Methodology Simple and inexpensive DNA extraction protocol for studying the bacterial composition of sludges used in microbial fuel cells

Abstract

ABSTRACT. Bacteria oxidize organic matter and nutrients to produce electric energy in microbial fuel cells (MFC) -a technology of increasing importance because of its sustainability. To improve the performance of MFCs, it is necessary not only to gain a better understanding of MFC engineering designs, but also to improve the understanding of the composition of the microbial communities in MFCs. Fast and efficient DNA extraction protocols that are suitable for extracting diverse bacterial genomes are necessary to identify the bacterial diversity present in MFCs and to further monitor the dynamic changes of microbial communities. This study focused on testing different direct cell lysis protocols to extract DNA from a microbial sludge harvested DNA extraction of bacterial consortium in MFC from an MFC. The protocol that achieved the best results was based on a previous study, but was modified by eliminating a chaotropic salt and the special columns used for nucleic acid purification. The efficiency of this less expensive and more straightforward protocol was confirmed by PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis analysis, which confirmed the extraction of multiple genomes. The sequences of 10 clones revealed the presence of phyla, Proteobacteria, Firmicutes and Actinobacteria, comprising both Gramnegative and Gram-positive bacteria. Some of these bacteria were identified at the genus level, e.g., Clostridium, Pseudoxanthomonas, Tistrella, and Enterobacter; these genera have been described in active sludges from wastewater treatment, supporting the congruency of our results. Therefore, this protocol is a useful tool for analysis of the bacteria responsible for energy production in MFCs.

Characterization of anode and anolyte community growth and the impact of impedance in a microbial fuel cell

“…The DNA extraction protocol for identifying the bacterial composition of sludge used in microbial fuel cells was used as described previously by Canto-Canché et al . [ 41 ]. Metagenomic DNA was sent to the Research and Testing Laboratory facility in Lubbock, Texas (USA) for pyrosequencing of the 16S rRNA gene.…”

Comparative analysis of microbial diversity in batch reactor with direct interspecies electron transfer system using an effective and inexpensive method mgDNA extraction

“…immersed in a liquid phase such as natural water streams, culture media, or wastewaters (Dang et al, 2017;González-Gamboa et al, 2018;González-Muñoz et al, 2018;González-Paz et al, 2020). Therefore, high concentration and variety of impurities predominate in the samples, the main reason why lengthy procedures, expensive DNA isolation kits and purification columns are required (Canto-Canché et al, 2013).…”

“…The above mentioned biofilms contain organic molecules such as nucleic acids and proteins, and also inorganic substances such as metals and nutrients which are sequestered within the exopolysaccharide matrix (Baishya et al, 2020;Flemming & Wingender, 2010). Additionally, the biofilms are supported on different conductive materials (Erable et al, 2010), and in many cases, the isolation of the microbial community from the supporting material is barely impossible (Canto-Canché et al, 2013). Carbonaceous and porous materials as granular activated carbon, carbon felt, fibers or cloth, layered corrugated carbon, among others, are cheap and conductive supporting materials frequently used in anaerobic reactors (Barua & Dhar, 2017).…”

“…High chemical oxygen demand (COD), presence of organic acids, metals and other contaminants carried by carbon supporting materials are also frequent (Zhang et al, 2018). Therefore, to isolate DNA from complex samples as microbial electrosynthesis (MES) bioelectrodes, anaerobic digesters for bioenergy production and environmental samples used as inoculum, commercial DNA isolation Kits are usually employed (Kiseleva et al, 2015;Yang et al, 2017), which are expensive in comparison with homemade DNA isolation protocols (Canto-Canché et al, 2013). Additionally, in many cases purification columns are required to achieve good quality mgDNA, which are also expensive and produce DNA losses (Canto-Canché et al, 2013;Rousseau et al, 2016).…”

The applications of the 16S rRNA gene in microbial ecology: current situation and problems

“…ccbb.utexas.edu / ) 是分析 rRNA 二级结构的数据库。 EzTaxon鄄e 收集了已培养或未培养微生物的 16S rRNA 序 列信息,常用于鉴定新分离的菌株 [30] 。 probeBase 可用来检索与 16S rRNA 有关的探针 [31] [37] 。 由于指纹图谱技术的操作相对较简单,在环境微生物动态监测和分析等方面得到了广泛应 66] 。 利用微生物的功能基因作为探针开发出的功能基因芯片-GeoChip [67] ,已广泛 能够克服这类问题,比如近些年来开发出专门用于检测人体微生物相关菌群的 HITChip [71] 及用于检测口腔微 生物的 OC Chip [72] 等。 在定量分析方面,由于某些菌 rRNA 基因操纵子是多拷贝的,而使测定的信号总量代 表的是 16S rRNA 基因的总拷贝数,只有使用改进的算法才能更精确地进行定量分析 [73] [74] ,第三代测序技术的进步将使测序更加快速准确,且能大大增加读长,而且可以直接 [82] 。 针 对不同的环境样品,应该根据文献报道的相关结果对 DNA 提取方法进行优化。 Canto 等在研究污泥微生物时 使用不同的细胞直接裂解方法,获得了一种实用有效的 DNA 提取方法 [83] ;Kuczynski 等建议在研究复杂微生 物群落时应使用多种方法进行细胞裂解 [84] ;Flores 等研究了一种新的直接 PCR 法以简化 DNA 提取步骤 [85] 。 对于粘性土壤,可以加入 skim milk 以提高 DNA 的提取效率 [86] 。 用试剂盒提取的 DNA,进行 qRT鄄PCR 实验 时的平行性往往优于自配试剂的提取方法。 在进行样品总 RNA 提取过程中, 要特别注意污染和降解问 题 [87] ,一些 RNA 酶抑制剂可以用于样品运输过程中的保存,以防止 RNA 降解,如 RNA later 和 Methanol / HEPES 溶液 [88] 等。 必须注意的是,要使结果分析重复性和精确性较高,在提取以及后面的操作过程中都要 严格控制污染问题,因为相比在测序和分析过程中引入的偏差,实验污染等问题可能对结果影响更大。…”