[Link].

Sci (2014) 3(5): 377-387

ISSN: 2319-7706 Volume 3 Number 5 (2014) pp. 377-387

[Link]

Original Research Article

Optimization and production of Polyhydroxybutarate(PHB) by
Bacillus subtilis G1S1from soil

[Link]*

Department of Biotechnology, [Link] and [Link] Arts College, Kadi, Gujarat, India
*Corresponding author

ABSTRACT

Polyhydroxybutyrate (PHB), a lipid-like polymer of 3-hydroxybutyrate, is a

Keywords representative member of polyhydroxyalkanoates (PHAs) formed in many
bacteria.12 bacterial isolates were found to be the most promising PHA
Bacillus sp. accumulating bacteria among 15 isolates screened from soil. Screening for PHA
Biopolymer, was done by Sudan black staining. Bacillus subtilis G1S1 shows PHA maximum
optimization production under the different carbon and nitrogen condition. PHA extraction was
PHA, FTIR carried out by chloroform digestion method. Biochemical and 16s rRNA analysis
spectroscopy, showed that PHA producing bacteria belong to Bacillus subtilis G1S1 genera with
Sudan black B Maximum production of PHA was analyzed by U.V spectrophotometer and finally
it was characterized by FTIR spectroscopy.

Introduction
Plastics have been an integral part of our Biodegradable plastics which also known
lives. From automobiles to medicine, as green plastics are plastics made from
plastics are utilized in almost every biodegradable polymer materials.
manufacturing industry in the world. Polyhydroxybutyrate (PHB) is a suitable
Approximately 25 million tons of plastics source for biodegradable polymer material
are produced by the plastics industry every because their fully degradability and non-
year (Wong et al., 2002). pollutant characteristics. Braunegg et al.
(1998) defined biodegradability as the
The development of biodegradable plastics capability to be broken down, especially
has become one of the major concerns in into innocuous products by the action of
the present society because the disposal of living things, known as microorganism.
the plastics has pointed out their major Bacteria and fungi are the natural
weaknesses. Plastics are produced from occurring microorganisms that are capable
non-renewable resources such as to degrade PHA to carbon dioxide and
petrochemical and are not compatible with water through secreting enzymes. It can
natural carbon cycles because of their non- also be degraded through non-enzymatic
degradable characteristics. hydrolysis.

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Recently, a large scale production of Poly- the bacteria in sequential action of three
-hydroxybutyrate (PHB) by bacteria has enzymes. 3-ketothiolase (phbA gene)
become a subject of increasing interest. catalyses the formation of a carbon-carbon
PHB is a useful biodegradable polymer bond by condensation of two acetyl-CoA
which can be used as a thermoplastic (Masamune et al, 1989) NADPH
(Holmes, 1985). Biopolymers are possible dependent acetoacetyl-CoA reductase
alternatives to the traditional, non- (phbB gene) catalyses the stereoselective
biodegradable petrochemical derived reduction of acetoacetyl-CoA formed in
polymers. In terms of molecular weight, the first reaction to R-3- hydroxybutyryl
brittleness, stiffness and glass transition CoA. The third reaction of this pathway is
temperature, the PHB homopolymer is catalyzed by the enzyme PHB synthase
comparable to some of the more common (phbC gene) that catalyzes the
petrochemical-derived thermoplastics, polymerization of R-3- hydroxybutyryl-
such as polypropylene (Barham, 1990). CoA to form PHB. The EC number is yet
to be assigned to PHA synthase
Poly- -hydroxybutyrate (PHB) belongs (Steinbüchel et al 1991). Figure 2.2
to the class of biodegradable plastics showed the biosynthetic pathway of PHB
PHAs. PHB was first among the family of from acetyl-CoA PHB is a partially
PHAs to be detected by Lemoigne in 1926 crystalline polymer which has material
as a constituent of bacterium Bacillus properties similar to polypropylene (PP)
megaterium (Lemoigne, 1926). and polyethylene (PE) (Holmes et al.,
Approximately 150 different 1988; Lee, 1996).Therefore, PHB has been
hydroxyalkanoic acids are at present considered as one of the most promising
known as constituents of these bacterial biodegradable plastics and as an
storage polyesters (Steinbüchel and alternative to petrochemical plastics. This
Valentin 1995). Polyhydroxyalkanoates is due to their biocompatibility,
(PHAs), a family of bacterial polyesters, biodegradability and versatile properties
are formed and accumulated by various make it an eco-friendly substitute for
bacterial species under unbalanced growth synthetic polymers (Brandl et al., 1998).
conditions. PHAs have thermomechanical In addition, PHB has more advantages
properties similar to synthetic polymers because it is far less permeable than PE
such as polypropylene, but are truly and PP, this known as a better material for
biodegradable in the environment (Lee et food packaging needless to use
al, 1996). The molecular structure of PHB antioxidant. However, industrial
are describes in Figure 1.1. PHB act as an application of PHB has been hampered
energy storage facility, and are developed owing to its low thermal stability and
when the bacteria s surroundings include excessive brittleness upon storage
excess carbon, and a deficiency of another (Matsusaki et al., 2000). Due to the poor
nutrient. physical properties of PHB, the
incorporation of a second monomer unit
Synthesis route / Production of PHB into PHB can significantly enhance its
properties. This has led to an increased
PHB are produced by many genera of interest to produce hetero-polymers with
bacteria as inclusion bodies to serve as improved qualities. The incorporation of
carbon source and electron sink. PHB is 3-hydroxyvalerate (3HV) into the PHB has
synthesized from acetyl-CoA produce by results in a poly-(3-hydroxybutyrate-co-3-

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Figure 1.1: Structure of PHB

Biosynthetic pathway of PHB from acetyl-CoA (Taguchi et al.)

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 [Link] (2014) 3(5): 377-387

hydroxyvalerate) [P(3HB-co-3HV)] which [Link] of PHB production under

is more flexible and tougher than PHB, defferent carbon and Nitrogen source
and more easier to degrade when 5. Characterization of PHA by FTIR
discarded into the natural environment. Spectroscopy

Application of PHB Materials and Methods

Economic and technological barriers are the Screening of PHB producing bacteria.
main concerns regarding largescale
microbial production of PHAs and poly- - Screening and Isolation of bacterial
hydroxybutyrate (PHB). Byrom cited that strains: 1 gm soil samples were inoculated
large scale production of poly- - into 250 ml flask containing 100 ml sterile
hydroxybutyrate(PHB) by bacteria has Nutrient broth. Flasks were incubated at
become a subject of increasing interest 37°C for 24 hrs on rotary shaker at 120
(Byrom, 1897).Applications focus in rpm. Isolation was carried out on Nutrient
particular on packaging such as containers agar plate, MRS plate and Ashby s
and films (Bucci and Tavares, 2005). It is
Mannitol Agar.
also processes into toners for printing
applications and adhesives for coating
applications (Madison and Huisman, 1999).
Fifteen bacterial colonies were isolated
PHB could replace some of the more and preserved using nutrient agar medium.
traditional, non biodegradable polymers. All isolates were screened for PHB by
Polymer blends is expected to be more staining with Sudan black B stain (0.3 in
widely accepted. It is cited that such blends 70% alcohol and observed under
will greatly increase the spectrum of microscope X100x). Hartman (1940) was
possible applications by expanding the range the first to suggest the use of Sudan black
of available physical properties. PHB in B, as a bacterial fat stain. Subsequently,
combination with other biocompatible and Burdon et al, (1942a) confirmed the
nontoxic polymers would also have an greater value of this dye and modified the
enhanced scope in biomedical applications procedure for demonstrating intracellular
(Christi et al, 1999). fatty material in bacteria by preparing
microscopic slides of bacteria stained with
The success in the biodegradable plastic alcoholic Sudan black B solution and
strategy largely depends on the isolation of counterstained with safranin. The selected
potent PHA producing bacteria and isolates were then identified on the basis
optimizing culture parameters for of their morphological, cultural and
maximum PHA biosynthesis. Keeping biochemical characteristics.
these points in view, the following
objectives were addressed in the present Optimization of culture medium and
study. conditions

1. Isolation of PHB producing bacteria To observe the effects of culture conditions

from soil samples. for maximum bacterial polymer production,
2. Screening for high PHB producers from cultures were incubated at different
the isolated bacteria. incubation period (4, 8, 16, 24, 30, 36, 40
3. Identification of Bacterial Strain by 16S and 48 h) at 37°[Link] production of
rRNA Method bacterial polymer under different carbon and
nitrogen were also studied using liquid

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complete medium Glucose, Fructose, Analytical methods

sucrose ,maltose and lactose were used as
carbon source, whereas Ammonium sulfate, PHA production was estimated as
Malt extract, Peptone and Yeast extract were previously described by Law and Slepecky
tested for their ability to utilize nitrogen (1961). Briefly, 3 ml of bacterial culture
source. grown in N-free medium was transferred
to glass centrifuge tubes (tubes were
Cell cultivation washed with acetone and methanol to
remove plasticizers if plastic tubes were
For large-scale growth, Inoculums was used) and centrifuged at 5000 rpm for 10
prepared in nutrient broth medium at 370C min. The cell pellet was suspended in 1 ml
and transferred to 500 mL of nutrient broth of standard alkaline hypochlorite solution
in a wide-necked 1 L culture flask, and incubated at 37°C for 1 - 2 h for
incubated at 37°C for 48h with continuous complete digestion of cell components
gentle shaking. except PHA. The mixture was centrifuged
to collect PHA granules. The sediment
Harvesting was washed twice with 10 ml of distilled
water and centrifuged. The PHA granules
After incubation, cells were harvested by in the sediment were washed twice with
centrifugation at 8000 rpm for 12 min, three portions of acetone, methanol and
washed in sterile water and recentrifuged diethyl ether, respectively. The polymer
similarly. Pellets were collected granule was dissolved with boiling
aseptically, dried at 60°C chloroform and the chloroform was
allowed to evaporate. Finally, the granules
PHA Extraction were mixed with 10 ml of concentrated
H2SO4 and the tube was capped and heated
The PHA was directly extracted using the for 10 min at 100°C in a water bath. The
solvent chloroform. First, the bacterial concentration of PHA was determined
cultures were harvested and then lipids from an established standard graph in
were removed from the cell pellet-using which the absorbance was plotted against
methanol (40 times the volume of cell the concentration of crotonic acid. The
pellets) after cells were incubated at 95°C presence of PHA was confirmed by the
for 1 h. Then it was filtered to remove the presence of a peak obtained between 230 -
methanol completely and the sediment 240 nm (Figure 2).
granules were incubated in an oven at
65°C till dry. Chloroform was added to the Fourier transform-infrared
dried granules and was incubated at 95°C spectroscopy (FT-IR analysis)
for 10 min. after cooling; the mixture was
gently mixed overnight. The solution was The PHA extracted from the organism was
then filtered to get the debris. Finally, the analyzed by FT-IR spectroscopy (JASCO
PHA was precipitated from the debris with FT/IR). It was used under the following
7:3 (v/v) mixtures of methanol and water. conditions: spectral range, 4000-400 cm-1
The precipitated PHA was then washed to confirm the functional groups of the
with acetone and dried. It was analyze by extracted polymer.
U.V. spectrophotometer by using crotonic
acid as a standard (Law , Slepecky et al
1961)

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Identification by 16S rRNA among 15 isolates, 2 were Gram positive

cocci, 8 were Gram positive bacilli and 5
DNA extraction were Gram negative bacilli. 15 bacterial
isolates were found to be positive for
The bacteria were grown in (LB) medium Sudan black staining which indicated PHA
at 25°C for 18 h. After growing, they were accumulation in bacterial cells. Gram
suspended by mixing with a vortex mixer reaction and Morphological analysis
in suspension buffer, (TE) buffer (10 mM revealed that the PHA producing strains
Tris, 1 mM EDTA)in pH in 8.0. Genomic belong to Staphylococcus, Bacillus,
DNA was obtained from pure cultures by Pseudomonas and Escherichia genera.
lysozyme-proteinase K-sodium dodecyl From These fifteen isolates, AS3-2 gave
sulfate (SDS) treatmentfollowed by good biopolymer production which is
phenol-chloroform extraction and Bacillus genes with respect of
subsequent ethanol precipitation (Braker et morphological, Biochemical, colony
al., 2003). The purity and concentration of character and 16s rRNA sequencing
the DNA preparations were determined technique.
spectrometrically. Electrophoresis of the
extracted DNA was carried out on 0.8 % The strain of Bacillus subtilis GISI was
agarose gels at 3.0 Vcm- out on 0.8 % screened for PHB production in Nutrient
agarose gels at 3.0 Vcm-1 in TAE buffer. medium where glucose as a carbon source.
Time-course analysis (Figure 1) indicated
16S rRNA amplification that PHB was a growth-associated product
and its accumulation significantly
The DNA amplifications were performed increased when the culture reached
with general methods (Sambrook et al., stationary phase (about 48-60 hrs). The
2001). Total bacterial 16S rRNA genes maximum values were achieved at 48 h
were amplified by PCR using the universal cultivation. After 52 hrs, a slight decrease
primer. The 16S rRNA sequences were in the level PHB content. This indicated
compared with all accessible sequences in that the presence of an intracellular PHB
databases using the BLAST server at depolymerase. Concentration of PHB
NCBI (National Centre of Biotechnology decreased significantly after 60 hrs
Information). The sequences were aligned cultivation due to nutrient depletion and
with those belonging to representative cells consumption of PHB as a carbon
organisms of the L-subclass of source. The lower total dry cell weight of
Furmicutes. The strain GISI was assigned the bacteria corresponded with high
to a genus based on the obtained 16S amount of PHA production within 48 hrs
rRNA gene sequence similarities Bacillus cultivation these was similar Ralstonia
Sp. eutropha (recent name Cupriavidus
eutropha), which accumulated PHB at the
Results and Discussion stationary phase (Madison et al 1999).

Fifteen isolates were isolated from soil of Dry cell weigh

KADI region. With the reference to gram
reaction eight isolates shows gram positive Culture sample (10 ml) was centrifuged
and nine isolates shows gram negative (15,000 rmp, 15 min, 4oC) and the cell
(Table 1). Gram staining showed that pellet was washed with deionized water,

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recovered by centrifugation again, and extract and peptone (Lee et al. [Link]
dried (105oC, 24 h) to constant weight. 2012)
Bacillus sp.G1S1 gave the highest values
for PHB concentration(1.41 ± 0.20 g/l and 16s rRNA sequence
55.0% of DCW, respectively) The PHA
content was within the range of 50-90% of G1S1 was identify by 16s rRNA technique
DCW produced by commercial PHA and it is confirmed that it is Bacillus Sp.
producing bacteria such as Cupriavidus The sequence of this species is
eutropha or recombinant E. coli approximately 98 % similarity with B.
(Steinbüchel, 2001). Chen et al 1991 subtilis . GenBank: HE577170.1
studied PHA in 11 different Bacillus spp.
and found PHB consisting 50% (w/v) of Similar sequence(s) found in database. <
dry cell weight of the bacteria. Family of BLAST hit with highest
sequence identity: Bacillaceae
Optimization of Carbon and Nitrogen
Bacillus subtilis subsp. Spizizenii
Effect of carbon sources on production of
PHB, G1S1 isolates have shown Family = Bacillaceae; Sequence identity =
maximum production of PHB in media 98.1%; Alignment length = 964 (86.7%);
with fructose and Glucose. However with E-Value = 0.0; Accession = AF074970
maltose yield was not significant. Glucose Bacillus vallismortis
and fructose, being monosaccharides were Family = Bacillaceae; Sequence identity =
readily utilizes by bacteria, hence their 98.1%; Alignment length = 997 (89.7%);
growth and subsequent production of E-Value = 0.0; Accession = AB021198
PHbwas higher. In case of sucrose and Bacillus subtilis subsp. subtilis
maltose, complexity of the carbon Family = Bacillaceae; Sequence identity =
increased and hence PHA yields were low. 98.1%; Alignment length = 997 (89.7%);
Similar conclusion was made by E-Value = 0.0; Accession = AJ276351
Chandrashekharaiah et al. However, for Brevibacterium halotolerans
the PHB production by Rhizobium Family = Brevibacteriaceae; Sequence
bacteria, sucrose was found to be better identity = 98.1%; Alignment length = 985
source of carbon (88.6%);
E-Value = 0.0; Accession = AM747812
It has been reported that the complex
nitrogen sources increased the yield of FTIR Analysis for Functional group
PHB by Bacillus megaterium. Growth identification
conditions including pH, temperature,
carbon and nitrogen sources play an The functional groups of the extracted
important role in the production rate of PHA granules were identified as C=O
PHA. G1S1 was gave good PHA group by FT-IR spectroscopy the results of
production on yeast extract (Table 1) . FT-IR spectroscopy are shown in Figures
Similar results were obtained from 3. The IR spectroscopic analysis gave
cultivation of Bacillus licheniformis, further insights into the chemical structure
Anaerobiospirillum succiniproducens and of the polymer and reflects the monomeric
Phaffia rhodozyma in the presence of units. In this study, the functional groups
yeast extract, and a combination of yeast of the polymer PHA was confirmed as

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U.V analysis of PHA

0.8

O.D. (nm)
0.6
O.D in nm
0.4

0.2

0
0 50 100 150 200 250 300
U.V. range

Figure.2 PHA analyses by U.V.

Table.1 Production of PHA of the G1S1 strain in media with different carbon and nitrogen sources

G1S1
Carbon Sources DCW (g/L) PHB (g/L)
Glucose 0.765 0.020
Sucrose 0.30 0.016
Maltose 0.38 0.014
fructose 0.60 0.018
Lactose 0.10 0.007

Nitrogen sources
Yeast extract 0.654 0.016
Meat extract 0.30 0.012
malt extract 0.38 0.014
peptone 0.49 0.015
(NH4)2SO4 0.14 0.010

Effect Of "C" on PHA G1S1 Effect Of "N" on PHA G1S1

1
IDENTIFICATION 16S rRNA 1
O .D at 540 n m
O .D at 540 n m

0.8 0.8
0.6 0.6
Series1 Series1
0.4 0.4
0.2
0.2
0
0
0.78 0.39 0.46 0.55 0.41
se
e

se

e
e
os

os

Yeast Meat Malt PeptoneA mmonium

os

to

to

ct
luc

cr

al

uc

La
M
Su

Extract Extract Extract Sulphate

G

Fr

"C" source "N" source

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Family of BLAST hit with highest sequence identity: Bacillaceae

Bacillus subtilis subsp. spizizenii

Figure.4 FT-IR spectrum of PHA produced by [Link]. Accumulation = 16; Zerofilling = Off;
Gain = 2; Resolution
= 4cm-1; Apodization = Cosine; Scanning speed = 2 mm/sec; 1. 3422.81, 2 2959.01 3. 2932.70, 4.
2852.45,
5. 2465.48, 6 2065. 7 1714.63, 8. 1419.35, 9 1384.73, 10. 1317, 4. 11 1239.3. 12 1070.46 13 673.3

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 [Link] (2014) 3(5): 377-387

C=O groups by FT-IR spectroscopy. The Braunegg, G. Lefebvre, G. Genser, K. F.

result obtained by this is exactly similar to 1998. Polyhydroxyalkanoates,
that of other researchers (De Smet et al., Biopolyesters from Renewable
1983; Castillo et al., 1986) 2983 m Resources: Physiological and
(CH,CH2,CH3) ; 2933 m (CH, CH2,CH3); Engineering Aspects. J Biotechnol. 65:
1720 (ester C=O valence); 1639 (thioester 127 161
C=O valence); 1380 m; 1302 m; 1260 m Bucci DZ, Tavares LBB 2005 PHB
(CH2-S) ; 1162 s (ester C-O); The IR packaging for the storage of food
spectrum reflects both monomeric units in products. Polymer testing. 24: 564
addition a strong absorption band at 1714 571
cm " was detected in G1S1, as is expected Burdon KL, Stokes JC et al 1942a Studies
for the C=O . All absorptions due to the of the common aerobic spore-forming
PHB moiety appeared in the spectrum, and Bacilli staining for fat with Sudan
in addition a strong absorption band at Black B [Link] of Bacteriology,
1639 cm " was detected a thioester bond 43: 717-724
(ShahKR., 2012). Byrom D., Polymer synthesis by
microorganisms: technology and
Among 12 strains of the bacteria , G1S1 economics, TIBTECH, 5, 156-167
identified with 16s rRNA sequence. This 1987.
bacteria gave the highest value of specific Byrom, D.: Industrial production of
growth with PHA production. PHA was copolymer from Alcaligenes eutrophus
characterized by FTIR which gave ester . In: Dawes, E.A., ed: Novel
containing group which consisted of Biodegradable Microbial Polymers,
Polyhydroxybutyric acid (G1S1). Bacillus Kluwer, Dordrecht, 1990, pp. 113±117
species isolated 1from the soil samples can Castillo RF, Valera FR, Ramos JG,
be employed in the industrial production Berraquero FR 1986. Accumulation of
of PHA. Poly _ hydroxybutyrate by
Halobacteria. Appl. Environ.
Acknowledgement Microbiol. 51: 214-216.
Chandrashekharaiah, P.S. and Jagadeesh,
These project financially supported by K.S.: Asian J. Microbiol. Biotech. Env.
GUJCOST under Sci-tech scheme. We are Sc., 10: 117-121 2008.
very thankful to GUJCOST. Chen GQ, Konig KH, Lafferty RM.
Occurrence of poly-D--3-
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