Respirometric measurement of microbial activity with OxiTop® Control System
The goal of the measurement
The state and activity of soil can be characterized by the continuous measurement of CO2-production of the adequately prepared soil. With the aid of the respiration measurement we may answer the questions:
- whether the soil was contaminated,
- whether the contamination was toxic and inhibited the microflora of the soil,
- whether the microflora has adapted to the contamination and functioned actively,
- whether the microflora could be activated,
- what were the technological parameters required for the optimal function of the microflora.
The CO2 production is proportional to the quantity of the degraded hydrocarbons and it is the measure of biological oxidation, so the system is suitable for the survey of the soil’s biological state, and for the characterization and monitoring of biodegradation. It is also an appropriate measurement tool of the differences in the type, age and concentration of organic contaminant according to the soil type, and it helps in deciding whether additional nutrients or additives (for ex.: detergents) are needed for the success of biodegradation technology. In the case of carbonate containing soils, the released CO2 may influence interfere with the measurement. The respirometric measurement to be performed during the laboratory exercise will examine soil respiration continuously for 5 days in a closed vessel.
- Measuring principle
Our test is a pressure measurement. If oxygen is consumed in a closed vessel at a constant temperature, while the emerging CO2 is sequestrated by an absorbent (NaOH), a negative pressure develops. The OxiTop measuring head measures and stores the pressure data during the whole duration of a measurement once started. To the manometric measurement of oxygen consumption the following conditions must be fulfilled:
The (biologically active) sample must be contained in an impermeable vessel.
Above the sample must be enough space to provide satisfactory amount of oxygen for the biological degradation. The CO2 absorbing agent must be placed in the vessel so that it could not get in contact with the sample. Pressure measurement tool must be placed into the reaction vessel. The vessel must be stored at constant temperature during the measurement time.
- The OxiTop Control closed vessel respirometric system
The OxiTop Control device is a tool for the measurement of respiration intensity. The system measures the consumption of oxygen by the respiration of anaerobic microbes in the sample. Soil microbes consume oxygen and produce CO2, which is sequestrated by NaOH, causing pressure drop in the vessel proportional to the quantity of the sequestrated CO2.
- Required tools and materials
OxiTop-C measuring heads
OxiTop OC110 Controller
ACHAT OC PC communicating software
AK 540/B data transfer wire for RS 232 port
Environmental sample (soil)
- Implementation of the measurement
We place 100 g of soil sample into the measuring vessels (250 ml of volume), and other additives if needed. After homogenizing the samples we fasten the perforated pots containing 1–1,5 g NaOH onto the vessels, and screw the OxiTop measuring heads on the top to close the system. After that we set the measurement parameters on the Control Panel (measurement mode, time, measuring limits), and start the measurement by pointing the Control Panel to the measuring heads within 40 cm distance to achieve infrared connection between the two devices. Flash of red light signals the start of the measurement. Constant temperature and a dark environment are required during the whole runtime, so that external parameters do not disturb the measured pressure values. The measured data are collected in the measuring head, and after the measurement time expires, we can obtain the whole dataset using the Control Panel, and transfer them into an Excel sheet with the aid of data transfer wire and processing software ACHAT OP PC.
Main parameters of the measurement:
Volume of reactor vessel: 250 ml
Soil quantity: 100 g
Moisture content of soil samples: 15–18 %
Temperature: 21.5 oC
Measurement mode: pressure change Δp [hPa]
Measuring limit: 300 hPa
Measuring time: 5 days
NaOH quantity: 1.5 g
- Evaluation of data
The OxiTop measuring head measures and stores the pressure data during five days at every 20 minutes. Since temperature is constant, the pressure changes are caused by the soil respiration solely. Soil microbes consume oxygen and produce CO2, which is sequestrated by NaOH, resulting pressure drop in the vessel proportional to the quantity of sequestrated CO2. The results can be interpreted as follows:
1. The pressure drop in every vessel shows that the soil samples are active, and the biodegradation of organic contaminants has started.
2. On the two steep curves we can observe that diesel oil can be degraded faster and easier by the microbes than the mazout components of high molecular weight.
3. Biodegradation starts faster in freshly contaminated soils than in the case of older contaminations, since in case of fresh contamination the proportion of easily accessible components is higher, while in the older contamination it is rich in hardly biodegradable components
4. The fact that biodegradation is intense in old diesel contaminated soil indicated also the presence of active microflora adapted to the contamination.
5. In the case of old mazout contamination the activity is even lower than in the control soil. This implies that the hydrocarbons are hardly or not at all available in the soil therefore re-activation of the microflora is required.
6. Pressure values illustrate well the processes taking place in the soil, and the adaptive capacity of microorganisms. Microbes adapted easily to the easily degradable diesel oil (higher oxygen demand and pressure drop). In these samples we can see that at a certain point the pressure drop stops –due to the saturation in NaOH – for this reason we need more NaOH in the case of such an active respiration.
Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science (2013) Environmental Toxicology, Manual of the laboratory practices, http://envirotox.hu/wp-content/uploads/2017/10/Laboratory_practices_manu...