Biochemical Engineering Journal 189 (2022) 108715

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Polyhydroxybutyrate production in one-stage by purple phototrophic

bacteria: Influence of alkaline pH, ethanol, and C/N ratios
Virginia Montiel-Corona , Germán Buitrón *
Laboratory for Research on Advanced Processes for Water Treatment, Unidad Académica Juriquilla, Instituto de Ingeniería, Universidad Nacional Autónoma de México,
Blvd. Juriquilla 3001, Querétaro 76230, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: This work aimed to evaluate the effect of alkaline pH, nutrient limitation, nitrogen source, C/N ratio, and ethanol
Polyhydroxybutyrate addition on polyhydroxybutyrate (PHB) production by a photoheterotrophic mixed culture enriched in Rho­
Ethanol dopseudomonas palustris. The results demonstrated the natural alkalinization of the culture medium was enough
Purple bacteria
to induce PHB accumulation in the mixed culture. The combination of alkaline pH, nitrogen, sulfur, or phosphate
Alkaline pH
deficiency negatively affected polymer production. The incorporation of glutamate for biomass synthesis was
Nutrients limitation
better than that of ammonium, which had a positive effect on PHB production. Interestingly, ethanol addition
improved the PHB accumulation by 25 %, but without consumption. PHB production improved at low C/N
ratios, where 27 ± 0.9% of the carbon source was transformed into biopolymers. The evaluated factors allowed
the biomass PHB content to increase by 60%, reaching PHB production of 219 ± 5 mg/L. The PHB production by
the photoheterotrophic mixed culture is associated with growth, allowing the PHB production in one stage. That
has significant advantages in technical-practical terms, such as reducing the time of the process, dispensing with
the separation of biomass from the feast phase to the famine phase, and requiring a single reactor.

1. Introduction
Approximately 90 genera of microorganisms can accumulate poly­
hydroxyalkanoates (PHA) as energy reserves and protection against
stress factors [1]. Although there are other biopolymers, PHA have
unique properties and advantages, mainly due to its rapid biodegrada­
tion and medical applications [2]. Currently, the PHA production cost is
higher than that of petrochemical-origin polymers [3], so to promote its
use, it is necessary to reduce production costs. Thus, the focus of the
research today is on developing more efficient and practical processes,
such as a process under a permanent feast regime in one stage [4] using
organic waste streams as substrate and mixed cultures that can accu­
mulate higher levels of PHA [3–5].
Purple non-sulfur bacteria (PNSB) recently received attention due to
their ability to accumulate high percentages of PHA, use complex sub­
strates, and grow under anaerobic conditions. The elimination of the
aeration system is significant, considering the high energy demand that
increases the production cost [2,4]. In addition, PNSB can produce other
high-value-added substances together with PHA. Although these ca­
pacities make the use of PNSB for PHA production attractive, they have
been little studied for this purpose (compared to hydrogen production)
[2]. In the works reported on the subject, several differences in the
growing conditions have been reported to stimulate PHA production by
PNSB. PHA production by PNSB depends on the type of culture, carbon
source, nitrogen source, C/N ratio, limitations of phosphorus (P), sulfur
(S), nitrogen (N), and micronutrients, pH, light intensity, among others
[2,6].
Regarding cultures, for technical and economic reasons, using PNSB
mixed cultures is convenient [3]. These microorganisms exist in acti­
vated sludge systems, wastewater treatment plants, rivers, paddy fields,
and aquatic sediments [7]. For their enrichment, Winogradsky columns
have been used successfully to obtain PNSB [5,8]. The carbon, oxygen,
and sulfur gradients that develop in the Winogradsky columns allow the
stratified growth of microorganisms and their easy separation [9].
Regarding nutrients such as N, S, and P, some studies have reported
that their individual or sequential limitation stimulates poly­
hydroxybutyrate (PHB) accumulation [10–12]. However, the nutrient
limitation is inappropriate for PHB production by marine purple
non-sulfur bacteria, as reported by Higuchi-Takeuchi and Numata [13].
That indicates that the effect of nutrient limitation depends on the strain

* Corresponding author.
E-mail address: GBuitronM@iingen.unam.mx (G. Buitrón).

https://doi.org/10.1016/j.bej.2022.108715
Received 12 July 2022; Received in revised form 21 October 2022; Accepted 3 November 2022
Available online 5 November 2022
1369-703X/© 2022 Elsevier B.V. All rights reserved.
V. Montiel-Corona and G. Buitrón
type and cannot be generalized to all species of PNSB. PHA production
under nutrient sufficiency allows the utilization of a one-stage process
instead of a two-stage process. In a classical two-stage process, biomass
must be separated after the first stage (feast phase) to transfer to the
starvation phase, complicating the production process.
One of the induction mechanisms for PHB production by PNSB is the
high light intensity [4]. The pH in the culture media is decisive in the
PHB production process. Some microorganisms can carry out the process
at neutral pH; in other cases, alkaline pH improves PHB production [14].
Suzuki et al. [15], using Rhodobacter sphaeroides RV suggested the in­
duction of PHB production by growing the biomass at neutral pH and
raising the pH to a suboptimal value. However, the ideal would be to use
microorganisms that do not require such changes.
The nitrogen source also plays an essential role in PHB production. It
has been reported that the nitrogen source does not affect PHB pro­
duction [16] or that ammonia favors PHB production rather than
glutamate [17]. In contrast, others recently reported the opposite [12,
18]. Such results can be related to the different PNSB cultures used, as
suggested by Sali and Mckey [6]. Therefore, defining the appropriate
nitrogen source to induce polymer production in each culture and the
carbon/nitrogen ratio (C/N) is crucial. PHB production by PNSB is often
accompanied by simultaneous hydrogen production, promoted by a
high C/N ratio. The C/N ratio can define which product is obtained in a
higher proportion: PHB or hydrogen [19].
Hydrogen and PHB production are routes that compete for reducing
power in the PNSB, and frequently, the same factors that affect either of
these pathways usually affect the other. Oh et al. [20] and Kim et al. [21]
reported that adding ethanol improved hydrogen production in Rhodo­
bacter sphaeroides. However, the PHB production was not evaluated, and
there is no information on the ethanol effect on PHB production. It is
more than interesting to define the impact of ethanol on polymer pro­
duction, given that residues from the fermentation industry used for
PHA production may contain high concentrations of ethanol [18]. Ef­
fluents from chain elongation technology enriched in medium-chain
carboxylic acids (MCCA) are being used for PHA production [22], and
these effluents often contain significant concentrations of residual
ethanol [23].
This work aims to select a mixed photoheterotrophic culture
enriched in PNSB with the ability to accumulate PHB and to evaluate the
effect of alkaline pH, the limitations of S, N, and P, the nitrogen source
(ammonium and glutamate), ethanol addition, and different C/N ratios
on PHB production using acetate as carbon source.
2. Materials and methods
2.1. Purple phototrophic bacteria
The photoheterotrophic mixed culture was obtained using a
Winogradsky column prepared as described by Montiel-Corona et al.
[5]. Activated sludge collected in a wastewater treatment plant in
Querétaro, Mexico, was used as inoculum. The activated sludge (500
mL) supplemented with 12.5 g CaSO4, 12.5 g CaCO3, and 25 g of
sawdust was added to the bottom of a glass cylinder of 4 cm diameter
and 1 L capacity. The rest of the cylinder was filled with water from the
activated sludge settled. The column was tapped and placed next to a
tungsten lamp (30 cm) for about 15 days. After that period, when a
reddish band formed below the surface of the water column, a sample
was collected and transferred to 120 mL vials with 85 mL of modified
medium for Rhodospirillaceae (German Collection of Microorganisms
and cell cultures GmbH_DSMZ: https://www.dsmz.de). The modified
Rhodospirillaceae medium contained (in g/L) yeast extract, 0.30; sodium
acetate, 2.00; (NH4)-acetate, 0.5; NH4Cl, 0.40; KH2PO4, 0.50; MgSO4,
0.19; NaCl, 0.4; CaCl20.2 H2O, 0.05; Fe(III) citrate, 0.01; L-cysteinium
chloride, 0.30; thiamine, 0.001; nicotinic acid, 0.001; trace element
solution, 0.4 mL. The trace element solution contained (in g/L)
ZnSO40.7 H2O, 0.1; MnCl20.4 H2O, 0.03; H3BO3, 0.30; CoCl20.6 H2O,
2
Biochemical Engineering Journal 189 (2022) 108715
0.20; CuCl20.2 H2O, 0.01; NiCl20.6 H2O, 0.02 and NaMoO40.2 H2O,
0.03. The pH was adjusted to 6.8. The medium was poured into 120 mL
vials fitted with rubber stoppers and flushed with helium to create
anaerobic conditions; finally, the medium was sterilized at 120 ◦ C for 15
min. The inoculated vials were incubated at 3 kLux in a photoregulated
chamber using tungsten lamps, and the temperature was maintained at
30 ◦ C. A total of eight subsequent subcultures were performed. The
resulting enriched culture named WWTP_W2 was used for PHB pro­
duction. For the PHB production test, the WWTP_W2 culture was grown
in Rhodospirillaceae medium and used as inoculum until it reached a
concentration of 1000–1200 mg DW/L (after about 48 h).
2.2. Test for PHB production with WWTP_W2 culture
An experiment with 4 g/L of sodium acetate as a carbon source and
0.5 g NH4Cl/L as a nitrogen source (as proposed by Carlozzi et al. [12])
was carried out using a modified Rhodospirillaceae medium to evaluate
the ability of the WWTP_W2 mixed culture to produce PHB. NH4-acetate
was removed from the modified Rhodospirillaceae medium, and L-cys­
teinium chloride (0.3 g/L) was kept. For one treatment, the initial pH
was adjusted to 6.8, and then it was not controlled; another treatment
was operated with pH control, which was maintained between 7.5 and
7.8 by adding a solution of HCl 0.2 N. The experiments were carried out
in triplicate in 150 mL serum bottles with 120 mL of work volume, 110
mL of medium, and 10 mL of inoculum (equivalent to 90–98 mg dry
weight (DW)/L). The serum bottles were fitted with rubber stoppers, and
helium was used for flushing the medium and headspace to maintain
anaerobic conditions. All serum bottles were then inoculated with 10 mL
of WWTP_W2 culture and incubated at 3500–3800 Lux in a photo­
regulated chamber using tungsten lamps (75 W), and the temperature
was maintained at 28–32 ◦ C. The treatments were conducted in batches
using simultaneously one-stage experiments, i.e., the growth and PHB
production. A sample was taken every 2 or 3 days to determine biomass
and PHB production, acetate removal, and pH. The experiment
concluded when PHB production ceased and PHB consumption began.
2.3. Microbial community characterization
After the test of PHB production, the biomass sample of the mixed
culture was recovered by centrifugation at 14,000 rpm for 10 min. Ac­
cording to the manufacturer’s instructions, genomic DNA was extracted
from the biomass sample using the DNeasy PowerSoil Kit (QIAGEN,
Germany). DNA was quantified by spectrophotometry using a NANO­
Drop 2000c (Thermo Scientific, USA). DNA with an absorbance 260/280
ratio of 1.89 was submitted to the Integrated Microbiome Resource
Laboratory (IMR) located at Dalhousie University, Canada, for Illumina
MiSeq sequencing analysis employing primers targeting the variable
region V3-V4 of the 16 S rDNA gene of bacteria (341 F 5′ -
CCTACGGGNGGCWGCAG-3′ and 805 R 5 ´-GACTACHVGGGTATC­
TAATCC-3′ ). The sequences were analyzed with the DADA2 methodol­
ogy in the R environment, according to Callahan et al. [24]. Forward and
reverse reads were filtered and truncated to 290 and 180 nucleotides,
respectively. The SILVA database v. 138 was used for the taxonomic
classification of bacterial amplicon sequence variants (ASVs). The rela­
tive abundances of ASVs were reported as a percentage of the total
sequencing read count.
2.4. Experiments under nutrient deficiency
Due to the poor growth of PNSB cultures under nutrient-deficient
conditions [10,12,17] and as a conventional process under a feast/f­
amine regimen, a two-stage process for PHB production was used. In the
first stage, sufficient cell growth was achieved (1216 ± 13 mg DW/L),
and in the second stage, the nutrient limitation was established. The
inoculum grew in Rhodospirillaceae medium for 48 h (with nutrient
sufficiency as Section 2.1) and was centrifuged at 6000 rpm for 8 min.
V. Montiel-Corona and G. Buitrón
The pellet was resuspended in the same medium deficient in nitrogen,
phosphorus, and sulfur, as applicable. The initial sodium acetate con­
centration was 4 g/L, as Touloupakis et al. [25] reported. NH4Cl (0.5
g/L) and L-cysteinium chloride (0.3 g/L) were used as nitrogen sources
for the experiments under P and S limitations. The initial pH was
adjusted to 6.8, and then the pH was not controlled. The experiments
were carried out in triplicate in 150 mL serum bottles with 120 mL of
work volume and inoculum equivalent to 1216 ± 13 mg DW/L. The
treatments were flushed with helium to establish anaerobic conditions.
The incubation conditions were the same as in Section 2.2.
2.5. Nitrogen source
The influence of ammonium and glutamate as nitrogen sources on
PHB production was evaluated in a batch experiment using the modified
Rhodospirillaceae medium (under nutrient sufficiency and one-stage).
NH4-acetate was removed from the medium, and L-cysteinium chlo­
ride (0.3 g/L) was kept. In this case, a Carbon/Nitrogen ratio (mol/mol)
of 16 was selected according to Kim et al. [16]. The NH4Cl and mono­
sodium glutamate concentrations were 0.5 g/L and 1.58 g/L, respec­
tively. The concentration of both substances was adjusted so that the
contribution in moles of nitrogen was the same, 9.34 mM of N. Adding
the nitrogen from L-cysteinium, the total concentration was 11.05 mM
of N/L. Acetate was used as a carbon source. The carbon from the
glutamate molecule was considered to establish the C/N ratio of 16.
Therefore, 7.4 g/L of acetate were added for the ammonium treatments
and 5.49 g/L for the glutamate treatments. The initial pH was adjusted
to 6.8, and then the pH was not controlled. The experiments were car­
ried out in triplicate in 150 mL serum bottles with 120 mL of work
volume, 110 mL of medium, and 10 mL of inoculum (equivalent to
89–93 mg DW/L). The incubation conditions were the same as in Section
2.2.
2.6. Experiments with ethanol
The experiment with ethanol was performed in a modified Rhodo­
spirillaceae medium at a carbon-to-nitrogen ratio of 16 (as the Section
2.5). The treatment “Glutamate + ethanol” was supplemented with 1.58
g/L of glutamate, 4.08 g/L of sodium acetate, and 0.789 g/L of ethanol.
The treatment “NH4 + ethanol” was supplemented with 0.5 g/L of
NH4Cl, 6.0 g/L of acetate, and 0.789 g/L of ethanol. The carbon from
acetate, ethanol, and glutamate was considered to establish the C/N
ratio of 16. The incubation and experiment conditions were the same as
in Section 2.2 (batch and one-stage). This experiment was simultaneous
to the experiment in Section 2.5.
2.7. Experiments at different carbon-to-nitrogen ratios
Once the effect of ethanol and the nitrogen source was defined, the
best C/N ratio was established. C/N (mM/mM) ratios of 12, 13, 14, 16,
18, and 20 were tested. Glutamate was used as a nitrogen source at a
constant concentration (1.58 g/L), 0.789 g/L of ethanol was added to all
treatments, and the sodium acetate concentration was 2.26, 2.72, 3.17,
4.08, 4.98, and 5.89 g/L for C/N ratios of 12, 13, 14., 16, 18, and 20,
respectively. The carbon from acetate, ethanol, and glutamate was
considered to establish the C/N ratios. The incubation and experiment
conditions were the same as in Section 2.5 (batch and one-stage).
2.8. Analytical methods
The samples were centrifuged at 14,000 rpm for 10 min, and the
biomass pellets were dried at 105 ◦ C until constant weight to determine
the biomass concentration as dry weight (DW). Quantitative PHB
determination was carried out according to Brandl et al. [26]. Dried cells
(approximately 10 mg) were weighed in screw-top glass test tubes, and
2 mL of chloroform, 1.7 mL of methanol, and 0.3 mL of sulfuric acid
3
Biochemical Engineering Journal 189 (2022) 108715
were added for the methanolysis reaction. The tubes were subjected to
100 ◦ C for 140 min. After cooling to room temperature, 1 mL of distilled
water was added, and the mixture was shaken in a vortex for 1 min. After
1 h, the organic phase (bottom layer) was filtered through a 0.45 µm
nylon syringe filter. The samples were analyzed in an Agilent 6890 gas
chromatograph (Santa Clara, CA) equipped with a DB-FFAP column (15
m by 0.32 mm by 0.5 µm; Agilent) and a flame ionization detector. One
microliter of each sample was injected. Nitrogen was used as the carrier
gas at a 2.5 mL/min flow rate. The oven was held at 60 ◦ C for 1 min,
heated to 120 ◦ C at 10 ◦ C/min, then to 250 ◦ C at 45 ◦ C/min, and held for
5 min. The injector and detector temperatures were 250 and 270 ◦ C,
respectively. A PHBV standard (Sigma Aldrich) was used to generate
calibration curves.
The residual acetic acid concentration was determined by gas chro­
matography. The samples were centrifuged at 14,000 rpm for 10 min;
the supernatant was filtered through a 0.45 µm membrane and subse­
quently analyzed using a gas chromatograph (Agilent 7890) equipped
with a flame ionization detector and a 15-m-long (0.53 mm i.d.) Zebron
DB-FFAP column. The injector and detector temperatures were 190 and
210 ◦ C, respectively. The column temperature was 45 ◦ C for 1.5 min and
increased to 135 ◦ C at a ramp rate of 8 ◦ C/min. Nitrogen was used as the
carrier gas at a flow rate of 9.5 mL/min.
The ammonium ion concentration was determined by the phenol-
hypochlorite method described in Weatherburn [27]. Extracellular
polymeric substances (EPS) as carbohydrates and proteins were quan­
tified according to Arcila and Buitrón [28].
The hydrogen gas volume was measured by the water displacement
method. The H2 content in the serum bottle headspace was determined
by TCD gas chromatography (SRI 8610 C) equipped with a silica-gel
packed column 6′ X 1/8", with N2 as a carrier gas (20 mL/min). The
column temperature was 40◦C for 4 min, increased to 110 ◦ C at a rate of
20 ◦ C/min, and was held for 3 min
2.9. Calculations
The biomass PHB content was calculated as a percentage of dry
weight:
PHB content in dry-biomass (% w/w) = (mg PHB/mg DW)*100
where the biomass expressed as dry weight (DW) includes the mi­
crobial active biomass and PHB.
The PHB yield per substrate consumed was calculated by dividing the
amount of PHB produced by the amount of acetate consumed:
Y (%) = (Cmol PHB/Cmol Acetate)*100
PHB/Ac
Where PHB and acetate are expressed as moles of carbon.
PHB productivity = Final PHB concentration/time, where time is the
day in which the maximum PHB production was reached. The experi­
ments under nutrient deficiency include both the cell growth and star­
vation periods.
One-way analysis of variance (ANOVA) test and Tukey’s test in
pairwise comparisons were performed to evaluate the statistical signif­
icance of the data. P-values less than 0.05 were accepted to be
significant.
3. Results and discussion
3.1. Microbial composition of WWTP_W2 mixed culture
Fig. 1 (A) shows the microbial community composition at the genus
level for the WWTP_W2 culture. The culture presented a higher relative
abundance of the Rhodopseudomonas genus, 72 %. Other genera detected
were Proteocatella spp., Wolinella spp., and Arcobacter spp. (with relative
abundances of 6.7 %, 2.5 %, and 1.96 %, respectively). Proteocatella is a
hydrolytic protein genus present in anaerobic sludge [29], Wolinella is a
V. Montiel-Corona and G. Buitrón
Fig. 1. (A). Microbial community composition at the genus level for WWTP_W2 culture. The figure shows the relative abundance of the major microbial groups.
“Others” includes genera with < 0.25% relative abundance; "NA" are microorganisms identified up to family or order but not up to the genus level. (B) Relative
abundance of Rhodopseudomonas species present in the mixed culture. “NA” are microorganisms identified up to the genus level but not to the species level.
genus of non fermentative bacteria present in the rumen of animals [30],
and the members of the genus Arcobacter are ubiquitous and harbors in
the gastrointestinal tract of many animals and humans [31]. Other
genera with low relative abundances were Anaerostignum (1.33 %),
Proteiniclasticum (0.95 %), and Anaerovorax (0.81 %), all of them har­
bors in the gastrointestinal tract of animals and have been detected in
activated sludge from urban wastewater plants [32–34]. Except for
Rhodopseudomonas, the other genera present have not been related to
PHA production [35].
Regarding Rhodopseudomonas genus, several species of purple non-
sulfur bacteria belong to this genus. This genus is abundant in
wastewater-activated sludge [7], hence the presence of these microor­
ganisms in the mixed culture obtained. A species-level analysis was
performed to identify the Rhodospseudomonas species in the mixed cul­
ture (Fig. 1B). The identified species were Rhodopseudomonas faecalis,
Rhodopseudomonas harwoodiae, Rhodopseudomonas palustris, Rhodop­
seudomonas pentothenatexigens, and Rhodopseudomonas thermotolerans.
Of the total bacteria belonging to the Rhopseudomonas genus, 90% was
the specie Rhodopseudomonas palustris, a PNSB widely known for accu­
mulating PHB and producing hydrogen. Among the PNSB that can
accumulate PHB, this bacterium has a relatively low ability to accu­
mulate the polymer. In contrast, Rhodobacter sphaeroides and Rhodo­
bacter capsulatus can accumulate up to 80 % by weight; for
Rhodopseudomonas palustris, no more than 50 % by weight has been
reported [5,12,36]. However, a percentage of 50 % is not negligible
compared to aerobic systems. Furthermore, R. palustris stands out for its
ability to degrade a wide range of complex substrates [2,6].
Rhodopseudomonas faecalis, Rhodopseudomonas harwoodiae, Rhodop­
seudomonas pentothenatexigens, and Rhodopseudomonas thermotolerans
4
Biochemical Engineering Journal 189 (2022) 108715
have been reported as hydrogen producers [37,38]. There are no reports
on PHB production with these species. Due to their low relative abun­
dance in the WWTP_W2 cultures (from 0.03 % to 0.17 %), PHB pro­
duction can be attributed to Rhodopseudomonas palustris (see Section
3.2). Another purple non-sulfur bacterium detected was Rhodobacter
capsulatus of the Rhodospirillaceae genus, with a relative abundance of
0.05 %. Although this species can accumulate high percentages of PHB,
its contribution to PHB production was probably low due to its low
relative abundance in the WWTP_W2 culture.
In principle, the Rhodospirillaceae culture medium was selected to
favor the growth of Rhodobacter sphaeroides or Rhodobacter capsulatus.
However, Rhodopseudomonas palustris predominated in the mixed cul­
ture obtained; the same result was obtained by Zhao et al. [39], who also
used the Rhodospirillaceae medium for enrichment. In other studies,
using Beibl and Pfennig’s and PSB medium, they isolated R. palustrsis
[40–42], and the common denominator was the inoculum source of the
activated sludge. That is indicative that the predominance of R. palustris
in activated sludge is high.
Using Winogradsky columns and activated sludge to obtain a culture
enriched in PNSB was effective. Activated sludge can contain up to 102 –
105 viable cells of PNSB per milligram of activated sludge [7]. On the
other hand, the stratified growth of microorganisms in the microenvi­
ronments that develop in the columns [9] made it possible to obtain a
mixed culture with a high percentage of PNSB. The traditional enrich­
ment method is often inefficient for the enrichment-isolation of PNSB
due to the slow growth rate of PNSB compared to other bacteria [8]. In
the Winogradsky column, ascending oxygen gradients (from the bottom
to the top) and a descending gradient of sulfur and carbon (from the
bottom to the top) are formed, allowing sulfate-reducing
V. Montiel-Corona and G. Buitrón
microorganisms to develop at the bottom of the column. Also, green
sulfur, purple sulfur, and purple non-sulfur bacteria grew in different
layers observing microalgae growing on the surface. The space-time
separation in the development of microbial communities facilitates the
recovery of PNSB.
3.2. Alkaline pH and PHB production in the WWTP_W2 mixed culture
Among the culture conditions that positively affect PHB production
is the alkaline pH [17,43,44]. That was the first condition tested in the
WWTP_W2 culture to induce PHB production. Since this stress factor is
easy to implement, the acetate consumption tends to alkalinize the
medium without requiring the addition of an external agent [5,16,17].
Fig. 2 shows the kinetics of biomass production, acetate consumption,
PHB content, PHB production, and pH for the treatment without pH
control. The treatment reached maximum PHB production (115
± 7 mg/L), biomass production (1275 ± 12 mg DW/L), and acetate
consumption (2344 ± 68 mg/L) on the third day. Notably, the pH
increased from 6.8 to 9.6, and its increase was parallel to polymer
production (Fig. 2B). The acetate consumption to produce biomass
alkalinized the medium; therefore, polymer production was induced.
Slight increases in pH can improve PHB production, Khatipov et al. [17]
reported that an increase in pH from 6.8 to 7.5 tripled the PHB accu­
mulated. Fig. 2B shows that on day two, the pH passed from 6.8 to 7.3,
and concomitantly 45 mg/L of PHB were produced. On day 3 the pH was
9.6, the polymer production reached 115 mg/L, and the PHB content in
dry biomass was 9 ± 0.4 % (w/w). In the treatment with pH control
(data not shown), the PHB accumulated was 50 % lower than in the
treatment without pH control (4.4 % ± 0.2 % w/w), which confirms the
alkalinization of the medium was responsible for improving PHB pro­
duction in the WWTP_W2 culture. Higher environmental pH values can
favor the entry of organic acids (such as acetic acid) into the cell, which
can, in turn, be converted into PHB. As Zhou et al. [44] mentioned, this
might partly explain why alkaline pH favors PHB accumulation. Another
explanation is related to hydrogen production. Hydrogen and PHB
production are routes that compete for the reducing power in the PNSB.
Some PNSB produce hydrogen simultaneously with PHB, as observed by
Carlozzi and Touloupakis [45]. However, there are reports that alkaline
pH enhances PHB production due to the inhibition of hydrogen pro­
duction [17]. In PNSB, hydrogen production is associated with nitro­
genase, which loses activity at alkaline pH [46]. In this work, there was
no hydrogen production in both treatments (under pH control and
non-pH control), which might be related to those mentioned above and
to ammonia’s presence. Nitrogenase is an enzyme that catalyzes
hydrogen production under ammonium or molecular nitrogen limitation
Fig. 2. Kinetics of biomass production, PHB content in dry-biomass, acetate consumption (A), pH and PHB production (B) for the WWTP_W2 culture (under nutrient
sufficiency). Every point represents the average of three determinations and its standard deviation.
5
Biochemical Engineering Journal 189 (2022) 108715
[47]. In both treatments, the initial ammonium concentration was
130 mg NH+ 4 /L, and, at the end of the process, there was 40–20 mg
NH+ 4 /L, a concentration sufficient to inhibit the hydrogen production
process [47].
Polymer production is associated with WWTP_W2 culture growth.
The increase in biomass concentration and PHB accumulation coincided
(Fig. 2A), which can be an advantage by reducing the time in which the
polymer is produced. In other cases, it has been observed that the
biomass is produced first and then the PHB, delaying the harvest time of
the polymer. Carlozzi et al. [12], using acetate and ammonium under
nutrient-sufficient conditions, did not detect PHB production during the
first three days of culture, where 75 % of biomass production was
reached, and PHB production began between days 4 and 5.
In conventional PHB production processes under a feast/famine
regime, the accumulated PHB is used by the bacteria to survive when the
carbon source becomes limiting. In this work, after the third day, the
accumulated polymer consumption began despite no carbon source
limitation. That can be related to the PHB degradation storage strategy
for coping with the stress caused by the increase in pH [14]. Although
the pH can induce PHB production, once synthesized, it can be
degraded-transformed to survive at that suboptimal pH [14].
3.3. PHB production under nitrogen, phosphate, and sulfur deficiency
There are two groups of PHA-producing bacteria. In the first group,
the bacteria require nutrient or carbon source limitations to accumulate
PHA. In the second group, the microorganisms do not require nutrient
limitation and accumulate the polymer during the growth phase [1].
Some PNSB strains belong to the first group, such as Rhodopseudomonas
palustris 42OL [10], Rhodospirillum rubrum [11], and Rhodopseudomonas
sp. S16-VOGS3 [12] and others such as Rhodovulum sulfidophilum, Rho­
dovulum euryhalinum, Rhodovulum imhoffii, and Rhodovulum visakha­
patnamense belong to the second group [13].
The WWTP_W2 culture was subjected to PHB production under ni­
trogen, phosphate, and sulfur deficiency to establish under what con­
ditions the WWTP_W2 culture produces PHB and to determine if,
together with the increase in pH (Section 3.2), the nutrients limitation
can improve the PHB yields.
The PHB content in dry biomass was 6.8 ± 0.20 %, 3.8 ± 0.03 %,
and 4.8 ± 0.12 % for the treatments with N, P, and S deficiency,
respectively (Fig. 3). The four treatments reached maximum PHB pro­
duction, biomass production, and acetate consumption on the third day
(not counting the biomass production phase) and under nutrient-
sufficient treatment (Fig. 2). In the treatments with N, P, and S defi­
ciency, biomass production increased by 301, 792, and 934 mg/L,
V. Montiel-Corona and G. Buitrón Biochemical Engineering Journal 189 (2022) 108715

Final pH

9.3
9.1
9.0
PHB productivity
(mg/L-d)

20
17
19
PHB production
(mg/L)

98 ± 3
83 ± 2
93 ± 3
Y PHB/Ac
Fig. 3. Kinetics of PHB content in dry-biomass under nitrogen, phosphate and

6.85
7.04
sulfur deficiency.

12.5
(%)

Time: The data presented correspond to the day the maximum PHB production was reached, including both the cell growth and starvation period.
respectively, directly related to acetate consumption (Table 1). Vin­
cenzini et al. [10], Khatipov et al. [17], and Carlozzi et al. [12] also

PHB content in dry biomass

observed biomass production despite the nutrient limitation, although
the increase was low. Nitrogen, a macronutrient used for protein syn­
thesis, influences cell growth. Nitrogen deficiency in the cultivation
medium caused the lowest biomass production (301 mg DW/L) and
acetate consumption (39 %); however, the highest PHB content in dry

6.8 ± 0.2
3.8 ± 0.3
4.9 ± 0.1
(% w/w)
biomass (6.5 % w/w), compared to sulfur and phosphorus deficiency
(Table 1). That indicates that more significant stress was caused in the
mixed culture, with 12.5 % of the carbon consumed transformed into
PHB. Phosphorus is used for amino acids and phospholipid synthesis,

Acetate removal
being present in proteins, DNA, ARN, and ATP, and is essential for cell
growth and metabolic energy transfer processes [48]. Phosphorus defi­
ciency caused the lowest PHB content, YPHB/Ac, and PHB production in
PHB production under nitrogen, phosphate and sulfur deficiency using acetate as the carbon source.a.

39 ± 6
52 ± 1
63 ± 2
the WWTP_W2 culture (Table 1). Sulfur deprivation affects the synthesis
(%)

of amino acids such as cysteine and methionine, thus preventing protein

production and cell reproduction [11]. In the sulfur deprivation treat­
ment, lower carbon transformation into PHB was observed than in the Every entry represents the average of three determinations and its standard deviation.
Biomass production

nitrogen-deficiency treatment (7.04 %). However, the higher biomass

production (934 vs. 301 mg/L) improved the total PHB production
(mg DW/L)

reaching similar concentrations to Nitrogen-deficiency treatment

(Table 1).
301
792
934

Under nutrient deficiency, several species of PNSB stop growing, but

they can maintain their metabolic activity, and due to the stress caused,
they initiate PHB accumulation [10,11]. However, in the case of the
Final biomass

WWTP_W2 culture, where PHB production is dependent on cell growth,

(mg DW/L)

1517 ± 46
2008 ± 58
2150 ± 70

the stress caused by nutrient limitation did not improve PHB production.
The PHB accumulated under nutrient deficiency was lower than the
treatment with nutrient sufficiency, which reached 9 ± 0.4% (w/w)
(Fig. 2A, Section 3.2). In these experiments, as in Section 3.2, the pH of
the treatments was not controlled; the final pH was 9.3, 9.1, and 9.0 for
Initial biomass

the treatments with N, P, and S deficiency, respectively. Probably, the

(mg DW/L)

1216 ± 13
1216 ± 13
1216 ± 13

WWTP_W2 culture cannot withstand two stress factors (pH and nutrient
deficiency). Although it would be interesting to corroborate this hy­
pothesis by carrying out experiments under N, P, and S deficiency with
pH control, in a practical way, the natural alkalinization of the medium
is a more straightforward strategy than the control of the nutrient
(days)
*Time

deficiency. That has a significant advantage for a continuous process,

5
5
5

where the PHB production is conducted in one stage, rather than the
classical procedure where the culture is grown first in a feast regimen for
biomass production and then under a nutrient limitation regimen to
N-deficiency
P-deficiency
S-deficiency
Treatment

induce polymer production. Few studies [13,45] have reported PHB

Table 1

production in a one-stage process using a pure culture of bacteria

(Rhodovulum euryhalinum, Rhodovulum imhoffii, Rhodovulum
a
*

6
V. Montiel-Corona and G. Buitrón
visakhapatnamense or Rhodovulum sulfidophilum). Even fewer studies
have used mixed cultures [4,18], as in the present work where one-stage
growth was associated with PHB production in a mixed culture enriched
in Rhodopseudomonas palustris.
It is important to note that the effect of nutrient deficiency on PHB
accumulated depends on the type of strain, the type of limited nutrient,
and the carbon source. For example, Higuchi-Takeuchi and Numata [13]
tested PHB production under nitrogen and phosphate limitations using
acetate as a carbon source. The PHB content in dry biomass did not
increase under nitrogen-limited conditions in the four PNSB tested
(Rhodovulum sulfidophilum, Rhodovulum euryhalinum, Rhodovulum
imhoffii, and Rhodovulum visakhapatnamense). Under phosphate limita­
tion, R. sulfidophilum exhibited higher PHB content in dry biomass, but it
grew poorly and reduced total PHB production.
Carlozzi and Touloupakis [45] using Rhodovulum sulfidophilum
DSM-1374 obtained in batch growth regime high PHB concentrations
under nutrient sufficiency when using acetate and lactate as carbon
sources. In contrast, they observed improved PHB production under
N-deficiency with malate and succinate, which means that the effect of
nutrient limitation is a function of the type of carbon source, the type of
microorganism, and the pH, as was observed in the present work.
3.4. Effect of nitrogen source on PHB production by the WWTP_W2
mixed culture
Glutamate and ammonium, two compounds commonly used as ni­
trogen sources for PNSB culture, influence PHB production, and their
effects depend on the strain type. An experiment was conducted to
determine the most convenient nitrogen source for PHB production by
the WWTP_W2 culture. Table 2 shows the results obtained. Regarding
polymer production, in both treatments (NH4 and Glutamate), the PHB
accumulated was the same (9.2%), which indicates that the WWTP_W2
culture has the same capacity to accumulate polymer regardless of the
nitrogen source. However, given that PHB production is linked to
biomass production, the PHB concentration in the treatment “2. Gluta­
mate” was 36% higher (156 mg PHB/L culture vs. 99 mg PHB/L culture)
than in treatment “1. NH4Cl” due to having a higher concentration of
biomass (1700 mg DW/L vs. 875 mg DW/L). For the WWTP_W2 culture,
the incorporation of glutamate for biomass synthesis was better than
ammonium. Incorporating the inorganic nitrogen source (NH4Cl) into
organic compounds involved the consumption of a higher percentage of
acetate for forming the carbon fraction of the synthesized proteins for
biomass production; this can be deduced from the registered acetate
consumption. In the treatment with ammonia, the acetate consumption
was higher than in the treatment with glutamate. This fact negatively
affected the PHB yield based on acetate consumed (YPHB/Ac), which was
a third of that obtained in the treatment "Glutamate" (6.1 % vs 17.6 %)
(Table 2).
As in this work, Kim et al. [16] observed a similar PHB content in
biomass when using ammonium sulfate and glutamate as nitrogen
sources and acetate as a carbon source, the final biomass concentration
was similar with both nitrogen sources, but the growth was faster with
Table 2
PHB production using ammonium and glutamate as nitrogen sources with and without adding ethanol.a.
Treatment Biomass Acetate Ethanol
consumption consumption
(mg DW/ (mg/L) (mg/L)
L)
1. NH4Cl 875 ± 82 2568 ± 7 – 9.2 ± 0.2
2. Glutamate 1700 ± 23 1688 ± 71 – 9.2 ± 0.1
3. NH4Cl + Ethanol 1358 ± 35 2974 ± 120 0.0 ± 0.0
4. Glutamate 1700 ± 47 1528 ± 71 0.0 ± 0.0
+ Ethanol
a
The data presented correspond to day 4, when the maximum PHB production was reached. Every entry represents the average of three determinations and its
standard deviation.
Biochemical Engineering Journal 189 (2022) 108715
glutamate. Other works have reported that the nitrogen source posi­
tively affects PHB accumulated but not biomass production. For
example, Carlozzi et al. [12] obtained the same biomass concentration
using ammonium and glutamate, but the percentage of PHB accumu­
lated with glutamate was 7.5% and 0.8% with ammonium chloride;
Policastro et al. [18] reported the same trend, and Khatipov et al. [17]
obtained higher PHB production with ammonium. The lack of a general
trend may be related to the different PNSB cultures used [6]. Therefore,
defining the appropriate nitrogen source to induce polymer production
in each culture is essential.
3.5. Effect of ethanol on PHB production by the WWTP_W2 mixed culture
Ethanol is a substance that can cause stress in microorganisms and
thereby induce PHB production [49]. However, its effect on PHB pro­
duction by purple non-sulfur bacteria has not been documented. It is
essential to determine its effect since substrates such as the effluents
from the chain elongation process, enriched in medium-chain carboxylic
acids, are used for PHB production [22]. These often contain significant
concentrations of residual ethanol [23].
Table 2 shows the results of adding ethanol to treatments 3 and 4
with ammonium and glutamate, respectively. In the treatment named
“NH4Cl + ethanol”, ethanol had no positive effect on the PHB accu­
mulated. The PHB content in dry biomass was 9.2 %, the same as
treatment “NH4Cl without ethanol”. However, ethanol positively
affected the treatment "Glutamate + ethanol", and the PHB content in
dry biomass was 25 % higher than that of treatment "Glutamate" without
ethanol. The higher PHB production and low acetate consumption (37 %
of the acetate fed) in the treatment "Glutamate + ethanol" allowed to
reach a YPHB/Ac of 25.7 % (Table 2).
Fig. 4 shows the kinetics of PHB content in dry biomass, acetate, and
ethanol consumption for the treatments supplemented with ethanol. On
day 4 the maximum acetate consumption was reached, but, notably,
there was no removal of ethanol in both experiment. Treatments 2 and 4
were repeated to corroborate the results of this experiment. The results
showed the same trend. Ethanol improved PHB content by 20 %, but it
was not consumed. Additionally, treatment 4 was tested with 1580 mg/
L (0.2 % v/v) and 2370 mg/L (0.3 % v/v) of ethanol (data not shown),
the PHB content in dry biomass was not significantly different from that
obtained in the treatment with 790 mg ethanol/L (0.1 % v/v). Obruca
et al. [49] also obtained the best PHB yields with Cupriavidus necator (a
bacterium that is not a PNSB) at concentrations of less than 0.5 % (v/v)
ethanol. From 1 %, they observed a decrease in biomass production and
PHB yield.
It is well documented that PHB production is a defense mechanism of
microorganisms against stress factors, and ethanol is one of the micro­
bial inhibitors that cause stress [49]. At a sublethal dose, this substance
can damage the integrity of the cell membrane or alter the activity of
membrane-bound enzymes [20]. Cupriavidus necator (a microorganism
conventionally used for PHA production under a feast/famine regimen)
showed a significant increase in PHB production (30%) in the presence
of ethanol (0.5% v/v). Obruca et al. [50] demonstrated that the addition
PHB content in dry Y PHB/ PHB PHB Final
biomass Ac production productivity pH
(% w/w) (%) (mg/L) mg/L-d
6.1 82 ± 8 21 9.4
17.6 156 ± 3 39 9.2
9.1 ± 0.2 8.1 123 ± 3 31 9.1
12.1 ± 0.1 25.7 206 ± 6 52 9.2
7
V. Montiel-Corona and G. Buitrón Biochemical Engineering Journal 189 (2022) 108715

Fig. 4. Kinetics of biomass production, PHB content in dry-biomass, acetate and ethanol consumption for the treatments supplemented with ethanol. Every point
represents the average of three determinations and its standard deviation.

of ethanol during the cultivation of Cupriavidus necator increases the

production of acetyl-CoA (primary substrate to initiate PHB synthesis)
and NAD (P)H (a reduced coenzyme necessary for the activity of
acetoacetyl-CoA reductase), due to the action of alcohol dehydrogenase,
that slightly inhibited the tricarboxylic acid cycle favoring the flow of
acetyl-CoA toward the PHB biosynthetic pathway.
In the case of the WWTP_W2 culture, there was no ethanol con­
sumption during the process, so it is unlikely that it followed the same
mechanism as Cupriavidus necator for PHB production. Although PNSB
does not consume ethanol, it can act through other mechanisms, as has
been shown by Oh et al. [20] and Kim et al. [21]. For example, other
products that compete for reducing power during PHB production by
PNSB are hydrogen and extracellular polymeric substances (EPS) [21].
Oh et al. [20] observed an increase in nitrogenase activity in the pres­
ence of ethanol, and Kim et al. [21] observed a lower EPS concentration
and higher hydrogen production in treatments with 0.5% (v/v) ethanol
than in treatments without ethanol. They concluded that ethanol sup­
presses EPS production and thereby enhances hydrogen production. In
the present work, since there is no hydrogen production, the EPS for­
mation could compete with PHB production. EPS production (in the
form of soluble extracellular proteins and carbohydrates) was quantified
in the treatments with and without ethanol to evaluate the previous
assumption. Fig. 5 shows the results obtained. In the “Glutamate
+ ethanol” treatment, the extracellular protein and carbohydrate pro­
duction was lower than in the “Glutamate” treatment without ethanol.
The same trend was observed for the treatments with NH4Cl for extra­
cellular soluble proteins. Ethanol decreased EPS formation, favoring the
use of the carbon source for polymer production. Another mechanism by
which ethanol could influence PHB production is by affecting
PHB-depolymerase. It has been reported that ethanol can inhibit some
extracellular depolymerase. For example, Papaneophytou et al. [51]
observed a 22 % reduction in the activity of the purified extracellular
mcl-PHA depolymerase from Thermus thermophilus in ethanol presence.
Depolymerases are among the proteins associated with the PHB granules
of the microorganisms, and ethanol could also act on them [52].

3.6. Effect of the carbon/nitrogen (C/N) ratio on PHB production
In the PHB production process, the C/N ratio plays a determining
role in yields. It has been shown that optimal ratios maximize PHA yields
[53]. The C/N ratio determines which products are obtained in a more
significant proportion, PHB or hydrogen [19]. In general, it is known
that excess carbon and high C/N ratios favor PHB production. For the
WWTP_W2 culture, C/N ratios (mM/mM) of 12, 13, 14, 16, 18, and 20
were tested using glutamate and acetate as nitrogen and carbon sources,
respectively. One mL/L of ethanol (790 mg/L) was added for all
Fig. 5. EPS production in treatments with and without adding ethanol using
ammonium and glutamate as nitrogen sources. Bars that do not share the same
letter significantly differ at a 95 % confidence level. Every point represents the
average of three determinations and its standard deviation.
treatments. Table 3 shows that at low C/N ratios (12− 16), the PHB
content in dry biomass was higher than in the treatments with C/N ratios
of 18 and 20. The means comparison by Tukey’s test showed no sig­
nificant difference between treatments C/N 12–16 was found, but these
were significantly different from treatments C/N 18 and 20. At low C/N

8
V. Montiel-Corona and G. Buitrón Biochemical Engineering Journal 189 (2022) 108715

Table 3
PHB production at different C/N ratios.a.
Treatment *Time Biomass Acetate fed Acetate PHB content in dry YPHB/Ac PHB PHB Average
removal biomass production productivity comparisons
PHB content
(days) (mg DW/L) (mg/L) (mg/L) (% W/W) (%) (mg/L culture) (mg/L-d) Tukey test**

C/N 12 3 1367 ± 30 2260 1561 ± 51 16.0 ± 0.8 27 219 ± 5 73 A

± 0.9
C/N 13 3 1383 ± 51 2720 1593 ± 13 14.7 ± 0.1 24 203 ± 8 68 A
± 0.2
C/N 14 3 1430 ± 13 3170 1666 ± 61 14.9 ± 0.8 24 213 ± 2 71 A
± 0.9
C/N 16 3 1350 ± 38 4080 1789 ± 27 14.4 ± 0.2 21 194 ± 6 65 A
± 0.3
C/N 18 5 1478 ± 62 4980 1992 ± 38 10.1 ± 0.4 14 150 ± 7 30 B
± 0.3
C/N 20 5 1283 ± 60 5887 1937 ± 93 10.3 ± 0.5 13 132 ± 7 26 B
± 0.7
a
Every entry represents the average of three determinations and its standard deviation.
*
Time day in which the maximum PHB production was reached
**
Treatments that do not share the same letter significantly differ at a 95% confidence level.

ratios (12− 16), the average PHB content was 15 % (w/w), which is 47%
higher than the average of 10.2 % (w/w) obtained with C/N ratios of 18
and 20.
Comparing the C/N = 16 treatment with ammonium as a nitrogen
source without ethanol (Table 2) and the C/N = 12 treatment with
ethanol and glutamate as nitrogen source, the PHB content in dry
biomass was from 9.2% (w/w) to 16% (w/w), which represents an
improvement of 70 %.
All treatments reached similar biomass concentrations (except the C/
N 20 treatment). The explanation is that the same glutamate concen­
tration was used in all treatments, which acted as a limiting reagent. At
high C/N ratios, the time to reach maximum PHB production was on the
fifth day, while at low C/N ratios, it was on the third day. Low C/N ratios
not only improved the PHB content but also decreased the time to
harvest and improved productivity passing from 26 mg PHB/L-d at high
C/N ratios to 73 mg PHB/L-d at low C/N ratio. In other studies, the time
to obtain the polymer was 7 days [12] and 18 days [19]. Another
advantage of using low C/N ratios was the substrate’s conversion effi­
ciency into the polymer; at low C/N ratios, the conversion of consumed
acetate to PHB was 27 %− 24 %, while at high C/N ratios, it was 13–14
% (Table 3). The combination of two stress factors (alkaline pH and high
C/N ratios) could cause higher PHB production at low C/N ratios with
the WWTP_W2 culture and not at the commonly reported high C/N ra­
tios. It would be interesting to test the PHB production at different C/N
ratios with pH control for future work.
Unlike this work, Demiriz et al. [19] observed simultaneous
hydrogen and PHB production and a better PHB content in biomass by
increasing the C/N ratio; the PHB content improved from 0.1% (w/w)
with a C/N ratio of 9–20% (w/w) with a C/N ratio of 61. High C/N ratios
decreased hydrogen production and enhanced PHB production in Rho­
dobacter capsulatus DSM 1710 under anaerobic conditions [19].
Results obtained in this study for the best conditions (PHB produc­
tion and content 219 mg/L and 16 %, respectively) were like those ob­
tained by others using a mixed consortium (203 mg/L) [18] or higher
than when a single culture of Rhodopseudomonas was used [53]. How­
ever, higher titers were obtained (410 mg/L) using a pure culture of
Rhodopseudomonas palustris grew on butyrate or coniferyl alcohol,
compounds with higher molecular weight than acetate [54].
Strategies that could be implemented in future work to improve PHB
production are related to light intensity, improving the light/dark cycles
[4,42], the use of complex carbon sources [54], and different growth
regimes [45]. The main advantage of using PNSB for PHB production is
related to the polymer production in a one-stage process under anaer­
obic conditions, the simultaneous generation of other substances with
high added value [2], and their capacity to produce PHA from
recalcitrant substances such as lignin derivatives [54]. One of the critical
challenges to increasing PHA production is to improve biomass yield,
which is directly affected by the distribution of light into the culture
broth and, therefore, the reactor design. As Brown et al. [55] reported,
genetic engineering tools could be used for PHA overproduction.
Finally, it should be noted that high PHA contents (60–70 %) can be
achieved with mixed cultures [4,44], opening the possibility of oper­
ating in non-sterile conditions and reducing energy consumption in the
process. Additionally, mixed cultures can be more robust than pure
cultures against variations in the composition of residual substrates and
the presence of native microbiota, as shown with a mixed culture of
photobacteria and microalgae [4]. The application of PHA for medical
purposes may justify the use of pure cultures and sterile conditions;
however, the use of mixed cultures could be a more economically viable
alternative for the manufacture of daily commodities.
4. Conclusions
The Winogradsky column effectively obtained a mixed culture of
PNSB enriched in Rhodopseudomonas palustris with a relative abundance
of 72 %. Alkaline pH induces PHB production in the mixed culture ob­
tained. The combination of alkaline pH, nitrogen, sulfur, or phosphate
deficiency negatively affected polymer production. The natural alka­
linization of the culture medium is a more straightforward strategy to
implement than the nutrient deficiency, which means that PHB can be
produced in one stage, representing a great advantage to implementing
the process. Ethanol addition enhanced PHB production by acting as a
stress factor, reducing EPS production and improving the use of the
carbon source for PHB synthesis. Glutamate as a nitrogen source
enhanced biomass production and PHA concentration compared to
ammonium. Low C/N ratios improved the PHB content in dry biomass,
YPHB/Ac, and reduced the time to harvest. With the evaluated strategies,
it was possible to improve the PHB content by up to 60 %.
CRediT authorship contribution statement
V. Montiel-Corona: Conceptualization, Investigation, Methodology,
Data curation, Writing – original draft. G. Buitrón: Conceptualization,
Supervision, Resources, Investigation, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

9
V. Montiel-Corona and G. Buitrón Biochemical Engineering Journal 189 (2022) 108715

Data Availability concentration and nitrogen source, J. Environ. Manag. 271 (2020), 111006,
https://doi.org/10.1016/j.jenvman.2020.111006.
[19] Ö.B. Demiriz, G. Kars, M. Yücel, I. Eroglu, U. Gündüz, Hydrogen and poly-β-
Data will be made available on request. hydroxybutyric acid at various acetate concentrations using Rhodobacter capsulatus
DSM 1710, Int. J. Hydrog. Energy 44 (32) (2019) 17269–17277, https://doi.org/
Acknowledgments 10.1016/j.ijhydene.2019.02.036.
[20] E.K. Oh, E.J. Kim, H.J. Hwang, X. Tong, J.M. Nam, M.S. Kim, J.K. Lee, The
photoheterotrophic H2 evolution of Rhodobacter sphaeroides is enhanced in the
This work was supported by Direccion General de Asuntos del Per­ presence of ethanol, Int. J. Hydrog. Energy 37 (21) (2012) 15886–15892, https://
sonal Academico de la UNAM DGAPA-UNAM (PAPIIT IT102522). Vir­ doi.org/10.1016/j.ijhydene.2012.08.029.
[21] D.H. Kim, J.H. Lee, S. Kang, P.C. Hallenbeck, E.J. Kim, J.K. Lee, M.S. Kim,
ginia Montiel thanks DGAPA for the award of a postdoctoral scholarship. Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by
The authors acknowledge the technical assistance of J.A. Juárez- ethanol addition and analysis of soluble microbial products, Biotechnol. Biofuels 7
Camacho, F.R. Chavez-Vega, J. Pérez-Trevilla, G. Moreno-Rodríguez (1) (2014) 79, https://doi.org/10.1186/1754-6834-7-79.
[22] R. Iglesias-Iglesias, A. Portela-Grandío, L. Treu, S. Campanaro, C. Kennes, M.
and E. Valenzuela-Reyes C. Veiga, Co-digestion of cheese whey with sewage sludge for caproic acid
production: role of microbiome and polyhydroxyalkanoates potential production,
References Bioresour. Technol. 337 (2021), 125388, https://doi.org/10.1016/j.
biortech.2021.125388.
[23] M. Roghair, T. Hoogstad, D.P. Strik, C.M. Plugge, P.H. Timmers, R.A. Weusthuis,
[1] Z.A. Raza, S. Abid, I.M. Banat, Polyhydroxyalkanoates: characteristics, production,
M.E. Bruins, C.J.N. Buisman, Controlling ethanol use in chain elongation by CO2
recent developments and applications, Int. Biodeterior. Biodegrad. 126 (2018)
loading rate, Environ. Sci. Technol. 52 (3) (2018) 1496–1505, https://doi.org/
45–56, https://doi.org/10.1016/j.ibiod.2017.10.001.
10.1021/acs.est.7b04904.
[2] V. Montiel-Corona, G. Buitrón, Polyhydroxyalkanoates from organic waste streams
[24] B.J. Callahan, P.J. McMurdie, M.J. Rosen, A.W. Han, A.J.A. Johnson, S.P. Holmes,
using purple non-sulfur bacteria, Bioresour. Technol. 323 (2021), 124610, https://
DADA2: high-resolution sample inference from Illumina amplicon data, Nat.
doi.org/10.1016/j.biortech.2020.124610.
Methods 13 (7) (2016) 581–583, https://doi.org/10.1038/nmeth.3869.
[3] M. Koller, L. Maršálek, M.M. de Sousa Dias, G. Braunegg, Producing microbial
[25] E. Touloupakis, E.G. Poloniataki, D.F. Ghanotakis, P. Carlozzi, Production of
polyhydroxyalkanoate (PHA) biopolyesters in a sustainable manner, New
biohydrogen and/or poly-β-hydroxybutyrate by Rhodopseudomonas sp. using
Biotechnol. 37 (2017) 24–38, https://doi.org/10.1016/j.nbt.2016.05.001.
various carbon sources as substrate, Appl. Biochem. Biotechnol. 193 (1) (2021)
[4] J.C. Fradinho, M.A.M. Reis, A. Oehmen, Beyond feast and famine: Selecting a PHA
307–318, https://doi.org/10.1007/s12010-020-03428-1.
accumulating photosynthetic mixed culture in a permanent feast regime, Water
[26] H. Brandl, R.A. Gross, R.W. Lenz, R.C. Fuller, Pseudomonas oleovorans as a source of
Res. 105 (2016) 421–428, https://doi.org/10.1016/j.watres.2016.09.022.
poly (β-hydroxyalkanoates) for potential applications as biodegradable polyesters,
[5] V. Montiel-Corona, S. Revah, M. Morales, Hydrogen production by an enriched
Appl. Environ. Microbiol. 54 (8) (1988) 1977–1982.
photoheterotrophic culture using dark fermentation effluent as substrate: Effect of
[27] M.W. Weatherburn, Phenol-hypochlorite reaction for determination of ammonia,
flushing method, bicarbonate addition, and outdoor–indoor conditions, Int. J.
Anal. Chem. 39 (8) (1967) 971–974.
Hydrog. Energy 40 (30) (2015) 9096–9105, https://doi.org/10.1016/j.
[28] J.S. Arcila, G. Buitrón, Influence of solar irradiance levels on the formation of
ijhydene.2015.05.067.
microalgae-bacteria aggregates for municipal wastewater treatment, Algal Res. 27
[6] S. Sali, H.R. Mackey, The application of purple non-sulfur bacteria for microbial
(2017) 190–197, https://doi.org/10.1016/j.algal.2017.09.011.
mixed culture polyhydroxyalkanoates production, Rev. Environ. Sci. Biotechnol.
[29] M. Barragán-Trinidad, J. Carrillo-Reyes, G. Buitrón, Hydrolysis of microalgal
20 (4) (2021) 959–983, https://doi.org/10.1007/s11157-021-09597-7.
biomass using ruminal microorganisms as a pretreatment to increase methane
[7] A. Hiraishi, H. Kitamura, Distribution of phototrophic purple non-sulfur bacteria in
recovery, Bioresour. Technol. 244 (2017) 100–107, https://doi.org/10.1016/j.
activated sludge systems and other aquatic environments, Jpn. Soc. Sci. Fish. 50
biortech.2017.07.117.
(11) (1984) 1929–1937, https://doi.org/10.2331/suisan.50.1929.
[30] N. Chu, D. Wang, H. Wang, Q. Liang, J. Chang, Y. Gao, Y. Jiang, R.J. Zeng, Flow-
[8] D. Dong, H. Sun, Z. Qi, X. Liu, Improving microbial bioremediation efficiency of
electrode microbial electrosynthesis for increasing production rates and lowering
intensive aquacultural wastewater based on bacterial pollutant metabolism
energy consumption, Engineering (2021), https://doi.org/10.1016/j.
kinetics analysis, Chemosphere 265 (2021), 129151, https://doi.org/10.1016/j.
eng.2021.09.015.
chemosphere.2020.129151.
[31] N. Shange, P. Gouws, L.C. Hoffman, Campylobacter and Arcobacter species in food-
[9] I. Babcsányi, F. Meite, G. Imfeld, Biogeochemical gradients and microbial
producing animals: prevalence at primary production and during slaughter, World
communities in Winogradsky columns established with polluted wetland
J. Microbiol. Biotechnol. 35 (9) (2019) 1–16, https://doi.org/10.1007/s11274-
sediments, FEMS Microbiol. Ecol. 93 (8) (2017) 1–11, https://doi.org/10.1093/
019-2722-x.
femsec/fix089.
[32] K. Zhang, L. Song, X. Dong, Proteiniclasticum ruminis gen. nov., sp. nov., a strictly
[10] M. Vincenzini, A. Marchini, A. Ena, R. De Philippis, H2 and poly-
anaerobic proteolytic bacterium isolated from yak rumen, Int. J. Syst. Evol.
β-hydroxybutyrate, two alternative chemicals from purple non sulfur bacteria,
Microbiol 60 (9) (2010) 2221–2225, https://doi.org/10.1099/ijs.0.011759-0.
Biotechnol. Let. 19 (8) (1997) 759–762, https://doi.org/10.1023/A:
[33] Y. Zhu, J. Xu, X. Cao, Y. Cheng, Characterization of functional microbial
1018336209252.
communities involved in different transformation stages in a full-scale printing and
[11] M.R. Melnicki, E. Eroglu, A. Melis, Changes in hydrogen production and polymer
dyeing wastewater treatment plant, Biochem. Eng. J. 137 (2018) 162–171, https://
accumulation upon sulfur-deprivation in purple photosynthetic bacteria, Int. J.
doi.org/10.1016/j.bej.2018.05.026.
Hydrog. Energy 34 (15) (2009) 6157–6170, https://doi.org/10.1016/j.
[34] K. Jayanama, A. Phuphuakrat, P. Pongchaikul, P. Prombutara, H. Nimitphong,
ijhydene.2009.05.115.
S. Reutrakul, S. Sungkanuparph, Association between gut microbiota and
[12] P. Carlozzi, A. Giovannelli, M.L. Traversi, E. Touloupakis, T. Di Lorenzo, Poly-3-
prediabetes in people living with HIV, Curr. Res. Microb. Sci. 3 (2022), 100143,
hydroxybutyrate and H2 production by Rhodopseudomonas sp. S16-VOGS3 grown in
https://doi.org/10.1016/j.crmicr.2022.100143.
a new generation photobioreactor under single or combined nutrient deficiency,
[35] C. Liu, H. Wang, W. Xing, L. Wei, Composition diversity and nutrition conditions
Int. J. Biol. Macromol. 135 (2019) 821–828, https://doi.org/10.1016/j.
for accumulation of polyhydroxyalkanoate (PHA) in a bacterial community from
ijbiomac.2019.05.220.
activated sludge, Appl. Microbiol. Biotechnol. 97 (21) (2013) 9377–9387, https://
[13] M. Higuchi-Takeuchi, K. Numata, Acetate-inducing metabolic states enhance
doi.org/10.1007/s00253-013-5165-6.
polyhydroxyalkanoate production in marine purple non-sulfur bacteria under
[36] R.G. Kranz, K.K. Gabbert, T.A. Locke, M.T. Madigan, Polyhydroxyalkanoate
aerobic conditions, Front. Bioeng. Biotechnol. 7 (2019) 118, https://doi.org/
production in Rhodobacter capsulatus: genes, mutants, expression, and physiology,
10.3389/fbioe.2019.00118.
Appl. Environ. Microbiol. 63 (8) (1997) 3003–3009, https://doi.org/10.1128/
[14] S. Obruca, P. Sedlacek, M. Koller, The underexplored role of diverse stress factors
aem.63.8.3003-3009.1997.
in microbial biopolymer synthesis, Bioresour. Technol. 326 (2021), 124767,
[37] P. Phankhamla, J. Sawaengkaew, P. Buasri, P. Mahakhan, Biohydrogen production
https://doi.org/10.1016/j.biortech.2021.124767.
by a novel thermotolerant photosynthetic bacterium Rhodopseudomonas
[15] T. Suzuki, A.A. Tsygankov, J. Miyake, Y. Tokiwa, Y. Asada, Accumulation of poly-
pentothenatexigens strain KKU-SN1/1, Int. J. Hydrog. Energy 39 (28) (2014)
(hydroxybutyrate) by a non-sulfur photosynthetic bacterium, Rhodobacter
15424–15432, https://doi.org/10.1016/j.ijhydene.2014.07.091.
sphaeroides RV at different pH, Biotechnol. Lett. 174 (4) (1995) 395–400, https://
[38] G.J. Xie, B.F. Liu, D.F. Xing, J. Ding, J. Nan, H.Y. Ren, N.Q. Ren, The kinetic
doi.org/10.1007/BF00130796.
characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-
[16] M.S. Kim, D.H. Kim, J. Cha, J.K. Lee, Effect of carbon and nitrogen sources on
53 and its application for enhancing continuous hydrogen production, Int. J.
photo-fermentative H2 production associated with nitrogenase, uptake
Hydrog. Energy 37 (18) (2012) 13718–13724, https://doi.org/10.1016/j.
hydrogenase activity, and PHB accumulation in Rhodobacter sphaeroides KD131,
ijhydene.2012.02.168.
Bioresour. Technol. 116 (2012) 179–183, https://doi.org/10.1016/j.
[39] Y. Zhao, Y. Chen, Nano-TiO2 enhanced photofermentative hydrogen produced
biortech.2012.04.011.
from the dark fermentation liquid of waste activated sludge, Environ. Sci. Technol.
[17] E. Khatipov, M. Miyake, J. Miyake, Y. Asada, Accumulation of poly-β
45 (19) (2011) 8589–8595, https://doi.org/10.1021/es2016186.
hydroxybutyrate by Rhodobacter sphaeroides on various carbon and nitrogen
[40] L. Yin, X. Li, Y. Liu, D. Zhang, S. Zhang, X. Luo, Biodegradation of cypermethrin by
substrates, FEMS Microbiol. Lett. 162 (1) (1998) 39–45, https://doi.org/10.1111/
Rhodopseudomonas palustris GJ-22 isolated from activated sludge, Fresenius
j.1574-6968.1998.tb12976.x.
Environ. Bull. 21 (2) (2012) 397–405.
[18] G. Policastro, V. Luongo, M. Fabbricino, Biohydrogen and poly-β-hydroxybutyrate
production by winery wastewater photofermentation: effect of substrate

10
V. Montiel-Corona and G. Buitrón
[41] B.V. Kumar, V.V. Ramana, Treatment of industrial effluents by a phototrophic
purple non-sulfur bacterium Rhodopseudomonas palustris JA190, Asian J. Microbiol.
Biotechnol. Environ. Sci. 1583 (2013) 585–588.
[42] V. Montiel-Corona, S. Le Borgne, S. Revah, M. Morales, Effect of light-dark cycles
on hydrogen and poly-β-hydroxybutyrate production by a photoheterotrophic
culture and Rhodobacter capsulatus using a dark fermentation effluent as substrate,
Bioresour. Technol. 226 (2017) 238–246, https://doi.org/10.1016/j.
biortech.2016.12.021.
[43] M. Villano, M. Beccari, D. Dionisi, S. Lampis, A. Miccheli, G. Vallini, M. Majone,
Effect of pH on the production of bacterial polyhydroxyalkanoates by mixed
cultures enriched under periodic feeding, Process. Biochem. 45 (5) (2010)
714–723, https://doi.org/10.1016/j.procbio.2010.01.008.
[44] W. Zhou, D.I. Colpa, B. Geurkink, G.J.W. Euverink, J. Krooneman, The impact of
carbon to nitrogen ratios and pH on the microbial prevalence and
polyhydroxybutyrate production levels using a mixed microbial starter culture, Sci.
Total Environ. 811 (2022), 152341, https://doi.org/10.1016/j.
scitotenv.2021.152341.
[45] P. Carlozzi, E. Touloupakis, Bioplastic production by feeding the marine
Rhodovulum sulfidophilum DSM-1374 with four different carbon sources under
batch, fed-batch and semi-continuous growth regimes, New Biotechnol. 62 (2021)
10–17, https://doi.org/10.1016/j.nbt.2020.12.002.
[46] A.S. Tsygankov, L.T. Serebryakova, D.A. Sveshnikov, K.K. Rao, I.N. Gogotov, D.
O. Hall, Hydrogen photoproduction by three different nitrogenases in whole cells
of Anabaena variabilis and the dependence on pH, Int. J. Hydrog. Energy 22 (9)
(1997) 859–867, https://doi.org/10.1016/S0360-3199(96)00242-X.
[47] H. Koku, I. Eroğlu, U. Gündüz, M. Yücel, L. Türker, Aspects of the metabolism of
hydrogen production by Rhodobacter sphaeroides, Int. J. Hydrog. Energy 27 (2002)
1315–1329, https://doi.org/10.1016/S0360-3199(02)00127-1.
[48] M.A. Yaakob, R.M.S.R. Mohamed, A. Al-Gheethi, G.A. Ravishankar, R.R. Ambati,
Influence of nitrogen and phosphorus on microalgal growth, biomass, lipid, and
11
Biochemical Engineering Journal 189 (2022) 108715
fatty acid production: an overview, Cells 10 (2) (2021) 393, https://doi.org/
10.3390/cells10020393.
[49] S. Obruca, I. Marova, Z. Svoboda, R. Mikulikova, Use of controlled exogenous
stress for improvement of poly (3-hydroxybutyrate) production in Cupriavidus
necator, Folia Microbiol 55 (1) (2010) 17–22, https://doi.org/10.1007/s12223-
010-0003-z.
[50] S. Obruca, I. Marova, M. Stankova, L. Mravcova, Z. Svoboda, Effect of ethanol and
hydrogen peroxide on poly (3-hydroxybutyrate) biosynthetic pathway in
Cupriavidus necator H16, World J. Microbiol. Biotechnol. 26 (7) (2010) 1261–1267,
https://doi.org/10.1007/s11274-009-0296-8.
[51] C.P. Papaneophytou, E.E. Velali, A.A. Pantazaki, Purification and characterization
of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from
Thermus thermophilus HB8, Polym. Degrad. Stab. 96 (4) (2011) 670–678, https://
doi.org/10.1016/j.polymdegradstab.2010.12.015.
[52] A. Sznajder, D. Jendrossek, Biochemical characterization of a new type of
intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic
activity on native PHB granules, Appl. Microbiol. Biotechnol. 89 (5) (2011)
1487–1495, https://doi.org/10.1007/s00253-011-3096-7.
[53] T.O. Ranaivoarisoa, R. Singh, K. Rengasamy, M.S. Guzman, A. Bose, Towards
sustainable bioplastic production using the photoautotrophic bacterium
Rhodopseudomonas palustris TIE-1, J. Ind. Microbiol. Biotechnol. 46 (9–10) (2019)
1401–1417, https://doi.org/10.1007/s10295-019-02165-7.
[54] A. Alsiyabi, B. Brown, C. Immethun, D. Long, M. Wilkins, R. Saha, Synergistic
experimental and computational approach identifies novel strategies for
polyhydroxybutyrate overproduction, Metab. Eng. 68 (2021) 1–13, https://doi.
org/10.1016/j.ymben.2021.08.008.
[55] B. Brown, C. Immethun, A. Alsiyabi, D. Long, M. Wilkins, R. Saha, Heterologous
phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production
from lignocellulosic biomass, Metab. Eng. Commun. 14 (2022) (2022), e00191,
https://doi.org/10.1016/j.mec.2021.e00191.