Real-Time Online Monitoring for Assessing Removal of Bacteria by Reverse Osmosis

Rigorous monitoring of microbial water quality is essential to ensure the safety of recycled water after advanced treatment for indirect and direct potable reuse. This study evaluated real-time bacterial monitoring for assessing reverse osmosis (RO) treatment for removal of bacteria. A strategy was employed to monitor bacterial counts online and in real time in the RO feed and permeate water using a real-time continuous bacteriological counter. Over the course of 68 h pilot-scale testing, bacterial counts were monitored in real time over approximate ranges from 1 × 10³ to 4 × 10⁴ and from 4 to 342 counts/mL in the RO feed (ultrafiltration-treated wastewater) and permeate, respectively. The results indicate that the bacteriological counter can track the variations in bacterial counts in the RO feed and permeate. Bacterial concentrations were confirmed by epi-fluorescence microscopy for total bacterial counts. A high correlation (<i>R</i>² = 0.83) was identified between the online bacterial counts and epi-fluorescence counts in the RO feed; a negligible correlation was observed for RO permeate. In this study, we evaluated a real-time bacteriological counter (i.e., counts per milliliter every second) to ensure continuous removal of bacterial contaminants by RO treatment

Feeding of ambroxol ameliorates neurodevelopmental defects and ER stress in the mutated hGBA induced <i>Drosophila</i> eye.

<p>Ambroxol can recover morphological defects and decrease ER stress in transgenic flies. (A) Less fluorescence emitted by the eye imaginal discs of hGBA^RecNciI transgenic combinations treated with, than without 1 mM Ambroxol. (B) Values generated by different transgenic combinations at fixed quantities of fluorescence intensity (n = 12–43 eye imaginal discs of third instar larvae per transgenic combination). Error bars represent SE. *Significant difference compared with controls (all without Ambroxol) (***P<0.001; Student's t test). (C) Ambroxol (1 mM) decreases expression levels of dBiP mRNA in the heads of hGBA^RecNciI transgenic combinations (n  =  about 30 fly heads per transgenic combination). Internal control was dRpL32. Error bars represent SE. (D) Eye phenotypes of hGBA^RecNciI transgenic combinations incubated without or with 1 mM Ambroxol. Size and shape of ocelli were uniform, and layout uniformity was more similar to that of normal fly eyes treated with 1 mM Ambroxol. (E) Size histograms of ocelli in hGBA^RecNciI transgenic combinations treated with or without 1 mM Ambroxol. (n = 6–10 flies per transgenic combination; about 400 ocelli each). Dispersion analysis showed significant differences from hGBA^RecNciI transgenic combinations treated with and without 1 mM Ambroxol (F = 2.07–3.35; P<0.001; Levene's test).</p

Generation of transgenic flies carrying hGBA variants.

<p>(A) Sequence of hGBA. Blue and red fonts show R120W and RecNciI mutations, respectively. (B) Expression levels of hGBA mRNA confirmed by quantitative RT-PCR (n  =  about 30 fly heads per transgenic combination) with dRpL32 as internal control. Error bars represent SE. (C) Levels of hGBA protein confirmed by Western blotting (n  =  about 100 fly heads per transgenic combination). Total amounts of hGBA protein were decreased in hGBA^R120W, and significantly decreased in hGBA^RecNciI transgenic combinations, compared with hGBA^WT transgenic combination.</p

Neurodevelopmental defects in the <i>Drosophila</i> eye caused by expression of hGBA carrying the RecNciI mutation.

<p>We investigated the effects of overexpression to mutated hGBAs in fly eyes. (A) Phenotype of eyes overexpressing hGBA^WT transgenic combination do not significantly differ from those of GMR control. Phenotype of eyes overexpressing hGBA^R120W transgenic combinations occasionally differed in terms of morphology in some flies compared with control. Eye morphology is obviously affected in hGBA^RecNciI transgenic combinations compared with control. (B) Size histograms of ocelli in transgenic combinations (n = 3–5 flies each, about 100 ocelli each). Dispersion analysis showed obvious differences in variance of the sizes of ocelli between the hGBA^RecNciI transgenic combinations and the GMR control (F = 29.50–37.19; P<0.001; Levene's test).</p

Endoplasmic reticulum (ER) stress detected in the mutated hGBA induced <i>Drosophila</i> eye.

<p>We used xbp1-EGFP as an ER stress marker in which EGFP is expressed in frame only after ER stress <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069147#pone.0069147-Ryoo1" target="_blank">[31]</a>. (A) Weak fluorescence is generated in eye imaginal discs expressing the hGBA^WT construct. The eye imaginal discs of hGBA^R120W transgenic combinations emitted more fluorescence than that of hGBA^WT transgenic combination. The eye imaginal discs of hGBA^RecNciI transgenic combinations emitted the most intense fluorescence. (B) Values generated by different transgenic combinations with fixed quantities of fluorescence intensity (n = 7–15 eye imaginal discs from third instar larvae per transgenic combination). Error bars represent SE. *Significant difference compared with values from GMR control (***P<0.001; Student's t test). (C) Endoplasmic reticulum stress marker gene, dBiP (major ER chaperone) mRNA expression in hGBA^R120W and hGBA^RecNciI transgenic combinations was upregulated (n  =  about 30 fly heads per transgenic combination). Internal control was dRpL32. Error bars represent SE. *Significant difference compared with GMR control (*P<0.05; Student's t test). (D) High levels of hGBAs are expressed in whole bodies of heat-shocked flies. Expression levels of dBiP mRNA of hGBA^R120W and hGBA^RecNciI transgenic combinations were also upregulated (n  =  about 30 flies per transgenic combination). Internal control was dRpL32. Error bars represent SE. *Significant difference compared with hs control (*P<0.05; **P<0.01; ***P<0.001; Student's t test).</p

Primer sequences for Quantitative RT-PCR.

<p>Human GBA primers were designed at Universal Probe Library Assay Design Center (Roche Applied Science).</p><p>Primers for dBiP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069147#pone.0069147-Plongthongkum1" target="_blank">[32]</a> and dRpL32 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069147#pone.0069147-Takehana1" target="_blank">[35]</a> were as described in respective citations.</p