JOURNAL OF PURE AND APPLIED MICROBIOLOGY, December 2014. Vol. 8(6), p.

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Production of Polyhydroxyalkanoates by Sugar Cane

Rhizospheric Soil Bacterial Isolates

S. Dash, S. Mohapatra*, D. P. Samantaray and A.K. Sethi

Department of Microbiology, OUAT, Bhubaneswar-3, India.

(Received: 21 July 2014; accepted: 14 September 2014)

In this research work, eight bacterial isolates were obtained from the rhizospheric
soil region of sugarcane. Then the isolates were screened for the presence of PHA granule
by following Sudan black staining of which, four bacterial isolates showed positive to
Sudan black staining. However, the bacterial isolates showing PHA granule were Gram
positive rod. Biochemical characterization revealed that all the bacterial isolates belongs
to genus Bacillus. A negative trend in cellulase and pectinase activity were observed in
case of all the PHA producing bacterial isolates. The optimum temperature and pH of all
the bacterial isolates were found be 7 and 370C respectively. Under optimized condition
Bacillus licheniformis produces 53.01% of polyhydroxyalkanoates in MSM culture
medium. The production of PHA was found to be increased along with the increase in the
biomass. The FTIR analysis of the extracted PHA showed the distinct peaks corresponding
to C=O groups and the spectroscopic analysis gave proper insight for the chemical structure
of PHB by reflecting the monomeric units. Further studies are required for elucidation of
structure of the monpmer and to reduce the cost of production.

Key words: Rhizospheric, Sudan-black, Biochemical, Polyhydroxyalkanoates & MSM

Polyhydroxyalkanoates (PHAs) are a commercial production of polymers. However

complex class of biopolymers, synthesized by chemical synthesis of these polymers is difficult
bacteria as storage compounds for carbon and and not economically feasible. Hence, micro-
energy. These are synthesized in the presence of organisms are the alternate source from which the
excess carbon and with at least one nutrient polymers can be obtained at low cost and high
essential for growth such as nitrogen, phosphorus, purity. PHA accumulation is one of the responses
sulphur or oxygen present in limiting towards stress experienced by microorganisms
concentration1. PHAs exhibit a high degree of residing at different ecological niches such as
polymerization and molecular weights up to several estuarine sediments, marine habitat, rhizosphereric
million dalton2. It is biodegradable, insoluble in soil, groundwater sediments and sewage. These
water, nontoxic, biocompatible, piezoelectric and environments are often rich in organic contents
thermoplastic. These features make them suitable and less rich in nutrient contents support the
for several applications in the packaging industry, microbial population actively involved in PHA
medicine, pharmacy, agriculture, food industry1,3. accumulation to meet the metabolic energy
PHAs can be obtained from renewable resources, requirements during starvation period4. Though
thus a remarkable intrest has been increased for different types of PHAs are available however
material properties and potential application of the
* To whom all correspondence should be addressed. PHAs vary depending on the monomer
Tel: 8984493886; composition5. Many Gram negative and Gram
E-mail: swatismile016@[Link] positive bacteria are capablility to produce PHAs,
2 DASH et al.: SUGAR CANE RHIZOSPHERIC SOIL BACTERIAL ISOLATES

however Gram positive bacteria lack of LPS lyer prescribed biochemical tests, enzymatic, sugar
and are consider to be the most potential microbes utilization and antibiotic sensitivity test required
for PHAs production5. The genus Bacillus is by Bergy’s manual of determinative bacteriology9
common with many other PHA-accumulating Gram- and PIBWin software10.
positive bacteria6,7 accumulates co-polymers of Enzymatic activities of the bacterial isolates
3HB when grown on different substrates. All the PHA producing bacterial isolates
Moreover in this research work Bacillus species were screened on pseudo-selective media for
were isolated from rhizospheric soil region of sugar production of various industrially important
cane and studied for PHAs production. extracellular enzymes like amylase, cellulase,
pectinase, gelatinase, caesinase, lipase and
MATERIALSAND METHOD chitinase, DNAase following standard
microbiological methods.
Sample collection Estimation pH tolerance
Representative soil samples were The pH tolerance test was conducted to
collected form rhizospheric region of sugar cane find out the optimum pH for growth of bacterial
for selective isolation of PHA producing bacteria. isolates. Selected bacterial cultures were revived
The samples were collected in sterile plastic bottle in nutrient broth. Ten ml of nutrient broth was taken
and were transported to the laboratory aseptically in different tubes and the pH was adjusted from 5-
for further analysis. 9 with help of 1N HCl, 1N NaOH and digital pH
Isolation and preservation of bacterial isolates meter. 100µl of the overnight culture was dispensed
The samples were processed in the into the test tubes and incubated at 37°C for 24
laboratory for isolation of bacteria using standard hours. Then the CFU/ml was counted and the
procedures of serial dilution and spread plating. optimum pH was determined.
Colonies of distinguished morphologies were Estimation temperature tolerance
individually picked and sub-cultured and Bacterial isolates were selected basing on
preserved at 4°C for further use. their presence of PHA granule in the cytoplasm in
Screening of PHA producing bacterial isolates order to find out the optimum temperature for their
Sudan Black B staining was used for growth. Selected bacterial cultures were revived in
detecting the existence of PHA in cytoplasm of nutrient broth. Ten ml of nutrient broth was taken
bacterial cells by8. However, before screening, the in different tubes and the temperature was varied
isolates were induced to accumulate PHA in a from 230C to 510C. 100µl of the overnight culture
nitrogen-limiting medium (MSM) for 24 hours. Smear was dispensed into the test tubes and incubated
was made on a clean glass slide, after drying, 0.3% at different temperature (230C, 30°C, 37°C, 440C,
of Sudan black was added. Then slide was washed 510C) for 24 hours. Then the CFU/ml was counted
gently with distilled water after 10 minutes. The and the optimum temperature was determined.
dried slides were engrossed in xylene for few PHA production by bacterial isolates
seconds and allowed to flood with 0.5% of Safranin PHA production and extraction by the
for 30 sec, the slide were washed gently and selected bacterial isolates was observed by
observed under light microscope. following sodium hypochlorite11. The selected
Identification and characterization of bacterial bacterial isolates were grown in conical flask
isolates containing minimal salt medium on a shaker at 37ºC
The morphological and physiological for 4days with an agitation rate of 125 rpm. After
properties of the isolates were investigated on the 4days, the bacterial culture was centrifuged at
basis of their colony characteristics on the Minimal 6500×g, then the supernatant was discarded and
salt agar that contained NaCl (3.0 g L-1), KH2PO4 the pellet was transferred into pre-weighed
(1.5 g L-1), K2HPO4 (1.5 g L-1), MgSO4.5H2O (1.0 g petriplates by dissolving it in distilled water. The
L-1), glucose (10.0 g L-1), ammonium nitrate (0.5 g L- plates were dried at 80ºC in hot air oven. The dried
1
), agar agar (5.0 g L-1) and Gram’s reaction. After weight of the pellet was taken in order to know the
the microscopic examination the Gram positive rods weight of biomass. Then sodium hypochlorite
were processed for identification by the standard solution was added to the dried pellet in 1:5 ratios

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 DASH et al.: SUGAR CANE RHIZOSPHERIC SOIL BACTERIAL ISOLATES 3

to remove non PHA materials, transferred to the positive to Sudan black staining. Then the bacterial
centrifuge tubes and kept in shaker for 30 minutes isolates showing PHA granule were subjected to
at 37ºC. After incubation, the samples were Gram’s reaction and found that, all the bacterial
centrifuged at 6500×g for 15 minutes. The isolates were Gram positive rod. Then the PHA
supernatant was discarded, the pellet was washed producing bacterial isolates were identified on the
with the distilled water and wash with diethlyether basis of biochemical tests and other standard
and acetone in 1:1 ratio to remove sodium microbiological tests (Table 1). The identified Gram
hypochlorite solution by centrifugation. Then the positive PHA producing bacterial isolates were
pellet was collected, 10 ml of chloroform was added Bacillus licheniformis ID score (0.980), Bacillus
to the pellet and filtered into the pre-weighed cereus, Bacillus licheniformis ID score (0.999) and
petriplates. The chloroform gets evaporated which Bacillus badius. Similar findings were also
leaves the PHA film in the petriplates. The weight observed 13,14 while isolated sixteen different
of the PHA film was observed and the PHA species of Bacillius form rhizospheric soil region
production was calculated by following formula, of different plants and Bacillus cereus from rubber
% of PHA production = (Weight of PHA/ Weight plants respectively. Moreover Bacillius species
of biomass) × 100. are the predominat soil ihabitig bacteria which can
Characterization of PHA by FTIR analysis grow by utilising cheap raw material for their growth
The functional group present in the and development. This results are also in
extracted PHA was determined by FTIR agreement with the literature regarding finding a
spectroscopy along with the standard PHB great quantity of microorganisms in sugar cane
procured from Sigma. PHA sample was mixed with Table 1: Biochemical characterization
2 % KBr. The mixtures were compressed into of bacterial isolates
translucent sample discs and fixed in the FTIR
spectrometer (Perkin-Elmer RX I) under the Sl. Biochemical test B1 B4 B6 B8
following conditions: spectral range, 4000–400 cm) No.
1; window material, CsI; 16 scans; resolution 4 cm)
1 Growth at 10% NaCl - - + -
1; the detector was a temperature-stabilized, coated
2 Hippurate hydrolysis - - - -
FR-DTGS detector for analysis12 . 3 Anaerobic growth - + + +
4 VP test - + + -
RESULTS AND DISCUSSION 5 Citrate reductase - - - -
6 Starch hydrolysis - + + -
Identification and characterization of bacterial 7 Oxidase reductase + + + +
isolates 8 Casein hydrolysis + + + -
A total of eight bacterial isolates were 9 Uerase hydrolysis + + + +
obtained from the rhizospheric soil region of 10 Nitrate hydrolysis + + + +
11 Esculin hydrolysis + + + -
sugarcane. Out of all, four bacterial isolates showed
12 Growth at 50ºC + + + +
Table 2. Enzymatic activity of bacterial isolates

Enzyme B. licheniformis B. cereus B. licheniformis B. badius

ID score(0.980) ID score(0.999)

Amylase - + + -
Caseinase + + + -
Chitinase - - - -
Gelatinase + + + +
Lipase + - - +
Cellulase - - - -
Pectinase - - - -
DNAse + - - +

- : Negative, +: Positiv

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4 DASH et al.: SUGAR CANE RHIZOSPHERIC SOIL BACTERIAL ISOLATES

crops able to accumulate PHAs due to selective observed hydrolysis of starch, cellulose, casein,
pressure caused by high carbon : nitrogen ratio15,16. lipid, gelatine, pectin and chitin for characterization
Enzymatic activities of the bacterial isolates of bacterial isolates of soil sample. The hydrolysis
A negative trend in sugar utilization such of various sugars by the bacterial isolates may be
as cellulose, pectin and chitin was observed in due to availability of different sugar in the
case of all the bacterial isolate, however the rhizospheric region of sugar cane plant. Moreover
bacterial isolates showed positive to gelatin (Table it is a positive sign for PHA production by the
2). Three of the bacterial isolates showed positive selected bscterisl isolates using various sugars as
result for caesinase activity except Bacillus badius. source of carbon and energy.
Bacillus cereus and Bacillus licheniformis showed Effect of pH on growth of bacterial isolates
positive to amylase while negative result against The result of pH tolerance (Table 3). of
DNase and lipase but Bacillus licheniformis and the bacterial isolates such as Bacillus
Bacillus badius showed positive for lipase activity licheniformis, Bacillus cereus, Bacillus
while negative for amylase activity. This findings licheniformis & Bacillus badius suggested that,
can be corroborate with the findings of 17 who the optimum pH for growth of the bacterial isolates

Table 3. Effect of pH on growth of bacterial isolates

Sl. Bacterial (CFU/ml) (CFU/ml) (CFU/ml) (CFU/ml) (CFU/ml)

No. isolates pH 5 pH 6 pH 7 pH 8 pH 9

1. B. licheniformis IDscore (.980) 4.1 X 103 6.0 X 105 8.4 X 105 5.1 X 105 5.0 X 105
2. B. cereus 1.99 X 105 2.5 X 105 8.9 x 106 8.2 X 105 6.9 X 105
3. B. licheniformis IDscore(0.999) 1.05 X 105 6.5 X 105 9.6 X 107 7.5 X 104 9.7 X 103
4. B. badius 1.57 X 104 2.21 X 104 4.6 X 107 2.95 X 105 2.7 X105

Table 4. Effect of temperature on growth of bacterial isolate

Sl. Bacterial (CFU/ml) (CFU/ml) (CFU/ml) (CFU/ml) (CFU/ml)

No. isolates 230C 30°C 37°C 440C 510C

1. B. licheniformis IDscore(0.980) 4.2 X 104 3.5 X 106 8.2 X 105 7.6 X 104 5.3 X 103
2. B. cereus 5.8 X 105 4.1 X 107 8.7 X 106 8.0 X 105 7.2 X 103
3. B. licheniformis IDscore(0.999) 3.5 X 104 1.08 X 107 9.4 X 106 7.2 X 105 4.7 X 103
4. B. badius 3.8 X 105 1.65 X 107 4.4 X 106 8.2 X 104 3.2 X 103

Table 5. PHA production by bacterial isolates

[Link]. Bacterial isolates Biomass (gm.) PHA (gm.) % of PHA

1 B. licheniformis IDscore (0.980) 0.825 0.437 53.01%

2 B. cereus 0.532 0.2487 46.76%
3 B. licheniformis IDscore (0.999) 0.669 0.2148 32.11%
4 B. badius 0.706 0.292 41.48%

Table 6. Peaks obtained by FTIR and their corresponding annotations

Peaks (cm-1) 3618 2331 1675 1714 1532

Bonds O-H stretch, O-H stretch –C=C– stretch C=O stretch N–Oasymmetric
H-bonded stretch
Corresponding Alcohols, Carboxylic alkenes Alpha, Nitro
functional Phenols Acids betaunsaturated compounds
groups esters

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 DASH et al.: SUGAR CANE RHIZOSPHERIC SOIL BACTERIAL ISOLATES 5

by18,19,20. In addition to that,21 reported that, the

PHA production in Bacillus sp. was found to be
optimum with 0.5 g/l of nitrogen source, 120 rpm of
agitation and 6.4 g/l of biomass inocolum which in
accordance to the result obtained during the present
study.
Characterization of PHA by FTIR analysis
The functional groups of the extracted
PHA samples from the potent isolate Bacillus
licheniformis (ID score 0.998) showed (Table 6)
the characteristic peaks at 3618 cm-1 (H-bonded
Fig. 1. FTIR analysis of the PHA sample extracted O-H stretch), 2331 cm-1 (O-H stretch), 1675 cm-1 (–
from B. licheniformis IDscore (0.980) C=C– stretch), 1714 cm-1 (C=O stretch), 1532 cm-
showingcharacteristic peak for PHA. 1 (N–Oasymmetric stretch), High intense peaks
were obtained at 3618 cm-1, 2331 cm-1, 1714 and
were 7 and the CFU count were 8.4x105, 8.9x106, 1675 cm-1, (Fig.1) however less intense peaks found
9.6x107 and 4.6x107 respectively. However moderate at 3839 cm-1, 3735 cm-1. The spectroscopic analysis
growth was observed in pH 6 and 8. Again mild gave proper insight for the chemical structure of
growth was also observed in pH 5 and 9. This PHB by reflecting the monomeric units, which is
result corresponds to that of17,13 they observed predominantly present in the PHA polymer. The
that PHA producing bacterial isolates grew better result obtained in this study is well within the result
at pH 7. This might be a result of molecular obtained by the previous workers 22,23,24,25. The IR
adaptation of these bacterial isolates as well as spectrum indicates the presence of monomeric units
increases the activity of enzyme for optimal growth. with a strong absorption band at 1714 cm -1
Effect of temperature on growth of bacterial isolate corresponding to C=O valence vibration of the
Temperature tolerance of the bacterial thio-ester bond.
isolates revealed (Table 4) that, optimum
temperature for growth of all the bacterial isolates CONCLUSION
were 300C and the CFU count were 3.5x106, 4.1x107,
1.08x107 and 1.65x107 respectively. However The isolate characterized in this study B.
moderate growth was observed at 370C and mild licheniformis IDscore (0.980) possesses the
growth was observed at 23 0C, 44 0C & 51 0 C potential for the production of PHB (53.01%) in
respectively. This result corresponds to that of17,13,1 vitro. It is pertinent to mention that Bacillus species
they observed that PHA producing bacterial are dominant bacteria in industry for a variety of
isolates grew better in between 250C to 300C. This reasons including grow in chief raw material, rapid
might be a result of adaptation of these bacterial growth rate leading to short fermentation cycle
isolates to the natural habitat which was ranges times, secretion of hydrolytic enzymes and
between 250C to 350C. production of co-polymers from structurally
PHA production by bacterial isolates unrelated sources. Thus, the potential bacteria B.
Under optimized condition the selected licheniformis IDscore (0.980) is further
bacterial isolates such Bacillus licheniformis, investigated to increase the productivity of PHB
Bacillus cereus, Bacillus badius and Bacillus by supplementation of chief raw material and
licheniformis were produced 0.825gm, 0.532gm, reduction in the cost of upstream & downstream
0.669gm and 0.706gm of cell biomass respectively. processing making the whole process more cost-
The amount of PHA extracted from these bacterial effective.
biomass were 0.437gm, 0.2487gm, 0.2148gm, and
0.292gm per liter of MSM. However Bacillus ACKNOWLEDGEMENTS
licheniformis (ID score 0.998) is the highest PHA
(53.01%) producting bacterial isolates (Table 5). We are thankful to Prof. B. B. Mishra
The obtained result falls within the results obtained (HOD, Microbiology), Dr. N. Sahoo, Dr. S. Acharya

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6 DASH et al.: SUGAR CANE RHIZOSPHERIC SOIL BACTERIAL ISOLATES

(In-charge of Central laboratory, OUAT) & Dr. G. 10. Bryant, T. N. PIBWin software for probabilistic
S. Acharya for providing central laboratory facility identification. J. Appl. Microbiol., 2004; 97
during the period of study. We are also thankful to (6):1326-1327.
Mr. M. S. Choudhury, WII, Deradhun and Mr. H. 11. Moreno, N., Gutierrez, I., Malagon, D., Grosso,
V., Revelo, D., Suarez, D., Gonzalez, J.,
R. Dash, NIT, Rourkela, India for his unconditional
Aristizabal, F., Espinosa, A. and Montoya, D.
support during this study. The authors have no Bioprospecting and Characterization of poly-
conflict of interest to declare ß-hydroxyalkanoates (PHAs) producing bacteria
isolated from Colombian sugarcane producing
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