Quantification of microaerobic growth of Geobacter sulfurreducens

Geobacter sulfurreducens was originally considered a strict anaerobe. However, this bacterium was later shown to not only tolerate exposure to oxygen but also to use it as terminal electron acceptor. Research performed has so far only revealed the general ability of G. sulfurreducens to reduce oxygen, but the oxygen uptake rate has not been quantified yet, nor has evidence been provided as to how the bacterium achieves oxygen reduction. Therefore, microaerobic growth of G. sulfurreducens was investigated here with better defined operating conditions as previously performed and a transcriptome analysis was performed to elucidate possible metabolic mechanisms important for oxygen reduction in G. sulfurreducens. The investigations revealed that cell growth with oxygen is possible to the same extent as with fumarate if the maximum specific oxygen uptake rate (sOUR) of 95 mgO2 gCDW-1 h-1 is not surpassed. Hereby, the entire amount of introduced oxygen is reduced. When oxygen concentrations are too high, cell growth is completely inhibited and there is no partial oxygen consumption. Transcriptome analysis suggests a menaquinol oxidase to be the enzyme responsible for oxygen reduction. Transcriptome analysis has further revealed three different survival strategies, depending on the oxygen concentration present. When prompted with small amounts of oxygen, G. sulfurreducens will try to escape the microaerobic area; if oxygen concentrations are higher, cells will focus on rapid and complete oxygen reduction coupled to cell growth; and ultimately cells will form protective layers if a complete reduction becomes impossible. The results presented here have important implications for understanding how G. sulfurreducens survives exposure to oxygen

Long-Term Behavior of Defined Mixed Cultures of Geobacter sulfurreducens and Shewanella oneidensis in Bioelectrochemical Systems

This work aims to investigate the long-term behavior of interactions of electrochemically active bacteria in bioelectrochemical systems. The electrochemical performance and biofilm characteristics of pure cultures of Geobacter sulfurreducens and Shewanella oneidensis are being compared to a defined mixed culture of both organisms. While S. oneidensis pure cultures did not form cohesive and stable biofilms on graphite anodes and only yielded 0.034 ± 0.011 mA/cm2 as maximum current density by feeding of each 5 mM lactate and acetate, G. sulfurreducens pure cultures formed 69 μm thick, area-wide biofilms with 10 mM acetate as initial substrate concentration and yielded a current of 0.39 ± 0.09 mA/cm2. Compared to the latter, a defined mixed culture of both species was able to yield 38% higher maximum current densities of 0.54 ± 0.07 mA/cm2 with each 5 mM lactate and acetate. This increase in current density was associated with a likewise increased thickness of the anodic biofilm to approximately 93 μm. It was further investigated whether a sessile incorporation of S. oneidensis into the mixed culture biofilm, which has been reported previously for short-term experiments, is long-term stable. The results demonstrate that S. oneidensis was not stably incorporated into the biofilm; rather, the planktonic presence of S. oneidensis has a positive effect on the biofilm growth of G. sulfurreducens and thus on current production

Long-Term Behavior of Defined Mixed Cultures of Geobacter sulfurreducens and Shewanella oneidensis in Bioelectrochemical Systems

This work aims to investigate the long-term behavior of interactions of electrochemically active bacteria in bioelectrochemical systems. The electrochemical performance and biofilm characteristics of pure cultures of Geobacter sulfurreducens and Shewanella oneidensis are being compared to a defined mixed culture of both organisms. While S. oneidensis pure cultures did not form cohesive and stable biofilms on graphite anodes and only yielded 0.034 ± 0.011 mA/cm2 as maximum current density by feeding of each 5 mM lactate and acetate, G. sulfurreducens pure cultures formed 69 μm thick, area-wide biofilms with 10 mM acetate as initial substrate concentration and yielded a current of 0.39 ± 0.09 mA/cm2. Compared to the latter, a defined mixed culture of both species was able to yield 38% higher maximum current densities of 0.54 ± 0.07 mA/cm2 with each 5 mM lactate and acetate. This increase in current density was associated with a likewise increased thickness of the anodic biofilm to approximately 93 μm. It was further investigated whether a sessile incorporation of S. oneidensis into the mixed culture biofilm, which has been reported previously for short-term experiments, is long-term stable. The results demonstrate that S. oneidensis was not stably incorporated into the biofilm; rather, the planktonic presence of S. oneidensis has a positive effect on the biofilm growth of G. sulfurreducens and thus on current production

Production and Characterization of Bacterial Cellulose Separators for Nickel-Zinc Batteries

The need for energy-storing technologies with lower environmental impact than Li-ion batteries but similar power metrics has revived research in Zn-based battery chemistries. The application of bio-based materials as a replacement for current components can additionally contribute to an improved sustainability of Zn battery systems. For that reason, bacterial cellulose (BC) was investigated as separator material in Ni-Zn batteries. Following the biotechnological production of BC, the biopolymer was purified, and differently shaped separators were generated while surveying the alterations of its crystalline structure via X-ray diffraction measurements during the whole manufacturing process. A decrease in crystallinity and a partial change of the BC crystal allomorph type Iα to II was determined upon soaking in electrolyte. Electrolyte uptake was found to be accompanied by dimensional shrinkage and swelling, which was associated with partial decrystallization and hydration of the amorphous content. The separator selectivity for hydroxide and zincate ions was higher for BC-based separators compared to commercial glass-fiber (GF) or polyolefin separators as estimated from the obtained diffusion coefficients. Electrochemical cycling showed good C-rate capability of cells based on BC and GF separators, whereas cell aging was pronounced in both cases due to Zn migration and anode passivation. Lower electrolyte retention was concluded as major reason for faster capacity fading due to zincate supersaturation within the BC separator. However, combining a dense BC separator with low zincate permeability with a porous one as electrolyte reservoir reduced ZnO accumulation within the separator and improved cycling stability, hence showing potentials for separator adjustment

Robustness and Plasticity of Metabolic Pathway Flux among Uropathogenic Isolates of <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa</i> is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using ¹³C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain <i>P. aeruginosa</i> PAO1 and 17 clinical isolates. All <i>P. aeruginosa</i> strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. <i>P. aeruginosa</i> recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that <i>P. aeruginosa</i> metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.</p></div

Metabolic properties of uropathogenic <i>P. aeruginosa</i> isolates and <i>P. aeruginosa</i> PAO1.

<p>Integration of flux phenotypes into the theoretical flux space on basis of anabolism (considering the biomass yield coefficient Y_x/s, given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>) and of NADPH metabolism (considering the NADPH balance as described below). In order to evaluate individual flux phenotypes, experimental values for biomass yield and apparent NADPH excess, respectively, (black squares) are integrated into the flux space created by elementary flux mode analysis (grey squares). Most strains localize close to the growth optimum. (A), Correlation of biomass formation and NADPH metabolism (B), contribution of isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase to supply of reducing power (C), correlation of biomass formation and flux through the oxidative pentose phosphate pathway (D). NADPH metabolism was inspected by balancing of the redox cofactor. For this purpose, the NADPH formation flux by concerted action of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and malic enzyme was balanced with the NADPH consumption flux for anabolism. For all strains, NADPH formation was higher than consumption. The resulting apparent excess flux of NADPH supply is presented here. The full flux data sets for all strains are presented in Supporting Information. The flux values are normalized to the corresponding glucose uptake rate for each strain (set to 100%).</p

In vivo carbon flux distributions in central metabolism of uropathogenic <i>P. aeruginosa</i> isolates during growth on glucose.

<p>Flux is given as average flux of all strains and is expressed as a molar percentage of the average glucose uptake rate of all strains (8.6 mmol g⁻¹ h⁻¹, calculated from the individual rates in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone-0088368-t001" target="_blank">Table 1</a>). Open arrows indicate flux toward biomass. For reversible reactions, the direction of net flux is indicated by a dashed arrow. The errors given for each flux reflect the corresponding 90% confidence intervals. The full flux data sets are presented in Supporting Information. Metabolic and isotopic steady states are ensured by constant stoichiometry, kinetics, and the constant ¹³C-labeling patterns of recruited metabolites during cultivation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s001" target="_blank">Figure S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s005" target="_blank">Figure S5</a>). Abbreviations are as follows: Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), glyoxylate (Gly) shunt, and tricarboxylic acid (TCA) cycle.</p

Metabolic adaption of pathogenic <i>P. aeruginosa</i>.

<p>The network representation integrates changes of flux determined for uropathogenic isolates and <i>P. aeruginosa</i> PAO1 (this work) and changes in transcription transcript level previously determined for successive isolates of <i>P. aeruginosa</i> from patients with chronic cystic fibrosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368-Hoboth1" target="_blank">[11]</a>. The variation of metabolic flux among strains is indicated by color (black = conserved, green = changed). Genes with changed or unchanged transcript levels are shown in green or black, respectively. Abbreviations are as follows: Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), glyoxylate (Gly) shunt, and tricarboxylic acid (TCA) cycle.</p

Metabolic flux in <i>P. aeruginosa</i> PAO1 and select gram-negative and gram-positive bacteria.

<p>Flux refers to the Entner-Doudoroff pathway (EDP), Embden-Meyerhof-Parnas pathway (EMPP), pentose phosphate pathway (PPP), and glyoxylate (Gly) shunt and reflects relative values normalized to the corresponding glucose uptake rate, defined as 100%.</p><p>* functional pathway not encoded.</p><p>** n.d. = not determined.</p

Kinetics and stoichiometry of glucose-grown <i>P. aeruginosa</i> PAO1 and uropathogenic <i>P. aeruginosa</i> isolates obtained from patients with catheter-associated urinary tract infection or urinary tract infections.

<p>The corresponding cultivation profiles for all strains are provided in Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088368#pone.0088368.s003" target="_blank">Figure S3</a>). Values indicate means and standard deviations of three biological replicates. By-products in the supernatants were not detected; concentrations were below the detection limit (1 µM for amino acids and 10 µM for organic acids).</p