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.
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).
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, NH
4+-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 CH
3, CH
2, 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.