Biotechnological Conversion of Glycerol from Biofuels to 1,3-propanediol Using Escherichia Coli

In the face of shortage of fossil fuel supplies and climate warming triggered by excessive carbon dioxide emission , alternative resources for chemical industry have gained considerable attention. Renewable resources and their derivatives are of particular interest. Glycerol, which constitutes one of the by-products during bio-diesel production, is such a substrate. Thus, generated excess glycerol may become an environmental problem, since it cannot be disposed of in the environment. The most promising products obtained from glycerol are polyols, including 1,3-propanediol, an important sub-strate in the production of synthetic materials, e.g. pol-yurethanes, unsaturated polyesters, and epoxy resins. Glycerol can be used as a carbon and energy source for microbial growth in industrial microbiology to produce 1,3-propanediol. This paper is a review of metabolic pathways of native producers and E. coli with the acquired ability to produce the diol via genetic manipulations. Culture conditions during 1,3-PDO production and genetic modifications of E. coli used in order to increase efficiency of glycerol bioconversion are also described in this paper. Currently, the primary trend in the fuel and chemical sector is to produce biofuels and chemical compounds using renewable energy sources. Biodiesel is a biofuel produced from vegetable oils or animal fats used to power compression-ignition (Diesel) engines as an ad-mixture or complete fuel. Biofuels are renewable energy sources, in contrast to fossil fuels such as crude oil, natural gas or coal. The increasing oil prices and the fact that it is a non-renewable energy source have resulted in the need to use alternative energy sources. Biodiesel is a biodegradable fuel, which considerably reduces emissions of pollutants and toxins to the atmosphere and the natural environment, being its advantage over regular diesel oil, which combustion generates considerable emissions of these substances (Xu et al., 2003; Chen et al., 2008). It is formed as a result of transesterification of triglycerides (TG), mainly methanol (González-Pajuelo et al., 2004; Mu et al., 2006). At present, the greatest practical importance as a raw material in its production is attributed to rapeseed oil (Poland) and soybean oil (Brazil). For many years the European Union has been the primary producer of biodiesel worldwide (Demirbas & Balat, 2006). In several countries of Western Europe (Austria, France, Germany) and in Scandinavia methyl esters of fatty acids have been used for a long time (Fig. 1). Apart from methyl esters in the production of bio-diesel the glycerol fraction is also produced, which depending …

Currently, the primary trend in the fuel and chemical sector is to produce biofuels and chemical compounds using renewable energy sources.Biodiesel is a biofuel produced from vegetable oils or animal fats used to power compression-ignition (Diesel) engines as an admixture or complete fuel.Biofuels are renewable energy sources, in contrast to fossil fuels such as crude oil, natural gas or coal.The increasing oil prices and the fact that it is a non-renewable energy source have resulted in the need to use alternative energy sources.Biodiesel is a biodegradable fuel, which considerably reduces emissions of pollutants and toxins to the atmosphere and the natural environment, being its advantage over regular diesel oil, which combustion generates considerable emissions of these substances (Xu et al., 2003;Chen et al., 2008).It is formed as a result of transesterification of triglycerides (TG), mainly methanol (González-Pajuelo et al., 2004;Mu et al., 2006).At present, the greatest practical importance as a raw material in its production is attributed to rapeseed oil (Poland) and soybean oil (Brazil).For many years the European Union has been the primary producer of biodiesel worldwide (Demirbas & Balat, 2006).In several countries of Western Europe (Austria, France, Germany) and in Scandinavia methyl esters of fatty acids have been used for a long time (Fig. 1).
Data for the years 2015, 2020 -the estimate by OECD-FAO; Source: Organisation for Economic Co-operation and Development -Food and Agriculture Organization, Agricultural Outlook 2011-2020, OECD-FAO 2011.Apart from methyl esters in the production of biodiesel the glycerol fraction is also produced, which depending on production parameters and the used substrate varies in terms of pure glycerol contents (Papanikolaou et al., 2008).The components found in the glycerol fraction, i.e. free fatty acids, methanol, catalyst, soaps and salts, make it useless for the pharmaceutical or cosmetics industries.The glycerol fraction accounts for 10% produces ester, which means that from 1 ton produced biodiesel we obtain 100 kg waste glycerol.Excessive amounts of this waste have led to a decrease in glycerol prices (Yazdani et al., 2007;Zhang, 2011;Yang et al., 2012).Waste glycerol from the production of biofuels has rapidly become a competitive raw material in relation to sugars in the production of valuable chemicals, including 1,3-propanediol, in the biotechnological sector (Dharmadi et al., 2006).The use of waste glycerol, being a derivative of a renewable energy source (e.g.longer-chain fatty acids of plant origin), as a source of carbon and energy for biotechnological processes would reduce production costs of biodiesel and would contribute to an increased share of renewable energy in the production of the desirable and extensive applications of chemical compounds (Ragauskas et al., 2006).In the past, glucose was mainly used in fermentation processes due to its lower price in relation to glycerol, while at present excess waste glycerol has contributed to greater profitability of processes with its application (Nakamura & Whited, 2003).
1,3-propanediol is produced mainly by chemical methods.However, it is possible to produce this compound using microorganisms in the fermentation processes of numerous carbon sources, e.g.glycerol.Pure glycerol does not pose a hazard and is extensively used in different branches of industry, while glycerol from the production of biofuels, containing numerous contaminants, is a noxious waste (Dharmadi et al., 2006).Purification of waste glycerol is not profitable and its potential conversion to a valuable compound contributes to the solution of the problem of its bothersome management.Production of 1,3-PDO from waste glycerol is cheaper than the chemical method used to generate this compound (Rehman et al., 2008).Results of studies concerning the effect of waste glycerol on bacterial growth and 1,3-PDO production show that there is no considerable difference in comparison to pure glycerol (Papanikolaou et al., 2004).The potential to use non-purified waste glycerol from biofuel production to synthesize 1,3-PDO is a tremendous advantage of this process.This solution meets economic, ecological, social and ethical requirements imposed on contemporary biotechnology in the production of important molecules.There are "natural producers" of 1,3-PDO, as well as heterologous hosts of the 1,3-PDO synthesis pathway (Homann et al., 1990;Gonzalez-Pajuelo et al., 2006).Both the former and the latter are used in studies on the production of 1,3-PDO.Generation of increasingly efficient bacterial strains, optimization of glycerol conversion and 1,3-PDO purification will facilitate future advances in valuable technologies applying live organisms.

CHARACTERISTICS OF 1,3-PROPANEDIOL
1,3-propanediol (1,3-PDO), also referred to as trimethylene glycol, is an organic compound belonging to the group of diols, with the formula C 3 H 8 O 2 (Fig. 2), molar mass of 76.09 g/mol and density of 1.0597 g/ cm 3 .Under normal conditions it is a colorless liquid, readily soluble in water.1,3-propanediol has many valuable properties, thanks to which for many years it has found many applications in different branches of industry (Igari et al., 2000).1,3-propanediol is mainly used as a monomer in the synthesis of biodegradable polyesters, polyurethanes and polyethers in the chemical industry, providing them with greater strength, while it is also contained in various products of the cosmetics, textile and medical industries.Countries of eastern and central Europe such as Poland, Turkey and Russia are those areas in this continent, which use and produce increasing amounts of polyurethanes (analysts in the team from IAL Consultants estimated that the present use in Europe, the Middle East and Africa is over 5 million ton annually).This compound provides specific properties to solvents, resins, laminates, detergents and cosmetics.1,3-PDO is used to produce polymers applied in bulletproof vests, and as a biocide (e.g.2-nitro-1,3-PDO, a commercially available biocide protecting against contaminants) (Menzel et al., 1997;Zeng & Biebl, 2002;Zhu et al., 2002;Cheng et al., 2007;Sauer et al., 2008).It has been known for over 120 years that 1,3-propanediol is a product formed during microbial fermentation of glycerol.The molecule was identified for the first time in 1881 by a chemist, August Freund, as being a product of glycerol fermentation by Clostridium pasteurianum (Nakas et al., 1983;Biebl et al., 1999).In turn, in 1914 Voisenet described the production of 1,3-propanediol by Bacillus ssp.also in the fermentation process.In 1928 researchers from Delft University of Technology initiated studies on the production of 1,3-propanediol using Enterobacteriaceae, continued later by a team of Mickelson and Werkman, researchers from Iowa State University of Science and Technology, Ames, Iowa (USA).In the 1990's advances were made in biotechnological 1,3-PDO production from glucose and glycerol as well as chemical 1,3-PDO production methods (Katrlík et al., 2007;Drożdżyńska et al., 2011;Leja et al., 2011).A breakthrough in the use of 1,3-PDO was connected with commercialization in 1995 by the British-Dutch petrochemical company Shell, of a new aromatic polyester Corterra ® PTT poly(trimethylene terephthalate) based on 1,3-PDO (Shell Chemical Company Press release, 1995).This polymer is more durable, has greater tensile strength, is more elastic than polyester based on ethylene or butylene glycol, or 1,2-propanediol (1,2-PDO) (Chuah et al., 1995;Kurian, 2005;Liu et al., 2010).It is used as a fiber in the production of textiles and carpets (Chuah, 1996;Brown & Chuah, 1997).In 2000 one of the biggest multinational chemical companies, DuPont, the inventor of e.g.nylon and teflon, commercialized poly(trimethylene terephthalate) Sorona ® based on 1,3-propanediol produced in the fermentation process (Chotani et al., 2000;EMBO reports. White Biotechnology, 2003;Nakamura & Whited, 2003).It was one of the first advances on the world biotechnological market, applying a genetically modified Escherichia coli K-12 strain and a renewable energy source, i.e. corn glucose.
At present, there are two processes of chemical 1,3-PDO synthesis.DuPont uses acrylaldehyde to synthesize 1,3-PDO, while Shell uses ethylene oxide (Fig. 3).Acrylaldehyde is hydrated, then hydrogenated at high pressure in the presence of a catalyst, while in the case of ethylene oxide hydratation is preceded by hydroformylation (Cameron et al., 1998;Hao et al., 2006).Considering the fact that ethylene oxide is cheaper than acrylaldehyde, the chemical method used by Shell is economically more advantageous.However, obvious drawbacks of both these processes are related with the formed toxic intermediate products, the need to apply high pressure, high temperature, expensive catalysts, as well as moderate process efficiency and environmental noxiousness (Raynaud et al., 2003;Lin et al., 2005).For this reason starting from the 1990's attempts have been made to find biotechnological methods to produce 1,3-PDO from alternative energy sources (e.g. from waste glycerol generated by the production of biofuels) (Xiu et al., 2007;Yang et al., 2007).Already in the 20 th century the primary method to obtain specialist chemical compounds, fuels, raw materials for heavy organic synthesis or plastics was a chemical method, in which crude oil or its derivatives were processed (rectified).Since it constitutes a non-renewable energy source and additionally its processing is connected with generation of environmental pollutants, chemical companies started to search for eco-friendly solutions applying e.g.glucose or glycerol as the energy sources.Obviously, it is promoted by the advances made in genetics, biotechnology and metabolic engineering of microorganisms (Ragauskas et al., 2006).According to Festel Capital, the global share of biotechnological processes in the production of chemicals being substrates for the synthesis of polymers in 2015 will amount to 15% (www.festel.com),which in comparison to 2004 (3%) indicates the increasing importance of biotechnology of microorganisms and fermentation processes in the global industry, thus clearly indicating the direction for the world's development.

PRODUCTION OF 1,3-PDO FROM GLYCEROL USING BIOLOGICAL METHODS
In the last two decades progress has been made in biological methods of 1,3-PDO production from cheap renewable energy sources using newly developed strains in 1,3-PDO production (Deckwer, 1995;Zhu et al., 2002).Glycerol, analogous to glucose, is the source of carbon (production of biomass) and energy for microorganisms.Because of the greater degree of glycerol reduction in relation to glucose, its bioconversion to valuable chemical compounds is more efficient than bioconversion of a glucose (Dharmadi et al., 2006).1,3-PDO is a product characteristic of glycerol fermentation only and is not obtained from conversion of any other organic compounds (Lin, 1976).The greater the availability of reduced coenzymes for reduction, the greater is the production of 1,3-PDO, which is not metabolized, but instead, it is accumulated in the culture medium (Johnson & Lin, 1987).The physiological role of 1,3-PDO synthesis in glycerol metabolism consists in the regeneration of a coenzyme NAD + from the reduced form produced during the oxidative pathway, in which glycerol is first oxidized to dihydroxyacetone (DHA), followed by the phosphorylation and the resulting production of dihydroxyacetone phosphate (DHAP), which is subsequently transformed to pyruvate (Zhu et al., 2002;Forage & Lin, 1982) (Fig. 4).Commercialization of 1,3-PDO-based polymer produced by the biotechnological method by DuPont shows that there is a considerable demand for such product.This concept is realized by the research teams worldwide and subjected to competition in terms of scientific reports.Synthesis of 1,3-propanediol makes it possible for cells to reach the redox equilibrium using reduced coenzymes, formed during biomass production (Gonzalez et al., 2008).
cillus brevis and Lactobacillus buchnerii (Schutz & Radler, 1984), that have in their glycerol metabolizing pathway a branch, in which 1,3-PDO is formed in the process of fermentation.Citrobacter freundii and Klebsiella pneumoniae are good 1,3-PDO producers, but they are classified as the microbial risk group 2. For this reason protection of the production process is connected with the additional costs.In turn, C. butyricum is an extensively used organism in 1,3-PDO production, but the absolute anaerobic conditions required by that microorganism prevent its large-scale application (Willke & Vorlop, 2008).Despite being a potential pathogen.K. pneumoniae is the most frequently used microorganism in 1,3-PDO production.Efficiency of 1,3-PDO production is dependent on the applied K. pneumoniae strain (Huang et al., 2002).Production of 1,3-PDO from glycerol is run most typically under the anaerobic conditions, although some strains from the genera Klebsiella, Citrobacter or Enterobacter synthesize 1,3-PDO also in the microaerobic and aerobic fermentation (Biebl et al., 1998).Most natural 1,3-PDO producers metabolize glycerol in two pathways: redox and oxidative (Zhu et al., 2002).1,3-PDO is formed in the redox pathway.In the first stage of the redox pathway glycerol is dehydrated as a result of the action of the B 12 coenzyme dependent glycerol dehydratase (GDHt), at the formation of 3-hydroxypropionaldehyde (3-HPA) (Figs. 4, 5) (Knietsch et al., 2003).Next, the NADH+ H + -dependent 1,3-PDO dehydrogenase (1,3-PD DH, 1,3-PDO PDOR-oxidoreductase) reduces 3-HPA to 1,3-PDO, regenerating NAD + (Ahrens et al., 1998;Skraly et al., 1998).In the oxidative pathway NAD + -dependent glycerol dehydrogenase (GDH) catalyzes reactions of glycerol oxidation to dihydroxyacetone (DHA), which is then phosphorylated in the presence of the glycolytic enzyme ATP-dependent dihydroxyacetone kinase (DHAK) (Forage & Lin, 1982;Daniel et al., 1995).Reduced co-enzymes formed in the oxidative pathway are required for the redox pathways, in which 1,3-PDO is synthesized together with other products of the glycerol metabolism.DHAP is further transformed in the process of glycolysis and the pentose phosphate pathway.During fermentation of glycerol from DHAP pyruvate is formed, which in Enterobacteriaceae is metabolized to acetyl coenzyme A or acetyl lactate (Biebl et al., 1999).The primary end products formed during glycerol fermentation in Enterobacteriaceae include 1,3-PDO and acetate, although several other organic compounds such as formate, succinate, butanol, ethanol or 2,3-butanediol are also formed.Concentrations of end products to a considerable degree depend on the culture conditions and the applied bacterial strain (Homann et al., 1990;Barbirato et al., 1998).Catabolism of glycerol in natural 1,3-PDO producers is presented in Figs. 4  and 5.At the application of natural producers the efficiency of 1,3-PDO synthesis from glycerol ranges from approx.0.51 to 0.64 (mol 1,3-PDO /mol glycerol ) depending on the concentration and quality of used glycerol (Zhang et al., 2007).Zhang et al. (2008) investigated the effect of changing conditions on values of metabolic fluxes within the main nodes in glycerol metabolism based on K. pneumoniae.The distribution of metabolic fluxes within glycerol and dihydroxyacetone phosphate proved to be of low sensitivity to changes in external conditions, while the researchers showed that changes in the activity of enzymes involved in the metabolism of pyruvate and acetyl-CoA may contribute to the increase in 1,3-PDO production.
In K. pneumoniae and C. freundii genes encoding functionally interacting enzymes: glycerol dehydratase (dhaB), 1,3-propanediol dehydrogenase (dhaT), glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) are contained in the dha regulon (Forage & Lin, 1982;Zhu et al., 2002) (Fig. 6).The dha regulon is induced by the presence of dihydroxyacetone as well as glycerol (Tong et al., 1991).Glycerol dehydratase (GDHt), which in K. pneumoniae and C. freundii require for its activity the presence of the B 12 co-enzyme, facilitated dehydratation of glycerol with the formation of 3-HPA (Sun et al., 2003).In the presence of glycerol the B 12 co-enzyme is irreversibly degraded, thus it is necessary to exchange the inactive vitamin B 12 with its active form in the presence of ATP and Mg 2+ , which requires the action of the twosubunit factor reactivating glycerol dehydratase (Honda et al., 1980;Kajiura et al., 2007).

Examples of 1,3-PDO production by natural producers and modified natural producers
Mu and co-workers used a strain of K. pneumoniae DSM 2026 in the process of glycerol fermentation yielding 51.3-53 g/L of 1,3-PDO (Mu et al., 2006).In turn, a research team headed by Yang et al. (2007) obtained over 80 g/L diol with an efficiency of 0.62 mol 1,3-PDO / mol glycerol using a modified strain of K. oxytoca (the knockout gene of lactate dehydrogenase).A wild K. pneumoniae strain M5a1 used by a team headed by Cheng produced 58.8 g 1,3-PDO with a yield of 0.53 mol 1,3-PDO /mol glycerol (Cheng et al., 2007).Efficiency of 1,3-PDO production depends on the type of fermentation, used glycerol, applied strain, culture conditions and accumulation of metabolites.Example yields and productivity of bacterial strains depending on the type of fermentation towards 1,3-PDO are given in Table 1.

Synthesis of 1,3-PDO under microaerobic and aerobic conditions
It was commonly assumed that the synthesis of 1,3-PDO in the process of glycerol fermentation may only occur under anaerobic conditions.However, many research teams showed that it may be run under microaerobic as well as aerobic conditions (Huang et al., 2002;Ma et al., 2009).Researchers from China in their study presented 1,3-PDO production by K. pneumoniae under Arrows determine approximate length and direction of transcription for respective genes.Legend: dhaK, dhaL, dhaM, medium, small and large subunits of dihydroxyacetone kinase; dhaD,glycerol dehydrogenase; dhaR, transcription regulator; orfW, unknown product; dhaG, small subunit of the glycerol dehydratase reactivating factor; dhaT, 1,3-propanediol dehydrogenase (1,3-propanediol oxidoreductase); orfY, unknown product; dhaB, large subunit of glycerol dehydratase; dhaC, medium subunit of glycerol dehydratase; dhaE, small subunit of glycerol dehydratase; dhaF, large subunit of glycerol dehydratase reactivating factor; orf2c, unknown product; orf2a, unknown produkt; gdrB, small subunit of dehydratase glycerol reactivating factor; gdrA, large subunit of glycerol dehydratase reactivating factor; gldA, large subunit of glycerol dehydratase; gldB, medium subunit of glycerol dehydratase; gldC, small subunit of glycerol dehydratase; dhaS and dhaA, two-component signal transduction systemprobably sensor protein and response regulator, respectively (role analogous to dhaR); dhaB1, B 12 -independent glycerol dehydratase; dhaB2, protein activator; orf6 and orf7, probably antioxidant proteins.The figure was prepared based on (Seifert et al., 2001;Raynaud et al., 2003;Kubiak et al., 2012) and the NCBI database: GenBank U30903.1.varying aerobic conditions, obtaining e.g. in fed-batch culture the yield of 0.56 mol 1,3-PDO /mol glycerol (Ma et al., 2009).The team headed by Cheng presented results of anaerobic-aerobic strategy of 1,3-PDO production, receiving 70 g/L diol (Cheng et al., 2004).A report by Hao et al. (2008) also showed potential production of 1,3-PDO in Klebsiella under aerobic conditions.Thanks to the above, it is possible to reduce production costs as well as minimize accumulation of a toxic metabolite, 3-hydroxypropionaldehyde (3-HPA).In another study Hao et al. (2008a) reported an efficient 1,3-PDO production by strains of Citrobacter and Klebsiella under aerobic conditions, with one strain of K. pneumoniae producing as much as 60.1 g/L 1,3-PDO from glycerol.Reports may also be found in literature on 1,3-PDO production by modified strains of E. coli in the presence of oxygen.Under aerobic conditions E. coli modified with genes from Clostridium butyricum produced 3.7 g/L 1,3-PDO with a yield of 0.3 g 1,3-PDO /1 g glycerol (Dąbrowski et al., 2012).

E. coli as a host of heterologous 1,3-PDO synthesis pathway
Organisms, which in their glycerol metabolism do not have the 1,3-PDO synthesis pathway, include e.g.Escherichia coli, Saccharomyces cerevisiae and Clostridium acetobutylicum.Escherichia coli is a Gram-negative, facultative anaerobic bacterium belonging to the family Enterobacteriaceae, owing their name to their discoverer, an Austrian pediatrician and bacteriologist Theodor Escherich.At present, among bacterial systems it is one of the most extensively applied systems for the production of heterologous proteins.Escherichia coli is a model organism in today's experiments in genetic and metabolic engineering.It constitutes the best known and the cheapest system to test new gene constructs and in attempts at production of proteins and enzymes in bench scale and commercial scale systems.Its application in commercial projects is undisputable.Obviously, the most significant advantages of bacteria E. coli include a high degree of genome characterization (defined transcription and translation systems), availability of a broad spectrum of vectors and strains, as well as capacity of rapid growth on cheap culture media (successive generations are produced very fast) using at the same time simple selection factors, mainly antibiotics.Escherichia coli is a non-pathogenic species, for which culture methods are already well-established.Human albumin, growth hormones as well as enzymes extensively used in molecular biology are successfully produced in E. coli cells.Potential applicability of the above mentioned properties of this host facilitates modification, use of genetic engineering methods, glycerol metabolism in E. coli cells leading to the acquisition of capacity to produce 1,3-PDO.

Glycerol metabolism in E. coli
In E. coli glycerol is transported to cells by facilitated diffusion, where the critical role is played by integral membrane protein of GlpF (Voegele et al., 1993;Lu et al., 2003).Only eight bacterial genera from the family Enterobacteriaceae (e.g.Citrobacter and Klebsiella) may ferment glycerol and at the same time produce 1,3-PDO, as they are equipped with glycerol dehydrogenase I and 1,3-PDO dehydrogenase (Bouvet et al., 1995).The capacity to synthesize 1,3-PDO has not been identified in E. coli and glycerol metabolism itself was, as it had been assumed before, dependent on the availability of external electron acceptors (Bouvet et al., 1994;Booth, 2005).Glycerol metabolism under aerobic conditions (or the presence of other electron acceptors, e.g.fumarate) occurs in the presence of glycerol transporter encoded by gene glpF, glycerol kinase (GK) and two glycerol-3-phosphate G-3-P dehydrogenases (G3PDHs) (Schryvers & Weiner, 1982;Borgnia & Agre, 2001;Walz et al., 2002;Booth, 2005) (Fig. 7).In the presence of oxygen active dehydrogenase, a-G3PDH (a-aerobic), encoded by the glpD gene, is required for glycerol metabolism, while in the presence of fumarate an-G3PDH dehydrogenase (ananaerobic), encoded by gene glpABC, catalyses conversion of glycerol-3-phosphate (G-3-P) to DHAP (Fig. 7).At a lack of oxygen or another electron acceptor (e.g.fumarate) the above mentioned dehydrogenases were not active, which contributed to the accumulation of G-3-P in amounts inhibiting bacterial growth, thanks to which it was stated that E. coli are not capable of glycerol fermentation (Booth, 2005).However, further studies concerning glycerol metabolism in E. coli conducted by Dharmadi et al. (2006) showed that E. coli may ferment glycerol when external electron acceptors are not available, and that it is capable of synthesizing 1,2-propanediol (1,2-PDO) (Gonzalez et al., 2008).Earlier attempts at observing glycerol fermentation in E. coli under anaerobic conditions failed due to culture conditions and culture medium composition, which had a negative effect on glycerol fermentation (e.g.pH, concentrations of glycerol, phosphate, potassium, accumulation of H 2 ) at pH 7.5 (Gonzalez et al., 2008).Strains devoid of genes encoding aerobic and anaerobic glycerol-3-phosphate dehydrogenase, respectively, were capable of fermenting glycerol, which indicated the existence of an alternative pathway metabolizing glycerol at the lack of electron acceptors (Murarka et al., 2008).
The glycerol metabolism pathway, including biosynthesis of 1,2-propanediol from glycerol in E. coli, was described by Gonzalez et al. (2008).The glycerol metabolism pathway in E. coli was presented in Fig. 7.In order to identify key enzymes of glycerol metabolism in E. coli, the 1,2-propanediol synthesis pathway and external conditions significantly affecting this process, Gonzalez et al. (2008) and Murarka et al. (2008), applied several genetic and biochemical approaches.Gonzalez et al. (2008) has demonstrated the effect of the gene encoding glycerol dehydrogenase type II (GlyDH-II; GldA) in E. coli on glycerol metabolism under anaerobic conditions.Glycerol dehydrogenase type II transforms glycerol to dihydroxyacetone (DHA), as well as hydroxyacetone (HA) to 1,2-propanediol (1,2-PDO) (Gonzalez et al., 2008).Next, DHA in E. coli undergoes phosphorylation catalyzed by PEP-dependent dihydroxyacetone kinase DHAK (Paulsen et al., 2000) and it is not released to the culture medium.A study by Gonzalez describes a model of glycerol fermentation in E. coli, which includes synthesis of 1,2-PDO (utilization of reduced coenzymes) and synthesis of ethanol (generation of ATP in substrate phosphorylation).The presented model is composed of the oxidative pathway comprising genes encoding glycerol dehydrogenase type II and dihydroxyacetone kinase (DHAK) leading to dihydroxyacetone phosphate (DHAP) (an intermediate product of glycolysis) and the redox pathway using first of all the activity of methylglyoxal synthase (MGS), aldehyde reductase (AOR) and glycerol dehydrogenase type II, with DHAP as the substrate, leading to the synthesis of 1,2-propanediol (Fig. 7).The pathways leading to 1,2-PDO and ethanol, and other minor products obtained from the reduction of pyruvate and acetyl-CoA, ensure the maintenance of redox equilibrium and ATP synthesis.The synthesis of 1,2-PDO and glycerol metabolism are significantly affected by pH of fermentation (Dharmadi et al., 2006;Murarka et al., 2008).Researchers also indicated the crucial role of formate:H 2 (FHL) lyase and F o F 1 -ATP synthase.In the process of glycerol fermentation in E. coli FHL catalyzes oxidation of formate to carbon dioxide and hydrogen, thanks to which it ensures CO 2 supply required for the cell growth, while it also participates in the establishment of proton motive force (PMF) (Hakobyan et al., 2005;Dharmadi et al., 2006;Gonzalez et al., 2008).ATP F o F 1 -synthase uses PMF produced by FHL in an acid medium, while in an alkaline environment PMF produced independently from the activity of FHL.The use of PMF by ATP F o F 1synthase is necessary for the generation of energy and metabolite transport, and thus -also key processes for glycerol fermentation in E. coli (Gonzalez et al., 2008).Apart from ethanol, other metabolites are also formed, such as succinate, lactate, formate and acetate (in greater amounts, when external electron acceptors are present) (Dharmadi et al., 2006).
The literature on the subject contains results of several more or less efficient attempts at the production of 1,3-propanediol from glycerol by genetically modified E. coli.In a report by Tong and Cameron (1992) a strain E. coli AG1 was transformed with the dha regulon from K. pneumoniae and the yield of 1,3-PDO production in co-fermentation (glycerol + glucose) was 0.63 mol 1,3-PDO /mol glycerol .The authors suggested that an addition of glucose which use constitutes the additional source of reducing co-factors, improves 1,3-PDO production.According to those authors, elimination of pathways leading to ethanol, succinate and lactate could also improve efficiency of 1,3-PDO synthesis.A research team headed by Skraly published results concerning 1,3-PDO production by E. coli containing in vector pSL301 controlled by promoter trc the dhaB and dhaT genes from K. pneumoniae.This strain during fedbatch fermentation, following induction of IPTG expression, produced 6.3 g/L 1,3-PDO from 9.3 g/L glycerol with a yield of 0.82 mol 1,3-PDO /mol glycerol (Skraly et al., 1998).A team headed by Zhu obtained a strain of E. coli capable of glycerol conversion to 1,3-propanediol through overexpression of the dha regulon from K. pneumoniae and C. freundii.However, efficiency was not spectacular because of the accumulation of toxic metabolites, e.g.glycerol-3 phosphate and methylglyoxal (MG) (Da Silva et al., 2009).For this reason, it was attempted to upgrade 1,3-PDO production by limiting the accumulation of these compounds.In order to reduce MG concentration they transformed E. coli with a gene coding glyoxylase I coming from a strain of Pseudomonas putida and the 1,3-PDO regulon from K. pneumoniae induced with IPTG, which caused a considerable reduction of the inhibitory metabolite and 1,3-PDO production increased by 50% under anaerobic conditions (efficiency of 1,3-PDO synthesis increased from 0.33 mol 1,3-PDO / mol glycerol to 0.52 mol 1,3-PDO /mol glycerol (Zhu et al., 2001).In another experiment recombinant bacteria E. coli containing the dha regulon from K. pneumoniae were used in 1,3-PDO production, adding fumarate as an exogenous electron acceptor, which increases anaerobic activity of G-3-P dehydrogenase, thus reducing the concentration of G-3-P (inhibitor of 1,3-PDO production) and improving cell growth and efficiency of 1,3-PDO synthesis (Zhu et al., 2002).An addition of fumarate was also investigated in the case of K. pneumoniae, in which it had an advantageous effect on GDHt activity by an enhanced activation of the reactivating factor, which resulted from the increased ATP production in the presence of fumarate, and in turn improved 1,3-PDO production (Lin et al., 2005).In E. coli an isoenzyme of 1,3-PD DH was identified, encoded by the yqhD gene (Emptage et al., 2003).An important characteristic of yqhD is connected with lesser sensitivity to oxygen and capacity to use coenzymes NADH and NADPH, thanks to which it catalyzes more efficiently the transformation of 3-HPA to 1,3-PDO (Sulzenbacher et al., 2004;Zhuge et al., 2010).Zhang and co-workers (2005) (a team from China) modified E. coli JM109 with IPTG-induced plasmids pUCtac-dhaB and pEtac-yqhD, containing under the tac promoter glycerol dehydratase from C. freundii and alcohol dehydrogenase from E. coli.Under aerobic conditions the recombinant strain produced 38 g/L 1,3-PDO from 50 g/L glycerol.In turn, a strain E. coli JM109 containing in the pHsh vector (thermal induction) the yqhD gene (being an isoenzyme, 1,3-propanediol oxidoreductase, naturally found in E. coli) and the dhaB gene from C. freundii produced 41.1 g/L 1,3-PDO from 62 g/L glycerol with an efficiency of 0.81 mol 1,3-PDO /mol glycerol (Zhang et al., 2006a).In this report the authors showed that production may be considerably increased using a mathematical model defining optimal concentrations of nutrients in the culture medium.Wang et al. (2007) in the modification of E. coli used two plasmids, i.e. pET28a carrying glycerol dehydratase and 1,3-PDO dehydrogenase, and pET22 carrying a gene encoding factor reactivating glycerol dehydratase.A strain of K. pneumoniae was the donor of genes under the T7 promoter.The recombinant strain E. coli in fed-batch fermentation on glycerol with an addition of glucose produced 8.6 g/L 1,3-PDO from 14.3 g/L glycerol at the efficiency of 0.68 mol 1,3-PDO /mol glycerol .Ma and co-workers described the generation of a recombinant strain E. coli JM 109 incorporating genes dhaB and dhaT from K. pneumoniae in pACYCDuet-1, each controlled by a separate T7 promoter.The microorganism under aerobic conditions produced 11.3 g/L 1,3-PDO from 40 g/L glycerol with an efficiency of 0.35 mol 1,3- PDO /mol glycerol (Ma et al., 2009a).A study by Tang et al. (2009) describes the highest to date 1,3-PDO production from glycerol.The authors transformed E. coli with the thermally induced pDY220 plasmid, carrying genes of glycerol dehydratase (independent from the B 12 coenzyme) together with the reactivating cofactor from C. butyricum and 1,3-PDO oxidoreductase yqhD from E. coli.In the two-stage (aerobic/30ºC-anaerobic 42ºC) fed-batch fermentation they finally obtained 104.4 g/L 1,3-PDO at a yield of 90.2% g/g glycerol conversion to 1,3-PDO.In another publication genes of the dha regulon, i.e. dhaB, dhaT, gdrA and gdrB from K. pneumoniae, were incorporated in E. coli and S. cerevisiae W303-1A.Modified E. coli produced 8-9 g/L 1,3-PDO from 40 g/L glycerol (plus 30 g/L glucose) at the efficiency of 0.26 mol 1,3- PDO /mol glycerol , while no such production was observed in W303-1A (Ma et al., 2010).A report by Qi et al. (2011) presented another attempt at 1,3-PDO production by a strain E. coli JM109 modified with plasmid pSE380 carrying a gene encoding GDHt from K. pneumoniae and the yqhD gene from E. coli BL21, localized under the trc promoter.They showed that glycerol dehydratase exhibits higher activity at 45ºC and pH 9 than at 37ºC and pH 7. The obtained production amounted to 28 g/L 1,3-PDO.A team headed by Dąbrowski constructed a recombinant strain of E. coli transforming it with genes from the dha regulon (dhaB1, dhaB2 i dhaT) coming from the C. butyricum strain 2CR371.5, which under aerobic conditions produced 3.7 g/L 1,3-PDO with an efficiency of 0.36 mol 1,3-PDO /mol glycerol (Dąbrowski et al., 2012).

FACTORS LIMITING 1,3-PDO PRODUCTION
The primary causes for the low efficiency and productivity of glycerol fermentation to 1,3-PDO are connected with the conditions, under which fermentation is run, i.e. physiological temperature, atmospheric pressure as well as the type of fermentation, typically being batch fermentation (Zeng & Biebl, 2002).Increasing the density of bacterial cells as well as application of fed-batch fermentation or continuous fermentation with immobilized cells instead of batch fermentation constitute an important step in the optimization of the process (Saxena et al., 2009).In Enterobacteriaceae we may observe accumulation of 3-hydroxypropionaldehyde, which is a toxic metabolite having an adverse effect on bacterial growth and 1,3-PDO production (Zeng et al., 1993;Biebl et al., 1998).Minor products formed during glycerol fermenta-Glycerol from biofuels to 1,3-propanediol using Escherichia coli tion, such as ethanol, 2,3-butanediol (in K. pneumoniae), lactate or acetate, may also negatively influence the efficiency of 1,3-PDO biosynthesis (Zeng et al., 1994;Menzel et al., 1997).Targeted mutagenesis and different genetic engineering strategies offer a chance to overcome this problem (Zeng & Biebl, 2002).It would be an interesting strategy to shut the synthesis pathway for acetate, which is the most toxic metabolite for bacterial cells; however, it has not been attempted to date.Inactivation of the ldhA gene encoding lactate dehydrogenase (LDH) in K. pneumoniae improved efficiency of 1,3-PDO synthesis (Xu et al., 2009a), alike the inactivation of the aldA gene in K. pneumoniae YMU2 encoding aldehyde dehydrogenase (ALDH), catalyzing conversion of acetyl-CoA to ethanol, which resulted from greater availability of NADH 2 cofactors for 1,3-PDO oxidoreductase in K. pneumoniae (Zhang et al., 2006).Many research teams analyzed the effect of the substrate and products of glycerol metabolism on 1,3-PDO production.Xiu et al. (2004) showed that an advantageous effect on the efficiency of 1,3-PDO synthesis, productivity and final concentration may be provided by the two-stage structure of the bioprocess, using two bioreactors, with a greater dilution rate applied in the second bioreactor.Numerous bacteria developed the mechanism of tolerance to different high concentrations of the substrate or products, and a method to produce such strains is their culture at high concentrations of metabolites, where the most resistant remain and the others are eliminated by a natural selection (Saxena et al., 2009).Efficiency of 1,3-PDO synthesis is significantly affected by the availability of NADH 2 cofactors, formed in the oxidative pathway leading to minor products.3-hydroxypropionaldehyde (3-HPA) formed in the redox pathway is a toxic metabolite, which limits 1,3-PDO production (Hao et al., 2008).Accumulation of this compound in the medium reduces GDHt activity, which results in a deteriorated catabolism of glycerol (Barbirato et al., 1996).Accumulation of 3-HPA is caused by an inappropriate ratio of GDHt activity to 1,3-PD DH, and it is suggested that increasing the activity of 1,3-PD DH would have a positive effect on 1,3-PDO production (Chen et al., 2009).High concentration of 1,3-PDO also has an inhibitory effect on bacterial cells, which results from the generally harmful effect of alcohols on the cell growth.The use of 1,3-PDO dehydrogenases, which also catalyze the reaction from 1,3-PDO to 3-HPA may lead to additional accumulation of 3-HPA, thus the potential application of oxidoreductases with lower activity from 1,3-PDO to 3-HPA, would reduce accumulation of the toxic metabolite (an example in this respect may be HOR oxidoreductase -hypothetical oxidoreductase from K. pneumoniae) (Wang et al., 2003).

PURIFICATION OF 1,3-PDO FROM SPENT MEDIUM
Spent medium contains numerous different components such as water, glycerol residue, acetate, succinate, lactate, proteins, polysaccharides, nucleic acids, etc., which hinders further utilization of 1,3-PDO, and at the same time is a challenge for further commercialization of the process.There are several methods of 1,3-PDO purification described to date in literature and applied in practice.These include reactive extraction, evaporation, distillation, membrane filtration or ion-exchange chromatography.However, each is burdened with certain drawbacks (Xiu & Zeng, 2008).An efficient purification process most frequently combines several of the above mentioned techniques.It is also possible to utilize organ-ic products applying other strains (from methanogenesis) with no negative effect on the production of 1,3-propanediol (Szymanowska-Powałowska et al., 2013).It seems essential to develop a technology with a greater efficiency, with lower energy consumption and ensuring greater purity of the desired product (Xiu & Zeng, 2008).

CONCLUSIONS
Nowadays, there is a lager industrial need to exchange the chemical way of various chemicals synthesis for biotechnological -more ecological and sustainable manner.The 1,3-PDO production from glycerol is an desirable alternative to the traditional production from non-renewable resources.Despite few technical barriers and some economical challenges, E. coli are promising as a biomaterial used for 1,3-PDO synthesis from glycerol, which is widely exploited in this direction (imitating 1,3-PDO synthesis from glucose by E. coli, which has already had many patents).The selection of E. coli bacteria for the production of 1,3-PDO seems fully justified, since they are thoroughly studied model organisms, easy to culture, rapidly proliferating in cheap media, and non-pathogenic.For these reasons E. coli bacteria comprise one among the best heterologous system entirely sufficient for such use i.e. the production of valuable chemical compounds.
Currently used variants include also microorganisms naturally producing 1,3-PDO (despite their invasiveness), while the emphasis is placed on the use of recombinant microorganisms.In already optimized processes, in fedbatch or continuous cultures using natural producers, typically production level is 40-50 g/L at a yield of 0.6 mol 1,3-PDO /mol glycerol , max.60-70 g/L 1,3-PDO with a comparable yield (Saxena et al., 2009).In the case of recombinant E. coli maximum production of slightly over 100 g/L 1,3-PDO from glycerol was reported at a glycerol conversion rate of 90.2% (g/g) in a two-stage optimized culture (Tang et al., 2009).Thus, obtaining higher efficiency with the use of recombinant E. coli rather than natural producers is feasible and it is possible to use even antibiotic selection, as evidenced by the above mentioned report.
For efficient biosynthesis of 1,3-PDO by E. coli not only genetic manipulations, optimization of culture conditions and media composition need to be conducted, but also emphasis should be put on means of selection for gaining particular strains which will be more resistant for majority stress factors.

Figure 1 .
Figure 1.Production of biodiesel in selected countries in the years 2008-2020 (in thousand tons).

Figure 4 .
Figure 4. Catabolism of glycerol in natural 1,3-PDO producers.Enzymes of the dha regulon are marked in rectangles.Pyruvate is reduced to different organic compounds depending on the microorganism and fermentation conditions.GDHt, glycerol dehydratase; 1,3-PDDH, 1,3-propanediol dehydrogenase; GDH, glycerol dehydrogenase; DHAK, dihydroxyacetone kinase.The blue colour marks the redox pathway, which incorporated in the Escherichia coli strain gives them capacity to produce 1,3-PDO.The figure was prepared based on(Biebl, 2001).