#
Effect of N_{2} on Biological Methanation in a Continuous Stirred-Tank Reactor with Methanothermobacter marburgensis

^{*}

^{†}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Instrumentation

^{−1}. Gases were fed with thermal mass flow controllers EL-FLOW select (Bronkhorst Deutschland Nord GmbH, Kamen, Germany) from compressed gas bottles into the fermenter via two ring gas spargers on the bottom. Temperature and pressure were both set constant at $\mathrm{\vartheta}=65\text{}{}^{\circ}\mathrm{C}$ and $p=1\mathrm{bar}\left(\mathrm{g}\right)$ with a pressure control valve EL-PRESS (Bronkhorst Deutschland Nord GmbH, Kamen, Germany). ${\mathrm{CO}}_{2}$, ${\mathrm{H}}_{2}$, and ${\mathrm{CH}}_{4}$ were measured in the off-gas with BCP gas sensors via infrared (IR) (carbon dioxide and methane) and thermal conductivity (hydrogen) (BlueSens gas sensor GmbH, Herten, Germany). The pH value was measured with a CPS11D (Endress+Hauser Messtechnik GmbH & Co. KG, Weil am Rhein, Germany) and regulated with ${\mathrm{H}}_{2}\mathrm{S}{\mathrm{O}}_{4}$ (${c}_{{\mathrm{H}}_{2}{\mathrm{SO}}_{4}}=1$ mol L${}^{-1}$) and ${\left(\mathrm{N}{\mathrm{H}}_{4}\right)}_{2}{\mathrm{CO}}_{3}$ (${c}_{{\left(\mathrm{N}{\mathrm{H}}_{4}\right)}_{2}\mathrm{C}{\mathrm{O}}_{3}}=1$ mol L${}^{-1}$). Redox potential (ORP) was measured with an EasyFerm Plus ORP Arc 120 (Hamilton Bonaduz AG, Bonaduz, Switzerland).

#### 2.2. Nutrition Media, Microorganism, and Gases

_{12}, 5 $\mathrm{m}$ $\mathrm{g}$ ${\mathrm{L}}^{-1}$ aminobenzoic acid, 5 $\mathrm{m}$ $\mathrm{g}$ ${\mathrm{L}}^{-1}$ $\mathrm{\alpha}$-lipoic acid, ad. 1000 $\mathrm{m}$ $\mathrm{L}$ distilled water] and filled up with distilled water. The reactor was flushed with ${\mathrm{CO}}_{2}$ for 180 $\mathrm{min}$ to obtain an anaerobic atmosphere. A total of 20 $\mathrm{m}$ $\mathrm{L}$ of $\mathrm{N}{\mathrm{a}}_{2}\mathrm{S}\xb79{\mathrm{H}}_{2}\mathrm{O}$ ($c=1$ mol L${}^{-1}$) was added to reduce the remaining ${\mathrm{O}}_{2}$, and 20 $\mathrm{m}$ $\mathrm{L}$ cell suspension of Methanothermobacter marburgensis (DSM-2133, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were added to the reactor. Then ${\mathrm{H}}_{2}$ was added until a ratio of ${\mathrm{H}}_{2}$:${\mathrm{CO}}_{2}$ 4:1 was reached. ${\mathrm{H}}_{2}$ had a nominal purity of $\ge 99.9\%$ (Linde AG, München, Germany), and ${\mathrm{CO}}_{2}$ (later referred to as C100) had a nominal purity of $99.8$% (Westfalen AG, Münster, Germany). To get a sufficient cell concentration of M. marburgensis, the organisms were cultured for $t=5$ d. The doubling time of M. marburgensis, according to the literature, is ${t}_{d}=2$ h [19,20]. In this work, a doubling time of about ${t}_{d}=2.4$ h was observed. Experiments were performed with different ${\mathrm{CO}}_{2}$ -containing premixed feed gases. ${\mathrm{CO}}_{2}$/${\mathrm{N}}_{2}$ mix gases BIOGON© C20 (${\mathrm{CO}}_{2}$ ($20\pm 2$)%, rest ${\mathrm{N}}_{2}$), C30 (${\mathrm{CO}}_{2}$ ($30\pm 3$)%, rest N2), and C40 (${\mathrm{CO}}_{2}$ ($40\pm 4$)%, rest ${\mathrm{N}}_{2}$) were obtained in pressure gas bottles from Linde AG. The resulting composition of the feed gases containing the ${\mathrm{CO}}_{2}$/${\mathrm{N}}_{2}$ mix and ${\mathrm{H}}_{2}$ is shown in Table 2. In addition, an anaerobe solution of $\mathrm{N}{\mathrm{a}}_{2}\mathrm{S}\xb79{\mathrm{H}}_{2}\mathrm{O}$ ($c=0.5$ mol L${}^{-1}$) was added periodically to the reactor to maintain the supply of sulfur for the growth of M. marburgensis [21].

#### 2.3. Calculations

## 3. Results

## 4. Discussion

## Author Contributions

## Funding

## Conflicts of Interest

## Abbreviations

ADP | Adenosine diphosphate |

ATP | Adenosine triphosphate |

C20 | Gas mix with approx. ${x}_{{\mathrm{CO}}_{2}}=0.2$ and ${x}_{{\mathrm{N}}_{2}}=0.8$ |

C30 | Gas mix with approx. ${x}_{{\mathrm{CO}}_{2}}=0.3$ and ${x}_{{\mathrm{N}}_{2}}=0.7$ |

C40 | Gas mix with approx. ${x}_{{\mathrm{CO}}_{2}}=0.4$ and ${x}_{{\mathrm{N}}_{2}}=0.6$ |

C100 | Gas with approx. ${x}_{{\mathrm{CO}}_{2}}=1$ |

CSTR | Continuous stirred-tank reactor |

DSMZ | Leibniz-Institut DSMZ-Deutsche Sammlung von Mikrooganismen und Zellkulturen GmbH |

IR | Infrared |

ORP | Oxidation reduction potential |

PSA | Pressure swing adsorption |

UNFCCC | United Nations Framework Convention on Climate Change |

WW | Water washing |

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**Figure 1.**Price per certificate (European Union Allowance) for the allowance to emit the greenhouse gas equivalent of 1 $\mathrm{t}$ ${\mathrm{CO}}_{2}$ from 1 January 2010 to 31 March 2019 [18].

**Figure 2.**Concentrations of ${\mathrm{H}}_{2}$, ${\mathrm{CO}}_{2}$, $\mathrm{C}{\mathrm{H}}_{4}$ (measured), and ${\mathrm{N}}_{2}$ (calculated) in the off-gas when using feed gases with different ${\mathrm{CO}}_{2}$ mole fractions: (

**A**) C100 (${x}_{\mathrm{C}{\mathrm{O}}_{2}}=1$), (

**B**) C20 (${x}_{\mathrm{C}{\mathrm{O}}_{2}}=0.186$), (

**C**) C30 (${x}_{\mathrm{C}{\mathrm{O}}_{2}}=0.262$), (

**D**) C40 (${x}_{\mathrm{C}{\mathrm{O}}_{2}}=0.360$).

**Figure 3.**Conversion rate ${r}_{c}$ with respect to $\mathrm{C}{\mathrm{H}}_{4}$ content in off-gas ${c}_{C{H}_{4},off}$ for each feed gas composition. Markers: the “×” show mean values the processes were running at, the “+” show theoretical maximum.

Source | ${\mathbf{CO}}_{2}$ | ${\mathbf{CH}}_{4}$ | ${\mathbf{N}}_{2}$ | Other |
---|---|---|---|---|

weak gas (PSA) | 91% to 99% | 1% to 8% | 0% to 3% | 0% to 1% |

weak gas (WW) | 11% to 22% | 0.1% to 0.3% | 58% to 63% | 17% to 20% |

coal seam | 24% | 90% to 95% | 1% to 8% | 0% |

inactive coalmine | 8% to 15% | 60% to 80% | 5% to 32% | 0% |

blast furnace | 23% | 0% | 49% | 28% |

**Table 2.**Composition of mixed gases and volumetric amounts of ${\mathrm{H}}_{2}$, ${\mathrm{CO}}_{2}$, and ${\mathrm{N}}_{2}$ in the feed gas.

$\mathbf{C}{\mathbf{O}}_{2}$-Containing Feed Gas | Measured Mole Fraction | Resulting Composition of the Whole Feed Gas | |||
---|---|---|---|---|---|

${\mathit{x}}_{{\mathbf{N}}_{2}}$ | ${\mathit{x}}_{{\mathbf{CO}}_{2}}$ | ${\mathbf{H}}_{2}$ | ${\mathbf{CO}}_{2}$ | ${\mathbf{N}}_{2}$ | |

C20 | 0.816 | 0.184 | $42.4$% | $10.6$% | $47.0$% |

C30 | 0.738 | 0.262 | $51.2$% | $12.8$% | $36.0$% |

C40 | 0.640 | 0.360 | $59.0$% | $14.8$% | $26.2$% |

C100 | 0 | 1 | $80.0$% | $20.0$% | 0% |

**Table 3.**Overview of the mean feed and off-gas concentrations and flows using pure ${\mathrm{CO}}_{2}$ (

**C100**). Concentration of ${\mathrm{CO}}_{2}$, ${\mathrm{H}}_{2}$, and $\mathrm{C}{\mathrm{H}}_{4}$ were measured values, ${\mathrm{N}}_{2}$ was calculated.

${\mathbf{H}}_{2}$ | $\mathbf{C}{\mathbf{O}}_{2}$ | ${\mathbf{N}}_{2}$ | $\mathbf{C}{\mathbf{H}}_{4}$ | ${\mathit{r}}_{\mathit{c}}$ | |
---|---|---|---|---|---|

${c}_{feed}$ | 80% | 20% | 0% | 0% | 0 |

${\dot{V}}_{feed}$ | 300 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | 75 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | 0 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | 0 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | |

${c}_{off}$ | $0.7$% | $2.7$% | 0% | $96.6$% | $99.3$% |

${\dot{V}}_{off}$ | $0.5$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $2.1$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | 0 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | $74.5$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} |

**Table 4.**Overview of the mean feed and off-gas concentrations and flows using

**C20**. Concentration of ${\mathrm{CO}}_{2}$, ${\mathrm{H}}_{2}$, and $\mathrm{C}{\mathrm{H}}_{4}$ were measured values, ${\mathrm{N}}_{2}$ was calculated.

${\mathbf{H}}_{2}$ | $\mathbf{C}{\mathbf{O}}_{2}$ | ${\mathbf{N}}_{2}$ | $\mathbf{C}{\mathbf{H}}_{4}$ | ${\mathit{r}}_{\mathit{c}}$ | |
---|---|---|---|---|---|

${c}_{feed}$ | $42.4$% | $10.6$% | $47.0$% | 0% | 0 |

${\dot{V}}_{feed}$ | 220 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | 55 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | $243.9$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | 0 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | |

${c}_{off}$ | $0.6$% | $3.9$% | $80.5$% | $15.0$% | $84.9$% |

${\dot{V}}_{off}$ | $2.0$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $12.1$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $243.9$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $46.7$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} |

**Table 5.**Overview of the mean feed and off-gas concentrations and flows using

**C30**. Concentration of ${\mathrm{CO}}_{2}$, ${\mathrm{H}}_{2}$, and $\mathrm{C}{\mathrm{H}}_{4}$ were measured values, ${\mathrm{N}}_{2}$ was calculated.

${\mathbf{H}}_{2}$ | $\mathbf{C}{\mathbf{O}}_{2}$ | ${\mathbf{N}}_{2}$ | $\mathbf{C}{\mathbf{H}}_{4}$ | ${\mathit{r}}_{\mathit{c}}$ | |
---|---|---|---|---|---|

${c}_{feed}$ | $51.2$% | $12.8$% | $36.0$% | 0% | 0 |

${\dot{V}}_{feed}$ | 300 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | 75 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | $211.3$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | 0 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | |

${c}_{off}$ | $1.0$% | $4.7$% | $71.6$% | $22.7$% | $90.9$% |

${\dot{V}}_{off}$ | $3.1$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $14.3$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $211.3$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $68.2$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} |

**Table 6.**Overview of the mean feed and off-gas concentrations and flows using

**C40**. Concentration of ${\mathrm{CO}}_{2}$, ${\mathrm{H}}_{2}$, and $\mathrm{C}{\mathrm{H}}_{4}$ were measured values, ${\mathrm{N}}_{2}$ was calculated.

${\mathbf{H}}_{2}$ | $\mathbf{C}{\mathbf{O}}_{2}$ | ${\mathbf{N}}_{2}$ | $\mathbf{C}{\mathbf{H}}_{4}$ | ${\mathit{r}}_{\mathit{c}}$ | |
---|---|---|---|---|---|

${c}_{feed}$ | $59.0$% | $14.8$% | $26.2$% | 0% | 0 |

${\dot{V}}_{feed}$ | 300 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | 75 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | $133.3$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | 0 $\mathrm{m}$ $\mathrm{L}$ $\mathrm{min}$^{−1} | |

${c}_{off}$ | $1.5$% | $5.4$% | $62.7$% | $30.5$% | $89.9$% |

${\dot{V}}_{off}$ | $3.2$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $12.0$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $133.3$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} | $67.4$$\mathrm{m}$$\mathrm{L}$ $\mathrm{min}$^{−1} |

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**MDPI and ACS Style**

Hoffarth, M.P.; Broeker, T.; Schneider, J. Effect of N_{2} on Biological Methanation in a Continuous Stirred-Tank Reactor with *Methanothermobacter marburgensis*. *Fermentation* **2019**, *5*, 56.
https://doi.org/10.3390/fermentation5030056

**AMA Style**

Hoffarth MP, Broeker T, Schneider J. Effect of N_{2} on Biological Methanation in a Continuous Stirred-Tank Reactor with *Methanothermobacter marburgensis*. *Fermentation*. 2019; 5(3):56.
https://doi.org/10.3390/fermentation5030056

**Chicago/Turabian Style**

Hoffarth, Marc Philippe, Timo Broeker, and Jan Schneider. 2019. "Effect of N_{2} on Biological Methanation in a Continuous Stirred-Tank Reactor with *Methanothermobacter marburgensis*" *Fermentation* 5, no. 3: 56.
https://doi.org/10.3390/fermentation5030056