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.
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.
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.
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