Microflora and mineralization activity in the bottom sediment taken from two distinct sites in Lake Biwa were investigated.From the colony formation process, the following three common features for soil bacteria were also confirmed in the sediment bacteria:1)the number of colonies increased not continuously but intermittently with incubation time;2)the number of colonies formed on diluted nutrient agar medium was considerably higher than that on nutrient agar;3)the total number of bacteria counted by epifluorescence microscopy was 100 to 10000 times higher than the number of colonies formed on both media.The following features were also found for sediment bacteria:1)the deeper the layer of the sediment, the smaller the number of colonies;2)the southern basin sediments yielded more colonies than northern basin sediments;3)the number of bacteria that can form colonies increased from spring to summer in the sediment sample contrary to the smaller count of total bacteria in the summer sample.From the bacterial activity of glucose mineralization the following points were revealed:1)the upper layer of the sediments are richer in aerobes than facultative anaerobes;and 2)under oxygen-deficient conditions even the upper layer of the sediments becomes rich in facultative anaerobes.As revealed in this study, the combination of observing the colony-forming process on agar medium and the measuring the glucose-mineralizing activity of the bacteria in the sediments seems to be useful for understanding the complex microflora in the sediments and changes relating to various factors, such as, nutrient and oxygen concentration, and temperature.

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A marine bacterium which grows on agar as its sole carbon and energy source shows an agar liquefying ability, was isolated from sea mud and identified as Pseudoalteromonas sp.An extracellular agarase was purified to homogeneity through a four-step process:ultra-filtration(10kDa<enzyme<0.2μm), two series of DEAE-Sephacel chromatography, and Sephacryl S-300 gel filtration.The enzyme was estimated to have a molecular mass of 56kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration, and be composed of a 37kDa protein and 19kDa of sugar, namely a glycoprotein.The optimum conditions for its action were 30ºC, pH of 6.5-7.5, and 0mM NaCl.Km to agarose was determined as 6.28mM.The enzyme was active at 15-65ºC, pH 4.0-9.0 and 0-4, 000mM salt concentration(NaCl).It was found to be an endo-type agarase which hydrolyses agar and agarose to yield galactose, neoagarobiose and a trace amount of neoagarohexaose.In addition, the enzyme had both α- and β-galactosidase activities.

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Throughout the year, the microbial biomass carbon(μg g^-1 dry soil)at a depth of 5-10cm in a forest or an arable soil classified as Andept was almost constant at 730 or 180, respectively.The biomass carbon of a forest or an arable soil classified as Ochrept was 1, 500 or 440, respectively.To examine the reasons for the lower size of biomass carbon of the arable soil compared to that of the forest soil, we studied the effect of soil agitation, temperature or moisture on the microbial biomass carbon of Andept samples incubated in a laboratory.Soil agitation for half a minute with a frequency of twice a week did not change the microbial biomass carbon of the forest or the arable soil during an incubation period of 3 months.On the other hand, the temperatures above 30ºC or the moisture levels below 35% significantly reduced the biomass carbon of the forest soil after a 2-week-incubation.However, periodic incubation at 40ºC for 4 hours and 25ºC for 20 hours daily, keeping soil moisture at 60%, did not reduce the biomass carbon of the forest soil after a 2-week-incubation.The moisture levels below 35% were observed in the arable soil at a depth of 0-3cm for a few weeks in the field, and the reduced biomass carbon of the air-dried forest soil did not recover after the soil was re-wetted.Therefore, severe desiccation seems to be one of the reasons for the lower size of microbial biomass carbon in the arable soil.

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It has been reported that higher plate counts for bacterial communities in river epilithon were yielded on agar plates by supplementation with sodium pyruvate, compared to the counts without supplementation.To support the credibility of this phenomenon, studies were conducted regarding the bacterial communities in several aquatic environments.Consequently, degrees of the ratio of colony-forming unit(CFU)on agar plates supplemented with sodium pyruvate to that without supplementation were grouped into two categories:a 2 to 3 magnitude group and a 5 to 6 magnitude one.According to cluster analysis of the water quality, higher degrees of the ratio were observed at environments that had a DOC concentration around 1ppm and had a low NO₃^- concentration.The compositions of the abundantly-appearing bacterial colonies were suggested to be different from that of those appearing on the agar plates without supplementation of sodium pyruvate.

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Sulfate-reducing bacteria(SRB)were quantified in 8 lowland paddy soils of Burkina Faso by MPN method using lactate and sulfate as substrates.Bacterial densities ranged from 10² to 10⁶g^-1 dry soil before soil flooding and from 10⁷ to 10⁹g^-1 dry soil on rice repining and heading stage.SRB populations decreased slightly 30 days before flooding, and increased after 2 weeks of flooding in most soils.In soil near rice roots, populations increased gradually with fluctuations from transplanting day to rice flowering and maturity stages, and decreased from rice maturity to harvest with the drainage of water.

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Recent molecular phylogenetic analyses based on nucleotides directly obtained from environmental samples have indicated that the microbial diversity of naturally occurring microbial communities is much greater than previously assumed based on standard cultivation and isolation methods.Although the early investigations were conducted focusing on the identification and diversity of uncultivated bacterioplankton in oligotrophic ocean and coastal water environments, increasing attention has been paid to the phylogenetic diversity and ecological significance of uncultivated or unknown microorganisms belonging to the domains of Bacteria and Archaea in various environments.Extreme environments that were previously believed“abiotic”are now viewed as one of the most important targets for molecular exploration because these areas have turned out to harbor a great diversity of microorganisms, so-called“extremophiles”, having novel abilities and metabolisms that sustain them, while adapting to extraordinary conditions and that might provide great insights into understanding the origins of life and early evolution on primitive earth.Here, we present the short history of the molecular phylogenetic approach to naturally occurring microbial communities mainly in extreme environments introducing significant discoveries and findings.We also discuss improvement and new techniques for this approach, and the significant role played in future research.

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The use of Denaturing Gradient Gel Electrophoresis(DGGE)has been widely recognized in microbial ecology and its importance has increased recently.DNA extracted from the environment is amplified by PCR using a set of primers in which one of the two contains the 40 base GC-rich sequence at the 5′ -end prior to DGGE. The PCR amplified DNA fragment migrates in a DGGE gel depending on its melting behavior. Moreover, the DNA resolved by DGGE can be promoted for further analyses, such as hybridization and sequencing without any cloning step. For these reasons, PCR-DGGE is one of the powerful tools available for the detection and phylogenetic analysis of environmental microbes. Although this method is easy and reproducible, some knowledge and technique are required to obtain reliable data. This technical series introduces protocols for the PCR-DGGE method with the theory presented to beginners as comprehensibly as possible. In addition, we discussed the application and limitation of the PCR-DGGE approach to microbial ecology.

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