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Edifying the strategy for the finest extraction of succinoglycanfrom Rhizobium radiobacter strain CAS
Prasad Andhare1 . Dweipayan Goswami2 . Cedric Delattre3,4 .
Guillaume Pierre3,4 . Philippe Michaud3,4 . Hilor Pathak1
Received: 20 December 2016 / Accepted: 18 May 2017 / Published online: 26 May 2017
� The Korean Society for Applied Biological Chemistry 2017
Abstract Succinoglycan is an industrially important
exopolysaccharide (EPS) that is produced by certain bac-
teria. There are several procedures to extract this EPS,
though the efficiency of all the available procedures is
questionable and any improvement in the extraction effi-
cient can greatly benefit the industry. Here we emphasize
on optimization and development of new modus operandi
to efficiently extract succinoglycan from liquid bacterial
culture. Also, we studied the effect of different extraction
methods on production, rheological and structural proper-
ties of succinoglycan. Eighteen different chemical and
physical methods were tested for succinoglycan extraction
from Rhizobium radiobacter CAS isolates with the prin-
ciple of extracting EPS by precipitating it, where only
eleven methods could precipitate the succinoglycan.
Comparing the extraction yield of all methods, biopolymer
extracted by acetone (3014 mg/L) was maximum followed
by cetyl-trimethyl-ammonium-bromide (CTAB 2939 mg/
L) and vacuum evaporation (2804 mg/L) methods. Upon
comparison of rheological property of recovered succino-
glycan, it was found that at shear rate 50 s-1 EPS recov-
ered using acetone and CTAB methods tends to make the
solution highly viscous with a viscosity of 150 and
146 mPa s, respectively. In agreement with these results,
power law equation showed that EPS extracted by acetone
and CTAB had high consistency index (k) and low flow
behavior index (g). The current results showed that the
physicochemical methods for EPS extraction significantly
affect the structural composition of, though succinoglycan
extracted using acetone and CTAB showed minimum
structural abrasion.
Keywords Exopolysaccharide � Extraction � Power law �Rhizobium � Succinoglycan
Introduction
Exopolysaccharide (EPS) is high molecular weight com-
pounds formed by polymerization of homo- and hetero-
monomeric sugar residues with diverse commercial
applications in industries owing to their virtues and phys-
ical properties such as pseudoplasticity, thixotropy, vis-
cosity, gelling and emulsifying activity [1, 2]. EPS is
referred source of ‘‘green’’ chemistry and synthesis for its
valuable properties in view of potential applications in
food, cosmetics and pharmaceutical industries [3, 4]. To
list few, alginate, chitosan, curdlan, dextran, levan, pullu-
lan, succinoglycan xanthan, etc. are some commercially
important microbially derived EPSs.
Succinoglycan is an economically important high
molecular weight EPS produced extracellularly by Si-
norhizobium, Rhizobium, Agrobacterium, Alcaligenes and
Pseudomonas [5–13]. It is commercially used in cosmetics
and home care products, fertilizer formulations, pharma-
ceuticals, food industries, for enhanced oil recovery as an
emulsifying agent, gelling agent, stabilizing agent,
& Hilor Pathak
1 P.D. Patel Institute of Applied Sciences, Charotar University
of Science and Technology (CHARUSAT), Changa,
Gujarat 388421, India
2 Department of Biochemistry and Biotechnology, St. Xavier’s
College (Autonomous), Ahmedabad, Gujarat 380009, India
3 Universite Clermont Auvergne, Universite Blaise Pascal,
Institut Pascal, BP 10448, 63000 Clermont-Ferrand, France
4 CNRS, UMR 6602, IP, 63171 Aubiere, France
123
Appl Biol Chem (2017) 60(3):339–348 Online ISSN 2468-0842
DOI 10.1007/s13765-017-0286-8 Print ISSN 2468-0834
texturing agent and thickening agent. All such properties of
succinoglycan are attributed due to its high consistency
under extreme operational condition such as high temper-
ature, salt or ionic concentration, pressure and high shear
rate [9, 14–18].
Considering the commercial importance of microbial
EPS, numerous universal extraction methods are available
for the precipitation, separation and extraction of microbial
EPS [19]; however, under different scenario including
culture conditions and type of EPS, the extraction proce-
dure has to be modified. Key points considered in EPS
extraction include (1) approaches to release and precipitate
EPS, with minimal structural abrasions, and (2) minimal
alteration in characteristics and property of extracted EPS
[20–23]. Owing to this, different extraction procedures
have to be employed to extract different EPS. For instance,
optimized extraction protocol for xanthan may not work
efficiently for succinoglycan. Regardless of several EPS
extraction techniques in the literature, it is necessary to
optimize modus operandi from the standpoint of yield and
composition. Also, after optimizing the extraction proce-
dure for a particular EPS, the quality and structural abra-
sions of extracted EPS have to be accessed [24, 25].
Under present study, we conducted an extensive opti-
mization study of extraction procedures for succinoglycan
extraction from culture suspension of Rhizobium
radiobacter strain CAS (GenBank accession number:
KJ191122.1). The succinoglycan produced by R.
radiobacter strain CAS was earlier characterized by FT-IR
and NMR spectroscopy and was found to be composed of
glucose and galactose in a molar ratio of 6.99:1 as con-
firmed by HPAEC and published by Andhare et al. [26]. To
optimize the extraction procedure for succinoglycan, thir-
teen chemical procedures and five physical methods were
employed for the extraction of succinoglycan produced by
R. radiobacter strain CAS with a major goal of offering a
comparative of yield from an array of extraction protocols.
Thus, we vividly studied the effect of various extraction
methods on the production yield and structural abrasions of
extracted succinoglycan. To our knowledge, this significant
piece of information is completely missing in the docu-
mented literature on the subject till now.
Materials and methods
Extraction of succinoglycan from bacterial culture
using chemical methods
Multiple Erlenmeyer flask each containing 100-ml Bush-
nell Hass broth (pH 7.0) and 1% sucrose with seed culture
of CAS was inoculated in the concentration 5% (v/v) with
an interval of 2%. The O.D. of seed broth of R. radiobacter
CAS strain was maintained at 1.0 at 600 nm for the initial
equal inoculum concentration. Flasks were incubated in
rotary shaker at 150 rpm at 30 �C for 4 days. Optimization
of growth condition and inoculum size is published by
Andhare et al. [26]. Wide range of chemical solvents, salts
and physical methods were used individually or/and in
combination to precipitate and extract out the succinogly-
can from the culture broth [6]. The overview of each
extraction scheme is illustrated in Fig. 1.
Methodology for extraction of succinoglycan using
chemical methods includes separation of cell biomass by
centrifugation at 94009g at 25 �C for 10 min, followed by
the addition of two volumes of chemical solvents in the
culture supernatant to extract succinoglycan by precipita-
tion, which was recovered using centrifugation at
94009g for 5 min at room temperature. Thirteen chemical
methods to extract succinoglycan from culture supernatant
included addition of two volumes chilled acetone [27], two
volumes chilled ethanol [8], two volumes chilled methanol
[28], two volumes chilled iso-propyl alcohol [11], two vol-
umes chilled n-propyl alcohol, cetyl-trimethyl-ammonium-
bromide (CTAB 2% w/v, 100 ml) [29], two volumes chilled
butanol, two volumes chilled iso-amyl alcohol [12], sodium
hydroxide (NaOH 1 M, 100 mL) [30], glutaraldehyde
(Sigma 25%, 100 mL) [31], formaldehyde (36.5%, 100 mL)
[32], ethylenediaminetetraacetic acid (EDTA 2%, 100 mL)
[33] and formaldehyde–NaOH (50 mL each) [30]. A dia-
grammatic summary of all the aforementioned eleven
extraction methods is shown in Fig. 1. The chemical method
which showed maximum precipitation of succinoglycan was
further optimized by combining physical treatments to the
original chemical extraction procedures.
Selection of the best succinoglycan extraction
procedure combining physical treatments
to chemical method
Four-day-old culture of R. radiobacter CAS growing in
liquid Bushnell Hass broth containing sucrose as a carbon
source was used. Briefly, cell biomass was removed from
the culture broth by centrifugation at 94009g at 25 �C for
10 min to get cell-free culture supernatant. Thereafter, five
physical treatments were individually given to culture
supernatant, which included (1) boiling [31], (2) heating
[21], (3) sonication, (4) sonication–boiling and (5) vacuum
evaporation [34]. After providing physical treatment to the
culture supernatant, succinoglycan was extracted by pre-
cipitation, using twice the volume of acetone. Here we
opted to use acetone because it yielded maximum extrac-
tion of succinoglycan from all the previously accessed
chemical solvents.
The exopolysaccharide precipitated was collected by
centrifugation at 94009g for 5 min at room temperature
340 Appl Biol Chem (2017) 60(3):339–348
123
and dried in hot air oven for overnight at 50 �C [35]. Dried
EPS was grind to fine powder and preserved for rheology
and structural elucidation studies.
Rheology studies of extracted succinoglycan
Different extraction conditions by physical and chemical
methods may alter the rheological behavior of extracted
Fig. 1 Flowchart for CAS EPS extraction methods. Acetone was used as solvent to precipitate EPS in physical methods (EPS
exopolysaccharide, CTAB cetyl-trimethyl-ammonium-bromide)
Appl Biol Chem (2017) 60(3):339–348 341
123
succinoglycan. As a consequence, the molecular conforma-
tions and rheological profile are influenced by the harsh
treatments employed to extract EPS [13]. Rheological analysis
will provide an insight to the abrasion caused to succinoglycan
by the harsh treatments. The ideal extraction procedure is one
which causes minimum abrasion to the complex structure of
EPS. Accordingly, rheological analysis of extracted succino-
glycan was carried out in Anton Paar Rheolab QC using cup
and bob geometry. Viscosity of all the succinoglycan samples
extracted using different methods was measured.
Aqueous solution of 1% (w/v) EPS in distilled water was
prepared and was left overnight in refrigerator for complete
hydration. This aqueous solution of EPS was used as a
sample for rheological analysis. A shear rate sweep from
0.01 to 1000 s-1 [18] was applied for 5 min with 60 points at
a time interval of 5 s each, and rheological data were col-
lected at 25 �C. The RheoPlus software from Anton Paar was
used for operating the instrument and recording results. The
relationship between apparent viscosity and shear rate was
studied by computing the consistency coefficient (k) and
flow behavior index (g) values in power law equation:
ga = k� (n-1), where ga is apparent viscosity (mPa s), � is
shear rate (s-1), k is consistency index (mPa) and n is the flow
behavior index (dimensionless).
Structural characterization of extracted EPS by FT-
IR analysis
Fourier transform infrared (FT-IR) spectroscopic analysis
was performed as it can reveal the structural abrasions
caused by different extraction methods. Succinoglycan
extracted from acetone, CTAB and vacuum evaporation
methods showed comparatively higher yield than other
methods and henceforth selected for structural analysis by
FT-IR. Also, FT-IR spectra of standard succinoglycan pro-
duced in laboratory from Rhizobium meliloti strain M5N1
[36] were performed for better comparison and were con-
sidered as a standard reference. Infrared spectroscopy of EPS
was recorded using Nicolet 6700 infrared spectrophotome-
ter. FT-IR analysis of extracted EPS was registered in the
mid-IR region of 400–4000 cm-1 with a scan speed of 32.
KBr disk was prepared by compressing the sample and KBr
in ratio 2:100, and spectra were recorded. The spectra
obtained were analyzed as stated by Lin et al. [37].
Results
Extraction of succinoglycan by physicochemical
methods
Yield of EPS from the microbial source is important for
industry. So any progress which can enhance the recovery
and overall yield of EPS is inevitable from the industrial
perspective. Under present study, an effort is made to
enhance the overall yield of succinoglycan from a micro-
bial source. After optimizing the yield of succinoglycan,
the effect of extraction procedure on the structural abrasion
and rheological behavior was studied. The comprehensive
research was carried involving succinoglycan extraction
from our strain R. radiobacter strain CAS (GenBank
accession number: KJ191122.1), using eighteen different
methods as shown in Fig. 1. Briefly, succinoglycan was
precipitated from the 4-day-old culture supernatant of R.
radiobacter strain CAS grown in Bushnell Hass broth
containing sucrose as carbon source (10 gm/L) using twice
the supernatant volume in all studies for equal comparison.
We have previously proved that R. radiobacter strain CAS
produced EPS.
We found that yield of succinoglycan was poor when
butanol, iso-amyl alcohol, NaOH, glutaraldehyde,
formaldehyde and formaldehyde–NaOH were used. The
yield of succinoglycan extracted from R. radiobacter strain
CAS by eleven processes is summarized in Fig. 2. Out-
comes showed that the EPS production was importantly
dependent upon the extraction method. Higher quantity of
EPS was extracted when acetone (3014 mg/L) followed by
CTAB (2939 mg/L) and vacuum evaporation (2804 mg/L)
was used as precipitating agents. Total quantity of EPS
produced by acetone extraction was 5.8 times higher than
the most commonly used precipitating agent, ethanol
(519 mg/L). The difference in succinoglycan yield in ele-
ven physicochemical methods varied in the range of
519–3014 mg/L. Ethanol is widely reported that ethanol
can efficiently precipitate succinoglycan with the volume
required being thrice or more to the volume of supernatant,
whereas here we showed that acetone was required in much
lesser quantity and yield was several folds high in com-
parison with ethanol.
Firstly, we suggested that acetone extraction could
prove efficient for EPS produced by Rhizobium sp. Sec-
ondly, when succinoglycan was extracted by adding one
tenth of the culture supernatant volume of CTAB (2%
w/v), the yield was high. To the best of our knowledge,
this is the first report suggesting the use of CTAB to
extract biopolymer from R. radiobacter. Lastly, vacuum
evaporation also presented potential prospective for EPS
extraction, as it reduced the overall acetone volume
required to precipitate EPS. It extracted 2804 mg/L EPS
which was higher than rest of the methods employed in
the present study. Thus, we conclude that acetone pre-
cipitation aided with vacuum evaporation is the best
extraction procedure to recover succinoglycan. Further-
more, this procedure also stands out to be economical as
large amount of acetone is not required to achieve effi-
cient extraction.
342 Appl Biol Chem (2017) 60(3):339–348
123
Rheology studies of extracted EPS
The flow curves of succinoglycan for different extraction
methods reported the effect of shear rate on apparent vis-
cosity in Fig. 3. With increase in shear rate, the apparent
viscosity considerably decreased indicating shear thinning
(pseudoplastic) behavior. Among the eleven different
methods tested, precipitation with acetone and CTAB was
highly efficient in extraction of succinoglycan and the
extracted succinoglycan using these methods had greater
viscosity. This indicates that precipitation with acetone and
CTAB does not erode the complexity of succinoglycan. At
shear rate 51, viscosity produced by EPS extracted by
acetone and CTAB methods was 150 and 146 mPa s,
respectively. The coefficients of this equation along with
regression coefficient (R2) for extracted all succinoglycan
samples are presented in Table 1. The relevance of power
law model and high level of relation between measuring
points to evaluate flow properties of extracted EPS were
suggested by regression coefficient (R2) with higher range
of 0.95–0.99. The consistency coefficient ‘k’ defines the
inclusive range of viscosities of resulted EPS from differ-
ent procedures. The highest consistency index (k) and the
lowest flow behavior index values (g) were reported in
succinoglycan extracted by acetone and CTAB. Our results
showed the effect of extraction methods influenced con-
sistency index and elevated the biogum precipitation where
succinoglycan extracted using acetone and CTAB showed
k value equal to 2760.9 and 2871.9, respectively. Flow
behavior indices, on the other hand, had decreased signif-
icantly for acetone (0.241) and CTAB (0.235) methods.
The g values for all extracted succinoglycan solutions were
between 0 and 1, ranging from 0.235 to 0.743, revealing
shear thinning behavior as lower values of exponent g(close to zero) are more pseudoplastic products. The flow
behavior coefficient denotes the degree of pseudoplastic
nature of extracted EPS by different approaches. To our
knowledge, this is the first study explaining the effect of
Fig. 2 Extraction of succinoglycan using different physicochemical
methods. Extraction by acetone, vacuum evaporation and CTAB
resulted in high EPS yield than other methods. [Analysis of variance
(ANOVA) was carried out to identify significant extraction enhance-
ment as compared to the minimum yielding method (MYM) (i.e.,
ethanol extraction) the EPS extraction values were compared at
significance levels of 5, 1 and 0.1% LSD]. p value has been calculated
using one way ANOVA and its interpretation is as follows: ns
(p value greater than 0.05), non-significance as compared to the
minimum yielding method (MYM) (i.e. ethanol extraction); *(p value
between 0.05 and 0.01), significant at 5% as compared to MYM;
**(p value between 0.01 and 0.001), significant at 1% as compared to
MYM; ***(p value less than 0.001) significant at 0.1% as compared
to MYM
Fig. 3 Effect of extraction
method on rheology of CAS
EPS (CTAB cetyl-trimethyl-
ammonium-bromide)
Appl Biol Chem (2017) 60(3):339–348 343
123
physicochemical extraction methods on consistency index
(k) and flow behavior index values (g) of succinoglycan.
Structural characterization of extracted EPS by FT-
IR analysis
Succinoglycan extracted using acetone, CTAB and vacuum
evaporation showed efficient biogum production, so only
these samples were analyzed using FT-IR. Also, FT-IR
spectra of these samples were compared with the spectra of
standard succinoglycan. Comparison of FT-IR spectra of
extracted succinoglycan with standard succinoglycan is
illustrated in Fig. 4 to analyze extraction methods capa-
bility to uphold the EPS structure and its properties. FT-IR
spectra of acetone-extracted succinoglycan, CTAB-ex-
tracted succinoglycan and standard succinoglycan are
superimposable as these spectra are similar. These findings
suggest that acetone and CTAB extraction methods did not
affect the glycosidic bond and sugar ring of carbohydrate;
however, succinoglycan extracted using vacuum evapora-
tion method had dissimilar spectra as observed by its dis-
torted spectra with weak peaks. Except for vacuum
evaporated succinoglycan, other spectrums had pro-
nounced band between 3399 and 3405 cm-1 characteristics
for O–H symmetrical stretching vibration for carbohydrate
compounds and involved in EPS water solubility according
to data explained by Karbowiak et al. [38]. The detailed
band assignment of main absorption peaks is shown in
Table 2. Comparison of spectra for acetone and CTAB
with succinoglycan showed that extraction methods had no
effect on functional groups and structure of extracted
biopolymer and both spectra comprised hydroxyl and car-
boxyl which correspond mostly to occurrence of carbohy-
drate as shown in Fig. 4 and Table 1. On the contrary, FT-
IR of EPS extracted by vacuum evaporation had weak
signals and prominent carbohydrate peaks were absent
indicating the structural abrasion. Consistent with this,
rheological analysis of vacuum evaporation precipitate
EPS had high flow behavior index (g) and low consistency
index (k) confirming the effect of extraction method on
conformation and property of exopolymers.
Discussion
EPS is a commercially important product as it is widely
used in food and pharmaceutical industry though they are
majorly used as gums, stabilizers, thickeners, emulsifiers,
etc. EPS is a polymer of monosaccharides produced by
microbes by utilizing available sugars from the environ-
ment [39]. Bacteria accumulate this EPS forming a layer
called as capsule above cell wall. Bacteria produce diverse
EPS with different complexity and structure. Property of
EPS to act as a gelling agent, thickeners, etc. depends on its
Table 1 Comparison of flow behavior index (g) and consistency
index (k) of succinoglycan powder solutions derived from eleven
physicochemical methods
Methods j (mPa s) g R2
Sonication and boiling 693 0.44 0.99
n-Propyl alcohol 1392 0.31 0.99
Sonication 468 0.48 0.99
Iso-propyl alcohol 1175 0.34 0.99
CTAB 2872 0.24 0.99
Acetone 2761 0.24 0.99
Vacuum evaporation 16 0.74 0.97
Heating 100 0.48 0.95
Dry heating 745 0.37 0.99
Ethanol 1654 0.30 0.99
Methanol 881 0.36 0.99
Fig. 4 Chemical
characterization and comparison
of EPS extracted by acetone,
CTAB and vacuum evaporation
methods with standard
succinoglycan by FT-IR (CTAB
cetyl-trimethyl-ammonium-
bromide)
344 Appl Biol Chem (2017) 60(3):339–348
123
structure and molecular weight [4]. Thus, while extracting
EPS from a microbial source, distortion in its structure
should be avoided. The technique which can extract EPS
without causing any significant harm to its structure is
commercially viable [40]. Also, at a commercial platform
the technique should provide high yield along with main-
taining structural integrity of EPS [41]. Under present
study, once such effort is made to enhance the extraction
yield of succinoglycan without causing any significant
distortion in its structure.
It is well known that strains belonging to Rhizobium
produce succinoglycan [36, 42, 43]. Strains of Rhizobium
possess 11 essential genes (exoY, exoF exoA, exoL, exoM,
exoO, exoU, exoW exoP, exoQ and exoT) which are needed
to produce succinoglycan [44]. Under present study, we
used one such strain R. radiobacter strain CAS which
produced succinoglycan from which we performed
extraction of succinoglycan using several chemical and
physical techniques.
Out of 11 extraction procedures employed, acetone,
CTAB and vacuum evaporation yielded efficient extraction
of succinoglycan. Even though use of ethanol is well cited
and most routinely used precipitating agent for extraction
of microbial EPS [40, 45], we showed that it is not an
efficient extracting agent specifically for succinoglycan. It
is widely reported that ethanol can efficiently precipitate
several microbial EPS with the volume required being
thrice or more to the volume of supernatant [41], whereas
here we showed that acetone was required in much lesser
quantity and yield was several folds high as compared to
ethanol. Use of CTAB to extract microbial EPS in general
is well known [29], though here we showed the exact
proportion of CTAB required for optimum extraction
specific for succinoglycan. We also showed that out of
several EPS extracting agents, CTAB is one among the best
for microbial succinoglycan. The third best technique in
terms of succinoglycan extraction was vacuum evapora-
tion. This is the first report for the use of vacuum evapo-
ration to specifically extract succinoglycan from microbial
source. Similar use of vacuum evaporation to recover EPS
produced by Lactobacillus rhamnosus was performed by
Polak-Berecka et al. [46] though the authors did not
compare the yield of the extracted EPS with other methods,
nor the extracted EPS was succinoglycan.
Only studying the yield of succinoglycan using different
procedures is not sufficient so we studied the physical
property of viscosity and rheology exhibited by succino-
glycan extracted using the most efficient extraction pro-
cedures (extraction using acetone, CTAB and vacuum
evaporation). The reason for performing such experiment is
to understand the quality (in terms of polysaccharide
complexity) of extracted succinoglycan, as the treatments
used to extract succinoglycan might cause physical harm to
the polymeric structure of EPS which is directly reflected
on the complexity of the structure. More the physical harm
to the EPS, lesser would be the structural complexity and
lesser would be the viscosity and vice versa. So, greater the
viscosity exhibited by extracted EPS, the lesser is the
structural abrasion caused by the extraction treatments
[47]. It is important in industrial point of view to know
which extraction method yields these properties as rheo-
logical assets affect the thickening, gelling and flow
behavior and processing operation of products [47].
Studying the rheology of extracted EPS was to observe the
consistency index (k) and the lowest flow behavior index
values (g) exhibited by this EPS. Rheology study of all
tested samples followed the power law model ga = k� (n-1)
demonstrating distinctive shear thinning behavior in good
Table 2 Structural characterization of extracted EPS by acetone and CTAB methods and comparison with standard succinoglycan by FT-IR
analysis
Wavenumber (cm-1) Band assignment References
Acetone CTAB Std. succinoglycan
3408 3405 3399 Stretching vibration of –OH group of sugar poly
hydroxyl groups
Yan et al. [52]
2921 2917 2918 –CH stretching of CH2/CH3 groups in carbohydrate
backbone
Liu et al. [53]
1726 1724 1726 C=O stretching of acetyl ester Castellane et al. [48, 49]
1628 1625 1615 –COO- asymmetrical stretching vibration of carboxylic
groups
Ruiz et al. [54]
1402 1402 1402 –COO- symmetrical stretching vibration of carboxylic
groups or bending tendency of symmetrical CH3
groups within acetyl and pyruvyl groups
Kwon et al. [55]
1066 1066 1069 C–C or C–O stretching vibration or asymmetrical C–O–
C stretching band resulting from sugar backbone
Botelho et al. [56]
Key functional groups observed from FT-IR spectra of extracted EPS were studied
Appl Biol Chem (2017) 60(3):339–348 345
123
agreement with previous published studies by Castellane
et al. [48, 49]. The flow behavior coefficient denotes the
degree of pseudoplastic nature of extracted EPS by dif-
ferent approaches, conferring distinctive viscosity proper-
ties to manipulate and control the rheological nature of
aqueous solutions as related by Moretto et al. [50]. The
succinoglycan extracted using acetone and CTAB showed
enhanced k value and the lowest g value. According to the
finding of Gomez-Dıaz and Navaza [51], enhanced k value
is associated with high water binding ability, and this
means succinoglycan extracted using acetone and CTAB
could efficiently bind water and could remain hydrated.
This property is attributed only if the extracted EPS is
complex and unharmed by the extraction procedures as
small and simple microbial EPS cannot bind water and
exhibit high viscosity [50]. However, vacuum evaporation
to precipitate EPS had high flow behavior index (g) and
low consistency index (k) which shows structural abrasion.
To our knowledge, this is the first study explaining the
effect of physicochemical extraction methods on consis-
tency index (k) and flow behavior index values (g) of
succinoglycan.
Lastly, succinoglycan extracted from acetone, CTAB
and vacuum evaporation methods was analyzed by FT-IR
spectroscopy to study the structural abrasion. On compar-
ing the spectra of these extracted succinoglycan samples
with standard succinoglycan, the signature peaks were
unaltered in the succinoglycan extracted using acetone and
CTAB [36]. However, this was not true in case of suc-
cinoglycan extracted using vacuum evaporation. Thus,
acetone and CTAB can efficiently extract succinoglycan
without causing any significant structural abrasion.
This study reported that extraction methods significantly
affect the amount and chemical composition of succino-
glycan extracted from R. radiobacter strain CAS.
Methodology consisting acetone and interestingly CTAB
extraction was the most efficient of all other methods
evaluated in this study extracting 3024 and 2939 mg/L of
succinoglycan, respectively. Cationic nature of CTAB
found effective in extraction of an anionic CAS succino-
glycan. Rheological studies to observe further decipher that
acetone and CTAB used for extraction were the best
methods for succinoglycan precipitation produced by Rhi-
zobium sp. in consideration with industrial implication as
rheological properties are significantly valuable in appli-
cation point of view. The power law model well elucidated
that succinoglycan extracted by acetone and CTAB meth-
ods depicted increased viscosity behavior as non-Newto-
nian shear thinning fluid complemented by small flow
behavior index (g) and high consistency value (k). FT-IR
observation also suggests that acetone and CTAB were the
best methods for EPS extraction produced by Rhizobium
sp. as they did not cause any structural damage to
succinoglycan.
Acknowledgments The author would like to thank Dr. Cedric
Delattre, Universite Clermont Auvergne, Universite Blaise Pascal,
Institut Pascal, BP 10448, F-63000 Clermont-Ferrand, France, for
providing standard succinoglycan for research work. The authors are
also thankful to Charotar University of Science and Technology for
providing the laboratory facilities for the present study.
Funding Authors did not receive any funding for the research.
Compliance with ethical standards
Conflict of interest All the authors of the manuscript declare that
they have no conflict of interest.
Ethical statement This article does not contain any studies with
human participants or animals performed by any of the authors.
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