Nitrogen fixation by immobilized Azotobacter chroococcum

A nitrogen-fixing bacterium, Azotobacter chroococcum, was immobilized in 2% agar gel. The optimum partial oxygen pressure, pO₂, of immobilized cells was 0.2 atm, wherea s that of native cells was 0.05 atm. When continual nitrogen fixation was performed under aerobic conditions, the nitrogenase activity of immobilized cells increased with increasing time. On the other hand, the activity of native cells decreased rapidly. Increase of nitrogenase activity was attributed to growth of the bacteria in the gel matrix. The production rate of total nitrogen compounds by the immobilized bacteria was also increased during the first 4 days. Nitrogen compounds produced by the immobilized cells were mainly amino acids such as γ-aminobutyrate, glutamate and arginine.     

Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100-21

3-Cyanopyridinase activity, i.e. the ability to convert 3-cyanopyridine to nicotinic acid plus ammonia, was induced in stationary phase cultures of Nocardia rhodochrous LL100-21 by the addition of 2-, 3-, or 4-cyanopyridine or benzonitrile; the latter nitrile gave maximum induction. Harvested bacteria possessing 3-cyanopyridinase activity could stoichiometrically convert 3-cyanopyridine at concentrations of up to 0.5 to nicotinic acid. Both 3-cyanopyridine and nicotinic acid inhibited the hydrolysis of 3-cyanopyridine by intact bacteria. Bacteria immobilized in calcium alginate beads and used in column bioreactors retained 3-cyanopyridinase activity for over 150 h when continuously supplied with 0.3 3-cyanopyridine.     

Properties of cyanogen bromide-activated, Agarose-immobilized catechol 1,2-dioxygenase from freeze-dried extracts of Nocardia sp. NCIB 10503

Catechol 1,2-dioxygenase [catechol: oxygen 1,2-oxidoreductase (decyclizing); EC 1.13.11.1], the aromatic intradiol ring-cleaving enzyme of Nocardia sp. NCIB 10503 prepared by freeze-drying cell-free extracts, was covalently attached to cyanogen bromide-activated Agarose. The properties of the immobilized enzyme were compared to those of the free enzyme preparation. Immobilization was shown to increase the thermal stability of the enzyme. The pH-activity profile was altered by immobilization. Various explanations for this phenomenon are discussed. The V_max and K_m of the enzyme were not significantly affected on immobilization. The enzyme had a broader substrate specificity than any previously reported catechol 1,2-dioxygenase, and this was largely unaltered by immobilization. The properties of the preparations are compared to those of other (free) catechol 1,2-dioxygenases. The results presented show that the immobilization of catechol 1,2-dioxygenase offers an attractive means for the production of cis,cis-muconate and novel substituted analogues.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

In vitro capsaicin production by immobilized cells and placental tissues of Capsicum annuum L. grown in liquid medium

Capsaicin, from green pepper fruits is used in formulated foods and in pharmaceuticals. Cell cultures of Capsicum annuum L. were obtained from seedlings on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. In vitro-grown cells and placental tissues from fruits were immobilized in calcium alginate. Immobilized cells and placental tissues produced capsaicin which leached out into the medium. Immobilized placental tissue exhibited greater potentiality for capsaicin synthesis than immobilized cells. Production reached a level of 1345 μg capsaicin g⁻¹ of immobilized placenta on the 14th day of culture. Production of capsaicin, on replenished nutrient medium in immobilized placenta was 2400 μg on the 30th day. Ferulic acid fed to immobilized placenta at 2.5 mM level increased capsaicin production by 2-fold by the 5th day of the culture period. Of the elicitors used, curdlan was effective on capsaicin production in immobilized cells. Extracts of Aspergillius niger and Rhizopus oligosporus stimulated capsaicin production in immobilized placental tissues.     

Reduced temperature sensitivity of immobilized Nitrobacter agilis cells caused by diffusion limitation

A dynamic model describing substrate consumption and growth of immobilized biomass was extended to predict and study the effect of variations in temperature. This was done by including temperature relations for all the individual parameters that are influenced by temperature.

A sensitivity analysis was executed to identify the parameters that determine the temperature sensitivity of the overall process in the different cultivation phases. The model was evaluated by cultivating immobilized Nitrobacter agilis cells in an airlift loop reactor at various temperatures between 6–30°C. The dynamics of the system were studied by imposing temperature changes during the cultivation. The model describes the effect of low temperatures on the macroscopic consumption rate fairly well. As predicted, immobilized cells are less sensitive to temperature changes than suspended cells. The macroscopic consumption rate of immobilized N. agilis cells at 10°C is around 70% of the macroscopic consumption rate at 30°C while in the case of suspended cells, this consumption rate is only 15%.     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.     

Evaluation of immobilized cytochrome P-448 from Saccharomyces cerevisiae using permeabilized whole cell, microsomal fraction and highly purified reconstituted forms, with benzopyrene-3-monooxygenase activity

Cytochrome P-448 from Saccharomyces cerevisiae in permeabilized whole cell, microsomal fraction and in a highly purified reconstituted benzopyrene-3-monooxygenase (EC 1.14.14.1) system have been immobilized on various supports. Calcium alginate was found to be especially useful and the kinetics of hydroxylation were close to that of the free enzyme system with all three forms of enzyme, even with permeabilized whole yeast cells (V _max of 664 pmol 3-hydroxybenzo(a)pyrene produced per h per nmol cytochrome P-448 compared with 1000 for free highly purified reconstituted enzyme system). Only the highly purified reconstituted form was successfully immobilized by BrCN-activated Sepharose-4B or by acrylamide. Both of these supports stabilized the highly purified reconstituted cytochrome P-448 benzopyrene-3-monooxygenase activity in prolonged storage at 4°C. Applications for various immobilized enzymes and cells are assessed.     

Comparison between immobilized Kluyveromyces fragilis and Saccharomyces cerevisiae coimmobilized with beta-galactosidase, with respect to continuous ethanol production from concentrated whey permeate

Kluyveromyces fragilis immobilized in calcium alginate gel was compared to Saccharomyces cerevisiae coimmobilized with beta-galactosidase, for continuous ethanol production from whey permeate in packed-bed-type columns. Four different whey concentrations were studied, equivalent to 4.5, 10, 15, and 20% lactose, respectively. In all cases the coimmobilized preparation produced more ethanol than K. fragilis. The study went on for more than 5 weeks. K. fragilis showed a decline in activity after 20 days, while the coimmobilized preparation was stableduring the entrire investigation. Under experimental conditions theoretical yields of ethanol were obtained from 4.5 and 10% lactose substrates with the coimmobilized system. Using 15% lactose substrate, theoretical yields were only obtained when a galactose-adapted immobilized S. cerevisiae column was run in series with the coimmobilized column. Then a maximum of 71 g/L ethanol was produced with a productivity of 2.5 g/L h. The coimmobilized column alone gave a maximum ethanol concentration of 52 g/L with a productivity of 4.5 g/L h, whereas immobolized K. fragilis only produced 13 g/L ethanol with a productivity of 1.1 g/L h. It was not possible to obtain theoretical yields of ethanol from the highest substrate concentration.     

An enzyme coimmobilized with a microorganism: the conversion of cellobiose to ethanol using beta-glucosidase and Saccharomyces cerevisiae in calcium alginate gels

The efficiency of beta-glucosidase and Saccharomyces cerevisiae in directly converting cellobiose to ethanol was studied for various combinations of the two catalytic species, both free and immobilized, in order to elucidate the advantages of using a coimmobilized system. The coimmobilized preparation was superior to a combination of separately immobilized biocatalysts. However, in this preparation, one-half the enzyme activity was lost within a week when incubated at the operational temperature in the absence of substrate. In continuous experiments, an 80% conversion of cellobiose to ethanol was obtained using the coimmobilized preparation, compared to 40% using separately immobilized biocatalysts when applying a dilution rate of 0.1 h(-1) in a packedbed reactor. The immobilized biocatalysts showed no decline in productivity during two weeks of continuous operation.     

Synthesis of -Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for -DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of -DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l⁻¹ of -DOPA was obtained in 20 h (productivity 0.39 g l⁻¹ h⁻¹). Cells immobilized in a carrageenan gel showed higher -DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.     

Continuous stereospecific Δ-3-keto-steroid reduction by PAAH bead-entrapped Clostridium paraputrificum cells

The development of a continuous anaerobic process for stereospecific Δ⁴-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ⁴, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested.     

Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel. II. Optimizing SDS-degrading activity and stability

Conditions were established for optimizing the surfactant (SDS)-degrading activity of Pseudomonas C12B immobilized in polyacrylamide gel beads. Optimum activity was obtained by using immobilized cells derived from stationary phase of batch cultures and incubating with SDS at 30°C at pH 6.5. Half-saturation of the degradation system was achieved at an SDS concentration of 0.23 m . Biocatalyst stability was highest for beads maintained in basal salts medium, retaining 91% of initial activity after 161 d. In Tris/HCl buffer or distilled water, the stability was much lower, although in all cases the stability of immobilized cells was higher than that of free cells under equivalent conditions. Biocatalyst beads “inactivated” by sequential incubation in three batches of distilled water containing only SDS could be reactivated by transferring beads to nutrient medium. Beads packed in a glass column and operated in a continuous up-flow mode using SDS/basal salts eluant produced 100% hydrolysis when run at retention times above 60 min. The system was highly stable in the continuous flow mode; when operated at a residence time of 55 min (initially giving 98% degradation), the extent of degradation decreased only slightly to 93% over a continuous operation period of 3 weeks.     

玉米芯、竹炭及油枯吸附-海藻酸钠包埋施氏假单胞菌PFS-4对二氯喹啉酸的降解

采用玉米芯、竹炭及油枯吸附-海藻酸钠包埋对分离到的施氏假单胞菌PFS-4进行复合固定.采用正交试验对固定化条件进行优化,研究了固定化菌剂及游离菌体对二氯喹啉酸的降解效果.结果表明: 固定化菌剂制备的最佳条件为:海藻酸钠质量分数为4%、吸附载体比例(玉米芯∶竹炭∶油枯)为1∶2∶1、CaCl₂质量分数为3%、交联时间4 h.固定化菌剂在温度为30 ℃、初始pH=7的条件下,经6 d培养后,对浓度为800 mg·L^-1的二氯喹啉酸降解率为91.4%,而游离菌体的降解率为72.8%.将游离菌体和固定化菌剂用于实际污水及土壤处理时,固定化菌剂对水中及土壤中二氯喹啉酸去除率仍能分别达到84.2%和74.3%.研究结果表明,载体及其联结方式对土壤中二氯喹啉酸去除产生显著影响,翻动频率与土壤中二氯喹啉酸的去除率呈显著正相关.因此,玉米芯、竹炭及油枯吸附-海藻酸钠复合固定施氏假单胞菌PFS-4对不良环境具有较好的缓冲性能,对二氯喹啉酸污染水体及土壤原位生态修复具有潜力.     

Bacterivory in the common foraminifer Ammonia tepida: Isotope tracer experiment and the controlling factors

The majority of sediment dweller foraminifera are deposit feeders. They use their pseudopodia to gather sediment with associated algae, organic detritus and bacteria. Uptake of bacteria by foraminifera have been observed but rarely quantified. We measured uptake of bacteria by the common foraminifera Ammonia tepida using ¹⁵N pre-enriched bacteria as tracers. In intertidal flats, seasonal, tidal and circadian cycles induce strong variations in environmental parameters. Grazing experiments were performed in order to measure effects of abiotic (temperature, salinity and irradiance) and biotic (bacterial and algal abundances) factors on uptake rates of bacteria. In mean conditions, A. tepida grazed 78 pgC ind^− 1 h^− 1 during the first eight hours of incubation, after which this uptake rate decreased. Uptake of bacteria was optimal at 30 °C, decreased with salinity and was unaffected by light. Above 7 × 10⁸ bacteria ml wt sed^− 1, uptake of bacteria remained unchanged when bacterial abundance increased. Algal abundance strongly affected algal uptake but did not affect uptake of bacteria. As uptake of bacteria represented 8 to 19% of microbes (algae plus bacteria) uptake, Ammonia seemed to be mainly dependant on algal resource.     

Growth of the sponge Pseudosuberites (aff.) andrewsi in a closed system

Explants of the Indo-Pacific sponge Pseudosuberites aff. andrewsi were fed with the microalgae Chlorella sorokiniana and Rhodomonas sp. It was microscopically observed that these algae were ingested and digested by the sponge cells, suggesting that they were consumed by the sponges. The algae were further used for two growth experiments with five explants of P. aff. andrewsi and four explants of P. andrewsi. Growth was measured as the increase in projected body area. The explants showed considerable growth (up to 730% in 54 days for P. aff. andrewsi and up to 680% in 22 days for P. andrewsi), which is much higher than previously reported growth rates for sponges. Growth started after a stationary phase of 5–20 days in which the projected body area did not increase. The growth of P. aff. andrewsi appeared to be linear and was inhibited at the end of the experiment. Two explants of P. andrewsi showed exponential growth instead of linear growth. Hence, no general statements about the growth kinetics of these sponges can be made at this time. However, the high growth rates found in this study suggest a promising future for cultivation of sponges in closed systems.     

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g⁻¹ h⁻¹. Composition of mixture of peptides was dependent on pH, concentrations of Na⁺ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.     

表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*

透明颤菌血红蛋白基因vgb在多种研究和工业发酵菌中异源表达很好的解决了高密度发酵中的溶氧率问题。酿酒酵母是经典的真核模型,且在发酵工业中具有重要的应用价值,但vgb在酿酒酵母中异源表达对细胞生长的影响并不清楚。以ADH1为启动子构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb的异源表达质粒YEplac195-ADH1pr-vgb,并转化至酿酒酵母BY4741。通过生长敏感性实验,发现在发酵碳源和非发酵碳源中,vgb的异源表达均抑制了菌株生长。接着,通过2',7'-二氯荧光黄双乙酸盐和PI染色和脂质过氧化产物检测分析,发现过表达vgb的酿酒酵母细胞中活性氧(ROS)的积累、细胞膜通透性改变以及脂质过氧化。结果表明,酿酒酵母中过表达vgb改变细胞的氧化状态促进活性氧的累积,氧化应激导致菌株的生长抑制。     

A photochemical fuel cell system using Anabaena N-7363

Summary A blue-green algae, Anabaena N-7363, was immobilized in 2% agar gel. The hydrogen productivity of the immobilized algae was three times higher than that of free algae. The maximum hydrogen production rate by the immobilized blue-green algae was 0.52 moles h^–1 g^–1 (of wet gel) in the medium without nitrogen sources under illumination (10,000 lux). The oxygen evolved was then removed by a reactor containing aerobic bacteria. A photo-current of 15–20 mA was continuously produced for 7 days by the photochemical fuel cell system consisting of the immobilized Anabaena reactor, the oxygen-removing reactor and the hydrogen-oxygen fuel cell. The conversion ratio of hydrogen to current was from 80% to 100%.     

毛竹入侵马尾松林的土壤菌群多样性变化

为揭示毛竹入侵邻域林分过程中对土壤细菌群落多样性变化的影响,以马尾松林为研究对象,采集纯毛竹林、竹针混交林和常绿针叶(马尾松)混交林3类样地的混合土样,基于高通量测序技术分析土壤细菌群落多样性和结构变化.结果表明: 研究期间共得到细菌类群39门88纲134目160科511属;在门的分类水平上,与竹针混交林、常绿针叶混交林相比,纯毛竹林酸杆菌门所占比例显著较低,而放线菌门、拟杆菌门、TM7和衣原体门所占比例较高;在属的分类水平上,相对于纯毛竹林,竹针混交林和常绿针叶混交林均有多属表现出所占比例显著上升或下降,单独出现在竹针混交林或常绿针叶混交林且所占比例在0.005%～0.1%的非优势属有130属;α多样性指数均表现为常绿针叶混交林>竹针混交林>纯竹林,且纯竹林与两者均有显著差异,而常绿针叶混交林和竹针混交林之间差异不显著.PCoA分析表明,毛竹入侵对土壤细菌的种群多样性和群落结构产生了影响;所占比例小于0.1%的非优势菌群门分类水平的百分比特征与土壤环境梯度(水溶性有机氮和硝态氮)之间有显著相关性,两者对毛竹入侵马尾松林后土壤细菌群落的非优势种群影响巨大,具有重要的参考价值.