Temperature acclimation and the expression of contractile protein isoforms in the skeletal muscles of the common carp (Cyprinus carpio L.)

Summary Common carp (Cyprinus carpio L.) were acclimated to either 2, 5, 8, 11, 15, 20, or 23°C for 12 weeks (12 h light: 12 h dark). Fish did not feed after 6 weeks at temperatures below 8°C. Skinned fibres were prepared from fast myotomal muscle by freeze-drying. Measured at 0°C unloaded contraction velocity (Vmax) and maximum isometric tension generation (Po) were 2–3 times higher in the 11°C-than 23°C-acclimated groups, and had intermediate values in 15 °C-acclimated fish. Po and Vmax at 0°C were not significantly different for carp maintained at 2, 5, 8, or 11°C. Measured at the acclimation temperature of each group Vmax and Po were 51% and 71% lower for fibres from 2°C- than 23°C-acclimated fish. The results indicate a partial capacity adaptation of muscle power output in fish acclimated between 11°C and 23°C. At 8°C the ATPase activity of myofibrils was 2 times higher in fish acclimated to 8°C than to 20°C. The effects of temperature acclimation on the protein composition of myofibrils was investigated using one- and two-dimensional electrophoresis. Peptide maps of purified myosin heavy chains and actin prepared by proteolytic digestion with either Staphylococcus aureus V8 protease or chymotrypsin were similar for both acclimation groups. The molecular weights and isoelectric points of the major isoforms of tropomyosin, troponin C, troponin I, troponin T, and myosin light chains (MLC1, MLC2 and MLC3) were also similar in 8°C- and 20°C-acclimated carp. A 20 kDa molecular weight protein with a pI intermediate between that for MLC2 and MLC3 was found in myofibrils and single fibres from carp acclimated to 8°C but was not present in carp acclimated to 20°C. It is suggested that this band corresponds to a myosin light chain isoform unique to cold-acclimated fish. Evidence was also obtained that myofibrils from warm-acclimated fish contained a second minor isoform of troponin I.     

An integrated UASB-sludge digester system for raw domestic wastewater treatment in temperate climates

To improve the performance of an upflow anaerobic sludge blanket (UASB) reactor treating raw domestic wastewater under temperate climates conditions, the addition of a sludge digester to the process was investigated. With the decrease in temperature, the COD removal decreased from 78% at 28 °C to 42% at 10 °C for the UASB reactor operating alone at a hydraulic retention time of 6 h. The decrease was attributed to low hydrolytic activity at lower temperatures that reduced suspended matter degradation and resulted in solids accumulation in the top of the sludge blanket. Solids removed from the upper part of the UASB sludge were treated in an anaerobic digester. Based on sludge degradation kinetics at 30 °C, a digester of 0.66 l per liter of UASB reactor was design operating at a 3.20 days retention time. Methane produced by the sludge digester is sufficient to maintain the temperature at 30 °C.     

Thermoregulatory behavior of a tropical marine fish: Canthigaster jactator (Jenkins)

Ten Canthigaster, jactator, tropical marine puffers from Hawaii, were tested individually for 3-day periods in electronic shuttle-boxes (Ichthyotrons) to determine their ability to thermoregulate behaviorally. These fish thermoregulated with a degree of precision comparable to that of temperate freshwater fishes: range 23–31°C, S.D. 1.8–2.4°C, S.E. 0.3–0.5° C. The modal final thermal preferendum was 27°C, comparable to temperate warmwater fishes. The mean preferred temperature did not differ significantly between night (26.5°C) and day (26.9°C); the 24-hr mean was 26.7°C. Apparently at least some tropical marine fishes are capable of thermoregulatory behavior similar to that of temperate freshwater fishes.     

Calcium regulatory proteins and temperature acclimation of actomyosin ATPase from a eurythermal teleost (Carassius auratus L.)

Summary Goldfish (Carassius auratus) were acclimated for 5 months at temperatures of either 2°C or 31°C. Natural actomyosin was prepared from white myotomal muscle and its Mg²⁺Ca²⁺ ATPase activity determined. Temperature acclimation results in adaptations in substrate turnover number and thermodynamic activation parameters of the ATPase. When assayed at 31°C the Mg²⁺Ca²⁺ ATPase of natural actomyosin was 4 times higher in 31°C than 2°C acclimated fish. Arrhenius plots of natural actomyosin ATPase from cold acclimated fish show a break in slope at 15–18°C. In contrast, the temperature dependence of warm acclimated actomyosin was linear. Activation enthalpy (H ) of the ATPase, calculated over the range 0–16°C, was approximately 8,000 cal/mole lower in 2°C than 32°C acclimated fish.In contrast, desensitised actomyosins from which the calcium regulatory proteins have been removed show a linear temperature dependence in the range 0–32°C and have similar properties in 2°C and 31°C acclimated fish. Cross-hybridisation of regulatory proteins (tropomyosin-troponins complex) from cold-acclimated fish to desensitised actomyosin from warm-acclimated fish alters the ATPase towards that of cold-acclimated natural actomyosin and vice versa. The results suggest that the regulatory proteins can influence the kinetics of the ATPase and, furthermore, that they are involved in the acclimation of the actomyosin to different cell temperatures.     

Characterization of the combined effects of enzyme,pH and temperature treatments for removal of pathogens from sewage sludge

The combined effects of enzyme, pH and temperature treatments for removal of pathogens from sewage sludge were investigated. An adjustment to pH 10 for 24-h at 23 °C or a 3-h hold at 50 °C resulted in a 100% kill of coliforms but a significant count of Salmonella species still survived. However, pH adjustment to 12 with a hold of 48-h at 23 °C or pH adjustment to 10 or 12 and a 3-h hold at 60 °C was required to achieve a 100% kill of Salmonella species. Although protease enzyme treatment at 40 °C with or without alkali treatment eliminated coliforms completely, Salmonella counts were reduced by two to three orders of magnitude. However, complete elimination of pathogens was observed when enzymatic treatment was provided at 50 °C. Application of alkaline protease, through its combined beneficial effects in pathogen reduction, solids reduction and improved solids settling, has potential as an effective procedure for processing of sewage sludge.     

Automated Sampling Procedures Supported by High Persistence of Bacterial Fecal Indicators and Bacteroidetes Genetic Microbial Source Tracking Markers in Municipal Wastewater during Short-Term Storage at 5°C

Because of high diurnal water quality fluctuations in raw municipal wastewater, the use of proportional autosampling over a period of 24 h at municipal wastewater treatment plants (WWTPs) to evaluate carbon, nitrogen, and phosphorus removal has become a standard in many countries. Microbial removal or load estimation at municipal WWTPs, however, is still based on manually recovered grab samples. The goal of this study was to establish basic knowledge regarding the persistence of standard bacterial fecal indicators and Bacteroidetes genetic microbial source tracking markers in municipal wastewater in order to evaluate their suitability for automated sampling, as the potential lack of persistence is the main argument against such procedures. Raw and secondary treated wastewater of municipal origin from representative and well-characterized biological WWTPs without disinfection (organic carbon and nutrient removal) was investigated in microcosm experiments at 5 and 21°C with a total storage time of 32 h (including a 24-h autosampling component and an 8-h postsampling phase). Vegetative Escherichia coli and enterococci, as well as Clostridium perfringens spores, were selected as indicators for cultivation-based standard enumeration. Molecular analysis focused on total (AllBac) and human-associated genetic Bacteroidetes (BacHum-UCD, HF183 TaqMan) markers by using quantitative PCR, as well as 16S rRNA gene-based next-generation sequencing. The microbial parameters showed high persistence in both raw and treated wastewater at 5°C under the storage conditions used. Surprisingly, and in contrast to results obtained with treated wastewater, persistence of the microbial markers in raw wastewater was also high at 21°C. On the basis of our results, 24-h autosampling procedures with 5°C storage conditions can be recommended for the investigation of fecal indicators or Bacteroidetes genetic markers at municipal WWTPs. Such autosampling procedures will contribute to better understanding and monitoring of municipal WWTPs as sources of fecal pollution in water resources.     

Temperature dependent (37-15°C) anaerobic digestion of a trichloroethylene-contaminated wastewater

The impact of a trichloroethylene (TCE) contaminated wastewater on the microbial community structure of an anaerobic granular biomass at 15 °C compared to 37 °C was investigated. Four expanded granular sludge bed (EGSB) bioreactors (R1-R4) were employed in pairs at 37 and 15 °C. The influents of one of each pair were supplemented with increasing concentrations of TCE (max. 60 mg l⁻¹). At 37 °C, stable operation was maintained with 88% COD removal and >99% TCE removal at maximum influent TCE concentrations. R3 performance decreased at influent TCE concentration of 60 mg l⁻¹, although TCE removal rates of >97% were recorded. Archaeal community analysis via clone library and quantitative polymerase chain reaction (qPCR) analysis, and bacterial community analysis via denaturing gradient gel electrophoresis (DGGE), indicated that temperature resulted in a greater change in community structure than the presence of TCE, and clones related to cold adaptation of biomass were identified at 15 °C.     

Effects of different ambient temperatures on physiological responses during exercise and recovery with special reference to energy requirement

Observation of the physiological responses was made on seven young male subjects ages 27–31, during pedalling a bicycle ergometer at the constant work load of 600 kg. m/min for 20 min and recovery in 35°C with 50% RH, in 30°C with 50% RH and in 23°C with 50% RH. Heart rate, respiratory volume, total oxygen intake and energy requirement were increased with an increase in ambient temperature, while blood pressures were lower in a hot environment than in cooler environments. In 35°C, oxygen intake during exercise, oxygen debt and anaerobic fraction of oxygen debt had increased when compared with those in 23°C. Thus it is inferred that the energy requirement, the oxygen debt and the anaerobic fraction of the oxygen debt for a fixed work had increased more in a hot environment than in a comfortable environment. Factors which caused differences in the physiological reactions during exercise and recovery in different conditions are discussed.     

Oxidative stress and antioxidant defense responses by goldfish tissues to acute change of temperature from 3 to 23 °C

The effects of a rapid transfer from a low (3 °C) to a warm (23 °C) temperature on oxidative stress markers and antioxidant defenses were studied in the brain, liver and kidney of the goldfish, Carassius auratus. Cold-acclimated fish were acutely moved to 23 °C and sampled after 1, 6, 12, 24, 48 or 120 h of warm temperature exposure. Lipid peroxide levels increased quickly during the first few hours at 23 °C, but thiobarbituric acid-reactive substances changed little. Protein carbonyl content was reduced by 20–40% in the liver over the entire experimental course, but increased transiently in the kidney. The content of high-molecular mass thiols decreased by two-thirds in the brain and was affected slightly in other organs. By contrast, total low-molecular mass thiols (e.g. glutathione and others) increased transiently. Activities of the primary antioxidant enzymes—superoxide dismutase and catalase—were generally unaffected in goldfish organs, whereas glutathione-dependent enzymes were elevated in the brain and kidney after 24–48 h at 23 °C. Glutathione peroxidase increased by 1.5–2.3-fold and glutathione-S-transferase by 1.7-fold. Hence, a short-term exposure to warm temperature disturbed several oxidative stress markers, but only slightly affected the activities of antioxidant enzymes. However, comparison of the current data for cold-acclimated winter fish with the same parameters in summer fish suggests that longer exposure to high ambient temperature requires the enhancement of activities of glutathione-dependent enzymes for maintaining the steady-state levels lipid peroxidation and protein oxidation in goldfish tissues.     

Capillarisation,oxygen diffusion distances and mitochondrial content of carp muscles following acclimation to summer and winter temperatures

Summary Many species of fish show a partial or complete thermal compensation of metabolic rate on acclimation from summer to winter temperatures. In the present study Crucian carp (Carassius carassius L.) were acclimated for two months to either 2° C or 28° C and the effects of temperature acclimation on mitochondrial content and capillary supply to myotomal muscles determined.Mitochondria occupy 31.4% and 14.7% of slow fibre volume in 2°C- and 28° C-acclimated fish, respectively. Fast muscles of coldbut not warm-acclimated fish show a marked heterogeneity in mitochondrial volume. For example, only 5 % of fast fibres in 28° C-acclimated fish contain 5 % mitochondria compared to 34 % in 2° C-acclimated fish. The mean mitochondrial volume in fast fibres is 6.1 % and 1.6 % for coldand warm-acclimated fish, respectively.Increases in the mitochondrial compartment with cold acclimation were accompanied by an increase in the capillary supply to both fast (1.4 to 2.9 capillaries/fibre) and slow (2.2 to 4.8 capillaries/fibre) muscles. The percentage of slow fibre surface vascularised is 13.6 in 28° C-acclimated fish and 32.1 in 2° C-acclimated fish. Corresponding values for fast muscle are 2.3 and 6.6 % for warm and cold-acclimated fish, respectively. Maximum hypothetical diffusion distances are reduced by approximately 23–30 % in the muscles of 2° C-compared to 28° C-acclimated fish. However, the capillary surface supplying 1 ³ of mitochondria is similar at both temperatures.Factors regulating thermal compensation of aerobic metabolism and the plasticity of fish muscle to environmental change are briefly discussed.     

A kinetic evaluation of anaerobic treatment of swine wastewater at two temperatures in a temperate climate zone

A static granular bed reactor (SGBR) was used to treat swine wastewater at 24 and 16 °C. At 24 °C, the organic loading rate (OLR) was 0.7-5.4 kg COD/m³ day and the average chemical oxygen demand (COD) removal efficiency was 88.5%, respectively. Meanwhile, at 16 °C, the OLR was 1.6-4.0 kg COD/m³ day and the average COD removal efficiency was 68.0%, respectively. The SGBR acted as a bioreactor as well as a biofilter. After backwashing, the recovery of COD removal was not a function of an OLR but recovery time, while that of TSS removal was not a function of either recovery time or the OLR. The maximum substrate utilization rate (k_max) ratio was 1.89 between 24 and 16 °C, and the half velocity constant (K_s) ratio was 1.22, and the maximum specific growth rate (μ_max) ratio was 4.71. In addition, the temperature-activity coefficient in this study was determined to be 1.09.     

Structural and circulatory responses to hypoxia in the secondary lamellae ofSalmo gairdneri gills at two temperatures

Summary The first gill arch ofSalmo gairdneri was fixed from normoxic (O₂ saturation of the water >90%) and hypoxic (O₂ saturation 25–30% for 5 days) fish at 10 °C and 18 °C, and from fish after one-day recovery from hypoxia at 18 °C. The secondary lamellae of the gills were analysed with morphometric methods for structural, haemotological and circulatory changes. During hypoxia a marked vascular distension took place at both temperatures. At both temperatures the vascular distension coincided with a shortening of the diffusion distance (36% at 10 °C and 21% at 18 °C) and a swelling of the erythrocytes (60% at 10 °C and 42% at 18 °C). The effects of these changes on the oxygen uptake of the gills are discussed.     

Microalgae-mediated brewery wastewater treatment: effect of dilution rate on nutrient removal rates,biomass biochemical composition,and cell physiology

Microalgae have been used to remove nitrogen, phosphorus, and chemical oxygen demand (COD) from brewery wastewater (BWW). The microalga Scenedesmus obliquus was grown on BWW, using bubble column photobioreactors that operated under batch and continuous regimes. For the first time, the cell physiological status cell membrane integrity and enzymatic activity was monitored during the microalgae based BWW treatment, using flow cytometry. All the cultivations batch and continuous displayed a proportion of cells with intact membrane >?87%, although the continuous cultivations displayed a lower proportion of cells with enzymatic activity (20–40%) than the batch cultivations (97%). The dilution rate of 0.26 day^?1 was the most favorable condition, since the microalgae cultivation attained the maximum biomass productivity (0.2 g ash-free dry weight day^?1) and the total nitrogen and COD removal rates were the highest (97 and 74%, respectively), while the phosphorous removal rate was the third (23%).     

Vertical movement and behavior of the ocean sunfish, Mola mola, in the northwest Atlantic

Pop-up satellite archival tags (PSATs) were attached to 31 ocean sunfish, Mola mola. in the Northwest Atlantic between 2005 and 2008, in order to examine their vertical movement and behavior. Tags remained attached from 7 to 242 days, with a mean attachment period of 107.2 ± 80.6 (SD) days. Fish spent greater than 30% of their time in the top 10 m of the water column, and over 80% of time in the top 200 m. The maximum depth recorded by any fish was 844 m. Temperatures experienced by tagged fish ranged from 6 to 30 °C. Vertical behavior of M. mola changed over short-term and seasonal scales. Ocean sunfish in northeastern US waters in the summer months inhabited shallower depths and spent more time at the surface than those that moved south in the winter and spring. This shift from shallow to deeper depths was especially apparent when fish entered the Gulf Stream, where they spent little time at the surface and dove to depths of 400-800 m. A diel pattern was observed in vertical behavior. Tagged fish spent more time at depth during the day and inhabited shallower waters at night. There was no observed relationship between the amount of time per day that fish spent in cold water (< 10 °C) and the amount of time fish spent near the surface (0-6 m), indicating a lack of evidence for M. mola basking at the surface as a mechanism for behavioral thermoregulation.     

Metabolic response of juvenile gray snapper (Lutjanus griseus) to temperature and salinity: Physiological cost of different environments

Juvenile gray snapper (Lutjanus griseus) occupy a wide range of estuarine and nearshore habitats that differ in physico-chemical properties. To quantify the energetic cost of inhabiting these different habitats, routine metabolism of individual gray snapper was measured in the laboratory at 20 combinations of temperature (18, 23, 28, and 33 °C) and salinity (5, 15, 25, 35, and 45 psu). An open, flow-through respirometer was used, enabling trials to be run for long periods (∼16 h), while maintaining water quality (dissolved O₂>70% saturation), and providing fish sufficient time to habituate to the chambers undisturbed. Video recordings of fish in the respirometer chambers were analyzed to quantify the spontaneous activity rate of individuals. Analysis of covariance, using fish weight and mean activity rate as covariates, indicated significant temperature and salinity effects on oxygen consumption. Oxygen consumption was significantly higher at high salinities, and the salinity effect was temperature dependent. A polynomial equation describing oxygen consumption as a function of temperature and salinity indicated the increase due to salinity from 5 to 45 psu at high temperatures (30-33 °C) was equivalent to a 3 °C increase in temperature. At intermediate temperatures (24-26 °C), the increase due to salinity from 5 to 45 psu was less dramatic, equivalent to a 2 °C increase in temperature. At the lowest temperatures (18 °C), salinity did not have a significant effect on oxygen consumption. The increased metabolic costs in high salinities (∼7% at the high temperature) represent a significant energy cost for juveniles, that would need to be balanced by lower predation risk or greater food availability to result in similar juvenile production compared to lower salinity environments.     

Stability of a downflow anaerobic fixed-film reactor to feed change

Summary A stepped-loading start-up regime utilising volatile fatty acids as carbon and energy sources was applied to a downflow anaerobic fixed-film reactor operated at 37°C. A steadystate was attained with over 90% COD reduction at an organic loading rate of 6.1 Kg COD/m³·day. A complex wastewater coming from a citrus processing plant was then used to test the feasibility of feeding changes as well as the stability of the reactor performance. A final COD reduction of 60% was achieved with hydraulic retention times down to 1.5 days.     

Responses of pea and wheat to textile wastewater reclaimed by suspended sequencing batch bioreactors

Availability of good quality water for crop irrigation is a big challenge in developing countries due to limited resources of clean water. Textile industry consumes a huge amount of water during dyeing process and consequently it releases high strength wastewater into wastewater streams. The present study was designed with the objective to use textile wastewater treated in sequencing batch bioreactor for irrigation purpose. Wastewater containing 100 mg/L reactive black-5 azo dye amended with different co-substrates was treated using mixed liquor suspended solids (MLSS) and two previously isolated dye-degrading bacterial strains (Psychrobacter alimentarius KS23 and Staphylococcus equorum KS26). About 90% color and COD removal in case of dye-containing wastewater amended either with mineral salts + yeast extract or only yeast extract was achieved in 24 h after treatment with mixed culture (MLSS + KS23 + KS26). The treated wastewater was applied for irrigation of pea and wheat plants under controlled conditions. Untreated dye-contaminated wastewater was used as a control for comparison. A significant positive effect of treated dye wastewater amended with different co-substrates on the seed germination index, root and shoot length and biomass was observed in response to application of dye-containing wastewater treated with MLSS and dye-degrading bacterial strains compared to untreated control. Results of this study reveal that the dye-degrading microbial cultures could be used to enhance the treatment efficiency of dye-contaminated wastewater that can be utilized for irrigation of crops and biomass production.     

The effects of pH and temperature on orthophosphate removal by immobilized Chlorella vulgaris

Summary When Chlorella vulgaris was immobilized in calcium alginate beads, it removed more than 90% phosphate (10mg P/L) added to artificial wastewater at pH 3 to 9 and from 10 to 30°C. Free cells, however, only removed 40–60% of added phosphate at low pH (3–5) and at 10°C. Immobilized C. vulgaris is shown to have great potentialities for removing phosphate from low pH wastewater and at low temperature.     

Thermal transitions in dimyristoylphosphatidyleholine foam bilayers

Thermal transitions in the system dimyristoylphosphatidylcholine/water/ethanol/sodium chloride were studied in the temperature range 10–31 °C. The water-ethanol dispersions were investigated by differential scanning calorimetry and the foam bilayers by the microinterferometric method for investigation of thin liquid films. Calorimetry showed that an increase in ethanol content (up to 47.5 vol.% — the concentration used in the experiments with foam bilayers) did not significantly influence the temperature of the main phase transition and led to the disappearance of the pretransition. The microinterferometric study of the foam bilayer thickness showed that there were two thermal transitions — at 13 and 23 °C. An Arrhenius type dependence was obtained for the critical concentration of dimyristoylphosphatidylcholine (DMPC) in the solution, which was necessary for the formation of the foam bilayer. A steep change in the slope of the linearized Arrhenius dependence was found at 23 °C. Values of the binding energy of a DMPC molecule in the foam bilayers were calculated using the hole-nucleation theory of stability and permeability of bilayers. It was proved that the phase transition at 23 °C was due to melting of the hydrocarbon tails of phospholipid molecules. The low-temperature phase transition was assumed to be due to a change in the tilt of the hydrocarbon tails. These experiments demonstrate for the first time the occurrence of phase transitions in foam bilayers. Correspondence to: D. Exerowa     

Effect of seed sludge and operation conditions on performance and archaeal community structure of low-temperature anaerobic solvent-degrading bioreactors

Two laboratory-scale expanded granular sludge bed (EGSB) anaerobic bioreactors (R1 and R2) were inoculated with biomass from different mesophilic (37 °C) treatment plants, and used for the treatment of an organic solvent-based wastewater at 9–14 °C at applied organic loading rates (OLRs) of 1.2–3.6 kg chemical oxygen demand (COD) m⁻³ d⁻¹. Replicated treatment performance was observed at 10–14 °C, which suggested the feasibility of the process at pilot-scale. Stable and efficient COD removal, along with high methane productivity, was demonstrated at 9 °C at an applied OLR of 2.4 kg COD m⁻³ d⁻¹. Clonal libraries and fluorescence in situ hybridization (FISH) indicated that the seed sludges were dominated (>60%) by acetoclastic Methanosaeta-like organisms. Specific methanogenic activity (SMA) profiles indicated shifts in the physiological profiles of R1 and R2 biomass, including the development of psychrotolerant methanogenic activity. Acetoclastic methanogenesis represented the primary route of methane production in R1 and R2, which is in contrast with several previous reports from low-temperature bioreactor trials. A reduction in the abundance of Methanosaeta-like clones (R2), along with the detection of hydrogenotrophic methanogenic species, coincided with altered granule (sludge) morphology and the development of hydrogenotrophic SMA after prolonged operation at 9 °C.