High Conversion of Methyl Acetate Hydrolysis in a Reactive Dividing Wall Column by Weakening the Self-Catalyzed Esterification Reaction

We show the superiority of reactive dividing wall column (RDWC) to the single reactive distillation (RD) column in improving the conversion of reactant, taking the hydrolysis of methyl acetate (MA) as example. It is difficult to achieve above 99% conversion for MA in a traditional reactive distillation column (TRDC) due to the existence of self-catalyzed methanol (MeOH)-acetic acid (HAc) esterification reaction in the column bottom. In this work, more than 99% conversion of MA hydrolysis was realized experimentally in an RDWC by separating MeOH from the hydrolysis mixture. The effects of several operation parameters on hydrolysis conversion were systematically investigated, including feedwater–MA mole ratio, heat duty, mole flow rate of feed MA, and vapor distribution. The simulation results by Aspen Plus showed that RDWC has several improvements in MA hydrolysis over TRDC, including lower energy consumption, lower water–MA mole ratio, and larger production capacity. With the increase in MA conversion, the superiorities became more obvious and contribute to the weaker self-catalyzed MeOH–HAc esterification reaction in the RDWC

Adhesion and immune induction by <i>E. coli</i> expressing or lacking curli and/or cellulose.

<p>(<b>A</b>) Adhesion to renal epithelial cells A498 was measured after 30 min. Curliated strains adhered significantly better to renal epithelial cells than strains lacking curli, independent of the expression of cellulose (^###<i>P</i><0.0001, <i>t</i>-test). Cellulose expression decreased the number of cell-associated bacteria in curliated (^***<i>P</i><0.0001, <i>t</i>-test) and non-curliated strains (^**<i>P</i> = 0.001, <i>t</i>-test). Results from three independent experiments in quadruplicates are shown as mean and standard deviation. Similar results were obtained for bladder epithelial cells (data not shown). (<b>B</b>) Induction of IL-8 was measured in culture supernatants of renal epithelial cells A498 stimulated with <i>E. coli</i> for 24 h. Curliated bacteria induced a significantly stronger IL-8 response than the mutants lacking curli in the presence (^##<i>P</i> = 0.001, <i>t</i>-test) and absence (^###<i>P</i><0.0001, <i>t</i>-test) of cellulose. In curliated bacteria, the expression of cellulose reduced IL-8 induction (^**<i>P</i> = 0.001, <i>t</i>-test). Results from three independent experiments in quadruplicate are shown as mean and standard deviation. Similar results were obtained for bladder epithelial cells (data not shown). (<b>C+D</b>) The phenotype of <i>E. coli</i> No. 12 could be restored by complementation of its mutants. Cellulose expression in strain B23 is inducible by aTc (left panels) and reduces adherence and IL-8 induction (^***<i>P</i><0.0001 and ^**<i>P</i> = 0.007, respectively, <i>t</i>-test). The curli subunits CsgA and CsgB are expressed from pWSK29-<i>csgBA</i> in strain WE11_1 (right panels) and increases adherence and IL-8 induction compared to WE11_1 carrying the vector pWSK29 only (^*<i>P</i> = 0.048 and ^**<i>P</i> = 0.003, respectively, <i>t</i>-test). Results in A498 cells are shown as mean and standard deviation. Data from three experiments in quadruplicate for adherence and from two experiments in triplicate for IL-8 induction are presented. (<b>E</b>) Mice were infected with the isogenic <i>E. coli</i> strains for 1 h. The curliated mutant were isolated from kidneys in significantly higher numbers than the double knockout (^#<i>P</i> = 0.026, Mann-Whitney U test). Individual values from <i>n</i> = 8–10 mice/group and medians are shown. (<b>F</b>) Levels of MIP-2 were measured in kidney tissue of infected mice after 16 h. In the absence of cellulose, the curliated mutant strain induced higher levels of MIP-2 compared to the non-curliated strain (^##<i>P</i> = 0.001, Mann-Whitney U test). Expression of cellulose reduced the induction of MIP-2 in the presence (^***<i>P</i><0.0001, Mann-Whitney U test) and absence of curli (^**<i>P</i> = 0.001, Mann-Whitney U test). Individual values from <i>n</i> = 5–10 mice/group and medians are shown.</p

LL-37 binds to recombinant polymerized CsgA and isolated wild-type curli.

<p>(<b>A</b>) Western blot analysis of supernatants after precipitation of LL-37 with curli. By adding polymeric CsgA (pol CsgA) or wild-type curli (wt curli) to a solution of 0.1 µM LL-37, the levels of LL-37 decreased in the supernatants after centrifugation. (<b>B</b>) Surface plasmon resonance. LL-37 exhibits a stronger association and lower dissociation rates to both polymeric (upper panel) and monomeric CsgA (lower panel) compared to the control peptides sLL-37 and VIP.</p

LL-37 prevents formation of biofilm by <i>E. coli</i> in vitro.

<p>(<b>A</b>) Different concentrations of LL-37 and the control peptides sLL-37 and VIP were added to the curli-expressing mutant. (<b>B</b>) At 2.5 µM, LL-37 caused more than 80% reduction of biofilm production, whereas the same concentration of the control peptides gave a reduction of only ∼10%. Mean and standard deviation from data of two separate experiments in triplicates are shown. The difference between LL-37 versus sLL-37 or VIP at 2.5 µM was statistically significant (^***<i>P</i> = 0.001, <i>t</i>-test). Similar results were obtained for the wild-type strain expressing both curli and cellulose (data not shown).</p

Oligos and plasmids used for mutant construction and complementation.

a<p>AmpR: resistance to ampicillin; Km^R: resistance to kanamycin.</p>b<p>Underlined sequences: restriction enzyme recognition sites; bold font: sequence for amplification of the pKD46 resistance/<i>km</i>RExTET cassette.</p

The monomeric form of CsgA remains stable in the presence of LL-37.

<p>(<b>A</b>) Monomeric CsgA was incubated for 20 h at 37°C without or with LL-37. When CsgA is incubated together with LL-37, the CsgA monomer is visible after SDS-PAGE, whereas the monomer is not detected in the absence of LL-37. When polymeric CsgA is treated with formic acid (FA), the CsgA monomer is detectable, excluding degradation of CsgA. The bands of ∼30 and ∼60 kDa are most likely the dimer and tetramer of CsgA, respectively. (<b>B</b>) The stability of the monomeric form of CsgA in the presence of LL-37 was also confirmed with CD spectroscopy after 60 h incubation. The CD spectrum reveals that CsgA alone exhibits a fiber-like structure with weak beta-sheet conformation and a decreased solubility. In contrast, CsgA incubated together with LL-37 displays an unstructured, random coil structure. The spectrum of LL-37 alone was subtracted from the spectrum of CsgA + LL-37.</p

Curli increase the resistance to the antimicrobial peptide LL-37.

<p>(<b>A</b>) Bladder epithelial cells T24 were infected with bacteria for 30 min and adherent bacteria were subjected to LIVE/DEAD staining. Curli and cellulose expression enhanced bacterial resistance to antimicrobial properties (^###<i>P</i><0.001 for curliated strains versus non-curliated strains, ^*<i>P</i> = 0.023 and ^**<i>P</i> = 0.003 for cellulose expressing strains with or without curli, <i>t</i>-test). Combined data from four experiments are shown. (<b>B+C</b>) Bacteria were exposed to conditioned medium of bladder epithelial cells T24 stimulated with phenylbutyrate to enhance LL-37 production. Curli expression enhanced bacterial survival over 30 min (^##<i>P</i> = 0.006, <i>t</i>-test) (<b>B</b>). Results from three experiments in triplicate are shown. Conditioned medium was incubated with neutralizing anti-LL-37-antibodies (nAb) or isotype control antibodies (Co) prior to bacterial inoculation. Neutralizing of LL-37 had no effect on viability of the curliated strain (left) but enhanced viability of the double knockout (right, ^*<i>P</i> = 0.047, <i>t</i>-test) (<b>C</b>). Results from four experiments in triplicate are shown. (<b>D–G</b>) The susceptibility to LL-37 and mCRAMP of <i>E. coli</i> strains expressing or lacking curli or cellulose was tested by the broth dilution method. The expression of curli increased the resistance to both LL-37 (<b>D+E</b>) and mCRAMP (<b>F+G</b>). A significant difference of bacterial growth was observed at 10 µM LL-37 between curliated and non-curliated strains (^###<i>P</i><0.001, <i>t</i>-test). The curliated strains were also significantly more resistant to 5 µM mCRAMP than bacteria not producing curli (^#<i>P</i><0.05, <i>t</i>-test). An increased resistance to both cathelicidins was not observed for cellulose. Mean and standard deviation from data of two separate experiments in triplicates are shown. The IC₅₀ is indicated by a broken line.</p