Enzyme activity down to -100 degrees C

The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to -97 degrees C and -100 degrees C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below -100 degrees C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.     

Effects of methanol cryosolvents on the structural and catalytic properties of bovine trypsin

The effect of aqueous methanol cryosolvents on the catalytic and structural properties of bovine trypsin has been investigated. The low freezing points and low viscosities of methanol-based cryosolvents are desirable for a variety of cryoenzymological experiments. Increasing concentrations of methanol caused increases in the values of kcat and Km for the hydrolysis of N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at 0 degrees C and a small increase in Ki for inhibition by benzamidine. Based on product analysis the increase in kcat with increasing methanol concentration at pH* 4.0 and 6.5 can be completely accounted for by nucleophilic competition of methanol for the acyl enzyme intermediate. This observation indicates that deacylation is the rate-limiting step under these conditions. The effect of increasing methanol concentration on kcat/Km for the above ester substrate and N alpha-benzoyl-L-arginine p-nitroanilide was similar. Incubation experiments indicated that trypsin was quite stable in 70% methanol at 0 degrees C and below. The Arrhenius plot for the catalytic reaction in 70% methanol was linear over the 0 to -40 degrees C range, indicating no change in rate-determining step nor temperature-induced structural perturbation. No evidence for structural effects induced by methanol or temperature were detected by monitoring the intrinsic fluorescence and absorbance. We conclude that aqueous methanol cryosolvents are satisfactory for cryosolvent studies of trypsin.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Cryoenzymology of the hammerhead ribozyme.

The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and -33 degrees C. Eyring plots are linear down to -27 degrees C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.     

Studies on the cryopreservation of Dictyocaulus viviparus (Nematoda) third-stage larvae

Various cooling (0.1-5,100 degrees C min-1) and warming (20-6,800 degrees C min-1) rates, stepped cooling schedules and four cryoprotective additives (dimethyl sulphoxide, methanol, ethanediol and glycerol) were investigated in cryopreservation studies with Dictyocaulus viviparus third-stage larvae. Exsheathment with sodium hypochlorite was essential to achieve significant survival. With uninterrupted cooling, highest survival (30% normally motile) was achieved with rates of 10-70 degrees C min-1. Survival was higher (50-75%) using 1 degree C min-1 to -10 degrees C followed by plunging into liquid nitrogen. The optimum warming rate was 6,800 degrees C min-1. The use of cryoprotectants led to marginally lower survival while varying the suspending media had no significant effect on survival. X-irradiated, exsheathed third-stage larvae cryopreserved by the optimum protocol yielded 38.3 +/- 4.2% survival. Two calves each infected with 45,000 (15,000 viable) exsheathed, unirradiated, cryopreserved third-stage larvae harboured 494 worms (1.1% infectivity) and 355 worms (0.8%) at necropsy. Numbers of first-stage larvae in the faeces reached 420/g and 105/g respectively 27 days after infection.     

Ribonuclease structure and catalysis: effects of crystalline enzyme, alcohol cryosolvents, low temperatures, and product inhibition

In order to determine the necessary conditions to stabilize intermediates in ribonuclease A catalysis at subzero temperatures for structural studies, we have examined the suitability of alcohol-based cryosolvents. On the basis of thermal denaturation transition curves, the enzyme is in the native conformation in high concentrations of ethanol and methanol, provided the temperature is suitably low. The effects of methanol on the catalytic properties for the hydrolysis for mono- and dinucleotide substrates also are consistent with the absence of adverse effects of the cosolvent. Significant methanolysis occurs in the presence of methanol as cosolvent. The kinetics of 2',3'-CMP hydrolysis are complicated by severe competitive product inhibition, both in aqueous and in methanolic solvents, accounting for the previously observed effect of substrate concentration on the observed Km. Computer-aided analysis allowed the determination of the inhibition constant as a function of experimental parameters. The reaction of ribonuclease A with 2',3'-CMP was investigated at subzero temperatures. The turnover reaction could be made negligible at temperatures below -60 degrees C at pH 3-6 in 70% methanol and below -35 degrees C at pH 2.1. The rate of the catalytic reaction with crystalline enzyme was compared to that of enzyme in solution for both 2',3'-CMP and the dinucleotide CpC. The rates were 50- and 200-fold slower, respectively, in the crystal. These investigations allowed calculation of the necessary conditions for NMR and X-ray diffraction experiments on the trapped enzyme--substrate intermediate.     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.     

Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways.

Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.     

Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and -2 degrees C in different concentrations of methanol and sucrose

The hatching performance of common carp (Cyprinus carpio L.) embryos was examined after 12-72-h storage at 4 and -2 degrees C using different concentrations of sucrose (0.1, 0.25, 0.5 and 1.0 M or 3.42, 8.55, 17.10 and 34.2%), methanol (MeOH) (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 M or 1.6, 3.2, 4.8, 6.4, 8.0, 9.6 and 11.2%), or varying concentrations of methanol in 0.5 M (17.10%) sucrose. For sucrose, 0.5 M (17.10%) showed the maximum survival (41+/-1% (12 h) to 11+/-1.5% (72 h)) at 4 degrees C. No survival was observed at -2 degrees C with any concentration of sucrose. At both temperatures employed, hatching was higher with mixed combination of methanol (1.5 M or 4.8%) and 0.5 M (17.10%) sucrose (4 degrees C: 41+/-1.5% (12 h), 38+/-1.2% (72 h); -2 degrees C: 33+/-1.7% (12 h), 28+/-1.2% (72 h)) compared to methanol alone (4 degrees C: 38+/-1.5% (12 h), 35+/-2.5% (72 h); -2 degrees C: 31+/-2.5% (12 h), 25+/-2% (72 h)). The combination of 1.5 M (4.8%) methanol and 0.5 M (17.10%) sucrose produced the best results among all the concentrations tested at both temperatures.     

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70 degrees C) conditions in series with a methane reactor operated at mesophilic (35 degrees C), thermophilic (55 degrees C), and hyperthermophilic (65 degrees C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22-46% at 70 degrees C, while it was 21-29% at 55 degrees C. The average protein solubilization was 44% at 70 degrees C. The double bond fatty acid removal ratio at 70 degrees C was much higher than at 55 degrees C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70 degrees C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55 degrees C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70 degrees C and methanogenesis at 55 degrees C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.     

Optimization of procedures for counting viruses by flow cytometry

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

Thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate reduction in methanol and formate-fed UASB reactors

The feasibility of thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate-reducing processes was investigated in three lab-scale upflow anaerobic sludge bed (UASB) reactors fed with either methanol or formate as the sole substrates and inoculated with mesophilic granular sludge previously not exposed to high temperatures. Full methanol and formate degradation at temperatures up to, respectively, 70 and 75 degrees C, were achieved when operating UASB reactors fed with sulfate rich (COD/SO4(2-)=0.5) synthetic wastewater. Methane-producing archaea (MPA) outcompeted sulfate-reducing bacteria (SRB) in the formate-fed UASB reactor at all temperatures tested (65-75 degrees C). In contrast, SRB outcompeted MPA in methanol-fed UASB reactors at temperatures equal to or exceeding 65 degrees C, whereas strong competition between SRB and MPA was observed in these reactors at 55 degrees C. A short-term (5 days) temperature increase from 55 to 65 degrees C was an effective strategy to suppress methanogenesis in methanol-fed sulfidogenic UASB reactors operated at 55 degrees C. Methanol was found to be a suitable electron donor for sulfate-reducing processes at a maximal temperature of 70 degrees C, with sulfide as the sole mineralization product of methanol degradation at that temperature.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Field and in vitro assay of three methods for freezing ram semen

Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25 ml straws (final cellular concentration: 100 x 10(6) spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35 degrees C. The sample was cooled to 5 degrees C (-0.30 degrees C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5 degrees C for 2h. It was then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Method M2: The sample was diluted with a specific solution at 35 degrees C (final concentration of glycerol 3%), cooled to 5 degrees C (-0.20 degrees C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 degrees C (-0.25 degrees C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1h at 5 degrees C and then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values (P<0.05) for kinetic parameters: average path velocity (VAP) (81.3 and 85.2 microm/s), straight-line velocity (VSL) (72.8 and 77.3 microm/s) and linearity (LIN) (66.6 and 68.8%). Method M2 showed the lowest kinetic parameters of motility (VAP 74.4, VSL 67.3 and LIN 62.5) and the highest percentage of cells with damaged plasma membrane (53.8%). Method M1 gave the worst results in viability and acrosome status assessed using fluorescence probes (31.3%-dead cells with damaged acrosomes-versus 25.4% in M2 and 23.3% in M3). A field trial carried out on fertility showed a significantly higher percentage of pregnant or lambing ewes (P<0.05) with Method M3 (67.3% versus 51.1% for M1 and 58.8% for M2). We concluded that the use of a simple dilution medium (test-fructose-glycerol-egg yolk) with the addition of glycerol (to 2% at 35 degrees C and to 4% at 5 degrees C) in two steps together with a programmable biofreezer was a productive method for freezing ram semen.     

Evaluation of the effects of various culture conditions on Cr(VI) reduction by Shewanella oneidensis MR-1 in a novel high-throughput mini-bioreactor

The growth and Cr(VI) reduction by Shewanella oneidensis MR-1 was examined using a mini-bioreactor system that independently monitors and controls pH, dissolved oxygen (DO), and temperature for each of its 24, 10-mL reactors. Independent monitoring and control of each reactor in the cassette allows the exploration of a matrix of environmental conditions known to influence S. oneidensis chromium reduction. S. oneidensis MR-1 grew in minimal medium without amino acid or vitamin supplementation under aerobic conditions but required serine and glycine supplementation under anaerobic conditions. Growth was inhibited by DO concentrations >80%. Lactate transformation to acetate was enhanced by low concentration of DO during the logarithmic growth phase. Between 11 and 35 degrees C, the growth rate obeyed the Arrhenius reaction rate-temperature relationship, with a maximum growth rate occurring at 35 degrees C. S. oneidensis MR-1 was able to grow over a wide range of pH (6-9). At neutral pH and temperatures ranging from 30 to 35 degrees C, S. oneidensis MR-1 reduced 100 microM Cr(VI) to Cr(III) within 20 min in the exponential growth phase, and the growth rate was not affected by the addition of chromate; it reduced chromate even faster at temperatures between 35 and 39 degrees C. At low temperatures (<25 degrees C), acidic (pH < 6.5), or alkaline (pH > 8.5) conditions, 100 microM Cr(VI) strongly inhibited growth and chromate reduction. The mini-bioreactor system enabled the rapid determination of these parameters reproducibly and easily by performing very few experiments. Besides its use for examining parameters of interest to environmental remediation, the device will also allow one to quickly assess parameters for optimal production of recombinant proteins or secondary metabolites.     

Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures.

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.     

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.     

Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.