Yuan-Yuan Cheng

Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin

Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32.

A photometric high-throughput method for identification of electrochemically active bacteria using a WO3 nanocluster probe.

Electrochemically active bacteria (EAB) are ubiquitous in environment and have important application in the fields of biogeochemistry, environment, microbiology and bioenergy. However, rapid and sensitive methods for EAB identification and evaluation of their extracellular electron transfer ability are still lacking. Herein we report a novel photometric method for visual detection of EAB by using an electrochromic material, WO3 nanoclusters, as the probe. This method allowed a rapid identification of EAB within 5 min and a quantitative evaluation of their extracellular electron transfer abilities. In addition, it was also successfully applied for isolation of EAB from environmental samples. Attributed to its rapidness, high reliability, easy operation and low cost, this method has high potential for practical implementation of EAB detection and investigations.

A plate-based electrochromic approach for the high-throughput detection of electrochemically active bacteria

Electrochemically active bacteria (EAB) have the ability to transfer electrons to electron acceptors located outside the cell, and they are widely present in diverse environments. In spite of their important roles in geochemical cycles, environmental remediation and electricity generation, so far, only a limited number and types of EAB have been isolated and characterized. Thus, effective and rapid EAB identification methods are highly desirable. In this protocol, we describe a photometric protocol for the visualization and high-throughput identification and isolation of EAB. The protocol relies on the fast electron acquisition and color change ability of an electrochromic material, namely a tungsten trioxide (WO3) nanorod assembly. The extracellular electron transfer (EET) from EAB to the WO3 nanorod assembly probe is accompanied by a bioelectrochromic reaction made evident by the color change of the probe. This protocol enables researchers to rapidly identify EAB and evaluate their EET ability either qualitatively with the naked eye or quantitatively by image analysis. We have also successfully used this protocol to isolate EAB from environmental samples. The time needed to complete this protocol is ∼2 d, with the actual EAB identification process taking about 5 min.

Photoautotrophic cathodic oxygen reduction catalyzed by a green alga, Chlamydomonas reinhardtii

Biofuel cells (BFCs) use enzymes and microbial cells to produce energy from bioavailable substrates and treat various wastewaters, and cathodic oxygen reduction is a key factor governing the efficiency of BFCs. In this study, we demonstrated that a green alga, Chlamydomonas reinhardtii, could directly mediate oxygen reduction. Cyclic voltammogram analysis revealed that the C. reinhardtii biofilm formed on a solid electrode was responsible for oxygen reduction without dosing of electron mediator. Furthermore, 4‐electron oxygen reduction pathway was found in this self‐sustained, light‐responded BFC. The results of this study could expand our understanding and viewpoints of biocathode catalysis, which is essential for novel catalyst design and development for BFCs. Biotechnol. Bioeng. 2013; 110: 173–179.

Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors.

Quorum sensing (QS) and quorum quenching (QQ) are two antagonistic processes coexisting in various bacterial communities in bioreactors, e.g., activated sludge for biological wastewater treatment. Although QS signal molecules are detected in activated sludge reactors and known to affect sludge properties and reactor performance, there has been no direct evidence to prove the endogenous existence of QQ effects in activated sludge. In this study, for the first time, acyl homoserine lactones-degrading enzymatic activity, a typical QQ effect, was discovered in activated sludge and found to considerably affect the QS detection results. The coexistence of QS and QQ bacteria in activated sludge was further confirmed by bacterial screening and denaturing gradient gel electrophoresis analysis. The method developed in this study could also be used to evaluate QQ activities in bioreactors, and a possible way is provided to tune bioreactor performance through balancing the QS and QQ processes.

Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization

Autoinducer-2 (AI-2), as a small-molecular-weight organic molecule secreted and perceived by various bacteria, enables intra- and inter-species communications. Quantitative determination of AI-2 is essential for exploring the bacterial AI-2-related physiological and biochemical processes. However, current strategies for sensitive detection of AI-2 require sophisticated instruments and complicated procedures. In this work, on the basis of the derivatization of AI-2 with 2,3-diaminonaphthalene, a simple, sensitive and cost-effective high-performance liquid chromatography with fluorescence detector (HPLC-FLD) method is developed for the quantitative detection of AI-2. Under the optimized conditions, this method had a broad linear range of 10-14,000 ng/ml (R(2)=0.9999), and a low detection limit of 1.0 ng/ml. Furthermore, the effectiveness of this approach was further validated through measuring the AI-2 concentrations in the cell-free culture supernatants of both Escherichia coli and Vibrio harveyi.

Promotion of Iron Oxide Reduction and Extracellular Electron Transfer in Shewanella oneidensis by DMSO

The dissimilatory metal reducing bacterium Shewanella oneidensis MR-1, known for its capacity of reducing iron and manganese oxides, has great environmental impacts. The iron oxides reducing process is affected by the coexistence of alternative electron acceptors in the environment, while investigation into it is limited so far. In this work, the impact of dimethyl sulphoxide (DMSO), a ubiquitous chemical in marine environment, on the reduction of hydrous ferric oxide (HFO) by S. oneidensis MR-1 was investigated. Results show that DMSO promoted HFO reduction by both wild type and ΔdmsE, but had no effect on the HFO reduction by ΔdmsB, indicating that such a promotion was dependent on the DMSO respiration. With the DMSO dosing, the levels of extracellular flavins and omcA expression were significantly increased in WT and further increased in ΔdmsE. Bioelectrochemical analysis show that DMSO also promoted the extracellular electron transfer of WT and ΔdmsE. These results demonstrate that DMSO could stimulate the HFO reduction through metabolic and genetic regulation in S. oneidensis MR-1, rather than compete for electrons with HFO. This may provide a potential respiratory pathway to enhance the microbial electron flows for environmental and engineering applications.

Redox properties of extracellular polymeric substances (EPS) from electroactive bacteria.

Although the capacity for electroactive bacteria to convert environmental metallic minerals and organic pollutants is well known, the role of the redox properties of microbial extracellular polymeric substances (EPS) in this process is poorly understood. In this work, the redox properties of EPS from two widely present electroactive bacterial strains (Shewanella oneidensis and Pseudomonas putida) were explored. Electrochemical analysis demonstrates that the EPS extracted from the two strains exhibited redox properties. Spectroelectrochemical and protein electrophoresis analyses indicate that the extracted EPS from S. oneidensis and P. putida contained heme-binding proteins, which were identified as the possible redox components in the EPS. The results of heme-mediated behavior of EPS may provide an insight into the important roles of EPS in electroactive bacteria to maximize their redox capability for biogeochemical cycling, environmental bioremediation and wastewater treatment.

Roles of 3,3',4',5-tetrachlorosalicylanilide in regulating extracellular electron transfer of Shewanella oneidensis MR-1.

Microbial extracellular electron transfer (EET) is critically involved in many pollutant conversion processes in both natural environment and engineered bioelectrochemical systems (BES), but typically with limited efficiency and poor controllability. In this study, we discover an important role of uncouplers in affecting the microbial energy metabolism and EET. Dose of lower-concentration 3,3′,4′,5-tetrachlorosalicylanilide (TCS) in the anolyte promoted the current generation and substrate degradation of an MFC inoculated with Shewanella oneidensis MR-1. However, higher TCS dosage caused obvious microbial inhibition. Our results suggest a previously unknown role of uncouplers in regulating the microbial EET. In addition, the underlying mechanisms of such processes are investigated. This work broadens our view about the EET behaviors of microorganisms in real water environment where uncouplers are usually present, and suggests a possible new approach to regulate microbial EET in BES.

Estimates of abundance and diversity of Shewanella genus in natural and engineered aqueous environments with newly designed primers

Shewanella species have a diverse respiratory ability and wide distribution in environments and play an important role in bioremediation and the biogeochemical cycles of elements. Primers with more accuracy and broader coverage are required with consideration of the increasing number of Shewanella species and evaluation of their roles in various environments. In this work, a new primer set of 640F/815R was developed to quantify the abundance of Shewanella species in natural and engineered environments. In silico tools for primer evaluation, quantitative polymerase chain reaction (qPCR) and clone library results showed that 640F/815R had a higher specificity and coverage than the previous primers in quantitative analysis of Shewanella. Another newly developed primer pair of 211F/815cR was also adopted to analyze the Shewanella diversity and demonstrated to be the best candidate in terms of specificity and coverage. We detected more Shewanella-related species in freshwater environments and found them to be substantially different from those in marine environments. Abundance and diversity of Shewanella species in wastewater treatment plants were largely affected by the process and operating conditions. Overall, this study suggests that investigations of abundance and diversity of Shewanella in various environments are of great importance to evaluate their ecophysiology and potential ecological roles.