Lorem ipsum dolor sit amet, pro menandri efficiendi in, eam ne omnium sapientem definitionem, choro scriptorem cum in. Duo choro placerat ne, ex ullum aliquip disputando per. Mutat laudem sea eu, ad quas labores theophrastus vim. Erant exerci laoreet vel ex, te his mucius consulatu consequat. Mea no option erroribus, nominati scribentur has et, pro at dicat dicit constituam. Affert doctus nam ut, novum homero indoctum vel in, elit regione virtute per te. Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.

Duo choro placerat ne, ex ullum aliquip disputando per. Mutat laudem sea eu, ad quas labores theophrastus vim. Erant exerci laoreet vel ex, te his mucius consulatu consequat. Mea no option erroribus, nominati scribentur has et, pro at dicat dicit constituam. Affert doctus nam ut, novum homero indoctum vel in, elit regione virtute per te. Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.

Week 1: May 1 - May 5

We spent our first week in the lab doing basic inventory and organization of our supplies. We familiarized ourselves with the lab equipment and carried out some of the basic protocols we would be using during the summer. We practiced making LB agar plates, chemically competent e.coli cells, plasmid minipreps and colony PCRs. We also completed the required lab safety and biosafety training. Then, we started researching Alberta’s wastewater treatment process; we conducted preliminary literature review of VFA production and PHB extraction. We met with Dr. Peter Dunfield, a professor of microbiology at the University of Calgary, to evaluate some of the more novel aspects of the project. We decided early on that we would try to avoid PHB extraction using solvents, especially chloroform, in our final process. Consequently, we brainstormed ideas about binding PHB granules to beads after the engineered E.coli autolysed.

Week 2: May 8 - May 12

We continued practicing lab protocols and and focused on taking a step back and evaluating our project within the scope of iGEM. We reviewed previous teams’ work and explored avenues of improvement and innovation. As a result we started moving toward a secretion based system as opposed to a lysis system. Concerns were also raised about the use of pure cultures with sludge; we began to address nutrient removal as an important step before the sludge could be inoculated with the pure culture.The engineering team began to outline the early drafts of the final process from VFA production to PHB characterization.

Week 3: May 15 - May 19

During this week, we discussed the role of our group in the project with the other sub-groups and divided the work into subcategories: extraction and purification of PHB, quantification and characterization of PHB, process scale-up and modelling. Objectives and milestones were then developed for each subcategory. Extraction and purification of PHB is one of the major contributors to high costs of PHB production [1]. We will therefore focus on finding novel extraction and purification methods that will be feasible at large scale and will test proposed methods in the lab. The process development group will also support the synthesis group by quantifying and characterizing the PHB that they produce in the lab. This week, we started looking into potential protocols and the availability of resources for lab-scale quantification and characterization of PHB. When designing a PHB production process, we will also consider scalability and feasibility at large scale. Our group will conduct a cost analysis for large scale PHB production using the proposed process. Finally, the group will also develop a quantitative model to be integrated with the project. Together with a representative from the synthesis group, we brainstormed a list of quantitative models that can potentially help in the project.

Early in the week, we met with Dr. Jason de Koning, an Assistant Professor, Departments of Biochemistry & Molecular Biology, and Medical Genetics at the University of Calgary to discuss modelling that can be integrated with our project. We also met with Dr. Christine Sharp, a postdoctoral fellow from the Energy Bioengineering Group supervised by Dr. Marc Strous. Their group is working on PHB production using alkaline soda lake biomass and design their own bioreactors.

Week 4: May 22 - May 26

At the start of the week, the group met with Dr. Nashaat Nassar, an Assistant Professor in Chemical and Petroleum Engineering to discuss PHB extraction and purification techniques. He recommended using a combination of coagulation and settling. We also met with Dr. Saurabh Sarma, a postdoctoral fellow in Civil Engineering, to discuss VFA production in wastewater treatment plants (WWTP). He provided suggestions on integrating our process with the desired applications. We also discussed the composition of VFAs commonly found in WWTP and parameters that impact VFA composition. Dr. Sarma also suggested reaching out to Daniel Larson, a laboratory technician in Civil Engineering, who might be able to assist us with measuring VFAs and PHB using gas chromatography.

The entire team visited one of Calgary’s WWTP this week, where we learned about the current wastewater treatment process. After the tour, we met with representatives from ACWA (Advancing Canadian Water Assets) Research Facility at the Pine Creek Wastewater Treatment plant, who are working on wastewater treatment research. However, they work with the output from the wastewater treatment plant, while most of our questions focused on the wastewater treatment process itself. They directed us to the City of Calgary chemistry laboratory that performs analysis on intermediate samples from various stages of the process. We have contacted representatives from the City of Calgary.

This week we also worked on researching into potential models that can be implemented with the project focusing on background information, questions that each type of model can answer, general procedure, required resources and pros and cons, which was the first modelling milestone.

Week 5: May 29 - June 2

This week our team focused on determining the feasibility of the 4 proposed applications for the project: production of PHB on Mars using human waste as a feedstock, integrating PHB production with a wastewater treatment plant, integrating PHB production with leachate treatment, and integrating PHB production in developing countries. Our sub-group calculated the approximate amount of PHB that would be expected in each scenario.

We approximated that about 40 - 90 kg of PHB can be produced on Mars per year with a crew of 6 astronauts. A crew of 6 will generate about 6 tonnes of solid organic waste over 2.5 years [2]. The COD content in feces reported in literature was found to be 354 mg COD per g wet [3]. One study looking at PHA production from food waste estimated the yield to be 0.05 g of PHA produced per g of COD applied [4]. Another study reported 0.11 kg of PHA produced per kg of effluent COD in a PHA production process using activated sludge as a feedstock [5]. Another member of the team approximated the production to be 41 kg/year assuming the average COD content of human excretions to be 61.75 g/cap/day [6], the COD to VFA ratio being 0.74 [7] and the VFA to PHB conversion being 0.38 g PHA/g VFA [7]. According to NASA, the cost of shipping supplies to space is 27 000$ per pound on the new SpaceX Dragon spacecraft. This meaning that the costs saved by producing 41kg of PHA in space would be 2 440 000$. The range of PHB that can be produced in space is based on the two PHA yield values found in literature. We contacted a 3D printing company. They use various different methods to create 3D models and one of the techniques they work with is called selective laser sintering, or SLS. SLS 3D printing has been found to be effective at printing with PHB. [21] We contacted Howard at 4G Vision Tech who ran an analysis for us on various 3D models we found online. According to his calculations, we are able to create approximately 50 hydroponic systems and 20 general tools, such as wrenches, hammers, and scissors.

The integration of PHB production in leachate treatment would likely be unfeasible due to low volumes of leachate that are usually produced at landfills. In Calgary, a single landfill generates about 100,000 L of leachate per day. Although COD content in leachate is usually higher than in wastewater, the estimated amount of PHB produced in Calgary was about 8000 kg/year, based on COD content of 1977 mg/L in Calgary leachate [8]. This would result in about $40,000 in profits, assuming a price of $5 per kg of PHB [9]. Another member of the team performed similar calculations assuming the 0.38 gPHB/gVFA production and received 22 250kg PHB/year, thus giving the potential profit of 111 126$/year. The cost of implementing such PHB production process will likely be magnitudes larger. Leachate treatment in China is a more promising alternative. China generates a larger amount of leachate compared to many other countries [10]. Additionally, the COD content in Hong Kong, China ranges from 15,700 to 50,000 mg/L for young landfills [10], which is 8 to 25 times greater than in Calgary. We estimated that about 900,000 - 3,000,000 kg of PHB can be produced per year in Hong Kong depending on COD content and using PHA yield of 0.11 kg of PHA per kg of COD. PHB production from leachate was also considered for Vancouver, which generates 2,225,978 cubic meters of leachate per year [11]. Based on our estimates, about 3,100,000 kg of PHB can be produced per year assuming the COD content of about 13,000 mg/L [12].

For the wastewater treatment plant, it would be possible to produce about 28,100,000 kg of PHB per year, based on our estimates. The Pine Creek Wastewater Treatment Plant in Calgary processes about 1 million cubic meters of waste per day. The COD content and PHA yield were assumed to be 1000 mg/L and 0.11 kg of PHA per kg of COD, respectively, for the calculations.

Developing countries

We envisioned incorporating PHB production into scaled-down wastewater treatment systems in small communities that lacked established treatment methods. Sale of the PHB would provide monetary incentive to construct a wastewater treatment system, which in turn would help reduce disease caused by poor sanitation. Therefore, we needed to calculate the revenue our system could make. PHB production was shown to be feasible with mixed cultures naturally present in activated sludge. Hence, we also had to show that the increase in PHB production as a result of using a pure culture was worth the increased expense incurred through required sterilization. We assumed a community size of 2000 people and 3.113 x 10-3 m3 /day/person for per capita solid waste generation in developing countries. [13] We then found that COD content in municipal waste was higher in developing countries and used 601 mg/L for our calculations. [14] Using the COD content we calculated PHB production from mixed cultures (0.11 g/gCOD) and pure cultures (0.88g/gCOD, which was calculated by assuming the 80% DCW accumulation) [15]. We found that extra revenue generated from more efficient PHB production using pure cultures (price of PHB was taken to be $5/kg [16]), was $2000 which could not justify the cost of sterilization which ranged around $100,000. [16].

While the developing country application was alluring in terms designing novel prototypes integrating systems like decentralised wastewater treatment (DEWATS), the sterilization cost acted as a significant deterrent toward using synthetic biology for incentivising wastewater treatment in developing countries.

Week 6: June 5 - June 9

This week, our team decided to pursue PHB production from human waste on Mars as the main application for our project. Our subgroup then revisited the different aspects that we will be working on and prioritised tasks. We will first focus on researching potential extraction and purification techniques for our process and choosing the top 3 methods for further testing in the lab. At the same time, we will also look at VFA production from feces. The first steps would be to make synthetic poop or obtain feces samples, determine desired conditions that we would like to test for VFA production, determine how VFAs can be separated from feces and test a protocol for quantifying VFAs. For safety reasons, it is preferred to prepare synthetic feces in the lab rather than working with real samples. We found a number of recipes for preparing synthetic feces [17], [18] and purchased required materials.

Week 7: June 12 - June 16

This week, we continued researching purification and extraction methods that can be used for our PHB production process. One proposed method that we will try at lab scale includes the following steps:

1. Centrifuge collected harvest to settle the bacteria and large particles
2. Collect the supernatant from a previous step and pass through a 0.2 µm filter to filter out smaller particles
3. Use an ultracentrifuge to settle the PHB granules in the supernatant obtained from a previous step
4. Wash the PHB granules and dry

This week, we also met with professors in the Chemical and Petroleum Engineering department at the Schulich School of Engineering to discuss extraction and purification techniques. One idea that resulted from these meetings was to grow bacteria in a biofilm with continuous collection of produced PHB. With this set up, a 0.2 µm filter might be enough to filter out the dead bacteria and we won’t need to centrifuge harvest to separate the bacteria. However, growing cells in biofilms might require a large surface area, which is not ideal for the space application where compact systems are desired. Another idea is to continuously produce PHB using a membrane reactor with pores that are small enough to retain the bacteria, but large enough to let the PHB through. With this idea, continuous production of PHB may be achieved with a compact system. We will also evaluate electrocoagulation as a method for extraction and purification of PHB.

Additionally, we planned the first experiments with VFA production from synthetic feces. We plan to detect VFAs using gas chromatography. The first step is to test the gas chromatography method and determine the amount of VFAs present in synthetic feces. To test this, we will take 4 samples of synthetic feces and spike 3 of the sample with known concentrations of VFAs.

The first experiment with Centrifuge collected harvest to settle the bacteria and large particles

1. Collect the supernatant from a previous step and pass through a 0.2 µm filter to filter out smaller particles
2. Use an ultracentrifuge to settle the PHB granules in the supernatant obtained from a previous step
3. Wash the PHB granules and dry

VFA production will aim to answer the following questions:

• Is it possible to make VFAs by inoculating synthetic feces with E. coli?
• How much VFAs can we make per amount of input feces and what types of VFAs will be made?
• How long does it take to reach maximum VFAs composition?
• Does temperature have an impact on VFAs production?
• Does the addition of yeast to synthetic feces make a difference in VFAs production?

The proposed conditions for the first VFAs experiment are summarized in Table 1. We plan to run the fermentation process for 5 days. Samples for VFA analysis will be collected on day 1 and day 5.

Table 1: Proposed experimental conditions for the first experiment for VFA production from synthetic feces

Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.

Lorem ipsum dolor sit amet, pro menandri efficiendi in, eam ne omnium sapientem definitionem, choro scriptorem cum in. Duo choro placerat ne, ex ullum aliquip disputando per. Mutat laudem sea eu, ad quas labores theophrastus vim. Erant exerci laoreet vel ex, te his mucius consulatu consequat. Mea no option erroribus, nominati scribentur has et, pro at dicat dicit constituam. Affert doctus nam ut, novum homero indoctum vel in, elit regione virtute per te. Veri discere in duo, blandit eloquentiam reprehendunt quo ne. Mucius deleniti nam no, has eu justo facilisi torquatos. An usu denique gubergren, eum ei errem mucius. Ex mel erat cotidieque, referrentur definitiones ut sed. Eam maiorum expetenda et, dolore prompta virtute ne vis, cu facete suscipit eos. Discere corrumpit gubergren vis te, est agam nemore id, ad eam eligendi tincidunt. Partiendo facilisis ullamcorper ius ei, mea eu alterum democritum. Pericula referrentur quo ut, quem omnes molestie et est. Ea per debet omittantur. Has viderer patrioque temporibus an, putent posidonium dissentiet duo et. Qui docendi propriae definitionem id, graece primis vis ne. Id cum eruditi apeirian, eu solet semper dictas sea. Ne usu impedit dolorem salutandi, ea duo duis detraxit. Has clita delicatissimi eu. In vitae pertinax tincidunt vim, eam eu partem nominavi concludaturque. Eum graece vocibus ei, enim brute nominavi ex eum, natum simul definitionem mei cu. Patrioque honestatis ea eam, eu quot splendide nam. In vix ignota intellegat. At pri iuvaret appetere, usu probo mediocritatem ei. Nec cu probo volumus albucius, amet prodesset vix at. Ne vim esse eloquentiam, graecis salutatus no quo, fierent probatus recusabo vix te. Splendide posidonium in sea, mel saepe disputando te. Ei solum nemore facete pro. Vis discere nusquam oporteat ad, ei nam eripuit vivendo sensibus, pertinax mandamus elaboraret no per.