Difference between revisions of "Team:UESTC-China/Model h2"

In order to find the best fermentation conditions of hydrogen, we designed the experiment and obtained the hydrogen production function by using the experimental data. The optimal fermentation conditions and maximum yield were obtained for the optimal solution of the function. Then we tried again under the best fermentation conditions. The result shows that the yield has obviously increased.

Hydrogen is the richest and lightest element in the universe and has great potential for development. Hydrogenases catalyze one of the simplest chemical reactions, \({\rm{2}}{{\rm{H}}^{\rm{ + }}}{\rm{ + 2}}{{\rm{e}}^{\rm{ - }}} \leftrightarrow {{\rm{H}}_{\rm{2}}}\), but their structures are very complex. We try to establish the kinetic model of the hydrogen production process and verify that the modified Escherichia coli has high hydrogen production ability.

Similar to the process of butanol fermentation, hydrogen fermentation also has three factors: temperature, inoculation quantity, initial pH. The kinetic equation of hydrogen synthesis was established and the factors affecting hydrogen fermentation were analyzed to optimize the process of hydrogen synthesis in order to increase the production of hydrogen.

The range and standardized values of the temperature \({{\rm{x}}_{\rm{1}}}\), inoculation quantity \({{\rm{x}}_{\rm{2}}}\)and initial pH \({{\rm{x}}_{\rm{3}}}\) , are shown in the following table:

Code	Variable	Low level(-1)	Zero level(0)	High level(+1)
\[{{\rm{x}}_{\rm{1}}}\]	Temperature（℃）	32	37	42
\[{{\rm{x}}_{\rm{2}}}\]	Inoculation quantity（%）	0.6	1	1.4
\[{{\rm{x}}_{\rm{3}}}\]	Initial pH	5	6	7

The test points of the BBD test design and the corresponding experimental data are shown in the following table:

Test number	\[{z_1}\]	\[{z_2}\]	\[{z_3}\]	\[y\](ml/36h)
1	0	0	0	1.587
2	0	0	0	1.588
3	0	0	0	1.587
4	0	1	1	1.12
5	0	1	-1	0.864
6	-1	0	1	2.139
7	1	-1	0	1.227
8	0	0	0	1.587
9	-1	-1	0	1.213
10	-1	1	0	1.857
11	1	0	-1	1.116
12	1	1	0	1.264
13	0	-1	1	1.489
14	0	-1	-1	1.242
15	-1	0	-1	1.355
16	0	0	0	1.588
17	1	0	1	1.102

Multivariate regression analysis of the experimental data was performed using Design-Expert11 software, and the following third-order polynomial equations were found to explain the generation of hydrogen:

The variance analysis of the regression analysis results is shown in the following table:

Source	Sum of Squares	df	Mean Square	F-value	P-value
Model	0.0522	12	0.0043	7398.36	< 0.0001
\[{z_1}\]	0	1	0	107.2	< 0.0001
\[{z_2}\]	0.0012	1	0.0012	2454	< 0.0001
\[{z_3}\]	0.0188	1	0.0188	3753	< 0.0001
\[{z_1}{z_2}\]	0.0006	1	0.0006	1250	< 0.0001
\[{z_1}{z_3}\]	0	1	0	4.5	< 0.0001
\[{z_2}{z_3}\]	0.0004	1	0.0004	722	< 0.0001
\[{z_1}^2\]	0.0135	1	0.0135	27001.18	< 0.0001
\[{z_2}^2\]	0.0012	1	0.0012	2398.03	< 0.0001
\[{z_3}^2\]	0.0003	1	0.0003	590.66	< 0.0001
\[{z_1}{z_2}{z_3}\]	0.0038	1	0.0038	7569	< 0.0001
\[{z_1}^2{z_3}\]	0.0067	1	0.0067	13340.25	< 0.0001
\[{z_1}{z_2}^2\]	0.0005	1	0.0005	930.25	< 0.0001
Pure Error	0	4	0
Cor Total	1.58	16
Coefficient of determination \[{R^2} = 0.9997\]			Adequate Precision=167.3256

The variance analysis of the regression analysis results is shown in the following table: the F value of the model is 7398.36, P<0.0001, indicating that the model fits well, and the model's goodness of fit R^2 is 0.9998, indicating that the regression model 99.98% explains the response. Only 0.02% of the model's changes cannot be explained by the model. Moreover, accuracy is used to describe the signal-to-noise ratio of the model [4]. The signal-to-noise ratio is 167.3256. Usually, the ratio is greater than 4, and the model can be applied to guide the experimental design and also to indicate an accurate signal. From the significant coefficients of the regression model, it was found that each factor had a significant effect on the yield of hydrogen.

The optimization of the model makes the partial derivative of the equation, and the derivative is 0, which constitutes a ternary equation system. By solving the equations, the optimal fermentation conditions are obtained [5]. The optimal results of the model: the temperature was 30.222 ° C, the initial substrate concentration was 1.128 %, the initial pH was 6.868, and the maximum yield of hydrogen was 2.162 ml / 36h.

The kinetic response surface of hydrogen synthesis, the three-dimensional response surface and the corresponding two-dimensional contour map are shown in Fig.1. The figure shows that three levels of each of the two independent factors explore their effect on hydrogen synthesis in an optimal range. It can be seen from the figure that each response surface has a distinct peak, indicating that the optimal fermentation conditions for hydrogen are within the selected experimental design range.

Figure（a）and（b）show the effect of initial substrate and temperature on hydrogen fermentation. Hydrogen production increased first and then decreased with the increase of temperature. Similarly, hydrogen production increased first and then decreased with the increase of initial substrate. The amount of hydrogen biosynthesis is more affected by the initial substrate than the temperature. Figure（c）and（d）show the effect of initial pH and temperature on hydrogen fermentation. Hydrogen production increased with the increase of initial pH. The amount of hydrogen biosynthesis is more affected by the initial pH than the temperature. Figure（e）and （f）show the effect of initial substrate and initial pH on hydrogen fermentation. The amount of hydrogen biosynthesis is more affected by the initial substrate than initial pH.

In summary, the three independent factors affect the size of butanol: initial substrate concentration > initial pH > temperature. Only when these three significant influence factors are in the optimal region, the amount of hydrogen synthesis can be maximized.

According to the prediction results of the regression equation, the model was further verified under the optimal conditions of hydrogen fermentation. The verification results are shown in Fig.2. After 36h, the hydrogen production reached a maximum of 2.162 ml, which was 77.08% higher than the highest hydrogen production before optimization, and the maximum synthesis rate of hydrogen was significantly improved.

Hydrogenase catalyzes one of the simplest chemical reactions,\({\rm{2}}{{\rm{H}}^{\rm{ + }}}{\rm{ + 2}}{{\rm{e}}^{\rm{ - }}} \leftrightarrow {{\rm{H}}_{\rm{2}}}\), but their structure is very complex[1]. So we consider simplifying the model. A molecular dynamics model was established to simulate the hydrogen production process. By comparing the theoretical hydrogen production with that in other literatures, it is proved that the hydrogen production of our constructed Escherichia coli is relatively high.

Because the hydrogenase catalytic reaction is very complex, we simplify the reaction to the enzymatic redox reaction shown below.

This reaction can be written as follows:

Using MATLAB 2014a to solve the above differential equations, the following parameters are used:

Parameter	Variable Name	Value	Reference
Michaelis Constant for HydA	\[k_m^{Hyda}\]	800mM	[2]
Catalysed rate of reaction for HydA	\[k_{cat}^{Hyda}\]	29000s^(-1)	[2]
Inhibition constant for \({{\rm{H}}_{\rm{2}}}\)	\[k_i^{{{\rm{H}}_{\rm{2}}}}\]	16 mM	[2]

Using MATLAB 2014a to solve the above differential equations, the following parameters are used:

Parameter	Variable Name	Value	Reference
Michaelis Constant for HydA	\[k_m^{Hyda}\]	800mM	[2]
Catalysed rate of reaction for HydA	\[k_{cat}^{Hyda}\]	29000s^(-1)	[2]
Inhibition constant for \({{\rm{H}}_{\rm{2}}}\)	\[k_i^{{{\rm{H}}_{\rm{2}}}}\]	16 mM	[2]

The result is shown in Fig.4: the maximum hydrogen production is 1.63mmol/g（gulose）,Hydrogen production rate per unit conversion is 1.8 \(\mu {\rm{mol}}\)(mg protein) \(^{{\rm{ - 1}}}{{\rm{h}}^{{\rm{ - 1}}}}\).

By comparing the results with the butanol production in other literatures, it is found that the hydrogen production of the constructed bacteria is in a high position.

System	\({{\rm{H}}_{\rm{2}}}\) production rate (converted units)	Reference
Expression of the Ralstonia eutropha SH operon	0.3μmol\({{\rm{H}}_{\rm{2}}}\) (mg protein)\(^{ - 1}{{\rm{h}}^{{\rm{ - 1}}}}\)	[3]
Expression of [NiFe]-hydrogenase 2 from Citrobacter sp. SG	2.6μmol\({{\rm{H}}_{\rm{2}}}\) (mg protein)\(^{ - 1}{{\rm{h}}^{{\rm{ - 1}}}}\)	[3]
Expression of HxEFUYH hydrogenase and the maturation proteins HypABCDEF and HoxW from Synechocystis sp. PCC 6803	0.004μmol\({{\rm{H}}_{\rm{2}}}\) (mg protein)\(^{ - 1}{{\rm{h}}^{{\rm{ - 1}}}}\)	[3]

[1] Gönül Vardar‐Schara, Toshinari Maeda, and Thomas K. Wood. Metabolically engineered bacteria for producing hydrogen via fermentation. Microb Biotechnol. 2008 Mar; 1(2): 107–125.

[2] Pierre-Pol Liebgott, Fanny Leroux, Bénédicte Burlat, Sébastien Dementin, Carole Baffert, Thomas Lautier, Vincent Fourmond, Pierre Ceccaldi, Christine Cavazza, Isabelle Meynial-Salles, Philippe Soucaille, Juan Carlos Fontecilla-Camps, Bruno Guigliarelli, Patrick Bertrand, Marc Rousset & Christophe Léger. Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase. Nature chemical biology, 2009(6):63-70.

[3] Toshinari Maeda, Kien Trung Tran, Ryota Yamasaki3, Thomas K. Wood. Current state and perspectives in hydrogen production by Escherichia coli: roles of hydrogenases in glucose or glycerol metabolism. Applied Microbiology and Biotechnology, 2018（102）: 2041-2050.