Pentachlorophenol degradation by Pseudomonas aeruginosa

Five Pseudomonas species were tested for ability to degrade pentachlorophenol (PCP). Pseudomonas aeruginosa completely degraded PCP up to 800 mg/l in 6 days with glucose as co-substrate. With 1000 mg PCP/l, 53% was degraded. NH₄ ⁺ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO₂, with release of Cl^-.The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India.     

The effect of acid rain,soil temperature and humidity on C-mineralization rates in organic soil layers under spruce

The biological activity in organic soil layers under spruce was determined by measuring rates of carbon dioxide emission. Under laboratory conditions, biological activity was found to be optimal at temperatures ranging from 20°C to 35°C and at water contents ranging from 40% to 60%. Weakly acidic to neutral pH values of organic materials stimulated microbial CO₂ formation whereas high acidity (pH<2.4) inhibited it. Low CO₂ emission rates were observed at pH<2.4 and therefore populations of microorganisms highly resistant to acid must be present in the organic materials. The O_L horizon was found to contribute 50% of the total potential C-mineralization with the lower horizons O_F and O_H contributing 25% each. In simulating acid rain experiments, analysis of water percolating through the organic layers was shown to cause constant leaching of cations at the pH 3 to 6.5 range. In this respect, the O_L horizon exerts a buffering effect. It can be postulated that the acidification of soil organic material under spruce by acid rain inhibits C-mineralization and decreases the release of mineral nutrients. In the long run this can be expected to affect the turnover of mineral nutrients in forest ecosystems.     

Effect of carbon dioxide and hydrostatic pressure on the pH of culture media and the growth of methanogens at elevated temperature

Summary High pressure/high temperature investigations on thermophilic methanogens require specific precautions to provide well-defined pH conditions in their culture media. Applying CO₂ as carbon source, sufficient buffering capacity of the culture medium is of crucial importance in investigations involving elevated pressures. In order to separate pressure effects on the growth and reproduction of thermophilic methanogens from pressure-induced protonation/deprotonation and increased solubility of gaseous components, direct pH measurements in common culture media in the absence and in the presence of CO₂ were performed at elevated temperature (65° C), and at pressures up to 100 MPa. Neutral phosphate buffer at high pressure shows a significant downward shift of its pH which is strongly enhanced in the presence of CO₂. In minimal media containing acetate, carbonate, formate and phosphate in 100 mM concentrations, 120 mM HEPES is found to provide optimum pH stability: near neutrality the pH change upon CO₂ saturation in the absence and in the presence of HEPES amounts to pH=2.10 and 0.41, respectively; the corresponding pressure dependences are pH/100 MPa=-0.26 and -0.07. As taken from these results, the apparent pressure dependence of the optimum growth ofMethanococcus thermolithotrophicus at 65° C in minimal medium reflects the pH shift below the cutoff point of growth (pH 5.5), rather than pressure-induced growth inhibition. At constant pH, elevated pressure up to 400 MPa is found to increase the rate and yield of growth; at the same time, alterations in the phenotype of the bacterium are observed.     

Respiration and oxygen transport in soybean nodules

Summary The respiration rate of individual soybean (Glycine max Merr.) nodules was measured as a function of pO₂ and temperature. At 23°, as the pO₂ was increased from 0.1 to 0.9 atm, there was a linear increase in respiration rate. At 13°, similar results were obtained, except that there was an abrupt saturation of respiration at approximately 0.5 atm pO₂. When measurements were made on the same nodule, the rate of increase in respiration with pO₂ was the same at 13° and 23°. Additional results were that 5% CO in the gas phase had no effect on respiration, except for a small decrease in the pO₂ at which respiration became saturated. Also, nodules still attached to the soybean root displayed the same respiratory behavior as detached nodules. A model for oxygen transport in the nodule is presented which explains these results quantitatively. The essence of the model is that the respiration rate of the central tissue of the nodule is almost entirely determined by the rate of oxygen diffusion to the respiratory enzymes. Evidence is given that the nodule cortex is the site of almost all of the resistance to oxygen diffusion within the nodule.     

The carbon dioxide system in the Elbe estuary

The pCO₂ distribution in thesurface waters of the Elbe estuary and German Bight was investigated during high river discharge in April 1997. pCO₂ values of about 1100 atm foundin the upper part of the estuary are, comparedto other European rivers, only moderate. In theouter estuary, the enhanced river dischargecreates a pronounced river plume reflected in decreased surface salinity.However, pCO₂ in this area(250 atm) is below atmospheric values dueto primary production. Comparison with resultswe obtained during other measuringcampaigns shows that in the outer estuarysupersaturation of CO₂ with respect to theatmosphere occurs only in early spring duringthe time of highest river discharge. In latespring and summer, intensive primary productionwithin the surface layer leads to a pronouncedundersaturation (141 atm), despite atemperature increase of more than 20 °Cfrom winter to summer. Undersaturation isenforced by stable stratification of the watercolumn during the productive seasons thatdiminishes the entrainment of CO₂from below the thermocline into the surfacewater. In winter, CO₂ is close toequilibrium with the atmosphere and controlledby physical processes. Our data suggest thatthe Elbe estuary represents a sink foratmospheric CO₂ over the investigatedseasons and that carbon cycling in this areais dominated by biological processes.     

Effect of<Emphasis Type="Italic"> p</Emphasis>CO<Subscript>2</Subscript> and temperature on the boron isotopic composition of the zooxanthellate coral<Emphasis Type="Italic"> Acropora</Emphasis> sp.

Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO₂ conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO₂ effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO₂ (438 µatm), the ¹¹B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of ¹¹B measured for corals cultured at higher pCO₂ (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The ¹¹B of corals cultivated at both high and normal pCO₂ conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli     

Fructose utilization by the cyanobacterium Anabaena variabilis studied using whole filaments and isolated heterocysts

We have investigated the utilization of [¹⁴C]-fructose by whole filaments and isolated heterocysts of Anabaena variabilis ATCC 29413, a strain which is capable of fructose-dependent heterotrophic growth. The experimental conditions were chosen such that both transport and subsequent metabolism were studied. The apparent K_m for fructose was 60 mM, close to the results of previous studies. Rates of fructose utilization were the same in light and darkness. When photosynthetic CO₂ fixation was possible, almost all the label appeared as cell-carbon. In darkness or in the presence of DCMU appreciable amounts of label were released as CO₂. Isolated heterocysts with high rates of endogenous metabolism were not capable of utilizing added fructose at significant rates. The effects of oxygen concentration on the metabolism of added fructose in darkness showed that uptake was saturated at low pO₂ values. Increasing the pO₂ values lead to an increase in the ratio between the lable released as CO₂ and that recovred as cell-carbon. These results suggest that fructose is taken up only by the vegetative cells but carbon derived from added fructose can be released as CO₂ as a result of respiration in the heterocysts. Fructose utilization was inhibited by uncouplers. The greatest inhibition was found when both (delta) (psi) and (delta) pH were abolished. High concentrations of erythrose inhibited fructose utilization. None of the other potential analogs tested had any effect.     

Carbon dioxide desorption from fermentation broth by use of oxygen vectors

The ability of oxygen vector to extract produced carbon dioxide has been tested in an anaerobic fermentation. During the continuous culture of Clostridium acetobutylicum at pH 4.6 and at a dilution rate of 0.124 h^–1, a feed composed of an emulsion of 18.5% by volume of Forane F66E was able to extract about 9% of the total CO₂ produced under CO₂ partial pressure equal to 0.42 atm. A theoretical evaluation of the extracted amount, based on the hypothesis of total saturation of the vector by carbon dioxide, has lead to very good agreement.List of Symbols [AA] g/l acetic acid concentration - [BA] g/l butyric acid concentration - D 1/h Q _w /V dilution rate - [ETH] g/l ethanol concentration - H _w Henry constant of CO₂ for water at 37°C (=23.91 mmol/(l atm)) - H _F Henry constant of CO₂ for Forane at 37°C (=83.4 mmol/(l atm)) - H _i g/mol molar mass of componenti - P _i atm partial pressure of gasi - W _w l/h aqueous flow - Q_f 1/h Forane flow - mmol/(lh) dissolved CO₂ flow in aqueous effluent - mmol/(lh) CO₂ gas flow - mmol/(lh) CO₂ gas flow without Forane - mmol/(lh) CO₂ gas flow with Forane - mmol/(lh) total CO₂ production - r _X g/(lh) biomass production rate - r _G mmol/(lh) total gas flow - mmol/(lh) hydrogen production - mmol/(lh) nitrogen flow - r _S mmol/(lh) glucose input - V 1 fermentor volume     

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with ¹⁴C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to ¹⁴CO₂ by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.     

Clostridium autoethanogenum,sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide

A strictly anaerobic, gram-positive, sporeforming, rod-like, motile bacterium was enriched from rabbit feces, and isolated using carbon monoxide as sole source of energy and carbon. The isolate metabolizes CO with ethanol, acetate and CO₂ as end-products. Other substrates used as carbon and energy sources include CO₂ plus H₂, pyruvate, xylose, arabinose, fructose, rhamnose, and l-glutamate. The optimum temperature for growth is 37°C. The optimum pH for chemolithotrophic growth lies around 5.8 to 6.0 Sulfate is not reduced. Growth is inhibited either by penicillin, chloramphenicol, tetracyclin or ampicillin, each at 100 g per ml. The isolate has a DNA-base composition of 25.9±0.6% guanine plus cytosine. The isolate represents a new species of Clostridium for which the name Clostridium autoethanogenum is proposed. The type strain is strain JA1-1     

Isolation of thermophilic obligately autotrophic hydrogen-oxidizing bacteria,similar to Hydrogenobacter thermophilus,from Icelandic hot springs

Thermophilic obligately autotrophic hydrogen-oxidizing bacteria were isolated from several alkaline hot springs in Iceland. The bacteria were Gram negative rods, 0.4–0.5 m in diameter and 3–4 m long but 6–7 m long cells without septa were often seen. Long and short laments are formed. Spores, flagella or lipid granules were not observed. Strains H1 and H12 grew optimally at 70° C and pH 6.5 under mixture of air plus 0.6 atm H₂ and 0.1 atm CO₂. The cells contained cytochromes and carotenoid-like pigments. They would not grow on agar or silicia gel plates. The cells would not grow heterotrophically on organic substrates and were inhibited by most of these same organic compounds and agar in low concentrations. They were very sensitive to common antibiotics. The role of these bacteria in the hot spring ecosystem is discussed.     

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.     

Exogenous inorganic carbon sources for photosynthesis in seawater by members of the Fucales and the Laminariales (Phaeophyta): ecological and taxonomic implications

Summary Characteristics of inorganic carbon assimilation by photosynthesis in seawater were investigated in six species of the Fucales (five Fucaceae, one Cystoseiraceae) and four species of the Laminariales (three Laminariaceae, one Alariaceae) from Arbroath, Scotland. All of the algae tested could photosynthesise faster at high external pH values than the uncatalysed conversion of HCO ₃ ^- to CO₂ can occur, i.e. can use external HCO ₃ ^- . They all had detectable extracellular carbonic anhydrase activity, suggesting that HCO ₃ ^- use could involve catalysis of external CO₂ production, a view supported to some extent by experiments with an inhibitor of carbonic anhydrase. All of the algae tested had CO₂ compensation concentrations at pH 8 which were lower than would be expected from diffusive entry of CO₂ supplying RUBISCO as the initial carboxylase, consistent with the operation of energized entry of HCO ₃ ^- and / or CO₂ acting as a CO₂ concentrating mechanism. Quantitative differences among the algae examined were noted with respect to characteristics of inorganic C assimilation. The most obvious distinction was between the eulittoral Fucaceae, which are emersed for part of, or most of, the tidal cycle, and the other three families (Cystoseiraceae, Laminariaceae, Alariaceae) whose representatives are essentially continually submersed. The Fucaceae examined are able to photosynthesise at high pH values, and have lower CO₂ compensation concentrations, and lower K_1/2 values for inorganic C use in photosynthesis, at pH 8, than the other algae tested. Furthermore, the Fucaceae are essentially saturated with inorganic C for photosynthesis at the normal seawater concentration at pH 8 and 10°C. These characteristics are consistent with the dominant role of a CO₂ concentrating mechanism in CO₂ acquisition by these plants. Other species tested have characteristcs which suggest a less effective HCO ₃ ^- use and CO₂ concentrating mechanism, with the Laminariaceae being the least effective; unlike the Fucaceae, photosynthesis by these algae is not saturated with inorganic C in normal seawater. Taxonomic and ecological implications of these results are considered in relation to related data in the literature.     

Inorganic carbon sources and biomass regulation in intensive microalgal cultures

Three freshwater and one marine algal species were grown under inorganic carbon limitation in laboratory continuous cultures. Comparisons were made between HCO₃^? alkalinity and bubbled CO₂ as carbon sources. HCO₃^? alkalinity was an excellent source of inorganic carbon below specific pH levels, but chemical precipitation at high pH placed an upper limit on productivity that was far lower than potential light-limiting levels. With bubbled CO₂ it was possible to achieve light limitation. The main factor controlling productivity was the mass flux of inorganic carbon added to the culture, which is the product of gas flow rate and influent P level. Small bubbles were more efficient than large bubbles at low gas flow rates and P levels, but led to froth flotation of algal cells and concomitant reductions in productivity at high bubble rates. At 1% CO₂ productivity was still dependent on mass fluxes of added carbon, but was independent of bubble size. At high bubble rates with 1% CO₂ narcosis was evident. Maximum yields occurred at intermediate dilution rates when inorganic carbon was supplied via bubbled gas.     

Interaction of increasing atmospheric carbon dioxide and soil nitrogen on the carbon balance of tundra microcosms

Summary Natural cores of vegetation and soils of arctic tundra were collected in frozen condition in winter near Barrow, Alaska (71°20N). These cores were used as microcosms in a phytotron experiment to measure the interactions, if any, between increasing atmospheric CO₂ concentration and fertilization by ammonium nitrate on net ecosystem CO₂ exchange and net yield of tundra vegetation. Increased soil N significantly enhanced net ecosystem CO₂ uptake. The effect of increased CO₂ concentration had little or no effect on mean net ecosystem carbon balance of the tundra microcosms. Added N significantly increased leaf area and phytomass of vascular plants in the microcosms while increased atmospheric CO₂ had no effect on these parameters. We conclude that atmospheric CO₂ is not now limiting net ecosystem production in the tundra and that its direct effects will be slight even at double the present concentration. the most probable effects of carbon dioxide in the coastal tundra will be through its indirect effects on temperature, water table, peat decomposition, and the availability of soil nutrients.     

Will changes in soil organic carbon act as a positive or negative feedback on global warming?

The world's soils contain about 1500 Gt of organic carbon to a depth of 1m and a further 900 Gt from 1--2m. A change of total soil organic carbon by just 10% would thus be equivalent to all the anthropogenic CO₂ emitted over 30 years. Warming is likely to increase both the rate of decomposition and net primary production (NPP), with a fraction of NPP forming new organic carbon. Evidence from various sources can be used to assess whether NPP or the rate of decomposition has the greater temperature sensitivity, and, hence, whether warming is likely to lead to an increase or decrease in soil organic carbon.Evidence is reviewed from laboratory-based incubations, field measurements of organic carbon storage, carbon isotope ratios and soil respiration with either naturally varying temperatures or after experimentally increasing soil temperatures. Estimates of terrestrial carbon stored at the Last Glacial Maximum are also reviewed. The review concludes that the temperature dependence of organic matter decomposition can be best described as: d(T) = exp[3.36 (T – 40)/(T + 31.79)] where d(T) is the normalised decomposition rate at temperature T (in °C). In this equation, decomposition rate is normalised to 1 at 40 °C.The review concludes by simulating the likely changes in soil organic carbon with warming. In summary, it appears likely that warming will have the effect of reducing soil organic carbon by stimulating decomposition rates more than NPP. However, increasing CO₂ is likely to simultaneously have the effect of increasing soil organic carbon through increases in NPP. Any changes are also likely to be very slow. The net effect of changes in soil organic carbon on atmospheric CO₂ loading over the next decades to centuries is, therefore, likely to be small.     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase

The substrate specificity factor, V _cK_o/V_oK_c, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO₂/O₂ specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K _c [Km(CO₂)] and V _c [Vmax (CO₂)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K _o [Ki (O₂)] was unchanged and V _o [Vmax (O₂)] increased by a factor of 5 over these temperatures. The CO₂ compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C₃ plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO₂ and O₂.Abbreviations RuBPC/O(ase) ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - CO₂ compensation concentration     

Regulation of carbonic-anhydrase activity,inorganic-carbon uptake and photosynthetic biomass yield inChlamydomonas reinhardtii

The regulation of carbonic anhydrase by environmental conditions was determined forChlamydomonas reinhardtii. The depression of carbonic anhydrase in air-grown cells was pH-dependent. Growth of cells on air at acid pH, corresponding to 10 m CO₂ in solution, resulted in complete repression of carbonic-anhydrase activity. At pH 6.9, increasing the CO₂ concentration to 0.15% (v/v) in the gas phase, corresponding to 11 M in solution, was sufficient to completely repress carbonic-anhydrase activity. Photosynthesis and intracellular inorganic carbon were measured in air-grown and high-CO₂-grown cells using a silicone-oil centrifugation technique. With carbonic anhydrase repressed cells limited inorganic-carbon accumulation resulted from non-specific binding of CO₂. With air-grown cells, inorganic-carbon uptake at acid pH, i.e. 5.5, was linear up to 0.5 mM external inorganic-carbon concentration whereas at alkaline pH, i.e. 7.5, the accumulation ratio decreased with increase in external inorganic-carbon concentration. It is suggested that in air-grown cells at acid pH, CO₂ is the inorganic carbon species that crosses the plasmalemma. The conversion of CO₂ to HCO ₃ ^- by carbonic anhydrase in the cytosol results in inorganic-carbon accumulation and maintains the diffusion gradient for carbon dioxide across the cell boundary. However, this mechanism will not account for energy-dependent accumulation of inorganic carbon when there is little difference in pH between the exterior and cytosol.