Identification performance of MALDI-ToF-MS upon mono- and bi-microbial cultures is cell number and culture proportion dependent.

Biotyping using matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectroscopy (MS) has revolutionized microbiology by allowing clinicians and scientists to rapidly identify microbes at genus and species levels. The present study extensively assesses the suitability and reliability of MALDI-ToF biotyping of 14 different aerobic and anaerobic bacterial species as pure and mixed cultures. Reliable identification at species level was possible from biomaterial of older colonies and even frozen biomaterial, although this was species dependent. Using standard instrument settings and direct application of biomaterial onto the MALDI-ToF target plates, it was determined that the cell densities necessary for completely reliable identification of pure cultures varied between 2.40 × 108 and 1.10 × 1010 viable cell counts (VCCs) per mL, depending on the species. Evaluation of the mixed culture algorithm of the Bruker Biotyper® software showed that the performance of the algorithm depends greatly on the targeted species, on their phylogenetic distance, and on their ratio of VCC per mL in the mixed culture. Hence, the use of MALDI-ToF-MS with incorporation of the mixed culture algorithm of the software is a useful pre-screening tool for early identification of contaminants, but due to the great variability in performance between different species and the usually unknown percentage of the possible contaminant in the mixture, it is advisable to combine this method with other microbiology methods.

Co-digestion of wheat and rye bread suspensions with source-sorted municipal biowaste.

Acidification of wheat bread (WBS), rye bread (RBS) and fresh biowaste suspensions (FBS), leading to lactate+acetate, lactate+acetate+n-buyrate, and acetate+propionate+n-butyrate, respectively, and biogas production as well as population dynamics were investigated. Co-fermentation of FBS (14 kg m(-3) d(-1) organic loading rate (OLR)) with WBS or RBS was stable up to an OLR of 22 kg m(-3) d(-1) and resulted in up to 3 times as much biogas. During co-fermentation at more than 20 kg m(-3) d(-1) OLR the total population increased more than 2-fold, but the originally low share of propionate-oxidizing bacteria significantly decreased. The proportion of methanogens also decreased. Whereas the proportion of Methanosarcinales to Methanomicrobiales in biowaste and biowaste+WBS remained constant, Methanosarcinales and in particular Methanosaeta spec. in the biowaste+RBS assay almost completely disappeared. Methanomicrobiales increased instead, indicating propionate oxidation via acetate cleavage to CO2 and hydrogen.

Effect of moisture of municipal biowaste on start-up and efficiency of mesophilic and thermophilic dry anaerobic digestion.

Methane production from biowaste with 20-30% dry matter (DM) by box-type dry anaerobic digestion and contributing bacteria were determined for incubation at 20, 37 and 55 °C. The same digestion efficiency as for wet anaerobic digestion of biowaste was obtained for dry anaerobic digestion with 20% DM content at 20, 37 and 55 °C and with 25% DM content at 37 and 55 °C. No or only little methane was produced in dry anaerobic reactors with 30% DM at 20, 37 or 55 °C. Population densities in the 20-30% DM-containing biowaste reactors were similar although in mesophilic and thermophilic biowaste reactors with 30% DM content significantly less but phylogenetically more diverse archaea existed. Biogas production in the 20% and 25% DM assays was catalyzed by Methanosarcinales and Methanomicrobiales. In all assays Pelotomaculum and Syntrophobacter species were dominant propionate degraders.

Characterization of the bacterial archaeal diversity in hydrocarbon-contaminated soil.

A polyphasic approach combining culture-based methods with molecular methods is useful to expand knowledge on microbial diversity in contaminated soil. Microbial diversity was examined in soil samples from a former industrial site in the European Alps (mainly used for aluminum production and heavily contaminated with petroleum hydrocarbons) by culture-dependent and culture-independent methods. The physiologically active eubacterial community, as revealed by fluorescence-in-situ-hybridization (FISH), accounted for 6.7% of the total (DAPI-stained) bacterial community. 4.4% and 2.0% of the DAPI-stained cells could be attributed to culturable, heterotrophic bacteria able to grow at 20°C and 10°C, respectively. The majority of culturable bacterial isolates (34/48) belonged to the Proteobacteria (with a predominance of Alphaproteobacteria and Gammaproteobacteria), while the remaining isolates were affiliated with the Actinobacteria, Cytophaga-Flavobacterium-Bacteroides and Firmicutes. A high fraction of the culturable, heterotrophic bacterial population was able to utilize hydrocarbons. Actinobacteria were the most versatile and efficient degraders of diesel oil, n-alkanes, phenol and PAHs. The bacterial 16S rRNA gene clone library contained 390 clones that grouped into 68 phylotypes related to the Proteobacteria, Bacteroidetes, Actinobacteria and Spirochaetes. The archaeal 16S rRNA gene library contained 202 clones and 15 phylotypes belonging to the phylum Euryarchaeota; sequences were closely related to those of methanogenic archaea of the orders Methanomicrobiales, Methanosarcinales, Methanobacteriales and Thermoplasmatales. A number of bacterial and archaeal phylotypes in the clone libraries shared high similarities with strains previously described to be involved in hydrocarbon biodegradation. Knowledge of the bacterial and archaeal diversity in the studied soil is important in order to get a better insight into the microbial structure of contaminated environments and to better exploit the bioremediation potential by identifying potential hydrocarbon degraders and consequently developing appropriate bioremediation strategies.