High Concentrations Of Galactose Research Articles

Preparation of low lactose prebiotic milk using β‐galactosidase from lotus seeds (Nelumbo nucifera)

SummaryA novel β‐galactosidase (NelnuGal) was isolated from Lotus (Nelumbo nucifera) seeds for the first time in this study. The enzyme was purified to homogeneity using hydrophobic interaction chromatography and ion exchange chromatography. Purified NelnuGal appeared as a monomer with a molecular mass of 50 kDa on SDS‐PAGE gel and zymogram. It was optimally active at pH 4.0 and stable in a broad pH range of 4–10. NelnuGal displayed temperature optimum of 50 °C and thermostability from 20 to 50 °C. It was resistant to inhibition by most of the metal ions tested, especially Ca+2 and Mg+2 making it useful in hydrolysis of milk. Remarkably, NelnuGal was not inhibited by high concentrations of galactose, which is end‐product of lactose hydrolysis. The purified NelnuGal displayed lactose hydrolysis and transgalactosylation properties. Addition of lotus seed powder to milk and whey resulted in lactose hydrolysis, forming galactose, glucose and galactooligosaccharides. Therefore, the addition of lotus seed powder to milk and whey can be applied for reducing the lactose present in dairy products which will also have potential prebiotic properties.

Ligand dose-dependent activation of signaling pathways through the gustatory receptor NlGr11 linked to feeding efficacy in Nilaparvata lugens.

Insects often face both conditions with sufficient nutrients and conditions of undernutrition in the field. Through gustatory receptors, insects sense nutrients and regulate their physiological functions such as feeding and reproduction. However, it remains unclear whether signaling pathways activated by gustatory receptors depend on the concentration of nutrients and whether the difference in signaling pathways directly affects insects' physiological functions. Herein, we found that a sugar gustatory receptor, NlGr11, from the brown planthopper (BPH), Nilaparvata lugens, activated Gprotein-coupled signaling and ionotropic pathways when bound to high galactose concentration. BPHs subsequently demonstrated longer feeding times, feeding loads, and higher vitellogenin (NlVg) expression than BPHs exposed to high galactose concentrations, which only activated the ionotropic pathway. For the first time, our findings link plant nutrient conditions, signaling pathways activated by nutrients, and their gustatory receptors, and nutrient dose-dependent feeding efficacy and vitellogenin (Vg) expression in an insect. This will help us to better understand the molecular mechanism for insect feeding strategies on plants at different stages of nutritional conditions.

Assessment of metal exposure (uranium and copper) in fatty acids and carbohydrate profiles of Calamoceras marsupus larvae (Trichoptera) and Alnus glutinosa leaf litter.

Physiological changes were explored in fatty acids (FA) and carbohydrate (CHO) composition in the shredder Calamoceras marsupus larvae (Trichoptera) and leaf litter (C. marsupus food) exposed to copper and uranium under natural and experimental conditions. We measured FA and CHO content in leaf litter and larvae specimens from reference and impacted streams, and exposed for 5 weeks to four realistic environmental concentrations of copper (35 μg L-1 and 70 μg L-1) and uranium (25 μg L-1 and 50 μg L-1). Regarding FA, (1) leaf litter had a reduced polyunsaturated FA (PUFA) content in metal treatments, s (14 to 33% of total FA), compared to natural conditions (≥39% of total FA). Leaf litter exposed to uranium also differed in saturated FA (SFA) composition, with lower values in natural conditions and higher values under low uranium concentrations. (2) C. marsupus had/showed low PUFA content under Cu and U exposure, particularly in high uranium concentrations. Detritivores also decreased in PUFA under exposure to both metals, particularly in high uranium concentrations. On the other hand, (1) microorganisms of the biofilm colonizing leaf litter differed in CHO composition between natural (impacted and reference) and experimental conditions, with glucose and galactose being consistently the most abundant sugars, found in different amounts under copper or uranium exposure; (2) CHO of detritivores showed similar high galactose and fucose concentrations in contaminated streams and high copper treatments, whereas low copper treatment showed distinct CHO profiles, with higher mannose, glucose, arabinose, and fucose concentrations. Our study provides evidence of metal exposure effects on FA and CHO contents at different trophic levels, which might alter the quality of food flow in trophic webs.

The Effect of Acute Oral Galactose Administration on the Redox System of the Rat Small Intestine.

Galactose is a ubiquitous monosaccharide with important yet incompletely understood nutritive and physiological roles. Chronic parenteral d-galactose administration is used for modeling aging-related pathophysiological processes in rodents due to its ability to induce oxidative stress (OS). Conversely, chronic oral d-galactose administration prevents and alleviates cognitive decline in a rat model of sporadic Alzheimer’s disease, indicating that galactose may exert beneficial health effects by acting in the gut. The present aim was to explore the acute time-response of intestinal redox homeostasis following oral administration of d-galactose. Male Wistar rats were euthanized at baseline (n = 6), 30 (n = 6), 60 (n = 6), and 120 (n = 6) minutes following orogastric administration of d-galactose (200 mg/kg). The overall reductive capacity, lipid peroxidation, the concentration of low-molecular-weight thiols (LMWT) and protein sulfhydryls (SH), the activity of Mn and Cu/Zn superoxide dismutases (SOD), reduced and oxidized fractions of nicotinamide adenine dinucleotide phosphates (NADPH/NADP), and the hydrogen peroxide dissociation rate were analyzed in duodenum and ileum. Acute oral administration of d-galactose increased the activity of SODs and decreased intestinal lipid peroxidation and nucleophilic substrates (LMWT, SH, NADPH), indicating activation of peroxidative damage defense pathways. The redox system of the small intestine can acutely tolerate even high luminal concentrations of galactose (0.55 M), and oral galactose treatment is associated with a reduction rather than the increment of the intestinal OS. The ability of oral d-galactose to modulate intestinal OS should be further explored in the context of intestinal barrier maintenance, and beneficial cognitive effects associated with long-term administration of low doses of d-galactose.

A Comparative Study of Maize and Miscanthus Regarding Cell-Wall Composition and Stem Anatomy for Conversion into Bioethanol and Polymer Composites

Due to an increasing demand for environmentally sustainable products, miscanthus and maize stover represent interesting lignocellulosic resources for conversion into biofuels and biomaterials. The overall purpose was to compare miscanthus and maize regarding cell-wall composition and stem anatomy for conversion into bioethanol and polymer composites using partial least squares regressions. For each of the two crops, six contrasted genotypes were cultivated in complete block design, and harvested. Internodes below the main cob for maize, and on the first aboveground internode for miscanthus, were analyzed for biochemistry and anatomy. Their digestibility was predicted using crop-specific near infrared calibrations, and the mechanical properties were evaluated in stem-based composites. On average, the internode cross-section of miscanthus anatomy was characterized by a thick rind (26.2%) and few but dense pith-bundles (3.5 nb/mm2), while cell-wall constituted 95.2% of the dry matter with high lignin (243.2 mg/g) and cellulose concentrations (439.7 mg/g). Maize internode-anatomy showed large cross-sections (397.5 mm2), pith with the presence of numerous bundles and non-lignified-pith fractions (22.3% of the section). Its cell-wall biochemistry displayed high concentrations of hemicelluloses, galactose, arabinose, xylose, and ferulic acid. Cell-wall, lignin, and cellulose concentrations were positively correlated with rind-fraction and pith-bundle-density, which explained strong mechanical properties as shown in miscanthus. Hemicelluloses, galactose, arabinose, and ferulic acid concentrations were positively correlated with pith fraction and stem cross-section, revealing high digestibility as shown in maize. This underlines interesting traits for further comparative genetic studies, as maize represents a good model for digestibility and miscanthus for composites.

Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic Sphingomonas strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll a quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll a and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic Sphingomonas bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation.IMPORTANCE Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium Sphingomonas sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in Sphingomonas may have other functions not linked to bioenergetics.

Mycoplasma genitalium Biofilms Contain Poly-GlcNAc and Contribute to Antibiotic Resistance.

Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluor-coupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial “towers” seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.

Lactose-Induced Chronic Diarrhea Results From Abnormal Luminal Microbial Fermentation and Disorder of Ion Transport in the Colon.

Diarrhea is one of the major abdominal symptoms in lactose-intolerant subjects. The changes in the large intestinal luminal environment and disorder of the epithelial ion transport in lactose-induced diarrhea remain unclear. The present study aimed to investigate the effect of an incremental high-lactose diet (IHLD, 30%/40%/50%) on luminal microbiota, microbiota-derived metabolite concentrations and colonic ion transport. Gut microbiota were analyzed by 16S rRNA amplicon sequencing and the concentration of SCFAs by gas chromatography, galactose, lactose and lactic acid through assay kit; Ussing chamber was performed to detect basal and stimulated ion transport; The expression and location of SCFA transporters, the Na-H exchanger 3(NHE3), cystic fibrosis transporter regulater (CFTR) and NKCC1 in the colon mucosa were analyzed by western and immunostaining. The concentrations of lactose, galactose and lactic acid of the cecal content were markedly increased (P < 0.01) and SCFA concentration was significantly decreased (P < 0.01). This was associated with depletion of the Lachnospiraceae NK4A136 group and Ruminococcaceae UCG-005 and increased relative abundance of Lactobacillus, escherichia-shigella and megamonas in the cecal microbiota. The expression of monocarboxylate transporter 1 was decreased in the colonic mucosa of the IHLD group. Low NHE3 expression and phosphorylation levels, and decreases in delta basal short circuit current after apical Na+ removal in the colonic mucosa of the IHLD group contributed to Na+ accumulation in the lumen and decrease stimulated Cl– secretion with low CFTR and NKCC1 expression would compensate for water and electrolyte loss during the diarrhea process. These results indicated that the persistence of the diarrhea state was maintained by abnormal colonic microbiota fermentation leading to high concentrations of lactose, galactose and lactic acid and low SCFAs in the lumen, and decreased Na+ absorption with the low NHE3 expression and phosphorylation levels.

Microwave-Assisted Degradation of Biomass with the Use of Acid Catalysis

The aim of the study was to assess the effectiveness of microwave pretreatment combined with acid catalysis in the decomposition of various types of biomass (pine and beech chips and hemp stems). It was clearly demonstrated that sulfuric acid was a catalyst enabling the most effective decomposition of the tested plant biomass, guaranteeing the highest concentrations of simple sugars released. Acid catalysis with 1% v/v sulfuric acid combined with microwave radiation provided high glucose concentrations of 89.8 ± 3.4, 170.4 ± 2.4 and 164.6 ± 4.6 mg/g for pine chips, beech chips and hemp stems, respectively. In turn, the use of nitric acid promoted the degradation of hemicellulose, which resulted in high concentrations of galactose and xylose, i.e., 147.6 ± 0.6, 163.6 ± 0.4 and 134.9 ± 0.8 mg/g of pine chips, beech chips and hemp stems, respectively, while glucose levels remained relatively low. It was also demonstrated that the undesirable dehydration of sugars such as glucose and xylose is more pronounced in sulfuric acid than nitric acid processes. The use of H2SO4 and increased pressure generated 5-hydroxymethylfurfural (5-HMF) and furfural at a concentration of ca. 12 and 6 mg/g, 10 and 45 mg/g and 14 and 30 mg/g, of pine chips, beech chips and hemp shoots, respectively. Our studies confirmed the usefulness of the combined use of microwaves and acid catalysis in the degradation of softwood, hardwood and non-wood plant biomass. It should be emphasized that obtaining high concentrations of released simple sugars (as potential substrates in biosynthesis), while maintaining low levels of toxic by-products (inhibitors), requires precise selection of process parameters such as pressure, exposition time and type of acid catalyst.

Improvement of bioethanol production by Saccharomyces cerevisiae through the deletion of GLK1, MIG1 and MIG2 and overexpression of PGM2 using the red seaweed Gracilaria verrucosa

Gracilaria verrucosa can be used as a biomass source for bioethanol production with a high yield due to the high carbohydrate content. A monosaccharide concentration of 48.6 g/L was obtained from G. verrucosa using 0.2 M HNO3 at 150 °C for 10 min with a combined severity factor (Ro) of 0.47. The addition of Tween 20 with CTec2 increased the affinity of G. verrucosa slurry to the enzyme with a Km value of 1.14 g/L compared with the Km value of 1.82 g/L using CTec2 alone and reduced enzyme loading. To improve galactose consumption, the deletion of repressor genes including GLK1, MIG1, and MIG2 and the overexpression of PGM2 were investigated. The strain with the deletion of three repressor genes and overexpression of PGM2 produced 24.8 g/L ethanol (YEtOH = 0.46) and showed an enhanced galactose consumption rate (0.24 g/L/h) compared with the rate of the control strain (0.04 g/L/h). The deletion of three repressor genes and overexpression of PGM2 reduced the repression of GAL genes and increased the transcription level of GAL genes. However, the galactose consumption rate of this strain was lower than that of the strain adapted to a high concentration of galactose.

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

A total monosaccharide concentration of 47.0g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyperthermal acid hydrolysis with 0.2M HCl at 140°C for 15min 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 16.5g/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 22.0g/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.

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.

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, 358.3mM H2SO4, and temperature of 142.6°C for 11min. After hyper thermal acid hydrolysis, enzymatic saccharification was carried out. The total monosaccharide concentration was 45.1g/L, 72.2% of the theoretical value of the total fermentable monosaccharides of 62.4g/L based on 120g dry weight/L in the G. amansii slurry. To increase ethanol production, 3.8g/L 5-hydroxymethylfurfural (HMF) in the hydrolysate was removed by treatment with 3.5% (w/v) activated carbon for 2min 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, 22g/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 36h compared to traditional fermentation.

Bioethanol Production from Soybean Residue via Separate Hydrolysis and Fermentation.

Bioethanol was produced using polysaccharide from soybean residue as biomass by separate hydrolysis and fermentation (SHF). This study focused on pretreatment, enzyme saccharification, and fermentation. Pretreatment to obtain monosaccharide was carried out with 20% (w/v) soybean residue slurry and 270mmol/L H2SO4 at 121°C for 60min. More monosaccharide was obtained from enzymatic hydrolysis with a 16U/mL mixture of commercial enzymes C-Tec 2 and Viscozyme L at 45°C for 48h. Ethanol fermentation with 20% (w/v) soybean residue hydrolysate was performed using wild-type and Saccharomyces cerevisiae KCCM 1129 adapted to high concentrations of galactose, using a flask and 5-L fermenter. When the wild type of S. cerevisiae was used, an ethanol production of 20.8g/L with an ethanol yield of 0.31g/g consumed glucose was obtained. Ethanol productions of 33.9 and 31.6g/L with ethanol yield of 0.49g/g consumed glucose and 0.47g/g consumed glucose were obtained in a flask and a 5-L fermenter, respectively, using S. cerevisiae adapted to a high concentration of galactose. Therefore, adapted S. cerevisiae to galactose could enhance the overall ethanol fermentation yields compared to the wild-type one.

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 373mM H2SO4 at 121°C for 59min. Glucose was obtained from enzymatic hydrolysis. Enzymatic saccharification was performed with a mixture of 16U/mL Celluclast 1.5L and Viscozyme L at 45°C for 48h. 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 0.09g/(Lh) with an ethanol yield of 0.25. Ethanol productivity of 0.16 and 0.19g/(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.

D-Galactose Causes Motor Coordination Impairment, and Histological and Biochemical Changes in the Cerebellum of Rats.

Classical galactosemia is an inborn error of carbohydrate metabolism in which patients accumulate high concentration of galactose in the brain. The most common treatment is a galactose-restricted diet. However, even treated patients develop several complications. One of the most common symptoms is motor coordination impairment, including affected gait, balance, and speech, as well as tremor and ataxia. In the present study, we investigated the effects of intracerebroventricular galactose administration on motor coordination, as well as on histological and biochemical parameters in cerebellum of adult rats. Wistar rats received 5μL of galactose (4mM) or saline by intracerebroventricular injection. The animals performed the beam walking test at 1 and 24h after galactose administration. Histological and biochemical parameters were performed 24h after the injections. The results showed motor coordination impairment at 24h after galactose injection. Galactose also decreased the number of cells in the molecular and granular layers of the cerebellum. The immunohistochemistry results suggest that the cell types lost by galactose are neurons and astrocytes in the spinocerebellum and neurons in the cerebrocerebellum. Galactose increased active caspase-3 immunocontent and acetylcholinesterase activity, decreased acetylcholinesterase immunocontent, glutathione, and BDNF levels, as well as caused protein and DNA damage. Our results suggest that galactose induces histological and biochemical changes in cerebellum, which can be associated with motor coordination impairment.

Evaluation of Galactose Adapted Yeasts for Bioethanol Fermentation from Kappaphycus alvarezii Hydrolyzates.

Bioethanol was produced from Kappaphycus alvarezii seaweed biomass using separate hydrolysis and fermentation (SHF). Pretreatment was evaluated for 60 min at 121°C using 12% (w/v) biomass slurry with 364 mM H2SO4. Enzymatic saccharification was then carried out at 45°C for 48 h using Celluclast 1.5 L. Ethanol fermentation with 12% (w/v) K. alvarezii hydrolyzate was performed using the yeasts Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 with or without prior adaptation to high concentrations of galactose. When non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 11.5 g/l, 6.7 g/l, and 6.0 g/l of ethanol were produced, respectively. When adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 15.8 g/l, 11.6 g/l, and 13.4 g/l of ethanol were obtained, respectively. The highest ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and YT% = 84.3%, which was obtained using adapted S. cerevisiae.

Evaluation of hyper thermal acid hydrolysis of Kappaphycus alvarezii for enhanced bioethanol production

Hyper thermal (HT) acid hydrolysis of Kappaphycus alvarezii, a red seaweed, was optimized to 12% (w/v) seaweed slurry content, 180mM H2SO4 at 140°C for 5min. The maximum monosaccharide concentration of 38.3g/L and 66.7% conversion from total fermentable monosaccharides of 57.6g/L with 120gdw/L K. alvarezii slurry were obtained from HT acid hydrolysis and enzymatic saccharification. HT acid hydrolysis at a severity factor of 0.78 efficiently converted the carbohydrates of seaweed to monosaccharides and produced a low concentration of inhibitory compounds. The levels of ethanol production by separate hydrolysis and fermentation with non-adapted and adapted Kluyveromyces marxianus to high concentration of galactose were 6.1g/L with ethanol yield (YEtOH) of 0.19 at 84h and 16.0g/L with YEtOH of 0.42 at 72h, respectively. Development of the HT acid hydrolysis process and adapted yeast could enhance the overall ethanol fermentation yields of K. alvarezii seaweed.

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

A total monosaccharide concentration of 39.6g/L, representing 74.0% conversion of 53.5g/L total carbohydrate from 80g 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 18.3g/L with Y EtOH of 0.46 and 13.4g/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.

Thermal Acid Hydrolysis Pretreatment, Enzymatic Saccharification and Ethanol Fermentation from Red Seaweed, Gracilaria verrucosa

In this study, the red seaweed Gracilaria verrucosa was used as a bioethanol producing biomass. G. verrocosa has a high content of easily degradable carbohydrates, making it a potential substrate for the production of liquid fuels [8]. The carbohydrates in G. verrucosa can be categorised according to their chemical structures: alginate, carrageenan, and agar. Carrageenan and agar, which are plentiful in the seaweed, can be used as a source of galactose and glucose. Various pretreatment techniques have been introduced to enhance the overall hydrolysis yield, and can be categorized into physical, chemical, biological, enzymatic or a combination of these [1]. Dilute acid hydrolysis is commonly used to prepare seaweed hydrolysates for enzymatic saccharification and fermentation for economic reasons [13]. However, thermal acid hydrolysis pretreatment for 3,6anhydrogalactose from G. verrucosa have produced 5hydroxymethylfurfural (HMF), an inhibitory compound for ethanol production [8]. One of the problems encountered in G. verrucosa fermentation has been high concentrations of NaCl due to its origin from sea water [3]. High-salt stress to yeasts is a significant impediment on the production of ethanol from seaweed hydrolysates. Salt stress in yeasts results in two phenomena: ion toxicity and osmotic stress [11]. Defense responses to salt stress are based on osmotic adjustments by osmolyte synthesis and cation transport systems for sodium exclusion [20]. The preferential utilization of glucose over non-glucose sugars by yeast often results in low overall ethanol production and yield. When yeast grows on a mixture of glucose and galactose, the glucose is metabolized first, whereas The seaweed, Gracilaria verrucosa, was fermented to produce bioethanol. Optimal pretreatment conditions were determined to be 12% (w/v) seaweed slurry and 270 mM sulfuric acid at 121C for 60 min. After thermal acid hydrolysis, enzymatic saccharification was carried out with 16 U/ml of mixed enzymes using Viscozyme L and Celluclast 1.5 L to G. verrucosa hydrolysates. A total monosaccharide concentration of 50.4 g/l, representing 84.2% conversion of 60 g/l total carbohydrate from 120 g dw/l G. verrucosa slurry was obtained by thermal acid hydrolysis and enzymatic saccharification. G. verrucosa hydrolysate was used as the substrate for ethanol production by separate hydrolysis and fermentation (SHF). Ethanol production by Candida lusitaniae ATCC 42720 acclimated to high-galactose concentrations was 22.0 g/l with ethanol yield (YEtOH) of 0.43. Acclimated yeast to high concentrations of specific sugar could utilize mixed sugars, resulting in higher ethanol yields in the seaweed hydrolysates medium.