Fourth-generation biofuels seek to adapt the raw material to improve efficiency in CO, capture and storage. They take advantage of synthetic biology of algae and cyanobacteria (Flays and Ducat, 2015), which is a young but strongly evolving research field. Synthetic biology is based on the design and construction of new biological parts, devices and systems, and the re-design of existing, natural biological systems for useful purposes. It is becoming possible to design a photo synthetic, iion-pliotosynthetic chassis, either natural or synthetic, to produce high-quality biofuels (Scaife et al., 2015). For the first-, second- and third-generation biofuels, the raw material is either biomass or waste, both being results of “yesterday’s photosynthesis” (yet not from fossil resources). While these biofuels often are very usefi.il in a certain region or country, they are always limited by the availability of the corresponding organic raw material,
i.e., the biomass, which limits their application on a global scale (Inganas and Sundstrom, 2016).
Fourth-generation biofuels will be based on raw materials that are essentially inexhaustible, cheap and widely available. Photosynthetic water splitting (water oxidation) into its constituents by solar energy can become a large contributor to fuel production on a global scale, both by artificial photosynthesis (Wijffels et al., 2013) and by direct solar biofuel production technologies. Not only the production of hydrogen but also the production of reduced carbon-based biofuels is possible by concomitant enhanced fixation of atmospheric CO, and innovative design of synthetic metabolic pathways for fuel production. The generation of “designer bacteria” with new useful properties requires revolutionary scientific breakthroughs in several areas of fundamental research.
Fourth-generation biofuels are produced (i) by designer photosynthetic microorganisms to produce photobiological solar fuels, (ii) by combining pliotovoltaics and microbial fuel production (electro biofuels) or (iii) by synthetic cell factories or synthetic organelles specifically tailored for production of desired high-value chemicals (production currently based on fossil fuels) and biofuels (Wijffels et al., 2013).
Designer microorganisms in production of solar biofuels
Key improvements in photon-to-fiiel conversion efficiency as well as in the quality of the fuel will be based on generation of designer microorganisms. They produce a selected photobiological solar fuel, which is a non-fossil fuel made by direct solar energy conversion in photosynthetic microorganisms (algae or cyanobacteria), and are engineered, when uecessaiy, to secrete the fuel. Photobiological solar fuel is made in photo synthetic cells from solar energy using only water, or water and CO, as raw materials, depending whether the produced fuel is hydrogen- or carbon-based fuel, respectively (Hays and Ducat,
2015). Knowledge based on powerful biochemical and biophysical insights in natural pliotosynthetic light harvesting, water splitting, electron transfer, hydrogeuases and carbon metabolism is critical for the development of direct photobiological solar fuels. Microorganisms will be optimized for fuel production with synthetic biology approaches, metabolic engineering and organism design based on knowledge acquired by genomic research, molecular systems biology and extensive modelling research (Halfinaim et al., 2014).
The most advanced research made towards direct photobiological solar fuel production is based on research with unicellular algae and cyanobacteria. Cyanobacteria are suitable as pliotosynthetic chassis for their well-developed genetic transformation technologies as well as for extensive knowledge on their light harvesting and electron transfer processes, on then metabolome and advanced modelling research of the entire cell. Cyanobacteria har e been genetically engineered to produce various fuels and chemicals (e.g., H,. ethanol, isobutanol, isoprene, lactic acid) (Savakis and Hellingwerf, 2015). Introduction of various fermentative metabolism pathways to cyanobacteria cells by synthetic biology approaches has made it possible to produce biofuels directly from solar energy and Calvin-Benson cycle intermediates (Rabaey and Rozendal, 2010). Furthermore, the efficient secretion of products from the cells will increase the production capacity.
Breakthroughs in direct photobiological solar fuel production call for further research in design of light-harvesting systems, modelling and simulation of biological reactions and systems as well as in development of synthetic biology tools and production systems. During the coming 10-20 years, it is expected that various photobiological solar fuels will gradually enter into the market (Hays and Ducat, 2015).
It is possible, through synthetic biology approaches, to establish new-to-nature hybrid production organisms that use renewable electricity and carbon sources for the production of commodity chemicals and biofuels. A newly emerging field relies on the capability of certain microbes for direct electron capture from electrodes (e.g., from solar cells or any renewable electricity source) to assimilation of the reducing equivalents into metabolism, along with CO, utilization (Torella et al., 2015). The new advanced technologies based on the combination of photovoltaics and microbial electrosynthesis are called electrobiofuels. The microbial electrosynthesis is based on the concept of capturing the energy from the electrodes, i.e., via solar cells or any other renewable source of energy. With this system, energy from solar cells can be turned into storable energy sources (electrofuels). They allow renewables from all sources to be stored conveniently and efficiently as a liquid fuel.
Recently (Aro. 2016), a novel and scalable integrative bio electrochemical production system for isopropanol was demonstrated. The solar water-splitting catalyst, based on earth-abundant metals, was used to provide energy for growth of a bacterium. Ralstonia eutropha. In metabolically engineered Ralstonia. the energy from water splitting could be diverted for production of isopropanol. Authors claim the highest bio electrochemical fuel yield reported so far.
From current biorefineries to synthetic factories in production of solar biofuels
The design of synthetic factories and cell organelles for enhanced biofuel production is a future technology and is recently entering intense developmental phase at the basic level of biofuel research.
Likewise, the synthetic biology technology itself is still young and only a few truly synthetic examples have been realized by now (Cameron et al., 2014). For optimal solutions, one needs to go beyond the known possibilities offered by biology by speeding up evolution and screening a massive number of artificial biological combinations. Thus, automation, microfabricatiou and measurement technologies are an integr al part of a successful synthetic biology platform. Most importantly, engineering principles should be used to guide modelling, design and standardization of the synthetic biological systems and the entire development process so that optimization can progress from the ctment trial-and-error situation to the design of truly programmable biofuel production systems (Cameron et ah. 2014).
Synthetic biology, after reaching maturity as a technology, will give tools and concepts that will make biology frilly engineerable. and thereby make it possible to take full advantage of the diversity, functionality and specificity that biology can offer. Such research and technology development programmers should be established for construction of synthetic factories and organelles towards efficient biofuel production (Biogas from Waste and Renewable Resources, 2011).