Microfluidic reactors for artificial photosynthesis of carbohydrates using Calvin cycle

Abstract

Natural photosynthesis (NPS) in green plants relies on Calvin cycle to convert CO2 into carbohydrates, but the energy efficiency is extremely low. The first step of Calvin cycle catalysed by the enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), though most important in plants, poses to be the major bottleneck due to its low enzymatic efficiency. This PhD research aims to produce basic food materials by realizing Calvin cycle in vitro using microreactors. The core technique here is the immobilization of RuBisCO into the microreactors, which enables to overcome the long-standing problems of enzymes and inherits many merits of microfluidics. This research developed three designs of microfluidic reactors for RuBisCO immobilization to produce the glucose precursor, 3-phosphglycerate (3-PGA) using the first step of Calvin cycle. It is original and new since little similar work can be found in literature. The first design is a PDMS microfluidic reactor (PMR), which directly immobilizes RuBisCO on the inner surface by physical adsorption. It produces 3-PGA at the production rate of 0.011 nmol·min-1 and presents a great feasibility in 3-PGA production. But the reusability is quite weak. Only ~20.3% of the initial activity can be attained after 5 cycles of reuse. The second design (RI-DPMR) utilizes polydopamine to modify the inner surface of microreactors for RuBisCO immobilization via covalent binding. It greatly increases the surface area from a nanoscopic aspect and significantly enhances the 3-PGA production rate to be as high as 0.122 nmol·min-1, presenting an 11-fold enhancement as compared to that of the first design. Experimental results also show that the immobilized RuBisCO presents enhanced storage stability, thermal stability and reusability as compared to the free RuBisCO in solution. In the third design (RI-SPMR), the microreactor is formed by compressing porous PDMS sponge into a confined space. Polydopamine is used for RuBisCO immobilization as well. This monolithic structure provides much larger surface area for RuBisCO immobilization, ensuring the highest 3-PGA production rate (0.715 nmol·min-1, 65-fold enhancement as compared to the first design) of the three designs. In summary, three designs of microfluidic reactors have been developed to improve the surface area and the enzyme immobilization technique for glucose precursor synthesis. Compared with the bulk reaction using free enzyme in solution, the microfluidic reactors have shown enhanced thermal stability, storage stability and reusability. Moreover, the use of microfluidic reactors allows easy control of reaction by changing the flow rate and continuous production of 3-PGA with small amount of RuBisCO. Once scaled up, they would enable to produce large amount of basic food materials in a low-cost, convenient and durable way, which would relieve the food crisis and prepare for future space colonization. Furthermore, the ideas can be applied to other bio-enzymatic systems, expanding the application of enzyme engineering and microfluidics in industry.