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Polyhydroxyalkanoates (PHAs) Large Scale Manufacturing – Process Modeling

and Techno-Economic Assessment (TEA) using SuperPro Designer.

Preprint · March 2022

DOI: 10.13140/RG.2.2.34587.54569

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 Manufacturing of
Polyhydroxyalkanoates (PHA)
Process Modeling and Evaluation using
SuperPro Designer®
by

Rafael da Gama, Nikiforos Misailidis and Demetri Petrides

March 2022

This is the ReadMe file of a SuperPro Designer example that analyzes the production of
polyhydroxyalkanoates (PHAs), which are biodegradable bioplastics that have the potential to replace
traditional plastics in various applications. The bioconversion process utilizes bacteria Cupriavidus necator
in 300 m3 fermentors, operating in fed-batch mode, using soybean oil as the main carbon source. After
fermentation, the intracellular PHA granules are released by cell disruption and purified with a surfactant /
enzyme treatment. The plant analyzed in this example produces 8,300 metric tons of PHAs per year. The
flowsheet of the process is appended to the bottom of this document. You may test-drive the model by
downloading the functional evaluation edition of SuperPro Designer from the downloads page of our
website (www.intelligen.com). All the files of this example can be found in the Examples \ Bio-Materials \
BioPolymer folder. The default installation path of the SuperPro Designer Examples folder follows below.

C:\ Users \ Public \ Public Documents \ Intelligen \ SuperPro Designer \ v12 \ Process Library \ Examples

If you have any questions regarding this example or SuperPro Designer in general, please send an email
message to dpetrides@intelligen.com

INTELLIGEN, INC.
Simulation, Design, and Scheduling Tools
For the Process Manufacturing Industries
www.intelligen.com
Introduction

Polyhydroxyalkanoates (PHAs) are a family of polyesters naturally produced by a variety of bacteria and
archaea primarily for the purpose of carbon and energy storage. They are usually synthesized when there
is an excess of carbon source relative to other key nutrients such as nitrogen or phosphorus, and are
accumulated under the form of intracellular granules [1–4]. PHAs are also involved in stress resistance
mechanisms, conferring a protective effect against cold conditions and osmotic pressure [5].

The interest in PHAs has grown over the last decades because some of them have physical and chemical
properties similar to fossil-based plastics such as polypropylene and polyethylene, while being renewable
and biodegradable [6]. In fact, PHAs can replace synthetic plastics in various packaging applications and
disposable consumer goods, such as food and beverage packaging [7], disposable cups, plates and cutlery,
electronic accessories [8], shampoo bottles, diapers [9], etc. In addition, PHAs can be used in the
biomedical sector, as surgical sutures, implants, and tissue-engineering scaffolds [10,11]; in the
pharmaceutical sector, as controlled release carriers for drug delivery [2,8]; and in agriculture, as mulching
films [6,10,11] and carriers for the controlled release of fertilizers and pesticides [6,8,9].

The molecular structure of PHAs is depicted in Figure 1:

Figure 1: (a) General molecular formula of PHAs. Typically, x=1–8, and n ranges from 100 to 1000 s. (b)
Some commonly synthesized short-chain-length PHA monomers (SCL-HA) and middle-chain-length PHA
monomers (MCL-HA). 3HB: 3-hydroxybutyrate, 3HV: 3-hydroxyvalerate, 3HHx: 3-hydroxyhexanoate, 3HO:
3-hydroxyoctanoate, 3HD: 3-hydroxydecanoate, 3HDD: 3-hydroxydodecanoate. Insert: TEM image
showing thin sections of recombinant C. necator PHB−4 cells that contain large amounts of P(3HB-co-
5 mol% 3HHx). The bar represents 0.5 μm. Picture extracted from [12]. Licensed under CC BY 4.0.

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Each monomer is composed of an alkyl chain with a hydroxyl group at one end and a carboxyl group at the
other end (hence a hydroxyalkanoic acid). In addition, there is a side chain at the carbon attached to the
hydroxyl group, making the monomer chiral (with a few exceptions). The side chain is typically made up of
saturated alkyl chains but side chains composed of unsaturated, substituted or aromatic groups also exist
[13]. More than 150 different monomers have been reported so far, which shows the diversity of PHAs and
hints at their potential for different applications. In addition, PHAs may form homopolymers, random
copolymers or block copolymers depending on the microorganism, substrates, and culture conditions,
which further increases their diversity of structures, functions and properties [10]. PHAs are categorized
according to the number of carbons in their monomers: polymers constituted by monomers of 3 to 5 carbons
are classified as short chain length (SCL), while those based on monomers of 6 to 14 carbons are classified
as middle chain length (MCL) [3]. This classification is used for both homopolymers and copolymers.
Copolymers composed of both SCL and MCL monomers are also possible [8]. SCL polymers tend to have
a substantial degree of crystallinity and to be thermoplastic, stiff, and brittle [8], while MCL polymers tend
to be amorphous, elastic, soft and sticky [6]. SCL PHAs are produced by a wide range of microorganisms,
such as Cupriavidus necator (formerly named Ralstonia eutropha), and Azohydromonas lata (formerly
named Alcaligenes latus). Poly 3-hydroxybutyrate (PHB) is the most representative SCL PHA; in fact, when
growing on simple sugars and lipids, SCL PHA-producing bacteria usually synthesize PHB [1]. MCL PHAs
are synthesized mainly by bacteria from the genus Pseudomonas [10] and are usually produced as random
copolymers [1].

PHA Biosynthesis

Three major biosynthetic pathways have been proposed for the production of PHAs, as schematically
shown in Figure 2:

• Route A converts sugars, lipids, and amino acids into PHB and other SCL PHAs via acetyl-CoA.
This is a well-characterized pathway observed in bacteria such as C. necator.
• Route B is linked to fatty acid catabolism (β-oxidation) and leads to the production of MCL PHAs.
• Route C is linked to de novo fatty acid synthesis and leads to the production of MCL PHAs as well
[9,14].

As a consequence, the monomeric composition of PHAs depends on the biosynthetic pathways available
in the producing microorganism, as well as on the carbon sources utilized [1]. Starch-based sugars,
sucrose, and vegetable oils are the main carbon sources employed industrially [6,7,14], although the
theoretical yield of PHA on sugars is substantially lower than that on vegetable oils [15–17]. In addition, the
production of industrially significant PHAs such as the copolymers of 3-hydroxybutyrate and 4-
hydroxybutyrate (P3HB4HB) and the copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV)
normally requires structurally related substrates such as propionic acid and γ-butyrolactone, respectively.

2
These copolymers have lower crystallinity than PHB and as a result exhibit better physical properties in
terms of material processing [1].

Figure 2: Major pathways for the biosynthesis of polyhydroxyalkanoates. Picture extracted from [14].
Licensed under CC BY 4.0.

Given that PHA biosynthesis overlaps with central metabolism, competing for key precursors for cell growth
such as acetyl-CoA, vigorous PHA production and biomass growth cannot happen at the same time in most
organisms [4]. Notable exceptions to this rule are Azohydromonas lata and recombinant strains of
Escherichia coli [3]. As a consequence, microbial culture for PHA production is usually conducted in batch
or fed-batch, in two phases: 1) biomass growth in a balanced medium; and 2) PHA synthesis in an
unbalanced medium (typically by restricting the availability of nutrients such as nitrogen or phosphorus).
The scientific literature also describes continuous PHA production using two (or more reactors), in which
the first reactor is devoted to biomass growth and the second reactor is devoted to PHA synthesis [1,3,14].

3
Established PHA manufacturers usually employ C. necator or recombinant E. coli for polymer biosynthesis.
However, the use of halophilic bacteria or archaea to produce PHA has attracted considerable attention
lately. These microbes can be cultivated in high-salt media such as seawater under non-sterile conditions,
which simplifies the production process and cuts costs [6,10,14]. Moreover, downstream processing may
be streamlined as well given that cell disruption can often be achieved by subjecting cells to a hypotonic
shock [18,19]. A few companies in China already produce PHA using halophiles, including PhaBuilder,
Medpha, and COFCO [10].

Mixed microbial consortia (MMC) can also be used to produce PHAs. In this case, a selective pressure is
applied to the culture medium with the aim of enriching the culture with PHA-producing organisms. This is
typically achieved by performing “feast and famine” cycles that stimulate the accumulation of carbon and
energy reserves such as PHA. MMC presents significant advantages over monoseptic culture: it can utilize
various types of cheap substrates such as food waste and industrial effluents; and it can operate under
non-sterile conditions. Usually, these substrates are first converted into volatile fatty acids (VFAs) by
anaerobic digestion, and then fed to PHA-producing microorganisms. Despite the significant advantages
of MMC, the heterogeneity of the product in terms of monomeric composition and molecular weight as well
as the quality variation between batches pose challenges to this production method [1,20]. The Full Cycle
Bioplastics startup company (https://fullcyclebio.com/) has developed a technology for PHA production
based on MMC [21].

A rather unique PHA production process has been developed by Newlight Technologies
(https://www.newlight.com/). Their technology utilizes marine microorganisms that, under aerobic
conditions, can convert greenhouse gases such as methane and carbon dioxide into PHB [14,22].

PHA Recovery

After microbial culture, it is necessary to recover PHA from within the cells and purify it. There are two basic
methods to accomplish PHA recovery: 1) solvent extraction 2) cell lysis with removal of non-polymer cell
mass (NPCM). In the solvent extraction method, a suitable solvent is mixed with the fermentation broth.
The solvent breaks the cells by disrupting the cell membrane, and then solubilizes the PHA granules
forming a non-aqueous phase [14]. This phase can be separated from the aqueous phase by filtration or
centrifugation, and the PHA can be recovered by evaporating the solvent or by precipitating the polymer
upon addition of an antisolvent [23]. Halogenated solvents such as chloroform are excellent for this recovery
method, leading to very high recovery rates and product purity, with limited degradation of the polymer
molecular weight (MW). In fact, solvent extraction with chloroform is considered as the benchmark for other
recovery methods. Nevertheless, chloroform and similar halogenated solvents are highly toxic and thus
industrially disfavored on safety and environmental grounds [18,23]. For that reason, several publications
in the literature describe PHA recovery using greener solvents such as acetone, cyclohexanone, ethylene
carbonate, 1,2-propylene carbonate, ethyl acetate, butyl acetate [18,23], and 1,3-dioxolane [14]. In many

4
cases, however, extraction with greener solvents must be performed at elevated temperatures to produce
good recovery and purity results, which leads to polymer degradation (i.e. lower MW) [23] .

In the cell lysis method, the cell components surrounding the PHA granules are removed by physical,
chemical and/or enzymatic methods. Typically, the cell membrane is disrupted by mechanical methods
[7,24,25], alkali, acids, oxidizing agents (e.g., sodium hypochlorite), surfactants and/or enzymes, and the
PHA granules are subsequently separated by centrifugation or filtration [23]. The polymer can be further
purified by treatment with surfactants and/or enzymes (e.g., proteases or lipases) [24]. The combination of
surfactants and sodium hypochlorite appears to be particularly effective for PHA recovery; however, sodium
hypochlorite may lead to a large reduction in MW, and generate toxic halogenated compounds [18,23].

PHA Industrialization

The industrialization of PHAs has a long history. British company ICI was one of the first to produce PHA
at large scale, introducing PHBV into the market in 1982 [26]. German company Biomer started producing
PHB at pilot scale in 1991 [22]. Another pioneer was the American company Metabolix, a spin-off from MIT
founded in 1992 [27]. In 2006, Metabolix formed an ambitious joint-venture with ADM called Telles, which
launched a manufacturing plant with a capacity of 50,000 MT/yr in 2009 [22]. ADM considered the market
adoption rate of the biopolymer too slow, however, and the joint venture was dissolved in 2012. Metabolix
sold its microbial PHA technology to Korean company CJ CheilJedang in 2016 and has focused on plant
biotechnology since then, under the name of Yield10 Bioscience. One of Yield10’s businesses is the
production of PHAs in plants [28].

Several companies currently produce PHAs at large scale, as indicated in Table 1. The table also shows
the type of PHA produced, the microorganism, and the carbon source employed. Currently, only PHB and
a few copolymers of 3HB are industrially relevant. Most manufacturers employ C. necator, recombinant E.
coli or Halomonas sp. for PHA synthesis. Production capacities are relatively small when compared with
total plastic production, which exceeds 380 million metric tons/year [29]. However, the market for PHA is
predicted to grow from $62 million to $121 million between 2020 and 2025, which corresponds to a
compound annual growth rate of 14.2% [30]. In fact, major market players such as Danimer, RWDC and
Kaneka are planning on expanding their production capacity in the near future [28,31].

The main obstacle to a more widespread use of PHAs as bioplastics is their high manufacturing cost.
According to a report published in 2017, the market price of PHA is about $6/kg, while the cost of oil-based
polymers is 1-2 $/kg [32]. The average sales price of Danimer Scientific’s formulated products (which
include both PHA and PLA) was $2.85/lb ($6.3/kg) in Q1/2021 [33]. A significant component of the price of
PHA is the cost of raw materials, which can account for more than 50% of the unit production cost.
Moreover, 70 – 80% of the cost of raw materials is due to the carbon source [34]. For that reason, a great
deal of research over the last years has focused on the utilization of waste carbon sources such as

5
lignocellulosic biomass, cheese whey, animal fats, waste frying oil, etc. with the aim of reducing
manufacturing costs. In general, however, these carbon sources lead to low titers due to their low
concentration and the presence of inhibitory compounds. Consequently, pretreatment and concentration
steps are crucial to improve product yield and volumetric productivity in this case [20]. Another possibility
is to focus on high value-added segments such as the biomedical sector. Tepha, TerraVerdae Bioworks
and PolyFerm have followed this strategy [14].

6
Table 1: Industrial production of PHAs. PHB = poly (3-hydroxybutyrate); PHBV = poly (3-hydroxybutyrate-co-3-hydroxyvalerate); P3HB4HB = poly
(3-hydroxybutyrate-co-4-hydroxybutyrate); PHBHx = poly (3-hydroxybutyrate-co-3-hydroxyhexanoate); P4HB = poly (4-hydroxybutyrate). Compiled
from [6,7,10,14,22,35].

Company Type of PHA Microorganism Substrate Capacity (MT/yr)

TianAn Biologic Materials PHB, PHBV Cupriavidus necator Glucose from corn or cassava 2,000
(China) starch
Tianjin GreenBio Materials P3HB4HB, PHBV Escherichia coli Sugars 10,000
(China)
Ecomann Biotechnology (China) P3HB4HB Escherichia coli Sugar / Glucose 10,000
Bluepha (China) PHBHx Cupriavidus necator, salt “Alternative carbon sources, 1,000
lake isolate including crops and kitchen waste”
COFCO (China) PHB Halomonas sp. ? 1,000
PhaBuilder (China) P3HB4HB Halomonas sp. Glucose, γ-butyrolactone 1,000-10,000
Medpha P3HB4HB Halomonas sp. ? 100
CJ CheilJedang P3HB4HB Escherichia coli ? 5,000
(Korea/Indonesia)
Kaneka PHBHx Cupriavidus necator Vegetable Oils 5,000
(Japan)
Danimer Scientific (USA) PHBHx ? Vegetable Oils 10,000
RWDC Industries PHBHx Cupriavidus necator Vegetable Oils / Sugar 5,000
(USA/Singapore)
Biomer PHB Azohydromonas australica Glucose from corn starch 900
(Germany)
bio-on* PHB, PHBV Cupriavidus necator Sugar beet by-products and 2,000
(Italy) molasses
Full Cycle Bioplastics (USA) ? MMC Organic waste ?
Newlight Technologies (USA) PHB marine microorganisms Methane, carbon dioxide 50
Tepha (USA) P3HB4HB, P4HB E. coli Sugars, 4HB precursors < 10
TerraVerdae Bioworks (Canada) ? ? Methanol ?
Polyferm (Canada) mcl-PHA Cupriavidus necator, Sugars and vegetable oils < 10
Aeromonas hydrophila,
Pesudomonas putida
* Declared bankruptcy in late 2019 [28]; current situation is unclear.

1
Process Description

This example models the industrial production of a polyhydroxyalkanoate (PHA) by Gram-negative bacteria.
The bacteria are cultivated in four staggered fermentors of ≈300 m3 each using soybean oil as the main
carbon source. Each batch produces ≈230 m3 of broth containing ≈160 g/L of total biomass, of which ≈75%
corresponds to PHA (in the form of intracellular granules). The broth is then pasteurized, and the cells are
disrupted by a combination of alkaline lysis and high-pressure homogenization to release the PHA granules
from within the cells. The granules are then recovered by centrifugation, neutralized, and treated with a
surfactant and a proteolytic enzyme to remove lipid and protein impurities. The polymer is then recovered
by rotary vacuum filtration, washed with water and ethanol, and finally dried in a rotary dryer. Approximately
24.5 MT of dry PHA is produced per batch, and a new batch starts every 24 h, so that the annual production
rate is ≈8,300 MT/yr.

For reporting and analysis purposes, the process has been divided into three sections:

• Fermentation (green icons)

• Primary Recovery (dark red icons)
• Purification (blue icons)

Flowsheet sections in SuperPro are simply sets of related unit procedures (processing steps). For
information on how to specify flowsheet sections and edit their properties, please use the Help tool (Help
Index Section). The contents of each process section are described in greater detail next. The flowsheet
of the model is appended to the bottom of this document.

Fermentation

In the Fermentation Section, Gram-negative bacteria Cupriavidus necator are cultivated in four staggered
300-m3 fermentors (P-20 / FR-101) to synthesize PHA. The microbial culture occurs in fed-batch mode,
using soybean oil as the main carbon source, for 80 h. It is conducted at 30 °C, with an average aeration
rate of 0.6 VVM (volume of air per volume of liquid per minute), at pH = 7. The fermentation starts with a
medium volume of 200 m3 having the composition shown in Table 2.

1
Table 2: Batch medium composition

Component Concentration (g/L)

Soybean Oil 20.0
KH2PO4 1.2
MgSO4*7H2O 1.4
NH4Cl 4.0
Na2HPO4*12H2O 11.0
Water q.s.

Extra soybean oil (approximately 26 m3) is added to the culture in fed-batch mode. Aqueous ammonia (NH3
29% w/w) and sodium hydroxide (NaOH 20% w/w) are added to the culture as well, for pH control. Ammonia
is added during the first 20 h of microbial culture, while sodium hydroxide is added during the remaining
60 h. This scheme is employed because the microbial culture for PHA production is typically divided into
two phases: 1) a biomass growth phase; and 2) a PHA production phase. During the first phase, ammonia
serves as nitrogen source for vigorous cell growth. In the second phase, the nitrogen supply is cut off by
replacing ammonia with sodium hydroxide, so that the bacteria are stimulated to synthesize PHA (after all,
PHA is a carbon and energy storage material that does not contain nitrogen).

To inoculate the production fermentation step, a seed train was included in the Fermentation section. The
seed train is composed of three serial seed fermentation steps (P-17 / SFR-101, P-18 / SFR-102 & P-19 /
SFR-103), carried out in fermentors of increasing size (260 L, 2.6 m3 and 26 m3, respectively). The broth
of P-17 inoculates P-18; the broth of P-18 inoculates P-19; and the broth of P-19 inoculates the production
fermentation step (P-20). The seed fermentation steps are similar to the main fermentation in many
respects: they are conducted at 30 °C, aerated with 0.6 VVM, and start with the same culture medium.
Extra soybean oil is added in fed-batch mode, and aqueous ammonia is added as well for pH control.
However, the seed fermentations take only 15 h and make no use of sodium hydroxide, given that their
sole objective is to grow cells.

The main fermentation and the seed fermentation steps are modeled by similar stoichiometric reactions.
The stoichiometry for the main fermentation step is provided by Eq. (1):

100 Soybean Oil + 3.4 NH3 + 125 O2 → 25 Biomass + 75 PHA + 81.62 CO2 + 46.78 H2O (1)

The “Biomass” component above represents dry cell mass excluding PHA. PHA, in turn, was specified as
100% intracellular (see the “Modeling Tips” at the end of this document for further details on how to handle
intracellular components). Moreover, the water content of Biomass was set to 75%. Eq. (1) was obtained
by elemental balance, assuming empirical formulas for Soybean Oil (C17.78H32.41O2)[36], Biomass
(CH1.74O0.46N0.19), and PHA (C4H6O2)[37], as well as specific yields of Biomass and PHA on Soybean Oil

2
(0.25 and 0.75, respectively). The conversion rate of this reaction was assumed to be 99%, and the heat
released, 3,700 kcal/kg of O2.

The stoichiometry for the seed fermentation steps is provided by Eq. (2):

100 Soybean Oil + 10.2 NH3 + 137.16 O2 → 75 Biomass + 25 PHA + 91.35 CO2 + 46.78 H2O (2)

Eq. (2) was also obtained by elemental balance but assuming different yields of Biomass and PHA on
Soybean Oil (0.75 and 0.25, respectively), given that nitrogen is available throughout the seed cultures.
The conversion rate of this reaction was assumed to be 99%, and the heat released, 3,700 kcal/kg of O2.

In both the main fermentation and seed fermentation operations, a reaction for the dissociation of
ammonium chloride (NH4Cl) was also included, so that the ammonia present in that salt was available for
cell growth as well.

Other than the fermentation procedures described above, the Fermentation Section includes the
preparation of medium components and aqueous ammonia. Soybean oil and water are sterilized and stored
using dedicated heat sterilizers and storage tanks. The medium salts are prepared as a concentrated (2X)
solution in a blending tank, heat-sterilized and stored in another tank. Aqueous ammonia is also stored in
a dedicated tank. Each one of these components (water, soybean oil, salt solution, and ammonia) is
distributed to the main fermentation and seed fermentation steps through dedicated Flow Distribution
procedures. In addition, the Fermentation Section includes Air Filtration, Gas Compression, and Flow
Distribution procedures that supply sterile air to all the fermentors.

By the end of each seed fermentation step, the Biomass concentration is approximately 30 g/L. By the end
of the main fermentation step, the Biomass and PHA concentrations are approximately 40 g/L and 120 g/L
(i.e. the total biomass concentration is about 160 g/L). In addition, the total broth volume by the end of the
main fermentation is approximately 230 m3. This broth is pasteurized at 70 °C (P-21 / PZ-104) to prevent
degradation of the polymer and sent to a storage tank (P-22 / HT-105) that acts as a buffer between the
batch Fermentation Section and the continuous Recovery Section.

Primary Recovery

The downstream portion of the process, which comprises the Primary Recovery and Purification Sections,
operates in continuous mode, as opposed to the Fermentation Section that operates in batch / semi-
continuous mode for the most part. Primary recovery starts with centrifugation (P-24 / DS-201) to separate
the cells from spent media; it is assumed that 97% of the Biomass is removed in the heavy stream, which
has a wet solids concentration of 600 g/L. Given that PHA has been specified as an intracellular component,
it is removed along with Biomass.

3
Next, the heavy stream is diluted with 1 volume of purified (RO) water (P-25 / MX-202) and treated with
NaOH 20% w/w to a final concentration of 0.03 mol/L. The resulting suspension is mixed in a continuous
stirred tank (P-29 / BT-201) with a residence time of 1 h, and subsequently sent to a high-pressure
homogenization step (P-30 / HC-201) that operates at a pressure drop of 700 bar. The combination of
alkaline treatment and high-pressure homogenization leads to a highly effective cell disruption process,
releasing the PHA granules from inside the cells. The material balance in the homogenizer is represented
by Eq. (3):

100 Biomass → 50 Proteins + 30 Cell Debris + 20 Impurities (3)

The conversion rate of this reaction is assumed to be 100%. It should be stressed that the “Biomass”
component represents the non-polymer cell mass (NPCM), and therefore PHA does not appear in the
equation. However, SuperPro does consider that PHA is released by the homogenization step (see the
“Modeling Tips” at the end of this document for more details).

Right after homogenization, the PHA granules are separated from cell debris and other cell components by
centrifugation (P-31 / DS-202). It is assumed that 97% of the PHA granules are removed in the heavy
phase, that has a solids concentration of 600 g/L. Next, the solids stream is diluted to a PHA concentration
of 100 g/L (P-32 / MX-204) and neutralized with HCl 20% w/w through a neutralization procedure (P-33 /
BT-202). The residence time in the neutralization tank is 1 h, and the neutralization reaction is given by Eq.
(4):

1 NaOH + 1 HCl → 1 NaCl + 1 H2O (4)

The conversion of the neutralization reaction is assumed to be 100%.

It should be noted that throughout the Primary Recovery Section, the temperature is kept under 30 °C to
minimize PHA degradation.

Purification

After neutralization, protein and lipid impurities are removed from PHA by treatment with a non-ionic
surfactant and a proteolytic enzyme. The suspension is first heated to 50 °C using a plate & frame heat-
exchanger (P-34 / HX-301). Next, the surfactant and protease are added to final concentrations of 0.1%
and 0.01%, respectively, and the suspension is mixed in a continuous stirred tank (P-37 / BT-301) with a
residence time of 3 h.

Subsequently, the polymer is recovered by rotary vacuum filtration (P-38 / RVF-301). 98% of the polymer
is retained by the filter, and the resulting cake has a loss-on-drying (LOD) value of 20%. In this processing
step, the cake is also washed with 2 volumes of RO Water.

4
The polymer is then submitted to a second rotary vacuum filtration step (P-41 / RVF-302) where 98% of the
PHA is retained, forming a cake with an LOD of 20%. This cake is washed with 2 volumes of ethanol 92%
w/w. Lastly, the polymer is dried with hot air in a rotary dryer (P-44 / RDR-301). The hot air is produced by
burning natural gas (P-43 / GBX-301). The final product has a purity of 99.5%. Approximately 1 metric ton
of dried PHA is produced per hour (24.5 MT/batch).

The Purification Section also includes procedures for ethanol recovery. The ethanol and water evaporated
by the dryer are condensed (P-45 /HX-302) and combined with the ethanol solution used to wash the
second rotary vacuum filter, forming a new solution with approximately 75% of ethanol and 23% of water
(w/w). This solution is sent to a distillation column (P-48 / C-301) to remove impurities and increase the
ethanol content to approximately 92%. This is achieved by operating the column with 12 theoretical stages,
a bottom to feed ratio of 0.33, and a reflux ratio of 2. The resulting distillate (containing 92% ethanol) is
cooled down and recycled back to wash the second rotary vacuum filter. A flow-adjusting procedure (P-39
/ FAD-301) was added to manage the supply of the makeup ethanol (33 L/h).

Process Scheduling and Cycle Time Analysis

The overall batch time of this process is approximately 7 days. This is the time elapsed from the start of a
given batch (i.e., media preparation) to the end of that batch (drying the product). However, the time
between consecutive batches, called Recipe Cycle Time (RCT), is only 24 h; this is possible because the
downstream procedures are continuous, many upstream procedures are shorter than 1 day, and multiple
(staggered) equipment units are used for those that are longer. The user can find the Batch Time and the
minimum RCT for the process in the Recipe Scheduling Information dialog (Tasks Recipe Scheduling
Information…). In this dialog, the user may also specify the RCT that he or she desires (as long as it is
larger than the minimum RCT, which is approximately 23 h in this process).

To visualize the process schedule, the user may click on Charts Equipment Occupancy Multiple
Batches. This will generate the Equipment Occupancy Chart (EOC) presented in Figure 3. This chart
displays the utilization of each equipment item over time. A total of 12 consecutive batches of PHA are
shown, and each one is associated with a different color. The three process sections – Fermentation,
Primary Recovery, and Purification – are also shown in the chart.

The Fermentation Section indicates that four fermentors are employed in the main fermentation step (FR-
101, FR-101b, FR-101c, FR-101d), operating in an alternating fashion. For instance, the first batch is
fermented in FR-101; the second batch, in FR-101b; the third batch, in FR-101c; and the fourth batch, in
FR-101d. These equipment units are said to operate in Stagger Mode. This configuration enables the plant
to initiate a new batch every 24 h, even though the main fermentation step takes longer than three days.
Further details on how to specify equipment in Stagger Mode can be found in the Farnesene example,
located in the …Examples \ Bio-Materials folder.

5
On the other hand, the downstream equipment items have a blockish appearance in the EOC; in fact, there
are no visible gaps between the bars of consecutives batches. This is because all the downstream
equipment items operate in continuous mode.

Another view of the process schedule is provided by the Operations Gantt chart (in the MS Project style).
That chart displays detailed scheduling information for one or multiple batches. The Gantt chart for a single
batch is generated by selecting Charts Gantt Charts Operations GC. Figure 4 displays a portion of
that Gantt chart, illustrating the scheduling of operations in the Fermentation Section. The golden bar
indicates the duration of the entire recipe, whereas the dark blue and cyan bars represent the duration of
procedures and operations, respectively.

The Gantt chart enables users to visualize the execution of a batch process in detail. It also facilitates
editing of batch recipes. Double-clicking on any of its bars brings up the dialog of the corresponding entity
(e.g., operation, procedure, recipe, etc.). The simulation calculations can then be redone, and the chart can
be updated by clicking on the refresh button of the chart ( ).

Furthermore, SuperPro can export its scheduling data to MS Project by selecting File Export to MS
Project XML File. Likewise, SuperPro can export its recipe data to SchedulePro by selecting File Export
to SchedulePro’s Recipe DB. SchedulePro is a resource management, production planning and
scheduling tool available from Intelligen. Please consult the Help facility for information on these two
exporting options (Help Index Exporting).

6
 Fermentation

Primary Recovery

Purification

Figure 3: Equipment Occupancy Chart (EOC) for twelve batches.

7
Figure 4: Operations Gantt Chart (part of one batch).

8
Material Balances

Table 3 displays overall process data such as batch size, annual production rate and number of batches
per year. This table was extracted from the RTF version of the Materials & Streams report, which can be
generated by selecting Reports Materials & Streams from the main menu bar of SuperPro. The format
of the report can be specified through the dialog that is displayed when you select Reports Options from
the main menu bar. The batch size is 24.5 MT, and the total number of batches per year is 337 which leads
to a Unit Production Rate of approximately 8,300 MT/yr. This process scale is close to the current capacity
of the largest industrial manufacturers (provided in Table 1 in the Introduction).

Table 3: Overall process data.

OVERALL PROCESS DATA

Annual Operating Time 49.00 wk

Unit Production Ref. Rate 8,270.45 MT MP/yr
Batch Size 24.54 MT MP
Recipe Batch Time 6.97 day
Recipe Cycle Time 1.00 day
Number of Batches per Year 337.00
MP = Total Flow of Stream 'S-94'

Table 4, which was also extracted from the Materials & Streams report, displays the raw material
requirements in MT/year, MT/batch, and MT/MT of MP (“MP” stands for main product, which is PHA in this
case). It shows that air, water, RO water, soybean oil, and ammonia are the materials used in large
quantities in this process (the demand for Salt Solution 2X and CIP-Caustic is also large, but both are
mostly composed of water). It is interesting to note that the consumption of fresh ethanol is relatively small:
575 kg per batch, i.e. only 23 kg per MT of PHA. Even though very large volumes of ethanol are used in
each batch, most of the solvent is recovered by distillation and recycled.

9
Table 4: Material requirements.

BULK MATERIALS (Entire Process)

Material MT/yr MT/batch MT/MT MP

Air 366,887 1,088.684 44.361
Biomass 0 0.000 0.000
CIP-Caustic 5,680 16.855 0.687
Ethyl Alcohol 194 0.575 0.023
HCl (20% w/w) 52 0.155 0.006
NaOH (20% w/w) 975 2.892 0.118
Natural Gas 197 0.586 0.024
NH3 29% (w/w) 1,371 4.069 0.166
Protease 9 0.025 0.001
RO Water 215,131 638.371 26.012
Salt Sln 2X 36,711 108.933 4.439
Soybean Oil 12,462 36.981 1.507
Surfactant 86 0.254 0.010
Water 27,992 83.064 3.385
TOTAL 667,747 1,981.445 80.739

Cost Analysis

SuperPro Designer performs thorough cost analysis, estimating capital costs (CAPEX) as well as operating
costs (OPEX), and generates the following three pertinent reports (through the Reports menu): the
Economic Evaluation Report (EER), the Cash Flow Analysis Report (CFR), and the Itemized Cost Report
(ICR). Table 5 displays the Executive Summary of the Economic Evaluation Report.

The total capital investment for this facility is approximately $67 million. This includes equipment purchase
and installation costs; other costs related to plant construction; startup and validation costs; and the working
capital required for this project. Plant construction costs such as the cost of buildings and piping are
estimated through multipliers in SuperPro Designer, and these were modified in this example to more
accurately represent the capital costs associated with an industrial biotechnology facility that produces a
low value-added product. For more information on capital cost estimations, please refer to the Industrial
Enzymes example in the …Examples / Bio-Materials folder or consult the Help tool (Help Search 
Capital Investment Dialog: DFC Tab).

Table 5 also displays the annual operating cost (AOC) and the unit production cost, which stand at $43
million and $5.2/kg of PHA, respectively. Assuming that buyers pay $7.0/kg of PHA, a premium price for its
elevated purity and environmentally friendly process, this project leads to a gross margin of 26%, a return
on investment (ROI) of 22% and a Payback Time of 4.5 years.

10
Table 5: Executive summary.

EXECUTIVE SUMMARY (2022 prices)

Total Capital Investment 66,916,000 $

Capital Investment Charged to This Project 66,916,000 $
Operating Cost 43,011,000 $/yr
Revenues 57,893,000 $/yr
Batch Size 24.54 MT MP
Cost Basis Annual Rate 8,270 MT MP/yr
Unit Production Cost 5.2 $/kg MP
Unit Production Revenue 7.0 $/kg MP
Gross Margin 25.71 %
Return On Investment 22.03 %
Payback Time 4.54 years
IRR (After Taxes) 14.53 %
NPV (at 9.0% Interest) 23,110,000 $
MP = Total Flow of Stream 'S-94'

Figure 5 displays a breakdown of the AOC, which was also extracted from the Economic Evaluation Report.
Raw materials and facility-dependent costs are the main contributors to the AOC, accounting for 30% and
27% of the total, respectively. Utilities and labor costs are also significant, accounting for 18% each,
respectively.

Figure 5: Annual operating cost breakdown.

11
Table 6, which was also extracted from the Economic Evaluation Report, provides a breakdown of raw
material costs. It shows that soybean oil is by far the largest component, accounting for approximately 73%
of the total raw material cost. This was to be expected given that soybean oil is the main substrate for PHA
synthesis, and most raw materials used in the downstream portion of the process are either inexpensive or
used in small quantities (such as protease). The cost of ethanol is low thanks to the recovery strategy
employed in this process.

Table 6: Breakdown of material costs.

MATERIALS COST - PROCESS SUMMARY

Unit Cost Annual Annual Cost

Bulk Material %
($) Amount ($)
Air 0.000 366,886,635 kg 0 0.00
Biomass 0.000 66 kg 0 0.00
CIP-Caustic 0.015 5,680,133 kg 84,634 0.65
Ethyl Alcohol 1.400 63,383 gal(STP) 88,736 0.68
HCl (20% w/w) 0.024 52,154 kg 1,252 0.01
NaOH (20% w/w) 0.104 974,683 kg 101,367 0.77
Natural Gas 0.600 197,481 kg 118,489 0.90
NH3 29% (w/w) 0.146 1,371,342 kg 199,818 1.52
Protease 25.000 8,568 kg 214,210 1.63
RO Water 5.000 215,131 MT 1,075,654 8.21
Salt Sln 2X 0.017 36,710,507 kg 1,149,754 8.77
Soybean Oil 0.350 27,475,096 lb 9,616,284 73.37
Surfactant 5.000 85,684 kg 428,421 3.27
Water 1.000 27,992 MT 27,992 0.21
TOTAL 13,106,611 100.00

NOTE: Bulk material consumption amount includes material used as:

- Raw Material
- Cleaning Agent
- Heat Transfer Agent (if utilities are included in the operating cost)

In conclusion, the results of the cost analysis suggest that the present PHA manufacturing process would
be an attractive investment. It should be noted, however, that many assumptions underlie this kind of
analysis, such as the market demand and selling price of the polymer; the prices of key raw materials such
as soybean oil; the yields of biomass and PHA in the fermentation steps; product losses in the primary
recovery and purification steps; etc. As a result, the actual economics of such an investment may be
substantially better or worse than the current projection. A useful exercise is to perform “what-if” analyses
with SuperPro to determine the impact of various changes (e.g., a higher yield of PHA on the substrate, a
lower cost of soybean oil, etc.). This allows the user to understand the potential risks and rewards of a
project under different sets of assumptions. What-if scenarios can be evaluated individually (by simply

12
changing parameter values manually and re-running the simulation to see the results), or they can be
automated through MS Excel. For information on how to drive SuperPro Designer through MS Excel and
automate sensitivity analysis, please consult the examples in the …Examples \ COM folder. Related
information is available in the Help facility of the tool, which can be accessed by selecting Help  COM
Interface and Library. Math optimization and Monte Carlo simulation can be performed in a similar
manner.

Modeling Tips

Handling intracellular components

In SuperPro Designer, it is possible to represent the water and other internal components of cells. This
feature was used in this example to represent PHA as an intracellular component.

First, the primary biomass component must be specified for the process. This is done by clicking on Tasks
 Pure Components  Register / Edit, View Properties… and then selecting the appropriate cellular
component in the dropdown menu. In the present example, Biomass was selected, as depicted in Figure
6.

Figure 6: Pure Component Registration Dialog. The “Primary Biomass” frame is highlighted in red.

13
Next, right-click on the fermentation (or cell culture) procedure, select Operation Data  Ferment to open
the fermentation operation dialog, and switch to the Reactions tab (see Figure 7). Right below the Reaction
Sequence frame, a Primary Biomass Water Content frame will have appeared. As the name implies,
here the user specifies which component represents intracellular water, and the mass percentage of water
in the cell; typically, the cell water content ranges from 60 – 80%.

Figure 7: Reactions tab of a fermentation operation dialog. The “Primary Biomass Water Content” frame is
highlighted in red.

In order to specify that certain components other than water are intracellular, the user must select the
appropriate fermentation reaction in the Reaction Sequence frame and click on the Edit Stoichiometry
button ( ). In the Stoichiometry Balance for Fermentation window that pops up, on the right side
(Products), a new column labeled Extra Cell % will have appeared to the right of the Mass / Molar Coeff.
column (see Figure 8). In this column, the user can specify the extent to which each component is
extracellular (except for the primary biomass and water components, which are automatically set by the

14
program) in terms of mass percentage. For instance, the Extra Cell % of PHA was specified as 0, indicating
that the polymer is completely intracellular.

Figure 8: Fermentation Stoichiometry Dialog. The "Extra Cell %" column is highlighted in red.

After mass and energy balances are done for the process, an “Extra Cell %” column will also appear in
every stream dialog. As such, it is possible to know the extent to which each component is intracellular for
any given stream. Figure 9 displays the dialog for the stream coming out of the fermentor (S-51).

15
Figure 9: Stream dialog for stream S-51. The Extra Cell % column is highlighted in red.

The use of this feature has implications on procedures that consider cells as particulate components, such
as centrifugation. For instance, right-click on the centrifugation procedure P-24 / DS-201, click on
Operation Data  Centrifuge and switch to the Mat. Balance tab (see Figure 10). Biomass is marked as
a particulate component, 97% of which is removed in the heavy stream, whereas Water and PHA are not
marked as particulate components. However, the intracellular water and PHA are automatically removed
along with the Biomass component to the heavy stream, given that they were specified as intracellular
components. Moreover, the mass of intracellular water and the mass of PHA are considered in the
particulate concentration in the solids stream, which was set to 600 g/L. In other words, the wet cell weight
rather than the dry cell weight is specified in this case.

16
Figure 10: Material Balance tab for a centrifugation operation.

This feature also affects homogenization / milling procedures that break cells open. For instance, right-click
on the homogenization procedure P-30 / HG-201, click on Operation Data  Homogenize and switch to
the Mat. Balance tab (see Figure 11). Here, the mass balance describes the conversion of non-polymer
cell mass into cell debris, proteins, and impurities upon cell lysis. The Homogenization Extent of
Reaction, in turn, indicates the extent of cell disruption and, by extension, how much intracellular water
and PHA are released to the medium. In the present example, the Release was set to 100%, so that all the
intracellular water and PHA granules are released upon homogenization. The user may verify that by
inspecting any stream contents after the homogenization step; all the water and PHA will be indicated as
extracellular.

17
Figure 11: Material Balance tab for a homogenization operation. The "Homogenization Extent of Reaction"
frame is highlighted in red.

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