Soil geochemical and structural heterogeneities are coupled in influencing the fate and transport of viruses in soil profiles. Only the soil layer at a certain depth, where viruses have least mobility, determines the total flux and distance of virus transport. However, few studies have addressed the dependence of virus transport on soil depths. This dependence is mostly due to the gradients of geochemistry, soil structure, and solution chemistry along the soil profile. We employed intact soil columns (5 cm in length, 4.5 cm in inner diameter) from three depths (5-10 cm, 35-40 cm, and 65-70 cm) to investigate the transport of two very different model viruses: 1) MS2; a hydrophobic, negatively charged, less aggregated phage of E. coli, and 2) 43X174; a hydrophilic, positively charged (pH=6.58 +/- 0.21), highly aggregated phage of E. coli as a function of depth. The breakthrough of both viruses increased with soil depth in both 10 mM and 40 mM NaCl solutions. The mobility of MS2 was greater than 43X174 at 5-10 cm and 35-40 cm depths, but the mobility of 43X174 increased much faster than MS2 with soil depth and surpassed MS2 mobility in the soil of 65-70 cm depth. The effect of ionic strength on the mobility of nanosized MS2 decreased with soil depth because of decreased soil organic matter (SOM) content. The decreased SOM lessened ionic strength sensitivity via a hydrophobicity-reduced Lewis acid-base attraction mechanism. In comparison, 43X174 viruses showed an overall weaker response to ionic strength change since they were aggregated into microsized particles in the experimental solutions and thereby subjected to mechanical straining in the intact soils. The elevated straining effect counteracted the electrostatically facilitated transport of 43X174. These findings suggest that soil amendments with organic matter might be low cost, effective measures for reducing virus transport through soil profiles.

Transport of pathogenic bacteria from land surface to groundwater is largely influenced by rainfall intensity and geochemical and structural heterogeneities of subsurface sediments at different depths. It has been assumed that the change in rainfall intensity has different effects on bacterial transport as a function of soil depth. In this study, repacked and intact column systems were used to investigate the influences of pore water velocity on the transport of Escherichia coli 652T7 through a loamy soil collected from varying soil depths. The soils differed in geochemical properties and soil structures. The concentrations of bacteria in soil and liquid samples were measured using plate counting method. The breakthrough percentages of E. coli 652T7 increased with pore water velocity at each depth in both intact and disturbed soils. Among the different soil depths, the largest velocity effect was observed for the transport through the top soil (0-5 cm) of both disturbed and intact soil profiles. This depth-dependent effect of pore water velocity was attributed to down gradients of soil organic matter (SOM) and iron oxide contents with depth because SOM and iron oxides were favorable for bacterial attachment on soil surfaces. In addition, less bacteria broke through the disturbed soil than through the intact soil at the same depth, and the pore water velocity effect was stronger with the disturbed than intact soils. Specifically, the maximum C/C0 (i.e., ratio of effluent to influent concentration) doubled (i.e., from 0.36 to 0.76) in the 0-5 cm intact soil columns and tripled (i.e., from 0.16 to 0.43) in the 0-5 cm repacked soil columns. This structure-dependent effect of pore water velocity was attributed to larger pore tortuosity and a narrower range of pore sizes in the disturbed soil than in the intact soil. These findings suggest that change in pore water velocity could trigger bacterial remobilization especially in surface soils, where more bacteria are retained relative to deep soils.

The United States (U.S.) aims to reduce half of food loss and waste (FLW) by 2030. To achieve this goal, the public, academic, and political attentions on FLW have been increasing, and a series of actions have been implemented. However, the actions lack consideration on the categorical priority of FLW mitigation in relation to environmental footprints. In this article, we compare the FLW of three main plant food categories (i.e., grains, vegetables, and fruits) and their water and carbon footprints during 1970-2017. The vegetable FLW doubled during the period, reaching 3.39 x 1010 kg in 2017, which was 5- and 2-fold higher than the FLW of grains and fruits, respectively. The FLW of vegetables, grains, and fruits contributed 29%, 47%, and 24% to the total blue water wasted through FLW. The total carbon dioxide emissions generated by plant FLW were contributed by vegetables with 50%, grains with 31%, and fruits with 19%. Canonical correspondence analysis indicates that vegetable FLW had a higher positive correlation with urbanization, household incomes, gross domestic product, and high-income population than grain FLW, whereas fruit FLW was not influenced by these socioeconomic factors. Therefore, we suggest that the FLW mitigation should be prioritized on vegetables. Specific strategies include local food sourcing, shortening food miles, building food belts, and developing controlled-environment agriculture. Our data-based comparisons provide valuable insights into food policy improvement for achieving the 2030 reduction goal of the U.S., but the insights could be improved by considering the influences of foods imported from other nations.

Due to the public health concerns of endocrine-disrupting chemicals, there is an increasing demand to develop improved high-throughput detection assays for enhanced exposure control and risk assessment. A substrate-free, autobioluminescent HEK293ARE/Gal4-Lux assay was developed to screen compounds for their ability to induce androgen receptor (AR)-mediated transcriptional activation. The assay was validated against a group of 40 recommended chemicals and achieved an overall 87.5% accuracy in qualitatively classifying positive and negative AR agonists. The HEK293ARE/Gal4-Lux assay was demonstrated as a suitable tool for Tier 1 AR agonist screening. By eliminating exogenous substrate, this assay provided a significant advantage over traditional reporter assays by enabling higher-throughput screening with reduced testing costs while maintaining detection accuracy.

Cadmium-tolerant fungus, designated as strain FMS2, was isolated from metal-polluted soil collected from Lahore, Pakistan, and its role in cadmium bioremediation was investigated. Strain FMS2 was identified as Penicillium chrysogenum through sequence analysis of the internal transcribed spacer (ITS) regions. Physicochemical growth factors and maximum tolerance level to cadmium were determined. Cadmium uptake and bioremoval potential were analyzed by atomic absorption spectrophotometry, scanning electron microscopy and energy-dispersive spectroscopic analysis, whereas its distribution inside the fungal cell was detected by scanning transmission electron microscopy and electron energy loss spectroscopy. P. chrysogenum FMS2 was found to be resistant to cadmium with a maximum tolerance level up to 1000 mg l−1. Strain FMS2 can grow under a wide range of temperature (15–35 °C) and pH conditions (4.0–12.0). The cadmium removal capacity of strain FMS2 was observed as 49 ± 0.8% within 15 days after exposure. Distinct white patches were observed in scanning electron micrographs with clear peaks of cadmium in energy-dispersive spectroscopic spectra indicating cadmium biosorption. The scanning transmission electron micrographs and electron energy loss spectra of strain FMS2 cells revealed cytoplasmic localization of cadmium.

Technologies enabling on-site environmental detection or medical diagnostics in resource-limited settings have a strong disruptive potential compared to current analytical approaches that require trained personnel in laboratories with immobile, resource intensive instrumentation. Handheld devices, such as smartphones, are now routinely produced with CPUs, RAM, wireless data transfer capabilities, and high-resolution complementary metal oxide semiconductor (CMOS) cameras capable of supporting the capture and processing of bioluminescent signals. In theory, combining the capabilities of these devices with continuously bioluminescent human cell-based bioreporters would allow them to replicate the functionality of more expensive, more complex, and less flexible platforms while supporting human-relevant conclusions. In this work, we compare the performance of smartphone (CMOS) and night vision (image intensifier) devices with in vivo (CCD camera), and in vitro (photomultiplier tube) laboratory instrumentation for monitoring signal dynamics from continuously bioluminescent human cellular models under toxic, stable, and induced expression scenarios. All systems detected bioluminescence from cells at common plating densities. While the in vivo and in vitro systems were more sensitive and detected signal dynamics representing cellular health changes earlier, the night vision and smartphone systems also detected these changes with relatively similar coefficients of variation and linear detection capabilities. The smartphone system did not detect transcriptional induction. The night vision system did detect transcriptional activation, but was less sensitive than the in vivo or in vitro systems and required a stronger induction before the change could be resolved.

Pathogenic bacteria, widely present in septic tanks, sewage sludges, and wastewater, can move through the subsurface environment to pollute drinking water. Bacterial transport behaviors vary with soil properties and are subject to soil pore networking. In this study, we investigated the transport of Escherichia coli 652T7 through intact and disturbed soils collected from different soil depths (0-5 cm, 5-10 cm, 10-15 cm, and 15-20 cm). The results obtained under steady-state saturated flow conditions demonstrated significant influences of soil depth and soil structure on the transport of E. coli 652T7. The breakthrough percentages of E. coli 652T7 from the intact soil cores increased with soil depth from 36 % at 0-5 cm to 63 % at 5-10 cm, 83 % at 10-15 cm, and 100 % at 15-20 cm. A similar trend was observed for the breakthrough from the repacked soil cores except with lower percentages (i.e., 16 % at 0-5 cm, 49 % at 5-10 cm, 76 % at 10-15 cm, and 86 % at 15-20 cm). Such an increase with soil depth was attributed to a combined effect of decreases in soil organic matter content (from 3.84 % to 2.47 %), free iron oxides (from 142.25 mg kg(-1) to 110.66 mg kg(-1)), and zeta potential (from -17.6 mV to -29.0 mV) with soil depth. The larger breakthrough percentages of E. coli 652T7 from the intact than the repacked soil cores are speculated due to the existence of larger macropores, lower pore connectivity density, and reduced access to attachment sites in the intact soils than in the disturbed soils. Overall, this study suggests that tillage and organic amendments might be effective measures for reducing bacterial movement in soils.

This study investigated the transport and retention of bioluminescent Escherichia coli strain 652T7 under different pore water velocities (8.7 cm h(-1) and 13.0 cm h(-1)) and pore water saturations (85% and 100%) utilizing a noninvasive, real-time bioluminescent imaging technique. Under saturated flow conditions, the concentrations of retained bioluminescent E. coli 652T7 decreased exponentially with distance from the source at the lower velocity but decreased non-exponentially at the higher velocity. Under unsaturated flow conditions, pore water velocity had no significant effect on bacterial breakthrough concentration; however, the concentrations of retained cells were maximal at a significant distance from the source (non-monotonic). The distance from source of the maximum concentration increased from 2.4-cm at 1.05 pore volumes to 4.3-cm at 3.15 pore volumes, indicating slow translation of bacterial down-gradient under unsaturated flow conditions. That conditions were modestly unfavorable to attachment at the solid-water interface (SWI) was indicated by deposition rate coefficients being greater (by a factor of four) for simulations versus experiments, and by significant repulsive barriers to attachment at both the SWI (260 kT) and the air-water interface (AWI, fully repulsive). The inferred slow translation under unsaturated flow conditions therefore reflects either accumulation without arrest in the secondary minimum at the SWI and/or capillary interaction at the AWI. This non-invasive bioluminescence method yielded real-time quantitative observation of bacterial distribution from source and demonstrated contrasting transport behaviors previously obtained solely via more laborious methods with limited spatio-temporal observation.

3D cell culture models represent an attractive approach to decode intracellular and intercellular signaling, providing biologically relevant information and predictive data. Bioluminescent reporter gene assays and bioluminescence imaging in 3D cell models are very promising bioanalytical tools for several applications. Here we report a very straightforward method for bioluminescence imaging and bioluminescent reporter gene assays in 3D cell-culture models. Both the assays can be easily implemented in laboratories equipped with basic cell culture facilities and instrumentation for bioluminescence detection, that is, low-light detectors connected to inverted microscopes and luminometers, without the need for additional equipment.

In this study, we show the repetitive detection of toluene on a tapered optical fiber element (OFE) with an attached layer of Pseudomonas putida TVA8 bioluminescent bioreporters. The bioluminescent cell layer was attached on polished quartz modified with (3-aminopropyl)triethoxysilane (APTES). The repeatability of the preparation of the optical probe and its use was demonstrated with five differently shaped OFEs. The intensity of measured bioluminescence was minimally influenced by the OFE shape, possessing transmittances between 1.41% and 5.00%. OFE probes layered with P. putida TVA8 were used to monitor liquid toluene over a two-week period. It was demonstrated that OFE probes layered with positively induced P. putida TVA8 bioreporters were reliable detectors of toluene. A toluene concentration of 26.5 mg/L was detected after <30 min after immersion of the probe in the toluene solution. Additional experiments also immobilized constitutively bioluminescent cells of E. coli 652T7, on OFEs with polyethyleneimine (PEI). These OFEs were repetitively induced with Lauria-Bertani (LB) nutrient medium. Bioluminescence appeared 15 minutes after immersion of the OFE in LB. A change in pH from 7 to 6 resulted in a decrease in bioluminescence that was not restored following additional nutrient inductions at pH 7. The E. coli 652T7 OFE probe was therefore sensitive to negative influences but could not be repetitively used.

Abstract Background Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous. Results We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20–30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system. Conclusions Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.

Bioluminescent yeast assays BLYES and BLYAS are whole-cell bioassays that utilize genetically modified Saccharomyces cerevisiae bioreporters to detect estrogenic and androgenic activities, respectively. The bioreporter strains chromosomally express human estrogen receptor alpha (BLYES) or androgen receptor (BLYAS) and contain a reporter plasmid expressing the complete bacterial luciferase gene cassette (luxCDABE) under the control of an estrogen- or androgen-responsive promoter. Exposure to endocrine-disrupting compounds activates the receptor which subsequently turns on the expression of the reporter genes, resulting in dose-dependent bioluminescence (i.e., light) emission. These yeast whole-cell bioassays provide rapid, cost-effective, and high-throughput detection of endocrine-disrupting activities in environmental samples. This protocol will provide a detailed description of the standard assay procedures as well as a framework for data analysis.

A whole-cell optical fiber sensor of toluene was prepared by the adsorption of Pseudomonas putida TVA8, the bacterial strain producing light in contact with toluene, in the form of biofilm. Bioreporter immobilization was facilitated by (3-Aminopropyl)triethoxysilane (APTES) treatment on the wider end of tapered quartz optical fiber element (OFE). Repeatability of preparation and response of the active part of the biosensor was demonstrated in five trials with different OFE geometries in which the bioluminescence (BL) was induced with toluene solution (26.5 mg L-1) for 2 weeks. Longevity of inductions of the biosensor was shown in over 135 days trial, when the biosensor was repetitively induced with toluene solution (26.5 mg L-1) 68 times. The intensities of BL gradually decreased due to release of the adsorbed cells and they were only temporarily restored by the addition of nutrients. The intensities of BL induced with contaminated ground water were lower than in the mineral medium (MSM) with the same content of toluene. Ideal shape of OFE, which maximizes the detected signal, was calculated to be a Frustrum cone. The developed sensor is plausible for multiple and fast online monitoring of the presence of a pollutant in remote localities but not its concentration.

This chapter explores the history of the bioengineering advances that have been applied to common luciferase enzymes and the improvements that have been accomplished by this work. The primary focus is placed on firefly luciferase (FLuc), Gaussia luciferase (GLuc), Renilla luciferase (RLuc), Oplophorus luciferase (OLuc; NanoLuc), and bacterial luciferase (Lux). Beginning with the cloning and exogenous expression of each enzyme, their step-wise modifications are presented and the new capabilities endowed by each incremental advancement are highlighted. Using the historical basis of this information, the chapter concludes with a prospective on the overall impact these advances have had on scientific research and provides an outlook on what capabilities future advances could unlock.

A synthetic bacterial luciferase-based autobioluminescent bioreporter, HEK293(ERE/Gal4-Lux), was developed in a human embryonic kidney (HEK293) cell line for the surveillance of chemicals displaying endocrine disrupting activity. Unlike alternative luminescent reporters, this bioreporter generates bioluminescence autonomously without requiring an external light-activating chemical substrate or cellular destruction. The bioreporter's performance was validated against a library of 76 agonistic and antagonistic estrogenic endocrine disruptor chemicals and demonstrated reproducible half maximal effective concentration (EC50) values meeting the U.S. Environmental Protection Agency (EPA) guidelines for Tier 1 endocrine disrupting chemical screening assays. For model compounds, such as the estrogen receptor (ER) agonist 17-estradiol, HEK293(ERE/Gal4-Lux) demonstrated an EC50 value (7.9x10(-12) M) comparable to that of the current EPA-approved HeLa-9903 firefly luciferase-based estrogen receptor transcription assay (4.6x10(-12) M). Screening against an expanded array of common ER agonists likewise produced similar relative effect potencies as compared with existing assays. The self-initiated autobioluminescent signal of the bioreporter permitted facile monitoring of the effects of endocrine disrupting chemicals, which decreased the cost and hands-on time required to perform these assays. These characteristics make the HEK293(ERE/Gal4-Lux) bioreporter potentially suitable as a high-throughput human cell-based assay for screening estrogenic activity.

Chemotaxis and haptotaxis are important biological mechanisms that influence microbial movement toward concentrated chemoattractants in mobile liquids and along immobile surfaces, respectively. This study investigated their coupled effect, as induced by naphthalene (10 mg L-1), on the transport and retention of two pollutant-degrading bacteria, Pseudomonas fluorescens 5RL (Pf5RL) and Pseudomonas stutzeri DQ1 (PsDQ1), in quartz sand and natural soil. The results demonstrated that PsDQ1 was not chemotactic, whereas Pf5RL was chemotactic at 25 degrees C but not at 4 degrees C due to the restricted movement. In a quartz sand column, haptotaxis did not play a role in increasing the transport of Pf5RL as compared with chemotaxis. Compared with a naphthalene-free soil column, Pf5RL broke through naphthalene-presaturated soil columns to reach a stable effluent concentration 0.5 pore volumes earlier due to advective chemotaxis occurring behind the plume front in the bulk solution. Pf5RL also demonstrated greater retention (e.g., a doubled rate of attachment and a one-third smaller breakthrough percentage) due to along-surface haptotaxis and near-surface chemotaxis occurring in less mobile water near the soil surface. However, both chemotaxis and haptotaxis were weakened when Pf5RL co-transported with naphthalene due to reduced adsorption of naphthalene on the soil. This study suggests that surface adsorption of naphthalene can mediate the relative importance of advective chemotaxis (facilitating initial breakthrough), near-surface chemotaxis (increasing bacterial collision), and haptotaxis (increasing bacterial residence time).

AIM:Escherichia coli is the conventional choice as the host strain for whole-cell bioreporter construction due to its well-understood genetics and well-established cloning protocols. However, for real-world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole-cell bioreporter for detection of bioavailable mercury by replacing E. coli with a wild-type bacterial host derived from a soil environment. MATERIALS AND RESULTS:In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4lux , detected mercury (HgCl2 ) at a minimum concentration of 0·2 µg l-1 with a linear response profile being maintained between a range of 0·4-1600 µg l-1 (R2  = 0·9604) with a peak bioluminescent response occurring within 1 h after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. Enterobacter cloacae DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2  = 0·9623). CONCLUSIONS:The results indicated that E. cloacae DWH4lux could detect mercury in quantities below the US Environmental Protection Agency's permitted limit values (2 µg l-1 ). Hence, it is concluded that E. cloacae DWH4lux has the potential to serve as an effective whole-cell bioreporter for the environmental monitoring of mercury contamination. SIGNIFICANCE AND IMPACT OF THE STUDY:This study provides new insight into the recruitment of mercury-tolerant bacterial hosts derived from environmental samples over the conventional lab-based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future.