# Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) using microalgae-derived starch and levulinic acid

Gabriel Brouchon, Pablo Alvarez, Alexandre Six, Pierre Lemechko, Alexandra Dimitriades-Lemaire, Gatien Fleury, Jean-François Sassi, Stéphane Bruzaud

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Gabriel Brouchon, Pablo Alvarez, Alexandre Six, Pierre Lemechko, Alexandra Dimitriades-Lemaire, et al.. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) using microalgae-derived starch and levulinic acid. Polymer Degradation and Stability, 2025, 233, pp.111176. 10.1016/j.polymdegradstab.2025.111176. hal-05369575

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# Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) using microalgae-derived starch and levulinic acid

Gabriel Brouchon<sup>a</sup>, Pablo Alvarez<sup>b</sup>, Alexandre Six<sup>b</sup>, Pierre Lemechko<sup>c</sup>,

Alexandra Dimitriades-Lemaire<sup>b</sup>, Gatien Fleury<sup>b</sup>, Jean-François Sassi<sup>b</sup>, Stéphane Bruzaud<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Biosynthesis

Microalgae

Starch

Polyhydroxyalkanoates

## ABSTRACT

A microalgae cultivation process was developed in order to produce starch-rich biomass, which can be converted into glucose after starch extraction and hydrolysis. Associated to levulinic acid, biosynthesis processes were carried out with microalgae-derived starch glucose, resulting in the production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBHV) with different contents in HB and HV units, with HV units reaching up to 32 %. The thermal and mechanical characteristics were measured as a function of HV proportion and are discussed in terms of chemical structure/morphology/properties relationships, showing that these properties can be modulated by adjusting the ratio between HB and HV units. The crystallinity of PHBHV copolymers gradually decreases as HV unit content increases and tends to drastically increase the ductility of materials with an elongation at break equal to about 800 % for PHBHV with 32 % in HV units. This work emphasizes the possibility to precisely design tailor-made PHBHV with tunable functional properties, by playing on the mixture of substrates initially introduced. Moreover, it addresses crucial societal considerations by using raw materials that do not compete with the food industry.

## 1. Introduction

Polyhydroxyalkanoates (PHA) constitute a large family of bacterial polyesters which have emerged as promising biopolymers in order to replace some conventional petroleum-based plastics [1]. The physico-chemical PHA characteristics depend on their chemical composition, itself influenced by the nature of the carbon source used for the bacteria growth and the PHA production, which offers a wide range of materials covering many areas of application (packaging, films, cosmetic products, coatings, biomedical devices, tissue engineering implants, carbon source for denitrification in wastewater treatment, etc.) [2,3]. PHA can be fine-tuned to align with the specific requirements of each industry, enabling the customization of material properties such as ductility, elasticity, thermal conductivity and transparency.

Among all the available PHA types, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) stands out for its adaptable functional properties (mechanical, thermal, barrier...) by playing on the proportions of HB and HV units [4,5]. Copolymers of PHBHV can lead to

lower crystallinity and thus less brittleness of the material with the increasing proportion of HV monomer units in the PHBHV chain. The interplay between physico-chemical properties, microstructure, crystallization kinetics, molecular weight and mechanical properties has already been reported [5-7]. Moreover, these PHBHV have been demonstrated to be highly biodegradable, especially in the marine environment, which offers an extremely interesting additional functionality to these PHA for applications that require rapid biodegradation [8,9]. However, the conventional PHA production relies heavily on the use of sugars derived from food crops considered as first-generation biomass feedstock, such as glucose and sucrose, which raises concerns about resource competition, ethical acceptability and environmental sustainability. The second carbon substrate used to generate HV units in PHBHV is often propionic or valeric acid coming from resources of petrochemical origin [5,10]. Valeric acids can be also found in mixes of volatile fatty acids (VFA) obtained from anaerobic digestion of agro-industrial coproducts but much effort must be made to manipulate the composition of VFA mixtures by means of fermentation conditions.

<footnote><sup>a</sup> Université Bretagne Sud, Institut de Recherche Dupuy de Lôme (IRDL), UMR CNRS 6027, 56321, Lorient, France</footnote>

<footnote><sup>b</sup> CEA, MicroAlgae Processes Platform, CEA de Cadarache, 13108, St Paul Lez Durance, France</footnote>

<footnote><sup>c</sup> Institut Régional des Matériaux Avancés (IRMA), 2 allée Copernic, 56270, Ploemeur, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: stephane.bruzaud@univ-ubs.fr (S. Bruzaud).</footnote>
The percentage of VFA with an odd number of carbons obtained depends on the agro-industrial residues used, their intrinsic variability and the fermentation process used [11,12].

In this context, studies must continue on these polymers, particularly towards the search for raw materials which do not compete with food resources and the occupation of arable land. In the same way as biofuels, numerous reservations are expressed about biobased polymers, or plastics in the broad sense, with regard to the availability of agricultural resources, competition in the use of resources as well as the risks of deforestation and exhaustion of water resources that they could generate. Even if these criticisms are not always justified (according to European Bioplastics, the areas intended for the cultivation of raw materials for the biosourced plastics industry would occupy, in 2023, 0.99 million hectares, equivalent to 0.021 % of the cultivable areas on a global scale [13]), to respond to this, the use of substrates coming from second or third generation biomasses must be encouraged, which could provide molecules of interest (starches, sugars, fatty acids, vegetable oils, etc.) for the biotechnology or chemistry sectors through environmentally and ethically responsible approaches.

To address these challenges, researchers have been exploring alternative feedstocks for PHA production. Microalgae are a promising third generation biomass feedstock and as replacement of conventional fossil-based polymers in the biobased economy. Microalgae-derived starch, represents a potential source of carbon for PHA synthesis. Currently, starch for bioplastic applications is produced only from terrestrial plants, alternative feed stocks that do not compete with food production as algae should therefore be prioritised [14]. Microalgae may potentially produce starch with a lower water and land footprint than conventional terrestrial crops [15].

Additionally, levulinic acid, derived from various biomass feedstocks in the context of second-generation biofuels, has recently gained attention as a carbon precursor for biobased polymers. Levulinic acid is a versatile platform chemical with the potential to be converted into a range of valuable compounds, including monomers for polymer production [16,17]. Leveraging the carbon content of levulinic acid for PHBHV synthesis not only adds value to biomass conversion processes but also contributes to the development of a circular bioeconomy with a ethically and responsible approach.

This article aims to explore the feasibility of producing PHBHV by employing microalgae starch as main carbon source, associated to levulinic acid as co-susbtrate and 3-HV precursor in order to modulate the chemical PHBHV structure and their resulting properties.

# 2. Materials et methods

## 2.1. Starch-rich microalgae cultivation

Around 500 g of starch-rich microalgae biomass was processed and transformed into glucose to provide the carbon substrate for the PHA-bacteria.

The microalgae strain used was *Chlorella vulgaris* CCALA924 (CCALA, Czech Republic). This strain was previously identified as a promising starch producer [18]. The starch accumulation was targeted by means of nitrogen starvation in the stationary growth phase, this process has been recently described [18–20]. The culture medium was a modified version of the Beijerinck medium, containing 1.2 g NaNO₃ L⁻¹, 50 mg.L⁻¹ CaCl₂·2H₂O, 100 mg.L⁻¹ MgSO₄·7H₂O, 106 mg.L⁻¹ K₂HPO₄, 53 mg.L⁻¹ KH₂PO₄, 11.4 mg.L⁻¹ BO₃H₃, 22 mg.L⁻¹ ZnSO₄·7H₂O, 5.6 mg.L⁻¹ MnCl₂·4H₂O, 4.9 mg.L⁻¹ FeSO₄·7H₂O, 1.6 mg.L⁻¹ CoCl₂·6H₂O, 1.57 mg. L⁻¹ CuSO₄·5H₂O and 1.1 mg.L⁻¹ MoO₄·2H₂O (NH₄)₆·4H₂O. Stock cultures of *C. vulgaris* were maintained autotrophically in an automated incubator ZWYC 290 A (LABWIT Scientific, Melbourne, Australia) under constant environmental conditions (photoperiod: 16-h light and 8-h darkness, agitation: 150 rpm, 2 vol% CO₂).

The production of *C. vulgaris* starch-rich biomass was carried out in 4 different laboratory pilot flat-panel airlift photobioreactors with static

mixers (Subitec GmbH, Stuttgart, Germany) with a capacity of 28 L each. These reactors are described by Vogel et al. [21]. The 4 photobioreactors were placed around a sodium-vapour lamp that provided a light intensity of 200 µE/m²/s with a photoperiod of 20:4 Day:Night (h). Temperature was controlled by means of a thermostatic water bath where the 4 photobioreactors were partially immerged at a constant temperature of 25 °C. Agitation of the reactors was provided by gas injection through a pierced silicone tube placed at the bottom of each reactor giving a constant homogeneous aeration at a flow of 200 L/h with compressed air and enriched at 2 % with CO₂. The pH of the reactors was stabilized at around 7.5 thanks to the constant provision of CO₂. Reactors were operated in batch cycles of around 8 days ensuring that the biomass microalgae generated in the first 4 days was able to deplete initial nitrogen concentration from culture medium in order to trigger starch accumulation on the 4 last days. Then, microalgae biomass was harvested letting inside the reactor 10 to 15 % of total culture volume to inoculate the next batch. The biomass produced in successive batches was concentrated by centrifugation at 15,900 g for 10 min at 5 °C in a 6 L batch centrifuge (Avanti J-26XP, Beckman Coulter, USA) and stocked frozen until enough biomass was available for starch extraction.

## 2.2. Microalgae biomass and starch content characterization

Microalgae concentration in the reactors was determined from 4 to 5 mL of culture suspension, samples were centrifuged at 4500 g for 10 min at 4 °C and the pellets were washed with pure water and centrifuged again to eliminate any remaining mineral solutes. The washed pellets were transferred to pre-weighed and pre-dried aluminum cups and dried in a ventilated oven at 105 °C for 24 h The weight loss upon drying was used to calculate biomass concentration. The supernatant of the centrifuged culture was recovered to determine major nutrients, i.e. nitrate and phosphate, concentration by ion chromatography (940 Professional IC Vario, Metrohm, Switzerland).

Starch accumulation in microalgae biomass was evaluated via the quantification of total carbohydrates following a colorimetric method first developed by Dubois et al. [22] and recently adapted [20]. 1–3 mg samples of lyophilized microalgae biomass were firstly digested in 1.25 M H₂SO₄ (0.5 mL.mg⁻¹ DW) at 100.5 °C for 3 h, then diluted in ultra-pure water to a final volume of 0.5 mL and then mixed with 500 µL of 5 % (w/v) phenol solution and 2.5 mL of 95 % H₂SO₄. Light absorption at 483 nm was determined in a spectrophotometer (UV–Vis Epoch2, BioTek Instruments, USA) and the values were referred to a calibration curve made on glucose.

## 2.3. Starch extraction and hydrolysis into glucose

Starch was separated from the microalgae biomass as previously stated according to the process developed by Six et al. [23]. Briefly several starch-enriched biomass batches were defrosted, merged and diluted to 10 % DW in pure water. The suspended biomass was first disrupted with high pressure homogenization (HPH, CF2 Cell Disruptor, Constant Systems Ltd., England) at 250 MPa for 5 cycles and then, the cell lysate was centrifuged at 15,900 g for 10 min at 5 °C (Avanti J-265 XP, Beckman Coulter, USA). After centrifugation three different phases were observed, a green liquid supernatant on top, a green pellet in the middle and a white starch pellet in the bottom. The starch pellet was recovered, while the other two phases were discarded.

The starch pellet was characterized in terms of DW and resuspended in water at 30 %, then starch was enzymatically hydrolysed into glucose in to steps, a first liquefaction with α-amylase (Sigma-Aldrich, A6380) and subsequent saccharification process with amyloglucosidase (Sigma-Aldrich, A7095). The reactions took place sequentially in a 2 L vessel with thermostat jacket and agitated via magnetic stirring at the bottom. First, the α-amylase reaction was conducted at 500U/g starch, pH 6.0 and 80 °C during 8 h. Then, the amyloglucosidase reaction was conducted at 250U/g starch, pH 4.0 and 40 °C during 8 h. For these
calculations the quantities of starch in biomass where considered equal to the measured total carbohydrates in biomass.

At the end of each enzyme-related phase the broth was boiled to ensure enzyme inactivation, boiling caused protein precipitation and hydrolysed-starch broth was filtered through 750nkDa membrane (Repligen Minikros S06-E750-05-N) in a tangential filtration device (KrosFlo Research IIi) to remove any residual suspended or precipitated solid. At the end of the hydrolysis and filtration processes, around 400 mL of glucose-rich hydrolysate (317.2 mg glucose.L⁻¹) were obtained, this glucose-rich medium was sterile filtered by 0.2 µm and freeze until PHA bacterial growth test.

## 2.4. PHA production and growth conditions

The strain used to produce PHA was Cupriavidus necator (DSMZ 545). It was stored in glycerol 25 % at -80 °C and 2 mL tubes were used for each preculture. The medium used for Erlenmeyer culture and bioreactor was modified Zobell medium (MZM) with 10 g.L⁻¹ Bactotryptone (Gibco), 5 g.L⁻¹ yeast extract (Gibco) and 5 g.L⁻¹ sea salts (Instant Ocean SW, Aquarium System) at pH 7.5. All media and bioreactor were sterilised at 121 °C for 20 min.

Precultures were grown overnight in 100 mL Erlenmeyer flasks at 180 rpm and 30 °C. Growth comparison experiment was done in the same conditions with the addition of commercial glucose (Thermos Scientific) or starch hydrolysate at 30 g.L⁻¹.

Bioreactor productions were performed in a 5 L bioreactor (GPC-BIO, MINIPROLAB, France) with a final volume of 2 L MZM supplemented with 30 g.L⁻¹ glucose at the start of the run. Cultivation was performed at 30 °C, pH was set at 7 and automatically controlled with 2 M NaOH and HCl solutions. Agitation and air flow were set at 500–1000 rpm and 2–5 L.min⁻¹, respectively and controlled based on dissolved oxygen measurement with a setpoint of 30 %. Feed pulses containing levulinic acid (LA) were added to the reactor during growth using a peristaltic pump (Masterflex), with the feed rate manually adjusted to maintain 0.1 g LA.g.X⁻¹.h⁻¹ and ensuring that each pulse does not exceed 5 g.L⁻¹ of LA in the reactor. The feed was designed to have excess carbon and limited nutrient while maintaining minimal biomass growth, based on the following constants for C. necator: yield on glucose 0.45 gₓ·gGᵢC⁻¹, yield on phosphate 55 gₓ·gP⁻¹, and yield on nitrogen 7 gₓ·gN⁻¹ [12]. The feed contained per litre: 582 g carbon source with varying ratio of glucose and LA, 24.1 g Bacto Tryptone and 12 g Bacto Yeast Extract. The feed solution was prepared with a maximum of 291 g.L⁻¹ LA. For the highest ratio glucose to LA tested (1:1) the feed contained then 291 g.L⁻¹ LA and 291 g.L⁻¹ glucose. The reactor was harvested when dissolved oxygen no longer remained below 30 % due to overfeeding or that reactor could no longer be run due to timing reasons.

Cell density during growth was measured by absorbance at 600 nm and by dry weight measurement of a 50 mL samples, centrifugated at 8000 G for 10 min at 4 °C and freeze-dried.

## 2.5. PHA extraction and film preparation

After production, the bioreactor volume was centrifuged (13,000 g, 10 min at 4 °C) and the pellet was freeze-dried before being crushed to powder and placed in chloroform at 30 g.L⁻¹ and left overnight on a hot plate at 50 °C with stirring. The resulting suspension was then filtered under vacuum on a sintered glass filter (pore size 40–100 µm) to remove biomass particulates. The filtrate was precipitated by dripping into cold ethanol (0–10 °C) with a dissolved PHA/ethanol ratio of 1:5. The white precipitate was collected and re-solubilised in chloroform at 30 g.L⁻¹ before being poured into glass Petri dishes of 9 cm diameter (25 mL/ plate) and allowed to dry with a lid under a flow hood for 48 h, then the lid was removed and the films were left to dry for 72 h extra.

## 2.6. Characterization of PHBHV produced

### 2.6.1. Gas chromatography (GC)

Chemical PHBHV composition, i.e. molecular ratios of HV to HB units, was measured by GC analysis (Hewlett Packard, 5890 series II). Samples propanolysis was performed with 2 to 15 mg of PHA, mixed in 1 mL of 1 g.L⁻¹ benzoic acid (internal standard) solution in chloroform. Once dissolved, 1 mL of 1-propanol and HCl (80/20 %, v/v) is added, and the sealed tube is left at 80 °C for 20 h. Then 1 mL of distilled water is added and vigorously shaken. After decanting, the organic phase is sampled and filtered on MgSO₄ and glass wool. GC analysis is performed on a HP-5 column (Agilent technologies, 50mx0.530 mm, 2.65 µm) with the following program: 3 min at 70 °C, 10 °C.min⁻¹ to 240 °C, 10 min at 240 °C. The injector and detector are set at 250 and 320 °C, respectively.

### 2.6.2. Steric exclusion chromatography (SEC)

Molecular weights and polydispersity index (PDI) were measured using SEC (Agilent 1260 infinity 2) and PS standards. Samples from film casting were dissolved into chloroform at 20 g.L⁻¹ and filtered on a 0,45 µm PTFE membranes before injection into the SEC apparatus.

### 2.6.3. Tensile test

Young's modulus, tensile strength and elongation at break were measured by tensile test (MTS Criterion model 4). Dried solvent-casted films were cut into A5 sized specimen and kept at control room temperature and humidity (23 °C and 50 %) for a minimum of 24 h and a maximum of 7 days prior to measurement. Tensile tests were performed at a tensile speed of 10 mm.min⁻¹ on 5 specimen minimum per sample using a 500 N cell. The thickness of each specimen was measured prior to testing. If the specimen did not have a uniform thickness along its length, an average thickness was taken by taking 3-point measurements and varied between 50 and 200 µm. Young's modulus, tensile strength and elongation at break were calculated automatically by the MTS software.

### 2.6.4. Differential scanning calorimetry (DSC)

Thermal properties of PHBHV were assessed by DSC (3 Star System, Mettler Toledo). Samples of 5–10 mg were taken from the film castings described above and placed in sealed aluminium pans (crucibles ME-26,763, Mettler Toledo). The heating and cooling sequence is set to heat from 25 to 190 °C at 10 °C.min⁻¹, hold at 190 °C during 2 min, cool down to -40 °C at 10 °C.min⁻¹, hold at -40 °C during 2 min, then heat to 190 °C at 10 °C.min⁻¹. The melting temperature (T<sub>m</sub>) and melting enthalpy (ΔH<sub>m</sub>) were calculated from the thermograms of first and second heating phase.

### 2.6.5. Fourier transform infrared spectroscopy (FTIR)

Crystallinity of each polymer films produced was evaluated using FTIR (Bruker, Vertex70v) according to the same protocol previously described [24].

# 3. Results and discussion

## 3.1. Microalgae biomass production

The process to produce starch-rich C. vulgaris was run in different laboratory pilot photobioreactors in order to attain the targeted biomass requirements of 500 g before transferring to the starch extraction phase. All batches followed the same production route with a first nutrient replete growth phase and a second nitrogen deprived starch-accumulation phase. However, these batches presented differences in microalgae and total carbohydrates production yields and must not be considered as replicates due to the complexity of laboratory pilot-scale operations.

Growth and total carbohydrates accumulation profiles of two of these batches are discussed here. On the first batch (Fig. 1(a)), the initial
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** *C. vulgaris* growth (black solid lines) and starch accumulation expressed as total carbohydrates (grey points) in two different 28 L pilot photobioreactors (Fig. 1a: Run 1; Fig. 1b: Run 2), the transition from biomass-growth phases in nutrient-replete medium to starch-accumulation phases in nitrogen-depleted medium is triggered by nitrate concentration (black dash lines) in the medium.</figcaption></figure>

185 mg N—NO₃/L were consumed in the first 5 days (118 h, nutrient-replete biomass-growth phase), while the nitrogen-depleted starch-accumulation phase lasted until day 8 (186 h). It can be observed that starch accumulation was triggered with the absence of nitrogen in the medium, rising from 27.62 % (±1.5) in the growth phase to 65.27 % (±0.76) at the end of the accumulation phase. Biomass raised from 2.9 g/L at the end of the growth phase to 4.72 g/L at the end of the starch accumulation phase. The growth of *C. vulgaris* in nitrogen-depleted conditions is correlated with a decrease in the proteins content [20, 25] therefore using proteins as internal nitrogen-stocks to maintain growth. Similarly, on the second batch (Fig. 1(b)), the initial 199 mg N—NO₃/L were consumed in the first 5 days (115 h, nutrient-replete biomass-growth phase) and the total carbohydrates content raised from 32.47 % (±0.87) on day 5 to 52.33 % (±1.14) on day 7 at the end of the starch accumulation phase, with a final biomass concentration of 3.4 g/L.

## 3.2. PHBHV production

PHA biosynthesis was done with the model strain *C. necator*. To produce PHBHV, the feed used is a combination of glucose and levulinic acid. The molar ratio between the two will govern the percent of HV content in the copolymer produced as HV units can only come from levulinic acid incorporation. First, to cope with reduced availability of microalgae starch hydrolysate, a growth comparison with commercial glucose and starch hydrolysate glucose was performed and showed no significant variation in growth rate between the two conditions in Erlenmeyer flasks, the two curves being almost superimposable (Fig. 2). This result allows us to consider glucose from microalgae starch as a relevant source of carbon, in the same way as commercial glucose. It validates the microalgae cultivation process, as well as the extraction and hydrolysis processes previously developed to produce this new source of glucose with a high degree of purity.

To allow production of PHBV from a mixture of LA and glucose, a conservative feed rate is required as the *C. necator* strain is sensitive to low concentrations of LA, with 10 g.L⁻¹ completely inhibiting growth [26,27]. The conservative feeding strategy used was designed to keep LA concentrations below this level for each pulse. As a representative production run, when glucose to LA ratio of 1:1 was used in the feed, a 40 h fermentation fed-batch produced 104 g of dried biomass in 70 h, from which 73 g of PHBHV was extracted with a final HV content of 30 mol% and an overall productivity of 0.52 g.L⁻¹.h⁻¹, which is in line with other attempts to produce PHBHV from LA and glucose combined. The total carbon input was 162 g glucose and 102 g LA, giving a yield of PHBHV produced over the carbon substrate input of 0.28 g/g. The yield of HV

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Growth comparison on two glucose sources over time between starch hydrolysate from microalgae and commercial glucose. Error bar is standard deviation of 3 biological replicates (P = 0.05).</figcaption></figure>

units produced from LA reached 0.23 g/g.

Biomass samples were taken during fermentation, from t₀ to 70 h of fermentation to follow cell density by cell dry weight measurement (CDW). The maximum growth rate of 0.37h⁻¹ was reached between t₀ and 17 h and decreased to 0.01h⁻¹ before the bioproduction was stopped.

The maximal LA feeding rate of 0.1 g LA g.X⁻¹.h⁻¹ being below optimal, the oxygen in the reactor was never depleted (Fig. 3), confirming that the feed consumption rate was higher than the feed rate used here. The molecular percentage of HV versus HB increases over time and seems to plateau close to 30 mol%. This is in line with HV mol % obtained in literature with a C/N ratio of 19 as used here, the maximum of 50 mol% being obtained when feeding LA alone [27]. Each pulse of feed showed two phases in the oxygen consumption rate, with the first phase showing lower dissolved oxygen in the reactor (~20%) and a second phase with the DO increasing to about 30%. This confirms the strain preference of feed, favouring LA over glucose, with LA consumption rate being higher than that of glucose [28].

After extraction procedure, the resulting dissolved PHA was formed into thin films by solvent casting and further analysed (Table 1). A total of 7 samples from different production batch with varying LA to glucose
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Growth pattern of a fed-batch 3 L bioreactor of *C. necator* with levulinic acid and glucose feed (1:1 g/g). Evolution of dissolved oxygen (DO), cell dry weight (CDW) and HV mol% (PHBHV) in polymer accumulation).</figcaption></figure>

<table><caption>**Table 1**

Physico-chemical characteristics of the PHBHV produced.</caption><thead><tr><th>% HV in PHBHV</th><th>Mass average molar mass (Eq. PS)</th><th>Polydispersity index</th><th>Young modulus (MPa)</th><th>Tensile strength at break (MPa)</th><th>Elongation at break (%)</th></tr></thead><tbody><tr><td>32</td><td>5.3 × 10<sup>5</sup></td><td>3.9</td><td>337±20</td><td>20±3</td><td>736±95</td></tr><tr><td>32</td><td>5.1 × 10<sup>5</sup></td><td>4.4</td><td>319±10</td><td>21±2</td><td>792±65</td></tr><tr><td>30</td><td>5.4 × 10<sup>5</sup></td><td>3.8</td><td>237±6</td><td>15±2</td><td>534±84</td></tr><tr><td>30</td><td>5.9 × 10<sup>5</sup></td><td>4.1</td><td>286±13</td><td>16±4</td><td>614±59</td></tr><tr><td>17</td><td>6.3 × 10<sup>5</sup></td><td>4.4</td><td>634±116</td><td>16±4</td><td>164±41</td></tr><tr><td>13</td><td>5.9 × 10<sup>5</sup></td><td>3.1</td><td>995±49</td><td>25±1</td><td>172±66</td></tr><tr><td>0</td><td>NA</td><td>NA</td><td>1418 ±170</td><td>27±4</td><td>6 ± 3</td></tr></tbody></table>

ratio were analysed, varying HV mol% from 0 to 32 mol%.

The evolution of the molecular weights of the PHBHV produced shows that they were relatively similar whatever the content in HV and ranged from 5 × 10⁵ and 6 × 10⁵ Eq. PS. Polydispersity index were of 4.1 ± 0.8, which are relatively high but which are current values for this type of biosynthesis [5].

FTIR spectroscopy analysis was performed in order to get access to the crystallinity of the different samples as FTIR is sensitive to the polymer organization [24]. We found a regular decrease of polymer crystallinity to reach about 60 % for the PHBHV containing 30 mol% in HV (Fig. 4). The decrease in crystallinity with increased HV was expected as the introduction of 3HV partially limits the chain crystallization processes and increases the amorphous phase of the copolymer produced.

### 3.3. Thermal properties

Due to the evolving morphology of PHA after casting [29], thermal properties of the formed films were measured between 6 and 13 days after waiting for the stabilization of the morphology. A set of 4 representative samples were chosen at various HV mol% to be analysed in DSC (Fig. 5). Characteristic values are determined from the first heating to observe the effect of the elaboration process on the final properties of the polymers.

The thermograms show that the melting temperature ($T_m$) has important variations between samples. In general, two melting peaks were observed at 162 °C and 172 °C, with melting enthalpies varying significantly between samples. A third $T_m$ is observed at 80 °C, in particular at 32 mol% HV. Melting phenomenon could be very complex; an apparent multiple peak could also be the result of an exotherm peak superimposed on an endotherm peak (crystalline perfection through

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Crystallinity measured by FTIR as absorbance at 1228 cm⁻¹ over absorbance at 1180 cm⁻¹ according to the percent of HV in co-polymer. Error bar is standard deviation over 3 measurements of the same solvent-casted film (P = 0.05).</figcaption></figure>

melting/recrystallization) works [30–32]. The interpretation of the multiple melting peaks is not straightforward and can also be explained by other complex phenomenon occurring in semi-crystalline polymer such as crystal thickening, crystal perfecting or recrystallisation that occur simultaneously with fusion and influenced by the heating rate [33, 34]. However, these separated melting peaks can also result from the presence of two copolymer fractions, which is reflected in the high dispersity index measured in the samples. Monomeric composition was not tested in this sample set but might reveal several polymer types depending on the ratio of LA to glucose and the feeding rate applied, which dictates the polymer chain elongation pattern. Levulinic acid was described to produce both HB and HV monomers by entering two separate metabolic pathways, via the acetyl-CoA and propionyl-CoA. LA contributes then to having a more random repartition of monomer during chain elongation by the enzyme PhaC, which can integrate both 3-hydroxybutyryl-CoA and 3-hydroxyvaleryl-CoA. Feeding pulse rate can also have an impact on monomeric composition distribution as the presence of LA inhibits the metabolic integration of glucose [28]. Because the feeding rate was adjusted manually according to physiological response of the culture as oxygen demand, this might have introduced variations of the monomeric sequence and could explain the variations observed in thermal properties between samples.

Analysis of the DSC curves suggests that the evolution of crystallinity correlates with the results obtained previously in FTIR spectroscopy, which showed a progressive decrease in the crystallinity of PHBHV with the increase in the rate of HV units in the copolymer. These data emphasize the crucial role of the steric hindrance of the lateral chain (HB vs HV) in the thermal properties of the polymer material.

### 3.4. Mechanical properties

Traction tests were carried out on a series of sample between 6 and 9 days after casting to avoid any change in polymer morphology.

A typical strain-stress curve is presented in Fig. 6, where the thermoplastic behaviour of the PHBHV with 30 % in HV is visible, and the mechanical properties are shown in Fig. 7.

As HV mol% increases from 0 to 32 mol%, a decrease in Young's modulus (from ~1400 MPa to ~300 MPa) and tensile strength (from ~27 MPa to ~15 MPa) was observed, as well as a drastic increase in elongation at break (from 5 to over 750 %) (Fig. 7). Due to their high crystallinity of PHB and to a lesser extent to that of PHBHV copolymers with 13 and 17 % in HV, samples exhibited a low elongation at break (6 ± 3 %, 172±41 %, 164±66 % for PHB, PHBHV 13 % and 17 % in HV,
<figure><img src="image_9.png" /><figcaption>Fig. 5. DSC thermograms of the first heating phase of 4 different PHBV films at 0, 13, 17 and 32 HV mol%. All films were obtained with the same fermentation protocol, with varying ratios of LA and glucose, and by the same solvent-casting method.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 6. Typical stress-strain curve of a PHBHV (with 30 % in HV) film.</figcaption></figure>

respectively), together with relatively high Young's modulus (1418 ±170 %, 99±49 %, 634±116 % for PHB, PHBHV 13 % and 17 % in HV, respectively) and high tensile strength (27±4 %, 25±1 %, 16±4 % for PHB, PHBHV 13 % and 17 % in HV). Increasing the HV content to 30 or 32 % resulted in a huge increase of the elongation at break (614±59 % and 792±65 % for PHBHV with 30 and 32 %, respectively). This increase in the elongation at break is associated to a strong decrease in Young's modulus and tensile strength.

Despite the variations in methodology for testing elongation at break, we believe these values of elongation at break are among the highest reported in literature for PHBV from C. necator DSMZ 545, but are not uncommon [5,26,35]. This extreme elongation at break could be due to the film forming method –solvent casting –as films prepared by solvent casting have a specific spherulitic morphology [7]. In addition, publications report presence of P4HV when using levulinic acid as a precursor for PHBV production with C. necator [36], with the use in some cases of mutant strains [37]. The presence of 4HV, even at very low

monomeric composition, could explain the mechanical properties observed. However, like others [38] we were unable to detect it our GC chromatograms. It is hence unlikely that the presence of 4HV in trace concentration would significantly influence the mechanical properties.

The predictable response of tested variables with increased content of 3HV contrasts with the variations observed in DSC. This indicated that monomeric distribution could influence thermal properties more than mechanical ones and that mechanical properties can be well predicted based on HV mol% alone.

As the time between solvent casting and tensile test is relatively short (6–9 days), the shelf life of one of the samples produced (32 HV mol% sample) was tested by casting several films at the same time and measuring the elongation at break every 3–4 weeks for 11 weeks (Fig. 8).

Over the course of 11 weeks, only the Young modulus changed significantly, from an average of 328±19 to 421±11 MPa. Tensile strength and elongation at break did not change significantly between 0 and 11 weeks of testing. This confirms that the tested properties are not due to too early stress assays but to monomeric composition and casting method. It also validates the relatively long shelf life of the co-polymer once purified, which is of primary importance for large scale commercialisation as a stable product is a highly desired feature.

The changes observed in tensile and thermal properties, even at low HV mol% are of great interest for various applications in 3D printing, cosmetics, textile and others. PHB is indeed brittle due to its high crystalline structure which limits its range of applications. Therefore, adjusting the PHB properties by introducing HV units at a controlled ratio holds great potential, as it allows for tuneable elasticity, flex resistance, melting temperature and transparency. This will make it easier for formulation to incorporate of PHA materials and expand their range of applications [2].

## 4. Conclusion

This pioneering work demonstrates that it is possible to produce PHA by developing eco-responsible and ethically acceptable biosynthesis processes. Indeed, the carbon substrates used in this study can be classified among second-generation biomasses (levulinic acid) and third-generation biomasses (microalgae). It is therefore possible to replace
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Tensile assays according to percentage of HV in copolymer (mol%). *a* = Young modulus (MPa), *b* = tensile strength (MPa), *c* = Elongation at break (%). Error bar is standard deviation over 5–7 specimens all made from the same solvent-casted –9 cm diameter –film (*P* = 0.05).</figcaption></figure>

commercial glucose by microalgae starch hydrolysate glucose.

Under these conditions, the PHBHV copolymers obtained exhibit very interesting mechanical characteristics in terms of elasticity when the HV content is significant and exceeds 30 %, as the elongation at break can reach >700 % for copolymers with 32 % in HV. This is a very promising point because it allows to consider improving the elastic PHA properties, the rigidity of commercial PHA being one of their main

<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Evolution of mechanical properties of a 32 % HV mol sample over time. *a* = Young modulus (MPa), *b* = tensile strength (MPa), *c* = Elongation at break (%). Error bar is standard deviation over 5–7 specimen all made from the same solvent-casted –9 cm diameter –film (*P* = 0.05). Films were stored at 20 °C and 50 % humidity in the tensile assay room until tested.</figcaption></figure>
drawbacks.

More broadly, this study opens up many perspectives since it validates the possibility to design tailor-made PHBV by playing on the proportions of HB and HV units and then, to precisely predict the functional PHA properties according to a given specification for an adequate application and end-of-life [9].

CRediT authorship contribution statement

**Gabriel Brouchon:** Writing – original draft, Investigation. **Pablo Alvarez:** Writing – original draft, Project administration, Investigation. **Alexandre Six:** Investigation. **Pierre Lemechko:** Investigation. **Alexandra Dimitriades-Lemaire:** Investigation. **Gatien Fleury:** Investigation. **Jean-François Sassi:** Project administration. **Stéphane Bruzaud:** Writing – review & editing, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was performed within the framework Nenu2PHAr project (https://nenu2phar.eu/), funded by the European Union's Horizon 2020 Research and Innovation programme and the Bio Based Industries Joint Undertaking (BBI-JU) under grant agreement #887474. The BBI-JU receives support from the European Union's Horizon 2020 Research and Innovation programme and the Bio Based Industries Consortium.

Data availability

No data was used for the research described in the article.

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