Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Mixotrophic and heterotrophic growth of Beggiatoa alba in continuous culture

Beggiatoa alba strain B18LD was grown in continuous culture under heterotrophic conditions on acetate or acetate and asparagine and under mixotrophic conditions on acetate plus either 1 mM sodium sulfide or 1 mM sodium thiosulfate. Considerable differences were observed between the yields and the cell compositions of heterotrophic and mixotrophic cultures at all dilution rates tested. The dry weight yield per gram acetate utilized was approximately three times higher in the acetate-sulfide mixotrophic culture than in the acetate heterotrophic culture, whereas the poly--hydroxybutyric acid and carbohydrate contents were much higher in the heterotrophic cultures. The high yields (0.52–0.75, corrected for the weight of the sulfur) obtained with the mixotrophic cultures imply that the acetate was utilized mainly for biosynthesis. Thus, the oxidation of sulfide supplied energy. The addition of catalase to the chemostat cultures increased yields slightly, but it was insufficient to explain the differences between the heterotrophic and the mixotrophic cultures.     

Growth kinetics of Aeromonas hydrophila in freshwaters supplemented with various organic and inorganic nutrients

Freshwaters of varying natural nutrient enrichment were used as growth media for the culture of an autochthonous, heterotrophic, freshwater bacterium, Aeromonas hydrophila. The growth rate of the bacterium in eutrophic waters was increased to the greatest extent by adding carbon, as glucose; generation times decreased by up to 65%. Additions of carbon and phosphorus increased the maximal cell densities by over 25-fold. In oligotrophic waters, bacterial growth was most strongly promoted by the simultaneous additions of carbon (as glucose) and phosphorus (as KH₂PO₄). In these waters, stationary phase densities were increased as much as 100-fold, with a corresponding 70% increase in growth rate. These data provide at least a partial explanation for the previously observed correlation between A. hydrophila densities and the trophic states of freshwaters.The authors are with the Department of Microbiology, Morrill Hall, University of Rhode Island, Kingston, Rhode Island 02881, USA     

Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture

Production of astaxanthin by sequential heterotrophic-photoautotrophiccultivation of a green alga, Haematococcus pluvialis was investigated.This involved cultivating the cells heterotrophically to high cellconcentration, followed by illumination of the culture for astaxanthinaccumulation. The optimum pH and temperature for heterotrophic biomassproduction were 8 and 25 ^°C, respectively. There was no significantdifference in the specific growth rate of the cells when acetateconcentration was varied between 10 mM and 30 mM. However, cellgrowth was inhibited at higher acetate concentrations. A pH stat methodwas then used for fed-batch heterotrophic culture, using acetate as theorganic carbon source. A cell concentration of 7 g L^-1 wasobtained. Higher cell concentration could not be obtained because the cellschanged from vegetative to cyst forms during the heterotrophic cultivation.However, by using repeated fed-batch processes, the cells could bemaintained in the vegetative form, leading to more than two times increasein cell number output rate. When the vegetative cells were transferred tophotoautotrophic phase, there was a sharp decrease in the cell number andonly very few cells encysted and accumulated astaxanthin. On the otherhand, when the shift from heterotrophic to photoautotrophic condition wasdone when most of the cells had encysted, there was still a decrease in cellnumber but astaxanthin accumulation was very high. The astaxanthinconcentration (114 mg L^-1) and productivity (4.4 mg L^-1d^-1) obtained by this sequential heterotrophic-photoautotrophiccultivation method are very high compared to the data in the literature.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Biomass production in mixotrophic culture of Euglena gracilis under acidic condition and its growth energetics

When Euglena gracilis was grown in the heterotrophic condition with glucose and (NH₄)₂SO₄ as the carbon and nitrogen source, a high cell yield (4.28–4.48 g l^–1) was obtained and the culture pH decreased to 1.6–2. The biomass production in the heterotrophic culture was compared to those in the autotrophic and mixotrophic cultures. Autotrophic growth was 4.7–6.3% of the heterotrophic one, whereas about 15–19% higher growth was obtained in the mixotrophic culture. Moreover, good production of chlorophyll (39.4 mg l^–1) and carotenoids (13.8 mg l^–1) were attained in the mixotrophic culture, giving the highest fermenter productivity with respect to biomass as well as chlorophyll and carotenoids. Through an energetic analysis in the mixotrophic culture, it was estimated about 25–28% of the total ATP requirement is formed in the photochemical reactions. This resulted in an improved biomass production in the mixotrophic culture of E. gracilis.     

In vitro production studies with a wild-type Helicoverpa baculovirus

The potential use of a wild-type Helicoverpa baculovirus as a biopesticide, using insect cell culture for its production, has been investigated. A Helicoverpa zea cell line was adapted to grow in suspension culture using a serum-free medium, SF900II and serum supplemented SF900II. The serum supplemented cells were infected with a wild-type nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV), at different stages of growth, in conditioned and tresh medium, to determine the effect of cell density on polyhedra production. Cultures infected at low cell densities, produced similar yields of virus (20–40 PIB/cell), irrespective of medium conditions. However, in infections which occurred at high cell densities, there was a 16-fold improvement in cell specific yields, when the spent medium was renewed with fresh medium prior to infection. Results indicated that only 60–70% of the viable cells in a culture produced polyhedra as a result of infections.     

Production of α-tocopherol by sequential heterotrophic–photoautotrophic cultivation of Euglena gracilis

Photoautotrophic cultivation of Euglena gracilis results in cells with high α-tocopherol content but the final cell concentration is usually very low due to the difficulty of supplying light efficiently to the photobioreactor. On the other hand, Euglena grows heterotrophically to high cell concentrations, using various organic carbon sources, but the α-tocopherol contents of heterotrophically grown cells are usually very low. Sequential heterotrophic/photoautotrophic cultivation, by which cells are grown heterotrophically to high cell concentrations and then transferred to photoautotrophic culture for accumulation of α-tocopherol was therefore investigated for efficient α-tocopherol production. In batch culture, using glucose as the organic carbon source, the cellular α-tocopherol content increased from 120 μg g⁻¹ at the end of heterotrophic phase to more than 400 μg g⁻¹ at the end of the photoautotrophic phase. By using ethanol as the organic carbon source during the heterotrophic phase, adding corn steep liquor as a nitrogen source and optimizing light supply during the photoautotrophic phase, the α-tocopherol content of the cells at the end of the photoautotrophic phase increased to 1700 μg g⁻¹. A system consisting of a mini-jar fermentor (for the heterotrophic phase) and an internally illuminated photobioreactor (for the photoautotrophic phase) was then constructed for continuous sequential heterotrophic/photoautotrophic cultivation. The cells were continuously cultivated heterotrophically in the mini-jar fermentor and the effluent was continuously passed through the photobioreactor for α-tocopherol accumulation. In this way, it was possible to produce 7 g l⁻¹ cells containing about 1100 μg α-tocopherol per g-cell continuously for more than 420 h. The continuous process resulted in α-tocopherol productivity of 100 μg l⁻¹ h⁻¹ which is about 9.5 and 4.6 times higher than those obtained in batch photoautotrophic culture and batch heterotrophic cultures, respectively.     

Comparison of secondary product accumulation in photoautotrophic,photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures

Summary Photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures were compared for the constitutive accumulation of secondary metabolites and the elicitor-induced formation of the phytoalexin capsidiol. Nicotine and chlorogenic acid were found in high amounts in the heterotrophic cultures and in moderate concentrations in photomixotrophic but not in photoautotrophic cells. Nicotinic acid-N-glucoside occured in all culture types; in photoautotrophic and photomixotrophic cells the formation of N-methylnicotinic acid (trigonelline) was also observed. Treatment with a fungal elicitor led to substantial accumulation of capsidiol in heterotrophic and photomixotrophic cells and in only low levels in photoautotrophic cultures. Elicitor-treated photomixotrophic cells showed a pronounced increase in cell wall-bound phenolics. The levels of nicotine, nicotinic acid-N-glucoside and trigonelline were not affected by elicitation.Abbreviations hcc heterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture - fr.wt. freshweight - nic-N-glc nicotinic acid-N-glucoside - PMG Phytophthora megasperma f. sp. glycínea - HPLC high performance liquid chromatography - GC gas chromatography - TLC thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - Kin kinetin - BAP 6-benzylaminopurine - NAA -naphthylacetic acid     

Heterotrophic metabolism and diazotrophic growth ofNostoc sp. fromCycas circinalis

The cyanobiont ofCycas circinalis (identified asNostoc sp.) was isolated and its heterotrophic metabolism was studied in free culture under nitrogen-fixing conditions. Morphology, growth rate, nitrogenase activity, biochemical composition, efficiency of assimilation of organic carbon and molecular nitrogen were determined under different conditions of energy and carbon supply. The study has revealed the high potential of the heterotrophic metabolism in this symbiotic cyanobacterium. Although low rates of metabolic activities were attained under heterotrophic conditions, the efficiencies of organic carbon utilization (0.48 g cell-carbon per g glucose-carbon in chemoheterotrophy, from 0.65 to 0.74 under photoheterotrophy) and of N₂ assimilation (35.0 mg N₂ fixed per g glucose used in chemoheterotrophy, from 58.3 to 61.9 under photoheterotrophy) displayed by this organism were among the highest ever found in diazotrophically grown microorganisms. The isolate fromC. circinalis was able to grow indefinitely in the dark under nitrogen-fixing conditions, maintaining a well balanced biosynthetic activity and the capacity to resume photosynthetic metabolism quickly. The significance of the heterotrophic potential of this symbioticNostoc is discussed.     

Modeling of recycling oxic and anoxic treatment system for swine wastewater using neural networks

A recycling reactor system operated under sequential anoxic and oxic conditions for the treatment of swine wastewater has been developed, in which piggery slurry is fermentatively and aerobically treated and then part of the effluent is recycled to the pigsty. This system significantly removes offensive smells (at both the pigsty and the treatment plant), BOD and others, and may be cost effective for small-scale farms. The most dominant heterotrophic were, in order,Alcaligenes faecalis, Brevundimonas diminuta andStreptococcus sp., while lactic acid bacteria were dominantly observed in the anoxic tank. We propose a novel monitoring system for a recycling piggery slurry treatment system through the use of neural networks. In this study, we tried to model the treatment process for each tank in the system (influent, fermentation, aeration, first sedimentation and fourth sedimentation tanks) based upon the population densities of the heterotrophic and lactic acid bacteria. Principal component analysis (PCA) was first applied to identify a relationship between input and output. The input would be microbial densities and the treatment parameters, such as population densities of heterotrophic and lactic acid bacteria, suspended solids (SS), COD, NH₄ ⁺-N, or-tho-phosphorus (o-P), and total-phosphorus (T-P). Then multi-layer neural networks were employed to model the treatment process for each tank. PCA filtration of the input data as microbial densities was found to facilitate the modeling procedure for the system monitoring even with a relatively lower number of input. Neural networks independently trained for each treatment tank and their subsequent combined data analysis allowed a successful prediction of the treatment system for at least two days.     

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.     

Interrelationships between <Emphasis Type="Italic">Dunaliella</Emphasis> and halophilic prokaryotes in saltern crystallizer ponds

Thanks to their often very high population densities and their simple community structure, saltern crystallizer ponds form ideal sites to study the behavior of halophilic microorganisms in their natural environment at saturating salt concentrations. The microbial community is dominated by square red halophilic Archaea, recently isolated and described as Haloquadratum walsbyi, extremely halophilic red rod-shaped Bacteria of the genus Salinibacter, and the unicellular green alga Dunaliella as the primary producer. We review here, the information available on the microbial community structure of the saltern crystallizer brines and the interrelationships between the main components of their biota. As Dunaliella produces massive amounts of glycerol to provide osmotic stabilization, glycerol is often postulated to be the most important source of organic carbon for the heterotrophic prokaryotes in hypersaline ecosystems. We assess here, the current evidence for the possible importance of glycerol and other carbon sources in the nutrition of the Archaea and the Bacteria, the relative contribution of halophilic Bacteria and Archaea to the heterotrophic activity in the brines, and other factors that determine the nature of the microbial communities that thrive in the salt-saturated brines of saltern crystallizer ponds. Three-letter abbreviations for names of genera of Halobacteriaceae conform the recommendations of the ICSP Subcommittee on the Taxonomy of Halobacteriaceae.     

Axenic Culture of the Heterotrophic Dinoflagellate Pfiesteria shumwayae in a Semi-Defined Medium

ABSTRACT. A semi-defined, biphasic culture medium was developed that supported the axenic growth of three strains of the heterotrophic dinoflagellate Pfiesteria shumwayae . Maximum cell yields and division rates in the semi-defined medium ranged from 0.1 × 10⁵ to 4.0 × 10⁵ cells/ml and 0.5 to 1.7 divisions/day, respectively, and depended on the concentration of the major components in the medium as well as the P. shumwayae strain. The medium contained high concentrations of certain dissolved and particulate organic compounds, including amino acids and lipids. Pfiesteria shumwayae flagellated cells were attracted to insoluble lipids present in the medium and appeared to feed on the lipid particles, suggesting that phagocytosis may be required for growth in axenic culture. Development of a semi-defined medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of the biochemical requirements of P. shumwayae , needed to advance understanding of the nutritional ecology of this species. Further, this medium provides an economical, simplified method for generating high cell densities of P. shumwayae in axenic culture that will facilitate controlled investigations on the physiology and biochemistry of this heterotrophic dinoflagellate.     

Neural network modeling for nutrient dynamics in a recycling piggery slurry treatment system

A recycling reactor system operated under sequential anoxic and oxic conditions was evaluated, in which the nutrients of piggery slurry were anaerobically and aerobically treated and then a portion of the effluent was recycled to the pigsty. The most dominant aerobic heterotrophs from the reactor were Alcaligenes faecalis (TSA-3), Brevundimonas diminuta (TSA-1) and Abiotrophia defectiva (TSA-2) in decreasing order, whereas lactic acid bacteria, LAB (MRS-1, etc.) were most dominantly observed in the anoxic tank. Here we have tried to model the nutrient removal process for each tank in the system based on population densities of heterotrophic and LAB. Principal component analysis (PCA) was first applied to delineate a relationship between input (microbial densities and treatment parameters such as population densities of heterotrophic and LAB, suspended solids (SS), COD, NH₄ ⁺–N, ortho-phosphorus, and total phosphorus) and output. Multi-layer neural networks using an error back-propagation learning algorithm were then employed to model the nutrient removal process for each tank. PCA filtration of microbial densities as input data was able to enhance generalization performance of the neural network, and this has led to a better prediction of the measured data. Neural networks independently trained for each treatment tank and the combined analysis of the subsequent tank data allowed a successful prediction of the treatment system for at least 2 days.     

Effects of mosquito larvae removal with Bacillus thuringiensis israelensis (Bti) on natural protozoan communities

The protein crystals produced by Bacillus thuringiensis israelensis (Bti) are used against the larvae of pestiferous flood-water mosquitoes in ephemeral wetlands. Although mosquito larvae are considered important predators on protozoans and bacteria, it is not known how a distinct reduction of mosquito larvae density in natural wetlands caused by application of Bti may indirectly affect these microbial communities. Here we show, in a large scale experiment in six natural wetlands, that the densities of heterotrophic protozoans was on an average 4.5 times higher in wetland areas treated with Bti than in control areas. In addition, the taxonomic richness of heterotrophic protozoans increased on an average of 60% in areas with Bti application compared to control areas. The increase in protozoan density and richness was fairly consistent among sites of different wetland habitats. We discuss the potential implications of our results for other parts of the ecosystem. Handling editor: K. Martens     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

Factors promoting the establishment of primary cultures of liver cells fromXenopus larvae

Summary The requirements for establishment and survival of primary cultures of larval amphibian liver cells were investigated.Plating efficiency was found to be enhanced by a collagen substrate, by diluted conditioned medium from an adultXenopus kidney cell line and by high initial cell densities. Plating efficiency was highest at a tonicity of 165–220 mOsm/kg. In cultures with undiluted conditioned medium the increase in cell number was 50–60% greater than in controls, where it was about 2-fold between day 3 and 6 of culture. Conditioned medium from theXenopus kidney cell line is assumed to contain at least two components, which are effective at different concentrations and stimulate either plating efficiency and cell aggregation or cell proliferation.In cultures without collagen sheets, cell flattening is greatly reduced, indicating that cell shape is also dependent upon the substrate.     

The lipids in photoautotrophic and heterotrophic cell suspension cultures of Chenopodium rubrum

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。