Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
Isolation of Bacillus sp. C042 capable of producing polyhydroxybutyrate (PHB) by using
unhydrolysed waste from rice bran oil production as carbon source
Wimol Chobchuenchom
Faculty of Medical Technology, Rangsit University,
Patumthani 12000, Thailand, E-mail: wimol@rsu.ac.th
Submitted date month, year; accepted in final form date month, year (To be completed by RJAS)
Abstract
Fifty soil samples obtained from various locations of Thailand including industrial sites, refuse sites, garden
and community areas were screened for Polyhydroxyalkanoates (PHAs) - producing microorganisms. Each soil samples
was diluted in distilled water and soil suspension was poured on MSA agar supplemented with 3% (w/v) unhydrolysed
waste from rice bran oil production by cold press method. The microorganisms which grew on the medium were further
screened for PHAs production by using Sudan Black B staining. The production of biopolymer was confirmed by using
Gas Chromatography-Mass Spectrometry (GC-MS) by comparing to standard polyhydroxybutyrate (PHB). The PHB
content was measured by using GC and percentage of PHB content per cell dry weight was calculated. It was found that
55 of 336 isolates (16.4%) which grew on MSA supplemented with 3% unhydrolysed waste from rice bran oil
production were positive for PHAs by using Sudan Black B staining. And the isolate designated C042 showed the
highest potential for PHAs production and was used for further experiment. Biopolymer was extracted from C042 dried
cells by using chloroform. And the capability of the isolate for PHB production was confirmed by using GC-MS and it
was found that biopolymer was PHB by comparing the mass spectrum to standard PHB. The isolate was gram positive
bacilli with spore forming and further identified by using mass spectrometry based proteomics. And it was preliminary
classified in Genus Bacillus, therefore the isolate was designated Bacillus sp. C042. The percentage of PHA production
per cell dry weight of Bacillus sp. C042 was 8.0 (w/w) when the cells were grown on MSA agar supplemented with
15% (w/v) unhydrolysed waste from rice bran oil production.
Keywords: Polyhydroxyalkanoates (PHAs), Biopolymer, Rice bran oil waste
_______________________________________________________________________________________________
บทคัดย่อ
ดำเนินกำรแยกจุลชีพที่สำมำรถสังเครำะห์
บริ เวณที่ทิ้งขยะ
แหล่งเกษตรกรรม
Polyhydroxyalkanoates
(PHAs)
ได้จำกตัวอย่ำงดินที่เก็บจำกแหล่งอุตสำหกรรม
และแหล่งชุมชนในประเทศไทย
จำนวน
50
ตัวอย่ำง
โดยกำรนำตัวอย่ำงดินมำละลำยในน้ ำกลัน่ ที่ปรำศจำกเชื้อแล้วทำกำรเพำะเชื้อบนอำหำรชนิด minimal salt agar (MSA) ที่มีกำกของเหลือทิ้ง
จำกกำรผลิตน้ ำมันรำข้ำวด้วยวิธีบีบเย็นที่ไม่ได้ผำ่ นกำร hydrolysis ผสมอยูร่ ้อยละ 3 (น้ ำหนัก/ปริ มำตร) ทำกำรตรวจคัดกรองเพื่อหำเชื้อจุลชีพที่สร้ำง
PHA โดยกำรย้อมสี Sudan black B ตรวจยืนยันควำมสำมำรถในกำรสังเครำะห์ PHAs ด้วยวิธี gas chromatography-mass spectrometry (GC-MS)
เทียบกับสำรมำตรฐำน
polyhydroxybutyrate
(PHB)
และหำร้อยละของปริ มำณ
PHB
ต่อน้ ำหนักเซลล์แห้งของจุลชีพที่คดั เลือก
จำกผลกำรทดลองพบว่ำจุลชีพที่ให้ผลบวกกับกำรย้อมสี Sudan Black B มีจำนวน 55 ไอโซเลท จำกทั้งหมด 336 ไอโซเลท (ร้อยละ 16.4)
ที่สำมำรถเจริ ญได้บน MSA ที่มีกำกของเหลือทิ้งจำกกำรผลิตน้ ำมันรำข้ำวด้วยวิธีบีบเย็นที่ไม่ได้ผำ่ นกำร hydrolysis ผสมอยูร่ ้อยละ 3
โดยพบว่ำไอโซเลต
C042
มีศกั ยภำพสู งที่สุดในกำรสร้ำง
เมื่อทำกำรสกัดสำรไบโอโพลิเมอร์จำกเซลแห้งของ C042
PHAs
จึงเลือกไอโซเลต
C042ในกำรศึกษำขั้นต่อไป
ด้วยคลอโรฟอร์ม และตรวจยืนยันด้วยวิธี GC-MS เทียบกับสำร PHB มำตรฐำน
พบว่ำไอโซเลต C042 สำมำรถสร้ำง PHB โดย mass spectrum ของสำรสกัดคลอโรฟอร์มของ C042 ตรงกับสำร PHB มำตรฐำน
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
และเมื่อตรวจกำรติดสี แกรม
และกำรย้อมสปอร์ของ
C042
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
พบว่ำเป็ น
gram
positive
bacilli
with
spore
forming
และเมื่อทำกำรทดสอบเพื่อแยกชนิดด้วยวิธี mass spectrometry based proteomics สรุ ปในเบื้องต้นได้วำ่ เป็ น Genus Bacillus ดังนั้นจึงให้ชื่อเป็ น
Bacillus sp. C042 เมื่อทำกำรหำปริ มำณของ PHB ที่เชื้อสำมำรถสร้ำง พบว่ำร้อยละของปริ มำณ PHB ต่อน้ ำหนักเซลล์แห้งเป็ น 8.0 (น้ ำหนัก/น้ ำหนัก)
เมื่อเพำะเลี้ยงเชื้อในอำหำรชนิด MSA ที่มีกำกของเหลือทิ้งจำกกำรผลิตน้ ำมันรำข้ำวด้วยวิธีบีบเย็นที่ไม่ได้ผำ่ นกำร hydrolysis ผสมอยูร่ ้อยละ 15
(น้ ำหนัก/ปริ มำตร)
คำสำคัญ: Polyhydroxyalkanoates (PHAs), ไบโอโพลิเมอร์ , ของเหลือทิ ง้ จากการผลิตนา้ มันราข้ าว
1. Introduction
The use of plastics has grown rapidly over the past few decades, with about 150 million tons of
plastic materials being consumed yearly worldwide. At least half of this amount is used in short-term
application such as packaging materials. The petroleum-derived synthetic plastic can persist in the
environment for an average of 100 years, results in a serious pollution problem. Accumulation of plastics in
the environment has become a worldwide problem. Moreover, methods for the adequate disposal of plastics
are problematic. In landfills, the degradation rates of plastics are extremely slow. Incineration is expensive
and may generate toxic by-products (Leda et al, 2009). Recycling can be done but is very tedious. The
sorting of the wide variety of discarded plastics is a very time-consuming process (Shilpi and Ashok, 2005).
An attempt to decrease the environment impacts of plastics is to replace conventional petroleum derived
plastics with biodegradable ones.
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by microbes that have
received interest in recent years, as they have been considered to be good substitutes for petroleum-derived
synthetic plastics (Mamtesh et al., 2009). Among PHAs, various bacteria commonly produce
polyhydroxybutyrate (PHB). PHB is a biopolymer that can be used as a biodegradable thermoplastic
material for waste management strategies and biocompatibility in the medical devices, medicine, pharmacy,
veterinary and food packaging. It is important to mention, PHAs present the advantage of being
biocompatible. Biocompatibility is characterized by the absence of any toxic compound generated during
polymer degradation, as well as by the shape and surface porosity of the material. Especially for PHB, since
PHB is a normal blood constituent and is found in the cell envelope of eukaryotes (Wiggam et al., 1997).
Therefore, PHAs are potential candidate for use in medical applications, such as in wound management
(suture, skin substitutes), vascular system devices (heart values, vascular grafts), orthopaedy (scaffolds for
cartilage engineering, screws, bone graft substitutes), micro – and nanospheres for controlled drug delivery,
urological stents and others (Chen et al., 2005). But commercial production of PHAs is limited by the high
cost of production compared to synthetic plastics (Okwuobi and Ogunjobi, 2012). Due to the large impact
of the carbon source price on PHAs production cost, one of the important approaches to reduce cost is to
use wastes and by-products as material for cultivation of microbes (Choi and Lee, 1999). And production
of PHAs from inexpensive carbon sources by solid-state fermentation or by mix culture also proposed with
the aim of reducing the cost or increasing the yeilds. (Leda et.al., 2009; Queirós et al., 2014).
A number of inexpensive and renewable carbon substrates such as molasses, whey, wheat bran,
rice bran, starch and starchy wastewaters, waste frying oil and so on for production of PHAs are widely
studied which targeted to bring down the cost of the polymer to 2.20 – 0.99 $/kg or lower in order to
compete with petroleum-derived synthetic plastics (Sangkharak and Prasertsan, 2012; Rawia et al., 2013;
Morgan-Sagastume et al., 2014; Queirós et al., 2014). If based on expensive pure substrates such as
glucose, even the most efficient processes will not enable PHAs to compete on the basis of price with
petroleum-based polymers. And productivity and PHA content are usually lower for bacteria grown in
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
crude, inexpensive substrates (Choi and Lee, 1999), so the development of efficiency processes based on
crude carbon sources, such as agro-industrial by-products and wastes, remains a challenge to be pursued
(Leda et al., 2009). For application of agro-industrial residues for bacterial PHAs production as well as
copolymers, attempts were done by using hydrolysed agro-industrial by-products or waste as carbon source
such as hydrolysed potato starch, wheat bran, extruded rice bran and extruded corn starch (Huang et al.,
2006). Hydrolysis of agro-industrial residue normally performed by amylase, Alcalase and lipase enzymatic
reaction for starch, protein and lipid, respectively (Shamala et al., 2012). In 2012, Shamala and et al.
studied on production of –amylase and PHA-copolymer simultaneously by Bacillus sp. using
unhydrolysed corn starch as a carbon substrate and enzyme hydrolysed wheat bran and rice bran as
enhancers of growth and metabolite production and the results indicated the potentiality of Bacillus sp.
CFR-67 to co-produce PHAs and –amylase from these agro-industrial residues.
In Thailand, there are so many agro-industrial wastes such as the wastes from rice bran oil
produced by using cold press method. After rice bran oil product was extracted from rice bran, a lot of
wastes were left behind. This is a preliminary study on isolation of microbes from a various soil samples
obtained in Thailand that capable of utilizing unhydrolysed waste from rice bran oil cold press-production
as carbon source and being able to produce PHB.
2. Objectives
This study aimed to isolate the microorganisms capable of producing PHB from soil samples in Thailand
by using unhydrolysed waste from rice bran oil cold press-production as carbon source.
3. Materials and Methods
3.1 Isolation procedures
Fifty soil samples were collected from various locations in Thailand included industrial sites,
refuse sites, garden and community areas. Each samples was diluted in sterilized distilled water and plated
onto minimal salt agar (MSA) supplemented with 3% unhydrolysed waste from rice bran oil production by
cold press method (URB) using standard pour plate technique. For preparation of MSA + URB, briefly, 1.0
g Ammonium sulphate,2.0 g Potassium dihydrogen phosphate, 0.6 g Di-Sodium hydrogen phosphate, 0.2 g
Magnesium sulphate heptahydrate, 0.75 g Citric acid, 2.0 g Sodium acetate, 0.05 g Yeast extract 15.0 g
Agar and URB were mixed in 1 L distilled water to obtain the final concentration (w/v) at 3,6,9,12 and 15
%. Sterilization of MSA+URB was carried out by autoclaving at 121 o C for 15 minutes. The plates were
incubated at 30 o C for 48 hours. Representative colonies were obtained and purified by repeated streaking
on MSA+3%URB. Each colony of pure culture was individually picked based on distinct morphological
characteristics.
3.2 Screening method for PHB accumulation in bacteria
The pure isolates were grown on MSA+3 % (w/v) URB plates and incubated at 30 o C for 48
hours. Under sterilization technique, each isolates was smeared on glass slide and air dried. Then, the dried
slides were heat-fixed and stained with Sudan Black solution using a modified method that previously
described (Okwuobi and Ogunjobi, 2012). Briefly, heat fixed slides were stained with 0.3% Sudan black B
in 95% Ethanol (w/v) for 10 minutes and rinsed off with slowly running water, counterstained with 0.5%
aqueous safranine for 5 seconds and rinsed off with slowly running water. The slides were air dried and
viewed under oil immersion lenses. The PHA granules appeared as blue-black granules inside pink cells
referred to positive cells and only pink cells for those that were negative.
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
3.3 Identification of isolated- PHB producing bacterium
The positive PHB producing bacterial isolate, C042 was identified by using gram staining, spore
staining and mass spectrometry- based proteomics. Mass spectrometry- based proteomics was kindly
performed by Microbiology laboratory of Nopparat Rajathanee hospital, Bangkok, Thailand.
3.4 Extraction and identification of PHB
Bacillus sp C042 was cultured on MSA+ 3%URB at 30 o C for 48 hours. Then the cells were
harvested and washed once with distilled water by centrifugation at 5000 rpm for 30 min and the pellet was
dried at 105 o C to obtain a constant weight in a hot air oven (Memmert). Twenty milligrams of dried cells
were added with 2 mL chloroform and 2 mL of 3% H 2SO4 (v/v) in methanol supplemented with 10 mg/mL
benzoic acid as the internal standard and then heated at 80 o C for 3.5 hours with mixing at 30 minutes
intervals. After that, the tube of mixture was placed on ice and let it cool down. Then, 2 mL of distilled
water was added, mixed vigorously by vortex for 5 minutes and let the tube stand overnight. Chloroform
layer was transferred into vial and used for identification of PHB by using Gas Chromatography - Mass
Spectrometry (GC-MS). The authentic PHB was prepared as same as the above. The identification of PHB
was performed at Scientific equipment center, Prince of Songkla University by using Gas ChromatographyMass Spectrometry (Perkin Elmer Clarus 500 GC / MS 17) with capillary column (MS), 30 m x 0.25 mm
coated with DB-5, 0.25m film thickness: column oven temperature of 80 ° C to be 150 ° C at the rate of
10 ° C /min., injection port temperature 230 ° C, constant pressure of carrier gas (helium), flow rate 1
mL/min. One L of sample was injected into the GC-MS for identification of PHB compared to PHB
standard (Fluka).
3.5 Extraction and quantitation of PHB content
After incubation Bacillus sp. C042 cells on MSA+ 3%, 6%, 9%, 12% or 15% URB, the cells were
harvested, washed twice with minimal salt broth and followed with distilled water by centrifugation at 2500
rpm for 10 minutes and lyophilized. PHB from dried cell was extracted by incubating 50 mg dried cell with
2 mL chloroform and 2 mL methanol which acidified by 3% H2SO4 (v/v) supplemented with 10 mg/mL
benzoic acid as the internal standard at 100 o C for 4 hours. Let the mixture cool down for overnight and
then 1 mL distilled water was added, mixed vigorously for 1-4 minutes and then let the mixture stand at
room temperature for overnight. Chloroform layer was transferred to vial and kept at -20 o C until
quantitation of PHB content. Gas chromatography was carried out on Gas chromatography instrument
(Agilent technologies 7890A) with capillary column HP5, 25 m x 0.32 mm, 0.52 m film thickness,
injection port temperature 250 o C (isothermal), column oven temperature of 100 o C to be 250 o C at the
rate of 5 o C/min., detector (FID) temperature 250 o C, split ratio 50:1, constant pressure of carrier gas
(helium) at flow rate 1mL/min. Sample was dissolved in chloroform and 1 L of sample was injected to GC
instrument by auto loading system.
4. Results
4.1 Isolation of PHB producing microbes from soil samples
From the total number of 50 soil samples obtained from various places of Thailand, there were 336
isolates could grow on MSA+3%WRB after incubation at 30 o C for 48 hours. The isolates were screened
for their PHB production by using Sudan Black B staining. And it was found that there were 55 isolates
from these 336 isolates (16.4 %) that showed the positive cells for PHA granules which appeared as blueblack granules inside pink cells. Among them, the isolate named C042 showed the highest percentage of
positive cells as shown in figure 1.
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
(A)
(B)
Figure 1 Sudan Black staining-photomicrograph of isolate Bacillus sp. C042, showing the PHB granules
produced in form of dark granules inside the bacterial cells (A) compare to negative cells of Escherichia
coli (B).
4.2 Identification of isolate C042 by mass spectrometry- based proteomics
The isolate C042 was found to be gram positive bacilli with spore forming. By using mass
spectrometry- based proteomics which was kindly performed by Microbiology laboratory of Nopparat
Rajathanee hospital, Bangkok, Thailand. It was found that the strain C042 was in genus Bacillus and
showed identification score of 2.3, 1.9 and 1.7 when compared to Bacillus cereus, Bacillus thuringinesis
and Bacillus subtilis, respectively. Because of the limitation of spectrometry- based proteomics data for
Bacillus, all of the identification score were less than 2.5 for available proteomics datas of Bacillus. Thus,
the strain C042 was preliminary identified to be Bacillus sp. and designated name as Bacillus sp. C042.
4.3 Identification of PHB produced by Bacillus sp. C042
PHB produced by Bacillus sp. C042 was identified by using GC-MS. The results of GC-MS
analysis obtained from Bacillus sp. C042 and standard PHB were represented in figure 2 and 3,
respectively. It was found that the mass spectrum of polymer extract at retention time 14. 30 from Bacillus
sp. C042 was corresponded to standard PHB.
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
(A)
(B)
Figure 2 GC chromatogram of the extract obtained from Bacillus sp. C042 at the peak of 14.03 (A) and
standard PHB at the peak of 13.69 (B). (Benzoic acid internal standard peak at 17.40 and 17.25,
respectively)
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
(A)
(B)
Figure 3 Mass spectrum of the extract obtained from Bacillus sp. C042 at the peak of 14.03 (A) compared
to standard PHB at the peak of 13.69 (B). (Benzoic acid internal standard peak at 17.40 and 17.25,
respectively)
4.4 PHB production by Bacillus sp. C042 using unhydrolysed waste from rice bran oil production by cold
press as carbon source
Bacillus sp. C042 was cultured on MSA supplemented with unhydrolysed waste from rice bran oil
production by cold press method at various concentrations as the followings; 3, 6, 9, 12 and 15 % (w/v).
After incubation period of 48 hours at 30 o C, the cells were harvested, lyophilized and extracted for
polymer by using chloroform technique. The PHB content was quantitated by using GC and the percentage
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
of PHB content per cell dry weight was calculated. And it was found that the percentage of PHB content per
cell dry weight of the cells cultured on MSA supplemented with unhydrolysed waste from rice bran oil
production by cold press method at 3, 6, 9, 12 and 15 % were 1.8, 2.6, 4.8, 5.6 and 8.0, respectively. The
example of GC chromatogram was illustrated in Figure 4.
Figure 4 GC chromatogram of the extract obtained from Bacillus sp. C042 cultured on MSA+ 15 % of
unhydrolysed waste from rice bran oil production by cold press method, the first peak is PHB peak, the
second peak is benzoic acid internal standard peak and the last peak is the peak of other compound or
artifacts.
5. Discussion
Recent studies have focused on the use of renewable carbon sources for the production of PHAs
that can reduce the production costs of PHAs biopolymer (Waqas et al., 2010). Thus, we explored the
potential of unhydrolysed waste obtained from rice bran oil production by using cold press method for use
as low cost carbon source for bacterial media. The soil samples used for isolation of PHB produced bacteria
were collected from various locations in Thailand which were considered as rich sites for organic carbon
source such as refuse site, industrial site, garden sites and community areas. The 336 isolates grew on MSA
supplemented with 3% of unhydrolysed waste from rice bran oil production by cold press method were
screened for PHB production. Fifty-five from 336 isolates showed PHB accumulation in the cells on Sudan
Black B staining. One of positive isolates, isolate C042 a positive result with nearly all of the cells (figure
1). Thus, this isolate was used for further studying and one of its polymer in extract was preliminarily
identified to be PHB by using GC-MS when compared to the standard PHB (figure 2 and 3). However, the
characterization of other biopolymers produced by this isolate should be done further. Though wide range
of the microbes can produce PHB, this polymer is crystalline and brittle. Due to relatively poor physical
properties of PHB monopolymer, extensive efforts are being directed towards the synthesis of copolymer
that have better properties. Incorporation of 3- and 5- carbon monomers into a polymer consisting mainly of
PHB leads to a decrease in crystallinity and melting point compared to PHB monopolymer. The
copolymers such as poly(hydroxybutyrate-co-hydroxyvalerate) and others have better mechanical
properties and are useful for various applications (Shilpi and Ashok, 2005). It was found that the
copolymer production depends on the carbon substrate provided for cultivation and the type of PHAsynthesis genes present in the microbes (Shamala et al., 2012). For sustained commercialization it is
essential to explore economic substrates for bacterial growth and copolymer production (Anil et al., 2007).
Rangsit Journal of Arts and Sciences, July-December 2014
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RJAS Vol. 4 No. 2, pp. C-1-C-5
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There have been reports on supplementation of organic acid such as propionic acid etc., to the bacterial
media can result in increased copolymer production (Jain, 2001), however this may increase the cost of
copolymer production. On the other hand, many literatures reported on copolymer production by using
hydrolysed agro-industrial wastes or by-products (Shamala et al., 2012) which can increase the cost too.
Therefore, the microbiological production of biodegradable copolymers, the utilization of cheap carbon
sources as substrates for bacterial growth strongly requires the identification of suitable microbial strains
able to produce and accumulate a variety of novel PHA without other supplementations or processes.
With limitation of our mass spectrometry- based proteomics data, the isolate C042 could not be identified
its specie exactly. In this study, the highest identification score for Bacillus cereus was less than 2.5. So,
C042 was preliminary identified as Bacillus sp. by using mass spectrometry- based proteomics and gram
stain. It was designated Bacillus sp. C042. The isolate could grow up on minimal salt agar supplemented
with unhydrolysed waste from rice bran oil production by cold press method at various concentrations as
the followings; 3, 6, 9, 12 and 15 % (w/v). And show the higher % PHA content per cell dried weight when
the more unhydrolysed waste from rice bran oil production by cold press method were supplemented which
were 1.8, 2.6, 4.8, 5.6 and 8.0, respectively.
The genus Bacillus, in common with many other PHA-accumulating gram-positive bacteria, accumulates
co-polymers of PHAs when grown on different substrates or wastes (Valappil et al. 2007; Anish et al.,
2014). Nowaday, it reveals that gram negative bacteria such as Ralstonia, Alcaligenes latus and
recombinant E. coli are among those which have been exploited for industrial scale PHB production. The
outer membrane of them contains lipo-polysaccharides (LPS) which are endotoxin and are toxic for human
beings. The purification of PHAs is more complicated due to the presence of endotoxin (Mamtesh et al.,
2009). Gram – positive bacteria lack lipopolysaccharide and are better sources of PHAs for use in
biomedical applications (Valappil et al., 2008). In 2014, Laranja and et al. found that PHB accumulating
Bacillus spp. could improve the survival, growth and robustness of Penaeus monodon (Fabricius, 1798)
postlarvae.
Therefore, Bacillus offers the advantage of lacking endotoxin. Some Bacillus such as Bacillus megaterium
has high possibility that accumulates different types of PHAs (Valappil et al., 2008). Bacillus subtilis
contains expression of self-lysing genes on completion of PHAs biosynthetic process for easy and timely
recovery and it can use agro-industrial as feed. These enable it to be a strong contender in the future as and
industrial PHAs producer (Mamtesh et al., 2009).
6. Conclusions
Bacillus sp. C042 was isolated from soil sample obtained from Thailand by using unhydrolysed
waste from rice bran oil cold press production as carbon source. It was screened by using Sudan Black B
staining for its capability of PHB production. And its chloroform extract was finally confirmed for at least
one of the polymers to be PHB by using GC-MS. The percentage of PHB content per cell dry weight when
the cells were cultured on MSA supplemented with 15 % (w/v) unhydrolysed waste from rice bran oil
production was 8.0. It seems reasonable to carry further investigation on other PHAs copolymers
production by this strain and optimized condition for increase PHB and perhaps other polymers yields in
laboratory scale bioreactor.
7. Acknowledgements
This study was financially supported by the research institute of Rangsit University, research fund
2014 (grant no. 25/2556).
8. References
Rangsit Journal of Arts and Sciences, July-December 2014
Copyright © 2011, Rangsit University
RJAS Vol. 4 No. 2, pp. C-1-C-5
ISSN 2229-063X (Print)/ISSN 2392-554X (Online)
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Rangsit Journal of Arts and Sciences, July-December 2014
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