A Complete Analysis On Pha Production And Their Rates Usingsunflower Oil And Mixed Cultures: Hydraulic Retention Times (Hrt) Effects

Jurnal Sains Kimia (Suplemen)
Vol 9, No.3, 2005: 31-41

A COMPLETE ANALYSIS ON PHA PRODUCTION AND THEIR
RATES USINGSUNFLOWER OIL AND MIXED CULTURES:
HYDRAULIC RETENTION TIMES (HRT) EFFECTS
Salim M R Md_Din, M. F1., Yunus, S.,1 Ujang, Z.1, Salim M. R1., van Loosdrecht, M.C.M2,
Ahmad, A.3, Marpongahtun 4.
1

IPASA, Faculty of Civil Engineering, University Technology of Malaysia, 81310 Skudai Johor, Malaysia.
Delft University of Technology, Kluyver Laboratory for Biotechnology, Department of Biochemical Engineering,
Julianalaan 67, NL-2628 BC Delft, The Netherlands.
3
Microbiology Department, Faculty of Science, University Technology of Malaysia, 81310 Skudai Johor, Malaysia.
4
Dept. of Chemistry, Faculty of Mathematic and Sciences, University North Sumatera, 20155 Medan, Indonesia
2

Abstract
Abilities of four HRT/SRT experiments were conducted to assess the optimal conditions for PHA

production using commercial fatty acid; sunflower oil (SO). Since the systems were conducted in a fast
regime (uptake rate), no idle or settling phase is adapted resulting HRT ≈ SRT (HRT/SRT). First, the
effect of carbon source was investigated. The system was dependent on microbial activity and operating
conditions, particularly HRT/SRT, COD/N, growth and accumulation conditions. The target was to
maintan the bacterial growth, so the capability of storage polymer (polyhydroxyalkanoates, PHAs) will
significant in the survival period (limiting condition of nutrient). The PHA constituents such as HV and
HH were also examined in order to compare the polyester production in commercial oil compositions.
The typical composition of SO mainly contains long-chain-fatty-acid (LCFA) under unsaturated fatty acid
(C14:1 – C18:3) and therefore, the bacterial not much utilize it. Here also, the highest PHA productions can
reach up to 33.77% of dry weight because the capability of storage mechanisms. Substrate uptake rates
were found not to be proportional to the cell growth, suggesting that only a small fraction of the cell
biomass was responsible for the main part of the substrate uptake. Based on these experiments, new
fabrication of experiment will usefulness for the development of renewable carbon sources (especially
from oil manufacturing) and sufficient for PHA production.
Key words: sunflower oil (SO), polyhydroxybutyrate (PHB), feast-famine regimes, mixed cultures, fatty
acids component.

INTRODUCTION
Mixed culture or co-culture systems
have been recognized to be important for

several fermentation processes. There are
several researchers claimed the integrity and
effectiveness system using mixed culture.
Unfortunately, they only emphasize the mixed
culture using two or three well-known bacterial.
The mixed culture using unknown bacterial
consortium is not quite established previous.
The idea of PHA production using mixed
culture was ignited owing to PHA role as an
metabolic intermediate of wastewater treatment
and as a biodegradable plastic. Activated sludge
(from organic waste) as a well known mixed
culture process is able to store PHA as carbon
and energy storage material under transition

32

conditions due to discontinuous feeding regime
and variation in electron acceptor presence.
Microorganisms which are able to quickly store

and consume substrate in a more balanced way
have a strong competitive advantage over
organisms without capacity of substrate storage
(Md Din et al., 2004a; van Loosdrecht et al.,
1997).
Oleochemical-based (SOs) derived
from agricultural products have a number of
potential advantages when compared to their
mineral counterparts. They always possess
lower
toxicity,
relatively
higher
biodegradability and are renewable while the
availability of petroleum chemical is finite. On
the other hand, SO composition consists of
similar fatty acids components as POME one.
Unfortunately, the characteristics of fatty acids

A Complete analysis on PHA production

(Salim M R)
are typically different from each other. Palm oil
contains more monounsaturated (40%) than
polyunsaturated (10%) and saturated (50%)
component. For comparison, SO contributes
more polyunsaturated (77%) compared to
monounsaturated (13%) and saturated (10%)
fatty acids. Among the fatty acids, oleic acid is
particularly stable to thermal-oxidation, due to
the presence of only one unsaturation in its
structure. Typically, SO compositions are
constituted up to 24% of oleic acid, while
linoleic acid is present up to 64.9%. Thus, this
vegetable oil (SO) is a very interesting substrate
for the synthesis of esters, which possess
application in pre-determined PHB production
under highly substrate compositions.
Depending on the substrate given the
organisms can include a wide variety of 3hydroxy fatty acids in PHA. Since the first
finding of PHB by Lemoigne et al. (1926), more

than 100 different monomer units have been
identified as constituents of PHA in above 300
different
microorganisms
(Lee,
1996b)
including 3-hydroxyalkanotes of 3-12 carbon
atoms with large variety of R-pendant groups,
4-hydroxyalkanoates of 4-8 carbon atoms, 5hydroxypentanoates, 5-hydroxyhexanoate and
6-hydroxydodecanoate. However, only a few of
these PHAs have been produced in amounts
sufficient to enable the characterization of their
material properties and to develop potential
applications. PHB has been known to be useful
biodegradable polymer which can be used as a
thermoplastic (Byrom, 1987; Holmes., 1985;
Doi, 1990). PHB is also accumulates by
numerous microorganisms and is the bestcharacterized PHA (Lee, 1996; Steinbüchel and
Füchtenbusch, 1998).A major problem in
commercialization of PHAs as substitutes for

conventional petrochemical-based polymers is
the high production cost of these compounds
(Byrom, 1987; Choi and Lee, 1999).
Many carbon sources, in particular
organic acid can be utilized by several bacterial
(even consortium) and theoretical approach for
the biosynthesis of PHB from various carbon
sources has been proposed (Yamane, 1993). The
importance of bacterial storage polymers in
general and poly-β-hydroxybutyrate (PHB) in
particular for carbon substrate conversion in
activated sludge processes is well recognized
(Chiesa et al., 1985; Kohno et al., 1991; van
Loosdrecht et al., 1997; Krishna and van
Loosdrecht, 1999). The presence of storage
compounds such as PHB and glycogen in
activated sludge bacteria and mostly in pure
culture has been extensively repeated (Doi et
al., 1990; Pagni et al., 1992; Beccari et al.,

1998). There are three kinds of polymer store
intracellular, which is, glycogen, PHA and
polyphosphate. However, only glycogen and
PHA are the main reported bacterial storage
polymers (van Loosdrecht et al., 1997). In this
study, we predominantly assume that only PHA
(generally refers to PHB as the main copolymer
storage) occurs in all of the storage phases. PHB
has been reported from several researchers to be
the more common storage polymer under
conditions of carbon sources excess (van den
Eynde et al., 1984; Smolders et al., 1995).
In the present study, we considered
such a mixed culture system, where SO as
volatile fatty acids from oil manufacturing were
converted to medium-chain-carbon (up to shortchain-carbon)
by
significant
bacterial
consortium and then stored as PHAs (or

PHB/HV) in one fed-batch system. Very little
attention has been made in the past on the
combining the typical high concentration of
fatty acid and mixed cultures. The goal of this
study, we try to investigate and stimulate the
strategy to enhance the productivity of PHA
production. Without depending any co-pure
bacterial the system was developed according to
competence
and
dominant
bacterial
accumulation.
To achieve a high productivity of a
desired bioproduct production, fed-batch
cultures are usually carried out with the control
of the nutrient feeding by monitoring dissolved
oxygen (DO), pH or carbon sources (Ryu et al.,
1997) as a feedback parameter. In our attention,
we tried to evaluate the nitrogen (as a primary

nutrient
limitation)
to
determine
the
productivity of PHB accumulation. In this case,
however, high-cell-density fermentation was
impossible due to the significant cell lysis
caused by the toxicity of NaOH solution,
excessively added to control pH. Furthermore,
more unsaturated fatty acid has been used and
the bacterial cell unable to degrade (or even
store) them easily. Until now, the potentially of
PHB production using mixed cultures (oily
material as substrate) currently was not quite
beneficial but interesting to investigate more.
MATERIAL AND METHODS
Experiment operation
The experiments were performed in a
double-jacketed laboratory fermentor with

working volume of 2 l. The fermentor was
equipped with pH and O2 electrode. The
composition of SO was evaluated and typically
the compounds consists more LCFA than
SCFA, as shown in Table 1. A mixed culture of

33

Jurnal Sains Kimia (Suplemen)
Vol 9, No.3, 2005: 31-41
heterotrophic
organisms
from
sewage
wastewater was used as inoculums. For
determination of the steady-state system, some
parameters were used; as representative to the
confirmation conditions likes TOC, cell dried
weight (CDW) and O2 profiles. Those
observations should be in constant value and

indicated that the organisms are capable
(metabolism activity) in the dynamic system.
During steady state, in one cycle a much higher
NH4+ (non-limiting nutrients) were added than
during normal operation. The cycle was
modified to ensure the production of growth till
up to 48 hour per cycle. The pH was maintained

at 7.00 ± 0.1 using 2N HCl or 2N NaOH. The
temperature was controlled almost at 30oC by
using a water-jacketed and a thermostat bath. In
steady state the process was extensively
monitored (pH, DO) and sampel (COD, TOC,
NH4+, PO42-, PHB/HV, CDW and ash content).
The well-aerated reactors were operated with
airflow of 2.39 l/min controlled by a mass-flow
controller and stirred with two standard
geometry six-blade turbines. Almost of the
process are conducted in turbulence regime to
ensure the well mass transfer and mixtures of oil
content using 1000 rpm approximately.

Table 1: Fatty acid composition (wt%) of sunflower oil (SO)
Systematic name
Trivial name
Hexadecanoic acid
Palmitic acid
Octadecanoic acid
Stearic acid
cis-9-Octadecenoic acid
Oleic acid
cis-9, cis-12Linoleic acid
Octadecadienoic acid
Cis-9, cis-12, cis-15Linolenic acid
Octadecatrienoienoic acid
Others (14:0, 20:0, 20:1ω5,
22:0, 24:0
Inoculation, medium and fed-batch technique
The working volume for this
cultivation is 2 l and the same batch reactor also
been used for others experimental work. The
proportion ratio for inoculums is 1:9 for POME
and distilled water respectively. The mixed
cultures were first grown in the same batch of
SBR that mentioned above and cultivated it for
approximately 36 hr at 30oC. A portion of the
preculture medium was transferred to the
bioreactor at 10% of the working volume after
the cells had reached the late exponential stage.
The seed of cultures for fermentation were
prepared for at least 24 hours in 2 l flasks each
containing 1.1 l seed of inoculums, 0.8 l
(solution A) of mineral medium and 0.1 l
sunflower oil as carbon sources.
Regarding the PHA harvesting and the
economics of PHA production, bacterial
biomass should be maintained at a high
concentration during the peak production of

Omega name
16:0
18:0
18:1ω9

Sunflower oil
5.7
4.1
23.5

18:2ω6

64.9

18:3ω3

0.2
1.5

PHA (during growth phase) in order to
maximize its productivity. However, there were
large decreases in the total biomass during the
nutrient limiting conditions especially when the
system is applied to SO cultivation. It is not
surprising to observe the rate of decline of the
biomass during the nutrient limitation phase
because N & P (nitrogen and phosphorus) are
essential nutrients required for cellular growth
by all living organisms.
In order to measure the SO
concentration in culture broth, 2 ml of the
culture broth was adopted to a screw tube and
then mixed with 5 ml hexane. After vigorous
shaken for 1 min, 1 ml of hexane layer was
transferred to pre-weighted tube, and then dried
at 37oC until the hexane phase evaporated. The
SO concentration was estimated as the amount
of extract by hexane according to the
predetermined calibration curve

Table 2: Nutrient adaptation for fed-batch control system during growth condition
Micronutrient*
FeCl3.6H2O
H3BO3
CuSO4.5H2O
MnCl2.4H2O

34

Concentration
(g/l)**
1.50
0.15
0.03
0.12

Macronutrient*
NH4Cl
KH2PO4
MgSO4.7H2O

Concentration
(g/l)
15.5
7.59
0.2

A Complete analysis on PHA production
(Salim M R)
Micronutrient*

Concentration
(g/l)**
0.06
0.12
0.15
10.0
0.18

Macronutrient*

Concentration
(g/l)

Na2MoO4.2H2O
ZnSO4.7H2O
CoCl2.6H2O
EDTA
KI
* Applied during growth condition
** Based on Visniac and Santer solution

Air

DO sensor
Stirrer

pH sensor
Mineral
Discharge
Carbon feed

1N, HCl
1N, NaOH
To
waterbath

Blade
turbines

Working Volume,
2l
Discharge Level,
11ll

From
waterbath
Figure 1: Diagram for SBR system (with feast and famine regime)
Analytical Procedures
As the sole carbon source, the fatty
acids of SO contained various LCFAs. About
0.15 g cells (in dry weight) were re-suspended
in 100 ml mineral solution and cultivated in a
rotary shaker at 200 rpm and 30oC for almost 24
hours. SO was added at the pre-determined
concentrations to examine their toxic effect on
cell growth and PHA synthesis. Batch
fermentation was further carried out in a 2 l
bioreactor for the time courses. About 3.4 g cell
mass harvested from the nutrient-rich culture
was re-suspended in 1 liter mineral solutions
that contained LCFA. In that case, the initial
concentrations of LCFA were controlled at 1.5 –
2.0 g/l. DO concentration was maintained at
higher than 20% of air saturation by aeration
and agitation.
The
dissolved
oxygen
(DO)
concentration in the reactor was measured
online with a DO- electrode as percentage of air
saturation. Sampels taken from the reactor for
analysis of acetate, NH4-N, PO4-P, TOC and

COD and volatile fatty acids (VFAs) were
immediately centrifuged and filtered using 0.45
μm filters to separate the bacterial cells from the
liquid. The centrifugation was performed using
Sorval RC-5B for 15 minute at 9000 rpm at 4oC.
The carbon concentration in the supernatant was
measured by gas chromatography, while NH4-N
and PO4-P concentration in the supernatant were
measured at 630 nm and 520 nm respectively
with auto analyzers (Skalar 5010). The
supernatant of VFAs were measured with GC
according to the type of carbon chain. The
quantification of CDW was performed using the
volatile suspended solids (VSS) technique
according to the Dutch Standard. Culture
(10mL) was centrifuge under high rotation
(rpm) to ensure the separation between pellet
and supernatant. The ash content of the biomass
was determined according to Dutch Standard
Method (NEN6621) (NNI. NEN) oxygen
content of the biomass was obtained by
subtracting the percentages of C, H, N, S and
ash from 100%. Sampel taken from the reactor
for the PHB determination were added to 10 ml

35

Jurnal Sains Kimia (Suplemen)
Vol 9, No.3, 2005: 31-41
tubes containing 2 drops of formaldehyde in
order to stop all biological activity immediately.
The PHB content of the washed and dried
biomass was determined by extraction,
hydrolyzation, and esterification in a mixture of
hydrochloric
acid,
1-propanol,
and
dichloroethane at 100oC. The resulting organic
phase was extracted with water to remove any
free acids. The proplyesters were analyzed by
GC. Benzoic acid was used as an internal
standard throughout the procedure. The PHA
content (or PHB content) was given as a
percentage of the total biomass dry weight (%
PHA or%PHB).

Results and Discussion
In our approach, the medium of SO
was re-concentrated based on the solubility data
from mixtures of Triton-X 100 solution. Then,
we calibrated the reasonable concentration
using different dilution factors. It was found that
SO solution could only be concentrated up to

120 ml/l by pre-treatment of Triton-X 100. The
feeding solution containing 120 ml/l was
sufficient to allow high density of cells and
relatively high concentration of PHB. Regarding
this, we preferred a good enrichment of cultures
when the SO solution was acclimatized for a
long period.
The initial DO was maintained up to
30% of air saturation. When the CDW reached
20 g/l, the DO was lowered to only 4%. Even
though a higher cell concentration of 25 g/l was
achieved without removing culture broth by
employing favourable SO concentration, the
final PHA content was still lower than 40%.
The PHB content is a very important factor,
which contributes significantly to the cost of
production of PHA in large scale cultivation.
Therefore, we examined the effect of the DO
during the fed-batch culture to achieve high
PHA content and cell concentration at the same
time. It was considered more significant than
cycle length study.

Table 3: Experiments the selected cultivation of fed-batch system under different cycle lengths
Experiment code
HRT/SRT
Oxygen saturation
Feast/Famine
Temperature
(h)
(%)
(h)
(oC)
53.3
20/4
HRTso – 53.3
80.0
24/12
HRTso – 80.0
4 – 35
30
106.7
27/21
HRTso – 106.7
213.3
25/71
HRTso – 213.3
somewhat higher by increasing the cultivation
Table 4 shows the suggested strategy
periods. However, the degradation rates (on PHA production. Yet, a great accumulation
kfPHAfamine) were almost the same in a range of
of PHB appeared which reincreased for at least
0.2 – 0.6 x 10-3 h-1. The overall observed
more than 24 hours. It showed that a sufficient
biomass yield was slightly higher in the normal
time during the active growth phase was
cycles (i.e. 24 hours). The biomass growth rate
important to achieve high final cell and PHB
in the feast period relative to the overall
concentrations. The behaviour of the
biomass growth rate was lower in the normal
microorganism in the cycles with high SO
cycles compared to the prolonged cycles with
dosage was comparable to the behaviour during
long adaptation periods. It must be noticed that
the normal steady state cycles (data not shown).
error in the determination of biomass growth in
The specific production rates during feast and
were
the feast period was relatively large.
famine
conditions
(qpfeast/-qpfamine)
Table 4: Specific observations based on kinetic rates under feast and famine condition
qpfeast/C-mol/C-mol. h
Time Degradation =
-3
qpfamine
x 10
kfPHAfeast/Experiments
cycle
-kfPHAfamine
feast
famine
C-mol/CkfPHAfamine
qp
-qp
(h)
x 10-2 (h-1)
mol
HRTso-53.3
24
0.612
24.408
14.678
1.663
0.211
HRTso-80.0
36
0.209
8.342
6.220
1.341
0.500
48
0.213
20.624
10.611
1.944
0.751
HRTso-106.7
96
0.505
21.551
9.783
2.203
0.283
HRTso-213.3
Data from a single aerobic cycle in the
aerobic SBR (the cycle length experiment) was
performed three times (Table 5). The specific

36

growth rates in the feast (μfeast) and famine
(μfamine) periods are the average growth rates.
The term μoverall means the cycle average

A Complete analysis on PHA production
(Salim M R)
specific growth rate, which is in fact the
reciprocal of the cycle length. Concerning these
data, it is clear that with the exception of the
SRT, it is easy to make difference between a
feast and famine condition. In comparing the
shorter and longer cultivation periods, it appears
that the specific growth rate in HRTso-213.3 can
be 10 times higher than in HRTso-53.3.
Obviously, the biomass takes an advantage (for
PHA storage) during feast period compared

with famine period because the ratio of specific
growth rates (μ/μoverall) seems high during this
period. In general, the ratio of biomass
accumulation depends on the prolonged cycle of
cultivation and adjustment of growth rate.
Obviously, when the cultivation is fixed to
warm condition, it may increase suddenly
because of the excited external energy for
biomass.

Table 5: Specific growth rates for aerobic SBR condition at different length of cultivation periods
Experiments
μfeast (h-1)
μfeast/μfamine
HRTso-53.3
0.729 ± 2.26
0.030 ± 0.36
HRTso-80.0
4.038 ± 1.26
0.020 ± 1.12
HRTso-106.7
1.368 ± 2.03
0.021 ± 1.03
HRTso-213.3
10.758 ± 2.14
0.065 ± 0.33
(Note: standard deviations are follows after plus/minus sign)

μfeast/μoverall
0.843 ± 0.226
1.603 ± 1.19
1.155 ± 2.55
1.830 ± 1.45

production rate. The behaviour at HRTso-213.3
cultivation is unexplainable because the COD
utilization appears to be lowered as compared to
the others. The result reached to only 0.02
mmol/l. h and then also inhibited the COD
production rate at the same value. Obviously,
there is no need for biomass growth but only
maintenance and storage is required.

VFA/TFA

%PHB/CDWmax

4.50

3.50
3.00
2.50
2.00
1.50
1.00

HRTso-80.0

0.00
HRTso-53.3

0.02

0.01

0.05
HRTso-213.3

0.005

HRTso-106.7

(C-mol/C-mol)

Puptake/VFA, VFA/TFA

4.00

10.000
9.000
8.000
7.000
6.000
5.000
4.000
3.000
2.000
1.000
0.000

max

Puptake/VFA

%PHB/CDW

The composition of the unknown
component was calculated using COD
distributions. The composition suggested an
extreme low degree of reduction (data not
shown) that is not typical for fatty acid
components. Because no experiment has been
carried out (acidification stage), COD analysis
was considered as the best explanation to the
total reduction and accumulation. Figure 3
shows the behaviour of COD utilization and

0.50

μfamine/μoverall
1.157 ± 1.44
0.397 ± 2.28
0.845 ± 1.34
0.170 ± 2.31

Experiment(s)

Figure 2: Behaviour of P and VFA utilization for PHB accumulation at one cycle measurement

37

0.14

0.14

0.12

0.12

0.10

0.10

0.08

0.08

0.06

0.06

0.04

0.04

0.02

0.02

0.00

COD production rate
(mmol/l. h)

COD utilization rate
(mmol/l. h)

Jurnal Sains Kimia (Suplemen)
Vol 9, No.3, 2005: 31-41

0.00
HRTso-53.3 HRTso-80.0 HRTso-106.7 HRTso-213.3
Experiment(s)
COD utilization rate
COD production rate

Figure 3: COD utilization and production rate based on biomass accumulation and cultivation at different cycle
length cultivation
whether this qpfeast/-qsfeast ratio is constant which
can be applied within certain limitations, or that
it is just a coincidence that constant values
appeared in Figure 4. In fact in this case the
qpfeast/-qsfeast ratio has a value very close to the
maximum yield coefficient of PHA on
substrate. Most of data in Figure 4 and Figure 5
show that the catabolic reaction appeared high
in feast period, compared with famine.
However, at HRTso-106.7, the yield resulted in
an invert value to reach at 0.879 C-mol/C-mol.
At HRTso-80.0, the value in feast (YSXfeast) and
famine (YSXfamine) occurs at the same level
(0.554 C-mol/C-mol). Unfortunately, the Yp/x
(0.07 C-mol/C-mol) obtained a lower result
compared to others.

(-)qsfeast

qp feast

qpfeast/-qsfeast

0.35

1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00

(-)qs feast , qpfeast
(C-mol/C-mol. h)

0.30
0.25
0.20
0.15
0.10
0.05
HRTso213.3

HRTso106.7

HRTso80.0

HRTso53.3

0.00

Experiment(s)

Figure 4: Specific substrate and production rates for different length of cultivation

38

qpfeast /-qs feast (C-mol/C-mol)

PHA conversion has been studied in
only a limited number of cases in literature.
Figure 4 shows reported specific substrate
uptake rates and specific PHA production rates
and their ratio in the feast period. As reported
earlier, the ratio qpfeast/-qsfeast indicates which
fraction of the substrate is stored. The substrate
uptake rates and PHA production rates were
somewhat higher in the long cycle’s cultivation
(72 and 96 hours). The overall observed
substrate uptake rate in the feast period was
high for almost 0.3 C-mol/C-mol. h at HRTso106.7 as compared with others. In general, the
ratio depends on the accumulation period and
COD fraction distributions. The results are also
showed a constant value from system HRTso53.3 to HRTso-80.0. It can be questioned

A Complete analysis on PHA production
(Salim M R)
YSXfeast

YSX famine

YP/X feast
1.00

1.20

Ysx feast , Ysx famine
(C-mol/C-mol)

0.80
0.70

0.80

0.60
0.50

0.60

0.40
0.40

0.30
0.20

0.20

Yp/x feast (C-mol/C-mol)

0.90
1.00

0.10
HRTso213.3

Experiment(s)

HRTso106.7

HRTso80.0

0.00
HRTso53.3

0.00

Figure 5: Catabolic and anabolic yield coefficient during feast and famine condition at different length of
cultivation.
The results of experiment HRTso-53.3
till HRTso-213.3 are shown in Figure 6. All
parameters measured at the beginning and at the
end of aerobic phase were compared
simultaneously between “standard” and
“prolonged” experiments. The results obtained
from the “standard” cycle are considered as
typical for the particular operating system
(Brdjanovic et al., 1997). The pattern and all
concentrations of all monitored parameters were
highly similar and the biomass compartment

(i.e. CDW, VSS and ash) decreased slightly
when the experiment delayed too long.
However, without conciliation the degradation
factor, the biopolymer compartment (i.e. PHA,
PHB and PHV) achieved a high value especially
during 48 hours of cultivation. The depleted of
PHA contents occurred after 48 hours of
cultivation (e.g. 96 hours) indicates that the
extended of cultivation period are negligible for
biomass accumulation and storage capability.

Figure 6: Biomass compositions during “standard” and “prolonged” SBR cycle of experiment (a) HRTso-53.3,

(b)

4000
3000
2000
1000
0
start

end

start

5000
4000
3000
2000
1000
0

end

start

Standard aerobic phase, 12 h Prolonged aerobic phase, 24
Experiments

Ash
PHH
Residual biomass
fPP

0.9
0.8
0.7
0.6
0.5

(c)

6000
5000
4000

0.4
0.3
0.2
0.1
0.0

3000
2000
1000
0
start

end

start

end

Standard aerobic phase, 12 h
Prolonged aerobic phase, 48 h
Experim ents

CDW
PHB
PHA
VSS/CDW

VSS
PHV
Poly-P
fPHB

Ash
PHH
Residual biomass
fPP

CDW
PHB
PHA
VSS/CDW

start

VSS
PHV
Poly-P
fPHB

7000
Concentrations (mg/l)

Concentrations (mg/l)

7000

VSS
PHV
Poly-P
fPHB

end

end

Standard aerobic phase, 12 h
Prolonged aerobic phase,36 h
Experim ents

VSS/CDW, f PHB, f PP

CDW
PHB
PHA
VSS/CDW

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

6000

Ash
PHH
Residual biomass
fPP

0.9
0.8
0.7

(d)

6000
5000

0.6
0.5
0.4
0.3

4000
3000
2000

0.2
0.1
0.0

1000
0
start

end

start

VSS/CDW, fPHB, fPP

5000

Concentrations (mg/l)

Concentrations (mg/l)

6000

7000

VSS/CDW, fPHB , fPP

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

VSS/CDW, fPHB, fPP

(a)
7000

end

Standard aerobic phase, 12 h
Prolonged aerobic phase, 96 h
Experim ents

CDW
PHB
PHA
VSS/CDW

VSS
PHV
Poly-P
fPHB

Ash
PHH
Residual biomass
fPP

(b) HRTso-80.0, (c) HRTso-106.7 and (d) HRTso-213.3. Note: Poly-P(mgP/l)=(CDW-VSS)-VSS*5/95,
PHA(mgPHA/l)=PHB+PHV, Residual biomass (mg biomass/l)=CDW-PHB-Poly-P, fPHB (g PHB/ g active
biomass)=PHB/active biomass, fPP (g P/g active biomass)=Poly-P*0.35/active biomass

39

Jurnal Sains Kimia (Suplemen)
Vol 9, No.3, 2005: 31-41
1.8
1.6
1.4
Δ fPHA

1.2
1

Exponential Association Model

0.8
0.6
y = 30.115 (0.0458 - exp (-0.085x))

0.4

R2 = 0.7903

0.2
0
40

90

140

190

240

HRT/SRT (h)

Figure 7: Dependence of the amount of PHA produced on HRT/SRT conditions. (♦) experiments used for the
fitting the points, (—) model equation developed from fittings.
The experiments of HRT/SRT have
been conducted individually to stimulate the
“standard” productivity of PHA (∆fPHA) using
SO as substrates. The predictive model (Figure
7) has been used to generate the model equation
in a single fed-batch culture. Since there are
significant interactions, the model found from
those experiments could be used for further
studies such as formulation, optimization, factor
analysis, or simulation without any bias. Further
verification experiment (with this formulation)
was not performed because this formulation was
for the robust process under the assumed
optimized condition. Moreover, no higher
results were expected from this experiment. The
final optimal formula obtained from different
experiment conditions, showed higher PHA
productivity at more than 90 hours of
HRT/SRT. However, the stationary behaviours
have been observed indicating the regular fedbatch cultivation is not much affected the PHA
production rate.
The results (Table 6) indicated that
peak PHA content (as well as PHB content) can
typically be obtained faster during the second
limitation of P concentration (famine period),
but the maximum PHA content obtained will

probably be less than during the first
accumulation phase (feast period). It was noted
that the nutrient limitation experiments of this
study (especially P) required significantly
longer time to obtain maximum PHA content
than was required during nitrogen limitation
experiments (Chinwetkitvanich et al., 2004).
Based on the result obtained by Md Din et al.
(2004b), it is probable that, even though the
influent contained no phosphorus, the biomass
still had phosphorus stored within it that had to
be depleted before PHA accumulation would
begin. The maximum polymer content in LCFA
was maintained in the longer period, therefore
possibly to break-down, since the degradation
of unsaturated fatty acids taken into account.
However, the possibility of the enzymatic
polymerization under LCFA has been
established using P. oleovorans (pure culture)
which clearly demonstrates that P. oleovorans is
able to produce variety of PHA, with unit
ranging from 6 to 11 carbon atoms, depending
on the substrate used for growth (Brandl et al.,
1988). No further experiment has been made to
confirm these mechanisms but it still significant
for overall results based on value-obtained (ratio
of VFAs conversion over fatty acids added)

Table 6: Accumulation of PHA content in HRT/SRT conditions under acclimatization of biomass concentration
and specific growth rate.

Experiment

Variable

HRTso-53.3
HRTso-80.0
HRTso-106.7
HRTso-213.3

Cycle
length

Biomass, Cx
(C-mM)
feast
famine
1998.57
310.00
1957.14
247.14
1927.14
364.29
1925.71
181.43

CONCLUSION
Since oil fed mostly contains a high
concentration of fatty acid (LCFA), the

40

Specific growth
rate, μ (h-1)
feast
famine
0.030
0.042
0.020
0.005
0.021
0.015
0.065
0.006

PHAmax
(C-mM)

%PHA/
CDW

265.19
255.51
387.95
387.95

11.41 ± 0.68
22.31 ± 1.85
33.77 ± 3.10
33.73 ± 2.36

oxidization of carbon-chain should be taken into
various accumulations. Continuous culture in a
chemostat system is a common method to study
the kinetics of cell growth and product

A Complete analysis on PHA production
(Salim M R)
formation under steady-state conditions, which
reveals the clear relationship between cells and
the environment. Since a high final PHB content
in cells is desired and can only be achieved in
the not-actively-growing cells, an optimal
process should have a variation in the cell
growth rate by controlling the feeding of growth
nutrients with time.
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