Journal of Bioscience and Bioengineering VOL. 117 No. 3, 343e350, 2014 www.elsevier.

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Aerobic utilization of crude glycerol by recombinant Escherichia coli for simultaneous production of poly 3-hydroxybutyrate and bioethanol
Pramod Shah,1 Feng-Shen Chiu,1 and John Chi-Wei Lan1, 2, *
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li, Taoyuan 320, Taiwan1 and Bio-renery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li, Taoyuan 320, Taiwan2 Received 15 March 2013; accepted 30 August 2013 Available online 17 October 2013

Crude glycerol, an inevitable byproduct during biodiesel production, is emerging as a potential feedstock for fermentation, due to its availability and a reasonable price. Biological utilization of abundant crude glycerol to several value added products is contemporary research area with benecial features. Solving the problem of proper disposal and raising economic viability of biodiesel industries. Several researches have been directed toward the production of numerous products by using Escherichia coli, an ideal organism for heterologous expression of various foreign proteins. In this fashion, recombinant E. coli strains were constructed for the simultaneous production of poly 3-hydroxybutyrate (P3HB) and bioethanol from crude glycerol. The incorporation of aldehyde reductase (Alrd) and aldehyde dehydrogenase (AldH) in recombinant strain showed 2-fold increment in crude glycerol utilization under aerobic condition. Moreover, these two enzymes introduced an alternative pathway leading toward the potential production of bioethanol which was more than redox-balancing steps. Acetate was accumulated as an intermediate product. Subsequently, acetate was utilized as substrate in the second pathway, which directly converted acetyl-CoA to P3HB. This strategy demonstrated a potential production manner of bioethanol as an extracellular product and P3HB as water insoluble inclusion bodies inside E. coli. The maximum production of bioethanol and P3HB in the recombinant strain was 0.8 g LL1 (17.4 mmol LL1) and 30.2% (w/w dry cell weight), respectively, which were higher than the parental strain. 2013, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Escherichia coli; Aerobic fermentation; Crude glycerol; Bioethanol; Poly 3-hydroxybutyrate; Aldehyde reductase; Aldehyde dehydrogenase]

The alarming rate of consumption and combustion of fossil fuels to support global energy demand is continuously causing depletion of its limited stocks and severe impact on health and environment (1). Therefore, increasing enthusiasm is focused toward the generation of alternative energy sources. Utilization of biomass for biofuel production has created unexceptional opportunities in dealing with increasing demand and escalating prices of fossil fuel. Biofuel are renewable energy with rather high efciency and its sustainability and biodegradability contribute to the reduction of carbon dioxide emission and other harmful gases during combustion (2,3). The production capacity and market values of biodiesel and bioethanol are gaining lots of attention than other categories of biofuels (4). The appropriate method applied for the production of biodiesel is a homogeneous base-catalyze trans-esterication of triglycerides existing in vegetable oils, animal fats, or waste cooking oils with methanol (5). This process separates fatty acids moiety from triglycerides resulting in the generation of an inevitable byproduct, glycerol. One mole of glycerol is produce with the production of every 3 mol of methyl esters (i.e., biodiesel), resulting in

* Corresponding author at: Bio-renery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li, Taoyuan 320, Taiwan. Tel.: 886 34638800x3550; fax: 886 34559373. E-mail address: lanchiwei@saturn.yzu.edu.tw (J.C.-W. Lan).

10% by weight (wt %) of the total crude glycerol produce (6). In recent years, the mandatory demand of biodiesel has ooded the market with excessive crude glycerol. As a result, the price of crude glycerol has drastically plummeted enforcing burden on economic viability in the biodiesel industry. Crude glycerol alone account for 13e14 % of the total production costs (7). As a consequence of it, several biodiesel companies need governmental aids for supporting its operation (4). Biodiesel companies are seeking new alternative for this crude glycerol to higher-value added products. Biological utilization of crude glycerol serves as a feedstock in various fermentation processes with the production of several value-added products such as 1,3-propanediol (PDO), dihydroxy-acetone (DHA), bioethanol, butanol, propionic acid, succinic acid, and so on (8). Utilization of crude glycerol in fermentation processes account in reduction of manufacturing costs and higher yields of products than other sugar substrates. Among several alternative of value-added products during the fermentation process, the production of bioethanol is a redox-balance pathway (9,10). Utilization of crude glycerol by Escherichia coli, a model organism in bioindustries has long been proved under aerobic condition with minimal ethanol production. In recent years, anaerobic and micro-aerobic fermentation of crude glycerol has been well established for the higher yield of bioethanol by the construction of different recombinants E. coli (10e17). A high production of bioethanol metabolically requires a signicant re-engineering of

1389-1723/$ e see front matter 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.08.018

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TABLE 1. Microbial strains, plasmids and vectors used in this study. Strain/Plasmid/Primer Strain R. eutropha H16 E. coli BL21 E. coli BL21_ pARD33-BHB2 Plasmids pGEM-T easy pBAD33 pGEM-T easy_alrd pGEM-T easy_aldH pARD33 pBHB2 Primers Alrd primer Forward Reverse AldH primer Forward Description Wild-type Wild-type pARD33 and pBHB2 Source BCRCa-13036 BCRCa-51878 This study

metabolic pathway to derive bioethanol exclusively from acetylCoA with ATP as the energy source and NADPH as the sources of reducing equivalents (14,18). In this work, the knowledge of aerobic fermentation has been used to engineer E. coli for the effective conversion of crude glycerol to bioethanol with aldehyde reductase and aldehyde dehydrogenase. Aldehyde reductase (Alrd, EC 1.1.1.21) is an NADPH-dependent oxidoreductase enzyme which catalytically reduces various aldehydes and carbonyls, including monosaccharide to corresponding polyalcohol (19). Aldehyde dehydrogenase (AldH, EC 1.2.1.3), on the other hand, catalyzes the NADH-dependent oxidoreductase activity, oxidizing aldehydes to corresponding carboxylic acids (20). These two enzymes provided alternative pathways for the utilization of glycerol through glycolysis and further, established the conversion of acetate, the end product of glycolysis, to ethanol. It has been observed that under aerobic condition, acetic acid is major product which exits as acetate ion (21). Accumulation of acetate is toxic to organism thus a specic approach was considered for the conversion of acetyl-CoA to poly 3-hydroxybutyrate (P3HB), a second value-added product. These pathways eliminated the accumulation of acetate. Poly-hydroxyalkanoates (PHAs) are another value-added product gaining lots of attention as an ingredient for thermoplastics. With its biodegradable nature and a variety of other material properties, they are suitable for several applications (22,23). PHAs are carbon and energy reserve accumulated in many bacteria as intracellular inclusions under excess carbon reserve or unfavorable condition. These intracellular inclusions enhance the tness of bacteria and also contribute to redox-balance (24). P3HB is a well known member of PHAs family which has been widely studied. However, the production of P3HB in large scale is limited to substrates cost (25). Therefore, the use of crude glycerol for this purpose can be an interesting opportunity. Synthesis of P3HB from glycerol as carbon source has been evaluated in several microbial strains along with different fermentation strategy (26,27). Ralstonia eutropha, a well known organism for the production of PHAs contains phaCAB operon for the conversion of acetyl-CoA to P3HB. It has been established that R. eutropha can accumulate more than 80% of P3HB content (28). The recombinant E. coli constructed by harboring phaCAB operon from R. eutropha (phaCABRE) has been shown to accumulate 67% of P3HB content, using 2e4% glucose as carbon source (29). In the meantime, bacterial hemoglobin gene (vgb gene) from Vitreoscilla hemoglobin has been noted to enhance cell density, protein production and bioremediation by increasing oxygen availability in the culture (30). The co-expression of phaCABRE and vgb genes in E. coli was revealed to enhance P3HB production (31). The aim of the current study was to generate desirable products from crude glycerol through alternate pathways provided by two plasmids. Our attempt resulted in simultaneous production of an extracellular and an intracellular product, bioethanol and P3HB, respectively.
MATERIALS AND METHODS Bacteria strains and plasmid used The wild-type strains, R. eutropha H16 and E. coli BL21(lDE3), were kindly provided by Bioresource Collection and Research Center (BCRC), Taiwan. E. coli BL21(lDE3) was used as a host for high level expression of cloned genes. It contained T7 RNA-polymerase gene in a lysogenic and inducible form. This strain is characterized by two protein decomposition enzyme genes lon and ompT that has been articially damage, making it suitable for the use of enzymes as a foreign gene expression (32). All strains used in this study along with plasmids and primers required for the construction of recombinant strains are listed in Table 1. The molecular biology methods applied here are according to the manufacturers protocols. Construction of single and double plasmid Plasmid pBAD33 was kindly provided by Prof. Soo Po-Chi (Tzu Chi University, Taiwan). This plasmid contained PBAD promoter of araBAD (arabinose) operon and its regulatory gene, araC, followed

Cloning vector Low level expression vector pGEM-T easy :: alrdRe pGEM-T easy :: aldHRe pBAD33 :: alrd/aldH pBluescript II KS :: phaCABRE

Promega Prof. Su Boqi (Tzu Chi University) This study This study This study The Yuan Ze PHA team

GAGGTACCAAGGAGGA AATGAAGCAAGTCAC GAGTCGACAGCTCAAA GCATTTCCAG AGTCGACAAGGAGAAT ATGCGAGAAGTCCC CGA GAAAGCTTTCAACGCAG AAGCAGGCGCAA

This study This study

This study

Reverse
a

This study

BCRC: Bioresource Collection and Research Center (Taiwan).

by multiple cloning site (MCS2) and M13 intragenic region with chloramphenicolresistance (cmR) gene, resulting in a high level expression system (33). Polymerase Chain Reaction (PCR) was performed for the amplication of Alrd and AldH from genomic DNA of R. eutropha H16 on Select BioProducts (Edison, NJ, USA). The reagents used were purchased from New England Biolabs (Ipswich, MA, USA) and Promega Corporation (Fitchburg, WI, USA). Expression vector pARD33 was constructed as shown in Fig. 2A, by cloning Alrd and AldH genes in cloning vector pBAD33 with restriction sites of kpnI/SalI and SalI/HindIII, respectively. The plasmid, pBHB2, was kindly provided by the Yuan Ze PHA team at Yuan Ze University (Taiwan) as mentioned in Table 1. This plasmid contains P3HB synthesis genes phaCABRE, from R. eutropha H16 and a bacterial hemoglobin gene (vgb gene) from V. hemoglobin with ampicillin-resistance gene (AmpR). The three main enzymes that has been well established for the conversion of acetyl-CoA to the P3HB in R. eutropha are: phaA (b-ketothiolase; EC 2.3.1.9), phaB (NADPH-dependent acetoacetyl-CoA reductase; EC 1.1.1.36) and phaC (PHA polymerase; EC 2.3.1) (28). For this plasmid, Isopropyl b-D-1-thiogalactopyranoside (IPTG) acted as an inducer for protein regulation and was purchased from SigmaeAldrich Corporation (St. Louis, MO, USA). The expression vectors pARD33 and pBHB2 were transformed into E. coli BL21(lDE3) individually for the construction of recombinant strains with single plasmid: E. coli BL21_pARD33 and E. coli BL21_pBHB2, respectively (as shown in Fig. 2B and C). The size of amplied Alrd and AldH genes, plasmids pBAD33, pARD33 and pBHB2 were conrmed by running 0.8% agarose gel, stained with ethidium bromide and visualized under UV light in Dolphin-Doc Plus (Wealtec Corp., NV, USA). The E. coli BL21_pARD33-BHB2 was constructed with transformation of two plasmids (pBHB2 and pARD33) one after another in single host, E. coli BL21(lDE3) as shown in Fig. 2D. The constructed recombinant strains were subsequently cultured and applied for further study. Culture medium The LuriaeBertani (LB) medium was used to prepare LB broth and LB plates. LB broth contained: 10.0 g L1 of tryptone, 5.0 g L1 of yeast 1 extract and 10.0 g L of NaCl, whereas LB plates contained ingredient of LB broth with addition of 15.0 g L1 of agar. Appropriate antibiotics were used at following concentrations: 50 mg ml1 chloramphenicol (Cm) and 100 mg ml1 Ampicillin (Amp) obtained from the SigmaeAldrich. The crude glycerol used in this study was provided by Greatec Green Energy Company, a local biodiesel plant in Taiwan. Greatec Green Energy produces biodiesel from rened waste cooking oil using sodium methoxide as catalyst. The crude glycerol was composed of 80% crude glycerol, 11% water, 9% metal ions and other impurities. All trials were conducted using the same batch of crude glycerol. The minimal dene medium used in this study contained 30 g L1 crude glycerol, 6.70 g L1 of Na2HPO4-7H2O, 2.50 g L1 of (NH4)2SO4, 1.50 g L1 of KH2PO4, 0.20 g L1 of MgSO4-7H2O, 10.00 mg L1 of CaCl2 and 5.0 ml of trace metal solution (6.84 g L1 of H3BO3, 6.00 g L1 of Na2EDTA, 0.86 g L1 of MnCl2-4H2O, 0.29 g L1 of FeCl3-6H2O, 0.06 g L1 of ZnCl2, 0.026 g L1 of CoCl2-6H2O and 0.002 g L1 of CuSO4-5H2O) and 2 g L1 of yeast extract. Most of the chemicals were obtained from SigmaeAldrich. To prevent reaction between chemicals; trace metal solution, crude glycerol, yeast extract and salts were autoclaved separately for 15 min at 121  C while antibiotics were sterile ltered.

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FIG. 1. Metabolic pathways involved in glycerol metabolism in Escherichia coli. Abbreviations: Alrd, aldehyde reductase (EC 1.1.1.21); AldH, aldehyde dehydrogenase (EC 1.2.1.3); phaA, b-ketothiolase (EC 2.3.1.9), phaB, NADPH-dependent acetoacetyl-CoA reductase (EC 1.1.1.36) and phaC, PHA polymerase (EC 2.3.1).

Culture condition Each recombinant strain was grown from one loop-full stock culture, streaking on LB plates with appropriate selection marker and incu bated for overnight at 37 C. Single colony from plate was used to inoculate 10 mL LB broth with specied selection marker and was incubated in a rotary shaker (200 rpm) at 37  C for 24 h. Batch cultures were conducted in 500 mL shake ask with 100 mL working volume. A pre-culture (3% v/v) were used to inoculate 100 ml of minimal dene medium and incubated in rotary shaker with a speed of 200 rpm at 37  C for 48e72 h. Samples were drawn periodically from the post inoculation at regular intervals of time. Assay of expression enzymes The expression of Alrd and AldH proteins were analyzed on a 12% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) in a vertical slab gel apparatus, followed by Coomassie Brilliant Blue staining. The protein concentrations in the supernatant and cell pellet extract after sonication of wet cell-pallets were determined by using the Bradford method (34) with bovine serum albumin (BSA) as a standard. The enzyme activities were calculated from the linear slope of increasing absorption of NAD/NADP reduced to NADH/NADPH (mM mL1) at 340 nm, respectively. The Alrd activity was performed in a 1 mL mixture at 37  C by using 4.67 mM D,L-glyceraldehyde as substrate in a buffering solution comprised of 0.25 M sodium phosphate, 0.38 M ammonium sulfate, 0.11 mM NADPH and 0.1 mM EDTA at pH 6.8 (35). The activity of AldH was performed in a 1 mL mixture containing 50 mM sodium phosphate buffering solution at pH 7.5, 50e100 mL of crude cell extract or 10e15 mg of puried enzyme, 0.5 mM NAD and 1 mM D,L-glyceraldehyde as substrate (36). One unit of enzyme activity is dened as the amount of protein that produces 1 pmol NADH or NADPH per minute. Analytical methods Biomass was measured by measuring dry cell weight which in turn was correlated with optical density. Cell growth was monitored by

measuring the optical density of culture at 600 nm, on UltraSpec 2100 pro UVeVisible spectrophotometer (GE Healthcare, Wauwatosa, WI, USA). In this case, original medium was used as the blank. Three consecutive samples were drawn and analyzed each time for the accuracy of work. The concentration of bioethanol and residual crude glycerol along with acetate was analyzed on a LC-2000 plus series high performance liquid chromatography (HPLC) systems (Jasco-Analytical Instruments, Japan) equipped with an ion exclusion column ICEeIONe300 (Transgenomic Labs, Omaha, NE, USA) operated at 60  C. Firstly, the samples were ltered through 0.45 mm of HEPA lters (Billerica, MA, USA) and 10 ml was auto-injected to the HPLC. The refractive index (RI-2031 plus) detector at 35  C was employed to analyze these samples. The samples were eluted at 0.4 mL min1 with 5 mM sulfuric acid (0.0085 N H2SO4) prepared in HPLC grade water. Data were processed and analyzed using the ChromNAV software. P3HB was recovered from the dry cell mass through solvent extraction and the P3HB content (wt %) was expressed as percentage of P3HB mass in dry cell mass. Briey, 20 mg of P3HB-containing cell dry weight was mix with 2 mL of chloroform and 2 mL of methanol solution containing sulfuric acid (1 mL of H2SO4 and 99 mL of methanol). The solution was incubated at 100  C for 15 h, and then 1 mL of 1 M NaCl was added to facilitate phase separation. After recovery of organic (chloroform) phase, 20 mL of gas chromatograph internal standard (GCIS) was added, and 300 mL of bottom organic layer was collected as sample. For analysis, 0.5 mL of sample was injected into a Shimadzu gas chromatograph GC-2014 system (Shimadzu Scientic Instruments, Columbia, MD, USA) equipped with a ame ionization detector (GC-FID) and a Phenomenex Zebron ZB-5 capillary column (30 m 0.25 mm 0.25 mm). Oven temperature was held at 100  C. Both injector and detector temperatures were set at 220  C. Nitrogen was used as the carrier gas and diphenyl ether was used as internal standard. Data were processed and analyzed by using Peak-ABC software. The

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FIG. 2. Construction and transformation of plasmids. (A) Cloning of AldH and Alrd to plasmid pBAD33 for the construction of pARD33. (BeD) Transformation of the constructed plasmids in Escherichia coli BL21(lDE3).

total P3HB content, yield of biomass and products were calculated according to the following equations: P3HB content w=w gram of P3HB=gram of total biomass produced 100 (1) Yield of biomass : Yx=s gram of biomass produced=gram of crude glycerol consumed (2) Yield of product : Yp=x gram of product produced=gram of dry cell weight DCW (3) Substrate consumption rate : ds=t total substrate consumed mass=volume= time interval (4)

RESULTS AND DISCUSSION Effect of pBAD33 plasmid on crude glycerol utilization The role of plasmid pBAD33 was marked in the strain E. coli BL21_pBAD33. Fig. 3A and Table 2 depicted the results in terms of cell growth and crude glycerol utilization, respectively. The E. coli BL21(lDE3), which was employed as control, showed better growth than E. coli BL21_pBAD33. However, crude glycerol consumption was noted to be similar in both strains, i.e., around 50%. This result proved that cloning vector had no signicant effect upon cell growth and crude glycerol utilization. Impact of Alrd consumption With and the AldH upon conrmation crude glycerol of ineffective

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glycerol concentrations for E. coli BL21_pARD33 and E. coli BL21(lDE3) were noted to be 1.13 and 12.54 g L1, respectively. The comparisons among different recombinant strains are summarized in Table 2. The results indicated that the presence of Alrd and AldH dramatically improved in crude glycerol utilization. Fig. 1 shows an alternative pathway introduced by Alrd and AldH without occluding the original pathway of crude glycerol metabolism. The Alrd catalyzes the oxidation of crude glycerol to D-glyceraldehyde, governing the release of NADPH (35). However, AldH catalyzes the NADH-dependent conversion of D-glyceraldehyde to D-glycerate with reduction of NAD to NADH (37,38). The aerobic condition was important for the reduction of NADP/NAD to NADPH/NADH, respectively. The accumulated D-glycerate enters the glycolysis pathway for the generation of pyruvate and later acetyl-CoA under aerobic condition. This alternative pathway furnished with the improvement of crude glycerol utilization in E. coli BL21_pARD33. Effects of Alrd and AldH in strains harboring single and double plasmid The presence of Alrd and AldH showed a 2fold increment in crude glycerol consumption by E. coli BL21_pARD33 harboring the single plasmid (Table 2). The E. coli BL21_pARD33-BHB2 harboring two plasmids showed similar results in term of crude glycerol utilization and total crude glycerol consumption rate with E. coli BL21_pARD33. After 48 h, the crude glycerol consumption was reported to be 96.1% and 98.2% (w/w) in E. coli BL21_pARD33 and E. coli BL21_pARD33BHB2, respectively. Nevertheless, higher biomass was observed in E. coli BL21_pARD33-BHB2 due to the elimination of the toxic effect of acetate and conversion of acetyl-CoA to P3HB which was redox-balancing pathways. Presence of vgb gene was another reason for the production of higher biomass which increases the oxygen availability in the batch culture (30,31). The recombinant strains harboring the plasmid pBHB2, i.e., E. coli BL21_pBHB2 and E. coli BL21_pARD33-BHB2 were distinguished in terms of biomass and crude glycerol utilization. Higher biomass and crude glycerol utilization were observed in E. coli BL21_pARD33-BHB2 whereas only 59.73% of crude glycerol was utilized by E. coli BL21_pBHB2. Higher crude glycerol utilization (98.2 %) was the reason for the higher biomass production in E. coli BL21_pARD33-BHB2. This result demonstrated that Alrd and AldH had beneciary action in terms of crude glycerol consumption (Table 2). Acetic acid as a competitive by-product Under aerobic condition, instead of oxygen availability, the production of acetate is dependent on the rate of crude glycerol utilization and biomass production (21). The clear explanation for the maximum consumption of crude glycerol in E. coli BL21_pARD33 and other recombinant strains were enlightened through HPLC analysis. Acetate is toxic to E. coli in higher concentration, thus its accumulation retarded the cell growth and hampered the consumption of crude glycerol. Continuous accumulation of acetate was observed in E. coli BL21(lDE3) for 24 h with stationary value thereafter (Fig. 3C) this retarded the further growth. In E. coli BL21_pARD33, high accumulation of acetate was observed at 9th hour of culture presumably due to overfeeding of abundant crude glycerol facilitated with Alrd and AldH. Declination in the acetate concentration was noted between 9 and 24 h, which resulted in acceleration of bioethanol production through Alrd and AldH (Fig. 3B). The lowest value of acetate was observed at 24 h. By nullifying the toxicity of acetic acid, E. coli BL21_pARD33 was able to further utilize crude glycerol. After 24 h, a slight increment in the accumulation of acetate was noticed with respect to the limitations in metabolic pathways and also oxygen availability. This resulted in no further increment in biomass without occluding bioethanol production.

FIG. 3. Outcomes of crude glycerol utilization by E. coli strains in terms of dry cell weight, ethanol production and acetate accumulation: (A) cell growth prole in term of dry cell weight, (B) bioethanol production at different time periods, and (C) production of acetic acid during cell culture.

phenomenon on crude glycerol utilization from original vector, pBAD33, the effect of expression vector, pARD33 containing Alrd and AldH was investigated in E. coli BL21_pARD33. Fig. 3A demonstrated that the prole of cell growth of E. coli BL21_pARD33 was similar to that of E. coli BL21(lDE3). However, the crude glycerol utilization was nearly 2-fold higher in E. coli BL21_pARD33 (Table 2) than the latter. The residual crude

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TABLE 2. Summarization of results observed during aerobic utilization of crude glycerol by E. coli strains.

J. BIOSCI. BIOENG.,

Strain E. E. E. E. E. coli coli coli coli coli (WT) (pBAD33) (pARD33) (pBHB2) (pARD33-BHB2)

DCW (g L1) X 4.16 3.71 4.92 5.58 6.47

Maxm EtOH concn. (g L1) p1 0.10 0.09 0.65 0.00 0.80

PHB cont. (w/w) 15.6 30.2

PHB concn. (g L1) p2 0.87 1.95

Consumption of crude glycerol(s) cont. (w/w) 55.13 52.91 96.1 59.73 98.2

Yx/s (g g1) 0.27 0.25 0.18 0.31 0.23

Yp1/x (g g1) 0.02 0.02 0.13 0.00 0.12

Yp2/x (g g1) 0.15 0.30

ds/t (g L1 h1) 0.32 0.31 0.58 0.37 0.60

En dash (e) denotes no data found; whereas X, p1, p2 and s denote dry cell weight (DCW), ethanol production, P3HB production and substrate, respectively. Y denotes yields whereas ds/t is total substrate consumption rate.

Signicantly, lower accumulation of acetate was observed in E. coli BL21_pBHB2 as an overow mechanism which exceeded the transfer rate of acetyl-CoA to P3HB. In E. coli BL21_pARD33-BHB2, negligible amount of acetate was noted due to well balanced metabolic pathways for the conversion of acetyl-CoA to P3HB and generation of bioethanol from over accumulation of acetate. This resulted in higher conversion of crude glycerol in term of biomass, bioethanol production and P3HB accumulation. Bioethanol as an extracellular product The bioconversion of crude glycerol leads to the diversity of metabolic products due to the complexity of metabolism in micro-organisms (10). In reference to Fig. 1, the acetyl-CoA produced in glycolysis pathway, proceed through two metabolic routes. Firstly, acetyl-CoA acted as substrate in tricarboxylic acid cycle (TCA cycle) for the generation of energy to support cell functions and growth. The second pathways led to the conversion of acetyl-CoA to acetate, catalyzed by acetyl-CoA synthetase. The reversed pathway was catalyzed by acetyl-CoA hydrolase which converted acetate to acetyl-CoA (39). The production of bioethanol from acetate or acetyl-CoA was the redox-balancing pathways that regenerated necessary equivalents of NAD and NADP. In E. coli BL21(lDE3) the production of bioethanol from acetyl-CoA is governed by two enzymes: acetaldehyde dehydrogenase and alcohol dehydrogenase (40). Under aerobic condition, the presence of oxygen inhibits the pathways for the conversion of bioethanol. As a results, 0.10 g L1 bioethanol was only produced in E. coli BL21(lDE3) culture (Fig. 3B). However, the presence of Alrd and AldH provided secondary metabolic pathway for the utilization of excess acetate to bioethanol. Here, AldH catalyzed the reduction of acetate to acetaldehyde (37), which was further catalyzed to bioethanol by Alrd. The higher metabolism of crude glycerol by E. coli BL21_pARD33 was due to further utilization of intermediate acetate to bioethanol with the help of Alrd and AldH. Fig. 3C shows the amount of acetic acid accumulated in E. coli BL21_pARD33 culture (0.57 g L1) which was nearly 4-fold lower than that of E. coli BL21(lDE3) (2.14 g L1). Moreover, the high production of bioethanol was noted in the recombinant strains harboring ethanol producing enzymes Alrd and AldH, i.e., E. coli BL21_pARD33 and E. coli BL21_pARD33-BHB2 with the maximum concentration of 0.65 g L1 and 0.8 g L1, respectively, at 48 h (Fig. 3B). The total production of bioethanol was nearly 8-fold higher in E. coli BL21_pARD33-BHB2 than that of E. coli BL21(lDE3) which

has been summarized in Table 2. The yield of bioethanol was comparatively low in E. coli BL21_pBAD33 and E. coli BL21(lDE3) (0.02 g g1 DCW). Notably, no bioethanol production was observed in E. coli BL21_pBHB2 due to the lack of Alrd and AldH. Nevertheless, acetyl-CoA was presumably employed for P3HB production that balanced the redox pathway in E. coli BL21_pBHB2. The production of bioethanol was not associated with the growth in biomass, as production of bioethanol was even observed in the stationary phase where biomass concentration remained constant (Fig. 3B). Intracellular production of P3HB The P3HB production was accompanied through acetyl-CoA with a sequential enzymatic pathway of phaCABRE operon. In order to eliminate the over accumulation of acetate from crude glycerol digestion in E. coli BL21_pARD33, the second metabolic pathway was introduced through plasmid pBHB2 in E. coli BL21_pARD33-BHB2 as mentioned in materials and methods section. The phaCAB operon eliminated the production of acetate by the conversion of acetylCoA to P3HB. The total P3HB content accumulated in E. coli BL21_pBHB2 and E. coli BL21_pARD33-BHB2 were analyzed to be 15.6% and 30.2% (w/w), respectively (Table 2). The P3HB yield was enhanced by 2-fold from 0.15 to 0.30 g g1 DCW with total P3HB concentration from 0.87 to 1.95 in E. coli BL21_pARD33-pBHB2 than E. coli BL21_pBHB2. Anaerobic utilization of crude glycerol and products formed In accordance with the results shown in Table 2, very slow rate of glycerol utilization and biomass production were demonstrated under anaerobic condition (Table 3). The anaerobic cultivation conditions were similar to aerobic condition, despite of purging nitrogen gas for the purpose of anaerobic circumstance. Under this circumstance, the growths of strains without Alrd and AldH were lowered due to imbalance of the redox potential. In E. coli BL21(lDE3), the bioethanol production was noted to be 5-fold higher than that of aerobic condition. This result was in accordance with the several references (9,11,14,15,17) showing that anaerobic condition was more appropriate for bioethanol production from glycerol. Under anaerobic condition, no signicant improvement in the yield of bioethanol production was noted in the strains harboring Alrd and AldH. Anaerobically, higher biomass was observed in E. coli BL21_pARD33-BHB2 than other strains under same condition. Moreover, 2-fold higher bioethanol production was noted in

TABLE 3. Summarization of results obtained under anaerobic condition in E. coli strains. Strain E. E. E. E. coli (WT) coli (pARD33) coli (pBHB2 coli (pARD33BHB2) DCW (g L1) X 0.37 0.41 0.26 0.80 Maxm EtOH concn. (g L1) p1 0.48 0.53 0.17 0.42 PHB cont. (w/w) e e e 31.5 PHB concn. (g L1) P2 e e e 0.17 Consumption of crude glycerol(s) cont. (w/w) 4.19 5.71 2.65 7.42 Yx/s (g g1) 0.28 0.22 0.31 0.35 Yp1/x (g g1) 1.30 1.29 0.65 0.53 Yp2/x (g g1) e e e 0.27 ds/t (g L1 h1) 0.03 0.04 0.02 0.05

En dash (e) denotes no data found; whereas X, p1, p2 and s denote dry cell weight (DCW), ethanol production, P3HB production and substrate, respectively. Y denotes yields whereas ds/t is total substrate consumption rate.

VOL. 117, 2014 E. coli BL21_pARD33-BHB2 under aerobic condition than anaerobic. In spite of no bioethanol production in E. coli BL21_pBHB2 under aerobic condition, slightly bioethanol production was noted anaerobically. This was for sure to balance the redox-equivalent Pathways. However no P3HB was accounted because of signicantly low biomass. The production of bioethanol under anaerobic culture was lower than the aerobic culture in the recombinant strains harboring Alrd and AldH. This showed the beneciary role of Alrd and AldH for the production of bioethanol aerobically. Improvement of crude glycerol utilization under aerobic condition yielded in higher production of bioethanol and P3HB. Total crude glycerol consumption rate (ds/t) was noted to be nearly 10-fold lower in anaerobic than aerobic condition (Table 2). Use of crude glycerol over pure glycerol Crude glycerol is a cheap and abundant material containing unreacted methanol, soap, salts, water and solid organic materials acquire during biodiesel production (40). Presence of these impurities in crude glycerol has been expected to inuence the negative effect in bioconversion process. Hence, for achieving a higher yield of bioethanol, many researchers focused on using pure glycerol under anaerobic or micro-aerobic conditions due to high glycerol content and negligible amount of impurities. Several grades of rened glycerol have been commercialized that differ in glycerol content. Purication of crude glycerol is an expensive process due to no market value of crude glycerol. Therefore, adopting the process of purication for crude glycerol or using commercially puried glycerol for the production of value added products is an infeasible route in bio-industry (13e18). Table 4 summarizes the production of bioethanol by different strains of E. coli on pure and crude glycerol, under different cultivation condition. Under aerobic condition, the complete utilization of crude glycerol can be achieved with higher biomass and low bioethanol yield (12,14). The production of only bioethanol was not a feasible proposition due to high operational cost and no effective use of biomass. Hence, the strategy discussed here resulted in the effectual use of biomass for the accumulation of P3HB.

SIMULTANEOUS REFINERY OF CHEMICALS FROM GLYCEROL

349

Awareness to environmental and energy issues have led to the development of biomass-based bioconversion processes. Production of fuels and chemicals from crude glycerol is cost-effective process which has been evaluated in recent years. E. coli can utilize crude glycerol for the production of bioethanol under aerobic as well as anaerobic condition (Table 2) (13,14). Bioethanol is only the secondary product of the crude glycerol fermentation under any condition. As a result, the yield of bioethanol production is relatively low than other metabolites. Hence, several modications have been made with the help of biotechnology and molecular biology for the improvement of bioethanol production from crude glycerol. In this work, the natural pathway for crude glycerol utilization was enhanced by the addition of Alrd and AldH, which further provided the alternative and effective route for bioethanol production in E. coli BL21_pARD33 (Fig. 1). Under aerobic condition, acetate was the major product derived through acetyl-CoA. The phaCAB operon used acetyl-CoA as a substrate for the production of P3HB which reduced the formation of acetate (28). Higher yield of bioethanol was observed under aerobic condition in the strains harboring Alrd and AldH. This result was contradictory with the general belief that high bioethanol can be only produced under anaerobic condition. This was probably due to the role of Alrd and AldH in aerobic condition which provided with addition pathway for the production of bioethanol and also balanced the redoxequivalents. The production of bioethanol and P3HB at the same time facilitated with high degree of value-added products from crude glycerol under both aerobic and anaerobic condition. However, higher yields of bioethanol and P3HB were noted under aerobic condition along with complete utilization of crude glycerol in E. coli BL21_pARD33-BHB2 at 48 h of post-culture. Considering the huge crude glycerol surplus, our nding established a new model for the conversion of crude glycerol under aerobic condition by using an E. coli-based platform. The total production of bioethanol and P3HB content was noted to be 0.8 g L1 and 30.2% (w/w), respectively, in E. coli BL21_pARD33BHB2.

ACKNOWLEDGMENTS
TABLE 4. Summary table showing utilization of pure and crude glycerol for bioethanol production by different strains of E. coli under different conditions. Strain Pure glycerol Escherichia coli MG1655 MG1655 (ATCC 700926) MG1655 SS1 BL21 BW25113 SY04 (pZSKL0MgldA) CT1061LER EH05 [pZSKLMgldA] BW25113 MG1655 TCS099 e50rep1/ pLOI297 TCS099/pLOI297 Crude glycerol Escherichia coli MG1655 (pZSKLMgldA) SY04 (pZSKLMgldA) SY04 (pZSKLMgldA) BL21_WT BL21_pARD33 BL21_pARD33-BHB2 Condition Initial glycerol Ethanol yields concn. (g L1) (mol mol1) Source

Anaerobic Anaerobic Anaerobic Anaerobic Anaerobic Anaerobic Anaerobic Micro-aerobic Micro-aerobic Micro-aerobic Micro-aerobic Micro-aerobic Aerobic

10 10 10 20 20 20 10 30 60 20 20 40 40

0.10 0.76 0.92 1.00 0.99 0.59 1.04 1.02 0.84 0.66 0.52 0.98 0.01

13 16 12 15 15 15 9 17 14 14 14 18 18

This research was supported by National Science Council (Taiwan) with funding number 99-2622-E-155-002-CC2 and 1012221-E-155-042. We thankfully acknowledge help from Prof. Shaw-Shan Wang (Distinguished Professor, Department of Chemical Engineering and Materials Science, Yuan Ze University) for improving English grammar, verb usage, sentence structure and general readability of the manuscript revision.

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