Hanna Miettinen

Evaluation of surface contamination and the presence of Listeria monocytogenes in fish processing factories.

The main objective of this study was to determine the level of surface contamination in fish processing factories and the presence of Listeria in the factory environment and products. Another objective was evaluation of the different hygiene-monitoring methods. Total aerobic heterotrophic and enterobacteria, yeast and mold samples were collected and ATP levels measured in 28 factories. The number of well or adequately washed and disinfected factories was small (2 of 28), in terms of total aerobic heterotrophic bacterial counts on the surfaces. Most surfaces contaminated with bacteria were heavily contaminated. Results of the ATP and the total bacteria contact agar slide methods were poorly correlated (r = 0.21) although 68% of the samples were categorized as good to moderate or unacceptable with both methods. The Listeria-positive surface samples usually contained increased numbers of total bacteria (70.9%). The contamination of products and raw fish together with Listeria spp. was 45% and with Listeria monocytogenes 12%. Cold smoked fish was the most contaminated, with 75% Listeria spp. and cold salted fish with 20% L. monocytogenes. Listeria innocua was found in the samples more than twice as often as L. monocytogenes.

Revealing the unexplored fungal communities in deep groundwater of crystalline bedrock fracture zones in Olkiluoto, Finland.

The diversity and functional role of fungi, one of the ecologically most important groups of eukaryotic microorganisms, remains largely unknown in deep biosphere environments. In this study we investigated fungal communities in packer-isolated bedrock fractures in Olkiluoto, Finland at depths ranging from 296 to 798 m below surface level. DNA- and cDNA-based high-throughput amplicon sequencing analysis of the fungal internal transcribed spacer (ITS) gene markers was used to examine the total fungal diversity and to identify the active members in deep fracture zones at different depths. Results showed that fungi were present in fracture zones at all depths and fungal diversity was higher than expected. Most of the observed fungal sequences belonged to the phylum Ascomycota. Phyla Basidiomycota and Chytridiomycota were only represented as a minor part of the fungal community. Dominating fungal classes in the deep bedrock aquifers were Sordariomycetes, Eurotiomycetes, and Dothideomycetes from the Ascomycota phylum and classes Microbotryomycetes and Tremellomycetes from the Basidiomycota phylum, which are the most frequently detected fungal taxa reported also from deep sea environments. In addition some fungal sequences represented potentially novel fungal species. Active fungi were detected in most of the fracture zones, which proves that fungi are able to maintain cellular activity in these oligotrophic conditions. Possible roles of fungi and their origin in deep bedrock groundwater can only be speculated in the light of current knowledge but some species may be specifically adapted to deep subsurface environment and may play important roles in the utilization and recycling of nutrients and thus sustaining the deep subsurface microbial community.

Prevalence of Listeria monocytogenes in, and Microbiological and Sensory Quality of, Rainbow Trout, Whitefish, and Vendace Roes from Finnish Retail Markets

The prevalence of Listeria monocytogenes in retail roe, as well as the microbiological and sensory qualities of the roe, were studied for three fish species under three different storage conditions. A total of 147 Finnish rainbow trout (Oncorhynchus mykiss), whitefish (Coregonus lavaretus), vendace (Coregonus albula), and burbot (Lota lota) roe samples were bought fresh, frozen, or frozen-thawed from Finnish retail markets. The overall prevalence of L. monocytogenes was 5%; however, the prevalence of the pathogen in fresh roe was 18%. Fresh-bought roe tested positive for Listeria spp. and for L. monocytogenes, respectively, 5 and 20 times as often as did frozen and frozen-thawed roe products combined. The microbiological quality (analyzed as total aerobic heterotrophic bacteria and coliform bacteria) of 78% of the roe samples was unacceptable. Frozen roe samples were found to have the best microbiological quality. According to the results of a sensory evaluation, at least one sensory attribute (appearance, odor freshness, texture, and freshness of taste) was unacceptable for 29% of the roe samples studied. The sensory quality of roe samples bought fresh was better than that of roe samples bought frozen or frozen-thawed. From the results of this study, it is concluded that both the microbiological and the sensory qualities of roe at the retail level need to be improved.

Microbiome composition and geochemical characteristics of deep subsurface high-pressure environment, Pyhäsalmi mine Finland

Pyhäsalmi mine in central Finland provides an excellent opportunity to study microbial and geochemical processes in a deep subsurface crystalline rock environment through near-vertical drill holes that reach to a depth of more than two kilometers below the surface. However, microbial sampling was challenging in this high-pressure environment. Nucleic acid yields obtained were extremely low when compared to the cell counts detected (1.4 × 104 cells mL−1) in water. The water for nucleic acid analysis went through high decompression (60–130 bar) during sampling, whereas water samples for detection of cell counts by microscopy could be collected with slow decompression. No clear cells could be identified in water that went through high decompression. The high-pressure decompression may have damaged part of the cells and the nucleic acids escaped through the filter. The microbial diversity was analyzed from two drill holes by pyrosequencing amplicons of the bacterial and archaeal 16S rRNA genes and from the fungal ITS regions from both DNA and RNA fractions. The identified prokaryotic diversity was low, dominated by Firmicute, Beta- and Gammaproteobacteria species that are common in deep subsurface environments. The archaeal diversity consisted mainly of Methanobacteriales. Ascomycota dominated the fungal diversity and fungi were discovered to be active and to produce ribosomes in the deep oligotrophic biosphere. The deep fluids from the Pyhäsalmi mine shared several features with other deep Precambrian continental subsurface environments including saline, Ca-dominated water and stable isotope compositions positioning left from the meteoric water line. The dissolved gas phase was dominated by nitrogen but the gas composition clearly differed from that of atmospheric air. Despite carbon-poor conditions indicated by the lack of carbon-rich fracture fillings and only minor amounts of dissolved carbon detected in formation waters, some methane was found in the drill holes. No dramatic differences in gas compositions were observed between different gas sampling methods tested. For simple characterization of gas composition the most convenient way to collect samples is from free flowing fluid. However, compared to a pressurized method a relative decrease in the least soluble gases may appear.

Pasteurization of rainbow trout roe: Listeria monocytogenes and sensory analyses

D- and z-values for a mixture of four Listeria monocytogenes strains originating from the roe of different fish species were determined in rainbow trout (Oncorhynchus mykiss) roe. The D60- and D63-values obtained were 1.60 and 0.44 min, respectively, and the z-value was 5.36 degrees C accordingly. In pilot-scale experiments, rainbow trout roe (100 g) was vacuum packaged into glass jars and pasteurized both at 62 and 65 degrees C for 10 min. These treatments were enough to destroy 10(8) CFU/ g of L. monocytogenes cells, which was the highest possible Listeria cell count to grow in roe. On the basis of the determined z-value and calculation of pasteurization values, these experimental pasteurizations were found to theoretically destroy at least 45 log units of L. monocytogenes cells in rainbow trout roe. In addition, these pasteurization treatments did not significantly affect the sensory quality of the roe. The sensory quality of pasteurized vacuum-packaged rainbow trout roe stored at 3 degrees C was evaluated as good after 6 months of storage and not statistically different from the control that was frozen from the same roe lot as the pasteurized roe samples. Pasteurization of rainbow trout roe was proven to be an appropriate method for ensuring product safety with regard to L. monocytogenes and to stabilizing the sensory and microbial quality of roe. However, the safety risk caused by spore-forming bacteria still exists in pasteurized roe. Therefore, it has to be stored below 3 degrees C.

Evidences for Microbial Precipitation of Calcite in Speleothems from Krem Syndai in Jaintia Hills, Meghalaya, India

ABSTRACT Speleothems from Krem Syndai, Meghalaya in Northeast India were studied for their microbial diversity using 16S rDNA-based phylogenetic approach and conventional microbiological techniques along with geochemistry, mineralogy and in vitro experiments to understand participation of microorganisms in CaCO3 precipitation. Speleothems imaged by scanning electron microscopy showed round coccoid-like, sporangia-like and spinose calcified structures, numerous broken cocci shells with spotted interiors inside a calcite crystal, honeycomb long reticulate, smooth, flat, twisted, ribbon-like, tubular, beaded, microbe-mineralized filaments and extracellular polymeric substances (EPS). Fourier spectroscopy indicated the presence of various organic compounds. δ13C and δ18O isotopic ratios of speleothems ranged from −4.65 to −7.34‰ and −3.06 to −6.80‰, respectively. Total number of microbial cells using SYBR Gold was high. Fluorescence in situ hybridization (FISH) indicated approximately 3 × 105 to 5 × 105 cells g sed–1 in the speleothems out of which the number of microbes belonging to Eubacteria ranged from 1.8 × 105 to 3.6 × 105 cells, g sed–1. FISH showed ∼45% active microbial cells of the total cell number in samples. DNA-based high-throughput amplicon sequencing revealed 19 bacterial phyla in the speleothem. Approximately 42% of the sequences were similar to Proteobacteria (Alphaproteobacteria: 22.4%, Betaproteobacteria: 8.9%, Gammaproteobacteria: 8.6%). Sequences similar to Nitrospiraceae (22.8%) had the highest proportion of sequences belonging to a single family. Bacterial strains isolated from the speleothems raised alkalinity and precipitated calcite in the laboratory cultures which was confirmed by X-ray diffraction (XRD) analyses. These isolates belonged to Bacillus spp., Actinomycetes spp., Streptomyces spp., Pseudomonas spp., Micrococcus spp., Staphylococcus spp., Xanthobacter spp. and Arthrobacter spp. Overall, the results showed unequivocal evidence of bacterial fingerprints during CaCO3 precipitation in the cave.

Diversity and functionality of archaeal, bacterial and fungal communities in deep Archaean bedrock groundwater

The diversity and metabolic functions of deep subsurface ecosystems remain relatively unexplored. Microbial communities in previously studied deep subsurface sites of the Fennoscandian Shield are distinctive to each site. Thus, we hypothesized that the microbial communities of the deep Archaean bedrock fracture aquifer in Romuvaara, northern Finland, differ both in community composition and metabolic functionality from the other sites in the Fennoscandian Shield. We characterized the composition, functionality and substrate preferences of the microbial communities at different depths in a 600 m deep borehole. In contrast to other Fennoscandian deep biosphere communities studied to date, iron-oxidizing Gallionella dominated the bacterial communities, while methanogenic and ammonia-oxidizing archaea were the most prominent archaea, and a diverse fungal community was also detected. Potential for methane cycling and sulfate and nitrate reduction was confirmed by detection of the functional genes of these metabolic pathways. Organotrophs were less abundant, although carbohydrates were the most preferred of the tested substrates. The microbial communities shared features with those detected from other deep groundwaters with similar geochemistry, but the majority of taxa distinctive to Romuvaara are different from the taxa previously detected in saline deep groundwater in the Fennoscandian Shield, most likely because of the differences in water chemistry.

Evaluation of Long-Term Post Process Inactivation of Bioleaching Microorganisms

The H2020 BioMOre project (www.biomore.info, Grant Agreement #642456) tests the feasibility of in-situ bioleaching of copper in deep subsurface deposits in the Rudna Mine, Poland. Copper is leached using biologically produced ferric iron solution, which is recycled back to the in-situ reactor after re-oxidation by iron-oxidizing bacteria (IOB). From a post operational point of view, it is important that the biological processes applied during the operation can be controlled and terminated. Our goal was to determine the possibility to use natural saline mine water for the inactivation of introduced IOB remaining in the in-situ reactor after completion of the leaching process of the Kupferschiefer ore. Aerobic and anaerobic microcosms containing acid-leached (pH 2) sandstone or black shale from the Kupferschiefer in the Rudna mine were further leached with the effluent from an iron-oxidizing bioreactor, at a temperature of 30°C, for 10 days, to simulate in-situ leaching. After the removal of the iron solution, residing IOB were inactivated by filling the microcosms with saline water (65 g L-1 Cl-) originating from the mine. The saline water completely inactivated the IOB and the naturally occurring saline water of the mine can be used for long-term post process inactivation of bioleaching microorganisms.