A simple and fast method for determining colony forming units.

AIMS: To develop a flexible and fast colony forming unit quantification method that can be operated in a standard microbiology laboratory. METHODS AND RESULTS: A miniaturized plating method is reported where droplets of bacterial cultures are spotted on agar plates. Subsequently, minicolony spots are imaged with a digital camera and quantified using a dedicated plug-in developed for the freeware program IMAGEJ. A comparison between conventional and minicolony plating of industrial micro-organisms including lactic acid bacteria, Eschericha coli and Saccharomyces cerevisiae showed that there was no significant difference in the results obtained with the methods. CONCLUSIONS: The presented method allows downscaling of plating by 100-fold, is flexible, easy-to-use and is more labour-efficient and cost-efficient than conventional plating methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for rapid assessment of viable counts of micro-organisms similar to conventional plating using standard laboratory equipment. It is faster and cheaper than conventional plating methods.

Degradation of methanethiol by methylotrophic methanogenic archaea in a lab-scale upflow anaerobic sludge blanket reactor.

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30 degrees C to H2S, CO2, and CH4. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter-1 day-1. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.

Metabolic interactions in methanogenic and sulfate-reducing bioreactors.

In environments where the amount of electron acceptors is insufficient for complete breakdown of organic matter, methane is formed as the major reduced end product. In such methanogenic environments organic acids are degraded by syntrophic consortia of acetogenic bacteria and methanogenic archaea. Hydrogen consumption by methanogens is essential for acetogenic bacteria to convert organic acids to acetate and hydrogen. Several syntrophic cocultures growing on propionate and butyrate have been described. These syntrophic fatty acid-degrading consortia are affected by the presence of sulfate. When sulfate is present sulfate-reducing bacteria compete with methanogenic archaea for hydrogen and acetate, and with acetogenic bacteria for propionate and butyrate. Sulfate-reducing bacteria easily outcompete methanogens for hydrogen, but the presence of acetate as carbon source may influence the outcome of the competition. By contrast, acetoclastic methanogens can compete reasonably well with acetate-degrading sulfate reducers. Sulfate-reducing bacteria grow much faster on propionate and butyrate than syntrophic consortia.

Interspecies electron transfer in methanogenic propionate degrading consortia.

Propionate is a key intermediate in the conversion of complex organic matter under methanogenic conditions. Oxidation of this compound requires obligate syntrophic consortia of acetogenic proton- and bicarbonate reducing bacteria and methanogenic archaea. Although H(2) acts as an electron-carrier in these consortia, evidence accumulates that formate plays an even more important role. To make energy yield from propionate oxidation energetically feasible for the bacteria and archaea involved, the concentrations of H(2) and formate have to be extremely low. On the other hand, the diffusion distance of these carriers has to be small to allow high propionate conversion rates. Accordingly, the high conversion rates observed in methanogenic bioreactors are due to the fact that the propionate-oxidizing bacteria and their methanogenic partners form micro-colonies within the densely packed granules.

Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans.

The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H2 and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate.