Poly-R-hydroxyalkanoates (PHAs) are polymers produced by a vast number of bacterial species under stress conditions. PHAs exhibit different thermal and mechanical properties that depend on their molecular structure. In this work, PHAs were produced using avocado oil as the carbon source.
Poly-R-hydroxyalkanoates (PHAs) are polymers synthesized by a large number of bacterial species as a response to unbalanced nutritional conditions [
Many PHAs have main chains formed from monomers with different numbers of carbon atoms. Short-length-chain PHAs (
Renewable carbon sources, such as sucrose, cellulose, and triacylglycerol, have served as substrates for PHA synthesis. Extensive studies have been conducted on the use of inexpensive substrates, including starch, glycerol, soybean oil, sugar cane bagasse, molasses, and activated sludge, to reduce the production cost of PHB [
One organism that has been extensively used in the synthesis of PHAs is
The use of fatty acids, such as those present in vegetable oils, as a carbon source drives the
A simplified representation of fatty acid metabolism in
Mexican avocado
The fatty acids composition in Mexican avocado mainly includes palmitic, stearic, oleic, linoleic, heptanoic, nonanoic, and heptadecanoic acids [
The growth medium contained, per litre of water, 10 g fructose, 1.57 g NH4SO4, 5.66 g NaH2PO4·12H2O, 1.5 g KH2PO4, 0.2 g MgSO4·7H2O, 10 mg CaCl2·2H2O, 20 mg FeSO4·7H2O, and 1 mL of trace element solution (0.3 g H3BO3, 0.2 g CoCl2·6H2O, 0.1 g ZnSO4·7H2O, 30 mg MnCl2·4H2O, 30 mg Na2MoO4·2H2O, 20 mg NiCl2·6H2O, and 10 mg CuSO4·5H2O, in HCl 0.1 N solution); the pH was adjusted to 7.
Commercial Mexican avocado oil for PHA synthesis was obtained from a single batch (the same production number) (Ahuacatlán, Mexico) to ensure a homogeneous chemical composition of the substrate.
Experiments were conducted in duplicate using 200 mL of growth media in 500 mL flasks at 30°C, pH 7.0, in an incubator with rotational agitation at 200 rpm (New Brunswick Innova 4300, USA). A 10% v/v of the seed culture was used to inoculate the growth medium to obtain 0.13 g L−1 (±0.1) of initial biomass (
A fermentation procedure consisting of three different stages was carried out as follows.
Batch cultivation at an initial carbon/nitrogen (C/N) ratio of 14 using the growth medium: carbon depletion in the medium (3 g L−1 of fructose) determined the length of the stage.
A fed-batch stage to increase biomass density at C/N ratio of 6.5: two additions of fructose and ammonium were made. The time of addition was determined as the point when the fructose remaining in the media reached approximately 3 g L−1.
PHA production under nitrogen limitation: avocado oil was added to the culture at the beginning of the stage, at 30 h. Different concentrations were tested: 5, 10, 15, 20, and 25% (v/v).
Control experiments consisted of additional flasks prepared using fructose as the carbon source for the three-stage fermentation.
Fermentation samples were taken every two hours and immediately centrifuged at 10000 rpm for 10 min at 4°C. Fructose and ammonium were analysed in the supernatant, and the bottom pellet (biomass) was washed thoroughly with distilled water before lyophilizing for gravimetric estimation of the dry cell weights (DCW).
Fructose consumption in the fermentation media was quantified using a 3,5-dinitrosalicylic acid (DNS) method [
The intracellular polymer was extracted from the lyophilized biomass using chloroform (1 g of biomass per 50 mL of solvent) at 60°C for 30 min with constant stirring. After incubation, PHA dissolved in the chloroform phase was filtered to eliminate cellular debris and then precipitated with hexane. The residual solvent in the polymer was removed by evaporation [
Dimensionless biomass yield (
Fatty acid methyl esters were derived from acid methanolysis of PHA at 100°C for 4 h by incubating 100 mg of PHA, 2 mL of chloroform, 2 mL of methanol (20% of HCl), and benzoic acid (as an internal standard) in borosilicate glass tubes with screw caps at 100°C for 4 h. After cooling, distilled water was added (1 mL), the tubes were vortexed for 60 s, and the lower phase containing the resulting methyl esters was recovered for analysis [
FTIR was performed within wavenumber ranges from 600 to 4000 cm−1 (BUCK Scientific, model 530, USA). PHA was dissolved in chloroform before pouring the solution onto KBr plates to form the polymer films.
DSC curves were obtained using a differential scanning calorimeter (Mettler Toledo DSC 823) according to López-Cuellar et al. [
The representative profiles of the PHAs synthesized by
Profiles of fructose (grey circles), ammonium (green circles), biomass (orange circles), and poly-R-hydroxyalkanoate (PHA) (blue circles) production during the cultivation of
Stage
Stage
Synthesis of PHAs (Stage
The results of the 50 h, three-stage fermentation are summarized in Table
Final yields of poly-R-hydroxyalkanoates obtained from a three-stage fermentation of
Substrate |
|
|
PHA |
|
PHA | Productivity |
---|---|---|---|---|---|---|
(% v/v) | (g L−1) | (g L−1) | (g L−1) | (%) | (g L−1 h−1) | |
Fructose | — | 5.25 (±0.01) | 4.04 (±0.02) | 1.21 (±0.009) | 76.87 (±1.03) | 0.081 |
|
||||||
Fructose, avocado oil | 5 | 4.45 (±0.02) | 2.64 (±0.03) | 1.82 (±0.012) | 59.21 (±1.12) | 0.053 |
10 | 4.63 (±0.05) | 3.14 (±0.04) | 1.49 (±0.017) | 67.69 (±1.38) | 0.063 | |
15 | 4.74 (±0.04) | 3.27 (±0.03) | 1.47 (±0.013) | 69.04 (±1.43) | 0.065 | |
20 | 4.91 (±0.04) | 3.48 (±0.04) | 1.43 (±0.015) | 70.83 (±1.31) | 0.070 | |
25 | 4.61 (±0.03) | 3.07 (±0.06) | 1.54 (±0.011) | 66.63 (±1.29) | 0.061 |
Conversely, control experiments (0% fed oil) produced 77% of the polymer, almost reaching the typical 80–85% PHA accumulation reported for the strain [
From gas chromatography, the chemical composition of the PHAs was determined using benzoic acid (internal standard) as evaluation base. Representative chromatograms of the evaluated methyl esters are presented in Figure
Chromatogram obtained from gas chromatography (GC) of (a) poly(3-hydroxybutyrate) (PHB) standard, (b) poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) standard with 12 mol% HV content, and (c) polymer produced from 20% v/v of avocado oil (7 mol% of HV). Benzoic acid was used as internal standard.
The most abundant monomer detected in all samples was 3HB monomer ranging from 92.8 to 98.94% for samples fed with avocado oil, as summarized in Table
Thermal properties and chemical composition of the poly-R-hydroxyalkanoates (PHAs) produced in
Substrate |
|
|
|
Monomeric composition of |
||
---|---|---|---|---|---|---|
3HB | 3HV | 3HA | ||||
Fructose | — | 175.15 | 67.05 | 100.00 | — | — |
|
||||||
Fructose, avocado oil | 5 | 173.22 | 56.68 | 98.94 | 1.06 | |
10 | 169.71 | 55.32 | 97.15 | 2.77 | 0.08 | |
15 | 168.22 | 54.29 | 96.64 | 3.27 | 0.09 | |
20 | 159.57 | 51.81 | 92.83 | 7.01 | 0.16 | |
25 | 164.31 | 53.26 | 95.14 | 4.75 | 0.11 |
The spectra recorded from the PHAs, depicted in Figure
Fourier transform infrared spectroscopy (FTIR) spectra of the poly-R-hydroxyalkanoates (PHAs) produced by the addition of avocado oil: (A) 0 (control); (B) 5, (C) 10, (D) 15, (E) 20, and (F) 25 percent [v/v]. (a) 600 to 4000 cm−1, (b) 2500 to 4000 cm−1.
The most prominent peak, located around 1720 cm−1, was related to the ester carbonyl group (C=O). The bands located in the region of 2800 to 3000 cm−1 corresponded to the methyl-methylene groups. The presence of these peaks was due to the symmetric and asymmetric stretching of the CH3 and CH2 groups and these peaks were related to the monomeric units in the lateral chain. Besides the C=O group, an asymmetrical C-H bending vibration in CH3 group shows an absorption band at 1453 cm−1, whereas C-O-H bond shows a peak at 1378 [
The recorded thermograms obtained during the second run of the DSC analysis of the PHAs are shown in Figure
Thermograms obtained during the second run of differential scanning calorimetry (DSC) of the poly-R-hydroxyalkanoates (PHAs) produced by the addition of avocado oil at (a) 0 (control), (b) 5, (c) 10, (d) 15, (e) 20, and (f) 25 percent [v/v].
The thermal properties of the control experiment (0% fed oil) were also estimated and compared against a PHB reference standard.
Different PHAs were synthesized by
Previous flask studies have typically been conducted in batch mode [
Comparison of studies reporting PHAs production in
Strain | Substrate | Scale | Control strategy |
|
Biomass | PHA | PHA | Productivity | Reference |
---|---|---|---|---|---|---|---|---|---|
produced | (g L−1) | (g L−1) | (%) | (g L−1 h−1) | |||||
|
Plant |
Flask | Batch | P(3HB) | 3.6–4.3 | 2.9–3.4 | 79–81 | 0.04–0.05 | Fukui and Doi [ |
|
Plant |
P(3HB-co-3HHX) | 3.5–3.6 | 2.7–2.9 | 76–81 | 0.04 | |||
|
Fructose | Flask | Batch | P(3HB) | 3.4 | n.a | 55 | n.a | Dennis et al. [ |
Palmitate | P(3HB-co-3HV-co-3HHX) | 0.51 | n.a | 58 | n.a | ||||
Oleate | P(3HB-co-3HHX) | 1.44 | n.a | 57 | n.a | ||||
|
Centrifuged fermented organic waste | Flask | Batch | P(3HB-co-3HV) | 2.77 | 1.13 | 40.0 | 0.025 | Ganzeveld et al., [ |
|
Bagasse hydrolysate | Flask | Batch | n.a | 6 | 3.9 | 65 | 0.08 | Yu and Stahl [ |
|
Palm oil | Flask | Batch | P(3HB-co-3HV) | 3.6 | 2.66 | 74 | n.a | Liu et al. [ |
|
|
Flask | Fed batch | P(3PHB) | 5,25 | 4.04 | 76.87 | 0.081 | This study |
Fructose, avocado oil | P(3HB-co-3HV) | 4.45–4.91 | 2.64–3.48 | 59–70 | 0.053–0.07 |
A maximum biomass yield was reached with 20% v/v oil in the medium. Flasks with 25% v/v oil showed a decrease in cellular density and polymer accumulation. This yield decrease could be related to oxygen transfer limitations or a substrate inhibition [
C
In the present study, PHAs were composed mainly of 3HB monomers, followed by 3HV ranging from 1 to 7 mol%, and small quantities of 3HA (more than 5 carbons). Interestingly, all samples fed with avocado oil contained identifiable 3HV monomers and the presence of these monomers was highly correlated with the oil concentration in the medium. In some manner, the particular fatty acid composition of the avocado oil seems to promote the formation of 3HV precursors. Although Ganzeveld et al. [
The thermal properties of the PHAs were enhanced by the presence of 3HV monomers in the polymer. As reported by Babel and Steinbüchel [
Partial results of this manuscript were presented as an abstract at the 9th Congress of FEBiotec (Annual Congress of Biotechnology).
The authors declare that they have no conflicts of interest.
Araceli Flores-Sánchez received grant-aided support from CONACyT (no. 417745). This research was partially funded by CONACyT (CONACyT-INFR-2015-254437 and CONACyT-CB-2014-239553). The authors acknowledge Alba-Flores Joel’s (CINVESTAV) technical assistance during experimental development. Support of Piliado-Hernández D.M. (ITESM) and González-Bret K. during the writing of this manuscript was also appreciated.