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Maximizing Omega-3 production in Chlorella sp. KLSc59 through central composite design-driven cultivation and cellulase-assisted extraction
(2026-10-01)
Pansomsuay, Rawirat
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Vachiraroj, Nuttapon
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Pandhal, Jagroop
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Sirikwanpong, Sukrit
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Maneeruttanarungroj, Cherdsak
Chlorella sp. KLSc59 is a promising freshwater microalga for omega-3 oil production, but its application is limited by low biomass yield and inefficient lipid recovery. This study developed an integrated strategy to improve biomass production and omega-3 recovery. Carbon and nitrogen source screening in modified Tris-Acetate-Phosphate (TAP) medium identified acetic acid (CH<inf>3</inf>COOH) and ammonium nitrate (NH<inf>4</inf>NO<inf>3</inf>) as the best-performing carbon and nitrogen sources. Central Composite Design (CCD) was then applied to optimize CH<inf>3</inf>COOH and NH<inf>4</inf>NO<inf>3</inf> concentrations and pH. The resulting M2 formulation (33.9 mM CH<inf>3</inf>COOH, 16.1 mM NH<inf>4</inf>NO<inf>3</inf>, pH 6.6) gave the highest specific growth rate (0.20 h−1) and biomass concentration (1.16 g L−1) among the tested cultivation conditions. A second CCD was applied to optimize cell disruption by varying cellulase loading and sonication duration, identifying D6 (1,000 International Units (IU) g−1 DCW cellulase; 150 s sonication) as the best-performing extraction condition within the tested range for total and omega-3 fatty acid recovery. Among the tested comparative conditions, M2CaD6 yielded the highest omega-3 content and yield, increasing from 0.37 to 6.22 µg mg−1 DCW and from 0.74 to 14.90 mg L−1, respectively, corresponding to 17- and 20-fold increases over the previous TAP-CaCl<inf>2</inf> cultivation and extraction method (PD0). Reducing NH<inf>4</inf>NO<inf>3</inf> in M2 by half increased linoleic acid content, whereas higher light intensity (50 µmol m−2 s−1) reduced cellular total fatty acid accumulation. Overall, coordinated optimization of cultivation, CaCl<inf>2</inf> induction, and enzymatic–mechanical disruption improved omega-3 production from Chlorella sp. KLSc59 and provides a basis for further process development.
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Purification and Identification of Antimicrobial Protein from the Green Alga Tetraspora Sp. CU2551
(2026-02-01)
Klinsanit, Tanaporn
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Tonawut, Yothawut
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Laokua, Natwikar
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Pansomsuay, Rawirat
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Khetkorn, Wanthanee
Antimicrobial resistance (AMR) is an escalating global health threat, necessitating the discovery of novel antimicrobial agents. Algae-derived proteins and peptides have gained increasing attention for their bioactive potential; however, standardized protocols for investigating antimicrobial peptides (AMPs) in green algae, particularly Tetraspora sp. CU2551, remain limited. This protocol describes a step-by-step workflow for the isolation, purification, and characterization of antimicrobial intact proteins from the green alga Tetraspora sp. CU2551. The procedure integrates optimized protein extraction, chromatographic fractionation, electrophoretic separation, antimicrobial activity screening, and protein identification. Protein extraction conditions are first optimized to reduce background inhibitory effects originating from buffer components. When the identity of the active peptide is unknown, a pull-down assay is applied to assess protein binding across different ion-exchange resins and to guide the selection of an appropriate purification matrix. DEAE-Sepharose ion-exchange chromatography is a suitable method for enriching antimicrobial protein fractions. All fractions are systematically evaluated for antimicrobial activity and analyzed by SDS-PAGE. The protein band corresponding to the highest antimicrobial activity, along with a distinct electrophoretic profile, is excised and subjected to LC-MS/MALDI-TOF analysis for protein identification. The workflow is further complemented by in silico analyses to predict antimicrobial peptide-related properties using publicly available bioinformatic tools. This protocol provides a versatile framework for antimicrobial protein discovery and can be readily adapted to other algal species and related biotechnological applications.
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Discovery of Streptomyces marinisediminis sp. nov., a new thiolutin producing actinomycete isolated from Thai marine sediment
(2025-12-01)
Pansomsuay, Rawirat
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Fukasem, Poowadon
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Pittayakhajonwut, Pattama
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Intaraudom, Chakapong
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Suriyachadkun, Chanwit
Thiolutin and 2,2-dioxidothiolutin are sulfur-containing antibiotics discovered through a targeted high-throughput screening strategy designed to identify compounds with antimicrobial activity. The producing strain, JHD1T, is an actinomycete isolated from marine sediment collected in Chonburi Province, Thailand. Chemotaxonomic characteristics and 16S rRNA gene sequence analysis indicated that strain JHD1T belongs to the genus Streptomyces. Taxonomic analysis using a polyphasic approach further supported its placement within the genus Streptomyces. The strain exhibited low similarity to its closest known relatives, Streptomyces alkaliterrae OF1T and Streptomyces chumphonensis KK1-2T, with average nucleotide identity based on MuMmer ultra-rapid aligning tool (ANIm) values of 85.7–86.2%, average amino acid identity (AAI) values of 71.8–78.1%, and digital DNA–DNA hybridization (dDDH) values of 23.4–25.0%, all well below the established thresholds for species delineation. These results reveal that strain JHD1T represents a novel species within the genus Streptomyces, for which the name Streptomyces marinisediminis sp. nov. is proposed. The crude extract obtained from the culture broth of strain JHD1T exhibited strong antimicrobial activity against Gram-positive bacteria and led to the discovery of two promising secondary metabolites, thiolutin and 2,2-dioxidothiolutin, along with 8-amino-2H-benz[e][1,3]oxazine-2,4(3H)-dione, benadrostin, 2,3-dihydroxybenzamide, and 2-hydroxy-3-methoxybenzamide. In this study, 2,2-dioxidothiolutin was structurally confirmed for the first time by X-ray crystallographic analysis. Interestingly, thiolutin demonstrated strong anti-human small cell lung cancer (NCI-H187) activity, with an IC₅₀ value of 0.35 µg/mL, and also showed cytotoxicity against human breast cancer (MCF-7) cells, with an IC₅₀ value of 5.61 µg/mL.
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Genome Characterization for the Antimicrobial Potential of Streptomyces samsunensis SA31, a Rhizospheric Actinomycete of Cymbopogon citratus (DC) Stapf.
(2025-01-01)
Wongsariya, Karn
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Duangupama, Thitikorn
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Pansomsuay, Rawirat
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Thanaboripat, Dusanee
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Thawai, Chitti
Streptomyces species are promising resources of bioactive compounds that play a vital role in medicinal biotechnology. Streptomyces sp. SA31 was isolated from the rhizospheric soil of Cymbopogon citratus (DC) Stapf. The strain was identified as Streptomyces samsunensis based on genome-based taxonomic analysis. The draft genome of strain SA31 contained 11,850,342 bp with a high percentage of G+C at 71.0% and carried 9,350 predicted protein-encoding sequences (CDSs). Furthermore, the secondary metabolite biosynthesis gene clusters in the genome of strain SA31 were predicted by antiSMASH. In silico analysis showed 71 predicted biosynthetic gene clusters (BGCs) responsible for antimicrobial secondary metabolite synthesis. Most gene clusters were involved in the biosynthesis of polyketide synthase. The genome of strain SA31 harbored seven types of polyketide biosynthesis gene clusters that might be associated with antimicrobial activity. Additionally, the genome of strain SA31 contained unexplored secondary metabolite biosynthesis gene clusters. In vitro antimicrobial assay showed that an ethyl acetate extract from the culture broth of strain SA31 could inhibit the growth of Staphylococcus aureus ATCC 25923, Kocuria rhizophila ATCC 9341, Bacillus subtilis ATCC 6633, Candida albicans ATCC 10231, and Aspergillus flavus IMI 242684, implying that the genome of strain SA31 contained potential BGCs for the production of antimicrobial secondary metabolites and led to the isolation of geldanamycin and 17-O-demethylgeldanamycin. Therefore, it can be proved that the rhizosphere-associated soil of C. citratus (DC) Stapf. is a rich habitat for actinomycetes that are capable of producing promising biologically active compounds.
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Streptomyces zingiberis sp. nov., an endophytic actinobacterium isolated from the root tissue of Zingiber montanum
(2023-06-01)
Pansomsuay, Rawirat
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Duangupama, Thitikorn
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Pittayakhajonwut, Pattama
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Intaraudom, Chakapong
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Suriyachadkun, Chanwit
An endophytic actinobacterium, designated strain PLAI 1-29T, was isolated from the root tissue of Zingiber montanum collected from Pathum Thani province, Thailand. Strain PLAI 1-29T was characterized using a polyphasic taxonomic approach. It typically exhibited morphological and chemotaxonomic properties of the genus Streptomyces. Strain PLAI 1-29T produced a spiral spore chain on aerial mycelium and grew at 15–40 °C, pH 6–10 on International Streptomyces Project 2 agar. The maximum NaCl concentration for growth was 9% (w/v). Cells of strain PLAI 1-29T presented ll-diaminopimelic acid, arabinose, galactose and ribose. The detected phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphati-dylinositol and phosphatidylinositol mannoside. The major menaquinones were MK-9(H<inf>6</inf>) and MK-9(H<inf>8</inf>). The major cellular fatty acids were iso-C<inf>16:0</inf>, anteiso-C<inf>15:0</inf> and anteiso-C<inf>17:0</inf> . The genome-based taxonomic details revealed the assignment of strain PLAI 1-29T to the genus Streptomyces and exhibited low threshold values for the delineation of a novel species by average nucleotide identity–blast (84.0%), average amino acid identity (80.0%) and digital DNA–DNA hybridization (27.6%) with its closest type strain, Streptomyces xinghaiensis S187T. Furthermore, several differential physiological and biochemical characteristics were detected between strain PLAI 1-29T and the closest type strain. Based on the combined phenotypic and genomic features, strain PLAI 1-29T (=TBRC 7645T=NBRC 113170T) is considered to represent a new Streptomyces species, for which we propose the name Streptomyces zingiberis sp. nov.
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Micromonospora solifontis sp. nov., an actinobacterium isolated from hot spring soil
(2023-05-01)
Duangupama, Thitikorn
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Pansomsuay, Rawirat
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Pittayakhajonwut, Pattama
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Intaraudom, Chakapong
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Suriyachadkun, Chanwit
An actinobacterium strain, PPF5-17T, was isolated from hot spring soil collected from Chiang Rai province, Thailand. The strain exhibited morphological and chemotaxonomic properties similar to those of members of the genus Micromonospora. Colonies of PPF5-17T were strong pinkish red and turned black after sporulation in ISP 2 agar medium. Cells formed single spores directly on the substrate mycelium. Growth was observed from 15 to 45 °C and at pH 5–8. Maximum NaCl concentration for growth was 3% (w/v). PPF5-17T was found to have meso-diaminopimelic acid, xylose, mannose and glucose in the whole-cell hydrolysate. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannosides were observed as the membrane phospholipids. MK-10(H<inf>6</inf> ), MK-9(H<inf>6</inf> ), MK-10(H<inf>4</inf> ) and MK-9(H<inf>4</inf> ) were the major menaquinones. The predominant cellular fatty acids were iso-C<inf>15:0</inf>, iso-C<inf>17:0</inf>, anteiso-C<inf>17:0</inf> and iso-C<inf>16:0</inf> . PPF5-17T shared the highest 16S rRNA gene sequence similarity with Micromonospora fluminis LMG 30467T (99.3%). A genome-based taxonomic study revealed that PPF5-17T was closely related to Micromonospora aurantinigra DSM 44815T in the phylogenomic tree with an average nucleotide identity by blast (ANIb) of 87.7% and a digital DNA–DNA hybridization (dDDH) value of, 36.1% which were below the threshold values for delineation of a novel species. Moreover, PPF5-17T could be distinguished from its closest neighbours, M. fluminis LMG 30467T and M. aurantinigra DSM 44815T, with respect to a broad range of phenotypic properties. Thus, PPF5-17T represents a novel species, for which the name Micromonospora solifontis sp. nov. is proposed. The type strain is PPF5-17T (= TBRC 8478T = NBRC 113441T).
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Micromonospora thermarum sp. nov., an actinobacterium isolated from hot spring soil
(2023-04-01)
Pansomsuay, Rawirat
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Duangupama, Thitikorn
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Pittayakhajonwut, Pattama
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Intaraudom, Chakapong
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Suriyachadkun, Chanwit
An actinomycete, designated strain HSS6-12T, was isolated from hot spring sediment collected from Ranong province, Thailand. The strain showed taxonomic characteristics consistent with those of members of the genus Micromonospora. HSS6-12T produced a single spore directly on the substrate mycelium, and no aerial mycelium was detected. The isomer of diamino acid presented in cell wall peptidoglycan was meso-diaminopimelic acid. Arabinose, xylose, glucose, and ribose were detected in whole-cell hydrolysates. MK-10(H<inf>4</inf>), MK-9(H<inf>4</inf>), and MK-10(H<inf>6</inf>) were major menaquinones. Major cellular fatty acids were iso-C<inf>16:0</inf>, iso-C<inf>15:0</inf>, and iso-C<inf>17:0</inf>. Phospholipid profile was composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylinositolmannosides. 16S rRNA gene analysis revealed that HSS6-12T shared the highest 16S rRNA gene sequence similarity with Micromonospora inositola DSM 43819T (99.3%). In contrast, the genome analysis showed that HSS6-12T formed a tight taxonomic position in a phylogenomic tree with Micromonospora endolithica DSM 44398T. Moreover, the average nucleotide identity-blast, the digital DNA-DNA hybridization, and the average amino acid identity values between HSS6-12T and M. inositola DSM 43819T and M. endolithica DSM 44398T were 83.1–84.0%, 27.5–28.7%, and 80.4–82.2%, respectively, indicating that HSS6-12T was different species with both closely related Micromonospora-type strains. In addition, HSS6-12T could be discriminated from its closely related type strains by many physiological and biochemical characteristics. Thus, HSS6-12T could be considered a novel species of the genus Micromonospora, and the name Micromonospora thermarum is proposed for the strain. The type strain is HSS6-12T (= BCC 41915T = JCM 17127T).
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Gordonia aquimaris sp. nov., a novel marine actinobacterium isolated from seawater in the upper gulf of Thailand
(2023-03-01)
Pansomsuay, Rawirat
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Duangupama, Thitikorn
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Pittayakhajonwut, Pattama
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Intaraudom, Chakapong
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Suriyachadkun, Chanwit
An actinobacterium strain, SW21T, was isolated from seawater collected in the upper Gulf of Thailand. Cells were Gram-stain-positive, aerobic and rod-shaped. Growth was observed from 15 to 37 °C and at pH 6–8. Maximum NaCl for growth was 14% (w/v). meso-Diaminopimelic acid, arabinose, galactose, glucose, rhamnose and ribose were detected in the whole-cell hydro-lysate. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside were detected as the phospholipids in the cells. The major menaquinones were MK-9(H<inf>2</inf> ) and MK-7(H<inf>2</inf> ). The major cellular fatty acids were C<inf>16:0</inf>, C<inf>18:1</inf> ω9c, C<inf>18:0</inf> and C<inf>18:0</inf> 10-methyl (TBSA). The 16S rRNA gene sequence data supported the assignment of strain SW21T to the genus Gordonia and showed that Gordonia mangrovi KCTC 49383T (98.7%) was the closest relative. Moreover, the average nucleotide identity-blast (85.5%) and digital DNA–DNA hybridization (30.7%) values between strain SW21T and its closest neighbour were below the threshold values for delineation of a novel species. The combination of genotypic and phenotypic data indicated that strain SW21T is representative of novel species of the genus Gordonia. The name Gordonia aquimaris sp. nov. is proposed for strain SW21T. The type strain is SW21T (=TBRC 15691T=NBRC 115558T).

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