Impact of Different Bacterial Strains on the Production , Composition , and Properties of Novel Polyhydroxyalkanoates Using Crude Palm Oil as Substrate

Polyhydroxyalkanoates (PHAs) are a group of biodegradable polymers produced from renewable sources by prokaryotic biocatalysts, accumulated intracellularly for energy and carbon storage. In the present study, production and characterization of PHAs synthetized by Cupriavidus necator (IPT 026 and IPT 027) and Burkholderia cepacia (IPT 119 and IPT 400) were evaluated using crude palm oil (C16:0 = 26.44 %, C18:1 = 54.50 %, C18:2 = 13.41 %) as substrate (15 g L –1 crude palm oil, pH 7.0, 180 rpm, 72 h). All strains were able to synthesize novel PHA copolymers (0.10–1.45 g L–1), and IPT 027 displayed the highest production. Copolymers monomeric composition (Mw = 173.78–389.30 kDa) was comprised mostly of hydroxyhexadecanoate (41.43–53.15 %) and hydroxy-9-octadecenoate (14.91–29.61 %). PHAs were predominantly amorphous, showed low polydispersity, and good thermal stability (Tonset ≥ 283 °C), which increased proportionally to crystallinity. Crude palm oil constitutes an emerging alternative for PHAs production, and microorganism strains strongly affect polymer accumulation, monomeric composition, molar mass, and properties.


Introduction
Polyhydroxyalkanoates (PHAs) are a group of bio-polyesters that have been largely studied due to their physicochemical characteristics and biodegradability.Their properties are comparable to those exhibited by traditional synthetic polyesters (petroleum based) and they are decomposed by naturally occurring prokaryotes, such as archaea and bacteria, into carbon dioxide and water 1,2,3 .
The biopolymers synthesis occurs by numerous prokaryotes, intracellularly.PHAs yields and properties are very dependent on the synthesizer microbe, commonly, a feast/famine feed strategy is used to enrich PHA accumulation within the producer microorganism cells, stimulating growth phase (feast period) and then shifting to favor polymer accumulation (famine period), where limitation of at least one nutrient necessary for cell multiplication (such as N, P, Mg or Fe) is applied 4,5 .
High costs of PHAs production is a limiting factor to their applications and competitiveness against synthetic plastics.Therefore, these expenses should be reduced to enhance economical sustainability.An approach to attenuate this problem is to use alternative and cheap substrates for the bioconversion, since customary ones such as sucrose, glucose and starch represent a major expense in the biomaterials production chain, among other problematic matters associated with their use 6,7 .
Substrates such as sugarcane molasses, fermented cheese whey, hemicellulose hydrolysates, and industrial wastes are being reported as alternative low-cost carbon sources for PHA production 8,9,10 .Additionally, plant oil biomass and its related products have been reported to be very suitable carbon sources for PHA synthesis using a variety of microorganisms, revealing to be better substrates for the polymer production than sugars, carbon sources more traditionally used 11,12,13,14 .
Plant oils contain higher carbon content per weight than sugars, suggesting a PHA yield of at least two times higher 15 .They are catabolized via β-oxidation cycle to produce polymers of different chain lengths 12 .The fatty acids are first converted to enoyl-CoA in the mentioned cycle, and then transformed to R-3-hydroxyacyl-CoA, polymerization precursor, by R-3-hydroxyacyl-CoA hydratase.Lastly, PHA polymerization is catalyzed by PHA synthase 16,17 .
Palm oil is one of the most important crops of agricultural oil production in the world, since its yield is about five times greater than that of oilseeds, such as soybean and rapeseed, per unit of planted area 18 .
Although the palm oil industry causes some controversies due to its impact on the environment and ecosystems, the rising and unyielding pressure of population growth and the need for food, biofuels and bioplastics motivate an immediate step for good management practices of environmentally friendly operations (plantation and milling) while intensifying the oil yields for a more sustainable production and fulfilling the future demand 19,20 .
In this context, the present study aimed to produce and characterize PHA using crude palm oil (Elaeis guineensis) as an alternative substrate for the submerged cultivation of whole cell biocatalysts Burkholderia cepacia (IPT 119 and IPT 400) and Cupriavidus necator (IPT 026 and IPT 027), intending to implement more viable biotechnological methods following market demand for biopolymers with suitable technological properties.

Bacterial strains
Cupriavidus necator IPT 026 and IPT 027, and Burkholderia cepacia IPT 400 and IPT 119 were supplied by the Institute for Technological Research (IPT), in São Paulo, Brazil.The bacteria were grown at 33 °C in nutrient broth, maintained on nutrient agar (NA) at 4 °C, and transferred to new plates every 15 days.

Main carbon source
Crude palm oil (Elaeis guineensis) was acquired in the trade market of Salvador-BA, Brazil and stored at 4 °C, protected from light, for preservation.

Chemical composition of the substrate
Total carbon (C) content was determined according to Nelson and Sommers 21 publication.Fatty acid profile was determined by the capillary column gas chromatographic method according to Joseph and Ackman 22 and Nascimento et al. 23 Separation of the methyl esters in the fatty acids was performed using gas chromatography (Varian 3800) with a flame ionization detector (GC-FID) and a fused silica gas chromatography capillary column EliteWAX (30 m × 0.32 mm × 0.25 μm).Quantification of fatty acids, expressed in milligrams per 100 g sample, was performed by the addition of an internal standard (C23:0 Sigma®, USA) according to Joseph and Ackman 22 and calculated using Eq.(1).

Concentration
where: AFA = area of fatty acid methyl ester peak in the chromatogram of the sample; MIS = weight (in milligrams) of the internal standard added to the sample; F = correction factor of fatty acid methyl ester to fatty acid; CTL = percentage composition of total lipids from the sample; AIS = area of internal standard fatty acid methyl ester peak in the chromatogram of the sample; M = sample mass (in milligrams); FFA = correction factor response of each fatty acid methyl ester ionization detector, relative to C23:0.

Shaker flask cultivation
Tests were executed in triplicate in 250-mL flasks containing 50 mL of nutrient broth medium and 2 mL of pre-culture inoculum, which was incubated at 30 °C in a shaking flask without baffles for 24 h (best cell concentration, 1011 UFC mL -1 ) at 150 rpm.FC cultivations were executed in flasks containing 80 mL nitrogen-non-limited mineral me-dium with addition of 10 % v/v of the inoculum culture and incubated at 30 °C and 150 rpm in orbital shaker for 24 h.SCs were run in flasks containing 80 mL nitrogen-limited mineral medium with the addition of 10 % v/v of FC and incubated at 35 °C and 180 rpm in orbital shaker for 72 h.

PHA recovery and separation
Cell cultures were harvested by centrifugation at 15,700 ×g for 30 min at 5 °C (HITACHI, model CR 22G), washed twice with distilled water, transferred into round bottom flasks (50 mL), and frozen at -80 °C for subsequent lyophilization (LIOBRAS model L101) at -42 °C for 24 h.PHA extraction from the freeze-dried cells was performed using chloroform at 60 °C for 2 h with vigorous stirring on a magnetic stirrer plate with heating (model IKAHS 7) 25 .Biomass and PHA production (obtained after extraction) were calculated using a gravimetric method and expressed in g L -1 .

Fourier transform infrared spectroscopy (FTIR)
PHA functional groups characterization was determined by FTIR spectroscopy (PerkinElmer Spectrum 100, Waltham, Massachusetts, USA) between the wave numbers of 4000 cm −1 and 400 cm −1 using a single-bounce attenuated total reflection (ATR) accessory with a Zinc selenide (ZnSe) crystal.

Thermal characterization
Thermogravimetric analysis (TGA) (Perkin-Elmer Model Pyris 1TGA Waltham, Massachusetts, USA) was performed to determine the initial degradation temperature (T onset ) and the maximum decomposition temperature (T max ).Five milligrams of PHA were placed in a platinum tray (cross-sectional area of 2.47 • 10 -5 m 2 ) and heated at the rate of 10 °C min -1 from 25 °C to 600 °C under a nitrogen flow rate of 40 mL min -1 .

X-ray diffraction analysis
Crystallinity and crystal peaks of the PHA sample were measured by X-ray diffraction.The X-ray diffractograms of the samples were obtained on a SHIMADZU (XRD-6000, USA) with graphite-filtered CuKa radiation (λ = 1.5433Å) operated at 40 kV and 30 mA in the region from 5 to 80° (2θ) at a rate of 2° min -1 .The percentage of crystallinity was calculated from the diffracted intensity measured by XRD according to the Vonk's method 26 .

Determination of PHA molar mass distribution
Molar masses, expressed by the weight average molar mass (M w ), the number average molar mass (M n ) and the polydispersity index (PDI = M w /M n ), were obtained by size-exclusion chromatography (SEC) according to Campos et al. 25 and Ribeiro et al. 27 High performance liquid chromatography (HPLC, PerkinElmer 200) with an autosampler and refractive index detector (PerkinElmer), a column Shodex KD 807 (30 cm × 78 mm × 5 μm) with an exclusion volume of 2 × 10 8 and an oven temperature of 35 °C were employed for separation.The polymer samples were dissolved in chloroform to a concentration of 7 mg mL −1 .In the mobile phase, chloroform was employed at 1 mL min −1 .A standard curve was created using polystyrene standards with a range size of 68-1,670,000 g mol −1 (Polystyrene High Mw Standards Kit Polymer Standards Service, USA).

PHA composition
The composition of the hydroxyalkanoates was determined using gas chromatography-mass spectrometry (GC-MS; Clarus 500 PerkinElmer) with the TurboMass software version 4.5.0 and the NIST 98 spectra library.Approximately 0.04 g of the dry PHA was subjected to methanolysis based on the method published by Braunegg et al. 28 with the modifications proposed by Brandl et al. 29 Analysis conditions were established according to Campos et al. 25 The hydroxyalkanoates were identified by comparison of retention time with poly(R)-3-hydroxybutyric acid (Sigma-Aldrich) as the control.The mass spectra were compared with the NIST 98 spectra library.

Data treatment
Data treatment was performed using the tools available in the software Statistica 8.0 (Statsoft Inc., Tulsa).Statistical significance was calculated by Tukey's test.

Substrate chemical composition and medium carbon nitrogen ratio
Elaeis guineensis is a plant that has its origin in Africa and stands out for having high oil production per unit area, popularly known as oil palm or African oil palm, it is a monocotyledon species of the Arecales order that belongs to the subfamily Arecoideae 30 .Oil palm Southeast Asia plantations cover over 107,000 km 2 , being a large-scale commer-cial tree crop with fundamental economic importance 31 .
Table 1 presents the chemical composition determined for the crude palm oil used as sole carbon source in the bio-catalyzed submerged cultivations for PHA synthesis studied in this paper.It is possible to observe that the highest fractions in the oil are comprised of Palmitic (C16:0) and Oleic (C18:1ω9) fatty acids.The substrate fatty acids composition is known to exert a major influence in PHA chain length and structure, since they may function as PHA copolymers 32,33 .
, the substrate studied showed a similar composition to that reported by Mozzon et al. 35 , displaying a high percentage of unsaturated fatty acids.
For fermentation, the cultivation medium (SC) was supplemented with a limiting (NH 4 ) 2 SO 4 supply to obtain a carbon-to-nitrogen ratio (C:N) approximately equal to 114:1.This value is within the C:N range documented in literature for PHA production using the nitrogen limitation approach.Reported C:N values range from 20:1 to 180:1, depending largely on the bacterial strain used for bioconversion 36,37,38 .

Palm oil bioconversion
The effect of Burkholderia cepacia IPT 400 and IPT 119, and Cupriavidus necator IPT 026 and IPT 027, for PHA production was tested in submerged cultivations using crude palm oil as substrate in limiting nitrogen conditions, Table 2. Microorganism and substrate are variables of great importance in PHA bioconversion and their combined influence can largely influence the polymer production sustainability 9 .
Thus, it was inferred that, regarding polymer synthesis, C. necator IPT 027 presented the highest PHA performance, 1.45 g L -1 (Table 2).In general, C. necator strains presented a better polymer synthesis activity compared to B. cepacia, with an average production 5 times higher.C. necator bacteria also presented the best biomass growth; these data reveal better adaptation of these strains to the palm oil substrate offered than that observed for B. cepacia.
Polyesters percentage extraction were significantly lower for B. cepacia bacteria, revealing that the cultivation conditions established triggered growth phase in a more effective manner than polymer accumulation (Table 2).This result suggests revaluation of the nitrogen limitation strategy (famine period) used for the mentioned strains in this study, indicating that a more severe nitrogen restriction must be set, since the limitation of this nutrient generates a cellular trauma that leads to PHA accumulation 39 .
Variance analysis (ANOVA) showed that the different microorganisms studied presented greater impact on biomass production (F = 273.1,p < 0.0001) than on PHA synthesis (F = 155.5,p < 0.0001).This phenomenon reflects high variation in the strains development in the same medium and cultivation conditions, revealing very distinct growth behaviors.

Fatty acid
Average (%) C4:0 0. Applying the same concentration of oil used in the present study, Wong et al. 24 described 2.60 g L -1 of PHA production by recombinant C. necator Re2160/pCB113 with crude palm kernel oil and 3.30 g L -1 with coconut oil (by the same strain).Altaee et al. 40 reported 0.54 g L -1 of PHA in their culture medium using palm oil as substrate and Rhodococcus equi as biocatalyst.Mozejko and Ciesielski 41 published 2.24 g L -1 PHA production with saponified waste palm oil and Pseudomonas sp.Gl01.
These data evidence and confirm high dependence of PHA production on the microorganism used for the bioconversion and the substrate offered, validating and encouraging the investigations on performance of new microorganisms and cheap carbon sources towards the sustainability of PHA production chain.

Biopolymers characterization
All biopolymers produced by C. necator and B. cepacia strains in submerged cultivations with crude palm oil were submitted to thermal, chemical and physical characterizations in order to evaluate the properties associated with their natural atomic structures and their capacity to attain standards that would endorse their commercial application.
The polymers functional groups characterization, presented in Fig. 1, was completed by spectroscopy in the infrared spectra (FTIR).All samples scanned showed the characteristic bands documented in the scientific literature related to PHAs structure, confirming the production of these polyesters.
The transmittance observed at 1728 cm −1 is associated with the axial deformation of C=O carbonyl group 42 .The C-H carbon-hydrogen bond stretching of CH 3 and CH 2 groups were present at the wave numbers 2931 cm -1 and 2854 cm -1 , in this order 42,43 .
Infrared transmittance at 3440 cm -1 was assigned to the hydroxyl group of the polymer chain 45 .The vibration band of the carbonyl of the ester group (C-C) is noted at 972 cm -1 45 .
The bands 1048 cm -1 and 1288 cm -1 are associated, correspondingly, to the asymmetric and the symmetric stretching vibration of the C-O-C group 46 .Wagging CH 3 is observed at the wave number 1381 cm -1 45 .
In Fig. 2, it is possible to see the chromatograms obtained by gas chromatography-mass spectrometry of the PHA samples produced by all the strains assessed in this study to determine the polymers monomeric composition, displayed in Table 3. Mass spectra of each peak were compared against NIST library for polymeric units identification.
Identified was the presence of five different monomeric units composing the structure of all copolymers produced in this study (Table 3).All strains evaluated produced polymers with major presence of hydroxyhexadecanoate and hydroxy-9octadecenoate.It is important to note that the major fractions of fatty acids in the palm oil substrate used in this study were composed of 16 and 18 carbons  In concordance with the data exposed, Thompson and He 32 , and Srivastava and Tripathi 33 reported that the presence of fatty acids in the fermentative medium is known to greatly influence PHA chain length and structure.It has been published that microorganisms use short, medium, and long chains of fatty acids as carbon sources to obtain polyunsaturated fatty acids, which may function as PHA copolymers.
X-ray diffractograms of the PHAs produced with C. necator and B. cepacia strains are displayed in Fig. 3, along with their crystallinity index (I c ), which is an indication of the percentage amount of polymer chains organized in the form of crystals in the material structure.
Crystallinity is an important property that largely influences polymer mechanical characteristics, such as yield stress, elastic modulus, and impact resistance.This parameter is also crucial for polymer processing and its desirable value must not be much higher than 50 %, or else brittleness and rigidity starts to cause complications related to the material applications 47,48,49 .
It is possible to observe (Fig. 3) the diffraction peaks at 2θ for all polymer samples assessed, and their intensity variations due to the different producer microorganisms from which they originated.De-spite that, all PHA presented peaks in 13.56°, 16.96°, 21.89° and 25.52°, typically associated with a semi-crystalline arrangement in polyesters 43 .
I c of the PHAs studied ranged from 48.47 % (C.necator IPT 026) to 27.80 % (B.cepacia IPT 400), demonstrating that the microorganism strain had significant impact in the polyesters structure arrangement.In general, the biomaterials produced by C. necator bacteria exhibited crystallinity indexes superior to those produced by B. cepacia, however, all polymers synthesized in this study showed predominant amorphous phase.This indicates good performance concerning their structural morphology in regard of the biosynthesis conditions set and the biocatalysts used 48,49 .
The processing of biopolymers is also challenged by their often low thermal stability, since it restricts the temperatures and time by which these materials can be processed 50,51 .PHAs thermal behaviors are displayed in the thermogravimetric curves (TGA) and its associated derivatives (DTGA) in Fig. 4. TGA curves depict the mass variation of the polymer samples as a function of temperature increase, which is related to the loss of volatile components.DTGA curves, first derivatives of the TGA, show the rate of thermal decomposition of the sample.
Thermal degradation of all polymers studied occurred in only one mass loss event (Fig. 4), indicating the presence of no impurities remaining from the extraction and separation processes on those materials 36 .Initial degradation temperatures (T onset ) and maximum decomposition temperatures (T max ) of the thermogravimetric events obtained from TGA/ DTGA curves for the PHAs produced from crude palm oil are presented in Table 4.
All polymers produced demonstrated initial thermal degradation above 283 °C, with emphasis on the PHA synthetized by C. necator IPT 026, which exhibited the best thermal stability with an initial degradation temperature of 311.86 °C, a maximum decomposition temperature of 334.42 °C and a total weight loss of 99.35 %.Similarly to what was found for the last strain, Ribeiro et al. 52 reported a PHA with initial degradation temperature of 316.7 °C in the bioconversion (Burkholderia cepacia IPT 438) of glycerol, and Campos et al. 25 described a T onset of 306.8 °C for PHA produced by C. necator IPT 026 in consumption of crude glycerol.
The performances of all biopolymers synthesized in the present study were substantially superior to those found in PHAs produced by Lysinibacillus sp.(T onset = 114.7 °C), reported by Mohapatra et al. 53 , Pseudomonas pseudoflava (T onset = 140-170 °C), reported by Reddy et al. 1 and Bacillus megaterium (T onset = 192.23 -281.83°C), published by Ribeiro et al. 27 Observed was a linear correlation between initial temperature of degradation and crystallinity index for the polymers evaluated (R 2 = 0.91).T onset increases with the increase in I c according to Eq. (2).
T onset = 1.434I c + 243.28 R 2 = 0.9071 (2)   This correlation is reasonable and probably due to the ordered conformation of crystals in the polymer structure, maintained/linked by a group of chemical bonds stronger than those observed for amorphous conformations.Thus, an elevated amount of crystallinity would offer high resistance to thermal degradation once an elevated number of chemical bonds would have to be broken for the polymer to decompose 54 .Fig. 5 displays the HPLC-RI chromatograms of the PHAs separation.Size-exclusion chromatography was used to obtain maximum, average and minimum molecular mass (M w ), number average molecular mass (M n ), and polydispersity (PDI) of the PHAs produced in this study, Table 5.A calibration curve was built using standard polystyrene with different values of M w as a function of column retention time (t).This curve was used to calculate the samples M w, and is presented in Eq. (3).log(M w ) = -0.8364t+ 14.831 R 2 = 0.9917 (3)   The biocatalyst variable played an important role in the weight average molecular mass of the copolymers produced, since this response parameter varied largely depending on the bacterial strain.The highest (389.296kDa) and the lowest (173.781kDa) weight average molecular mass was observed for the PHAs produced by B. cepacia strains, IPT 400 and IPT 119, respectively.
Gumel et al. 55 , studying the substrate effect in PHA production from Pseudomonas putida, found weight average molecular masses ranging from 55 to 77 kDa, while Ribeiro et al. 27 , using different crude glycerol sources for PHA production, reported a range of 1400 to 3740 kDa, in submerged cultivations of Bacillus megaterium.Pan et al. 7 reported M w of 450.8 kDa by Burkholderia cepacia using a detoxified sugar maple hemicellulosic hydrolysate, and Campos et al. 25 described a range of 510 kDa to 780 kDa, by Cupriavidus necator using crude glycerol.This illustrates the great variety of molecular masses reported in literature in response to the use of different biocatalysts, substrates and cultivation conditions.
Regarding polymer polydispersity (PDI) (Table 5), C. necator and B. cepacia bacteria produced materials with good homogeneity of molecular masses, representing good bacterial biosynthesis consistency.The PDI values identified for the polymeric samples in this study ranged from 1.67 to 2.46, revealing the production of materials far more uniform than those reported by Sang-Hyeop et al. 56 in their investigations with PHA production from wastewater sludge (PDI = 3.44), and Gahlawat and Soni 44 , with their P(3HB-co-3HV) copolymer production by C. necator DSM 545 (PDI = 4.3) using waste glycerol.

Conclusions
The bacterial strains investigated were able to use crude palm oil as substrate to synthesize novel PHAs mostly constituted of long side chain monomers -C16 and C18, which is a rare feature in the polymers field.All copolymers produced presented variable monomeric composition and molar masses, with predominant amorphous molecular arrangements, high thermal stability, and low polydispersity.C. necator exhibited the highest performance in biosynthesis.C. necator and B. cepacia bacterial strains presented great effects in PHAs production and properties, confirming the importance of the biocatalysts variable in the bioprocess.Medium composition and fermentation conditions can be optimized to maximize biomass and biopolymer production.Additionally, the understanding of biochemical and cellular mechanisms of PHA production and accumulation, associated with new techniques of polymer extraction can provide valuable insights to overcome the hurdles in the utilization of these bioplastics.

F
i g . 1 -FTIR spectra of PHA produced by C. necator and B. cepacia strains in submerged cultivation with crude palm oil Ta b l e 3 -Monomeric profile of the PHAs produced by C. necator and B. cepacia bacteria, using crude palm oil as substrate, obtained by gas chromatography-mass spectrometry in the NIST library

F i g . 2 -
Chromatograms obtained by gas chromatography-mass spectrometry of PHA produced by C. necator and B. cepacia strains in submerged cultivation with crude palm oilF i g .3 -X-ray diffractograms of PHA produced by C. necator and B. cepacia strains in submerged cultivation with crude palm oil

F i g . 4 -
TGAs (a) and DTGs (b) of crude palm oil and PHAs produced by C. necator and B. cepacia strains in submerged cultivationTa b l e 4 -Initial degradation (T onset ) and maximum decomposition temperatures (T max ), along with total weight loss of PHAs produced by C. necator and B. cepacia strains using crude palm oilMicroorganism strain T onset (°C) T max (°C) Total mass loss (%)

F i g . 5 -
HPLC-RI chromatograms of PHAs produced by the C. necator and B. cepacia strains from crude palm oil in submerged cultivationsTa b l e 5 -Molar mass of PHA produced by the C.