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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
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52
73
75
47
100
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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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