Quantification of Moss-Associated Cyanobacteria Using Phycocyanin Pigment Extraction

Abstract

In the boreal forest, cyanobacteria can establish associations with feather moss and realize the biological nitrogen fixation (BNF) reaction, consisting in the reduction of atmospheric dinitrogen into bioavailable ammonium. In this ecosystem, moss-associated cyanobacteria are the main contributors to BNF by contributing up to 50% of new N input. Current environmental changes driven by anthropogenic activities will likely affect cyanobacteria activity (i.e., BNF) and populations inhabiting mosses, leading to potential important consequences for the boreal forest. Several methods are available to efficiently measure BNF activity, but quantifying cyanobacteria biomass associated with moss is challenging because of the difficulty to separate bacteria colonies from the host plant. Attempts to separate cyanobacteria by shaking or sonicating in water were shown to be poorly efficient and repeatable. The techniques commonly used, microscopic counting and quantitative PCR (qPCR) are laborious and time-consuming. In aquatic and marine ecosystems, phycocyanin (PC), a photosynthesis pigment produced by cyanobacteria, is commonly used to monitor cyanobacteria biomass. In this study, we tested if PC extraction and quantification can be used to estimate cyanobacteria quantity inhabiting moss. We report that phycocyanin can be easily extracted from moss by freeze/thaw disturbance of cyanobacteria cells and can be quickly and efficiently measured by spectrofluorometry. We also report that phycocyanin extraction is efficient (high recovery), repeatable (relative SD < 13%) and that no significant matrix effects were observed. As for aquatic systems, the main limitation of cyanobacteria quantification using phycocyanin is the difference of cellular phycocyanin content between cyanobacteria strains, suggesting that quantification can be impacted by cyanobacteria community composition. Nonetheless, we conclude that phycocyanin extraction and quantification is an easy, rapid, and efficient tool to estimate moss-associated cyanobacteria number.

New insights into the drivers of moss‐associated nitrogen fixation and cyanobacterial biomass in the eastern Canadian boreal forest

“…In our study, we reported a significant positive exponential relationship between moss‐associated N 2 ‐fixation and cyanobacterial biomass (Figure 3). Several studies demonstrated a positive linear relationship between N 2 ‐fixation and the quantity of cyanobacteria on moss in Sweden (DeLuca et al, 2007; Rousk, DeLuca, & Rousk, 2013), Chile (Arróniz‐Crespo et al, 2014) and Canada (Renaudin et al, 2021; Rousk, Degboe, et al, 2017), confirming that N 2 ‐fixation and cyanobacterial biomass are linked. It has been hypothesized that this relationship is the result of the moss controlling the number of colonizing cyanobacteria as a function of its N demand (Bay et al, 2013; Leppänen et al, 2013).…”

“…A positive linear relationship between the number of cyanobacteria living on moss and N 2 -fixation activity was reported for various moss species at numerous locations (Arróniz-Crespo et al, 2014;DeLuca et al, 2007;Renaudin et al, 2021;Rousk, Degboe, et al, 2017;, suggesting that higher N 2 -fixation is correlated with higher cyanobacterial biomass on moss. While significant, this relationship only partially explained variability (9%-50%) in N 2 -fixation and is not always observed (Goth et al, 2019).…”

“…Several authors have suggested that mosses can control cyanobacteria colonization based on their N needs by attracting cyanobacteria when N is limiting (Bay et al, 2013) and inhibiting colonization in N‐rich environments (DeLuca et al, 2007; Gundale et al, 2011; Leppänen et al, 2013). A positive linear relationship between the number of cyanobacteria living on moss and N 2 ‐fixation activity was reported for various moss species at numerous locations (Arróniz‐Crespo et al, 2014; DeLuca et al, 2007; Renaudin et al, 2021; Rousk, Degboe, et al, 2017; Rousk, DeLuca, & Rousk, 2013), suggesting that higher N 2 ‐fixation is correlated with higher cyanobacterial biomass on moss. While significant, this relationship only partially explained variability (9%–50%) in N 2 ‐fixation and is not always observed (Goth et al, 2019).…”

“…Moss N 2 -fixation was estimated using the acetylene reduction assay (ARA; Hardy et al, 1968) on four replicated subsamples per species, site and time of sampling, according to Renaudin et al (2021). Moss shoots were cleaned, and brown parts were discarded.…”

The moss traits that rule cyanobacterial colonization

“…The leaf size and number of cyanobacterial cells for 1755 leaves (90 for A. turgidum , 360 for P. schreberi , 360 for T. nitens and 945 for H. splendens ) were measured. Microscopic counting, rather than using proxies of cyanobacterial quantity ( Renaudin et al ., 2021 ), enabled us to link leaf traits with cyanobacterial colonization.…”