Growth inhibition of Clostridium butyricum
VPI 3266 by raw glycerol, obtained from the biodiesel
production process, was evaluated. C. butyricum presents
the same tolerance to raw and to commercial
glycerol, when both are of similar grade, i.e. above 87%
(w/v). A 39% increase of growth inhibition was observed
in the presence of 100 g l 1 of a lower grade raw
glycerol (65% w/v). Furthermore, 1,3-propanediol production
from two raw glycerol types (65% w/v and 92%
w/v), without any prior purification, was observed in
batch and continuous cultures, on a synthetic medium.
No significant differences were found in C. butyricum
fermentation patterns on raw and commercial glycerol
as the sole carbon source. In every case, 1,3-propanediol
yield was around 0.60 mol/mol glycerol consumed

Andrade, J. C.

González-Pajuelo, M.

Vasconcelos, I.

English

Repositório Institucional da Universidade Católica Portuguesa

M. Gonzalez-Pajuelo Æ J. C. Andrade Æ I. VasconcelosProduction of 1,3-propanediol by Clostridium butyricum VPI 3266using a synthetic medium and raw glycerolAbstract Growth inhibition of Clostridium butyricumVPI 3266 by raw glycerol, obtained from the biodieselproduction process, was evaluated. C. butyricum pre-sents the same tolerance to raw and to commercialglycerol, when both are of similar grade, i.e. above 87%(w/v). A 39% increase of growth inhibition was ob-served in the presence of 100 g l1 of a lower grade rawglycerol (65% w/v). Furthermore, 1,3-propanediol pro-duction from two raw glycerol types (65% w/v and 92%w/v), without any prior puriﬁcation, was observed inbatch and continuous cultures, on a synthetic medium.No signiﬁcant diﬀerences were found in C. butyricumfermentation patterns on raw and commercial glycerolas the sole carbon source. In every case, 1,3-propanediolyield was around 0.60 mol/mol glycerol consumed.Keywords Clostridium butyricum Æ Raw glycerol Æ1,3-PropanediolIntroduction1,3-Propanediol, an emerging bulk chemical, is amonomer for producing plastics with special properties,such as biodegradability, and is the base of the newpolyester PTT [3, 16, 21]. 1,3-Propanediol is currentlyderived from acrolein, a petroleum derivative andharmful reagent, making the monomer productionprocess relatively expensive and dangerous [3]. As abiological alternative to the chemical process, it hasbeen shown that some bacteria are able to produce 1,3-propanediol from glycerol [2, 8–10, 17, 19], andClostridium butyricum is probably the best producerdescribed. One approach towards develop an econom-ically viable process for the microbial production of1,3-propanediol is to use low price renewable feedstockas substrates. In recent years, there has been anincreasing interest in biodiesel as a less polluting,alternative fuel [4, 7, 11]. Biodiesel is produced by thetransesteriﬁcation of plant seed oils and yields glycerolas the main by-product (about 10% by weight). Since50% of the entire cost of the microbial production of1,3-propanediol is due to the price of raw materials [6],raw glycerol from biodiesel production processes maybe an interesting renewable carbon source for C. bu-tyricum if glycerol costs were signiﬁcantly lower thancurrent bulk prices. In this work, growth inhibition ofC. butyricum VPI 3266 by diﬀerent grades and types ofglycerol were evaluated. Two raw glycerol types (65%w/v and 92% w/v), by-products of diﬀerent biodieselproduction processes, were used without any priorpuriﬁcation in order to evaluate their potential toxiceﬀect on growth and 1,3-propanediol production by C.butyricum VPI 3266 in batch and continuous cultureswith a synthetic medium.Materials and methodsOrganismClostridium butyricum VPI 3266 (Virginia PolytechnicInstitute Culture Collection, Blackburn, Va.) wasmaintained in the synthetic medium described below,with 0.2 M MOPS (3-[N]-morpholino]propanesulphonicacid), in spore form, at 20C. This strain is available inother culture collections as C. butyricum NCIMB 7423(National Collections of Industrial and Marine Bacteria,Aberdeen, Scotland, UK) and C. butyricum CECT 361(Coleccio´n Espan˜ola de Cultivos Tipo; Universitat deValencia, Valencia, Spain).M. Gonza´lez-Pajuelo Æ J. C. Andrade Æ I. Vasconcelos (&)Escola Superior de Biotecnologia,Universidade Cato´lica Portuguesa,Rua Dr. Anto´nio Bernardino de Almeida,4200 Porto, PortugalE-mail: ivasc@esb.ucp.ptTel.: +351-2-25580049Fax: +351-2-25090351Culture mediaExperiments to study growth inhibition were per-formed in reinforced clostridial medium (RCM, Ox-oid). The synthetic medium used contained (per litreof deionised water): glycerol 30–60 g; KH2PO4 0.5 g;K2HPO4 0.5 g; MgSO4Æ7H2O 0.2 g; CoCl2Æ6H2O0.01 g; FeSO4Æ7H2O 0.01 g; biotin, 0.04 mg; p-amino-benzoic acid 8 mg; acetic acid, 2 g. The pH of themedium was adjusted to 6.5 with 6 N NH4OH. Forbatch cultures without pH regulation, MOPS wasadded to the synthetic medium (ﬁnal concentration,0.2 M). The feed medium for continuous cultures wasthe synthetic medium described above, without aceticacid, and with 0.028 g l1 FeSO4Æ7H2O (instead of0.01 g l1), 1.5 g l1 NH4Cl and 1 ml H2SO4 17.4 M;the pH of the medium was not adjusted in this case.Commercial glycerol, a puriﬁed 87% (w/v) gradeglycerin, or raw glycerol, a by-product of biodieselproduction processes without any prior puriﬁcation,were used as carbon source. Commercial glycerol waspurchased from Riedel-de Hae¨n (Seelze, Germany).Raw glycerol, from the transesteriﬁcation process forbiodiesel production using rapeseed oil was kindlysupplied by Novance (Compie`gne, France) and con-tained the following components (as provided by thesupplier): glycerol, 65% (w/v); sodium salts, less than5% (w/v); non-glycerol organic matter, less than 1%(w/v); metals, less than 1,000 ppm; heavy metals, lessthan 5 ppm. A second type of raw glycerol from abiodiesel production process, with a higher grade(92% w/v), was kindly provided by Diester Industrie(Grand Couronne, France). Commercial and rawglycerol grades were conﬁrmed by HPLC analysis.Batch fermentations without pH regulationAfter preparation, the culture medium was boiled, dis-tributed in 100 ml ﬂasks and sparged with O2-freenitrogen for 15 min. The ﬂasks were then sealed withbutyl rubber stoppers and autoclaved (121C, 20 min).The media were inoculated (10% v/v) with a cell sus-pension in exponential growth phase. Cultures weregrown at 35C.Continuous culturesContinuous cultures were performed in a 2-l bioreac-tor (Biostat MD; Braun, Melsungen, Germany) with aworking volume of 1,250 ml. To create anaerobicconditions, the sterilised synthetic medium in the ves-sel was ﬂushed with sterile O2-free nitrogen until roomtemperature was attained. After sterilisation, the feedmedium was also sparged with sterile O2-free nitrogen,until it reached room temperature. A growing cultureon the synthetic medium with MOPS, taken at theearly exponential growth phase, was used as inoculum(10% v/v). The culture was ﬁrst grown batchwise andcontinuous feeding was started once the exponentialgrowth phase was reached. During the experiments,the feed medium was maintained under N2 at 30 mbar(3,000 Pa), to avoid O2 entry. All tubing was made ofbutyl rubber and the bioreactor gas outlet was pro-tected with a pyrogallol arrangement [20]. The culturewas stirred at 200 rpm, temperature was set to 35Cand pH was maintained constant by automatic addi-tion of 6 N NH4OH.Measurement of growth inhibitionGrowth inhibition was measured in cultures performedin tubes containing 9 ml RCM, with diﬀerent glycerolconcentrations (0, 20, 40, 65, 100 g l1), inoculatedwith 1 ml cells in early exponential growth phase andincubated at 35C. For this study, commercial glycerol(87% w/v) and the two types of raw glycerol (65% w/v and 92% w/v) were tested. Experiments were per-formed in duplicate. Growth was monitored every 2 hby optical density (OD) measurement at 620 nm. Themaximum speciﬁc growth rate (l) of C. butyricum VPI3266, on the diﬀerent types and concentrations ofglycerol, was determined from the slope of the leastsquare regression lines of the logarithm of OD vs timedata.Inhibition (%) in each case was determined from thefollowing equation:% Inhibition = (1 li/l0) 100where, li is the maximum speciﬁc growth rate inexperiment i and l0 is the maximum speciﬁc growth rateof the control experiment (no glycerol added to themedium).Analytical proceduresCell concentration was measured turbidometrically, at620 nm, and correlated with cell dry weight determineddirectly. Glycerol, 1,3-propanediol, ethanol and acetic,butyric and lactic acid concentrations were determinedby HPLC (System Gold; Beckman, Fullerton, Calif.).Separation was performed on a Bio-Rad Aminex HPX-87H column (300 mm ·7.8 mm; Bio-Rad, Richmond,Calif.) and detection was achieved by refractive index.Operating conditions were as follows: mobile phase,sulphuric acid 0.5 mM; ﬂow rate 0.5 ml/min; tempera-ture 30C [20].1,3-Propanediol volumetric productivity (Q1,3-Pdiol)and speciﬁc formation rate (q1,3-Pdiol) in chemostat cul-tures were calculated as Q1,3-Pdiol= C1,3-Pdiol·D and q1,3-Pdiol=C1,3-Pdiol·D/X respectively, where C1,3-Pdiol is the1,3-propanediol mass concentration, D is the dilutionrate of the chemostat and X is the cell mass concentra-tion.ResultsGrowth inhibition of C. butyricum VPI 3266by diﬀerent types of glycerolThe eﬀect of two types of raw glycerol (65% w/v and92% w/v) and of commercial glycerol (87% w/v) on thegrowth of C. butyricum VPI 3266 is presented in Fig. 1.No inhibitory eﬀect was observed with 20 g l1 of anykind of glycerol. The percentage of growth inhibitionincreased linearly with commercial and raw glycerolconcentrations between 20 and 100 g l1 . Up to100 g l1, the percentages of growth inhibition bycommercial and 92% (w/v) raw glycerol were very sim-ilar (0, 21, 41 and 62% of growth inhibition for glycerolconcentrations of 20, 40, 60 and 100 g l1, respectively).The inhibition eﬀect was more evident when 65% (w/v)raw glycerol was used; a growth inhibition of 86% wasobserved when the medium contained 100 g l1 of thistype of glycerol.Batch cultures of C. butyricum VPI 3266on a synthetic medium with raw glycerolThe ability of C. butyricum VPI 3266 to produce 1,3-propanediol from commercial and raw glycerol typeswas shown in batch cultures without pH regulation.Samples for product analysis were taken 24 h afterinoculation, when the pH of the medium was between5.0 and 5.2. The results in Table 1 show that 1,3-pro-panediol was the major measured fermentation end-product. Organic acids, such as acetic and lactic acid,were also produced; however, butyric acid, an organicacid usually synthesised by C. butyricum, could not bequantiﬁed from these experiments (as it could not beseparated from MOPS by HPLC analysis).Continuous cultures of C. butyricum VPI 3266on a synthetic medium with raw glycerolThe possibility of producing 1,3-propanediol from rawglycerol by C. butyricum was also shown in continuouscultures. Commercial glycerol and the two types of rawglycerol (65% w/v and 92% w/v) were used as the solecarbon source at two diﬀerent concentrations (30 and60 g l1). The results are shown in Table 2.1,3-Propanediol was the major fermentation end-product. Acetic and butyric acid were also synthesised.Lactic acid production was not observed in continuouscultures, in contrast to results obtained in batch fer-mentations. Glycerol consumption was around 100%,whether the glycerol concentration in the feed tank was30 g l1 or 60 g l1 . In every case, 1,3-propanediol yieldwas around 0.60 mol 1,3-propanediol/mol glycerolconsumed. The ﬁnal 1,3-propanediol titre increased withthe initial glycerol concentration, 30 g l1 being ob-tained from 60 g l1 of glycerol as carbon substrate. Thefermentation patterns of C. butyricum grown on raw orcommercial glycerol were similar.DiscussionIn this work, the inhibitory eﬀect of raw glycerol, frombiodiesel production processes, on the growth of C.butyricum VPI 3266 was shown. Petitdemange et al. [15]Fig. 1 Growth inhibition ofClostridium butyricum VPI 3266by diﬀerent types of glycerol.Filled circles Commercialglycerol (87% w/v), dashedregression line; ﬁlled trianglesraw glycerol (92% w/v), dottedregression line, ﬁlled squaresraw glycerol (65% w/v), solidregression lineshowed the eﬀect of commercial glycerol on the growthof C. butyricum DSM 5431. At 100 g l1 commercialglycerol, growth was inhibited by 59%. This value isclose to those obtained here with commercial and 92%raw glycerol. Although these authors also showed thatsome strains from bacterial culture collections were un-able to grow on raw glycerol, in our experimentsC. butyricum VPI 3266 was able to grow on two diﬀerenttypes of raw glycerol. C. butyricum VPI 3266 seems tohave the same tolerance to raw and to commercialglycerol when both have a similar grade (above 87%).An appreciably higher growth inhibition of C. butyricumVPI 3266 was observed when 65% raw glycerol wasused. Due to its production process, raw glycerol con-tains other substances such as sodium salts and heavymetals in concentrations that might interfere with celldivision.The eﬀect of raw glycerol on batch and continuouscultures was minimal, and it did not interfere with 1,3-propanediol production. In every case, the consumptionof raw glycerol yielded about 0.60 mol 1,3-propanediol/mol glycerol consumed. These results are similar tothose obtained by Saint-Amans et al. [18] with 30 g l1commercial glycerol, where 14 g l1 1,3-propanediol,0.4 g l1 acetate and 5.1 g l1 butyrate were obtained incontinuous fermentation, leading to a yield of 0.65 mol1,3-propanediol/mol glycerol consumed. Petitdemangeet al. [15] also showed that four of ten isolated strains ofC. butyricum were able to grow on raw glycerol. One ofthese strains, C. butyricum E5, produced 58 g/l 1,3-propanediol, in fed-batch culture, from 109 g l1 rawglycerol in a medium containing 1 g l1 yeast extract.Another newly isolated strain of C. butyricum was alsoshown to consume raw glycerol (65% grade) in a med-ium containing 1 g l1 yeast extract [13]; this strain,C. butyricum F2b, yielded around 0.66 mol 1,3-pro-panediol/mol glycerol consumed in a continuous culturefed with 90 g l1 glycerol.Recently, the use of raw glycerol has also been re-ported for lipid production by Yarrowia lipolytica [12]and for butanol production by Clostridium acetobutyli-cum ATCC 824 [1].In the present work, the collection strain C. butyricumVPI 3266 was able to produce 1,3-propanediol from rawglycerol on a synthetic medium. It was shown that whenraw glycerol fermentations were compared to a com-mercial glycerol fermentation, no signiﬁcant diﬀerenceswere observed concerning ﬁnal product concentrationand 1,3-propanediol yield and volumetric productivity.These facts show that raw glycerol is an interestingsubstrate for 1,3-propanediol biological production. Theeconomical viability of this process is dependent on rawTable 1 Batch cultures of Clostridium butyricum VPI 3266 on commercial and raw glycerol (synthetic medium without pH regulation,35C)Type of glycerol(grade w/v)Initial glycerolconcentration (g l1)Glycerolconsumed (%)Products (mol/mol glycerol consumed)1,3-Propanediol Lactic acid Acetic acidCommercial (87%) 39.6 41 0.58 0.026 0.072Raw (92%) 44.1 47 0.51 0.023 0.073Raw (65%) 33.5 54 0.56 0.011 0.087Table 2 Continuous cultures of C. butyricum VPI 3266 on diﬀerent types of glycerol. Synthetic medium, dilution rate (D) =0.1 h1,pH 6.5, 35C. Y1,3-Pdiol 1,3-propanediol yield (mol/mol glycerol consumed), Q1,3-Pdiol 1,3-propanediol volumetric productivity, q1,3-Pdiol1,3-propanediol speciﬁc formation rateType of glycerolCommercial glycerol(87% w/v)Raw glycerol(92% w/v)Raw glycerol(65% w/v)Feed glycerol (g l1) 30.2 58.0 35.0 60.6 30.8 62.1Residual glycerol (g l1) 0.48 0.18 0.55 0.19 0.30 0.20Biomass (g l1) 1.15 2.18 1.33 2.71 1.09 2.46Product concentration (g l1)1,3-Propanediol 14.8 29.7 16.4 30.0 15.8 31.5Acetate 0.47 2.50 0.65 2.42 0.62 2.38Butyrate 3.88 5.31 4.50 5.61 4.09 5.61Y1,3-Pdiol (mol/mol) 0.60 0.62 0.57 0.60 0.62 0.61Q1,3-Pdiol (g l1 h1) 1.54 2.98 1.67 3.02 1.59 3.15q1,3-Pdiol (g g dry weight1 h1) 1.32 1.36 1.25 1.11 1.46 1.28Carbon recovery (%)a 97 97 96 96 96 96aFor calculation of carbon recovery, carbon dioxide concentration was estimated from end-product concentrations based on Pap-outsakis [14]glycerol price and availability. As a carbon feedstock forfermentations, glycerol could be competitive if its price isnot higher than US$ 0.3/kg [5].Acknowledgements This work was ﬁnancially supported by theEuropean FAIR project (Contract No. FAIR-CT96-1912). M.Gonza´lez-Pajuelo was supported by PRAXIS XXI with a PhDgrant (BD/16036/98).References1. Andrade JC, Vasconcelos I (2003) Continuous cultures ofClostridium acetobutylicum: culture stability and low-gradeglycerol utilization. Biotechnol Lett 25:121–1252. Biebl H, Marten S, Hippe H, Deckwer W-D (1992) Glycerolconversion to 1,3-propanediol by newly isolated clostridia.Appl Microbiol Biotechnol 36:592–5973. Biebl H, Menzel K, Zeng A-P, Deckwer W-D (1999) Microbialproduction of 1,3-propanediol. Appl Microbiol Biotechnol52:289–2974. Chowdhury J, Fouhy K (1993) Vegetable oils: from table to gastank. Chem Eng 100:35–395. Claude S, Heming M, Hill K (2000) Commercialisation ofglycerol. In: Stelter W, Kerckow B, Hagen M (eds) Proceedingsof chemical-technical utilisation of vegetable oils conference,20–21 June, Bonn, pp 129–146. ISBN 3 00 006959 36. Deckwer W-D (1995) Microbial conversion of glycerol to 1,3-propanediol. FEMS Microbiol Rev 16:143–1497. Eggersdorfer M, Meyer J, Eckes P (1992) Use of renewableresources for non-food materials. FEMS Microbiol Rev103:355–3648. Heyndrickx M, de Vos P, Vancanneyt M, de Ley J (1991) Thefermentation of glycerol by Clostridium butyricum LMG 1212t2and 1213t1 and Clostridium pasteurianum LMG 3285. ApplMicrobiol Biotechnol 34:637–6429. Homann T, Tag C, Biebl H, Deckwer W-D, Schink B (1990)Fermentation of glycerol to 1,3-propanediol by Klebsiella andCitrobacter strains. Appl Microbiol Biotechnol 33:121–12610. Menzel K, Zeng A-P, Deckwer W-D (1997). High concentra-tion and productivity of 1,3-propanediol from continuous fer-mentation of glycerol by Klebsiella pneumoniae. EnzymeMicrob Technol 28:82–8611. Muniyappa PR, Brammer SC, Noureddini H (1996) Improvedconversion of plant oils and animal fats into biodiesel and co-product. Bioresour Technol 56:19–2412. Papanikolaou S, Aggelis G (2002) Lipid production by Yarr-owia lipolytica growing on industrial glycerol in a single-stagecontinuous culture. Bioresour Technol 82:43–4913. Papanikolaou S, Ruiz-Sanchez P, Pariset B, Blanchard F, FickM (2000) High production of 1,3-propanediol from industrialglycerol by a newly isolated Clostridium butyricum strain.J Biotechnol 77:191–29814. Papoutsakis ET (1984) Equations and calculations for fer-mentations of butyric acid bacteria. Biotechnol Bioeng 26:174–18715. Petitdemange E, Durr C, Abbad Andaloussi S, Raval G (1995)Fermentation of raw glycerol to 1,3-propanediol by new strainsof Clostridium butyricum. J Ind Microbiol 15:498–50216. Potera C (1997) Genecor&Dupont create ‘‘green’’ polyester.Genet Eng News 17:1717. Saint-Amans S, Perlot P, Goma G, Soucaille P (1994) Highproduction of 1,3-propanediol from glycerol by Clostridiumbutyricum in a simply controlled fed-batch system. BiotechnolLett 17:211–21618. Saint-Amans S, Girbal L, Andrade J, Ahrens K, Soucaille P(2001) Regulation of carbon and electron ﬂow in Clostridiumbutyricum VPI 3266 grown on glucose-glycerol mixtures.J Bacteriol 183:1748–175419. Solobov M, Smiley KL (1960) Metabolism of glycerol by anacrolein-forming lactobacillus. J Bacteriol 79:261–26620. Vasconcelos I, Girbal L, Soucaille P (1994) Regulation ofcarbon and electron ﬂow in Clostridium acetobutylicum grownin chemostat culture at neutral pH on mixtures of glucose andglycerol. J Bacteriol 176:1443–145021. Witt U, Mu¨ller R-J, Widdecke H, Deckwer W-D (1994) Syn-thesis, properties and biodegradability of polyesters based on1,3-propanediol. Makromol Chem Phys 195:793–802

Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium and raw glycerol

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Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium and raw glycerol

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