Parallel bioreactor system for accessible and reproducible anaerobic culture

Abstract

When working with anaerobic bacteria it is important to have the capability to perform parallel bioreactor growth experiments that are both controllable and reproducible, although capital and consumables costs for commercially available systems are often prohibitively high. Hence, a three-vessel parallel bioreactor system was designed and constructed that has the capabilities for batch and fed batch processes and can also be set up for continuous culture at a fraction of the cost of commercial systems. This system carries over many of the same functionalities of those systems with a higher price point of entry, including in-line monitoring of temperature, pH, and redox poise. To validate the performance of this system Clostridium saccharoperbutylacetonicum was grown under conditions that promote ABE fermentation, an established industrial process used to produce the solvents acetone, butanol and ethanol. Measurements of cell density, pH, and redox poise all confirmed reproducible culture conditions for these parallel vessels, and solvent quantitation via GCMS verified consistent metabolic activities for the separate cultures. In future, this system will be of interest to researchers that require high performance parallel fermentation platforms but where commercial systems are not accessible.

Deletion of glyceraldehyde‐3‐phosphate dehydrogenase (gapN) in Clostridium saccharoperbutylacetonicum N1‐4(HMT) using CLEAVE™ increases the ATP pool and accelerates solvent production

“…Deletion of gapN was also hypothesised to elevate ATP levels, so the concentrations of ATP were also monitored during fermentation in bespoke bioreactors (Monaghan et al ., 2021 ) and were found to be higher in the ∆ gapN strain compared to wild type for the majority of fermentation period (Fig. 5 ).…”

“… Fermentation measurements for ∆gapN (o) and wild type (•) C. saccharoperbutylacetonicum N1‐4(HMT). Cultures were grown on 40 g l −1 glucose in YETM media in bespoke bioreactors (Monaghan et al ., 2021 ); 0.1 M MES was supplemented as a pH buffering agent and the following parameters were monitored: (A) OD 600 ; (B) pH; (C) redox potential of media (mV vs. NHE); (D) Glucose concentrations in growth media. Data points are mean values of a total of six repeats including three biological repeats.…”

“… ATP measurements for ∆ gapN (o) and wild type (•) strains grown in 40 g l −1 glucose in YETM media in bespoke bioreactors (Monaghan et al ., 2021 ); 0.1 M MES was supplemented as a pH buffering agent. Data points are mean values of a total of six repeats including three biological repeats.…”

“…ABE fermentation was carried out as recently described using a bespoke anoxic bioreactor system (Monaghan et al ., 2021 ). Briefly, 1 l Pyrex Quickfit culture vessels were used (SciLabware, Hartlepool, United Kingdom) with 500 ml culture volumes at 32°C and media consisted of yeast extract tryptone media (YETM) (40 g l −1 glucose, 2.5 g l −1 yeast extract, 2.5 g l −1 tryptone, 0.5 g l −1 ammonium sulphate and 0.025 g l −1 iron sulphate) at pH 6.2, supplemented with 0.1 M 2‐(N‐morpholino)ethanesulfonic acid (MES) free acid (Merck) for pH control.…”

“…Acids and solvents from culture supernatants were quantitated using Gas Chromatography Mass Spectrometry (GCMS) using an Agilent 6890N instrument as previously described (Monaghan et al ., 2021 ). The GC was equipped with a Phenomenex 7HG‐6013‐11 Zebron column.…”