Mix-method toolbox for monitoring greenhouse gas production and microbiome responses to soil amendments.

In this study, we adopt an interdisciplinary approach, integrating agronomic field experiments with soil chemistry, molecular biology techniques, and statistics to investigate the impact of organic residue amendments, such as vinasse (a by-product of sugarcane ethanol production), on soil microbiome and greenhouse gas (GHG) production. The research investigates the effects of distinct disturbances, including organic residue application alone or combined with inorganic N fertilizer on the environment. The methods assess soil microbiome dynamics (composition and function), GHG emissions, and plant productivity. Detailed steps for field experimental setup, soil sampling, soil chemical analyses, determination of bacterial and fungal community diversity, quantification of genes related to nitrification and denitrification pathways, measurement and analysis of gas fluxes (N2O, CH4, and CO2), and determination of plant productivity are provided. The outcomes of the methods are detailed in our publications (Lourenço et al., 2018a; Lourenço et al., 2018b; Lourenço et al., 2019; Lourenço et al., 2020). Additionally, the statistical methods and scripts used for analyzing large datasets are outlined. The aim is to assist researchers by addressing common challenges in large-scale field experiments, offering practical recommendations to avoid common pitfalls, and proposing potential analyses, thereby encouraging collaboration among diverse research groups.•Interdisciplinary methods and scientific questions allow for exploring broader interconnected environmental problems.•The proposed method can serve as a model and protocol for evaluating the impact of soil amendments on soil microbiome, GHG emissions, and plant productivity, promoting more sustainable management practices.•Time-series data can offer detailed insights into specific ecosystems, particularly concerning soil microbiota (taxonomy and functions).

Carbon and Nutrients from Organic Residues Modulate the Dynamics of Prokaryotic and Fungal Communities.

Inputs of carbon (C) and nutrients from organic residues may select specific microbes and shape the soil microbial community. However, little is known about the abiotic filtering of the same residues with different nutrient concentrations applied to the soil. In our study, we explored how applying organic residue, vinasse, as fertilizer in its natural state (V) versus its concentrated form (CV) impacts soil microbiota. We conducted two field experiments, evaluating soil prokaryotic and fungal communities over 24 and 45 days with vinasse (V or CV) plus N fertilizer. We used 16S rRNA gene and ITS amplicon sequencing. Inorganic N had no significant impact on bacterial and fungal diversity compared to the control. However, the varying concentrations of organic C and nutrients in vinasse significantly influenced the soil microbiome structure, with smaller effects observed for V compared to CV. Prokaryotic and fungal communities were not correlated (co-inertia: RV coefficient = 0.1517, p = 0.9708). Vinasse did not change the total bacterial but increased the total fungal abundance. A higher C input enhanced the prokaryotic but reduced the fungal diversity. Our findings highlight vinasse's role as an abiotic filter shaping soil microbial communities, with distinct effects on prokaryotic and fungal communities. Vinasse primarily selects fast-growing microorganisms, shedding light on the intricate dynamics between organic residues, nutrient concentrations, and soil microbes.

Dynamics and resilience of soil mycobiome under multiple organic and inorganic pulse disturbances.

Disturbances in soil can cause short-term soil changes, consequently changes in microbial community what may result in long-lasting ecological effects. Here, we evaluate how multiple pulse disturbances effect the dynamics and resilience of fungal community, and the co-occurrence of fungal and bacterial communities in a 389 days field experiment. We used soil under sugarcane cultivation as soil ecosystem model, and organic residue (vinasse - by-product of sugarcane ethanol production) combined or not with inorganic (organic residue applied 30 days before or together with mineral N fertilizer) amendments as disturbances. Application of organic residue alone as a single disturbance or 30 days prior to a second disturbance with mineral N resulted in similar changes in the fungal community. The simultaneous application of organic and mineral N as a single pulse disturbance had the greatest impact on the fungal community. Organic amendment increased the abundance of saprotrophs, fungal species capable of denitrification, and fungi described to have copiotrophic and oligotrophic lifestyles. Furthermore, the changes in the fungal community were not correlated with the changes in the bacterial community. The fungal community was neither resistant nor resilient to organic and inorganic disturbances over the one-year sampling period. Our findings provide insights on the immediate and delayed responses of the fungal community over one year to disturbance by organic and inorganic amendments.

Strategies to mitigate the nitrous oxide emissions from nitrogen fertilizer applied with organic fertilizers in sugarcane.

Vinasse is a major byproduct of the sugarcane biofuel industry, recycled in the fields. However, there is evidence that the application of vinasse with mineral nitrogen (N) fertilizers in sugarcane enhances the emission of greenhouse gases (GHGs). Therefore, strategies are needed to decrease the environmental impacts caused by both inputs. We carried out three sugarcane field experiments by applying N fertilizer (ammonium nitrate) with types of vinasses (concentrated-CV and standard-V) in different combinations (vinasses with N fertilizer and vinasses one month before or after mineral N fertilization). The gases nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) were measured in one experiment fertilized in the beginning (fall/winter = dry season) and two experiments fertilized in the end (spring = rainy season) of the harvest season. Sugarcane fields were sinks rather than sources of CH4, while total carbon emitted as CO2 was similar between seasons and treatments. The effect of mineral fertilization and vinasses (CV and V) on N2O emissions was highly dependent on soil moisture (rain events). The N2O-N fertilizer emission factor (EF) varied from 0.07% to 0.51%, whereas the average EF of V and CV were 0.66% and 0.34%, respectively. On average across the three experiments, the combination of vinasse (CV or V) with N fertilizer increased the N2O emissions 2.9-fold compared to that of N fertilizer alone. For CV + N, the EF was 0.94% of the applied N and 0.23% of the ammonium nitrate-N, and for V + N (EF = 0.47%), increased emissions were observed in two out of three experiments. The strategy of anticipating or postponing vinasse application by one month with respect to mineral N reduced the N2O emissions by 51% for CV, but not for V. Therefore, to avoid boosting N2O emissions, we suggest applying vinasses (CV and V) before or after mineral N fertilization.

Resilience of the resident soil microbiome to organic and inorganic amendment disturbances and to temporary bacterial invasion.

BACKGROUND: Vinasse, a by-product of sugarcane ethanol production, is recycled by sugarcane plantations as a fertilizer due to its rich nutrient content. However, the impacts of the chemical and microbial composition of vinasse on soil microbiome dynamics are unknown. Here, we evaluate the recovery of the native soil microbiome after multiple disturbances caused by the application of organic vinasse residue, inorganic nitrogen, or a combination of both during the sugarcane crop-growing season (389 days). Additionally, we evaluated the resistance of the resident soil microbial community to the vinasse microbiome. RESULTS: Vinasse applied alone or 30 days prior to N resulted in similar changes in the soil microbial community. Furthermore, the impact of the application of vinasse together with N fertilizer on the soil microbial community differed from that of N fertilizer alone. Organic vinasse is a source of microbes, nutrients, and organic matter, and the combination of these factors drove the changes in the resident soil microbial community. However, these changes were restricted to a short period of time due to the capacity of the soil community to recover. The invasive bacteria present in the vinasse microbiome were unable to survive in the soil conditions and disappeared after 31 days, with the exception of the Acetobacteraceae (native in the soil) and Lactobacillaceae families. CONCLUSION: Our analysis showed that the resident soil microbial community was not resistant to vinasse and inorganic N application but was highly resilient.

Recycling organic residues in agriculture impacts soil-borne microbial community structure, function and N2O emissions.

Recycling residues is a sustainable alternative to improve soil structure and increase the stock of nutrients. However, information about the magnitude and duration of disturbances caused by crop and industrial wastes on soil microbial community structure and function is still scarce. The objective of this study was to investigate how added residues from industry and crops together with nitrogen (N) fertiliser affect the microbial community structure and function, and nitrous oxide (N2O) emissions. The experimental sugarcane field had the following treatments: (I) control with nitrogen, phosphorus, and potassium (NPK), (II) sugarcane straw with NPK, (III) vinasse (by-product of ethanol industry) with NP, and (IV) vinasse plus sugarcane straw with NP. Soil samples were collected on days 1, 3, 6, 11, 24 and 46 of the experiment for DNA extraction and metagenome sequencing. N2O emissions were also measured. Treatments with straw and vinasse residues induced changes in soil microbial composition and potential functions. The change in the microbial community was highest in the treatments with straw addition with functions related to decomposition of different ranges of C-compounds overrepresented while in vinasse treatment, the functions related to spore-producing microorganisms were overrepresented. Furthermore, all additional residues increased microorganisms related to the nitrogen metabolism and vinasse with straw had a synergetic effect on the highest N2O emissions. The results highlight the importance of residues and fertiliser management in sustainable agriculture.