Our low environmental impact assessment for plant-based manufacturing should compare favorably with fermentation based approaches to producing Griffithsin . In the latter, the complexities of purification suggest less efficient utilization of materials and higher disposal volumes, although a side-by-side environmental analysis between the two platforms was not conducted in this study.Upstream, Griffithsin expression rates were based on empirical findings using TMV whole virion as the expression vector, which can achieve typically 0.5–1.0 g Griffithsin/kg plant biomass . An average pilot-scale expression rate of0.52 g/kg was used in our model . Although this expression level is quite good for TMV, higher Griffithsin expression levels can be achieved with different technology. For example, Nomad Bioscience GmbH has achieved Griffithsin expression in N. benthamiana exceeding 2.5 g Griffithsin/kg FW biomass using NomadicTM agrobacterial vectors applied to plants either through vacuum infiltration or agrospray , albeit these results were obtained in small-scale studies. The utilization of such an induction process instead of TMV virions could further improve process economics. For example, even with the same recovery efficiency of 70% assumed in the current model,tomato grow bags the output of Griffithsin at the higher expression level would be 1.75 g API/kg plant material, instead of the current 0.37 g/kg; this represents more than 4.7- times the modeled output of protein per kg biomass.
Under such conditions, the costliest parts of the current process, namely biomass production and upstream procedures, would be lowered by the reduced biomass needs to produce the required 20 kg/year of API. Although a full analysis of the cost of agrobacterial inoculation for Griffithsin production needs to be conducted, we know from similar analyses that economics can be favorably impacted by higher expression efficiencies. We can therefore envision that by using a more efficient induction process the per-dose production cost could be less than the current $0.32. Still other gene expression methods can be considered, including using transgenic plants expressing Griffithsin either in constitutive or inducible systems , which could also lead to higher API accumulation in host plant biomass and potentially lower COGS . Increasing expression yield upstream might shift costs to downstream operations to handle process streams with higher concentrations of API. Definition of the comparative cost benefits of these improvements relative to the current process modeled awaits a subsequent evaluation. From a process standpoint, improvements in the efficiency of lighting technologies and/or incorporating solar panels would reduce upstream utilities costs, one of the major contributors to the upstream operating costs. Improving hydroponic nutrient utilization through recycling and minimizing runoff in the simulation model will reduce raw material costs as well as aqueous waste disposal costs, thereby reducing the COGS. In the downstream portion of the process consumables play a major role, particularly dead-end filters and plate-and frame filters; if these could be replaced with tangential flow filtration systems that utilize reusable, cleanable ceramic filters, downstream operating costs could be further reduced. At the time of this writing, such systems were being considered and their impact on Griffithsin COGS will be the subject of a future analysis.
Fisheries and aquaculture are a growing industry, and seafood consumption has been increased from an average of 9.9 kg per capita in the 1960s, to 20.3 kg per capita in 2017. Pathogenic Vibrio spp., specifically, Vibrio parahaemolyticus, are the leading causes of seafood-associated disease in U.S., and 45% of the seafood-borne outbreaks are related to molluscan shellfish. Vibrio spp. are natural inhabitants of estuaries and coastal marine environments. They can be found in water, sediments, and all flora and fauna in coastal environments, including freshly harvested seafood. Another aquatic important microorganism is Aeromonas hydrophila which can cause disease in both fish and humans, affecting seafood safety, quality, and causing severe losses for production and marketing. The importance of controlling these pathogenic bacteria is highlighted by the fact that the aquaponics industry isgrowing globally, and the number of aquaponics producers in the U.S. has continued to grow. Since most fresh produce is consumed raw, the potential for cross-contamination and transfer of pathogenic bacteria from aquaculture water to edible parts of the plants represents a serious risk to public health. Controlling zoonotic fish diseases and food borne pathogens in recirculating aquaculture systems and aquaponics using antibiotics, chemical sanitizers, and pesticides, is challenging due to the sensitivity of the microbial community in bio-filters which oxidize ammonia to nontoxic nitrate, chemical residue concerns in both fish and plants, and regulatory stand points. These challenges motivated researchers to develop novel chemical-free, or bio-based antimicrobial approaches. For example, the application of light-based interventions has emerged recently as an alternative approach to inactivate bacteria.
The photo sensitizer curcumin, in combination with light, has been used for inactivation of various pathogenic and spoilage microorganisms. Exposure of curcumin to light radiation results in photo oxidative generation of reactive oxygen species , which have strong antimicrobial properties. Recent reports have documented curcumin-mediated photo oxidative DNA damage as a mechanism for bacterial inactivation. Light emitting diodes and UV-A are two main safe sources of lights for photo dynamic inactivation which have been used to improve sanitation of food products. Nanobubble technology represents another promising antimicrobial approach that has been recently described. Cavitative collapse of nanobubbles generates reactive oxygen species, as well as a physical insult to microbial cell structures. Due to nanobubbles’ unique properties, this technology has been applied in various areas of advanced science and technology including engineering, medical, agricultural, and food sectors; for cleaning surfaces; dental hygiene; wound cleaning; removing bacteria from fresh produce ; inactivation of norovirus; and removing microbial biofilms. Nanobubbles can exist in both bulk solution and at liquid–solid interfaces, and due to their unique physical properties , nanobubbles can remain stable for up to 24 h, resulting in a supersaturated bubble phase. However, only a few studies have investigated the antimicrobial properties of nanobubbles alone or in combination with chemicals and other nonthermal processes such as ultrasound. Thus, to address the potential challenges in water sanitation in Recirculating Aquaculture Systems and aquaponics, we propose the use of two technologies including curcumin-mediated photo sensitization and nanobubbles with ultrasound to inactivate V. parahaemolyticus and A. hydrophila. The efficacy of light -activated curcumin at different concentrations and exposure time and temperature against these bacteria was evaluated. Antimicrobial properties of synergistic approaches of nanobubbles–ultrasound were also determined at different exposure times at room temperature. This study illustrates the potential of light activated food grade antimicrobial materials, such as curcumin,grow bags garden and a novel chemical-free approach combining nanobubbles and ultrasound for water sanitation in intensive RAS and aquaponics systems to reduce reliance on chemical-based approaches.The efficacy of curcumin activated by UV-A and LED lights was studied against V. parahaemolyticus and A. hydrophila. Samples with curcumin were prepared by adding 5 mL fresh V. parahaemolyticus and A. hydrophila cell suspensions to 5 mL curcumin solution . The final concentration of the cells was 106 cfu/mL. Samples were placed into sterile 6-well clear polystyrene microplates and were treated by UV-A for 5 and 15 min, and LED for 15 and 30 min at 4 and 22 ◦C. After exposure, V. parahaemolyticus and A. hydrophila were cultured on TCBS and TSA , and incubated at 35 and 37 ◦C for 48 h, respectively. Bacteria with curcumin without exposure to lights, and bacteria without curcumin exposed to lights were used as control groups. All the experiments were repeated at least two times in triplicate . The pH of the water samples was adjusted to 5.6 using 1 N citric acid. RAS-aquaponics water sanitation was simulated using water from a seventy liter pilot-scale RAS-aquaponics system . Before conducting the experiment, to ensure that there were no Vibrio sp. and Aeromonas in the RAS-aquaponics water, 100 mL RAS water was filtered using 0.45 µm, and a filter was placed on TCBS and selective Aeromonas medium , and no bacterial colonies were observed. The pH of the water samples was adjusted to 5.6 using 1 N citric acid.
Then, samples were prepared as mentioned previously, by mixing 5 mL of the pH-adjusted aquaponics water with bacterial cell suspensions to obtain 106 cfu/mL and 10 mg/L curcumin concentrations, which were treated with light sources for 5, 10, and 20 min. The synergistic antimicrobial activity of nanobubbles and ultrasound was studied against V. parahaemolyticus and A. hydrophila. A nanobubble solution was provided using deionized water and pure oxygen gas using a Moleaer 25 L nanobubble generator . Our preliminary experiments showed that nanobubbles produced by pure oxygen showed stronger anti-biofilm and antimicrobial properties as compared to nanobubbles generated by pure carbon dioxide or air. A total of 1 mL of the previously washed bacterial cell suspensions was added to 9 mL deionized water containing nanobubbles. The solutions were exposed to ultrasound for 5, 10, and 15 min. Bacteria in PBS with ultrasound and bacteria in nanobubbles without ultrasound were used as control. Bacteria were also exposed to LED light for 15 and 30 min with curcumin . The results indicated that increasing the curcumin concentration from 1 to 10 mg/L resulted in increasing the efficacy of the combination approach to inactivate bacteria. Curcumin concentration demonstrated a strong impact on the reduction in V. parahaemolyticus and A. hydrophila regardless of the light source. Overall, the results indicated that V. parahaemolyticus was less resistant to light-activated curcumin compared to A. hydrophila. A higher reduction was also observed for V. harveyi compared to A. salmonicida, and V. parahaemolyticus exhibited a faster rate of inactivation compared to Staphylococcus aureus and Lactobacillus plantarum in the presence of 405 as well as 470 nm LED illumination. Gram-negative bacteria are more resistant to light-activated curcumin, compared to Gram-positive bacteria, which is mainly because of the Gram-positive bacteria outer wall structure permeability for curcumin. The outer-wall structure of Gram-positive bacteria contains up to 100 peptidoglycan layers, which display a relatively high degree of porosity, which is permeable to molecules such as curcumin. However, on the contrary, a higher sensitivity of Gram-negative bacteria compared to Gram-positive bacteria has also been reported. Furthermore, our results indicated that UV-A 400 nm caused a higher bacterial reduction compared to LED 470 nm. After 5 min of light treatment, a greater than 5 log cfu/mL of V. parahaemolyticus and a greater than 4 log cfu/mL reduction in A. hydrophila were achieved by UV-A light. However, LED 470 nm only reduced 3 and 3.5 log cfu/mL of V. parahaemolyticus and A. hydrophila, respectively. Similar results have been previously observed by other researchers, where at an equal radiant energy dosage of 1100 J/cm2 , the 395 nm LED treatment was more effective with a 2.48 log cfu/g reduction, than the 455 nm with a 1.6 log cfu/g reduction in Salmonella in wheat flour. Kumar et al. also reported low antimicrobial properties of 460 nm compared to 405 nm, regardless of bacterial species and illumination temperature. This can be explained by the fact that UV-A can generate ROS and directly cause microbial cell damage compared to visible light, and UV-A light may excite curcumin molecules compared to LED 470 nm. UV-A has been used to activate curcumin for treating E. coli and L. innocua in water and fresh produce. Moreover, mainly LED or visible light were used in combination with photosensitizers for inactivating bacteria including Vibrio sp. and Aeromonas sp.. The results of these experiments illustrate the potential of using the photo dynamic inactivation approach for food production systems for which using antibiotics, therapeutics, and pesticides is restricted such as intensive indoor aquaculture systems, aquaponics, and the oyster industry.Similar to the room temperature experiment, curcumin and UV-A light together caused a more than 5 log cfu/mL reduction in V. parahaemolyticus and a more than 4 log cfu/mL reduction in A. hydrophila at 4 ◦C . Compared to UV-A treatment, LED 470 nm only caused a 4.5 and 2.5 log cfu/mL reduction in V. parahaemolyticus and A. hydrophila, respectively at 4 ◦C . Temperature control is a critical food safety and quality step during seafood harvesting and processing. It has been reported that antimicrobial properties of UV-A light-activated bio-based compounds were inhibited at refrigerated temperatures against E. coli O157:H7 and L. innocua, which is due to the lower bacterial metabolism rates at a lower temperature.