Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Advancements and current challenges in the
sustainable downstream processing of bacterial
polyhydroxyalkanoates
Md Ariful Haque1,a, Anshu Priya1,a, Zubeen J. Hathi1,a,
Zi-Hao Qin1, Srinivas Mettu2 and Carol Sze Ki Lin1
Abstract
Diminishing sources of synthetic plastics and their unsustain-
able production processes have increased the demand for
alternative biodegradable and sustainable polymers. Bacterial
biopolymer-producing factories can carry out large-scale pro-
duction of such alternatives using improved fermentation
techniques, such as fed-batch and pulsed feeding of inducers,
that can increase bacterial biopolymer accumulation. However,
the successive downstream processing (DSP) techniques still
pose challenges in making the production process both
economically and environmentally sustainable. These chal-
lenges are mostly associated with biomass pre-treatment, the
use of solvents, and the embedded parameters of the DSP
techniques. Conventional halogenated/chlorinated solvents
can be substituted with green solvents to yield PHAs of high
purity (98%) for high-end applications and to establish a sus-
tainable circular economy. As an economically and environ-
mentally sustainable approach, the use of recycled waste as a
substrate and greener extraction solvents for bacterial
biopolymer production should be further explored for the effi-
cient replacement of synthetic plastic production.
Addresses
1
School of Energy and Environment, City University of Hong Kong,
Hong Kong
2
Chemical and Environmental Engineering, School of Engineering,
RMIT University, Melbourne, Victoria 3000, Australia
Corresponding author: Lin, Carol Sze Ki (carollin@cityu.edu.hk)
a
First author with equal contribution.
Current Opinion in Green and Sustainable Chemistry 2022,
36:100631
This review comes from a themed issue on Selected papers from 5th
EUGSC Conference (2021)
Edited by Konstantinos Triantafyllidis, Alina M. Balu and Florent
Allias
Available online 2 May 2022
For complete overview of the section, please refer the article
collection - Selected papers from 5th EUGSC Conference (2021)
https://doi.org/10.1016/j.cogsc.2022.100631
2452-2236/© 2022 Elsevier B.V. All rights reserved.
Keywords
Biopolymer, Fermentation, Polyhydroxyalkanoates, Downstream
processing, Sustainable production.
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Green and Sustainable Chemistry
Introduction
Petroleum-based plastics have significantly penetrated
the market due to their low cost and high versatility.
However, these synthetic plastics take a long time to
degrade, causing harmful effects on the environment
and wildlife, especially in the form of microplastics that
accumulate in the oceans and enter the food chain. The
raw materials used for the production of synthetic
plastics are limited, making the production of such
plastics unsustainable. Biopolymers are produced intra-
cellularly by microbes via an enzyme-assisted biosyn-
thetic process that links up sugars, amino acids, or
hydroxy fatty acids to yield high-molecular weight
compounds [1]. Polyhydroxyalkanoates (PHAs) are a
type of biopolymer produced intracellularly by bacteria
and some archaea under a nitrogen-limiting and carbon-
excess condition [2]. The biosynthetic production of
bacterial biopolymers and the harnessing of their unique
properties for various applications [3e5], including
novel antibacterial drugs and bio-based materials, have
been studied extensively. PHAs are biodegradable and
environment friendly; hence, blending them with syn-
thetic polymers to produce plastics with desirable
properties could be a lucrative strategy [2].
The accumulation of PHA biopolymers can be achieved
through various fermentation techniques, such as
dynamic fed-batch fermentation with alternating
repeated periods of feast and famine, which can be
achieved under aerobic or anaerobic conditions [6], and
pulsed volatile fatty acid-feeding regime that stimu-
lates the PHA-synthesising bacteria to accumulate
PAHs intracellularly throughout multiple feast and
famine cycles. Recent findings suggest this feed-on-
demand process is a promising approach even for
industrial-scale production [6].
Biosynthetically produced bacterial PHAs are extracted
and recovered through a series of downstream process-
ing (DSP) techniques. The most commonly used DSP
techniques include biomass pre-treatment (mechanical
and chemical), cell lysis-assisted extraction using
various solvents, enzymatic, supercritical fluid extrac-
tion, and further purification (Figure 1) by solvent for
example chloroform [7,8].. Conventional extraction
Current Opinion in Green and Sustainable Chemistry 2022, 36:100631
2 5th EUGSC Conference (2021)
Figure 1
A schematic process flow of PHAs extraction and recovery from bacterial biomass.
methods generally use solvents that are toxic, expensive,
and non-recyclable, making these methods environ-
mentally unsustainable [8].
Therefore, a green and sustainable technique is desir-
able for the production of PHA biopolymers. A green
technique is defined as a green extraction and recovery
process that consumes low energy, uses green solvents
and renewable natural products, and produces safe and
high-quality products [6,9,10]. Green techniques close
the loop of linear solvent utilisation processes to create
circular zero-waste processes, engendering a transition
from a linear to a circular economy. Figure 1 shows the
schematic diagram of a recovery technique that can be
applied to achieve sustainable PHA production using
green solvents.
This review describes the challenges encountered in the
downstream recovery and purification of PHA bio-
polymers. Although several conventional extraction
methods could be used for PHA recovery, a well-defined
green and sustainable extraction process is warranted
and has yet to be established. This review provides in-
sights into the potential green and sustainable recovery
Current Opinion in Green and Sustainable Chemistry 2022, 36:100631
techniques for the production of bacterial biomass-
derived PHA biopolymers.
Downstream processes for high-purity
PHAs
Cell disruption and biomass separation
Several cell disruption methods, such as bead beating,
ultrasonication, homogenisation, and gamma irradia-
tion, have been applied in PHA extraction. Cell lysis
through bead beating is a common method for recov-
ering intracellular products such as enzymes, DNA, and
biopolymers using cell-wall-disrupting agents such as
glass beads that induce solid shear forces in a non-
specific manner. The method has the advantage of
being feasible to use even at low biomass concentration
to yield high-purity biopolymer of interest. Gutt et al.
performed a comparative assessment of the efficacy of
various DSP methods in disrupting similar quantities of
Cupriavidus necator biomass [11]. They found that cell
wall disruption by mechanical bead beating in combi-
nation with sodium dodecyl sulphate (SDS) treatment
resulted in the complete recovery of a biopolymer with
a more than 90% purity in 2 h. High-pressure homog-
enisation (HPH) is another cell disruption method that
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 Downstream processing of polyhydroxyalkanoates Haque et al. 3

satisfies most of the DSP criteria applicable for large-
scale production. During this process, the fermenter
both flows through a narrow chamber and is subjected
to high pressure and shear forces, which leads to cell
disruption. One study reported achieving a 98% yield of
P(3-HB) (Poly 3-hydroxy butyrate) with 95% purity
from a Methylobacterium sp. using HPH and 5% w/v SDS
[12]. However, one disadvantage of using HPH is the
release of DNA during cell lysis, which reduces the
purity of the recovered product. To overcome this
drawback and simplify the DSP, methods such as hy-
pochlorite treatment, addition of nucleases, and heat
treatment are adopted. Another method for PHA re-
covery from a fermentation broth is cell disruption by
bead milling and ultrasonication. This is followed by
the addition of surfactants and coagulants to the
fermentation broth under alkaline conditions. The
PHAs can then be separated by centrifugation or
vacuum filtration after 1 h of alkaline treatment. The
process flow, ease of operation, and low-cost input
make the combination of bead milling and homogeni-
sation one of the most reliable, scalable, and sustain-
able techniques for harvesting and purifying PHAs
[13]. Other less widely used methods of cell lysis are
described in Table 1.
Green and sustainable approaches for PHAs
extraction and recovery
The downstream recovery and purification of PHA
biopolymers accounts for half of the PHAs production
cost [22]. In the case of fermentative production using
microbes, the biomass is first separated through
centrifugation or filtration and then subjected to
extraction and purification, where PHA is first extrac-
ted by solubilising the cellular material or the polymer
of interest, and subsequently purified according to the
requirements of the final product. Extraction strategies
such as solvent extraction, supercritical fluid extrac-
tion, chemical disruption methods (based on bio-
surfactants) in combination with enzymes, and

Table 1

Tabular summary of cellular disruption and pre-treatment methods for PHAs biopolymer extraction.

Method Advantages Drawbacks Organism studied Recovery of References

polyhydroxyalkanoates
(PHAs) in percent

Bead milling Effective even with low Several repetitions are Cupriavidus necator 95% [11,13]
biomass required Haloferax mediterranei
Scalable Pre-treatment step is
essential
High-pressure Provides a high yield High biomass Alcaligenes. latus 98% [12,14]
homogenisation Scalable concentration is required Methylobacterium sp.
(HPH) Leads to PHA V49
micronisation
Parameter optimisation
is time consuming
Genetically induced cell Based on a phage- Despite conclusive Alcanivorax 90% [15,16]
lysis based lysis system and results, the genetically borkumensis SK2
allows the efficient and induced cell lysis Pseudomonas putida
gentle release of PHAs method has not been CA-3
implemented at an Bacillus megaterium
industrial scale
Pre-treatment for cell Straightforward process Works mainly for H. mediterranei 90% [17,18]
fragility with few steps halophiles (osmotic Azotobacter vinelandii
Low risk of polymer lysis)
damage during the In a few cases, only
extraction process partial lysis is observed,
leading to a low yield of
biopolymer
Ultrasonication Low cost Effective when used in Cupriavidus. 60–90% [19,20]
High extraction yield combination with HPH or taiwanensis
bead beating methods C. necator
In some cases, the
higher purity of the
polymer comes at the
cost of a reduced
molecular weight
Gamma irradiation Ease of polymer Scarcity of literature on Bacillus. flexus 54% [21]
extractability this technique
Mainly used with
solvents

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4 5th EUGSC Conference (2021)
biological approaches using insects and other animals
have been explored in the past [23]. Despite the
availability of several types of extraction methods,
solvent-based extraction is the most widely used and
reliable recovery method. However, large amounts of
expensive halogenated solvents (e.g. chloroform and
dichloromethane) that pose risks to the environment
are required for this extraction method [24]. For
example, chlorinated solvents are known to have
adverse effects on the ecological footprint of the overall
production process. As a result, the production of
PHBV biopolymers is fraught with environmental
concerns. Furthermore, the solubility of a polymer, an
important parameter affecting polymer recovery, is
influenced by factors such as polymer concentration,
temperature, and pressure [25]. Therefore, haloge-
nated solvents, such as chloroform and dichloro-
methane, are commonly used because of their high
efficiency in polymer extraction. In some studies, non-
halogenated solvents, such as dimethyl carbonate, ethyl
acetate, and propylene carbonate, have been shown to
perform better extraction than halogenated solvents,
affording satisfactory extraction yields [26]. Reis et al.,
investigated several parameters to optimise a green
extraction and purification strategy for PHAs using
dimethyl carbonate and 1-butanol, respectively. They
observed only minor differences in the PHA yields for
different extraction times or biomass to solvent ratios.
Furthermore, purification with 1-butanol resulted in
increased PHA purity from 91.2% to 98%. Although
higher extraction yields can be obtained using chloro-
form or dichloromethane as solvents, the resulting
extraction process has a low economic and environ-
mental sustainability. In summary, these studies have
opened up several promising research directions to
explore a circular economy for the bioplastic industry.
Factors affecting PHAs recovery
Both the carbon source and microorganism species used
to produce PHAs influence the PHAs recovery effi-
ciency. A review by Policastro et al. [27], reported that
microbes that produce a low PHA yield lead to a large
amount of carbon source wastage, and a large amount of
energy is required for subsequent PHA extraction,
which ultimately reduces the total recovery efficiency.
For an economically viable fermentation process, a
theoretical productivity of not less 0.48 g/g PHA is
suggested by Koller [28]. Mongili et al. [29], reported
that the state of biomass affects Poly 3-hydroxybutyrate-
co-3-hydroxy valerate (PHBV) recovery during extrac-
tion, with wet bacterial biomass being more suitable for
PHBV extraction than dry biomass.
Pulse-feeding with fermentation fluid containing vola-
tile fatty acids is effective to achieve high PHBV accu-
mulation. This fermentation strategy uses the metabolic
pathways of acetyl Co-A to 3HB and of acetyl Co-A and
Current Opinion in Green and Sustainable Chemistry 2022, 36:100631
propionyl Co-A synthase to 3HV [6,30]. Final yield of
PHAs is directly proportional to the intracellular accu-
mulation and biomass production.
A high accumulation of PHAs by manipulating the
carbon/nitrogen (C/N) ratio can also influence the final
PHA yield. For instance, a C/N ratio of 25e35 achieved a
47% PHA yield and was thus recommended to be highly
suited for most of the PHA-synthesising bacterial strains
[31,32].
PHBV recovery from bacterial cells and its purity are also
influenced by the wet/dry state of the biomass subjected
to DSP. For instance, Perez et al. [24] reported that the
recovery and purity of PHBV are lower from wet biomass
(recovery 87.9%, data not available for purity) than from
dry and lyophilised biomass (recovery: 92.1% and 91.4%,
respectively; and purity: 95.1% and 98.3%, respectively)
[24].
In the review by Perez et al. [24], the suitability of
different extraction techniques was summarised in the
following order based on their advantages, limitations,
and impacts on both humans and the environment:
Solvent extraction > Green solvent > Chemical
digestion > Supercritical fluid extraction > Biological
recovery. The influence of various factors on PHA re-
covery and yield from bacterial biomass is depicted in a
flow diagram (Figure 2).
Current challenges in PHA recovery
The biopolymer production process faces several chal-
lenges that impede the growth and expansion of the
PHA market (Figure 3) [33]. The main challenge
encountered in PHA production and acceptance is its
economical non-competitiveness with petrochemical
plastics. Some of the conventionally used substrates,
such as purified carbon sources, sugars, oils, and fatty
acids, constitute a major part of the PHA production
cost [34]. As a low-cost alternative, using waste as a
carbon substrate may be profitable, but due to the
heterogeneous composition of waste and the presence of
impurities in it, the overall quality and quantity of the
PHA produced may be compromised [35,36].
Another major challenge in the PHA production chain is
the complex DSP of the extracted PHA. Several toxic
reagents, such as chloroform, methanol, and sodium
hypochlorite, are used in the recovery process without
subsequent solvent recycling, which poses a threat to
the environment and increases the production cost [37].
Further, the use of mechanical extraction techniques
either alone or in combination with chemical extraction
techniques also contributes to the production cost
[37,38]. The presence of various contaminants such as
extraction solvents in the recovered PHBV has also been
reported to increase the purification cost [17]. Further,
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Figure 2
A summary of the characteristics of various methods used for PHAs extraction and recovery from bacterial biomass.
Figure 3
Major challenges in polyhydroxyalkanoate (PHA) recovery.
the preservation of the initial molecular weights of the
polymer without compromising its purity and integrity
for various applications is another major hindrance [17].
Life cycle assessments (LCAs) of the PHA production
process have revealed that the toxic extraction solvents
have a high carbon footprint. Sodium hypochlorite
extraction has a carbon footprint of 29.46 kg CO2 eq/h,
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Downstream processing of polyhydroxyalkanoates Haque et al. 5
whereas NaOH and H2SO4 have a carbon footprint of
4.08 kg CO2 eq/h and 6.27 kg CO2 eq/h, respectively
[39]. Techno-economic studies have reported that the
industrial feasibility of the production process and the
plant capacity also influence the production cost. For
example, an increase in PHA plant capacity from 2000
tonne/year to 10,000 tonne/year has been reported to
Current Opinion in Green and Sustainable Chemistry 2022, 36:100631
6 5th EUGSC Conference (2021)
decrease the unit production cost from $4.29/kg to
$2.71/kg [40].
Conclusions
Despite the increasing awareness of plastic waste
accumulation and the need for alternative biopolymers,
the extraction and purification methods for biopolymers
such as PHAs are still based on conventional approaches
that use toxic and expensive solvents. Studies have
shown that the sustainability factor of biopolymer pro-
duction can be significantly improved by using organ-
isms such as halophiles that have no detrimental effect
on the environmental and from which harvesting, and
purification of biopolymers is less expensive. Strategies
suggested in this review, such as real-time LCA of the
fermentation and extraction processes, can significantly
reduce the time and challenges associated with the
purification of biopolymers. Given increasing environ-
mental concern about petrochemical plastics, there is a
need for their replacement with bioplastics. PHAs are
promising biopolymers, but their economic non-
competitiveness with petrochemical plastics impedes
their production and acceptance at a large scale. The
DSP of PHAs is complex and significantly influences the
ecological footprint. Therefore, further studies on sus-
tainable development goals, such as improving the
health of humans and the environment, are warranted to
inform the sustainable production of biopolymers such
as PHAs to efficiently replace the synthetic plastics
currently available in the market. There is an urgent
need for extensive research on the development of a
simple, effective, and economical DSP approach that is
environmentally sustainable.
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.
Acknowledgement
The authors are grateful for the financial support pro-
vided by the Hong Kong Research Grants Council via
the Collaborative Research Fund account C1105-20G.
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