Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts

Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were cultured in a carbon-rich medium at different temperatures to investigate whether growth parameters, lipid accumulation, and fatty acid (FA) composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to a lower temperature decreased their specific growth rate, but did not affect their biomass yield (Y_X/S). Obligate and facultative psychrophiles exhibited the highest Y_X/S. Acclimation to lower temperature decreased the lipid yield (Y_L/X) in mesophilic yeasts, but did not affect Y_L/X in facultative psychrophilic ones. Similar Y_L/X were found in both groups of psychrophiles, suggesting that lipid accumulation is not a distinctive characteristic of adaptation to permanently cold environments. The unsaturation of FAs was one major adaptive feature of the yeasts colonizing permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expense of linoleic acid, whereas it was scarce or absent in all the other strains. Increased unsaturation of FAs was also a general acclimatory response of facultative psychrophiles to a lower temperature. These results improve the knowledge of the responses enabling psychrophilic yeasts to cope with the cold and may be of support for potential biotechnological exploitation of these strains.     

Thermally adaptive tradeoffs in closely related marine bacterial strains

Time series studies have shown that some bacterial taxa occur only at specific times of the year while others are ubiquitous in spite of seasonal shifts in environmental variables. Here, we ask if these ubiquitous clades are generalists that grow over a wide range of environmental conditions, or clusters of strain‐level environmental specialists. To answer this question, vibrio strains isolated at a coastal time series were phylogenetically and physiologically characterized revealing three dominant strategies within the vibrio: mesophiles, psychrophiles and apparently generalist broad thermal range clades. Thermal performance curves from laboratory growth rate experiments help explain field observations of relative abundances: the mesophilic clade grows optimally at temperatures 16°C higher than the psychrophilic clade. Strains in the broad thermal range clade all have similar optimal growth temperatures but also exhibit temperature‐related tradeoffs with faster growth rates for warm temperature strains and broader growth ranges for strains from cool temperatures. Moreover, the mechanisms of thermal adaptation apparently differ based on evolutionary time scales: shifts in the temperature of maximal growth occur between deeply branching clades but thermal performance curve shape changes on shorter time scales. Thus, apparently ubiquitous clades are likely not generalists, but contain subclusters with distinct environmental preferences.     

Growth Temperatures and Temperature Characteristics of Aeromonas

Six of the 13 Aeromonas hydrophila, 1 of 10 A. shigelloides, and none of 10 A. salmonicida were found to be psychrophiles. All of the rest of the strains were mesophiles. The mu values (temperature characteristics) could not be used to distinguish psychrophiles from mesophiles.     

Method of temperature-gradient incubation and its application to microbiological examinations

Nakae, Toshitaka (Tohoku University, Sendai, Japan). Method of temperature-gradient incubation and its application to microbiological examinations. J. Bacteriol. 91:1730-1735. 1966.-An apparatus for incubating organisms by means of a temperature-gradient system under steady-state conduction of liquid was devised. This "temperature-gradient bath" consists of a cylindrical temperature-gradient compartment mounted between two circulating water compartments which are regulated separately at constant temperature, the upper compartment being warmer than the lower compartment. A series of different, constant temperatures in the temperature-gradient compartment can be established simultaneously. Water is a suitable heat-transfer liquid for this purpose. Incubation tubes containing solid media are inserted vertically into the temperature-gradient bath and incubated. Visible growth of the organism appears in a definite portion of the agar column, corresponding to its temperature range for growth. The apparatus was successfully applied to the rapid and simultaneous estimation of maximal, minimal, and optimal temperatures for microbial growth, as well as for the simultaneous isolation of psychrophiles, mesophiles, and thermophiles.     

Relationship of critical temperature to macromolecular synthesis and growth yield in Psychrobacter cryopegella

Most microorganisms isolated from low-temperature environments (below 4 degrees C) are eury-, not steno-, psychrophiles. While psychrophiles maximize or maintain growth yield at low temperatures to compensate for low growth rate, the mechanisms involved remain unknown, as does the strategy used by eurypsychrophiles to survive wide ranges of temperatures that include subzero temperatures. Our studies involve the eurypsychrophilic bacterium Psychrobacter cryopegella, which was isolated from a briny water lens within Siberian permafrost, where the temperature is -12 degrees C. P. cryopegella is capable of reproducing from -10 to 28 degrees C, with its maximum growth rate at 22 degrees C. We examined the temperature dependence of growth rate, growth yield, and macromolecular (DNA, RNA, and protein) synthesis rates for P. cryopegella. Below 22 degrees C, the growth of P. cryopegella was separated into two domains at the critical temperature (T(critical) = 4 degrees C). RNA, protein, and DNA synthesis rates decreased exponentially with decreasing temperatures. Only the temperature dependence of the DNA synthesis rate changed at T(critical). When normalized to growth rate, RNA and protein synthesis reached a minimum at T(critical), while DNA synthesis remained constant over the entire temperature range. Growth yield peaked at about T(critical) and declined rapidly as temperature decreased further. Similar to some stenopsychrophiles, P. cryopegella maximized growth yield at low temperatures and did so by streamlining growth processes at T(critical). Identifying the specific processes which result in T(critical) will be vital to understanding both low-temperature growth and growth over a wide range of temperatures.     

Effect of temperature on growth parameters of psychrophilic bacteria and yeasts

Three bacterial (Pedobacter heparinus, Pedobacter piscium, Pedobacter cryoconitis) and three yeast strains (Saccharomyces cerevisiae, Leucosporidiella creatinivora, Rhodotorula glacialis) of different thermal classes (mesophiles and psychrophiles) were tested for the effect of temperature on a range of growth parameters, including optical density, viable cell numbers, and cell dry mass, in order to determine the temperature conditions under which maximum biomass formation is obtained. Maximum values of growth parameters obtained at the stationary growth phase of the strains were used for statistical calculation. Temperature had a significant (P ≤ 0.05) effect on all growth parameters for each strain; correlations between the growth parameters were significant (P ≤ 0.05–0.01). The maximum growth temperature or the temperature at which microbial growth was fastest was in no case the temperature at which the investigated strains produced the highest amount of biomass. All tested psychrophilic bacteria and yeast strains produced highest amounts of cells (as calculated per mg cell dry mass or per OD₆₀₀ unit) at 1°C, while cell numbers of mesophiles were highest at 20°C. Thus, cultivation temperatures close to the maximum growth temperature are not appropriate for studying psychrophiles.     

Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture.

Detailed comparison of growth kinetics at temperatures below and above the optimal temperature was carried out with Escherichia coli ML 30 (DSM 1329) in continuous culture. The culture was grown with glucose as the sole limiting source of carbon and energy (100 mg liter(-1) in feed medium), and the resulting steady-state concentrations of glucose were measured as a function of the dilution rate at 17.4, 28.4, 37, and 40 degrees C. The experimental data could not be described by the conventional Monod equation over the entire temperature range, but an extended form of the Monod model [mu = mu(max) x (s - s(min))/(Ks + s - s(min))], which predicts a finite substrate concentration at 0 growth rate (s(min)), provided a good fit. The two parameters mu(max) and s(min) were temperature dependent, whereas, surprisingly, fitting the model to the experimental data yielded virtually identical Ks values (approximately 33 microg liter(-1)) at all temperatures. A model that describes steady-state glucose concentrations as a function of temperature at constant growth rates is presented. In similar experiments with mixtures of glucose and galactose (1:1 mixture), the two sugars were utilized simultaneously at all temperatures examined, and their steady-state concentrations were reduced compared with to growth with either glucose or galactose alone. The results of laboratory-scale kinetic experiments are discussed with respect to the concentrations observed in natural environments.     

Distinctions in adenylate metabolism among organisms inhabiting temperature extremes

Microbiota from multiple kingdoms (e.g., Eubacteria, Fungi, Protista) thrive at temperature optima ranging from 0–20°C (psychrophiles) to 40–85°C (thermophiles). In this study, we have monitored changes in adenylate levels and growth rate as a function of temperature in disparate thermally adapted organisms. Our data indicate that growth rate and adenylate levels increase with temperature in mesophilic and thermophilic species, but rapid losses of adenosine 5-triphosphate (ATP) occur upon cold or heat shock. By contrast, psychrophilic species decrease adenylate levels but increase growth rate as temperatures rise within their viable range. Moreover, psychrophilic ATP levels fell rapidly upon heat shock, but dramatic gains in ATP (~20–50%) were observed upon cold shock, even at sub-zero temperatures. These results suggest that energy metabolism in thermophiles resembles that in mesophiles, but that elevated adenylate nucleotides in psychrophiles may constitute a compensatory strategy for maintaining biochemical processes at low temperature.     

Amino-acid interactions in psychrophiles, mesophiles, thermophiles, and hyperthermophiles: insights from the quasi-chemical approximation

We investigate the mechanisms used by proteins to maintain thermostability throughout a wide range of temperatures. We use the quasi-chemical approximation to estimate interaction strengths for psychrophiles, mesophiles, thermophiles, and hyperthermophiles. Our results highlight the importance of core packing in thermophilic stability. Although we observed an increase in the number of charged residues, the contribution of salt bridges appears to be relatively modest by comparison. We observed results consistent with a gradual loosening of structure in psychrophiles, including a weakening of almost all types of interactions.     

Manipulations of AMP metabolic genes increase growth rate and cold tolerance in Escherichia coli: implications for psychrophilic evolution

Diverse organisms have adapted to thrive at low temperatures (i.e., <20 °C, termed psychrophiles), colonizing the majority of earth's biosphere. In contrast with mesophiles (20-40 °C thermal range), all observed psychrophiles increase intracellular adenosine 5'-triphosphate concentrations as temperatures decline; this phenomenon has been described as an important compensatory mechanism to deal with decreases in thermal energy and molecular motion. We considered purine metabolic pathways in class gammaproteobacteria (n = 115) to investigate metabolic and evolutionary bases of this process. A survey of the KEGG database indicated that psychrophilic purine metabolic pathways tend to be enriched with de novo adenosine 5'-monophosphate (AMP) synthetic enzymes, whereas mesophiles tend to be enriched with AMP degradative enzymes. Function of the observed psychrophilic pathway structure was tested by engineering the mesophilic gammaproteobacterium Escherichia coli to reflect psychrophilic purine metabolism, specifically by expressing adenylosuccinate synthetase (purA) from the psychrophilic gammaproteobacterium, Psychrobacter cryohalolentis, in an AMP nucleosidase (amn)-deficient background. Modified E. coli was capable of growing up to ～70% faster at low temperatures and became up to ～10-fold more cold tolerant relative to wild type. These findings highlight potentially important transitional steps in psychrophilic evolution.     

Cold adaptation of microorganisms

Psychrophilic and psychrotrophic microorganisms are important in global ecology as a large proportion of our planet is cold (below 5 degrees C); they are responsible for the spoilage of chilled food and they also have potential uses in low-temperature biotechnological processes. Psychrophiles and psychrotrophs are both capable of growing at or close to zero, but the optimum and upper temperature limits for growth are lower for psychrophiles compared with psychrotrophs. Psychrophiles are more often isolated from permanently cold habitats, whereas psychrotrophs tend to dominate those environments that undergo thermal fluctuations. The molecular basis of psychrophily is reviewed in terms of biochemical mechanisms. The lower growth temperature limit is fixed by the freezing properties of dilute aqueous solutions inside and outside the cell. In contrast, the ability of psychrophiles and psychrotrophs to grow at low, but not moderate, temperatures depends on adaptive changes in cellular proteins and lipids. Changes in proteins are genotypic, and are related to the properties of enzymes and translation systems, whereas changes in lipids are genotypic or phenotypic and are important in regulating membrane fluidity and permeability. The ability to adapt their solute uptake systems through membrane lipid modulation may distinguish psychrophiles from psychrotrophs. The upper growth temperature limit can result from the inactivation of a single enzyme type or system, including protein synthesis or energy generation.     

Studies on psychrophilic bacteria in two lakes of different trophy

The number of bacteria capable of growth at low temperatures in two lakes was found to be subject to considerable variation throughout the year although the changes were not correlated with changes in ambient temperature. A correlation was, however, observed between temperature and ratio of number of psychrophiles to "total" count. All of the psychrophilic bacteria isolated from the studied lakes were able to grow at temperatures below 0 degrees C. Microorganisms isolated from eutrophic lakes were characterized by more pronounced stenothermy than those isolated from lake with low tropic level. These bacteria represented a type resembling a type resembling obligatory or ,,true" psychrophiles. Most of the isolates belonged to the genus Pseudomonas and to the Flavobacterium--Cytophaga group.     

Competition of marine psychrophilic bacteria at low temperatures

The occurrence of obligately and facultatively psychrophilic bacteria in the marine environment suggests that environmental conditions exist which can favour each of these groups in competitive processes. Differences were found in the way in which temperature affected the growth rates of obligate and facultative psychrophiles. Maximum specific growth rates of a number of obligately and facultatively psychrophilic bacteria were determined in batch culture and competition experiments were carried out in a chemostat at growth-limiting substrate concentrations. From the results the relation between the specific growth rate and the concentration of the growth-limiting substrate for both types of organisms at different temperatures was deduced. Both at low and high substrate concentrations obligate psychrophiles grew faster than facultative psychrophiles at the lower temperature extreme (? 4 C). These results suggest that obligately psychrophilic chemoorganotrophs are responsible for mineralization processes in cold natural environments such as ocean waters and the arctic and antarctic regions. In these environments they can successfully compete with facultative psychrophiles because they can grow faster.     

THE MICROBIOLOGICAL EXAMINATION OF BUTTER

SUMMARY: Butter made on farms and at creameries was examined microbiologically within 24 hr of production. The overall care exercised in the manufacture of the butter was reflected in the results obtained with the various tests used. Colony counts of psychrophiles, coli-aerogenes organisms, and yeasts and moulds were the most useful and economical tests for providing information on the standard of hygiene during manufacture, and they gave some indication of the potential keeping quality of the butter. The following satisfactory levels are recommended for creamery butter: psychrophiles <10³/g, coli-aerogenes organisms <10/g and yeasts and moulds <50/g.     

Downward-Shifting Temperature Range for the Growth of Snow-Bacteria on Glaciers of the Tibetan Plateau

Fifty-seven snow-bacteria strains were isolated from the snow of the Zadang and Mengdagangri Glaciers located in the central and southern part of the Tibetan Plateau, respectively. 16S rRNA gene sequence analysis showed that strains isolated from the Zadang Glacier belonged to the Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, and were dominated by the Firmicutes. Strains from the Mengdagangri Glacier belonged to the Actinobacteria, Alphaproteobacteria and Gammaproteobacteria, and were dominated by the Actinobacteria. Sixty-one percent of the isolates were colored with pigment. Sixty-nine percent of isolates from the Zadang Glacier were psychrotolerants, and there were no psychrophiles. We compared the growth-temperature range of 26 snow-bacteria strains to their closest mesophilic type strains and found that 46% of them had an optimum growth-temperature at or lower than 20°C, and 65% were all able to grow at 0°C. However, only 5.3% of mesophilic strains had optimum growth-temperatures at or lower than 20°C, and 9% could grow at 0°C. Snow-bacteria shift their growth-temperature downward; and doing this, in terms of the minimum and optimum temperatures for growth, might be an important strategy for them to adapt to low temperature after they have been deposited on the glacier. Our results suggested that, in order to adapt from mesophilic environments to a cold habitat, snow-bacteria widen their temperature range for growth, convert from mesophiles to psychrotolerants, but not to psychrophiles. In addition, eight isolates formed pigmented colonies, while their mesophilic counterparts were achromogenic. This helped us to confirm through comparative analysis that pigmented microorganisms were more abundant in high-altitude glaciers than in mesophilic environments.     

Effect of growth temperature on cellular fatty acids in sulphate-reducing bacteria

The effect of growth temperature on the cellular fatty acid composition of sulphate-reducing bacteria (SRB) was studied in 12 species belonging to eight genera including psychrophiles and mesophiles. Most of these species were of marine origin. The investigated SRB with the exception of four Desulfobacter species exhibited only a minor increase in the proportion of cis-unsaturated fatty acids (by < or = 5% per 10 degrees C) when the growth temperature was decreased; psychrophiles maintained their typically high content of cis-unsaturated fatty acids (around 75% of total fatty acids) nearly constant. The four Desulfobacter species, however, increased the proportion of cis-unsaturated among total fatty acids significantly (by > or =14% per 10 degrees C; measured in late growth phase) with decreasing growth temperature. The ratio between unsaturated and saturated fatty acids in Desulfobacter species changed not only with the growth temperature, but also with the growth state in batch cultures at constant temperature. Changes of cellular fatty acids were studied in detail with D. hydrogenophilus, the most psychrotolerant (growth range 0-35 degrees C) among the mesophilic SRB examined. Desulfobacter hydrogenophilus also formed cis-9,10-methylenehexadecanoic acid (a cyclopropane fatty acid) and 10-methylhexadecanoic acid. At low growth temperature (12 degrees C), the relative amount of these fatty acids was at least threefold lower; this questions the usefulness of 10-methylhexadecanoic acid as a reliable biomarker of Desulfobacter in cold sediments.     

Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation

Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.     

Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation.

Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.     

Amino acid contacts in proteins adapted to different temperatures: hydrophobic interactions and surface charges play a key role

Thermophiles, mesophiles, and psychrophiles have different amino acid frequencies in their proteins, probably because of the way the species adapt to very different temperatures in their environment. In this paper, we analyse how contacts between sidechains vary between homologous proteins from species that are adapted to different temperatures, but displaying relatively high sequence similarity. We investigate whether specific contacts between amino acids sidechains is a key factor in thermostabilisation in proteins. The dataset was divided into two subsets with optimal growth temperatures from 0–40 and 35–102°C. Comparison of homologues was made between low-temperature species and high-temperature species within each subset. We found that unspecific interactions like hydrophobic interactions in the core and solvent interactions and entropic effects at the surface, appear to be more important factors than specific contact types like salt bridges and aromatic clusters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.