Autotrophic denitrification by nitrate-dependent Fe(II) oxidation in a continuous up-flow biofilter

A continuous-upflow biofilter packed with sponge iron was constructed for nitrate removal under an anaerobic atmosphere. Microbacterium sp. W5, a nitrate reducing and Fe(II) oxidizing strain, was added to the biofilter as an inoculum. The best results were achieved when NO₃ ^?-N concentration was 30 mg/L and Fe²⁺ was 800 mg/L. Nitrite in influent would inhibit nitrate removal and aqueous Fe²⁺ resulted in encrustation. Fe(II)EDTA would prevent cells from encrustation and the maximum nitrogen removal efficiency was about 90 % with Fe(II)EDTA level of 1100 mg/L. Nitrate reduction followed first-order reaction kinetics. Characteristics of biofilms were analyzed by X-ray fluorescence spectroscopy.     

Cost-effective IMTA: a comparison of the production efficiencies of mussels and seaweed

This paper compares the biofilter capacity and cost-effectiveness of blue mussels (Mytilus edulis) and seaweed for use in integrated multi-trophic aquaculture (IMTA) based on experiences in Ireland and Denmark. This comparison shows that weight for weight, mussels are a better biofilter than seaweed with regard to the amount of nitrogen assimilated. Furthermore, in optimized systems, areal requirement for mussels is similar to the cultivation of the same tonnage (1,000 t) of seaweed (approximately 8 ha). The cost-effectiveness of a mussel biofilter is €11–30 kg^?1 nitrogen (N) removed based on various examples compared to production costs of €209–672 removed and €1,013 kg^?1 N removed, respectively, for Laminaria digitata and Alaria esculenta from extrapolated laboratory and field trials. However, commercial seaweed (Saccharina latissima) producers claim that production costs are less than €10–38 kg^?1 N removed. These up-scaled and commercial figures make the seaweed cost competitive to mussels for removal of nitrogen. Disadvantages such as predators (e.g. eider ducks) and biofouling should also be taken into account before choice of biofilter is made. These drawbacks can reduce overall biofilter capacity and biomass value as a consequence of biomass spoilage or loss. However, disadvantages may be mitigated by seasonal choice of cultivation and harvest times. Cultivation technologies and harvesting methods may be improved together with breeding to improve the cost-efficiency of the biofilter, especially in the newer European seaweed cultivation. Furthermore, upscaling of IMTA to commercial proportions, other than the Danish example, would allow more real data on production costs and revenues.     

Functional rigidity of a methane biofilter during the temporal microbial succession

Temporal microbial succession was investigated in relation to the performance of a methane biofilter. A laboratory-scale biofilter packed with perlite was operated for 108 days, without a deliberate biomass control. The system performance was stable over the period with a mean elimination capacity of 1,563 g m^?3 day^?1, despite a temporal deterioration (45–56 days). Ribosomal-tag pyrosequencing showed that bacterial communities at days 14–28 were distinct from those of days 68–108. The accumulation of nonviable substances strongly coincided with the community change (R ²?>?0.97). Rhodobacter, Hydrogenophaga, and Methylomonas were dominated in the earlier period, while Methylocaldum and Methylococcus were abundant in the later period. The methanotrophic proportion gradually increased to 41 %, and type I methanotrophs became predominant over time. However, community structure and methanotrophic population density stably retained over time, allowing the system to keep the similar performance. Therefore, the perlite biofilter system was functionally rigid against the temporal microbial succession.     

The beneficial effect EDTA on development of mouse one-cell embryos in chemically defined medium.

An improved methology for culturing noninbred (ICR) mouse one-cell embryos is described. The successful development of one-cell embryos into blastocysts in chemically defined (Whitten's) medium was significantly enhanced by the presence of EDTA. More than 70% of ICR one-cell embryos developed into blastocysts in Whitten's medium in the presence of 10.8 μM EDTA, while, without EDTA, only 15–30% of embryos reached blastocyst stage. A concentration of 10.8 μM EDTA also promoted the development of 65–90% of inbred $\text{C57BL}\text{6}$ one-cell embryos in Whitten's medium. This beneficial role of EDTA is probably related to the chelation of some metal ion(s) other than Ca²⁺ or Mg²⁺.     

Role of Thiobacillus thioparus in the biodegradation of carbon disulfide in a biofilter packed with a recycled organic pelletized material

This study reports the biodegradation of carbon disulfide (CS₂) in air biofilters packed with a pelletized mixture of composted manure and sawdust. Experiments were carried out in two lab-scale (1.2 L) biofiltration units. Biofilter B was seeded with activated sludge enriched previously on CS₂-degrading biomass under batch conditions, while biofilter A was left as a negative inoculation control. This inoculum was characterized by an acidic pH and sulfate accumulation, and contained Achromobacter xylosoxidans as the main putative CS₂ biodegrading bacterium. Biofilter operation start-up was unsuccessfully attempted under xerophilic conditions and significant CS₂ elimination was only achieved in biofilter A upon the implementation of an intermittent irrigation regime. Sustained removal efficiencies of 90–100 % at an inlet load of up to 12 g CS₂ m^?3 h^?1 were reached. The CS₂ removal in this biofilter was linked to the presence of the chemolithoautotrophic bacterium Thiobacillus thioparus, known among the relatively small number of species with a reported capacity of growing on CS₂ as the sole energy source. DGGE molecular profiles confirmed that this microbe had become dominant in biofilter A while it was not detected in samples from biofilter B. Conventional biofilters packed with inexpensive organic materials are suited for the treatment of low-strength CS₂ polluted gases (IL <12 g CS₂ m^?3 h^?1), provided that the development of the adequate microorganisms is favored, either upon enrichment or by inoculation. The importance of applying culture-independent techniques for microbial community analysis as a diagnostic tool in the biofiltration of recalcitrant compounds has been highlighted.     

Effect of pH and Chelating Agents on the Heat Resistance and Viability of Salmonella typhimurium Tm-1 and Salmonella senftenberg 775W in Egg White

Supplementation of egg white at pH 8.9 with 5 mg of disodium ethylenediaminetetraacetic acid (EDTA) per ml resulted in a kill of Salmonella typhimurium Tm-1 of greater than 10⁶ per ml after 28 days at 2 C. While at 28 C, supplementation with 7 mg of EDTA per ml resulted in approximately a 10⁶ kill in less than 24 hr. Kena supplementation at 40 mg/ml of egg white resulted in a kill of S. typhimurium Tm-1 of greater than 10⁶ after approximately 60 hr of storage at 28 C. This is in contrast to no reduction in viable count in unsupplemented egg white stored at 2 C and a 100-fold increase in viable count in that stored at 28 C. Supplementation of egg white with EDTA at 7 mg/ml or with Kena at 10 mg/ml also affected the heat resistant characteristics of the two organisms at 52.5 C, reducing the time required to kill 90% of the population (D value) at any pH by a factor of 2 to 6. There was a synergistic effect between EDTA and lactic acid when lactic acid was used to adjust EDTA-supplemented egg white to an acidic pH (5.3) which greatly decreased the heat resistance of Salmonella senftenberg 775W (from 100D to D).     

Spin concentration measurements of high-spin (g′ = 4.3) rhombic iron(III) ions in biological samples: theory and application

Electron paramagnetic resonance (EPR) signals at g′ = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe³⁺ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g′ = 4.3 intensity standard and for accurately quantifying the amount of Fe³⁺ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308–315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe³⁺–EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu²⁺–EDTA and aqua VO²⁺, both S = 1/2 ions, and of Fe³⁺–transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of ±3.6, ±5.3 and ±2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe³⁺–desferrioxamine of known Fe³⁺ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.     

Pseudomonas aeruginosa contamination of mouth swabs during production causing a major outbreak

Background

MUC7 12-mer (RKSYKCLHKRCR), a cationic antimicrobial peptide derived from the human low-molecular-weight salivary mucin MUC7, possesses potent antimicrobial activity in vitro. In order to evaluate the potential therapeutic application of the MUC7 12-mer, we examined the effects of mono- and divalent cations, EDTA, pH, and temperature on its antimicrobial activity.

Methods

Minimal Inhibitory Concentrations (MICs) were determined using a liquid growth inhibition assay in 96-well microtiter plates. MUC7 12-mer was added at concentrations of 1.56–50 μM. MICs were determined at three endpoints: MIC-0, MIC-1, and MIC-2 (the lowest drug concentration showing 10%, 25% and 50% of growth, respectively). To examine the effect of salts or EDTA, a checkerboard microdilution technique was used. Fractional inhibitory concentration index (FICi) was calculated on the basis of MIC-0. The viability of microbial cells treated with MUC7 12-mer in the presence of sodium or potassium was also determined by killing assay or flow cytometry.

Results

The MICs of MUC7 12-mer against organisms tested ranged from 6.25–50 μM. For C. albicans, antagonism (FICi 4.5) was observed for the combination of MUC7 12-mer and calcium; however, there was synergism (FICi 0.22) between MUC7 12-mer and EDTA, and the synergism was retained in the presence of calcium at its physiological concentration (1–2 mM). No antagonism but additivity or indifference (FICi 0.55–2.5) was observed for the combination of MUC7 12-mer and each K⁺, Na⁺, Mg²⁺, or Zn²⁺. MUC7 12-mer peptide (at 25 μM) also exerted killing activity in the presence of NaCl, (up to 25 mM for C. albicans and up to 150 mM for E. coli, a physiological concentration of sodium in the oral cavity and serum, respectively) and retained candidacidal activity in the presence of KCl (up to 40 mM). The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60°C did not affect the activity.

Conclusion

MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity. These results suggest that, especially in combination with EDTA, it could potentially be applied as an alternative therapeutic agent for the treatment of human oral candidiasis.     

Removal characteristics of hydrogen sulphide and methanethiol by Thiobacillus sp. isolated from peat in biological deodorization

Thiobacillus sp. HA43 as a dominant strain was isolated from a H₂S-acclimated peat biofilter seeded with aerobically-digested sludge of night soil. Strain IIA43 degraded both H₂S and methanethiol (MT) without lag-time, but degraded neither dimethy sulphide (DMS) nor dimethyl disulphide (DMDS). The removal characteristics for sulphur compounds (H₂S, MT, DMS and DMDS) by strain HA43 well reflected the removal behaviour of the H₂S-acclimated peat biofilter where this strain was isolated. The specific H₂S and MT uptake rates of strain HA43 in batch culture were determined as 1.22 × 10⁻¹² and 8.53 × 10⁻¹⁴ g-S·cell⁻¹·h⁻¹, respectively. The maximum removal rates (V_m = g-S·kg-dry peat⁻¹·d⁻¹) for H₂S and MT by peat biofilter inoculated by strain HA43 were obtained as follows: V_m(H₂S)− 11.3, V_m(MT) = 0.21 in sterilized peat; V_m(H₂S) = 12.4, V_m(MT)− 0.27 in non-sterilized peat; V_m(H₂S) = 33.0, V_m(MT) = 0.27 in peat with aerobically-digested sludge of night soil. The peat biofilter inoculated with strain HA43 enhanced the maximum removal rate for H₂S 6-fold compared with the biofilter without strain HA43.     

Qualitative and quantitative changes in Locusta haemolymph proteins and lipoproteins during ageing and adipokinetic hormone action

Changes in haemolymph proteins and lipoproteins during adipokinetic hormone action have been studied using polyacrylamide gel electrophoresis (PAGE) and a heparin/EDTA precipitation technique. During hormone action, the formation of A⁺ takes place at the expense of Ayellow and C_L-proteins, which decrease in free concentration in the haemolymph. Ayellow is heparin precipitable, whereas A⁺ precipitates with EDTA after prior treatment with heparin. After injection of adipokinetic hormone, heparin-precipitable protein (HPP) decreases after a delay of 10–15 min, but heparin/EDTA precipitable protein (HEPP) increases immediately. These changes occur in response to extracts of corpora cardiaca and to synthetic adipokinetic hormone, and are dose-dependent. Both the lipid and the C_L-protein content of the HEPP rise as its protein content increases. A⁺ formation does not occur in fifth-instar nymphs or newly emerged adults, but this response to adipokinetic hormone develops slowly as the adults mature.     

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra‐weak photon emission (UPE). We investigated the UPE from the Fe²⁺–EDTA (ethylenediaminetetraacetic acid)–AA (ascorbic acid)–H₂O₂ (hydrogen peroxide) system with a multitube luminometer (Peltier‐cooled photon counter, spectral range 380–630 nm). The UPE, of 92.6 μmol/L Fe²⁺, 185.2 μmol/L EDTA, 472 μmol/L AA, 2.6 mmol/L H₂O₂, reached 1217 ± 118 relative light units during 2 min measurement and was about two times higher (P < 0.001) than the UPE of incomplete systems (Fe²⁺–AA–H₂O₂, Fe²⁺–EDTA–H₂O₂, AA–H₂O₂) and medium alone. Substitution of Fe²⁺ with Cr²⁺, Co²⁺, Mn²⁺ or Cu²⁺ as well as of EDTA with EGTA (ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid) or citrate powerfully inhibited UPE. Experiments with scavengers of reactive oxygen species (dimethyl sulfoxide, mannitol, sodium azide, superoxide dismutase) revealed the dependence of UPE only on hydroxyl radicals. Dimethyl sulfoxide at the concentration of 0.74 mmol/L inhibited UPE by 79 ± 4%. Plant phenolics (ferulic, chlorogenic and caffec acids) at the concentration of 870 μmol/L strongly enhanced UPE by 5‐, 13.9‐ and 46.8‐times (P < 0.001), respectively. It is suggested that augmentation of UPE from Fe²⁺–EDTA–AA–H₂O₂ system can be applied for detection of these phytochemicals.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: III. Large bleb formation caused by Sr2+, ionophores X537A and A23187, and compound 4880

Fertilized eggs of Ilyanassa obsoleta form a protuberance which resembles a normal polar lobe when injected with Sr²⁺ or Ca²⁺ by microiontophoresis. Eggs also form a lobe-like protuberance when exposed to any of three drugs: compound $\text{48}\text{80}$, ionophore X537A, and ionophore A23187. Protuberances form more quickly and at lower drug concentrations if additional exogenous Ca²⁺ is added, whereas higher concentrations of Mg²⁺ do not have such an effect. When eggs are exposed to these drugs in Ca²⁺-, Mg²⁺-“free” seawater, with or without 10 mM EDTA, the eggs are still able to undergo extensive shape changes and form protuberances. Drug-induced shape changes are prevented by cytochalasin B, but will still occur in the presence of colchicine. Approximately 75% of Ilyanassa eggs are capable of forming and resorbing their third polar lobe and undergoing cytokinesis in Ca²⁺-, Mg²⁺-“free” artificial seawater (even containing 10 mM EDTA), solutions which by atomic absorption spectroscopy are shown to contain low concentrations of Ca²⁺ (3–5 μM) and Mg²⁺ (1.0–3.5 μM). The data suggest that if Ca²⁺ is required for normal polar lobe formation and cytokinesis, it is derived from intracellular sources or is required in only very low exogenous concentrations (i.e., less than 10^?2 μM free Ca²⁺, in the presence of 10 mM EDTA).     

Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals

We have prepared monoclonal antibodies (MoAbs) with the specific ability to bind metal chelates such as ¹¹¹In benzyl EDTA. One, 10, 50 and 100 μg MoAb CHA255 K_b = 4 × 10E9 was complexed with ¹¹¹In BLEDTA II, BLEDTA IV, and benzyl EDTA and injected i.v. in Balb/c mice with KHJJ tumor. The biological half-life by whole body counting was profoundly altered for all three compounds; from minutes to hours with 10 μg; to days with 100 μg. Tumor uptake increased 50 fold at 24 h with increasing MoAb but satisfactory tumor concentrations (3% per g) and tumor/blood ratios (1.8:1) were obtained with an amount equivalent to 7 mg for a human. Blood level and whole body activity were decreased 30–50% within 3 h or i.v. injection of a “flushing” dose of unlabeled indium benzyl EDTA, increasing tumor/blood ratios to 50:1.     

The application of a micro-algal/bacterial biofilter for the detoxification of copper and cadmium metal wastes

In the present study the potential of a biofilter containing a mixture of dried micro-algal/bacterial biomass for removing heavy metals (Cu²⁺, Cd²⁺) from dilute electroplating waste was tested. The biomass was produced in an artificial stream using the effluent of a municipal waste water treatment plant as a nutrient source, with the additional benefit of reducing phosphorus and nitrogen loadings. Baseline batch experiments determined that optimum adsorption for both metals (80–100%) were achieved with the deionized-H₂O conditioned biomass at initial pH 4.0. Other biosorption variables (contact time, initial metal concentration) were also tested. Biosorption data were fitted successfully by the Langmuir model and results showed a high affinity of the used biomass for both metals (q_max 18–31 mg metal/g.d.w). Flow-through column experiments containing Ca-alginate/biomass beads showed that metal adsorption depends also on flow-rate and volume of treated waste. Desorption of both metals with weak acids was very successful (95–100%) but the regeneration of the columns was not achieved due to the destabilization of beads.     

On the toxic effects of tetraethyl lead and its derivatives on the chrysophyte poterioochromonas malhamensis. IV. influence of lead antidotes and related agents

The unicellular alga Poterioochromonas malhamensis was exposed to 12.5 μM of inorganic or triethyl lead and simultaneously treated with lead antidotes and related agents at concentrations of 12.5, 31.25 and 62.5 μM. With increasing concentrations some of the antidotes alone slightly to severely inhibited algal growth (BAL¹, CaNa₂EDTA, EDTA, Na₂EDTA), whereas others (DPA, EGTA, DIZO) were non-toxic at the concentrations tested. EGTA and CaNa₂EDTA, at all concentrations tested, completely suppressed the growth inhibition caused by inorganic lead; Na₂EDTA and EDTA were protective at the lower or medium concentrations, but DIZO. DPA and BAL considerably enhanced lead toxicity with increasing concentrations. None of the tested agents was able to reduce the toxic effects of triethyl lead. All antidotes markedly increased inhibition of algal growth caused by triethyl lead and some were even lethal to the poisoned algae either at the highest (Na₂EDTA, EDTA, DPA) or at all concentrations used (DIZO, BAL). P. malhamensis proved to be a highly sensitive and valuable tests system and the results obtained exhibited striking parallels to medical and clinical experience in therapy of human poisoning with inorganic and organic lead compounds.     

Performance evaluation and microbial community analysis of the composite filler micro-embedded with Pseudomonas putida for the biodegradation of toluene

The composite filler micro-embedded with Pseudomonas putida (P. putida) was prepared and the biodegradation performance of the filler was evaluated in a biofilter. Five phases were set up to evaluate the performance of the biofilter under different toluene inlet loadings and transient shock loadings. In particular, the microbial community structure in the biofilms and fillers was measured by sequence analysis of the 16S rRNA gene. The results show that the biofilter packed with the composite fillers was suitable for the biodegradation of toluene. The biofilter could start up quickly with high removal efficiency (RE), and remain above 90 % RE when the empty bed residence time (EBRT) was 18 s and the inlet loading rates were not higher than 41.4 g/(m³·h). Moreover, the biofilter could tolerate substantial transient shock loadings. The high removal efficiency and elimination capacity contributed to rich bacterial communities for the efficient degradation of toluene. The dominant microbial communities at the phylum level were mainly Firmicutes, Actinobacteria and Proteobacteria. It is noteworthy that the abundance of Bacteroidetes at phylum level and Chungangia and Stenotrophomonas at genus level increased significantly during the re-start period.     

Physicomechanical Characterization and Optimization of EDTA–mPEG and Avicel®–EDTA–mPEG In Situ Melt Dispersion Mini-Pellets

The purpose of this study was to develop a physicomechanically customizable oral metal chelatory in situ hot melt dispersion mini-pellet entity which could be utilized within a binary drug delivery system. Avicel^® RC/CL type R-591 was included within the in situ hot melt dispersion mini-pellet formulations to determine the physicomechanical effect this compound would have on the mini-pellet formulations. The physicomechanical properties of the hot melt in situ mini-pellet formulations were mathematically fitting to regression curves. Physicomechanical adjustment of the in situ hot melt dispersion mini-pellet formulations could be mathematically predicted with the derived regression curve equations. The addition of Avicel^® RC/CL type R-591 increased the physicomechanical properties such as matrix hardness and increased total disintegration of the in situ hot melt dispersion mini-pellet formulations. The utilization of a physicomechanically customizable oral metal chelatory in situ hot melt dispersion mini-pellet entity within a binary drug delivery system would to achieve a synergistically enhance the activity of a drug-carrying entity or a permeation enhancing entity within a single drug delivery unit. The experimental results indicated that weights of the pellets that achieved optimal hardness ranged between 35 and 45 mg. The melt–dispersion formulations disintegrated within shorter time periods and maintained higher ethylenediaminetetraacetic acid (EDTA) concentrations whereas melt–dispersion formulations which included Avicel^® had superior physicomechanical properties. Disintegration times ranged between 1,000 s for melt–dispersions containing EDTA and methyloxy polyethylene glycol 2000 (mPEG) only, to >6,000 s for melt–dispersions comprising EDTA, mPEG, and Avicel^®.     

The synthesis of 3-nonaprenyl-4-hydroxybenzoate in rat liver mitochondria: the effect of Ca2 , Mg2+, chelators, and bacitracin on the activity of p-hydroxybenzoate-polyprenyl transferase

The formation of the first intermediate in ubiquinone-9 biosynthesis, 3-nonaprenyl-4-hydroxybenzoate (NPHB), by the enzyme p-hydroxybenzoate:polyprenyl transferase, has been studied in isolated rat liver mitochondria using solanesol pyrophosphate and p-hydroxybenzoate as the substrates. Phosphate buffer (100 mm) is inhibitory but at 20 mm inhibition is not apparent compared to other buffers at the same concentration. With various buffers at low concentration (20 mm) both EDTA and Mg²⁺ stimulate formation of NPHB while Ca²⁺ inhibits. Release of Ca²⁺ inhibition can be achieved by the addition of Mg²⁺, or EDTA, or EGTA, with EGTA being less effective than EDTA. When Mg²⁺, Ca²⁺, and EDTA are present together, a two- to threefold increase in activity of the enzyme is observed. The antibiotic bacitracin inhibits the synthesis of NPHB and the inhibition is increased when divalent cations are present. EGTA is more effective than EDTA in overcoming inhibition due to bacitracin. The possibility that these effects are partially due to alteration of mitochondrial membrane conformation as well as a direct effect on the enzyme is evaluated. The possible role of polyprenylphosphates in mitochondrial membrane function is discussed.     

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q _OO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O₂ at a rate of 4 µl hr^-1 dry wt^-1. Q _OO2 was increased 50% by ADH (100 U/liter) or by cyclic 3'',5''-AMP (10 mM/liter). Na⁺-free Ringer''s depressed the Q _OO2 by 40%. The Q _OO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na⁺-free Ringer''s but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na⁺ lack or ADH. The intracellular Na⁺ and K⁺ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H₂O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na⁺ concentrations. Cells unresponsive to ADH and Na⁺ lack had high sucrose spaces and low transcellular membrane gradients of Na⁺, K⁺, and Cl^-. The results suggest that trypsin and EDTA disaggregation damage the active Na⁺ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.     

Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

Deuterated styrene ([²H₈]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [²H₈]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [²H₈]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.