Advances in biotechnological production of 1,3-propanediol

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Biochemical Engineering Journal 64 (2012) 106– 118

Contents lists available at SciVerse ScienceDirect

Biochemical Engineering Journal

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dvances in biotechnological production of 1,3-propanediol

uneet Kaur, A.K. Srivastava ∗, Subhash Chandepartment of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

r t i c l e i n f o

rticle history:eceived 16 September 2011eceived in revised form 6 March 2012ccepted 7 March 2012vailable online 16 March 2012

eywords:lycerol

a b s t r a c t

1,3-propanediol (1,3-PD) is a chemical compound with myriad applications particularly as a monomerfor the production of polyesters, polyethers and polyurethanes. It is a raw material for the production ofbiodegradable plastics, films, solvents, adhesives, detergents, cosmetics and medicines. Various strategieshave been employed for the microbial production of 1,3-PD which include several bioprocess cultivationtechniques facilitated by natural and/or genetically engineered microbes. Though 1,3-PD is produced innature by the bioconversion of glycerol its production directly from sugars like glucose has been alsomade possible by the development of recombinant strains. This review presents the “state of the art”

,3-propanediolioprocess designodelling

olytrimethylene terephthalateownstream processing

in the biotechnological production technologies of 1,3-PD particularly with respect to bioprocess engi-neering methods. It also highlights the significance of mathematical model-based approach for designingvarious bioreactor operating strategies to facilitate the improvement in 1,3-PD production. Attempt hasalso been made to focus on the protocols used for downstream processing of 1,3-PD and the associatedproblems. Finally concluding remarks on the future outlook on biobased 1,3-PD to reduce the dependenceon disappearing fossil fuels are presented.

© 2012 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062. 1,3-PD: a speciality chemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073. Production of 1,3-PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

3.1. Synthesizing 1,3-PD via the chemical way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073.2. Synthesizing 1,3-PD via the biological way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

3.2.1. Metabolic pathway to 1,3-PD: role of genes and their encoded enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083.2.2. Bottlenecks in the biological route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093.2.3. Process development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

3.3. Downstream processing of biotechnologically produced 1,3-PD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1144. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

. Introduction The roster comprises cosmetics, food, fuels, lubricants, plastics,beverages, fibres and medicines. Of these, the production of bio-fuels, particularly biodiesel has attracted a great deal of attention.
In the recent years concern over the biological production of
ommercially important metabolites has been burgeoning. Thiss mainly attributed to the escalating global energy and environ-

ental problems which have stimulated researchers worldwide toevise methods for producing almost everything via the green way.

Abbreviations: 1,3-PD, 1,3-propanediol; 3-HPA, 3-hydroxypropanaldehyde; 2,3-D, 2,3-butanediol; DHAP, dihydroxyacetone phosphate; D, dilution rate.∗ Corresponding author. Tel.: +91 11 26591010; fax: +91 11 26582282.

E-mail addresses: [email protected] (G. Kaur), [email protected],[email protected] (A.K. Srivastava), [email protected] (S. Chand).

369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2012.03.002

Biodiesel (fatty acid alkyl ester) produced by transesterification offats and oils has been considered as a renewable, biodegradable andnon-toxic fuel [1]. A predominant by-product of the biodiesel plantis glycerol. Glycerol is a chemical compound of immense impor-tance both as an end product and a starting material for a myriadother useful products [2]. Raw glycerol is currently used in largescale cosmetic and food manufacturing industry besides being used
as the feedstock for a number of interesting compounds (acrolein,dihydroxyacetone, methanol, propionic acid, succinic acid, etc.)which are produced either chemically or biologically [2,3]. Hugeamounts of glycerol are available (1 gallon of crude glycerol is left
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ineering Journal 64 (2012) 106– 118 107

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G. Kaur et al. / Biochemical Eng

ehind for every 10 gallon of biodiesel produced) against a mea-re demand with the result often leading to a severe loss to thendustry [4]. Besides this surplus amounts of waste glycerol areenerated by soap industries and alcohol beverage manufacturingnits thereby necessitating a ‘search’ for methods befitting the dis-osal of the same. An interesting way is the conversion of this ‘wasteroduct’ to a ‘high-value added’ product. This would not only sus-ain the utility of an important commodity like glycerol but also

ake these production plants more economical. Bioconversion oflycerol into propionic acid, succinic acid, citric acid, single cell oil,utanol, hydrogen as well as 1,3-PD has been investigated [5–8].ith the increasing emergence of novel applications of 1,3-PD the

emand for its biological production has been on a rise. 1,3-PD isajorly used to make a new class of polymers with enhanced func-

ionality. The market for 1,3-PD is growing very rapidly, as newroducts are developed to capitalize upon the functionality of theolymers that can be synthesized from 1,3-PD [9,10]. This marketffers a significant opportunity to develop new, cost-competitiverocesses to produce 1,3-PD which would avoid the use of moreetroleum, provide substantial energy savings and afford signifi-ant market penetration for the burgeoning bio-products industry.

This report describes the current status of the biotechnologi-al production of 1,3-PD particularly with an aim to optimize theroduction and have economic separation protocols for a varietyf applications. Attempt has been made to describe and discuss inetail both previous and recent published work on 1,3-PD produc-ion in order to delineate the progress made in 1,3-PD bioprocesstrategies and purification techniques over the years. More impor-antly, the review ‘proposes’ a different approach of mathematical

odel-based designing of nutrient feeding strategies for improvedroduction of 1,3-PD and discusses the results obtained by theuthors by the use of this method. Finally some perspectives anduture outlook on microbial production of 1,3-PD are given underhe light of current status of research.

. 1,3-PD: a speciality chemical

1,3-PD is a colourless viscous organic compound with the for-ula C3H8O2 and a non-flammable, low toxicity liquid which isiscible with water, alcohols and ethers so that it can be eas-

ly transported. It has a history of being a high priced specialityhemical. It has many desirable properties for participation in poly-ondensation reactions. It is a unique alternative compound forndustrial chemical formulations. However, high cost [11] and lim-ted availability of 1,3-PD in the past were two important factors

hich restricted its applications mainly as a solvent and in the pro-uction of dioxanes [12]. This scenario changed (1995–1998) afterhe annunciation of commercialization of a 1,3-PD based polyester,amed polytrimethylene terephthalate by DuPont and Shell whichermed it as SORONATM and CORTERRATM, respectively [13,14].his copolyester is a condensation product of 1,3-PD and tereph-halic acid and is principally used in the manufacture of carpet andextile fibres but also finds applications as engineering thermoplas-ics, films and coatings [15]. The fabrics have stretch resilience, lowtatic generation, colour fastness, good stretch recovery, stain resis-ance, etc. [13]. In thermoplastic urethanes (TPU), use of 1,3-PDan lead to improved thermal and hydrolytic as well as thermalimensional stability [16]. 1,3-PD can be used to modify polyesterystems. This property is particularly beneficial for the productionf powder coatings where partial substitution with 1,3-PD can givemproved flexibility without adversely affecting other key proper-
ies such as storage stability and outdoor weather ability. In engineoolant formulations, 1,3-PD demonstrates improved heat stabil-ty, less corrosion especially to lead solder, and lower toxicity thanthylene glycol coolants.
Fig. 1. Applications of 1,3-PD.

1,3-PD has a multitude of other applications as well (Fig. 1). Thebiodegradable nature, higher light stability and solubility of 1,3-PD-based polyesters in most common solvents add to their alreadygrowing list of applications [17,18]. 1,3-PD can be formulated intolaminates, solvents, mouldings, adhesives, resins, detergents, cos-metics, deodorants and other end uses [19]. Solvent uses of 1,3-PDinclude water based inks such as ink-jet and screen inks. 1,3-PD isan important intermediate for organic synthesis, can be used in var-ious types of medicines (vitamin H and immunosuppressive drugs),insect repellents, fragrances, etc. Thus the production of biobased1,3-PD is extremely important.

3. Production of 1,3-PD

3.1. Synthesizing 1,3-PD via the chemical way

The conventional modus operandi for the production of 1,3-PDconsisted of the methods which were followed by the two chemicalcompanies instrumental for the upsurge in the interest in 1,3-PD.

DuPont started with acrolein which was converted to 3-hydroxypropionaldehyde (3-HPA) by hydration. This was followedby hydrogenation in second step to give 1,3-PD [20]. Shell on theother hand followed the method of hydroformylation of ethyleneoxide to 3-hydroxypropanal. This was subsequently extracted andhydrogenated for the production of 1,3-PD [12].

However, several drawbacks of these chemical methods [21]like requirement of high pressure and high temperature, use ofexpensive catalyst, release of toxic intermediates, dependence onnon-renewable materials, low yield and complexity [22] called forthe biological route which would give a cleaner product with notoxic intermediates thus expanding the spectrum of its utility.

3.2. Synthesizing 1,3-PD via the biological way

Pursuing the biological route to 1,3-propanediol is particularlyattractive since it utilizes renewable feedstock and cultivationsat normal temperature and pressure leading to no generation oftoxic by-products. The production of 1,3-PD occurs from glycerolin nature with the latter being the only substrate which can be fer-mented to 1,3-PD [23]. Other advantages of using glycerol includehighly reduced nature of the carbon atoms in glycerol thereby yield-ing more reducing equivalents than glucose or xylose [24], lower
capital and operational costs.
The list of microorganisms competent in performing glycerol to1,3-PD bioconversion comprises Klebsiella (K. pneumoniae and K.oxytoca), Clostridia (C. butyricum and C. pasteurianum), Enterobacter

108 G. Kaur et al. / Biochemical Engineering Journal 64 (2012) 106– 118

Fig. 2. Metabolic pathway of glycerol conversion. Rounded rectangles—NADH + H+ consuming products; Ellipse—NADH + H+ producing intermediates and products; Box—keyg , 1,3-b 3-hy

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enes of dha regulon. Name of genes shown in italics. GDHt—Glycerol dehydrataseutanediol, DHA—Dihydroxyacetone, DHAP—Dihydroxyacetone phosphate, 3-HPA—

E. agglomerans), Citrobacter (C. fruendii) and Lactobacilli (L. bre-is and L. buchneri) [25–39]. Besides glycerol other carbon sourcesuch as glucose, corn hydrolysate and sugarcane molasses have alsoeen used for the production of 1,3-PD [40–42]. However therere no native 1,3-PD producers which can directly convert sugarso 1,3-PD. Therefore the attempts with sugars as substrate haveeen made with either multistage fermentations [42,43] involving aombination of microorganisms or genetically engineered microor-anisms [40,41] carrying genes for conversion of sugar to glycerolnd then glycerol to 1,3-PD.

.2.1. Metabolic pathway to 1,3-PD: role of genes and theirncoded enzymes

The pathway to 1,3-PD using glycerol as a substrate is a cou-led oxidation–reduction process [44]. In this process, generationf energy in the form of ATP and reducing equivalents in the formf NADH + H+ occurs in the oxidative branch while regeneration ofAD+ concomitant with the formation of reduced product 1,3-PDccurs in the reductive branch (Fig. 2). NADH + H+ is generated dur-ng glycolytic reactions (oxidative branch) which also yield severalther by-products.

In the reductive branch, glycerol is first dehydrated to-HPA, a reaction catalyzed by coenzyme B12-dependent glyc-rol dehydratase (GDHt), which is then reduced to 1,3-PD byADH + H+-dependent 1,3-PD dehydrogenase (1,3-PD DH) byADH + H+ utilization. More dehydratases have been described,hich are B12-independent such as GDHt in C. butyricum [2]. Dif-

erent types of by-products are formed by different organismsn the oxidative branch. While butyric acid and acetic acid arehe major by-products given by C. butyricum [33], butanol is pro-uced by C. pasteurianum [45]. Besides these ethanol, lactic acid,uccinic acid, 2,3-butanediol (2,3-BD) constitute those producedy the Enterobacteria [44]. The reactions up to the formationf pyruvate are common to all the microorganisms [46]. Glyc-rol is dehydrogenated to dihydroxyacetone (DHA) with the help
f NAD-dependent glycerol dehydrogenase (GDH) which is thenhosphorylated by DHA kinase (DHAK) to dihydroxyacetone phos-hate (DHAP), one of the metabolites in the glycolytic pathway.here is a net production of two NADH + H+ molecules and one
PD DH—1,3-propanediol dehydrogenase, PEP—Phosphoenolpyruvate, 2,3-BD—2,3-droxypropanaldehyde, 1,3-PD—1,3-propanediol.

ATP molecule during the conversion of glycerol to pyruvate [47].An alternative route of glycerol degradation is found in Klebsiellasp. when grown under aerobic conditions. Glycerol is phosphory-lated to sn-glycerol-3-phosphate by a glycerol kinase which is thenconverted to DHAP by NAD-dependent glycerol 3-phosphate dehy-drogenase [48]. In Lactobacillus sp. the oxidative branch of glycerolmetabolism is missing and therefore these microorganisms need tobe provided with an additional carbon source in order to fulfil therequirements of energy and reducing equivalents [49].

The enzymes of the 1,3-PD pathway are encoded by dha operonwhich has been characterized in K. pneumoniae [49], C. fruendii[50], and C. butyricum [51]. Glycerol dehydratase considered asa rate limiting enzyme of the pathway [25] is coenzyme B12-dependent and consists of three polypeptides encoded by threeORFs- dhaB, dhaC and dhaE, respectively (alternate nomenclatureis dhaB1, dhaB2 and dhaB3) [52] that catalyzes the conversion ofglycerol to 3-HPA. Inactivation of coenzyme B12 occurs upon itsinteraction with glycerol which leads to cessation of catalysis bydehydratase. This inactivation is caused due to the irreversiblecleavage of Co C bond of the coenzyme which then results in theformation of 5′-deoxyadenosine and alkylcobalamine-like species.The latter binds tightly to dehydratase and renders it inactive. Theresumption of dehydratase activity occurs only after the dissoci-ation of dehydratase-bound inactive B12 is mediated by anotherenzyme glycerol dehydratase reactivase. Regeneration of coen-zyme B12 from inact-B12 is mediated by two subunits of thereactivating factor (dhaF-dhaG) of GDHt encoded by dhaF and dhaGgenes [41] (Fig. 3). The coenzyme B12-independent GDHt of C.butyricum however functions via a different mechanism. It utilizesS-adenosylmethionine instead of adenosylcobalamin to catalyzethe reaction. Moreover it is oxygen sensitive and therefore cannotbe used in aerobic systems for 1,3-PD production unlike coenzymeB12-dependent GDHt. The second reaction of the reductive branchis catalyzed by 1,3-PD dehydrogenase encoded by dhaT gene [53].An isoenzyme of 1,3-PD DH called as 1,3-PD oxidoreductase has
also been reported from Escherichia coli [54] which is coded byyqhD gene and has been found to have higher oxidoreductive activ-ity compared to 1,3-PD DH. This enzyme is an NADP-dependentdehydrogenase unlike 1,3-PD DH which is NAD-dependent.

G. Kaur et al. / Biochemical Engineering Journal 64 (2012) 106– 118 109

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.2.2. Bottlenecks in the biological routeAlbeit the biological production of 1,3-PD propounds an eco-

riendly route to the important chemical; it suffers from certainrawbacks which need to be overcome to make this method eco-omically feasible. These can be described as low yield, inhibitiony both substrate and product [27,31,55], simultaneous formationf by-products [47,56], cost of the substrate and other mediumomponents [35]. The drawbacks of the microbial path to 1,3-PDave been cogitated by several researchers and efforts have beenade to make this endeavour a successful one. The solutions to

he problems encompassing the biological route can be catego-ized as Process development and Strain improvement. This reviewescribes only the bioprocess strategies for improving the produc-ion of 1,3-PD.

.2.3. Process developmentOne of the approaches to optimize the microbial production of

,3-PD from glycerol is process optimization. Different bioreactorperating strategies have been implemented by several researchersn order to maximize 1,3-PD production (Table 1). A detailedescription of these strategies is given below. Of all the microor-anisms capable of producing 1,3-PD from glycerol, Enterobacteriand Clostridia appear to be the best producers in terms of high yieldnd productivity offered by the two [57–59].

Batch cultivation as a primary investigation approach was usedy Günzel et al. [60] and Barbirato et al. [28] to demonstrate theeasibility of scale up of 1,3-PD production to industrial reactorizes and establish the best organism for the same, respectively. C.utyricum indeed was the most promising candidate for industrialioprocess owing to its higher 1,3-PD conversion yield, no accumu-

ation of 3-HPA and shorter fermentation times as compared to K.neumoniae, C. fruendii and E. Agglomerans [28]. Usage of raw glyc-rol (67%, w/w) for 1,3-PD production by C. butyricum CNCM1211as attempted by Himmi et al. [33]. A high 1,3-PD concentration of

5 g/L from 121 g/L raw glycerol could be obtained using only 4 �g/Liotin. Biebl et al. [61] investigated the use of C. butyricum DSM431 for the production of 1,3-PD. It featured a concentration of9.5 g/L 1,3-PD with consumption of 52 g/L glycerol thereby giving

1,3-PD yield of 0.56 mol/mol. Batch experiments without pH regu-ation were carried out by González-Pajuelo et al. using C. butyricumPI3266 [62]. A 1,3-PD yield of 0.58 mol/mol with a consumption of1% glycerol was obtained in this work. A high 1,3-PD concentration

try in 1,3-PD pathway.

(61 g/L) and productivity (1.7 g/L/h) was achieved by batch cultiva-tion of K. pneumoniae DSM2026 [63]. Though batch fermentationappears as a simple strategy for production of the desired metabo-lite, it suffers from the problems of substrate limitation towardsthe end of fermentation coupled with product inhibition whichare inherently involved in the 1,3-PD production process. Thus thisrequires the use of better cultivation strategies which may facili-tate cultivation under non-limiting and non inhibitory cultivationsand thereby improve the overall productivity of the fermentation.Table 2 summarizes the results of batch 1,3-PD fermentation.

3.2.3.1. Fed batch fermentation. The inverse relation between highinitial substrate concentration and growth rate of the microor-ganism meant fed-batch cultivation could be used in order toachieve high concentration and/or productivity of 1,3-PD [64].Though reasonably high 1,3-PD concentrations have been achievedby researchers using fed-batch cultivation, only few reports aredealing with this mode of reactor operation primarily due to thepossibility of unlimited trials to achieve the optimum concentra-tion in a suitable fed-batch fermentation. The process optimizationefforts also require suitable sterilizable on-line, real time sensorsfor the measurement of key process variables (e.g. substrate) forbetter process control, which are largely non-existent. As a resultthere is scope of further improvement in the feeding strategies forenhanced 1,3 PD production. Some literature reported fed-batchstrategies are summarized below [64–71].

A simple fed-batch system for high production of 1,3-PD fromglycerol by C. butyricum VPI3266 was devised by Saint-Amans et al.[64] which coupled the feeding of the substrate to the volumet-ric C02 produced during the course of fermentation. A productionof 65 g/L 1,3-PD with a productivity of 1.21 g/L/h and a yield of0.69 mol 1,3-PD per mol glycerol consumed could be achievedby this process with an elimination of substrate inhibition. Cou-pled feeding of glycerol and ammonium to alkali consumptionwas attempted by Reimann and Biebl [65] using C. butyricum DSM5431 and its product tolerant mutants. A considerable shorteningof the cultivation times was reported which could be attributedto the maintenance of substrate at a non-limiting level and thus
faster growth. An integrated bioconversion of raw glycerol to 1,3-PD was developed and evaluated using an isolated high substrateand product tolerant strain of C. butyricum IK124 [66]. A fed-batch strategy combining a low base-driven glycerol addition with


Table 1Different fermentation strategies for 1,3-PD production.

Type of fermentation Organism used 1,3-PD (g/L) Yield (mol/mol) Q (g/L/h)a Ref. no.

Fed-batch C. butyricum VPI3266 65 0.69 1.21 [64]C. butyricum DSM5431; C. butyricum mutant 2/2 70.4; 70.5 0.68; 0.66 1.4; 0.9 [65,65]C. butyricum IK124; C. butyricum IK124 87.0; 77.5 0.65; 0.67 1.9; 1.2 [66,66]C. butyricum AKR102a 76.2 0.62 2.3 [67]C. butyricum VPI1718 67.9 0.67 0.78 [68]C. butyricum VPI1718 70.8 0.66 0.70 [69]K. pneumoniae ME-308 70 0.70 0.97 [70]K. pneumoniae ME-303 71.58 0.65 1.93 [71]

Continuous K. pneumoniae DSM2026 35.2–48.5 0.61 4.9–8.8 [73]C. butyricum VPI3266 16–30 0.60 1.5–3.0 [62]C. butyricum VPI3266 30 0.65 10.3 [57]C. butyricum F2b 31–48 0.67 2.9–5.5 [74,75]C. butyricum VPI3266 30.1 0.63–0.67 1.87 [68]

Continuous with cell recycling C. butyricum DSM5431 26.6 – 16.4 [76]

Cell immobilization C. freundii 16.4 0.57 8.2 [36]K. pneumoniae 14.8 0.47 2.96 [59]

51.8 0.39 1.08 [59]13.6 0.43 4.49 [59]

K. pneumoniae DSM4799 48–51 – 1.16 [78]C. beijernickii NRRL B-593 31 0.79 12 [79]

Multi-stage C. freundii 41 0.62 1.8 [30]C. butyricum F2b 41–46 0.67 3.4 [74]C. butyricum F2b 43.5 0.49 1.33 [6]K. pneumoniae 74.07 0.62 1.08 [80]

P. farinosa/K. pneumonia 2.5 0.12 0.034 [43]E. coli/K. pneumoniae 14.1 0.64 2.01 [43]

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S. cerevisiae/C. acetobutylicum DG1 (pSPD5)

a 1,3-PD productivity.

onstant on-line glycerol measurement was followed in the study. high final 1,3-PD concentration of 87 g/L with an overall 1,3-D productivity of 1.9 g/L/h was achieved using refined glycerol.oreover the use of raw glycerol also featured a high 1,3-PD con-

entration and productivity of 80.1 g/L and 1.8 g/L/h, respectively,hich were comparable to the results obtained using refined glyc-

rol. Usage of potato nitrogen concentrate, a cheap effluent fromtarch industry as a replacement of yeast extract in medium alongith raw glycerol was also investigated in fed-batch fermentation.

t yielded 77.5 g/L 1,3-PD with a slight prolongation of lag phasehereby resulting in a lower 1,3-PD productivity of 1.2 g/L/h.

Production of 1,3-PD from crude glycerol in 1 L and 200 Lcale fermentors using another isolate C. butyricum AKR102a wasttempted recently [67]. Fed-batch fermentation yielded 1,3-PDoncentration and productivity (76.2 g/L and 2.3 g/L/h, respec-ively) comparable to the results obtained using pure glycerol
93.7 g/L and 3.3 g/L/h, respectively). The difference in 1,3-PD pro-uction was more pronounced when 1,3-PD concentration in theroth had increased beyond 60 g/L. This was attributed to theresence of impurities such as fatty acids, heavy metal ions and
able 2,3-PD production by batch cultures.

Organism Glycerol type 1,3-PD (g/L) Yield (m

C. butyricum CNCM1211 Raw glycerolb 63.4 0.69

C. butyricum CNCM1211 Raw glycerolc 65.4 0.66

C. butyricum DSM5431 Pure glycerol 29.5 0.56

C. butyricum VPI3266 Pure glycerol;Raw glycerold;Raw glycerole

7.35; 8.26; 7.93 0.58; 0.5

K. pneumoniae DSM2026 Pure glycerol 61 0.53

a 1,3-PD productivity.b Obtained from transesterification process of rapeseed oil.c Obtained from transesterification process of colza oil.d Obtained from transesterification process of rapeseed oil (purity 92%, w/v).e Obtained from transesterification process of rapeseed oil (purity 65%, w/v).

25.5 0.67 0.16 [42]

salts which are inherent constituents of crude glycerol. Scale upto 200 L scale was also attempted in this study. A reasonablyhigh 1,3-PD concentration of 61.5 g/L with a 1,3-PD productivityof 2.11 g/L/h could be achieved in 200 L fermenter. These resultsappeared promising to further optimize the process for large scale1,3-PD production. In a recent report fed-batch cultivation of C.butyricum VPI1718 using pulse feeding of concentrated crude glyc-erol under non-sterile conditions was attempted [68]. A maximum1,3-PD concentration of 67.9 g/L could be obtained under theseconditions at the end of 87 h of fermentation. Using the samestrain the impact of anaerobiosis strategy and reactor geometryon 1,3-PD production was also investigated [69]. It was observedthat continual sparging with nitrogen during fed-batch cultiva-tion as opposed to self-generated anaerobiosis yielded differentresults with respect to 1,3-PD production and acid formation. Undercontinual sparging conditions 70.8 g/L 1,3-PD could be produced
by C. butyricum VPI1718. This 1,3-PD concentration significantlydecreased to 30.5 g/L in the latter strategy. Moreover lactic acid pro-duction was characteristically high in self-generated anaerobiosisfed-batch conditions which negatively affected both biomass and
ol/mol) Q (g/L/h)a Residual glycerol (g/L) Ref. No.

1.85 0 [28]1.67 0 [33]2.3 – [61]

1; 0.56 0.31; 0.34; 0.33 23.37; 23.4; 15.4 [62]; [62]; [62]

1.7 14.2 [63]

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,3-PD production. Thus anaerobiosis strategy was found to haven important effect on the biochemical behaviour of the cultureuring 1,3-PD production.

Ji et al. [70] developed and analyzed three different pH-controleeding strategies to achieve the highest production of 1,3-PD using. pneumoniae ME-308. First two attempts of feeding glycerol andH3 in two separate lines and then together as a mixture did notrove beneficial as they featured either glycerol limitation or inhi-ition (due to the lack of proper monitoring system for glycerol) or

relatively high concentration of either 2,3-BD (pH 6.3) or lactatepH 7.3) which competed with 1,3-PD for NADH + H+ thereby reduc-ng the final concentration of the latter. Finally the last fed-batchpproach of fluctuating the pH value between 6.3 and 7.3 periodi-ally by feeding glycerol-NH3 mixture and 30% H2SO4 limited they-product formation, reduced unconsumed glycerol in the brotht the end of fermentation and gave a high concentration of 70 g/Lf 1,3-PD.

Recently fed-batch fermentation using hemicellulosicydrolysates (corn straw) as cosubstrate along with glycerol

or 1,3-PD production has been reported by Jin et al. [71]. In theed-batch cultivation of K. pneumoniae, the redox state variationas regulated online by the feeding rate (maintaining xylose

oncentration at 5–8 g/L). It featured a 1,3-PD concentration of1.58 g/L, yield and productivity of 0.65 mol/mol and 1.93 g/L/h,espectively, which represented an improvement of 17.8%, 25%nd 17.7%, respectively, as compared to the use of glycerol alone.

.2.3.2. Continuous culture. The usual approach to elimination ofroduct inhibition is the removal of the inhibitory product fromhe fermentation broth by either Plug flow or Continuous Stirredank Reactor (CSTR). Continuous cultures offer a distinct advantagef high productivity but usually not very high product concentra-ions are obtained primarily due to significantly high feeding andithdrawal rates thereby making downstream processing a true

ottleneck. One of the earliest studies on continuous cultures fornderstanding the regulation of glycerol bioconversion were car-ied out by Abbad-Andaloussi et al. [25] using C. butyricum DSM431. From the determination of concentrations of by-products,ADH + H+, intermediates like acetyl CoA and enzymes of theathway it was established that the enzyme thiolase probably par-itioned the flux between acetate and butyrate since low levelsf this enzyme were found. On the other contrary glycerol dehy-ratase appeared to be the rate limiting enzyme of 1,3-PD pathways reflected by high intracellular concentrations of NADH + H+

nd high activity of 1,3-PD DH in the cells with the only limita-ion imposed by GDHt. In yet another study the role of pyruvate

etabolism in regulation of reduced equivalents in glycerol bio-onversion by K. pneumoniae DSM2026 was investigated usingontinuous cultures by Menzel et al. [72]. The study establishedhe involvement of pyruvate dehydrogenase (PDH) and absencef pyruvate: Ferredoxin oxidoreductase in 1,3-PD fermentationy means of enzyme assays. In vitro PDH activity of continuousultures was found to increase with increasing inlet glycerol con-entrations and decrease with increasing cell growth rates. Laterenzel et al. [73] also investigated the effects of inlet glycerol

oncentration and dilution rate (D) in continuous cultures of K.neumoniae DSM2026. The study showed that 1,3-PD productiony K. pneumoniae was a function of D as it decreased with increase

n the latter. A high 1,3-PD concentration of 35.2–48.5 g/L with pro-uctivity between 4.9 and 8.8 g/L/h could be obtained at D rangingrom 0.1 h−1 to 0.25 h−1. Significantly lower 1,3-PD concentrationsere observed at glycerol limiting conditions, which reached high

oncentrations of 48 g/L only when the glycerol supply was inxcess in the medium. González-Pajuelo et al. [62] performed con-inuous cultivation with two raw glycerol types (65%, w/v and2%, w/v) using C. butyricum VPI3266 at two different glycerol

ng Journal 64 (2012) 106– 118 111

concentrations (30 g/L and 60 g/L) and D = 0.1 h−1. Complete glyc-erol consumption and a yield of 0.60 mol 1,3-PD/mol glycerol wasobserved for both the glycerol concentrations with an increase infinal 1,3-PD titre upon increase in initial glycerol concentration.However, there were no reports in literature demonstrating highsubstrate consumption rates in continuous cultivations carried outat high D till in 2005 [57], the same group reported a produc-tion of up to 30 g/L 1,3-PD from 60 g/L feed glycerol at a high Dof 0.3 h−1 leading to a volumetric productivity of 10.3 g/L/h whichwas the highest ever reported for continuous cultivations using C.butyricum. It was also reported that the 1,3-PD yield remained ata high, constant value (0.65 mol/mol) regardless of substrate feedconcentration and/or D. Cultivation of C. butyricum F2b on rawglycerol (65%, w/w) in single stage continuous cultures at differ-ent D and several inlet glycerol concentrations was attempted byPapanikolaou et al. [74,75]. A constancy in 1,3-PD yield at all inletglycerol concentrations was observed in these studies. High 1,3-PD concentrations ranging from 31 to 48 g/L could be obtainedat low D particularly using 60 and 90 g/L glycerol concentrationswhich decreased slightly as D increased beyond 0.13 h−1. Similarlyalmost complete consumption (95–99%) of glycerol was observedat low D and low inlet glycerol concentrations whereas significantamounts of unconsumed glycerol were left behind in the fermenta-tion broth at high D (>0.13 h−1). Continuous cultures featured lowacetate yields at low D particularly at inlet glycerol concentrationsof 30 and 90 g/L which reached higher values when D was increasedbeyond 0.08 h−1. A rather different trend could be seen for butyrateyield which remained constant at low and medium D and thendecreased significantly at D greater than 0.2 h−1. In a recent reportfeasibility of production of 1,3-PD through non-sterilized contin-uous cultures of C. butyricum VPI3266 on biodiesel-derived crudeglycerol (purity 81%, w/w) was investigated [68]. The aim of thestudy was aimed at reducing the investment and energy costs of theprocess. At an inlet glycerol concentration of 30 g/L and D of 0.06 h−1

it was possible to maintain the system at steady state for 16 dayswhich featured an accumulation of 13.9 g/L 1,3-PD. Microscopic andpolymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE) examinations revealed the presence of single organ-ism in steady state cultures and therefore reinforced the stabilityof the proposed system. Continuous cultures at high glycerol con-centrations (80 g/L) and different D indicated an almost constantbiomass production (∼0.8 g/L) with an increase in 1,3-PD produc-tion at lower D. D of 0.04 h−1 gave the highest 1,3-PD concentration(30.1 g/L) while the highest productivity (1.87 g/L/h) with 23.4 g/L1,3-PD could be achieved at a D of 0.08 h−1.

3.2.3.3. Continuous culture with cell recycling. Continuous culturewith cell recycling ensures high cell concentration in the bioreactorwhich may give rise to high product concentrations at significantlyhigher D. The potential use of the above bioreactor cultivation strat-egy in improving 1,3-PD concentration, rates and productivity isnot very well established. Reimann et al. [76] reported the firstattempt of continuous cultivation with cell recycling using hollowfibre modules made of polysulphone for improved bioconversionof glycerol. The usage of two glycerol concentrations—32 g/L and56 g/L resulted in increased cell density in accordance with theretention ratio giving a 4–5-fold increase for 32 g/L glycerol con-centration and a 3.4–4-fold increase for a glycerol concentrationof 56 g/L, as compared to the continuous cultivation without cellrecycling. However, the system failed at higher glycerol concen-tration of 92 g/L due to the inability of cells to grow at such high
substrate concentration. With a glycerol concentration of 56 g/L andD of 1.0 h−1, steady state could be achieved at a retention ratio of10 which featured a 1,3-PD concentration of 26.6 g/L. The cultiva-tion however could not be continued at a D higher than 1.0 h−1 due

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o membrane plugging. The productivity was at its highest valuet a D of 0.7 h−1 for both substrate concentrations (32 and 56 g/L).owever it decreased when D was increased beyond 0.7 h−1 whichas particularly due to inhibition by the fermentation products

t higher D values. It was shown by the authors that inhibitionmposed by the product was the major limitation of this method

hich was further exacerbated by the clogging of the membrane.t appears that enhancement of product concentration and produc-ivity can be attempted by modified reactor design with a “spinlter” device operating at higher D while retaining the growingells in the bioreactor. This reactor design would feature removalf cell free broth and the inhibitory product from the reactor andherefore will be ideally suited for the 1,3-PD production system.

.2.3.4. Immobilization. Another strategy which could be industri-lly important is immobilization. It offers several advantages suchs reuse of the biocatalyst, easier downstream product process-ng, continuous operation at high cell density with resultant higheaction rates leading to high productivity. Hitherto, the appli-ation of this technique for the production of 1,3-PD has beenimited. Cells of C. freundii were immobilized on POLYHIPETM poly-

ers and modified polyurethane foam for production of 1,3-PDy Griffith and Bosley [77] and Pflugmacher and Gottshalk [36],espectively. The latter could obtain a 2-fold increase in produc-ivity (8.1 g/L/h) and 16.3 g/L 1,3-PD using this method. Zhao et al.59] investigated the use of microcapsules made of NaCS/PDMDAACSodium cellulose sulfate/poly-dimethyl-diallyl ammonium chlo-ide) for encapsulation of K. pneumoniae in order to produce 1,3-PD.he initial trials as shake flask fermentations using encapsulatednd free K. pneumoniae cells showed an enrichment of biomassn the microcapsule (6.48 g/L capsule vs 2.62 g/L free cell culture)ut lesser 1,3-PD accumulation (13.1 g/L vs 17.65 g/L) which wasttributed to mass transfer resistance through the microcapsuleembrane. A comparison of the performance of encapsulated cells

n batch, fed-batch and continuous cultivation yielded interestingesults as it was found that the encapsulated cells performed evenetter under different bioreactor operating conditions as comparedo the shake flask cultivation. Various constraints with respect to,3-PD concentration were eliminated upon cultivation of encapsu-

ated cells in the bioreactor. Firstly the average amount of biomasser litre of medium in the bioreactor was larger than in shake flaskhich increased the glycerol uptake rate of the encapsulated cells.oreover controlled neutral pH conditions and high cell density

nside the microcapsule favoured anaerobic conditions and there-ore increased the reduction of glycerol and hence 1,3-PD yield.ncreased substrate tolerance of encapsulated cells (due to the

ass transfer resistance of microcapsule membrane) allowed highnitial glycerol concentration (120 g/L) to be taken inside the biore-ctor thereby improving 1,3-PD concentration. Batch cultivationeatured glycerol consumption in only 5 h leading to an accumu-ation of 14.8 g/L 1,3-PD and giving a productivity of 2.96 g/L/h.ultivation of encapsulated cells in fed-batch mode resulted in1.86 g/L 1,3-PD with a productivity of 1.08 g/L/h which establishedhe stability of their catalytic activity upon fresh medium additions.ontinuous cultures, on the other hand resulted in a much higherroductivity (4.49 g/L/h) with increased D (0.33 h−1) but the final,3-PD concentration was compromised (13.6 g/L). 1,3-PD produc-ivity in continuous cultivation increased by 1.5-fold and 4.15-folds compared to batch and fed-batch cultivation, respectively. Thisresented NaCS/PDMDAAC microcapsule as a desirable immobi-

ization system for continuous glycerol to 1,3-PD bioconversion.Jun et al. [78] produced 1,3-PD from raw glycerol (purity 80%,

/w) by fed batch cultivation with both suspended and immo-ilized cells of K. pneumoniae DSM 4799. It was reported thataw glycerol yielded better 1,3-PD productivity (1.51 g/L/h) asompared to 0.84 g/L/h obtained from pure glycerol when fed

ng Journal 64 (2012) 106– 118

batch cultivations were performed using suspended cells. Repeatedfed batch fermentations conducted with immobilized cells usinghydrophobic polyurethane media in fibrous bed reactor couldprovide sufficient biomass at the beginning of fed-batch therebyeliminating the need of preculture, resulting in significant reduc-tion of lag phase and thus improving the productivity of the process.The productivity of 1,3-PD increased from 1.06 g/L/h in first cycleto 1.61 g/L/h in the fourth which could be attributed to successfulcell immobilization.

In a recent study by Gungormusler et al. [79] continuous pro-duction of 1,3-propanediol using immobilized cells of Clostridiumbeijernickii NRRL B-593 and raw glycerol without purification wasattempted. It was reported that a hydraulic retention rate (HRT)of 2 h gave the best volumetric production rate of 1,3-PD withboth suspended cells and those immobilized on ceramic rings andpumice stones. Moreover, a 2.5-fold increase in productivity couldbe achieved as a result of cell immobilization which further sup-ported the use of this technique for improved 1,3-PD production.

3.2.3.5. Multi-stage fermentation. This cultivation strategy offersthe advantage of using carbon sources other than glycerol and/ordifferent operating conditions in different steps of the productionprocess. This type of cultivation strategy has been investigated byseveral researchers for improving the bioconversion of glycerol to1,3-PD. A two step 1,3-PD fermentation was carried out using C.freundii by Boenigk et al. [30]. In this approach an exponentiallygrowing culture was obtained by operating the first fermentorunder conditions of glycerol limitation while a reduction in D onthe second fermentor facilitated increase in 1,3-PD production. Themethodology could feature a concentration of 42 g/L 1,3-PD with aproductivity of 1.8 g/L/h. Papanikolaou et al. [74] carried out two-stage continuous fermentation with a newly isolated C. butyricumstrain F2b using raw glycerol (main by-product of fuel-ester pro-duction process, purity 65%, w/w). This cultivation strategy utilizeda high D in the first fermentor in order to increase the volumetricproductivity of 1,3-PD and a lower D in the second stage to obtainan increased concentration of the product. The report presented ahigh productivity of 7.2 g/L/h and significant amounts (31–45 g/L)of unused glycerol in the fermentation broth of first fermentor withless biomass concentration in the second (compared to first) irre-spective of D. This was attributed to bacterial autolysis in stagetwo. A global overall productivity of 3.4 g/L/h was obtained usingthis method. In a similar investigation in 2008 [6] operation of firstbioreactor at D = 0.11 h−1 and second bioreactor at 0.04 h−1 facili-tated in achieving a final 1,3-PD concentration of 43.5 g/L in stagetwo cultivation. Lesser biomass concentration (1.4 g/L vs 2.2 g/L),lower 1,3-PD yield and lower carbon recovery (76% vs 95%) wasobserved in stage two as compared to stage one. This was proba-bly due to incomplete reduction of 3-HPA to 1,3-PD which led to3-HPA toxicity to cells. An overall 1,3-PD productivity of 1.33 g/L/hcould be obtained in this study. In an attempt to achieve 3-HPAdetoxification in K. pneumoniae with simultaneous high produc-tion of 1,3-PD Zheng et al. [80] used two-stage fed batch cultivationstrategy. Maintaining an initial glycerol concentration of 40 g/L andagitation rate of 250 rpm in batch cultivation with the subsequentfeeding performed at 300 rpm gave a maximum concentration of74.07 g/L 1,3-PD and a productivity of 3.08 g/L/h.

Hartlep et al. [43] studied microbial production of 1,3-PD fromglucose in a two-stage process using two different combinations ofmicroorganisms. In stage one, either an osmotolerant yeast Pichiafarinosa or a recombinant E. coli strain harboring GPD1 and GPP2(glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate
phosphatise, respectively) genes was used for glycerol productionfrom glucose. In stage two, K. pneumoniae DSM 2026 convertedglycerol thus produced to 1,3-PD. Maintenance of a low glucoseconcentration (20–30 g/L) in stage I along with an increase in

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ormation of arbitol in stage I. However, a low growth rate of K.neumoniae was observed in stage II the exact reason for whichould not be established. On the contrary, the process with recom-inant E. coli and K. pneumoniae in stage one and two, respectively,ppeared to be a technically feasible system. It was reported thatpon growth of E. coli on relatively low glucose concentration theame produced only glycerol and almost no acetate which had pos-tive effects on 1,3-PD production in stage two.

In a recent study Mendes et al. [42] reported the highest val-es achieved so far for 1,3-PD production using a two-step process.onversion of sugars—glucose or sugar cane molasses into glyceroly a recombinant substrate tolerant S. cerevisiae followed by itsiotransformation into 1,3-PD by a recombinant non-pathogenic. acetobutylicum DG1 (pSPD5) in the same bioreactor was inves-igated in this study. With 103 g/L initial glucose concentration, anal 1,3-PD concentration of 25.5 g/L and a 1,3-PD productivity of.16 g/L/h could be obtained in this study. The investigators alsoighlighted the potential use of molasses for glycerol conversion byeast which gave comparable results as glucose except inhibition oflostridial growth in step two upon use of high sugar concentrations77.8 and 101.3 g/L) in step one.

.2.3.6. Mixed cultures. The prospect of using mixed cultures forhe production of 1,3-PD has also been explored. The conversion oflycerol into hydrogen gas and 1,3-PD was investigated by anaer-bic fermentation using heat-treated mixed cultures from fourifferent sources (tomato soil, wheat soil, compost, and sludge) byelembo et al. [81]. A comparison of fermentation with glycerolnd glucose as substrate showed that though the former yielded.69 mol of produced 1,3-PD per mol of consumed glycerol the lat-er produced more hydrogen (1.06 mol H2/mol glucose vs 0.28 mol2/mol glycerol). Comparable results from biodiesel-derived glyc-rol (0.31 mol H2/mol glycerol and 0.59 mol 1,3-PD/mol glycerol)ndicated the feasibility of integrating biodiesel production with,3-PD and improving the economy of the biodiesel plant.

Organic acids such as acetic acid, butyric acid and formic acidre formed as by-products in the fermentation of glycerol to 1,3-PDhich inhibit the growth of C. butyricum and deteriorate its ability

o biotransform glycerol to 1,3-PD. In an attempt to address thisroblem, a novel mixed culture of C. butyricum and a methane bac-erium Methanosarcina mazei was proposed by Bizukojc et al. [82].t was believed that since the organic acids particularly acetic andormic acids were utilized efficiently by M. mazei, a combination of. butyricum and M. mazei could be used as a mixed culture in whichhe organic acids released by 1,3-PD producer (C. butyricum) coulde used by M. mazei for efficient energy production. This wouldacilitate in relieving the inhibition imposed on the process (byhese acids) and thus help in improving the overall biotransfor-

ation process in the favour of 1,3-PD production. Two-speciesetabolic models were proposed and used for examining several

ultivation scenarios with respect to the acid-scavenging efficiencyf M. mazei. It was reported that maximum methanogenesis andess extensive growth of M. mazei were the best conditions foremoval of toxic acids from the system by it.

.2.3.7. Kinetics of 1,3-PD fermentation and physiological modelling.

. butyricum has been considered as a microorganism of indus-rial value in the production of 1,3-PD from glycerol particularlyecause of high fermentation yields and titre and relatively sim-le fermentation conditions [64]. However, inhibition from both
ubstrate and products reduces the overall growth and productormation rates [29,64]. Thus a thorough understanding of the inhi-ition kinetics imposed on the process is extremely important toesign inhibition-free cultivations in order to achieve maximum
ng Journal 64 (2012) 106– 118 113

productivity. Biebl [29] used pH-auxostat to measure product inhi-bition in glycerol fermentation to 1,3-PD with C. butyricum DSM5431. It was reported that at pH 6.5 growth was totally inhibitedat a concentration of 60 g/L 1,3-PD, greater than 80 g/L glycerol,27 g/L acetic acid and 19 g/L butyric acid. Thereafter Zeng et al.[58] proposed mathematical models describing the growth of C.butyricum and K. pneumoniae under substrate and/or product inhi-bition. It was revealed that the critical concentrations leading tono growth of the microorganisms were 0.35 g/L for undissoci-ated acetic acid, 10.1 g/L for total butyric acid, 16.6 g/L for ethanol,71.4 g/L for 1,3-PD, and 187.6 g/L for glycerol. Colin et al. [31] stud-ied the inhibition imposed by 1,3-PD on C. butyricum and reportedthat the maximum specific growth rate was inversely proportionalto the initial concentration of 1,3-PD and that the strain toleratedhigher 1,3-PD concentration during fermentation (81.3 g/L arisingout of initial addition and produced during cultivation) than theinitial 1,3-PD concentration (68 g/L). This finding by Colin et al.[31] was somewhat supported by a Papanikolaou et al. [6] whoreported that the biomass concentration remained almost con-stant in continuous cultures of C. butyricum F2b even when 1,3-PDconcentrations inside the chemostat vessel had reached 84.2 g/L.This demonstrated a high 1,3-PD tolerance of this natural isolate.Clostridial growth inhibition by different types and concentra-tions of glycerol was also tested by González-Pajuelo et al. [62]using C. butyricum VPI 3266. It was demonstrated that inhibitionincreased from 21% to 62% with increase (20–100 g/L) in concen-tration of both commercial (87%, w/v) and raw glycerol (92%, w/v).However with raw glycerol 65% (w/v) an inhibition of 86% wasobserved against 62% inhibition observed in the case of other twoglycerol types. Besides the inhibition caused by substrate and prod-uct(s) of 1,3-PD fermentation, it is important to investigate thepossible inhibitory effects of impurities which are found in rawglycerol derived from biodiesel plant, especially when its valoriza-tion to 1,3-PD has already attracted a great deal of attention. Thisindeed was attempted by Chatzifragkou et al. [83] who examinedthe impact of salts (NaCl, K2HPO4, Na2HPO4), fatty acids (oleic,stearic) and methanol present in raw glycerol on growth and 1,3-PD production by C. butyricum VP1718. It was observed that whileNaCl had an evident inhibitory effect when present at a glycerolconcentration of 4.5% (w/w of glycerol) in 200 mL flasks, no signif-icant inhibition could be seen in bioreactor trials at even higherconcentrations (30%, w/w of glycerol). This could be due to bet-ter control of environmental conditions in the latter. Additionallyphosphoric salts had no inhibitory effects. Presence of 2% (w/w)of oleic acid in glycerol totally inhibited microbial growth with 1%being the growth threshold. It was also proved in the study thatthe observed inhibition was due to the presence of double bond infatty acid as no negative impact on growth was found upon addi-tion of stearic acid in the bioreactor. An important constituent ofraw glycerol–methanol also did not affect either microbial growthor 1,3-PD production in batch cultures irrespective of its concentra-tion in the medium. Similar results as that of batch cultivation wereobtained upon addition of methanol at steady state in continuousculture conditions.

Due to inhibition by substrate as well as product it becomesrather difficult and tricky to design fresh nutrient feeding strate-gies as it features a scenario of cultivation when the culture iseither starving or inhibited during the entire cultivation period. Theconventional trial and error approach of nutrient feed design forelimination of substrate/product inhibition without the limitationof substrate for high productivity cultivation is laborious, frustrat-ing, time-consuming and inefficient. In such a case, a mathematical
model-based approach appears to be intelligent, fast, reliable andproductive. It features off-line description of substrate and prod-uct inhibition kinetics of cultivation and is sensitive to varyingsubstrate/product concentrations emerging out of feeding of fresh


Table 3Optimized values of 1,3-PD model parameters in various literature reports.

�max (h−1)a Ks (g/L)b K1,3-PD/X (g/g)c Q (g/L/h)d Organism Reference No.

0.527 – 17.14 6.47 C. butyricum F2b [88]0.39–0.43 – – 3.4 C. butyricum F2b [74]0.67 0.005 C. butyricum DSM5431 [58]0.71 0.026 – – K. pneumoniae DSM2026 [58]

0.65 0.026 3.78* K. pneumoniae DSM2026 [89]8.7# K. pneumoniae DSM2026 [89]

a Maximum specific growth rate.b Saturation constant for glycerol.c 1,3-PD yield (gram per gram dry cell weight).d 1,3-PD productivity.

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utrient feed. The model has the ability to simulate (on computer)he consequence of feeding of different concentrations of freshutrients and their rates which not only eliminates the limitationf substrate but also addresses the problem of inhibition of prod-cts [84]. Therefore a number of model simulations can be done onhe computer to optimize the right nutrient feeding strategy whichould result in “the” highest 1,3-PD concentration. This feeding

trategy can then be implemented experimentally.The above methodology was followed in the investigations

onducted in our laboratory for glycerol to 1,3-PD fermentation.t was possible to enhance 1,3-PD concentration from 25.8 g/Lbtained in batch [39] to 61.2 g/L in model-based fed-batch cultiva-ion conditions. As has been demonstrated by other fermentation

etabolite production such as sorbose [85], gibberellic acid [86],oly(�-hydroxybutyrate) [87], etc. 1,3-PD concentration and/orroductivity can be further enhanced by altering different feed-

ng and/or cultivation strategies in fed-batch cultivation (s) e.g., byptimizing the Start/Stop time of nutrient feeding, concentrationf substrate in the feed bottle, the feeding profile, etc. The modelould be further used to design other strategies such as continuousultivation, continuous cultivation with cell recycling, etc. to fur-her optimize the fermentation. However the utility of the above

entioned approach for 1,3-PD process optimization lies in theeliberate selection of only those nutrient feeding profiles for biore-ctor operation which could be adapted in the industrial conditionsithout any difficulty.

Mathematical models have also been applied by Papanikolaout al. [88] to predict the kinetic behaviour of C. butyricum F2bor growth on raw glycerol and 1,3-PD production. A Contois-ype model was found to fit the experimental data on 1,3-PDroduction in continuous cultures of C. butyricum with a highalue of R2 (97.155). The estimated values of model parame-ers (obtained by non-regression analysis and Marquardt iterativeearch algorithm) compared well with the results reported in lit-rature thus confirming the suitability of raw glycerol for 1,3-PDioconversion. More importantly the maximum value of produc-ivity obtained by the model (6.47 g/L/h) was close to the highestalues reported in literature. This proved the potential applica-ion of the model for 1,3-PD process optimization. Xiu et al. [89]ttempted mathematical-model guided analysis and optimizationf glycerol bioconversion in one and two-stage anaerobic culturesf K. pneumoniae DSM2026. It was reported that the highest volu-etric 1,3-PD productivity (3.78 g/L/h) in batch culture could be

chieved with an initial glycerol concentration of 88.32 g/L and.1 g/L inoculum concentration. On the other hand, optimal con-itions for continuous fermentation were a D of 0.29 h−1 and feed
oncentration of 67.252 g/L which could result in twice the 1,3-PDroductivity of 8.7 g/L/h as compared to batch. Theoretical analysisevealed that a two-stage process with a higher D in second biore-ctor would be favourable for high 1,3-PD production. Optimized
values of 1,3-PD model parameters from various studies have beensummarized in Table 3.

3.2.3.8. Metabolic engineering approach for 1,3-PD production.Although the focus of the present article is production of 1,3-PD by native 1,3-PD producers, some classical reports on the useof genetically engineered microorganisms capable of producing1,3-PD either from glycerol or directly from glucose are worth men-tioning. The most important and indeed successful examples ofthe application of metabolic engineering for 1,3-PD production hasbeen the development of an engineered E. coli strain by DuPont andGenencor International, Inc., USA which could directly convert lowcost feedstock D-glucose (corn hydrolysate) to 1,3-PD. By an intel-ligent combination of heterologous gene expression, gene deletion,appropriate control of flux distribution while maintaining the redoxand energy balance and modification of substrate uptake mecha-nism 1,3-PD could be produced at a titre of 135 g/L, a productivityof 3.5 g/L/h and a weight yield of 51% in a 10 L fed-batch fermenta-tion [41]. In yet another attempt with E. coli, 1,3-PD pathway genes(dhaB1 and dhaB2) from C. butyricum and yqhD gene from E. coliwere tandemly arrayed under the control of a constitutive, temper-ature sensitive promoter to construct a novel operon for expressionin E. coli K-12 ER2925 [40]. This engineered strain was then usedin a novel two-stage fermentation process which involved a shiftin temperature from 30 to 42 ◦C between the two stages for fastgrowth and efficient production of 1,3-PD from glycerol. It pre-sented a high 1,3-PD concentration of 104.4 g/L and a productivityof 2.6 g/L/h. C. acetobutylicum has been popularly used for solventproduction (acetone–butanol–ethanol) by researchers worldwide.However, it could not grow on glycerol due to its inherent inabil-ity to regenerate sufficient NADH for the growth and metabolism.It was genetically engineered by González-Pajuelo et al. [90] bythe introduction of 1,3-PD pathway from C. butyricum to create amutant C. acetobutylicum DG1 (pSPD5). The recombinant organismcould grow on glycerol and produce 84 g/L 1,3-PD with a yield of0.65 mol/mol glycerol in fed-batch cultivation. Studies with contin-uous cultures demonstrated a higher concentration and volumetricproductivity of 54.73 g/L and 3 g/L/h, respectively, in comparison tothat obtained from C. butyricum.

3.3. Downstream processing of biotechnologically produced1,3-PD

The recovery of 1,3-PD from complex fermentation broth rep-resents a true bottleneck in the development of a commerciallyviable bioprocess. This could be mainly attributed to its high
boiling point and presence of two hydroxyl groups which makeit strongly hydrophilic and therefore complicate its extraction.Nonetheless various methods have been applied for separation of1,3-PD from the fermentation broth (Table 4). The application of

G. Kaur et al. / Biochemical Engineering Journal 64 (2012) 106– 118 115

Table 4Different downstream processing methods for 1,3-PD and associated problems.

Reference No. Separation method Problems

[91,92] Evaporation, Vacuum distillation Requirement of large amounts of energy, desalination, low productyield

[94] Ion-exclusion using polystyrene sulphate in Na form Requirement of energy due to dilution of 1,3-PD in broth[95] Ion-exclusion using charcoal column and acidic cation exchange

polystyrene resinRequirement of energy due to dilution of 1,3-PD in broth

[96] Process chromatography –[97] Cyclic sorption and desorption by zeolite Requirement of dewatering step, high chance of contamination[98] Liquid–liquid extraction Requirement of large amount of solvent[100] Reactive extraction Non-specificity of reaction[93] Reactive extraction Requirement of electrodialysis for desalination of broth[101] Aqueous two-phase extraction Requirement of large amounts of methanol, difficulty of separation of

two alcohols

rlao

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idi(t

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[102] Aqueous two-phase extraction

[103] Ultrafiltration, activated charcoal, vacuum distillation,chromatography

elatively simple approaches of evaporation and vacuum distil-ation for the recovery of 1,3-PD [91,92] has been attempted butppeared unattractive and uneconomical due to the requirementf large amounts of energy, desalination and low product yield.

The use of ion exclusion for extraction of 1,3-PD has beeneported. The method consisted of desalination of the fermentedroth by the aid of strongly cationic and weakly basic anionic resinsith subsequent passage over a cationic exchange resin for purifi-

ation of 1,3-PD [93]. Chromatographic column packed with cationxchange resin was used by Hilaly and Binder [94] and Roturiert al. [95] for the recovery of 1,3-PD. While the use of polystyreneulfonate in Na form was contrived by Hilaly and Binder [94] toeparate 1,3-PD from impurities the latter used a charcoal col-mn and strong acidic cation exchange resin of polystyrenesulfoniccid type to produce a protein-free broth which contained 90.5 g/L,3-PD. However both the aforementioned methods diluted 1,3-PD

n the broth thereby demanding more energy than simple con-entional processes. Besides this, the prospect of using processhromatography to avert feedback inhibition of cell growth androduct formation has been investigated by Wilkins and Lowe [96]or in situ removal of 1,3-PD.

Corbin and Norton [97] disclosed a different approach involv-ng on-line separation using cyclic sorption and desorption of theesired product 1,3-PD by a resin zeolite. The method entailed an

ndispensible dewatering step and a high chance of contaminationdue to uninterrupted link between the bioreactor and the separa-ion equipment).

Liquid-liquid extraction employing a suitable solvent could besed for energy-efficient separation of the metabolite of inter-st from a dilute solution and therefore has been attempted forurification of 1,3-PD as well. Malinowski [98] experimented with

iquid–liquid extraction to establish its utility for the recovery of,3-PD. A thorough verification revealed that it was probably notn efficient method owing to the low partition coefficient of 1,3-PDnto organic solvents. The unavoidable requirement of addition ofarge amounts of solvent for the separation of 1,3-PD again madehe process undesirable [99]. An approach which has been investi-ated quite a lot as a possible solution to the problems resultingrom the hydrophilic nature of 1,3-PD was reactive extraction.he method involved a reaction between 1,3-PD and acetalde-yde catalyzed by a Dowex or Amberlite ion-exchange resin toive 2-methyl-1,3-dioxane (2MD). This was followed by extrac-ion using an organic solvent such as toluene [100] and conversiono 1,3-PD by hydrolyzation. An overall 1,3-PD conversion yieldf 98% could be achieved by this method. However the acetalde-
yde used in this method could also react with other substances
n the fermentation broth like ethanol, glycerol, etc. The possibilityf this reaction reduced the specificity of this method for 1,3-PDnly. An improvement in aforementioned method was therefore

––

attempted by Hao et al. [93] by using other aldehydes like propi-onaldehyde, butyraldehyde, etc., for reaction with 1,3-PD to formhighly hydrophobic acetals. These acetals being miscible with thealdehydes enabled their use both as reactant and extractant. How-ever, an indispensible need for electrodialysis to desalinate thebroth made this method unattractive.

Recently the use of aqueous two-phase systems (ATPS) forefficient extraction of 1,3-PD from complex broth has been inves-tigated. An ATPS composed of 46% (v/v) ethanol and saturatedammonium sulphate was found to give a high partition coeffi-cient of 4.77 and 93.7% recovery of 1,3-PD with simultaneousextraction of 2,3-BD, acetoin, residual glycerol and removal of cellsand proteins from the broth [101]. High extraction efficiency ofthis ATPS could be attributed to high valent charge of the anionand strong polarity of the solvent. However requirement of largeamounts of methanol for salt removal and difficulty of separation oftwo alcohols restricted its industrial application. Therefore anotherATPS composed of methanol/phosphate where methanol could beeffectively used both as extractant (for 1,3-PD) and solvent (forcrystallizing the salt) was suggested and studied in detail by theLi et al. [102]. A higher partition coefficient (38.3) of the systemcomposed of 35% (v/v) methanol, saturated concentration of phos-phate and pH 10.7 could give a recovery of 98.1% 1,3-PD. Efficientand easy removal of salt (94.7%) by just adjusting the pH to 4.5 fol-lowed by addition of 1.5 volume of methanol could be achieved bythis ATPS.

A novel method of downstream processing of 1,3-PD has beenrecently developed which involves the purification of 1,3-PD inthree simple steps: [103] removal of biomass and proteins by theuse of microfiltration and activated charcoal, respectively, concen-tration of the broth by vacuum distillation followed by separationof 1,3-PD by chromatography. The authors have reported an overall1,3-PD yield of 75.47% by using this protocol [104].

4. Concluding remarks

The microbial production of 1,3-PD is an exciting method ofvalorizing ‘waste’ glycerol from the biodiesel plant and thus pro-ducing an industrially important raw material by biological routewhile minimizing the dependence on rapidly disappearing fos-sil fuels. The economic biological production of 1,3-PD needs tobe addressed urgently. Enormous industrial applications of 1,3-PDparticularly in textiles and cosmetics and its production from arenewable source present it as an eco-friendly and economicallyfeasible process.

One of the major factors governing the economic viabilityof any bioprocess is the cost of the starting material, which inthis case is glycerol. The escalating global energy demands anda predilection for clean, biodegradable and inexhaustible fuels is

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xpected to increase the already growing biofuel industry man-fold in times to come. This would mean greater availability ofwaste’ glycerol for conversion to high valued products. Thoughuropean Union leads the world in biodiesel production, it is nothe only one with such tremendous production capacity. In factiodiesel production is already manifested by developing coun-ries such as Brazil, Argentina and China. Skyrocketing crude oilrices (crude oil priced at USD 116.32 per barrel) (Ref 2011),uge amounts of crude glycerol generated as ‘waste’ from theiodiesel plants throughout the globe and therefore further decline

n glycerol prices is expected to make the glycerol-based 1,3-PDroduction cost competitive with the petrochemically produced,3-PD and also with sugar-based 1,3-PD production. The usef bioprocess engineering strategies such as fed-batch, continu-us cultivation with/without cell recycling, mixed cultures andmmobilized cells and/or enzymes deserve further research andevelopment efforts. Besides this, the application of mathemat-

cal modelling for designing various reactor operating strategiesor improved concentration and/or productivity of 1,3-PD is a sim-le yet attractive and result-yielding approach. It is particularly

nteresting considering the various constraints such as substrateimitation and substrate/product inhibition imposed on 1,3-PDrocess. Therefore it is believed that with the development ofppropriate bioprocess engineering strategies and availability ofower-priced glycerol in the near future, the production of biobased,3-PD using native strains would prove both economical androductive.

cknowledgement

The Senior Research Fellowship (SRF) award by Indian Councilf Medical Research (ICMR), Govt. of India, New Delhi for the execu-ion of the project is gratefully acknowledged by one of the authorsMs. Guneet Kaur).

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