title: Insights into Clostridium phytofermentans biofilm formation: aggregation, microcolony development and the role of extracellular DNA.
abstract: Biofilm formation is a critical component to the lifestyle of many naturally occurring cellulose-degrading microbes. In this work, cellular aggregation and biofilm formation of Clostridium phytofermentans, a cellulolytic anaerobic bacterium, was investigated using a combination of microscopy and analytical techniques. Aggregates included thread-like linkages and a DNA/protein-rich extracellular matrix when grown on soluble cellobiose. Similar dense biofilms formed on the surface of the model cellulosic substrate Whatman no. 1 filter paper. Following initially dispersed attachment, microcolonies of ~500 µm diameter formed on the filter paper after 6 days. Enzymic treatment of both the biofilm and cellular aggregates with DNase and proteinase resulted in significant loss of rigidity, pointing to the key role of extracellular DNA and proteins in the biofilm structure. A high-throughput biofilm assay was adapted for studying potential regulators of biofilm formation. Various media manipulations were shown to greatly impact biofilm formation, including repression in the presence of glucose but not the β(1→4)-linked disaccharide cellobiose, implicating a balance of hydrolytic activity and assimilation to maintain biofilm integrity. Using the microtitre plate biofilm assay, DNase and proteinase dispersed ~60 and 30 % of mature biofilms, respectively, whilst RNase had no impact. This work suggests that Clostridium phytofermentans has evolved a DNA/protein-rich biofilm matrix complementing its cellulolytic nature. These insights add to our current understanding of natural ecosystems as well as strategies for efficient bioprocess design.
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Trevor R
authors.initials: TR
authors.lastname: Zuroff
authors.affiliation: Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Weimin
authors.initials: W
authors.lastname: Gu
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Fore
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Susan B
authors.initials: SB
authors.lastname: Leschine
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Curtis
mesh_terms: D001422:Bacterial Adhesion; D018441:Biofilms / Q000254:growth & development*; D002482:Cellulose / Q000378:metabolism; D003013:Clostridium / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D002482:Cellulose; D003851:Deoxyribonucleases; D010447:Peptide Hydrolases