Microbial community analyses of two 1,4-dioxane degrading consortia enriched from uncontaminated soils [16s rRNA]

Two consortia (Consortium A and Consortium B) that can use 1,4-dioxane (a groundwater contaminant of emerging concern) as the sole carbon source were enriched from Rice University (Houston, TX, USA) campus soil. Phylogenetic analysis by 16S rRNA sequencing revealed the dominant genus in both of the consortia is Mycobacterium (56% in Consortium A and 49% in Consortium B). The predominance of Mycobacterium spp, in these consortia support the notion that this is an important and commonly encountered genus of dioxane degraders. Among other genera present that make at least 2% of these consortia, only Afipia encompasses a strain (i.e., Afipia sp. D1) that was reported to degrade dioxane as sole carbon and energy source. A nested PCR analysis using two degenerate primers to target the hydroxylase alpha subunit of groups 3 to 6 SDIMOs was performed to gain insights into which enzymes were responsible for dioxane degradation by these consortia. The purified products obtained from the second PCR run were sequenced and compared to genes databases (NCBI) encompassing all of the currently reported SDIMOs. The dominant SDIMO genes in Consortium A corresponded to a group-6 putative propane monooxygenase-like SDIMO (98.8%); while in Consortium B, SDIMO genes from both groups 5 (47.3%) and 6 (51.9%) were observed. In both consortia, the relative abundance of thmA/dxmA gene was negligible (0.03%), which is consistent with the negative amplification of these genes as verified in qPCR. Overall, the high relative abundance of group-6 putative propane monooxygenases in our two consortia suggests the novel finding that group 6-SDIMOs could also play an important role in dioxane degradation. This underscores the need for further research on genes and enzymes involved in dioxane biodegradation to develop novel biomarkers that can be useful for forensic analysis and performance assessment of bioremediation and natural attenuation at dioxane-impacted sites. DNA was extracted from bacteria biomass harvested in exponential growth phase, when half or more of the added dioxane (100 mg/L) was consumed. Total DNA extractions were performed using the UltraClean® Microbial DNA Isolation Kit (MO BIO, Carlsbad, CA, USA) according to the manufacturer's protocol. The V4 region of the 16S rRNA gene was amplified by PCR using the forward 515F and reverse 806R primers. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) by Illumina MiSeq paired-end sequencing (approximately 2×300 bp as the read length). Sequence data were processed using MR DNA analysis pipeline. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against the RDPII (http://rdp.cme.msu.edu) and NCBI (www.ncbi.nlm.nih.gov) databases.Previously designed degenerate primers NVC57, NVC58, NVC65 and NVC66 to target conserved regions in the soluble di-iron monooxygenases (SDIMO) alpha subunit gene (groups 3 to 6) were used to examine the presence and diversity of SDIMO genes in these two consortia. A nested PCR strategy was used to increase the PCR product yield. In the first run, the PCR mixture contained 1 µL of NVC65 and NVC58 primer mixture (10 µM), 20 ng of the extracted genomic DNA, 12.5 µL of KAPA HiFi HotStart ReadyMix (2X) (KAPA Biosystems, Wilmington, MA, USA), and nuclease-free water to yield a total volume of 25 µL. PCR was performed in a Bio-Rad Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following temperature profile: initial denaturation (94°C, 5 min), then 29 amplification cycles (94°C for 30 s, 55°C for 30 s, 72°C for 1 min per kb) and a final extension (72°C for 5 min). The length of the PCR products in the first run was checked by 1% agarose gel and DNA bands of the correct size (1100 bp) were excised and purified. 20 ng of the purified PCR product was used as the DNA template in the second run, with the second set of primers (NVC57 and NVC66). The purified product (420 bp) from the second PCR was sent to MR DNA (www.mrdnalab.com, Shallowater, TX, USA) for Illumina MiSeq paired-end sequencing (approximately 2×300 bp as the read length). Sequence data were processed using MR DNA analysis pipeline. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). A database including all of the currently reported SDIMO genes on NCBI was created and used to taxonomically classify the final OTUs. Overall design: To investigate the microbial community composition of two 1,4-dioxane degrading consortia enriched from uncontaminated soils, 16S rRNA gene sequencing were processed using Illumina Miseq technology. The results show that the main species in these two consortia are Mycobacteria spp.本研究从美国德克萨斯州休斯顿市莱斯大学校园土壤中富集得到两个可将1,4-二氧六环（1,4-dioxane，一种新兴关注地下水污染物）作为唯一碳源的菌群（菌群A与菌群B）。通过16S rRNA测序（16S rRNA sequencing）进行系统发育分析发现，两个菌群的优势菌属均为分枝杆菌属（Mycobacterium）：菌群A中占比56%，菌群B中占比49%。分枝杆菌属在这两个菌群中的主导地位佐证了该菌属是一类重要且常见的二氧六环降解菌的观点。在其余占比不低于2%的菌属中，仅阿菲波菌属（Afipia）包含有被报道可将二氧六环作为唯一碳源与能源进行降解的菌株（即阿菲波菌属菌株D1，Afipia sp. D1）。为探究这两个菌群降解二氧六环所依赖的酶类，本研究使用靶向3至6族可溶性二铁单加氧酶（soluble di-iron monooxygenases, SDIMO）羟化酶α亚基的简并引物，开展了巢式PCR（nested PCR）分析。将第二轮PCR获得的纯化产物进行测序，并与涵盖当前所有已报道SDIMO的基因数据库（NCBI，美国国家生物技术信息中心）进行比对。菌群A中占主导的SDIMO基因对应于6族推定丙烷单加氧酶样SDIMO（占比98.8%）；而菌群B中则同时存在5族（47.3%）与6族（51.9%）的SDIMO基因。两个菌群中thmA/dxmA基因的相对丰度均可忽略不计（仅0.03%），这与定量PCR（qPCR）验证得到的该基因未获得扩增的结果一致。总体而言，两个菌群中6族推定丙烷单加氧酶的高相对丰度提示了一项新发现：6族SDIMO也可能在二氧六环降解过程中发挥重要作用。这一结果强调了需要针对二氧六环生物降解相关基因与酶开展进一步研究，以开发可用于受二氧六环污染场地的法医分析、生物修复（bioremediation）与自然衰减（natural attenuation）效果评估的新型生物标志物。本研究在指数生长期（exponential growth phase，此时已有一半及以上添加的100 mg/L二氧六环被消耗）收集细菌菌体并提取DNA。总DNA提取采用UltraClean®微生物DNA分离试剂盒（UltraClean® Microbial DNA Isolation Kit，MO BIO，美国加利福尼亚州卡尔斯巴德市），严格遵循制造商的操作流程。使用正向引物515F与反向引物806R对16S rRNA基因的V4区进行PCR扩增。测序工作由MR DNA（www.mrdnalab.com，美国德克萨斯州肖洛特市）通过Illumina MiSeq双端测序（Illumina MiSeq paired-end sequencing，读长约2×300 bp）完成。序列数据采用MR DNA分析流程进行处理。操作分类单元（Operational taxonomic units, OTUs）通过3%差异度（即97%相似性）聚类定义。最终OTUs的分类学注释通过BLASTn比对核糖体数据库项目II（RDPII，http://rdp.cme.msu.edu）与NCBI数据库完成。此前设计的靶向可溶性二铁单加氧酶α亚基基因（3至6族）保守区域的简并引物NVC57、NVC58、NVC65与NVC66，被用于检测这两个菌群中SDIMO基因的存在与多样性。本研究采用巢式PCR策略以提升PCR产物的产量。第一轮PCR反应体系包含1 μL浓度为10 μM的NVC65与NVC58引物混合液、20 ng提取的基因组DNA、12.5 μL KAPA HiFi HotStart预混液（2×，KAPA Biosystems，美国马萨诸塞州威尔明顿市），并加入无核酸酶水将总体积补至25 μL。PCR反应在Bio-Rad（伯乐）公司的热循环仪中进行，反应程序如下：初始变性（94℃，5 min），随后29个扩增循环（94℃ 30 s，55℃ 30 s，72℃ 每kb 1 min），最后终延伸（72℃，5 min）。通过1%琼脂糖凝胶（agarose gel）电泳检测第一轮PCR产物的长度，切取并纯化对应正确长度（1100 bp）的DNA条带。取20 ng纯化后的第一轮PCR产物作为DNA模板，使用第二轮引物对（NVC57与NVC66）开展第二轮PCR。将第二轮PCR获得的纯化产物（420 bp）送至MR DNA（www.mrdnalab.com，美国德克萨斯州肖洛特市），采用Illumina MiSeq双端测序（读长约2×300 bp）进行测序。序列数据采用MR DNA分析流程进行处理，OTUs通过3%差异度聚类定义，并通过比对NCBI中涵盖当前所有已报道SDIMO基因的自建数据库完成分类学注释。整体实验设计：为探究从未污染土壤中富集得到的两株二氧六环降解菌群的微生物群落组成，采用Illumina MiSeq技术对16S rRNA基因进行测序分析。结果显示，这两个菌群中的主要物种为分枝杆菌属（Mycobacterium spp.）。