SCBT 691-3: Biotechnology Seminar 1-3 2010 L.02 November 25 ...

SCBT 691-3: Biotechnology Seminar 1-3 2010
L.02 November 25 th , 2010 (9:00-12:00 A.M.)
Speaker: Mr. Perapat Potinoun
I.D. 5237144 SCBT/M
Advisor: Dr. Nuttawee Niamsiri
Title: Statistical Optimization and Culture Conditions for Growth and Polyhydroxybutyrate (PHB)
Accumulation from Cassava Starch by Isolated Bacillus sp.
Polyhydroxyalkanoates (PHAs) are a class of natural polymers that are produced by a wide variety
of bacteria as their intracellular energy storage compounds. They are biodegradable and process similar
material properties to various petrochemical-based plastics. However, its higher cost of production is still
one of the important factors hampering the commercialization of PHAs. Thus, research on improving the
productivity of PHAs from bacterial system is still needed in order to help reducing the overall PHAs
production cost.
Our main goals are to find suitable media compositions as well as culturing conditions for
maximizing both cell growth and PHAs production from cassava starch by the PHA-producing bacteria of
interest previously isolated in our laboratory. Thus far, four different Bacillus megaterium strains; S0-1,
S1-2, S50-3 and S50-3 have been selected and studied for their growth profile as well as their PHB
production capability. Thus far, S1-2 was found to be the best PHA producer that can produce
polyhydroxybutyrate (PHB) up to 50% PHB content per cell dried weight with biomass of 4.5 g/L from
glucose. Consequently, it was selected for the further studies for its growth and PHB production from
various carbon substrates representing different stages of digested cassava starch; glucose, maltose, dextrin,
liquefied starch, saccharified starch and gelatinized starch that were provided at the same C:N ratio. We
found that S1-2 could produce PHB from the saccharified cassava starch better than liquefied and
gelatinized cassava starch, resulting in similar biomass and %PHB content to pure glucose. Currently, the
effect of different nitrogen source on growth and PHB production by S1-2 is investigated when provided
together with the saccharified cassava starch. For the nitrogen source selection, ammonium sulfate and
ammonium chloride gave the highest growth and PHB production among various nitrogen sources (i.e.
ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate and urea). Ammonium
sulfate was selected, due to its less toxicity than ammonium chloride at high concentration. Then, other
important media compositions and culturing conditions will then be optimized based upon statistical
designed experiments using Plackett-burman design and response surface methodology in shake-flasks as
well as in bioreactors.
Reference: Speaker’s research work
Speaker: Miss Naruwan Phenprom
Advisor: Assoc. Prof. Dr. Apinya Assavanig
I.D. 5237142 SCBT/M
Title: Proteinase-producing halophilic lactic acid bacteria isolated from fish sauce fermentation and their
ability to produce volatile compounds

Halophilic lactic acid bacteria were isolated from fish sauce mashes fermented at 1 to 12 months.
Seven outof sixty-four isolates were selected according to their proteolytic activity and growth at 25%
NaCl for characterization and investigation of volatile compound production. All selected isolates were
Gram-positive cocci with pairs/tetrads and grew at 0–25% NaCl, pH 4.5–9.0. Results of 16S rRNA gene
sequence analysis showed 99% homology to Tetragenococcus halophilus ATCC 33315. The restriction
fragment length polymorphism (RFLP) patterns of all isolates were also similar to those of T. halophilus
ATCC 33315. These isolates were, thus, identified as T. halophilus. All isolates hydrolyzed fish protein in
the medium containing 25% NaCl. Intracellular aminopeptidase of 7 isolates exhibited the highest activity
of 2.85–3.67 U/ml toward Ala-p-nitroanilide (Ala-pNA). T. halophilus strains MS33 and M11 showed the
highest alanyl aminopeptidase activity (Pb0.05), and produced histamine in mGYP broth containing 5 and
25% NaCl in the level of 6.62–22.55 and 13.14–20.39 mg/100 ml, respectively. Predominant volatile
compounds of fish broth containing 25% NaCl inoculated with T. halophilus MS33 and MRC5-5-2 were 1-
propanol, 2-methylpropanal, and enzaldehyde, corresponding to major volatile compounds in fish sauce. T.
halophilus appeared to play an important role in volatile compound formation during fish sauce
fermentation.
Reference:
Udomsil, N., Rodtong, S., Tanasupawat , S., & Yongsawatdigul, J. (2010). Proteinase-producing
halophilic lactic acid bacteria isolated from fish sauce fermentation and their ability to produce
volatile compounds. International Journal of Food Microbiology, 141, 186–194.
Speaker: Miss Onsaya Patanun
I.D. 5237145 SCBT/M
Advisor: Assoc. Prof. Dr. Jaranya Narangajavana
Title: Genome-wide prediction and identification of microRNAs and their target genes in cassava
In the past several decades, many studies have been discovered that microRNAs regulate various
stages of development and physiological process in plants. Nevertheless, the miRNAs whose function
involved in the switching from fibrous root to storage root are not yet known. To unravel miRNAs
associated with storage root development, approximately 400 mature miRNAs sequences from Arabidopsis
thaliana and Populus trichocarpa were used as query sequences to blast against the cassava genome in
Phytozome database. All blast hits were further screened by using online Mfold program to analyze their
secondary structure. At present, 13 potential miRNAs in cassava reported in MicroPC database were
collected and also 10 miRNA were identified from the cassava genome. We totally collected 18 miRNA in
cassava. Interestingly, we also found 5 new cassava miRNA, which are miR156, 160, 164, 167, and 396
based on sequence homolog of the mature miRNA sequence. All miRNA target genes were also predicted.
Some candidate miRNAs were further confirmed by using experimental approaches. The expression
analysis of miRNAs and their target genes are in progress. The identified miRNAs should enable
investigation of the role of miRNAs in storage root development. The better understandings of the miRNAs
in storage root development would be beneficial in helping to develop cassava variety with desired
properties.
Reference: Speaker’s research work

Speaker: Miss Chattri Khomkrit
I.D. 5237147 SCBT/M
Advisor: Dr. Thipa Asvarak
Title: Control of sheath blight disease in rice by thermostable secondary metabolites of Trichothecium
roseum MML003
Fungal plant diseases are the main major causes of crop production yield loss worldwide. Chemical
fungicides are extensively used but human and an environment may have an affect from those chemicals
too. Using antagonistic microorganism and its secondary metabolites to control plant diseases are friendlier
and less toxic than chemical treatment, and are becoming a very interesting approach in the present. In this
study Trichothecium roseum MML003 was investigated for biocontrol mechanisms against Rhizoctonia
solani, rice sheath blight pathogen, both in vitro and in vivo experiments. On petri dish Trichothecium
roseum MML003 inhibited growth of Rhizoctonia solani with an existing of a clear zone on GPYEA
medium. The culture filtrates of Trichothecium roseum can inhibit mycelium growth and sclerotia
formation and germination, indicating that bioactive activity is due to antibiotic production. Crude
metabolites of Trichothecium roseum MML003 showed reduction of sheath blight disease in pot
experiments. The crude antifungal metabolites were also characterized. Thus, this study provided evidence
of using fungal bioactive metabolites to control plant disease.
Reference:
Jayaprakashvel, M., Selvakumar, M., Srinivasan, K., Ramesh, S., & Mathivanan, N. Control of sheath
blight disease in rice by thermostable secondary metabolites of Trichothecium roseum MML003.
European Journal of Plant Pathology, 126: 229–239.
Speaker: Miss Porawee Pramoolkit
I.D. 5237147 SCBT/M
Advisor: Assoc. Prof. Dr. Jaranya Narangajavana
Title: Expression and functional analyses of microRNA417 in Arabidopsis thaliana under stress
conditions
MicroRNAs (miRNAs) are 20-25 nucleotides-long RNA transcripts that are capable of suppressing
target gene expression by either mRNA cleavage or translational repression. Although many recent reports
propose that miRNAs play diverse roles in growth, development, morphogenesis, and stress responses of
plants, the biological roles of many miRNAs remain to be verified. Here, we investigated stress responsive
expression patterns and functional roles of miRNA417 in seed germination and seedling growth of
Arabidopsis thaliana under various abiotic stress conditions. miRNA417 was expressed constantly
throughout the entire growth stages and ubiquitously in all organs including stems, roots, leaves, and
flowers. The expression of miRNA417 was regulated by salt stress, dehydration stress, orabscisic acid
treatment. To examine the biological roles of miRNA417 in stress responses, the transgenic Arabidopsis
plants that constitutively overexpress miRNA417 under the control of cauliflower mosaic virus 35S
promoter were generated, and their phenotypes were analyzed under stress conditions. Results showed that
seed germination of the transgenic plants was retarded compared with the wild-type plants in the presence
of high salt or abscisic acid. These results imply that miRNA417 plays a role as a negative regulator of
seed germination in Arabidopsis plants under salt stress conditions.
Reference:
Jung, H.J., & Kang, H. (2007). Expression and functional analyses of microRNA417 in Arabidopsis
thaliana under stress conditions. Plant Physiology and Biochemistry 45, 805-811.