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aDepartment of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
bDepartment of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
cDepartment of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
dDepartment of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino,Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan.
ï€ª Corresponding author.
Telephone: +603-89467515. Fax: +603- 89467510
Effect of carbon to nitrogen ratio on accumulation of P(3HB-co-3HV) and its properties by Comamonas sp. EB172 from organic acids as substrates
Fed-batch cultivation has been used in laboratory and pilot scale bioreactor to obtain maximum growth and product yield. pH- stat fed- batch cultivation has been applied widely in biosynthesis of polyhydroxyalkanoate (PHA) when organic acids is used as carbon sources. This work aims to study effect of carbon to nitrogen ratio and organic nitrogen sources on the growth and poly(3-hydroxybutyrate-co-3-hydrovalerate) P(3HB-co-3HV) in 2 L fermenters using mixed organic acids by Comamonas sp. EB172. Cell growth, acids uptake, PHA yield and polymer properties are greatly influenced by the carbon to nitrogen ratio applied to culture. The polmer produced were characterized and recorded. The number average molecular weight (Mn) of P(3HB-co-3HV) copolymer reached the highest at 838 x103 Da with polydispersity index (PI) 1.8, when supplemented with yeast extract and mixed organic acids. Tensile strength, elongation to break and Young`s modulus of the copolymer containing 6-8 mol% 3HV were 13-15 MPa, 160-339 % and 0.26-0.34 GPa, respectively comparable to polypropylene (PP) and polyethylene (PE).
Keywords: Comamonas sp. EB172; poly(3-hydroxybutyrate-co-3-hydroxyvalerate); mixed organic acids; carbon to nitrogen ratio
Bio-based plastics such as polyhydroxyalkanoates (PHAs) are gaining much interest from the polymer researchers as these materials share the thermal and mechanical properties similar to petrochemical based-plastics. A lot of research and development have been carried out in order to select suitable microorganisms, to find appropriate substrates from renewable and cheaper carbon source and to improve the fermentation and downstream processes in order to make PHAs more economically viable and competitive, comparable to conventional plastics1 One of the limiting factors for the economic production of PHAs is the cost of feedstock, which contributes up to 40% of the total operating costs.3 Several successful studies have been reported on PHA production from cheap and renewable carbon sources, for example, date syrup, mahua flowers and palm oil mill effluent (POME).3-5 These potential substrates could be useful in reducing the overall PHA production cost.
We have recently reported on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV) production from a locally isolated bacterium Comamonas sp. EB172, which was successfully isolated from an anaerobic digester treating POME. 6-8 This bacterium exhibited PHA accumulation when organic acids from anaerobically treated POME were used as carbon sources. The strain was further tested for its physiological and genotypic properties in order to discriminate this strain with the other existing strains in the genus of Comamonas species. Based on the phenotypic and genotypic properties of Comamonas sp. EB172, it was proven that the strain is novel.7 The potential of this microorganism to accumulate PHA in various types of organic acids and at different initial medium pH have been tested, as well as the suitability of organic acids from anaerobically treated POME to be used as substrate for the PHA fermentation. 8 We have reported that the strain could successfully convert the organic acids from anaerobically treated POME into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV) copolymers with various fractions of poly(3-hydroxyvalerate) [P(3HV)]. The physical and thermal properties of the polyesters produced from Comamonas sp. EB172 have also been reported.8
Apart from our study, there is limited number of reports on the PHA accumulation by Comamonas species. It has been reported that Comamonas acidovorans was able to accumulate P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB) copolymers under specific carbon and fermentation conditions.9-10 A study by Thakor et al. (2003)11 showed that poly(3-hydroxybutyrate) [P(3HB)] homopolymer can be obtained from naphthalene by Comamonas testosteronii under specific growth requirement. These examples of PHA production from Comamonas sp. focused on PHA production from synthetic substrates. The ability of Comamonas sp. to produce PHA from renewable carbon source, especially organic acids has not been widely discussed. Since Comamonas sp. EB172 is a newly characterized strain, it is necessary to monitor the factors affecting fermentation conditions with respect to their growth, PHA production and P(3HV) incorporation in the copolymer. Thus, the present study is aimed at obtaining optimal fermentation conditions by monitoring the suitability of carbon to nitrogen ratio on the growth and PHA production by Comamonas sp. EB172 in 2 L fermenter by fed- batch cultivation using technical grade mixed organic acids. The types of polymer produced are presented.
Bacterial strain, cultivation and pH -stat fed-batch cultivation processin 2 L fermenter
The inoculum preparation, media composition and cultivation conditions for Comamonas sp. EB172 are similar to those reported previously, unless otherwise stated. 8 The pre-grown cells from the growth stage (10% v/v) were transferred into 900 mL of mineral salts medium (MSM) containing 3 g L-1 of technical grade organic acids mixtures. The MSM composition is similar to that previously reported until otherwise stated.7 The technical grade organic acids solution with the concentration of 500 g L-1 was prepared from mixtures of acetic: propionic and butyric acids at ratio 5:3:2 for feeding during fed-batch fermentation. The preparation of the acid mixtures were mimicked to original ration of acids obtained from anaerobically treated palm oil mill effluent. The initial medium pH was set at pH 7.0 with 2 M NaOH. (NH4)2SO4 was used as inorganic nitrogen source and the carbon / nitrogen ratio (C/N) were set at 5, 15 and 25 respectively. Organic nitrogen sources such as peptone and yeast extract were also used in this study and supplemented into the organic acid mixtures at 40 g L-1 and 20 g L-1 respectively. The fermentation processes were conducted in batch mode for 10-12 h before pH-stat fed-batch process was applied. The pH was maintained at pH 7.0 by feeding in the organic acids mixture (500 g L-1) with suitable C/N ratio. Dissolved oxygen tension (DOT) in the fermenter was maintained at 40% air saturation using cascade mode by gradually increasing the impeller speed. The cultures were incubated at 30 °C for 35 h for all experiments. The cultures were incubated at 30 °C for 35 for all experiments.
Organic acids concentration from the broth media was determined by high performance liquid chromatography (HPLC) (Shimadzu, LC-10 AS) as being described previously.3 PHA content and composition of the lyophilized cells was determined using gas chromatography (Agilent, model 7890A) using ID- BP1 capillary column, 30 x 0.32 x 0.25 µm (SGE). A total of 20-25 mg of lyophilized cells was subjected to methanolysis in the presence of methanol and sulfuric acid in the ratio of [85%: 15% (v/v)]. The resulting hydroxyacyl methyl esters were then analyzed according to standard method. 12
PHA in the cells were extracted using solvent extraction method by using chloroform as solvent. The extracted PHA samples (0.5-1.5 g) were soaked in 50-100 mL chloroform for 24-48 h and until completely dissolved. Films were prepared by a solvent-casting technique from chloroform solutions of the polymer using borosilicate glass petri dishes (Duran, Germany) as casting surfaces. The films were dried to constant weight in vacuum at room temperature. All samples were stored at -20°C until further analysis.
Number average molecular weight (Mn) and weight average molecular weight (Mw) were determined by gel permeation chromatography (GPC) and thermal properties of the polymer were determined by differential scanning colorimetry (DSC) (TA Instruments). For DSC analysis, 5-7 mg of copolymer samples were weighed and heated from 20 to 200 °C at heating rates 10 °C min-1 and held for 1 min. The first scan was conducted to eliminate the history of polymer properties. The samples were then fast cooled from 200 °C to -30 °C. The second scan was to reheat the samples to 200 °C at the same heating rates and the second scan was used in evaluating the thermal properties of the copolymers.8
The PHA film with average 0.25-0.35 mm of thickness and 3.0-3.3 mm of width were prepared from 2.0 g of extracted PHA samples. The samples were cut into dumbbell shape using a dumbbell cutter (Die BS 6476). The thicknesses of the samples were measured using a thickness gauge. The tensile strength, Young`s modulus, and elongation to break were determined by using Instron Universal Testing Machine (Model 4301) (USA) at 5 mm min-1 of crosshead speed. The results obtained from the computer system (using Merlin software), such as stress, strain, and elongation at break, were recorded. The results were expressed as a plot of tensile strength (MPa), tensile modulus (GPa), and elongation at break (%). Mechanical tensile data were calculated from the stress-strain curves on an average of four specimens.
RESULTS AND DISCUSSION
Effect of C/N and organic nitrogen source on the cell growth and P(3HB-co-3HV) production in the 2 L fermenterFed-batch cultivation of Comamonas sp. EB172 using different C/N ratio and organic nitrogen sources was carried out and the results are depicted in Fig. 1. The highest CDW formation was recorded at 14.5 g L-1 when 20 g L-1 of YE was supplemented in the feeding solution containing 500 g L-1 of technical grade mixed organic acids (Fig. 1a). This was followed by C/N 5 (9.5 g L-1), C/N 15 (8.1 g L-1), 40 g L-1 of peptone (6.0 g L-1) and C/N 25 (5.1 g L-1). Organic acids uptake profile of all experiments is shown in Fig. 1b. Approximately 40 mL (≈ 20 g) of acids were consumed in the experiment when YE was used as nitrogen source. It was observed that the organic acid uptake was correlated with the CDW formation. Different trend was observed on the remaining NH3-N (mg L-1) concentration in the broth solution after 30 h of fermentation period (Fig. 1c). Obviously, NH3-N concentration was kept accumulating up to 1300 mg L-1 when C/N 5 was applied to fermentation process. However, NH3-N concentration from C/N 15, 25 and peptone was maintained at values 200-400 mg L-1. The NH3-N concentration was low when YE was used as nitrogen source and totally exhausted towards 17 h of fermentation period. PHA accumulation was detected as early as 10 h but was considered low (less than 10 wt.%) in the log phase of the growth. The highest PHA accumulation was recorded at 53 (wt.%) when C/N 15 feeding solution was fed into the bioreactor (Fig. 1d). This was followed by C/N 5 (48 wt.%), peptone (45 wt.%), YE (43 wt.%) and C/N 25 (40 wt.%). From the results obtained it is suggested that Comamonas sp. EB172 was a growth associated PHA producer as PHA was accumulated during growth stage. This was supported by high amount of remaining NH3-N concentration when C/N 5 feeding solution was used.
Optimization of carbon to nitrogen (C/N) is necessary in order to monitor the requirement of growth and PHA accumulation in bacterial cells. Several studies have been reported that suitable C/N ratio is crucial for obtaining optimal growth and PHA production yield.13-15 Feeding solution with C/N 10 was fed at first stage of growth and was succeeded with C/N 50 to promote accumulation of P(3HB-co-3HV) by A. euthrophus using propionic acid and resulted in 64 g L-1 of CDW and 58 (wt.%) of accumulation respectively.14 Comamonas sp. EB172 seems to accumulate PHA during growth stage and thus, it may be grouped in the same group as Azotobacter beijerinckii and Alcaligenes latus, which are known to be the growth associated PHA producer.13 Even though the accumulation of PHA by Comamonas sp. EB172 was not restricted under nitrogen-rich medium, the imposed of nitrogen limitation in the second stage of fermentation period may enhance the PHA accumulation. Wang and Lee,13 reported that PHB accumulation was enhanced by 27 % in A. latus when nitrogen limitation was imposed to culture medium. C/N ratio is indeed an important factor in order to obtain higher biomass formation and PHA accumulation as proven by the results from this study. Our current results showed that up to 14.6 g L-1 of cells could be obtained from 0.4 g L-1 inocula, which is better than our previous report (9.6 g L-1 of cells from 1 g L-1 of inocula).8 This finding shows the highest cell dry biomass obtained so far from a genus of Comamonas with accumulation of PHA abilities.
Thermal and physical properties of P(3HB-co-3HV) copolymer produced.
Table 1 shows PHA yield, number average molecular weight (Mn), polidispersity index, melting temperature(Tm) and mechanical properties of of polymers produced at different C/N ratio. The highest conversion (0.25 g g-1) of the organic acids to PHA was recorded when YE was used as organic nitrogen source. This was followed by C/N 15, and peptone. This suggested that YE can be a suitable organic nitrogen source for growth and PHA accumulation in Comamonas sp. EB172, and at the same time gives higher PHA yield. In overall, the melting temperature, Tm for all samples was slightly reduced when higher mol% of 3HV incorporated in the P(3HB-co-3HV) copolymer.. P(3HB-co-3HV) copolymers are semi-crystalline polymers like PHB homopolymer. Their physical and mechanical properties are greatly influenced by the incorporation of 3HV unit into P(3HB-co-3HV) copolymer. It was reported that, the presence of the 3HV monomer unit in the P(3HB-co-3HV) copolymer, restricted the crystal formation and slower the spherulite growth rate.2,16-17 In this study, incorporation of 3HV monomer in the range of 6-8 (mol%) have reduced the Tm from 163 to 157 °C in comparison to P(3HB) homopolymer (170 °C).18 This study was in agreement with previous reports where incorporation of 3HV into P(3HB-co-3HV) copolymer improves the size of processing window and thermal stability of the polymer.9,18
The Mn and polydispersity index (Mw/Mn) recorded in this study were in the range of 622 to 838 x103 Da and 1.8 to 2.0, respectively. The polymer obtained from fermentation with YE as organic nitrogen source resulted in the highest molecular weight. The Mn obtained from this study was improved in comparison to our study reported earlier in which 153 - 412 x103 Da of Mn was recorded.8 This might be due to the different process or fermentation strategy applied. The high molecular weight of the copolymer obtained from Comamonas sp. EB172 exhibited high degree of polymerization that is suitable for commercialization purpose whereas Mw of below 200 x103 is brittle material and below 400 x103 resulted in poor mechanical properties.19
Meanwhile, the toughness of the polyester was recorded in the range from 13.2 to 15.1 MPa. The elasticity of the polymers produced by Comamonas sp. EB172 was recorded within the range of 160 to 339 and shared similar properties to those of polypropylene and polyethylene (Table 1). Young`s modulus results on the other hand showed that flexibility of the P(3HB-co-3HV) copolymer was influenced by the incorporation of the 3HV monomer unit. The higher the 3HV units (mol%) incorporated in the copolymer, the more flexible the polyester materials. The polymer containing 8 mol% of 3HV unit showed the highest flexibility (0.34 GPa). When it comes to product application, the mechanical test values of the polymers are normally considered. P(3HB) and P(3HB-co-3HV) copolymers were reported to have similar mechanical properties to petroleum-based plastics, polypropylene and polyethylene.2,9 Reduced tensile (MPa) and Young`s modulus (GPa) values exhibit tougher and more flexible polyester materials. The higher P(3HV) incorporated in the copolymers resulted in higher percentage of elongation to break and improved the mechanical properties.2,18 The mechanical properties of the copolymers can also be influenced by having different molecular weights and ageing factor.16,18 However, this results showed for the first time the mechanical properties of the P(3HB-co-3HV) copolymer produced from Comamonas sp. EB172.
Comamonas sp. EB172 which is an acid-tolerant strain is suitable for PHA production using renewable mixed organic acids derived from POME, making this strain a suitable candidate for industrial applications. Controlled parameters like carbon to nitrogen ratio was crucial for the PHA accumulation as well as regulation of P(3HV) mole fraction when using mixed organic acids. Supplementation of YE as organic nitrogen source in the feed solution further improved the PHA yield. Furthermore, thermal and mechanical properties, as well as the molecular weight of PHA produced from this strain exhibited promising potential polymers for a wide range of applications.
This work was performed with a financial support from Ministry of Science Technology and Innovation (MOSTI) Malaysia. The authors would like to acknowledge Ministry of Higher Education (MOHE), Malaysia for financial support, Federal Land and Development Authority (FELDA) Malaysia, and Japan Society for the Promotion of Science (JSPS) for technical assistance.