Metabolites produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. enable a Gram-positive bacterium to achieve extracellular electron transfer

Previous studies revealed the abundance of Pseudomonas sp. in the microbial community of a microbial fuel cell (MFC). These bacteria can transfer electrons to the electrode via self-produced phenazine-based mediators. A MFC fed with acetate where several Pseudomonas sp. were present was found to be rich in a Gram-positive bacterium, identified as Brevibacillus sp. PTH1. Remarkably, MFCs operated with only the Brevibacillus strain in their anodes had poor electricity generation. Upon replacement of the anodic aqueous part of Brevibacillus containing MFCs with the cell-free anodic supernatants of MFCs operated with Pseudomonas sp. CMR12a, a strain producing considerable amounts of phenazine-1-carboxamide (PCN) and biosurfactants, the electricity generation was improved significantly. Supernatants of Pseudomonas sp. CMR12a_Reg, a regulatory mutant lacking the ability to produce PCN, had no similar improvement effect. Purified PCN, together with rhamnolipids as biosurfactants (1 mg L⁻¹), could clearly improve electricity generation by Brevibacillus sp. PTH1, as well as enable this bacterium to oxidize acetate with concomitant reduction of ferric iron, supplied as goethite (FeOOH). When added alone, PCN had no observable effects on Brevibacillus’ electron transfer. This work demonstrates that metabolites produced by Pseudomonas sp. enable Gram-positive bacteria to achieve extracellular electron transfer. Possibly, this bacterial interaction is a key process in the anodic electron transfer of a MFC, enabling Brevibacillus sp. PTH1 to achieve its dominance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The direct electrocatalysis of phenazine-1-carboxylic acid excreted by Pseudomonas alcaliphila under alkaline condition in microbial fuel cells

In this paper, we reported a kind of exoelectrogens, Pseudomonas alcaliphila (P. alcaliphila) strain MBR, which could excrete phenazine-1-carboxylic acid (PCA) to transfer electron under alkaline condition in microbial fuel cells (MFCs). The electrochemical activity of strain MBR and the extracellular electron transfer mechanism in MFCs were evaluated by cyclic voltammetry (CV) and electricity generation curve measurement. The results indicated a soluble mediator was the key factor for extracellular electron transfer of strain MBR under alkaline condition. The soluble mediator was PCA detected by gas chromatography-mass (GC-MS) analyses.     

Stable operation of microbial fuel cells at low temperatures (5–10 °C) with light exposure and its anodic microbial analysis

Performances of microbial fuel cells (MFCs) were studied at 5–10 and 25–30 °C. Results showed stable operation of the MFCs at low temperatures with only slight reductions of voltage and power generation (11 versus 14 % for double-chamber MFC, while 14 versus 21 % for single-chamber MFC, 1,000 Ω) compared to those at mesophilic temperatures. MFCs operated at low temperatures showed lower COD removal rates accompanied by higher coulombic efficiencies (CEs). PCR-DGGE analysis revealed that psychrotrophic microbes (mainly Arcobacter, Pseudomonas, and Geobacter) dominated on anodes of the MFCs at low temperatures. Interestingly, light-induced red substances appeared on anode of the MFCs operated at low temperature and were proven to be the main anodic microbes (Arcobacter and Pseudomonas). Co-existence of the aforementioned microbes could assist stable low-temperature operation of the MFCs. Cyclic voltammetry analysis supported the results of the CE and DGGE. Stable performance of MFCs at low temperatures might be achieved by the control of anodic bacteria.     

Correlation Between Antifungal Agent Phenazine-1-Carboxylic Acid and Pyoluteorin Biosynthesis in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18

Biosynthesis and secretion of two different types of antifungal compound [phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in Pseudomonas sp. M18] contribute to its suppression of soil-borne root pathogens. To better understand the correlation between two antifungal agents in secondary metabolism, a DNA fragment covering partial pltC and pltD coding sequences was obtained by screening the genomic library of Pseudomonas sp. M18. A mutant, M18T, was then constructed by insertion of the aacC1 gene cassette (encoding gentamycin resistance). With the same methods, one PCA biosynthetic gene cluster was insertionally inactivated and a mutant M18Z1 was created. The mutant strain M18T produces no Plt and the same amount of PCA in comparison with the wild-type strain M18. The mutant M18Z1, however, produces less PCA but more Plt than the wild-type strain M18. According to the documented data on strain M18, it is suggested that production of PCA is not influenced by Plt yield, but Plt biosynthesis is influenced by an alteration of PCA production.     

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.     

Biotechnological potential of a rhizosphere <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> strain producing phenazine-1-carboxylic acid and phenazine-1-carboxamide

Bacterial phenazine metabolites belong to a group of nitrogen-containing heterocyclic compounds with antimicrobial activities. In this study, a rhizosphere Pseudomonas aeruginosa strain PA1201 was isolated and identified through 16S rDNA sequence analysis and fatty acid profiling. PA1201 inhibited the growth of various pathogenic microorganisms, including Rhizotonia solani, Magnaporthe grisea, Fusarium graminearum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Staphylococcus aureus. High Performance Liquid Chromatography showed that PA1201 produced high levels of phenazine-1-carboxylic acid (PCA), a registered green fungicide ‘Shenqinmycin’ with the fermentation titers of 81.7 mg/L in pigment producing medium (PPM) and 926.9 mg/L in SCG medium containing soybean meal, corn steep liquor and glucose. In addition, PA1201 produced another antifungal metabolite, phenazine-1-carboxaminde (PCN), a derivative of PCA, with the fermentation titers of 18.1 and 489.5 mg/L in PPM and SCG medium respectively. To the best of our knowledge, PA1201 is a rhizosphere originating P. aeruginosa strain that congenitally produces the highest levels of PCA and PCN among currently reported P. aeruginosa isolates, which endows it great biotechnological potential to be transformed to a biopesticide-producing engineering strain.     

Role of phenazine-enzyme physiology for current generation in a bioelectrochemical system

Pseudomonas aeruginosa produces phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO), which aid its anaerobic survival by mediating electron transfer to distant oxygen. These natural secondary metabolites are being explored in biotechnology to mediate electron transfer to the anode of bioelectrochemical systems. A major challenge is that only a small fraction of electrons from microbial substrate conversion is recovered. It remained unclear whether phenazines can re-enter the cell and thus, if the electrons accessed by the phenazines arise mainly from cytoplasmic or periplasmic pathways. Here, we prove that the periplasmic glucose dehydrogenase (Gcd) of P. aeruginosa and P. putida is involved in the reduction of natural phenazines. PYO displayed a 60-fold faster enzymatic reduction than PCA; PCA was, however, more stable for long-term electron shuttling to the anode. Evaluation of a Gcd knockout and overexpression strain showed that up to 9% of the anodic current can be designated to this enzymatic reaction. We further assessed phenazine uptake with the aid of two molecular biosensors, which experimentally confirm the phenazines’ ability to re-enter the cytoplasm. These findings significantly advance the understanding of the (electro) physiology of phenazines for future tailoring of phenazine electron discharge in biotechnological applications.     

一株耐盐产电菌Shewanella algae E-1的分离及其产电特性分析

【背景】产电微生物的种类和电化学活性机制对微生物燃料电池的产电性能有着重要的影响。【目的】从海水中分离获得一株耐盐产电微生物,研究其产电特性并鉴定种属信息。【方法】以取自南海的海水为接种液启动并运行阳极液中含有不同盐浓度的微生物燃料电池,从富集的阳极生物膜上分离得到一株纯培养的微生物菌株,命名为E-1。通过接种于阳极液中添加不同盐浓度的微生物燃料电池中对其产电特性进行分析,并利用形态学观察、Biolog分析和16SrRNA基因序列比对相结合的方法进行种属鉴定。【结果】菌株E-1在无外源添加和外源添加6.6%NaCl条件下产生的功率密度分别为51.69 m W/m2和26.56 m W/m2,这与其良好的耐盐能力相关。菌株E-1被鉴定为海藻希瓦氏菌(Shewanella algae),表现出多样的底物利用能力,生长的温度范围为25-40°C,pH范围为5.0-10.0。【结论】这是首次对Shewanella algae种内微生物产电性能及其在微生物燃料电池中应用的报道,丰富了产电微生物的多样性,菌株E-1能够在较高盐浓度条件下表现出良好的产电性能,为微生物燃料电池在海水资源化处理方面的应用提供新的实验材料。     

假单胞菌M18 relA 突变株的构建及其对吩嗪-1-羧酸合成的调控

假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素两种抗生素的植物根际分离细菌。RelA催化合成的效应分子ppGpp能介导细菌因营养饥饿引起的应激反应。以M18菌株染色体DNA为模板,PCR扩增获得relA基因,通过庆大霉素抗性片段插入失活与同源重组技术,构建假单胞菌M18的relA突变菌株M18RAG。在PPM培养基中进行PCA发酵分析,发现突变菌株M18RAG的PCA产量显著升高,约为野生型菌株的1.5-2倍。relA基因反式互补实验以及phzA′-′lacZ翻译融合测定结果,均进一步证明了RelA对PCA生物合成及其基因表达具有抑制作用。     

Bioelectricity enhancement via overexpression of quorum sensing system in Pseudomonas aeruginosa-inoculated microbial fuel cells

Low electron transfer efficiency from bacteria to electrodes remains one of the major bottlenecks that limit industrial applications of microbial fuel cells (MFCs). Elucidating biological mechanism of the electron transfer processes is of great help in improving the efficiency of MFCs. Here, we reported that Pseudomonas aeruginosa could use different electron shuttles in a MFC under different quorum sensing (QS) expression patterns. An electron shuttle (rather than phenazines) with a high mid-point potential of 0.20 V (vs. Ag/AgCl–KCl saturated electrode) was found to be the dominating shuttle in a wild-type P. aeruginosa strain. Strikingly, upon genetic overexpression of rhl QS system in this wild-type strain, the electron shuttle was substituted by phenazines (pyocyanin and phenazine-1-carboxylate, with a low mid-point potential of −0.17 V and −0.28 V, respectively), which directly resulted in an increase of about 1.6 times of the maximum current of the rhl overexpressed strain over the wild-type strain. Our result implied that manipulating electron transfer pathways to improve MFCs’ efficiency could be achieved by rewiring gene regulatory circuits, thus synthetic biology strategies would be adopted.     

Differential Regulation of rsmA Gene on Biosynthesis of Pyoluteorin and Phenazine-1-carboxylic Acid in Pseudomonas sp. M18

Summary Plant growth promoting rhizobacteria (PGPR) strain Pseudomonas sp. M18 can produce two different types of antibiotics, pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). The global regulator RsmA is a translational repressor of secondary metabolism in many prokaryotes. A chromosomally rsmA inactivated mutant strain M18R was constructed to study the regulatory mechanism of Plt and PCA biosynthesis and enhancement of Plt or PCA production in Pseudomonas sp. M18. The accumulation of Plt increased six-fold over that of the wild-type strain whereas PCA production was not significantly affected in cultures of M18R. Plt production was inhibited completely but PCA biosynthesis was not altered after complementation with rsmA gene in trans in the strain of M18R. The differential activity of rsmA gene on these two operons was further confirmed by the analysis of β-galactosidase activities from translational phzA′-′lacZ and pltA′-′lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway and pltA is the first gene of the pyoluteorin biosynthesis pathway. The results indicate that RsmA can control Plt production negatively but not PCA production in M18, and show that the global regulator RsmA does not repress the biosynthesis of all secondary metabolites.     

Development of platforms for functional characterization and production of phenazines using a multi-chassis approach via CRAGE

Phenazines (Phzs), a family of chemicals with a phenazine backbone, are secondary metabolites with diverse properties such as antibacterial, anti-fungal, or anticancer activity. The core derivatives of phenazine, phenazine-1-carboxylic acid (PCA) and phenazine-1,6-dicarboxylic acid (PDC), are themselves precursors for various other derivatives. Recent advances in genome mining tools have enabled researchers to identify many biosynthetic gene clusters (BGCs) that might produce novel Phzs. To characterize the function of these BGCs efficiently, we performed modular construct assembly and subsequent multi-chassis heterologous expression using chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE allowed rapid integration of a PCA BGC into 23 diverse γ-proteobacteria species and allowed us to identify top PCA producers. We then used the top five chassis hosts to express four partially refactored PDC BGCs. A few of these platforms produced high levels of PDC. Specifically, Xenorhabdus doucetiae and Pseudomonas simiae produced PDC at a titer of 293 mg/L and 373 mg/L, respectively, in minimal media. These titers are significantly higher than those previously reported. Furthermore, selectivity toward PDC production over PCA production was improved by up to 9-fold. The results show that these strains are promising chassis for production of PCA, PDC, and their derivatives, as well as for function characterization of Phz BGCs identified via bioinformatics mining.     

Power generation from cellulose using mixed and pure cultures of cellulose-degrading bacteria in a microbial fuel cell

Microbial fuel cells (MFCs) have been used to generate electricity from various organic compounds such as acetate, glucose, and lactate. We demonstrate here that electricity can be produced in an MFC using cellulose as the electron donor source. Tests were conducted using two-chambered MFCs, the anode medium was inoculated with mixed or pure culture of cellulose-degrading bacteria Nocardiopsis sp. KNU (S strain) or Streptomyces enissocaesilis KNU (K strain), and the catholyte in the cathode compartment was 50mM ferricyanide as catholyte. The power density for the mixed culture was 0.188mW (188mW/m(2)) at a current of 0.5mA when 1g/L cellulose was used. However, the power density decreased as the cellulose concentration in the anode compartment decreased. The columbic efficiencies (CEs) ranged from 41.5 to 33.4%, corresponding to an initial cellulose concentration of 0.1-1.0g/L. For the pure culture, cellobioase enzyme was added to increase the conversion of cellulose to simple sugars, since electricity production is very low. The power densities for S and K strain pure cultures with cellobioase were 162mW/m(2) and 145mW/m(2), respectively. Cyclic voltammetry (CV) experiments showed the presence of peaks at 380, 500, and 720mV vs. Ag/AgCl for the mixed bacterial culture, indicating its electrochemical activity without an external mediator. Furthermore, this MFC system employs a unique microbial ecology in which both the electron donor (cellulose) and the electron acceptor (carbon paper) are insoluble.     

Isolation,Identification, and Degradation Characteristics of Phenazine-1-Carboxylic Acid–Degrading Strain <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. DP58

A phenazine-1-carboxylic acid (PCA)–degrading bacterium, strain DP58, was isolated from pimiento rhizosoil. Based on morphology, physiologic tests, 16S rDNA sequence, and phylogenetic characteristics, it was identified as Sphingomonas sp. The PCA-degradation experiments were conducted both in Luria-Bertani and inorganic salt medium at 28°C. The relationship between bacterium growth and PCA degradation suggested that strain DP58 could use PCA as the sole source of carbon and nitrogen and was able to completely degrade PCA in 40 hours. Newly isolated strain DP58 represents the first bacterium that can degrade PCA.     

Effect of electron mediators on current generation and fermentation in a microbial fuel cell

Effects of select electron mediators [9,10-anthraquinone-2,6-disulfonic acid disodium salt (AQDS), safranine O, resazurin, methylene blue, and humic acids] on metabolic end-products and current production from cellulose digestion by Clostridium cellulolyticum in microbial fuel cells (MFCs) were studied using capillary electrophoresis and traditional electrochemical techniques. Addition of the mediator resazurin greatly enhanced current production but did not appear to alter the examined fermentation end-products compared to MFCs with no mediator. Assays for lactate, acetate, and ethanol indicate that the presence of safranine O, methylene blue, and humic acids alters metabolite production in the MFC: safranine O decreased the examined metabolites, methylene blue increased lactate formation, and humic acids increased the examined metabolites. Mediator standard redox potentials (E ₀) reported in the literature do not coincide with redox potentials in MFCs due presumably to the electrolytic complexity of media that supports bacterial survival and growth. Current production in MFCs: (1) can be effected by the mediator redox potential while in the media, which may be significantly shifted from E ₀, and (2) depended on the ability of the mediator to access the bacterial electron source, which may be cytoplasmic. In addition, some electron mediators had significant effects on metabolic end-products and therefore the metabolism of the organism itself. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Single cell protein from bagasse pith by a mixed bacterial culture

A spontaneous association of Cellulomomas sp. with another bacterial strain was studied for its capabilities for single cell protein (SCP) production from bagasse pith. The associated strain was identified as Pseudomonas sp. and further characterized for its physiological properties. The effect of the initial proportions of both strains, the way of propagation, and the effect of pH on the growth of the mixed culture on bagasse pith was studied. Separate propagation of both strains before the fermentation step (“controlled mixed culture”), a range of proportions Cellulomonas-Pseudomonas from 4:1 to 100: 1, and pH 7.0, were found to be the most appropriate conditions of growth. A mutualistic symbiotic relationship was demonstrated to take place between both strains during the mixed growth on bagasse pith, the Cellulomonas supplying the carbon source (glucose produced from bagasse degradation) to the Pseudomonas, and the latter producing the vitamin supplements necessary for the Cellulomonas growth, allowing the growth of the mixed culture in a minimal medium, without any growth factor supplement. Fed-batch cultivation of the mixed culture on this substrate was successful, giving rise to high biomass production (19.4 g/l), thus increasing the productivity of the system. Due to its improved productivity, high biomass production, inexpensiveness of the culture medium, (without any vitamin supplement), and good stability, this culture presents economical advantages and constitutes an attractive choice for lignocellulosic substrate utilization.     

Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin

The microbial fuel cell (MFC), is a promising environmental biotechnology for harvesting electricity energy from organic wastes. However, low bacterial membrane permeability of electron shuttles is a limiting factor that restricts the electron shuttle‐mediated extracellular electron transfer (EET) from bacteria to electrodes, thus the electricity power output of MFCs. To this end, we heterologously expressed a porin protein OprF from Pseudomonas aeruginosa PAO1 into Escherichia coli, which dramatically increased its membrane permeability, delivering a much higher current output in MFCs than its parental strain (BL21). We found that the oprF‐expression strain showed more efficient EET than its parental strain. More strikingly, the enhanced membrane permeability also rendered the oprF‐expression strain an efficient usage of riboflavin as the electron shuttle, whereas its parental strain was incapable of. Our results substantiated that membrane permeability is crucial for the efficient EET, and indicated that the expression of synthetic porins could be an efficient strategy to enhance bioelectricity generation by microorganisms (including electrogenic bacteria) in MFCs. Biotechnol. Bioeng. 2013; 110: 408–416. © 2012 Wiley Periodicals, Inc.     

The significance of the initiation process parameters and reactor design for maximizing the efficiency of microbial fuel cells

Microbial fuel cells (MFCs) can be used for electricity generation via bioconversion of wastewater and organic waste substrates. MFCs also hold potential for production of certain chemicals, such as H₂ and H₂O₂. The studies of electricity generation in MFCs have mainly focused on the microbial community formation, substrate effect on the anode reaction, and the cathode’s catalytic properties. To improve the performance of MFCs, the initiation process requires more investigation because of its significant effect on the anodic biofilm formation. This review explores the factors which affect the initiation process, including inoculum, substrate, and reactor configuration. The key messages are that optimal performance of MFCs for electricity production requires (1) understanding of the electrogenic bacterial biofilm formation, (2) proper substrates at the initiation stage, (3) focus on operational conditions affecting initial biofilm formation, and (4) attention to the reactor configuration.     

Enhanced production of 2-hydroxyphenazine in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> GP72

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 μg ml⁻¹, compared with just 5 μg ml⁻¹ for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.