Evaluation of Antioxidant Activity of Residue from Bioethanol Production Using Seaweed Biomass.

This study explores the potential of producing bioethanol from seaweed biomass and reusing the residues as antioxidant compounds. Various types of seaweed, including red (Gelidium amansii, Gloiopeltis furcata, Pyropia tenera), brown (Saccharina japonica, Undaria pinnatifida, Ascophyllum nodosum), and green species (Ulva intestinalis, Ulva prolifera, Codium fragile), were pretreated with dilute acid and enzymes and subsequently processed to produce bioethanol with Saccharomyces cerevisiae BY4741. Ethanol production followed the utilization of sugars, resulting in the highest yields from red algae > brown algae > green algae due to their high carbohydrate content. The residual biomass was extracted with water, ethanol, or methanol to evaluate its antioxidant activity. Among the nine seaweeds, the A. nodosum bioethanol residue extract (BRE) showed the highest antioxidant activity regarding the 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity, ferric reducing antioxidant power (FRAP), and reactive oxygen species (ROS) inhibition of H2O2-treated RAW 264.7 cells. These by-products can be valorized, contributing to a more sustainable and economically viable biorefinery process. This dual approach not only enhances the utilization of marine resources but also supports the development of high-value bioproducts.

Circular bioeconomy in action: Upscaling cutlassfish waste for eco-friendly recombinant protein production.

The fish processing industry generates a significant amount of waste, and the recycling of this waste is an issue of global concern. We sought to utilize the heads of cutlassfish (Trichiurus lepturus), which are typically discarded during processing, to produce peptone, which is an important source of amino acids for microbial growth and recombinant protein production. Cutlassfish head muscle (CHM) were isolated, and the optimal protease and reaction conditions for peptone production were determined. The resulting peptone contained 12.22 % total nitrogen and 3.19 % amino nitrogen, with an average molecular weight of 609 Da, indicating efficient hydrolysis of CHM. Growth assays using Escherichia coli have shown that cutlassfish head peptone (CP) supports similar or superior growth compared to other commercial peptones. In addition, when recombinant chitosanase from Bacillus subtilis and human superoxide dismutase were produced in E. coli, CP gave the highest expression levels among six commercial peptones tested. In addition, the expression levels of chitosanase and superoxide dismutase were 20 % and 32 % higher, respectively, in CP medium compared to the commonly used Luria-Bertani (LB) medium. This study demonstrates the potential of using cuttlassfish waste in the production of microbial media, thereby adding significant value to fish waste. The results contribute to sustainable waste management practices and open avenues for innovative uses of fish processing by-products in biotechnological applications.

Brown Seaweed Byproduct Extracts Improve Intestinal Motility and Auto-Inflammation in Mice with Loperamide-Induced Constipation.

Sargassum fusiforme and Sargassum fulvellum are types of brown algae used for their nutritional value and medicinal properties, including anti-inflammatory, antioxidant, and anticancer effects. Despite their importance in various industries, many seaweed byproducts containing dietary fiber and polysaccharides are discarded in landfills. These byproducts can be recycled and repurposed for different applications. In this study, we investigated the impact of S. fusiforme food processing byproducts (MbP-SFF) and S. fulvellum food processing byproducts (MbP-SFV) on improving intestinal motility and reducing inflammation in mice with constipation induced by loperamide. To evaluate this, mice were orally administered 500 mg/kg/day of the byproducts once daily for 8 days. Constipation was induced by 5 mg/kg/day of loperamide for two days after oral administration for 6 days. Each sample contained approximately 70% carbohydrates. MbP-SFF had 52.0% mannuronic acid and 18.8% guluronic acid, while MbP-SFV had 36.9% mannuronic acid and 32.9% guluronic acid. These byproducts enhanced fecal excretion and intestinal motility by modulating inflammatory responses. Furthermore, they restored the balance of the gut microbiota disrupted by loperamide, increasing beneficial Bifidobacterium and reducing harmful Staphylococcus aureus. Overall, MbP-SFF and MbP-SFV improved intestinal motility and inflammation by influencing the gut microbiota and inflammatory responses in a loperamide-induced mouse model. These byproducts show potential as ingredients in functional foods aimed at enhancing gut health, potentially reducing waste disposal costs and addressing environmental concerns associated with their utilization.

Enhanced Photosynthetic Pigment Production Using a Scaled-Up Continuously Circulated Bioreactor.

Microalgae have gained attention as a promising source of chlorophylls and carotenoids in various industries. However, scaling up of conventional bubble columns presents challenges related to cell sedimentation and the presence of non-photosynthetic cells due to non-circulating zones and decreased light accessibility, respectively. Therefore, this study aimed to evaluate the newly developed continuously circulated bioreactor ROSEMAX at both laboratory and pilot scales, compared to a conventional bubble column. There was no significant difference in the biomass production and photosynthetic pigment content of Tetraselmis sp. cultivated at the laboratory scale (p > 0.05). However, at the pilot scale, the biomass cultured in ROSEMAX showed significantly high biomass (1.69 ± 0.11 g/L, dry weight, DW), chlorophyll-a (14.60 ± 0.76 mg/g, DW), and total carotene (5.64 ± 0.81 mg/g, DW) concentrations compared to the conventional bubble column (1.17 ± 0.11 g/L, DW, 10.67 ± 0.72 mg/g, DW, 3.21 ± 0.56 mg/g, DW, respectively) (p ≤ 0.05). Flow cytometric analyses confirmed that the proportion of Tetraselmis sp. live cells in the culture medium of ROSEMAX was 32.90% higher than that in the conventional bubble column, with a photosynthetic efficiency 1.14 times higher. These results support suggestions to use ROSEMAX as a bioreactor for industrial-scale applications.

Enhancement of catabolite regulatory genes in Saccharomyces cerevisiae to increase ethanol production using hydrolysate from red seaweed Gloiopeltis furcata.

Glucose and galactose are monosaccharides obtained from Gloiopeltis furcata (Red algae). A total monosaccharide yield of 62.3 g/L was obtained from G. furcata using thermal acid hydrolysis and enzymatic saccharification. Activated carbon was used to eliminate hydroxymethylfurfural (HMF) from the hydrolysate. Previously obtained monosaccharides are used for ethanol production by Saccharomyces cerevisiae. S. cerevisiae consumes glucose first, then galactose. The methods for reducing fermentation time and increasing the ethanol yield coefficient using the simultaneous consumption of glucose and galactose have been evaluated. Gal3p and Gal80p of S. cerevisiae act as signal transducers that govern the galactose inducer Gal4p mediated transcriptional activation of the Gal gene family. Gal80p binds to Gal4p for transcription deactivation. Therefore, Gal80p was deleted for Gal4p expression without interruption.

Enhancement of Galactose Uptake from Kappaphycus alvarezii Using Saccharomyces cerevisiae through Deletion of Negative Regulators of GAL Genes.

This study was aimed at enhancing galactose consumption from the red seaweed Kappaphycus alvarezii. The optimal pretreatment condition of thermal acid hydrolysis was treated with 350 mM HNO3 for 60 min at 121 °C. The enzymatic saccharification with a 1:1 mixture of Celluclast 1.5 L and Viscozyme L showed the maximum yield of glucose; 42-g/L monosaccharide concentration was obtained with the highest yield of pretreatment and enzymatic saccharification (EPS) and the lowest inhibitory compound concentration. The deletion of the GAL80, MIG1, CYC8, or TUP1 gene was performed to improve the galactose consumption rate. The strains with the deletion of the MIG1 gene (mig1Δ) showed higher galactose consumption rate and ethanol yield than other strains. High transcription levels of regulatory genes revealed that the mig1Δ relieved glucose repression. These results show that the mig1Δ enhances galactose consumption rate from K. alvarezii.

Enhancement of Galactose Uptake from Kappaphycus alvarezii Hydrolysate Using Saccharomyces cerevisiae Through Overexpression of Leloir Pathway Genes.

A total 42.68 g/L monosaccharide with 0.10 g/L HMF was obtained from 10% (w/v) Kappaphycus alvarezii with thermal acid hydrolysis using 350 mM HNO3 at 121 °C for 60 min and enzymatic saccharification with a 1:1 mixture of Viscozyme L and Celluclast 1.5 L for 72 h. To enhance the galactose utilization rate, fermentation was performed with overexpression of GAL1 (galactokinase), GAL7 (galactose-1-phosphate uridyltransferase), GAL10 (UDP-glucose-4-epimerase), and PGM2 (phosphoglucomutase 2) in Saccharomyces cerevisiae CEN.PK2 using CCW12 as a strong promoter. Among the strains, the overexpression of PGM2 showed twofold high galactose utilization rate (URgal) and produced ethanol 1.4-fold more than that of the control. Transcriptional analysis revealed the increase of PGM2 transcription level leading to enhance glucose-6-phosphate and fructose-6-phosphate and plays a key role in ensuring a higher glycolytic flux in the PGM2 strain. This finding shows particular importance in biofuel production from seaweed because galactose is one of the major monosaccharides in seaweeds such as K. alvarezii.

Comparison of Ethanol Yield Coefficients Using Saccharomyces cerevisiae, Candida lusitaniae, and Kluyveromyces marxianus Adapted to High Concentrations of Galactose with Gracilaria verrucosa as Substrate.

The red seaweed Gracilaria verrucosa has been used for the production of bioethanol. Pretreatment for monosaccharide production was carried out with 12% (w/v) G. verrucosa slurry and 500 mM HNO3 at 121°C for 90 min. Enzymatic hydrolysis was performed with a mixture of commercial enzymes (Cellic C-Tec 2 and Celluclast 1.5 L; 16 U/ml) at 50°C and 150 rpm for 48 h. G. verrucosa was composed of 66.9% carbohydrates. In this study, 61.0 g/L monosaccharides were obtained from 120.0 g dw/l G. verrucosa. The fermentation inhibitors such as hydroxymethylfurfural (HMF), levulinic acid, and formic acid were produced during pretreatment. Activated carbon was used to remove HMF. Wildtype and adaptively evolved Saccharomyces cerevisiae, Candida lusitaniae, and Kluyveromyces marxianus were used for fermentation to evaluate ethanol production.

Correction to: Butanol and butyric acid production from Saccharina japonica by Clostridium acetobutylicum and Clostridium tyrobutyricum with adaptive evolution.

Unfortunately, the author name was wrongly published as Pailin Sukwang.instead of Pailin Sukwong.

Lipid and unsaturated fatty acid productions from three microalgae using nitrate and light-emitting diodes with complementary LED wavelength in a two-phase culture system.

In this study, Pavlova lutheri, Chlorella vulgaris, and Porphyridium cruentum were cultured using modified F/2 media in a 1 L flask culture. Various nitrate concentrations were tested to determine an optimal nitrate concentration for algal growth. Subsequently, the effect of light emitted at a specific wavelength on biomass and lipid production by three microalgae was evaluated using various wavelengths of light-emitting diodes (LED). Biomass production by P. lutheri, C. vulgaris, and P. cruentum were the highest with blue, red, and green LED wavelength with 1.09 g dcw/L, 1.23 g dcw/L, and 1.28 g dcw/L on day 14, respectively. Biomass production was highest at the complementary LED wavelength to the color of microalgae. Lipid production by P. lutheri, C. vulgaris, and P. cruentum were the highest with yellow, green, and red LEDs' wavelength, respectively. Eicosapentaenoic acid production by P. lutheri, C. vulgaris, and P. cruentum was 10.35%, 10.14%, and 14.61%, and those of docosahexaenoic acid were 6.09%, 8.95%, and 11.29%, respectively.

Enhancement of bioethanol production from Gracilaria verrucosa by Saccharomyces cerevisiae through the overexpression of SNR84 and PGM2.

A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.

Optimization of light intensity and photoperiod for Isochrysis galbana culture to improve the biomass and lipid production using 14-L photobioreactors with mixed light emitting diodes (LEDs) wavelength under two-phase culture system.

The optimal light intensity and photoperiod required to produce high biomass and lipid contents in Isochrysis galbana cultured in a 14-L bioreactor with LED wavelengths was studied. The cell biomass production was monitored in the first phase comprising of mixed blue (465 nm) and red (640 nm) LED wavelengths, then green (520 nm) LED were used in the second phase for lipid production. The optimal light intensity was 400 µmol/m2/s giving a maximum cell biomass of 1.05 g dcw/L and total lipid content of 65.2% (w/w) cultured under 12:12 h L/D cycle. The optimal light intensity of 400 µmol/m2/s was applied at different L/D cycles, the maximum cell biomass (1.25 g dcw/L) and lipid content (71.1% w/w) were obtained at 18:6 h L/D cycle. Stearic acid was the main fatty acid ranging from 42.91 (500 µmol/m2/s) to 65.57% w/w (100 µmol/m2/s) and 53.84 (18:6 h) to 65.44% w/w (24:0 h).

Enhancement of galactose consumption rate in Saccharomyces cerevisiae CEN.PK2-1 by CRISPR Cas9 and adaptive evolution for fermentation of Kappaphycus alvarezii hydrolysate.

Ethanol ferrmentation of Kappaphycus alvarezii hydrolysates was performed using wild-type (WT) Saccharomyces cerevisiae CEN.PK2-1, hexokinase 2 deleted (Δhxk2) and adapted strain on high galactose concentrations. The WT and Δhxk2 strains produced 8.9 and 14.67 g/L of ethanol with yield coefficient (YEtOH) of 0.20 and 0.33 (g/g), respectively. However, neither the WT nor Δhxk2strain could utilize all of the galactose, leaving 16.4 and 6.2 g/L of galactose in the fermentation broth, respectively. Therefore, fermentation with S. cerevisiae CEN.PK2-1 adapted to galactose was carried out to increase the ethanol yield coefficient (YEtOH), producing a maximum ethanol concentration of 20.0 g/L with a YEtOH of 0.44 (g/g). Ethanol concentration of adapted strain was 1.36-2.25 times higher than WT and Δhxk2 strains. The adapted yeast exhibited the highest transcript levels of GAL genes. The yeast strain via adaptive yeast strain produced ethanol with a higher titer and yield due to a modular activation of GAL genes than WT or the hxk2 deleted strains.

Butanol and butyric acid production from Saccharina japonica by Clostridium acetobutylicum and Clostridium tyrobutyricum with adaptive evolution.

Optimal conditions of hyper thermal (HT) acid hydrolysis of the Saccharina japonica was determined to a seaweed slurry content of 12% (w/v) and 144 mM H2SO4 at 160 °C for 10 min. Enzymatic saccharification was carried out at 50 °C and 150 rpm for 48 h using the three enzymes at concentrations of 16 U/mL. Celluclast 1.5 L showed the lowest half-velocity constant (Km) of 0.168 g/L, indicating a higher affinity for S. japonica hydrolysate. Pretreatment yielded a maximum monosaccharide concentration of 36.2 g/L and 45.7% conversion from total fermentable monosaccharides of 79.2 g/L with 120 g dry weight/L S. japonica slurry. High cell densities of Clostridium acetobutylicum and Clostridium tyrobutyricum were obtained using the retarding agents KH2PO4 (50 mM) and NaHCO3 (200 mM). Adaptive evolution facilitated the efficient use of mixed monosaccharides. Therefore, adaptive evolution and retarding agents can enhance the overall butanol and butyric acid yields from S. japonica.

Detoxification of Hydrolysates of the Red Seaweed Gelidium amansii for Improved Bioethanol Production.

In this study, bioethanol was produced from the seaweed Gelidium amansii as biomass through separate hydrolysis and fermentation (SHF) processes. The SHF processes examined in this study include thermal acid hydrolysis pretreatment, enzymatic saccharification, detoxification, and fermentation. Thermal acid hydrolysis pretreatment was conducted using H2SO4, with a slurry content of 8-16% and treatment time of 15-75 min. The optimal conditions for thermal acid hydrolysis pretreatment were 12% (w/v) seaweed slurry content and 180 mM H2SO4 at 121 °C for 45 min, at which 26.1 g/L galactose and 6.8 g/L glucose were produced. A monosaccharide (mainly glucose) was also obtained from the enzymatic saccharification of thermal acid hydrolysate using 16 U/mL Celluclast 1.5 L enzyme at 45 °C for 36 h. Detoxification was performed using the adsorption method with activated carbon, the overliming method with Ca (OH)2, and the ion exchange method with polyethyleneimine. Among those detoxification methods, activated carbon showed the best performance for hydroxymethylfurfural removal. Ethanol fermentation was performed using 12% (w/v) seaweed hydrolysate with Saccharomyces cerevisiae adapted to galactose as well as various detoxification treatments.

Application of the Severity Factor and HMF Removal of Red Macroalgae Gracilaria verrucosa to Production of Bioethanol by Pichia stipitis and Kluyveromyces marxianus with Adaptive Evolution.

Gracilaria verrucosa, red seaweed, is a promising biomass for bioethanol production due to its high carbohydrate content. The optimal hyper thermal (HT) acid hydrolysis conditions are 12% (w/v) G. verrucosa with 0.2 M H2SO4 at 130 °C for 15 min, with a severity factor of 1.66. This HT acid hydrolysis produces 50.7 g/L monosaccharides. The maximum monosaccharide concentration of 58.0 g/L was achieved with 96.6% of the theoretical monosaccharide production from 120 g dry weight/L G. verrucosa slurry after HT acid hydrolysis and enzymatic saccharification. Fermentation was carried out by removing an inhibitory compound and via yeast adaptation to galactose. Both Pichia stipitis and Kluyveromyces marxianus adapted to galactose were excellent producers, with the ethanol yield (YEtOH) of 0.50 and 29.0 g/L ethanol production. However, the bioethanol productivity with Pichia stipitis adapted to galactose is higher than that with Kluyveromyces marxianus adapted to galactose, being 0.81 and 0.35 g/L/h, respectively. The results from this study can be applied to industrial scale bioethanol production from seaweed.

Acetone, butanol, and ethanol production from the green seaweed Enteromorpha intestinalis via the separate hydrolysis and fermentation.

Acetone, butanol, and ethanol (ABE) were produced following the separate hydrolysis and fermentation (SHF) method using polysaccharides from the green macroalgae Enteromorpha intestinalis as biomass. We focused on the optimization of enzymatic saccharification as pretreatments for the fermentation of E. intestinalis. Pretreatment was carried out with 10% (w/v) seaweed slurry and 270-mM H2SO4 at 121 °C for 60 min. Monosaccharides (mainly glucose) were obtained from enzymatic hydrolysis with a 16-U/mL mixture of Celluclast 1.5 L and Viscozyme L at 45 °C for 36 h. ABE fermentation with 10% (w/v) E. intestinalis hydrolysate was performed using the anaerobic bacteria Clostridium acetobutylicum with either uncontrolled pH, pH controlled at 6.0, or pH controlled initially at 6.0 and then 4.5 after 4 days, which produced ABE contents of 5.6 g/L with an ABE yield (YABE) of 0.24 g/g, 4.8 g/L with an YABE of 0.2 g/g, and 8.5 g/L with an YABE of 0.36 g/g, respectively.

Improved fermentation performance to produce bioethanol from Gelidium amansii using Pichia stipitis adapted to galactose.

This study employed a statistical method to obtain optimal hyper thermal acid hydrolysis conditions using Gelidium amansii (red seaweed) as a source of biomass. The optimal hyper thermal acid hydrolysis using G. amansii as biomass was determined as 12% (w/v) slurry content, 358.3 mM H2SO4, and temperature of 142.6 °C for 11 min. After hyper thermal acid hydrolysis, enzymatic saccharification was carried out. The total monosaccharide concentration was 45.1 g/L, 72.2% of the theoretical value of the total fermentable monosaccharides of 62.4 g/L based on 120 g dry weight/L in the G. amansii slurry. To increase ethanol production, 3.8 g/L 5-hydroxymethylfurfural (HMF) in the hydrolysate was removed by treatment with 3.5% (w/v) activated carbon for 2 min and fermented with Pichia stipitis adapted to high galactose concentrations via separate hydrolysis and fermentation. With complete HMF removal and the use of P. stipitis adapted to high galactose concentrations, 22 g/L ethanol was produced (yield 0.50). Fermentation with total HMF removal and yeast adapted to high galactose concentrations increased the fermentation performance and decreased the fermentation time from 96 to 36 h compared to traditional fermentation.

Acetone-Butanol-Ethanol Production from Waste Seaweed Collected from Gwangalli Beach, Busan, Korea, Based on pH-Controlled and Sequential Fermentation Using Two Strains.

The optimal conditions for acetone-butanol-ethanol (ABE) production were evaluated using waste seaweed from Gwangalli Beach, Busan, Korea. The waste seaweed had a fiber and carbohydrate, content of 48.34%; these are the main resources for ABE production. The optimal conditions for obtaining monosaccharides based on hyper thermal (HT) acid hydrolysis of waste seaweed were slurry contents of 8%, sulfuric acid concentration of 138 mM, and treatment time of 10 min. Enzymatic saccharification was performed using 16 unit/mL Viscozyme L, which showed the highest affinity (Km = 1.81 g/L). After pretreatment, 34.0 g/L monosaccharides were obtained. ABE fermentation was performed with single and sequential fermentation of Clostridium acetobutylicum and Clostridium tyrobutyricum; this was controlled for pH. A maximum ABE concentration of 12.5 g/L with YABE 0.37 was achieved using sequential fermentation with C. tyrobutyricum and C. acetobutylicum. Efficient ABE production from waste seaweed performed using pH-controlled culture broth and sequential cell culture.

Enhancement of Ethanol Production via Hyper Thermal Acid Hydrolysis and Co-Fermentation Using Waste Seaweed from Gwangalli Beach, Busan, Korea.

The waste seaweed from Gwangalli beach, Busan, Korea was utilized as biomass for ethanol production. Sagassum fulvellum (brown seaweed, Mojaban in Korean name) comprised 72% of the biomass. The optimal hyper thermal acid hydrolysis conditions were obtained as 8% slurry contents, 138 mM sulfuric acid, and 160°C of treatment temperature for 10 min with a low content of inhibitory compounds. To obtain more monosaccharides, enzymatic saccharification was carried out with Viscozyme L for 48 h. After pretreatment, 34 g/l of monosaccharides were obtained. Pichia stipitis and Pichia angophorae were selected as optimal co-fermentation yeasts to convert all of the monosaccharides in the hydrolysate to ethanol. Co-fermentation was carried out with various inoculum ratios of P. stipitis and P. angophorae. The maximum ethanol concentration of 16.0 g/l was produced using P. stipitis and P. angophorae in a 3:1 inoculum ratio, with an ethanol yield of 0.47 in 72 h. Ethanol fermentation using yeast co-culture may offer an efficient disposal method for waste seaweed while enhancing the utilization of monosaccharides and production of ethanol.