Morphological and Ecophysiological Adaptations of the Marine Oligochaete Tubificoides benedii to Sulfidic Sediments

SYNOPSIS. The marine oligochaete worm Tubificoides benedii inhabitscoastal tidal sediments in which sulfide can reach toxic concentrations.The role of external ironsulfide deposition in sulfide detoxificationis discussed together with a review of morphological and ecophysiologicaladaptations of T. benedii to sulfide. The body wall of T. benediiturns black in the presence of sulfide. Histochemical studiesand micro-X-rayanalyses provide evidence for the reaction ofiron in the mucus layer above the cuticle of the worm with environmentalsulfide to produce ironsulfide. The deposited ironsulfides areeither reoxidized or shed off through moulting, a process otherwiseunknown in oligochaetes. However, calculations on the diffusionrate of sulfide into T. benedii show that the deposition ofironsulfides do not play an important role in sulfide detoxification.The first and last few segments of T. benedii are not blackenedby sulfide and do not appear to precipitate sulfide. The diffusionrate of sulfide through these segments is so rapid that internalsulfide concentrations reach levels inhibitory to cytochromec oxidase, the key enzyme of aerobic respiration, within minutes.When internal sulfide concentrations increase to toxic levels,reliance on an anaerobic metabolism represents a successfulmechanism of sulfide tolerance in T. benedii. Metabolic adaptationsto hypoxia and sulfide include the maintenance of aerobic pathwaysdespite low oxygen or high sulfide concentrations and the abilityto gain energy through anaerobic pathways when oxygen and/orsulfide concentrations become limiting     

The ecological significance of sulfur in the energy dynamics of salt marsh and coastal marine sediments

Sulfur is an important element in the metabolism of salt marshes and subtidal, coastal marine sediments because of its role as an electron acceptor, carrier, and donor. Sulfate is the major electron acceptor for respiration in anoxic marine sediments. Anoxic respiration becomes increasingly important in sediments as total respiration increases, and so sulfate reduction accounts for a higher percentage of total sediment respiration in sediments where total respiration is greater. Thus, sulfate accounts for 25% of total sediment respiration in nearshore sediments (200 m water depth or less) where total respiration rates are 0.1 to 0.3gCm^–1 day^–1 , for 50% to 70% in nearshore sediments with higher rates of total respiration (0.3 to 3gCm^–2 day^–1), and for 70% to 90% in salt marsh sediments where total sediment respiration rates are 2.5 to 5.5gcm^–2 day^–1 .During sulfate reduction, large amounts of energy from the respired organic matter are conserved in inorganic reduced sulfur compounds such as soluble sulfides, thiosulfate, elemental sulfur, iron monosulfides, and pyrite. Only a small percentage of the reduced sulfur formed during sulfate reduction is accreted in marine sediments and salt marshes. When these reduced sulfur compounds are oxidized, energy is released. Chemolithoautotrophic bacteria which catalyze these oxidations can use the energy of oxidation with efficiencies (the ratio of energy fixed in organic biomass to energy released in sulfur oxidation) of up to 21–37% to fix CO₂ and produce new organic biomass.Chemolithoautotrophic bacterial production may represent a significant new formation of organic matter in some marine sediments. In some sediments, chemolithoautotrophic bacterial production may even equal or exceed organoheterotrophic bacterial production. The combined cycle of anaerobic decomposition through sulfate reduction, energy conservation as reduced sulfur compounds; and chemolithoautotrophic production of new organic carbon serves to take relatively low-quality organic matter from throughout the sediments and concentrate the energy as living biomass in a discrete zone near the sediment surface where it can be readily grazed by animals.Contribution from a symposium on the role of sulfur in ecosystem processes held August 10, 1983, at the annual meeting of the A.I.B.S., Grand Forks, ND; Myron Mitchell, convenor.     

Fermentative degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by a defined coculture of strictly anaerobic bacteria

Degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) under strictly anaerobic conditions was studied in enrichment cultures from marine and freshwater sediments. In all cases, dipicolinic acid was completely degraded. From an enrichment culture from a marine sediment, a defined coculture of two bacteria was isolated. The dipicolinic acid-fermenting bacterium was a Gram-negative, non-sporeforming strictly anaerobic short rod which utilized dipicolinic acid as sole source of carbon, energy, and nitrogen, and fermented it to acetate, propionate, ammonia, and 2CO₂. No other substrate was fermented. This bacterium could be cultivated only in coculture with another Gram-negative, non-sporeforming rod from the same enrichment culture which oxidized acetate to CO₂ with fumarate, malate, or elemental sulfur as electron acceptor, similar to Desulfuromonas acetoxidans. Since this metabolic activity is not important in substrate degradation by the coculture, the basis of the dependence of the dipicolinic acid-degrading bacterium on the sulfur reducer may be sought in the assimilatory metabolism.     

Environmental and functional limits to muscular exercise and body size in marine invertebrate athletes

Many similarities exist between the key characteristics of muscular metabolism in marine invertebrates and those found in vertebrate striated muscle, even though there are important phosphagens and glycolytic end products that differ between groups. Lifestyles and modes of locomotion also vary extremely among invertebrates thereby shaping the pattern of exercise metabolism. In accordance with the limited availability of integrated ecological and physiological information the present paper reports recent progress in the exercise physiology of cephalopods, which are characterized by high rates of aerobic and anaerobic energy turnover during high velocity hunts or escapes in their pelagic environment, and a sipunculid worm, which mostly uses anaerobic resources during extended marathon-like digging excursions in the hypoxic marine sediment. Particular attention is paid to how lifestyle and oxygen availability in various marine environments shapes the use and rates of aerobic and anaerobic metabolism and acidosis as they depend on activity levels and energy saving strategies. Whereas aerobic scope and, accordingly, use of ambient oxygen by blood oxygen transport and skin respiration is maximized in some squids, aerobic scope is very small in the worm and anaerobic metabolism readily used upon muscular activity. Until recently, it was widely accepted that the glycolytic end product octopine, produced in the musculature of these invertebrates, acted as a weak acid and so did not compromise acid-base balance. However, it has now been demonstrated that octopine does cause acidosis. Concomitant study of tissue energy and acid-base status allows to evaluate the contribution of glycolysis, pH and free ADP accumulation to the use of the phosphagen and to the delayed drop in the Gibb's free energy change of ATP hydrolysis. The analysis reveals species specific capacities of these mechanisms to support exercise beyond the anaerobic threshold. During high intensity anaerobic exercise observed in squid, the levels of ATP free energy change finally fall to critical minimum levels contributing to fatigue. Maintenance of sufficiently high energy levels is found at low but extended rates of anaerobic metabolism as observed in the long term digging sipunculid worm. The greatest aerobic and anaerobic performance levels are seen in squid inhabiting the open ocean and appear to be made possible by the uniform and stable physicochemical parameters (esp. high O(2) and low CO(2) levels) that characterize such an environment. It is suggested that at least some squid operate at their functional and environmental limits. In extremely different environments, both the worm and the squids display a tradeoff between oxygen availability, temperature, performance level and also, body size.     

The composition of animal cells: solutes contributing to osmotic pressure and charge balance

The cytoplasmic solutes of vertebrates and invertebrates, other than Na, K and Cl, are surveyed in relation to their influence on ionic regulation through osmolality and charge balance. The most abundant include MgATP, phosphagens, amino acids, various other nitrogen and phosphorus compounds and sometimes anaerobic end products and antifreeze agents. Differences in muscle osmolality, e.g. between marine and non-marine animals, affect mainly nitrogenous solutes of no net charge, such as certain amino acids, taurine, betaine, trimethylamine oxide and urea. The high osmolality of axoplasm in marine invertebrates is due more to anions such as aspartate, glutamate and isethionate.     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

Peculiarities of organization of tissue metabolism in molluscs with different tolerance to external hypoxia

Oxygen consumption, content of several carbohydrate metabolites, and activities of their coupled enzymes were studied in bivalve molluscs with different tolerance to oxygen deficit: Mytilus galloprovincialis Lam. (black morpha) and Anadara Inaequivalvis Br. It has been shown that under conditions of external normoxia the anadara resistance to hypoxia preserves anaerobic orientation of metabolism. Its tissues are distinguished by high activities of malate and lactate dehydrogenases with the decreased content of glucose and the increased level of lactate. In several organs the succinate thiokinase and fumarate reductase reactions are realized, which is indicated by elevated activities of the alanine and aspartate aminotransferases. The anaerobic orientation of protein metabolism is added by a high pool of free amino acids and the elevated urea content in the mollusc tissues. The total orientation of metabolism in the anadara tissues rules out a significant lactate accumulation and determines low requirement of its organism in oxygen.     

Contribution of Sediment Respiration to Summer CO2 Emission from Low Productive Boreal and Subarctic Lakes

We measured sediment production of carbon dioxide (CO₂) and methane (CH₄) and the net flux of CO₂ across the surfaces of 15 boreal and subarctic lakes of different humic contents. Sediment respiration measurements were made in situ under ambient light conditions. The flux of CO₂ between sediment and water varied between an uptake of 53 and an efflux of 182 mg C m⁻² day⁻¹ from the sediments. The mean respiration rate for sediments in contact with the upper mixed layer (SedR) was positively correlated to dissolved organic carbon (DOC) concentration in the water (r² = 0.61). The net flux of CO₂ across the lake surface [net ecosystem exchange (NEE)] was also closely correlated to DOC concentration in the upper mixed layer (r² = 0.73). The respiration in the water column was generally 10-fold higher per unit lake area compared to sediment respiration. Lakes with DOC concentrations <5.6 mg L⁻¹ had net consumption of CO₂ in the sediments, which we ascribe to benthic primary production. Only lakes with very low DOC concentrations were net autotrophic (<2.6 mg L⁻¹) due to the dominance of dissolved allochthonous organic carbon in the water as an energy source for aquatic organisms. In addition to previous findings of allochthonous organic matter as an important driver of heterotrophic metabolism in the water column of lakes, this study suggests that sediment metabolism is also highly dependent on allochthonous carbon sources.     

Effect of different killing techniques on early labeled photosynthetic products in c(4) plants

The choice of leaf-killing technique was found to affect significantly the distribution of label among early labeled photosynthetic products in two C₄ plants, Portulaca oleracea and Zea mays. The major effect of these procedures was on the amount of amino acids present, particularly alanine, and the ratio of malate to aspartate. Killing Portulaca leaves in alcohol generally results in more alanine and the predominance of malate over aspartate. When the leaves are killed by immediate freezing, however, aspartate contained more radioactivity than malate, and alanine was present in much reduced amounts. The various methods also differ in the relative amounts of C₃ cycle compounds and other, secondary intermediates which were obtained.     

Aspartate in the intestine: dual service as anaerobic electron acceptor and nitrogen source

Fumarate was previously known to serve as an anaerobic electron acceptor by E. coli when colonizing the mammalian intestine, but the source of that fumarate was elusive. In this issue, Unden and coworkers demonstrate that l -aspartic acid is the source of fumarate that drives anaerobic respiration by colonized E. coli (Schubert et al., 2021). Moreover, Schubert et al., establish that E. coli is able to grow anaerobically by using aspartate as a sole source of nitrogen. These groundbreaking findings indicate that a single amino acid – aspartate – supports anaerobic respiration and acquisition of nitrogen by E. coli in the intestine.     

The energy production of juvenileArenicola marina (Polychaeta) under anoxic and hypoxic conditions

The mode of anaerobic energy production of juvenileArenicola marina (0-generation) was investigated under experimental conditions and in the biotope. Under experimental anaerobic conditions, juvenileA. marina produce energy by the pathways known from the adults and other euryoxic invertebrates with succinate and the volatile fatty acids, acetate and propionate, as main end products. However, the juvenile lugworms are less resistent to anoxia than the adults. The reasons for this might be their small glycogen stores and their limited ability to reduce the metabolic rate. Nevertheless, on the tidal flats the juveniles settle particularly in the area next to the high tide line, which offers such extreme conditions that adult lugworms cannot live there. This different behaviour can be explained by the dissimilar ability to use oxygen at very low partial pressures. Juveniles maintain an aerobic energy metabolism even at a P_{WO 2} of 15 Torr at which adults are forced to produce energy exclusively by the less effective anaerobic mode. In the field, no indications of an anaerobic energy metabolism were detected in juveniles even after an exposure of 8 hours.     

Utilization of malate or aspartate by a yeast lacking malate enzyme

Summary Hansenula anomala, a yeast lacking malate enzyme, was able to grow in media containing malate or aspartate as sole carbon and energy sources. Both aspartate--ketoglutarate transaminase and pyruvate kinase activities changed their levels when the yeast was grown on different carbon sources. Pyruvate kinase activity was increased by fructose 1,6-diphosphate.These results indicate that in this yeast malate enzyme is not indispensable for the formation of pyruvate from malate or aspartate and that C₄ dicarboxylic acids may provide pyruvate through the combined action of phosphoenolpyruvate carboxykinase and pyruvate kinase. It is also concluded that aspartate--ketoglutarate transaminase and pyruvate kinase are under regulatory control in Hansenula anomala.     

Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Steep vertical gradients of oxidants (O₂ and NO₃⁻) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.     

Anaerobic energy metabolism in the oligochaeteLumbriculus variegatus Müller

Summary Anoxia tolerance, glycogen degradation, free amino acid pool, adenylate energy charge and the accumulation and excretion of end products were monitored inLumbriculus variegatus Müller throughout 48 h of anoxia. A transition period lasting about 4 h could be distinguished from subsequent events during which malate, present in high amounts in the resting animals, is utilized, probably by conversion to succinate. Up to the 12th hour of anoxia there is an increase in concentration of free amino acids, except aspartate. Glutamate increases rapidly during the first half hour but decreases thereafter. Beginning with the second hour of anoxia the alanine concentration increases at the same rate glutamate concentration decreases, but the source of nitrogen during the first hour is unknown. It is argued that the nitrogen required for the synthesis of some of the amino acids is ultimately derived from proteolysis. After about 3 h of anoxia propionate and acetate are synthesized. At first these acids accumulate in the tissues, but after 4–6 h they are excreted into the surrounding medium. Acetate is excreted over the whole experimental period at a constant rate, whereas the excretion rate of propionate decreases slowly with time. The propionate/acetate ratio is in excess of 2. Classic malate dismutation is by far the most important mechanism in the maintenance of redox balance. Depletion of glycogen stores appears to play an important role in determining anoxic survival time. Due to extremely low activity of PEPCK the ratio of the specific activities of PK and PEPCK is very high. Further, the kinetic properties of pyruvate kinase do not support the assumption of a shift of the glycolytic carbon flow at the PEP level.Abbreviations PK Pyruvate kinase - PEPCK phosphoenolpyruvate carboxykinase - PEP phospho(enol)pyruvate - FBP fructose-1,6-bisphosphate - AEC adenylate energy charge - EMP-scheme Embden-Meyerhof-Parnas scheme of glycolysis - f _w fresh body weight - dw dry body weight     

Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus ferrooxidans

Formate-grown cells of the obligately chemolithoautotrophic acidophile Thiobacillus ferrooxidans were capable of formate- and elemental sulfur-dependent reduction of ferric iron under anaerobic conditions. Under aerobic conditions, both oxygen and ferric iron could be simultaneously used as electron acceptors. To investigate whether anaerobic ferric iron respiration by T. ferrooxidans is an energy-transducing process, uptake of amino acids was studied. Glycine uptake by starved cells did not occur in the absence of an electron donor, neither under aerobic conditions nor under anaerobic conditions. Uptake of glycine could be driven by formate- and ferrous iron-dependent oxygen uptake. Under anaerobic conditions, ferric iron respiration with the electron donors formate and elemental sulfur could energize glycine uptake. Glycine uptake was inhibited by the uncoupler 2,4-dinitrophenol. The results indicate that anaerobic ferric iron respiration can contribute to the energy budget of T. ferrooxidans.     

Investigations of the anaerobic metabolism of the polychaete wormNereis diversicolor M.

Summary The anaerobic metabolism ofNereis diversicolor M. was studied during various periods of experimental anaerobiosis.The degradation of glycogen is shown to be the main source of anaerobic energy production. During first hours of anaerobiosis, aspartate, in addition to glycogen, is metabolized in considerable quantities.Five acids were found to accumulate as end-products: alanine, D-lactate, succinate, acetate and propionate (Table 2).Alanine is accumulated only during the first hours of anaerobiosis. The increase in alanine is correlated with a decrease in aspartate.D-Lactate is the main end-product during the first 24 h of anaerobiosis, and continues to be produced even during prolonged anaerobiosis. In accordance with lactate production,Nereis diversicolor possesses a high glycolytic capacity (Table 4).The major end-products of long term fermentation are propionate and acetate. In contrast to other end-products, these acids are excreted in substantial amounts.Abbreviations GAPDH glyceraldehydephosphate dehydrogenase, EC 1.2.1.12 - LDH lactate dehydrogenase, EC 1.1.1.27 - GOT aspartate aminotransferase, EC 2.6.1.1 - GPT alanine aminotransferase, EC 2.6.1.2 - MDH malate dehydrogenase, EC 1.1.1.37 Supported by Deutsche Forschungsgemeinschaft (Gr 456/5 and Gr 456/6)     

Use of carbon monoxide and hydrogen by a bacteria–animal symbiosis from seagrass sediments

The gutless marine worm Olavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO₂) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O. algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H₂) might also be used as energy sources. We provide direct evidence that the O. algarvensis symbiosis consumes CO and H₂. Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3‐symbiont, uses the energy from CO oxidation to fix CO₂. Pore water analysis revealed considerable in‐situ concentrations of CO and H₂ in the O. algarvensis environment, Mediterranean seagrass sediments. Pore water H₂ concentrations (89–2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36‐fold higher than previously known from shallow‐water marine sediments. Pore water CO concentrations (17–51 nM) were twice as high as in the overlying seawater (no literature data from other shallow‐water sediments are available for comparison). Ex‐situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.     

Diel variations in carbon isotopic composition and concentration of organic acids and their impact on plant dark respiration in different species

Leaf respiration in the dark and its C isotopic composition (δ¹³C_R) contain information about internal metabolic processes and respiratory substrates. δ¹³C_R is known to be less negative compared to potential respiratory substrates, in particular shortly after darkening during light enhanced dark respiration (LEDR). This phenomenon might be driven by respiration of accumulated ¹³C‐enriched organic acids, however, studies simultaneously measuring δ¹³C_R during LEDR and potential respiratory substrates are rare. We determined δ¹³C_R and respiration rates (R) during LEDR, as well as δ¹³C and concentrations of potential respiratory substrates using compound‐specific isotope analyses. The measurements were conducted throughout the diel cycle in several plant species under different environmental conditions. δ¹³C_R and R patterns during LEDR were strongly species‐specific and showed an initial peak, which was followed by a progressive decrease in both values. The species‐specific differences in δ¹³C_R and R during LEDR may be partially explained by the isotopic composition of organic acids (e.g., oxalate, isocitrate, quinate, shikimate, malate), which were ¹³C‐enriched compared to other respiratory substrates (e.g., sugars and amino acids). However, the diel variations in both δ¹³C and concentrations of the organic acids were generally low. Thus, additional factors such as the heterogeneous isotope distribution in organic acids and the relative contribution of the organic acids to respiration are required to explain the strong ¹³C enrichment in leaf dark‐respired CO₂.     

Non-morphogenic metabolism during shoot induction in radiata pine cotyledon explants

Summary The effects of excision, light and cytokinin (N⁶-benzyladenine) on¹⁴C-acetate metabolism in cotyledons ofPinus radiata (D. Don) were determined.¹⁴CO₂ was released and the distribution of radioactivity into lipids, sugars, organic acids and amino acids was determined. While light and cytokinin generally caused some increase in metabolism, the effect of excision, i.e., wounding, was most pronounced. Specific metabolites examined (citrate, malate, succinate, alanine, aspartate, glutamate and glutamine) were at least 50% greater in¹⁴C-labeling in excised cotyledons as compared to intact seedlings. This enhancement of wound metabolism would mask possible morphogenically-related changes occurring at that time. This research was supported by the Natural Sciences and Engineering Research Council of Canada Grant A-6467 to T.A. Thorpe.     

CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.