Way to the detection of Mars life]

In this review, I would like to introduce how we can detect the possible life on Mars. Even though the quantitative estimation of the possibility of biogenesis on Mars is difficult, Dr. McKay and his colleagues work has thrown a tiny light for this possibility. Considering Mars environmental conditions, the possible life is microorganisms. The detection of microorganisms in natural environments is not easy even on Earth due to the premature detection technique. We have developed a method based on the fluorescence microscopic technique. This method proved to be successful for the detection of terrestrial microorganisms. Even some pre-biotic cells can be detected. We are developing a miniature detection apparatus which meet the required standard for installing on the Mars landers. We also propose the ground based experiments using Martian meteorites or pseudo-Martian rocks.     

Progress in developing a new detection method for the harmful algal bloom species,Karenia brevis,through multiwavelength spectroscopy

Multiwavelength spectroscopy is a rapid analytical technique that can be applied to detect, identify, and quantify microorganisms such as Karenia brevis, the species known for frequent red-tide blooms in Florida's coastal waters. This research will report on a model-based interpretation of UV–vis spectra of K. brevis. The spectroscopy models are based on light scattering and absorption theories, and the approximation of the frequency-dependant optical properties of the basic constituents of living organisms. Absorption and scattering properties of K. brevis, such as cell size/shape, internal structure, and chemical composition, are shown to predict the spectral features observed in the measured spectra. The parameters for the interpretation model were based upon both reported literature values, and experimental values obtained from live cultures and pigment standards. Measured and mathematically derived spectra were compared to determine the adequacy of the model, contribute new spectral information, and to establish the proposed spectral interpretation approach as a new detection method for K. brevis.     

Biofouling on the walls of a spent nuclear fuel pool with radioactive ultrapure water

Microbial activity in spent nuclear fuel pools which contain ultrapure and radioactive water has been previously observed. The aim of the present research was to isolate and identify the microorganisms attached to the nuclear pool wall of a Spanish nuclear power plant. Amplification of 16S rDNA fragments from the culturable microorganisms by PCR using universal primers for the domain 'Bacteria', followed by Denaturing Gradient Gel Electrophoresis analysis revealed the presence of six different bacteria. The complete gene for 16S rDNA of each one was sequenced and identified as belonging to three different phylogenetic groups, viz. beta-Proteobacteria, Actinomycetales and the Bacillus/Staphylococcus group. A fungus was also found and identified as Aspergillus fumigatus by sequencing the D2 region of the large subunit rDNA gene. The isolation of these microorganisms in oligotrophic and radioactive conditions is of great interest due to the possibility of their use in bioremediation processes of radionuclide-contaminated environments.     

A novel and cost effective methodology for qualitative screening of alkalo-thermophilic cellulase free xylano-pectinolytic microorganisms using agricultural wastes

Qualitative screening of alkalo-thermophilic cellulase free xylano-pectinolytic microorganisms was done on agricultural residues. Since xylan is an expensive substrate for the isolation of xylanase producing microorganisms, the possibility of using wheat bran for screening of these microorganisms was investigated. Screening was carried out on wheat bran for the selection of xylanolytic microorganisms, on waste paper for the evaluation of cellulase free xylanolytic microorganisms, and on citrus peel for screening of pectinolytic microorganisms. Qualitative analysis of xylanase, pectinase and cellulase activities depicted that the zones obtained on nutrient agar medium containing agricultural residues were apparent and comparable with the zones obtained on nutrient agar medium containing commercial substrates. A strategy of using cost effective wheat-bran, wastepaper and citrus-peel for the isolation of cellulase free xylano-pectinolytic microorganisms is a novel and promising method and will ultimately bring down the cost of screening of these enzyme producing microorganisms.     

Rapid identification of Candida species by using nuclear magnetic resonance spectroscopy and a statistical classification strategy

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.     

Bacterial synthesis,purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels

An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives (¹⁵N-, ¹⁵N-/¹³C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly α-helical conformation. The existence of cross-peaks for all glycines of the ¹⁵N-HSQC NMR spectra as well as relatively small line widths (∼20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.     

细菌生物被膜检测与分析方法

细菌生物被膜(Biofilm,BF)由物体表面集聚生长的微生物群落和自身分泌的胞外物质构成,是造成细菌产生多重耐药性的原因之一。可靠、简单和快速的BF检测方法有助于有效预防和治疗相关疾病。基于不同原理的检测与分析方法已广泛用于BF研究中,本文从生物学方法、物理方法和化学方法等方面对BF检测方法进行总结,重点阐述显微镜技术在BF检测中的应用。并介绍了近年来发展的拉曼光谱、质谱成像、MALDI-TOF-MS等新技术,同时比较其优点和局限性,以便研究者找到最合适和最新的研究方法。     

Investigation of the microbial community in a microbiological additive used in a manure composting process

The objectives of this study were to investigate the fate of microorganisms by using cultivation methods as well as DNA analyses in a commercial microbiological additive (MA) in the course of the composting. Almost all the predominant species in the microbial succession during composting process determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were in disagreement with those determined by the clone library method. None of the microbial species in the composting stages corresponded to the microorganisms identified in the MA either by the cultivation method or DNA analysis. The results in regard to predominant microorganisms of the MA detected from the liquid medium by the PCR-DGGE did not correspond with those detected from the MA itself and composting processes. Although no evidence was found that predominant species in the MA itself dominate in the composting process, predominant species diversity in the MA itself was markedly changed after culturing at different thermophilic temperatures. These results suggested that cultivable microorganisms in the MA did not become predominant in the composting process: however, some microorganisms that are detected from the MA itself by the DNA analysis may act effectively in the composting process.     

Biofouling on the Walls of a Spent Nuclear Fuel Pool with Radioactive Ultrapure Water

Microbial activity in spent nuclear fuel pools which contain ultrapure and radioactive water has been previously observed. The aim of the present research was to isolate and identify the microorganisms attached to the nuclear pool wall of a Spanish nuclear power plant. Amplification of 16S rDNA fragments from the culturable microorganisms by PCR using universal primers for the domain ‘Bacteria’, followed by Denaturing Gradient Gel Electrophoresis analysis revealed the presence of six different bacteria. The complete gene for 16S rDNA of each one was sequenced and identified as belonging to three different phylogenetic groups, viz. β-Proteobacteria, Actinomycetales and the Bacillus/Staphylococcus group. A fungus was also found and identified as Aspergillus fumigatus by sequencing the D2 region of the large subunit rDNA gene. The isolation of these microorganisms in oligotrophic and radioactive conditions is of great interest due to the possibility of their use in bioremediation processes of radionuclide-contaminated environments.     

Methodology for determination of the state of microbial culture by degree of spatial organization of whole cells and their external structures

The proposed methodology for determination of biosystems state is based on registration of changes in the space-time parameters of cells and their external structures in a population of microorganisms from different taxonomic groups by the method of noninvasive layer-by-layer analysis using internal reflection spectroscopy. The dependence between the state of an organism and the changes in spatial organization of whole cells and their surface layers have been revealed. The proposed method allows estimation of the character and degree of dynamics of biosystem variations irrespective of the kind of influence. This method can be used for assessing the state of other organisms by the differences between values A for the surface structures and cells of these organisms.     

Sprayed-On Acrylic Plastic,from the Commercial Type of Pressurized Can,for Impressions of Plant Epidermal Surfaces

A synthetic aromatic polymer has been used for preparing replicas of different microorganisms. This method of preparing highly concentrated (9.6 k) microbiological samples for scanning electron microscopy was compared with a standard method. The micrographs of the replicated samples are satisfactory. This method is rapid, cost effective and produces good results, especially in the case of spore-forming mycelial microorganisms.     

daime, a novel image analysis program for microbial ecology and biofilm research

Combinations of microscopy and molecular techniques to detect, identify and characterize microorganisms in environmental and medical samples are widely used in microbial ecology and biofilm research. The scope of these methods, which include fluorescence in situ hybridization (FISH) with rRNA-targeted probes, is extended by digital image analysis routines that extract from micrographs important quantitative data. Here we introduce daime (digital image analysis in microbial ecology), a new computer program integrating 2-D and 3-D image analysis and visualization functionality, which has previously not been available in a single open-source software package. For example, daime automatically finds 2-D and 3-D objects in images and confocal image stacks, and offers special functions for quantifying microbial populations and evaluating new FISH probes. A novel feature is the quantification of spatial localization patterns of microorganisms in complex samples like biofilms. In combination with '3D-FISH', which preserves the 3-D structure of samples, this stereological technique was applied in a proof of principle experiment on activated sludge and provided quantitative evidence that functionally linked ammonia and nitrite oxidizers cluster together in their habitat. This image analysis method complements recent molecular techniques for analysing structure-function relationships in microbial communities and will help to characterize symbiotic interactions among microorganisms.     

The role of microorganisms in mediating and facilitating the uptake of plant nutrients from soil

Summary No root systems in nature are without a microbial population. These may be freeliving or symbiotic.The incidence and nutrition of the freeliving microorganisms is discussed. Shortage of substrate makes it unlikely that the N-fixers in the population can fix useful amounts of N. There is a possibility that P supply is improved, but an analysis of possible processes shows them to be rather unlikely, and evidence for them to be poor. Manganese and iron uptake can be altered by microbial activity. Growth of plants can be affected by non-nutritional bacterial effects.The ecology of Rhizobium in the soil is briefly discussed, and the varying needs of different identified strains is stressed.Mycorrhizal infection of plants leads to large growth increases in appropriate conditions. This is almost always linked to increased P uptake, but zinc and copper nutrition can also be improved. The processes involved are briefly discussed. Rapid and extensive infection is important; it is very sensitive to temperature. New modelling methods are now becoming available to measure the behaviour of the fungal infections. The microorganisms require C compounds from the plant, and new measurements of this cost are discussed. The possibility of practical use of mycorrhizal fungi seem to be improving.Keynote address     

Sheen Screen, a Miniaturized Most-Probable-Number Method for Enumeration of Oil-Degrading Microorganisms

Sheen Screen is a miniaturized method for enumerating oil-degrading microorganisms. The technique relies on the ability of oil-degrading microorganisms to emulsify oil when provided as a sole carbon source in 24-well tissue culture plates. Sediments that actively respire hydrocarbons have high numbers of Sheen Screen-positive microorganisms.     

Measurement of protein biomass by Fourier transform infrared-photoacoustic spectroscopy

A relatively new analytical technique, Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), provides spectra of bacteria, fungi, and other microorganisms in solid states not suitable for conventional absorption spectroscopy. In this paper the feasibility of quantitative measurement of protein biomass on solid substrates by FTIR-PAS is examined and discussed. By measuring photoacoustic absorption bands from amide groups in the protein of microorganisms, the increase in biomass that occurs during growth was monitored directly and accurately. Incorporation of polyacrylonitrile into the sample as an internal standard was shown to be a convenient method for improving both the reliability and the range of detection by photoacoustic spectroscopy. Results of FTIR-PAS measurements of known quantities of microbial mass in simulated growth experiments suggest that the technique may be especially suitable for assays of microorganisms used in solid-state biosyntheses of drugs, hormones, and other biological agents.     

On-line biomass measurements in bioreactor cultivations: comparison study of two on-line probes

Two on-line probes for biomass measurement in bioreactor cultivations were evaluated. One probe is based on near infrared (NIR) light absorption and the other on dielectric spectroscopy. The probes were used to monitor biomass production in cultivations of several different microorganisms. Differences in NIR probe response compared to off-line measurement methods revealed that the most significant factor affecting the response was cell shape. The NIR light absorption method is more developed and reliable for on-line in situ biomass estimation than dielectric spectroscopy. The NIR light absorption method is, however, of no significant use, when the cultivation medium is not clear, and especially in processes using adsorbents or solid matrix for the microorganism to grow on. The possibilities offered by dielectric spectroscopy are impressive, but the on-line probe technology needs to be improved.     

Preparation, characterization, and biotransformation of the inclusion complex of phytosterols and hydroxypropyl-beta-cyclodextrin by Mycobacterium neoaurum

The inclusion complex of hydroxypropyl-beta-cyclodextrin (HBbetaCD) and phytosterols (PSs) was prepared and characterized by thermogravimetric analysis (TGA) and infrared (IR) spectroscopy. Biotransformation of the inclusion complex of phytosterols and hydroxypropyl-beta-cyclodextrin (PSs-HBbetaCD) by Mycobacterium neoaurum to 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione [AD(D)] was studied. The TGA and IR results indicated that the thermal stability of PSs was improved in the complex with HBbetaCD. Biotransformation improved the solubility of PSs in the aqueous medium a lot because the AD(D) production was increased remarkably compared with the control, but growth of the bacteria was inhibited in the presence of HBbetaCD. The optimal inclusion ratio, ultrasonic treating time, dosage, and time of addition of PSs-HBbetaCD complexe were found to be 2:1, 10 min, 1.5 g/30 ml medium, and 48 h after incubation, respectively. This inclusion technique not only increased the availability of the substrates for the microorganisms, but also the capability of these microorganisms to produce AD(D) from PSs.     

Screening method for detection of hydrocarbon-oxidizing bacteria in oil-contaminated water and soil specimens

Toxic action of crude oil on the living world and ecosystems in general is a global problem of both aquatic and terrestrial environments. Bearing in mind the possibility of biodegradation of this toxicant, the procedures of determining counts and activity of cultivable microorganisms, and especially of bacteria responsible for degradation processes, are of great significance. The aim of this work was to study the possibility of modifying some solid media by adding triphenyltetrazolium chloride reagent as an indicator of the dehydrogenase activity, to develop a simple screening method for a simultaneous assessment of the count and activity of cultivable hydrocarbon-oxidizing bacteria in the oil-contaminated environments. The modified method appeared to be rapid and very suitable for the intended purposes.     

Possible entry of fragments of a bacterial genome into the trophozoite nucleus of the dysentery amoeba

The use of F?lgen-like electron microscopy reaction has revealed particles in the cytoplasm and nucleus of the dysentery amoeba trophozoites which in their size and resistance to acidic hydrolysis and in their affinity for uranilacetate are similar to tightly packed elements of the bacterial genome. On this basis a conclusion was drawn that some fragments of genomes of phagocytic bacteria can be preserved in the amoebal cell. The community of microorganisms consisting of trophozoites of the dysentery amoeba and bacteria of the intestinal flora is supposed to be a suitable model for studying the possibility of penetration of transgenetic elements from procaryote to eucaryote.