A downstream process control tool based on the redox dye resazurin for rapid and accurate measurement of microbial metabolic activity.

Many sophisticated water treatment plants need a reliable, fast, and economical microbial load detection method. We refined a colorimetric assay using the redox dye resazurin to assess viable microorganisms. Here, we have used a mixed bacterial suspension of significant multi-drug-resistant coliform bacteria isolated from hospital wastewater and constructed a resazurin reduction calibration curve which could accurately predict the level of microbial contamination. The number of viable microorganisms was calculated from calibration curve in terms of log colony forming units (CFU) per milliliter. Ultrasonication disinfection of bacterial suspension for a duration of 50 min measured by resazurin assay depicted a reduction of 16.94%, 26.48%, and 37.69% at 410 W, 580 W, and 700 W, respectively. A synergistic effect of the combined methods of ultrasonication and heat disinfection treatments on raw wastewater and secondary wastewater effluent was observed and was also evaluated using both resazurin assay and standard plate count method. For raw wastewater, about 1.8 log reduction was observed for ultrasonication alone and 4 log CFU/mL reduction for thermosonication. In the secondary wastewater effluent, a reduction of 2.9 and 3.2 log CFU/mL was recorded for ultrasonication and thermosonication respectively. Resazurin microbial viability test results were highly comparable with conventional colony plate count for all treatment procedures, suggesting its appropriateness for quick and reliable wastewater sample microbial viability monitoring.

Terpinolene as an enhancer for ultrasonic disinfection of multi-drug-resistant bacteria in hospital wastewater.

The present study reports for the first time, a novel disinfection method that combines ultrasonication with a natural biocide terpinolene to inhibit tough and opportunistic antimicrobial-resistant (AMR) microorganisms isolated from hospital wastewater treatment plant (HWWTP). The enhancement of the disinfection process was evaluated for the effect of ultrasonication power, operating temperature, and inoculum size. A hybrid methodology combining terpinolene with traditional physico-chemical method of acoustic cavitation delivered efficient disinfection of the secondary effluent of field scale HWWTP, amended with a higher inoculum size of multi-drug-resistant coliform bacteria Enterobactor sp., Citrobacter freundii, and Klebsiella pneumonia. A bacterial load of 6.4 log CFU/mL was completely eliminated in 25 min. The present study also reports that due to the hybrid process, a very small concentration of 0.312 mM (0.25 × Minimum Inhibitory Concentration or MBC) of terpinolene was enough to completely disinfect the multi-drug-resistant coliforms. The leakage of intracellular nucleic acids during the disinfection process suggested disruption of cell membrane as the primary mechanism of disinfection followed by disruption of cellular metabolic function measured by respiratory chain dehydrogenase activity. Moreover, this study is the first to prove that terpinolene remained stable even after the cavitation process, thus revealing possibilities of recycling of the natural compound for wastewater disinfection. The results of the present research suggest that using terpinolene as a bio-additive can efficiently eliminate hazardous multi-drug-resistant bacteria and drastically reduce operational time and cost thus rendering it suitable to replace conventional wastewater disinfection.

Protection from metal toxicity by Hsp40-like protein isolated from contaminated soil using functional metagenomic approach.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil.

The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic microbiota inhabiting in these environments by using metatranscriptomics approach was the focus of this study. Size fractionated eukaryotic cDNA libraries (library A, < 0.5 kb, library B, 0.5-1.0 kb, and library C, > 1.0 kb) were constructed from RNA isolated from the metal contaminated soil. The library C was screened for Cadmium (Cd) tolerant genes by using Cd sensitive yeast mutant ycf1Δ by functional complementation assay, which yielded various clones capable of growing in Cd amended media. One of the Cd tolerant clones, PLCg39 was selected because of its ability to grow at high concentrations of Cd. Sequence analysis of PLCg39 showed homology with DHHC palmitoyl transferases, which are responsible for addition of palmitoyl groups to proteins and usually possess metal coordination domains. The cDNA PLCg39 was able to confer tolerance to Cd-sensitive (ycf1Δ), Copper-sensitive (cup1Δ) and Zn-sensitive (zrc1Δ) yeast mutants when grown at different concentrations of Cd (40-100 µM), Cu (150-1000 µM) and Zn (10-13 mM), respectively. The DHHC mutant akr1Δ transformed with PLCg39 rescued from the metal sensitivity indicating the role of DHHC palmitoyl transferase in metal tolerance. This study demonstrated that PLCg39 acts as a potential metal tolerant gene which could be used in bioremediation, biosensing and other biotechnological fields.

Metatranscriptomics: an approach for retrieving novel eukaryotic genes from polluted and related environments.

Metatranscriptomics, a subset of metagenomics, provides valuable information about the whole gene expression profiling of complex microbial communities of an ecosystem. Metagenomic studies mainly focus on the genomic content and identification of microbes present within a community, while metatranscriptomics provides the diversity of the active genes within such community, their expression profile and how these levels change due to change in environmental conditions. Metatranscriptomics has been applied to different types of environments, from the study of human microbiomes, to those found in plants, animals, within soils and in aquatic systems. Metatranscriptomics, based on the utilization of mRNA isolated from environmental samples, is a suitable approach to mine the eukaryotic gene pool for genes of biotechnological relevance. Also, it is imperative to develop different bioinformatic pipelines to analyse the data obtained from metatranscriptomic analysis. In the present review, we summarise the metatranscriptomics applied to soil environments to study the functional diversity, and discuss approaches for isolating the genes involved in organic matter degradation and providing tolerance to toxic metals, role of metatranscriptomics in microbiome research, various bioinformatics pipelines used in data analysis and technical challenges for gaining biologically meaningful insight of this approach.

Heavy metal hypertolerant eukaryotic aldehyde dehydrogenase isolated from metal contaminated soil by metatranscriptomics approach.

Constant addition of heavy metal pollutants in soil resulting from anthropogenic activities can prove detrimental to both macro and micro life forms inhabiting the ecosystem. The potential functional roles of eukaryotic microbes in such environment were explored in this study by metatranscriptomics approach. Sized eukaryotic cDNA libraries, library A (<0.5 kb), library B (0.5-1.0 kb), and library C (>1 kb) were constructed from the soil RNA and screened for copper (Cu) tolerance genes by using copper sensitive yeast mutant strain cup1Δ. Screening of the cDNA libraries yielded different clones capable of growing in Cu amended medium. In the present investigation, one of the transcripts PLCc38 from the library C was characterized and tested for its ability to tolerate different heavy metals by using metal sensitive yeast mutants. Sequence analysis PLCc38 showed homology with aldehyde dehydrogenase (ALDH) and capable of tolerating high concentrations of Cu (150-1000 µM). Aldeyde dehydrogenases are stress response enzymes capable of eliminating toxic levels of aldehydes generated due to abiotic environmental stresses. The cDNA PLCc38 also provided tolerance to wide range of Cd (40-100 µM), Zn (10-13 mM) and Co (2-50 mM) concentrations. Oxidative stress tolerance potential of PLCc38 was also confirmed in presence of different concentrations of H2O2. This study proves that PLCc38 is a potent gene associated with metal tolerance which could be used to revegetate heavy metal polluted soil or as a biomarker for detection of metal contamination.