Soil N availability and microbial biomass
Different biochar application rates significantly affected soil N availability (Table 1). Compared with CK, biochar application increased the soil TN content by 4.7–32.3%. Soil TN in BC3 and BC4 increased significantly (p < 0.05) and were significantly (p < 0.05) higher than in BC1 and BC2. NN contents in BC1, BC2, BC3, and BC4 were 6.4%, 9.5%, 11.6%, and 12.5% lower, respectively, than in CK. AN contents in BC2, BC3, and BC4 were 57.4%, 58.9%, and 101.5% higher, respectively, than in CK. MBN and AN trends were consistent for all treatments. In addition, with increasing amounts of biochar applied, the NN/TN indicators exhibited a decreasing trend, with CK > BC1 > BC2 > BC3 > BC4. However, the changes in AN/TN were the opposite, and the MBN/TN trend was similar to that of NN/TN.
Table 1
Effects of different biochar application rates on soil nitrogen availability
Treatment | TN(g·kg− 1) | NN(mg·kg− 1) | AN(mg·kg− 1) | MBN(mg·kg− 1) | NN/TN | AN/TN | MBN/TN |
CK | 2.32 ± 0.03 b | 132.38 ± 2.45 a | 8.64 ± 0.52 c | 51.35 ± 1.02 c | 57.64 ± 0.01 a | 3.73 ± 0.85 b | 22.15 ± 0.26 ab |
BC1 | 2.43 ± 0.20 b | 128.91 ± 4.23 b | 9.63 ± 0.76 c | 53.20 ± 1.16 c | 52.03 ± 0.01 b | 3.84 ± 1.68 ab | 23.90 ± 1.25 a |
BC2 | 2.50 ± 0.09 b | 119.80 ± 4.87 bc | 13.60 ± 0.90 b | 61.24 ± 3.27 b | 48.85 ± 0.03 b | 4.44 ± 2.61 ab | 23.97 ± 0.82 a |
BC3 | 3.07 ± 0.08 a | 116.95 ± 4.79 c | 13.73 ± 1.43 b | 62.80 ± 1.00 ab | 38.64 ± 0.01 c | 4.35 ± 1.80 ab | 20.44 ± 0.79 b |
BC4 | 3.04 ± 0.01 a | 115.81 ± 2.56 c | 17.41 ± 1.05 a | 64.84 ± 1.30 a | 38.16 ± 0.01 c | 4.75 ± 1.04 a | 21.79 ± 0.45 b |
Values are presented as mean ± SD (n = 4), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan's multiple range test. Abbreviations: TN, total inorganic N; NN, nitrate nitrogen; AN, ammonium nitrogen; MBN, microbial biomass of nitrogen. |
Effects of biochar on soil microbial diversity and community structure
Microbial richness and diversity indices
We observed 741965 quality sequences, with an average of 22752 sequences per sample. The average base length was 416 bp for the bacterial 16S rRNA. The coverage index of soil amended with biochar was 97%, indicating that the dataset included all sequences between V3 and V4 regions, and that the sequence data volumes were reasonable (Fig. 1). The number of public OTUs processed by each treatment was 2039, 70.0%, 66.6%, 65.2%, 67.2%, and 66.0% of the total OTUs from CK, BC1, BC2, BC3, and BC4, respectively.
The alpha diversity of bacteria communities were positively affected by the application of biochar rates(Table 2), and biochar treatments significantly increased the Ace, Chao, and Shannon indices. Compared with CK, the Ace and Chao indices increased in BC1 were 11.1% and 11.5%, respectively. However, the differences in the richness index in the other biochar treatments were not significant. With increased biochar application, the Shannon index of the soil bacteria increased, among which the different biochar treatments reached significant differences compared to CK. In contrast, the biochar treatments significantly decreased the Simpson index related to CK. The Simpson indices in treatments BC3 and BC4 were significantly lower by 72% and 60%, respectively, than in CK.
Table 2
Effects of biochar application rates on the alpha diversity of the bacterial community
Treatment | Ace | Chao | Shannon | Simpson | Coverage |
CK | 2932 ± 116 b | 2911 ± 105 b | 5.93 ± 0.34 b | 0.025 ± 0.016 a | 0.977 ± 0.002 a |
BC1 | 3257 ± 127 a | 3245 ± 117 a | 6.31 ± 0.06 a | 0.012 ± 0.002 ab | 0.975 ± 0.002 a |
BC2 | 3157 ± 182 ab | 3117 ± 193 ab | 6.32 ± 0.12 a | 0.012 ± 0.004 ab | 0.974 ± 0.004 a |
BC3 | 3083 ± 209ab | 3106 ± 173 ab | 6.39 ± 0.11 a | 0.007 ± 0.001 b | 0.974 ± 0.007 a |
BC4 | 3050 ± 78 ab | 3052 ± 96 ab | 6.39 ± 0.12 a | 0.010 ± 0.003 b | 0.972 ± 0.004 a |
Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan's multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4%. |
Effects of biochar on soil bacterial community composition
Analyses based on the 16S rRNA data indicate that the main bacterial phyla in the soil samples were Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, and Bacteroidetes. Their total relative abundance was 81.60–84.93%. The relative abundances of Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, and Bacteroidetes were 28.78–32.26%, 24.92–32.67%, 5.96–10.84%, 4.98–8.97%, and 5.53–7.14%, respectively (Fig. 2). The relative abundance of Proteobacteria in BC3 was 4.4% higher than in CK. Compared to CK, the relative abundance of Proteobacteria was significantly reduced in BC4. The relative abundances of Chloroflexi and Acidobacteria increased significantly with increased biochar application. Compared to CK, BC4 increased the relative abundances of these two phyla by 79.0% and 61.9%, respectively. For the other less abundant bacterial phyla, the application of biochar reduced the relative abundances of Firmicutes, Gemmatimonadetes, and Patescibacteria. However, BC3 and BC4 increased the relative abundance of Nitrospirae, which increased further with increasing biochar application. Compared to CK, BC4 increased the relative abundance of Nitrospirace by 95.3%.
Effect of biochar on the principal components of soil bacterial communities
A PCA was performed on the soil bacterial communities with regard to the different biochar application rates, from which two principal factors was extracted (Fig. 3). The total interpreted amount was 80.47%, of which PC1 and PC2 comprised 51.23% and 29.24%, respectively. The soil samples from CK were distributed in the negative areas of PC2. BC1, BC2, BC3, and BC4 gradually changed from the negative area to the positive area of PC2, and were mainly distributed in the positive area of PC2. This indicates that the different biochar application rates had a significant effect on the soil bacterial community.
Effect of Biochar on Predictive Functional Profiling of Bacterial Communities Related to Soil Nitrification and Denitrification Using 16S rRNA Marker Gene Sequences
N cycling processes in the soil need to be coordinated by various enzymes in each branch (data URL: https://www.genome.jp/). According to the N metabolism pathway diagram, the corresponding relationship between the enzymes and genes related to N metabolism can be obtained. In addition, the enzymes involved in the soil nitrification and denitrification can be obtained by comparing sequence data to enzyme nomenclature (Table 3). By analyzing the changes in related enzymes for different biochar application rates, we found that ammonia monooxygenase (1.14.99.39) and hydroxylamine dehydrogenase (1.7.2.6), which are involved in the ammonia oxidation process, were significantly different. Compared to CK, the ammonia oxygenase (1.14.99.39) in BC2 decreased by 20.1%, while BC3 and BC4 had increased gene expressions of 19.3% and 22.9%, respectively. Hydroxylamine dehydrogenase (1.7.2.6) exhibited the same trend as the ammonia oxygenase (1.14.99.39). In the N denitrification process, the gene expressions of nitrite reductase (1.7.2.1), nitric oxide reductase (1.7.2.5), and nitrous oxide reductase (1.7.2.4) also changed to varying degrees. As the amount of biochar application increased, the expressions of enzyme genes increased. The copy number of 16S marker gene of related nitrite reductase, nitric oxide reductase nitrite reductase (1.7.2.1), nitric oxide reductase (1.7.2.5), and nitrous oxide reductase (1.7.2.4) in BC3 were 13.7%, 16.4%, and 16.0% greater, respectively, than in CK.
Table 3
Number of copies of 16S marker gene related enzymatic functions to nitrification and denitrification processes in the soil treatments (gene copies/g soil)
Enzyme name | Enzyme Commission (EC) number | CK | BC1 | BC2 | BC3 | BC4 |
Ammonia monooxygenase | 1.14.99.39 | 274 ± 17 b | 243 ± 28 b | 219 ± 10 c | 327 ± 23 a | 337 ± 38 a |
Hydroxylamine dehydrogenase | 1.7.2.6 | 258 ± 26 ab | 229 ± 17 b | 223 ± 30 b | 311 ± 39 a | 322 ± 54 a |
Nitrite reductase (NO-forming) | 1.7.2.1 | 3546 ± 131 b | 3242 ± 134 b | 3409 ± 244 b | 4031 ± 166 a | 3318 ± 128 b |
Nitrate reductase | 1.7.99.4 | 24968 ± 480 a | 25178 ± 505 a | 25469 ± 915 a | 25044 ± 405 a | 26160 ± 886 a |
Nitric-oxide reductase (cytochrome c) | 1.7.2.5 | 2095 ± 88 c | 2121 ± 98 bc | 2322 ± 77 a | 2439 ± 108 a | 2282 ± 74 ab |
Nitrous-oxide reductase | 1.7.2.4 | 1803 ± 85 bc | 1739 ± 24 c | 1897 ± 82 b | 2092 ± 99 a | 1899 ± 72 b |
Values are mean plus standard deviation (n = 3), and data with different lowercase letters are significantly different at p < 0.05 according to Duncan's multiple range test. Treatments CK, BC1, BC2, BC3, and BC4 had biochar dosages of 0%, 0.5%, 1%, 2%, and 4%. |
Correlations between soil bacterial community composition, soil N availability, and related enzyme gene expression
A redundant analysis (RDA) was used to analyze the correlations between soil N availability, the related enzyme functions, and the bacterial community composition. The first and second ordination axes explained 38.6% and 19.9% of the total variability, respectively (Fig. 4a). Regarding soil N availability, the main factors influencing the first ordination axis were NN (-0.5821), AN (0.5327), and NN/TN (-0.5312). The main factors influencing the second ordination axis were AN/TN (-0.3854) and NN (0.3464). This indicates that AN, NN/TN, and NN were the main factors affecting the structure of the soil bacterial community. Regarding enzymatic functions, the first and second ordination axes explained 26.5% and 24.9% of the total variability, respectively (Fig. 4b). The main factors influencing the first ordination axis were nitric oxide reductase (cytochrome c) (-0.5245), nitrous-oxide reductase (-0.2721), and ammonia monooxygenase (0.1272). The main factors influencing the second ordination axis were nitrite reductase (0.7263) and hydroxylamine (0.6929). This indicates that nitrite reductase, hydroxylamine, and nitric oxide reductase (cytochrome c) were the main factors affecting the structure of the soil bacterial community.