Survival kinetics of lactic acid starter cultures during and after freeze drying

Survival kinetics of lactic acid starter cultures were modeled considering the microorganism and external medium interfacial area as the critical factors determining the resistance of the microorganisms to freeze-drying. Surviving fraction of the microorganisms increased with the increasing biomass concentration during freeze-drying, and this is attributed to the mutual shielding effect of the microorganisms against the severe conditions of the external medium. Survival of the microorganisms over the storage period after freeze drying was enhanced by the presence of dead microorganisms which reduce the interfacial area between the live cells and the external medium. Streptococcus thermophilus was found to be more resistant to freeze-drying conditions than Lactobacillus bulgaricus. Storage under vacuum or nitrogen was superior to storage under air. Poor survival rates under air was attributed to oxygen diffusion into the dry cells through the interfacial area.     

The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight

The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments.     

Anhydrobiosis: Inside yeast cells

Under natural conditions yeast cells as well as other microorganisms are regularly subjected to the influence of severe drought, which leads to their serious dehydration. The dry seasons are then changed by rains and there is a restoration of normal water potential inside the cells. To survive such seasonal changes a lot of vegetative microbial cells, which belong to various genera and species, may be able to enter into a state of anhydrobiosis, in which their metabolism is temporarily and reversibly suspended or delayed. This evolutionarily developed adaptation to extreme conditions of the environment is widely used for practical goals – for conservation of microorganisms in collections, for maintenance and long storage of different important strain-producers and for other various biotechnological purposes. This current review presents the most important data obtained mainly in the studies of the structural and functional changes in yeast cells during dehydration. It describes the changes of the main organelles of eukaryotic cells and their role in cell survival in a dry state. The review provides information regarding the role of water in the structure and functions of biological macromolecules and membranes. Some important intracellular protective reactions of eukaryotic organisms, which were revealed in these studies and may have more general importance, are also discussed. The results of the studies of yeast anhydrobiosis summarized in the review show the possibilities of improving the conservation and long-term storage of various microorganisms and of increasing the quality of industrially produced dry microbial preparations.     

Cosmic vacuum prevents radiopanspermia

The paper describes the new physical mechanism for the explanation of the irreversible damage of vegetative cells and spores of microorganisms (m/o) under space thermovacuum conditions (tvc) (vacuum+high temperatures), developed by the authors based on the published experimental data of various authors. The suggestion was made that this mechanism could inactivate most vegetative cells and spores of the m/o at the initial stage of their spontaneous migration into the cosmos from the platforms where life has originated and evolved. The authors believe the Earth and Earth-like planets to be such platforms. Such a mechanism could restrict the application of the radiopanspermia hypothesis to the explanation of the origin of life on the Earth.     

Can microorganisms survive high-temperature heating during the interplanetary transfer by meteorites?

The question about the tolerance of microorganisms to high-temperature heating at the moments when fragments are launched from the surface rocks of cosmic objects and enter an atmosphere is among the major aspects of the problem of life transfer through cosmic space. The heating process in the course of such events was modeled here, and the survival of microorganisms was revealed by the example of two E.coli K12 mutant strains. Samples of these cultures in desiccated state were exposed to short heating pulses. Heating took place under extremely low pressure. It was experimentally showed that bacteria survive at a temperature up to 250°C with duration ～10 s under vacuum conditions, meanwhile the same heating under atmospheric pressure leads to complete sterilization.     

Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity

Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90). Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s. Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions. This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses. As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S. cerevisiae and B. subtilis. However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells. Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.     

Can microorganisms survive upon high-temperature heating during the interplanetary transfer by meteorites?

One of the most important aspects of the problem of life transfer in the cosmic space is the resistance of microorganisms to high-temperature heating during the launch and entry into the atmosphere. The high-temperature limits of the survival of microorganisms were studied under conditions modeling the laungh from the Mars and the landing on the Earth. Two strain of E. coli K12 exposed to short heating pulse were studied in order to tind out if they could resist high temperature while being in the desiccated state. The procedure was performed in vacuum. It was found that a fraction of bacteria survive heating pulses up to 250 degrees C in vacuum, while similar heating at normal atmospheric pressure leads to the total sterilization of samples.     

The protective role of pigments against UV rays in fungi isolated from the mesosphere

Five among six species of microorganisms isolated from the mesosphere contained pigments which made them more resistant to the action of UV as compared to pigmentless microorganisms in the atmosphere of Earth. UV irradiation in the atmosphere is supposed to select resistant pigmented forms, so that they predominate in the mesosphere. To confirm this assumption, mutants of Aspergillus niger, Penicillium notatum and Circinella muscae were sported by irradiating them four times and then subjecting to stepwise selection. These mutants either synthesized pigments at a very low rate or did not produce them at all. No significant differences were found by studying the biomass, mycelium and sporeforming organs of the parent cultures and their mutants. However, their resistance to UV was not the same. Addition of the pigment apsergillin, isolated from the conidia of Aspergillus niger, to a suspension of the pigmentless (mutant) conidia of Penicillium notatum, the spores of Circinella muscae, and the vegetative cells of Micrococcus albus, before their irradiation with UV, considerably increased their resistance to this factor.     

Survival of bacteria in the Artificial Mars unit

The survival of bacteria was studied in the "Artificial Mars" apparatus reproducing a complex of physical extreme factors. Bacteria isolated from microbiocenoses subjected to the action of chemical extreme factors (hydrogen peroxide, catalytically active minerals) were shown to be most resistant among soil heterotrophic bacteria. Cells in the resting state caused by dehydration survived better than vegetative cells. It has been concluded that microorganisms quite different in their physiological requirements and ecological properties can exist in the habitats extreme for life conditions.     

Resin straw as an alternative system to securely store frozen microorganisms

Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.     

Survival of sulfate-reducing bacteria after oxygen stress, and growth in sulfate-free oxygen-sulfide gradients

Abstract The survival after oxygen stress was studied with eight species of sulfate-reducing bacteria. In the absence of sulfide all species tolerated 6 min of aeration without loss of viability. Even after 3 h of aeration the viability of four species ( Desulfovibrio vulgaris, D. desulfuricans, D. salexigens and Desulfobacter postgatei ) was not impaired. Four other species were sensitive to 3 h of aeration: the surviving fractions of Desulfotomaculum ruminis, D. nigrificans and Desulfococcus multivorans were about 1%, that of Desulfotomaculum orientis about 0.01%. Formation of spores resulted in oxygen resistance of D. orientis . Reducing agents did not protect the vegetative cells of this strain against oxygen toxicity. In contrast, sulfhydryl group-containing agents increased the oxygen sensitivity considerably.
Growth of sulfate- and sulfur-reducing bacteria in oxygen-sulfide gradients in agar tubes was studied. In the gradients these strictly anaerobic bacteria revealed oxygen-dependent growth in sulfate- and sulfur-free medium. Three sulfate-reducing bacteria that could not use thiosulfate or sulfur as electron acceptor failed to grow in oxygen-sulfide gradients. Obviously, not directly molecular oxygen, but oxidation products of sulfide, such as thiosulfate or sulfur, were used as electron acceptors and were continuously regenerated in a cycling process from sulfide by autoxidation. The conceivable ecological significance of a short sulfur cycle driven by autoxidation of sulfide is discussed.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Catabolite regulation of antibiotic biosynthesis

Conclusion In view of the possible mediation of carbon catabolite repression of antibiotic biosynthesis by phosphorylated substances, the concept of the role of phosphorus in the regulation of secondary metabolism should be re-evaluated. Many conclusions are based on an analogy with the effect of phosphorus in animal or plant cells (for review cf. Martin 1977). However, in contrast with plant and animal physiology, the production physiology of actinomycetes, typical soil microorganisms, was studied under nonphysiological conditions of nutrient over-supply in a submerged culture. This is analogous e.g. to space biology which studies the physiology of organisms in the state of weightlessness. Both disciplines have an extremely high significance but the elucidation of basic biological regularities is to be carried out back on Earth. In conclusion we may state that a critical evaluation of our contemporary knowledge seem to support the hypothesis that the antibiotic biosynthesis in actinomycetes, as well as the spore formation in bacilli — both regulated by the mechanism of catabolite repression — has its ecological significance, i.e. makes it possible for the organism to survive under conditions when vegetative growth is limited. For this reason this aotivity was preserved in the course of evolution as a protective mechanism of microbial populations and remained a part of the genome of many species.     

Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.     

Microbiological coal desulphurization

Sulphur compounds present in coal impose severe limitations on its utilization since sulphur-containing gases emitted into the atmosphere upon direct combustion of coal cause serious environmental pollution problems. Removal of sulphur compounds from coal by microbial action has many advantages over physical and chemical desulphurization methods. The potential use of various microorganisms for the removal of sulphur compounds from coal is presented. Environmental conditions and major process variables affecting the process performance are identified and their possible effects are discussed. Various process schemes for microbial desulphurization (MDS) of coal are suggested. It is concluded that microbial methods have a high potential in removing sulphur compounds from coal. However, more research and development work is needed in this field to overcome present technological problems.     

Responsibility of Hydrogen Peroxide for the Lethality of Resting Escherichia coli B Cells Exposed to Alternating Current in Phosphate Buffer Solution

Surviving fractions of Escherichia coli B exposed to an alternating current (AC) of 50 Hz in a phosphate buffer solution of pH 7.0 at 29°C were closely related to the amount of H₂O₂ formed in cell suspensions. At a definite current density, the amount of H₂O₂ in the suspensions or in buffer solution without cells increased with increasing AC-exposure time under aerobic conditions. On the other hand, the formation of H₂O₂ on AC-exposure was not detected under anaerobic conditions. It was considered that H₂O₂ was formed on the surface of carbon electrodes by AC-electrolytic reduction of dissolved oxygen. The amount of H₂O₂ formed decreased with increasing concentration of cells suspended or of catalase added to the suspension. When the formation of H₂O₂ was significantly suppressed, surviving fractions of cells exposed to AC remained almost unchanged. Growth conditions, modifying the intracellular level of catalase of E. coli, affected the sensitivity of cells to AC-exposure.     

Development of lyophilization procedure ensuring survival of bifidobacteria and preservation of their probiotic potential upon long-term storage

Survival of bifidobacteria and preservation of their morphological characteristics after 12-month storage of lyophilized cells was studied for the strains of Bifidobacterium bifidum and B. animalis isolated and maintained in the microbial collection of the Department of Microbiology, Moscow State University. A combined approach to pre-lyophilization treatment of microorganisms and subsequent storage was developed in order to improve cell survival. Compared to the standard cryoprotector concentrations, sucrose and glucose (5% and higher) in skim milk, as well as freezing at?70°C with subsequent storage at the same temperature resulted in improved survival of bifidobacteria. Under such conditions, the number of viable cells (CFU) after 12 months of storage was two to three orders of magnitude higher than in the case of the standard lyophilization procedure. Investigation of dynamics of resistance of reactivated clones to such gastrointestinal stress factors as gastric juice and bile acids revealed preservation of these properties at all storage modes. However, since the number of surviving cells decreased during storage according to the standard procedure, the number of stress-affected cells was correspondingly lower. Reactivated cultures exhibited high resistance to oxygen, with survival decreasing to 35% of the initial level.     

The vegetation adventivisation through perspective of modern ecological ideas

Results of study of vegetation adventivisation (increase in proportion of invasive species) correspond to the theory of present ecology that denies general universal laws. Diverse features of invasive species play different role under various ecological conditions and at various time and space scale. The invasibility of communities under various conditions is determined by combination of different biotic and abiotic factors though it is obvious that most of invasive species are characterized with the high seed production, well developed vegetative propagation, windblown pollination, high plasticity and effective use of resources, low consumption by herbivores. The definition of an "ideal invasive species" or an "ideal invasible community" is impossible. The regularities of vegetation adventivisation can be observed clearly only at very large scale.