As the focus of our project revolves around the new durable platform for enzyme production, investigating possible industrial setups is central to our project. After an initial examination of the current production designs, it became clear that each of our projects would require a separate implementation strategy. While UTEX strains can be cultured using an existing framework, working with cyanophages calls for creative solutions with a careful risk assessment.
We have outlined herein two different industrial scalable strategies for cyanobacteria enzyme production. The first technique referred to as the hunting technique, involves using wild type cyanobacteria, gathered from the seas, in order to cause intracellular enzymatic production. Such an industrial plant would necessitate a location close to sea water. The second technique involves a more traditional factory setting used in industry. Extracellular secretion techniques are widely used for enzymatic production in organisms such as E. coli or yeast. The medium delivery and enzyme extraction do not vary greatly from heterotrophic organism production, however, being photosynthetic, an appropriate light source is necessary for cyanobacteria.
Cyanophages - indirect manipulation
Use of bacteriophages in fermentation technology is a novelty and thus most of our plant design focuses exploration of this technique. Below is an overview of the proposed operations involved that are further discussed in the subsequent sections. Although the yield calculations found in the Model tab suggest that the industrial scalability of utilizing phages for enzymatic production is not economically viable, it nevertheless serves as an interesting thought experiment to imagine a large scale operating facility. Moreover, such a design could be implemented in similar processing facilities or serve as an inspirational blueprint for other projects.
Overview of the production stages:
- Piping seawater to the facility
- Addition of cyanophages
- Infection and target enzyme production
- Separation of the product
- Biocontainment and bioresistivity prevention
- Dewatering and wastestream discharge
The key benefit of our phage technology is the use of seawater as a medium of the fermentation broth.
Addressed challenge: Freshwater scarcity: in recent years the water beds of many countries marked a decreasing trend  and multiple NGOs put this freshwater crisis into the spotlight. Although Belgium is a small country with a relatively big coastline and many rivers, the level of groundwater is one of the lowest in Europe . At the same time, Belgium has one of the highest industrial production volumes per capita in the world .
Solution: In this setting, we find it essential to avoid freshwater use in industrial applications. Seawater is readily available and would remain accessible even in the changing climate. This is specifically relevant for coastal countries with an arid climate.
To locate the most optimal possible locations for a plant using our technology, we first conducted a literature assessment to map where the highest concentrations of cyanobacteria are present. We found average year temperature to be the primal determinant for cyanobacterial population densities. According to Flombaum et al. , the annual mean global abundances of Synechococcus is 7.0 ± 0.3 × 1026 cells . The highest regional concentrations can be found around Asia Pacific.
Figure 1: Global Synechococcus distribution; adapted from the group of Flombaum et al. 
Seawater piping system
Calls to use more energy from renewable sources: After signing the Paris Climate Agreement Belgium pleaded “to reduce CO2 emissions in Belgium by 35% by 2030 and to increase the share of renewable energy to 18.3%.”. Belgium sometimes faces energy shortages in winter months and currently looks into alternatives for expanding its energy grid, particularly as the future of its nuclear reactors is uncertain under the new government. To assure energy security for our plant operation that’s independent of government regulations, such as quota on energy use during peak hours, we propose going off-grid.
Solution: As our plant is necessarily located at the coast to guarantee access to seawater, we can take advantage of energy generated by water movement to power our piping system.
Looking deeper into the problem, we analyzed ways to reduce energy use for water displacement. One promising solution would be harvesting the tidal energy and constructing a system where we install double acting pumps to fetch the water to our production facility. Alternatively we can rely on wave energy which is more unpredictable, however allows for installation of smaller buoyant pumps that take little space and are movable. An extra constraint for this case is the shape of the seafloor. Preferably, the continental shelf would deepen quickly as shallow coastal shelf induces friction which leads to loss of energy of the waves. Taking the US as an example, it would be optimal to use such pumps on the West coast rather than East coast. 
As our theoretical data showed a very low production yield, it is therefore futile to discuss product separation through membranes and dewatering methods as industry requires relatively high product concentrations for such operations. If future improvements, for instance an additional step of culturing cyanobacteria performed in a quasi-steady-state operation, would significantly increase the biomass fraction, shear- or polymer-induced flocculation methods, can be investigated. This would allow to separate the fermentation broth into two fractions. The upper one, containing majority of broth volume can be discarded. The lower, concentrated fraction with the sedimented flocculant would be further processed in the dewatering unit using centrifugation to produce the wet cake. A difficulty that would arise in this process is that after bursting, the bacteria release a range of compounds for which it may be difficult to find a common flocculating agent.
UTEX - direct manipulation
As previously mentioned, the main problem of cyanobacteria is their relatively low growth rate, a challenge which the UTEX strain can address. We wanted to see whether it can offer a viable alternative to current enzymatic processes. The core advantage of our technique is that relying on photosynthetic organisms, the energy is harvested from the Sun. However this introduces irregularity to the system as sunlight intensity is beyond our control, thus greatest enzymatic production would occur during summer months for temperate-climate countries on the Northern hemisphere.
We understand that industry embraces changes more easily if they require less effort and can be implemented in the existing infrastructure. For this reason our investigation around UTEX centers around exploiting its rapid growth.
A major difference with cyanophage plan, is that this time we do culture our cyanobacteria instead of working with a “ready-made” medium. Majority of classical fermentors are made of stainless steel as they were designed for broths with microorganisms such as yeast or E.coli which do not require sunlight to grow. Cyanobacteria production needs a photobioreactor and since we are working with GM organisms, a closed system is mandatory as opposed to many outdoors open tank plants used for algae production. Below is a comparison of two types of closed photobioreactors:
|GLASS TUBES||POLYMER BAGS|
|Note: Some outdoor tubular systems can be also made of polyethylene|
To make our idea industrially viable, large scale testing would be necessary as moving a system from carefully controlled lab conditions to the field always introduces unforeseen variables. In order to be able to transition smoothly from a pilot plant to a permanent facility, we can rely on polymer bags in the initial stages as their lower cost would encourage optimization. The bags should be hung in a rigid structure however, so they resemble cassettes which width can later serve as diameter for polyethylene/glass tubing.
Choice of an appropriate tubing material depends on the heat produced during bacterial growth. As mentioned before, a major problem for our system would be that tubes act as photo receivers, meaning that light not used in photosynthesis is converted into thermal energy. Hence well- insulating materials should be avoided for tubing. If no strain is applied, thermal conductivity of polyethylene stays nearly constant at around 0.45 - 0.52 W m-1 K-1.over a large temperature range. For glass, it depends on the composition (high silicate vs high lead/barium), but generally is around 0.9 -1.2 W m-1 K-1. Should the pilot plant show that overheating annihilates our culture, we can introduce some cooling solutions. We can use solutions that already exist for algae plants, i.e. water bath immersion of the polymer bags or sprinkling the tubes with cool water .
At the moment, UTEX 2973 requires a lab-grade medium for optimal growth creating very high costs. In comparison, E. coli strains used in enzymatic production are supplied with low cost media such as corn starch or maltose. Nonetheless, if we assume that R&D could drive the cost down, the advantage of solar powered light and CO2 negative absorption would create a very appealing production alternative.
Figure 2: A cartoon model of an envisioned industrial plant; the greenhouse allows for optimal sunlight exposure, while the nearby building serves as the filtration center.
To ensure easier biocontainment, the tubing/bags would be set up under a greenhouse structure, creating a closed system. The floor of the facility should be painted white of with a reflective coating. Use of a closed system also enables easier temperature control and in case of using sprinklers for cooling purposes, also a possibility to recycle water through a closed-loop system. Besides certain nutrients needed for the medium, photosynthetic organisms need carbon dioxide supply to obtain carbon for their metabolic processes. To provide this CO2 we could use carbonation towers and to circulate it through the medium using pumps. If the pumps are strong enough, there may be no need for additional stirrers. Overall, the process should be kept at steady state to keep the modified culture alive as engineering a new one would be costly and time-consuming. To make our design even more durable, we can locate our plant close to chemical processing plants that produce carbon dioxide as a waste product, which we can then directly use in our operations . During our experiments, the natural daytime was not used for culturing, hence no insight can be made as to whether UTEX bacteria exhibited a strong circadian rhythm. Should they have a similar one to algae, then the optimal harvest time would be late afternoon .
Membrane technology is readily used within the enzymatic production industry as it offers a low cost, effective filtration mechanism. In reference to the TRIZ 40 Matrix results found under the Entrepreneur section, we consulted literature and specialists about potential membrane use. All membranes require a gradient of pressure, or concentration to cause the separation of desired and undesired material, or as it is referred to within the field, separation between the retentate and permeate . A major hurdle stems from membrane fouling, as membranes can become covered with retentate at the surface obstructing permeability of other substances. Utilizing this technology is an important consideration in the category of future research for this project as enzyme filtration is necessary for industrial production.
Both projects use different secretion mechanisms, the cyanophages lyse of intracellular expressed enzymes whereas UTEX 2973 involves extracellular secretion, causing a need for two different membrane types to be considered. When working with cyanophages, these biomaterials are the smallest units that must be retained for biocontainment reasons; having a diameter of 50nm requires membrane pore size of diameter 50nm or less. These types of membranes fall under the section of ultrafiltration. In contrast, UTEX 2973 excretes enzymes so the cyanobacteria are objects that must be retained. The approximate size of such prokaryotes is around 2um by 0.5 um due to their rod shape, requiring membrane pore diameter sizes in the vicinity of 0.5um falling under microfiltration. Important to point out that the latter technique has a vast edge in terms of economic viability as microfiltration membranes are cheaper. Moreover, lyse bacteria will cause accumulation of humic acid which must be filtered out as well along from the phages of interest, adding another reason for the low industrial feasibility of such system.
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