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Fe and N co-doped walnut shell biochar (Fe,N-BC) was prepared through a one-pot pyrolysis procedure by using walnut shells as feedstocks, melamine as the N source, and iron (III) chloride as the Fe source. Moreover, pristine biochar (BC), nitrogen-doped biochar (N-BC), and α-Fe 2 O 3 -BC were synthesized as controls. All the prepared materials were characterized by different techniques and were used for the activation of peroxymonosulfate (PMS) for the degradation of sulfamethoxazole (SMX). A very high degradation rate for SMX (10 mg/L) was achieved with Fe,N-BC/PMS (0.5 min -1 ), which was higher than those for BC/PMS (0.026 min -1 ), N-BC/PMS (0.038 min -1 ), and α-Fe 2 O 3 -BC/PMS (0.33 min -1 ) under the same conditions. This is mainly due to the formation of Fe 3 C and iron oxides, which are very reactive for the activation of PMS. In the next step, Fe,N-BC was employed for the formation of a composite membrane structure by a liquid-induced phase inversion process. The synthesized ultrafiltration membrane not only exhibited high separation performance for humic acid sodium salt (HA, 98%) but also exhibited improved self-cleaning properties when applied for rhodamine B (RhB) filtration combined with a PMS solution cleaning procedure. Scavenging experiments revealed that 1 O 2 was the predominant species responsible for the degradation of SMX. The transformation products of SMX and possible degradation pathways were also identified. Furthermore, the toxicity assessment revealed that the overall toxicity of the intermediate was lower than that of SMX., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 μmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis., Competing Interests: Competing interests statement:W.T., I.P.T., and W.E.H. have filed a provisional patent application with the UK Patent Office related to this work.

Microbial rhodopsin, a significant contributor to sustaining life through light harvesting, holds untapped potential for carbon fixation. Here, we construct an artificial photosynthesis system which combines the proton-pumping ability of rhodopsin with an extracellular electron uptake mechanism, establishing a pathway to drive photoelectrosynthetic CO 2 fixation by Ralstonia eutropha (also known as Cupriavidus necator) H16, a facultatively chemolithoautotrophic soil bacterium. R. eutropha is engineered to heterologously express an extracellular electron transfer pathway of Shewanella oneidensis MR-1 and Gloeobacter rhodopsin (GR). Employing GR and the outer-membrane conduit MtrCAB from S. oneidensis, extracellular electrons and GR-driven proton motive force are integrated into R. eutropha's native electron transport chain (ETC). Inspired by natural photosynthesis, the photoelectrochemical system splits water to supply electrons to R. eutropha via the Mtr outer-membrane route. The light-activated proton pump - GR, supported by canthaxanthin as an antenna, powers ATP synthesis and reverses the ETC to regenerate NADH/NADPH, facilitating R. eutropha's biomass synthesis from CO 2 . Overexpression of a carbonic anhydrase further enhances CO 2 fixation. This artificial photosynthesis system has the potential to advance the development of efficient photosynthesis, redefining our understanding of the ecological role of microbial rhodopsins in nature., (© 2023. The Author(s).)

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO 2 to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO 2 fixation. A light-driven proton pump comprising Gloeobacter rhodopsin (GR) and its cofactor retinal have been assembled in Ralstonia eutropha ( Cupriavidus necator ) H16. In the presence of light, this strain fixed inorganic carbon (or bicarbonate) leading to 20% growth enhancement, when formate was used as an electron donor. We found that an electrode from a solar panel can replace organic compounds to serve as the electron donor, mediated by the electron shuttle molecule riboflavin. In this new autotrophic and photo-electrosynthetic system, GR is augmented by an external photocell for reductive CO 2 fixation. We demonstrated that this hybrid photo-electrosynthetic pathway can drive the engineered R. eutropha strain to grow using CO 2 as the sole carbon source. In this system, a bioreactor with only two inputs, light and CO 2 , enables the R. eutropha strain to perform a rhodopsin-dependent autotrophic growth. Light energy alone, supplied by a solar panel, can drive the conversion of CO 2 into biomass with a maximum electron transfer efficiency of 20%.

Nitric oxide (NO) is an important disease biomarker found in many chronic inflammatory diseases and cancers. A well-characterized nitric sensing system is useful to aid the rapid development of bacteria therapy and synthetic biology. In this work, we engineered a set of NO-responsive biosensors based on the P norV promoter and its NorR regulator in the norRVW operon; the circuits were characterized and optimized in probiotic Escherichia coli Nissle 1917 and mini SimCells (minicells containing designed gene circuits for specific tasks). Interestingly, the expression level of NorR displayed an inverse correlation to the P norV promoter activation, as a strong expression of the NorR regulator resulted in a low amplitude of NO-inducible gene expression. This could be explained by a competitive binding mechanism where the activated and inactivated NorR competitively bind to the same site on the P norV promoter. To overcome such issues, the NO induction performance was further improved by making a positive feedback loop that fine-tuned the level of NorR. In addition, by examining two integration host factor (IHF) binding sites of the P norV promoter, we demonstrated that the deletion of the second IHF site increased the maximum signal output by 25% (500 μM DETA/NO) with no notable increase in the basal expression level. The optimized NO-sensing gene circuit in anucleate mini SimCells exhibited increased robustness against external fluctuation in medium composition. The NO detection limit of the optimized gene circuit pPnorVβ was also improved from 25.6 to 1.3 nM in mini SimCells. Moreover, lyophilized mini SimCells can maintain function for over 2 months. Hence, SimCell-based NO biosensors could be used as safe sensor chassis for synthetic biology.

The ability to directly modify native and established biofilms has enormous potential in understanding microbial ecology and application of biofilm in 'real-world' systems. However, efficient genetic transformation of established biofilms at any scale remains challenging. In this study, we applied an ultrasound-mediated DNA delivery (UDD) technique to introduce plasmid to established non-competent biofilms in situ. Two different plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway were introduced into established bacterial biofilms in microfluidic flow (transformation efficiency of 3.9 ± 0.3 × 10 -7 cells in biofilm) and microbial fuel cells (MFCs), respectively, both employing UDD. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD-treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD-treated Shewanella oneidensis MR-1 biofilms generated significantly (P < 0.05) greater (61%) bioelectricity production (21.9 ± 1.2 µA cm -2 ) in MFC than a wild-type control group (~ 13.6 ± 1.6 µA cm -2 ). The effects of UDD were amplified in subsequent growth under selection pressure due to antibiotic resistance and metabolism enhancement. UDD-induced gene transfer on biofilms grown in both microbial flow cells and MFC systems was successfully demonstrated, with working volumes of 0.16 cm 3 and 300 cm 3 , respectively, demonstrating a significant scale-up in operating volume. This is the first study to report on a potentially scalable direct genetic engineering method for established non-competent biofilms, which can be exploited in enhancing their capability towards environmental, industrial and medical applications., (© 2021 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Biological wax esters offer a sustainable, renewable and biodegradable alternative to many fossil fuel derived chemicals including plastics and paraffins. Many species of bacteria accumulate waxes with similar structure and properties to highly desirable animal and plant waxes such as Spermaceti and Jojoba oils, the use of which is limited by resource requirements, high cost and ethical concerns. While bacterial fermentations overcome these issues, a commercially viable bacterial wax production process would require high yields and renewable, affordable feedstock to make it economically competitive and environmentally beneficial. This review describes recent progress in wax ester generation in both wild type and genetically engineered bacteria, with a focus on comparing substrates and quantifying obtained waxes. The full breadth of wax accumulating species is discussed, with emphasis on species generating high yields and utilising renewable substrates. Key areas of the field that have, thus far, received limited attention are highlighted, such as waste stream valorisation, mixed microbial cultures and efficient wax extraction, as, until effectively addressed, these will slow progress in creating commercially viable wax production methods., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Electrochemically active biofilms are capable of exchanging electrons with solid electron acceptors and have many energy and environmental applications such as bioelectricity generation and environmental remediation. The performance of electrochemically active biofilms is usually dependent on c-type cytochromes, while biofilm development is controlled by a signal cascade mediated by the intracellular secondary messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP). However, it is unclear whether there are any links between the c-di-GMP regulatory system and the expression of c-type cytochromes. In this study, we constructed a S. oneidensis MR-1 strain with a higher cytoplasmic c-di-GMP level by constitutively expressing a c-di-GMP synthase and it exhibited expected c-di-GMP-influenced traits, such as lowered motility and increased biofilm formation. Compared to MR-1 wild-type strain, the high c-di-GMP strain had a higher Fe(III) reduction rate (21.58 vs 11.88 pM of Fe(III)/h cell) and greater expression of genes that code for the proteins involved in the Mtr pathway, including CymA, MtrA, MtrB, MtrC and OmcA. Furthermore, single-cell Raman microspectroscopy (SCRM) revealed a great increase of c-type cytochromes in the high c-di-GMP strain as compared to MR-1 wild-type strain. Our results reveal for the first time that the c-di-GMP regulation system indirectly or directly positively regulates the expression of cytochromes involved in the extracellular electron transport (EET) in S. oneidensis, which would help to understand the regulatory mechanism of c-di-GMP on electricity production in bacteria., (© 2020 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli , Pseudomonas putida , and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome., Competing Interests: Competing interest statement: C.F., I.P.T., H.Y., Z.C., and W.E.H. have filed a provisional patent application with the UK Patent Office related to this work., (Copyright © 2020 the Author(s). Published by PNAS.)

Pollution in rapidly urbanising cities and in delta systems is a serious problem that blights the lives and livelihoods of millions of people, damaging and restricting potable water supply and supplies to industry (Whitehead et al, 2015, 2018). Employing new technology based on luminescent molecular biosensors, the toxicity in the rivers around Dhaka in Bangladesh, namely the Turag, Tongi, Balu and Buriganga, has been assessed. Samples taken at 36 sites during medium and low flow conditions and during the Bishwa Ijtema Festival revealed high levels of cell toxicity, as well as high concentrations of metals, particularly aluminium, cadmium, chromium, iron, zinc, lithium, selenium and nickel. Chemical analysis also revealed low dissolved oxygen levels and anoxic conditions in the rivers at certain sites. The bacterial molecular biosensors were demonstrated to be fast, with results in 30 min, robust and a highly sensitive method for the assessment of water toxicity in the field. Furthermore, the biosensor toxicity analysis correlated with the metals data, and a multivariate regression relationship was developed relating toxicity to key metals, such a selenium, zinc and chromium. The resulting model has been validated against split samples and the Bishwa Ijtema Festival data. The combination of modelling and the molecular biosensor technology provides a new approach to detecting and managing pollution in urban river systems., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this work, five Crassulacean Acid Metabolism (CAM) species from the five different genera (Agave, Ananas, Euphorbia, Kalanchoe, and Opuntia) were selected as alternative feedstocks and their biochemical methane potentials (BMP) were investigated. Batch assays were performed using sludge and rumen fluid as inocula under uncontrolled pH and at mesophilic temperature (39 °C). Mean methane yields from the CAM plants inoculated with AD sludge ranged from 281 to 382 ml/gVS. These values were not significantly different from the methane yield obtained from maize, a feedstock for biomethane and volatile fatty acid (VFA), suggesting that CAM plants may be viable as bioenergy crops on poor-quality soils in areas with low rainfall that are unsuitable for cultivation of food crops., (Copyright © 2019. Published by Elsevier Ltd.)

A simple aspirin-inducible system has been developed and characterized in Escherichia coli by employing the P sal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that the effector-free form, SalR r , can compete with the effector-bound form, SalR a , binding the P sal promoter and repressing gene transcription. The induction of the P sal promoter was compared in two different gene circuit designs: a simple regulation system (SRS) and positive autoregulation (PAR). Both regulatory circuits were induced in a dose-dependent manner in the presence of 0.05 to 10 µM aspirin. Overexpression of SalR in the SRS circuit reduced both baseline leakiness and the strength of the P sal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on the SalR r /SalR a competitive binding model not only fit the observed experimental results in SRS and PAR circuits but also predicted the performance of a new gene circuit design for which weak expression of SalR in the SRS circuit should significantly improve induction strength. The experimental result is in good agreement with this prediction, validating the SalR r /SalR a competitive binding model. The aspirin-inducible systems were also functional in probiotic strain E. coli Nissle 1917 and SimCells produced from E. coli MC1000 ΔminD These well-characterized and modularized aspirin-inducible gene circuits would be useful biobricks for synthetic biology. IMPORTANCE An aspirin-inducible SalR/ P sal regulation system, originally from Acinetobacter baylyi ADP1, has been designed for E. coli strains. SalR is a typical LysR-type transcriptional regulator (LTTR) family protein and activates the P sal promoter in the presence of aspirin or salicylate in the range of 0.05 to 10 µM. The experimental results and mathematical simulations support the competitive binding model of the SalR/ P sal regulation system in which SalR r competes with SalR a to bind the P sal promoter and affect gene transcription. The competitive binding model successfully predicted that weak SalR expression would significantly improve the inducible strength of the SalR/ P sal regulation system, which is confirmed by the experimental results. This provides an important mechanism model to fine-tune transcriptional regulation of the LTTR family, which is the largest family of transcriptional regulators in the prokaryotic kingdom. In addition, the SalR/ P sal regulation system was also functional in probiotic strain E. coli Nissle 1917 and minicell-derived SimCells, which would be a useful biobrick for environmental and medical applications., (Copyright © 2019 Chen et al.)

It is of great significance to understand CO 2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13 C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13 C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO 2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for β-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO 2 fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The emergence and widespread distribution of antimicrobial resistant (AMR) bacteria has led to an increasing concern with respect to potential environmental and public health risks. Culture-independent and rapid identification of AMR bacteria in-situ in complex environments is important in understanding the role of viable but non-culturable and antibiotic persistent bacteria and in revealing potential pathogens without waiting for colony formation. In this study, a culture-independent and non-destructive phenotyping approach, so called Raman Deuterium Stable Isotope Probing (Raman-DIP), was developed to identify AMR bacteria in the River Thames. It is demonstrated that Raman-DIP was able to accurately identify resistant and susceptible bacteria within 24 hours. The work shows that, in the River Thames, the majority of the bacteria (76 ± 2%) were metabolically active, whilst AMR bacteria to carbenicillin, kanamycin and both two antibiotics were 35 ± 5%, 28 ± 3%, 25 ± 1% of the total bacterial population respectively. Raman activated cell ejection (RACE) was applied to isolate single AMR bacteria for the first time, linking AMR phenotype (reistance to antibiotics) and genotype (DNA sequence). The sequences of the RACE sorted cells indicate that they were potential human pathogens Aeromonas sp., Stenotrophomonas sp. and an unculturable bacterium. This work demonstrates Raman-DIP and RACE are effective culture-independent approach for rapid identification of AMR bacteria at the single cell level in their natural conditions.

This work serves as a proof-of-concept for bacterially derived SimCells (Simple Cells), which contain the cell machinery from bacteria and designed DNA (or potentially a simplified genome) to instruct the cell to carry out novel, specific tasks. SimCells represent a reprogrammable chassis without a native chromosome, which can host designed DNA to perform defined functions. In this paper, the use of Escherichia coli MC1000 ∆minD minicells as a non-reproducing chassis for SimCells was explored, as demonstrated by their ability to act as sensitive biosensors for small molecules. Highly purified minicells derived from E. coli strains containing gene circuits for biosensing were able to transduce the input signals from several small molecules (glucarate, acrylate and arabinose) into the production of green fluorescent protein (GFP). A mathematical model was developed to fit the experimental data for induction of gene expression in SimCells. The intracellular ATP level was shown to be important for SimCell function. A purification and storage protocol was developed to prepare SimCells which could retain their functions for an extended period of time. This study demonstrates that SimCells are able to perform as 'smart bioparticles' controlled by designed gene circuits.

Metalworking fluids (MWFs) are used as lubricants and coolants in the manufacturing operations. Their biodeterioration, while in operation, is a widespread problem leading to poor performance and worker health issues. Adding biocides, though effective in reducing microbial growth, leads to the production of more recalcitrant wastewaters that are difficult to dispose or recycle on-site. Increasing environmental concerns have led to robust legislation for reducing/eliminating the use of toxic biocides in MWFs, stimulating a growing interest in the development/application of alternative biological preservation strategies. In this study, inducing nutrient imbalance was investigated for controlling microbial growth in MWFs. Phosphorus was immobilized employing insoluble La 2 O 3 to form LaPO 4 . Concentrations of La 2 O 3 greater than 0.08%w (%w = weight percent) completely inhibited microbial growth (from 1.4 × 10 7 to 0 CFU/mL) and hindered biodegradation. Raman spectroscopy suggested that La 2 O 3 converted intracellular phosphorus into LaPO 4 . The growth inhibition potentials of both 0.06%w La(NO 3 ) 3 and La 2 O 3 were found to be superior to formaldehyde. The antimicrobial property of La 2 O 3 (i.e., inhibition) was tenable by adding sufficient phosphate, acting as an on/off switch for controlling microbial growth in MWFs. This technology offers the potential to reduce/eliminate the use of biocides in MWFs, improves the feasibility of end-of-life biological treatment, and closes the water loop.

Cell sorting coupled with single-cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial 'dark matter'. Single-cell Raman spectra (SCRSs) are label-free biochemical 'fingerprints' of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman-activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid-containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE-based single-cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single-cell genomics has been demonstrated., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Nanoparticulate delivery of biocides has the potential to decrease levels of exposure to non-target organisms, and miminize long-term exposure that can promote the development of resistance. Silica nanoparticles are an ideal vehicle since they are inert, biocompatible, biodegradable, and thermally and chemically stable. Encapsulation of biocides within nanoparticulates can improve their stability and longevity and maximize the biocidal potential of hydrophobic volatile compounds. Herein, we have shown that the plant secondary metabolites allyl isothiocyanate and cinnamaldehyde demonstrated increased antimicrobial activity against Escherichia coli in planktonic form, when packaged into mesoporous silica nanoparticles. Furthermore, the biocide-loaded nanoparticles showed activity against Pseudomonas aeruginosa biofilms that have inherent resistance to antimicrobial agents. The delivery platform can also be expanded to traditional biocides and other non-conventional antimicrobial agents., (© 2017 The Author(s).)

In this study, physical and chemical approaches were employed to distinguish between passive and active mechanisms in biofilms removing contaminants in waste waters and their relative merits were assessed. Respiration, post-exposure recovery and scanning electron microscopic analysis demonstrated that both ultraviolet (UV) treatment (300 mJ/cm(2)) and sodium azide (10 mM) completely inhibited metabolic activity at 5 and 24 h exposure, respectively, whilst not damaging the integrity of the biofilms. Amongst the commonly used chemical inhibitors, only sodium azide showed complete inhibition after 24 h incubation with only about 10% (±4%) of biofilm carbon released into the bulk solution, compared to 33-41% (±8%) when exposed to 5 mM and 10 mM 2,4-dinitrophenol (DNP) and 69-80% (±5%) when exposed to 2% and 5% w/v formalin, respectively. Biofilm inhibition with UV and sodium azide was found to be equally effective at inhibiting biofilms for treatment of triethanolamine (TEA) and benzotriazole (BTA): the results confirming that the dominant removal mechanism was biodegradation. However, the rates of glucose removal by sodium azide-inhibited biofilms were similar to controls, suggesting that chemical inhibitors were not effective for distinguishing the removal mechanisms of simple sugars. Statistically similar amounts of metal were removed by biofilms treated with UV and sodium azide in zinc, copper and cadmium single-systems: the results indicated that the removal mechanism is predominantly a passive biosorption process., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

The microbial assessment of pollutant toxicity rarely includes behavioral responses. In this study, we investigated the tactic response of Pseudomonas putida G7, a representative of soil bacterium, towards engineered zero-valent iron nanoparticles (nZVIs), as a new end-point assessment of toxicity. The study integrated the characterization of size distribution and charge of nZVIs and tactic reaction response by means of inverted capillary assay and computer-assisted motion analysis of motility behavior. Iron nanoparticles (diameter ≤ 100 nm) were prepared in the absence of oxygen to prevent aggregation, and then exposed in aerobic conditions. We first demonstrate that iron nanoparticles can elicit a negative tactic response in bacteria at low but environmentally-relevant, sub-lethal concentrations (1-10 μg/L). Cells were repelled by nZVIs in the concentration gradients created inside the capillaries, and a significant increase in turning events, characteristic of negative taxis, was detected under exposure to nZVIs. These tactic responses were not detectable after sustained exposure of the nanoparticles to oxygen. This new behavioral assessment may be prospected for the design of sensitive bioassays for nanomaterial toxicity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hybrid approaches for the remediation and detoxification of toxic recalcitrant industrial wastewater were investigated. The focus was waste metalworking fluid, which was selected as a representative model of other waste streams that are toxic, recalcitrant and that require more sustainable routes of safe disposal. The hybrid approaches included biodegradation, electron beam irradiation and zero-valent nano iron advanced oxidation processes that were employed individually and in sequence employing a factorial design. To compare process performance operationally exhausted and pristine metalworking fluid were compared. Sequential hybrid electron beam irradiation, biological, nanoscale zero-valent iron and biological treatment lead to synergistic detoxification and degradation of both recalcitrant streams, as determined by complementary surrogates and lead to overall improved COD removal of 92.8 ± 1.4% up from 85.9 ± 3.4% for the pristine metalworking fluid. Electron beam pre-treatment enabled more effective biotreatment, achieving 69.5 ± 8% (p = 0.005) and 24.6 ± 4.8% (p = 0.044) COD reductions., (Copyright © 2016. Published by Elsevier Ltd.)

The activity of methanogens and related bacteria which inhabit the coal beds is essential for stimulating new biogenic coal bed methane (CBM) production from the coal matrix. In this study, the microbial community structure and methanogenesis were investigated in Southern Qinshui Basin in China, and the composition and stable isotopic ratios of CBM were also determined. Although geochemical analysis suggested a mainly thermogenic origin for CBM, the microbial community structure and activities strongly implied the presence of methanogens in situ. 454 pyrosequencing analysis combined with methyl coenzyme-M reductase (mcrA) gene clone library analysis revealed that the archaeal communities in the water samples from both coal seams were similar, with the dominance of hydrogenotrophic methanogen Methanobacterium. The activity and potential of these populations to produce methane were confirmed by the observation of methane production in enrichments supplemented with H2 + CO2 and formate, and the only archaea successfully propagated in the tested water samples was from the genus Methanobacterium. 454 pyrosequencing analysis also recovered the diverse bacterial communities in the water samples, which have the potential to play a role in the coal biodegradation fueling methanogens. These results suggest that the biogenic CBM was generated by coal degradation via the hydrogenotrophic methanogens and related bacteria, which also contribute to the huge CBM reserves in Southern Qinshui Basin, China.

The dynamics of bacterial and eukaryotic community associated with each step of a water purification plant in China was investigated using 454 pyrosequencing and qPCR based approaches. Analysis of pyrosequencing revealed that a high degree diversity of bacterial and eukaryotic communities is present in the drinking water treatment process before sand filtration. In addition, the microbial compositions of the biofilm in the sand filters and those of the water of the putatively clear tanks were distinct, suggesting that sand filtration and chlorination treatments played primary roles in removing exposed microbial communities. Potential pathogens including Acinetobacter, Clostridium, Legionella, and Mycobacterium, co-occurred with protozoa such as Rhizopoda (Hartmannellidae), and fungi such as Penicillium and Aspergillus. Furthermore, this study supported the ideas based on molecular level that biofilm communities were different from those in corresponding water samples, and that the concentrations of Mycobacterium spp., Legionella spp., and Naegleria spp. in the water samples declined with each step of the water treatment process by qPCR. Overall, this study provides the first detailed evaluation of bacterial and eukaryotic diversity at each step of an individual potable water treatment process located in China., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Tightening regulations regarding the use of biocides have stimulated interest in investigating alternatives to current antimicrobial strategies. Plant essential oils and their constituent compounds are promising candidates as novel antimicrobial agents because of their excellent ability in killing microbes while being non-toxic to humans at antimicrobially-active concentrations. Allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde (CNAD), citral, and thymol were investigated for their antibacterial activity against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The five compounds were screened via disc diffusion assay and broth microdilution method, by which inhibition zone diameters, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs) were determined. AIT and CNAD displayed the greatest inhibitory effects against all species tested, with AIT yielding MICs of 156.25mg/L and MBCs of 156.25 to 312.5mg/L, and CNAD yielding MICs of 78.125 to 156.25mg/L and MBCs of 78.125 to 312.5mg/L. Based on these results, AIT and CNAD were selected for closer examination of their toxic effects. Two complementary bioluminescence-based bacterial biosensors, E. coli HB101&#95;pUCD607&#95;lux and Acinetobacter baylyi ADP1&#95;recA&#95;lux, were employed to examine the dose-response relationships and mechanism of action of AIT and CNAD. This is the first reported study to employ a lux-based biosensor assay coupled with parallel plate count experiments to demonstrate that AIT and CNAD not only damaged cell membranes, but also disrupted cellular metabolism and energy production in bacteria. It is also the first to use genotoxicity-sensing whole-cell bioreporters to demonstrate that neither AIT nor CNAD induced expression of the universal DNA repair gene, recA. This suggests that AIT and CNAD were not genotoxic. As an antimicrobial agent, it is advantageous that the compound be genetically non-damaging so that toxicity towards higher multicellular organisms and resistance development can be minimized. Thus, AIT and CNAD may be of high value as novel antimicrobial agents., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Thiodiglycol (TDG) is both the precursor for chemical synthesis of mustard gas and the product of mustard gas hydrolysis. TDG can also react with intermediates of mustard gas degradation to form more toxic and/or persistent aggregates, or reverse the pathway of mustard gas degradation. The persistence of TDG have been observed in soils and in the groundwater at sites contaminated by mustard gas 60 years ago. The biotransformation of TDG has been demonstrated in three soils not previously exposed to the chemical. TDG biotransformation occurred via the oxidative pathway with an optimum rate at pH 8.25. In contrast with bacteria isolated from historically contaminated soil, which could degrade TDG individually, a consortium of three bacterial strains isolated from the soil never contaminated by mustard gas was able to grow on TDG in minimal medium and in hydrolysate derived from an historical mustard gas bomb. Exposure to TDG had little impacts on the soil microbial physiology or on community structure. Therefore, the persistency of TDG in soils historically contaminated by mustard gas might be attributed to the toxicity of mustard gas to microorganisms and the impact to soil chemistry during the hydrolysis. TDG biodegradation may form part of a remediation strategy for mustard gas contaminated sites, and may be enhanced by pH adjustment and aeration.

In metal machining processes, the regulation of heat generation and lubrication at the contact point are achieved by application of a fluid referred to as metalworking fluid (MWF). MWFs inevitably become operationally exhausted with age and intensive use, which leads to compromised properties, thereby necessitating their safe disposal. Disposal of this waste through a biological route is an increasingly attractive option, since it is effective with relatively low energy demands. However, successful biological treatment is challenging since MWFs are chemically complex, and include biocides specifically to retard microbial deterioration whilst the fluids are operational. In this study remediation of the recalcitrant component of a semi-synthetic MWF by a novel hybrid ozone-bacteriological treatment, was investigated. The hybrid treatment proved to be effective and reduced the chemical oxygen demand by 72% (26.9% and 44.9% reduction after ozonation and biological oxidation respectively). Furthermore, a near-complete degradation of three non-biodegradable compounds (viz. benzotriazole, monoethanolamine, triethanolamine), commonly added as biocides and corrosion inhibitors in MWF formulations, under ozonation was observed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Disposal of operationally exhausted metal working fluids (MWF) through a biological route is an attractive option, since it is effective with relatively low energy demands. However, it is enormously challenging since these fluids are chemically complex, including the addition of toxic biocides which are added specifically to retard bio-deterioration whilst the fluids are operational. Nano-sized elemental iron represents a new generation of environmental remediation technologies. Laboratory scale batch studies were performed to test the degradation ability of a semi-synthetic metalworking fluid (MWF) wastewater (which was found to be resistant to initial bacterial treatment in specifically established bioreactors) by employing a novel hybrid approach. The approach was to combine the synergistic effects of nano zerovalent iron (nZVI) induced oxidation, followed by biodegradation, specifically for the remediation of recalcitrant components of MWF effluent. Addition of nZVI particles to oxygenated wastewater resulted in oxidation of organic contaminants present. Our studies confirmed 78% reduction in chemical oxygen demand (COD) by nZVI oxidation at pH 3.0 and 67% reduction in neutral pH (7.5), and 85% concurrent reduction in toxicity. Importantly, this low toxicity made the nZVI treated effluent more amenable for a second stage biological oxidation step. An overall COD reduction of 95.5% was achieved by the novel combined treatment described, demonstrating that nZVI oxidation can be exploited for enhancing the biodegradability of a recalcitrant wastewater in treatment processes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Landfills represent a major source of methane in the atmosphere. In a previous study, we demonstrated that earthworm activity in landfill cover soil can increase soil methane oxidation capacity. In this study, a simulated landfill cover soil mesocosm (1 m × 0.15 m) was used to observe the influence of earthworms (Eisenia veneta) on the active methanotroph community composition, by analyzing the expression of the pmoA gene, which is responsible for methane oxidation. mRNA-based pmoA microarray analysis revealed that earthworm activity in landfill cover soil stimulated activity of type I methanotrophs (Methylobacter, Methylomonas, Methylosarcina spp.) compared to type II methanotrophs (particularly Methylocystis spp.). These results, along with previous studies of methanotrophs in landfill cover soil, can now be used to plan in situ field studies to integrate earthworm-induced methanotrophy with other landfill management practises in order to maximize soil methane oxidation and reduce methane emissions from landfills., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Disposal of operationally exhausted metalworking fluids (MWFs) is enormously challenging. In this study the feasibility of employing a sequential Fenton-biological oxidation for the treatment of recalcitrant components of MWF wastewater was investigated. A statistical experimental design was employed to address Fenton reagent (H₂O₂, Fe²⁺) dose optimisation which ensured minimal concentrations of the reagents, thus making the treatment environmentally less toxic to subsequent biological steps and economically viable. This was achieved by employing a five-level-two-variable central composite experimental design. The results demonstrated that Fenton pre-treatment of the MWF effluent greatly improved biodegradability index (BOD₅)/COD increased from 0.160 to 0.538) with a synchronous lowering in the toxicity of the wastewater, making the recalcitrant component more amenable to subsequent biological treatment. An overall decrease of 92% and 86% in chemical oxygen demand (COD) and total organic carbon (TOC), respectively, was achieved by the two-step treatment method developed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study, we investigated the tactic response of Pseudomonas putida G7, a representative soil bacterium, towards silver nanoparticles (AgNPs). The study integrated the characterization of surface area and size distribution of AgNPs, toxicity determinations, based on ATP production, and assessment of the repellent reaction by means of an inverted capillary assay ('chemical-in-pond' method), and changes in the motility behaviour determined by computer-assisted motion analysis. Our data demonstrate, for the first time, that nanoparticles can elicit a negative tactic response in bacteria at low but environmentally relevant, sublethal concentrations. Data obtained by the chemical-in-pond method indicated that cells exposed to 0.1 mg l(-1) of two AgNPs preparations, differing in particle size (maximum diameter ≤ 100 nm and ≤ 150 nm respectively), were repelled in the gradients created inside the capillaries. However, cells exposed to similar low concentration of AgNO3 did not demonstrate any detectable repellent response, although it reduced cell viability by 20%, a decrease comparable to that caused by AgNPs. Computer analysis of swimming behaviour of cells exposed to AgNPs (0.2 mg l(-1) ) revealed a significant increase in turning events, as compared with unexposed controls, which is characteristic of bacterial repellent response. Greater AgNPs concentrations (up to 100 mg l(-1) ) also induced changes in the swimming behaviour, although they did not induce any detectable repellent response as determined by the chemical-in-pond assays. In contrast, AgNO3 failed to induce the repellent swimming behaviour within the wide range of concentrations tested (0.001-100 mg l(-1) ), and caused a significant inhibition of cell motility at a concentration above 0.1 mg l(-1) . The evidence presented here suggests there are likely to be alternative mechanisms by which nano-scale silver induces a repellent response, which is more direct than the toxic response of macro-forms of silver, attributed to ion formation and exposure., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The fate of Ti was examined in an activated sludge plant serving over 200,000 people. These studies revealed a decrease of 30 to 3.2 μg/L of Ti < 0.45 μm from influent to effluent and a calculated Ti presence of 305 mg/kg DW in wasted sludge. Thus, using sludge as a fertiliser would result in a predicted deposition of up to 250 mg/m² of Ti to soil surfaces using a recommended maximal agricultural application rate. Given the major use of TiO₂ in many industrial and domestic applications where loss to the sewer is possible, this measured Ti was presumed to have been largely TiO₂, a proportion of which will be nanoparticle sized. To assess the behaviour of engineered nanoparticle (ENP) TiO₂ in sewage and toxicology studies, Optisol (Oxonica Materials Ltd) and P25 (Evonik Industries AG), which are representative of forms used in sunscreen and cosmetic products, were used. These revealed a close association of TiO₂ ENPs with activated sludge. Using commercial information on consumption, and removal rates for sewage treatment, predictions were made for river water concentrations for sunscreen TiO₂ ENPs for the Anglian and Thames regions in Southern England. The highest predicted value from these exercises was 8.8 μg/L for the Thames region in which it was assumed that one in four people used the recommended application of sunscreen during a low flow (Q95) period. Ecotoxicological studies using potentially vulnerable species indicated that 1000 μg/L TiO₂ ENP did not affect the viability of a mixed community of river bacteria in the presence of UV light. Direct exposure to TiO₂ ENPs did not impair the immuno-effectiveness of earthworm coelomocyte cells at concentrations greatly above those predicted for sewage sludge., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The fate of nanomaterials when they enter the environment is an issue of increasing concern and thus it is important to know how they interact with natural organic molecules since this may have a significant impact on the particles' behavior. Because of our poor knowledge in this regard, the interaction of ZnO nanoparticles with amino acids of contrasting surface charge, including Histidine (HIS), Glycine (GLY), Aspartic acid (ASP) and Glutamic acid (GLU) which occur commonly in natural habitats, such as the plant root zone, was investigated over a range of pH conditions and concentrations. The addition of the individual amino acid led to significant changes in nanoparticle colloidal zeta potential stability, particle size distribution and the extent of agglomeration. Variations in pH resulted in considerable changes in nanoparticle surface charge and hydrodynamic size. In general, the particle size distribution decreased as the amino acid concentration increased, with more acidic conditions exacerbating this effect. In addition, increased concentrations of amino acids resulted in more stable nanoparticles in aqueous suspensions. Histidine had the greatest effect on colloidal stability, followed by Glycine, Aspartic acid and finally Glutamic acid. This study illustrates how nanoparticle behavior may change in the presence of naturally occurring amino acids, an important consideration when assessing the fate of nanoparticles in the environment. Additionally, utilization of amino acids in industrial processes could reduce particle agglomeration and it could lead to a way of employing more sustainable reagents., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Zero-valent iron (ZVI) nanoparticles are of interest because of their many potential biomedical and environmental applications. However, these particles have recently been reported to be cytotoxic to bacterial cells. The overall objective of this study was to determine the impact of 100mg/L ZVI nanoparticles on the diversity and structure of an indigenous river water bacterial community. Response during exposure for 36 days was determined by denaturing gel gradient electrophoresis (DGGE) analysis of bacterial 16S rRNA genes, amplified from extracted DNA, and viable and total cell abundances were determined by plate counting and fluorescent microscopy of DAPI-stained cells. Changes in river water chemistry were also monitored. Addition of ZVI nanoparticles led to a rapid decrease in oxidation-reduction potential (ORP) (+196 to -281 mV) and dissolved oxygen (DO) concentration (8.2-0.6 mg/L), both of which stabilized during the experiment. Interestingly, both viable and total bacterial cell abundances increased and pH decreased, characteristic of an active microbial community. Total community structure was visualized using rank-abundance plots fitted with linear regression models. The slopes of the regression models were used as a descriptive statistic of changes in evenness over time. Importantly, despite bacterial growth, addition of ZVI nanoparticles did not influence bacterial community structure., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Succinic acid is a platform molecule that has recently generated considerable interests. Production of succinate from waste orange peel and wheat straw by consolidated bioprocessing that combines cellulose hydrolysis and sugar fermentation, using a cellulolytic bacterium, Fibrobacter succinogenes S85, was studied. Orange peel contains D-limonene, which is a well-known antibacterial agent. Its effects on batch cultures of F. succinogenes S85 were examined. The minimal concentrations of limonene found to inhibit succinate and acetate generation and bacterial growth were 0.01%, 0.1%, and 0.06% (v/v), respectively. Both pre-treated orange peel by steam distillation to remove D: -limonene and intact wheat straw were used as feedstocks. Increasing the substrate concentrations of both feedstocks, from 5 to 60 g/L, elevated succinate concentration and productivity but lowered the yield. In addition, pre-treated orange peel generated greater succinate productivities than wheat straw but had similar resultant titres. The greatest succinate titres were 1.9 and 2.0 g/L for pre-treated orange peel and wheat straw, respectively. This work demonstrated that agricultural waste such as wheat straw and orange peel can be biotransformed to succinic acid by a one-step consolidated bioprocessing. Measures to increase fermentation efficiency are also discussed.

Iron (Fe) nanoparticles are increasingly being employed for the remediation of Chlorinated Aliphatic Hydrocarbon (CAH) contaminated sites. However, these particles have recently been reported to be cytotoxic to bacterial cells, and may therefore have a negative impact on exposed microbial communities. The overall objective of this study was to investigate the impact of Fe nanoparticles on the biodegradation of CAHs by an indigenous dechlorinating bacterial community. Also, to determine the most appropriate combination and/or application of bimetallic (Ni/Fe) nanoparticles and dechlorinating bacteria for the remediation of CAH contaminated sites. Addition of Fe nanoparticles to groundwater collected from a CAH contaminated site in Derby, UK, led to a decrease in the oxidation-reduction potential (ORP) and an increase in pH. The biological degradation rate of TCE was observed to progressively decrease in the presence of increasing Fe nanoparticle concentrations; which ranged from 0.01 to 0.1 gL(-1), and cease completely at concentrations of 0.3 gL(-1) or above. Concentrations greater than 0.3 gL(-1) led to a decline in viable bacterial counts and the inhibition of biological sulphate reduction. The most appropriate means of combining bimetallic (Ni/Fe) nanoparticles and indigenous dechlorinating bacteria was to employ a two step process: initially stimulating the biodegradation of TCE using acetate, followed by the addition of bimetallic nanoparticles to degrade the remaining cis-1,2-DCE and VC., (2010. Published by Elsevier Ltd. All rights reserved.)

The use of nano-scale particles as a means of environmental remediation still provides a comparatively novel approach for the treatment of contaminated waters. The current study compares the reactivity of micro-scale Fe, nano-scale Fe and nano-scale Ni/Fe (nickel/iron) particles specifically for dechlorination of solutions containing 350 mg L(-1) of TCE (concentration measured at a contaminated site in Derbyshire, UK). The results indicated that employing 1 g L(-1) of reactive material for dechlorination in the monometallic form (both micro- and nano-scale) exhibited very little reduction capability compared with the bimetallic Ni/Fe nano-scale particles, containing 28.9% Ni (in molar), which achieved complete dechlorination of the TCE in solution within 576 h. Experiments were also performed to determine the optimum bimetallic composition of the Ni/Fe particles for TCE reduction. This revealed that 3.2% Ni was the optimum Ni/Fe molar ratio for both maximum dehalogenation performance and minimum release of Ni into solution. Using particles of the most effective bimetallic composition, experiments were carried out to determine the concentration required for optimal TCE reduction. Over the range of nano-scale particle concentrations tested (0.1-9 g L(-1)), reduction rates of TCE increased with greater TCE:nano-scale particle ratios. However, a concentration range of 1-3 g L(-1) was selected as the most appropriate for site remediation, since more concentrated solutions demonstrated only small increases in rates of reaction. Finally, in order to test the long term performance and reactivity of the 3.2% Ni/Fe bimetallic nano-scale particles, weekly spikes of 350 mg L(-1) TCE were injected into a 3 g L(-1) nano-scale particle batch reactor. Results showed that the bimetallic nano-scale particles had the ability to reduce 1750 mg L(-1) TCE and remained active for at least 13 weeks., ((c) 2010 Elsevier Ltd. All rights reserved.)

Up to comparatively recently orange peel and the associated residual remnants of membranes resulting from juice extraction represented a significant disposal problem, especially in those regions where orange cultivation is a major industry. However, recent research has demonstrated that orange peel waste represents a potentially valuable resource that can be developed into high value products. These developments are critically reviewed in this article. This includes a summary of the chemical composition of the substrate and an assessment of the range of applications in which the peel is deployed. Utilization as a substrate to produce animal feed, fertilizer, essential oils, pectin, ethanol, methane, industrial enzymes, and single cell protein is discussed. The applications described together with those that will no doubt be developed in the future, represent great opportunities to harness the economical benefit of this agro-industrial waste and to develop even more efficient and sustainable systems. A scheme of integrated utilization of orange peel in a biorefinery approach is discussed together with some prediction of further necessary research.

Aims: To examine plant terpenoids as inducers of TCE (trichloroethylene) biotransformation by an indigenous microbial community originating from a plume of TCE-contaminated groundwater., Methods and Results: One-litre microcosms of groundwater were spiked with 100 micromol 1(-1) of TCE and amended weekly for 16 weeks with 20 microl 1(-1) of the following plant monoterpenes: linalool, pulegone, R-(+) carvone, S-(-) carvone, farnesol, cumene. Yeast extract-amended and unamended control treatments were also prepared. The addition of R-carvone and S-carvone, linalool and cumene resulted in the biotransformation of upwards of 88% of the TCE, significantly more than the unamendment control (61%). The aforementioned group of terpenes also significantly (P < 0.05) allowed more TCE to be degraded than the remaining two terpenes (farnesol and pulegone), and the yeast extract treatment which biotransformed 74-75% of the TCE. The microbial community profile was monitored by denaturing gradient gel electrophoresis and demonstrated much greater similarities between the microbial communities in terpene-amended treatments than in the yeast extract or unamended controls., Conclusions: TCE biotransformation can be significantly enhanced through the addition of selected plant terpenoids., Significance and Impact of the Study: Plant terpenoid and nutrient supplementation to groundwater might provide an environmentally benign means of enhancing the rate of in situ TCE bioremediation.

In a study to optimize bacterial whole cell biosensors (bioreporters) for the detection of environmental contaminants, we constructed a toxicity sensing strain Acinetobacter baylyi ADP1&#95;recA&#95;lux. The ADP1&#95;recA&#95;lux is a chromosomally based bioreporter which makes the sensing system stable and negates the need for antibiotics to maintain the trait. The AOP1&#95;recA&#95;lux is activated to express bioluminescence when it is exposed to DNA damaging toxicants. Since the ADP1&#95;recA&#95;lux constantly expresses a baseline level of bioluminescence, false negative results are avoided. The host strain, A. baylyi ADP1, is an ideal model strain typical of water and soil bacteria occurring in the natural environment, and it is more robust than E. coli in terms of viability, maintenance, and storage. The expression of reporter genes - luxCDABE cloned from Photorhabdus luminescens - is robust in a broad range of temperature (10-40 degrees C). The ADP1&#95;recA&#95;lux was used to detect a variety of toxic or potentially toxic compounds including mitomycin C (MMC), methyl methanesulfonate, ethidium bromide, H2O2, toluene, single-wall nanocarbon tubes (SWNCT), nano Au colloids (20 nm), pyrene, beno[a]pyrene, and UV light. These exposures revealed that the ADP1&#95;recA&#95;lux was able to detect both genotoxicity and cytoxicity, qualitatively and quantitatively. The optimal induction time of the ADP1&#95;recA&#95;lux bioreporter was 3 h, and the detection limits for MMC and benezo[a]pyrene were 1.5 nM and 0.4 nM, respectively. The ADP1&#95;recA&#95;lux was also used to detect toxicity of groundwater contaminated by a mixture of phenolic compounds, and the bioreporter toxicity detection was in a good agreement with chemical analysis. The optimized whole cell bioreporter ADP1&#95;recA&#95;lux could be valuable in providing a simple, rapid, stable, quantitative, robust, and costly efficient approach for the detection of toxicity in environmental samples.

This study investigated the use of electrokinetics in unsaturated soil to promote biodegradation of pentachlorophenol through increased contact between bacteria and contaminant. Soil microcosms, contaminated with approximately 100 mg kg(-1) pentachlorophenol (containing [(14)C]-PCP as a tracer), and inoculated with a specific pentachlorophenol-degrading bacterium (Sphingobium sp. UG30-1 x 10(8) cfu g(-1)) were subjected to constant and regularly reversed electric currents (10 mA). The former caused large pH and moisture content changes due to water electrolysis and electroosmotic effects, with subsequent negative impacts on biodegradation parameters including enzyme activity and contaminant mineralisation (as measured by (14)CO(2) evolution rate). The reversed field caused little change in pH and moisture content and led to more rapid contaminant mineralisation, lower soil contaminant concentration in the majority of the microcosms and increased soil enzyme activity (with the exception of soil immediately adjacent to the anode). The presence of an electric field, if suitably applied, may therefore enhance contaminant biodegradation in unsaturated soil.

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope ((13)C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 muM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.

In the United Kingdom, landfills are the primary anthropogenic source of methane emissions. Methanotrophic bacteria present in landfill biocovers can significantly reduce methane emissions via their capacity to oxidize up to 100% of the methane produced. Several biotic and abiotic parameters regulate methane oxidation in soil, such as oxygen, moisture, methane concentration and temperature. Earthworm-mediated bioturbation has been linked to an increase in methanotrophy in a landfill biocover soil (AC Singer et al., unpublished), but the mechanism of this trophic interaction remains unclear. The aims of this study were to determine the composition of the active methanotroph community and to investigate the interactions between earthworms and bacteria in this landfill biocover soil where the methane oxidation activity was significantly increased by the earthworms. Soil microcosms were incubated with 13C-CH4 and with or without earthworms. DNA and RNA were extracted to characterize the soil bacterial communities, with a particular emphasis on methanotroph populations, using phylogenetic (16S ribosomal RNA) and functional methane monooxygenase (pmoA and mmoX) gene probes, coupled with denaturing gradient-gel electrophoresis, clone libraries and pmoA microarray analyses. Stable isotope probing (SIP) using 13C-CH4 substrate allowed us to link microbial function with identity of bacteria via selective recovery of 'heavy' 13C-labelled DNA or RNA and to assess the effect of earthworms on the active methanotroph populations. Both types I and II methanotrophs actively oxidized methane in the landfill soil studied. Results suggested that the earthworm-mediated increase in methane oxidation rate in the landfill soil was more likely to be due to the stimulation of bacterial growth or activity than to substantial shifts in the methanotroph community structure. A Bacteroidetes-related bacterium was identified only in the active bacterial community of earthworm-incubated soil but its capacity to actually oxidize methane has to be proven.

A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.

Environmental contamination with recalcitrant toxic chemicals presents a serious and widespread problem to the functional capacity of soil. Soil bacteria play an essential role in ecosystem processes, such as nutrient cycling and decomposition; thus a decrease in their biomass and community diversity, resulting from exposure to toxic chemicals, negatively affects the functioning of soil. Plants provide the primary energy source to soil microorganisms and affect the size and composition of microbial communities, which in turn have an effect on vegetation dynamics. We have found that transgenic tobacco plants overexpressing a bacterial nitroreductase gene detoxify soil contaminated with the high explosive 2,4,6-trinitrotoluene (TNT), with a significantly increased microbial community biomass and metabolic activity in the rhizosphere of transgenic plants compared with wild type plants. This is the first report to demonstrate that transgenic plants engineered for the phytoremediation of organic pollutants can increase the functional and genetic diversity of the rhizosphere bacterial community in acutely polluted soil compared to wild type plants.

Aims: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater., Methods and Results: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal., Conclusion: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident., Significance and Impact of the Study: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.

In this study we examine the effects of polycyclic aromatic hydrocarbons (PAHs) on the ability of the hyperaccumulator plant Alyssum lesbiacum to phytoextract nickel from co-contaminated soil. Planted and unplanted mesocosms containing the contaminated soils were repeatedly amended with sorbitan trioleate, salicylic acid and histidine in various combinations to enhance the degradation of two PAHs (phenanthrene and chrysene) and increase nickel phytoextraction. Plant growth was negatively affected by PAHs; however, there was no significant effect on the phytoextraction of Ni per unit biomass of shoot. Exogenous histidine did not increase nickel phytoextraction, but the histidine-extractable fraction of soil nickel showed a high correlation with phytoextractable nickel. These results indicate that Alyssum lesbiacum might be effective in phytoextracting nickel from marginally PAH-contaminated soils. In addition, we provide evidence for the broader applicability of histidine for quantifying and predicting Ni phytoavailability in soils.

Single-cell Raman microspectroscopy has the potential to report on the whole-cell chemical composition of bacteria, reflecting metabolic status as well as growth history. This potential has been demonstrated through the discriminant functional analysis of Raman spectral profiles (RSP) obtained from the soil and plant-associated bacterium Pseudomonas fluorescens SBW25, grown in vitro using defined media, and in planta using 3-month-old sugar beets (Beta vulgaris var. Roberta). SBW25 in vitro RSP data showed significant variation between those cells grown on different amino acids, sugars, TCA cycle intermediates, rich King's B, and culture media derived from the sugar beet phytosphere. Raman analysis was also able to follow the transition of SBW25 starved of carbon over a period of days, and SBW25 in planta RSP data also showed variation with significant differences between bacteria recovered from soil and the rhizosphere. SBW25 whole-cell chemical composition, and therefore growth and metabolic history, could be interpreted by coanalyzing in vitro and in planta RSP data. SBW25 recovered from the phytosphere was found to be more similar to SBW25 grown in vitro on Fru or Asp, rather than on Glc or Arg, and quite dissimilar to that resulting from carbon starvation. This suggests that SBW25 growth in the phytosphere is generally neither carbon-catabolite-repressed nor carbon-limited. These findings demonstrate that the analysis of single-cell RSP can differentiate between isogenic populations of bacteria with different metabolic histories or after recovery from different parts of their natural environment. In addition, Raman analysis is also capable of providing biologically relevant biochemical inferences, which might then be tested to uncover the mechanistic basis (biochemical-metabolic-genetic) differentiating bacteria growing in complex environments and exposed to different conditions.

Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100mg kg(-1) oven dry soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close to the acidic anode where PCP accumulated (36d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and sustainability of this remediation strategy.

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in non-amended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.