The influence on enzyme activity of storage of tissue blocks at −70° C

Summary Blocks of tissue from various organs of the rat have been chilled by precipitate immersion in n-hexane cooled to –70° C, and then stored at –70° C. At various intervals (up to 14 days) after chilling, cryostat sections were prepared from these blocks and assayed for the activity of a variety of enzymes. Enzyme activity was measured by scanning and integrating microdensitometry. With the exception of acid phosphatase and cytochrome oxidase, all enzymes assayed were stable for at least 7 days after storage at –70° C and most were stable for 14 days. Storage of fresh-frozen sections at –30° C in the cabinet of the cryostat, for up to 24 h, had little effect on enzyme activity.     

The effect of cold storage at 4 °C on the Rooting of chestnut cuttings

This article reports the effect of cold storage at 4 °C from December to April on chestnut cuttings. Rooting was found to increase after four months preceded by treatment with 4000 mg l^-1 IBA. ThePhaseolus test of the biological activity of extracts of the cuttings showed the inhibition exerted by the acid fraction of the fresh (December) cuttings to have been replaced by a rootpromoting effect in cuttings stored for 4 months at low temperature. Cold storage thus seems to favour rooting.     

The biochemical diversity of life near and above 100°C in marine environments

Hyperthermophilic micro-organisms grow at temperatures above 90 °C with a current upper limit of 113 °C. They are a recent discovery in the microbial world and have been isolated mainly from marine geothermal environments, which include both shallow and deep sea hydrothermal vents. By 16S rRNA analyses they are the most slowly evolving of all extant life forms, and all but two of the nearly 20 known genera are classified as Archaea (formerly Archaebacteria). Almost all hyperthermophiles are strict anaerobes. They include species of methanogens, iron-oxidizers and sulphate reducers, but the majority are obligate heterotrophs that depend upon the reduction of elemental sulphur (S°) to hydrogen sulphide for significant growth. The heterotrophs utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S° has been proposed. Two S°-reducing enzymes have been purified from the cytoplasm of one hyperthermophile (T(opt) 100 °C) that is able to grow either with and without S°. However, the mechanisms by which S° reduction is coupled to energy conservation in this organism and in obligate S°-reducing hyperthermophiles is not known. In the heterotrophs, sugar fermentation is achieved by a novel glycolytic pathway involving unusual ADP-dependent kinases and ATP synthetases, and novel oxidoreductases that are ferredoxin- rather than NAD(P)-linked. Similarly, peptide fermentation involves several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Several of these oxido-reductases contain tungsten, an element that is rarely used in biological systems. Tungsten is present in exceedingly low concentrations in normal sea water, but hydrothermal systems contain much higher tungsten concentrations, more than sufficient to support hyperthermophilic life.     

The heat shock response of an antarctic alga is evident at 5°C

A subtidal seaweed collected in antarctic waters, Plocamium cartilagineum (L. Dix.), displayed induction of mRNAs encoding the 70 kDa heat shock protein (HSP70) and the ubiquitin polyprotein (UBI) when incubated at 5°C. Maximal induction of HSP70 mRNA was observed when the alga was incubated at 10°C for 1 h. Incubations at higher temperatures or for longer periods reduced the amount of HSP70 mRNA detected. Incubations at 20°C or greater resulted in cell death. These data indicate that dispite the unusually low temperature of induction, this macrophyte exhibits a heat shock response similar to that of other organisms at temperatures 5 to 10°C above usual growth conditions.     

The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C

Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 μM MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P > 0.05) throughout the trial. Although the findings of the present study revealed that 1 μM MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen.     

The effect of sulfate reduction on the thermophilic (55°C) methane fermentation process

Summary The continuously operated suspended growth anaerobic contact system was utilized to estimate the effect of sulfate reduction on the thermophilic (55°C) methane fermentation process. Results indicated that reduction in methanogenesis in the presence of sulfate was due to two separate, but related, processes;i.e. competitive and sulfide inhibition. Although prevention of competitive inhibition would be difficult under normal fermenter operation, sulfide inhibition could be minimized by environmental selection of sulfide tolerant microbial populations through biomass recycle and pH control. Stable fermenter operation was achieved at soluble sulfide concentrations as high as 330 mg/l soluble sulfide. Using batch fermenters, a maximum thermophilic sulfate reduction rate of 3.7 mg SO₄ ^2––S/g volatile solids (VS)-day was estimated. The importance of reporting sulfate reduction rates on a biomass basis is demonstrated by a simple population adjustment kinetic model.This research study was conducted at the Department of Agricultural Engineering, Cornell University, Riley Robb Hall, Ithaca, NY 14853, U.S.A.     

The impact of ten years at -20°C on gas exchange in five lichen species

Summary We examined the relative ability of multiple factors to explain variation in two interrelated life-history traits, interclutch interval and annual reproductive success, in feral pigeons. Seasonal influences, brood size, and female body mass and tarsus length explained significant amounts of variation in interclutch interval in this population, while female plumage phenotype was insignificant. These results are discussed in terms of resource allocation and responses to environmental heterogeneity. Multivariate selection analysis revealed strong directional fecundity selection on body mass, and correlated selection response on bill length. A prospective selection analysis based on estimates of the genetic variance-covariance matrix revealed that the mean change in a trait often differed in sign from the directional selection estimate. The relationship between annual reproductive success and these two traits was found only in melanic females, suggesting that selection differentials may covary with plumage pattern.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter⁻¹) and ammonia N concentrations (1.3 g liter⁻¹). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH₄ production (r_Pearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H₂ partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH₄ production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H₂ scavengers while yielding the highest CH₄ production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments.     

Biosynthetic capacity of hepatocytes after storage at 4°C

Capacity to synthesize glucose, urea, and ketone bodies is well maintained in hepatocytes after storage for at least 24 h at 4 degrees C. Substrates and albumin are the only requirements.     

Changes in productivity and carbon storage of grasslands in China under future global warming scenarios of 1.5°C and 2°C

Aims The impacts of future global warming of 1.5°C and 2°C on the productivity and carbon (C) storage of grasslands in China are not clear yet, although grasslands in China support ~45 million agricultural populations and more than 238 million livestock populations, and are sensitive to global warming.     

The effect of ice formation on the function of smooth muscle tissue stored at −21 or −60 °C

The mechanisms by which single cells are injured during freezing are relatively well understood, but it is likely that additional factors apply to tissues and organs, factors that may be responsible for the poor suecess of attempts to cryopreserve complex multicellular systems. One such factor may be the formation of extracellular ice.

This study was designed to discover whether ice formation as such is detrimental to the contractile recovery of pieces of mammalian smooth muscle after storage at subzero temperatures. Strips of taenia coli muscle were equilibrated with 2.56 M Me₂SO in a buffered solution, cooled at either 0.3 or 2 °C/min to ?21 °C and then held at this temperature in the frozen state. Other muscle strips were bathed in a solution the composition of which mimicked that of the unfrozen phase of the previous solution at ?21 °C; it contained 4.49 M Me₂SO and 1.75 times the normal concentration of salts, and muscles equilibrated with this solution were also cooled at either 0.3 or 2 °C/min to ?21 °C, and then held unfrozen for the same length of time.It was shown that exposure to ?21 °C and the increased concentration of solutes had little effect on the contractile recovery of the muscles, whereas ice formation was damaging. Furthermore, the rate of cooling had a marked effect upon functional recovery in the frozen muscles, and this could be correlated with the known effect of these cooling rates on the pattern of ice formation in the tissue. The effect was also seen in muscles frozen at ?60 °C. Improved buffering increased the functional recovery of all groups, but the effect of ice, and of cooling rate in the presence of ice, was confirmed. These findings may have significant implications for attempts to cryopreserve complex tissues and organs.     

Individualising the exposure of −110 °C whole body cryotherapy: The effects of sex and body composition

The purpose of this study was to investigate the effects of whole body cryotherapy (WBC) on a range of thermoregulatory measures. We also sought to examine the influence of sex and body composition. A convenience sample of 18 healthy participants (10 males and 8 females) (27±6 yr) volunteered for this study. Temperature (core, tympanic, skin and mean body), heart rate, blood pressure, and thermal comfort and sensation were recorded pre- and post- (immediately and every 5 min until 35 min post) exposure to a single bout of WBC (30 s at −60 °C, 150 s at 110 °C). Anthropometric data (height, weight, body surface area, body mass index, fat mass and fat free mass) were also recorded. No significant differences in temperature (core, tympanic, skin and mean body), heart rate, blood pressure, or thermal comfort / sensation were observed between male and females at baseline. Immediately post WBC mean body (male:31.9±0.8 °C; female:31.0±0.9 °C; ∆ mean body temperature:0.9±0.1 °C; P≤0.05, d=0.64) and mean skin (male:22.1±2.2 °C; female:19.6±2.8 °C; ∆ mean skin temperature:−2.5±0.6 °C; d=0.99, P≤0.05) temperature was significantly different between sexes. Sex differences were also observed in regional skin temperature (male thigh, 20.8±1.1 °C; female thigh, 16.7±1.1 °C, ∆ mean thigh skin temperature:−4.1 °C; d=3.72; male calf, 20.5±1.1 °C; female calf, 18.2±1 °C, ∆ mean calf skin temperature:−2.3±0.1 °C; d=3.61; male arm, 21.7±1 °C; female arm, 19±0.4 °C, ∆ mean arm skin temperature: −2.7±0.3 °C; d=3.54; P≤0.05). Mean arterial pressure was significantly different over time (P≤0.001) and between sexes (male 0 mins:94±10 mmHg; female 0 mins:85±7 mmHg; male 35 mins:88±7 mmHg; female 35 mins:80±6 mmHg; P≤0.05). Combined data set indicated a strong negative relationship between skin temperature and body fat percentage 35 min’ post WBC (r=−0.749, P≤0.001) and for core temperature and body mass index in males only (r=0.726, P≤0.05) immediately after WBC. There were no significant differences between sexes in any other variables (heart rate, tympanic and perceptual variables). We observed sex differences in mean skin and mean body temperature following exposure to whole body cryotherapy. In an attempt to optimise treatment, these differences should be taken into account if whole body cryotherapy is prescribed.     

Metabolic differences inBacillus stearothermophilus grown at 55°C and 37°C

Summary Bacillus stearothermophilus was adapted to grow at 55°C and 37°C in a complex medium with almost equivalent yields in cell mass. In both temperature ranges the maximum specific growth rates (μ_max) were identical. Cellular extracts of this bacterium showed remarkable differences in the activity levels of several enzymes, depending on the respective growth temperature. High activities of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase were observed in bacteria from thermophilic cultures (55°C) and the respiratory quotient exceeded 1.0. Under anaerobic conditions at 55°C μ_max was the same as in aerobic cultures. No alcohol dehydrogenase was detected in cells from mesophilic cultures (37°C), however, and the level of glyceraldehyde-3-phosphate dehydrogenase was also extremely low under mesophilic conditions. Succinate dehydrogenase and isocitrate dehydrogenase activity appeared to be higher in bacteria grown at 37°C; the resspiratory quotient was always lower than 1.0. At 37°C, acetoin formation was observed regularly, a fermentation product which was never detected in 55°C-cultures. Under anaerobic conditions at 37°C a very low growth rate was found. When adapted to grow at 37°C or 55°C,B. stearothermophilus is apparently able to use different catabolic systems.     

The energy balance in farm scale anaerobic digestion of crop residues at 11–37 °C

Crop residues can be used for biogas production in farm scale reactors. Use of a process temperature below mesophilic conditions reduces the need for heating as well as investment and operating costs, although it may also reduce the methane yield. In the present study the effect of temperature on net energy output was studied using sugar beet tops and straw as substrates for two pilot-scale reactors. Digestion was found to be stable down to 11 °C and optimal methane yield was obtained at 30 °C. The methane yield and process performance was studied at 15 °C and 30 °C as organic loading rates were increased. It was found that the highest net energy production would be achieved at 30 °C with a loading rate of 3.3 kg VS m⁻³ day⁻¹. Running a low-cost process at ambient temperatures would give a net energy output of 60% of that obtained at 30 °C.