Light dazzles from the black box: Whole-cell biosensors are ready to inform on fundamental soil biological processes

Whole-cell biosensors are natural or engineered microorganisms producing signals in response to specific stimuli. This review introduces the use of whole-cell biosensors for the study of the soil system, discuss the recent developments and some current limitations and draws future prospects of the whole-cell biosensors for application to the study of the agro-ecosystems. The review focuses mainly on the lux- and gfp-inserted whole-cell biosensors producing bioluminescence and multicoloured fluorescent proteins, which allow an easy and reproducible detection of the signals from a large number of prokaryotic and eukaryotic soil-borne microorganisms. This review also points out how the whole-cell biosensors indicate the bioavailability of selected analyte, an information that cannot be straight forwardly extrapolated using the chemical methods of soil analysis. However, regardless of the immense progress in biotechnology and genetics that allows to construct whole-cell biosensors for virtually detecting any chemical at ultra low concentrations, the soil still remains the most extreme natural system to be studied with these biotechnological analytical tools. Although a lack of standardization for most of the constructed whole-cell biosensors along with the scarce knowledge of their performance concur to prevent their use in the official methods of soil and environmental analysis, owing to their stability and selectivity we restate that the whole-cell biosensors are ready to provide information on the main processes occurring in soil, and represent unprecedented sensitive tools for improving agriculture and for soil monitorin

Development of a Fully Automated Flow Injection Analyzer Implementing Bioluminescent Biosensors for Water Toxicity Assessment

This paper describes the development of an automated Flow Injection analyzer for water toxicity assessment. The analyzer is validated by assessing the toxicity of heavy metal (Pb2+, Hg2+ and Cu2+) solutions. One hundred μL of a Vibrio fischeri suspension are injected in a carrier solution containing different heavy metal concentrations. Biosensor cells are mixed with the toxic carrier solution in the mixing coil on the way to the detector. Response registered is % inhibition of biosensor bioluminescence due to heavy metal toxicity in comparison to that resulting by injecting the Vibrio fischeri suspension in deionised water. Carrier solutions of mercury showed higher toxicity than the other heavy metals, whereas all metals show concentration related levels of toxicity. The biosensor’s response to carrier solutions of different pHs was tested. Vibrio fischeri’s bioluminescence is promoted in the pH 5–10 range. Experiments indicate that the whole cell biosensor, as applied in the automated fluidic system, responds to various toxic solutions

Synthetic biology tools for environmental protection

Synthetic biology transforms the way we perceive biological systems. Emerging technologies in this field affect many disciplines of science and engineering. Traditionally, synthetic biology approaches were commonly aimed at developing cost-effective microbial cell factories to produce chemicals from renewable sources. Based on this, the immediate beneficial impact of synthetic biology on the environment came from reducing our oil dependency. However, synthetic biology is starting to play a more direct role in environmental protection. Toxic chemicals released by industries and agriculture endanger the environment, disrupting ecosystem balance and biodiversity loss. This review highlights synthetic biology approaches that can help environmental protection by providing remediation systems capable of sensing and responding to specific pollutants. Remediation strategies based on genetically engineered microbes and plants are discussed. Further, an overview of computational approaches that facilitate the design and application of synthetic biology tools in environmental protection is presented

Microbial Bioremediation of some Heavy Metals in Soils: An updated review

Nowadays, due to industrialization and extraction of natural resources, soil and water pollution is one of the major global concerns. During the recent era of environmental protection, the use of microorganisms for the recovery of heavy metals from soil, sediments and water as well as employment of plants for landfill applications has generated growing attention. The role of microorganisms and plants in biotransformation of heavy metals into nontoxic forms is well-documented, and understanding the molecular mechanism of metal accumulation has numerous biotechnological implications for bioremediation of metal-contaminated sites. The food and water we consume are often contaminated with a range of chemicals and heavy metals, such as gold, copper, nickel, zinc, lead, cadmium, arsenic, chromium, and mercury that are associated with numerous diseases. Human activities like metalliferous mining and smelting, agriculture, waste disposal or industry discharge these metals which can produce harmful effects on human health when they are taken up in amounts that cannot be processed by the organism.  Many studies have demonstrated that microbes have the ability to remove heavy metals from contaminated soils. Among others some of the microorganisms that play great role in bioremediation of heavy metals are Pseudomonas spp.Alcaligenes spp., Arthrobacter spp., Bacillus spp., Corynebacterium spp., Flavobacterium spp., Azotobacter spp., Rhodococcus spp.Mycobacterium spp., Nocardia spp., Methosinussp.,Methanogens, Aspergilusniger, Pleurotusostreatus , Rhizopusarrhizus, Stereumhirsutum, Phormidiumvalderiumand Ganodermaapplantus. The encouraging evidence as to the usefulness of microorganisms and their constituents for the remediation of heavy metals from contaminated soils is reviewed in this article. Keywords: Bioremediation, Biotransformation, Heavy metals, Microbial, Toxicity.

New naphthalene whole-cell bioreporter for measuring and assessing naphthalene in polycyclic aromatic hydrocarbons contaminated site

A new naphthalene bioreporter was designed and constructed in this work. A new vector, pWH1274_Nah, was constructed by the Gibson isothermal assembly fused with a 9 kb naphthalene-degrading gene nahAD (nahAa nahAb nahAc nahAd nahB nahF nahC nahQ nahE nahD) and cloned into Acinetobacter ADPWH_lux as the host, capable of responding to salicylate (the central metabolite of naphthalene). The ADPWH_Nah bioreporter could effectively metabolize naphthalene and evaluate the naphthalene in natural water and soil samples. This whole-cell bioreporter did not respond to other polycyclic aromatic hydrocarbons (PAHs; pyrene, anthracene, and phenanthrene) and demonstrated a positive response in the presence of 0.01 μM naphthalene, showing high specificity and sensitivity. The bioluminescent response was quantitatively measured after a 4 h exposure to naphthalene, and the model simulation further proved the naphthalene metabolism dynamics and the salicylate-activation mechanisms. The ADPWH_Nah bioreporter also achieved a rapid evaluation of the naphthalene in the PAH-contaminated site after chemical spill accidents, showing high consistency with chemical analysis. The engineered Acinetobacter variant had significant advantages in rapid naphthalene detection in the laboratory and potential in situ detection. The state-of-the-art concept of cloning PAHs-degrading pathway in salicylate bioreporter hosts led to the construction and assembly of high-throughput PAH bioreporter array, capable of crude oil contamination assessment and risk management

Bioremediation of PCB-contaminated marine sediments: From identification of indigenous dehalorespirers to enhancement of microbial reductive dechlorination

Marine sediments are the main accumulation reservoir of organic recalcitrant pollutants such as polychlorinated biphenyls (PCBs). In the anoxic conditions typical of these sediments, anaerobic bacteria of the phylum Chloroflexi are able to attack these compounds in a process called microbial reductive dechlorination. Such activity and members of this phylum were detected in PCB-impacted sediments of the Venice Lagoon. The aim of this work was to investigate microbial reductive dechlorination and design bioremediation approaches for marine sediments of the area. Three out of six sediment cultures from different sampling areas exhibited dechlorination activities in the same conditions of the site and two phylotypes (VLD-1 and VLD-2) were detected and correlated to this metabolism. Biostimulation was tested on enriched dechlorinating sediment cultures from the same site using five different electron donors, of which lactate was the best biostimulating agent; complementation of microbial and chemical dechlorination catalyzed by biogenic zerovalent Pd nanoparticles was not effective due to sulfide poisoning of the catalyst. A new biosurfactant-producing strain of Shewanella frigidimarina was concomitantly obtained from hydrocarbon-degrading marine cultures and selected because of the low toxicity of its product. All these findings were then exploited to develop bioremediation lab-scale tests in shaken reactors and static microcosms on real sediments and water of the Venice lagoon, testing i) a bioaugmentation approach, with a selected enriched sediment culture from the same area, ii) a biostimulation approach with lactate as electron donor, iii) a bioavailability enhancement with the supplementation of the newly-discovered biosurfactant, and iv) all possible combinations of the afore-mentioned approaches. The best bioremediation approach resulted to be a combination of bioaugmentation and bioremediation and it could be a starting point to design bioremediation process for actual marine sediments of the Venice Lagoon area