Bioresource Technology Reports 18 (2022) 101065

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Bioresource Technology Reports

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Green solvent extraction and properties characterization of Poly

(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesized by mixed
microbial consortia fed fermented dairy manure
Maryam Abbasi a, Erik R. Coats b, Armando G. McDonald a, *
a
Department of Forest, Rangeland and Fire Sciences, University of Idaho, Moscow, ID 83844-1133, USA
b
Department of Civil and Environmental Engineering, University of Idaho, Moscow, ID, USA

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

Keywords: The aims of this study were to assess the large-scale extraction and recovery of poly(3-hydroxybutyrate-co-3-
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) hydroxyvalerate) (PHBV) biosynthesized by mixed microbial consortia (MMC) that was fed fermented dairy
Polymer Extraction manure at the pilot level using green solvents (ethanol, cyclohexanone (CYC), and dimethyl carbonate (DMC)).
Green Solvent
The reflux extraction procedure with CYC for 3 h at 130 ◦ C and DMC for 4 h at 90 ◦ C showed the highest recovery
Thermal properties
Rheology
(32.3%) with an overall pure yield of 30.0%. A modified Soxhlet extraction process was also evaluated and gave
Mechanical properties lower crude (18–20%) and purified (12.7–13.8%) PHBV yields. The molar mass of extracted PHBV with DMC in
reflux was 7.07 × 105 g⋅mol− 1 which was comparable to the control. The thermal, mechanical, and rheological
behavior of extracted PHBV were also examined. Mechanical properties result showed that strength and modulus
values of the polymers extracted in the large Soxhlet extractor were lower than those extracted by reflux.

1. Introduction of petrochemical based plastics is limited by consideration of the

required purity for the plastic waste recovered and due to quality and
Due to their versatility, petroleum-derived polymers have played a material fatigue (Koller, 2019). The depletion of fossil fuel sources and
significant role in modern day life. However, the high rate of plastic the consequences of poor degradability of the petrochemical plastics has
waste generation and the resistance to biodegradation of petrochemical increased the demand to replace them with sustainable bio-derived and
based plastics has caused severe environmental problems. A variety of biodegradable plastics like polyhydroxyalkanoates (PHA) (Zheng et al.,
methods for disposal of petrochemical based plastics have been used 2020).
including landfilling, incineration, and even just depositing in the PHAs are biodegradable polyesters that can be produced through
environment. Almost 80% of plastic wastes are disposed of in landfills. microbial biosynthesis on renewable feedstocks. PHAs accumulate
This creates an increasing pile of plastic wastes and their distribution on intracellularly as an energy and carbon storage under stressed growth
the planet has made considerable environmental pollution (Koller, conditions in the presence of excess carbon source (Yu and Chen, 2006;
2019). Depositing plastic wastes into the oceans causes harm to marine Koller et al., 2013; Choi and Lee, 1999; Kim et al., 2003). PHAs, and
and endangers human health through the introduction of microplastic specifically the most known copolymer poly(3-hydroxybutyrate-co-3-
particles (< 5 mm) into the food cycle (Koller, 2019; Campanale et al., hydroxyvalerate) (PHBV), show properties similar to conventional
2020). The annual global production of petrochemical plastics was 330 petrochemical-based synthetic plastics and after disposal are entirely
megatons (Mt) in 2019, from which 150 Mt. of plastic waste enters the biodegraded to water and carbon dioxide by microorganisms (Choi and
oceans (Koller, 2019). Disposing of plastic waste in landfills ends with Lee, 1999; Koller et al., 2011). However, the widespread production of
contamination of terrestrial and aquatic environments, through their PHBV bioplastics is hampered by their high production cost, including
migration via the wind or water systems, into the oceans (Koller, 2019; the upstream and downstream recovery after fermentation, compared to
Worm et al., 2017; Guo et al., 2020). While incineration of plastic wastes commodity petroleum-derived plastics (Zheng et al., 2020; Yu and Chen,
can be used for energy production it has led to the formation and release 2006; Koller et al., 2013; Choi and Lee, 1999). It has been reported using
of greenhouse and toxic gaseous emissions (Koller, 2019). The recycling mixed microbial cultures (MMCs), which do not require a sterile

* Corresponding author at: Department of Forest, Rangeland and Fire Sciences, University of Idaho, 875 Perimeter Drive MS 1133, Moscow, ID 83844-1133, USA.
E-mail address: [email protected] (A.G. McDonald).

https://doi.org/10.1016/j.biteb.2022.101065
Received 1 March 2022; Received in revised form 18 April 2022; Accepted 19 April 2022
Available online 25 April 2022
2589-014X/© 2022 Elsevier Ltd. All rights reserved.
M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

environment, and the potential of broader metabolism compared to stability, environmental impact, environmental health, and life cycle
single strain has provided the opportunity to use a wide variety of low- score of different solvents are considered (Pérez-Rivero et al., 2019;
cost carbon sources (Samorì et al., 2015a). A variety of natural feed­ Henderson et al., 2011; Alder et al., 2016). Considering the GSK solvent
stocks including municipal and industrial wastewater (Dionisi et al., guide, green solvents having a score > 5 in environmental impact, safety
2004; Amanat et al., 2021; Lorini et al., 2021), vegetable oils (Moralejo- and health, and the literature (García et al., 2019; Samorì et al., 2015b;
Gárate et al., 2014; Campanari et al., 2017), agricultural and forestry Rosengart et al., 2015) were used to select a suitable extraction solvent
wastes (Guho et al., 2020; Amanat et al., 2021; Yin et al., 2020;, and food for PHA. Specifically, the green solvents evaluated were DMC, CYC, and
industry wastes (Colombo et al., 2016; Hilliou et al., 2016; Morgan- ethanol, with CHCl3 as control for PHBV yield and recovery using the
Sagastume et al., 2020) have been used as main nutritional substrate. reflux extraction method. A modified Soxhlet extraction method at small
The downstream processing, including pre-treatment, extraction, and large-scale was also evaluated. PHBV composition, molar mass,
and purification of the PHBV biopolymer, covers more than 50% of the thermal, mechanical, and rheological properties of the extracted PHBVs
total PHBV production cost (Guo et al., 2020; Samorì et al., 2015a; were determined to establish whether extraction/purification influ­
Haddadi et al., 2019; Pérez-Rivero et al., 2019; López-Abelairas et al., enced their properties.
2015). Two general strategies have been adopted for the separation of
PHBV granules from cellular biomass: PHBV solubilization, and cellular 2. Materials and methods
biomass dissolution (Yu and Chen, 2006; Samorì et al., 2015b). It is
reported that the direct extraction of PHBV granules from biomass 2.1. Materials
serves as the best method (Koller et al., 2013). A variety of methods for
PHBV extraction based on the two aforementioned strategies have been CYC (≥ 99%) and DMC (99%) were obtained from Alfa Aesar (Ward
studied including solvent extraction, extraction using chelate- surfac­ Hill, MA, USA). Ethanol (EtOH, 99%) and acetone (99%) were technical
tant, chemical extraction, biological extraction, enzymatic extraction grade. CHCl3 (99.8%) was from EMD Millipore (Burlington, MA, USA).
and mechanical disruption (Haddadi et al., 2019; Jacquel et al., 2008). PHBV rich biomass was produced in a pilot-scale operation consist­
Among different PHBV extraction methods, the solvent extraction ing of (i) sequencing batch dairy manure fermenter (760 L) to produce
method, especially halogenated solvents, including chloroform (CHCl3), volatile fatty acids (VFA), (ii) an enrichment reactor (680 L) which was
methylene chloride, and 1,2-dichloroethane, have been widely studied fed VFAs to produce a MMC culture that can produce PHBV via a feast-
(Guho et al., 2020; Pérez-Rivero et al., 2019; Ramsay et al., 1994). famine regime and (iii) PHBV production reactor (450 L) which was fed
Halogenated solvent extraction provides the extracted polymer with VFAs to produce and accumulate PHBV and reported in detail in a
high purity and low endotoxin content without polymer degradation. previous study (Guho et al., 2020). The lyophilized PHBV rich biomass
However, it provides the extracted polymer with low recovery, is (PHBV content of 31.1% (w/w); HV molar ratio 0.29 ± 0.03) was used
harmful to the environment, is not economical, and also affects the for the subsequent extraction studies in reflux and small Soxhlet
natural morphology of the polymer, thereby limiting the use of PHA in extractor. The lyophilized PHBV rich biomass (PHBV content of 24.5%–
specific applications like durable fibers production (Pérez-Rivero et al., 35.6% (w/w); and HV molar ratio 0.26 ± 0.00 to 0.32 ± 0.00) was used
2019; Kynadi and Suchithra, 2014; Jacquel et al., 2008). In view of the for the extraction studies in large Soxhlet extractor. The operational
adverse effects of halogenated solvents, researchers have now focused conditions such as solvent type (EtOH, DMC, CYC, and CHCl3), tem­
on green solvents for PHBV recovery such as esters, alcohols, amides, perature (58–130 ◦ C), contact time (30–300 min), and extraction steps
cyclic and acyclic ketones. Many successful small laboratory-scale (1 to 3 times) in reflux, small and large Soxhlet extractor were studied.
studies have been performed on PHA extraction with green solvents
like dimethyl carbonate (DMC), cyclohexanone (CYC), methanol, ani­ 2.2. PHBV solvent extractions
sole, and ionic liquids (Rosengart et al., 2015; Samorì et al., 2015b).
Solvent extraction can provide the polymer with a high purity, but the 2.2.1. Small scale reflux method
high volume of required solvent, high required temperature, and po­ Lyophilized biomass (20 g) was washed with acetone (400 mL, 3×)
tential human and environment toxicity are the hindrance through for 24 h to remove lipids, filtered and air dried. The lipid-free biomass
commercializing this method. While the process of extraction with was extracted with either EtOH, DMC, CYC, and CHCl3 in reflux with
surfactants and chelating agents is simple, it needs a high surfactant dose constant stirring to obtain crude PHBV. Table 1 gives extraction process
which increases recovery cost, requires large volume of water, and conditions used and yields. A polymer/solvent <5% (w/v) was required
provides the polymer with low purity and significantly reduced molar to have a suitable viscosity to process effectively (Choi and Lee, 1999).
mass (Righi et al., 2017; Jacquel et al., 2008; Haddadi et al., 2019; In the 1st step 300–600 mL solvent was used. In the 2nd and 3rd steps
Samorì et al., 2015a). Chemical extraction methods yield polymers with 250–400 mL and 150 mL of solvent were used, respectively. The extracts
a high molar mass but with a low recovery and purity. In comparison, were hot filtered (P8 filter paper, Fisher Scientific) in a heated bath to
relatively inexpensive biological extraction methods result in polymers remove cell debris. Extracts from steps 1–3 were combined, evaporated
with a high molar mass but low yield and endotoxin materials as by- to dryness (rotary evaportator) under vacuum (60 ◦ C) to a constant
products. However, the biological materials involved in this extraction weight and the crude polymer yield recorded. The purification process
need to be pretreated. Mechanical and enzymatic extraction methods on crude PHBVs was variable depending on the solvent used for
are environmentally friendly and have low contamination, however extraction. The purification process for PHBV's extracted with each
they are expensive and complex processes (Haddadi et al., 2019; Bhat­ solvent is given below.
tacharyya et al., 2012). Unfortunately, PHBV extraction studies have The crude PHBV from CHCl3 extraction was dissolved in CHCl3 and
been performed at small scale (≤ 3 g of biomass) and there is a gap in then added dropwise to cold petroleum ether (700 mL), under constant
studies using larger-scale extractions for commercialization and stirring, and PHBV recovered as a precipitate. The precipitated PHBV
comprehensive characterization of extracted PHBV (Samorì et al., was further dissolved in CHCl3 and added dropwise to cold methanol
2015b; García et al., 2019; Rosengart et al., 2015). (700 mL), under constant stirring, and pure PHBV recovered as a white
The aim of this study was to evaluate green solvents for the extrac­ precipitate, vacuum dried and yield recorded. The crude PHBV's from
tion of PHBV in small and moderate scale from PHBV rich biomass ob­ DMC and CYC extraction were dissolved in CHCl3 and added dropwise to
tained from a pilot-scale bioprocess using MMC fed fermented dairy cold methanol (700–1200 mL) under constant stirring. The precipitated
manure (Guho et al., 2020). The Glaxo Smith Kline (GSK) solvent sus­ PHBV was vacuum dried (60 ◦ C), and yield recorded. The crude PHBV
tainability guide helps scientists in an objective assessment of using from DMC extraction only required one purification step, while the CYC
solvents (Alder et al., 2016). Various factors including reactivity and required two methanol purification steps.

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

Table 1
Operational condition, yields, and purity of PHBV extraction with dimethyl carbonate (DMC), cyclohexanone (CYC), ethanol (EtOH) and CHCl3 control on lipid-free
biomass in reflux, small and medium scale Soxhlet extractor.
Sample Solvent Apparatus Extraction steps temperature Time PHBV Yield (%) Purity

(◦ C) (h) Crude Pure (%)

CHCl3 CHCl3 Reflux 2 58–60 3,1 30.6 ± 2.2 23.1 ± 0.0 94.9 ± 2.8
CYC-1 CYC Reflux 1 120 1 27.1 22.88 87.0 ± 2.8
CYC-2 CYC Reflux 3 120 1,1,1 27.2 26.36 99.3 ± 0.8
CYC-3 CYC Reflux 2 130 2,1 35.5 ± 0.1 32.3 ± 1.0 98.2 ± 1.0
CYC-4 CYC Reflux 2 130 3,1 32.8 ± 0.3 30.6 ± 1.0 96.3 ± 2.5
DMC-1 DMC Reflux 2 90 3,1 33.5 ± 0.8 30.0 ± 1.0 98.4 ± 1.2
DMC-2 DMC Reflux 2 90 4,1 30.1 ± 0.1 25.4 ± 2.4 96.4 ± 1.6
DMC-3 DMC Reflux 2 90 1,1, 31.1 26.59 95.4 ± 1.2
EtOH -1 EtOH Reflux 1 77 0.5 3.7 – –
EtOH -2 EtOH Reflux 1 77 1 3.5 – –
EtOH -3 EtOH Reflux 1 77 5 8.9 – –
DMC-S1 DMC small Soxhlet 1 40 4 16.6 13.3 99.0 ± 2.6
DMC-S2 DMC small Soxhlet 1 40 24 17 14 98.9 ± 1.1
DMC-S3 DMC modified small Soxhlet 1 60–75 4 20.8 18.5 96.4 ± 2.5
CHCl3-S4 CHCl3 small Soxhlet 1 40 4 19.1 14.2 99.4 ± 1.7
CHCl3-S5 CHCl3 small Soxhlet 1 40 24 36.8 29.7 99.9 ± 1.2
DMC-S6 DMC modified large Soxhlet 1 60–75 11 19 12.7 97.2 ± 3.5
DMC-S7 DMC modified large Soxhlet 1 60–75 12 18.2 12.7 96.1 ± 1.5
DMC-S8 DMC modified large Soxhlet 1 60–75 14 20 13.8 94.0 ± 4.8

The PHBV extraction with ethanol was performed in three different et al., 2020).
treatments (Table 1). Lyophilized biomass (21 g) was suspended in EtOH The number (Mn) and weight average (Mw) molar mass and the
(700 mL), homogenized for 1–4 min, and then refluxed (77 ◦ C). The polydispersity index of PHBV samples (2 mg⋅mL− 1) were determined, in
EtOH extract was filtered to recover the biomass, acetone was added duplicate, by size exclusion chromatography (SEC) multi-angle light
(350–700 mL) to the filtrate, and PHBV was recovered after centrifu­ scattering (MALS) analysis. Separation was achieved using a Jordi DVB
gation (7200 rpm, − 10 ◦ C, for 10 min), vacuum dried, and yield linear mixed bed column (7.8 mm × 300 mm) on elution with CHCl3
recorded. (0.5 mL⋅min− 1) coupled to a refractive index (Waters model 2414,
Crude PHBV yields were based on ash free lyophilized biomass Milford, MA, USA) and MALS (PostNova Analytics PN3609, 658 nm,
weight basis. Pure PHBV was calculated from GCMS analysis according Landsberg, Germany) detectors. A refractive index increment (DN/DC)
to the literature (Guho et al., 2020; Braunegg et al., 1978). value of 0.034 for PHB in CHCl3 was used (Guho et al., 2020). Data were
analyzed using the NovaSEC v1.5.0 software. Calibration of the system
2.2.2. Small scale Soxhlet method was performed using a narrow polystyrene standard (Malvern, Mw =
Acetone washed biomass samples (≤5 g) were Soxhlet extracted with 105,268 g⋅mol− 1).
1
either DMC or CHCl3 (150 mL) for 4 and 24 h (Table 1). In extraction H-NMR spectra of PHBV in CDCl3 were recorded on a 300 MHz
with DMC, a modified Soxhlet extraction process was also employed to Bruker spectrometer. Spectra were analyzed using TopSpin V3.6.2
improve extraction efficiency by heating the extraction unit thimble software. The 1H-NMR spectra obtained for 3HV and 3HB were used for
zone to 60–75 ◦ C using heating tape. The extracts were rotary evapo­ determination of 3HV molar fraction through the ratio of the integrated
1
rated, dried in a vacuum oven (60 ◦ C) to record the crude yield and H peak areas of the 3HV methyl resonance and the sum of 3HB + HV
purified with the same method explained for PHBV extracted with DMC methyl resonance (Wei et al., 2014; Guho et al., 2020).
and CHCl3 in the reflux, respectively and yields recorded. FTIR spectra of PHBV samples were obtained using a Nicolet-iS5
spectrometer (Thermo-Scientific, Madison, WI, USA), 64 scans, with
2.2.3. Large scale Soxhlet method an attenuated total reflection (ATR) probe (ZnSe crystal). Spectra were
The lyophilized PHBV rich biomass (415 to 680 g) (S6; PHBV content baseline corrected and averaged using the Omnic v9.0 software. To
of 24.5% (w/w); HV molar ratio 0.27 ± 0.00, S7; PHBV content of determine carbonyl index (IC=O), which reflects the crystallinity of
35.6% (w/w); HV molar ratio 0.32 ± 0.00, and S8; PHBV content of PHBV biopolymers, the normalized spectra were curve-fitted using Igor
32.7% (w/w); HV molar ratio 0.26 ± 0.00) were used for the extraction Pro 9 software (WaveMetrics) (Wei and McDonald, 2015; Wei et al.,
in the large Soxhlet extractor. Acetone washed biomass (391 to 649 g) 2015a, 2015b). The area (A) of each band gained from curve fitting was
was placed in a polypropylene filter bag and Soxhlet extracted with DMC integrated by the software. The IC– –O was calculated as the ratio of the
(5–7 L) for 11–12 h using a modified extractor in which the extraction areas (A) under carbonyl (C=O) bands in 1720–1740 cm− 1 of PHBV
bag zone was heated to 60–75 ◦ C with heating tape (Table 1). The pu­ (IC=O = A1720/A1740).
rification steps were the same as the DMC extracted material in reflux Differential scanning calorimetry (DSC) experiments on PHBV sam­
(4.5–7 L methanol). ples (5 mg) were performed using a Q200 DSC (TA Instruments, New
Castle, DE, USA) with refrigerated cooling 40 ◦ C (3 min) then ramped to
2.3. Analytical methods 180 ◦ C (3 min) at 10 ◦ C⋅min− 1 and subsequently cooled to − 50 ◦ C (3
min) at − 10 ◦ C⋅min− 1, and the heating-cooling cycles repeated. The
PHBV composition was performed on purified PHBV (1 mg), in glass transition (Tg), melting (Tm) and crystallization temperatures (Tc),
triplicate, by methanolysis (3% H2SO4 in methanol (2 mL) and CHCl3 (2 and the degree of crystallinity (Xc%) were obtained from the peak
mL, containing 0.25 mg⋅mL− 1 benzoic acid as internal standard)) in a maximum of the exotherm and endotherm of the second heating/cool­
digestion test tube and the mixture heated at 100 ◦ C for 4 h. The 3HV/ ing scan. The Xc% of PHBV samples was calculated using the melting
3HB ratio as their methyl ester derivatives were analyzed by GC–MS enthalpy of 100% crystallized PHBV at 146 J⋅g− 1 (Wei et al., 2014). Data
(Trace1300-ISQ7000, Thermo Scientific) on a ZB-1 capillary column were analyzed using TA Universal analysis v4.5A software.
(30 m × 0.25 mm, Phenomenex, Torrance, CA USA) with a temperature The thermal stability of PHBV was determined by thermogravimetric
profile of 40 ◦ C (1 min) ramped to 200 ◦ C (10 min) at 5 ◦ C⋅min− 1 (Guho analysis (TGA) on a Perkin Elmer TGA-7 instrument (Shelton, CT, USA)

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

(5 mg, from 30 ◦ C to 800 ◦ C at 20 ◦ C⋅min− 1 under nitrogen (30 reflux method are given in Table 1. Among the polymers extracted by
mL⋅min− 1)). Data was analyzed using the Pyris v13.3 software. reflux, PHBV extracted with CYC-3, CYC-4 and DMC-1 methods showed
The rheological behavior of PHBV (25 mm Ø × 1.2 mm) was studied the highest crude yield (35.5%, 32.8% and 33.5%) and the highest pure
using a parallel plate rheometer (Bohlin CVO 100 NF, NJ, USA) from yield (32.3%, 30.6% and 30.0%), respectively. These crude yields were
0.01 to 100 Hz (0.0628–628 rad⋅s− 1), 0.5% strain, and 180 ◦ C. Data slightly higher than the crude yield of PHBV extracted with CHCl3
were analyzed (power-law models) using the Bohlin rheology v6.51.0.3 (30.6%) by reflux. Also, the pure PHBV yield extracted with CYC and
and Microsoft Excel software. DMC, except CYC-1 (25.4%–32.3%), was higher than the pure PHBV
PHBV (0.3 g) samples were hot pressed at 160–180 ◦ C in a preheated yield with CHCl3 (23.1%). The crude yields of DMC extracted PHBV
die-press mold (Ø 25 mm) to obtain 0.5 mm thick discs. Samples were were higher than those reported in the literature (crude yield 20%, and
cut into strips (10 × 2.5 × 0.5 mm3). Tensile (gauge length 10 mm) and purity 98% for 60 min, at 90 ◦ C, without pretreatment) (Samorì et al.,
three-point bending (10 mm span length) tests were each performed at 2015b).
room temperature on a DMA Q800 (TA instruments, New Castle, DE, While EtOH was introduced as a promising green solvent for PHA
USA) with controlled force (3 N⋅m− 1) and constant strain (1%⋅min− 1) extraction and shows good pure yield and purity in lab-scale (82% and
until yield, respectively. Tensile strength, Young's modulus, elongation 82%, respectively) (García et al., 2019), the crude yield in this study was
to break, flexural strength, and flexural modulus were determined from low (3–9%). Due to the low crude yield, experiments with EtOH were
the obtained stress-strain curve using Universal Analysis V4.5A soft­ not continued. The composition (3HV molar fraction) of the reflux EtOH
ware. At least five test specimens were measured per sample. extracted PHBVs were determined by GC–MS (3HV = 0.22 to 0.28) and
1
H NMR spectroscopy (3HV = 0.21 to 0.25) (Table 2). These extracted
3. Result and discussion PHBV samples have 3HV molar fraction values that were comparable to
the cellular biomass (~0.29).
3.1. PHBV yields The reflux extraction yields were influenced by extraction time and
temperature. Extraction with DMC was at a lower temperature (90 ◦ C)
PHBV rich biomass was produced at pilot scale by MMC fed fer­ than CYC (120–130 ◦ C) and similar extraction time (240 min) but
mented dairy manure (Guho et al., 2020). The removal of lipid resulted in similar yields. By increasing the extraction temperature from
extractable material from the lyophilized biomass was achieved using 120 to 130 ◦ C for CYC a 50% improvement in yield was obtained; this is
acetone (5–7% of the biomass weight). PHBV was then extracted on the consistent with previous observations (Fiorese et al., 2009). Increasing
acetone washed biomass. The yields of crude and pure PHBV from the the extraction time from 60 to 180 min for CYC did not improve crude

Table 2
3HV composition, molar mass, and thermal properties of the various purified PHBV samples obtained from different extraction methods.
Solvent/ 3HV molar fractionb Molar Massb Thermal propertiesb
Sample
Purified PHBV Mw Mn PDIa DSC FTIR TGA
name
Tg Tm1 Tm2 Tc Xc IC=O Tonset Td Tendset
1
GC–MS H NMR (×105 (×105 (◦ C) (◦ C) (◦ C) (◦ C) (%) (◦ C) (◦ C) (◦ C)
(mol. (mol. g⋅mol− 1) g⋅mol− 1)
mol− 1) mol− 1)

CHCl3 0.25 ± 0.24 ± 7.18 ± 6.35 ± 1.1 ± –7.9 NA 161.9 71.2 ± 19.7 2.35 259 291 298 ±
0.01 0.01 0.22 0.44 0.0 ± 0.4 ± 0.2 1.5 ±5 ±4 4
CYC-1 0.22 ± 0.21 ± 3.09 ± 2.94 ± 1.05 –7.1 147.7 163.3 80.9 ± 19.6 2.20 283 310 317 ±
0.01 0.01 0.35 0.28 ± 0.02 ± 0.5 ± 0.2 ± 0.1 0.6 ±0 ±1 1
CYC-2 0.23 ± 0.22 ± 3.22 ± 2.88 ± 1.12 –7.0 148.8 163.7 73.5 ± 21.2 2.38 295 321 327 ±
0.00 0.01 0.12 0.07 ± 0.02 ± 0.2 ± 0.4 ± 0.1 0.3 ±1 ±1 1
CYC-3 0.24 ± 0.24 ± 2.54 ± 2.35 ± 1.08 –7.0 151.6 164.7 86.4 ± 19.1 2.28 287 314 319 ±
0.01 0.00 0.19 0.16 ± 0.01 ± 0.5 ± 2.7 ± 0.9 10.4 ±2 ±2 2
CYC-4 0.24 ± 0.23 ± 2.03 ± 1.82 ± 1.12 –7.2 149.5 164.0 81.34 20.1 2.29 293 317 323 ±
0.00 0.01 0.16 0.22 ± 0.06 ± 0.1 ± 0.3 ± 0.2 ± 0.3 ±2 ±3 3
DMC-1 0.25 ± 0.25 ± 7.08 ± 5.91 ± 1.20 –7.4 153.9 162.4 82.5 ± 17.8 2.24 283 314 319 ±
0.00 0.00 0.53 0.34 ± 0.03 ± 0.2 ± 0.5 ± 1.5 2.3 ±0 ±2 2
DMC-2 0.28 ± 0.24 ± 6.45 ± 5.15 ± 1.26 –7.7 153.1 162.0 84.1 ± 18 2.14 285 315 321 ±
0.00 0.01 0.32 0.33 ± 0.1 ± 0.8 ± 0.5 ± 1.8 7.7 ±4 ±3 3
DMC-3 0.28 ± 0.24 ± 6.27 ± 5.39 ± 1.16 –7.5 NA 161.4 78.5 ± 18.5 2.36 285 316 323 ±
0.00 0.00 0.30 0.15 ± 0.02 ± 0.4 ± 0.4 1.0 ±3 ±2 3
DMC-S1 0.33 ± 0.31 ± 5.7 ± 0.2 4.3 ± 0.1 1.33 –5.9 NA 156.9 51.3 ± 1.4 – 273 304 310 ±
0.00 0.00 ± 0.02 ± 0.4 ± 0.7 0.4 ±1 ±0 1
DMC-S2 0.36 ± 0.35 ± 5.4 ± 0.1 4.3 ± 0.1 1.26 –5.3 NA 157.2 49.2 ± 1.3 – 266 300 306 ±
0.00 0.00 ± 0.01 ± 0.2 ± 0.4 0.3 ±2 ±1 0
DMC-S3 0.27 ± 0.26 ± 6.4 ± 0.1 4.8 ± 0.2 1.35 –7.9 NA 162.0 75.2 ± 16 – 264 299 305 ±
0.01 0.02 ± 0.04 ± 0.2 ± 0.5 0.8 ±0 ±1 2
CHCl3-S4 0.25 ± 0.24 ± 7.4 ± 0.1 5.7 ± 0.1 1.3 ± –8.6 NA 157.8 97.9 ± 17.9 – 262 294 301 ±
0.00 0.01 0.02 ± 0.5 ± 0.4 0.3 ±1 ±1 1
CHCl3-S5 0.25 ± 0.25 ± 8.5 ± 0.9 7.3 ± 0.9 1.16 –6.9 156.1 165.6 95.4 ± 18 – 264 298 303 ±
0.01 0.01 ± 0.01 ± 0.5 ± 0.3 ± 0.1 0.0 ±0 ±2 1
DMC-S6 0.27 ± 0.25 ± 22.5 ± 1.6 21.9 ± 1.5 1.03 –4.0 NA 164.5 45.8 ± 5.60 2.06 288 310 317 ±
0.00 0.01 ± 0.01 ± 0.1 ± 04 1.1 ±2 ±2 1
DMC-S7 0.30 ± 0.29 ± 8.6 ± 0.3 8.0 ± 0.3 1.07 –6.0 NA 165.7 77.7 ± 13.8 2.04 285 310 317 ±
0.00 0.01 ± 0.01 ± 0.2 ± 0.2 0.2 ±3 ±1 2
DMC-S8 0.30 ± 0.28 ± 9.8 ± 0.2 9.2 ± 0.1 1.06 –5.8 NA 162.5 46.7 ± 5.7 2.22 284 310 317 ±
0.00 0.02 ± 0.01 ± 0.6 ± 0.1 0.5 ±1 ±2 1
a
PDI = Mw / Mn.
b
Values are mean ± standard deviation.

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

PHBV yield, but did increase pure PHBV yield and purity; this obser­ 3.3. FTIR spectral analysis
vation is consistent with others (Fiorese et al., 2009). The reduction of
PHBV yield at longer extraction times may be attributed to the degra­ FTIR spectroscopy was used to analyze the chemical features of the
dation of polymer chains (Rosengart et al., 2015; Hahn et al., 1994; various purified PHBV samples extracted with either CYC, DMC, and
Fiorese et al., 2009). CHCl3. Overall, the spectra for all the PHBV samples were similar. The
Issues observed with using CYC were (i) its strong odor, even bands at 1720 cm− 1 with a shoulder at 1740 cm− 1 having the highest
working in the fume hood, (ii) the hot filtration step raised safety con­ intensity belong to carbonyl (C=O) stretching, which corresponded to
cerns, and (iii) an additional purification step required to obtain crystalline and amorphous components of PHBV, respectively (Guho
reasonable purity, and therefore using CYC in subsequent trials was not et al., 2020; Bai et al., 2015; Wei et al., 2015a, 2015b; Xu et al., 2021).
further pursued. Due to the lengthy process of PHA recovery using the The band at 1275 cm− 1 was assigned to saturated ester linkage (C–O
reflux method, a more efficient/streamlined extraction process, Soxhlet stretch) (Pramanik et al., 2014; Bai et al., 2015), while the band vibra­
extraction, was investigated. The crude and pure PHBV yields for the tion at 977 cm− 1 presents the C–C stretching vibration (Aguiar et al.,
Soxhlet extraction process are given in Table 1. Using a small Soxhlet 2018). A band at 1178 cm− 1 was also present and assigned to C–O–C
extractor, with CHCl3 as a control for 24 h, the crude PHBV yield was stretching in an amorphous structure (Zhou et al., 2019). The bands
37% (pure PHBV yield 30%). These results were comparable to those of between 1054 and 1129 cm− 1 and 1226 cm− 1 were assigned to the C–O
polyhydroxybutyrate (PHB) extracted with CHCl3 from single strains stretching (Bai et al., 2015). The four bands at 1378 (-CH3), 1456 (-CH3),
(recovery 31%, and purity 92% at 37 ◦ C (Valappil et al., 2007), and 2933 (–CH2), and 2975 cm− 1 (-CH3) are attributed to C–H bending
recovery 27%, and purity 95% at 25 ◦ C (Ramsay et al., 1994)). However, vibrations (Bai et al., 2015; Pramanik et al., 2014; Aguiar et al., 2018).
using DMC as solvent the PHBV yields were low (~17%), likely due to The carbonyl index (IC=O) of the PHBV biopolymers was calculated
the extraction temperature around 30 ◦ C in the thimble. The Soxhlet from the ratios of fitted bands of crystalline PHBV regions at 1720 cm− 1
extraction unit was then modified to heat the extraction thimble to amorphous PHBV regions at 1740 cm− 1 (Table 2) (Wei and McDo­
chamber to about 60–75 ◦ C and this resulted in a crude PHBV yield nald, 2015; Wei et al., 2015a, 2015b). All the samples extracted with
increase to 21% (pure PHBV yields 14–18%). Increasing the extraction different solvents by reflux (CHCl3, CYC-1 to CYC-4, and DMC-1 to DMC-
time from 4 to 24 h also improved PHBV yields. This result is in 3) showed similar IC– –O which ranged between 2.14 and 2.38, while the
agreement with the literature (crude yield 17% and purity 87%) (Samorì samples DMC-S6 to DMC-S8 extracted using the large Soxhlet showed
et al., 2015a). The PHBV 3HV molar ratios (determined by GC–MS) lower IC– –O values (1.97–2.04). These results agree with Xc by DSC for
using the small Soxhlet unit were between 0.25 and 0.36 (Table 2). PHBV extracted with various solvents in reflux and with DMC using the
Due to the ease of using the Soxhlet apparatus with DMC, a larger large Soxhlet (18–21% by reflux and 5.6–13.8% by Soxhlet) (Wei and
extractor with a heated extraction chamber was employed to extract McDonald, 2015; Wei et al., 2015a, 2015b).
PHBV from the biomass. Crude and purified PHBV yields of 18–20% and
13–14% were obtained, respectively (Table 1). The low yields could be 3.4. Thermal analysis of PHBV
attributed to the lower extraction temperature (60–75 ◦ C) as compared
to the reflux method (90 ◦ C). Further work is required to optimize yields The thermal properties (Tg, Tm, Tc and Xc) of purified PHBV samples
and will be part of a future study. The 3HV molar ratios were between were determined by DSC. The second heating and cooling DSC ther­
0.27 and 0.3 (Table 2). The Soxhlet extraction method was simpler to mograms are shown in Fig. 1. The Tg, Tm, and Xc values were determined
operate, DMC (80%) could be readily recovered, without the need for from the second heating cycle, while the Tc was determined on the
hot filtration, however the yields were lower. second cooling cycle (Table 2). The results show that PHBV extracted
with CYC, DMC-1, DMC-2 and CHCl3-S5 have two melting points – a
low-temperature melting peak (Tm1, 147 to 156 ◦ C) and a high-
3.2. PHBV molar mass determination temperature melting peak (Tm2, 156 to 165 ◦ C). The Tg values from
PHBV extracted by reflux with DMC, CYC, and CHCl3 were similar (− 7
The molar mass (Mn and Mw) of extracted and purified PHBV samples to − 8 ◦ C), while the Tg from the small Soxhlet extractor with DMC and
was determined by SEC-MALS and the results are given in Table 2. The CHCl3 was − 5 to − 8 ◦ C. PHBV extracted with DMC using the large
Mw and Mn values for the CHCl3 reflux extracted PHBV control were Soxhlet unit had a Tg from − 4 to − 6 ◦ C. The extraction method had no
7.18 × 105 and 6.35 × 105 g⋅mol− 1, respectively; the PHBV Mw was considerable influence on the Tm values and were comparable with
6–9% lower than observed by Wei et al. using PHBV-rich biomass ob­ published studies (Koller et al., 2013). In a separate study, PHB
tained from a bench-scale bioreactor fed fermented dairy manure (Wei extracted with CHCl3 had a Tm of 173–176 ◦ C while PHB extracted with
et al., 2014). The DMC reflux extracted PHBV samples were comparable EtOH had a Tm of 179 ◦ C (García et al., 2019). This result shows the
(Mw 6.3–7.1 × 105 g⋅mol− 1 and Mn 5.2–5.9 × 105 g⋅mol− 1) to that of the improvement effect of HV monomer in widening the processing window
control. In contrast the CYC reflux extracted PHBV had lower Mw of extracted PHBV comparing PHB (Shang et al., 2012).
(2.0–3.2 × 105 g⋅mol− 1) and Mn (1.8–2.9 × 105 g⋅mol− 1) values. The The presence of two melting peaks could be attributed to the isodi­
lower Mw and Mn for the CYC extracted PHBV is likely due to hydrolysis morphism phenomena during the heating of PHBV (Guho et al., 2020;
at 120–130 ◦ C and this phenomenon has been reported (Rosengart et al., Bluhm et al., 1986; Lv et al., 2016). The isodimorphism phenomena in
2015; Fiorese et al., 2009). The PDI for the reflux extracted PHBV semi-crystalline PHBV with some higher HV content crystals present will
samples ranged between 1.05 and 1.26. have a higher amorphous phase ratio and thus will be melted (Tm1) first.
The Mw of the Soxhlet (small and large) extracted PHBV samples was The lower HV content crystals present having a higher crystallinity ratio
between 5.4 and 14.3 × 105 g⋅mol− 1 and in the range obtained using the require more energy to melt and thus melt at a higher temperature (Tm2)
reflux extraction method. The large Soxhlet extracted PHBV with DMC (Souza Junior et al., 2019; Gahlawat and Soni, 2017; Hasan et al., 2018).
had the lowest PDI (1.01–1.07), while the PDI values for PHBV extracted Other factors that can lead to two melting points include differences in
with the small Soxhlet extractor were higher (1.16–1.35). The PHBV crystalline morphology (thickness, perfection, lamellar stability, or
sample DMC-S6 had a significantly higher Mw and Mn than other distribution), relaxation of the rigid amorphous phase, physical aging,
extracted PHBV samples by Soxhlet extraction, which cannot be and presence of crystallites with different molar masses (Souza Junior
explained. According to the literature, continuous extraction with green et al., 2019; Laycock et al., 2014). The data also suggests that the Xc of
solvents such as carbonates, esters, and ketones can recover polymers the extracted PHBV by reflux were similar (17%–21%), resulting in
with high molecular weight (1.1–1.4 × 106 g⋅mol− 1) and constant PDI similar mechanical properties. The Xc of the polymers extracted with
(1.4–3.9) at lab-scale (Pagliano et al., 2021). DMC using the small Soxhlet extractor (DMC–S1, and –S2) was <3%,

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

Fig. 1. DSC thermograms of (a) the second heating cycle and (b) the second cooling cycle of the various purified PHBV samples.

which was significantly lower than the crystallinity of PHBV with by cross-linking with dicumyl peroxide, with a similar decrease in IC– –O
similar HV% extracted with CHCl3 (Dai et al., 2007; Serafim et al., from 1.3 to 0.39 (Wei et al., 2015a, 2015b; Wei and McDonald, 2015).
2008). The crystallinity of 34% has been reported for PHBV with 35% The Tc for the reflux extracted PHBV ranged between 71 and 86 ◦ C
HV extracted with CHCl3 (Dai et al., 2007). In another study, for PHBV (Table 2). PHBV extracted with CHCl3 using the small Soxhlet extractor
extracted with CHCl3 having the HV monomer content of 4.6% the Xc of showed the highest Tc (95–98 ◦ C) vs. PHBV extracted using other
17.8% and for PHBV with 72% HV content the Xc of 6.7% is reported extraction conditions. PHBV extracted with DMC using the Soxhlet
(Serafim et al., 2008). However, PHBV extracted using the heated (DMC–S1, –S2, –S6, and –S8) had the lowest Xc (1–6%) and Tc
Soxhlet system (DMC–S3) showed higher Xc (16%) which is close to (45–51 ◦ C). The PHBV samples DMC-S3 and –S7 showed similar Tc
that obtained with CHCl3 (~18%). The Xc of PHBV extracted with DMC (75–77 ◦ C), which was slightly lower than PHBV extracted with DMC
using the large Soxhlet extractor with heating (DMC–S6, –S7, and under reflux (78–84 ◦ C). Suhazsini et al. obtained a Tc value of 108.9 ◦ C
–S8) was significantly lower (6–14%) than that obtained using the for PHBV (16.6% HV) extracted with NaClO and CHCl3 (Suhazsini et al.,
small Soxhlet extractor (16%). This is likely due to the higher HV con­ 2020). Melendez-Rodriguez et al. observed a Tc between 41 and 82 ◦ C
tent of these polymers (0.27–0.3). for CHCl3 extracted PHBV with varying HV contents (20–60%)
The IC––O, from FTIR spectroscopy, of all the samples extracted with (Melendez-Rodriguez et al., 2021). In another study, the Tc for PHBV
different solvents in reflux (CHCl3, CYC-1 to CYC-4, and DMC-1 to DMC- (20% HV) fiber and film cultured using fruit pulp biowaste was 65.6 and
3) ranged between 2.14 and 2.38, while the samples DMC-S6 to DMC-S8 71.2 ◦ C (Melendez-Rodriguez et al., 2018), respectively. The presence of
extracted using the large Soxhlet showed lower IC– –O values (1.97–2.04). an additional exothermic peak in the heating cycle of the DMC-S6 and
A poor correlation (R2 = 0.43) between IC– –O and Xc by DSC for the
–S8 implies that the nucleation and crystal formation were slower for
extracted PHBVs was observed. Published work on PHBV extracted these samples compared to the others (Fig. 1a) (Chotchindakun et al.,
using different methods showed Xc values between 54.4% and 64.1% 2021). A reduction in Tc corresponds to a reduced melting enthalpy,
and IC––O values between 0.74 and 0.78, with samples having higher Xc meaning a lower Xc (Chotchindakun et al., 2021).
exhibiting higher IC––O (Valappil et al., 2007). In another study, the IC–O
– The thermal degradation of PHBV was performed by TGA. The
for CHCl3 extracted PHBV (0–47% HV) ranged between 0.81 and 0.99, thermograms of purified PHBV samples showed a significant 1-step
and crystallinity determined by X-ray diffraction (62 to 69%) showed degradation due to a nonradical random chain scission mechanism
that the IC––O values were nearly independent of HV content (Bloem­ comprising a six-membered ring transition state (Da Silva Moreira Thiré
bergen et al., 1986). The Xc of PHBV can be decreased from 40 to 2.5% et al., 2011; Chen et al., 2013; Liu et al., 2009). The thermal onset

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

(Tonset), thermal end set (Tendset), and degradation temperature (Td) of The η* of PHBV sample CYC-1 was shown to decrease more rapidly
the purified PHBV samples are given in Table 2. PHBV extracted with with ω than the other PHBV samples. This might be ascribed to the lower
CHCl3 in reflux had a significantly lower Tonset (259 ◦ C) vs. the polymers purity (87%) of the polymer and partial plasticizing effect of impurities
extracted using different conditions. Also, Tonset, Tendset, and Td were present in the polymer (Hilliou et al., 2016). The MW of the polymers
lower in PHBV extracted with DMC (DMC–S1, –S2, and –S3) and extracted with DMC by reflux and the large Soxhlet extractor and CHCl3
CHCl3 (CHCl3 –S4, and –S5) using the small Soxhlet extractor than by reflux was considerably higher than the MW of the polymers extracted
other PHBV extracted with different solvents in reflux and with DMC in with CYC by reflux. In addition, their zero-shear viscosity was lower
large scale Soxhlet extractor. than the polymers extracted with CYC by reflux, which can be attributed
Tonset of the PHBV extracted with DMC and CHCl3 in the small to the simultaneous effect of higher crystallinity which increases the η*
Soxhlet extractor ranged between 262 and 273 ◦ C, while Td ranged from and lower Mw which decreases the η* in polymers extracted with CYC
294 to 304 ◦ C and Tendset ranged from 301 to 310 ◦ C. For PHBV extracted (Lim and Hoag, 2013).
with CYC and DMC in reflux and large scale Soxhlet extractor, the Tonset, To assess the shear thinning behavior quantitatively, the rheological
Td, and Tendset were 283–295 ◦ C, 310–321 ◦ C, and 317–327 ◦ C, respec­ data were fitted to the Power–law (Ostwald–de Waele) model, repre­
tively. The highest reported Td for natural P3HB is 290 ◦ C (Ramsay et al., senting η* as a function proportional to some power of the ω (Modi et al.,
1994). The thermal stability (as assessed by Td) of PHBV is attributed to 2016). The relationship between steady shear and dynamic shear vis­
the HV content of PHB (Carrasco et al., 2006). Weight loss of PHBV CYC- cosity has been established using the Cox–Merz rule, which is applicable
1 started at lower temperature than the other polymers which is possibly for PHA, PHB, and PHBV (Modi et al., 2016). A modified Power-law
due to the polymer being still embedded in the proteinaceous cellular model was obtained by exchanging the steady shear terms with dy­
residue and the higher impurity compared with extracted material namic viscosity terms that is expressed as:
(Samorì et al., 2015b). The TGA thermograms of most PHBV samples
|η* (ω) | = K(ω)n− 1
(1)
showed at >95% weight loss and associated with thermal degradation
(Shang et al., 2012). The large difference between Tm (by DSC) and Td
where, K is the consistency coefficient and n is the non-Newtonian or
(by TGA) for PHBV provides the processability window for this material
flow behavior index. These parameters were determined from the dou­
and thus makes them suitable for molding and extrusion for marketable
ble log plots of η* vs. ω (Fig. 2). For n < 1 the material is pseudo-plastic,
products (Liu et al., 2019; García et al., 2019; Koller et al., 2013).
when n = 1 the material is Newtonian, and n > 1 the material is dilatant
(Emadzadeh et al., 2015; Gupta, 2005; Modi et al., 2016). However, one
drawback of this model is that at the low and high shear regions the
3.5. Rheology
power law model deviates considerably from the experimental data and
should not be extended to these ranges (Emadzadeh et al., 2015; Gupta,
Rheology provides information on how to process (e.g., extrusion)
2005). The goodness of fit for the models R2 was used. All the PHBV
polymer melts by measuring the flow (viscosity) behavior as a function
samples had R2 > 0.96. The K and n parameters, and η* at ω = 6.28
of shear rate (Bhat and Kandagor, 2014). The complex viscosity (η*) of
rad⋅s− 1 for the various PHBV samples are given in Table 3. The Pow­
the PHBV samples extracted using the reflux and Soxhlet methods at
er–law model fit the data well, with K values ranging from 3.39 to 31.2
180 ◦ C versus shear rate (ω), also known as flow curves, are shown in
Pa.s. The n values ranged from 0.738 to 0.850 showing pseudo-plastic
Fig. 2. The PHBV flow curves show a reduction in η* with ω which is
behavior. Values from this study were considerably different to those
indicative of shear thinning behavior typical for non-Newtonian fluid
reported in the literature for PHBV (5% HV content) at 160 ◦ C (K = 1.35
(Liao et al., 2009). At high shear rate the anisotropic polymer chains
× 106 Pa.s and n = 0.0823) (Modi et al., 2016; Modi et al., 2013). Dif­
with high molar mass will disentangle and align and thus less molecular
ferences in measurement temperature (160 vs. 180 ◦ C) may be a
interaction among polymer chains occurs resulting in a higher free
contributing factor in the different K values.
volume which leads to a reduction in viscosity (Modi et al., 2013; Liao
For comparative purposes, the η* of the PHBV samples at ω = 6.28
et al., 2009).

Fig. 2. Complex viscosity (η*) versus shear rate (ω) plots for the various extracted PHBV samples (180 ◦ C) and power-law fitted models (dotted lines).

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

Table 3 (14.8–18.6 MPa), while the flexural modulus large Soxhlet extracted
1
Power–Law parameters (K and n) and complex viscosity (η*) at ω = 6.28 rad⋅s− (DMC–S6, –S7, and -S8; 0.31 to 0.41 GPa) was lower than the reflux
(1 Hz) determined at 180 ◦ C for the various PHBV samples. extracted samples (0.56–0.9 GPa). Flexural strength and modulus values
Solvent/Sample name K (Pa.s) n η* (Pa.s) of compression molded PHBV (28.2 MPa and 1.28 GPa) (Persico et al.,
CYC-1 17.7 0.738 10.95
2011) and injection molded 8% HV PHBV (34.5 MPa and 3.2 GPa)
CYC-2 24.6 0.829 17.97 (Jiang et al., 2008a) have been published, respectively. Again, strength
CYC-3 31.2 0.829 27.77 and modulus values were inversely related to HV content (Lim and
CYC-4 25.0 0.850 18.94 Hoag, 2013).
DMC-1 6.09 0.776 4.03
DMC-2 9.26 0.771 6.08
DMC-3 7.20 0.779 4.8 3.7. Relationships between PHBV properties
CHCl3 3.39 0.756 2.16
DMC-S6 8.38 0.819 6.01 The parameters of solvent type, extraction time and temperature in
DMC-S7 14.9 0.829 10.84 two different extraction apparatus were studied here. Changes in these
DMC-S8 10.2 0.815 7.23
parameters effected on the yields and HV content of the extracted PHBV.
The effect of extraction methods on yields is discussed earlier.
rad⋅s− 1 = 1 Hz was determined and ranged from 2.16 Pa.s to 27.8 Pa.s. Comparing methods, PHBVs extracted in small and large Soxhlet had a
The polymers extracted with CYC showed a higher η* than the other higher range of HV content. This can be because of the lower tempera­
samples ranging from 11.0 Pa.s to 27.8 Pa.s. The PHBV extracted with ture of the solvent in contact with biomass in Soxhlet than reflux which
CHCl3 by reflux showed the lowest η* at 2.16 Pa.s. The η* of the PHBV provides a heating energy that is enough for extraction of mostly
samples extracted with DMC in reflux and large Soxhlet, except for amorphous high HV content structure than crystallized high HB content
DMC–S7, were similar (4.03–7.23 Pa.s). The η* of DMC-S7 sample was structure. By increasing the extraction time to optimum the HV content
similar to CYC-1 sample. The difference in the η* may be attributed to and Mw of the extracted PHBVs increased. When extraction time
the difference in HV content of the samples. The lower η* is associated exceeded the optimum level, the HV content and the Mw of the extracted
with PHBV of higher HV content. PHBVs decreased. The previous studies have shown that the longer
extraction time can result in the hydrolysis of the polymer chains.
3.6. Mechanical properties Because polymer granules decompose rapidly, the molecular weight
reduction can be minimized by shortening extraction time (Mohammadi
The mechanical properties (tensile and flexural) of the various et al., 2012; Choi and Lee, 1999).
extracted PHBV samples were determined and the results are given in Generally, regardless of extraction method the HV content of the
Table 4. The tensile strength of the polymers extracted with various extracted polymers influenced the Mw, PDI, Xc and IC––O values. Thermal
solvents in reflux ranged between 6.5 MPa and 12.4 MPa and the properties including Tm, Tc, and Tg decreased with HV content. This can
Young's modulus of the samples ranged between 0.25 GPa and 0.86 GPa. be attributed to the fact that an increase in the HV content of the
The tensile strength values of 29.1 MPa for solution cast PHBV (5% HV) polymer increases the amorphous structure and reduces the nucleation
(Thiagamani et al., 2019), 27.1 MPa for injection molded PHBV (8% HV) of PHBV and initiates crystallization at a lower temperature (Chotch­
(Jiang et al., 2008a), and 14.1 MPa for solution cast PHBV (12% HV) indakun et al., 2021).
(Jiang et al., 2008b) have been reported. Furthermore, the Young's The η* of PHBVs extracted with CYC (CYC-1 to − 4) and DMC (DMC-1
modulus of 2.19 GPa for solution cast PHBV (5% HV) (Thiagamani et al., to- 3 and DMC-6 to − 8) increased with HV content.
2019), 3.5 GPa for injection molded PHBV (8% HV) (Jiang et al., 2008a), The mechanical properties of PHBV showed a negative relationship
and 0.82 GPa for solution cast PHBV (12%) HV (Jiang et al., 2008b) with HV content (R2 ≥ 0.63) which is due to the higher amorphous
have also been documented. Strength and modulus values were content (Lim and Hoag, 2013).
inversely related to HV content. The Young's modulus of PHBV from the
large Soxhlet extractions (DMC–S6, –S7, and –S8) ranged from 0.25 4. Conclusions
to 0.4 GPa and were lower than PHBV extracted by the reflux method
(0.41–0.86 GPa). Protocols for green solvent (CYC, DMC, and EtOH) extraction of
The flexural strength of large Soxhlet extracted PHBV (DMC–S6, PHBV from fermented manure by MMC was successful. DMC was a
–S7, and -S8; 7.3 to 9.3 MPa) was lower than the reflux extracted PHBV suitable solvent for PHBV extraction with >80% recovery, and one pu­
rification step from the crude extract but lower PHBV yields. Modified
Table 4
Soxhlet extraction used exhibited suitable extraction, ease of use, and
Tensile and flexural properties of the various extracted PHBV samples1,2. workup. Molar mass, polydispersity, and mechanical properties were
comparable for PHBV extracted with DMC and CHCl3 (control) but
Solvent/ Tensile Young's Flexural Flexural
Sample strength modulus strength modulus
higher melt viscosity (η*) for the former. DMC solvent was efficiently
used on a large lab-scale extraction but needs further research optimi­
MPa GPa MPa GPa
zation to improve yields for greener industrial extraction processes.
CHCl3 9.5 ± 0.6b 0.47 ± 0.02bc 17.0 ± 0.6ab 0.73 ± 0.07bc
CYC-1 6.5 ± 1.3b 0.86 ± 0.17a 16.8 ± 0.5abc 0.90 ± 0.07a
CRediT authorship contribution statement
0.77 ±
CYC-2 12.4 ± 1.1b 0.61 ± 0.08b 18.6 ± 1.1a
0.15abc
CYC-3 10.3 ± 1.3b 0.56 ± 0.08b 17.1 ± 0.8ab 0.71 ± 0.04bc Maryam Abbasi: Conceptualization; Methodology; Validation;
CYC-4 11.8 ± 0.8b 0.59 ± 0.05b 17.5 ± 0.7a 0.78 ± 0.06b Formal analysis; Investigation; Writing.
DMC-1 11.2 ± 1.3b 0.52 ± 0.07bc 15.5 ± 0.5bc 0.67 ± 0.05cd Erik R. Coats: Supervision; Funding acquisition; Writing.
DMC-2 9.9 ± 0.7b 0.51 ± 0.03bc 17.2 ± 0.4a 0.74 ± 0.02bc
Armando G. McDonald: Supervision; Project administration; Fund­
DMC-3 10.0 ± 1.6b 0.41 ± 0.06c 14.8 ± 0.4c 0.56 ± 0.05d
DMC-S6 8.8 ± 0.6b 0.40 ± 0.04a 9.3 ± 0.9d 0.41 ± 0.04e ing acquisition; Writing; Methodology; Conceptualization.
DMC-S7 8.0 ± 0.8a 0.30 ± 0.06a 7.3 ± 0.7d 0.31 ± 0.03e Declaration of competing interest.
DMC-S8 9.6 ± 0.3b 0.25 ± 0.03a 7.3 ± 0.5d 0.32 ± 0.02e The authors declare that they have no known financial or personal
1
Values are mean ± standard deviation. conflicts that might influence the work in this article.
2
Statistical differences of means using a Tukey test (P-value <0.05) and
different superscript letters (a, b, c, d, e) shows significance.

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M. Abbasi et al. Bioresource Technology Reports 18 (2022) 101065

Declaration of competing interest Bioeng. 62 (5), 546–553. https://doi.org/10.1002/(SICI)1097-0290(19990305)62:

5<546::AID-BIT6>3.0.CO;2-0.
Chotchindakun, K., Pathom-Aree, W., Dumri, K., Ruangsuriya, J., Pumas, C., Pekkoh, J.,
The authors declare that they have no known competing financial 2021. Low Crystallinity of Poly (3-Hydroxybutyrate-co-3-Hydroxyvalerate)
interests or personal relationships that could have appeared to influence Bioproduction by Hot Spring Cyanobacterium Cyanosarcina spAARL T020. Plants 10
the work reported in this paper. (3), 503. https://doi.org/10.3390/plants10030503.
Colombo, B., Sciarria, T.P., Reis, M., Scaglia, B., Adani, F., 2016. Polyhydroxyalkanoates
(PHAs) production from fermented cheese whey by using a mixed microbial culture.
Bioresour. Technol. 218 (2016), 692–699. https://doi.org/10.1016/j.
Acknowledgement
biortech.2016.07.024.
Da Silva Moreira Thiré, R.M., Arruda, L.C., Barreto, L.S., 2011. Morphology and thermal
The authors would like to acknowledge the financial support from (i) properties of poly(3-hydroxybutyrate-co-3. hydroxyvalerate)/attapulgite
USDA-NIFA award number 2018-67022-27894, (ii) Idaho State Board of nanocomposites. Mater. Res. 14 (3), 340–344. https://doi.org/10.1590/S1516-
14392011005000046.
Education IGEM award number IGEM19-001, (iii) the University of Dai, Y., Yuan, Z., Jack, K., Keller, J., 2007. Production of targeted poly(3-
Idaho Equipment and Infrastructure Support (EIS) Awards Program hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate
from the Office of Research and Economic Development (ORED) RISE as sole carbon source. J. Biotechnol. 129 (3), 489–497. https://doi.org/10.1016/j.
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