Biological treatment of phenolic wastes: Comparison between free and immobilized cell systems

Summary Phenol degradation by free and immobilized cells ofFusarium flocciferum was studied in a chemostat at steady-state conditions. For the free cell system the dilution rates varied from 0.02 to 0.13h^–1, with a total phenol removal up to 0.08h^–1. Wash-out seemed to set in at 0.11h^–1. The immobilized cells showed virtually complete phenol utilization at 1g/l, over a period of four months. At D=0.2h^–1 and above 1g/l phenol, the complete phenol removal is not achieved: a progressive increase in the outlet concentration was observed attaining a value of 284mg/l at 1.5g/l.     

Degradation of phenol by a defined mixed culture immobilized by adsorption on activated carbon and sintered glass

Summary A defined mixed culture of the yeast Cryptococcus elinovii H1 and the bacterium Pseudomonas putida P8 was immobilized by adsorption on activated carbon and sintered glass, respectively. Depending on its adsorption capacity for phenol the activated carbon system could completely degrade 17 g/l in batch culture, whereas the sintered glass system was able to degrade phenol up to 4 g/l. During semicontinuous degradation of phenol (1 g/l) both systems reached constant degradation times with the fourth batch that lasted 8 h when using the activated carbon system and 10 h in the sintered glass system. In the course of continuous degradation of phenol the activated carbon system reached a maximum degradation rate of 9.2 g l^–1 day^–1 compared to 6.4 g l^–1 day^–1degraded by the sintered glass system. 2-Hydroxymuconic acid semialdehyde could be identified and quantitatively determined as a metabolite of phenol degradation by P. putida P8. Increased membrane permeability under the influence of phenol was demonstrated by the examination of K⁺ efflux from P. putida P8. Offprint requests to: H.-J. Rehm     

Biological monitoring of hexamethylene diisocyanate by determination of 1,6-hexamethylene diamine as the trifluoroethyl chloroformate derivative using capillary gas chromatography with thermoionic and selective-ion monitoring

A GC method using a novel derivatization reagent, 2′,2′,2-trifluoroethyl chloroformate (TFECF), for the derivatization of primary and secondary aliphatic amines with the formation of carbamate esters is presented. The method is based on a derivatization procedure in a two-phase system, where the carbamate ester is formed. The method is applied to the determination of 1,6-hexamethylene diamine (HDA) in aqueous solutions and human urine, using capillary GC. Detection was performed using thermionic specific detection (TSD) and mass spectrometry (MS)—selective-ion monitoring (SIM) using electron-impact (EI) and chemical ionization (CI) with ammonia monitoring both positive (CI)⁺ and negative ions (CI)⁻. Quantitative measurements were made in the chemical ionization mode monitoring both positive and negative ions. Tetra-deuterium-labelled HDA (TDHDA; H₂NC²H₂(CH₂)₄C²H₂NH₂) was used as the internal standard for the GC—MS analysis. In CI⁺ the m/z 386 and the m/z 390 ions corresponding to the [M + 18]⁺ ions (M = molecular ion) of HDA—TFECF and TDHDA—TFECF were measured; in CI⁻ the m/z 267 and the m/z 271 ions corresponding to the [M — 101]⁻ ions. The overall recovery was found to be 97 ± 5% for a HDA concentration of 1000 μg/l in urine. The minimal detectable concentration in urine was found to be less than 20 μg/l using GC—TSD and 0.5 μg/l using GC—SIM. The overall precision for the work-up procedure and GC analysis was ca. 3% (n = 5) for 1000 μg/l HDA-spiked urine, and ca. 4% (n = 5) for 100 μg/l. The precision using GC—SIM for urine samples spiked to a concentration of 5 μg/l was found to be 6.3% (n = 10).     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

Phenol degradation by microorganisms adsorbed on activated carbon

Summary The phenol degradation by Candida sp. and Pseudomonas sp. immobilized on activated carbon was investigated. Thanks to its great adsorptive surface, activated carbon is suited as supporting material for microorganisms and also provides a high adsorption capacity for phenol.The immobilization by adsorption avoids any unphysiological treatment of the microorganisms. One gram activated carbon adsorbed in 10 h about 4×10⁹ Pseudomonas cells and 3×10⁸ Candida cells. While the free cells did not tolerate more than 1.5 g/l phenol, the adsorbed microorganisms survived at temporary high phenol concentrations up to 15 g/l, and they degraded about 90% of the adsorbed phenol.The activated carbon operated like a depot: the adsorbed phenol diffused out of the carbon and could be metabolized by the microorganisms. The results give an explanation of the stimulating effect of activated carbon in the treatment of waste waters observed until now.     

Synthesis of l-tyrosine by immobilized Escherichia intermedia cells

Summary Cells of Escherichia intermedia were immobilized by entrapment in a polyacrylamide gel and used for the enzymatic production of l-tyrosine from phenol, pyruvate, and ammonia. A preparation containing 50 mg of cells/g of gel retained 60% of its original activity. The effect of temperature, pH and substrate concentration on the activity of free cells was almost identical with the effect on immobilized cells. Phenol showed inhibition and inactivation of the catalyst at high concentration. Synthesis of l-tyrosine (up to 10 g/l) was demonstrated in batch reactors with high conversion yields (95–100%) and a maximal productivity of 2 g/l/h. In continuous reactor the catalyst showed a very high operational stability (more than 54 days without losses).     

Improved exopolysaccharide production in fed-batch fermentation of Aureobasidium pullulans, with increased impeller speed

Summary Aureobasidium pullulans — when grown in fed-batch fermentation on 190 g glucose/l produced 68 g exopolysaccharide/l with a productivity of 0.42 g/l-h. When the impeller speed was increased from 280 rpm to 340 rpm, the exopolysaccharide increased to 101 g/l, the productivity was 0.90 g/l-h and the conversion ratio was 63 % (w/w).     

Effects of Diazinon on bluegill sunfish, Lepomis macrochirus, gills: scanning electron microscope observations

Gills of bluegill sunfish, Lepomis macrochirus, exhibited varied degrees of structural damage following a 24-h exposure to sublethal concentrations (15 μg/l, 30 μg/l, 45 μg/l, 60 μg/l and 75 μg/l) of Diazinon [O,O-diethyl-O-(2-isopropyl-6-methyl-4 pyrimidinyl ester or phosphorothioate]. Exposure to 15 μg/l and 30 μg/l resulted in exocytosis of some material to the cell surface and perforations of the microridges. At higher doses (above 45 μg/l), the extrusion was reduced and the cells were swollen. Compared to control values, the thickness of the microridge on the gill arch and on the gill filament generally increased with exposure to Diazinon. Also, the distance between microridges decreased with increased exposure concentrations. At 60 μg/l, gill arch microridges fused and some ridges of gill filaments disappeared. At 75 μg/l exposure, epithelial cells of the gill arch became obscured with severe cellular extrusions and the lamellar surfaces swelled. The mucus extrusion, lamellar swelling and reduced microridges may be related to a defence mechanism which reduces the water surface around the gill and increases the barrier distance for diffusion of toxicants from outside to the blood capillaries. Although this mechanism protects the fish from toxicants, it also reduces the oxygen supply which leads to suffocation of the fish.     

Kinetics of bio-oxidation of a medium comprising phenol and a mixture of organic contaminants

The kinetics of bio-oxidation by a microbial ensemble of a model mixture of contaminants that mimicked the ground-water pollution plume at an existing contaminated site was investigated. Phenol at 50 mg/l and a mixture of ten organic contaminants (MOC) (benzene, tetrachloromethane, trichloroethylene, toluene, o-xylene, 1,4-dichlorobenzene, o-cresol, nitrobenzene, naphthalene and 2,6-dichlorophenol) at individual concentrations ranging from 150 g/l to 600 g/l were the components of the model mixture. The microbial ensemble consisted of at least three Pseudomonas spp. isolated from the polluted site. Patterns of oxygen uptake rate (OUR) for the oxidation of phenol alone and with added MOC were treated mathematically. The values for kinetic parameters that gave the best fit to the data were respectively 11.29 and 15.03 ml O₂ h^–1 (mg protein)^–1 for the OUR maximum (OUR_max), 75.89 mg/l and 33.66 mg/l for the saturation constant (K _s), 105.92 mg/l and 36.44 mg/l for the inhibitor constant (K _i), and 89.66 mg/l and 35.02 mg/l the substrate minimum inhibitory concentration (S _mic). This study also scrutinised interference between the two components of the model mixture of contaminants (phenol and MOC) on the basis of variations in kinetic patterns. MOC was shown to be toxic at milligram per litre levels. The microbial ensemble increased phenol oxidation in response to MOC, possibly to obtain the energy to overcome this toxic effect. This was indicated by an acceleration of phenol oxidation in response to increasing concentrations of MOC and higher OUR_max for oxidation of phenol in the presence of MOC. The toxicity of MOC also resulted in enhanced vulnerability of the microbial ensemble to a phenol inhibitory effect, indicated by the diminution of K _i and S _mic. The microbial ensemble showed high resistance to inhibition by the sole presence of phenol possibly because of adaptation to toxic features of MOC during the processes of enrichment and cultivation.     

Production of xylitol from concentrated wood hydrolysates by Debaryomyces hansenii: Effect of the initial cell concentration

Summary Eucalyptus globulus wood hydrolysates were concentrated by vacuum evaporation to increase their xylose contents, treated with activated charcoal, supplemented with nutrients and used as culture media for xylitol production by Debaryomyces hansenii NRRL Y-7426. The susceptibility of hydrolysates to fermentation was strongly dependent on the initial cell concentration: media containing 58–78 g xylose/l were hardly consumed in batch experiments starting with 16 g cells/l, whereas 39–41 g xylitol/l were achieved in fermentations carried out with similar concentration of the carbon source and initial cell concentrations of 50–80 g/l).     

In vitro culture of Cuminum cyminum regeneration of flowering shoots from calli of hypocotyl and leaf explants

Calli from hypocotyl explant of Cuminum cyminum L. (Cumin) grew rapidly on Gamborg's B₅ basal medium with the following supplements, (i) 0.5 mg/l — 2,4-D (ii) 4 mg/l — NAA plus 2 mg/l — Kinetin and (iii) 0.2 mg/l — NAA plus 0.2 mg/l — BAP, whereas calli from leaf explant in these media grew slowly. Hypocotyl and leaf calli produced roots when transferred to basal medium only and shoots in basal medium with 0.5 mg/l NAA and 0.1 mg/l BAP. Ninety percent of the shoots produced roots when they were transferred to half strength MS inorganic salts supplemented with 0.5 mg/l each of IBA and NAA.Fifty to sixty percent of rootless as well as rooted shoots produced terminal umbellate flowers on this medium.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

Determination of cholesterol and cortisone absorption in polyurethane : I. Methodology using size-exclusion chromatography and dual detection

A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 μl, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 μg/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 μg (1 μg/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 μg/ml with a lower limit of 0.015 μg (0.1 μg/ml). The results show that after 44 h, 2037 μg/g cholesterol and 3131 μg/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer.     

Determination of therapeutic and toxic concentrations of doxepin and loxapine using gas—liquid chromatography with a nitrogen-sensitive detector, and gas chromatography—mass spectrometry of loxapine

A gas—liquid chromatographic procedure is presented for the determination of therapeutic and toxic serum levels of doxepin and loxapine, using a nitrogen—phosphorus-sensitive detector. Amitriptyline is used as the internal standard. The method is accurate, sensitive and specific with no derivatization required prior to analysis. An advantage of the procedure is the small serum sample size needed for analysis and the selectivity and sensitivity of the detector, with the limit of detection being 3 and 2 μg/l for doxepin and loxapine, respectively. Nine cases of doxepin and loxapine misuse are presented. Serum doxepin concentrations ranged from 113 to 439 μg/l, with a loxapine concentration of 192 μg/l observed in one patient. The presence of the tricyclics was identified and confirmed by gas chromatography—mass spectrometry and the mass spectrum of loxapine is reported.     

Electron histochemical study of 1:2-glycol groups in experimental hamster amyloid

Summary Leishmania-induced amyloid of hamster kidneys was studied by the periodic acid — thiocarbohydrazide — osmium tetroxide method which is analogous to the PAS reaction.The amyloid fibrils failed to give a reaction for 12-glycol groups. The ultrastructural distribution of mucosubstances containing 12-glycol groups was found to be localized in the ground substance between the fibrils.The reaction was unaffected by -amylase digestion, and chloroform-methanol extraction of lipids; it was dependant upon periodic acid oxidation.     

Feinbau und Entwicklung der Calciumoxalat-Kristallzellen mit suberinähnlichen Kristallscheiden in der Rinde und im sekundären Holz vonAcacia Senegal Willd

Zusammenfassung Die Kristallkammerfasern vonAcacia bestehen aus vielen hintereinanderliegenden Zellen mit je einem Kristall. Ein erwachsener Kristall ist von einer Scheide umhüllt, deren am weitesten in das Lumen vorragender Teil schwer kontrastierbar ist, in 50% Schwefelsäure nicht quillt, Primärfluoreszenz aufweist, aber keine Lamellierung zeigt. Dieses füllkorkähnliche Material geht mit faserigen Fortsätzen in die übrige Kristallscheide aus Cellinmaterial über. Die wachsenden Kristalle scheinen in einem Kompartiment zu liegen, dessen Membran meist dicht dem Kristall anliegt, an manchen Stellen aber ER-ähnliche Ausläufer in die Zelle entsendet. Kleine Kristalle liegen in einer Vakuole, sind darin aber nochmals von einem Cytoplasmabelag überzogen, der an manchen Stellen mit dem übrigen Cytoplasma in Verbindung steht. Plasmareste und Membranen in größeren Kristallvakuolen sind vermutlich osmotisch bedingte Fixierungsartefakte. Es wird angenommen, daß der Kristall im Oxalatkompartiment wächst, während das Calcium durch die cytoplasmatische Phase herantransportiert wird.

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Ultrastructure and development of the calcium oxalate crystal cells with suberin-like crystal sheaths in the bark and secondary xylem ofAcacia Senegal Willd

Summary Each chamber of a chambered crystalliferous cell is an independent cell containing one crystal. In a fully developed cell, the crystal sheath consists of celluloso-pectic wall material (cellin) where it is attached to the cell wall. Where the sheath is farthest away from its place of attachment, its interior layer shows low contrast after any conventional staining or after the Thiéry reaction for polysaccharides. Section treatment with 50% sulphuric acid did not result in swelling or dissolution of this material which normally ought to dissolve if it were callose. This non-reactive material resembles filling cork (Wattendorff 1969, 1976 a) und shows autofluorescence in the light microscope. It is fixed with fibre-like prolongations in the cellin part of the sheath and covered by a tertiary cellin layer.A crystal seems to grow in an extraplasmatic compartment. Its membrane adheres closely to the crystal in most places, but sometimes it appears to be prolonged into an endoplasmic reticulum-like system. Small crystals may lie in vacuoles, but within these vacuoles they are again covered by a thin cytoplasmic layer which in some places is connected with the bulk of the cytoplasm. Plasmatic remnants and membranes in the crystal vacuoles of the literature might be artifacts induced by differences of osmotic pressure during fixation. It is supposed that the crystal compartment contains the oxalate ion while the calcium can be transported within the cytoplasmic compartment which is in close contact with the crystal, through its membrane.

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Symbole für alle abbildungen und bildtexte A Kristallvakuole, wahrscheinlich unterschiedlicher Herkunft — Crystal vacuole of probably various origin - a kleine membranerfüllte Vakuole — small membrane-filled vacuole - B Skiereiden, Bastfasern — sclereids, bast fibres - C Cellinwand (Primär- und Sekundärwand aus Cellulose; Pektin etc.) — pecto-cellulosic wall - cy Cytoplasmabelag — cytoplasmic layer - d Dictyosom — dictyosome - er (glattes) Endoplasmaretikulum oder ähnliches Membransystem — (smooth) endoplasmic reticulum or similar membrane system - g girlandenförmige Abhebung einer Biomembran — festooned detachment of a membrane - h Hechtsche Fäden, Anschnitte — Hecht's threads in section - K Kristallaussparung — Crystal space, the crystal itself lost by preparation - k kleine Kristallaussparung — small crystal space - L Zell-Lumen — cell lumen - m Mitochondrion — mitochondrion - nu Nukleus — nucleus - p Plastide — plastid - pd Plasmodesmen — plasmodesms - R Rindenparenchym — bark parenchyma - rer rauhes Endoplasmaretikulum — rough endoplasmic reticulum - S Kristallscheide (=Kristallhülle) aus nicht reaktivem Material, füllkorkähnlich — crystal sheath (=crystal envelope) of material similar to filling cork - Sk Schnittkontrast — section staining - Sc Kristallscheidenteil aus Cellinwandmaterial — part of the crystal sheath of pectocellulosic material - s schräge Grenzfläche zwischen Kristallaussparung und Einbettungsmittel — view on oblique border between crystal hole and embedding material - T Tertiärwandmaterial, überS, Sc abgelagert — tertiary wall material, deposited overS, Sc - Pfeil — arrow - Pfeilspitze — arrowhead Herrn Prof. Dr.Adolf Falles, Anatomisches Institut Freiburg, zum 65. Geburtstag gewidmet.     

Immunocytochemical identification of neuroendocrine markers in human cardiac paraganglion — like structures

Summary Paraganglion-like structures (PLS) containing chromaffin-positive cells have been reported to be present in the adult human heart. The present work was initiated in order to evaluate the densitity of these structures in the interatrial septum and to study the presence of immunoreactivity of their cells to NSE and PGP 9,5 antibodies, two neuroendocrine markers. Six hundred 6-m paraffin serial sections were obtained from the upper third of the interatrial septum from six adult human hearts. From 2 to 12 paraganglia were found in each case, and their principal cells stained positively with NSE and PGP 9,5 antibodies. Depending on how these PLS related to other cardiac structures, four different types were identified: Type I — True paraganglia (located adjacent to ganglia or nerve fibers); Type II — Free paraganglia (immersed in the interatrial adipose tissue, without evident connection to other structures); Type III — Intraganglionic paraganglia (located within the nervous ganglia); Type IV — Intramyocardic paraganglia (small nests of immunoreactive cells closely related to myocardiocyte bundles). These cardiac paraganglia, which probably belong to the visceral-autonomic group, may have a role in the regulation of the cardiac function and in the adaptive mechanisms of the heart. Its is also possible that they originate functioning and non-functioning tumours.Work supported by grants from FINEP and CNPq (Brazil)     

Over-expression system for secretory phospholipase D by<Emphasis Type="Italic"> Streptomyces lividans</Emphasis>

The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces lividans using a shuttle vector—pUC702—for Escherichia coli and S. lividans. The transformant was found to secrete a large amount of PLD (about 2.0×10⁴ U/l, 42 mg/l) when cultured in a jar fermentor. Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5×10⁴ U/l, 118 mg/l) after about 60 h. In contrast to the original producer, Stv. cinnamoneum, which secretes only a small amount of PLD (about 1.1×10³ U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.