A Diverse Soil Microbiome Degrades More Crude Oil than Specialized Bacterial Assemblages Obtained in Culture

Abstract

Soil microbiome modification may alter system function, which may enhance processes like bioremediation. In this study, we filled microcosms with gamma-irradiated soil that was reinoculated with the initial soil or cultivated bacterial subsets obtained on regular media (REG-M) or media containing crude oil (CO-M). We allowed 8 weeks for microbiome stabilization, added crude oil and monoammonium phosphate, incubated the microcosms for another 6 weeks, and then measured the biodegradation of crude oil components, bacterial taxonomy, and functional gene composition. We hypothesized that the biodegradation of targeted crude oil components would be enhanced by limiting the microbial taxa competing for resources and by specifically selecting bacteria involved in crude oil biodegradation (i.e., CO-M). Postincubation, large differences in taxonomy and functional gene composition between the three microbiome types remained, indicating that purposeful soil microbiome structuring is feasible. Although phylum-level bacterial taxonomy was constrained, operational taxonomic unit composition varied between microbiome types. Contrary to our hypothesis, the biodegradation of C10 to C50 hydrocarbons was highest when the original microbiome was reinoculated, despite a higher relative abundance of alkane hydroxylase genes in the CO-M microbiomes and of carbon-processing genes in the REG-M microbiomes. Despite increases in the relative abundances of genes potentially linked to hydrocarbon processing in cultivated subsets of the microbiome, reinoculation of the initial microbiome led to maximum biodegradation.

Importance: In this study, we show that it is possible to sustainably modify microbial assemblages in soil. This has implications for biotechnology, as modification of gut microbial assemblages has led to improved treatments for diseases like Clostridium difficile infection. Although the soil environment determined which major phylogenetic groups of bacteria would dominate the assemblage, we saw differences at lower levels of taxonomy and in functional gene composition (e.g., genes related to hydrocarbon degradation). Further studies are needed to determine the success of such an approach in nonsterile environments. Although the biodegradation of certain crude oil fractions was still the highest when we inoculated with the diverse initial microbiome, the possibility of discovering and establishing microbiomes that are more efficient in crude oil degradation is not precluded.

FIG 1

Shifts in microbiome structure through time by microbiome type. (a) Metrics for operational taxonomic unit (OTU) richness, diversity, and evenness in the three microbiome types, both before and after incubation with crude oil. Bars represent standard errors or estimated standard errors in the case of the Chao richness estimator. Principal-coordinate analysis (PCoA) ordinations of Bray-Curtis dissimilarity at the phylum (b) and OTU (c) levels. Shapes colored in white represent the means of replicate DNA extractions from the initial inocula. For the REG-M and CO-M inocula, they represent the means of the two and three media types used to produce them, respectively, as bacteria from each media type were added in equal amounts. Bar charts showing mean Bray-Curtis distances between microbiome types and to the median of all samples at each time point are shown at both the phylum (d) and OTU (e) levels. INIT, sterile soil reinoculated with the initial soil; REG-M, sterile soil reinoculated with bacteria cultured on regular media; CO-M, sterile soil reinoculated with bacteria cultured on media containing crude oil; week 0, after 8 weeks stabilization in soil; week 6, after 6 weeks incubation with crude oil.

FIG 2

Shifts in taxonomy through time by microbiome type. (a) Bubble plots showing the mean relative abundances of the most abundant phyla and the 20 most abundant Actinobacteria OTUs (across week 0 and week 6 combined) by microbiome type. Bubbles are shown for the initial inocula, week 0 (pre-crude oil), and week 6 (post-crude oil). Actinobacteria are highlighted to indicate that this phylum converged strongly by week 6 across microbiome types and was by far the dominant bacterial group. The abundance of Actinobacteria at week 6 did not differ significantly across microbiome types, while those of all other phyla and genera shown did (P < 0.05; multiple ANOVA with P value correction using the Benjamini-Hochberg false discovery rate). (b) Dot plot with 95% confidence intervals showing changes in OTU abundance from week 0 to week 6 for each microbiome type. The OTUs displayed are those with a relative abundance that increased significantly between time points by at least 1% in at least one microbiome type. The abundance of each OTU for each microbiome type at week 0 is indicated in a bar plot at the bottom of the figure. (c) Ternary plots of OTUs shared between all three microbiome types and across all time points to demonstrate treatment fidelity over time. The relative abundance of each OTU across microbiome types was normalized to 1 at each time point, so that the comparison represents the abundance of an OTU in relation to those of the other microbiome types, as opposed to other OTUs within a microbiome type. Colors represent the inoculum in which the OTU had the highest relative abundance (INIT, blue; REG-M, gold; CO-M, red). OTUs that are closer to the points of the triangle have higher relative abundances in the microbiome type represented by that point than the other microbiome types, whereas points close to the center have similar relative abundances in all microbiome types. (d) Proportion of total sequences for each microbiome type (INIT, blue; REG-M, gold; CO-M, red) at each time point that are classified as one of the 137 OTUs that were shared across time points and across microbiome types. INIT, sterile soil reinoculated with the initial soil; REG-M, sterile soil reinoculated with bacteria cultured on regular media; CO-M, sterile soil reinoculated with bacteria cultured on media containing crude oil.

FIG 3

Functional profiles of the three microbiome types at week 6 and from the initial source soil, based on shotgun metagenomic sequencing. (a) Principal-component analysis (PCA) ordination on SEED subsystem functional profiles produced by MG-RAST (level 3). (b) Regression of Shannon diversity of level 3 SEED functions against Shannon diversity of bacterial OTUs. Colors are as in panel a. (c, d) Scatterplots comparing the relative abundances of functional categories between INIT and CO-M and INIT and REG-M microbiomes. A comparison between REG-M and CO-M is not shown, due to the high degree of similarity between the CO-M and INIT profiles. Lines extending from points in the scatterplots indicate standard deviations. (e) Histogram and confidence intervals of functions found to be significantly different between INIT and REG-M, with an effect size (difference in relative abundance) of at least 0.25. These functions are found within the orange-colored area of panel d. There were no significant differences of this size between INIT and CO-M. (f) Relative abundances of functional categories related to carbon processing across microbiome types and within the initial source soil. The top three histograms are level 1 categories from SEED subsystem annotations, whereas the bottom histogram (alkane hydroxylases) was created by combining all GenBank alkane hydroxylase annotations using the All Annotations search function in MG-RAST. INIT, sterile soil reinoculated with the initial soil; REG-M, sterile soil reinoculated with bacteria cultured on regular media; CO-M, sterile soil reinoculated with bacteria cultured on media containing crude oil.

FIG 4

PCA plot produced using STAMP analysis of PICRUSt-projected functional profiles (level 3, KEGG orthology). This plot shows that the week 6 profiles group together with the profiles from the crude oil plus M9 media, whereas the pre-crude oil profiles group together with the profiles from all other media types (TSA and glucose, with or without crude oil added).

FIG 5

Percent degradation of C₁₀ to C₅₀ hydrocarbons after 6 weeks of microcosm incubation following the addition of crude oil and monoammonium phosphate. Bars represent standard errors. Different letters over columns indicate significant differences between treatments based on a Tukey HSD post hoc test following one-way ANOVA. The sterile (noninoculated) controls were rerun after the initial experiment, as the original controls experienced contamination. The incubation conditions were the same, and the C₁₀ to C₅₀ loss was compared to that of paired baseline samples that were frozen at the outset of this incubation. For treatments, n = 10, and for sterile controls, n = 3. INIT, sterile soil reinoculated with the initial soil; REG-M, sterile soil reinoculated with bacteria cultured on regular media; CO-M, sterile soil reinoculated with bacteria cultured on media containing crude oil.