Overall, a comparative metagenomic analysis on the microbiomes of lichen and moss biocrusts in the Mu Us Sandland is presented in our study, revealing distinct differences in taxonomic and functional diversity of microbiomes in C and N cycles between two types of biocrust and supplying useful information for understanding biochemical processes and nutrient cycling in the desert.
Effect of biocrust type and (bio-)chemical parameters on the microbiome
Our results suggest that biocrust type greatly affects the microbiome’s species composition, gene, and metabolic diversity (Fig. 2). Well-developed biocrusts have an abundance of Actinobacteria, Bacteroidetes, and Acidobacteria [47, 48], consistent with the alkaline character of desert soils [49–51], especially in the Tabernas desert . According to Vetrovsky et al.  and Qiao et al. , Actinobacteria can degrade complex compounds, such as polysaccharides and phenolic compounds, and Bacteroidetes can degrade xylan to improve the nutritional status of biocrusts. Acidobacteria may be a vital contributor to degrade the polymorphs in plant residues . Critical for biocrust formation, Proteobacteria could secrete extracellular polysaccharides for bonding sand particles to prevent soil wind erosion and contribute the most to N2-fixation in the early stages of biocrust formation. [55, 56]. Kidron et al.  indicated that Cyanobacteria rapidly colonized the soil surface due to their rapid growth, migration, and strong adaptability to extreme environments. However, being self-sufficient for carbon and nitrogen assimilation [58–60]. Moss biocrusts had more abundant Actinobacteria, Bacteroidetes, and Acidobacteria than lichen biocrusts, while lichen biocrusts had more abundant Proteobacteria and Cyanobacteria than moss biocrusts (Table S4). The results indicated that biocrusts at the early succession stage are rich in carbon and nitrogen-fixing micro-organisms that aim to accumulate topsoil nutrients, while biocrusts at the late succession stage are dominated by micro-organisms that mainly degrade complex compounds to provide effective nutrients for vascular plant establishment [27, 31, 61].
The genera Actinoplanes, Streptomyces, Pyrinomonas, Micromonospora, Microvirga, Conexibacter, and Microcoleus are found in dry and extreme arid habitats  and were presented in the two biocrust types analyzed in this study (Table S4). In particular, the two biocrust types differed significantly in the abundance of Microcoleus, a dominant photosynthetic organism in Cyanobacteria biocrusts . According to the Random Forest analysis, Geminocystis and Truepera were the biomarkers distinguishing biocrust type (Fig. S1B). Geminocystis, found in lakes, belongs to Cyanophyta and fixes carbon and nitrogen . Truepera, a bulbous bacterium that resists harsh environments , belongs to Deinococcus-Thermus and may efficiently digest soil organic matter .
As changes in the biomass and soil physiochemical environment markedly altered the composition and structure of microbial communities, we predicted that the different physiochemical environments of biocrusts would result in selective microbial growth, which would be seen as a shift in microbial community structure and accompanying gene content. Consistent with Yang et al.  and Fierer and Jackson , our research found that soil biogeochemical properties were closely related to microbial communities. The analyses revealed that DOC and SOC were the main environmental drivers of microbial community β-diversity in both biocrust types (Fig. S1A and S2), which is consistent with previous studies [49, 68–71]. In addition, DOC and SOC had strong positive correlations with the microbial community structure in both biocrust types. In this study, moss biocrusts had significantly higher organic carbon, soluble carbon, and MBC than lichen biocrusts (Table 1), supporting our previous research revealing the carbon sink’s role in soil carbon input in moss biocrusts .
Biocrust microbiome and its influence on carbon and nitrogen fixation and transformation
Our second significant finding was biocrust types differed in the relative abundance of CAZymes and enzymes associated with carbon and nitrogen metabolism (Table S5, Fig. 4). Moss biocrusts had significantly more glycoside hydrolases (e.g., glycoside hydrolases, carbohydrate-binding module) and polysaccharide lyases (e.g., polysaccharide lyases) than lichen biocrusts, indicating that moss biocrust microbiomes secrete more biodegradable carbohydrate enzymes. In contrast, lichen biocrust had more glycosyltransferases and carbohydrate esterases, suggesting that the glycosyltransferases in microbiomes catalyzed the attachment of activated sugars to different receptor molecules, such as proteins, nucleic acids, oligosaccharides, and lipids, secreting carbohydrate esterases to produce glycosidic bonds . The microbiomes in both biocrust types used different substrates to decompose organic carbon. Moss biocrusts usually used carbohydrates as a carbon source, while lichen biocrusts used aromatic compounds. The microbiomes in moss biocrusts reduced NO3− and nitrite to NO or NH3, explaining the lower NO3− and higher NH4+ than lichen biocrusts. The microbiomes in lichen biocrusts fixed N2 and synthesized amino acids, further promoting microbial growth and reproduction, explaining the higher MBN content than moss biocrusts.
The metagenome profile of the carbon- and nitrogen-related metabolic pathways also differed between lichen and moss biocrusts. In addition, the high abundance of genes associated with amino acid biosynthesis (e.g., lysine and arginine) in both biocrust types (Table S6) indicates their importance for microbiome adaptation to desert environments and life processes in general . Biocrusts are a major source of SOC in drylands [17, 73, 74]. Interestingly, our results show a clear dominance of respiration over carbon fixation in microbial communities associated with both biocrust types (Table S6), in contrast to other findings in the Tengger Desert, where mostly carbon fixation metabolic pathways were found in bacterial and moss biocrusts . In our results, the most important pathways in both biocrusts were glycolysis and the citrate cycle, which are essential for microbial respiration . These differences could be due to changes in the soil microbial populations and thus metabolism due to the enormous spatial heterogeneity in arid desert soils.
The metagenomic data revealed that the rbcL and rbcS genes, encoding the rate-limiting enzyme-RuBisCo, were deficient, with no significant difference between lichen and moss biocrusts (Table S7). The key coxL, coxS, and coxM genes in the carbon cycle, encoding carbon monoxide dehydrogenases—a bidirectional enzyme allowing organisms to both make use of CO as a source of energy (CO oxidation) and CO2 as a source of carbon (CO2 fixation) —differed slightly between biocrust types. Interestingly, these genes were 9-fold (lichen crust) to 18-fold (moss crust) more abundant than RuBisCo genes (Table S7). Thus, the high abundance of CO dehydrogenases in biocrusts suggests that these genes contribute to CO2 fixation through the Calvin cycle or chemosynthetic carbon fixation in some deserts, such as the Antarctic desert and Tengger Desert [28, 76, 77].
Nitrogen availability is the most important factor limiting the primary production of biocrusts in arid environments except for water [78, 79] and largely depends on the microbiomes driving N cycling . Studies have shown that nitrogen inputs in desert environments strongly depend on the nitrogen-fixing microbiomes in biocrusts [26, 81, 82]. The Low diversity and abundance of genes in N2 fixation pathways in our study (Table S9) is a distinguishing feature of biocrust microbiomes . More than 80% of the nitrogen in the soil is transformed into available nitrogen in the form of ammonium and nitrate nitrogen by the mineralization mediated by microbiomes for being absorbed and utilized by plants . We found that the conversion of urea to NH4+ (ammonification) by the ureC gene and N2 to NH4+ (N fixation) by the nifH, nifD, and nifK genes have high levels of potential functionality in providing nitrogen for biocrusts in the nitrogen cycle. Consistent with previous literature, we found a high level of denitrification in biocrusts (Fig. 5, Table S9), revealing that denitrification may enable N2O and NO production and prevent NO3− production . Both assimilatory and dissimilatory NO3−reduction were identified in these samples (Table S9). The microbiomes of lichen and moss biocrusts were rich in assimilatory and dissimilatory NO3− reduction genes, respectively (Table S9). NO3− was reduced to nitrite by nasA, nasB, narB, and NR genes for assimilatory NO3− reduction or napA and napB for dissimilatory NO3− reduction, and further reduced to NH4+ by nirA for assimilatory NO3− reduction or nirB, nirD, nrfA, and nrfH for dissimilatory NO3− reduction (Fig. 5). The conversion of NO3− to NH4+ (assimilatory or dissimilatory NO3− reduction) could increase soil NH4+ derived from NO3− during desert revegetation over time. It may be used for the amino acid synthesis of the microbiome in moss crusts and provide an available nitrogen source for moss crusts growth. However, because the hzo gene was not detected during biocrust succession in this study, the conversion of and NH4+ to N2 (anammox) was likely not to be active in the N cycle of biocrusts in arid and semi-arid regions [31, 81].
Combined ammonification, NO3− reduction (assimilative or dissimilatory), and denitrification accounted for 93.96% and 91.49% of the gene abundance for nitrogen-metabolism-related pathways in lichen and moss biocrusts, respectively (Fig. 5, Table S9). Therefore, the transformation of NO3− to NH4+ may explain the high amino acid biosynthesis, keeping the nitrogen bio-bound and avoiding nitrogen losses due to leaching and denitrification. The reproduction and colonization of biocrusts regulate plant-available nitrogen content and promote nitrogen mineralization and availability in soil, which is an important biological factor affecting soil nitrogen cycling.