April - June 2007 - Kasetsart University

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326 the basal medium and placed in a 125 ml Erlenmeyer flask. All experiments were carried out in triplicate flasks. Cultivation was initiated by addition of 1 ml of inoculum (adjusted cells concentration to 1.0 at OD 600 nm). Incubation was done on a rotary shaker (Sac Science-ENG LTD, Part) at 140 rpm at room temperature for 4 days. To test different media, the basal media was modified in the following manner: 1. The carbon source replaced by either glucose, fructose, sucrose, glucose syrup and agricultural products i.e. molasses, and sugar cane juice (Sahakarnnamtan Co. Ltd., Chonburi, Thailand). 2. The nitrogen sources replaced by peptone, soybean meal, skimmed milk, ammonium sulfate, potassium nitrate, sodium nitrate, monosodium glutamate (MSG). 3. Sea salt concentration (salinity) 0- 200% of sea water. The effect of temperature on growth and DHA production were also determined by using temperature gradient incubator (Model TN-3, Toyo, Kagaku Sangyo Co., Ltd., Tokyo, Japan) set at 15, 20, 25, 30 and 35°C. Optimization of growth and astaxanthin production by thraustochytrid mutant The mutant was cultivated in a 125 ml Erlenmeyer flask containing 30 ml of GYC medium (Marvelisa et al., 2003) and kept in an incubator shaker at 180 rpm for 10 days at 25°C. Light was provided by fluorescent lamps at the intensity of 2 kLux with light:dark periods at 16:8 hrs. Effects of carbon sources such as sugar cane juice, molasses and maltose:glucose (1:1, w/w) contained the same carbon equivalent as 2% glucose were studied. Environmental conditions such as light intensity at 0, 5 and 10 kLux and temperature (as described above) were also determined. <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) Analytical procedures Growth was determined as the dry weight of the cells (drying conditions). Lipid was extracted by the modified method of Bligh and Dyer (1959), followed by transmethylation according to Holub and Skeaff (1987). Fatty acid methyl esters were analyzed in a gas-liquid chromatography (GC-14B; Shimadzu, Tokyo, Japan) equipped with flame ionization detector and a split injector at 1:40 ratio using capillary column in 30 m length, 0.25 mm internal diameter, 0.25 mm. film thickness (AT-WAX, Alltech Associates Inc, USA). Fatty acids were identified by comparing retention times with authentic standards from Sigma by using C-R6A Chromatopac Data Integrator (Shimadzu, Japan). The astaxanthin content was determined by the method modified from Fontana et al. (1996). The concentration was quantified by using absorbance values at 479 nm calculated with the specific absorption coefficient a (1cm,1%) = 1600 as proposed by Anderwes et al. (1976). Isomers of astaxanthin were identified by thin layer chromatography according to Donkin (1976) compared with reference standards extracted from Haematococcus pluvialis. These determinations were confirmed by HPLC (model HP1100, Agilent Technology) following the procedure of Marvelisa et al. (2003). RESULTS AND DISCUSSION Identification of thraustochytrid BR2.1.2 by 18S rDNA sequence analysis The corrected partial sequence of 18S rDNA of thraustochytrid BR2.1.2 was 912 bases in length after gaps, inserts and ambiguous positions had been removed and was deposited in DDBJ as accession number 794133. A phylogenetic tree was constructed from an alignment of the BR2.1.2 sequence with those from related species obtained from GenBank by the NJ method (Figure 1). It was clearly seen that
BR2.1.2 formed the same clade with Thraustochytrium aggregatum and Schizochytrium limacinum but with slight distance. Hence the strain BR2.1.2 was finally identified as Schizochytrium limacinum. Effect of culture conditions on growth and DHA production by S. limacinum BR2.1.2 1. Carbon source Among the various carbon sources tested, S. limacinum BR2.1.2 exhibited highest growth rates in 3% fructose and glucose with 14.3 and 13.4 g/l of CDW, respectively (Figure 2A). DHA yields were 392.5 and 362.1 mg/l with DHA <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) 327 contents at 49.1 and 49.7% of TFA, respectively. Although, relatively good growth rates were obtained in complex carbon sources, i.e. molasses (10.5 g/l) and sugar cane juice (11.5 g/l), DHA production was low. Sucrose and glucose syrup were poorer carbon source for this organism. The results coincided with those of Wu et al. (2005) as glucose syrup contained mainly oligosaccharides that could not support growth for many microorganisms. Although glucose was slightly inferior compared to fructose, it is considered to be the good carbon source, because it was ready available and substantially cheaper. Figure 1 Phylogenetic tree reconstruction based on 18S rDNA sequence by neighbour-joining (NJ) method. The number at each branch shows bootstrap values 1000 replications.
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April - June 2007 Volume 41 Number
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KASETSART JOURNAL NATURAL SCIENCE T
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Kasetsart J. (Nat. Sci.) 41 : 205 -
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harvesting time which started from
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y Chen and Paull (2000). Figure 1 a
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pineapple fruit allowed the accumul
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Kasetsart J. (Nat. Sci.) 41(2) 215
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Cluster analysis Cluster analysis u
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Table 3 (Continued) 1 2 3 4 5 6 7 8
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CONCLUSION AFLP markers classified
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difference of soil texture used in
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under wet soil condition planting.
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tropics. Following the expansion, w
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sealed in a plastic box with 1 cm w
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moisture content increment after so
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Frequency Frequency 30 20 10 0 20.0
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School, Kasetsart University. The a
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for combining ability of lines (Lam
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selective mass sibbing within indiv
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Although most of S 2-interfamily hy
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composite sets as used in this stud
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days. The numbers of calli producin
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the report of Ching (1982) showing
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clearly amplified bands generated b
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caused by either the recombination
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Kuhlmann, V. and B. Foroughi-Wehr.
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Xanthomonas fuscans subsp. aurantif
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was collected and washed with 70% e
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expected size was amplified with 35
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eaction technique has been used for
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Schaad, N.W., D. Opgenorth and P. G
Page 75 and 76: MATERIALS AND METHODS Model descrip
Page 77 and 78: Kasetsart J. (Nat. Sci.) 41(2) 277
Page 79 and 80: calculated TF value calculated TF v
Page 81 and 82: sincere gratitude and deep apprecia
Page 83 and 84: 1989). In monogastrics, the inclusi
Page 85 and 86: of all LLS samples in this study we
Page 87 and 88: Melbourne, Parkville, Victoria. Goe
Page 89 and 90: considered responsible for low prod
Page 91 and 92: Kasetsart J. (Nat. Sci.) 41(2) 291
Page 93 and 94: mineral supplementation suggested b
Page 95 and 96: Body weight chane (kg/head) 32 30 2
Page 97 and 98: CONCLUSION AND RECOMMENDATION With
Page 99 and 100: SAS. (Statistical Analysis System).
Page 101 and 102: genes covering a variety of desirab
Page 103 and 104: mg/l NAA and 0.5 mg/l zeatin) incub
Page 105 and 106: 5. Purification by various sucrose
Page 107 and 108: as the culture period increased. So
Page 109 and 110: culture, pp. 1-20. In L.C. Fowke an
Page 111 and 112: Kasetsart J. (Nat. Sci.) 41 : 311 -
Page 113 and 114: GC. Analytical methods Total carboh
Page 115 and 116: ash. The crude hot water polysaccha
Page 117 and 118: spirulan (H-SP), and a desulfated c
Page 121 and 122: cells often displayed an increased
Page 123 and 124: LITERATURE CITED Bell, T.A. and D.V
Page 125: for alternative sources of supply.
Page 129 and 130: supplement with glutamate (Iida et
Page 131 and 132: optimal for S. limacinum BR2.1.2 fo
Page 133 and 134: fluorescent lamp at 1.5 kLux develo
Page 137 and 138: Wisconsin, USA). Total extracted RN
Page 139 and 140: RESULTS Cloning and sequencing of m
Page 141 and 142: Expression of rmIL-2 and purificati
Page 143 and 144: other strains of mice have differen
Page 145 and 146: dose interleukin-2 therapy promotes
Page 147 and 148: The chitosano-oligosaccharides are
Page 149 and 150: enzyme and cells were maximized by
Page 151 and 152: of crude chitosanases. Fortunately,
Page 153 and 154: Relative activity (%) 100 80 60 40
Page 155 and 156: Uchida, K. Tateishi, O. Shida and K
Page 157 and 158: MATERIALS AND METHODS 1. Materials
Page 159 and 160: without coating and coated with pec
Page 161 and 162: in Table 5-7. It was found that the
Page 165 and 166: the cultures at 10,200 × g for 15
Page 167 and 168: incubation and reached the final pH
Page 169 and 170: y lysine- and tyrosine-decarboxylas
Page 171 and 172: during the stages of ripening as su
Page 175 and 176: As the 25 companies were divided in
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All the data comes up with informat
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as piece “A” or straight as pie
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et al. (1998); Zeng (2000); and Tal
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p 1, i * Kasetsart J. (Nat. Sci.) 4
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c ≤ c2 jq2 j n q p k p * 2, j , ,
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algorithm would consider RM 5, prio
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the maximum deviation of about 10%.
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Stock Quantities Using Heuristic Op
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which dynamically and automatically
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2. Framework architecture and compo
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Risk (VaR) calculation which was im
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Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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April - June 2007 - Kasetsart University

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