Eri Nasuno

Isolation of Biofilm-Forming Bacteria from the Secondary Effluent of the Wastewater Treatment Plant and its Ability to Produce N-Acylhomoserine Lactone as Quorum Sensing Signal

Bacterial quorum sensing (QS) is one of the cell-to-cell communication systems. N-Acyl-homoserine lactones (AHLs) are the most common QS signals and responsible for biofilm formation in gram-negative bacteria. Inactivation of QS, also referred to as quorum quenching, has been regarded as a popular strategy to control membrane bioreactor (MBR) operations because biofouling in MBR wastewater treatment systems is often caused by biofilm formation. In the present study, 24 bacterial strains were isolated from the secondary effluent of a wastewater treatment plant and AHL productivity and biofilm formation were evaluated on 8 out of 24 isolates. The isolated 8 strains can produce AHLs that only possess a long (C8-C14) acyl side chain. The representative 3 strains were selected from these isolates based on the higher activity of biofilm formation. The AHL separation analysis with a thin layer chromatography clearly showed that N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) existed in the secondary effluent sample, while no C6-HSL producing strain was isolated. C8-HSL was identified as the product of isolate No. 6. These results suggest that the secondary effluent probably contains various cell-to-cell signaling molecules derived not only from the inhabitants but also from the other microorganisms involved in the activated sludge for the biological pre-treatment. In this secondary effluent, AHL trapping techniques can be proposed as one of the acceptable strategies for the control of the QS systems because the remaining AHLs have relatively long acyl side chain and low concentrations.

Quick detection of cell to cell communication in gram negative bacteria by colour change of polymer matrix entrapping reporter bacteria

Abstract Quick and easy bioassay system for detection of N-acyl-L-homoserine lactones (AHLs) became possible by determining colour change of polymer matrix surfaces entrapping Chromobacterium violaceum CV026 cells, which accumulates purple pigment violacein inside cells through cell–cell communication system termed as quorum sensing (QS). The AHL is popularly produced as one of the QS signals to regulate various cell functions, and its concentration increase can lead to activate the QS. Sensitive detection of producing AHLs could be realised on the polymer surfaces as the gradual changing purple because the AHL synthase defective strain CV026 could respond to produce purple violacein in the presence of AHLs derived from other bacteria. The b* coordinate representing the difference between yellow and blue chromas in the CIE-L*a*b* colour space was obviously negatively correlated with absorbance at 585 nm of butanol extracted violacein from cells irrespective of culture time and initial concentration of cells at inoculation. Time evolution of the chroma at the matrix surface expressed in a*, b* plane was investigated to generate a master curve even when the QS was activated with different concentrations of N-hexanoyl-L-homoserine lactone or N-octanoyl-L-homoserine lactone. As compared with the master curve for the culture broth in a*, b* plane, the matrix surface meaningfully expands since the refractive measurement can give intensive signals at the matrix surface during the initial period of the sequential QS activation. The proposed biosensor can detect the AHL within 3–5 h when the AHL mediated QS system of unknown bacteria is about to be activated, where the conventional agar plate assay needs >16 h.

Novel quorum quenching enzymes identified from draft genome of Roseomonas sp. TAS13

Roseomonas sp. strain TAS13 isolated from an activated sludge sample degrades N-acylhomoserine lactones (AHLs) that are widely utilized as a signal in bacterial quorum sensing systems. The draft genome of Roseomonas sp. TAS13 contains 816 contigs (total 5,078,941 bp) which carries 4760 protein-coding genes and 52 tRNA genes (DDBJ/EMBL/GenBank accession numbers BDLP01000001 through BDLP01000816).

Enhancement effects of cationic contaminants from bacteria on cake layer formation and biofouling on an RO membrane

The model cationic molecule prodigiosin interacted with a polyamide/polysulfone composite reverse osmosis (RO) membrane, resulting in a reduction of the membrane permeation rate. Prodigiosin is an antibacterial agent produced by Serratia marcescens that is frequently isolated from activated sludge of domestic or industrial wastewater. Such molecules respectively secreted or leaked from live or dead cells are thought to affect membrane biofouling. In this study, a cell suspension containing prodigiosin-producing S. marcescens AS-1 wild-type or the non-producing AS-1ΔspnI strain was fed to the thin RO membrane to determine the occlusion ratio on the membrane. Cationic prodigiosin enhanced membrane biofouling by clogging the pores and enhanced the accumulation of the cake layer. The effects remarkably recovered the occlusion ratio after removing the cake layer by feeding with water. After temporary pressure relief, the occlusion ratios for AS-1 and AS-1ΔspnI were recovered to stable levels from approximately 70 to 49% and 23%, respectively. Zetapotential analysis supported the neutralization effects leading to the accumulation of bacterial cells under applied high pressure for RO membrane permeation.

Inhibition of Antimicrobial Prodigiosin Production in Serratia marcescens Using Cyclodextrin-Immobilized Microgel Beads Prepared by a Non-Contact Jet Dispensing System

Some gram-negative bacteria possess N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) as one of the cell-to-cell communication systems for regulation of various cell functions. Increase in the AHL concentration above its threshold leads to simultaneous activation of target gene expression in each cell. The AHL can diffuse in and out of the cells through the cell membrane. Artificial reduction of the AHL concentration can inhibit the AHL complex formation with its receptor inside the cell. In this study, the AHL that diffused out into the culture broth was captured on α-cyclodextrin (CD), which possesses a hydrophobic cavity and a hydrophilic shell. The α-CD-immobilized calcium alginate gel beads, which were prepared using a non-contact jet dispenser, were immersed in the culture medium during the cell growth of Serratia marcescens AS-1, which was used as the model opportunistic human pathogen. The trapping of AHLs on immobilized α-CDs due to hydrophobic interaction of the AHL acyl-chain led to an effective inhibition of AHL-mediated prodigiosin production to approximately 2% in the presence of 27 wt% gel beads in the culture broth. The results of our study suggest that CD has high potential to regulate QS involved in matter production, biofilm formation, and virulence factor expression.

Color Transition Properties of Water Dispersible Polydiacetylene Particles Conjugated with the Thermosensitive Polymer

An assembly of diacetylene molecules is necessary to progress a topochemical reaction as UV-irradiated polymerization. In this study, we attempt to regularly assemble 10, 12- pentacosadiynoic acid (PCDA) by freezing the solution, followed by lyophilization. Dried PCDA assembly was easily dispersed in aqueous solution by sonication. Irradiation of UV light can promote polymerization reaction of PCDA judging from a color change of the solution into blue. A color transition of the polyPCDA-dispersed solution from blue to red was observed at approximately 56°C due to distortion of its π-conjugated system. A color transition temperature could be controlled by hybridization with thermosensitive hydroxypropyl cellulose, of which lower critical solution temperature appears around 38-39°C. By synchronizing to coli-globule transition of HPC in aqueous media, blue-red transition response of water-dispersed polyPCDA could be induced by changing conformation of surrounded polymer.