K2HPO4 and KH2PO4 were obtained from Fisher Chemicals Ltd., Loughborough, UK. Chloroform (HPLC grade), n-hexane and methanol were obtained from Rathburn Chemicals Ltd., Walkerburn, UK. Sulphuric acid was purchased from Scientific & Chemical Supplies Ltd., Bilston, UK. Methyl benzoate, KNO3, (NH4)2SO4, MgSO4.7H2O, NaCl, CuSO4.5H2O, MnSO4.5H2O, ZnSO4.5H2O, CaCl2, FeSO4 and (NH4)6Mo7O24
.4H2O were purchased from BDH Chemicals Ltd., Poole, UK.
Pure vegetable oil, from rapeseed, was obtained from a local supermarket. Waste frying oil was provided by a food establishment, which deep-fries chips and chicken. Heated oil was obtained by heating pure vegetable oil in an open container at 180°C for 1 week. 180°C is a common frying temperature (Costa et al. 2001). All oils were sterilised separately in a steam autoclave and added directly to fermentations. Since the oils were either refined or subjected to prolonged heating, only few spores were present in the oils. Speadplating of 1 ml of oil on TSA showed no colonies after incubation at 30°C for 24 hours.
Tryptone Soya Broth (TSB) contains distilled water, 17 g/l Tryptone, 3 g/l Soy Peptone, 5 gl/l NaCl, 2.5 g/l K2HPO4 and 2.5 g/l D-glucose.
Tryptone Soya Agar (TSA) contains distilled water, 15 g/l Tryptone, 5 g/l Soy Peptone, 5 g/l NaCl and 12 g/l Agar No. 2.
Basal Salts Medium (BSM) contains distilled water, 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, 10 ml/l Trace elements solution. Trace element 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, 2 mg/l (NH4)6Mo7O24.4H2O.
Basal Salts Agar (BSA) contains all ingredients of BSM and additionally 15 g/l Agar No. 2.
Nitrogen Concentration in Media
It has been reported that the nitrogen concentration in bacteriological media highly influences the production of intracellular PHAs (Khanna and Srivastava 2005b). From EDX (Energy Dispersive X-Ray) experiments it was found that TSB contains around 4.4 g/l of nitrogen. Most of this is probably available from short amino acids. From the chemical formulas of its constituents it was calculated that BSM contained approximately 0.35 g/l of nitrogen. The main nitrogen sources in BSM are nitrate and ammonium.
Cupriavidus necator H16 (NCIMB 10442, ATCC 17699) was obtained from NCIMB, Aberdeen, United Kingdom, and subcultured on Tryptone Soya Agar (TSA).
25 ml of Tryptone Soya Broth (TSB) was inoculated from a single colony and incubated for approximately 24 hours at 30°C. Cultures were checked for purity by Gram staining and observed under a microscope at 1000 ×.
Batch fermentations were performed in Erlenmeyer flasks of 500 ml. The flasks were filled with 250 ml of medium, including the 25 ml TSB inoculum, resulting in an inoculation ratio of 10% (v/v). The initial viable cell number was 2.107-1.108 cfu/ml. Either a quantity of pure vegetable oil, heated vegetable oil or waste frying oil was added to the fermentation medium. The initial oil concentrations were 20 g/l. After addition of the oil, but before inoculation, the sterile medium was sonicated for 10 minutes to achieve a homogenised mixture.
All flasks were incubated in a rotary incubator (150 rpm) at 30°C and fermentations were stopped after certain times. All experiments were done in triplicate. The fermentation broth was centrifuged at 2500 × g for 10 minutes. The bacterial pellet was lyophilised and the supernatant kept for analysis.
Viable Cell Count and Total Cell Dry Weight
After ten-fold serial dilutions the number of viable cells was assessed by spread plating of 100 μl on TSA plates. To determine the total cell dry weight, a known volume of the fermentation broth was centrifuged at 2500 × g for 10 minutes and the supernatant was separated off. The bacterial pellet was lyophilised for 24 hours and weighed.
Determination of PHB content by Gas Chromatography
The amount of PHB in the bacterial biomass was determined using the method developed by Braunegg et al (1978). Additions made to the original method by Jan et al. (1994) and Yunji Xu (Koutinas et al. 2007) were implemented.
Freeze-dried biomass was grinded to a powder and put into a gas-tight screw-capped tube. 2 ml acidified methanol (3% v/v sulphuric acid, 2.5 g/l methyl-benzoate) and 2 ml chloroform were added. The methyl-benzoate was used as an internal standard to improve accuracy. The tube was closed and kept at 90-100°C for 3 hours. After rapid cooling, 4 ml of distilled water was added to achieve a good phase separation. The sample was vortexed for 10 seconds and the chloroform-phase was then filtered through a PTFE-filter and 1 μl was injected into a gas chromatograph (GC). The used GC was fabricated by the Thermo Finnigan Corporation, Milan, Italy. It had a Restek capillary column (Rtx-5MS) 30 m × 0.25 mm × 0.25 μm. Gas flow: 1.5 ml/ min. Carrier gas: He and make-up gas: N2. Column temperature range: 100-160°C. Temperature gradient: 15°C/ min. Inlet temperature: 250°C. Split ratio: 40. Detector temperature: 300°C. Detector type: flame ionisation.
A calibration curve was constructed using pure PHB from solvent extraction. The PHB content is determined from the peak-areas of the methyl-3-hydroxybutyrate and the internal standard.
Up to 5 grams of lyophilised biomass was transferred into an extraction thimble. In a soxhlet extractor the PHAs ware extracted with approximately 250 ml of chloroform during 3 hours.
The hot solution of polymer was concentrated by evaporation. Subsequently the solution was precipitated in n-hexane (1:4, v:v) with a dropping pipette for purification. The polymer precipitated as a white substance.
Fatty acid analysis
1 mg of pure oil and 1 mg of waste oil were added to separate clean glass tubes. 1 ml of saponification reagent was added, consisting of a 150 g/l NaOH-solution in methanol/distilled water 1:1 (v:v). The tubes were closed with a PTFE-lined cap and mixed on a vortex shaker for 10 seconds. The tubes were then placed in a water bath at 100°C for 30 minutes.
After cooling, 2 ml of methylation agent was added. The methylation agent was a 13:11 (v:v) mixture of 6.00 N hydrochloric acid and methanol. The tubes were vortexed and kept at 80°C for 10 minutes.
Again the tubes were cooled to room temperature and 1.25 ml of extraction solvent (a solvent with even volumes of hexane and methyl tert-butyl ether) was added. For 10 minutes the tubes were then mixed slowly, end-over-end, to achieve a good mass transfer to the solvent. The organic (top) phase was washed with a sodium hydroxyl solution and injected in a gas chomatograph (GC). The used GC had a 25 m × 0.2 mm capillary column and a temperature trajectory of 170-270°C was used at a rate of 5°C/min. Gas flow: 1.5 ml/ min. Carrier gas: He and make-up gas: N2. Detector type: flame ionisation. Fatty acids were identified with the Sherlock identification system.
Nuclear Magnetic Resonance (NMR)
Samples were dissolved in deuterated chloroform (CDCl3) and 1H NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at 400 MHz.
Size Exclusion Chromatography (SEC)
Size exclusion chromatography (SEC) was used to assess the molecular weight distribution of polymers in solution based on a standard curve (Wong et al. 2004). The SEC system consists of a Polymers Labs (UK) pump connected to a PLgel, 10 μm, MIXED column. Chloroform (HPLC grade) was used as the eluent at 1 ml/min. Before use the column was calibrated with polystyrene standards of molecular weights between 580 and 3,000,000 Da.
PHB concentration and statistical analysis
In one-stage batch fermentations bacterial cells grow in cell number and mass and simultaneously accumulate intracellular polymer. The PHB concentration, which is found by multiplying the total cell dry weight (g/l) by the PHB content (wt%), is the quantity that gives an indication when the broth contains an optimal amount of PHB. Fermentation data were fitted with second-order polynomial functions. Two-way ANOVA was used to compare data sets in GraphPad Prism. The highest PHB data points were compared to the next lower ones with a Student t-test.