Metagenomics is a branch of genomics that aims to investigate complex microbial communities directly from environmental samples, without the need for laboratory cultivation. This methodol has had a significant impact across various fields, including human health, evolutionary biology, microbial ecology, agriculture, and biotechnology. However, the field faces significant challenges, including the complexity of analyzing large volumes of data, high computational resource requirements, limitations in current metagenomic tools and species identification, all of which hinder the full exploration of metagenomic data. To address these challenges, this dissertation aims to develop a metagenomic tool for taxonomic classification. To this end, a systematic review of state-of-the-art metagenomic tools was conducted, evaluating their classification algorithms and limitations. A review of taxonomic databases was also carried out, leading to the development of a standardized dataset representative of all domains of life, enabling fair comparison and validation of new methods. Based on these foundations, HYMET (Hybrid Metagenomic Tool) was introduced, an innovative classifier that combines optimized k-mer screening with a smart alignment system that dynamically switches between high-precision and efficient tools depending on the genomic characteristics of the sample. HYMET is based on the dynamic construction of a reference database specific to each sample and a robust weighted classification algorithm. The results demonstrate HYMET’s superior performance, being mutation-resistant and capable of classifying all domains with F1 scores above 0.90, while maintaining computational efficiency and processing datasets in under an hour. HYMET operates with minimal resource consumption: the main installation requires only 2.82 GB, dynamically adjusting storage usage between 5–30 GB, with efficient CPU (13.83%) and memory (10.91%) utilization. This dissertation not only advances the state-of-the-art in metagenomic classification but also lays the groundwork for future innovations, driving progress in the study of microbial life.

Global climate change has the potential to seriously and adversely affect marine ecosystem functioning. Numerous experimental and modeling studies have demonstrated how predicted ocean acidification and increased ultraviolet radiation (UVR) can affect marine microbes. However, researchers have largely ignored interactions between ocean acidification, increased UVR and anthropogenic pollutants in marine environments. Such interactions can alter chemical speciation and the bioavailability of several organic and inorganic pollutants with potentially deleterious effects, such as modifying microbial-mediated detoxification processes. Microbes mediate major biogeochemical cycles, providing fundamental ecosystems services such as environmental detoxification and recovery. It is, therefore, important that we understand how predicted changes to oceanic pH, UVR, and temperature will affect microbial pollutant detoxification processes in marine ecosystems. The intrinsic characteristics of microbes, such as their short generation time, small size, and functional role in biogeochemical cycles combined with recent advances in molecular techniques (e.g., metagenomics and metatranscriptomics) make microbes excellent models to evaluate the consequences of various climate change scenarios on detoxification processes in marine ecosystems. In this review, we highlight the importance of microbial microcosm experiments, coupled with high-resolution molecular biology techniques, to provide a critical experimental framework to start understanding how climate change, anthropogenic pollution, and microbiological interactions may affect marine ecosystems in the future.

During the European Commission's Framework Six Programme, HERMES, we investigated three main areas along the European margin, each characterized by the presence of seep-related structures exhibiting different intensity of activity and biological diversity. These areas are: (1) the Nordic margin with the Hakon Mosby mud volcano and many pockmarks, (2) the Gulf of Cadiz, and (3) the eastern Mediterranean with its hundreds of mud volcanoes and brine pool structures. One of the main goals of the HERMES project was to unravel the biodiversity associated with these seep-associated ecosystems, and to understand their driving forces and functions, using an integrated approach. Several multidisciplinary research cruises to these three areas provided evidence of high variability in ecosystem processes and associated biodiversity at different spatial scales, illustrating the "hotspot" nature of these deep water systems.

<jats:p>Agricultural soils are responsible for ecological functions and services that include primary production of food, fiber and fuel, nutrient cycling, carbon cycling and storage, water infiltration and purification, among others. Fungi are important drivers of most of those ecosystem services. Given the importance of fungi in agricultural soils, in this study, we aimed to characterize and analyse the changes of the soil fungal communities of three cropping systems from Madeira Island, where family farming is predominant, and investigate the response of fungi and its functional groups to soil physicochemical properties. To achieve that, we sequenced amplicons targeting the internal transcribed spacer 1 (ITS1) of the rRNA region, to analyse soil samples from 18 agrosystems: 6 vineyards (V), 6 banana plantations (B) and 6 vegetable plantations (H). Our results showed that alpha diversity indices of fungal communities are similar in the three cropping systems, but fungal composition and functional aspects varied among them, with more pronounced differences in B. Ascomycota, Basidiomycota, and Mortierellomycota were the main phyla found in the three cropping systems. Agaricomycetes and Sordariomycetes are the predominant classes in B, representing 23.8 and 22.4%, respectively, while Sordariomycetes (27.9%) followed by Eurotiomycetes (12.3%) were the predominant classes in V and Sordariomycetes (39.2%) followed by Tremellomycetes (8.9%) in the H. Saprotrophs are the fungal group showing higher relative abundance in the three cropping systems, followed by plant pathogens. Regarding symbionts, endophytes were highly observed in B, while mycorrhizal fungi was predominant in V and H. The structure of fungal communities was mainly correlated with soil content of P, K, N, Fe, and Cu. In addition, we identified bioindicators for each cropping system, which means that cultivated crops are also drivers of functional groups and the composition of communities. Overall, the three cropping systems favored diversity and growth of taxa that play important roles in soil, which highlights the importance of conservative management practices to maintain a healthy and resilient agrosystem.</jats:p>

The effects of sewage sludge selectively enriched with Cd and Zn, both singly and in combination, on the bacterial, fungal, Alphaproteobacteria and Actinobacteria communities of a soil under arable or grassland management were studied with a PCR-DGGE approach. The effects of Cd and Zn were evaluated after a short time (7 d) when the Cd and Zn solubility were low and the C availability was high, and again after 180 d when the labile sludge C was mineralized and the effects of heavy metals predominated. In the arable soil all treatments induced significant short-term changes in the studied microbial groups, and long-term changes were observed in Actinobacteria and fungal communities. In the grassland soil, all treatments induced significant short-term changes in the studied microbial groups except for Alphaproteobacteria and fungi, and long-term effects on the actinobacteria and fungal communities. It was concluded that incorporation of Cd- and Zn-rich sludge into soils may have both short- and long-term effects on various bacterial phylogenetic groups whereas the metals may be better tolerated by the dominant soil fungi. In this study the impact was greater in arable than in grassland soil.

Background: Exploring different microbial sources for biotechnological production of organic acids is important. Dutch and Thai cow rumen samples were used as inocula to produce organic acid from starch waste in anaerobic reactors. Organic acid production profiles were determined and microbial communities were compared using 16S ribosomal ribonucleic acid gene amplicon pyrosequencing. Results In both reactors, lactate was the main initial product and was associated with growth of Streptococcus spp. (86% average relative abundance). Subsequently, lactate served as a substrate for secondary fermentations. In the reactor inoculated with rumen fluid from the Dutch cow, the relative abundance of Bacillus and Streptococcus increased from the start, and lactate, acetate, formate and ethanol were produced. From day 1.33 to 2, lactate and acetate were degraded, resulting in butyrate production. Butyrate production coincided with a decrease in relative abundance of Streptococcus spp. and increased relative abundances of bacteria of other groups, including Parabacteroides, Sporanaerobacter, Helicobacteraceae, Peptostreptococcaceae and Porphyromonadaceae. In the reactor with the Thai cow inoculum, Streptococcus spp. also increased from the start. When lactate was consumed, acetate, propionate and butyrate were produced (day 34). After day 3, bacteria belonging to five dominant groups, Bacteroides, Pseudoramibacter_Eubacterium, Dysgonomonas, Enterobacteriaceae and Porphyromonadaceae, were detected and these showed significant positive correlations with acetate, propionate and butyrate levels. Conclusions The complexity of rumen microorganisms with high adaptation capacity makes rumen fluid a suitable source to convert organic waste into valuable products without the addition of hydrolytic enzymes. Starch waste is a source for organic acid production, especially lactate.

Background & Aim: Floating treatment wetlands (FTW) are a nature-based solution for water treatment and also delivered a wide range of other ecosystem services. The establishment of a FTW relies, mainly, on the presence of plants in a floating structure, where direct contact of the biological components enables the uptake and removal of contaminants from the water, through phytoremediation processes (Calheiros et al, 2023). However, there is still a lack of knowledge concerning the performance and the impact on biodiversity of this NBS. The aim of this study was to assess the biodiversity associated with a FTW installed in 2018 in a freshwater pond using cork agglomerate as floating platform. Methods: The flora comprised specimens of Iris germanica, Acorus gramineus, Caltha palustris and Typha latifolia. Samples from the platform biofilm and plant rhizosphere were analyzed by 16S rRNA sequencing technique to identify culturable bacterial communities and they were characterized on their ability to produce plant growth- promoting substances (PGPS). Macroinvertebrates were identified and biodiversity indexes calculated. Results: Preliminary results identified a variety of bacterial communities and the presence of strains with production of PGPS. Macroinvertebrates were classified mainly in the order Odonata, with more than 80% belonging to genus Coenagrion. Conclusions: Further research should explore the use of the bacterial strains with best production of PGPS in phytoremediation strategies and with plants undergoing climate change conditions. This study highlights the use of FTW as a water management technology and to promote biodiversity, as its aesthetic value.

Traditional foods are increasingly valued by consumers, whose attention and purchase willingness are highly influenced by other claims such as ‘natural’, ‘sustainable’, and ‘clean label’. The purpose of the present study was to evaluate the impact of a novel non-thermal food processing method (i.e., HPP-assisted biocontrol combining mild high hydrostatic pressure, listeriophage Listex, and pediocin PA-1 producing Pediococcus acidilactici) on the succession of bacterial communities and quality of a fermented sausage model. A comparative analysis of instrumental color, texture, and lipid peroxidation revealed no significant differences (p > 0.05) in these quality parameters between non- and minimally processed fermented sausages throughout 60-day refrigerated storage (4 °C). The microbiota dynamics of biotreated and untreated fermented sausages were assessed by 16S rRNA next-generation sequencing, and the alpha and beta diversity analyses revealed no dissimilarity in the structure and composition of the bacterial communities over the analyzed period. The innovative multi-hurdle technology proposed herein holds valuable potential for the manufacture of traditional fermented sausages while preserving their unique intrinsic characteristics.

Microbial monitoring of constructed wetlands (CWs) treating domestic wastewater is generally scarce, despite the need of more knowledge about its biocenosis. The sanitation quality of a wastewater treated in a CW is a crucial aspect, mainly when the receiving water body is used as a swimming and/or recreation area. The present study was carried out in a horizontal subsurface flow CW planted with Phragmites australis receiving pre-treated domestic wastewater (mean flow 50 m3 day−1), from a population of about 300 inhabitants. The monitoring programme undertaken during the first year operation, revealed removal efficiencies of 61% BOD5, 44% COD, and 65% TSS for inlet water with ca. 90 mg L−1 BOD5, 157 mg L−1 COD, and 17 mg L−1 TSS. Total Coliform (TC) and Faecal Coliform (FC) bacteria were removed from wastewater (mean inlet values of 5 × 106 CFU 100 mL−1 TC and of 9 × 105CFU 100 mL−1 FC), with efficiencies of 92 and 97%, respectively. The dynamics of microbial communities established in the system assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), had revealed a high bacterial diversity within the system, with no relevant differences in composition at the CW inlet and outlet but exhibiting temporal differences in bacterial communities.

The reuse of treated wastewater could be a promising measure to attenuate the water scarcity burden. In agriculture, irrigation with wastewater may contribute to improve production yields, reduce the ecological footprint and promote socioeconomic benefits. However, it cannot be considered exempt of adverse consequences in environmental and human health. Apart from the introduction of some biological and chemical hazardous agents, the disturbance of the indigenous soil microbial communities and, thus, of vital soil functions impacting soil fertility may occur. The consequences of these disturbances are still poorly understood. This chapter summarises the physicochemical and microbiological alterations in soil resultant from irrigation with treated wastewater that are described in scientific literature. These alterations, which involve a high complexity of variables (soil, wastewater, climate, vegetal cover), may have impacts on soil quality and productivity. In addition, possible health risks may arise, in particular through the direct or indirect contamination of the food chain with micropollutants, pathogens or antibiotic resistance determinants. The current state of the art suggests that irrigation with treated wastewater may have a multitude of long-term implications on soil productivity and public health. Although further research is needed, it seems evident that the analysis of risks associated with irrigation with treated wastewater must take into account not only the quality of water, but other aspects as diverse as soil microbiota, soil type or the cultivated plant species.