Endophytic yeast diversity in leaf tissue of rice,corn and sugarcane cultivated in Thailand assessed by a culture-dependent approach

Endophytic yeasts are yeast that can colonize healthy plant tissues without causing any damage to the host plant. This work aimed to explore the diversity of endophytic yeasts in leaf tissue of main agricultural crops (rice, corn and sugarcane) in Thailand, by a culture-dependent approach. A total of 311 leaf samples, consisting of rice (n = 100), corn (n = 109) and sugarcane (n = 102). From the tissue of rice (n = 92), corn (n = 76) and sugarcane (n = 78) leaf samples, 117, 118 and 123 yeast strains were respectively isolated and identified based on the D1/D2 region of the large subunit (LSU) rRNA gene sequence analysis to be yeast species in both the phyla Basidiomycota and Ascomycota. Higher numbers of basidiomycetous yeast than ascomycetous yeast were detected in the leaf tissue of the three crops. Pseudozyma (Dirkmeia) churashimaensis (Ustilaginales) was the most prevalent yeast species in the rice and corn leaves with relative frequencies (RF) of 35.9 % and 17.8 %, respectively. Whereas the predominant species in the sugarcane leaves was Meyerozyma caribbica (Saccharomycetales) with an RF of 14.6 %. In addition, six new yeast species and one new yeast genus were proposed. Our findings suggest that these plant species are good sources from which new yeast species may be isolated.     

Pseudozyma vetiver sp. nov., a novel anamorphic ustilaginomycetous yeast species isolated from the phylloplane in Thailand

Three strains representing one novel yeast species were isolated from the phylloplanes of the vetiver grasses (DMKU-LV90 and DMKU-LV99^T) and sugarcane (DMKU-SP260) collected in Thailand by leaf washing followed by a plating technique. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer region (ITS), the three strains were found to represent a single novel anamorphic ustilaginomycetous yeast species in the genus Pseudozyma. The name Pseudozyma vetiver sp. nov. is proposed for this novel species. The type strain is DMKU-LV99^T (BCC 61021 = CBS 12824). The novel species showed phylogenetic relationships to the other members of the genus Pseudozyma and to teleomorphic fungal genera, namely Ustilago, Sporisorium and Anomalomyces in Ustilaginaceae, Ustilaginales. The three strains showed identical sequences both in the D1/D2 and ITS regions. The Pseudozyma species closest to the novel species in terms of pairwise sequence similarity in the D1/D2 region was Pseudozyma pruni but with 2.3 % nucleotide substitutions (14 nucleotide substitutions and no gaps out of 606 nt). The novel species and P. pruni differed by 10.9 % nucleotide substitutions (75 nucleotide substitutions and 31 gaps out of 691 nt) in the ITS region. The phylogenetic analysis based on the combined sequences of the ITS region and the D1/D2 region of the LSU rRNA gene showed that the novel species was found to be most closely related to Pseudozyma fusiformata but with 2.9 % nucleotide substitutions in the D1/D2 region and 7.4 % nucleotide substitutions in the ITS region.     

Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov., two novel methylotrophic yeast species from phylloplane in Thailand

Three strains (KM03^T, KM13 ^T and KM15) representing two novel methylotrophic yeast species were isolated from the external surface of plant leaves, which were collected from Kanchanaburi province, Thailand, by three-consecutive enrichments in methanol broth. Strain KM03^T was isolated from phylloplane of a mango tree (Mangifera indica) and two strains, KM13^T and KM15, were obtained from phylloplane of different wine grapes (Vitis vinifera). The sequences of the D1/D2 region of the large subunit (LSU) rRNA gene of the two strains (KM13^T and KM15) were identical and differed markedly from that of strain KM03^T. In terms of pairwise sequence similarity of the D1/D2 region the closest species to the strains KM13^T and KM15 were Candida suzukii (CBS 9253^T) and Candida nitratophila (CBS 2027^T) but with 2.1 % nucleotide substitutions. Strain KM03^T differed from Ogataea wickerhamii (CBS 4307^T), its closest relative, by 2.3 % nucleotide substitutions. Phylogenetic analysis based on the D1/D2 sequences placed the three strains in the Ogataea clade. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analyses of the D1/D2 and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the three strains represent two novel Ogataea species although formation of ascospores was not observed. Ogataea kanchanaburiensis sp. nov. is proposed for strain KM03^T (=BCC 47626^T = NBRC 108603^T = CBS 12673^T). Two strains, KM13^T and KM15, were assigned to Ogataea wangdongensis sp. nov. (type strain KM13^T = BCC 42664^T = NBRC 107778^T = CBS 12674^T). GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 and the ITS regions of O. kanchanaburiensis KM03^T are AB734090 and AB734093, respectively, of O. wangdongensis KM13^T are AB734091 and AB734094, respectively, and of O. wangdongensis KM15 are AB734092 and AB734095, respectively.     

<Emphasis Type="Italic">Papiliotrema phichitensis</Emphasis> f.a., sp. nov., a novel yeast species isolated from sugarcane leaf in Thailand

Strain DMKU-SP105^T representing a novel yeast species was isolated from the external surface of a sugarcane leaf (Saccharum officinarum L.) collected from a sugarcane plantation field in Phichit province, Thailand. On the basis of sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the strain DMKU-SP105^T differed by 7–16 substitutions in the D1/D2 region of LSU rRNA gene and 6–22 substitutions in the ITS region from a group of related species, Papiliotrema aspenensis, Papiliotrema odontotermitis, Papiliotrema rajasthanensis and Papiliotrema laurentii. A phylogenetic analysis based on the concatenated sequences of ITS region and the D1/D2 region of the LSU rRNA gene indicated that strain DMKU-SP105^T belongs to the laurentii clade of Papiliotrema in the Tremellales and is distinct from other related species in the clade. It therefore represents a novel species of the genus Papiliotrema although the formation of basidiospores was not observed. The name Papiliotrema phichitensis f.a., sp. nov. is proposed. The type is DMKU-SP105^T (=?CBS 13390^T?=?BCC 61187^T?=?NBRC 109699^T).     

Simulation and optimization of fed-batch culture for ethanol production from molasses

Two simulation methods for ethanol production from molasses by a flocculating yeast, Saccharomyces cerevisiae AM12, were investigated and molasses feeding was optimized. The first method was based on a deterministic model with fixed kinetic parameters and the second was based on regression analysis. The amount of ethanol produced in a fed-batch culture with multiple additions of molasses was simulated by both of these two methods. Simulated results of a fed-batch culture were compared with those of a simple batch culture by a model of regression analysis. The intermittent addition of molasses gave better production than a single addition at the beginning; more frequent addition may further improve production. The experimental results suggested the same. The effect of the amount of the added molasses on ethanol production was investigated by simulation. Repeated batch culture with and without intermittent addition of molasses in each batch was also done.List of Symbols C _e deviation of calculated results from experimental results - F m³ volume of feed medium added to the fermentor - P kg/m³ concentration of ethanol - P _M kg total amount of ethanol - S kg/m³ concentration of sugar - S ₀ kg/m³ concentration of sugar in the molasses feed medium - S _M kg total amount of sugar - V m³ culture volume - X kg/m³ concentration of cells - X _M kg total amount of cells - x _c calculated data - x _e experimental data - h^–1 specific rate of growth - kg-sugar/(kg-cell h) specific rate of sugar consumption - kg-ethanol/(kg-cell h) specific rate of ethanol production     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

Candida siamensis sp. nov., an anamorphic yeast species in the Saturnispora clade isolated in Thailand

Three strains of a novel anamorphic yeast species were isolated from natural samples collected in Thailand, including strain EF10 isolated from sediment in a mangrove forest, strain ST-473 from rotten wood, and strain ST-479 from a wild mushroom ( Hygrophorus sp.). Analysis of the D1/D2 domains of the large-subunit rRNA gene sequence revealed that the sequence of strain EF10 differed from the other two strains (ST-473 and ST-479), which had identical sequences, by only one nucleotide (nt) substitution in 535 nt. The closest species in terms of pairwise sequence similarity was Saturnispora mendoncae , with 32 nt substitutions and seven gaps in 535 nt. Phylogenetic analysis demonstrated that the three strains form a cluster with S. mendoncae and the other eight species in the Saturnispora clade. Phenotypic characteristics of the three strains were the same including proliferation by multilateral budding, absence of ascospores and negative diazonium blue B and urease reactions. The major ubiquinone was Q-7. On this basis, the three strains were assigned to a single novel species of the genus Candida , for which the name Candida siamensis sp. nov. is proposed. The type strain is EF10^T (=BCC 29901^T=NBRC 104878^T=CBS 11022^T).     

Yamadazyma siamensis sp. nov., Yamadazyma phyllophila sp. nov. and Yamadazyma paraphyllophila sp. nov., three novel yeast species isolated from phylloplane in Thailand and Taiwan

Four strains representing three novel anamorphic yeast species were isolated from the external surface of sugarcane leaves (DMKU-RK254^T), corn leaves (DMKU-RK548^T), bean leaves (K129) in Thailand and hengchun pencilwood leaves (TrB1-1^T) in Taiwan. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene, the internal transcribed spacer (ITS) region, the actin gene (ACT1) and the elongation factor 2 gene (EF2), the four strains were determined to represent novel Yamadazyma species although formation of ascospores was not observed. Strain DMKU-RK254^T was determined to be related to Candida diddensiae, Candida naeodendra and Candida kanchanaburiensis but with 1.8, 1.8 and 2.0 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, respectively. It was assigned to Yamadazyma siamensis sp. nov. (type strain DMKU-RK254^T = BCC 50730^T = NBRC 108901^T = CBS 12573^T). The sequences of the D1/D2 region of the LSU rRNA gene, the ITS region, ACT1 gene and EF2 gene of two strains (DMKU-RK548^T and K129) were identical but differed from that of strain TrB1-1^T by 0.6, 1.0, 3.3 and 5.9 % nucleotide substitutions, respectively. Therefore, the two strains (DMKU-RK548^T and K129) and strain TrB1-1^T were assigned to be two separate species. The closest species in terms of pairwise sequences similarity of the D1/D2 region to the two novel species was Yamadazyma philogaea but with 1.1–1.7 % nucleotide substitutions. The two strains (DMKU-RK548^T and K129) were assigned to Yamadazyma phyllophila sp. nov. (type strain DMKU-RK548^T = BCC 50736^T = NBRC 108906^T = CBS 12572^T) and the strain TrB1-1^T was named Yamadazyma paraphyllophila sp. nov. (type strain TrB1-1^T = BCRC 23030^T = CCTCC AY 204005^T = CBS 9928^T).