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).

The Serpulidae are a large family of sedentary polychaetes, characterized by a calcareous habitation tube, which they cannot leave. The calcium carbonate tube is in the form of both aragonite and calcite, in fairly constant ratio for each taxon. Tubes are cemented firmly to any hard substrate (in only few species tubes are free). Although in the majority of the species the tubes encrust the substrate for all their length, the distal part may eventually detach and grow erectly. Certain species in dense populations build tubes vertical to the substrate in clumps and cement the tubes to each other. This gives serpulids the capability of forming reef-life structures when densely settling. Despite the relative smallness of the individual tubes (rarely longer than 15cm and wider than 1cm), such reef-like structures may cover tens of m2, with a layer more than 1m thick. Serpulid reefs can be divided roughly into seven groups, according to the building modality and the type of habitat they occupy: (i) pseudocolonies; (ii) littoral belts; (iii) subtidal to deep-water reefs; (iv) reefs in coastal lakes and harbours; (v) brackish water reefs; (vi) tapestries in freshwater caves; (vii) biostalactites inside marine caves. The role of serpulid reefs in the ecosystems they inhabit is multifarious and may be distinguished in functions (biomass and production, benthic pelagic coupling, resistance and resilience, reproductive and survivorship strategies, trophodynamics, bioconstruction, living space and refuge, nursery, sediment formation and retention, food for other species, carbonate deposition and storage) and services (water clearance, reef associated fishery, cultural benefits). On the other hand, many serpulids are important constituents of biological fouling, and their calcareous masses damage submerged artefacts, causing huge economic costs. Positive and negative roles of serpulid reefs need to be compared with common metrics; the overall balance, however, is still to be assessed.

With rapid global urbanization, anthropogenic activities alter aquatic biota in urban rivers through inputs of dissolved organic carbon (DOC) and nutrients. Microorganisms-mediated global element cycles provide functions in maintaining microbial ecology stability. The DOC (bottom-up control) and microbial predation (top-down control) may synergistically drive the competition and evolution of aquatic microbial communities, as well as their resistance and resilience, for which experimental evidences remain scarce. In this study, laboratory sediment-water column experiments were employed to mimic the organic carbon-driven water blackening and odorization process in urban rivers and to elucidate the impact of DOC on microbial ecology stability. Results showed that low (25-75 mg/L) and high DOC (100-150 mg/L) changed the aquatic microbial community assemblies in different patterns: (1) the low DOC enriched K-selection microorganisms (e.g., C39, Tolumonas and CR08G) with low biomass and low resilience, as well as high resistance to perturbations in changing microbial community assemblies; (2) the high DOC was associated with r-selection microorganisms (e.g., PSB-M-3 and Clostridium) with high biomass and improved resilience, together with low resistance detrimental to microbial ecology stability. Overall, this study provided new insight into the impact of DOC on aquatic microbial community stability, which may help guide sustainable urban river management.

Traditionally, resistance and resilience are associated with good ecological health, often underpinning restoration goals. However, degraded ecosystems can also be highly resistant and resilient, making restoration difficult: degraded communities often become dominated by hyper-tolerant species, preventing recolonization and resulting in low biodiversity and poor ecosystem function. Using streams as a model, we undertook a mesocosm experiment to test if degraded community presence hindered biological recovery. We established 12 mesocosms, simulating physically healthy streams. Degraded invertebrate communities were established in half, mimicking the post-restoration scenario of physical recovery without biological recovery. We then introduced a healthy colonist community to all mesocosms, testing if degraded community presence influenced healthy community establishment. Colonists established less readily in degraded community mesocosms, with larger decreases in abundance of sensitive taxa, likely driven by biotic interactions rather than abiotic constraints. Resource depletion by the degraded community likely increased competition, driving priority effects. Colonists left by drifting, but also by accelerating development, reducing time to emergence but sacrificing larger body size. Since degraded community presence prevented colonist establishment, our experiment suggests successful restoration must address both abiotic and biotic factors, especially those that reinforce the \'negative\' resistance and resilience which perpetuate degraded communities and are typically overlooked.

Climate change will alter precipitation patterns with consequences for soil C cycling. An understanding of how fluctuating soil moisture affects microbial processes is therefore critical to predict responses to future global change. We investigated how long-term experimental field drought influences microbial tolerance to lower moisture levels (\"resistance\") and ability to recover when rewetted after drought (\"resilience\"), using soils from a heathland which had been subjected to experimental precipitation reduction during the summer for 18 years. We tested whether drought could induce increased resistance, resilience, and changes in the balance between respiration and bacterial growth during perturbation events, by following a two-tiered approach. We first evaluated the effects of the long-term summer drought on microbial community functioning to drought and drying-rewetting (D/RW), and second tested the ability to alter resistance and resilience through additional perturbation cycles. A history of summer drought in the field selected for increased resilience but not resistance, suggesting that rewetting after drought, rather than low moisture levels during drought, was the selective pressure shaping the microbial community functions. Laboratory D/RW cycles also selected for communities with a higher resilience rather than increased resistance. The ratio of respiration to bacterial growth during D/RW perturbation was lower for the field drought-exposed communities and decreased for both field treatments during the D/RW cycles. This suggests that cycles of D/RW also structure microbial communities to respond quickly and efficiently to rewetting after drought. Our findings imply that microbial communities can adapt to changing climatic conditions and that this might slow the rate of soil C loss predicted to be induced by future cyclic drought.

The longitudinal structure of the environmental and phytoplankton variables was investigated in the Ebro River (NE Spain), which is heavily affected by water abstraction and regulation. A first exploration indicated that the phytoplankton community did not resist the impact of reservoirs and barely recovered downstream of them. The spatial analysis showed that the responses of the phytoplankton and environmental variables were not uniform. The two set of variables revealed spatial variability discontinuities and river fragmentation upstream and downstream from the reservoirs. Reservoirs caused the replacement of spatially heterogeneous habitats by homogeneous spatially distributed water bodies, these new environmental conditions downstream benefiting the opportunist and cosmopolitan algal taxa. The application of a spatial auto-regression model to algal biomass (chlorophyll-a) permitted to capture the relevance and contribution of extra-local influences in the river ecosystem.

Heavy metals are some of the most persistent and potent anthropogenic environmental contaminants. Although heavy metals may compromise microbial communities and soil fertility, it is challenging to causally link microbial responses to heavy metals due to various confounding factors, including correlated soil physicochemistry or nutrient availability. A solution is to investigate whether tolerance to the pollutant has been induced, called Pollution Induced Community Tolerance (PICT). In this study, we investigated soil microbial responses to a century-old gradient of metal ore pollution in an otherwise pristine subarctic birch forest generated by a railway source of iron ore transportation. To do this, we determined microbial biomass, growth, and respiration rates, and bacterial tolerance to Zn and Cu in replicated distance transects (1 m-4 km) perpendicular to the railway. Microbial biomass, growth and respiration rates were stable across the pollution gradient. The microbial community structure could be distinguished between sampled distances, but most of the variation was explained by soil pH differences, and it did not align with distance from the railroad pollution source. Bacterial tolerance to Zn and Cu started from background levels at 4 km distance from the pollution source, and remained at background levels for Cu throughout the gradient. Yet, bacterial tolerance to Zn increased 10-fold 100 m from the railway source. Our results show that the microbial community structure, size and performance remained unaffected by the metal ore exposure, suggesting no impact on ecosystem functioning.

To study the stability and succession of sediment microbial and macrobenthic communities in response to anthropogenic disturbance, a time-series sampling was conducted before, during, and 1 year after dredging in the Guan River in Changzhou, China, which was performed with cutter suction dredgers from 10 April to 20 May 2014. The microbial communities were analyzed by sequencing bacterial 16S rRNA and eukaryotic 18S rRNA gene amplicons with Illumina MiSeq, and the macrobenthic community was identified using a morphological approach simultaneously. The results indicated that dredging disturbance significantly altered the composition and structures of sediment communities. The succession rates of communities were estimated by comparing the slopes of time-decay relationships. The temporal turnover of microeukaryotes (w = 0.3251, P < 0.001 [where w is a measure of the rate of log(species turnover) across log(time)]) was the highest, followed by that of bacteria (w = 0.2450, P < 0.001), and then macrobenthos (w = 0.1273, P < 0.001). During dredging, the alpha diversities of both bacterial and microeukaryotic communities were more resistant, but their beta diversities were less resistant than that of macrobenthos. After recovery for 1 year, all three sediment communities were not resilient and had reached an alternative state. The alterations in sediment community structure and stability resulted in functional changes in nitrogen and carbon cycling in sediments. Sediment pH, dissolved oxygen, redox potential, and temperature were the most important factors influencing the stability of sediment communities and ecosystem multifunctionality. This study suggests that discordant temporal turnovers and nonresilience of sediment communities under dredging resulted in functional changes, which are important for predicting sediment ecosystem functions under anthropogenic disturbances.
Understanding the temporal turnover and stability of biotic communities is crucial for predicting the responses of sediment ecosystems to dredging disturbance. Most studies to date focused on the bacterial or macrobenthic community, only at two discontinuous time points, before and after dredging, and hence, it was difficult to analyze the community succession. This study first compared the stabilities and temporal changes of sediment bacterial, microeukaryotic, and macrobenthic communities at a continuous time course. The results showed that discordant responses of the three communities are mainly related to their different biological inherent attributes, and sensitivities to sediment geochemical variables change with dredging, resulting in changes in sediment ecosystem multifunctionality.

Advanced oxidation (AO) coupled with biodegradation is an emerging treatment technology for wastewaters containing biologically recalcitrant and inhibitory organics, including those containing chlorinated aromatic compounds. The composition of the AO effluent organics can vary significantly with reaction conditions, and this composition can affect the performance of subsequent biodegradation. Three synthetic effluents were used to mimic varying degrees of AO of 2,4,5-trichlorophenol: 4-chlorocatechol to mimic light transformation, 2-chloromuconic acid to mimic moderate transformation, and acetate to mimic extensive transformation. The substrates were fed to identical chemostats and analyzed at steady state for removal of chemical oxygen demand (COD) and dissolved organic carbon (DOC), biomass concentration, and bacterial diversity. The chemostat fed acetate performed best at steady state. The 2-chloromuconic acid chemostat was next in terms of steady-state performance, and the 4-chlorocatechol reactor performed worst, correlating with degree of AO transformation. A spike of 100 μM 2,4,5-trichlorophenol was then applied to each chemostat. The chemostat fed 4-chlorocatechol exhibited the best resistance to the perturbation in terms of maintaining consistent community structure and biomass concentration, whereas the performance of the acetate-fed chemostat was severely impaired in these categories, although it quickly regained capacity to remove organics near pre-perturbation levels demonstrating good resilience. The opposing trends for steady-state versus perturbed performance highlight tradeoffs inherent in coupled chemical-biological systems.