Experimental procedures for studying microbial reactions under high hydrogen gas saturations in microcosms

Abstract

This methodology is proposed to investigate the response of microbial communities through analysis of headspace composition under high saturations of hydrogen. Changes in headspace composition will be related to specific communities and environmental conditions that will influence their response and result in changes in gases produced or potential changes in the liquid phase of microcosms pertaining to the hydrogen consumption rate through microbial metabolic processes. A step-by-step procedure is documented here.•Methodology includes an easy setup utilising common laboratory equipment.•The method showed minor appreciable loss of hydrogen from the microcosm setup/storage and the use of exetainers for gas measurements.•Actively studied microbial hydrogen consumption across 18 days at 30 °C and 50 °C This method is useful for the first instances in scientific studies towards understanding species or microbial communities found in environments with high percentages of hydrogen: underground hydrogen storage sites, hydrogen pipelines, and hydrogen leakage into subsurface soils.

Keywords: Acetogenesis; Hydrogen; Methanogenesis; Microbial metabolism; Microbiology studies under high saturations of hydrogen in headspace in microcosms; Microcosm studies; Sulfate reducing bacteria.

Graphical abstract

Fig. 1

An illustration of a microcosm in a Wheaton crimp seal bottle during headspace flushing and after inverting for reduced hydrogen loss during incubation. (a) Injection gas needle, 19 G (1.1 × 50 mm), attached to gas canister of original headspace mix 50 % hydrogen and 50 % nitrogen; (b) Exit/venting needle, 21 G (0.8 × 38 mm), for flushed headspace to escape and avoid over pressurisation; (c) Aluminium crimp seal; (d) Butyl rubber stopper; (e) Basal media with additional micronutrients/vitamins and sourced inocula; (f) Headspace starting mix, 50 % hydrogen and 50 % nitrogen.

Fig. 2

Hydrogen was tested from distilled water and different puncturing routines in microcosms. Time series at time point 0, immediately after microcosm creation, and time point end (day 18). NP = non-punctured rubber stoppers over time until the final sample day 18. P = punctured microcosm at the same sample period of the 18-day experiment.

Fig. 3

Blue Line = Negative control of distilled water. Orange line = Microcosms containing sulfate. Green line = Microcosms not containing sulfate. Red line = Microcosms containing nitrogen in headspace and sulfate. Purple line = Microcosms containing nitrogen in headspace and no sulfate. A) Recorded hydrogen in mmoL at 30 °C. B) Recorded hydrogen in mmoL at 50 °C. C) Methane in mmoL at 30 °C.D) Methane in mmoL at 50 °C. E) Carbon Dioxide in mmoL at 30 °C. F) Carbon dioxide in mmoL at 50 °C. G) pH across microcosms at 30 °C. H) pH across microcosms at 50 °C.

Fig. 4

Standard linear line for H₂ % of 1 % and 50 %.