Enumeration of viable Escherichia coli by real-time PCR with propidium monoazide.

A photo-inducible DNA-binding dye, propidium monoazide (PMA), was used to distinguish viable and dead Escherichia coli cells. Microscopic observations using a combination of the dyes 4',6-diamidino-2-phenylindole and PMA indicated that PMA stained only dead cells, with membrane damage, red. Mixtures of viable and heat-treated E. coli cells were subjected to real-time polymerase chain reaction (PCR) with PMA treatment. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells in the mixtures of viable and heat-treated cells, as long as the ratio of dead cells to viable cells was no greater than 10. In the wastewater treatment plants, total, viable and culturable E. coli were enumerated by real-time PCR, real-time PCR coupled with PMA treatment and the most probable number method using EC-MUG medium, respectively. The concentrations of viable E. coli in the wastewater treatment plants were much higher than those of culturable cells. In addition, viable cells were even more chlorine resistant than culturable ones.

Behaviors of physiologically active bacteria in water environment and chlorine disinfection.

Direct microscopic methods using several fluorescent staining were applied to estimate the proportion of physiologically active bacteria in the water environment and evaluate the efficacy of disinfection with chlorine. 4',6-diamidino-2-phenylindole (DAPI) was used to determine total bacterial numbers, and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was chosen for direct detection of respiring bacteria. BacLight kit was used to assess bacterial membrane integrity. Bacteria with growth potential were enumerated using the DVC method and microcolony technique. The total bacterial number in river was 8 x 10(6)-3 x 10(10) cells/mL, and colony forming units on R2A medium were 1 x 10(4)-4 x 10(5) cfu/mL. In the case of wastewater treatment plant, 1-10% of total bacterial cells could form colonies. Physiologically active bacteria in river and wastewater treatment plant determined by fluorescent staining were much higher than those obtained by plate counting. The effect of chlorine on the physiological viability of Escherichia coli was also investigated. Microscopic viable bacteria were even more chlorine resistant than culturable bacteria. The inactivation rate coefficients of direct viable bacteria were one-second to third those of culturable bacteria.

Phylogenetic relationships of filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N bacteria) isolated from wastewater-treatment plants and description of Thiothrix eikelboomii sp. nov., Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.

The relationship of mixotrophic and autotrophic Thiothrix species to morphologically similar chemoorganotrophic bacteria (e.g. Leucothrix species, Eikelboom type 021N bacteria) has been a matter of debate for some years. These bacteria have alternatively been grouped together on the basis of shared morphological features or separated on the basis of their nutrition. Many of these bacteria are difficult to maintain in axenic culture and, until recently, few isolates were available to allow comprehensive phenotypic and genotypic characterization. Several isolates of Thiothrix spp. and Eikelboom type 021N strains were characterized by comparative 16S rRNA sequence analysis. This revealed that the Thiothrix spp. and Eikelboom type 021N isolates formed a monophyletic group. Furthermore, isolates of Eikelboom type 021N bacteria isolated independently from different continents were phylogenetically closely related. The 16S rRNA sequence-based phylogeny was congruent with the morphological similarities between Thiothrix and Eikelboom type 021N. However, one isolate examined in this study (Ben47) shared many morphological features with the Thiothrix spp. and Eikelboom type 021N isolates, but was not closely related to them phylogenetically. Consequently, morphology alone cannot be used to assign bacteria to the Thiothrix/type 021N group. Comparative 16S rRNA sequence analysis supports monophyly of the Thiothrix/type 021N group, and phenotypic differences between the Thiothrix spp. and Eikelboom type 021N bacteria are currently poorly defined. For example, both groups include heterotrophic organisms that deposit intracellular elemental sulfur. It is therefore proposed that the Eikelboom type 021N bacteria should be accommodated within the genus Thiothrix as a new species, Thiothrix eikelboomii sp. nov., and three further new Thiothrix species are described: Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.

Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion.

The characteristics of the degradation of cellulose, soluble starch, and glucose in the acidogenic phase and the effects of the substrate loading rate and biological solids retention time on the methanogenic phase of anaerobic digestion were investigated. The results obtained from continuous experiments using laboratory-scale anaerobic chemostat reactors elucidated the true rate-limiting step of anaerobic digestion. The specific rate of substrate utilization decreased in the following order: glucose, soluble starch, acetic acid, and cellulose. The rate of the hydrolysis of cellulose was so low that this was shown to be the rate-limiting step in overall anaerobic digestion. Among methanogenic bacteria Methanosarcina would provide a higher substrate utilization rate than Methanothrix, and the maximum allowable substrate loading rate in the methanogenic phase was 11.2 g acetic acid/L day.