Bioremediation of diesel-oil-contaminated alpine soils at low temperatures

Bioremediation of two diesel-oil-contaminated alpine subsoils, differing in soil type and bedrock, was investigated in laboratory experiments at 10 °C after supplementation with an inorganic fertilizer. Initial diesel oil contamination of 4000 mg kg⁻¹ soil dry matter (dm) was reduced to 380–400 mg kg⁻¹ dm after 155 days of incubation. In both soils, about 30 % of the diesel oil contamination (1200 mg kg⁻¹ dm) was eliminated by abiotic processes. The residual decontamination (60 %–65 %) could be attributed to microbial degradation activities. In both soils, the addition of a cold-adapted diesel-oil-degrading inoculum enhanced biodegradation rates only slightly and temporarily. From C/N and N/P ratios (determined by measuring the contents of total hydrocarbons, NH₄ ⁺ N, NO₃ ⁻ N and PO₄ ³⁻ P) of soils␣it could be deduced that there was no nutrient deficiency during the whole incubation period. Soil biological activities (basal respiration and dehydrogenase activity) corresponded to the course of biodegradation activities in the soils. Received: 9 September 1996 / Accepted: 7 December 1996     

Effect of alginite amendment on microbial activity and soil water content in forest soils

The effect of the amendment with alginite, an organic rock originating from the biomass of fossilized unicellular algae, on microbial activity of forest soils was tested using a pot experiment. Five variants of soil-alginite mixtures were tested in three replicates with two forest soils: a loose sandy soil and a sandy loam. Gravimetric moisture closely correlated with the dose of alginite in both soils. Basal respiration and catalase activity increased with the dose of alginite in the sandy soil, but not in the sandy loam, where the highest response was observed at intermediate doses of alginite. The correlations of microbial activity parameters with moisture in the sandy soil were also much closer than in the sandy loam. The amendment with alginite was thus effective in improving some of the selected microbial activity indicators, but the optimum dose of alginite strongly depends on soil texture.     

Mechanisms of heat inactivation in Salmonella serotype Typhimurium as affected by low water activity at different temperatures

AIMS: To determine the effect of reduced water activity (a(w)) on thermal inactivation of Salmonella serotype Typhimurium at different temperatures and its mechanism. METHODS AND RESULTS: D-value determinations at a range of different temperatures showed that heating at reduced a(w) (0.94, produced by addition of glucose or sodium chloride to nutrient broth) was protective at temperatures above 53-55 degrees C but sensitizing below this temperature. Using selective enumeration media to determine injury, it was shown that at lower heating temperatures cells survived at high a(w) with cytoplasmic injury whereas at low a(w) these cells were killed. At higher temperatures ribosome degradation was a more important cause of death and was inhibited by low a(w) heating media thereby providing greater heat resistance. CONCLUSIONS: The observed change in behaviour reflects the different reactions responsible for thermal death at different temperatures and their different response to reduced a(w). SIGNIFICANCE AND IMPACT OF THE STUDY: This work qualifies the previous assumption that reduced a(w) is protective and suggests that the efficacy of low temperature pasteurization regimes may be increased by reduced a(w).     

Limits to life at low temperatures and at reduced water contents and water activities

Liquid water is generally considered an absolute requisite for functional life; consequently, life is expected to function only over the range of temperatures that permit its existence. These limits, however, do not apply to cell survival. Some cells can survive the closest attainable approach to 0 K, and some can survive the loss of over 99% of their water.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

Temperature relationships of fungi isolated at low temperatures from soils and other substrates

A study of the temperature relationships of 20 mycelial and yeast fungi which had been isolated at low temperatures from soils and from abattoirs indicated that few fungi can be regarded as truly psychrophilic. Only 1 species failed to grow at 25C. Although all the species investigated were able to grow at 4C and can therefore be considered as potential spoilage organisms on refrigerated foods, their optimal growth temperature was either 15C or 25C. The 20 species could be divided into 4 groups in relation to their temperature relationships, particularly their optimal temperatures and their ability to grow at 30C.A number of fungi able to grow at 4C has been isolated from soils and from abattoirs in New Zealand (1, 2). Several mycelial fungi can show reasonable growth below 10C, and some even down to –10C, but, when laboratory studies have been performed, most of these fungi prove to have an optimal growth temperature at 25C or above (3). These fungi, however, can be a cause of serious spoilage of a variety of refrigerated foods and even at the temperature currently used for meat storage ( –12C), occasional consignments of meat are encountered which show the characteristic spots of fungal contamination. During investigations into the occurrence of these fungi in an abattoir, (1), 9 mould and 4 yeast species were isolated which could grow at 4C, and further studies into the reservoirs of these fungi in the outside environment resulted in the isolation of a further 8 mould and 2 yeast species (2). This paper reports on investigations into the temperature relationships of 20 of these species.     

Measured and predicted mineralization of clover green manure at low temperatures at different depths in two soils

Predictive models of the temporal mineralization pattern of organic residues may help in development of strategies to synchronize N mineralization with the crop demand and minimize off-season losses. In the present investigation, two double first-order models with temperature as a driving variable were tested against data on decomposition and N mineralization, respectively, in two field experiments with green manure. On 15 November 1984, mesh bags with red clover (Trifolium pratense L.) shoot material were placed at five depths (0–30 cm) on a sandy-loam and a loam site in south-eastern Norway. 167 days after burial, 73% of the initial clover nitrogen remained on the surface, 62% at 5-cm depth, and 56% at 30-cm depth. The differences among buried samples largely persisted throughout the experimental period (1.5 years). The decomposition rate slowed down appreciably after day 270, when the amount of N in buried bags averaged 33% of the initial N. The effect of site was small and varied during the experiment. The decay model, which was derived from laboratory incubations, predicted the initial observations of remaining clover material fairly well. Later, predicted and measured values diverged because recalcitrant residues decomposed more extensively in the field than in the laboratory. The N mineralization model was tested against net N mineralization from white-clover (T. repens L.) green manure ploughed down in late October. The course of the net N mineralization was well described when disregarding an over-prediction (6–12% of applied clover N), which may be due to N losses not accounted for in the model. The predictions were sensitive to the kind of function applied for correction of decay rates at temperatures below 0° C. The results showed that decomposition of clover green manure is rapid, even at temperatures below 5° C. N-rich plant material, therefore, should be worked into the soil as late as possible in the autumn or, preferably, remain on the soil surface until spring in order to reduce the probability of N losses.     

The physical state of water at low temperatures in plasma with different water contents as studied by differential thermal analysis and differential scanning calorimetry

Differential thermal analysis (DTA) and differential scanning calorimetry (DSC) have been used for a study of the physical state of water at low temperatures in freeze-dried blood plasma rehydrated to different water contents. Measurement of the amplitude of the specific heat change accompanying the glass transition, the quantity of ice formed during rewarming, and the total ice content in the sample provided a determination of the extent of crystallization in water at low temperatures. Even after slow cooling or annealing, a fraction of water remained incorporated in an amorphous phase. It is suggested that the unfreezable water (0.47 g per g of dry substance) could be divided into two more or less distinct species: 0.25 g per g is the minimum water content above which the system can gain enough mobility to give rise to a detectable glass transition.     

Improving the catalytic activity of a thermophilic enzyme at low temperatures

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.     

Anaerobic on-site treatment of kitchen waste in combination with black water in UASB-septic tanks at low temperatures

Anaerobic on-site treatment of a mixture of black water and kitchen waste (BWKW) was studied using two-phased upflow anaerobic sludge blanket (UASB) septic tanks at the low temperatures of 20 and 10 degrees C. Black water (BW) was also treated alone as reference. The two-phased UASB-septic tanks removed over 95% of total suspended solids (TSS) and 90% of total chemical oxygen demand (COD(t)) from both BWKW (effluent COD(t) 171-199mg/l) and BW (effluent COD(t) 92-100mg/l). Also, little dissolved COD (COD(dis)) was left in the final effluents (BW 48-70mg/l; BWKW 110-113mg/l). Part of total nitrogen (N(tot)) was removed (BW 18% and BWKW 40%) and especially at 20 degrees C ammonification was efficient. A two-phased process was required to obtain the high removals with BWKW at 10 degrees C, while with BW a single-phased process may have sufficed even at 10 degrees C. BWKW also produced more methane than BW alone. Sludge in phases 1 of BW and BWKW treatment was not completely stabilised after 198d of operation.     

Oil degradation by basidiomycetes in soil and peat at low temperatures

A total of 17 basidiomycete strains causing white rot and growing on oil-contaminated substrates have been screened. Three strains with high (Steccherinum murashkinskyi), average (Trametes maxima), and low (Pleurotus ostreatus) capacities for the colonization of oil-contaminated substrates have been selected. The potential for degrading crude oil hydrocarbons has been assessed with the use of fungi grown on nonsterile soil and peat at low temperatures. Candida sp. and Rhodococcus sp. commercial strains have been used as reference organisms with oil-degrading ability. All microorganisms introduced in oil-contaminated soil have proved to be ineffective, whereas the inoculation of peat with basidiomycetes and oil-degrading microorganisms accelerated the destruction of oil hydrocarbons. The greatest degradation potential of oil-aliphatic hydrocarbons has been found in S. murashlinskyi. T. maxima turned out to be the most successful in degrading aromatic hydrocarbons. It has been suggested that aboriginal microflora contributes importantly to the effectiveness of oil-destructing microorganisms. T. maxima and S. murashkinskyi strains are promising for further study as oil-oxidizing agents during bioremediation of oil-contaminated peat soil under conditions of low temperatures.     

Phase diagram of 1,2-dioleoylphosphatidylethanolamine (DOPE):water system at subzero temperatures and at low water contents.

The phase behavior of partially hydrated 1, 2-dioleoylphosphatidylethanolamine (DOPE) has been studied using differential scanning calorimetry and X-ray diffraction methods together with water sorption isotherms. DOPE liposomes were dehydrated in the H(II) phase at 29 degrees C and in the L(alpha) phase at 0 degrees C by vapor phase equilibration over saturated salt solutions. Other samples were prepared by hydration of dried DOPE by vapor phase equilibration at 29 degrees C and 0 degrees C. Five lipid phases (lamellar liquid crystalline, L(alpha); lamellar gel, L(beta); inverted hexagonal, H(II); inverted ribbon, P(delta); and lamellar crystalline, L(c)) and the ice phase were observed depending on the water content and temperature. The ice phase did not form in DOPE suspensions containing <9 wt% water. The L(c) phase was observed in samples with a water content of 2-6 wt% that were annealed at 0 degrees C for 2 or more days. The L(c) phase melted at 5-20 degrees C producing the H(II) phase. The P(delta) phase was observed at water contents of <0.5 wt%. The phase diagram, which includes five lipid phases and two water phases (ice and liquid water), has been constructed. The freeze-induced dehydration of DOPE has been described with the aid of the phase diagram.     

Heat resistance of native and demineralized spores of Bacillus subtilis sporulated at different temperatures

Demineralization reduced heat resistance of B. subtilis spores, but the pattern and magnitude of the reduction depended on sporulation temperature and on heating menstruum pH. The differences in heat resistance of native spores caused by sporulation temperature almost disappeared after demineralization. Demineralized spores were still susceptible to the heat-sensitizing effect of acidic pH.     

Properties of ternary phospholipid/dimethyl sulfoxide/water systems at low temperatures

X-ray diffraction, neutron diffraction and differential scanning calorimetry were used to investigate phase transitions in the ternary system phospholipid/dimethyl sulfoxide (DMSO)/water under cooling for three homologous phospholipids: dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). Below the temperature of ice formation from -40 to -113 degrees C, a new lamellar phase of DPPC and DSPC was found at and above a DMSO molar fraction of X(DMSO) = 0.05. Below X(DMSO) = 0.05 only a single dehydrated Lc-phase exists after ice formation. The new phase has an increased membrane repeat distance and coexists with a dehydrated Lc-phase. DPPC with a DMSO molar fraction of X(DMSO) = 0.07 shows a membrane repeat distance of the new phase of d = 6.61 +/- 0.03 nm. The value of d increases at the increase of X(DMSO). The new phase was not observed in the ternary system with DMPC. No correlation between the new phase and the glass transition of bound water in the intermembrane space was detected. The new phase was detected only in the systems with excess of water. The creation of the new phase demonstrates the specific DMSO interaction with hydrocarbon chains.     

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

Transesterification of divinyladipate with glucose at various temperatures by an alkaline protease of Streptomyces sp.

The transesterification of 1 M divinyladipate with 0.25 M glucose in dimethylformamide (DMF) catalyzed by 5 mg ml^–1 alkaline protease (24 units mg^–1 min^–1) from Streptomyces sp. gave 6-O-vinyladipoyl d-glucose as the main product with yields are between 60 and 90%. The optimum temperature for the reaction was about 50 °C.     

Changes in the activity of enzymes of phenylpropanoid metabolism in tomatoes stored at low temperatures

A large increase in the activity of an enzyme involved in chlorogenic acid metabolism, hydroxycinnamyltransferase occurs in tomatoes stored at low temperatures. In contrast, the activity of the enzyme remains constant or falls slightly during normal ripening at 20°. The rise in activity occurs at temperatures below 10° and fails to occur at 15° or 20°. This increase in activity during low temperature storage occurs with fruit at all stages of ripening from mature green to fully ripe. The hydroxycinnamyltransferase of chilled tomatoes falls rapidly on transfer to 20° with a lag of about 4–8 hr and within 48 hr returns to that of unchilled fruit. The effects of such warming treatments are reversible since when a chilling period is resumed following warming to 20°, the rise in hydroxycinnamyltransferase activity is also resumed. Of the 5 other enzymes of phenylpropanoid metabolism studied, only PAL shows a similar increase in activity during low temperature storage although the activity of the other enzymes was maintained at higher levels in fruit at 2° than at 20°. The possible relationship between the behaviour of hydroxycinnamyltransferase activity at various temperatures and the known susceptibility of tomatoes to chilling injury is discussed.     

Glass transition temperatures of cassava starch-whey protein concentrate systems at low and intermediate water content

Glass transition temperatures of cassava starch (CS)-whey protein concentrate (WPC) blends were determined by means of differential scanning calorimetry (DSC) in a water content range of 8-20% (dry basis, d.b.). Water equilibration in the samples was carried out by storing them at room temperature (25 °C) during four weeks. Physical aging and phase segregation were observed in some samples after this storage period depending on the water content. Both, first DSC heating scans and tan δ curves of CS-WPC blends with intermediate water content (10-18%), showed two endothermic thermal events. The first one appeared at around 60 °C and was independent of water content. The second one was detected at higher temperatures and moved towards the low-temperature peak as the water content increased. The results can be explained by a phase segregation process that can take place when the samples are conditioned below their glass transition temperatures. The Gordon-Taylor equation described well the plasticizing effect of water on the blends. WPC was also found to decrease the glass transition temperature, at constant water content, an effect attributed to additional water produced during browning reactions in the blends.     

Nitrogen fixation by white clover when competing with grasses at moderately low temperatures

It was the aim of this study to determine the way in which low temperature modifies the effect of a competing grass on nitrogen fixation of a forage legume. White clover (Trifolium repens L.) was grown in monoculture or in different planting ratios with timothy (Phleum pratense L.) or perennial ryegress (Lolium perenne L.) in growth chambers at either 7.5/5°C (LoT) or 15/10°C (HiT) average day/night temperatures, and with 2.5 or 7.5 mM¹⁵N-labelled nitrate in the nutrient solution.Competition with grass led to a marked increase in the proportion of clover nitrogen derived from symbiosis (% N_sym). This increase was slower at LoT where % N_sym was reduced considerably; it was closely related to the reduction in the amount of available nitrate as a result of its being utilized by the grass.Nitrogen concentration in white clover herbage and dry matter yield per clover plant were reduced, for the most part, when a competing grass was present. The amount of nitrogen fixed per plant of white clover decreased markedly with temperature. Low temperature consequently accentuated competition for nitrate. The capacity of white clover to compete successfully was limited by its slower growth and nitrogen accumulation.