The guanidine thiocyanate-high EDTA method for total microbial RNA extraction from severely heavy metal-contaminated soils.

Molecular analyses relying on RNA, as a direct way to unravel active microbes and their functional genes, have received increasing attention from environmental researchers recently. However, extracting sufficient and high-quality total microbial RNA from seriously heavy metal-contaminated soils is still a challenge. In this study, the guanidine thiocyanate-high EDTA (GTHE) method was established and optimized for recovering high quantity and quality of RNA from long-term heavy metal-contaminated soils. Due to the low microbial biomass in the soils, we combined multiple strong denaturants and intense mechanical lysis to break cells for increasing RNA yields. To minimize RNAase and heavy metals interference on RNA integrity, the concentrations of guanidine thiocyanate and EDTA were increased from 0.5 to 0.625 ml g-1 soil and 10 to 100 mM, respectively. This optimized GTHE method was applied to seven severely contaminated soils, and the RNA recovery efficiencies were 2.80 ~ 59.41 µg g-1 soil. The total microbial RNA of non-Cr(VI) (NT) and Cr(VI)-treated (CT) samples was utilized for molecular analyses. The result of qRT-PCR demonstrated that the expressions of two tested genes, chrA and yieF, were respectively upregulated 4.12- and 62.43-fold after Cr(VI) treatment. The total microbial RNA extracted from NT and CT samples, respectively, reached to 26.70 µg and 30.75 µg, which were much higher than the required amount (5 µg) for metatranscriptomic library construction. Besides, ratios of mRNA read were more than 86%, which indicated the high-quality libraries constructed for metatranscriptomic analysis. In summary, the GTHE method is useful to study microbes of contaminated habitats.

Metatranscriptomic analysis reveals active microbes and genes responded to short-term Cr(VI) stress.

Heavy metals have been severely polluting the environment. However, the response mechanism of microbial communities to short-term heavy metals stress remains unclear. In this study, metagenomics (MG) and metatranscriptomics (MT) was performed to observe the microbial response to short-term Cr(VI) stress. MG data showed that 99.1% of species were similar in the control and Cr(VI) treated groups. However, MT data demonstrated that 83% of the microbes were active in which 58.7% increased, while the relative abundance of 41.3% decreased after short-term Cr(VI) incubation. The MT results also revealed 9% of microbes were dormant in samples. Genes associated with oxidative stress, Cr(VI) transport, resistance, and reduction, as well as genes with unknown functions were 2-10 times upregulated after Cr(VI) treatment. To further confirm the function of unknown genes, two genes (314 and 494) were selected to detect the Cr(VI) resistance and reduction ability. The results showed that these genes significantly increased the Cr(VI) remediation ability of Escherichia coli. MT results also revealed an increase in the expression of some rare genera (at least two times) after Cr(VI) treatment, indicating these rare species played a crucial role in microbial response to short-term Cr(VI) stress. In summary, MT is an efficient way to understand the role of active and dormant microbes in specific environmental conditions.

Organismic-level acute toxicology profiling of reactive azo dyes.

In the present study, organismic-level acute toxicology profile of three reactive azo dyes, viz. Reactive Blue 221, Reactive Red 195, and Reactive Yellow 145, was investigated, by using bacterial (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis), fungal (Trichoderma asperellum, Aspergillus flavus, Fusarium fujikuroi, and Rhizoctonia solani), plant (Raphanus sativus, Triticum aestivum, Sorghum bicolor, and Phaseolus mungo), and aquatic (Artemia salina and Daphnia magna) specimens. Microbial test organisms (all the six bacteria and two fungi, i.e., T. asperellum and A. flavus) and D. magna were found to be relatively more sensitive towards the reactive azo dyes and their mixture, as the EC50 values were in the range of 80-330, 135-360, and 108-242 ppm for bacteria, fungi, and D. magna, respectively (but the effect was not acutely toxic). Moreover, the effect of dye mixture was comparable tothe individual dyes in almost all the tested microbial specimens. For plant seeds, the dye mixture was found to be relatively more inhibitory towards T. aestivum and R. sativus than the individual dyes. For S. bicolor and P. mungo seeds, the effect of the dye mixture was almost identical to the individual dyes. However, in all cases, EC50 values were in the range of 950-3500 ppm, which indicates a non-toxic effect on plant seed germination potential. Likewise, the dyes and their mixture were not acutely toxic for Artemia salina larvae (more sensitive to the dye mixture) and Daphnia magna neonates (EC50, 516-950 and 108-242 ppm, respectively).