Cultivating <Emphasis Type="Italic">in vitro</Emphasis> coconut palms (<Emphasis Type="Italic">Cocos nucifera</Emphasis>) under glasshouse conditions with natural light,improves <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis nursery survival and growth

Plantlets of coconut were cultured in vitro under three different ambient conditions including a standard culture room, a culture room inside a glasshouse with natural light but controlled temperature, and a standard glasshouse with natural light and natural fluctuations of temperature. Plantlets from the 3 treatments were compared in terms of growth, plant survival as well as net photosynthesis and efficiency of PSII (Fv/Fm ratio) both at the end of the in vitro stage and at 3 stages of ex vitro acclimatization. At the end of the in vitro stage, plantlets cultured in vitro under glasshouse conditions showed the best performance showing the highest photosynthesis rate, dry weight and number of leaves. Plantlets from the standard culture room showed the lowest photosynthesis and growth rate. After 6 months of ex vitro acclimatization, plantlets originally grown in vitro under glasshouse conditions maintained better field survival and growth rates in terms of fresh weight, dry weight and leaf number than plantlets originally grown in vitro in the standard culture room. Although more studies are required to define the reason for this effect, it is clear that the conditions of standard culture rooms are not the best for in vitro cultivation of coconut and perhaps other tropical species.     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

Excretory overexpression of <Emphasis Type="Italic">Paenibacillus pabuli</Emphasis> US132 cyclodextrin glucanotransferase (CGTase) in <Emphasis Type="Italic">Escherichia coli</Emphasis>: gene cloning and optimization of the culture conditions using experimental design

The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24°C, an induction-starting A₆₀₀ nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).     

High throughput culture and gametogenesis induction of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophyte clones

In anticipation of the application of a new sporeling-raising method using gametophyte clones to Laminaria commercial cultivation in China, techniques of mass culture and gametogenesis induction of L. japonica gametophyte clones were developed, as a mass of fertile gametophytes is a prerequisite for sporeling-raising with the new method. Gametophyte clones which were subjected to fed-batch culture exhibited a classical logistic growth curve. Growth rates decreased gradually after 2 months of culture, and were negatively correlated to cell density. UNOVA also showed that only cell density has a significant effect on the growth of gametophyte clones under the experimental conditions. Based on the dynamics models revealed, a culture strategy only directed at the control of cell density was adopted. By this strategy, a total of 36 kg wet weight from an initial weight of 0.75 kg was achieved after 3 months culture in 100 20-L bottles. The final average density reached 24 g L⁻¹. For the subsequent gametogenesis induction, amplificatory male and female gametophyte clones were cut, mixed and cultured in bottles under the same conditions used in amplification except for a change of photoperiod from continuous irradiance to 10 h light: 14 h dark cycle. Egg discharge occurred 10 days after the mixed culture and increased gradually with the culture duration. Most gametophytes gave rise to sporophytes 20 days after induction. Large-scale culture of gametophyte clones and gametogenesis induction for commercial cultivation in 2003–2005 have been conducted successfully.     

Bacterial consortium proteomics under 4‐chlorosalicylate carbon‐limiting conditions

In this study, the stable consortium composed by Pseudomonas reinekei strain MT1 and Achromobacter xylosoxidans strain MT3 (cell numbers in proportion 9:1) was under investigation to reveal bacterial interactions that take place under severe nutrient‐limiting conditions. The analysis of steady states in continuous cultures was carried out at the proteome, metabolic profile, and population dynamic levels. Carbon‐limiting studies showed a higher metabolic versatility in the community through upregulation of parallel catabolic enzymes (salicylate 5‐hydroxylase and 17‐fold on 2‐keto‐4‐pentenoate hydratase) indicating a possible alternative carbon routing in the upper degradation pathway highlighting the effect of minor proportions of strain MT3 over the major consortia component strain MT1 with a significant change in the expression levels of the enzymes of the mainly induced biodegradation pathway such as salicylate 1‐hydroxylase and catechol 1,2‐dioxygenase together with important changes in the outer membrane composition of P. reinekei MT1 under different culture conditions. The study has demonstrated the importance of the outer membrane as a sensing/response protective barrier caused by interspecies interactions highlighting the role of the major outer membrane proteins OprF and porin D in P. reinekei sp. MT1 under the culture conditions tested.     

Production of <Emphasis Type="Italic">Anti-Helicobacter pylori</Emphasis> metabolite by the lichen-Forming fungus <Emphasis Type="Italic">Nephromopsis pallescens</Emphasis>

The present study was conducted to evaluate the antibacterial activity of lichen-forming fungi (LFF) against Helicobacter pylori, and to optimize the culture conditions of LFF for maximum production of natural antibiotics against H. pylori. To accomplish this, a screening assay was first conducted among 19 species of LFF. The extract of Nephromopsis pallescens (KOLRI-040516) exhibited the strongest anti-ff. pylori activity. Bioautograghic TLC and HPLC analysis identified usnic acid as the main antibacterial substance produced by JV. pallescens. The growth of JV. pallescens and production of antibacterial substances produced by the fungus were then investigated under several culture conditions including the culture media, initial medium pHs, incubation temperatures, and the degree of aeration. The results indicated that culture in MY medium with an initial pH of 6.0, a temperature of 15°C and a low degree of aeration supported the largest usnic acid production of the fungus (16.4 ug usnic acid/g dry biomass). Especially, aeration was found to be an important factor that affect both growth and usnic acid production of N. pallescens.     

Metabolite profiling of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis> reveals evolutionary conservation of osmoprotective substances

The moss Physcomitrella patens is suitable for systems biology studies, as it can be grown axenically under standardised conditions in plain mineral medium and comprises only few cell types. We report on metabolite profiling of two major P. patens tissues, filamentous protonema and leafy gametophores, from different culture conditions. A total of 96 compounds were detected, 21 of them as yet unknown in public databases. Protonema and gametophores had distinct metabolic profiles, especially with regard to saccharides, sugar derivates, amino acids, lignin precursors and nitrogen-rich storage compounds. A hydroponic culture was established for P. patens, and was used to apply drought stress under physiological conditions. This treatment led to accumulation of osmoprotectants, such as altrose, maltitol, ascorbic acid and proline. Thus, these osmoprotectants are not unique to seed plants but have evolved at an early phase of the colonization of land by plants.     

The effect of light quality on anther culture of <Emphasis Type="Italic">Citrus clementina</Emphasis> Hort. ex Tan.

This preliminary research reports results on the influence of light quality on anther culture of Citrus clementina Hort. ex Tan., cultivar Nules. After one month of cultivation in darkness, four light qualities were tested: White, Red, Far-Red and Blue. Continuous Darkness and White light under photoperiod of 16 hrs were used as a control conditions. Gametic embryoids and embryogenic callus were obtained only under photoperiodic conditions of White light, suggesting that the alternation of light and dark can be used for the process of gametic embryogenesis in Citrus.     

Phénolamides et induction florale de Cichorium intybus dans différentes conditions de culture en serre ou in vitro

Phenolamides and floral induction of Cichorium intybus in different conditions of culture in glass-room or in vitro. Three complexes between phenols and amines (phenolamides) have been found in Cichorium intybus L., a plant with an absolute requirement of vernalisation followed by long days for flowering. Upon hydrolysis, these complexes (A, B and C) liberate aromatic amines whose exact identification is in progress, but which are closely related to dopamine, tyramine and serotonin, respectively. In a first series of experiments, phenolamides were studied in the buds of plants grown in the greenhouse under varying conditions. Only buds from plants which flower in long days contained large amounts of these compounds. Much smaller amounts were found in buds at the end of vernalisation (at 2–4°C) before long-day treatment as well as in buds kept in the vegetative state after vernalisation by being grown in short days (8 h light) or in total darkness. In a second series of experiments, phenolamides were studied in bud-forming calli induced in vitro on explants of tuberised root. After sixteen days of culture in continuous light, large quantities of phenolamide were found in the buds and calli of the upper part of the explant, while the lower part which never produces buds contained much less. Buds formed under continuous light produce inflorescences in approximately one month. Various other culture conditions make it possible to maintain the explants in the vegetative state. This can be obtained by short-day conditions, or otherwise under continuous illumination by decreasing the sugar or increasing the NAA levels in the medium. After 13 days of culture, the phenolamide levels were much lower under all of these conditions, than under conditions favourable to floral induction. Compound C is absent or present in trace amounts in vegetative buds. The significance of the differences observed between floral and vegetative buds is supported by the sensitivity of the analytical techniques used. The accumulation of phenolamides in tissues of Cichorium intybus appears to be closely linked to floral induction. Under continuous light it begins very early in young buds and even in the calli that bear these buds.     

Competitive displacement of the native species <Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> by an alien species <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> (Nematoda: Aphelenchida: Aphelenchoididae): a case of successful invasion

The presence of alien invasive species has serious negative impact on endemic biodiversity, especially on native species that occupy the same niche in the ecosystem. To study the influence of the alien invasive species Bursaphelenchus xylophilus on its native sister species B. mucronatus, the two nematode species were mix-cultured in a fungal mat and mix-inoculated into a susceptible host. By comparing the propagation parameters of both species under competitive and noncompetitive conditions it was shown that the propagation level of B. xylophilus was clearly higher than that of B. mucronatus under laboratory culture. Furthermore, the propagation capacity of B. xylophilus under competitive conditions was much higher than that under noncompetitive conditions, both in laboratory culture and with host inoculation. Bursaphelenchus xylophilus also excluded B. mucronatus when the two species were cultured as a mixture for a longer time. The relative abundance ratios of the two species in natural pinewoods were also determined by random sampling of dying pine trees from regions with different invasion histories. It was noted that with an increase in invasion years the distribution frequency of B. xylophilus increased while that of B. mucronatus decreased. Experimental tests verified our hypothesis that because of its high fecundity and strong competitive ability, the invasive species B. xylophilus out-competed the native species B. mucronatus and displaced it in natural ecosystems. The successful invasion of B. xylophilus is attributed to competitive displacement, which may be one of the ecological invasive mechanisms.     

EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC,AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE1

Heterotrophic growth of microalgae presents significant economic advantages over the more common autotrophic cultivation. The efficiency of growth and nitrogen, phosphorus, and glucose uptake from synthetic wastewater was compared under heterotrophic, autotrophic, and mixotrophic regimes of Chlorella vulgaris Beij. immobilized in alginate beads, either alone or with the bacterium Azospirillum brasilense. Heterotrophic cultivation of C. vulgaris growing alone was superior to autotrophic cultivation. The added bacteria enhanced growth only under autotrophic and mixotrophic cultivations. Uptake of ammonium by the culture, yield of cells per ammonium unit, and total volumetric productivity of the culture were the highest under heterotrophic conditions when the microalga grew without the bacterium. Uptake of phosphate was higher under autotrophic conditions and similar under the other two regimes. Positive influence of the addition of A. brasilense was found only when light was supplied (autotrophic and mixotrophic), where affinity to phosphate and yield per phosphate unit were the highest under heterotrophic conditions. The pH of the culture was significantly reduced in all regimes where glucose was consumed, similarly in heterotrophic and mixotrophic cultures. It was concluded that the heterotrophic regime, using glucose, is superior to autotrophic and mixotrophic regimes for the uptake of ammonium and phosphate. Addition of A. brasilense positively affects the nutrient uptake only in the two regimes supplied with light.     

Effect of culture medium composition on the activity of extracellular lectins of <Emphasis Type="Italic">Lentinus edodes</Emphasis>

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. The lectin activity 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 day 15–18 of culturing at pH 8.0–9.0.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 200–203.Original Russian Text Copyright © 2005 by Tsivileva, Nikitina, Garibova.     

Lipid accumulation in <Emphasis Type="Italic">Schizochytrium</Emphasis> G13/2S produced in continuous culture

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h⁻¹, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h⁻¹, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Optimization of feed conditions in a 2.5-l fed-batch culture using rice oil to improve cephalosporin C production by <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> M25

Summary Rice oil significantly affected cephalosporin C production in a 2.5-l bioreactor culture of Cephalosporium acremonium M25. To improve cephalosporin C production, the feed conditions of rice oil were optimized. Reducing the feed rate of rice oil improved cephalosporin C production to 1.01 g/l when the consumption rate of rice oil decreased. Overall, under optimal feed conditions in the 2.5-l fed-batch culture, cephalosporin C production increased about four times compared to before optimization.     

Effect of blue light on indoor seedling culture of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Phaeophyta)

Saccharina japonica is a brown alga that has been commercially cultured on a large scale in China. Integrating the light condition under seawater and the adaptation of Saccharina to this condition, it is expected that blue light would be beneficial to Saccharina culture system. Consequently, the detailed effect of blue light on the key stages during indoor seedling culture of S. japonica was investigated in this study. Irradiances and light qualities had little effect on zoospore attachment and germination. Egg formation occurred sooner under blue light than white light. Under optimum irradiances, 95 ± 4% female gametophytes gave rise to eggs in 6 d under blue light, while it took 12 d for over 90% formation of eggs under white light. Over a culture period of 3 weeks, mean sporeling length and width under blue light was 1.39 and 1.56 times of that under white light, respectively, while the mean sporeling size obtained under red light was only 25% of that under white light. The higher growth rate under blue light was largely due to higher photosynthetic efficiency, as indicated by chlorophyll fluorescence of photosystem II. In addition, the mean ratio of sporeling width to length under blue light was significantly higher than that under white light. These results suggest that blue light would be superior to white light for indoor seedling culture of S. japonica. Based on these findings, an improved S. japonica seedling culture system is discussed.