Savitreea siamensis sp. nov., an ascomycetous yeast species in the family Saccharomycetaceae discovered in Thailand.

Four yeast strains, representing a novel anamorphic species, were isolated in Thailand. The two strains (ST-3660T and ST-3647) were obtained from two different estuarine water samples in a mangrove forest. Strain DMKU-FW1-37 was derived from a grease sample, and another strain (TSU57) was isolated from a fruiting body of Phallus sp. Pairwise sequence analysis showed that the four strains had identical or differed by only one nucleotide substitution in the D1/D2 domains of the large subunit (LSU) rRNA gene, and differed by one to three nucleotide substitutions in the internal transcribed spacer (ITS) regions. Savitreea pentosicarens is the most closely related species to the four strains, but with 9-10 (1.57-1.72 %) nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 29-31 (4.22-4.45 %) nucleotide substitutions in the ITS regions. Phylogenetic analyses based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene showed that the four strains form a well-separated lineage from S. pentosicarens with high bootstrap support, confirming that they represent a distinct species. Therefore, the four strains are assigned as representives of a novel species of the genus Savitreea, for which the name Savitreea siamensis sp. nov. is proposed. The holotype is TBRC 4481T and the ex-type is PYCC 9794T (=ST-3660T). The MycoBank number of the novel species is MB 851951.

Phylogenomic delineation of two new species of ascomycetous yeasts, Wickerhamiella koratensis sp. nov. and Wickerhamiella limtongiae sp. nov., and proposal of two synonyms, Wickerhamiella infanticola and Wickerhamiella tropicalis.

Two novel ascomycetous yeast species of the genus Wickerhamiella are proposed based on isolates obtained in Thailand from food waste and the fruiting body of a polypore fungus, and on a combination of conventional DNA-barcode sequence analyses and whole-genome phylogenies. We focus on a particular subclade of the genus Wickerhamiella that contains species found in anthropic environments and describe Wickerhamiella limtongiae sp. nov. (DMKU-FW31-5T=PYCC 9022T=TBRC 15055T), found on food waste samples. In an adjacent clade, we describe Wickerhamiella koratensis sp. nov. (DMKU-KO16T=PYCC 8908T=TBRC 14869T), which represents the closest relative of Wickerhamiella slavikovae and was isolated from the fruiting body of Bjerkandera sp. In the subclade of W. limtongiae sp. nov., we propose that Wickerhamiella infanticola should be regarded as a synonym of Wickerhamiella sorbophila and that Wickerhamiella tropicalis should be regarded as a synonym of Wickerhamiella verensis.

Lipase Production by Limtongozyma siamensis, a Novel Lipase Producer and Lipid Accumulating Yeast.

Lipase is a well-known and highly in-demand enzyme. During the last decade, several lipase optimization studies have been reported. However, production costs have always been a bottleneck for commercial-scale microbial enzyme production. This research aimed to optimize the conditions for lipase production by Limtongozyma siamensis DMKU-WBL1-3 via a One-Factor-At-a-Time (OFAT) approach combined with statistical methods while using a low-cost substrate. Results suggest that low-cost substrates can be substituted for all media components. An optimal medium was found, using response surface methodology (RSM) and central composite design (CCD), to consist of 0.50% (w/v) sweet whey, 0.40% (w/v) yeast extract (food grade), and 2.50% (v/v) palm oil with the medium pH adjusted to 4 under shaking flask cultivation. From an economic point of view, this work was successful in reducing production costs while increasing lipase productivity. The medium costs were reduced by 87.5% of the original cost while lipase activity was increased by nearly 6-fold. Moreover, lipase production was further studied in a 2-L stirred-tank fermentor. Its activity was 1,055.6 ± 0.0 U/ml when aeration and agitation rates were adjusted to 1 vvm and 170 rpm, respectively. Interestingly, under this optimal lipase production, the yeast showed accumulated lipids inside the cells. The primary fatty acid is a monounsaturated fatty acid (MUFA) that is typically linked to health benefits. This study hence reveals promising lipase production and lipid accumulation by L. siamensis DMKU-WBL1-3 that are worthy of further study.

Kazachstania surinensis f.a., sp. nov., a novel yeast species isolated from Thai traditional fermented food.

Seven yeast strains (UBIF12-1, UBFB13-1, SRFS56-3, SRFS57-2, SKFS62-1, SKFS66-1 and SKFS67-1) representing a single anamorphic novel yeast species were isolated from traditional Thai fermented foods in Ubon Ratchathani, Surin and Sisaket in the northeast part of Thailand. The results of analysis of the sequences of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region indicated that the seven strains showed zero to one nucleotide substitutions in the sequences of the D1/D2 region of the LSU rRNA gene, and zero to four nucleotide substitutions in the ITS region. Kazachstania humilis CBS 5658T was the most closely-related species, but with 0.7-0.9% nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, and 2.0-2.2% nucleotide substitution in the ITS region. The results of a phylogenetic analysis based on the concatenated ITS and D1/D2 regions confirmed that the seven strains represented a single species of the genus Kazachstania distinct from the other recognized species of the genus. Furthermore, the morphological, biochemical and physiological properties of the seven strains not only indicated that they represented members of the genus Kazachstania, but that they were separated from K. humilis and K. pseudohumilis, the two most closely related species in the phylogenetic tree. Therefore, the seven strains were identified as representing a novel species, for which we propose the name Kazachstania surinensis f.a., sp. nov. The holotype is TBRC 15053T (isotype: SRFS57-2 and PYCC 9021). The MycoBank number of the novel species is 841892.

Magnusiomyces siamensis sp. nov., a yeast-like fungus isolated from food waste.

Three yeast strains, DMKU-GTSP8-6, DMKU-GTSP8-14T and DMKU-JED8-73, were isolated from food waste in Thailand. Based on the phenotypic characteristics and sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, it was revealed that the three strains clustered with the Magnusiomyces/Saprochaete clade. These strains were distinguished from the closely related species Saprochaete quercus CBS 750.85, Magnusiomyces ovetensis CBS 192.55T, Magnusiomyces starmeri CBS 780.96T, Saprochaete chiloensis CBS 8187T and Magnusiomyces ingens CBS 517.90T by 11.4, 13.1, 11.9, 11.2 and 12.6 % sequence divergence in the D1/D2 domain and by 34.6, 34.5, 33.6, 33.2 and 34.9 % sequence divergence in the ITS region, respectively. The new species, which does not produce ascospores, is described as Magnusiomyces siamensis. The holotype of Magnusiomyces siamensis is TBRC 15056T, and the isotypes are DMKU-GTSP8-14T and PYCC 9023T.

Description of Crinitomyces reliqui gen. nov., sp. nov. and Reassignment of Trichosporiella flavificans and Candida ghanaensis to the Genus Crinitomyces.

The systematic position of 16 yeast strains isolated from Thailand, Hungary, The Netherlands, and the Republic of Poland were evaluated using morphological, physiological, and phylogenetic analyses. Based on the similarity of the D1/D2 domain of the LSU rRNA gene, the strains were assigned to two distinct species, Trichosporiella flavificans and representatives of a new yeast species. Phylogenetic analyses revealed that Candida ghanaensis CBS 8798T showed a strong relationship with the aforementioned two species. The more fascinating issue is that Candida and Trichosporiella genera have been placed in different subphyla, Saccharomycotina and Pezizomycotina, respectively. The close relationship between Trichosporiella flavificans, Candida ghanaensis and the undescribed species was unexpected and needed to be clarified. As for morphological and physiological characteristics, the three yeast species shared a hairy colony appearance and an ability to assimilate 18 carbon sources. Based on phylogenetic analyses carried out in the present study, Crinitomyces gen. nov. was proposed to accommodate the new yeast species, Crinitomyces reliqui sp. nov. (Holotype: TBRC 15054, Isotypes: DMKU-FW23-23 and PYCC 9001). In addition, the two species Trichosporiella flavificans and Candida ghanaensis were reassigned to the genus Crinitomyces as, Crinitomyces flavificans (Type: CBS 760.79) comb. nov. and Crinitomyces ghanaensis (Type: CBS 8798) comb. nov., respectively.

Pichia bovicola sp. nov., a yeast species associated with small-intestinal content of cattle.

Three yeast strains, DMKU-MP6-4T, DMKU-MP2-6 and DMKU-MP5-1, were isolated from the small-intestinal content or Pia of cattle in Thailand during the investigation of yeast diversity in this habitat. According to the D1/D2 domain of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) region sequence analysis, these strains represent a novel yeast species in the genus Pichia. The species produced one to four ascospores per ascus with spherical to ellipsoidal shape and heterogenous in terms of size. These three strains were identical and differed from their closely related species, Pichia exigua NRRL Y-10920T by 2% (six nucleotide substitutions and five gaps) in the D1/D2 domain of the large subunit rRNA gene, while ITS region sequences differed by 3.1% (16 nucleotide substitutions and 27 gaps), 3.7% (19 nucleotide substitutions and 28 gaps) and 3.1% (16 nucleotide substitutions and 27 gaps) for DMKU-MP6-4T, DMKU-MP2-6 and DMKU-MP5-1, respectively. The name, Pichia bovicola, is proposed to accommodate these species. The holotype is DMKU-MP6-4T (TBRC 15616T=PYCC 8905T).

Savitreea pentosicarens gen. nov., sp. nov., a yeast species in the family Saccharomycetaceae isolated from a grease trap.

Two strains (DMKU-GTCP10-8 and CLIB 1740) representing a novel anamorphic yeast species were isolated from a grease sample collected from a grease trap in Thailand and from an unidentified fungus collected in French Guiana, respectively. On the basis of phylogenetic analysis based on the combined D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, Lachancea fermentati CBS 707T was the closely related species with 12.8 % sequence divergence (70 nucleotide substitutions and three gaps in 571 nucleotides) and 28.1 % sequence divergence (93 nucleotide substitutions and 90 gaps in 651 nucleotides) in the D1/D2 domain of the LSU rRNA gene and the ITS region, respectively. Phylogenetic analysis based on the concatenated sequences of the five genes including the small subunit rRNA gene, the D1/D2 domain of the LSU rRNA gene, the ITS region, translation elongation factor-1 alpha (TEF1) and RNA polymerase II subunit 2 (RPB2) genes confirmed that the two strains (DMKU-GTCP10-8 and CLIB 1740) were well-separated from other described yeast genera in Saccharomycetaceae. Hence, Savitreea pentosicarens gen. nov., sp. nov. is proposed to accommodate these two strains as members of the family Saccharomycetaceae. The holotype is S. pentosicarens DMKU-GTCP10-8T (ex-type strain TBRC 12159=PYCC 8490; MycoBank number 835044).

Wickerhamiella osmotolerans sp. nov. and Wickerhamiella tropicalis sp. nov., novel ascomycetous yeast in the family Wickerhamiellaceae.

Seven yeast strains, DMKU VGT1-14T, DMKU VGT1-19T, DMKU-JMGT1-28, DMKU-JMGT1-32, DMKU VGT2-06, DMKU VGT2-19 and DMKU VGT6-14, were isolated from a grease trap in Thailand and two strains, SJ-1 and SN-102 were isolated from the sea surface microlayer in Taiwan. On the basis of phenotypic characteristics and sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, these strains represented two novel yeast species of the genus Wickerhamiella. In terms of pairwise sequence similarity, four strains, DMKU VGT1-14, DMKU-JMGT1-32, DMKU VGT6-14 and SN-102, were closely related to Wickerhamiella infanticola NRRL Y-17858T but differed by 13 nucleotide substitutions with one gap (2.46 %) in the D1/D2 domain of the LSU rRNA gene and 15 nucleotide substitutions with 23 gaps (4.2 %) in the ITS region. The strains DMKU VGT1-19T, DMKU-JMGT1-28, DMKU VGT2-06, DMKU VGT2-19 and SJ-1, differed from the type strain of the most closely related species, Wickerhamiella sorbophila NRRL Y-7921T, by nine nucleotide substitutions with one gap (1.66 %) in the D1/D2 domain of the LSU rRNA gene and nine nucleotide substitutions with 17 gaps (2.52%) in the ITS region. Hence, the names Wickerhamiella osmotolerans sp. nov. and Wickerhamiella tropicalis sp. nov. are proposed to accommodate these species in the genus Wickerhamiella. The holotypes are W. osmotolerans DMKU VGT1-14T (ex-type strain TBRC 11425=PYCC 8359=CGMCC 2.6179; Mycobank number 833394) and W. tropicalis DMKU VGT1-19T (ex-type strain TBRC 11426=PYCC 8360=CGMCC 2.6180; Mycobank number 833393).

Grease Waste as a Reservoir of Lipase-Producing Yeast and Description of Limtongella siamensis gen. nov., sp. nov.

A total of 175 yeast isolates were obtained from grease samples. Based on the D1/D2 region of the large subunit (LSU) ribosomal RNA (rRNA) gene analysis, 150 yeast isolates were identified as belonging to 36 described yeast species, whereas 25 isolates required more analysis. Among the described species, Rhodotorula mucilaginosa was the only Basidiomycetous yeast, whereas 149 isolates were identified as belonging to 35 described species of 15 genera in the phylum Ascomycota, and Candida tropicalis was the most abundant species. A study of lipase production indicated that strain DMKU-JMGT1-45 showed volumetric activity of 38.89 ± 9.62 and 155.56 ± 14.70 U/mL when grown in yeast extract malt extract (YM) and YM supplemented with 1% olive oil, respectively. In addition, this strain intracellularly accumulated lipid, of which the fatty acid profile revealed the major fatty acids to be 39.9% oleic acid (C18:1), 27.61% palmitoleic acid (C16:1) and 14.97% palmitic acid (C16:0). A phylogenetic analysis of the combined multi-locus gene sequences showed that the strains DMKU-JMGT1-45T and DMKU-JMGT4-14 formed a well-separated lineage and could not be assigned to any of the currently recognized genera of the Saccharomycetales. Limtongella siamensis gen. nov., sp. nov. is therefore proposed to accommodate these two strains as members of the order Saccharomycetales.

Biosynthetic Pathway of Indole-3-Acetic Acid in Basidiomycetous Yeast Rhodosporidiobolus fluvialis.

IAA biosynthetic pathways in a basidiomycetous yeast, Rhodosporidiobolus fluvialis DMKU-CP293, were investigated. The yeast strain showed tryptophan (Trp)-dependent IAA biosynthesis when grown in tryptophan supplemented mineral salt medium. Gas chromatography-mass spectrometry was used to further identify the pathway intermediates of Trp-dependent IAA biosynthesis. The results indicated that the main intermediates produced by R. fluvialis DMKU-CP293 were tryptamine (TAM), indole-3-acetic acid (IAA), and tryptophol (TOL), whereas indole-3-pyruvic acid (IPA) was not found. However, supplementation of IPA to the culture medium resulted in IAA peak detection by high-performance liquid chromatography analysis of the culture supernatant. Key enzymes of three IAA biosynthetic routes, i.e., IPA, IAM and TAM were investigated to clarify the IAA biosynthetic pathways of R. fluvialis DMKU-CP293. Results indicated that the activities of tryptophan aminotransferase, tryptophan 2-monooxygenase, and tryptophan decarboxylase were observed in cell crude extract. Overall results suggested that IAA biosynthetic in this yeast strain mainly occurred via the IPA route. Nevertheless, IAM and TAM pathway might be involved in R. fluvialis DMKU-CP293.

Production of Indole-3-Acetic Acid by Enterobacter sp. DMKU-RP206 Using Sweet Whey as a Low-Cost Feed Stock.

In this study, we investigated Indole-3-acetic acid (IAA) production by a rice phylloplane bacteria, Enterobacter sp. DMKU-RP206, using sweet whey as a feed stock instead of lactose. We succeeded in using sweet whey for Enterobacter sp. DMKU-RP206 to produce 3,963.0 mg IAA/l with the optimal medium containing 1.48% sweet whey, 1.42% yeast extract and 0.88% L-tryptophan. The medium pH was adjusted to 6 and the culture conditions were shaking at 200 rpm on an orbital shaker at 30°C for 3 days. We also evaluated the effect of IAA in culture filtrates of Enterobacter sp. DMKU-RP206 on the promotion of jasmine rice growth in a pot experiment. Compared with the negative control (without IAA), the result showed that biosynthetic IAA produced by Enterobacter sp. DMKU-RP206 significantly increased the growth of jasmine rice (Oryza sativa L. cv. KDML105) in terms of length and dry weight of shoot. This work thus reveals the impact of IAA produced by Enterobacter sp. on the promotion of jasmine rice growth.