Assessing Phytoplankton and Bacterioplankton Production During Early Spring in Lake Erken, Sweden

The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5°C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The ¹⁴C and O₂ methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO₃ N assimilation ratio was 1.57. The total integrated incorporation of [¹⁴C]bicarbonate into particulate material (>1 μm) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 × 10⁹ to 1.52 × 10⁹ cells liter⁻¹ (~0.5 μg of C liter⁻¹ day⁻¹). Estimates of bacterioplankton production from rates of [³H]thymidine incorporation were ca. 1.2 to 1.7 μg of C liter⁻¹ day⁻¹. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 μg of C liter⁻¹ day⁻¹, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [³H]thymidine incorporation method.     

Effects of Temperature on Two Psychrophilic Ecotypes of a Heterotrophic Nanoflagellate, Paraphysomonas imperforata

Two different psychrophilic types of the heterotrophic nanoflagellate Paraphysomonas imperforata were isolated from Newfoundland coastal waters and the Arctic Ocean. When fed bacteria without food limitation, both isolates were able to grow at temperatures from -1.8 to 20°C, with maximum growth rates of 3.28 day^-1 at 15°C and 2.28 day^-1 at 12.3°C for the Newfoundland and the Arctic isolates, respectively. Ingestion rates increased with temperature from 14 to 62 bacteria flagellate^-1 h^-1 for the Newfoundland isolate and from 30 to 99 bacteria flagellate^-1 h^-1 for the Arctic isolate. While temperature did not affect cell yields (number of protozoa produced divided by number of bacteria consumed), it affected flagellate sizes. This differential effect of temperature on cell yield and cell size resulted in a changing gross growth efficiency (GGE) in terms of biovolume; colder temperatures favored higher GGEs. The comparison of Q₁₀ values for growth rates and ingestion rates between the isolates shows that the Arctic isolate is better adapted to extremely cold temperature than the Newfoundland isolate. At seawater-freezing temperature (-1.8°C), the estimated maximum growth rates and maximum ingestion rates are 0.81 day^-1 and 30 bacteria flagellate^-1 h^-1 for the Arctic isolate and 0.54 day^-1 and 12 bacteria flagellate^-1 h^-1 for the Newfoundland isolate. Our findings about psychrophilic nanoflagellates fit the general characteristics of cold-water-dwelling organisms: reduced physiological rates and higher GGEs at lower temperatures. Because of the large and persistent differences between the isolates, we conclude that they are ecotypes adapted to specific environmental conditions.     

Aggregation and Sedimentation of Thalassiosira weissflogii (diatom) in a Warmer and More Acidified Future Ocean

Increasing Transparent Exopolymer Particle (TEP) formation during diatom blooms as a result of elevated temperature and pCO₂ have been suggested to result in enhanced aggregation and carbon flux, therewith potentially increasing the sequestration of carbon by the ocean. We present experimental results on TEP and aggregate formation by Thalassiosira weissflogii (diatom) in the presence or absence of bacteria under two temperature and three pCO₂ scenarios. During the aggregation phase of the experiment TEP formation was elevated at the higher temperature (20°C vs. 15°C), as predicted. However, in contrast to expectations based on the established relationship between TEP and aggregation, aggregation rates and sinking velocity of aggregates were depressed in warmer treatments, especially under ocean acidification conditions. If our experimental findings can be extrapolated to natural conditions, they would imply a reduction in carbon flux and potentially reduced carbon sequestration after diatom blooms in the future ocean.     

Grazing, Growth, and Ammonium Excretion Rates of a Heterotrophic Microflagellate Fed with Four Species of Bacteria

We studied aspects of the population growth of a microflagellate, Monas sp., isolated from Lake Kinneret, Israel. The protozoan growth rates, rates of ingestion of bacteria, and final population yields generally increased with increasing bacterial concentrations, although the exact relationship varied depending on the species of bacteria used as food. Grazing rates decreased hyperbolically with increasing food density. Gross growth efficiencies and ammonia excretion rates were similar over a range of food densities among the four species of bacteria. Population doubling times and ammonia excretion rates were lowest, and growth efficiencies were highest, at temperatures between 18 and 24°C. Under optimum conditions, the microflagellates had average population doubling times of 5.0 to 7.8 h, average growth efficiencies of 23.7 to 48.7%, and average ammonia excretion rates of 0.76 to 1.23 μmol of NH₄⁺ per mg (dry wt) per h.     

Adaptation of Phytoplankton-Degrading Microbial Communities to Thermal Reactor Effluent in a New Cooling Reservoir

In water column and sediment inocula from a nuclear reactor cooling reservoir, natural phytoplankton substrate labeled with ¹⁴C was used to determine aerobic and anaerobic mineralization rates for a range of temperatures (25, 40, 55, and 70°C) expected during reactor operation. For experiments that were begun during reactor shutdown, aerobic decomposition occurred at temperatures of <55°C. After 2 months of reactor operation, aerobic rates increased substantially at 55 and 70°C, although maximum rates were observed at temperatures of ≤40°C. The temperature range for which maximum anaerobic mineralization (i.e., the sum of CH₄ and CO₂) was observed was 25 to 40°C when the reactor was off, expanding to 25 to 55°C during reactor operation. Increased rates at 55°C, but not 70°C, correlated with an increase in the ratio of cumulative methane to carbon dioxide produced over 21 days. When reduced reactor power lowered the maximum temperature of the reservoir to 42°C, aerobic decomposition at 70°C was negligible, but remained substantial at 55°C. Selection for thermophilic decomposers occurred rapidly in this system in both aerobic and anaerobic communities and did not require prolonged exposure to elevated temperatures.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

Effect of altering the root-zone temperature on growth, translocation, carbon exchange rate, and leaf starch accumulation in the tomato

Tomato seedlings (Lycopersicon esculentum Mill. cv Vendor) were grown hydroponically with their root systems maintained at a constant temperature for a 2-week period commencing with the appearance of the first true leaf. Based on fresh and dry weight and leaf area, the optimal root-zone temperature for seedling growth was 30°C. The carbon exchange rate of the leaves was also found to increase with rising root-zone temperature up to 30°C. However, a more complex relationship seems to exist between root-zone temperature and the accumulation of ¹⁴C-labeled assimilates in the roots; inasmuch as there is no enhancement in this accumulation at the most growth promoting root-zone temperatures (22-30°C).     

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

Interaction Effects of Light,Temperature and Nutrient Limitations (N,P and Si) on Growth,Stoichiometry and Photosynthetic Parameters of the Cold-Water Diatom Chaetoceros wighamii

Light (20-450 μmol photons m^-2 s^-1), temperature (3-11°C) and inorganic nutrient composition (nutrient replete and N, P and Si limitation) were manipulated to study their combined influence on growth, stoichiometry (C:N:P:Chl a) and primary production of the cold water diatom Chaetoceros wighamii. During exponential growth, the maximum growth rate (~0.8 d^-1) was observed at high temperture and light; at 3°C the growth rate was ~30% lower under similar light conditions. The interaction effect of light and temperature were clearly visible from growth and cellular stoichiometry. The average C:N:P molar ratio was 80:13:1 during exponential growth, but the range, due to different light acclimation, was widest at the lowest temperature, reaching very low C:P (~50) and N:P ratios (~8) at low light and temperature. The C:Chl a ratio had also a wider range at the lowest temperature during exponential growth, ranging 16-48 (weight ratio) at 3°C compared with 17-33 at 11°C. During exponential growth, there was no clear trend in the Chl a normalized, initial slope (α*) of the photosynthesis-irradiance (PE) curve, but the maximum photosynthetic production (P_m) was highest for cultures acclimated to the highest light and temperature. During the stationary growth phase, the stoichiometric relationship depended on the limiting nutrient, but with generally increasing C:N:P ratio. The average photosynthetic quotient (PQ) during exponential growth was 1.26 but decreased to <1 under nutrient and light limitation, probably due to photorespiration. The results clearly demonstrate that there are interaction effects between light, temperature and nutrient limitation, and the data suggests greater variability of key parameters at low temperature. Understanding these dynamics will be important for improving models of aquatic primary production and biogeochemical cycles in a warming climate.     

Cultured Ovules as Models for Cotton Fiber Development under Low Temperatures

Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a “growth ring” for each time the temperature cycled to 22 or 15°C. Rings were rarely detected when the low point was 28°C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34°C/22°C cycling developed similarly to constant 34°C controls, but 34°C/22°C and 34°C/15°C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34°C/22°C cycling, but both parameters were reduced at the end of the experiment under 34°C/15°C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule-specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response.     

Effect of temperature on nitrogenase functioning in cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30°C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20°C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30°C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15°C to a maximum around 35°C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38°C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35°C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15°C and 47°C.     

Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophyll a liter⁻¹). At this time bacterial abundance abruptly decreased from 2.8 × 10⁶ to 0.75 × 10⁶ ml⁻¹, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Psychrotolerant Bacteria Isolated from Arctic Soil That Degrade Polychlorinated Biphenyls at Low Temperatures

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus Pseudomonas. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile Burkholderia cepacia LB400 were determined at 7, 37, and 50°C. Total removal values of Aroclor 1242 at 7°C by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 μg of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37°C than at 7°C but as much as 90% lower at 50°C than at 37°C. In contrast, rates of PCB removal by LB400 were higher at 50°C than at 37°C. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.     

Effects of Temperature, Water Activity, and Syrup Film Composition on the Growth of Wallemia sebi: Development and Assessment of a Model Predicting Growth Lags in Syrup Agar and Crystalline Sugar

We investigated the effects of temperature, water activity (a_w), and syrup film composition on the CFU growth of Wallemia sebi in crystalline sugar. At a high a_w (0.82) at both high (20°C) and low (10°C) temperatures, the CFU growth of W. sebi in both white and extrawhite sugar could be described using a modified Gompertz model. At a low a_w (0.76), however, the modified Gompertz model could not be fitted to the CFU data obtained with the two sugars due to long CFU growth lags and low maximum specific CFU growth rates of W. sebi at 20°C and due to the fact that growth did not occur at 10°C. At an a_w of 0.82, regardless of the temperature, the carrying capacity (i.e., the cell concentration at t = ∞) of extrawhite sugar was lower than that of white sugar. Together with the fact that the syrup film of extrawhite sugar contained less amino-nitrogen relative to other macronutrients than the syrup film of white sugar, these results suggest that CFU growth of W. sebi in extrawhite sugar may be nitrogen limited. We developed a secondary growth model which is able to predict colony growth lags of W. sebi on syrup agar as a function of temperature and a_w. The ability of this model to predict CFU growth lags of W. sebi in crystalline sugar was assessed.     

Higher Thermal Acclimation Potential of Respiration but Not Photosynthesis in Two Alpine Picea Taxa in Contrast to Two Lowland Congeners

The members of the genus Picea form a dominant component in many alpine and boreal forests which are the major sink for atmospheric CO₂. However, little is known about the growth response and acclimation of CO₂ exchange characteristics to high temperature stress in Picea taxa from different altitudes. Gas exchange parameters and growth characteristics were recorded from four year old seedlings of two alpine (Picea likiangensis vars. rubescens and linzhiensis) and two lowland (P. koraiensis and P. meyeri) taxa. Seedlings were grown at moderate (25°C/15°C) and high (35°C/25°C) day/night temperatures, for four months. The approximated biomass increment (ΔD²H) for all taxa decreased under high temperature stress, associated with decreased photosynthesis and increased respiration. However, the two alpine taxa exhibited lower photosynthetic acclimation and higher respiratory acclimation than either lowland taxon. Moreover, higher leaf dry mass per unit area (LMA) and leaf nitrogen content per unit area (N_area), and a smaller change in the nitrogen use efficiency of photosynthesis (PNUE) for lowland taxa indicated that these maintained higher homeostasis of photosynthesis than alpine taxa. The higher respiration rates produced more energy for repair and maintenance biomass, especially for higher photosynthetic activity for lowland taxa, which causes lower respiratory acclimation. Thus, the changes of ΔD²H for alpine spruces were larger than that for lowland spruces. These results indicate that long term heat stress negatively impact on the growth of Picea seedlings, and alpine taxa are more affected than low altitude ones by high temperature stress. Hence the altitude ranges of Picea taxa should be taken into account when predicting changes to carbon fluxes in warmer conditions.     

Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches,Nearby Wastewater Effluents,and Bird Fecal Droppings

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Effect of Temperature and Prey Availability on Growth of Paramoeba invadens in Monoxenic Culture

Paramoeba invadens Jones 1985 is a pathogenic marine amoeba responsible for mass mortalities of sea urchins (Strongylocentrotus droebachiensis) of Nova Scotia between 1980 and 1983. A direct relationship between temperature and sea urchin paramoebiasis has been shown in previous laboratory and field studies. This study examined the effect of prey availability and temperature on the growth of P. invadens in monoxenic culture (with the marine bacterium Pseudomonas nautica). At 15°C, the specific growth rate of P. invadens increased with bacterial prey concentration and was highest at 10⁸ bacterial cells ml⁻¹. Growth rate of P. invadens was maximal at 15 to 20°C (which corresponds to annual sea temperature maxima in the natural environment) and the minimum generation time was 19.41 h at 20°C. At 10 and 12°C, generation times were 91.18 and 73.39 h, respectively; at 2 and 5°C, there was no growth. P. invadens did not survive in monoxenic culture at 27°C. Growth rates of P. invadens in vitro were positively correlated with time to morbidity of infected S. droebachiensis.     

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.