Response of indigenous heterotrophic groundwater bacteria to low organic substrate availability

Abstract

Groundwater is one of the least studied environments, yet many people rely on groundwater for their sole drinking water supply. Little is known about the indigenous microflora, but it is believed to be similar to oceanic planktobacteria due to the low nutrient concentrations occurring in both ecosystems. That is, groundwater microorganisms are atypically small, mostly Gram-negative cells. Also like the oceanic planktobacteria, they may have no affinity for surface attachment and may rely on dissolved low molecular weight organic substrates in dilute solution for their nutrition. Periods of metabolic dormancy may occur when natural substrate concentrations drop below the level required to sustain vegetative cell function. In these studies total cells present were determined by 4'6-diamidino-2-phenylindole (DAPI) epifluorescent counts. The percentage of those bacteria which were metabolically active was determined by a modification of the 2-(p-iodophenyl)-3-(p- nitrophenyl)- 5-phenyl tetrazolium chloride (INT) reduction method. Advantages of this method over others include more specific fluorochrome staining, ease of transfer of the cells to the slide, time saved, and ease of microscopic viewing. Heterotrophic uptake of aspartate, succinate, glucose and fructose by indigenous bacteria was measured and calculations of maximum uptake velocity (V_max) and a constant (K_t + S_n) equalling the natural substrate concentration (S_n) plus the half-saturation concentration (K_t) were made based on net assimilation (cellular retention) of radiolabeled substrate. Total counts by DAPI staining were 4-12.1 x 10⁴ cells/ml of which 17.4 to 20.85% were metabolically active (INT+). Mean maximum uptake velocities ranged from 1.73 to 2000 nmol/l/hr with aspartate being taken up at the highest rate followed by fructose, succinate and glucose.