Sample collection Nine different fermented sample sources were used for sample collection. They were collected from various natural fermented sources and locations in Bangladesh, where temperatures fluctuate between 35 and 40 C during summer. Selected sample sources were: Tari (overnight natural fermented palm juice/sap collected from the xylem tissue of palm tree, Phoenyx sylvestries, at around 35–40 C), Pantavat (Pv, an overnight natural fermented rice in tap water at around 35–38 C), Boiled potato (Bp, Boiled potato was exposed in open air where various starch-containing garbage are available), Watermelon juice (Wm, from street vendors), Sugarcane juice (Sc, from street vendors), Sugar molasses (Sm, from sugar mill), Decomposed food materials (Df, from food markets), Municipal solid waste (Msw) and Municipal liquid waste (Mlw). All of the samples were inoculated into Yeast-Peptone-Dextrose (YPD) agar plates and incubated at 37 C for two days. Yeast-like colonies were isolated for further study.
Isolation of thermotolerant microorganisms: Thermotolerant microorganisms were isolated by an enrichment technique in a media containing Sugarcane juice (8% total sugars), 0.05% (NH4)2SO4 and 4% (v/v) ethanol and with pH 4.5. Medium pH was adjusted by 1 N HCl. After inoculation, cultures were incubated for three days in a rotary shaker at a predetermined temperature with shaking speed of 160 rpm. Enriched cultures were then streaked on YPD agar plates containing the same medium and incubated at 30, 37, 40, 42, 45 and 50 C. Purified yeast cultures were kept on YPD agar plates and slants and then stored at 4 and ¡20 C, respectively. Thermotolerant yeast strains were screened based on their growth performances at the abovementioned temperatures in YPD agar plates.
Screening of thermotolerant microorganisms under various physiological conditions Thermotolerance of the isolates was determined by growing them on temperatures ranging from 30 to 50 C. Microscopic studies were used to identify strain morphologically, after growing them under various physiological conditions. Furthermore, sequencing of yeast 26S rDNA (D1/D2 region) was used for further identification/screening of microorganisms, according to the method developed by Kurtzman and Fell [5]. Morphological and physiological characterizations of isolated yeasts were carried out in medium containing various carbon sources and at various temperatures and pH. Three different carbon sources, YPD/Yeast– Peptone–Xylose (YPX)/Yeast–Peptone–Arabinose (YPA) medium (10 g/L yeast extract and 20 g/L peptone) were prepared and supplemented with 20 g/L of Dextrose/ Xylose/Arabinose, which were designated as YPD, YPX and YPA, respectively. At first, one loop-full yeast colony was inoculated from a fresh YPD plate into test tube containing 3 mL of YPD broth and incubated at 30 8C for 24 h in a shaking water bath (incubator). Then yeast cells were streaked separately on the YPD, YPX and YPA agar plates and incubated at 30 8C for 48 h. The cell morphology of thermotolerant yeast and their appearance on solid medium (YPD agar plate) were examined, after incubating at 37 C for two days. The appearance of the cultures (texture, colour and surface of colonies) was recorded. Cells from a young actively growing culture were inoculated into test tube containing 5 mL of YPD broth, incubated at 37 C for four days. The culture was examined to determine the growth of thermotolerant yeast visually on the surface of YPD liquid medium. In order to identify the optimum growth temperature and pH, YPD agar plates were streaked with thermotolerant yeasts from actively growing culture and incubated overnight at various temperatures (30, 37, 40, 42, 45, 48 and 50 C) and pH (4.5, 5.0, 5.5 and 6.0). YPD broth was prepared in several conical flasks and pH was adjusted by concentrated sulphuric acid (H2SO4) or 2 N NaOH, where necessary. For the measurement of growth curve, 25 mL (200-fold dilution) from a young actively growing culture was inoculated into test tubes containing 5 mL of YPD or YPX broth and then incubated at various temperatures (25–45 8C) for at least 96 h [6]. During incubation, cell suspension was collected in every 4 h interval, the optical density was measured in a spectrophotometer (Specord UV/Visible Spectrophotometer, Analytic Jena, Germany) at 600 nm against the corresponding broth medium as blank.
Fluorescent microscopic observation For microscopic imaging, cells from liquid culture were harvested by mild centrifugation (5000 rpm for 5 min). In brief, cells were washed, fixed and stained with 5 mg/mL DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride). The fixed cells (5 mL) were dropped into the well of a 10-well multi-test microscope slide (76 £ 26 mm with 24 £ 60 mm coverslip; Matsunami glass Ind., Ltd., Japan) and air dried at 27 C. Immunofluorescent microscope was used for characterization of thermotolerant yeasts morphologically, specially shape, size and visualization of nucleoid area of cell.
Selection of thermotolerant microorganisms for ethanol production Bioethanol production was conducted independently under various medium temperatures, pH and carbon sources as described in the results section below. Inoculums were prepared by transferring one loop full of 24 h culture grown on a plate of YPD agar to an Erlenmeyer flask containing 50 mL of a sugar cane juice medium as described earlier. The inoculums were transferred at the rate of 0.5% to the screening medium, followed by incubation on a rotary shaker at various temperatures ranging from 30 to 42 C in 250 mL Erlenmeyer flasks containing 100 mL of a basal Sugarcane juice medium composed of Sugarcane juice supplemented with Glucose up to 18% total sugars and 0.05% (NH4)2SO4.