Team:USP-EEL-Brazil/Application



This section was created as proof of concept project we idealized. Also, it is based on information and concerns we got from our Integrated HP and come collaborations.

Our application is divided on three topics: Scale-up that presents a model of how our project would work on real conditions; Biosafety brings the concern we gained for the safety in our system’s application; Legislation is the result of our determination to see how our project fits into our country’s reality.



Scale Up

As the project try to solve a real problem, we couldn’t just find a solution, we have to make it real. To analyze the possibility of a real scale plant, we collected data about Lorena’s wastewater treatment station (the small city where we live, where we study and where we’re trying to improve life’s quality around), and we did a scale up based on the feed and effluent of this treatment station. At the end of the page, you’ll be able to find the structural data about the wastewater treatment station that was provided to us by SABESP (distribution and water treatment company of São Paulo).

Our wastewater treatment is composed of 2 modules of 2 pounds. They’re added in series with a facultative pond after the aerobic pond. The total hydraulic residence time of the system (considering both ponds) is 13,8 days. The medium flux feed is 150 L/s and the effluent is 150L/s. All of that can be confirmed at the document on the end of the page.

Using some data from our bibliographic references and some models we developed, we could find viable dimensions for the application of the reactor in our city.

According to Hofman & Schlosser (2015), the PH laccase could be used to apply into a system of a 2L bench enzymatic membrane reactor. They used only 100U/L to remove 4mg/L of EDC, while Lloret (2012) used 500U/L or 2000U/L, and is a “low” quantity of enzyme to use. We’ve scaled up the 2L EMR with the 100U/L and compared with the other parameters (used by Lloret). We established a proportionality between initial concentration and initial activity to helps us on the scaling.

The stirring would be done by a vertical blade, which can be ideal for non-viscous and slightly viscous fluids, once the feed composition of the reactor isn’t constant along the year. Our system would be implemented at the end of the regular system, after the pond treatments, having a feed of 150 L/s. Our system is shown in Figure 1.

Figure 1: Application of the laccase system on Lorena’s wastewater treatment station.

For the scale-up we considered to keep the power per unit volume of medium. For cylindrical tanks, the power number is described by equation (1).

Where N is the rotation frequency, Di is the impeller diameter, P is the power and ρ is the density of reaction mixture. We considered that the density of the reaction system is very close to the density of the water, making possible to use ρ=1kg/m3 and µ=1.002Pa/s.

We’ve also used the equation (2), (3), (4) and (5) to establish relations about the bench scale and real scale system. The indication 1 is for the bench reactor and 2 is for the real.

With the conditions of 250rpm, V=2L, and HRT=4h, the regime is laminar. According to the Re=0.2772, it’s possible to find the Np=60, that implies P=35.56W for the bench scale. Using a relation between laccase concentration and initial activity, we find a constant k, as shown in the equation (6), and used on equation (7). As we plan to use the same concentration of laccase (100U/L), the denominator is equal to 1.

The EDCs concentration expected in real scale was used as a sum of all data Auriol (2007) published. When the volume is found, we can apply equation (8) to find the power needed on the reactor, once we’re keeping the Np.

With the expression (9), the diameter D2 can be found. Using the equation (4), the Di,2 can be found too.

And this way, it’s easy to find the height and the agitation of the tank (equations (2) and (5)). We also maintained the HRT of 4h.

With this scale up we found that with a cylindrical reactor with the conditions shown in Table 1.

Table 1: dimensions for our EMR application.

As we found that oxygenation is better than aeration with the models, we would apply that on real conditions. But as we know we live in a small city, our advice is to apply aeration and use a larger concentration of enzyme, as 500U/L or something like that.

Check our information for the Sabesp water treatment here

REFERENCES

Auriol, M., Filali-Meknassi, Y., Tyagi, R. D., & Adams, C. D. (2007). Laccase-catalyzed conversion of natural and synthetic hormones from a municipal wastewater. Water Research, 41(15), 3281–3288. doi:10.1016/j.watres.2007.05.008

Hofmann, U., & Schlosser, D. (2015). Biochemical and physicochemical processes contributing to the removal of endocrine-disrupting chemicals and pharmaceuticals by the aquatic ascomycete Phoma sp. UHH 5-1-03. Applied Microbiology and Biotechnology, 100(5), 2381–2399. doi:10.1007/s00253-015-7113-0

Lloret, L., Eibes, G., Feijoo, G., Moreira, M. T., & Lema, J. M. (2012). Degradation of estrogens by laccase from Myceliophthora thermophila in fed-batch and enzymatic membrane reactors. Journal of Hazardous Materials, 213-214, 175–183. doi:10.1016/j.jhazmat.2012.01.082

Introduction

The focus of our scaled-up application was for a reactor that would be applied on the WWTP. We believe that the use of purified laccases in a reactor is the safest form for applications in sewage treatment station or water treatment station, since the effluent treatment process itself deactivates the enzyme in the chlorination step, preventing any type of contamination of the treated effluent.

Yet, as our enzymes would be obtained from a genetically modified E. coli, we saw as a concern the need for a biosafety measure. So, we searched for killswich mechanisms that could fit our proposal.

Unfortunately, we were unable to test the planned killswitch, so all bacteria produced in the laboratory, after protein production and cell lysis, were treated with sanitary water to ensure safety standards. However, for future applications, we designed a simple mechanism to end our E. coli in large-scale processes.



BBa_R0040

The plasmid pSB1AT3, gives a new resistance to Tetracycline for our E.coli. Therefore, during the enzymatic production process, we add anhydrotetracycline in the medium and the Tet operator will be deactivated by the presence of the antibiotic. That is why we chose the BBa_R0040 promoter.

In the absence of anhydrotetracycline, the promoter is ON. "In contrast to tetracycline, anhydrotetracycline is a particularly useful inducer. It binds Tet R with an ~35-fold higher binding constant and thus allows to operate at very low concentrations. At the same time, its antibiotic activity is ~100-fold lower and concentrations of <50 ng/ml as required for the full induction of P LtetO-1 have no effect on the growth of E. coli."

With our promoter ON, begins the production of a lysozyme that acts in two steps for the activation of ColicinE7 that will generate cell death.

BBa_K117000 and Colicin E7

Colicin E7 is a type of Colicin, a bacteriocin made by E. coli which acts against other nearby E. coli to kill them with its DNase Activity; it digests the cell's genome in specific locations, ultimately leading to the death of the cell. After synthesis inside the E. coli cell, the colicin binds its Colicin Immunity Protein, Im7, to its nuclease domain, to prevent the host cell from being killed by its activity.


• First step: Release of ColE7 from ColE7—Immunity complex This function causes the detachment of ColE7 from Immunity protein, hence switches on the endonuclease activity of ColE7

• Second stage: Partial lysis of host cell membrane This function induces the lysis of host cell, exposing the interior cell contents and hence allows free diffusion of ColE7 proteins towards pathogenic bacteria strains.

Introduction

Since the LACQUASE project has its scope focused on the improvement of water quality in Brazil and countries with similar water and sanitary structures, a study was carried out on the current legislation regarding the water treatment process. We aim to adapt our project to meet the quality standards, resolutions and environmental guidelines imposed.

The company Sabespis a Brazilian company that holds the concession of public basic sanitation services in the State of São Paulo, being responsible for the treatment of water in the city of Lorena. In this sense, the company informed us about the criteria and emission parameters of the treated effluents and we had access to the resolutions CONAMA No.357 and Portaria de consolidação No.5 anexo 20.

Analyzing the resolutions

CONAMA is the National Council for the Environment, and its Resolution No.357 dictates water quality rules and specifies the maximum composition of certain organic and inorganic compounds that may be present in each type of water. One of main articles from the resolution is the follows:

Art. 14. Class 1 fresh water shall observe the following conditions and standards:
    I - water quality conditions:
    1. non-verification of chronic toxic effects to organisms, according to the criteria established by the competent environmental body or, in its absence, by a renowned national or international institutions, by a standardized ecotoxicological test or other recognized methodology.
    2. floating materials, including non-natural foams: virtually absent;
    3. oils and greases: virtually absent;
    4. substances that communicate taste or odor: virtually absent;
    5. dyes from anthropic sources: virtually absent;
    6. objectionable solid waste: virtually absent;
    7. thermotolerant coliforms: for the use of primary contact recreation should be obeyed the standards set in the CONAMA Resolution No. 274 of 2000. For other uses, a limit of 200 thermotolerant coliforms per 100 milliliters should not be exceeded 80% or more, of at least 6 samples, collected during a period of one year, bimonthly. The E. Coli can be determined in place of the thermotolerant coliform parameter of according to limits established by the competent environmental agency;
    8. BOD 5 days at 20 ° C to 3 mg / L of oxygen;
    9. OD, in any sample, of not less than 6 mg / L O2;
    10. turbidity up to 40 nephelometric turbidity units (UNT);
    11. true color: natural color level of the body of water in mg Pt / L; and pH: 6.0 to 9.0.

Looking at article 14, it can be seen that the LACQUASE project meets and improves several of the proposed requirements. Degrading endocrine disruptors, that are toxic to organisms, degrade compounds that give estrogenicity in dyes from anthropogenic sources without altering the BOD, color, and the smell of water are possible conditions using the enzyme laccase in the treatment of effluents.

Observing article 4, topic (a) and (b), it says that effluents treatment has the function of:
  1. preserve the natural balance of aquatic communities;
  2. preserve aquatic environments in protected areas of integral protection.

It is noted that these topics also meet the LACQUASE project, since we aim to reduce the levels of estrogens in the waters, wich affect directly on aquatic life.

In addition, a table is also provided wich shows the maximum amount of some organic and inorganic components that may be in the water for consumption.

Throuth the table, we can observe that there are still large amount of compounds with estrogenicity that can be degraded to a version less harmful to human being. In addition, the worst part is that the legislation doesn't mention anything about estrogens or endocrine disruptors.

In terms of the population’s health, Sabesp follows the federal Portaria de consolidação No.5 anexo 20. The rule deals with the importance of water for the health of people and shows the competences and obligation of the Brazilian state together with the companies in the treatment and distribution services.

In this sense, we highlight article 22:

Art. 22.- The analytical methodologies for determining the parameters foreseen in this Annex must comply with the latest national or international standards, such as:
  1. Standard Methods for the Examination of Water and Wastewater, from American Public authorship, Health Association (APHA), American Water Works Association (AWWA) and Water Environment Federation (WEF);
  2. United States Environmental Protection Agency (USEPA);
  3. Standards published by International Standardization Organization (ISO);
  4. Methodologies recommended by the World Health Organization (WHO).

Knowing that Sabesp meets the performance and safety of international standards for these applications, we can conclude and ensure the viability of LACQUASE as a way to improve the water treatment because we studied Sabesp norms, and it clearly will fit with our parameters.

Conclusion

Although there is a false idea that water resources are infinite, less than 3% of the world's water is potable, of which more than 99% is frozen in the polar regions or in rivers and subterranean lakes, which makes it difficult to use for the man.

Analyzing the main article of the current water constitutions in Brazil, it is noted that the Brazilian State and water companies play a fundamental role in guaranteeing water quality for the entire population. The existence of laws and regulatory standards such as these are intended to define the criteria and procedures that water must have from its origin to the final consumer.

In this sense, the study of new and more efficient treatment techniques is extremely important to keep water clean, since civilization is in constant technological change, accompanied by the emergence of new pollutants and compounds that can be harmful to human health.

All information conteined here can be accessed both from website of Ministry of Health and in Ministry of Environment. In addition, the translations made are for the purposes of the project, that is why, they do not have official dots, just didactic.


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