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ImpactofanaerobiosisstrategyandbioreactorgeometryonthebiochemicalresponseofClostridiumbutyricum...

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Impact of anaerobiosis strategy and bioreactor geometry on the biochemicalresponse of Clostridium butyricum VPI 1718 during 1,3-propanediol fermentation

Afroditi Chatzifragkou a, George Aggelis b, Michael Komaitis a, An-Ping Zeng c, Seraphim Papanikolaou a,⇑a Department of Food Science and Technology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greeceb Unit of Microbiology, Department of Biology, Division of Genetics, Cell and Development Biology, University of Patras, 26504 Patras, Greecec Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 15 Denickestrasse, D-21073 Hamburg, Germany

a r t i c l e i n f o

Article history:Received 28 June 2011Received in revised form 5 September 2011Accepted 7 September 2011Available online 13 September 2011

Keywords:Clostridium butyricum1,3-PropanediolFed-batchLactate dehydrogenaseCrude glycerol

a b s t r a c t

The impact of anaerobiosis strategy on 1,3-propanediol production during cultivation of Clostridium butyr-icum VPI 1718 in different size bioreactors was studied. In batch trials with N2 gas infusion, the fermentationwas successfully accomplished, regardless of initial glycerol concentration imposed and bioreactor geom-etry. However, in the absence of N2 continual sparging, significant variations concerning the biochemicalresponse of the strain were observed. Specifically, at 1-L bioreactor, the absence of N2 infusion at high initialglycerol concentration induced lactate dehydrogenase activity and thus lactic acid synthesis, probably dueto partial blockage of phosphoroclastic reaction caused by insufficient self-generated anaerobiosis environ-ment. During fed-batch cultivation with continual N2 sparging, the strain produced �71 g L�1 of 1,3-pro-panediol, whereas under self-generated anaerobiosis, 1,3-propanediol pathway was evidently restricted,as only 30.5 g L�1 of 1,3-propanediol were finally produced. Apparently, N2 infusion strategy paired withbioreactor geometry can alter the biochemical behavior of the particular strain.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

1,3-Propanediol (PDO) represents a specialty chemical, knownfor more than a century, with a wide spectrum of industrialapplications, such as composite, adhesive, antifreeze, laminateand aliphatic polyester. However, the launch of a new 1,3-propane-diol-based polyester with a number of excellent physical proper-ties, namely polytrimethylane terephthalate (acknowledged bythe acronyms PTT or 3GT) in recent years, attracted much interestin the field of novel polymer production (Papanikolaou, 2009; Zengand Biebl, 2002). By taking into account the fact that the chemicalproduction of 1,3-propanediol requires handling of hazardousstarting materials and catalysts (e.g. acrolein, rubidium, etc.), aswell as extreme operating conditions such as high pressure andtemperature, the production of this organic compound via biotech-nological routes constitutes an attractive and continuously devel-oping option (Biebl et al., 1999; Papanikolaou, 2009; Zeng andBiebl, 2002).

The biotechnological production of PDO using glycerol assubstrate is achieved by a number of anaerobic or facultativeanaerobic prokaryotic microorganisms belonging principally to theClostridium genus or the Enterobacteriaceae family (Barbirato et al.,

1997; Chen et al., 2003; Gonzalez-Pajuelo et al., 2004; Mu et al.,2006; Papanikolaou et al., 2000, 2004, 2008; Petitdemange et al.,1995; Zeng et al., 1994). The metabolic reactions that are relatedwith glycerol break-down under anaerobic conditions by Clostrid-ium sp. strains are mainly divided into two pathway branches; areductive and an oxidative one (Fig. 1). In the reductive branch,glycerol is dehydrated to 3-hydroxypropionaldehyde (3-HPA) andthen further reduced to PDO by consumption of reducing co-fac-tors (NADH2). In the oxidative branch, glycerol is oxidized viaDHA to pyruvic acid by glycolysis reactions, resulting in the forma-tion of by-products such as butyric, acetic and lactic acid. As it canbe seen by the biochemical reactions that are depicted in Fig. 1, thefermentation yield of PDO is strongly depended on the activity ofthe metabolic pathways which compete the pathway glycerol toPDO in terms of NADH2 co-factors re-generation, such as the met-abolic pathway glycerol to butyric acid and, to a lesser extent, theone of glycerol to lactic acid. On the contrary, the pathway glycerolto acetic acid does not require the consumption of reducing co-factors, thus resulting in higher PDO yields (for reviews describingthe biochemistry of PDO fermentation see: Biebl et al., 1999;Papanikolaou, 2009; Zeng and Biebl, 2002).

Continuous energetic crisis, potential exhaustion of conven-tional fuels and several important environmental issues imposed(e.g. rise of green-house gases emission through combustion of fos-sil fuels, global warming problem, etc.) have resulted in increasingdemands for renewable fuels, the application of which on a largecommercial scale is strongly recommended by several authorities.

0960-8524/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2011.09.023

⇑ Corresponding author. Address: Laboratory of Food Microbiology and Biotech-nology, Department of Food Science and Technology, Agricultural University ofAthens, Greece. Tel./fax: +30 210 5294700.

E-mail address: spapanik@aua.gr (S. Papanikolaou).

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This entire situation resulted in the notable increment of biodieselproduction the last decade (Papanikolaou, 2009; Zeng and Biebl,2002). Taking into account that biodiesel derives from triacylglyc-erol trans-esterification which results in both the synthesis of fattyesters (in most cases fatty acid methyl-esters) and the side-produc-tion of glycerol, the significant expansion of biodiesel has led intothe generation of very large quantities of glycerol, as the principalby-product of this process. This rise in biodiesel-derived crudeglycerol surplus has rendered the possibility of industrial applica-tion of PDO fermentation a very promising bioprocess, as crudeglycerol has been proved as an inexpensive and excellent startingmaterial for PDO production (Chatzifragkou et al., 2010, 2011;Günzel et al., 1991; Papanikolaou et al., 2000, 2004, 2008). A num-ber of researchers have investigated the capability of microorgan-isms belonging to Clostridium and Klebsiella species to produce PDOin scaled-up fermentation conditions, aiming to achieve high finalconcentrations and productivities (Cheng et al., 2007; Günzel et al.,1991; Liu et al., 2007; Zheng et al., 2009). However, in order for thisbioprocess to be financially profitable, there is still need to furtherreduce the operation costs. Moreover, the industrial application ofPDO production requires the utilization of non-pathogenic and eas-ily handling microorganisms.

Aim of this study was to access and evaluate the biochemical re-sponse of C. butyricum VPI 1718 growth under anaerobic condi-tions, either ensured by N2 gas sparging in the growth mediumor under self-generated anaerobic conditions imposed via micro-bial metabolism (a practice that is currently applied in industrialconditions) in laboratory bioreactors of different size and geome-try. Insights concerning the effects of the different anaerobiosisstrategy imposed on glycerol assimilation and on actual yields ofPDO are given and thoroughly discussed.

2. Methods

2.1. Microorganism and media

C. butyricum VPI 1718 strain (Chatzifragkou et al., 2010, 2011),kindly provided by the Biochemical Engineering Division, GBF –National Research Center for Biotechnology (Braunschweig,Germany) was used in the present study. The microorganism wasmaintained in Reinforced Clostridial Medium (RCM), in 50-mLanaerobic flasks) at T = 4 �C. The anaerobic flasks were, in fact, ser-um bottles, sealed with butyl septum and aluminum ring, after theaddition of 25 mL of RCM medium and were infused with N2 gasfor 10 min under an analytical evaporator. Before any experiment,a heat-shock (T = 80 �C/10 min) was performed, in order to stimu-late the germination of the spores. Then, precultures of the post-germination culture were performed in previously sterilized andN2 infused 200-mL anaerobic flasks, filled with 50 mL of preculturemedium (composition defined in the following Culture media sec-tion) and incubated at T = 35 �C for 12 h. The composition of thepreculture medium used in order to set the microorganism inexponential phase had as follows (per L of deionized water) (Biebl,1991): 3.4 g K2HPO4; 1.3 g KH2PO4; 2.0 g (NH4)2SO4; 0.2 g,MgSO4�7H2O; 0.02 g CaCl2�2H2O; 2.0 g CaCO3; 1.0 g yeast extract;20.0 g glycerol; 2.0 mL trace element solution; 1.0 mL Fe solution.The composition of the Fe solution per L was: 5 g FeSO4�7H2O;4 mL HC1 (37%). The trace element solution per L consisted of:70 mg ZnC12; 0.1 g MnCl2�4H2O; 60 mg H3BO3; 0.2 g CoC12�2H2O;20 mg CuC12�2H2O; 25 mg NiC12�6H2O; 35 mg Na2MoO4�2H2O;0.9 ml HC1 (37%). The fermentation medium used in all experi-ments contained (per L of deionized water) (Reimann and Biebl,1996): 2 g NH4Cl; 0.75 g KCl; 1.38 g NaH2PO4; 0.28 g Na2SO4;

Fig. 1. Metabolic pathways of glycerol catabolism in Clostridium sp. strains. 3-HPA: 3-hydroxypropionaldehyde; GDHt: glycerol dehydratase; GDH: glycerol dehydrogenase;DHAk: di-hydroxyacetone kinase; PDOR: 1,3-propanediol oxidoreductase; FDox (or red): ferredoxine oxido-reductase. Pathways described by Zeng and Biebl (2002) andPapanikolaou et al. (2008, 2009).

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0.42 g citric acid; 1.0 g yeast extract; 0.26 g MgCl2�6H2O; 2.9 mgCaCl2�H2O; 1.0 mL Fe solution (FeCl3�6H2O, 1 g L�1); 2.0 mL traceelement solution; 0.1 g Desmophen (anti-foaming agent). As car-bon source, crude glycerol provided by ‘‘ADM Industries’’(Hamburg, Germany), originating from rapeseed oil trans-esterifi-cation was used in various initial concentrations and had the fol-lowing composition (in w/w): glycerol, 81%; water, 10–12%;potassium salts, 5.0–6.0%; free-fatty acids, 1.0% and methanol,<0.1%.

2.2. Batch and fed-batch cultures

Trials were preformed in three bioreactors of different size andgeometry. Specifically, bioreactors of 1- and 3-L of total volume(New Brunswick Scientific Co, New Jersey, USA) with working vol-umes of 0.8 L and 2 L, respectively, as well as a 4.5-L bioreactor(MBR Bioreactor AG, Zurich, Switzerland), with working volumeof 3 L, were used. For the purposes of the experiment, the height-to-diameter ratio (HR/DR) for each bioreactor used in the presentstudy was calculated. Therefore, in the case of 1-L bioreactor, theheight-to-diameter ratio (HR/DR) was 1.45, for the 3-L bioreactorthe HR/DR ratio was equal to 2.01, while the corresponding ratioin the case of the 4.5-L bioreactor was found to be 3.6. Beforeany experimental work conducted, it was deemed necessary toevaluate the loss of the active volume in both bioreactors due toN2 gas infusion. For this reason the bioreactors were filled with fer-mentation medium, sparged constantly with N2 gas at the workingflow (0.1 vvm) for approximately 100 h and the volume level wasmeasured every 6 h. Indeed, the loss in active volume was esti-mated to be at �0.1% for both bioreactors and therefore, the calcu-lation of the obtained experimental values included the volumeloss. Biodiesel-derived glycerol was used as the sole carbon source,at initial glycerol concentrations (Glol0) ranging from 20 to60 g L�1. In all cases, sterilized culture medium was inoculatedwith 10% (v/v) of post-germinated cell suspension in exponentialgrowth phase with a sterile 20 mL syringe under aseptic condi-tions, whilst anaerobiosis was ensured by infusion of pure N2 priorto inoculation under aseptic conditions, at a rate of 0.4 vvm for30 min. Thereafter, two different anaerobiosis strategies were fol-lowed: in the first approach, pure N2 infusion was performedthroughout the fermentation process at a constant flow rate of0.1 vvm, whilst in the second approach, N2 gas supply was stoppedafter inoculation and the system was left in a self-generatedanaerobiosis regime, under which anaerobiosis was ensuredthrough gas production from microbial metabolism (i.e. decarbox-ylation of pyruvic acid resulting in the production of CO2 andpotentially H2). Culture conditions were as follows: incubationtemperature T = 35 �C; agitation rate at 150 for 1-L fermentorand 200 rpm for 3- and 4.5-L fermentor, respectively; pH was ad-justed and automatically regulated at 6.8 by addition of NaOH 5 M.

Fed-batch experiments were performed in a 1-L bioreactor(New Brunswick Scientific Co, New Jersey, USA), with a workingvolume of 0.8 L. The culture medium contained crude glycerol(81% w/w purity) as carbon source, at initial concentration of20 g L�1. In terms of anaerobiosis strategy, both regimes as de-scribed above were separately investigated. The feed containing75% (w/w) crude glycerol and 1% (w/w) yeast extract was freshlyprepared and sterilized prior to its addition in the fermentor. Cul-ture was stirred at 150 rpm and incubated at T = 35 �C and pH 6.8as described above.

2.3. Preparation of cell free extract and enzyme assay

Culture samples were withdrawn aseptically with a syringe andcells were harvested by centrifugation in a Hettich Micro 12–24centrifuge (Tuttlingen, Germany) for 15 min (9000g, at 4 �C). The

cell pellet was washed twice with sterilized, cold phosphate buffersaline (PBS, pH = 7.4) and suspended in PBS buffer with addition of0.1 mM of protease inhibitor mix (Serva). Cells were disrupted inan ice water jacket by an ultrasonic device (VCX 400, Sonics &Materials Inc, Vibra Cell, Danbury, USA) within six repeatedbatches of 30 s, with 1 min intervals and cell debris were removedby centrifugation in a Hettich Micro 12–24 centrifuge (Tuttlingen,Germany) for 1 h (9000g, at 4 �C). Then, crude cell extract wasimmediately assayed for enzyme activity. Accordingly, proteinconcentration in the cell-free extract was determined accordingto Bradford (1976) method.

Lactate dehygrogenase (LHD) activity was determined by theUV-method described by Vassault (1987). Briefly, LHD activitywas correlated to the amount of pyruvate consumed bycontinuously monitoring the decrease of absorbance at 339 nmdue to oxidation of NADH. The reaction was performed at 30 �C,in masked 3-mL cuvettes placed in a Hitachi U-2000 spectropho-tometer (Hitachi Ltd, Tokyo, Japan). The reaction mixture contained80 mM Tris–sodium chloride (pH = 7.2), 200 mM NaCl, 0.2 mMNADH, 1.6 mM pyruvate as the substrate of the enzymatic reactionand 0.5 mL cell free extract. One unit of LHD activity is defined asthe amount of enzyme that catalyses the oxidation of 1 lmol ofNADH per minute, at 30 �C.

2.4. Analytical procedures

Samples of 5 mL volume were withdrawn every two hours dur-ing batch and fed-batch fermentations. Biomass production(X, g L�1) was determined both turbidometrically by means of opti-cal density – OD at 650 nm in a Hitachi U-2000 (Hitachi Ltd, Tokyo,Japan) spectrophotometer and by cell dry weight – CDW (aftercells dehydration at 110 �C/24 h), so that a correlation betweenOD and CDW could be directly established, the equation of whichis given below (Eq. (1)):

DCW ðin g L�1Þ ¼ 0:275� OD650nm � 0:062; r2 ¼ 0:985 ð1Þ

Glycerol (Glol, g L�1), 1,3-propanediol (PDO, g L�1), butyric acid(But, g L�1), acetic acid (Ace, g L�1) and lactic acid (Lac, g L�1) weredetermined by HPLC analysis (Waters 600E, Milford, MA, USA)with an Aminex HPX-87H (300 mm � 7.8 mm, Bio-Rad, CA, USA)column coupled to a differential refractometer (RI Waters 410)and a UV detector (Waters 486). Operating conditions were as fol-lows: sample volume 20 lL; mobile phase 0.005 M H2SO4; flowrate 0.6 mL min�1; column temperature T = 65 �C. For data consis-tency check, a total carbon recovery (RC) calculation was carriedout at the end of batch and fed-batch cultures according to Papa-nikolaou et al. (2000). Specifically, in these calculations, the drybacterial mass was considered to be represented by the chemicalformula C5H7NO2. Moreover, for one mole of acetic acid produced,one mole of CO2 was synthesized while the formation of one moleof butyric acid resulted in the production of two moles of CO2. Alldata presented are the average of two independent experimentsperformed under the same culture conditions.

3. Results and discussion

3.1. Batch cultures of C. butyricum with different anaerobiosis regimes

In a first approach, 1,3-propanediol fermentation was carriedout in batch bioreactor presenting a volume of 1-L, with crudeglycerol as the sole carbon source at initial substrate concentra-tions (Glol0) ranging from 20 to 60 g L�1. The anaerobiosis strategy,which was initially followed, anticipated the continuous infusion

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of N2 gas throughout the fermentation process. The kinetic dataobtained from this experimental work are presented in Table 1.In any case, the bacterial strain in question proved capable of totalglycerol consumption, while PDO production was satisfactory, cor-responding to 35.1 g L1 at Glol0 = 60 g L�1. Accordingly, the yield of1,3-propanediol formation per glycerol consumed (YPDO/Glol) wassatisfactory, ranging between 0.56 and 0.57 g per g of consumedsubstrate, close to the maximum theoretical value of 0.60 g g�1

(Zeng et al., 1994). As far as organic acids production is concerned,butyric acid was the predominant one with maximum final con-centration of 8.8 g L�1 at Glol0 = 60 g L�1, followed by low amountsof acetic and lactic acid in all batch experiments (Table 1).

In a second anaerobiosis strategy N2 gas infusion was inter-rupted after inoculation and the culture was left in order to achievea self-generated anaerobiosis environment (van Andel et al., 1985).Trials were undertaken with the same Glol0 concentrations asbefore (ranging from 20 to 60 g L�1), in order to obtain comparableresults as far as the biochemical response of the strain under theimposed anaerobiosis regime is concerned. At Glol0 up to 30 g L�1,no significant differences concerning the maximum production of1,3-propanediol and organic acids were observed (Table 1). How-ever, in cultures with Glol0 = 40 g L�1, maximum butyric acid pro-duction was reduced to 5.1 g L�1 (compared to 6.0 g L�1 achievedin the continual supply of N2 gas), while lactic acid productionwas enhanced, reaching 3.9 g L�1 at the end of the fermentation.Furthermore, when Glol0 = 60 g L-1 were imposed, noticeable bio-chemical variations were identified. Specifically, even after a pro-longation of the fermentation time of more than 60 h, the straindid not manage to completely assimilate the available substrate,since 13.0 g L�1 of glycerol remained unconsumed at the end ofthe fermentation. Signs of growth restriction were also detected,as maximum biomass formation was only 0.5 g L�1, compared to1.0 g L�1 achieved in trials with constant N2 gas infusion. As a con-sequence, PDO formation was reduced to only 25.1 g L�1, accompa-nied by a decreased conversion yield YPDO/Glol of 0.42 g g�1 (seeTable 1). Lactic acid was found to be the predominant organic acid

produced corresponding to 10.2 g L�1, while butyric acid produc-tion seemed to be also affected by the imposed cultivation condi-tions, as only 4.7 g L�1 were detected in the fermentationmedium. It should be noted that the C. butyricum strain employedin the present study secretes lactic acid as a by-product of 1,3-pro-panediol fermentation, whereas it produces H2 in negligibleamounts (Biebl and Spröer, 2002). Moreover, Simon (1947) has re-ported that in the case of C. acetobutylicum, the lactic acid pathwayis not normally operational and can be only activated as a less effi-cient mechanism for the oxidation of NADH2 equivalents when thegeneration of molecular hydrogen through the phosphoroclasticreaction is blocked. Therefore, this observation could account forthe ability of C. butyricum VPI 1718 used in the present study to se-crete lactic acid; yet, in the case of N2 gas infusion batches, maxi-mum lactic acid production did not exceed 1.4 g L�1 (Table 1).

As a next step, a series of batch experiments were carried out in a3-L bioreactor, in which both anaerobiosis strategies were followed.Data obtained concerning the aforementioned kinetics are pre-sented in Table 2. In any case, C. butyricum was capable of accom-plishing successfully the 1,3-propanediol fermentation, appearingto be unaffected by the presence or absence of N2 gas throughoutthe fermentation process. Final PDO concentration was alike andcomparable in both cultivation conditions. Only in the case ofself-generated anaerobiosis experiment with Glol0 = 60 g L�1, a fairincrease in lactic acid production was observed, equal to 2.5 g L�1.Nevertheless, the conversion yield YPDO/Glol was found to be stable,with the corresponding values between 0.55 and 0.56 g per g ofconsumed substrate. It may be presumed that in the case of thestrain cultivation at 1-L bioreactor, the combination of N2 gas ab-sence, the low height-to-diameter ratio (1.45 against 2.01 in the3-L bioreactor), as well as the excess-glycerol conditions seemedto positively affect the production of lactic acid for the particularstrain. By taking into account the difference in geometry of thetwo bioreactors employed, the low height-to-diameter ratio inthe case of 1-L bioreactor generally offers a relatively large surfacearea across which gases can potentially easily diffuse. On the

Table 1Experimental results of C. butyricum VPI 1718 during batch cultivation in 1-L bioreactor, at various initial crude glycerol concentrations, under two different anaerobiosis regimes.

Anaerobiosis Time (h) Glolcons (g L�1) X (g L�1) PDO (g L�1) But (g L�1) Ace (g L�1) Lac (g L�1) YPDO/Glol (g g�1) RC (% w/w)

12 20.2 0.6 11.3 3.2 0.4 0.3 0.55 89.4By N2 infusion 19 30.5 0.6 17.6 4.7 0.5 0.8 0.57 92.9

29 38.5 0.8 21.4 6.0 0.6 1.0 0.55 91.753 60.8 1.0 35.1 8.8 1.5 1.4 0.57 94.3

12 20.3 0.6 11.4 2.6 0.4 0.3 0.55 97.1Self-generated 18 30.4 0.7 16.7 4.4 0.6 1.1 0.55 90.1

30 38.9 0.7 20.5 5.1 0.8 3.9 0.52 92.561 46.4 0.5 25.1 4.7 0.2 10.2 0.42 100.1

Presentation of fermentation time (h), glycerol consumption (Glolcons, g L�1), biomass (X, g L�1), 1,3-propanediol (PDO, g L�1), butyric acid (But, g L�1), acetic acid (Ace, g L�1),lactic acid (Lac, g L�1) and 1,3-propanediol produced per glycerol consumed (YPDO/Glol, g g�1). Culture conditions: incubation temperature 35 �C, agitation rate 150 rpm, pH 6.8.Determinations were conducted in duplicate.

Table 2Experimental results of C. butyricum VPI 1718 during batch cultivation in 3-L bioreactor, at various initial crude glycerol concentrations, under two different anaerobiosis regimes.

Anaerobiosis Time (h) Glolcons (g L�1) X (g L�1) PDO (g L�1) But (g L�1) Ace (g L�1) Lac (g L�1) YPDO/Glol (g g�1) RC (% w/w)

12 20.3 0.9 11.4 2.6 0.4 0.3 0.56 87.4By N2 infusion 18 30.1 1.1 16.7 4.1 0.6 1.1 0.55 91.2

28 40.7 1.2 22.5 4.6 0.9 1.2 0.55 87.854 61.0 1.1 33.6 5.9 1.1 1.4 0.55 84.1

12 19.6 0.9 10.9 2.5 0.5 0.4 0.56 87.7Self-generated 18 29.4 1.1 16.1 3.6 0.6 0.4 0.55 86.1

29 39.1 0.9 21.6 5.7 0.8 1.2 0.55 91.252 58.5 1.1 32.1 5.8 0.8 2.5 0.55 85.8

Presentation of fermentation time (h), glycerol consumption (Glolcons, g L�1), biomass (X, g L�1), 1,3-propanediol (PDO, g L�1), butyric acid (But, g L�1), acetic acid (Ace, g L�1),lactic acid (Lac, g L�1) and 1,3-propanediol produced per glycerol consumed (YPDO/Glol, g g�1). Culture conditions: incubation temperature 35 �C, agitation rate 200 rpm, pH 6.8.Determinations were conducted in duplicate.

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contrary, the 3-L bioreactor used in the present study had a higherratio (2.01), allowing the gases to remain longer within the liquidphase, and providing less opportunity for gas transfer to occur(Matthews, 2008). For this reason, in the absence of N2 continualgas sparging, the anaerobiosis achieved in the 1-L bioreactor wasprobably insufficient. Therefore, when higher initial glycerol con-centrations are imposed, causing a prolongation of microbial lagphase, the insufficient anaerobiosis environment (also due to thedelayed onset of the microbial metabolic activity) seems to imposea partial blockage in the level of phosphoroclastic reaction (Fig. 1).Specifically, it could be stated that the activity of NADH-ferredoxinreductase was potentially inhibited (it is known that the above-mentioned enzyme is relatively oxygen-sensitive – Papanikolaou,2009), leading into intracellular increment of NADH2 co-factorsand pyruvic acid. Under this specific regime, the pathway pyruvateto lactate could be favored, yielding eventually into increasedamounts of lactic acid accumulation into the culture medium. Thisfinding can be also enforced by the fact that the increment of initialglycerol concentration resulted in increase of lactate/butyrate ratio,whereas the yield of PDO production (YPDO/Glol) was gradually de-creased (Fig. 2). Besides that, growth repression was also noticed,most probably due to the high concentration of lactic acid itself inthe culture medium (more than 10 g L�1 at the corresponding batchcultivation in the 1-L bioreactor).

In order to fortify and validate the previously presented find-ings, an additional experiment was held in a 4.5-L bioreactor withincreased height-to-diameter ratio (HR/DR = 3.6) and high initialglycerol concentration (Glol0 = 60 g L�1). N2 infusion was stoppedafter inoculation, so as to induce a self-generated anaerobiosisenvironment inside the bioreactor. As shown in Fig. 3, microbialgrowth was sufficient, accompanied by total substrate consump-tion. Final 1,3-propanediol was 32.1 g L1, with the correspondingbioconversion yield YPDO/Glol equal to 0.53 g g�1 (Fig. 3a). As far asorganic acid production is concerned, butyric acid was alwaysfound the predominant one, reaching finally 4.8 g L�1, while lacticand acetic acid were found, as expected, in lower amounts insidethe culture medium (Fig. 3b). Judging from the above results, itseems that the conditions that favor the production of non-negligi-ble amounts of lactic acid due to insufficient anaerobiosis environ-

ment are met predominantly in small size laboratory bioreactorsunder the absence of continuous N2 gas infusion, for the particularC. butyricum strain.

3.2. Lactate dehydrogenase activity

Judging from the results obtained by C. butyricum under the twoanaerobiosis regimes, it was deemed necessary to further investi-gate the enzyme associated with the production of lactic acid,namely lactate dehydrogenase (LDH). An attempt to detect andmeasure the activity of the enzyme in question was made, duringbatch cultivations of the strain in 1- and 3-L bioreactor, under self-generated anaerobiosis environment. For this purpose, two differ-ent Glol0 concentrations were implemented, corresponding to 30and 60 g L�1 respectively. As depicted in Fig. 4, no actual enzymeactivity was detected in cultures performed on a 3-L fermentor,regardless of the initial substrate concentration implemented. On

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the contrary, LDH activity was detected during cultivations of C.butyricum on a 1-L fermentor, in which noticeable amount of lacticacid was produced (more than 10 g L�1). In particular, batch trialswith Glol0 = 60 g L�1 yielded the maximum LDH activity that corre-sponded to 0.067 U per lg of intracellular protein (Fig. 4).

Generally, strains belonging to Clostridium genus usually con-tain the enzyme of lactate dehydrogenase (Andreesen et al.,1989; Gheshlaghi et al., 2009). However, lactate is not consideredas a major metabolic by-product of 1,3-propanediol fermentationwhen carried out by Clostridium species, as mainly acetic and buty-ric acid are the usual end-fermentation by-products of the partic-ular bioprocess (Biebl et al., 1999; Papanikolaou et al., 2000,2004, 2009; Petitdemange et al., 1995; Zeng and Biebl, 2002). Infact, a considerable constancy of the conversion of glycerol intoPDO, acetic acid and butyric acid regardless of the Glol0 concentra-tion employed has been reported for several C. butyricum strains,suggesting the presence of mechanisms that precisely partitionthe carbon flow between biosynthesis of the above-mentioned or-ganic acids and the formation of PDO during glycerol break-down(Abbad-Andaloussi et al., 1995; Petitdemange et al., 1995; Papa-nikolaou et al., 2000, 2004, 2008).

Gonzalez-Pajuelo et al. (2006) investigated the enzymes in-volved into the metabolic pathway of glycerol assimilation and1,3-propanediol formation and found that in C. butyricum VPI3266, lactate dehydrogenase activity was less than 0.002 U permg of protein since the particular strain did not produce lactic acid.On the contrary, noticeable LHD activity was detected in C. acet-obutylicum DG1 (0.19 U mg�1), justifying the production of lacticacid by the aforementioned strain. On the other hand, Forsberg(1987) studied the ability of Clostridium species to produce PDOfrom glycerol, and in the case of C. butyricum B593, lactic acidwas found to be a non-negligible end-product of 1,3-propanediolfermentation. Lactic acid occurs as a non-negligible by-product of1,3-propanediol fermentation carried out by prokaryotic bacteriabelonging to the enteric group, such as Klebsiella pneumoniae, Citro-bacter freundii and Enterobacter agglomerans, under aerobic or mi-

cro-aerobic conditions (Biebl et al., 1999; Chen et al., 2009; Liu etal., 2007; Zeng and Biebl, 2002). In the case of Clostridium species,lactate is produced only if there is some blockage of the usualfermentation pathways. Such a situation can arise if furtherbreak-down of pyruvate is inhibited by iron limitation, by carbonmonoxide gassing or by the availability of substrate in excess(Andreesen et al., 1989; Gheshlaghi et al., 2009). In the presentinvestigation, the glycerol excess conditions, as well as the lowerheight-to-diameter ratio, seemed to be the key-factors for LDHexpression and, thus, secretion of non-negligible amounts of lacticacid in the fermentations carried out in the absence of continual N2

gas supply into the medium.

3.3. Fed-batch cultures of C. butyricum with different anaerobiosisregimes

Aiming to further investigate the biochemical phenomena ob-served during batch cultivation of the microorganism under differ-ent anaerobiosis regimes imposed, it was decided to performanother type of cultivation for the implementation of 1,3-propane-diol fermentation, namely a fed-batch mode. In this case, only the1-L bioreactor was used, under conditions of either N2 continualsupply or under self-generated anaerobiosis regime. When con-stant N2 gas infusion was performed, after a cultivation of morethan 100 h, the strain was capable of producing 70.8 g L�1 of 1,3-propanediol (Fig. 5a), with a corresponding bioconversion yieldYPDO/Glol of 0.55 g per g of consumed glycerol. In fact, this maxi-mum 1,3-propanediol concentration can be considered as one ofthe highest reported among wild-type bacterial strains in the inter-national literature. It should be stated that when the particularC. butyricum VPI 1718 strain was cultivated under non-sterile fer-mentation conditions in fed-batch mode, it was proved capableof producing 67.9 g L�1 of PDO (Chatzifragkou et al., 2011). More-over, Hirschmann et al. (2005) reached 87.7 g L�1 of PDO by awild-type Clostridium sp. IK124 strain under optimized fed-batchoperation. Besides that, high 1,3-propanediol productions of thesame range (70–85 g L�1) in fed-batch operation conditions havebeen also reported for other bacterial strains, such as K. pneumo-niae and K. oxytoca; yet, the majority of these strains are addressedas mutants, characterized by increased resistance to PDO or de-creased production of undesirable fermentation by-products ( i.e.lactic or butyric acid) that are antagonistic ones to the pathwayglycerol to 1,3-propanediol as regards NADH2 co-factors re-gener-ation (Abbad-Andaloussi et al., 1995; Jin et al., 2011; Liu et al.,2007; Yang et al., 2007).

Finally, a fed-batch cultivation was conducted without continualN2 sparging into the growth medium (meaning, thus, under ‘‘insuf-ficient’’ anaerobiosis imposed by the self-generated anaerobiosisenvironment) in order to compare the biochemical behavior ofthe strain in terms of growth and 1,3-propanediol formation duringthese fermentation conditions. The kinetics of growth, glycerolassimilation, PDO formation and organic acid production by C.butyricum are depicted in Fig. 5b. It was observed that until the first20 h of fermentation, the principle organic acid produced was thatof butyric. However, during evolution of the fed-batch fermenta-tion, lactic acid concentration was increased as opposed to buty-rate, being therefore the dominant organic acid in the culturemedium (Fig. 5b). It is characteristic that at the end of the processmore than 11 g L�1 of lactic acid were produced, whereas butyricacid production was reduced to 5.9 g L�1 and acetic acid was onlyfound in traces (<0.1 g L�1). This fact, combined with the absenceof N2 infusion, had a demonstrable negative effect on the fermenta-tion, since PDO production was significantly reduced to 30.5 g L�1,while glycerol assimilation virtually ceased after 80 h of fermenta-tion, a time point in which nearly 70 g L�1 of glycerol wereconsumed by the microorganism. Accordingly, growth formation

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Fig. 4. Lactate dehydrogenase activity as functions of fermentation time in C.butyricum VPI 1718, under self-generated anaerobiosis environment, in 1-L (filledsymbols) and 3-L bioreactor experiments (open symbols), with initial glycerolconcentration of 30 (circles) and 60 g L�1 (diamonds) in each case. Cultureconditions as described in Tables 1 and 2.

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was restricted, since maximum biomass formation was only0.8 g L�1, compared to 1.6 g L�1 yielded in the respective fed-batchexperiment with continual N2 supply, most probably due to thehigh presence of lactic acid in the culture medium. Thus, the con-ducted fed-batch trials indicate that for the given microorganism,the induction of lactated dehydrogenase activity can be secondarilyattributed to the existence of high glycerol concentrations in thefermentation medium and principally at the geometry/size of thebioreactor employed, as well as the anaerobiosis strategy that is fol-lowed each time. Furthermore, it can be assumed that under condi-tions of self-generated anaerobiosis, the metabolic pathway ofpyruvic acid reduction to lactic acid antagonizes the need in re-duced co-factors (NADH2) with butyric acid formation, due to par-tial blockage of pyruvate to acetyl-CoA conversion pathway and oflimited activity of NADH-ferredoxin reductase enzyme in the path-way of phosphoroclastic reaction, resulting eventually in increasedamounts of lactate.

4. Conclusions

The impact of anaerobiosis strategy upon growth and PDO pro-duction by C. butyricum was tested in the present study. In the ab-sence of N2 gas infusion, the size and geometry of the bioreactor, aswell as the concentration of the substrate contributed into thealteration of the biochemical behavior of the particular strain.These cultivation conditions were found to favor lactic acid secre-tion, which was found as the predominant and antagonistic by-product of 1,3-propanediol in this fermentation, while signs ofgrowth restrictions were also detected. However, these phenom-ena were found to diminish when large-sized bioreactors wereoperated.

Acknowledgements

Financial support has been kindly provided by the EU (FP7 Pro-gram ‘‘PROPANERGY – Integrated bioconversion of glycerine intovalue-added products and biogas at pilot plant scale’’, Grant num-ber: 212671).

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