Production and Purification of Remazol Brilliant Blue R Decolorizing Peroxidase from the Culture Filtrate of Pleurotus ostreatus

An extracellular H(inf2)O(inf2)-requiring Remazol brilliant blue R (RBBR) decolorizing enzymatic activity was found in the culture medium of Pleurotus ostreatus. The enzymatic activity was maximally obtained in idiophase, and the optimum C/N ratio was 24. High C/N ratios repressed the enzymatic activity, and addition of veratryl alcohol had no effect on the production of enzyme. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q chromatography. The purification of RBBR decolorizing peroxidase, as judged by the final specific activity of 6.00 U/mg, was 54.5-fold, with a yield of 9.9%. The molecular mass of the native enzyme determined by gel permeation chromatography was found to be about 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a monomer with a molecular mass of 71 kDa. The enzyme was optimally active at pH 3.0 to 3.5 and at 25(deg)C. Under standard assay conditions, the apparent K(infm) values of the enzyme toward RBBR and H(inf2)O(inf2) were 10.99 and 32.97 (mu)M, respectively. The enzyme had affinity toward various phenolic compounds and artificial dyes, and it was inhibited by Na(inf2)S(inf2)O(inf5), potassium cyanide, NaN(inf3), and cysteine. The absorption spectrum of the enzyme exhibited maxima at 407, 510, and 640 nm. The addition of H(inf2)O(inf2) to the enzyme resulted in an absorbance decrease at 407 and 510 nm.     

Occurrence of a Highly Heat-Sensitive Spore Subpopulation of Bacillus coagulans STCC 4522 and Its Conversion to a More Heat-Stable Form

The profile of the survival curves, at different heating temperatures, of B. coagulans STCC 4522 sporulated at 52(deg)C has been studied, focusing on the early moments of treatment. A highly heat-sensitive spore subpopulation that includes more than 90% of the total spore population has been found. This heat-sensitive spore fraction was inactivated after 2 s of treatment at 111(deg)C. Its heat resistance was as much as 200-fold lower than that of the heat-resistant spore fraction (D(inf111(deg)C) of 0.01 min for the heat-sensitive spore fraction compared with D(inf111(deg)C) of 2 min for the heat-resistant fraction). The shape of the survival curve at 108.5(deg)C was modified after a sublethal heat shock at 80(deg)C for 3.5 h, resulting in a straight-line survival curve. The temperature of treatment also influenced the shape of the survival curves. The conversion of the highly heat-sensitive spore subpopulation to a more heat-stable form is discussed.     

Acetogenic Capacities and the Anaerobic Turnover of Carbon in a Kansas Prairie Soil

To assess the anaerobic capacities of a temperate grassland soil, a Kansas prairie soil was incubated anaerobically as either soil-water (1:2) suspensions or as soil microcosms at 78% soil water-holding capacity. Prairie soil formed acetate and CO(inf2) as the two main initial carbonaceous products from the anaerobic turnover of endogenous organic matter. Metabolic capacities of soil suspensions and microcosms were similar. Rates of acetate formation from endogenous organic matter in soil-water suspensions incubated at 40, 30, and 15(deg)C approximated 3.3, 2.4, and 1.1 (mu)g of acetate per g (dry weight) of soil per h, respectively. Supplemental H(inf2) and CO(inf2) were subject to consumption with the apparent concomitant synthesis of acetate in both soil suspensions and soil microcosms. In soil microcosms, rates of H(inf2)-dependent acetogenesis at 30 and 55(deg)C were nearly equivalent. The uptake of supplemental H(inf2) was not coupled to methanogenesis under any condition examined. These anaerobic activities were relatively stable when soils were subjected to either aerobic drying or alternating periods of O(inf2) enrichment. On the basis of the formation of nitrogen (N(inf2)), denitrification was engaged during anaerobic incubation periods; nitrous oxide (N(inf2)O) was also formed under certain conditions. Although extended incubation of soil induced the delayed methanogenic turnover of acetate, acetate was subject to immediate turnover under either O(inf2)- or nitrate-enriched conditions. These studies support the following concepts: (i) obligately anaerobic bacteria such as acetogenic bacteria are stable to periods of aerobiosis and are active in the anaerobic microsites of oxic soils, and (ii) acetate synthesized in anaerobic microsites of oxic terrestrial soils constitutes a trophic link to both aerobic and anaerobic microbial communities.     

Stable-Isotope Analysis of a Combined Nitrification-Denitrification Sustained by Thermophilic Methanotrophs under Low-Oxygen Conditions

To simulate growth conditions experienced by microbiota at O(inf2)-limited interfaces of organic matter in compost, an experimental system capable of maintaining dual limitations of oxygen and carbon for extended periods, i.e., a pO(inf2)-auxostat, has been used. (sup15)N tracer studies on thermophilic (53(deg)C) decomposition processes occurring in manure-straw aggregates showed the emission of dinitrogen gas from the reactor as a result of simultaneous nitrification and denitrification at low pO(inf2) values (0.1 to 2.0%, vol/vol). The N loss was confirmed by nitrogen budget studies of the system. Depending on the imposed pO(inf2), 0.6 to 1.4 mmol of N/day (i.e., 20 to 40% of input N) was emitted as N(inf2). When the pO(inf2) was raised, the rates of both nitrification and denitrification increased instantaneously, indicating that ammonia oxidation was limited by oxygen. In auxostats permanently running at pO(inf2) >= 2% (vol/vol), the free ammonium pool was almost completely oxidized and was converted to nitrite plus nitrate and N(inf2) gas. Labelling of the auxostat with [(sup13)C]carbonate was conducted to reveal whether nitrification was of autotrophic or heterotrophic origin. Incorporation of (sup13)CO(inf2) into population-specific cellular compounds was evaluated by profiling the saponifiable phospholipid fatty acids (FAs) by using capillary gas chromatography and subsequently analyzing the (sup13)C/(sup12)C ratios of the individual FAs, after their combustion to CO(inf2), by isotope ratio mass spectrometry. Apart from the observed label incorporation into FAs originating from a microflora belonging to the genus Methylococcus (type X group), supporting nitrification of a methylotrophic nature, this analysis also corroborated the absence of truly autotrophic nitrifying populations. Nevertheless, the extent to which ammonia oxidation continued to exist in this thermophilic community suggested that a major energy gain could be associated with it.     

Purification and Characterization of a Glucoamylase from Humicola grisea

A thermostable extracellular glucoamylase from the thermophilic fungus Humicola grisea was purified to homogeneity. Its molecular mass and isoelectric point were 74 kDa and 8.4, respectively. The enzyme contained 5% carbohydrate, showed maximal activities at pH 6.0 and 60(deg)C, and was stable at 55(deg)C and pH 6.0 for 2 h. The K(infm) of soluble starch hydrolysis at 50(deg)C and pH 6.0 was 0.14 mg/ml. The purified enzyme was remarkably insensitive to glucose.     

Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174

An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly.     

Effects of Environmental Parameters on the Formation and Turnover of Acetate by Forest Soils

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.     

Adaptation of Psychrophilic and Psychrotrophic Sulfate-Reducing Bacteria to Permanently Cold Marine Environments

The potential for sulfate reduction at low temperatures was examined in two different cold marine sediments, Mariager Fjord (Denmark), which is permanently cold (3 to 6(deg)C) but surrounded by seasonally warmer environments, and the Weddell Sea (Antarctica), which is permanently below 0(deg)C. The rates of sulfate reduction were measured by the (sup35)SO(inf4)(sup2-) tracer technique at different experimental temperatures in sediment slurries. In sediment slurries from Mariager Fjord, sulfate reduction showed a mesophilic temperature response which was comparable to that of other temperate environments. In sediment slurries from Antarctica, the metabolic activity of psychrotrophic bacteria was observed with a respiration optimum at 18 to 19(deg)C during short-term incubations. However, over a 1-week incubation, the highest respiration rate was observed at 12.5(deg)C. Growth of the bacterial population at the optimal growth temperature could be an explanation for the low temperature optimum of the measured sulfate reduction. The potential for sulfate reduction was highest at temperatures well above the in situ temperature in all experiments. The results from sediment incubations were compared with those obtained from pure cultures of sulfate-reducing bacteria by using the psychrotrophic strain ltk10 and the mesophilic strain ak30. The psychrotrophic strain reduced sulfate optimally at 28(deg)C in short-term incubations, even though it could not grow at temperatures above 24(deg)C. Furthermore, this strain showed its highest growth yield between 0 and 12(deg)C. In contrast, the mesophilic strain ak30 respired and grew optimally and showed its highest growth yield at 30 to 35(deg)C.     

Inhibition of Methane Oxidation by Methylococcus capsulatus with Hydrochlorofluorocarbons and Fluorinated Methanes

The inhibition of methane oxidation by cell suspensions of Methylococcus capsulatus (Bath) exposed to hydrochlorofluorocarbon 21 (HCFC-21; difluorochloromethane [CHF(inf2)Cl]), HCFC-22 (fluorodichloromethane [CHFCl(inf2)]), and various fluorinated methanes was investigated. HCFC-21 inhibited methane oxidation to a greater extent than HCFC-22, for both the particulate and soluble methane monooxygenases. Among the fluorinated methanes, both methyl fluoride (CH(inf3)F) and difluoromethane (CH(inf2)F(inf2)) were inhibitory while fluoroform (CHF(inf3)) and carbon tetrafluoride (CF(inf4)) were not. The inhibition of methane oxidation by HCFC-21 and HCFC-22 was irreversible, while that by methyl fluoride was reversible. The HCFCs also proved inhibitory to methanol dehydrogenase, which suggests that they disrupt other aspects of C(inf1) catabolism in addition to methane monooxygenase activity.     

Effect of Metal Cations on the Viscosity of a Pectin-Like Capsular Polysaccharide from the Cyanobacterium Microcystis flos-aquae C3-40

The properties of purified capsular polysaccharide from the cyanobacterium Microcystis flos-aquae C3-40 were examined by capillary viscometry. Capsule suspensions exhibited similar viscosities between pH 6 and 10 but were more viscous at pH <=4 than at pH 6 to 11. At pH 7, a biphasic effect of metal ion concentration on capsule viscosity was observed: (i) capsule viscosity increased with increasing metal ion concentration until a maximal viscosity occurred at a specific concentration that was a reproducible characteristic of each metal ion, and (ii) the viscosity decreased with further addition of that ion. Because the latter part of the biphasic curve was complicated by additional factors (especially the precipitation or gelation of capsule by divalent metal ions), the effects of various metal chlorides were compared for the former phase in which capsule viscosity increased in the presence of metal ions. Equivalent increases in capsule viscosity were observed with micromolar concentrations of divalent metal ions but only with 10 to 20 times greater concentrations of Na(sup+). The relative abilities of various metal salts to increase capsule viscosity were as follows: CdCl(inf2), Pb(NO(inf3))(inf2), FeCl(inf2) > MnCl(inf2) > CuCl(inf2), CaCl(inf2) >> NaCl. This pattern of metal efficacy resembles known cation influences on the structural integrity of capsule in naturally occurring and cultured M. flos-aquae colonies. The data are the first direct demonstration of an interaction between metal ions and purified M. flos-aquae capsule, which has previously been proposed to play a role in the environmental cycling of certain multivalent metals, especially manganese. The M. flos-aquae capsule and the plant polysaccharide pectin have similar sugar compositions but differ in their relative responses to various metals, suggesting that capsular polysaccharide could be a preferable alternative to pectin for certain biotechnological applications.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.     

Spatial Patterns of Rhizoplane Populations of Pseudomonas fluorescens

Geostatistical analysis was used to compare rhizoplane colonization patterns of an antibiotic-producing biological control bacterium versus a non-antibiotic-producing mutant strain. Pea seeds were inoculated with Pseudomonas fluorescens 2-79RN(inf10) or P. fluorescens 2-79-B46 (a phenazine-deficient Tn5 mutant of P. fluorescens 2-79RN(inf10)) (10(sup8) CFU/pea), planted in sterile sand, and incubated at 20(deg)C. After 3 days, seedlings were prepared for scanning electron microscopy. Photomicrographs (x1,000) of the root surface were taken at the seed-root junction and at 0.5-cm intervals to the root tip. Bacterial counts on the root surface were made in 5- by 5-(mu)m sample units over an area which was 105 by 80 (mu)m. Coordinates and number of bacteria were recorded for each sample unit. Spatial statistics were calculated by covariance for the following directions: omnidirectional, 0, 45, 90, and 135(deg). The ranges of spatial influence and nugget (estimator of spatially dependent variation) were determined. For both P. fluorescens 2-79RN(inf10) and P. fluorescens 2-79-B46, spatial structure was evident along the entire root, particularly in the 0(deg) direction (along the root length) (e.g., range = 24 (mu)m, nugget = 0.52). The degree of spatial dependence observed indicated aggregation of bacterial cells. No differences were detected in the spatial patterns of colonies of P. fluorescens 2-79RN(inf10) and P. fluorescens 2-79-B46, indicating that the lack of phenazine production did not influence spatial patterns on the rhizoplane.     

Modelling frost resistance of Scots pine seedlings using temperature,daylength and pH of cell effusate

The annual course of frost resistance (LT₅₀) and the pH of the cell effusate in needles of two-year-old Scots pine seedlings were monitored in a field experiment in Oulu, Northern Finland (65° N, 25° E) during 1995. The aim of the work was to to develop model to predict the annual variation in frost resistance by pH of the cell effusate and meteorological data. The seedlings were covered with a fibre cloth shelter which transmitted sufficient light for them to experience the photoperiod, but prevented the accumulation of snow over them. The shelter above the seedlings was removed at the beginning of May and erected again at the end of September. The seedlings were watered only for the time when the shelter was removed, and received fertilizer only during the previous summer (1994).Frost resistance was only -5° C during the growing season but more than -100° C during the winter rest period. It was about -10° C at the end of August, increased to -55° C in the next three weeks, and reached -100° C at the beginning of October. The pH of the cell effusate was lowest during the growing season and highest in winter, the difference being about one and half pH unit. Needles exposed to -196° C showed pH from 4.0 in summer to 5.5 in winter, while pH of the non-frozen needles varied from 5.0 to 6.5, respectively. Seasonal variation in frost resistance was explained by a regression model well (R² = 0.9) when day length, minimum air temperature and pH were entered as variables.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Anaerobic Capacities of Leaf Litter

Leaf litter displayed a capacity to spontaneously form organic acids, alcohols, phenolic compounds, H(inf2), and CO(inf2) when incubated anaerobically at 20(deg)C either as buffered suspensions or in a moistened condition in microcosms. Acetate was the predominant organic product formed regardless of the degree of litter decomposition. Initial rates of acetate formation in litter suspensions and microcosms approximated 2.6 and 0.53 (mu)mol of acetate per g (dry weight) of litter per h, respectively. Supplemental H(inf2) was directed towards the apparent acetogenic synthesis of acetate. Acetoclastic methanogenesis was induced by partially decomposed litter after extended lag phases; freshly fallen litter did not display this capacity.     

Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures

The effect of temperature and O₂ saturation on the production of recombinant proteins -galactosidase and human glucocerebrosidase by Spodoptera frugiperda cells (Sf9) infected with recombinant Autographa californica nuclear polyhedrosis virus was investigated. The rates of cell growth, glucose consumption, O₂ consumption and product expression were measured at temperatures between 22° C and 35° C. The results indicated that possible O₂ limitation may be alleviated without compromising the maximum cell yield by lowering the incubation temperature from 27° C to 25° C. The expression level of the recombinant proteins at 27° C was similar to that obtained at 22° C and 25° C; lower protein yields were obtained at 30° C. An increase in temperature from 22° C to 27° C led to earlier production of the proteins and to an increase in the proportion of the product released outside the cells. Correspondence to: J. Shiloach     

Effect of liming on EUF-nutrient fractions in the soil,on nutrient contents of grape leaves and on grape yield

Summary In pot experiments with vine, liming significantly raised EUF-Ca 20°C as well as EUF-Ca 80°C values of an acid clay soil (pH 4.2). This resulted in a marked rise in Ca contents of vine leaves (Table 2). High amounts of K fertilizers without lime raised mainly the EUF-Ca 20°C values whereas the EUF-80°C values remained at a low level (Table 3). Liming lowered the EUF-K 20°C values and as a result the ratio EUF-K 80°C C/EUF-K 20°C increased from 0.7 to 1.0.High K applications raised the K content of the leaves at flowering stage but at grape ripening a marked decrease in K content was observed (Table 5). In contrast, the application of both lime and K fertilizer raised the K content of leaves at both flowering and ripening. Grape yield increased as well (Table 11).Liming raised the EUF-P values of the soil and to a lesser extent the P contents of leaves (Tables 6 and 7).High K applications without lime raised the Mn contents of leaves (Table 9), Exchange processes due to K fertilizer addition were reflected in increased EUF-Mn values (Table 9).The highest yield (three-year average) was obtained in a high K treatment (22 g K₂O/pot) in combination with lime (40 g CaO/pot).     

Oxidation and Assimilation of Atmospheric Methane by Soil Methane Oxidizers

The metabolism of atmospheric methane in a forest soil was studied by radiotracer techniques. Maximum (sup14)CH(inf4) oxidation (163.5 pmol of C cm(sup-3) h(sup-1)) and (sup14)C assimilation (50.3 pmol of C cm(sup-3) h(sup-1)) occurred at the A(inf2) horizon located 15 to 18 cm below the soil surface. At this depth, 31 to 43% of the atmospheric methane oxidized was assimilated into microbial biomass; the remaining methane was recovered as (sup14)CO(inf2). Methane-derived carbon was incorporated into all major cell macromolecules by the soil microorganisms (50% as proteins, 19% as nucleic acids and polysaccharides, and 5% as lipids). The percentage of methane assimilated (carbon conversion efficiency) remained constant at temperatures between 5 and 20(deg)C, followed by a decrease at 30(deg)C. The carbon conversion efficiency did not increase at methane concentrations between 1.7 and 1,000 ppm. In contrast, the overall methane oxidation activity increased at elevated methane concentrations, with an apparent K(infm) of 21 ppm (31 nM CH(inf4)) and a V(infmax) of 188 pmol of CH(inf4) cm(sup-3) h(sup-1). Methane oxidizers from soil depths with maximum methanotrophic activity respired approximately 1 to 3% of the assimilated methane-derived carbon per day. This apparent endogenous respiration did not change significantly in the absence of methane. Similarly, the potential for oxidation of atmospheric methane was relatively insensitive to methane starvation. Soil samples from depths above and below the zone with maximum atmospheric methane oxidation activity showed a dramatic increase in the turnover of the methane assimilated (>20 times increase). Physical disturbance such as sieving or mixing of soil samples decreased methane oxidation and assimilation by 50 to 58% but did not alter the carbon conversion efficiency. Ammonia addition (0.1 or 1.0 (mu)mol g [fresh weight](sup-1)) decreased both methane oxidation and carbon conversion efficiency. This resulted in a dramatic decrease in methane assimilation (85 to 99%). In addition, ammonia-treated soil showed up to 10 times greater turnover of the assimilated methane-derived carbon (relative to untreated soil). The results suggest a potential for microbial growth on atmospheric methane. However, growth was regulated strongly by soil parameters other than the methane concentration. The pattern observed for metabolism of atmospheric methane in soils was not consistent with the physiology of known methanotrophic bacteria.     

Xylanase production by a new alkali-tolerant isolate of Bacillus

The xylanolytic system of an alkali-tolerant Bacillus sp. consists of several xylanases ranging from 22 to 120 kDa and pI values from 7.0 to 9.0. Crude xylanase retained 72% of initial activity after 5 h at pH 9.0 and 45°C. Xylanase production was induced by xylose and xylan and was maximum at 42°C and pH 7.8. Crude xylanase released xylotriose and xylotetraose as main products of xylan hydrolysis. Xylose was not detected. © Rapid Science Ltd. 1998