Probing Saltern Brines with an Oxygen Electrode: What Can We Learn about the Community Metabolism in Hypersaline Systems?

Abstract

We have explored the use of optical oxygen electrodes to study oxygenic photosynthesis and heterotrophic activities in crystallizer brines of the salterns in Eilat, Israel. Monitoring oxygen uptake rates in the dark enables the identification of organic substrates that are preferentially used by the community. Addition of glycerol (the osmotic solute synthesized by Dunaliella) or dihydroxyacetone (produced from glycerol by Salinibacter) enhanced respiration rates. Pyruvate, produced from glycerol or from some sugars by certain halophilic Archaea also stimulated community respiration. Fumarate had a sparing effect on respiration, possibly as many halophilic Archaea can use fumarate as a terminal electron acceptor in respiration. Calculating the photosynthetic activity of Dunaliella by monitoring oxygen concentration changes during light/dark incubations is not straightforward as light also affects respiration of some halophilic Archaea and Bacteria due to action of light-driven proton pumps. When illuminated, community respiration of brine samples in which oxygenic photosynthesis was inhibited by DCMU decreased by ~40%. This effect was interpreted as the result of competition between two energy yielding systems: the bacteriorhodopsin proton pump and the respiratory chain of the prokaryotes. These findings have important implications for the interpretation of other published data on photosynthetic and respiratory activities in hypersaline environments.

Keywords: Halobacteria; Haloquadratum; Salinibacter; bacteriorhodopsin; halophilic; hypersaline; oxygen; salterns.

Figure 1

A crystallizer pond of Salt of the Earth, Ltd., Eilat, Israel.

Figure 2

Changes in the respiration rate by the microbial community from a saltern crystallizer pond in Eilat following addition of glycerol (A); dihydroxyacetone (B); Na-acetate (C) or NaOH-neutralized fumaric acid (D). Portions of 630 mL NaCl-saturated brine from a crystallizer pond sampled in May 2011, and containing 2.9 × 10⁷ prokaryote cells/mL and 1200 Dunaliella salina cells/mL, were incubated in the dark in completely filled 630-mL Plexiglas chambers, each provided with a Yellow Springs Instrument optical oxygen electrode (Pro20 Lab/Field Dissolved Oxygen Meter) and a magnetic stirring bar, the temperature being controlled at 30–31 °C. The oxygen concentration was recorded every 5 min. At the time indicated by arrows, the carbon sources were added to a final concentration of 1 mM by injection of 0.63 mL of 1 M solutions of the respective compounds, and the effect of the substrate was estimated by the change in the oxygen uptake rate.

Figure 3

Kinetics of oxygen evolution and oxygen consumption in the light and in the dark by Eilat crystallizer brine, sampled in May 2015, at 35 °C in the presence and in the absence of DCMU. Chambers (630 mL) equipped with oxygen and temperature sensors and surrounded by a water jacket for temperature control were completely filled with brine from a crystallizer pond of the Eilat salterns. After temperature equilibration in the light (200–220 μmol quanta/m²·s,) for 70 min, changes in dissolved oxygen concentration were recorded. At the time indicated by the arrows, DCMU (5 μM) was added from a 5 mM solution in ethanol, and illumination was turned off and on as indicated by the white (light) and black (dark) bars at the upper part of each panel. The slopes from which the kinetics of net photosynthesis and respiration were calculated are indicated by the dashed lines, all based on data collected at a temperature of 35 ± 0.3 °C. The brine had a density of 1.202 g/mL at the in situ temperature of 35 °C, contained ~3.5 × 10⁷ prokaryotes/mL with >70% flat square cells, 2170 Dunaliella cells/mL, 0.8 μg/L chlorophyll a, 0.28 mg/L β-carotene, 0.098 mg/L bacterioruberin carotenoids, and ~3.6 nmol/L bacteriorhodopsin and other retinal proteins. From Extremophiles Vol. 20, 2016, p. 75, A. Oren et al. [18], with permission of Springer.