Co-feeding glucose with either gluconate or galacturonate during clostridial fermentations provides metabolic fine-tuning capabilities.

Clostridium acetobutylicum ATCC 824 effectively utilizes a wide range of substrates to produce commodity chemicals. When grown on substrates of different oxidation states, the organism exhibits different recycling needs of reduced intracellular electron carrying co-factors. Ratios of substrates with different oxidation states were used to modulate the need to balance electron carriers and demonstrate fine-tuned control of metabolic output. Three different oxidized substrates were first fed singularly, then in different ratios to three different strains of Clostridium sp. Growth was most robust when fed glucose in exclusive fermentations. However, the use of the other two more oxidized substrates was strain-dependent in exclusive feeds. In glucose-galacturonate mixed fermentation, the main products (acetate and butyrate) were dependant on the ratios of the substrates. Exclusive fermentation on galacturonate was nearly homoacetic. Co-utilization of galacturonate and glucose was observed from the onset of fermentation in growth conditions using both substrates combined, with the proportion of galacturonate present dictating the amount of acetate produced. For all three strains, increasing galacturonate content (%) in a mixture of galacturonate and glucose from 0 to 50, and 100, resulted in a corresponding increase in the amount of acetate produced. For example, C. acetobutylicum increased from ~ 10 mM to ~ 17 mM, and then ~ 23 mM. No co-utilization was observed when galacturonate was replaced with gluconate in the two substrate co-feed.

Biofunctionalized Cellulose Nanofibrils Capable of Capture and Antiadhesion of Fimbriated Escherichia coli.

In this study, naturally derived cellulose nanofibrils (CNFs), a renewable and easily modified nanomaterial with low cytotoxicity, were rendered bioactive via one-step functionalization with mannopyranoside (CNFs-mannose) for use as a new glyconanomaterial platform for control of bacterial pathogenesis. The recognition affinity of the bioactive surfaces toward fimbriated Escherichia coli was assessed using genetically engineered strains as well as wild-type (WT) MG1655 bacteria. The results revealed high surface coverages of FimH+ (with overexpressed FimH) and WT bacteria on the films of CNFs-mannose due to specific interaction between prevalent mannose on nanofibrils and FimH receptors on E. coli fimbriae. The CNFs-mannose nanofibrils were capable of capturing E. coli from a liquid suspension, as demonstrated either by the nanofibril clusters or by the cellulose filter papers impregnated with CNFs-mannose. More importantly, CNFs-mannose efficiently inhibited adhesion of both FimH+ and WT E. coli to mannosylated surfaces even at a very low concentration, resulting in over 95% reduction of bacterial adhesion. Furthermore, the bioactive nanofibrils showed effective disruption of nonspecific binding of bacteria to abiotic surfaces in flow channel tests. These findings highlight the potential of cellulose nanofibrils as a biocompatible polyvalent nanoscale scaffold and exemplify sugar grafted nanofibrils as novel and effective tools in control of bacterial pathogenesis, bacterial removal, as well as in many other applications.

Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1-butanol tolerance using a metagenomic DNA library.

Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real-time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi-genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1-butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP-PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP-PCR enabled high efficiency library cloning (with no DNA shearing or end-polishing) and the inclusion un-culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent-tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real-time and is adaptable to high-throughput screening, making it relevant to metabolic engineering.

Near-real-time analysis of the phenotypic responses of Escherichia coli to 1-butanol exposure using Raman Spectroscopy.

Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.