Microbial co-occurrence network analysis of soils receiving short- and long-term applications of alkaline treated biosolids

Agricultural soils are inherently disturbed systems where organic matter additions are considered to enhance microbial community structure and resilience. High-throughput sequencing of community was applied to soils receiving annual applications of an alkaline stabilized biosolid (ATB), at four increasing rates over 10 years, as an environmental stressor in contrast to a one-time application of ATB ten years prior. Bacterial community structure was more greatly influenced by annual ATB applications relative to fungi and eukaryotes. Specifically, higher relative abundances of Proteobacteria, Acidobacteria, Bacteroidetes, and Chloroflexi were measured in annual ATB rates relative to the single ATB rates and the control. High rates of annual ATB applications resulted in lower bacterial alpha-diversity, as well as fungal and eukaryotic Shannon diversity, but single ATB or lower rates of ATB applied annually showed increased alpha -diversity relative to the control. Soil microbiome responses to annual ATB and single ATB rates were also examined using co-occurrence network analysis. High rates and frequency of ATB application resulted in a decrease in network interactions, lower average number of neighbors, and reduced network density compared to control soils. A concomitant increase in network diameter and characteristic path length further suggests annual additions of ATB led to a more adapted, but less cooperative, state in the microbiome. The data suggest a more universal functional response of microbiomes to the stressors compared to community structure and local diversity. In particular, beta-analysis and network analysis were both able to resolve significant effects on soil microbiomes 10 years post-application of low rates of ATB. Community complexity and stability were increased by single low rate of ATB additions and decreased by single high rate and annual moderate rates of ATB additions. These results provide insights into the effects that ATB additions have on soil community after only one-time use and after annual additions over a decade.