PETR: A novel peristaltic mixed tubular bioreactor simulating human colonic conditions.

A novel bioreactor simulating human colonic conditions for in vitro cultivation of intestinal microbiota is presented. The PEristaltic mixed Tubular bioReactor (PETR) is modular designed and periodically kneaded to simulate intestinal peristalsis. The reactor is introduced, characterized from a bioprocess engineer's perspective and discussed in its ability to mimic colon conditions. PETR provides physiological temperature and appropriate anaerobic conditions, simulates intestinal peristalsis, and has a mean residence time of 32.8 ± 0.8 h comparable to the adult human colon. The single-tube design enables a time-constant and longitudinally progressive pH gradient from 5.5 to 7.0. Using a dialysis liquid containing high molecular weight polyethylene glycol, the integrated dialysis system efficiently absorbs short chain fatty acids (up to 60%) and water (on average 850 mL d-1 ). Cultivation of a typical gut bacterium (Bifidobacterium animalis) was performed to demonstrate the applicability for controlled microbiota cultivation. PETR is unique in combining simulation of the entire colon, peristaltic mixing, dialytic water and metabolite absorption, and a progressive pH gradient in a single-tube design. PETR is a further step to precise replication of colonic conditions in vitro for reliable and reproducible microbiota research, such as studying the effect of food compounds, prebiotics or probiotics, or the development and treatment of infections with enteric pathogens, but also for further medical applications such as drug delivery studies or to study the effect of drugs on and their degradation by the microbiota.

Core fluxome and metafluxome of lactic acid bacteria under simulated cocoa pulp fermentation conditions.

In the present work, simulated cocoa fermentation was investigated at the level of metabolic pathway fluxes (fluxome) of lactic acid bacteria (LAB), which are typically found in the microbial consortium known to convert nutrients from the cocoa pulp into organic acids. A comprehensive (13)C labeling approach allowed to quantify carbon fluxes during simulated cocoa fermentation by (i) parallel (13)C studies with [(13)C6]glucose, [1,2-(13)C2]glucose, and [(13)C6]fructose, respectively, (ii) gas chromatography-mass spectrometry (GC/MS) analysis of secreted acetate and lactate, (iii) stoichiometric profiling, and (iv) isotopomer modeling for flux calculation. The study of several strains of L. fermentum and L. plantarum revealed major differences in their fluxes. The L. fermentum strains channeled only a small amount (4 to 6%) of fructose into central metabolism, i.e., the phosphoketolase pathway, whereas only L. fermentum NCC 575 used fructose to form mannitol. In contrast, L. plantarum strains exhibited a high glycolytic flux. All strains differed in acetate flux, which originated from fractions of citrate (25 to 80%) and corresponding amounts of glucose and fructose. Subsequent, metafluxome studies with consortia of different L. fermentum and L. plantarum strains indicated a dominant (96%) contribution of L. fermentum NCC 575 to the overall flux in the microbial community, a scenario that was not observed for the other strains. This highlights the idea that individual LAB strains vary in their metabolic contribution to the overall fermentation process and opens up new routes toward streamlined starter cultures. L. fermentum NCC 575 might be one candidate due to its superior performance in flux activity.

Industrial biotechnology of Pseudomonas putida and related species.

Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.

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.

Robustness and plasticity of metabolic pathway flux among uropathogenic isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa 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 P. aeruginosa PAO1 and 17 clinical isolates. All P. aeruginosa 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. P. aeruginosa 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 P. aeruginosa metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.

Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions.

Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections.

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min(-1)), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105±16 µm up to 246±67 µm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.