响应面法优化火麻仁油微胶囊喷雾干燥工艺的研究

为了制备包埋率高、稳定性好的火麻仁油微胶囊,拓展其在食品领域的应用范围,以火麻仁油为芯材、单双脂肪酸甘油酯为乳化剂、酪蛋白酸钠为壁材、固体玉米糖浆为填充剂、柠檬酸钠为缓冲盐、抗坏血酸棕榈酸钠为抗氧化剂,通过喷雾干燥法制备60%载油率的火麻仁油微胶囊,以微胶囊包埋率为响应值,在单因素实验的基础上,以干物浓度、进风温度、出风温度为实验因素,采用Box-Behnken响应面分析法进行优化。随后通过扫描电镜观察火麻仁油微胶囊表面形态结构,以确定包埋效果。并利用油脂氧化分析仪检测火麻仁油微胶囊的氧化稳定性。研究确定微胶囊的最佳工艺条件为：干物浓度42%、进风温度168 ℃、出风温度74 ℃,在此条件下制备得到的火麻仁油微胶囊包埋率可达92.15%。通过扫描电镜观察到火麻仁油微胶囊表面圆滑无裂痕,表明火麻仁油微胶囊包埋效果比较理想。经油脂氧化分析仪测定,与对照组（火麻仁油）相比,试验组（火麻仁油微胶囊）的氧化诱导期时间较长,能够达到30 h以上,说明通过对火麻仁油进行微胶囊包埋可以较大程度地提高油脂的稳定性。研究结果为火麻仁油在食品工业领域的开发和应用提供了理论支持。     

On the similarity in the effects of different cryopreservation conditions on the growth and development of plants

The morphological characteristics of 35 wild plant species were studied after freezing of seeds under the conditions of deep, fast, and programmed freezing (-196 degrees C) and non-deep freezing (-10 degrees C). The seeds were stored frozen for a month. The seeds of all the species were characterized by a low humidity. The field and laboratory seed germination capacity, leaf growth, the quantity and length of shoots, the quantity of generative organs, and the variability of these characteristics were studied. It was shown that the direction of changes under different cooling conditions was the same except for the laboratory germination capacity of some species. The direction was determined by the species features rather than cooling conditions.     

Hydrogeological controls on microbial activity and habitability in the Precambrian continental crust

Earth's deep continental subsurface is a prime setting to study the limits of life's relationship with environmental conditions and habitability. In Precambrian crystalline rocks worldwide, deep ancient groundwaters in fracture networks are typically oligotrophic, highly saline, and locally inhabited by low-biomass communities in which chemolithotrophic microorganisms may dominate. Periodic opening of new fractures can lead to penetration of surface water and/or migration of fracture fluids, both of which may trigger changes in subsurface microbial composition and activity. These hydrogeological processes and their impacts on subsurface communities may play a significant role in global cycles of key elements in the crust. However, to date, considerable uncertainty remains on how subsurface microbial communities may respond to these changes in hydrogeochemical conditions. To address this uncertainty, the biogeochemistry of Thompson mine (Manitoba, Canada) was investigated. Compositional and isotopic analyses of fracture waters collected here at ~1 km below land surface revealed different extents of mixing between subsurface brine and (paleo)meteoric waters. To investigate the effects this mixing may have had on microbial communities, the Most Probable Number technique was applied to test community response for a total of 13 different metabolisms. The results showed that all fracture waters were dominated by viable heterotrophic microorganisms which can utilize organic materials associated with aerobic/facultative anaerobic processes, sulfate reduction, or fermentation. Where mixing between subsurface brines and (paleo)meteoric waters occurs, the communities demonstrate higher cell densities and increased viable functional potentials, compared to the most saline sample. This study therefore highlights the connection between hydrogeologic heterogeneity and the heterogeneity of subsurface ecosystems in the crystalline rocks, and suggests that hydrogeology can have a considerable impact on the scope and scale of subsurface microbial communities on Earth and potentially beyond.     

Response of microbial communities of Lake Baikal to extreme temperatures

The survival rate, metabolic activity, and ability for growth of microbial communities of Lake Baikal have been first studied after exposure to extremely low temperatures (freeze-thawing) for different lengths of time. It has been shown that short-term freezing (1–3 days) inhibits the growth and activity of microbial communities. The quantity of microorganisms increased after 7-and 15-day freezing. In the periods of maximums, the total number of microorganisms in the test samples was twice as high as in the control. It was established that after more prolonged freezing the microorganisms required more time after thawing to adapt to new conditions. In the variants with 7-and 15-day freezing, the activities of defrosted microbial communities were three or more times higher than in the control. The survival rate and activity of Baikal microorganisms after freeze-thawing confirms the fact that the Baikal microbial communities are highly resistant to this type of stress impact.     

Response of microbial communities of Lake Baikal to extreme temperatures

The survival rate, metabolic activity, and ability for growth of microbial communities of Lake Baikal after exposure to extremely low temperatures (freeze-thawing) for different lengths of time have been first studied. It has been shown that short-term freezing (1-3 days) inhibits the growth and activity of microbial communities. The quantity of microorganisms increased after 7- and 15-day freezing. In the periods of maximums, the total number of microorganisms in the test samples was twice as high as in the control. It was established that after more prolonged freezing the microorganisms required more time after thawing to adapt to new conditions. In the variants with 7- and 15-day freezing, the activities of defrosted microbial communities were three or more times higher than in the control. The survival rate and activity of Baikal microorganisms after freeze-thawing confirms the fact that the Baikal microbial communities are highly resistant to this type of stress impact.     

Impact of ploughing on soil seed bank dynamics in temporary pools

We examined the impact of ploughing on soil seed banks of plant communities living in temporary marshes located in agricultural fields. The quantity, quality and vertical distribution of seeds were quantified under ploughed or unploughed treatment at community level. We also focussed on a typical semi-aquatic species, Damasonium alisma, to investigate the impact of ploughing at population level. We used two complementary techniques to study seed banks: hand sorting and seedling emergence. We found that species richness of seeds, seed abundance and germination ability were strongly affected by ploughing at community level. Concerning D. alisma, most of the seeds (56%) were stored in the two deepest soil layers among the four considered in ploughed pools. Moreover, the germination rate was higher for buried seeds (84%) than for seeds collected at the surface (33.6%). These patterns were almost inverted in unploughed pools. Our results agree with the temporal storage effect generally suggested to describe the seed bank property of plant communities. But in addition, we showed that ploughing induces a spatial storage effect in accumulating species and individuals in the seed banks that favourably influence community dynamics. We conclude that, in contrast to what is usually thought, ploughing disturbance can be of benefit for such ephemeral wetland vegetation.     

Immunolabeling of grapevine flavescence dorée MLO in salivary glands of Euscelidius variegatus: a light and electron microscopy study

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.     

Microbial communities of frozen soils of the Tuimaada Valley in Central Sakha

This study determined the characteristic features of microbial communities inhabiting frozen soils of Central Sakha. All groups of microorganisms were present in high numbers comparable with the microbial densities in steppe soils of Transbaikalia; their distribution along soil profiles followed a specific pattern with no decrease in abundance with depth. In summer, the dominant groups of the microbial pool were actinomycetes and oligonitrophilic bacteria, while in autumn it was heterotrophic bacteria. A typical feature of the frozen soils of Central Sakha was the gradual penetration of microorganisms into deeper soil horizons during the vegetation season and their accumulation in the suprapermafrost horizon by autumn.     

Associations between the White Sea invertebrates and oxygen-evolving phototrophic microorganisms

Eleven species of White Sea invertebrates (sponges, actinians, hydroids, polychaetes, and nudibranch mollusks) were tested for the presence of associated oxygen-evolving phototrophic microorganisms (OPM) (microalgae and cyanobacteria). Two main approaches were applied: (a) light and electron microscopy of intact samples and fixed preparations of invertebrates, and (b) isolation of microorganisms from samples of invertebrates after mild surface sterilization. The obtained results lead to conclusions on the formation of multicomponent associations by White Sea invertebrates and OPM based on the following data: (1) isolation of 27 cultures of OPM from eight species of invertebrates (sponges, hydroids, polychaete trochophore larva), (2) specificity of association between epibiontic communities of microorganisms and macroorganisms within the same biotope, and (3) spatial integration of micro- and macropartners resulting in the formation of morphological structures within the interorganismic contact zones.     

Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding

The analysis of ancient DNA (aDNA) provides archaeologists and anthropologists with innovative, scientific and accurate data to study and understand the past. In this work, ancient seeds, found in the "Mora Cavorso" archaeological site (Latium, Central Italy), were analyzed to increase information about Italian Neolithic populations (plant use, agriculture, diet, trades, customs and ecology). We performed morphological and genetic techniques to identify fossil botanical species. In particular, this study also suggests and emphasizes the use of DNA barcode method for ancient plant sample analysis. Scanning electron microscope (SEM) observations showed seed compact structure and irregular surface but they did not permit a precise nor empirical classification: so, a molecular approach was necessary. DNA was extracted from ancient seeds and then it was used, as template, for PCR amplifications of standardized barcode genes. Although aDNA could be highly degraded by the time, successful PCR products were obtained, sequenced and compared to nucleotide sequence databases. Positive outcomes (supported by morphological comparison with modern seeds, geographical distribution and historical data) indicated that seeds could be identified as belonging to two plant species: Olea europaea L. and Cornus mas L.     

Extreme environments and exobiology

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.     

Palaeomicrobiology: current issues and perspectives

Palaeomicrobiology is an emerging field that is devoted to the detection, identification and characterization of microorganisms in ancient remains. Data indicate that host-associated microbial DNA can survive for almost 20,000 years, and environmental bacterial DNA preserved in permafrost samples has been dated to 400,000-600,000 years. In addition to frozen and mummified soft tissues, bone and dental pulp can also be used to search for microbial pathogens. Various techniques, including microscopy and immunodetection, can be used in palaeomicrobiology, but most data have been obtained using PCR-based molecular techniques. Infections caused by bacteria, viruses and parasites have all been diagnosed using palaeomicrobiological techniques. Additionally, molecular typing of ancient pathogens could help to reconstruct the epidemiology of past epidemics and could feed into current models of emerging infections, therefore contributing to the development of appropriate preventative measures.     

Immunohistochemical staining on hydroxyethyl-methacrylate-embedded tissues

Hydroxyethyl-methacrylate (GMA) embedding has recently been proposed for light microscopy studies. In the present investigation extracellular protein antigens were localized on GMA-embedded renal biopsy tissue. Conventionally frozen sections were compared with GMA sections from 55 renal specimens for the detection of extracellular protein antigens. Sections were directly stained with fluorescein- or peroxidase-conjugated antisera against immunoglobulin (Ig) G, IgA, IgM, C3, C1q, and fibrinogen. Results obtained using these two methods showed a 74-89% agreement, depending on the antigen under study. Some discrepancy between GMA and frozen sections was observed in three cases of renal amyloidosis and those cases presenting focal or trace reactions; the differences did not, however, influence the diagnosis. Prerequisites for antigen recovery on GMA sections were a) choice of fixative; b) abrupt dehydration of specimens; and c) treatment of sections with nonspecific protease. The improved localization and the lower background staining obtained led to easy and immediate detection of antigens on GMA sections despite the reduced antigenicity due to the embedding process.     

Errata corrige

Abstract

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as “absolute extreme.” Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.     

Relationships between Soil Organic Status and Microbial Community Density and Genetic Structure in Two Agricultural Soils Submitted to Various Types of Organic Management

The effects of soil organic management on indigenous microorganisms were studied by comparing mulching straw (S), conifer compost (CC), and conifer bark (CB) as well as grass landing with grass (G), clover (Cl), and fescue (F) in a silty–clay soil (Macon), and by incorporating vine shoot (VS) and single and double doses of farmyard manure (FM) and mushroom manure (MM) in a calcareous sandy soil (Chinon). Soil physicochemical and microbial characteristics were assessed at each site at two depths by sampling at 0–5 and 5–20 cm for the Macon site and 0–10 and 10–20 cm for the Chinon site. Changes in the quantity of soil organic matter (SOM), through an increase in C_org and N_org contents, and in its quality, through modifications in the C/N and humic acid/fulvic acid ratios, were essentially recorded at the surface layer of treated plots with differential magnitudes according to the inputs and soil type. Quantitative modifications in microbial communities were assessed by means of C-biomass measurements and resulted in an increase in microbial densities fitted with the increase of C_org and N_org contents. However, the deduced C incorporation in microbial biomass was negatively correlated with the C/N ratio, demonstrating a strong influence of the type of organic management on the rate of microbial processes. Qualitative modifications in microbial communities were evaluated by the characterization of the genetic structure of bacterial and fungal communities from DNA directly extracted from the soil, using bacterial and fungal automated ribosomal intergenic spacer analysis. Organic amendments led to changes in the bacterial and fungal communities of both sites. However, the magnitude and the specificity of these changes were different between sites, organic amendments, and microorganisms targeted, revealing that the impact of organic management is dependent on the soil and organic input types as well as on the particular ecology of microorganisms. A co-inertia analysis was performed to specify the role of the quantity and quality of SOM on the modifications of the genetic structure. A significant costructure was only observed for Macon plots at 0–5 cm between the bacterial genetic structure and the SOM characteristics, demonstrating the influence of the relative amount of the different humic substances (humic and fulvic acids) on microbial composition.     

Scanning Electron Microscopy of Microorganisms on Chicken Skin

1. Scanning electron microscopy was used to examine microorganisms in situ on chicken skin. They developed in a layer of material on the surface of the skin. The observations explain the relative ineffectiveness of carcass cleaning procedures and the increased recovery of microorganisms by destructive compared with non-destructive sampling techniques.     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

Susceptibility of biofilms to Bdellovibrio bacteriovorus attack

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of Bdellovibrio as low as 10(2) PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of B. bacteriovorus to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh Bdellovibrio; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by Bdellovibrio but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments B. bacteriovorus was able to decrease the biomass of both E. coli and Pseudomonas fluorescens biofilms as determined by phase-contrast and epifluorescence microscopy.     

Microbial communities adhering to the obverse and reverse sides of an oil painting on canvas: identification and evaluation of their biodegradative potential

In this study, we investigated and compared the microbial communities adhering to the obverse and the reverse sides of an oil painting on canvas exhibiting signs of biodeterioration. Samples showing no visible damage were investigated as controls. Air samples were also analysed, in order to investigate the presence of airborne microorganisms suspended in the indoor atmosphere. The diversity of the cultivable microorganisms adhering to the surface was analysed by molecular techniques, such as RAPD analysis and gene sequencing. DGGE fingerprints derived from DNA directly extracted from canvas material in combination with clone libraries and sequencing were used to evaluate the non-cultivable fraction of the microbial communities associated with the material. By using culture-dependent methods, most of the bacterial strains were found to be common airborne, spore-forming microorganisms and belonged to the phyla Actinobacteria and Firmicutes, whereas culture-independent techniques identified sequenced clones affiliated with members of the phyla Actinobacteria and Proteobacteria. The diversity of fungi was shown to be much lower than that observed for bacteria, and only species of Penicillium spp. could be detected by cultivation techniques. The selected strategy revealed a higher microbial diversity on the obverse than on the reverse side of the painting and the near absence of actively growing microorganisms on areas showing no visible damage. Furthermore, enzymatic activity tests revealed that the most widespread activities involved in biodeterioration were esterase and esterase lipase among the isolated bacterial strains, and esterase and N-acetyl-β-glucosaminidase among fungi strains.