Production Of Pha (Poly Hydroxy Alkanoates) by Ralstonia Eutropha on Hydrolysed Sago Starch as Main Substrate using Fed-Batch Cultivarion Method
Proceedings of the 1" Illternational Conference 011 Natural Resources Engineering & Technology 2006
24-25'" Jul)' 2006; Putrajaya, Malaysia, 153-157
Production of Pha (Poly Hydroxy Alkanoates) by Ralstonia Eutropha on
Hydrolysed Sago Starch as Main Substrate using Fed-Batch Cultivation
Method
Khaswar Syamsu*, Anas M_ Fauzi, Liesbetini Hartoto, Ani Suryani, Nur Atifah
Dept of Agroilldllstrial Technology alld Research Center for JJioresollrces and Biotechnology. Bogor Agriciiltura1 Univer,S
(J
25
tl,OO
20
3,00
15
2,00
ｾ＠
c
---
1.00
co
C"I
::l
1/)'
- - . . - Cell growth
G>
(J
C"I
"-
c
8
-:;-
10
Substrate uptake
iU
::l
"'0
5
'iii
0
0::
G>
0,00
o
12
24
48
36
60
72
84
96
Time (hour)
Fig 1. Growth and subsrrate consumption of R. eutropha in batch cultivation with the initial
hydrolysed sago starch concentration of25 giL, and elN ratio of 10: 1.
3.2.
Fed-Batch Cultivation
Based on the previous research results in batch cultivation, feedings of substrates andlor
limiting the aeration were started from 48th hour. The hypotheses to be tested whether
feeding of carbon source andlor limiting other essential nutrients (nitrogen, oxygen,
phosphate) at stationary phase can induce formation and accumulation ofPHA.
The results (Table 1.) show that, in all treatments, feeding of substrates andlor limiting the
aeration can induce the formation and accumulation of PHA as indicated by the increasing
specific yield of PHA and PHA conl.;entr31.icn. These treatments may have caused
unbalanced growth in which the carbon source was in excess while other essential nutrients
(nitrcgcn, phosphat, oxygen) wcre limited. Compared to control (batch cultivation). feeding
of hydrolysed sago starch only (F2) gave the best results, namely increasil1g the PHA
formation as indicated by the increasing of specific product yield from 32.65% to 76.54%
(increasing of more than two folds), and increasing the product accumulation as indicated by
the increasing of product concentration at the end of fed batch from 1.44 giL to ;'.72 giL
(increasing of more than two folds). In addition, biomass was only slightly increased from
4,4 I giL to 4.86 giL (increasing about 10%). Comparison between all treatments for biomass,
product and specific product yield is summarized in Table !.
155
Proceedings of the
II
t" Illtemational Conferellce all Nailiral Resources Engineering & Technology 2006
24_25 Ih luly 2006; PU/r(u"aya, Malay:;ia, 153-157
Table I
Biomass, Product (PHA) and Specific Product Yield of PH A produced by R. eutropha in
batch and fed batch cultivation with and without limiting essenstial nutrients.
Treatments
Batch (control)
. BatchAo
Fed batch
Fl
I F2
F3
F4
F5
F6
I F7
Biomass (g I Product (g dry
PHAlL)
.drr ceil/L1 I
4,41 ± 0,83 ' 1,44±0,27
I Specific Product Yield
I
{% w/wl
' 32.65 ± 6.56
3,40 ± 0,39
1,81 ± 0,57
51,15 ± 12.32
3,34 ± 0,11
4.86 ± 0,14
4,26 ± 1,45
3,67 ± 0,28
3,65 ± 0,10
4,58 ± 0,24
3,40 ± 0,01
2,15 ± 0,03
3,72 ± 0,24
2,68 ± 0,67
2,12 ± 0,05
2,18 ｾ＠ 0,01
1.85 ± 0.06
1,62 ± 0,09
64.37 ± 2.30
76,54 ± 5.41
63.36 ± 2.29
157.77 ± 4.61
59,73
1.66
40.39 ± 2.49
47,65 ± 2.65
I
II
I
i
i
I
Notes:
(Ao) Limitation of oxygen supply at stationary phase, without substrate feeding;
(Fl) Feeding of complete media with the same composition as batch media, with aeration;
(F2) Feeding of hydrolsed sago starch, with aeration;
(F3) Feeding of hydrolysed sago starch without aeration;
(F4) Feeding of hydrolysed sago starch +MgS04.7H20, with aeration;
(F5) Feeding of hydrolysed sago starch+MgS04.7H20 without aeration;
(F6) Feeding of hydrolysed sago starch+ MgS04.7H20+(NH4hHP04 with aeration;
(F7)Feeding of hydrolysed sago starch+ MgS04. 7H20+(NH4hHP04 wiihout aeration.
The results show that the more complete the media is fed (F6 and P7), the lower the PHA is
produced. On the contrary, the more limited the media is fed (F2 and F3) which was basicaUy
only hydrolysed sago starch as carbon source, the more the PHA is produced Different from
the limitation of nitrogen, phosphate, and sulphur, however, limitation of oxygen (Ao, F3, F5,
F7) may have a negative impact to the production of cell and hence the PHA as a product.
Tanaka et al. (1993) who used the two stage cultivation method involving LactDcoccus
lactis 10-1 and Alcaligenes eutrophus to produce PHB using xylose as main substrate
produced biomass of 8.5 giL and PHB at the specific yield of 55% [6]. The specific product
yield in this current research (76.54%) is higher than that of1 anaka et al (1993). However, it
may be due to the biomass produced in this current research (4.86 giL) is lower than that of
Tanaka et al (1993).
Kim et al. (1994) produced PHB by R. eutropha using automatic fed batch technique with
amonium limitation [7]. Glucose concentration was controlled at the range of 10-20 gIL. The
maximum specific product yield obtained was also 76% with the productivity of 2.42 g/L.h.
Compared to the results of Kim et al. (1994), specific product yield in this current research
(76.54%) was nearly the same. Further, Ryu et al (1997) was able to obtain the specific
product yield of up to 80% by using high density fedbatch of Alcaligenes eutrophus in a 60 L
bioreactor [8].
156
"
Proceedings of the 1" Infernaljollal Conference 011 Nalural Resources Engineering & Technology 2006
24-25'1> July 2006; PUlrajaya. Malaysia, 153·157
4.0
Conclusions
These results have proven that hydrolysed sago starch can also be used as carbon source to
produce PHA by Ralstonia eutropha. A higher formation and accumulation of PHA can be
achieved by feeding of an excess carbon source '.'.-hi!.:: limiting other essential nutritions
through fed batch cultivation method. Fedbatch cultivation strategy can increase both
product concentration and specific product yield.
Acknowledgement
This research is financially supported by the Indonesiu.H Ministrj for Research and
Technology, Indonesia through Integreted alld Strategic Research Grant. Funding from
Project B, Department of Agroindustria! Technology, IPB for adively partipatlog in an
international conference is apreciateo.
References
[I] Van Wegen, RJ., Y. Ling, and A.P.J. Middleberg. 1998. Industria! production of polyhydroxyalkanoates
using Eschericia coli: an economic analysis. Trans fchemE, val 76, part A., pp. 417-426.
[2] Lee, S.Y., J. Choi, K. Han dan J.Y. Song. 1999. Removal of endotoxin during purification of poly(3hydroxybutyrate) from gram-negative bacteria. Applied and Environmental Microbiology 65(6):2762-2764
(3] Brandl, H., R.A. Gross, R.W. Lenz Jan R.C. Fuller. 2001. Plastics from bacteria and for bacteria: poly(Bhydroxyalkanoates) as natural, lJiC'compatible, and biodegradahle polyesters. In : Babel, W. dan A.
Steinbiichel.(Eds). Biopolyester : Advances in Biochemical Engineering! Biotechnology. Vol 71. SpringerVerlag Berlin Heidelberg.
[4] Klem, 1.K. 1999. Alcaligenes. In: R.K. Robinson, C.A. Batt, P.P. Patel (Eds). Encyclopedia 0/ Food
Microbiology. Vol 1. 2000. Academic Press, London, UK.
(5] Hahn, S.K., Y.K. Chang dan S.Y. Lee. 1995. Recovery and characterizatian of poly (3-hydroxybutyric acid)
synthesized in AlcaligeneE eutrophus and recomhinant Escherichia coli. Applied alld &;virOl1l1lpntal
Microbiology 61(1 ):34-39.
[G] Tanaka, K., K.Katamune dan A. Ishizaki. 1993. l'ermentati':e production of poly-B-hydroxybutyric acid
from xylose by a two-stage culture method employing Lactocuccus lactis 10-1 and A!caligenes eutrophus.
Biotechnology Letters 15(12): 1217-1222.
[7] Kim B.S., S.c. Lee, S.Y Lee, H.N. Chang, Y.K. Chang dan S.l. Woo. 1994. Production of poly(3Hydroxybutyric acid) by fed-batch culture of Alcaligenes elltroplws with glucose concentration control.
Biotechnology Bioengineering. 43:892-898.
[8] Ryu, H.W., S.K. Hahn, YK. Chang dan H.N. Chang. 1997. Production of poIy{3.hydroxybutyrate) by hieh
cell density fed-batch culture of Alcaligenes eutrophus with phosphate limitation. Biotechnology
Bioengineering 55 :28-32.
157
24-25'" Jul)' 2006; Putrajaya, Malaysia, 153-157
Production of Pha (Poly Hydroxy Alkanoates) by Ralstonia Eutropha on
Hydrolysed Sago Starch as Main Substrate using Fed-Batch Cultivation
Method
Khaswar Syamsu*, Anas M_ Fauzi, Liesbetini Hartoto, Ani Suryani, Nur Atifah
Dept of Agroilldllstrial Technology alld Research Center for JJioresollrces and Biotechnology. Bogor Agriciiltura1 Univer,S
(J
25
tl,OO
20
3,00
15
2,00
ｾ＠
c
---
1.00
co
C"I
::l
1/)'
- - . . - Cell growth
G>
(J
C"I
"-
c
8
-:;-
10
Substrate uptake
iU
::l
"'0
5
'iii
0
0::
G>
0,00
o
12
24
48
36
60
72
84
96
Time (hour)
Fig 1. Growth and subsrrate consumption of R. eutropha in batch cultivation with the initial
hydrolysed sago starch concentration of25 giL, and elN ratio of 10: 1.
3.2.
Fed-Batch Cultivation
Based on the previous research results in batch cultivation, feedings of substrates andlor
limiting the aeration were started from 48th hour. The hypotheses to be tested whether
feeding of carbon source andlor limiting other essential nutrients (nitrogen, oxygen,
phosphate) at stationary phase can induce formation and accumulation ofPHA.
The results (Table 1.) show that, in all treatments, feeding of substrates andlor limiting the
aeration can induce the formation and accumulation of PHA as indicated by the increasing
specific yield of PHA and PHA conl.;entr31.icn. These treatments may have caused
unbalanced growth in which the carbon source was in excess while other essential nutrients
(nitrcgcn, phosphat, oxygen) wcre limited. Compared to control (batch cultivation). feeding
of hydrolysed sago starch only (F2) gave the best results, namely increasil1g the PHA
formation as indicated by the increasing of specific product yield from 32.65% to 76.54%
(increasing of more than two folds), and increasing the product accumulation as indicated by
the increasing of product concentration at the end of fed batch from 1.44 giL to ;'.72 giL
(increasing of more than two folds). In addition, biomass was only slightly increased from
4,4 I giL to 4.86 giL (increasing about 10%). Comparison between all treatments for biomass,
product and specific product yield is summarized in Table !.
155
Proceedings of the
II
t" Illtemational Conferellce all Nailiral Resources Engineering & Technology 2006
24_25 Ih luly 2006; PU/r(u"aya, Malay:;ia, 153-157
Table I
Biomass, Product (PHA) and Specific Product Yield of PH A produced by R. eutropha in
batch and fed batch cultivation with and without limiting essenstial nutrients.
Treatments
Batch (control)
. BatchAo
Fed batch
Fl
I F2
F3
F4
F5
F6
I F7
Biomass (g I Product (g dry
PHAlL)
.drr ceil/L1 I
4,41 ± 0,83 ' 1,44±0,27
I Specific Product Yield
I
{% w/wl
' 32.65 ± 6.56
3,40 ± 0,39
1,81 ± 0,57
51,15 ± 12.32
3,34 ± 0,11
4.86 ± 0,14
4,26 ± 1,45
3,67 ± 0,28
3,65 ± 0,10
4,58 ± 0,24
3,40 ± 0,01
2,15 ± 0,03
3,72 ± 0,24
2,68 ± 0,67
2,12 ± 0,05
2,18 ｾ＠ 0,01
1.85 ± 0.06
1,62 ± 0,09
64.37 ± 2.30
76,54 ± 5.41
63.36 ± 2.29
157.77 ± 4.61
59,73
1.66
40.39 ± 2.49
47,65 ± 2.65
I
II
I
i
i
I
Notes:
(Ao) Limitation of oxygen supply at stationary phase, without substrate feeding;
(Fl) Feeding of complete media with the same composition as batch media, with aeration;
(F2) Feeding of hydrolsed sago starch, with aeration;
(F3) Feeding of hydrolysed sago starch without aeration;
(F4) Feeding of hydrolysed sago starch +MgS04.7H20, with aeration;
(F5) Feeding of hydrolysed sago starch+MgS04.7H20 without aeration;
(F6) Feeding of hydrolysed sago starch+ MgS04.7H20+(NH4hHP04 with aeration;
(F7)Feeding of hydrolysed sago starch+ MgS04. 7H20+(NH4hHP04 wiihout aeration.
The results show that the more complete the media is fed (F6 and P7), the lower the PHA is
produced. On the contrary, the more limited the media is fed (F2 and F3) which was basicaUy
only hydrolysed sago starch as carbon source, the more the PHA is produced Different from
the limitation of nitrogen, phosphate, and sulphur, however, limitation of oxygen (Ao, F3, F5,
F7) may have a negative impact to the production of cell and hence the PHA as a product.
Tanaka et al. (1993) who used the two stage cultivation method involving LactDcoccus
lactis 10-1 and Alcaligenes eutrophus to produce PHB using xylose as main substrate
produced biomass of 8.5 giL and PHB at the specific yield of 55% [6]. The specific product
yield in this current research (76.54%) is higher than that of1 anaka et al (1993). However, it
may be due to the biomass produced in this current research (4.86 giL) is lower than that of
Tanaka et al (1993).
Kim et al. (1994) produced PHB by R. eutropha using automatic fed batch technique with
amonium limitation [7]. Glucose concentration was controlled at the range of 10-20 gIL. The
maximum specific product yield obtained was also 76% with the productivity of 2.42 g/L.h.
Compared to the results of Kim et al. (1994), specific product yield in this current research
(76.54%) was nearly the same. Further, Ryu et al (1997) was able to obtain the specific
product yield of up to 80% by using high density fedbatch of Alcaligenes eutrophus in a 60 L
bioreactor [8].
156
"
Proceedings of the 1" Infernaljollal Conference 011 Nalural Resources Engineering & Technology 2006
24-25'1> July 2006; PUlrajaya. Malaysia, 153·157
4.0
Conclusions
These results have proven that hydrolysed sago starch can also be used as carbon source to
produce PHA by Ralstonia eutropha. A higher formation and accumulation of PHA can be
achieved by feeding of an excess carbon source '.'.-hi!.:: limiting other essential nutritions
through fed batch cultivation method. Fedbatch cultivation strategy can increase both
product concentration and specific product yield.
Acknowledgement
This research is financially supported by the Indonesiu.H Ministrj for Research and
Technology, Indonesia through Integreted alld Strategic Research Grant. Funding from
Project B, Department of Agroindustria! Technology, IPB for adively partipatlog in an
international conference is apreciateo.
References
[I] Van Wegen, RJ., Y. Ling, and A.P.J. Middleberg. 1998. Industria! production of polyhydroxyalkanoates
using Eschericia coli: an economic analysis. Trans fchemE, val 76, part A., pp. 417-426.
[2] Lee, S.Y., J. Choi, K. Han dan J.Y. Song. 1999. Removal of endotoxin during purification of poly(3hydroxybutyrate) from gram-negative bacteria. Applied and Environmental Microbiology 65(6):2762-2764
(3] Brandl, H., R.A. Gross, R.W. Lenz Jan R.C. Fuller. 2001. Plastics from bacteria and for bacteria: poly(Bhydroxyalkanoates) as natural, lJiC'compatible, and biodegradahle polyesters. In : Babel, W. dan A.
Steinbiichel.(Eds). Biopolyester : Advances in Biochemical Engineering! Biotechnology. Vol 71. SpringerVerlag Berlin Heidelberg.
[4] Klem, 1.K. 1999. Alcaligenes. In: R.K. Robinson, C.A. Batt, P.P. Patel (Eds). Encyclopedia 0/ Food
Microbiology. Vol 1. 2000. Academic Press, London, UK.
(5] Hahn, S.K., Y.K. Chang dan S.Y. Lee. 1995. Recovery and characterizatian of poly (3-hydroxybutyric acid)
synthesized in AlcaligeneE eutrophus and recomhinant Escherichia coli. Applied alld &;virOl1l1lpntal
Microbiology 61(1 ):34-39.
[G] Tanaka, K., K.Katamune dan A. Ishizaki. 1993. l'ermentati':e production of poly-B-hydroxybutyric acid
from xylose by a two-stage culture method employing Lactocuccus lactis 10-1 and A!caligenes eutrophus.
Biotechnology Letters 15(12): 1217-1222.
[7] Kim B.S., S.c. Lee, S.Y Lee, H.N. Chang, Y.K. Chang dan S.l. Woo. 1994. Production of poly(3Hydroxybutyric acid) by fed-batch culture of Alcaligenes elltroplws with glucose concentration control.
Biotechnology Bioengineering. 43:892-898.
[8] Ryu, H.W., S.K. Hahn, YK. Chang dan H.N. Chang. 1997. Production of poIy{3.hydroxybutyrate) by hieh
cell density fed-batch culture of Alcaligenes eutrophus with phosphate limitation. Biotechnology
Bioengineering 55 :28-32.
157