Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.slsovier.comilocato/biotechady ELSEVIER Research review paper Glycerol: A promising and abundant carbon source for industrial microbiology Gervasio Paulo da Silva *“', Matthias Mack”, Jonas Contiero “* Deparment of Eduction ahi StateUnvrsy-Deh, BR 07, km 17, Senha do Bon BA 857-06, a vite or Tcl Mots Made nies of Ape eee deck 10 6813 Maem. Gera Deportes of Bochemsty ond Mera SS ana Sate Ue Unep. A 248 155, Bel Vs, i Cla 513506 00 Bra ARTICLE INFO ABSTRACT ‘Accepted 3 Jay 2008 ‘alae one 16 Aagust 2008 ‘eroleum isthe main energy source wilzed in the worl, but valli is Hmied and the search for ‘ew renewable energy sources i of majo interest. lofts, such 3s ethanol and biogesl are among the ‘ost promising sources fr te substitution offs fuels. Biodiesel can replace petroleum diesel ei is ‘produced from animal fats and vegetable ols, which generate about 10% (whw) glycerol as the main by- product. The excess glycerol enerated may become an environmental problem sine it cannot be dixporee On the environment. One ofthe posible applictions is its use as carbon an energy Source for mieroial Saas rowch in industrial microbiology. Giyerol bioconversion invaluable chemical, such as 13-propanedol yer ‘itydroxyaetone ethanol, suceinate ets dsesse inthis review artic Caton sauce “© 2008 sever ne. Alright reserved 1S tepanedol 1. troduction 30 2 Glycerol and badiese 3 5. microbial growth with glyeel as carbon and energy source 2 31. Glyeerolupake 2 32. Microbial metabolism of glcerol 2 4. Giyerol bioconversion in industrial microbiology. 33 43, 12-propanedio B 4.1L. Bacterial production of 13-PD0- u 42. Ditydroxyaetone 35 43. Suecine acd 36 45. hanol 36 46. Gime acl 7 47. Pigments 3 43. Polvhydronvalcanoate 7 49. Bosuracants 7 5. Conchsions a Acknowedgments 38 Reteences 38 * caexpning ator Te 155193505418. Fm etree: rpsbauned (a Sa. mmackOhsanahim de jeoaoreaes be Conseo) 1. Introduction 073457508 - sce fea mater © 2008 Hse ne Al igs reserved “The demand for biofuels is currently onthe rise worldwide and the use of biomass is one ofthe most promising alternatives, Brazil was aPd Sina a. seco races 27 (2008) 3-38 a pioneer in the use of clean (with respect to CO> balances) energy Sources (biofuel) when its government decided to replace gasoline With alcohol fuel in 1973, In the mid 1980s, about 95% of th automobiles produced in Brazil were changed to ethanol combustion, Flex fuel vehicles, which were engineered in 2003, account for more than 80% of the automobiles sold in Brazil The use of biofuels will cettanly continue to grow. as the availability of petroleum is clearly limited. Estimations point toa gradual decline in petroleum produc- tion starting in 2010 and, according ta some projections, the reserves are thought to become completely depleted by 2050 (Campbell and Lahertére, 1998). ‘Another biofuel originated from biomass is biodiesel, produced from vegetable olls and animal fts. Just as ethanol can replace gasoline in Otto cycle motors, biodiesel can replace the diesel in diesel engine motors. AL the present time, the European Union is the principal biodiesel producer, with 82% of biodiesel produced in th world in 200% (Demirbas and Balt, 2006). According tothe European Biodiesel Board (EBB, 2006), the estimated production of biodiesel in 2005 was about 32 million tons and the production capacity in 2006 ‘was estimated to be about 6069 milion tons. The USA is the second major world producer and produced about 250,000 tons of biodiesel in 2005 (E88, 2006), Biodiesel has been mainly used in Germany, which is currently the largest producer and consumer ofthis biofuel, exceeding 25 billion liters annually. A numberof other countries have recently adopted the use of biodiesel, usually as an admixture to petroleum diesel in differen: proportions. In Brazil, the addition of 2% biodiesel (#2) is obligatory since January of 2008, increasing to 5% (85) in 2013. Brazil will become a major producer and consumer of biodiesel for two easons: fist, the wse of alcohol to fuel cars has along tradition in the Brazilian culture and, secondly, the conditions for cultivating leaginous plant are extremely favorable in many areas. Furthermore, the agricultural know-how to grow these plants is available and also large areas of eultivatable land have not yet been explored Simple alcohol glycerol (12.3-propanetril, also glycerin or glvcer- ine) is the principal by-product obtained during transesterfcation of vegetable oils and animal fats (Solomon et al, 1995; Barbirao et al, 1987a.b, 1988; Colin et a, 2001) Glycerol is abundant in nature, since it isthe structural componentof many lipids tis also one ofthe principal compatible solutes, being widely produced in response to decreased extracellular water activity during osmoregulation in yeasts(Weng etl, 2001). Due tots ample occurence in nature, mary known microorgan- isms can naturally utilize glycerol asa sole carbon and energy source, “These microorganisms have attracted attention to the potential use in bioconversion of abundant glycerol produced from biodiesel (Soloman etal, 1995; Barbirato and Bories, 1997; Menzel et a 19973), Glycerol ‘may substitute traditional carbohydrates, such as sucrose, glucose and starch, n some industrial fermentation processes. Clycerol bioconversion adds significant valu to the productivity of the biodiese industry. Throughout this paper, examples of possible biotechnological production processes based on glycerol demonstrate that this simple chemical is a promising abundant new carbon source for industria! microbiology. Triglyerides 2 Clycerol and biodiesel Clycerol can be produced either by microbial fermentation or chemical synthesis from petrochemical feedstock. It can also be recovered from soap manufacturing. Inthe traditional process of the latter, glycerol is released as a by-product during the hydrolysis of fats. ‘This process is currently of less importance, since soap has been largely replaced by detergents (Wang etal. 2001) Biodiesel s produced from vegetable ols and animal fats through transesterification with, for instance, ethanol ot methanol (aleoho- lysis, generally catalyzed by NaOH or KOH (Fig. 1) and glycerol represents 10% (vjv) of the ester (Papanikolaou et al, 2002: Gonzélez- Pajuelo etal, 2004; Mu etal, 2006). Europe mainly uses rapeseed oil for biodiesel production. In Brazil, oils from soybean, sunflower, African oil palm (Elaes guineensis), castor oil and Jatropha curas are see, In some European countries, the production of glycerol has increased significantly due to biadiesel uptake. As a consequence, prices have fallen and the majority of companies that chemically produced glyceral have shut down business (Dharma etal, 2006; Deckwver, 1995). In the EU, some biodiesel companies have severe problems getting rid of excess glycerol and disposal i quite expensive, ‘The collapse of glycerol prices causes major problems to these companies (Wilke and Voriop, 2004; Dharmadi etal, 2006). IC is reported that approximately 75-85% of the final costs of biodiesel arse from the cost of raw material (Yuste and Dorado, 2005), Biodiesel s now widely accepted as a renewable fuel and a major goal for the future will be to reduce the costs related to biodiesel production. One possibility is to use inexpensive or low-cost raw ‘materials, such as vegetable oils used for frying (Yuste and Dorado, 2006). The reduction in taxes for biofuels i another important approach. Considering the inereasing need for renewable fuels throughout the world and the increasing demand and production of biodiesel, an excess of glycerol will be available in the world. Since lycerol can be used as a carbon source in industrial microbiology his by-product adds value to the productive chain of the biodiesel industry, contributing to their competitiveness Clycerol is present in many applications in the cosmetic, paint, automotive, food, tobaceo, pharmaceutical, pulp and paper, leather and textile industries. Italo is used as a feedstock forthe production fof various chemicals (Wang etal, 2001). New applications are being evaluated in the food industry, the polyglvcerol and polyurethane industry, the field of wood stabilizers and production of small ‘molecules, such as dihydroxyacetone, glyceric and hydroxypyruvie acids and glycerol carbonate (Claude, 1999). Glycerol has also been Considered as a feedstock for new industrial fermentations in the future (Wang et al, 2001). Thus, one of the many promisi applications for the use of glycerol sits bioconversion to high value Compounds through microbial fermentation, Glycerol is not only cheap and abundant, bu its greater degree of reduction than sugars offess the opportunity to obtain reduced chemicals, 2s succinate, ethanol, xyitl, propionate, hydrogen, ec. at higher yields than those obtained using sugars (Dharmadi et al, 2006), gh gmp ar I I CHy- HCH, $4. iF aa | OH OH OH a 4 ‘ Ethyl esters (Biodiesel) Fig 1 Tansesterifeton etn of gerd wih ethanol (sleholvi izing NOK a cat and enerng isl miro ft act exter a eral2 GP da Sina et itera bane 27 (2008) 30-38, 3. Microbial growth with glycerol as carbon and energy source 31, Glycerol uptake Like other small uncharged molecules, glycerol can cross the ‘cytoplasmic membrane through passive diffusion. However, cells limited 10 passive uptake have a growth disadvantage at low concentrations of substrate. Glycerol uptake is frequently cited as the only example of transport mediated by facilitated diffusion across the Escherichia coli nner membrane (Voegele etal, 1993). Facilitated diffusion is achieved by an integral membrane protein, the glycerol facilitator ipF (Heller etal, 1980; Voegele et al, 1993; Darbon etal 1899), Intracellular glycerol is subsequently converted to glycerol-3- phosphate by glycerol kinase (Gipk). Glycerol-3-phosphate remains {rapped in the cell until its further metabolized because it is not a substrate forthe glycerol facilitator (Voegele et a, 1983; Darbon et al 1999; Braun et al, 2000}, ClpF acts as a highly selective channel, also conducting poly- alcohols and urea derivatives, for which it is stereo-selective and enantioselective (Braun et al, 2000; Fu et al 2000). All these channels are strictly selective for non-ionic compounds, including hydroxide and hydronium ions, thus preventing the dissipation of the ‘membrane potential (Braun et al, 2000; Fu etal, 2000). The influx of glycerol mediated by Gip# is 100-to 1000-fold greater than expected for a transporter and is non-saturable ata glycerol concentration of 200 mt (Fu et al, 2000), In Saccharomyces cerevisiae, the flow of glycerol across the plasma ‘membrane is controlled either by passive diffusion, a channel protein tor an active uptake mechanism (Wang etal, 2001), 2, Microbial metabolism of glycerol ‘A number of microorganisms are able to grow anaerobically on slycerolas the sole carbon and energy source, such as Citrobacter reundi (Homann etal, 1960; Danie et al, 1985; Seifert etal. 2001), Klebsiella ‘pneumoniae (Forage and Foster, 1982; Tong et al, 1991; Menzel e al 1997); Bibl et al, 1998: Németh et al, 2002), Clestrdium pasteurianu (Laer et al, 1997; Macis et al, 1998; Bieb, 2001), Clostridium butyricum (Abbad-Andaloussi t al, 1995; Biel, 1991; Bibl et al. 1982; Himmi etal, 1999: Malaou and Marceak, 2001; Colin et al, 2007), Enterobacter agglomerans(Barbirato eal, 1996; Sarbrato and Bories, 1987; Barbirato cecal, 19972), Enterobacter aerogenes ito etal, 2005) and Lactobacillus reuter (Talasico etal, 1988, 1990) In Klebsiella, Citrobacter, Clostridium and Enterobacter, glycerol is ‘metabolized both oxidatively and reduetively (zhu et al, 2002)-In the oxidative pathway, the NAD'-dependent enzyme glycerol dehydrogenase (EC 1116) catalyzes the conversion of gycerol to ditydroxyacetone and the glycolytic enzyme dilydroxyacetone kinase (EC 271.28) phosphor vlates the latter product (Daniel et al, 1985; Luers etal, 1897: Macis etl, 1998), which s then funneled to glycolysis. The reducing pathway Js catalyzed by coenzyme B-dependent glycerol dehydratase (EC 42.130) and elated diol dekydratases (EC 4.21.28) (Toray etal, 1978: Forage and Foster, 1982; Knietsch et al, 2003), converting glycerol t03- hhydrexypropionaldehyde (Toraya et al, 1980; Tong etal, 1991; Seifert etal, 2001), and by the NADH+H"-dependent enzyme 13-propanediol dehydrogenase (13-propanediol-oxydoreductase, EC 111.202) reducing 3-tydroxypropionaldelyte to 13-propanediol and regenerating NAD” (ici tal 1998; kraly eta, 1998; Ahrens etal, 1998: Veiga da Cunha and Foster, 1982; Németh et al, 2003) (Fig. 2} The final 13-propanediol (GLYCEROL Glycerol dehydrogenase (aap) Glycerol dehydratase DIHYDROXY ACETONE ae Dihydroxyacetone: kinase (dhak) ADP [DIHYDROXYACETONE-P| nap» —! ADP wn, = : Sap v PHOSPHOENOLPYRUVATE| App are PYRUVATE] HYDROXY PROPIONALDEHYDE| 1,3-propanedio! dehydrogenase (dhar) NADH, NADY 1,3-PROPANEDIOL| Fig 2Fementative ater of gyeeradsnaion dependent an 13-70 formation The key eye of the dh elon and espective genes eae a el elon are ‘Shown Pyruvate wil be reduced to diferent ergaic compen depenges on mirorgansm and fermentation conan, regenerating NAD” (agape em Eat 125Pd ina a. seco Aras 27 (2008) 3-38 2 yer (GLYCEROL <"“""" » [DIHYDROXYACETONE) yc nase K~ ATP nop (GLYCEROL-3-PHOSPHATE eer 3-pospnate (— NAD® Sehysraerase NADH, DIHYDROXYACETONE-P| Fig. 3. Pathways lr growch and DHA production by ondnsin cero The membrane-ound sero éeyérognase least a excell production of DHA and uses O25 thefnal acreprorof secon rd reduced equsens mean af abgsinae snd eytecirome a DHA? ietbalze y mess aye pntrephonpate pay pee om (13-PD0) products highly specific for glycerol fermentation and cannot bbe obtained from any other anaerobic conversion (Homann et al, 199; Deckwer, 1995) ln K pneumoniae (Forage and Lin, 1982) and C.freundi, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 13-PD0 dehydrogenase (dha?}, glycerol dehydrogenase (dhaD}, and eliydroxyacetone kinase (dhoK) are encompassed by the dha regulon (zhu et al, 2002) (Hg. 2). The 13-PDO operon of butyricum is composed of three genes a diferent type of glycerol detydratase (haf), its activator protein (dha#2) and dhaT (Raynaud et al, 2003) tn this bacterium, glycerol dehydratase is extremely oxygen sensitiv, strongly associated with the cell membrane and vitamin-y. independent (Saint-Amans etal, 2001; Raynaud etal 2003; Gonzilez-Pajuelo etal, 2004, 2005a}, 2006) InS. cerevisiae and a numberof other yeasts, glycerol i degraded via Aitydroxyacetone of via glycerol-3-phosphate(Wanget al, 2001) Inthe later, glycerol is converted to ghterol-3-phosphate through glycerol kinase (EC 2.7130), which can be used either asa precutsor fo lipid biosynthesis orconversion to dihyéraxyacetone phosphate and can then either be transformed to glycealdeyde-3-phosphate by tise phosphate isomerase (EC 53.11) in glycolysis or can serve as a substrate forthe synthesis of other metabolites (Wang et a 2001), Similar pathway for ycerol oxidation (gp regulon) are present in K pneumoniae (Ruch et, 1974; Forage and Li, 1982), Gluconobaceraxydans (Bares etal, 1991 Claret etal, 1994) and € acetobutylicum (GonzSlez-Pajulo etal, 2006) (Fig. 3) According Ruch etal. (1974, the glyerol-3-phosphate pathway fs espansble forthe aerobic degradation of glycerol in K pneuoniae (formerly K. aerogenes), while the dihydroxyacetone pathway is responsible for the anaerobic degradation of this substrate, Fermentation from glycerol to ethanol or butanol by C: pasteuria- rum daes not depend on the formation of by-products (Bieb, 2001) since hydrogen carriers are completely regenerated in the pathway (Bieb! et a, 1998) Another example of redox-balanced process isthe Conversion of glycerol into succinic acid. although the pathways for ethanol and succinate are equivalent regarding the overall redox balance, the energetic contribution of the ethanologenic pathway is ‘much higher, as | ATP is produced per each molecule of glycerol converted into ethanol, while production of energy in the succinate pathway is limited tothe potential generation ofa proton motive force by fumarate reductase (Dharmadi etal, 2006) (ig. 4) 4. Glycerol bioconversion in industrial microbiology 41. 13-propanediol “The diglycol13-propanediol (Trimethylene glvcal, propylene glycol) isthe principal product obtained through glycerol fermentation. 13-PDO was first observed asa product ofthe fermentation of glycerol in 1881 (Werkman and Gillen, 1922) is one ofthe oldest known fermentation products, bu ite attention was paid to this microbial pathway for aver 4 century (Biebl et al, 1999}, 13-PDO presents several interest applications (Tong etal, 1991; Deckwer, 1985; Colin eal, 2000; Cher ft al, 2004; Lin et al, 2005). it can be used as a monomer fo polvcondensations to produce plastics with special properties, ie polyesters, polyethers and polyurethanes (Deckower, 1985; Colin etal, 2001; Wilke, 1999; Himmi eta, 1999; Yang etal, 2007; Du et a, 2006), as a monomer for cyclic compounds (Knietsch et al, 2003), as a polyglvcol-type lubricant andi also may serve asa solvent (Papaniko- laos etal, 2000}, 1-PDO is an emerging commodity chemical (Cameron et al 1998) and renewed interest in its biotechnological production has recently arisen (Abbad-Andalouss etal, 1995; Zhang etal, 20060), ‘The development of a new polyester, polypropylene terephthalate (PPr or polytrimethylene terephthalate ~ PTT) from 13-propanediol Fequites a drastic inerease in the production of this chemical (Raynaud et al, 2003; Gonzilez-Pajuelo et al. 2005b, 2006). PPT is 8 biodegradable polyester that has great potential fr use in carpet and textile manufacturing and is related to polyethylene terephtha- late (PET) and polybutylene terephthalate (PBT) (Hao et al, 2005), “The chemical companies Shell and DuPont hold patents related to ‘new commercial PPT polymers obtained from 1.3-propanediol, shell has developed Corterra® and DuPont has developea Sorona® and Hytel® PPT was developed and patented in 1941, but application was limited since the precursor 1,3-PDO was too expensive. Thus, many companies tried to develop new and more efficient technologies for 13-PDO production. In the new Shell process, 13-PDO is chemically obtained from the reaction of ethylene oxiée with carbon monoxide and hydrogen. In the DuPont development, 13-PDO is produced fermentatively from glucose by recombinant microorganisms (Biebl ct al, 1999; Chotani et al, 2000; Nakamura and Whited, 2003), According to Nakamura and Whited (2003), the comparison between slucose and glycerol considering substrate and yield has favored the direct glucose route, manly because glucose was cheaper. However, with the abundant surplus of glycerol from biodiesel production, the glucose route is probably more expensive now and glucose may be used for other applications, such as alcohol fermentation. Further- ‘more, glucose competes directly with food and feed production, which is not the case for giyceral, Over 10° tons of 13-PDO are produced yearly, mostly through chemical synthesis (Németh etal, 2003; Zhang et al, 2006a).13-PDO is chemically produced through two different routes: hydration of actolen and hydrformylation of ethylene oxide (Cameron eta, 1998; Hao etal, 2006; Yang etal, 2007). Chemical synthesis requires highCP da Sina to. eco Avan 27 (2008) 3-38, wap? 20H, [DTHYDROXYACETONE| DIHYDROXYACETONE-P) ‘(NADY | —— app v ADP ATP. PHOSPHOENOLPYRUVATE] >4e ADP NADH, NAD: NADH, Piruvate 2 C0, (AcTaTE (PYRUVATE)... [ACETOIN ei hone con gg nae oe (ACETATE <-"*“-.[ACETYL-CoA] mH neety-con eae sata er SUCCINATE [ACETOACETYL-Con] Noo, ane eae —— NADH, v a — ae (PROPIONATE) id BUTYRYL-Coal ETHANOL [BUTYRALDEHYDE > = [BUTYRATE| temperature, high pressure and expensive catalysts (Lin et al, 2005) and produces toxic intermediates (Raynaud et al, 2003; Gonzilez- Pajuelo et al, 2004, 2005a). Consequently, chemical synthesis is expensive and, thus, 13-PD0 still has a low market volume (Deckwer, 1995}, Because ofthe environmental benefits and use ofa renewable feedstock, the biotechnological synthesis of 13-PDO appears to be an attractive alternative to chemical synthesis (Hao etal, 2005) 4.1, Bacterial production of 13-PDO “The biotechnological production of 13-PDO from glycerol has been demonstrated for several bacteria, such as Lactobacillus brevis, Lbuchner,Bailuswelei,C.freundi pneumoniae. pateurianur, butyricum and & agglomerans (Nakas etal, 1983; Forsberg. 1987; Homann etal 1990; Veiga da Cunhaand Foster, 1992; Bibl, 1991; Biebl etal, 1992; Bibl and Marten, 1995; Daniel et a, 1995; Macis etalPd ina a. seco races 27 (2008) 3-38 3 13998; Deckwer, 1995; GonzSlez-Pajuelo etal, 2008; Chenget al, 2004; Yang etal, 2007), Complete conversion of glycerol to 13-PDO is not possible de to the requirement of an addtional reducing equivalent (Chotani et a, 2000) (Fi, 2) Clycerol fermentation by enterobacteriaceae results in the accu- mulation of two main products, 13-PDO and acetate, whereas the secondary products, lactate, formate, succinate and ethanol, are produced in variable amounts depending on the culture conditions (Homann et al, 1990; Barbirato et al, 1998). With C. buyricum, 13- PDO is the main product, with butyric acid and acetic acid as by- products in addition to CO, and Hz (Barbtato et al, 1998). The bacterium C. pasteurionum growing on glycerol produces a variety of ‘metabolic end products, such 25 n-butanol, 13-propanedial, ethanol, acetic aid, butyric acid and lati acid (Biel, 2001) (Fig 4) ‘According to Bicbl (1891), the concentrations that inhibit glycerol fermentation by C.butyrcum are 60 gil for 13-PDO, 27 g/L for acetate and 19 g/L for butyrate, and glycerol was supported until 80 gi. Barbirato et al, (1998) report that, in batch fermentations using butyricur containing 112 git industrial glycerin, the final 13-PDO concentration was 6344 gi, which is close to the maximal concentra- tion tolerated by this microorganism. The conversion yield was 0.69 mol/mol and maximal 13-PDO productivity was 1.85 giLh, with butyrate (75 gil) and acetate (81 g/L) as the only by-products obtained, Papanikolaou etal. (2000) investigated the production of 13-PDO by a newly isolated C. butyricum strain. The highest 13-PD0 concentration obtained in continuous culture was 31=48 gi, with 2 conversion yield of 055 g 13-PDOjg glycerol. Using a two-stage strategy, the maximum 13-PDO obtained was 41-46 gi, with 2 ‘maximum volumetric productivity of 4 g/L Mi et al. (2006) used pneumoniae to ferment crude glycerol. obtaining a concentration of 513-53 glL of 13-PDO with productivity of 17 g/ljh. The use of crude yerol should simplify the process and reduce operational costs. In fed-batch fermentation with sucrose as a co-substrate, Yang et al (2007) obtained up to 83.56 13-PDOJL with a yield of 6.62 molimol roland productivity of 1.61 giljh with K.xytoca MSal, a mutant Strain deficient in lati acd biosynthesis. Inthe first report on plot- scale production of 13-PDO using K pneumoniae Mai, Cheng et al (2007) obtained 58.8 g 13-PDO/L, yield of 0.53 mol/mol glycerol and Drodtctivity of 092 g/L “Metabolic engineering can be used! to maniptlate the pathways so that the formation of by-products is reduced or even eliminated (Cheng eal, 2005,2006)-The genetic manipulation ofthe microorganisms can also eliminate pathways that compete with the desired product or fenhance the production of the desired metabolites, The biological production of both glycerol and 13-propanediol is known, but it has rhever been demonstrated that a single organism can accomplish the entire process (Chotani et al, 2000). Thus, no natural microorganisms have yet been found that can directly convert glucose to 1.3-PDO (Hartep tal, 2002). Moreover, wild-type Ecol strains donot ferment iyerol to 1.3-PDO (Cameron etal, 1998) Some studies have developed Fecombinant microorganisms for 13-PDO production from glucose Hartlep etal, (2002) present a two-stage strategy forthe conversion of glucose to 12-PDO, First, a recombinant Ecol strain produces glycerol from o-glucase. Then glycerol is converted to 13-PDO by K. pneumonie. “Thistwo-stage process renders up to 60-70 iL 13-PDOJL Inthe DuPont and Genencor patent (Chotani et al, 2000), a process using 2 recombinant E col strain containing the genes from S. cerevisiae for seerol production and the genes from K. pneumoniae for 13-PDO production is described (US, Patent 5 599.689, 1997) This recombinant ‘microorganism reached 2 final L3-PDO concentration of 135 g[l-using glucose as substrate; the productivity was35 gil{hand the eficiency of Substrate conversion was 51% (Sanford etal, 2004). Te need to add large amounts of expensive vitamin B,(Raynaud et a, 2003) isa major limitation and has prevented the large-scale production of 1.3-PD0 using this fermentation proces (Yang etal, 2007). Stains of E col that are able to convert glycerol to 1.2-PDO have been constructed by overexpressing genes of the dha regulon from K. pneumoniae or C feundi However, the glycerol conversion is low, The main season for this low yield is a number of toxic by-products, such as glycerol-3-phosphate (Zhu et al, 2002). Gonzilez-Pajuelo al, (2005b) developed a recombinant C acerabusyicum DGI stain by introducing a plasmid (p$PDS) that contained the 13-propanediol pathway from C butyricum. This bacterium is considered the best ‘natural producer in terms of both yield and titer of 13-PDO produced, Recombinant C acetobutylicum DG! (p$PDS) produced 1104 mM 13- PDO in 2 fed-batch culture, with 2 yield of 0.65 molfmol glycerol consumed and productivity of 1.70 gi. The authors conclude that © acetobutylicum DG1 (pSPDS) can be used for the continuous industrial production of 13-PDO with high yields, titers and productivity from raw glycerol from biodiesel (Gonzslez-Pajuelo etal, 2005b, 2006). Zhang et al. (2006a) constructed a recombinant E.coli JM109 strain (pHsh-dhaB-yghD) containing the genes yqhD (coding for 13-propanediol oxidoreductase from wild-type E.coli and dha from C freundi in the temperature control expression plasmid vector pHsh. This recombinant strain produced up to 411 giLof 13- PDO in an optimized culture medium containing 618 g glvcerell, Cameron et al. (1998) deseribe several metabolic engineering approaches for 12 and 13-propanediol production 23-Butanedio is another glycol that an be produced from glycerl According to Syu(2001),23-butaneiol sa compound that can be added asa flavoring agent in food products when converted to diacetyl through ‘oxidation 23-Butanediol ean also be converted to 13-butadiene, which i used inthe production of synthetic rubber. Many other derivatives of T-butadiene are of commercial value, such as antifeeze agents, solvents and plastics, liquid fuel additives and polyurethanes fr drugs, cosmetic products nd lotions. The mechanisms that lead to the production of 13-PDO and 23-butanedol from giyerol by K preume- riae were discussed by Bebl etal (1998) and the highest 23-butanediol production oecurs at low pH and an excess of glycerol. 42. Ditydroxyacetone Diydroxyacetone is frequently used in the cosmetic industry and serves as a versatile building block for the organic synthesis of a variety of fine chemicals (Heat et al, 2003; Bauer etal, 2005). Due to rigorous safety requirements, the chemical production of DHA is rather expensive. Thus, DHA synthesis is performed more economic- ally using a microbial process (Hekmat et al, 2003) Itis produced via the oxidation of glycerol using the acetic acid bacterium 6. oxydans (Flickinger and Perlman, 1977; Nabe et al, 1879; Wethmar and Deckwer, 1999; Bauer et al, 2005) in 2 process that requires good ‘oxygenation and a medium containing yeast extract (Fickinger and Perlman, 1977). Wethmar and Deckwer (1999) developed 2 semi- synthetic medium that allows increased specific DHA. production, significantly reducing the yeast extract in the medium. ‘There are only two pathways for glycerol catabolism in G.oxydans (orieset a, 1991; Claret etal, 1954) Microbial growths ensured by 2 cytoplasmic pathway, in which glycerol is phosphorylated to giycerol- S-phosphate and then dehydrogenated to DHA-phosphate (DHA-P), Which is ATP- and NAD-dependent. DHA-P is catabolized in the pentose-phosphate pathway. DHA produetion occurs via a mem- brane-bound glycerol dehydrogenase, which appears to be the ony process responsible fr BHA synthesis and employs oxygen asthe final acceptor of reduced equivalents, without NADH involvement (Fig 3). Ce ofthe main problems of microbial DHA synthesis sth fact that both the substrate and product have an inhibitory effect on bacterial growth (Bories et al, 1991; Clare eta, 1994; Hekmat etal, 2003; Bauer etal, 2005). Hekiat etal (2003) propose a repeated-fed-batch mode, allowing 2 fully automated quast-continuous operation that can be ‘maintained aver a period of several months with no decrease in productivity thereby reducing processing costs. The productivity ofthe36 CP da Sina to / tecnalogy Abas 27 (2008) 3-38, process was increased by 75%, from 1.6 km/h to about 2.8 kei, but the authors observed that maximum productivity had not yet been achieved, Using this new process, Bauer et al-(2005) demonstrated that the culture was able to grow in up to a DHA concentration of 80 kglsn® Without any influence of product inhibition ¢. oxjdans lost its fegenetation capability at DHA concentrations above 160 kgity, but product formation was observed up toa maximum DHA concentration (0f 220 kg/m’ because intact membrane-bound glyceral debydrogenase \was sil active both inthe iceversibly growh-inhibited cells as wel as inthe cel debris. Gatgens etal. (2007) assessed the effect ofthe overexpression of slycerol dehydrogenase (ORES sIdAB) on glycerol oxidation, demon- Strating that growth on glycerol was significantly improved in the overexpression strains (OD 28-29) compared to the control strains (0D 18-20) Both the DHA formation rate and the final DHA Concentration were affected such that up to about 30 gil of DHA was accumulated by the overexpression strains, compared to 18-25 g} Lin control strains when 50 gil glycerol was supplied. The higher concentration of enzyme possibly reduced the velocity of total glycerol dehydrogenase inactivation and slowed down the inactiva- tion of glycerol axidation and cell viability, This also explains the higher DHA production rate in the strains overexpressing the sldAl gene 43, Sucinie acid Succinate has a specialty chemical market in industries producing food and pharmaceutical products, surfactants and detergents, green solvents, biodegradable plastics and ingredients to stimdlate animal and plant growth. Due to its structure as a linear saturated dicarboxylic acid, succinate can be used as an intermediate chemical and be converted to 14-butanediol, tetrahydrofuran, y-butyrolactone, adipic acid, n-methylpyrtolidane and linear aliphatic esters (Zeikus tal, 1999). An inereasing demand for succinic acid is expected a its use is extended to the synthesis of biodegradable polymers such as polybutyrate succinate (PBS) and polyamides (Song and Lee, 2005), Ranucei et al. (2000) describe the synthesis of a new biodegradable polymer, poly(1-propylene succinate), obtained through the thermal polycondensation of succinic acid with 1.3-PDO. Succinate is currently produced petrochemically from butane through maleic anhydride; only natural succinic acid sold in the food market is praduced by fermentation (Zeikus et al, 1999) Succinate is normally produced under anaerobic conditions throug Several diferent metabolic pathways, such as PEP and pyruvate catboxylation. Anaerobispirilum succniciproducens is one of the most efficient suecinate producers and uses the PEP carboxylation pathway Fig, 4), catalyzed by PEP carboxykinase (or PEP carboxylase), ‘malate dehydrogenase, fumarase and fumarate dehydrogenase (Lee etal, 2004), Lee etal. (2001) showed that A succiniipraducens can efficiently convert glycerol to succinate. A maximum of 19 g/L. of succinic acid was obtained by fermentation ofA, succiniciproducens when glycerol Was used as the sole cazbon source ina medium supplemented with yeast exact and 296 g/l wher glycerol was fed with glucose According with the authors, succinic acid production from giyceral presents several advantages over glicose, suc as high succinic acid Yield with reduced acetic acid formation, This is advantageous because acetic acid imposes dificulties with respect to downstream processes forthe recovery of succinic aid. Considering the costs of Separation and purification of succinate from fermentation broth containing mixed acid, the formation of by-products isa problem to be solved through metabolic engineering and fermentation process optimization (Song and Lee, 2005), Fumaric acd is a chemical product that has several industrial applications, such as an acdulant in the food industry, the mansfactr- ingof sizing resins forthe paper industry and isa promising candidate in the obtainment of polymers (Zhou et l,2002}. While no workhas been performed to evaluate the production of fumaric acid from glycerol re is possibility of also being obtained frm glycerol fermentation, asitisa direc precursor of sucinic aid (Fig 4). 44, Propionic acid Propionic acd is another substance synthesized ina similar pathway to that ofsuccnic acd andi derived directly ftom a metabolic pathway balanced with regard to redox-equivalents (Barbirato et al, 19973) (Fig 4), Propionateisused as an antifungal agent in food and feed and as ‘abasic chemical to produce cellulose-based plastics, herbicides, solvents fand perfumes (Barbitato et al, 19972), arthritis deugs, flavors and ‘thermoplastics (Hinvmi etal, 2000), The numerous industrial applica- tions of propionic acid account for an increasing interest in the evelopment ofa biotechnological production process based on the renewable resource glycerol (Babirato eta, 1997a, Kimmie al, 2000), Barbirato etal (1997a) assessed the production of propionate from slycerol by three bacterial strains: Prpionibacterium acidipropionii, Propionibacterum acres and Clostridium propianicum. Consieri fermentation time and conversion yield, the best strain for glycerol conversion to propionate was P.aeidpropionic. The fermentation profile of this bacterium revealed five end products, consisting of propionic aid as the major product (0.84 mol/mol and productivity of (018 iL)h}, with the following minimal by-products: suceinat acetate, formate and n-propanol. Productivity of up to 036 sikh \was obtained and the maximal propionic acid concentration was 42 g) using 80 gi glycerol in Une medium. Acetic acid formation was low ‘when compared to the amount observed during glicose fermentation by P.acidipropionic. As the efficiency of propionic acid extraction through distillation is strongly limited by acetic acid (as can be observed for succinic aid), the extremely low acetie acid concentra- tion obzained by using glycerol as substrate should greatly increase the yield of propionic aid recovered through distillation and simplify the distillation procedure (Barbitato et al, 19572). The authors conclude that glycerol is a promising substrate for propionic acid production both in terms of conversion yield and productivity, whieh fs similar or superior to that of lactic acid (035 gilt) or glucose (028 gilfh). Better efficiency for propionic acid production from slycerol could be expected because of its higher reduction level over Conventional substrates, 45, Ethanol Ethanol i primarily produced from sugarcane in Brazil from corn starch inthe USA and from sugar beets in the EU. Ina study to obtain solvents from algal biomass, Nakas et al. (1983) describe a soil bacterium tentatively classified as a member of the genus Bacillus, Which produces ethanol (final concentration 70-96 gil) from 2 slycerolenriched algal mixture. Jarvis et al. (1997) demonstrated that formate and ethanol are the major products of glycerol fermentation by Klebsiella plantcol isolated from the rumen. These authors state “the observation that formate and ethanol are the major glyeral fermentation products is an interesting one with respect to micro- biological ecology of the red deer rumen”. With the expected surplas of glycerol from biodiesel, this observation will become interesting also to industrial microbiology. demonstrating the importance of basic studies in microbiah ecology and diversity to the development of applied microbiology. DDnarmadi etal (2006) report that &. coli can ferment glycerol in a pH-dependent manner, being linked tothe availabilty of CO>, which 's produced under acidic conditions by the oxidation of formate by the enzyme formate hydrogen lyase. Glycerol (10 g/L) was almost completely consumed within 84h, with ethanol (86%) and succinic acid (7%) accounting for 93%{molar basis) ofthe products. Only minor amounts of acetate were produced. According the authors, Ecol isPd Sina a. seco races 27 (2008) 3-38 ” already a good biocatalyst for the conversion of glycezol into ethanol and hydrogen. lto et al.(2005) state that E aerogenes can be used for the high-yield production of ethanol from biodiesel wastes contain- ing glycerol. ln a synthetic medium containing biodiesel wastes with glycerol at 80 mM, glycerol was consumed in 24 h, yielding Hy at (0.89 moljmol glycerol and ethanol at 1.0 moljmol glycerol. The ethanol yield from glycerol fermentation described is low and future development is needed in order for this fermentation to be applicable, 46. Citric acd Citric aid is produced in large quantities by fermentation, with 2 slobal production rate estimated at about 14 milion tons in 2004, Citric acid is widely used to impart a pleasant, tatfruity Mavor to foods and beverages and is also used as an additive in detergents, pharmaceuticals, cosmetics and toiletries (Soecol et al, 2006). In general, citric aid is produced by submerged microbial fermentation fon molasses using Aspergillas niger. In recent yeats, considerable intrest has arisen in finding less expensive carbon sources for citric acid production (Soccol etal, 2006) Papanikolaou et al. (2002) found that the etre acid producer Yarrowia ipolytica grows on glycerol, concluding that raw glycerol may bea suitable substrate for citric acid production. This yeast produced up to 35 g.of citric acid when a high initial concentration of glycerol was used in the culture medium. Growth and citric acid production parameters on glycerol were similar to those obtained using glucose. Imandi et al (2007) describe a statistically optimized medium for ¥. liplytica NCIM 3589, The authors predict a final cittic acid concentration of 774 gil in the fermentation broth wien using raw glycerol a a substrate AZ. Pigments Some studies have been made using glycerol as carbon source for pigment production, such prodigiosin and astaxantin.Prodigiosin isa deep red pigment formed by the Gram-negative bacterium Serratia marcescens when cultivated on tich solid media. This pigment isan immunosupressor and has been reported to induce apoptosis of several cancer cell line, such a6 hematopoietic cancer cell, colon cancer cells, lymphatic cancer eells and chronic lymphocytic leukemia cells, but not in nonmalignant cells, which indicates that prodigiosin may have potential as new antineoplastic candidate (Montaner et al, 2000; Campas et al, 2003), Tao et al. (2005) demonstrated that glyceral allowed the highest prodigiosin produc- tion rate when used as main carbon source in the medium among several athers carbon sources, In a two-step Feeding strategy using glucose for growth and then glycerol for prodigiosin synthesis, @ 'S moreescens mutant produced about 583 mg prodigiosinL in 30h, with glycerol asthe sole carbon source in a 5-I bioreactor. ‘Astaxanthin (33'-dihydroxy-B,-carotene-44"-ione) isa red or orange pigment found in marine environments. It is used to feed animals such as salmon, trout freshwater) and crustaceans in order to five them a color that appeals to consumers. Synthetic astaxanthin is Currently incorporated at arate of approximately 40-150 mgik of feed. However, a natural source of astaxanthin is preferred for an apparent safe uilization as 2 food additive (Kusdiyantini tal, 1998), “The yeast Phafa rhodozyma produces astaxanthin and itis considered a potential source of this pigment. Kusdiyantini et al, (1998) demonstrated that glycerol is a potential substrate for astaxanthin prodtction in P. rhodozyma, obtaining a maximum total volumetric fstaxanthin concentration of 33,7 mail 4.8, Plyhydroxyaleanoate Polyaydroxyaleanoate (PHA) represents a complex class of natu- rally occurring bacterial polyesters that are synthesized intracellularly as carbon and energy reserve materials (Ashby et al, 2004) by ‘microorganisms belonging tothe Bacteria and Archaea domains ofife (Solaiman et al, 2008)- These reserve materials are synthesized when the microorganisms are grown under nutrient-limited conditions (Ashby et al, 2005), Since PHAs are biodegradable and biocompatible, they may replace petroleum-dlerived polymers, which are widely used in medicine, drug delivery, agriculturehorticuture, the ber in and the consumer care branch (Solaiman etal, 2008), Due to the cost of the fermentative substrate, esearch effort abound to exploit inexpensive fermentable raw material as ermenta- tive substrates for PHA production (Solaiman etal, 2006). Bormann and Roth (1999) describe the production of polyhydroxybutyrate (PHB) by Methylobacterim rhodesianum and Ralstonia eutropha in a medium containing glycerol and casein peptone or casamino acids, but the conversion was low. Ashby et al. (2004) used Pseudomonas sp. for PHA production from a co-product stream of biodiesel containing glycerol, free fatty acids and fatty acid methylester and showed that glycerol was the preferred substrate by P oleavorans. This sinceresting in that slycerol isthe most energetically favorable substrate forthe formation of acetyl-CoA, the precursor for PHB synthesis. The authors conclude that co-produt from biodiesel canbe used for PHA production without the need for separating and recovering glycerol and other components. Ashby eta. (2005) showed that up to 5% glycerol (iv) could be used for the production of both PHB by P oleovorans and medium-chain- length PHA by ?.corrugata under identical growth conditions allowing mixed culture fermentations to be used in the formation of PHA polymer blends with varying blend ratios Koller etal. (2005) obtained 16.2 gil of PHA in the glycerol liquid phase from the biodiesel industry, which contained about 70% glycerol (w/w). PHB from glycerol has low molecular weight, because the substrate eauses termination of chain propagation through covalent esterification of glycerol to PHB in a chain terminating position, thus rendering PHAS of low molecular weight (Ashby et al, 2005; Koller tal, 2005) This finding is certain to have an important implication wher using glycerol to produce PHA (Solaiman et al, 2006), 49, Biosurfactonts Research in the field of biosurfactants has expanded quite a lt in recent yeats, due to their potential us in areas suchas the food industry, agriculture, pharmaceutical industry, crude il recovery and bioreme- dition of contaminated sites (Sannat etal, 2000), The development of studies on biosurfactants has a considerable importance, mainly in view ofthe present concern for protecting the envionment Biosurfactants hhave many advantages over chemically manufactured surfactants. They are less toni, biodegradable and have unique surface-actve properties (ognolo, 1995), Nitschke ct al. (2005) found that Pseudomonas feruginosacan synthesize rharnnolipids from glycerol a the ole carbon source, but the yield was low when compared to using hydrophobic substrates. Zhang etal. (2005) produced 15.4 gil-shamnolipis us feruginosa growing on 2 basal mineral medium containing glycerol 2s the sole carbon source. Rahman et al.(2002) observed that aeruginosa 1DS10-129 produced 177 gil. rhamnolipids on glycerol. These results show that glycerel can be wsed forthe production of biosuractants. 5. Conclusions In general, the use of renewable waste substrates is an environ- ‘menta-tiendy choice that also contributes to the reduction of waste treatment costs and increases the economic value of by-products. Some chemical commodities currently produced from petroleum can, in principle, be produced biotechnologically from glycerol using miero- organisms. This bioconversion would directly benefit the environment by obtaining biodegradable polymers, promoting the use of biodiesel and reducing petroleum dependency. The development of processes far Converting inexpensive glycerol into higher value products is expectedx» GP da Siva to. iecnalogy Abanees 27 (2008) 30-38, fo make biodiesel production more economical and will thus help establish more birefineries(Dharmadi etal, 2006). This will also have an important social impact, s small farmers cultivate oleaginous plants ‘which in turn are the basis fr biodiesel production Future research will fecus on the isolation and characterization of microorganisms that can use glycerol as a carbon source and generate valuable molecules with ‘unusual properties, thereby broadening the potential applicationsof this cheap by-product of transesterfcation Glycerol is a versatile catbon and energy source with many possible applications in industrial fermentation. Most research has focused on the employment of glycerol in solvent production such as dikydroxyacetone and 13-propanediol. However, glycerol can also be used as a earbon source to oblain other valuable microbial products, such as recombinant proteins and enzymes, medicinal drugs, antibiotis and fine chemicals. ‘Acknowledgments ‘This work was supported by Banco do Nordeste do Brasil, ETENE] FUNDECI program (convénio BNB/Uneb 2007/058), Bahia State University (Uneb), Fundacio de Amparo 2 Pesquisa do Estado da Bahia (Fapesb) and Sdo Paulo State University (Unesp) References Ain das 5 Mango. 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