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ORIGINAL ARTICLE

The production of poly(3-hydroxybutyrate) [P(3HB)]


by a newly isolated Bacillus sp. ST1C using liquid
waste from biodiesel production
Wankuson Chanasit & Lukman Sueree & Brian Hodgson &
Kamontam Umsakul
Received: 19 June 2013 / Accepted: 29 October 2013 / Published online: 29 November 2013
#Springer-Verlag Berlin Heidelberg and the University of Milan 2013
Abstract A newly isolated poly(3-hydroxybutyrate)
[P(3HB)] producing strain, ST1C, was identified as Bacillus
aryabhattai based on its morphological, biochemical and
molecular characteristics. It synthesized and accumulated rel-
atively high amounts of P(3HB). The aim of this work was to
establish if it could convert an inexpensive liquid waste prod-
uct from the production of biodiesel, biodiesel liquid waste
(BLW), to P(3HB). Using a mineral salt medium (MSM)
containing 2.0 % (v/v) glycerol present in the BLW and both
normal batch and a draw and fill culture method, B.
aryabhattai ST1C produced a maximum P(3HB) content
and biomass concentration of 72.31 % dry cell weight
(DCW) and 7.24 g/L, respectively, over a 24 h cultivation
period in the draw and fill cultivation method. From 24 h to
the end of cultivation at 72 h both the P(3HB) content and the
biomass concentrations continuously reduced. Concentrations
of glycerol in the BLWin this MSMabove 3.0 %(v/v) or from
pure glycerol (PG) or with an added NaCl concentration of
greater than 3.0 % significantly reduced both the maximum
P(3HB) content and the biomass concentrations.
Keywords Poly(3-hydroxybutyrate) [P(3HB)]
.
Bacillus
aryabhattai
.
Biodiesel liquid waste (BLW)
Introduction
Polyhydroxyalkanoates or PHAs are considered to be strong
candidates for producing biodegradable polymer materials
because they possess many properties similar to various syn-
thetic thermoplastics and elastomers (Din et al. 2008). In
addition, upon disposal, they are completely degraded by
microorganisms to water and carbon dioxide under aerobic
conditions or to methane and water under anaerobic condi-
tions (Volova et al. 2010). Of the large PHA family, a homo-
polymer of 3-hydroxybutyrate, poly(3-hydroxybutyrate)
[P(3HB)], is the most widespread in nature and the best
characterized PHA compound (Obruca et al. 2011). P(3HB)
is synthesized by numerous bacteria as a reserve intracellular
carbon and energy storage material, usually when an essential
nutrient such as nitrogen or phosphorus is limiting growth in
the presence of an excess source of metabolizable carbon
(Sudesh et al. 2000; Merugu et al. 2012). The major problem
that has limited the commercialization of P(3HB) as a substi-
tute for conventional petrochemical-based plastics is its high
production costs. Hence, much effort has been devoted to
lowering the cost of P(3HB) by the isolation of more efficient
bacterial strains and/or the development of less costly sub-
strates, production processes and more economical recovery
methods (Chee et al. 2010).
One economic evaluation showed that the production ex-
penses for P(3HB) could be reduced by over a half if a
renewable waste material and/or a by-product from industry
Electronic supplementary material The online version of this article
(doi:10.1007/s13213-013-0755-1) contains supplementary material,
which is available to authorized users.
W. Chanasit
:
L. Sueree
:
K. Umsakul (*)
Department of Microbiology, Faculty of Science, Prince of Songkla
University, Hat-Yai, Songkhla, Thailand 90112
e-mail: kamontam.u@psu.ac.th
B. Hodgson
Faculty of Pharmaceutical Sciences, Prince of Songkla University,
Hat-Yai, Songkhla, Thailand 90112
Ann Microbiol (2014) 64:11571166
DOI 10.1007/s13213-013-0755-1
was used as the main carbon substrate for P(3HB) biosynthe-
sis (Mengmeng et al. 2009).
Biodiesel can nowreplace petroleumdiesel as a fuel source
as it is produced from renewable sources such as animal fats
and vegetable oils, but this process generates about 10.0 %(w/
w) glycerol as the main waste byproduct as it is the fatty acids
present as glycerol esters that are the required biodiesel prod-
ucts. The excess biodiesel liquid waste (BLW) generated may
become an environmental problem, when released as a waste
material. One possible way to eliminate this BLW would be to
refine the glycerol to a high purity before applying it in the
drug, food, beverage, chemical and synthetic material indus-
tries (Yang et al. 2012). However, the refining of BLWmay be
a costly process depending on the economy of production
scale and/or the availability of a glycerol purification
facility (Thompson and He 2006). One possible appli-
cation for this BLW would be to use it as a carbon and
energy source for supporting microbial growth and their
products in industrial microbiology (Silva et al. 2009).
There have been a number of attempts to convert BLW to
PHA using: Pseudomonas oleovorans NRRL B-14682 and
Pseudomonas corrugata 388 (Ashby et al. 2004),
Cupriavidus necator JMP134, Paracoccus denitrificans and a
number of different microorganisms (Mothes et al. 2007),
Cupriavidus necator DSM 545 (Cavalheiro et al. 2009),
Bacillus sonorensis, Halomonas hydrothermalis (Shrivastav
et al. 2010), Halomonas sp. KM-1 (Kawata and Aiba 2010),
Novosphingobium sp. THA_AIK7 (Teeka et al. 2010),
Burkholderia cepacia ATCC 17759 (Zhu et al. 2010),
Bacillus aryabhattai S4 (Tanamool and Kaewkannetra 2011).
The most successful among these was in a small scale cultiva-
tion, in which Burkholderia cepacia ATCC17759 accumulated
a P(3HB) content of up to 81.9 %of its cell dry weight (5.8 g/L)
that amounted to a 4.8 g/L P(3HB) concentration using a
mineral salt medium (MSM) supplemented with 3.0 % (v/v)
of a biodiesel-glycerol as a carbon source (Zhu et al. 2010). The
aims of the work reported here were to detect, isolate and
characterize bacteria that could convert BLW to relatively high
amounts of PHA, and to optimize the culture and operating
conditions for the most suitable isolate to produce the maximum
PHAyields from the BLWand to determine the composition of
the isolated PHA. This is the first report of the isolation of a B.
aryabhattai strain able to efficiently utilize all the glycerol
present in BLW prepared from waste cooking oil as a feedstock
into a relatively high cell mass and P(3HB) production.
Materials and methods
Isolation of the organism
Bacteria were isolated by adding 1 g of soil into 100 mL of
mineral salt medium (MSM) consisting in g/L: total glycerol
in BLW, 20; (NH
4
)
2
SO
4
, 1.0; KH
2
PO
4,
2.0; Na
2
HPO
4
, 0.6;
MgSO
4
7H
2
O, 1.0; and trace element solution 1 mL contain-
ing in g/L: CaCl
2
, 20; ZnSO
4
7H
2
O, 1.3; FeSO
4
7H
2
O, 0.2;
(NH
4
)
6
Mo
7
O
24
4H
2
O, 0.6; and H
3
BO
3
, 0.6; (Kulpreecha et al.
2009). The pH in the medium was adjusted to 7.0 before
sterilization. After 3 days at 30 C and shaking at 200 rpm,
0.1 mL aliquots from each enrichment culture were plated
onto MSM agar containing 2.0 % (v/v) of glycerol in the
BLW. Approximately 0.25 mg of the hydrophobic dye, Nile
Blue A stain powder (Sigma-Aldrich) was dissolved in 1 mL
of dimethyl sulfoxide (DMSO) and this Nile Blue A solution
was added to the MSM agar at a final concentration of 0.5 g/
mL to check for possible PHA deposits (Chaudhry et al.
2011). After 72 h incubation at 30 C, the agar plates were
observed under UV light. The colonies that produced strong
fluorescence because of their PHA granules were purified. All
selected colonies grew so the accumulation of dye had not
interfered with their viability.
The colony that seemed to have the deepest red fluorescent
color and, therefore, good PHA production was chosen for
further work. Good P(3HB) production was further confirmed
by checking for PHA granules using a phase contrast micro-
scope and a fluorescence microscope after staining with Nile
Blue A (Online Resource S1).
This new PHA accumulating bacterium was isolated from
soil obtained from a dense forest in the Hala-Bala mountain
range, Satun province in southern Thailand. The stock culture
was kept on a nutrient agar (NA) slant at 4 C and transferred
to other new NA slants every 2 weeks.
Identification of the new isolate using microbiological
methods and its 16S rRNA gene
The new PHA accumulating bacterium was first characterized
using standard morphological and biochemical tests. From
these tests it was identified as a Bacillus species and was
named Bacillus sp. ST1C. It was then further identified as
Bacillus aryabhattai ST1C using a full-length 16S rDNA
sequencing method. The genomic DNA from the Bacillus
sp. ST1C strain was prepared following the standard protocol
described by Krueger et al. (2012). The 16S rRNA gene
amplification was carried out using Taq polymerase with the
forward primer: 5-GAGTTT GAT CCT GGCTCAG-3 and
reverse primer: 5-GTT ACC TTG TTA CGA CTT-3. The
amplification program employed was the DNA Engine
Dyad Thermal Cycler (Bio-Rad Laboratories) and com-
prised one cycle at 94 C for 3 min, 25 cycles of denaturation
at 94 Cfor 1 min, annealing at 50 Cfor 1 min and elongation
at 72 C for 2 min, followed by a final amplification step at
72 C for 3 min. The amplification products were purified
using the Qiagen PCR purification kit and its sequence was
determined on an ABI Prism 3730XL DNA Sequencer
(Applied Biosystems, Foster City, California, USA). The
1158 Ann Microbiol (2014) 64:11571166
16S rRNA gene sequence analysis was carried out using the
NCBI-BLAST (National Centre for Biotechnology
Information http://www.ncbi.nml.nih.gov) program. The
DNA sequences were determined and aligned for
comparison with a program CLUSTAL X (version 1.8) in
the BioEdit Program. Alignment gaps and unidentified bases
were eliminated. Distance matrices for the aligned sequences
were calculated using the Kimuras two-parameter method
(Kimura 1980; Zakaria et al. 2010). A phylogenetic tree of
16S rRNA genes was constructed by the neighbor-joining
method of Saitou and Nei (Saitou and Nei 1987; Zakaria
et al. 2010). The robustness for individual branches was
estimated by 1,000 replication bootstrapping with the program
MEGAVersion 4.0.
Culture medium and conditions
Preparation of the pre-culture inoculum
Bacillus sp. ST1C was grown on an NA slant for 24 h then
suspended in 0.85 % (w/v) sodium chloride to an optical
density (660 nm) of 0.5. A 250 mL Erlenmeyer flask con-
taining 50 mL of pre-culture medium was inoculated with 2
mL of cell suspension; Basal Culture Medium (BCM)
(Kulpreecha et al. 2009) was adjusted to an initial pH of 7.0
and incubated on a rotary shaker at 200 rpm and 30 C. This
medium, analyzed by a CHNS-O analyzer (CHNS-O
analyzer, CE Instruments Flash EA 1112 Series, Thermo
Quest, Italy), had a C/N ratio of 4.7. This C/N ratio was
probably suitable to promote more cell growth because of its
relatively high nitrogen content.
Growth medium for PHA accumulation
A waste product from the manufacturing process for bio-
diesel production using waste cooking oil as feed stock was
named biodiesel liquid waste (BLW). This BLW was
analyzed by a CHNS-O analyzer as above prior to use as
a carbon source in the PHA production medium in order to
determine its carbon and nitrogen content. The approximate
glycerol content was measured by the free glycerol deter-
mination method as described by Bondioli and Della Bella
(2005; Teeka et al. 2010).
The PHA production was carried out in shake flasks using
the BLWor pure glycerol (PG from Sigma-Aldrich) as a sole
carbon source. After the Bacillus sp. ST1Chad been grown in
BCM pre-culture medium until it reached mid exponential
growth, 4.0 %(v/v) of the pre-grown inoculumwas inoculated
into a 250 mL Erlenmeyer flask containing 50 mL of MSM
(Kulpreecha et al. 2009) supplemented with 2.0 % (v/v) of
total glycerol in the BLWor 2.0 % (v/v) of PG with an initial
pH adjusted to 7.0. Aerobic conditions were maintained by
shaking at 30 C and 200 rpm. Samples were taken every 12 h
until 72 h of cultivation and then the biomass concentration,
PHA content, and the glycerol utilization in the cell free
culture medium were analyzed.
Effect of glycerol concentration in BLWon cell growth
and P(3HB) production
The effect of the glycerol concentrations in BLWon the PHA
production and growth of Bacillus sp. ST1Cwas performed in
MSM production medium supplemented with various con-
centrations of the total glycerol in the BLW (0.5, 1.0, 2.0, 3.0,
4.0, and 5.0 % (v/v)). The initial pH of the medium was
adjusted to 7.0 and cultivated under the same conditions as
described above.
Effects of supplementing the initial growth mediumwith extra
nutrients (draw and fill cultivation method) on cell growth
and P(3HB) production
The first draw and fill cultivation was carried out using
the same conditions as described above until 12 h of
cultivation. The original working volume before feeding
was 50 mL. Then 10 mL of MSM containing 2.0 % (v/v)
total glycerol in the BLW with 1.0 g/L ammonium sulfate
was added to the culture at 12 h and again at every 12 h
interval until 60 h. At the same time 10 mL of culture was
removed to promote the fresh medium to the cells and
wash out some of the older cells from the culture medium.
We called this system the draw and fill cultivation method
because extra nutrients were supplied and the culture was
removed every 12 h. This extra carbon and nitrogen might
enhance and prolong the cell growth and P(3HB) accu-
mulation. This did result in a small increase in cell dry
weight and P(3HB) but it was not significant.
As there was a possibility that addition of the first extra
substrate at 12 h may have exceeded the concentration of
glycerol that resulted in some inhibition and perhaps the
C/N ratio became less optimal so we changed the strategy.
Other workers had also shown that biodiesel-derived crude
glycerol at higher concentration of glycerol of 2.0 % (v/v)
inhibited cell growth and PHA accumulation (Shrivastav
et al. 2010; Wattanaphon and Pisutpaisal 2011; Sindhu
et al. 2011). MSM containing 2.0 % (v/v) total glycerol in
BLW was then used and the initial nitrogen content was
increased to 1.25 g/L to promote a little more cell growth
and then the MSM containing 0.5 % (v/v) total glycerol in
the BLWand 0.2 g/L ammonium sulfate were added at 12 h
and then at 12 h intervals until 60 h of cultivation was
reached. The lower amounts of glycerol ensured that its
inhibitory concentrations were not reached during the time
P(3HB) was accumulating.
Ann Microbiol (2014) 64:11571166 1159
Analytical methods
Dry cell weight The cell pellet obtained after centrifugation
(7,155 g at 4 C for 20 min) was dried at 80 C to a constant
cell weight.
PHA content Chloroform and acidified methanol (3.0 % (v/v)
H
2
SO
4
) at 2 mL each were added into 20 mg of dried cells and
then heated at 80 C for 3.5 h. After cooling at room temper-
ature, 2 mL of distilled water was added followed by vigorous
shaking and centrifugation (1,006 g for 10 min), the chlo-
roform portion containing the PHA methyl esters was trans-
ferred to a vial for analysis by Gas Chromatography (GC).
PHA was determined by the method described by Sun et al.
(2009). Benzoic acid was used as the internal standard.
Glycerol utilization
The glycerol concentration was measured by the free glycerol
determination method as described by Bondioli and Della
Bella (2005; Teeka et al. 2010). The working reagents pre-
pared for the experiment were 1.6 M acetic acid stock solution
and 4.0 M ammonium acetate stock solution. The 0.2 M
acetylacetone solution and 10 mM sodium periodate solution
were prepared in a 1:1 ratio of acetic acid and ammonium
acetate stock solution. The extraction solvent was prepared by
mixing an equal volume of distilled water with 95 % ethanol.
A mixture of hexane and extraction solvent was added to the
sample. Upon mixing, two layers were formed and the lower
layer was transferred into a new test tube. Then, 1.5 mL of the
extraction solvent and 1.2 mL of sodium periodate were
added. After that, 1.2 mL of acetylacetone solution was added
and the mixture was incubated in a water bath at 70 C for
1 min. An absorbance measurement was made at 410 nm
against a blank sample.
Results and discussion
Identification of the bacterial isolate
Of the 190 isolates obtained, only 11 showed strong fluores-
cence from accumulated PHA granules. Among them, a
Bacillus sp. ST1C isolate, seemed to have the most potential
to synthesize and accumulate PHA because it showed the
strongest fluorescent color under a fluorescence microscope
and the largest PHAgranules using phase contrast microscopy
(Online Resource S1).
The morphological and physiological characteristics of the
isolate were investigated (Online Resource S2). The isolate
was grown with various carbohydrates and alcohols as the
carbon source (API 50 CHB/20E Medium test kit,
bioMerieux, France, and API ZYM test kit, bioMerieux,
France). As a result of these observations, the new isolate
was determined to be a Bacillus sp. ST1C (with 97.7 %
identity to Bacillus megaterium). However, the biochemical
tests alone do not provide enough data to make a totally
accurate identification.
Further identification was then performed using 16S rDNA
analysis. A full length 16S rDNA sequence of 1,482 bps was
obtained by PCR. The BLASTX analysis revealed a 99.93 %
identity to the sequence of the 16S rRNA gene of Bacillus
aryabhattai B8W22
T
(accession no. EF114313). Lineage was
according to the phylogenetic tree generated (Fig. 1). The
isolate was identified as a strain of Bacillus aryabhattai
according to all the identification results. The isolate was then
deposited in GenBank under the code name Bacillus
aryabhattai ST1C (JX 524506).
Bacillus aryabhattai sp. nov. was first isolated in 2009 by
Shivaji et al. from the upper atmosphere (Shivaji et al. 2009).
Tanamool and Kaewkannetra (2011) were the first to report
that another isolate of Bacillus aryabhattai S4 from a sugar-
cane plantation soil produced PHA with a maximum content
of 57.62 % of its dry weight and productivity of 0.097 g/Lh
when cultivated in 20 g/L of initial total sugar present in a
sweet sorghum juice at pH 7.0, and 35 C.
Comparison of the 16S rDNA of our isolate STIC and the
standard strain (Bacillus aryabhattai B8W22
T
) showed a
99.93 % similarity which was higher than that of Bacillus
aryabhattai S4 that possessed only a 99.7 % similarity
(Tanamool and Kaewkannetra 2011).
Analysis of BLWand its effect on cell growth and P(3HB)
production
Before using this BLW as a carbon source for P(3HB)
production, the BLW was first analyzed by a CHNS-O
analyzer in order to determine the carbon and nitrogen
contents (Table 1). The carbon and nitrogen element av-
erages were 43.65 % and 0.15 %, respectively and the
average glycerol content in the initial BLW sample was
43.16 % (w/v). In general, glycerol was the most single
abundant component in the BLW (data from the special-
ized R & D center for alternative energy from palm oil
and oil crops, Faculty of Engineering, Prince of Songkla
University, Online Resource S3). From this result it was
concluded that this BLW was suitable for use as a carbon
source for P(3HB) biosynthesis because of its high carbon
and glycerol content whereas it had a very low nitrogen
content. The C/N ratio of the MSM containing 2.0 % (v/v)
total glycerol in the BLW used in the culture medium was
20. Moreover, the glycerol was likely to be an energeti-
cally favorable substrate for the formation of acetyl-CoA
(the precursor for P(3HB) synthesis). This can be seen in
1160 Ann Microbiol (2014) 64:11571166
the stoichiometry of the conversion of glycerol to acetyl
CoA (Ashby et al. 2004):
Glycerol 3NAD

ADP CoAAcetylCoA
3NADH 3H

ATP CO
2
H
2
O
The production of PHAby Bacillus aryabhattai ST1Cfrom
2.0 % (v/v) total glycerol in BLW was investigated (Fig. 2).
During the period of exponential growth, accumulation of
P(3HB) increased gradually and reached a maximum dry cell
weight (DCW) and P(3HB) content of 5.68 g/L and 57.76 %
DCW, respectively, at 24 h of cultivation simultaneously with
the almost complete utilization of glycerol (the most abundant
component in BLW) as indicated by the reduction of the
glycerol concentration to 0.75 g/L. After 36 h the accumula-
tion of P(3HB) significantly decreased to 40.77 % DCW due
presumably to the absence of glycerol and no alternative
readily metabolizable carbon compound; therefore, cells
started to degrade the intracellular PHA storage for cell
maintenance purposes whereas the DCWremained fairly con-
stant to 24 h then decreased to 3.59 g/L at 72 h. In addition,
some sporulation was observed but only after 24 h so perhaps
some P(3HB) was being used for the production of the spores
(data not shown). GC analysis of the polymer produced from
this B. aryabhattai ST1Cshowed that only the 3HBmonomer
unit was detected (Online Resource S4).
Effect of the glycerol concentration in BLWon cell growth
and P(3HB) production using normal batch fermentation
The effect of the glycerol concentration in BLW on cell
growth and P(3HB) accumulation were investigated at various
concentrations of MSM containing total glycerol in the BLW
that ranged from 0.5 % (v/v) to 5.0 % (v/v). Both cell growth
11
100
93
52
73
75
47
100
40
0.005
3
10
7
8
6
5
1
2
4
9 Bacillus koreensis BR030
T
(AY667496)
Bacillus megaterium IAM 13418
T
(D16273)
Bacillus flexus strain IFO 15715
T
(AB021185)
Bacillus aryabhattai B8W22
T
(EF114313)
Bacillus pocheonensis Gsoil420
T
(AB245377)
Bacillus nealsonii FO-092
T
(AF234863)
Bacillus circulans ATCC4513 (AY724690)
Bacillus firmus IAM 12464
T
(D16268)
Bacillus oceanisediminis H2
T
(GQ292772)
Bacillus aryabhattai ST1C (JX524506)
Lactobacillus arizonensis NRRL B-14768
T
(AJ965482)
Fig. 1 Neighbour-joining phylogenetic tree constructed on the basis of
16S rRNA gene sequences showing the phylogenetic relationships be-
tween Bacillus sp. ST1C and its close relationship to Bacillus
aryabhattai B8W22
T
(GenBank accession no. EF114313). The percent-
ages of replicate trees in which the associated taxa clustered together in
the bootstrap test (1,000 replicates) are shown next to the branches
Table 1 The percentages of carbon and nitrogen elements and glycerol
content in BLW collected at different times
BLW collection Elements
a
(%) Glycerol content
in BLW (%, w/v)
b
Carbon Nitrogen
1st collection (30 Sept 2011) 43.84 0.10 43.89
2nd collection (30 Nov 2011) 43.47 0.20 42.44
Averages 43.65 0.15 43.16
a
CHNS-O analyser, CE Instruments Flash EA 1112 Series, Thermo
Quest, Italy
b
The free glycerol determination method as described by (Bondioli and
Della Bella 2005)
Fig. 2 Time profiles of cell growth, P(3HB) production and glycerol
utilization by Bacillus aryabhattai ST1C grown in MSM containing
2.0 % (v/v) total glycerol in BLW as a sole carbon source. Each data
point represents a mean value of three independent experiments and a
vertical bar represents the standard deviation
Ann Microbiol (2014) 64:11571166 1161
and P(3HB) production were expected to increase with an
increasing amount of glycerol. However, at concentrations
above 3.0 % (v/v) glycerol, the amount of the dry cell weight
was reduced with a significant reduction of PHA productivity
(Table 2).
In comparison to other work the P(3HB) content of B.
aryabhattai ST1C (57.76 % DCW) was similar to that of
Bacillus aryabhattai S4 (57.62 % DCW). However, the cell
mass of 5.68 g/L DCWand productivity of 0.137 g/Lh when
using 2.0 % (v/v) of total glycerol in BLW was significantly
higher than for B. aryabhattai S4 cultivated with 20 g/L of
total sugar in a sweet sorghumjuice that produced only 3.02 g/
L DCW with a productivity of 0.097 g/Lh (Tanamool and
Kaewkannetra 2011). This indicated that the glycerol in BLW
was a good substrate for B. aryabhattai ST1C to grow and
produce P(3HB) (Ashby et al. 2004). However, a further
increase of the total glycerol concentration in BLW to 3.0 %
(v/v) produced a reduced cell growth and P(3HB) production
(Table 2). The cell biomass and P(3HB) biosynthesis de-
creased to 2.05 g/L and 46.58 % DCW, respectively, with a
PHA productivity of only 0.029 g/Lh that was produced
when B. aryabhattai ST1C was cultivated in 5.0 % (v/v)
glycerol. A similar observation had been made with Bacillus
sonorenis SM-P-1S that grew less on plates with 5.0 % (v/v)
of a Jatropha biodiesel byproduct but showed a luxuriant
growth on plates containing 1.0 % and 2.0 % (v/v) of the
same biodiesel byproduct (Shrivastav et al. 2010) whereas in
Bacillus sphaericus NII 0838 the maximum P(3HB) yield
was at a 1.0 % (v/v) glycerol concentration (Sindhu et al.
2011). The growth of Pseudomonas oleovorans was not
affected by increasing concentrations of a co-product stream
from a soy-based biodiesel production (CSBP) but the in-
creased concentration did produce a 100 % increase in the
yield of polymer (from0.2 g/L at 1.0 %(w/v) CSBP to 0.4 g/L
at 5.0 % (w/v) CSBP). In contrast, when the CSBP
concentration was increased for Pseudomonas corrugata
from 1.0 % to 5.0 % (w/v), the cell growth decreased from
2.1 g/L to 1.7 g/L but the polymer yields stabilized at 0.7 g/L
with an increase of the initial CSBP media concentration from
2.0 % to 5.0 % (w/v) (Ashby et al. 2004).
One possible reason for the reduction of cell biomass and
P(3HB) biosynthesis when using the biodiesel liquid waste as the
main carbon source was an increase in sodiumions (a catalyst in
the de-esteraification process). From a previous report, crude
glycerol from biodiesel production was contaminated with salts,
primarily sodium, at approximately 23 % (Hansen et al. 2009).
Also, sodium was found to have a particularly adverse effect on
both the growth rate and polymer yield due to osmoregulation
(Cavalheiro et al. 2009). These cell growth and polymer yield
results have indicated that the controls on PHA synthesis vary
and are species/even strain specific.
To study the effect of NaCl on cell growth and P(3HB)
production, 0.5 % and 3.0 % of NaCl were added into MSM
containing 2.0 % (v/v) PG. The sodium content in the BLW
Table 2 Maximum cell growth, P(3HB) accumulation and P(3HB)
productivity produced from Bacillus aryabhattai ST1C at various con-
centrations of glycerol in BLW using normal batch fermentation
Glycerol
concentration
in BLW
(%, v/v)
Maximum
DCW
(g/L)
Maximum
P(3HB)
concentration
(g/L)
Maximum
P(3HB)
content
(% DCW)
Maximum
P(3HB)
productivity
(g/Lh)
0.5 2.740.15 1.250.03 48.182.00 0.052
a
1.0 3.870.10 1.860.05 51.122.50 0.078
a
2.0 5.680.20 3.280.10 57.763.50 0.137
a
3.0 4.350.08 1.940.05 52.402.00 0.081
a
4.0 3.280.10 1.180.08 47.211.00 0.033
b
5.0 2.050.20 1.050.02 46.581.25 0.029
b
a
Maximum productivity at 24 h
b
Maximum productivity at 36 h
Fig. 3 a Cell growth and b P(3HB) production by Bacillus aryabhattai
ST1C when cultivated in MSM containing 2.0 % (v/v) PG, PG adding
0.5 %and 3.0 %of NaCl and 2.0 %(v/v) total glycerol in BLW. Each data
point represents a mean value of three independent experiments and a
vertical bar represents the standard deviation
1162 Ann Microbiol (2014) 64:11571166
used in this study was determined to be only 0.5 % by ICP-
OES analysis. The growth and P(3HB) content in both the
BLW and PG with added 0.5 % NaCl were almost identical
with a slightly higher yield with the PG (Fig. 3). From this it
was inferred that there was no inhibitory effect of BLW
containing 0.5 % sodium ion on cell growth and P(3HB)
production. In contrast, MSM with PG and added 3.0 %
NaCl had significantly less growth and polymer synthesis than
the others and produced only 9.26%DCW of PHA produced
and 1.96 g/L of biomass at the end of cultivation (Fig. 3) hence
in that case 3.0 % NaCl was inhibitory.
Glycerol in some bacteria serves to function as an intracel-
lular osmolyte for balancing external osmotic pressure. It
plays important roles in physiological processes such as com-
bating osmotic stress, managing cytosolic phosphate levels,
and maintaining the NAD+/NADH redox balance. In spite of
this at high concentrations it could suppress cellular metabo-
lism, and decrease the production of PHA. Some mixed
bacterial cultures are capable of growth in glycerol concentra-
tion up to 50 % and only a further increase above 50 % started
to inhibit cell growth with no growth at glycerol concentra-
tions of 60 and 70 % (Wattanaphon and Pisutpaisal 2011).
However, this is not the normal response of bacteria.
Pseudomonas corrugate cultures grew to high cell densi-
ties in media with glycerol concentration only up to 2.0 % w/v
glycerol. This is similar to the results reported here for
Bacillus aryabhattai ST1C. It can be assumed that when the
concentration of glycerol in the medium was increased above
2.0 % v/v the organisms began to feel the effects of osmotic
stress (as evidenced by lower biomass yields) that also
b
Carbon content in 2.0% (v/v) total glycerol in BLW
Carbon content in 3.0% (v/v) total glycerol in BLW
Glycerol utilization in 2.0% (v/v) total glycerol in BLW
Glycerol utilization in 3.0% (v/v) total glycerol in BLW
PHA produced in 2.0% (v/v) total glycerol in BLW
PHA produced in 3.0% (v/v) total glycerol in BLW
a
Fig. 4 Comparison of a cell
growth, b P(3HB) production and
substrate utilization by Bacillus
aryabhattai ST1C when
cultivated in MSM containing
2.0 % (v/v) and 3.0 % (v/v) total
glycerol in BLW. Each data point
represents a mean value of three
independent experiments and a
vertical bar represents the
standard deviation
Ann Microbiol (2014) 64:11571166 1163
decreased polymer production (Ashby et al. 2005). This as-
sumption was further confirmed by determining the reduction
of the carbon content in MSM containing 3.0 % (v/v) of total
glycerol in BLW during the fermentation process. The
percentage of total carbon content in the culture medium with
3.0 % (v/v) glycerol cultures decreased from 1.98 % at the
initial time (0 h) to 1.71 %at the end of cultivation (72 h) at the
same time the biomass and P(3HB) content continuously
Fig. 5 Comparison of a cell
growth and b P(3HB) production
by Bacillus aryabhattai ST1C
when cultivated in normal batch
fermentation: MSM containing
2.0 % (v/v) total glycerol in
BLW without extra feeding
and a draw and fill fermentation
(N-limitation): MSM
supplemented with 2.0 % (v/v)
total glycerol in BLW containing
1.0 g/L ammonium sulfate or
0.2 g/L ammonium sulfate was
fed every 12 h for 72 h. Each data
point represents a mean value of
three independent experiments
and a vertical bar represents the
standard deviation
Table 3 Comparison of the maximum biomass and P(3HB) production by different fermentation methods
Fermentation method Carbon concentration (%, v/v) and nitrogen
concentration (g/L)
max.* DCW (g/L) max*. P(3HB)
content (% DCW)
max*. P(3HB)
productivity (g/Lh)
Draw and fill fermentation Feed 0.5 % (v/v) total glycerol in BLW
+ 0.2 g/L (NH
4
)
2
SO
4
7.240.40 72.314.15 0.216
Feed 2.0 % (v/v) total glycerol in BLW
+ 1.0 g/L (NH
4
)
2
SO
4
6.220.15 64.443.40 0.193
Normal batch fermentation 2.0 % (v/v) total glycerol in BLW
+ 1.0 g/L (NH
4
)
2
SO
4
5.680.20 57.763.50 0.137
max* maximum
1164 Ann Microbiol (2014) 64:11571166
decreased from the maximum of 4.35 g/L and 52.40 % DCW
at 24 h to 1.41 g/L and 31.38 % DCWat 72 h, respectively. It
is interesting that the total carbon content in this culture
medium was still in the range of 1.71.8 % after 24 h whereas
the DCW and P(3HB) production steadily declined. It would
seem that this new Bacillus sp. ST1C was not able to readily
utilize glycerol at 3.0 % (v/v), hence the accumulated P(3HB)
was being slowly utilized. The glycerol content also remained
constant in the range of 57 g/L after 24 h. In contrast at 2.0 %
(v/v) total glycerol in the BLW, the percentage of the total
carbon content of the medium decreased from 1.42 % at the
initial time to 1.05 % at 24 h and then to 0.25 % at the end of
cultivation and the glycerol content decreased from 20 g/L to
less than 0.75 g/L at 24 h when the highest DCWand P(3HB)
was achieved. The remaining glycerol was then completely
consumed and the cells started to degrade P(3HB) for cell
maintenance and survival. These results support the above
assumption that the new Bacillus sp. ST1C efficiently metab-
olized the glycerol at 2.0 % (v/v). However, the further in-
crease of glycerol up to 3.0 % (v/v) lowered the biomass and
P(3HB) content (Fig. 4). More work would be required to
identify the cause of this effect of the higher glycerol content
as it inhibits glycerol utilization, cell growth and PHA
accumulation.
This, however, did raise the question about what might
happen to the growth and P(3HB) synthesis if one started
the culture at 2.0 % v/v glycerol concentration then added
more glycerol with or without nitrogen when the initial con-
centration had significantly reduced. Other workers have
shown that it was possible to increase the amount of growth
and PHA by feeding a fresh culture medium into a typical
batch fermentation (Sabra and Abou-zeid 2008; Ibrahim and
Steinbchel 2009; Kulpreecha et al. 2009; Pandian et al. 2010;
Obruca et al. 2011). When the normal medium, i.e. 2.0 %(v/v)
total glycerol in BLW with 1.0 g/L ammonium sulfate was
added to the culture 12 h after normal culture conditions then
at 12 h intervals until 60 h, the dry cell weight at 24 h
increased from 5.68 to 6.22 g/L and the P(3HB) content from
57.76 to 64.44 % DCW, so more P(3HB) was being produced
but it was not a huge gain. However, when MSM containing
0.5 % (v/v) total glycerol in BLW and 0.2 g/L ammonium
sulfate was fed 12 h after growth in the normal mediumthen at
12 h intervals, the cell growth and P(3HB) content at 24 h
significantly increased to 7.24 g/L and 72.31 % DCW, respec-
tively, with productivity of 0.216 g/Lh (Fig. 5). From these
results it can be concluded that the glycerol in BLW and the
nitrogen concentration both affected cell growth and P(3HB)
accumulation. Therefore, addition of glycerol concentration at
0.5 % v/v and a little more nitrogen led to an increase in the
biomass and P(3HB) content. The maximum cell growth and
P(3HB) production achieved from the normal batch fermen-
tation and the draw and fill cultivation method are compared
in Table 3. Further work will be required to find the optimum
conditions for P(3HB) production in scale up conditions but a
yield of P(3HB) from utilizing glycerol of more than 70 %
DCW in a 24 h period is probably worth further experimen-
tation. In the normal batch cultivation at 72 h the dry cell
weight reduced to 3.85 g/L and the PHA to 25.73 %, whereas
in the drawand fill cultivation method with 0.5 % v/v glycerol
and 0.2 g/L ammonium sulfate the dry cell weight and PHA
content decreased to 5.89 g/L and 53.71 % DCW, respective-
ly. For maximum production of cells and P(3HB) cultivation a
time somewhere between 24 and 36 h is probably optimum.
In conclusion, a newly isolated Bacillus aryabhattai ST1C
showed a high efficiency for production of P(3HB) from
glycerol present in BLW, a waste product from biodiesel
production. However, glycerol accounted for only 43 % of
the total carbon in the waste and there was little evidence that
the remaining carbon was being utilized (Figs. 4 and 5) for
PHAproduction or growth. So, although the BLWis a suitable
cheap and effective source of glycerol for PHA production,
there would still be the problem of what to do with the
remaining carbon. It would be of interest to determine if the
remaining carbon could be utilized by other bacteria even
perhaps to supplement the production of PHA.
Acknowledgments We would like to acknowledge gratefully the Spe-
cialized R & D Center for Alternative Energy from Palm Oil and Oil
Crops, Faculty of Engineering, Prince of Songkla University for the
supply of their biodiesel liquid waste. This research was financially
supported by Prince of Songkla University (SCI560117S) and the Devel-
opment and Promotion of Science and Technology Talents Project
(DPST) Scholarship, Thailand.
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