Table 2.

Studies on pH gradient in biofilm and the effect of carbon source.

Study method		Biofilm type	pH Range	Findings	Effect of carbon source	References
pH microelectrodes		Monospecies Citrobacter sp. biofilms (can be found in food and water)	From pH around 7 to the lowest pH of 5.12	The pH in phosphate-limited biofilm fell within the biofilm (pH shift of 0.4 and 1.2 pH units in 0–400 μm from the substratum), whereas that of carbon-limited biofilm remained constant.	Biofilm on phosphate-limiting medium became more acidic.	Allan et al. (1999)
Fluorescent dyes	C-SNARF-4 confocal microscopy	Dental biofilm multi-species	7 to 5.5–5.9	Heterogeneous pH in the basal layer of young dental biofilm model (7–100 μm), no obvious correlation between biofilm thickness and pH drop in the bottom layer.	Adding glucose caused pH drop.	Schlafer et al. (2011)
			7 to 4.5	Highly heterogeneous pH landscapes due to supply of glucose and buffering ions, bacterial acid production, bacterial acid consumption, and diffusion of acids within and out of biofilm.	The pH dropped in deeper layers of five-species laboratory biofilms (up to 70 μm) upon exposure to 0.4% glucose.	Dige et al. (2016)
	*TPE and CLSM with fluorescence lifetime imaging	Mixed culture oral biofilm	Within 10 min of exposure to glucose, pH fell from 7.0 ± 0.3 to 6.0 ± 0.4	Sharp gradients of pH within microbial biofilms.	Adding sucrose resulted in pH drop, especially in layers at depths of 35 and 70 μm.	Vroom et al. (1999)
Fluorescent nanoparticles	Silica nanoparticle sensors confocal microscopy	Mixed-culture wastewater biofilms	5 to >7	Heterogeneity of the pH profiles within these biofilms. The pH is more heterogeneous throughout the mixed culture than the axenic one at 77 μm from the substratum.	By adding glucose, pH becomes more acidic.	Hidalgo et al. (2009)
	pH-responsive nanoparticles confocal microscopy	P. aeruginosa (opportunistic pathogen) and Streptococcus mutants (oral pathogen)	3.5 to 7.5	pH gradient across the biofilm thickness and over time and an acidic core of microcolonies.	pH dropped after the addition of carbon sources (glucose and sucrose).	Hollmann et al. (2021)
		P. fluorescens (soil and water)	5 to 8	pH gradient at different depths of biofilm (0–40 μm).	Biofilm produced more acidic by-products under oxygen-limiting conditions found in the deepest regions of the biofilm.	Fulaz et al. (2019)

^*Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM).