identification and characterization of polyhydroxybutyrate … · aarthi.n, ramana.k.v...

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 5, 2011

© 2011 Aarthi.N et al., licensee IPA- Open access - Distributed under Creative Commons Attribution License 2.0

Research article ISSN 0976 – 4402

Received on December, 2010 Published on January 2011 744

Identification and Characterization of Polyhydroxybutyrate producing

Bacillus cereus and Bacillus mycoides strains Aarthi.N, Ramana.K.V

Food Biotechnology Division, Defence Food Research Laboratory, Siddharthanagar, Mysore

[email protected]

doi:10.6088/ijessi.00105020005

ABSTRACT

Polyhydroxybutyrate producing bacteria from garden soil were isolated and characterized for

their morphological, biochemical properties. Based on their 16S rRNA gene sequences, they

were identified as Bacillus mycoides DFC1, Bacillus cereus DC1, Bacillus cereus DC2,

Bacillus cereus DC3 and Bacillus cereus DC4. The bacteria were screened for PHB

production and compared for the intensity of fluorescence using Nile Blue A sulphate and

Nile red staining methods. The morphology and the extent of PHB accumulation was also

studied using Field Emission Scanning Electron Microscopy (FE-SEM). The PHB

production was found to be growth associated. The polymer production by the strains was

found to vary from 12.18% to 57.2 % content (w/w) of the dry cell weight. The highest PHB

yield was observed in Bacillus mycoides DFC1 accumulating as high as 1.83g/L, amounting

to 57.20% (w/w) of cell dry weight. The growth of the bacterium and PHB production using

several complex carbon source comprising wheat starch, corn starch, potato starch containing

media were also studied. The Bacillus mycoides DFC1 resulted in a high PHB yield of

1.28g/L in wheat starch containing medium at the end of 48h of growth. The polymer

produced was extracted and analyzed for its purity using Fourier Transform Infrared

spectroscopy (FTIR) and was confirmed to be PHB.

Key words: Polyhydroxybutyrate, PHB, Bacillus cereus, Bacillus mycoides, Starch, FESEM

and FTIR

.

1. Introduction

Synthetic plastics provide a range of utilities in the civilization of mankind, at the same time

the accumulation of these non-degradable plastics in the environment is a menacing

drawback increasing day by day. The continuous exhaustion of fossil fuels led to the research

for the production of biodegradable plastics from renewable sources. Furthermore, the

synthetic plastics cause deleterious effects to wild life and pose threat to environment and

other serene habitats. The production of biodegradable polymers from renewable resources is

the need of the hour, in the face of these ecological facts. Polyhydroxyalkanoate is one such

biodegradable microbial polymer which is accumulated in bacteria as intracellular storage

granules in the presence of excess carbon sources and limited nitrogen source (Anderson and

Dawes, 1990). The polymer is known to occur as intracellular granules in several genera of

microorganisms. The granules are synthesized by prokaryotes using fatty acids, sugars and

other carbon sources (Madison and Huisman, 1998). PHB is insoluble in water, resistant to

ultraviolet radiation and is impermeable to oxygen, and is very much suitable for use as food

packaging material. This polymer is readily degraded in the soil and sewage, and can be

processed using the extrusion technology that is currently used in making polyethylene or

polypropylene films (Byrom, 1987). The PHA content and its composition are influenced

Identification and Characterization of Polyhydroxybutyrate producing Bacillus cereus and Bacillus mycoides

strains

Aarthi.N, Ramana.K.V

International Journal of Environmental Sciences Volume 1 No.5, 2011 745

mainly by the strain of the microorganism, the type of substrate employed and its

concentration, and other growth conditions (Valappil, 2007a). To achieve a cost effective

PHA production, the availability of an efficient bacterial strain is a prerequisite and is a focus

of interest for many investigations.

In the present study, PHB accumulating bacteria from common garden soil were isolated,

identified and characterized using morphological, biochemical and molecular techniques.

They were identified as bacteria belonging to Bacillus mycoides and Bacillus cereus strains.

Growth and polymer production was studied employing simple media containing glucose,

and different complex starch as sole carbon source. The PHB production using inexpensive

carbon sources in the form of starch by the indigenous strains can be advantageous as the

complex starch substrates were used directly without involvement of any hydrolysis step. The

polymer production and yield among the native isolates was compared and the purity of the

extracted polymer was confirmed using FTIR spectroscopy.

2. Materials and Methods

2.1 Isolation of bacteria from soil

Soil samples 3.0-4.0 cm deep from surface was used for isolation of the bacteria. Around

1.0g of sample was serially diluted in sterile distilled water and plated onto nutrient agar

plates and incubated at 30˚C for 24 hours. Various colonies of different morphologies

including branched and rhizoidal forms were individually picked and sub cultured 3-4 times

on nutrient agar plates.

2.2 Maintenance of bacterial cultures

The bacteria were streaked on to nutrient agar slants, incubated at 30˚ C overnight and then

stored at 4˚C for further use.

2.3 Screening the bacteria for PHB production

The bacteria were initially grown in 1.0 %( w/v) nutrient broth glucose medium for inoculum

development. PHB production in shake flasks was studied using the modified basal mineral

salt medium (Ramsay et al, 1990) with appropriate carbon source. For shake flask

experiments, quantities of 50 ml medium in 250 ml capacity Erlenmeyer flasks sterilized by

autoclaving at (15 lb, 20 min) and cooled was used. They were inoculated with 1.0% (v/v)

inoculum of overnight culture and incubated at 37˚ C, 120 rpm/min for 48 h. The bacteria

were screened for PHB granule accumulation from 16 hours onwards using Nile blue A

sulphate (Ostle and Holt, 1982) and Nile red staining methods.

The bacteria positive for PHB production were selected by observing the granules under

fluorescence microscope, OLYMPUS Reflected Fluorescence System, (Olympus Corporation,

Japan) using BX-RFA fluorescence illuminator, fitted with Image Analyzer. Nile Red stain

was prepared from stock solution in acetone as described earlier (Greenspan, 1985). The Nile

red stained preparations were observed at excitation wavelength of 540nm and emission at

590nm respectively. Bacterial cultures showing substantial fluorescence were selected for

further study.


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2.4 Characterization of PHB producing isolates

Based on the fluorescence microscope study, five bacterial isolates namely DFC1, DC1, DC2,

DC3 and DC4 were characterized by growing at various temperatures i.e., 15˚, 25˚, 35˚ and

45˚C, for spore formation, catalase production, and growth at different pH 5.0, 6.0, 7.0, 8.0,

9.0 and 10.0. In addition, esculin, starch, casein hydrolysis and production of acid from

glucose, lactose, mannose, raffinose, and xylose were also tested.

2.5 Identification of bacteria by 16S r RNA gene

The genomic DNA from the isolates were isolated as per the standard protocol described

earlier (Sambrook, 1989). The 16S rRNA gene amplification was carried out using Taq

polymerase (Fermentas) using forward primer: 5’AGAGTTTGATCCTGGCTAG 3’ and

reverse primer: 5’AAGGAGGTGATCCAGCC 3’. Amplification was carried out using

Thermo cycler, eppendorf (Applied Biosystem, CA, USA). The amplification program

comprised of 1 cycle at 95˚C for 5min; 30 cycles of 94 ˚C/40 sec, 52˚C/1min, 72˚C/1min 30

sec with final extension at 72˚C/10min. The amplification products were purified using

Qiagen PCR purification kit according to the manufacturer’s instruction with elution in 30μl

of PCR water. The purified PCR products were sequenced by Eurofins Genomics, India. The

16S rRNA gene sequence analysis was carried out using NCBI-BLAST (National centre for

Biotechnology Information http://www.ncbi.nml.nih.gov) program.

2.6 Phylogenetic tree analysis

The 16S rRNA gene sequences of the strains DFC1, DC1, DC2, DC3 and DC4 were analyzed

using multiple sequence alignments generated by CLUSTAL W program (Thompson, 1994)

using BioEdit software version 5.09.04. Phylograms were constructed using neighbor-joining

analysis and unrooted trees were generated using TREEVIEW software (Page, 1996).

2.7 PHA production and Extraction

Based on Fluorescence microscope study, five isolates namely Bacillus mycoides DFC1,

Bacillus cereus DC1, Bacillus cereus DC2, Bacillus cereus DC3 and Bacillus cereus DC4

were further studied for PHB production. A simplified media containing only 1.0 %( w/v)

glucose, 0.5% (w/v) peptone and 0.25 %( w/v) NaCl was used for PHB production. In other

experiments the glucose was substituted for different complex starches like 1.0%(w/v)

soluble starch, wheat starch, corn starch and potato starch as carbon source for PHB

production. The shake flask studies were carried out in 500ml Erlenmeyer flask containing

100ml of the production medium. The flasks were inoculated with 1.0 %( v/v) of 24h pre-

grown culture, incubated at 37˚C in an incubator shaker at 120rpm for 48 hours. The PHB

accumulation was monitored from 16 hours onwards by Nile Red fluorescence staining of

PHB granules. At the end of 48 hours the biomass was harvested by centrifugation (Hareus

Biofuge centrifuge) at 11,963g for 10 minutes and was washed with double distilled water.

The biomass was kept in -20˚C overnight and later freeze dried under vacuum for 5 hours

using Heto Dry winner model DW3 lyophilizer. From the freeze dried biomass PHB

extraction was done using sodium hypochlorite-chloroform dispersion method as described

earlier (Hahn, 1994).


strains



2.8 Field Emission Scanning Electron Microscopy (FE-SEM) of PHB granules

The bacterial biomass prepared for PHB extraction as mentioned above was used to observe

for the extent of PHB accumulation using Field Emission Scanning Electron Microscopy FE-

SEM, Nova 600, Nano SEM (FEI, Germany).

2.9 FTIR analysis of the polymer

The polymer extracted was analyzed qualitatively in the MID IR region by FT-IR using

ThermoNicolet FT-IR spectrometer, model 5700 range from 4000cm-1

to 650cm-1

, using

single bounce ATR accessory with Zinc selenide crystal. 64 scans were averaged to get the

spectra. IR spectra were recorded with 4cm-1

resolution.

3. Results and Discussions

3.1 Isolation and screening of bacteria for PHB production:

Several bacteria isolated from soil sample were studied for PHB production as soil is rich in

microflora. Various colony morphologies including rhizoidal and branched colonies typical

for Bacillus sp were obtained. The bacteria were initially screened for the PHB production in

basal mineral salt broth and the ability to synthesize PHB granules was confirmed using Nile

blue and Nile red staining of PHB granules in the intracellular environment of the isolated

bacteria. Based on the intensity of the fluorescence observed in the staining methods the

potential PHB producers were identified (McCool, 1996). The granules were observed as

reddish-orange fluorescence at an emission wavelength of 580nm (Figure 1)

Figure 1: Fluorescence of PHB granules using Nile Red staining

3.1 1 Characterization of PHB producing isolates

Five PHB producing bacterial strains were further characterized by Gram staining,

morphological and biochemical tests as shown in Table 1 and 2. All the isolates were Gram

positive, rod shaped, spore formers. Growth was observed over a wide range of temperatures

(15˚C-45˚C). The Bacillus mycoides DFC1 utilized a wide range of sugars when tested for

sugar fermentation and exhibited good growth over wide range of pH (5.0-10.0). However,

sporulation was observed when the bacteria were grown at extremes of pH, i.e., pH 5.0 and

pH 10.0. All isolates are facultatively anaerobic. Hydrolysis of casein, lipid and 1.0 %( w/v)

soluble starch, was observed as zone of clearance by all the Bacillus mycoides and Bacillus

cereus strains by plate assay.


strains



Table 1: Morphological and Biochemical characterization of the isolates

Characteristics

DFC1

DC1

DC2

DC3

DC4

Morphology

Off white

Rhizoidal

Off white

branched

Highly

branched

Branched

Branched

Gram staining

+ + + + +

Spore formation + + + + +

Growth at

Temperature

(˚C)

15 - +

+

+

+

25

+

+

+

+

+

35 +

+

+

+

+

45 - - + + +

Aerobic conditions

+ + + + + +

Anaerobic conditions + + + + + +

Growth at pH

5.0 + - - - -

6.0 + w + w +

7.0 + + + + +

8.0 + - - - -

9.0

+ - - - -

10.0

+ - - - -

Catalase

+ + + + + +

Hydrolysis

Starch + + + + + +

Esculin + - - - - -

1. DFC1, DC1, DC2, DC3, DC4 - Isolates b) w- Weak

3.2 16S rRNA gene amplification and sequence analysis

Analysis of the 16S rRNA gene sequences on five isolates was performed using NCBI-

BLAST (National centre for Biotechnology Information http://www.ncbi.nml.nih.gov). The

complete sequences were aligned to the homologous sequence available for Bacillus strains.

The BLAST (NCBI) search using the sequences showed 99% homology to other GenBank

B .cereus 16S rRNA gene sequences. The sequences of the 16S rRNA gene of the isolated

strains (~1.4kb) was deposited in the GenBank sequence database and were given the

following accession numbers: Bacillus mycoides DFC1 (GQ344802), Bacillus cereus DC1

http://www.ncbi.nml.nih.gov/


strains



(GQ344803), Bacillus cereus DC2 (GQ344804), Bacillus cereus DC3 (GQ344805) and

Bacillus cereus DC4 (GQ344806).

Table 2: Carbohydrate Fermentation Tests

Carbohydrates

Isolates

DFC1

DC1

DC2

DC3

DC4

Dextrose + - - - -

Xylose + - - - -

Maltose + + + + +

Fructose

+ + + + +

Galactose +` - - - -

Raffinose

+ - - - -

Trehalose + + + + +

Melibiose

+ - - - -

Sucrose

+ + + - +

Arabinose + - - w -

Mannose + - + - +

Sodium gluconate + - - - -

Glycerol + + + w w

Glucosamine + - - - -

Dulcitol

+ - - - -

Mannitol

w - - - -

Adonitol - - + - -

α-methyl-D-mannoside + - - - -

Xylitol + - - - -

a) w-Weak

3.3 Phylogenetic analysis:

Phylogenetic tree analysis and sequence similarity calculations after neighbour joining

analysis showed strong homology with other Bacillus sp strains available in the database. The

phylogenetic tree analysis also showed that Bacillus cereus and Bacillus mycoides formed a

distinct clade as reported earlier (Ash et al, 1991) (Figure 2).


strains



Figure 2: Phylogenetic tree of the isolates and related bacteria with respective accession

numbers. The label at the internal nodes shows the distance and the bar 0.01 represents

substitution

3.4 Production of PHB using simplified media

In the present study, we have noticed that all the bacterial isolates were able to produce

substantial amounts of PHB during growth using the simplified media mentioned above

containing a single carbon and nitrogen source. The PHB accumulation was noticed as early

as 16 hours of incubation in the bacterial cells. The synthesis of PHB was noticed from the

log phase of growth and it continued until late exponential phase as the carbon source was

utilized for both growth and PHB production. The PHB extracted was calculated as

percentage yield of the cell dry weight obtained (Lee, 1995). The maximum yield of PHB

was observed in Bacillus mycoides DFC1 amounting to 1.8g/L from 3.2g/L of biomass

resulting 57.20% yield at the end of 48hrs(Figure 3).This strain was used for further studies.

The other isolates, Bacillus cereus DC1, Bacillus cereus DC2, Bacillus cereus DC3 and

Bacillus cereus DC4 produced 19.20%, 25.36%, 35.60% and 12.18% respectively (Figure 3).


strains



Figure 3: Cell dry weight and Percentage of PHB yield in Glucose Peptone Broth

Among the several complex nitrogen source studied, peptone gave maximum PHB yield

(data not shown). The substantial PHB production using the simplified glucose peptone

medium may be attributed to the presence of complex organic nitrogen source, peptone

favoring the growth as well as PHB accumulation (Page, 1992; Song et al., 1999; Thakur,

2002). Unlike the other gram negative bacteria reported such as Cupriavidus necator,

Pseudomonas aeruginosa, Methylobaterium extroquens, which require two stage cultivation

techniques, the gram positive Bacillus sp are known to accumulate PHB during growth phase.

Hence, in order to achieve a good PHB content, cultivation techniques to improve the

biomass should be adopted for Bacillus genera. In the present study, Bacillus mycoides DFC1

showed substantially higher biomass(3.2g/L) in turn, high PHB accumulation (1.83g/L)

(57.20%) among the isolates as well as when compared with other Bacillus sp strains like

Bacillus sp INT005 (35.30%) (Tajima, 2003), Bacillus cereus SPV (41.90%) (Valappil,

2007a), Bacillus cereus CFR06 (46.0%) (Halami, 2008) reported so far. The production of

PHB using several starch sources was also studied using the Bacillus mycoides DFC1 (Table

3). The B.mycoides DFC1 showed maximum PHB yield of 1.28g/L using wheat starch

amounting to 33.05% of cell dry weight. The yield is encouraging, since conditions used for

PHB production were not optimized, and PHB yield as high as 1.28g/L at the preliminary

level could be achieved. The PHB yield in other complex starch source studied like soluble

starch (0.64g/L), potato starch (0.30g/L), and corn starch (0.48g/L) was comparatively low.

The order of preference of carbon sources on the basis of PHB production by the bacterium is

glucose>maltose> wheat starch> soluble starch>corn starch>potato starch. This shows that

the ability of the bacterium to utilize different complex starch substrates is variable and is

dependent on several factors like nature of the substrate used and the type of enzyme

produced. In the present study, Bacillus mycoides DFC1 had shown preference for carbon

sources like, glucose, maltose and wheat starch. Strains of Bacillus genera are well

documented for their ability to utilize complex starch substrates resulting in the hydrolysis of

1, 4-α-linkages into simpler sugars like maltose and glucose, by way of producing amylases

like α-amylase and the de-branching enzymes such as pullulanases, favoring the bacterium

for its growth as well as for PHB production (Schulein and Pederson, 1984; Atkins and

Kennedy, 1985). PHB production using starch materials can also be useful to save energy

required for liquefaction and saccharification of the native starch. Furthermore, amylase

produced by these bacteria during growth process hydrolyze the native starch steadily even at

moderate temperatures is desirable for large scale applications.

0

10

20

30

40

50

60

70

B. myc

oide

sDFC

1

B. cer

eus.DC1

B. cer

eusD

C2

B. cer

eus.DC3

B. cer

eus.DC4

PH

B C

on

ten

t(%

)

0

0.5

1

1.5

2

2.5

3

3.5

4

CD

W &

PH

B (

g/L

)

PHB content CDW(g/l) PHB(g/L)


strains



Table 3: PHB production using starch sources in Bacillus mycoidesDFC1

Carbon source Cell dry weight(g/L) PHB(g/L) PHB content (%)

Maltose 4.7 0.3 1.650.42 41.60.51

Soluble starch 4.0 0.5 0.640.34 17.00.86

Potato starch 0.6380.3 0.0580.66 9.090.47

Wheat starch 3.89 0.6 1.28 0.34 33.050.58

Corn starch 3.45 0.4 0.4860.52 14.080.65

3.5 FE-SEM of PHB granules

The Field Emission Scanning Electron Microscopy (FE-SEM) was used to see the

predominance of PHB granules in the bacterial cells. The PHB granules were found as

electron dense granules of spherical to oblong shaped, while the bacterial cells were long and

rod shaped. Furthermore, the PHB granules showed the highly crystalline morphology under

FE-SEM (Figure 4&5). Due to freeze drying under vacuum, the nature of PHB granules were

probably transformed from amorphous to crystalline form during lyophilization as reported

earlier (Hahn, 1995). It is also possible that the PHB granules are released from the cell

content exposing the granule morphology in the FE-SEM photograph.

Figure 4: Field Emission Scanning Electron Microscopy of PHB granules and bacterial cells

of B.mycoides DFC1


strains



Figure 5: Field Emission Scanning Electron Microscopy of PHB granules showing its

crystalline morphology

3.6 FTIR analysis

FT-IR spectroscopy of the polymer produced using glucose and starch as substrates was

investigated along with PHB obtained from commercial source (Sigma cat no: 363502). The

polymer extracted showed the intense absorption characteristic for ester carbonyl v (C=O)

stretching groups at 1720 cm-1

in comparison with the standard polyhydroxybutyrate (Hong,

1999) (Figure 6).

S ample 1

P H B P ure

P H B S ample 1 - 19-03-09

P P H B

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

Ab

so

rba

nce

1000 1500 2000 2500 3000 3500

Wavenumbers (cm-1)

1720

1279

Figure 6: FTIR spectroscopy of the standard and extracted polymer showing absorption at

1720 cm-1

for C=O group

4. Conclusions

Among the several biodegradable polymers, polyhydroxyalkanoates are considered as

suitable biodegradable thermoplastics for applications in the food processing industry as

packaging material (Lee, 1995). PHB production using renewable carbon sources such as

starch, glucose and fatty acids attracts much importance as they are renewable materials in


strains



the nature. Isolation of new strains capable of utilizing the cheap carbon source is essential to

reduce the cost at industrial level (Steinbuchel, 1995). Bacillus sp are known for their rapid

growth on simple nutrients. Earlier, researches have focused their studies on the production

of PHAs using specialized media. Bacteria which are able to produce PHB in using complex

starch sources are rarely documented. In this study, isolation of bacteria which can utilize

starch and glucose in simple media containing only peptone as nitrogen source for PHA

production has been identified and characterized. The production of PHB was found to

increase along with the increase in the biomass. Further studies are required to optimize the

growth media to improve the PHB yield and to reduce the cost of production media along

with suitable PHB induction media components.

Acknowledgment

The authors thank Dr. A. S. Bawa, Director, Defence Food Research Laboratory, Mysore, for

providing all the facilities to carry out the work. The authors also thank Dr. V. A. Sajeev

Kumar and Dr. S. N. Sabapathy, Head Food Engineering and Packaging Discipline, for help

and valuable discussions. Aarthi N kindly acknowledges the Defence Research Development

Organisation Fellowship

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