Characterization of electrochemically active bacteria utilizing a high-throughput voltage-based screening assay

Metal reduction assays are traditionally used to select and characterize electrochemically active bacteria (EAB) for use in microbial fuel cells (MFCs). However, correlating the ability of a microbe to generate current from an MFC to the reduction of metal oxides has not been definitively established in the literature. As these metal reduction assays may not be generally reliable, here we describe a four- to nine-well prototype high throughput voltage-based screening assay (VBSA) designed using MFC engineering principles and a universal cathode. Bacterial growth curves for Shewanella oneidensis strains DSP10 and MR-1 were generated directly from changes in open circuit voltage and current with five percent deviation calculated between each well. These growth curves exhibited a strong correlation with literature doubling times for Shewanella indicating that the VBSA can be used to monitor distinct fundamental properties of EAB life cycles. In addition, eight different organic electron donors (acetate, lactate, citrate, fructose, glucose, sucrose, soluble starch, and agar) were tested with S. oneidensis MR-1 in anode chambers exposed to air. Under oxygen exposure, we found that current was generated in direct response to additions of acetate, lactate, and glucose.     

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The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3′,5′-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP–cyclic adenosine 3′,5′-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP–CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.     

Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.     

Characterization of Heavy Metal Resistance of Metal-Reducing Shewanella Isolates From Marine Sediments

This study focuses on heavy metal resistance of marine, benthic Mn(IV)- and Fe(III)-reducing bacteria and their potential to mobilize heavy metals from sedimentary phases, as hydrous ferric oxides (HFO) and Mn(IV)-oxides (δ -MnO ₂ ). One isolate was obtained from enrichments of metal-polluted sediment with δ -MnO ₂ (strain MB4, 99% similarity to S. marisflavi), and two strains were isolated from enrichments on HFO (strain FB18 and FS8, 98 and 97% 16S rRNA gene similarity to Shewanella collwelliana). The 16S rRNA sequences similar to isolates MB4 and FS8 were detected previously in DGGE profiles and clone libraries of the original sediment samples. Toxicity tests under aerobic conditions showed that the latter two ceased growth at 150 μ M Cu, but strain MB4 and reference strain S. oneidensis MR1 were more tolerant to copper; growth with 150 μ M Cu reached 56–58 ± 0.1% of maximal optical density, OD_max, in control cultures. Similar experiments conducted under anaerobic conditions with fumarate indicated no significant change in copper tolerance in strain MB4 (66 ± 3% OD_max at 150 μ M). Biphasic experiments with δ -MnO ₂ -reduction followed by use of fumarate, furthermore indicated that the presence of manganese oxides decreased bio-availability of copper through sorption processes, thereby alleviating the toxicity of copper to strain MB4 to some extent. Scanning electron microscopic images showed the initial amorphous Mn(IV)-oxides and newly formed, highly crystalline, lemon-shaped, particles making up the precipitate that remained after microbial reduction. Concomitant electron dispersive x-ray spectrometry confirmed presence of copper in the initial sample, yet detected no copper in the precipitate after microbial reduction, indicating that the Mn(IV)-reducing Shewanella strain MB4 mobilized copper adsorbed to δ -MnO ₂ .     

The Influence of Carbon Source on the Products of Dissimilatory Iron Reduction

We have examined the influence of carbon source on both the rate of iron reduction and the mineralogy of the reduction products with Shewanella putrefaciens strain W3-18-1. When pyruvate was the carbon source, the secondary products were spherules composed of siderite. When uridine was used as the carbon source, the products were hexagonal plate-like structures identified as iron carbonate hydroxide hydrate, also known as carbonate green rust, a precursor to fougerite. When lactate was used as the carbon source, products were a mixture of iron carbonate hydroxide and magnetite. In terms of reaction stoichiometry, there were differences in the amount of acetate produced depending on the starting organic carbon source. Incubation with pyruvate produced a relatively large amount of acetate compared to incubation with uridine and lactate. There were also differences in the final pH of the cultures. While the pH for incubations with lactate started at 8.6 and ended between 8.0–8.3, the pH of cultures incubated with uridine was found to be almost a full unit lower at the conclusion of the experiment (～7.4). Solubility diagrams based on the chemistry found in our experiments predict that the production of Fe²⁺ _(aq) should always lead to the formation of magnetite. However, strain W3-18-1 produced different minerals depending on the carbon source utilized as the electron acceptor.     

厌氧条件下Shewanella oneidensis MR-1对2,4-二硝基甲苯的还原转化

【目的】研究Shewanella oneidensis MR-1厌氧生物转化2,4-二硝基甲苯(2,4-DNT)的能力、转化过程和影响因素。【方法】以乳酸钠为电子供体, 2,4-DNT为电子受体, S. oneidensis MR-1为降解菌, 黄素为胞外电子载体, 设立四个不同的对照体系并监测各体系在转化过程中2,4-DNT及其产物的动态变化。同时研究不同2,4-DNT浓度下细胞的生长情况, 以及不同黄素浓度下2,4-DNT的降解情况。【结果】S. oneidensis MR-1菌能够高效还原转化2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT), 并将其进一步还原为2,4-二氨基甲苯(2,4-DAT), 黄素能加速转化过程。【结论】S. oneidensis MR-1菌具备高效还原转化2,4-DNT的能力, 为实际环境中硝基苯污染的原位修复提供科学依据。     

一种新的异育银鲫病原———腐败希瓦氏菌

【目的】江苏盐城一家养殖场的异育银鲫暴发疾病,通过对病原进行研究,旨在为该病的防治提供理论依据和参考。【方法】从病鱼体表病灶和内脏中分离出优势菌株,经人工感染试验证实为病原菌。采用传统的形态、生理生化表型鉴定与16S rDNA序列分析相结合的方法确定菌株的分类地位。运用K-B琼脂法对病原菌株进行药物敏感性测定。【结果】综合菌株形态、生理生化表型以及16S rDNA序列分析的结果,确定该分离株为腐败希瓦氏菌(Shewanella putrefaciens)。回接感染试验证实腐败希瓦氏菌即是导致此次异育银鲫发病死亡的致病原,其半数致死量(LD50)为2.1×103cfu/g。该株腐败希瓦氏菌对吡哌酸、萘啶酸、氟哌酸、氟啶酸、氟苯尼考、利福平、美满霉素、氟罗沙星、恩诺沙星、复达欣、菌必治、先锋Ⅳ、罗红霉素和左氟沙星等抗生素敏感。【结论】首次报道了异育银鲫一种新的病原,说明腐败希瓦氏菌作为一种潜在的新病原也可能会对异育银鲫的养殖造成威胁。     

Mineral Influence on Microbial Survival During Carbon Sequestration

Geologic carbon sequestration involves the injection of supercritical carbon dioxide into deep saline aquifers. Some of the CO₂ dissolves into the brines, perturbing water chemistry and water-rock interactions, and impacting microbial habitat and survival. In this study 3 model organisms were tested for their ability to survive high pressures of CO₂ exposure in batch cultures: the gram-negative Shewanella oneidensis (SO) strain MR-1, the gram-positive Geobacillus stearothermophilus (GS), and the methanogenic archaeon Methanothermobacter thermoautitrophicus (MT). Results indicate that GS can survive the highest pressures of CO₂ for the longest periods of time while SO is the most sensitive to CO₂ toxicity. Survival was then evaluated for SO with various minerals and rocks representative of deep saline aquifers to determine if minerals enhanced survival. Cultures were exposed to 25 bar of CO₂ for 2 to 8 h and were plated for viable cell counts. Results show that biofilm formation on the mineral surface is important in protecting SO from the harmful effects of CO₂ with quartz sandstones providing the best protection. The release of toxic metals like Al or As from minerals such as clays and feldspars, in contrast, may enhance microbial death under CO₂ stress.