Environmental sample collection for eDNA analysis v1

This document describes a series of protocols for the collection of environmental samples intended for the monitoring and surveillance of marine invasive species by means of eDNA metabarcoding analysis, as described in the associated publication (Fernandez et al. 2021: https://doi.org/10.1016/j.marpolbul.2021.112893).

RNA-Stable Isotope Probing v2

The following protocol describes how to perform an RNA-Stable Isotope Probing experiment. The scope of this protocol only covers the parts involving separating labelled RNA from unlabelled RNA using ultracentrifugation in a caesium trifluoroacetate density gradient and downstream quantification to evaluate whether the labelling and separation of the RNA were successful. Total RNA should be extracted from an environmental sample or an enrichment culture that was incubated with an isotopically-labelled substrate. Labelling can be of the carbon, oxygen or nitrogen in the RNA (or any combination of the 3). For environmental samples, we recommend extracting RNA using our protocol Total Nucleic Acids Extraction from Soil and purifying it using the Purification of RNA from Crude NA Extract protocol. This protocol is based on the following papers: Whiteley et al. (2007); Dumont et al. (2011); Angel and Conrad (2013). For a comprehensive discussion on how to design a SIP experiment and how to analyse the resulting data, we recommend referring to the recent book on the subject: Stable Isotope Probing: Methods and Protocols, especially chapters: 1-3 and 9-18.

RNA-Stable Isotope Probing v2

The following protocol describes how to perform an RNA-Stable Isotope Probing experiment. The scope of this protocol only covers the parts involving separating labelled RNA from unlabelled RNA using ultracentrifugation in a caesium trifluoroacetate density gradient and downstream quantification to evaluate whether the labelling and separation of the RNA were successful. Total RNA should be extracted from an environmental sample or an enrichment culture that was incubated with an isotopically-labelled substrate. Labelling can be of the carbon, oxygen or nitrogen in the RNA (or any combination of the 3). For environmental samples, we recommend extracting RNA using our protocol Total Nucleic Acids Extraction from Soil and purifying it using the Purification of RNA from Crude NA Extract protocol. This protocol is based on the following papers: Whiteley et al. (2007); Dumont et al. (2011); Angel and Conrad (2013). For a comprehensive discussion on how to design a SIP experiment and how to analyse the resulting data, we recommend referring to the recent book on the subject: Stable Isotope Probing: Methods and Protocols, especially chapters: 1-3 and 9-18.

Prevalence and epidemiological distribution of selected foodborne pathogens in human and different environmental samples in Ethiopia: a systematic review and meta-analysis

Bacterial Foodborne Pathogens (FBP) are the commonest cause of foodborne illness or foodborne diseases (FBD) worldwide. They contaminate food at any stages in the entire food chain, from farm to dining-table. Among these, the Diarrheagenic Escherichia coli (DEC), Non typhoidal Salmonella (NTS), Shigella spp. and Campylobacter spp. are responsible for a large proportion of illnesses, deaths; and, particularly, as causes of acute diarrheal diseases. Though existing studies indicate the problem may be severe in developing countries like Ethiopia, the evidence is commonly based on fragmented data from individual studies. A review of published and unpublished manuscripts was conducted to obtain information on major FBP and identify the gaps in tracking their source attributions at the human, animal and environmental interface. A total of 1753 articles were initially retrieved after restricting the study period to between January 2000 and July 2020. After the second screening, only 51 articles on the humans and 43 on the environmental sample based studies were included in this review. In the absence of subgroups, overall as well as human stool and environmental sample based pooled prevalence estimate of FBP were analyzed. Since, substantial heterogeneity is expected, we also performed a subgroup analyses for principal study variables to estimate pooled prevalence of FBP at different epidemiological settings in both sample sources. The overall random pooled prevalence estimate of FBP (Salmonella, pathogenic Escherichia coli (E. coli), Shigella and Campylobacter spp.) was 8%; 95% CI: 6.5–8.7, with statistically higher (P < 0.01) estimates in environmental samples (11%) than in human stool (6%). The subgroup analysis depicted that Salmonella and pathogenic E. coli contributed to 5.7% (95% CI: 4.7–6.8) and 11.6% (95% CI: 8.8–15.1) respectively, of the overall pooled prevalence estimates of FBD in Ethiopia. The result of meta-regression showed, administrative regional state, geographic area of the study, source of sample and categorized sample size all significantly contributed to the heterogeneity of Salmonella and pathogenic E. coli estimates. Besides, the multivariate meta- regression indicated the actual study year between 2011 and 2015 was significantly associated with the environmental sample-based prevalence estimates of these FBP. This systematic review and meta-analysis depicted FBP are important in Ethiopia though majority of the studies were conducted separately either in human, animal or environmental samples employing routine culture based diagnostic method. Thus, further FBD study at the human, animal and environmental interface employing advanced diagnostic methods is needed to investigate source attributions of FBD in one health approach.

The Promotive Effect of Cyanobacteria and Chlorella sp. Foliar Biofertilization on Growth and Metabolic Activities of Willow (Salix viminalis L.) Plants as Feedstock Production, Solid Biofuel and Biochar as C Carrier for Fertilizers via Torrefaction Process

The effect of foliar application of Cyanobacteria and Chlorella sp. monocultures on physiological activity, element composition, development and biomass weight of basket willow (Salix viminalis L.) and the possibility to prepare biofuel from it in the fortification process was studied. Triple foliar plant spraying with non-sonicated monocultures of Cyanobacteria (Anabaena sp. PCC 7120, Microcystis aeruginosa MKR 0105) and Chlorella sp. exhibited a considerably progressive impact on metabolic activity and development of plants. This biofertilization increased cytomembrane impermeability, the amount of chlorophyll in plants, photosynthesis productivity and transpiration, as well as degree of stomatal opening associated with a decreased concentration of intercellular CO2, in comparison to control (treatments with water, Bio-Algeen S90 or with environmental sample). The applied strains markedly increased the element content (N, P, K) in shoots and the productivity of crucial growth enzymes: alkaline or acid phosphorylase, total dehydrogenases, RNase and nitrate reductase. Treatments did not affect energy properties of the burnt plants. These physiological events were associated with the improved growth of willow plants, namely height, length and amount of all shoots and their freshly harvested dry mass, which were increased by over 25% compared to the controls. The effectiveness of these treatments depended on applied monoculture. The plant spraying with Microcystis aeruginosa MKR 0105 was a little more effective than treatment with Chlorella sp. and Anabaena sp. or the environmental sample. The research demonstrate that the studied Cyanobacteria and Chlorella sp. monocultures have prospective and useful potential in production of Salix viminalis L., which is the basic energy plant around the word. In this work, a special batch reactor was used to produce torrefaction material in an inert atmosphere: nitrogen, thermogravimetric analysis and DTA analysis, like Fourier-transform infrared spectroscopy. The combustion process of Salix viminalis L. with TG-MS analysis was conducted as well as study on a willow torrefaction process, obtaining 30% mass reduction with energy loss close to 10%. Comparing our research results to other types of biomasses, the isothermal temperature of 245 °C during thermo-chemical conversion of willow for the carbonized solid biofuel production from Salix viminalis L. biomass fertilized with Cyanobacteria and Chlorella sp. is relatively low. At the end, a SEM-EDS analysis of ash from torrefied Salix viminalis L. after carbonization process was conducted.

Complete Genome Sequences of Bacillus cereus Group Phages AaronPhadgers, ALPS, Beyonphe, Bubs, KamFam, OmnioDeoPrimus, Phireball, PPIsBest, YungSlug, and Zainny

Here, we report genome sequences of 10 Bacillus cereus group-infecting bacteriophages. Each virus was isolated from an environmental sample, contained a double-stranded DNA genome, and belonged to the Myoviridae family. Nine phages exhibit a conserved genome structure, and one phage appears novel in genome structure, sequence, and protein content.