Tolerance and Immobilization of Cobalt by Some Bacteria from Ferromanganese Crusts of the Afanasiy Nikitin Seamounts

Bacteria isolated from cobalt–enriched ferromanganese crusts on the Afanasiy Nikitin Seamounts in the Equatorial Indian Ocean were examined for their ability to tolerate, and immobilize cobalt in unamended seawater and seawater amended with 0.01% glucose. Retrievable bacterial counts in the form of CFU (colony forming units) on media supplemented with 1 mmol Co l^?1 (58 mg Co l^?1) and 1 mmol Mn l^?1 (54 mg Mn l^?1) were in the range of 1.71 × 10⁴ to 1.05 × 10⁵ gm^?1 (wet wt) of crust, respectively. Most of the isolates (14/24) were pigmented and showed taxonomic affinities to Flavobacterium sp. Two representative isolates were tested for their tolerance of cobalt. We observed that in amended medium, the isolates tolerated up to 1 mmol Co l^?1, whereas in unamended medium they tolerated upto 10 mmol Co l^?1. Microscopic observations of cultures incubated with 10 mmol Co l^?1 showed the occurrence of an extracellular slime layer, which may be responsible for immobilizing the cobalt from the liquid phase. In the unamended medium, the tolerance and stimulation in total cell counts was similar to that in amended medium or sometimes greater. Total cell counts peaked at 100 μmol Co l^?1 for incubations in unamended medium (1.1–2.5 × 10¹¹ cells l^?1) and at 0.1–1 μmol Co l^?1 for incubations in amended medium (1.5–2.6 × 10¹¹ cells l^?1). Counts of formazan-stained respiring cells of both the isolates in the unamended medium reached up to a maximum of 2.9–7.8 × 10¹⁰ l^?1 after incubation for 10 days at 23(±1)°. In the amended medium cell counts of respiring cells attained a maximum in the range of 4.6–15.8 × 10¹⁰ l^?1 at 100 μmol Co l^?1. The Co immobilization rate was on average 82 (± 87.9, n = 24) μmol of Co d^?1. Since the isolates were naturally occurring bacteria from crusts, they could be more environmentally acceptable and safe if used for metal recovery and bio-leaching.     

Cobalt Immobilization by Manganese Oxidizing Bacteria from the Indian Ridge System

Co immobilization by two manganese oxidizing isolates from Carlsberg Ridge waters (CR35 and CR48) was compared with that of Mn at same molar concentrations. At a lower concentration of 10 μM, CR35 and CR48 immobilized 22 and 23 fM Co cell(-1) respectively, which was 1.4 to 2 times higher than that of Mn oxidation, while at 10 mM the immobilization was 15-69 times lower than that of Mn. Scanning electron microscope and energy dispersive X-ray analyses of intact bacterial cells grown in 1 mM Co revealed Co peaks showing extracellular binding of the metal. However, it was evident from transmission electron microscope analyses that most of the sequestered Co was bound intracellularly along the cell membrane in both the isolates. Change in morphology was one of the strategies bacteria adopted to counter metal stress. The cells grew larger and thus maintained a lower than normal surface area-volume ratio on exposure to Co to reduce the number of binding sites. An unbalanced growth with increasing Co additions was observed in the isolates. Cells attained a length of 10-18 μm at 10 mM Co which was 11-15 times the original cell length. Extensive cell rupture indicated that Co was harmful at this concentration. It is apparent that biological and optimal requirement of Mn is more than Co. Thus, these differences in the immobilization of the two metals could be driven by the differences in the requirement, cell physiology and the affinities of the isolates for the concentrations of the metals tested.     

Longevity of Microbial Biocontrol Agents in a Planting Mix Amended with Glomus intraradices

Commercial sources of the biological control agents Bacillus subtilis , Trichoderma harzianum and Streptomyces griseoviridis and experimental single isolates of Serratia plymuthica , a Pseudomonas fluorescens parent and its lacZY mutant were evaluated for their survival and compatibility with the mycorrhizal fungus, Glomus intraradices , in a commercial planting mix. The control treatments were the unamended mix and mix amended with G. intraradices alone. All were applied to 128-cell Speedling styrofoam flats and planted with the tomato cultivar 'Sunny'. At four to five intervals during the growing period (6.5-8 weeks), each organism was quantified by dilution plating and G. intraradices infection (%) was determined at the end of each test. The number of Trichoderma isolates increased slightly within 2 weeks after application and then stabilized through the end of the test. Serratia and Streptomyces isolates declined throughout the test from about 7.75 log colony-forming units (CFU) g - 1 to numbers at the end (6.2 log CFU g - 1) similar to the controls. Bacillus isolates declined about 1 log CFU g - 1 in the first week but then stabilized. G. intraradices had no influence on numbers of these four genera. The Pseudomonas parent and its lacZY mutant declined about 1 log CFU g - 1 during the test with the mutant yielding higher CFU for each sampling period. Propagules of both Pseudomonas isolates were greater when mixed with G. intraradices than when alone. In these experiments, Bacillus and Trichoderma were the best survivors in a mix for potential use as biocontrol agents for tomato transplants.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M

AIMS: Exopolysaccharides (EPS) were produced by Lactobacillus rhamnosus RW-9595M during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: Cultures were conducted in supplemented whey permeate (SWP) medium containing 5 or 8% (w/w) whey permeate. For free-cell batch cultures in 8% SWP medium, very high maximum cell counts (1.3 x 10(10) CFU ml(-1)) and EPS production (2350 mg l(-1)) were measured. A high EPS production (1750 mg l(-1)) was measured after four cycles for a short incubation period of only 7 h. Several methods for immobilized biomass determination based on analysis of biomass components (proteins, ATP and DNA) were tested. The DNA analysis method proved to be the most appropriate under these circumstances. This method revealed a high maximum immobilized biomass of 8.5 x 10(11) CFU ml(-1) support during repeated immobilized cell cultures in 5% SWP. The high immobilized biomass increased maximum EPS volumetric productivity (250 mg l(-1) h(-1) after 7 h culture) compared with free-cell batch cultures (110 mg l(-1) h(-1) after 18 h culture). CONCLUSIONS: High EPS productions were achieved during batch cultures of Lact. rhamnosus RW-9595M in SWP medium, exceeding 1.7 g EPS per litre. Repeated-batch cultures with immobilized cells resulted in increased EPS productivity compared with traditional free-cell cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study clearly shows the high potential of the strain Lact. rhamnosus RW-9595M and immobilized cell technology for production of EPS as a functional food ingredient.     

Development of a Chemically Defined Medium for the Growth of Leuconostoc mesenteroides

A chemically defined medium for the growth of Leuconostoc mesenteroides was developed. This medium contained lactose, Mn(sup2+), Mg(sup2+), 12 amino acids, eight vitamins, adenine, uracil, and Tween 80. We showed the beneficial effect of aerobic conditions on growth and that potassium phosphate (135 mM) is a suitable buffer. The growth rate in this medium was 0.85 (plusmn) 0.10 h(sup-1) for the six strains examined, and cell densities up to 3.5 x 10(sup9) CFU/ml were attained.     

Characterization of subterranean bacteria in the Hungarian Upper Permian Siltstone (Aleurolite) Formation

The main purpose of this work was to study the microbiology of the Hungarian Upper Permian Siltstone (Aleurolite) Formation, to assess the safety of future underground repositories for nuclear waste. Sixty-seven air, groundwater, technical water, rock, and surface samples were collected aseptically from different depths. The number of aerobic and anaerobic isolates was 277. The mesophilic minimum and maximum CFU counts of the air samples were 1.07-5.84 x 10(2).mL-1 (aerobic) and 0.22-1.04 x 10(2).mL-1 (anaerobic), respectively; those of the water samples were 0.39-1.25 x 10(5).mL-1 (aerobic) and 0.36-3.9 x 10(3).mL-1 (anaerobic); those of the technical water samples were 0.27-5.03 x 10(6).mL-1 (aerobic) and 4 x 10(5)-->10(6).mL-1 (anaerobic); and those of the aleurolite samples were 2.32 x 10(2)-2.47 x 10(5).g-1 (aerobic) and 0.45-9.5 x 10(2).g-1 (anaerobic). In the groundwater, the thermophilic aerobic bacteria count was 0-2.4 x 10(2).mL-1 and the thermophilic anaerobic bacteria count was 0.43-4.6 x 10(4).mL-1. The gases produced by the 16 gas-forming isolates were CO2 (aerobic isolates), and CO2 and H2 (anaerobic isolates). About 20% of the aerobic isolates produced siderophores. The proportions of organic acid producers were lowest in aerobic and anaerobic isolates from the aleurolite, 13% and 14%, respectively. The highest proportions of acid producers in the aerobic and anaerobic isolates from the air samples were 63% and 54%. Altogether 160 of the aerobic isolates and 52 of the anaerobic isolates were spore formers. The radiosensitivity of the aerobic isolates was also determined; the D10 values of the sporeformers ranged between 0.8-2.44 kGy. Our results indicate that the sulfate-reducing bacteria and the production of complexing agents (siderophores) may contribute to the mobilization of radionuclides from underground repositories. As well, microbial gas production can influence the environmental conditions. The variability in bacterial radiotolerance indicates the biodiversity at this potential disposal site. These facts must be considered during the planning of a nuclear waste repository.     

Application of antisera raised against sulfate-reducing bacteria for indirect immunofluorescent detection of immunoreactive bacteria in sediment from the German Baltic Sea.

Polyclonal rabbit antisera raised against sulfate-reducing bacteria (SRB) could detect several distinct populations of bacteria in sediment from the German Baltic Sea. The depth distribution of immunoreactive bacteria was determined by an indirect immunofluorescence filter method. Anti-Desulfovibrio desulfuricans DSM 1926 serum showed maximum bacterial numbers at a depth of 18 cm, with a concentration of 60 x 10(6) cells cm-3. With anti-Desulfovibrio baculatus DSM 2555 serum, counts were highest at the same depth, approaching 0.7 x 10(6) cells cm-3. Other significantly smaller populations were observed. Anti-SRBStrain 1 (lactate,vibrio) maxima were at 0 to 4 cm and at 17 to 18 cm. Anti-SRBStrain 2 (lactate,vibrio) serum showed several local maxima. Anti-SRBStrain 3 (lactate,oval) serum detected one single peak at a depth of 10 to 12 cm. Also determined were rates of sulfate reduction, total bacterial counts by acridine orange staining, and the viable counts by dilution series on anaerobic lactate medium. The total bacterial counts were highest (180 x 10(6) cells cm-3) at 3 to 4 cm and dropped to 24 x 10(6) cells cm-3 at 10 to 11 cm but showed additional local maxima reaching 140 x 10(6) cells cm-3 at a depth of 17 to 18 cm. Viable counts probable number) were above 10(5) CFU cm-3 at 0 to 3.6 cm but remained below 10(3) CFU at 7.2 to 18 cm. The sulfate reduction rate was maximal (107 nmol cm-3 day-1) at a depth of 1 to 2 cm, dropped to 10 nmol cm-3 day-1 at 12 to 13 cm, and reached 38 nmol cm-3 day-1 at 17 to 18 cm.     

Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa

Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers, they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro. Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 x 10(6)/ml) were incubated for 30 min at 38 C with neutrophils (0,0.5 x 10(6),1 x 10(6), 5 x 10(6) and 10 x 10(6)/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H2O2 was measured at T30 with the Amplex Red assay kit. At T30, there was a significant (P < 0.01) increase in H2O2 with the addition of 5 x 10 and 10 x 10(6) neutrophils/ml activated by FMLP (0.76 +/- 0.3 and 0.99 +/- 0.4 microM, respectively, versus 0.0024 +/- 0.002 microM in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 +/- 5.1 and 48 +/- 6.0%, respectively, versus 80 +/- 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H2O2 with the addition of 5 x 10(6) and 10 x 10(6) neutrophils/ml activated by PMA (1.88 +/- 0.2 and 2.07 +/- 0.3 microM, respectively, versus 0.0009 +/- 0.0006 microM in sperm alone). The results of this study demonstrate that 5 x 10(6) activated neutrophils/ml are sufficient to impair equine sperm motility in vitro.     

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.     

Psychrotrophic bacteria from a coastal station in the Ross sea (Terra Nova Bay, Antarctica).

Seawater samples were collected from a fixed, coastal station in the Terra Nova Bay at different depths during the Xth Oceanographic Cruise in the 1994-95 Antarctic summer. Picoplanktonic abundance, estimated by direct counts in epifluorescence microscopy, ranged from 2.2 x 10(7) to 1.6 x 10(8) cells.l-1. The heterotrophic bacterial densities, evaluated on Marine Agar 2216 (Difco) after incubation at +4 degrees C for 21 days, ranged from 2 x 10(3) to 4.5 x 10(6) CFU.l-1. The qualitative composition of the heterotrophic bacterial community was studied on 64 morphological and biochemical characters of the 125 strains isolated. Heterotrophic, psychrotrophic isolates were tentatively identified at genus level as Pseudomonas, Vibrio, Acinetobacter, and Flavobacterium/Cytophaga. In order to compare the characteristics of the isolates with those previously studied during 1989/90, the synthetical indices of the structure and the metabolic potentiality of the heterotrophic bacterial community were processed. Results showed that the bacterial community was metabolically more active and more homogenous than that previously studied.     

Leishmania amazonensis: characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Does in vitro activation of postsynaptic alpha 2-adrenoceptor utilize intracellular Ca2+ for contraction in dog saphenous vein?

Dog saphenous vein spiral strips were employed to determine whether an intracellular source of Ca2+ is used for contraction upon activation of the alpha 2-adrenoceptor by B-HT 920 in Ca2+-free Krebs solution containing 50 microM EGTA. The studies were carried out in parallel with the activation of the alpha 1-adrenoceptor by phenylephrine (Phe) under the condition that B-HT 920 (10(-5) M) and Phe (2 x 10(-6) M) gave rise to a similar level of responses in Ca2+-containing Krebs solution. A similar level of responses to these agonists at equieffective concentrations in Ca2+-free medium were also observed. Such responses to Phe and B-HT 920 were inhibited by 10(-7) M rauwolscine and 10(-7) M prazosin, respectively, and were not affected by 10(-7) M nifedipine or 5 mM Mn2+. The responses to B-HT 920 (10(-5) M) and submaximal concentration of Phe (2 x 10(-6) M) in Ca2+-free medium were additive. However, if the vascular strips were first contracted maximally with 10(-4) M Phe in Ca2+-free medium to deplete the intracellular Ca store, subsequent addition of B-HT 920 failed to induce additional response. Our results strongly suggest that activation of alpha 2-adrenoceptor in dog saphenous vein in Ca2+-free medium indeed utilizes intracellular Ca2+ for contraction. We also found that the failure of earlier studies to demonstrate the contractile effects of B-HT 920 in dog saphenous vein was due to experimental artifacts derived from the use of high concentration of EGTA and artificial pH-buffering reagent.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

A microbial consortium isolated from a crude oil sample that uses asphaltenes as a carbon and energy source

A microbial consortium capable of mineralizing asphaltenes was obtained from the Maya crude oil. The enrichment system was built with a glass column reactor containing mineral medium supplied with asphaltenes as energy and carbon source. The consortium growth was evaluated in Casoy agar during 40 weeks. The steady-state phase of the enriched bacterial community was observed after 10 weeks when the culture reach 10(5) to 10(6) CFU ml(-1). The isolates belong to bacterial genus reported for degradation of other hydrocarbons and they were identified as Corynebacterium sp., Bacillus sp., Brevibacillus sp. and Staphylococcus sp. The bacterial consortium growth was evaluated by a viable counts during 14 days exposed to different aeration, temperature, salinity, and pH conditions. The ability of the consortium to mineralize asphaltenes was evaluated using the method of ISO 9439 in glass column reactors of 20 x 3.2 cm during 13 days. Temperatures of 55 degrees C and salinity of 1.8% were growth limiting. The respiration of the microbial consortium using asphaltenes as a sole carbon source (800 micromoles CO2 in 13 days) was significantly higher than those of the samples containing only the microbial consortium (200 micromoles CO2) or only asphaltenes (300 micromoles CO2). These results indicated the existence of asphaltenes-degradating microbes in the crude oil and confirmed that the consortium could mineralize asphaltenes in conditions of room temperature, salinity of 100 ppm, aeration of 1 l min(-1) and pH of 7.4.     

Optimal conditions for the production of sulfated polysaccharide by marine microalga Gyrodinium impudicum strain KG03

A marine microalga Gyrodinium impudicum strain KG03 produced sulfated exopolysaccharide designated as p-KG03, which showed a strong antiviral activity against encephalomyocarditis virus (EMCV). To optimize culture conditions for the production of p-KG03, mineral salts, vitamins, plant growth hormones, temperature, pH and light conditions were examined. From this study, M-KG03 medium for the maximum production of p-KG03 was suggested as follows; NH(4)Cl 75 microM, NaH(3)PO(4) 200 microM, NaHCO(3) 50 microM, Na(2)SO(4) 10 microM, FeCl(2) x 6H(2)O 10 microM, MnCl(2) x 4H(2)O 0.1 microM, vitamin B(12) 0.75 microg, naphthalene acetic acid (NAA) 7.5 microg and myo-inositol 200 mg per liter of aged sea water. The optimal temperature and pH were 22.5 degrees C and 8.0, respectively. The optimal light conditions of intensity and period were 150 microE m(-2) s(-1) and 16:8 h light:dark cycle. Finally, the cell growth and p-KG03 production were measured in one liter of M-KG03 medium with 1% CO(2) and 50 ml min(-1) of airflow using two liters airlift balloon type photobioreactor (ABTPR). At these optimal conditions, p-KG03 production and cell growth were 134.6+/-5.9 mg l(-1) and 123,076+/-1,597 cells ml(-1), respectively, representing a 7.7 and 5.1 times compared with f/2 medium with Erlenmeyer flask culture (p-KG03 production 17.5+/-1.3 mg l(-1) and cell growth 24,311+/-1,291 cells ml(-1)).     

Rate of oxygen consumption by isolated articular chondrocytes is sensitive to medium glucose concentration

Cartilage tissue engineering typically involves the culture of isolated chondrocytes within a scaffold material. The oxygen tension within the engineered tissue is known to be an essential parameter for implant success. This will be sensitive to the oxygen consumption behavior of the embedded chondrocytes, which remains to be characterized. We report that the oxygen consumption of bovine articular chondrocytes is sensitive to glucose deprivation below 2.7 mM, increasing from a basal level of 9.6x10(-16) to <18.4x10(-16) mol/cell.h in 1.3 mM glucose. Further studies examined the influence of selecting high (18.4 mM) or low (5.1 mM) glucose medium on the oxygen tension in 2 mm thick cellular agarose constructs. A relative upregulation of oxygen consumption was observed in constructs cultured in low glucose medium. This resulted in the near-anoxic oxygen concentration of 5 microM oxygen in constructs seeded with 40x10(6) cells/ml, compared to 57 microM in the corresponding high glucose culture. The upregulation of oxygen consumption generally corresponded to the inhibition of glycolysis, which is consistent with the Crabtree phenomenon. Medium osmolarity (316-600 mOsm) had minimal effects on chondrocyte oxygen consumption rate. In conclusion, glucose availability is a critical parameter that regulates the oxygen tension within tissue engineered constructs.     

The influence of chemical agents on the level of ionized [Ca2+] in squid axons

Squid giant axons injected with either aequorin or arsenazo III and bathed in 3 mM Ca (Na) seawater were transferred to 3 mM Ca (K) seawater and the response of the aequorin light or the change in the absorbance of arsenazo III was followed. These experimental conditions were chosen because they measure the change in the rate of Na/Ca exchange in introducing Ca into the axon upon depolarization; [Ca]o is too low to effect a channel-based system of Ca entry. This procedure was applied to axons treated with a variety of compounds that have been implicated as inhibitors of Na/Ca exchange. The result obtained was that the substances tested could be placed in three groups. (a) Substances that were without effect on Ca entry effected by Na/Ca exchange were: D600 at 10-100 microM, nitrendipine at 1-5 microM, Ba2+ and Mg2+ at concentrations of 10-50 mM, lidocaine at 0.1-10 mM, cyanide at 2 mM, adriamycin at a concentration of 3 microM, chloradenosine at 35 microM, 2,4-diaminopyridine at 1 mM, Cs+ at 45-90 mM, and tetrodotoxin at 10(-7). (b) Substances that had a significant inhibitory effect on Na/Ca exchange were: Mn2+, Cd2+, and La3+ at 1-50 mM, and quinidine at 50 microM. (c) There were also blocking agents and biochemical inhibitors whose action appeared to be the inhibition of nonmitochondrial Ca buffering in axoplasm rather than an inhibition of Na/Ca exchange. These were the general anesthetic l-octanol at 0.1 mM and 1 mM orthovanadate plus apyrase.