A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila

In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species.     

Salt adaptation in pseudomonads: characterization of glucosylglycerol-synthesizing isolates from brackish coastal waters and the rhizosphere

The compatible solute glucosylglycerol (GG) is widespread among cyanobacteria, but, until now, has been reported for only two species of heterotrophic bacteria. About 120 bacterial isolates from coastal regions of the Baltic Sea were screened by HPLC for their ability to synthesize GG. Positive isolates (26) were grouped by SDS-PAGE of whole-cell proteins and representative strains of each group were investigated by sequencing their 16S rRNA genes and phenotypic characterization. All GG-synthesizing isolates were shown to belong to the genus Pseudomonas (sensu stricto) and were assigned to 4 distinct groups, although none of the GG-synthesizing isolates could be unambiguously assigned to described species. The identity of GG was verified by 13C NMR analysis and enzymatic digestion with alpha- and beta-glucosidases. Besides GG, salt adapted cultures of the aquatic isolates accumulated the dipeptide N-acetylglutaminylglutamine amide (NAGGN) and glutamate. The accumulation of noncharged compatible solutes was also tested in previously identified pseudomonads isolated from the rhizosphere of oilseed rape and potato. The majority of these strains were fluorescent species of the genus Pseudomonas and accumulated trehalose and NAGGN when grown under salt stress conditions. However, rhizosphere isolates of Stenotrophomonas maltophilia synthesized GG and trehalose or only trehalose in a strain-dependent manner. These data indicate that the ability to synthesize GG is widely distributed among slightly or moderately halotolerant pseudomonads.     

Glucosylglycerate is an osmotic solute and an extracellular metabolite produced by<Emphasis Type="Italic">Streptomyces caelestis</Emphasis>

Streptomyces caelestis DSM 40084 produces two osmolytes, viz. 2-O-(alpha-D-glucopyranosyl)-zeta-glyceric acid (GG) and trehalose. Both compounds were isolated and identified by nuclear magnetic resonance spectroscopy and mass spectrometry. A very sensitive regulation of the cell osmolytes was demonstrated in exponentially growing cultures. The intracellular levels of GG and trehalose increased 2x in response to a step change of medium osmolarity caused by 0.3% NaCl. 1H NMR analysis of the cell extracts did not confirm the presence of additional osmolytes. GG is a S. caelestis metabolite commonly released from the cells; its concentration reached 3 g/L during the cultivation in a yeast extract--(NH4)2SO4-glycerol medium. This is the first report on the occurrence of the ionic osmolyte GG in the genus Streptomyces and on its free excretion to the medium.     

A relatively small change in sodium chloride concentration has a strong effect on adhesion of ocular bacteria to contact lenses

Adhesion of bacteria to hydrogel lenses is thought to be an initial step of ocular colonization allowing evasion of normal host defences. The salt concentration of media is an important parameter controlling microbial adhesion. Salinity varies from 0·97% NaCl equivalents in the open eye to 0·89% in the closed eye state. In this study, the effect of sodium chloride in the concentration range of 0·8–1·0% (w/v) NaCl on adhesion of ocular bacteria to soft contact lenses was investigated using a static adhesion assay. Pseudomonas aeruginosa was found to adhere to lenses in significantly greater amounts than Serratia marcescens, Flavobacterium meningosepticum, Stenotrophomonas maltophilia and Staphylococcus intermedius . Increasing NaCl from 0·8% to 1·0% (w/v) increased adhesion of all bacteria tested. This adhesion was strong since the organisms could not be removed by washing in low ionic buffer. Adhesion of these organisms did not correlate with their cell surface properties as determined by bacterial adhesion to hydrocarbons (BATH) and retention on sepharose columns.     

Stenotrophomonas interspecies differentiation and identification by gyrB sequence analysis

Stenotrophomonas species are found commonly in environmental and clinical samples; Stenotrophomonas maltophilia is an important opportunistic pathogen of humans. Traditional phenotyping protocols, as well as genotyping by 16S rRNA gene sequence analysis, do not reliably distinguish the species of Stenotrophomonas. Sequence analyses of two targeted PCR-amplified regions of the gyrB gene, which encodes the β-subunit of DNA gyrase, enabled resolution and identification of these species. Most type strains of the different species of Stenotrophomonas exhibited more than 7% dissimilarity in the gyrB gene sequences. Among these, strains identified as the same species exhibited sequence dissimilarities up to 4.6% and 5.9% for the two regions, respectively. Strains identified as S. maltophilia, with 16S rRNA gene sequence similarities > 99.0%, were grouped within a 'S. maltophilia complex'; these organisms exhibited gyrB similarities as low as 93%. Many of these strains possessed genomic DNA similarities with the type strain of S. maltophilia CCUG 5866(T) below 70%. These data, including gyrB sequence comparisons, indicate that strains identified as S. maltophilia may comprise distinct, new species.     

A variant of the hyperthermophile Archaeoglobus fulgidus adapted to grow at high salinity

A variant of Archaeoglobus fulgidus VC-16 was isolated from cultures obtained after a stepwise transfer from media containing 1.8-6.3% NaCl by a plating-independent, selected-cell cultivation technique, using a laser microscope. This variant, A. fulgidus VC-16S, had a higher growth rate throughout the salt range of the parental strain, but was also able to grow in media containing NaCl up to 6.3%, whereas the parental strain could not grow above 4.5% NaCl. Diglycerol phosphate (DGP), only encountered in the Archaeoglobales, was the major solute accumulated under supra-optimal salinities, whereas at supra-optimal growth temperatures di-myo-inositol phosphate was the predominant solute. The accumulation of compatible solutes during growth of variant VC-16S was lower than in the parental strain within 1.8-4.5% NaCl, but the levels of compatible solutes, including DGP, increased sharply in the variant at higher salinities (5.5 and 6.0%). This variant represents, at this time, one of the most halophilic hyperthermophiles known, and its ability to grow at high salinity appears to be due to the massive accumulation of DGP.     

The plant-associated bacterium Stenotrophomonas rhizophila expresses a new enzyme for the synthesis of the compatible solute glucosylglycerol

The rhizobacterium Stenotrophomonas rhizophila accumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme from S. rhizophila represented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned from Pseudomonas sp. strain OA146. The ggpPS gene was induced after a salt shock in S. rhizophila cells. After the salt-loaded cells reached stationary phase, the ggpPS mRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The complete ggpPS gene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis in S. rhizophila were found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.     

Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose

Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

斑点叉尾鮰一株致病菌的分离鉴定及系统发育分析

从发生急性流行性传染病的斑点叉尾肝、肾分离到一高致病性的菌株(CCF00024),经人工感染实验证实其为该病的病原菌。对该菌的形态、生理生化及16S rDNA序列分析结果表明,其为非发酵型,严格需氧,革兰氏阴性杆菌,极生多鞭毛,对除麦芽糖和甘露糖以外的多种糖类不能利用产酸,氧化酶阴性,DNA酶、蛋白酶、脲酶、赖氨酸脱羧酶阳性,MR阴性。在以该菌16S rDNA序列(GenBank登录号AY970826)和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,分离菌CCF00024与嗜麦芽寡养单胞菌(Stenotrophomonasmaltophilia)聚在一簇,特别是与S.maltophiliaM5-1的同源性最高,其序列相似性达99.6%,结合形态和生理生化特点将其鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)。     

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).     

Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge

An extremely halotolerant and alkaliphilic bacterium was isolated previously from deep-sea sediment collected at a depth of 1050 m on the Iheya Ridge. The strain, designated HTE831 (JCM 11309, DSM 14371), was Gram-positive, strictly aerobic, rod-shaped, motile by peritrichous flagella, and spore-forming. Strain HTE831 grew at salinities of 0-21% (w/v) NaCl at pH 7.5 and 0-18% at pH 9.5. The optimum concentration of NaCl for growth was 3% at both pH 7.5 and 9.5. The G+C content of its DNA was 35.8%.Low level (12-30%) of DNA-DNA relatedness between strain HTE831 and the species of these genera was found, indicating that HTE831 could not be classified as a member of a new species belonging to known genera. Based on phylogenetic analysis using 16S rDNA sequencing, chemotaxonomy, and the physiology of strain HTE831, it is proposed that this organism is a member of a new species in a new genus, for which the name Oceanobacillus iheyensis is proposed.     

Marinospirillum alkaliphilum sp. nov., a new alkaliphilic helical bacterium from Haoji soda lake in Inner Mongolia Autonomous Region of China

A new helical, alkaliphilic, gram-negative, chemoorganotrophic bacterium designated strain Z4T was isolated from Haoji soda lake in Inner Mongolia Autonomous Region, China. The isolate grows at salinities between 0.2% and 5.0% (w/v) NaCl and pH range 7.0-11.0, with an optimum at 2.0% (w/v) NaCl and pH 9.5. Its growth temperature ranges from 8 degrees to 49 degrees C with an optimum at 37 degrees C. The G+C content of the DNA is 46.8 mol%. The major isoprenoid quinone is ubiquinone 8 (Q-8). Phylogenetic analyses based on 16S rDNA sequence comparison indicates that strain Z4T is a member of the genus Marinospirillum. Phenotypic features and DNA-DNA homology of less than 20% with the described species of Marinospirillum support the view that strain Z4T represents a new species of the genus Marinospirillum. Strain Z4T (= AS 1.2746) is proposed as the type strain of a new species, named Marinospirillum alkaliphilum sp. nov.     

Description of Citricoccus nitrophenolicus sp. nov., a para-nitrophenol degrading actinobacterium isolated from a wastewater treatment plant and emended description of the genus Citricoccus Altenburger et al. 2002

A novel actinobacterium, designated PNP1(T), was isolated from a wastewater treatment plant at a pesticide factory by selective enrichment with para-nitrophenol. The strictly aerobic strain PNP1(T) grew with para-nitrophenol as the sole carbon and energy source. Metabolism of para-nitrophenol resulted in the stoichiometric release of nitrite. When incubated with both para-nitrophenol and acetate, para-nitrophenol was degraded and utilized as growth substrate prior to acetate. When grown on acetate (in the absence of ammonium) both nitrite and nitrate served as nitrogen sources, nitrate being quantitatively reduced to nitrite which accumulated in cultures during aerobic growth. Cells were coccoid and stained Gram-positive, were non-motile and did not form endospores. Colonies of strain PNP1(T) on agar medium were bright yellow, circular and smooth. The dominant menaquinone was MK-8(H(2)) (54%) and the major cellular fatty acid was anteiso C15:0 (75%). Strain PNP1(T) grew optimally at 27°C, at pH 8-8.5, at salinities 3% (w/v) NaCl, yet exhibited a substantial halotolerance with growth occurring at salinities up to 17% (w/v) NaCl. In addition to para-nitrophenol, a range of sugars, short chain fatty acids and alcohols served as electron donors for growth. The DNA G + C mol% was 68%. The genotypic and phenotypic properties suggest that strain PNP1(T) represents a novel species of the actinobacterial genus Citricoccus for which the name Citricoccus nitrophenolicus is proposed. It is the first member of this genus that has been reported to hydrolyze and grow on para-nitrophenol. The type strain is PNP1(T) (=DSM 23311(T) = CCUG 59571(T)).     

<Emphasis Type="Italic">Marinimicrobium haloxylanilyticum</Emphasis> sp. nov., a new moderately halophilic,polysaccharide-degrading bacterium isolated from Great Salt Lake,Utah

A new moderately halophilic, strictly aerobic, Gram-negative bacterium, strain SX15^T, was isolated from hypersaline surface sediment of the southern arm of Great Salt Lake (Utah, USA). The strain grew on a number of carbohydrates and carbohydrate polymers such as xylan, starch, carboxymethyl cellulose and galactomannan. The strain grew at salinities ranging from 2 to 22% NaCl (w/v). Optimal growth occurred in the presence of 7–11% NaCl (w/v) at a temperature of 35°C and a pH of 6.7–8.2. Major whole-cell fatty acids were C16:0 (30.5%), C18:0 (14.8%), C18:1ω7c (13.1%) and C12:0 (7.8%). The G+C content of the DNA was 60 ± 0.5 mol%. By 16S rRNA gene sequence analysis, strain SX15^T was shown to be affiliated to members of the gammaproteobacterial genus Marinimicrobium with pair wise identity values of 92.9–94.6%. The pheno- and genotypic properties suggest that strain SX15^T represents a novel species of the genus Marinimicrobium for which the name Marinimicrobium haloxylanilyticum is proposed. The type strain is SX15^T (= DSM 23100^T = CCUG 59572^T).     

Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum

A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).     

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.     

Geobacillus thermoleovorans subsp. stromboliensis subsp. nov., isolated from the geothermal volcanic environment

A novel thermophilic, aerobic, endospore-forming bacterium, designated strain PizzoT, was isolated from geothermal volcanic environment. Samples were collected from the Pizzo sopra la Fossa site at Stromboli Island (Eolian Islands, south of Italy) at the high altitude of 918 m. Cells of strain PizzoT were rod-shaped and stained Gram-positive. Growth was observed between 50 and 75 degrees C (optimum 70 degrees C) and at pH 5.0-8.0 (optimum pH 7.0). NaCl (0.4%, w/v) supported growth and among the hydrocarbons tested none induced growth. The G+C content of the DNA was 54.1 mol% and the sequence analysis of the 16S rRNA gene showed that the new isolate was phylogenetically closely related to the members of the Bacillus rRNA Group 5. DNA-DNA hybridization studies revealed a borderline similarity between the new isolate and Geobacillus thermoleovorans DSM 5366T (69.8%) and Geobacillus kaustophilus DSM 7263T (63.4%). On the basis of phylogenetic analysis and physiological traits of the isolate, it should be described as a new member of the Geobacillus thermoleovorans species and it is proposed that strain PizzoT can be classified as Geobacillus thermoleovorans subsp. stromboliensis, subsp. nov. (ATCC BAA-979T; DSM 15393T).     

Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30°C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80°C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.