The regreening of nitrogen-deficient Chlorella fusca

Chlorella fusca, strain 211-15, cells degreened in a nitrogen-deficient mineral growth medium in the light for 4–6 weeks were regreened for up to 24 hrs in a nitrogen rich medium that leads to synchronous cell division at 24–26 hrs. Structural changes in the plastid membranes during the regreening period were observed by thin section and freeze-fracture electron microscopy. Nitrogen-deficient plastids were found to have non-appressed lamellae, prolamellar body-like membrane aggregations, and only 2 types of freeze-fracture face. At this time no photosynthetic oxygen evolution could be demonstrated. After 6 hrs regreening the plastid lamellae had fused to form bands of appressed lamellae and the four types of freeze-fracture face, described previously, were visible. At this time photosynthetic oxygen evolution could be demonstrated. After 24 hrs regreening the plastids had an appearance typical of normally grown Chlorella and had commenced to divide. Supporting evidence for these developmental stages is presented from isolated chloroplast particle fractions. An unusual type of cell wall proliferation was observed in the nitrogen-deficient Chlorella cells that resulted in the laying down of several walls, each with a trilaminar component.     

The regreening of nitrogen-deficient Chlorella fusca II. Structural changes during synchronous regreening.

Chlorella fusca, strain 211-15, cells degreened in a nitrogen-deficient mineral growth medium in the light for 4-6 weeks were regreened for up to 24 hrs in a nitrogen rich medium that leads to synchronous cell division at 24-26 hrs. Structural changes in the plastid membranes during the regreening period were observed by thin section and freeze-fracture electron microscopy. Nitrogen-deficient plastids were found to have non-appressed lamellae, prolamellar body-like membrane aggregations, and only 2 types of freeze-fracture face. At this time no photosynthetic oxygen evolution could be demonstrated. After 6 hrs regreening the plastid lamellae had fused to form bands of appressed lamellae and the four types of freeze-fracture face, described previously, were visible. At this time photosynthetic oxygen evolution could be demonstrated. After 24 hrs regreening the plastids had an appearance typical of normally grown Chlorella and had commenced to divide. Supporting evidence for these developmental stages is presented from isolated chloroplast particle fractions. An unusual type of cell wall proliferation was observed in the nitrogen-deficient Chlorella cells that resulted in the laying down of several walls, each with a trilaminar component.     

Chlorophyll synthesis and intracellular fluctuations of 5-aminolaevulinate formation during the regreening of nitrogen-deficient Chlorella fusca

Orange, chlorophyll-deficient cells of Chlorella fusca were obtained by prolonged exposure (6 wk) to light and CO₂ (1.5% in air) in a nitrogen-sparse medium: growth ceased after 6 days, chlorophyll formation after 3 days, and then chlorophyll degradation followed with a drop in chlorophyll a:b ratio. When the 6-wk-old cells were exposed to light in a nitrogen-rich medium and sparged with CO₂ (1.5% in air) rapid chlorophyll synthesis ensued with preferential synthesis of chlorophyll a. Regreening under these conditions was complete in approximately 24–30 hr and during this period no cell division occurred. We were unable to demonstrate 5-aminolaevulinate synthase (EC 2.3.1.37) in cell-free extracts of regreening Chlorella but demonstrated aminolaevulinate formation by whole regreening cells incubated in the presence of laevulinate, an inhibitor of aminolaevulinate dehydratase (EC 4.2.1.24). Chlorophyll synthesis was almost completely inhibited by 100 mm laevulinate, and a stoichiometric relationship exists between aminolaevulinate formation and the chlorophyll deficit caused by the presence of laevulinate: thus, the use of the inhibitor provides a true indication of the ability of the cells to form aminolaevulinate.Using this technique we found that chlorophyll synthesis during regreening appears to be regulated by the availability of aminolaevulinate since there was a correlation between the rate of aminolaevulinate and chlorophyll synthesis: both reached a maximum about halfway through the regreening period. It was not possible to decide whether the availability of aminolaevulinate was limited by the level or activity of aminolaevulinate synthase or by the supply of succinyl CoA. Regreening of orange Chlorella was inhibited by cycloheximide. Regreening of Chlorella can occur in the dark if vigorously sparged with oxygen so differing from greening of higher plants which is light dependent.Both [1,4-¹⁴C]succinate and [2-¹⁴C]glycine were incorporated into aminolaevulinate by partly regreened Chlorella fusca cells incubated in the presence of laevulinate.     

The regreening of nitrogen-deficientChlorella fusca

The wildtype strain 211-15 of the green algaChlorella fusca appears orange after long incubation in nitrogen-sparse media. The cells regreen within 24 h when incubated in a nitrogen-rich medium in the light. During the regreening process the very low chlorophyll a: b ratio of 1.8 is increased to 3.3 indicating a preferential synthesis of chlorophyll a. Respiratory activity of the cells is high throughout the regreening period. Photosynthetic oxygen evolution occurs 4–6 h after the commencement of regreening. When regreening is completed under the defined conditions synchronized cell division occurs.     

Spectral effectiveness in chlorophyll and 5-aminolevulinic acid formation during regreening of glucose-bleached cells of Chlorella protothecoides

Regreening of glucose-bleached cells of Chlorella protothecoidesis stimulated by light. Spectral effectiveness in the processshowed maxima around 370, 440 and 480 nm, suggesting a flavoproteinas primary photoreceptor. Action spectra of ALA synthesis provedto be similar to those of chlorophyll formation, indicatingthat light stimulation of greening in this alga is regulatedat the first step of chlorophyll biosynthesis. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 27, 1978; )     

P-31 in-vivo NMR investigation on the function of polyphosphates as phosphate-and energysource during the regreening of the green alga Chlorella fusca

The green alga Chlorella fusca accumulates polyphosphates under conditions of nitrogen starvation while deassembling the photosynthetic apparatus. The polyphosphate content of cells regreening after resupply with nitrate under different culture conditions was investigated by P-31 in-vivo NMR spectroscopy. Neither phosphate deficiency nor anaerobiosis during the first hours of regreening inhibited the recovery of the cells. Polyphosphates were degraded during regeening. Differences in the amount of polyphosphates of phosphate supplied and deficient cells occurred only after more then 8 h. After 16 h phosphate deficient cells had still 75% of the polyphosphate content of phosphate suppled cells. In cells kept under anaerobic conditions polyphosphate degradation was much higher than in oxygen supplied cells. After 8 h they contained less than 50% of the polyphosphate content of oxygen supplied cells. These data suggest that polyphosphates serve as obligatory phosphate source during regreening and may be used as an energy source.Non standard abbreviations EDTA Ethylene diamine tetraacetic acid - FID Free induction decay - MOPSO 3-(N-morpholine)-2-hydroxy-propanesulfonic acid - NMR Nuclear magnetic resonance - PP Polyphosphates - PP₄ central phosphate groups of polyphosphates     

Gas metabolism in ovine rumen cultures on a nitrogen-deficient medium.

Inocula of rumen fluid from sheep were grown on a medium of low nitrogen content under an oxygen-free atmosphere containing N2. The cultures produced methane and carbon dioxide. Hydrogen gas was utilized and the partial pressure of N2 decreased by 24% in 48 h. Assuming this N2 to have been reduced to metabolically available nitrogenous compounds, these account for about 0.2% of the sheep's daily need.     

Light-induced postharvest regreening of pummelo fruit*

Regreening was observed and measured in harvested pummelo fruit stored in the light. At temperatures of 22 - 28°C, regular daylight was sufficient for regreening to occur. The addition of continuous fluorescent light intensified the process. Pre-stored fruit held in darkness at 11°C and non-stored fruit responded to both light conditions in a similar manner. Electron microscopy has shown that globular chromoplasts revert to chloroplasts during regreening. The similarities between regreening processes in preharvest and postharvest fruits are discussed.     

Oxidative assimilation treatment of a nitrogen-deficient toxic waste

Batch growth tests were performed under both replicating and nonproliferating (no nitrogen source in medium) conditions with acclimated heterogenous populations that utilized phenol as a sole source of carbon and energy. It was shown that the acclimated populations could efficiently remove the toxic waste component phenol under nonproliferating conditions by utilizing an oxidative assimilation mechanism. The phenol was assimilated and converted into nonnitrogenous storage products. During the assimilation process, the cells had a tendency to excrete some product (nonsubstrate) chemical oxygen demand (COD). Bench-scale oxidative assimilation units were operated by sequentially feeding a carbon source (phenol) and nitrogen source (ammonium sulfate) to heterogeneous populations. This demonstrated that, subsequent to the addition of the nitrogen source to the medium, the cells utilized the stored carbon for replication. Four of these units were operated at different phenol COD-to-ammonia-nitrogen ratios of 10:1, 20:1, 40:1, and 50:1. All of these units demonstrated excellent removal of phenol using an oxidative assimilation mechanism. These results suggested the feasibility of utilizing a continuous flow oxidative assimilation process for the treatment of nitrogen-deficient phenolic wastes. This process would be advantageous over conventional treatment processes in that it would realize a savings in chemical costs (ammonia as nitrogen source) and prevent leakage of excess ammonia from the system.     

Biological hydrogen production from nitrogen-deficient substrates

Dark fermentation of biomass using mixed bacterial cultures is one approach to producing renewable H(2). The objective of this work was to determine if this approach could be applied to N-deficient feedstocks using an N(2)-fixing mixed culture. A mixed culture produced up to 240 mL H(2)/g glucose (1.9 mol H(2)/mol glucose) from a medium initially lacking combined N. Yields from sugarcane were also promising: 170 mL H(2)/g volatile solids (7.5 mmol H(2)/g volatile solids). This approach could reduce economic and environmental costs of fermentative H(2) production, provide combined N for subsequent bioconversion stages, and improve effluent suitability for subsequent uses.     

The cryopreservation of Chlorella

The temperature at which Chlorella 211/8h was grown determined the response to a subsequent stress of freezing to and thawing from-196°C. Cells cultured at 20°C were the most sensitive to freezing injury; at both higher and lower growth temperatures resistance to damage induced by freezing developed. At all culture temperatures examined the freezing tolerance varied with the age of culture.     

The cryopreservation of Chlorella

Following a shift from autotrophic to heterotrophic nutrition, cells of Chlorella protothecoides become sensitive to the stresses of freezing and thawing. The injury then observed at slow rates of cooling cannot be explained by the cellular response to hypertonic solutions, and at faster cooling rates intracellular ice formation was not demonstrated to be damaging. These findings are at variance with suggested mechanisms of injury in other cellular systems. The results are compared with alterations in ultrastructure and in the composition of the cellular fatty acids.Abbreviations BHT butylated hydroxy toluene - TLC thin layer chromatography - AW-DMCS acid washed and silanized     

The regulation of synthesis of mitochondrial enzymes in regreening and division-synchronized Euglena cultures

The effect of light and carbon nutrition on the synthesis of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) in dark-grown resting (carbon deficient) and in phototrophic division-synchronized cultures of Euglena gracilis Klebs strain z were investigated. Exposure of dark-grown Euglena to white or red light produced a transient increase in the specific activities of citrate synthase and malate dehydrogenase but blue light (of equal energy) was ineffective. Citrate-synthase activity increased at the end of the light phase and in early dark phase in phototrophic cultures division-synchronized by a regime of 14 h light-10 h dark. The addition of ethanol or malate produced a twofold increase in citrate-synthase activity compared with phototrophic cultures. White and blue light, but not red light, produced a transient repression of the metabolite-induced increase in citrate-synthase activity in division-synchronized cultures. Since only red light could effect a transient increase in the specific activity of mitochondrial enzymes, and the blue-red plastid receptor should respond to both blue and red light, the synthesis of mitochondrial enzymes in regreening cultures may be under the control of a new photoreceptor responding only to red light. In division-synchronized phototrophic cells the primary effector of synthesis of mitochondrial enzymes is not light but carbon nutrition.     

Fatty-acid metabolism in senescing and regreening soybean cotyledons

Changes in fatty-acid metabolism were studied in soybean (Glycine max Merr.) cotyledons during senescence as well as in cotyledons which had been caused to regreen by removal of the epicotyl from the seedling. The activities of the enzymes acetyl-CoA synthetase (EC 6.2.1.1) and fatty-acid synthetase in plastids isolated from the cotyledons decreased during senescence but increased in response to regreening. These changes in enzyme activities followed the same pattern as changes in the quantities of chlorophyll and polyunsaturated fatty acids in this tissue. The in-vivo incorporation of [¹⁴C]acetate into total fatty acids in the senescing and regreening cotyledons did not vary markedly with age. In addition, the quantity of label in fatty acids did not decrease for as much as 60 h after the removal of the substrate. During this 60-h period however, there was substantial redistribution of the label among the individual fatty acids. While the labelling pattern of the individual fatty acids did not vary significantly with respect to age in the senescing cotyledons, there was a substantial increase in the synthesis of labelled polyunsaturated fatty acids in the regreening tissue. Thus, the incorporation of [¹⁴C]acetate into fatty acids did not reflect the changes in the quantities of the individual fatty acids in senescing tissue as well as they did in regreening tissue.     

A short-filament mutant of Anabaena sp. strain PCC 7120 that fragments in nitrogen-deficient medium.

Strain 129 is a fragmentation mutant of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Growing with fixed nitrogen, this mutant forms filaments that are much shorter than wild-type filaments. Following starvation for fixed nitrogen, strain 129 becomes nearly unicellular and forms few heterocysts, although electron microscopy suggests that proheterocysts form while fragmentation occurs. Starvation for sulfate, phosphate, iron, and calcium does not cause this fragmentation. The affected gene in strain 129, fraC, was cloned by complementation and characterized. It encodes a unique 179-amino-acid protein rich in phenylalanine. Insertional inactivation of the chromosomal copy of fraC results in a phenotype identical to that of strain 129, while complementation using a truncated version of FraC results in only partial complementation of the original mutant. Heterocysts could be induced to form in N-replete cultures of strain 129, as in wild-type cells, by supplying extra copies of the hetR gene on a plasmid. Thus, FraC is required for the integrity of cell junctions in general but is apparently not directly involved in normal differentiation and nitrogen fixation.     

Mobilisation of NADPH-glutamate dehydrogenase mRNA in regreening cultures of Euglena gracilis

Exposure of dark-grown resting Euglena gracilis Klebs strain Z Pringsheim to light results in a transient increase in the specific activity of NADPH-glutamate dehydrogenase. NADPH-glutamate dehydrogenase antibody was used to detect NADPH glutamate dehydrogenase resulting from the translation of total polyadenylated RNA and polysomal RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in cells at all stages of development and present on polysomes from dark-grown and regreening cells but not on polysomes from dark-grown resting cells. These results indicate that the light-induced increase in NADPH-glutamate dehydrogenase in dark-grown resting cells represent an increase in the rate enzyme synthesis resulting from the mobilisation of NADPH-glutamate dehydrogenase mRNA onto polysomes.     

Biomass content governs fermentation rate in nitrogen-deficient wine musts

Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.