Yeast and filamentous fungi have been used for centuries in diverse biotechnological processes. Fungal fermentation technology is traditionally used in relation to food production, such as for bread, beer, cheese, sake and soy sauce. Last century, the industrial application of yeast and filamentous fungi expanded rapidly, with excellent examples such as purified enzymes and secondary metabolites (e.g. antibiotics), which are used in a wide range of food as well as non-food industries. Research on protein and/or metabolite secretion by fungal species has focused on identifying bottlenecks in (post-) transcriptional regulation of protein production, metabolic rerouting, morphology and the transit of proteins through the secretion pathway. In past years, genome sequencing of some fungi (e.g. Aspergillus oryzae, Aspergillus niger) has been completed. The available genome sequences have enabled identification of genes and functionally important regions of the genome. This has directed research to focus on a post-genomics era in which transcriptomics, proteomics and metabolomics methodologies will help to explore the scientific relevance and industrial application of fungal genome sequences.

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Phytases are a group of enzymes capable of releasing phosphate from phytate, one of the most abundant forms of organic phosphate in the natural environment. Phytases can be found in many organisms; in bacteria, they are particularly described in g-proteobacteria. In recent years, bacterial phytases have been isolated, characterized and proposed as potential tools in biotechnology. Microbial phytases have been applied mainly to animal (swine and poultry) and human foodstuffs in order to improve mineral bioavailability and food processing. Here, we summarize the current knowledge of bacterial phytases and phytase-producing bacteria, as well as their potential biotechnological applications, including new fields poorly explored, such as fish nutrition, environmental protection and plant nutrition. Despite the recognized importance in biotechnology, information on bacterial phytases and phytase-producing bacteria is clearly limited and major efforts are required to improve the knowledge of phytases present in bacteria and their utilization.

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Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification have not been well characterized. In this study, we employed Stable Isotope Probing (SIP) to study succinate-assimilating denitrifiers in soil microcosms amended with nitrate and ¹³C-succinate. Microbial populations represented in ¹²C- and ¹³C-DNA fractions were different based on denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragment. A nearly full-length 16S rRNA gene was also amplified, cloned, and sequenced from ¹³C-DNA fraction. Both PCR-DGGE and clone library analyses revealed that Burkholderiales and Rhodocyclales dominated the succinate-assimilating population in denitrifying soil after 24 h incubation. Among these, novel Betaproteobacteria, possibly within the order Rhodocyclales, represented 43% of the clones obtained. Nitrite reductase genes, nirS and nirK, were also amplified and cloned from the ¹³C-DNA fraction. While most nirK clones in this study were similar to the nirK sequences from Rhizobiales, a majority of the nirS clones were similar to the nirS sequences from Burkholderiales and Rhodocyclales, consistent with the 16S rRNA gene analysis. These groups of bacteria, including the novel Betaproteobacteria, may play an important role in denitrification in rice paddy soil.

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The relationships between soil microbial properties and nitrous oxide emission were examined in upland soil under different tillage systems [no tillage (NT), rotary and plow tillage] and cover crop systems (fallow, cereal rye, and hairy vetch) in 2004 and 2005. Microbiological analyses included the determination of soil ergosterol as an indicator of fungal biomass, bacterial plate counting, and MPN estimations of ammonia oxidizers and denitrifiers. The combined practice of NT with rye-cover crop treatment increased fungal biomass but not bacterial populations in 0-10 cm deep soils. Such increase in fungal biomass was not found in 10-20 cm and 20-30 cm deep cover-cropped NT soil. The combined practice of NT with rye-cover cropping resulted in higher in situ N₂O emission rates compared with rotary- and plow-till treatments. N₂O flux was positively correlated with soil ergosterol content but not with denitrifier MPN and other soil chemical properties. These results suggested a significant contribution of fungi to N₂O emission in cover-cropped NT soils.

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The aim of the study was to survey cultivable heterotrophic bacteria from sea ice samples collected from Peter the Great Bay of the Sea of Japan, and to test the isolates for antagonistic activity. A total of 195 strains were isolated and investigated by a phenotypic approach. The 16S rRNA gene sequence analysis of thirty eight strains revealed primarily representatives of the class Gammaproteobacteria, phylum Bacteroidetes, and phylum Actinobacteria. Members of the class Alphaproteobacteria were a minor component. Most strains shared 98-99% sequence similarity to recognized species, including those recovered previously from Antarctic or Arctic sea ice or polar environments, Shewanella frigidimarina, Psychrobacter urativorans, Psychrobacter glacincola, Psychrobacter fozii, Pseudomonas veronii, or Pseudomonas proteolytica. At least seven bacterial groups may represent novel species within known genera. Five isolates have been previously described as the novel species Marinomonas primoryensis and Psychrobacter maritimus. Antagonistic activity assays revealed a number of strains-antagonists of the Pseudomonas fluorescens group, Bacillus and Nocardioides displaying remarkable antifungal and antibacterial activities. For the first time our findings show that sea ice offshore in the Sea of Japan represents an untapped source of bacterial biodiversity and microorganisms capable of antibacterial and antifungal metabolite production.

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The NAD⁺-dependent malic enzyme (DME) has been reported to play a key role supporting nitrogenase activity in bacteroids of Sinorhizobium meliloti. Genetic evidence for a similar role in Bradyrhizobium japonicum USDA110 was obtained by constructing a dme mutant. Soybean plants inoculated with a dme mutant did not show delayed nodulation, but formed small root nodules and exhibited significant nitrogen-deficiency symptoms. Nodule numbers and the acetylene reducting activity per nodule as a dry weight value 14 and 28 days after inoculation with the dme mutant were comparable to those of plants inoculated with wild-type B. japonicum. However, shoot dry weight and acetylene reducting activity per nodule decreased to ca. 30% of the values in plants with wild-type B. japonicum. The sucrose and organic acid (malate, succinate, acetate, α-ketoglutarate and lactate) contents of the nodules were investigated. Amounts of sucrose, malate and a-ketoglutarate increased on inoculation with the dme mutant, suggesting that the decreased DME and nitrogenase activities in the bacteroids resulted in a reduction in the consumption of these respiratory metabolites by the nodules. The data suggest that the DME activity of B. japonicum bacteroids plays a role in nodule metabolism and supports nitrogen fixation.

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Transgenic crops able to tolerate environmental stress are being developed throughout the world. However, little data is available on the impact of environmental stress-tolerant transgenic crops on soil microorganisms and biochemistry. Recently developed transgenic potato plants carrying an environmental stress-related gene, DREB1A, with a stress-inducible promoter, are being evaluated for growth performance in greenhouses. In this study, we investigated microbial diversity and soil function to assess potential environmental risks of these transgenic potato lines. Genotypic diversity of the 16S-23S rRNA intergenic spacer region and activity levels of four enzymes were used as indicators of microbial genetic diversity and soil function, respectively. Salinity had a major effect on both bacterial (88-93%) and fungal (54-55%) diversity, while the transgene had a relatively small effect on genotypic structure (0-5%) based on the analysis of variance. However, a few genotypes appeared only in soils planted with the transgenic lines. Some enzyme activities were found to differ significantly between the transgenic and non-transgenic lines, although the results were not repeatable in the second trial. These results suggest that abiotic growth environments had a stronger impact on soil microorganisms and biochemistry than did plant genotypes.

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The precise estimation of extracellular DNA, long enough to encode a gene, is valuable for determining its potential involvement in genetic transformation. Here, the applicability of real-time long PCR was examined by using target DNA of different lengths and transformation with competent cells to monitor the fate of plasmid DNA released into rivers. Detection limits of the PCR were 7 and 30 copies reaction^-1 for a plasmid (4.1 kbp), and 30 and 3×10⁴ copies reaction^-1 for lambda DNA (8.6 kbp and 15.5 kbp). The copy numbers of the plasmid obtained by the real-time long PCR were highly correlated with those determined by the transformation metod (R²=0.98). Real-time PCRs targeting a short fragment and full-length plasmid DNA were carried out to monitor fragmentation during 506 h of incubation. After 75 h, more than 100-fold larger amounts of the short fragments persisted compared to the full-length plasmid and the values remained constant in the following days. Real-time long PCR revealed that long DNA persisted in river water for prolonged periods of incubation and is thus useful to assess the fate of target DNA in natural water systems.

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The PCR amplification-based analysis of microbial diversity is subject to potential problems. In this study, to minimize the bias toward a 1:1 ratio in multitemplate PCR, a real-time PCR assay was carried out using a quenching fluorescence dye primer and amplification efficiency was monitored. Then terminal-restriction fragment length polymorphism (T-RFLP) profiling was performed using the PCR product with minimized PCR bias. This method was applied to an analysis of the diversity of the archaeal community in an upland rice field under different tillage systems and winter cover cropping. Terminal restriction fragments (T-RFs) of PCR-amplified archaeal 16S rRNA genes were assigned to the gene sequences recovered from the same soil by using an archaeal 16S rRNA gene clone library. Our results indicated that soil archaeal members were not influenced but the relative abundance of archaeal species particularly those belonging to Crenarchaeota which changed between the tillage and non-tillage treatments.

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The respiration rates per unit of microbial biomass (qCO₂) of live cells were evaluated in inorganic medium using glass beads to which glucose was added at a rate of 50 μg C vial^-1 day^-1. The qCO₂ of live cells (g CO₂-C (g biomass-C)^-1 day^-1) was 0.23 in a pure culture of bacteria, 0.67 in a mixed culture of bacteria and protozoa, 0.16 in a mixed culture of bacteria and fungi, and 0.20 in a mixed culture of bacteria, protozoa and fungi. A culture system containing glass beads is a useful tool to estimate qCO₂ of live cells.

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We developed a method for direct DNA isolation from phyllosphere microbial communities, designated Direct-DIP. This method comprises DNA extraction from non-shredded leaves with benzyl chloride, and DNA purification by gel filtration. Scanning electron microscopy showed that epiphytic microorganisms were completely removed from the leaf surface after benzyl chloride treatment, while microstructures of the leaf were not damaged. Clear DGGE profiles were obtained regardless of the plant species. Shannon diversity indices of DGGE profiles by Direct-DIP were higher than those by a conventional method. Our findings suggest that Direct-DIP is a rapid, simple, and cost-effective method of extracting DNA from phyllosphere microbial communities.

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