Advances in biotechnological production of 1,3-propanediol

Advances in biotechnological production of 1,3-propanediol

Biochemical Engineering Journal 64 (2012) 106–118 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal journal homepag...

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Biochemical Engineering Journal 64 (2012) 106–118

Contents lists available at SciVerse ScienceDirect

Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Review

Advances in biotechnological production of 1,3-propanediol Guneet Kaur, A.K. Srivastava ∗ , Subhash Chand Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

a r t i c l e

i n f o

Article history: Received 16 September 2011 Received in revised form 6 March 2012 Accepted 7 March 2012 Available online 16 March 2012 Keywords: Glycerol 1,3-propanediol Bioprocess design Modelling Polytrimethylene terephthalate Downstream processing

a b s t r a c t 1,3-propanediol (1,3-PD) is a chemical compound with myriad applications particularly as a monomer for the production of polyesters, polyethers and polyurethanes. It is a raw material for the production of biodegradable plastics, films, solvents, adhesives, detergents, cosmetics and medicines. Various strategies have been employed for the microbial production of 1,3-PD which include several bioprocess cultivation techniques facilitated by natural and/or genetically engineered microbes. Though 1,3-PD is produced in nature by the bioconversion of glycerol its production directly from sugars like glucose has been also made possible by the development of recombinant strains. This review presents the “state of the art” in the biotechnological production technologies of 1,3-PD particularly with respect to bioprocess engineering methods. It also highlights the significance of mathematical model-based approach for designing various bioreactor operating strategies to facilitate the improvement in 1,3-PD production. Attempt has also been made to focus on the protocols used for downstream processing of 1,3-PD and the associated problems. Finally concluding remarks on the future outlook on biobased 1,3-PD to reduce the dependence on disappearing fossil fuels are presented. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,3-PD: a speciality chemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of 1,3-PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Synthesizing 1,3-PD via the chemical way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Synthesizing 1,3-PD via the biological way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Metabolic pathway to 1,3-PD: role of genes and their encoded enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Bottlenecks in the biological route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Process development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Downstream processing of biotechnologically produced 1,3-PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In the recent years concern over the biological production of commercially important metabolites has been burgeoning. This is mainly attributed to the escalating global energy and environmental problems which have stimulated researchers worldwide to devise methods for producing almost everything via the green way.

Abbreviations: 1,3-PD, 1,3-propanediol; 3-HPA, 3-hydroxypropanaldehyde; 2,3BD, 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). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2012.03.002

106 107 107 107 107 108 109 109 114 115 116 116

The roster comprises cosmetics, food, fuels, lubricants, plastics, beverages, fibres and medicines. Of these, the production of biofuels, particularly biodiesel has attracted a great deal of attention. Biodiesel (fatty acid alkyl ester) produced by transesterification of fats and oils has been considered as a renewable, biodegradable and non-toxic fuel [1]. A predominant by-product of the biodiesel plant is glycerol. Glycerol is a chemical compound of immense importance both as an end product and a starting material for a myriad other useful products [2]. Raw glycerol is currently used in large scale 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]. Huge amounts of glycerol are available (1 gallon of crude glycerol is left

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

behind for every 10 gallon of biodiesel produced) against a meagre demand with the result often leading to a severe loss to the industry [4]. Besides this surplus amounts of waste glycerol are generated by soap industries and alcohol beverage manufacturing units thereby necessitating a ‘search’ for methods befitting the disposal of the same. An interesting way is the conversion of this ‘waste product’ to a ‘high-value added’ product. This would not only sustain the utility of an important commodity like glycerol but also make these production plants more economical. Bioconversion of glycerol into propionic acid, succinic acid, citric acid, single cell oil, butanol, hydrogen as well as 1,3-PD has been investigated [5–8]. With the increasing emergence of novel applications of 1,3-PD the demand for its biological production has been on a rise. 1,3-PD is majorly used to make a new class of polymers with enhanced functionality. The market for 1,3-PD is growing very rapidly, as new products are developed to capitalize upon the functionality of the polymers that can be synthesized from 1,3-PD [9,10]. This market offers a significant opportunity to develop new, cost-competitive processes to produce 1,3-PD which would avoid the use of more petroleum, provide substantial energy savings and afford significant market penetration for the burgeoning bio-products industry. This report describes the current status of the biotechnological production of 1,3-PD particularly with an aim to optimize the production and have economic separation protocols for a variety of applications. Attempt has been made to describe and discuss in detail both previous and recent published work on 1,3-PD production in order to delineate the progress made in 1,3-PD bioprocess strategies and purification techniques over the years. More importantly, the review ‘proposes’ a different approach of mathematical model-based designing of nutrient feeding strategies for improved production of 1,3-PD and discusses the results obtained by the authors by the use of this method. Finally some perspectives and future outlook on microbial production of 1,3-PD are given under the light of current status of research.

107

Fig. 1. Applications of 1,3-PD.

1,3-PD has a multitude of other applications as well (Fig. 1). The biodegradable nature, higher light stability and solubility of 1,3PD-based polyesters in most common solvents add to their already growing list of applications [17,18]. 1,3-PD can be formulated into laminates, solvents, mouldings, adhesives, resins, detergents, cosmetics, deodorants and other end uses [19]. Solvent uses of 1,3-PD include water based inks such as ink-jet and screen inks. 1,3-PD is an important intermediate for organic synthesis, can be used in various types of medicines (vitamin H and immunosuppressive drugs), insect repellents, fragrances, etc. Thus the production of biobased 1,3-PD is extremely important. 3. Production of 1,3-PD 3.1. Synthesizing 1,3-PD via the chemical way

2. 1,3-PD: a speciality chemical 1,3-PD is a colourless viscous organic compound with the formula C3 H8 O2 and a non-flammable, low toxicity liquid which is miscible with water, alcohols and ethers so that it can be easily transported. It has a history of being a high priced speciality chemical. It has many desirable properties for participation in polycondensation reactions. It is a unique alternative compound for industrial chemical formulations. However, high cost [11] and limited availability of 1,3-PD in the past were two important factors which restricted its applications mainly as a solvent and in the production of dioxanes [12]. This scenario changed (1995–1998) after the annunciation of commercialization of a 1,3-PD based polyester, named polytrimethylene terephthalate by DuPont and Shell which termed it as SORONATM and CORTERRATM , respectively [13,14]. This copolyester is a condensation product of 1,3-PD and terephthalic acid and is principally used in the manufacture of carpet and textile fibres but also finds applications as engineering thermoplastics, films and coatings [15]. The fabrics have stretch resilience, low static generation, colour fastness, good stretch recovery, stain resistance, etc. [13]. In thermoplastic urethanes (TPU), use of 1,3-PD can lead to improved thermal and hydrolytic as well as thermal dimensional stability [16]. 1,3-PD can be used to modify polyester systems. This property is particularly beneficial for the production of powder coatings where partial substitution with 1,3-PD can give improved flexibility without adversely affecting other key properties such as storage stability and outdoor weather ability. In engine coolant formulations, 1,3-PD demonstrates improved heat stability, less corrosion especially to lead solder, and lower toxicity than ethylene glycol coolants.

The conventional modus operandi for the production of 1,3-PD consisted of the methods which were followed by the two chemical companies instrumental for the upsurge in the interest in 1,3-PD. DuPont started with acrolein which was converted to 3hydroxypropionaldehyde (3-HPA) by hydration. This was followed by hydrogenation in second step to give 1,3-PD [20]. Shell on the other hand followed the method of hydroformylation of ethylene oxide to 3-hydroxypropanal. This was subsequently extracted and hydrogenated 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 of expensive catalyst, release of toxic intermediates, dependence on non-renewable materials, low yield and complexity [22] called for the biological route which would give a cleaner product with no toxic 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 particularly attractive since it utilizes renewable feedstock and cultivations at normal temperature and pressure leading to no generation of toxic by-products. The production of 1,3-PD occurs from glycerol in nature with the latter being the only substrate which can be fermented to 1,3-PD [23]. Other advantages of using glycerol include highly reduced nature of the carbon atoms in glycerol thereby yielding more reducing equivalents than glucose or xylose [24], lower capital and operational costs. The list of microorganisms competent in performing glycerol to 1,3-PD bioconversion comprises Klebsiella (K. pneumoniae and K. oxytoca), Clostridia (C. butyricum and C. pasteurianum), Enterobacter

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Fig. 2. Metabolic pathway of glycerol conversion. Rounded rectangles—NADH + H+ consuming products; Ellipse—NADH + H+ producing intermediates and products; Box—key genes of dha regulon. Name of genes shown in italics. GDHt—Glycerol dehydratase, 1,3-PD DH—1,3-propanediol dehydrogenase, PEP—Phosphoenolpyruvate, 2,3-BD—2,3butanediol, DHA—Dihydroxyacetone, DHAP—Dihydroxyacetone phosphate, 3-HPA—3-hydroxypropanaldehyde, 1,3-PD—1,3-propanediol.

(E. agglomerans), Citrobacter (C. fruendii) and Lactobacilli (L. brevis and L. buchneri) [25–39]. Besides glycerol other carbon sources such as glucose, corn hydrolysate and sugarcane molasses have also been used for the production of 1,3-PD [40–42]. However there are no native 1,3-PD producers which can directly convert sugars to 1,3-PD. Therefore the attempts with sugars as substrate have been made with either multistage fermentations [42,43] involving a combination of microorganisms or genetically engineered microorganisms [40,41] carrying genes for conversion of sugar to glycerol and then glycerol to 1,3-PD. 3.2.1. Metabolic pathway to 1,3-PD: role of genes and their encoded enzymes The pathway to 1,3-PD using glycerol as a substrate is a coupled oxidation–reduction process [44]. In this process, generation of energy in the form of ATP and reducing equivalents in the form of NADH + H+ occurs in the oxidative branch while regeneration of NAD+ concomitant with the formation of reduced product 1,3-PD occurs in the reductive branch (Fig. 2). NADH + H+ is generated during glycolytic reactions (oxidative branch) which also yield several other by-products. In the reductive branch, glycerol is first dehydrated to 3-HPA, a reaction catalyzed by coenzyme B12 -dependent glycerol dehydratase (GDHt), which is then reduced to 1,3-PD by NADH + H+ -dependent 1,3-PD dehydrogenase (1,3-PD DH) by NADH + H+ utilization. More dehydratases have been described, which are B12 -independent such as GDHt in C. butyricum [2]. Different types of by-products are formed by different organisms in the oxidative branch. While butyric acid and acetic acid are the major by-products given by C. butyricum [33], butanol is produced by C. pasteurianum [45]. Besides these ethanol, lactic acid, succinic acid, 2,3-butanediol (2,3-BD) constitute those produced by the Enterobacteria [44]. The reactions up to the formation of pyruvate are common to all the microorganisms [46]. Glycerol is dehydrogenated to dihydroxyacetone (DHA) with the help of NAD-dependent glycerol dehydrogenase (GDH) which is then phosphorylated by DHA kinase (DHAK) to dihydroxyacetone phosphate (DHAP), one of the metabolites in the glycolytic pathway. There is a net production of two NADH + H+ molecules and one

ATP molecule during the conversion of glycerol to pyruvate [47]. An alternative route of glycerol degradation is found in Klebsiella sp. when grown under aerobic conditions. Glycerol is phosphorylated to sn-glycerol-3-phosphate by a glycerol kinase which is then converted to DHAP by NAD-dependent glycerol 3-phosphate dehydrogenase [48]. In Lactobacillus sp. the oxidative branch of glycerol metabolism is missing and therefore these microorganisms need to be provided with an additional carbon source in order to fulfil the requirements of energy and reducing equivalents [49]. The enzymes of the 1,3-PD pathway are encoded by dha operon which has been characterized in K. pneumoniae [49], C. fruendii [50], and C. butyricum [51]. Glycerol dehydratase considered as a rate limiting enzyme of the pathway [25] is coenzyme B12 dependent and consists of three polypeptides encoded by three ORFs- dhaB, dhaC and dhaE, respectively (alternate nomenclature is dhaB1, dhaB2 and dhaB3) [52] that catalyzes the conversion of glycerol to 3-HPA. Inactivation of coenzyme B12 occurs upon its interaction with glycerol which leads to cessation of catalysis by dehydratase. This inactivation is caused due to the irreversible cleavage of Co C bond of the coenzyme which then results in the formation of 5 -deoxyadenosine and alkylcobalamine-like species. The latter binds tightly to dehydratase and renders it inactive. The resumption of dehydratase activity occurs only after the dissociation of dehydratase-bound inactive B12 is mediated by another enzyme glycerol dehydratase reactivase. Regeneration of coenzyme B12 from inact-B12 is mediated by two subunits of the reactivating factor (dhaF-dhaG) of GDHt encoded by dhaF and dhaG genes [41] (Fig. 3). The coenzyme B12 -independent GDHt of C. butyricum however functions via a different mechanism. It utilizes S-adenosylmethionine instead of adenosylcobalamin to catalyze the reaction. Moreover it is oxygen sensitive and therefore cannot be used in aerobic systems for 1,3-PD production unlike coenzyme B12 -dependent GDHt. The second reaction of the reductive branch is 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 by yqhD gene and has been found to have higher oxidoreductive activity compared to 1,3-PD DH. This enzyme is an NADP-dependent dehydrogenase unlike 1,3-PD DH which is NAD-dependent.

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

109

Fig. 3. Coenzyme B12 chemistry in 1,3-PD pathway.

3.2.2. Bottlenecks in the biological route Albeit the biological production of 1,3-PD propounds an ecofriendly route to the important chemical; it suffers from certain drawbacks which need to be overcome to make this method economically feasible. These can be described as low yield, inhibition by both substrate and product [27,31,55], simultaneous formation of by-products [47,56], cost of the substrate and other medium components [35]. The drawbacks of the microbial path to 1,3-PD have been cogitated by several researchers and efforts have been made to make this endeavour a successful one. The solutions to the problems encompassing the biological route can be categorized as Process development and Strain improvement. This review describes only the bioprocess strategies for improving the production of 1,3-PD. 3.2.3. Process development One of the approaches to optimize the microbial production of 1,3-PD from glycerol is process optimization. Different bioreactor operating strategies have been implemented by several researchers in order to maximize 1,3-PD production (Table 1). A detailed description of these strategies is given below. Of all the microorganisms capable of producing 1,3-PD from glycerol, Enterobacteria and Clostridia appear to be the best producers in terms of high yield and productivity offered by the two [57–59]. Batch cultivation as a primary investigation approach was used by Günzel et al. [60] and Barbirato et al. [28] to demonstrate the feasibility of scale up of 1,3-PD production to industrial reactor sizes and establish the best organism for the same, respectively. C. butyricum indeed was the most promising candidate for industrial bioprocess owing to its higher 1,3-PD conversion yield, no accumulation of 3-HPA and shorter fermentation times as compared to K. pneumoniae, C. fruendii and E. Agglomerans [28]. Usage of raw glycerol (67%, w/w) for 1,3-PD production by C. butyricum CNCM1211 was attempted by Himmi et al. [33]. A high 1,3-PD concentration of 65 g/L from 121 g/L raw glycerol could be obtained using only 4 ␮g/L biotin. Biebl et al. [61] investigated the use of C. butyricum DSM 5431 for the production of 1,3-PD. It featured a concentration of 29.5 g/L 1,3-PD with consumption of 52 g/L glycerol thereby giving a 1,3-PD yield of 0.56 mol/mol. Batch experiments without pH regulation were carried out by González-Pajuelo et al. using C. butyricum VPI3266 [62]. A 1,3-PD yield of 0.58 mol/mol with a consumption of 41% glycerol was obtained in this work. A high 1,3-PD concentration

(61 g/L) and productivity (1.7 g/L/h) was achieved by batch cultivation of K. pneumoniae DSM2026 [63]. Though batch fermentation appears as a simple strategy for production of the desired metabolite, it suffers from the problems of substrate limitation towards the end of fermentation coupled with product inhibition which are inherently involved in the 1,3-PD production process. Thus this requires the use of better cultivation strategies which may facilitate cultivation under non-limiting and non inhibitory cultivations and 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 high initial substrate concentration and growth rate of the microorganism meant fed-batch cultivation could be used in order to achieve high concentration and/or productivity of 1,3-PD [64]. Though reasonably high 1,3-PD concentrations have been achieved by researchers using fed-batch cultivation, only few reports are dealing with this mode of reactor operation primarily due to the possibility of unlimited trials to achieve the optimum concentration in a suitable fed-batch fermentation. The process optimization efforts also require suitable sterilizable on-line, real time sensors for the measurement of key process variables (e.g. substrate) for better process control, which are largely non-existent. As a result there is scope of further improvement in the feeding strategies for enhanced 1,3 PD production. Some literature reported fed-batch strategies are summarized below [64–71]. A simple fed-batch system for high production of 1,3-PD from glycerol by C. butyricum VPI3266 was devised by Saint-Amans et al. [64] which coupled the feeding of the substrate to the volumetric C02 produced during the course of fermentation. A production of 65 g/L 1,3-PD with a productivity of 1.21 g/L/h and a yield of 0.69 mol 1,3-PD per mol glycerol consumed could be achieved by this process with an elimination of substrate inhibition. Coupled feeding of glycerol and ammonium to alkali consumption was attempted by Reimann and Biebl [65] using C. butyricum DSM 5431 and its product tolerant mutants. A considerable shortening of the cultivation times was reported which could be attributed to the maintenance of substrate at a non-limiting level and thus faster growth. An integrated bioconversion of raw glycerol to 1,3PD was developed and evaluated using an isolated high substrate and product tolerant strain of C. butyricum IK124 [66]. A fedbatch strategy combining a low base-driven glycerol addition with

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Table 1 Different 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 C. butyricum DSM5431; C. butyricum mutant 2/2 C. butyricum IK124; C. butyricum IK124 C. butyricum AKR102a C. butyricum VPI1718 C. butyricum VPI1718 K. pneumoniae ME-308 K. pneumoniae ME-303

65 70.4; 70.5 87.0; 77.5 76.2 67.9 70.8 70 71.58

0.69 0.68; 0.66 0.65; 0.67 0.62 0.67 0.66 0.70 0.65

1.21 1.4; 0.9 1.9; 1.2 2.3 0.78 0.70 0.97 1.93

[64] [65,65] [66,66] [67] [68] [69] [70] [71]

Continuous

K. pneumoniae DSM2026 C. butyricum VPI3266 C. butyricum VPI3266 C. butyricum F2b C. butyricum VPI3266

35.2–48.5 16–30 30 31–48 30.1

0.61 0.60 0.65 0.67 0.63–0.67

4.9–8.8 1.5–3.0 10.3 2.9–5.5 1.87

[73] [62] [57] [74,75] [68]

Continuous with cell recycling

C. butyricum DSM5431

26.6



16.4

[76]

Cell immobilization

C. freundii K. pneumoniae

K. pneumoniae DSM4799 C. beijernickii NRRL B-593

16.4 14.8 51.8 13.6 48–51 31

0.57 0.47 0.39 0.43 – 0.79

8.2 2.96 1.08 4.49 1.16 12

[36] [59] [59] [59] [78] [79]

C. freundii C. butyricum F2b C. butyricum F2b K. pneumoniae

41 41–46 43.5 74.07

0.62 0.67 0.49 0.62

1.8 3.4 1.33 1.08

[30] [74] [6] [80]

P. farinosa/K. pneumonia E. coli/K. pneumoniae

2.5 14.1

0.12 0.64

0.034 2.01

[43] [43]

S. cerevisiae/C. acetobutylicum DG1 (pSPD5)

25.5

0.67

0.16

[42]

Multi-stage

a

1,3-PD productivity.

constant on-line glycerol measurement was followed in the study. A high final 1,3-PD concentration of 87 g/L with an overall 1,3PD productivity of 1.9 g/L/h was achieved using refined glycerol. Moreover the use of raw glycerol also featured a high 1,3-PD concentration and productivity of 80.1 g/L and 1.8 g/L/h, respectively, which were comparable to the results obtained using refined glycerol. Usage of potato nitrogen concentrate, a cheap effluent from starch industry as a replacement of yeast extract in medium along with raw glycerol was also investigated in fed-batch fermentation. It yielded 77.5 g/L 1,3-PD with a slight prolongation of lag phase thereby 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 L scale fermentors using another isolate C. butyricum AKR102a was attempted recently [67]. Fed-batch fermentation yielded 1,3-PD concentration and productivity (76.2 g/L and 2.3 g/L/h, respectively) 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 production was more pronounced when 1,3-PD concentration in the broth had increased beyond 60 g/L. This was attributed to the presence of impurities such as fatty acids, heavy metal ions and

salts which are inherent constituents of crude glycerol. Scale up to 200 L scale was also attempted in this study. A reasonably high 1,3-PD concentration of 61.5 g/L with a 1,3-PD productivity of 2.11 g/L/h could be achieved in 200 L fermenter. These results appeared promising to further optimize the process for large scale 1,3-PD production. In a recent report fed-batch cultivation of C. butyricum VPI1718 using pulse feeding of concentrated crude glycerol under non-sterile conditions was attempted [68]. A maximum 1,3-PD concentration of 67.9 g/L could be obtained under these conditions at the end of 87 h of fermentation. Using the same strain the impact of anaerobiosis strategy and reactor geometry on 1,3-PD production was also investigated [69]. It was observed that continual sparging with nitrogen during fed-batch cultivation as opposed to self-generated anaerobiosis yielded different results with respect to 1,3-PD production and acid formation. Under continual sparging conditions 70.8 g/L 1,3-PD could be produced by C. butyricum VPI1718. This 1,3-PD concentration significantly decreased to 30.5 g/L in the latter strategy. Moreover lactic acid production was characteristically high in self-generated anaerobiosis fed-batch conditions which negatively affected both biomass and

Table 2 1,3-PD production by batch cultures. Organism C. butyricum CNCM1211 C. butyricum CNCM1211 C. butyricum DSM5431 C. butyricum VPI3266

K. pneumoniae DSM2026 a b c d e

Glycerol type b

Raw glycerol Raw glycerolc Pure glycerol Pure glycerol; Raw glycerold ; Raw glycerole Pure glycerol

1,3-PD (g/L)

Yield (mol/mol)

Q (g/L/h)a

Residual glycerol (g/L)

Ref. No.

63.4 65.4 29.5 7.35; 8.26; 7.93

0.69 0.66 0.56 0.58; 0.51; 0.56

1.85 1.67 2.3 0.31; 0.34; 0.33

0 0 – 23.37; 23.4; 15.4

[28] [33] [61] [62]; [62]; [62]

61

0.53

1.7

14.2

[63]

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

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

1,3-PD production. Thus anaerobiosis strategy was found to have an important effect on the biochemical behaviour of the culture during 1,3-PD production. Ji et al. [70] developed and analyzed three different pH-control feeding strategies to achieve the highest production of 1,3-PD using K. pneumoniae ME-308. First two attempts of feeding glycerol and NH3 in two separate lines and then together as a mixture did not prove beneficial as they featured either glycerol limitation or inhibition (due to the lack of proper monitoring system for glycerol) or a relatively high concentration of either 2,3-BD (pH 6.3) or lactate (pH 7.3) which competed with 1,3-PD for NADH + H+ thereby reducing the final concentration of the latter. Finally the last fed-batch approach of fluctuating the pH value between 6.3 and 7.3 periodically by feeding glycerol-NH3 mixture and 30% H2 SO4 limited the by-product formation, reduced unconsumed glycerol in the broth at the end of fermentation and gave a high concentration of 70 g/L of 1,3-PD. Recently fed-batch fermentation using hemicellulosic hydrolysates (corn straw) as cosubstrate along with glycerol for 1,3-PD production has been reported by Jin et al. [71]. In the fed-batch cultivation of K. pneumoniae, the redox state variation was regulated online by the feeding rate (maintaining xylose concentration at 5–8 g/L). It featured a 1,3-PD concentration of 71.58 g/L, yield and productivity of 0.65 mol/mol and 1.93 g/L/h, respectively, which represented an improvement of 17.8%, 25% and 17.7%, respectively, as compared to the use of glycerol alone. 3.2.3.2. Continuous culture. The usual approach to elimination of product inhibition is the removal of the inhibitory product from the fermentation broth by either Plug flow or Continuous Stirred Tank Reactor (CSTR). Continuous cultures offer a distinct advantage of high productivity but usually not very high product concentrations are obtained primarily due to significantly high feeding and withdrawal rates thereby making downstream processing a true bottleneck. One of the earliest studies on continuous cultures for understanding the regulation of glycerol bioconversion were carried out by Abbad-Andaloussi et al. [25] using C. butyricum DSM 5431. From the determination of concentrations of by-products, NADH + H+ , intermediates like acetyl CoA and enzymes of the pathway it was established that the enzyme thiolase probably partitioned the flux between acetate and butyrate since low levels of this enzyme were found. On the other contrary glycerol dehydratase appeared to be the rate limiting enzyme of 1,3-PD pathway as reflected by high intracellular concentrations of NADH + H+ and high activity of 1,3-PD DH in the cells with the only limitation imposed by GDHt. In yet another study the role of pyruvate metabolism in regulation of reduced equivalents in glycerol bioconversion by K. pneumoniae DSM2026 was investigated using continuous cultures by Menzel et al. [72]. The study established the involvement of pyruvate dehydrogenase (PDH) and absence of pyruvate: Ferredoxin oxidoreductase in 1,3-PD fermentation by means of enzyme assays. In vitro PDH activity of continuous cultures was found to increase with increasing inlet glycerol concentrations and decrease with increasing cell growth rates. Later Menzel et al. [73] also investigated the effects of inlet glycerol concentration and dilution rate (D) in continuous cultures of K. pneumoniae DSM2026. The study showed that 1,3-PD production by K. pneumoniae was a function of D as it decreased with increase in the latter. A high 1,3-PD concentration of 35.2–48.5 g/L with productivity between 4.9 and 8.8 g/L/h could be obtained at D ranging from 0.1 h−1 to 0.25 h−1 . Significantly lower 1,3-PD concentrations were observed at glycerol limiting conditions, which reached high concentrations of 48 g/L only when the glycerol supply was in excess in the medium. González-Pajuelo et al. [62] performed continuous cultivation with two raw glycerol types (65%, w/v and 92%, w/v) using C. butyricum VPI3266 at two different glycerol

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concentrations (30 g/L and 60 g/L) and D = 0.1 h−1 . Complete glycerol consumption and a yield of 0.60 mol 1,3-PD/mol glycerol was observed for both the glycerol concentrations with an increase in final 1,3-PD titre upon increase in initial glycerol concentration. However, there were no reports in literature demonstrating high substrate consumption rates in continuous cultivations carried out at high D till in 2005 [57], the same group reported a production of up to 30 g/L 1,3-PD from 60 g/L feed glycerol at a high D of 0.3 h−1 leading to a volumetric productivity of 10.3 g/L/h which was the highest ever reported for continuous cultivations using C. butyricum. It was also reported that the 1,3-PD yield remained at a high, constant value (0.65 mol/mol) regardless of substrate feed concentration and/or D. Cultivation of C. butyricum F2b on raw glycerol (65%, w/w) in single stage continuous cultures at different D and several inlet glycerol concentrations was attempted by Papanikolaou et al. [74,75]. A constancy in 1,3-PD yield at all inlet glycerol concentrations was observed in these studies. High 1,3PD concentrations ranging from 31 to 48 g/L could be obtained at low D particularly using 60 and 90 g/L glycerol concentrations which decreased slightly as D increased beyond 0.13 h−1 . Similarly almost complete consumption (95–99%) of glycerol was observed at low D and low inlet glycerol concentrations whereas significant amounts of unconsumed glycerol were left behind in the fermentation broth at high D (>0.13 h−1 ). Continuous cultures featured low acetate yields at low D particularly at inlet glycerol concentrations of 30 and 90 g/L which reached higher values when D was increased beyond 0.08 h−1 . A rather different trend could be seen for butyrate yield which remained constant at low and medium D and then decreased significantly at D greater than 0.2 h−1 . In a recent report feasibility of production of 1,3-PD through non-sterilized continuous cultures of C. butyricum VPI3266 on biodiesel-derived crude glycerol (purity 81%, w/w) was investigated [68]. The aim of the study was aimed at reducing the investment and energy costs of the process. 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 days which featured an accumulation of 13.9 g/L 1,3-PD. Microscopic and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) examinations revealed the presence of single organism in steady state cultures and therefore reinforced the stability of the proposed system. Continuous cultures at high glycerol concentrations (80 g/L) and different D indicated an almost constant biomass production (∼0.8 g/L) with an increase in 1,3-PD production 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/L 1,3-PD could be achieved at a D of 0.08 h−1 .

3.2.3.3. Continuous culture with cell recycling. Continuous culture with cell recycling ensures high cell concentration in the bioreactor which may give rise to high product concentrations at significantly higher D. The potential use of the above bioreactor cultivation strategy in improving 1,3-PD concentration, rates and productivity is not very well established. Reimann et al. [76] reported the first attempt of continuous cultivation with cell recycling using hollow fibre modules made of polysulphone for improved bioconversion of glycerol. The usage of two glycerol concentrations—32 g/L and 56 g/L resulted in increased cell density in accordance with the retention ratio giving a 4–5-fold increase for 32 g/L glycerol concentration and a 3.4–4-fold increase for a glycerol concentration of 56 g/L, as compared to the continuous cultivation without cell recycling. However, the system failed at higher glycerol concentration 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 and D of 1.0 h−1 , steady state could be achieved at a retention ratio of 10 which featured a 1,3-PD concentration of 26.6 g/L. The cultivation however could not be continued at a D higher than 1.0 h−1 due

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to membrane plugging. The productivity was at its highest value at a D of 0.7 h−1 for both substrate concentrations (32 and 56 g/L). However it decreased when D was increased beyond 0.7 h−1 which was particularly due to inhibition by the fermentation products at higher D values. It was shown by the authors that inhibition imposed by the product was the major limitation of this method which was further exacerbated by the clogging of the membrane. It appears that enhancement of product concentration and productivity can be attempted by modified reactor design with a “spin filter” device operating at higher D while retaining the growing cells in the bioreactor. This reactor design would feature removal of cell free broth and the inhibitory product from the reactor and therefore will be ideally suited for the 1,3-PD production system. 3.2.3.4. Immobilization. Another strategy which could be industrially important is immobilization. It offers several advantages such as reuse of the biocatalyst, easier downstream product processing, continuous operation at high cell density with resultant high reaction rates leading to high productivity. Hitherto, the application of this technique for the production of 1,3-PD has been limited. Cells of C. freundii were immobilized on POLYHIPETM polymers and modified polyurethane foam for production of 1,3-PD by Griffith and Bosley [77] and Pflugmacher and Gottshalk [36], respectively. The latter could obtain a 2-fold increase in productivity (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/PDMDAAC (Sodium cellulose sulfate/poly-dimethyl-diallyl ammonium chloride) for encapsulation of K. pneumoniae in order to produce 1,3-PD. The initial trials as shake flask fermentations using encapsulated and free K. pneumoniae cells showed an enrichment of biomass in the microcapsule (6.48 g/L capsule vs 2.62 g/L free cell culture) but lesser 1,3-PD accumulation (13.1 g/L vs 17.65 g/L) which was attributed to mass transfer resistance through the microcapsule membrane. A comparison of the performance of encapsulated cells in batch, fed-batch and continuous cultivation yielded interesting results as it was found that the encapsulated cells performed even better under different bioreactor operating conditions as compared to the shake flask cultivation. Various constraints with respect to 1,3-PD concentration were eliminated upon cultivation of encapsulated cells in the bioreactor. Firstly the average amount of biomass per litre of medium in the bioreactor was larger than in shake flask which increased the glycerol uptake rate of the encapsulated cells. Moreover controlled neutral pH conditions and high cell density inside the microcapsule favoured anaerobic conditions and therefore increased the reduction of glycerol and hence 1,3-PD yield. Increased substrate tolerance of encapsulated cells (due to the mass transfer resistance of microcapsule membrane) allowed high initial glycerol concentration (120 g/L) to be taken inside the bioreactor thereby improving 1,3-PD concentration. Batch cultivation featured glycerol consumption in only 5 h leading to an accumulation of 14.8 g/L 1,3-PD and giving a productivity of 2.96 g/L/h. Cultivation of encapsulated cells in fed-batch mode resulted in 51.86 g/L 1,3-PD with a productivity of 1.08 g/L/h which established the stability of their catalytic activity upon fresh medium additions. Continuous cultures, on the other hand resulted in a much higher productivity (4.49 g/L/h) with increased D (0.33 h−1 ) but the final 1,3-PD concentration was compromised (13.6 g/L). 1,3-PD productivity in continuous cultivation increased by 1.5-fold and 4.15-fold as compared to batch and fed-batch cultivation, respectively. This presented NaCS/PDMDAAC microcapsule as a desirable immobilization system for continuous glycerol to 1,3-PD bioconversion. Jun et al. [78] produced 1,3-PD from raw glycerol (purity 80%, w/w) by fed batch cultivation with both suspended and immobilized cells of K. pneumoniae DSM 4799. It was reported that raw glycerol yielded better 1,3-PD productivity (1.51 g/L/ h) as compared to 0.84 g/L/ h obtained from pure glycerol when fed

batch cultivations were performed using suspended cells. Repeated fed batch fermentations conducted with immobilized cells using hydrophobic polyurethane media in fibrous bed reactor could provide sufficient biomass at the beginning of fed-batch thereby eliminating the need of preculture, resulting in significant reduction 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 cycle to 1.61 g/L/h in the fourth which could be attributed to successful cell immobilization. In a recent study by Gungormusler et al. [79] continuous production of 1,3-propanediol using immobilized cells of Clostridium beijernickii NRRL B-593 and raw glycerol without purification was attempted. It was reported that a hydraulic retention rate (HRT) of 2 h gave the best volumetric production rate of 1,3-PD with both suspended cells and those immobilized on ceramic rings and pumice stones. Moreover, a 2.5-fold increase in productivity could be achieved as a result of cell immobilization which further supported the use of this technique for improved 1,3-PD production. 3.2.3.5. Multi-stage fermentation. This cultivation strategy offers the advantage of using carbon sources other than glycerol and/or different operating conditions in different steps of the production process. This type of cultivation strategy has been investigated by several researchers for improving the bioconversion of glycerol to 1,3-PD. A two step 1,3-PD fermentation was carried out using C. freundii by Boenigk et al. [30]. In this approach an exponentially growing culture was obtained by operating the first fermentor under conditions of glycerol limitation while a reduction in D on the second fermentor facilitated increase in 1,3-PD production. The methodology could feature a concentration of 42 g/L 1,3-PD with a productivity of 1.8 g/L/h. Papanikolaou et al. [74] carried out twostage continuous fermentation with a newly isolated C. butyricum strain F2b using raw glycerol (main by-product of fuel-ester production process, purity 65%, w/w). This cultivation strategy utilized a high D in the first fermentor in order to increase the volumetric productivity of 1,3-PD and a lower D in the second stage to obtain an increased concentration of the product. The report presented a high productivity of 7.2 g/L/h and significant amounts (31–45 g/L) of unused glycerol in the fermentation broth of first fermentor with less biomass concentration in the second (compared to first) irrespective of D. This was attributed to bacterial autolysis in stage two. A global overall productivity of 3.4 g/L/h was obtained using this method. In a similar investigation in 2008 [6] operation of first bioreactor at D = 0.11 h−1 and second bioreactor at 0.04 h−1 facilitated in achieving a final 1,3-PD concentration of 43.5 g/L in stage two 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%) was observed in stage two as compared to stage one. This was probably due to incomplete reduction of 3-HPA to 1,3-PD which led to 3-HPA toxicity to cells. An overall 1,3-PD productivity of 1.33 g/L/h could be obtained in this study. In an attempt to achieve 3-HPA detoxification in K. pneumoniae with simultaneous high production of 1,3-PD Zheng et al. [80] used two-stage fed batch cultivation strategy. Maintaining an initial glycerol concentration of 40 g/L and agitation rate of 250 rpm in batch cultivation with the subsequent feeding performed at 300 rpm gave a maximum concentration of 74.07 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 from glucose in a two-stage process using two different combinations of microorganisms. In stage one, either an osmotolerant yeast Pichia farinosa 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 production from glucose. In stage two, K. pneumoniae DSM 2026 converted glycerol thus produced to 1,3-PD. Maintenance of a low glucose concentration (20–30 g/L) in stage I along with an increase in

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osmolarity by addition of NaCl resulted in a significant improvement in glycerol yield while simultaneously decreasing the formation of arbitol in stage I. However, a low growth rate of K. pneumoniae was observed in stage II the exact reason for which could not be established. On the contrary, the process with recombinant E. coli and K. pneumoniae in stage one and two, respectively, appeared to be a technically feasible system. It was reported that upon growth of E. coli on relatively low glucose concentration the same produced only glycerol and almost no acetate which had positive effects on 1,3-PD production in stage two. In a recent study Mendes et al. [42] reported the highest values achieved so far for 1,3-PD production using a two-step process. Conversion of sugars—glucose or sugar cane molasses into glycerol by a recombinant substrate tolerant S. cerevisiae followed by its biotransformation into 1,3-PD by a recombinant non-pathogenic C. acetobutylicum DG1 (pSPD5) in the same bioreactor was investigated in this study. With 103 g/L initial glucose concentration, a final 1,3-PD concentration of 25.5 g/L and a 1,3-PD productivity of 0.16 g/L/h could be obtained in this study. The investigators also highlighted the potential use of molasses for glycerol conversion by yeast which gave comparable results as glucose except inhibition of clostridial growth in step two upon use of high sugar concentrations (77.8 and 101.3 g/L) in step one. 3.2.3.6. Mixed cultures. The prospect of using mixed cultures for the production of 1,3-PD has also been explored. The conversion of glycerol into hydrogen gas and 1,3-PD was investigated by anaerobic fermentation using heat-treated mixed cultures from four different sources (tomato soil, wheat soil, compost, and sludge) by Selembo et al. [81]. A comparison of fermentation with glycerol and glucose as substrate showed that though the former yielded 0.69 mol of produced 1,3-PD per mol of consumed glycerol the latter produced more hydrogen (1.06 mol H2 /mol glucose vs 0.28 mol H2 /mol glycerol). Comparable results from biodiesel-derived glycerol (0.31 mol H2 /mol glycerol and 0.59 mol 1,3-PD/mol glycerol) indicated the feasibility of integrating biodiesel production with 1,3-PD and improving the economy of the biodiesel plant. Organic acids such as acetic acid, butyric acid and formic acid are formed as by-products in the fermentation of glycerol to 1,3-PD which inhibit the growth of C. butyricum and deteriorate its ability to biotransform glycerol to 1,3-PD. In an attempt to address this problem, a novel mixed culture of C. butyricum and a methane bacterium Methanosarcina mazei was proposed by Bizukojc et al. [82]. It was believed that since the organic acids particularly acetic and formic acids were utilized efficiently by M. mazei, a combination of C. butyricum and M. mazei could be used as a mixed culture in which the organic acids released by 1,3-PD producer (C. butyricum) could be used by M. mazei for efficient energy production. This would facilitate in relieving the inhibition imposed on the process (by these acids) and thus help in improving the overall biotransformation process in the favour of 1,3-PD production. Two-species metabolic models were proposed and used for examining several cultivation scenarios with respect to the acid-scavenging efficiency of M. mazei. It was reported that maximum methanogenesis and less extensive growth of M. mazei were the best conditions for removal of toxic acids from the system by it. 3.2.3.7. Kinetics of 1,3-PD fermentation and physiological modelling. C. butyricum has been considered as a microorganism of industrial value in the production of 1,3-PD from glycerol particularly because of high fermentation yields and titre and relatively simple fermentation conditions [64]. However, inhibition from both substrate and products reduces the overall growth and product formation rates [29,64]. Thus a thorough understanding of the inhibition kinetics imposed on the process is extremely important to design inhibition-free cultivations in order to achieve maximum

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productivity. Biebl [29] used pH-auxostat to measure product inhibition in glycerol fermentation to 1,3-PD with C. butyricum DSM 5431. It was reported that at pH 6.5 growth was totally inhibited at 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 inhibition. It was revealed that the critical concentrations leading to no growth of the microorganisms were 0.35 g/L for undissociated 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] studied the inhibition imposed by 1,3-PD on C. butyricum and reported that the maximum specific growth rate was inversely proportional to the initial concentration of 1,3-PD and that the strain tolerated higher 1,3-PD concentration during fermentation (81.3 g/L arising out of initial addition and produced during cultivation) than the initial 1,3-PD concentration (68 g/L). This finding by Colin et al. [31] was somewhat supported by a Papanikolaou et al. [6] who reported that the biomass concentration remained almost constant in continuous cultures of C. butyricum F2b even when 1,3-PD concentrations 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 concentrations of glycerol was also tested by González-Pajuelo et al. [62] using C. butyricum VPI 3266. It was demonstrated that inhibition increased from 21% to 62% with increase (20–100 g/L) in concentration of both commercial (87%, w/v) and raw glycerol (92%, w/v). However with raw glycerol 65% (w/v) an inhibition of 86% was observed against 62% inhibition observed in the case of other two glycerol types. Besides the inhibition caused by substrate and product(s) of 1,3-PD fermentation, it is important to investigate the possible inhibitory effects of impurities which are found in raw glycerol derived from biodiesel plant, especially when its valorization to 1,3-PD has already attracted a great deal of attention. This indeed was attempted by Chatzifragkou et al. [83] who examined the impact of salts (NaCl, K2 HPO4 , Na2 HPO4 ), fatty acids (oleic, stearic) and methanol present in raw glycerol on growth and 1,3PD production by C. butyricum VP1718. It was observed that while NaCl had an evident inhibitory effect when present at a glycerol concentration of 4.5% (w/w of glycerol) in 200 mL flasks, no significant inhibition could be seen in bioreactor trials at even higher concentrations (30%, w/w of glycerol). This could be due to better control of environmental conditions in the latter. Additionally phosphoric 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 that the observed inhibition was due to the presence of double bond in fatty acid as no negative impact on growth was found upon addition of stearic acid in the bioreactor. An important constituent of raw glycerol–methanol also did not affect either microbial growth or 1,3-PD production in batch cultures irrespective of its concentration in the medium. Similar results as that of batch cultivation were obtained upon addition of methanol at steady state in continuous culture conditions. Due to inhibition by substrate as well as product it becomes rather difficult and tricky to design fresh nutrient feeding strategies as it features a scenario of cultivation when the culture is either starving or inhibited during the entire cultivation period. The conventional trial and error approach of nutrient feed design for elimination of substrate/product inhibition without the limitation of substrate for high productivity cultivation is laborious, frustrating, time-consuming and inefficient. In such a case, a mathematical model-based approach appears to be intelligent, fast, reliable and productive. It features off-line description of substrate and product inhibition kinetics of cultivation and is sensitive to varying substrate/product concentrations emerging out of feeding of fresh

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Table 3 Optimized values of 1,3-PD model parameters in various literature reports. max (h−1 )a

Ks (g/L)b

0.527 0.39–0.43 0.67 0.71

– – 0.005 0.026

0.65

0.026

a b c d * #

K1,3-PD /X (g/g)c 17.14 – –

Q (g/L/h)d

Organism

Reference No.

6.47 3.4 –

C. butyricum F2b C. butyricum F2b C. butyricum DSM5431 K. pneumoniae DSM2026

[88] [74] [58] [58]

3.78* 8.7#

K. pneumoniae DSM2026 K. pneumoniae DSM2026

[89] [89]

Maximum specific growth rate. Saturation constant for glycerol. 1,3-PD yield (gram per gram dry cell weight). 1,3-PD productivity. Batch cultivation. Continuous cultivation.

nutrient feed. The model has the ability to simulate (on computer) the consequence of feeding of different concentrations of fresh nutrients and their rates which not only eliminates the limitation of substrate but also addresses the problem of inhibition of products [84]. Therefore a number of model simulations can be done on the computer to optimize the right nutrient feeding strategy which would result in “the” highest 1,3-PD concentration. This feeding strategy can then be implemented experimentally. The above methodology was followed in the investigations conducted in our laboratory for glycerol to 1,3-PD fermentation. It was possible to enhance 1,3-PD concentration from 25.8 g/L obtained in batch [39] to 61.2 g/L in model-based fed-batch cultivation conditions. As has been demonstrated by other fermentation metabolite production such as sorbose [85], gibberellic acid [86], poly(␤-hydroxybutyrate) [87], etc. 1,3-PD concentration and/or productivity can be further enhanced by altering different feeding and/or cultivation strategies in fed-batch cultivation (s) e.g., by optimizing the Start/Stop time of nutrient feeding, concentration of substrate in the feed bottle, the feeding profile, etc. The model could be further used to design other strategies such as continuous cultivation, continuous cultivation with cell recycling, etc. to further optimize the fermentation. However the utility of the above mentioned approach for 1,3-PD process optimization lies in the deliberate selection of only those nutrient feeding profiles for bioreactor operation which could be adapted in the industrial conditions without any difficulty. Mathematical models have also been applied by Papanikolaou et al. [88] to predict the kinetic behaviour of C. butyricum F2b for growth on raw glycerol and 1,3-PD production. A Contoistype model was found to fit the experimental data on 1,3-PD production in continuous cultures of C. butyricum with a high value of R2 (97.155). The estimated values of model parameters (obtained by non-regression analysis and Marquardt iterative search algorithm) compared well with the results reported in literature thus confirming the suitability of raw glycerol for 1,3-PD bioconversion. More importantly the maximum value of productivity obtained by the model (6.47 g/L/h) was close to the highest values reported in literature. This proved the potential application of the model for 1,3-PD process optimization. Xiu et al. [89] attempted mathematical-model guided analysis and optimization of glycerol bioconversion in one and two-stage anaerobic cultures of K. pneumoniae DSM2026. It was reported that the highest volumetric 1,3-PD productivity (3.78 g/L/h) in batch culture could be achieved with an initial glycerol concentration of 88.32 g/L and 0.1 g/L inoculum concentration. On the other hand, optimal conditions for continuous fermentation were a D of 0.29 h−1 and feed concentration of 67.252 g/L which could result in twice the 1,3-PD productivity of 8.7 g/L/h as compared to batch. Theoretical analysis revealed that a two-stage process with a higher D in second bioreactor would be favourable for high 1,3-PD production. Optimized

values of 1,3-PD model parameters from various studies have been summarized 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 1,3PD by native 1,3-PD producers, some classical reports on the use of genetically engineered microorganisms capable of producing 1,3-PD either from glycerol or directly from glucose are worth mentioning. The most important and indeed successful examples of the application of metabolic engineering for 1,3-PD production has been the development of an engineered E. coli strain by DuPont and Genencor International, Inc., USA which could directly convert low cost feedstock D-glucose (corn hydrolysate) to 1,3-PD. By an intelligent combination of heterologous gene expression, gene deletion, appropriate control of flux distribution while maintaining the redox and energy balance and modification of substrate uptake mechanism 1,3-PD could be produced at a titre of 135 g/L, a productivity of 3.5 g/L/h and a weight yield of 51% in a 10 L fed-batch fermentation [41]. In yet another attempt with E. coli, 1,3-PD pathway genes (dhaB1 and dhaB2) from C. butyricum and yqhD gene from E. coli were tandemly arrayed under the control of a constitutive, temperature sensitive promoter to construct a novel operon for expression in E. coli K-12 ER2925 [40]. This engineered strain was then used in a novel two-stage fermentation process which involved a shift in temperature from 30 to 42 ◦ C between the two stages for fast growth and efficient production of 1,3-PD from glycerol. It presented a high 1,3-PD concentration of 104.4 g/L and a productivity of 2.6 g/L/h. C. acetobutylicum has been popularly used for solvent production (acetone–butanol–ethanol) by researchers worldwide. However, it could not grow on glycerol due to its inherent inability to regenerate sufficient NADH for the growth and metabolism. It was genetically engineered by González-Pajuelo et al. [90] by the introduction of 1,3-PD pathway from C. butyricum to create a mutant C. acetobutylicum DG1 (pSPD5). The recombinant organism could grow on glycerol and produce 84 g/L 1,3-PD with a yield of 0.65 mol/mol glycerol in fed-batch cultivation. Studies with continuous cultures demonstrated a higher concentration and volumetric productivity of 54.73 g/L and 3 g/L/h, respectively, in comparison to that obtained from C. butyricum. 3.3. Downstream processing of biotechnologically produced 1,3-PD The recovery of 1,3-PD from complex fermentation broth represents a true bottleneck in the development of a commercially viable bioprocess. This could be mainly attributed to its high boiling point and presence of two hydroxyl groups which make it strongly hydrophilic and therefore complicate its extraction. Nonetheless various methods have been applied for separation of 1,3-PD from the fermentation broth (Table 4). The application of

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115

Table 4 Different downstream processing methods for 1,3-PD and associated problems. Reference No.

Separation method

Problems

[91,92]

Evaporation, Vacuum distillation

[94] [95]

Ion-exclusion using polystyrene sulphate in Na form Ion-exclusion using charcoal column and acidic cation exchange polystyrene resin Process chromatography Cyclic sorption and desorption by zeolite Liquid–liquid extraction Reactive extraction Reactive extraction Aqueous two-phase extraction

Requirement of large amounts of energy, desalination, low product yield Requirement of energy due to dilution of 1,3-PD in broth Requirement of energy due to dilution of 1,3-PD in broth

[96] [97] [98] [100] [93] [101] [102] [103]

Aqueous two-phase extraction Ultrafiltration, activated charcoal, vacuum distillation, chromatography

relatively simple approaches of evaporation and vacuum distillation for the recovery of 1,3-PD [91,92] has been attempted but appeared unattractive and uneconomical due to the requirement of large amounts of energy, desalination and low product yield. The use of ion exclusion for extraction of 1,3-PD has been reported. The method consisted of desalination of the fermented broth by the aid of strongly cationic and weakly basic anionic resins with subsequent passage over a cationic exchange resin for purification of 1,3-PD [93]. Chromatographic column packed with cation exchange resin was used by Hilaly and Binder [94] and Roturier et al. [95] for the recovery of 1,3-PD. While the use of polystyrene sulfonate in Na form was contrived by Hilaly and Binder [94] to separate 1,3-PD from impurities the latter used a charcoal column and strong acidic cation exchange resin of polystyrenesulfonic acid type to produce a protein-free broth which contained 90.5 g/L 1,3-PD. However both the aforementioned methods diluted 1,3-PD in the broth thereby demanding more energy than simple conventional processes. Besides this, the prospect of using process chromatography to avert feedback inhibition of cell growth and product formation has been investigated by Wilkins and Lowe [96] for in situ removal of 1,3-PD. Corbin and Norton [97] disclosed a different approach involving on-line separation using cyclic sorption and desorption of the desired product 1,3-PD by a resin zeolite. The method entailed an indispensible dewatering step and a high chance of contamination (due to uninterrupted link between the bioreactor and the separation equipment). Liquid-liquid extraction employing a suitable solvent could be used for energy-efficient separation of the metabolite of interest from a dilute solution and therefore has been attempted for purification of 1,3-PD as well. Malinowski [98] experimented with liquid–liquid extraction to establish its utility for the recovery of 1,3-PD. A thorough verification revealed that it was probably not an efficient method owing to the low partition coefficient of 1,3-PD into organic solvents. The unavoidable requirement of addition of large amounts of solvent for the separation of 1,3-PD again made the process undesirable [99]. An approach which has been investigated quite a lot as a possible solution to the problems resulting from the hydrophilic nature of 1,3-PD was reactive extraction. The method involved a reaction between 1,3-PD and acetaldehyde catalyzed by a Dowex or Amberlite ion-exchange resin to give 2-methyl-1,3-dioxane (2MD). This was followed by extraction using an organic solvent such as toluene [100] and conversion to 1,3-PD by hydrolyzation. An overall 1,3-PD conversion yield of 98% could be achieved by this method. However the acetaldehyde used in this method could also react with other substances in the fermentation broth like ethanol, glycerol, etc. The possibility of this reaction reduced the specificity of this method for 1,3-PD only. An improvement in aforementioned method was therefore

– Requirement of dewatering step, high chance of contamination Requirement of large amount of solvent Non-specificity of reaction Requirement of electrodialysis for desalination of broth Requirement of large amounts of methanol, difficulty of separation of two alcohols – –

attempted by Hao et al. [93] by using other aldehydes like propionaldehyde, butyraldehyde, etc., for reaction with 1,3-PD to form highly hydrophobic acetals. These acetals being miscible with the aldehydes enabled their use both as reactant and extractant. However, an indispensible need for electrodialysis to desalinate the broth made this method unattractive. Recently the use of aqueous two-phase systems (ATPS) for efficient extraction of 1,3-PD from complex broth has been investigated. An ATPS composed of 46% (v/v) ethanol and saturated ammonium sulphate was found to give a high partition coefficient of 4.77 and 93.7% recovery of 1,3-PD with simultaneous extraction of 2,3-BD, acetoin, residual glycerol and removal of cells and proteins from the broth [101]. High extraction efficiency of this ATPS could be attributed to high valent charge of the anion and strong polarity of the solvent. However requirement of large amounts of methanol for salt removal and difficulty of separation of two alcohols restricted its industrial application. Therefore another ATPS composed of methanol/phosphate where methanol could be effectively used both as extractant (for 1,3-PD) and solvent (for crystallizing the salt) was suggested and studied in detail by the Li et al. [102]. A higher partition coefficient (38.3) of the system composed of 35% (v/v) methanol, saturated concentration of phosphate and pH 10.7 could give a recovery of 98.1% 1,3-PD. Efficient and easy removal of salt (94.7%) by just adjusting the pH to 4.5 followed by addition of 1.5 volume of methanol could be achieved by this ATPS. A novel method of downstream processing of 1,3-PD has been recently developed which involves the purification of 1,3-PD in three simple steps: [103] removal of biomass and proteins by the use of microfiltration and activated charcoal, respectively, concentration of the broth by vacuum distillation followed by separation of 1,3-PD by chromatography. The authors have reported an overall 1,3-PD yield of 75.47% by using this protocol [104]. 4. Concluding remarks The microbial production of 1,3-PD is an exciting method of valorizing ‘waste’ glycerol from the biodiesel plant and thus producing an industrially important raw material by biological route while minimizing the dependence on rapidly disappearing fossil fuels. The economic biological production of 1,3-PD needs to be addressed urgently. Enormous industrial applications of 1,3-PD particularly in textiles and cosmetics and its production from a renewable source present it as an eco-friendly and economically feasible process. One of the major factors governing the economic viability of any bioprocess is the cost of the starting material, which in this case is glycerol. The escalating global energy demands and a predilection for clean, biodegradable and inexhaustible fuels is

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expected to increase the already growing biofuel industry manifold in times to come. This would mean greater availability of ‘waste’ glycerol for conversion to high valued products. Though European Union leads the world in biodiesel production, it is not the only one with such tremendous production capacity. In fact biodiesel production is already manifested by developing countries such as Brazil, Argentina and China. Skyrocketing crude oil prices (crude oil priced at USD 116.32 per barrel) (Ref 2011), huge amounts of crude glycerol generated as ‘waste’ from the biodiesel plants throughout the globe and therefore further decline in glycerol prices is expected to make the glycerol-based 1,3-PD production cost competitive with the petrochemically produced 1,3-PD and also with sugar-based 1,3-PD production. The use of bioprocess engineering strategies such as fed-batch, continuous cultivation with/without cell recycling, mixed cultures and immobilized cells and/or enzymes deserve further research and development efforts. Besides this, the application of mathematical modelling for designing various reactor operating strategies for improved concentration and/or productivity of 1,3-PD is a simple yet attractive and result-yielding approach. It is particularly interesting considering the various constraints such as substrate limitation and substrate/product inhibition imposed on 1,3-PD process. Therefore it is believed that with the development of appropriate bioprocess engineering strategies and availability of lower-priced glycerol in the near future, the production of biobased 1,3-PD using native strains would prove both economical and productive. Acknowledgement The Senior Research Fellowship (SRF) award by Indian Council of Medical Research (ICMR), Govt. of India, New Delhi for the execution of the project is gratefully acknowledged by one of the authors (Ms. Guneet Kaur). References [1] H. Fukuda, A. Kondo, S. Tamalampudi, Bioenergy: sustainable fuels from biomass by yeast and fungal whole-cell biocatalysts, Biochem. Eng. J. 44 (2009) 2–12. [2] V.E.T. Maervoet, M.D. Mey, J. Beauprez, S.D. Maeseneire, W.K. Soetaert, Enhancing the microbial conversion of glycerol to 1,3-propanediol using metabolic engineering, Org. Process. Res. Dev. 15 (2011) 189–202. [3] V.F. Wendisch, S.N. Lindner, T.M. Meiswinkel, Use of glycerol in biotechnological applications, in: G. Montero, M. Margarita (Eds.), Biodiesel-Quality, Emissions and By-Products, InTech, 2011, pp. 305–340. [4] Y. Xu, H. Liu, W. Du, Y. Sun, X. Ou, D. Liu, Integrated production for biodiesel and 1,3-propanediol with lipase-catalyzed transesterification and fermentation, Biotechnol. Lett. 31 (2009) 1335–1341. [5] G.P. Da Silva, M. Mack, J. Contiero, Glycerol: a promising and abundant carbon source for industrial microbiology, Biotechnol. Adv. 27 (2009) 30–39. [6] S. Papanikolaou, S. Fakas, M. Fick, I. Chevalot, M. Galiotou-Panayotou, M. Komaitis, I. Marc, G. Aggelis, Biotechnological valorization of raw glycerol discharged after bio-diesel (fatty acid methyl esters) manufacturing process: production of 1,3-propanediol, citric acid and single cell oil, Biomass Bioenerg. 32 (2008) 60–71. [7] A. Vlysidis, M. Binns, C. Webb, C. Theodoropoulos, Glycerol utilisation for the production of chemicals: conversion to succinic acid a combined experimental and computational study, Biochem. Eng. J. 58–59 (2011) 1–11. [8] A. Chatzifragkou, A. Makri, A. Belka, S. Bellou, M. Mavrou, M. Mastoridou, P. Mystrioti, G. Onjaro, G. Agellis, S. Papanikolaou, Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species, Energy 36 (2011) 1097–1108. [9] H. Liu, Y. Xu, Z. Zheng, D. Liu, 1,3-propanediol and its copolymers: research, development and industrialization, Biotechnol. J. 5 (2011) 1137–1148. [10] T.M. Carole, J. Pellegrino, M.D. Paster, Opportunities in the industrial biobased products industry, Appl. Biochem. Biotechnol. 113–116 (2004) 871–885. [11] P. Millet, Retournement de la situation de la glyce rine, Inform. Chim. 345 (1993) 102–104. [12] C.J. Sullivan, Propanediols Ullmann’s Encyclopedia of Industrial Chemistry, vol. A22, VCH, Weinheim, 1993, pp. 163–171. [13] J.V. Kurian, A new polymer platform for the future-SoronaR from corn-derived 1,3-propanediol, J. Polym. Environ. 13 (2005) 159–167. [14] Shell Chemical Company Shell Chemical Company announces commercialization of new polymer (Press Release), 1995.

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