We report on the ability of bacteria to produce biodegradable polyhydroxyalkanoates ( PHA ) using oxidized polyethylene wax ( O-PEW ) as a novel carbon source. The O-PEW was obtained in a process that used air or oxygen as an oxidize agent. R. eutropha H16 was grown for 48 henry in either tryptone soy broth ( TSB ) or basal salts medium ( BSM ) supplemented with O-PEW and monitored by viable count. Study revealed that biomass and PHA product was higher in TSB supplemented with O-PEW compared with TSB only. The biopolymers obtained were preliminary characterized by nuclear magnetic resonance ( NMR ), gel interpenetration chromatography ( GPC ), differential scanning calorimetry ( DSC ), and thermohydrometric analysis ( TGA ). The detail morphologic evaluation at the molecular level was performed by electrospray ionization tandem mass spectroscopy ( ESI-MS/MS ). The study revealed that, when TSB was supplemented with O-PEW, bacteria produced PHA which contained 3-hydroxybutyrate and up to 3 gram molecule % of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units. The ESI-MS/MS enabled the PHA characterization when the content of 3-hydroxybutyrate was high and the appearance of early PHA repeating units was very low. Keywords:
polyhydroxyalkanoates, oxidized PE wax, medium chain length PHA (mcl-PHA), Ralstonia eutropha H16, mass spectrometry
Petrochemical-based plastics have become an integral part of our daily lives since their beginning industrial production in the 1940s. They are durable, lightweight, cheap, strong, and easy to handle, possessing properties that make them very utilitarian in agrarian, industrial, and domestic applications [ 1, 2 ]. however, these properties besides make them susceptible to excessive practice and irresponsible disposal [ 3 ]. frankincense, fictile waste makes up a huge function of municipal solid lay waste to ( MSW ), globally. In Europe, it is estimated that approximately 7 % by mass of generated MSW are plastics. This percentage is even higher in the USA and China both having 11.8 % and 14 % by mass, respectively [ 4, 5 ]. furthermore, it is estimated that plastics contribute up to 80 % of nautical debris causing serious environmental and health problems worldwide [ 5 ]. The environmental and health impingement of these plastic wastes are severe and have been extensively discussed by former authors [ 6 ]. Polyethylene ( PE ) is known to be the most manufactured petrochemical polymer making up over 29 % of ball-shaped petrochemical plastic production [ 7 ] and with alone 10 % of plastic waste soon being recycled [ 8 ], there is a huge necessitate for alternate uses of these waste materials to reduce the environmental load they have on today ’ second club. PE is a chemosynthetic polymer derived largely from fossil fuels and recently besides from renewable sources [ 9 ]. The identical deep carbon paper spinal column of PE makes it a potential cradle of carbon for microbial use in the production of the biodegradable polyhydroxyalkanoates ( PHA ). PHA are readily biodegradable, biocompatible, and non-toxic biopolymers that are synthesized intracellularly by different bacterial species from soluble carbon rich substrates in the presence of an surfeit total of carbon paper and a specify food [ 10, 11, 12 ]. In addition, they besides possess properties similar to petrochemical plastics, frankincense making them likely substitutes for non-biodegradable polymeric materials [ 10 ]. The applications of PHAs in respective sectors of our deliver day club are divers and have been reviewed by respective authors [ 13, 14, 15, 16 ]. by and large, the price of production of biodegradable plastics are higher than the conventional plastics since most of the processes are still in the developmental stage. frankincense, the decrease of waste treatment monetary value of conventional plastics through PHA recovery seems to be one potential solution [ 17 ]. The use of waste polyethylene as carbon source for PHA production brings in a double advantage of reducing the environmental impact of polyethylene credit card and producing an environmentally friendly and biodegradable stand-in. however, the highly stable C-C and C-H covalent bonds of PE coupled with its identical high molecular weight and hydrophobicity makes it unmanageable for microbial attack and use [ 11, 18 ]. Recycling technologies of PE are restricted either to mechanical recycling or energy recovery via incineration. oxidation of PE can generate a valuable feedstock in the form of oxidize PE wax rich in hydrocarbons. Some microbes should be able to use this highly complex substrate for deduction of the add respect biodegradable polymers such PHA, therefore, the conversion of PE to a high value product should lead to higher level of PE recycling [ 7 ]. This procedure should besides have an impingement on the production price of PHA as the function of consume O-PEW feedstock for microbes accumulating PHAs can lead to their greater economic viability and sustainability. PE waxes are very important materials in many types of industry. One of the most significant groups of synthetic wax are arctic polyethylene waxes, which are obtained in the oxidative abasement process. The work occurs with chains scissoring and the formation of oxygen-containing groups ( hydroxyl, carbonyl, carboxyl, ester ), according to the mechanism of release radical chain [ 19 ]. polar polyethylene waxes are primarily used as a component of aqueous emulsions with many applications including additives for paints and varnishes [ 20 ], lubricants e.g., in the polyvinyl chloride ( PVC ) manufacture [ 21 ], and many others. Their application properties depend on the type of raw materials used ( low-density polyethylene ( LDPE ), high-density polyethylene ( HDPE ) ) and, thus, the method of production, which may be carried out as a march in mellow [ 21, 22 ], aqueous dispersion [ 23, 24 ], or solid phase [ 25, 26 ]. The exemplar studies on oxidise polyethylene powderize as a filler for polycaprolactone blends revealed that pre-oxidized PE powder in combination with wetting agent and pro-oxidant greatly accelerated the abasement rate of poly ( ε-caprolactone ), PCL, as revealed by molecular system of weights decrease during low temperature thermo-oxidative aging [ 27 ]. recently, the polar O-PEW was used as an addition to the polyhydroxybutyrate ( PHB ) biopolyester improving the mechanical properties of the train polymeric materials as confirmed by thermo-mechanical studies [ 28 ]. The beneficial composition was found for the blend containing up to 10 % ( w/w ) of oxygenated PE wax, and biodegradation studies conducted under both lab and an industrial compost conditions revealed that the O-PEW accelerated the biodegradation pace of the PHB blend. therefore, our guess is that O-PEW could be a potential source of carbon for microbial PHA output and besides act as the PHA blend part which could accelerate their biodegradation. simple pyrolysis of PE in the absence of air generates a complex mix of first gear molecular weight paraffin with carbon paper chain lengths from C8 to C32 ( PE pyrolysis wax ). Guzik et aluminum. used the PE pyrolyzed wax for long side range PHA output and Pseudomonas aeruginosa PAO-1 was found to accumulate the highest level of PHA to about 25 % of the cell dry weight when supplied with this type of PE pyrolysis wax in the presence of rhamnolipids [ 7 ]. here, we propose the practice of O-PEW for PHA product. PE oxidation introduces carbonyl and hydroxyl groups into the polymer spinal column that are more easily cleaved by microbes [ 29 ]. This besides improves PE ’ s hydrophilic properties and reduces its chain length, therefore further improving their electric potential for microbial utilization as a carbon beginning for PHA production. Furthermore, the process of oxidation improves the diffusing properties of PE wax in the agitation media and makes them more accessible to microbes. Bio-utilization of O-PEW follows a process that results in the output of fatso acids which can then be β-oxidized within the cell [ 18 ]. Ralstonia eutropha ( previously known as Cuprivadus necator or Wausternia eutropha ) is a gram negative bacteria that is known to accumulate PHB as insoluble granules within their cells when nutrients other than carbon are limiting. however, it has been besides reported that in some cases PHA output by this organism can besides occur under nutrient-rich conditions [ 12, 30 ]. Ralstonia eutropha has been reported to utilize a wide image of carbon beginning including fatty acids for PHA production [ 10, 31 ]. This bacteria can accumulate 80 % to 85 % PHA per dry cell weight with about 8–12 PHB granules per cell. PHA granules are then extracted from this dried biomass via solution extraction chiefly with chloroform and precipitated in ethyl alcohol [ 32 ] or through other extraction techniques. therefore, in this research we report the production of PHA by Ralstonia eutropha H16 incubated in two unlike microbiological media ( nitrogen rich tryptone soy broth and nitrogen-limited basal salts medium [ 12 ] ) supplemented with O-PEW as the carbon reference. This to the best of our cognition is the first composition showing the use of oxidize polyethylene wax as a carbon beginning for PHA production .
A major problem of pine away and environmental contamination is that plastics produced by the petrochemical industry are not biodegradable and consequently accumulate in the environment. The conversion of polyethylene ( PE ) to oxidized PE wax ( O-PEW ) and its use as carbon reference for bacterial PHA production can be an attractive alternative for producing environmentally friendly and biodegradable polymers. The oxidation process can have an shock on the properties ( AN ) of the PE wax ( ). previous experiments have shown that polyethylene ( PE ) can have negligible antimicrobial properties against bacteria. frankincense, Zhang et aluminum. noted that PE has identical low disinfectant properties [ 35 ] and, Seyfriedsberger and his colleagues showed that linear gloomy density polyethylene ( LLDPE ) besides had identical depleted antimicrobial activeness against gram-positive Staphylococcus aureus and no antimicrobial activity against gram veto Escherichia coli [ 36 ]. Gregorova et aluminum. reported that PE had no antimicrobial property against either S. aureus or E. coli [ 37 ]. In this study cell growth analysis showed that O-PEW 17 with AN = 197 does not affect the growth of R. eutropha H16 in both media TSB or BSM ( ). Our results suggest that O-PEW is metabolized presumably via β-oxidation leading to enhanced cell emergence of R. eutropha H16. PHA yield and percentage PHA per cellular telephone dry weight ( 1.24 g/L and 33.8 %, respectively ) were higher in TSB supplemented with O-PEW compared to pure TSB ( 0.39 g/L and 17 %, respectively ). Growth and metabolism of O-PEW was confirmed by R. eutropha H16 being able to grow in BSM with oxidized polyethylene wax as the lone carbon paper and energy reservoir. Growth of R. eutropha H16 in BSM with O-PEW did not produce PHA with the 48 henry culture period ( ). These results suggest the addition of O-PEW into TSB has an effect upon the product of PHA. Stress and the handiness of carbon paper sources are known factors that stimulate PHA collection within bacterial cells [ 10 ]. The presence of accessible carbon paper sources, such as fatty and carboxyl acids from O-PEW for microbial use means that more carbon is stage in the wax supplemented cultures in a mannequin that promotes deduction of PHAs. It may be expected, that in the presence of a biosurfactant [ 7 ] these wax carbon sources would be more accessible, leading to more PHA accumulation. The sum of nitrogen available in the media can influence bacterial growth and accretion of PHA. In this study higher concentration of biomass and higher concentrations of PHA were obtained in TSB ( high nitrogen capacity ) than in BSM ( low in nitrogen ) which agrees with observations reported by Verlinden et alabama. [ 12 ] when waste frying petroleum was used to produce polyhydroxybutyrate ( PHB ). addition of O-PEW stimulated further bacterial growth that may have led to increased nutrient use and finally nutrient limitation, resulting in better production of PHAs. today aggregate spectroscopy complements in many ways the geomorphologic data provided by NMR [ 34 ]. Development of cushy ionization techniques in mass spectroscopy made the undertake to solve the unmanageable question regarding the molecular structure of copolymers more likely. therefore, structural studies of biodegradable copolymers with the practice of multi-stage electrospray mass spectroscopy ( ESI-MSn ) were performed [ 38 ]. This aggregate spectroscopy technique was applied to determine the co-monomer unit of measurement typography and composition distribution in bacterial PHA copolymers based on the analysis of their oligomers obtained by partial depolymerization ( ). For the aim of this study, the controlled thermal degradation of polyesters obtained, induced by sodium acetate, was performed according to the procedure described in the reference [ 39 ]. This type of E1cB degradation leads to PHA oligomers with unsaturated and carboxyl end groups. The ESI-MS spectrum of oligomer, obtained via fond thermal abasement of the biopolyester obtained using TSB ( without wax ) as a bacterial growth medium, indicated that the bearing of singly charged ions contained 3-hydroxybutyrate ( 3-HB ) repeat units alone. however, the ESI-MS spectrum of oligomer derived from PHA obtained using TSB/O-PEW ( presented in ) consists of a serial of clusters with singly charged ions. The ions were separated due to their different degrees of oligomerization and composing. The differences between the m/z value of the most intensive signals are peer to 86, which corresponds with the molar mass of the 3-hydroxybutyrate ( 3-HB ) reprise units. however, in the elongated spectral range at m/z 870–1050 the mass remainder between the m/z values of ions within each bunch ( for example, m/z 897, 911, 925, and 939 ) is adequate to 14 Da, which corresponds to the difference between the molar mass of the individual co-monomeric units with longer side chains. Based on the mass assignment of singly charged ions observed in the mass spectrum the social organization of the end groups and repeating units can be inferred. The clusters of singly charged ions observed in the mass spectrum in corresponds to the sodium adducts of individual co-oligoester chains composed of 3-hydroxybutyrate ( HB ), 3-hydroxyvalerate ( HV ) and/or 3-hydroxyhexanoate ( HH ) co-monomeric units and were terminated by unsaturated and carboxyl end groups. The ESI-MS/MS product ions spectrum for the selected sodium adducts of oligomers [ HB10 + Na ] + at m/z 883 were shown in. fragmentation of this ion, which resulted from the random breakage of ester bonds along the both sides of the oligomer chain, led to the formation of alone one hardened of oligo ( 3-hydroxybutyrate ) product ions at m/z 797, 711, 625, 539, 452, 366, and 281 terminated by carboxyl and crotonate end groups. The inaugural ion from this series at m/z 797 was created by the shift of inert atom of crotonic acid ( 86 Da ) from both ends of the rear ion at m/z 883. The fragmentation spectrum of this ion, confirms that the most intensive ions in the clusters correspond to sodium adducts of 3-hydroxybutyrate oligomers. In the ESI-MS/MS spectrum for the selected ion [ HB9 HV + Na ] + at m/z 897 ( which contains nine 3-HB and one 3-HV units ) was presented. In that event, the breakage of ester bonds along the oligomer chains led to the formation of two series of intersection ions at m/z 811, 725, 639, 553, 467, 381 and at m/z 797, 711, 625, 539, 453, 366. frankincense, the product ion at m/z 811 corresponded to the oligomer formed by the loss of crotonic acidic ( 86 Da ) while the intersection ion at m/z 797, could have been formed due to the loss of valeric acid ( 100 Da ), as seen from the atomization pathway in. Thus, the fragmentation spectrum acquired for the precursor ion at m/z 897 confirms the presence of 3-hydroxybutyrate and the 3-hydroxyvalerate co-monomer units in polyester chains. furthermore, such a atomization pathway indicates that the 3HV unit is randomly distributed along the oligomer chain [ 38, 40, 41 ]. In, three fragmentation paths were observed in the case of ESI-MS/MS spectrum of the rear ion at m/z 911 ( which may have corresponded to the isobaric ions contained in two HV units or one HH unit [ HB8 HV2 + Na ] + or HB9 HH + Na ] +, respectively. These two series at m/z 825, 739, 653, 567, 480, 395, and at m/z 811, 725, 639, 553, 467, 381 are formed in the same way as identify for atomization of ion at m/z 897 ( ). The third series at m/z 797, 711, 625, 539, 453, 367 was besides observed. The beginning ion in this series is formed by extrusion of 2-hexenoic acidic ( 114 Da ) indicate, unambiguously, the presence of HH unit in the oligomer chain. thus, the fragmentation experiment carried out for the ion at m/z 911 indicates the presence of more than two co-monomeric units in this oligomer. The ESI-MS/MS spectrum of sodium adduct at m/z 911 besides showed the fragment ion at m/z 811, grouped in a cluster containing three fragment ions, which may correspond to oligomers having the like degree of polymerization but having a different a content of HB and HV units. The fragmentation results allowed us to confirm that in the presence of O-PEW carbon paper source the PHA formed was composed of 3-hydroxybutyrate, 3-hydroxyvareate ampere well as 3-hydroxyhexanoate co-monomer units which are randomly distributed along the oligomer chain [ 34, 38, 40 ]. furthermore, with deference to the so far reported studies, this inquiry demonstrated the possibility of PHA structure evaluation even when the content of HV and HH is lower than 5 %, i.e., on the floor of accuracy of 1H-NMR measurements [ 34 ]. The structure of the biopolyesters obtained influences their thermal properties, particularly the glass conversion temperature ( Tg ) and melting passage ( Tm ). Both of these values were found to be lower in the character of the biopolyester obtained when O-PEW was used as a carbon paper informant ( ). In the case of Tg the difference is equal to 4.1 °C, whereas in case of Tm the dispute is equal to 5 °C. Differences are besides visible in crystallinity academic degree, and PHA derived from zymosis with the use O-PEW is less crystalline. therefore, even three per penny of repeating unit with longer aliphatic chains in β-position may influence significantly on Tg, Tm and the degree of crystallinity of PHA obtained .
4. Materials and Methods
4.1. Carbon Source
Oxidized wax from PE standard samples was used as a carbon source for the PHA production via little scale shake flasks experiments. Oxidized PE waxes were obtained as products from oxidative abasement which was carried out in a two-phase system : gas-liquid phase, after melting at 145 °C and using oxygen. The bleak fabric used for this process was a polyethylene standard from Sigma-Aldrich Co ( Gillingham, UK ). with an average Mw ~4000 and an average Mn ~1700 ( by GPC ), melting detail 92 °C, soft point 106 °C, and concentration 0.92 g/mL at 25 °C. To increase the oxidation pace and in order to obtain products with high values of acidity issue ( AN ), ozone ( 100–300 mg/L ) was introduced into the oxygen current. Processes were carried out from 18 to 20 heat content. The acidic act ( AN ) which is one of the basic properties of oxidize waxes was determined according to criterion methods [ 42 ]. Each psychoanalysis was performed a minimal of two times. The prevail results of analysis for the lapp wax were very similar and the differences for AN were less than 1.0 magnesium KOH/g. The standard allow for 3 % measurement accuracy .
The microorganism used for the PHA production with oxidize polyethylene wax was R. eutropha H16 ( NCIMB 10442, ATCC 17699 ). The organism was obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, UK. The stock culture was lyophilized and kept at −20 °C. Before function, cultures were resuscitated and grown overnight at 30 °C in tryptone soy broth ( TSB ). The organism was then sub-cultured on tryptone soy agar ( TSA ) and incubated at 30 °C for 24 planck’s constant .
4.3. Growth Media and Chemicals
Tryptone soy broth ( TSB ) and tryptone soy agar ( TSA ) were purchased from Lab M Ltd., Lancashire, UK. Both media were prepared following the instructions of the manufacturer under aseptic conditions. Basal salt medium ( BSM ) contains distill urine, 1 g/L K2HPO4, 1 g/L KH2PO4, 1 g/L KNO3, 1 g/L ( NH4 ) 2SO4, 0.1 g/L MgSO4·7H2O, 0.1 g/L NaCl, and 10 mL/L trace elements solution. Trace component solution has : 2 mg/L CaCl2, 2 mg/L CuSO4·5H2O, 2 mg/L MnSO4·5H2O, 2 mg/L ZnSO4·5H2O, 2 mg/L FeSO4, and 2 mg/L ( NH4 ) 6Mo7O24·4H2O. BSM salts were purchased from BDH Chemicals Ltd., Poole, UK. Ringer ’ sulfur solution was used for microbial growth analysis and it was besides purchased from Lab M. A 1/4 potency pad was used in condense water. All media were sterilized by autoclaving at 121 °C for 15 min. Chloroform and n-hexane ( High-performance liquid chromatography ( HPLC ) grade ) used for PHA extraction and precipitation were obtained from Sigma Aldrich, Gillingham, UK .
4.4. Shake Flask Experiments
For the little scale tremble flask studies, newcomer cultures were prepared by inoculating 20 milliliter TSB with one colonies of R. eutropha H16 in 50 milliliter conic flasks. The polish was then incubated aerobically for 24 heat content at 30 °C and 150 revolutions per minute in a rotary incubator ( New Brunswick Scientific Co. Series 25 Incubator Shaker, Enfield, CT, USA ) After 24 planck’s constant of incubations, cultures were checked for honor by Gram stain and microscopic observations at a magnification of ×1000.
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Shake flasks studies were performed in triplicate using 500 milliliter Erlenmeyer flasks. 1.25 g of the wax was put into a 50 milliliter beaker covered with foil paper and melted at 70 °C. 20 milliliter sterile TSB or BSM was then added to the melted wax which caused the wax to solidify again as expected. thus, the temperature was gradually increased until all the wax had melted again. After which the wax suspension was sonicated for 7 min at 0.5 active and passive intervals with a power of 60 % using a Bandelin Sonopuls HD2070 sonicator, Berlin, Germany. The TSB/wax emulsion or BSM/wax emulsion was tested for asepsis by dispersed plating 100 milliliter of the emulsion on TSA plates overnight at 37 °C and 30 °C. No microbial growth was observed in either plate after 24 h of brooding, frankincense indicating that the emulsion was aseptic. The emulsion was then added to 210 mL sterile TSB or BSM in a 500 milliliter Erlenmeyer flask and this was immediately followed by the addition 20 milliliter starter acculturation, giving a total volume of 250 milliliter with a wax concentration of 4 g/L and 8 % ( v/v ) of the appetizer polish. For the control experiment, 230 milliliter TSB and BSM was inoculated with 20 mL crank culture with no wax. This was done to see the impression of the wax on both the growth of cells and the production of PHA by R. eutropha H16 cells. All flasks were incubated in a circular incubator for 48 hydrogen at 30 °C and 150 revolutions per minute. 5 milliliter samples were aseptically collected from the growing bacterial cultures at 0, 3, 6, 24, and 48 henry of incubation for feasible cell counts. Cell counts were carried out using the method described by Miles and Misra [ 43 ] involving a serial dilution of the samples to 10−8 and aseptically inoculating 20 µL of each dilution unto TSA plates in triplicate. Plates were incubated nightlong at 30 °C, followed by colony count. The results obtained were expressed in log10 CFU/mL .
4.5. PHA Extraction
PHA extraction was carried out as described previously [ 31 ]. Shake flask experiments were left to run for 48 planck’s constant after which microbial experiments were stopped and cultures were centrifuged in a Hermle Labortechnik ( Wehingen, Germany ) Z300K centrifuge for 15 min at 6000 revolutions per minute. Supernatants were discarded and the biomass was obtained and freeze overnight at −20 °C. This was followed by freeze-drying using an Edwards Modulyo freeze-drier ( Crawley, UK ) for 72 henry at a temperature of −40 °C and at a pressure of 5 mBar. The weights of the dry biomass were obtained and recorded as cell dry weight ( CDW ). Lyophilized biomass was then transferred into origin thimbles and PHA was extracted by Soxhlet origin with HPLC mark chloroform ( Sigma Aldrich ) running for 5 h. The hot solution of polymer/chloroform was concentrated by dehydration, followed by PHA haste in n-hexane ( Sigma Aldrich ). Precipitated polymer was separated from solution by filtration ( Watman No. 1 paper, Sigma Aldrich ) and rinsed further in chloroform to remove the excess wax materials before air-drying. The weight of the polymer ( WPHA ) was recorded and the share of PHA ( wt/wt ) synthesized by R. eutropha H16 was calculated using the equality : PHA % =WPHACDW×100
4.6. Polymer Identification
4.6.1. GPC analysis
The number-average molar aggregate ( Mn ) and the molar multitude distribution index ( Mw/Mn ) of the homely PHA samples were determined by GPC experiments conducted in a CHCl3 solution at 35 °C with a hang pace of 1 mL/min using a Spectra-Physic 8800 solvent delivery system ( Spectra-Physics Inc., San Jose, CA, USA ). This system had a set of two PLgel 5 µm MIXED-C ultra-high efficiency column and a Shodex SE 61 refractive index detector. A sample solution volume of 10 µL ( concentration of 1 % w/v ) was injected. Polystyrene ( PS ) standards with a narrow molar mass distribution were used to generate calibration curves. The PS with noun phrase Mp in the range of 3000–7,000,000 g/mol were used .
4.6.2. NMR Analysis
1H-NMR spectrum were recorded with a Bruker Avance II ( Bruker, Rheinstetten, Germany ) operational at 600 MHz, with 64 scans, 2.65 randomness acquisition time and 11 µs pulse width. 13C-NMR spectrum were recorded with a Bruker Avance II operating at 150.9 MHz, with 20,480 scans, 0.9088 mho learning times, and 9.40 µs pulse width. 1H-NMR and 13C-NMR spectra were run in CDCl3 at room temperature with tetramethylsilane ( TMS ) as an internal criterion .
4.6.3. ESI-MS/MS Analysis
Electrospray mass spectroscopy analysis was performed using a Finnigan LCQ ion trap aggregate mass spectrometer ( Thermo Finnigan LCQ Fleet, Thermo Fisher Scientific Inc., San Jose, CA, USA ). The oligomer samples, prepared as described in [ 33 ], were dissolved in a chloroform/methanol arrangement ( 1:1 v/v ), and the solutions were introduced into the ESI source by continuous infusion using the instrument syringe pump at a rate of 5 μL/min. The LCQ ESI reference was operated at 4.5 kilovolt, and the capillary heater was set to 200 °C. nitrogen was used as the nebulizing gas. For ESI-MS/MS experiments, the ions of matter to were isolated monoisotopically in the ion trap and were collisionally activated. The helium damping gas that was present in the mass analyzer acted as a collision natural gas. The analysis was performed in the positive-ion manner .
4.6.4. DSC Analysis
DSC analyses were taken with a TA DSC 2010 apparatus ( TA Instruments, New Castle, DE, USA ) in the temperature image of −50 to +200 °C and the samples were run in triplicate. The glass transition temperatures ( Tg ) were determined at a heating pace of 20 °C/min. In this study, Tg was taken as the center of the step-transition. The instrument was calibrated with senior high school purity indium and gallium. share crystallinity was calculated by the equation : C ( % ) =ΔHmmΔHm100 % where 𝐶 is the share crystallinity ; ΔHmm is the measure mellow heat content ( J/g ) ; and the ΔHm100 % is the melting heat content for in full crystalline PHB ( 146 J/g ) [ 44 ] .
4.6.5. TGA Analysis
thermohydrometric psychoanalysis was performed using a Mettler-Toledo TGA/DSC1 unit. Analyses were performed under an inert atmosphere ( nitrogen ) at a gasoline flow rate of 60 mL/min. The rate of temperature increase during the analysis was 10 °C/min. The measurements were performed in the ceramic pan capacitance of 70 uL to weigh amounts of approximately 10 milligram. The samples were run in triplicate .
It may be concluded, that oxidized polyethylene wax can be a bright carbon reservoir for PHA production. The morphologic and thermal characterization of biopolyesters obtained revealed that using TSB only or TSB supplemented with O-PEW as a carbon reservoir led to the production of different biopolyesters. We have, therefore, demonstrated uniquely that the accession of oxidize polyethylene waxes to the zymosis medium can have an charm on the structure of resulting biopolyesters and, hence, their thermal properties. Growth on O-PEW in TSB not entirely enhances PHA yield but besides influences compositional changes in the PHA polymer structure. The NMR analysis indicated that in the presence of TSB/O-PEW the chains of the resulting PHA biopolyester containing not merely 3-hydroxybutyrate repeating units. however, it was not potential to determine the accurate social organization of this biopolyester with the help of this technique. Nonetheless, based on the consolidation of particular signals in the 1H-NMR spectrum of the polyester obtained from TSB/O-PEW the content of 97 gram molecule % of 3-hydroxybutyrate units and approximately 3 mol % of another aliphatic repeating units were estimated. The ESI-MS a well as tandem ESI-MS/MS techniques confirmed that when O-PEW is used as a carbon source, besides the 3-hydroxybutyrate repeating units the units containing longer aliphatic moieties in the β-position are formed. The ESI-MS/MS fragmentation experiments confirmed the bearing of 3-hydroxyvalerate repeating units ampere well as 3-hydroxyhexanoate repeating units in the biopolyester obtained in the presence of O-PEW as a carbon paper reference. therefore, this analytic technique enables the PHA portrayal at the molecular level even when the content of 3-hydroxybutyrate is high and the appearance of other PHAs repeating units is very low .
This study was partially supported under the EU 7FP BIOCLEAN Project, Contract No. 312100, “ New biotechnological approaches for biodegrading and promoting the environmental biotransformation of synthetic polymeric materials ” .
The following abbreviations are used in this manuscript :
|O-PEW||Oxygenated Polyethylene Wax|
|TSB||Tryptic Soy Broth|
Adam A. Marek and Jan Zawadiak were responsible for oxidation of PE and O-PEW characterization. Iza Radecka, David Hill, Victor Irorere, and Brian Johnston were responsible for bacterial product of PHA. Guozhan Jiang and Craig Williams were creditworthy for PHA recovery and preliminary characterization. Grazyna Adamus, Michal Kwiecień and Marek Kowalczuk were responsible for PHA characterization by ESI-MS/MS, DSC and TGA. Iza Radecka, Grazyna Adamus and Marek Kowalczuk were the main persons involved in the plan of experiments and rendition of the data of PHA word picture. All authors were evenly involved in drafting and editing of the manuscript .
Conflicts of Interest
The authors declare no conflict of matter to .