Polysulfide as a possible substrate for sulfur-reducing bacteria

Because of its low solubility it is unlikely that elemental sulfur serves as the direct substrate for sulfur-reducing bacteria. To test the hypothesis that polysulfide may represent a soluble intermediate of sulfur reduction, the maximal polysulfide concentrations formed from elemental sulfur in aqueous sulfide solutions were measured at near neutral pH and at temperatures up to 90°C. The saturation concentrations decreased by two orders of magnitude when the pH was lowered from 7 to 6 at a given temperature, and increased about tenfold when the temperature was raised from 37°C to 90°C at a given pH. The dissolution of 0.1 mM zerovalent sulfur in 1 mM sulfide (H₂S+HS^–) required a pH of 7.5 at 20°C and of only 6.1 at 100°C. A comparison with the growth optima of sulfur-reducers suggests that polysulfide is present at sufficient concentration at the growth conditions of the Bacteria and the moderately acidophilic Archaea. Polysulfide is apparently not available at the growth conditions of the extremely acidophilic Archaea. Alternative mechanisms for the sulfur utilization under these conditions are discussed.Abbreviations MOPS Morpholinopropanesulfonate - PIPES 1,4 piperazine-N,N-bis(2-ethanesulfonate) - HEPES N-2-hydroxy-ethylpiperazine-N-ethanesulfonate     

Characterization of cellulase and xylanase activities of Streptomyces lividans

Summary The production of cellulases and of xylanase by Streptomyces lividans 1326 was studied under different growth conditions. The strain grew between 18°C and 46°C and is therefore thermotolerant. Submerged cultures of the microorganism, when grown on a defined salt medium containing xylan as main carbon source, exhibited an overall cellulolytic activity as determined by the filter paper test. S. lividans produced optimal levels of extracellular -1,4-glucan-glucanohydrolase (1 IU/ml) and large amounts of -1,4-xylanxylanohydrolase (50 IU/ml) at 40°C. A better production of both enzymes was observed when xylan instead of cellulose was used as substrate.The stability of the enzyme was found to be significantly greater than those of the cellulases and xylanases produced by other streptomycetes. The optimal incubation temperatures for the enzyme assays were 55°C and 60°C for CM-cellulase and xylanase respectively and optimal pH values were found in the range of pH 6–7.     

Degradation of the 32-kilodalton thylakoid-membrane polypeptide of Chlamydomonas reinhardi Y-1

Isolated thylakoid membranes of Chlamydomonas reinhardi Y-1 with the 32-kDa polypeptide either radioactively labelled or unlabelled were incubated in vitro under various conditions in order to gain information about the degradation of the 32-kDa polypeptide. The degradation was higher at pH 6 compared with pH 7 and pH 8 and exhibited a temperature maximum between 20° C and 25° C (pH 6, pH 8). A light-dependent part of the total degradation was linearly dependent on white light of energy fluence rate between 1 and 20 mW·cm^-2 at 25° C and leveled out at higher fluence rates. The degradation in light was only slightly stimulated by ATP but was reduced by 3-(3-4-dichlorophenyl)-1,1-dimethylurea. Adenosine-5-diphosphate and heparin (2.7 mM and 200 g per 100 l, respectively) known to inhibit kinases, caused a 50% decrease in degradation indicating that a phosphorylation step is involved in degradating the 32-kDa polypeptide. Out of various inhibitors specific for different types of proteases, only those for thiol- and endoproteases showed intense effects. These results point to a proteolytic degradation of the 32-kDa polypetide by a thylakoid-membrane-bound thiol-endoprotease. Its activity yields soluble breakdown products with relative molecular masses (M_rs) of 23, 16.5, 11.3 and 10.7 kDa, and these are accumulated in the in-vitro system. Partial proteolytic digestion of thylakoids with Staphylococcus aureus V8 protease results in at least two labelled breakdown products (M_rs 23, and 16.5 kDa). It is assumed that cleaving at identical amino-acid residues of the 32-kDa polypeptide by the thylakoid-membrane-bound thiolendoprotease and the V8 protease results in these two breakdown products. They are derived from subsequent cleavage at amino-acid residues 60–242 and 60–189 according to the deduced protein sequence (Erickson et al. 1984, EMBO J. 3, 2753–2762).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - LDS-PAGE lithiumdodecyl sulphate-polyacrylamide gel electrophoresis - M apparent molecular mass - PSII photosystem II - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Studies on the physiology of microbial degradation of pentachlorophenol

Summary The requirements and conditions for pentachlorophenol (PCP) biodegradation by a mixed bacterial culture was studied. The effects of oxygen, nutrients, additional carbon sources, pH and temperature are described. Up to 90% of PCP was degraded into CO₂ and inorganic chloride in 1 week at an input concentration of <600 M. Degradation continued when pO₂ was lowered to 0.0002 atm but ceased when pO₂ was further decreased to 0.00002 atm. Supplementary carbon sources, such as phenol, hydroxybenzoic acids or complex nutrients did not affect the biodegradation, but the presence of ammonium salts enhanced the rate of PCP degradation without affecting the yield of CO₂. The degrading organisms were shown to be procaryotic mesophiles; no degradation was shown at temperatures below +8° and above +50°C. The optimum pH for degradation was from 6.4 to 7.2 and at higher pH value (8.4) degradation was inhibited more than at lower pH (5.6).     

Environmental factors influencing methanogenesis in a shallow anoxic aquifer: a field and laboratory study

Summary The environmental factors influencing methanogenesis in a shallow anoxic aquifer were probed in a combined field and laboratory study. Field data collected over a year revealed that in situ rates of methane production were depressed in winter and elevated in summer. Over the same period, ground water pH values ranged from 6.0 to 7.8 while temperatures varied from 7–22°C. In situ methanogenesis was severely inhibited at temperatures < 13°C or by pH values < 7. The influence of these factors on microbial methane formation from both endogenous and exogenous substrates were tested in aquifer slurries adjusted to pH 5–9 and incubated at temperatures ranging from 5–45°C. Temperature optima for methane production from endogenous substrates varied as a function of pH, but the pH optimum was 8 at all temperatures. Optimal conditions for acetoclastic methanogenesis were found at pH 8 and 35°C. An analysis of variance revealed that pH, temperature, and a pH-temperature interaction are all significant variables influencing aquifer methanogenesis. In addition transient sulfate accumulations were also found to limit methane production in some areas. A comparison of field and laboratory methane production patterns suggest that pH, temperature, and sulfate accumultations are important, but not the only environmental variables influencing the mineralization of organic matter in shallow aquifers.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Effect of temperature on humus respiration rate and nitrogen mineralization: Implications for global climate change

Respiration and nitrogen mineralization rates of humus samples from 7 Scots pine stands located along a climatic transect across the European continent from the Pyrenees (42°40) to northern Sweden (66°08) were measured for 14 weeks under laboratory conditions at temperatures from 5 °C to 25 °C. The average Q₁₀ values for the respiration rate ranged from about 1.0 at the highest temperature to more than 5 at 10 °C to 15 °C in the northernmost samples. In samples from more northern sites, respiration rates remained approximately constant during the whole incubation period; in the southern end of the transect, rates decreased over time. Respiration rate was positively correlated with incubation temperature, soil pH and CN ratio, and negatively with soil total N. Regressions using all these variables explained approximately 71% of the total variability in the respiration rate. There was no clear relation between the nitrogen mineralization rate and incubation temperature. Below 15 °C the N-mineralization rate did not respond to increasing temperature; at higher temperatures, significant increases were found for samples from some sites. A regression model including incubation temperature, pH, N_tot and CN explained 73% of the total variability in N mineralization. The estimated increase in annual soil respiration rates due to predicted global warming at the high latitudes of the Northern Hemisphere ranged from approximately 0.07×10¹⁵ to 0.13×10¹⁵ g CO₂ at 2 °C and 4 °C temperature increase scenarios, respectively. Both values are greater than the current annual net carbon storage in northern forests, suggesting a switch of these ecosystems from net sinks to net sources of carbon with global warming.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Immobilization of alkaline protease from Conidiobolus macrosporus for reuse and improved thermal stability

Alkaline protease from Conidiobolus macrosporus was immobilized on polyamide using glutaraldehyde as a bifunctional agent. The immobilized enzyme was optimally active at a higher temperature of 50°C than the free enzyme (40°C ) and showed a ten-fold increased thermostability at 60°C compared to that of the free enzyme. The efficiency of immobilization was 58% under the optimal conditions of pH and temperature. There was a 14-fold decrease in the K _m of immobilized enzyme compared to the free enzyme. The immobilized enzyme was fully active even after twenty-two cycles of repeated use. It retained 80% activity at 50°C in presence of 8 M urea exhibiting its stability to the denaturant and was compatible with several commercial detergents.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

Production,isolation and characterization of extracellular protease of an alkaliphilic strain of Arthrobacter ramosus,MCM B-351 isolated from the alkaline lake of Lonar,India

An extracellular protease was produced by Arthrobacter ramosus isolated from the alkaline lake of Lonar, Buldhana District of Maharashtra, India when grown on a synthetic medium of pH 10 containing casein. The optimum conditions for production were 3.0% initial casein concentration, 2% inoculum of 1 × 10⁸ cells/ml, pH 9.0, temperature 30 °C and shaken culture conditions. The protease was purified by ammonium sulphate precipitation followed by Sephadex G-100 chromatography. Two proteases viz. Arthro I and Arthro II, having molecular weights 21 and 11.4 kDa respectively were isolated. The Arthro II fraction had K _m 395 g/ml and V _max 10.55 g/min for azocasein. The maximum activity of enzyme was at 55 °C and pH 8. It was thermostable (up to 80 °C), alkali stable (pH 12) and stable in commercial detergent. The enzyme may contain a thiol group at the active site.     

Cytochemical detection of catalase with 3,3′-diaminobenzidine

Summary The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5° C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the mildest fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25° C) the maximal pigment production was obtained at pH 10.5 but incubation at 45° C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H₂O₂ in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

Ammonia inhibition of the maximum growth rate (μm) of hydrogenotrophic methanogens at various pH-levels and temperatures

Summary A method for the quantitative measurement of the maximum growth rate (_m) of hydrogen-consuming methanogenic populations was applied to assess the toxicity of ammonia¹ under various pH and temperature conditions. The maximum uninhibited growth rate of the hydrogenotrophic population present in sludge from an industrial anaerobic wastewater treatment system appeared to be 0.126 h^-1 at pH=7 and 37°C. At 350 mM ammonia the maximum growth rate had decreased to almost half that value. At a temperature of 29°C the maximum growth rates in the ammonia range tested appeared to be approximately 60% of that at 37°C, while increasing ammonia concentrations caused a similar maximum growth rate decline. At 37°C an increase of the pH to 7.8 appeared to enhance ammonia inhibition of the maximum growth rate. Increased propionate concentrations (tested up to 60 mM) appeared to have no effect on ammonia inhibition.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2±0.2 M and 11.2±0.2 M at 5 °C and 20°C respectively.This revised version was published online in October 2005 with corrections to the Cover Date.     

High-speed autoradiography of 3H-thymidine-labelled nuclei

Summary High-speed autoradiography with stripping film of ³H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20°C, (b) exposure of cell preparations and blanks for various times at either –70°C or +20°C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time 1 h at either temperature and relative humidity 20–30%. A linear relationship between autoradiographic efficiency and exposure time of 1–7 h was found at either temperature, although the efficiency of autoradiographs exposed at –70°C was by approximately 30% higher than that of autoradiographs exposed at +20°C. Background values of autoradiographs dried with a fan and exposed for 1/4–7h at either –70°C or +20°C were 0.6–0.8 grain/100 m². Theoretical calculations and experimental data showed that high-speed autoradiographs are 30–50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.This investigation was supported in part by MR II.1 grant. The technical assistance of Mrs. S. Bie is gratefully acknowledged     

Thermal stability characteristics of cellulase enzymes produced by Sporotrichum thermophile

Summary The thermal stability characteristics of the cellulase enzymes present in culture filtrates of the thermophilic fungus Sporotrichum thermophile were investigated at different temperatures and at different times of exposure. Maximum enzymic activities under assay conditions were found at 68°C for the filter paper activity (FPA) and the Cx activity (carboxymethylcellulose), while the maxima for the C₁ activity (cotton) and -glucosidase activity (cellobiose) were found to be at 55°C and 72°C respectively. Culture filtrates were exposed to a given constant temperature for varying lengths of time to a maximum of 48 hrs. and then analyzed for residual enzymic activities under assay conditions. The exposure temperatures studied were 50°C, 60°C and 65°C. After 48 hrs. exposure time at 50°C the residual activities for the FPA, Cx and -glucosidase were found to be 88%, 98% and 93% of the original activities respectively.     

Fatty acid-binding to erythrocyte ghost membranes and transmembrane movement

Summary Palmitate binding to human erythrocyte ghost membranes has been investigated with ghost preparations suspended in 0.2% albumin solutions. Free unbound palmitate in the extracellular water phase was measured in equilibrium studies using albumin-filled acid loaded ghosts as small semipermeable bags. The apparent dissociation constant of binding to the membrane is 13.5 nM and the binding capacity 19 nmoles per 7.2 × 109 cells.The 0°C exchange efflux kinetics of palmitate from albumin-filled ghosts is described by a model, which provides estimates of the rate constant of membrane transfer, k₃ = 0.024 s^–1, independent of the molar ratio of palmitate to albumin () and of a mean dissociation rate constant of the palmitate-albumin complex, k₁ = 0.0015 s^–1 at 0.2, allowing for a heterogeneity of the palmitate binding to albumin.The values of a third kinetically determined dependent model constant, Q, the ratio of palmitate bound to the membrane inner surface to palmitate on intracellular albumin, are not different from the Q values obtained by equilibrium experiments.The temperature dependences of k₁ and k₃ in the interval 0°C to 15°C give activation energies of 96 and 103 kJ/mole, respectively. The 0°C exchange efflux increases about 2 fold in response to a rise of pH from 6 to 9. The results suggest a carrier mediated palmitate flux at low with a V_max about 2 pmoles min^–1 cm^–2 at 0°C pH 7.3.