2. 412 M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421
mixed cultures occurs under transient conditions of carbon sup-
ply (Majone et al., 1996; van Loosdrecht et al., 1997), referred
to as feast-famine or aerobic dynamic feeding (ADF), which
generates an unbalanced cell growth. During the carbon excess
phase, external substrate uptake is mainly driven towards inter-
nalpolymerstorage.Aftersubstrateexhaustion,theaccumulated
polymer can be used as an energy and carbon source for cell
growth and maintenance.
Unlike pure cultures, mixed cultures do not store carbohy-
drates as PHAs but rather as glycogen (Dircks et al., 2001).
Volatile fatty acids (VFA), however, can be stored as PHAs by
mixed cultures. Most studies on PHA production by mixed cul-
tures were carried out using synthetic organic acids (e.g., acetate,
propionate, butyrate, and valerate) (Beccari et al., 2002; Dionisi
et al., 2004; Serafim et al., 2004; Lemos et al., 2006). Serafim
et al. (2004) showed that mixed microbial cultures subjected to
dynamic feeding conditions using acetate as the carbon source
may accumulate PHB up to 65% cell dry weight. Only few works
have been published describing PHA production by mixed cul-
tures using waste-based or low cost substrates (Rhu et al., 2003;
Dionisi et al., 2005; Bengtsson et al., 2007). PHA synthesis from
such carbohydrate-rich substrates requires a previous anaerobic
fermentation step to transform the sugar content of the substrate
into VFA.
One of the main challenges in the mixed culture PHA pro-
duction process is culture selection. Selection of a stable culture
with a high PHA storage capacity is of major importance for the
effectiveness of these processes. Almost all the studies describ-
ing PHA production by mixed cultures perform culture selection
and PHA accumulation in two separate steps (Dionisi et al.,
2004; Serafim et al., 2004; Lemos et al., 2006). Only few works
report the use of a three-step production process, in which an
anaerobic fermentation step precedes the culture selection and
polymer accumulation steps. Dionisi et al. (2005) proposed a
PHA production process from olive oil mill effluents (OME),
in which a PHA accumulating culture is selected on a mixture
of synthetic organic acids, followed by PHA batch production
using fermented OME and the selected culture. Bengtsson et al.,
2007 developed a three-step process for the production of PHA
from paper mill effluents, including the continuous fermenta-
tion of the paper mill effluent followed by culture selection on
the fermented effluent and PHA production using the fermented
effluent and the selected culture. At this point, it is not clear
which of the strategies for culture selection allows for the higher
process PHA yield and productivity.
In this study, the use of sugar cane molasses (a by-product
of the sugar refinery industry with very high sugar content –
over 50% dry weight) was investigated for the production of
PHAs by mixed microbial cultures. A three-step process was
used, comprising (1) continuous molasses acidogenic fermen-
tation, (2) selection of a PHA-accumulating culture under feast
and famine conditions, and (3) batch PHA accumulation using
the selected culture and the fermented molasses. Two differ-
ent strategies were used: conducting culture selection with a
synthetic substrate (acetate) and accumulation with fermented
molasses or using the fermented molasses as feedstock for both
steps. The effect of acidogenic fermentation pH (resulting in
different organic acids profiles) on polymer storage yield and
monomer composition was evaluated using the acetate-selected
culture. Strategies of reactor operation for the selection of a
PHA-accumulating culture on fermented molasses were devel-
oped.
2. Materials and methods
2.1. Feed preparation
The sugar cane molasses used in this study was obtained
from an industrial sugar manufacturing plant (Refinaria de
Açúcares Reunida, Porto). The molasses has a very high sugar
content (total sugars make up 54% w/w of the cane molasses),
composed mainly of sucrose (62%), and fructose (38%).
The molasses were first diluted (1:3) to reduce its viscosity
and allow it to be pumped. The diluted molasses pH was
adjusted (5 ± 0.05 to 7 ± 0.05) with a 0.5 M NaOH solution and
the solution (410 g/l or 220 g sugars/l) kept at 4 ◦C in a contin-
uously stirred refrigerated vessel. A mineral nutrients solution,
which was used to further dilute the molasses – to 10 g sugars/l
(344 Cmmol/l) –, was added separately to the reactor (Fig. 1) in
order to prevent the molasses from fermenting beforehand. The
mineral nutrients medium included a nitrogen and a phosphorus
source (NH4Cl and KH2PO4/Na2HPO4). The solution was
prepared in tap water and pH was adjusted (5 ± 0.05 to 7 ± 0.05)
with a 0.5 M NaOH solution. The mineral nutrients solution
ammonia and phosphate concentrations (173–307 mgN/l
and 59 mgP/l) were adjusted to keep the C/N/P ratios (on
a molar basis) at 100:6:1 (steady-states 1–3) and 100:3:1
(steady-state 4).
2.2. Experimental set-up
The set-up consists of three bench-scale reactors and a hollow
fibre membrane filtration module (Fig. 1). The molasses acido-
genic fermentation was carried out in a continuous stirred tank
reactor (CSTR) under anaerobic conditions. PHA-accumulating
culture selection was carried out in an aerobic sequenc-
ing batch reactor (SBR) and PHA accumulation in a batch
reactor.
2.2.1. Continuous acidogenic fermentation reactor
The CSTR (working volume of 1140 ml) was inoculated with
sludge from a full-scale anaerobic digester receiving mixed
domestic-industrial wastewater (Beirolas) and acclimatised to
the molasses feed. During the initial acclimatisation period, the
inoculum was fed with diluted molasses (10 g sugars/l), and
nutrients (21 Nmmol/l and 2 Pmmol/l) and the reactor was oper-
ated discontinuously until acidification was observed. After that,
reactor operation was switched to continuous mode and biomass
concentration and fermentation end products were monitored
until a steady state was attained. Two peristaltic pumps were
used to continuously feed the reactor: one for the molasses feed
and one for the mineral nutrients solution. The diluted molasses
feed (410 g/l) and the mineral nutrients solution flow rates (5
and 109 ml/h, respectively) were adjusted to keep the reactor
3. M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421 413
Fig. 1. Three-step PHA production process from sugar cane molasses by mixed cultures using either fermented molasses – full line – or acetate – dashed line – for
culture selection.
hydraulic retention time at 10 h and the initial substrate concen-
tration at 10 g sugars/l (344 Cmmol/l).
The reactor was operated at three different pH values (7, 6,
and 5). pH was controlled using a pH controller which switched
on and off the supply of 0.5 M NaOH solution to the reac-
tor. Mixing was kept at 400 rpm and temperature at 30 ◦C.
Oxidation–reduction potential was monitored.
The effluent was withdrawn by overflow and collected in
a continuously stirred refrigerated tank (at 4 ◦C). The effluent
was clarified using a filtration set-up composed of a peristaltic
pump and an ultra filtration hollow fibre membrane module
(5 × 105 MW cut-off). The clarified effluent was kept at 4 ◦C
prior to its use in PHA batch accumulation assays or as a
feedstock for the culture selection SBR. Samples were taken
daily from the reactor, from the diluted molasses feed and
from the mineral nutrients solution in order to monitor sug-
ars, organic acids, (and other possible fermentation end-products
such as, ethanol), ammonia, phosphate, and volatile suspended
solids (VSS). The gas produced was measured using a mass
flow meter.
2.2.2. PHA-accumulating culture selection reactor
The PHA-accumulating culture selection on fermented
molasses was conducted in a sequencing batch reactor (SBR)
(working volume of 1000 ml) operated under ADF conditions.
The reactor was inoculated with a PHA-accumulating mixed
culture acclimatised to acetate (Serafim et al., 2004). The SBR
12 h cycles consisted of four discrete periods: fill (12.5 min); aer-
obiosis (feast and famine) (11 h); settling (38.5 min), and draw
(9 min). The hydraulic retention time (HRT) was kept at one
day. A purge of mixed liquor (100 ml) was performed daily
in order to keep the sludge retention time (SRT) at 10 days.
The reactor was fed with clarified effluent from the continuous
acidogenic fermentation reactor produced during steady-state
4 (pH 6 and C/N ratio of 100:3, see Table 1 for composi-
tion). Five hundred milliliter of clarified fermented molasses
were pumped into to the reactor at the beginning of each cycle
using a peristaltic pump. A second peristaltic pump was used
to withdraw the reactor effluent (500 ml per cycle) following
the settling phase. Ammonium and phosphate were added to
the clarified fermented molasses feed so as to make up initial
reactor concentrations of 1.4 or 2.5 Nmmol/l and 0.32 Pmmol/l,
respectively. Thiourea was also added to this feedstock in order
to inhibit nitrification. Feed pH was adjusted to 8 ± 0.05. Air
was supplied by an air pump through a ceramic diffuser. Mixing
was kept at 500 rpm. pH and dissolved oxygen were monitored.
Pumping (fill and draw), aeration, and mixing were automati-
cally controlled by a software program developed in our research
group. In addition, the software was also used to acquire pH
and dissolved oxygen data. At given cycles, samples were taken
periodically from the reactor in order to determine VFA and
ammonium uptake, TOC removal, PHA storage, and biomass
growth. The reactor stood in a temperature-controlled room
(23–25 ◦C).
The PHA-accumulating culture selected with acetate is
described in Serafim et al. (2004). Reactor conditions are gen-
erally those described for the culture selected on fermented
molasses, except that acetate was used as the carbon source.
2.2.3. PHA batch accumulation assays
PHA accumulation assays were carried out using clarified
fermentedmolassesproducedunderdifferentCSTRsteady-state
conditions (see Table 2 for composition) and sludge from PHA-
accumulating cultures selected under ADF conditions either
5. M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421 415
using acetate as the carbon source (Serafim et al., 2004) or
fermented molasses.
The accumulation assays were carried out in a 600 ml
working volume vessel. Approximately 200 ml of sludge
were collected from the acetate-selected or the fermented
molasses-selected SBR at the end of the cycle, immediately
after the withdrawing phase and added to 400 ml of clari-
fied fermented molasses whose pH was previously adjusted to
seven.
Air was supplied by an air pump through a ceramic diffuser
and mixing was provided by magnetic stirring. During these
batch tests, organic acids take up, ammonium consumption,
PHA storage, biomass growth, and oxygen uptake rate (OUR)
were monitored over time. In order to determine the OUR, the
mixed liquor was recirculated (using a peristaltic pump) through
a respirometer, where mixing was provided using magnetic
stirring and an oxygen electrode was inserted. Recirculation
was stopped at given intervals and the decrease in dissolved
oxygen concentration in the respirometer was registered and
used to determine the OUR. pH was monitored. The accumu-
lation assays were conducted in a temperature-controlled room
(23–25 ◦C).
2.3. Analytical procedures
Biomass concentration was determined using the volatile sus-
pended solids (VSS) procedure described in standard methods
(APHA, 1995).
Lactate, acetate, propionate, butyrate, and valerate con-
centrations were determined by high performance liquid
chromatography (HPLC) using a Merck-Hitachi chromatog-
rapher equipped with a UV detector and Aminex HPX-87H
pre-column and column from BioRad (USA). Sulphuric acid
0.01 M was used as the eluent at a flow rate of 0.6 ml/min
and 50 ◦C operating temperature. The detection wavelength
was set at 210 nm. The organic acids concentrations were
calculated through a calibration curve using 25–500 mg/l
standards.
Sugars (sucrose, fructose, and glucose) and ethanol con-
centrations were determined by HPLC using the same
Merck-Hitachi chromatographer and a Merck-Hitachi differ-
ential refractometer detector (RI-71). The columns and elution
conditions used were the same as stated above for organic acid
determination, except for the eluent flow rate, which was in
this case 0.5 ml/min. The sugars and alcohol concentrations
were calculated through a calibration curve using 25–500 mg/l
standards.
Total sugars were determined using the Morris method
(1948) Morris (1948) with modifications by Koehler (1952),
Bailey (1958), and Gaudy (1962). Samples were digested with
an anthrone reagent (0.125 g anthrone in 100 ml sulphuric
acid) at 100 ◦C, and absorbance was measured at 625 nm.
Sucrose standards (0–100 mg/l) were used to determine total
sugars.
PHAs were determined by gas chromatography using the
method described in Serafim et al. (2004) and Lemos et al.
(2006). Hydroxybutyrate and hydroxyvalerate concentrations
are calculated using P (HB-HV) (70%/30%) (Sigma) standards
and corrected using a heptadecane internal standard.
Ammonia concentration was determined using an ammo-
nia gas sensing combination electrode ThermoOrion 9512.
A calibration curve was obtained with NH4Cl standards
(0.01–10 Nmmol/l).
Total organic carbon (TOC) from clarified samples was
analysed in a Shimadzu TOC automatic analyser. Potassium
hydrogen phthalate standards (20–500 mgC/l), sodium hydro-
gen carbonate, and sodium carbonate standards (1–25 and
20–500 mgC/l) are used to produce the TC and IC calibration
curves.
Gel permeation chromatography (GPC) was conducted in
a size exclusion chromatography (SEC) apparatus (Waters),
including a solvent delivery system composed by a model 510
pump,aRheodyneinjector,andarefractiveindexdetectormodel
2410. The operating temperature was 30 ◦C, using chloroform
as eluent with a flow rate of 1 ml/min. A series of three Waters
Ultrastyragel columns, 103, 104, and 105 Å was used. Sample
injection volumes of 150 l were used.
2.4. Calculations
The sludge PHA content was calculated as a percentage of
VSS on a mass basis (% PHA = PHA/VSS*100, in g PHA/g
VSS), where VSS includes active biomass (X) and PHA.
The maximum specific substrate uptake (-qS in Cmmol
VFA/Cmmol X h) and PHA storage rates (qP in Cmmol
HA/Cmmol X h) were determined by adjusting a linear function
to the experimental data of VFA and PHA concentrations plotted
over time, calculating the first derivative at time zero and divid-
ing the value thus obtained by the active biomass concentration
at that point. VFA concentration corresponds to the sum of all
the organic acids concentrations (VFA, in terms of Cmmol/l, is
equal to HAc, HProp, HBut, HVal, HLac, in Cmmol/l). PHA
concentration (in Cmmol/l) corresponds to the sum of HB and
HV monomers concentrations (in Cmmol/l).
The yields of PHA (YP/S in Cmmol HA/Cmmol VFA) and
active biomass (YX/S in Cmmol X/Cmmol VFA) on substrate
consumed were calculated by dividing the amount of PHA
formed or the active biomass formed by the total amount of
organic acids consumed, respectively.
The respiration yield on substrate (YO2/S in Cmmol/Cmmol
VFA) was calculated by integrating the curve of the experi-
mental OUR (in mmol O2/l h) over time (during the period of
VFA consumption) and dividing the value thus obtained by the
amount of substrate consumed (in Cmmol VFA/l). Oxygen is
expressed as carbon assuming that 1 mol CO2 is formed for
1 mol O2 consumed.
The material mass balance for a given accumulation study
can be represented by:
ΔY = YP/S + YX/S + YO2/S
The global yield (Y) accounts for all the carbon recovered
from the material mass balance (it is one if all the carbon is
recovered).
6. 416 M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421
3. Results and discussion
3.1. Acidogenic reactor: impact of pH and C/N ratio on
VFA production
A 1 litre CSTR fed with sugar cane molasses (initial sub-
strate concentration of 10 g/l total sugars) has been operated for
a period of two years. The effect of pH on organic acid produc-
tivity and concentration profiles was evaluated. Three different
pH values (7, 6, and 5) were tested, resulting in different sugar
to organic acid yields and different organic acids concentration
profiles (Table 1). The organic acids produced included volatile
fatty acids – acetate, propionate, butyrate, and valerate – and
lactate. No ethanol production was observed.
Sugar-to-organic acid conversion yield as well as the spe-
cific productivity did not vary significantly with pH, although
slightly higher yields were obtained under more acidic condi-
tions. The lower sugar to organic acids conversion yield obtained
at pH 7 might be explained by some methanogenic activity pos-
sibly occurring at this pH. These results agree well with those
of Fang and Liu (2002) who studied the effect of pH on hydro-
gen production from glucose by a mixed culture in a CSTR
operated at 6 h HRT. The authors observed a small variation
of the organic acid yields on glucose with pH (0.44, 0.46, and
0.48 Cmmol VFA/Cmmol glucose, at pH 7, 6, and 5, respec-
tively). The higher values obtained with molasses relatively to
those determined for glucose can be explained by the molasses
organic fraction other than sugars. In fact, sugars represent
about 71% of the total organic fraction (TOC) of the molasses.
Part (48%) of the non-sugar TOC fraction of the molasses is
consumed during the fermentation process. Another possible
reason for the difference between the conversion yields obtained
with molasses and those reported for glucose is the fact that
in the later case, conditions were optimized for hydrogen pro-
duction, whose metabolic pathway is associated with substrate
decarboxylation.
Unlike organic acids productivity and yield on substrate, the
organic acids distribution was strongly affected by pH (Table 1).
At pH 7 and 6, volatile fatty acids (acetate, propionate, butyrate,
and valerate) concentrations decreased with increasing VFA
chain length. Acetate was the more concentrated species, mak-
ing up more than 50% of the organic acids produced. At pH 5,
acetate was still the more concentrated, but both butyrate and
valerate surpassed propionate. Lactate was produced at pH 7
and 5. Acetate and propionate concentrations decreased from pH
7 to 5, while butyrate and valerate concentrations significantly
increased in the same pH range. The fact that lower operating pH
favoured the production of longer chain fatty acids was expected,
since under these more acidic conditions there are more reduc-
ing equivalents available to be incorporated into the fatty acid
chains (Zoetemeyer et al., 1982).
The effect of pH on VFA concentration profiles was previ-
ously studied with pure sugars (Zoetemeyer et al., 1982; Fang
and Liu, 2002 and Khanal et al., 2004) and complex substrates
(Reis et al., 1991, using molasses slops). In all of these studies,
a similar trend of organic acids distribution as a function of pH
was observed.
The C/N/P ratio was initially set at 100:6:1 Cmol:Nmol:Pmol
(steady-states 1–3) to ensure the fermentation reaction was car-
ried out in excess nutrient conditions. Residual nitrogen of
10–12 Nmmol/l (in the form of ammonia) and phosphorus of
0.20–0.25 Pmmol/l (in the form of phosphate) were present in
the fermentation reactor effluent. To assess the effect of the C/N
ratioandsimultaneouslyproduceanammoniafreeeffluent(tobe
used in the PHA production step), the C/N ratio was increased
from 100:6 Cmol:Nmol in steady-state 2 to 100:3 Cmol:Nmol
in steady-state 4 (both operated at pH 6). The ammonia con-
centration in the effluent of the acidogenic reactor decreased to
0.1 Nmmol/l. The organic acid distribution was not significantly
altered by the change in C/N ratio (Table 1). However, the sugar-
to-organic acids conversion yield and specific production rate
suffered a slight decrease.
3.2. PHA accumulation using fermented molasses and a
culture selected with acetate
PHA batch accumulation assays were carried out using
sludge from a mixed microbial culture grown on acetate under
feast-famine conditions. Clarified fermented molasses produced
under different acidogenic fermentation conditions (pH 7–5)
were used as substrate. The aim of this study was to evaluate
the effect of the organic acids concentration profile on PHA
production (yield on substrate and polymer composition) by a
culture selected with acetate, which previously showed a high
capacity of PHA storage. Reactor operating conditions used to
select for this culture were described in Serafim et al. (2004).
3.2.1. PHA accumulation from fermented molasses
Fig. 2 shows the results of a typical batch study using clarified
fermented molasses as the carbon source for PHA accumulation.
Acetate and propionate were consumed at higher rates than the
remaining organic acids, thereby being first exhausted. Butyrate
and valerate uptake rates increased after acetate and propionate
exhaustion. The oxygen uptake rate dropped after acetate and
propionate exhaustion, only increasing again after a short period,
suggesting an adaptation of the microbial culture to the substrate
Fig. 2. PHA batch accumulation test using a culture selected on acetate under
ADF conditions and clarified fermented molasses produced at pH 5.
7. M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421 417
shift. Further decreases were observed in the oxygen uptake rate
as the remaining organic acids were exhausted. PHA production
was mainly associated with acetate and propionate consumption,
and less with butyrate and valerate uptake (the major part of the
later VFA was only consumed after the first acids were exhausted
and polymer production increased but slightly). It was expected
that the acetate-acclimatised culture would preferentially take up
acetate and propionate rather than butyrate and valerate (Lemos
et al., 2006). Active biomass growth occurred during the feast
phase, in parallel with polymer storage.
3.2.2. Effect of the organic acids concentration profiles on
PHA production
Three PHA batch accumulation assays were conducted with
clarified fermented molasses produced at pH 7, 6, and 5. The
organic acid concentrations used in these studies (listed in
Table 2) suffered a dilution relatively to their respective concen-
trations in the acidogenic reactor effluent (presented in Table 1),
since only 400 ml of fermented molasses were used in a batch
reactor with a liquid volume of 600 ml.
Different PHA storage yields (YP/S) on fermented molasses
were obtained in the three batch tests (Table 3). The lower value
was obtained for the fermented molasses produced at pH 7, and
the higher for that produced at pH 5. At the latter batch test,
the ratio of carbon to ammonia (21 Cmol/Nmol) was relatively
higher than that used in the other two assays (14 Cmol/Nmol),
which explains the higher storage yield and the lower growth
yield obtained. Furthermore, these differences may also reflect
the VFA composition fed to each reactor. For the first two assays,
carried out at the same C/N ratio, the polymer storage yields
increased with the combination of the acetate and propionate
molar fractions, the two acids, which were shown to be the major
VFA contributors for PHA synthesis.
The polymer storage yields obtained with clarified fermented
molasses were slightly lower than those reported for the same
culture using pure substrates (0.58 and 0.51 Cmmol HA/Cmmol
VFA for acetate and an acetate/propionate mixture, in Serafim
et al., 2004 and Lemos et al., 2006, respectively) and in the
range of those reported for other fermented waste substrates.
Dionisi et al. (2005) reported a polymer storage yield of 1 g
PHA/g VFA, expressed as COD, from fermented olive oil mill
effluents. The authors attribute this unexpectedly high yield to
carbon sources other than VFA, which may contribute to the
metabolism of PHA synthesis. Bengtsson et al., 2007 reported
a polymer storage yield of 0.33 Cmmol HA/Cmmol VFA using
fermented paper mill effluents as a feedstock for PHA storage
under excess nutrient conditions.
The preference of VFA consumption was consistent in
the three assays conducted with fermented molasses. Acetate
was consumed at a higher rate (0.37, 0.19, and 0.16 Cmmol
HAc/Cmmol X h, for fermented molasses produced at pH 7, 6,
and5,respectively)thanpropionate(0.12,0.05,and0.10 Cmmol
HProp/Cmmol X h, from pH 7, 6, and 5, respectively), butyrate
(0.01, 0.04, and 0.05 Cmmol HBut/Cmmol X h, from pH 7, 6,
and 5, respectively) and valerate (0.01, 0.02, and 0.02 Cmmol
HVal/Cmmol X h, from pH 7, 6, and 5, respectively). The
butyrate and valerate uptake rates increased after acetate and
propionate exhaustion. The maximum specific PHA production
rates and substrate uptake rates (Table 3) were similar in the
accumulation studies with the fermented molasses produced at
pH 6 and 5, but higher for pH 7. The latter result reflects the fact
that in this study, the acetate uptake rate was much higher than
that observed for the pH 6 and pH 5 studies.
The maximum specific PHA accumulation rates obtained
with fermented molasses fall between those reported for the
same culture using acetate and an acetate/propionate mixture
(0.47 and 0.52 Cmmol HA/Cmmol X h, respectively, Serafim et
al., 2004 and Lemos et al., 2006) and those obtained using propi-
onate, butyrate or valerate as sole carbon sources (0.10, 0.07, and
0.08 Cmmol HA/Cmmol X h, respectively, Lemos et al., 2006).
Furthermore, the maximum specific PHA production rates are
lower than that obtained from fermented OME (0.52 Cmmol
HA/Cmmol X h, Dionisi et al., 2005), but much higher than
that reported for fermented paper mill effluents (0.06 Cmmol
HA/Cmmol X h, Bengtsson et al., 2007).
The highest storage yield obtained (with fermented molasses
produced at pH 5) corresponds to a polymer yield on molasses
of 0.22 g PHA/g molasses (0.26 g PHA/g COD) and to a volu-
metric productivity of 0.43 g PHA/l h. These values are within
the range of what has been reported for other low-cost carbon
substrates used for PHA production by mixed cultures (an esti-
mate of 0.11 g PHA/g COD from paper mill effluents, Bengtsson
et al., 2007) and pure cultures (0.11 to 0.33 g PHA/g substrate
and 0.05 to 0.90 g PHA/l h, Kim, 2000). Although the maxi-
Table 3
PHA batch accumulation assays using a culture selected with acetate and clarified fermented molasses produced at different pH and with different residual ammonia
concentrations, or using a simulated feed composed of synthetic organic acids
Substrate CSTR
pH
Xi (g/l) VSSmax
(g/l)
VFA
(Cmmol/l)
NH4
+
(Nmmol/l)
YX/S YP/S YO2/S -qS qP PHA composition
mol HB:mol HV
Effect fermentation
pH
Fermented
molasses (high N)
7 2.0 2.8 100 7.0 0.34 0.37 0.16 0.53 0.28 60:40
6 2.6 4.6 112 8.3 0.37 0.45 0.20 0.30 0.20 69:31
5 3.0 5.5 165 7.8 0.23 0.50 0.18 0.33 0.23 47:53
Effect of residual
NH4
+
Fermented
molasses (low N)
6 3.5 4.6 109 0.1 0.004 0.62 0.23 0.26 0.23 62:38
Effect of the
molasses matrix
Synthetic VFA
mixture
– 2.0 3.4 120 0.1 0.004 0.62 0.28 0.39 0.37 69:31
Xi is active biomass (or biomass minus PHA); YX/S in Cmmol X/Cmmol VFA; YP/S in Cmmol HA/Cmmol VFA; YO/S in Cmmol/Cmmol VFA; qS in Cmmol
VFA/Cmmol X h; qP in Cmmol HA/Cmmol X h.
8. 418 M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421
Fig. 3. Polymer composition dependence on organic acid concentration profiles
(accumulation studies listed in Table 3 and three experiments not shown in this
work).
mum accumulation capacity was not determined, a polymer cell
content of about 30% (PHA per cell dry weight) was observed
in all batch accumulation tests conducted with fermented
molasses.
Polymer composition (HB:HV molar ratio) varied with the
organic acids concentration profiles. The molar fraction of 3HB
monomers is directly proportional to the molar fraction of
acetate and butyrate in the feed, while the molar fraction of
3HV monomers is directly proportional to the molar fraction
of propionate, valerate, and lactate (Fig. 3). This is generally
consistent with the metabolic pathways for carbon consumption
and polymer storage that have been reported for pure cultures
and which are generally used to predict the behaviour of mixed
PHA-accumulating microbial cultures (Lemos et al., 2006).
As the organic acid molar fractions varied from 0.79/0.21 to
0.51/0.49 (Ac + But)/(Prop + Val + Lac), the polymer composi-
tion varied from 69:31 to 47:53 mol HB:mol HV. These assays
clearly demonstrate that the polymer composition can be manip-
ulated by acting on the acidogenic fermentation operating pH.
Tailored polymer synthesis may be controlled through a very
simple operating parameter of the pre-fermentation required
in PHA production from carbohydrate-rich carbon sources by
mixed cultures.
3.2.3. Effect of the ammonium concentration and of the
molasses matrix on PHA accumulation
Once it was determined that increasing the C/N ratio in the
acidogenic fermentation reactor (from 17 to 33 Cmol/Nmol)
did not significantly alter the organic acid concentration profile
in the fermented molasses, the effect of the residual ammo-
nia concentration on PHA accumulation was evaluated. For
that, PHA batch accumulation studies were conducted using
clarified fermented molasses produced in steady-states 2 and
4 (both operated at pH 6 but with C/N ratios of 100:6 and
100:3, respectively), which presented similar organic acids con-
centration profiles (listed in Table 2) but different residual
ammonium concentrations (8.3 and 0.1 Nmmol/l of NH4
+).
Results are summarized in Table 3. The growth yields, calcu-
lated based on the ammonia uptake, were 0.37 and 0.004 Cmmol
X/Cmmol VFA, for the fermented molasses with high and
low ammonia concentrations, respectively. The polymer stor-
age yield was considerably higher in the later case (0.62 Cmmol
HA/Cmmol VFA versus 0.45 Cmmol HA/Cmmol VFA). In
the first case, the significant amount of ammonia available
for growth limited the fraction of carbon driven toward poly-
mer storage. Furthermore, the specific PHA production rate is
also higher in the case of low ammonia concentration. These
results are in agreement with those obtained by Serafim et al.
(2004).
The polymer storage yield thus obtained was lower than
that reported for the same acetate selected culture (0.79 Cmmol
HA/Cmmol VFA) using acetate and low ammonia concentra-
tion (90 Cmmol/l acetate and 0.7 Nmmol/l NH4
+), but similar to
those obtained with fermented paper mill effluents under nutri-
ent limiting conditions (0.55–0.67 Cmmol HA/Cmmol VFA,
Bengtsson et al., 2007).
In order to evaluate if this lower performance is due to the
VFA composition or to the molasses matrix, one batch accumu-
lation assay was conducted with a synthetic acid mixture with
similar organic acid concentration profiles (listed in Table 2)
and similar ammonia concentration to the one above described.
Results are shown in Table 3. The storage yield obtained with the
simulated feed (0.62 Cmmol HA/Cmmol VFA) was the same as
that obtained with the real fermented effluent but the maximum
specific VFA uptake (-qS) and PHA production (qP) rates were
higher.
The major difference between the two tests was in the
preference of VFA consumption. In the assay conducted with
fermented molasses, a preferential uptake of acetate and propi-
onate was observed (as previously shown in Fig. 2), while in the
assay carried out with the simulated feed, all the organic acids
(acetate, propionate, butyrate, and valerate) were consumed
simultaneously and the maximum PHA content was attained
when practically all of the organic acids were fully exhausted.
The preference of VFA consumption observed for the fermented
molasses (and which was not observed with the synthetic organic
acid mixture) seems to be associated with lower substrate uptake
and PHA production rates. Organic or inorganic compounds in
the molasses matrix may be responsible for this difference in
substrate consumption.
Although the influence of the molasses matrix on PHA stor-
age is not yet fully understood, the observations of the present
work demonstrate that caution is necessary when extrapolating
results obtained with synthetic substrates to infer what might
be expected when using complex substrates such as fermented
waste streams.
3.3. Culture selection and PHA production with clarified
fermented molasses
In this study, culture selection and PHA accumulation were
carried out with fermented molasses obtained in steady-state 4
of the acidogenic reactor (pH 6 and C/N ratio of 100:3). These
studies aim at evaluating the performance of a process using fer-
mented molasses as the feedstock for both culture selection and
PHA accumulation. Results are compared with those obtained
with the acetate-selected culture.
9. M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421 419
3.3.1. Culture selection
A sequencing batch reactor (SBR) was started up to select
for a PHA-accumulating culture under ADF conditions using
clarified fermented molasses as a feedstock. The SBR was inoc-
ulated with a PHA-accumulating culture grown on acetate under
“feast and famine” conditions (Serafim et al., 2004). Cycle
length (12 h) and reactor operating conditions (see Material and
methods) were similar to those used in the acetate selection
SBR.
In order to keep the process as simple as possible, the organic
acids produced on steady-state 4 (composition described in
Table 1) were fed to the SBR, resulting in an organic loading
rate of 75 Cmmol VFA per cycle. The fermented molasses was
supplemented with ammonia and phosphate (initial reactor con-
centrations of 1.4 Nmmol/l and 0.32 Pmmol/l) in concentrations
similar to those supplied to the original acetate-selected culture.
Fig. 4(a) shows organic acids and ammonia uptake, TOC
removal and PHA production in a cycle of the enrichment SBR,
one week after reactor inoculation. Fig. 4(b) shows reactor per-
formance over time in terms of PHA storage yield and maximum
PHA cell content per cycle. After 10 days (one sludge age), the
storage yield had dropped to about half of its initial value. The
PHA cell content was, from the reactor start-up, considerably
higher than the PHA content (the amount of polymer accumu-
lated in each cycle), indicating that the polymer accumulated in
each cycle was not being consumed during the respective famine
phase. This was due to a gradual decrease (from cycle to cycle) in
the VFA uptake rate, which decreased the length of the famine
phase. Furthermore, total organic carbon analysis on clarified
mixed liquor samples taken throughout the cycle indicated that
the organic fraction, other than VFA, continued to be consumed
after the VFA were exhausted, thereby shortening the length of
the famine phase. Eventually, the feast/famine ratio required to
stimulate the development of PHA accumulating organisms was
compromised. After 20 days of reactor inoculation, the PHA
content, which had been decreasing over time, was almost zero,
indicating that PHA was no longer being produced. The reactor
operating conditions led to the selection of organisms without
the capacity to accumulate PHA.
After this initial failure, a new reactor was started up with the
same culture used to inoculate the former one and the organic
loading rate was lowered to 30 Cmmol VFA per cycle (by dilut-
ing the acidogenic fermentation reactor effluent by 2/5) in order
to decrease the ratio of the feast to famine duration. Once again,
the culture gradually lost its PHA storing capacity (results not
shown).
Assuming that the cause of failure of culture selection was
related with the feast to famine length ratio, a third reactor was
inoculated with the same culture used in the two previous reac-
tors and operated at the organic loading rate of 30 Cmmol VFA
per cycle and at an initial ammonia concentration of 2.5 Nmmol/l
toensurethatthisnutrientwaspresentthroughouttheSBRcycle.
The presence of ammonia throughout the cycle was expected to
allow the polymer-accumulating organisms to grow during the
faminephaseusingtheinternallystoredpolymerandtheexternal
ammonia, thereby providing them with a competitive advantage
over non-accumulating organisms (namely those with the capac-
ity to degrade the non VFA organic fraction of the fermented
molasses).
Fig. 4 (c) shows organic acids and ammonia uptake, TOC
removal and PHA production in the enrichment SBR (20 days
after reactor inoculation). Cell growth and PHA storage were
observed during the feast phase along with carbon and ammonia
uptake. In the famine phase (after the organic acids were fully
Fig. 4. SBR cycles (VFA and NH4 uptake, TOC removal and PHA storage – Ysto refers to YP/S) and performance over time (PHA cell content, storage yield, and
VSS) in a culture selection SBR fed with fermented molasses using initial concentrations of either 75 Cmmol/l VFA and 1.4 Nmmol/l NH4
+ (a and b) or 30 Cmmol/l
VFA and 2.5 Nmmol/l NH4
+ (c and d).
10. 420 M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421
exhausted), ammonia continued to be slowly consumed along
with the internally stored polymer, providing carbon, energy, and
nutrients for cell growth. The residual TOC remained constant
after VFA exhaustion.
This approach resulted in the selection of a culture with
a stable PHA accumulating capacity (Fig. 4.d). During the
50-day period of reactor operation, the PHA cell content ini-
tially decreased as the polymer that was present in the cells
at the time of reactor inoculation was consumed. The PHA
content remained constant over time demonstrating a stable
accumulating performance. Approximately one SRT after reac-
tor inoculation, the PHA cell content and the PHA content
values converged, indicating that the polymer produced in one
cycle was consumed during the respective famine phase. From
that time on, both values remained constant at approximately
10% (g PHA per g cell dry weight). This low value can be
attributed to the low organic loading (30 Cmmol VFA per
cycle) combined with a relatively high cell concentration (about
4.4 g/l).
A different strategy to select for a PHA accumulating cul-
ture was followed by Serafim et al. (2004) with acetate and
Lemos et al. (2006) with propionate, in which ammonia was
only present in the feast phase of the SBR cycle. The fact that a
sole substrate was used resulted in a very well defined feast to
famine ratio, thereby ensuring that organisms able to accumu-
late PHA predominate in the system. Indeed, results indicate that
the reactor operation conditions for selection of PHA accumu-
lating organisms are different when using synthetic substrates
rather than real complex substrates such as fermented waste
streams.
3.3.2. PHA production
Polymer production using the culture thus selected (sludge
harvested 50 days after reactor inoculation) was evaluated
in a batch reactor fed with clarified fermented molasses
produced under steady-state 4 conditions (pH 6; C/N of
100:3 Cmol:Nmol).
Unlike what had been observed with the acetate-enriched cul-
ture, the organic acids were simultaneously consumed (Fig. 5),
probably because the selected culture was acclimatised to a
mixture of substrates rather than to a single substrate. Co-
polymers of P (HB-co-HV) were produced. Gel permeation
chromatography (GPC) confirmed the presence of copolymers
(chromatograms with unimodal behavior were obtained).
The organic acids distribution and residual ammonia in the
clarified fermented molasses (composition described in Table 2)
used in these studies were similar to those fed to the acetate-
selected culture under ammonia limiting conditions. Both the
Fig. 5. PHA batch accumulation test using a culture selected on fermented
molasses under ADF conditions and clarified fermented molasses produced at
pH 6.
storage yield and polymer production rate (Table 4) were lower
than those obtained for the acetate-selected culture (0.44 ver-
sus 0.62 Cmmol HA/Cmmol VFA and 0.12 versus 0.23 Cmmol
HA/Cmmol X h, respectively). It is worth noting that the respi-
ration yield (YO2/S) of the fermented molasses selected culture
was higher than that of the acetate selected culture. In addi-
tion, unlike what was observed with the acetate-selected culture,
acetate did not begin to be consumed immediately after reactor
feeding (a lag phase was observed in the acetate uptake curve).
In fact, though the initial acetate concentrations in the accumula-
tion tests were similar for the two cultures (70 and 68 Cmmol/l),
their respective initial specific acetate uptake rates were signif-
icantly different (0.19 and 0.04 Cmmol VFA/Cmmol X h). The
combination of these factors seems to indicate that inhibition
by the acetate occurs in the fermented molasses accumulating
culture.
To verify this hypothesis, a second batch accumulation test
was carried out using a lower initial fermented molasses con-
centration (61 Cmmol VFA/l), corresponding to a decrease in
initial acetate concentration from 68 to 37 Cmmol/l. Although
a short lag phase on acetate uptake was still observed (Fig. 5),
the polymer storage yield increased to 0.59 Cmmol HA/Cmmol
VFA which is close to that obtained for acetate-selected
culture (0.62 Cmmol HA/Cmmol VFA), but the maximum
specific substrate uptake and polymer production rates
(0.21 Cmmol VFA/Cmmol X h and 0.14 Cmmol HA/Cmmol
X h, respectively) were lower (0.26 Cmmol VFA/Cmmol X h
and 0.23 Cmmol HA/Cmmol X h). Inhibition by acetate was
already reported by Serafim et al. (2004) but only for concentra-
tions higher than 90 Cmmol/l. Results seem to indicate that the
culture selected using fermented molasses is more susceptible
Table 4
PHA batch accumulation assays using a culture selected with fermented molasses and clarified fermented molasses as substrate
Xi (g/l) VSS (g/l) VFA (Cmmol/l) NH4
+ (Nmmol/l) YX/S YP/S YO2/S -qS qP PHA composition mol HB:mol HV
2.9 3.9 91 0.9 0.05 0.44 0.32 0.27 0.12 79:21
3.5 4.5 61 0.1 0.01 0.59 0.26 0.21 0.14 83:17
Xi is active biomass (or biomass minus PHA); YX/S in Cmmol X/Cmmol VFA; YP/S in Cmmol HA/Cmmol VFA; YO/S in Cmmol/Cmmol VFA; qS in Cmmol
VFA/Cmmol X h; qP in Cmmol HA/Cmmol X h.
11. M.G.E. Albuquerque et al. / Journal of Biotechnology 130 (2007) 411–421 421
to inhibition by acetate than the original acetate-selected cul-
ture. Nevertheless, in the accumulation step, this inhibition can
be overcome by supplying the fermented molasses by pulses of
small amounts of substrate.
4. Conclusions
A three-step process for PHA production by mixed cultures
from sugar cane molasses was successfully established.
The main conclusions obtained in this work can be summa-
rized as follows:
(1) the fermentation reactor pH influences the composition of
volatile fatty acids produced, which in turn has a direct
effect on the composition of the polymer produced (copoly-
mers from 69:31 to 47:53 mol HB:mol HV were obtained).
This result indicates that polymer composition may be
manipulated simply by controlling the operating pH in the
acidogenic fermentation reactor;
(2) high ammonia concentration showed to negatively affect
the PHA production in the accumulation step but stimulate
the development of PHA accumulating organisms in the
selection step;
(3) a stable PHA accumulating culture was obtained with fer-
mented molasses as a feedstock, when a low organic loading
and high ammonia concentration were used, thus providing
the culture with the selective pressure for PHA accumula-
tion;
(4) thecultureselectedonfermentedmolassesshowedaslightly
lower PHA accumulation performance (lower polymer stor-
age yield, production rate, and higher susceptibility to
acetate inhibition) than the acetate-selected culture. Nev-
ertheless, in terms of total process cost, the cost of the
pure substrate can outbalance the lower polymer yield and
production rate of the fermented molasses;
(5) although the fully integrated three-stage PHA production
process is still at a very early stage of development, this work
illustrates the advantage of using this type of side stream
production process, which offers the possibility of using
different conditions and/or strategies to optimise culture
selection and polymer accumulation.
Acknowledgments
This work was supported by Fundação para a Ciência e
a Tecnologia (FCT, Portugal) through the project POCI/BIO/
55789/2004. M.G.E. Albuquerque acknowledges FCT for grant
SFRH/BD/ 17141/2004. The authors acknowledge Refinarias
de Açúcar Reunidas (RAR) for the supply of the sugar cane
molasses. The contributions of Prof. Ana Ramos (polymer char-
acterization), Dr. Luisa Serafim and Dr. Mário Eusébio are also
greatly acknowledged.
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