Spatial and temporal changes of charosphere hotspots with or without nitrogen additions

The charosphere is a thin soil one surrounding the biochar with highly active biochemical functions. Yet, little is known about the spatial and temporal distribution of charosphere hotspots. In this study, repacked soil cores were incubated with a central layer of biochar (pristine or acid-modified) with or without nitrogen (N) additions for 30 days and sliced at the millimeter scale for analyzing soil pH, mineral N, bacterial and fungal communities as well as the putative functions. We aimed to determine gradient distributions (in millimeter scale) of charosphere affected by biochar under different N additions. Our results showed narrower gradient changes (3 mm) of microbial community composition and wider shifts (6 mm) in pH and inorganic N contents in charosphere. The pristine biochar increased the soil pH up to 1.5 units in the charosphere, and subsequently boosted the relative abundance of Proteobacteria, Acidobacteria, and Zygomycota. With N addition, both the biochar site and charosphere were observed with decreased complexity of microbial networks, which might imply the limited microbial functionality of charosphere. These results will advance the understanding and prediction of biochar’s environmental impacts in soil. The charosphere hotspots of soil pH and mineral N content reached 6 mm. Soil microbial compositions fluctuated within 3 mm of charosphere. Pristine biochar site fostered higher fungal diversity than that in bulk soils. Lower bacterial diversity in pristine biochar site was found. The microbial network complexity declined with N addition in charosphere. The charosphere hotspots of soil pH and mineral N content reached 6 mm. Soil microbial compositions fluctuated within 3 mm of charosphere. Pristine biochar site fostered higher fungal diversity than that in bulk soils. Lower bacterial diversity in pristine biochar site was found. The microbial network complexity declined with N addition in charosphere.


Graphical abstract 1 Introduction
Soil is a complex matrix with spatially heterogeneous distribution of organic matter (OM) at different spatial scales (from micrometers to meters) (Liang et al. 2019;Peth et al. 2014;Schlueter et al. 2022) as well as of nutrients and microorganisms (Delin and Strömberg 2011;Vedere et al. 2020).Particularly, the soil microenvironments (rhizosphere, detritusphere, charosphere, etc.) are microbial hotspots, with even orders of magnitude faster rates of biochemical processes and much stronger interactions between microbes and their environment than those in the bulk soil (Kuzyakov and Blagodatskaya 2015).The charosphere refers to biochar and the adjacent soil where physicochemical conditions are greatly influenced and microbial activities are stimulated or inhibited (Quilliam et al. 2013;Wang et al. 2017).The rates and dynamics of biogeochemical processes in microbial hotspots determine the process rates at the macroscale (Kuzyakov and Blagodatskaya 2015).A couple of biochar relevant studies explicitly took spatial heterogeneity and soil-biochar structure into account to reveal the detailed mechanisms of biochar's influences on soil carbon (C) and nitrogen (N) turnover (Yu et al. 2019;Zhu et al. 2022).It was demonstrated that unraveling the underlying mechanisms requires matching knowledge of the spatial and temporal distribution of those charosphere hotspots (Woolet and Whitman 2020).
Biochar is used as an organic material in agro-ecological environmental field (Shaheen et al. 2019).When straw-based biochar is added to soil, its high alkalinity would contribute to the gradient changes of soil pH (Zhu et al. 2022).The charosphere was previously investigated in laboratory experiments by adding biochar with different types or localized applications (Buss et al. 2018).It has been established that the soil pH was influenced over an area of a few millimeters around the biochar particles, and more rapidly over larger distances with biochar produced from higher temperature.Some studies revealed that the EC value of biochar was one of the main factors determining the extent of the charosphere (Chen et al. 2021).Furthermore, the biochar's porous nature and high adsorption capacity usually lead to spatial redistribution of soil solutes, such as gradient distribution of soil dissolvable organic carbon and ammonium, resulting in microbial hotspots associated with C and N (Yu et al. 2019).Additionally, the acid-modified biochar, without releasing soluble compounds or alkaline substances from the biochar into the soil, has been revealed to effectively improve soil nutrient availabilities and plant growth in recent studies (Peiris et al. 2022;Sahin et al. 2017).Practical knowledge of the spatiotemporal characteristics of hotspots induced by acid-modified biochar could greatly advance our understanding of the benefits of biochar application.However, no study has been performed to evaluate the properties of such charosphere.
The application of ammonium fertilizer and urea significantly impacts soil oxygen status and nutrient availabilities mainly by nitrification (Song et al. 2019;Zhu et al. 2015).Consequently, it can modify the interactions between biochar and charosphere microorganisms.However, we do not know to what extent nor how far the biochar modifies the surrounding bacterial communities with or without N addition.In order to investigate N-related functional genes in charosphere, Yu et al. (2019) designed a multi-sectional box and separated the charosphere into discreet divisions at millimeter scale using nylon mesh.Such nylon meshes may cause physical disturbances for the movementof soil nutrients and microorganisms, particularly for the colonization of hyphal network (Steinberg et al. 2018).Additionally, fungi are considered as another important contributor to the C and N turnover process after exogeneous organic carbon addition (Vedere et al. 2020).However, how the diversity and the relative abundances of both bacteria and fungi are influenced by the charosphere have not been fully explored yet.
In this study, we adopted a novel approach with minimized disturbance on soil.The specific objectives were to: (1) quantify the volumetric characteristics of the physicochemical properties involved in the charosphere of pristine and acid-modified biochar with and without N additions; (2) investigate the variations in soil bacterial and fungal diversity and community structure due to changes in physicochemical properties of charosphere; (3) examine the potential functional groups related to the C and N turnover processes with biochar additions.We hypothesized that: 1. pristine biochar would induce a wider charosphere zone through diffusion of alkali substances and other minerals relative to acid-modified biochar.2. lower diversity but stronger connectivity of bacteria and fungi community are expected to be observed at the biochar sites because of the limited nutrients and higher pH as compared to those in bulk soils, and would facilitate the retention of soil organic C and mineral N.

Preparation of soil and biochar
Soil samples were collected from the 8-year fallow land at the Shangzhuang Experimental Station (N 40° 08′ 21″, E 116° 10′ 52″) of China Agricultural University.This region is characterized by a temperate humid monsoonal climate with annual average precipitation and temperature of 628.9 mm and 12.5 °C, respectively.The soil type is fluvisol and the texture is classified as sandy loam, which consists of 28% sand, 52% silt and 20% clay.The soil collected from 5 to 20 cm was sieved through a 0.5 mm sieve and pre-incubated at room temperature (20 °C) for 7 days to restore microbial activity.The basic characteristics of soil are shown in Table 1.
Pristine biochar was produced from maize straw, which was air-dried and ground to powder (2 -3 mm), then anaerobically pyrolyzed in a muffle furnace at 500 °C for 2 h.The biochar was treated with acid solutions to remove the alkalinity and dissolvable minerals of the biochar (Zhang et al. 2013).Briefly, 80 g of biochar was mixed and shaken in 800 mL of 1 M: 1 M HCl-HF solution for 10 h.The treated biochar was rinsed with deionized water several times to remove residual acid and soluble salts until reaching a neutral pH.The pristine biochar and acid-modified biochar were remarked as BC and ABC, respectively.The obtained biochar was crushed and sieved to 0.25 -0.5 mm for further use.

Experimental setup and soil sampling
Six treatments included: biochar (BC), acid-modified biochar (ABC), biochar with urea addition (BC-N), acidmodified biochar with urea addition (ABC-N), control, which is soil cores without biochar (CK), control with urea addition (CK-N).There were three replicates for each treatment.Soil cores were assembled by placing the soil into a customized cylinder at a bulk density of 1.3 g cm −3 .For the urea addition treatments, urea was dissolved in the deionized water, then we sprayed this urea solution into the soil and mixed soil with the solution thoroughly right before the soil repacking process, and the N addition rate was 100 mg Nper kg of dry soil.Each core consisted of 0.6 g of biochar or acid-modified biochar with the thickness of 2 mm and 55.6 g of dry soil on either side corresponding to a field application rate of 2.8 × 10 4 kg ha −1 (Sun et al. 2020), then the three layers were compacted.The resulting core of soil had a total thickness of 42 mm with a diameter of 50 mm (Fig. 1a).In order to reduce the heterogeneity of water content distribution in soil cores, we assembled the soil cores as follows: briefly, after the first soil compartment was repacked, its water content was adjusted by adding deionized water to reach 60% of the water holding capacity (WHC).Then the biochar compartment was packed and its water content was also adjusted to 60% of WHC.Finally, the other soil compartment was assembled and and its water content was adjusted to 60% of WHC.To keep a constant-moisture content in soil column, deionized water equal to the mass loss was added by measuring the mass of each soil column every other day during the incubation period.Then all soil cores were placed horizontally in the climate chamber to maintain the uniform water content distribution throughout the incubation.
Soil was sampled after 3, 7, 14, 21 and 30 days (referred as T1, T2, T3, T4 and T5, respectively).Each soil core was sectioned into 1.0 mm thick slices from the biochar site outwards and divided into a total of 23 discrete sections by sterilized cutting device (Fig. 1b).The both sides of the biochar with the same distance were mixed and marked as charosphere (Yu et al. 2019;Zhu et al. 2022).The remaining 10-mm thick layer outward to the charosphere was sectioned separately.A digital pH meter was used to measure soil pH in a suspension with soil to water ratio of 1:5.The cation exchange capacity (CEC) was determined by the ammonium acetate compulsory displacement method, adapted to biochar (Gaskin et al. 2008).We extracted mineral N in soil with 0.5 M K 2 SO 4 solution.An automated flow-injection analyzer (AA3, Bran+Luebbe Corp, Wrexham, UK) was used to determine the concentration of NH 4 + -N and NO 3 − -N in the K 2 SO 4 extracts with the modified Berthelot reaction and cadmium reduction method, respectively.

DNA extraction and sequencing
The total DNA for each sample from biochar site, charosphere and bulk soil was extracted directly using the Fast DNA SPIN Kit (MP Biomedicals LLC, Ohio, USA) following the manufacturer's instructions.The quality and concentration of extracted DNA were evaluated by agarose gel electrophoresis (1.0% agarose gel, 120 V, 30 min) and spectrophotometer (NanoDrop ND-2000, Wilmington, DE, USA), and then diluted 50-fold subsequently as DNA template for PCR reaction.Bacterial and fungal sequencing libraries from 180 DNA samples were prepared.The hypervariable V4 region of eukaryotic 16S rRNA gene was amplified using the forward primer 515F (5′-GTG CCA GCMGCC GCG GTAA-3′) and reverse primer 806R (5′-GGA CTA CVSGGG TAT CTAAT-3′) (Zhou et al. 2016).Amplifications of ITS1 regions of genes were performed using the universal primer ITS1 (5′-CTT GGT CAT TTA GAG GAA GTAA-3′) (Gardes and Bruns 1993) and the reverse primer ITS2 (5′-TGC GTT CTT CAT CGA TGC -3′) (Innis et al. 1990).Both the forward primer and reverse primer for bacterial and fungal sequencing libraries had a sample-specific10~12-bp barcode that was used to distinguish samples.The 50.0 µL PCR mixture with a 2.0 µL DNA template, 13.0 µL double distilled water, 25.0 µL Premix Taq DNA polymerase (TaKaRa Biotech, USA), and 5.0 µL of each primer, was used for bacteria.And then the amplifications were under the following conditions: denaturation at 94 °C for 5 min (1 cycle), 30 cycles denaturation at 94 °C for 40 s, primer annealing at 56 °C for 60 s, extension at 72 °C for 60 s, and a final extension period of 10 min at 72 °C on a Veri-tiTM 96 Thermal Cycler (ABI, Vernon, USA).For the amplification of the fungal ITS1 region, total volume of 50.0 μL PCR mixture consisted of 17.0 μL double distilled water, a 2.0 μL DNA samples as template, 25 μL Premix Taq DNA polymerase (TaKaRa Biotech, USA), 2.0 μL forward primer Barcode, 2.0 μL reverse primer and 2.0 μL 0.5% BSA solution.PCR reactions were performed with the following conditions: denaturation at 95 °C for 5 min (1 cycle), 30 cycles denaturation at 95 °C for 45 s, primer annealing at 50 °C for 50 s, extension at 72 °C for 45 s, and a final extension of at 72 °C for10 min on the same VeritiTM 96 Thermal Cycler (ABI, Vernon, USA).Triplicate PCR products for each of 180 samples and negative and positive controls were conducted through the experiment and confirmed by gel electrophoresis (1.0% agarose gel, 120 V, 30 min).Afterwards, the 16S rRNA and ITS gene sequencings were carried out on an Illumina Miseq (Nova PE250) platform (Illumina, Inc., San Diego, CA, USA) according to the manufacturer's protocols.

Bioinformatics
Sequenced paired-end reads were assembled and quality controlled with VSEARCH v2.3.4 (Rognes et al. 2016).Raw data were processed (denoising, removing chimera sequences) and analyzed (barcode assignment, primer clipping and classification) following the previously described methods (Edgar et al. 2011;Li et al. 2019) in a galaxy instance (www.freeb ioinfo.org).We assigned sequences to samples based on the primer and barcode regions and clustered into operational taxonomic units (OTUs) using UPARSE (Edgar 2013) with the 97% sequence similarity cutoff.Taxonomic assignments for the clustered OTUs were performed using the RDP classifier for bacteria (http:// rdp.cme.msu.edu/) and the SILVA (Release 123) (Quast et al. 2013) for fungal OTUs.Alpha diversity indices including Chao1 (Anne Chao 1984), Simpson index (Hunter and Gaston 1988), Pielou's evenness (Jeffers and Pielou 1971) and observed species were calculated for each sample.Bray-Curtis distance matrixes for bacterial and fungal microbiota were determined and imaged by Principal Coordinate Analysis (PCoA).
In this study, we predicted the functional profiles of bacterial communities by FAPROTAX based on the normalized OTU table annotated against RDP database (Louca et al. 2016).Moreover, we applied 'WGCNA' R package (version 1.70.3) to construct the bacterial-fungal co-occurrence network based on the Person coefficient matrix (Langfelder and Horvath 2012).OTUs were screened by relative abundances greater than 0.01% and the top 75% of the median absolute deviation.'Modules' was clustered by merging the individual branches with a distance less than 0.25.The nodes and the edges in the network represented OTUs and the correlations between pairs of OTUs, respectively.P-values were adjusted by Benjamini and Hochberg false discovery rate (FDR) test (Benjamini et al. 2006), and the adjusted P-values had a 0.001 cutoff.The network properties were validated by 'igraph' R package and described by Gephi-0.9.2 soft.

Statistical analysis
The three-way analysis of variance (ANOVA) with SPSS 22.0 software (IBM Corp, Armonk, NY, USA) was applied to examine the effects of biochar type, N addition and charosphere distance on the soil biochemical properties, while graphs were produced using the software OriginPro 2021 (OriginLab, USA) and GraphPad Version 9.3.1 software (Prism, USA).Significantly different means were separated using the Duncan Multiple Range Test (DMRT) at 5% level of probability (P < 0.05).
Analysis of PERMANOVA using the adonis test in Vegan v.2.5-7 with R software was used to evaluate the significant differences between samples from biochar and charosphere.The separation degree of between-group and within-group mean rank similarities was marked by statistic global R. To identify the taxon differences of fungi among various systems, linear discriminant analysis, coupled with effect size (LEfSe), and LDA score (log 10), was used (Segata et al. 2011).All plots were performed using the R package Vegan v.2.5-7 and ggplot2 v. 3.3.5 in software R.

Temporal variation and spatial distribution of pH in the charosphere
Biochar had significant impacts on temporal changes of soil pH (Fig. 2a).The mean soil pH value in charosphere was significantly increased with pristine biochar addition, and N addition enhanced this trend in the treatments of both biochar types.The soil pH of the charosphere increased gradually throughout incubation time and reached a pH mean value of 7.79 on day 14 in BC treatment, and subsequently declined.Furthermore, N addition in BC increased the soil pH up to about 1.5 units higher than that of the initial soil.In contrast, the soil pH in the charosphere of acid-modified biochar was slightly changed by 0.1 -0.2 units.
The biochar type and N addition greatly influenced the soil pH gradients in charosphere (Fig. 2b).Spatially, the soil pH value generally decreased away from the biochar site, and the maximum pH value was more than 8.5 at the 1 mm from the boundary of the pristine biochar site.The pH gradients surrounding the biochar site in the near charosphere zone (~ 4 mm) were steeper than in the far charosphere zone (5 -10 mm) and controls (pH 7.17).Subsequently, the zone with pH gradients induced by pristine biochar expanded to more than 6 mm after 14 days.The acid-modified biochar led to insignificant changes in the soil pH in the charosphere, but a significantly lower soil pH value than that in BC.The urea addition (BC-N, ABC-N) further increased soil pH by about 0.1 and 0.2 units on day 14, respectively.The soil NH 4 + -N in charosphere gradually increased away from the biochar site, which was contrast to the change of the soil pH in the charosphere (Fig. 3a).Without N additions, higher NH 4 + -N contents were observed in ABC treatment with higher CEC in charoshphere at later periods.Both the pristine and acid-modified biochar co-applied with urea resulted in significantly higher NH 4

Soil NH
+ -N content in charosphere.The concentrations of soil NH 4 + -N from the near charosphere zone (0 -6 mm) increased gradually, then reached a maximum value at 250 mg kg −1 after 14 days (BC-N).The maximum values of soil NH 4 + -N concentrations in ABC-N were about 40 mg kg −1 lower than that in BC-N treatment.In addition, a significant correlation between NH 4 + -N content and distance in the linear regression model was found during the 30 incubation days (Additional file 1: Fig. S1a).Soil NO 3 − -N concentrations in charospheres of both biochar types were not significantly different from those in the controls at all distances, and fluctuated in the range of 60-80 mg kg −1 (Fig. 3b).Urea addition resulted in higher concentrations of soil NO 3 − -N in charosphere zone.The soil NO 3 − -N contents in charosphere zone with BC-N treatment reached the highest level on day 21, while the soil NO 3 − -N content in ABC-N increased gradually during the entire incubation period.Similarly, there was a close linear relationship between NO 3 − -N concentration and incubation time (Additional file 1: Fig. S1b).

Bacterial and fungal community compositions based on high-throughput sequencing
Significant differences of species richness and relative abundance for both bacteria and fungi were observed among biochar types, incubation times, and distances from the biochar site (Figs. 4 and 5).The bacterial richness in the pristine biochar site was lower than that in charosphere and controls.The charosphere (1 -4 mm) closer to the pristine biochar site had the lower bacterial richness at the early stage (day 3, T1).With the addition of urea, the bacterial richness was decreased in charosphere compared to the corresponding treatment without N addition for all sampling times.Additionally, the most dominant bacterial phyla in pristine biochar sites were Proteobacteria and Acidobacteria, whereas Proteobacteria, Acidobacteria and Actinobacteria were dominant in charosphere.With the addition of urea, the relative abundance of Proteobacteria and Gemmatimonadetes increased near the biochar site but no shifts were found near the acid-modified biochar site.
The richness of fungi in the pristine biochar site was significantly higher than that in charosphere and controls soil.Acid-modified biochar and urea addition attenuated the differences of fungal richness among biochar site and charosphere.With urea addition, the fungal richness at the distance of 3 mm from the biochar site increased with the increase of incubation time.Moreover, the relative abundance of dominant fungi in the pristine biochar site had no significant difference compared to controls.Still, clear differences were found in charosphere, with the increased relative abundance of Zygomycota and the sampling time.Compared with the pristine biochar site, the acid-modified biochar site had a higher relative abundance of Ascomycota and a lower relative abundance of Basidiomycota, whereas ABC-N treatment gathered more Basidiomycota.
PCA ordinations and ANOSIM tests showed that the bacterial and fungal community compositions from biochar site and charosphere exhibited a clear separation (P = 0.001; Additional file 1: Fig. S2).In addition, redundancy analysis (RDA) was performed to examine bacterial and fungal community variations for distances away from the biochar site and sampling time with the edaphic parameters (Additional file 1: Fig. S3).Specifically, the bacterial and fungal communities were significantly separated after incubation of 3 days and 30 days (P = 0.001) across the first principal coordinate at all scenarios.Most of the microbial communities were also separated among the sampling zones (D 1 , D 3 and D 5 ) of charosphere.

General properties of bacterial-fungal co-occurrence patterns
To further characterize the effect of biochar type on charosphere, we measured the co-occurrence patterns of bacterial-fungal communities (Fig. 6 and Additional file 1: Fig. S4).The connectivity of bacteria and fungi at biochar site were stronger than those in charosphere in all scenarios (Additional file 1: Table S1).The nodes of bacteria and fungi in biochar site were higher than the charosphere, which was decreased both in the pristine biochar treatment (from 1240 to 980) and acid-modified biochar (from 1600 to 1082) with the addition of urea.Comparing to the charosphere of pristine biochar (BC), the nodes of bacteria and fungi in pristine biochar with urea (BC-N) were increased (from 292 to 772), but decreased in acid-modified biochar with urea (ABC-N) (from 723 to 261).A higher average degree usually represents a greater network complexity.The network complexity was higher in biochar site than that in the charosphere, except acid-modified biochar treatment (ABC).Urea addition increased the network complexity of biochar site but decreased the network complexity of charosphere in the pristine biochar treatment, whereas the network complexity of both acid-modified biochar site and the corresponding charosphere were decreased with the urea addition.In addition, the average clusting coefficient (degree of nodes tending to cluster together) in charosphere was higher than that in the biochar site in all treatments.

Functional profile of bacteria and fungi affected by biochar and N addition
Functional annotation of taxa was examined by FAPRO-TAX based on normalized OTU tables.Eight-four functional categories were related to the bacterial communities in the present study.Three-way PERMANOVA analysis of those functions showed that the overall effects of the biochar type and N addition were significant (P < 0.05).Here, we focused only on the putative functions involved in the C and N turnover processes (Fig. 7), including hydrocarbon degradation, chitionlysis, xylanolysis, cellulolysis, fermentation, aerobic ammonia oxidation, aerobic nitrite-oxidation, denitrification, nitrogen fixation and nitrate ammonification.
The relative abundance of the bacteria linked in hydrocarbon degradation was slightly lower in biochar site than in the charosphere in BC treatment.The N addition (BC-N vs BC) increased the capacity of hydrocarbon degradation in BC treatments, but not in ABC treatments (ABC-N vs ABC).The abundance of chitinolysis function was significantly boosted in charosphere in BC treatment, and increased gradually from biochar site to charosphere in BC-N, whereas the acid-modified biochar significantly decreased the abundance of chitinolysis function in charosphere compared to controls.Different from chitinolysis, the process of xylanolysis was significantly increased by N addition in charosphere.Particularly in pristine biochar treatments, the N addition enhanced the function of xylanolysis (BC-N vs BC).However, such effect was not observed in acid-modified biochar treatments (ABC-N vs ABC).The relative abundances of both cellulolysis and fermentation functions in biochar site were relatively higher in ABC treatments than in BC treatments.The N addition elevated the capacity of fermentation in BC treatments, but not in ABC treatments.
The biochar type and N addition had significant interactions on the N turnover processes.Compared to the control, pristine biochar significantly decreased the relative abundance of aerobic ammonia oxidation function in charosphere and such negative effect was further enhanced by N addition (BC-N), while little effect was found in the charosphere of ABC and ABC-N.Similarly, N addition had negative effect on the process of aerobic nitrite oxidation at charosphere in BC.The BC-N treatment resulted in a lower relative abundance of denitrificater in biochar site and 1 mm of charosphere, compared to the control.However, all charosphere had the lower relative abundances of denitrificater in ABC-N.Additionally, both biochar types substantially increased the relative abundance of functional bacteria involved in N fixation in the biochar site.
Biomarkers with significantly different fungal taxon responses to the charosphere hotspots were selected by linear discriminant analysis coupled with effect size (LEfSe) analysis (Fig. 8), and these biomarkers' main functions were summarized (Table 2).Higher relative abundance of biomarkers was found in biochar site than in the charosphere in all treatments, which was consistent with the bacterial-fungal co-occurrence network in which fungi played a non-negligible role in biochar site.Comparing to the pristine biochar, acid-modified biochar had a higher relative abundance of Ascomycota and Sordariomycetes, while Mortierellaceae, Mortierella and Zygomycota were gathered in pristine biochar.On the other hand, urea addition attracted the colonization of Nectriaceae, Fusarium, Hypocreales, Sordariomycetes and Ascomycota in pristine biochar site, whereas Nectriaceae and Ascomycota in acid-modified biochar site.

The extent of charosphere
In the present study, both biochar types and N additions altered soil biochemical properties and microbial community compositions in charosphere (Chen et al. 2021;Yu et al. 2021).Generally, the size of soil zones influenced by biochar shifted with the parameter assessed, being narrower for microbial community composition and wider for soil pH and inorganic N content.Particularly, the spatio-temporal variations of soil pH in the charosphere were closely related to biochar type.Elevated soil pH in the charosphere of pristine biochar treatment was observed up to 6 mm from the biochar site.The previous study showed that the increased soil pH was up to 4.5 mm from the boundary of the biochar patch under unsaturated condition within a few hours (Zhu et al. 2022).At the same time, increased soil pH with a longer distance (~ 10 mm) away from the biochar site was reported under water-logged conditions (Yu et al. 2021).Besides the importance of soil water content, Chen et al. ( 2021) reported the ability of pristine biochar to raise soil pH was significantly correlated with the biochar EC value, which was attributed to the main factors determining the size of charosphere and its pH gradient.As expected, the acid-modified biochar contained negligible alkali or acidic substances and had a very low EC value; thus, its application did not result in a significant spatial gradient of soil pH.These differences likely result from the transfer of soluble alkalis from biochar to the soil, influenced by biochar type, diffusion coefficients, soil structure and moisture conditions (Fidel et al. 2017).With the strong alkalinity of pristine biochar, soil pH gradients of charosphere were established rapidly and attenuated gradually after a couple of weeks under the buffering equilibrium between biochar and soil.
Several studies have shown that microbial community composition changes in the biochar-soil interfaces (Dai et al. 2017;Yu et al. 2021).In agreement with these studies, microbial community composition in the present study differed between biochar sites and different layers of the charosphere.The pristine biochar induced relatively lower bacterial diversity but higher fungal diversities in the biochar site.Distinct gradients of microbial diversities were maintained up to 3 mm away from the biochar site.Soil pH and C availability generally limit the growth rates and reproduction strategies of microorganisms (Hoyle et al. 2008;Zhalnina et al. 2015).Hence, the C availability and pH distributions are probably the main drivers of differential microbial diversity and community composition in the various layers of the charosphere.RDA analysis further confirmed that the edaphic chemical properties, including pH, NO 3 − (P < 0.01) and NH 4 + (P < 0.01), significantly affected bacterial and fungal community structure.Acid-modified biochar resulted in neutral pH value in charosphere and increased the relative abundance of Proteobacteria, Gemmatimonadetes, and Ascomycota.However, the pristine biochar increased  as much as 1.5 units of the pH value in charosphere, which was beneficial for the colonization of microbes with higher pH tolerance, such as Proteobacteria (Dhakar and Pandey 2016), Acidobacteria (Xiong et al. 2012), and Zygomycota.This species-differentiation between the biochar site and charosphere further induced the distinct bacteria-fungi networks.Regarding C availability, biochar contains both labile substrates and recalcitrant C (Pathy et al. 2020), and those different C sources probably contribute to the differentiation of microbial species, consequently influencing the variations of microbial community structure and bacterial-fungal network (Xiong et al. 2021a;Yu et al. 2019).For instance, the most abundant bacterial phyla, Proteobacteria and Acidobacteria, in pristine biochar site could play key roles in plant C decomposition (Tiwari et al. 2016).Ascomycota mainly degrades the labile fraction of crop residues in the early stage of the decomposition process, while Basidiomycota mainly degrades the recalcitrant organic matter later (Challacombe et al. 2019;Francioli et al. 2016).Moreover, biochar can be used for fungi as physical growth matrix, and fungal hyphae could grow on the inner and outer surfaces of biochar particles (Hammer et al. 2014).Therefore, the biochar site (both C sources and physical habitats) was beneficial for higher fungal richness and fungal-bacterial complexity than charosphere (< 3 mm).Furthermore, biochar has relatively high water holding capacity, which could absorb water from the surrounding soil (Edeh et al. 2020), and result in redistribution of water contents in charosphere.Therefore, differences in soil moisture along the charosphere are likely to be another important factor to identify the differential community composition in the surrounding of biochar site.
Further study investigating the detailed moisture gradients in charosphere could be crucial for revealing the microbial hotspots in charosphere.
The charosphere zone influenced by biochar was the widest for ammonium contents (Fig. 3).NH 4 + -N was decreased in the vicinity of biochar site compared to controls.These decreases of NH 4 + -N occurred up to 6 mm from the biochar site in the BC-N treatment, which was further than the decrease of bacterial diversity (up to 3 mm).The ammonium adsorption capacity of both biochar and soil clay minerals (Wang et al. 2021) could contribute to the extent of NH 4 + -N declining gradients.The acid-modified biochar may have higher ammonium adsorption capacity due to increases in carboxyl and hydroxyl groups on biochar surface (Wang et al. 2020b), therefore, it resulted in sharper gradients of ammonium decline in ABC-N.Such a decrease of ammonium gradients in charosphere indicated that biochar might inhibit the processes of ammonium production (mainly urea hydrolysis) or increase ammonium consumption, which is discussed further in the next section.

Impacts of biochar types and N addition on putative N and C cycling functions in charosphere
The addition of biochar and N altered the soil available C and N content, and those nutrient gradients between the biochar site and charosphere, consequently, induced the shifts in microbial community structure and the relevant microbial-driven functions (Xu et al. 2015).As mentioned in the earlier section, the ammonium declining gradients in charosphere implied the biochar's influences on ammonium production and consumption.
As the main process for ammonium production in the treatments with N addition, urea hydrolysis was likely influenced by the application of biochar.The possible inhibition of urease activity was observed by Liu et al. (2018), who revealed that the biochar's inhibition may be the result of oxidative reactions with free radicals on the biochar surface or oxidative reactions with reactive oxygen species promoted by free radicals.In addition, strong physical adsorption of urea on biochar surfaces could possibly result in inhibited or delayed hydrolysis of urea (Saha et al. 2017).The pore structure of biochar was greatly improved and the surface adsorption sites were increased by the acid modification, which would enhance the capacity of biochar on urea adsorption, thereafter slowing down the urea hydrolysis as well as the ammonium production.The above-mentioned processes would all contribute to the higher soil inorganic N content (NH 4 + -N and NO 3

−
-N) at 30 days of the acid-modified biochar treatment.
Besides the urea hydrolysis, biochar and N addition could possibly influence the ammonium consumption processes, including ammonia volatilization, nitrification and N immobilization.Both types of biochar application lowered the soil inorganic N availability in charosphere, partially by stimulating N immobilization (Nguyen et al. 2017).These results are in line with other studies, reporting greater N immobilization by the microorganisms after biochar addition likely due to the high C:N ratio of biochar (Bruun et al. 2012;Kirkby et al. 2014).As discussed in the previous section, significant localized effects on soil pH were induced by biochar application, and such high pH conditions could affect the equilibrium dynamics of ammonium dissociation, consequently increasing the risk of ammonia volatilization (Zhao et al. 2013).However, the high CEC of biochar could possibly increase ammonium adsorption thereby decreasing ammonia volatilization (Mandal et al. 2016).In addition, biochar addition may decrease soil redox potential (Eh) as the increased soil pH, and low Eh may be beneficial for maintaining NH 4 + -N form (Richardson 2005) in the charosphere, leading to a high potential for ammonia loss due to the sufficient substrate and high soil pH.
It was worth noting that biochar might inhibit nitrification process, indicated by the lower relative abundances of both function groups of aerobic ammonia oxidation and aerobic nitrite oxidation in the biochar site.Particularly, the pristine biochar in combination with N addition decreased the relative abundance of nitrification relevant process in the charosphere compared to controls.Yao et al. (2022) confirmed that biochar and N addition inhibited the autotrophic nitrification rate mainly by greatly reducing the abundance of amoA-AOB gene, and the presence of polyaromatic hydrocarbons in biochar might also contribute to the inhibition of the nitrification process (Thangarajan et al. 2018).Additionally, it was demonstrated that nitrification would be ceased when pH was lower than 5.0, and occurred rapidly when pH was over 6.0 (Sahrawat 2008).The liming effect of alkaline biochar, which depends on the nature of feedstock materials and the pyrolysis conditions (Bolan et al. 2023), could contribute to the increased nitrification in acidic soils (Ulyett et al. 2014); however, the effects of biochar on nitrification in alkaline soils (as in the present study) remain inconsistent (Cayuela et al. 2014).
The biochar's influences on denitrification are complicated.The addition of biochar into soil alters various geochemical parameters in soil, closely related to the diversity, and abundance of soil microbial communities with denitrification, such as N forms (NO 3 − /NH 4 + ) and availability, pH and oxygen saturations (Cayuela et al. 2014).Since the pH in the ABC treatment was marginally changed, the observed decreases in the abundance of denitrifiers in biochar sites were unlikely to be caused by liming effect .Van Zwieten et al. (2015) claimed that biochar application may promote heterotrophic microbial respiration and growth on the surface of biochar particles, thus it could contribute to forming anoxic microsites within soil particles and aggregates, consequently enhancing complete denitrification.On the other hand, the porous structure of biochar may improve the aeration and maintain relatively sufficient O 2 availability (Zhu et al. 2022); consequently, it was beneficial for forming oxic conditions in the biochar-soil interfaces, which would inhibit the denitrification.Such contrasting impacts would complicate the biochar's influence on denitrification.Further study on detailed pore size distribution and moisture gradients, as well as gene expression activities in charosphere would be helpful to untangle the microscale impacts of biochar on denitrification.
Additionally, both types of biochar increased the relative abundance of microorganisms capable of nitrogen fixation in biochar sites.The biochar amendment alters several environmental parameters, such as elevated pH, slightly lower concentrations of NO 3 − and NH 4 + , which might stimulate the abundance and activity of nitrogen fixation microorganisms (Atkinson et al. 2010;Harter et al. 2014).Meanwhile, assimilation of the biochar-derived C requires additional sources of N, which might favor microorganisms capable of N fixation under conditions of limited N availability.Thus, the interplay of these parameters is likely to be responsible for the elevated abundance of nitrogen fixation bacteria in the biochar sites.Another key factor influencing N fixation is the O 2 contents, which could be investigated with detailed gradients in biochar treatment in further studies.
There were distinct responses of N-related fungal groups between the biochar site and charosphere, revealed by the potential function of fungal biomarkers selected by LEfSe analysis (Table 2), including N-fixation, denitrification, and N mineralization (Crenshaw et al. 2008;Green et al. 2008;Challacombe et al. 2019).Mortierellaceae, Mortierella and Zygomycota gathered in pristine biochar site, which were reported to have the ability to improve the content of available N (Ning et al. 2022;Ozimek and Hanaka 2021;Shang et al. 2020).Whereas the acid-modified biochar had the lower relative abundance of those biomarkers.The urea addition attracted the colonization of Nectriaceae, Fusarium, Hypocreales, Sordariomycetes and Ascomycota, which were reported to participate in the processes of denitrification (Deng et al. 2021).Those distinct fungal communities harbored in biochar did not perform the same functions as the harbored bacteria, and such diversified performances would likely contribute to inconsistent functions of biochar addition on N turnover.
The putative C degradation processes in charosphere hotspots would greatly be impacted by the biochar type and N addition (Fig. 7).The biochar had varied effects on different C degradation processes.One of the main factors impacting organic C degradation was biochar's adsorption capacity, which was promoted by the large surface area and enriched functional groups of biochar (Ding et al. 2018;Lehmann et al. 2021).Recent studies revealed that biochar's adsorption of organic C would rather be selective (Hill et al. 2019).Therefore, such selective adsorption in biochar site could lead to conditions that favor one particular organism over another even expanded to charosphere, resulting in distinct ecological functions.In the present study, the functions of hydrocarbon degradation, chitinolysis and xylanolysis were suppressed in the pristine biochar site at BC, which were in line with the decreased relative abundances of Planctomycetes, Actinobacteria, and Firmicutes (Dedysh and Ivanova 2019;Lacombe-Harvey et al. 2018;Veliz et al. 2017).Those decline of C-related functions could slow down the C mineralization, potentially contributing to soil C sequestration.The N addition enhanced such suppress of chitinolytic in charosphere, but offset the inhibition or even promoted the xylanolysis in charosphere.Therefore, the interaction of biochar and N addition might have diversified functions on C degradation.
Moreover, several studies have revealed that bacterialfungal co-occurrence network coupled with microbial diversity (Banerjee et al. 2016;Wagg et al. 2019) can provide insight into species that share similar ecological niches like C turnover (Freilich et al. 2018).The microbial diversity was observed to be closely associated with microbial network complexity, with higher phylogenetic diversity related to complex networks (Wagg et al. 2019).The degradation of recalcitrant biochar would require cooperation among varied microbial guilds, which resulted in a complex microbial network in biochar sites.The higher complexity of bacteria-fungi co-occurrence networks has been shown to indicate a stronger cooperation of different microbes, which may contribute to greater resilience against environmental disturbances (Ling et al. 2016), and to benefit ecosystem functioning (Wagg et al. 2019).The enhanced microbial network complexities could, to some extent, support that biochar sites might be the reservoir of the C and N pools, whereas the decreased complexity of microbial network in charosphere may imply conservative microbial functionalities, such as relatively weak C degradation and retarded nitrification, under current conditions.More direct and detailed evidences of such implications are needed with further research efforts.

Conclusions
The present study investigated the spatio-temporal evolution of key chemical and biological properties in charosphere with and without N additions.The pristine biochar induced a rapidly established charosphere of a few millimeters thick, with a decreasing gradient of pH and an increasing gradient of NH 4 + -N away from the biochar site.The increased pH in pristine biochar site elucidated the dissimilarity of keystone bacteria and fungi in charosphere, forming diverse co-occurrence patterns and functional profiles.We found that bacterial richness decreased closer to the pristine biochar site with dominant phyla of Proteobacteria and Acidobacteria.In contrast, the fungal richness was increased in the pristine biochar site with a relative high abundance of Ascomycota and Zygomycota.Regardless the type of biochar, the charosphere has decreased complexity of microbial networks with urea application, which might imply the conservative microbial functionality of charosphere.The observed lower relative abundance of C degradation communities and nitrification-related groups in biochar site and charosphere by FAPROTAX revealed the import role of biochar application on C and N turnover processes.Overall, our results revealed the distinct spatial distribution and network pattern of bacterial and fungal communities in the biochar site and charosphere in millimeter scale, which could advance our understanding of the dynamic changes of soil C and N in response to biochar application.

Fig. 1
Fig. 1 Schematic of incubation and sampling equipment

Fig. 2
Fig.2(a) Mean value of soil pH in the charosphere during the incubation period, the horizontal solid and dotted line mark the initial soil pH with (pH 7.28) or without (pH 7.17) N addition.(b) The variation of soil pH value in the charosphere of BC and ABC with or without N addition on day 14 (T3)

Fig. 3
Fig. 3 The variations of soil (a) NH 4 + -N and (b) NO 3 − -N gradients in the charosphere with or without N addition

Fig. 4 Fig. 5
Fig.4Species richness of bacteria and fungi among biochar types, incubation times, and distances away from the biochar site.T1, T3 and T5 represent the sampling time on days 3, 10, and 20, respectively.D0 is the biochar site.D1, D3 and D5 are the charosphere sections away from the biochar site for 1, 3 and 5 mm, respectively.CK and CK-N represent the sample of controls without and with N addition, respectively.The small letters represent significant differences among distances within the same sampling time (P < 0.05)

Fig. 6
Fig.6Bacterial and fungal co-occurrence networks based on operational taxonomic units (OTUs) at the species level in biochar site (D0) and charosphere (D1D3D5).Node size is proportional to the relative abundance.Bacteria and fungi are colored by red and green, respectively

Fig. 7
Fig. 7 Variations in the relative abundance of putative microbial functions associated with C and N cycling in response to biochar type and N addition.Different letters indicate significant differences (one-way ANOVA, P < 0.05, Duncan's multiple-range test) among distance within the different treatments.The bars represent standard errors (n = 9), except for biochar site in BC treatment, n = 8.The solid and dotted lines indicate the initial values of each parameter with or without N application, respectively

Fig. 8
Fig.8The significantly different abundant taxa exposed to pristine biochar and acid-modified biochar by

Table 1
Chemical properties of soil and biochar

Table 2
The main functional list of fungal biomarkers picked by LEfSe