Usually, the conventionally employed volume is comprised between 400 mL and
500 mL. This gives good results also in terms of reproducibility. In the case of
volumetric and manometric measurement of the gas, the volume of the vessels can
be also higher and comprised between 500 mL and 1000 mL, respectively.
The vessels have to be locked with butyl rubber or silicon septs to obtain gastight
conditions. The abovementioned septs, in the case of manometric measurement of
the produced biogas, need to have enough dense to be pierced many times with a
needle to allow the biogas to evacuate from the bottle and be sampled. If the biogas
volume and composition is measured with a device which is connected to the vessel,
the piping has to grant that no leakage and no air infiltration should happen. To avoid
any leakage, tests should be carried out before starting the incubation test. When
composing the mixture to be inserted in the vessel to perform the BMP tests, usually
the quantity of inoculum is higher than that of the substrate; for this reason, the
inoculum in this case not only represents the origin of microbia but also provides
micro and macro-elements, vitamins, and nutrients to the same, together with an
important buffering capacity. If not sufficient, micro and macro-elements can also be
supplied or integrated (Angelidaki et al. 2009). If the measurement of the alkalinity
is below 3 gCaCO 3 L
À1 , then it has to be controlled by adding sodium bicarbonate to
the inoculum, to reach at least 3 gCaCO3 L
À1
. During the test preparation, to avoid
changes in the carbonate balance (Koch et al. 2015) considering the quantity of
carbon dioxide that dissolves in water, flushing has to be performed using a gas
mixture of N 2 and CO 2 , in which the volumetric concentration of CO 2 resembles that
expected for the concentration of CO 2 in the biogas (e.g., 20–40v% CO 2 ; remaining
concentration represented by N 2 ). When preparing the mixture to be digested, a
concentration of volatile solids of about 20 to 60 gVS L
À1 is recommended. In all the
vessels, the volatile solids added through the inoculum should be the same. If the
sample is very dry, this can be diluted using deionized water. The inoculum to
substrate ration (ISR) is an important parameter to be considered in the BMP test;
this is calculated as the ration of the volatile solids contained in the inoculum to the
volatile solids contained in the substrate. To avoid acidification and inhibition
processes, it is recommended that the concentration of volatile solids in the inoculum
should be higher than that in the substrate (e.g., ISRs of 2 and 4 are optimal for easily
degradable substrates, to avoid accumulation of VFA). An ISR less than or equal to
one can be applied for less degradable substrates, such as lignocellulosic organic
matter. Anaerobic digestion can be performed both at thermophilic and mesophilic
conditions. Usually, the same conditions of the anaerobic digestion which has
provided the inoculum should be adopted. During incubation, mixing is needed. If
the substrate is mixed with continuous mechanical movement, this should be
performed at moderate intensity. In most of the cases, manual mixing can be
performed once a day to avoid the formation of scum layers.
10 Substrate Characterization in the Anaerobic Digestion Process
327
500 mL. This gives good results also in terms of reproducibility. In the case of
volumetric and manometric measurement of the gas, the volume of the vessels can
be also higher and comprised between 500 mL and 1000 mL, respectively.
The vessels have to be locked with butyl rubber or silicon septs to obtain gastight
conditions. The abovementioned septs, in the case of manometric measurement of
the produced biogas, need to have enough dense to be pierced many times with a
needle to allow the biogas to evacuate from the bottle and be sampled. If the biogas
volume and composition is measured with a device which is connected to the vessel,
the piping has to grant that no leakage and no air infiltration should happen. To avoid
any leakage, tests should be carried out before starting the incubation test. When
composing the mixture to be inserted in the vessel to perform the BMP tests, usually
the quantity of inoculum is higher than that of the substrate; for this reason, the
inoculum in this case not only represents the origin of microbia but also provides
micro and macro-elements, vitamins, and nutrients to the same, together with an
important buffering capacity. If not sufficient, micro and macro-elements can also be
supplied or integrated (Angelidaki et al. 2009). If the measurement of the alkalinity
is below 3 gCaCO 3 L
À1 , then it has to be controlled by adding sodium bicarbonate to
the inoculum, to reach at least 3 gCaCO3 L
À1
. During the test preparation, to avoid
changes in the carbonate balance (Koch et al. 2015) considering the quantity of
carbon dioxide that dissolves in water, flushing has to be performed using a gas
mixture of N 2 and CO 2 , in which the volumetric concentration of CO 2 resembles that
expected for the concentration of CO 2 in the biogas (e.g., 20–40v% CO 2 ; remaining
concentration represented by N 2 ). When preparing the mixture to be digested, a
concentration of volatile solids of about 20 to 60 gVS L
À1 is recommended. In all the
vessels, the volatile solids added through the inoculum should be the same. If the
sample is very dry, this can be diluted using deionized water. The inoculum to
substrate ration (ISR) is an important parameter to be considered in the BMP test;
this is calculated as the ration of the volatile solids contained in the inoculum to the
volatile solids contained in the substrate. To avoid acidification and inhibition
processes, it is recommended that the concentration of volatile solids in the inoculum
should be higher than that in the substrate (e.g., ISRs of 2 and 4 are optimal for easily
degradable substrates, to avoid accumulation of VFA). An ISR less than or equal to
one can be applied for less degradable substrates, such as lignocellulosic organic
matter. Anaerobic digestion can be performed both at thermophilic and mesophilic
conditions. Usually, the same conditions of the anaerobic digestion which has
provided the inoculum should be adopted. During incubation, mixing is needed. If
the substrate is mixed with continuous mechanical movement, this should be
performed at moderate intensity. In most of the cases, manual mixing can be
performed once a day to avoid the formation of scum layers.
10 Substrate Characterization in the Anaerobic Digestion Process
327