Anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate.

In the presence of added sulfate, 2,4-dichlorophenol and 4-chlorophenol were transformed stoichiometrically to 4-chlorophenol and phenol, respectively, in anaerobic freshwater lake sediments between 18 and 40 degrees C. The concomitantly occurring sulfate reduction reduced the initial sulfate concentration from 25 mM to about 6 to 8 mM and depressed methane formation.     

Chlorophenol degradation coupled to sulfate reduction.

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Chlorophenol degradation coupled to sulfate reduction

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Effect of specific inhibitors on the anaerobic reductive dechlorination of 2,4,6-trichlorophenol by a stable methanogenic consortium

The transformation of 2,4,6-trichlorophenol (TCP) into 4-chlorophenol (4CP) was studied using a stable methanogenic enrichment culture derived from an anaerobic fixed bed reactor. Using acetate as a growth substrate, different inhibitors of methanogenesis exhibited distinct effects on TCP dechlorination. Whereas reductive dechlorination activity was not affected by 2% ethylene in the gas phase, 25 mM bromoethanesulfonic acid (BESA) had a direct inhibitory effect on this process. The choice of BESA as a specific inhibitor for identifying the subpopulations involved in reductive dechlorination of chloroaromatics is thus questionable. Inhibitors of sulfate reduction such as molybdate (20 mM) and selenate (20 mM) had a direct inhibitory effect on reductive dechlorination independently of the presence of sulfate in the medium supplemented with acetate as growth substrate. Consequently much more care must also be taken with these inhibitors to prove that reductive chlorination is coupled to sulfate reduction.     

Anaerobic Degradation of Chloroaromatic Compounds in Aquatic Sediments under a Variety of Enrichment Conditions

Anaerobic degradation of monochlorophenols and monochlorobenzoates in a variety of aquatic sediments was compared under four enrichment conditions. A broader range of compounds was degraded in enrichments inoculated with sediment exposed to industrial effluents. Degradation of chloroaromatic compounds was observed most often in methanogenic enrichments and in enrichments amended with 1 mM bromoethane sulfonic acid. Degradation was observed least often in enrichments with added nitrate or sulfate. The presence of 10 mM bromoethane sulfonic acid prevented or inhibited degradation of most compounds tested. Primary enrichments in which KNO(3) was periodically replenished to maintain enrichment characteristics degraded chlorobenzoates, but not chlorophenols. In contrast, primary enrichments in which Na(2)SO(4) was periodically replenished failed to degrade any chloroaromatic compounds. Upon transfer to fresh medium, none of the sulfate enrichments required the presence of Na(2)SO(4) for degradation, while only two nitrate enrichments required the presence of KNO(3) for degradation. As a class of compounds, chlorophenols were degraded more readily than chlorobenzoates. However, as individual compounds 3-chlorobenzoate, 2-chlorophenol, and 3-chlorophenol degradation was observed most often and with an equal frequency. Within the chlorophenol class, the relative order of degradability was ortho > meta > para, while that of chlorobenzoates was meta > ortho > para, In laboratory transfers, 2-chlorobenzoate, 3-chlorobenzoate, and 2-chlorophenol degradation was most easily maintained, while degradation of para-chlorinated compounds was very difficult to maintain.     

Anaerobic degradation of halogenated phenols by sulfate-reducing consortia.

Sulfidogenic consortia enriched from an estuarine sediment were maintained on either 2-, 3-, or 4-chlorophenol as the only source of carbon and energy for over 5 years. The enrichment culture on 4-chlorophenol was the most active and this consortium was selected for further characterization. Utilization of chlorophenol resulted in sulfate depletion corresponding to the values expected for complete mineralization to CO2. Degradation of 4-chlorophenol was coupled to sulfate reduction, since substrate utilization was dependent on sulfidogenesis and chlorophenol loss did not proceed in the absence of sulfate. Other sulfur oxyanions, sulfite or thiosulfate, also served as electron acceptors for chlorophenol utilization, while carbonate, nitrate, and fumarate did not. The sulfidogenic consortium utilized phenol, 4-bromophenol, and 4-iodophenol in addition to 4-chlorophenol. 4-Fluorophenol, however, did not serve as a substrate. 4-Bromo- and 4-iodophenol were degraded with stoichiometric release of halide, and 4-[14C]bromophenol was mineralized, with 90% of the radiolabel recovered as CO2.     

Reductive dechlorination of halogenated phenols by a sulfate-reducing consortium

A sulfidogenic consortium enriched from an estuarine sediment utilized 4-chlorophenol as a sole source of carbon and energy. Reductive dechlorination as the initial step in chlorophenol degradation by the sulfate-reducing consortium was confirmed with the use of chloro-fluorophenols. Both 4-chloro-2-fluorophenol and 4-chloro-3-fluorophenol were dechlorinated, resulting in stoichiometric accumulation of 2-fluorophenol and 3-fluorophenol, respectively. The fluorophenols were not degraded further. Furthermore, phenol was detected as a transient intermediate during degradation of 4-chlorophenol in the presence of 3-fluorophenol. Reductive dechlorination was inhibited by molybdate and did not occur in the absence of sulfate. These results indicate that 4-chlorophenol is reductively dechlorinated to phenol under sulfate-reducing conditions and mineralization of the phenol ring to CO₂ is coupled to sulfate reduction.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

Biodegradation of 4-chlorophenol by entrappedAlcaligenes sp. A 7-2

Summary The degradation of 4-chlorophenol by free and by Ca-alginate-immobilized cells ofAlcaligenes sp. A 7-2 has been studied. Increasing concentrations of 4-chlorophenol (0.4–0.55 mM) were better tolerated and more quickly degraded by the immobilized organisms than by free cells. The capability for haloarene-degradation is inducible. In semicontinuous fermentation at pH 7 a minimal degradation time of 5 h for degrading 0.2 mM 4-chlorophenol was reached. Fermentation temperature was shown to be important for inducing the degradation capability, but to be less important for the degradation rate by induced organisms. High-frequency feeding of small amounts of 4-chlorophenol (0.05 mM) was more favourable than low-frequency feeding of larger amounts (0.15 mM).Continuous fermentation with unbuffered medium allowed a degradation rate of about 2 mmol·l^-1·d^-1; with buffered medium a higher degradation rate of nearly 4 mmol·l^-1·d^-1 was reached, but the Ca-alginate beads dissolved.     

Anaerobic dechlorination and mineralization of pentachlorophenol and 2,4,6-trichlorophenol by methanogenic pentachlorophenol-degrading granules

Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized¹⁴C-labeled PCP to¹⁴CH₄ and¹⁴CO₂. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of¹⁴C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of¹⁴CH₄ and¹⁴CO₂ from [¹⁴C]PCP. 2,4,6-trichlorophenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chlorophenol or 2-chlorophenol to phenol at pH 7.0–7.2.     

Degradation of mono-chlorophenols by a mixed microbial community via a meta- cleavage pathway

A mixed microbial community, specially designed todegrade a wide range of substituted aromaticcompounds, was examined for its ability to degrademono-chlorophenols as sole carbon source in aerobicbatch cultures. The mixed culture degraded 2-, 3-, and4 -chlorophenol (1.56 mM) via a meta- cleavagepathway. During the degradation of 2- and3-chlorophenol by the mixed culture, 3-chlorocatecholproduction was observed. Further metabolism was toxicto cells as it led to inactivation of the catechol2,3-dioxygenase enzyme upon meta- cleavage of3-chlorocatechol resulting in incomplete degradation.Inactivation of the meta- cleavage enzyme led toan accumulation of brown coloured polymers, whichinterfered with the measurement of cell growth usingoptical denstiy. Degradation of 4-chlorophenol by themixed culture led to an accumulation of5-chloro-2-hydroxymuconic semialdehyde, themeta- cleavage product of 4-chlorocatechol. Theaccumulation of this compound did not interfere withthe measurement of cell growth using optical density.5-chloro-2-hydroxymuconic semialdehyde was furthermetabolized by the mixed culture with a stoichiometricrelease of chloride, indicating complete degradationof 4-chlorophenol by the mixed culture via ameta- cleavage pathway.     

Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge.

We investigated the anaerobic biodegradation of mono- and dichlorophenol isomers by fresh (unacclimated) sludge and by sludge acclimated to either 2-chlorophenol, 3-chlorophenol, or 4-chlorophenol. Biodegradation was evaluated by monitoring substrate disappearance and, in selected cases, production of 14CH4 from labeled substrates. In unacclimated sludge, each of the monochlorophenol isomers was degraded. The relative rates of disappearance were in this order: ortho greater than meta greater than para. For the dichlorophenols in unacclimated sludge, reductive dechlorination of the Cl group ortho to phenolic OH was observed, and the monochlorophenol compounds released were subsequently degraded. 3,4-Dichlorophenol and 3,5-dichlorophenol were persistent. Sludge acclimated to 2-chlorophenol cross-acclimated to 4-chlorophenol but did not utilize 3-chlorophenol. This sludge also degraded 2,4-dichlorophenol. Sludge acclimated to 3-chlorophenol cross-acclimated to 4-chlorophenol but not to 2-chlorophenol. This sludge degraded 3,4- and 3,5-dichlorophenol but not 2,3- or 2,5-dichlorophenol. The specific cross-acclimation patterns observed for monochlorophenol degradation demonstrated the existence of two unique microbial activities that were in turn different from fresh sludge. The sludge acclimated to 4-chlorophenol could degrade all three monochlorophenol isomers and 2,4- and 3,4-dichlorophenol. The active microbial population in this sludge appeared to be a mixture of populations present in the 2-chlorphenol- and 3-chlorophenol-acclimated sludges, both of which could utilize 4-chlorophenol. Experiments with 14C-radiolabeled p-chlorophenol, o-chlorophenol, and 2,4-dichlorophenol demonstrated that these compounds were converted to 14CH4 and 14CO2.     

Biodegradation of phenolic compounds by sulfate-reducing bacteria from contaminated sediments

The biodegradation of phenolic compounds under sulfate-reducing conditions was studied in sediments from northern Indiana. Phenol, p-cresol and 4-chlorophenol were selected as test substrates and added to sediment suspensions from four sites at an initial concentration of 10 mg/liter. Degradative abilities of the sediment microorganisms from the four sites could be related to previous exposure to phenolic pollution. Time to onset of biodegradation of p-cresol and phenol in sediment suspensions from a nonindustrialized site was approximately 70 and 100 days, respectively, in unacclimated cultures. In sediment slurries from three sites with a history of wastewater discharges containing phenolics, time to onset of biodegradation was 50–70 days for p-cresol and 50–70 days for phenol in unacclimated cultures. In acclimated cultures from all four sites, the length of the lag phase was reduced to 14–35 days for p-cresol and 25–60 days for phenol. Length of the biodegradative phase varied from 25 to 40 days for phenol and 10 to 50 days for p-cresol and was not markedly affected by acclimation. Substrate mineralization by sulfate-reducing bacteria was confirmed with radiotracer techniques using an acclimated sediment culture from one site. Addition of molybdate, a specific inhibitor of sulfate reduction, and bacterial cell inactivation inhibited sulfate reduction and substrate utilization. None of the sites exhibited the ability to degrade 4-chlorophenol, nor were acclimated phenol and p-cresol degrading cultures from a particular site able to cometabolize 4-chlorophenol.Correspondence to: D. Dean-Ross     

Biotransformation of halophenols by a thermophilic Bacillus sp.

The thermophilic Bacillus sp. A2 transformed various halophenols. 2-Chlorophenol, 2-bromophenol, 3-bromophenol and 2-fluorophenol were transformed under resting cell conditions at 60°C to 3-chlorocatechol, 3-bromocatechol, 4-bromocatechol and 3-fluorocatechol, respectively. The hydroxylation of 3-bromophenol occurred at the proximal and distal position relative to the halogen substituent. In complex medium this strain completely transformed 2-chlorophenol and 2-bromophenol at concentrations up to 1 mM. Concomitantly, an accumulation of oxygen-and temperature sensitive halocatechols was observed. 3-Chlorocatechol possesses a half-life of 11.5 h at 60°C and is therefore readily decomposed during incubation. The hydroxylating system was present in phenolgrown cells but not in glucose-grown cells. The hydroxylase activity could also be induced by 2-chlorophenol. The product, 3-chlorocatechol, is not a substrate for the catechol 2,3-dioxygenase.Abbreviations 2-CP 2-chlorophenol - DCP dichlorophenol - TCP trichlorophenol - tetraCP tetrachlorophenol - MIC minimal inhibitory concentration - CF chloride-free - CFG chloride-free plus glucose - CFGY chloride-free plus glycerol - CFP chloride-free plus phenol - CAM chloramphenicol     

Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.     

Sulfate transport in human lung fibroblasts (IMR-90)

Sulfate transport in a fibroblast cell line derived from human lung (IMR-90) occurred mainly via high- and low-affinity, SITS-sensitive pathways and to a lesser extent by an SITS-insensitive mechanism. In low-ionic-strength media (sucrose substituted for salts) the apparent Km of the carrier-mediated sulfate influx was 1 mM. At 0.3 mM, the sulfate concentration normally found in human serum, the contribution of the SITS-insensitive pathway was negligible. In physiological salts solution, an SITS-sensitive, high-affinity (Km 34 +/- 14 microM) sulfate influx system was observed at extracellular sulfate concentrations less than 100 microM. Between 100 and 500 microM sulfate, the range normally found in human serum, sulfate influx occurred via an SITS-sensitive, low-affinity pathway and to a small extent by an SITS-insensitive mechanism. Extracellular chloride inhibited the influx and stimulated the efflux of sulfate. Bicarbonate and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux. Phosphate, arsenate, or Na+ did not affect sulfate uptake. These results indicate that in human lung fibroblast IMR-90 cells sulfate is transported mainly via an SO4(2-)/Cl- exchange system independent of the phosphate or Na+ transport. Since sulfate concentration as high as 50 mM only slightly increased sulfate efflux, SO4(2-)/SO4(2-) exchange is probably a minor component of sulfate uptake.     

Utilization and cooxidation of chlorinated phenols byPseudomonas sp. B 13

Pseudomonas sp. B13 was grown in continuous culture on 4-chlorophenol as the only carbon source. Maximum growth rate of 0.4h^-1 was observed at a substrate concentration of >0.01 mM and <0.15 mM. In addition to the enzymes of phenol catabolism, high specific 1,2-dioxygenase activities with chlorocatechols as substrates were found. The isomeric monochlorinated phenols were also totally degraded by 4-chlorophenol grown cells. (+)-2,5-Dihydro-4-methyl- and (+)-2,5-dihydro-2-methyl-5-oxo-furan-2-acetic acid were formed in high yield as dead-end catabolites from cooxidation of cresoles.Several dichlorophenols except 2,6-dichlorophenol were removed from the culture fluid by chlorophenol grown cells. Ring cleavage of chlorinated catechols were shown to be one of the critical steps in chlorophenol catabolism. A catabolic pathway for isomeric chlorophenols is discussed.Non-Standard Abbreviations HPLC High performance liquid chromatography - DHB Dihydrodihydroxybenzoate 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid     

Regeneration of bacteriorhodopsin in mixed micelles

Regeneration of bacteriorhodopsin from bacterioopsin and all-trans-retinal was studied in a mixed micelle system consisting of dodecyl sulfate, CHAPS and a water-soluble phospholipid dihexanoylphosphatidylcholine (hex2-PhosChol). Regeneration to approximately 40,000 M-1.cm-1 extinction at 550 nm (epsilon 550) was obtained with either 2.3 mM or 6.5 mM CHAPS along with 6.9 mM dodecyl sulfate and 4.5 mM hex2-PhosChol in 0.16 M NaCl and 40 mM phosphate (pH 6.0). Without CHAPS, the regeneration in 4.5 mM Hex2-PhosChol gave epsilon 555 = 27,800; without PhosChol, the 1:3 CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 20,000; and without PhosChol the nearly equimolar CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 10,000. The composition of the mixed micelles was estimated from fluorescence spectroscopy using pyrene butyryl hydrazine. The molecular weight was estimated by molecular seive chromatography to be 87,100 for 2.3 mM CHAPS, 6.9 mM dodecyl sulfate and 0.67 mM hex2-PhosChol; and 83,200 for 7.0 mM CHAPS, 6.9 mM dodecyl sulfate, and 1.1 mM hex2-PhosChol. These results are consistent with the idea that at low concentrations of CHAPS and dodecyl sulfate, CHAPS organizes the dodecyl sulfate into disk shaped bilayer micelles that are favorable for bacterioopsin refolding. However, a high concentration of either detergent inhibits regeneration. Added hex2-PhosChol can overcome the inhibitory effects of high concentrations of either CHAPS or dodecyl sulfate.     

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.