Abstract graphic file with name ao0c02501_0006.jpg In this study, we found that biofilm formation is a critical factor affecting the activity of Aspergillus niger SJ1 xylanase. Xylanase activity increased 8.8% from 1046.88 to 1147.74 U/mL during A. niger SJ1 immobilized fermentation with biofilm formation. Therefore, we carried out the work of genomic analysis and biofilm-related time-series transcriptome analysis of A. niger SJ1 for better understanding of the ability of A. niger SJ to produce xylanase and biofilm formation. Genome annotation results revealed a complete biofilm polysaccharide component synthesis pathway in A. niger SJ1 and five proteins regarding xylanase synthesis. In addition, results of transcriptome analysis revealed that the genes involved in the synthesis of cell wall polysaccharides and amino acid anabolism were highly expressed in the biofilm. Furthermore, the expression levels of major genes in the gluconeogenesis pathway and mitogen-activated protein kinase pathway were examined. 1. Introduction The filamentous fungus of Aspergillus consists of more than 250 species of saprophytic fungi. Aspergillus niger, Aspergillus terreus, and Aspergillus oryzae are commercially used for the production of enzymes, pharmaceuticals, and traditional Asian foods.^[38]1,[39]2A. niger is one of the safest fungal species that has received approval by the Food and Drug Administration (FDA) in the United States. It is also a pivotal fungal strain in industrial enzyme production, such as for cellulase, xylanase, protease, amylase, and lipase. Industrial enzymes produced by A. niger play an eminent role in starch processing, brewing, beverage production, animal feed production, and papermaking industry.^[40]3,[41]4 Xylanase has become increasingly important in the recent years owing to its utility in commercial applications, including in pulp bleaching, food and animal feed industries, fuel processing, textile industries, and water management.^[42]5 Moreover, the production of xylanase by A. niger offers the advantages of high yield, protein post-translational modification, strong extracellular secretion, and safety.^[43]6 The technology of immobilized fermentation was developed on the basis of usage of an immobilized enzyme in the 1960s and 1970s.^[44]7 As early as in 1995, Kang et al. studied immobilized fermentation of A. niger and reported that the lignocellulase activity was significantly higher for immobilized cells in a bubble column than for a shake-flask culture.^[45]8 Fungal biofilm is a highly structured microbial community involving interactions between cells or between cells and substrate, and it is itself encapsulated in self-produced protective extracellular polymeric substances.^[46]9 Biofilm is the main form of microbial growth that has been widely studied in clinical practice. Biofilm cells, in contrast to planktonic cells, have demonstrated a greater ability to resistant fungal invasion.^[47]10,[48]11 As biofilms confer valuable resistance in addition to other beneficial properties on cells, their application in industrial processes is paramount. Our previous laboratorial studies have revealed that biofilms are certainly beneficial in ethanol production by Saccharomyces cerevisiae, citrate production by A. niger, nuclease p1 production by Penicillium citrinum, and in the anaerobic treatment of sewage.^[49]12−[50]14 In general, Aspergillus biofilm formation involves the steps of spore adhesion, mycelial growth, and biofilm maturation. Overall, the extracellular matrix (ECM) composed of extracellular polysaccharides, eDNA, polyol, melanin, and hydrophobin is produced on the surface of hyphae after mycelium germination.^[51]15 A few studies have reported the molecular mechanisms and functional genomics of Aspergillus biofilms. The A. niger wild-type strain ATCC 9029, high-yield citrate strain ATCC 1015, high-yield enzyme strain CBS 513.88, and spore-free amylase-producing industrial strain SH2 have been tested for whole-genome sequencing. The method of examining the relevant regulatory mechanisms in the formation of A. niger biofilms is well established.^[52]16−[53]18 Iwashita suggested that a physiological and morphological change, in response to a specific solid-state signal of water activity and unknown transcription factors, can result in high enzyme production during the solid-state fermentation process as this change can create differences in the expression of a large number of genes.^[54]19 Bruns et al. conducted the first comprehensive analysis of the transcriptome and proteome of Aspergillus fumigatus biofilm in 2010.^[55]10 They found that genes encoding hydrophobin and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated at the mature biofilm stage, but metabolic activity seems to be reduced. Moreover, in 2012, Gibbons et al. found that the genes overexpressed in A. fumigatus biofilm cells were involved in the transport, secondary metabolism, and cell wall and cell surface functions.^[56]20 In this study, we analyzed the transcriptome data of biofilm cells and free mycelia in our attempt to understand the differences in the process of yielding xylanase between immobilized and free mycelia forms of A. niger. 2. Results and Discussion 2.1. A. niger SJ1 Immobilized Fermentation In the optimized corn cob-based medium in which the biofilm carrier PAF201 was added, the xylanase activities of immobilized A. niger SJ1 reached 1147.74 U/mL, which was an 8.8% increase in comparison with 1046.88 U/mL by free mycelial fermentation. The same trend was recorded in the fermentation medium supplemented with xylan as a carbon source (fermentation medium 2) ([57]Figure [58]1 A). In contrast to A. niger KKS immobilized in a bubble column, A. niger SJ1 immobilized onto PAF201, having the same tendency that improved the xylanase activity in shake-flask culture. Villena and Gutierrez-Correa reported that A. niger (ATCC 10864) biofilms showed improved cellulase activity on polyester cloth. Although the mycelial biomass fixed on polyester cloth in a shaker and a bioreactor was lower than that in free mycelia state, it showed higher cellulase activity.^[59]21 During the fermentation process of A. niger SJ1, almost no difference was noted in the dry weight of total cells between the free and immobilized fermentation processes ([60]Figure [61]1B). This result confirms that the xylanase activity was increased not due to the increase in biomass, rather because of the addition of PAF201 carrier, which changed the growth state of the mycelia into a so-called biofilm. As a consequence, this phenomenon motivated subsequent study of the difference in gene expressions between mycelia immobilized on a carrier and free-growing mycelia. Figure 1. [62]Figure 1 [63]Open in a new tab Fermentation parameter detection. (A) Xylanase activity in different fermentation modes of medium 1 and medium 2 (p < 0.01). (B) Dry weight about immobilized cells and free mycelia in immobilized and free cell fermentation. The values are the means and standard deviations of three independent experiments. ***p < 0.001, **p < 0.01, and *p < 0.05 by Student’s t-test. 2.2. Genomic Feature and Comparative Genomics of A. niger SJ1 The results of sequencing by Single Molecule, Real-Time (SMRT) Sequencing technology on the PacBio Sequel platform were assembled by SMRT Analysis (v2.2 software). The genome size was 35.78 Mb, and the GC content accounted for 49.33% of the whole genome ([64]Table [65]1 ). The draft genome sequence of A. niger SJ1-merged 15 contigs into one circle. The sequencing raw data has been deposited to the NCBI database (accession number SRP173743). Table 1. Genome Statistics of A. niger SJ1. feature value genome size (bp) 35,782,651 G + C (bp) 17,651,443 N50 (bp) 4,076,075 tRNA genes 269 DNA scaffolds 15 total genes 10,750 protein coding gene 9425 annotated genes 10,728 average exon length (bp) 482 average intron length (bp) 123 [66]Open in a new tab Through the analysis of genome annotation, some genes involved in polysaccharide biosynthesis and biofilm formation were detected in A. niger SJ1, such as α-(1,3)-glucan coding genes Ags1 and Ags2, β-(1,3)-glucan coding gene Fks1, galactomannan (GM) and galactosaminogalactan (GAG) synthetic and secretory genes Uge3, Uge5, Ugm1, Gtb3, Agd3, Ega3, and Sph3, chitin synthesis-related genes ChsA, Chs1, ChsC1, ChsC2, and ChsD. Glycolysis and gluconeogenesis pathways that are closely related to the synthesis of these polysaccharides were also traced. As ECM does not exist in fungal colonies grown under classical liquid-shaking conditions,^[67]22 the expression levels of these genes involved in the synthesis of extracellular polysaccharides were also observed in biofilms. The signaling pathways that regulate biofilm formation in yeasts, include the Ras-cAMP pathway, the glucose-inhibitory pathway, and the MAPK-dependent filamentous growth pathway.^[68]23 The MAPK signaling pathway, probably associated with Aspergillus biofilm, was also detected in the A. niger SJ1 genome, which was the focus of our transcriptome analysis. To study the genome of A. niger SJ1 in detail, we first identified 628 CAZymes families (e < 10^–5) that were actively involved in carbohydrate metabolism. A total of five proteins were identified for their involvement in A. niger SJ1 xylanase production, including endo-1,4-beta-xylanase A, endo-1.4-b-xylanase B, endo-1,4-beta-xylanase F1, endo-1,4-beta-xylanase 5, and endo-1,4-beta-xylanase B precursor, respectively. To analyze the mechanism of A. niger SJ1 producing xylanase, the completed CAZy annotation results may help analyze the carbohydrate enzymes secreted by A. niger SJ1. Second, we compared the A. niger SJ1 with A. niger ATCC 1015 (citric acid production), A. niger An76 (lignocellulolytic enzymes production), and A. niger CBS513.88 (glucoamlyase production). [69]Figure [70]2 A displays the comparison of the genomes of the A. niger SJ1-merged genome and the other three reported A. niger genomes. Multiple genome sequence alignments of the above strains were also performed using Mauve 2.3.1. The figure of collinearity analysis is illustrated in [71]Figure [72]2B, and the areas with lines represent the collinear regions between species. The ratio of the length of A. niger SJ1 and other strains was calculated, which can be aligned to the total length of the A. niger SJ1 sequence. The similarity between A. niger SJ1 and A. niger CBS513.88 as well as between A. niger SJ1 and A. niger ATCC 1015 was nearly 80%. On the other hand, the similarity between A. niger SJ1 and A. niger An76 was higher, up to 96.53%. Genome-wide comparative analysis results showed that A. niger SJ1 and A. niger An76 share high DNA sequence similarity, which is consistent with the fact that A. niger SJ1 is an effective producer of xylanase. However, its regulatory factors and mechanisms remain unclear.^[73]24 Figure 2. [74]Figure 2 [75]Open in a new tab (A) Genome-wide comparative analysis of A. niger SJ1 and several other related reports of A. niger strains. (B) Figure of collinearity analysis. The Locally Collinear Blocks connected by corresponding color lines, and the upper and lower collinear blocks are similar in forward and reverse directions. White areas indicate no homology between genomes. The degree of similarity is shown from the outside to the inside of the circle diagram. Each genome is represented by a unique color, and the color is from deep to light to white, which in turn indicates the degree of similarity. 2.3. Scanning Electron Microscopy Analysis and Differentially Expressed Genes The biofilm features were characterized by scanning electron microscopy (SEM). [76]Figure [77]3 illustrates the growth of cells on the carrier PAF201 at different stages of biofilm formation. The actual biofilm formation process on PAF201 was intuitively observed at different time points and under different magnifications. Notably, in the mature biofilm formation stage, a dense network structure akin to a spider’s web, filled the gaps in PAF201, showing similar characteristics to the A. fumigatus biofilm structure.^[78]25 Figure 3. [79]Figure 3 [80]Open in a new tab SEM analysis of free cells and immobilized cells at 6, 12, and 24 h. (A,D,G) was at 6 h, (B, E, H) was at 12 h, and (C,F,I) was at 24 h and (A–C) is 1.00 mm scale, (D–F) is 500 μm scale, and (G–I) is 100 μm scale. Based on the results recorded at 6, 12, and 24 h from transcriptome sequencing at different stages of biofilm formation, we calculated FPKM (reads per kilobase of exon model per million mapped reads) to quantify all gene expression differences between biofilm cells and free mycelia at different periods. A large number of genes were found to be upregulated at 6 h and 12 h ([81]Figure [82]4 A), as per the screening of significant differentially expressed genes (DEGs). The results of this comparison were the same as expected earlier. In comparison to that in free mycelium, the expression level of several genes in the biofilm cells was increased, indicating that the gene activity was increased significantly at the early stage of biofilm formation. Most of the commonly upregulated genes in the biofilm were involved in transcriptional and translational regulations, which reflects the establishment of different transcription and translation programs between the two growth conditions. The raw transcriptome reads have been deposited to the NCBI Sequence Read Archive database (accession number SRP174380). Figure 4. [83]Figure 4 [84]Open in a new tab Functional enrichment of DEGs and different expression of genes in free mycelia and biofilm cells. (A) Number of DEGs between free mycelia and biofilm cells. (B) Heat map of GO enrichment classification differences between free mycelia and biofilm cells. (C) Top 20 significantly enriched pathways of DEGs from KEGG pathway. (D) Heat map of the qRT-PCR result about the genes mentioned. 2.4. Gene Ontology Functions and KEGG Pathway Analysis The analysis of DEGs enrichment under the International Standard Classification System Gene Ontology (GO) ([85]http://www.geneontology.org/) for Gene Function, and all cellular components, molecular functions, and biological processes involved are listed in [86]Table S2. To identify the genes with a possible association with biofilms, the molecular functions of biofilm cells and those of free mycelia were examined at different time points. The activity of a large number of transporters, especially transmembrane transporters, was found to increase at 6 h in the biofilm cells. The structural molecular activity and oxidoreductase activity were upregulated at 12 h, while the oxidoreductase activity and catalytic activity pathway were upregulated at 24 h ([87]Figure [88]4 B). Cell components and the biological process DEGs at different time points were mainly concentrated on components of membrane and transmembrane transport. The KEGG pathway analysis ([89]http://www.genome.jp/kegg/pathway.html) was also applied to identify the major biochemical metabolic pathways and signal transduction pathways involved in significant DEGs between biofilm cells and free mycelia (Q-values < 0.05). The relevant enrichment results are detailed in [90]Table S2, and the top 20 significantly enriched pathways of DEGs are given in [91]Figure [92]4 C. The study of carbohydrate and amino acid metabolisms suggested most important differences at different time points. Particularly, a large number of genes involved in the glycolysis/gluconeogenesis pathway were noted to be upregulated in the 12 h enrichment medium. Among all, the key genes regulating gluconeogenesis, namely Fbp1 and PckA,^[93]26 were significantly upregulated by 6.33-fold and 7.53-fold, respectively; this data provided a basis for the conjecture of the synthesis of the cell wall and biofilm-related polysaccharides. The glycolysis pathway is illustrated in [94]Figure [95]5. Figure 5. [96]Figure 5 [97]Open in a new tab Schematic representation of the A. niger mycelial cell walls and the metabolic processes of gluconeogenesis and cell wall polysaccharides. The orange arrows show the gluconeogenesis pathway, the pale blue arrows show the cell wall polysaccharide synthesis pathway. Abbreviations: Glc, Glucose; GlcNAc, N-acetylglucosamine; and GalNAc, N-acetylgalactosamine. The fungal MAPK signaling pathway has attracted much attention in the research world because of its crucial role in the signal transduction from the cell surface to the nucleus.^[98]27 Three kinases in MAPK regulate diverse important cellular physiological/pathological processes such as cell growth, differentiation, and environmental stress adaptation.^[99]28,[100]29 Analysis of RNA-seq data revealed that most of the MAPK signal pathway genes were upregulated at 6 h (10 genes); nearly 20 expressions of genes involved in the MAPK signal pathway were significantly different at 12 and 24 h; and the expression of Hog1 was upregulated by approximately 2.3-times, probably because Hog1 plays an important role in biofilm formation. de Assis et al. reported that cAMP-dependent protein kinase A (PKA) activity and the high-osmolarity glycerol response pathways could synergistically regulate cell wall carbohydrate mobilization to maintain the normal cell wall morphology.^[101]30 In the same year, the authors reported that MAP kinases and phosphatases in the MAPK signaling pathway played an important role in regulating the cell wall components, ECM production, cell adhesion, and biofilm formation.^[102]31 Although not all MAPK-associated kinases were upregulated in our RNA-seq data, Hog1 and PtcB phosphatase, which promotes Hog1 phosphorylation, were both upregulated in biofilm cells. It has been reported that the A. fumigatusPtcB deletion strain induces increased chitin and β-1,3-glucan production but is more sensitive to cell wall damage with impaired biofilm formation.^[103]32 At the same time, the MAP kinase MpkA that responds to the cell wall stress pathway is also upregulated, the downstream RlmA transcription factor expression is increased, and RlmA contributes to the formation of biofilm components such as chitin and melanin. Because the main substrates of the MAPK signaling pathway are transcription factors, it is plausible that the MAPK signaling pathway is the key pathway that affects the formation of Aspergillus biofilm. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data relevant to this section are presented in [104]Figure [105]4 D. 2.5. Transcriptional Profile of Genes Related to Cell Wall Polysaccharide Formation Aspergillus cell wall is mainly composed of polysaccharides.^[106]33,[107]34A. niger cell wall is composed mainly of carbohydrate (73–83%) and contains glucan, chitin, GM, and GAG.^[108]35 The main metabolic processes of cell wall polysaccharides are given in [109]Figure [110]5 . As seen in [111]Figure [112]2I, the germinated mycelium was cross-linked to form a network structure, and the surface of the network showed the presence of ECM, especially in the overlap of multiple hyphae. ECM is rich in polysaccharides such as GM, GAG, and glucan.^[113]36 Extracellular polysaccharides provide structural scaffolds for the attachment of other biomolecules and play a vital role in the maintenance and function of biofilms.^[114]37 The presence of the 3 α-(1,3)-glucans genes are well known in the A. fumigatus genome, namely, Ags1, Ags2, and Ags3.^[115]25 These 3 genes were upregulated in A. fumigatus biofilm, which demonstrated the features of α-(1,3)-glucans in hyphal adhesion.^[116]20 According to our RNA-seq data, both Ags1 and Ags2 showed high expression in A. niger SJ1 biofilm. RNA-seq data are presented in [117]Tables S1, and the qRT-PCR results are given in [118]Figure [119]4 D. GAG is a linear heteropolysaccharide linking galactose and GalNAc via α-1,4 glycosidic bond linkages.^[120]38,[121]39 It binds to the outer cell wall and is present in the ECM of the Aspergillus biofilm. The cell wall-related GAG functions as the main adhesion point of A. fumigatus, thereby mainly acting on adhesion to plastics, fibronectin, and epithelial cells for mediating the biofilm formation .^[122]40 It has been reported that MedA, which controls the binding of fungi to host cells and basement membrane components and which is required in biofilm formation, has a defect in the production of GAG in a deficient strain of a previously described developmental transcription factor.^[123]41,[124]42 Deletion of the UDP-galactose mutase Ugm1, which is required for the production of galactofuranose, has been reported to increase the yield of GAG.^[125]43 There also exists a synthetic pathway for GAG in A. niger SJ1, which is similar to that in A. fumigatus, wherein UDP-glucose 4-epimerases, Uge3 and Uge5, are the key genes involved in the synthesis of GAG, showing a relatively high trend in biofilm cells. The expression levels of MedA and StuA did not show any significant differences, probably because of the presence of other molecular functions as transcription factors. However, Ugm1 showed lower expression levels in biofilm cells, probably because of the involvement of Ugm1 in the GM synthesis pathway. Moreover, GM shares the substrate UDP-GalNAc with GAG, which also proves that GAG production is higher in biofilm cells. RNA-seq data are presented in [126]Table S1 and qRT-PCR results in [127]Figure [128]4 D. 2.6. Amino Acid Anabolism Analysis of the RNA-seq data revealed that the expression levels of most amino acids in the biofilm cells and free mycelia were modified to some extent and that most amino acid metabolic pathways in the biofilm cells became more active. The KEGG pathway-enrichment analysis suggested that the metabolic pathways of tryptophan, leucine, isoleucine, tyrosine, glycine, serine, and threonine had changed to some extent at different time points. Moreover, a large amount of amino acid metabolism was reportedly upregulated in Candida albicans biofilms.^[129]44 The RNA-seq data suggested that the TCA cycle was downregulated at 24 h, which resulted in the acceleration of amino acid accumulation, reflecting that biofilm cells possess a higher degree of activity. In conclusion, with the rapid development of high-throughput sequencing technology, the advent of SMRT sequencing technology enabled whole-genome sequencing of A. niger SJ1 at a much faster pace. Comparative analysis of the whole genome of several representative A. niger strains suggested a similarity between A. niger SJ1 and A. niger An76 of 96.53%. A. niger SJ1 possesses the capability of forming biofilms and producing xylanase enzyme. We found that the addition of PAF201 carrier to two different mediums as a support material during biofilm formation improved the activity of xylanase. The outcomes of the fermentation experiment in this study laid a strong foundation for the analysis of transcriptome sequencing of biofilm cells on PAF201 carrier and that of free mycelia at different time points in biofilm formation. This paper is also the first of its kind to report the completion of biofilm-related time-series transcriptome sequencing in A. niger. We visually observed the biofilm formation by A. niger SJ1 through SEM at different predetermined time-points. The analysis of RNA-seq data revealed that biofilm cells were more active than free mycelia with respect to extracellular polysaccharide and amino acid metabolisms. Meanwhile, RNA-seq analysis revealed that the biofilm cells possessed greater gene expression characteristics in the gluconeogenesis and MAPK signaling pathways; this observation provides a direction for the future research on A. niger SJ1 biofilms. The present preliminary study has provided a theoretical explanation for the production of xylanase by A. niger SJ1 and has confirmed the key role of biofilm in the industrial fermentation process of A. niger. It has also provided a theoretical basis for the subsequent industrial fermentation of A. niger and other filamentous fungi in the future. 3. MATERIALS and METHODS 3.1. Strains and Cultivation Conditions A. niger SJ1 was stored in our laboratory as an industrial strain and preserved in the China Center for Type Culture Collection (Wuhan, China) with the deposit number CCTCC M201911. A. niger SJ1 was cultured for 3 days at 30 °C on potato dextrose agar media until spore maturation. The plates were then washed with sterile distilled water and diluted to yield 1 × 10^6 spores/mL count. The collected spores were stored at 4 °C until further analysis. In this experiment, wheat bran extract was used for the preparation of seed and fermentation media. In brief, 40 g of wheat bran was added to 800 mL of distilled water and treated at 121 °C for 1 h, followed by filtration and dilution to obtain 1 L of wheat bran extract for the preparation of subsequent media. The ability of A. niger spores to adsorb on a carrier material is critical for biofilm formation. The carrier PAF201 was processed into the shape of a cube with a side length of 3 mm in our laboratory for the fermentation of A. niger. This carrier is composed of polyurethane and carbon black.^[130]12 Both the seed and fermentation media (100 mL) were added to 500 mL flasks for the preparation of the wheat bran extract. The seed medium was composed of 1% sucrose; fermentation medium 1 was composed of 2.81% 90 mesh corncob powder, 0.42% NaNO[3], 0.1% KH[2]PO[4], 0.05% MgSO[4]·7H[2]O, and 0.05% Tween 80; and fermentation medium 2 contained soluble xylan instead of corncob powder as in fermentation medium 1. The seed medium of immobilized fermentation was added to 0.1 g of the carrier, which is different from free-cell fermentation. To initiate both kinds of fermentation processes, 6 mL of freshly harvested A. niger spores were inoculated to the seed culture medium and incubated for 24 h. Then, 5 mL of the seed culture suspension was transferred to the free-cell fermentation medium. In addition, the carrier containing the mycelia was transferred to the immobilized fermentation medium. The same culture condition was maintained at 30 °C at 220 rpm. The fermentation period was 5 days. 3.2. Detection of Xylanase Activity and the Determination of Cultivation Parameters Xylanase activities were detected by Acidic Xylanase Activity Assay Kit (Solarbio, Beijing, China). The fermentation broth was centrifuged at 8000 xg at 4 °C for 15 min, and the supernatant was taken. Xylanase can degrade xylan into reducing oligosaccharides and monosaccharides at pH 4.8 and further develop color reaction with 3,5-dinitrosalicylic acid in 50 °C water for 30 min. The value of spectrophotometer OD[540] is converted into xylanase activity. One unit (U) of xylanase activity is defined as the amount of enzyme required to release 1 μM reducing sugar per minute of reaction under standard assay conditions.^[131]45 The biomass was quantified on the dry weight basis of hyphae.^[132]46 In brief, after the centrifugation process, the hyphal precipitate was washed twice with distilled water and then dried to a constant weight in an oven at 100 °C for 24 h; this dry weight was recorded after subtracting the weight of empty centrifuge tube. The hyphal precipitate was then transferred to the centrifuge tube with 6 M NaOH and then dried again in an oven at 100 °C until a constant weight was obtained, followed by weighing and calculating the dry weight as mentioned earlier. The final weight was the dry weight of the free hyphae in the medium. 3.3. Genomic DNA Preparing, Sequencing, and Assembly A. niger SJ1 spores (1 × 10^6) were inoculated in fresh yeast extract peptone dextrose medium and cultured at 30 °C/200 rpm for 10 h, followed by harvesting of the mycelium through centrifugation and washing (thrice) with phosphate buffered saline (PBS). The collected hyphae were crushed by liquid nitrogen grinding method, and the genomic DNA extraction was performed using the TaKaRa MiniBEST Plant Genomic DNA Extraction Kit as per the manufacturer’s instructions. The extracted genomic DNA was tested and sequenced on the PacBio Sequel platform based on SMRT sequencing technology, and the raw sequencing data was filtered and processed using the P_Fetch and P_Filter modules of SMRT Analysis (v2.2) software. The parameters were set to: minSubReadLength = 500, minLength = 100, readScore = 0.75. The valid data obtained was assembled using the assembly software hierarchical genome-assembly process (HGAP). HGAP uses Subreads to perform the assembly, and the errors are corrected by Arrow (smrtlink 5.1) to achieve the final assembly data. 3.4. Genome Annotation and Whole Gene Comparison Analysis We first predicted the location and structure of genes by HomoloGene prediction, De novo prediction (software: Augustus, Genscan, and GlimmerHMM), cDNA/EST, third-generation full-length transcriptome, second-generation RNA-seq prediction, and MAKER software. The gene sets predicted by various methods were integrated into a non-redundant and a more complete gene set, and the final reliable gene set was obtained by integrating the BUSCO data using the HiCESAP process. Finally, the proteins in the gene set were functionally annotated with the help of the foreign protein database (SwissProt, TrEMBL, KEGG, InterPro, GO, and NR) to determine the biological function of the products and the metabolic pathways involved. A total of three closely related species were selected, namely A. niger An76, A. niger ATCC1015, and A. niger CBS 513.88. First, all scaffolds of A. niger SJ1 were merged into a scaffold, and the genomic data of SJ1 were blast compared with the genome sequences of A. niger An76, A. niger CBS 513.88, and A. niger ATCC 1015 (blastn parameter: e-value 1 × 10^–5). The result of the comparison can be entered into the Circos software for drawing ([133]http://circos.ca/, version number: v0.62). The mauve tool was used to draw a collinear graph of the four species. The protein data was annotated with the CAZy database (Carbohydrate-Active enZYmes Database, [134]http://www.cazy.org/), and the gene set was compared with the CAZy database V5.0 using hmmscan ([135]http://hmmer.janelia.org/search/hmmscan) to obtain the carbohydrate-active enzyme annotation information corresponding to the gene; threshold setting: if the comparison length is >80 amino acid, the comparison parameter setting expectation value e-value will be 1 × 10^–5, otherwise the e-value would be <1 × 10^–3, and the ratio of coverage to HMM model will be >0.3. We used transcriptome sequencing data to correct the genome sequencing data to improve the accuracy of genomic sequencing. HISAT2 was used to compare the sequencing results, and Stringtie was used to reconstruct the transcriptional information. Then, Maker software was used to integrate the transcriptional and genome annotation information to determine the boundary information. 3.5. SEM Analysis Fresh A. niger spores were inoculated in the seed medium containing the carrier and then the immobilized carrier was taken out at 6 h (adhesion, cell aggregation, and ECM production), 12 h (conidial germination into hyphae and development), and 24 h (biofilm maturation). The carrier was subsequently rinsed thrice with PBS, and the treated carrier was stored frozen in a −80 °C refrigerator. Next, the carrier was dried at a low temperature using a vacuum dryer, then gold was sputter-coated, and the cells on the carrier were observed using the Hitachi S-4800 field emission scanning electron microscope.^[136]47 3.6. RNA Preparation, cDNA Library Construction, and Transcriptome Analysis Biofilm cells and free mycelia were collected at different time points, washed thrice with PBS, and subjected to RNA extraction from cells after breaking the wall of liquid nitrogen using the TaKaRa MiniBEST Universal RNA Extraction Kit in conformance with the manufacturer’s instructions. Next, cDNA was obtained by reverse transcription of RNA after quality control with the HiScript Q RT SuperMix for quantitative reverse transcription-PCR (qRT-PCR). The FPKM method was used to calculate the expression level of the target gene after Illumina sequencing and quality control. In our study, the EdgeR package was used for DEGs, and the screening threshold was false discovery rate (FDR) of <0.05, log[2]FC [fold change (condition 2/condition 1)] for a gene) > 1 or log[2]FC < −1. DEGs were then subjected to enrichment analyses for GO functions and the KEGG pathway, FDR ≤ 0.05. Assays were performed in triplicate, and the experiments were repeated thrice. 3.7. Confirmation of RNA-Seq Results by qRT-PCR RNA extraction and cDNA synthesis were performed as described in the earlier section. To confirm the results of the transcriptomic analysis, actin gene was selected as the internal reference gene, and Primer 5 software was used to select primers. The primers used in the analysis are listed in [137]Table S1. The qRT-PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems; USA) along with the SYBR Green PCR Master Mix (Applied Biosystems; USA). The reaction was carried out following the manufacturer’s instructions, and each sample included three parallel and negative controls. The expression level of the gene was calculated according to the comparative 2^–ΔΔCt method. Acknowledgments