Conversion of Glycerol into Polyhydroxybutyrate(PHB)UsingEscherichia coli

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1 Conversion of Glycerol into Polyhydroxybutyrate(PHB)UsingEscherichia coli Endah Fitriani Rahayu a, Wega Trisunaryanti b, Karna Wijaya b Abstract Conversion of glycerol into polyhydroxybutyrate (PHB) usingescherichia coli bacteria had been evaluated. The bacteria was cultured in lactose medium, consist of % of lactose, peptone, and NaCl, then incubated at 7 C for h. The bacteria culture was then added into the g/l, g/l, g/l, g/l, and 5 g/l glycerol media and then fermented for, 8, 7, and 6 h, then followed by centrifugation and solid phase was dried and weighed as a biomass. After centrifugation, the filtrate was analysed to determine the remaining glycerol. The PHB was extracted from the biomass using chloroform, and then analysed by FT-IR, H-NMR, C-NMR and viscometer. Monomer unit of the polymer was produced by esterification of the polymer then analysed by GC. Analysis results of FT-IR, H-NMR, C-NMR, and GC showed that the PHB can be synthesized from glycerol usingescherichia coli. The PHB production was increase following the increase of fermentation time and initial glycerol concentration, with the highest PHB percentage was 9.7%. The optimum molecular weight of the PHB (77 g/mol) was achieved at initial glycerol concentration of g/l and fermentation time of 96h. Keywords:Escherichia coli; fermentation; glycerol; polyhydroxybutyrate a Department of Chemistry, Semarang State University b Department of Chemistry, GadjahMada University *Corresponding author address: endahfitrianirahayu@yahoo.com Introduction Polyhydroxybutyrate is a short chain polyhydroxyalkanoates, it has thermoplastic characteristic [5], high resistance, and high oxygen permeability []. Polyhydroxybutyrate (PHB) is represent a class of compounds with physical-chemical characteristics similar to polypropylene, but are environmentally compatible and totally biodegradable. Polyhydroxybutyrate can be synthesized by numerous bacterial species from many carbon substrates. Some carbon source that can be used to produce polyhydroxybutyrate are sugars [], oleic acid [6], and glycerol [;7]. Although it is feasible for these carbon source to be used to produce polyhydroxybutyrate in the laboratory, high production costs in large scale commercial production and the cost of feedstock fermentation can account for up to 5% of overall production cost. Among several carbon source, glycerol is the potential source for fermentation process that produce polyhydroxybutyrate. Glycerol is a major byproduct of the biodiesel industry that approximately % of the final weight of biodiesel production. As biodiesel production has increased dramatically from. million gallons in998 to million gallons in [], crude glycerol generated from trans esterification of vegetable oil has also been produced in large quantities. Recent publications [7] described fermentation of biodiesel-glycerol to polyhydroxybutyrate by Burkholderiacepacia with polyhydroxybutyrate production of 8.9% of total dry microbial biomass during 96 hours. in addition, Ashby et al., demonstrated the production of polyhydroxybutyrate by Pseudomonas oleovorans with glycerol as carbon source. This research revealed that crude glycerol can be used to produce.% polyhydroxybutyrate of total dry cell biomass. in order to examine alternative uses for biodiesel waste glycerol, small scale fermentations were initially performed byescherichia coli in shake flasks and analysed for polyhydroxybutyrate production using glycerol as a carbon source. Material and Methods Bacterial strains and culture conditions Escherichia coli were cultured in lactose medium, including % of each lactose, peptone, and NaCl. Bacterial culture was incubated at 7 C for hours. 56 P a g e Green Chemistry Section : Physical Chemistry, Endah Fitriani Rahayu, et al.

2 Synthesis polyhydroxybutyrate (PHB) Glycerol as a carbon source was initially added into medium. Bacterial culture % (v/v) added into glycerol medium and was then fermented at 7 C. After then followed by centrifugation at g for min. The biomass was subsequently washed with distilled water and centrifuged to remove supernatant. The remaining glycerol analysed by the previous procedure [].Thepolymerwas then extracted from biomass using chloroform and stirred for h, then precipitated by soaking it into cold methanol. Polyhydroxybutyrate characterization The polymer was analysed using proton NMR, carbon NMR, XRD, and molecular mass of the polymer were determined from viscosity of polyhydroxybutyrate solution. The methyl ester monomer was determined by gas chromatography under the following procedure: 5 mg of dry biomass was mixed with ml of H SO /methanol volume ratio (5/85) and ml chloroform, heated at C for min. The product of esterification was then characterized by GC. Result and Discussion PHB produced was characterized with FT-IR spectrometer, NMR 5MHz spectrometer to determine the structure, XRD and GC were used to determine crystallinity and yield. FT-IR FT-IR spectrum of glycerol (a), polymer (b), and esterification product of polymer were presented in Figure. Glycerol spectrum was showed absorption band at 79,9 cm - of O-H stretching vibration. The C-H stretching appears at 99,5 cm - and C-H bending appears at - cm -.Stretching vibrations of C-O groups in glycerol has the absorption band at - cm -.FT-IR spectrum of polymer revealed that the O-H stretching vibration was appeared in the wavenumber of. cm -.The spectrum had characteristic absorption bands of C-H, C=O, andc-o that are at 9,9 cm -,658,78 cm -,and 7, cm -, respectively.absorption bands C-H bending has also observed in the - cm -.The methyl ester monomers spectrum showed absorption bands at 6,7 cm -, 9,9 cm -, 65.7 cm -, and,65 cm -, each of which are vibrations of the O-H groups, C-H, C=O, and C-O.The three FT-IR spectrumrevealedthat PHB had been produced in this study. NMR Figure.FT-IR spectra of (a) glycerol, (b) PHB, (c) PHBmonomer End-capped polyhydroxybutyrate was analysed by H- NMR and C-NMR spectroscopy present in Figure (H- NMR) and Figure (C-NMR). Gas Chromatography Analysed with gas chromatography shows that the monomer of polymer resulted is -hydroxybutyric acid. The retention time of polymer Sampleis compare with the standard. Peak of polyhydroxybutyrate appear at retention time,7 min. X-Ray Diffraction To identify the PHB produced inescherichia coli its XRD pattern were recorded from -6 of θ, as seen in Figure. It can be seen that the characteristic peak values of θ of. ;. ; 5. ;. ;. dan 5.5 found in the PHB Sample were also in the standard PHB. The spectrum and molecular structure at Figure showed the characteristic resonances of PHB: chemical shift, ppm for C, C at chemical shift 5, ppm, C at chemical shift at. ppm. This results confirm that the polymer is polyhydroxybutyrate. Beside, other peaks showed the glycerol and metoxy end-capping of polyhydroxybutyrate. That was appeared resonance at, ppm to C and C6;,5 ppm to C7 and C8;,6 ppm untuk C9. The recomendedpolymerstructure is shows at the front of spectrum. Carbon NMR spectral analysis also showed the polymer structure as Figure. Chemical shift appears at 6.8 ppm for C:. ppm for C; 5. ppm for C and C6;. ppm and 57. ppm for C and C; 7.8 ppm for the C5, and 56, ppm for C9. Green Chemistry Section : Physical Chemistry, Endah Fitriani Rahayu, et al. P a g e 57

3 % PHB (b/b) converted glycerol (%) Dry cell weight (g/l) Figure.H-NMR spectrum of PHB glis g/l Fermentation time (h) Figure 5.The effect of fermentation time on dry cell weight At the beginning of fermentation process, glycerol was converted lower than the percentage ofphb produced.during 8 h fermentation time, there isother nutrients nitrogen are available.afterthat, possible amount of nitrogen discharged in the media because there are very limited. Figure.C-NMR spectrum of PHB The effect of fermentation time on converted glycerol (A) and %PHB (B) are presented in Figure 6. The percentage of PHB wassignificantlyincreasead as well as converted glycerol. The maximum PHB was producedat 96 h, which is 9,% PHB was produced Fermentation time(h) glis g/l Figure.Chromatogram of methylesther monomer of PHB (top), metil-hydroxybutyrate standard (bottom) Effect of fermentation time Figure 5 presence the effect of fermentation time on the production of PHB. Figure 5 revealedthat the longer of the fermentation time caused higher dry cells are produced until 96h.Fermentation time had a positive effect on cell growth inescherichia coli fermentation time (h) glis g/l Figure 6.The effect of fermentation time on converted glycerol (top) and %PHB (bottom) 58 P a g e Green Chemistry Section : Physical Chemistry, Endah Fitriani Rahayu, et al.

4 MW (x g/mol) dry cell weight (g/l) MW (x g/mol) % PHB Converted glycerol (%) The effect of fermentation time on molecular weight of PHB was shown in Figure 7. Molecular weight of PHB was increased with the increasing of fermentation time, until the initial glycerol concentration g/l in the media. At the initial glycerol concentration 5 g/l, molecular weight of PHB was decreased from h to 96h. This occurs because the Escherichia colicells has osmotic stress and begin to decreased enzyme activities in the formation of PHB.Maximum molecular weight of PHB obtained at 96h Figure 7.The effect of fermentation time on molecular weight of PHB The effect of Initial Glycerol Concentration The effect of the initial concentration of glycerol on bacteria growth was presented in Fig8. The greater dry cells weight as the large concentrations of glycerol were added into the media. Optimum dry cell weight was 5,6 g/l, produced at the initial concentration of glycerol 5 g/l fermentation time (h) glis g/l jam 5 6 initial concentration of gliserol (g/l) Figure 8.The effect of the initial concentration of glycerol on dry cell weight (b/b) Figure 9 shown the effect of initial glycerol concentration on converted glycerol and %PHB. The dry cell weight produces was increased following the increasing of initial glycerol concentration. The highest dry cell weight was produced at initial glycerol concentration 5 g/l Figure 9.The effect of glycerol concentration on converted glycerol and %PHB The effect of fermentation time on molecular weight of PHB was shown in Figure. Molecular weight of PHB was increased when the fermentation time was increased, until the initial glycerol concentration g/l at the media. At the initial glycerol concentration 5 g/l, molecular weight of PHB was decreased from fermentation time h to 96h. 8 6 Figure. Effect of initial concentration to molecular weight PHB Conclusion jam 5 initial concentration of glycerol (g/l) 5 6 initial concentration of glycerol (g/l) jam 5 6 initial cencentration of glycerol (g/l) jam The PHB can be synthesized using Escherichia coli bacteria and glycerol as carbon source through the fermentation process.the highest percentage of PHB produced in fermentation time of 96 hours and the concentration of glycerol 5 g/l.the highest molecular Green Chemistry Section : Physical Chemistry, Endah Fitriani Rahayu, et al. P a g e 59

5 weight PHB achieved on the initial concentration of glycerol g/l and the fermentation time of 96 hours. References. Amarilla, F; Fernandes, E.G; Mata, D, Quagliano, J.C; dan Miyazaki, S.S.. WorldJ. Microbiol. Technol. 7, 9-. Ashby, R.D.; Solaiman, D.K.Y.; Foglia, T.A.. J.Polym. Env, Vol., No., July.. Ashby, R.D.; Solaiman, D.K.Y.; Strahan, G.D.. J. Am. Oil. Chem.Soc, DOI.7/s Atifah, N; Fauzi, A.M; Hartoto, L; Suryani, A; Syamsu, K. 7. JurnalSainsdanTeknologi Indonesia Vol.9 No. Hlm, Chee, J.Y; Yoga, S.S; Lau, N.S; Ling, S.C; Abed, R.M.M; Sudesh, K.. Current Research, Technology, and Education Topics in Applied Microbiology and Microbiol Biotechnology. 6. Djamaan, A., Majid, M.I.A., Noor, M.A.M.. MajalahFarmasi Indonesia (), Nakas, J.P; Zhu, Chengjun; Nomura, C.T; Perotta, J.A; Stipanovic, A.J. 9. J. Biotechnol. Prog.,Vol 6, No.. 6 P a g e Green Chemistry Section : Physical Chemistry, Endah Fitriani Rahayu, et al.

Lactic acid production from rice straw using plant-originated Lactobacillus rhamnosus PN04

Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2016, 8(5):590-594 Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5 Lactic acid production from rice straw using plant-originated