Effect of Oxygen on the Light-enhanced Dark Carbon Dioxide Fixation in Chlorella Cells

With Chlorella ellipsoidea cells, the effect of oxygen was investigated on the products of enhanced dark ¹⁴CO₂ fixation immediately following preillumination in the absence of CO₂. When the reaction mixture was made aerobic by bubbling air (CO₂-free) throughout preillumination and the following dark ¹⁴CO₂ fixation periods, the initial fixation product was mainly 3-phosphoglyceric acid. When nitrogen gas had been used instead of air, only about one-half of the total radioactivity in the initial fixation products was in 3-phosphoglyceric acid and the rest in aspartic, phosphoenolpyruvic, and malic acids. The percentage distribution of radioactivity incorporated in these initial products rapidly decreased during the rest of the dark period. Concurrent with the decrease in the initial ¹⁴CO₂ fixation products, some increase was observed in the radioactivities of the sugar phosphates. The maximal radioactivity incorporated in sugar mono- and diphosphates accounted for only 10% of total ¹⁴C, under either the aerobic or anaerobic conditions. Under anaerobic conditions most of the ¹⁴C incorporated was eventually transferred to alanine, whereas the main end products under aerobic conditions were aspartate and glutamate. The pattern of ¹⁴CO₂ fixation products was unaffected by the atmospheric condition during the period of preillumination. The preferential flow of the fixed carbon atom to alanine or aspartate depended on the presence or absence of oxygen during the period of dark CO₂ fixation.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

Transport and Partitioning of CO(2) Fixed by Root Nodules of Ureide and Amide Producing Legumes

Nodulated and denodulated roots of adzuki bean (Vigna angularis), soybean (Glycine max), and alfalfa (Medicago sativa) were exposed to ¹⁴CO₂ to investigate the contribution of nodule CO₂ fixation to assimilation and transport of fixed nitrogen. The distribution of radioactivity in xylem sap and partitioning of carbon fixed by nodules to the whole plant were measured. Radioactivity in the xylem sap of nodulated soybean and adzuki bean was located primarily (70 to 87%) in the acid fraction while the basic (amino acid) fraction contained 10 to 22%. In contrast, radioactivity in the xylem sap of nodulated alfalfa was primarily in amino acids with about 20% in organic acids. Total ureide concentration was 8.1, 4.7, and 0.0 micromoles per milliliter xylem sap for soybean, adzuki bean, and alfalfa, respectively. While the major nitrogen transport products in soybeans and adzuki beans are ureides, this class of metabolites contained less than 20% of the total radioactivity. When nodules of plants were removed, radioactivity in xylem sap decreased by 90% or more. Pulse-chase experiments indicated that CO₂ fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO₂ fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO₂ fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen.     

Evidence of in situ uptake and incorporation of bicarbonate and amino acids by a hydrothermal vent mussel

The hydrothermal vent mussel Bathymodiolus sp. is demonstrated to incorporate inorganic CO₂ from sea water. After ≈24 h incubation with H¹⁴CO⁻₂ the major part of the radioactivity is incorporated into macromolecules mostly in proteins but also in a notable lipidic fraction. 77 to 98% of this radioactivity is found in the gill and autoradiographs show that CO₂ fixation is only observed in cells containing high concentrations of bacteria. The results endorse the hypothesis that the associated bacteria might provide a nutritional source for the mussel.The mussel is also able to absorb and incorporate dissolved amino acids. Heterotrophic processes involving dissolved organic matter may interfere with the autotrophic pathways. Beside its capability of feeding on particulate material, the mussel may be thus able to live on reduced carbon and nitrogen compounds synthesized by its associated bacteria as well as on dissolved organic compounds present in sea water. The effective participation of the different processes is probably related to the ecological conditions experienced by the mussel in vent areas.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

Veränderliche Markierungsmuster bei 14CO2-Fütterung von Bryophyllum tubiflorum zu verschiedenen Zeitpunkten der Hell-Dunkelperiode

Summary The distribution of radioactivity between the products of ¹⁴CO₂ light fixation in phyllodia of Bryophyllum tubiflorum could be influenced experimentally by manipulating the malic acid content of the cells. Accelerating the deacidification of the tissue during the light period by application of higher light intensities accelerated the increase of malate labelling and the decrease of the sucrose labelling after ¹⁴CO₂ light fixation under our standard conditions (10 min preillumination, 15 min ¹⁴CO₂ light fixation, 8000 lux).In other experiments different malate contents of the tissues were induced by treating the phyllodia with different temperatures during the night period. In the morning, phyllodia with low malate content transferred most of the label into malate, and phyllodia with high malate content incorporated most of the ¹⁴C radioactivity into sugars. However, this was true only after preillumination of 1 hour. When the phyllodia fixed ¹⁴CO₂ without preillumination, no differences in the labelling patterns between acidified and non-acidified phyllodia could be observed.In experiments using leaf tissue slices of Bryophyllum daigremontianum we could again observe that malate was labelled more heavily in the deacidified tissue than in the acidified controls, with less radioactivity being transferred into phosphate esters and sugars. The rates of ¹⁴CO₂ light fixation were identical in tissue slices with high and low malate content. However, the rates of CO₂ dark fixation in the acidified samples were clearly lower than those in the deacidified ones. The low rate of CO₂ dark fixation in acidified samples could not be inhibited by an inhibitor of PEP-carboxylase as the high CO₂ dark fixation rate of the deacidified tissue could be inhibited.The results are discussed in relation to the feed back inhibition of PEP-carboxylase in vivo by malate. Compartmentation also seemed to be involved in controlling the flow of carbon during CO₂ light fixation in succulent tissue.     

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.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

Carbon assimilation by the autotrophic thermophilic archaebacterium Thermoproteus neutrophilus

Growth of Thermoproteus neutrophilus at 85°C was studied using an improved mineral medium with CO₂, CO₂ plus acetate, CO₂ plus propionate, or CO₂ plus succinate as carbon sources; sulfur reduction with H₂ to H₂S was the sole source of energy. None of the carbon compounds added was oxidized to CO₂. The organism grew autotrophically with a generation time of 9–14 h, up to a cell density of 0.5 g dry weight per liter (2×10⁹ cells/ml). Propionate did not stimulate, succinate slightly stimulated the growth rate. Acetate, even at low concentrations (0.5 mM), stimulated the growth rate, the generation time being shortened to 3–4 h. Acetate provided 70% of the cell carbon, which shows that Thermoproteus neutrophilus is a facultative autotroph. The path of these carbon precursors into cell compounds was studied by ¹⁴C long-term labelling and investigation of enzyme activities. Propionate could not be used as a major carbon source and was incorporated only into isoleucine, probably via the citramalate pathway. Acetate was a preferred carbon source which suppressed autotrophic CO₂ fixation: acetate grown cells exhibited an incomplete citric acid cycle in which 2-oxoglutarate dehydrogenase was present, but fumarate reductase was repressed. The succinate incorporation pattern and enzyme pattern indicated that autotrophic CO₂ fixation proceeded via a yet to be defined reductive citric acid cycle.     

The CO2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic,aerobic hydrogen-oxidizing bacterium,Hydrogenobacter thermophilus

The incorporation of ¹⁴CO₂ by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with ¹⁴CO₂, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO₂ fixation.In cell-free extracts of Hydrogenobacter thermophilus, the two key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase could not be detected. The key enzymes of the reductive tricarboxylic acid cycle, fumarate reductase and ATP citrate lyase were present. Activities of phosphoenolpyruvate synthetase and pyruvate carboxylase were also detected. The referse reactions (dehydrogenase reactions) of -ketoglutarate synthase and pyruvate synthase could be detected by using methyl viologen as an electron acceptor.These findings strongly suggested that a new type of the reductive tricarboxylic acid cycle operated as the CO₂ fixation pathway in Hydrogenobacter thermophilus.     

Influence of CO2-HCO3− Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO₂-HCO₃⁻ growth conditions and suggest that there exists a threshold level of CO₂ for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO₂-HCO₃⁻ growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K′) was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO₂-HCO₃⁻ as a consequence of regulating electron sink metabolism.     

Root respiration associated with nitrogenase activity (c(2)h(2)) of soybean, and a comparison of estimates

Root respiration associated with symbiotic fixation in soybean (Glycine max [L.] Merr.) was estimated by four methods.

Averaged over the life of the plant, the root respires 5.8 milligrams C per milligram N accumulated from fixation. When nitrogenase (C₂H₂) activity and root respiration were decreased by treating roots briefly with 1.0 atmosphere O₂, the respiration associated with nitrogenase was estimated as 2.10 micromoles CO₂ per micromole C₂H₄.

When nitrogenase activity and respiration were decreased by addition of nitrate, the respiration associated with fixation was calculated as 2.90 micromoles CO₂ per micromole C₂H₄. Removing nodules from roots decreased fixation and root respiration, and the ratio was 4.08 micromoles CO₂ per micromole C₂H₄. When soybean plants were kept in prolonged darkness, then returned to light, the associated drop and recovery of respiration and nitrogenase activity had a ratio of 4.36 micromoles CO₂ per micromole C₂H₂.

     

Untersuchungen zur Photosynthese-Induktion bei Chlorella mit Hilfe von radioaktivem CO2

Summary ¹⁴CO₂ fixation and radioactive products in Chlorella cells were examined during the induction period. Chlorella vulgaris (strain 211-11f) grown under low CO₂ concentration (0.03 vol.-%) was used in our experiments. No transients in ¹⁴CO₂ fixation could be observed under the conditions used (0.7 vol.-% CO₂). Autoradiographic studies of the kinetics of appearance of labelled products showed that at the beginning of the illumination period radioactivity appeared first in intermediates of the Calvin cycle (e.g. phosphate esters). After 2 min smaller amounts of radioactivity were incorporated in malate and aspartate. In another series of experiments the influence of iodoacetamide (0.001 M) on ¹⁴CO₂ fixation was examined. In Chlorella cells inhibited with iodoacetamide radioactivity was located in malate, aspartate and 3-phosphoglycerate only. Results were discussed with reference to the participation of two different carboxylation pathways in the formation of the transients. Also the possible role of photorespiration in these transients was discussed.     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.     

ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria

Köhler P. B.,Ryant C. and Behm Carolyn A. 1978. ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria. International Journal for Parasitology8: 399–404. Succinate decarboxylation was measured by the formation of ¹⁴CO₂ from 1,4-¹⁴C-succinate in a particle free, dialysed mitochondrial extract from liver fluke. It has an absolute requirement for Mg²⁺ and CoA. ATP, ADP and inorganic phosphate are essential for optimal activity. Ap₅A, an inhibitor of adenylate kinase, and glutathione are also necessary. GTP supports decarboxylation as well as ATP, provided ADP is also present. The formation of CO₂ and propionate greatly exceeds the amount of ATP and CoA initially present in the reaction mixture. A net, substrate-level phosphorylation of ADP occurs, the amount of ATP formed being equivalent to the production of CO₂ or propionate. This system is inhibited in flukes incubated in vitro with mebendazole.It is concluded that ATP is required to spark the fermentation system when succinate is the initial substrate and intermediate substrates are absent; that the terminal step in propionate formation is catalysed by a transferase which transfers CoA from propionyl CoA to succinate; and that ATP formation is coupled to the decarboxylation of methylmalonyl-CoA. A reaction scheme is presented.     

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L

Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO₂ fixation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH¹⁴CO₃, and the kinetics of ¹⁴C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO₂ fixation is similar in the different mesophyll tissues from which these cells were derived.     

Fate of Immediate Methane Precursors in Low-Sulfate, Hot-Spring Algal-Bacterial Mats

The fates of acetate and carbon dioxide were examined in several experiments designed to indicate their relative contributions to methane production at various temperatures in two low-sulfate, hot-spring algal-bacterial mats. [2-¹⁴C]acetate was predominantly incorporated into cell material, although some ¹⁴CH₄ and ¹⁴CO₂ was produced. Acetate incorporation was reduced by dark incubation in short-term experiments and severely depressed by a 2-day preincubation in darkness. Autoradiograms showed that acetate was incorporated by long filaments resembling phototrophic microorganisms of the mat communities. [³H]acetate was not converted to C³H₄ in samples from Octopus Spring collected at the optimum temperature for methanogenesis. NaH¹⁴CO₃ was readily converted to ¹⁴CH₄ at temperatures at which methanogenesis was active in both mats. Comparisons of the specific activities of methane and carbon dioxide suggested that of the methane produced, 80 ± 6% in Octopus Spring and 71 ± 21% in Wiegert Channel were derived from carbon dioxide. Addition of acetate to 1 mM did not reduce the relative importance of carbon dioxide as a methane precursor in samples from Octopus Spring. Experiments with pure cultures of Methanobacterium thermoautotrophicum suggested that the measured ratio of specific activities might underestimate the true contribution of carbon dioxide in methanogenesis.     

Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation

The ability of cysts of the brine shrimp, Artemia salina, to incorporate ¹⁴CO₂ into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H₂O/g dried cysts, the “critical hydration” for CO₂-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of ¹⁴CO₂ incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO₂-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H₂O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.     

Whey Fermentation by Anaerobiospirillum succiniciproducens for Production of a Succinate-Based Animal Feed Additive

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with Anaerobiospirillum succiniciproducens. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO₂, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO₂, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO₂, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.     

Non-phototrophic CO 2 fixation by soil microorganisms

Although soils are generally known to be a net source of CO₂ due to microbial respiration, CO₂ fixation may also be an important process. The non-phototrophic fixation of CO₂ was investigated in a tracer experiment with ¹⁴CO₂ in order to obtain information about the extent and the mechanisms of this process. Soils were incubated for up to 91 days in the dark. In three independent incubation experiments, a significant transfer of radioactivity from ¹⁴CO₂ to soil organic matter was observed. The process was related to microbial activity and could be enhanced by the addition of readily available substrates such as acetate. CO₂ fixation exhibited biphasic kinetics and was linearly related to respiration during the first phase of incubation (about 20–40 days). The fixation amounted to 3–5% of the net respiration. After this phase, the CO₂ fixation decreased to 1–2% of the respiration. The amount of carbon fixed by an agricultural soil corresponded to 0.05% of the organic carbon present in the soil at the beginning of the experiment, and virtually all of the fixed CO₂ was converted to organic compounds. Many autotrophic and heterotrophic biochemical processes result in the fixation of CO₂. However, the enhancement of the fixation by addition of readily available substrates and the linear correlation with respiration suggested that the process is mainly driven by aerobic heterotrophic microorganisms. We conclude that heterotrophic CO₂ fixation represents a significant factor of microbial activity in soils.