IV Biomass Energy

Laboratory practices ..

POTENTIAL PRODUCTION OF BIOGAS FROM MIXTURES OF WASTES

This working document serves as support for the conduct of laboratory sessions. For a
better understanding, the theoretical bases of a viable biological process to be applied to obtain
energy from waste are presented in a summarized form. In addition, procedures for developing
trials are presented.

1. Basic concepts on anaerobic digestion.

Anaerobic organic matter digestion treatment is widely used to reduce the volume of
biosolids (sludge) produced in a wastewater treatment plant. The treatment of wastewater is
usually carried out using activated sludge technology, which generates a large amount of sludge
that must be stabilized in the same plant.
Through the anaerobic biological process, the organic matter content of a waste can be
reduced and biogas generated. No air supply is required and the biogas generated can be used
to heat the digester itself.
Biogas consists mainly of methane (60-80%) and carbon dioxide (40-20%) and can be
used as fuel for the generation of thermal and/or electrical energy. There is a maximum limit of
methane that can be generated from the removal of organic matter (as COD) and it is 0.35 m3
CH4/kg COD removed.
However, the slowness of the anaerobic process makes it necessary to work with high
residence times in the reactors, which is why large reactors are used.
This anaerobic digestion process can be applied to any biodegradable organic waste,
hence its interest since it is viable to integrate into industrial plants to generate fuel from wet
waste.

2. Stages of anaerobic organic matter digestion treatment

This biological process is quite complex since it is carried out by the action of different
trophic groups of bacteria. Figure 1 shows the typical stages of the process, which are as follows:

(a) Hydrolysis. Complex and insoluble organic matter is transformed by extracellular

enzymes excreted by bacteria. Most simple and soluble substances are formed.

(b) Acidogenesis. Simple organic substances are used as substrates by a set of bacteria
known as acidogenic, because they produce mainly short-chain organic acids from soluble
polymers. Other products are H2, CO2 and alcohols.

(c) Acetogenesis. The products of the previous phase are metabolized by acetogenic
bacteria, which result in the formation of acetic acid.

(d) Methanogenesis. Finally, biogas (mainly methane and carbon dioxide) is formed
from acetic acid (70%) and from H2+CO2 (30%). The bacteria that act at this stage are known as
methanogens and are the most sensitive to environmental conditions (pH, temperature). They
also suffer processes of inhibition and are those of slower growth; therefore they are the limiters
of the global process, or also it is said that they are the controllers of the speed of elimination
of soluble organic matter. This last phase is the most important, as it is here that the reduction
of the organic matter content of the waste and the generation of the product of energy interest
really takes place. In the previous phases only transformations from more complex forms to
simpler ones have taken place.

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Soluble and insoluble complex substances

(Carbohydrates, fats, proteins)

Hydrolysis

Simpler and more soluble substances

Acidogenesis

H2 CO2 Organic acids Acetic Acid Alcohols

Acetogenesis
Acetic Acid

Methanogenesis

Biogas

Figure 1: Stages of the anaerobic digestion process of organic matter

Factors influencing the anaerobic digestion process

The main variables that affect the process are:

- Temperature (>35ºC very favourable)

- pH (close to 7)
- availability of nutrients (N, P, S, others).
- the mixture is also very important to avoid the formation of
microenvironments and with it the appearance of processes of inhibition of
the bacterial metabolism.

3. Production of fuels from biomass

Biomass, such as sludge or sewage sludge, can and should be considered a renewable
energy source, since it is viable to be transformed to produce electricity, heat and fuel for
transport.
In the European society envisaged in Horizon 2020, solid waste and sludge generated in
the treatment of wastewater from different sources will be managed in an integrated system
where waste can be recycled or purified for various uses. The main objective pursued in Europe
is the progressive reduction of the quantity of waste and, in the case of solid waste, to reduce
considerably those deposited in landfills, which would lead to an increase in the percentage of
recycled organic material.
Figure 2 illustrates how solid waste from different sources and sewage sludge can be
managed through an integrated system. By means of this scheme, different sectors can be
connected to achieve efficient energy use.
Special attention should be paid to the anaerobic digestion process, where
biodegradable waste from various sources can be transformed into biogas, allowing it to be used
for different purposes, such as heating processes and electricity generation.

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Figure 2: Scheme of an integrated waste management system for energy production

Biogas in the form of compressed gas such as liquid, after improvement, can be used as
fuel for vehicles, and can therefore replace considerable amounts of fossil fuels.
In addition to biogas, there is another final product of anaerobic digestion called
digestate. The digestate is the fraction of solids or semisolids from the original input that remains
in the digesters and is not processed by bacteria. This properly treated fraction can be used as a
biofertilizer for the agricultural sector or as a soil conditioner for industrial purposes.
On the other hand, biogas can be easily integrated wherever there is natural gas
infrastructure, which increases the use of biogas, including for transport purposes. But also, the
production and use of biogas can function as an isolated solution for the transformation of waste
into energy in cities without gas infrastructure, such as the production of heat and fuel for
vehicles.
Biomass of plant origin can be used for biogas production. This biomass can come from
natural biomass, produced by natural ecosystems, from residual biomass, generated as waste in
the production processes of the agricultural, forestry and industrial sectors and from biomass
produced from energy crops.
Lignocellulosic biomass of vegetable origin is made up of plant tissues whose cells have
a cell wall made up of a network of cellulose microfibrils forming layers covered with
hemicellulose on which lignin is deposited (Figure 3). This cellular wall allows to create and
support the structure of the plants to capture the solar radiation, giving them a greater
mechanical resistance and to possible pathogens. This is why it is so difficult to use them to
obtain compounds derived from biomass. Pre-treatment methods are required to access them,

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but none of available techniques is capable of completely isolating each component without
partially modifying it or degrading it.

Figure 3: Structure of plant tissues.

For the use of vegetable waste with biogas production, it is necessary several pre-
treatments mechanical (grinding), chemical (hydrolysis) and/or enzymatic (hydrolysis) to
facilitate the degradation of cellulose and hemicellulose. Enzymatic hydrolysis is an interesting
process, due to mild operational condition comparing the extreme conditions applied for
chemical processes (treatment with acids or concentrated bases and high temperature).
In enzymatic hydrolysis, it requires the action of different types of enzymes that are
capable of releasing glucose, easy to metabolize by bacteria. However, there are no bacteria
that have these enzymes in sufficient quantities to make the process efficient in a single
fermentative step along with the generation of biogas, therefore, enzymatic hydrolysis should
be performed as a pre-treatment.

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4. Objective of laboratory practice

We will work with a mixture of residues: vegetable residues submitted to a pre-

treatment of crushing + enzymatic hydrolysis and sludge resulting from the biological
process of wastewater treatment. Both residues will be given to the students, who will
have to carry out the indicated procedures to reach the following objectives:

a) Determination of the feasibility of using a waste for the generation of biogas.

b) Application of chromatographic methods to determine the methane content of the
biogas produced and the concentration of volatile fatty acids in the digestate.

5. Materials and procedure

MATERIALS
- Glass bottles for anaerobic cultures
- Pipettes
- pH meter
- Incubator
- Gas chromatograph with TCD detector and GC coupled to FID detector
- Gas sampling syringe.

REAGENTS
- Waste
- Solution NaOH and HCl 0.10 N.

PROCEDURE
- Transfer 20 mL from the residue to an anaerobic culture bottle.
- Add 2 mL of anaerobic sludge.
- Bubbles with nitrogen for 2 min to ensure anaerobic conditions.
- Close the bottle and incubated at 37ºC with a disposable syringe on the top.
- At the end of the incubation period, take 0.5 mL of biogas with a gas-sampling syringe.
Inject the gas into the sampling port of CG –TCD to analyze the percentage of methane
and carbon dioxide of the biogas generated. Measure the volume of gas reading the
syringe plus the head space of the culture bottle.
- Take 2 mL of the liquid from the incubation bottle and filter with a 0.45 µm membrane.
Introduced this into a vial (0.5 mL minimum) to measure the fatty acids by GC-FID.

Taking as volume the free space of the bottle plus the reading of the syringe and using the
calibration lines indicated below, estimate the potential generation of energy from the
incubated residue, taking as valid the relation for the calorific content of the biogas: 55.5 kJ/g of
methane. Express the potential generation in kJ/m3 of waste.

Injection volume of gaseous sample = 0.5 mL.

Concentration of each gas in mmol/L at 25 ºC and 1 atm.
Calibrated straight CH4: Peak area 1 = 47754.32 x Concentration - 1810.06 r2= 0.995
Straight calibrated CO2: Peak area 2 =9528.70 x Concentration - 1405.02 r2= 0.998