Polyhydroxyalkanoates (PHAs) Production from Volatile Fatty Acids (VFAs) from Organic Wastes by Pseudomonas oleovorans

Abstract

This study aimed to investigate the production of polyhydroxyalkanoates (PHAs), a biodegradable polymer from organic wastes by Pseudomonas oleovorans. Volatile fatty acids (VFAs) from acidogenic fermentations of chicken manure (VFAs-CM) and potato peels (VFAs-PP), rich in organic matter majorly acetic (49.9%), butyric (15%) and propionic acids (11.1%) were utilized as substrates for microbial processes. During 72 h of cultivations, samples were withdrawn at intervals and analyzed for cell growth parameters, PHAs accumulation and polymeric properties. The highest biopolymer accumulation (0.39 g PHAs/g DCW) was achieved at 48 h of cultivation from medium containing VFAs-PP as the sole source of carbon. On characterization, the produced biopolymers were shown to be semi-crystalline of carbonyl C=O group. Additionally, thermogravimetric analysis (TGA) showed that the produced biopolymers demonstrated the capability to withstand thermal degradation above prescribed temperatures at which cross-linking isomerization reaction occurs, which is a vital property denoting the thermal stability of biopolymer.

1. Introduction

The use of synthetic polymers for a variety of products is widespread in modern society; however, these polymer products when discarded contribute heavily to pollution due to their persistence and accumulation as solid waste in the environment [1]. Synthetic plastics signify a large fraction of solid waste and its accumulation poses serious concerns in forms of environmental contamination, toxicity to the ecosystem and human health [2]. Poor waste management in landfills causes solid waste leakage into waterways and ocean leading to marine plastic pollution; causing critical environmental issues globally. Hence, production and usage of biopolymer is preferable as a viable alternative to synthetic polymers.

Polyhydroxyalkanoates (PHAs) is a group of biodegradable polymers synthesized by a variety of microorganisms under unbalanced growth conditions as intracellular storage compounds in discrete granular form in their cytoplasm [3]. They possess many advantages compared to synthetic polymers such as hydrophobicity, inertness, thermoplastic- processability, relatively high melting point, and optical purity [4,5]. The possibility of their production from renewable sources by various microorganisms as well as their biodegradability has further made them attractive alternative to synthetic polymers [6,7].

Commercial production of PHAs has raised serious concerns because major PHAs are manufactured by utilizing purified sugars, edible vegetable oils, food crops, etc., thereby competing directly with food supply [8]. In addition, the cost of carbon sources for PHAs production account for up to approximately 50% of the overall production cost, thereby making the cost of PHAs production relatively higher than fossil fuels-derived polymers [9]. Thus, there is a need for utilization of cheap biomass resources and wastes as substrates for PHAs synthesis [8,10]. Furthermore, carbon sources do not only affect the economics of PHAs manufacturing, they also affect the cell growth, productivity, redox potential of cell metabolism, molecular mass, carbon yield, quality, and compositions of polymer [11]. Thus, selection and cost of carbon sources can be considered as the main influencing parameters for commercial-scale PHAs production [1,12].

Potato (Solanum tuberosum) is the fourth largest food crop in the world after rice, wheat and maize, and is a very important part of human diets [13]. Worldwide use of potatoes is increasingly shifting away from fresh towards machined products, and this leads to huge amounts of potato peels (PP) being produced as waste. Additionally, poultry waste generation is increasing daily as a result of exponential growth of livestock industry. Chicken manure is rich in biodegradable organic matter together with nutrients and micronutrients [14]. It is a major source of noxious gases responsible for odor and airborne diseases [15]. Thus, valorization of these wastes as carbon sources constitutes a viable strategy for cost-effective and eco-friendly microbial PHAs synthesis, coupled with simultaneous waste disposal [1].

Anaerobic digestion is a biological process used for processing organic wastes to biogas. The process consists of four main steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis [16]. During the acidogenesis phase, products formed in the hydrolysis phase can be converted to volatile fatty acids (VFAs) and minor products [17,18]. With appropriate methods, VFAs can be converted to value-added products such as alcohols, biohydrogen, microalgal lipids, bioelectricity, aldehydes, ketones, alkanes and polyhydroxyalkanoates (PHAs) [16,18,19,20].

The aim of this study was to investigate production of polyhydroxyalkanoates (PHAs) from volatile fatty acids streams derived from organic wastes by Pseudomonas oleovorans. The work was divided into two stages. In the first stage, synthetic volatile fatty acids (acetic, butyric, propionic, valeric, and isovaleric acid) were used separately as sole sources of carbon for cultivation and growth of Pseudomonas oleovorans to evaluate its potential substrate preferences and feasibility of PHAs accumulation; glucose was used as reference carbon source. The process was then repeated for bacterial cultivation in media containing VFAs-rich streams from previous acidogenic fermentations [12,21] of chicken manure and potato peels as carbon sources in pure cultures. Biomass growth, cell dry weight and PHAs accumulation was investigated in both cultures. Furthermore, the produced biopolymers from both media constituents were subjected to characterization using FTIR, DSC and TGA for functional groups and thermal evaluation.

2. Materials and Methods

2.1. Bacteria Strain Cultivation

Pseudomonas oleovorans ATCC 29347 (American Type Culture Collection, Manassas, VA, USA) was used for this study. The bacterial culture was preserved in Lysogeny broth (LB) agar plates composed of (in g/L) agar 15, tryptone 10, NaCl 5, and yeast extract 5. New plates were prepared monthly by streaking method and followed by incubation at 30 °C for two days and storage at 4–6 °C until use. Cultivation was performed according to [22]. Minimal salts medium and volatile fatty acids were con, stituted in 250 mL Erlenmeyer flasks to make 100 mL cultivation medium with final carbon source concentration of 5 g/L in each flask and then sterilized at 121 °C for 15 min, allowed to cool and inoculated with one loopful each of bacteria. Flasks were then transferred to gyratory incubator shaker (New Brunswick Scientific Co., Enfield, CT, USA) for cultivation at 30 °C, pH 7.0 and 121 rpm for 72 h. Experiments were conducted in triplicates and samples were withdrawn for analysis at 24 h intervals. The remaining total flask medium volumes were used for cell mass quantification, PHAs extraction, quantification and characterization at 24 h, 48 h and 72 h of cultivation.

2.2. Minimal Salt Medium (MSM) and Substrate Preparation

Minimal salt medium (MSM) used in this study was prepared according to [23], with the following composition (in g/L): Na₂HPO₄·2H₂O 4, KH₂PO₄ 3.6, (NH₄)₂SO₄ 3, MgSO₄·7H₂O 0.5, CaCl₂·H₂O 0.05, NaCl 0.02, carbon source 5, trace metals, and vitamins. The vitamin solution (added at 1 mL/L) contained 0.05 g/L of d-biotin, 0.2 g/L of p-aminobenzoic acid, 1 g/L of nicotinic acid, 1 g/L of Ca-pantothenate, 1 g/L of pyridoxine, 1 g/L of thiamine, and 25 g/L of m-inositol, while the trace metal solution (added at 1 mL/L) contained 3 g/L of EDTA,0.9 g/L of CaCl₂·H₂O, 0.9 g/L of ZnSO₄·7H₂O, 0.2 g/L of H₃BO₃, 0.19 g/L of MnCl₂·4H₂O, 0.08 g/L of Na₂MoO₄·2H₂O, 0.06 g/L of CoCl₂·2H₂O, 0.06 g/L of CuSO₄·5H₂O, and 0.002 g/L of KI [23]. Glucose and synthetic VFAs (SVFAs) media were prepared separately and tested as the sole sources of carbon for cultivation. All media were autoclaved (Systec, Linden, Germany) at 121 °C for 20 min.

2.3. Substrate Preparation

Glucose and synthetic VFAs (acetic acid, butyric acid, propionic acid, valeric acid and isovaleric acid) were prepared separately and tested separately as sole sources of carbon for cultivation. All media were autoclaved (Systec, Linden, Germany) at 121 °C for 20 min. The VFAs-rich streams used in this study: (i) VFAs-rich stream from chicken manure; (VFAs-CM) and (ii) VFAs-rich stream from potato peels; (VFAs-PP) were obtained from previous acidogenic fermentation [3,21] of chicken manure and potato peels separately in immersed membrane bioreactors. The VFA-rich streams were sterilized and analyzed for their composition using gas chromatograph (GC-FID; Clarus 590, Perkin Elmer) and high- performance liquid chromatograph (HPLC) (Waters Corporation, Milford, CT, USA), respectively. Ammonium nitrogen (NH₄⁺-N) was analyzed using the Ammonium 100 test kit (Nanocolor, MACHEREY-NAGEL GmbH & Co. KG, Dueren, Germany). These VFAs-rich streams mixed with minimal salt medium were constituted separately into defined cultivation media as sources of carbon for growth of Pseudomonas oleovorans ATCC 29347. Substrate optimization was done for all carbon sources (evaluating 1, 2, 5 and 10 g/L substrate composition) and the optimum substrate concentration was employed for subsequent cultivation media. Cultivation medium in all experiments was adjusted to pH 7.0 by addition of 2 M NaOH or 2 M HCl.

2.4. Analytical Methods

Substrate consumption from cultivation medium was evaluated by high-performance liquid chromatograph (HPLC) (Waters Corporation, Milford, CT, USA) using a hydrogen-based column (Aminex HPX87, BioRAD Laboratories, Munich, Germany), H₂SO₄ (5 mM) as eluent at a rate of 0.6 mL/min, and a refractive index detector (Waters Corporation, Milford, CT, USA). For carbon consumption in VFAs streams, an ultraviolet (UV) absorption detector operating at 210 nm wavelength (Waters 2487, Waters Corporation, Milford, CT, USA) was used in series with the refractive index detector. All samples were kept in 2 mL Eppendorf tubes, centrifuged (Fresco 21) and then filtered (using 0.2 µm pore size filters) into vials for HPLC analysis.

For biomass growth, one mL sample was withdrawn from the cultivation medium at specified intervals and analyzed in the spectrophotometer at a wave length of 600 nm for optical density and cell growth (OD) measurement. For cell dry weight, bacteria cells were harvested by centrifugation at 10,000× g for 10 min (Megafuge 8, Thermo Fisher Scientific GmbH, Dreieich, Germany). The cells were then washed three times with distilled water, followed by centrifugation and drying to constant weight in an oven at 70 °C (TERMAKS, Bergen, Norway). Dry cell weight (DCW) is then reported as grams of cells per liter of medium (g/L) and as grams of cells per gram (g/g) of consumed carbon source, respectively.

PHAs extraction from cultivation medium was performed according to [24]. Bacterial cells were first collected from medium by centrifugation at 10,000× g for 10 min, followed by rinsing three times with distilled water, and then drying in the oven. The dried cells were dissolved in chloroform in 250 mL screw-capped borosil bottles and incubated at 40 °C for 36 h with vigorous agitation. The contents of the bottles were then filtered using Whatman filter paper to remove the cell debris. The chloroform solution containing dissolved polymer was then transferred to 250 mL flask and precipitated by adding cold absolute ethanol. The precipitate was then kept at 4 °C for 1–2 h. Final PHAs recovery was done by centrifugation at 14,000× g for 10 min at 4 °C. Extracted PHAs were placed in pre-weighted aluminum cups and dried to constant weight in an oven at 70 °C (TERMAKS, Bergen, Norway). The percentage PHAs based on dry cell weight was quantified by Equation (1).

$$\mathrm{PHAs}\%=\frac{\mathrm{PHAs}\mathrm{weight}}{\mathrm{Cell}\mathrm{dry}\mathrm{weight}}\times 100$$

(1)

Fourier Transform Infrared spectroscopy (FTIR) analysis was conducted on the extracted PHAs samples for functional groups by directly scanning the samples 32 times in the mid-infra-red region in a spectrum range of 400 to 4000 cm⁻¹ at resolution 4 cm⁻¹ using Nicolet OMNIC 4.1 software. The obtained spectra were then analyzed by Essential FTIR (eFTIR, Madison, WI, USA).

Thermal properties of the extracted PHAs sample were determined by differential scanning calorimeter (Q500 TA instruments, Waters LLC, New Castle, DE, USA). Three milligrams of each sample were heated from −40 °C to 225 °C at a rate of 10 °C/min. Measurement was conducted in triplicate under nitrogen atmosphere. The crystallinity of extracted biopolymer [25] was calculated using Equation (2);

$${\mathrm{X}}_{\mathrm{c}}=∆{\mathrm{H}}_{\mathrm{m}}/{\mathsf{\Delta }\mathrm{H}}_{\mathrm{PHB}}^{\mathrm{o}}$$

(2)

where, X_c = crystallinity of extracted biopolymer. Δ${\mathrm{H}}_{\mathrm{m}}$ = measured enthalpy of melting of extracted polymer (in J/g), ${\mathsf{\Delta }\mathrm{H}}_{\mathrm{PHB}}^{\mathrm{o}}$ = enthalpy of melting of pure PHB crystals (146 J/g).

Degradation temperature (Td) of extracted PHAs was determined using Thermogravimetric analyzer (TGA, Mettler Toledo stare TGA/DSC 1, Q500 TA instruments, Waters LLC, New Castle, DE, USA) at a ramp of 20 °C/min in nitrogen atmosphere from 30 to 500 °C. The weight curve was analyzed at different stages for degradation parameters such as drying, volatility and sublimation [16].

3. Results

In this study, production of biodegradable polymer from synthetic volatile fatty acids as well as volatile fatty acids derived from organic wastes by Pseudomonas oleovorans was studied. Results of substrate concentration optimization showed that carbon concentration of 5 g/L was the optimum. This concentration was employed for further cultivation in all media tested. The produced biopolymers were later characterized using FTIR, DSC and TGA analyses.

3.1. Substrate Consumption, Cell Growth and PHAs Accumulation in Synthetic VFAs Media

In the first stage of this work, synthetic volatile fatty acids (acetic, butyric, propionic, valeric and isovaleric acid) (SVFAs) were constituted separately into defined media as the sole sources of carbon for cultivation and growth of Pseudomonas oleovorans ATCC 29347 to evaluate its substrate preferences, and feasibility of PHAs accumulation; glucose was used as a reference carbon source (Figure 1a).

The pH profiles of the cultivation media showed an increasing trend generally from the initial pH value (7.0) at the beginning of cultivations, except for propionic acid medium where downward trend was observed (Figure 1b). For biomass growth, maximum values for cell accumulation were observed in all media at 48 h of cultivation, while further increase in fermentation time led to lower values (Figure 1c). The resulting DCW and PHAs followed the trend (1.45, 0.95, 0.85, 0.65, 0.52, 0.35 g/L) for DCW (

Table 1. Dry cell weight and PHAs accumulation by P. oleovorans in synthetic VFAs media.

Substrates	Dry Cell Weight (g/L)			PHAs (g/L)			PHAs Content (g/g)			PHAs Content (%)
Time (h)	24	48	72	24	48	72	24 h	48	72	24, 48, 72
Glucose	0.53	1.45	0.96	0.15	0.16	0.10	0.28	0.11	0.10	28, 11, 10
Acetic	0.42	0.95	0.79	0.11	0.12	0.09	0.26	0.13	0.11	26, 13, 11
Butyric	0.36	0.85	0.72	0.08	0.11	0.06	0.22	0.12	0.08	22, 12, 8
Propionic	0.15	0.35	0.18	0.02	0.03	0.01	0.13	0.09	0.06	13, 9, 6
Valeric	0.21	0.52	0.38	0.04	0.07	0.03	0.19	0.13	0.08	19, 13, 8
Isovaleric	0.28	0.65	0.46	0.06	0.10	0.05	0.21	0.15	0.11	21, 15, 11

Abbreviation: PHAs = Polyhydroxyalkanoates; SVFAs = Synthetic volatile fatty acids.

and Figure 2a) and (0.28, 0.26, 0.22, 0.21, 0.19, 0.09 g/g) for PHAs (Table 1 and Figure 2b) in glucose, acetic, butyric, isovaleric, valeric and propionic acids media, respectively. This showed that glucose was the most preferable substrate for growth and PHAs accumulation compared to other carbon sources.

3.2. Substrate Consumption, Cell Growth and PHAs Accumulation in VFAs-Rich Streams Media

The process was repeated for Pseudomonas oleovorans cultivation in media containing VFAs-rich streams obtained from previous acidogenic fermentations of chicken manure (VFAs-CM) and potato peels (VFAs-PP) as sole sources of carbon in pure cultures. The VFAs-rich streams were analyzed for metabolites composition and the results showed that acetic, butyric and propionic acids dominated the streams composition (

Table 2. Composition of VFAs-rich streams used in this study.

Components	VFAs-CM (g/L)	(%)	VFAs-PP (g/L)	(%)
Acetic acid	4.789	50.82	5.718	67.85
Butyric acid	1.529	16.22	1.092	12.96
Isobutyric acid	0.0025	0.027	0.0078	0.093
Propionic acid	1.066	11.31	1.005	11.92
Valeric acid	0.069	0.73	0.038	0.45
Isovaleric acid	0.268	2.84	0.167	1.98
(NH4+-N)	1.700	18.04	0.400	4.75

).

The pH profiles of the cultivation media were in the range (7–10) (Figure 1d). The initial pH of cultivation media was set at 7.0 at the start of fermentation, and samples were withdrawn every 24 h intervals for pH measurement (

Table 3. Dry cell weight and PHAs accumulation by Pseudomonas oleovorans at varied concentrations of VFAs-rich streams used in this study.

Substrates	Dry Cell Weight (g/L)			PHAs (g/L)			PHAs (g/g)			PHAs (%)
Time (h)	24	48	72	24	48	72	24	48	72	24	48	72
VFAs-CM(g/L)	0.45	1.03	0.32	0.12	0.29	0.10	0.27	0.28	0.31	27	28	31
3	0.75	1.16	1.02	0.23	0.38	0.17	0.31	0.33	0.17	31	33	17
5	1.24	1.64	1.45	0.20	0.58	0.21	0.16	0.35	0.14	16	35	14
VFAs-PP (g/L)	0.56	1.19	0.61	0.13	0.32	0.12	0.23	0.27	0.20	23	10	20
3	1.1	1.62	1.3	0.11	0.39	0.25	0.01	0.24	0.19	10	24	19
5	1.4	2.11	1.82	0.28	0.82	0.18	0.21	0.39	0.10	20	39	10

Abbreviations: PHAs = Polyhydroxyalkanoates; VFAs-CM = Volatile fatty acids stream from Chicken manure. VFAs-PP = Volatile fatty acids stream from Potato peels.

).

Substrate Consumption in Cultivation Media at Varied Concentrations

For media containing VFAs-CM as sole sources of carbon, total substrate consumption except propionic acid was observed in all medium at 72 h cultivation (Figure 3a–c). The effects of varied VFAs concentrations on cell growth and PHAs accumulation were tested. For media containing VFAs-PP as sole sources of carbon, rapid and total substrate consumption was observed at substrate concentrations 1 and 3 (g/L) before 72 h (Figure 3d,e), while at substrate concentration 5 g/L, substrate (majorly propionic acid) remained in the cultivating medium at 72 h cultivation (Figure 3f).

3.3. PHAs Characterization

After drying, extracted biopolymer films were subjected to FTIR analysis for spectra evaluation and function groups (Figure 4).

Extracted biopolymers were subjected to differential scanning calorimetry (DSC) analysis for melting temperatures determination. DSC thermograms and thermal parameters data were presented in Figure 5a–d and

Table 4. Comparison of DSC data for produced biopolymers with commercial PHB.

Polymer Sample	Tm °C	ΔHm	Xc	Suspected PHBV
Commercial PHB	165.57	93.44	0.64	PHB
PHAs fromSVFAs	113.84	75.92	0.52	PHB8V
VFAs-CM	152.70	99.28	0.68	PHB5V
VFAs-PP	152.92	94.9	0.65	PHB5V

Abbreviations: PHAs = Polyhydroxyalkanoates; SVFAs = Synthetic volatile fatty acids, VFAs-CM = Volatile fatty acids stream from Chicken manure; VFAs-PP = Volatile fatty acids stream from Potato peel, T_m = Melting temperature of extracted biopolymer, ΔH_m = enthalpy of melting of biopolymer, X_c = Crystallinity of biopolymer calculated using ΔH_m.

.

Extracted biopolymers were further subjected to TGA analysis for degradation and thermal stability evaluation. Data for degradation parameters and TGA spectra are presented in

Table 5. Thermogravimetric analysis (TGA) data of produced PHAs in this study.

Sample	DehydrationTemperature (°C)	Moisture Loss (Dehydration) (%)	Volatiles Loss (°C)	Volatiles Mass (%)	Thermal Degradation Td (°C)	Mass Loss (%)	Inert Residue (%)
Commercial PHB	-	-	-	-	271.94	58.46	10.67
PHAs from SVFAs	138.80	6.2	382.75	7.78	382.75	84.03	2.17
PHAs from VFAs-CM	114.10	1.33	364.68	1.08	364.68	79.47	8.4
PHAs from VFAs-PP	100	-	256.33	50.02	385.50	44.89	-

PHAs = Polyhydroxyalkanoates; SVFAs = Synthetic volatile fatty acids; VFAs-CM = Volatile fatty acids stream from Chicken manure; VFAs-PP = Volatile fatty acids stream from potato peels.

and Figure 6.

4. Discussion of Results

In this present study, production, characterization and evaluation of PHAs by Pseudomonas oleovorans using volatile fatty acids were investigated. Substrate optimization was studied to evaluate optimum substrate concentration among the varied concentrations tested (1 g/L, 3 g/L, 5 g/L and 10 g/L). Result showed that carbon concentration 5 g/L was the optimum and this was adopted for all media preparations. In all media tested, Pseudomonas oleovorans utilized glucose and synthetic fatty acids as sources of carbon for cell growth, although with variations in rates of carbon consumption. In synthetic media, glucose was observed to be the most preferable substrate followed by acetic, butyric, valeric, and isovaleric acid, with the least consumption in propionic acid (Figure 1a).

For dry cell weight (DCW) and PHAs accumulation, Pseudomonas oleovorans gave greater cell and PHAs accumulation in glucose medium compared to other synthetic acids media tested (Figure 2a,b). For bacteria cultivation in media containing volatile fatty acids rich streams from chicken manure (VFAs-CM) and volatile fatty acids rich stream from potato peels (VFAs-PP) as sole sources of carbon, Pseudomonas oleovorans accumulated higher dry cell weight (DCW) and PHAs in medium containing VFAs-PP as the sole carbon source (Figure 2c,d). In all media tested, it was observed that PHAs accumulation started at the log phase and maximum synthesis of PHAs was reached at stationary phase of growth. PHAs then decreased after the stationary phase due to intracellular utilization of the PHAs as energy and carbon reserves (Figure 1b,d).

Comparison of PHAs yield by Pseudomonas oleovorans in this study with PHAs/PHB yield by Pseudomonas oleovorans and other bacteria in previous studies utilizing various waste streams as sole sources of carbon showed that Pseudomonas oleovorans cultivation on volatile fatty acids from organic wastes as sole sources of carbon gave greater PHAs yield (

Table 6. Comparison of PHAs/PHB yield from various substrates.

S/N	Microorganism	Substrate	Mode	PHA/PHB Yield (%)	Reference
1	P. oleovorans	Glucose	Batch	28	This study
		VFAs from Potato peels	Batch	39	This study
		VFAs from Chicken Manure	Batch	35	This study
2	P. oleovorans	Jatropha Curcas Oil	Batch	26	[26]
3	P. oleovorans	Glucose	Batch	6.8	[27]
4	P. oleovorans	Sodium Octanoate	Batch	13	[8]
5	P. oleovorans	n-alkanoic acid	Batch	30	[18]
6	C. necator	Glucose	Fed-batch	15	[28]
7	C. necator	Waste Glycerol	Fed-batch	14	[28]

).

The pH profiles of the cultivation media showed an increasing trend generally from the initial pH value (7.0) at the beginning of cultivations in all media except in propionic acid medium where a downward trend was observed (Figure 1c). The acidic trend in medium containing propionic acid as the sole carbon source could be responsible for low biomass growth, CDW and PHAs accumulation in propionic acid medium (Figure 2a,b). Thus, it can be deduced that pH range greater than 7.0 favored increased cells growth and PHAs accumulation.

Ammonium analysis of the VFAs-rich streams showed that VFAs-PP contained 470 mg/L ammonium while VFAs-CM contained 1700 mg/L (Table 2). Based on CDW and PHAs accumulation, it can be deduced that reduced ammonia composition of volatile fatty acids stream from potato peels favored cell growth, cell dry weight and polyhydroxyalkanoates (PHAs) accumulation in media containing VFAs-PP as the sole source of carbon compared to media containing VFA-CM as the sole source of carbon. Additionally, it was observed that since pH of the cultivation media was not controlled throughout the cultivation process, NH₄⁺-N composition of the media streams may account for the observed pH range (7–10) of the cultivation media (Figure 1e). Hence, it can be deduced that alkaline pH favored cell growth and PHAs accumulation (Figure 2c,d).

FTIR spectra of all extracted PHAs showed strong absorption near 1600 cm⁻¹ and 3200–3500 cm⁻¹, which can be attributed to carbonyl C=O group and hydroxyl O-H groups stretching. Other common stretches of CH₃, CH₂, CH=CH, C-C, N-H stretch and CH groups were observed at the peaks 3020 cm⁻¹, 1450–1600.76, and 2800–2900 cm⁻¹. The vibrational band in the range 1410–1446 cm⁻¹ was due to –CH bending, while the peak at 1049 indicates stretching of C-O in the presence of ester bond. The functional groups of extracted PHAs around 1600 cm⁻¹ was identified as carbonyl group, attributed to stretching of C=O bonds not only on carboxylic groups, but also in ester groups [3]. This indicated formation of ester linkages involving carboxylic and hydroxyl groups of polymer chains. These findings were similar to those by previous researchers [27,29,30] on polyhydroxyalkanoates production by Pseudomonas oleovorans cultivated in various medium. All extracted PHAs showed crystallinity band near 1180 cm⁻¹ (C-O-C). This substantiated the identification of extracted PHAs as carbonyl C=O group (Figure 4).

DSC analysis is used for evaluating melting temperatures (T_m) of materials. In this study, DSC thermograms (Table 4 and Figure 5a) showed melting peaks of commercial PHB between 160 and 170 °C indicating characteristics peak of homopolymer, while PHAs extracted from synthetic VFAs and VFAs-rich streams showed melting points lower than 160 °C, characteristic of copolymers [31]. Melting temperatures (T_m) 113.84 °C, 152.70 °C and 152.92 °C were obtained for biopolymers extracted from synthetic medium, VFAs-CM and VFAs-PP, respectively (Table 4 and Figure 5b–d). This is an indication of the incorporation of HV units in the produced biopolymer chain [32]. Furthermore, the melting points of produced biopolymers in this study were similar to melting temperature values of PHAs and PHB produced in previous studies [32,33], where melting temperatures 129 °C, 151 °C and 145 °C were recorded. It has to be noted that synthetic polymers used for general purpose engineering and high temperature specialized products have melting temperatures around 100, 150 and 300 °C, respectively [30,34]. Thus, the produced biopolymers in this study can be utilized for general purpose engineering usage.

Furthermore, the double melting points presented by VFAs-PP is a confirmation of its copolymer property [3]. According to [35], multiple melting behavior of a polymer is usually linked either to the process of melting or to melting of crystals with different lamella thickness; and/or different crystal morphology for PHB and its copolymers with various hydroxy valerate content [1]. Based on calculated DSC data from this study (Table 4), produced biopolymers in this study can be categorized as PHB8V, PHB5V and PHB5V for PHAs extracted from SVFAs, VFAs-CM and VFAs-PP, respectively [5]. Additionally, calculated values of crystallinity based on enthalpies of melting, ΔH_m (Equation (2)) 0.64, 0.52, 0.68 and 0.65 for commercial PHB, biopolymers extracted from SVFAs, VFAs-CM and VFAs-PP, respectively (Table 4), confirmed the produced biopolymers to be semi-crystalline in nature.

Thermogravimetric analysis provides information relating to thermal stability performance and weight loss of polymers under varying degradation temperatures. Degradation of PHAs took place in three well define steps. Step 1; weight loss due to moisture, step 2; weight loss due to volatile material loss and step 3; weight loss due to maximum degradation [10]. For biopolymer extracted from synthetic VFAs, weight loss occurred in three steps; (i) from 100–138.80 °C, moisture loss (6.2%), (ii) from 138.80–382.75 °C, volatile materials loss (7.78%) and (iii) at 382.75 °C, thermal degradation occurred till 454.09 °C leaving ash residue of 2.17% (Table 5 and Figure 6b).

For biopolymers extracted from VFAs-CM, weight loss occurred in three steps; (i) from 100–114.10 °C, moisture loss (1.33%), (ii) from 114.10–364.68 °C, volatile materials loss (1.08%), and (iii) at 364.68 °C, thermal degradation occurred till 434.35 °C, leaving an ash residue of 8.4% (Table 5 and Figure 6c). In the case of biopolymer extracted from VFAs-PP, double melting peaks were observed at 256.33 °C and 387.50 °C, after which maximum thermal degradation occurred till 434.28 °C, leaving no ash residue (Table 5 and Figure 6d). The observed weight loss in all produced biopolymers during TGA analysis can be explained by cross-linking isomerization reactions, which confirms thermal stabilities of the biopolymers. If cross linking reactions occurred during thermal degradation of PHAs, an exothermic peak would be detected in the thermogram at temperatures around 431 °C [2]. This is a vital property required for specialized applications of biopolymers for specialized usage. This is because a major barrier to some applications of PHAs was thermal instability due to an inability to cross-link. The produced biopolymers in this study showed this vital property, confirming their suitability for specialized usage. Information such as moisture content, thermal decomposition, melting and degradation temperatures are essential for handling of polymer-based products.

5. Conclusions

A biodegradable polymer was produced using VFAs-rich streams from organic wastes by Pseudomonas oleovorans. The highest biopolymer accumulation (0.39 g PHAs/g CDW; 39%) was achieved within 72 h of cultivation in media containing VFAs from potato peels, followed by (0.35 g PHAs/g DCW) in VFAs from chicken manure as sole sources of carbon compared to accumulation (0.28 g PHAs/g DCW; 28%) in glucose medium. The results of FTIR analysis showed the functional group of produced biopolymers as carbonyl C=O group. Thermal analyses (DSC and TGA) showed the produced biopolymers as semi-crystalline possessing thermal stability with capacity for cross-linking isomerization. This study demonstrated the potentials of VFAs from organic wastes as cheap sources of carbon for biopolymer production, which can be explored for industrial-scale processing.