Evaluation of stable isotope fingerprinting techniques for the assessment of the predominant methanogenic pathways in anaerobic digesters.

Laboratory biogas reactors were operated under various conditions using maize silage, chicken manure, or distillers grains as substrate. In addition to the standard process parameters, the hydrogen and carbon stable isotopic composition of biogas was analyzed to estimate the predominant methanogenic pathways as a potential process control tool. The isotopic fingerprinting technique was evaluated by parallel analysis of mcrA genes and their transcripts to study the diversity and activity of methanogens. The dominant hydrogenotrophs were Methanomicrobiales, while aceticlastic methanogens were represented by Methanosaeta and Methanosarcina at low and high organic loading rates, respectively. Major changes in the relative abundance of mcrA transcripts were observed compared to the results obtained from DNA level. In agreement with the molecular results, the isotope data suggested the predominance of the hydrogenotrophic pathway in one reactor fed with chicken manure, while both pathways were important in the other reactors. Short-term changes in the isotopic composition were followed, and a significant change in isotope values was observed after feeding a reactor digesting maize silage. This ability of stable isotope fingerprinting to follow short-term activity changes shows potential for indicating process failures and makes it a promising technology for process control.

NBDT (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene)--a novel fluorescent dye for studying mechanisms of toluene uptake into vital bacteria.

Uptake of small hydrophobic substances such as toluene into bacteria is widely assumed to occur by passive diffusion. Some toluene degrading bacteria, however, are described to contain uptake systems which may be involved in the transport of this compound. In this study, a fluorescently labeled toluene analogue dye (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene; NBDT), flow cytometry, and shot gun proteome analysis were used to follow toluene uptake into bacteria in more detail. The new dye has excitation peaks at 444 and 475 nm and an emission peak at 537 nm. The toluene-degraders P. putida mt-2 and P. putida F1 as well as P. putida KT2440 and E. coli K12 as negative controls were included. To enable quantification of NBDT uptake, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to inactivate NBDT efflux pumps. The porin inhibitor cadaverine was added to study the porin-mediated influx of toluene. Cadaverine reduced NBDT uptake by toluene-grown P. putida mt-2 and F1 by 25% and 42%, respectively, thus revealing an involvement and possibly a regulatory function of porins in the uptake of the toxic substrate toluene. Shot gun proteome measurements gave evidence for the presence of toluene transporting porins in P. putida mt-2 grown on toluene but not when grown on glucose.