Most frequent questions and answers
When cycles of high gas production followed by plateau levels are periodically observed, it is recommended to check the water level in the thermostatic water bath. When the amount of water falls below the recommended level, there is a risk of temperature drop in the incubated reactors. This can lead to less or no gas production and therefore the graph appears as multiple steady-states. The user needs to check the water level of the thermostatic water bath regularly, e.g. twice per week. In cases of an experiment running under thermophilic conditions, more frequent checks are suggested. The level of water should reach the plastic glass lid of the thermostatic water bath.
The control experiments are usually performed for testing the quality and activity of inoculum using a standard substrate (e.g. cellulose, starch or gelatin). Cellulose is commonly used as a reference substrate for energy crops or waste from agriculture, starch for food residue, whereas gelatin is used for household waste rich in meat products. The average methane yields for cellulose, starch and gelatin are reported in the literature as 350±29, 350±33, and 380±2 NmL CH4/g VSadded at an ISR of 2 (Raposo et al 2011). However, according to more recent publications, the methane yield for cellulose and starch should be within 85-90% of the theoretical value (i.e., 415 Nml/gVS), which means above 350 NmL/gVS (Holliger et al 2016).
The VS parameter provides an estimation of the organic material content in a sample. It is expressed either i) relative to the total amount of wet sample VS% (w/w) or ii) relative to the total solids VS% (VS/TS). The first definition should be applied when a BMP test is performed with the help of the AMPTS II and the AMPTS II Light. This means that the entered data for the inoculum and substrate concentration should be based on the organic material content relative to the initial weight of wet sample. The user of the AMPTS II and AMPTS II Light has to enter the VS values (VS%, w/w) for the inoculum and substrate together with inoculum to substrate ratio (ISR), as well as the total amount of mixture in the Experiment page of the AMPTS II or AMPTS II Light software. The software will automatically calculate and present the values for the weight of the substrate and inoculum which needs to be added to each test flask.
The biogas produced during an AD process contains water vapour and the fractional volume of water vapour is a function of the AD process temperature. With the AMPTS II and AMPTS II Light, the biogas produced in each bioreactor passes through individual CO2-absorption units containing an alkaline solution before reaching the detection unit. Due to temperature drops in the water saturated biogas exiting the bioreactor, condensation water can be formed in the gas tubes which connect to the CO2-absorption bottles. This phenomenon cannot be avoided since biogas which is produced at a high temperature (e.g. 37 or 50 oC) is coming into contact with a gas tube kept at a lower temperature (i.e., room temperature). Part of the water accumulated in the gas tube is transferred by the gas produced in the bioreactor into the CO2-absorption bottles. The level of liquid in the bottles can increase from 80 to 100 mL during one batch test. This additional liquid is not affecting the efficiency of the CO2-absorption step and gives no negative impact on the accuracy and precision.
To eliminate the necessity for periodically examining the water level in the thermostatic water bath and to minimise water loss due to evaporation, some laboratories use silicone oil (e.g. LABOTHERMOL S from NeoLab) to replace water. While this is an option, water is more cost effective, cleaner and easier to work with and does not attach to objects it comes in contact with. If an older version of the thermostatic water bath lid is used for 500 ml reactors, it is recommended to update to the latest version. It contains 15 sealing rings, which minimises the evaporation issue.
There may be various reasons for no gas registration. Some of them may relate to the experiment itself, others may concern instrument setup. It is very important to isolate the issue before it is possible to find the right solution. Test if the flow cell is the problem by manually lifting the flow cell with your hand and seeing if the opening of the flow cell is registered by the software or not. If the software can register the opening of flow cell, the data acquisition of the instrument is intact. Next, test the gas tightness of the test line from the bioreactor to the inlet of the gas detection unit. If there are no symptoms of gas leakage for the entire test line, you need to figure out if there are any biological issues (e.g. very low activity of inoculum, substrate inhibition, etc).
AMPTS II was developed for monitoring gases with low water solubility (e.g. CH4, H2, N2), but not biogas, where high concentrations of water soluble carbon dioxide (CO2) is present. The solubility of CO2 is much higher than the solubility of CH4 in water (0.74 vs. 0.03 l/l) and it is a technical challenge to measure a low quantity of highly water soluble gases accurately. Even if the literature reports that acidified water (pH 2), mineral oils, saturated NaCl or acidified saturated NaCl can decrease the solubility of CO2 in water, this is usually done by a factor of a maximum of 3 and will not completely solve the solubility issue. Therefore, the acidified water will not be a suitable solution for measuring the volume of biogas to meet the high-quality demand of scientific work. This challenge has been solved with our design of flow cell and flow cell chamber utilised in the Gas Endeavour – an instrument specifically developed to meet the measurement demands of water soluble gases or ultra-low quantities of gas. Please contact us for more information regarding our new solutions for biogas potential measurements and for measuring any water soluble gases.
For high precision and accurate test data, the experiment should be set-up correctly: high amount of inoculum-substrate mixture (e.g. 400 g in 500 ml bottles), low bioreactor headspace volume, sufficient substrate quantity and suitable inoculum to substrate ratio (in the range of 2 to 4). This will lead to a high volume of gas production at a high flow rate and will allow for a high signal to background noise ratio.
AMPTS II, its light version and Gas Endeavour have an embedded server running a full operating system stack. An externally connected computer, tablet or smartphone is used for control and visualisation of the experimental data. Unplugging the Ethernet cable or shutting down / restarting a computer that is connected to the instrument has no effect on the data being registered and there is no risk that it will interrupt ongoing experiments.
In order to investigate the true biomethane potential or biodegradability of substrates with large particles, you may use a grinding machine to get a more homogenised sample for batch fermentation tests. The simplest version is the blender used in regular households. There are also mill homogenisers for laboratory and industrial applications. According to the literature, samples such as corn silage might be ground through a Wiley mill followed by grinding with a Udy mill.
For flushing the reactor headspace prior to an anaerobic batch tests, each bioreactor bottle has to be flushed with a flush gas separately. There is no practical way to flush all of the reactors at the same time. The flush gas should contain an oxygen free gas (e.g. N2 or a mixed gas based on 60% CH4 and 40% CO2 or 60% N2 and 40% CO2). In most cases, the flush gas is stored in a gas pressurised bottle equipped with a double stage pressure regulator, e.g. a first stage of 0-200 bar and a second stage of 0-30, 0-5 or 0-1.5 bar. Before flushing a reactor, the gas tubes from the CO2-absorption unit must be disconnected. The gas source has to be connected to the gas tube with the tubing clamp by using a plastic tube connector / reduction adapter. The tubing clamp from the reactor should then be opened and the reactor can be flushed gently with a low gas flow for 30 to 60 seconds. In order to visualise the gas flow rate from the gas source, the gas tube which was disconnected from the CO2-absorption unit can be immersed into a beaker filled with water in order to use the bubble formation as a guide. At the end of the flushing step, stop the flush gas, close the tubing tube clamp, take the gas tube from the water and connect it to the CO2-absorption unit. Disconnect the flush gas source and repeat the procedure for the remaining reactors.