RESUMO
The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA(®) SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil(®) (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed ß- and γ-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits.
Assuntos
Bactérias/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Microbiologia do Solo , Regiões Árticas , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Kit de Reagentes para Diagnóstico/economiaRESUMO
A population shift of ammonia-oxidizing bacteria (AOB) was described within a bench-scale activated sludge process treating an industrial wastewater in a previous report (Kuo et al. in Environ Eng Sci 23:507-520, 2006). In this investigation, transcriptional levels (amoA mRNA-based) of the three AOB groups (i.e., RI-27, B2-3, and Nitrosomonas nitrosa) identified in the treatment process were determined by quantitative real-time reverse transcription (RT-PCR) assays to circuitously evaluate AOB ammonia-oxidizing activity and to assess the presumed correlation between cellular activity and the dominant (greatest number) AOB population. Results demonstrated that the AOB group with higher amoA mRNA levels dominated the overall AOB population in the wastewater treatment process. Although AOB population dominance did not correlate well with transcripts at a normalized cellular level (amoA mRNA/DNA ratio), overall amoA mRNA levels did reflect the activity of distinct AOB groups under different N-loading conditions. Thus, an additional molecular parameter (amoA mRNA) was successfully utilized to assess timely shifts in AOB population structure that may impact nitrification treatment performance.
Assuntos
Amônia/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Oxirredução , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Esgotos/microbiologiaRESUMO
Conditions were developed for the reproducible production, isolation and characterization of a novel microbial extracellular polysaccharide believed to be involved in transient viscous bulking at an industrial wastewater treatment plant. The exopolysaccharide was extracted from cell-free culture supernatants of Thauera sp. strain MZ1T grown on a minimal medium with succinate. The purified polymer was found to be approximately 260 kDa in size by gel-permeation chromatography. The GC-MS analysis of the alditol acetate and per-O-trimethylsilyl methyl glycoside derivatives revealed that the exopolysaccharide was composed of four monosaccharides including: rhamnose, galacturonic acid, N-acetylglucosamine and N-acetylfucosamine. Glucose, which also appeared at low levels, is most likely from a co-eluting glucan. The FTIR and NMR spectroscopic analyses further revealed the presence of esterified component groups on the polymer. These results represent the first published description of a polysaccharide from a member of the genus Thauera, and lay the foundation for a deeper understanding of the factors potentially involved in zoogloeal cluster formation and viscous bulking.
