Download Synthesis and characterization of poly-β-hydroxybutyrate

Indian Journal of Biotechnology
Vol 5, July 2006, pp 276-283
Synthesis and characterization of poly-β-hydroxybutyrate from
Bacillus thuringiensis R1
D Rohini1, S Phadnis2 and S K Rawal1*
1
2
Plant Tissue Culture Division and Process Development Division, National Chemical Laboratory, Pune 411 008, India
Received 20 February 2005; revised 27 July 2005; accepted 25 September 2005
A poly-β-hydroxybutyrate (PHB) accumulating, Gram positive Bacillus thuringiensis R1 was isolated from the soil
samples. The growth of the organism and PHB accumulation in the presence of different carbon sources was studied. While
maximum dry cell mass accumulation (3.90 to 4.11g/L) resulted in the presence of glycerol (1% v/v), least biomass formation (1.28g/L) was in the presence of 1%(v/v) lactic acid. Glycerol also supported maximum accumulation of PHB (34.18 to
34.23%) on cell dry mass basis. PHB accumulation in the presence of table sugar (sucrose) and molasses was 28.23 % and
23.06%, respectively. No PHB accumulation was observed in the presence of acetic acid or ethanol. B. thuringiensis R1
cells grown to log phase in the basal medium in the absence of a additional carbon source and then reinoculated into the
same medium supplemented with 1% (v/v) glycerol or 1% (w/v) table sugar accumulated 64.10% and 31.36% PHB of the
dry cell mass, respectively. The recovered polymer was characterized by NMR, FTIR, GPC, DSC and TGA.
Keywords: Bacillus thuringiensis, β-hydroxybutyric acid, carbon sources, biphasic studies, physical characterization
IPC Code: Int. Cl.8 A01N63/00; C08G63/00; C12R1/07
Introduction
Polyhydroxyalkanoates (PHAs) are biodegradable
polyesters and elastomers, which accumulate as
cytoplasmic inclusions in certain bacteria during
unbalanced
growth
conditions1-3,
usually
characterized by an excess carbon supply and the lack
of one or more essential nutrients4. About 150
different hydroxyalkanoic acids have been identified
as constituents of bacterial polyesters5. The first PHA,
a homopolymer of poly-β-hydroxybutyrate (PHB),
was discovered by Lemoigne in 19256. It is a
biodegradable plastic and an elastomer that
accumulates in several bacterial species under
limiting growth conditions. PHB also acts as a carbon
storage compound and a sink for reducing
equivalents7. PHB and its co-polymers have been
industrially produced since 1982 as substitutes for
petroleum based plastics. Global environment
concerns and solid waste management problems have
generated much interest in the development of
biodegradable plastics that retain the desired physical
and chemical properties of the conventional synthetic
plastics. PHAs have found wide ranging applications
as biodegradable and biocompatible polymers8. One
——————
*Author for correspondence:
Tel.: 91-20-5393382 Extn. 2220; Fax: 91-20-5893438
E-mail: [email protected]
of the major stumbling blocks in large-scale synthesis
and wide commercialisation of PHAs is the high
production cost compared with the conventional
petrochemical derived plastic materials9. Studies have
shown that the raw material costs (mainly carbon
source) contribute most significantly to the overall
production cost of the PHAs (up to 50% of the total
operating costs)10. Ralstonia eutropha, Alcaligenes
latus and recombinant Escherichia coli harbouring the
Ralstonia eutropha PHA biosynthetic genes
accumulate PHB in presence of glucose or sucrose1,9.
However,
Streptomyces
aureofaciens
and
recombinant E.coli expressing the PHB biosynthetic
genes from S. aureofaciens have been shown to
accumulate high amounts of PHB in the presence of
glycerol11-15. Bacillus thuringiensis, better known for
its insecticidal δ-endotoxin, has been reported to
accumulate PHB16. Other Bacillus species e.g., B.
mycoides RCJ B-01717, B. megaterium18, B. subtilis,
B. firmus, B. sphaericus and B. pumilus16 have also
been shown to accumulate polyhydroxyalkanoates in
varying amounts. The present study reports synthesis
and accumulation of higher amounts of PHB (64.1%)
by a B. thuringiensis isolate that is higher than any of
the Bacillus species isolates reported earlier. The
physical and chemical characterization of the
accumulated polymer is also described.
ROHINI et al: SYNTHESIS OF POLY-β-HYDROXYBUTYRATE BY B. THURINGIENSIS R1
Materials and Methods
Isolation and Identification
Soil samples were collected from different locations of the National Chemical Laboratory, Pune
Campus. One gram of each sample was suspended in
sterile deionised water and volume made up to 100
mL. The samples were shaken vigorously for 2 min.
The supernatant was serially diluted from 101 to 108
times with 0.9% NaCl and 0.1 mL of the diluted samples was plated on nutrient agar medium plates. The
plates were incubated at 37°C for 24-48 h.
Analytic Method for PHB Producing Strain
Each bacterial colony obtained on the nutrient agar
medium plates was picked and grown in 5 mL of nutrient broth with shaking for 24 h at 37°C. These cultures were then used as the inoculum (1% v/v) for a
50 mL basal medium containing (w/v): yeast extract
(1%), peptone (1%), Na2HPO4 (0.1%), MgSO4
(0.02%) and glycerol (1%v/v) as the carbon source.
The cultures were grown for 24-48 h at 37°C with
shaking at 150 rpm. After incubation, the cell cultures
were centrifuged at 4000×g for 10 min at 15oC. The
cell pellet was washed with sterile deionised water.
An aliquot of the cell pellet was stained with Nile
blue-A and observed for orange fluorescence microscope at an excitation wavelength of 460 nm19. One
such colony showing orange fluorescence was identified as B. thuringiensis R1 by colony morphology,
Gram staining and biochemical characteristics.
Culture Conditions
The B. thuringiensis R1 cells were grown for 70 h
at 37°C with shaking at 150 rpm in a basal medium
containing glycerol (1%v/v). Samples were harvested
at different time intervals; the O.D. at 600 nm and dry
weight was recorded. To study the effect of various
carbon sources on the growth of B. thuringiensis R1
and PHB accumulation, cells were cultivated for 36 h
(stationary phase) in the basal medium supplemented
with glycerol, glucose, molasses, table sugar (cane
sugar), fructose, maltose, lactose, liquid glucose (byproduct of jaggery industry containing 80% glucose),
acetic acid, lactic acid, ethanol and βhydroxybutyrate. Each carbon source was used at a
final concentration of 1% (w/v) or (v/v) in the medium except for β-hydroxybutyrate that was used to a
final concentration of 0.50% (w/v) in the medium. For
biphasic growth studies the B. thuringiensis R1 cells
were grown in the basal medium for 36 h, harvested
277
and reinoculated in the basal medium supplemented
individually with glycerol (1%v/v), molasses
(1%w/v), table sugar (1%w/v) and β-hydroxybutyrate
(0.50%w/v). The dry cell mass was used for GC
analysis to identify and quantify the accumulated
PHA.
Extraction of Polymer from Cells
Modified method of Hahn et al.20 was used to recover the polymer from B. thuringiensis R1 cells.
Briefly, the cells were harvested by centrifugation at
2000×g, washed twice with deionized water and
freeze-dried under vacuum. The lyophilized cell pellet
was shaken for 90 min at 37°C with chloroform and
30% sodium hypochlorite (1:1). The dispersion was
centrifuged at 4000×g, at room temperature for 10
min. Lower chloroform phase that contained the solubilised polymer was recovered and the polymer precipitated by the addition of 4 volumes of methanol.
The precipitated polymer was washed with acetone,
dried and used for physical characterization.
Gas Chromatography Analysis
Acidic propanolysis of dried cell pellet was carried
out as described previously21. About 100 mg of the
dry cell pellet was taken in a tightly sealable vial.
Two mL of 1,2-dichloroethane (DCE), 2 mL of acidified propanol (1 vol concentrated HCl + 4 vol
n-propanol) and 200 μL of the internal standard (2.0 g
benzoic acid in 50 mL n-propanol) were added to the
pellet and incubated at 85°C for 4 h. The mixture was
shaken from time to time. After cooling to room temperature, a mixture of 2 mL DCE and 2 mL acid propanol was added. The sample was centrifuged at
10,000×g for 5 min. the clear supernatant was collected and an aliquot was injected into the gas chromatograph. GC analysis was performed using a Shimadzu GC 17-A gas chromatograph. A 0.32 mm diameter BP1 capillary column (J&W Scientific Co.,
USA) of 25 m length was used. The analysis started at
80°C for 5 min followed by a 7°C/min rise in temperature to the final temperature of 200°C. Nitrogen
(5 mL/min) was used as the carrier gas. Quantitative
evaluation was effected by the peak areas of hydroxybutyl and benzoyl esters formed.
Physical Characterization of the polymer
Scanning Electron Mmicroscopy
PHA sample purified from B. thuringiensis R1
cells was sonicated at 20 KHz for 3 cycles of 5 min
each. A drop of suspension was dried on a brass stub
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INDIAN J BIOTECHNOL, JULY 2006
under the IR lamp and later coated with gold using
Polaron sputter unit. SEM photographs were taken
with a Leica Stereoscan 440 Scanning electron microscope equipped with a Phoenix EDAX attachment.
1
H NMR Spectroscopy
The 1H NMR of the extracted polymer was carried
out using Bruker Ac200 at 24°C22. The polymer was
solubilized in CDCl3.The samples were analysed in 5
mm sample tubes in chloroform-d. The spectra were
referenced to internal tetramethylsilane.
FTIR Analysis
A polymer sample (2 mg) was thoroughly mixed
with KBr (Spectroscopic grade). A 15 mg KBr treated
pellet was dried at 100°C for 4 h23. FTIR spectrum
was taken using a Perkin-Elmer (USA) model 1720
Fourier Transform IR spectrometer.
Determination and Distribution of Molecular Weight by GPC
The molecular weight and molecular weight distribution of the polymer was determined by gelpermeation chromatography (GPC) using a Waters
515 pump with four Stryagel HR columns. Monodisperse polystyrene and chloroform were used as the
molecular weight standard and the mobile phase, respectively. For each analysis, 250 μL of 0.1% (w/v)
polymer solution in CHCl3 was injected.
Differential Scanning Calorimetry
To determine the morphological state of the polymer, the melt temperature and the enthalpy of fusion
were measured using a Perkin-Elmer Differential
Scanning Calorimeter (DSC-7). Samples of 10-15 mg
were encapsulated in aluminium pans and heated from
0 to 200°C at a rate of 20°C/min. The melting temperature (Tm) was taken at the peak of the melting
endotherm.
Thermogravimetric Analysis of PHA
Thermogravimetric analysis of the polymer sample
was analysed using Rheometric TG analyser
(Rheometric Scientific, USA). The analysis was carried out under nitrogen flow rate of 15mL/min with
scanning rate of 10°C/min. The temperature was increased until weight loss was 20%. After cooling rapidly to room temperature (100°C/min), the residue
was recovered. This residue was completely soluble
in chloroform.
Results
One particular bacterial isolate from the soil samples when stained with Nile blue A and viewed under
an excitation wavelength of 460 nm, showed characteristic orange fluorescence, indicating the possible
presence of PHA(s) in the cells. Based on biochemical
characteristics, the isolate was characterized and identified as the endospore forming, Gram-positive Bacillus thuringiensis, and designated as the strain R1 (Table 1).
The B. thuringiensis R1 cells grown for 24 h in the
basal medium containing glycerol (1%v/v) were harvested. GC analysis of the dried and esterified cells
revealed the accumulation of PHB. The cells were
then cultured in the presence of glycerol (1%v/v) for
70 h to study growth and PHB accumulation patterns.
The pH of the medium dropped from 7.2 to 5.5 during
this period. B. thuringiensis cells entered stationary
phase of growth after 36 h of incubation (Fig. 1a).
Table 1—Morphological and physiological characteristics of the
strain Bacillus thuringiensis R1
Characteristics
Response
Gram staining
Spore staining
Cell shape
Density
Elevation
Margin
Motility
Fluorescence and pigments
Indole test
Methyl red test
Voges Proskauer test
Citrate utilization
Casein hydrolysis
Urea hydrolysis
Nitrate reduction
Catalase test
Cytochrome oxidase test
Acid production from
Glucose
Sucrose
Mannitol
Arabinose
Xylose
Meso-inositol
Raffinose
Rhamnose
Salicin
Galactose
Positive
Positive
Rods
Opaque
Convex
Wavy
Negative
Nil
Negative
Positive
Negative
Positive
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
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ROHINI et al: SYNTHESIS OF POLY-β-HYDROXYBUTYRATE BY B. THURINGIENSIS R1
Maximum dry cell biomass (3.90g/L of the medium)
and PHB accumulation (1.4g/L; 34.18% of the dry
cell mass) coincided with the advent of the stationary
phase of growth. B. thuringiensis cell dry biomass
formation and PHB accumulation, however, varied
with the carbon source (Fig. 1b). In the presence of
molasses, lactose and table sugar it was 3.11, 2.67 and
2.01g/L, respectively. Least cell growth was observed
in the presence of ethanol and lactic acid. Dry cell
mass in the presence of monomer β–hydroxybutyric
acid (0.5%w/v) was 1.5g/L. PHB accumulation in the
presence of table sugar, molasses and fructose was
28.23%, 23.06% and 19.76% (0.57; 0.72 and 0.34 g/L
of media), respectively. PHB accumulation in the
presence of lactose was 12.72% (0.34 g/L) and 4.61%
(0.06 g/L) in the presence of lactic acid. Only 9.23%
(0.13 g/L) PHB accumulated in the presence of glucose in the medium. Acetic acid and ethanol did not
support any PHB accumulation. Marginal accumulation of PHB (2.10% on cell dry mass basis; 0.03 g/L)
was observed with the incorporation of maltose in the
basal medium. PHB accumulation was 52.50% of dry
cell mass (0.79 g/L) in the presence of β–
hydroxybutyric acid monomer in the medium.
Under biphasic growth conditions, B. thuringiensis
cell dry weight in the presence of glycerol (1%v/v)
increased from 4.11 to 6.11g/L of the medium and
PHB accumulation from 34.23 to 64.10% of dry cell
mass (1.41 to 3.91g/L). When table sugar was used in
the biphasic study there was an increase in dry weight
from 2.01 to 2.70g/L and PHB content from 28.23 to
31.36% of dry cell mass (0.57 to 0.85g/L). There was
no increase of PHB content in the biphasic growth of
B. thuringiensis when either the monomer β–
hydroxybutyric acid or molasses were used as the carbon source (Table 2).
GC analysis of the dried and esterified B. thuringiensis R1 cells grown for 36 h in the presence of
different carbon sources, revealed two peaks. One
corresponding to the propyl ester of 3hydroxybutyrate (3-HB) and the other to the propyl
ester of the internal standard benzoic acid (Fig. 2).
PHB granules isolated from the B. thuringiensis
cells observed under scanning electron microscope
showed stable spherical configuration with an average
diameter of 5 microns (Fig. 3). The NMR spectra
identified the polymer as an isotactic homopolymer
(Fig. 4). The spectrum revealed the presence of three
groups of signals characteristic of PHB homopolymer.
The doublet at 1.3 ppm was attributed to the methyl
Fig. 1a—Growth of Bacillus thuringiensis R1 in basal medium
with glycerol (1%v/v)
Fig. 1b—Effect of different carbon sources on growth and
accumulation of polyhydroxy butyrate in B. thuringiensis R1 at 36
h. 1 Glycerol, 2 Glucose, 3 Molasses, 4 Table sugar, 5 Fructose, 6
Maltose, 7 Lactose, 8 Liquid glucose, 9 Acetic acid, 10 Lactic
acid, 11 Ethanol and 12 β–hydroxybutyric acid.
Table 2—Biphasic growth studies in Bacillus thuringiensis R1
Carbon source
Glycerol
Table sugar
Molasses
β-hydroxybutyrate
Dry wt. (g/L)
PHB accumulation
after
(% dry cell mass) after
1st 36 h 2nd 36 h of 1st 36 h 2nd 36 h of
of Phase I Phase II of Phase I Phase II
4.11
2.01
3.15
1.50
6.11
2.70
3.18
1.58
1.41
0.57
0.73
0.79
3.91
0.85
0.73
0.83
group coupled to one proton; the doublet of the quadruplet at 2.57 ppm to the methylene group adjacent to
an asymmetric carbon atom bearing a single proton
and the multiplet at 5.28 ppm to the methyne group.
Chloroform-d gave a chemical shift signal at 7.25
ppm. FTIR analysis revealed two absorption bands at
1280 cm-1 and 1735 cm-1 corresponding to C=O and
C-O stretching groups, respectively (Fig. 5).
The gel permeation chromatography analysis of the
polymer isolated from B. thuringiensis R1 cells (Fig.
6) revealed that the polydispersity index (Q) (defined
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INDIAN J BIOTECHNOL, JULY 2006
as Mn/Mw), number average molecular weight (Mn),
weight average molecular weight (Mw) were 1.77,
5.8526×104 and 1.0385×105, respectively. The melting
temperature and the enthalpy of fusion of the polymer
were 165.6°C and 84.1 J/g, respectively. The polymer
degraded rapidly between 225 and 270°C with a peak
at 261°C (Fig. 7).
Fig. 4—1H NMR spectra of the polymer recovered from B.
thuringiensis R1 cells.
Fig. 2—Gas chromatograms of the propyl ester of the authentic
Polyhydroxybutyrate sample (A) and the B. thuringiensis R1 cells
grown in medium containing glycerol (B) Propyl ester of PHB
and benzoic acid are represented by peaks 1 and 2, respectively.
Fig. 5—FTIR spectra of polymer purified from B. thuringiensis
R1.
Fig. 3—Scanning electron microscopy of the PHB granules
obtained from B. thuringiensis R1.
Fig. 6—GPC of the polymer purified from B. thuringiensis R1
done. Monodisperse polystyrene and chloroform were used as the
molecular weight standard and the mobile phase respectively. For
each analysis 250 μL of 0.1% (w/v) polymer solution in CHCl3
was injected.
ROHINI et al: SYNTHESIS OF POLY-β-HYDROXYBUTYRATE BY B. THURINGIENSIS R1
281
Fig. 7—DSC of the polymer purified from B. thuringiensis R1.
Discussion
B. thuringiensis better recognised for the production of insecticidal δ-endotoxin has been reported to
accumulate PHB16. However, no study has been carried out to guage the influence of media and culture
conditions on the accumulation of PHAs by this microorganism. The present study highlights the accumulation of PHB by B. thuringiensis R1. Maximum
cell mass formation and the accumulation of the
polymer coincided with the beginning of stationary
phase of the cell growth cycle. Thereafter, the amount
of accumulated PHB declined possibly due to PHB
metabolism24. GC analysis of the B. thuringiensis R1
cells revealed that the cells supported synthesis of the
PHB homopolymer when supplied with a single carbon source. However, glycerol supported maximum
cell growth as well as PHB accumulation (except for
when the hydroxybutyrate monomer was used). The
utilization of glycerol for growth and PHB formation
ensures conversion of glycerol to acetyl CoA via either glycolysis or the methylglyoxal pathways13.
Some of the Bacillus species have been reported to
accumulate 6-36% PHB of the cell dry mass16. PHB
accumulation of 64.1% in the biphasic growth cycle
of B. thuringiensis R1 cells is the highest recorded for
this bacterium so far. This indicates that while the
first phase is used for the development of the biomass,
the second phase in the presence of glycerol is prefer-
entially utilized for PHB production by the cells.
Glycerol, which is the by-product of soap industry,
can be utilized as the carbon source for efficient PHB
production by the B. thuringiensis R1 cells. Table
sugar and molasses being cheap sources from the
massive sugar industry in India may also be used for
cost effective production of PHB on a large-scale.
This is the first report of utilization of glycerol, table
sugar or molasses for the production of PHB by B.
thuringiensis cells. Borah et al17 reported the use of
sucrose as the cheaper source for the production of
PHB by B. mycoides RLJ B-017. Similarly, B. Jma525
(25-35%) and B. megaterium18 (40.8%) accumulate
PHB during fermentation with molasses. B. megaterium18 has also been reported to accumulate PHB in
the presence of glucose (39.9%). Belma et al16 have
reported PHB accumulation of 8.0 and 7.5% in B.
thuringiensis strains D1 and D2, respectively in glucose containing medium. In the present study, glucose
did support high accumulation of PHB (9.2%). This
may be possibly the result of medium acidosis since
the cell mass formation was also low. It is also documented that glucose in the medium represses the expression of phosphotransbutyrylase (Ptb) gene that is
needed for PHB production26. Glucose is metabolized
through the EMP pathway to yield pyruvate and acetate as the main products. Acetate is partially converted into PHB, which is consumed during sporula-
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INDIAN J BIOTECHNOL, JULY 2006
tion in Bacillus sp.27. Although acetate is a precursor
in acetyl-CoA synthesis1 it did not support PHB accumulation by the B. thuringiensis strain R1. Though
there was increased accumulation of PHB (52.5%)
with the use of β-hydroxybutyrate (sodium salt) as the
carbon source in the medium, it is not feasible to use
the monomer as a carbon source owing to its high
cost. Growth of the B. thuringiensis culture in two
phases resulted in the increase of PHB production
when glycerol or table sugar was used as the carbon
source. This indicates that the fully grown culture of
B. thuringiensis could utilize the carbon source supplied in the second phase, essentially for the accumulation of PHB. This biphasic growth for PHB accumulation may be used as a strategy for increased PHB
production on a commercial scale in B. thuringiensis
as reported in other bacteria.
The NMR signal multiplicity by a proton as a quadruplet or octet in case of protons of CH2 group was
obtained due to proton coupling in isotactic form unlike in syndiotactic where duplet signal is obtained due
to coupling. Similar spectral signatures have been reported for PHB isolated from B. cereus28 and B. circulans22. Rozsa et al22 have shown that the FTIR absorption band at about 1730 cm-1 is a characteristic of carbonyl group and a band at about 1280-1053 cm-1 characterizes the valence vibration of the carboxyl group.
These are characteristics of the polyhydroxybutyrate.
The high enthalpy of fusion (84.1 J/g) suggests
high crystalline nature of the recovered PHB that was
calculated to be of 60-65 % assuming the enthalpy of
fusion of 100% crystalline sample to be 146 J/g. The
melting temperature of PHB (165.6°C) in the present
study was slightly lower than reported for PHB from
B. cereus28 (170°C) and B. circulans22 (173°C). The
difference between the melting temperature and the
decomposition temperature (261°C) was high enough
to facilitate processing of the polymer.
Acknowledgement
RD thanks University Grants Commission, India
for the grant of the research fellowship.
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