Because of the mismatch in the volumes of fuel needed versus the volume of each individual therapeutic needed, it will be necessary to have a large number of crops, each producing the same bio-fuel precursor and different high-value products, which will be agronomically challenging. Biofuel production from lignocellulosic biomass relies on the microbial bioconversion of cell wall sugars and components into fuels and products . A major hurdle to efficient bioconversion is the recalcitrance of the feedstock material and the inhibitory effect that lignin has on this process. Cell-wall engineering has shown promise for decreasing overall recalcitrance by increasing the ratio of C6/C5 sugars, reducing lignin content, and reducing the acetylation of cell-wall polymers that limit the conversion efficiency of the feedstock material. While lignin is a major contributor of feedstock recalcitrance, it is also a promising substrate for specialized microbes that convert these aromatic polymers into usable products. The introduction of specialized microbial hosts into various processing systems has the potential to optimize the conversion of all lignocellulosic feedstock components into products with economic value, limiting the waste streams for bio-fuel production and increasing the viability for their use on a global scale. The synergistic application of these various strategies has the potential to make lignocellulosic bio-fuels economically viable while shifting the current paradigm of what an effective bio-fuel/bio-product production system achieves.
Through a multidisciplinary approach across all sectors,4×4 grow table we have the potential to revolutionize the manufacturing of bio-fuels/bio-products from lignocellulosic biomass ushering in a new era of green technologies. While the first and second generations of bio-fuels use light and CO2 to produce biomass in crops that is later fed to microbes, third-generation or algal bio-fuels combine energy capture and fuel production within a single cell of photosynthetic cyanobacteria and algae . Having the entire fuel-production process take place in one organism makes the process more direct and efficient with no energy invested in non-fermentable parts such as plant stems, roots, and leaves. The solar energy conversion in cyanobacteria and algae is higher than that in plants, reaching an efficiency of 3% in microalgae compared to less than 1% in most crops. Furthermore, many species can grow in wastewater or marine environments with simple nutritional requirements and therefore do not compete for land use with agriculture. It is estimated that microalgae can produce oil at a yield of 100,000 L/hectare/year, while palm and sunflower oil can only reach 1,000–6,000 L/hectare/year. Algal fermentation could also lead to 9,000 L/hectare/year of bioethanol production, compared to 600 L/hectare/year derived from corn. Despite these favorable comparisons, attempts at large-scale cultivations have struggled with high production costs. Unlike agriculture, which has been optimized over millennia by humans, the technology for mass scale cultivation of photosynthetic microorganisms is still in its early developmental stage. The cultivation can be done in either an open system like a raceway pond, or in a closed system such as a photobioreactor.Ideotypes are theoretical archetypes of crops which serve as a practical framework for plant breeders to critically evaluate what traits they should be targeting for specific applications.
With advances in plant biotechnology and a growing urgency to adopt more sustainable practices across our economy, new uses for crops as bioenergy feedstocks may pivot our definition of an ideal crop that is engineered for biomass and bioenergy production, in contrast to food production. Although there is a plethora of specific applications to which plant engineering efforts can contribute, here we highlight recent advances in two broad areas of research: increasing available plant biomass and engineering production of higher value co-products. Before our ability to genetically engineer plants, plant breeders were constrained to breeding and selecting from the morphological, physiological, and metabolic repertoire already preexisting in plant genomes. Initially, such efforts were focused on breeding out deleterious traits or on a narrow aim such as yield. Fifty years ago, the concept of an ideotype was proposed as an alternative regime. The ideotype is an idealized form of a particular crop, which could then be a target to breed towards, rather than merely breeding away from deleterious traits. This shift in mentality provided a much-needed framework to help set goals and target traits for plant breeding efforts. A useful ideotype must be ‘theoretically capable of greater production than the genotype it is to replace and of such design as to offer reasonable prospect that it can be bred from the material available. The discovery and development of plant genetic engineering technologies such as Agrobacterium-mediated and biolistic transformation expanded the scope of possible ideotypes, as plant engineering efforts can now draw on a much larger effective pool of genetic material, expanding from interfertile germplasm to all sequenced and characterized genes from across the tree of life.
Feedstock crops are harvested primarily for biomass, which is then used as a substrate for downstream processes . Thus, it becomes useful to frame plant carbon partitioning in terms of biomass composition, and what production or deposition of small molecules or polymers would be present in feedstock ideotypes. Using new synthetic biology tools to redesign carbon flow in plants, one may alter and optimize the composition of biomass and bioproducts in a way that cannot be achieved through conventional breeding methods, ultimately improving the scalability and feasibility of renewable feedstock crops. The ideotype for each crop may vary depending on its economics, growing region, and intended application. Here, we focus on carbon allocation as a metabolic/ physiological trait that may be modified to increase the utility and value of feedstock crops. Specifically, we focus on two aspects: 1) traits that may alter overall plant biomass and the usability of this biomass and 2) traits that may enhance the value of feedstock crops with the production of higher value co-products, paying special attention to advances within the last two years. The plant cell wall is a complex network of polymers and is one of the most effective carbon sequestering systems on the planet, with annual production of land plants estimated at 150–170 billion metric tons per year71. Cell walls represent a massive and largely untapped supply of six carbon sugars in the form of cellulose . However, cell walls are naturally recalcitrant to degradation and fermentation, limiting their use as chemical feedstocks rather than bulk materials. Lignin is a main inhibitor of saccharification in woody crops and hemicellulose limits saccharification yields in monocot biomass crops. Many engineering efforts have focused on decreasing lignin and improving fermentation characteristics. We are only beginning to explore ways to modify the composition and deposition of plant cell wall components to improve their ability to serve as biomass feedstocks. One strategy for reducing lignin accumulation uses 3-dehydroshikimate dehydratase from Corynebacterium glutamicum, which converts a lignin precursor into protocatechuate. Transgenic expression of QsuB in Arabidopsis thaliana plastids reduced lignin accumulation and improved saccharification yield by 25-100% depending on treatment method. Moreover, the six-carbon/five-carbon sugar ratio of the biomass also affects saccharification yields, with higher ratios performing better. The most highly accumulated C5 sugar is xylose, but xylan synthesis mutants show dwarfism due to xylem vessel collapse. This phenotype has been rescued by returning xylan synthesis specifically to vessel tissue,cannabis drying system leading to a 42% increase in saccharification yield compared to wild type. Acetylated cell wall components are converted during fermentation to acetic acid, which inhibits fermentation. RNA-interference has been used to decrease expression of genes responsible for acetylation, nearly tripling saccharification yields. Gene stacking has been used to generate engineered lines that contain multiple aforementioned traits. This demonstrates how modern bioengineering strategies can be used in tandem to modify the cell wall composition, a step towards engineering the optimum bioenergy crop ideotype. While ideotype specifics will vary by crop and intended application, in general an idealized biomass cell wall will have a high C6/C5 sugar ratio, low lignin concentration, and provide a favorable substrate for fermentation. Beyond modifying the molecular composition of the cell wall, others have also focused on engineering upstream metabolic processes to increase rates of photosynthesis, carbon fixation, and biomass production. Plants often absorb more photons than they can use for photosynthesis, leading to non-photochemical quenching that dissipates excess energy as heat but does not contribute to biomass. Mutation of light harvesting complex components results resulted in a 25% biomass increase in Nicotiana tabacum under field conditions78. It is also possible to modulate the NPQ process to shift more quickly from a heat-producing to a photosynthetic state, restoring energy capture via production of NADPH and ATP. Engineered N. tabacum over expressing the genes coordinating NPQ relaxation showed increases of ~15% in plant height, leaf area, and total biomass accumulation in field conditions.
These are promising results, as most plants use similar mechanisms making this technology applicable to bioenergy crops dependent on the maximum accumulation of lignocellulosic biomass. Another key process that limits the theoretical maximum for biomass accumulation is photorespiration. The primary cost of photorespiration stems from the process plants use to ‘recycle’ the unintended product formed via the oxygenase activity of RuBiSCO, leading to loss of both carbon and nitrogen. An alternative photorespiratory bypass based on the 3-hydroxypropionate bicycle was successfully engineered into cyanobacteria by expressing six heterologous genes from Chloroflexus aurantiacus. This bypass not only limits losses from photorespiration, it also fixes additional carbon and can supplement the Calvin-Benson cycle. Other photorespiratory bypasses have been demonstrated to work in planta yielding more than a 25% increase in biomass in field trials. Thus, the ability to modify both the rate of carbon fixation and the fate of carbon deposition in the form of various cell wall polymers have been shown to be complementary processes for increasing the accessible feedstock sugars from future feedstock plant crops. Lignocellulosic bioproduction offers a much larger potential supply of biomass than food-based fuels such as corn-ethanol, and reduces the conflict between food and fuels, materials, and other products which may be produced from biomass crops. Future biomass crop ideotypes should therefore be designed to ensure the use of lignocellulosic material is cost effective. Lignocellulosic bio-fuels have been slow to achieve commercial viability, in part due to low fuel prices and the chemical recalcitrance of lignocellulosic matter. A promising strategy to make lignocellulosic bio-fuels economically competitive is the co-production of higher value products directly in feedstock crops, which can be separated from the bulk carbon fuel source during processing. This can be achieved in two ways: either feedstocks for lignocellulosic bio-fuels can be modified so as to produce a higher value side product, or lignocellulosic bio-fuel can be produced from side products of other agricultural processes. The former is amenable to feedstock bioengineering efforts to optimize for bio-fuel purposes and will be discussed here. The ideotype of co-product crops will depend on the specific crop, but one important component is that the co product sells for more than the cost of extraction. Co-product value and market size tend are often inversely correlated, as shown in Figure 4.The base use of most biomass crops is production of ethanol, but plants have been engineered to produce co-products such as higher value fuels, commodity chemicals, and high value small molecules. Higher value fuel products include lipids for bio-diesel and jet fuel. Biodiesel-grade lipids have recently been produced in engineered sorghum that accumulates 8% dry weight oil in leaves in the form of lipid droplets85. These droplets can be extracted using simple, cheap techniques during the standard processing pipeline for lignocellulosic bio-fuels, minimizing additional purification costs. Jet fuel is also a high-volume product with an annual market size of 290 billion liters in 2015, with prices usually ranging around $1 per liter. There is no practical alternative available for liquid aviation fuels, which account for a small but rapidly growing fraction of total anthropogenic greenhouse gas emissions- currently 2.3% and growing at approximately 6% per year. Jet fuels have been produced from the oilseed crop camelina, and efforts are underway to increase jet fuel yield. Another promising high-volume side product is 1,5-pentanediol, a commodity chemical used in polyester and polyurethane production. The present market value is around $6000/ton, with a market size of 18 million USD. Using plants as a production chassis for high value low volume products has received substantial attention in recent years, with several analyses suggesting plants may allow for cheaper production of edible vaccines, bulk enzymes, and monoclonal antibodies than alternative systems.