Microbial activity, dissolved organic C and total extractable N
C addition - for all amendments - increased microbial activity 125-211% (\({\chi }_{4}^{2}\) = 886.47; P < 0.001; Figure 1A). There was a significant interaction between C amendment and soil moisture (\({\chi }_{16}^{2}\) = 67.98; P < 0.001; Figure 1B) which was primarily due to α-pinene marginally increasing activity above both LMW-DOC compounds at 25% WHC, and oxalate at 35% WHC (Pairwise P < 0.1). Further, at 25% WHC, the two VOC treatments elicited 26-40% higher cumulative microbial activity rates than the two LMW-DOC treatments (Figure 1B). Additionally, methanol increased microbial activity above both LMW-DOC compounds at 25%, 35%, and 45% WHC, but only above glucose at 60% WHC, while all C amendments had statistically equal activities at the highest moisture level (70% WHC).
In general, C addition - averaged across moisture levels - decreased total extractable N (\({\text{F}}_{4}\) = 33.97; P < 0.001; Figure 1C). Total extractable N concentrations were also affected by an interaction between C and moisture level (\({\text{F}}_{16}\) = 1.91; P = 0.029; Figure 1D). Methanol reduced total extractable N at all moisture levels except 45% WHC, while α-pinene decreased extractable N at 25%, 35%, and 60% WHC, and glucose only had an effect at 70% WHC. Oxalate did not significantly affect total extractable N concentrations at any moisture level.
C additions only marginally affected DOC (\({\chi }_{4}^{2}\) = 8.85; P = 0.06), which was driven by DOC in methanol-amended soils being 11% lower than in glucose amended soils (pairwise P = 0.09; Supplemental Figure 1). And, there was no moisture effect, or interaction between moisture and carbon source on DOC (\({\chi }_{16}^{2}\) = 19.39; P = 0.249).
Microbial community composition
There was a main effect of C amendment on Shannon Diversity (\({\chi }_{4}^{2}\) = 18.40; P = 0.001; Figure 2A), which was 2.9-3.5% lower in the methanol treatment than the control and the two LMW-DOC treatments. This decrease in diversity was driven by lower ASV evenness, i.e., Simpson index (\({\chi }_{16}^{2}\) =19.40; P < 0.001; Supplemental Figure 2A), which was marginally reduced in the methanol treatment by 0.5-0.7% (all pairwise P < 0.1). There was also a significant interaction between C amendment and moisture on Shannon Diversity (\({\chi }_{16}^{2}\) = 33.01; P = 0.007; Figure 2B). Specifically, at 25% WHC, α-pinene had 8.1-9.0% lower diversity than the control (pairwise P < 0.1), and all other C treatments (all pairwise P < 0.05). This reduction in diversity was also driven by lower ASV evenness, with α-pinene having 1.0-1.8% lower Simpson diversity than the other treatments at 25% WHC (\({\chi }_{16}^{2}\) = 28.48; P = 0.028; Supplemental Figure 2B).
PERMANOVA analysis of microbial ASVs using Bray-Curtis distances also identified both an overall C effect (Pseudo \({\text{F}}_{4}\) = 2.98, P = 0.001), and an interactive effect of moisture and C amendment (Pseudo \({\text{F}}_{16}\) = 1.41, P = 0.001). Pairwise PERMANOVA of microbial community compositions indicated that all C treatments were significantly different from the control when averaged across all moisture levels (Figure 2C). The most apparent differences in Bray-Curtis distances visualized with non-metric multidimensional scaling (NMDS), were clear separation of methanol from the other C treatments at all moisture levels, and α-pinene at low moisture only (Figure 2D).
Across all treatments, the ten most abundant phyla were the Proteobacteria (41.3%), Acidobacteria (16.4%), Bacteroidetes (10.1%), Verrucomicrobia (7.7%), Actinobacteria (7.2%), Planctomycetes (3.4%), Chloroflexi (2.5%), Firmicutes (2.3%), Nitrospirae (2.0%), and Thaumarchaeota (1.9%) (Figure3A). To link specific prokaryotic taxa to microbial functional responses (i.e. microbial activity, and total extractable N) we performed best-subsets multiple regression. The best-subsets regression model for predicting microbial activity - using the most abundant 10 phyla as candidate predictor variables - contained Chloroflexi, Nitrospirae, Proteobacteria, and Verrucomicrobia. Higher Verrucomicrobia abundance was associated with lower microbial activity while the other three phyla were associated with higher microbial activity (Table 1). The best-subsets regression model for predicting total extractable N contained Actinobacteria, Bacteroidetes, and Thaumarchaeota, of which Actinobacteria was associated with lower total extractable N while the other two were associated with higher total extractable N (Table 1).
Table 1: Best-subsets multiple regression models of predictor phyla for
each functional response.
Response
|
Predictors in Top Model
|
Coefficients
|
adj. R2
|
Microbial Activity
|
Chloroflexi
|
0.389
|
19.3
|
Nitrospirae
|
0.515
|
Proteobacteria
|
0.486
|
Verrucomicrobia
|
-0.538
|
intercept
|
5.47
|
Total Extractable N
|
Actinobacteria
|
-0.007
|
14.7
|
Bacteroidetes
|
0.006
|
Thaumarchaeota
|
0.013
|
intercept
|
0.09
|
All four phyla in the best-supported microbial activity GLM model responded significantly to C amendment (Figure 3B): Proteobacteria (\({\chi }_{4}^{2}\) = 44.60; P < 0.001), Nitrospirae (\({\text{F}}_{4}\) = 44.60; P = 0.029), Chloroflexi (\({\text{F}}_{4}\) = 2.48; P = 0.049), and Verrucomicrobia (\({\text{F}}_{4}\) = 2.03; P = 0.096). Methanol increased the relative abundance of Proteobacteria 6.4-16.4% relative to the control and all other C treatments, and α-pinene increased Proteobacteria 9.4% more than control. In contrast, methanol reduced the relative abundance of Verrucomicrobia by 12.4% relative to the control. Chloroflexi were significantly affected by C amendments, driven by oxalate-treated soils having relative abundance 13.2% higher than methanol-treated soils. Proteobacteria (\({X}_{16}^{2}\) = 28.54; P = 0.027), and Nitrospirae (\({\text{F}}_{16}\)= 3.36; P < 0.001) were affected by a significant interaction between C amendment and moisture level (Supplemental Figure 3). Notably, at 25% WHC α-pinene increased Proteobacteria relative abundance by 27-28% above the control and the two LMW-DOC treatments, while at 60% WHC, methanol addition increased the relative abundance of Nitrospirae by 44.9% above the control and 32-34% above the other C treatments. However, at the highest moisture, both glucose (pairwise P = 0.054), and methanol (pairwise P <0.001) reduced Nitrospirae relative abundance relative to the control (Figure 3B), by 21%, and 34% respectively.
All of the phyla retained in the best-subsets regression model for predicting total extractable N exhibited a significant C amendment response: Thaumarchaeota (\({\text{F}}_{4}\) = 4.25; P = 0.003), Actinobacteria (\({X}_{4}^{2}\) = 25.57; P < 0.001), and Bacteroidetes (\({\text{F}}_{4}\) = 2.25; P < 0.069). However, none of those phyla had a significant interaction with moisture. Methanol marginally reduced Thaumarcheota relative abundance by 24.6% below the control (pairwise P = 0.075). Likewise, there was higher relative abundance of Thaumarcheota in the oxalate treatment than in both methanol and α-pinene (pairwise P < 0.05). Oxalate marginally increased relative abundance of Actinobacteria relative to the control (pairwise P = 0.091), and both glucose and oxalate treatments had ~31% greater relative abundance of Actinobacteria than the methanol treatment (pairwise P < 0.05; Figure 3C). Methanol marginally reduced the relative abundance of Bacteroidetes by ~15.9% below the control (pairwise P = 0.058; Figure 3C).