Evaluation of thermotolerant and ethanol-tolerant Saccharomyces cerevisiae as an alternative strain for bioethanol production from industrial feedstocks.

Despite the fact that yeast Saccharomyces cerevisiae is by far the most commonly used in ethanol fermentation, few have been reported to be resistant to high ethanol concentrations at high temperatures. Hence, in this study, 150 S. cerevisiae strains from the Thailand Bioresource Research Center (TBRC) were screened for ethanol production based on their glucose utilization capability at high temperatures. Four strains, TBRC 12149, 12150, 12151, and 12153, exhibited the most outstanding ethanol production at high temperatures in shaking-flask culture. Among these, strain TBRC 12151 demonstrated a high ethanol tolerance of up to 12% at 40 °C. Compared to industrial and laboratory strains, TBRC 12149 displayed strong sucrose fermentation capacity whereas TBRC 12153 and 12151, respectively, showed the greatest ethanol production from molasses and cassava starch hydrolysate at high temperatures in shaking-flask conditions. In 5-L batch fermentation, similarly to both industrial strains, strain TBRC 12153 yielded an ethanol concentration of 66.5 g L-1 (58.4% theoretical yield) from molasses after 72 h at 40 °C. In contrast, strain TBRC12151 outperformed other industrial strains in cell growth and ethanol production from cassava starch hydrolysis at 40 °C with an ethanol production of 65 g L-1 (77.7% theoretical yield) after 72 h. Thus, the thermotolerant and ethanol-tolerant S. cerevisiae TBRC 12151 displayed great potential and possible uses as an alternative strain for industrial ethanol fermentation using cassava starch hydrolysate. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03436-4.

Characterization of cold-active trehalose synthase from Pseudarthrobacter sp. for trehalose bioproduction.

Trehalose is a functional sugar that has numerous applications in food, cosmetic, and pharmaceutical products. Production of trehalose from maltose via a single-step enzymatic catalysis using trehalose synthase (TreS) is a promising method compared with the conventional two-step process due to its simplicity with lower formation of byproducts. In this study, a cold-active trehalose synthase (PaTreS) from Pseudarthrobacter sp. TBRC 2005 was heterologously expressed and characterized. PaTreS showed the maximum activity at 20 °C and maintained 87% and 59% of its activity at 10 °C and 4 °C, respectively. The enzyme had remarkable stability over a board pH range of 7.0-9.0 with the highest activity at pH 7.0. The activity was enhanced by divalent metal ions (Mg2+, Mn2+ and Ca2+). Conversion of high-concentration maltose syrup (100-300 g/L) using PaTreS yielded 71.7-225.5 g/L trehalose, with 4.5-16.4 g/L glucose as a byproduct within 16 h. The work demonstrated the potential of PaTreS as a promising biocatalyst for the development of low-temperature trehalose production, with the advantages of reduced risk of microbial contamination with low generation of byproduct.

Integrated Whole-Cell Biocatalysis for Trehalose Production from Maltose Using Permeabilized Pseudomonas monteilii Cells and Bioremoval of Byproduct.

Trehalose is a non-conventional sugar with potent applications in the food, healthcare and biopharma industries. In this study, trehalose was synthesized from maltose using whole-cell Pseudomonas monteilii TBRC 1196 producing trehalose synthase (TreS) as the biocatalyst. The reaction condition was optimized using 1% Triton X-100 permeabilized cells. According to our central composite design (CCD) experiment, the optimal process was achieved at 35°C and pH 8.0 for 24 h, resulting in the maximum trehalose yield of 51.60 g/g after 12 h using an initial cell loading of 94 g/l. Scale-up production in a lab-scale bioreactor led to the final trehalose concentration of 51.91 g/l with a yield of 51.60 g/g and productivity of 4.37 g/l/h together with 8.24 g/l glucose as a byproduct. A one-pot process integrating trehalose production and byproduct bioremoval showed 53.35% trehalose yield from 107.4 g/l after 15 h by permeabilized P. moteilii cells. The residual maltose and glucose were subsequently removed by Saccharomyces cerevisiae TBRC 12153, resulting in trehalose recovery of 99.23% with 24.85 g/l ethanol obtained as a co-product. The present work provides an integrated alternative process for trehalose production from maltose syrup in bio-industry.

Screening, Cloning, Expression and Characterization of New Alkaline Trehalose Synthase from Pseudomonas monteilii and Its Application for Trehalose Production.

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40°C, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

Application of raw starch degrading enzyme from Laceyella sacchari LP175 for development of bacterial cellulose fermentation using colored rice as substrate.

Brown and black rice substrates were applied for sugar syrup production by the hydrolysis of raw starch degrading enzyme (RSDE) from Laceyella sacchari LP175 (300 U/mL) and commercial glucoamylase (GA, 2.0 U/mL) at 50 °C for 12 h using a simplex centroid mixture design. Results indicated that 300 g/L of substrates, consisting of 255 g/L Leum Pua glutinous rice and 45 g/L Black Jasmine rice, gave the highest sugar syrup production at 124.6 ± 2.52 g/L with 2.00 ± 0.05 mg GAE/mL of total phenolic content (TPC), equivalent to 0.42 ± 0.01 g/g rice sample and 6.67 ± 0.15 mg GAE/g rice sample, respectively. The obtained sugar syrup was used as the substrate for production of bacterial cellulose (Nata) by Komagataeibacter xylinus AGR 60 in a plastic tray at room temperature for 9 days. The fermentation medium containing 200 mL of rice syrup (25 g/L), 2.0 g of ammonium sulfate [(NH4)2SO4] and 0.4 mL glacial acetic acid yielded 1.1 ± 0.08 cm thickness with 8.15 ± 0.12 g of dry weight. The obtained bacterial cellulose from colored rice was characterized compared with bacterial cellulose from the conventional coconut juice by scanning electron microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR) which demonstrated that the sugar syrup from colored rice could use as substrate for a novel bacterial cellulose as a healthy product in the future through microbial enzyme technological process. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02673-3.

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production.

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8 g:10 g:2 g yielded the highest enzyme production of 201.6 U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5 × 106 spores/mL inoculum, which gave the highest enzyme activity of 389.5 U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2 g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300 g raw cassava chips/L with cane molasses.