Method for Indirect Quantification of CH4 Production via H2O Production Using Hydrogenotrophic Methanogens

Abstract

Hydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. Methanogens exhibit extraordinary ecological, biochemical, and physiological characteristics and possess a huge biotechnological potential. Yet, the only possibility to assess the methane (CH4) production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH4. In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH4 production potential we developed a novel method for indirect quantification of the volumetric CH4 production rate by measuring the volumetric water production rate. This method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was estimated by determining the difference in mass increase in a quasi-isobaric setting. This novel CH4 quantification method is an accurate and precise analytical technique, which can be used to rapidly screen pure cultures of methanogens regarding their volumetric CH4 evolution rate. It is a cost effective alternative determining CH4 production of methanogens over CH4 quantification by using gas chromatography, especially if applied as a high throughput quantification method. Eventually, the method can be universally applied for quantification of CH4 production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens.

Keywords: Archaea; anaerobic cultivation; hyperthermophile; methane; methanogenesis; psychrophile; standard operation procedure (SOP); water.

Figure 1

Schematic illustration of the method. The figure illustrates the SOP: start with weighing the serum bottles ①, determine the head space pressure of the serum bottles②, flush and purge to remove CH₄ from the headspace of the serum bottles③, weigh the serum bottles ④, incubate the serum bottles ⑤, start again with step ①; taking a sample Ⓧ for OD measurement or cell counting is optional after step ④, whereas step ③ and ④ would have to be repeated after sampling for OD measurement.

Figure 2

The diagram shows the change of serum bottle mass and serum bottle headspace pressure during one experimental run.

Figure 3

(A) Measurement of the serum bottle headspace pressure with a digital manometer; (B) parallel gassing of the serum bottle headspace; (C) gassing manifold.

Figure 4

Mass gain for M. villosus, M. okinawensis and M. marburgensis. For M. villosus an experiment with a daily gassing event [marked M. villosus (1)] and one experiment with two gassing events per day [marked as M. villosus (2)] were performed. For M. marburgensis cultivations at 55°C and at 65°C were performed. Negative controls are not shown.

Figure 5

Total cumulative mass gain for M. villosus, M. okinawensis and M. marburgensis. For M. villosus an experiment with a daily gassing event [marked M. villosus (1)] and one experiment with two gassing events per day [marked as M. villosus (2)] were performed. For M. marburgensis cultivations at 55°C and at 65°C were performed. Negative controls are not shown.

Figure 6

Model for WER and MER determination using serum bottle headspace pressure and Δ water mass quantification.