Exopolysaccharide production by the epipelic diatom Cylindrotheca closterium: effects of nutrient conditions

During the stationary phase of a batch culture of the epipelic diatom Cylindrotheca closterium, accumulation of exopolysaccharides and intracellular carbohydrates was observed. When nitrogen was added to the culture in the stationary phase, growth was resumed and the accumulation of exopolysaccharides was delayed. This indicated that nitrogen depletion caused cessation of growth, and stimulated exopolysaccharide accumulation. Exopolysaccharide accumulation was also stimulated when cells were either resuspended in medium lacking N or P, or when they were inoculated in medium with low concentrations of N or P. Growth was not immediately affected by low N or P concentrations. S depletion only resulted in exopolysaccharide accumulation when growth was affected. Si or Fe depletion did not stimulate exopolysaccharide accumulation, even when growth rates were lowered. Apparently, stimulation of exopolysaccharide accumulation is dependent on the type of nutrient depletion. Intracellular storage carbohydrates did not accumulate when cells were incubated at low N or P concentrations. Cells grown with ammonium as nitrogen source produced more carbohydrates (both extracellular and intracellular) than cells grown with nitrate as nitrogen source, indicating that both exopolysaccharides and intracellular carbohydrates accumulated as a result of overflow metabolism.     

高粱苗(Sorghum vulgare)水分亏缺时所累积的游离脯氨酸的来源

在完全营养液中,用Glu或Ala替代Ca(NO_3)_2为氮源时,在水分胁迫条件下可得到同样程度的Pro累积量。但是用Ser为氮源时,在正常水分条件下Glu和Ala的相对含量较少,Ser和Gln相对含量较高,在水分胁迫条件下,Pro的累积受到抑制。在缺钾营养液中培养时,植株游离氨基酸中Glu和Ala相对量较少,Ser和Gln相对量较高,在水分胁迫条件下,Pro累积也受到抑制。在缺钾营养液中,用Glu或Ala替代Ca(NO_3)_2为氮源时,在水分胁迫条件下Pro的累积可达到甚至超过完全营养液中Pro的相对量。     

培养基和培养条件对栀子悬浮细胞合成多糖的影响

对栀子悬浮细胞合成多糖的调控因子研究表明 :B5为最适培养基 ;5～10d继代周期的细胞可以保持良好的生长状态和多糖的合成能力 ;80g L的鲜细胞的接种量有利于栀子细胞的生长和多糖的合成 ;使用单一碳源时 ,葡萄糖比蔗糖对细胞生长更有益 ,但葡萄糖成本高 ,因而混合碳源 45g L(葡萄糖 :蔗糖 =1∶1)是最佳配方 ;氮源种类对细胞生长和多糖合成没有明显的影响 ,但氮源浓度是主要因素 ,40～50mmol L是最佳浓度 ,同时运用悬浮细胞生产栀子多糖可以通过在不同时间收获的细胞来避免提取时黄色素的干扰 ,具有很好的实际意义。     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.     

The effect of glucose and ammonium sulfate on kinetic acidification by heterogeneous mixed culture

Summary The effect of glucose and ammonium sulfate concentration on the kinetics of lactic acid formation by a heterogeneous mixed culture was evaluated by the sole product formation using the Gompertz model, which can be used to define culture media composition taking into account product accumulation and acidification rate constant. A compromise between ionic inhibition and nitrogen limitation was found by using ammonium sulfate as nitrogen source. The sugar tolerance was similar to that in exenic cultures.     

Turbidostat culture of yeast during transition from a resting state to active growth

The age components of a Saccharomyces cerevisiae 14 culture and the kinetics of its growth were studied afer the quiescent state at the onset active growth. The following factors induced the quiescent state: the cessation of a chemostat flow for 24 h, growth inhibition with 2,4-dinitrophenol (DNP) for 24 h, the storage of a culture growing on agar in a refrigerator during 24 h. The process of transition from the point of growth activation to the maximum rate of growth was then studied in turbidostat. This process took the shortest time in a refrigerated culture as well as in a culture that had been limited with a phosphorus source and in a culture limited with a nitrogen source and grown in chemostat at D = 0.26 h-1. The process was longer in cultures that had been either limited with glucose or inhibited with DNP and longest in a chemostat culture limited with a nitrogen source at D = 0.15 and 0.05 h-1. The rate of initial mitosis phases in the yeast is presumed to exert the greatest effect on the duration of this transition process.     

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837)

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.     

多次补氮和增强蓝光促进三角褐指藻积累岩藻黄素

本研究旨在优化多次补氮和增强蓝光模式，以促进光发酵三角褐指藻(Phaeodactylum tricornutum)积累岩藻黄素。结果表明，在摇瓶中将含有胰蛋白胨和尿素的混合氮源(1:1,Nmol/Nmol；总氮浓度为0.02 mol/L)分6次加入培养系统是最佳的多次补氮模式；在5 L发酵罐中实施两阶段调光模式培养，在第二阶段增强蓝光(R:G:B=67.1:16.7:16.3)后，细胞密度、生物量生产率以及岩藻黄素的含量、产量和生产率分别达到1.12×108cells/mL、330mg/(d·L)、19.62mg/g、69.71mg/L和6.97mg/(d·L)。与红蓝光(R:G:B=70.9:18.3:10.9)下分6次补氮的一阶段培养相比，岩藻黄素含量显著提高了7.68%(P<0.05)，但生产率无显著差异(P>0.05)；与红蓝光(R:G:B=70.9:18.3:10.9)下一次性加入氮源的一阶段培养相比，岩藻黄素含量和生产率显著提高了45.98%和48.30%(P<0.05)。因此，本研究开发的多次补氮和增强蓝光的两阶段培养模式，有效促进了岩藻黄素积累、提高了...     

Bioconversion of dethiobiotin to biotin by a cell-free system of a bioYB transformant of Bacillus sphaericus

Abstract Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.     

油菜愈伤组织中烯丙基硫苷的积累和某些外源碳氮化合物对其含量的影响

油菜愈伤组织在培养中生长34d的增殖量最大,增殖倍数为7．08倍,烯丙基硫苷的含量在第6天最高;外源L-缬氨酸明显促进烯丙基硫苷的积累,其浓度为2和4mmol·L^-1时烯丙基硫苷含量分别达到98．5和97．40mg·g^-1（DW）;蔗糖最有利于油菜愈伤组织的生长和其中烯丙基硫苷的积累,浓度以30g·L^-1。的蔗糖为最佳。     

Pattern of dry-matter accumulation,and nitrogen concentration and uptake as influenced by levels and methods of nitrogen application in rainfed-upland rice

Summary Dry-matter accumulation, and concentration and uptake of nitrogen increased with increasing level of nitrogen at all the stages of crop growth. The differences in nitrogen concentration due to nitrogen levels were greatest at panicle initiation stage and started becoming narrower with the advancement in crop age. Split application of nitrogen with its heavier fractions at tillering and panicle initiation stages either through soil alone or soil+foliage (1/3+1/3+1/3) resulted in higher dry-matter accumulation, and higher nitrogen concentration and uptake than other methods. The crop, on an average, removed 61 kg N/ha. Plants accumulated nearly 15% of the total absorbed nitrogen, up to tillering, 50% up to panicle initiation and 85–90% up to heading. Proportionately lesser nitrogen uptake and dry-matter accumulation at post-heading stage is an indicative of a major constraint for production efficiency of rainfed-upland rice culture.     

不同氮源及氮浓度对真眼点藻纲微藻生长及油脂积累的影响

以真眼点藻纲8株微藻(类波氏真眼点藻(Eustigmatos cf. polyphem)、大真眼点藻(Eustigmatos magnus)、波氏真眼点藻(Eustigmatos polyphem)、魏氏真眼点藻(Eustigmatos vischeri)、斧形魏氏藻(Vischeria helvetica)、点状魏氏藻(Vischeria punctata)、星形魏氏藻(Vischeria stellata)和眼点拟微绿球藻(Nan-nochloropsis oculata))为研究材料, 用3种氮源(硝酸钠、碳酸氢铵或尿素)和4种氮浓度(18、9、6和3 mmol) 在改良的BG-11培养基中对藻细胞进行培养。比较分析这8株微藻在不同培养条件下的藻液pH、生物量、油脂含量、脂肪酸组成的差异, 从而筛选出适合该类微藻生长和油脂积累的最适氮源与最佳氮浓度。结果表明, 这8株微藻均能在3种氮源中生长, 但是随着培养时间延长, 以碳酸氢铵和尿素为氮源时藻液pH逐渐降低, 其变化范围为5.0—6.0, 而以硝酸钠为氮源时藻液pH保持在7.0—8.0, 变化不大。当以尿素为氮源培养时, 能获得较高的生物量, 但是不同藻株在不同尿素浓度时达到最高生物量。最高生物量是波氏真眼点藻(E. polyphem)在9 mmol时达到, 为10.96 g/L。总脂含量分析发现, 在低氮浓度下均能促进8株微藻油脂的积累, 真眼点藻属中的魏氏真眼点藻(E. vischeri)在8株藻中获得最高油脂含量, 达到59.24%。进一步对脂肪酸分析发现, 8株微藻总脂肪酸含量为细胞干重的50%—58%, 主要脂肪酸组成为豆蔻酸(C14鲶0)、棕榈酸(C16鲶0)、棕榈油酸(C16鲶1)、油酸(C18鲶1)和二十碳五烯酸(C20鲶5), 其中拟微绿球藻(N. oculata)细胞中棕榈酸的含量最高占总脂肪酸50%左右; 其他7株微藻细胞中棕榈油酸的含量较高, 其占总脂肪酸含量范围在40%—60%。8株微藻均表现出较高的生物量与油脂积累能力, 以尿素为氮源, 氮浓度为6 mmol时更有利于该类微藻生物量和油脂的积累。总体来说, 真眼点藻纲的微藻是一类极具潜力适合于微藻生物燃料生产的微藻, 而真眼点藻属藻株表现更为明显的优势。     

Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth.     

剩余污泥热碱解及其用于微生物油脂生产的探索

为了将剩余污泥中的蛋白质成分进行资源化利用,采用热碱解法进行处理,并尝试将处理后得到的上清液作为氮源培养圆红冬孢酵母用于微生物油脂的合成。结果表明,pH 13下处理5 h的条件对于污泥减量以及含氮物质析出最为有效,而污泥在pH 10处理5 h时得到的上清液则相对更适于酵母培养。在剩余污泥中添加NaOH调节初始pH为10,在60 ℃下处理5 h后中和体系pH值至7,得到的上清液经微孔膜过滤后用于配制限氮培养基可以使菌体生长良好,并且细胞内油脂含量达35％。     

Exopolysaccharide production by the cyanobacterium Anabaena sp. ATCC 33047 in batch and continuous culture

The halotolerant, filamentous, heterocystous cyanobacterium Anabaena sp. ATCC 33047 released, during the stationary growth phase in batch culture and, at low dilution rate, in continuous culture, large amounts of an exopolysaccharide (EPS) to the culture medium. Different environmental, nutritional and physical parameters affected production and accumulation of the EPS. The presence of either a combined nitrogen source or NaCl at high concentration led to decreased EPS production, without affecting cell growth. In contrast, generation of the EPS was markedly enhanced in response to an increase in either air flow rate, temperature or irradiance. In continuous culture, accumulation of EPS in the medium increased in response to a decrease in the dilution rate, with maximal EPS productivity being reached at a dilution rate of 0.03 h⁻¹.     

Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under the conditions of submerged culture was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and pH of culture medium. The activity of lectin in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C : N ratio, (9.5-12): 1)) on the day 15-18 of culturing at pH 8-9.     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

Effect of nitrogen source and nitrogen concentration on the production of pyruvate by Torulopsis glabrata

The effect of nitrogen sources including yeast extract, peptone, soybean hydrolyzate and some inorganic nitrogen sources, as well as the nitrogen concentration on the fermentative production of pyruvate by Torulopsis glabrata WSH-IP12 was investigated. The addition of yeast extract greatly inhibited pyruvate accumulation, while peptone was shown to be the most favorable nitrogen source. In flask culture, 15 g l(-1) peptone was needed to consume 80 g l(-1) glucose with 23.4 g l(-1)of pyruvate accumulated. Pyruvate production was markedly dependent on the ratio of carbon to nitrogen (C:N), its production was improved by increasing the concentration of glucose and peptone proportionally and reduced by exclusively increasing the glucose concentration. In a glucose fed-batch culture, cell growth and pyruvate production slowed after 28 h. However, cell growth and pyruvate production recovered after further nitrogen, in the form of peptone and ammonium sulfate, was added to the culture. A final concentration of pyruvate of 54.5 g l(-1) was achieved at 64 h (yield to glucose consumed of 0.471 g g(-l)). By using aqueous ammonia instead of potassium hydroxide for pH control, 57.3 g l(-1) pyruvate with a yield of 0.498 g g(-1) was produced by 55 h. This result further indicates that nitrogen level plays an important role in the production of pyruvate.     

The effect of the cultivation conditions on the growth and pigmentation of Serratia marcescens

The influence of cultivation conditions on the growth and pigmentation of S. marcescens was studied. The cultures under study grew in media containing glycerin, glucose or acetate and organic or mineral nitrogen. Pigment formation occurred in a medium with organic nitrogen and glycerin or acetate, but not glucose. Sodium chloride inhibited the growth of cultures, but at a concentration not exceeding 4-5% increased the accumulation of prodigiosin. Prodigiosin was accumulation by the culture growing at 28 degrees C, while at 37 degrees C no accumulation of the pigment occurred. The illumination of the growing culture with visible light decreased the intensity of its pigmentation. Prodigiosin apparently plays an important role in the metabolism of S. marcescens and is linked with the energy exchange of the cell.     

Carbon accumulation in <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> induced by nitrogen limitation

Background

Oleaginous microorganisms, such as bacterium, yeast and algal species, can represent an alternative oil source for biodiesel production. The composition of their accumulated lipid is similar to the lipid of an oleaginous plant with a predominance of unsaturated fatty acid. Moreover this alternative to conventional biodiesel production does not create competition for land use between food and oleo-chemical industry supplies. Despite this promising potential, development of microbial production processes are at an early stage. Nutritional limited conditions, such as nitrogen limitation, with an excess of carbon substrate is commonly used to induce lipid accumulation metabolism. Nitrogen limitation implies modification of the carbon-to-nitrogen ratio in culture medium, which impacts on carbon flow distribution in the metabolic network.

Results

The goal of the present study is to improve our knowledge of carbon flow distribution in oleaginous yeast metabolism by focusing carbon distribution between carbohydrate and lipid pools in order to optimize microbial lipid production. The dynamic effects of limiting nitrogen consumption flux according to carbon flow were studied to trigger lipid accumulation in the oleaginous yeast Rhodotorula glutinis. With a decrease of the specific nitrogen consumption rate from 0.052 Nmol.Cmol_X^?1.h^?1 to 0.003 Nmol.Cmol_X^?1.h^?1, a short and transitory intracellular carbohydrate accumulation occurred before the lipid accumulation phase. This phenomenon was studied in fed-batch culture under optimal operating conditions, with a mineral medium and using glucose as carbon source. Two different strategies of decreasing nitrogen flow on carbohydrate accumulation were investigated: an instantaneous decrease and a progressive decrease of nitrogen flow.

Conclusions

Lipid production performance in these fed-batch culture strategies with R. glutinis were higher than those reported in the previous literature; the catalytic specific lipid production rate was 0.07 Cmol_lip.Cmol_X*^?1.h^?1. Experimental results suggested that carbohydrate accumulation was an intrinsic phenomenon connected to the limitation of growth by nitrogen when the nitrogen-to-carbon ratio in the feed flow was lower than 0.045 Nmol.Cmol^?1. Carbohydrate accumulation corresponded to a 440% increase of carbohydrate content. These results suggest that microbial lipid production can be optimized by culture strategy and that carbohydrate accumulation must be taken account for process design.