Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov. and Actinomycetospora aurantiaca sp. nov., endophytic actinobacteria isolated from wild orchid (Aerides multiflora Roxb).

Three novel mycelium-forming actinobacteria, designated OC33-EN06T, OC33-EN07T, and OC33-EN08T, were isolated from wild orchid (Aerides multiflora Roxb), collected from a hill evergreen forest in Northern Thailand. Strains OC33-EN06T and OC33-EN07T showed the highest 16S rRNA gene similarity with Actinomycetospora lutea TT00-04T, 99.17 and 99.45%, respectively. Strain OC33-EN08T showed high similarity with four species, namely 'Actinomycetospora termitidis Odt1-22T' (99.37%), Actinomycetospora chiangmaiensis DSM 45062T (99.02%), Actinomycetospora corticicola 014-5T (99.02%), and Actinomycetospora soli SF1T (98.81%). Comparative genome analysis of OC33-EN06T, OC33-EN07T, and OC33-EN08T with the closely related type strains showed that average nucleotide identity (ANI) based on blast, ANI based on MUMmer, and average amino acid identity values were less than 95% and the digital DNA-DNA hybridization values were less than 70%, all below the thresholds for species demarcation. The digital G+C content of OC33-EN06T, OC33-EN07T, and OC33-EN08T were 74.5, 74, and 74 mol%, respectively. These three strains developed bud-like chains of non-motile cylindrical spores with a smooth surface. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars contained ribose, arabinose, and galactose. The predominant menaquinone was MK-8(H4). The phospholipid profile included phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. Based on comparative analysis of genotypic, phenotypic and chemotaxonomic data, strains OC33-EN06T (=TBRC 18349T=NBRC 116543T), OC33-EN07T (=TBRC 18350T=NBRC 116544T), and OC33-EN08T (=TBRC 18318T=NBRC 116542T) represent the type strains of three novel species of the genus Actinomycetospora for which the names Actinomycetospora aeridis sp. nov., Actinomycetospora flava sp. nov., and Actinomycetospora aurantiaca sp. nov., are proposed.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Paeniglutamicibacter quisquiliarum sp. nov., isolated from midden soil waste.

A Gram-stain-positive, irregular short-rod and non-motile bacterium, designated strain ABSL32-1T, was isolated from a soil sample collected from the Suphan Buri municipal solid waste disposal area. According to the results of a polyphasic taxonomic study, a novel species belonging to the genus Paeniglutamicibacter was described. Strain ABSL32-1T grew optimally at 20-25 °C and at pH 6.0-8.0 in the presence of 1 % (w/v) NaCl. The whole-cell sugars were ribose, mannose and glucose. The peptidoglycan structure contained A4α peptidoglycan (Lys-Glu; A11.54). The polar lipids contained digalactosyldiacylglycerol, diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids and two unidentified lipids. The major menaquinones were MK-9 and MK-10. The major cellular fatty acid was anteiso-C15 : 0 (70.1 %). Based on 16S rRNA gene sequence analysis, strain ABSL32-1T showed the highest similarity to Paeniglutamicibacter sulfureus DSM 20167T (99.5 %), followed by Paeniglutamicibacter antarcticus SPC26T (99.0 %) and Paeniglutamicibacter psychrophenolicus AG31T (98.8 %). The genome of strain ABSL32-1T is 4.4 Mbp with a DNA G+C content of 66.0 mol%. The average nucleotide identity values between strain ABSL32-1T and the type strains P. sulfureus DSM20167T, P. antarcticus SPC26T and P. psychrophenolicus AG31T were 86.6, 74.7 and 83.6 %, respectively. On the basis of phenotypic, chemotaxonomic and genotypic properties, strain ABSL32-1T is proposed to represent a novel species to be named Paeniglutamicibacter quisquiliarum sp. nov. The type strain is ABSL32-1T (=TBRC 14976T=NBRC 115252T).

Biodegradation of crude oil by immobilized Exiguobacterium sp. AO-11 and shelf life evaluation.

Exiguobacterium sp. AO-11 was immobilized on bio-cord at 109 CFU g-1 carrier for the removal of crude oil from marine environments. To prepare a ready-to-use bioremediation product, the shelf life of the immobilized cells was calculated. Approximately 90% of 0.25% (v/v) crude oil removal was achieved within 9 days when the starved state of immobilized cells was used. The oil removal activity of the immobilized cells was maintained in the presence of oil dispersant (89%) and at pH values of 7-9. Meanwhile, pH, oil concentration and salinity affected the oil removal efficacy. The immobilized cells could be reused for at least 5 cycles. The Arrhenius equation describing the relationship between the rate of reaction and temperature was validated as a useful model of the kinetics of retention of activity by an immobilized biocatalyst. It was estimated that the immobilized cells could be stored in a non-vacuum bag containing phosphate buffer (pH 7.0) at 30 °C for 39 days to retain the cells at 107 CFU g-1 carrier and more than 50% degradation activity. These results indicated the potential of using bio-cord-immobilized crude oil-degrading Exiguobacterium sp. AO-11 as a bioremediation product in a marine environment.

Paeniglutamicibacter terrestris sp. nov., isolated from phenanthrene-degrading consortium enriched from Antarctic soil.

A novel bacterium, designated strain ANT13_2T, was isolated from a phenanthrene-degrading consortium enriched from a soil sample collected near the Great Wall Station located in the southwestern area of King George Island, Antarctica. Following a polyphasic taxonomic study, a novel species belonging to the genus Paeniglutamicibacter was described. The strain was a Gram-stain-positive bacterium that exhibited a rod-coccus growth cycle. Strain ANT13_2T grew aerobically at an optimum temperature of 20-25 °C and at pH 7.0-8.0. Ribose, arabinose and glucose were detected as whole-cell sugars. The predominant menaquinone was MK-9. The diagnostic phospholipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified phospholipid. The predominant cellular fatty acids were anteiso-C15 : 0 (67.7 %) and anteiso-C17 : 0 (11.2 %). The DNA G+C content of the genomic DNA was 60.6 mol%. Based on 16S rRNA gene sequence analysis, strain ANT13_2T showed the highest similarities to Paeniglutamicibacter antarcticus SPC26T (98.9 %) followed by Paeniglutamicibacter gangotriensis Lz1yT (98.4 %), Paeniglutamicibacter sulfureus DSM 20167T (98.3%) and Paeniglutamicibacter kerguelensis KGN15T (97.9 %). The average nucleotide identity values between strain ANT13_2T and the type strains of P. antarcticus SPC26T and P. gangotriensis Lz1yT were 73.8 and 77.5 %, respectively, which are well below the 95-96 % species circumscription threshold. On the basis of this polyphasic taxonomic study, strain ANT13_2T is proposed to represent a novel species to be named Paeniglutamicibacter terrestris sp. nov. The type strain is ANT13_2T (=TBRC 11756T=NBRC 114615T).

Actinomycetospora endophytica sp. nov., isolated from wild orchid (Podochilus microphyllus Lindl.) in Thailand.

A novel endophytic actinomycete, designated strain A-T 8314T, was isolated from a wild orchid, Podochilus microphyllus Lindl., collected from Trat Province, Thailand. The taxonomic position of strain A-T 8314T was established using a combination of genotypic and phenotypic analyses. The isolate was a Gram-positive bacterium that developed bud-like spore chains. Strain A-T 8314T grew aerobically at an optimum temperature of 20-25 °C and an optimal pH 6.0. The cell wall contained meso-diaminopimelic acid, and the whole-cell sugars were ribose, arabinose and galactose. The predominant menaquinone was MK-8 (H4). The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylmonomethylethanolamine, hydroxy-phosphatidylmonomethylethanolamine and hydroxyl phosphatidylethanolamine. The predominant cellular fatty acid was iso-C16 : 0. The DNA G+C content of the genomic DNA was 73.2±0.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A-T 8314T belonged to the genus Actinomycetospora, and was most closely related to Actinomycetospora chiangmaiensis YIM 0006T (98.8 %) and Actinomycetosporacorticicola 014-5T (98.6 %). The DNA-DNA relatedness values that distinguished A-T 8314T from its closest species were below 70 %. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, it was concluded that the new isolate represents as a novel species, for which the name Actinomycetospora endophytica sp. nov is proposed. The type strain is A-T 8314T (=TBRC 5722T=NBRC 113235T).

Polycladomyces subterraneus sp. nov., isolated from soil in Thailand.

A thermophilic poly(L-lactide)-degrading Gram-stain-positive filamentous bacterial strain that develops single spores on the aerial mycelium was isolated from forest soil at Srinagarind Dam, Kanchanaburi Province, Thailand. The results of a polyphasic taxonomic study showed that our isolate had characteristics typical of members of the genus Polycladomyces. The isolate grew aerobically at an optimum temperature of 50-55 °C and optimal pH 6-7. Meso-diaminopimelic acid was present as the diagnostic diamino acid in the peptidoglycan but no characteristic sugars are detected. The predominant menaquinone was MK-7. The diagnostic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine diphosphatidylglycerol, phosphatidylglycerol and phosphatidylserine. The predominant cellular fatty acid was iso-C15 : 0. The DNA G+C content of strain KSR 13T was 53.4 mol%. The 16S rRNA gene sequence analysis also indicated that strain KSR 13T belonged to the genus Polycladomyces, being most closely related to Polycladomyces abyssicola JIR-001T (99.2 %). The DNA-DNA relatedness values that distinguished KSR 13T from P. abyssicola JIR-001T were 17.8-32.1 %, which were significantly below the 70 % cutoff value recommended for species delineation. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, the new isolate is proposed as a novel species and named Polycladomyces subterraneus sp. nov. The type strain is KSR 13T (=BCC 50740T=NBRC 109332T).

Antagonistic activity of endo-ß-1,3-glucanase from a novel isolate, Streptomyces sp. 9X166, against black rot in orchids.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its ß-1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58 U mg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80 kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was ß-1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type ß-1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing ß-1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a ß-1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.