Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida     

Effects of supraoptimal temperatures on the myxomycete Phyrasum polycephalum

Summary The effects of heat shock on the protoplasmic streaming, respiration and leakage of plasmodial constituents absorbing at 260 nm (products of nucleic acid metabolism), 280 nm (products of protein metabolism), and 415 nm (the yellow pigments of the plasmodia) were studied in plasmodia of the myxomycete Physarum polycephalum.Plasmodia grown on a semidefined medium displayed a lower primary thermoresistance of the protoplasmic streaming, and had a lower Q ₁₀ coefficient of the heat injury of this function compared to those grown on rolled oats. They are able to repair thermal injuries during heating. The primary thermoresistance of the protoplasmic streaming is not changed during the mitotic cycle.A 10 min heating at 32°C lowers the rate of protoplasmic streaming and results in a leakage of plasmodial pigments. After a 10 min exposure at 37–38°C the protoplasmic streaming is stopped, the respiration reduced, and products of nucleic acid metabolism are detectable in the heating fluid. Leakage of protein metabolits was observed after 10 min heatshocks at 41°C. A heating of the plasmodia to 47–50°C caused the highest level of leaked substances and the complete cessation of respiration.In contrast to higher plants, the respiration and leakage of the pigments are thermolabile indicators of the condition of Physarum polycephalum plasmodia.     

Interactions between ethylene-bis-nitrourethane and Gibberellic acid during the germination of lettuce seed

Summary Exposing lettuce seed (variety Grand Rapids) to temperatures from 30°–42° C inhibits subsequent germination at 25° C. This inhibition can be overcome by the addition of gibberellic acid (GA) either during or after the heat treatment. Ethylene-bis-nitrourethane (EBNU) and ethylene dinitramine (EDNA), although without effect when applied alone, have been shown to increase the activity of GA when present in admixture during the heat treatment. The compounds act synergistically only when the seeds are kept at 30° and 35° C for 72 hours or longer.EBNU and EDNA do not increase the effectiveness of GA in breaking natural or heat induced dormancy in lettuce seeds.     

Removal of RNA by heat treatment

Summary Eight bacterial strains were subjected to a discontinuous heat shock treatment aimed at causing a degradation of RNA. The treatment involved a 10 s to 10 min exposure to 65°C and then an incubation period of up to 3 h at 50°C. At intervals the cells were analyzed for RNA, DNA and protein. Whereas the contents of protein and DNA were not affected, RNA was degraded. An almost complete degradation of RNA occurred inAlcaligenes eutrophus H 16 — PHB^–4 andEscherichia coli K 12; only about 50% of the cellular RNA were degraded inPseudomonas putida andP.flava GA; inCorynebacterium autotrophicum 7 C,Nocardia opaca 1 b and coryneform strains 11 X and 30.1 b RNA degradation occurred only to a small extent.A continuous flow system for the treatment of cell suspensions by heat shock followed by incubation at an elevated temperature was developed. The results confirmed those obtained by batch-wise heat treatment.     

Alteration of Ion Channels in the Plasmalemma of Nitella flexilisCells during Long-Term Hyperthermia

The conventional microelectrode technique was applied to study changes in conductance and activation characteristics of potassium and chloride channels in the plasmalemma of characean alga Nitella flexilis(L.) Agardz. during long-term heat treatment. Measurements were conducted at 18–20°C after preliminary exposure of cells to 33°C for 1–25 days. The conductance of outward- and inward-rectifying potassium channels, as well as the currents of excitable chloride channels, decreased after 2–3 days of heat treatment. By the 15th–17th days, the conductance of potassium channels was reduced by a factor of 3–5, whereas the peak values of the chloride current, associated with the action potential, was reduced by a factor of 8–10. These heat-induced changes were long lasting: the restoration of the initial parameters of transport systems after transferring cells to chilling or room temperature occurred within several days. Moreover, the recovery at chilling temperatures (8–10°C) proceeded nearly two times longer than at room temperature. Prolonged hyperthermia accelerated activation and deactivation of outward-rectifying potassium channels and caused the shift of their activation curve towards positive potentials by 35–40 mV. Analysis of current–voltage relations showed that the inward current in inward- and outward-rectifying potassium channels was reduced to a greater extent than the outward current. At the same time, both inward and outward currents of chloride channels were reduced to an equal extent. It is assumed that the changes observed are involved in thermal adaptation and account for the decrease in the intracellular concentrations of potassium and other cations and anions, which represents a nonspecific response of plant cells to stress.     

Induced autolysis ofSaccharomyces cerevisiae: Morphological effects,rheological effects,and dynamics of accumulation of extracellular hydrolysis products

Changes in the internal structure ofSaccharomyces cerevisiae cells and accumulation of proteins and nucleic components in extracellular fluid as decomposition products were studied in the 40–75°C temperature range under the effect of various membranotrophic additives. Autolysis was shown to be a two-step process: The first step consists of the restructuring of cell endostructures and the activation of lytic enzymes, which is accompanied by reduction of cell volume and system viscosity; the second step directly follows the first step and consists of hydrolysis of cell components and release of hydrolysis products into extracellular space. Duration of the first step depends on the temperature and the plasmolyzer. Hydrophilic additives (ethanol, ethyl acetate) were most effective during the first step at 60–65°C, whereas hydrophobic additives (lecithin, lauric acid) were most effective at 55°C. In the second step, the temperature optimum of protease activity in the control (without additives) was 60°C, that of nuclease activity was 70°C. Additives reduce the temperature optimum of endoenzymatic activity. Cell morphology was studied at various stages of autolysis by electron and phase-contrast microscopy.     

Cheese whey: an alternative growth and protective medium for<Emphasis Type="Italic"> Rhizobium loti</Emphasis> cells

Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at –20 °C and –80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.In memoriam of Sylvio Cortina Vicepresident of Fundación COREPRO     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

Methanotrophs of the Psychrophilic Microbial Community of the Russian Arctic Tundra

In tundra, at a low temperature, there exists a slowly developing methanotrophic community. Methane-oxidizing bacteria are associated with plants growing at high humidity, such as sedge and sphagnum; no methanotrophs were found in polytrichous and aulacomnious mosses and lichens, typical of more arid areas. The methanotrophic bacterial community inhabits definite soil horizons, from moss dust to peat formed from it. The potential ability of the methanotrophic community to oxidize methane at 5°C enhances with the depth of the soil profile in spite of the decreasing soil temperature. The methanotrophic community was found to gradually adapt to various temperatures due to the presence of different methane-oxidizing bacteria in its composition. Depending on the temperature and pH, different methanotrophs occupy different econiches. Within a temperature range from 5 to 15°C, three morphologically distinct groups of methanotrophs could be distinguished. At pH 5–7 and 5–15°C, forms morphologically similar to Methylobacter psychrophilus predominated, whereas at the acidic pH 4–6 and 10–15°C, bipolar cells typical of Methylocella palustris were mostly found. The third group of methanotrophic bacteria growing at pH 5–7 and 5–10°C was represented by a novel methanotroph whose large coccoid cells had a thick mucous capsule.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Growth and biochemical composition with emphasis on the fatty acids of Tetraselmis sp.

Summary The influence of temperature (15–32°C) and the ratio of nitrogen to phosphorus (N/P) in the culture medium (0.5–80) on the growth kinetics and protein, chlorophyll, lipid and fatty acid content of the marine microalga Tetraselmis sp. have been studied. Below an N/P of 20, growth was determined by N limitation and above 20 by P limitation. Protein increased with a rise in N content at any test temperature. The chlorophyll content increased with temperature, with maximum values at 25°C. The lipid content decreased with increasing N/P ratio above 20°C. The polyunsaturated fatty acid content tends to be inversely proportional to the growth rate within the N/P range 20–80. The quotient of the n ₃ and n ₆ polyunsaturated-fatty-acid fractions, an indicator of the nutritive value of microalgae, was found to be within the range 2–3. These values were obtained either between 25 and 28°C independent of the N/P ratio used at 20°C for N/P ratios higher than 40.0. Offsprint requests to: Emilio Molina     

Delineation of human chromosome contour by heat treatment and hematoxylin staining

An unusual form of staining was observed as a result of heat treatment: chromosome contour became easily recognizable. Chromosome contour is optimally delineated by heat treatment of metaphase preparations in 0.06 M phosphate buffer, pH 6.8–7.0 at 95° C for 10 min, and subsequent staining with Heidenhain's hematoxylin. This result is obtained by a process similar to that in which Giemsa has been reported to stain particular foci considered to represent selective staining of constitutive heterochromatin. Mordanting with 3% ferrous ammonium sulfate produced maximum staining. However, hematoxylin (Harris) also stained the periphery of chromosomes. A transitional temperature existed between 70° C and 80° C in which contour became evident. When metaphase preparations were heated at 90–95° C, contour occurred on some chromosomes within 3 min and was demonstrated in 100% of the chromosomes analysed after 10 min of heat treatment.Supported in part by a grant to C. Romero-Sierra from the Defence Research Board of Canada (DRB 9350-24) and to E. A. MacKinnon from the Medical Research Council of Canada (MA-4135).     

Effects of high ambient temperatures on the metabolism of West African dwarf goats. II

32 West African dwarf goats were exposed in respiration chambers to temperature treatments of 20, 25, 30, 35, 35, 35, 30, 25, 20°C. Each treatment lasted three days. 16 goats were kept in individual pens (I); the others in two group pens of eight animals each (G). During each treatment, heat production and activity were recorded continuously over 48 hours. In addition, feed and water intake, rectal temperature, skin temperature and respiratory rate were measured during each treatment.Compared to 20°C, at 35°C rectal temperature increased from 39.0°C to 39.9°C, respiratory rate from 30 to 260 times. min^–1 and skin temperature from 37.1°C to 39.5°C. Hay intake decreased by 40%; concentrates (30 g. kg^–0.75. d^–1) were always completely consumed. Heat production was higher for the G animals at 20°C and higher for the I animals at 35°C. These differences in heat production between the two groups were reflected in differences in rectal and skin temperature and in respiratory rate but only very slightly in differences in hay intake.Tissue insulation was 0.014 K. m². W^–1 at 30°C and 35°C and 0.022 K. m². W^–1 at 20°C.It is concluded that the reactions of these dwarf goats to high ambient temperatures are not different in principle from those of other domestic ruminants and that they do not exhibit a specific suitability or unsuitability for ambient temperatures as prevailing in West Africa.     

Cadmium tolerance in tobacco cell culture and its relevance to temperature stress

Growth of unselected tobacco (Nicotiana tabacum W38) cell suspension cultures was reduced by 50–200 M cadmium (Cd) in the culture medium and cells were killed by 400 M Cd. Tolerance to Cd was increased either by using rapidly growing cells or by culturing cells at higher densities. Cell lines tolerant to 2 mM Cd were established by progressively elevating levels of Cd in the culture medium. The Cd tolerance was not due to differences in uptake between unselected and Cd-tolerant cell lines, and the tolerance to Cd was not lost during long term culture in the absence of Cd. Cd-tolerant cells also showed higher tolerance to heat shock (37.5°C, 2–8 hours) and cold treatments (4°C, 1–7 days) than the unselected cells.     

Phenotypic changes in the chemistry of Aspergillus nidulans: Influence of culture conditions on mycelial composition

A quantitative study was made of macromolecular (nucleic acids, protein), carbohydrate and mineral (magnesium, potassium and phosphorus) components of Aspergillus nidulans in glucose limited chemostat cultures, under varying conditions of dilution rate, temperature, pH and NaCl concentration.The overall mineral content showed greatest variation in response to changes in culture salinity, which also affected the mycelial carbohydrate content. Concomitant and opposite changes in the conent of cations and carbohydrates under conditions of increasing salinity may be interpreted in terms of mycelial osmoregulation. Slight variations in DNA content but gross fluctuations in the level of RNA were noted under the different cultural conditions examined. Co-ordinate changes in RNA and Mg²⁺ contents were evident only under certain conditions: dilution rate from 0.05–0.07 h^-1 or temperature from 22–30° C. The constant molar stoichiometry between RNA and Mg²⁺ characteristic of unicellular microorganisms was not a feature of fungal growth. The protein content was most affected by shifts of temperature and reached minimal values at 25 and 50° C.The growth environment had a marked influence on the protein synthesising activity of RNA, which increased eightfold as the dilution rate was increased from 0.02–0.175 h^-1, doubled within the temperature range 20–30° C and fell by 50% between 40 and 50° C. These observations are discussed in the context of the constant ribosomal efficiency in protein synthesis hypothesis.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

Sensible and latent heat loss from the body surface of Holstein cows in a tropical environment

The general principles of the mechanisms of heat transfer are well known, but knowledge of the transition between evaporative and non-evaporative heat loss by Holstein cows in field conditions must be improved, especially for low-latitude environments. With this aim 15 Holstein cows managed in open pasture were observed in a tropical region. The latent heat loss from the body surface of the animals was measured by means of a ventilated capsule, while convective heat transfer was estimated by the theory of convection from a horizontal cylinder and by the long-wave radiation exchange based on the Stefan–Boltzmann law. When the air temperature was between 10 and 36°C the sensible heat transfer varied from 160 to –30 W m^–2, while the latent heat loss by cutaneous evaporation increased from 30 to 350 W m^–2. Heat loss by cutaneous evaporation accounted for 20–30% of the total heat loss when air temperatures ranged from 10 to 20°C. At air temperatures >30°C cutaneous evaporation becomes the main avenue of heat loss, accounting for approximately 85% of the total heat loss, while the rest is lost by respiratory evaporation.Part of first authors doctoral thesis     

The activity of two heat shock loci of Drosophila hydei in tissue culture cells and salivary gland cells as analyzed by in situ hybridization of complementary DNA

Complementary DNA was made to poly A⁺ nuclear or polysomal RNA isolated from heat shock tissue culture cells of Drosophila hydei. A number of loci other than the four major heat shock loci are labelled after in situ hybridization of these cDNA preparations, while solution hybridization indicated that only about 10% of the cDNA was specific for heat shocked cells. Removal of the fraction of cDNA which could react with 25° C RNA and subsequent in situ hybridization of heat shock specific cDNA indicated that locus 4–81 B, a major salivary gland heat shock locus, is also active at 25° C in tissue culture cells, while locus 4–85 B is specifically activated by heat shock in tissue culture cells. This latter locus is not seen to be clearly puffed in salivary glands, but was shown to be active in that tissue both by direct autoradiography of salivary gland chromosomes after 3H-uridine labeling and by hybridization of cDNA to chromosomal RNA.     

High-speed autoradiography of 3H-thymidine-labelled nuclei

Summary High-speed autoradiography with stripping film of ³H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20°C, (b) exposure of cell preparations and blanks for various times at either –70°C or +20°C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time 1 h at either temperature and relative humidity 20–30%. A linear relationship between autoradiographic efficiency and exposure time of 1–7 h was found at either temperature, although the efficiency of autoradiographs exposed at –70°C was by approximately 30% higher than that of autoradiographs exposed at +20°C. Background values of autoradiographs dried with a fan and exposed for 1/4–7h at either –70°C or +20°C were 0.6–0.8 grain/100 m². Theoretical calculations and experimental data showed that high-speed autoradiographs are 30–50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.This investigation was supported in part by MR II.1 grant. The technical assistance of Mrs. S. Bie is gratefully acknowledged