I been working on an experiment to study the heavy metal removal by isolated bacteria. Briefly, bacteria were inoculated in a conical flask with minimal medium and heavy metal at different concentration. 2-days and 4-days old culture were later aliquoted and centrifuged to remove the bacterial pallet. The supernatant that contain remaining heavy metal from biosorption were diluted 100x with deionized water and analyzed for heavy metal using ICp-MS.
Based on the result, concentration of heavy metal in day 4 sample were higher than day 2. But based on previous studies, the concentration of heavy metal should have decreased as the heavy metal will undergo biosorption process by inoculated bacteria.
Are there any possibilities that could cause this condition?
Your opinion and suggestion will be greatly appreciated.
Restating your question, what could cause metal concentration in supernatant of your metal biosorption process to increase after 4 compared to after 2 days? Was day 2 supernatant metal concentration lower than initial (day 0) concentration? If so, the most likely explanation would seem to be cell lysis or disruption by day 4 that did not occur on or by day 2. Cell lysis could be related to bacterial cell death due to culture conditions. Disruption could be related to centrifugation conditions. Since centrifugation conditions were presumably the same on days 2 and 4, there may have been a combined age (mechanical weakening of cells) and centrifugation effect on day 4 that did not occur on day 2. Did you determine cell biomass (dry mass of bacterial pellet, or cell count) on days 2 and 4?
Thank you for your reply. The day 2 metal concentration were lower than the initial concentration.I did not perform any cell count for this experiment. Interestingly, even reading for the control (minimal medium + heavy metal only) showed an increase in heavy metal concentration, but lower than for the cultured sample.
What did metal concentration from day 0 to day 2 do in the control?
From day 2 to day 4 metal increased in your control (medium+metal only) AND in your biosorption treatment, but the increase was greater in your biosorption treatment. How large were the (absolute and relative) increases in both the control and the biosorption culture(s)?
Can you share a data summary? Be much more efficient to see all the relationships at the same time.
Sounds like some longterm contamination by used vessels, flasks, centrifuge tubes, etc. The longer the contact time and the more changes to different vessels the higher the contamination risk. Which elements did you use; which conc levels, which pH and matrix load? As an essential prerequisite to be on the save side avoiding biasing processes of adsorption/desorption/contamination by vessel material and to turn out your experiments well you should take utmost care of this issue and use exclusively PFA material in all steps. Otherwise you go a reasonable risk to produce the observed strange results. Of course there could be other reasons, e.g. due to machine and handling problems in ICP-MS analysis (less probable (?)).
Good luck
D. Zachmann
Hi Bryce. I attached the excel file containing the data here for your reference.
The tested heavy metals are zinc and manganese. The concentration used are 200, 400, 600, 800 and 1000 ppm and the pH of the growth medium is 6.4. Thank you for your suggestion Dieter
Hello Pranesha,
considering your Excel data (suppose it is Zn (?))I feel corroborated that there is a problem with contamination; especially at the 200-ppm-experiment, but also the 1000 ppm experiment looks strange. On the other hand, the other experiments show indeed a slight conc increase at day 4. Possibly there is another (additional (?)) source of biasing your experiments and you should have a look whether or not there is a change in pH. If you take glass vessel materialI adsorption/retention of metal to the glass walls will occur to some degree. Adsorption is very sensitive to changes in pH. If there would be a slight decrease in pH (< 0.1 pH units) this would cause desorption and increase of metal in solution. Therefore, as already recommended you should use PFA-material where adsorption (and contamination by memory effects) can be excluded.
Good luck
D. Zachmann
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Pranesha,
Dieter's suggestions are all good. However, the size of the apparent variation in your data (100-300 ppm) is substantial and relatively consistent. I converted your results to proportional differences for each sample day compared to Day 0 [i.e., (Day2-Day0)/Day0 and Day4-Day2)/Day0] and noticed that if you consider all the data except the 200 ppm treatment data, the proportional results are quite similar. There was, relative to Day 0, a 19-28% (average 22.6%) decrease from Day 0-Day 2 and a 0-7% (average 3.7%) increase from Day 2-Day 4. Even the control treatment decreased by 20% from Day0-Day2 and increased by 7% from Day2-Day4. That is, the control acted effectively exactly as the biosorption treated samples except the 200-ppm treatment. And, the 200-ppm treatment on Day 2 showed an increase in metal above the Day 0 level, which presumably must be an analytical error of >100 ppm. This seems to suggest a systemic source of error not related to the biological treatment. I would suggest you look into the ICP-MS analytical instrumentation used. Are you using an auto-sampler or auto-calibration of the ICP-MS? These automated systems are subject to errors that can show up as generally consistent or wildly random errors over a run of a set of samples. All such instruments should have instructions/methods for manual evaluation of system function and accuracy. I suggest you check the instrumentation and verify proper function and accuracy before trying to identify . If you find problems, inform other researchers who have been using the same instrumentation. Their data could be affected by similar errors that will not necessarily be as obvious in effect as yours.
In addition to the great ideas given above, I have some concern about the metal content of the isolated solid after the 2 day biosorption. The decrease in your metal content in the supernatant liquid must be accompanied with an increase in the metal content of the solid isolate. There could be a death of the bacteria followed by desorption of the adsorbed metal ions after the 2 days treatment. Such a suggestion assumes that the control flask showed no change in the metal ion content, to make sure that your system does not contribute negatively.... Good luck.
I will check through the machine again and possibilities for desorption.
Thank you very much for all the suggestions and opinions, greatly appreciate it.
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