Achievement

The Xylose Utilization Index (XUI)

In the total solution experiment, we strive to measure the carbon fixation amount of each sample. After reading numerous publications, we found out that previous researches determine the efficiency of carbon fixation via measuring the decrease of carbon dioxide concentration in the closed system or measure the weight percentage of ¹⁴C radioisotope in the dry cell. However, due to biosafety constrain of our lab, we can barely use the radioisotope. Measuring the decrease of carbon dioxide concentration in the closed system is also impractical for us since we have too much test samples. A new method to measure multiple samples in the short period of time is developed by our team. We are able to evaluate the fixation efficiency of each sample with optical density O.D. 600 and xylose consumption. We have measure various construction to prove that the enzyme of our construction is necessary for carbon fixation.

The test samples below were incubated in a modified M9 medium which substitutes xylose for glucose. 1/1000 of Luria-Bertani (LB) medium was added to support trace elements. Since the concentration of LB medium is too low, it doesn’t contribute to the carbon source of the bacteria.

We defined a new index, Xylose Utilization Index (XUI), to describe the potential of carbon fixation. We can compare this index of each strain to find out the strain that has highest capacity of carbon fixation.

To define the XUI, we firstly made two assumptions:

1. O.D. 600 of the sample has linear relationship to dry cell weight (biomass). Optical density is frequently used as a means of describing the cell density in the broth. We measured the dry cell weight of samples in different O.D. value and discovered that it has linear relationship. We conclude that we can utilize O.D. value to estimate the dry cell weight. 1 O.D. of BL21 (DE3) strain per litre yields the dry cell weight of 0.8 gram.


Fig 1. shows the dry cell weight of BL21 (DE3) incubated in modified M9 xylose medium. A linear relationship between O.D. and dry cell weight is observed.

2. The elemental formula of E. coli should be fixed or varies within a small range. Although the formula may have variations in different growth condition, we assume that such error can be ignore during the following calculation.

Combining these two assumptions, we can conclude that in a fixed O.D. 600 value, the composite weight of carbon is also fixed. Thus, O.D. 600 can be considered equivalent to carbon weight of the bacteria.

After these two assumptions, the XUI is designed to evaluate the carbon fixation ability of each strain. The definition of the index is xylose consumption over O.D. 600. O.D. 600 measurement can be viewed as the weight of carbon of the bacteria. The index shows the ratio of xylose consumption per biomass. For wild type E. coli, it only consumes xylose (the sole carbon source provided by the medium) as its carbon source. Although some native E. coli pathway may utilize CO₂ (such as lipid synthesis), the amount is too small to be considered. As for engineered strain, carbon dioxide can be utilized as its carbon source. By producing same amount of carbon biomass, it requires less xylose. We can thus compare the XUI of each strain to determine the strain that fix carbon. The less the XUI in the sample, the more possibility that it fix carbon.

$${XUI = {{xylose \ consumption \ (g/l)} \over {O.D. 600}}}$$

We use the XUI to compare the carbon fixation efficiency of each strain and prove the function of each system. For the experiment result, please view the Result(hyperlink) page.

Carbon Fixation amount estimation

To find out how much and how efficient genetically engineered E. coli can fix carbon dioxide, we use the material balance concept to evaluate the heterotrophic CO₂ fixation process. Consider a system composed of a single component, the general material balance can be written as: $${\{Input\ to\ the\ system\}\ –\ \{Output\ to\ the\ system\}\ =\ \{Accumulation\ in\ the\ system\}}$$ A system can be defined as an arbitrary portion of a process considered for analysis, in which in this case, is an engineered carbon capturing E. coli.

The engineered E. coli BL21 (DE3) is cultured in M9 medium with formula adjusted so that xylose is the sole carbon source. The aforementioned M9 Medium contains 4 (g/l) xylose and 1/1000 LB medium (the carbon consumed from LB medium can be ignored). By applying the law of conservation of mass, which states that mass may neither be created nor destroyed, the material balance for carbon in an engineered E. coli may simply be written as $${\{C_{CO_2}\ in\}\ +\ \{C_{xylose}\}\ -\ \{C_{CO_2}\ out\}\ -\ \{C_{waste}\}\ =\ \{C_{biomass}\}...(1)}$$ Considering the difficulties in measuring carbon in E. coli metabolic waste and that C_waste would be positive, the equation reduces to $${\{C_{CO_2}\ in\}\ -\ \{C_{CO_2}\ out\}\ ≥\ \{C_{biomass}\}\ -\ \{C_{xylose}\}...(2)}$$ Let {C_CO2 net}= {C_CO2 in} - {C_CO2 out}, equation (2) further simplifies to $${\{C_{CO_2}\ net\}\ ≥\ \{C_{biomass}\}\ -\ \{C_{xylose}\}...(3)}$$ If C_waste is very small and negligible, we can obtain the net amount of carbon dioxide fixed over time. If, on the contrary, C_waste cannot be neglected, equation (3) allows us to estimate the minimum net amount of carbon dioxide fixed.

C_biomass can be calculate by multiplying O.D. 600 to DCW and mass percent of carbon in E. coli biomass. The O.D. 600 of engineered E. coli is measured after a 12-hour cultivation and the result obtained is 0.45O.D. . Yin Li et al. reported that dry cell weight (DCW) of E. coli is $${0.35g\over L ∙ 𝑂.𝐷. 600}$$ , determined by experiment. E. coli biomass contains 48% of carbon by mass. $${C_{biomass}\ =\ 0.4511\ ×\ 0.35\ ×\ 48\%}$$ $${=\ 0.0758\ g/L}$$

On the other hand, C_xylose can be calculated by multiplying the amount of xylose consumed per unit volume of broth to the mass percent of carbon in xylose. Xylose consumption is calculated by using a DNS kit that measures the concentration of reducing sugar and the result obtained is 0.1723g of xylose consumed per liter of M9 medium. Carbon mass percentage of xylose is 40%. $${C_{xylose}\ =\ 0.1723\ ×\ 40\%\ =\ 0.0689\ g/L}$$ By equation (3) $${C_{CO_2\ net}\ =\ 0.0758\ -\ 0.0689}$$ $${=\ 0.0069\ g/L}$$ Since the E. coli has been cultured for 12 hours, we can calculate the rate of carbon fixation by $${Rate\ of\ carbon\ fixation\ =\ {𝐶_{𝐶𝑂_2\ 𝑛𝑒𝑡}\over 12}}$$ $${=\ {0.0069\over 12}}$$ $${=\ 0.575\ {mg\over L ∙hr}}$$ To find out how much carbon in biomass comes from the carbon in CO2 captured by the heterotrophic microbes, we can divide equation (3) by the mass percentage of carbon in biomass:

$${{{ \{ CO_{2 net}} \} \over \{ {C_{biomass}} \} } \geq {1 - { \{ {C_{xylose}} \} \over \{ {C_{biomass}} \} }}}$$

We can thus calculate the ratio with our experiment results:

$${{Ratio \ of \ carbon \ in \ CO_2 \ fixed \ to \ carbon \ in \ biomass} = {1 -{0.0689 \over 0.0758}} = 9.1 \%}$$

References

1. Gong, F., Liu, G., Zhai, X., Zhou, J., Cai, Z., & Li, Y. (2015). Quantitative analysis of an engineered CO₂-fixing Escherichia Coli reveals great potential of heterotrophic CO₂ fixation. Biotechnology for Biofuels,8(1). doi:10.1186/s13068-015-0268-1
2. Stockar, U. V., & Liu, J. (1999). Does microbial life always feed on negative entropy? Thermodynamic analysis of microbial growth. Biochimica Et Biophysica Acta (BBA) - Bioenergetics,1412(3), 191-211. doi:10.1016/s0005-2728(99)00065-1