Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.     

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.     

Critical influence of Eschscholzia californica cells nutritional state on secondary metabolite production

In this study, we investigated the influence of initial internal nutrient concentrations at the time of elicitation on the ability of Eschscholzia californica (EC) cells to produce alkaloids. Three EC cell suspensions cultivated in culture media differing in their PO4(3-) and NO3- contents were sampled daily for 12 days and analyzed for extracellular and intracellular nutrient concentrations. The ability of the cells to produce alkaloids was tested along the three cell suspension cultures. Sampled cells were then further cultured for 7 days in a production medium containing the elicitor and an extraction resin. The alkaloid production of the cells was measured 7 days post-elicitation. In the low-N medium, starch, glucose, and phosphate contents in the biomass was increased by 470, 1624 and 70%, respectively, 10 days after inoculation compared to the control culture in standard B5 medium. Cell concentration was significantly reduced from 10.3 to 8.6 millions cell/mL on this low-N medium compared to the control, nevertheless alkaloid production was multiplied by 39 at day 10 when cells were elicited. Cells grown on the low-N or low-P media accumulated 83% and 188% more carbon, respectively, than control cells at the end of the culture. This intracellular C was mainly stored in the form of starch in low-P medium and both in the form of starch and glucose in the low-N medium. The ability of EC cells to produce alkaloids upon elicitation was shown to be strongly dependent on the initial intracellular C and P content at the time of elicitation. This suggests that reproducibility and productivity during EC cell culture could be enhanced by manipulating the intracellular C and P content at the time of elicitation.     

Morphological diversity ofMortierella alpina: Effect of consumed carbon to nitrogen ratio in flask culture

The influence of the consumed carbon to nitrogen (C/N) ratio on mycelial morphology was investigated in cultures ofMortierella alpina using shake flasks. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of the carbon and nitrogen sources was 50 g/L. The whole mycelia and filamentous mycelia exhibited no relationship with the consumed C/N ratio below a consumed C/N ratio of 20 in the presence of either excess carbon or excess nitrogen. However, when the consumed C/N ratio increased higher than 20, the mycelial sizes increased in proportion to the consumed C/N ratio. However, the area ratio of filamentous mycelia to total mycelia was found to be independent of the consumed C/N ratio, and remained constant at 0.82. In the case of a fixed consumed C/N ratio of 20, the whole mycelia and filamentous mycelia increased in proportion to the degree of the medium strength, yet the area ratio of filamentous mycelia to total mycelia remained unchanged at 0.76. Accordingly, these results show that fungal morphology and mycelial size are both affected by the ratio of carbon to nitrogen. The findings of the current study will be helpful in obtaining the efficient production of useful bioproducts from fungal cultures.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.     

Effect of nitrogenous resource on growth,biochemical composition and ultrastructure of Isochrysis galbana (Isochrysidales,Haptophyta)

The nitrogenous resource used to promote algal growth has cost implications for mass culture processes. The present study therefore aimed to determine the effect of different nitrogenous resources (nitrate, ammonium and urea) on various performance parameters (growth, final cell yield, pigmentation, lipid yield and cellular and sub‐cellular characteristics) in Isochrysis galbana. Growth rate was unaffected by nitrogenous resource, but the final cellular yield in the nitrate and urea treatments far exceeded that evident in the ammonium treatments. The reduced cell yield in ammonium treatments and the earlier onset of the stationary phase was brought about by nitrogen‐starvation due to an increase in pH and resultant ammonia volatilization. This starvation initiated an early onset of lipid accumulation, chlorophyll depletion and an increase in the carotenoid to chlorophyll ratio relative to the other nitrogen (N) source treatments. Hence, in spite of being potentially the preferred source of N by algae (due to its reduced state), ammonium‐nitrogen is undesirable for mass culture. The performance parameters of Isochrysis grown in urea (an organic N source) and nitrate (an inorganic N source) were similar, but lipid accrued earlier in cells grown in medium supplemented with urea. This is advantageous for lipid acquisition for the production of biodiesel since it would reduce the duration of photobioreactor runs. Urea is easily available and considerably cheaper than all the other N sources tested and is thus recommended as the nitrogenous resource for large‐scale culture of I. galbana for biodiesel production.     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Effect of C/N ratio and aeration on the fatty acid composition of heterotrophicChlorella sorokiniana

The effect of the carbon to nitrogen (C/N) ratio of the medium and the aeration rate on the lipid content and fatty acid composition ofChlorella sorokiniana was investigated using heterotrophic, batch culture. Both parameters had a significant effect. A C/N ratio of approximately 20, was found to indicate a change from carbon to nitrogen limitation forC. sorokiniana. Cell lipid content was at a minimum at this value and increased at both higher and lower C/N values. Low C/N ratios favoured a high proportion of trienoic fatty acids at the expense of monoenoic acids. Aeration enhanced cell growth, fatty acid yield and the synthesis of unsaturated dienoic and trienoic fatty acids, but reduced cell lipid content. The results demonstrate that the fatty acid composition and lipid content of heterotrophically-grown microalgae can be favourably manipulated by varying culture conditions.     

三分三悬浮培养细胞中苯丙氨酸、鸟氨酸和莨菪碱至东莨菪碱的生物转化

在三分三悬浮培养中分别补充前体苯丙氨酸、鸟氨酸和莨菪碱时,悬浮细胞的生长速率及东莨菪碱的转化率均以补充苯丙氨酸者为最高,分别比未加前体的对照增加50.2％和4.0％。 在上述处理培养两周后于培养液中分别加入70ppm的放线菌酮抑制蛋白质合成时,虽然细胞中东莨菪碱的转化按克干重计算均有明显增加,但是由于细胞生长受强烈抑制不利于东莨菪碱的总的转化,按每瓶总干重计算的转化率均比对照低。 在分别补充三种前体并培养两周后使营养缺乏时,细胞生长比对照稍有降低,但细胞中东莨菪碱的转化却显著增加。特别是补充莨菪碱并使营养缺乏时,大大利于莨菪碱转化为东莨菪碱,转化率高达27.4％。既降低了培养成本又提高了东莨菪碱产量,有利于未来的工业生产。     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

基质碳氮比对广叶绣球菌生长发育的影响

培养基质碳氮比显著影响食用菌菌丝生长及子实体生长发育。广叶绣球菌是一种珍贵的食药用真菌,前期研究发现其生长发育过程中对碳源的需求较多。本研究采用松木屑、复合氮源作为栽培原料,研究基质碳氮比对绣球菌菌丝生长及子实体生长发育的影响。结果表明：6种不同碳氮比（C/N）的栽培基质对菌丝生长速度无显著影响,但是随着C/N比值的增加,子实体形成期逐渐延长,子实体形成率呈先增大后减小的趋势。当基质C/N在48.8-55.9之间时,采收率较高,在80-90d内,达到采收标准的菌袋比例分别为83.7%和81.4%。当基质C/N为43.4时,鲜菇产量最高,但外观品质受影响。综合考虑生产成本及效率,采用松木屑和复合氮源作为栽培原料、适宜的基质C/N为48.8时,绣球菌子实体形成期为64d,子实体形成率为95%且较为集中,鲜菇平均产量为231g/袋（76%集中于200-260g/袋）,整个生产周期为84d。本研究结果表明绣球菌具有“高碳低氮”的营养生理特性。     

Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH ₄ ⁺ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH ₄ ⁺ and prevented the growth of algae by rapidly assimilating all NH ₄ ⁺ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH ₄ ⁺ , thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.     

碳源和氮源对彩绒革盖菌液体发酵合成漆酶的影响

研究了碳源、氮源对彩绒革盖菌液体发酵合成漆酶的影响。结果表明,在所选的几种碳、氮源中,麸皮为试验菌株合成漆酶的较好碳源;酵母浸膏、酒石酸铵、蛋白胨均是比较理想的氮源。不同的碳氮比对彩绒革盖菌漆酶的产量有着显著的影响,高碳高氮是其生产漆酶的最佳条件。在适宜的培养条件下该菌株能合成高活力的漆酶,其酶活力可达298 U/mL以上,产酶周期为6~7 d。     

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures.