Alteration of biomass and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots by media sterilization method and sugars

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

Modeling sucrose hydrolysis in dilute sulfuric acid solutions at pretreatment conditions for lignocellulosic biomass

Agricultural and herbaceous feedstocks may contain appreciable levels of sucrose. The goal of this study was to evaluate the survivability of sucrose and its hydrolysis products, fructose and glucose, during dilute sulfuric acid processing at conditions typically used to pretreat lignocellulose biomass. Solutions containing 25g/l sucrose with 0.1-2.0% (w/w) sulfuric acid concentrations were treated at temperatures of 160-200 degrees C for 3-12min. Sucrose was observed to completely hydrolyze at all treatment conditions. However, appreciable concentrations of fructose and glucose were detected and glucose was found to be significantly more stable than fructose. Different mathematical approaches were used to fit the kinetic parameters for acid-catalyzed thermal degradation of these sugars. Since both sugars may survive dilute acid pretreatment, they could provide an additional carbon source for production of ethanol and other bio-based products.     

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

Effect of Sugars on Rabbit Serum Albumin Stability and Induction of Secondary Structure

The effect of sugars (sucrose, maltose, and glucose) on the thermal and chemical denaturation of rabbit serum albumin (RSA) has been examined by viscosity and far UV circular dichroism measurements. The viscosity measurements indicate a change in the reduced viscosity from 4.18 to 16.23 ml/g in the temperature range from 20 to 90°C. The T _m value for RSA obtained by viscosity measurements in the absence of sugar was found to be 63.2°C, but this value increased to 68.4, 70.3, and 73.2°C in the presence of 0.5 M sucrose, 0.5 M glucose, and 0.5 M maltose, respectively. Further, the stability of RSA in the presence of 0.5 M sugars was also investigated by measuring the mean residue ellipticity at 222 nm (MRE₂₂₂) using chemical (0-6 M guanidine hydrochloride) and thermal (20-90°C) transition processes. At the midpoint of the chemical denaturation, the increase in the MRE values at 222 nm in the presence of 0.5 M sugars were of the same order as the increase in the T _m values, i.e., maltose > glucose > sucrose. Interestingly, a mixture of 0.25 M glucose and 0.25 M fructose showed a cumulative effect on the thermal as well as chemical stability as compared to 0.5 M sucrose alone. In the case of both thermal and chemical denaturation, there was an increase in the MRE₂₂₂ values upon addition of various sugars, this indicating induction of secondary structure in the protein.     

Regulation of invertase in Aspergillus nidulans: effect of different carbon sources

Aspergillus nidulans produces an extracellular beta-D-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the beta-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate. On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C degrees. After this time the level of detectable invertase in the cultures declined. A proportion of the enzyme was secreted during the linear growth phase of the fungus. Various sugars were investigated for induction of invertase, but only the two beta-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level. Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium. After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level. The repressive effect of other sugars, e.g. glucose and xylose, on invertase production was also demonstrated in this experimental system.     

Factors influencing artemisinin production from shoot cultures of Artemisia annua L.

Artemisinin, an anti-malarial drug isolated from the annual wormwood Artemisia annua L., has a marked activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum, and is useful in treatment of cerebral malaria. Shoot cultures of Artemisia annua L. were established on Murashige and Skoog basal medium which contained (per litre) 30 g sucrose, 0.5 mg 6-benzyladenine and 0.05 mg naphthaleneacetic acid. Using an optimized combination of sucrose (30 g/l), nitrate (45 mM), inorganic phosphate (200 mg/l), gibberellic acid (7 mg/l) and the ratio of NH₄ ⁺-N to NO₃ ^–-N of 1:3, artemisinin production reached 26.7 mg/l after 30 days. This procedure provides a potential alternative for production of artemisinin from in vitro tissue cultures.     

Changes in the pool of soluble sugars induced by dehydration at the heterotrophic phase of growth of wheat seedlings

Loss of dehydration tolerance coincides with a shift from heterotrophy to autotrophy during post-germination growth of spring wheat seedlings. This critical stage falls on the fifth day following imbibition. Till the sixth day of experiment light had no effect on dry weight of the seedlings but the survival of six day old seedlings was reduced by half upon dehydration. Germinating seeds in the presence of 5 mM glucose, fructose, mannose or sucrose did not promote seedling growth but either increase (glucose, fructose) or decreased (mannose, sucrose) the survival of dehydrated seedlings. Protection against dehydration by the former sugars was correlated, irrespective of the seedling age, with the decrease of sugar pool in seeds and increase in shoots (coleoptile and first leaf) and roots. The opposite changes were provoked by the sugars hampering seedling survival. Generally, survival of wheat seedlings was not correlated with the size of soluble sugar pool but its distribution and composition. Lower mobilisation of soluble sugars in seed, lower proportion of reduced sugars to sucrose and higher share of raffinose is characteristic for the tolerant four day old seedlings and those grown in the media containing glucose or fructose. The results presented indicate that higher proportion of reduced sugars to sucrose and lower share of raffinose in six day old seedlings seems to be associated with the loss of dehydration tolerance of these seedlings, despite heterotrophic character of growth.     

Different effects of galactose and mannose on cell proliferation and intracellular soluble sugar levels in <Emphasis Type="Italic">Vigna angularis</Emphasis> suspension cultures

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.     

Effect of some disaccharides, hexoses and pentoses on nitrate reductase, glutamine synthetase and glutamate dehydrogenase in excised pea roots

The effects of exogenous sucrose, lactose, d -glucose, d (-)fructose, d -galactose, d -mannose, l -sorbose, l -arabinose and d -xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O₂ consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO₃. GS reaches its maximum at lower sugar concentrations, this fact being especially clear-cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O₂ consumption which is most pronounced with NR. The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism.     

Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology

Optimization of the fermentation medium for maximization of actinorhodin production by Streptomyces coelicolor A3(2) was carried out. Response surface methodology (RSM) was applied to optimize the medium constituents. A 2⁴ full-factorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. sucrose, glucose, yeast extract (YE) and peptone, and to design a minimum number of experiments. The P-values of the coefficients for linear, quadratic and cross-product effect of sucrose and glucose concentration were <0.0001, suggesting that these were critical variables having the greatest effect on the production of actinorhodin in the complex medium. The optimized medium consisting of 339 g/l sucrose, 1 g/l glucose, 1.95 g/l YE and 2.72 g/l peptone predicted 195 mg/l of actinorhodin which was 32% higher than that of the unoptimized medium. The amounts of glucose, YE and peptone required were also reduced with RSM.     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

Sweet potato: A novel substrate for pullulan production by Aureobasidium pullulans

A strain Aureobasidium pullulans AP329, was used for the production of pullulan by employing hydrolysed sweet potato as cultivation media. Hydrolysis with α-amylase alone resulted in the lowest yields of pullulan. In contrast continuous hydrolysis with pullulanase and the β-amylase in sweet potato itself gave higher yields, but prolonged hydrolysis with amyloglucosidase decreased the yield. The maximum pullulan yield (29.43 g/l) was achieved at the dextrose equivalent value of 45 and pH of 5.5 for 96 h. As a substitute of sucrose, hydrolysed sweet potato was found to be hopeful and the yield of pullulan was higher than that of glucose and sucrose. The molecular weight of pullulan obtained from hydrolysed sweet potato media was much higher than that of sucrose and glucose media. Results of this work indicated that sweet potato was a promising substrate for the economical production of pullulan.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

甘油影响红曲菌产色素的碳源选择性探究

红曲菌(Monascus spp.)是我国重要的药食同源微生物,红曲色素(Monascus pigments,MPs)是其主要次级代谢产物之一。有研究表明,甘油可促进红曲菌产MPs,但作用机制不明。以丛毛红曲菌(Monascus pilosus)MS-1为实验菌株,考察甘油与葡萄糖或蔗糖复合对红曲菌产MPs的影响。在不含碳源的合成培养基中,将甘油与葡萄糖或蔗糖复合,采用分光光度法和高效液相色谱法等分析MPs的产量和组分、生物量及发酵液pH。当甘油与葡萄糖复合,添加甘油后发酵液pH、生物量无显著变化(P0.05),总色价显著降低(P0.05)。当2 g/L或40 g/L甘油与蔗糖复合,发酵液pH显著降低而生物量及总色价显著增加(P0.05)。当40 g/L甘油与蔗糖复合时,总色价是仅以蔗糖为碳源时的16.5倍,且MPs同系物数量明显增多(P0.05)。在合成培养基条件下,甘油促进红曲菌产MPs具有碳源种类的选择性。该结果可为研究甘油影响红曲菌产MPs的作用机制提供参考,为甘油用于MPs生产提供依据。     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Sugar preferences and feeding strategies in the hawkmoth Macroglossum stellatarum

Hummingbird hawkmoths were tested for their preferences for different types of sugar. In triple choice tests, moths sucked for longer periods from sucrose than from fructose and glucose. Naive moths released in a large flight cage and monitored over 24 days, drank on average 137 l sucrose, 67 l fructose and 7 l glucose daily. In an independent test, moths spent more time feeding from sucrose than from fructose and more time feeding from fructose than from glucose. Animals in hibernation made less but longer feeding bouts, whereas animals that fed every day and newly eclosed animals were more likely to make more but shorter visits to feeders. The hawkmoths learned to associate colour with the preferred sugar. In a dual choice test, flower-naive moths chose blue rather than yellow artificial flowers. After the initial test, these animals received sucrose from the yellow and glucose from the blue feeders. A week later they chose yellow more frequently than blue indicating that they had learned to associate a colour with the preferred type of sugar.