DOI: 10.3303/CET2293028 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Paper Received: 20 January 2022; Revised: 2 March 2022; Accepted: 28 April 2022 
Please cite this article as: Marchetti A., Lorini L., Salvatori G., Gianico A., Majone M., Villano M., 2022, Polyhydroxyalkanoates Production by 
Mixed Microbial Cultures in Sequencing Batch Reactors Operated Under Different Feeding Conditions, Chemical Engineering Transactions, 93, 
163-168  DOI:10.3303/CET2293028 
  

 CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL. 93, 2022 

A publication of 

 

The Italian Association 
of Chemical Engineering 
Online at www.cetjournal.it 

Guest Editors: Marco Bravi, Alberto Brucato, Antonio Marzocchella 

Copyright © 2022, AIDIC Servizi S.r.l. 

ISBN 978-88-95608-91-4; ISSN 2283-9216 

Polyhydroxyalkanoates Production by Mixed Microbial 

Cultures in Sequencing Batch Reactors Operated under 

Different Feeding Conditions 

Angela Marchettia, Laura Lorinia, Gaia Salvatoria, Andrea Gianicob, Mauro Majonea, 

Marianna Villanoa,* 

aDepartment of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy 
bWater Research Institute, National Research Council (CNR-IRSA), Via Salaria km 29.300, 00015 Monterotondo (Rome), Italy 

marianna.villano@uniroma1.it 

The production of polyhydroxyalkanoates (PHA) by mixed microbial cultures (MMC) requires a multistage 

process, whereby the microbial selection of PHA-storing microorganisms plays a key role on the overall 

performance. A strategy to favor the microbial selection consists in the alternance of excess (feast phase) and 

absence (famine phase) of the external carbon source. In this work, three runs of a lab-scale Sequencing Batch 

Reactor (SBR) operated under different working conditions for the establishment of the feast and famine (F/F) 

regime were analyzed. A fixed organic loading rate of 4.25 gCOD (Chemical Oxygen Demand)/Ld, and a fixed 

cycle length of 12 h were applied to the SBR. The F/F regime consisted of fully aerobic dynamic (ADF) or 

aerobic/anoxic (AE/ANOX) conditions. Results showed an intracellular PHA content as high as 40 ± 2 (%, w/w) 

when ADF conditions were applied with the organic feeding solution made of acetate (85 % on COD basis) and 

propionic (15%) acids. The hydroxyvalerate content in the stored polymer increased (from 25 ± 1 to 41 ± 3, % 

w/w) by increasing the propionic fraction (up to 35%) in the feeding solution. The AE/ANOX condition resulted 

in a lower PHA-storing ability which warrants further investigations. 

1. Introduction 

In recent years, the scientific and industrial attention is being focused on the possibility to replace plastic 

materials with bio-alternatives, having identical or similar properties to conventional ones, but with a lower 

environmental impact (Kourmentza et al., 2017). Among the others, polyhydroxyalkanoates (PHA) are 

particularly interesting substitutes since they are biologically produced, bio-based, and completely 

biodegradable in the environment. Notably, PHA are a family of polyesters with a wide range of thermal and 

mechanical properties, which depend on the length and composition of the side chain (Melendez-Rodriguez et 

al., 2021). Even though industrial PHA production involves pure microbial cultures, mixed microbial cultures 

(MMC)-based processes are recently being investigated at pilot scale since can be integrated into wastewater 

treatment plants to develop an urban biorefinery (Incocciati et al., 2020; Valentino et al., 2021). Also, novel 

processes integrating the side stream treatment of nitrogen removal via nitrite have been proposed (Frison et 

al., 2015). In general, MMC-PHA production implies multistage processes which typically include the acidogenic 

fermentation of waste organic feedstocks to obtain volatile fatty acids (VFA), the selection of PHA-storing 

microorganisms from activated sludge and the polymer accumulation to maximize the intracellular PHA content 

(Reis et al., 2011). The microbial selection stage plays a pivotal role on the overall process performance. A 

common strategy to favour the microbial selection consists in the establishment of dynamic feeding conditions, 

such as the alternance of excess (feast phase) and deficiency (famine phase) of external carbon substrates, 

that induce growth kinetics limitations favoring the accumulation of PHA. In this way, a selective pressure is 

imposed on the microbial culture triggering competition between bacteria that are able to store the externally 

fed carbon source in the form of polymer granules and bacteria that are unable to do so. The former would grow 

during the famine phase using the stored PHA, hence outcompeting non-PHA-storing bacteria (Daigger and 

Grady, 1982; Frigon et al., 2006). The F/F conditions can be easily established in Sequencing Bach Reactors 

163

mailto:marianna.villano@uniroma1.it


(SBR) by applying proper operating conditions (Kourmentza et al., 2017). Here, the performance of a lab-scale 

SBR on the microbial selection of PHA-storing microorganisms has been investigated by using a synthetic 

mixture of VFA containing acetic and propionic acid as carbon source, with the F/F regime established under 

either fully aerobic (ADF) or aerobic/anoxic (AE/ANOX) dynamic conditions.  

2. Material and Methods 

2.1 SBR operative conditions 

This study was performed by using an SBR operated at an applied organic load rate (OLR) of 4.25 gCOD 

(Chemical Oxygen Demand)/Ld, made of a synthetic mixture of acetic (HAc) and propionic (HPr) acid, and at a 

fixed cycle length of 12 h, corresponding to 2 cycles per day. The SBR (1 L working volume, T = 25 °C) was 

inoculated with activated sludge collected from a municipal wastewater treatment plant, stirred by a mechanical 

impeller, and aerated with O2 supplied with air pumps connected to ceramic diffusors. The hydraulic retention 

time (HRT) was set at 1 day and corresponded to the sludge retention time (SRT), since no settling phase was 

performed. Three working conditions (referred to as run 1, run 2, and run 3) were investigated by changing the 

ratio between the organic acids in the carbon feeding solution and the applied feast and famine (F/F) strategy. 

In all cases, an uncoupled carbon and nitrogen supply was applied, with a COD/N ratio between 33 and 35 

gCOD/gN-NH4+. As shown in Figure 1, the phases of each SBR cycle consisted of carbon feeding (C feed, 0.42 

L/cycle), reaction I, withdrawal of the mixed liquor (W, 0.50 L/cycle), nitrogen feeding (N feed, 0.08 L/cycle), and 

reaction II. In run 1, fully aerobic dynamic conditions (referred to as ADF) were imposed to guarantee the FF 

regime, with HAc and HPr accounting for 85% and 15% of the overall COD (Lorini et al., 2020). 

 

  

Figure 1: Scheme of the SBR cycle during either the (a) ADF strategy; (b) or the AE/ANOX feeding strategy 

This feed composition was maintained in run 2 in which, however, the F/F regime consisted of the alternance of 

an aerobic feast phase and an anoxic famine phase (referred to as AE/ANOX). The anoxic conditions were 

established by the absence of oxygen and the presence of nitrite (supplied as sodium nitrite) as electron 

acceptor. The nitrite load accounted for 2 g NO2-/Ld (corresponding to about 0.61 gN/Ld) and this was supplied 

simultaneously with ammonium (as ammonium sulphate). Run 3 was performed under ADF conditions, but with 

the carbon source consisting of 65% and 35% (on COD basis) of HAc and HPr, respectively. 

 

   

Figure 2: (a) Schematic representation of the SBR aimed at the selection PHA-producing microorganisms; (b) 

pictures of the reactor on the first day of operation; (c) and after the establishment of the F/F regime 

The feast phase was aerobic during all the SBR runs and comprised both the C feed phase and the Reaction I 

phase. The reaction II phase was either aerobic (run 1 and run 3) or anoxic (run 2). The duration of each phase 

of the SBR cycles was controlled by a software or digital timers. Nevertheless, the feast phase duration is strictly 

dependent on the rise in the dissolved oxygen concentration in the medium, which consequently determines the 

Cycle length: 12hFeed composition:•Run 1(ADF) and Run 2(AE/ANOX) à85% acetic acid, 15% propionic acid•Run 3(ADF) à65% acetic acid, 35% propionic acidC feed (10 min) W (5min)N feed (5 min)Reaction I AEReaction II AEC feed (10 min) N feed (5min)Reaction I AE Reaction II ANOXW (5min)(a)(b)

164



duration of the famine phase. A schematic representation of the SBR is reported in Figure 2a, along with two 

pictures of the reactor in correspondence to the first day of operation (after inoculation, Figure 2b) and after the 

establishment of steady state conditions (Figure 2c). The change in the colour of the mixed liquor is linked to 

the occurrence of the microbial selection due to the establishment of the F/F regime. 

2.2 Analytical Methods 

The measurement of biomass dry weight was determined as volatile suspended solids (VSS) according to 

standard methods (APHA, 2005). The oxygen profile was measured and registered by an Oxi 3310 (WTW) 

sensor equipped with a DO probe (CellOx 325, WTW). Ammonia quantification was carried out on filtered liquid 

samples (0.45 μm porosity) by using the Nessler spectrophotometric method (APHA, 2005), with the absorbance 

of colored reacted samples measured at 420 nm wavelength (SHIMADZU Spectrophotometer UV-1800). Nitrite 

was quantified by using ion chromatography (Dionex ICS-1000 instrument). Liquid samples for the determination 

of organic acids were filtered (0.45 μm porosity) and injected (1 μL) into a gas chromatograph (Dani Master, 

Milan, Italy) equipped with a flame ionization detector (FID). Organic acids concentrations were converted into 

COD according to the oxidation stoichiometry of 1.07 gCOD/g acetate and 1.51 gCOD/g propionate. Analytical 

determination of PHA was made on 5.0 mL of sample of mixed liquor (without filtration) immediately treated with 

1.0 mL of a NaClO solution (5 % active Cl2) and stored at −20 °C for the following analysis. For the PHA 

quantification samples were extracted and hydrolyzed and the released monomers were esterified into 3-

hydroxyacyl methyl esters using an acidified methanol solution (3% v/v H2SO4) to be quantified by gas-

chromatography (GC-FID Perkin Elmer 8410), as reported elsewhere (Lorini et al., 2020). The relative 

abundance of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) monomers was made by using a commercial 

P(HB/HV) copolymer with a HV content of 5 % (w/w) (Sigma–Aldrich, Milan, Italy). The stoichiometry conversion 

factors of 1.67 gCOD/gHB monomer and 1.92 gCOD/gHV monomer were used to convert the PHA 

concentration in terms of COD. The characterization of the SBR performance was evaluated using the data 

obtained from these analyses. In particular, the intracellular PHA content was calculated as the ratio between 

polymer and VSS concentration (%, w/w) determined in correspondence to the end of the feast phase and the 

PHA composition (%, w/w) was determined as the ratio between HV and (HB + HV) monomers. 

3. Results and discussion 

3.1 SBR operation under ADF and AE/ANOX conditions 

In this study, an SBR aimed at the selection of PHA-storing microorganisms from an activated sludge has been 

operated under feast and famine (F/F) conditions, with uncoupled carbon (C) and nitrogen (N) supply. The 

obtained results (run 2 and run 3) have been compared to the results of a previous study (run 1) (Lorini et al., 

2020). During all the SBR runs, the C source consisted of a synthetic mixture of HAc and HPr. The N source 

(as ammonium sulphate) was fed at the beginning of the famine phase (i.e., after VFA depletion) to be used for 

microbial growth both in fully aerobic (ADF) conditions (run 1 and run 3) and in aerobic/anoxic (AE/ANOX) 

conditions (run 2), wherein N in the form of sodium nitrite was also supplied to serve as the electron acceptor 

during the famine phase. This uncoupled strategy provides an additive means (with respect to the traditional 

F/F regime) to favour the selection of PHA-accumulating bacteria (Lorini et al., 2020). Indeed, only the bacteria 

able to store the carbon source as intracellular PHA during the feast phase can grow in the following famine 

phase, using the provided nutrients and the stored polymer as C and N sources. During each SBR run, the 

exact time in correspondence to the end of the feast phase was identified from the recorded profiles of the 

dissolved oxygen (DO) concentration in the reaction medium. The trend of the DO concentration during a typical 

SBR cycle is reported in Figure 3, for both the ADF (run 3) and AE/ANOX (run 2) conditions (Figure 3a and 

Figure 3b, respectively). From the DO profile, it was possible to recognise the various phases of the SBR cycle 

characterizing the microbial selection process through the F/F regime, numbered (in red) from 1 to 5 in the 

graphs. At the beginning of each cycle, a sudden decrease of the DO concentration occurred from values 

ranging between 6 and 7 mgO2/L (characteristic of the end of the previous cycle) to values of approximately 1 

mgO2/L. This decrease was due to the increased microbial activity triggered by the C source supply during the 

feast phase. When the organic substrate was depleted, a sudden increase in the DO concentration to values 

close to the initial ones occurred, indicating the end of the feast phase, that represents a characteristic moment 

of the SBR working cycle in which the maximum value of PHA concentration in the reactor is typically reached, 

prior to being subsequently consumed as the only carbon source for microbial growth. Under the ADF condition 

(Figure 3a), with oxygen provided during the whole SBR cycle, once the feast phase was over the oxygen 

concentration resumed to the initial value of about 7 mgO2/L and remained approximately constant during the 

entire famine phase. This because the oxygen request for microbial growth by using the intracellular stored PHA 

is lower than that required for the removal of the external substrate during polymer accumulation. As for the 

AE/ANOX condition (Figure 3b), after a sudden increase in its value in correspondence to the end of the feast 

165



phase, the DO profile presented an abrupt decrease until 0 mgO2/L due to the interruption of aeration in the 

SBR to guarantee anoxic conditions in the famine phase. Indeed, in this case, nitrate replaced oxygen as 

electron acceptor to support the microbial growth on the stored polymer (Frison et al., 2015). However, as shown 

in Figure 3b, the oxygen supply was resumed 10 minutes before the end of the cycle to establish the aerobic 

conditions required for the feast phase in the following cycle.  

 

  

Figure 3: Typical profile of dissolved oxygen (DO) concentration in the SBR operated under: (a) aerobic dynamic 

feeding (ADF, run 3); (b) or aerobic/anoxic (AE/ANOX, run 2) feeding strategy 

3.2 SBR performance under different working conditions 

The performance of the three SBR runs has been evaluated in terms of average values of main process 

parameters (e.g., duration of the feast phase, intracellular PHA content, and polymer composition). As for the 

duration of the feast phase, average data are reported in Figure 4a as the ratio between the length of the feast 

phase and the length of the whole SBR cycle (set at 12 hours). The lower obtained value (equal to 21 ± 0.6 %, 

h/h) falls within the range that guarantees an optimal performance in the microbial selection process under ADF 

conditions (run 1). Indeed, as reported in the literature, the threshold value for this ratio corresponds to 

approximately 25 % for ADF processes with coupled feeding of carbon and nitrogen sources (Reis et al., 2011) 

and to 29% for ADF processes with uncoupled C and N feeding (Lorini et al., 2020). Data obtained for run 2 and 

run 3 (i.e., 31 ± 2 % and 33 ± 1 %, respectively) are close but slightly higher that the abovementioned threshold 

value, suggesting a low reduction in the selective pressure required to enrich MMC in PHA-producing 

microorganisms. However, it should be mentioned that in run 2 the end of the aerobic phase (imposed to a 

maximum value of 33% of the whole cycle length) was referred to as the end of the feast phase, even though 

organic acids were not always completely depleted in the reaction medium. The length of the feast phase plays 

a pivotal role in the performance of the selection reactor. If it is too long and the following famine phase too 

short, the non-PHA-accumulating bacteria will be able to survive, being in contact with the external carbon 

source for a long time and the metabolic pathway will be more directed towards growth, negatively affecting 

storage performance (Reis et al., 2011). Indeed, the duration of the feast phase affected the average value of 

the intracellular PHA content obtained for the three runs (Figure 4b). In run 1 the obtained lower length of the 

feast phase reflected in a higher PHA content (in correspondence to the end of the feast phase) with respect to 

the other runs, accounting for 40 ± 2 (%, w/w) (Lorini et al., 2020). Lower values were observed for run 2 and 

run 3, which resulted in 11 ± 1 and 16 ± 1 (%, w/w), respectively. The intracellular polymer content has an impact 

on the following biopolymer extraction steps, since an increase in the extraction costs is due to low contents as 

well as to a high presence of non-PHA-storing microorganisms (Tan et al., 2014). In terms of composition of the 

stored PHA, a poly(3-hydroxybutyrate/3-valerate) copolymer, P(HB/HV), was obtained in all the investigated 

conditions, due to the presence of both HAc and HPr in the synthetic carbon feeding solution. The P(HB/HV) 

copolymer has improved properties over the polyhydroxybutyrate (PHB) homopolymer, that is the most 

extensively studied PHA. As reported in the literature (Reis et al., 2011), the incorporation of short-chain 

monomers other than 3-hydroxybutyrate (HB), such as 3-hydroxyvalerate (HV), affects polymer crystallinity as 

well as the thermal and mechanical properties. The extent of the effect is related to the HV content in the 

copolymer (Melendez-Rodriguez et al., 2021) which, in turn, is linked to the final PHA applications (Muneer et 

al., 2020). Figure 4b shows the average value of the HV content in the polymer stored during the three SBR 

runs. In particular, run 1 and run 2 exhibited a comparable value of the HV content, accounting for 25 ± 1 and 

23 ± 1 (%, w/w), respectively; whereas the maximum obtained value (equal to 41 ± 3 %, w/w) was reached 

during the operation of run 3. This because, even though the VFA composition remained qualitatively unchanged 

for the three SBR runs, the percentage of HPr was the same (15% on COD basis) for run 1 and run 2, but 

significantly higher (35%) for run 3. HAc is involved in the synthesis of both the HB and HV monomers, while 

166



HPr is mainly involved in the production of the HV monomer (Serafim et al., 2008). Therefore, as expected, the 

higher HV content obtained in run 3 than in the other runs is primarily associated to the higher percentage of 

HPr in the organic feeding solution fed to the SBR, pointing out the fact that the PHA composition is independent 

on the strategy used to apply the F/F regime, either fully aerobic (run 1) or aerobic/anoxic (run 2). Furthermore, 

under ADF conditions (run 1 and run 3), a higher concentration of HPr in the VFA feeding solution likely led to 

a lower intracellular polymer content. The kinetics of substrate consumption, and therefore the kinetics of 

polymer storage during the feast phase, may have been slowed down in run 3 as PHA storing microorganisms, 

in contact with a higher concentration of HPr than in run 1, took longer time to synthesise the more complex HV 

monomer, thus affecting the length of the feast phase but also the total intracellular PHA content. Indeed, the 

metabolism of PHA production is dependent on the substrate composition since the amount of energy (e.g., as 

ATP) required for the conversion of different organic acids into PHA is different. In fact, HAc is converted to 

acetyl-CoA and subsequently to PHB, while HPr is converted to propionyl-CoA and then to PHV (Dias et al., 

2005). Therefore, HAc needs less ATP to produce one C-mol of PHA compared to HPr (Wang et al., 2017). 

 

  

Figure 4: (a) Average value of the length of the feast phase to the overall SBR cycle length; (b) intracellular 

polymer content and HV content in the stored polymer at the end of the feast phase 

Table 1 summarizes the average values of the main parameters characterizing the SBR performance. The 

amount of polymer stored during each run (ΔPHA) was calculated as the difference between the maximum (in 

correspondence to the end of the feast phase) and minimum (end of the famine phase) values of PHA 

concentration in the reactor. The greater this difference, the greater the ability of bacteria to grow on the stored 

polymer. In addition, once the famine phase is over the bacteria, having consumed the stored polymer, will again 

be ready to store the external carbon source in the form of PHA, implying a favourable microbial selection. The 

greater (ΔPHA) value detected for run 1 is a further indication of the good selective pressure obtained during its 

operation, characterized by a fully aerobic F/F regime and 15% of propionic acid in the organic feeding solution. 

However, by operating the F/F regime under AE/ANOX conditions without changing the VFA composition (run 

2), the value of (ΔPHA) significantly decreased. Indeed, during the operation of run 2, when acids were still 

present in the reaction medium during the anoxic phase, they were consumed along with ammonium, that was 

not further consumed after VFA depletion. This resulted in a higher value of ammonium (about six-fold increase 

with respect to run 1) detected at the end of the cycle, that was used for microbial growth during the following 

aerobic phase in presence of the carbon source, negatively affecting the polymer storage.  

Table 1: Main parameters characterizing the SBR performance under the different working conditions 

Run ΔPHA  

(mg/L) 

PHA end feast 

(wt/wt, %) 

HV end feast 

(wt/wt, %) 

N-NH4+ end cycle 

(mgN/L) 

NO2- end cycle 

(mgNO2-/L) 

Run 1 (Lorini et al, 2020) 572 ± 54 40 ± 2 25 ± 1 14.0 ± 2 N/A 

Run 2 (This study) 35.3 ± 7 11 ± 1  23 ± 1 83.0 ± 8 42.3 ± 20 

Run 3 (This study) 232 ± 29 16 ± 1 41 ± 3 20.2 ± 3 N/A 

 

Finally, the nitrite ion was almost completely consumed (over 95%) by the end of the cycle, being used as 

electron acceptor. Overall, during the operation of run 2, the SBR performance was unstable and the AE/ANOX 

feast and famine strategy needs to be further investigated and optimized in terms of PHA production. This also 

because it presents several advantages, such as the possibility to integrate the production of PHA with the 

nitrification/denitrification process, as well as the possibility to save, during the anoxic famine phase part of the 

oxygen required in the ADF processes (Frison et al., 2015). 

0

10

20

30

40

Run 1 Run 2 Run 3

Le
n

g
th

 o
f 

fe
a

st
 p

h
a

se
 (

%
, 

h
/h

) (a)

0

10

20

30

40

50

Run 1 Run 2 Run 3

P
H

A
 a

n
d

 H
V

 c
o

n
te

n
t 

(%
, 

w
/w

)

Intracellular PHA HV(b)

167



4. Conclusions 

This study compared the performance of a lab-scale SBR aimed at the selection of PHA-storing microorganisms, 

wherein the required feast and famine (F/F) regime was established under either fully aerobic (ADF) or 

aerobic/anoxic (AE/ANOX) conditions. Three SBR runs were performed at a fixed cycle length of 12 h and an 

applied organic load rate of 4.25 gCOD/Ld, consisting of a synthetic mixture of acetic (from 65% to 85% of the 

overall COD) and propionic (from 15% to 35%) acids. A higher storage ability was observed with ADF relative 

to the AE/ANOX conditions, and the highest obtained intracellular PHA content accounted for 40 ± 2 (%, w/w). 

As for the PHA composition, the obtained results highlighted that it is independent on the strategy used to apply 

the F/F regime. However, as expected, an increase of propionic acid in the feeding solution resulted in an 

increase of the HV content (up to 41 ± 3 %, w/w) in the stored polymer. In AE/ANOX conditions, over 95% of 

nitrite was used as electron acceptor during the anoxic famine phase, but the process was unstable thus 

indicating the need for further investigation and optimization in terms of PHA production. 

Acknowledgements 

This research was supported by Sapienza University of Rome (Ateneo 2019 grant n. RM11916B87F9FBFB).  

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	Polyhydroxyalkanoates Production by Mixed Microbial Cultures in Sequencing Batch Reactors Operated under Different Feeding Conditions