A mysterious wikipedia quote : does phosphate have an influence on prodigiosin secretion by S. marcescens ?

Picture from upload.wikimedia.org : Serratia marcescens

 

Wikipedia, a large and free dictionary, enriched by everyone is full of surprise. To find an idea about my project on microbiology in the Petnica Science Center, I did some research about one bacteria available in the lab : Serratia marcescens. The latter produce a red pigment that is also a secondary metabolite. To put word differently, it plays a role in the development of the bacteria but it isn't a vital one. During my research on wikipedia, it says that a “low quantity of phosphate” increase the concentration of pigment. This point was interesting me to create my project. Thus, I looked at the article related to this information. However, it isn't available on Internet. After more researches, I found other references to this article and also some old articles that made some similar experimentations.

Thus, to check if the wikipedia reference has disappeared for good reasons, I chose to work on the impact of phosphate on the prodigiosin secretion by S. marcescens. 

How ? I chose eight different concentrations of phosphate based on the work of K. Bahadur and S. Ranganayaki : 0g, 0.003g, 0.0045g, 0.006g, 0.0075g, 0.009g, 0.0105g, 0.0120g and 0.0135g that I put on 3 mL. I did three replicates for each concentrations. To check if the phosphate have an impact on the pigment, I did a negative control with two tested concentrations without pigment : one at 0.003g and an other at 0.0135g of phosphate. To obtain colony without pigment, I put during one night the tube in an incubator at 44°C while for the other one I put the temperature at 30°C.

Then to estimate the density of pigment, I put all the tube in a spectrophotometer. According to literature, the wavelength for the pigment is 532 nm. However, each tube didn't have the same concentration of cell. It is crucial to have this information because if the number of cell is hight, the quantity of pigment alss and conversely. Thus, I also measured the optical density of the bacteria, with a wavelength at 600nm. Next, I calculated the ratio pigment density per bacteria density to quantify the quantity of pigment produced per cell.

Ratio pigment density per bacteria density according to the concentration, in red the negative controle without pigment and in blue the other tube with pigment

Here the result : in red the negative control and in blue the ratio with pigment. The first point interesting is that without phosphate there are less pigment than cell. Then, the best quantity of phosphate is 0.006g. However, it is interesting to notice that the ratio decrease for 0.009g and increase again at 0.0105g. Thus, ion phosphate could interfere with the bacteria and slow down the production of pigment. 

Furthermore, an unexpected result is showing by this graph : the tube without pigment … produced pigment ! According to literature it is unexpected. However, it is fun to imagine that temperature could have an impact on the activity of the phosphate. To put word differently, at 44 °C the phosphate could help the bacteria to produce pigments while without the bacteria couldn't. Especially, the amount of pigment is higher than at 30 °C. Moreover, a higher quantity of phosphate seems to increase the production of prodigiosin. To test this hypothesis it could be interesting to put 0g of phosphate in one tube without pigment. Indeed, we could know if the presence of pigment is causes by the phosphate or if at 44°C pigment is produce.

Finally, to improve this experimentation, two things could be change. For instance, we can estimate the number of phosphate ion produce. It is interesting because we can know the level of saturation of the bacteria. Furthermore, we could extract the pigment from the bacteria to estimate more exactly the quantity of pigment. In fact, the wavelength of the bacteria and the pigment are close. Indeed, this overlapping created a bias because the bacteria could absorbed a little bit at 532nm.

To put in a nutshell, this humble work seems to be agree with the mysterious article quote by wikipedia. However, it could be interesting to improve it in order to increase our knowledge about the influence of phosphate on the bacteria.

If you want to know more :

- about prodigioson : https://en.wikipedia.org/wiki/Prodigiosin

- about secondary metabolite : https://en.wikipedia.org/wiki/Secondary_metabolite

- about Serratia marcescens : http://bacterioblog.over-blog.com/article-3817367.html

- about some characteristic of our bacteria :

1) J Bacteriol. 1971 May;106(2):438-43.Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens.Williams RP, Gott CL, Qadri SM, Scott RH.

2) Jpn J Microbiol. 1958 Apr;2(2):197-201.A study of the influence of milk, phosphate and calcium carbonate on the formation of 2,3 butanediol in Serratia marcescens cultures.BAHADUR K, RANGANAYAKI S.

- And if you would try to find the mysterious article : M. Todd-Guay and P.H. Demchick. 1995. Role of prodigiosin in phosphate-starved Serratia marcescens. Abstract of the Annual Meeting, American Society for Microbiology.