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 decreases below the recommended level, there is a risk of temperature drop in those incubated reactors, which leads to less or no gas production and therefore the graph appears as multiple steady-states. The user needs to check the water level of thermo water bath in regular time base, e.g. twice per week. in case of experiment under thermophilic condition, more frequent check is suggested. The level of water should be up to the plastic glass lid from 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, gelatin). Cellulose is commonly used as reference substrate for energy crops or waste from agriculture, starch for waste based potatoes and cereal grains, 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 ISR of 2 (Raposo et al 2011). However, according with the latest 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).
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 AMPTS II and AMPTS II Light. This means that the entering data for the inoculum and substrate concentration is 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 of the weight of substrate and inoculum which need to be added in each test flask.
The biogas produced during an AD process contains water saturated vapor and the fractional volume of water vapor is a function of AD process temperature. In AMPTS II and AMPTS II Light, the biogas produced in each bioreactor passes through individual CO2-absorption unit containing alkaline solutions before reaching the detection unit. Due to temperature drops when water saturated biogas flow out from bioreactor, a part of water vapor condensates can be formed into the Tygon tube which connects the test flasks to the CO2-absorption unit. This phenomenon of water vapor condensation cannot be avoided since biogas which is produced at high temperature (e.g. 37 or 50 oC) is coming in contact with gas tube kept at lower temperature (i.e., room temperature). The “block of liquid” accumulated into the Tygon tube is pushed by gas continuously produced in the bioreactor and transferred into the bottle for CO2-absorption. The level of liquid in the bottles with alkaline solution can increase from 80 to 100 mL during one batch of experimental test, depending on the process temperature and volume of biogas produced during a batch test. This “block of liquid” is not affecting the efficiency of CO2-adsorption step and no negative impact on the accuracy and precision on the gas volume and flow measurements.
To eliminate the necessity for periodically check of the water level in the thermostatic water bath during a batch fermentation test and to minimise water loss due to its evaporation, some of the laboratories use silicone oil (e.g., LABOTHERMOL S from NeoLab) to replace water. The price of such a silicone oil is approx. 60 €/L. Even though water is free of charge, much clean and more user friendly to clean items which come in contact with, customers may have their own choice to use such silicone oil.
There may be various reasons for no gas registration. Some of them may relate to experiment itself, others may concern instrument setup. It is very important to isolate problems before finding the right solution. In term of instrument setup, you should make sure that the unit is receiving gas to the gas detection unit. You can check if the flow cell is the problem or the detection unit by manually lifting the flow cell with your hand, and see if the opening of flow cell can be registered by the software or not. If the software can register openings of flow cell, the data acquisition of the instrument does intact with the mechanical movement of flow cell. Then you need to check the gas tightness of the test line from the bioreactor to the inlet of gas detection unit. If there is no symptom of gas leakage for the whole test line, you need to figure out if there is any biological issue (e.g. very low activity of inoculum, substrate inhibition, etc).
AMPTS II was developed for monitoring the accumulated volume of gases with low water solubility (e.g. CH4, H2, N2), but not biogas where high concentration of water soluble carbon dioxide (CO2) presents. Solubility of CO2 is much higher than the solubility of CH4 in water (0.74 vs. 0.03 l/l) and it is always a technical challenge to measure volume of high water soluble gases accurately in low quantity when water presents no matter what measuring principles to be used. Even if the literature reports that the 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 max 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 for scientific work. The challenge for measuring water soluble gases has solved with our new design of flow cell and flow cell chamber utilised in new Gas Endeavour - a smart instrument specially developed meet the measurement demands of water soluble gases or ultra low quantity of gas volume. Contact us for more information of our new solution for biogas potential or measuring any gas soluble gases.
For high precision and accuracy 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 2 to 4). This will lead to a high volume of gas production at high flow rate and allow a high signal to background noise rate.
Both AMPTS II and AMPTS II Light have an embedded micro-processor that runs web-based data logging program. Any external connected computer, tablet and smart phone is used for control and visualise the experimental data. Unplugging ethernet cable connection, shutting down or restarting a computer that is connected to the instrument has no effect and any risk to interrupt ongoing experiment and lost data registration.
In order to investigate true biomethane potential or biodegradability of substrates with large size particles, you may use grinding machine to get more homogenised sample for batch fermentation tests. The simplest one is the home used blender. There are also mill homogeniser for laboratory and industrial application. According to the literature, samples such as corn silage might be grounded through Wiley mill followed by grinding with Udy mill. A brief description of the working principle of the Udy cyclone sample mill is presented at the following link.
For flushing the reactor headspace prior anaerobic batch tests, each bioreactor bottle has to be flushed with a flush gas separately. There is no way to flush all of reactors at the same time. The flush gas should contain an oxygen free gas (e.g. N2 or a mixed gases 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: i) First stage 0-200 bar; ii) Second stage: 0-30, 0-5 or 0-1.5 bar. Before flushing reactors with the selected gas to create anaerobic condition, the Tygon tubing from the CO2-adsorption unit must be disconnected. The gas source has to be connected to the Tygon tubing with the red clamp by using a plastic tubing connector/reduction adapter. The red clump from the reactors should be opened and the reactors can be flushed with a low gas flow gently over 0.5 – 1 minute. In order to control the gas flow rate from the gas source, the Tygon tubing which was disconnected from the CO2-absorption unit might be immersed into a beaker with water to visualise the bubble formation for flow control. At the end of the flushing step, stop the flush gas, close the red tube clamp, take the Tygon tubing from the water and connect it to the CO2-adsorption unit. Disconnect the flush gas source and repeat the procedure for all the reactors.