The measure of microbial respiration
Microbial activity is dependent on respiration and just as our respiration rates increase in response to work so does the microbial. Given this it is possible to calculate the microbial biomass by measuring respiration, in terms of CO2 production.
The method outlined here is a relatively routine lab procedure involving titration and whilst it requires specialist accurate equipment, chiefly a burette and involves the use of laboratory grade reagents, including barium chloride and chloroform as well as ethanol it is possible for any competent person to perform.
Providing a laboratory grade burette and reagents are used and sufficient care is taken to execute the procedure accurately then it should be possible to estimate the microbial respiration rates under specific conditions. These rates can then be used to estimate the potential nitrogen mineralisation rates of the soil under similar conditions.
To measure microbial respiration rates we need to construct a set of microbial respirometers. In addition to the respirometers needed for the replicates of two separate treatments at least two additional respirometers will be needed for the controls. The standard method is to use a 250ml conical flask to which a bung has been fitted with attached to the bottom, a small hook. Into the flask will be placed 50g of moist soil and from this hook will be suspended a vial containing a measured amount of Sodium Hydroxide (NaOH), commonly referred to as caustic soda. The flask will be sealed by the bung and left for a given period (ten days).
The two treatments are one untreated soil and one fumigated with chloroform. The treated flask is inoculated with untreated soil at the commencement and both treatments and the controls are set in motion. It is unfortunate that the method requires the use of chloroform as a fumigant as steam sterilisation in a pressure cooker [autoclave] would similarly kill off the soil microbial population. One even questions if autoclaving is totally necessary since few soil organisms can survive temperatures above 75 C. and the few that can are unlikely to number many. I would not be surprised that if a pressure cooker was unavailable that placing a prepared flask into a water bath and gently boiling that water for 30 minutes would achieve the same results. The flask and its contents would need to be allowed to cool to room temperature before being inoculated but the soil would be sterile or as good as.
Within the moist conditions of the flask the microbial biomass will mineralise any organic matter respiring in the process and producing CO2. the control will scavenge any CO2 from within the flask and we will be able to use this figure to account for the corresponding CO2 in the air of our two treated flasks. The untreated soil sample will respire as normal using the soil organic carbon as a food source whilst the sterilised and inoculated soil will also use the remains of the original soil biomass as well as the residual organic content for nutrition.
Thus the three flasks result in increasing amounts of CO2 production with the relative differences corresponding to microbial activity and this being dependent on substrate suitability. The amount of increased CO2 production in the treated flask over the untreated is as a consequence of the mineralisation of the original microbial biomass in the treated sample and so corresponds directly with the original soil biomass.
The CO2 evolution is measured after ten days by titrating the NaOH solution (which has an affinity for CO2 to form sodium carbonate (Na CO3) and water (H2O)) against a quantity of HCl. The sample though is first prepared by treating with barium chloride to precipitate the carbonate and leave only the residual NaOH to react with the HCl. The resulting solution is then titrated against hydrochloric acid (HCl) until a change in pH is noted by the Phenolphthalein indicator, which changes colour at pH8.7.
The amount of hydrochloric acid required to change the pH corresponds with the amount of NaOH remaining in the vial after the experiment. From this it can be calculated the amount of CO2 that was absorbed over the ten days and comparison between the titration results of the treated and untreated soil samples allows us to estimate the original microbial soil mass. The controls are used to account for the CO2 present in the air of the flasks at the start.
From this we can potentially estimate the microbial respiration rates for the whole site and from that approximate the amount of carbon being consumed. If we also know the Nitrogen content of the soil we can calculate a C:N ratio and using our estimate for the soil microbial biomass estimate the nitrogen mineralisation rates. There are a lot of estimates here, a lot of buts, and fudges and whilst it would be nice to be more accurate and even nicer if the soil would conform to being measured with a simple ruler; we do not have such a luxury.
In addition to microbial respiration it is also possible to measure general enzyme activity and to use this to estimate the size of the microbial biomass producing it. The most common of these enzyme tests being for catalyse, which is a broad microbial enzyme, and more recently sulphatyse and phosphatyse concentrations to estimate both general microbial activity and the potential availability of sulphur and phosphorus in the soil solution. However as well as being relatively complex microbial procedures thwart with difficulties and variables, the value of even accurate measurements is still very difficult to qualify.