Physiological aspects of free and immobilized Aspergillus niger cultures producing citric acid under various glucose concentrations

Similarities and differences between cultures of free and immobilized Aspergillus niger were identified under various glucose concentrations. Growth and citric acid production rates were compared, and the macro-morphology and fine structure of the mycelia examined to determine which parameters were significant in the production of citric acid. With free cultures the diameter of pellets was inversely related to glucose concentration, while biomass levels were lower for immobilized cultures than the equivalent free cultures. Rates of citric acid production were higher with immobilized mycelium, especially at higher glucose levels. The morphology that characterized high citric acid productivity was that of swollen hyphal tips which were seen at concentrations over 100 kg/m³ glucose in both free and immobilized mycelium. Although there is a characteristic morphology associated with high productivity it does not account for the difference observed between free and immobilized mycelia. The increased glucose uptake and productivity was not due to an increased surface area either, since the immobilized system was slightly lower in total surface area than the equivalent free cultures. The major difference was in the mean diffusion path in the two systems.     

The kinetics of simultaneous glucose and fructose uptake and product formation by Aspergillus niger in citric acid fermentation

The kinetics of substrate uptake and product formation in the process of citric acid accumulation by Aspergillus niger on sucrose as a sole carbon source are presented. The experiments are aimed at studying if glucose and fructose obtained from the hydrolysis of sucrose are equivalent carbon sources for A. niger and how the presence of the two different carbon substrates might influence the citric acid formation process. Beet sugar was used as a sole carbon source in the first series of experiments conducted in two types of bioreactors: stirred tank and air-lift. The fructose uptake rate was significantly lower than the glucose uptake rate in the late idiophase. A substrate utilisation breakpoint occurred when a large amount of citric acid was accumulated in the fermentation broth. A similar phenomenon was also detected in repeated fed-batch fermentation. This phenomenon was confirmed by the second series of parallel shake culture runs, in which fungal growth and citric acid accumulation by A. niger was simultaneously tested on the media containing the following carbon sources: sucrose, glucose and fructose, with and without addition of concentrated citric acid solution. Finally, it was shown that high concentration of citric acid strongly depleted fructose uptake rate.     

Biotransformation of glucose to gluconic acid by Aspergillus niger—study of mass transfer in an airlift bioreactor

A glucose–gluconic acid biotransformation system was suggested for the experimental study of oxygen transfer in bioreactors. This biosystem was used for the investigation of the effect of the flow rate and biomass concentration on the volumetric oxygen transfer coefficient k_La in a 10 dm³ internal-loop airlift bioreactor. For this purpose, the fermentation broth of the mycelial strain Aspergillus niger was employed, representing a three-phase system, where bubbles come into contact with dense rigid pellets. The results showed that the presented biotransformation system can be successfully utilised for the determination of the oxygen transfer rate in airlift bioreactors. The experiments showed a strong positive influence of the air flow rate on the rate (r_Glu), specific rate of gluconic acid production (k_Glu/X) as well as on the volumetric oxygen transfer coefficient (k_La). This confirmed an expected limitation of production rate by the oxygen transport from the gas to the liquid phase in the whole range of air flow rates applied. Moreover, consistent curves of the production rate r_Glu and k_La values vs. biomass concentration c_X (amount of enzymes) were observed. These exhibited a local maximum for c_X equal to 6.68 g dm⁻³. On the other hand, the specific production rate monotonously decreased with increasing biomass concentration. A decline of k_La values at higher c_X values was attributed to a bubble coalescence promoting effect of mycelial pellets.     

Enhancement of citric acid production by Aspergillus niger using n-dodecane as an oxygen-vector

Due to the significant oxygen requirement during citric acid production and the relatively low solubility of oxygen in water, aeration is critical. The potential use of n-dodecane as an oxygen-vector for improvement of citric acid production by Aspergillus niger was studied. The volumetric fraction of oxygen-vector has a great influence on the volumetric oxygen transfer coefficient k_La. With the addition of an oxygen-vector to the fermentation medium with a final concentration of 5%, the k_La value reached a maximum value (130 h⁻¹), which is twice that of the control experiment. The addition of 5% (v/v) n-dodecane enhanced citric acid accumulation, reduced residual sugar concentration and stimulated mycelial growth. Adding n-dodecane had no adverse effects on the cells of A. niger. The results of enzyme assays indicated that no significant differences were observed between the activity of citrate synthase of two kinds of mycelial cell-free extracts.     

Glyoxyl agarose as a new chromatographic matrix

At alkaline pHs, glyoxyl agarose is able to immobilize most of the proteins contained in a crude extract. However, due to its special immobilization features, at pH 7.0 only proteins that contain at least two exposed low pK amino groups in the same plane were immobilized (β-galactosidase from Escherichia coli, catalase from bovine liver, and IgG from rabbit). However, with many other proteins, even multimeric ones, immobilization could not be achieved (e.g.: glucose oxidase from Aspergillus niger and Penicillium vitale; catalase from Micococcus sp., A. niger and bovine liver; alcohol oxidase from Pichia pastoris, Hansenula sp. and Candida boidinii, β-galactosidase from Thermus sp., etc.). Elution of the attached proteins under mild conditions was not simple, if the number of protein-support bonds was very high, only boiling in SDS allowed the elution of the proteins. However, using glyoxyl agarose 4BCL with only 20 μmol of aldheyde groups/g of support, proteins could be fully eluted by competition with amino compounds (e.g., Tris buffer). In this first approach, we have tried to take advantage of this specific immobilization at pH 7.0 to purify multimeric proteins, using a β-galactosidase from E. coli as a model. The enzyme could be eluted from the support using Tris–HCl buffer as eluting agent, with a high yield (80%) and a high purification factor (32).     

黑曲霉木质纤维素降解酶基因的可变剪接分析及验证

黑曲霉Aspergillus niger因能够产生大量的木质纤维素降解酶而在木质纤维素资源利用中发挥重要作用。目前,有关黑曲霉基因组中与木质纤维素降解相关的基因是否存在可变剪接的情况尚不清楚。本研究以黑曲霉CBS513.88菌株为研究对象,采用rMATS和ABLas两种方法对黑曲霉在葡萄糖为唯一碳源(G组)和小麦秸秆为唯一碳源(WS组)下的56个木质纤维素降解酶基因的可变剪接事件进行分析,并通过RT-PCR扩增和内含子特异性扩增对3个典型基因的可变剪接体进行了验证。结果表明,ABLas可变剪接分析算法相较于rMATS分析算法更为准确,ABLas分析算法显示G组和WS组共有21个木质纤维素降解酶基因出现了可变剪接,可变剪接类型以内含子保留(IR)为主,占所有可变剪接事件的82.85%。另外,G组和WS组发生可变剪接的木质纤维素降解酶基因也有所不同：G组发生可变剪接的基因为13个,WS组发生可变剪接的基因为14个,两组都发生可变剪接的基因为6个,这表明黑曲霉木质纤维素降解酶基因的可变剪接在不同生长条件下存在差异,另一方面,黑曲霉中众多可变剪接体的存在也为开发新型的木质纤维素降解酶资源提供基础。     

查尔酮衍生物对黑曲霉线粒体结构及功能的影响

本研究旨在探讨查尔酮衍生物对黑曲霉线粒体结构和功能的影响,评估查尔酮衍生物对黑曲霉的抗真菌效果。采用不同浓度查尔酮衍生物处理黑曲霉菌丝体,通过透射电子显微镜观察线粒体结构;并进一步对黑曲霉线粒体的活性氧、丙二醛水平、三羧酸循环相关酶活性和线粒体膜电位的变化进行测定。结果表明,查尔酮衍生物以剂量依赖的方式诱导黑曲霉线粒体结构损伤,导致线粒体总脱氢酶、琥珀酸脱氢酶和ATP酶活性改变,线粒体膜电位降低,丙二醛和活性氧水平显著升高。这些发现表明查尔酮衍生物可以破坏线粒体膜通透性,导致线粒体结构破坏,从而损害线粒体功能,达到抗真菌效果。     

Vertical Rotating Immobilized Cell Reactor of the bacteriumZymomonas mobilis for stable long-term continuous ethanol production

Summary Vertical Rotating Immobilized Cell Reactor was designed and built for glucose conversion into ethanol. Immobilized biomass units withZ. mobilis cells attached into polyurethane foam discs were fixed along a rotating shaft inside the bioreactor. The effect of rotation speed on the concentration of immobilized biomass was studied. Stability of the bioreactor over long-term operation was dependent on the concentration of the immobilized biomass. With fermentation carried out at 6 rpm a constant active immobilized cell concentration of only 34.5 g/l was maintained and used to convert up to 140 g glucose/l into more than 70 g ethanol/l with a volumetric ethanol productivity of 63 g/l/h.     

Practical reactor systems for yeast cell immobilization using biomass support particles

The technique of cell immobilization using porous support particles (biomass support particles) has been successfully applied to yeast cells. Two reactor configurations exploiting the use of these particles have been developed and assessed for use in aseptic yeast fermentations. A liquid-fluidized bed fermenter has been devised for use with particles denser than the fermentation liquor whilst a gas-stirred circulating bed fermenter proved suitable for particles of essentially neutral buoyancy. Both systems have been operated successfully for extended periods of continuous operation. The utilization of biomass support particle technology in such reactors provides a practical and robust system for immobilized cell reactors. This technology offers significant opportunities for further development.     

耐热黑曲霉3.316菌株全基因组测序及分析

黑曲霉3.316是一株耐热型丝状真菌,最高生长温度达47℃,在工业发酵中有着巨大的应用潜力。为了更加充分地在工业发酵中利用其耐热特性,需要对菌株信息进行全面了解。通过PacBio Sequel测序平台的CLR测序方式对黑曲霉3.316菌株进行全基因组测序。结果表明,基因组最后得到15个contigs,总长度为34 956 132bp,GC含量为49.21%,预测到10 032个编码基因,在GO、KOG、KEGG数据库分别有6 901、2 118和9 494个基因得到注释。通过分析比较得到黑曲霉耐热性与抗氧化相关基因超氧化物歧化酶（SOD）、过氧化氢酶（CAT）有密切关系,这为后续研究黑曲霉3.316耐热特性提供可靠信息,同时为应对工业发酵中的高能耗、高生产成本以及高温环境的现状提供优良菌株。     

Commercial lipase immobilization on Accurel MP 1004 porous polypropylene

Three commercial lipases (CLs), A Amano 6 (from Aspergillus niger), M Amano 10 (from Mucor javanicus), and R Amano (from Penicillium roqueforti) - called lipase A, M and R respectively - were characterized in terms of carbohydrate content, protein content and enzymatic activity (p-nitrophenylacetate assay). All the CL preparations contained different proteins as observed from electrophoresis. Lipases were immobilized on Accurel MP1004 porous polypropylene by physical adsorption.The Immobilization process caused a loss of enzymatic activity. The retained activity was similar for lipase M and R (about 15%). In contrast, lipase A retained only the 1.3% of the specific activity of the free lipase. The retained activity of lipases M and R seems to be due to a feature of the support, while the lower activity a of lipase A may be attributed to a strong structure distortion caused by lipase-support interaction.     

Immobilization of the Solanum tuberosum epoxide hydrolase and its application in an enantioconvergent process

Five supports have been evaluated for the immobilization of the epoxide hydrolase from Solanum tuberosum (StEH) by adsorption. The highest immobilization yield (90-99%) and the maximum EH (epoxide hydrolase) activity (0.6 U g^-1 wet support) were obtained by ionic adsorption onto DEAE-cellulose. Although the activity recovered upon immobilization of StEH onto DEAE-cellulose was low, a notable stabilization factor of 6.9 at 65°C was obtained. In addition, the immobilized StEH showed a higher temperature for maximal activity (57°C) and the optimal pH (5.0) was shifted one unit towards the acidic region as compared to the free enzyme. Immobilized StEH was successfully reused in six consecutive hydrolytic kinetic resolutions of rac-pCSO without noticeable loss in activity. Finally, the sequential use of immobilized StEH with the immobilized EH from Aspergillus niger (AnEH) in a repeated batch reactor, operated for five cycles, enabled the enantioconvergent preparation of the corresponding (R)-diol, which was thus obtained with an ee of 89% and an overall yield of 100%.     

Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone

In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.     

Specificity and kinetic parameters of recombinant Microdochium nivale carbohydrate oxidase

A new carbohydrate oxidase from Microdochium nivale heterologously expressed in Aspergillus oryzae (rMnO) has been characterized. The carbohydrate oxidase is a flavoenzyme which oxidizes glucose and other mono- or oligosaccharides. It shows a broad substrate specificity towards carbohydrates reacting with aldoses in the 1-position. The rMnO oxidizes the β-form of -glucose, and the product of -glucose oxidation is -gluconic acid.

The mechanism of carbohydrate oxidation by oxygen and artificial electron acceptors has been described by a ping-pong scheme. Compared to Aspergillus niger glucose oxidase (GOx) the reactivity of rMnO at pH 7.0 is significantly lower; k_cat is 20, k_ox 11 and k_red 22 times less, using oxygen as electron acceptor. Also with other two electron acceptors, like DPIP, the activity is low. However, compared to oxygen the rMnO shows 2–10 times higher activity towards some artificial single electron acceptors (AAs). The enzyme activity increases at higher ionic strength of the solution, if positively-charged AAs are used.

The high activity towards AAs and low rate for oxygen as well as broad specificity to carbohydrates indicates that rMnO may have some advantages compared to the most used GOx in connection with enzyme use for analytical devices and for biotechnological purposes.     

无载体固定化酵母细胞木薯淀粉质原料酒精连续发酵研究

以木薯粉糖化液为发酵底物,在总发酵体积(有效)为15L的悬浮床生物反应器中,对一株粟酒裂殖酵母变异株进行一级和二级连续发酵研究。结果表明,二级连续发酵系统可明显改善一级系统的不足,并取得了平均流加糖液浓度150g/L,发酵强度为97g/L.h,流出液酒精浓度727g/L,残糖浓度374g./L,总糖利用率达90%的较好结果;整个系统在连续一个月的运行中从未发现染菌现象,发酵操作稳定。     

Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Screening and mutagenesis of moulds for the improvement of glucose oxidase production

The strain of Aspergillus niger G most effective for producing glucose oxidase (see β- -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was selected out of 110 moulds belonging to 15 different species by the method of test-tube microculture. Conidia of the selected strain were further subjected to mutagenesis with both u.v. and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and the products were analysed for glucose oxidase activity with our own diffusion plate method. Among 960 strains isolated after mutagenesis only 12 showed higher activity (from 1.5 to 18%) than the starting strain A. niger G.     

Characterization of Hydrogen Peroxide and Superoxide Degrading Pathways of Aspergillus Niger Catalase: A Steady-State Analysis

The oxidized intermediates generated upon exposure of Aspergillus niger catalase to hydrogen peroxide and superoxide radical fluxes were examined with UV-visible spectrophotometry. Hydrogen peroxide and superoxide radical were generated by means of glucose/glucose oxidase and xanthine/xanthine oxidase systems. Serial overlay of absorption spectra in the Soret (350-450 nm) and visible regions (450-700 nm) showed that the decomposition of hydrogen peroxide by the catalase of Aspergillus niger can proceed through one of two distinct pathways: (i), the normal “catalatic” cycle consisting of ferric catalase → Compound I → ferric catalase; (ii), a longer cycle where superoxide radical transforms Compound I to Compound II which is then converted to the resting ferric enzyme via Compound III. The latter sequence of reactions ensures that the catalase of Aspergillus niger restores entirely its activity upon exposure to low levels of superoxide radicals due to the actions of oxidases.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Continuous production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke