Esn

Ma

b

a

ARR2AA

KC1BNC

1

ehppSth2top(Vt

DG

0h

Journal of Biotechnology 163 (2013) 408– 418

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology

j ourna l ho me pag e: www.elsev ier .com/ locate / jb io tec

nhanced 1,3-propanediol production by a newly isolated Citrobacter freundiitrain cultivated on biodiesel-derived waste glycerol through sterile andon-sterile bioprocesses

aria Metsoviti a, An-Ping Zengb, Apostolis A. Koutinasa, Seraphim Papanikolaoua,∗

Department of Food Science and Technology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, GreeceInstitute of Bioprocess and Biosystems Engineering, Hamburg University of Technology (TUHH), 15 Denickestrasse, D-21073 Hamburg, Germany

r t i c l e i n f o

rticle history:eceived 6 October 2012eceived in revised form4 November 2012ccepted 28 November 2012vailable online 7 December 2012

eywords:rude glycerol

a b s t r a c t

The production of 1,3-propanediol (PD) by a newly isolated Citrobacter freundii strain [FMCC-B 294(VK-19)] was investigated. Different grades of biodiesel-derived glycerol were employed. Slightly lowerPD biosynthesis was observed in batch experiments only when crude glycerol from waste-cooking oiltrans-esterification was utilized and only at elevated initial substrate concentrations employed. Batchbioreactor cultures revealed the capability of the strain to tolerate elevated amounts of substrate (glycerolup to 170 g/L) and produce quantities of PD in such high substrate concentrations. Nevertheless, maxi-mum PD quantities (45.9 g/L) were achieved at lower initial glycerol concentrations (∼100 g/L) employed,suggesting some inhibition exerted due to the increased initial substrate concentrations. In order to

,3-Propanedioliodieselon-sterile bioprocessitrobacter freundii

improve PD production, a fed-batch fermentation was carried out and 68.1 g/L of PD were produced (thehighest PD quantity achieved by C. freundii strains so far) with yield per glycerol consumed ∼0.40 g/g andvolumetric productivity 0.79 g/L/h. Aiming to perform a more economical and eco-friendlier procedure,batch and fed-batch fermentations under completely non-sterile conditions were carried out. Duringnon-sterilized fed-batch process, 176 g/L of raw glycerol were converted to 66.3 g/L of PD, suggesting thepotentiality of the non-sterile fermentation by C. freundii FMCC-B 294.

. Introduction

1,3-Propanediol (PD) is a platform chemical presenting sev-ral industrial applications. Due to its special properties, PDas an important role in many synthetic reactions, especiallyoly-condensations, which result in the synthesis of poly-esters,oly-ethers and poly-urethanes (Biebl et al., 1999; Lee et al., 2004;axena et al., 2009). Plastics based on this monomer, besidesheir bio-degradability, exhibit better product properties andigher light stability than those produced by 1,2-propanediol,,3-butanediol or ethylene glycol (Papanikolaou, 2009). Of par-icular interest is the use of PD as a monomer for the synthesisf polytrimethylene-terephthalate (PTT), a novel polymer withroperties comparable to Nylon and wide applications in carpets

Corterra) and textile fibers (Sorona) (Celinska, 2010; Willke andorlop, 2008). Moreover, besides its utilization as a base-unit for

he synthesis of biodegradable plastics, PD can present various

∗ Corresponding author at: Laboratory of Food Microbiology and Biotechnology,epartment of Food Science and Technology, Agricultural University of Athens,reece. Tel.: +30 210 5294700; fax: +30 210 5294700.

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

168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jbiotec.2012.11.018

© 2012 Elsevier B.V. All rights reserved.

interesting applications in the chemical industry; this chemicalcompound can be efficiently used as a polyglycol-type lubricantand its addition can significantly improve the properties in varioussolvent systems (increased flexibility in blending ester quats),adhesives, laminates, resins (low intrinsic viscosity) and cosme-tology products (long-lasting but not sticky moisturizing effect)(Papanikolaou, 2009; Zeng and Biebl, 2002).

Traditionally, bulk production of PD has been performedthrough chemical synthesis based on the utilization of petrochem-ical substrates, mainly propylene (“Degussa” process) or ethylene(“Shell” process) oxide as staring materials. However, increasingworldwide demand and consequent decreasing availability ofpetroleum, as well as appearance of reaction intermediates andutilization catalysts that could potentially provoke severe healthproblems (e.g. acroleïne, rubidium, etc.) together with extremeoperating conditions (e.g. significantly high pressure employed invarious of the steps employed in the processes) elaborated in thePD production through chemical process, have rendered as a veryimportant priority the discovery, elaboration and application in

large-scale operations of alternative eco-friendly ways of synthesisof PD, therefore initiating the research dealing with the biotechno-logical production of this compound (Anand et al., 2010; Liu et al.,2010; Saxena et al., 2009). In fact, in order for PD to be synthesized

Biotec

tutiadbwtgi2weaaimepeo“Ygao2Ymcf

sam2IotddpvtsBtdoogroa2M2e

npsv(f

M. Metsoviti et al. / Journal of

hrough microbial fermentations, glycerol should absolutely besed as (sole or co-) substrate of the process. Glycerol fermenta-ion to PD, well known for many years (this type of conversions known since 19th century – see Freund, 1881), is attractinggain attention, due to the constant increase in conventionaliesel prices, as well as the excess of crude glycerol deriving fromiodiesel production. In fact, 1 kg of glycerol becomes availableith the synthesis of 10 kg of biodiesel from various oils. During

he last decade, continuously enhanced biodiesel production,enerated a glut of crude glycerol with a subsequent reduction ints price (Anand et al., 2010; Papanikolaou, 2009; Zeng and Sabra,011). Moreover, significant quantities of glycerol-containingastes can be generated through oleochemical facilities, or bio-

thanol and/or alcoholic beverages production units (Yazdanind Gonzalez, 2007). Although pure glycerol has many industrialpplications, the utilization of biodiesel-derived crude glycerols still limited due to its composition. Impurities such as residual

ethanol, catalysts, salts, free fatty acids and methyl esters restrictmployment in the chemical and pharmaceutical industry withoutre-treatment, while the cost of purification makes the applicationconomically unfeasible (Metsoviti et al., 2012a). Therefore insteadf a functional co-product, crude glycerol is now considered as awaste stream”, with related disposal cost (Papanikolaou, 2009;azdani and Gonzalez, 2007). As a result, conversion of crudelycerol to higher value products could be an economical as wells ecological resolution for biodiesel industries and potentiallyleochemical and/or bio-ethanol-producing plants (Dobson et al.,011; Papanikolaou et al., 2000; Papanikolaou and Aggelis, 2003;azdani and Gonzalez, 2007). Given that the substrate cost foricrobial PD production can reach up to half of the total production

ost, crude glycerol could constitute a cost-effective and abundanteedstock for this bioconversion (Zeng and Sabra, 2011).

Several species of the Enterobacteriaceae family such as Kleb-iella pneumoniae, Klebsiella oxytoca and Citrobacter freundii as wells species of the genus Clostridium have been reported to fer-ent glycerol into PD (for state-of-the-art reviews see: Kaur et al.,

012a; Papanikolaou, 2009; Saxena et al., 2009; Tracy et al., 2012).n most cases, glycerol is subjected to assimilation under anaer-bic conditions via two parallel metabolic pathways encoded byhe dha regulon: through the oxidative pathway glycerol is dehy-rogenated to dihydroxyacetone (DHA) by the enzyme glycerolehydrogenase, which, after phosphorylation, is converted to DHA-hosphate. Furthermore, via EMP glycolysis it can be converted toarious end-products including acetic acid, lactic acid, ethanol, etc.,he formation of which is strain-dependent (for relevant metabolicchemes see Papanikolaou, 2009; Papoutsakis, 2008; Zeng andiebl, 2002). Through the reductive pathway, glycerol is dehydratedo 3-hydroxypropionaldehyde, reaction catalyzed by glycerol dehy-ratase, which is then reduced to PD by the enzyme 1,3-glycerolxidoreductase. On the other hand, although a significant numberf reports deal with the biotechnological formation of PD from purelycerol, only the last years there has been a rise in the number ofeports that deal with the utilization of crude glycerol as a substrate,r the effects of its impurities in order for PD to be produced (Anandnd Saxena, 2012; Chatzifragkou et al., 2010, 2011; Hiremath et al.,011; Jun et al., 2010; Kaur et al., 2012b; Metsoviti et al., 2012b;u et al., 2006; Papanikolaou et al., 2000, 2004, 2008; Ringel et al.,

012; Rossi et al., 2012; Sattayasamitsathit et al., 2011; Wilkenst al., 2012).

In our previous study, after a screening of a relatively highumber of prokaryotic microbial strains in relation with theirotentiality to ferment crude glycerol, a newly isolated C. freundii

train FMCC-B 294 (VK-19) was revealed to be capable to con-ert crude glycerol to PD, in a relatively high conversion yieldsMetsoviti et al., 2012a). The objective of the current study was tourther investigate the dynamics of PD production from this strain

hnology 163 (2013) 408– 418 409

through evaluation of the impact of initial glycerol concentrationinto the medium, in addition to the effect of non-sterile conditionsduring batch and fed-batch bioprocesses.

2. Materials and methods

2.1. Microorganism and medium

C. freundii FMCC-B 294 (VK-19), isolated from minced beef undermodified atmosphere packaging conditions (Doulgeraki et al.,2011; Metsoviti et al., 2012a), was used throughout this study.Long-term storage took place at −80 ◦C in Tryptic Soy Broth, sup-plemented with 20% glycerol (Sigma Chemical Co, St Louis, MO,USA). Before experimental use, strain was sub-cultured twice inTryptic Soy Broth and incubated at 30 ◦C for 24 h. Pre-cultures ofC. freundii were conducted in 100-mL anaerobic flasks contain-ing 40 mL Tryptic Soy Broth and incubated for 20 h, at 30 ± 1 ◦Cwithout agitation. Culture medium contained per L: carbon source20 g; peptone 5 g; meat extract 5 g; yeast extract 2.5 g; K2HPO4 2 g;CH3COONa 5 g; MgSO4 0.41 g and MnSO4 0.05 g. The carbon sourcesused were analytical grade (pure) glycerol 99% (w/w) and threetypes of biodiesel derived raw glycerol provided by: (1) “ArcherDaniels Midland Company (ADM)”, Hamburg, Germany [purity 81%(w/w); impurities composed of: 11–12% (w/w) water, 5–6% (w/w)potassium salts, 1% (w/w) free-fatty acids and less than 0.2% (w/w)methanol] (glycerol feedstock derived from trans-esterification ofrapeseed oil); (2) “ELIN-Biofuels SA”, Athens, Greece [purity 90%(w/w); impurities composed of: 5% (w/w) water, 5% (w/w) ash andless than 0.2% (w/w) methanol] (glycerol feedstock derived fromtrans-esterification of waste cooking oils); (3) “GF Energy ABEE”,Korinthos, Greece [purity 85.5% (w/w); impurities composed of:10% (w/w) water, 4–5% (w/w) potassium salts and 0.7% (w/w) ash](glycerol feedstock derived from trans-esterification of mixturesof edible fatty materials like soybean oil and cottonseed oil). Itis evident that when crude glycerol was utilized as substrate atvarious initial glycerol concentrations (Gly0), the appropriate cal-culations taking into consideration the purity of the feedstockswere performed, in order to achieve the requested carbon sub-strate concentration. The initial pH of the medium was 6.2 ± 0.1after autoclaving.

2.2. Culture conditions

Batch and fed-batch bioreactor fermentations were conductedin a 1.2-L bioreactor (New Brunswick Scientific, USA), in which theworking volume was adjusted at 0.9 L. Cultures under sterile andnon-sterile conditions were inoculated with 5% (v/v) and 10% (v/v),respectively, of a 20-h pre-culture inoculum. Agitation performedat 150 ± 5 rpm, the incubation temperature was T = 30 ◦C and thepH was maintained at 7.0 by automatic addition of 5 M NaOH. Con-tinuous gassing with N2 at flow rate 0.1 LPM provided anaerobicconditions for all trials performed. In fed-batch fermentation, glyc-erol concentration was initially at 40 g/L and after it was below5 g/L, pulses with concentrated raw glycerol (60%, v/v) and yeastextract (1%, w/v) were added into the medium.

2.3. Analytical methods

Cell concentration (X, g/L) was determined through a linearequation of cell dry weight (90 ± 5 ◦C until constant weight) andoptical density (OD) at 650 nm (Hitachi U-2000 Spectrophotome-ter, Japan). Cells were collected by centrifugation (9000 × g/15 min,

9 ◦C) in a Hettich Universal 320-R (Germany) centrifuge and washedtwice with distilled water. Finally, concentrations of glycerol,1,3-propanediol and organic acids were determined with High Per-formance Liquid Chromatography (HPLC) analysis (Waters 600E)

4 Biotec

wualrttc

3

3

ucoedtc∼ttctptsoouot

m1cPtw(YeveoqsmggsnbtC“cf

3u

e

10 M. Metsoviti et al. / Journal of

ith an Aminex HPX-87H (300 mm × 7.8 mm, Bio-Rad, USA) col-mn coupled to a differential refractometer (RI Waters 410) and

UV detector (Waters 486). Operating conditions were as fol-ows: sample volume 20 �L; mobile phase 0.005 M H2SO4; flowate 0.6 mL/min; column temperature T = 65 ◦C. All data points inables and figures presented are the mean values deriving fromwo independent experiments performed under the same cultureonditions.

. Results

.1. Product formation by different grades of crude glycerol

As revealed in our previous study (Metsoviti et al., 2012a), C. fre-ndii FMCC-B 294 (VK-19) efficiently converted biodiesel-derivedrude glycerol, with the major fermentation product being PD. Inrder to extend this observation, three different types of crude glyc-rol feedstock (with purities 90%, 85.5 and 81%, w/w, deriving fromifferent biodiesel production plants) were tested, while a “con-rol” experiment using pure glycerol was conducted. Initial glyceroloncentration (Gly0) was adjusted at relatively low quantities, i.e.20 g/L, in order to avoid inhibition phenomena deriving from both

he substrate (glycerol) itself and the impurities, the concentra-ion of which would have been higher when more elevated Gly0oncentrations would have been employed. Final product concen-ration, total glycerol consumption and fermentation duration areresented in Table 1. The achieved results of all cultures, showedhat glycerol was completely and rapidly consumed after 9–12 h,uggesting that the strain was capable to adapt to the impuritiesf crude glycerol feedstock very rapidly. In the culture performedn “Crude 1” (purity 81.0%, w/w) higher biomass production (X val-es of 1.8 g/L) was observed, as compared to all of the other typesf glycerol feedstocks used (X = 1.4 g/L). It seems that impurities inhis type of glycerol slightly favored cell growth.

The predominant metabolic compound throughout the four fer-entations was PD and maximum concentration achieved was

0.6 g/L in case of “Crude 1”, while 11.4 g/L were formed in theontrol experiment with pure glycerol. Almost equal quantities ofD ranging between 10.2 and 10.6 g/L were formed when differentypes of crude glycerol were utilized as carbon sources. Like-ise, the conversion yield of PD produced per glycerol consumed

YPD/Gly) was almost equal for all trials performed; specificallyPD/Gly was 0.53 g/g in all three fermentations in which crude glyc-rol was employed as C. freundii substrate, with the respectivealue being 0.54 g/g in the control experiment with pure glyc-rol. Moreover PD production was accompanied by the synthesisf small amounts of acetic and lactic acid (Table 1), as well as trivialuantities of formic acid [concentration less than 0.5 g/L (data nothown)]. From all the above-mentioned analysis it may be sum-arized that under the experimental conditions imposed, all three

rades of crude glycerol proved to be suitable substrates for cellrowth and PD production by C. freundii FMCC-B 294 (VK-19). Feed-tock impurities (salts, methanol, etc.) did not seem to have anyegative impact on glycerol assimilation by the strain, whilst theiosynthesis of PD also did not seem to be severely curtailed byhe different feedstocks used, at the Gly0 concentration employed.rude glycerol with purity 81.0% (w/w) (“Crude 1” deriving fromArcher Daniels Midland Company”) was selected for the forth-oming experiments, due to its slightly enhanced biomass and PDormation, compared with the other types of crude glycerol.

.2. Impact of initial glycerol concentration on product formation

nder sterile and non-sterile conditions

In order to evaluate whether the initial concentration of glyc-rol and the impurities of glycerol feedstock could affect PD

hnology 163 (2013) 408– 418

production by C. freundii, batch-bioreactor fermentations withconstantly increasing initial glycerol (Gly0) concentrations wereperformed with all other fermentations parameters remaining con-stant. Therefore, Gly0 quantities ranging from ∼40 up to ∼170 g/Lwere performed, it may be assumed, therefore, that in severalcases, indeed very high Gly0 concentrations (e.g. >100 g/L) wereemployed. Moreover, as recently proposed in investigations per-formed by our research team (Chatzifragkou et al., 2011; Metsovitiet al., 2012b) 1,3-propanediolic fermentation can efficiently andsuccessfully be performed under completely non-sterile fermenta-tion conditions (microorganisms tested were strains of the speciesClostridium butyricum and K. oxytoca), and this event can noticeablydecrease the total cost of the bioprocess, since it can significantlyreduce the investment cost for the bioreactor set-up and the opera-tion costs at an industrial level (e.g. energy for substrate treatmentand sterilization whereas large-scale bioreactors are subjected toin situ sterilization). To this end, additional experiments using C.freundii were performed under non-sterile conditions, using simi-lar Gly0 concentrations employed in the sterile cultures, aiming atrendering much more environmentally friendly the proposed bio-process. As given in Table 2, with the rise of Gly0 up to 100 g/L, totaland quick assimilation of glycerol in both sterile and non-sterileprocess was observed. Beyond this value, glycerol consumptionrate declined, while fermentation time was prolonged. In partic-ular when the medium contained 170 g/L of glycerol, the amountof non-consumed glycerol was ∼48% (w/w) of the initial concentra-tion, and fermentation time was ∼2.5 times higher compared withthe experiment at Gly0 ∼ 100 g/L, in which the highest amount ofglycerol was consumed.

Regarding product formation, PD production progressivelyincreased, when the Gly0 concentration increased up to a levelof 100 g/L. Maximum PD concentration of 45.9 g/L was achievedat Gly0 = 100 g/L, under sterile conditions, after 38 h of fermenta-tion, which corresponded to a yield YPD/Gly value of 0.47 g/g and aproductivity of 1.2 g/L/h. Similarly at non-sterile processes compa-rable high PD production was observed and after 38 h of culture43.5 g/L of PD were accumulated into the medium. Furthermorethroughout all non-sterilized trials, periodical microscopic obser-vations after Gram coloration indicated that almost exclusively C.freundii cells were found into the fermentation medium. Addition-ally results from HPLC analysis showed that the same metaboliccompounds were produced during sterile and non-sterile pro-cesses, irrespective the Gly0 concentration, confirming once againthat C. freundii was the predominant microorganism, during theexperiments.

In both sterile and non-sterile processes at Gly0 > 100 g/L(namely at 150 and 170 g/L), PDmax quantities were reduced tolower levels compared with trials with Gly0 ∼ 100 g/L. Meanwhile,PD yield per glycerol consumed reached a maximum value of0.53 g/g at the lowest Gly0 and steadily and linearly decreased to0.40 g/g at Gly0 ∼ 170 g/L (Fig. 1). Even more noticeable was thedecrease of PD volumetric productivity (Fig. 2). More precisely, amaximum productivity of 1.53 g/L/h was observed at Gly0 ∼ 80 g/Land subsequently declined to 0.36 g/L/h at Gly0 ∼ 170 g/L. Thesefindings imply that PD formation was significantly impeded byhigh substrate concentration. On the other hand, lactic acid forma-tion gradually increased, when glycerol was added to the medium.In particular, the highest concentration, 28.9 and 27.6 g/L wasobtained at Gly0 ∼ 170 g/L, under sterile and non-sterile conditions,respectively. In the same way, yield of lactic acid produced perglycerol consumed, increased from value 0.11 to 0.33 g/g and 0.09to 0.33 g/g, under sterile and non-sterile conditions, correspond-

ingly. In relation to acetic acid formation, no inhibition phenomenawere noticed. Low quantities (in terms of both absolute values – g/Land relative values – %, w/w, on glycerol consumed) were secretedinto the medium thorough the experiments (ranging from 3.0 up

M. Metsoviti et al. / Journal of Biotechnology 163 (2013) 408– 418 411

Table 1Effect of type of glycerol on biomass and product formation by C. freundii FMCC-B 294 (VK-19). Culture conditions: growth on batch bioreactor experiments, initial glycerolconcentration (Glyc0) ∼ 20 g/L, T = 30 ◦C, pH 7.0, agitation at 150 ± 5 rpm. All data points are the mean values deriving from two independent experiments performed underthe same culture conditions.

Glycerol (purity %, w/w) Duration (h) Glyc (g/L) X (g/L) PD (g/L) Ace (g/L) Lac (g/L) YPD/Gly (g/g)

Pure (99.0%) 9 20.2 1.4 11.4 1.8 1.6 0.54“Crude 1” (81.0%) 10 20.1 1.8 10.6 1.8 1.7 0.53“Crude 2” (90.0%) 12 19.6 1.4 10.4 1.9 1.8 0.53“Crude 3” (85.5%) 10 19.3 1.4 10.2 1.8 1.9 0.53

Glyc: glycerol consumed; X: biomass produced; PD: 1,3-propanediol produced; Lac: lactic acid produced; Ace: acetic acid produced.

Table 2Effect of initial glycerol concentration on growth and product formation during batch cultures of C. freundii FMCC-B 294 (VK-19), under sterile and non-sterile conditions.All data points are the mean values deriving from two independent experiments performed under the same culture conditions.

Gly0 (g/L) Glyc (g/L) Duration (h) X (g/L) PD (g/L) Lac (g/L) Ace (g/L)

Sterile conditions∼40 40.5 14 1.9 21.5 4.6 3.0∼80 80.7 26 2.5 39.9 14.6 4.6

∼100 98.0 38 2.3 45.9 20.3 3.7∼150 96.6 70 1.8 38.2 26.3 3.5∼170 88.7 96 1.8 35.1 28.9 3.5

Non-sterile conditions∼40 42.5 14 2.2 21.6 4.1 3.8∼80 79.2 27 2.5 40.2 15.9 4.9

∼100 97.1 38 2.3 43.5 21.2 3.9∼150 95.6 70 1.8 35.7 26.9 3.5

1.8

G : lacti

tf

Gtpi

Fcfai

∼170 87.8 96

lyc: glycerol consumed; X: biomass produced; PD: 1,3-propanediol produced; Lac

o 4.9 g/L), irrespective of the initial glycerol concentration and theermentation mode.

Biomass production reached a maximum of 2.5 g/L atly ∼ 80 g/L and declined to 1.8 g/L, when higher Gly concen-

0 0

rations were imposed (specifically from 150 up to 170 g/L),resumably due to substrate inhibition exerted by the relatively

ncreased concentration found into the medium. This conclusion

30

35

40

45

50

55

0 40 80 12 0 160 20 0

Sterile conditionsNon-sterile conditions

% Y

PD

/Gly (g/

g)

Initial Glycerol concentration (g/L)

ig. 1. Profile of conversion yield of 1,3-propanediol (PD) produced per glycerolonsumed during growth of Citrobacter freundii strain FMCC-B 294 (VK-19) on dif-erent initial glycerol concentrations, in batch bioreactor experiments under sterilend non-sterile conditions. All data points are the mean values deriving from twondependent experiments performed under the same culture conditions.

33.4 29.6 3.5

c acid produced; Ace: acetic acid produced.

was also enhanced by the calculation of lag time (tL) of the cul-tures for all the experiments with different Gly0 concentrations; tLgradually increased from 4 to 12 h, with highest value observed atGly0 = 170 g/L (data not presented). Moreover, the specific growth

rate (�) for all of the performed trials was evaluated by fittingthe equation ln(X/X0) = f(t) on the experimental data found withinthe early exponential growth phase of each of the cultures. Values

0

0,5

1

1,5

2

2,5

0 40 80 12 0 160 20 0

Sterile conditionsNon-Sterile conditions

1,3

-Pro

pan

edio

l Pro

duct

ivity

(g/L/h)

Initial Glyce rol concentration (g/L)

Fig. 2. Profile of 1,3-propanediol productivity, during growth of Citrobacter freundiistrain FMCC-B 294 (VK-19) on different initial glycerol concentrations, in batchbioreactor experiments under sterile and non-sterile conditions. All data points arethe mean values deriving from two independent experiments performed under thesame culture conditions.

412 M. Metsoviti et al. / Journal of Biotec

0

0,1

0,2

0,3

0,4

0,5

0 40 80 12 0 160 20 0

Spec

ific

gro

wth

rat

e (h

-1)

Initial Glycerol concentration (g/L)

F2t

fl�ve

8Pdtiswwtftww(ot(Piaticfita8tqtsse

d

4. Discussion

ig. 3. Specific growth rate (�) during growth of Citrobacter freundii strain FMCC-B94 (VK-19) on different initial glycerol concentrations, in batch bioreactor fermen-ations.

uctuated between 0.09 and 0.42 h−1, with noticeable decrease of values observed when Gly0 concentrations increased (the �max

alue was observed at Gly0 ∼ 20 g/L) (Fig. 3), providing once morevidence of substrate inhibition against the strain.

Taking into consideration that a Gly0 value of approximately0 g/L resulted in both maximum biomass (2.5 g/L) and maximumD (1.53 g/L/h) productivity, an additional experiment was con-ucted with another grade of crude glycerol, in order to examinehe strain’s ability to adapt, at an elevated Gly0, to the differentmpurities found in crude glycerol of various sources. The feed-tock with purity 90.0% (w/w) (derived from biodiesel in whichaste cooking oil was the starting material) was selected as theorst case, because previous experiments at Gly0 ∼ 20 g/L showed

hat this feedstock had the longest fermentation duration, there-ore implying that more time was required for the strain to adapto impurities (see Section 3.1). As depicted in Fig. 4a, 36.5 g/L of PDere formed after 30 h of fermentation. This PDmax concentrationas approximately 9% lower than the value achieved with the 81.0%

w/w) purity glycerol feedstock (derived from trans-esterificationf non-thermally treated rapeseed oil as the starting material forhe synthesis of biodiesel), under sterile and non-sterile conditionsFig. 4b and c). Thus, a slight negative effect on the biosynthesis ofD was observed, due to the presence of the thermally treated oilmpurities. However, this PDmax quantity can still be characterizeds satisfactory. In all three cultures (Fig. 4a–c), glycerol assimila-ion rate is comparable (rGly ∼ 3 g/L/h), irrespective of the nature ofmpurities of the glycerol feedstock or the conditions under whichultures were performed (sterile or non-sterile). As far as the pro-le of other metabolic compounds is concerned, the culture withhe 90% (w/w) purity glycerol resulted in the biosynthesis of lactatet slightly lower quantities compared to the sterile trial with the1.0% (w/w) purity glycerol. On the other hand, the non-asepticrial was accompanied by the synthesis of slightly higher lactateuantities compared to the results achieved under sterile condi-ions (see Fig. 4b and c). In any case, it can be concluded that thetrain is capable of converting crude glycerol to PD, without beingtrongly affected by the type of impurities found in the waste glyc-

rol feedstock.

From the aforementioned results two conclusions can beeducted: firstly although microbial growth, glycerol consumption

hnology 163 (2013) 408– 418

and PD production observed in indeed high Gly0 concentration tri-als (e.g. 150 or 170 g/L), increment of Gly0 concentration in therange of 150–170 g/L, resulted in a substrate inhibition toward thestudied C. freundii strain. A second important remark refers to trialsperformed during non-sterile processes, which resulted in similarproduct formation profiles with the respective sterilized processesfor equivalent Gly0 concentrations imposed.

3.3. Fed-batch process under sterile and non-sterile conditions

C. freundii strain FMCC-B 294 (VK-19) was able to grow inbatch cultures even at Gly0 concentrations as high as 170 g/L, atboth sterile and non-sterile conditions. Since fed-batch processis an effective fermentation mode to avoid substrate inhibitionphenomena, it was decided to perform fed-batch fermentations inorder to improve glycerol consumption and enhance PD produc-tion. An initial glycerol concentration of 40 g/L was selected as itwas observed that this value resulted in the optimum combina-tion of high PD yield (0.53 g/g) and productivity (1.53 g/L/h) (seeTable 2). When glycerol concentration was lower than ∼5 g/L con-centrated, crude glycerol was added into the medium. The kineticprofile of the fermentation performed under aseptic conditions isdepicted in Fig. 5a and b. During the first 24 h of the fermentationnearly half of the total amount of crude glycerol was consumed(∼90 g/L). Specific glycerol consumption rate (qS) reached maxi-mum value 1.15 g/g/h, 15 h after the inoculation. Once the thirdfeed was added into the medium, glycerol uptake rate graduallydeclined, and finally after 86 h of fermentation 172 g/L were con-verted to a variety of metabolic compounds (Table 3). Moreover, theconcentration of biomass remained almost constant throughoutthe culture 24 h after inoculation. On the other hand, ethanol grad-ually started to be accumulated into the fermentation medium after24 h from inoculation, with its final concentration being 5.5 g/L.In the bioprocess performed under aseptic conditions, PD was thepredominant metabolite throughout the fermentation and a max-imum quantity of this compound of 68.1 g/L was observed at theend of the fed-batch experiment. A remarkable volumetric produc-tivity of ∼1.62 g/L/h was obtained during the first 24 h, decreasingto the value of 0.79 g/L/h, by the end of the process (Table 4).Likewise conversion yield of PD produced per glycerol consumedreached the highest value 0.50 g/g throughout the first 24 h of fer-mentation, and gradually reduced to 0.40 g/g, at the end of thefermentation (Table 4). By far, the metabolite the concentrationof which presented remarkable values after PD was that of lac-tic acid, with a final maximum concentration of ∼30 g/L, whilelower quantities of acetic (7.3 g/L) and formic acid (1.3 g/L) werefound into the medium. Similar results concerning the final con-centrations of the metabolic products and glycerol consumptionwere observed during the fermentation under non-sterile condi-tions as shown in Fig. 6a and b. After 93 h of fermentation, ∼176 g/Lof glycerol had been converted to 66.3 g/L of PD and 33.7 g/L oflactic acid (Table 3). Maximum biomass production 3.3 g/L wasobtained in both experimental conditions. As also observed in batchfermentations under non-sterile conditions lactic acid productionwas slightly higher than sterile process. Finally regular microscopicobservations after Gram coloration proved that C. freundii was thepredominant microorganism found into the fermentation medium.Comparing, finally, overall results of sterile and non-sterile fed-batch experiments it can be concluded that non-sterilized process,is an appropriate method for high PD formation and crude glycerolassimilation by C. freundii strain FMCC-B 294 (VK-19).

Glycerol fermentation to PD has been principally demonstratedby bacteria belonging to the species K. pneumoniae and Cl. butyricum

M. Metsoviti et al. / Journal of Biotechnology 163 (2013) 408– 418 413

0

10

20

30

40

50

60

70

80

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35

Gly (g/L ) PD (g/L )Lac (g/L )Ace (g/L )

Form (g/L )X (g/L)

Gly

cero

l (g/

L)

Bio

mas

s (X

, g/

L) &

Pro

duct

for

mat

ion

(g/L

)

Time (h)

0

10

20

30

40

50

60

70

80

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30

Gly (g/L ) PD (g/L)

Lac (g/L )

Ace (g/L)

Form (g/L)

X (g/L)

Gly

cero

l (g/

L)

Bio

mas

s (X

, g/

L) &

Pro

duct

for

mat

ion (g/

L)

Time (h)

0

10

20

30

40

50

60

70

80

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30

Gly (g/L )

a b

c

PD (g/L)Lac (g/L )Ace (g/L)

Form (g/L )X (g/L)

Gly

cero

l (g/

L)

Bio

mas

s (X

, g/

L) &

Pro

duct

for

mat

ion (g/

L)

Time (h)

Fig. 4. Kinetics of glycerol consumption and biomass (X – g/L), 1,3-propanediol (PD – g/L), lactic acid (Lac – g/L), acetic acid (Ace – g/L) and formic acid (Form – g/L) productionduring growth of Citrobacter freundii strain FMCC-B 294 (VK-19) on Gly0 ∼ 80 g/L (purity 90%, w/w, glycerol feedstock deriving from used cooking oil as starting material forthe synthesis of biodiesel) (a), on Gly0 ∼ 80 g/L (purity 81.0%, w/w, glycerol feedstock deriving from rapeseed oil used as starting material for the synthesis of biodiesel) (b),and on Gly0 ∼ 80 g/L (purity 81.0%, w/w, glycerol feedstock deriving from rapeseed oil used as starting material for the synthesis of biodiesel under non-sterile conditions) inbatch bioreactor fermentation. Culture conditions: T = 30 ◦C, pH 7.0 ± 0.2, growth on 1.2-L bioreactor.

Table 3Comparison of glycerol consumption, maximum biomass (Xmax) and final product concentrations, in fed-batch fermentations by C. freundii FMCC-B 294 (VK-19), under sterileand non-sterile conditions. All data points are the mean values deriving from two independent experiments performed under the same culture conditions.

Conditions Time (h) Glyc (g/L) X (g/L) PD (g/L) Lac (g/L) Ace (g/L) EtOH (g/L) Form (g/L)

Sterile 86 172.3 3.3 68.1 29.8 7.3 5.5 1.3Non-sterile 93 175.8 3.3 66.3 33.7 6.9 8.3 0.9

Glyc: glycerol consumed; X: biomass produced; PD: 1,3-propanediol produced; Lac: lactic acid produced; Ace: acetic acid produced; EtOH: ethanol produced; Form: formicacid produced.

414 M. Metsoviti et al. / Journal of Biotechnology 163 (2013) 408– 418

Table 4Comparison of conversion yield of PD produced per glycerol consumed and PD productivities at different stages of fed-batch fermentations by C. freundii FMCC-B 294 (VK-19),under sterile and non-sterile conditions. All data points are the mean values deriving from two independent experiments performed under the same culture conditions.

Fermentation time (h) YPD/Gly (g/g)a PD volumetric productivity (g/L/h)a

Sterile process Non-sterile process Sterile process Non-sterile process

0–12 0.50 0.50 1.42 1.4412–24 0.48 0.48 1.62 1.5824–36 0.44 0.43 1.61 1.2936–48 0.41 0.39 1.27 1.0148–86 0.40 0.38

0–86 0.40 0.38

a Calculation of conversion yields and volumetric productivities was based on the spec

0

5

10

15

20

25

30

35

40

0

10

20

30

40

50

60

70

0 20 40 60 80 10 0

Gly (g/L) PD (g/L)

Gly

cero

l (G

ly, g/

L)

1,3

-Pro

pan

edio

l (P

D, g/

L)

Time (h)

a

b

0

5

10

15

20

25

30

0 20 40 60 80 10 0

Lac (g/L )Ace (g/L)

EtOH (g/L)X (g/L)

Bio

mass

(X

, g/

L) &

Pro

du

ct F

orm

ati

on (g/

L)

Time (h)

Fig. 5. Kinetics of glycerol consumption and 1,3-propanediol (PD – g/L) production(a), and biomass (X – g/L), ethanol (EtOH – g/L), lactic acid (Lac – g/L) and aceticacid (Ace – g/L), production (b), during growth of Citrobacter freundii strain FMCC-B294 (VK-19) on crude glycerol in fed-batch bioreactor experiments, under sterileconditions. Culture conditions: T = 30 ◦C, pH 7.0 ± 0.2, growth on 1.2-L bioreactor.

0.79 0.790.79 0.71

ific time intervals.

(Chatzifragkou et al., 2011; Chen et al., 2003; Cheng et al., 2004;Gonzalez-Pajuelo et al., 2004; Hiremath et al., 2011; Ji et al., 2009;Jun et al., 2010; Liu et al., 2007; Ma et al., 2009; Mu et al., 2006;Papanikolaou et al., 2000, 2004, 2008; Petrov and Stoyanov, inpress; Ringel et al., 2012; Rossi et al., 2012; Sattayasamitsathitet al., 2011; Saxena et al., 2009; Sotenko et al., in press; Wilkenset al., 2012; Xu et al., 2009; Zeng, 1995, 1996). Fewer reportsdeal PD-producing microorganisms which belong to the speciesK. oxytoca (Metsoviti et al., 2012b; Yang et al., 2007), Clostrid-ium diolis (Otte et al., 2009; Kaur et al., 2012a,b,c), Clostridiumacetobutylicum (Gonzalez-Pajuelo et al., 2005), Clostridium saccha-robutylicum (Gungormusler et al., 2010), Enterobacter agglomerans(Barbirato et al., 1995, 1996), Lactobacillus diolivorans (Pfügl et al.,2012), Pantoea agglomerans (Casali et al., 2012) and Halanaerobiumsaccharolyticum (Kivistö et al., 2012). A limited number of stud-ies deal with PD production by the genus Citrobacter; strains ofthe species C. freundii (Anand and Saxena, 2012; Barbirato et al.,1998; Boenigk et al., 1993; Casali et al., 2012; Homann et al., 1990;Metsoviti et al., 2012a) have been employed in this conversion andcrude glycerol rarely was used as substrate, while recently strains ofthe species Citrobacter werkmanii (Maervoet et al., 2012) have beenrevealed capable to perform the 1,3-propanediolic fermentation.

Our previous investigation revealed a natural strain namelyC. freundii FMCC-B 294 (VK-19) (Metsoviti et al., 2012a) thatcould break-down waste glycerol and produce quantities of PD.The present study validated the ability of this strain to toler-ate highly elevated amounts of crude glycerol (up to 170 g/Lof glycerol into the bioreactor) and produce significant quanti-ties of PD. Studies have shown that increased initial substratequantities may have an inhibitory effect on growth and glycerolassimilation and consequently on PD formation, and this effecthas been attributed to several reasons, such as the increasedosmotic pressure and the toxic effect of glycerol itself toward themicroorganism (Papanikolaou et al., 2000; Papanikolaou, 2009) orthe presence of crude glycerol’s impurities into the fermentationmedium (Petitdemange et al., 1995; Chatzifragkou et al., 2010). Inthe current study, at low Gly0 concentrations (e.g. 20 g/L) the strainadapted very quickly to the impurities and there was no signif-icant variation of the cultures duration between pure and crudeglycerol. The increase of Gly0 led to a further increase of the lagtime. In particular, when Gly0 exceeded 100 g/L, substrate inhibi-tion was observed. On the other hand, when raw glycerol was addedgradually inside the fermentor during the fed-batch process, andprogressive accumulation of feedstock impurities into the culturemedium occurred, no virtual inhibition on glycerol consumptionwas observed due to that fact, indicating that the strain was ableto adapt to impurities and therefore consume high glycerol quanti-ties in fed-batch mode. Thus, as far as the batch fermentations areconcerned, it seems that the elevated initial glycerol concentration

(>100 g/L) potentially has the effect of increasing osmotic pressureinto the medium or increasing the toxic effect of glycerol towardthe bacterial strains, resulting in somehow decreased fermenta-tion performances. In any case, the ability of the strain, itself, to

M. Metsoviti et al. / Journal of Biotec

0

5

10

15

20

25

30

35

40

0

10

20

30

40

50

60

70

0 20 40 60 80 10 0

Gly (g/L) PD (g/L)

Gly

cero

l (G

ly, g/

L)

1,3

-Pro

pan

edio

l (P

D, g/

L)Time (h)

0

5

10

15

20

25

30

35

0 20 40 60 80 10 0

Lac (g/L)Ace (g/L)

EtOH (g/L)X (g/L)

Bio

mass

(X

, g/

L) &

Pro

du

cts

For

mati

on (g/

L)

Time (h)

a

b

Fig. 6. Kinetics of glycerol consumption and 1,3-propanediol (PD – g/L) production(a), and biomass (X – g/L), ethanol (EtOH – g/L), lactic acid (Lac – g/L) and acetic acid((c

pei4afTatch

Ace – g/L), production (b), during growth of Citrobacter freundii strain FMCC-B 294VK-19) on crude glycerol in fed-batch bioreactor experiments, under non-sterileonditions. Culture conditions: T = 30 ◦C, pH 7.0 ± 0.2, growth on 1.2-L bioreactor.

resent growth, PD production and substrate assimilation at indeedlevated substrate concentrations (e.g. Gly0 ∼ 150 or 170 g/L), is ofmportance. On the contrary, batch cultures of K. pneumoniae DSM799 with Gly0 = 100 g/L in the feed, were impeded, most prob-bly by the presence of impurities, and during the first 24 h ofermentation no PD production was observed (Jun et al., 2010).he researchers also reported that for Gly0 higher than 60 g/L, the

dded glycerol was not completely consumed after 48 h of fermen-ation. This result corroborates the findings of Biebl (1991) whoonsidered that increasing substrate amounts (concentrations asigh as 9–11%, w/v) did not allow an efficient cell growth of Cl.

hnology 163 (2013) 408– 418 415

butyricum strains. Likewise, in order to obtain glycerol consump-tion higher than 100 g/L, Günzel et al. (1991) performed fed-batchoperations starting with initial glycerol concentrations of 20 or50 g/L (studies conducted with Cl. butyricum DSM 5431). LowerGly0 employed concentrations were sufficient for growth inhibi-tion by another newly isolated K. pneumoniae strain (Gungormusleret al., 2011). Likewise, Ringel et al. (2012) reported that strain Cl.butyricum AKR92a was unable to tolerate 150 g/L of crude glyc-erol, although satisfactory results were achieved at initial glycerolquantity of 100 g/L.

The effect of different grades and origins of crude glycerol wasalso examined in our study. Three biodiesel-derived glycerol feed-stocks (purities 81.0%, 85.5% and 90.0%, w/w), produced by usingthermally and non-thermally treated oils as starting material, wereemployed as substrates at initial glycerol concentration 20 g/L andC. freundii was able to grow on all cultures. The resulting PD yieldwas the same in all three trials (YPD/Gly = 0.53 g/g – see Table 1)and the final PD concentration also had no significant variation(10.2–10.6 g/L). At this low Gly0, the strain apparently presentsthe same tolerance to different grades of crude glycerol and dif-ferent feedstock impurities. Two of the feedstocks were also testedat initial glycerol concentration 80 g/L. The first feedstock (purity81.0%, w/w), produced by using rapeseed oil as starting material forbiodiesel synthesis, resulted in a final PD concentration of 39.9 g/L,while the second feedstock (purity 90.0%, w/w), produced fromused cooking oil, had a final PD concentration of 36.5 g/L. Therefore,the origin of the fatty material used in the production of biodieselappears to have an impact on the bioprocess. Prolongation of fer-mentation duration was also observed when the medium containedcrude glycerol deriving from cooking oil trans-esterification (purity90.0%, w/w), irrespective of the value of Gly0. This variation could bedue to the presence of different types of impurities found in crudeglycerol feedstock deriving from thermally treated oils (e.g. ben-zopyrenes and other cyclic hydrocarbons), which negatively affectthe PD fermentation efficiency (see Fig. 4a and b). The aforemen-tioned results suggest that glycerol origin rather than the purityitself affects the efficiency of the 1,3-propanediolic fermentation.

Gonzalez-Pajuelo et al. (2004) also studied two different gradesof crude glycerol (purity 65.0% and 92.0%, w/v) on batch cultures ofCl. butyricum VPI 3266 and calculated growth inhibition based onthe maximum specific growth rate. They showed that growth inhi-bition effect was more evident when the 65% (w/v) crude glycerolwas used as a substrate; in fact when the medium contained 100 g/Lof this type of glycerol, growth inhibition reached 86%. Althoughlower grade crude glycerol had an inhibitory effect on cell growth,no significant variations were found on PD production in termsof absolute values (g/L) on batch and continuous cultures. Theirconclusion was therefore that glycerol origin did not interfere onPD final concentration, but only on cell growth. In the study byChatzifragkou et al. (2010) the effect of several glycerol feedstockimpurities was studied toward the fermentation performances ofCl. butyricum VPI 1718. Unsaturated free-fatty acids (FFAs) in crudeglycerol were reported to have a noticeable negative effect on cellgrowth. Venkataramanan et al. (2012) came to similar conclusionsconcerning the unsaturated FFAs found in crude glycerol feedstocksas far as growth and metabolite production of Cl. pasteurianum ATCC6013 was concerned. Anand and Saxena (2012) demonstrated thatbiodiesel derived crude glycerol had a strong inhibitory effect onthe growth of a C. freundii strain; five different grades of crudeglycerol were tested and PD synthesis diminished, irrespective ofthe glycerol feedstock employed. With the aim of overcoming thisobstruction, they treated crude glycerol with non-polar solvents

and after removing FFAs and fatty acids methyl esters (FAMEs)from crude glycerol, the strain was able to produce PD in amountscomparable to pure glycerol. Pre-treatment of crude glycerol hasalso been proposed by Moon et al. (2010) in order to avoid growth

416 M. Metsoviti et al. / Journal of Biotechnology 163 (2013) 408– 418

Table 51,3-Propanediol production from different bacterial strains, cultivated on media composed of crude glycerol.

Strain PD (g/L) YPD/Gly (g/g) Crude glycerol purity (%, w/w) Fermentation type Reference

Citrobacter freundiia 25.6 0.51 a Batch Anand and Saxena (2012)FMCC-B 294 (VK-19) 68.1 0.40 81.0 Fed-batch Present study

Klebsiella oxytocaFMCC-197 50.1 0.40 81.0 Fed-batch Metsoviti et al. (2012b)

Klebsiella pneumoniaeDSM 4799 80.2 0.45 80.0 Fed-batch Jun et al. (2010)DSM 2026 53.0 a 85.0 Fed-batch Mu et al. (2006)SU6 25.0 a a Shake flasks Sattayasamitsathit et al. (2011)BLh-1 9.4 0.34 83.0 Batch Rossi et al. (2012)

Clostridium butyricumAKR102a 76.2 0.51 55.0 Fed-batch Wilkens et al. (2012)VPI 1718 67.9 0.55 81.0 Fed-batch Chatzifragkou et al. (2011)E5 58.4 0.54 95.0 Fed-batch Petitdemange et al. (1995)F2b 48.1 0.55 65.0 Continuous (1 stage) Papanikolaou et al. (2000)F2b 47.1 0.53 65.0 Batch Papanikolaou et al. (2008)F2b 43.5 0.49 65.0 Continuous (2 stages) Papanikolaou et al. (2008)AKR 91b 38.0 a 48.0 DWP Ringel et al. (2012)VPI 3266 31.5 0.50 65.0 Continuous Gonzalez-Pajuelo et al. (2004)VPI 3266 30.0 0.50 92.0 Continuous Gonzalez-Pajuelo et al. (2004)DSM 5431 25.0 0.50 a Batch Rehman et al. (2008)

Clostridium sp.

iIcpft

hic(2ioerbrsbsyuBbwopc(cPptsaiat

IK 124 80.1 0.56 a

a Non-designated.

nhibition due to the impurities found into the industrial feedstock.n that study, five Clostridium strains were unsuccessfully tested onrude glycerol derived from waste vegetable-oil-based biodieselroduction; once pre-treated crude glycerol was utilized, both PDormation and productivity were improved, and values similar torials with pure glycerol were obtained.

PD concentrations from crude glycerol in the range of 58–80 g/Lave been achieved by the “classical” PD-producing microorgan-

sms K. pneumoniae and Cl. butyricum, with fed-batch cultures beingonsidered as the most appropriate for this type of bioprocessMetsoviti et al., 2012b; Chatzifragkou et al., 2011; Wilkens et al.,012; Jun et al., 2010). Several techniques have been employed

n order to improve final PD concentration, such as inactivationf genes responsible for the by-products formation or over-xpression of genes involved in PD synthesis (for a state-of-the-arteview see: Celinska, 2010). The highest PD formation so far haseen achieved by DuPont and Genencor International Inc., utilizingecombinant Escherichia coli strains with glucose employed as theole substrate; therefore, in fed-batch processes (utilization of 15-Lioreactors), the genetically modified E. coli KLP23/pAH48/pDT29train achieved a PDmax quantity of ∼68 g/L, with a conversionield of PD produced per glucose consumed of 0.24 g/g and a vol-metric productivity of ∼1.8 g/L/h (Emptage et al., 2003, 2006).y using the E. coli strain KLP23/pAH48/pKP32 equally in fed-atch experiments, the authors reported a PDmax value of 112 g/Lith a conversion yield of 0.26 g/g and a volumetric productivity

f ∼2.3 g/L/h (Emptage et al., 2003). The strain RJ8/pAH48/pDT29roduced ∼50 g/L of 1,3-propanediol with conversion yield on glu-ose consumed ∼0.35 g/g and volumetric productivity of ∼1.2 g/L/hEmptage et al., 2006). Finally, fed-batch cultivation of the geneti-ally engineered RJ8/pAH48/pKP32 strain on glucose resulted in aDmax quantity of 129 g/L with concomitant conversion yield of diolroduced per sugar consumed of 0.34 g/g and volumetric produc-ivity of PD produced of 1.7 g/L/h (Emptage et al., 2003, 2006). In ourtudy, 68.1 g/L of PD were produced during a fed-batch process by

wild-type C. freundii strain. Potentially, this value could slightlyncrease with another glycerol feeding into the bioreactor, althought that stage, as previously reported, qS was remarkably lowerhat the one reported at the beginning of the fed-batch process,

Fed-batch Hirschmann et al. (2005)

presumably due to accumulation of compounds that could poten-tially inhibit the microbial growth like 1,3-propanediol and lacticacid, the concentration of which was rather elevated at the end ofthe process (see Table 3); in any case, in the current submission,PDmax concentration achieved was rather high and comparable tothe ones documented in literature so far, and, to the best of ourknowledge, the highest one achieved by C. freundii strains (Table 5).The production of PD was accompanied by the synthesis of lacticacid, as the principal bioprocess by-product, while smaller quanti-ties of ethanol, acetic and formic acid were also secreted. Accordingto Biebl et al. (1999) the highest coefficient yield of PD producedper glycerol consumed is achieved when acetic acid is the onlyby-product during glycerol fermentation to PD; the maximum the-oretical value is 0.71 moles/moles (=0.59 g/g) (Papanikolaou, 2009;Zeng, 1996). Once other by-products (specifically if they are antago-nistic to the recycling of NADH2 equivalents) are also synthesized,PD yield is reduced; one such product is lactic acid (Biebl et al.,1999). In our study, lactic acid formation reached almost 44% of thefinal PD concentration, during the fed-batch experiment. Thus, PDcoefficient yield was lower than the maximum theoretical, but inany case the achieved value of 0.40 g PD per g glycerol consumedtogether with a PDmax value of 68.1 g/L can be considered as rathersatisfactory.

The enzyme responsible for lactate synthesis during glycerolmetabolism is that of lactate dehydrogenase, encoded by theldhA gene (Papanikolaou, 2009). Potential inactivation of this genein C. freundii FMCC-B 294 (VK-19), creating a lactate-deficientmutant, could certainly increase even more the (already elevated)PDmax concentration achieved, increasing also the conversion yieldYPD/Gly, since a supplementary portion of NADH2 equivalents wouldbe recycled through the glycerol → 1,3-propanediol pathway. Suchan approach has been carried out in the study of Xu et al. (2009),who created a d-lactate-deficient recombinant strain of K. pneumo-niae HR 526 by knocking out the ldhA gene encoding in the synthesisof lactate dehydrogenase. In experiments using pure glycerol as

feedstock, PD concentration increased from 95.4 g/L (utilization ofthe wild strain) to ∼102 g/L (genetically engineered strain) (it isnoted that in both instances very high PDmax concentrations wereachieved). The same technique was also performed in the study

Biotec

oMpf∼eg

acghiAedo2p5c8gg(geccgmarce(ibPco

fcmscvpPwebmPstfuw

emcbc

M. Metsoviti et al. / Journal of

f Yang et al. (2007), using the strain K. oxytoca M5al; the strain5al formed ∼39 g/L of PD, while its lactate deficient mutant LDH3

roduced ∼56 g/L. When sucrose was added as co-substrate in theermentation broth, PD concentration significantly improved to58 g/L for the strain M5al and ∼83 g/L for the mutant LDH3 (Yangt al., 2007). To the best of our knowledge, inactivation of the ldhAene has not been reported yet for C. freundii strains.

Throughout our study it was proved that crude glycerol was suitable substrate for PD production by C. freundii. In literature,ertain confusion exists concerning the effect of pure and crudelycerol on cell growth and PD production; several studies reportedigher PD concentrations obtained with pure glycerol, whereas

n some cases crude glycerol was beneficial for PD biosynthesis.mongst the highest PD yield values documented so far in lit-rature is ∼94 g/L by using natural Cl. butyricum strain AKR 102,uring its culture on pure glycerol, however the respective valuen raw glycerol was remarkably lower, i.e. ∼76 g/L (Wilkens et al.,012). Mu et al. (2006) performed fed-batch cultures of the strain K.neumoniae DSM 2026 and achieved PDmax values of 61.9 g/L and3.0 g/L when pure and raw glycerol, respectively, were used asarbon sources. Hirschmann et al. (2005) reported PDmax values of7.7 g/L and 80.1 g/L by Clostridium sp. IK 124 when this microor-anism was cultivated in fed-batch experiments using pure and rawlycerol, respectively, as carbon sources. On the contrary, Jun et al.2010) revealed that PD production was more effective with rawlycerol than with pure. Thus, 80.2 and 63.3 g/L of PD were formed,mploying crude and pure glycerol respectively, during a fed-batchulture with suspended cells of K. pneumoniae DSM 4799. This valueorresponds to the highest PD concentration reported using rawlycerol. In the study by Hiremath et al. (2011) fed-batch experi-ents were performed with pure and crude glycerol and similar

mounts of PD were produced in both cases (58.0 and 56.0 g/L,espectively), however the fermentation with crude glycerol wasompleted in 92 h, compared to the fermentation with pure glyc-rol, which lasted only 72 h. In the study by Petitdemange et al.1995) several Cl. butyricum strains were isolated and, in certainnstances, microorganisms were not able at all to grow on mediaased on raw glycerol feedstocks. Finally, significant quantities ofD (74–85 g/L) have been recently synthesized by Lb. diolivoransultivated on MRS medium in which carbon sources were mixturesf pure glycerol and analytical-grade glucose (Pfügl et al., 2012).

The feasibility of batch and fed-batch cultures under non-sterileermentation conditions was also tested and the absence of asepticonditions proved to have no considerable effect on glycerol fer-entation by C. freundii. Compared to the results achieved under

terile conditions, trivial differences were detected on final productoncentrations; PD formation in terms of absolute values and con-ersion yield YPD/Gly was similar and only the respective volumetricroductivity was slightly enhanced in the sterile fed-batch culture.Dmax quantity achieved under non-aseptic conditions (66.3 g/L)as comparable to that of 67.9 g/L reported by Chatzifragkou

t al. (2011) during a non-sterilized fed-batch fermentation of Cl.utyricum VPI 1718. The absence of sterile cultivation conditionsay prove as a usable aspect for the industrial application of the

D formation process. In all our experiments performed under non-terile conditions, periodic microscopic observations confirmedhat C. freundii was the predominant microorganism found into theermentation medium. Further, no other “unusual” metabolic prod-cts, which could indicate the presence of other microorganisms,ere found in the fermentation broth.

The implementation of non-sterile conditions minimizes thenergy cost for sterilization, as well as the overall cost for equip-

ent. Together with the utilization of low-cost biodiesel-derived

rude glycerol as substrate, this implementation will help micro-ial conversion of crude glycerol to PD to become an economicallyompetitive bioprocess. In the current study, a cost-effective and

hnology 163 (2013) 408– 418 417

environmentally friendly bioprocess was developed for the conver-sion of elevated quantities of raw glycerol into high amounts of PDby a natural C. freundii strain.

Acknowledgement

This research was financially supported by the EU (FP7 Program“Integrated bioconversion of glycerine into value-added productsand biogas at pilot plant scale”, acronym “PROPANERGY”, GrantNumber: 212671).

References

Anand, P., Saxena, R.K., 2012. A comparative study of solvent-assisted pretreatmentof biodiesel derived crude glycerol on growth and 1,3-propanediol productionfrom Citrobacter freundii. New Biotechnology 29, 199–205.

Anand, P., Saxena, R.K., Yadav, S., Jahan, F., 2010. A greener solution for darker sideof biodiesel: utilization of crude glycerol in 1,3-propanediol production. Journalof Biofuels 1, 83–92.

Barbirato, F., Camarasa, C., Claret Grivet, J.P., Bories, A., 1995. Glycerol fermenta-tion of 1,3-propanediol producing microorganism: Enterobacter agglomerans.Applied Microbiology and Biotechnology 43, 786–793.

Barbirato, F., Soucaille, P., Bories, A., 1996. Physiologic mechanisms involved inaccumulation of 3-hydroxypropionaldehyde during fermentation of glycerolby Enterobacter agglomerans. Applied and Environmental Microbiology 62,4405–4409.

Barbirato, F., Himmi, E.H., Conte, T., Bories, A., 1998. 1,3-Propanediol production byfermentation: an interesting way to valorize glycerin from the ester and ethanolindustries. Industrial Crops and Products 7, 281–289.

Biebl, H., 1991. Glycerol fermentation of 1,3-propanediol by Clostridium butyricum.Measurement of product inhibition by use of pH-auxostat. Applied Microbiologyand Biotechnology 35, 701–705.

Biebl, H., Menzel, K., Zeng, A.P., Deckwer, W.D., 1999. Microbial production of 1,3-propanediol. Applied Microbiology and Biotechnology 52, 289–297.

Boenigk, R., Bowien, S., Gottschalk, G., 1993. Fermentation of glycerol in continu-ous cultures of Citrobacter freundii. Applied Microbiology and Biotechnology 38,453–457.

Casali, S., Gungormusler, M., Bertin, L., Fava, F., Azbar, N., 2012. Development of abiofilm technology for the production of 1,3-propanediol (1,3-PDO) from crudeglycerol. Biochemical Engineering Journal 64, 84–90.

Celinska, E., 2010. Debottlenecking the 1,3-propanediol pathway by metabolic engi-neering. Biotechnology Advances 28, 518–530.

Chatzifragkou, A., Dietz, D., Komaitis, M., Zeng, A.P., Papanikolaou, S., 2010. Effectof biodiesel-derived waste glycerol impurities on biomass and 1,3-propanediolproduction of Clostridium butyricum VPI 1718. Biotechnology and Bioengineer-ing 107, 76–84.

Chatzifragkou, A., Papanikolaou, S., Dietz, D., Doulgeraki, A.I., Nychas, G.J.E., Zeng,A.P., 2011. Production of 1,3-propanediol by Clostridium butyricum growing onbiodiesel-derived crude glycerol through a non-sterilized fermentation process.Applied Microbiology and Biotechnology 91, 101–112.

Chen, X., Zhang, D., Qi, W., Gao, S., Xiu, Z.L., Xu, P., 2003. Microbial fed-batchproduction of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobicconditions. Applied Microbiology and Biotechnology 63, 143–146.

Cheng, K.K., Liu, D.H., Sun, Y., Liu, W.B., 2004. 1,3-Propanediol production by Kleb-siella pneumoniae under different aeration strategies. Biotechnology Letters 26,911–915.

Dobson, R., Gray, V., Rumbold, K., 2011. Microbial utilization of crude glycerol forthe production of value-added products. Journal of Industrial Microbiology andBiotechnology 39, 217–226.

Doulgeraki, A.I., Paramithiotis, S., Nychas, G.J.E., 2011. Characterization of the Entero-bacteriaceae community that developed during storage of minced beef underaerobic or modified atmosphere packaging conditions. International Journal ofFood Microbiology 145, 77–83.

Emptage, M., Haynie, S.L., Laffend, L.A., Pucci, J.P., Whited, G.M., 2003. Process for thebiological production of 1,3-propanediol with high titer. US Patent US 6,514,733B1.

Emptage, M., Haynie, S.L., Laffend, L.A., Pucci, J.P., Whited, G.M., 2006. Process for thebiological production of 1,3-propanediol with high titer. US Patent US 7,067,300B2.

Freund, A., 1881. Über die bildung und darstellung von trimethylene-alkohol ausglycerin. Monatshefte für Chemie 2, 636–641.

Gonzalez-Pajuelo, M., Andrade, J.C., Vasconcelos, I., 2004. Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium andraw glycerol. Journal of Industrial Microbiology and Biotechnology 34, 442–446.

Gonzalez-Pajuelo, M., Meynial-Salles, I., Mendes, F., Andrade, J.C., Vasconcelos, I.,Soucaille, P., 2005. Metabolic engineering of Clostridium acetobutylicum for the

industrial production of 1,3-propanediol from glycerol. Metabolic Engineering7, 329–336.

Gungormusler, M., Gonen, C., Ozdemir, G., Azbar, N., 2010. 1,3-Propanediol produc-tion potential of Clostridium saccharobutylicum NRRL B-643. New Biotechnology27, 782–788.

4 Biotec

G

G

H

H

H

J

J

K

K

K

K

L

L

L

M

M

M

M

M

M

O

P

P

propanediol production and the new trends. In: Scheper, T. (Ed.), Advances

18 M. Metsoviti et al. / Journal of

ungormusler, M., Gonen, C., Azbar, N., 2011. 1,3-Propanediol production potentialby a locally isolated strain of Klebsiella pneumoniae in comparison to Clostrid-ium beijerinckii NRRL B593 from waste glycerol. Journal of Polymers and theEnvironment 19, 812–817.

ünzel, B., Yonsel, S., Deckwer, W.D., 1991. Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2 m3. AppliedMicrobiology and Biotechnology 36, 289–294.

iremath, A., Kannabiran, M., Rangaswamy, V., 2011. 1,3-Propanediol productionfrom crude glycerol from jatropha biodiesel process. New Biotechnology 28,19–23.

irschmann, S., Baganz, K., Koschik, I., Vorlop, K.D., 2005. Development of an inte-grated bioconversion process for the production of 1,3-propanediol from rawglycerol waters. Landbauforschung Völkenrode 55, 261–267.

omann, T., Tag, C., Biebl, H., Deckwer, W.D., Schink, B., 1990. Fermentation ofglycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Applied Micro-biology and Biotechnology 33, 121–126.

i, X.J., Huang, H., Zhu, J.G., Hu, N., Li, S., 2009. Efficient 1,3-propanediol productionby fed-batch culture of Klebsiella pneumoniae: the role of pH fluctuation. AppliedBiochemistry and Biotechnology 159, 605–613.

un, S.A., Moon, C., Kang, C.H., Kong, S.W., Sang, B.I., Um, Y., 2010. Microbial fed-batch production of 1,3-propanediol using raw glycerol with suspended andimmobilized Klebsiella pneumoniae. Applied Biochemistry and Biotechnology161, 491–501.

aur, G., Srivastava, A.K., Chand, S., 2012a. Advances in biotechnological productionof 1,3-propanediol. Biochemical Engineering Journal 64, 106–118.

aur, G., Srivastava, A.K., Chand, S., 2012b. Mathematical modelling approach forconcentration and productivity enhancement of 1,3-propanediol using Clostrid-ium diolis. Biochemical Engineering Journal 68, 34–41.

aur, G., Srivastava, A.K., Chand, S., 2012c. Determination of kinetic parameters of1,3-propanediol fermentation by Clostridium diolis using statistically optimizedmedium. Bioprocess and Biosystems Engineering 35, 1147–1156.

ivistö, A., Santala, V., Karp, M., 2012. 1,3-Propanediol production and toleranceof a halophilic fermentative bacterium, Halanaerobium saccharolyticum subsp.saccharolyticum. Journal of Biotechnology 158, 242–247.

ee, S.Y., Hong, S.H., Lee, S.H., Park, S.J., 2004. Fermentative production of chemicalsthat can be used for polymer synthesis. Macromolecular Bioscience 4, 157–164.

iu, H.J., Zhang, D.J., Xu, Y.H., Mu, Y., Sun, Y.Q., Xiu, Z.L., 2007. Microbial productionof 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobicconditions up to a pilot scale. Biotechnology Letters 29, 1281–1285.

iu, H., Ou, X., Zhou, S., Liu, D., 2010. Microbial 1,3-propanediol, its copolymerizationwith terephthalate, and applications. In: Chen, G.Q. (Ed.), Plastics from Bacteria:Natural Functions and Applications. Microbiology Monographs, vol. 14. BerlinHeidelberg, pp. 405–425.

a, B.B., Xu, X.L., Zhang, G.L., Wang, L.W., Wu, M., Li, C., 2009. Microbial productionof 1,3-propanediol by Klebsiella pneumoniae XJPD-Li under different aerationstrategies. Applied Biochemistry and Biotechnology 152, 127–134.

aervoet, V.E.T., Beauprez, J., De Maeseneire, S.L., Soetaert, W., De Mey, M.,2012. Citrobacter werkmanii, a new candidate for the production of 1,3-propanediol: strain selection and carbon source optimization. Green Chemistry14, 2168–2178.

etsoviti, M., Paramithiotis, S., Drosinos, E.H., Galiotou-Panayotou, M., Nychas,G.J.E., Zeng, A.P., Papanikolaou, S., 2012a. Screening of bacterial strains capable ofconverting biodiesel-derived raw glycerol into 1,3-propanediol, 2,3-butanedioland ethanol. Engineering in Life Sciences 12, 57–68.

etsoviti, M., Paraskevaidi, K., Koutinas, A., Zeng, A.P., Papanikolaou, S., 2012b. Pro-duction of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolatedKlebsiella oxytoca strain growing on biodiesel-derived glycerol based media.Process Biochemistry 47, 1872–1882.

oon, C., Ahn, J.H., Kim, S.W., Sang, B.I., Um, Y., 2010. Effect of biodiesel-derived rawglycerol on 1,3-propanediol production by different microorganisms. AppliedBiochemistry and Biotechnology 161, 502–510.

u, Y., Teng, H., Zhang, D.J., Wang, W., Xiu, Z.L., 2006. Microbial production of1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodieselpreparation. Biotechnology Letters 28, 1755–1759.

tte, B., Gruwalt, E., Mahmoud, O., Jennewein, S., 2009. Genome shuffling in Clostrid-ium diolis DSM 15410 for improved 1,3-propanediol production. Applied andEnvironment Microbiology 75, 7610–7616.

apanikolaou, S., 2009. Microbial conversion of glycerol into 1,3-propanediol: glyc-erol assimilation, biochemical events related with 1,3-propanediol biosynthesisand biochemical engineering of the process. In: Aggelis, G. (Ed.), Microbial Con-

versions of Raw Glycerol. Nova Science Publishers Inc., New York, pp. 137–168.

apanikolaou, S., Aggelis, G., 2003. Modelling aspects of the biotechnological valo-rization of raw glycerol: production of citric acid by Yarrowia lipolytica and1,3-propanediol by Clostridium butyricum. Journal of Chemical Technology andBiotechnology 78, 542–547.

hnology 163 (2013) 408– 418

Papanikolaou, S., Ruiz-Sanchez, P., Pariset, B., Blanchard, F., Fick, M., 2000. Highproduction of 1,3-propanediol from industrial glycerol by a newly isolatedClostridium butyricum strain. Journal of Biotechnology 77, 191–208.

Papanikolaou, S., Fick, M., Aggelis, G., 2004. The effect of raw glycerol concentra-tion on the production of 1,3-propanediol by Clostridium butyricum. Journal ofChemical Technology and Biotechnology 79, 1189–1196.

Papanikolaou, S., Fakas, S., Fick, M., Chealot, I., Galiotou-Panayotou, M., Komaitis,M., Marc, I., Aggelis, G., 2008. Biotechnological valorisation of raw glyceroldischarged after biodiesel (fatty acid methyl esters) manufacturing process: pro-duction of 1,3-propanediol, citric acid and single cell oil. Biomass and Bioenergy32, 60–71.

Papoutsakis, E.T., 2008. Engineering solventogenic clostridia. Current Opinion inBiotechnology 19, 420–429.

Petitdemange, E., Dürr, C., Abbad-Andaloussi, S., Raval, G., 1995. Fermentation ofraw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. Journalof Industrial Microbiology and Biotechnology 15, 498–502.

Petrov, K., Stoyanov, A. Accelerated production of 1,3-propanediol from glycerol byKlebsiella pneumoniae using the method of forced pH fluctuations. Bioprocessand Biosystems Engineering, in press

Pfügl, S., Marx, H., Mattanovich, D., Sauer, M., 2012. 1,3-Propanediol production fromglycerol by Lactobacillus diolivorans. Bioresource Technology 119, 133–140.

Rehman, A., Matsumura, M., Nomura, N., Sato, S., 2008. Growth and 1,3-propanediolproduction on pre-treated sunflower oil bio-diesel raw glycerol using a strictanaerobe Clostridium butyricum. Current Research in Bacteriology 1, 7–16.

Ringel, A.K., Wilkens, E., Hortig, D., Willke, T., Vorlop, K.D., 2012. An improvedscreening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations. Applied Microbiologyand Biotechnology 93, 1049–1056.

Rossi, D.M., de Costa, J.B., de Souza, E.A., do Carmo Ruaro Peralba, M., Ayub, M.A.Z.,2012. Bioconversion of residual glycerol from biodiesel synthesis into 1,3-propanediol and ethanol by isolated bacteria from environmental consortia.Renewable Energy 39, 223–227.

Sattayasamitsathit, S., Prasertsana, P., Methacanonc, P., 2011. Statistical optimiza-tion for simultaneous production of 1,3-propanediol and 2,3-butanediol usingcrude glycerol by newly bacterial isolate. Process Biochemistry 46, 608–614.

Saxena, R.K., Anand, P., Saran, S., Isar, J., 2009. Microbial production of 1,3-propanediol: recent developments and emerging opportunities. BiotechnologyAdvances 27, 895–913.

Sotenko, M.V., Rebros, M., Sans, V.S., Loponov, K.N., Davidson, M.G., Stephens, G., Lap-kin, A.A. Tandem transformation of glycerol to esters. Journal of Biotechnology,in press

Tracy, B.P., Jones, S.W., Fast, A.G., Indurthi, D.C., Papoutsakis, E.T., 2012. Clostridia:the importance of their exceptional substrate and metabolite diversity forbiofuel and biorefinery applications. Current Opinion in Biotechnology 23,364–381.

Venkataramanan, K.P., Boatman, J.J., Kurniawan, Y., Taconi, K., Bothun, G.D., Scholz,C., 2012. Impact of impurities in biodiesel-derived crude glycerol on the fer-mentation of Clostridium pasteurianum ATCC 6013. Applied Microbiology andBiotechnology 93, 1325–1355.

Wilkens, E., Ringel, A.K., Hortig, D., Willke, T., Vorlop, K.D., 2012. High-level produc-tion of 1,3-propanediol from crude glycerol by Clostridium butyricum AKR102a.Applied Microbiology and Biotechnology 93, 1057–1063.

Willke, T.H., Vorlop, K.D., 2008. Biotransformation of glycerol into 1,3-propanediol.European Journal of Lipid Science and Technology 110, 831–840.

Xu, Y.Z., Guo, N.N., Zheng, Z.M., Ou, X.J., Liu, X.J., Liu, D.H., 2009. Metabolism in 1,3-propanediol fed-batch fermentation by a d-lactate deficient mutant of Klebsiellapneumoniae. Biotechnology and Bioengineering 104, 965–972.

Yang, G., Tian, J., Li, J., 2007. Fermentation of 1,3-propanediol by a lactate deficientmutant of Klebsiella oxytoca under microaerobic conditions. Applied Microbiol-ogy and Biotechnology 73, 1017–1024.

Yazdani, S.S., Gonzalez, R., 2007. Anaeobic fermentation of glycerol: a path to eco-nomic viability for the biofuels industry. Current Opinion in Biotechnology 18,213–219.

Zeng, A.P., 1995. A new balance equation of reducing equivalents for data consistencycheck and bioprocess calculation. Journal of Biotechnology 43, 111–124.

Zeng, A.P., 1996. Pathway and kinetic analysis of 1,3-propanediol production fromglycerol fermentation by Clostridium butyricum. Bioprocess Engineering 14,169–175.

Zeng, A.P., Biebl, H., 2002. Bulk chemicals from biotechnology: the case of 1,3-

in Biochemical Engineering and Biotechnology, vol. 74. Heidelberg, Berlin,Springer, pp. 239–259.

Zeng, A.P., Sabra, W., 2011. Microbial production of diols as platform chemicals:recent progress. Current Opinion in Biotechnology 22, 749–757.