Nomad Bioscience has reported successful substitution of the agroinfiltration step with “agrospray,” a technique in which a suspension containing the Agrobacterium inoculant is admixed with a small amount of surfactant and sprayed onto the leaves of host plants. This approach eliminates the necessity to grow plants in containers , a requirement imposed by the mechanics of the vacuum infiltration treatment in current procedures. Concomitantly, it also eliminates the cost of setting up and operating commercial-scale vacuum chambers, robotic tray manipulators, biomass conveyer systems, and so forth. Thus, this new approach should enable large-scale field inoculation of plants with agrobacteria and the production of bio-logics with more favorable economics. While we modeled the costs of producing cellulases via the agrospray approach, the sheer volume of enzymes needed for commercial-scale cellulosic ethanol processes necessitated a large investment in inoculum production infrastructure, including multiple fermentation trains and associated processing equipment. Further, the most efficient method of inoculating large areas was by aerial spraying, a procedure that not only entailed higher cost but that would also face regulatory uncertainties over spraying GM bacteria. We opted instead for an alternative model using trans genic N. tabacum plants, each line of which carries an ethanol-inducible gene for one component enzyme of the cellulase complex. Synthesis of the cellulase is triggered by application of a dilute solution of ethanol onto the leaves,grow table a process that has been demonstrated in small scale using a double-inducible viral vector.
We assumed that the dilute ethanol solution would be applied via ground irrigation systems that are currently used in agricultural practices, instead of aerial tankers. It was also assumed that the ethanol would be taken off as a side stream from the associated ethanol production facility that uses the cellulase enzymes. In so doing, we obviated the need to produce multiple inocula of GM bacteria and deliver them via aerial spraying. We were also able to model higher biomass density as well as higher expression yields of the enzymes in planta. These changes resulted in multiple economic benefits and were therefore adopted in our calculations.Issues that are important in PMP, such as mammalian-like glycosylation or other post translational modifications, high purity, or specific formulation, are not relevant in the manufacture of cellulases and hence we modeled the use of conventional Nicotiana species in the production of the several enzymes necessary for complete saccharification of feedstock.In this case study, we modeled the use of stable transgenic N. tabacum varieties, each modified to express one cellulase protein upon induction with dilute ethanol. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the non-induced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter.
In greenhouse studies, recombinant proteins have been expressed at up to 4.3 g/kg FW leaf biomass in the ethanol-inducible hosts, but seed lines for field application have yet to be developed. In our modeling, we assumed that each transgenic line would have been already field tested and available for implementation. We also assumed that large-scale stocks of each transgenic seed would need to be produced and have included this unit operation in our cost calculations. Because cellulases are needed in different ratios to effect saccharification of different feed stocks, we assumed that seeds would be mixed at the appropriate ratios and that the seed mixtures would be planted directly in the field. At maturity, what one would expect is a field of plants representing all the needed cellulase classes in the appropriate ratio for the intended feed stock. The current method of hydroponic cultivation of seedlings for transplantation to open fields, a common commercial tobacco cultivation practice to ensure germination and plants with good leaf size and quality, was substituted by direct seeding for more favorable economics. For example, traditionally tobacco may be grown at 12,000–16,000 plants/ha depending on variety. Higher-density seedling production for nontraditional uses of tobacco has been reported, targeting planting densities of over 86,000 plants/ha. While transplanting ensures germination and quality, there is an economic limit to the scale at which it can be deployed with highly cost-sensitive AI, leading to interest in direct seeding practices. Experimental high density cultivation studies via direct seeding have reported 400,000 to over 2 million plants/ha and biomass yields exceeding 150 mt/ha. Our modeling included these higher-density practices to determine economic impact.In contrast to typical PMP products, the cellulases would not be extracted after accumulation; rather, the plants would be mechanically harvested and transported to a centralized facility for silaging and storage. Since the cellulase enzymes need to be continuously supplied to the saccharification process in the bioethanol plant and the harvested tobacco is only available for a limited period during the year, the silage inventory would increase during the tobacco-harvesting period and would decrease during the fall/winter.
Cellulase activity in the ensilaged biomass is expected to be stable during the off-season storage. For feedstock conversion, cellulase-containing biomass would be mixed with pretreated lignocellulosic feedstock under controlled conditions to effect saccharification. Although not considered in this economic analysis, this feedstock replacement could also reduce corn stover feedstock requirements and associated costs. After separation of solids, the sugar solution would be fermented conventionally into ethanol, followed by distillation. The overall process we modeled is based on the US National Renewable Energy Laboratory process described by Humbird et al, with substitution of fungal cellulase production in the NREL model by the cellulases stored as silage described herein. Design premises for this process, specific assumptions used in modeling,ebb flow table and the resultant cost calculations are presented .Table 3 shows the total capital investment and annual operating costs for the plant-made rBuChE facility at an expression level of 500 mg/kg FW plant biomass . The annual operating costs are shown with and without facility dependent costs to simulate a new facility and use of an existing facility, respectively. Table 4 shows the resulting rBuChE cost per dose for both cases. Table 3 shows the breakdown of the capital investment and operating costs for the plant-made rBuChE and indicates that the unit production costs are estimated to be about $234/dose if facility dependent costs are not included in the annual operating costs or about $474/dose if these costs are included. Most of the capital cost and a significant portion of the operating costs are associated with the recovery and purification of rBuChE. Our base case assumed rBuChE expression of 500 mg/kg FW because that is a target expression level in ongoing research at several institutions. If a currently achievable level of 100 mg/kg FW is used instead , the costs increase to $1,210/dose and $430/dose when including and excluding facility dependent costs, respectively. In any scenario examined, the production costs in plants are significantly lower than the estimated production costs for blood-derived BuChE . We recognize that additional modification or formulation of the plant-produced enzyme might be necessary or desirable prior to adoption for human use and that such additional modifications would increase the cost of the AI. For example, Geyer et al. reported improved pharmacokinetics of PEGylated plant-produced BuChE relative to the nonmodified enzyme. However, because consensus on the preferred options for modification has not yet been reached, we omitted these additional steps from our calculations.The following premises and assumptions were used for evaluation of cellulase bio-manufacturing in open fields. Due to the fact that this process is specialized and due to the scale and input requirements of a modern bio-fuels operation, our analysis included the construction of a new, dedicated manufacturing facility to provide the required cellulase enzymes for a large scale cellulosic ethanol facility . Figure 4 shows the process operations required for cellu lase enzyme production on a per-batch basis. The flow sheet on the top shows the blending tank needed for preparation of the ethanol induction solution to be applied in the field, and the flow sheet on the bottom shows the transport and storage operations following harvest of the transgenic tobacco.Table 6 shows the total capital investment and annual operating costs for the production of 2.87 million kg of cellulase enzymes per year at an expression level of 4 g cellulase/kg FW tobacco biomass and a plant density of 130 metric tons of biomass per hectare per year.
The table also indicates the corresponding costs obtained from the JBEI model for fungal fermentation-based production of approximately the same amount of cellulase enzymes per year . For the base case study, the plant-based system results in a >30% reduction in unit production costs for the cellulases as well as an 85% reduction in the required capital investment. For the plant-based cellulase production system, the major contributors to the unit production cost were the costs associated with tobacco cultivation , the costs associated with ethanol spraying , followed by the costs associated with ethanol dilution, transporting and storage , and seed costs . The differences in total capital investment and annual operating costs for the two cellulase production platforms are not surprising, since the fungal fermentation area alone requires twelve 288,000-L fermenters along with the seed train necessary to provide the inoculum for the production fermenters. The differences between the two systems would be expected to be even larger if the total capital investment included additional factors for associated piping, instrumentation, insulation, electrical facilities, buildings, yard improvements, and auxiliary facilities because these would be reflected in the facility dependent component of the annual production costs. Figure 5 shows the effect of biomass density on the unit production costs for cellulase enzyme using the ethanol induced tobacco system and indicates, as expected, that the cost of goods decreases as tobacco biomass density increases. In agronomic studies with field-seeded tobacco cultivated at high density, biomass yields exceeding 150 mt/ha have been achieved; higher field densities may be possible with selected varieties and specialized agronomic practices.At the eastern boundary of the Sahel lies the Greater Horn of Africa , a region of northeastern Africa with highly food-insecure countries that are particularly vulnerable to interannual and decadal swings in precipitation totals . Both the GHA and Sahel receive the bulk of their precipitation during the June–September boreal summer season. These regions experienced a significant decline in JJAS precipitation during the 1970s and 1980s due to anomalous warming of the South Atlantic and Indian Oceans and subsequent shifts in moisture transports and upper-air flow . Following the 1980s, warming in the North Atlantic appears to have caused a precipitation recovery throughout much of the Sahel . In the GHA, however, the extent of post-drought recovery is unclear. Recent studies suggest drying over GHA may have continued into the 2000s , but the cause of this potential drying and decoupling of precipitation trends in the GHA from those of the Sahel remains unexplored. Previous studies have identified a variety of drivers of JJAS precipitation and drought in the GHA. While JJAS wind trajectories toward the GHA are quite variable from year to year, the rainforest region of the Congo Basin appears to be the main moisture source to Sudan and much of the Ethiopian Highlands, and the Indian Ocean is the main moisture source for eastern portions of Ethiopia and Kenya . South of the center of the North African surface low, deep convection and enhanced moisture transport from the south and west fuel monsoonal precipitation during JJAS in the Sahel and GHA. Historically, for both the Sahel and GHA, the primary driver of interannual and longer-term variability of summer precipitation is the north-south displacement of jets, zone of maximum convection, and southern boundary of the thermal low over northern Africa , all of which appear to be influenced by tropical sea-surface temperature anomalies . Some research also suggests that increased tropospheric aerosol concentration works to suppress the northward migration of the JJAS rainbelt into northern Africa, potentially reducing precipitation in the GHA . Further, relationships exist between the strength of the Indian monsoon and GHA precipitation . Overall, drought in the GHA tends to occur when there is a reduction in the amount of moisture reaching the GHA from the tropical Atlantic Ocean and Congo Basin region.