Original Design

The purpose of our iGEM project is to modify the secondary sewage treatment process in a way that eliminates the inefficiencies created by the dynamic between aerobic nitrifying bacteria and heterotrophic denitrifying bacteria. Sewage Treatment systems employ processes designed around bacteria that occupy specific niches to treat human wastewater. The Biological Nutrient Removal stage in wastewater treatment uses bacteria capable of nitrification and denitrification to remove dissolved nitrogen from secondary effluent sewage. The process of denitrification has been found in both aerobic and anaerobic organisms, and major steps of the denitrification pathway have been elucidated in multiple strains (Potter; Brons; Costa; Gavira; Hasegawa; Moreno-Vivián) . Denitrification is the progressive anaerobic reduction of nitrates to nitrites to nitric oxide, nitrous oxide, and eventually nitrogen gas (Peng et al 2006). Nitrification involves the oxidation of ammonia into hydroxylamine and hydroxylamine into nitrites (Arp et al 2002). Our primary goal was to bring the capabilities of nitrification and denitrification into a single cell.

Our search for organisms that possess the ability to nitrify and denitrify aerobically came up with three species of interest: Paracoccus denitrificans, Pseudomonas aeruginosa, and Nitrosomonas europaea. Between these organisms are the genes for comprehensive denitrification from ammonia and nitrates to nitrogen gas. Pseudomonas aeruginosa is a well characterized facultative anaerobe that can reduce nitrates and nitrites during anaerobic respiration. There are BioBricks made using genes from P. aeruginosa and it has been used in multiple iGEM projects (SCU_China; DTU-Denmark; BYU_Provo; NYMU-Taipei). The main drawback to P. aeruginosa is that it cannot reduce nitrates and nitrites under aerobic conditions. Some strains are also known human pathogens making it a potential Risk Group 2 organism (Palmer; U.S. Dept. of Energy). Paracoccus denitrificans is a denitrifying chemolithoautotroph (Harms; U.S. Dept. of Energy). It can survive and multiply in both aerobic and anaerobic conditions and is shown to be capable of partial denitrification in aerobic conditions (Baumann; Takaya). Most common molecular biological techniques work on Pc. denitrificans and most broad-range vectors and promoters used in E. coli also work in Pc. denitrificans and it has even been shown to possess minor nitrification capabilities (Crossman et al 1997). Nitrosomonas europaea is an obligate chemolithoautotroph with a strictly aerobic metabolism. It feeds off the oxidation of ammonia to nitrites in a process called nitrification (U.S. Dept. of Energy). It is often found in aerated sewage where there is a high concentration of dissolved nitrogen in the form of ammonia. The genes responsible for nitrification in N. europaea are mostly characterized and available as a BioBrick in the iGEM Parts Registry (DTU-Denmark). N. europaea is an efficient nitrifier, but it cannot survive under anoxic conditions. This presents a challenge for sewage treatment plants when heterotrophic denitrifiers consume oxygen, necessitating continued mixing and aeration of the sewage during secondary treatment to maintain the N. europaea component.

We have chosen Pc. denitrificans as a prospective chassis because it is a well characterized organism and is amenable to genetic modification. Most importantly, it is an efficient denitrifying bacteria. Sewage treatment plants currently use a multi-strain, multi-step system that employs a mixture of bacteria called sludge, which is known to contain N. europaea, Pc. denitrificans, and P. aeruginosa, to remove dissolved nitrates, nitrites, and ammonia. In current systems, the presence of multiple strains in a single reaction adds the risk that one strain outcompetes others and the reactor breaking down or becoming less efficient (Castillo; Heufelder). The sludge must be moved between two separate chambers in the Modified Bardenpho Process (Heufelder 2017), one being anoxic and the other oxygenated. Alternatively, in Sequential Batch Processing a single chamber is intermittently aerated to sustain both the aerobic and anaerobic microbes present in sludge through fluctuations in the dissolved oxygen content.

Early research suggested that Pc. denitrificans was effective in some sewage treatment designs as a co-culture with N. europaea (Kokufuta; Uemoto). In this design, small amounts of ethanol must be added daily to act as an electron donor so that nitrification reaction can be sustained. Instead, we propose a single-organism sewage treatment system achieved by cloning the entire amo operon from N. europaea to express the nitrification circuit in Pc. denitrificans when under aerobic conditions (Fig. 2, 3). Our initial intent was to tie expression of nitrification genes to dissolved oxygen levels in the sewage. By design, our device would need to increase the expression of nitrification genes in response to increasing oxygen concentrations.

Having a single organism that is capable of both denitrification and nitrification will simplify the maintenance of a bioreactor and increase the efficiency of the wastewater treatment process. Pc. denitrificans is not restricted by the oxygen supply, so it is a superior competitor for space and resources under hypoxic or anoxic conditions when compared with N. europaea, which can only inhabit certain zones where the dissolved oxygen content is high enough. In the previous system, nitrates had to leave N. europaea and be absorbed by Pc. denitrificans before being converted into nitrogen gas (Kokufuta; Uemoto). By confining the nitrites to a single cellular compartment (Fig. 2, 3), they can build up and encounter denitrification enzymes sooner, making the process faster. The oxidative process of nitrification produces reduced electron acceptors sometimes in the form of quinone that can then be used as donors in the reductive denitrification steps. This will negate the need for regular infusions of electron donors. Figure 2: Device Implementation During the Aerobic Cycle: Atmospheric oxygen is aerated into the sequential batch reactor as sewage is pumped in from secondary processing. Under aerobic conditions, the chassis will nitrify ammonia. Denitrification nitrates and nitrites at partial capacity, producing more nitrous oxide than nitrogen. During the aerobic cycle, the sludge is also mixed.

Figure 3: Device Implementation During the Anaerobic Cycle: Mixing of the sludge is ceased along with aeration and secondary sewage influx to minimize oxygen influx. Pc. denitrificans consumes the remaining oxygen. Under anaerobic conditions it performs comprehensive denitrification to nitrogen gas. The effluent tertiary sewage is then released.

Early Characterization

Early in the project timeline, we plan to characterize the growth properties of Pc. denitrificans by creating a standard growth curve in both LB media and recommended ATCC media for Pc. denitrificans. The goal is to construct a growth curve of the relatively uncharacterized chassis for use in future protocols that require Pc. denitrificans to be at a certain stage in its growth. The use of both LB and paracoccal media is for two main goals: first, LB is needed so that Pc. denitrificans and E. coli be directly compared. Second, the ATCC media is needed so that Pc. denitrificans growth can be characterized under ideal conditions. After these are established, work towards characterizing part activity within the chassis will begin. Growth curves will also be constructed for N. europaea so that comparisons can be made between the final device and the original nitrifying strain it borrows from.

Paracoccus Transformation

Our search for relevant methods for paracoccus transformation yielded few results. Most literature involves the transformation of Pc. denitrificans by conjugation using a donor E. coli strain. Due to time constraints and limits in technical expertise, we had to find an alternative method. This method came in the form of an electroporation method developed by Holo et al for general use and more specifically in lactococcal strains. Work by Barak et al picked up on this research and reported successful transformation of Pc. denitrificans using a modified version of the original protocol. We plan to attempt to replicate the results reported by Barak et al and Holo et al to transform Pc. denitrificans with our biobricks of interest.

Comparative Expression

Once we have successfully transformed Pc. denitrificans, we will move on to transform in with reporter constructs designed to help characterize the behavior of certain promoter elements in Pc. denitrificans. Our initial plan is to tie a standard GFP coding sequence to a termination sequence and ribosome binding site. Next, we plan to create composite biobricks by using restriction digests on the prefix of this construct. Digested promoter sequences for VhB, Mn-SOD, and a T7 constitutive promoter will all be tied to the same RBS, CDS, and termination sequence, thus direct comparisons between promoter activity can be made. We plan to measure promoter activity as a function of fluorescence emitted by transformed cells. The measurements will be made using a fluorometer or comparable equipment. We plan to measure fluorescence of each transformation against a media background. Samples will then be compared to control untransformed bacteria. We plan to use a qPCR detection system to measure fluorescence of transformed cells using the protocol and guidelines set by Utermark and Karlovsky (2006).

Circuit Design

The finished device will carry the coding sequences for amoABC, HAO, Cc554, and Ccm552 from N. europaea (Fig. 5). All ammonia oxidation genes from N. europaea are iGEM BioBricks and make up the composite part BBa_K1067005. The amoABC gene cluster encodes the proteins for ammonia monooxygenase (AMO), a heterotrimeric enzyme that catalyzes the oxidation of ammonia into a hydroxylamine intermediate (DTU-Denmark; Arp et al 2002). HAO encodes hydroxylamine oxidoreductase (HAO), which oxidizes hydroxylamine to nitrite. The genes for cytochromes A and X (as annotated in BBa_K1067005) encode for Cc554 and Ccm552, two c-type cytochromes. Cc554 accepts an electron from HAO and donates it to Ccm552, which then donates the electron to quinone or other more terminal electron acceptors. Together, these genes catalyze the conversion of ammonia into nitrites. The denitrification circuit will be placed behind a strong RBS and an Mn-SOD oxygen-inducible promoter (BBa_K258005). Figure 5: Ammonia Oxidation Circuit: Our circuit for ammonia oxidation is mostly derived from BBa_K1067005. RBS sequences and an oxygen-sensitive VHb promoter have been added upstream of the coding sequences (CDS). An additional promoter has been added at the midpoint to minimize the risk of premature RNA polymerase drop-off.

Device Testing

We planned to quantify the activity of our device both in E. coli and in Pc. denitrificans so that we could justify the decision to use a paracoccal strain instead of a standard chassis organism. We would also quantify the responses of untransformed E. coli, Pc. denitrificans, and N. europaea after being treated with an “ammonia spike”. Our assay would be conducted in a erlenmeyer flask on top of a hotplate set to 30 C and a magnetic stirrer. We plan to track the levels of nitrates, ammonia, dissolved oxygen, and the OD 600 of each culture over time after introduction of excess ammonia. Ideally, data from the growth curve assays will inform the project and allow us to grow cultures to mid-exponential phase for maximum responsiveness to excess ammonia. The responses of each strain to an ammonia strike would then be standardized against the corresponding reported OD 600 so that the ability of a unit of biomass to oxidize ammonia and reduce nitrates can be estimated and compared to the results of other strains.