Evidence for 18O labeling of photorespiratory CO2 in photoautotrophic cell cultures of higher plants illuminated in the presence of 18O2

The ¹⁸O-enrichment of CO₂ produced in the light or during the post-illumination burst was measured by mass spectrometry when a photoautotrophic cell suspension of Euphorbia characias L. was placed in photorespiratory conditions in the presence of molecular ¹⁸O₂. The only ¹⁸O-labeled species produced was C¹⁸O¹⁶O; no C¹⁸O¹⁸O could be detected. Production of C¹⁸O¹⁶O ceased after addition of two inhibitors of the photosynthetic carbon-oxidation cycle, aminooxyacetate or aminoacetonitrile, and was inhibited by high levels of CO₂. The average enrichment during the post-illumination burst was estimated to be 46 ± 15% of the enrichment of the O₂ present during the preceding light period. Addition of exogenous carbonic anhydrase, by catalyzing the exchange between CO₂ and H₂O, drastically diminished the ¹⁸O-enrichment of the produced CO₂. The very low carbonio-anhydrase level of the photoautotrophic cell suspension probably explains why the ¹⁸O labeling of photorespiratory CO₂ could be observed for the first time. These data allow the establishment of a direct link between O₂ consumption and CO₂ production in the light, and the conclusion that CO₂ produced in the light results, at least partially, from the mitochondrial decarboxylation of the glycine pool synthesized through the photosynthetic carbon-oxidation cycle. Analysis of the C¹⁸O¹⁶O and CO₂ kinetics provides a direct and reliable way to assess in vivo the real contribution of photorespiratory metabolism to CO₂ production in the light.     

Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii

The oxyhydrogen reaction (the reduction of O₂ to water by H₂) in the presence of CO₂ was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of ¹⁴CO₂ incorporation into acid-stable products in the dark. The endogenous rate of CO₂ uptake (50-125 nmol/mg chlorophyll per h) was increased about 3- to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H₂ was replaced by N₂ or by air. Decrease in CO₂ uptake by dl-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO₂ incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanol-p-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO₂ uptake could have been by “product inhibition” of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] J Gen Physiol 26: 241-267) that CO₂ fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration.     

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.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

Brassinosteroids accelerate recovery of photosynthetic apparatus from cold stress by balancing the electron partitioning,carboxylation and redox homeostasis in cucumber

The aim of this study was to examine the role of brassinosteroids (BRs) in protecting the photosynthetic apparatus from cold‐induced damage in cucumber (Cucumis sativus) plants. Recovery at both high light (HL) and low light (LL) after a cooling at 10/7°C induced irreversible inhibition of CO₂ assimilation, photoinhibition at photosystem I (PSI) and inhibition of enzyme activities of Calvin cycle and ascorbate (AsA)‐reduced glutathione (GSH) cycle, followed by accumulation of H₂O₂ and malondialdehyde. However, cold‐induced photoinhibition at PSII was fully recovered at LL but not at HL. Meanwhile, recovery at HL increased electron flux to O₂‐dependent alternative pathway [Ja(O₂‐dependent)]. Foliar application of 24‐epibrassinolide (EBR) accelerated recovery from photoinhibition of PSII but not of PSI. EBR also significantly increased CO₂ assimilation, activity of Calvin cycle enzymes and electron flux to carbon reduction [Je(PCR)], with a concomitant decrease in Ja(O₂‐dependent); meanwhile EBR increased the activity of enzymes in AsA‐GSH cycle and cellular redox states. However, the positive effect of EBR on plant recovery was observed only at HL, but not LL. These results indicate that BR accelerates the recovery of photosynthetic apparatus at HL by activation of enzymes in Calvin cycle and increasing the antioxidant capacity, which in turn mitigate the photooxidative stress and the inhibition of plant growth during the recovery.     

Inhibition by calcium of senescence of detached cucumber cotyledons: effect on ethylene and hydroperoxide production

The effect of Ca on senescence was followed in detached cucumber (Cucumis sativus L.) cotyledons floating on various solutions in the dark. Compared with those in water, cotyledons in 10⁻⁴ molar CaCl₂ exhibited reduced chlorophyll loss and H₂O₂ production, reduced and delayed ethylene production, and did not undergo a burst in CO₂ production. In contrast, Mg had little effect on cotyledon senescence, whereas K stimulated chlorophyll loss but did not increase H₂O₂ accumulation of ethylene and CO₂ production. This reduction in the rate of senescence by Ca could also be achieved by increasing the endogenous levels of Ca in the cotyledons before excision, although the reduction was less than that with Ca in the external solution. The addition of H₂O₂ to the solutions on which cotyledons were floated stimulated chlorophyll breakdown, but effects on ethylene and CO₂ were not consistent.     

Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: I. Carbon Dioxide Photoassimilation and Glycolate Synthesis

The influence of pH upon the O₂ inhibition of ¹⁴CO₂ photoassimilation (Warburg effect) was examined in intact spinach (Spinacia oleracea) chloroplasts. With conditions which favored the Warburg effect, i.e. rate-limiting CO₂ and 100% O₂, O₂ inhibition was greater at pH 8.4 to 8.5 than at pH 7.5 to 7.8. At pH 8.5, as compared with 7.8, there was an enhanced ¹⁴C-labeling of glycolate, and a decrease of isotope in some phosphorylated Calvin cycle intermediates, particularly triose-phosphate. The ¹⁴C-labeling of starch was also more inhibited by O₂ at higher pH. The enhanced synthesis of glycolate during ¹⁴CO₂ assimilation at higher pH resulted in a diminution in the level of phosphorylated intermediates of the Calvin cycle, and this was apparently a causal factor of the increased severity of the Warburg effect.     

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO₂-free buffer at 21% O₂, showed a decrease (30-70%) in CO₂-dependent O₂ evolution and ¹⁴CO₂ uptake. This photoinhibition was observed only when the O₂ concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO₂ concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO₂ concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O₂ evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO₂ concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O₂ evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O₂-dependent photoinhibition of light saturated CO₂ fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O₂ for photoinhibition of CO₂ fixation capacity in isolated chloroplasts may be explained by an effect of O₂ in allowing metabolic depletion of Calvin cycle intermediates.     

Ribulose bisphosphate oxygenase activity from freshly ruptured spinach chloroplasts

Suspensions of freshly lysed spinach chloroplasts, in which ribulose bisphosphate carboxylase displays an in vivo K_m [CO], exhibited a ribulose bisphosphate-dependent uptake of oxygen. The kinetic properties of this oxygenase activity were examined at air levels of CO₂ (10 μm) and O₂ (240 μm). The pH optimum was 8.6–8.8 and the K_M [ribulose bisphosphate] was 45 μm. At 240 μm O₂, the oxygenase activity is inhibited one-half by 25 μm CO₂. The apparent K_m(O₂) is large, somewhere between 1 and 2 atm. The phosphoglycolate phosphatase activity of the chloroplasts was in great excess, suggesting that phosphoglycolate formed by the oxygenase would be quickly hydrolyzed to glycolate for possible metabolism by photorespiration.A comparison of the pH dependence of both the carboxylase and oxygenase activities at air levels of CO₂ and O₂ suggests that the pH of the chloroplast stroma could regulate their relative activities and that the oxygenase activity is sufficient to account for glycolate production during photosynthesis. It is predicted that at pH 7.8, about 40% of the carbon assimilated by the Calvin cycle would go through glycolate.     

Calvin Cycle Mutants of Photoheterotrophic Purple Nonsulfur Bacteria Fail To Grow Due to an Electron Imbalance Rather than Toxic Metabolite Accumulation

Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO₂-fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO), prevents photoheterotrophic growth unless an electron acceptor is provided or if cells can dispose of electrons as H₂. Such observations led to the long-standing model wherein the Calvin cycle is necessary during photoheterotrophic growth to maintain a pool of oxidized electron carriers. This model was recently challenged with an alternative model wherein disrupting RubisCO activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate (RuBP) (D. Wang, Y. Zhang, E. L. Pohlmann, J. Li, and G. P. Roberts, J. Bacteriol. 193:3293-3303, 2011, http://dx.doi.org/10.1128/JB.00265-11). Here, we confirm that RuBP accumulation can impede the growth of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) RubisCO-deficient (ΔRubisCO) mutants under conditions where electron carrier oxidation is coupled to H₂ production. However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H₂ production is permitted. Thus, the Calvin cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H₂ production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated.     

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.     

Starch and Sucrose Synthesis in Phaseolus vulgaris as Affected by Light, CO(2), and Abscisic Acid

Phaseolus vulgaris L. leaves were subjected to various light, CO₂, and O₂ levels and abscisic acid, then given a 10 minute pulse of ¹⁴CO₂ followed by a 5 minute chase with unlabeled CO₂. After the chase period, very little label remained in the ionic fractions (presumed to be mostly carbon reduction and carbon oxidation cycle intermediates and amino acids) except at low CO₂ partial pressure. Most label was found in the neutral, alcohol soluble fraction (presumed sucrose) or in the insoluble fraction digestable by amyloglucosidase. Sucrose formation was linearly related to assimilation rate (slope = 0.35). Starch formation increased linearly with assimilation rate (slope = 0.56) but did not occur if the assimilation rate was below 4 micromoles per square meter per second. Neither abscisic acid, nor high CO₂ in combination with low O₂ (thought to disrupt control of carbon metabolism) caused significant perturbations of the sucrose/starch formation ratio. These studies indicate that the pathways for starch and sucrose synthesis both are controlled by the rate of net CO₂ assimilation, with sucrose the preferred product at very low assimilation rates.     

Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L

Activities of key enzymes of the Calvin cycle and C₄ metabolism, rates of CO₂ fixation, and the initial products of photosynthetic ¹⁴CO₂ fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv `Toria.' Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C₄ metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of ¹⁴CO₂ assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO₂ during light. However, respiratory losses were very high during the dark period.     

Intermediates Of Photosynthesis in Acetabularia mediterranie Chloroplasts

The chloroplast fraction isolated from Acetabularia mediterranie was exposed to ¹⁴CO₂ as NaH¹⁴CO₃ in light and darkness, and soluble radioactive compounds were analyzed at frequent intervals. The behavior of Calvin cycle intermediates indicates that this cycle was responsible for much of the carbon fixation in the chloroplasts. However, a substantial part of recently fixed carbon was metabolized via glycolic and glyceric acids. Possible pathways for their metabolism are discussed. Some carboxylation of C₃ acids was suggested by the behavior of phosphoenolpyruvate and malate. A number of amino acids were formed. Small amounts of such compounds as citrate, succinate, and fumarate not usually associated with photosynthesis might have been derived from a low level of mitochondrial contamination. About one-third of the carbon fixed in light was present in acid-labile insoluble compounds other than polysaccharides or proteins. Dark fixation of CO₂ was very small compared with photosynthesis.     

Postillumination burst of carbon dioxide in crassalacean Acid metabolism plants

Immediately following exposure to light, a postillumination burst of CO₂ has been detected in Crassulacean acid metabolism plants. A detailed study with pineapple (Ananas comosus) leaves indicates that the postillumination burst changes its amplitude and kinetics during the course of a day. In air, the postillumination burst in pineapple leaves generally is exhibited as two peaks. The postillumination burst is sensitive to atmospheric CO₂ and O₂ concentrations as well as to the light intensity under which plants are grown. We propose that the CO₂ released in the first postillumination burst peak is indicative of photorespiration since it is sensitive to either O₂ or CO₂ concentration while the second CO₂ evolution peak is likely due to decarboxylation of organic acids involved in Crassulacean acid metabolism.     

Oxygen exchange in leaves in the light

Photosynthetic O₂ production and photorespiratory O₂ uptake were measured using isotopic techniques, in the C₃ species Hirschfeldia incana Lowe., Helianthus annuus L., and Phaseolus vulgaris L. At high CO₂ and normal O₂, O₂ production increased linearly with light intensity. At low O₂ or low CO₂, O₂ production was suppressed, indicating that increased concentrations of both O₂ and CO₂ can stimulate O₂ production. At the CO₂ compensation point, O₂ uptake equaled O₂ production over a wide range of O₂ concentrations. O₂ uptake increased with light intensity and O₂ concentration. At low light intensities, O₂ uptake was suppressed by increased CO₂ concentrations so that O₂ uptake at 1,000 microliters per liter CO₂ was 28 to 35% of the uptake at the CO₂ compensation point. At high light intensities, O₂ uptake was stimulated by low concentrations of CO₂ and suppressed by higher concentrations of CO₂. O₂ uptake at high light intensity and 1000 microliters per liter CO₂ was 75% or more of the rate of O₂ uptake at the compensation point. The response of O₂ uptake to light intensity extrapolated to zero in darkness, suggesting that O₂ uptake via dark respiration may be suppressed in the light. The response of O₂ uptake to O₂ concentration saturated at about 30% O₂ in high light and at a lower O₂ concentration in low light. O₂ uptake was also observed with the C₄ plant Amaranthus edulis; the rate of uptake at the CO₂ compensation point was 20% of that observed at the same light intensity with the C₃ species, and this rate was not influenced by the CO₂ concentration. The results are discussed and interpreted in terms of the ribulose-1,5-bisphosphate oxygenase reaction, the associated metabolism of the photorespiratory pathway, and direct photosynthetic reduction of O₂.     

Photosynthetic activities of isolated bundle sheath cells in relation to differing mechanisms of C-4 pathway photosynthesis.

Photosynthetic activities of bundle sheath cell strands isolated from several C₄ pathway species were examined. These included species that decarboxylate C₄ acids via either NADP-malic enzyme (Zea mays, NADP-malic enzyme-type), NAD-malic enzyme (Atriplex spongiosa and Panicum miliaceum, NAD-malic enzyme-type) or phosphoenolpyruvate carboxykinase (Chloris gayana and Panicum maximum, phosphoenolpyruvate carboxykinase-type). Preparations from each of these species fixed ¹⁴CO₂ at rates ranging between 1.2 and 3.5 μmol min^?1 mg^?1 of chlorophyll, with more than 90% of the ¹⁴C being assimilated into Calvin cycle intermediates. With added HCO₃^? the rate of light-dependent O₂ evolution ranged between 2 and 4 μmol min^?1 mg^?1 of chlorophyll for cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species but with Z. mays cells there was no O₂ evolution detectable. Most of the ¹⁴CO₂ fixed by Z. mays cells provided with H¹⁴CO₃^? plus ribose 5-phosphate accumulated in the C-1 of 3-phosphoglycerate. However, 3-phosphoglycerate reduction was increased several fold when malate was also provided. Cells from all species rapidly decarboxylated C₄ acids under appropriate conditions, and the CO₂ released from the C-4 carboxyl was reassimilated via the Calvin cycle. Malate decarboxylation by Z. mays cells was dependent upon light and an endogenous or exogenous source of 3-phosphoglycerate. Bundle sheath cells of NAD-malic enzyme-type species rapidly decarboxylated [¹⁴C]malate when aspartate and 2-oxoglutarate were also provided, and [¹⁴C]aspartate was decarboxylated at similar rates when 2-oxoglutarate was added. Cells from phosphoenolpyruvate carboxykinase-type species decarboxylated [¹⁴C]aspartate when 2-oxoglutarate was added and they also catalyzed a slower decarboxylation of malate. Cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species evolved O₂ in the light when C₄ acids were added. These results are discussed in relation to proposed mechanisms for photosynthetic metabolism in the bundle sheath cells of species utilizing C₄ pathway photosynthesis.     

Photosynthetic carbon metabolism of isolated corn chloroplasts

Chloroplasts have been isolated from 4- to 6-day-old corn (Zea mays) leaves capable of assimilating 45 micromoles CO₂ per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of ¹⁴C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix CO₂ at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate.     

The role of carbon dioxide in the formation of end-products by Hymenolepis diminuta

The hypothesis that ambient CO₂ levels determine the end-products of energy metabolism excreted by Hymenolepis diminuta was tested by incubating the parasite in a range of CO₂ concentrations and measuring internal concentrations of adenine nucleotides and the excretion of organic acids. The strain of H. diminuta used was found to excrete mainly lactic acid and acetic acid. Succinic acid production was generally less than 5–10% of the total. At high CO₂ concentrations, the rate of excretion of lactic acid decreased while that of succinic acid increased, which conforms with the hypothesis. Acetic acid excretion did not vary significantly over the range of CO₂ concentrations used. Other results did not support the hypothesis. High CO₂ levels reduced the total amounts of acids excreted and the rate of succinic acid excretion was so small as to be ineffective in preventing the accumulation of H⁺ ions. When present in the incubation medium, succinic acid was taken up by H. diminuta. Lactic and acetic acid excretion was always sufficient to limit the accumulation of H⁺ ions. The conditions of incubation were shown not to be responsible for the low rates of succinic acid excreted. Incubation conditions and metabolic end-products were found to affect the rates of excretion of organic acids. There is thus a need, in work of this nature, to regulate and specify experimental conditions and to stipulate the strain of parasite used. The hypothesis was rejected and it was suggested that the energy metabolism of parasitic helminths is adapted to fluctuating O₂ and CO₂ tensions.