Regulation of Chloroplast Development by Nitrogen Source and Growth Conditions in a Chlorella protothecoides Strain

Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6-bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6-phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Metabolic aspects of LPP cyanophage replication in the cyanobacterium Plectonema boryanum

Cyanophage LPP1G is reproduced at the same yield in heterotrophic conditions (dark, glucose) as in photoautotrophic conditions; aerobiosis is required for dark cyanophage replication. Exogenous glucose is not required for the cyanophage replication in the dark in heterotrophically grown cells. In photoautotrophically grown cells, the maximum burst size in dark and glucose is delayed for a period corresponding to glucose uptake induction. Cyanophage LPP2SPI replication occurs in conditions where only Photosystem I operates. Of photosynthesis parameters tested, only CO₂ photoassimilation is affected during cyanophage LPP1G infection under photoautotrophic conditions.     

STIMULATION OF GROWTH IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) BY HUMIC ACIDS UNDER IRON LIMITED CONDITIONS1,2

Stimulatory affects of humic acids of molecular weight 30,000 or greater on iron-starved Scenedesmus obliquus (Turp.) Kütz. in association with bacteria were studied by growth and Fe uptake experiments. Humic acids stimulated growth of Fe-starved cells by decreasing the lag phase and extending the growth phase. Humic acids stimulated increased algal growth in medium containing EDTA as well as in medium containing no EDTA, indicating humic acids are not stimulating algal growth under Fe limiting conditions by creating a soluble Fe pool. Humic acids decreased Fe availability to Fe-starved S. obliquus. Iron bound to humic acids is unavailable for uptake by Fe-starved cells indicating growth stimulation is not due to chelation effects alone. Stimulation of growth is not a membrane phenomenon as humic acids show the same stimulatory effect when in contact with cells or separated by dialysis membrane. Humic acids also stimulate growth in the dark, with and without aeration, indicating use as a heterotrophic substrate. A photoheterotrophic mechanism is indicated by increased algal growth caused by illuminating cultures, containing humic acids but excluding CO₂.     

HETEROTROPHY AND PHOTOHETEROTROPHY BY ANTARCTIC MICROALGAE: LIGHT-DEPENDENT INCORPORATION OF AMINO ACIDS AND GLUCOSE 1

Diatoms isolated from the benthic, planktonic and sea ice microbial communities in Mc Murdo Sound, Antarctica assimilated ambient concentrations of dissolved amino acids and glucose in both the light and dark. Uptake of amino acids but not glucose was influenced by the iucubation irradiance and amino acid uptake rates were up to 250 times greater than those of glucose. Amino acids were incorporated into proteins and other complex polymers and the rates of assimilation and patterns of polymer synthesis were similar to those of the light-saturated photosynthetic incorporation of inorganic carbon. This suggests that these diatoms can use exogenous amino acids to synthesize the essential macromolecules for heterotrophic growth. The assimilation of dissolved organic substrates could supplement light-limited growth during the austral spring and summer as well as potentially support the heterotrophic growth of these diatoms throughout the aphotic polar winter.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.     

Differential growth and biochemical composition of photoautotrophic and heterotrophic <Emphasis Type="Italic">Isochrysis maritima</Emphasis>: evaluation for use as aquaculture feed

The growth and biochemical composition of photoautotrophic and heterotrophic Isochrysis maritima in 50 L of Walne’s medium were compared. Heterotrophic I. maritima fed with 0.02 M glucose had a 4.6-fold higher maximum cell density (38.17 ± 0.23 × 10⁶ cells mL^?1) than photoautotrophic cells (8.29 ± 0.70 × 10⁶ cells mL^?1). The carbohydrate content was slightly higher in heterotrophic cells at all growth stages (mid-exponential, 40.8%; early stationary, 48.3%; and late stationary, 47.6%), but there was no significant effect on the protein content under either trophic condition. The total saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were higher under heterotrophic conditions than those under photoautotrophic conditions. However, because omega-3 PUFAs are the most essential element in feed nutrition, low results for eicosapentaenoic acid (EPA) (0.28 ± 0.06%) and docosahexaenoic acid (DHA) (3.22 ± 0.26%) in the heterotrophic cells compared to the photoautotrophic cells (EPA: 0.44 ± 0.11%; DHA: 8.58 ± 0.73%) plus a low omega-3/6 PUFAs ratio (heterotrophic: 0.16–0.47; photoautotrophic: 2.60–2.88) and high value of (SFA + MUFA)/PUFA (heterotrophic: 5.50–6.81; photoautotrophic: 2.64–3.60) showed that this species is not suitable for aquaculture feed when cultivated under heterotrophic conditions.     

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.     

索罗金小球藻异养转自养过程中基因表达的全局调控

为提高异养条件下索罗金小球藻(Chlorella sorokiniana)蛋白质含量,扩大该藻株在食品和饲料领域的应用,研究发现当异养条件下培养的C. sorokiniana GT-1细胞转入光自养培养条件后,蛋白质含量显著提高。通过转录组学分析揭示了C. sorokiniana GT-1在异养转自养过程中基因表达发生全局变化,其中糖酵解途径与磷酸戊糖途径上调,氮转运和同化途径中的关键酶的编码基因明显上调,且谷氨酸族氨基酸和丙酮酸族氨基酸的生物合成途径的多个酶在转录水平上显著增强。研究还发现在异养条件下藻细胞仍然可以表达部分光合作用蛋白的编码基因,当转入光自养条件后24h内绝大多数光合作用相关蛋白编码基因的转录被激活。结果表明在异养转自养条件过程中蛋白质含量的升高与氮的吸收及利用增加、还原能合成的增强、部分氨基酸的合成上调及光合作用蛋白质的大量合成有关。研究为后续如何通过培养条件优化或代谢工程改造提高C. sorokiniana GT-1产蛋白质的能力提出了新的思路。     

Role of heterotrophic nutrition in growth of the alga Scenedesmus obliquus in high-rate oxidation ponds.

The green alga Scenedesmus obliquus readily adapted to heterotrophic growth in the dark, utilizing glucose as the sole carbon source. Heterotrophic cells differed significantly from photoautotrophic cells with respect to several physiological properties such as the rate of photoassimilation of CO2, rate of incorporation of glucose, and chlorophyll a concentration. Oxidation pond cells shared features common to both photoautotrophic and heterotrophic cells. Approximately 15 percent of oxidation pond algal carbon was derived from glucose assimilated directly without first being oxidized by bacteria. Bacteria seem to play a minor role in biological oxygen demand reduction in high-rate oxidation ponds, and their role is probably confined to degradation of biopolymers, thus producing substrates for algal consumption.     

Glutamate Uptake and Metabolism in C-6 Glioma Cells: Alterations by Potassium Ion and Dibutyryl cAMP

Abstract: Uptake and metabolism of glutamate was studied in the C-6 glioma cell line grown in the absence or presence of dibutyryl cyclic AMP (dbcAMP). Glutamate and aspartate uptake were competitive in cells grown under both conditions. Increased [K⁺] in the medium caused a significant decrease in the uptake of both amino acids. A small part of this decrease (<25%) was due to an enhanced efflux of tissue amino acid. The effects of increased [K⁺] were observed whether or not the [Na⁺] in the medium was concomitantly decreased. In cells grown in the presence of 1 mM dbcAMP for 48 h, glutamate uptake and metabolism were altered. Tissue levels of glutamate, aspartate, glutamine, GABA, and alanine were generally less in treated than in naive cells. When incubated with 50 μM [U-¹⁴C]glutamate, there was significantly less incorporation of radioactivity into treated cells with time, resulting in greatly lowered specific radioactivities of glutamate, aspartate, and GABA. However, the rate of labeling of glutamine was greatly increased; this was consistent with the previously observed doubling in glutamine synthetase activity in dbcAMP-treated C-6 cells. Tissue glutamate decarboxylase activity was halved in treated cells, accounting for the large decrease in GABA labeling. The metabolic data suggested a decreased uptake of exogenous glutamate; in studies on initial rates of uptake, the V_max of high-affinity glutamate uptake was decreased by 40%. This decrease was of the same order of magnitude as that observed in the metabolic experiments. Thus, in this glial model, both rapid, acute changes and slower, long-term changes in neuroactive amino acid metabolism were observed. Each of these conditions mimics a stimulus of neuronal origin, and the resulting changes could modulate extrasynaptic activity of neuroactive amino acids.     

DNA synthesis and nuclear division during formation of infection structures by bean rust uredospore germlings

Plectonema boryanum can grow in the dark with ribose, sucrose, mannitol, maltose, glucose, or fructose. Cell doubling times with 10 mM substrate are the following: 5 days with ribose, 6 days with sucrose or mannitol, 10 days with maltose, 12 days with glucose, and 13 days with fructose; with ribose plus 0.1% casamino acids it is 2.5 days. Dark-grown cells appear morphologically similar to light-grown cells. Cells grown in the dark for several years remain pigmented and resume photoautotrophic growth when placed in the light. Dim light (85 lux) increases the growth rate with ribose and with ribose plus casamino acids to nearlytwice that of the dark rate. In moderate light, growth takes place with ribose even in the presence of 1x10^-5 M DCMU.     

Lipids in plant tissue cultures. VIII: Reversible changes in the composition of lipids and their constituent fatty acids in response to alternate shifts in the mode of carbon supply

When heterotrophic cell cultures of red goosefoot (Chenopodium rubrum) turned photoautotrophic, their contents of various glycolipids and phospholipids increased. The total lipids and the individual lipid classes, especially monogalactosyldiacylglycerols, became richer in linolenic and poorer in linoleic acids. When photoautotrophic cell cultures were rendered heterotrophic again a reversal of changes occurred; both the composition of lipids and the patterns of their constituent fatty acids became similar to those of the starting heterotrophic cultures.The results indicate that the biosynthesis of linolenic acid in photoautotrophic cell cultures involves mainly desaturation of linoleic acid and that chain extension of hexadecatrienoic acid is possibly another, though minor pathway. Monogalactosyldiacylglycerols are apparently the substrates preferred for linolenic acid biosynthesis, whereas various phospholipids are the substrates preferred for linoleic acid biosynthesis.During a growth period of 6 weeks, the levels of polyunsaturated fatty acids in the lipids from both heterotrophic and photoautotrophic cell cultures decrease with time, whereas the proportions of palmitic acid increase.     

Role of heterotrophic nutrition in growth of the alga Scenedesmus obliquus in high-rate oxidation ponds

The green alga Scenedesmus obliquus readily adapted to heterotrophic growth in the dark, utilizing glucose as the sole carbon source. Heterotrophic cells differed significantly from photoautotrophic cells with respect to several physiological properties such as the rate of photoassimilation of CO2, rate of incorporation of glucose, and chlorophyll a concentration. Oxidation pond cells shared features common to both photoautotrophic and heterotrophic cells. Approximately 15 percent of oxidation pond algal carbon was derived from glucose assimilated directly without first being oxidized by bacteria. Bacteria seem to play a minor role in biological oxygen demand reduction in high-rate oxidation ponds, and their role is probably confined to degradation of biopolymers, thus producing substrates for algal consumption.     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Application of process mass spectroscopy to the detection of metabolic changes in plant tissue culture

There is a clear need in the area of plant cell culture for methods of on-line estimation of culture parameters. The introduction of plant cells into culture can result in a loss of their photoautotrophic character so that they are largely heterotrophic. As a result, fermentation off-gas analysis may not be confounded by photosynthetically-related O₂ production. In this study performance of a suspension culture of Syringa vulgaris, in a pneumatically agitated bioreactor of in-house design, was investigated. The effect of light on growth, carbohydrate metabolism and the respiratory quotient (RQ), determined by process mass spectroscopy, was studied. Yield coefficients for cells grown in the light and dark were similar although the patterns of carbohydrate uptake were quite different. Maximum biomass yields were higher in this bioreactor than normally observed in shake flasks. The RQ was dynamic during the course of the fermentation, peaking during the transition from the lag phase to the growth phase. It is suggested that the RQ may prove useful as an on-line parameter for monitoring transitions in cellular metabolism during plant cell culture fermentations.Abbreviations RQ respiratory quotient - v.v.m. volume of gas fed to fermenter per unit volume per minute - Y_X/S growth yield coefficient based on total carbohydrate     

GROWTH,PHYSIOLOGY, AND ULTRASTRUCTURE OF A DIAZOTROPHIC CYANOBACTERIUM,CYANOTHECE SP. STRAIN ATCC 51142, IN MIXOTROPHIC AND CHEMOHETEROTROPHIC CULTURES1

The growth, physiology, and ultrastructure of the marine, unicellular, diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, was examined under mixotrophic and chemoheterotrophic conditions. Several organic substrates were tested for the capacity to support heterotrophic growth. Glycerol was the only substrate capable of enhancing mixotrophic growth in the light and supporting chemoheterotrophic growth in the dark. Dextrose enhanced mixotrophic growth but could not support chemoheterotrophic growth. Chemoheterotrophic cultures in continuous darkness grew faster and to higher densities than photoautotrophic cultures, thus demonstrating the great respiratory capacity of this cyanobacterial strain. Only small differences in the pigment content and ultrastructure of the heterotrophic strains were observed in comparison to photoautotrophic control strains. The chemoheterotrophic strain grown in continuous darkness and the mixotrophic strain grown in light/dark cycles exhibited daily metabolic oscillations in N₂ fixation and glycogen accumulation similar to those manifested in photoautotrophic cultures grown in light/dark cycles or continuous light. This “temporal separation” helps protect O₂-sensitive N₂ fixation from photosynthetic O₂ evolution. The rationale for cyclic glycogen accumulation in cultures with an ample source of organic carbon substrate is unclear, but the observation of similar daily rhythmicities in cultures grown in light/dark cycles, continuous light, and continuous dark suggests an underlying circadian mechanism.