Improved gellan gum production by a newly-isolated Sphingomonas azotifigens GL-1 in a cheese whey and molasses based medium

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na₂HPO₄ 14.58 g/L and KH₂PO₄ 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na₂HPO₄ 9.71 g/L and KH₂PO₄ 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 10⁶ and 0.89 × 10⁶ Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.     

Enhanced gellan gum production by hydrogen peroxide (H2O2) induced oxidative stresses in Sphingomonas paucimobilis

In this study, the effect of H₂O₂-induced oxidative stress on gellan gum production and cell growth were investigated. Gellan gum production was improved and cell growth was inhibited by H₂O₂. A multiple H₂O₂ stresses with different concentrations were developed to optimize gellan gum production. A maximal gellan gum yield (22.52 g/L), which was 35.58 % higher than the control, was observed with 2, 2, 3, 4 mmol/L H₂O₂ added at 6, 12, 18, 24 h, respectively. Moreover, UDP-glucose pyrophosphorylase activity and glucosyltransferase activity were increased with H₂O₂ stresses. This new strategy of multiple H₂O₂-induced oxidative stresses would be further applied to gellan gum production in future study.     

木薯粉同步糖化发酵(SSF)产丁二酸

【目的】通过优化产琥珀酸放线杆菌GXAS137同步糖化发酵木薯粉产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本。【方法】在单因素试验的基础上,先利用Plackett-Burman试验设计筛选出影响丁二酸发酵的重要参数,再采用正交试验确定重要参数的最佳水平。【结果】价格低廉玉米浆可用作氮源,影响丁二酸产量的重要参数是木薯粉、玉米浆、碱式碳酸镁和糖化酶浓度。最佳条件为(g/L):木薯粉100,玉米浆14,糖化酶2.0 AGU/g底物,碱式碳酸镁75。优化后丁二酸产量达到69.31 g/L,丁二酸得率为90.01%,生产强度为1.44 g/(L·h)。与初始条件(52.34 g/L)相比,丁二酸浓度提高了32.42%。并利用1.3 L发酵罐对SSF与SHF两种发酵工艺进行了比较,SSF丁二酸产量(72.21 g/L)远高于SHF(56.86 g/L)。【结论】产琥珀酸放线杆菌同步糖化发酵木薯粉丁二酸产量高,生产成本低,具有较好的工业化应用前景。     

Effect of triton X-100 on compactin production from<Emphasis Type="Italic">Penicillium citrinum</Emphasis>

Glucose alone was found to be the most effective carbon source for producing compactin. An initial glucose concentration of 40 g/L gave the highest compactin concentration of 250 mg/L. Among the various nitrogen sources, when 5 g/L of pharmamedia and soybean meal as the sole nitrogen source were used, respectively, the compactin concentration was higher than 250 mg/L. Especially, in the case of the mixture of 6 g/L of pharmamedia and 8 g/L of soybean meal, the compactin concentration was 400 mg/L. To select the best surfactant for effective compactin production, various surfactants were investigated. When Triton X-100 was used, the maximum compactin concentration was 445 mg/L. With the initial concentration ranging from 1.5 to 2.0 g/L, the compactin concentration was the highest at 465–450 g/L. The cell concentration was similar to that of the control without the addition of Triton X-100. On the other hand, when the above 4.0 g/L of Triton X-100 were used, the cell concentration decreased. Using the based results the continuous fed-batch cultures by adding the Triton X-100 were carried out for 10 days in an air-lift bioreactor. When 1.5 g/L of Triton X-100 was added to the culture broth at 0, 4, and 8 days of culture, respectively, the compactin production was increased with the increase of culture time. The maximum compactin concentration after 10 days of culture was 1,200 mg/L, which was about 2.0-fold higher than that of the control without the addition of Triton X-100.     

少动鞘脂单胞菌产结冷胶发酵培养基的响应面法优化

通过单因素试验分析不同碳源、氮源、无机盐对（Sphingomonas paucimobilisFJAT-5627）产胶量的影响,确定最适碳源、氮源、无机盐,并在单因素筛选试验的基础上,利用Box-Benhnken设计和响应面分析法对碳源、氮源和无机盐进行优化,得到少动鞘脂单胞菌产生结冷肢发酵培养基最佳优化组合.实验结果表明,少动鞘脂单胞菌产胶量发酵最适碳源、氮源和无机盐分别为淀粉、豆饼粉和KH₂PO₄.响应面法得到产胶量（Y）与碳源淀粉（x₁）、氮源豆饼粉（x₂）和无机盐KH₂PO₄（x₃）的回归方程为:Y=13.87+0.54x₁+0.22x₂-0.42x₃-3.26x₁²-1.85x₂²-1.51x₃²+0.053x₁x₂+0.067x₁x₃+0.4x₂x₃.优化培养基组合为:淀粉浓度为30g/L,豆饼粉浓度为5 g/L,KH₂PO₄的浓度为0.7g/L,且此组合下少动鞘脂假单胞发酵得到结冷胶可达23.87g/L.     

Factors influencing β-glucosidase production, activity and stability inNectria catalinensis

The influence of different cultivation conditions on β-glucosidase production and of some parameters on the activity and stability of this enzyme were studied inNectria catalinensis. Maximal β-glucosidase production was achieved with ammonium nitrate (0.5 g N/L) as nitrogen source. Tween 80, Tween 20 and Triton X-100 increased β-glucosidase yields, Tween 80 was the most effective for enzyme release and growth at a concentration of 3.4 mmol/L. On the other hand, Tween 20 and Triton X-100 had an inhibitory effect onN. catalinensis growth. A temperature of 23°C and an initial pH of cultures of 6.5 were optimal for biomass and β-glucosidase production. Under optimal cultural conditions (ammonium nitrate, 0.5 g N/L; Tween 80, 3.4 mmol/L; 23°C; initial pH 6.5) the β-glucosidase yield was increased more than five fold respect to the initial state. Optimal temperature for β-glucosidase activity was 45°C, the initial activity dropped 60 % after 6 h of incubation at this temperature. Optimal pH for enzyme activity was 5.3. At this pH the β-glucosidase was completely stable after 3 d of incubation. TheV andK _m values calculated from Lineweaver-Burk and Eadie-Hofstee plots were 0.23 μmol 4-nitrophenol per min per mg of protein and 0.25 mmol 4-nitrophenol β-d-glucopyranoside per L, respectively. The activation energy according to Arrhenius plot was 49.6 KJ/mol.     

An industrial medium for improved production of carotenoids from a mutant strain of Phaffia rhodozyma

A medium based on less expensive nutrient sources, such as corn starch hydrolyzate (hydrol), corn steep liquor (CSL), urea and potassium phosphate was used for the growth of the yeast Phaffia rhodozyma 2A2N strain. A central composite experimental design has been employed to derive a statistical model on the effect of hydrol and CSL on carotenoid production. An initial concentration of sugars as glucose equivalent 73?g/l in hydrol and 43?g/l CSL were found optimal for the maximization of final carotenoid production in shake flask cultures. The carotenoid production was increased by adding urea and phosphate sources. Laboratory scale fermentation was performed with the optimized medium and total carotenoid production of 52.4?mg/l was obtained using constant fed-batch culture.     

Studies on root nodules of leguminous plants bioproduction of indole acetic acid by a Rhizobium sp. from a twiner Clitoria ternatea L.

The Rhizobium sp. isolated from the root nodules of Clitoria ternatea L., a leguminous twiner, produced a high amount of IAA (16.4 μg/ml) from tryptophan in an unsupplemented basal medium. The production of IAA started simultaneously with the growth and had no different growth and production phase. The growth and production were parallel and increased up to 45–50 h. The IAA production by the Rhizobium sp. was increased by 520% when the medium was supplemented with fructose (1.5%), MnSO₄ (1.0 μg/ml), riboflavin (0.10 μg/ml) and Triton X-100 (0.01%). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Characterization and evaluation of corn steep liquid in acetone-butanol-ethanol production by Clostridium acetobutylicum

Corn steep liquor (CSL) obtained from a commercial starch manufacturing process was analyzed and tested as a complex nutrient source for ABE (acetone, butanol, and ethanol) production by Clostridium acetobutylicum PJC4BK_AdhE1. CSL contained a wealth of trace elements and nitrogenous components, proteins and amino acids. ABE production increased the content of CSL was raised up to 6% (v/v) in medium and then decreased at higher contents. In 6% CSL-containing medium, C. acetobutylicum PJC4BK_AdhE1 produced 21.4 g/L of ABE with a yield of 0.41 g/g in 18 h of fermentation. Although the final titer of ABE was similar in CSL containing media and Clostridial Growth Medium (CGM, containing yeast extract and asparagines as complex nutrients), the yield and productivity of ABE in 6% CSL-containing medium were found to be higher than 32 and 26%, respectively.     

Optimization of D-xylose to xylitol biotransformation by Candida guilliermondii cells permeabilized with Triton X-100

Abstract

The effect of NADP⁺ and glucose-6-phosphate (G6P) on the biotransformation of D-xylose to xylitol by cells of Candida guilliermondii permeabilized with surfactant Triton X-100 was evaluated. The experimental runs were performed with 12 g L^?1 of permeabilized cells and a reaction medium composed of Tris–HCl buffer (0.1 M pH 7), D-xylose (57 g L^?1), and MgCl₂.6H₂O (5 mM). The levels of NADP⁺ (from 0.0 to 1.7 mM) and G6P (from 0.00 to 0.17 M) were varied according a 2²-full factorial composed design. Under optimized conditions (NADP⁺ 0.5 mM and 0.05 M G6P), the xylitol volumetric productivity (Q_P) and yield factor (Y_P/S) predicted were 1.86 ± 0.03 g L^?1 h^{? 1} and 0.64 ± 0.03 g g^?1, respectively. These values were 94% and 19% higher than those obtained with unpermeabilized cells under fermentation conditions (0.97 g L^?1 h^?1 and 0.53 g g^?1, respectively). On the basis of the results, it can be concluded that xylitol production by biotransformation with cells of C. guilliermondii permeabilized with Triton X-100 is a promising alternative to the fermentative process.     

Comparison of Nostocean hormogonium induction and its motility on solid plates between agar and gellan gum at varying gel matrix concentrations

To establish a sensitive bioassay for Nostocean hormogonium induction, we compared the effectiveness of the morpho-differentiation induction on two gelled plates, agar and gellan gum, for anacardic acid C15:1-Δ⁸ decyl ester (1) (100 nmol/disc). On BG-11₀ (nitrogen-free) medium-based 0.6 and 0.8% agar plates, Nostoc sp. strain Yaku-1 isolated from a coralloid root of Cycas revoluta in Yakushima Island showed clear morpho-differentiation from filamentous aggregates into hormogonia, and the induced hormogonia dispersed within 24 h; however, similar hormogonium formation was not observed at agar concentrations of 1.0% or higher. Conversely, hormogonium induction was considerably more pronounced on gellan gum plates than those on agar plates through concentrations ranging from 0.6 to 1.6% even after 12 h of incubation, particularly active on the 0.8–1.0% gellan gum plates. Thus, gellan gum plates can achieve clear results within 12 h and are thus highly useful for primary screening for hormogonium-inducing factors (HIFs).     

Influence of alkali-treated cornsteep liquor containing medium on protein A production byStaphylococcus aureus

Staphylococcus aureus cultivated in liquid media containing untreated cornsteep liquor (CSL) and alkali-treated CSL produced similar biomass yields (6.5–6.9 g/L). However, contents of protein A in the biomass was 0.5% and 1.56% for untreated CSL and treated CSL, respectively. Addition of treated CSL at 20 g/L achieved optimal enhancement of protein A production (0.11 g/L). Probable factors associated in treated CSL for the enhanced protein A production are discussed.     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

响应面法优化Burkholderiasp．SYBCLIP—Y发酵产低温脂肪酶条件的研究

用响应面法对Burkholderiasp．SYBCLIP—Y液体发酵产低温脂肪酶的发酵条件进行了快速优化。首先利用Plackett—Burman设计对影响其产酶相关因素进行评估并筛选出具有显著效应的三个因素：牛肉膏,橄榄油,TritonX-100;用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,确定出牛肉膏,橄榄油,TritonX-100的最佳浓度分别为：牛肉膏31．8g／L、橄榄油21mL／L、TritonX-10036．55mL／L,优化后脂肪酶的酶活达到61．52U／mL,是优化前的2．62倍。     

Fermentative production of succinic acid from glucose and corn steep liquor byAnaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens requires expensive complex nitrogen sources such as yeast extract and polypeptone for its growth and succinic acid production. It was found thatA. succiniciproducens was able to grow in a minimal medium containing glucose when supplemented with corn steep liquor (CSL) as the sole complex nitrogen source. The concentration of CSL had a significant effect on the glucose consumption byA. succiniciproducens. When 10–15 g/L of CSL was supplemented, cells were grown to an OD₆₆₀ of 3.5 and produced 17.8 g/L succinic acid with 20 g/L glucose. These results are similar to those obtained by supplementing yeast extract and polypeptone, thereby suggesting that succinic acid can be produced more economically using glucose and CSL.     

Evaluation of Saccharomyces cerevisiae Y5 for ethanol production from enzymatic hydrolysate of non-detoxified steam-exploded corn stover

Saccharomyces cerevisiae Y5 was used to produce ethanol from enzymatic hydrolysate of non-detoxified steam-exploded corn stover, with and without a nitrogen source, and decreasing inoculum size. The results indicated that the ethanol concentration of 44.55 g/L, corresponding to 94.5% of the theoretical yield was obtained after 24 h, with an inoculum size of 10% (v/v) and nitrogen source (corn steep liquor, CSL) of 40 mL/L. With the same inoculum size, and without CSL, the ethanol concentration was 43.21 g/L, corresponding to 91.7% of the theoretical value after 60 h. With a decreased inoculum size of 5% (v/v), and without CSL, the ethanol concentration was 40.00 g/L, corresponding to 85.8% of the theoretical value after 72 h. The strain offers the potential to improve the economy of cellulosic ethanol production by simplifying the production process and reducing the costs associated with the process such as water, capital equipment and nutrient supplementation.     

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.     

Optimization of a corn steep medium for production of ethanol from synthesis gas fermentation by <Emphasis Type="Italic">Clostridium ragsdalei</Emphasis>

Fermentation of biomass derived synthesis gas to ethanol is a sustainable approach that can provide more usable energy and environmental benefits than food-based biofuels. The effects of various medium components on ethanol production by Clostridium ragsdalei utilizing syngas components (CO:CO₂) were investigated, and corn steep liquor (CSL) was used as an inexpensive nutrient source for ethanol production by C. ragsdalei. Elimination of Mg²⁺, NH₄ ⁺ and PO₄ ³⁻ decreased ethanol production from 38 to 3.7, 23 and 5.93 mM, respectively. Eliminating Na⁺, Ca²⁺, and K⁺ or increasing Ca²⁺, Mg²⁺, K⁺, NH₄ ⁺ and PO₄ ³⁻ concentrations had no effect on ethanol production. However, increased Na⁺ concentration (171 mM) inhibited growth and ethanol production. Yeast extract (0.5 g l⁻¹) and trace metals were necessary for growth of C. ragsdalei. CSL alone did not support growth and ethanol production. Nutrients limiting in CSL were trace metals, NH₄ ⁺ and reducing agent (Cys: cysteine sulfide). Supplementation of trace metals, NH₄ ⁺ and CyS to CSL (20 g l⁻¹, wet weight basis) yielded better growth and similar ethanol production as compared to control medium. Using 10 g l⁻¹, the nutritional limitation led to reduced ethanol production. Higher concentrations of CSL (50 and 100 g l⁻¹) were inhibitory for cell growth and ethanol production. The CSL could replace yeast extract, vitamins and minerals (excluding NH₄ ⁺). The optimized CSL medium produced 120 and 50 mM of ethanol and acetate, respectively. The CSL could provide as an inexpensive source of most of the nutrients required for the syngas fermentation, and thus could improve the economics of ethanol production from biomass derived synthesis gas by C. ragsdalei.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.