Conversion of glycerol to ethanol and formate by Raoultella Planticola

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1 Conversion of glycerol to ethanol and formate by Raoultella Planticola Li Z.A.D 1., Chong W.K., Mathew, S., Montefrio, M.J.F. and Obbard J.P. 2 Division of Environmental Science and Engineering, National University of Singapore, WS2 #4- ABSTRACT 15, 1 Engineering Drive 3, S Formate and ethanol production from crude glycerol by Raoultella planticola was evaluated in this study. With an uptake of 4 % glycerol, we could achieve a final product of 1.7g/L formate and 2.2 g/l ethanol. Further acclimatization work for the organism with varying concentrations of glycerol resulted in a 9% conversion of glycerol to 1.g/L of formate. INTRODUCTION The fuels used for transportation still predominantly come from fossil sources (Yazdani and Gonzalez, 27). The growing concerns over their high cost, sustained availability, security and contribution to global warming has led to a search for technologies to produce fuel from renewable carbon sources, such as plant biomass. The last few years have seen a significant rise in biodiesel and bioethanol production as an alternative to fossil fuels (Dharmadi et.al., 26) Bioethanol is produced by microbial fermentation of sugars derived from corn, sugarcane or sugar beet, but there is growing interest in lingo-cellulosic sources. Biodiesel is produced by the transesterification of vegetable oils or animal fats with an alcohol to produce fatty acid methyl esters, using chemical or enzymatic catalysts. Biodiesel has advantages over bioethanol in that it has a higher energy density and does not require extensive engine modifications to the user vehicle. However, glycerol is an inevitable byproduct of the process. Although glycerol has its uses in cosmetics and food, the growth of the biodiesel industry has resulted in glycerol prices declining to the extent that it is no longer economical to recover it. Once considered a desirable co-product, glycerol has now become a waste stream with a high disposal cost associated to it. Developing new uses of glycerol is thus desirable. Recent studies of the conversion of waste glycerol to various compounds through microbial actions have been reported. Ito et.al., 25 and Sakai and Yagishita (25) converted glycerol to butanol, hydrogen and ethanol. There has also been focus on the production of 1,3-propanediol in studies by Papanikolaou, 2, and Mu, 26, as it is a valuable polymer raw material. There have been no papers on the anaerobic fermentation of waste glycerol for methane production. There are a few informal reports of its use online (Addison and Hiraga, 28), but these 1 Student 2 Professor

2 have not been focused studies. Methane production is a potential avenue as natural gas utilization technology and distribution is already well-developed. Our earlier experiments (Wong, 27) explored the use of glycerol digestion in the presence of wastewater sludge. The experiment found a negative effect on methane production with increasing amounts of glycerol added. The final ph of the product was found to be less than 5.5. It was suggested that the products of glycerol dissimilation by acidogenic bacteria i.e. acetic, propionic and other volatile acids - were the cause of the low ph. It was therefore proposed to convert the glycerol to volatile acids, so that the addition of the acids to the sludge could be controlle, and maintain the neutral ph required. Glycerol can be metabolized to a number of volatile acids and alcohols, as shown in figure 1. Figure 1. Products of Glycerol Metabolism (Biebl 1999) Anaerobic fermentation of glycerol by E. Coli has been carried out (Dharmadi, 26). E. coli produced ethanol at 3 g/l, and smaller quantities of succinate and acetate, from an initial amount of 1 g/l of glycerol. Formate produced was completely converted to CO 2 and H 2 by the bacteria. As it

3 is preferable to produce substrates in the liquid state, a literature search was conducted to identify bacteria that could accumulate formate. R. Planticola was found to produce 1.5 and 1.4 g/l of ethanol and formate, respectively, from an initial amount of 12 g/l of glycerol in? (Jarvis, 1997). In this study we investigate the fermentation of glycerol by Raoutella planticola to produce formate and ethanol. Formate can be used as a substrate for methanogenesis and ethanol recovered for use as fuel.

4 MATERIALS AND METHODS Microorganism The microorganism used in this study was Raoultella Planticola, obtained from American Type Culture Collection (#33531). The bacteria were maintained on Nutrient Broth plates (NB plates) at 4 o C. Culture Conditions Culture medium was based on a modified version of phosphate buffered medium (Jarvis 1997). The medium contained the following per liter: KH 2 PO g, K 2 HPO g, NH 4 Cl 1. g, MgCl 2.6H 2 O.2 g, CaCl 2.2H 2 O.1 g, 1 ml of trace mineral solution, and.1 g of yeast extract. The trace mineral solution contained the following per liter: Nitrilotriacetic acid.15 g, FeSO 4.7H 2 O.1 g, MnCl 2.4H 2 O.1 g, CoCl 2.6H 2 O.17 g, CaCl 2.2H 2 O.1 g, ZnCl 2.1 g, NiSO 4.6H 2 O.26 g, CuCl 2.2H 2 O.25 g, H 3 BO 3.1 g, Na 2 MoO 4.2H 2 O.1 g, NaCl 1. g, Na 2 SeO 3.16 g. Pure glycerol was added as the major carbon source at 2.5 or 1 g/l. Culture medium with glycerol is referred to as glycerol medium (GM). The pre-cultivation of the bacteria was carried out anaerobically in 25 ml serum vials containing 2mL of Jarvis modified medium. The cultures were incubated at 37 o C without agitation. A 1% (v/v) inoculum was inoculated into the experimental vials and samples were withdrawn at regular intervals. Analytical Methods Cell growth in liquid culture was monitored by measuring optical density at 6 nm. Glycerol, ethanol and formate were measured using HPLC (Shimadzu), with an ion-exclusion Metacarb H Plus 3 x 7.8 mm column (Varian, Palo Alto, CA) run at 4 o C. The mobile phase was 2 mm H 2 SO 4, running isocratically at.4 ml/min. A refractive index detector was used. To confirm the identity of formate and ethanol as products, addition of a prepared standards to samples was completed. Acclimatization Acclimatization was carried out after the first 1 g/l glycerol run, as the amount of total glycerol taken up was less than 5%. The first set of acclimatization, with a set consisting of 5 sub-cultures, was carried out on plates of NB, NB with glycerol at 5 g/l, GM at 5 g/l with acetate, GM at 5 g/l without acetate, and GM with acetate but without glycerol. No growth occurred on the plates with acetate but without glycerol, so all subsequent cultures were carried out without acetate. Due to poor growth on the first set, the second set of acclimatization was carried out with GM at 2.5 g/l. This acclimatized batch was subsequently used to inoculate the run at 2.5 g/l.

5 RESULTS AND DISCUSSION?? 14 A 2.5 Glycerol (g/l) Growth (OD) and Products (g/l) Time (h) 3.5 B Glycerol (g/l) Growth (OD) and Formate (g/l) Time (h) Figure 2. Fermentation of glycerol by R. Planticola. Profile for glycerol consumption (squares), cell growth as measured by O.D. (diamonds), and formate (triangles) and ethanol (crosses) accumulation. A: Initial glycerol at 1 g/l, with acetate. B: Initial glycerol at 2.5 g/l, without acetate. needs improving

6 With the starting glycerol at 1 g/l (A), glycerol consumption, growth and accumulation of products took place in the 12-36th hour. Glycerol uptake was 4%. Final product concentrations were correspondingly low, with formate at 1.7 g/l and ethanol at 2.2 g/l. This is a yield, of mol of product per mol of glycerol consumed, of about.7 for formate and.9 for ethanol. These results are higher than that reported by Jarvis, where the initial amount of glycerol was 12 g/l. Glycerol uptake was 5%, yielding 1.5 and 1.4 g/l of formate and ethanol, a yield of.5 for both. In the subsequent study at 2.5 g/l, glycerol consumption, growth and accumulation of products took place earlier and for a longer period, in the 8-48th hour. Glycerol uptake was 9%. The final formate concentration was at 1. g/l, a yield of about.65 mol per mol of glycerol consumed. Ethanol could not be measured in this run due to a problem encountered with the HPLC. With glycerol at 1 g/l, Enterobacter Aerogenes (Ito et al, 25) and Klebsiella Pneumoniae (Mu, 26) showed a more rapid and complete uptake of glycerol. E. Aerogenes completely consumed glycerol within 6 hours, while K. Pneumoniae consumed 9% in 1 hours. The bacteria also showed a better tolerance for a high concentration of glycerol. E. Aerogenes could completely consume glycerol of up to 25 g/l, while K. Pneumoniae could tolerate 4 g/l. Papanikolaou showed that a particular strain of Clostridium Butyricum could almost completely consume up to 9 g/l of glycerol in continuous culture conditions. E. Aerogenes produced ethanol at 1 g/l in a fed batch culture. This suggests the possibility that ethanol is not inhibiting the consumption of glycerol in our study. Dharmadi et al found that E. Coli ferments glycerol in a ph dependent manner. In E. Coli, formate produced is converted to CO 2 and H 2 by the action of Formate Hydrogen Lyase (FHL). It was shown that CO 2 is required for glycerol fermentation. When FHL activity is suppressed, the fermentation of glycerol is inhibited. In our study, the final formate and ethanol concentrations are similar, suggesting that formate was not decomposed to CO 2 and H 2. Jarvis reported that the concentration of formate decreased marginally over several hundred hours, with a stoichiometric increase in CO2 and H2. It is plausible that the uptake of glycerol by Raoultella Planticola is limited by its ability to break down formate to CO 2 and H 2. Glycerol Formate Ethanol Volts Figure 3. HPLC profile for the supernatant of samples

7 CONCLUSIONS Raoultella Planticola is able to convert glycerol to the desired ethanol and formate, but the current product concentrations are too low to be used as substrate for methane production. Further acclimatization work will be carried out with higher glycerol concentrations in an effort to raise the production of formate and ethanol. REFERENCES Addison K. and Hiraga M., "Glycerine: Journey to Forever", retrieved 28 Jan 28 from " Biebl H., Menzel K., Zeng A.P., Deckwer W.D. (1999), "Microbial production of 1,3-propanediol", Applied Microbiology and Biotechnology, Vol 52, No. 3, p Dharmadi Y., Murarka A., Gonzalez R. (26), "Anaerobic Fermentation of Glycerol by Escherichia coli: A New Platform for Metabolic Engineering", Biotechnology and Bioengineering., Vol. 94, No. 5, p Ito T. et al. (25), "Hydrogen and Ethanol Production from Glycerol-Containing Wastes Discharged after Biodiesel Manufacturing Process", Journal of Bioscience and Bioengineering Vol. 1, No.3, p Mu Y. et al. (26), "Microbial Production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel", Biotechnology Letters, Vol 28, No. 21, p Papanikolaou S. et al. (2), "High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain", Journal of Biotechnology, 77, p Sakai S. and Yagishita T. (27), "Microbial Production of Hydrogen and Ethanol from Glycerol- Containing Wastes Discharged from a Biodiesal Fuel Production Plant in a Bioelectrochemical Reactor with Thionine", Biotechnology and Bioengineering, Vol. 98, No. 2, p Wong J. (27), "Fermentation of Biodiesel Glycerol by Saccharomyces Cerevisiae and Anaerobic Sludge", National University of Singapore, Department of Chemical & Biomolecular Engineering, Undergraduate Final Year Project. Yazdani S.S. and Gonzalez R. (27), "Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry", Current Opinion in Biotechnology 27, 18: