Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.

BACKGROUND: Despite the recognition that microbial biofilms play a role in environmental degradation of bioplastics, few studies investigate the relationship between bioplastic biodegradation and microbial colonisation. We have developed protocols based on a combination of confocal laser scanning microscopy and contact angle goniometry to qualitatively and quantitatively map surface changes due to biofilm formation and biopolymer degradation of solvent cast poly(3‐hydroxyalkanoate) films in an accelerated in vitro biodegradation system.RESULTS: A significant regression relationship between biofilm formation and polymer biodegradation (R2 = 0.96) was primarily conducted by cells loosely attached to the film surfaces (R2 = 0.95), rather than the strongly attached biofilm (R2 = 0.78). During biodegradation the surface rugosity of poly(3‐hydroxybutyrate) and poly[(3‐hydroxybutyrate)‐co‐(3‐hydroxyvalerate)] increased by factors of 1.5 and 1.76, respectively. In contrast, poly(3‐hydroxyoctanoate) films showed little microbial attachment, negligible weight loss and insignificant changes in surface rugosity.CONCLUSION: A statistically significant link is established between polymer weight loss and biofilm formation. Our results suggest that this degradation is primarily conducted by cells loosely attached to the polymer rather than those strongly attached. Biofilm formation and its type are dependent upon numerous factors; the flat undifferentiated biofilms observed in this study produce a gradual increase in surface rugosity, observed as an increase in waviness. Copyright © 2008 Society of Chemical Industry

Biopolymers derived from natural resources are potential alternatives to recalcitrant synthetic plastics; however, studies investigating the degradability of these biopolymers in natural environments are relatively few. This study compares the environmental degradation of polymers described as ‘green plastics’ in garden soil in terms of weight loss, topographical changes and biofilm attachment. Poly(3‐hydroxybutyrate) (PHB) and poly[(3‐hydroxybutyrate)‐co‐(3‐hydroxyvalerate)] (P(HB‐co‐8HV)), (copolymer containing 8 mol% HV) films degraded rapidly, losing 50% of their initial weight in 50 days. In contrast, after burial for 380 days, the medium chain length polyhydroxyoctanoate (PHO) lost 60% of its weight, poly(D,L‐lactide) (PDLL) 18% and poly[(D,L‐lactide)‐co‐glycolide] (PDLLG) 35%. Polystyrene (PS) and ethyl cellulose (EC) showed no significant degradation. Both weight loss and biofouling occurred in the following sequence: P(HB‐co‐8HV) = PHB > PHO > PDLLG > PDLL > PS = EC. The surface rugosity and surface areas of PHB and P(HB‐co‐8HV) increased three‐ and twofold, respectively, during degradation, indicating surface erosion. The surface rugosity of PHO increased twofold and the surface area increased by 25%. This in situ study demonstrates a quantifiable relationship between biofilm attachment, surface rugosity and polymer degradation. PHB and P(HB‐co‐8HV) showed greater biofouling and increased surface rugosity, and degraded significantly faster than the other polymers studied. Copyright © 2009 Society of Chemical Industry

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