Segregation of the Anodic Microbial Communities in a Microbial Fuel Cell Cascade.

Metabolic interactions within microbial communities are essential for the efficient degradation of complex organic compounds, and underpin natural phenomena driven by microorganisms, such as the recycling of carbon-, nitrogen-, and sulfur-containing molecules. These metabolic interactions ultimately determine the function, activity and stability of the community, and therefore their understanding would be essential to steer processes where microbial communities are involved. This is exploited in the design of microbial fuel cells (MFCs), bioelectrochemical devices that convert the chemical energy present in substrates into electrical energy through the metabolic activity of microorganisms, either single species or communities. In this work, we analyzed the evolution of the microbial community structure in a cascade of MFCs inoculated with an anaerobic microbial community and continuously fed with a complex medium. The analysis of the composition of the anodic communities revealed the establishment of different communities in the anodes of the hydraulically connected MFCs, with a decrease in the abundance of fermentative taxa and a concurrent increase in respiratory taxa along the cascade. The analysis of the metabolites in the anodic suspension showed a metabolic shift between the first and last MFC, confirming the segregation of the anodic communities. Those results suggest a metabolic interaction mechanism between the predominant fermentative bacteria at the first stages of the cascade and the anaerobic respiratory electrogenic population in the latter stages, which is reflected in the observed increase in power output. We show that our experimental system represents an ideal platform for optimization of processes where the degradation of complex substrates is involved, as well as a potential tool for the study of metabolic interactions in complex microbial communities.

A novel finding that Streptomyces clavuligerus can produce the antibiotic clavulanic acid using olive oil as a sole carbon source.

AIMS: This study aims to establish whether commercially available food oils can be used by Streptomyces clavuligerus as sole carbon sources for growth and clavulanic acid production. METHODS AND RESULTS: Batch cultures in bioreactors showed that Strep. clavuligerus growth and clavulanic acid yields in a P-limited medium containing 0.6% (v/v) olive oil were respectively 2.5- and 2.6-fold higher than in a glycerol-containing medium used as control. Glycerol- and olive oil-grown cells present different macromolecular composition, particularly lipid and protein content. CONCLUSIONS: Streptomyces clavuligerus uses olive oil as the sole carbon and energy source for growth and clavulanic acid production. Yields and production rates in olive oil are comparable to those reported for oil-containing complex media. Differences in yields and in the macromolecular composition indicate that different metabolic pathways convert substrate into product. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of oils being used as the sole carbon source by Strep. clavuligerus. Apart from economic benefits, interesting questions are raised about Strep. clavuligerus physiology. Defined culture media allow physiological studies to be performed in the absence of interference by other compounds. Understanding how Strep. clavuligerus catabolises oils may have an economic impact in clavulanic acid production.

Transcriptomic analysis identifies growth rate modulation as a component of the adaptation of mycobacteria to survival inside the macrophage.

The adaptation of the tubercle bacillus to the host environment is likely to involve a complex set of gene regulatory events and physiological switches in response to environmental signals. In order to deconstruct the physiological state of Mycobacterium tuberculosis in vivo, we used a chemostat model to study a single aspect of the organism's in vivo state, slow growth. Mycobacterium bovis BCG was cultivated at high and low growth rates in a carbon-limited chemostat, and transcriptomic analysis was performed to identify the gene regulation events associated with slow growth. The results demonstrated that slow growth was associated with the induction of expression of several genes of the dormancy survival regulon. There was also a striking overlap between the transcriptomic profile of BCG in the chemostat model and the response of M. tuberculosis to growth in the macrophage, implying that a significant component of the response of the pathogen to the macrophage environment is the response to slow growth in carbon-limited conditions. This demonstrated the importance of adaptation to a low growth rate to the virulence strategy of M. tuberculosis and also the value of the chemostat model for deconstructing components of the in vivo state of this important pathogen.

Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism.

An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D = 0.03 h(-1)) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D = 0.01 h(-1)) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence.

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.

Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris.

Simple cyclic fed-batch culture (cfbc), consisting of a constant medium feed with periodic withdrawals of culture, resulted in a product yield (13.4 mg protein per gram biomass) similar to that obtained using the complex multiphase industrial production strategy (13.7 mg protein per gram biomass). In cfbc, productivity was ultimately limited by the rate at which the cells could assimilate methanol. Glycerol was inhibitory to growth at high concentrations. However, product yield continued to increase as the glycerol concentration was increased. In chemostat culture, dissolved oxygen concentration influenced product yield independently of any detectable influence on cell growth.

Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans.

The carbon metabolism of derivatives of Streptomyces lividans growing under phosphate limitation in chemostat cultures and producing the antibiotics actinorhodin and undecylprodigiosin was investigated. By applying metabolic flux analysis to a stoichiometric model, the relationship between antibiotic production, biomass accumulation, and carbon flux through the major carbon metabolic pathways (the Embden Meyerhoff Parnas and pentose-phosphate pathways) was analyzed. Distribution of carbon flux through the catabolic pathways was shown to be dependent on growth rate, as well as on the carbon and energy source (glucose or gluconate) used. Increasing growth rates promoted an increase in the flux of carbon through glycolysis and the pentose-phosphate pathway. The synthesis of both actinorhodin and undecylprodigiosin was found to be inversely related to flux through the pentose-phosphate pathway.

The Vancomycin biosynthetic pathway is induced in oxygen-limited Amycolatopsis orientalis (ATCC 19795) cultures that do not produce antibiotic.

Vancomycin biosynthetic capacity was stimulated in oxygen-limited bioreactor culture (6.4% DOT) of Amycolatopsis orientalis, coinciding with a down-regulation of the culture growth and protein synthesis rates. However no Vancomycin was detected. ATP production was similar in oxygen-limited and oxygen-sufficient cultures (400-150 µmole(ATP). g(biomass)(-1)). Our findings suggest a critical requirement for oxygen flux through the Vancomycin biosynthetic pathway, which is only met under nutrient-limited conditions in which oxygen is in excess.

Current topics in signal transduction in bacteria.

Among the signal transfer systems in bacteria two types predominate: two-component regulatory systems and quorum sensing systems. Both types of system can mediate signal transfer across the bacterial cell envelope; however, the signalling molecule typically is not taken up into the cells in the former type of system, whereas it usually is in the latter. The Two-component systems include the recently described (eukaryotic) phosphorelay systems; quorum sensing systems can be based upon autoinducers of the N-acylated homoserine lactones, and on autoinducers of a peptidic nature. A single bacterial cell contains many signalling modules that primarily operate in parallel. This may give rise to neural-network behaviour. Recently, however, for both types of basic signal transfer modules, it has been demonstrated that they also can be organised in series (i.e. in a hierarchical order). Besides their hierarchical position in the signal transduction network of the cell, the spatial distribution of individual signalling modules may also be an important factor in their efficiency in signal transfer. Many challenges lie hidden in future work to understand these signal transfer processes in more detail. These are discussed here, with emphasis on the mutual interactions between different signal transfer processes. Successful contributions to this work will require rigorous mathematical modelling of the performance of signal transduction components, and -networks, as well as studies on light-sensing signal transduction systems, because of the unsurpassed time resolution obtainable in those latter systems, the opportunity to apply repeated reproducible stimuli, etc. The increased understanding of bacterial behaviour that already has resulted--and may further result--from these studies, can be used to fine-tune the beneficial activities of bacteria and/or more efficiently inhibit their deleterious ones.

Bacillus thuringiensis growth and toxicity. Basic and applied considerations.

Despite the known importance of the composition of culture media and culture conditions on Bacillus thuringiensis growth and toxicity, very few reviews are concerned with this subject. This article reviews some aspects of the microbiology of Bacillus thuringiensis, and how toxicity is affected by the composition of growth media and bioreactor operation.

Bacillus thuringiensis growth, sporulation and δ-endotoxin production in oxygen limited and non-limited cultures.

The production of crystals and spores ofBacillus thuringiensis var.israelensis was studied under different aeration conditions. The results with 4 l batch cultures showed that for O2 non-limited, cultures cell yield, toxin production and spore count were constant for all oxygen transfer rates (OTR). Under O2 limitation, °-endotoxin concentrations and spore counts were lower than those obtained in non-limited cultures. In addition, δ-endotoxin yields diminished under O2 limitation, suggesting that the toxin synthesis mechanism could have been affected.

Organic and inorganic nitrogen source ratio effects onBacillus thuringiensis var.israelensis delta-endotoxin production.

The influence of different organic and inorganic nitrogen source combinations and C∶N ratios was studied in connection with growth and protein production ofBacillus thuringiensis var.israelensis. Protein production was assumed to be proportional to delta-endotoxin production. Delta-endotoxin concentration increased when media were supplemented with (NH4)2SO4, but the delta-endotoxin: biomass dry weight ratio was unaffected by different C∶N ratios. Organic nitrogen source, yeast extract, could be partially replaced by (NH4)2SO4 with a significant increase in delta-endotoxin production.