The main objective was to evaluate organic residues, examine their potential as media and to investigate the viability of certain parameters such as proline and protease production. NB was used for (+) control and PBS for (-) control. All study results were obtained after 24 hours.
Proline
We evaluated various organic waste materials found in the environment. The focus was on the production of proline and protease, which are commercially important and necessary to protect the environment.
Excluding the control media, Gram (+) bacteria showed higher proline production than Gram (-) bacteria. B. cereus produced 4.996 μg / ml proline in the presence of PLW (Fig. 1). The lowest production was seen in P. aeruginosa with 0.24 μg / ml in the presence of TWW (Fig. 7). However, on average Gram (-) bacteria in this study showed higher proline production. In our previous study in LB medium, we obtained 5.071 μg / ml in E. coli (Fig. 3) and 1.337 μg / ml in P. aeruginosa (Fig. 7). In the presence of 100 mM KCl in the same medium, we obtained 2.395 μg / ml in E. coli (Fig. 3) and 9.226 μg /ml in P. aeruginosa (Fig. 7).
In our study, the highest production was observed in B. cereus with molasses at 4.381 μg/ml and in the presence of WCW (Fig. 1) in S. aureus with 2.074 μg/ml (Fig. 5), excluding NB and PBS media used for control. The most efficient medium for E. coli was eggshell with 1.982 μg / ml (Fig. 3). For P. aeruginosa, it was TCW medium with 3.27 μg/ml (Fig. 7). Considering all environments, the most efficient in the study were eggshell and OWW. The most effective waste materials for proline production were TCW with 1.738 μg/ml average and molasses with 1.250 μg/ml average (Fig. 1). The most ineffective waste was TWW with an average of 0.859 μg / ml (Fig. 5). The most effective proline producing bacteria were P. aeruginosa with an average of 2.551 μg / ml (Fig. 7). All bacteria produced an average of 1.738 μg / ml proline in the presence of TCW ((Fig. 1 and 3). The lowest amount of organic waste was obtained in the presence of TWW with an average of 0.859 μg / ml. While P. aeruginosa produced 2.551μg / ml proline on average in all environments (Fig. 7), S. aureus produced the lowest amount with 0.993 μg / ml (Fig. 5). Since no research similar to our study was found in the literature, no comparison could be made with other studies. This study was conducted with different biological waste materials. We believe that microbiological proline production can be achieved practically and economically thus benefiting the environment.
As has been shown in previous tests (mostly on plants), proline is a response to stress in living organisms. For unknown reasons, negative changes were observed in E. coli and B. cereus. The biggest changes were seen in S. aureus (4.4-fold) (Fig. 5) followed by P. aeruginosa with 2.9-fold (Fig. 7). WCW did not materially increase stress response. This may result from the lack of adequate components for proline production in WCW and the weakness of the protein-produced metabolic pathways in bacteria.
When the control media are excluded, the lowest proline production was in TWW at 0.24 μg / ml (Fig. 1) and the highest in TCW at 3.27 μg / ml (Fig. 7). The difference was 13.6-fold.
When we look at proline production in the presence of organic waste, the highest value (+) were reached with 12.38 µg / ml in NB medium (Fig. 5), which we used as control. 0.69 µg / ml were obtained in (-) control PBS (Fig. 5). Inhibition was seen in TWW (0.24 µg / ml) (Fig. 1) and molasses (0.55 µg / ml) (Fig. 3). In the presence of the remaining organic waste materials (OWW, TCW, and WCW), values of 1.26, 3.27 and 2.02 µg/ml was obtained, respectively (Fig. 5). However, no value could be obtained in the presence of WFO (not shown on the graph). This is interesting and may be due to the high carbohydrate load. These results show that the protein load in organic waste is important in proline production. TCW, WCW, and OWW are organic wastes that support proline production.
Protease
Protease is a virulence factor and a secondary metabolite. No protease production was detected in our study on EW and PLW so they are not shown on the graph. Unlike proline production, protease activity was higher in Gram (-) bacteria. However, the highest value in (-) control PBS was 0.104 U/ml while no results in (+) control NB were obtained in these experiments.
In our previous study with LB medium in E. coli and P.aeruginosa, 0.401 U / ml and 0.2976 U / ml values were obtained, respectively. In the presence of 100 mM KCl, 0.9327 U / ml were obtained in E. coli and 0.295 U / ml in P. aeruginosa.
In our study, the highest protease production in WCW with high protein load was in E. coli (Fig.4). However, when we looked proportionally, it was clear that the highest difference was in P. aeruginosa at 67.5 times (Fig. 8). Why did E. coli produce the most despite this huge difference? This may have resulted from the metabolic load caused by the large genome of P. aeruginosa close to yeasts (Fig. 8). The lowest rate was seen in S. aureus (0.31) (Fig. 6) and the lowest difference in B. cereus (3.3-fold) (Fig. 2). We think this is because B. cereus is soil bacteria and protease activity may not be much needed in the soil environment. However, other bacteria may have higher protease activity than B. cereus because of their ability to survive in living organisms.
The highest values were observed in P. aeruginosa at 51.347 U / ml (Fig. 8) and in E. coli in the presence of molasses at 47.182 U / ml (Fig. 4). The highest subsequent rate was 27.297 U / ml in the presence of OWW (Fig. 7). B. cereus reached the highest value in the presence of TCW with 8.394 U / ml (Fig. 2). S. aureus reached 3.717 U / ml (Fig. 6), again in the presence of molasses. The most efficient medium was molasses with a high carbohydrate load. The highest average for all four bacteria, 24.841 U / ml, was reached in the presence of molasses with a high hydrate load. Protease production in the last three environments was the lowest: WFO 0.261 with B. cereus (Fig. 2), 0.170 U/ml P. aeruginosa (Fig. 8) in the same organic waste and 0.159 U / ml S. aureus (Fig. 6). The lowest average was in the presence of WFO with 0.215 U / ml, excluding controls (Fig. 6). E. coli yielded the highest mean value of 13.326 U / ml in all media (Fig. 4). The lowest average production was in S. aureus at 1.9 U / ml (Fig. 6). The three most successful bacteria were P. aeruginosa with 51.35 U / ml (Fig. 8) and E. coli with 41.182 U / ml in the presence of molasses and E. coli with 27.297 U / ml in the presence of OWW (Fig. 4). The most efficient production waste environment was molasses with an average of 24.81 U / ml (Fig. 6) and OWW with an average of 8.288 U / ml (Fig. 8). When all waste materials were evaluated separately, the most effective production among bacteria was E. coli with an average of 13.326 U / ml (Fig. 4).
Protease activity was approximately 23-fold higher than proline activity. The OD600 value (data not shown) increased by 314% and the increase in the number of living cells was reached 1,816%. According to these results, cells may be more viable and transparent.
We believe that protease production from organic waste is also important for environmental protection so we focused on the organic wastes mentioned above. Unlike proline production, the highest production was in the presence of molasses (51.35 U / ml) (Fig. 6). The OD600 (1.36) value of molasses was found to be the highest in this study. The lowest value was (-) in control PBS (0.04 U / ml). The most significant values were then observed in TCW (3.84 U / ml) (Fig. 2) and WCW (2.93 U / ml) (Fig.4). Except for the control, the lowest value was 0.17 U / ml in WFO with a high oil load (Fig. 6).