Similar to calcium localization, decreased antioxidant potential in the apoplast has been shown to be correlated with BER symptom development. Ascorbic acid treatment of discs inhibited BER symptom development, representing the first causative evidence that increasing antioxidant potential in the apoplast incurs a protective effect against BER development.The pericarp disc system used with immature green tomatoes has multiple potential uses for future research. As demonstrated by the respiration data presented here, using this method in combination with non-destructive measurements can allow for active monitoring of the biological changes in the discs as symptoms develop. Similarly, performing destructive analyses on half of each disc while allowing the remaining half of the disc to continue the storage progression may allow for a better understanding of why some discs and tissues develop symptoms while others do not. However, care should be taken to reduce the risk of handling induced symptoms if this technique is pursued. Finally, and most beneficial, treating pericarp tissues directly can provide causative evidence for testing hypotheses that are currently supported by correlative evidence. Ascorbic acid and increased antioxidant capacity generally,draining pots have been proposed as incurring a protective effect against BER development. Results presented here provide causative evidence to support ascorbic acid’s protective effect. Similarly, much of the evidence for calcium’s role at the tissue level has been correlative in nature. Results presented here provide the first causative evidence for calcium’s protective effect against BER development at the cellular level. This method can be used to explore the mechanisms of BER development.
It would be particularly interesting to test the effect of calcium transport and receptor inhibitors. Inhibitors of enzymes involved in ROS metabolism could also be tested. Varied timing of such treatments during symptom development could also improve knowledge of the timing of events involved in BER development. This method could also be used to screen new BER treatments before larger scale testing in the field or greenhouse. Additionally, fruit phytotoxicity of non-BER specific field treatments could also be assessed using the pericarp disc system. It is worth noting, however, that treatments with effects at the whole plant level may not have the same effects in the pericarp system. Similarly, without access to xylem or phloem nutrient sources, cell expansion in the pericarp disc system is likely halted or abnormal. Thus, any treatment that acts through regulating cellular expansion may not be effective. However, this system is very effective in assessing treatments that disrupt pathways leading to cell death during blossom-end rot development.Chlamydomonas reinhardtii is a single-celled green alga that has been determined to be able to grow in the absence of light, and therefore does not require photosynthesis, by utilizing carbon containing substrates such as acetate. This is known as heterotrophic growth, where growth and propagation occur under dark conditions with metabolism of external carbon sources. This growth cannot be considered entirely decoupled from photosynthesis, however, because essentially all of these carbon substrates are derived from photosynthesis, including petroleum products which are the result of ancient photosynthesis. A new, carbon fixing electrocatalytic process developed at the University of Delaware has been shown to be able to fix CO2 and CO into acetate rich product streams.
This technology utilizes a copper nanosheet cathode and an IrO2 anode to catalyze the reduction of these single carbon substrates, demonstrating a relatively high efficiency of approximately 54% conversion into acetate. Due to the high acetate content of the product stream, when incorporated into a common algal growth media, Tris-Acetate-Phosphate , a new media can be produced that can possibly harbor algal growth. This process can be powered entirely by electricity and thus, photovoltaic technology can be employed and direct comparisons regarding efficiency given a fixed solar footprint can be made. By combining these two processes, there lies potential for developing an artificial photosynthetic system that can possibly match or exceed the efficiencies of conventional plant or algal growth, offering unforeseen advantages. Without the reliance on light, inconsistencies of sunlight due to climate variations can be remedied; this is a commonly discussed advantage of hydroponic agriculture1 . This project has significant implications for introducing alternative methods of agriculture that can aid in the battle against food shortage without further expanding agricultural lands. The first experiment conducted was testing the growth of the algae on a modified version of TAP media, where the acetate was exchanged with the acetate contained within a simulated, chemically identical effluent, as the effluent produced by the University of Delaware, was not yet accessible. The purpose of this experiment was to observe if media produced utilizing the effluent can harbor algal growth in heterotrophic conditions. The amount of effluent added to supplement acetate was enough to replicate the typical acetate concentration used to grow algae . This also came with the potentially cytotoxic chemicals included with the effluent and was labeled as TP-Effluent media. The positive controls for this experiment were TAP-dark, and TAP-light, where the cultures were grown on TAP media in the absence and presence of light, respectively.
TAP media is typically sterilized using an autoclave, but it was discovered that the effluent contained heat or pressure sensitive chemicals that would drastically increase the pH of solution and develop white, crystalline precipitate following auto-claving. Due to this issue, after adjusting media pH utilizing 5.0M HCl and 5.0M NaOH to approximately 7.23, the media was instead vacuum filtered for both the positive controls and experimental cultures. Inside of a biohood, the corresponding media solutions were placed into 125 mL, pre-autoclave sterilized Erlenmeyer flasks, topped with pink caps for sterile air flow and wrapped in aluminum foil for the dark cultures. The flasks were then inoculated with 21gr- Chlamydomonas reinhardtii strain from an agar-plate preculture, placed under a light source and left to grow for 1 week.Growth parameters were measured by aliquoting 1.0 mL of culture for cell count and OD750 analysis. For cell culture analysis, a Biorad TC20TM automated cell counter was used, where 10 µL of culture was placed into both A/B sides of the slides, measured, and averaged. These parameters were measured at time 0, and at 1 week. The second experiment aimed to determine cytotoxic chemicals in the effluent and TP Effluent media, in order to develop potential treatment methods to improve growth. This was done by omitting chemical constituents from the simulated effluent and examining if growth improved. Because there were four chemical candidates for growth inhibition , each of the experimental effluent solutions omitted one candidate, labeled -KHCO3, – Ethanol, -1-Propanol, and -Propionic Acid, respectively. Preparation of the media was identical to that of the first growth experiment. The inoculation protocol for this experiment was slightly different than the first,large plastic garden pots instead using liquid pre-cultures of 21gr+ Chlamydomonas reinhardtii grown under light for 7-days. The inoculation of the control and experimental cultures was to achieve 5.0✕105 cells/mL cell density. The cell density of the pre-cultures was determined using the Biorad TC20TM automated cell counter, diluted to the target density and inoculated into pre-autoclaved flasks similar to that of the first experiment, but instead with 50 mL of media. All of the samples were grown in triplicate, with the positive controls; TP-effluent, TAP, and the experimental flasks; KHCO3, – Ethanol, -1-Propanol, and -Propionic Acid. The growth parameters used for this experiment differed slightly from the previous experiment, with the cell counting method being the same as before, but now with the inclusion of optical density measurement at 750 nm with a Molecular Devices QuickDropTM Spectrophotometer. This is a common method of quantifying algal growth. This measurement was blanked with a small aliquot of TAP media with no algae. Microscopic analysis was also performed using an Olympus BX51 Fluorescence Microscope. For each of these methods, 1.0 mL of culture was taken from each flask at time 0, and every 48 hours subsequently until 16 days total growth. On day 16, the dry biomass of the cultures by separating the cells from the media, baking overnight, and weighing the cell mass.The third experiment was the Media Optimization Experiment, where effluent and thus acetate and cytotoxic chemical concentrations were altered to investigate the thresholds of algal growth on TP-effluent media. The percentages of acetate used in this experiment were 25%, 50%, 75% and 100% . TAP and TP were included as positive and negative controls. This experiment sought to determine how algal growth can be affected with lower concentrations of effluent, predicted to worsen growth due to less acetate but also improve growth due to lower concentrations of cytotoxic chemicals.
The same effluent recipe from the drop out experiment was used in this experiment. Inoculation and culturing protocols were very similar to that of the “Drop-Out” experiment, with the same growth parameter measurement methods, consisting of cell counts and OD750 every 48 hours for a 16-day period and dry biomass measurements at the very end of the experiment. The same strain of 21gr+ Chlamydomonas reinhardtii was used. For this experiment, the algae were grown in the dark, by wrapping the flasks with aluminum foil. The fourth and final experiment was the growth experiment where the actual effluent derived from the electrocatalytic reduction process was tested. The inoculation protocol was similar to previous experiments, but cell counts for this experiment were done manually with a hemocytometer for higher fidelity. The other growth parameters measured were the OD750 and dry biomass. To optimize growth for this experiment, the culture flasks were placed on a shaker with a controlled temperature of 30°C. Effluents of various compositions were tested in this experiment, as the collaborating laboratory was able to produce various kinds of different compositions, some containing entirely different chemicals. The same strain of 21gr+ Chlamydomonas reinhardtii was grown in the dark using aluminum foil. Although the different effluents contain different acetate concentrations, the effluent was added to the media so that the final medias had the same acetate concentration . The first growth of the algae with effluent proved to be unconvincing, with poor growth even in traditional photosynthetic conditions. There was also noticeable clumping of the cells inside the effluent flasks, suggesting cell stress. This indicated that there was a strong cytotoxic component in the media, and therefore in the simulated effluent as well. In the drop-out experiment, after 16 days of growth, the results strongly suggested that the cytotoxic component in the effluent was potassium bicarbonate, or KHCO3. In Figure 2A, the appearance of the culture grown in media with KHCO3 omitted was similar to the positive control, TAP. This growth appeared to be lush and dark green, while the other flasks had relatively patchy growth. This could be, however, the result of inadequate mixing. The OD750 and cell count assays demonstrated similar findings, with the TAP and -KHCO3, cultures growing considerably better than the other experimental cultures. For cell density, the results even suggested that the -KHCO3 cultures grew better than standard TAP, although the errors for this proved to be very large. Dry biomass collected at the end of the growth period showed that the TAP, -KHCO3 and -propanoic acid cultures grew best. This was unexpected considering the OD and cell count measurements. By analyzing microscopic images, the TAP and -KHCO3 cells were dispersed and not clumped. All KHCO3 containing cultures had both lower cell density and cell clusters. This refilected what was observed in the initial growth experiment. For the media optimization experiment, it was found that for medias containing TPEffluent, 75% TP-Effluent and 50% TP-Effluent, there were not significant differences in cell viability over the course of a 16-week growth period, shown in Figures 3B and 3C. The 25% TPEffluent showed weaker growth, possibly because the cells were too quickly depleted of a carbon source, limiting their growth even when the presence of cytotoxic chemicals was reduced. The dry biomass measurement showed highly unexpected results, with the TP-Effluent having more biomass accumulation than even TAP. One explanation for this result is that the residual chemicals in the effluent were not properly washed away or evaporated during the dry biomass collection process. KHCO3 is a salt that could have been retained in the dry biomass, which could explain the relatively large dry biomass measurement for the TP-Effluent cultures.