Enhancement of galactose uptake for bioethanol production from Eucheuma denticulatum hydrolysate using galactose-adapted yeasts.

Eucheuma denticulatum is a red macroalgae with a high carbohydrate content. The fermentable sugars from E. denticulatum were obtained through sequential thermal acid hydrolysis, enzymatic saccharification, and detoxification. Thermal acid hydrolysis of E. denticulatum was optimized under the condition of 10% (w/v) slurry content and 300 mM HNO3 at 121 â„ƒ for 90 min. The maximum monosaccharide concentration after thermal acid hydrolysis was 31.0 g/L with an efficiency (ETAH) of 44.7%. By further enzymatic hydrolysis of pretreated biomass solution under 20 U/mL Cellic CTec2 at 50 â„ƒ and 160 rpm for 72 h, the maximum monosaccharide concentration reached 79.9 g/L with an efficiency of 66.2% (ES). To remove 5-hydroxymethylfurfural (5-HMF), a fermentation inhibitor, absorption using 2% activated carbon was performed for 2 min. Ethanol fermentation was performed using wild-type and high galactose-adapted strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, and Candida lusitaniae. As a result, galactose-adapted strains showed higher ethanol production than wild-type strains. Especially, the fermentation result by adaptively evolved S. cerevisiae produced the highest ethanol of 37.6 g/L and with YEtOH of 0.48 g/g. Moreover, the transcript level of MIG1 in the galactose-adapted strain was slightly lower than that in the wild-type strain. The application of adaptive evolution of microorganisms was efficient for bioethanol production.

Valorization of microalgae into 5-hydroxymethylfurfural by two-step conversion with ferric sulfate.

Microalgae are known as renewable, potential, and sustainable feedstocks for biofuel production. The present work investigated the efficient valorization of green microalgae Chlorella sp. to produce sugars and 5-hydroxymethylfurfural (5-HMF) using thermochemical conversion with a metal-salt (ferric sulfate) as catalyst using a statistical approach and two-step conversion. A statistical approach with a Box-Behnken design was introduced to optimize the conversion for producing sugars. As a result of optimization, 86.46% sugar yield (68.32% glucose yield) was achieved under the condition of 5% biomass and 0.6 g-catalyst/g-biomass at 155 °C and 40 min. Two-step thermochemical conversion was introduced to produce 5-HMF from microalgae. In the first step, sugars were produced from the above optimum condition; in the second step, sugar hydrolysates were converted into 5-HMF by thermochemical conversion without an additional catalyst. In two-step conversion, the maximum 5-HMF yield (37.23%) was achieved at 170 °C and 60 min from the sugar hydrolysate of microalgae obtained from the first-step thermochemical conversion with ferric sulfate. In conclusion, the microalgae as biomass and ferric sulfate as catalyst have availability and the potential to produce biosugars and platform chemicals.

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.

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.

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.

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.

Effects of light-emitting diode (LED) with a mixture of wavelengths on the growth and lipid content of microalgae.

Integrations of two-phase culture for cell growth and lipid accumulation using mixed LED and green LED wavelengths were evaluated with the microalgae, Phaeodactylum tricornutum, Isochrysis galbana, Nannochloropsis salina, and Nannochloropsis oceanica. Among the single and mixed LED wavelengths, mixed LED produced higher biomass of the four microalgae, reaching 1.03 g DCW/L I. galbana, followed by 0.95 g DCW/L P. tricornutum, 0.85 g DCW/L N. salina, and 0.62 g DCW/L N. oceanica than single LED or fluorescent lights at day 10. Binary combination of blue and red LEDs could produce the high biomass and photosynthetic pigments in the four microalgae. The highest lipid accumulation during second phase with the exposure to green LED wavelengths was 56.0% for P. tricornutum, 55.2% for I. galbana, 53.0% for N. salina, and 51.0% for N. oceanica. The major fatty acid in the four microalgae was palmitic acid (C16:0) accounting for 38.3-47.3% (w/w) of the total fatty acid content.

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.

Industrial robustness linked to the gluconolactonase from Zymomonas mobilis.

The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l-1) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l-1) together with sorbitol (65 g l-1) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l-1, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.

Hyper-thermal acid hydrolysis and adsorption treatment of red seaweed, Gelidium amansii for butyric acid production with pH control.

Optimal hyper-thermal (HT) acid hydrolysis conditions for Gelidium amansii were determined to be 12% (w/v) seaweed slurry content and 144 mM H2SO4 at 150 °C for 10 min. HT acid hydrolysis-treated G. amansii hydrolysates produced low concentrations of inhibitory compounds and adsorption treatment using 3% activated carbon. An adsorption time of 5 min was subsequently used to remove the inhibitory 5-hydroxymethylfurfural from the medium. A final maximum monosaccharide concentration of 44.6 g/L and 79.1% conversion from 56.4 g/L total fermentable monosaccharides with 120 g dw/L G. amansii slurry was obtained from HT acid hydrolysis, enzymatic saccharification, and adsorption treatment. This study demonstrates the potential for butyric acid production from G. amansii hydrolysates under non-pH-controlled as well as pH-controlled fermentation using Clostridium acetobutylicum KCTC 1790. The activated carbon treatment and pH-controlled fermentation showed synergistic effects and produced butyric acid at a concentration of 11.2 g/L after 9 days of fermentation.

Bioethanol production from Gracilaria verrucosa using Saccharomyces cerevisiae adapted to NaCl or galactose.

This study examined the pretreatment, enzymatic saccharification, and fermentation of the red macroalgae Gracilaria verrucosa using adapted saccharomyces cerevisiae to galactose or NaCl for the increase of bioethanol yield. Pretreatment with thermal acid hydrolysis to obtain galactose was carried out with 11.7% (w/v) seaweed slurry and 373 mM H2SO4 at 121 °C for 59 min. Glucose was obtained from enzymatic hydrolysis. Enzymatic saccharification was performed with a mixture of 16 U/mL Celluclast 1.5L and Viscozyme L at 45 °C for 48 h. Ethanol fermentation in 11.7% (w/v) seaweed hydrolysate was carried out using Saccharomyces cerevisiae KCTC 1126 adapted or non-adapted to high concentrations of galactose or NaCl. When non-adapted S. cerevisiae KCTC 1126 was used, the ethanol productivity was 0.09 g/(Lh) with an ethanol yield of 0.25. Ethanol productivity of 0.16 and 0.19 g/(Lh) with ethanol yields of 0.43 and 0.48 was obtained using S. cerevisiae KCTC 1126 adapted to high concentrations of galactose and NaCl, respectively. Adaptation of S. cerevisiae KCTC 1126 to galactose or NaCl increased the ethanol yield via adaptive evolution of the yeast.

Efficient utilization of Eucheuma denticulatum hydrolysates using an activated carbon adsorption process for ethanol production in a 5-L fermentor.

A total monosaccharide concentration of 37.8 g/L and 85.9% conversion from total fermentable monosaccharides of 44.0 g/L from 110 g dw/L Eucheuma denticulatum slurry were obtained by thermal acid hydrolysis and enzymatic saccharification. Subsequent adsorption treatment to remove 5-hydroxymethylfurfural (5-HMF) using 5% activated carbon and an adsorption time of 10 min were used to prevent a prolonged lag phase, reduced cell growth, and low ethanol production. The equilibrium adsorption capacity (q e) of HMF (58.183 mg/g) showed high affinity to activated carbon comparing to those of galactose (2.466 mg/g) and glucose (2.474 mg/g). The efficiency of cell growth and ethanol production with activated carbon treatment was higher than that without activated carbon treatment. Fermentation using S. stipitis KCTC7228 produced a cell concentration of 3.58 g dw/L with Y X/S of 0.107, and an ethanol concentration of 15.8 g/L with Y P/S of 0.48 in 96 h.

Potential of phosphoric acid-catalyzed pretreatment and subsequent enzymatic hydrolysis for biosugar production from Gracilaria verrucosa.

This study combined phosphoric acid-catalyzed pretreatment and enzymatic hydrolysis to produce biosugars from Gracilaria verrucosa as a potential renewable resource for bioenergy applications. We optimized phosphoric acid-catalyzed pretreatment conditions to 1:10 solid-to-liquid ratio, 1.5 % phosphoric acid, 140 °C, and 60 min reaction time, producing a 32.52 ± 0.06 % total reducing sugar (TRS) yield. By subsequent enzymatic hydrolysis, a 68.61 ± 0.90 % TRS yield was achieved. These results demonstrate the potential of phosphoric acid to produce biosugars for biofuel and biochemical production applications.

Evaluation of ethanol production and bioadsorption of heavy metals by various red seaweeds.

The objective of this study was to evaluate ethanol production and bioadsorption with four red seaweeds, Gelidium amansii, Gracilaria verrucosa, Kappaphycus alvarezii and Eucheuma denticulatum. To produce ethanol, thermal acid hydrolysis, enzymatic saccharification and fermentation was carried out. After pretreatment, 38.5, 39.9, 31.0 and 27.5 g/L of monosaccharides were obtained from G. amansii, G. verrucosa, K. alvarezii and E. denticulatum, respectively. Ethanol fermentation was performed with Saccharomyces cerevisiae KCCM 1129 adapted to 80 g/L galactose. The ethanol productions by G. amansii, G. verrucosa, K. alvarezii and E. denticulatum were 18.8 g/L with Y EtOH = 0.49, 19.1 g/L with Y EtOH = 0.48, 14.5 g/L with Y EtOH = 0.47 and 13.0 g/L with Y EtOH = 0.47, respectively. The waste seaweed slurries after the ethanol fermentation were reused to adsorb Cd(II), Pb(II) and Cu(II). Using langmuir isotherm model, Cu(II) had the highest affinity for waste seaweeds with the highest q max and electronegativity values among three heavy metals.

Effects of galactose adaptation in yeast for ethanol fermentation from red seaweed, Gracilaria verrucosa.

A total monosaccharide concentration of 39.6 g/L, representing 74.0 % conversion of 53.5 g/L total carbohydrate from 80 g dw/L (8 % w/v) Gracilaria verrucosa slurry, was obtained by thermal acid hydrolysis and enzymatic saccharification. G. verrucosa hydrolysate was used as a substrate for ethanol production by 'separate hydrolysis and fermentation' (SHF). The ethanol production and yield (Y EtOH) from Saccharomyces cerevisiae KCCM 1129 with and without adaptation to high galactose concentrations were 18.3 g/L with Y EtOH of 0.46 and 13.4 g/L with Y EtOH of 0.34, respectively. Relationship between galactose adaptation effects and mRNA transcriptional levels were evaluated with GAL gene family, regulator genes of the GAL genetic switch and repressor genes in non-adapted and adapted S. cerevisiae. The development of galactose adaptation for ethanol fermentation of G. verrucosa hydrolysates allowed us to enhance the overall ethanol yields and obtain a comprehensive understanding of the gene expression levels and metabolic pathways involved.

Enhanced ethanol production by fermentation of Gelidium amansii hydrolysate using a detoxification process and yeasts acclimated to high-salt concentration.

A total monosaccharide concentration of 59.0 g/L, representing 80.1 % conversion of 73.6 g/L total fermentable sugars from 160 g dw/L G. amansii slurry was obtained by thermal acid hydrolysis and enzymatic hydrolysis. Subsequent adsorption treatment using 5 % activated carbon with an adsorption time of 2 min was used to prevent the inhibitory effect of 5-hydroxymethylfurfural (HMF) >5 g/L in the medium. Ethanol production decreased with increasing salt concentration using C. tropicalis KCTC 7212 non-acclimated or acclimated to a high concentration of salt. Salt concentration of 90 psu was the maximum concentration for cell growth and ethanol production. The levels of ethanol production by C. tropicalis non-acclimated or acclimated to 90 psu high-salt concentration were 13.8 g/L with a yield (YEtOH) of 0.23, and 26.7 g/L with YEtOH of 0.45, respectively.

Characterization of salt-tolerant ß-glucosidase with increased thermostability under high salinity conditions from Bacillus sp. SJ-10 isolated from jeotgal, a traditional Korean fermented seafood.

The ß-glucosidase gene, bglC, was cloned from Bacillus sp. SJ-10 isolated from the squid jeotgal. Recombinant BglC protein overexpression was induced in Escherichia coli. The optimal pH and temperature of the enzyme, using p-nitrophenyl-ß-D-glucopyranoside (pNPßGlc) as a substrate, were pH 6 and 40 °C, respectively. Enzymatic activity increased by 3.3- and 3.5-fold in the presence of 15% NaCl and KCl, respectively. Furthermore, enzyme thermostability improved in the presence of NaCl or KCl. At 45 °C in the presence of salts, the enzyme was stable for 2 h and maintained 80% activity. In the absence of salts, BglC completely lost activity after 110 min at 45 °C. Comparison of the kinetic parameters at various salt concentrations revealed that BglC had approximately 1.5- and 1.2-fold higher affinity and hydrolyzed pNPßGlc 1.9- and 2.1-fold faster in the presence of 15% NaCl and KCl, respectively. Additionally, the Gibb's free energy for denaturation was higher in the presence of 15% salt than in the absence of salt at 45 and 50 °C. Since enzymatic activity and thermostability were enhanced under high salinity conditions, BglC is an ideal salt-tolerant enzyme for further research and industrial applications.

Conversion of red-algae Gracilaria verrucosa to sugars, levulinic acid and 5-hydroxymethylfurfural.

This study employed a statistical methodology to investigate the optimization of conversion conditions and evaluate the reciprocal interaction of reaction factors related to the process of red-algae Gracilaria verrucosa conversion to sugars (glucose, galactose), levulinic acid and 5-hydroxymethylfurfural (5-HMF) by acidic hydrolysis. Overall, the conditions optimized for glucose formation included a higher catalyst concentration than did those for galactose, and these conditions for galactose were similar to those for 5-HMF. Levulinic acid production, meanwhile, was optimized at a higher reaction temperature, a higher catalyst concentration, and a longer reaction time than was glucose, galactose or 5-HMF production. By this approach, the optimal yields (and reaction conditions) for glucose, galactose, levulinic acid, and 5-HMF were as follows: glucose 5.29 g/L (8.46 wt%) (reaction temperature 160 °C, catalyst concentration 1.92%, reaction time 20 min), galactose 18.38 g/L (29.4 wt%) (160 °C, 1.03%, 20 min), levulinic acid 14.65 g/L (18.64 wt%) (180.9 °C, 2.85%, 50 min), and 5-HMF 3.74 g/L (5.98 wt%) (160.5 °C, 1%, 20 min).

The high reutilization value potential of high-salinity anchovy fishmeal wastewater through microbial degradation.

To provide an option for the reutilization of high-salinity anchovy fishmeal wastewater (FMW), generated during the anchovy fishmeal manufacturing processes, its potential for biodegradation was assessed in 1-l five-neck flasks using a halotolerant and proteolytic microbial consortium. During the first 41 h of biodegradation, the pH, DO, ORP, and dry-sludge weight decreased as the total cell number of the microbial consortium increased steadily; the COD(Cr)/TN ratios remained between 4.0 and 5.5, respectively, indicating the stable metabolic degradation of organic matter. The ORP tended to increase after 41 h, and the unpleasant fishy smell disappeared once positive ORP values were achieved. The removal percentages of COD(Cr) and TN were 59.0 and 54.4%, respectively, and the dry-sludge weight decreased from 115.5 to 68.0 g, with a degradation rate of 0.59 g h(-1), during the 80 h experiment. The supernatant from the culture of the anchovy FMW at 70 h (culture supernatant) was phytotoxin-free, and the level of total amino acids was 8.04 g 100 g(-1), comparable to that of commercial fertilizers. In hydroponic cultures containing red bean and barley, the culture supernatant demonstrated a good fertilizing ability. The culture supernatant also exhibited a high degree of antioxidant activity, with a 52.3% hydroxyl radical-scavenging activity and 0.16 reducing power (at OD 700 nm). Moreover, the culture supernatant inhibited DNA damage from hydroxyl radicals, enhancing the reutilization value of anchovy FMW. This report presents the first description of high-salinity anchovy FMW possessing a high reutilization value potential both for agriculture and medicine.