MBR-004: THE FIRST BIONIC SOLAR CELL THAT PRODUCES IN-SITU HUMAN LIFE SUPPORT

RESOURCES USING CO2 AND SUNLIGHT Mohammed bin Rashid (MBR) Space Settlement Challenge

Final Report

December 2018

This project received seed funding from the Dubai Future Foundation through Guaana.com open

research platform

Performed and Prepared by:

Dr. Tara Karimi Chief Science Officer - Cemvita Factory Houston, TX

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THE FIRST BIONIC SOLAR CELL THAT PRODUCES IN-SITU HUMAN LIFE SUPPORT RESOURCES USING CO2 AND SUNLIGHT

EXECUTIVE SUMMARY One of the major challenges of a Mars mission is the production of human life support supplies including a nutritious source of food. Currently, almost all supplies are supported from the earth. Dependance on earth is a risk for long-duration space missions. Therefore, development of innovative solutions for in-situ resource utilization (ISRU) is critical to provide a self-regenerative food system for long-duration space missions. One such ISRU solution can be modeled after natural photosynthesis in plants that can harvest the abundant solar energy to convert CO2 and water to glucose and oxygen. CO2 is an ideal feedstock for ISRU since astronauts breathe out 1kg/day/person of CO2, and 96% of the air consists of CO2 on Mars.

In this study, we designed a bionic plant which mimics the photosynthesis process. Our enzyme-based synthetic pathway absorbs sunlight, CO2, and water to generate glucose and oxygen. Glucose is a base nutrient and a critical feedstock for almost all biomanufacturing processes. Results of the first phase of our research indicated the feasibility of glucose production utilizing an enzyme-based system which mimics photosynthesis. In the next step, we will focus on the large-scale glucose production and the generation of other human life support resources including pharmaceutics.

INTRODUCTION Photosynthesis provides the base energy requirements for sustaining of life on earth. However, growing plants in space is very difficult due to microgravity and harsh environmental conditions. An alternative approach would be a synthetic system which mimics the natural photosynthesis process by bioengineering of enzymatic processes in a more controlled environment. Figure 1 provides a summary of the photosynthesis reaction and Figure 2 provides a schematic image representative of photochemical and enzymatic pathways involved in natural photosynthesis in plants Including:

• 1- light-dependent phase: in the first step of photosynthesis solar energy is used for hydrolysis of water. Oxygen is released to the environment and hydrogen is used for the next step CO2 reduction reactions.

2- light-independent phase: in this stage CO2 reduction leads to the production of glucose.

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Figure 1. Photosynthesis equation

Figure 2. Schematic image representative of two main stages of photosynthesis in plants

Here, we designed a bionic plant which simulates the photosynthesis in plants to produce glucose as a base nutrient and a major feedstock in biomanufacturing processes. The system comprises two units:

1- The first unit simulates the light-dependent photosynthesis reactions by absorption of sunlight and water hydrolysis which leads to the production of hydrogen and oxygen molecules. Oxygen is either released or captured to be used in the oxygen recycling system. Hydrogen is collected for use in the sequential CO2 fixation reactions.

2- The second unit simulates the biosynthetic reactions of photosynthesis (through CO2 fixation process). At this stage, CO2 is absorbed from the atmosphere and reduced by hydrogen molecules (released from the water-splitting reaction). Glucose is then reduced as the output product through a set of enzymatic reactions. Glucose can be eaten directly or used as a feedstock for a wide range of downstream biomanufacturing processes including the production of different nutrients, pharmaceutics, and polymers.

Extensive research has been done on artificial photosynthesis. However, the major focus has been on producing solar fuels (Zhang et al., 2011; Nocera, 2012, Liu, et al., 2016; Li, et al., 2017; Sakamoto, et al., 2017; Schlager et al., 2017). In other attempts, artificial photosynthesis has been applied as a biomanufacturing method to produce chemicals (Zhang et al., 2011; Nocera, 2012). These methods also use rare and expensive metals like platinum, rhenium, and iridium as catalysts. Very limited research has been done on the manufacturing of food and human life-supplies with artificial photosynthesis.

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We have applied the existing knowledge in biomanufacturing and with inspiration from photosynthesis in plants, we suggest an innovative approach for in situ production of human life supplies, starting with glucose. To this end, we designed a synthetic biochemical pathway applying methods of synthetic biology. The main objective of this study was to find out if we could replicate the photosynthesis reactions using controlled biochemical pathways to absorb sunlight, carbon dioxide, and water from the environment and produce oxygen and glucose.

EXPERIMENTAL DESIGN In this study we divided the photosynthesis reactions into three steps:

1- Production of oxygen and hydrogen by water-splitting reaction (Figure 3-A).

2- CO2 fixation and biosynthesis reaction (Figure 3-B).

3- Regeneration reaction and recharging of initial substrates.

For sustainability purposes, we regenerated the main substrates (e.g. Ribulose 1,5-bisphosphate from the light- independent reactions) and co-factors (e.g. NADH) through enzymatic reaction. We used CO2 and sunlight as the source of feedstock and power. Figure 4-A illustrates a lab-scale prototype of our water hydrolysis system. We then used the output hydrogen, generated from the water-splitting reaction, for reduction of CO2.

Figure 3 (A-B). A schematic image representative of photo and biochemical reactions that are involved in designing of Cemvita's bionic solar cell. 1- In the first step, we hydrolyzed the water-applying a pair of graphite electrodes and a power supply (A). 2- In the second step, we

simulated the light-independent reactions of photosynthesis through a synthetic enzyme pathway (B).

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Figure 4 (A-B). Image illustrates a Lab-scale prototype of water hydrolysis unit (A) and the lab-scale enzyme-based bioreactor unit (B).

METHODS AND PROCEDURES • Production of oxygen and hydrogen through water-splitting reaction

To simulate the first stage of photosynthesis (and to accelerate the efficiency of hydrogen and oxygen production reaction), we applied an electrochemical method for the water-splitting reactions and utilized a solar panel to provide the energy required. We performed the water-splitting reaction at low voltage starting from 1.9 V and gradually increased up to 5 V. To increase the conductivity, we prepared a NaCl solution (0.9%) and initiated the reaction using a pair of graphite-based electrodes at voltage 1.9 V. We adapted the design of electrodes and experimental conditions for water-splitting reaction from microbial electrosynthesis systems (Liu et al., 2018; Hass et al., 2018; Rabaey and Rozendal, 2010).

CO2 Fixation and Biosynthesis Reactions To perform the CO2 fixation and glucose biosynthesis reactions, we designed an enzyme-based pathway which mimics the light-independent reactions of photosynthesis. Figure 5 shows the enzymatic-based pathway to produce glucose from CO2 and hydrogen. The reaction cocktail included enzymes, cofactors, and critical substrates for in-vitro production of glucose. We applied the following enzymes in the CO2 fixation and glucose synthesis reactions; Rubisco, Aldolase, Fructose 1,6-Bisphosphate, Fructose 6-Phosphatase, Glucose 6-Phosphatase, Adenylate Cyclase, AMP- Phosphotransferase, and Phosphoglycerate Kinase. Ribulose 1,5-Bisphosphate was also used as the initial substrate. ATP and NADPH were also added to the reaction cocktail to initiate the process. We injected CO2 into the reaction with the flow rate of 100 ml/min and used NADP+ as a carrier of hydrogen between water-splitting reaction and CO2 fixation/reduction process.

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Figure 5. Schematic shows the photoelectrical and biochemical reactions involved in synthetic pathways of glucose production.

The first enzymatic reaction was operated by Rubisco which plays a major role in carbon dioxide fixation/reduction. The initial product of carbon dioxide reduction is glyceraldehyde 3-phosphate (GA3P), which is a simple 3C sugar. In the next stage, conjugation of two molecules of GA3P leads to the production of 6C- sugars. To produce 6C- sugars (including fructose and glucose), Aldolase was added to the reaction. Aldolase causes the conjugation of two molecules of GA3P to generate a 6C- sugar (Fructose 6-phosphate). Phosphoglucoisomerase was also added to the reaction to convert Fructose 6-phosphate to Glucose 6-phosphate. Finally, Glucose 6-phosphatase converts Glucose 6-Phosphate to glucose. Figure 5 illustrates a schematic image representative of the above-mentioned biochemical reactions for in-vitro reduction of CO2 and production of glucose molecules.

We prepared the reaction buffer by adding 12.11 mg/ml Tris Base, 0.48 mg/ml MgCl2, 6.6 mg/ml KHCO3, and 0.78 mg/ml dithiothreitol (DTT) in deionized water. Then, we added ATP (with the final concentration of 0.5 mg/ml). Next, we added the following carbon-fixation enzymes/reagents to the reaction: 0.177 mg/ml of NADPH (reduced nicotinamide adenine dinucleotide phosphate), 10 U/ml of phosphoglycerate kinase (PGK), 10 mg/ml of ribulose bisphosphate carboxylase (Rubisco), 1 U/ml Aldolase, 10U/ml Fructose 1,6-Bisphosphatase, 10 U/ml Phosphoglucoisomerase, 0.1 U/ml Glucose 6-Phosphatase, 10U/ml Adenylate kinase, 10U/ml Phosphotransferase, 0.08 mg/ml glyceraldehyde 3- phosphate dehydrogenase (GAPDH), and 0.116 mg/ml of Ribulose 1,5-bisphosphate (RuBP). The solution was shaken vigorously for 30 seconds. The reaction solution was then transferred to a bioreactor chamber. We injected CO2 into the reaction solution with the flow rate of 100 ml/min (Figure 4-B). In addition, we inserted a pair of graphite electrodes in the solution. Electrodes were separated from each other by a layer of Nafion membrane 117 and the process was initiated by connection to the power supply.

Regeneration reactions 1- Regeneration of NADPH The progress of the first stage of photosynthesis depends on the water-splitting reaction. To increase the efficiency of water-splitting reaction, several synthetic approaches including electrochemical, photochemical, and enzymatic methods have been applied. Here, in the first phase of this study, we applied an electrochemical method to split water molecules and produce oxygen and hydrogen. Oxygen was released to the atmosphere in our current study. However, it can also be isolated for storage or

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use. Hydrogen reacts with NAD(P)+ to produce NAD(P)H which functions as a hydrogen-carrier to transfers hydrogen to the next stage CO2 reduction reactions.

Regeneration of NADH is critical for the progress of photosynthesis reactions. Otherwise continuous addition of NAD(P)H to the reaction is not sustainable. According to the results of previous studies, different methods can be applied to reduce and regenerate NAD(P)H molecules including, enzymatic methods, chemical, electrochemical, and photochemical methods (Zhang et al., 2011, Uppada et al., 2014; Wang et al., 2017; Ali 2018). Given the high efficiency and simplicity of electrochemical methods, in the first phase of this study, we selected this method to split water molecules. To this end, we used a pair of graphite electrodes and water-splitting reaction started at 1.9 Volts. We supported the required power supply for the reaction from solar energy using a solar panel that can provide a varying output of up to 12 Volts and a power equivalent to 4.25 Watts. In addition to the above- mentioned electrochemical method, in the next phase of this study, we will apply a photochemical catalyst (carbon nitride) for water-splitting reaction.

2- Regeneration of ATP molecules ATP is the energy currency of the cell and therefore required for almost all biosynthesis processes in the biological systems. During natural photosynthesis, ATP is produced by F0F1 ATP synthase which is a part of light-dependent reactions of photosynthesis. ATP provides the required energy for the progress of CO2 fixation/reduction. Production of ATP in cell-free systems (applying F0F1 ATPase) has low efficiency due to the complexity of the natural structure of F0F1 ATP synthase on thylakoid membrane. Therefore, development of alternative low-cost methods for regeneration of ATP is demanding for cell-free photosynthesis and biomanufacturing processes.

In this study, we regenerated ATP applying a two-step synthetic enzymatic reaction including polyphosphate kinase (PPK), Adenylate kinase (ADK) and AMP Phosphotransferase (PPT). In this reaction, we used polyphosphate or Poly (Pi)n as a source of phosphate. Polyphosphate is an attractive source of phosphoryl group for ATP regeneration because of the low cost and the high stability of its chemical structure. Figure 6 illustrates a scheme of ATP regeneration reaction applying PPT and ADK. In the first step, PPK can regenerate ATP by using exogenous polyphosphate and ADP. Sub-sequentially, one molecule of ATP can be generated from two ADP molecules. This reaction is mediated by polyphosphate-independent Adenylate Kinase (ADK) and yields one AMP molecule. In the next step, AMP is converted to ADP by PPT by using one phosphate group from Poly (Pi)n.

The PPT/ADK system provides an attractive alternative to existing enzymatic ATP regenerative systems and has the cost advantage since AMP and Polyphosphate are both inexpensive substrates. ATP-regeneration by PPT/ADK system has previously been applied as a cost-effective solution in different scaled cell-free biomanufacturing processes (Zhang et al., 2011). In this study, we applied this PPT/ADK system in a synthetic enzymatic pathway for CO2 fixation and glucose synthesis.

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Figure 6. Schematic image representative of ATP regeneration from phosphate-Poly (Pi)n mediated by polyphosphate: AMP phosphotransferase (PPT) and Adenylate Kinase (ADK). (Image adapted from Zhang et al., 2011).

To optimize the experimental conditions, glucose synthesis was evaluated at different environmental conditions including different ranges of temperature (25, 30, and 37, 40, 45. And 50 0C), pH and concertation. We collected samples at different time points (after 6, 12, and 24, 36 and 48 hours) and stored at -200C until the analytical assays.

Glucose Analytical Assays For total carbohydrate and glucose measurement assay, we used Abcam carbohydrate assay and Megazyme, D- Glucose assay kits. Abcam carbohydrate assay kit was based on the Phenol-sulfuric acid method. Carbohydrates (including glucose) were hydrolyzed in the presence of sulfuric acid and converted to furfural or hydroxy furfural. Furfural compounds were detected by addition of the developer solution which caused the formation of dark- orange chromogen. The chromogen was quantified by measurement of absorbance at 490 nm.

In addition, we applied Megazyme assay kit for more specific detection of glucose level. Megazyme assay kit is designed based on two enzymatic reactions for specific detection of glucose. Before performing of glucose assay, firstly we heated up all the samples to 60 0C for 10 min to deactivate the existing enzymes (from the glucose synthesis pathway) in the solution. Then we cooled the samples on ice for 15 min for measurement of glucose content. Megazyme assay were done by two sequential enzymatic reaction which cause the conversion of glucose to glucoronate-6-phosphate. In the first reaction, glucose converts to glucose 6 phosphate (G-6-P) by Hexokinase (HK) and in the presence of ATP. In the second reaction, in the presence of Glucose 6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized by NADPH to gluconate-6-phosphate with the formation of NADPH. The outcome product (which was stoichiometric with the amount of glucose) was measured in absorbance 340 nm.

RESULTS AND DISCUSSION

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We observed the initiation of the water hydrolysis reaction at low voltage starting from 1.9 V. Formation of hydrogen and oxygen bubbles were observed around cathode and anode immediately after the connection to the power supply. Figure 3 illustrates the water hydrolysis reaction in the lab-scale hydrogen producing system. After the operation of the experiment in the bioreactor unit (containing all enzymes and cofactors), we evaluated the production of glucose at different time points (6, 12, 24 hours) using the glucose colorimetric measurement assay. Results of glucose measurement assay confirmed the glucose production under the present experimental conditions (Figure 7).

Figure 7. Image illustrating the formation of orange color by the glucose colorimetric assay in multiple samples obtained from glucose

synthesis reaction after 24 hours compared to the control group.

Figure 8 illustrates the trend of glucose production by time at 370C and pH 7.4. We observed Glucose production at levels of 2, 5.8 and 10.6, 18.4 and 39.3 mg/ml after 6, 12, and 24, 36, and 48 hours under the present experimental conditions. Figure 9 illustrates the effect of temperature on glucose production in enzyme-based photosynthesis reactions at pH 7.4, after 24 hours. We observed the optimal glucose biosynthesis reaction to be at 370C. Results of this study indicated that glucose production rate enhanced significantly by increasing the temperature from 20 to 370C. By increasing the temperature up to 400C we observed no significant difference in the reaction rate. We observed a significant decline in glucose synthesis when the temperature increased more than 400C. This can be related to the denaturation of enzymes beyond 400C. Figure 10 shows the effect of pH on glucose production in enzyme-based photosynthesis reactions in 10% enzyme mix solution after 24 hours. We observed the highest glucose production at pH 8.

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Figure 8. Graph shows the trend of glucose production by time in an enzyme-based reaction in at 370C and pH 7.4.

Figure 9. Graph shows the effect of temperature on glucose production in the enzyme-based photosynthesis reactions, pH 7.4 and after 24 hours.

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Figure 10. Graph shows the effect of pH on glucose production in enzyme-based photosynthesis reactions at 37 0C after 24 hours.

In conclusion, the results of this study revealed the feasibility of glucose synthesis from CO2 in a cell-free enzyme- based system. Similar results were reported by Schwander et al., 2016, developing a synthetic enzymatic pathway containing seventeen enzymes to convert CO2 into glucose. In the present study, glucose synthesis pathway was composed of nine critical enzymes. This makes the process more cost-effective compared to the previous efforts.

The main advantage of this cell-free biomanufacturing system compared to the bacterial cell-culture, especially for space application, is that the system does not require the time-consuming cell-culture processes (including the continuous monitoring of cell growth and sub-culture systems). In addition, compared to the chemical-based CO2 reduction method (which relies on the application of chemical catalysts), all reactions of an enzyme-based system can be operated in mild environmental conditions. In the present study, we achieved the CO2 reduction at 370C, while performing similar CO2 reduction reactions applying electrochemical methods requires significant energy consumption to achieve and maintain the high temperature. For instance, Sabatier reaction which is a well- defined electrochemical method for absorption and processing CO2 typically runs at 300-400°C (Guerral, et al., 2018).

The main concern about the application of enzymes in biomanufacturing processes is related to their functional stability at industrial scale. Methods of bioinformatics and genetic engineering have been applied extensively in bioengineering of industrial enzymes to generate new isoforms with higher structural and functional stability (Sigh et al., 2013: Damborky et al., 2014; Rigoldi et al., 2018; Silva et al., 2018). Also, immobilization of enzymes and biocatalysts on the surface of solid matrices (such as carbon nanotubes) or encapsulation in hydrogels have been applied to increase the shelf-life of industrial enzymes (Mohammad et al, 2015; Zucca et al., 2016; Satagopn, et al., 2017). Methods of enzymes’ stabilization and immobilization are very well established in the food, beverage, and pharmaceutics industries (Rigoldi et al., 2018: Silva et al., 2018). However, these methods have been less applied for the generation of enzyme-based artificial photosynthesis and CO2 utilization systems. The objective of this study at phase one was to determine the feasibility of glucose production through a hybrid photo- electrochemical and enzyme-based system which mimics photosynthesis. Our goal was to apply the existing knowledge in biomanufacturing and by inspiration from photosynthesis to offer an innovative approach for in-situ resource utilization.

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NEXT STEPS In the first phase of this study, our focus was on design and optimization of reactions for the glucose synthesis. In the next phase, we will continue our work with the scaleup study. To this end, we also provided a photobioreactor setup for growing of cyanobacteria and large-scale production of photosynthesis enzymes to be used in the next step cell-free enzyme-based biomanufacturing processes (Figure 11).

Figure 11: Cemvita Factory’s photobioreactor for large-scale production of enzymes of photosynthesis pathway in cyanobacteria.

We have also designed a hydrogel-based enzyme immobilization system to increase the stability of enzymes. We applied Calcium Alginate which is a linear polysaccharide derived from marine brown seaweed and algae. Alginate is a biocompatible polymer and has been applied widely in food and pharmaceutics industry (Blandino et al., 2001; Chakrabarti et al., 2003; Al-Mayah et al., 2012; Zucca et al., 2016). Figure 12- A, B illustrates a batch of Calcium Alginate beads that were generated for enzymes’ immobilization.

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Figure 12. image illustrates the prototype design of Cemvita’s photobioreactor (A) and Calcium Alginate beads (B). Alginate calcium beads will

be loaded with the enzymes of photosynthesis (glucose synthesis) pathway from Cyanobacteria (C)

In the next step we will continue our work with the scaleup study. Alginate loaded beads will be applied for CO2 fixation and biomanufacturing processes. Enzyme immobilization is critical for large-scale production of biomaterials and can improve the efficiency of biomanufacturing by increasing the structural and functional stability of enzymes, recycling of enzymes, and enhanced isolation of products from reactants.

In addition to glucose, in the next phase of this study we will work on biomanufacturing of other critical biomolecules including amino acids and vitamin as well as probiotics and critical pharmaceutics for deep space exploration and settlement applications.

CONCLUSION In summary, results of this study indicated the feasibility of glucose production, applying the combination of electrochemical and enzyme-based methods which mimic photosynthesis. Application of this bionic plant in large- scale provides a sustainable solution for in-situ production of human life support resources for long duration space missions and settlement of Mars. Same approach can also be deployed to achieve energy sustainability on earth via CO2 Utilization.

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