Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z

Glycolate was excreted from the 5% CO₂-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O₂ under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO₂ after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O₂ and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO₂ but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

Glycolate Formation and Excretion by Chlorella pyrenoidosa and Netrium digitus

Conditions are described whereby suspensions of Chlorella pyrenoidosa and Netrium digitus photosynthetically biosynthesize and excrete glycolate continuously in high yields. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate-linked enzymes, increased the excretion of glycolate approximately 4-fold in 1 hour (8 millimolar) and 20-fold in 4 hours (40 millimolar) in the presence of 0.2% CO₂ in air. The amount of glycolate excreted in the presence of aminooxyacetate and an atmosphere of 0.2% CO₂ in air equaled or exceeded the amount excreted in 0.2% CO₂ in O₂ minus aminooxyacetate. CO₂ and light were required for glycolate excretion. Aminooxyacetate also stimulated photosynthetic glycolate excretion in an atmosphere of 0.2% CO₂ in nitrogen or helium, although the stimulation was not as great as when air or O₂ was present.

The excreted glycolate was converted to H₂ and CO₂ by the combined action of glycolic oxidase and the formic hydrogenlyase complex found in Escherichia coli in total conversion yields of 80%.

     

The relationship between carbonic anhydrase activity and glycolate excretion in the blue-green alga Coccochloris peniocystis

Summary The rate of glycolate excretion by Coccochloris peniocystis Kütz. cells incubated under conditions of low bicarbonate concentration and high light intensity was linear for only the initial 15 min of incubation and no additional glycolate accumulated in the medium after 20 min. Excretion was maximal in cells grown on 5% CO₂ in air when transferred to an incubation medium containing no added bicarbonate. The inhibitor INH (isonicotinyl hydrazide) had no measurable effect on the amount of glycolate released whereas HPMS (-hydroxy-2-pyridyl methanesulfonate) stimulated excretion 3-fold. Cells transferred to air from growth on 5% CO₂ in air increased in carbonic anhydrase activity, while a decrease occurred in the cells' ability to excrete glycolate. Cells grown on air and switched to 5% CO₂ in air showed an increase in their ability to excrete glycolate with a concomitant decrease in carbonic anhydrase activity. Diamox, a specific inhibitor of carbonic anhydrase, was found to stimulate excretion with both airgrown and 5% CO₂-grown cells which had been off 5% CO₂ for approximately 30 min. The rate of carbon fixation by 5% CO₂-grown cells put on air was found to rise over a 110 min period, corresponding to both the induction period of carbonic anhydrase and the period of decline in the ability of the cells to excrete glycolic acid. These results suggest that the absence of carbonic anhydrase in 5% CO₂-grown cells causes a stimulation of glycolate excretion when these cells are transferred to a low bicarbonate medium, because of an increased rate of glycolate formation due to the oxidation of ribulose diphosphate by molecular oxygen at low internal CO₂ concentrations.Abbreviations INH isonicotinyl bydrazide - HPMS -hydroxy-2-pyridyl methanesulfonate     

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic ¹⁴CO₂ fixation by Chlamydomonas reinhardtii Dangeard, (−) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO₂ or low CO₂ (air). After 2 minutes with air-grown cells, [¹⁴C]glycolate increased from 0.3% of the total ¹⁴C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial ¹⁴CO₂ fixation was into C₄ acids. Initial products of ¹⁴CO₂ fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.     

Glycolate Excretion and the Oxygen to Carbon Dioxide Net Exchange Ratio during Photosynthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells were grown in high (5% v/v) or low (0.03% v/v) CO₂ concentration in air. O₂ evolution, HCO₃⁻ assimilation, and glycolate excretion were measured in response to O₂ and CO₂ concentration. Both low- and high-CO₂-grown cells excrete glycolate. In low-CO₂-grown cells, however, glycolate excretion is observed only at much lower CO₂ concentrations in the medium, as compared with high-CO₂-adapted cells. It is postulated that the activity of the CO₂-concentrating mechanism in low-CO₂-grown cells is responsible for the different dependence of glycolate excretion on external CO₂ concentration in low- versus high-CO₂-adapted cells.     

Inhibition of photorespiration and increase of net photosynthesis in isolated maize bundle sheath cells treated with glutamate or aspartate

Net photosynthetic ¹⁴CO₂ fixation by isolated maize (Zea mays) bundle sheath strands was stimulated 20 to 35% by the inclusion of l-glutamate or l-aspartate in the reaction mixture. Maximal stimulation occurred at a 7.5 mm concentration of either amino acid. Since the photosynthetic rate and the glutamate-dependent stimulation in the rate were equally sensitive to a photosynthetic electron transport inhibitor, 3-(p-chlorophenyl)-1,1-dimethylurea, it was concluded that glutamate did not stimulate CO₂ fixation by supplying needed NADPH (NADH) through glutamate dehydrogenase. Treatment of the bundle sheath strands with glutamate inhibited glycolate synthesis by 59%. Photorespiration in this tissue, measured as the O₂ inhibition of CO₂ fixation (the Warburg effect), was inhibited by treatment with glutamate. The stimulation in net photosynthetic CO₂ fixation probably results from the decrease in photorespiratory CO₂ loss. This metabolic regulation of the rate of glycolate synthesis and photorespiration observed with isolated bundle sheath strands could account for the inability to detect rapid photorespiration in the mature intact maize leaf.     

Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic ¹⁴CO₂ fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O₂ level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O₂ concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO₂ per gram fresh weight per hour (a 60% increase) when the O₂ level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO₂ per gram fresh weight per hour (a 22% increase). The decrease in the O₂ sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.

The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O₂ concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO₂ fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants

Mass spectrometric techniques were used to trace the incorporation of [¹⁸O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C₃ plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [¹⁸O]oxygen at the CO₂ compensation point were heavily labeled with ¹⁸O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of ¹⁸O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [¹⁸O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.

The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of ¹⁸O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.

The carboxyl oxygens of 3-phosphoglycerate also became labeled with ¹⁸O in 20- and 40-minute feedings with [¹⁸O]oxygen to intact leaves at the CO₂ compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 ± 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO₂ compensation point.

Oxygen uptake sufficient to account for about half of the rate of ¹⁸O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO₂ compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.

These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.

     

Erratum

Glycolate synthesis was inhibited 40–50% in illuminated tobacco leaf disks, which have rapid rates of photorespiration, when floated on 20 mm potassium glycidate (2,3-epoxypropionate), an epoxide similar in structure to glycolate. The inhibitor also decreased the release of photorespiratory CO₂ about 40%, and the specificity of glycidate was demonstrated by the 40–50% increase in rate of photosynthetic CO₂ uptake observed in its presence. The importance of glycolate synthesis and metabolism in the production of photorespiratory CO₂ and the role of glycolate in diminishing net photosynthesis in species with rapid rates of photorespiration was thus further confirmed. L-(or 2S)-Glycidate was slightly more active than DL-glycidate, but glycidate was more effective as a specific inhibitor in leaf tissue than several other epoxide analogs of glycolate examined. The products of photosynthetic ¹⁴O₂ fixation after 3 or 4 min of uptake were proportionately altered in the presence of glycidate, and the specific radioactivity of the [¹⁴C]glycolate produced was closer to that of the ¹⁴CO₂ supplied. Glycidate inhibited glycolate synthesis in tobacco leaf disks irreversibly, since the degree of inhibition was the same for at least 2 hr after the inhibitor solution was removed. Glycidate also blocked glycolate synthesis in maize leaf disks, tissue with low rates of photorespiration, but large increases in net photosynthesis were not observed in maize with glycidate, because glycolate synthesis is normally only about 10% as rapid in maize as in tobacco. The demonstration of increases in net photosynthesis of 40–50% when glycolate synthesis (and photorespiration) is blocked with glycidate indicates in an independent manner that the biochemical or genetic control of photorespiration should permit large increases in plant productivity in plant species possessing rapid rates of photorespiration.     

Glycolate Metabolism in Low and High CO(2)-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by O-Labeling

Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring ¹⁸O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO₂-grown cells than in air-grown cells when both were assayed under the same O₂ and CO₂ concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O₂ levels and inhibited by high CO₂ levels. Glycolate synthesis in 1.5% CO₂-grown Chlorella, when exposed to a 0.035% CO₂ atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O₂ concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O₂ concentration was lowered to 2% or the CO₂ concentration was raised to 1.5%. Glycolate excretion was also sensitive to O₂ and CO₂ concentrations in 1.5% CO₂-grown cells and the glycolate that was excreted was ¹⁸O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory ¹⁸O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

An investigation of glycolate excretion in two species of blue-green algae

Summary The amount of ¹⁴C-glycolate excreted by Oscillatoria sp. and Anabaena flos-aquae is less than 1% of the ¹⁴C fixed by the algae during photosynthesis. Transfer of cells grown on 5% CO₂ in air to a medium of low bicarbonate concentration or treatment of the cells with isonicotinyl hydrazide (INH) during photosynthesis, caused little increase in glycolate excretion. -Hydroxysulfonates failed to stimulate massive excretion of glycolate. Although these blue-green algae excreted little glycolate, a significant proportion of the photosynthetically fixed carbon was excreted in the form of basic, neutral and acidic compounds, and such excretion was greater in 5% CO₂-grown cells than in air-grown cells.     

Influence of the glucose input concentration on the kinetics of metabolite production by Klebsiella aerogenes NCTC 418: Growing in chemostat culture in potassium- or ammonia-limited environments

Thiobacillus neapolitanus grown in minerals medium in a thiosulfate-limited chemostat excreted 15% of all the carbon dioxide fixed as ¹⁴C-organic compounds at a dilution rate (D) of 0.03 h^-1. At D=0.36 h^-1 this excretion was 8.5%. Up to a D of 0.2h^-1 glycolate was the major excretion product. Glycolate excretion was maximal at a pO₂ of 100% air saturation (a.s.) and not detectable at a pO₂ of 5% (a.s.). Increasing the pCO₂ of the gassing mixture to 5% (v/v), at a pO₂ of 50% a.s. resulted in a lowering of the glycolate excretion from 3.5% of the total CO₂ fixed to 1.8%. These results indicate that glycolate excretion in T. neapolitanus is due to oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase. HPMS (2-pyridylhydroxymethanesulfonate), an inhibitor of glycolate metabolism, did not stimulate the glycolate production in T. neapolitanus. Glycolate excretion was not observed in thiosulfate-limited chemostat cultures of the obligately chemolithotrophic Thiomicrospira pelophila or in thiosulfate- or formate-grown cultures of the facultatively chemolithotrophic Thiobacillus A2.Abbreviation HPMS 2-pyridylhydroxymethanesulfonate     

Glycolate from microalgae: an efficient carbon source for biotechnological applications

Glycolate is produced in autotrophic cells under high temperatures and C_i‐limitation via oxygenation of ribulose‐1,5‐bisphosphate. In unicellular algae, glycolate is lost via excretion or metabolized via the C₂ cycle by consuming reductants, ATP and CO₂ emission (photorespiration). Therefore, photorespiration is an inhibitory process for biomass production. However, cells can be manipulated in a way that they become glycolate‐producing ‘cell factories’, when the ratio carboxylation/oxygenation is 2. If under these conditions the C₂ cycle is blocked, glycolate excretion becomes the only pathway of photosynthetic carbon flow. The study aims to proof the biotechnological applicability of algal‐based glycolate excretion as a new biotechnological platform. It is shown that cells of Chlamydomonas can be cultivated under specific conditions to establish a constant and long‐term stable glycolate excretion during the light phase. The cultures achieved a high efficiency of 82% of assimilated carbon transferred into glycolate biosynthesis without losses of function in cell vitality. Moreover, the glycolate accumulation in the medium is high enough to be directly used for microbial fermentation but does not show toxic effects to the glycolate‐producing cells.     

Carbon dioxide fixation in isolated kalanchoe chloroplasts

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO₂ per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO₂ fixation were primarily those of the photosynthetic carbon reduction cycle.     

Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO₂ (1200 microliters per liter) than in ambient CO₂ (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO₂. Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO₂. The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO₂ compared with 50% in the ambient atmosphere. While CO₂ enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO₂.