Growth-substrate dependent dechlorination of 1,2-dichloroethane by a homoacetogenic bacterium

A rod shaped, gram positive, non sporulating Acetobacterium strain was isolated that dechlorinated 1,2-dichloroethane (1,2-DCA) to ethene at a dechlorination rate of up to 2 nmol Cl^- min^-1 mg^-1 of protein in the exponential growth phase with formate (40 mM) as the substrate. Although with other growth substrates such as pyruvate, lactate, H₂/CO₂, and ethanol higher biomass productions were obtained,the dechlorination rate with these substrates was more than 10-fold lower compared with formate growing cells. Neither cell extracts nor autoclaved cells of the isolatedAcetobacterium strain mediated the dechlorination of 1,2-DCA at significant rates. The addition of 1,2-DCA to the media did not result in increased cell production. No significant differences in corrinoid concentrations could be measured in cells growing on several growth-substrates. However, these measurements indicated that differences in corrinoid structure might cause the different dechlorination activity. The Acetobacterium sp. strain gradually lost its dechlorination ability during about 10 transfers in pure culture, probably due to undefined nutritional requirements. 16S rDNA analysis of the isolate revealed a 99.7% similarity with Acetobacterium wieringae. However, the type strains of A. wieringae and A. woodii did not dechlorinate 1,2-DCA.     

Evidence of Substantial Carbon Isotope Fractionation among Substrate, Inorganic Carbon, and Biomass during Aerobic Mineralization of 1,2-Dichloroethane by Xanthobacter autotrophicus

Carbon isotope fractionation during aerobic mineralization of 1,2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of ¹³C in residual 1,2-DCA was observed, with a mean fractionation factor α ± standard deviation of 0.968 ± 0.0013 to 0.973 ± 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor α to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of ¹³C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S_N2 nucleophilic substitution. S_N2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.     

Effect of competitive terminal electron acceptor processes on dechlorination of cis-1,2-dichloroethene and 1,2-dichloroethane in constructed wetland soils

Thermodynamic calculations were coupled with time-series measurements of chemical species (parent and daughter chlorinated solvents, H(2), sulfite, sulfate and methane) to predict the anaerobic transformation of cis-1,2-dichloroethene (cis-1,2-DCE) and 1,2-dichloroethane (1,2-DCA) in constructed wetland soil microcosms inoculated with a dehalorespiring culture. For cis-1,2-DCE, dechlorination occurred simultaneously with sulfite and sulfate reduction but competitive exclusion of methanogenesis was observed due to the rapid H(2) drawdown by the dehalorespiring bacteria. Rates of cis-1,2-DCE dechlorination decreased proportionally to the free energy yield of the competing electron acceptor and proportionally to the rate of H(2) drawdown, suggesting that H(2) competition between dehalorespirers and other populations was occurring, affecting the dechlorination rate. For 1,2-DCA, dechlorination occurred simultaneously with methanogenesis and sulfate reduction but occurred only after sulfite was completely depleted. Rates of 1,2-DCA dechlorination were unaffected by the presence of competing electron-accepting processes. The absence of a low H(2) threshold suggests that 1,2-DCA dechlorination is a cometabolic transformation, occurring at a higher H(2) threshold, despite the high free energy yields available for dehalorespiration of 1,2-DCA. We demonstrate the utility of kinetic and thermodynamic calculations to understand the complex, H(2)-utilizing reactions occurring in the wetland bed and their effect on rates of dechlorination of priority pollutants.     

Complete lab-scale detoxification of groundwater containing 1,2-dichloroethane

The suspected carcinogenic solvent 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C₂ groundwater pollutant on earth. However, an efficient reductive in situ detoxification technology for this compound is not known. Detoxification results of 1,2-DCA with the recently isolated anaerobic bacterium Desulfitobacterium dichloroeliminans strain DCA1 are presented. First, it was verified that strain DCA1 could compete for nutrients in the presence of fast-growing Enterococcus faecalis; the latter was observed in the enrichment culture from which strain DCA1 was isolated. Subsequently, lab-scale bioaugmentation of the strain to groundwater containing 40 mg 1,2-DCA/l indicated that the bacterium has strong metabolic activity under prevailing environmental conditions, converting the pollutant into ethene. During exponential growth, the maximum 1,2-DCA dechlorination rate exceeded 350 nmol chloride released per min per mg total bacterial protein. Growth and dechlorination within the community with autochthonous bacteria indicated a high competitive strength of strain DCA1. Interestingly this dechlorination process does not produce any toxic byproducts, such as vinyl chloride. Furthermore, complete groundwater detoxification happens within a short time-frame (days) and is robust in terms of bacterial competition, oxygen tolerance, high ionic strength, and pH range.     

Anaerobic dechlorination of trichloroethene,tetrachloroethene and 1,2-dichloroethane by an acetogenic mixed culture in a fixed-bed reactor

An anaerobic enrichment culture with glucose as the sole source of carbon and energy plus trichloroethene (TCE) as a potential electron acceptor was inoculated with material from a full size anaerobic charcoal reactor that biologically eliminated dichloromethane from contaminated groundwater (Stromeyer et al. 1991). In subcultures of this enrichment complete sequential transformation of 10 µM TCE viacis-dichloroethene and chloroethene to ethene was reproducibly observed. Maintenance of this activity on subcultivation required the presence of TCE in the medium. The enrichment culture was used to inoculate an anaerobic fixed-bed reactor containing sintered glass Raschig elements as support material. The reactor had a total volume of 1780 ml and was operated at 20 °C in an up-flow mode with a flow rate of 50 ml/h. It was fed continuously with 2 mM glucose and 55 µM TCE. Glucose was converted to acetate as the major product and to a minor amount of methane; TCE was quantitatively dehalogenated to ethene. When, in addition to TCE, tetrachloroethene or 1,2-dichloroethane were added to the system, these compounds were also dehalogenated to ethene. In contrast, 1,1,1-trichloroethane was not dehalogenated, but at 40 µM severely inhibited acetogenesis and methanogenesis. When the concentration of TCE in the feed was raised to 220 µM, chloroethene transiently accumulated, but after an adaptation period ethene was again the only volatile product detected in the effluent. The volumetric degradation rate at this stage amounted to 6.2 µmol/l/h. Since complete transformation of TCE occurred in the first sixth of the reactor volume, the degradation capacity of the system is estimated to exceed this value by factor of about ten.Abbreviations CA chloroethane - 1,1-DCA 1,1-dichloroethane - 1,2-DCA 1,2-dichloroethane - 1,1-DCE 1,1-dichloroethene - c-DCE cis-1,2-dichloroethene - t-DCE trans-1,2-dichloroethene - PCE tetrachloroethene, perchloroethene - 1,1,1-TCA 1,1,1-trichloroethane - TCE trichloroethene - VC chloroethene, vinyl chloride     

Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H₂/CO₂ converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.Abbreviations BrES 2-bromoethanesulfonic acid - CA chloroethane - 1,2-DCA 1,2-dichloroethane - F₄₃₀ Ni(II)tetrahydro-(12, 13)-corphin with an uroporphinoid (III) ligand skeleton     

The accumulation of fixed biomass increases the observed growth yield of a nitrifying biofilm

The observed growth yield (Y _obs) of a nitrifying biofilm metabolizing ammonia in a continuous flow reactor was constant below a fixed biomass concentration of 40–50 g COD-biomass cm^–2. Beyond this range, an increased Y _obs with the additional accumulation of fixed biomass could be due to a considerable accumulation of inactive materials within the nitrifying biofilm.     

Microbial oxidation of 1,2-dichloroethane under anoxic conditions with nitrate as electron acceptor in mixed and pure cultures

Many organisms have been found to readily oxidize the prevalent contaminant 1,2-dichloroethane (1,2-DCA) to CO2 under aerobic conditions. Some organisms have also been isolated that can reduce 1,2-DCA to ethene via dihaloelimination under anaerobic, fermentative conditions. However, none have been described that can metabolize 1,2-DCA under anoxic, nitrate-reducing conditions. In microcosms prepared from aquifer material and groundwater samples from a contaminated site in eastern Louisiana, USA, 1,2-DCA was observed to degrade with nitrate as the terminal electron acceptor. Nitrate-dependent enrichment cultures were developed from these microcosms that sustained rapid 1,2-DCA degradation rates of up to 500 microM day(-1). This degradation was tightly coupled to complete reduction of nitrate via nitrite to nitrogen gas. A novel 1,2-DCA-degrading organism belonging to the Betaproteobacteria (affiliated with the genus Thauera) was isolated from this enrichment culture. However, degradation rates were much slower in cultures of the isolate than observed in the parent mixed culture. Complete mineralization of 1,2-DCA to CO2 was linked to cell growth and to nitrate reduction in both enrichment and isolated cultures. Monochloroacetate, a putative metabolite of 1,2-DCA degradation, could also be mineralized by these cultures.     

Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater

The microbial community of a groundwater system contaminated by 1,2-dichloroethane (1,2-DCA), a toxic and persistent chlorinated hydrocarbon, has been investigated for its response to biostimulation finalized to 1,2-DCA removal by reductive dehalogenation. The microbial population profile of samples from different wells in the aquifer and from microcosms enriched in the laboratory with different organic electron donors was analyzed by ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA genes. 1,2-DCA was completely removed with release of ethene from most of the microcosms supplemented with lactate, acetate plus formate, while cheese whey supported 1,2-DCA dehalogenation only after a lag period. Microbial species richness deduced from ARISA profiles of the microbial community before and after electron donor amendments indicated that the response of the community to biostimulation was heterogeneous and depended on the well from which groundwater was sampled. Sequencing of 16S rRNA genes separated by DGGE indicated the presence of bacteria previously associated with soils and groundwater polluted by halogenated hydrocarbons or present in consortia active in the removal of these compounds. A PCR assay specific for Desulfitobacterium sp. showed the enrichment of this genus in some of the microcosms. The dehalogenation potential of the microbial community was confirmed by the amplification of dehalogenase-related sequences from the most active microcosms. Cloning and sequencing of PCR products indicated the presence in the metagenome of the bacterial community of a new dehalogenase potentially involved in 1,2-DCA reductive dechlorination.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Autohydrogenotrophic Denitrifying Microbial Community in a Glass Beads Biofilm Reactor

A glass bead biofilm reactor was operated using H₂ as an electron donor to remove nitrate at 150 mg NO₃–N l⁻¹ to below detection level. The microbial community in the glass beads biofilm reactor was investigated by using denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. In DGGE analysis of the biofilm, five bands were dominant and indicated the presence of eight β-proteobacteria, one γ-proteobacteria and twelve clostridia. An unculturable Hydrogenophaga sp., which is a new genus of hydrogen-oxidizing bacterium was dominant in microbial community of the biofilm reactor.     

Nitrogen and phosphorus removal by wetland mesocosms subjected to different hydroperiods

The effect of hydroperiod on nutrient removal efficiency from simulated wastewater was investigated in replicate wetland mesocosms (area, 2 m², planted with Scirpus californicus). Alternate draining and flooding of sediments (pulsed discharge) increased nutrient removal efficiency compared to the continuous-flow “control”. Average PO₄³⁻ removal efficiency was 20–30% higher in wetland mesocosms that drained twice daily compared to the control. Inorganic N removal efficiency was less affected than phosphate removal by hydroperiod variation. At the higher NH₄⁺ loading rate (1.83 g N m⁻² day⁻¹), inorganic N removal efficiency was consistently 5–20% higher in pulsed-discharge wetland mesocosms than in the control. At the lower NH₄⁺ loading rate (0.9 g N m ⁻² day ⁻¹), pulsed-discharge hydrology had no effect on inorganic N removal efficiency. Twice-daily drainage exhibited average inorganic N removal efficiencies of 96% (lower N loading rate) and 87% (higher N loading) and average phosphate removal efficiencies of 81% (lower P loading) and 90% (higher P loading). Mass balance data from the continuous-flow treatment revealed that the aquatic macrophyte Scirpus californicus was the most important nutrient sink, assimilating 50% of the NH₄⁺ and PO₄³⁻ supply. The high plant productivity in the mesocosms (15.6 kg m⁻² year⁻¹) occurred under conditions of high light (high edge per mesocosm area) and high root contact with nutrient-rich influent (shallow, sandy substrate) and may overestimate plant uptake in larger wetlands. The addition of a nitrification-inhibitor (N-Serve) indicated that 34% of the NH₄⁺ supply was transformed to NO₃⁻ by nitrifying bacteria.     

Enhanced nitrite build-up in proportion to increasing alklinity/NH4+ ratio of influent in biofilm reactor

When the alkalinity/NH₄ ⁺ratio increased from 4.1 to 9.4, the ammonium removal rate increased from 45 to 90 mg NO_x-N l^–1 h^–1. An increase in alkalinity/NH₄ ⁺ratio was a major reason for higher pH and free ammonia (FA) concentration in the reactor. The high concentration of FA showed a selective inhibition for Nitrobacter, which caused enhanced nitrite build-up in a biofilm reactor.     

PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B<Subscript>12</Subscript>-dependent 1,2-propanediol degradation by<Emphasis Type="Italic"> Salmonella enterica</Emphasis> serovar Typhimurium LT2

Salmonella enterica forms polyhedral bodies involved in coenzyme-B₁₂-dependent 1,2-propanediol degradation. Prior studies showed that these bodies consist of a proteinaceous shell partly composed of the PduA protein, coenzyme-B₁₂-dependent diol dehydratase, and additional unidentified proteins. In this report, we show that the PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica. A PCR-based method was used to construct a precise nonpolar deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal medium and expressed 105-fold less propionaldehyde dehydrogenase activity (0.011 mol min^–1 mg^–1) than did wild-type S. enterica grown under similar conditions (1.15 mol min^–1 mg^–1). An Escherichia coli strain was constructed for high-level production of His₈-PduP, which was purified by nickel-affinity chromatography and shown to have 15.2 mol min^–1 mg^–1 propionaldehyde dehydrogenase activity. Analysis of assay mixtures by reverse-phase HPLC and mass spectrometry established that propionyl-CoA was the product of the PduP reaction. For subcellular localization, purified His₈-PduP was used as antigen for the preparation of polyclonal antiserum. The antiserum obtained was shown to have high specificity for the PduP protein and was used in immunogold electron microscopy studies, which indicated that PduP was associated with the polyhedral bodies involved in 1,2-propanediol degradation. Further evidence for the localization of the PduP enzyme was obtained by showing that propionaldehyde dehydrogenase activity co-purified with the polyhedral bodies. The fact that both Ado-B₁₂-dependent diol dehydratase and propionaldehyde dehydrogenase are associated with the polyhedral bodies is consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. enterica on 1,2-propanediol.     

Microfluorometric analysis of Cl permeability and its relation to oscillatory Ca signalling in glucose-stimulated pancreatic β-cells

The cytoplasmic concentrations of Cl⁻([Cl⁻]_i) and Ca²⁺ ([Ca²⁺]_i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic β-cells isolated from ob/ob mice. Steady-state [Cl⁻]_i in unstimulated β-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl⁻ into β-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl⁻]_i either in the absence or presence of furosemide inhibition of Na⁺, K⁺, 2 Cl⁻ co-transport. Glucose-induced oscillations of [Ca²⁺]_i were transformed into sustained elevation in the presence of 4,4′ diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid (H₂DIDS). A similar effect was noted when replacing 25% of extracellular Cl⁻ with the more easily permeating anions SCN⁻, I⁻, NO₃⁻ or Br⁻. It is concluded that glucose stimulation of the β-cells is coupled to an increase in their Cl⁻ permeability and that the oscillatory Ca²⁺ signalling is critically dependent on transmembrane Cl⁻ fluxes.     

A Novel Reductive Dehalogenase, Identified in a Contaminated Groundwater Enrichment Culture and in Desulfitobacterium dichloroeliminans Strain DCA1, Is Linked to Dehalogenation of 1,2-Dichloroethane

A mixed culture dechlorinating 1,2-dichloroethane (1,2-DCA) to ethene was enriched from groundwater that had been subjected to long-term contamination. In the metagenome of the enrichment, a 7-kb reductive dehalogenase (RD) gene cluster sequence was detected by inverse and direct PCR. The RD gene cluster had four open reading frames (ORF) showing 99% nucleotide identity with pceB, pceC, pceT, and orf1 of Dehalobacter restrictus strain DSMZ 9455^T, a bacterium able to dechlorinate chlorinated ethenes. However, dcaA, the ORF encoding the catalytic subunit, showed only 94% nucleotide and 90% amino acid identity with pceA of strain DSMZ 9455^T. Fifty-three percent of the amino acid differences were localized in two defined regions of the predicted protein. Exposure of the culture to 1,2-DCA and lactate increased the dcaA gene copy number by 2 log units, and under these conditions the dcaA and dcaB genes were actively transcribed. A very similar RD gene cluster with 98% identity in the dcaA gene sequence was identified in Desulfitobacterium dichloroeliminans strain DCA1, the only known isolate that selectively dechlorinates 1,2-DCA but not chlorinated ethenes. The dcaA gene of strain DCA1 possesses the same amino acid motifs as the new dcaA gene. Southern hybridization using total genomic DNA of strain DCA1 with dcaA gene-specific and dcaB- and pceB-targeting probes indicated the presence of two identical or highly similar dehalogenase gene clusters. In conclusion, these data suggest that the newly described RDs are specifically adapted to 1,2-DCA dechlorination.     

Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

Ammonia exposure of Chasmagnathus granulata (Crustacea, Decapoda) Dana, 1851: accumulation in haemolymph and effects on osmoregulation

In order to study lethal and sublethal effects of ammonia to the estuarine crab Chasmagnathus granulata in the presence of an additional stress factor such as salinity, we determined the LC₅₀ (96 h) of ammonia at 20‰ and in response to osmotic stress (5–40‰) and evaluated ammonia accumulation in the haemolymph of C. granulata and ammonia effects on osmo- and ion-regulation of this species through determinations of the haemolymph Na⁺, Ca²⁺, Cl⁻ and osmotic concentration. The LC₅₀ values (96 h) of total ammonia (NH₃+NH₄⁺) were 10.10, 17.85 and 14.0 mM for crabs maintained at 5, 20 or 40‰ salinity, respectively, suggesting that this crab is fairly resistant to ammonia. The haemolymph ammonia concentration augmented with ambient ammonia during a 6-h exposure to sublethal ammonia concentrations which were not enough to reach equilibrium between external and haemolymph ammonia. At 20‰ salinity, following a 96-h exposure to sublethal concentrations, a significant decrease (P<0.05) of haemolymphatic chloride concentration was registered at 3.3 and 5.5 mM of total ammonia. At 40‰ salinity, a significant increase (P<0.05) of the haemolymph osmotic pressure was apparent at 5.5 mM total ammonia. We postulate that C. granulata gives priority to NH₃ formation as a mechanism to eliminate it by simple diffusion. The differential Na⁺ and Cl⁻ regulation of crabs maintained at 20‰ salinity could modify the strong ion difference, augmenting pH, which in turn should lead the NH₄⁺/NH₃ equilibrium towards NH₃.     

Chloride conductance and mitochondria-rich cell density in isolated skin of Rana catesbeiana acclimated to various environments

The Cl⁻ conductance in isolated skin of frogs (Rana catesbeiana) acclimated to 30 mM solutions of NaCl, Na₂SO₄, MgCl₂ and distilled water (DW) was studied. Transepithelial potential difference (PD_trans), short-circuit current (I_SC) and total conductance (G_t) were measured under conditions such that there was Cl⁻ flux in the presence and absence of Na⁺ transport. The Cl⁻ content of the mucosal solution was acutely replaced with SO₄²⁻ or gluconate to evaluate the effect of removal of Cl⁻ conductance on electrophysiological parameters. Mitochondria-rich cell density (D_MRC) was also measured. Skins from frogs acclimated to NaCl and Na₂SO₄ showed the lowest and the highest D_MRC, respectively, but no difference could be found between the skins from frogs acclimated to DW and MgCl₂ indicating that D_MRC is not unconditionally dependent on environmental Cl⁻ in this species. Frogs acclimated to NaCl showed marked differences when compared to the other groups: the highest G_t, probably represented by a higher paracellular conductance; the lowest transepithelial electrical potential difference which remained invariant after replacement of mucosal Cl⁻ with SO₄²⁻ or replacement of mucosal Cl⁻ with gluconate and an inwardly oriented positive current in the absence of bilateral Na⁺.