Yasushi Kitagawa

Rapid detection and identification of beer-spoilage lactic acid bacteria by microcolony method.

We evaluated a microcolony method for the detection and identification of beer-spoilage lactic acid bacteria (LAB). In this approach, bacterial cells were trapped on a polycarbonate membrane filter and cultured on ABD medium, a medium that allows highly specific detection of beer-spoilage LAB strains. After short-time incubation, viable cells forming microcolonies were stained with carboxyfluorescein diacetate (CFDA) and counted with muFinder Inspection System. In our study, we first investigated the growth behavior of various beer-spoilage LAB by traditional culture method, and Lactobacillus lindneri and several L. paracollinoides strains were selected as slow growers on ABD medium. Then the detection speeds were evaluated by microcolony method, using these slowly growing strains. As a result, all of the slowly growing beer-spoilage LAB strains were detected within 3 days of incubation. The specificity of this method was found to be exceptionally high and even discriminated intra-species differences in beer-spoilage ability of LAB strains upon detection. These results indicate that our microcolony approach allows rapid and specific detection of beer-spoilage LAB strains with inexpensive CFDA staining. For further confirmation of species status of detected strains, subsequent treatment with species-specific fluorescence in situ hybridization (FISH) probes was shown as effective for identifying the CFDA-detected microcolonies to the species level. In addition, no false-positive results arising from noise signals were recognized for CFDA staining and FISH methods. Taken together, the developed microcolony method was demonstrated as a rapid and highly specific countermeasure against beer-spoilage LAB, and compared favorably with the conventional culture methods.

Microbial Analyses by Fluorescence in situ Hybridization of Well-Settled Granular Sludge in Brewery Wastewater Treatment Plants.

The characteristics of granular sludge from full-scale upflow anaerobic sludge blanket reactors used for the treatment of brewery wastewater were investigated. Fluorescence in situ hybridization (FISH) analyses of settled granules from a reactor that had been treating brewery wastewater stably at COD removal rates of over 90% for more than 6 months showed that a methanogen of the genus Methanosaeta was predominant near the granule surface and that Bacteria were not abundant. The center of the granules was composed of dead or resting cells, or both, which were used as a support for active archaeal and bacterial cells near the surface. Periodic analysis of granules from full-scale plants showed that granules containing methanogens deep within them tended to float. Granules with a Bacteria layer on the surface also tended to float. On the basis of these findings, well-settled granules are considered to have methanogens that develop near the granule surface so that the gases generated during methane fermentation are readily released.

Modified Multiplex PCR Methods for Comprehensive Detection of Pectinatus and Beer-Spoilage Cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.

Factual Analysis of Granule Flotation in Brewery Wastewater Treatment Plants by the Fluorescence in situ Hybridization Method.

The factors that change the microbial distribution and consequently the flotation of brewery granules were investigated using laboratory-scale upflow anaerobic sludge blanket (UASB) reactors and the fluorescence in situ hybridization (FISH) method. The startup operations of laboratory-scale UASB reactors fed with acetate-based synthetic wastewater, in which the loading rate was maintained at 0.1 gCOD/gVSS/d (Run 1) and increased in a stepwise manner from 0.1 gCOD/gVSS/d to 1.0 gCOD/gVSS/d (Run 2), generated methanogen colonies near the granule surface, while the overloading operation at 1.0 gCOD/gVSS/d from the startup (Run 3) resulted in the formation of methanogen colonies deep in the granules. In each run, a proportion of the granules floated when overloaded at 2.0 gCOD/gVSS/d and circulation was stopped. The ratio of floating granules increased as the methanogen-growing region increased. On the other hand, the Bacteria layer on the granule surface, which is also considered as a possible cause of granule flotation, was not formed by the inflow of other organic acids such as propionate and lactate. Glucose caused formation of a 5-microm-thick surface Bacteria layer, but the granules were still resistant to flotation. Interfusing of air under glucose feeding caused the formation of a Bacteria layer over 50 microm thick leading to granule flotation.

HorC, a hop-resistance related protein, presumably functions in homodimer form.

To determine whether two HorC molecules coordinately form a single unit, the functional properties of covalently linked dimers of HorC encoded by tandemly fused horC genes were studied. Lactobacillus brevis introduced with the fused horC genes and a single horC gene exhibited same degree of resistance to hop compounds and cetyltrimethylammonium bromide. This suggests that HorC functions as a homodimer.