Production Of 1,3-propanediol By Clostridium Butyricum P50b1 Using Commercial And Raw Glycerol From Biodiesel Production.

PRODUCTION OF 1,3-PROPANEDIOL BY CLOSTRIDIUM BUTYRICUM

P50B1 USING COMMERCIAL AND RAW GLYCEROL FROM BIODIESEL PRODUCTION

WIEN KUSHARYOTO1,*, DIAN ANDRIANI1, MARTHA SARI1, NUNIK SULISTINAH2, BAMBANG SUNARKO2

1

Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI) Cibinong Science Center, Jalan Raya Bogor Km. 46, Cibinong-Bogor 16911

Tel.: 021-8754587 Fax.: 021-8754588 2

Research Center for Biology, Indonesian Institute of Sciences (LIPI) Cibinong Science Center, Jalan Raya Bogor Km. 46, Cibinong-Bogor 16911

Tel.: 021-87907612 Fax.: 021-87907612

* Corresponding Author. E-mail: wien.kyoto@yahoo.com

ABSTRACT

Production of biodiesel (fatty acid methyl esters) by transesterification of palm oil generates glycerol as the main by-product. Different strains of Clostridium butyricum

are able to produce 1,3-propanediol (1,3-PD) from glycerol. A new strain of C. butyricum was obtained during a screening for natural producers of 1,3-propanediol, and designated C. butyricum P50B1. Growth inhibition of C. butyricum P50B1 by commercial glycerol and raw glycerol from a palm oil-based biodiesel production process was evaluated. The strain exhibited the same tolerance to raw glycerol (87% w/v) and to commercial glycerol (87% w/v). Furthermore, 1,3-propanediol production from commercial and raw glycerol, without any prior purification, was observed in batch and fed-batch cultures on a synthetic medium. No significant differences were found in C. butyricum P50B1 fermentation patterns on raw and commercial glycerol as the sole carbon source. For both types of cultures, the conversion yield obtained was around 0.6 mol of 1,3-propanediol formed per 1 mol glycerol consumed. The highest end-concentration of 1,3-propanediol achieved during fed-batch cultures was around 57 g∙l‾1, with productivity of 0.95 g∙l‾1∙ h‾1.

Keywords: 1,3-propanediol, biodiesel, Clostridium butyricum P50B1, palm oil, raw glycerol

1. INTRODUCTION

Biodiesel (fatty acid methyl esters), which is derived from triglycerides by transesterification with short chain alcohols, has attracted considerable attention during the past few decades as a renewable, biodegradable, and non-toxic fuel. Synthesis of biodiesel generates a main by-product, glycerol, which represents 10% (w/v) of the transesterification products. If biodiesel became a significant product of an

oleochemical unit operation, capture of even a relatively small portion of the current non-renewable diesel market would result in a large increase in the amount of glycerol available for the marketplace. With increasing supplies of glycerol will come a decrease in cost. Therefore, the oleochemical sectors will need to find ways to move this unavoidable co-product to market, and to seek new product introductions based on glycerol in order to better balance supply and demand.

Microbial conversion of glycerol to 1,3-propanediol (1,3-PD) is particularly attractive in that the process is relatively easy and does not generate toxic by-products. 1,3-PD has numerous applications in polymers, cosmetics, foods, lubricants, and medicines. Industrial 1,3-PD production has attracted attention as an important monomer to synthesize a new type of polyester PTT, (polytrimethylene terephthalate).

Several bacterial groups ferment glycerol and produce 1,3-PD in significant quantities. They include species of Citrobacter, Klebsiella (Menzel et al., 1997; Ahrens

et al., 1998), Clostridium (Abbad-Andaloussi et al., 1995; Reimann et al., 1998; Biebl

et al., 1999) and Lactobacillus (Schutz and Radler, 1984). Currently, Clostridium butyricum VPI 3266 is considered as probably the best natural 1,3-PD producer, since production of 1,3-PD by this strain is not a B12-vitamin dependent process (Saint Amans et al., 2001).

Recent studies on the prodiction of 1,3-PD have been conducted using pure or commercial glycerol, since in industrial or raw glycerol, the salts released from the transesterification of oils, exert significant inhibitory effects on microbial cell growth. Only few isolated C. butyricum strains have been reported to grow on technical glycerol (Petitdemange et al., 1995). However, since 50% of the entire cost of the production of 1,3-PD is due to the price of raw materials, raw glycerol from biodiesel production processes may be an interesting renewable carbon source for the microbial conversion. Recently, we reported on the utilization of raw glycerol from palm oil-based biodiesel production as carbon source for 1,3-PD production by C. butyricum NRRL B-1024 (Kusharyoto et al., 2010)

In this study, the ability of a newly isolated C. butyricum P50B1 to produce 1,3-PD was investigated. Raw glycerol from palm oil-based biodiesel production was used as sole carbon and energy source. The inhibitory effects of commercial and raw glycerol

on cell growth, as well as the formation of the major metabolites of the strain (1,3-PD, acetate and butyrate) were studied in anaerobic batch and fed-batch cultures.

2. MATERIALS AND METHODS

2.1. Microorganism and growth medium

The bacterium C. butyricum P50B1 was isolated in a screening for natural producers of 1,3-PD as previously described (Andriani et al., 2010) and maintained in Reinforced Clostridial Medium (RCM, Oxoid) on agar plates. Precultures and bioreactor cultures were carried out in M9-Medium containing (per liter): 50 g glycerol; 9.09 g KH2PO4; 0.535 g NH4Cl; 0.123 g MgSO4 · 7H2O; 0.017 g CaSO4 · 2H2O; 0.01 g FeSO4 · 7H2O; 2.0 g yeast extract; 1.0 ml resazurine 0,1 %; 0.25 g L-cysteine · HCl, and 10 ml of trace elements solution DSMZ 144. The pH-value of the medium was adjusted to pH 7.0 by the addition of 5 M NaOH. The carbon sources used were commercially available glycerol (Merck, purity 87% w/v) and raw glycerol (PT Sumi Asih, Jatimulya-Bekasi) obtained as by-product of a palm oil-based biodiesel production (purity 87% w/v).

2.2. Measurement of growth inhibition

The experiments for the measurement of growth inhibition were performed in flasks containing 50 ml RCM and commercial or raw glycerol at concentrations of 0, 20, 40, 60 and 100 g∙l‾1, r e sp e c t iv e l y. Each flask was inoculated with 1 ml cells in early exponential growth phase, and incubated at 37°C. Growth was monitored every 2 h by optical density (OD) measurement at 660 nm. The maximum specific growth rate of C. butyricum P50B1 on the different types and concentrations of glycerol, was determined from the slope of the least square regression lines of the logarithm of OD vs time data.

Inhibition (%) in each case was determined from the following equation: % Inhibition = (1 – µi/µ0)  100

where µi is the maximum specific growth rate in experiment i and µ0 is the maximum specific growth rate of the control experiment (glycerol concentration 0 g∙l‾1).

2.3. Batch fermentations without pH-regulation

Batch fermentations were carried out in 500 ml Schott bottles containing 300 ml M9 medium at 37°C. The medium contained 50 g∙l‾1 glycerol (commercial or raw glycerol). Preculture was prepared by the inoculation of a bacterial colony into 30 ml medium, and incubated for 24 h at 37°C without agitation. Fermentation was initiated by the inoculation of the preculture into the medium. In order to ascertain the anaerobiosis during the first fermentation steps of the culture (until 5 h after inoculation), nitrogen gas was flushed into the medium at a rate of ± 0.05 l ∙ h‾1. and the bottles were tightly closed thereafter.

2.3. Fed-batch fermentations

Fed-batch fermentations were carried out in a 5 liter bioreactor (Eyela) containing a total medium volume of 2.5 l. The first preculture was prepared by the inoculation of a bacterial colony into 25 ml medium, and incubated for 24 h at 37°C without agitation. This preculture was inoculated into a second preculture containing 225 ml medium, and incubated for 24 h at 37°C on a shaker. Fermentation was initiated by the inoculation of the second preculture into the fermentation medium. Initial glycerol concentration was 50 g∙l‾1 and additional glycerol was pumped into the medium during fermentation at a rate of ± 0.01 g∙l‾1∙ min‾1. The incubation temperature was 37°C, the agitation speed 200 rpm, and the pH was adjusted to 7.0 ± 0.1 by automatic addition of 5 M NaOH. An anaerobic environment in the bioreactor was maintained by sparging with nitrogen gas at a rate of ± 0.05 l ∙ h‾1.

2.4. Analytical procedures

Cell concentration was measured turbidometrically at 660 nm. Glycerol concentrations during fermentation were assayed enzymatically using the enzymes glycerol kinase and glycerol-3-phosphate oxidase as previously described (Kusharyoto

acid) was carried out with a Shimadzu HPLC system equipped with a refractive index detector. Separation was performed on a Rezex ROA column (300 mm  7.8 mm; Phenomenex Inc.) using 0.05 M sulphuric acid as mobile phase at a rate of 0.5 ml ∙ min‾1 and column temperature of 60ºC.

3. RESULTS AND DISCUSSION

3.1. Growth inhibition of C. butyricum P50B1 by glycerol

The effect of commercially available glycerol (87% w/v) and of raw glycerol (87% w/v) on the growth of C. butyricum P50B1 is depicted in Fig. 1. No inhibitory effect was observed with 20 g∙l‾1 of any kind of glycerol. The percentage of growth inhibition increased linearly with commercial and raw glycerol concentrations between 20 and 100 g∙l‾1. Up to 100 g∙l‾1 the percentages of growth inhibition by commercial and raw glycerol were very similar.

Fig. 1: Growth inhibition of Clostridium butyricum P50B1 by commercial and raw glycerol

Previously, the inhibitory effect of increasing glycerol concentration on the growth of C. butyricum DSM 5431 (Petitdemange et al., 1995), C. butyricum VPI 3266 (Gonzalez-Pajuelo et al., 2004) and C. butyricum NRRL B-1024 (Kusharyoto et al., 2010) has been observed as well. At 100 g∙l‾1 glycerol, growth was inhibited by around 60%, either using commercial glycerol raw glycerol (92% or 87% purity). This value is

Glycerol concentration [g . _l-1_]

20 40 60 80 100

Gro

wth

inh

ibit

ion

[%

]

0 20 40 60 80

Commercial glycerol Raw glycerol

close to those obtained in these experiments using 87% commercial glycerol and 87% raw glycerol, respectively. Apparently, C. butyricum P50B1 has the same tolerance to raw and to commercial glycerol when both have a similar grade of at least 87%.

3.2. Batch fermentations

The effect of raw glycerol on the production of 1,3-PD by C. butyricum P50B1 was studied in batch fermentations without pH regulation using commercial and raw glycerol as the sole carbon source, respectively. The medium contained glycerol at a concentration of 50 g∙l‾1, and the initial pH value of the medium was adjusted to 7.0. End-products of fermentation were analyzed 52 h after inoculation.

Based on HPLC analysis, the residual concentration of commercial glycerol in the fermentation broth was 17.4 g∙l‾1, which corresponded to glycerol utilization of 65%, while the residual concentration of raw glycerol was 18.9 g∙l‾1 or similar to glycerol consumption of 62%. The final concentration of 1,3-PD obtained by the fermentation of commercial glycerol was 16.8 g∙l‾1, with a molar yield of 0.62 mol 1,3-PD formed for every mol glycerol consumed. Using raw glycerol as the carbon source, a final concentration of 15.1 g∙l‾1 1,3-PD was obtained, which corresponded to a molar yield of 0.59 mol 1,3-PD/mol glycerol consumed (Tabel 1). Thus, there was no significant difference in the molar yields of 1,3-PD in batch fermentation using either commercial or raw glycerol.

Table 1: Batch fermentation without pH-regulation of commercial and raw glycerol by Clostridium butyricum P50B1

Type of glycerol grade (w/v)

Glycerol

consumed [g∙l‾1]

End-concentration of 1,3-PD [g∙l‾1]

Molar yields [mol 1,3-PD/mol glycerol]

Commercial (87%)

32.6 (65%) 16.8 0.62

3.3. Fed-batch fermentations

The ability of C. butyricum P50B1 to produce 1,3-PD from commercial and raw glycerol, respectively, was also shown in fed-batch fermentations. The initial concentration of glycerol in the medium was 50 g∙l‾1 and the pH value of the medium was maintained at pH 7.0 ± 0.1. During the fermentation, commercial and raw glycerol (80% w/v) was added into the culture at a rate of 0.01 g∙l‾1∙ min‾1, respectively. Cell-density and glycerol consumption during fed-batch fermentations are depicted in Fig. 2.

Fig. 2: Cell growth and glycerol consumption by Clostridium butyricum P50B1 in fed-batch fermentation using commercial and raw glycerol as carbon source, respectively

1,3-PD was the major fermentation end-product, with acetic, butyric and lactic acid as the main by-products. In every case, the glycerol utilization was similar, being 81% for commercial glycerol and 79% for raw glycerol. The final concentrations of 1,3-PD were almost equal, being 33.9 g∙l‾1 with commercial glycerol, and 31.7 g∙l‾1 with raw glycerol. In case of commercial glycerol, the molar yield was 0.60 mol 1,3-PD/mol glycerol with a productivity of 0.77 g∙l‾1∙h‾1, while using raw glycerol the molar yield was 0.57 mol 1,3-PD/mol glycerol with a productivity of 0.66 g∙l‾1∙h‾1. Thus, our results showed that there were only slight differences in the molar yields of 1,3-PD and productivities for the cultures using commercial and raw glycerol. The fermentation patterns of C. butyricum P50B1 grown on commercial or raw glycerol were also similar (Table 2).

C ultu re [hou rs]

0 10 20 30 40 50

OD at 660 nm 0 1 2 3 4 5 Gly cer ol c on cen tra tio n [ g

. l -1] 10 20 30 40 50 60

OD (com m . glycerol) OD (raw glycerol)

Concentration of com m . glycero l Concentration of raw glycerol

Table 2: Fed-batch cultures of Clostridium butyricum P50B1 on commercial and raw glycerol at 37°C

Type of glycerol

Commercial glycerol Raw glycerol

Glycerol total [g∙l‾1] 86.60 85.2

Glycerol consumed [g∙l‾1] 70.20 (81%) 68.5 (80%) End-product concentration [g∙l‾1]

1,3-PD 35.6 33.2

Lactic acid 1.25 0.93

Acetic acid 2.46 4.81

Butyric acid 12.6 10.9

Molar yields

[mol 1,3-PD/mol glycerol] 0.61 0.59

Productivity [g∙l‾1∙h‾1] 0.71 0.66

In order to increase the final concentration of 1,3-PD in fermentation broth, fed-batch fermentation was performed with a higher feeding rate of raw glycerol (80 g∙l‾1) at 0.02 g∙l‾1∙ min‾1 and feeding of yeast extract at a concentration of 40 g∙l‾1. The final concentration of 1,3-propanediol which could be achieved during this fermentation was around 57 g∙l‾1, with a molar yield of 0.53 mol 1,3-PD/mol glycerol and productivity of 0.95 g∙l‾1∙ h‾1 (Table 3).

Table 3: Fed-batch fermentation of Clostridium butyricum P50B1 on raw glycerol at 37°C with feeding rate of raw glycerol at 0.02 g∙l‾1∙ min‾1 and feeding of yeast extract at a concentration of 40 g∙l‾1.

Type of glycerol Raw glycerol

Glycerol total [g∙l‾1] 140

Glycerol consumed [g∙l‾1] 130.5 (87%)

End-product concentration [g∙l‾1]

1,3-PD 57.5

Lactic acid 0.35

Acetic acid 6.53

Butyric acid 9.73

Molar yields [mol 1,3-PD/mol glycerol] 0.53

The results obtained in the present work are similar to those obtained previously with 30 g∙l‾1 commercial glycerol, where 14 g∙l‾1 1,3-PD, 0.4 g∙l‾1 acetate and 5.1 g∙l‾1 butyrate were obtained in continuous fermentation by C. butyricum VPI 3266, leading to a yield of 0.65 mol 1,3-PD/mol glycerol (Saint-Amans et al., 2001). Petitdemange et al. (1995) also reported that an isolated strain, C. butyricum E5, could produce 58 g∙l‾1 1,3-PD from 109 g∙l‾1 raw glycerol in fed-batch culture. Another newly isolated strain, C. butyricum F2b, was also shown to consume raw glycerol (65% grade) in a medium containing 1 g∙l‾1 yeast extract, and yielded around 0.66 mol 1,3-PD/mol glycerol consumed in a continuous culture fed with 90 g∙l‾1 glycerol (Papanikolau et al., 2000). All of the results showed that the effect of raw glycerol on batch, fed-batch and continuous cultures was minimal, and it did not interfere with 1,3-PD production.

4. CONCLUSION

In the present work, 1,3-PD could be produced in anaerobic batch and fed-batch fermentations of raw glycerol from palm-oil based biodiesel production by a newly isolated C. butyricum P50B1. No significant differences were observed with regards to 1,3-PD final concentration, 1,3-PD yield and volumetric productivity in fermentations usimg either raw glycerol or commercial glycerol. These facts reveal that raw glycerol from palm-oil based biodiesel production is an interesting feedstock or f biotechnological production of 1,3-PD. However, the economical viability of this process will be largely dependent on raw glycerol price and its availability.

5. ACKNOWLEDGEMENT

The authors would like to thank PT Sumi Asih, Jatimulya-Bekasi for providing with raw glycerol samples, and Suri Handayani for technical assistance. This work was supported by a grant from Competitive Research Program of LIPI.

6. REFERENCES

Abbad-Andaloussi, S., C. Manginot, C. Durr, J. Amine, E. Petitdemange and H. Petitdemange (1995) Isolation and characterization of Clostridium butyricum

DSM-5431 mutants with increased resistance to 1,3-propanediol and altered production of acids. Appl. Environ. Microbiol. 61: 4413-4417

Ahrens, K., K. Menzel, A.P. Zeng and W.-D. Deckwer (1998) Kinetic, dynamic and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture. III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation. Biotechnol. Bioeng. 59: 544–552

Andrade, J.C. and I. Vasconcelos (2003) Continuous cultures of Clostridium acetobutylicum: culture stability and low grade glycerol utilization. Biotechnol. Lett. 25: 121–125

Andriani, D., W. Kusharyoto, B. Prasetya, G.-T. Jeong, D.-H. Park, T. Willke and K.D. Vorlop (2010) Screening for natural producers capable of producing 1,3-propanediol from glycerol. ASEAN-Korea Symposium and Workshop on Biorefinery Technology. Jakarta, 17-18 February 2010.

Biebl, H., K. Menzel, A. P. Zeng and W.-D Deckwer (1999) Microbial production of 1,3-propanediol. Appl. Microbiol. Biotechnol. 52: 289-297

Gonzalez-Pajuelo, M., J. C. Andrade and I. Vasconcelos (2004) Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium and raw glycerol. J. Ind. Microbiol. Biotechnol. 31: 442-446

Gonzalez-Pajuelo, M., J. C. Andrade and I. Vasconcelos (2005) Production of 1,3-propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity. J. Ind. Microbiol. Biotechnol. 32: 391-396

Himmi, E. H, A. Bories and F. Barbirato (1999) Nutrient requirements for glycerol conversion to 1,3- propanediol by Clostridium butyricum. Bioresorce Technol.

67: 123-128

Kusharyoto, W., M. Sari, N. Sulistinah and B. Sunarko (2010) Microbial conversion of glycerol to 1,3-propanediol by Clostridium butyricum NRRL B-1024. ASEAN-Korea Symposium and Workshop on Biorefinery Technology. Jakarta, 17-18 February 2010.

Menzel K, A-P Zeng and W-D Deckwer (1997) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microbiol. Technol. 28: 82–86

Mu, Y., H. Teng, D-J. Zhang, W. Wang and Z-L. Xiu (2006) Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations. Biotechnol. Lett. 28: 1755–1759

Papanikolaou, S., P. Ruiz-Sanchez, B. Pariset, F. Blanchard and M. Fick (2000) High production of 1,3-propanediol from industrial glycerol by a newly isolated

Clostridium butyricum strain. J. Biotechnol. 77: 191–298

Petitdemange, E., C. Durr, S. A. Andaloussi and G. Raval (1995) Fermentation of raw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. J. Ind. Microbiol. 15: 498–502

Reimann, A., S. Abbad-Andaloussi, H. Biebl and H. Petitdemange (1998) 1,3-Propanediol formation with product tolerant mutants of Clostridium butyricum

DSM 5431 in continuous culture: productivity, carbon and electron flow. J. Appl. Microbiol. 84: 1125–1130

Saint-Amans, S., L. Girbal, J. Andrade, K. Ahrens and P. Soucaille (2001) Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures. J. Bacteriol. 183:1748–1754

Schutz, H. and F. Radler (1984) Anaerobic reduction of glycerol to 1,3-propanediol by

Lactobacillus brevis and L. buchneri. Syst. Appl. Microbiol. 5: 169–178.

Xu Y.Y., W. Du, D.H. Liu and J. Zeng (2003) A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium. Biotechnol. Lett. 25: 1239–1241

Zeng A. P. (1996) Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum. Bioprocess Eng. 14: 169-175

close to those obtained in these experiments using 87% commercial glycerol and 87% raw glycerol, respectively. Apparently, C. butyricum P50B1 has the same tolerance to raw and to commercial glycerol when both have a similar grade of at least 87%.

3.2. Batch fermentations

The effect of raw glycerol on the production of 1,3-PD by C. butyricum P50B1 was studied in batch fermentations without pH regulation using commercial and raw glycerol as the sole carbon source, respectively. The medium contained glycerol at a concentration of 50 g∙l‾1, and the initial pH value of the medium was adjusted to 7.0. End-products of fermentation were analyzed 52 h after inoculation.

Based on HPLC analysis, the residual concentration of commercial glycerol in the fermentation broth was 17.4 g∙l‾1, which corresponded to glycerol utilization of 65%, while the residual concentration of raw glycerol was 18.9 g∙l‾1 or similar to glycerol consumption of 62%. The final concentration of 1,3-PD obtained by the fermentation of commercial glycerol was 16.8 g∙l‾1, with a molar yield of 0.62 mol 1,3-PD formed for every mol glycerol consumed. Using raw glycerol as the carbon source, a final concentration of 15.1 g∙l‾1 1,3-PD was obtained, which corresponded to a molar yield of 0.59 mol 1,3-PD/mol glycerol consumed (Tabel 1). Thus, there was no significant difference in the molar yields of 1,3-PD in batch fermentation using either commercial or raw glycerol.

Table 1: Batch fermentation without pH-regulation of commercial and raw glycerol

by Clostridium butyricum P50B1

Type of glycerol grade (w/v)

Glycerol

consumed [g∙l‾1]

End-concentration of 1,3-PD [g∙l‾1]

Molar yields [mol 1,3-PD/mol glycerol]

Commercial (87%)

32.6 (65%) 16.8 0.62

3.3. Fed-batch fermentations

The ability of C. butyricum P50B1 to produce 1,3-PD from commercial and raw glycerol, respectively, was also shown in fed-batch fermentations. The initial concentration of glycerol in the medium was 50 g∙l‾1 and the pH value of the medium was maintained at pH 7.0 ± 0.1. During the fermentation, commercial and raw glycerol (80% w/v) was added into the culture at a rate of 0.01 g∙l‾1∙ min‾1, respectively. Cell-density and glycerol consumption during fed-batch fermentations are depicted in Fig. 2.

Fig. 2: Cell growth and glycerol consumption by Clostridium butyricum P50B1 in fed-batch fermentation using commercial and raw glycerol as carbon source, respectively

1,3-PD was the major fermentation end-product, with acetic, butyric and lactic acid as the main by-products. In every case, the glycerol utilization was similar, being 81% for commercial glycerol and 79% for raw glycerol. The final concentrations of 1,3-PD were almost equal, being 33.9 g∙l‾1 with commercial glycerol, and 31.7 g∙l‾1 with raw glycerol. In case of commercial glycerol, the molar yield was 0.60 mol 1,3-PD/mol glycerol with a productivity of 0.77 g∙l‾1∙h‾1, while using raw glycerol the molar yield was 0.57 mol 1,3-PD/mol glycerol with a productivity of 0.66 g∙l‾1∙h‾1. Thus, our results showed that there were only slight differences in the molar yields of 1,3-PD and productivities for the cultures using commercial and raw glycerol. The fermentation patterns of C. butyricum P50B1 grown on commercial or raw glycerol were also similar (Table 2).

C ultu re [hou rs]

0 10 20 30 40 50

OD at 660 nm 0 1 2 3 4 5 Gly cer ol c on cen tra tio n [ g

. l -1] 10 20 30 40 50 60

OD (com m . glycerol) OD (raw glycerol)

Concentration of com m . glycero l Concentration of raw glycerol

Table 2: Fed-batch cultures of Clostridium butyricum P50B1 on commercial and raw glycerol at 37°C

Type of glycerol

Commercial glycerol Raw glycerol

Glycerol total [g∙l‾1] 86.60 85.2

Glycerol consumed [g∙l‾1] 70.20 (81%) 68.5 (80%) End-product concentration [g∙l‾1]

1,3-PD 35.6 33.2

Lactic acid 1.25 0.93

Acetic acid 2.46 4.81

Butyric acid 12.6 10.9

Molar yields

[mol 1,3-PD/mol glycerol] 0.61 0.59

Productivity [g∙l‾1∙h‾1] 0.71 0.66

In order to increase the final concentration of 1,3-PD in fermentation broth, fed-batch fermentation was performed with a higher feeding rate of raw glycerol (80 g∙l‾1) at 0.02 g∙l‾1∙ min‾1 and feeding of yeast extract at a concentration of 40 g∙l‾1. The final concentration of 1,3-propanediol which could be achieved during this fermentation was around 57 g∙l‾1, with a molar yield of 0.53 mol 1,3-PD/mol glycerol and productivity of 0.95 g∙l‾1∙ h‾1 (Table 3).

Table 3: Fed-batch fermentation of Clostridium butyricum P50B1 on raw glycerol at 37°C with feeding rate of raw glycerol at 0.02 g∙l‾1∙ min‾1 and feeding of yeast extract at a concentration of 40 g∙l‾1.

Type of glycerol Raw glycerol

Glycerol total [g∙l‾1] 140

Glycerol consumed [g∙l‾1] 130.5 (87%)

End-product concentration [g∙l‾1]

1,3-PD 57.5

Lactic acid 0.35

Acetic acid 6.53

Butyric acid 9.73

Molar yields [mol 1,3-PD/mol glycerol] 0.53

The results obtained in the present work are similar to those obtained previously with 30 g∙l‾1 commercial glycerol, where 14 g∙l‾1 1,3-PD, 0.4 g∙l‾1 acetate and 5.1 g∙l‾1 butyrate were obtained in continuous fermentation by C. butyricum VPI 3266, leading to a yield of 0.65 mol 1,3-PD/mol glycerol (Saint-Amans et al., 2001). Petitdemange et al. (1995) also reported that an isolated strain, C. butyricum E5, could produce 58 g∙l‾1 1,3-PD from 109 g∙l‾1 raw glycerol in fed-batch culture. Another newly isolated strain, C. butyricum F2b, was also shown to consume raw glycerol (65% grade) in a medium containing 1 g∙l‾1 yeast extract, and yielded around 0.66 mol 1,3-PD/mol glycerol consumed in a continuous culture fed with 90 g∙l‾1 glycerol (Papanikolau et al., 2000). All of the results showed that the effect of raw glycerol on batch, fed-batch and continuous cultures was minimal, and it did not interfere with 1,3-PD production.

4. CONCLUSION

In the present work, 1,3-PD could be produced in anaerobic batch and fed-batch fermentations of raw glycerol from palm-oil based biodiesel production by a newly isolated C. butyricum P50B1. No significant differences were observed with regards to 1,3-PD final concentration, 1,3-PD yield and volumetric productivity in fermentations usimg either raw glycerol or commercial glycerol. These facts reveal that raw glycerol from palm-oil based biodiesel production is an interesting feedstock or f biotechnological production of 1,3-PD. However, the economical viability of this process will be largely dependent on raw glycerol price and its availability.

5. ACKNOWLEDGEMENT

The authors would like to thank PT Sumi Asih, Jatimulya-Bekasi for providing with raw glycerol samples, and Suri Handayani for technical assistance. This work was supported by a grant from Competitive Research Program of LIPI.

6. REFERENCES

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