id4058218 pdfMachine by Broadgun Software - a great PDF writer! - a great PDF creator! - http://www.pdfmachine.com http://www.broadgun.com JBaBnuary 200ii9 ooTTeecchhnnoollVolooume g3g Issuyye 1 Trade Science Inc. An Indian Journal FULL PAPER BTAIJ, 3(1), 2009 [24-29] Polyhydroxyalkanoate (PHA) production from Amphibacillus sp. Pushpa S.Murthy*, M.Madhumathi Plantation Products, Spices and Flavour Technology Dept., Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore-570020, Karnataka, (INDIA) Tel : 91-821-2512352; Fax: 91-821-2517233 E-mail : [email protected] Received: 31st October, 2008 ; Accepted: 5th December, 2008 ABSTRACT KEYWORDS Many bacteria accumulate Polyhydroxyalkanoate (PHA) as an energy re- Polyhydroxyalkanoate (PHA); source. The microbial thermoplastics are regarded as potentially useful poly- Amphibacillus sp.; ester replacing petroleum-derived thermoplastics. Polyhydroxy butyrate Polymer industry soil, (PHB) is the best-known member of the PHA series of polyesters. PHB pro- ducing strain of Amphibacillus sp was isolated from polymer industry soil ’s manual of determi- and identified by comparison with keys given in Bergey native bacteriology. The initial yield of PHA was 39.90 0.5% in the produc- tion medium. Different carbon and nitrogen sources were tested for PHB production by this bacterium Maximum yield of PHA was observed with glucose (82.20 0.4 %) followed by fructose (47.09 0.51%) and mannose (47.09 0.4%), arabinose (41.42 0.5 %). Sucrose, being a cheaper substrate, was used for further studies. Among the nitrogen sources beef extract and tryptone promoted PHB synthesis with 42.42 0.3 % and 41.65 0.02 % respectively. Growth conditions for production of PHA by the Amphibacillus sp were studied. The optimized conditions with 30 0.50C of temperature and pH 7.0 0.1 with 3 % inoculum at 250 rpm at 48 h of fermentation using sucrose as the carbon source yielded 67.03 0.5% of PHB. The PHB pro- duced was analyzed and confirmed by FTIR and NMR. 2009 Trade Science Inc. - INDIA INTRODUCTION economically feasible by further understanding the poly- -hydroxybutyrate (PHB) accumulation process and Many bacteria accumulate polyhydroxyalkanoate improving the productivity. A number of Bacillus sp, (PHA) as an energy resource[21]. PHAs have physical have been reported to accumulate 9-44.5% dry cell and chemical properties similar to those of synthetic poly- weight (DCW) PHA[1,5,7,1113,22,19]. Biotechnological mers and are also biodegradable[11]. PHA accumula- processes for the fermentative production of poly (3HB) tion usually occurs in the presence of excess carbon and poly (3HB-co3HV) using strains of Alkaligenes and with a limitation of one essential nutrient such as latus and Ralstonia eutropha have been established nitrogen, phosphorus, sulfur, magnesium, potassium or since late 1970s for the manufacture of various prod- iron[14]. Efforts are being made to make this process ucts. To obtain a novel isolate that can grow on a cheaper BTAIJ, 3(1) January 2009 Pushpa S.Murthy and M.Madhumathi 25 FULL PAPER substrate, we have attempted to isolate bacteria that culture was maintained at 40C. accumulate PHA from a soil. In this paper, we describe Characterization of the bacterium the isolation and characterization of gram-positive bac- terium Amphibacillus sp which had 40-80 % PHA The morphological and physiological properties of productivity and influence of nutritional and environmen- the isolate were investigated according to Bergeys [6] tal conditions on the growth and PHB accumulation. manual of determinative bacteriology . Structural analysis MATERIALS AND METHODS The polymer analysis of PHA were carried by FTIR and NMR. Along with the standard PHB Chemicals (Sigma,Germany). The FTIR was performed using All the chemicals and reagents used in the studies perkinn Elmer, FTIR spectrometer, Spectrum-2000. -1 were of analytical grade obtained from standard com- The IR source with 400-4000cm intensity was used. panies. The NMR spectra of PHA were obtained in deuter- ated chloroform (CDCl3) for protons using BRUKER Isolation of bacterial strain by enrichment tech- AVANCE 500 ultra shield spectrometer. The 1H NMR nique spectra were recorded using standard PHB with a 30 Various bacterial species were isolated from the soil pulse, 10.50 s, 10,000Hz spectral width and a 3 sec located near the polymer industry located near Mysore, repetition rate. – India. The selected isolates were purified by dilution Production of PHA from various carbon and nitro- streaking to obtain single colonies, and were grown in gen sources nutrient medium in order to produce large amount of cells. After 24 h, the cells were inoculated (10% v/v) to Different sugars like sucrose, glucose, fructose, a sterilized liquid culture medium 100ml (gl-1): galactose, lactose, xylose, maltose, arabinose, some of Na HPO 2H O, 2.2, KH PO , 1.5; (NH ) SO , 1.5; the sugar alcohols such as dulcitol, mannitol, were in- 2 4 2 2 4 4 2 4 vestigated for the PHA production by the isolated strain. MgSO47H2O, 0.2; Sucrose, 20; pH 7.The inoculated flasks were incubated at 300C at 250 rpm for 24h. Each Likewise organic and inorganic nitrogen sources such of the purified isolates was screened for PHA accumu- as beef extract, yeast extract, peptone and tryptone, lation after 24 hours of the growth and stained with potassium nitrate, ammonium nitrate, ammonium phos- phatewere also studied with sucrose (2.0%) along with Sudan black stain to detect the presence of intracellular PHA granules. Only the isolates, which could synthe- mineral salts and trace elements were incubated for 48h 0 size PHA, was selected and studied for PHA produc- of fermentation at 30 C at 250 rpm. The cell mass and tion. PHB content was determined. PHA production Optimization of PHA production The PHA production was carried out in duplicate The influence of pH, temperature, duration, aera- in 500ml Erlenmeyer flasks in a liquid medium (100ml) tion and inoculum was determined to obtain maximum mentioned under inoculum preparation. Overnight yield of PHA production from the bacterial strain. Vari- grown cultures of the isolated bacteria were inoculated ous pH levels i.e. 6.0,6.5,7.0 and 7.5 was studied. 0 and incubated at 250 rpm and 300C for 72h. The cell Fermentation temperature at 30, 37 and 42 C and aera- biomass was collected by centrifugation. The cell pellet tion for bacterial growth in terms of shaking (250rpm) was washed with distilled water and dried. The PHA and static conditions along with different fermentation content was determined after sodium hypochlorite hy- time at 24, 48 and 72h was investigated. Also the in- drolysis and chloroform extraction. PHA (%) was de- oculum concentration at 1, 2, 3 % were studied with fined as the percentage of the PHB to dry cell weight sucrose as the carbon source to obtain the best yield of (DCW). The isolate that produced maximum yield of PHA. The PHA production was estimated as mentioned PHA was considered for further investigation. The stock earlier. BiioTechnollogy An Indian Journal 26 PHA production fro.m Amphibacillus sp. BTAIJ, 3(1) January 2009 FULL PAPER TABLE 1 : Morphological and taxonomical characteristics of amphibacillus sp. e Characterization c Observations n morphology a t – i Medium Rods, 1.2 2.0 m e Shape, Size ( m), Motility s 3.0, motile n a Gram staining, Sudan Staining, r T +, +, + Spore formation % Features of colonies: (Plate) Small, circular, whitish Features of the colony cream Wavenumbers(cm-1) Pigmentation No pigmentation Figure 1: FTIR of standard PHB Margin, Elevation Irregular, Flat Growth in Nutrient broth Uniform with less turbidity e Flocculent Evenly dispersed in medium c n a Pellicle / sediment Sediment t i Growth in m e Faint growth only in 3% s Nacl:3%,5%,7%,10% n a Growth on Macconkey - r T Optimum pH and temperature 7.0, 30oC % Physiological Characteristics - /Biochemical properties Wavenumbers(cm-1) Catalase - Figure 2: FTIR of PHB from Amphibacillus sp Indole + MR-VP ++ -- 0 Starch, Citrate production, perature at 37 C. Based on the results obtained from - Oxidase various biochemical test performed (TABLE 1) and Casein, Nitrate reduction - Bergeys manual of determinative bacteriology, the strain TSI, Litmus reduction, Gelatin, + was identified and designated as Amphibacillus sp. H2S Aerobic Conditions + Production of PHA Utiltisation of sugars: Arabinose, Sucrose, Starch, Amphibacillus sp produced 39.90 0.3% dry Glucose, Fructose, Galactose, cell weight (DCW) of PHA in the production media Inulin, Maltose, Arabinose, + with respect to dry cell mass. Sucrose was rapidly uti- Dulcit, Rhamnose ,Mannose, lized during the growth and PHB accumulation was Xylose, Lactose, Mannitol, Nanonoic acid, Hexanoic acid, observed. Amphibacillus xylanus has been reported Heptanoic acid to produce PHA[12]. No other species apart from Positive: + and Negative: - A.xylanus is reported. In the recent past, species of RESULTS AND DISCUSSION the genus Amphibacillus such as A. sediminis, A.fermentum, A.tropicus are reported[23] as a new iso- Screening of bacteria accumulating PHA lates isolated from sea and sludge. Our isolate with ge- We attempted to isolate bacteria that accumulate nus Amphibacillus, could be a new species and is po- PHA from polymer industry soil. Forty-six strains were tential producer of PHA and is of industrial interest. isolated by enrichment culture method and were classi- Structural analysis fied according to their morphology and their ability to The FTIR analysis of the polymer suggests very synthesis PHA was determined. The isolate with the prominent peak at the 1724-1760 cm-1. The major best yield was further studied for nutritional and bio- peaks obtained from our sample corresponds to 1739.4 chemical characterization. cm-1, which is very close to peak 1740cm-1ester car- The isolated strain was gram positive, rod shaped bonyl groups when compared with the standard PHB cell (1.22 m-2.0 m) and motile.