Showing papers by "Frederik Hammes published in 2005"
TL;DR: It was shown that the flow-cytometric enumeration method could be used to establish complete growth curves for a natural microbial consortium growing on AOC, and such kinetic data provide much clearer insight into the actual growth potential of a water.
Abstract: The concentration of easily assimilable organic carbon (AOC) largely determines the microbiological stability of drinking water. However, AOC determination is often neglected in practice due to the complex and tedious nature of the conventional bioassay. The three major drawbacks of the conventional method are (1) a long assay time of 9-12 days, (2) the use of a labor-intensive enumeration technique (plating on growth media), and (3) limited information supplied by the use of selected pure cultures (Pseudomonas fluorescens P-17 and Spirillum NOX) for measuring a complex pool of natural bioavailable carbon compounds. A new method is proposed here, in which plating was replaced with fluorescence staining of total nucleic acids combined with flow cytometry as a rapid and straightforward growth enumeration method. This approach also allowed for the detection of inactive and/or unculturable microorganisms. Hence, the conventionally used pure cultures were replaced in the new AOC assay with a natural microbial consortium. It was shown that the flow-cytometric enumeration method could be used to establish complete growth curves for a natural microbial consortium growing on AOC. Compared to the end-point measurements of the conventional method, such kinetic data provide much clearer insight into the actual growth potential of a water.
296 citations
TL;DR: This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed, and shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.
Abstract: The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.
31 citations