Impact of different nitrogen additions on microbes and exopolysaccharides excretion in cyanobacterial biocrusts

Recently, it has been found that nitrogen (N) deposition strongly affects the coverage of biocrusts. However, we know little about the response of exopolysaccharides (EPSs), the key cementing material in the formation and stability of biocrusts, to N deposition. Three N-sources including nitrate, ammonia and urea were added to biocrusts at three rates (2 mg/g, 4 mg/g, 8 mg/g) to evaluate the effect of N additions on the growth of biocrusts and the abundance of EPS. Our results showed 2 mg/g of nitrate–N had no obvious effect on the cyanobacterial biomass, while 4 and 8 mg/g of nitrate–N inhibited the growth of Microcoleus vaginatus, the dominant cyanobacterium in biocrusts, but promoted other cyanobacteria growth. Ammonia-N and urea-N strongly decreased the cyanobacterial biomass, indicated by chlorophyll-a and 16 s rRNA gene copy-numbers. On the whole, N additions had a positive impact on the α-biodiversity of biocrusts. However, Ammonia-N and urea-N shifted the bacterial communities from more Cyanobacteria to more Proteobacteria and Actinobacteria. Notably, lesser-N (2 mg/g) promoted the excretion of EPSs, while greater-N (8 mg/g) had the opposite effect, and the total proportion of rhamnose and fucose in EPSs decreased in all treatment groups. N additions (except 2 mg/g of nitrate–N) reduced cyanobacterial biomass and affected the bacterial communities in biocrusts, which would obstruct the development and succession of biocrusts. Meanwhile, the simultaneous reductions of the EPSs contents and proportion of rhamnose and fucose in EPSs may further reduce stability and persistence of cyanobacterial biocrusts, after N additions.


Introduction
Most terrestrial ecosystems (forest, grassland, cropland, etc.) in the world are limited by nitrogen (N), which is a crucial element controlling the species composition, diversity, productivity and ecological function of ecosystems (LeBauer and Treseder 2008; Xia and Wan 2008;Zhou et al. 2017). In addition to biological N fixation, N deposition is also an important N input form for terrestrial ecosystems. In the past century, due to the continuous intensification of human activities, the contents of reactive N (mainly nitrogen oxides and ammonia) in the atmosphere significantly increased (Victor 2011). Recent studies have shown that 100 Tg of ammonia and nitrogen oxides are emitted from land to atmosphere annually (Fowler et al. 2013), and part of the N in the atmosphere re-enters the land in two ways: dry and wet deposition. Therefore, for areas with relatively developed industries, N deposition shows a trend of continuous increase. In fact, increased N deposition poses a growing threat to terrestrial ecosystems, including soil acidification, altering soil microbial community structure and even reducing plant productivity (Cheng et al. 2019;Freedman et al. 2015;Holland et al. 2005). From 1980 to 2015, the total N deposition in arid and semi-arid regions of China increased by nearly 200%, from4kg N/(ha·y) to 12 kg N/(ha·y), of which the ratio of ammonia-N to nitrate-N was about 2:1 (Liu et al. 2011;Yu et al. 2019). Although a comprehensive understanding of the response of dryland ecosystems to N deposition is lacking, dryland ecosystems may be particularly sensitive to increasing N deposition, since the N utilization rate in drylands is at a lesser level and decreases with the intensification of drought (Bobbink et al. 2010).
Exploring the response of biocrusts to N deposition in dryland is undoubtedly an important part to understand the change of the dryland ecosystems to N deposition. Biocrust is a common ecological component in drylands, formed by various organisms such as cyanobacteria, lichens, mosses, etc., which combines with soil particles due to the entanglement of filaments, hyphae, and exopolysaccharides (EPSs). Biocrusts play an important role in stabilizing the soil surface, improving nutrient and water conditions, and accelerating ecological restoration Lan et al. 2014;Mager and Thomas 2011;Warren et al. 2019). Unfortunately, due to the sensitivity of biocrusts and drastic climate changes caused by human activities (without considering N deposition), it is predicted that the coverage of biocrusts will decrease by 25-40% over the next 65 years (Rodriguez-Caballero et al. 2018). However, in the context of continued increase in N deposition in drylands, the stabilization and coverage of biocrusts could face more severe challenges.
Collectively, based on the composition of dominant organisms, biocrusts can be divided into different successional stages, such as cyanobacterial crusts, lichen crusts, moss crusts, and biocrusts in different successional stages provide different ecological services (Miralles et al. 2020;Su et al. 2009). In the succession process of biocrust communities, cyanobacteria are often the pioneers in the soil surface, creating material conditions for the emergence of subsequent green algae, lichen and moss (Zaady et al. 2000). Some studies have shown that increasing N deposition negatively affects the lichen-biocrust and reduced their spatial coverage in semiarid ecosystems (Benvenutto-Vargas and Ochoa-Hueso 2020). However, though cyanobacteria-biocrusts are the basis for the succession of lichen-biocrusts and moss-biocrusts, few studies have reported the impact of N deposition on them. The structure of cyanobacteria-biocrusts is relatively simple (Lan et al. 2012), thus the effect of N deposition may be more obvious for cyanobacterial biocrusts. Biological components of biocrusts play a key role in N dynamics, mostly through cyanobacterial N fixation or N leaching prevention (Castillo-Monroy et al. 2010), while excess N might drive a shift in bacterial community in biocrusts towards loss of cyanobacteria (Wang et al. 2015;Zhong et al. 2015). Once the community structure of biocrusts has changed tremendously, the development and succession of the biocrusts may also be affected.
EPSs are not only an important metabolite assisting the development of biocrusts (Belnap 2006;Chen et al. 2009;Chrismas et al. 2016;Ehling-Schulz and Scherer 1999;Frosler et al. 2017), but also an important indicator for evaluating the stability of biocrusts (Chamizo et al. 2019;Rossi et al. 2018;Veluci et al. 2006). Generally, EPSs in biocrusts are divided into released exopolysaccharides (RPS) and capsular exopolysaccharides (CPS) according to their degree of adhesion to microbial cells and soil particles. RPS is loosely attached to the cells or soil particles, which makes great contributions in biocrust stability, hydrologic condition and regulation of heterotrophic microbial community structure (Rossi et al. 2012b). As an important barrier, CPS is firmly attached to microbial cells, contributing to UV protection, anti-oxidation, etc., and enhancing the stress resistance of microorganisms . Due to different ecological functions between RPS and CPS, the changes of their contents not only reflect the current state of biocrusts to a certain extent but will also provide guidance for us to understand the sustainability and persistence of biocrusts in the future. However, the current studies on the relationship between N and EPSs are mostly limited to certain specific cyanobacteria. For example, Brull et al. (2000) found that N significantly affected the ratio of EPSs to cyanobacterial biomass in Nostoc commune. Qian et al. (2021) also observed that N regulated EPSs excretion of Microcoleus vaginatus (M. vaginatus) acting as an environmental signal. For some other cyanobacteria, the amounts of EPS excretions varied according to the different N source additions (De Philippis and Vincenzini 1998). Notably, little is known about the effect of N on the excretion of EPSs in biocrusts. Therefore, we investigated the response of biocrusts to the different N (nitrate-N, ammonia-N, urea-N) inputs in this study. Also, the effects and regulation of N on the stabilization of biocrusts were explored from three aspects: biocrusts' biomass, excretion and metabolism patterns of EPSs and the variation of bacterial community structure.
Considering the complexity and variability that happens in field experiments, this study utilized an incubator with constant temperature and light with biocrusts collected from the Gurbantunggut Desert to investigate how N addition affects biocrusts. We hypothesized that 1) greater N would hinder the development of biocrusts and decrease the biocrust's stability because greater N inhibits cyanobacterial growth and affect the excretion of EPSs in biocrust; 2) we also hypothesized that the tress caused by ammonium would stimulate production EPSs to moderate the stress of biocrusts, because in previous experiments, the dominant species in biocrusts-M. vaginatus did not grow well in environments with ammonia-N as the sole N source; 3) The addition of N would affect the enzymes related to EPS synthesis, thus resulting in the difference of EPSs monosaccharide composition; 4) finally, we hypothesized that compared with the two kinds of inorganic N (nitrate-N and ammonia-N), the effects of urea-N on the growth of biocrusts is different, because the inorganic N is more easily used by microorganisms than organic N.

Study site and sample collection
The study site is located in the center of the Gurbantunggut Desert (43°35′N, 88°51′E), Xinjiang, China, where the mean annual temperature is 6.6 °C ~ 8.1 °C and July is the hottest month (23 °C ~ 26 °C). The rainfall mainly occurs in spring and early summer, and the annual precipitation is 110 ~ 150 mm (Liu et al. 2020). Due to the influence of precipitation patterns, ephemeral plants such as Haloxyloseon ammodendron, Artemisia sphaerocephala and Artemisia absinthium are widely distributed in the desert. Biocrusts are widespread on 1 3 Vol:. (1234567890) the surface there, and the formation and growing of which are usually during the moist, cool periods of spring and autumn (Zhou et al. 2020). In October 2020, ten 2 m × 2 m plots with only cyanobacteriabiocrusts and no vegetation were established, and about 80 g samples were collected in each plot. At the time of sampling, the plots were separated by areas with shrubs and other small plants, strongly reducing the interferences between the sampling points. Following the principle of random sampling, the cyanobacteria-biocrusts were sampled by trowel and carefully put into plastic culture dishes, then quickly sealed to prevent to sample breakage. Tools and containers used in the sampling process had been sterilized in advance.

Experimental design
Samples were stored in small plastic containers and carried to the laboratory for re-incubation. The experiment design consists of a control group (control group) and 9 N treatment groups, with three replicates for each treatment. The nine treatment groups representing the addition of different N sources (nitrate-N, ammonia-N, urea-N) and different N contents (lesser: 2 mg/g, medium: 4 mg/g, greater: 8 mg/g) are hereafter designated nitr2, nitr4, nitr8, ammon2, ammon4, ammon8, urea2, urea4 and urea8, respectively. Application concentrations were set up to double the TN of samples and mimic the local N deposition (Liu et al. 2020). Each N agent was blended in appropriate amount of water and evenly sprayed onto the biocrusts. In order to eliminate the influence of water on the experiment, the amount of water added was 10% of the weight of the biocrusts (based on advance measurements this was approximately half the saturated water content). The biocrusts were placed in an incubator under constant temperature (25 ± 2 °C) and light conditions for three weeks, illuminated with cool white fluorescent light at 40 μ E/(m 2 ·s). During the incubation period, 5 mL of water was added to biocrusts every 5 days to maintain a moisture content (8%-10%) of the biocrusts, thereby ensuring that the biocrusts could grow efficiently without dormancy. Samples were taken on day 1, day 10, and day 21 for analysis of chlorophyll-a (Chl-a), EPSs, and soluble N forms in biocrusts.

Biocrusts characteristics analysis
The Chl-a of biocrusts was determined by UV spectrophotometry. These samples were ground and put into a 10 mL centrifuge tube and 5 mL of acetone was added and shaken thoroughly and left overnight at 4℃. Samples were then centrifuged at 8000 r/min for 10 min and the supernatants were spectrophotometrically quantified according to Qian et al. (2021). For the biocrust's N analysis, the available N was determined by the alkali diffusion method (Evans and Belnap 1999), the contents of total soluble N, nitrate-N (Ferree and Shannon 2001) and ammonia-N (Solórzano 1969) were analyzed colorimetrically after the extraction of the biocrusts with 1 M KCl. EPSs were extracted with a method modified from Chen et al. (2014) and Rossi et al. (2012a). About 10 g samples were extracted three times with distilled water for 1 h at 30 ℃ in constant temperature oscillator (200 r/min). Extracts were centrifuged at 6000 g for 15 min, then the supernatants were collected and stored for RPS analysis. The remaining sediment was mixed with deionized water again and heated in a water bath for 6 h at 80 °C to extract the CPS and the same was extracted 3 times (Ge et al. 2014). The solution of RPS and CPS was concentrated by rotary evaporator with the rotation rate of 120 r/min at 60 °C and until the volume became 1/4 of the original. The concentrate was mixed with ethanol (95%) and kept overnight at 4•C to obtain the crude RPS and CPS. The sediment was collected by centrifuging and subsequently re-dissolved in deionized water and treated with Sevag reagent to remove the proteins from the crude RPS and CPS Zha et al. 2014). Finally, the treated liquid was subjected to dialysis operation by dialysis bag (3 KD) for 48 h to remove the residual impurities, and purified RPS and CPS were obtained (Delattre et al. 2016). RPS and CPS were freeze-dried for storage, and their contents were tested by the phenol-sulfuric acid method Dubois et al. 1951). The monosaccharide composition of the EPSs was determined by GC/MS, (Agilent Technologies, CA, USA). Briefly, EPSs was hydrolyzed with trifluoroacetate, and then hydroxylamine hydrochloride in pyridine, acetic anhydride were added successively for derivatization reaction. Finally, the derivatized samples were diluted with chloroform and were obtained for GC/MS analysis (Qian et al. 2021).

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Vol.: (0123456789) The total DNA was extracted according to the instructions of the EZNA® soil kit (Omega Bio-tek, Norcross, GA, US). The quality and quantity of the DNA samples were checked by agarose electrophoresis detection and NanoDrop2000. The composition and diversity of bacterial communities were assessed by Illumina PE300 sequencing analysis of the 16S rRNA gene. The universal primers 338F (5'-ACT CCT A CGG GAG GCA GCA G-3') and 806R (5'-GGA CTA CHVGGG TWT CTAAT-3') were selected for the PCR amplification with the program: 95 °C predenaturation for 3 min, 27 cycles (95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s), and finally 72 °C for 10 min (PCR instrument: ABI GeneAmp® 9700). The amplification system was 20ul, 4ul 5*FastPfu buffer, 2ul 2.5 mM dNTPs, 0.8ul primer (5uM), 0.4ul FastPfu polymerase; 10 ng DNA template. Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, USA) according to the standard protocols of Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China). In addition, the regulation mechanism of N on the EPSs' monosaccharide composition was also explored ( Fig. S5 and Table S2) by analyzing the activities of enzymes related to EPSs synthesis (Han et al. 2017;Knowles and Plaxton 2003;Stal and Moezelaar 1997).

Statistical analysis
Two-way ANOVA analysis and post-hoc test (LSD) was used to determine the effects of N-sources and N-contents on the development of biocrusts and excretion of EPSs. The significance is divided into: not significant (ns), significant at p < 0.05(*), p < 0.01(**) and p < 0.001(***), respectively. Statistical significance of correlations between enzyme activities and monosaccharide composition was based on correlation test (PEARSON). The significant correlation was defined as correlation coefficient (R 2 > 0.5) and significant effect (P < 0.05). Values shown are the means of 3 or 4 replicates ± SD (standard deviation) and the data was analyzed by SPSS statistics version 2.0. A Non-metric Multidimensional Scaling (NMDS) analysis based on the Bray-Curtis dissimilarity matrix was created to visualize differences in community composition for each sample, based on the type and properties of our data.

The biomass of cyanobacteria in biocrusts under different N levels
In this experiment, the N-sources, N-contents and their interaction had similar effects on the Chl-a and the cyanobacterial gene copy numbers of biocrusts (p < 0.001, Table S1). The specific results are as follows: Chl-a of biocrusts in the nitrate-N groups and control group showed an upward trend during reincubation (Fig. 1A). The Chl-a in the control group was the highest, 5.63 μg/g, followed by that in nitr2 group, 5.5 μg/g. However, the Chl-a in the ammonia-N groups (Fig. 1C) and urea-N groups decreased with incubation time (Fig. 1C) and the lowest Chl-a appeared in ammon4 group, 2.56 μg/g. Considering the difference of initial Chl-a content in different biocrusts, the ratio of Chl-a content in N treatment groups to Chl-a content in control group (response ratio) at different time could better reveal the effects of N additions on the change of photosynthetic biomass (Fig. 1D). The results also showed that the response ratio of the nitrate-N groups to the control group maintained around 1, while the ammonia-N groups decreased significantly, especially for ammon4 and ammon8, which from the 0.95 and 1.35 (1 th d) to 0.45 and 0.72 (21 th d), respectively. In contrast, the decrease in Chl-a of biocrusts was smaller in the urea-N groups.
Notably, in the nitr4 and nitr8 groups, the gene copy-numbers of cyanobacteria increased significantly (p < 0.001), while the cyanobacterial gene copy-numbers decreased first and then increased with the increase of N additions in the ammonia-N and urea-N groups ( Fig. 2A). Yet for M. vaginatus, a common dominant species in biocrusts, the gene copy-numbers decreased significantly in all treatment groups (Fig. 2B): nitrate-N groups (p < 0.05), ammonia-N groups (p < 0.001), urea-N groups (p < 0.01). The addition of nitrate-N promoted the growth of cyanobacteria in biocrusts, but excess nitrate-N also inhibited the growth of M. vaginatus.

The Changes and losses of N in biocrusts
The total soluble N in the control group increased significantly, from 0.155 mg/g to 0. 408 mg/g ( Fig. 3A; p < 0.01), after the laboratory experiments. In the 1 3 Vol:. (1234567890) nitrate-N groups and ammonia-N groups, the main types of N are the same as the source of N additions. During the incubation, N gradually transformed into other forms of N. The order of ammonia-N contents in each experimental group was: ammonia-N groups > urea-N groups > nitrate-N groups, which might be consistent with the development of cyanobacteria in the biocrust. However, total soluble N of biocrusts decreased to varying degrees in the different N treatment groups (Fig. 3). Among them, total soluble N of the ammonia-N groups and the urea-N group dropped significantly, which was particularly obvious for the greater-N (N8) groups (p < 0.001). In nitr8 group, total soluble N decreased from 6.40 mg/g to 4.24 mg/g, while the change of total soluble N in ammonia-N8 and urea-N8 groups decreased from 6.37 to 2.81 mg/g and 6.31 to 2.86 mg/g, respectively ( Fig. 3D, H, L). These results suggested that some N may be lost from the biocrust into the sand below the surface (Fig. S1).

Contents of RPS and CPS under different N levels
In this experiment, the N-sources, N-contents and their interaction have significant effects on the contents of EPSs in biocrusts (p < 0.001, Table S1). Interestingly, the nitrate-N groups and the ammonia-N groups formed similar EPSs excretion patterns under the influence of different N sources (nitrate-N and ammonia-N). After lesser-N addition, the contents of RPS increased in the nitr2 group and ammon2 group, which were 126.5 μg/g, 123.7 μg/g, respectively. However, the RPS decreased significantly after the greater-N additions (Fig. 4A, p < 0.001). The CPS increased significantly (p < 0.001) in N2 and N4 groups, but remained nearly unchanged in the N8 groups. Strangely, the excretion pattern of EPSs in the urea-N groups was completely different from the nitrate-N groups and ammonia-N groups, in which the contents of RPS decreased, but the contents of CPS increased significantly (p < 0.001; Fig. 4A, B). Additionally, in all experimental groups, CPS/RPS also increased and the values were greater than 1 after N additions, which suggested that the N affected the balance between CPS and RPS (Fig. 4C). The highest contents of EPSs (RPS + CPS) were found in lesser-N groups (Fig. 4D), which also showed that lesser-N promoted the excretion of EPSs instead of greater-N.
The similar change trend of EPSs in the nitrate-N groups and the ammonia-N groups may be caused by different mechanisms. Figs. S2 and S3 showed that both the RPS and CPS (per unit of Chl-a) in biocrusts added with lesser or medium ammonia-N are significantly increased (p < 0.001). Especially in the ammon2 group, the contents of RPS and CPS (per unit of Chl-a) were almost twice that of the control group. However, in nitrate-N groups, the medium and lesser levels of N did not drastically increase RPS and CPS (per unit of Chl-a), and the RPS and CPS (per unit of Chl-a) in the greater-N group significantly decreased (Figs. S2 and S3; p < 0.001). Based on comprehensive consideration of RPS and CPS, in addition to the ammonia-N, the urea-N also slightly promoted the increase of EPSs (per unit of Chl-a; Fig. S4).

Monosaccharide composition of EPSs
The results showed that rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose were all detected while fructose and some uronic acids were absent or below detection limits. These complex monosaccharide compositions are typical characteristics of cyanobacterial EPSs (Rossi et al. 2018) and have been affected by N-sources, N-contents and their interactions (Table S2). As shown in Tables 1 and 2, the RPS and CPS in control group, the proportion of glucose was the highest, higher than 30%, followed by mannose, accounting for about 15%. In general, the proportion of glucose and mannose in CPS were higher than that in RPS, and the proportion of fucose and rhamnose were less. After different N additions, the proportion of rhamnose in RPS (all samples) and CPS (expert from urea4 group) decreased significantly (p < 0.001). Similarly, the proportion of glucose also reduced significantly after N additions. On the contrary, the proportion of mannose increased significantly (p < 0.01) after N additions.

Responses of biocrusts α-diversity and β-diversity under different N levels
To explore the effects of the different N sources on biocrust's α-diversity, the richness indicator (Chao 1) and the diversity indicator (Shannon index) were surveyed based on the OTU matrix. Shannon's diversity index increased significantly after different N additions   Fig. 5A). Overall, Shannon index increased first and then decreased in nitrate-N groups and ammonia-N groups, while in the nitr4 group, nitr8 group and urea8 group, the Chao1 index increased significantly (p < 0.05, Fig. 5B). No difference in Chao1 index was found after ammonia-N addition. In order to identify the variations of community structure and co-occurrence patterns between different N treatment groups, NMDS was applied to analyze the distribution of bacterial structure at OTU. After 21d, our findings showed bacterial microbiomes change across the N gradients, which were based on the distribution of prokaryotic composition at the OTU level in each N source (Fig. 6A, B, C). Under the same N source, the effect of different N contents on bacterial community was obvious. Moreover, the changes caused by different nitrogen sources cannot be ignored. In particular, the bacterial community after urea-N addition was significantly different from the other two N treatment groups (Fig. 6D), which was fairly close to the control group (points representing nitrate-N groups and ammonia-N groups were further away from control group).

Change of relative abundance of microbial taxa
In the bacterial community (phylum level), Cyanobacteria, Proteobacteria, Actinobacteria, Chloroflexi and Bacteroides accounted for more than 70% of the entire bacterial community. In control group, Cyanobacteria was the most abundant phylum, accounting for 46.5% of the total reads. Proteobacteria (21.7%) was the next most dominant phylum, followed by Actinobacteria (14.7%), Chloroflexi (2.93%), Bacteroidetes (4.81%) and other phyla (Fig. 7). The bacterial community compositions in the biocrusts shifted in response to N additions. After the N additions, the relative abundance of Cyanobacteria in the biocrusts was significantly decreased (p < 0.001). Especially in the urea8 group, the relative abundance of Cyanobacteria dramatically decreased from 46.84% to 21.3%. Meanwhile, the relative abundance of Actinobacteria significantly increased from 14.75% to about 25% and 35% in ammonia-N groups and urea-N groups, respectively (p < 0.001). In addition, Proteobacteria and Chloroflexi were also more prevalent. At the genus level, in the control group, Microcoleus was the most abundant genus belonging to Cyanobacteria, accounting for 32.4%, followed by Rubellimicrobium (8.46%), Micrococcaceae (4.54%), Chroococcidiopsaceae (3.38%), Geodermatophilus (2.15%) and other genera (Fig. 7). The relative abundance of Microcoleus significantly decreased to 5.9% in ammon4 group (p < 0.001). The relative abundance of Geodermatophilus increased after the N additions (including all samples), especially in urea2 group, from 2.15% to 6.95%. The relative abundance of no-rank-Micrococcaceae also increased in most groups (except for nitr8), especially in urea8 group, from 4.54% to 9.57%. The gene copy-numbers of bacteria in biocrusts were detected by qPCR and were also affected by the N additions (p < 0.001; Fig. 7C, F, M). In nitrate-N groups, the bacterial copy-numbers of all biocrusts were significantly higher than control group, which was different from in ammonia-N and urea-N groups, where only greater-N could increase the bacterial biomass significantly (p < 0.001).

N additions decreased M. vaginatus and total photosynthetic biomass
In the laboratory, a significant difference in cyanobacterial biomass was found among different N addition groups. As the unique pigment of photosynthetic autotrophs (Lan et al. 2015;Ye et al. 2020), Chl-a is usually used as an indicator to evaluate the cyanobacterial biomass in biocrust. Continued decline in Chl-a indicated obvious inhibition of cyanobacterial biocrusts growth in ammonia-N groups and urea-N groups, while the opposite tendency was found in control group and nitrate-N groups. Notably, the significant reduction of Chl-a response ratio in ammonia-N groups and urea-N groups accurately indicated that the growth of cyanobacteria was inhibited (p < 0.01). The biomass of cyanobacteria was further quantified by qPCR, and the results showed that cyanobacterial gene copy-numbers in ammonia-N groups (p < 0.001) and urea-N groups (p < 0.01) were significantly less than that of the control group, and the opposite result was found in medium and greater nitrate-N groups.
As the pioneer and dominant microbes in biocrusts, M. vaginatus is widely distributed in arid and semi-arid regions (Redfield et al. 2002) and excrete a large amount of EPSs, playing an important role in biocrusts formation and stabilization (Chamizo However, compared to controls, the biomass of dominant genus M. vaginatus, indicated by gene copy number was significantly decreased after N additions, especially in ammonia-N groups and urea-N groups. Deserts are N-limited areas, and the lack of N restricts the survival of many organisms (Elbert et al. 2012). As the most abundant cyanobacteria, M. vaginatus cannot fix N by itself like other cyanobacteria (Flores et al. 2019;. In fact, C/N exchange with symbiotic heterotrophic N-fixing bacteria (Couradeau et al. 2019) provides M. vaginatus with the required N . This characteristic is considered an important reason for M. vaginatus dominance and its rapid formation of biocrusts in global drylands . Some studies have shown that provision of N will result in loss of heterotrophic diazotrophs that are symbiotic with M. vaginatus . Moreover, N additions directly led to the significant increase of N in biocrusts, providing equal N utilization opportunities for all biocrust microorganisms, in return their growth probably affected the accumulation of M. vaginatus biomass. The decrease in the proportion of M. vaginatus and the significant increase of 16 s rRNA gene copy numbers supported this speculation. Additionally, the toxic effects caused by ammonium might be another important reason that led to the inhibition of M. vaginatus and other biocrust organisms in ammonia-N groups (Collos and Harrison 2014;Dai et al. 2014;Patterson and Bolis 1995).

Effects of N additions on excretion and monosaccharide composition of EPSs in biocrusts
As an important binding material between cyanobacteria filaments and sand grains, it is considered that the higher the EPSs contents, the more stable the biocrusts (Belnap 2003; Mager and Thomas 2011). EPSs mainly consist of RPS and CPS according to their degree of adhesion to microbial cells and soil particles . RPS strengthen the connection between soil particles and cyanobacteria filaments and could increase the stability of biocrusts, while CPS play an important role in protecting The results showed that lesser-N promoted the excretion of EPSs significantly (p < 0.001). However, the contents of RPS decreased significantly after the addition of greater or medium N, which may be caused by the decline of EPSs-produced cyanobacteria and growth of heterotrophic bacteria (Figs. 4A and 7). In dryland soils, EPSs serve as the main organic carbon source for the growth of heterotrophic microorganisms (Chamizo et al. 2019;Stuart et al. 2016). After N additions, the significant increase of heterotrophic bacterial biomass, indicated by 16S rRNA gene copies numbers (Fig. 7) might lead to the increasing consume of C source, as a result led to the decrease of EPSs.
Many studies have shown that the ratio of CPS to RPS in natural biocrusts is about 1 (Chamizo et al. 2019(Chamizo et al. , 2020Chen et al. 2014), which may be the result of balanced distribution of EPSs in achieving the two main ecological functions: gluing soil particles and reducing environmental stress. In this study, the CPS/RPS in all treatment groups increased significantly, the highest CPS/RPS occurred in nitr4 (1.86), amon4 (1.98) and urea8 (2.52), respectively. Especially in urea-N groups, the CPS/RPS increased with the increasing of N additions amount, which was different from the other two groups. On one side, urea is always enzymatically hydrolyzed into ammonium salts before being absorbed and utilized by organisms (Burton and Prosser 2001), and this part of energy consumption may affect the excretion of EPSs. On the other side, different bacterial community structures are likely to cause differences in EPSs excretion and consumption, which will be discussed below. In addition, the contents of EPSs (including RPS and CPS) in biocrust showed similar changes in the trend between the nitrate-N groups and ammonia-N groups, but the regulation mechanisms might be different. The main reason for the greater EPSs in the ammonia-N groups may be due to the greater excretion efficiency induced by stress caused by ammonia-N addition (Cho et al. 2001;Leung and Wu 2007), while the greater EPSs in nitrate-N groups might be caused by higher M. vaginatus biomass (Fig. S4) (Delattre et al. 2016).
After analysis of the monosaccharide composition of EPSs, we found that the proportion of glucose in RPS in this experiment was significantly higher than some artificial biocrusts Hu et al. 2003) and field biocrusts (Hu et al. 2003). The results also indicated that RPS may be a good carbon source for bacteria, because glucose has good biodegradability (Chamizo et al. 2019). After N addition, the proportion of mannose in EPSs (including RPS and CPS) increased significantly (Tables 1 and 2, p < 0.01), which was also indicative of heterotrophic bacterial proliferation because this sugar widely exists in the EPSs excreted by many bacteria in soil (Fischer et al. 2003;Kielak et al. 2017;Zhai et al. 2021). The proportion of rhamnose and fucose in RPS was significantly higher than that in CPS (Tables 1 and 2, p < 0.001), which indicated the better hydrophobicity of RPS (Colica et al. 2015). A relatively higher ratio of RPS with strong binding ability implies a greater stability and lesser risk of erosion of biocrusts (Rossi et al. 2018). However, the sum of proportions of rhamnose and fucose in EPSs was significantly lower in N treatment groups than in control group (p < 0.001), which further revealed the risk of N addition on biocrusts' stability.
In addition, by analyzing the changes of four enzymes selected from the pathway of EPSs synthesis in cyanobacteria (related to EPSs' monosaccharide composition), we found there were correlations between these enzymes with certain monosaccharides (Fig. S5). Combined with the monosaccharide composition of EPSs detected previously (typical characteristics of cyanobacterial EPSs), which demonstrated that cyanobacteria might also be the main producer for EPSs in biocrusts after N additions. These results were consistent with the mainstream view derived from current research (Pereira et al. 2009;Rossi et al. 2012a). Although the monosaccharide composition of EPSs and the activity of related enzymes displayed disparities in different N treatment groups (Table S1), they did not show a clear response pattern to N additions.
Effects of N additions on bacterial community structure and biocrust stability N additions significantly increased the α-diversity of biocrusts (Fig. 5A, p < 0.01), which was not consistent with some other studies (Ling et al. 2017;Zeng et al. 2016). However, there are also some reports demonstrating that N additions have no or negative impact on bacterial diversity (Rong et al. 2022;Wang et al. 2016;Yuan et al. 2013). It is worth noting that the response of bacterial diversity to N additions may be more dependent on the durations of the experiments and the types of field environments (Rath et al. 2019;Zhang et al. 2021). Therefore, we speculate that the increase of biodiversity of biocrusts in this experiment may be due to the much milder environmental factors (temperature, moisture and light) in laboratory. The mild environment conditions provided the basis for the reproduction of some microorganisms which accounted for a small proportion or multiplied difficultly in the wild. Furthermore, the effects of different N-sources on the abundance of bacteria were not completely consistent. Based on the NMDS analysis, bacterial communities in biocrusts were sensitive to N changes, including quantities and types. Interestingly, regardless of the contents, the community structure of urea-N groups was more similar to that of the control group, but different from that of nitrate-N groups and ammonia-N groups (Fig. 6D), which was basically consistent with the changes in α-diversity of biocrusts mentioned above. In terms of N sources, inorganic N is easier to be used by microorganisms than organic N, so it may be easier to affect the microbial community of biocrusts. However, the addition of urea-N not only improved the N-level in the biocrust, but also provided the element C, which may keep the relative balance of C/N in the urea-N groups (Marsh et al. 2005;Smith and Ferry 2000). Therefore, this may also be the reason why the urea-N led to the different excretion of EPSs and community structure of biocrusts. However, the mechanism of the difference of biocrusts' response to organic N needs to be further explored.
At the genus level, Microcoleus was dominant in biocrusts. In fact, Microcoleus was further identified as the species M. vaginatus. However, N additions (mainly ammonia-N and urea-N) severely inhibited the reproduction and development of M. vaginatus, accompanied by a significant reduction in EPSs excretion, which undoubtedly had a negative impact on the persistence and stabilization of biocrusts. Rubellimicrobium, second most dominant genus in biocrusts, is considered to be thermophilic and has strong resistance to solar radiation (Miralles et al. 2020). Their strong resistance allows them to occupy more harsh places, thus avoiding competition with other bacteria. Geodermatophilus has been identified as prevailing as one of the soil microorganisms in the incipient biocrusts (Miralles et al. 2020), which may play a key role in drought stress (Karray et al. 2020). In our observation, Chroococcidiopsaceae were also found, which usually appears in relatively mature biocrusts (Miralles et al. 2020). Although their relative abundance in biocrusts was lesser, the three bacteria mentioned above were important for the survival of biocrusts in greater-aridity and greater-UV regions. However, their nearly constant relative abundance did not mean that N additions had little negative effect on the drought resistance and UV resistance characteristic of biocrusts, because of the declining cyanobacteria in biocrusts after N additions.
Soil biota has a positive effect on aggregation, and copiotrophs (e. g. Proteobacteria, Bacteroidetes and Actinobacteria) generally make a substantial contribution to the stability of aggregates (Merino-Martin et al. 2021). However, Proteobacteria and Actinobacteria had limited effects on the stability of biocrusts. Instead, filamentous cyanobacteria and EPSs are the most important biotic factor and metabolites affecting the stability of biocrusts, respectively (Gupta and Germida 2015). Some persistent microaggregates combine into larger microaggregates through the cyanobacterial filaments and microaggregates bond with each other through transient binding agents such as EPSs to form macroaggregates (Costa et al. 2018), which are the material basis for the formation of biocrusts. In this study, the bacterial biomass increased but the cyanobacteria biomass decreased in most treatment groups, especially in the ammonia-N groups and urea-N groups (Fig. 7). The decline of cyanobacteria undoubtedly would affect the development of biocrusts and may even hinder their succession process. Moreover, the reduction of cyanobacterial biomass and massive reproduction of heterotrophic bacteria would additionally lead to the decrease of EPSs (Figs. 4 and 7), thus breaking the associations of organic matter with soil particle in biocrusts , weakening the stability of biocrusts. On the contrary, nitr2 did not inhibit the growth of cyanobacteria while improving EPSs, which indicated that lesser-level of nitrate-N may have a positive effect on the stability of biocrusts.
Overall, our results were consistent with our hypothesis 1 and 4, that greater-N (especially ammonia-N) addition did inhibit the growth and development of biocrusts, indicated by the cyanobacterial biomass, EPS contents and community structure. NMDS also showed that the bacterial community structure in the urea-N groups was significantly different from the other two groups, which might lead to its distinctive EPSs excretion patterns. However, further exploration is needed to determine whether the specificity of urea-N is because it provides both N and C, thus maintaining the relative balance between C and N in the environment. Regarding hypothesis 2, lesser level of ammonia-N did promote the excretion of EPSs, while the greater level of ammonia-N caused the decline of EPSs, which was not completely consistent with our hypothesis. The inhibition of the growth of cyanobacteria and the massive reproduction of heterotrophic bacteria (RPS decomposition) probably led to the decrease of EPSs. For hypothesis 3, although there was correlation between four enzymes and some monosaccharide compositions in EPS (Fig. S5), the change of enzyme did not show a clear response mode for different N addition (Table S3). Therefore, the regulation of N on monosaccharides of EPS in biocrusts needs to be further explored.

Conclusions
Our study confirms that the stability and persistence of cyanobacterial biocrusts are threatened by greater N additions by exploring the changes in photosynthetic biomass, microbial community composition and EPSs. These results showed that N additions resulted in an obvious shift towards more Proteobacteria and Actinomycete-dominated bacterial communities from Cyanobacteria-dominated after 21d re-incubation. The addition of greater-N increased the total biomass of bacteria in biocrusts detected by qPCR, while significantly inhibiting both the biomass and relative abundance of M. vaginatus, especially in ammonia-N groups and urea-N groups. The accumulation of EPSs in biocrusts after N additions was hindered, which was not only conducive to the stability of biocrust, but also may affect the subsequent development and succession of biocrusts. Furthermore, the N addition decreased the proportion of rhamnose and fucose in EPSs, which would further reduce adhesion of soil particles by EPSs. Considering the importance of filamentous cyanobacteria and EPSs in biocrusts, cyanobacterial biocrusts coverage may face challenges, in the context of continuous increasing N deposition in drylands.