, it is surprising that in literature only sporadic reports
are available which describe the screening of exopolysaccharide producing
organisms for specific applications.

To date, most of the biopolymers which have been developed for a range of
commercial applications have been found as a result of empirical experimentation and
the full extent of their physicochemical properties has been determined only after the
product has been made commercially available (Sutherland, 1996). Hence, from a
literature survey, it appeared that many reports were available on evaluation of
 42

already known and commercially available exopolysaccharides such as xanthan and

gellan for specific applications. Also, recently, emphasis has been placed on EPS of
biofilm forming bacteria (Weiner et al., 1995). Sutherland (1996) envisaged that it
might be possible in future to replace the empirical approach in development of EPS
for specific industrial applications with a more pragmatic appraisal of the specific
physical and chemical properties needed. Precisely, conforming to the above view, the
present study was undertaken with a more rational approach for screening and
evaluation of a suitable EPS for specific applications.

The present investigation focused on the development of an algal alginate-like EPS as

a suitable substitute for algal alginate for specific applications. Algal alginate is an
anionic and uronic acid based EPS and viscosity-curri-pesudoplasticity based its major
application is for “reactive printing” in the textile industry. The final test of suitability
of an EPS for specific application is subjecting it to the actual conditions that prevail
during the intended applications. Hence, in the present study, it was envisaged that the
initial screening of an anionic polysaccharide and its subsequent evaluation using a
variety of criteria with respect to, (i) intended applications, and (ii) production related
aspects might lead to development of a suitable substitute for sodium alginate. Hence,
a systematic screening program was undertaken where at the secondary
screening level itself, the selected EPS were assessed with respect to specific
desirable properties/performance keeping specific application under
consideration. Such a rational approach for development of a microbial
exopolysaccharide for major applications has not been reported in the literature so far.

2.3 MATERIALS AND METHODS

2.3.1 Screening of exopolysaccharide producing organisms

In general, the exopolysaccharide producing cultures were screened on following

medium containing (g/1): Sucrose, 15.0; K2HPO4, 0.5; MgS04.7H20, 0.2;
CaCl2.2H20, 0.1, and NaCl, 0.2 with or without yeast extract (0.1 g/1)
supplementation. To the above medium either KNO3 (1.0 g/1) or monosodium
glutamate (0.2 g/1) was added as a nitrogen source. pH of the medium was adjusted to
7.0 and sterilized by autoclaving at 10 psi for 20 min. The plates after inoculation
 43

were incubated at 30+1 °C for 48 h and the colonies producing copious amount of

exopolysaccharide were selected.

Two different approaches were used for screening of Azotobacter sp. also from soil.
In the first approach, to a soil sample of sufficient size to fill a small petri dish, 1.0 g
of both CaCC>3 and mannitol were added. After mixing, 200 pi of a 10% (w/v)
solution of both K2HPO4 and MgS04.7H20 were added. Subsequently, enough water
was added to prepare a soft paste that was not water saturated. The mixture was put in
the bottom half of the petridish and the surface was smoothed with a knife. The
petridish was then placed (without lid) into a larger petridish on a wet filter paper,
which ensured a moist atmosphere. After 2 to 3 days of incubation at 30±1°C,
whatever mucoid colonies developed on soil surface, were isolated on Ashby’s
Mannitol Agar having following composition (g/1): Mannitol, 15.0; K2HP04, 0.50;
CaS04, 0.10, NaCl, 0.20; Na2Mo04.2H20, 0.05, and MgS04.7H20 0.20. pH of the
medium was adjusted to 7.6 and autoclaved at 10 psi for 20 min.

In the second approach, the soil suspension was streaked on to (i) Burk’s medium and
Ashby’s Mannitol Agar. The plates after inoculation were incubated at 30±1°C for 48
h. Burk’s medium contained (g/1): Sucrose, 20.0; K2HPO4, 0.80; KH2PO4, 0.20;
MgS04.7H20, 0.20; NaCl, 0.20; CaS04, 0.10, 0.1 ml of Fe-Mo Mixture (FeCl3.6H20

1.45 g and Na2MoC>4. 2H2O, 0.253 g in 100 ml), H3BO3, 10 fig; ZnSC>4, 10 jig;
MJ1SO4,1 |ig; CUSO4.5H2O, 0.3 jig; KI, 0.1 (ig and Agar 25.0. pH of the medium was
adjusted to 7.3 and autoclaved at 10 psi for 20 min.

2.3.2 Maintenance and growth

The isolates were maintained on the agar slopes containing (g/1): Sucrose, 15.0;
KN03> 1.0; K2HPO4j0.5; MgS04.7H20, 0.2; CaCl2.2H20,0.1, and NaCl, 0.2. pH of
the medium was adjusted to 7.0 and sterilized by autoclaving at 10 psi for 20 min.

2.3.3 EPS production at shake flask level

Cultivation of EPS producing isolates was routinely carried out in the medium
containing (g/1): sucrose or mannitol, 15.0; KN03,1.0 or mono sodium glutamate,
0.2; K2HPO4, 0.5; MgS04.7H20, 0.2; CaCl2 .2H20, 0.1, and NaCl, 0.2. pH of the
 44

medium was adjusted to 7.0 and sterilized by autoclaving at 10 psi for 20 min. The
flasks containing the above medium (50 ml in 250 ml Erlenmeyer flasks) were
inoculated with the suspension of cells (Afioo= 0.5) of isolates at 15.0% (v/v) level and
incubated on a rotary shaker (at 200 rpm) at30±l°C for 96 h.

2.3.4 Extraction of exopolysaccharide

The viscous fermentation broth was diluted 20 times with distilled water and
centrifuged at 10,000 g. The EPS from the supernatant was precipitated by adding
three volumes of acetone. The precipitates were subsequently dialyzed for 48 h with
three changes of water and dried at 50±1°C to constant weight.

2.3.5 Precipitation of exopolysaccharide using cetyl pyridinium chloride

To one ml of various EPS solutions (1% w/v), 1.0 ml of aqueous solution (2% w/v) of
cetyl pyridinium chloride (CPC) was added and after mixing using a vortex mixer,
formation of precipitates was observed visually.

2.3.6 Determination of viscosity and shear stress

Viscosity and shear stress were measured using Brookfield LV DV 11+ viscometer
equipped with a small sample adapter (SSA-8R) and spindle number 16 at 30+l°C
unless otherwise mentioned. Consistency index K and flow index n were determined
using the Power law model from the plot of log shear rate versus log shear stress as
described by Margaritis and Pace (1985).

2.3.7 Biochemical tests

Biochemical tests were carried out as mentioned previously either using ATB 32E
system (Freney et al., 1991) or using standard media for biochemical tests prepared
in-house as per Bergey’s Manual of Systematic Bacteriology (Noel and Krieg, 1984)
and as described by Smibert and Krieg (1981). Either 55 pi (in case of ATB 32E
system) or 2 loopfuls (in case of in-house prepared media) of the suspension of isolate
BE1 cells (A6oo = 0.5) in normal saline was inoculated into the respective media and
the results were noted visually after incubation for 24 h at 30±1°C. For determining
salt sensitivity of the culture, to Luria-Bertani (LB) broth, NaCl was added at a final
concentration of 1.0% (w/v) and 10.0 % (w/v). Either organic acids (0.20 % w/v) or
 45

ethanol 1.0% (v/v) or meso-inositol 1% (w/v) were incorporated in a basal medium as

described by Graham and Parker (1964) containing (g/1): MgS04.7H20, 0.25 g;
CaCl2. 2H20, 0.025 g; Na2HP04.2H20, 1.2 g; KH2P04, 0.55 g; NaCl, 0.25 g; FeS04
(NH4)2S04. 6H20, 4 mg; ZnS04.7H20, 0.16 mg; CuS04.5H20, 0.08 mg; 0.5
mg, and MnS04.H20, 0.4 mg. pH of the medium was adjusted to 7.0 and sterilized by
autoclaving at 10 psi for 20 min.

2.3.8 Antibiotic sensitivity tests

Sensitivity of various exopolysaccharide producing isolates to antibiotics was carried
out on Luria agar using the Octadiscs (Himedia, India). The antibiotic containing
discs were placed on Luria agar plates pre-inoculated with the respective cultures and
after incubation for 48 h at 30±1°C, results were noted in terms of zone of inhibition.

2.3.9 Assessment of EPS for textile printing application

2.3.9.1 Preparation of print paste
Stock print paste having following composition was prepared by dissolving all the
ingredients in distilled water (100 ml) and mixing at high speed: resist salt, 1.0 g;
NaHCOa, 1.5 g, and urea, 10.0 g. Sodium alginate (used as a control thickener) was
added at concentration of 9.0% (w/v) to attain a target print paste viscosity of 9600 cP
(a shear rate of 2.9 sec'1, spindle no. 16, 30+1 °C). To the stock print paste, respective

dyes were added to a final concentration of 3.0% (w/v) and dissolved through high­
speed stirring.

2.3.9.2 Screen printing

Laboratory scale screen-printing on cotton fabric (white poplin) was carried out by
applying 50 g print paste on a printing screen with a rubber squeeze. All the prints
were dried at 80°C for 5 min, followed by steaming for 10 min in a laboratory
steamer. Washing of the steamed fabrics was carried out first in cold water and then in
warm water (15 min at 90°C) till no more colour came off. A wash in detergent
solution was also given to remove any unbound dye before air-drying the fabrics.
 46

23.93 Measurement of colour strength

Colour strength of the printed fabrics was determined by reflectance
spectrophotometry using the Data Colour Spectroflash reflectance spectrophotometer
as reported earlier (Kanik and Hauser, 2004). The concentration of the dye on the
surface of the fabric could be derived from reflectance value using Kubelka Munk
equation:
K/S= (1-R)2/ 2R

Where K= measure of light wavelength, S=measure of light absorption, and R =

reflectance, K/S values of printed samples were calculated at the wavelength of
maximum absorption. The colour strength value obtained when sodium alginate was
used as thickener was taken as 100%.

2.3.10 16S rRNA analysis

Genomic DNA of the isolate BE1 was prepared using a method of as described by
Spanevello et al. (2002) in which achromopeptidase (final concentration 0.8 pg/pl)
was used to improve cell lysis following lysozyme treatment. Thirty ml of a 48-h-old
culture wgs centrifuged at 10000 g for 5 min. The pellet was resuspended in 487 pi
TE buffer (10 mM Tris.HCl, pH 7.4; 1 mM EDTA, pH 8.0), 8 pi lysozyme (50
mg/ml) and 40 pi achromopeptidase (10 mg/ml) and incubated for 1 h at 37°C. Thirty
pi of 10% SDS and 3 pi of proteinase K (20 mg/ml) were added and the mixture was
further incubated at 50°C for 1 h. Subsequently, 100 pi of NaCl (5 M) and 80 pi of
10% CTAB/0.7 M NaCl were added, the solution was vortexed and incubated at 65°C
for 10 min. DNA was purified from the suspension by first extracting with equal
volumes of chloroform:isoamyl alcohol (24:1) and then with
phenol: chloroform;isoamyl alcohol (25:24: 1). Chromosomal DNA was recovered by
adding 450 pi of 2-propanol and centrifuging at 10,000 g for 15 min. The
chromosomal DNA pellet was then washed with 250 pi of 70% ethanol, dried and
resuspended in 100 pi TE buffer. Chromosomal DNA integrity was checked by
agarose gel electrophoresis and ethidium bromide staining and visualized by UV
fluorescence.

16S rRNA gene amplification and sequencing was carried out as described previously
(Andrews and Patel, 1996). The 16S rRNA gene of isolate BE1 was amplified by
 47

PCR prepared in sterile 0,2 ml thin-walled tubes. Each reaction consisted of: lOx PCR
buffer (50 mM Tris.HCl, pH 8.3, 20 mM MgCl2, 2.5 mg BSA/ml), 5 pi; 20 mM
dNTPs, 0.5 pi; 50 pM forward primer Fdl (5-CCG
AATTCGTCGACAACAGACTTTGATCCTGGCTCAG-3') and reverse primers
Rdl(S'-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3') (Redbum and
Patel, 1993), 1 pi; chromosomal DNA, 200 ng; 1 U Taq polymerase; and sterile
double-distilled water, 40.3 pi. The PCR thermal cycling was carried out in a Rapid
Cycler (Idaho Technology) using the following parameters: 94°C for 2 min followed
by 30 cycles of 94°C for 1 min, 50°C for 1 min and 74°C for 1.3 min with a slope of
9.9. PCR products were purified using QiaQuick PCR Purification spin columns
according to the manufacturer's instructions (Qiagen). The PCR product was analyzed
on a 96-lane ABI377 DNA sequencer (Applied Biosystems).

Sequences were assembled into one contiguous sequence and corrected using the
sequence editor BIOEDIT (Hall, 1999). The most homologues sequence to the 16S
rRNA gene sequence of isolated BE1 were identified in the Gene Bank database using
the BLAST and aligned manually. Positions of sequence uncertainties and alignment
were omitted from the analysis. Pair wise evolutionary distances based on 1405
unambiguous nucleotides were determined by the method of Jukes and Cantor (1969)
using DNA dist program. The phylogenetic tree was constructed by neighbour joining
method (Saitou and Nei, 1987) using PHYLIP suite of programs (Felsenstein, 1993).

2.3.11 3-ketolactose production test

The ketolactose production test was performed as described by De Lay and Bemaerts,
(1963). A pure culture of the isolate was grown for 48 h at 30±1°C on agar slants
containing (g/1): glucose, 20.0; yeast extract, 10.0; CaCC>3, 20.0, and agar 25.0. A
loopful of the bacterial growth from agar slant was deposited as a small heap (about
0.5 mm in diameter) on a solid medium (in a petridish) containing (g/1): lactose, 10.0;
yeast extract, 1.0, and agar, 25.0. After incubation for 48 h at 28±1°C, the plates with
growth of the isolate BE1 was flooded with a shallow layer of Benedict’s reagent
(sodium citrate, 86.5 g; NaaCCh, 50.0 g, and CUSO4.5H2O, 8.7 g in 100 ml distilled
water) and left at room temperature. Formation of ketolactose surrounding the cell
mass was indicated by a yellow ring of CU2O.
 48

2.3.12 Acetylene reduction assay

Nitrogenase activity of the isolate BE1 was tested by the method as described earlier
by Sharma et al. (1985). Cells were grown on Ashby’s Mannitol Agar slants for 48 h
at 30±1°C. Cotton pings of slants were replaced by serum caps and 1.0 ml of
acetylene gas was injected into the tubes and incubation continued for 2 h at 30±1°C.
From the headspace, 1.0 ml of gas was withdrawn and analyzed by gas
chromatography for the presence of ethylene using Shimadzu 14-B gas
chromatograph equipped with Chromosorb 105 column as described earlier (Sharma
et al., 1985; Mody and Modi, 1987).

2.3.13 Comparison of strains by SDS-PAGE of cell-free extracts

The whole-cell protein profiles of Rhizobium radiobacter BE1 and Rhizobium

radiobacter ATCC 19358 were obtained by the method as described by Perez et al.
(2000). Cells were grown in synthetic medium (50 ml ini 250 ml Erlenmeyer flasks)
having following composition (g/1): sucrose, 15.0; KNO3,1.0; K^HPCH,0.5; MgSC>4.
7H20,0.2; CaCl2.2H20,0.1, and NaCl, 0.2. pH of the medium was adjusted to 7.0 and
sterilized by autoclaving at 10 psi for 20 min. Flasks after inoculation with cells of
both R.radiobacter BE1 and the type strain Rradiobacter ATCC 19358 separately
both at (Afioo = 0.5), were incubated at 30±1°C for 24 h. Cells were harvested by
centrifugation at 10,000 g for 30 min. The cell pellets obtained were washed twice
with saline (0.85% w/v) and resuspended in distilled water.; The A600 of the washed
cell suspensions were adjusted to 0.5. To 1.0 ml of the above suspensions, 100 jxL of
SDS-PAGE sample treatment buffer (150 mM Tris.HCl, pH 6.8; 4% SDS; 20% (w/v)
glycerol, 10% (v/v) p-mercapthoethanol, and 0.005% (v/v) bromophenol blue) was
added and boiled for 10 min. The above suspension was centrifuged twice at 10,000 g
for 10 min in order to obtain the cell free extracts.

Whole cell extracts (25 pL) were loaded into polyacrylamide slab gels and analyzed
by SDS-PAGE [10% (w/v) acrylamide in the resolving gel and 4% (w/v) acrylamide
in the stacking gel] by the method of Laemmli (1970). Molecular weight markers
(Bangalore Genei, India) contained a mixture of: phosphorylase b (97.4 KDa), bovine
albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soyabean
trypsin inhibitor (20.1 kDa) and lysozyme (14.3 KDa). Gels were run at 20 mA, fixed
 49

in a mixture of 50% (v/v) methanol and 10% (v/v) acetic acid, stained for 1 h with
0.1% (w/v) coomassie blue R-250 [in 40% (v/v) methanol + 10% (v/v) acetic acid]
and then destained using destaining solution consisting of 40% (v/v) methanol+10%
(v/v) acetic acid.

2.3.14 Carrot disc infectivity assay

Carrot-root slices were used as a second host to estimate the relative infectivity of
different Agrobacterium strains as described by Lippincott and Lippincott, 1969.
Carrot roots purchased at a local market were cleaned, peeled, wiped with 95% (v/v)
ethanol and soaked in 0.9% sodium hypochlorite for 15 min, rinsed three times with
sterile distilled water and drained between sterile paper towels. Slices (about 5 mm
thick) were cut from the middle one-third of each surface disinfected carrot using
asceptic technique and placed in petri dishes containing 1% (w/v) agar in distilled
water. 0.05 ml of a suspension (Agoo - 0.5) of either Rhizobium radiobacter BE1 or
Rhizobium radiobacter ATCC 19358 (in 0.85% w/v NaCl) was inoculated on to each
disc. Control discs received 0.05 ml of sterile saline (0.85% w/v). The inoculated and
control carrot discs (in triplicates) were incubated in dim light at 27+1 °C. Tumour
and root formation on the discs was scored after 3 weeks of incubation.

2.3.15 Chemicals
All chemicals and dyes used were of analytical grade and were obtained locally.
Antibiotic discs were obtained from Hi Media, Mumbai, India. Enzymes and protein
molecular weight markers were obtained from Bangalore Genei, India; SRL limited,
Mumbai, India and Sigma Chemical co. Missouri, USA.

2.3.16 Reproducibility
All the experiments have been carried out in triplicates and have been performed three
times.
 50

2. RESULTS AND DISCUSSION

2.4.1 Primary screening

Initially, extensive screening was undertaken to isolate bacterial cultures producing
highly mucoid colonies on medium having high C:N ratio. Although thermophilic
organisms may have advantages such as reduction in cooling requirements of the
fermenter during production, they are less preferred due to their low productivity and
slow growth rate (Sutherland, 1983). In fact, all the commercially produced EPS
today are from aerobic mesophilic organisms. Hence, in the present study, for
isolation, the cultures were routinely incubated at or near 30°C and under aerobic
condition. Samples from different ecological niches such as terrestrial and hypersaline
soil, fresh water and sea water, garden soil, soil from rhizosphere, aquatic
environment (fresh water and marine), root surface of plants known to contain high
concentration of sugars (such as sugarcane), decaying fruits and vegetables, activated
sludge, biofilms on surfaces and samples from environments where sugar rich waste
was discarded, were used as sources of EPS producing organisms.

It has been reported, in the case of lactic acid bacteria, the carbon source added to the
screening medium played an important role in the production of EPS and the use of
several carbohydrates for screening would improve the detection of EPS producing
strains (Ruas-Modiedo de Los Reyes-Gavilan, 2005). Hence, in the present study, the
samples were plated out on synthetic medium (with or without yeast extract
supplementation) containing a variety of carbon substrates (each separately) such as
sucrose, mannitol, lactose, glucose, starch, inulin or cellulose and either KNO3 or
monosodium glutamate as nitrogen source. In all, 150 bacterial isolates producing
large and highly mucoid colonies were obtained. Interestingly, the garden soil and
rhizosphere of plants having fleshy roots yielded more number of EPS producing
organisms compared to hypersaline soil and water samples from which only a few
isolates were obtained.

2.4.2 Secondary screening

It has been rightly suggested that there is still a lack of suitable and specific
techniques for the selection of good potential polysaccharide-producing strains on the
 51

basis of colony morphology and the only technique to test for the ability of an
organism to produce EPS is cultivation under shake flask condition and estimating the
viscosity of the culture broth (Galindo, 1993). Additionally, it has been observed
earlier (Fyfe, 1983; Galindo, 1993) and also experienced during the present study that
although colonies of microorganisms producing polysaccharides might have a mucoid
nature and thus can be detected macroscopically, no direct correlation existed between
colony morphological characteristics (such as mucoid nature) on solid medium and
the ability of a culture to produce exopolysaccharide in liquid medium.

Hence, in the case of Xanthomonas campestris strains, Galindo (1993) suggested that
the only way to measure the xanthan-producing ability of a strain in terms of the
quality and quantity of xanthan produced was (i) cultivation under defined conditions
in liquid medium, and (ii) measuring the xanthan yield and the viscosity of the culture
broth. Similarly, in another report, the bacterial isolates obtained from polluted
ground water had mucoid mode of growth in liquid medium indicating EPS
production by these bacteria (Fusconi and Godinho, 2002; Guezennec et al., 1994).
Hence, when the 150 bacterial isolates obtained in the present study were further
tested for their ability to produce viscosity in respective synthetic medium under
shake flask conditions, culture broths of 52% of the isolates showed viscosity more
than 100 cP (at a shear rate of 2.9 sec'1) and were selected for further testing. In a

previous study also, using a similar approach, Butyrivibrio strains which either did not
grow well in the defined medium containing glucose (1% w/v) or which did not
produce enough EPS for viscosity measurements were eliminated from further studies
(Ha et al., 1991).

The recovery of microbial exopolysaccharide is a crucial step in determining both the

cost and functional properties of the finished product (Smith and Pace, 1982). In the
case of microbial EPS, this component of the cost can make up to 50% of the total
cost. Currently, the only method used for recovery of exopolysaccharides
commercially is their precipitation from the viscous culture broth by use of low
molecular weight alcohols or acetone. When precipitated using solvents, in
comparison to powdery precipitates, the formation of fibrous precipitates allowed
easy recovery of the EPS from the culture broth. Of the total number of cultures tested
for EPS production in liquid culture, in case of 27% of the cultures, precipitates of
 52

EPS having fibrous nature (apart from good viscosity) were obtained when 3 volumes
of acetone were added to the culture broth.

Furthermore, since Ihe objective of the present study was to isolate anionic EPS, each
EPS sample was tested for its anionic nature qualitatively. It has been suggested that
the precipitation of a polysaccharide by quaternary ammonium compounds such as
cetyl pyridiniumchloride (CPC) and cetyl trimetylammoniumbromide (CTAB)
indicated anionic nature of that polysaccharide (Scott, 1965; Danishefsky et al., 1970;
Kumar et al., 2004). Although all the EPS samples tested in the present study could be
precipitated using CPC, thereby indicating their anionic nature, the quality of
precipitates obtained varied considerably. The exopolysaccharide of isolates M8, EC
and El showed powdery precipitates when CPC was added to the respective EPS
solutions at a final concentration of 1% (w/v). Over all, the EPS of eight different
cultures were found to satisfy the set selection criteria viz. high viscosity in liquid
medium, ease of precipitation using acetone and anionic nature. Although all the
above cultures prqduced mucoid colonies on synthetic medium, they were found to be
different on the basis of their colony appearance on plates (table 1) and their
sensitivity to various antibiotics as given in table 2. ,

2.4.3 Rheology of exopolysaccharides of various isolates

The utility of an EPS for its various applications such as thickener and viscosifying
agent relies on the viscosity of its solution at a defined concentration. Hence, the
selected EPS samples were further evaluated in terms of the viscosity of their solution
at a concentration of 1% (w/v) as shown in table 3. Among the EPS samples tested,
an EPS of isolatelBEl showed the highest viscosity of 6010 cP (at a shear rate: of
2.9 sec* and at 30±1°C). In the present study, the exopolysaccharide samples of the

isolates selected were named as “EPS/ designation:of the isolate”. For example, an
EPS of isolate BE1 was designated here as EPS/BE1 and the EPS of isolate AM as
EPS/AM and so on.

In textile printing, the viscosity of the print paste may vary depending upon the
thickener added even when the different thickeners were used at identical
concentration. For testing of suitability of novel polymerfs) as thickener for textile
 53

printing, the print paste having similar viscosity should be used while printing on the
fabric (Teli et al„ 1987).

Table 1. Colony appearance and source of EPS producing isolates

colony
Isolate appearance Source

vv
BE1 •. *v Rhizosphere soil
• -0

AM
Rhizosphere soil

EC
Bffl
Hi
,/v
Rhizosphere of aquatic plant

H6 Hypersaline soil
S;

Garden soil

mgp
E3

El Garden soil

B2 Fertile soil

The colony appearance of seven of the eight selected isolates were observed on
synthetic medium containing sucrose (15.0 g/1) and KNCb (1.0 g/1) as carbon and
nitrogen sources, respectively. The plates after inoculation were incubated at 30±1°C
for 48 h and the ability of isolates to produce EPS was noted visually on the basis of
mucoid nature of their colonies.
 54

Table 2. Antibiotic sensitivity profiles of exopolysaccharide producing isolates

Isolates —»

BE1 El E3 AM B2 EC M8 H6
Antibiotic
4
+ + + 4 + + +
Amoxyclav (10 pg) +

4 + + + . + + +
Cephalexin (10 jig) -
+ + + + + + + +
Ciprofloxacin (10 jig)
+ + + 4 4 +
Clindamycin (2 pg , - -
4 - - 4 +
Cloxacillin (5 pg) -
4- 4 4 + + + 4 +
Co-trimaxazole (25 pg)
+ + - - + + + -
Erythromycin (15 pg)
+ + + + + + + +
Tetracyclin (10 pg)
+ + - “ + + 4 -
Ampicillin (10 pg)
+ + + + + + + +
Carbenicillin (100 pg)
+ + + + + + + +
Cephatoxime 30 pg
+ + + + + + + “
Chloramphenicol (30 pg)

Gentamycin (10 pg) + + + + + + + +

4 4 4 4 + + 4 +
Norfloxacin (300 pg)
+ 4 + 4
Oxacillin (5 pg) - - -
4 4 4 4 + • + +
Ceftriaxone (30 pg)
4 4 4 + +
Ceflazidime (30 pg) - - -

Lincomycin (2 pg) + 4 + + + + + +

Netilmicin (30 pg) + + + + + + + +

+ + + + + + 4 4
Ofloxacin (2 pg)

Vancomycin (30 pg) + + ** “ + + +

-

Sensitivity of the isolates to the antibiotics was tested by placing discs containing
antibiotics (HiMedia Laboratories, India) on Luria agar plates pre-seeded (100 pi of
A60o= 0.5), with individual bacterial cultures. Plates were incubated at 30±1°C for 72
h and the zone of inhibition was observed visually. Key: (+) sensitive; and (-)
resistant to the particular antibiotic
 55

Table 2 (contd.). Antibiotic sensitivity profiles of exopolysaccharide producing

isolates

'\Isolates -»
BE1 El E3 AM B2 EC M8 H6
Antibiotic\.
4 4 4 4 4 4 4
Colistin
4 4 4 4 4 4
Nitrofurantoin - ~
4 4- 4 4 4 4 4
Augmentin -

4 4 4 4 4 4 4
Tobramycin +
4- 4 4 4
Cephalothin - - -

4 4 4 4 4 4 4 4
Amikacin
4 4 4 4 4
Fusidic acid + -4 -

4* 4 4 4 4
Methicillin - “
4 4 4 4 4 4
Novobiocin -
“
4 4 4 4
Penicillin G -
-
-
4 4 4 4 4 4 4 4
Streptomycin
4 4 4 4 4 4 4 4
Tetracyclin

Sensitivity of the isolates to the antibiotics was tested by placing discs containing
antibiotics (HiMedia Laboratories, India) on Luria agar plates pre-seeded (100 pi of
Afi0o= 0.5) with individual bacterial cultures. Plates were incubated at 30+1 °C for 72
h and the zone of inhibition was observed visually. Key: (+) sensitive and (-) resistant
to the particular antibiotic

Table 3. Comparison of viscosity of EPS solutions of different isolates

EPS samples Viscosity (cP)

M8 2544
EC 3096
E3 3420
B2 1248
BE1 6010
El 168
AM 528
H6 5268

Solutions of various exopolysaccharides were prepared in distilled water at a

concentration of 1.0% (w/v). Viscosity of the solutions was measured (at a shear rate
of 2.9 sec'1) using Brookfield LV DV n+ viscometer equipped with a small sample
adapter (SSA-8R) and spindle number 16 at 30±1°C.
 56

Sodium alginate when used in the print paste at a concentration of 9% (w/v) showed a
viscosity of 9600 cP (at a shear rate of 2.9 sec’1 and 30+1 °C) and good print

performance in terms of sharpness and uniformity of the prints were obtained using
the above print paste containing reactive dye Procion Orange H-2R (3% w/v). Hence,
in order to evaluate the selected EPS samples for their suitability as a thickener in
reactive textile printing, initially, the concentration of respective EPS samples
required to attain a viscosity of 9600 cP was determined using Procion Orange H-2R.
Depending upon the EPS used, with an increase in the concentration of respective
EPS in the print paste, the viscosity of the print paste was also found to increase as
/

shown in figure 1. The concentration of respective EPS required to obtain a print

paste viscosity of 9600 cP (referred to as printable viscosity) as determined from the
figure 1 is given in table 4. It is evident from table 4, that for attaining a printable
viscosity of 9600 cP, EPS/BE1 was required at a very low concentration of 0.95%
(w/v) whereas EPS/El was required at a concentration of 4.9% (w/v).

Figure 1. Effect of concentration of EPS samples on viscosity of printing paste

viscosity (cP)

0 1 2 3 4 5 6
concentration (% w/v)

Viscosity of the solutions was measured (at a shear rate of 2.9 sec'1) using Brookfield
LV DV H+ viscometer equipped with a small sample adapter (SSA-8R) and spindle
number 16 at 30±1°C. (•) EPS/BE1, (o) EPS/AMI, (T) EPS/E3, (V) EPS/H6, (■)
EPS/El, (□) EPS/M8, (♦) EPS/EC, and (0) EPS/B2.
 57

Table 4. Concentration of EPS required to get printable viscosity

Exopolysaccharides Concentration of EPS (% w/v) in print

paste required to attain printable
viscosity of 9600 cP

Sodium alginate 9.0

EPS/BE1 0.95
EPS/AM 4.5
EPS/E3 2.8
EPS/H6 3.0
EPS/El 4.9
EPS/M8 1.8
EPS/EC 1.9
EPS/B2 3.5

Printable viscosity using sodium alginate (9.0 % w/v) as thickener and the reactive
dye Procion Orange H-2R (3.0% w/v) was found to be 9600 cP. Viscosity of the print
pastes was measured (at a shear rate of 2.9 sec'1) using Brookfield LV DV n+
viscometer equipped with a small sample adapter (SSA-8R) and spindle number 16 at
30±1°C.

The printable viscosity in case of EPS/BE1 was attained at nearly 10 times less
concentration as compared to sodium alginate, which was added to the print paste
at a concentration of 9.0%. For other EPS samples, 1.8 to 4.5% (w/v) a concentration
of was required to attain the printable viscosity. For textile printing, a print paste
having high degree of pseudoplasticity is desirable. Hence, when the flow behaviour
of the print pastes containing individual EPS samples and Procion Orange H-2R was
studied, all the print pastes showed pseudoplastic behaviour and followed the Power-
law model. The consistency index K, which is measure of viscosity, and the flow
index n, which is a measure of degree of pseudoplasticity for print pastes containing
EPS of the selected isolates was found to vary depending upon the EPS sample used
(table 5).

The print paste containing EPS/BE1 as a thickener (0.95% w/v) showed the highest
consistency index (110 dynes sn/cm2) and highest pseudoplastic nature (n = 0.082).

The print paste containing EPS/EC was the least pseudoplastic with a flow index of
0.911. The print paste containing AM was found to show lowest consistency index of
0.76 dynes S“/cm2. The high viscosity at low concentration compared to sodium
 58

alginate, high degree of pseudoplasticity and consistency index obtained in case

of print pastes containing EPS/BE1 suggested that it could be used as a thickener
for reactive textile printing.

Table 5. Consistency index K and flow index n for print paste containing various
thickeners

Consistency
Exopolysaccharide Index^T Flow index n
(dynes s"/cm2)
EPS/EC 1.5 0.911
EPS/AM 0.76 0.807
EPS/BE1 110 0.082
EPS/B2 13 0.317
EPS/H6 78 0.212
EPS/M8 15 0.470
EPS/E3 41.0 0.251
EPS/El 3.4 0.542

Shear stress of a solution of EPS (1.0% w/v) was measured at different shear rates
using Brookfield LV DV13+ viscometer equipped with a small sample adapter (SSA-
8R) and spindle no. 16 at 30±1°C. Values of consistency index K and flow index n
were determined using Power law model from the plot of log shear rate versus log
shear stress as described by Margaritis and Pace (1985).

2.4.4 Evaluation of the EPS of the selected isolates for use as a thickener in textile
printing
The use of sodium alginate in reactive textile printing is mainly due to its desirable
chemical (very low reactivity with reactive dyes) and rheological nature. Hence, when
evaluating a substitute for sodium alginate, it was desirable that at the screening level
itself those polymers which did not meet the required chemical and rheological nature
and satisfactory print performance were eliminated from further studies. When the
exopolysaccharide of the selected isolates were evaluated as thickener using three
different reactive dyes belonging to both the Procion and Remazole brands, the colour
strength of the printed fabrics obtained were found to vary with the type of EPS used
in the print paste (figures 2 and 3). In case of Procion Red H-8B, the print paste
 59

containing EPS/BE 1 showed the highest and even better colour strength than that
obtained using print paste containing sodium alginate, whereas that containing
EPS/El, EPS/E3 and EPS/B2 gave less than 80% colour yield.

Figure 2. Colour strength of fabrics printed using various exopolysaccharides as

thickener and Procion dyes
Relative colour strength (%)

LU W £ tN U
CD UJ
CC

EPS samples

The different EPS were incorporated in the print paste at following concentrations (%
w/v): EPS/BE 1, 0.95; EPS/El, 4.9; EPS/E3, 2.8; EPS/AM, 4.5; EPS/B2, 3.5;
EPS/EC, 1.9; EPS/M8, 1.8; and EPS/H6, 3.0. Each dye was added to the print paste
at a concentration of 3% (w/v). Printing on cotton fabric (white poplin) was carried
out by applying 50 g print paste on a screen with a rubber squeeze. All prints were
dried at 80° C for 5 min, followed by steaming for 10 min and washed first with cold
water and warm water till no colour came off from the fabric and air dried. Colour
strength of the printed fabric samples was measured using Data Colour Spectroflash
reflectance spectrophotometer. The concentration of the dye on the surface of the
fabric could be derived from reflectance using Kubelka Munk equation. (■) Procion
Red H-8B, (■) Procion Orange H-2R and (■) Procion Purple H-3R. Colour strength
obtained of fabric samples printed using sodium alginate as thickener was taken as
100%.

Although, both EPS/AM and EPS/BE 1 showed identical colour strength in case of
Procion Red H-8B, in case of Procion Orange H-2R, EPS/AM showed moderately
poor colour strength in comparison to EPS/BE 1. In case of Procion Purple, except in
case of the print paste containing EPS/El and EPS/E3, no significant difference in the
colour strength was observed when other exopolysaccharides were present as
thickeners. In case of Remazole Red 5B, EPS/BE 1, EPS/H6 and EPS/AM gave the
highest colour strength. In case of Remazole Orange 3R. EPS/BE 1 gave 10% colour
strength and except El and E3, other EPS showed more or less 80% colour strength.
 60

In case of Remazole violet 5R, highest colour strength was obtained in case of
EPS/BE 1 followed by EPS/H6. Irrespective of the type and shade of the reactive dye
used in the print paste, EPS/El and EPS/E3 always showed poor performance.

Figure 3. Colour strength of fabrics printed using various EPS as thickener and
Remazole dyes

120
Relative colour strength (%)

§
<N
Q

0
BE1 El E3 AM B2 EC M8 H6

EPS samples

The different EPS were incorporated in the print paste at following concentrations (%
w/v): EPS/BE1,0.95; EPS/El, 4.9; EPS/E3, 2.8; EPS/AM, 4.5; EPS/B2, 3.5; EPS/EC,
1.9; EPS/M8, 1.8; and EPS/H6, 3.0. Each dye was added to the print paste at a
concentration of 3% (w/v). Printing and measurement of color strength was carried
out as described previously for Procion dyes. (■) Remazole Red 5B, (■) Remazole
Orange 3R and (■) Remazole Violet 5R. Colour strength of fabric sample obtained
using sodium alginate as thickener was taken as 100%.

In conclusion, EPS/BE1 was found to be the most suitable thickener due to

following salient characteristics: (i) exhibited high viscosity 6010 cP at a shear
rate of 2.9 sec'1 and 30±1 °C) and best pseudoplasticity compared to EPS of other

isolates tested, (ii) could produce printable viscosity at nearly 10 times less
concentration (i.e. 0.95% w/v) as compared to sodium alginate, (iii) when tested
as a thickener for reactive printing, yielded high colour strength with different
type and shade of the dyes, (iv) anionic nature, and (v) formed fibrous
precipitates in presence of 3 volume of acetone. Hence, studies were undertaken to
characterize the isolate BE1 in terms of its taxonomic status, similarities and
differences in comparison to the type strain and pathogenicity to plants.
 61

2.4.5 Identification of the isolate BE1

On Luria Agar, the bacterial isolate BE1 (a gram negative rod shaped bacterium)
formed medium sized, round, smooth, raised and translucent colonies with an entire
margin and without any slime formation. On synthetic medium containing sucrose
(15.0 g/1) and KNO3 (1.0 g/1) as carbon and nitrogen sources respectively, large (3 to
4 mm), raised, opaque and mucoid colonies of BE1 with copious amounts of slime
formation were observed. In either of the above mentioned media, no pigmentation of
the colonies was noted. Initially, for identification of the isolate, using the classical
approach, various characteristics were studied and the results are as shown in table 6.

2.4.5.116S rRNA gene sequence analysis

In the recent edition (second) of Bergey’s Manual of Systematic Bacteriology, the
organization of content follows a phylogenetic framework based on analysis of the
nucleotide sequence of the 16S rRNA rather than a phenotypic structure adopted in its
earlier editions (Boone and Catenholz, 2001). Hence, 16S rRNA gene sequence
analysis of isolate BE1 was carried out in order to determine its taxonomic position.
On the basis of the 16S rRNA gene sequence, a phylogenetic analysis of isolate BE1
was performed within the representatives of the Domain Bacteria. A comparison of
1405 unambiguously : aligned 16S rRNA sequences of isolates BE1 with the 16S
rRNA sequences type strains extracted from the GenBank database revealed that the
isolate BE1 belonged to genus Rhizobium with its closest member being
Rhizobium radiobacter DSM 30147T or ATCC 19358 (similarity greater than 99%).

The dendrogram (figure 4) generated using the neighbour joining method (Saitou and
Nei, 1987) and Jukes and Cantor evolutionary distance matrix (Jukes and Cantor,
1969) showed the phylogenetic position of strain BE1 within the radiation of the
Order Rhizobiales, Class Alphaproteobacteria, Phylum Proteobacteria. The
Mesorhizobium and Sinorhizobium clusters representing up to 4 strains per cluster are
shown as filled triangles. The G+C content of the 16S rRNA gene was found to be
4.75%. Using a similar approach novel species of Microvirga subterrana and
Fervidobacterlum gondwanense were identified previously (Andrews and Patel, 1996;
Kanso and Patel, 2003).
Table 6. Characteristics of isolate BE1
 62

Characteristics Result

Gram-negative short
Gram reaction rods occurring
mostly in single.
Endospore formation -

Motility +
Fluorescence under UV -

Growth temperature (°C)

4 -

10 -

15 -

22 +
26 +
30 +
37 +
42 +
55 -

65 -

Growth pH
5.0 +
• 5.7 +
+
6.8
+
8.0
9.0 +

11.0 +

Growth in presence of NaCl (% w/v)

2.5 -

' 5.0 -

7.0 -

8.5 -

9.0 -

10.0 -

Growth under anaerobic

condition
Lactose fermentation when -

grown on MacConkey agar

Indole production -

Methyl red production +

Voges Proskauer test -

Citrate utilization +

The biochemical tests were performed according to the methods described by

Smibert mid Krieg, 1981; Noel and Rrieg, 1984; and Freney et al., 1991.
 63

Table 6 (contd.) Biochemical characteristics of isolate BE1

Characteristics
Result
Gas production from glucose -

Casein hydrolysis -

Urea hydrolysis -

Esculine hydrolysis +
Nitrate reduction -

Nitrite reduction +
H2S production - •

Cytochrome oxidase +
Catalase production ■ +
Oxidation/Fermentation -

Gelatin liquefaction -

DNAse production -

Phenylalanine deamination -

Adonitol fermentation -

Arabinose fermentation -

Cellobiose fermentation -

Dextrose fermentation -

Dulcitol fermentation -

Fructose fermentation -

Galactose fermentation -

Inositol fermentation -

Inulin fermentation -

Lactose fermentation ■ -

Maltose fermentation -

Mannitol fermentation -

Mannose fermentation -

Melibiose fermentation -

Raffinose fermentation
Rhamnose fermentation -

Salicin utilization -

Starch utilization -

Sorbitol fermentation -

Glucuronate utilization -

Trehalose fermentation -

Malonate utilization -

Ornithine decarboxylase
-
activity
Lysine decarboxylase activity -

Para Phenylalanine deaminase

-
activity
Saccharose utilization -

The biochemical tests were performed according to the methods described by

Smibert and Krieg, 1981, Noel and Krieg, 1984, and Freney et a!., 1991.
 64

Table 6 (contd.) Biochemical characteristics of Rhizobium radiobacter BE1

characteristics Result

5-Ketogluconate utilization -
Palationase activity -
Galacturonate utilization -
Colistin production -

Coumarate utilization -

Tetrathionate reductase activity -

O-Nitrophenyl N- acetyl P-D- -
Glucoseaminide
P-N-acetyl p-D- “

Galactopyranoside utilization
a-Galactosidase activity +
Indoxyl phosphate production -

The biochemical tests were performed according to the methods described by

Smibert and Krieg, 1981; Noel and Krieg, 1984; and Freney et al., 1991.

2.4.S.2 Brief taxonomic description of Rhizobium radiobacter

The taxonomy of genus Agrobacterium had been highly confusing and controversial
(De Ley et al., 1966). As per the recent classification, Rhizobium radiobacter is the
revised name for Agrobacterium radiobacter (Young et al., 2001). Earlier,
Agrobacterium was a separate genus within the family Rhizobiaceae (Kersters and
DeLay, 1984; Jordan, 1984). Of the various Agrobacterium species, Agrobacterium
tumefaciens included tumourigenic and Agrobacterium rhizogenes included
rhiozogenic strains with a wide host specificity. However, Agrobacterium rubi and
Agrobacterium vitis induced tumourigenic reactions in Rubus sp. and Vitis sp. only.
Importantly, Agrobacterium radiobacter comprised non-pathogenic Agrobacterium
strains.

Young et al., (2001) arguing convincingly that (i) Agrobacterium is an artificial genus,
(ii) Agrobacterium, Allorhizobium and Rhizobium formed a monophyletic group
based on comparative 16S rDNA analyses, and (iii) they shared common phenotypic
characteristics, amalgamated all species of Agrobacterium Conn 1942 and
Allorhizobium undicola (de Lajudie et al., 1998) into a single genus Rhizobium.
Hence, today, the new combination of Rhizobium radiobacter encompasses the
 65

strains previously allocated to Agrobacterium radiobacter and Agrobacterium

tumefaciens (Young et al., 2001).

Figure 4. Phylogenetic position of Rhizobium radiobacter as determined by

16S rRNA analysis.

2%
1---------

Mesorblzobium

Smorhizobiiun

— Rhizobium giardinii CIP1Q55Q3TIB6344

j— Rhizobium rluzogeites IFO 13257T D14501
'-------- Rhizobium tropici CIAT899T TJ89832

Rhizobium hainanense DSM11917TU71078

r Rhizobium gatlicumMSDJ1109T U86343
-Li
Rhizobium mongolense USDA1844T U89817
-t Rhizobium leguminosarum IAM12609T D14513
Rhizobium etli CFN 42TU289R5
Rhizobium galegae ATCC 43677TD11343
------------- Rhizobium uudicola LMG H875TY17047
■ Rhizobium vitis NCPPB 3554TD01258
- Rhizobium nibi 1AM13569T D14503
Rhizobium radiobacter DSM30147TD01256
STRAIN BE!
— Bradyrhizobium.iapoiucumIiMG6138TX66024

Dendrogram was constructed from 1405 unambiguous nucleotides by using the

neighbour joining method (Saitou and Nei, 1987) and Jukes and Cantor evolutionary
distance matrix (Jukes and Cantor, 1969) shows the phylogenetic position of strain
BE1 within the radiation of the Order Rhizobiales, Class Alphaproteobacteria,
Phylum Proteobacteria. The most homologuos sequences to the 16S rRNA gene
sequence of isolate BE1 were identified in the Gene Bank database using the BLAST.
The genus/species name is followed by the culture collection/accession number and
the GenBank sequence accession number. Bar, represents 2 nucleotide exchanges per
100 nucleotides. Mesorhizobium and Sinorhizobium clusters representing up to 4
strains per cluster are shown as filled triangles. The outgroup sequence used in the
analysis was Bradyrhizobium japonicum.

Although, Young et al. (2001), were the authors of Agrobacterium and Rhizobium
chapters in the second edition of Bergey’ Manual of Systematic Bacteriology
(Kuykendall, 2005), they inform in a recent paper (Young et al., 2003), that the above
 66

chapters were prepared before the amalgamation of the two genera and hence
Rhizobium and Agrobacterium were retained as separate genera.

In summary the phylogenetic position of strain BE1 is as follows:

Domain Bacteria
Phylum Proteobacteria
Class Alphaproteobacteria
Order Rhizobiales
Family Rhizobiaceae
Genus Rhizobium
Species radiobacter

2.4.5.3 Comparison of biochemical characteristics of Rhizobium radiobacter BE1

and reference strain R. radiobacter ATCC 19358
When the major biochemical characteristics unique to Rhizobium radiobacter were
tested, no significant differences were observed in the biochemical characteristics of
Rhizobium radiobacter BE1 and the reference strain Rhizobium radiobacter ATCC
19358 (table 7). It has been observed previously that, all Agrobacterium radiobacter
strains and majority of the Agrobacterium tumefaciens strains (85%) produce 3-
ketolactose from sugars by oxidation at carbon-3 of the glycosyl moiety and other
Rhizobia do not produce this compound (Bemaerts and De Ley, 1963; De Ley et al.,
1966; Clark, 1969). Rhizobium radiobacter BE1 (similar to the reference strain)
produced 3-ketolactose confirming that it belonged to the previously existing
Agrobacterium group. However, when the EPS production capacity was tested on
synthetic medium containing sucrose (15.0 g/1) and KNO3 (1.0 g/1) as carbon and
nitrogen sources respectively, the colonies of reference strain were not mucoid
whereas colonies of strain BE1 produced copious amount of exopolysaccharides
indicating that the strain BE1 had different physiological characteristics with respect
to EPS biosynthesis.
 67

Table 7, Comparison of characteristics of Rhizobium sp.

Result
Characteristics
Rhizobium radiobacter Rhizobium
ATCC 19358 radiobacter BE1
Growth at 35 °C + +
Growth at 28 °C + +
In the presence of 2% (w/v) NaCl + 4•
3-ketolactose production + +
Acid production from meso-inositol - -

Acid production from ethanol - -

Alkali production from Na malonate - -

Growth factor requirement Nil Nil

Phytopathogenicity Nil Nil

Growth at different temperatures and in presence of 2% NaCl was observed in Luria-

Bertani broth visually. 3-ketolactose production test was carried out as described by
De Lay and Bemaerts (1963). Formation of ketolactose was visually detected by the
presence of a yellow zone surrounding the colonies after flooding with Benedict’s
reagent. Formation of acid and alkali were tested by incorporating the respective
substrates in medium as described by Graham and Parker (1964). Phytopathogenicity
was determined using the carrot disc infectivity assay as described by Lippincott and
Lippincott (1969) by observing the formation of either tumours or roots after
incubation of inoculated carrot discs for 3 weeks in dim light at 27+1 °C.

2.4.6 Comparison of whole cell protein profiles of Rhizobium radiobacter BE1

and R. radiobacter ATCC 19358

DNA finger printing, lipopolysaccharide (LPS), lipid and protein profiles are well
known methods to study similarity and differences between different genera (Hillis et
al., 1996; Boone and Castenholz, 2001). Recently, a reference map for Agrobacterium
tumefaciens proteins has been presented which contained more than 300 entries as a
basis for comparison of the proteome under specific experimental conditions (Rosen
et al., 2004). In a previous report, in case of Salmonella mutants, whole-cell lysates
reflected unique LPS profiles indicating that they were separate chemotypes
(Hitchcock and Brown, 1983). Hence, in order to delineate if any difference in whole
cell protein profiles existed between the Rhizobium radiobacter BE1 and Rhizobium
radiobacter ATCC 19358, SDS-PAGE analysis of the whole cell proteins was carried
out (figure 5). The whole cell protein profile of R.radiobacter BE1 strain was almost
 68

similar to that exhibited by the reference strain, except that strain BE1 did not show a

band at 33.8 kDa.

Figure 5. SDS-PAGE of whole cell protein extracts of Rhizobium radiobacter BE1 and
R. radiobacter ATCC 19358

1 2 3
KDa

97.4
66.0 mm

43.0 mm *

29.0

20.1 m ■

14.3 '

To 1.0 ml of the R.radiobacter ATCC 19358 and R.radiobacter suspensions (A60o =

0.5), 100 pL of SDS-PAGE sample treatment buffer was added and boiled for 10 min.
Whole cells and cell fragments were separated by centrifuging twice at 10 000 g for
10 min in order to obtain the cell free extracts. Whole cell extracts (25 pL) were
loaded onto 10% polyacrylamide slab gels and analyzed by SDS-PAGE by the
method of Laemmli (1970). Lane 1. Molecular weight markers. Lane 2. Whole cell
extract of Rhizobium radiobacter ATCC 19358 and Lane 3. Whole cell extract of
Rhizobium radiobacter BE1. Gels were run at 20 mA, fixed in a mixture of 50% (v/v)
methanol and 10% (v/v) acetic acid, stained for 1 h with 0.1% (w/v) Coomassie blue
R-250 (in 40% (v/v) methanol + 10% (v/v) acetic acid) and then destained using
destaining solution consisting of 40% (v/v) methanol and 10% (v/v) acetic acid.
Molecular masses of the marker proteins are indicated on the left-hand side. Arrow
(near lane 2) indicates the band at 33.8 kDa, which is present in cell-free protein
extracts of Rhizobium radiobacter ATCC 19358 and absent in case of cell-free
protein extract of Rhizobium radiobacter BE1 (lane 3).

Three prominent bands observed at 60.6 KDa, 56.3 KDa and 50.48 KDa in case of
R. radiobacter ATCC 19358 were also present in R.radiobacter BE1. However, the
molecular weight of the above proteins in case of Rhizobium radiobacter BE1
appeared to be higher at 65.2, 60.6 and 54.3 KDa, respectively. Moreover, the band at
36.3 KDa was less intense in case of R.radiobacter BE1. In past, protein finger
 69

printing has been used for characterization of two novel isolates, Lactobacillus
acidifarinae and Lactobacillus zytnae (Vancanneyt et al., 2005) and taxonomic
characterization of denitrifying bacteria that degrade aromatic compounds (Song et al.,
1999).

2.4.7 Carrot disc infectivity assay

As it has been discussed earlier, as per previous classification, the strain BE1
belonged to A. radiobacter biovar 1, which included non-phytopathogenic strains.
However, after the recent revision and amalgamation of Agrobacterium with
Rhizobium by Young et al. (2001), the tumourigenic strains of A.tumefaciens were
also merged into a single species Rhizobium radiobacter. In fact, as per the recent
classification, Agrobacterium tumefaciens does not exist as a separate species.
Formation of roots or tumor on wounded stems of Daucus carota (carrot) has been
used as a diagnostic tool to distinguish between the pathogenic and non-pathogenic
strains of Agrobacterium (Lippincott and Lippincott, 1969; Jordan, 1984). Hence, to
determine whether the Rhizobium radiobacter BE 1 was phytopathogenic, when carrot
disc infectivity assay was carried out, in case of both Rhizobium radiobacter BE1 and
Rhizobium radiobacter ATCC 19358, even after 3 weeks of incubation, no root or
tumour formation occurred (figure 6) indicating that the strain BE1 belonged to non-
pathogenic biovar of Rhizobium radiobacter.

Figure 6. Carrot disc infectivity assay

Discs of carrot roots (about 5 mm thick) were surface sterilized using ethanol and
0.9% hypochlorite and placed in petri dishes containing 1% (w/v) agar prepared in
distilled water. 0.05 ml of a 48 h culture of R.radiobacter and Rhizobium radiobacter
ATCC 19358 was inoculated onto carrot discs individually (in triplicates) and were
incubated in dim light at 27+l°C. Control discs received 0.05 ml of normal saline
(0.85% w/v NaCl). (A) Control, (B) Carrot disc inoculated with Rhizobium
radiobacter ATCC 19358, and (C) Carrot disc inoculated with Rhizobium
radiobacter BEI. Either tumour or root formation on the discs was scored after 3
weeks of incubation.
 70

2.5 CONCLUSION
The exopolysaccharide of Rhizobium radiobacter BE1 holds promise as a substitute
for sodium alginate in textile printing with good rheological properties and high
colour strength compared to EPS of other isolates evaluated.

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