Studies in the Amazon are being intensified to evaluate the alterations in the microbial communities of soils and sediments in the face of increasing deforestation and land-use changes in the region. However, since these environments present highly heterogeneous physicochemical properties, including contaminants that hinder nucleic acids isolation and downstream techniques, the development of best molecular practices is crucial. This work aimed to optimize standard protocols for DNA extraction and gene quantification by quantitative real-time PCR (qPCR) based on natural and anthropogenic soils and sediments (primary forest, pasture, Amazonian Dark Earth, and várzea, a seasonally flooded area) of the Eastern Amazon. Our modified extraction protocol increased the fluorometric DNA concentration by 48%, reaching twice the original amount for most of the pasture and várzea samples, and the 260/280 purity ratio by 15% to values between 1.8 to 2.0, considered ideal for DNA. The addition of bovine serum albumin in the qPCR reaction improved the quantification of the 16S rRNA genes of Archaea and Bacteria and its precision among technical replicates, as well as allowed their detection in previously non-amplifiable samples. It is concluded that the changes made in the protocols improved the parameters of the DNA samples and their amplification, thus increasing the reliability of microbial communities' analysis and its ecological interpretations.

Microorganisms are critical indicators of soil quality due to their essential role in maintaining ecosystem services. However, anthropogenic activities can disrupt the vital metabolic functions of these microorganisms. Considering that soil biology is often underestimated and traditional assessment methods do not capture its complexity, molecular methods can be used to assess soil health more effectively. This study aimed to identify the changes in soil microbial diversity and activity under different cocoa agroforestry systems, specially focusing on taxa and functions associated to carbon and nitrogen cycling. Soils from three different cocoa agroforestry systems, including a newly established agroforestry with green fertilization (GF), rubber (Hevea brasiliensis)–cocoa intercropping (RC), and cocoa plantations under Cabruca (cultivated under the shave of native forest) (CAB) were analyzed and compared using metagenomic and metaproteomic approaches. Samples from surrounding native forest and pasture were used in the comparison, representing natural and anthropomorphic ecosystems. Metagenomic analysis revealed a significant increase in Proteobacteria and Basidiomycota and the genes associated with dissimilatory nitrate reduction in the RC and CAB areas. The green fertilization area showed increased nitrogen cycling activity, demonstrating the success of the practice. In addition, metaproteomic analyses detected enzymes such as dehydrogenases in RC and native forest soils, indicating higher metabolic activity in these soils. These findings underscore the importance of soil management strategies to enhance soil productivity, diversity, and overall soil health. Molecular tools are useful to demonstrate how changes in agricultural practices directly influence the microbial community, affecting soil health.

The Amazonian floodplain forests are dynamic ecosystems of great importance for the regional hydrological and biogeochemical cycles and provide a significant contribution to the global carbon balance. Unique geochemical factors may drive the microbial community composition and, consequently, affect CH ₄ emissions across floodplain areas. Here we provide the first report of the in situ seasonal dynamics of CH ₄ cycling microbial communities in Amazonian floodplains. We asked how abiotic factors may affect both overall and CH ₄ cycling microbial communities and further investigated their responses to seasonal changes. We collected sediment samples during wet and dry seasons from three different types of floodplain forests, along with upland forest soil samples, from the Eastern Amazon, Brazil. We used high-resolution sequencing of archaeal and bacterial 16S rRNA genes combined with real-time PCR to quantify Archaea and Bacteria, as well as key functional genes indicative of the methanogenic (methyl coenzyme-M reductase – mcr A) and methanotrophic (particulate methane monooxygenase – pmo A) metabolisms. Methanogens were found to be present in high abundance in floodplain sediments and they seem to resist to dramatic seasonal environmental changes. Methanotrophs known to use different pathways to oxidise CH ₄ were detected, including anaerobic archaeal and bacterial taxa, indicating that a wide metabolic diversity may be harboured in this highly variable environment. The floodplain environmental variability, which is affected by the river origin, drives not only the sediment chemistry, but also the composition of the microbial communities. The results presented may contribute to the understanding of the current state of CH ₄ cycling in this region.