NOVEL METHOD FOR DETERMINATION OF GAS CONSUMPTION IN

NOVEL METHOD FOR DETERMINATION OF GAS CONSUMPTION IN Cupriavidus necator:

ASSESSING FEASABILITY OF CO2 FIXATION FROM BIOMASS-DERIVED SYNGAS

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI’I AT MĀNOA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

MOLECULAR BIOSCIENCES AND BIOENGINEERING

DECEMBER 2012

By

Allexa R. Dow

Thesis Committee:

Jian Yu, Chairperson Dulal Borthakur Sean Callahan

Allexa Dow MBBE Thesis

2

ABSTRACT

Synthesis gas (syngas) is an industrially important feed stock for electricity, hydrogen and liquid

fuel production. Biomass-derived syngas has high CO2 levels and alternative methods of CO2 removal are

needed to implicate biomass gasification systems. This research identified and tested a possible method

of biological CO2 fixation from syngas. The chemolithoautotrophic organism Cupriavidus necator

produces PHB and offers a valuable product as a result of CO2 fixation. The energy efficiency of biological

CO2 fixation using chemolithoautotrophic growth supported by hydrogen, oxygen and carbon dioxide was

theoretically analyzed and then tested in C. necator using a novel experiment. The experimental design

included cultivating C. necator in a mineral solution inside of plastic gas sampling bags under constant

temperature and pressure. The gas composition of the bag was monitored over time using a GC/TCD

system. To determine the consumption of the individual gasses H2, O2 and CO2 the total moles of the gas

mixture in the bag was monitored using the inert gas methane (CH4). The efficiency of

chemolithoautotrophic growth was determined from gas consumption. It was found that there was a

large discrepancy between the maximum theoretical energy efficiency and the actual efficiency. It was

found that the gas consumption requirements of C. necator are 1:1.8:5.8 (mol CO2 : mol O2 : mol H2). This

hydrogen requirement is almost three times more than the theoretically possible, and results in less than

30% of hydrogen consumed for growth being stored as reduced CO2, or bacterial biomass.


3

TABLE OF CONTENTS

ABSTRACT ........................................................................................................................................... 2

LIST OF TABLES ................................................................................................................................... 5

LIST OF FIGURES .................................................................................................................................. 6

CHAPTER 1. INTRODUCTION AND OBJECTIVES ..................................................................................... 7

1.1 Introduction ................................................................................................................................. 7

Syngas .......................................................................................................................................... 7

Chemolithoautotrophic growth of C. necator: The Calvin cycle and hydrogenase enzymes .............. 8

Theoretical energy efficiency of chemolithoautotrophic growth ................................................... 10

1. MBH activity: ATP formation from hydrogen oxidation .................................................... 11

2. SH activity: NAD(P)H formation from hydrogen oxidation ................................................ 12

3. ATP and NAD(P)H requirement for CO2 fixation via the CBB cycle ..................................... 12

4. Energy efficiency of CO2 fixation ...................................................................................... 12

1.2 Objectives................................................................................................................................... 15

CHAPTER 2. MATERIALS AND METHODS ............................................................................................ 16

2.1 Chemicals, media and cultures ..................................................................................................... 16

2.2 Bioreactor cultivation .................................................................................................................. 17

2.3 Determination of PHB percentage in FDCM .................................................................................. 18

CHAPTER 3. 16S RNA ANALYSIS ......................................................................................................... 19

3.1 Isolation of DNA and amplification of 16S RNA gene ..................................................................... 19

3.2 Species identification using sequence of 16S RNA gene ................................................................ 19

CHAPTER 4. CHEMOLITHOAUTOTROPHIC BIOREACTOR CULTIVATION ................................................ 20

4.1 Specific growth rate and doubling time ........................................................................................ 20

4.2 Comparison of cellular performance with previous findings .......................................................... 21


4

4.3 PHB accumulation throughout cultivation- Implications of nutrient deficiency .............................. 23

CHAPTER 5. METHOD FOR EVALUATING H2, O2 AND CO2 CONSUMPTION ............................................ 25

5.1 Theoretical method development ................................................................................................ 25

5.2 Experimental design .................................................................................................................... 27

5.3 Gas chromatography method for H2, O2 and CO2 analysis ............................................................. 30

5.4 Protocol for measuring molar change of H2, O2 and CO2 during chemolithoautotrophic growth ..... 30

Inoculum preparation ........................................................................................................... 30

Inoculating gas bag cultures .................................................................................................. 31

Gas sampling with the GC machine ........................................................................................ 32

Determination of bag volume ................................................................................................ 33

Incubation ............................................................................................................................ 33

5.5 Calibration curve relating GC absolute area to volume percentage for H2, O2 and CO2 ................... 33

CHAPTER 6. H2, O2 AND CO2 CONSUMPTION DURING CHEMOLITHOAUTOTROPHIC GROWTH.............. 35

6.1 Data analysis ............................................................................................................................... 35

6.2 Results: Gas consumption ratios of chemolithoautotrophic growth .............................................. 37

6.3 Results: Efficiency of CO2 fixation ................................................................................................. 41

6.4 Results: Total molar consumption of H2, O2, and CO2 .................................................................... 42

6.5 Results: Rate of CO2 fixation ........................................................................................................ 43

6.6 Testing repeatability of H2/CO2 and H2/O2 ratio measurements using ANOVA ............................... 44

CHAPTER 7. DISCUSSON AND FUTURE RESEARCH ............................................................................... 46

7.1 Discussion ................................................................................................................................... 46

7.2 Future Research-Improving CO2 fixation efficiency with genetically engineered C. necator ............ 47

REFERENCES ..................................................................................................................................... 50


5

LIST OF TABLES

Table 1. Calculating percentage of hydrogen recovered as biomass depending on the P/2e- ratio

of the microbial cell .............................................................................................................. 13

Table 2. Summary of H2 and CO2 volume percentages in syngas products from various biomass

gasification agents and coal................................................................................................... 14

Table 3. Bioreactor growth parameters ............................................................................................. 21

Table 4. Results of gas consumption during chemolithoautotrophic growth in a closed-system

bag culture for five independent replicates ........................................................................... 38

Table 5. Total molar consumption of the individual gasses H2, O2, and CO2 during

chemolithoautotrophic growth ............................................................................................. 42

Table 6. Data used from gas consumption experiment to calculate rate of CO2 consumption

expressed in gCO2/gFDCM ∙hr-1 ............................................................................................. 43

Table 7. Results of ANOVA testing for H2/CO2 and H2/O2 ratios obtained from averages across

three sampling periods for five replicates .............................................................................. 45


6

LIST OF FIGURES

Figure 1. The MBH and SH operon organization and structural models of the two

energy-conserving hydrogenase enzymes from C. necator ..................................................... 10

Figure 2. Bacterial growth curve from bioreactor cultivation .............................................................. 20

Figure 3. PHB accumulation throughout bioreactor cultivation ........................................................... 23

Figure 4. Method of closed-system chemolithoautotrophically cultivated C. necator for

analysis of gas consumption .................................................................................................. 28

Figure 5. Apparatus to measure volume of gas sampling/culture bag at any point during

gas consumption experiment ................................................................................................ 29

Figure 6. Calibration curve between water displacement measured in millimeters and

actual volume in milliliters .................................................................................................... 29

Figure 7. Method of bag culture inoculation using a syringe barrel with a conical tip .......................... 32

Figure 8. Calibration equations showing correlation between the volume percentage

of standard gas mixtures and the absolute area detected by the GC machine ......................... 34

Figure 9. Flowchart of data analysis applied to each set of successive gas samples obtained

during a single gas consumption experiment ......................................................................... 36

Figure 10. Average H2/CO2 and H2/O2 ratios of chemolithoautotrophic growth for each of the

five replicates as well as the average of all replicates over time intervals 1-3 .......................... 39


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CHAPTER 1. INTRODUCTION AND OBJECTIVES

1.1 Introduction

Syngas is an energy-rich gas captured from gasification, a controlled process of burning biomass, and

has been produced for over a century as a fuel source. This gas consists mainly of hydrogen (H2) and

carbon monoxide (CO) but depending on the gasification agent carbon dioxide (CO2) is largely present as

well. H2 and CO are energy-rich molecules captured during syngas production, but CO2 inhibits their

downstream reactivity and must be removed from the syngas. Biomass-derived syngas has high CO2

levels and is therefore undesirable to syngas engineers. The potential to replace current methods of CO2

removal from syngas with a process of microbial CO2 fixation is investigated.

The organism used in this research is the facultative chemolithoautotroph, Cupriavidus necator.

Microbial CO2 fixation using this organism could be an attractive CO2 removal technique due to the

production of bacterial biomass containing the bio-plastic substrate poly-3-hydroxybutyrate (PHB) in

large quantities, which can be extracted and transformed into renewable bio-plastic products or biofuels.

The purpose of this research is to evaluate the cellular efficiency of CO2 fixation in C. necator. First the

theoretical efficiency of CO2 fixation was determined based on the requirements of the Calvin cycle (CO2

fixation pathway) and potential hydrogen consumption of the two distinct energy-conserving

hydrogenase enzymes. Next, a novel experiment was designed and implemented to test the efficiency of

CO2 fixation. The efficiency of CO2 fixation is represented as the molar requirement of hydrogen for CO2

fixation and is presented as the H2/CO2 ratio.

Syngas

Syngas is the gaseous product obtained from a high temperature (750-850°C) controlled burn, called

gasification, of virtually any carbon source. Usually the carbonaceous gasification agents are natural gas,

naphtha, residual oil, petroleum coke, coal, and biomass [21, 28]. Recently however, many efforts have

been focused on the production of syngas from renewable sources of biomass, including lignocellulosic

biomass, biomass char, cyanobacterial blooms, bagasse, rice husks, glycerin and sewage sludge to name a

few [1, 8, 9, 15, 32, 34]. Although commonly referred to as syngas, the gas mixture obtained from

gasification can obtain vastly different ratios of the major product gasses H2, CO and CO2. The ratio of

CO/CO2 in syngas largely depends on the elemental composition of the gasification agent, and is an

important parameter for process engineers to understand and control [1].


8

Syngas is commonly used for hydrogen production, but it is also an important substrate for the

chemical catalytic synthesis of hydrocarbons and alcohols through industrial processes like Fischer-

Tropsch (F-T) synthesis [28]. Current technologies depend on the gasification of fossil fuels, especially

coal. Coal is preferred as a gasification agent because it has consistent elemental composition and is less

oxidized than biomass, resulting in a higher CO/CO2 ratio of the syngas product. During biomass

gasification the formation of CO2 is markedly increased due to the oxidized nature of biomass. Maximal

chemical transformation of syngas in downstream processes (F-T synthesis) requires a purified gas with a

fixed H2:CO ratio of approximately 2.15, therefore CO2 is removed from the syngas in multiple ways,

including re-carbonation to CaCO3 and post-combustion CO2 removal, prior to F-T synthesis [17, 26, 30,

33]. These CO2 removal processes typically account for 60-70% of the gasification plant’s capital costs

[10]. The inconsistency of syngas composition and the resulting increased CO2 fraction resulting from

lignocellulosic biomass gasification remains the largest hurdle for the development of a unified biomass

gasification process [17].

Syngas can be generated using a wide variety of raw biomass resources, although the decreased

CO/CO2 ratio and variable elemental composition of renewable biomass has largely restricted its use for

syngas production. Chemical technologies associated with fuel production from biomass-derived syngas

require specific H2/CO ratios and use gasification conditions (temperature, pressure, induction of water-

gas shift reaction) to control the syngas composition favoring the formation of CO over CO2, regardless of

markedly reduced gas yields [28]. Additionally, post-gasification CO2 removal drastically increases capital

costs and results in a complete loss of the CO2 fraction of the syngas. Efficient gasification plants should

allow for maximum gas yields and focus less on the specific syngas composition resulting from

gasification. Such a transition would be facilitated by a system to harness the CO2 fraction of the syngas in

an economical manner.

Chemolithoautotrophic growth of C. necator: The Calvin cycle and hydrogenase enzymes

Chemolithoautotrophic growth is characterized by an organism’s ability to gain energy in the form of

ATP from hydrogen-oxidation, and carbon for biomass generation from CO2 fixation. The process of

microbial CO2 fixation from syngas is plausible for organisms such as C. necator because the energy, H2,

and carbon, CO2, for chemolithoautotrophic growth are both products of gasification. Using C. necator

specifically to remove CO2 from syngas could offer additional value to the gasification plant due to the

accumulation of poly-3-hydroxybutyric acid (PHB), which can be extracted from cellular biomass and used

as a bio-plastic substrate [19, 27].


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C. necator is a facultative lithoautotrophic ß-proteobacterium, all genes necessary for this alternative

chemolithoautotrophic growth strategy are encoded on a native megaplasmid, and therefore C. necator

is thought to have acquired this characteristic in response to the scarce environmental availability of

hydrogen and CO2 [18, 25]. In reducing environments, C. necator can fix CO2 into bacterial cell mass via

the Calvin-Bensham-Bassham (CBB) cycle [6]. The energy for this process is provided by the cleavage of

elemental hydrogen into protons and electrons through a redox reaction performed by

Hydrogen:acceptor oxioreducatases, or hydrogenases, the enzymes that achieve hydrogen-oxidation

biologically [12].

C. necator possesses two functionally distinctly hydrogenases. These enzymes have been

characterized based on their cellular location. The membrane-bound hydrogenase (MBH) is located on

the periplasmic side of the inner cellular membrane and the soluble hydrogenase (SH) forms aggregates

in the cytoplasm [11]. Both of these enzymes are multimeric proteins encoded in a regulated gene

cluster known as the hox genes residing on the megaplasmid pHG1 in C. necator [11]. The MBH is linked

to the respiratory chain through association with a cytochrome-b like oxidase and is responsible for the

generation of a proton gradient as a result of hydrogen-oxidation into protons and electrons [3, 11]. The

SH however is unique because it is composed of a hydrogenase module and a FMN-accommodating

NADH dehydrogenase moiety which allows for the reduction of NAD+ at the direct expense of H2 [11]. The

organization of the hox gene cluster and the structure of the two hydrogenases including the Ni-Fe active

sites are shown in figure 1. The regulation of this gene cluster is controlled by a two-component sensor

kinase regulatory system with a regulatory hydrogenase (hoxA) being the NtrC family-like response

regulator, which can activate the σ54 RNA Polymerase, enabling transcription of the hox gene cluster

when hydrogen is present in the environment [11].


10

Figure 1. The MBH and SH operon organization (upper part) and structural models of the two energy-

conserving hydrogenase enzymes from C. necator (lower part). The unusual catalytic site-containing

proteins found in both hydrogenases are hoxG in the MBH and hoxH in the SH. Figure from Freidrich and

Lens 2005 [11].

Theoretical energy efficiency of chemolithoautotrophic growth

Existing knowledge about ATP generation and carbon fixation can be used to calculate the

maximum potential energy efficiency for microbial carbon fixation via the CBB Cycle. The CO2 fixation

efficiency of chemolithoautotrophic growth is represented by the molar amount of hydrogen required for

fixation of one mole of CO2. This theoretical analysis offers a range of CO2 fixation efficiencies to be

expected from our bacteria and will be used as a baseline for comparison when the actual CO2 fixation

efficiency is tested using our novel experimental design.

C. necator is a hydrogen-oxidizing bacterium, thereby obtaining the electrons required for both

energy and reducing equivalent formation from H2. The chemical energy from H2 is harnessed and stored

by two distinct hydrogenase enzymes, either as an electrochemical proton gradient or as NAD(P)H. The

MBH supplies electrons directly to the electron transport chain for oxidative phosphorylation leading to

ATP production, while the SH produces reducing equivalents in the form of NAD(P)H. The ATP/H2 (P/2e-)

ratio of chemolithoautotrophic growth in C. necator represents the energy required for carbon fixation

via the CBB cycle with hydrogen oxidation providing the energy for carbon fixation.


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1. MBH Activity: ATP formation from hydrogen oxidation

The periplasmically oriented MBH catalyzes the oxidation of hydrogen present in the

environment before it diffuses through the cellular membrane according to the following reaction:

H2 2H+ + 2e- [1]

The electrons generated from the MBH directly enter the electron transport chain at the level of

cytochrome b according to the following oxidation-reduction reaction [3]. The final electron acceptor is

O2, leading to the formation of water:

½ O2 + 2H+ + 2e- H2O (∆G’= -237.1 KJ/mol) [2]

Generation of a proton gradient to drive the F1F0-type ATPase resulting in formation of ATP from

ADP and Pi is achieved by activity of the MBH in two ways. As a result of equation 1, protons are

generated directly in the periplasm. Electrons from equation 1 enter the electron transport chain

through cytochrome b and proton translocation from the cytoplasm to the periplasm occurs as the

electrons are transferred between cytochromes. The reaction of ATP formation is simplified by the

following equation (equation 3):

ADP + Pi ATP (∆G’= +30.5 KJ/mol) [3]

The total amount of ATP formation is a product of the change in Gibbs free energy (equation 4),

and for ATP generation to be driven by free energy change, the value of n should be less than 8 (equation

5). The variable n represents the number of ATP formed per H2 (2e-) oxidized by the MBH, and is

represented by the ratio P/2e-. As is seen in equation 5, the maximum theoretical P/2e- ratio may be as

high as 8, but in bacterial cells this ratio is often lower. There have been many explanations offered for

the large difference observed between potential and actual ATP synthesis efficiencies, although no

distinct underlying cause has been demonstrated.

H2 + ½ O2 + n(ADP+Pi) H2O + nATP (∆G’= -237.1 + 30.5n Kcal/mol) [4]

0= -237.1 + 30.5n, 30.5n=237.1, n=237.1/30.5, n=7.77, n=8 [5]


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2. SH Activity: NAD(P)H formation from hydrogen oxidation

The soluble hydrogenase resides in the cytoplasm where NAD(P)+ is reduced thorough hydrogen

oxidation. The SH splits dihydrogen into protons and electrons through the reaction of equation 1 but

can channel the electrons to reduce NAD(P)+. One proton is released during the reduction of NAD(P)+ and

is available for transport to the periplasm through the electron transport chain (equation 6). Overall, the

oxidation of one hydrogen molecule by the SH reduces one NAD(P)+ molecule into NAD(P)H.

NAD(P)+ + 2H+ + 2e- NAD(P)H + H+ [6]

3. ATP and NAD(P)H requirement for CO2 fixation via the CBB cycle

Rubisco is the key enzyme of the CBB cycle, catalyzing carbon fixation from one molecule of

ribulose-1,5-bisphosphate (5 carbons) into two molecules of 3-phosphoglycerate (3 carbons). One ATP is

required per molecule of 3-phosphoglycerate formed, and one ATP is required for the regeneration of

ribulose-1,5-bisphosphate. Each of the two molecules of 1,3-bisphosphoglycerate formed from 3-

phosphoglycerate requires an NADPH for their reduction into glyceraldehyde-3-phosphate, resulting in

the release of Pi. Therefore, the CBB cycle requires 3 ATP and 2 NADPH for fixation of one CO2 molecule.

The net result of CO2 fixation via the CBB cycle is given in equation 7.

CO2 + 2NADPH + 3ATP CH2O [7]

4. Energy efficiency of CO2 fixation

Combining equations 4, 6 and 7 will give us the theoretical hydrogen requirement for CO2 fixation

via the CBB Cycle. However the ratio of P/2e-, given by equation 4 determines the energy efficiency of

the process. The percentage of hydrogen energy that is recovered as reduced biomass during

chemolithoautotrophic growth of C. necator depends on the ratio between the energy consumed by

hydrogen oxidation and the energy stored in reduced bacterial biomass, equations 4 and 6 respectively.

The energy consumed by hydrogen oxidation is represented by the P/2e- ratio, where a lower P/2e- ratio

leads to remarkably lower energy recovery in the form of reduced biomass (table 1). The energy

efficiency of the cell is the quotient of the energy released during carbohydrate oxidation and the energy

consumed during hydrogen oxidation according to the following equations 8 and 9 (∆H⁰f is the enthalpy

of formation (H2O) or combustion (CH2O) at standard conditions, 25⁰C, 1atm):


13

H2 + ½ O2 H2O (∆H⁰f = -285.8 kJ/mol) [8]

CH2O + O2 CO2 +H2O (∆H⁰f = -467.5 kJ/mol) [9]

Table 1. Calculating percentage of hydrogen recovered as biomass depending on the P/2e- ratio of the

microbial cell. P/2e- ratio represents the amount of ATP molecules produced per hydrogen molecule, H2.

H2/CO2 ratio of CO2 fixation through the CBB cycle requires 3 ATP and is largely affected by the P/2e-

ratio. H2/NADPH ratio is the requirement of hydrogen for generation of two NADPH needed by CBB cycle.

The H2/CO2 ratio represents the total potential requirements of the CBB cycle depending on the P/2e-

ratio of the cell. The hydrogen energy required for CO2 fixation is the product of the H2/CO2 ratio and the

energy of hydrogen oxidation (equation 8). The percentage of this energy recovered as biomass is the

energy of carbohydrate oxidation (equation 9) divided by the input hydrogen energy. The red box

indicates the lowest H2/CO2 ratio theoretically obtainable for chemolithoautotrophic growth using the

CBB cycle.

P/2e- (mol/mol) 8 6 4 3 2 1 0.5

H2/CO2 ratio of CO2 fixation (3ATP/[ P/2e-])

0.4 0.5 0.75 1 1.5 3 6

H2/NADPH (mol/mol) Reducing equivalent requirement of CBB

2 2 2 2 2 2 2

H2/CO2 (mol/mol) 2.4 2.5 2.75 3 3.5 5 8

Hydrogen Energy for CO2 fixation (kJ) (285.8kJ)(H2/CO2)

678.8 714.5 786 857.4 1000.3 1429 2286.4

Energy recovery as biomass (%) (467.5kJ)/ Hydrogen energy for CO2 fixation (kJ)

68.8% 65.4% 59.4% 54.5% 46.7% 32.7% 20.4%

From table 1 it is observed that the efficiency of chemolithoautotrophic growth in terms of

hydrogen energy recovered as biomass can vary from 68% at the theoretical maximum to about 30% or

even lower if the bacterial cell is not efficiently producing ATP from hydrogen. The theoretical

chemolithoautotrophically grown microbial cell can obtain 50% hydrogen energy recovery as reduced

biomass at a P/2e- ratio of about 2.5. This implies that the cell would consume almost three times the

molar amount of hydrogen for fixation of one mole of CO2 and half of this hydrogen energy would be

recovered as biomass. Relating to the CO2 fixation from syngas, if the cell grew at P/2e- of 2.5,

theoretically it would require almost 30% of the hydrogen in syngas to completely fix 10% CO2. Table 2

shows some typical syngas compositions obtained from the gasification of biomass agents, highlighting

the relative percentages of H2 and CO2 available in the syngas products. Syngas engineers must


14

determine how much of the hydrogen will be removed from the syngas for microbial CO2 fixation, and

according to expected syngas compositions (table 2), how much hydrogen, if any will be left over after

the CO2 has been consumed.

Table 2. Summary of H2 and CO2 volume percentages in syngas products from various biomass gasification agents and coal. The gas composition of H2 and CO2 is calculated excluding the syngas product N2. The gas composition reported represents the volumetric percentage of H2 or CO2 from the total of the products: CO, H2, CO2, CH4 and occasionally CnHm when reported. Information is a compilation of data from table 2, Alauddin et.al., 2010 [1] as well as coal-derived syngas composition information from table 3c, Chiesa et. al., 2005 [7].

Gasification Agent

Gas composition (vol%) excluding N2 H2/CO2 ratio of

syngas Hydrogen (H2) Carbon Dioxide (CO2)

Coal 36.8 9 6.50

Almond Shell 52.2 16.9 3.09

Wood Pellets 35.9 12.9 2.78

Larch Wood 56 29.2 1.92

Pine Sawdust 38.4 27.6 1.39

Coconut Shell 38.7 29.9 1.29

Olive Kernel 37.9 30.7 1.24

Bagasse 22 32 0.69

Rice Husk 9.2 33.3 0.28

Observing the boxes highlighted in red from tables 1 and 2 it can be seen that microbial CO2 fixation

from syngas must occur at a theoretically maximal level of ATP conversion from hydrogen for this process

to be performed efficiently. At a theoretically maximal level of hydrogen conversion to ATP the cell

should require about 2.4 times the molar amount of hydrogen than CO2 (red box, table 1). According to

table 2, only the most optimum parameters yield biomass-derived syngas with a H2/CO2 ratio above 2.4.

If this process of microbial CO2 fixation were employed to remove the CO2 fraction of the syngas, the

cellular efficiency of hydrogen conversion to ATP in C. necator will be the defining factor of process

efficiency, determining how much hydrogen will remain in the syngas product after CO2 fixation.


15

1.2 Objectives

The purpose of this research is to demonstrate the chemolithoautotrophic growth capacity and

gas requirements of our environmentally isolated strain of C. necator. There are four core objectives of

this research arising from the process of microbial CO2 fixation from syngas:

1. Species identification of current CO2 fixing lab strain. What is the process of CO2

fixation in our microbe?

2. What is the doubling time of this bacterium grown chemolithoautotrophically?

3. What is the molar requirement of hydrogen for CO2 fixation? Is the process of

carbon fixation theoretically efficient?

4. What is the total molar consumption of H2, O2 and CO2 during chemolithoautotrophic

growth?


16

CHAPTER 2. MATERIALS AND METHODS

2.1 Chemicals, media and cultures

Cultures of C. necator were grown heterotrophically on Yeast-Peptone-Meat (YPM) media. YPM

contains 5g/L yeast extract, 2.5g/L peptone, 2.5g/L meat extract and 1 g/L ammonium sulfate, and 15g/L

agar when solid media is used. Chemolithoautotrophic cultures of C. necator were cultivated in mineral

salts medium and incubated under an H2:O2:CO2 atmosphere of 7:1:1 [24]. The chemolithoautotrophic

gas atmosphere was created from compressed H2, O2 and CO2 using a gas proportioner flowmeter system

with three separate flow tubes and one mixing tube to control the flow rate of each gas separately (Cole-

Parmer). The actual gas composition was tested via gas chromatography with the same methodology and

operating conditions as described in chapter 5 (Varian GC, Carboxen PLOT 1006 column, thickness

0.15mm x 30m, Sigma-Aldrich). Mineral salts medium contains 2.3g/L KH2PO4, 2.9g/L Na2HPO4 ∙ 2H2O,

1g/L NH4Cl, 0.5g/L MgSO4∙ 7H2O, 0.5 g/L NaHCO3, 0.01 g/L CaCl2∙ 2H2O, 0.05 g/L ferric ammonium citrate,

1ml/L trace element solution and 15g/L agar when solid media is used [24]. To avoid precipitation, the

ferric ammonium citrate is prepared and autoclaved separately and added to the media once cool. Trace

element solution contains 0.6g/L H3BO3, 0.4g/L CoCl2∙ 6H2O, 0.2g/L ZnSO4∙ 7H2O, 0.06g/L MnCl2∙ 4H2O,

0.06g/L NaMoO4∙ 2H2O, 0.04g/L NiCl2∙ 6H2O and 0.02g/L CuSO4∙ 5H2O [24]. All chemicals and nutrients

were purchased from Sigma Aldrich.

A laboratory strain of C. necator was stored at 4⁰C on YPM agar slant tubes. This stock served as

the inoculum for 5ml YPM test tubes. Inoculated 5mL YPM test tubes were incubated at 30⁰C in an orbital

rotary incubator shaken at 200 rpm for 24 hours. One 5mL YPM culture was used to seed 100 mL of

mineral salts media in a 1L heavy-duty, vacuum-resistant polypropylene bottle (Nalgene, Thermo-

Scientific). Bottles were equipped with 3-port filling/venting caps enabling aseptic refilling of the

H2:O2:CO2 gas mixture during cultivation (Nalgene, Thermo-Scientific). Bottles were refilled twice daily

with the H2:O2:CO2 gas mixture during chemolithoautotrophic growth. The inlet gas was directed through

a membrane filter with a 55cm diameter and a pore size of 0.1µm immediately before entering the

bottles. Cultures from chemolithoautotrophically derived bottle cultures were aseptically transferred into

gas sampling bags with a sterile syringe for gas analysis experiments. All OD measurements were taken at

620nm on a Beckman DU-530 spectrophotometer and diluted accordingly so the absorbance was recorded

between 0.05 and 0.5.


17

2.2 Bioreactor cultivation

The chemolithoautotrophic growth capacity of C. necator was assessed in an open system, 2L

bioreactor (New Brunswick Scientific, Model # Q191900). The 2L reactor was inoculated with 100mL of

chemolithoautotrophically generated seed culture at an OD of about 3, leading to an initial bioreactor OD

of around 0.3. The H2:O2:CO2 atmosphere of 7:1:1 was mixed in the gas proportioner and fed into the

bioreactor at a total flow rate of 200mL/min. The gas enters the bioreactor through a membrane filter

with a 55cm diameter and a pore size of 0.1µm. The gas travels to the bottom of the bioreactor where it

enters the culture and an agitator increases gas mixing in solution, improving the mass transfer from gas

to liquid phase of the three essential gasses. The gas in the headspace of the bioreactor goes through a

condenser off the top of the bioreactor and leaves through an efflux port, again equipped with a

membrane filter. Constant gas flow through the bioreactor was insured by monitoring escape of gas from

the slightly submerged efflux port tube. A dissolved O2 sensor in the bioreactor controls the agitation

speed leading to increased oxygen supply when the dissolved oxygen level drops below 21%. The pH in

the bioreactor was maintained at 6.8 throughout cultivation by the action of an automatic pump

connected to 15% ammonia hydroxide (NH4OH), looped to the pH meter and added drop wise whenever

the pH dropped below 6.8. The temperature was maintained at 30⁰C with a heating jacket around the

culture vessel.

Culture samples were taken for OD measurements using a sampling loop in the bioreactor. A

50mL syringe was used to displace air and culture from the loop and the first 10mL of culture expelled

was used to clear the loop of old culture and disposed. The culture collection vessel was washed with

distilled water, dried and then used to collect another 10mL of culture. This sample was transferred to a

pre-weighed 15mL conical tube using a 10mL pipette. The cells were harvested by centrifugation at

8,000xg for 10 minutes, the liquid decanted and stored and the cell pellets were frozen for further use.

The OD was recorded from each sample after 10mL was removed. The frozen cell pellets were

gathered at the end of the bioreactor cultivation and freeze-dried for 24 hours. The dry cell pellets were

weighed in their conical tubes and the previously recorded tube weight was subtracted from this value

yielding the cell mass in grams harvested from 10mL of culture at a specifically recorded OD. The OD and

time course of the bioreactor cultivation was used to generate the specific growth rate and doubling time

of chemolithoautotrophic growth, using equations 10 and 11 where m stands for the cell density and t

stands for time:


18

Specific growth rate: µ=ln(mt2/mt1)/t2-t1 [10]

Doubling time (t2/t1): td= ln(2)/µ [11]

2.3 Determination of PHB percentage in FDCM

PHB content of FDCM was tested by organic solvent extraction of the polyester from FDCM

followed by analysis with a GC machine equipped with an FID detector. 50-100mg of FDCM was added to

2mL methanol solution (H2SO4 3% v/v; 10mg/ml benzoic acid as internal standard) in a test tube. 2mL of

chloroform was added and the tube shaken. The test tube was incubated at 100⁰C for 4 hours, and

cooled to ambient temperature. 1mL distilled water was added and vortexed vigorously for 2-3 minutes.

The organic and aqueous phases were allowed to separate. The bottom chloroform phase containing the

PHB fraction of the cells was transferred to a GC sampling vial using a long thin glass Pasteur pipette.

PHB samples were tested on the FID detector of the GC after separation on a Select™ Biodiesel

FAME capillary column (3m length x 0.32mm x 0.25µm, Varian, Varian 450-GC). The method for

compound separation in the column was as follows: helium is the carrier gas, air and hydrogen are used

for combustion in the FID which detects at 275⁰C, sample injection temperature is 250⁰C, initial split state

is 50, column oven starts at 100⁰C for 1 minute and increases 15⁰C a minute to 250⁰C and holds for 4

minutes totaling 15 minutes. The retention time of 3HBME is about 4 minutes and the retention time of

the standard BAME is about 5.1 minutes. Previously, internal standard calibrations have been performed

equating the area ratio of 3-hydroxybutyrate methyl ester (3HBME) and the internal standard benzoic

acid methyl ester (BAME) to the PHB percentage. The areas of 3HBME and BAME are recorded for each

sample and the following equations are used to determine the PHB%:

PHB (mg) = (3HBME/BAME)/0.0172 [12]

PHB wt% = PHB (mg)/FDCM (mg) x 100 [13]


19

CHAPTER 3. 16S RNA ANALYSIS

Our lab strain of bacteria was isolated from soil samples collected in Hawai’i and has previously

been shown to grow chemolithoautotrophically and produce PHB. These physiological characteristics

suggest that we have an isolate of C. necator. However, in order to theoretically examine the CO2 fixation

energy requirements in this organism we needed to confirm the identity of our microbial strain

molecularly. This molecular identification will distinguish the correct method of CO2 fixation to be used

for the theoretical analysis of CO2 fixation efficiency.

3.1 Isolation of DNA and amplification of 16S RNA gene

Chromosomal DNA was extracted from YPM overnight liquid cultures of the lab strain with a

commercially available chromosomal DNA preparation kit (ISOLATE Genomic DNA Mini Kit, Bioline). A

25µL Polymerase Chain Reaction (PCR) was run for 30 cycles, 55⁰C annealing temperature and a 4-minute

extension time with 2X Accuzyme (Bioline). The universal bacterial primers, forward primer fD1: 5’-AGA

GTT TGA TCC TGG CTC AG-3’ and reverse primer rD1: 5’- AAG GAG GTG ATC CAG CC-3’ were

used and the PCR product was visualized on a 1% agarose gel using ethidium bromide [31]. A 1.5kB

fragment of DNA representing the amplified 16S RNA gene was cut out of the gel and the DNA was

isolated with a gel extraction kit (QIAquick Gel Extraction Kit, Quiagen).

3.2 Species identification using sequence of 16S RNA gene

The same forward and reverse primers were used for sequencing by the Advanced Studies in

Genomics, Proteomics and Bioinformatics (ASGPB) program at UH Mānoa. The forward and reverse

sequencing results were combined into a full-length contig with the Gene Construction Kit software

program (GCK). This full-length sequence was submitted to a nucleotide BLAST search from the

nucleotide database on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

The results confirmed with greater than 99% nucleotide identity that our isolate is in fact C.

necator. This microbe was previously named Ralstonia eutropha and there are multiple strains with

complete genome sequences available for R. eutropha. The strain identified as the best match to the

sample sequence was the common strain R. eutropha H16. It has been shown that CO2 fixation in this

organism occurs via the Calvin cycle [6].


20

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 70D

ou

blin

g ti

me

(h

)

OD

62

0n

m

Cultivation time (hours)

OD

Doubling time

CHAPTER 4. CHEMOLITHOAUTOTROPHIC BIOREACTOR CULTIVATION

4.1 Specific growth rate and doubling time

An open-system, continuously gas-fed bioreactor design was used to characterize the

chemolithoautotrophic growth capacity for our strain of C. necator (figure 2). The bioreactor was

monitored continually over a period of 3 days until the culture became nutrient limited, leading to the

cessation of growth at which point the OD became stable at 60. The OD and cell yield in grams were

recorded throughout the cultivation period, and the specific growth rate as well as doubling time of this

strain was calculated for each sampling period (figure 2). The growth rates and doubling times were

calculated using OD measurements and equations 10 and 11 respectively.

Figure 2. Bacterial growth curve from bioreactor cultivation. The bioreactor was continuously fed gas substrates throughout cultivation. Doubling times are based on OD measurements.

During the first 30 hours of cultivation the cells had an average doubling time of 5.7 hours with a

standard deviation of 2.01 hours. The shortest doubling time observed was 2.81 hours (Table 3). After 40

hours of cultivation there was a distinguishable increase in the doubling time of the culture until it

reached stationary phase after about 65 hours. The average doubling time during this period was 30

hours with a standard deviation of 15.8 hours. When the culture reached stationary phase after 65 hours

the doubling time increased to 99 hours, the OD became stable and cessation of growth was observed.


21

The biomass from the bioreactor culture was harvested and 47.28 g of FDCM was obtained from 2L total

culture volume after 3 days of cultivation.

Table 3. Bioreactor growth parameters. The specific growth rate is calculated from the OD of the culture at each sampling period using equation 10. The doubling time is calculated from µ using equation 11. The fastest doubling time observed during cultivation is highlighted in red.

4.2 Comparison of cellular performance with previous findings

The bioreactor cultivation yielded a cell density within the expected range for C. necator. We

obtained 24g/L FDCM from our bioreactor and previously 25g/L FDCM was reported for high density

autotrophic cultivation of C. necator [23]. Additionally, it was witnessed that our culture stopped growing

at an OD of 60, which was also corroborated by previous observations [23]. It was found by the Repaske

group that after an OD of 44 was reached; it became impossible to manually adjust the addition of

nutrients and gas concentrations necessary to continue promoting exponential growth, and from OD 44-

60 growth was no longer exponential. This was also witnessed during our bioreactor; after the OD

reached 44 the doubling time of the culture quickly increased from an average of about 6.5 hours to 30

hours and continued increasing until reaching stationary phase.

Cultivation time (h)

OD620nm Cell yield

FDCM (g/L)

Specific growth rate [µ]

(OD/OD*h)

Doubling time (h)

0 0.237 3.3 n/a n/a

3 0.326 3.4 0.1 6.93

9 0.58 3.7 0.096 7.22

21 11.1 6 0.246 2.81

25 18 9 0.121 5.73

31 27.6 14 0.071 9.76

43 45.4 19 0.04 17.3

47 50.6 20 0.027 25.67

54 56 23 0.0145 47.8

66 60.8 24 0.007 99.02

69 61.6 24 0 n/a

72 60.6 24 0 n/a


22

According to Repaske, the automatic demand of NH4OH could be used to indicate proper growth

of the culture. The first indication of an impending nutritional deficiency was the decrease in the

frequency of NH4OH addition [23]. If the deficiency was not corrected, NH4OH addition stopped [23]. This

observation was also made during our bioreactor cultivation. Once the OD reached 44 the frequency of

NH4OH addition decreased drastically, and was no longer required once the OD reached 60. This suggests

that a nutrient deficiency was experienced by our bioreactor after an OD of 44 was reached, at which

point the doubling time of the culture continued increasing drastically until cessation of cell growth was

observed. This implies that the average maximum growth rate of our bioreactor during the period of no

nutrient limitation was about 6.5 hours; the fastest turnover observed during this time was 2.8 hours.

Our bioreactor experiment showed the ability to grow our strain to high densities as previously

reported, however during our experiment it took three days to reach an OD of 60, whereas previously

this yield was achieved in just one day [23]. In the past, doubling times of 2 hours could be maintained

throughout cultivation to an OD of 44 until an increase in the growth rate was observed due to nutrient

limitation or NH4+ excess [23, 29]. For chemolithoautotrophic growth in bioreactors the most difficult

aspect of providing gas substrates to the cells is overcoming the extremely poorly soluble hydrogen and

oxygen gasses. In practice, immense gas aeration and liquid flow throughout the bioreactor are required

to increase the gas to liquid surface area interface, facilitating the mass transfer of gasses to liquid phase.

The bioreactor design used in this study is not specifically equipped to increase aeration in the

liquid culture. The liquid agitation in our bioreactor was controlled by the dissolved O2 level. When the

cell density was low and the corresponding oxygen concentration was high, the agitator only functioned

at about half speed. The agitator began to function at full speed responding to decreasing dO2 levels at

an OD of about 10. It is seen in table 3 that the maximum doubling time was achieved by the bioreactor

at an OD of about 10. Before this OD was reached, it is believed the slow agitation speed resulted in

decreased mass-transfer due to restricted interaction of gas to liquid phases. When the agitation

occurred at maximum speed (900rpm) and there were no limiting nutrients in the bioreactor, the fastest

doubling time of 2.8 hours was observed. Maximal doubling times of 2.5 hours have been reported for

chemolithoautotrophic growth C. necator [23]. When mass transfer was at the maximum level

obtainable, we witnessed comparable growth rates to previous reports. To maintain growth rates at

maximum levels throughout cultivation, future bioreactor designs must focus on increasing the mass to

liquid surface area, facilitating mass transfer and the availability of gas substrates to growing cells.


23

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70

% P

HB

OD620nm

4.3 PHB accumulation throughout cultivation- Implications of nutrient deficiency

Polyhydroxyalkanoates (PHAs) are a class of polyesters formed in some bacteria as an energy

reserve, in C. necator when grown chemolithoautotrophically, the alkanoate monomeric constituent of

the polymer is butyric acid making the polyester polyhydroxybutyrate (PHB) [22]. When culture

conditions are nutrient rich the cells devote metabolism to cell growth, however when nutrients become

limiting metabolism is shifted towards PHA synthesis. Early stages of nutrient deficiency in

chemolithoautotrophic bioreactor cultures could be identified by a shift towards PHA metabolism [23].

Limitation of PO43-, SO4

2-, Mg2+, or NH4+ caused the cell to shift metabolism towards PHB production[23].

Additionally, iron limitation could be visualized by the production of a yellow pigment in the medium

[23].

Figure 3. PHB accumulation throughout bioreactor cultivation. The PHB content of the cells was tested at each sampling period from FDCM of the 10mL culture sample. Equations 12 and 13 were used to equate GC data to PHB%.

It was observed in our bioreactor that a large increase in the PHB composition of the cells was

observed after an OD of 44 was reached. This also indicates that a nutrient deficiency was experienced by

the cells in the bioreactor after an OD of 44, and the continuation of growth lead to PHB accumulation up

to 50% dry cell weight. A deficiency in nitrogen as a result of decreasing NH4OH additions after the OD of

44 was the likely nutrient limitation leading to PHB accumulation. Additionally, the supernatant of the


24

culture samples were observed for each sample. A faint yellow pigment was observed at OD 44 and the

color increased throughout cultivation to bright yellow by OD 60. Anti-foam was also needed during the

end of cultivation to keep the culture from overflowing out the gas outlet. These two observations

indicate that iron was also limiting in the bioreactor around an OD of 44.

The combined effects of multiple nutrient deficiencies around OD 44 are likely the cause for the

large increase in PHB accumulation during this period. The increase in PHB % correlates directly with the

decrease in growth rate of the culture (figures 2 and 3). From the PHB analysis it is clear that nutrient

deficiency was experienced after OD 44, leading to the decrease in growth rate until cell division ceased.

In order to increase the capacity for high density autotrophic bioreactor cultivation, anticipated

nutritional supplements should be made according to the cellular requirements and the amount of

biomass generated over time.


25

CHAPTER 5. METHOD FOR EVALUATING H2, O2 AND CO2 CONSUMPTION

The focus of this research is to investigate the potential for microbial CO2 fixation from syngas.

Syngas is often created for hydrogen production and is the largest source of hydrogen in the United

States. Therefore, the amount of hydrogen required to fix the CO2 present in the syngas through

microbial chemolithoautotrophic growth is an integral piece of information required for the economic

analysis of this proposed CO2 fixation process. Process engineers will not accept this sustainable upgrade

if the microbial CO2 fixation process requires the majority of the hydrogen from the syngas. To easily and

readily determine the hydrogen requirement of chemolithoautotrophic growth, a novel experiment was

designed and implemented using a closed-system cultivation of C. necator with the analysis of the head-

space gas composition over time.

5.1 Theoretical method development

The ideal gas law can be used to determine the change in moles of any ideal gas over time using

the following equations:

Ideal gas law: P V = n R T [14]

Change in moles (n): ∆n = nf - no [15]

In our system we will work at ambient pressure and the temperature is held constant at 30⁰C for

bacterial cultivation. Therefore these two variables from the ideal gas law cancel out when combining

equation 14 for an initial and final sample. Combining equations 14 and 15 will give the change in volume

for each gas in the system (i), with the assumption that for an ideal gas, molar percentage equals volume

percentage. The equation for molar change of each gas in this system (i) depends on the total molar

change of the system over time and is as follows:

Combining equations 14 and 15: ∆ni = {nf [vol. %]i.f – no [vol. %]i.o} [16]

Factoring out no: ∆ni = no {nf/no [vol. %]i.f – [vol. %]i.o} [17]

Equation 17 tells us the parameters that are required in the experimental design to give the

change in moles for any gas between two sampling periods. Since the temperature of the system is held

constant, the only two parameters that can change in a closed-system are the pressure and volume of

the system. Holding one of these factors constant will enable us to use the ideal gas law to determine the


26

change in moles of a closed-system over time. In this experiment the pressure will be held constant at

ambient pressure, therefore the volume change in the system will be measured, and the total moles will

be calculated from the volume of the system.

The term no expresses the total moles in the system after the initial gas sample is taken and is

calculated from equation 14 using the initial volume of the system at each sample and the constants;

pressure (1 atm), temperature (303.15 K) and R (0.08206 L∙atm/mol∙K), the ideal gas constant. The total

moles in the system must be determined to express the molar consumption of each gas as well as the

CO2 fixation rate during chemolithoautotrophic growth. However, the total moles are not needed to

determine the gas consumption ratio between two different gasses during one time interval. To

determine the ratio of consumption for individual gas (i) to gas (j), equation 17 is combined for each gas

(i) and (j) and becomes:

⁄ [ ] [ ]

[ ] [ ]

As seen above, to obtain a ratio of gas consumption (i/j) for any culture during the same time period the

total moles in the system (no) cancel out because the total moles in the system are equal regardless of

what gasses are being compared. Therefore, to determine the ratio of H2/CO2 for chemolithoautotrophic

growth, only the initial and final gas samples are required and this ratio is not affected by inaccuracies in

volumetric measurement necessary to calculate no.

The term nf/no represents the ratio of molar change between the initial and final gas samples.

This term will be used to equate the molar percentage from the final sample to the molar percentage in

the initial sample for each gas. The term nf/no must therefore be calculated from an inert gas in the

system that is neither created nor destroyed during cultivation. The measurement of nf/no from the inert

gas can then be multiplied by the final molar % for each sample, equating moles from the initial sample

to moles in the final sample as given in equation 17.

In this experiment, methane (CH4) was chosen as the inert gas because it is neither consumed nor

created by C. necator and is readily available in the lab as natural gas. The standard gas methane is used

as follows to obtain nf/no:

Methane is inert: ∆nCH4 = nCH4f - nCH4o = 0 [18]


27

No volume change in CH4: nf/no [vol. %]CH4f – [vol. %]CH4o = 0 [19]

nf/no = [vol. %]CH4o / [vol. %]CH4f [20]

Calculation of nf/no depends on the volume ratio of CH4 initial to CH4 final. Since this is a ratio, the

actual volume does not need to be calculated from the absolute area given by the GC machine. Instead

the ratio can be calculated from the absolute area of CH4, thereby eliminating the need to create a

calibration between absolute area and volume percentage for the inert standard CH4. The final equation

giving the variable nf/no therefore becomes:

nf/no = [area]CH4o / [area]CH4f [21]

To analyze the ratio of H2/CO2 consumed by C. necator during lithoautotrophic growth, the

change in volume over a specific period of time for the gasses H2 and CO2 in a closed system must be

evaluated. After the derivation of equations 14-21, it is apparent that in order to calculate the molar

change in gas composition between two samples one must know; the volume percentage of each gas in

both the initial and final gas samples, the total moles present in the system initially and the ratio of molar

change between gas samples determined from the gas standard. Using the ideal gas law (equation 14),

the total number of moles present initially can be calculated from the volume, pressure and temperature

of the system. The measurements that must be taken for each sampling period are therefore; a gas

sample injection in to the GC machine which gives both nf/no as well as the volumetric concentration of

each individual gas and the initial volume of the system taken immediately after the initial gas injection

was made for each time period.

5.2 Experimental design

The theoretical method development yielded an analysis technique that can be used to measure

the change in each gas H2, O2, and CO2 in a closed-system over time. The requirements of the system are

that the gas can be sampled without letting any ambient air into the system and the volume of the

system is free to change and can be measured at any time. A culture vessel capable of satisfying these

two requirements is the integral component of this system.

In this experiment chemolithoautotrophic cultures of C. necator were cultivated in 0.6L Tedlar

gas sampling bags equipped with a barbed on-off valve (Discovery Sciences, VWR). The gas sampling bags

are created especially for injecting clean gas samples into the GC machine. When the barbed valve is


28

inserted into the sampling port on the GC machine an air-tight seal is made and gas can only flow out of

the bag when mechanically forced through the GC machine by squeezing the bag (figure 4). Using this

technique ambient air is never allowed to enter the bag, as the valve is only opened once inserted into

the GC machine and closed again before removal.

Figure 4. Method of closed-system chemolithoautotrophically cultivated C. necator for analysis of gas consumption. Gas sampling was achieved by connecting the bag to the GC machine, twisting open the valve on the bag, expelling gas from the bag manually until the GC machine automatically takes a gas sample of fixed volume, and twisting the valve closed again. This technique uses the gas sample to purge the sampling loop before the sample is taken and maintains a closed-system throughout gas sampling. The bag was removed from the GC machine after sampling and the volume was recorded through water displacement. The bag was stored between sampling periods at 30⁰C in a rotary incubator at 100 rpm.

In addition to being equipped for clean, accurate GC gas injection, the gas sampling bags are

flexible and can change volume. This property of the gas sampling bags made them the ideal candidate

for a culture vessel in this experiment. The volume of the bag was measured in a four liter beaker,

retrofitted with a ruler up the side and heavily-weighted scaffolding to hold the gas bag at the bottom of

the beaker (figure 5). The beaker was filled about ¾ full with 30⁰C water, kept in a 30⁰C water bath and

the water level was recorded in millimeters using the ruler inside the beaker. The bag was attached to a

scaffold at the bottom of a beaker and after the water settled the level was again recorded in millimeters.

The volume of displacement was calculated from a calibration curve relating millimeters to milliliters in

the apparatus (figure 6). The volume of gas within the bag at any time was determined by subtracting the

volume of the vacuum sealed bag alone (38.5mL) as well as the 15mL culture volume from the total

volume of water displaced by the bag.

Liquid culture

GC sampling bag valve

Gas sampling bag filled with H

2, O

2, CO

2 and CH

4


29

Figure 5. Apparatus to measure volume of gas sampling/culture bag at any point during gas consumption experiment. The water in the displacement apparatus was kept at 30⁰C using water from a heated water bath. The initial water level was brought to 100-120mm and recorded (A). The bag was attached to the scaffold on the bottom of the apparatus using a very short piece of tubing to fit over the bag valve with a loop to attach to the scaffold hook. The bag was completely submerged underwater, the water was allowed to settle and the final water level was recorded (B). The change in water level can be observed by the measurements circled in A and B, the total water displacement was 30mm.

Figure 6. Calibration curve between water displacement measured in millimeters and actual volume in milliliters. The calibration was performed by bringing the water level up to 100mm in the apparatus and adding 50mL of water, measured in a graduated cylinder, and recording the water level in mm. This was done repeatedly until 500mL had been added. The calibration was performed with the water level at 100mm because this was the range at which the water level was used for volumetric displacement. The scaffolding holding the bag at the bottom of the beaker was left in the apparatus during calibration and was completely submerged; therefore the volume of the scaffold was accounted for by starting measurements at 100mm.

y = 19.204x R² = 0.9981

0

100

200

300

400

500

600

0 5 10 15 20 25 30

Dis

pla

cem

en

t vo

lum

e (

mL)

∆ H2O level (mm)

A B


30

5.3 Gas chromatography method for H2, O2 and CO2 analysis

In order to detect the permanent gasses of chemolithoautotrophic growth in C. necator with the

GC machine a new method was developed. A Varian 450-GC machine was equipped with a Carboxen

PLOT 1006 column, thickness 0.15mm x 30m (Sigma-Aldrich) for sample separation and fed into a

thermal conductivity detector for the evaluation of gas concentration. The gas injection sample loop is

250µL. The carrier gas is nitrogen. Nitrogen and hydrogen have largely different thermal conductivities

making the analysis of large amounts of hydrogen much more accurate than with helium. However,

nitrogen and carbon dioxide have very similar thermal conductivities making this compound the least

accurately detected in this system, although we observed repeatedly accurate detection of carbon

dioxide using this method.

The method for gas separation in the column is as follows; from time zero to two minutes the

column oven is 35⁰C and the column pressure is 5.3psi. At two minutes, after the detection of H2 and O2

the column temperature is increased 25⁰C/min and the column pneumatics are increased 0.7psi/min for

five minutes so the final oven temperature is 100⁰C and the final column pressure is 7.4psi to decrease

the elution time of CO2 from the column. Because hydrogen is detected the most sensitively using

nitrogen, the initial range of the TCD detector is set at 0.5 and is changed after 1.6 minutes to 0.05 for a

more sensitive detection of O2 and CO2. Using this method hydrogen elutes from the column at

1.5minutes, oxygen elutes at 1.7 minutes and carbon dioxide elutes at 4.5 minutes.

The peaks of each gas component are integrated using the software Galaxie. The integration

events were defined within a small window during which the gasses eluted from the column. The

program calculates the area of each curve using an algorithm to detect a threshold change in slope of the

chromatogram and integrates the area within this threshold giving the absolute area in µV/min for each

gas substrate.

5.4 Protocol for measuring molar change of H2, O2 and CO2 during chemolithoautotrophic growth

Inoculum preparation

Cultures of C. necator were generated chemolithoautotrophically in 1L heavy-duty, vacuum-

resistant polypropylene bottles as described in chapter 2 for two days. On day three, the OD of the

culture was about 3 and this density gave the best results in bag cultures. The bottle cultures were

continuously fed gas substrates at the same concentration to be used in the gas consumption experiment


31

for 30 minutes to 1 hour prior to being transferred into bags. This was done to ensure that the cells were

not experiencing gas limitation, mainly oxygen, when transferred to bags. Employing this technique vastly

increased the accuracy and repeatability of cellular performance in bag cultures.

Inoculating gas bag cultures

Transferring growing cells to the gas bag was done with as little perturbation to normal growth

conditions as possible. As the inoculum culture was continuously bubbling with gas substrates the gas

bags were prepared. First, the bags were vacuum sealed; expelling all ambient air from the system, and

the valve was tightened. At this point leaky bags could be identified because they would not hold a seal.

The leaky bags were thrown away; only bags that could hold a vacuum for five minutes were used.

After being vacuum sealed, the pre-mixed gas containing H2, O2 and CO2 flowing into the

inoculum culture was used to fill the bags about half full. Next, a 100mL gas tight syringe was used to

measure 50mL of natural gas (Hamilton Company). The gas syringe has a PTFE luer lock male fitting

terminator that could be inserted into the sampling valve with a gas-tight seal. The syringe was fitted into

the barbed gas sampling bag valve, the valve was opened and the 50mL of natural gas was injected slowly

into the bag. The valve was then closed and the syringe removed. All the bags necessary for one

experiment were prepared to this point collectively; however inoculation only occurred immediately

before the initial gas sample was taken for each bag. This ensured all replicates had cultures experiencing

the exact same conditions before being transferred to the bags.

Inoculation of the gas bags was done using a sterile 25mL syringe barrel with a conical tip as a

funnel to insert liquid culture through the sample valve of the bag (figure 7). The male fitting on the

syringe barrel could be inserted into the gas sampling valve with a secure seal. A sterile graduated

cylinder was used to measure 15mL of culture. The culture was transferred to the sterile syringe barrel,

and the bag valve was twisted open. The valve was shut immediately after the culture was transferred to

ensure no air entered the bag. The inoculated bag was filled the remainder of the way with the pre-mixed

gas substrate.


32

Gas sampling with the GC machine

The initial gas sample was taken immediately after filling the bag all the way up with gas directly

following inoculation. Usually there was a slight amount of pressure in the bags after being filled with

gas. Upon the initial gas sampling, the bag was allowed to purge gas through the GC machine until it was

not able to displace mineral oil out of the GC efflux port, ensuring the bag did not hold any more pressure

than ambient conditions at the start of the analysis.

Gas was sampled from the bag by connecting the barbed valve of the bag into a receptor port for

gas sampling on the GC machine (figure 4). This port was fitted to the bag valve using a ferrule to increase

the sample tubing from ⅛” to ¼”. As little tubing as possible was used to marry the GC injection port and

gas sampling valve to eliminate “dead” volume from the system. Gas was manually expelled from the bag

through the machine for 10 seconds prior to initiating the gas sample injection into the column to purge

the gas sampling loop with the desired gas composition before injection. After injection, the bag valve

was closed before being removed from the GC machine to maintain a closed-system throughout

cultivation.

Figure 7. Method of bag culture inoculation using a syringe barrel with a conical tip. The inoculum was measured volumetrically using a graduated cylinder before being transferred into the bag through the syringe barrel. The valve was only slightly opened and shut immediately after all the liquid was pulled into the bag.


33

Determination of bag volume

Throughout the experiment, directly after a gas sample was taken, the volume of the bag was

measured with a water displacement technique using a 4L beaker (figure 5). The water level of the

displacement apparatus was brought to about ¾ full and recorded. The bag was attached to a scaffold at

the bottom of the displacement apparatus and fully submerged in 30⁰C water. The water level was

allowed to settle and the water level was again recorded. The bag was immediately taken out of the

apparatus, dried off and incubated. A calibration curve was used to equate volume displacement in

millimeters to milliliters after data was collected (figure 6).

Incubation

Bags were incubated at 30⁰C in an orbital rotary incubator at 100rpm. Bags are not ideal culture

vessels and if adequate turbulence was not experienced in the 15mL of media during cultivation, the cells

settled and did not perform normally. Also, if too much turbulence was experienced, the culture volume

was spread throughout the bag and inconsistent results were observed. To maintain culture turbulence

similar to that in a bottle or flask it was found that the bags must be laid flat, if not the bacterial cells

would settle in the corners of the bag. To achieve adequate movement of the media during cultivation

the bags were laid flat atop flask holders in the rotary incubator and secured using two large pieces of

tape. This ensured that the culture remained metabolically active, that gas was transferred from gas to

liquid phase in the media uniformly and greatly improved the repeatability of the experimental results.

Incubation at 30⁰C occurred between gas samples which were taken every 2 hours.

5.5 Calibration curve relating GC absolute area to volume percentage for H2, O2 and CO2

In this experiment the gas composition in the headspace of actively growing cultures is measured

using a gas chromatography machine for sample separation and a TCD for detection of the permanent

gasses H2, O2, CO2 and CH4. TCD detectors register a change in resistance between the reference gas and

sample gas, the strength of the signal is related to the quantity of the gas in the sample stream. This

signal is a measurement in μV and represents the Y-axis of the chromatogram obtained as a result of GC

analysis. Before the sample gas reaches the TCD detector it flows through a column and the individual gas

components are separated due to their affinity for the material within the column. Therefore each gas

elutes into the TCD detector at a different retention time that is represented on the X-axis of the

chromatogram in minutes. A peak is formed when each gas component is detected by the TCD over a

short period of time during which it was eluding from the column. The area defined under any peak


34

y = 8951.1x R² = 0.9959

y = 3341.1x R² = 0.9977

y = 13853x R² = 0.9772

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0 20 40 60 80 100

Ab

solu

te A

rea

(µV

)

Volume Percentage (%)

Hydrogen

Oxygen

CO2

generated from GC analysis is integrated by the computer software Galaxie, which is associated with the

GC machine and reports the absolute area of each compound represented in μV/min.

The absolute area reported by the GC machine can be correlated to a volume percentage of any

specific gas when a calibration equation is generated. The slope of the calibration equation will represent

the volume percentage of any absolute area detected for each specific gas and is known as the f-value for

that gas. Standard gas mixtures representing a range of known volumes for each gas H2, O2 and CO2 were

generated individually. Gas standards were generated using a gastight syringe to create mixtures of pure

gas with a background gas (nitrogen), giving a desired volumetric percentage of the sample gas in each

standard sample. The gas standards were made in a 0.6L gas sampling bag with a GC injection valve. With

this technique ranges of gas standards suitable to the volumetric concentrations used in the experiment

were created. A calibration curve relating absolute area (µV/min) to a volume percentage was obtained

individually for the gasses H2, O2 and CO2. Regression equations were generated from 20, 24, and 17

independently generated gas standards for hydrogen, oxygen, and carbon dioxide respectively.

Correlation coefficients of greater than R2=0.99 were obtained for hydrogen and oxygen while a

correlation coefficient greater than R2=0.97 was obtained for carbon dioxide (figure 8).

Figure 8. Calibration equations showing correlation between the volume percentage of standard gas

mixtures and the absolute area detected by the TCD on the GC machine. A regression line was forced

through the origin for each gas H2 (red), O2 (blue) and CO2 (green), and the slope of this line is the f-value

for that gas. The f-values and regression coefficients for each gas are given in the colored boxes next to

the corresponding regression line.


35

CHAPTER 6. H2, O2 AND CO2 CONSUMPTION DURING CHEMOLITHOAUTOTROPHIC GROWTH

The gas requirements of chemolithoautotrophic growth in our isolate of C. necator were

determined using the novel experiment designed in chapter 5. The closed-system analysis allowed us to

determine the molar gas composition of the growing culture over time. From this data the ratio of H2/CO2

consumed by the culture was calculated after every gas sampling event. The total moles of H2, O2 and CO2

gas consumed during chemolithoautotrophic growth is determined. Additionally, the rate of CO2 fixation

was also calculated and is expressed in CO2 (g)/FDCM(g) ∙ hour -1. The information gathered from the gas

consumption experiments gives an indication of the performance capacity of this organism with regards

to CO2 fixation from syngas.

6.1 Data analysis

The results of the gas bag experiments give the volumetric concentrations of each gas, H2, O2 and

CO2 at the beginning and end of each sampling period (2 hour intervals). The volumetric percentage of

each gas can be converted into total moles of each gas with the experimental design. To report the

volumetric percentage of each gas at one sampling period, the data needs to be analyzed in two ways.

The absolute area detected by the GC machine (measured in µV/min) is first converted to a volume

percentage using a calibration curve (figure 8). Because the total moles in the bag change between the

initial and final gas samples required for one sampling period, the final gas composition must be

expressed in terms of the initial gas composition by using the ratio of the inert standard gas (CH4)

between samples, nf/no in equation 17. Once the final volume percentage has been multiplied by the

ratio of molar change, it can be subtracted from the initial volume percentage to find the change in

volume percentage of each individual gas (i) between any two samples. The total moles in the bag can be

calculated from the volume of the bag initially at each sampling interval using equation 14, and is used to

find the total molar consumption of each individual gas using equation 17.

The gas consumption experiments were conducted for six hours, gas samples were taken every

two hours, yielding three sampling periods. The flowchart in figure 9 shows the data manipulation

applied to the raw data collected during the experiment in order to obtain the molar consumption of H2,

O2 and CO2 during all three sampling periods. The final gas compositions of sample one become the initial

values for sample two. The volume of the bag is recorded immediately after each gas sample is taken,

giving the initial volume for the system at the beginning of the sampling period. This technique was

repeated for each sampling period. Figure 9 shows the data manipulation required for any two


36

consecutive gas samples, and was performed for samples one and two, samples two and three and

samples three and four respectively. The resulting data showed the change in volume percentage for

each gas H2, O2 and CO2 over three distinct sampling periods. This data can also be expressed as the total

moles of H2, O2 and CO2 consumed during each sampling period. The rate of CO2 fixation can be

calculated from the molar gas consumption data.

Figure 9. Flowchart of data analysis applied to each set of successive gas samples obtained during a single gas consumption experiment. The three pieces of data necessary for the analysis are illustrated at the top of the figure; an initial and final gas sample recorded as a chromatogram and the initial volume of the bag measured by water displacement. The f-values for H2, O2 and CO2 are given in figure 8. The total moles in the bag was calculated from the ideal gas law (equation 14) at experimental conditions (v=L, 1atm, 303.15K [30⁰C], R=0.08206 L∙atm/mol∙K) with the equation n = L/24.876. The yellow box highlights how the H2/CO2 ratio can be calculated from the change in volume detected by the GC machine and is not affected by the volume of the system. The total moles of gas consumed are calculated from the volumetric measurement of the bag using water displacement. This analysis was applied to any two successive gas samples during a gas consumption experiment.


37

6.2 Results: Gas consumption ratios of chemolithoautotrophic growth

The gas consumption experiments were conducted under the same conditions; 15mL of seed

culture, 30⁰C incubation with 100rpm shaking in a rotary incubator for all replicates. During one

experiment 3-5 bags were tested and only bags that grew properly and did not leak were used for

analysis. The data reported characterizes the replicates which grew well and consumed a significant

volume of gas throughout the experiment, indicating healthy growth conditions and normal gas

consumption ratios. The experiments were performed for six hours with gas samples taken every two

hours. It was observed that the data after six hours of incubation experienced large fluctuations in gas

consumption ratios and was likely due to oxygen limitation because the lower limit of 5% O2 was rapidly

approached in the closed-system at this time. The data is presented as the change in gas composition

over six hours which is believed to represent the cellular performance under the best conditions without

nutrient limitation. The average starting gas concentration of all replicates was 62%:13%:13% H2:O2:CO2

with a standard deviation of 3.5%:2.7%:1.4%. The final O2 level never dropped below 5% in any of the five

replicates and the average ending O2 concentration was 9.5% with a standard deviation of 3.8%.


38

Table 4. Results of gas consumption during chemolithoautotrophic growth in a closed-system bag

culture for five independent replicates. Every time interval (∆t1-∆t3) represents 2 hours of incubation.

∆t1 is 2 hours after inoculation, ∆t2 is four hours and ∆t3 is six hours after inoculation. The doubling time

was calculated from the initial and final OD in the bag using equation 11. The change in volume

percentage for the gasses H2, O2 and CO2 is given for each two hour time interval. The H2/CO2 as well as

H2/O2 ratios are calculated at each sampling period using the change in volume at that time interval. The

CO2 : O2 : H2 (mol:mol:mol) ratio is also calculated from volume percentage. The cells at the bottom right

represent the average and standard deviation of all three ratios from all replicates at every time interval

(n=15 samples). The control bag was tested under the same conditions with the exception that 15mL of

hydrogen media was used instead of bacterial culture.

Replicate #

Doubling time (h)

Time interval

∆ vol% H2/CO2 H2/O2 CO2 : O2 : H2

H2 O2 CO2

1 7.06

∆t1 8.3 2.4 1.3 6.4 3.4 1 : 1.9 : 6.4

∆t2 8.2 2.5 1.3 6.1 3.2 1 : 1.9 : 6.1

∆t3 5.1 1.9 0.8 6.1 2.7 1 : 2.3 : 6.1

2 13.41

∆t1 9.3 2.9 1.8 5.1 3.2 1 : 1.6 : 5.1

∆t2 8.9 2.7 1.2 7.3 3.3 1 : 2.2 : 7.3

∆t3 6.5 2.2 1.1 6.2 3.0 1 : 2.1 : 6.2

3 8.47

∆t1 5.3 2.0 0.9 6.1 2.7 1 : 2.3 : 6.1

∆t2 5.6 2.1 0.8 7.3 2.7 1 : 2.7 : 7.3

∆t3 6.9 2.9 1.0 7.1 2.4 1 : 2.9 : 7.1

4 6.43

∆t1 6.7 2.0 1.0 6.5 3.3 1 : 2.0 : 6.5

∆t2 10.7 3.0 2.0 5.4 3.6 1 : 1.5 : 5.4

∆t3 11.5 3.8 1.7 6.6 3.0 1 : 2.2 : 6.6

5 6.68

∆t1 7.8 2.2 1.6 5.1 3.6 1 : 1.4 : 5.1

∆t2 8.3 2.3 1.4 6.0 3.6 1 : 1.6 : 6.0

∆t3 10.7 3.7 1.6 6.5 2.9 1 : 2.2 : 6.5

Control (no bacteria)

n/a

∆t1 0.1 0.2 0.1 Average ± Stdev

∆t2 0.4 0.01 0.03 6.23 ±0.70

3.11 ±0.38

1 : 2.1 : 6.2 ±0 : 0.4 : 0.7 ∆t3 0.5 0.09 0.02


39

6.23

3.11

0

1

2

3

4

5

6

7

8

H2/CO2 H2/O2

All replicates Replicate 1 Replicate 2

Replicate 3 Replicate 4 Replicate 5

Figure 10. Average H2/CO2 and H2/O2 ratios of chemolithoautotrophic growth for each of the five replicates as well as the average of all replicates over time intervals 1-3. The average ratios were calculated from the corresponding ratio of each replicate at time intervals one two and three. The average of all replicates across all three time intervals is expressed in grey on the far left column and the average value is labeled on the chart. The replicates are ordered from 1-5 left to right for both ratios. The average H2/CO2 and H2/O2 ratios and standard deviations for all replicates over three sampling periods equaling six hours of cultivation are 6.23±0.7 and 3.11±0.38 respectively.

The results of the gas consumption experiments were used to find the H2/CO2 and H2/O2 ratio of

chemolithoautotrophic growth in our strain of C. necator. The H2/CO2 ratio is especially interesting as this

tells us how much hydrogen will be required to fix CO2 in this organism, and directly relates to the

amount of hydrogen energy recovered as biomass. This information is integral for syngas engineers as

mentioned before because it indicates the efficiency of CO2 fixation from syngas. It is important to note

that determination of the H2/CO2 and H2/O2 ratios depend solely on the GC data and calibration

equations for each gas as highlighted in yellow in figure 9. This implies that these ratios are not affected

by any inaccuracies in measurement of bag volume obtained from water displacement. The overall ratio

of gas consumption during chemolithoautotrophic growth is reported as CO2:O2:H2. The data is expressed

in terms of the molar percentage of CO2, therefore the ratio gives the molar amount of O2 and H2

required for fixation of one mole of CO2. During this experiment the average ratio of

chemolithoautotrophic growth in C. necator was 1 : 2.1 : 6.2.


40

It was observed throughout the development of the gas consumption methodology that the

results obtained during the first time period represented the most efficient growth. For this reason the

total cultivation time was reduced to six hours with two hour sampling periods to maintain experimental

conditions adequate to promote normal cell growth and performance throughout the entire experiment.

However, the same trend of decreasing cellular efficiency was still witnessed over six hours, although the

extent of fluctuation was markedly reduced from previous observations. Therefore it is likely that in our

experimental design the most accurate estimates of the true gas consumption ratios of

chemolithoautotrophic growth in C. necator should be obtained from the average ratios across all five

replicates after the initial 2 hour sampling period only. Averaging the H2/CO2 ratio of ∆t1 for all five

samples gives 5.8± 0.7. Averaging the H2/O2 ratio of ∆t1 for all five samples gives 3.2± 0.3. Combining the

average ratios between all five replicates for ∆t1 samples only gives the average ratio of gas consumption

being 1 : 1.8 : 5.8.

The results of our gas consumption experiments are comparable to results found previously in C.

necator. There have been two independent research groups in the past that have attempted to identify

the ratio of gas consumption during chemolithoautotrophic growth for C. necator. One group used an

open-system bioreactor design and the other used a closed-system, recycled gas bioreactor [2, 29]. In

both experimental designs the gas composition of the inlet and outlet gas streams from the bioreactor

were used to determine the amount of gas consumed by the culture and was converted into a

consumption ratio of carbon dioxide : oxygen : hydrogen.

The Amman group found the average ratio of chemolithoautotrophic growth in C. necator to be

1 : 1.7 : 5.7 [2]. The Ishizaki group found the average ratio of chemolithoautotrophic growth in C. necator

to be 1 : 1.5 : 5.2 [29]. We found the average ratio of chemolithoautotrophic growth in C. necator to be

1 : 1.8 : 5.8. Our results are in very good agreement with results published previously using different

experimental designs. The correspondence in gas consumption ratios across three independent and

methodologically distinct research initiatives suggests that the data portrays the true cellular gas

demands of chemolithoautotrophic growth in C. necator.


41

6.3 Results: Efficiency of CO2 fixation

It was found that the average ratio of gas consumption of H2/CO2 is 5.8. Recalling the theoretical

efficiency of CO2 fixation calculated in chapter 1 and presented in table 1, the H2/CO2 ratio of

chemolithoautotrophic growth determines the percentage of hydrogen energy recovered as bacterial

biomass. According to the results found in this experiment and in the past, it appears that C. necator does

not grow efficiently under lab-induced chemolithoautotrophic conditions. In fact, the cell appears to

grow quite inefficiently with a P/2e- ratio of below 1 (table 1). The amount of hydrogen energy devoted

to generating recoverable bacterial biomass at this P/2e- ratio is less than 30% of the total hydrogen

energy consumed during growth.

These results indicate that the current methods of simulating chemolithoautotrophic growth in

the wild-type C. necator do not yield CO2 fixation efficiencies attractive to syngas engineers. As is seen in

table 2, biomass-derived syngas does not contain enough hydrogen to allow for complete CO2 fixation

using C. necator. Even coal-derived syngas will be almost completely quenched of hydrogen after CO2

fixation. Additionally, only 30% of the hydrogen can be recovered as biomass, and only about 50-60% of

this biomass will be the valuable product PHB. Clearly for this process of CO2 removal from syngas to

become economical and viable, ways to improve the P/2e- ratio of chemolithoautotrophic growth must

be discovered. A possible way to improve the CO2 fixation efficiency of this strain is through genetic

engineering. A hypothesis is made in chapter 7 as to how this might be possible by decreasing the activity

of the membrane-bound hydrogenase.


42

6.4 Results: Total molar consumption of H2, O2, and CO2

Another piece of information gleaned from this experiment is the total molar consumption of

hydrogen, oxygen and carbon dioxide during chemolithoautotrophic growth. This can be found using

equation 17. For this data, the volume of the bag initially is used to determine the total moles in the bag

(no) required by equation 17. The total molar consumption is important as it indicates how much syngas

could be processed in a reasonable sized bioreactor. The molar consumption of each gas is reported as

the total millimoles consumed throughout the six hour experiment. The total moles are then divided by

the average cell mass in the bag determined from the initial and final OD measurements. This data gives

the molar consumption for each gas per gram of cell mass over six hours of cultivation (table 5).

Table 5. Total molar consumption of the individual gasses H2, O2, and CO2 during chemolithoautotrophic growth. The data is represented as the millimoles of gas consumed per gram FDCM over six hours of cultivation. The average OD for each replicate was calculated using the initial and final OD samples and the amount of FDCM was determined using the conversion y=0.4135x (y is FDCM in g/L, x is OD) and adjusted for the 15mL volume used in the experiment.

Replicate Total moles consumed during experiment (millimoles/gram FDCM)

H2 O2 CO2

1 112 35 18

2 96 30 16

3 109 42 16

4 157 47 26

5 156 47 27

Average 126 40 20


43

6.5 Results: Rate of CO2 fixation

The gas consumption experiments can also be used to determine the rate of CO2 fixation in our

strain of C. necator. The change in volume percentage for each gas throughout the experiment can be

converted into the number of moles consumed using equation 17 and the volume of the bag measured

through water displacement. Figure 9 shows how the moles of any gas consumed during one sample

period are calculated. In order to express a rate of CO2 fixation, the total moles of CO2 consumed over the

six hour experiment was determined for each replicate by adding the moles of CO2 consumed over all

three sampling periods. Consumption of CO2 is converted from moles into grams by multiplying the

molecular weight of CO2, 44.01g/mol by the total moles consumed (table 6). The average OD in the bag

during the experiment was determined from the initial and final OD sample and is converted into grams

of FDCM using the previously established equation y=0.4135x where x is OD and y is FDCM in g/L. From

the FDCM in g/L it was determined how many grams were present in 15mL of culture. The total grams of

CO2 consumed per bag are divided by the average cell mass of the bag and then divided by six hours. This

data represents the average rate of CO2 fixation per dry cell mass per time and is expressed as

gCO2/gFDCM ∙ hr-1.

Table 6. Data used from gas consumption experiment to calculate rate of CO2 consumption expressed in gCO2/gFDCM ∙hr-1 (red column). Total grams of CO2 consumed was divided by the average grams of FDCM in 15mL and divided by the six hours between ODo and ODf.

Replicate #

Total CO2

consumption ∆t1 to ∆t3 (mmol)

gCO2 consumed (44.01gCO2/mol)

Average OD [(ODf-ODo)/2]

FDCM in 15mL of

culture (g)

gCO2/

gFDCM ∙hr-1

1 65.3 0.0287g 5.80 0.036g 0.133

2 77.1 0.0339g 7.80 0.048g 0.118

3 43.7 0.0192g 4.39 0.027g 0.119

4 78.6 0.0346g 4.86 0.030g 0.192

5 78.5 0.0345g 4.74 0.029g 0.199


44

Table 6 shows the CO2 fixation rates for all five replicates over the course of the six hour gas

consumption experiment. The average CO2 fixation rate was determined to be 0.15 grams CO2/gFDCM ∙

hr-1 with a standard deviation of 0.04. The gas bag experiment was not designed to optimize mass

transfer and increase growth rates. Therefore the CO2 fixation rates found in this experiment are

expected to be slower than previously reported, although the relative growth rate of the culture can be

used to compare the difference in CO2 fixation rates found in the literature.

The only group to offer an estimate of the CO2 fixation rate in C. necator performed cultivation in

an open-system bioreactor during the late sixties [2]. They were able to determine the amount of CO2

consumed by their bioreactor and reported that 30mL/min CO2 was consumed by a 4-liter reactor with a

constant cell density of 1.65g/L (dry weight). This rate of CO2 fixation equates to 0.48 gCO2/gFDCM∙ hr-1.

However the rate of CO2 fixation determined will depend on the doubling time of the culture, if the

culture is growing faster, more CO2 will be consumed per gram of cells per hour. To compare our results

with those found previously we must consider the doubling time of the two cultures.

Table 4 shows the doubling times for all five replicates. Replicate 2 was inoculated much higher

than the other replicates in order to investigate the effect of cell density on bag performance and gas

consumption data. Because replicate 2 was inoculated high and does not have a doubling time similar to

the other four replicates it was omitted from the average doubling time which was calculated using

replicates 1,3,4,and 5. The average doubling time of the four replicates in this gas consumption

experiment was 7.16 hours with a standard deviation of 0.9 hours. The average doubling time of the

open-system batch culture experiment by Amman was calculated using the average cell yield in g/hr

reported in the literature and was determined to be 2 hours. In our experiment the average doubling

time was about 3.5 times slower than Amman’s group, therefore it is expected that the CO2 consumption

rate calculated in this experiment should be about 3.5 times slower than that found by Amman. The

average CO2 fixation rate calculated in this experiment is 3.2 times slower than the CO2 fixation rate

determined by the Amman group.

6.6 Testing repeatability of H2/CO2 and H2/O2 ratio measurements using ANOVA

The average values reported for the H2/CO2 and H2/O2 ratios came from the average of

measurements taken at each sampling period for the five replicates. In order to determine whether the


45

difference between the averages found for each replicate are statistically different from one another we

will test the data using the analysis of variance (ANOVA) test. Using ANOVA we can test the null

hypothesis that there is more difference in the variance between groups than within groups. The null

hypothesis states that there is no statistical difference between the average ratios obtained from each

replicate, indicating that cellular performance was similar for all replicates and that the average

calculated does not depend on the group the data was from. The two assumptions of ANOVA are that the

data is normally distributed and that the variances of the replicates are approximately equal. Both of

these assumptions are fulfilled for data from the H2/CO2 and H2/O2 ratios. We will test the null hypothesis

at the level of α=0.05, meaning a p-value greater than 0.05 fails to reject the null hypothesis.

Using the data from table 6 which expresses the average and standard deviations of H2/CO2 and

H2/O2 ratios for replicates 1-5, ANOVA tests were conducted. For each test the number of treatments was

five and the number of groups was three. There were 14 total degrees of freedom for each ANOVA test.

The p-values were found to be 0.58 for H2/CO2 and 0.075 for H2/O2. In both cases the ANOVA failed to

reject the null hypothesis. The variation observed within the data for each of the five replicates was not

statistically different from the variation between averages from the five replicates. This implies that the

results of this experiment were repeatable and the average values reported for the H2/CO2 and H2/O2

ratios are an accurate representation of the data collected during this experiment.

Table 7. Results of ANOVA testing for H2/CO2 and H2/O2 ratios obtained from averages across three sampling periods for five replicates. The averages and standard deviations given were used for the ANOVA analysis. The null hypothesis that Ho: µ1=µ2=µ3=µ4=µ5 was tested at the α=0.05 level and was failed to be rejected for both the H2/CO2 and H2/O2 ratios tested. Note: replicate 3 is a clear outlier, if this data is omitted from the ANOVA analysis the p-values for both ratios are greater than 0.5.

Replicate # Average H2/CO2 Std. Dev. H2/CO2 Average H2/O2 Std. Dev. H2/O2

1 6.17 0.16 3.11 0.385

2 6.18 1.08 3.14 0.18

3 6.83 0.19 2.60 0.16

4 6.15 0.70 3.31 0.31

5 5.83 0.74 3.37 0.41

P-value from ANOVA 0.58 0.075


46

CHAPTER 7. DISCUSSON AND FUTURE RESEARCH

7.1 Discussion

Using a novel experimental design this research has offered valuable insights on the CO2 fixation

efficiency for our strain of C. necator. The four core objectives were fulfilled; the strain was identified

using 16S RNA sequencing, the specific growth rate and doubling time, H2/CO2 ratio, total molar gas

consumption and CO2 fixation rate during chemolithoautotrophic growth were determined. The data

collected during this experiment will be used to continue improving this microbial process of CO2 fixation

from syngas. However more importantly, the method created during this experiment will be a valuable

tool in the future for analysis of microbial gas consumption.

This method offers a quick, easy, cheap and repeatable way to determine the CO2 fixation

efficiency and gas consumption requirements for chemolithoautotrophic growth in C. necator. In the

future if attempts are made to improve the CO2 fixation efficiency of this strain through genetic

engineering this method can be used as the assay for gas consumption. Additionally, the method can be

adopted to assay gas consumption in other bacteria using different gas substrates as well; the only

change required is the GC methodology used to separate and detect the specific gasses on the GC

machine.

As discussed in the introduction, syngas is an industrially important source of hydrogen. The

feasibility of a wide-scale renewable biomass gasification initiative depends on the discovery of an

economic method of CO2 fixation to harness the increased fraction of this undesired molecule. The

microbial fixation of CO2 from syngas will only be useful if there is an appreciable amount of hydrogen

remaining after CO2 removal has been achieved.

It is demonstrated in tables 1 and 2 that the CO2 fixation efficiency of C. necator must occur at a

theoretically optimal level for the process of microbial CO2 fixation from syngas to be feasible. It was

determined that the H2/CO2 ratio of chemolithoautotrophic growth in our strain was 5.8. This is

equivalent to the production of less than one ATP per hydrogen molecule. This CO2 fixation efficiency is

much lower than theoretically expected. Even with coal-derived syngas which yields the highest H2/CO2

ratios, there is just barely enough hydrogen present in the syngas to allow for complete CO2 fixation at

this level of cellular CO2 fixation efficiency. For the future development of this CO2 fixation process it

must be discovered why the cell is functioning at such a low P/2e- level. The P/2e- ratio must be improved


47

for this method of CO2 fixation so that the cells require less hydrogen for growth and more of the input

hydrogen energy is recovered as biomass that can later be turned into valuable product (PHB).

Another aspect of this microbial CO2 fixation strategy that must be very-well tailored to promote

optimal growth rates and CO2 fixation rates is the bioreactor design. Nutrient requirements can be

predicted and impending nutrient deficiencies can be witnessed during batch cultures. To avoid nutrient

deficiencies at any time during continuous cultivation, each essential nutrient should be monitored via

probes in the bioreactor and automatic pumps should provide the necessary nutrients when required.

However, even with no nutrient deficiencies, the mass-transfer rates of the three gasses H2, O2 and CO2

from gas to liquid phase will determine the amount of gas available to the cells and is directly correlated

with the growth rate of the culture. Hydrogen and oxygen have very poor mass transfer coefficients and

are not easily dissolved in water. Poor mass transfer in our bioreactor and especially the bag cultures is

the reason why we could not achieve doubling times of 2 hours like previously reported. Improving the

mass transfer of H2 and O2 using membranes to increase aeration and gas to liquid surface area for mass

exchange are the focus of some groups studying syngas fermentation techniques [20].

Another step in designing more economical microbial CO2 fixation techniques from syngas using

C. necator will come from a better understanding of the molecular mechanisms determining the CO2

fixation efficiency in this organism. Genetic engineering could be used to increase the intrinsic CO2

fixation efficiency of this microbe.

7.2 Future Research-Improving CO2 fixation efficiency with genetically engineered C. necator

Due to previous experimental evidence and knowledge about the well-studied bacterium C.

necator, there is reason to believe that cellular performance under syngas conditions is different than in

environmental conditions. Hydrogen is scarcely available in the aerobic environmental conditions

indicative of C. necator niches. The presence of a periplasmically oriented membrane-bound

hydrogenase (MBH) directly linked to the electron transport chain is thought to confer hydrogen

scavenging ability to this organism allowing for direct ATP generation anytime the substrate (H2) is

available [18]. However syngas conditions are predominantly represented by hydrogen, which is present

in copious amounts compared to environmental conditions. If the MBH has no regulation for the amount

of hydrogen oxidized, there is the potential for protons to be generated faster than ATP synthase can


48

produce ATP for the anabolic and anaplerotic reactions of the cell. An accumulation of unused protons in

the periplasm could result in the decreased ability to maintain a proton gradient across the membrane,

leading to energy spilling and a decrease in potential cellular efficiency.

The presence of two functionally distinct hydrogenase enzymes enables C. necator to exploit

hydrogen availability by converting H2 into utilizable reducing equivalents and energy at all times during

its presence. The expression of two unique hydrogenase enzymes simultaneously controlled by the same

response regulator indicates that this organism has the ability to readily change its proteome in response

to H2 availability. Because natural environments promoting lithoautotrophic growth in C. necator are

hydrogen deprived, hydrogen exploitation is advantageous. However, hydrogen exploitation may be

disadvantageous to the process of biological conversion of syngas into liquid fuels.

When cultivated lithoautotrophically, only the three gasses required for growth: H2, CO2, and O2

are supplied as the gas substrate. The concentration of these gasses in the feed-gas stream to the culture

can be manipulated in the laboratory and in practice an excess of hydrogen is provided to

lithoautotrophically grown cultures (H2:O2:CO2, 7:1:1 vol/vol) due to poor mass-transfer of hydrogen. The

hox gene cluster allows C. necator to scavenge alternative sources of energy under conditions of

hydrogen availability, but perhaps this system is over-productive in lithoautotrophic growth conditions

simulating syngas where hydrogen is present in excess.

The initial characterization of the two hydrogenase enzymes in C. necator involved independently

complementing a megaplasmid-cured and therefore lithoautotrophic deficient strain with the soluble and

particulate hydrogenase gene complexes on separate overexpression vectors [14]. C. necator mutants

devoid of a soluble NAD+ reducing hydrogenase are greatly impaired in lithoautotrophic growth while

mutants devoid of the respiratory chain-linked, membrane bound hydrogenase were not affected in

growth with hydrogen [14]. These results indicate that the SH alone can provide the NADPH reducing

power and reverse electron flow required to maintain a proton gradient for ATP production supporting

CO2 fixation via the CBB cycle under lithoautotrophic conditions. It was later found that activity of the SH

in MBH deficient mutants could compensate for the loss of the primary energy-generating MBH system

and accommodate for the altered electron flow by increasing the NADH dehydrogenase activity by 65%

from wild-type levels [16]. Without MBH activity, the electron flow of lithoautotrophic growth proceeds

through only one of four cytochrome oxidases, whereas it proceeds through two in wild-type cells [16].


49

The observation that the MBH deficient mutant was not affected in lithoautotrophic growth

supports the theory that the MBH may be consuming hydrogen for ATP production faster than the cell

can use the NADPH reducing equivalents available for cell growth. It is also evident that the hydrogenase

function of the SH alone can supply enough NAD(P)H to drive the CBB cycle and donate some NADH to

transfer electrons to the electron transport chain for the generation of a proton motive force. During

heterotrophic growth the proton motive force of ATP generation is maintained through the donation of

electrons from NADH, therefore it is not unreasonable that the NADH-generating activity of the SH can

also support this mode of proton gradient generation during conditions of unlimited hydrogen

availability.

Hydrogen is the most valuable product of syngas. Removing CO2 from syngas using C. necator

would be less efficient than possible if the MBH is oxidizing H2 faster than it can be utilized biologically.

Therefore, it will be very interesting to study the CO2 fixation capacity of a MBH deficient mutant using

the method created in this research. The resulting effect of reduced electron flow through the branched

electron transport chain of C. necator may drastically decrease the amount of hydrogen consumed during

lithoautotrophic growth.

During this research we attempted to create a genetic mutant of our strain by deleting the hoxG

gene using an R6K ORI derived suicide vector. We chose to delete hoxG because it is the functional H2-

dependent subunit of the MBH and ∆hoxG mutants have been shown to have completely abolished H2-

oxidizing activity of the MBH [4]. Due to a limitation of available vectors and the capacity for this strain to

become spontaneously resistant to ampicillin and spectinomycin we were unable to isolate a ∆hoxG

mutant. However, this strain has already been created by a German research group and could be

evaluated for CO2 fixation efficiency [4].


50

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