Roberta Gorra

Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation.

Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America. The information collected using DGGE has made it possible to confirm the existing knowledge on food fermentation and spoilage. However, in some cases, new evidence that helps scientists to fully comprehend a specific microbial ecosystem has emerged. In this review, the roadmap of culture independent methods in food microbiology will be summarized, focusing on the DGGE technique. Examples of how this approach is useful to obtain a better understanding of microbial diversity are reported for several kinds of fermented food, such as fermented sausage, cheese and wine. The future of culture independent methods in food microbiology, with the increasing availability of next generation sequencing techniques, is also discussed.

Enrichment and cultivation of prokaryotes associated with the sulphate–methane transition zone of diffusion‐controlled sediments of Aarhus Bay, Denmark, under heterotrophic conditions

The prokaryotic activity, diversity and culturability of diffusion-controlled Aarhus Bay sediments, including the sulphate-methane transition zone (SMTZ), were determined using a combination of geochemical, molecular (16S rRNA and mcrA genes) and cultivation techniques. The SMTZ had elevated sulphate reduction and anaerobic oxidation of methane, and enhanced cell numbers, but no active methanogenesis. The prokaryotic population was similar to that in other SMTZs, with Deltaproteobacteria, Gammaproteobacteria, JS1, Planctomycetes, Chloroflexi, ANME-1, MBG-D and MCG. Many of these groups were maintained in a heterotrophic (10 mM glucose, acetate), sediment slurry with periodic low sulphate and acetate additions (~2 mM). Other prokaryotes were also enriched including methanogens, Firmicutes, Bacteroidetes, Synergistetes and TM6. This slurry was then inoculated into a matrix of substrate and sulphate concentrations for further selective enrichment. The results demonstrated that important SMTZ bacteria can be maintained in a long-term, anaerobic culture under specific conditions. For example, JS1 grew in a mixed culture with acetate or acetate/glucose plus sulphate. Chloroflexi occurred in a mixed culture, including in the presence of acetate, which had previously not been shown to be a Chloroflexi subphylum I substrate, and was more dominant in a medium with seawater salt concentrations. In contrast, archaeal diversity was reduced and limited to the orders Methanosarcinales and Methanomicrobiales. These results provide information about the physiology of a range of SMTZ prokaryotes and shows that many can be maintained and enriched under heterotrophic conditions, including those with few or no cultivated representatives.

NaOH-Debittering Induces Changes in Bacterial Ecology during Table Olives Fermentation

Limited information is available on the impact of the NaOH treatment on table olive fermentations, and for this reason a polyphasic approach has been adopted here to investigate its effect on the fermentation dynamics and bacterial biodiversity. The microbial counts of the main groups involved in the transformation have not shown any differences, apart from a more prompt start of the fermentation when the olives were subjected to the NaOH treatment. The data produced by culture-independent analyses highlighted that the fermentation of table olives not treated with NaOH is the result of the coexistence of two different ecosystems: the surface of the olives and the brines. A sodium hydroxide treatment not only eliminates this difference, but also affects the bacterial ecology of the olives to a great extent. As proved by high-throughput sequencing, the fermentation of the olives not treated with NaOH was characterized by the presence of halophilic bacteria, which were substituted by Lactobacillus at the later stages of the fermentation, while enterobacteria were dominant when the olives were treated with sodium hydroxide. Higher biodiversity was found for Lactobacillus plantarum isolated during untreated fermentation. Different biotypes were found on the olive surface and in the brines. When the debittering process was carried out, a decrease in the number of L. plantarum biotypes were observed and those originating from the surface of the olive did not differentiate from the ones present in the brines.

Complex coupled metabolic and prokaryotic community responses to increasing temperatures in anaerobic marine sediments: critical temperatures and substrate changes

The impact of temperature (0–80°C) on anaerobic biogeochemical processes and prokaryotic communities in marine sediments (tidal flat) was investigated in slurries for up to 100 days. Temperature had a non-linear effect on biogeochemistry and prokaryotes with rapid changes over small temperature intervals. Some activities (e.g. methanogenesis) had multiple ‘windows’ within a large temperature range (∼10 to 80°C). Others, including acetate oxidation, had maximum activities within a temperature zone, which varied with electron acceptor [metal oxide (up to ∼34°C) and sulphate (up to ∼50°C)]. Substrates for sulphate reduction changed from predominantly acetate below, and H2 above, a 43°C critical temperature, along with changes in activation energies and types of sulphate-reducing Bacteria. Above ∼43°C, methylamine metabolism ceased with changes in methanogen types and increased acetate concentrations (>1 mM). Abundances of uncultured Archaea, characteristic of deep marine sediments (e.g. MBGD Euryarchaeota, ‘Bathyarchaeota’) changed, indicating their possible metabolic activity and temperature range. Bacterial cell numbers were consistently higher than archaeal cells and both decreased above ∼15°C. Substrate addition stimulated activities, widened some activity temperature ranges (methanogenesis) and increased bacterial (×10) more than archaeal cell numbers. Hence, additional organic matter input from climate-related eutrophication may amplify the impact of temperature increases on sedimentary biogeochemistry.

Driving factors of soil microbial ecology in alpine, mid-latitude patterned grounds (NW Italian Alps)

Patterned ground (PG) is one of the most evident expressions of cryogenic processes affecting periglacial soils, where macroscopic, repeated variations in soil morphology seem to be associated with small-scale edaphic and vegetation gradients, potentially influencing also microbial communities. While for high-latitude environments only few studies on PG microbiology are available, the alpine context, where PG features are rarer, is almost unexplored under this point of view. We followed a double approach, based on denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR), in order to investigate microbial community composition and abundance of phylogenetic markers and functional genes (bacterial and archaeal amoA) within single PG features and among different sites from four areas in the Western Italian Alps, characterized by different lithotypes. Bacterial, archaeal, and fungal community composition was quite homogeneous within single features, with more differences among samples collected from different lithologies. The abundance of phylogenetic and functional markers was uniform at different sites, except for the highest altitude one showing the lowest bacterial, archaeal, and ammonia-oxidizing archaea abundance. Nevertheless, at a small-scale level, a concentric distribution of microbial markers was described within single features, paralleling soil chemical property trends. These first results support the hypothesis that microbial ecology in alpine, periglacial ecosystems is driven by a complex series of environmental factors, such as lithology, altitude, and cryogenic activity, acting simultaneously on community shaping both in terms of diversity and abundance.

Prokaryotic Diversity and Distribution in Different Habitats of an Alpine Rock Glacier-Pond System

Rock glaciers (RG) are assumed to influence the biogeochemistry of downstream ecosystems because of the high ratio of rock:water in those systems, but no studies have considered the effects of a RG inflow on the microbial ecology of sediments in a downstream pond. An alpine RG-pond system, located in the NW Italian Alps has been chosen as a model, and Bacteria and Archaea 16S rRNA genes abundance, distribution and diversity have been assessed by qPCR and Illumina sequencing, coupled with geochemical analyses on sediments collected along a distance gradient from the RG inflow. RG surface material and neighbouring soil have been included in the analysis to better elucidate relationships among different habitats.Our results showed that different habitats harboured different, well-separated microbial assemblages. Across the pond, the main variations in community composition (e.g. Thaumarchaeota and Cyanobacteria relative abundance) and porewater geochemistry (pH, DOC, TDN and NH4+) were not directly linked to RG proximity, but to differences in water depth. Some microbial markers potentially linked to the presence of meltwater inputs from the RG have been recognised, although the RG seems to have a greater influence on the pond microbial communities due to its contribution in terms of sedimentary material.