Extended Abstract FINAL

1
Anaerobic digestion monitoring under high ammonia concentrations – A
Case Study
Isabel de Sousa Baeta Paixão
Master Degree in Biological Engineering, Instituto Superior Técnico, Lisbon, Portugal
Tratolixo E.I.M., Anaerobic Digestion Plant, Mafra, Portugal
June 2016
Abstract
Anaerobic Digestion (AD) is a microbiological process that provides an answer to two of the most important
problems of the XXI century: clean energy production and sustainable waste management. The AD process at Tratolixo
plant, in Mafra, transforms the biodegradable organic matter present in the pre-sorted municipal solid waste plus source
collected biodegradable waste into biogas, under mesophilic regime, in three parallel digesters with a working volume of
3150m3
/each. The objective of this work was to monitor and analyse of the most important operational parameters and
waste degradation inhibitors at the AD Plant process, in an effort to understand their influence in biogas production and
quality, especially regarding ammonia concentrations. This parameter was measured by direct Kjeldahl distillation method,
in order to assess also the reliability of the methods used in the plant. Main results highlight that an average biogas
productivity of 600 Nm3
/ t VS is being achieved at a feeding loading rate of 7.2 t VS/m3
.d. Waste feeding TS content average
is 50% of which 35% are VS. The process depends mostly on waste load and composition, available alkalinity and pH as
well as feeding and temperature selected regimes. Ammonia nitrogen even at 4±1g/L was not inhibiting methanogens
mainly due to operating temperature and pH. AD plant has been successful in obtaining a good biogas yield and quality
(57% CH4). Liquid effluent is sent to an in house WWTP with a soluble COD content of 25g/L, which includes VFA ranging
from 2-4g/L, and ammonia nitrogen content within 2-4g/L.
Keywords: Anaerobic Digestion, Ammonia Inhibition, Ammonia Quantification, COD, AD Monitoring
1. Introduction
Fossil fuels have been powering the world’s
economy since the Industrial Revolution. However, being a
finite resource, prices have been suffering severe increases,
following the tendency of demand. The burning of fossil fuels
have been accounted as the main cause for Greenhouse
Gas Emissions (GHG), leading to global warming and
climate change, which have been in the spotlight recently
due to increased public and political awareness regarding
environmental issues.
As population increases and searches for a higher
standard of living, the tendency is to consume more goods
(Tchobanoglous G. K., 2002). Consequently, Municipal Solid
Waste (MSW) production has been growing severely. Until
recently, the most widely spread method of MSW disposal
was landfilling, but despite being the easiest and least
expensive way for disposal, many environmental risks and
constraints are associated with it, such as soil pollution and
GHG emissions. In addition, as a significant portion of waste
can be reused and recycled, by promoting these practices
we are contributing to minimize the amount of waste
accumulated, and in need of disposal, while simultaneously
turning waste materials into resources, lowering
environmental impacts and reducing the amount of energy
consumed in making new goods. This is the basis for the
Circular Economy Strategy.
Anaerobic Digestion (AD) is a process by which the
Organic Fraction of Municipal Solid Waste (OFMSW) is
decomposed by microorganisms, resulting in biogas with
high energy potential, and a nutrient rich digestate that can
be used as a soil conditioner. OFMSW is the largest fraction
of MSW (accounting from 35% to 50% (Castro, 2015)), and
has been recognized for several decades as a valuable
resource due to its potential of conversion into useful sub-
products. While producing added-value products, such as
biogas which can be used to generate clean energy, from
materials that would be otherwise be thrown away, the
process allows for a carbon neutral cycle (Vögeli, 2014), also
reducing the amount of waste sent to landfill and its
subsequent GHG emissions, complying with European goals
and legislation.
Biogas is generally composed of 48-65% of
Methane (CH4), 36-41% of Carbon Dioxide (CO2), about 17%
of Nitrogen, and traces of O2, H2S and other gases (Khalid,
2011). Biogas can be produced from energy crops, such as
maize, or biodegradable wastes, including sewage and food
waste. Different sources lead to different specific
compositions. Its yield is affected by several factors. Europe
had almost 8 million tons of organic treatment capacity in 244
plants, accounting for 25% of Europe’s biological treatment
(Adekunle, 2015).
1.1. The AD Process
1.1.1. Hydrolysis
In this step, organic polymers and other insoluble
organic molecules are broken down into smaller soluble
compounds by strict anaerobes and facultative bacteria.
Nitrogen plays an important role in AD, being necessary for
the formation of new biomass and ammonia, which is
released in this step and is a major contributor to pH

2
stabilization in the reactor, despite its inhibitory potential
which occur at high concentrations (Fricke, 2007).
1.1.2. Acidogenesis
In this step, the components previously formed
suffer further degradation by facultative and obligatory
anaerobic bacteria (Adekunle, 2015), being converted into
short-chain volatile fatty acids (VFA), from C1 to C5, which
highly inhibit the AD process beyond a certain concentration.
1.1.3. Acetogenesis
In this step, products previously produced that
cannot be directly converted into CH4, such as VFA, are
converted by homoacetogenic bacteria into hydrogen and
CO2, to be further used as substrates by the methanogenetic
bacteria. This conversion of VFA is of great importance,
given that when accumulation occurs the process suffers
severe inhibitory effects. From a thermodynamics point of
view, these acetogenic reactions are endergonic.
However, acetogenic bacteria are obligatory H2
producers, and constantly reduce exergonic Hydrogen (H2)
to CO2 and acetic acid. Therefore, the acetate formation from
VFA is thermodynamically viable solely under a very low H2
partial pressure (Deublein, 2008). The acetogens therefore
rely on a symbiosis with methanogens, which can only
survive under high H2 partial pressure. They remove the
products formed by acetogens from the medium, keeping the
pressure low enough to suit acetogens (Adekunle, 2015),
(Deublein, 2008).
1.1.4. Methanogenesis
In this step, CH4 and CO2, which are the primary
components of biogas, are produced by methanogens under
strict anaerobic conditions. H2 is removed from the substrate
and its partial pressure in the medium is kept low, which
allows the conversion reactions of VFA in the previous step,
minimizing its accumulation and its related inhibitory effects.
1.2. Operational and Control Parameters
Several parameters influence the AD process. The most
important are VFA concentration, pH, alkalinity, temperature,
ammonia concentrations, organic load and biogas quality
and production.
1.2.1. Volatile Fatty Acids (VFA)
VFA act as a substrate for methanogenic bacteria
and are essential to the process. VFA concentration in
digesters depend on the digester feed, and generally range
between 200-2000 mg/L acetate equivalent (Mata-Alvarez,
2003). However, more important than the real VFA value is
a sudden and steady increase in the digester’s effluent.
Accumulation causes microbial stress at increased
concentrations, especially in their dissociated form,
penetrating inside the cellular membrane as lipophilics,
denaturating cell proteins and causing severe problems
(Deublein, 2008), (Di Berardino, 2006). Labatut and Gooch
stated that for concentrations between 1500 and 2000 mg/L
acetate equivalent, biogas production suffers inhibitory
effects. (Labatut, 2012). McCarty and McKinney proposed a
VFA concentration under 2000 mg/L acetate equivalent and
a pH of about 7.5 as optimal process conditions (McCarty,
1964). The inhibitory effect is intensified by pH decreases.
Higher concentrations are also related to peaks in
loading. If too much VFA is fed to the digester within a short
period of time, inhibitory acidification takes place. Therefore,
increases in load should be preferably performed in small
increments rather than suddenly, providing enough time to
the microorganisms to acclimate themselves.
1.2.2. Alkalinity and pH
Methanogens are extremely sensitive to acid
accumulation and consequently to pH variations. Some
studies state that the optimal pH value for anaerobic
digestion is between 5.5 and 8.5 (RISE-AT, 1998). Lay et al
stated that methanogenesis only occurs at high-rate when
pH is neutral, demonstrating that CH4 rate production in high
moisture feeds (90-96%) is optimal at pH 6.8, functional at
pH between 6.6 and 7.8 and failure occurs for pH values
lower than 6.1 or higher than 8.3 (Lay, 1997). Its growth rate
decreases severely for pH levels below 6.6. Regarding
acetogens, the optimal pH level is 7, while for acidogens is
about 6 (Schön, 2009). pH is also related to NH3. An increase
in pH results in increased NH3 toxicity, due to a shift in the
NH3/NH4
+
equilibrium, favoring NH3 which is more toxic
(Chen, 2008). In addition, a decrease in pH leads to an
increase in VFA accumulation, leading again to a decrease
in NH3. Alkalinity is the medium’s capacity to resist changes
in pH, caused by increases in VFA, and is usually expressed
in terms of CaCO3. Typical values in digesters range
between 2000-4000 mg/L CaCO3. An unbalanced system
with fast acidification and slow methanogenesis may result
in a substantial accumulation of VFA and a severe decrease
in pH, therefore needing a greater amount of buffer. Alkalinity
variations occurs faster compared to pH, meaning that when
a variation in pH is detected, the buffering capacity (alkalinity)
of the system is already lost (Mata-Alvarez, 2003)
VFA and alkalinity concentrations show fast
variations when the system is upset, VFA tending to increase
while alkalinity decreases. Therefore, the ratio between
these two parameters is a valuable tool for stability
assessment. Appels et al stated that a molar ratio of at least
1.1:1 of alkalinity/VFA should be maintained for stable and
well-buffered digestion (Appels, 2008), while Mata-Alvarez et
al suggest a ratio of 0.3 (Mata-Alvarez, 2003).
1.2.3. Ammonia (NH3)
NH3 results from the anaerobic degradation of
nitrogen compounds. In general, concentrations below
200mg/L are beneficial to the process, since nitrogen is an
essential nutrient. In addition, it ensures sufficient buffer
capacity, increasing process stability (Rajagopal, 2013).
Several mechanisms regarding NH3 have been proposed,
such as changes in intracellular pH, increase of maintenance
energy requirements and inhibition of enzymes (Chen,
2008). NH3 in high concentrations has a powerful inhibitory
effect. It exists in the form of Free Ammonia (FA) and
(Ammonium) NH4
+
, and together they are the main forms of
inorganic ammonia in solution, in a pH dependent
equilibrium. High pH and temperature values shift the
equilibrium between NH3/NH4
+
towards the formation of FA
(Deublein, 2008).
Membrane-permeable FA has been identified as
the main inhibition cause (De Baere L. D., 1984). Associated
with NH3 instability is VFA accumulation, which, in turn, leads
to a decrease in pH, and thereby a decrease in FA (Chen,
2008). This relationship between VFA, pH and ammonia lead

3
to an “inhibited steady state”, a condition where the process
is stable but with low CH4 yields (Chen, 2008). Studies show
that methanogenic bacteria are the most sensitive to high
values of FA (Deublein, 2008). Loster and Lettinga showed
that as NH3 concentrations increased, methanogenic
bacteria lost about half of its activity, while acidogenic
population were hardly affected (Koster, 1988), (Chen,
2008). Some stains showed severe inhibition in
concentrations in the order of 4 g/L, while others showed
resistance up to 10g/L (Chen, 2008). Some studies relate
ammonia inhibition to Total Ammonia Nitrogen (TAN), which
is a combination of FA and NH4
+
. McCarty stated that when
TAN concentration exceeds 3000mg/L the process is
inhibited at any pH (McCarty, 1964). Calli et al and Labatut
reported inhibitory effects above 1500 mg/L for pH values
higher than 7.4 (Calli, 2005), while Kroeker et al reported
50% CH4 production for TAN ranging 1.7-14g/L (Kroeker,
1979), (Koster, 1988).
Methanogenic organisms can adapt themselves to
various environmental conditions, if given enough time to
adjust (Chen, 2008). Once adapted, they retain viability at
concentrations far beyond the initial inhibitory concentrations
(Kroeker, 1979). Koster and Lettinga reported that while
unacclimated methanogens failed to produce methane for
NH3 concentrations ranging 1.9-2g/L, after adaptation they
tolerated up to 11g/L (Koster, 1988). Adaptation to ammonia
concentrations above 5g/L have been reported in manure-
feeding systems (Labatut, 2012), (Calli, 2005). Other studies
state levels as high as 8-9g/L of TAN with no significant
decrease in CH4 production after acclimation.
1.2.4. Temperature
There are 3 ranges of temperature considered
optimal for digestion, and microbial populations can be
separated into categories: psychrophiles (<20ºC),
mesophiles (20-45ºC) and thermophiles (>45ºC) (Alves,
2007). Methanogenic bacteria present optimal grow in the
mesophilic range between 30-38ºC and in the thermophilic
range between 49-47 (Alves, 2007). An increase in
temperature usually has a positive effect on the metabolic
rate, however it results in higher FA. Studies show that high
concentrations of FA inhibit thermophilic digestions more
severely that mesophilic digestions (Wang, 2014), while
others state that thermophilic flora tolerates FA twice as
much when compared to mesophilic flora (Chen, 2008).
Gallert et al stated that under mesophilic conditions, FA
becomes inhibitory in concentrations ranging 80-150 mg/L,
at a pH of 7.5. However, for thermophilic conditions and FA
concentration of 620 mg/L, biogas yield showed a decrease
in 21% (Gallert, 2014). Wang et al noticed that increased
temperature resulted in increased pH and CH4 potential
(Wang, 2014).
1.2.5. Feed Characteristics
1.2.5.1. Total Solids (TS)
TS highly influences biogas production efficiency.
Wet AD (10-15% solids) leads to higher costs and reactor
volumes compared to dry AD (22-40% solids). Duan et al
stated that high-solids systems could reach higher CH4
production rates compared with low-solids systems, at the
same retention time (Duan, 2012). Abbassi-Guendouz et al
showed that CH4 production decreased with TS increasing
from 10 to 25% (Abbassi-Guendouz, 2012), while Yi et al
obtained an increase in production for higher TS, in food
waste (Yi, 2014), which points to the importance of substrate
type and its biodegradability.
1.2.5.2. Volatile Solids
Kayhaninan stated that VS matter provides a good
estimation on the biodegradability of the waste (Kayhanian,
1995). The extent of stabilization depends on the system and
the substrate’s physicochemical characteristics, ranging
from 30-42% for manure-only digesters (Labatut, 2012).
Residues with high VS contents and low non-biodegradable
matter (such as lignin) are best suited for AD (Verma, 2002).
1.2.6. Organic Loading Rate (OLR)
ORL measures the biological conversion of the
system, and is expressed as the ratio between feed and
reactor volume, in kg VS/m3
.day or COD/m3
.day (Verma,
2002), (TRATOLIXO E.I.M., 2008). Usually, as OLR
increases so does bacterial activity and biogas production,
as long as the increase is performed sufficiently low in order
to allow adaptation of microorganisms (TRATOLIXO E.I.M.,
2008). Higher than a certain level, which is the maximum
OLR the system can receive, VFA accumulation occurs due
to acidification, accompanied by a decrease in pH and biogas
yield.
1.2.7. Biogas content, Production Rate and Productivity
CH4 content is a good process indicator, affected by
all other parameters and inhibitors. In a well-operated
digester treating manure, CH4 content ranges between 58-
65%. Production is the most important parameter in
monitorization. Higher productions lead to higher energy
generation. Productivity is expressed in Nm3
/t, and is usually
considered an average. Usually, CH4 content, production
and productivity are fairly stable over time and, therefore, are
an important indicators of process disturbance. However,
neither production nor content alone can give the necessary
information to perform corrections to the process in time, in
case of failure.
1.3. The Anaerobic Digestion Plant (ADP) of TRATOLIXO
TRATOLIXO is an inter-municipal company,
responsible for the treatment, final deposition, re-use and
recycling, trading of transformed materials and other
services regarding waste, operating according to sustainable
principles and national and international legislation. The ADP
of Abrunheira, Mafra, with a biological treatment capacity of
75.000 tons of organic waste per year, allows the production
of 20.000 tons of compost, as well as biogas, resulting in 22.8
GWh of electrical energy injected in the national electrical
grid in 2015. TRATOLIXO’s ADP has been successful in
obtaining a good yield in biogas, despite its amount of
ammonia in the digesters, whose inhibitory effect does not
appear to have much influence in the process.
The operating parameters selected to provide
information about the biological processes stability are
biogas quality (% CH4), pH, VFA and VFA/Alkalinity. Hence,
set-points for each parameter were established, accordingly
to process optimal, alarm and critical states (green, yellow
and red), as described in table 1 (TRATOLIXO E.I.M., 2008).
Ammonia concentrations are also included, given the insight
valuable insight it provides, regarding inhibition.

4
Table 1 - Operating parameters and set-points according to
process optimal, alarm and critical states.
PARAMETER OPTIMAL ALARM CRITICAL
Quality (% CH4) >49 45-49 <45
VFA/Alkalinity
(acetate/CaCO3)
<0.6 0.6-1 >1
VFA (g/L acetate) <8 8-12 >12
pH >7.5 7-7.5 <7
NH3 (g/L) <5 5-6 >6
1.4. Aim of Study
This study had three objectives. The first was a real
experience in an ADP. To better understand how the process
runs, the AD process was followed during 15 weeks, which
involved all process indicators, from sample collection to
feeding substrate and digestate characterization. While at
the ADP, a fully laboratory work was undertaken, including
content quantification of the main potential inhibitors, e.g.
VFA and ammonia. The obtained results, complemented
with other data gently supplied by Tratolixo, were interpreted,
as well as their impact on the process performance regarding
biogas production rate and quality (% CH4).
The second objective relates to NH3 content,
especially in terms of operating test reliability. NH3
measurement analytical techniques used at the ADP were
compared to digestate analysis of Kjeldahl nitrogen, or total
organic nitrogen, assessing the biodegradability extent of
nitrogenous compounds and NH3 content by alkaline steam
distillation, followed by titration. Also, given that a quarterly
analysis performed by an external laboratory usually
measures NH3 in the press liquid fraction instead of directly
in the digestate, a comparison between substrates was
performed regarding NH3 measurements, in an effort to
identify any differences.
The third objective was to determine total and
soluble COD for the digestate and tank samples, in order to
characterize their variability, given that COD is only
determined once every quarter, and only for digestate
samples.
2. Materials and Methods
MSW, digestate, liquid effluents, organic load and
biogas production and quality were closely monitored during
a period of 15 weeks at the ADP. MSW, digestate and
effluent samples were collected every day, and
measurements of solid content were performed in all.
Digestate sample was collected after 20 minutes of
recirculation, and the following parameters were measured:
VFA, alkalinity, pH, temperature and ammonia. Ammonia
was measured at the ADP by the semi-quantitative
QUANTOFIX®
test once a week.
Tank samples were collected in the morning, plus a
second sample of tank 5 (which receives the effluent from
the centrifuges and sends it to the in-house WWTP) in the
afternoon, due to significant differences in TS content. Total
Dissolved Solids (TDS), Total Suspended Solids (TSS) and
Settleable Solids (S/S) were also measured for tank 5.
Biogas production was accounted as the sum of biogas used
by the motor-generators and burner, every day, and quality
was measured as the percentage of CH4 before filtering and
desulphurization, every day.
A separate set of digestate and tank samples were
recovered once a week, between week 4 and week 9, and
taken to the Environmental and Ecoprocess Engineering
Research Group, at Instituto Superior Técnico, in order to
determine total and soluble COD, as well as total organic
nitrogen by Kjeldahl Digestion and NH3 by alkaline steam
distillation.
2.1. Solid Content
All samples were evaporated and dried to constant
weight at 105º. VS content was determined by the weight
loss after igniting the dry samples at 550ºC. Glass and inert
materials under 0.5 mm in the ash residue were also
weighted after screening. The grab samples taken from tank
5 were centrifuged at 4200 rpm for 10 minutes. TDS and TSS
were measured by weight loss of the pellet and supernatant
fractions, respectively. S/S content was determined by
measuring the volume of settled solids after 24 hours in an
Imhoff cone. These procedures were performed according to
the ADP’s biological manual (TRATOLIXO E.I.M., 2008)
2.2. Chemical Oxygen Demand (COD)
The COD test was performed according to the
Standard Methods for the Examination of Water and
Wastewater (SMEWW), section 5220 B (APHA, 2005). The
procedure consisted in using a TR 420 Spectroquant
Digester from Merck, adding to each tube 1.5ml of sample,
1ml of potassium dichromate solution and 2ml of sulphuric
acid reagent. Each sample was tested in triplicate and left in
the digester for 2.5 hours, at 150ºC. The blank assays were
prepared similarly, using distilled water instead of sample.
The sample’s organic and inorganic matter suffers oxidation
by an excess of dichromate, in the presence of a catalyst. A
titration was performed in order to determine the excess
dichromate using the FAS solution 0.0125M as titrant. The
turning point of the titration occurs when the solution turns
from light blue to bright orange.
2.3. Ammonia Analysis
NH3 measurements were performed using the
Quantofix® kit (from Macherey-Nagel). A sample of the
digestate effluent was centrifuged at 4200 rpm for 10
minutes, with 10% (w/w) flocculant. 1 ml of supernatant was
collected and diluted 1:100. 5 ml of solution were placed in
the measurement vessel and added 10 drops of the
ammonia reagent. A test strip was dipped into the solution
and its colour compared to the colour scale of the kit (0, 10,
25, 50, 100, 200 and 400 mg NH4
+
/L.) The result is calculated
by equation 4, where DF stands for the dilution factor used.
Also, it is necessary to take into account the percentage of
flocculant used in the sample preparation and adjust the
result. The measuring range goes from 10-400 mg/L NH4
+
visually, and 10-350 mg/L NH4
+
reflectometrically.
𝑁𝐻4
+(𝑚𝑔. 𝐿−1) 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝑁𝐻4
+(𝑚𝑔. 𝐿−1) 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 × 𝐷𝐹 Eq. 1
2.3.1. Kjeldahl Digestion
This method is used for the quantitative
determination of organic nitrogen. Samples of the digestate
were collected in the morning and centrifuged at 4200 rpm
for 10 minutes, with 10% (w/w) flocculant. 5 ml of supernatant
were collected and diluted 1:20.
In the first week, samples were recovered and
digested by the Kjeldahl method, according to the SMEWW,
section 4500-Norg B (APHA, 2005). The method consists in
three steps: digestion, a steam distillation and a titration.
Only the distillation and titration steps were performed from

5
week 2 forward (SMEWW, section 4500-NH3 B (APHA,
2005)).
The digestion step is performed by adding sulphuric
acid (to heat the sample and digest it by oxidation),
potassium sulphate (to rise the boiling point) and a catalyst.
The heating was performed as described in table 2. Digestion
converts the nitrogen present in the sample into NH3 (other
than that which is in the form of nitrates or nitrites), and
organic matter into CO2 and H2O (McClements, 2016).
Ammonia is in the form of ammonium ion (NH4
+
), and binds
itself with the sulphate ion (SO4
2-
) remaining in solution.
Table 2 – Kjeldahl Digestion step heating scheme
Temperature (ºC) Time (min)
110 90
120 30
250 30
385 60
Cooling 60
This step was not performed from week 2 forward.
Regarding digestate samples, 6ml were added to 6ml of
concentrated sulphuric acid and 150 ml of ultrapure water
(milli-Q). The mixtures were made alkaline by addition of
sodium hydroxide, which converts the ammonium sulphate
into NH3 gas, according to equation 2 (McClements, 2016).
(𝑁𝐻4)2 𝑆𝑂4 + 2𝑁𝑎𝑂𝐻 → 2𝑁𝐻3 + 2𝐻2 𝑂 + 𝑁𝑎2 𝑆𝑂4 Eq. 2
NH3 was also quantified in samples collected from
tanks by alkaline steam distillation, followed by titration.
Flasks containing 1g of sample were placed into a distillation
unit (K-355, BUCHI); pH was raised up to 12 by adding
NaOH (32% w/v) and steam distilled for 10 minutes. The NH3
gas is released from the solution, moving into a receiving
flask containing 50ml of the indicative solution of boric acid
(prepared as described in Appendix II), which by lowering pH
converts NH3 gas into the ammonium ion and the boric acid
into the borate ion, according to equation 3 (McClements,
2016). In the presence of NH3, the boric acid solution turns
its colour from pale lavender to green.
𝑁𝐻3 + 𝐻3 𝐵𝑂3 → 𝑁𝐻4
+
+ 𝐻2 𝐵𝑂3
−
Eq. 3
In the titration step, the ammonium borate is titrated
with sulphuric acid 0.025N, until the solution turns lavender
again. The concentration of H+
required to reach the end-
point is equivalent to the concentration of nitrogen initially
present in the sample. The nitrogen concentration can be
determined by equation 4, where A is the titrant volume
titrated for the sample, B the titrant volume titrated for blank,
0.025 is for the titrant molarity and 14.01 is the nitrogen
molecular weight (g/mol).
𝑁𝐻3 − N(𝑚𝑔/𝐿 𝑜𝑟 𝑚𝑔/𝑘𝑔) =
(𝐴−𝐵)× 0.025×14.01
𝑚𝑙 𝑜𝑟 𝑤𝑡 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒
Eq. 4
Despite being the most widely used method due to
its universality, high precision and good reproducibility, it
does not give an absolute result since some of the nitrogen
present in the sample is in the form of proteins and other
compounds. Therefore, when using this method, the total
Kjeldahl nitrogen (TKN) results need to be considered as the
sum of NH3, nitrogen and ammonium.
2.4. Volatile Fatty Acids (VFA)
The digester’s samples were centrifuged at 4200
rpm for 10 minutes, with 10% (w/w) flocculant. 5 ml of
supernatant were collected and diluted 1:20. The solution
was then titrated with agitation, with sulphuric acid 0.1N, to
pH 5.1 and pH 3.5, and the alkalinity and VFA values were
determined by the following equations. These procedures
were performed according to the ADP’s biological manual,
based on the assumption that acid titration volume correlates
with the change in equilibrium of VFA and carbonate
alkalinity (Lützhoft, 2014), that both VFA and bicarbonates
are water soluble, and no other molecules interfere in the
measurements (TRATOLIXO E.I.M., 2008), (Lützhoft, 2014).
[𝐻2 𝑆𝑂4] 𝑝𝐻=5.1 =
𝑉1×𝑁
𝑉𝑆
Eq. 5
[𝐻2 𝑆𝑂4] 𝑝𝐻=3.5 =
𝑉2×𝑁
𝑉𝑆
Eq. 6
𝐴1 =
10−5.1−10−𝑝𝐻 𝑖
𝐾1+10−5.1 Eq. 7
𝐴2 =
10−3.5−10−𝑝𝐻 𝑖
𝐾1+10−3.5 Eq. 8
𝐵1 =
10−5.1−10−𝑝𝐻 𝑖
𝐾2+10−5.1 Eq. 9
𝐵2 =
10−3.5−10−𝑝𝐻 𝑖
𝐾2+10−3.5 Eq. 10
𝑉𝐹𝐴𝑖𝑜𝑛𝑖𝑧𝑒𝑑 =
([𝐻2 𝑆𝑂4] 𝑝𝐻=3.5 × 𝐵1) − ([𝐻2 𝑆𝑂4] 𝑝𝐻=5.1 × 𝐵2)
(𝐵1 × 𝐴2) − (𝐵2 × 𝐴1)
× 1000 (
𝑚𝑒𝑞
𝐿
)
Eq. 11
𝐴𝑙𝑘𝑎𝑙𝑖𝑛𝑖𝑡𝑦 =
([𝐻2 𝑆𝑂4] 𝑝𝐻=5.1 × 𝐴2) − ([𝐻2 𝑆𝑂4] 𝑝𝐻=3.5 × 𝐴1)
(𝐵1 × 𝐴2) − (𝐵2 × 𝐴1)
× 1000 (
𝑚𝑒𝑞
𝐿
)
Eq. 12
𝑇𝑜𝑡𝑎𝑙 𝑉𝐹𝐴 (𝑔 𝑎𝑐𝑒𝑡𝑎𝑡𝑒 𝑒𝑞. 𝐿−1
) =
𝑉𝐹𝐴𝑖𝑜𝑛𝑖𝑧𝑒𝑑 × 60
1000
Eq. 13
𝑇𝑜𝑡𝑎𝑙 𝐴𝑘𝑎𝑙𝑖𝑛𝑖𝑡𝑦 (𝑔 𝐶𝑎𝐶𝑂3 𝑒𝑞. 𝐿−1
) =
𝑉𝐹𝐴𝑖𝑜𝑛𝑖𝑧𝑒𝑑 × 60
1000
Eq. 14
Where:
Vs = sample volume,
V1 = titrant volume spent at pH 5.1,
V2 = titrant volume spent at pH 3.5
N = Acid normality (0.1)
K1 = 1.76x10-5
, the thermodynamic constant of VFA
K2 = 4.8x10-7
, the thermodynamic constant of bicarbonates
pHi = initial pH value, measured immediately before titration
2.5. Data Analysis
In order to understand performance and behaviour
of the process, all parameters were studied in a period of 15
weeks. Tables 3 and 4 present weekly operating conditions
regarding constraints.
Table 3 - Operating conditions at the ADP in the observed
period (15 weeks).
Week Date
dd/m
Operating conditions
1 03/1 No constraints observed
2 10/1 Equipment repair

6
Table 4 (continuation) - Operating conditions at the ADP in
the observed period (15 weeks).
Week Date
dd/m
Operating conditions
3. Results and Discussion
3.1. ADP Performance
3.1.1. Waste Feeding Characterization
Two types of waste are being processed at the
ADP, MSW and BW, figure A-I 1. On average, 250 tonnes of
waste is daily received, of which 30% is BW. Total solids
content and moisture of waste was almost constant and ca.
50%, in a weekly basis, figure A-I 2. Anaerobic digesters are
being operated with 30% TS and thus moisture is corrected
by recirculating liquid effluent from the centrifuges in the
dewatering stage. Inert content is quite high, which can
influence the HRT within digesters and equipment
performance, provoking pumps failure mainly due to
clogging. However, inert content is not unusual given the
typical quality of MSW. Glass and metal was also quite high,
ca. 30% of TS, interfering with digester walls integrity, screen
damaging, among other problems, associated with abrasion
phenomena.
3.1.2. Operating Parameters
Figure A-I 3 resumes MSW mass loading rate
during the observed period of time (15 weeks). Severe
variability can be noticed associated with daily operating
conditions. Waste (MSW and BW) as received is discharged
into a receiving pit, where homogenization is attempted prior
to digesters feeding. VS content of waste fed to each reactor
vary significantly due to the different amounts of type of
waste available, besides source generation rate, collection
route and season. In figure A-I 4, it is shown the VS content
of the feeding being noticeable the large variation within
weekdays. Moreover, it can be observed that two of the
reactors received substrate with same quality while a third
one was fed with waste with different composition. Thus, it
should be related to the digester’s feeding schedule. This
variability can lead to VFA accumulation in the system, which
will be discussed in more detail in the next sections. The daily
volumetric loading rate (VLR), expressed as kg VS/m3
.d,
figure A-I 5, is just a function of the amount of waste available
as digester’s working volumes remain almost constant
(estimated as 3150 m3
per digester, within the observed
period). Therefore, VLR follows daily and weekly variation
patterns according to weekday and season, averaging 7±2
kg VS/m3
.day.
3.1.3. Biogas Production and Quality
Daily biogas production pattern is similar to VFA
accumulation, figure A-I 6. It is noteworthy the minimum
values verified every Monday, associated with the lowest
values of VFA measured in the digestate. In this figure, VFA
accumulation in digester 2 is representative of all digesters.
On Saturday VFA are not monitored but it seems that VFA
start to diminish after Friday if it follows the same pattern
observed within the week. Two phenomena could contribute
to this behaviour such as the lack of feeding on Sunday and
different characteristics of the waste that is being fed on
Saturday.
Despite the low weekly production in week 5, that
was the week with less production variations. Weeks 4 and
10 showed higher variability. Average minimum weekly
production values were between 13.000 Nm3
in week 6 and
26.000 Nm3
, while average maximum weekly production
ranged between 22.000 Nm3
in week 5 and 41.000 Nm3
, in
week 10.
Analysing biogas productivity, as represented in
figure A-I 7, we can that it also presents variation within the
observed period. In some of the weeks, we can see minimum
values in the beginning of the week, and tendency to
increase towards the end of the week. This behaviour is
similar to the performance of the organic mass loading rate,
which is expected, given that biogas production is highly
influenced by load. Unexpected minimum values are also
related with periods of malfunctions and repairs in the
equipments, as described in table 4.
Figure A-I 8 represents biogas quality, quantified by
the content in CH4 expressed as percentage, which
averaged 57% in the observed period. Quality suffers
variations throughout the weeks, with a tendency to
decrease toward the end of the week. Increased quality
pattern tends to relate with a decrease in production.
3.1.4. Digestate Characterization
3.1.4.1. Solid Content
On average, VS content in the digestate was 35%
of TS while on feeding waste was around 50%, providing an
idea of the biodegradability of the waste, figure A-I 9.
The amount of VS removed was estimated as a
weekly average due to the lack of daily measurements of
inert content in the feeding and digestate, figure A-I 10. The
removal efficiency of VS was on average 50%, if outliers are
not taken into account.
3.1.4.2. Volatile Fatty Acids
Figure A-I 11 represent VFA content in the
digestate of the three digesters, and figure A-I 12 show
digester 2 in detail.
Minimum values were observed on the beginning of
the week (Monday or Tuesday) with a visible tendency to
increase by the end of the week (Thursday and Friday). VFA
peaks (accumulation) may be related with the increase in VS
of the waste loading, within the observed period.
We can also observe that the digestate presented
approximately a 50% reduction in VFA concentration over
the weekend. A change in feeding schedule (alternate
feeding days) or even a decrease in loading could be
alternatives, in an effort to make the best use of VFA amount
not consumed, or even a new smaller digester, especially to
receive digestate before it is send to the dewatering stage.

7
VFA values are a little high when compared to the
ones described in the literature review, however they all fall
within the ADP’s optimal state (green performance, in table
1). Protein content ability to absorb CO2 present (in biogas)
may have created an additional buffer effect which,
combined with the amount of alkalinity in the system, was
enough to sustain VFA variations. However, TAN is not being
quantified, and thus protein content is uncertain.
Moreover, since pH values were between 7.6-7.8,
VFA inhibition was not foreseen, even with 3.5 g acetate/L.
pH influence which will be further discussed in the next
section.
3.1.4.3. Alkalinity and pH
Peaks in VFA (figure A-I 12) relate to lower values
in Alkalinity, as expected. Figure A-I 13 shows alkalinity
variation in the digestate of DG2, which was considered
representative of the three digesters. In fact, analysing the
daily Alkalinity, we can observe the minimum values
occurring on Friday while maximum is verified on Monday.
pH values do not present severe variability, ranging
between 7.6-7.8, even with severe variations in VFA. This
could be explained by the system’s high buffer capacity,
provided by the amount of alkalinity, as shown in figure A-I
14. Also, as already discussed, VFA increases do not
present an inhibitory risk in these ranges of pH. Average
minimum pH values occur by the end of the week (Thursday
and Friday), in agreement with average minimum values in
Alkalinity, while maximum pH values occur in the beginning
of the week (Monday and Tuesday).
As described in the literature review,
methanogenesis was promoted by high concentrations of
bicarbonate alkalinity. Despite severe VFA variations, the
weekly biogas quality tended to be stable, following the
weekly tendency of alkalinity, figure A-I 15. The different
pattern found in the last three weeks can be due to an
increase of the OLR, which positively influenced biogas
quality, but also VFA concentration, which in turn promoted
a drop in alkalinity.
3.1.4.4. VFA/Alkalinity ratio
In the observed period, VFA/Alkalinity ratio has
suffered significant variations, figure A-I 16, but without major
disturbances in the process. The ratio value was always
lower than the ratios described in the literature review.
Minimum values tend to occur in the beginning of the week,
while maximum values occur in the end. Despite the
imbalance in the beginning of the week, the ratio
performance seemed fairly constant, between 0.17 and 0.24,
within the ADP’s optimal state range (Table 1), leading to the
conclusion that the system is well-buffered and capable on
enduring VFA variations without major disturbances.
3.1.4.5. Temperature
Temperature patterns in the three digesters are
similar, figure A-I 17, performing always in the mesophilic
range. Between weeks 5 to 7 a severe decrease in
temperature has occurred for all three digesters. In the same
period biogas production rocketed up. As this observation is
unique within the observed period, is difficult to relate this
fact with any inhibition effect around 40ºC comparatively to
the high performance observed at 39ºC. VFA also suffered
an increase but due to a stable value of alkalinity (and pH),
its inhibitory effects should have been minimal.
Its relationship with free ammonia inhibition should
be further investigated. For values close to 38ºC and a pH
range between 7.6-7.8, the amount of free ammonia in the
system is below 10%. Nonetheless, a more fine analysis
indicates that FA content is varying between 6 and 7.6%,
which denotes an increase of ca. 25% in FA content,
described in table 5.
Table 5 - Free Ammonia content, in percent, as a function of
pH and temperature (adapted from Thurston, 1979).
T (ºC)
pH
7.64 7.7 7.72
38.8 5.98 6.25
39.4 6.15 7
39.8 7.17 7.48
40 7.25 7.57
3.1.4.6. Ammonia
Figure A-I 18 shows the influence of ammonia in
biogas production (on the right) and in quality (on the left). As
previously stated in the literature review, ammonia beyond a
certain concentration inhibits methanogens and therefore a
decrease in biogas quality is expected. Ammonia was
measured at the ADP by the Quantofix® test, which will be
further analysed in the next sections, and despite a slight
decrease in quality in weeks 1 to 4, ammonia values were
equal in all weeks as both quality and production suffered
variations. The colour gradation of the Quantofix® test
(ranging visually from bright yellow to bright orange)
indicates progressive concentrations levels (0, 10, 25, 50,
100, 200 and 400 mg/L NH4
+
). Given this gradation scheme,
a visual measurement has a significant reading error, and
therefore there is a certain degree of uncertainty regarding
the ammonia measurements performed by this method.
3.1.4.7. COD Content
In all digesters, there was a decrease from weeks 4
to 7 in all digesters (for Total COD), and a subsequent
increase from week 8 to 9, as shown in figure A-I 19.
A fair amount of organic matter possibly
biodegradable could still be present, which could be digested
anaerobically before being discarded. Total COD could
present a high content in fibers, which are more difficult to
degrade, leading to its high values in all tanks. Lower COD
improve biogas efficiency and may facilitate solid removal by
settling.
3.1.5. Effluent Characterization
The final destination of the liquid effluent is an in-
house WWTP. It is received in tank 5 and characterized by
an ammonia content of ca. 3 g/L, a total COD content, around
50 g O2/L and, on average, a soluble COD content of 25 g
O2/L. In terms of solid content, is presents a TS content of
about 3.5 %, 70% of VS, 2.41% TDS and 0.34% TSS. 73.1%
of its solids settle after a period of 24h.
As the AD downstream processing are constituted
mainly by the dewatering step, all of these parameters were
also measured in the remaining effluents (tanks 1 to 4) in
order to understand their variations throughout the ADP
dewatering step.

8
3.1.5.1. Ammonia
In the dewatering step, ammonium nitrogen
decreases up to 50% as observed in figure A-I 20, which can
be explained by ammonia release into the building’s
atmosphere. The effluent sent to the WWTP contains about
2.5 g/L of ammonium nitrogen.
In addition to the weekly monitoring, another
ammonia test is performed every quarter, for quality
purposes. Due to method limitations, the substrate in which
they perform this measurements is the liquid fraction
collected from the presses (of the dewatering stage), and not
the digestate effluent. These results are usually a little lower
than the results measured at the ADP, ranging between 2.17
g/L and 5.20 g/L in the years 2015 and 2016. In an effort to
understand if this change in substrate had an impact on the
measurements, samples were also collected from the liquid
fraction of the presses in order to measure ammonia by the
Kjeldahl method, and values were compared to the digestate
effluent. Two samples were tested for two digesters and the
results are presented in figure A-I 21, DG1 on the left, and
DG3 on the right. pH values measured in situ during
sampling were the following: 7.7 for DG1 and 7.9 for the
press in week 6, 7.6 for DG3 and 7.9 for the press in week 7,
7.7 for DG1 and 7.9 for the press in week 8, and 7.7 for DG3
and 7.9 for the press in week 9. The liquid fraction is
extremely similar to the raw digestate since no treatment was
performed other than pressing.
The results do not differ very much for both
substrates for DG 1, while for DG 3 the pressed sample
showed higher ammonia concentrations, when compared to
the direct effluent. This result is merely indicative since only
two weeks were analysed for each sample, therefore more
studies should be performed in a wider period of time to fully
understand in which sample the results are more reliable.
3.1.5.2. COD Content
Tank 1 and 2 have the highest values of COD,
which is expected, as these tanks receive the effluents from
the presses and the sieves, respectively. COD does not
suffer extreme variations, except regarding tank 2 in week 8,
reaching a total COD of 206 gO2/L, figure A-I 22. COD for
tank 3 and 4 seem to have a similar behaviour, but reaching
higher values for tank 4 than for tank 3, figure A-I 23. Also,
these two tanks are the ones with the less constant tendency
when compared with the other tanks. Tank 5 behaved very
constantly, in the morning and in the afternoon.
3.1.5.3. Solid Content
Solid content was also measured in all dewatering
tanks. For tank 5, two separate samples were collected, one
in the morning and one in the afternoon, in order to measure
separately TS content. TDS, TSS and S/S were also
quantified in tank 5, in order to characterize this effluent
before being sent to the WWTP.
Figure A-I 24 illustrates TS and VS in tanks. Tank
1 and 2 have the highest values of TS, as it was also verified
regarding COD. Their TS content is fairly constant
throughout the weeks, always within project value range,
except for tank 1 in week 7, when it reached 19.1%, and tank
2 in week 12, when it reached 16%. TS contents of Tanks 3,
4 and 5, which receive effluents from three separate
centrifuges, were always within range. VS content in the
observed period was always below the limit values. Tanks 3
and 4 are the ones with the less constant tendency in TS (as
it was also observed regarding COD), and usually have high
TS contents, of about 11%. However, as their function is to
recirculate into the digesters to serve as dilution liquid, their
TS content need to be lower, about 9%. In order to achieve
that, both tank 3 and 4 are mixed with tank 5 effluent, which
has a TS content of about 4%, therefore obtaining the
desired content. VS content in the observed period was, as
observed for tank 1 and 2, always below the limit values.
In tank 5, which sends its effluent directly to the
WWTP, VS content was also below the limit values in the
observed period.
In terms of TS content for the morning and
afternoon samples, figure A-I 25, a 50% variation seems to
occur between the morning and afternoon samples, which
was expected, due to the washing cycles of equipment
(performed by night), which decreases TS content in the next
day’s morning samples. Therefore, the system could be
optimized with, for instance, an equalization basin.
Figure A-I 26 represents TSS and TDS contents as
well as S/S content, in the morning sample of tank 5. Since
the morning sample is the least concentrated sample of the
day, these values are underestimated.
TDS represents mainly dissolved salts, and ranged
between 1.97% for week 2 and 2.81% for week 12, with an
average of 2.41%, while TSS represents probably fibber
content, ranging between 0.15% in week 10 and 0.73% in
week 3, with an average of 0.34%.
S/S content was measured after 24 hours in an
Imhoff cone. This period of time was found to be appropriate
to properly read S/S, because the sample is extremely dark
and the reading is impossible to perform in the day of
collection. The objective is to closely monitor S/S values, in
an effort to minimize subsequent solid settling in the
equalization basin at the WWTP. We can observe that
suspended solids are fairly settleable in about 10% of its
volume in 24h, therefore eliminating about 50% of the COD
present on the sample. This effluent probably is not
compatible with the implementation of a gravity system at the
WWTP due to its high fermentation potential, which creates
gas bubbles, that when rising to the surface disturb
sedimentation.
3.2. Ammonia Quantification
In order to assess the reliability of the
measurements currently in use at the ADP, one-grab
samples of the digestate and effluents were collected,
between weeks 4 and 9, measured by the Quantofix® test
and compared with Kjeldahl digestion and steam distillation
measurements. One round of samples were submitted to
Kjeldahl digestion and then compared with the un-digested
results, as described in table 6.
Table 6 - Ammonia content (g/L) in the digestate by Kjeldahl
method. Comparison between digested samples and un-
digested samples.
Digester NH3-N (g/L)
Digested Un-digested
DG 1 3.63 3.60
DG 2 3.63 3.63
DG 3 3.59 3.63

9
Usually, if the measurements are not performed in
the same day as sampling, samples need to be preserved in
sulfuric acid, as to maintain a pH≤2, as described in the
literature (APHA, 2005). However, the optimization of the
methodology led to unforeseen delays in the measurements
and therefore all the samples were stored at 4ºC without
acidification, and so a certain degree of degradation need to
be taken into account.
Measurements performed by the Kjeldahl method
result in lower and more constant values, with an average of
3.7 ± 0.07 g/L for DG1, 4.0 ± 0.06 g/L for DG2 and 3.8 ± 0.09
g/L for DG3. The Quantofix ® method averaged 5.7 g/L, 5.5
g/L and 5.4 g/L, for DG1, DG2 and DG3, respectively,
possibly validating the hypothesis of visual error due to the
colour scheme of the Quantofix® test.
In conclusion, direct measurement of NH3-N by
distillation seems enough for ammonia control. Besides its
accuracy, the steam distillation step is relatively fast, taking
about 20 minutes to process each sample (10 minutes for the
analysis and another 10 minutes for cleaning), while the
digestion step takes more time (about 5 hours). However,
TAN quantification would be a step forward in the control of
the biodegradability of this complex substrate.
The investment to acquire the equipment would be
of about €10.000, however, given the accuracy and the
present difficulties in understanding the real ammonia
values, it could be a good alternative to the current
methodologies performed at the ADP. Also, it could be
interesting to validate the degree of protein degradation in
MSW, by performing a comparison between the amount of
N-organic compounds in MSW and in the digestate.
4. Conclusions and Future Perspectives
Process proved to be robust, highly buffered and
therefore capable of enduring VFA variations without major
disturbances in biogas production. The increased buffer
capacity denoted by pH prevented an eventual VFA toxicity
by accumulation during the week. Therefore, the reading of
pH without other parameters is not enough to ascertain
process stability.
The anaerobic digester’s are fed with 7.2 kg
VS/m3.
d, with an HRT of ca. 40 days, producing an average
of 600 Nm3
/t VS, leading to a 50% reduction in VS content,
as expected. This efficiency could be underestimated, since
most of the waste is unsorted waste, containing some plastic
that interferes with VS measures.
Quality behaved with an average of 57%, which is
within the expected range of values described in the
literature. The parameters that appear to have the most
influence in quality and production seem to be the feed and
mass load, which showed high variability throughout the
weeks, with a VS content of about 30%, leading to VFA
accumulation, which decreases over the weekend. Average
values of 3.5 g acetate/L in the digestate are reduced to 2.0
g/L. High inert content reduces retention time of the
biodegradable fraction and conducts to a low conversion
efficiency of VFA during the week. It is foreseen an
optimization of the process by reducing inert material
percentage or with the implementation of a new digester due
to the digestate’s high potential for a new digestion process,
in order to obtain extra CH4 production.
It was not observed any ammonia inhibition, in the
pH and temperature ranges of operation, probably due to the
presence of high-tolerant ammonia strains. All proteins
present seem to have been transformed, with no N-organic
compounds present in the digestate. It should be further
studied and taken into account protein and TAN in the
feeding and digestate, in order to determine protein
hydrolysis efficiency and its contribution to system buffer
capacity. Moreover, NH3-N content quantified by Kjeldahl
method showed a high discrepancy with operating values
taken with a commercial kit currently in use at the ADP.
Soluble COD can be attributed to the remaining
VFA and some non-degraded matter. Thus, the optimization
of the process seems critical to reduce the organic load of
the liquid effluents to be sent to the WWTP. This effluent also
presents a 50% variation in TS between the morning and the
afternoon, and the system could be improved with an
equalization basin. Solids are fairly settleable in 10% of the
volume, possibly not compatible with a gravity system at the
WWTP due to the high fermentation potential of the tank 5
effluent. However, it would be interesting to evaluate its BOD
content.
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Degree in Earth Resources Engineering. New York:
Columbia University.
VÖGELI, Y. L. (2014). Anaerobic Digestion of Biowaste in
Developing Countries: Practical Information and Case
Studies. Dübendorf, Switzerland: Swiss Federal Institute of
Aquatic Science and Technology (Eawag).
WANG, Q. K. (1999). Degradation of volatile fatty acids in
highly efficient anaerobic digestion. Biomass and Energy,
16:407-416.
WANG, X. L. (2014). Effects of Temperature and Carbon-
Nitrogen (C/N) Ratio on the Performance of Anaerobic Co-
Digestion of Dairy Manure, Chicken Manure and Rice Straw:
Focusing on Ammonia Inhibition. PLoS ONE, 9(5):e97265.
YI, J. D. (2014). Effect of Increasing Total Solids Contents on
Anaerobic Digestion of Food Waste under Mesophilic
Conditions: Performance and Microbial Characteristics
Analysis.PLoSONE,9(7):e102548.

11
APPENDIX
Figure A-I 1 - Characterization of the waste received at the ADP
Figure A-I 2 - TS and moisture (%, on the left) and TS content as VS, Inerts and Glass/metal (%, on the right) in the feed.
Figure A-I 3 - MSW mass loading rate (t/day) for the three digesters. The gaps represent Sunday, where no feeding was
performed.
0
20
40
60
80
100
120
140
160
180
200
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
WasteSources(t/d)
Total MSW Total BW
0
20
40
60
80
100
120
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
MSWmassloading(t/day)
Digester 1 (MSW) Digester 2 (MSW) Digester 3 (MSW)

12
Figure A-I 4 - VS content, in percent, in the three digesters. The gaps represent Sunday, where no feeding was performed.
Figure A-I 5 - Volumetric loading rate (kg VS/m3
.d) for the three digesters. Each value calculated considering the working
volume of each digester. The gaps represent Sunday, where no feeding was performed.
Figure A-I 6 - Biogas production (Nm3
/d), measured after cleaning. VFA accumulation (g acetate/L) in DG2.
0.23
0.27
0.31
0.35
0.39
0.43
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
VS(%) Digester 1 (MSW) Digester 2 (MSW) Digester 3 (MSW)
1
3
5
7
9
11
13
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
VolumetricLoadingrate
(kgVS/m3.d)
VLR 1 VLR 2 VLR 3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
10000
15000
20000
25000
30000
35000
40000
45000
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
VFADG2(gacetate/L)
BiogasProduction(Nm3)
Daily Biogas Production VFA DG2

13
Figure A-I 7 - Total Biogas productivity (Nm3
/t VS.day) for the three digesters. HRT – Hydraulic Retention Time, of about 40
days. The gaps represent Sunday, where no feeding was performed.
Figure A-I 8 - Influences of biogas production (Nm3
) in biogas quality (% CH4).
Figure A-I 9 - TS and moisture (%, on the left) and TS content as VS, Inerts and Glass/metal (%, on the right) in the digestate.
0
200
400
600
800
1000
1200
1400
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
Biogas(Nm3/tVS.d)
HRT~40days Daily
52
53
54
55
56
57
58
59
60
61
62
63
10000
20000
30000
40000
50000
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
Quality(%CH4)
Production(Nm3)
Biogas production Biogas quality
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 1011121314151617
Time (weeks)
Total Solids Moisture
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 1011121314151617
Time (weeks)
Volatile Solids Inerts > 0,5mm Glass and metal

14
Figure A-I 10 - VS removed in percent. Weekly average of the three digesters.
Figure A-I 11 - VFA content (g acetate/L) in the digestate of the three digesters. The gaps represent the weekends, where no
measurements were performed.
Figure A-I 12 - DG2 feeding VS content and VFA accumulation.
25
30
35
40
45
50
55
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
%VSremoved
Time (weeks)
0.15
0.20
0.25
0.30
0.35
0.40
0.45
2.2
2.7
3.2
3.7
4.2
4.7
5.2
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
VFADG2(gacette/L)
VSfeeding(%)
VFA DG2 VS DG2

15
Figure A-I 13 - pH and Alkalinity measurements in the digestate of digester 2. The gaps represent the weekends, where no
Figure A-I 14 - pH as a function of Alkalinity for the three digesters.
Figure A-I 15 - Average Alkalinity (g CaCO3/L) of the three digesters, measured in the digestate and its influence on total
biogas quality (% CH4).
16
17
18
19
20
21
22
7.0
7.2
7.4
7.6
7.8
8.0
8.2
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
Alkalinity(gCaCO3/L)
pH pH Alkalinity
7.55
7.60
7.65
7.70
7.75
7.80
7.85
15 16 17 18 19 20 21 22
pH
Alkalinity (g CaCO3/L)
DG1 DG2 DG3
16.5
17.0
17.5
18.0
18.5
19.0
19.5
20.0
52
54
56
58
60
62
64
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
Alkalinity(gCaCO3/L)
BiogasQuality(%CH4)
Biogas quality Alkalinity

16
Figure A-I 16 - VFA to Alkalinity ratio in the digestate of the three digesters. The gaps represent the weekends, where no
Figure A-I 17 - Temperature (ºC) in the digestate of the three digesters. The gaps represent the weekends, where no
Figure A-I 18 - Influence of ammonia (g/L, one grab sample) on biogas quality (% CH4, on the left) and on biogas production
(Nm3
, on the right), in the digestate of the three digesters.
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
VFA/Alkalinity
(gacetate/gCaCO3)
DG1 DG2 DG3
38.6
39.0
39.4
39.8
40.2
40.6
41.0
3/jan
10/jan
17/jan
24/jan
31/jan
7/fev
14/fev
21/fev
28/fev
6/mar
13/mar
20/mar
27/mar
3/abr
10/abr
Temperature(ºC)
DG1 DG2 DG3
54
56
58
60
62
0
2
4
6
8
0 5 10 15 20
Quality(%CH4)
NH3(g/L)
Time (weeks)
Quantofix ® Method Biogas Quality
2.E+04
3.E+04
4.E+04
5.E+04
6.E+04
0
2
4
6
8
0 5 10 15 20
Production(Nm3)
NH3(g/L)
Time (weeks)
Quantofix ® Method Biogas Average Production

17
Figure A-I 19 - Total COD (g O2/L, on the left) and soluble COD (g O2/L, on the right) in digesters. Samples collected from
inside the digesters after 20 minutes of recirculation.
Figure A-I 20 - Ammonia content (g/L) obtained by the Kjeldahl method (one grab sample), in the effluents of the dewatering
stage. Tank 1 receives effluent from presses and sends it to the sieves; Tank 2 receives from sieves and sends it to the
centrifuges. Centrifuge outlet are collected in tanks 3 and 4 from where liquid effluent can be recirculated to the digesters as
water balance. Tank 5 receives from centrifuges and sends to the WWTP the liquid effluent in excess.
Figure A-I 21 - Comparison between digestate and press liquid fraction in DG1 and DG3. Content in NH3-N (Kjeldahl Method).
50
100
150
200
250
300
4 5 6 7 8 9
TotalCOD(gO2/L)
Time (weeks)
DG1 DG2 DG3
0
40
80
120
160
200
4 5 6 7 8 9
SolubleCOD(gO2/L)
Time (weeks)
DG1 DG2 DG3
0
2
4
6
4 5 6 7 8 9
NH3-N(g/L)
Time (weeks)
Tank 01 Tank 02 Tank 03 Tank 04
0
2
4
6
4 5 6 7 8 9
NH3-N(g/L)
Time (weeks)
Tank 05 morning Tank 05 afternoon
2
3
4
5
6
5 6 7 8 9
NH3-N(g/L)
Time (weeks)
DG1 Press
2
3
4
5
6
5 6 7 8 9
NH3-N(g/L)
Time (weeks)
DG3 Press

18
Figure A-I 22 - Total COD (g O2/L, on the left) and soluble COD (g O2/L, on the right) in tanks 1, 2 and 5. One-grab samples
collected from inside the tanks, when the equipments are operating. Tank 1 receive effluent from presses an sends it to the
sieves, Tank 2 receives from sieves and sends it to the centrifuges, and Tank 5 receives from centrifuges and sends it to the
WWTP.
Figure A-I 23 - Total COD (g O2/L, on the left) and soluble COD (g O2/L, on the right) in tanks 3 and 4. One-grab samples from
inside the tanks, when the equipments are operating. Tanks 3 and 4 receive effluent from centrifuges in order to perform
recirculation into the digesters.
Figure A-I 24 - TS (%, on the left) and VS (%, on the right) contents in tanks from the dewatering stage.
0
50
100
150
200
250
4 5 6 7 8 9
TotalCOD(gO2/L)
Time (weeks)
Tank 01 Tank 02
0
25
50
75
100
125
4 5 6 7 8 9
SolubleCOD(gO2/L)
Time (weeks)
Tank 01 Tank 02
0
30
60
90
120
150
4 5 6 7 8 9
TotalCOD(gO2/L)
Time (weeks)
Tank 03 Tank 04
0
10
20
30
40
50
4 5 6 7 8 9
SolubleCOD(gO2/L)
Time (weeks)
Tank 03 Tank 04
0%
5%
10%
15%
20%
25%
0 5 10 15
TS(%)
Time (weeks)
Tank 1 Tank 2
Tank 3 Tank 4
30%
40%
50%
60%
70%
0 5 10 15
VS(%)
Time (weeks)
Tank 1 Tank 2
Tank 3 Tank 4
Tank 5 morning

19
Figure A-I 25 - Tank 5 TS content, measured in the morning and afternoon samples.
Figure A-I 26 - Weekly measurements of TDS and TSS (in percent, on the left) and S/S (ml/L, on the right), performed in the
morning sample of Tank 5. S/S was performed after 24h.
0%
1%
2%
3%
4%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (weeks)
TDS TSS
0
200
400
600
800
1000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
S/S(ml/L)
Time (weeks)

20
Figure A-I 27 - Comparison between ammonia measurements (g/L) obtained by the Quantofix® Method (one,-grab sample)
and the Kjeldahl method (one grab sample), in the digestate of the three digesters.

Extended Abstract FINAL

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