Science of the Total Environment 756 (2021) 143729
Contents lists available at ScienceDirect
Science of the Total Environment
j ourna l homepage: www.e lsev ie r .com/ locate /sc i totenvReviewA review of biopolymer (Poly-β-hydroxybutyrate) synthesis in microbes
cultivated on wastewaterAyesha Algade Amadu a, Shuang Qiu a, Shijian Ge a,⁎, Gloria Naa Dzama Addico b, Gabriel Komla Ameka c,
Ziwei Yu a, Wenhao Xia a, Abdul-Wahab Abbew a, Dadong Shao a, Pascale Champagne d, Sufeng Wang e
a Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Xiao LingWei 200,
Nanjing 210094, PR China
b Council for Scientific and Industrial Research (CSIR) – Water Research Institute (WRI), P.O. Box AH 38, Achimota Greater Accra, Ghana
c Department of Botany, University of Ghana, P.O. Box LG55, Legon, Accra, Ghana
d Department of Civil Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada
e School of Economics and Management, Anhui Jianzhu University, Hefei, Anhui 230601, PR ChinaH I G H L I G H T S G R A P H I C A L A B S T R A C T• Criteria for selecting appropriate PHB-
producing microbes were proposed.
• Biosynthesis and degradation pathway
of PHB was illustrated.
• Optimization of culture conditions for
improved PHB yield was highlighted.
• Feasibility of producing other metabo-
lites using wastewater was evaluated.
• Environmentally friendly extraction
methods and application of PHB were
reviewed.⁎ Corresponding author.
E-mail address: geshijian1221@njust.edu.cn (S. Ge).
https://doi.org/10.1016/j.scitotenv.2020.143729
0048-9697/© 2020 Elsevier B.V. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:
Received 24 July 2020
Received in revised form 4 November 2020
Accepted 5 November 2020
Available online 25 November 2020
Editor: Yifeng Zhang
Keywords:
Microbe
Poly-β-hydroxybutyrates
Resource recovery
PHA
PHB
WastewaterThe large quantities of non-degradable single use plastics, production and disposal, in addition to increasing
amounts ofmunicipal and industrialwastewaters are among themajor global issues known today. Biodegradable
plastics from biopolymers such as Poly-β-hydroxybutyrates (PHB) produced by microorganisms are potential
substitutes for non-degradable petroleum-based plastics. This paper reviews the current status of wastewater-
cultivated microbes utilized in PHB production, including the various types of wastewaters suitable for either
pure or mixed culture PHB production. PHB-producing strains that have the potential for commercialization
are also highlighted with proposed selection criteria for choosing the appropriate PHB microbe for optimization
of processes. The biosynthetic pathways involved in producing microbial PHB are also discussed to highlight the
advancements in genetic engineering techniques. Additionally, the paper outlines the factors influencing PHB
production while exploring other metabolic pathways and metabolites simultaneously produced along with
PHB in a bio-refinery context. Furthermore, the paper explores the effects of extraction methods on PHB yield
and quality to ultimately facilitate the commercial production of biodegradable plastics. This reviewuniquely dis-
cusses the developments in research on microbial biopolymers, specifically PHB and also gives an overview of
current commercial PHB companies making strides in cutting down plastic pollution and greenhouse gases.
© 2020 Elsevier B.V. All rights reserved.
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729Contents1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. Overview of biopolymer production routes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Research tendencies in Poly-β-hydroxybutyrate production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Current developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Selection of Poly-β-hydroxybutyrate-producing microbes suitable for wastewater cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Utilization of wastewater for Poly-β-hydroxybutyrate production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1. Poly-β-hydroxybutyrate production from industrial wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.2. Poly-β-hydroxybutyrate production from municipal wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.3. Co-production of Poly-β-hydroxybutyrate and other metabolites in wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.4. Biosynthesis and metabolic engineering of microbial Poly-β-hydroxybutyrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.4.1. Gene manipulation – nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.4.2. Gene manipulation – morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.4.3. Gene manipulation - non-native host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Factors influencing the accumulation and composition of Poly-β-hydroxybutyrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.1. Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5.2. Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.3. Feeding mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.4. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.5. Photoperiod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.6. Cycle length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6. Effects of extraction methods on Poly-β-hydroxybutyrate yield and purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
7. Challenges and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
7.1. Optimizing production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
7.2. Acclimatization of microbes for wastewater cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
7.3. Improving yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
CRediT authorship contribution statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Declaration of competing interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151. Introduction
Wastewater (WW) generation is an unavoidable aspect of daily life
and a consequence of industrialization and urbanization. Almost all
the water used in our homes and industries end up as municipal or in-
dustrial WW and, when released into the environment, creates a signif-
icant footprint such as eutrophication (Ge et al., 2015; Ranade and
Bhandari, 2014; Qiu et al., 2020b). TheseWWeffluents are often loaded
with contaminants that make it unsafe for discharge into the aquatic
environment. However, some WWs have been found to be excellent
nutrient sources for certain microbes (Das et al., 2018). Fast growing
eukaryotic algae, cyanobacteria and bacteria (hereafter collectively re-
ferred to as microbes) are the promising solution to biological nutrient
removal such as activated sludge system in wastewater treatment
plants (WWTPs) (Ge et al., 2014; Qiu et al., 2020b; X. Xu et al., 2020;
M. Xu et al., 2020). Not only is microbial WWT eco-friendly (Arroyo
and Molinos-Senante, 2018), it ensures the use of fewer resources for
microbial growth, and also offers opportunities for resource recovery
(Gabriel et al., 2018; Ge et al., 2017). Countless algal species used in
WWT are excellent bio-fixers (Demirbas, 2011; Kassim and Meng,
2017), thus they are able to remove carbon dioxide from the atmo-
sphere as well as inorganic nitrogen and phosphorus from the aquatic
environment. These nutrients support microbial growth (Chen et al.,
2020; Ge et al., 2018a; Ge and Champagne, 2017; Ge and Champagne,
2016) while producing valuable energy alternatives such as bio-
methane and bio-fuel, natural antioxidants, insect feed additives (Ge
et al., 2018b; Jochum et al., 2018; Qiu et al., 2019b, 2020a; Shen et al.,
2020), bio-fertilizers (Shweta and Samuel, 2015), or biopolymers
(Meixner et al., 2017).
Bio-plastics are plastics obtained from renewable biomass, unlike
their petroleum counterparts, and have been produced from first gener-
ation feed-stocks such as corn, sugar beet, or second-generation feed-
stock like lignocellulose materials in the past. Recently, however, more2
attention is paid to the usage of third generation feedstock such as mi-
crobes that do not compete with human food or animal feed, arable
land or freshwater (Ge et al., 2017; Qiu et al., 2019a). Microbial bio-
plastics are polymers synthesized within the cytoplasm of some cells
as water-insoluble “inclusion bodies” (Jiang et al., 2015). These inclu-
sion bodies have been described as “entirely natural and biodegradable”
(Hankermeyer and Tjeerdema, 1999; Sedlacek et al., 2019). They serve
as a carbon reservematerial especiallywhen cells are grownunder stress
(Kamravamanesh et al., 2018; Mendhulkar and Shetye, 2017). Notwith-
standing their role as storagematerial, they helpmicrobial cellsmaintain
their integrity, particularly through protection against sudden osmotic
imbalances (Sedlacek et al., 2019). Polyhydroxyalkanoate (PHA) is an
umbrella term that describes a group of naturally occurring polymers
of which Poly-3-hydroxybutyrate (P3HB) is the most studied (Jiang
et al., 2015;Mathuriya and Yakhmi, 2019; Urtuvia et al., 2014).Microbial
polymers have material properties that are suitable in many industrial
applications due to their similarity to conventional plastics. For instance,
good barrier properties have allowed for their use in the food industry for
packaging purposes (Khosravi-Darani, 2015; Markl et al., 2018). These
properties are based on theirmelting point (175 °C), glass transition tem-
perature (15 °C), molecular weight (5 × 105 Da), density (1.25 g/cm3),
tensile strength (40 MPa) and young's modulus (3.5 GPa) (Carofiglio
et al., 2017; Hempel et al., 2011; Sathya et al., 2018).
It is estimated that the production cost for PHB is four to nine
times higher than the price of polyethylene (Hempel et al., 2011;
Kamravamanesh et al., 2017). High preference for pure culture fer-
mentation, as well as substrate requirements and various culture
conditions also compound the high production cost problem. Culture
conditions like temperature, pH, light fluxes, nutrients and cycle
length must be optimized to achieve significant yields, while not
overlooking the important role the carbon source plays in the pro-
duction process (Sedlacek et al., 2019). Most of the carbon sources
utilized in the production of conventional PHB are raw material-
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729based, comprising solely of carbohydrates, such as sucrose, maltose,
glucose, starch and fatty acids along with their derivatives, methanol
and alkanes (Kamravamanesh et al., 2017).
In order to overcome the challenge of finding suitable non-
competitive and cost-effective carbon sources for PHB production, re-
cent research efforts have concentrated on coupling WWT with PHB
production. Wastewater from various sources is among the options
that have been investigated (Muhammadi et al., 2015; Raza et al.,
2018; Roland-holst and Heft-neal, 2013). The challenge, however, is
that, although sufficient amounts of PHB production from industrial
WW (both synthetic and natural) have been reported in some studies,
others have also noted that enrichment of theWWmediumwas neces-
sary to achieve similar results (Yuan et al., 2015). Nevertheless, coupling
microbial WWT with resource recovery is a viable solution to reducing
the WW footprint, as well as reducing the cost of production of benefi-
cial microbial bio-products such as bio-plastics.
1.1. Overview of biopolymer production routes
PHA synthesis occurs via different metabolic routes. Heterotrophic
bacteria for instance, are able to controlmetabolite flux for PHA accumu-
lation by using extracellular substrates. Contrarily, photoautotrophs such
as cyanobacteria have to begin with CO2 to synthesize their metabolites
(Asada et al., 1999; Carpine et al., 2015). These organisms draw energy
from sunlight to produce ATP without the need for oxygen. Such anaer-
obic conditions contribute to cost reduction in culture systems due to the
elimination of aeration (Fradinho et al., 2019). Under nitrogen-starved
conditions, amino acid synthesis is reduced, thus leading to an increase
in acetyl-CoA flux. Another enzyme, phosphoacetyltransferase activity
increases and finally PHB synthesis takes place due to the activation of
PHB synthase.
The photoautotrophic approach of producing PHB occurs in mi-
crobes such as Chlorella sp., Calothrix sp., purple- and green-non sul-
phur bacteria that use light as an energy source for photosynthesis.
Chemoautotrophs on the other hand utilize CO2 and energy from
chemical reactions. Methanotrophs are chemoautotrophic microbes
widely used inWWT and PHB production (AlSayed et al., 2018). Con-
trarily, the heterotrophic route of PHB production involves microbes
such as Synechocystis sp., Spirulina sp. and halophilic archaea that
utilize waste organic matter for PHB synthesis (Salgaonkar and
Bragança, 2017). Chemoheterotrophs use chemical energy and or-
ganic matter to synthesize PHB but it should be noted, that the
chemoheterotrophic method of PHB production in bacterial system
is expensive due to costly carbon sources such as acetate (a PHB pre-
cursor) needed for growth (Narancic et al., 2016). In contrast, the
production of PHB through the photoautotrophic cyanobacteria or
microalgae systems is a feasible alternative for low-cost PHB produc-
tion due to the inexpensive feedstock from light and CO2 (AlSayed
et al., 2018; Carpine et al., 2020; Löwe et al., 2017). There are in-
stances where PHB is produced both autotrophically and heterotro-
phically. For instance, X. Xu et al. (2020) and M. Xu et al. (2020)
demonstrated the ability of Cupriavidus necator to produce PHB via
heterotrophic and autotrophic approaches. Heterotrophic growth
in Cupriavidus necator takes place through the utilization of organic
substrates (Garcia-Gonzalez et al., 2015). Oxygen and NH+4 are con-
sumed during growth while producing CO2 as a side-product. At this
stage, PHB production is suppressed by excess NH+4 supply but under
nutrient limitation, the organic carbon source is used for PHB produc-
tion (Mozumder et al., 2014). Ranaivoarisoa et al. (2019) also evaluated
the PHB performance of Rhodopseudomonas palustris TIE-1 under pho-
toautotrophic, chemoheterotrophic and photoheterotrophic conditions.
The high PHByield under chemoheterotrophic conditions (aerobic)was
attributed to the supply of amino acids from peptone supplementation.
Overall, photoelectroautotrophy and photoferroautotrophy showed the
highest PHB electron yield and the highest specific PHBproductivity, re-
spectively. These results demonstrate the ability of R. palustris to yield3
the highest specific PHB productivity using Fe(II) as an electron donor
for photoautotrophy through new routes. These new routes serve as po-
tential substitutes for PHB bioproduction (Ranaivoarisoa et al., 2019).
Similarly, the growth performance of Chlorella vulgaris (a photoautotro-
phic microbe) measured under different CO2 concentrations and light
intensities using a novel microdroplet photobioreactor proved to be
better than that of a flask culture because of the reduced shading effects
and improved mass transfer (Sung et al., 2016). However, strains such
as Calothrix scytonemicola, Nostoc muscorum and Spirulina sp. LEB 18
perform poorly in photobioreactors due to biofilm formation (Carpine
et al., 2020). These scenarios prove that, aside the type of microbial
strain, reactor type also influences PHB production to a large extent.
Using mixed microbial cultures (MMC) for WW treatment coupled
with PHB production offers numerous advantages such as reduction in
cost associated with production (Fradinho et al., 2019; Yuan et al.,
2015), and utilization of complex substrates (Aslan et al., 2016; Chen
and Jiang, 2017) among others. To achieve this, various genetic engi-
neering and molecular biology techniques have been adopted. To en-
hance PHB production in microorganisms, techniques such as over-
expressing genes in natural producers (Ben et al., 2016) or introduction
or deletion of genes innon-PHB-producingmicrobes have been success-
fully accomplished (Wu et al., 2016). Advances in this area have allowed
for the alteration of biosynthetic pathways involved in PHB production,
which subsequently led to significantly higher PHB yields. Additionally,
these alterations have provided opportunities for the utilization of a
broader substrate range, otherwise unsuitable for microbial cultivation.
Thus, allowing successful exploitation of resources initially considered
as waste. However, due to the high cost associated with sterilization, it
is economically unwise to cultivate genetically engineered organisms
onwastewater. There have also been successful attempts at transferring
the capability for PHB synthesis from bacteria to higher plants (Poirier
et al., 1995; Suriyamongkol et al., 2007). Although this approach has
been demonstrated to be successful in many laboratory scale projects,
commercialization has yet to be cost-competitive.
The paper presents the biosynthesis and biodegradation pathway of
the biopolymer, PHB which is produced by microbes cultivated on
waste effluents, either in pure or mixed cultures. The conditions associ-
ated with wastewater cultivation such as nutrients, pH, light, cycle
length and strain type are also discussed along with some genetic engi-
neering approaches that have been successfully employed in the pro-
duction of PHB. The paper also highlights the commercial aspect of
PHB production and the challenges faced by the industry.
2. Research tendencies in Poly-β-hydroxybutyrate production
As far back as 1519, interests in the study of microalgae arose from
the discovery of Spirulina sp. in Spain due to its nutritional properties
(Soni et al., 2017). Since then, numerous research has gone into under-
standing the potential benefits of microalgae toman. This led to the dis-
covery of the PHB homopolymer by Lemoigne in 1920's (Kosseva and
Rusbandi, 2018). Many years down the line, in the 1950's Oswald
et al. (1957) pioneered the research to propose the use of microalgae
inwastewater bioremediation. This has set the pace for wastewater cul-
tivation of microbes for resource recovery. In this paper, the authors
have attempted to show the trends in PHB research over a 20-year pe-
riod through author keywords such as PHB, microbial bioplastics and
biosynthesis as topic searches for indexed articles published from
1999 to 2020. This summarized information provides an indication of
developments in research interests from a temporal perspective.
ELSEVIER ScienceDirect, SpringerLink, Taylor & Francis andWileyOnline
Library databases were used and data obtained from each database
using the keywords was analysed. The analysis revealed that out of
the searched literature, PHAs were the most frequently mentioned
keyword for the 20-year period (50%). These results indicate the level
of interest of research activities within the scientific community. Inter-
estingly, degradation declined from 5% in 1999–2003 period to 1% in
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729the 2014–2020 period which could indicate a shift from a focus on sin-
gle use plastic degradation to a more sustainable point of view of dura-
bility and re-usability for an eco-friendly bio-economy. Research in
industrial applications of PHB increased from 2% in 1999–2003 period,
doubled in 2004–2013 and continued to increase. Currently research in-
terest in 2020 stands at 7% in comparison to other keywords used in this
research (Fig. 1).
2.1. Current developments
Spirulina sp. and Chlorella sp. are common species that are commer-
cially cultivatedworldwide. They aremostly used as protein supplements
for human food, aquaculture feed andpigments in the cosmetics industry.
Dunaliella salina and Haematococcus pluvialis are also very popular due to
their pigments and antioxidants like carotenoids, astaxanthins and beta
carotene (de Jesus et al., 2018). Likewise, microbial biopolymers are of
much interest due to their environmental and economic potential. TheFig. 1. Trends in research related to microbial bioplastics from polyhydroxyalkanoates (PHA)
industrial applications with reference to the (a) increments of published literature, and pe
(e) 2009 to 2013, and (f) 2014 to 2020.
4
global PHAmarket is said to reachUS $ 93.5million in 2021, froman eval-
uated US $ 73.6 million in 2016 (Singh et al., 2019). This market size has
the potential to grow by US $ 18.66 million during 2020–2024 period
(Technavio, 2020). Currently, several biopolymer companies exist across
Asia, Europe and the Americas, producing PHA and its variants. Most of
these companies sell PHA as raw materials in resin or powder form
under brand names such as Sogreen™ (by Tianjin GreenBio, China),
Mirel® (by Metabolix, USA), Nodax™ (by MHG Bio, USA), Biocycle (by
Biocycles, Brazil), MINERV-PHA™ (by Bio-On, Italy) and VersaMer™ (by
Polyferm, Canada) among others. Metabolix, which is now Yield 10 Bio-
science has demonstrated the feasibility of using PHA polymers to en-
hance the performance of a widely used polymer such as polylactic acid
(PLA). Amorphous PHA copolymers have been used as plasticizers to
strengthen PLA which is often brittle, thus, bringing both ductility and
toughness. Another advantage of this blend is that, it does not compro-
mise the compostability of the polymer (Bioplastic News, n.d.). Another
exemplary company is Mango Materials, based in San Francisco, USA.and Poly-β-hydroxybutyrate (PHB), their biosynthesis, degradation, co-production and
rcentage research interest from (b) 1999 to 2020, (c) 1999 to 2003, (d) 2004 to 2008,
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729The company has been in existence since 2012, producing PHA pellets
under the brand name YOPP and YOPP+. The company situates its facto-
ries next to existing methane production facilities such as landfills,
WWTPs and agricultural facilities in order to convert the methane into
PHA, specifically, P3HB. This conversion is done by the natural PHA accu-
mulation ability of methanotrophs that are naturally selected in the culti-
vation system and not genetically engineered (Mango Materials, n.d.).
The consortia of methanotrophs are ancient, robust bacteria that with-
stand invasionby othermicroorganisms andhence, do not require expen-
sive sterilization processes. The use of methane as feedstock also adds to
cost reduction as well as scalability of the entire system. Currently, the
company works with biogas from the Silicon Valley Clean Water WWTP
in the San Francisco Bay Area to validate and scale their PHA production
process. Across board, it has been observed that the main challenges
faced bymost companies are improving strain characteristics through ge-
netic techniques, developing efficient methods of cultivation in terms
open ponds or closed reactors, contamination control and optimizing har-
vesting processes. Biomass harvesting alone, contributes between 20 and
30% of the overall biomass production price (Fasaei et al., 2018). Energy-
intensive processes such as drying also contribute significantly (20%) to-
wards the overall cost of production. It is important to also bear in mind
the type and volume of solvents used in the extraction processes. Not
only are the solvents usually toxic, they are also volatile,making it difficult
to recover and reuse. Novel extraction techniques such as enzymatic or
bioextraction systems are promising approaches for environmentally
friendly cell disruption (Costa et al., 2018).
3. Selectionof Poly-β-hydroxybutyrate-producingmicrobes suitable
for wastewater cultivation
The high diversity among PHB-producing microbes is attributed to
their existence in various ecological niches due to their ability to with-
stand various environmental conditions. Both prokaryotic and eukary-
otic PHB-producing microbes dwell in high organic matter habitats
such as dairy waste conditions (Obruca et al., 2011; Rodriguez-Perez
et al., 2018), oil processing wastes (Carofiglio et al., 2017), waste from
pulp and paper mill processes (Bengtsson et al., 2008; Bhuwal et al.,
2013; Jiang et al., 2012), agricultural wastes and wastewater treatment
plant (WWTP) activated sludge (Khardenavis et al., 2007; Sangyoka
et al., 2012). Among PHB-producing microbes, halophiles have been
favoured in PHB studies since they eliminate the need tomaintain asep-
tic conditions (Tohme et al., 2018), aswell as their robustness in harsher
environments. PHBs have also been found to aid in cell integrity by pro-
viding protection against sudden osmotic imbalances (Sedlacek et al.,
2019). Thus,microbes that are naturally exposed to harsh environmental
conditions could storemore PHB. Identifying PHB-producingmicrobes in
nature, involves collection and rapid screening using phenotypic- and
genotypic-based screening (Muhammadi et al., 2015). Viable colony
staining techniques have been proposed as a method of phenotypic-
screening of PHB-accumulating bacteria. Gram-staining methods were
used to characterise PHB-producing microbes from soil pelagia and
paper mill effluents (Bhuwal et al., 2013; Biradar et al., 2015) which al-
lows rapid detection of PHB-accumulating colonies through PCR tech-
niques (Gasser et al., 2009; Wang et al., 2014). Genotypic-screening
methods were subsequently developed as more appropriate tools to cir-
cumvent the numerous drawbacks of phenotypic-screening methods
(Biradar et al., 2015). Two of the most widely exploited microbes in
PHB research are the photosynthetic cyanobacteria, Synechococcus and
Synechocystis spp. (Arias et al., 2018b; Kamravamanesh et al., 2018;
Kavitha et al., 2016b) due to their PHB accumulating efficiency while
growing on WW (Burnap, 2015; Hollinshead et al., 2014), and the ease
of genetic manipulation due to the availability of their full genome se-
quence (Kanesaki et al., 2012).
Apart fromdistinguishingmicrobes based on their natural ecological
niches and cell wall structure, they can be grouped further into two
major categories based on the required culture conditions for polymer5
synthesis (Khanna and Srivastava, 2005). The first group requires that
an essential nutrient like magnesium, nitrogen, phosphorus or sulphur
becomes limiting for PHA synthesis from an excess carbon source. This
includes bacteria species such as: Ralstonia eutropha (now Cupriaviduc
necator), Protomonas extorquens and Pseudomonas oleovorans. The sec-
ond group accumulates the polymer during growth phase, thus, devoid
of nutrient limitation. Alcaligenes latus, a mutant strain of Azotobacter
vinelandii and the recombinant Escherichia coli (Khanna and Srivastava,
2005) fall within this category. Six nutrient limiting approaches based
on nitrogen (N) and phosphorus (P) in Halomonas smyrnensis were
tested. The highest levan and PHB yieldswere achieved under unlimiting
conditions (Tohme et al., 2018), indicating that,H. smyrnensis belongs to
the group of PHB-producers that do not require limitation of a nutrient
for PHB accumulation.
It is also important to consider the nutritional requirements of the
microbe in terms of metabolism. That is, whether the microbe is a pho-
toautotroph, chemoautotroph, photoheterotrophor chemoheterotroph.
Thus, in order to select the most appropriate microbe for optimal PHB
production, a roadmap is proposed in Fig. 2 based on; (1) cell wall struc-
ture (2) nutritional requirements (3) ecological niche and (4) number
of carbon atoms. The cell wall structure of microbes confers unique
properties on the microbe. Most PHB-producing bacteria in literature
have been found to be gram-negative, compared to the limited number
of Gram-positive bacteria (Muhammadi et al., 2015). Amajor advantage
of using gram-positive species for PHA production is the absence of the
immunogenic lipopolysaccharides (Philip et al., 2009). Lipopolysaccha-
rides make up majority of the impurities in purified PHAs from gram-
negative bacteria and have been known to induce strong immunogenic
reactions. Therefore, their absence in gram-positive PHA is a major ad-
vantage inmedical applications. Thenumber of carbon atoms in themo-
nomeric units of PHA can also be used as a criterion to group microbes
based on carbon chain length. PHA production is strain specific and
sometimes dependent on carbon substrate the microbe is exposed to.
The first group produces short-chain-length PHAs (SCL-PHAs – C3-
C5), while a second group, can accumulate medium-chain-length
PHAs (MCL-PHAs – C6-C14). Although themajority of bacteria accumu-
late either SCL- or MCL-PHAs, a third group of bacteria have been found
to synthesize PHA co-polymers containing both SCL- andMCL-PHA (C3-
C14) (Goh and Tan, 2012). The fourth group is capable of synthesizing
long-chain length PHAs (LCL-PHAs >C14) (Timm et al., 2004) and
these typically include the pseudomonads. Knowing the specific
carbon-chain length produced by themicrobewill also inform the choice
of solvent used in the extraction process, as solvent properties such as
concentration, action time andworking temperature are known to affect
the molecular weight of the polymer (Chen, 2009). Taking these factors
into consideration will facilitate optimized cultivation, harvesting and
extraction conditions for efficient decision making in the event of
scale-up.
4. Utilization of wastewater for Poly-β-hydroxybutyrate production
Microbial WW cultivation has been performed since the 1950s
(Hoffmann, 1998). This technology allows the integration of WWT
with microalgal biomass generation for resource recovery. Wastewater
has been used as a source of substrate for the production of biofuels,
lipids and biopolymers for decades (Monshupanee and Incharoensakdi,
2014; Patel et al., 2018; Rahman et al., 2015; Takeshita et al., 2014). Bio-
polymers such as PHBs have been successfully produced with various
waste streams on a laboratory scale (Kamravamanesh et al., 2017;
Meixner et al., 2016). Although the price of producing biopolymers
largely depends on the substrate cost (Roland-holst and Heft-neal,
2013), several reports have also estimated that the total cost of produc-
ing these polymers also depends on microbial yield and productivity,
culture conditions such as temperature, aeration, pH-value etc., and the
recovery and purification processes. The carbon source alone is said to
account for 25–45% of the total production costs (Nath et al., 2008).
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729
Fig. 2. (a) Common microalgal and bacterial strains (wild and engineered types) utilized in research and industrial applications for enhanced Poly-β-hydroxybutyrate production
(b) criteria for selecting Poly-β-hydroxybutyrate-storing microbes based on ecological, morphological, physiological and metabolic factors.Thus commercially produced polymers such as PHAs have utilized rela-
tively inexpensive substrates such as methanol, cheese whey, molasses,
olive oil mill WW, poultry waste etc. (Pisco et al., 2009) which can be
broadly classified under industrial and municipal WW sources. Essen-
tially, there are two principal routes to produce PHB from WW. These
are: (1) cultivation of pure culture of a microbe using theWW as carbon
source; and (2) utilization of openmixedmicrobial culture (MMC) that is
enriched in PHB-producing microbes by the selective pressure imposed
on the culture. The latter approach allows the integration of PHB
production withWW treatment by adjusting already widely used princi-
ples of biological WW treatment such as activated sludge processes.6
Environmentally friendlymethods ofwastemanagement such as anaero-
bic digestion produces effluents like digestate supernatant which provide
carbon, nitrogen and phosphorus sources for microalgal cultivation
(Hollinshead et al., 2014; Kovalcik et al., 2017; Meixner et al., 2016).
The effluent quality improvement parameters highlighted in Table 1 are
with emphasis on nitrogen, phosphorus and COD reduction.
4.1. Poly-β-hydroxybutyrate production from industrial wastewater
Food processing WW is often rich in fermentable nutrients like
lactose, lipids and soluble proteins, and can therefore serve as an
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729
Table 1
Microbial cultivation with wastewater as substrate for biopolymer production.
Strain Source Substrate Culture conditions Organic Nutrient PHB content General comments & References
Removal
Chlorella National Collection of Cheese whey WWb Stationary phase 94.2% (N), 6.54g/L (79.8%) Reduced CODc & BODd while fixing
pyrenoidosaa Industrial 92.54% (P) CO2 (Sathya et al., 2018)
Microorganisms
(NCIM), India
Pseudomonas National Chemical Sugar refinery waste (cane N/A N/A 62.44% CDWe PHB Economically improved productivity
aeruginosa Laboratory, Pune, India molasses) of 0.11 g/L/h (Tripathi et al., 2012)
Cupriavidus Soil Cane final molasses Exponential-Stationary N/A 2.86 ± 0.82 g 27% Hydrothermal acid pre-treated
necator Phase molasses as carbon source led to
highest growth (Sen et al., 2019)
Synechocystis cf. Culture Collection of Digestate supernatant Exponential Phase N/A 78% PHB Nutrient concentrations (TNf, Pg) after
salinaWislouch Autotrophic Organisms harvesting, were below detection
(No. 192) (CCALA) limits (Meixner et al., 2016)
Bacillus subtilis Soil samples from Sugar industry WW Stationary Phase N/A 51.80% PHB yield up to 4.991 g/L with sugar
nG220 Haryana and Uttar industry WW as sole nutrient source
Pradesh (India) (Singh et al., 2013)
MMCh (Activated TuamWWTPi Galway, Synthetic WW (acetate, N/A Effluent PO4–P 28.8–50% Maximum PHB - 28% (anaerobic) &
sludge) Ireland yeast extract) concentrations 50% (aerobic conditions) (Rodgers and
below 1 mg/L Wu, 2010)
Activated sludge Combined dairy and Rice & Jowar grain-based N/A N/A 40% - 42.3% Addition of DAHP increased PHB
food processing distillery spent wash (67%) (Khardenavis et al., 2007)
industry WWTP
Azohydromonas N/A Dairy industrialWW (Cheese Mid-exponential phase N/A P(3HB) 1.21 g/L; Pre-treated whey, suitable substrate
lata DSMZ Whey) P(3HV) 0.45 g/L for PHBs & PHVsj production
1123 (Sharifzadeh et al., 2010)
Sludge Sludge from the UC Cheese WW N/A 83% COD 3% by MLVSSk More glycogen & PHB produced by SFl
Davis WWTP oxidation removal type than RFm type (Goffredo et al.,
ditch efficiency 2009)
E. coli City of Logan, WWT Hydrolyzed microalgae Stationary phase N/A 31% PHB Maximum PHB accumulation up to 31
facility Supernatant with standard ± 8.9% (Rahman et al., 2015)
E. coliM9 growth media
Activated Sludge Winnipeg South End 1.MWWn N/A Phosphorus PHB fromMMW MWWused with carbon-rich
Water Pollution Control 2.Beef extract uptake (mg/L) (15%) industrial waste for PHB obtainment
Centre 3. Acetate MWW (33.2) BE (13%) (Yuan et al., 2015)
4. Glucose Beef Extract (BE) A (42%)
(23.2) G (40%)
Acetate (A)
(83.0)
Glucose (G)
(54.4)
Activated sludge N/A Food processing industrial N/A N/A 33% C/No ratio of 144 led tomaximum PHB
WW (acetic) (Kumar et al., 2004)
Activated sludge Kayseri domestic Simulated WW(Acetate) N/A N/A 55% PHB storage increased as the cycle
wastewater treatment length decreased (Ozdemir et al.,
plant (Turkey) 2014)
Bacillus Czech Collection of Cheese whey Stationary phase N/A 51.57% PHB yields improved by 40% after 1%
megaterium Microorganisms (Brno, introduction of ethanol (Obruca et al.,
CCM2037 Czech Republic) 2011)
Pseudodonghicola Red sea, Saudi Arabia Date syrup Stationary phase N/A 38.85% 4% NaCl, and peptone was the
xiamenensis preferred nitrogen source (Mostafa
et al., 2020)
Purple non-sulfur N/A Winery WW N/A COD & N 203 mg/L Co-production of H2 (468 mL/L) and
bacteria (mixed reduction PHB (203 mg/L) (Policastro et al.,
consortium) 2020)
Aulosira N/A Aquaculture WW Log phase Ammonia, 92 g/m2 (summer), Recirculatory WWT and PHB
fertilissima nitrite, and 89 g/m2 (rainy), 80 production (Samantaray et al., 2011)
phosphate g/m2 (winter)
reduction
a Algae,
b WW – Wastewater,
c COD – Chemical oxygen demand,
d BOD – Biological oxygen demand,
e CDW – Cell dry weight,
f TN – Total nitrogen,
g P – Phosphorus,
h MMC – Mixed microbial culture,
i WWTP– Wastewater treatment plant,
j PHV – Polyhydroxyvalerate,
k MLVSS – Mixed liquor volatile suspended solids,
l SF - static fill,
m RF – react fill,
n MWW – Municipal wastewater,
o C/N – Carbon-to-nitrogen ratio.
7
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729inexpensive substrate for microalgal cultivation (Raza et al., 2018).
Mixed cultures allow the exploitation of complex substrates. In the
WWT industry, microorganisms employed for phosphorus removal,
also known as phosphorus-accumulating organisms (PAOs), have the
capability to synthesize PHB as their source of energy (Yuan et al.,
2015). The reuse of organic matter in producing PHB from industrial
WW highly rich in carbon such as agricultural waste, brewery waste
and municipal WW, may considerably lower the cost (Anterrieu et al.,
2014). Furthermore, biopolymer production has been integrated into a
sugar factory WW treatment by mimicking factory processes in two
parallel sequencing batch reactors (SBRs). Both SBRs produced biomass
along with PHA production while maintainingWWT standards with re-
spect to carbon, nitrogen and phosphorus for the factory effluents. Cas-
sava starch manufacturing WW (CSW) has been successfully used by a
chemoautotrophic bacteria species,Cupriavidus sp. KKU38 as a substrate
suitable for PHB production. Acidogenic fermentation of CSW to obtain
volatile fatty acids (VFAs) was first conducted by Sangyoka et al. (2012)
and was found to be more efficient in producing PHB than raw CSW.
Variations in chemical oxygen demand:nitrogen:phosphorus ratio
(COD:N:P ratio) were also investigated, and an optimum ratio of
100:0.5:11 resulted in maximum PHB (85.53%). Clearly, apart from the
suitability of WW for PHB production, culture conditions have to be
well optimized in order to achieve maximum results.
4.2. Poly-β-hydroxybutyrate production from municipal wastewater
To a large extent, the concentration of WW constituents influences
PHB production (Kavitha et al., 2016a). For instance, in an instance
where glucosewas the only substrate used, majority of it was converted
by the succinate-propionate pathway to propionyl-CoA, resulting in
poly-3-hydroxyvalerate (PHV) (Ahn et al., 2009) with relatively low
PHB production. It was thus suggested that a mixture of municipal
WW and certain carbon-rich industrial wastes could be suitable
substrates for PHB production. The feasibility of mixing municipal
wastewater (MW) and magnesium (Mg2+)-enriched nickel laterite
ore wastewater (NLOWW) on the growth, cellular composition, photo-
synthetic activities, nutrient and Mg2+ removal ability of Chlorella
sorokiniana was demonstrated by Chen et al. (2020). This approach
was demonstrated as economically feasible with revenue of $75.6 per
kilogram biomass which could be applied in PHB research. Low PHB
production has been observed with beef extract 13% cell dry weight
(CDW) suggesting that PAO in this system could not effectively utilize
beef extract and that substrates high in amino acid were therefore not
ideal for PHB production (Yuan et al., 2015). Again, a method for the
treatment of municipal WW was developed by Basset et al. (2016), in-
corporating the selection of biopolymer-storing microorganisms under
aerobic conditions with nitritation/denitritation. The selection of these
microorganisms was successful and internally stored PHA facilitated
denitritation in the famine phase.
The challenge with using mixed cultures for PHB production is
the variation in carbon source preference, which sometimes leads
to overall low PHB productivity due to the presence of certain spe-
cies. Under carefully selected limiting conditions, dominance of a
specific strain(s) subsequently results in higher PHB yields. Thus,
majority of research on PHB production in mixed cultures usually en-
rich the cultures to eliminate the non-PHB storing population, leav-
ing a mixture of a few high yielding species which sometimes leads
to an almost-pure culture. For instance, Kourmentza et al. (2009) re-
ported that after several cycles of alternating between carbon and ni-
trogen limitation, Pseudomonas sp. dominated the culture. However,
the enrichedmixed culturewas found to bemore promising for PHAs
production from short-chain fatty acids compared to the individual
productivity of two isolated strains. The enriched culture also led to
higher yields of PHAs per VFAs consumed. Another challenge with
utilizing municipal wastewater (MWW) for PHA production is the
relatively low VFA content. Despite this challenge of VFA-poor8
MWW streams, a feast–famine approach has proven to be feasible
at laboratory and pilot-scale studies (Morgan-Sagastume et al.,
2014). The advantage of using mixed cultures in wastewater PHB pro-
duction is the ability to maintain the cultures under non-sterile condi-
tions. Pure cultures require stringent conditions in order to maintain
only the desired population. Nonetheless, techno-environmental assess-
ment of MMC PHA production fromMunicipal WWTP has a potential of
deliveringmore valuable renewable rawmaterials than the known bio-
gas and bioenergy of current technologies (Morgan-Sagastume et al.,
2016). Table 1 highlights some key research works done on microbial
PHBs achieved through WW cultivation. These key works demonstrate
the importance of simultaneous water quality improvements and valu-
able biopolymer production.
4.3. Co-production of Poly-β-hydroxybutyrate and other metabolites in
wastewater
Aside the sole production of PHBwithWW as nutrient source, other
valuable by-products can be obtained through a cell factory concept of
co-production. Co-production of PHB and other metabolites is a com-
mon phenomenon in a typical microbial system. It is feasible to use
the PHB pathway to manipulate other metabolic pathways (Kang
et al., 2010; Xu et al., 2016). However, the yield of one product over an-
other is dependent on the microbial strain, the culture conditions as
well as to a large extent, the research goals. Integrated approaches
aimed at producing high yields of multiple bio-products can be success-
fully achieved, as long as the biosynthesis pathways of the individual
products do not compete for substrates (Kumar and Kim, 2018). De-
pending onwhich bio-product is desired, growth conditions can bema-
nipulated to stimulate the accumulation of a specific bio-product over
another (Quagliano andMiyazaki, 1999). Tomake bio-processing easier,
one of the desired productsmust be “membrane-bound, secretory or ex-
tracellular” (Kumar and Kim, 2018) in order to allow for the maximum
utilization of resources, as well as easy downstream processing espe-
cially with regard to the simultaneous extraction of various metabolites.
Carotenoids such as astaxanthins and β-carotene are undoubtedly
important metabolites of high market value that can be co-produced
along with PHBs. Several industrial applications exist for the use of
these pigments in nutritional supplements, alternative medicine, food
etc. thus, showing a great potential in lowering manufacturing costs
due to this zerowaste approach. The bacterium Rhodobacter sphaeroides
is capable of utilizing waste effluents in PHB and hydrogen production
while concomitantly reducing COD levels (Eroǧlu et al., 2004; Ghimire
et al., 2016). Similarly, high hydrogen productionwas achieved through
co-production with PHB by R. palustris CGA676 utilizing agroindustrial
waste. The highest hydrogen production was observed in wheat bran
effluents (648.6 mL/L), while the highest PHB yield was obtained with
olive pomace (11.53% TS) (Corneli et al., 2016). The ability to obtain var-
ious products from a batch culture emphasizes the feasibility of cost re-
duction through coproduction of metabolites.
4.4. Biosynthesis and metabolic engineering of microbial Poly-β-
hydroxybutyrate
Biosynthesis of PHB occurs via a variety of well-established routes.
The most common PHB production route takes place: (i) within micro-
bial cell systems via a PHB-polymerase catalysed reaction through ge-
netically engineered recombinant microbes, (ii) during the anaerobic
digestion of biological wastes or (iii) through the utilization of trans-
genic plants (Hahn et al., 1999; Zinn et al., 2001). Of these, microbial
production is considered the major source of PHB. Due to the large
chemical diversity of the biopolymer, biosynthesis in microorganisms
varies widely (Suriyamongkol et al., 2007). Among the numerous
classes of microorganisms, bacteria are the most studied group of
PHB-producing microbes. Although other classes of microorganisms
such as green algae (Arias et al., 2019; Sathya et al., 2018) and diatoms
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729(Hempel et al., 2011) are known, bacteria, either wild type or geneti-
cally engineered types, have been reported to produce higher polymer
contents. Thus, majority of literature on the biosynthesis pathways
and genetic manipulations have focused on bacterial strains.
Under certain nutritional and environmental conditions, microbes
accumulate PHB through the hydrolytic activity of PHA depolymerase
(PhaZ) (Uchino et al., 2008). Although factors such as oxidative stress
(Koskimäki et al., 2016), protein synthesis rate and cellular energy de-
mand (Handrick et al., 2000) have been identified as possible stimulants
of PHB accumulation in microbes, till date, the mechanisms controlling
the accumulation process are yet to be fully understood (Teixeira et al.,
2019). The balance between these stimulants and the accumulation
process, is a crucial metabolic cycle (PHB cycle) (Trainer and Charles,
2006)which is very necessary formicrobial survival in the environment
(Sedlacek et al., 2019). Several studies have correlated PHB accumula-
tion with microbial survival under UV radiation, high temperature and
osmotic shock (Koskimäki et al., 2016; Ruiz et al., 2001; Sedlacek
et al., 2019). The intracellularly accumulated PHB is coated by an abun-
dance of structural proteins called Phasins that regulate the number and
size of the PHB granules within microbial cells (Jendrossek, 2009;
Mezzina and Pettinari, 2016).
The aim of many genetic manipulation approaches has been to in-
crease polymer content, to reduce cost or improve polymer quality.
These approaches have been improved upon over many years to meet
biotechnological needs.
4.4.1. Gene manipulation – nutrition
To overcome certain production hurdles for potential industrializa-
tion, several studies have successfully engineered certain microbial
strains to produce significant amounts of PHBs by broadening substrate
ranges (Wang et al., 2018), or engineering bacteriamorphology for easy
downstream separation (Chen and Jiang, 2017; Liu et al., 2011). PHB is
synthesized through a three-step pathway in bacteria (Steinbüchel
and Füchtenbusch, 1998) involving three key enzymes, namely β-
ketothiolase, NADP-specific acetoacetyl-CoA reductase, and PHB syn-
thase which are coded by phbA, phbB, and phbC, respectively.
By changing the fatty acid concentrations, co-monomer composi-
tions were easily regulated in Pseudomonas putida KTQQ20 which sub-
sequently produced “a novel diblock copolymer P3HHx-b-P(3HD-co-
3HDD) made up of 49mol% P3HHx and 51mol% P (3HD-co-3HDD)”
(Tripathi et al., 2013). Thus, a platformwas established to produce a bio-
polymer with adjustable monomer compositions through this β-
oxidation-weakened P. putida.
4.4.2. Gene manipulation – morphology
Engineering attempts have also been made towards modifying the
morphology of cells in terms of size and form modification, to increase
accumulation of inclusion bodies such as PHBs. The size and shape of
E. coli JM109SG cells were successfully modified bymaking them larger
and less fragile. Typical E. coli cells of about 0.5–2 μm increased their di-
ameters to 10 μm and changed from typical rod shapes to spherical,
leading to increased PHB production. However, these new cells grew
poorly compared to their parent cells (Jiang and Chen, 2015). Jiang
et al. (2015) observed that, cells reverted to rod shape uponmreB allele
insertion, thus, recommended that shape change induction be done
after the cells have already grown to high enough densities. Again, typ-
ical binary fission in E. coliwasmodified tomultiple fission by removing
fission-related genesminC andminD (Wu et al., 2016) (Fig. 3). Multiple
fission rings known as Z-rings were formed at several locations of a
lengthened cell, thus achieving more than the usual two daughter
cells through cell division. This led to higher CDW and over 80% PHB ac-
cumulation compared to control cells with normal binary fission. En-
larged morphology is generally known to increase PHB synthesis and
also promote separation of cells through gravity from the fermentation
broth (Jiang and Chen, 2015). Enhanced downstream processing and
subsequent cost reduction can be achieved through this technology.9
4.4.3. Gene manipulation - non-native host
Apart from genemanipulationwithin PHB-producers, phb genes can
be incorporated directly into non-native hosts. The successful incorpo-
ration of PHB genes from Ralstonia eutropha into Chlamydomonas
reinhardtii demonstrated the assimilation of part of a native biopolymer
synthesis pathway into a non-native host. PCR results confirmed the in-
tegration of both phbB and phbC genes into nuclear DNA of C. reinhardtii,
thus the double transgenic microalgae harbouring phbB and phbC genes
was obtained (Chaogang et al., 2010). Similarly, the full PHB pathway of
a bacteria (R. eutropha H16) was successfully integrated into a diatom
(Phaeodactylum tricornutum), achieving about 10.6% PHB (% CDW)
(Hempel et al., 2011). These successful examples highlight the possibil-
ity of inserting biochemical pathways into non-native hosts, to broaden
the avenues available for PHB production. With current engineering
technologies such as clustered regularly interspaced short palindromic
repeat (CRISPR), cell factories can be engineered using ideal microbial
strains to efficiently synthesize PHBs. Microbe-derived bio-plastics can
therefore become economically attractive through a bio-refinery
model, where multiple bio-products will be produced from a single mi-
crobial source, particularly if WW is utilized as the carbon source,
thereby valorizing the entire process. Fig. 4 illustrates the biosynthesis,
application and degradation of PHB (Hankermeyer and Tjeerdema,
1999; Pakalapati et al., 2018; Steinbüchel and Füchtenbusch, 1998).
5. Factors influencing the accumulation and composition of Poly-β-
hydroxybutyrate
Physiological processes within microbial cells are temperature, light
and nutrient dependent. Optimal growth yield occurs within specific
ranges which are successfully controlled in closed systems, but prove
challenging in open pond systems (Ge et al., 2017). Nutrient availability,
feeding mechanism, pH, cycle length, temperature and light are factors
that influence the ability of PHB-producingmicrobes to accumulate sub-
stantial amounts of the biopolymer, aswell as influence its composition.
The inherent ability of themicrobes to assimilate certain nutrients, syn-
thesize specific metabolites, as well as strain specificity should also be
considered in accounting for PHB yield. Themolecular mass of PHB pro-
duced in the cells of the bacteria, E. coli for instance depends strongly on
culture conditions (Suriyamongkol et al., 2007).
5.1. Strain
Apart from the fact that substrate cost is a major bottleneck in PHB
production (Chen and Jiang, 2017; Lee et al., 1999; Roland-holst and
Heft-neal, 2013), the mode of application and type of substrate, has a
huge impact on the performance of the microbial community. For in-
stance, Roja et al. (2019) observed faster growth rate in cyaonbacteria
species (Synechococcus, Leptolyngbya and Oscillatoria) than in green
microalgae (Chlorella) cultivated on ASN III medium. The maximum
growth of the cyaonbacteria species occurred on day 18 compared to
day 21 for green algae. The difference in the thermal stability of the ex-
tracted PHA also buttresses the point about strain specificity in situa-
tions where cultures conditions are kept the same. The use of MMCs
in activated sludge systems for PHB production is an approach that re-
duces the production cost as well as having the advantage of higher
biodegradability (87%). This is due to the consortium of organisms com-
pared to a single organism system in pure cultures (Moita and Lemos,
2012; Shalin et al., 2014). In a mixed system, cultures are usually sub-
jected to enrichment conditions in order to favour the microbes with a
higher biopolymer production rate. For instance, the impact of a non-
storing biomass on PHA production in a mixed culture was investigated
and the results revealed that, although Plasticicumulans acidivorans, a
known PHA producer has the potential of accumulating a good amount
of PHA, the presence of the non-storing population (Methylobacillus fla-
gellates) reduced the maximum PHA content of the culture to 66 wt%
from more than 80 wt% in an SBR (Marang et al., 2014). Between 84
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729
(a)
Multiple fission 
rings (Z-rings)
Cell shape 
control gene 
(mreB)
(b)
(c)  
phbC
phbB Double transgenic 
microbe
Fig. 3. Enhanced Poly-β-hydroxybutyrate production through genetic modifications for cost-effective bio-plastic production. (a) Modification of typical binary fission to multiple fission-
Four daughter cells instead of two. (b) Changing from typical rod shapes to spherical-Enhanced cell size. (c) Introduction or deletion of genes in non-PHB-producing microbes.and 90 wt% PHB content has also been achieved in an MMC system
dominated by proteobacteria, Plasticicumulans acidivorans and Thauera
selenatis (Jiang et al., 2011a). A feast/famine approachwas usedwith ac-
etate and lactate as substrates. This approach gives an indication of the
selective pressure of MMCs on microbial stains in PHB production. In
another study, the effect of the influent substrate concentration
(30–60 Cmmol VFA/L) on the selection of a PHA-storing culture was
assessed, using fermented sugar molasses. An influent substrate
concentration of 45 Cmmol VFA/L proved to be the best PHA-storing ca-
pacity, yielding about 74.6% due to a highly enriched PHA-storing
population of 88% (Albuquerque et al., 2010). The fact that neither sub-
strate concentration nor feast to famine ratio was limiting factors in
those conditions is noteworthy. Meaning that the PHA yield achieved
was as a result of the dominating population of PHA-producers.
5.2. Nutrients
Nutrient limitation is widely known to affect PHB production
(Kaewbai-ngam et al., 2016; Kamravamanesh et al., 2017). Particularly,
nitrogen (N) and phosphorus (P) limitation, which are common natural
stress conditions encountered bymicrobes have beenwell documented
and known to influence the accumulation of PHBs (Dutt and Srivastava,
2018; Kamravamanesh et al., 2018; Monshupanee and Incharoensakdi,102014; Takeshita et al., 2014). The combined effects of N and P defi-
ciency have resulted in the highest PHB accumulation in unicellular
cyanobacterium Synechocystis sp. PCC 6714 under photoautotrophic
conditions (Kamravamanesh et al., 2017), and have also led to dom-
inance of cyanobacteria over green algae Scenedesmus sp. in an SBR
with mixed consortia of microalgae (Arias et al., 2019). Further
confirming the influence of nutrients on strain type selectivity. Nu-
trient deprivation, especially nitrogen deprivation, leads to a signifi-
cantmetabolic reorganization. Evidently, bacterial PHB synthesis can
be induced by limiting oxygen or an essential nutrient like nitrogen,
phosphate, sulphate, magnesium, or potassium (Ansari and Fatma,
2016; Basset et al., 2016; S. S. Costa et al., 2018a; Nakaya et al.,
2015; Nath et al., 2008). In their absence, microorganisms cannot
produce amino acids or proteins, but instead synthesize and accu-
mulate PHB as discrete granules in the presence of excess carbon
(Muhammadi et al., 2015). They store this excess carbon until the
limitation is removed, at which time they may degrade and metabo-
lize the stored PHB (Hankermeyer and Tjeerdema, 1999). Metabolic
reorganization that occurs within the cells includes; loss of chloro-
phyll (chlorosis) (Sauer et al., 2001), a decrease in protein levels
(Depraetere et al., 2015) and increase in storage polymers like glyco-
gen and PHB in a sequential manner (Damrow et al., 2016). Gener-
ally, nutrient limitation, is more favourable for PHB accumulation
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729
Potential Waste Carbon Sources 
Cassava starch WW, Olive mill effluent, Milk/ice 
cream WW, Digestate supernatant, Sugar refinery WW, Biodegradation
Brewery WW, Rice grain distillery WW   
Biosynthesis O2
PHB
Microbial cell
Acetyl-CoA (2) PHB Depolymerase Depolymerization 
β-ketothiolase 
(phbA) Condensation
(R)-3-hydroxybutyric acid 
Acetoacetyl-CoA
NADPH+ H
β-Oxidase Oxidation
Acetoacetyl-CoA 
reductase (phbB) Reduction
NADP+ H2O
Acetyl-CoA
(R)-3-hydroxybutyryl-CoA 
PHB synthase Polymerization 
(phbC)
TCA 
Cycle
(Inclusion bodies)
Precursors (H2O, CO2) 
(Biological Building
Blocks)
Extraction PHBvia:
(Solvent, ozone, mechanical 
disruption, enzymatic digestion) Applications
Medicine
Controlled drug release, absorbable 
sutures, bone plates & bone marrow 
support, surgical pins
Agriculture Packaging Material 
Plant growth regulators, Disposable plates, cups,
regulated herbicides and cutlery, film& staples
pesticides discharge, fertilizers
Energy
Biofuels from 3-HB & 3-HA 
methyl esters, HTL
Fig. 4.Microbial Poly-β-hydroxybutyrate biosynthesis and degradation pathway with potential wastewater carbon sources and industrial applications.than their complete absence (Arias et al., 2018a; Cavaillé et al.,
2016), thus majority of studies in this area make reference to nutri-
ent limitation rather than deprivation.
Numerous reports on impacts of various forms of nitrogen on mi-
crobial growth (Carpine et al., 2015; Costa et al., 2018a; Dionisi et al.,
2005; Manna et al., 1999; Montiel-Jarillo et al., 2017; Nakaya et al.,
2015; Tavernier et al., 1997; Tohme et al., 2018) have reported
higher PHB yield with nitrate than with ammonia as the limiting11nutrient (Kamravamanesh et al., 2017). Higher PHB yield was ob-
served with organic nitrogen source (peptone) than with inorganic
nitrogen (NH4Cl) (Mostafa et al., 2020). The PHB contents of 134
PHB-producing strains of bacteria studied appeared to be subtly no-
ticeable under normal growth conditions. However, this significantly
increased in 63 strains which were put under nitrogen deprivation
(\\N). Higher than with phosphate deprivation, and/or potassium and
an all-nutrient deprivation (Kaewbai-ngam et al., 2016). Usually,
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729microalgae cellular growth decreases in nitrogen-limited medium,
indicating that nitrogen deficiency affectsmetabolic activity ofmicroor-
ganisms negatively and this subsequently translates to low biomass
production, as well as alteration of the biochemical composition
(Costa et al., 2018a). To overcome the bottleneck of reduced biomass
growth while trying to achieve high PHB content, additional carbon
sources are introduced to themediumduringnitrogen limitation. For in-
stance, acetate supplementation under N-deprivation has been shown
to increase PHB accumulation. In cyanobacteria, Synechosystis PCC
6803, a twofold increase was observed after addition of acetate. Glucose
supplementation significantly improved cellular growth rate and, thus,
improved PHB productivity (Ansari and Fatma, 2016; Monshupanee
et al., 2016). Adding acetate at the beginning of nitrogen deprivation
doubled the PHB levels within the cells and contributed 44–48% to
PHB synthesis, further demonstrating this (Dutt and Srivastava, 2018).
Gene expression related to assimilation of nitrogen and nitrate or
the transport of nitrite is known to be reduced by overexpression of ei-
ther SigE or Rre37 after nitrogen starvation. SigE andRre37 are transcrip-
tional regulators whose transcript and protein levels increase during
nitrogen starvation (Nakaya et al., 2015). Nutritional influence on
gene expression can further be understood through the biosynthetic
pathway of PHB, discussed in Section 4.4.1. In a mixed WW-borne mi-
crobial culture, phosphorus limitation resulted in a cyanobacteria-
dominated culture and noticeably higher levels of carbohydrate content
(43%–48%) than cultures with high loads of nitrogen and phosphorus
and carbon limitation (29%). Carbon uptake and the resultant produc-
tion of polymers from cyanobacteria are shown to be improved through
nutrient feeding strategies (Arias et al., 2018a).
5.3. Feeding mechanism
Theway inwhichmicrobes are fed in a controlled cultivation system
has a great influence on their bio-product accumulation capacity. Ge
et al. (2018b) compared single-dose initial feeding, multiple-dose step
feeding and single-dose exponential feeding under mixotrophic condi-
tions and noticed that feeding glycerol at the late exponential growth
stage resulted in the highest biomass and lipid productivities with var-
ied lipid compositions. Likewise, PHB production is usually more effi-
cient in a two-stage culture (Monshupanee et al., 2016; Ozdemir et al.,
2014). Cells are first grown in an enriched medium containing suitable
nutrients, once they generate enough biomass, they are centrifuged,
washed and transferred to a nitrogen-free medium. During step two,
absence of nitrogen or phosphorus, or both, leads to PHB accumulation
at the expense of further biomass generation. Conversely, in a one-step
culture condition, cells grown in a medium with glucose as the only
carbon source were used as inoculum in a nitrogen-limited medium
containing different carbon sources. The amount of nitrogen sup-
plied leads to limitations in cell multiplication thus, the biopolymer
is formed from the excess of carbon source available (Silva-queiroz
et al., 2009).
Aerobic dynamic feeding (ADF) conditions also showed promise of
significant capacity to store polyhydroxybutyrate (PHB). In a study
(Serafim et al., 2004), high substrate concentration supplied at once,
proved to be inhibitory for PHB storage mechanism in a mixed culture.
To avoid substrate inhibition, acetate was supplied differently including
180 Cmmol/L continuously fed and three pulses of 60 Cmmol/L each.
This approach increased the specific PHB storage rate in both cases,
yielding 56.2% and 78.5% PHB content respectively, indicating that
pulse feeding has a positive impact on PHB storage. Similarly, Fradinho
et al. (2014) observed that out of the six tested organic acids (malate,
citrate, lactate, acetate, propionate and butyrate), only three of the
VFAs enabled PHA production in a mixed photosynthetic culture. Ace-
tate and butyrate led to the formation of PHB while propionate pro-
duced a HB:HV copolymer with a 51% fraction of HV. These examples
reiterate the fact that feeding strategy could be optimized to achieve
specific polymer properties and obtain significant yields especially in a12mixed culture, by stimulating the dominance of a high polymer-
storing population.
WWTPs operated under anaerobic and aerobic cycles are capable of
PHA production due to the presence of glycogen accumulating Organ-
isms (GAOs) and PAOs. These microbes cycle PHA as part of their me-
tabolism by taking up carbon substrates for PHA synthesis while
consuming glycogen. Under anaerobic conditions, PAOs release phos-
phate, thus, acquiring energy for PHA accumulation.Meanwhile, during
aerobic conditions, phosphate is taken up in excess for the replenish-
ment of polyphosphate pool, and PHA is degraded for storing energy
(Serafim et al., 2008). Thus, in the presence of oxygen, both PAOs and
GAOs use stored PHA for growth, maintenance and glycogen pool
replenishment.
The behaviour of microbes subjected to feast-famine regime was
first proposed by Daigger and Grady (1982). The authors explained
that, when there is an absence of external substrate for a significant pe-
riod, the amount of intracellular components such as RNA and enzymes
required for cell growth decreases. After such long starvation period, in
the event that the culture is dosedwith an excess of carbon, the amount
of the available intracellular enzymes is much lower than that required
to reach the maximum growth rate. This is usually observed as slow
growth response. In such situations, PHA storage becomes thedominant
response mechanism.
5.4. pH
Physical growthparameters also play a crucial role in biopolymer ac-
cumulation capacity of microbes. The parameters mostly known to in-
fluence maximum PHB production include pH usually between 7.0
and 7.5 and an incubation temperature of 30 °C (Lathwal et al., 2015).
A pH of about 7.5 has been reported to be the optimal pH for many
microorganisms (Ansari and Fatma, 2016; Kavitha et al., 2016a;
Montiel-Jarillo et al., 2017; Touloupakis et al., 2016). Outside this
range, microalgae capacity to absorb CO2 is drastically reduced and
the cell's ability tomaintain the activity of the RuBisCO enzyme is inter-
fered (Sutherland et al., 2015). Such unfavourable conditions translate
into poor cell growthwhich subsequently affects the PHB storage capac-
ity. This is because, in a microbial culture system, cells would firstly
channel their energy into increasing biomass before accumulating stor-
age products like PHB. This phenomenon has been demonstrated in sev-
eral studies. For instance, acidic pH is known to be unfavourable for PHB
accumulation in Bacillus cereus SPV. Themaximum optical density (OD)
was 0.05 under acidic conditions (pH 3.0). However, at pH 6.8, the OD
was as high as 5.9 which yielded PHB 23% CDW. Getachew and
Woldesenbet (2016) also reported that the best PHB production from
Bacillus sp. was observed at an optimum pH of 7 at 37 °C. Similarly,
Mostafa et al. (2020) observed that the highest PHB accumulation by
Pseudodonghicola xiamenensis was achieved at pH 7.5–8.0 and pH 7–9
for PHB-producing bacteria (Bacillus sp.) (Thapa et al., 2019). Outside
this range, PHB accumulation reduced drastically. However, to over-
come certain cultivation issues, such as contamination, pH is usually in-
creased to saline concentrations. The effect of pHon Synechocystis sp. PCC
6803was observed by raising the pH to as high as 11 inmass cultures. Al-
though this technique led to contamination-free cultures, it also led to re-
duced lipid and increased carbohydrate contents (Touloupakis et al.,
2016). In a co-culture system where other by-products such as lipids
and starch are desired, this technique might not be favourable.
5.5. Photoperiod
Due to the ability of photosyntheticmicrobes to utilize sunlight as an
energy source, sunlight has been proposed as a cheap source of illumi-
nation for phototrophic mixed culture (PMC) production of PHA for
cost reduction (Fradinho et al., 2019). Light-harvesting complexes
allow certain microbes to capture enough light energy for biomass pro-
duction. This subsequently leads to accumulation of storage compounds
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729like PHB. Low light intensity was used to select PHA-producers in the
selection step and high light intensity of about 20 W/g was used to
achieve maximum PHA productivity rate (2.21 ± 0.07 Cmol PHB) in
the accumulation step (Fradinho et al., 2019). Similarly, Monshupanee
and Incharoensakdi (2014) also observed that high light intensity
(200 μmol photon m−2 s−1) was optimal for the co-production of GL,
LP and PHB in Synechocystis sp. In a cyanobacteria-dominated mixed
culture, maximum concentration of PHB (104 mg/L) was achieved
under continuous illumination in comparisonwith 12 h light/dark alter-
nation (Arias et al., 2018b). Similarly, 26.37% w/w PHB was obtained
faster (on day 7 instead of day 21) by applying 10:14 h light/dark pho-
toperiod conditions together with other optimized physicochemical
conditions such as pH of 7.5 and temperature of 30 °C in a study with
cyanobacteria (Ansari and Fatma, 2016). Contrarily, PHB production
was tripled in a dark fermentation culture under 30:30 min light/dark
conditions compared to continuous illumination (Montiel Corona
et al., 2017). Similarly, in another study alternating light/dark rhythm
under shaken conditions improved intracellular PHB accumulation in
Synechocystis sp. compared to continuous light (Koch et al., 2020).
These studies prove that, the effect of light (in terms of duration and in-
tensity) on PHB productivity and yield is species-specific. Much like a
feast-famine regime, light can also be alternated in order to achieve
the desired results in a two-stage system of selection and accumulation.
5.6. Cycle length
The duration of a culture influences the rate of biopolymer accumu-
lation. Several studies have demonstrated the effect of cultivation cycle
and length on PHB yield. For instance, aerobic microorganisms from ac-
tivated sludge mixed culture were reported to store 16, 18, 42, and 55%
PHB content after 12, 8, 4, and 2 hour cycles respectively (Ozdemir et al.,
2014). Similarly, an experiment of a mixed culture with a cycle length
range of 1–18 h at 20 °C and 30 °C, revealed that, over 75% (CDW)
PHB was accumulated by two dominating microbes; Zoogloea and
P. acidivorans. This correlated well with cycle length at a constant solid
retention time (SRT) (Jiang et al., 2011b). Both studies indicate that, to
achieve high PHB content, the cycle length should be decreased. Con-
trarily, Moralejo-Gárate et al. (2013) revealed that a longer cycle length
of 24 h was ideal for PHA production, whereas a shorter cycle length of
6 h favoured polyglucose production over PHA in the same dominating
microbe in the mixed culture. This phenomenon suggested a metabolic
rather than amicrobial competition response since twometabolic prod-
ucts were simultaneously achieved from the SBR. From these studies, it
can be inferred that the effect of cultivation length on PHB productivity
and final yield is dependent on the microbial community and the reac-
tor setup.
6. Effects of extraction methods on Poly-β-hydroxybutyrate yield
and purity
PHB-producing microbes can accumulate up to 90% of their own
weight as biopolymer (Bhuwal et al., 2013; Ozdemir et al., 2014) but
the polymer, within the cell is difficult to extract. Downstream process-
ing such as harvesting and extraction, are said to be responsible for
60–80% of the total production cost (Jacquel et al., 2008). Thus, in
order to achieve a significant recovery yield with the desired polymer
properties, extraction methods have to be optimized. Conventional
methods of PHB extraction include solvent, chemical, mechanical, en-
zyme and surfactant-chelate extraction. Over the years, different sol-
vents and cell disruption techniques have been developed and applied
in lysing cells to release the desired product.
Solvent extraction usually involves the soaking of microbial biomass
in a cocktail of solvents in a stepwisemanner and the subsequent recov-
ery of the polymer through precipitation. Cell wall strength plays a sig-
nificant role in the disruption of microbial cells for biopolymer recovery
and manipulation of the growth medium composition can lead to13alterations in microbial cell wall structure. Processing conditions such
as the type, concentration, action time and working temperature of sol-
vent have significant effects on the molecular weight, as well as extrac-
tion rate and purity of a biopolymer (Chen, 2009). These conditions
affect the costs, characteristics, and biopolymer monomeric composi-
tion, which subsequently impacts their applications in industry (Costa
et al., 2018b). Applications of the polymer in themedical industry for in-
stance, require toxin-free polymer with high purity compared to appli-
cations in single-use plastic bags. Extractionmethods are also known to
significantly (p<0.05) affect themolecularmass, degree of crystallinity
andmonomeric composition of the biopolymer (Costa et al., 2018b), in-
dicating that the extraction method is crucial in polymer recovery as
well as obtaining the desired characteristics for the intended industrial
applications. For instance, PHB extracted with a mixture of sodium hy-
pochlorite, diethyl ether and hot chloroform was used in cancer detec-
tion. The breast cancer cells (T47D) appeared to have a stronger
attachment for the PHB sheets compared to normal epithelial cells
(PCS-600-010) (Sabarinathan et al., 2018). This biocompatibility with
mammalian cells has allowed the application of PHA polymers espe-
cially PHB in surgical tools, wound dressing, bone repair and drug deliv-
ery (Bonartsev et al., 2019; Bunster, 2016). It is also widely used in the
agricultural sector (Tan et al., 2019). Generally, higher acid concentra-
tions have led to desirable mechanical strength properties. Although
the end results might be good, it is important to take note of the nega-
tive environmental and economic implications of the use of harsh
chemicals in extraction.
Due to the disadvantages of the aforementioned methods such as
toxicity, time-consumption and costliness, environmentally friendly
methods such as biological extraction (known as bioextraction) and hy-
drothermal conversion are being exploited. Bioextraction methods in-
clude bacteriophage-mediated lysis systems, predation systems and
mealwormdigestion systems. Thesemethods promise of cost reduction
and reduced harmfulness to the environment and human health
(Haddadi et al., 2019). Coupling WWT with PHB recovery is a step in
the right direction. Ceyhan and Ozdemir (2011) utilized hypochlorite
method of extracting PHB from Enterobacter aerogenes cultivated in do-
mestic wastewater and obtained a yield as high as 96.25%. Again, PHB-
containing biomass utilized in WWT was successfully transformed into
propylene through hydrothermal conversion (Li and Strathmann, 2019).
Extraction solvents such as anisole, cyclohexanone and phenetole have
also been tested by Rosengart et al. (2015) as sustainable industrial
solvents. Biopolymer recovery yields of 97% and 93% were achieved
with anisole and cyclohexanone, respectively, which were very similar
(96–98%) to yields obtained by chloroform extraction.
After the successful extraction of the biopolymer components from
cell biomass, it is necessary to purify the extract to eliminate impurities
such as solvents, bacteria, colour and odour. This also allows application
of the biopolymer in sensitive areas such as the medical industry as
mentioned earlier. Purification methods usually involve enzymes or
chelating agents in combination with hydrogen peroxide treatment
(Jacquel et al., 2008) and ozone (Horowitz and Brennan, 2005). Ozone
treatment has many advantages such as bleaching, deodorization, and
solubilization of impurities from the biopolymer. This could eventually
replace hydrogen peroxide treatment. Klasener et al. (2018) also pro-
posed an environmentally friendly approach to handling the biopoly-
mer extraction WW by utilizing it in further cultivation of microbes
that is, re-utilizing the aqueous phase; a technique currently gaining
grounds in microalgae research. This was demonstrated with Spirulina
LEB 18 which was successfully cultivated in extraction WW in their
study. Optimization of the extraction and purification processes of
PHB should consider the type of polymer-producing strain, expected
standard of purity, intended industrial application as well as type and
composition of the desired biopolymer. It is necessary to also consider
the use of green and recyclable harvesting techniques such as the use
of crystalline nanocellulose to reduce cost associated with harvesting
microalgae (Qiu et al., 2019a). Table 2 summarizes some key works on
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729
Table 2
Effects of extraction methods on yield and purity of PHBs.
Method Strain Extractant Cultivation conditions Yield Purity References
(%)
Organic Spirulina sp. Sodium hypochlorite, methanol, chloroform, Vertical tubular reactor with 6.1–9.8% w/w 63.51 (Costa et al.,
solvent acetone continuous agitation at 30 °C and 2018b)
extraction 93.62
Synechocystis KOH, ethanol, sodium acetate, amyloglucosidase, Autophototrophic/heterophototrophic Autophototrophic Ndeprivation N/A (Monshupanee
sp. PCC 6803 chloroform, methanol, KCL, acidic dichromate, growth in BG-11 50/200 μmol photon (2.4–13.5% w/w DW) and
H SO m−2 s−12 4 at 28 °C heterophototrophic 0.4% (w/v) Incharoensakdi,
glucose addition (3.3–9.2% w/w 2014)
DW)
Cupriavidus Acetone/ethanol/propylene carbonate (A/E/P, Autotrophic (CO2 as substrate and H2 83–92% 92–93 (Fei et al., 2016)
necator 1:1:1 v/v/v) as electron acceptor)
Nostoc Pre-treatment of biomass with methanol:acetone: 10:14 h light:dark periods with 0.4% NaCl and P deficiency yielded N/A (Ansari and
muscorum water:dimethylformamide [40:40:18:2 (MAD-I)] glucose (as additional carbon source), 26.37% PHB Fatma, 2016)
NCCU-442 with 2 h magnetic bar stirring followed by 30 h 30 °C
continuous chloroform soxhlet extraction
Cupriavidus Cyclohexanone, 120 °C 3 min Tryptic soy broth with vegetable oil as 82.3% 95 (Jiang et al.,
necator H16 sole carbon source, 35 °C, 72 h 2018)
Mechanical Bacillus flexus SDS sonication Modified basal mineral (MBM) 96.7 ≥96 (Arikawa et al.,
disruption medium 2017)
Pseudomonas Sonication and heptane (marginal non-solvent) Octanoic acid as sole carbon & energy 29.5/37.1% N/A (Ishak et al.,
putida Bet001 source 2015)
Enzyme Cupriavidus CelumaxVR Citrus molasses as carbon source and a 93.2% 94 (Neves and
extraction necator BC supply of propionic acid during N Müller, 2012)
limitation
Bioextraction
Bacteria Pseudomonas Bdellovibrio bacteriovorus (mutant strain) 30 °C 0.65–0.87 g/L N/A (Martínez et al.,
extraction putida Sodium octanoate as carbon source 2016)
system
Phage lysis Pseudomonas Bacteriophage Ke14 Modified P1 ammoniummineral salts 0.84 g/L N/A (Hand et al.,
system oleovorans medium contains with crude glycerol, 2016)
30 °C
Animal Cupriavidus Yellow mealworm (Tenebrio molitor) Palm olein (plant oil) & waste animal 55 wt% - from palm olein 94 (Ong et al.,
digestive necator fats 60 wt% - from waste animal fats 2018)
systembiopolymer extraction and Fig. 5 demonstrates the production stream
of PHB from wastewater-cultivated microbes through a bio-refinery
concept.
7. Challenges and future prospects
The major challenge faced in the PHB industry is of an economic na-
ture. Current commercial PHB production is done in batch fermenters
using bacteria that require large amounts of organic carbon sources
and salts, contributing to about 50% of production cost (Costa et al.,
2019). Microalgae are promising microbes in this field due to the fact
that they are photoautotrophic, utilize CO2 and light as their main en-
ergy source, thus, contributing to greenhouse gas emission reductions
(Garcia-Gonzalez et al., 2015). In addition to the fact that landfill degra-
dation of conventional plastics is slow and incineration of plastics gen-
erates toxic by-products, life cycle assessments (LCAs) mostly conclude
that PHB production using WW is economically feasible but still re-
mains uncompetitive with conventional bioplastics (Troschl et al.,
2018; Yates and Barlow, 2013). Thus, in order to successfully reduce
bio-plastics cost, and increase its competitiveness, a few issues have to
be addressed.
7.1. Optimizing production
Developing a zero-waste process such as a bio-refinery where mul-
tiple products are co-produced during cultivation, extraction WW is
reused and resultant biomass is utilized as biochar for soil conditioning.
These techniques are likely to lower production price. However, in
doing so, consideration has to be given to cultivation conditions, reactor
type, yield, quality, monomer properties and microbial strain especially
with regard to genetically modified microbes which are sensitive and
require specifically controlled conditions. Studies show that genes in-
volved in PHB production are post-transcriptionally regulated because14there is no differential expression in PHB biosynthesis genes during
the production of PHB under varied growth conditions. Certain mi-
crobes such as Calothrix scytonemicola TISTR 8095, Nostoc muscorum
CCAP 1453/9, and Spirulina sp. LEB 18, are not ideal for cultivation in
photobioreactors due to the formation of biofilms during growth.
Thus, reactor set-up is an important factor to consider for optimized
PHB production. To ensure the sustainability of PHB production, mi-
crobes that are capable of growing under different growth conditions
should be prioritized in order to enhance the viability of production pro-
cesses. For instance, Rhodopseudomonas palustris TIE-1 has demonstrated
metabolic flexibility because it can grow under chemoheterotrophic,
photoheterotrophic and photoautotrophic conditions or Cupriavidus
necator that can produce PHB both heterotrophically and autotrophi-
cally. Also the ability of the microbe to synthesize PHB under nutrient-
limited conditions (e.g. Synechocystis sp.) or under normal growth (e.g.
Cupriavidus necator) should be considered due to their unique ability of
phenotypic heterogeneity.
7.2. Acclimatization of microbes for wastewater cultivation
In trying to reduce production cost, open-pond systems would have
been ideal to incorporatewithWWTP, but this is challenging as it would
limit the type of PHB-producing microbes that could be used as well as
influence the downstream processing. Thus, it is important to select
WW sources for specific microbes that are naturally inclined to thrive
well under those conditions.
7.3. Improving yield
Currently, novel strategies in the field include the use of biological
methods in PHB recovery through entomology and botany. The former
involves the use of insects by feeding them on PHB-accumulating mi-
crobes and recovering the polymer via excretion. These insects could
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729
Fig. 5. Utilization of wastewater-cultivated microbial biomass in a bio-refinery concept for value added products, simultaneous wastewater treatment and carbon capture for a better
climate and bio-economy.subsequently be incorporated in fish feed, bio-fertilizers or other useful
bio-products. The latter involves the use of transgenic plants to produce
PHBs. Plant parts such as roots, stems, leaves and fruits should continue
to be exploited. Another successful approach that has been well docu-
mented and holds future prospects lies with the use of halophiles
which can withstand harsh environmental stresses as well as contami-
nation, potentially reducing the cost of production.
8. Conclusions
With regard to substrate choice, studies have shown that substrates
high in VFAs are suitable for bacterial PHB production. Microalgae such
as Chlorella sp. are also known to have high tolerance for VFAs. Thus
waste substrates such as anaerobic digester liquor with high VFA con-
tent would be ideal for PHB production in microalgae or in mixed cul-
tures and agro-industrial wastes like ensiled maize, which are high in
easily fermentable carbohydrates, would be a better substrate option
for hydrogen production in a co-production system. Producing biopoly-
mers such as PHB from wastewater-cultivated microbes for bioplastic
production can be regarded as a sustainable approach to wastewater
treatment.Majority of the plastic pollution seenworldwide, is as a result
of single-use plastics such as shopping bags which do not require strin-
gent sterilization efforts during production unlike those required in
medical applications. Thus, adopting mixed microbial cultures capable
of growing onwastewater seems like a reasonable approach to tackling
the plastic pollution problem due to the biodegradability of the poly-
mer. Additionally, the ability of autotrophic microalgae to utilize inor-
ganic carbon sources adds more value to the production chain as the
microbes can thrive on flue gases such as CO2 from industrial plants.
Aside cost-reduction, this also plays a critical role in sustainable environ-
mental bioremediation through the improvement of effluent quality.
Itwould be beneficial fromanenvironmental and economic perspec-
tive for future commercial PHB-producing plants to consider integrating
waste effluents rich in organic matter for simultaneous WWT and PHB
production. A two-stage system that takes advantage of metabolically15versatile microbes would be ideal. Such microbes are commercially via-
ble because they can grow under various conditions by increasing their
biomass within a short period when conditions are favourable and sub-
sequently accumulating PHBwhen nutrients are limited. This approach
has the potential to reduce cost through the utilization of abundant
resources like CO2 and light for autotrophic purposes and waste or-
ganic matter for heterotrophic methods and a combination of vari-
ous approaches.
CRediT authorship contribution statement
Amadu, Ayesha Algade: Writing- original draft, Visualization,
Data curation, Investigation. Qiu, Shuang: Writing - review &
editing, Visualization, Funding acquisition. Ge, Shijian: Conceptuali-
zation, Supervision, Writing - review & editing, Funding acquisition.
Addico, Gloria Naa Dzama: Writing - review & editing. Ameka, Gabriel
Komla: Writing - review & editing. Yu, Ziwei: Data curation, Investiga-
tion. Xia, Wenhao: Data curation, Investigation. Abbew, Abdul-Wahab:
Data curation, Investigation. Shao, Dadong: Writing - review & editing.
Champagne, Pascale:Writing - review& editing.Wang, Sufeng: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 influ-
ence the work reported in this paper.
Acknowledgement
This work was supported by the National Natural Science Foundation
of China (52000103, 51708294, and 21976089), Natural Science Founda-
tion of Jiangsu Province for Distinguished Young Scholars (BK20190022),
Natural Science Foundation of Jiangsu Province (BK20181303), China
Postdoctoral Science Foundation (2020M671402), and Fundamental
A.A. Amadu, S. Qiu, S. Ge et al. Science of the Total Environment 756 (2021) 143729Research Funds for the Central Universities (30920021117). Dr. ShijianGe
acknowledges the support of Distinguished Professorship of Jiangsu Prov-
ince and China Association for Science and Technology.
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