Kittiwongwattana, Chokchai

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Alternative Name

Kittiwongwattana, Chockchai

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School of Science

chokchai.ki@kmitl.ac.th

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Gel purification of gDNA for next-generation sequencing applications
(2022-07-01)
Utthiya, Supanut
;
Wonnapinij, Passorn
;
Napaumpaiporn, Pondpan
;
Kittiwongwattana, Chokchai
;
Sakulkoo, Jenjira
We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This method yields a substantial amount at microgram levels of gDNA per gel cleanup with high purity. An RNase A treatment step can be omitted. The quality of gDNA is suitable for next-generation sequencing, resulting in >10 Mb reads and high-quality read data (Phred score >28 up to 100 of 150 base reads). Furthermore, the gDNA can be kept intact in a gel slice for several days. This method has been tested for dictyostelids, bacteria and plants.
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Modified natural seawater as growth medium for the halotolerant cyanobacterium Aphanothece halophytica to increase lipid content for biodiesel production
(2025-02-01)
Thongtha, Sitthichai
;
Aryusuk, Kornkanok
;
Kittiwongwattana, Chokchai
;
Incharoensakdi, Aran
;
Phunpruch, Saranya
Biodiesel derived from cyanobacterial oils becomes attractive as an efficient renewable energy. The present study aims to optimize growth and lipid production of the halotolerant unicellular cyanobacterium Aphanothece halophytica cultivated in natural seawater. In this study A. halophytica was able to grow in natural seawater when supplemented with low concentration of NaNO<inf>3</inf>, whereas no growth occurred without supplementation. The specific growth rate of 0.230 day-1 and cell concentration of 25.17 x 106 cells mL-1 were achieved in A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO<inf>3</inf> and Turk Island salt solution (suitable natural seawater; SNSW) for 14 days. This growth rate was comparable to that of cells grown in normal BG11 plus Turk Island salt solution. The lipid content and fatty acid profiles of A. halophytica varied with changes in NaCl concentrations. The highest lipid content of 50.47 % and lipid productivity of 48.33 mg L-1 day-1 were obtained in cultures supplemented with 1.89 mmol C-atom L-1 glucose and 0.75 M NaCl. The optimal medium pH and cultivation temperature for lipid production was 7.5 and 25-35 °C, respectively. When cultivating A. halophytica in optimized SNSW with various NaCl concentrations, the highest contents of linoleic and linolenic acids, and the lowest contents of palmitic, stearic, and oleic acids were observed with 0.75 M NaCl. In contrast, cultures grown in optimized SNSW with 0.5 M NaCl showed fatty acid methyl ester profiles rich in monounsaturated fatty acids, which are favorable for high-quality biodiesel production.
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Correlation of Antifungal Activities and Nonribosomal Peptide Synthetase Gene Expression of Bacillus siamensis 1021
(2022-03-01)
Nuwong, Warinya
;
Kittiwongwattana, Chokchai
Members of the genus Bacillus were biocontrol agents against various phytopathogenic fungi. Several factors affected the regulation and production of nonribosomal peptides (NRPs) in Bacillus species. The aim of this study was to examine how culture media influenced the antagonistic activities and gene expression of Bacillus siamensis 1021. The bacterial supernatant from potato dextrose broth (PDB) exhibited higher inhibitory effects against mycelia of Pyricularia oryzae and Colletotrichum fructicola, when compared with those from nutrient broth (NB) and minimal salt medium (MSM). However, the biocontrol activities of these supernatants were not significantly different when they were tested against P. oryzae and C. fructicola conidia. Additionally, the bacterial supernatant from PDB significantly reduced the disease severity caused by P. oryzae in rice seedlings when compared with the water-treated control group. The genome of strain 1021 was sequenced, and biosynthetic gene clusters of nonribosomal peptides surfactin, fengycin and bacillibactin were identified. Effects of PDB, NB and MSM media on expression levels of core biosynthetic genes of surfactin (srfAA), fengycin (fenC) and bacillibactin (dhbE) gene clusters were determined by reverse-transcription quantitative PCR. fenC expression was significantly increased in PDB and corresponded with the antagonistic activities against fungal mycelia. Conversely, expression of the regulatory genes comA and codY in PDB were highly reduced in PDB, indicating their negative relation with fenC expression. This expression analysis indirectly suggested that fengycin was potentially the bioactive compound of B. siamensis 1021 against P. oryzae and C. fructicola.
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Medium Effect on Antagonistic Activity and Detection of Nonribosomal Peptide Synthetase Genes in Epiphytic Bacillus Strains
(2021-01-01)
Apimeteethamrong, Suchitra
;
Kittiwongwattana, Chokchai
The biosynthesis of non-ribosomal peptides (NRPs) in biocontrol bacteria was one of the major antagonistic mechanisms for their application in agriculture. Bacillus spp. 1021, 2211 and 3210 were previously shown to inhibit mycelial growth of the leaf blast fungus Pyricularia oryzae. Here, we aimed to further study the antagonistic mechanism in those three strains. Cell-free supernatants obtained from bacteria grown in potato dextrose broth (PDB) exhibited a higher degree of inhibition against P. oryzae when compared to those obtained from nutrient broth (NB). This indicated the effect of culture media in the production of extracellular antibiotic compounds by these strains. Phylogenetic analysis of their partial 16S rRNA gene sequences indicated a close relationship between the three strains and Bacillus siamensis KCTC13613T, Bacillus amyloliquefaciens DSM7T and Bacillus velezensis CR-502T. Complete genome sequences of these Bacillus species were analyzed on the antiSMASH server to identify the presence of NRP biosynthesis gene clusters. Non-degenerate primers were designed for the detection of the core biosynthesis genes for surfactin (srfAA), fengycin (fenC) and bacillibactin (dhbF). All three genes were amplified in strains 1021 and 2211, while only srfAA and dhbF were detected in strain 3210. Phylogenetic analysis of the deduced amino acid sequences indicated that the sequences of strain 1021 were distinct from those of strains 2211 and 3210. This result indirectly suggests the possibility of NRP production as the antagonistic mechanism of these three strains.
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Genetic relationship of Coccinia grandis (L.) Voigt accessions, based on RAPD and ISSR markers
(2022-01-01)
Kittiwongwattana, Chokchai
Coccinia grandis (L.) Voigt or ivy gourd is widely distributed in nature and has potential for medicinal uses. However, there is little information on the genetic diversity of wild populations in Thailand. Here, 15 ivy gourd accessions were collected from Bangkok and nearby provinces. Angled and lobed leaves were observed. DNA fingerprints were generated from twelve random amplified polymorphic DNA (RAPD) and twelve inter-simple sequence repeat (ISSR) primers. The level of polymorphism in the RAPD profiles was higher than that of the ISSR profiles. Dice's similarity coefficient from RAPD data ranged from 0.73 to 0.82 and was lower than that from the ISSR data (0.88-1.00). The unweighted pair group method with arithmetic mean dendrogram, based on combined RAPD and ISSR data, was similar to the RAPD dendrogram but distinct from the ISSR dendrogram. Two major clusters were found with eleven and four accessions. The genetic relationship between some accessions was relatively consistent with either their locations or leaf shapes. The result indicated that the RAPD and ISSR techniques were suitable for the genetic study of ivy gourd. It also provided the basis for future studies with a higher number of samples for a better understanding of ivy gourd genetic diversity in Thailand.
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Biocontrol Potential, Genome and Nonribosomal Peptide Synthetase Gene Expression of Bacillus velezensis 2211
(2023-01-01)
Niemhom, Nantawan
;
Kittiwongwattana, Chokchai
Members of the genus Bacillus produced a diverse group of antimicrobial compounds. Here, we presented the antifungal activity and genome sequence analysis of Bacillus sp. 2211, a potential plant-growth-promoting bacterium. Bacterial supernatants from strain 2211 cultures in nutrient broth (NB) and potato dextrose broth (PDB) suppressed the mycelial growth of Pyricularia oryzae, Colletotrichum aenigma, Colletotrichum fructicola and Fusarium oxysporum. The supernatants were also able to suppress spore germination of these fungi, except for F. oxysporum. However, the supernatant from PDB displayed a significantly higher inhibition activity than NB. Additionally, the supernatant from PDB significantly reduced the disease severity caused by P. oryzae on rice seedlings. The genome of strain 2211 was sequenced. The highest digital DNA-DNA hybridization (80.1%) and average nucleotide identity (97.57%) levels indicated that strain 2211 was a member of the species Bacillus velezensis. The phylogenomic analysis showed that it clustered with B. velezensis NRRL B-41580T, B. velezensis KACC 13105 and B. velezensis subsp. plantarum FZB42T. The gene expression analysis showed the up-regulation of nonribosomal peptide synthetase (NRPS) genes bmyA, fenB and dhbE in PDB, compared to NB. This work demonstrated that the culture media affected the antagonistic activity of strain 2211 possibly through the modification of NRPS biosynthesis genes.
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Lactobacillus plantarum JCM 1149 Growth Enhancement by using Chlorella sp. KLSc61-pretreated Cells
(2025-06-01)
Khanrin, Lalita
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Boonyakorn, Phonwimon
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Wongsariya, Karn
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Kittiwongwattana, Chokchai
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Maneeruttanarungroj, Cherdsak
Dry microalgal biomass was previously tested as prebiotic to enhance the growth of probiotic bacteria. However, the drying process could be ineffective for scaling up probiotic production. This study aimed to investigate the use of fresh, pretreated microalgal biomass to promote the growth of Lactobacillus plantarum JCM 1149. Chlorella sp. KLSc61 cells were pretreated by three different methods: physical treatment with microwave radiation at power levels of 300, 500, and 700 W; chemical treatment with 0.1 M citric acid and 0.5 M sodium hydroxide; and biological treatment with cellulase enzyme. The 2.5% pretreated Chlorella cells were then added to L. plantarum JCM 1149 culture, and the growth was observed at 37 °C for 24 h of incubation. The results showed that, during the log phase (6-10 h), Chlorella cells pretreated with microwave radiation at 700 W were the most effective in promoting L. plantarum JCM 1149 growth, which was 1.4- and 1.5-fold of L. plantarum JCM 1149 without adding Chlorella and with untreated cells, respectively. Extension of the pretreatment time by microwave radiation at 700 W from up to 2 min increased the growth of L. plantarum JCM 1149 up to 1.8-fold of pretreatment time by microwave radiation at 700 W 1 min, compared to the control groups. Additionally, increasing the amount of Chlorella biomass up to 5% (w/v) extended the log phase of L. plantarum JCM 1149 and increased cell accumulation during the stationary phase. Unlike dry microalgal biomass, the simplicity of fresh, pretreated Chlorella biomass shown in this study may facilitate large-scale, commercial production of L. plantarum strains as probiotics.
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Validation of reference genes for the normalization of RT-qPCR gene expression in Bacillus siamensis 1021 grown in different culture media
(2022-01-01)
Nuwong, Warinya
;
Kittiwongwattana, Chokchai
Background and Objectives: House-keeping genes are generally selected as reference genes in gene expression analysis. However, some genes may not be stably expressed across all experimental conditions. Thus, this study aimed to validate seven house-keeping genes for gene expression analysis in Bacillus siamensis 1021. Materials and Methods: Strain 1021 was grown in potato dextrose broth, nutrient broth and mineral salt medium. Re-verse-transcription quantitative PCR was used to determine Cq values of seven reference genes including gyrA, gyrB, ssb and dnaB, rpsU, gat_Yqey and udp in these media. Expression stability of these genes was analyzed, using geNorm and Normfinder applications. The target gene ftsZ was used for assessment of the best candidate genes. Results: Based on geNorm and Normfinder, ssb was the most-stably expressed gene, while udp was the least-stably expressed gene. Pairwise variation indicated the combination of ssb, gyrA, gyrB and gatB_Yqey was suitable for the normalization of ftsZ expression. ftsZ expression in potato dextrose broth and mineral salt medium was higher than that in nutrient broth. In contrast, the normalization against udp resulted in an under-and overestimation of ftsZ expression in potato dextrose broth and mineral salt medium, respectively. Conclusion: The combination of ssb, gyrA, gyrB and gatB_Yqey was the best candidate for normalization of target gene expression in B. siamensis 1021 in these media. This study emphasized the significance of reference gene validation for gene expression analysis and provided a guideline for future gene expression studies in B. siamensis.
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Chitinophaga oryzae sp. Nov., an epiphytic bacterium isolated from rice root surfaces
(2021-01-01)
Niemhom, Nantawan
;
Suriyachadkun, Chanwit
;
Kittiwongwattana, Chokchai
Two Gram-stain-negative, non-motile, rod-shaped bacterial strains were isolated from the surfaces of rice roots. They were designated as strains 1303T and 1310. Their colonies were circular, entire, opaque, convex and yellow. They were chitinaseand catalase-positive, reduced nitrate and grew at 16–37 °C (optimum, 30 °C), pH 5.0–10.0 (optimum, pH 7.0) and 0–2.0% NaCl (optimum, 1.0%). Based on the 16S rRNA gene sequence analysis, they were classified as members of the genus Chitinophaga. Results of phylogenetic and phylogenomic analyses indicated that they formed a cluster with Chitinophaga eiseniae YC6729T, Chitinophaga qingshengii JN246T, Chitinophaga varians 10-7 W-9003T and Chitinophaga fulva G-6-1-13T. When the genomic sequences of strains 1303T and 1310 were compared with their close relatives, the average nucleotide identity and digital DNA– DNA hybridization values were below the cut-off levels. Phosphatidylethanolamine was the major polar lipid. MK-7 was the major respiratory quinone. iso-C<inf>15:0</inf>, C<inf>16:1</inf> ω5c, iso-C<inf>17:0</inf> 3-OH and summed feature 3 (C<inf>16:1</inf> ω7c/C<inf>16:1</inf> ω6c) were the predominant fatty acids. Differential characteristics between both strains and their close relatives were also observed. Based on the distinctions in genotypic, phenotypic and chemotypic features, strains 1303T and 1310 represent members of a novel species of the genus Chitinophaga, for which the name Chitinophaga oryzae sp. nov. is proposed. The type strain is 1303T (=KACC 22075T=TBRC 12926T).
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Light-Emitting Diode Illumination Enhances Biomass, Pigment, and Lipid Production in Halotolerant Cyanobacterium Aphanothece halophytica
(2025-06-01)
Thongtha, Sitthichai
;
Kittiwongwattana, Chokchai
;
Incharoensakdi, Aran
;
Phunpruch, Saranya
Light characteristics, including spectrum and intensity, significantly impact cyanobacterial biomass production, pigment biosynthesis, and cellular metabolism, influencing the composition of various biochemical compounds. This study aimed to investigate the effects of light-emitting diode (LED) illumination on biomass, pigment, and lipid production in the unicellular halotolerant cyanobacterium Aphanothece halophytica, cultivated in a suitable natural seawater (SNSW) medium. The results revealed that LED light outperformed fluorescent light, with blue LED light, particularly at an intensity of 60 μmol photons m−2 s−1, significantly enhancing growth, pigment synthesis, and lipid accumulation. This resulted in a maximum cell density of 68.96 ± 1.52 × 106 cells mL−1, a specific growth rate of 0.302 ± 0.002 day−1, and a lipid productivity of 56.81 ± 0.75 mg L−1 day−1. White LED light produced lipids suitable for biodiesel, whereas blue, green, and red LEDs promoted the accumulation of polyunsaturated fatty acids (PUFAs), beneficial for food supplements. These findings highlight the potential of LED-based cultivation strategies for optimizing biomass and biochemical compound production in A. halophytica.

Kittiwongwattana, Chokchai

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