Multi-Parameter Laser Imaging Reveals Complex Microscale Biofilm Matrix in a Thick (4,000 μm) Aerobic Methanol Oxidizing Community

Abstract

Although methanol has frequently been used as an inexpensive supplementary carbon source to support treatment processes, knowledge of the resultant microbial biofilms, their 3D architecture, microenvironments, exopolymer chemistry and populations remains limited. We supplied methanol as a supplementary carbon source to biofilms developing in rotating annular reactors. Analysis of circulation waters (1.0 l d−1) indicated that dissolved organic carbon was reduced by 25%, NO3-nitrogen by 95%, and total phosphorus by 70%. Analyses of populations using culture based techniques and fluorescence in situ hybridization indicated enrichment of nitrifiers, denitrifiers, and methylotrophic bacteria relative to reference biofilms not receiving methanol. The biofilms that developed were up to 4,000 μm thick. Staining with fluor conjugated lectins in combination with nucleic acid stains, revealed the presence of discrete bacterial cells inside complex globular polymeric structures. These structures were in turn surrounded by an interstitial polymer containing a variety of bacterial cell types. The globular structures bound FITC-conjugated lectins, from Canavalia ensiformis and Ulex europeaus. The FITC-lectin of Phaseolus vulgaris bound the surface of the globular structures and more generally within the matrix. Chemical analyses of the polymer paralleled the results of lectin analyses indicating that the dominant neutral sugars were glucose, galactose, mannose, rhamnose, with fucose and ribose as minor constituents. Amino sugars were not detected. Dual channel imaging with pH sensitive probes indicated that pH gradients from pH 4 to 7 occurred across the globular microcolonies. Critically for the maintenance of aerobic conditions throughout the thick biofilm it was extensively penetrated by a fine fissure network revealed by the location of fluorescent latex microbeads as detected by confocal laser scanning microscopy. Microelectrode studies confirmed the absence of any detectable Eh gradients within the biofilm. However, mobility of various size-fractionated fluorescent probes indicated that the basal region was only penetrated by the lowest molecular weight probes with a hydrated radius of 2.2 nm or less. These observations indicate the selection of a unique, thick (>4,000 μm) microbial community in which a self-organized architecture promotes the maintenance of optimal conditions and metabolism throughout the biofilm community.

Matrix glycoconjugate characterization in multispecies biofilms and bioaggregates from the environment by means of fluorescently-labeled lectins

“…The lectins selected for FLBA can be combined with other fluorochromes as contrasting agents, e.g., for staining cell distribution with nucleic acid-specific fluorochromes. A smart combination of different staining and imaging approaches will allow visualization of a complex 3-dimensional biofilm landscape in multiple channels (Neu and Lawrence, 2015 ; Lawrence et al, 2016 , 2018 ).…”

Fluorescent nanosensors reveal dynamic pH gradients during biofilm formation

“…Alternatively, ratiometric dyes with a dual emission spectrum or a combination of pH-sensitive and reference dyes can be employed. These dyes can be applied in solution, or coupled to dextrans, planar surface (optodes), or particles (nanosensors) 54 . Nanosensors containing commercially available fluorophores have aroused increasing interest and are being widely implemented in approaches to pH measurement.…”