Engineering bionanoreactor in bacteria for efficient hydrogen production.

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.

Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology.

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.

Development of Aspirin-Inducible Biosensors in Escherichia coli and SimCells.

A simple aspirin-inducible system has been developed and characterized in Escherichia coli by employing the Psal 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, SalRr, can compete with the effector-bound form, SalRa, binding the Psal promoter and repressing gene transcription. The induction of the Psal 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 Psal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on the SalRr/SalRa 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 SalRr/SalRa 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/Psal 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 Psal 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/Psal regulation system in which SalRr competes with SalRa to bind the Psal promoter and affect gene transcription. The competitive binding model successfully predicted that weak SalR expression would significantly improve the inducible strength of the SalR/Psal 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/Psal 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.

Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean.

It is of great significance to understand CO2 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 CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for ß-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 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.

Phosphorus Depletion as a Green Alternative to Biocides for Controlling Biodegradation of Metalworking Fluids.

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 La2O3 to form LaPO4. Concentrations of La2O3 greater than 0.08%w (%w = weight percent) completely inhibited microbial growth (from 1.4 × 107 to 0 CFU/mL) and hindered biodegradation. Raman spectroscopy suggested that La2O3 converted intracellular phosphorus into LaPO4. The growth inhibition potentials of both 0.06%w La(NO3)3 and La2O3 were found to be superior to formaldehyde. The antimicrobial property of La2O3 (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.

A contribution of hydrogenotrophic methanogenesis to the biogenic coal bed methane reserves of Southern Qinshui Basin, China.

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.

Activation of peroxymonosulfate by Fe,N co-doped walnut shell biochar for the degradation of sulfamethoxazole: Performance and mechanisms.

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-Fe2O3-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-Fe2O3-BC/PMS (0.33 min-1) under the same conditions. This is mainly due to the formation of Fe3C 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 1O2 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.

Soil biotransformation of thiodiglycol, the hydrolysis product of mustard gas: understanding the factors governing remediation of mustard gas contaminated soil.

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.

Engineering artificial photosynthesis based on rhodopsin for CO2 fixation.

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 CO2 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 CO2. Overexpression of a carbonic anhydrase further enhances CO2 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.

Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation.

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO2 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 CO2 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 CO2 fixation. We demonstrated that this hybrid photo-electrosynthetic pathway can drive the engineered R. eutropha strain to grow using CO2 as the sole carbon source. In this system, a bioreactor with only two inputs, light and CO2, 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 CO2 into biomass with a maximum electron transfer efficiency of 20%.

Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells.

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 PnorV 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 PnorV 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 PnorV 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 PnorV 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 pPnorVß 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.

Bacterial wax synthesis.

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.

Genetic engineering biofilms in situ using ultrasound-mediated DNA delivery.

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 cm3 and 300 cm3 , 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.

Biorefinery of waste orange peel.

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.

Succinic acid production from orange peel and wheat straw by batch fermentations of Fibrobacter succinogenes S85.

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.

Elevated intracellular cyclic-di-GMP level in Shewanella oneidensis increases expression of c-type cytochromes.

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.

Anaerobic digestion of Crassulacean Acid Metabolism plants: Exploring alternative feedstocks for semi-arid lands.

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.

Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization.

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.

Ultrasound-mediated DNA transfer for bacteria.

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell-pilus-cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5alpha and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl(2), temperature of 22 degrees C, plasmid concentration of 0.8 ng/microl, P. putida UWC1 cell concentration of 2.5 x 10(9) CFU (colony forming unit)/ml and reaction volume of 500 microl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 +/- 2.3 x 10(-6) transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5alpha and P. fluorescens SBW25 with efficiencies of 1.16 +/- 0.13 x 10(-6) and 4.33 +/- 0.78 x 10(-6) transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.

Enhanced biodegradation of pentachlorophenol in unsaturated soil using reversed field electrokinetics.

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.