Optimization of aqueous-assisted extraction of polysaccharides from pumpkin (Cucurbita moschata Duch) and their biological activities

The pumpkin pulp contains a greater composition of edible polysaccharides and has reported with excellent biological applications. This research pertains to optimize the extraction of polysaccharides from the fleshy portion of the pumpkin using aqueous assisted extraction (AAE). The result showed that the optimal extraction condition of pumpkin polysaccharide was as follows: extraction temperature at 55 °C, pH 4.5, and enzyme concentration of 4000 µ/g for 80 min. Under the optimal extraction condition, the yield of pumpkin polysaccharide via AAE (15.4) was significantly higher. The biological activities of extracted polysaccharide including α-amylase inhibition (57.41% at 1000 µg/mL) and anti-inflammatory (50.41% at 25 µg/mL) activity increased significantly. Additionally, the antioxidant activities of extracted pumpkin polysaccharides including IC₅₀ values of DPPH and ABTS were 59.87% and 58.74%, respectively. The pumpkin polysaccharide has maximum inhibitory effects against bacterial strains especially for Escherichia coli than that of fungal strains. It is suggested that the aqueous assisted extraction of is a cost-effective promising method to decrease the processing time as well as enhancing extracted polysaccharide yield – times.     

He-Ne laser irradiation of folate-producing Candida utilis and optimization of culture conditions under submerged fermentation

In an attempt to obtain a microbial strain with higher yield of folate for industrial applications, we mutated the wild strain Candida utilis Y1.0 using a novel mutagenic process, i.e., irradiation by a helium–neon (He-Ne) laser with an output power of 20 mW and an exposure time of 20 min. The yield of folate in the mutated cells reached 1,102 ng/mL, which was 20.4-fold that of the wild strain. The mutant strain Y3.636 was relatively stable in terms of folate production through eight successive transfers of cultures and batch fermentation in a 3.7-L stirred-tank fermenter. Optimization further increased the yield of the mutant by 110 %, i.e., to 2,314?±?13 ng/mL. The optimal culture conditions for folate production were: cultivation in fermentation culture medium composed of 62.5 g/L glucose, 15 g/L corn liquor, 3 g/L (NH₄)₂SO₄, 3 g/L MgSO₄, and 1 g/L glutamic acid; inoculum size of 9 %; incubation at 28 °C and 196 rpm for 36 h. A time-course study of cell growth and folate production by mutant strain Y3.636 strongly suggested that folate production in C. utilis is growth-associated.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Optimization of Microbial Flocculant-Producing Medium for <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

This study aimed to improve microbial flocculant production by optimizing the components of a Bacillus subtilis CZ1003 culture medium. Using the flocculation rate as the dependent variable, single-factor experiments were performed and beef extract at a concentration of 9 g/L was found to be the optimal nitrogen source, while glucose at a concentration of 20 g/L was the optimal carbon source. KCl, MgCl₂, NaCl, and CaCl₂ at concentrations of 0.75, 2.5, 0.5, and 5.0 g/L, respectively, were the optimum inorganic salts, in order of flocculant production activity. Orthogonal experimental demonstrated that KCl played a dominant role for Bacillus subtilis production of bioflocculants, followed by NaCl and CaCl₂. Optimization experiments demonstrated that the optimal combination of the two salts was 0.75 g/L KCl and 0.5 g/L NaCl, resulting in a flocculation rate of 36.2% when included together at these concentrations. The final optimized medium consisted of 20 g/L glucose, 9 g/L beef extract, 0.75 g/L KCl, and 0.5 g/L NaCl. Compared with the initial medium, the optimized medium enhanced the flocculation activity from 12.1 to 36.2%, which equates to an increase of 199.17%. Meanwhile, the flocculant yield was increased from 0.058 g/L to 0.134/L, an increase of 131.03%. The optimized medium could be used to improve microbial flocculant production and provides a basis for further exploration.     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

<Emphasis Type="Italic">Tequilana weber</Emphasis> Agave Bagasse Enzymatic Hydrolysis for the Production of Fermentable Sugars: Oxidative-Alkaline Pretreatment and Kinetic Modeling

As a way to mitigate the harmful effects of fossil fuel utilization, the use of second-generation ethanol has been proposed. However, the microorganisms responsible for its production are not able to degrade structural polysaccharides, so their hydrolysis is necessary. Previously to this work, a factorial experimental design was carried out to investigate the relation between the NaOH and H₂O₂ concentrations with the yield of carbohydrates, and then this variable was optimized by using a response surface method. A study of the hydrolysis process was performed using enzymes to establish a process that maximizes the depolymerization of Agave tequilana fibers after an alkali-oxidative pretreatment with optimal reagents concentrations, this pretreatment was selected because it can remove almost the total content of lignin and destroys efficiently the crystallinity of cellulose fibers with a lower sugar losses and no production of toxic compounds. An orthogonal array using the novel enzymes Cellic CTec 3 and Cellic HTec 3 was performed to determinate the optimal combination of them, which has resulted in a concentration of 165.67 g/L at the supernatant with 82.21 % conversion and a yield of 352.18 g reducing sugars per kilogram of lignocellulosic material in dry basis. These results are 29 % better in comparison with the previous generation of enzymes with a reduction in the enzymatic charge of 82 %.     

Co-fermentation of cassava waste pulp hydrolysate with molasses to ethanol for economic optimization

Cassava waste pulp (CWP)–enzymatic hydrolysate was co-fermented with molasses (CWP-EH/molasses mixture) with the aim to optimize ethanol production by Saccharomyces cerevisiae TISTR 5606 (SC 90). The optimal fermentation conditions for ethanol production using this mixture were 245 g/L initial total sugar supplemented with KH₂PO₄ (8 g/L), at 30 °C for 48 h of fermentation under an oxygen-limited condition with agitation at 100 rpm, producing an ethanol concentration of 70.60 g/L (0.31 g ethanol/g total sugar). The addition of cassava tuber fiber (solid residue of CWP after enzymatic hydrolysis) at 30 g/L dry weight to the CWP-EH/molasses mixture increased ethanol production to 74.36 g/L (0.32 g ethanol/g total sugar). Co-fermentation of CWP-EH with molasses had the advantage of not requiring any supplementation of the fermentation mixture with reduced nitrogen.     

香菇液体发酵高产多糖菌株筛选

通过评价香菇野生菌株发酵产多糖性能,筛选高产香菇多糖菌株.以采自长白山野生香菇通过组织分离获得的6株菌株和2株人工栽培菌株为出发菌株,对不同发酵培养时间菌丝体生物量、胞内多糖含量、胞外多糖含量等进行测试分析,结果表明,8株菌株随着培养时间的延长,菌丝体生物量均有不同程度的增加,但胞内多糖含量和胞外多糖得率变化趋势不同,...     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

银耳子实体多糖高温高压提取工艺研究

通过对银耳子实体多糖高温高压提取工艺中,提取温度、料液比、提取时间,提取次数等影响因素的实验分析,确定银耳子实体多糖高温高压提取的最佳条件为:提取温度110℃(对应压力0.04 MPa);料液比为1∶70;提取时间为2 h;提取次数为2次,在此条件下,银耳子实体多糖提取率可达36.5%。     

Impact of pH and butyric acid on butanol production during batch fermentation using a new local isolate of Clostridium acetobutylicum YM1

The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (Y_P/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1.     

Fermentation characteristics and hypoglycemic activity of an exopolysaccharide produced by submerged culture of Stropharia rugosoannulata #2

We have investigated the mycelial growth and extracellular polysaccharide (EPS) production of Stropharia rugosoannulata #2 under optimal culture conditions in a 5-L stirred-tank fermenter. Maximum biomass growth (16.35 g/L) was achieved after 5 days of cultivation, whereas EPS reached its maximum level (10.83 g/L) after 8 days. The morphological parameters (i.e., mean diameter, circularity, roughness, compactness) of the fungal pellets and broth viscosity were also characterized. The compactness of the pellets was determined to be significantly and positively correlated with EPS content. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, included decreases in the plasma concentrations of glucose (37 %), total cholesterol (26 %), and triacylglycerol (24 %) and decreased aspartate aminotransferase activity (20 %). The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.     

Development of thin-film photo-bioreactor and its application to outdoor culture of microalgae

Photosynthetic microalgae have received much attention as a microbial source of diverse useful biomaterials through CO₂ fixation and various types of photo-bioreactors have been developed for efficient microalgal cultivation. Herein, we developed a novel thin-film photo-bioreactor, which was made of cast polypropylene film, considering outdoor mass cultivation. To develop optimal design of photo-bioreactor, we tested performance of three shapes of thin-film photo-bioreactors (flat, horizontal and vertical tubular shapes) and various parts in the bioreactor. Collectively, vertical tubular bioreactor with H/D ratio 6:1 and cylindrical stainless steel spargers showed the most outstanding performance. Furthermore, the photo-bioreactor was successfully applied to the cultivation of other microalgae such as Chlamydomonas reinhardtii and Chlorella vulgaris. The scalability of photo-bioreactor was confirmed by gradually increasing culture volume from 4 to 25 L and the biomass productivity of each reactor was quite consistent (0.05–0.07 g/L/day) during the cultivation of H. pluvialis under indoor and outdoor conditions. Especially, we also achieved dry cell weight of 4.64 g/L and astaxanthin yield of 218.16 mg/L through long-term cultivation (100 days) under outdoor condition in 15 L photo-bioreactor using Haematococcus pluvialis, which means that the astaxanthin yield from outdoor cultivation is equal or superior to that obtained from controlled indoor condition. Therefore, these results indicate that we can apply this approach to development of optimal photo-bioreactor for the large-scale culture of microalgae and production of useful biomaterials under outdoor condition.     

Hydrogen production by immobilized Chlorella vulgaris: optimizing pH, carbon source and light

The effects of light, pH and organic carbon sources were investigated on hydrogen production by algae. An optical fiber was examined as an internal light source. The optical fiber rendered prolonged lag time and total time of hydrogen production. The optimal pH to produce hydrogen for Chlorella sp. was 8.0. Glucose, fructose, sucrose and malt extract were compared as organic carbon sources. The optimal dose of each carbon source was 5 g/L for maximum hydrogen yield. Sucrose produced the largest hydrogen volume (1,315 ml/L), while the highest production rate (24 ml/L/h) was observed in the presence of fructose.     

Difference analysis of the enzymatic hydrolysis performance of acid-catalyzed steam-exploded corn stover before and after washing with water

The difference in the enzymatic hydrolysis yield of acid-catalyzed steam-exploded corn stover (ASC) before and after washing with water reached approximately 15 % under the same conditions. The reasons for the difference in the yield between ASC and washed ASC (wASC) were determined through the analysis of the composition of ASC prehydrolyzate and sugar concentration of enzymatic hydrolyzate. Salts produced by neutralization (CaSO₄, Na₂SO₄, K₂SO₄, and (NH₄)₂SO₄), sugars (polysaccharides, oligosaccharides, and monosaccharides), sugar-degradation products (weak acids and furans), and lignin-degradation products (ethyl acetate extracts and nine main lignin-degradation products) were back-added to wASC. Results showed that these products, except furans, exerted negative effect on enzymatic hydrolysis. According to the characteristics of acid-catalyzed steam explosion pretreatment, the five sugar-degradation products’ mixture and salts [Na₂SO₄, (NH₄)₂SO₄] showed minimal negative inhibition effect on enzymatic hydrolysis. By contrast, furans demonstrated a promotion effect. Moreover, soluble sugars, such as 13 g/L xylose (decreased by 6.38 %), 5 g/L cellobiose (5.36 %), 10 g/L glucose (3.67 %), as well as lignin-degradation products, and ethyl acetate extracts (4.87 %), exhibited evident inhibition effect on enzymatic hydrolysis. Therefore, removal of soluble sugars and lignin-degradation products could effectively promote the enzymatic hydrolysis performance.     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

Exopolysaccharide production by filamentous fungi: the example of Botryosphaeria rhodina

One-hundred and five fungal strains, belonging to 46 different species, were screened for exopolysaccharide production. Phytopathogenicity and, in particular, inability to produce conidia, were physiological characteristics positively associated and correlated with the fungal ability to produce polysaccharides. Among the 29 positive strains, Botryosphaeria rhodina DABAC-P82 was the most interesting reaching, when grown on optimal nitrogen source and concentration (NaNO₃ and 2.0 g l⁻¹, respectively) and culture medium pH (3.7), 17.7 g l⁻¹ of exopolysaccharide production after only 24 h of fermentation; yield and productivity were 0.69 g g⁻¹ and 0.73 g l⁻¹ h⁻¹, respectively. The purified polysaccharide was characterised as a homopolysaccharide of glucose with a molecular weight of 4.875·10⁶ Da. Studies of structural analysis indicated the presence of β-1,3 and β-1,6 linkages; the EPS structure was very similar to that of scleroglucan. This revised version was published online in August 2006 with corrections to the Cover Date.