Team:IISER-Tirupati India/Implementation

Implementation

The purpose of our engineered bacteria, Coli Kaze, is to eliminate antibiotics from Antibiotic polluted waste outlets efficiently. Thus, bacteria are engineered to precisely degrade the designated targets, antibiotics from animal farm waste. The by-product during the process can be used in agriculture process for clean application in organic food production farming and even enough to provide passive income to farmers in deals of organic solid and liquid fertilizers. This would also save them the space for landfill and improve farm hygiene and sanitation.

Figure 1: On overview of the proposed implementation of Coli Kaze

For ease of this purpose, Coli Kaze would be made available in a concentrated blend of E. coli spores formulated to treat sulfonamide polluted waste. Coli Kaze used as pure spores, dehydrated cultures, provide the greatest flexibility for use and application. These bacteria can grow over a wide temperature range of 5℃-55℃ and pH range of 6-10. The other method for same could be a pre-inoculated mix of viable Coli Kaze bacteria, which would be no different from powder blend of Colikaze in principle. It contains genetically modified single strains of BL21 DE3, which constitutively produces sulfonamide degrading enzyme, sulfonamide monooxygenase, and has the arabinose inducible kill switch to lyse open the cell to release the degrading enzymes.

Treatment of excreta will proceed in various phase for as follows:

Phase I (Pre-Treatment):

Y amount of innoculant (powdered blend/inoculation) is cultured in a sterilized culture tank of volume v, v’ (v’ = v/10) volume of LB broth to which culture is to be resuspended and is grown til the culture reaches OD₆₀₀ 0.5 in time t (can be estimated from the graph) at 37℃ and 200 RPM. Basic processing for animal excreta is initiated alongside to mark the end of the phase. X kg of excreta is passed through crude separation for macro solid waste removal, next it is diluted to achieve a relative density approximately one and made up to volume V litres (V = 100 x v’). This step ensures the maximum degradation of sulfonamide in excreta by substantially improving the mixability of Coli Kaze culture and slurry. The water for dilution can be from any freshwater source available near this facility.

Figure 2: Adapted from [1].

Phase II (Kill switch induction):

1.179 X 10^-3 M of L-arabinose is added to the culture tank, speeding at RPM 200. We wait for an optimised time of approximately one-two min. By this time, most cells in culture media lyse open releasing antibiotic degrading enzymes, and degraded fragments of bacterial DNA are released in media.


Figure 3: Amount of L-arabinose added to media Vs Volume of Growth Medium

Phase III (Incubation):

Cell lysate of volume v’, of OD₆₀₀=0.5, relative density approximately one, an estimated yield of enzyme 32.11 mg, temperature & pH of 37℃ and 7- 7.5 respectively, is added to the slurry of volume V, relative density nearly one and similar temperature and pH and are mixed in incubation tank/digester at twice RPM of culture tank, i.e. 400 RPM with controlled temperature and pH regulators, along with the suitable supply of O₂ through the inlet and CO₂ is removed via the outlet.

Detailed implementation plan of culture tank/bioreactor:

Bioreaction of growing cells would be performed in the BioProcess Container (BPC) in culture Tank. BPC is a single-use flexible container system. Each BPC has its impeller, which will be connected to the motor box to churn the culture while the pH probe and Temperature Probe will also be connected to BPC and monitored through the Control Panel. In the BPC. Kill switch would be induced by adding arabinose through various inlets and incubated. The cell lysate thus obtained after the induction of the kill switch, would be transferred to the digester tank using a vacuum pump draining out the whole BPC, where it would be mixed with a slurry of excreta—thus initiating the process of antibiotic degradation. After incubation, phase 3 is concluded when a suitable chemical test is performed for the processed slurry to check for the presence of hazardous antibiotic concentration. If the test result is satisfactory the processes slurry is ready to be processed further in phase IV, if not then the incubation is extended to a finite time until the range of antibiotics is not hazardous to be released.

Figure 4: Overview of the design of the BioProcess Container

This system would not only enable us to collect inoculum for the next round from BPC but, it would also add an additional level of security by preventing contamination in cell culture of our engineered bacteria. We can use a cocktail of engineered bacteria that degrades a wide variety of antibiotics. Also, as the kill switch is user modulated, it can be induced on the will of the user by adding arabinose to bacterial culture.

Figure 5: The complete Tank design for the working of Coli Kaze (Cell free system).

Phase IV (Manure Production):

Manures which are potentially valuable as fertilizers or soil conditioners are resources that need to be managed adequately. We obtain manure as a by-product of the antibiotic remediation process. After incubation, the slurry is left to be settled for 15 to 20 min resulting in the separation of supernatant, diluted liquid fraction, and precipitate, the solid fibre fraction. The diluted liquid fraction can be separated and stored. If needed, it can be concentrated further by heat separation of solid from a semi-liquid stream in two different fractions, one solid and other liquid by drum filtration.


Figure 6: Representation of drum filtration




Figure 7: Time of incubation (hours) v/s processed amount of excreta (Kg)


Figure 8: Estimate time of degradation of sulfonamides (minutes) v/s amount of enzyme (μM) v/s amount of excreta (Kg)

Particulate matter is concentrated in the solid fraction while the concentration of soluble compounds is almost uniformly distributed in the two fractions. While phosphorus and organic nitrogen are concentrated in the solid fraction, soluble nitrogen that is the ammonia or nitrates and potassium mass flow rates are higher for the liquid fraction. Solid fractions can be more easily exported to areas with low livestock density whereas liquid fraction can be further processed in situ.

   

	% DM	Total N (kg/ton)	NH4-N (kg/ton)	P (kg/ton)	K (kg/ton)
Untreated slurry	4.9	5.0	3.5	1.0	3.5
Fibre fraction	10.4	5.3	3.5	1.4	3.8
Liquid fraction (reject)	2.9	4.7	3.4	0.8	3.3

Table 1: Data From Drum Separator Applied To Swine Slurry Treatment

Additives: Acidification

The main objective of acidification of liquid manure is to lower the level of pH in the manure and thereby increase the concentration on ammonium at the expense of ammonia- which will result in the reduced free ammonia emission. We target to make pH as low as 5.5 - 6.0.

The solid phase manure at 60% water share is composted and piled in shade for a few weeks to achieve the moisture content of below 40% to limit the microbial activity. At this point, the final product is ready to be packed and used.


Table 2 : Tentative implementation operation schedule

(For operational example: v’=1L, V= 100L, X=15Kg)

Pros of this system:

Extra Bio-safety is assured as the bacterial culture doesn’t come in contact with users. Therefore, prevention of contamination is possible. Moreover, as the cell lysate has all the genetic material degraded, this would ensure no transfer of the antibiotic degrading genes into other bacteria in the slurry. Sterilization of Growth tank is not required, as BPCs are one-time use containers. As engineered E. coli is cultured in the growth chamber, which is free of other microbes in the excreta, there is no selection pressure. These engineered E.coli can be quickly grown to the carrying capacity of culture media. We could also use a cocktail of bacteria having different antibiotic degrading enzyme. As the engineered bacteria are killed before adding to the excreta, we would get a wider time window for degrading the antibiotics present in the excreta as there is no risk of the spread of antibiotic resistance genes.

Alternative Whole cell System

Here instead of lysing the bacterial cell and then adding them to slurry, we will add bacterial culture directly to slurry thus bacterial will take up antibiotic and degrade it and later inducing kill switch post degradation by inducing kill switch.

Detailed Implementation plan of whole cell system


Figure 9: The complete Tank design for the working of Coli Kaze (whole cell system)

Genetically engineered bacteria would be cultured in the growth chamber. The poultry excreta is collected and diluted in the treatment chamber. The cultured bacteria are then added to this diluted slurry, and incubated for an optimal amount of time. The antibiotics in the slurry would consequently be degraded, significantly reducing the level of the antibiotics below the PNEC (Predicted No-Effect Concentration) levels. Thus, arabinose is added to the slurry in the treatment chamber, resulting in the death of GM bacteria (Our Coli Kaze in this case). The processed slurry is thereby antibiotic-free, can be further processed and used as manure.

This system has its problems:

1. Our engineered E. coli is a lab strain, and it is overproducing certain specific proteins. As the poultry excreta consists of diverse species of microbes, there might be a competition for resources, and thus our bacteria may not grow efficiently in the presence of these microbes in the excreta.
2. The death rate of cells in natural conditions is relatively high. Thus, antibiotic degrading genes could be released into the slurry even before the kill switch is induced. Therefore increasing the chances of uptake of these genes by transformation into the other bacteria present in the slurry, resulting in the antibiotic resistance in bacteria, contradicting our aim of reducing antibiotic resistance among bacteria.
3. Naturally occurring coliphages can transduce the antibiotic degrading genes from engineered E. coli to other bacteria present in excreta and thus spread antibiotic resistance.



Rationale

The implementation of the whole-cell system is similar to the cell-free system except for the fact that we would be using cells that would take up the antibiotics and degrade them. For our purposes, we have considered the uptake rate to be instantaneous, and the system would function precisely as the cell-free system, but the degradation would be time-bound by HGT. The above 3D plot (Figure 8) would prove to be the most important in determining the use of the whole-cell system.

We have discussed the importance of α values that we have used in our modelling to take into consideration the fact that our enzymes wouldn’t be in ideal conditions. These would need to be determined experimentally to determine the kinetics of the enzyme. For the above graph we have used α₁ = 0.7 and α₂ = 0.8. But these would have to be determined for the system and slurry that would be used. A future direction for the use of the whole-cell system would be quick experimental methods to determine these alpha values. Once we have the enzyme kinetic equations ready, We would measure the concentration of antibiotics present in the slurry and wild bacterial population. Once we have an estimate of these values, we would run the HGT models by varying the amount of Coli Kaze that we add to the slurry. We would grow the bacteria in a separate tank and then mix the tank contents with the slurry. We use a sufficient amount of LB such that its nutrients are used up. Once bacterial growth is complete, this culture would be added to the slurry, as slurry won’t be enriched with furthermore nutrients limit the growth rate of Coli Kaze. For our simulations, we have used a growth rate of 0.03 hr^-1 and death rate 0.005 hr^-1 based on some freshwater lake data for the bacterial population. We run the simulations with different initial amounts of Coli Kaze and determine the amount of enzyme produced and the time taken for HGT. For our purposes, we have set the horizontal gene transfer threshold to be 1 cell/mL of slurry. We then see whether the time required for degradation is less than the time required for HGT transfer to reach its threshold. If the time is not lesser than the threshold, we would need to split the excreta into smaller batches with lesser amounts of antibiotics such that the degradation would be complete with a minimum amount of HGT.

Conclusion

From this implementation, we see that the whole-cell system would never be devoid of HGT, and it would be better to use a cell-free system as, in theory, it prevents HGT. However, in cases where the cellular physiology of the GMO is necessary for degradation of the antibiotic, we would require the whole-cell system as a backup for the degradation of antibiotics. The whole-cell system is still imperfect and is still subject to a lot of changes for proper implementation. However, this serves as a decent alternative when bacterial physiology is necessary for antibiotic degradation.

References:

1. Larentis, A.L., Nicolau, J.F.M.Q., Esteves, G.d.S. et al. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Res Notes 7, 671 (2014). https://doi.org/10.1186/1756-0500-7-671
2. Adapted from: Ymxfilter.com. 2020. Wedge Wire Trommel Screen, Wedge Wire Screen, Wire Screen Supplier.[Accessed 24 October 2020]
3. Scribd. 2020. Manure Processing Technologies | Agriculture | Anaerobic Digestion. [online] Available at: https://www.scribd.com/document/401721077/21010-technical-report-II-manure-processing-technologies-pdf [Accessed 24 October 2020].
4. Kim E, Lee DH, Won S, Ahn H. Evaluation of Optimum Moisture Content for Composting of Beef Manure and Bedding Material Mixtures Using Oxygen Uptake Measurement. Asian-Australas J Anim Sci. 2016;29(5):753-758. doi:10.5713/ajas.15.0875