Seasonal freeze ‒ thaw processes impact microbial communities of soil aggregates associated with soil pores on the Qinghai–Tibet Plateau

Background Seasonal freeze ‒ thaw (FT) processes alter soil formation and cause changes in soil microbial communi‑ ties, which regulate the decomposition of organic matter in alpine ecosystems. Soil aggregates are basic structural units and play a critical role in microbial habitation. However, the impact of seasonal FT processes on the distribution of microbial communities associated with soil pores in different aggregate fractions under climate change has been overlooked. In this study, we sampled soil aggregates from two typical alpine ecosystems (alpine meadow and alpine shrubland) during the seasonal FT processes (UFP: unstable freezing period, SFP: stable frozen period, UTP: unstable thawing period and STP: stable thawed period). The phospholipid fatty acid (PLFA) method was used to determine the biomass of living microbes in different aggregate fractions. Results The microbial biomass of 0.25–2 mm and 0.053–0.25 mm aggregates did not change significantly dur‑ ing the seasonal FT process while the microbial biomass of > 2 mm aggregates presented a significant difference between the STP and UTP. Bacterial communities dominated the microbes in aggregates, accounting for over 80% of the total PLFAs. The microbial communities of soil aggregates in the surface layer were more sensitive to the sea‑ sonal FT process than those in other soil layers. In the thawing period, Gram positive bacteria (GP) was more dominant. In the freezing period, the ratio of Gram‑positive to Gram‑negative bacterial PLFAs (GP/GN) was low because the enrichment of plant litter facilitated the formation of organic matter. In the freezing process, pores of 30–80 μm (mesopores) favored the habitation of fungal and actinobacterial communities while total PLFAs and bacterial PLFAs were negatively correlated with mesopores in the thawing process.


Introduction
Soil aggregates, as the basic functional unit of soil structure, impact soil physicochemical properties and biogeochemistry conditions (Ozbolat et al. 2023).Soil aggregates have important functions in organic matter stabilization, microbial biodiversity maintenance and nutrient cycling, with aggregate size and stability serving as important indicators of these functions (Six et al. 2001;Nie et al. 2014).Aggregates can be used to identify unique spatial structures acting as microbial microhabitats, which regulate the decomposition of organic matter (Bailey et al. 2012(Bailey et al. , 2013)).Not only can aggregates stabilize microbial compositions and enhance community interactions, but aggregate associations can also change microbial functions and traits through spatial confinement (Wilpszeski et al. 2019).
Microaggregates were found to support a slower growing, more diverse and less complex group of microbes than communities in macroaggregates (Lupwayi et al. 2001;Kuzykova and Blagodatskaya et al. 2015;Bach et al. 2018).Microaggregate microbial communities also change more dynamically across the sampling season than those of large macroaggregates (Upton et al. 2019).The heterogeneous microenvironments within soil aggregates, which are mostly shaped by soil pores, can influence microbial activities by affecting gas/solution transport and substrates accessibility (Ruamps et al. 2013;Kravchenko and Guber 2017).Yang et al. (2022) found that pores > 5 μm had positive effects on microbial diversity and abundance in an Ultisol.Pores 10-100 μm and 30-150 μm of soil aggregates were found to support microbial activities (Kravchenko et al. 2019;Liang et al. 2019).Nevertheless, the specific soil microbial biomass in different soil aggregates and its relationships with pore size distribution were not well revealed under freezethaw (FT) processes.
The FT process is a common abiotic disturbance to alpine soils.The process leads to changes in soil structure initially and causes redistribution of nutrients and mineral elements, which may further lead to niche differentiation (Ji et al. 2022).Through mechanisms including cell lysis in low-temperature stress and the promotion of microbial activity following thawing, FT could alter the soil microbial communities (Lipson et al. 2000;Grogan et al. 2004).Many studies have demonstrated the effect of FT cycles on microbial biomass, but their results are inconsistent.Previous studies have shown that FT cycles could decrease fungal biomass (Song et al. 2017), increase fungal biomass (Buckeridge and Grogan 2008), or have no impact on the diversity of bacterial and fungal communities (Liu et al. 2022).Through the abundant released organic matter in soil, some microbes can develop the high resistance to FT rapidly (Han et al. 2018;Walker et al. 2006).Therefore, the responses of microbial communities to FT processes are complex, and are closely associated with many factors including initial soil water content, soil temperature, soil texture, etc. (Wu et al. 2017).Also, FT processes can affect the variance of microbial abundance.Zhao and Hu (2023) found that freezing could decrease the variance in microbial biomass between soil horizons.However, most of these studies were conducted on homogenized samples or monoculture isolates rather than soil aggregates.How the seasonal FT process alters the microbial biomass and distribution of soil aggregates associated with soil pores in alpine ecosystems was not well understood.
The Qinghai-Tibet Plateau (QTP) has the largest permafrost coverage in the middle to low latitudes of the world, which is crucial to the global carbon cycling (Wu et al. 2010).In recent years, dramatic changes have occurred in the QTP as the depth and duration of FT processes decreased while the frequency of FT cycles increased (Liu et al. 2016).This has led to changes in community structure and function of soil microbes, thereby affecting element cycling at the regional scale (Zhao and Hu 2023).In this study, we investigated the changes in microbial communities of soil aggregates in typical ecosystems on the northeastern Qinghai-Tibet Plateau during the seasonal FT.The phospholipid fatty acid analysis (PLFA) method was used for determining microbial biomass and composition.We expected to reveal for soil aggregates: (1) how the soil microbial biomass changed; (2) changes in variability in microbial communities of different aggregates; and (3) the relationships between microbial communities and pore size distribution during the seasonal FT process.We hypothesized that: (i) the microbial abundance of aggregates was highest in the stable thawed period; and (ii) pores 30-80 μm posed positive impact on microbial abundance.

Study site and experimental design
The study was carried out in the Qinghai Lake Basin (36°15′N-38°20′N, 97°50′-101°20′E), which lies in the north-eastern part of Qinghai-Tibet Plateau.The region is located in a high-altitude, cold climate zone with a mean annual temperature of 0.1 °C and precipitation of 400 mm (Li et al. 2018).The Kobresia pygmaea meadow and the Potentilla fruticose shrubland were the two ecosystems selected for the study (Fig. 1).They jointly account for approximately 70% of the watershed's land area and are representative alpine ecosystems.The soil type was classified as Gelic Cambisols according to the FAO UNESCO system (IUSS Working Group WRB 2022).The soils in alpine meadow and alpine shrubland ecosystems are featured by the mattic epipedon which occurs in the soil surface layer (Zhi et al. 2017).Mattic epipedon is characterized by dense roots and plays a crucial role in carbon storage, soil water retention, and indication of alpine vegetation degradation (Yang et al. 2014).To get the replicates across ecosystems, three Kobresia pygmaea meadow sites and three Potentilla fruticose shrubland sites were selected.We tried to avoid the simple pseudo replication so that each sampling site has a certain distance with others (> 1 km).Three sites within each ecosystem have similar vegetation conditions.In every FT period, three sampling plots (1 m × 1 m) were set up at each site.
The seasonal FT process was separated into four periods according to Li et al. (2018) and the soil temperature obtained by the sensor Campbell Sci.109L at 0.5 Hz with 30 min averages: the unstable freezing period (UFP, as soil temperature starts to drop to 0 °C), the stable frozen period (SFP, with soil temperature completely below 0 °C), the unstable thawing period (UTP, as soil temperature starts to rise above 0 °C), and the stable thawed period (STP, with soil temperature completely above 0 °C) (Table 1).We recommend readers refer to Supplementary Fig. 1 for specific annual changes in soil temperature.

Field sampling and aggregate sieving
A total of 18 profiles were obtained before collecting soil samples.Every soil profile was dug directly within the plant patch to guarantee similar conditions.We divided the soil layers into 0-10 cm, 10-30 cm and 30-50 cm soil layers, representing surface soil, subsurface soil and subsoil, respectively (Chen et al. 2021a).Soil cores (approximately 1 kg) and bulk soil were collected in each soil layer for aggregate sieving and physicochemical properties measurements, respectively.Soil cores were immediately placed in an icebox and transferred to the laboratory.The temperature of the icebox was modified corresponding to the actual air temperature when sampling to maintain the conditions of samples similar to those when they were taken.At each sampling site, bulk soil samples from the same soil layer were thoroughly mixed.Particle size distribution was determined by the sieve-pipette method.The soil water content on a weight basis was determined using an oven-dried method.Soil pH measurements were conducted with an FE20 pH meter (Mettler Toledo, Columbus, USA) from slurries of samples at a soil:water ratio of 1:2.5 (w:w).Soil organic carbon (SOC) and total nitrogen (TN) contents were determined using a CN 802 elemental analyzer (VELP, Italy).
Dry sieving was conducted to separate bulk soils into soil aggregate fractions > 2 mm (large macroaggregates), 0.25-2 mm (small macroaggregates) and 0.053-0.25 mm (microaggregates) in diameter.Soil cores were gently broken by hand into 1-cm clods, and then soils were laid out between sheets of brown paper (Schutter and Dick 2002).Debris such as gravel and roots were removed from the samples.Two hundred grams of soil was placed on the top sieve and shaken for five  minutes by a sieve shaker (200 r/min).All the fractions were stored at − 20 °C for subsequent microbial analysis using the PLFA method.

Measurement of microbial characteristics
The PLFA method was used to quantify the microbial biomass and community structure of all aggregate fractions following the procedure described by Buyer and Sasser (2012) and Sharma and Buyer (2015).Briefly, lipids were extracted from 8 g of fresh soil using a mixed chloroform:methanol:citrate buffer (1:2:0.8,v/v/v).The phospholipids were separated from nonpolar lipids and derivatized into their corresponding fatty acid methyl esters before analysis.Methylated fatty acids were identified using a triple quadrupole tandem mass spectrometer (450 GC-MS/MS) produced by the Bruker Company, USA.The concentrations of phospholipids were quantified based on the internal standard of methyl nonadecanoate (19:0).The PLFA content of soil was expressed in nmol/g dry weight.The PLFA contents of the specific groups was estimated based on biomarkers as shown in Table 2. Total PLFAs, which included all identified PLFAs, were used to obtain the relative abundance of each group including bacterial, fungal, actinobacterial (Act.),Gram-positive (GP) bacterial, Gram-negative (GN) bacterial and arbuscular mycorrhizal fungal (AMF) communities.
In addition to measuring the PLFAs of aggregates, we analyzed the variability in microbial abundance of different aggregate fractions and of different soil depths during the seasonal FT process.The coefficient of variation (CV) was selected as it could represent the variations of soil characteristics (Adhiari et al. 2012).The CV was calculated as follows: where X i denotes the microbial abundance of soil aggre- gates (nmol/g) and SD represents the standard deviation.We calculated the CV of microbial biomass in the same aggregate fraction from 0-10 cm, 10-30 cm to 30-50 cm during the seasonal FT process to indicate the variance of different soil depths.We also calculated the CV of microbial biomass in the 0.053-0.25 mm, 0.25-2 mm and >2 mm aggregate fractions from the same soil layer to quantify differences in microbial biomass among aggregate fractions.

Measurement of pore size distribution of aggregates
The soil aggregates were scanned using a nanoVoxel-4000 X-ray three-dimensional microscopic CT (Sanying Precision Instruments Co., Ltd., China) with X-ray source parameters of voltage 80 kV and current 50 μA.During a 360° rotation, 2800 detailed and low-noise images were generated.The reconstructed images featured a 3.6 μm spatial resolution and 2800 × 2800 × 1500 voxels.Representative macroaggregates of all soil layers in every FT period were selected and scanned with a total of 144 aggregates.
The pore structure was reconstructed and visualized through Avizo 9.0 software following the procedure of Wang and Hu (2023).The Volume Edit tool was used to remove the fragments outside the aggregate.Mask extraction was conducted in the Segmentation module (Zhao et al. 2020).After acquiring the mask, we selected the pores in aggregates using the histogram thresholding method based on the global thresholding algorithm (Jaques et al. 2021;Yang et al. 2021).The complete grayscale histogram was used to segment the data.T min and (1) Table 2 The biomarkers corresponded with specific soil microbial groups
The two-dimensional images were transformed into 3D images by Volume Rendering tool in Avizo 9.0 software.Due to limited resolution of the images, pores smaller than 3.6 μm could not be distinguished.The soil pores larger than 3.6 μm were divided into four size classes: < 15 μm, 15-30 μm, 30-80 μm and > 80 μm (Wang and Hu 2023).The pore size distribution was categorized by equivalent diameter (EqD) and was featured by percentage of porosity.The equivalent diameter of pores can be obtained using the Volume Fraction tool in the Avizo software and was defined as the diameter of spherical particle with the same volume and was calculated by pore volume (Zhao and Hu 2023): where V represents the volume of pores.

Statistical analysis
All statistical analysis was conducted with IBM SPSS 25 software (SPSS Inc., USA).All experimental data were checked for normal distribution by the Shapiro-Wilk normality test.One-way ANOVA followed by Fisher's least significant difference (LSD) test was used to determine the differences in soil microbial biomass of aggregates during the seasonal FT periods.The variances of CV values were also determined with this method.Pearson correlations were conducted to evaluate the linkages between the soil microbial community and pore size distribution.

Total microbial biomass of aggregates
In alpine ecosystems, the total PLFAs of 0.25-2 mm aggregates and microaggregates did not change significantly during the seasonal FT process (Fig. 2a).In the alpine meadow (Fig. 2a), the total PLFAs of > 2 mm aggregates in STP was 20.68 nmol/g, which was significantly higher than that in UTP (12.60 nmol/g).In the alpine shrubland (Fig. 2b), the total PLFAs of > 2 mm peaked in the STP (25.90 nmol/g) and were lowest in the UTP (16.24 nmol/g) among four seasonal FT periods.Therefore, the seasonal FT process mainly affected the total microbial biomass of > 2 mm aggregates rather than that of other aggregate fractions.Following the seasonal FT process, the total PLFAs first decreased and then peaked in the stable thawed period.
Figure 3 depicts the profile distribution of the microbial biomass of soil aggregates during the seasonal FT process.The variation in microbial biomass in the four Fig. 2 The total microbial biomass of soil aggregates in the a alpine meadow and b alpine shrubland ecosystems during the seasonal FT process (UFP, unstable freezing period; SFP, stable frozen period; UTP, unstable thawing period; and STP, stable thawed period).Different lowercase letters denote significant differences among FT periods (P < 0.05) FT periods was obvious in the surface soil.In the alpine meadow, the total microbial biomass of all aggregate size fractions in the SFP and STP decreased with increasing soil depth (Fig. 3a).For > 2 mm aggregates, the microbial biomass in the UFP and UTP did not vary significantly among soil depths.For 0.25-2 mm and 0.053-0.25 mm aggregates, the microbial biomass in the UFP and UTP was highest in subsurface soil.In the shrubland, the difference in microbial biomass of soil aggregates between the surface soil and subsurface soil was less significant than that in the meadow (Fig. 3b).In the SFP and STP, decreasing trends in microbial biomass with increasing soil depth were found.Therefore, the profile distribution of total microbial biomass was affected by ecosystem types, FT periods and aggregate sizes.

Microbial community structure of aggregates
The microbial community of aggregates mainly consisted of bacteria and fungi, and the biomass of the bacteria accounted for over 80% of the microbial biomass of aggregates.The responses of different microbial groups to the seasonal FT were dependent on the aggregate size (Fig. 4).In the meadow, the bacterial and fungal PLFAs of > 2 mm aggregates decreased first and then increased following the seasonal FT process.The actinobacterial PLFAs of 0.25-2 mm aggregates was lowest in the UTP (0.61 nmol/g) and highest in the STP (1.01 nmol/g) among four seasonal FT periods.In the shrubland, the actinobacterial PLFAs of all aggregate fractions first decreased and then increased following the seasonal FT, peaking in the stable thawing period (Fig. 4g).The seasonal FT process did not impact the AMF PLFAs in both alpine meadow and alpine shrubland ecosystems.
Figure 5 depicts two critical ratios, namely the ratio of fungal biomass and bacterial biomss (F/B) and the ratio of GP bacteria and GN bacteria (GP/GN) of soil aggregates in the freezing period and the thawing period.In both ecosystems, the GP/GN values of all aggregate fractions in the thawing period were all significantly higher than those in the freezing period.In the alpine shrubland, the F/B values of all aggregate fractions in the freezing period were all significantly higher than those in the thawing period.No significant variance in F/B values was observed in the alpine meadow ecosystem.Fig. 4 The bacterial, fungal, actinobacterial and aubruscular mycorrhizal fungal PLFAs in the alpine meadow (a-d) and alpine shrubland (e-h) ecosystems during the seasonal FT process (UFP, unstable freezing period; SFP, stable frozen period; UTP, unstable thawing period; and STP, stable thawed period).Different lowercase letters denote significant differences among FT periods (P < 0.05).Different uppercase letters denote significant differences among aggregate fractions (P < 0.05)

Variations in microbial biomass of aggregates
The seasonal FT process did not significantly alter the variability of microbial biomass of microaggregates among different soil depths (Fig. 6).For > 2 mm aggregates, the CV value of microbial biomass altered as the value in the UFP (0.61) was significantly higher than those in the UTP (0.28) and STP (0.31).For 0.25-2 mm aggregates, the CV value in the UFP (0.49) was significantly higher than those in the UTP, STP and SFP.Therefore, for macroaggregates, the freezing process enhanced the microbial biomass variability in different soil depths.
Figure 7A and B depict the CV of microbial biomass of different aggregate fractions in the alpine meadow and shrubland ecosystems, respectively.The CV values in total, bacterial, fungal and Actinobacterial PLFAs gradually increased during the seasonal FT process and were highest in the stable thawed period.Overall, the thawing process increased the variability of microbial biomass among aggregate fractions.

Relationships between microbial abundance and pore size distribution
Table 3 demonstrates the correlations between microbial abundance and pore size distribution of macroaggregates.In the freezing process, the fungal PLFAs and actinobacterial PLFAs were positively correlated with pores 30-80 μm and negatively correlated with pores > 80 μm (P < 0.05).The AMF PLFAs were also negatively correlated with pores > 80 μm (P < 0.05).In the thawing process, total PLFAs and bacterial PLFAs were negatively correlated with pores 30-80 μm (P < 0.05).

Discussion
Seasonal FT processes altered the microbial community structure of aggregates The impact of FT processes on the microbial communities of aggregates can be dependent in aggregate size.In our study, the seasonal FT process only affected the total microbial biomass of > 2 mm aggregates rather than that of 0.25-2 mm and 0.053-0.25 mm aggregates (Fig. 2).This can be explained by the fact that > 2 mm aggregates have lower stability and are more vulnerable to water phase changes than other aggregate fractions, which can reshape microbial habitats.Previous studies have demonstrated significant changes in the stability and nutrients of > 2 mm aggregates after FT cycles (Chai et al. 2014;Li and Fan 2014).Apart from FT processes, many studies have reported changes in the internal microbial communities of > 2 mm aggregates due to external forces.Yang et al. (2019) reported that the higher soil respiration rates occurred in > 2 mm aggregates than in microaggregates.Jiang et al. (2011) Fig. 6 Coefficient of variation (CV) in microbial biomass in soil aggregates from different soil layers during the seasonal FT process (UFP, unstable freezing period; SFP, stable frozen period; UTP, unstable thawing period; and STP, stable thawed period).Different lowercase letters denote significant differences among FT periods (P < 0.05).* represents a significant difference in CV of aggregate fractions (P < 0.05) found that nutrient limitations in > 2 mm aggregates could lead to changes in microbial biomass of them.Therefore, aggregate size can mediate the microbial communities' responses to seasonal FT processes.Our results showed that total microbial PLFAs of > 2 mm aggregates peaked in the stable thawed period and were lowest in the unstable thawing period (Fig. 2).This is similar to previous findings.The microbial biomass of both subalpine soil and alpine swamp soil decreased first and then increased during the seasonal FT process (Chen et al. 2020).A significant decrease in microbial biomass occurred in early spring was partly because the microbial community dominated by bacteria had not fully established a tolerance to the cold environments (Edwards et al. 2006;Isobe et al. 2018).During the stable thawed period, the melting of ice crystals accelerated the release of unstable nutrients, increasing the amount of effective substrates (Sang et al. 2021), and thus stimulates the rapid growth and reproduction of microorganisms (Jefferies et al. 2010).Microbes can also acclimate to the environment Fig. 7 Coefficient of variation (CV) in microbial biomass in soil aggregate fractions in the a alpine meadow and b alpine shrubland ecosystems during the seasonal FT process (UFP, unstable freezing period; SFP, stable frozen period; UTP, unstable thawing period; and STP, stable thawed period).Different lowercase letters denote significant differences among FT periods (P < 0.05) via increased lipid membrane fluidity (Pastore et al. 2023).Therefore, the seasonal FT process significantly changed the total microbial biomass of > 2 mm aggregates, and the biomass of > 2 mm aggregates was highest in the stable thawed period.
Our study also revealed that the seasonal FT process had a greater impact on the microbial abundance of surface soil aggregates in the alpine meadow ecosystem (Fig. 3).This can be attributed to the mattic epipedon which is a special surface with intertwined roots and minerogenic matter.The mattic epipedon contains a large amount of organic matter, which leads to nutrient enrichment.The belowground biomass could lead to the more obvious change in aggregate stability in the topsoil than in other layers (Dong et al. 2023).Nutrient enrichment, as well as its strong seasonal variability affect microbial abundance (Chen et al. 2021b).The input of organic compounds could decrease the resistance of soil bacterial community to freeze-thaw disturbances through changes in their physiological and functional traits (Zhang et al. 2022).In the alpine shrubland ecosystem, the difference in the microbial biomass of aggregates between the 0-10 cm and 10-30 cm soil layers was not significant (Fig. 3).The extension of shrub roots created favorable nutrient conditions for microorganisms in the 10-30 cm soil layer.Therefore, nutrient conditions, which was shaped by belowground root development, could affect the profile distribution of microbial communities.
In the thawing period, GP/GN values of all aggregate fractions were significantly higher than that in the freezing period, indicating that GP was more dominant in the freezing period (Fig. 5a, b).GN mainly utilizes carbon from plant sources while GP mostly utilizes carbon from more stable organic matter (Fanin et al. 2019).So, the enrichment of plant litter facilitated the formation of plant-derived carbon in the freezing period, which led to the decrease in the GP/GN of soil aggregates.In the alpine shrubland, the freezing process increased the F/B value of all aggregate fractions (Fig. 5d).While bacteria communities are more suited to warm, humid conditions, fungi communities are better able to sustain their physiological and metabolic activity in cold, dry soil (Strickland and Rousk 2010).
Overall, the seasonal FT process led to changes in microbial communities especially of > 2 mm aggregates by affecting nutrient and aggregate stability.Considering the differences in microbial changes between aggregate components and soil depth, the complex interactions between soil properties and microbial communities need to be urgently explained under future global warming (Hicks et al. 2022).

Seasonal FT processes altered the variability of microbial communities of aggregates
Our results demonstrated that the CV values of microbial biomass in the same aggregate fractions from different soil layers were more significant in the freezing period (Fig. 6), indicating that the freezing process enhanced the variability of microbes of different soil depths.The freezing process led to major disparities in water and nutritional conditions among different soil layers, which also caused a major decrease in soil water infiltration (Gao et al. 2021;Ji et al. 2022).In turn, these differences in water and nutrient conditions cause significant differences in microbial biomass of aggregates between different soil layers.
CV values of microbial biomass among different aggregate fractions in the stable thawed period were significantly higher than those in the other periods (Fig. 7), which highlighted the impact of thawing process on enhancing the microbial variability among aggregate fractions.In general, plants affect soil microbial communities significantly and high plant activity in the stable thawed period can lead to the establishment of smaller ranges of microbial community hotspots and enhance variation (Wardle et al. 2004).At the aggregate scale, the thawing process involves the release of nutrients (Jefferies et al. 2010).Due to significant differences in nutrient contents among different aggregates (Wang and Hu 2023), the variability of the conditions for microbial activity is also enhanced, resulting in greater variance in microbial biomass among aggregate fractions in the stable thawed period.

The impact of pore size distribution on microbial biomass
The relationships between pore size distribution and microbial biomass differed in the seasonal FT process.In the freezing process, pores 30-80 μm favored the habitation of fungal and Actinobacterial communities while their abundance were negatively correlated with > 80 μm pores (Table 3).Fungi communities have high tolerance to cold and dry environments (Strickland and Rousk 2010).Actinobacteria grow by apical branching and share similar morphology with fungi (Wolf et al. 2013).Under freezing conditions, they tended to extend and move from larger pores to smaller pores (Xia et al. 2022).The enrichment of SOC in mesopores also provides preferable conditions for fungal and Actinobacterial growth (Kravchenko and Guber 2017).
In the thawing process, total PLFAs and bacterial PLFAs were negatively correlated with pores 30-80 μm, indicating that mesopores did not favor microbial habitation (Table 3).This is consistent with Liang et al. (2019).Vegetation grows rapidly in the thawing process, and soil aeration and moisture conditions improved.Bacterial communities were sensitive to water-filled conditions while pores 30-80 μm of aggregates favor water absorption (Lal and Shukla 2004;Xia et al. 2022).Therefore, this anaerobic environment was not conducive to the growth of the bacteria-dominated microbial communities.

Implications and limitations of the study
Changes in microbial communities induced by FT processes can be an important indicator for carbon and nitrogen dynamics under climate change (Robroek et al. 2013).FT could diminish the microbial C and N metabolic functionality which was closely associated with microbial abundance, further affected soil C and N pools and fluxes (Ji et al. 2022;Zong et al. 2023).Considering the important role of aggregates in SOC protection, inconsistent changes in microbial abundance from aggregates of different depths and sizes during the FT process can help us predict future carbon dynamics more accurately.
Also, as the index of soil fertility and ecosystem productivity, soil microbial biomass is a key soil driver of ecosystem functioning (Singh and Gupta 2018).Accurate understanding of soil-vegetation-microbial interactions will help us address the increasingly severe land degradation problem in alpine ecosystems (Li et al. 2021(Li et al. , 2023)).
In this study, we explored the changes in the microbial community structure of soil aggregates, as well as the distribution patterns among aggregate fractions during the seasonal FT process.However, to verify the precise impact of FT processes on the microbial communities of aggregates, the results obtained by field experiments and simulated FT cycles should be combined and incorporated into a comprehensive evaluation system for future predictions.Although this study explains the variability in microbial biomass among aggregate fractions, it is necessary to analyze the potential flow pathways of microbial communities and their accompanying functional changes through techniques such as isotope tracing.Additionally, soil microbial biomass assessment provides instantaneous measurements of microbial activities, which may vary in space and time if repeated measurements are done.More robust indicators of soil quality such as SOC fractions (e.g.microbial necromass carbon) could be applied in further studies.

Conclusions
Seasonal FT processes alter soil formation and cause changes in soil microbial communities, which regulate the decomposition of organic matter in alpine ecosystems.The impact of seasonal FT processes on the distribution of microbial communities in different aggregate fractions under climate change has been overlooked.The findings of this study revealed the impact of seasonal FT processes on microbial distributions of aggregates.The seasonal FT process significantly changed the total microbial biomass of > 2 mm aggregates due to the low stability of this fraction.The release of accumulated soil nutrients led to a relatively high microbial abundance in the stable thawed period.The enrichment of nutrients in surface soil caused a decrease in microbial biomass in aggregates with increasing soil depth.The GP/GN value in the thawing period was significantly higher than that in the freezing period.The freezing process caused a greater variability in microbial biomass of different soil depths.The thawing process enhanced the variation in microbial biomass among different aggregate fractions.Mesopores of aggregates supported the habitation of actinobacterial and fungal communities in the thawing process while they were not conducive to bacterial growth in the thawing process.This study has critical implications for evaluating soil microbial functions and reducing the uncertainty in global carbon cycle predictions under climate change.

Fig. 1
Fig. 1 Location, landscape and typical soil profiles of sampling sites in the alpine meadow and shrubland ecosystems

Fig. 3
Fig. 3 Profile distribution of total microbial biomass of the a > 2 mm, b 0.25-2 mm and c 0.053-0.25 mm aggregate fractions in the alpine meadow and the d > 2 mm, e 0.25-2 mm and f 0.053-0.25 mm aggregate fractions in the alpine shrubland ecosystem during the seasonal FT process (UFP, unstable freezing period; SFP, stable frozen period; UTP, unstable thawing period; and STP, stable thawed period)

Fig. 5
Fig.5 Gram-positive bacteria/Gram-negative bacteria (GP/GN) of soil aggregates in the a alpine meadow and b shrubland ecosystems and fungi/ bacteria (F/B) of soil aggregates in the c alpine meadow and d shrubland ecosystems in freezing periods and thawing periods.Significant differences are denoted by different lowercase letters (P < 0.05).Error bars represent standard deviations

Table 1
Classification of the seasonal FT periods Seasonal FT period Time range (month) Sampling timeUnstable freezing period September, October, November

Table 3
Correlations between microbial abundance and pore size distribution of macroaggregates *represents the correlation was significant at P = 0.05 level n = 24Note: