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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
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Kongsinkaew, Chatchol
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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
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Tangsattayatithan, Chutipol
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Chittapun, Supenya
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Phiphatbunyabhorn, Parivat
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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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Fed-batch strategies for growing β-carotene enriched yeast Rhodotorula paludigena using untreated molasses
(2025-11-01)
Kongsinkaew, Chatchol
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Chisti, Yusuf
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Ketudat-Cairns, Mariena
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Chittapun, Supenya
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Pornpukdeewattana, Soisuda
The β-carotene-rich yeast Rhodotorula paludigena CM33 was grown in a 22-L bioreactor using sugarcane molasses in combination with several distinct fed-batch strategies: DO (dissolved oxygen)-stat; stepwise feeding; and hybrid feeding (an initial exponential feeding phase followed by a DO-stat operation). The impacts of feeding strategies on biomass production, carotenoid production and nutrient conversion efficiencies were evaluated. The stepwise feeding proved most effective, achieving: a final dry biomass concentration of 73.1 ± 2.7 g L−1; a β-carotene titer of 181.9 ± 1.0 mg L−1; a biomass volumetric productivity of 1.33 ± 0.05 g L−1 h−1; and β-carotene productivity of 3.31 ± 0.02 mg L−1 h−1. However, with this feeding strategy the sugar and nitrogen conversion efficiencies were the lowest: the β-carotene yield on total sugar was 0.92 ± 0.00 mg g−1 and its yield on nitrogen was 5.47 ± 0.06 mg g−1. In contrast, the DO-stat operation was the most resource efficient, providing a biomass yield on sugar of 0.47 ± 0.02 g g−1, a biomass yield on nitrogen of 2.64 ± 0.05 g g−1, a β-carotene yield on sugar of 1.08 ± 0.04 mg g−1, and β-carotene yield on nitrogen of 6.08 ± 0.08 mg g−1. The hybrid strategy resulted in intermediate values of the various performance parameters. Raw sugarcane molasses proved to be excellent for high-cell-density culture of R. paludigena. A compromise between productivity and resource use efficiency may be required for commercial production of β-carotene especially if the substrate is expensive compared to the product.
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Natural versus Saccharomyces boulardii self-induced anaerobic coffee fermentation: Effects on physicochemical properties and microbial ecology, and their influence on volatile profiles and sensory attributes across roast levels
(2025-10-01)
Pholtaisong, Jatuphol
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Kongsinkaew, Chatchol
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On-Mee, Thapanut
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Chittapun, Supenya
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Pornpukdeewattana, Soisuda
This study investigates the effects of natural self-induced anaerobic fermentation (NSIAF) and Saccharomyces boulardii self-induced anaerobic fermentation (SSIAF) on Arabica coffee during wet processing. Over 24 h of fermentation, NSIAF exhibited greater microbial diversity, higher titratable acidity, and increased reducing sugar consumption, while SSIAF provided a more controlled microbial environment dominated by S. boulardii. Volatile compound analysis identified 207 compounds, with lighter roasts showing the greatest number of significantly different compounds between NSIAF and SSIAF treatments. Sensory evaluation revealed a higher cupping score for NSIAF at a light roast (82.08 ± 0.14) compared to SSIAF (81.58 ± 0.14), reflecting distinct flavor characteristics imparted by each fermentation process. Both methods achieved specialty coffee standards (≥80 points), highlighting the potential of NSIAF for complex and diverse profiles and the suitability of SSIAF for consistency and controlled fermentation.
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Thermal degradation kinetics and purification of C-phycocyanin from thermophilic and mesophilic cyanobacteria
(2025-02-01)
Chittapun, Supenya
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Suwanmanee, Kattiya
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Kongsinkaew, Chatchol
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Pornpukdeewattana, Soisuda
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Chisti, Yusuf
The natural blue colorant C-phycocyanin (C-PC) has many potential applications but its poor heat stability limits its commercial use. This study compares the production and thermal stability of C-PC from two cyanobacteria: the thermophilic Thermosynechococcus sp. TUBT-T01 and the mesophilic Synechococcus cedrorum TISTR8589. Thermosynechococcus sp. produced nearly 1.9-fold more C-PC than S. cedrorum. Batch adsorption using a chromatographic cationic ion exchange resin (Streamline Direct HST1) was used to effectively purify the C-PC. The equilibrium adsorption capacity (Q<inf>eq</inf>) of the resin for C-PC was the highest at pH 5. At this pH, the Q<inf>eq</inf> for the thermophilic C-PC was 5.5 ± 0.1 mg mL⁻¹, whereas for the mesophilic C-PC it was 1.5 ± 0.2 mg mL⁻¹. Purification increased the concentration of the thermophilic C-PC by 5.9-fold, and that of mesophilic C-PC by 4.2-fold. The purity ratios of the final products from the two cyanobacteria were similar at ∼2.2. At 60 °C and pH 7, the C-PC of Thermosynechococcus sp. had ∼12-times longer half-life than the mesophilic C-PC; however, the productivity of the thermophilic C-PC was comparatively low because of a low biomass productivity of Thermosynechococcus sp.
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Enhanced high β-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals
(2024-12-01)
Thumkasem, Namphet
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On-mee, Thapanut
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Kongsinkaew, Chatchol
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Chittapun, Supenya
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Pornpukdeewattana, Soisuda
This study assessed Rhodotorula paludigena CM33's growth and β-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and β-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a β-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Q<inf>x</inf>) of 0.91 g/L.h and remarkable β-carotene production rates (Q<inf>p</inf>) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale β-carotene production for aquatic animal nutrition.
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Large-scale production of paraprobiotic soy milk in stirred tank bioreactor: A dual-step fermentation approach
(2024-12-01)
Kongsinkaew, Chatchol
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Hongphankul, Kant
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Soontornkitlert, Thanakorn
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Surarit, Worawat
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Sutheerawattananonda, Manote
This study investigates the feasibility of using soy milk as a substrate for cultivating Lactobacillus casei and its subsequent conversion into paraprobiotics. The research focuses on optimizing the fermentation process from shake flasks to bioreactor scale and assessing the antioxidant properties and proximate compositions of the resulting paraprobiotic soy milk beverage. The study highlights a dual-step large-scale fermentation process comprising an anaerobic fermentation step followed by a thermal inactivation step using the in situ temperature control system of the bioreactor. Results indicated that soy milk enriched with 10 g/L glucose and fermented at 37 °C for 24 h on flask scale provided optimal conditions for L. casei growth, achieving a bacterial count increase of 8.80 ± 0.11 log CFU/mL and a titratable acidity of 0.89 ± 0.03 %. These optimal conditions were employed for bioreactor scale fermentation, attaining a bacterial count of 10.02 ± 0.06 log CFU/mL and a titratable acidity of 1.07 ± 0.04 % at 24 h. The thermal inactivation step at 90 °C for 30 min successfully converted the probiotic soy milk into a paraprobiotic form, maintaining its antioxidant activity at 62.12 ± 2.58 % radical scavenging activity. The final paraprobiotic product met the Food and Drug Administration (FDA) specifications for titratable acidity, presenting a viable alternative for soy-based functional beverages.
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Rhodotorula paludigena CM33 cultivation process development for high β-carotene single cell protein production
(2023-11-01)
Thumkasem, Namphet
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On-mee, Thapanut
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Chittapun, Supenya
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Pornpukdeewattana, Soisuda
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Ketudat-Cairns, Mariena
Rhodotorula yeast has potential in terms of biotechnology for the production of various compounds for the environmental, industrial, and medical sectors. It is well known to generate carotenoid pigments that are beneficially distinguished. This investigation aimed to optimize the growth of Rhodotorula paludigena CM33 and its production of β-carotene. Response Surface Methodology was employed to optimize temperature, ammonium sulfate, and ferric chloride concentrations in a 5L-bioreactor batch culture. The optimal conditions of 30 °C, 6.2 g/L of ammonium sulfate, and 75.0 mg/L of ferric chloride provided biomass of 17.71 g/L, a specific growth rate of 0.22 h−1, and β-carotene concentration of 40.29 mg/L. Upon scaling up the cultivation process, the 22L-bioreactor displayed similar growth patterns to those of the 5L-bioreactor. However, the 22L-bioreactor exhibited higher product yields in a shorter time period. The maximum biomass production of 18.96 ± 1.31 g/L and the maximum β-carotene production of 42.81 ± 0.61 mg/L were achieved at 28 h. This scaling-up strategy resulted in improved production efficiency. Additionally, the nutritional value of the cells was evaluated, suggesting their potential as animal feed supplements. R. paludigena CM33 demonstrated a β-carotene single-cell protein content, making it a valuable source for aquaculture and livestock feed. These findings highlight the potential of this yeast strain for large-scale production of β-carotene and single-cell protein, offering nutritional benefits for animal feed.
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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
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Piyapittayanun, Chanitchote
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Boonyaratanakornkit, Viroj
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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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