Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes

Abstract

Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solid-phase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.

Keywords: Anaerobic respiration; Bioreactor; Bioremediation; Environmental engineering; Geochemistry; Microbiology; Pilot-scale; Redox.

Figure 1.. Schematic of vertical down flow biochemical reactor columns.

The three ports, indicated by circles on the column, were designed for solid substrate retrieval in conjunction with flow along the z-axis of the columns. The five liquid ports are depicted in blue along the side of the column. For discrete retrieval of solid substrates, columns were temporarily tilted to a horizontal plane using a custom built rack to mitigate water pressure complications and resultant loss (Figures 5 and 6). As labelled, ‘MIW Inf.’ indicates where the mining influenced water is introduced. The effluent from these columns was collected at the bottom liquid port as visualized in Figure 4A.

Figure 2.. Examples of solid-phase matrix components deployed within columns.

A. Alfalfa hay; B. Woodchips; C. Sawdust; and D. Limestone. Alternative substrates such as sand or other organic solid-phase materials could have relevance to alternative applications such as studying subsurface flow or aquifer recharge.

Figure 3.. Removal of biogenic sulfide.

Produced gases traveled from the column headspace to a connected plastic bag. Sulfide was removed by reacting with a 10% NaOH solution.

Figure 4.. Sample ports for liquid and solid substrate retrieval.

A. 3-way valve to sample effluent; B. Intermediate liquid sampling port; C. Position of intermediate solid-phase sampling port in conjunction with liquid sampling ports; D. Side profile of a solid-phase port before substrate was added to columns, bags were lined up in the port adjacent to each other; E. Sacrificial sample bag containing solid-phase substrate utilized in experiments.

Figure 5.. Pilot scale deployment of apparatus detailing.

A. Column frame before columns; B. Column frame after columns and plumbing were established.

Figure 6.. Intact columns and supporting hardware in use during sampling process.

One of the columns is being tilted horizontally for sample bag retrieval to enable analysis during the experiment.