Figure 1.
Isolation and identification of TSH1.
(A). Ethanol fermentation capability of the screened strains. Eight strains isolated from soils in which sweet sorghum stocks were stored were cultured with crushed sweet sorghum as the substrate in 100-mL vials at 30°C for 7 hours. The ethanol content was determined by HPLC and is presented as the ethanol production rate in g/h per kg of crushed sweet sorghum (g/kg/h) in the top panel. Gas production analysis was performed with graduated syringes that were inserted into the rubber plugs of each vial before the start of the culture (bottom panel). The white arrows indicate the cumulative gas production during the 7-hour cultivation. The strain numbered “1” is TSH1, and 2–8 are the other strains screened. (B). Morphology of TSH1 grown on rich medium. TSH1 was cultured at 30°C for 12 hours on YPD plates. (C). Colony phenotype of TSH1 described in (B). (D). Cell morphology of TSH1. Images of vegetative cells grown on YPD medium were captured using a Nikon Ti-E Inverted Fluorescence Microscope. Magnification = 100×10. The bars represent 10 µm. (E). Cells of TSH1 in the budding (asexual reproduction) state. Cells formed buds on YPD medium after 12 hours at 30°C. The bars represent 10 µm. (F). TSH1 ascospore formation. TSH1 was grown on McClary medium for 7 days at 28°C. The bars represent 10 µm. (G). TSH1 ploidy analysis with the detection of MATa and MATα alleles. CK1 (S. cerevisiae Y294) was used as the haploid control and CK2 (S. cerevisiae BY4743) was used as the diploid control, and M represents the DNA ladder (from top to bottom: 750 bp, 500 bp, and 250 bp). (H). TSH1 is closely related to S. cerevisiae S288c. Phylogenetic tree reconstructed from the neighbor-joining analysis of the 18S rDNA gene and 26S rDNA sequence of TSH1. The bootstrap percentages over 50% (from 1000 bootstrap replicates) are shown. The reference sequences were from the species type strains retrieved from GenBank under the indicated accession numbers. The bars represent 0.01 substitutions per nucleotide position.
Table 1.
Assimilation and fermentation capability of different nutrient elements.
Figure 2.
TSH1 exhibits good tolerance to different stress conditions.
TSH1 was cultured under the indicated stress conditions until the growth curve reached the stationary phase. BY4743 served as the control strain. The growth curves of TSH1 (left panel) and BY4743 (middle panel) were constructed using OD600 values and fitted by a logistic growth equation. The absolute growth rate (AGR) was calculated and is shown in the right panel. The error bars represent SD (n = 3). (A)∼(C). Tolerance to a high culture temperature. (D)∼(F). Tolerance to ethanol inhibition. (G)∼(I). Tolerance to acetate inhibition. (J)∼(L). Tolerance to acidic pH.
Figure 3.
Temperature and moisture tolerance analysis in solid-state fermentation.
(A). High temperature tolerance of TSH1. Solid-state fermentation with TSH1 was performed at the indicated temperatures for 12 hours using crushed sweet sorghum stalks as the substrate. BY4743 served as the control strain. The ethanol concentrations were measured by HPLC at the start and end points of fermentation. The ethanol production rate represents the ethanol weight produced per kg of substrate with 70% moisture content per hour. The error bars represent the SD (n = 3). (B). Low moisture content tolerance of TSH1. The crushed sweet sorghum stalks were pretreated to achieve the different moisture contents and subsequently loaded into the solid-state fermentation flasks. The error bars represent SD (n = 3).
Figure 4.
The performance of TSH1 in the 50-L rotary-drum fermenter.
(A). Photograph of the 50-L rotary-drum fermenter. (B). Photograph of the lid of the 50-L rotary-drum fermenter. (C). Structure model of the 50-L rotary-drum fermenter. The fermenter consists of two lids and a tank. The tank and two lids are sealed with two sealing clasps, and handles are set on both lids; only one gas exit is fixed on the left side of the lid. Eight temperature sensors are evenly positioned on the tank to monitor the temperature at different positions, and a drive gear is fixed in the middle of the tank for rotation. (D). Structure model of the fermenter (crosscut view). The fermenter is covered with an insulation layer for heat preservation and a water jacket for temperature regulation. Many baffles are fixed on the inside wall to enhance material and heat transfer. (E). Solid-state fermentation analysis of TSH1 in the 50-L fermenter. Crushed sweet sorghum stalks (14 kg) inoculated with TSH1 were loaded and fermented at 30°C with a 0.5 rpm rotary speed for 12 hours. BY4743 served as the control strain. The ethanol concentrations were measured by HPLC at the start and end points of fermentation. The RTEY is defined as the ratio of ethanol weight produced compared to the theoretical yield based on sugar consumed (%). The error bars represent the SD (n = 3).
Table 2.
Ethanol rate and RTEY of step-up enlarged fermenters.
Figure 5.
Fermentation of TSH1 in the 550-m3 fermenter.
(A). Image of the 550-m3 fermenter in the workshop. (B). Structure model of the fermenter. The fermenter is fixed on a base supported by three support gears and rotates via a drive gear; a feed inlet and discharge outlet can cross the fermenter on conveyors. A 5-degree angle between the tank and base is applied to enhance substrate transfer. The surface of the fermenter is covered with a layer of insulation for heat preservation. (C). Residual sugar and ethanol yield during fermentation. Crushed sweet sorghum stalks (96 tons) were fermented by TSH1 at 30°C for 21 hours, and samples were collected at the indicated times. The residual sugar was measured by the DNS method and is represented as a percentage (by weight) of the total sugar (including sucrose, glucose, and fructose) that remained unfermented in 1 g of substrate. The ethanol yield was measured by HPLC and is represented as the percentage (by weight) produced by 1 g of substrate. The error bars represent the SD (n = 3).
Table 3.
Energy input and output based on 550-m3 fermenter data.
Table 4.
Economic analyses per ton of ethanol based on 550-m3 fermenter.