ISOLATION, IDENTIFICATION AND GENOMICS ANALYSIS OF ACTIVATED SLUDGE FLOC/BIOFLOC FORMING PSEUDODUGANELLA EBURNEA STRAINS
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摘要: 从云南省泸西县的污水处理厂分离到一株菌胶团形成菌YN12, 经过鉴定与象牙白伪杜擀氏菌(Pseudoduganella eburnea)10R5-21T模式株具有较近的亲缘关系, 属于同一物种。为揭示该菌株与其他活性污泥细菌间菌胶团形成机制及碳源利用方面的异同, 对该菌株进行全基因组测序、组装、注释及比较基因组学分析。结果表明: P. eburnea YN12株基因组大小约为5934 kb, G+C含量为63.9%, 包含5313个蛋白质编码序列, 具有与喜树脂动胶菌(Zoogloea resiniphila)MMB株、解叔丁醇水居菌(Aquincola tertiaricarbonis)RN12株及解壳聚糖松江菌(Mitsuaria chitosanitabida)XHY-A6株相似的胞外多糖生物合成途径、PrsK-PrsR双组分系统和PEP-CTERM胞外蛋白家族, 共同介导和调控的菌胶团形成机制。与后者相比, 菌株YN12中胞外多聚物(Extracellular polymeric substances, EPS)形成相关基因集中在大小约为72 kb的大型基因簇上, 且能吸收利用的碳源特别是单糖、二糖和多糖更为丰富多样。同时, 我们还从河流及养殖水体中也分离到象牙白伪杜擀氏菌, 这些菌株可用于生物絮团技术(Biofloc Technology), 改善水产养殖水质。Abstract: In activated sludge process the separation of sludge and water is achieved by gravity, and the floc-forming bacteria are predominated and play a central role in the water purification. In this study, a floc-forming bacterial strain YN12 was isolated from activated sludge of the municipal sewage treatment plant of Luxi County, Yunnan Province, and it was identified as Pseudoduganella eburnean and was phenotypically similar to the type strain 10R5-21T. In order to reveal and compare the floc-forming mechanism and carbon source utilization of the strain YN12 with those of other floc-forming bacteria, the genome sequencing, assembly, annotation and comparative genomics analysis were conducted. The results showed that the genome size of Pseudoduganella eburnea YN12 was about 5934 kb, the G+C content was 63.9%, and it contained 5313 protein coding sequences. Comparative analyses revealed a similar floc-forming and regulation mechanism to Zoogloea resiniphila MMB, Aquincola tertiaricarbonis RN12 and Mitsuaria chitosanitabida XHY-A6, which involves the exopolysaccharide biosynthesis, PrsK-PrsR two-component system and PEP-CTERM domain containing protein family. Meanwhile, these genes involved in the synthesis of extracellular polymeric substances (EPS) were concentrated on a large gene cluster of about 72 kb in size, and the YN12 strain was more functionally diverse in the absorption and utilization of carbon sources, particularly monosaccharides, disaccharides and polysaccharides. Moreover, two other Pseudoduganella eburnea strains had also been isolated from a river and an aquaculture pond. It is suggested that these strains could also be utilized in biofloc technology to improve the water quality and nutrient recycling in aquaculture.
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图 1 Pseudoduganella eburnea YN12菌胶团形成表型及显微镜图
A为振荡培养时菌株YN12的表型; B为菌株YN12静置后沉淀的菌胶团; C为YN12在普通光学显微镜下的表型, 亚甲基蓝染色; D是用微分干涉显微镜拍摄的菌株YN12; 显微镜照片标尺均为10 μm
Figure 1. Floc-forming phenotype and microscopical observation of P. eburnea YN12 strain
A. Agitated bacterial cultures of strain YN12; B. Settled bacterial cultures of strain YN12; C. Microscope image of methylene blue staining of strain YN12; D. Photographed with differential interference microscope of strain YN12; Bars: 10 μm
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图 2 Pseudoduganella eburnea YN12及近缘细菌基于16S rDNA序列的系统发育进化树
Figure 2. Phylogenetic tree based on 16S rDNA sequences of Pseudoduganella eburnea YN12 and closely related strains
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图 3 Pseudoduganella eburnea YN12胞外多聚物形成相关基因簇
Figure 3. The extracellular polymeric substances biosynthesis gene cluster of P. eburnea YN12
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图 4 从不同水体中分离纯化的象牙白伪杜擀氏菌菌胶团形成表型及显微镜图
A、B、C分别为菌株YN12、F1、SS14用微分干涉显微镜拍摄的照片; D、E从左至右依次为菌株YN12、F1、SS14; D为各菌株振荡时表型; E为各菌株静置后表型; 标尺为10 μm
Figure 4. Floc-forming phenotype and microscopic observation of the Pseudoduganella eburnean strains isolated from different water bodies
Panel A, B and C were the YN12, F1 and SS14 strains photographed with differential interference microscope, respectively; Panel D. Agitated bacterial cultures from panels A to C, from left to right; Panela E. Settled bacterial cultures from panels A to C, from left to right. Bars: 10 μm
表 1 基因组信息统计
Table 1 Genomic information statistics
版本信息Version P. eburnea YN12 基因组大小Genome size (bp) 5933582 G+C含量GC content (%) 63.9 Scaffold N50长度Length of scaffold N50 (bp) 451172 基因组L50 Genome L50 5 序列重叠群数目Number of contigs with PEGs 81 子系统数Number of subsystems 465 蛋白质编码序列Number of coding sequences 5313 RNA数目Number of RNAs 104 
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表 2 Pseudoduganella eburnea YN12与喜树脂动胶菌MMB中EPS合成相关基因簇表达产物的序列相似性
Table 2 The gene products of the extracellular polymeric substances (EPS) biosynthesis gene cluster of Pseudoduganella eburnea YN12 strain and the predicted orthologues in the closely related proteobacterial genomes (the polypeptide sequence identity was shown)
蛋白Protein 预测功能Predicted function Zoogloea resiniphila MMB 蛋白Protein 预测功能Predictedfunction Zoogloea resiniphila MMB Wzc EPS chain length determinant 48% FemAB-like protein Unknown 49% AsnB Asparagine biosynthesis 44% Wzy polymerase EPS unit polymerization 44% MltE Soluble lytic murein transgly-cosylases 42% AAA ATPase EPS export 46% CapK Unknown 55% Polysaccharide deacetylase Removal of acetyl group from EPS 57% EpsH Transmembrane protein involved in EPS biosynthesis 32% DegQ2 Periplasmic serine protease 43% AsnH Asparagine biosynthesis 66% WzxC Flipping ofEPS units 21% Wzz EPS chain length determinant 43% Outer membranePorin EPS export 22% Wza EPS export 58% Ugd UDP-glucose dehydrogenase 71% EpsB2 PEP-CTERM system associated sugar transferase 44% Uge Synthesis of UDP-D-galacturonate 22% PrsR PEP-CTERM box response regulator 66% PrsK PutativePEP-CTERM system histidine kinase 42% PrsT Putative PEP-CTERM system TPR-repeat lipoprotein 33% Diaminopimelate decarboxylase Lysine biosynthesis 63%
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表 3 Pseudoduganella eburnea YN12基于BioLog结果的碳源利用
Table 3 Utilization of carbon source based on BioLog results of Pseudoduganella eburnea YN12
碳源Carbon source 1 2 3 4 5 碳源Carbon source 1 2 3 4 5 糊精Dextrin + ND ND + + 肌醇Myo-Inositol + – – – – 麦芽糖D-Maltose + + + – + 甘油Glycerol – ND – + + 海藻糖D-Trehalose – ND ND – – 明胶Gelatin + ND ND – – 纤维二糖D-cellobiose + + + – – 甘氨酸-L-脯氨酸Glycyl-L-proline + ND ND – – 龙胆二糖Gentiobiose + ND ND – – L-丙氨酸L-alanine – + – + + 蔗糖Sucrose + + + – + L-精氨酸L-Arginine + ND ND – – 松二糖Turanose – ND ND – – L-天冬氨酸L-aspartic acid + – – + + 水苏糖Stachyose – ND ND – – L-谷氨酸L-glutamic acid + – – + + 棉子糖D-Raffinose – + – – – L-组氨酸L-Histidine + + + – + 乳糖α-D-Lactose – ND ND – – L-丝氨酸L-Serine + + + – – 蜜二糖D-Melibiose – + – – – 果胶Pectin + ND ND – + β-甲基-D-葡糖苷β-Methyl-D-glucoside – ND ND – + 葡糖酸D-Gluconic acid – ND ND – + 水杨苷D-Salicin – – + – – 苹果酸L-malic acid + ND ND + + N-乙酰-D-葡萄糖胺N-acetyl-D-glucosamine + ND + – – 柠檬酸Citrate – – – – + N-乙酰-D-半乳糖胺N-acetyl-D-galactosamine + ND ND – – 乳酸Lactic acid – + – + + 葡萄糖α-D-Glucose + + + – + 吐温40 Tween 40 + ND ND – + 甘露糖D-Mannose + + – – – 丁酸Butyric acids + ND ND + + 果糖D-Fructose + – – – + 丙酸Propionic acid – + ND – – 半乳糖D-Galactose + ND + – – 甲酸Formic acid – ND ND – + 岩藻糖Fucose – – – – – 乙酸Acetic acid – + – + + 鼠李糖L-Rhamnose – – – – – 乙酰乙酸Acetoacetic acid – ND ND – + 肌苷Inosine – ND ND – + 丙酮酸甲酯Methyl Pyruvate – ND ND + + 山梨醇D-Sorbitol – – – – + 羟基苯乙酸p-hydroxy-phenylacetic acid – ND ND + + 甘露醇Mannitol – – + – + 乳酸甲酯D-lactic acid methyl ester – ND ND + + 阿拉伯醇D-Arabitol – ND ND – + α-酮戊二酸α-Keto-glutaric acid – ND ND – – 注: 1. 菌株P. eburnea YN12; 2. 菌株P. eburnean 10R5-21T[11]; 3. 菌株P. violaceinigra YIM 31327T[8]; 4. 菌株Z. resiniphila MMB[6]; 5. 菌株A. tertiaricarbonis RN12[12]。“+”表示可以利用; “–”表示不能利用, “ND”表示没有相关数据Note: Strain: 1. P. eburnea YN12; 2. P. eburnean 10R5-21T[11]; 3. P. violaceinigra YIM 31327T[8]; 4. Z. resiniphila MMB[6]; 5. A. tertiaricarbonis RN12[12]. “+”, Positive reaction; “–”, Negative reaction; “ND”, No data available
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