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High-cell-density culture for recombinant xylanase production and its application in hydrolysis of mild alkaline pretreated rice straw for xylooligosaccharide production
(2026-08-01)
Laemthong, Tunyaboon
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Sukhumsirichart, Wasana
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Chittapun, Supenya
;
Kongsinkaew, Chatchol
;
Satipattarn, Atikant
Rice straw is an abundant lignocellulosic residue whose effective utilization requires coordinated enzyme performance and pretreatment conditions. In this study, recombinant xylanase production was integrated with mild alkaline pretreatment to enable efficient conversion of rice straw into xylooligosaccharides (XOS). Recombinant xylanase was produced by Escherichia coli using high-cell-density fed-batch cultivation, achieving a maximum activity of 207.95 U/mL in an enriched synthetic medium. Alkaline pretreatment was optimized using response surface methodology, identifying 6.75% (w/v) biomass loading, 193.17 mM NaOH, and 20 min as conditions that balanced solid recovery with sugar release during subsequent enzymatic hydrolysis. Hydrolysis of the pretreated rice straw yielded 6.13 mg/mL total XOS, mainly xylotriose (X3, 2.73 mg/mL; 44.56%), xylobiose (X2, 2.00 mg/mL; 32.62%), and xylotetraose (X4, 1.02 mg/mL; 16.57%), with only minor amounts of xylose and xylopentaose detected. The combined X2 and X3 fractions accounted for 77.18% of the total XOS. XOS with degrees of polymerization of 2–4 are recognized as effective prebiotics, particularly for beneficial intestinal bacteria such as Bifidobacterium spp. Overall, linking recombinant enzyme production with alkaline pretreatment improved conversion consistency and supported efficient XOS production from rice straw.
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Valorizing Red Seaweed Spent Biomass into Reducing Sugars for β-Carotene Production by Rhodotorula paludigena
(2026-05-01)
Kongsinkaew, Chatchol
;
Tangsattayatithan, Chutipol
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Chittapun, Supenya
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Phiphatbunyabhorn, Parivat
;
Laemthong, Tunyaboon
Seaweed bioactive extraction generates de-extracted residual solids that remain carbohydrate-rich but are often underutilized. This study developed an integrated valorization route for Gracilaria fisheri spent biomass to produce fermentable sugars for β-carotene production by Rhodotorula paludigena CM33. Reducing sugar production was optimized using response surface methodology (Box–Behnken design) by varying reaction time, sulfuric acid concentration, and biomass loading at 90 °C. The predicted optimum (47.39 min, 2.50% (w/v) H<inf>2</inf>SO<inf>4</inf>, and 7.13% (w/v) biomass) yielded 22.41 g/L reducing sugars and was validated experimentally at 22.22 ± 0.19 g/L, indicating that the model reliably predicted reducing sugar production. The optimized condition was scaled up in a 22 L bioreactor with sequential acid hydrolysis followed by enzyme-assisted hydrolysis, increasing reducing sugars from ~30 to ~40 g/L. FTIR and SEM analyses indicated progressive modification of the carbohydrate matrix across processing stages. Batch cultivation of R. paludigena on the hydrolysate showed that ammonium sulfate supplementation significantly increased biomass, whereas β-carotene titers were not significantly different. Repeated-batch operation on non-supplemented hydrolysate sustained production over four cycles with β-carotene titers of 13.75–17.27 mg/L, demonstrating the operational feasibility of the hydrolysate-based system. Overall, this work demonstrates a practical seaweed biorefinery approach to upgrade G. fisheri spent biomass into sugars and carotenoid-rich yeast biomass.
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Enhancing Antimicrobial Peptide Productivity in Pichia pastoris (Muts Strain) by Improving the Fermentation Process Based on Increasing the Volumetric Methanol Consumption Rate
(2023-03-01)
Kongsinkaew, Chatchol
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Chittapun, Supenya
;
Piyapittayanun, Chanitchote
;
Boonyaratanakornkit, Viroj
;
Sooksai, Sarintip
The instability of the protein expression in Pichia pastoris strains has been an issue for various peptide productions. Some modifications to the traditional fermentation process could potentially solve the problem. Here, we consider a four-stage fermentation process to express the CAP2 (cell-penetrating antimicrobial peptide 2) candidate in P. pastoris KM71H, a slow methanol utilization strain. During the fermentation process, CAP2 productivity is limited (6.15 ± 0.21 mg/L·h) by the low overall methanol consumption (approximately 645 g), which is mainly the result of the slow methanol utilization of the P. pastoris KM71H. To overcome this limitation, we increased the cell concentration two-fold prior to the induction stage. A fed-batch process with exponential and dissolved oxygen tension (DOT) stat feeding strategies was deployed to control the glycerol feed, resulting in an increase in cell concentration and enhancement of the volumetric methanol consumption rate. The improved fermentation process increased the overall methanol consumption (approximately 1070 g) and the CAP2 productivity (13.59 ± 0.24 mg/L·h) by 1.66 and 2.21 times, respectively. In addition, the CAP3 (cell-penetrating antimicrobial peptide 3) candidate could also be produced using this improved fermentation process at a high yield of 3.96 ± 0.02 g/L without any further optimization. Note that there was no oxygen limitation during the improved fermentation process operating at high cell density. This could be due to the controlled substrate addition via the DOT stat system.

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