The oxygen output streams indicated in Figures 1 and 2 represent net oxygen production by the plants due to photosynthesis. However, oxygen production was not included as part of the model since it does not impact the economics of the process. Figure 3 shows the downstream processes for recovery and purification of the rBuChE, which was modeled after the rBuChE lab purification scheme from vacuum infiltrated N. benthamiana described by Hayward and the purification methods described by Lockridge et al.. Major operations include plant harvesting, shredding, screw press/disintegration, ammonium sulfate precipitation, centrifugation, tangential flow microfiltration, tangential flow ultrafiltration, ion exchange chromatography, affinity chromatography, and diafiltration.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,growing lettuce hydroponically 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 non-modified 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 cellulase 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 ethanolinduced 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.Comparison of responses of Cleopatra with the more tolerant root stock cultivar Carrizo citrange showed a similar down regulation for several pathways at 12 mai, most notably pathways associated with arginine and proline metabolism, galactose metabolism, and propanoate metabolism. The similarity of metabolic responses of Cleopatra and Carrizo is surprising, but may be associated with the foliar disease symptoms which were similar in manifestation in both cultivars at this stage of infection. It is important to note that except for galactose metabolism, other pathways of carbohydrate metabolism as well as TCA cycle reactions were not affected in Carrizo, which may explain the better performance of this root stock cultivar under HLB pressure. In contrast, the tolerant root stocks US-897 and US-942 which did not show any disease symptoms at 12 mai responded to infection by changes in the amounts of only three metabolites. This suggests that different and root stock specific mechanisms are associated with tolerant responses to HLB. Due to concern for pollinating bees in citrus trees in the spring, spraying for psyllid control with harsher, but often more effective pesticides is suspended from when 5 to 10 % of the citrus flowers are open until 95 % petal fall is reached. After use of dormant sprays at least one spray on emerging flush is desired before bee-friendly sprays are applied during the ‘flowering period’. Data was not available for how long a period existed from various stages of vegetative growth until 5 to 10 % open flowers is reached. Hamlin, Valencia,
Sunburst and Murcott blocks were monitored during the 2015, 2016 and 2017 bloom periods to determine vegetative bud break, leaf feather stage and unfolded leaves as well as pinhead, popcorn, open and petal fall stages of flowering. In the first two years the average time period from bud break to 10 % open flowers was 39 and 45 days, respectively, and the days from 10 % leaf feathers until 10 % open flowers was 26 and 20 days. Round oranges and mandarins differed in time from bud break to full bloom in 2015, 72 versus 57 days, but were similar in 2016, 57 and 56 days. Petal fall occurred 8 to 20 days after full bloom. Weather data will be evaluated to determine if any differences in time for vegetative development between the three years was due to daily temperatures, similar to the temperature response for flower development. More details including 2017 data will be discussed,4x8ft rolling benches particularly as pertaining to the best time to apply the first vegetative spray before the 10 % open flower stage is reached.This is most pronounced in early- to mid-winter under Florida conditions and has consistently occurred over the last four years. This flowering reduces the available buds for normal spring flowering and probably contributes to carry over of fungal inoculum for post bloom fruit drop . The role of fall tree water stress on the induction of this off-season flowering, and the association of this tree water stress with debilitation from HLB will be discussed. Huang long bing is a devastating bacterial disease of citrus and has been detected in most citrus producing areas worldwide. However, genetic sources of resistance/tolerance to this disease are extremely limited and little information is known about the inheritance of HLB tolerance. Several F1 populations derived from crosses between an advanced breeding selection and commercial germplasm are being evaluated under high HLB disease pressure conditions in Fort Pierce and Gainesville, Florida. Pedigree analysis of this trait originating from Poncirus trifoliata fits a Mendelian dominant inheritance pattern. These populations present an opportunity for identifying loci controlling HLB tolerance in citrus. Genetic variability in insect vectors can be a valuable tool to study vector competence determinants and to select or establish non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested more than 20 isofemale lines of Asian citrus psyllid Diaphorina citri, vector of Candidatus Liberibacter asiaticus . CLas is the bacterial pathogen associated with huanglongbing, which is the most serious citrus disease worldwide. Individual ACP adult males and females were collected from Murraya plants in various parts of Florida and reared in pairs. After mating, females laid eggs on Murraya plants before being tested for CLas by qPCR. Nymphal progeny from non-infected parents were tested for CLas acquisition from infected citrus plants for three successive generations. So far, we identified one line as a ‘good acquirer’ with 26.8-48.6 % of CLas+ adults. We also identified several poor acquirer lines with 0-10.2 % CLas+ adults. The ‘good’ and ‘poor’ acquisition phenotypes were stable over the three successive generations, showing that variation in CLas acquisition has a genetic basis in the psyllid. ‘Good’ and ‘poor’ acquirer lines were selected and further tested for CLas transmission into healthy citrus, using our excised leaf assay method . Interestingly, CLas transmission assays indicated that Laurel 8 is also a good vector line and Laurel 16 is a poor vector line , which suggests that both acquisition and transmission of CLas are correlated at least in these ACP populations.
We are continuing these tests and studying the genetic composition of both good and poor vector lines using molecular biology methods to study vector competence determinants of CLas in ACP that could be valuable in developing new tools for combating this devastating citrus disease. As sequencing technologies rapidly advance, the successful application of other cutting edge molecular technologies has become increasingly reliant upon the availability of a complete and accurate genome sequence. In an effort to verify the accuracy of the Las bacterial genome while not being subject to the identical limitations encountered with the original sequencing method, a BAC library was constructed from the DNA of Asian citrus psyllids collected from Huanglongbing-affected citrus. Of the 61,440 clones contained within the BAC library, 27 clones specific for Candidatus Liberibacter asiaticus were sequenced in their entirety and used to reconstruct the genome. During the reassembly, a novel ~8.3 kb DNA fragment containing 9 putative ORFs was revealed. Although smaller segments of the 8.3 kb fragment were found dispersed amongst the other sequenced Liberibacter strains, 2 of the putative proteins did not share any homology with any previously identified Las protein. Moreover, 1 of these proteins is not homologous to any protein currently in the NCBI database. Further examination regarding the fragment demonstrated that it was absent in 20% of the Las-infected citrus samples and 5% of the Las-infected ACP samples collected from across the state of Florida, suggesting it is functionally important in the life cycle of the bacterium although not required for survival since the entire 8.3 kb fragment was absent in the genomic DNA used to sequence Ca. L. asiaticus strain psy62. Taken together, the data suggest that the fragment is involved in the generation of a heterogeneous population and may contribute to the virulence of the bacteria through a type of phase variation. It also demonstrates the plasticity of the Las genome and provides a possible marker for monitoring genetic shift in the bacterium.Huanglongbing -affected oranges are typically green or yellow in color, rather than orange, and the latter is more common in the Florida citrus groves. The yellow color is often associated with insufficient accumulation of carotenoids in the flavedo, lack of natural shine, and shriveled peel . The green color is an indicator of maturity due to HLB-associated phloem malfunction and resulting retarded growth and development of the fruit.