Dear Mr, Antoun,

I am using 10 ml of 2M KOH solution and titrating against 0.5 M HCl. During the experiment, I am keeping few sets  as a control without adding any soil.  when titrate against HCl, I am getting the same reading for both soil and control. But I can see clear difference in precipitation after the addition of BaCl2.

Hi Manjunatha,

As Hani Antoun said, you tritate with HCl the excess of KOH or NaOH that did not react with CO2. Using 10 ml of 2M KOH solution and titrating them against 0.5 M HCl, in the case of null trapped CO2 you will need 40 ml of HCl to titrate the 10 ml of KOH. As more trapped CO2, less volume of HCl you will need. Blanks indicate the trapped CO2 not induced by substrate respiration. The smaller it is, the less noise will be (in your case, around 40 ml of HCl to titrate 10 ml of KOH indicates around null noise). On the contrary, if you don't need HCl to neutralize the KOH, or you need a very low quantity, it means that all, or close to all, KOH has consumed to trap the CO2. If it's the case, as Hani Antoun proposed, you must either use a larger quantity of KOH, shorter the time of exposure, or maybe lesser quantity of  substrate.

I suppose that you use closed jars, is it true?

On the other hand, respiration indicates biomass heterotrophic activity. It is related to microbial biomass, although both concepts (biomass and activity) are different. Please, do not confuse them.

All the best

Hi Manjunatha

I have been doing that analysis for the past 2 years, but instead on KOH, I use 1M NaOH and the same molarity as you for HCl. I have been getting really good results. Do you end up using the same amount on HCl during titration for both the control and sample?If that is the case, that would mean there wasn't any microbial respiration during the incubation period. Do you wet the soil samples to eye field capacity before you incubate them? How long do you incubate your samples for?. For my research I first air-dry my samples then re-wet them to eye field capacity before incubation so as to spark the activity in the soil. After adding the BaCl2, do you see the milky (white precipitate) color on both the sample and the control? And do your samples turn to pink after you have added the phenolphthalein indicator?

Sorry for the questions but they will help me to understand your problem better and therefore attempt to answer it.

Hi,

Yes, I wet the soil before incubation. I incubate for 16 hrs. Yes, I get milky precipitate in both, but the  precipitation is higher in sample compared to control. Before titration I filter the milky precipitate and use equal amount of filtrate for titration. I titrate  for pink to colorless.

How do you filter it? your control isn't really supposed to have a milky color unless it has been exposed to atmospheric CO2. You ought to be very cautious with you control so that it doesn't get exposed to CO2. I know I have struggled with this as well, maybe it would be best if you send me your methodology and I will send you mine as well that way I think we can narrow down the problem.

Good luck with you research! 

Hi Portia Phohlo,

When we opened our control in lab, it might have captured the atmospheric carbon dioxide, hence I got little milky precipitate. But the differences in milky precipitate is visible between sample and control. I removed the precipitate using filter paper.

Dear Portia Phohlo,

Last time you mentioned about NaOH should not get exposed to atm CO2, when we are working in lab how can we control it?

In substrate-induced respiration (SIR) assays, the measurement of microbial activity through the quantification of produced CO2 is a common practice. These assays often involve the absorption of CO2 in a base, such as NaOH, and subsequent titration with an acid, like HCl, to determine the amount of CO2 produced. The lack of observed difference between a blank (control without substrate) and a sample (with substrate) after titration against HCl could be attributed to several factors, each of which must be considered systematically to troubleshoot and address the underlying issue.

1. Inadequate Substrate Concentration

• Issue: The substrate concentration may be insufficient to elicit a measurable microbial response. Microbial communities may require a threshold level of substrate before a detectable respiration increase occurs.
• Solution: Increase the substrate concentration, ensuring it is within the optimal range for the microbial community being studied.

2. Microbial Activity

• Issue: The microbial population in the sample may be too low or inactive, possibly due to suboptimal storage conditions, sample age, or the presence of inhibitors.
• Solution: Verify the microbial viability through alternative assays. Ensure the sample is fresh and stored under appropriate conditions to maintain microbial activity.

3. Experimental Setup and Titration Accuracy

• Issue: Errors in the experimental setup, including improper sealing of the reaction vessel, incorrect titrant concentration, or inaccurate titration technique, could lead to erroneous results.
• Solution: Double-check the experimental setup for any leaks or setup errors. Confirm the concentration of HCl and ensure accurate titration techniques are employed.

4. CO2 Absorption Efficiency

• Issue: Inefficient CO2 capture by the NaOH solution can occur if the volume or molarity of NaOH is insufficient.
• Solution: Ensure the NaOH solution has an adequate concentration and volume to absorb all the CO2 produced during the respiration process.

5. Interference or Contamination

• Issue: The presence of contaminants or interfering substances in either the blank or sample that could neutralize the acid or base, leading to inaccurate titration results.
• Solution: Prepare fresh reagents and ensure all glassware and instruments are clean to avoid contamination. Review all reagents for potential expiration or degradation.

6. Analytical Sensitivity

• Issue: The sensitivity of the titration method may not be sufficient to detect small differences in CO2 production, especially if the microbial activity is low.
• Solution: Consider increasing the sensitivity of the assay, possibly by using more sensitive detection methods for CO2 or adjusting the assay conditions to enhance microbial activity and CO2 production.

Conclusion

A lack of difference between the blank and sample in a substrate-induced respiration assay after titration against HCl suggests issues with either the experimental setup, microbial activity, substrate concentration, or analytical sensitivity. By methodically examining and addressing these potential factors, one can identify the root cause and take appropriate measures to rectify the problem, ensuring accurate and reliable measurement of microbial respiration in response to the substrate.

With this protocol list, we might find more ways to solve this problem.