Effect of vitamin on accumulation of PHB by Zobellella species under submerged fermentation process

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In the current context of eco-pollution, the substitute of conventional plastics with bioplastics is a big confront. On account of that, a non-growth associated PHB production study was conducted using Zobellella species under submerged fermentation process. As a result, Zobellella species showed the ability to accumulate 2.2g/L of PHB at pH 8. 0, temperature 37oC, salt concentration 2% in modified growth medium containing vitamins. Then, primary structural characterization of extracted polymer was conducted by FTIR analysis. The high intense peak was obtained at 1720.50 cm-1 corresponding to ester carbonyl (C=O) stretching vibration of PHB. This finding will be paved the way of researchers during selection of suitable vitamins containing inexpensive carbon sources for cost effective PHB production. Thus, extensive research is highly indispensible for bioconversion of vitamin containing raw materials to PHB.

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Nội dung Text: Effect of vitamin on accumulation of PHB by Zobellella species under submerged fermentation process

Int.J.Curr.Microbiol.App.Sci (2017) 6(2): 1310-1316 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 2 (2017) pp. 1310-1316 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2017.602.148 Effect of Vitamin on Accumulation of PHB by Zobellella Species under Submerged Fermentation Process S. Maity1, S. Das2 and D.P. Samantaray1* 1 Department of Microbiology, CBSH, OUAT, Bhubaneswar-3, Odisha-751003, India 2 Department of Life Science, LEnME, NIT, Rourkela, Odisha - 769008, India *Corresponding author ABSTRACT Keywords In the current context of eco-pollution, the substitute of conventional plastics with bioplastics is a big confront. On account of that, a non-growth associated PHB production Eco-pollution, study was conducted using Zobellella species under submerged fermentation process. As a PHB, Zobellella, result, Zobellella species showed the ability to accumulate 2.2g/L of PHB at pH 8. 0, FTIR, Vitamins. temperature 37oC, salt concentration 2% in modified growth medium containing vitamins. Then, primary structural characterization of extracted polymer was conducted by FTIR Article Info analysis. The high intense peak was obtained at 1720.50 cm-1 corresponding to ester Accepted: carbonyl (C=O) stretching vibration of PHB. This finding will be paved the way of 22 January 2017 researchers during selection of suitable vitamins containing inexpensive carbon sources for Available Online: cost effective PHB production. Thus, extensive research is highly indispensible for 10 February 2017 bioconversion of vitamin containing raw materials to PHB. Introduction In response to risk associated with found in Gram positive (Kalaivani and petrochemical based plastic waste and its Sukumaran 2015; Mohapatra et al., 2016 and effect on the environment, there has been Chaudhry et al., 2010) and Gram negative considerable interest on the development and bacteria (Ibrahim and Steinbuchel, 2009; production of an alternative, biodegradable Tortajada et al., 2013 and Poli et al., 2011). plastic. In the current context of eco- PHAs include poly- (R)-3-hydroxybutyrate pollution, the substitute of conventional (PHB), a copolymer (PHBV) of (R)-3- plastics with bioplastics is a big confront. hydroxybutyrate (3HB) and (R)-3- Moreover, their development became hydroxyvalerate (3HV) and a copolymer indispensable due to the depletion of fossil (PHBHHx) of 3HB and (R)-3- fuel reserves, high oil prices and increased hydroxyhexanoate (3HHx) are produced by greenhouse gas emissions. Reports also various microbes (Xiao and Jiao, 2011; suggested that burning of bioplastics Mohapatra et al., 2014). producing methane, which can be further used as source of energy (Versa and Savita, 2011). These PHAs has a wide range of applications Polyhydroxyalkanoates (PHAs) are group of such as plastics materials, medical implants, most common biopolymers or bioplastics drug delivery carriers, printing and 1310
Int.J.Curr.Microbiol.App.Sci (2017) 6(2): 1310-1316 photographic materials, nutritional PHB production under submerged supplements, drugs and fine chemicals (Chen, fermentation 2009). One-stage batch cultivation in shake flasks Wide-spread substitution of conventional method was conducted for accumulation of plastics has been limited due to their high PHB granule in the bacterial cell. The production costs, which hold back its Zobellella strain was subjected to PHB successful commercialization (Waltz, 2008). production under optimized growth Thus, additional efforts are needed for making conditions. Briefly, 1L of PHB production this process economically feasible by medium containing Glutamic acid 3.8 g/L; analysing inherent mechanism of PHAs Malic acid 2.7 g/L; Na2HPO41.5 g/L; KH2PO4 accumulation process and improving its 2.5 g/L; K2HPO4 2.5g/L; NaCl 20g/L; CaCl2, productivity. Therefore, exploitation of 2H2O 0.2 g/L; MgSo4 0.8g/L; MnSo4 0.001 improved cultivation medium, culture g/L; 7H2O 0.8 g/L; (NH4)2SO4 3.08 g/L; conditions, inexpensive carbon sources and Glucose 20 g/L was prepared with pH 8.0 and genetically modified microbes can be reduced then supplemented with filter (0.45µm) cost of PHB production (Castilho et al., 2009; sterilized vitamins solution (Biotin, Thiamine, Khanna and Srivastava, 2005). Thus, the Niacin, Riboflavin, Folic acid and Patnothenic objective of study is to determine the effect of acid; each 1mg/L) individually and in different vitamins on PHB production using different combination. Subsequently, high yielding bacterial isolates. This will be submerged fermentation process was carried paved the way of lead researchers regarding out with 15% of inoculum (overnight culture bioconversion of vitamin containing containing 1.5x108 cells/ml) at temperature inexpensive carbon sources for cost effective 37ºC for 72 h with an agitation rate of 150 PHB production. rpm. Materials and Methods Extraction and quantitative analysis of PHB Culture collection PHB extraction was carried out by sodium In our preceding work, the Gram-negative hypochlorite digestion and multi-solvent Zobellella strain was found to produce 2.2 g/L extraction method (Mohapatra et al., 2015). PHB from 3.0 g biomass (CDW) in modified Then, bacterial cells were harvested by growth medium under optimized growth centrifugation at 10,000g for 15 min, dried at condition. The preserved (-20oC) bacterial 50oC to obtain dry cell mass (DCM) and isolate was revived and induced synthesis of treated with sodium hypochlorite to digest the PHB granule in their cytosol using modified non PHAs materials (NPM). The mixture was growth medium (Mohapatra et al., 2015). then centrifuged and subsequently washed Then, occurrence of PHB granule in the twice with acetone, methanol and diethyl cytosol was detected by Sudan black ether (1:1:1) to remove sodium hypochlorite (Mohapatra et al., 2015) and Nile red staining and debris. Finally, the PHB were dissolved (Spiekerman et al., 1999). All the chemicals in boiling chloroform to remove all the and culture media used in the study are remaining organic solvents present with PHB procured from Merck Millipore and Hi-media pellets and subsequent evaporation by air Laboratories Private Limited. drying to yield dried form of extracted PHB. 1311
Int.J.Curr.Microbiol.App.Sci (2017) 6(2): 1310-1316 PHB production was also quantified using thiamine has higher effect on cellular growth standard formula. than other vitamin supplemented in the medium. Consequently, PHAs production was Characterization of PHB by FTIR Analysis found to be 0.062-1.835 g/L and was higher in presence of thiamin and lower with folic The functional groups present in the PHB acid. The PHAs production was 1.835 g/L and were analyzed by FTIR spectroscopy. PHB 1.790 g/L in presence of thiamin and biotin sample was mixed with 2% KBr and respectively. Notably, no significant compressed into translucent sample discs and difference of PHAs production was observed fixed in the FTIR spectrometer (Perkin-Elmer between thiamin and biotin as source of RX I). Scans were accomplished at spectral vitamin. However, percentage of PHAs range, 4000-400 cm-1; window material, CsI; production was 65.42% and 42.93% on 16 scans and resolution 4 cm-1. The detection supplementation of biotin and thiamin was conducted with a temperature stabilized, respectively. Hence biotin has a major role on coated FRDTGS detector (Dash et al., 2014). PHAs yield, which is not parallel to biomass production in case of Zobellella species. Results and Discussion Similarly, different combinatorial effect of vitamin on PHAs production results (Figure Although wide arrays of bacterial species 2) indicated that, the biomass and PHAs yield accumulate PHAs, however the major was varied between 2.601-3.847 g/L and limitations in the production of PHAs are 1.401-2.085 g/L respectively. The biomass selection of inexpensive materials and its and PHAs production was increased with composition, producer microbes, bioprocess supplementation of biotin and thiamin condition, fermentation process and high cost combination and decreased with riboflavin downstream processing. Among all selection and folic acid. However, higher percentage of inexpensive material and its composition is (68.14%) of PHAs production was obtained the predominant factor affecting PHAs on supplementation of multi-vitamin such as production bioprocess technology. As regards biotin and riboflavin in the production 50% of the production costs of PHAs is medium. From the above data, it is concluded depend upon cost of carbon sources and that biotin has a noteworthy role on PHAs vitamins supplemented in the production production nevertheless after supplementation medium (Gomma, 2014). In this small piece of biotin, thiamine, niacin, riboflavin, folic of research an attempt has been made to study acid and patnothenic acid production was the effect of different vitamin such as biotin, increased to 2.2 g/L by Zobellella species. thiamin, niacin, riboflavin, folic acid and patnothenic acid on PHAs accumulation by No comprehensible evident is available in the bacterial isolate. public domain regarding increase in PHAs production by Zobellella species on PHAs production supplementation of vitamin. In addition, vitamins including thiamin, riboflavin and From the result (Figure 1), it was found that biotin are essential growth factors for biomass production by the bacterial isolate different bacterial isolates (Du et al., 2011; ranged between 1.20-4.20 g/L in presence of Preciado et al., 2015). Thus, vitamin and different vitamins. The biomass production amino acids can be supplemented for efficient was increased with supplementation of PHAs production (Varsha and Savitha, 2011). thiamin and decreased with folic acid. Thus, In contrast to our observation, biotin is also 1312
Int.J.Curr.Microbiol.App.Sci (2017) 6(2): 1310-1316 able to switch the carbon flux of acetyl-CoA (Chowdhury et al., 2005; Sangkharak and towards biosynthesis of PHB by Prasertsan 2008). In our observation the Corynebacterium glutamicum thereby PHAs production was increased with increases production (Jin et al., 2009). supplementation of vitamins such as biotin, However, vitamin was no effect on PHB thiamin and riboflavin, which suggesting production by Pseudomonas pseudomallei selection of vitamin containing raw material and Rhodobacter sphaeroides 14F for cost effective production. Fig.1 Effect of vitamins on cell biomass and PHAs production Fig.2 Effect of vitamins (combination) on cell biomass and PHAs production 1313
Int.J.Curr.Microbiol.App.Sci (2017) 6(2): 1310-1316 Fig.3 FTIR analysis of PHAs extracted from Zobellella sp. FTIR spectral analysis of extracted PHAs thiamine and niacin may be used for cost effective PHB production. Thus, extensive The FTIR data indicated (Figure 3) different research is highly indispensible for characteristic peak at 1055.06 cm-1 and bioconversion of vitamin containing raw 1276.88 cm-1 corresponding to C-N stretch materials for PHB production. and C-C, C-H, O-H stretch respectively. On the other hand, the high intense peak was Acknowledgement obtained at 1720.50 cm-1 conferred to ester carbonyl (C=O) stretching vibration of PHB. The authors are thankful to Dr. B. B. Mishra, Thus, IR spectrum depicts correct insight for HOD, Microbiology and staff of Central the chemical configuration of PHB, which is laboratory, OUAT for providing laboratory the most common homopolymer of PHAs. facilities during the period of study. The This result is also similar to the IR spectrum authors have no conflict of interest to declare. obtained at 1720.50 cm-1 corresponding to (C=O) ester carbonyl group, characteristics of References PHB (Shah, 2012; Sharma and Bajaj, 2015). Castilho, L.R., Mitchell, D.A. and Freire, The present study was designed to determine D.M.G. 2009. Production of the effect of different vitamins on PHB polyhydroxyalkanoates (PHAs) from production using high yielding bacterial waste materials and by-products by isolates. As a result, Zobellella species submerged and solid-state fermentation. showed the ability to accumulate 2.2g/L of Biores. Tech., 100: 5996–6009. PHB in growth medium with vitamins which Chaudhry, W.N., Nazia, J., Ali, I., Ayaz, can increases the cost of production. M.H. and Hasnain, S. 2010. Screening However, this finding will be paved the way for polyhydroxyalkanoate (PHA)- of researchers during selection of suitable producing bacterial strains and inexpensive carbon sources for PHB comparison of PHA production from production. Raw materials (cane molasses, various inexpensive carbon sources. beet molasses, banana peel and fish waste) Ann. Microbiol., DOI 10.1007/s13213- containing different vitamin such as biotin, 010-0181-6. folic acid, pantothenic acid, riboflavin, Chen, G.Q. 2009. A microbial 1314
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