The colonization of liquid surfaces as floating biofilms or pellicles
is a bacterial adaptation to optimally occupy the airliquid
(A-L) niche. In aerobic heterotrophs, pellicle formation
is beneficial for the utilization of O2 and nonpolar organic
compounds. Pseudomonas alkylphenolica KL28, an alkylphenol
degrader, forms flat circular pellicles that are 0.3–
0.5 mm in diameter. In this study, we first monitored the pellicle
developmental patterns of multicellular organization
from the initial settlement stage. The pellicles developed by
clonal growth and mutants for flagella and pilus formation
established normal pellicles. In contrast, the mutants of an
epm gene cluster for biosynthesis of alginate-like polymer
were incompetent in cell alignment for initial two-dimensional
(2D) pellicle growth, suggesting the role of the Epm
polymer as a structural scaffold for pellicle biofilms. Microscopic
observation revealed that the initial 2D growth transited
to multilayers by an accumulated self-produced extracellular
polymeric substance that may exert a constraint force.
Electron microscopy and confocal laser scanning microscopy
revealed that the fully matured pellicle structures were densly
packed with matrix-encased cells displaying distinct arrangements.
The cells on the surface of the pellicle were relatively
flat, and those inside were longitudinally cross-packed. The
extracellular polysaccharide stained by Congo red was denser
on the pellicle rim and a thin film was observed in the open
spaces, indicative of its role in pellicle flotation. Our results
demonstrate that P. alkylphenolica KL28 coordinately dictates
the cell arrangements of pellicle biofilms by the controlled
growth of constituent cells that accumulate extracellular
polymeric substances.