Application of random mutagenesis to enhance

Application of random mutagenesis to enhance production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil Stanislav Obruca1*, Ondrej Snajdar2, Zdenek Svoboda3, Ivana Marova1 1

Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic 2

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic 3

Research Institute of Brewing and Malting, Malting Institute Brno, Mostecka 7, 614 00 Brno, Czech Republic *

Corresponding author: Stanislav Obruca, Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic. E-mail: [email protected], Tel: +420 541 149 486, Fax: +420 541 211 697

Abstract: Using random chemical mutagenesis we obtained mutant of Cupriavidus necator H16 which was capable of improved (about 35%) production of poly(3-hydroxybuytrate) (PHB) as compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably due to activation of these enzymes, we also observed an increase of NADPH/NADP+ ratio. It is likely that as a side effect of increasement of NADPH/NADP+ ratio activity of PHB biosynthetic pathway was enhanced, which supported accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of utilization of waste frying oils and production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for development of innovative fermentation strategies for production of PHA and also it might help to define novel targets for genetic manipulations of PHA producing bacteria.

Keywords Cupriavidus necator, polyhydroxyalkanoates, waste frying oils, oxidative stress, random mutagenesis, 2-methyl citrate cycle

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Introduction Polyhydroxyalkanoates (PHA) are linear polyesters which occur in many bacteria as storage compounds for carbon and energy. Due to their mechanical properties, PHA have attracted much attention as a biodegradable alternative to traditional petrochemical plastics (Kessler and Wilholt 1999). Mechanical properties of PHA strongly depend on monomer unit composition. Homopolymer of 3-hydroxybutyrate, poly(3-hydroxybutyrate) (PHB), which is the most ubiquitous PHA in nature, possesses highly crystalline structure that makes it relatively stiff and brittle. Nevertheless, mechanical properties of PHB can be significantly improved by incorporation of other monomer units into PHA structure.

For

instance, incorporation of

3-hydroxyvalerate

results

in material

(poly(3-

hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV)) with increased flexibility and decreased melting point (Sudesh et al. 2000). Various PHA producing bacterial strains such as Cupriavidus necator (Reinecke and Steinbuchel 2009), Alcaligenes latus (Yamate at al. 1996), Halomonas boliviensis (Guzman et al. 2009), Delftia acidovorans (Mothes and Ackermann 2005), Bacillus megaterium (Faccin et al. 2012), Burkholderia sacchari (Pradella et al. 2010) or genetically modified E. coli (Gao et al. 2012) have been considered as candidates for industrial production of PHA. The suitability of a bacterium for PHA production depends on many different factors including stability and safety of the organism, growth and accumulation rates, achievable cell densities and PHA contents, extractability of the polymer, molecular weights of polymer, range of utilizable carbon sources, costs of the carbon source and the other media components, and occurrence of by-products (Kessler and Wilholt, 1999). Apart from utilization of bacterial monocultures, production of PHA by mixed bacterial cultures can be also considered as an alternative approach (Sefarim et al. 2008). To maximize productivity of the process, various fermentation strategies including continuous, repeated batch and fed-batch cultivation modes are used to reach high cell density and PHA yields (Ienczak et al. 2013). Generally, the main obstacle preventing PHA from entering market massively is their production cost. Analysis and economic evaluation of the bacterial PHA productions suggested, that the cost of substrate (mainly carbon source) contributed the most significantly (up to 50%) to the overall production cost. Thus, cheap waste substrates attract attention of both scientific researches and 2

industrial companies in order to reduce PHA production cost and make this environmental friendly product also more economically feasible (Choi and Lee, 1997). Thus, waste frying oils and similar cheap fatty substrates for PHA production have recently attracted attention of several research groups (Obruca et al. 2010a; Budde et al. 2011; Mozejko et al. 2012). Fatty acids-based carbon sources are suitable substrates for PHA production, because they are metabolized via -oxidation pathway leading to formation of acetyl-CoA which is the crucial substrate of PHA biosynthetic pathway (Kessler and Wilholt 1999). The

Gram-negative

facultative

chemolithoautotrophic

hydrogen-oxidazing

bacterium

Cupriavidusnecator H16 (formerly Ralstonia eutropha, Wautersia eutropha and Alcaligenes eutrophus) represents one of the model organisms for PHA metabolism. Moreover, due to its capability of utilizing fatty acids, triacylglycerols and other substrates while accumulating high amounts of PHA (more than 70% of its cell dry weight), C. necator H16 is considered as a candidate for industrial production of PHA (Reinecke and Steinbuchel 2009). This bacterium is capable of accumulation of either homopolymer PHB or copolymer P(3HB-co-3HV); however, for accumulation of the later valerate, propionate or other propionyl-CoA generating substrate must be introduced into fermentation media (Ewering et al. 2006). The aim of this work was to employ random mutagenesis to gain PHA overproducing mutants of C. necator capable of utilizing waste frying oils as an economically feasible substrate. Further, we looked into the metabolic reasons of improved production of PHA. The understanding of metabolic processes in PHA overproducing mutants may help to define new targets for genetic manipulations and develop novel fermentation strategies for improvement of PHA production.

Materials and Methods Microorganisms and chemicals Cupriavidus necator H16 (CCM 3726) was obtained from Czech Collection of Microorganisms, Brno, Czech Republic. PHB none-producing strain Cupriavidus necator H16/PHB-4 (DSM-541) was purchased from Leibnitz Institute DSMZ-German Collection of Microorganism and Cell Cultures, Braunschweig, Germany. 3

NAD+, NADH and glyoxilic acid were purchased from Serva Electrophoresis GmbH (Germany). Nile Red, NADP+, NADPH, DL-isocitric acid trisodium salt hydrate, D-malic acid, glucose-6-phosphate, L-glutamic acid, oxidized glutathione, Coenzyme A, 5,5'-Dithiobis-(2Nitrobenzoic Acid) (DNTB), acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, oxalacetic acid, glutathione reductase, glucose-6-phosphate dehydrogenase and propionic acid anhydride were bought from Sigma Aldrich (Germany).

Chemical mutagenesis and selection of PHA producing mutants Erlenmeyer flasks (volume 250 ml) containing 50 ml of Mineral Salt (MS) medium (composition of MS medium: 3 g (NH4)2SO4, 1 g KH2PO4, 11.1 g Na2HPO4·12 H2O, 0.2 g MgSO4, 1 ml of microelement solution and 1 l of distilled water, the microelement solution composed of 9.7 g FeCl3, 7.8 g CaCl2, 0.156 g CuSO4·5 H2O, 0.119 g CoCl2, 0.118 g NiCl2, 0.062 g CrCl2 in 1 l of 0.1 mol l-1 HCl), 20 g l-1 of waste frying rapeseed oil and chemical mutagen ethyl methane sulphonate at concentration of either 0.1 or 1.0 g l-1 were inoculated by 5 ml of the wild-type strain of Cupriavidus necator H16 (24 h, 30°C, 150 rpm, biomass concentration of inoculum typically reached 2.5 g l-1). After 48 hours of cultivation, the cell suspensions were suitably diluted in sterilized physiological buffer (Na-phosphate buffer, 100 mol l-1, pH 7.4, 9 g l-1 NaCl) and seeded on agar plates. For selection of PHA positive mutants we employed modified Nile Red viable-colony staining protocol developed by Spiekermann et al. (1999). Briefly, the cells exposed to mutagen were inoculated onto the surface of agar plates containing MS medium (as described above only concentration of (NH4)2SO4 was reduced to 0.5 g l-1), 20 g l-1 fructose and 0.002 vol. of a solution of 0.25 mg Nile Red per ml of dimethylsulfoxide (DMSO). The sensitivity of staining was even improved by introduction of 1 mmol l-1 ethylenediaminetetraacetic acid (EDTA). Cells were incubated at 30°C for period of approx. 5 days. After the incubation, PHA rich mutants were easily distinguished when exposed to ultraviolet irradiation as colonies revealing typical orange fluorescence. We expected that intensity of fluorescence approximately correlated with PHA intracellular content. Hence, for further tests we selected the colonies showing the highest fluorescence intensity. Selected mutants were transferred onto petri dishes containing Nutrient Broth (NB) medium (10 g peptone, 10 g beef 4

extract, 5 g NaCl in 1 l of distilled water) and further tested for their ability to produce PHA on waste frying oil.

Cultivation in Erlenmeyer flasks Nutrient broth (NB) medium was used for inoculum development and culture preservation (agar plates). Mineral salt (MS) medium was used in all the production experiments. Waste rapeseed frying oil obtained from university canteen was used as a carbon source in the production media (20 g in 1 l of MS medium). Oil, salt solutions and microelement solutions were autoclaved separately (121°C, 25 min) and then aseptically reconstituted at room temperature prior to inoculation. The pH was adjusted at 7.0 using 1 M NaOH /H2SO4. Cultivations were performed in Erlenmeyer flasks (250 ml volume) containing 50 ml of the MS medium. Temperature was set at 30°C, agitation at 180 rpm. In order to produce copolymer P(3HB-co-3HV), precursors of 3HV (propanol and sodium propionate) were added after 24 h of cultivation in amounts needed to reach concentrations of 5 g l-1 of precursor. After 70 hours of cultivation, the cells were harvested (centrifugation, 8000× g, 5 min) and biomass as well as PHA yields were determined. All the cultivations were performed in triplicate.

Analysis of PHA To determine biomass concentration (expressed as dry cell weight, DCW) and PHA content in cells, samples (10 ml) were centrifuged and the cells were washed with 5% (v/v) Triton X (10 ml) and then distilled water. The biomass concentration was analysed as reported previously (Obruca et al. 2010b). PHA content in cells and monomer composition of PHA were analysed as methyl esters of particular 3-hydroxyacids by gas chromatography as reported by Brandl et al. (1988). P(3HB-co-3HV) with 12% content of 3HV (Sigma Aldrich, Germany) was used as a standard, benzoic acid (LachNer, Czech Republic) was used as an internal standard. To determine molecular weight of PHA, polyesters were extracted from the dried cells into chloroform by stirring for 24 h at 60°C. Solution was filtered in order to remove the residual cell material. Finally, pure PHA was obtained by non-solvent precipitation (five times the volume of chloroform) and filtration. The non-solvent used was a mixture of methanol and water (7:3 [vol/vol]). 5

Resulting PHA were used for determination of molecular weight by gel permeation chromatography [Agilent 1100 Series; refractive-index detector, USA; column PLgel Mixed B (300 9 7.5 mm;10 lm)].

Analysis of waste frying rapeseed oil Waste rapeseed frying oil utilized in this study as a sole carbon source for PHA production was obtained from university canteen at Faculty of Chemistry, Brno University of Technology. The fatty acid composition of oil was determined by gas chromatography after their conversion to fatty acids methyl esters (FAME) by following method. Approx. 70 mg of oil were dissolved in 3 ml of isooctane. After that 200 l of methanol solution of KOH (13.1 g of KOH per 100 ml of methanol) was added and the mixture was shaken. Subsequently, 1 g of NaHSO4 was introduced, mixture was shaken again and upper organic layer was analysed for FAME. The GC (Trace GC Ultra, Thermo Scientific) was equipped with a flame ionization detector using helium as carrier gas (1.2 mL/min). The sample injected (i.e. methyl esters) was separated in a SLB-IL100 (Supelco) column (60 m × 0.25 mm mm x 0,20 μm ). GC oven temperature was programmed at 185 °C for 40 min, then increased at a rate of 15 °C min−1 to 250 °C, and then maintained at 250 °C for 11.68 min. Injector temperature was programmed at 250 °C for 55 min, and detector temperature set at 250 °C. Supelco® 37 Component FAME Mix was used as a standard for identification of individual FAME. Further, (i) acidic value expressed in mg of KOH/g of sample, which is an indication of the free fatty acid content of the sample, (ii) its saponification value, expressed in mg of KOH/g of sample, which is the amount of alkali necessary to saponify a certain quantity of the sample, (iii) its iodine value, expressed as the number of centigrams of iodine absorbed per gram of sample, which is a measurement of the unsaturations of the sample were determined as described by Kartika et al. (2013). Results regarding characterization of waste frying oil used in this study are summarized in Table 1.

6

Preparation of cell-free extracts and enzyme assays The cells were suspended in 50 mmol l-1 phosphate buffer (pH 7) and disrupted by sonication (Sonopuls HD 3200, Bandeline, Germany) at 4°C. The cytosol was separated from the cell debris by centrifugation (10,000 g, 20 min., 4°C) and stored at -20°C. Activities of NAD-dependent isocitrate dehydrogenase (NAD-IDH) and NADP-dependent isocitrate dehydrogenase (NADP-IDH) were determined spectrophotometrically (340 nm) in reaction mixture consisting of 50 mmol l-1 phosphate buffer (pH 8.0) 1 mmol l-1 NAD+ or NADP+, 50 mmol l-1 isocitrate, 1 mmol l-1 MgSO4 and 100 mmol l-1 KCl (Wang et al. 2003). Similarly, activities of malate dehydrogenase (MDH) and malic enzyme (ME) were measured in reaction mixture consisting of: phosphate buffer pH 8.0, 1 mmol l-1 NAD+ in case of MDH or 1 mmol l-1 NADP+ in case of ME, 50 mmol l-1 malate, 10 mmol l-1 MgSO4 and 100 mmol l-1 KCl (Bruland et al. 2010). The activity of glucose 6-phosphate dehydrogenase (G6PD) was measured in following reaction mixture: phosphate buffer pH 7.4, 1 mmol l-1 NADP+, 5 mmol l-1 glucose-6-phosphate and 30 mmol l-1 MgCl2 (Singh et al. 2007). To determine activity of glutamate dehydrogenase (GDH) the following reaction mixture was used: 100 mmol l-1 phosphate buffer pH 7.4, 1 mmol l-1 NADP+, 5 mmol l-1 L-glutamic acid (Singh et al. 2007). Activity of glutathione reductase (GR) was assayed in following mixture: phosphate buffer pH 7.4, 0.4 mmol l-1 NADPH and 5 mmol l-1 oxidized glutathione (Singh et al. 2005). Activity of malate synthase (MS) was determined by monitoring the release of CoA during the enzymatic reaction (at 30°C) using 1 mmol l-1 5,5´-dithiobis(2-nitrobenzoic acid) at 412 nm in 50 mmol l-1 Tris-HCl buffer (pH 8.0) containing 0.2 mmol l-1 acetyl-CoA, 5 mmol l-1 glyoxylic acid and 100 mmol l-1 KCl (Wang et al. 2003). Similarly, activity of -ketohiolases (-KT) was assayed by following release of free CoA using DNTB. Reaction mixture for determination of activity of -KT1 contained phosphate buffer (50 mmol l-1, pH 7.4) 0.2 mmol l-1 acetyl-CoA and 1 mmol l-1 DNTB. When -KT2 capable of incorporation of propionyl-CoA into 3-hydroxyvaleryl-CoA was assayed, reaction mixture contained 0.2 mmol l-1 acetyl-CoA, 0.2 mmol l-1 propionyl-CoA and 1 mmol l-1 DNTB and the activity of -KT1 was subtracted from the results of the assay to estimate activity of KT2. Acetoacetyl-CoA reductase (AACoR) activity was determined in mixture containing 1 mmol l-1

7

acetoacetyl-CoA, 0.4 mmol l-1 NADPH and phosphate buffer (100 mmol l-1, pH 8.0), NADP+ formation was measured spectrophotometrically as a decrease of absorbance at 340 nm (Saito et al., 1977). PHB synthase activity was determined spectrophotometrically (412 nm) in mixture containing phosphate buffer (50 mmol l-1, pH 7,4) 0.2 mmol l-1 3-hydroxybutyryl-CoA and 1 mmol l-1 DNTB (Obruca et al. 2010b). Activity of 2-methylcitrate synthase was determined in mixture consisting of 50 mmol l-1 Tris-HCl buffer pH 8.0, 2 mmol l-1 oxaloacetate, 1 mmol l-1 propionyl-CoA and 2 mmol l-1 DNTB by measuring the absorbance at 412 nm (Ewering at al., 2006). All the samples were analysed in triplicate. In all these cases, final volume of reaction mixture was 250 L, the reactions were initiated by addition of cell extracts (25 L) and the changes of absorbance were read at 340 or 412 nm with regards to the enzyme analysed, temperature was set at 30°C (ELx800, Biotek, Germany). One unit of enzyme activity was defined as the conversion of one nmol of substrate per minute. The amount of soluble protein in cell extracts was determined by Hartree-Lowry method using bovine serum album as a standard (NanoPhotometer, Implen, Germany). Generally, concentrations of soluble proteins in cell extract varied in range of approx. 2.0 – 3.0 g/ml. Determination of NADPH/NADP+ ratios in cell extracts was performed immediately after cell disruption by spectrophotometric method described by Zhang et al. (2000).

Synthesis of propionyl-CoA Synthesis of propionyl-CoA was performed according to Ewering at al. (2006). Coenzyme A (10 mg) was solubilized in 0.5 mol l-1 K2CO3 and aliquots of 2 L of propionic acid anhydride were added into the solution (stirred on ice) till no free Coenzyme A was detected by spot test using 5 mmol l-1 DNTB as reaction agent. After that, the pH of mixture was set at 4.5 by addition of HCl and resulting propionyl-CoA was stored at -20°C. The concentration of propionyl-CoA was calculated considering complete Coenzyme A conversion.

8

Oxidative stress challenge The mutant as well as the wild-type strain was cultivated in MS medium containing waste frying oil as a sole carbon source as described above. At the 48th h of cultivation, 10 ml of culture was harvested; cells were centrifuged (8000×g, 5 min), resuspended in sterilized physiological buffer and diluted to reach cell count approx. 108 per ml. After that the viable cell count was measured (time 0) and 3% hydrogen peroxide was applied to reach concentration of 100 mmol l-1 of H2O2. Samples were withdrawn at times 5 and 30 min to estimate viable cell counts. Spread plate method was used to determine the number of viable bacteria in the samples. Cell suspension was suitably diluted by physiological buffer and 100 µL of the diluted cell suspension was placed on sterile agar plates with solid NB medium. Plates were incubated at 30°C in thermostat for 48 h. After that the plates containing 20 -200 colonies were selected and numbers of colony-forming units (CFU) were counted. Finally, number of viable cells expressed as CFU per ml in original suspension was calculated. Survival of the cells was expressed in %, the viable cell count at time 0 (before introduction of hydrogen peroxide) was taken as 100%. The experiment was performed twice, all the samples were analysed in triplicate.

Flow cytometry measurement To determine distribution of PHA among cell populations of the wild-type and the mutant strain, flow cytometry analysis according to Kacmar at al. (2005) was adopted. Cells were harvested at various times of cultivation (24, 48, 60 and 72h) in MS medium using 20 g l-1 waste oil as a substrate, centrifuged and washed by physiological buffer. After that the cells were fixed by cold ethanol (30%) for 15 minutes, pelleted, resuspended and diluted in physiological buffer to reach cell count approx. 5·106 per ml. Further, 5 l of Nile Red (0.05 mg ml-1 DMSO) was used to stain 1 ml of the cell suspension. Cells were placed in dark for 5 minutes and then analysed by flow cytometry (Apogee A50, Apogee, GB) using 488 nm laser for excitation and orange canal (FL2) (band pass filter 620 ± 30 nm) for fluorescence detection. PHA none-accumulating strain C. necator H16/PHB-4 was used as a negative control.

9

Batch cultivations in fermentor Fermentor vessel (5 L, BiostatBplus, Sartorius Stedim Biotech, Germany) containing 3.15 l of MS medium and waste rapeseed oil (20 g l-1) was inoculated with 350 ml 24 h culture grown on MS medium (20 g l-1 of waste rapeseed oil). The temperature was set at 30°C, pH was maintained at 7 by 2 mol l-1 NaOH/H2SO4. The dissolved oxygen (DO) concentration was monitored by an O2 electrode. DO value was maintained at the level of 50% to air saturation by varying the agitation speed automatically.

Results Mutagenesis and mutants screening Introduction of the mutagen into the fermentation media resulted in random mutagenesis influencing also PHA accumulation abilities of the mutants. Therefore, Nile Red viable colony staining described by Spiekermann at al. (1999), which sensitivity was even improved by introduction of 1 mmol l-1 EDTA, was used to distinguish PHA accumulating mutants. The fact that the intensity of the fluorescence is likely to depend on intracellular content of PHA (the higher PHA content the higher intensity of fluorescence) enabled us to reduce the number of mutants to be further screened by eliminating PHA none-accumulating mutants or mutants with low PHA content from further tests. In total, we obtained 54 mutants which were tested for their ability to produce PHB on waste frying oil. Unfortunately, many mutants lost their ability to grow and produce PHB in MS medium with waste frying oil as a sole carbon source. However, we isolated 4 mutants which revealed improved PHA productivity as compared to the wild-type strain. Among them the mutant EO1 seemed to be the most promising one, because its growth ability was not negatively influenced and, moreover, PHB yields were significantly higher (about 35%) than that of the wild-type strain. Furthermore, the mutant strain EO1 produced PHB of higher molecular weight than the wild-type strain (Tab. 2).

Characterization of mutant EO1 In order to understand the reasons why the mutant strain overproduced PHB, we measured activities of several intracellular enzymes such as those involved in PHB synthetic pathway, TCA 10

cycle, glyoxalate cycle, pentose phosphate pathway etc. (Tab. 3). We observed that the mutant strain revealed significantly increased specific activities of NADP-dependent isocitrate dehydrogenase (NADP-IDH), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH). These enzymes generate NADPH and, therefore, they are, apart from their other metabolic functions, considered being involved in stress response towards oxidative stress (Singh et al. 2005, Singh et al. 2007). Furthermore, the mutant strain also exhibited increased specific activities of enzymes of PHB biosynthetic pathway and higher NADPH/NADP+ ratio. Since we observed that the mutant strain showed increased specific activities of enzymes involved in oxidative stress response as well as increased NADPH/NADP+ ratio, which might indicate adaptation to oxidative stress conditions, we decided to expose the cells of the wild-type strain as well as the mutant strain to the hydrogen peroxide as a model oxidative stress causing agent. The mutant strain was capable of facing harmful conditions caused by hydrogen peroxide significantly better than the wild-type strain (Fig. 1) which was especially evident at the 5th min of the test (survival: 10.95 % wild-type strain, 70.58 % mutant strain). These results supported our hypothesis that the mutant strain revealed partial metabolic adaptation to oxidative stress. To determine distribution of PHB among the cell populations of the wild-type and the mutant strain, we performed flow cytometry analysis at various times of cultivation. Surprisingly, we observed that since the 48th h of cultivation, the wild-type strain population contained approx. 5 % of PHA negative cells. On the contrary, population of the mutant strain consisted only of PHA positive cells (Fig. 2), which indicated higher dynamics of PHA accumulation in the mutant strain. However, in the following periods of cultivation (60 and 72 h) we observed PHA negative subpopulation neither in the wild-type nor in the mutant strain.

Incorporation of 3HV precursors into P(3HB-co-3HV) In order to produce copolymer P(3HB-co-3HV) we introduced either propanol or sodium propionate into fermentation media. Both substances are metabolized via propionyl-CoA and; therefore, they can be incorporated into polymer in form of 3HV. Propanol significantly suppressed the growth of the mutant strain as compared to the wild-type strain which negatively influenced total 11

PHA yields (Tab. 3). On the other side, utilization of sodium propionate did not cause inhibition of growth of the mutant. Furthermore, the mutant was capable of accumulating higher amount of PHA with enhanced 3HV fraction which significantly improved coefficient of precursor transformation into 3HV (Y3HV/prec) in comparison with the wild-type strain (Tab. 4). To investigate metabolic backgrounds of improved 3HV incorporation into PHA in presence of propionate, we looked into the specific activities of selected enzymes which may influence metabolism of propionyl-CoA. The cells were cultivated in the presence of propionate as in the previous experiment but harvested and disrupted at the 48th h of cultivation (Tab. 5). In comparison with the wild-type strain, the mutant strain EO1 exhibited significantly (more than two-fold) enhanced specific activities of ME (which was in agreement with our previous observation, see Tab. 3) as well as 2-methylcitrate synthase and -KT2, which couples acetyl-CoA and propionyl-CoA resulting in formation of 3-hydroxyvaleryl-CoA. On the contrary, there was no statistically significant difference in specific activity of MDH between the wild-type and the mutant strain.

Production of P(3HB-co-3HV) in bioreactor The PHA production abilities of the mutant and the wild-type strain were also compared in bioreactor experiment in batch mode using 20 g l-1 of waste frying oil and 5 g l-1 of sodium propionate (applied at the 10th h of cultivation) (Tab. 6). After 30 h of cultivation we gained very similar yields of biomass (the wild-type strain 17.2 and the mutant strain 18.7 g l-1) but PHA content and; therefore, PHA yields were significantly higher in the mutant strain than in the wild-type strain (about 13 % and 21 %, respectively). Moreover, also in bioreactor the mutant strain confirmed its improved ability to incorporate propionate into P(3HB-co-3HV) which resulted in production of material containing 9.2 % of 3HV (6.3 % in the wild-type strain) and relatively high precursor incorporation coefficient (Y3HV/pec = 0.29). Therefore, the mutant EO1 seems to be superior to the wild-type strain in term of PHA productivity as well as in incorporation of propionate into P(3HB-co-3HV).

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Discussion Since the cost of PHA is the main obstacle preventing these perspective materials from entering everyday life of consumers, a lot of effort is made to improve PHA production. At first, utilization of cheap waste substrates is likely to enhance economic aspects of PHA production. Furthermore, isolation of new PHA-producing microorganisms or utilization of tools of genetic engineering may also enable economically feasible production of PHA. In addition, random mutagenesis using either chemical or physical mutagens can be employed to increase PHA accumulation abilities of PHA producing strains (Adwitiyia et al. 2009). Despite the fact that this approach may be considered being out of date, it has also several advantages that should be taken into account. Above all, unlike techniques of genetic engineering, random mutagenesis acts randomly and, therefore; it may help to reveal novel and unexpected consequences and/or connection in the metabolism. Because production of PHA is a complex process influenced by various metabolic pathways as well as environmental factor, we believe that random mutagenesis could be an efficient tool to obtain highly productive mutant strains for PHA production. The first condition to reach this goal is an effective selection of mutant strains with desired properties. A key limitation of random mutagenesis is labour-intensive process of overproducing mutants selection, because they are present in high background of cells of low interest. Hence, we employed viable colony staining technique developed by Spiekermann et al. (1999) which, based on the intensity of fluorescence, allowed us to omit none-perspective mutants and select only PHA producing colonies. Introduction of 1 mmol l-1 EDTA probably facilitated the stain to penetrate the cells which resulted in improved sensitivity of the staining. To our knowledge, this is the first report on utilization of Nile Red viable staining for mutant selection. Furthermore, since presence of Nile Red does not negatively influence the growth of bacteria (Spiekermann et al. 1999), very similar approach could be used also in liquid media and mutants might be selected by fluorescence-activated cell sorting, which would provide very powerful tool for selection of PHA overproducing mutant strains. In total, we isolated and screened 54 mutants. Among them the mutant designated as EO1 seemed to be the most promising one. Therefore, we decided to characterize the mutant in terms of 13

intracellular activities of the selected enzymes of the crucial metabolic pathways. We observed significantly enhanced specific activities of NADP-IDH, ME, G6PD and GD. Apart from their metabolic function within particular metabolic pathways, the activity of these enzymes is also associated with production of reduced cofactor NADPH, which creates reducing environment in cells and which is necessary for regeneration of glutathione and thioredoxin involved in detoxification of reactive oxygen species (Murakami et al. 2006). Therefore, due to their ability to increase NADPH/NADP+ ratio and create strong reducing environment in the cell, NADP-IDH, ME, G6PD and GD are considered being among crucial enzymes involved in oxidative stress response of bacteria (Singh et al. 2005). For instance, Singh et al. (2007) observed significant increase of specific activity of NADP-IDH (3 fold enhanced as compare to control), G6PD (4.5 fold), ME (1.75 fold) and GD (2.4 fold) in cells exposed to oxidative stress induced by menadione. Therefore, it is likely that the increase of specific activity of the mentioned enzymes was related to enhancement of NADPH/NADP+ ratio in the mutant strain . There are several reports that indicated NADPH/NADP+ ratio as the crucial factor influencing activity of PHA biosynthetic pathway at enzymatic level. Lee et al. (1996) identified high NADPH/NADP+ ratio as the most critical factor stimulating activity of AACoR and, subsequently, PHB synthesis in recombinant Escherichia coli. Furthermore, Kessler and Wilholt (2001) stated that NADPH, apart from its function as a cofactor of AACoR, partially inhibits TCA cycle which consequently stimulates flux of acetyl-CoA into PHA biosynthetic pathway. Therefore, it seems reasonably that improved PHB accumulation ability of the mutant strain is a side effect of its oxidative stress adaptation resulting in an increase of NADPH/NADP+ ratio. Moreover, our results are in agreement with our previous observations of positive influences of mild oxidative stress on PHB biosynthetic pathway of C. necator (Obruca et al. 2010b, Obruca et al. 2010c) and similar effect was also observed by Jung and Lee (2000). Moreover, the mutant EO1 revealed improved ability to face oxidative stress challenge in comparison with the wild-type strain. In our opinion, this was more likely result of its reputed oxidative stress adaptation than the consequence of the increased PHA intracellular content of the mutant. On the other side, flow cytometry analysis showed that at the 48th h of cultivation, population 14

of the wild-type strain, unlike the mutant, contained small portion of PHA-negative cells. It might also negatively influence its survival in presence of hydrogen peroxide, since the positive effect of presence of PHA in cytoplasm of bacteria on their survival under various stressful conditions has been reported by several authors. Ruiz et al. (2001) observed that PHA mobilization increased level of stress-related sigma factor RpoS in Pseudomonas oleovorans cells and thus increased its ability to face heat and oxidative stress. Further, polyhydroxyalkanoates as highly reduced storage compounds were identified by Ayub et al. (2009) as essential substances for maintenance of intracellular redox state during low temperature adaptation in the Antarctic bacterium Pseudomonas sp. 14-3. As was mentioned in introduction, mechanical and processing properties of material are strongly dependent upon monomer composition of PHA. Incorporation of 3HV results in formation of copolymer P(3HB-co-3HV) possessing improved flexibility and strength as compared to homopolymer PHB (Kessler and Witholt 1999). Incorporation of 3HV is implemented via feeding of propionyl-CoA generating precursors such as propionate, propanol, or valerate (Kessler and Wilholt 2001). Propionyl-CoA can be either incorporated into the copolymer or metabolized via 2methylcitrate cycle. The first is realized by the enzyme -ketothiolase. Due to the fact that ketothiolase encoded by the gene phbA is specific only for acetyl-CoA, this step must be catalysed by the enzyme encoded by the gene bktB which is placed out of phaCAB operon of C. necator (Slater et al. 1998). Metabolization of propionyl-CoA in 2-methylcitrate cycle results in formation of pyruvate which may be further decarboxylated to acetyl-CoA (Bramer and Steinbuchel 2001, Ewering at al. 2006). Because utilization of propionyl-CoA via 2-methylcitrate cycle dominates in C. necator, only small portion (less than 15% of carbon) of available propionyl-CoA is incorporated into P(3HB-co3HV) (Yu and Si 2004). Taking into account costs of 3HV precursors, the inefficiency of precursor incorporation into copolymer negatively influences economics of PHA production. The mutant EO1 was capable of incorporation of higher amounts of 3HV into the PHA than the wild-type strain (19.5 and 13.3 %, respectively) when propionate was employed as a precursor of 3HV and cultivation was performed in Erlenmeyer flasks. It positively influences both mechanical properties of material (the higher 3HV content, the higher flexibility of material) as well as economic aspects of PHA production, because higher coefficient of precursor incorporation (Yprec/3HV = 0.17 in wild-type strain and 0.29 in 15

EO1 mutant) was accompanied with higher PHA yields. This effect was even more obvious in experiments performed in bioreactor that confirmed improved production abilities of the mutant in terms of PHA yields and precursor incorporation. We hypothesize that the improved introduction of propionyl-CoA into P(3HB-co-3HV) by the mutant is associated with activation of NADPH generating enzymes such as ME. The first enzyme of 2-methylcitrate cycle, 2MCS couples propionyl-CoA and oxaloacetate while forming 2-methylcitrate (Ewering et al. 2006). Therefore; oxaloacetate, which is mainly formed by oxidation of malate by malate dehydrogenase, is the crucial substrate of the metabolic pathway. This was proved by Bramer and Steinbuchel (2002) who showed that MDH lacking mutant of C. necator was unable to grow on propionate as a sole carbon source. Furthermore, malate is also a substrate for ME that catalyses its oxidative decarboxylation providing pyruvate and NADPH (Bruland et al. 2010). Since the mutant EO1 revealed significantly increased specific activity of ME, it may cause reduction of pool of available oxaloacetate for 2MCS and; hence, partial inhibition of metabolitazation of propionyl-CoA in 2-methylcitrate cycle in spite of the fact that activity of 2MCS was significantly higher in the mutant strain than in the wild-type strain. This is in agreement with the observation of Yu and Li (2004), who identified malate as a node of C3/C4 pathway and TCA cycle depending on NADPH demands. Because the action of 2-methylcitrate cycle was probably partially inhibited in the mutant strain due to lower availability of oxaloacetate, more propionyl-CoA could have been built in P(3HBco-3HV) structure. In addition, the mutant EO1 exhibited higher specific activity of -KT2 responsible for production of 3-hydroxyvalerate-CoA, which indeed supported transformation of propionyl-CoA into 3-hydroxyvalerate-CoA. PHA accumulation abilities have been identified in many bacterial strains, however, only few of them can be considered as candidates for economically feasible industrial production of these polymers. Table 7 summarizes production of PHA from various oils by using different bacteria. Although various bacteria such as Chromobacterium sp. (Kimura et al. 1999), Burkholderia cepacia (Alias and Than 2005), Pseudomonas aeruginosa (Chan et al.2006) or Pseudomonas putida (Annuar et al. 2007) have been tested as PHA producers from oils, the highest yields have been reported for bacterial strain C. necator (Taniguchi et al. 2003, Obruca et al. 2010a) cultivated in fed-batch mode in 16

fermentor. Therefore, comparison of PHA producing abilities of the EO1 mutant strain with the wildtype strain of C. necator H16 indicates that the mutant can be considered as a candidate for PHA production using inexpensive waste frying oils as substrates. Nevertheless, because oxidative stress sensing and response of bacteria is very sophisticated and complex process involving many levels of regulation (Marles-Wright and Lewis, 2007; Moen et al. 2009; Calhoun and Kwon 2010, Rangel 2011), it would be very difficult to certainly identify the changes in the genome of the mutant EO1. However, metabolic characterization of mutant indicated that activation of NADPH generating enzymes enhances PHA accumulation in C. necator. Further, enhanced activity of ME is likely to reduce oxaloacetate level which results in higher efficiency of propionyl-CoA incorporation into P(3HB-co-3HV). We believe that these findings may help to define new targets for genetic engineering of PHA producing bacteria. For instance, enhancement of the activity of ME by its overexpression significantly increased production of lipids in Mucor circinelloides (Zhang et al. 2007). Similar strategy could be also used in C. necator in order to improve its ability to incorporate propionyl-CoA into PHA. Furthermore, introduction of mild oxidative stress conditions by addition of oxidative stress factors or by regulation of aeration and/or redox state of the culture may improve PHA production in the same manner as in the mutant EO1 also in other PHA accumulating bacteria. Acknowledgement: This work was supported by project “Centre for Materials Research at FCH BUT” No. CZ.1.05/2.1.00/01.0012 from ERDF and by the project "Excellent young researcher at BUT" No. CZ.1.07./2.3.00/30.0039.

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Mozejko J, Wilke A, Przybylek G, Ciesielski S (2012) Mcl-PHAs Produced by Pseudomona ssp Gl01 using fedbatch cultivation with waste rapeseed oil as carbon source. J Microbiol Biotechn 22: 371-377. Murakami K, Tsubouchi R, Fukayama M, Ogawa T, Yoshino M (2006) Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADPisocitrate dehydrogenase. Arch Microbiol 186: 385-392. Obruca, S., Marova, I., Snajdar, O., Mravcova, L. and Svoboda, Z. (2010a) Production of poly(3hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate. Biotech Lett 39: 1925-1932. Obruca S, Marova I, Stankova M, Mravcova L, Svoboda Z (2010b) Effect of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) biosynthetic pathway in Cupriavidus necator H16. World J Microb Biotech 26: 1261–1267. Obruca S, Marova I, Svoboda Z, Mikulikova R (2010c) Use of controlled exogenous stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus necator. Folia Microbiol 55:17-22. Pradella JGC, Taciro MK, Pataquiva AY (2010) High-cell-den-sity poly (3-hydroxybutyrate) production from sucrose using Burkholderia sacchari culture in airlift bioreactor. Bioresourc Technol 101:8355–8360. Rangel DEN (2011) Stress induced cross-protection against environmental challenges on prokaryotic and eukarytotic microbe. World J Microb Biotech 27: 1281–1296. Reinecke F, Steinbuchel A (2009) Ralstonia eutropha Strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. J Mol Microbiol Biotechnol 16:91-108. Ruiz JA, Lopez NI, Fernandez R, Mendez BS (2001) Polyhydroxyalkanoates degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms. Appl Environ Microb 67: 225-230. Saito T, Fukui T, Ikeda F, Tanaka Y, Tomita K. (1977) An NADP-linked acetoacetyl-CoA reductase from Zooglear amigera. Arch Microbiol 114: 211-217. Serafim LS, Lemos PC, Albuquerque MGE, Reis MAM (2008) Strategies for PHA production by mixed cultures and renewable waste materials. Appl Microbiol Biot 81: 615-628. Singh R, Beriault R, Middaugh J, Hamel R, Chenier D, Appanna VD, Kalyuzhnyi S (2005) Aluminum-tolerant Pseudomonas fluorescens: ROS toxicity and enhanced NADPH production. Extremophiles 9:367-373. Singh R, Mailloux RJ, Puiseux-Dao S, Apanna V. (2007) Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens. J Bacteriol 189: 6665-6675. Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ (1998) Multiple betaketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180: 1979–1987. Spierkermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinbuchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171: 73-80. Sudesh K, Abe H, Doi Y. (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25: 1503-1555. Taniguchi I, Kagotani K, Kimura Y (2003) Microbial production of poly(hydroxyalkanoates)s from waste edible oils. Green Chem 5: 545-548. Vidal-Mas J, Resina-Pelfort O, Haba E, Comas J, Maresa A, Vives-Rego J (2001) Rapid flow cytometry – Nile red assssment of PHA cellular content bad heterogenitity in cultures of Pseudomonas aeruginosa 4ZT2 (NCIB 40044) grown in waste frying oil. Antonie van Leeuwenhoek 80: 57-63. Wang ZX, Bramer C, Steinbuchel A (2003) Two phenotypypically compensating isocitrate dehydrogenase in Ralstonia eutropha. FEMS Microbiol Lett 227: 9-16. Yamane T, Fukunaga M, Lee YW (1996) Increased PHB production by high-cell-density fed-batch culture of Alcaligenes latus, a growth associated PHB producer. Biotechnol Bioeng 50:197–202. Yu J, Si YT (2004) Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short fatty acids. Biotechnol Prog 20: 1015-1024. Zhang Z, Yu J, Stanton RC (2000) A method for determination of pyridine nucleotides using a single extract. Anal Biochem 285: 163-167. Zhang Y, Adams IP, Ratledge C (2007) Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiol-SGM 153: 2013-2025.

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List of Figure Legends: Figure 1 Survival of wild type and mutant strain in presence of 100 mmol l-1 hydrogen peroxide. Triangles – the wild strain, circles – the mutant strain. Data represent results of two independent experiments, each sample was analysed in triplicate.

Figure 2 Flow cytometry analysis of cell populations of C. necator H16 (a) and its mutant EO1 (b) after 48 h of cultivation using waste frying oil as a sole carbon source. PHA none-producing mutant strain C. necator H16/PHB-4 was used as a negative control (black line). Arrow shows PHA negative subpopulation of the wild-type strain which was not present in the mutant strain at the same time of cultivation.

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Table 1 Characterization of waste frying rapeseed oil used as a sole carbon substrate within the study. Parameter Acidic Value Saponification Value Iodine Value

Value 3 189 75

Fatty acid Myristic acid (C14:0) Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Arachidic acid (C20:0) α-Linolenic acid (C18:3) cis-11-Eicosenoic acid (C20:1) Behenic acid (C22:0) Erucic acid (C22:1)

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Composition [%] 0.2 12.1 0.3 2.6 55.3 21.6 0.5 6.0 1.0 0.3 0.1

Table 2 Growth and PHB production of wild type of Cupriavidus necator H16 and its mutant strain EO1 in MS medium using waste rapeseed oil as a sole carbon source. Erlenmeyer flasks, waste frying oil 20 g l-1, 70 h of cultivation.

DCW (g l-1)

PHB (g l-1)

PHB (%)

Mn (Da·105)

Mw (Da·105)

Mw/Mn

Wild 7.9 ± 0.4 4.9 ± 0.3 62.3 ± 0.7 2.19 6.27 2.87 OE1 8.6 ± 0.6 7.6 ± 0.9 87.9 ± 3.0 2.94 7.24 2.46 Note: Results are in form: mean ± standard deviation, DCW stands for dry cell weight, Mw-weight average molecular weight, Mn- number average molecular weight

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Table 3 Specific activities of selected intracellular enzymes of Cupriavidus necator H16 and its mutant EO1. Cells were cultivated in MS medium, 20 g l-1 of waste frying oil, harvested and disrupted after 48 h of cultivation. Specific enzyme activity (U mg-1 of protein) Pathway TCA cycle

Enzyme/Parameter NAD-IDH

*

MDH MS NADP-IDH ME NADPH generating and oxidative stress response G6PD involved enzymes GDH GR* -KT PHB synthesis AACoR PHB synthase NADPH/NADP+ratio Note: Results are in form: mean ± standard deviation, Glyoxalate cycle

*

Wild Type

OE1

2.6 ± 0.6

3.40 ± 1.0

1325.6 ± 10.2 983.2 ± 12.5 24.1 ± 1.2 14.9 ± 0.4 29.5 ± 1.0 111.7 ± 6.8 123.5 ± 8.5 363.7 ± 6.8 4.9 ± 0.3 22.7 ± 0.2 6.5 ± 1.1 9.9 ± 1.1 14.1 ± 0.2 10.6 ± 2.4 12.7 ± 0.5 25.2 ± 0.7 916.1 ± 17.4 1040.8 ± 33.7 3.5 ± 0.6 15.2 ± 1.3 0.7 ± 0.1 1.2± 0.1 difference between the wild and the mutant

strain was not statistically significant (T-test,  = 0.05), IDH – isocitrate dehydrogenase, MDH – malate dehydrogenase, MS – malate synthase, ME – malic enzyme, G6PD – glucose-6-phosphate dehydrogenase, GDH – glutamate dehydrogenase, -KT – ketothiolase, AACoR – acetoacetyl-CoA reductase. 1 U of enzyme activity is defined as an amount of enzyme catalyzing conversion of 1 nmol of substrate per minute.

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Table 4 Production of P(3HB-co3HV) by Cupriavidus necator H16 and mutant EO1 employing propanol and sodium propionate as precursors of 3HV. Cells were cultivated for 70 h in Erlenmeyer flasks with MS medium, 20 g l-1 of waste frying oil, precursors were introduced at the 24th h of cultivation at concentration of 5 g l-1.

Precursor 5gl

Strain

-1

Propanol

DCW -1

Wild type EO1

PHA -1

PHA

3HV

(g l )

(g l )

(%)

(%)

6.2 ± 0.2 4.4 ± 0.1

4.5 ± 0.3 3.2 ± 0.1

72.8 ± 3.5 72.8 ± 0.2

10.62 ± 0.59 10.14 ± 0.03

Y3HV/prec* 0.07 0.05

Wild type 6.5 ± 0.6 4.2 ± 0.1 64.3 ± 0.7 13.3 ± 0.5 0.17 EO1 6.7 ± 0.1 4.9 ± 0.3 73.3 ± 1.4 19.5 ± 1.5 0.29 * Precursor incorporation coefficient Y3HV/prec was calculated as follow: Y3HV/prec=(moles of precursor Propionate

incorporated into copolymer)/(moles of precursor introduced into medium). Results are in form of mean ± standard deviation.

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Table 5 Specific activities of selected intracellular enzymes, cells were cultivated in MS medium with 20 g l-1 of oil, sodium propionate was introduced at the 24th h of cultivation, cells were harvested and disrupted after 48 h of cultivation.

Specific enzyme activity (U mg-1 of protein) Enzyme *

MDH ME 2MCS -KT2

Wild Type

OE1

1290.9 ± 14.4 159.3 ± 9.2 119.0 ± 16.5 5.6 ± 1.3

1156.7 ± 102.3 326.4 ± 14.6 197.1 ± 8.9 12.1 ± 1.8

Note: Results are in form: mean ± standard deviation,

*

difference between the wild and the mutant

strain was not statistically significant (T-test,  = 0.05), MDH – malate dehydrogenase, ME – malic enzyme, 2MCS – 2-methylcitrate synthase, KT2 – -ketothiolase coupling acetyl-CoA and propionyl-CoA, 1 U of enzyme activity is defined as an amount of enzyme catalyzing conversion of 1 nmol of substrate per minute.

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Table 6 Biomass and PHA yields and productivity coefficients obtained in bioreactor. MS medium, 20 g l-1 of oil, 5 g l-1 of sodium propionate (added at the 10th h of cultivation), 30 h of cultivation.

Wild OE1

DCW (g l-1)

PHA (g l-1)

PHA (%)

3HV (%)

YX/S

YP/S

Y3HV/prec

17.2 ± 0.8 18.7 ± 0.9

12.7 ± 0.5 16.0 ± 1.1

74.2 ± 0.7 85.5 ± 2.0

6.3 ± 0.2 9.2 ± 0.4

0.69 0.75

0.51 0.64

0.16 0.29

Biomass and product yields coefficients were calculated as biomass (YX/S)or PHA (YP/S) yields in grams per grams of substrate (oil + precursor) introduced. Precursor incorporation coefficient Y3HV/prec was calculated as follow: Y3HV/prec=(moles of precursor incorporated into copolymer)/(moles of precursor introduced into medium). Results are in form of mean ± standard deviation.

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Table 7 Overview of basic parameters of PHA productions from plant oils reported in literature. Oil

Strain

Cultivation mode

PHA

Sunflower oil Palm olein Palm oil Waste oil Olive oil Waste frying oil Palm oil Soybean oil Waste rapeseed oil Waste rapeseed oil Waste rapeseed oil

Chromobacterium sp. Burkholderia cepacia Pseudomonas aeruginosa Pseudomonas aeruginosa Alcaligenes sp. AK 202 Pseudomonas aeruginosa Pseudomonas putida Cupriavidus necator* Cupriavidus necator H16 Cupriavidus necator H16 Cupriavidus necator EO1

Batch, E. flasks Batch, E. flasks Batch, Ferm. Batch, E. flasks Batch, E. flasks Batch, E. flasks Batch, fermentor Fed-Batch, Ferm. Fed-Batch, Ferm. Batch, Ferm. Batch, Ferm.

PHB PHB MCL-PHA MCL-PHA PHB MCL-PHA MCL-PHA P(HB-co-HHx) P(HB-co-HV) P(HB-co-HV) P(HB-co-HV)

DCW (g l-1) 17.6 5.1 2.2 5.4 3.1 8.1 2.1 138.0 138.5 17.2 18.7

PHA (%) 49.8 43.1 36.0 5.7 47.0 37.0 70 74.0 75.9 74.2 85.5

PHA (g l-1) 8.8 2.2 0.8 0.3 1.5 3.0 1.5 102.1 105.1 12.7 16.0

Reference Kimura et al. 1999 Alias and Than 2005 Marsudi et al. 2008 Chan et al. 2006 Akiyama et al. 1992 Vidal-Mas et al. 2001 Annuar et al. 2007 Taniguchi et. al 2003 Obruca et al. 2010a This work This work

- recombinant strain of C. necator, E. flasks – Erlenmeyer flasks; Ferm. – Fermenor, MCL-PHA – medium chain length polyhydroxyalkanoates, P(HB-co-

*

HHx) – copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate.

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