Improved Bioproduction of 1-Octanol Using Engineered Synechocystis sp. PCC 6803.

1-Octanol has gained interest as a chemical precursor for both high and low value commodities including fuel, solvents, surfactants, and fragrances. By harnessing the power from sunlight and CO2 as carbon source, cyanobacteria has recently been engineered for renewable production of 1-octanol. The productivity, however, remained low. In the present work, we report efforts to further improve the 1-octanol productivity. Different N-terminal truncations were evaluated on three thioesterases from different plant species, resulting in several candidate thioesterases with improved activity and selectivity toward octanoyl-ACP. The structure/function trials suggest that current knowledge and/or state-of-the art computational tools are insufficient to determine the most appropriate cleavage site for thioesterases in Synechocystis. Additionally, by tuning the inducer concentration and light intensity, we further improved the 1-octanol productivity, reaching up to 35% (w/w) carbon partitioning and a titer of 526 ± 5 mg/L 1-octanol in 12 days. Long-term cultivation experiments demonstrated that the improved strain can be stably maintained for at least 30 days and/or over ten times serial dilution. Surprisingly, the improved strain was genetically stable in contrast to earlier strains having lower productivity (and hence a reduced chance of reaching toxic product concentrations). Altogether, improved enzymes and environmental conditions (e.g., inducer concentration and light intensity) substantially increased the 1-octanol productivity. When cultured under continuous conditions, the bioproduction system reached an accumulative titer of >3.5 g/L 1-octanol over close to 180 days.

Process and reactor design for biophotolytic hydrogen production.

The green alga Chlamydomonas reinhardtii has the ability to produce molecular hydrogen (H2), a clean and renewable fuel, through the biophotolysis of water under sulphur-deprived anaerobic conditions. The aim of this study was to advance the development of a practical and scalable biophotolytic H2 production process. Experiments were carried out using a purpose-built flat-plate photobioreactor, designed to facilitate green algal H2 production at the laboratory scale and equipped with a membrane-inlet mass spectrometry system to accurately measure H2 production rates in real time. The nutrient control method of sulphur deprivation was used to achieve spontaneous H2 production following algal growth. Sulphur dilution and sulphur feed techniques were used to extend algal lifetime in order to increase the duration of H2 production. The sulphur dilution technique proved effective at encouraging cyclic H2 production, resulting in alternating Chlamydomonas reinhardtii recovery and H2 production stages. The sulphur feed technique enabled photobioreactor operation in chemostat mode, resulting in a small improvement in H2 production duration. A conceptual design for a large-scale photobioreactor was proposed based on these experimental results. This photobioreactor has the capacity to enable continuous and economical H2 and biomass production using green algae. The success of these complementary approaches demonstrate that engineering advances can lead to improvements in the scalability and affordability of biophotolytic H2 production, giving increased confidence that H2 can fulfil its potential as a sustainable fuel of the future.