Integration of Microalgae Culturing Within The Wastewater Scheme

Integration of microalgae culturing within
the wastewater scheme
Oscar Mauricio Martínez Avila

Matr. 797677
Supervisor:
PhD. Eng. Elena Ficara
Politecnico di Milano
School of Industrial and Information Engineering
M.Sc. Energy Engineering
Piacenza, Italy
A.Y. 2013-2014
Acknowledgments
First of all I would like to thanks to Professor Elena Ficara for her valuable support, for the
time she devoted to supervising this project and all her opinions and suggestion, but
mainly for her kindness and comprehension with me during the development of this
project. Furthermore, I would like to acknowledge to Professor Valeria Mezzanote who
has also supported and advised any step of the project with her helpful indications. A
special thanks to Chiara, Enrica and Mattia, who have helped me in different ways, but
mainly thanks for holding me with my awful Italian.
Abstract V
Abstract
This document provides an analysis of the potential improvements involved in the
implementation of a microalgae culture integrated to the typical depurative cycle of a
wastewater treatment plant. In particular, it is of interest the integration with the anaerobic
digestion of the waste sludge, since the biogas produced from the anaerobic co-digestion
of these feeds can be used to generate onsite electrical power or thermal heat to offset
biomass processing and extraction processes. In fact, when both processes are
integrated and operated simultaneously, the benefits to wastewater treatment are
extended beyond the energy generation, including a considerably improvement of the
effluent quality. For this purpose a Pilot plant scale culture was tested in order to define
the operational parameter required for modeling this unit in an integrated scheme WWTP-
PBR. From the simulations, it is evident the increase on produced biogas due to the co-
digestion process, as well as the boost quality in the centrate stream. The outcomes
confirm the potential of this scheme for improving the energy balance of the WWTP, but it
is essential to go into deep in the optimization of the culture system, as well as the
harvesting process, in order to avoid possible negative effects.
Keywords: Microalgae, Anaerobic Digestion, WWTP, Mass balance, Integration

Contents VII
Table of Contents
Pg.
Abstract............................................................................................................................ V
List of figures.................................................................................................................. IX
List of tables ................................................................................................................... XI
List of Symbols and abbreviations .............................................................................. XII
Introduction ..................................................................................................................... 1
1. State of the art .......................................................................................................... 5

1.1 Current expectations on microalgal biomass .................................................... 5
1.2 Microalgae characteristics ............................................................................... 7
1.2.1 Microalgae growth ................................................................................. 9
1.2.2 Culture systems .................................................................................. 15
1.2.3 Productivity of microalgae cultures ...................................................... 24
1.3 Anaerobic digestion process .......................................................................... 26
1.3.1 Biochemistry of the AD process .......................................................... 28
1.3.2 Factors affecting AD process .............................................................. 31
1.3.3 Potential of microalgae species for producing methane....................... 34
1.4 Integration of microalgae growth with AD ....................................................... 37
2. Mathematical models for algae growth ................................................................. 41

2.1 Mass and energy balances on PBR ............................................................... 42
2.2 Kinetic models for nutrients ............................................................................ 43
2.3 Kinetic models related to light intensity .......................................................... 46
2.4 Multiple factors kinetics .................................................................................. 51
3. Experimental phase ............................................................................................... 53

3.1 Algae growth in batch operation..................................................................... 54
3.2 Algae growth in continuous operation ............................................................ 60
3.3 Anaerobic digestion tests ............................................................................... 65
3.4 Harvesting of produced algae ........................................................................ 70
4. Integrated Mass Balance ....................................................................................... 73

4.1 Wastewater section ....................................................................................... 76
4.2 Sludge treatment section ............................................................................... 80
4.3 Combined Heat and Power section ................................................................ 85
4.4 Main results of the model ............................................................................... 87
VIII Integration of microalgae culturing within the wastewater
5. Conclusions and Recommendations ....................................................................91
A. Appendix: Raw operational data in Bresso WWTP 2012-2013 .............................93
B. Appendix: Combustion reaction analysis in CHP unit .........................................99
C. Appendix: Description of the solution in Excel ..................................................103
D. Appendix: Description of the experimental systems and related procedures .107
Bibliography .................................................................................................................113
Contents IX
List of figures
Pg.
Figure 1-1. Energy feedstocks processing using microalgae. ............................................6
Figure 1-2. Photosynthetic activity behavior of microalgae. ...............................................9
Figure 1-3. Algae growth phases. ....................................................................................10
Figure 1-4. Biomass productivity as function of light intensity ..........................................12
Figure 1-5. Biomass productivity as a function of culture mixing ......................................12
Figure 1-6. Comparison of microalgal CO2 fixation ability. ...............................................14
Figure 1-7. Typical open system configurations. ..............................................................16
Figure 1-8. Typical raceway pond configuration...............................................................16
Figure 1-9. Tubular PBR of parallel runs..........................................................................18
Figure 1-10. Flat plate PBR configuration. .......................................................................18
Figure 1-11. Bubbling column PBR..................................................................................20
Figure 1-12. Main interactions accounted for photobioreactors design. ...........................20
Figure 1-13. Light pathway and light-darkness cycles......................................................21
Figure 1-14. CO2 Mass transfer coefficient between gas and liquid phases. ....................23
Figure 1-15. Volumetric productivity of some microalgae strains .....................................25
Figure 1-16. Mean Pvol and Pareal for different PBR configurations. ...................................26
Figure 1-17. Typical routes for processing anaerobic digestion products. ........................27
Figure 1-18. Biogas yield and methane content of different substrates. ...........................28
Figure 1-19. Stages of the anaerobic digestion process. .................................................29
Figure 1-20. Growth rate of microorganisms in each temperature range. ........................31
Figure 1-21. Theoretical Biological methane potential of some microalgae species .........36
Figure 1-22. Methane yield of some algae strains. ..........................................................36
Figure 1-23. Scheme of integration AD-microalgae-wastewater proposed by Uttley ........37
Figure 1-24. Scheme of integration AD-microalgae-wastewater by Yuang ......................38
Figure 1-25. Scheme of integration AD-microalgae-wastewater according Sahu .............39
Figure 1-26. Proposed scheme of integration in Bresso plant. .........................................40
Figure 2-1. Operating variables of a PBR for algae growth. .............................................41
Figure 2-2. Irradiance profile along a flat-plate PBR. .......................................................48
Figure 2-3. Incident radiation and its relationship with a given point inside the culture .....50
Figure 3-1. Methodology employed in the experimental part............................................53
Figure 3-2. Current location and appearance of the PBR pilot plant. ...............................55
Figure 3-3. Procedure for operating the pilot plant in batch mode. ...................................55
Figure 3-4. Evolution of TN and TP concentration during batch runs ...............................56
Figure 3-5. Evolution of N-NH4 concentration during batch runs. .....................................57
Figure 3-6. Evolution of solids content during batch test. .................................................57
X Integration of microalgae culturing within the wastewater
Figure 3-7. Absorbance of the medium during batch runs. .............................................. 58

Figure 3-8. N-NH4 removal on the different batch tests. .................................................. 58
Figure 3-9. TSS concentration change on the batch tests. .............................................. 59
Figure 3-10. Linearization of the kinetic growth using absorbance as reference variable. 60
Figure 3-11. Procedure for operating the pilot plant in continuous mode. ........................ 61
Figure 3-12. Solids content for the continuous operation. ................................................ 62
Figure 3-13. N-NH4 behavior during continuous operation. ............................................. 62
Figure 3-14. Absorbance of the culture medium during continuous operation ................. 63
Figure 3-15. TSS/Abs correspondence in the microalgae culture. ................................... 64
Figure 3-16. Estimated yield parameters for the continuous experiments........................ 65
Figure 3-17. Sketch of the bench scale anaerobic digestion system. .............................. 65
Figure 3-18. Procedure for operating the AD system in semi-batch mode. ...................... 66
Figure 3-19. Methane production during the AD tests. .................................................... 68
Figure 3-20. (a) Specific methane production and (b) organic loading rate of AD runs. ... 68
Figure 3-21. Feed and digestate solid contents. .............................................................. 69
Figure 3-22. Settleable solids of produced microalgae during continuous operation. ...... 71
Figure 3-23. Efficiency and solid concentrations after microalgal biomass harvesting. .... 72
Figure 4-1. Transformations included in the ASM1. ......................................................... 74
Figure 4-2. Transformations included in the ADM1 ......................................................... 75
Figure 4-3. Proposed scheme of integration inside Bresso plant ..................................... 76
Figure 4-4. Process units composing the wastewater section in Bresso plant. ................ 76
Figure 4-5. Streams of the wastewater section................................................................ 77
Figure 4-6. Units and streams composing the sludge treatment section. ......................... 80
Figure 4-7. Units of the section dedicated to sludge treatment. ....................................... 82
Figure 4-8. Units composing the subsection of microalgae culture. ................................. 83
Figure 4-9. Units and streams composing the CHP section............................................. 85
Figure 4-10. Effect of PBR implementation on the produced biogas................................ 88
Figure 4-11. Required area for the PBR implementation at different productivities .......... 90
Contents XI
List of tables
Pg.
Table 1-1. Main design characteristics of microalgal bioreactor systems. ........................17
Table 1-2. Generic description of mixing methods in PBR´s. ...........................................22
Table 1-3. Typical composition of biogas. ........................................................................27
Table 2-1. Kinetic models for nitrogen dependent algae growth. .....................................44
Table 2-2. Kinetic models for phosphorus dependent algae growth. ................................45
Table 2-3. Kinetic models for carbon dependent algae growth. .......................................46
Table 2-4. Kinetic models related to light intensity and algae growth. ..............................47
Table 2-5. Summary of specific growth rates of previous studies.....................................52
Table 3-1. Estimation of anaerobic digestion efficiency. ..................................................70
Table 4-1. Summary of degree of freedom analysis in wastewater section. .....................79
Table 4-2. Summary of degree of freedom analysis in sludge treatment section. ............81
Table 4-3. Limiting nutrients and maximum expected biogas production. ........................89
Contents XII
List of Symbols and abbreviations
Symbols with Latin letters
Symbol Definition SI Unit

A Area m2
Ci Concentration of component/species i Kg/m3; mg/L
h Plank constant J.s
I Irradiance W.m-2
I Light Intensity µmol.m-2s-1
kLa Global mass transfer coefficient in the liquid phase m.s-1
m Maintenance coefficient Jg-1s -1
mi Mass flow rate of stream i Kg.s -1
ni Molar flow rate of stream i Kmol.s -1
PArea Areal biomass productivity gb.m-2.day-1
PVol Volumetric biomass productivity gb.m-3.day-1
Pdirect Equivalent light path distance for direct irradiance m
Pdisp Equivalent light path distance for disperse irradiance m
Pel Electric power Kwh.s-1
Qi Volumetric Flow rate stream i m3. s -1
Ri Growth Rate Reduction factor i -
rrk Recycle Ratio section k -
V Volume m3
Xi Biomass concentration in the medium mg.L-1
Yi Yield coefficient component i -
Symbols with Greek letters
Symbol Definition SI Unit

ν Wavelength nm
η Removal efficiency, Unit efficiency -
µ Specific Growth Rate algae s -1
ρ density Kg.m-3
φ angle Rad
ϕ Polar coordinate inside a tubular reactor Rad
β Stoichiometric Air to fuel ratio -
ε Excess fraction of air -
Contents XIII
Abbreviations
Abbreviation Definition
AD Anaerobic Digestion
ASM1 Activated Sludge Model 1
ADM1 Anaerobic Digestion Model 1
BOD Biological Oxygen Demand
BMP Biological Methane Potential
CHP Combined Heat and Power Plant
COD Chemical Oxygen Demand
CV Coefficient of variance
DOF Degree Of Freedom
HRT Hydraulic Retention Time
LHV Low Heating Value
OCD Optimal Cell Density
OLR Organic Loading Rate
PAR Photosynthetically Active Radiation
PBR Photobioreactor
SMP Specific Methane Production
SRT Solid Retention Time
STS Settleable solids
TOE Tons of oil equivalent
TS Total Solids
TSS Total Suspended Solids
TVS Total Volatile Solids
VFA Volatile Fatty Acids
WWTP Wastewater Treatment Plant
Introduction
The effects of pollutants presented in water streams have wide and heterogeneous
characteristics. They affect the surrounded environment, the human health and also
increase the pressure over the resource. From this point of view, we realized that a proper
treatment of the polluted effluents reduces the adverse effects of those waste products,
and at the same time, we increase the chance of reusing these streams for valuable
purposes. On the other hand, a suitable design and operation of the treatment stations
should be guaranteed in order to fulfill the related environmental requirements while
keeping an economical and reliable processing.
In terms of wastewater treatment, a widespread amount of techniques have been

developed, and they are well established according to the particular characteristics of
influents and geographical location of the streams. Most of the technologies include a
primary stage of physical separation, a secondary phase based on a biological
degradation and a final step to further improve the effluent quality by means of physic-
chemical operations. Nevertheless a further consideration emerges from the main by-
product of this process, the sludge streams. This is commonly treated for stabilizing it and
to reduce its negative effect on the environment by means of composting, aerobic or
anaerobic digestion, generating a biosolid that is employed as feedstock in the production
of the so-called organic fertilizers.
From these treatments, municipal and industrial anaerobic digestion (AD) of organic
wastes is broadly practiced and recognized as a mature technology that additionally to
treat the sludge, allows producing biogas (Holm-Nielsen et al, 2009). Just in Europe
during 2007 energy from sewage sludge derived biogas was 887.2 Ktoe and it is
expected that in 2020 this will arrive to 3.4 Mtoe, representing 15% of the total biogas
energy production in the region (AEBIOM, 2009). This successful diffusion can be
attributable to the advantages AD presents, of which the broad diversity of feedstocks to
be employed in the process has played an important role.
AD can use a variety of organic materials to produce methane, such as food waste from
both domestic and industrial sources, farm manures and slurries, sewage sludge and
purpose grown crops for energy. However another AD feedstock largely investigated
since the fifties (Golueke et al, 1957) is the algal and microalgal biomass. Microalgae
have a high productivity and are able to produce large quantities of biomass in a more
efficient way than terrestrial crops. Depending on the system, photosynthetic efficiency of
these microorganisms can reach 4–5% of the solar energy compared to 1–2% for
2 Introduction
terrestrial plants (Ward et al, 2014). Furthermore, microalgae can grow in wastewater
containing high nutrient concentrations, recovering a substantial portion of them, helping
in this way with its treatment (Razzak et al, 2013).
In general microalgal biomass production counts with a significant amount of advantages,

reason why its culture has been adopted as source for third-generation biofuels. These
are also called advanced biofuels and are sourced from non-food crops, but the resulting
product is identical to its petroleum counterparts (Frigon et al, 2013). But, even if there is
no formal definition of which the most effective process for biofuel production from
microalgae is, AD carries out the simplest transformation process and according to some
investigations (Collet et al, 2011; Harun et al, 2011; Park et al, 2011), anaerobic digestion
is the most environmentally friendly and cost-effective method to get an energy product.
Thanks to the synergy presented between these processes, integration of microalgae

culture with anaerobic digestion has become a prominent alternative not just for
wastewater treatment, but as renewable energy source too. Thus, it is convenient to
assess the interactions between variables involved in the integration process, in order to
maximize the energy production as well as to improve the wastewater treatment system
(Alcántara et al, 2013).
While investigations like that presented by (Wang et al, 2010) and (Ji et al, 2013) have
focused on the effectiveness of microalgae for treating wastewater coming from different
steps inside the process, other like (Schenk et al, 2008), (Sforza, 2012) and (Cho et al,
2013) have studied some routes to foster microalgae growth in wastewater in order to
produce a biomass rich in lipids which can be further extracted for biodiesel processing.
Another line of investigation emphasis is the establishment of microalgae potential for
removing CO2 as suggested by (Martinez, 2009), (Ho et al, 2011) and (Arbib et al, 2014).
Finally, the explicit integration of microalgae growth with anaerobic digestion has been
explored in a minor degree by authors like (Uslenghi, 2011) and (Sahu et al, 2013) who
have tested the feasibility of growing microalgae in this medium, as a source for the AD
treatment.
In this work, an assessment of the mass balance inside an integrated scheme

“microalgae growth-wastewater treatment” has been developed taking as reference the
operation of the Bresso (Milano) municipal wastewater treatment plant. For this purpose,
the effectiveness of the growth was evaluated considering the culture of microalgae inside
a pilot plant cylindrical photobioreactor (PBR) using as feed the centrate obtained after
the anaerobic digestion process. On the other hand, the potential of microalgae for
producing methane through AD by bench tests was established, and those results were
used for developing a simple model for describing the interaction of each unit in the global
wastewater process.
In the first part of the document a review of the main characteristics of microalgae growth
and anaerobic digestion is presented, as well as a discussion about the integration
Introduction 3
process and its possible effects over the treatment performance. In Chapter 2 a
conceptual discussion about the growth rate kinetics was established, while in chapter 3 a
description of the methodology, materials and results of the experimental part that
complement the results of the mass balance are shown Afterward, the development of the
mathematical modeling of the mass balances in WWTP is described in chapter 4. In the
final fragment of the document, the conclusions derived from the performed analysis are
presented as well as the recommendations for future works.
This project has been developed as teamwork between Politecnico di Milano, Università
Degli Studi di Milano Bicocca with collaboration of AMIAQUE and Bresso WWTP, looking
for an improvement in the energy balance of the whole plant treatment. An additional aim
was the evaluation of the mass balance and the effect of the integration over the outlet
streams, particularly the biosolid stream after the anaerobic digestion.
1. State of the art
1.1 Current expectations on microalgal biomass
Our contemporary society has become an insatiable fossil fuels consumer, leaving
behind, a trail of social, political and economic problems, as well as several unsolved
pollution issues. According to (BP, 2013) we consumed around 16 TW of energy
worldwide during 2012 of which 85% came from combusting fossil fuels. This implies
human society is prone to three large threats: depleting of fossil fuel reserves, economic
disruptions due to competition for energy resources and global climate change produced
by the increment in CO2 levels in the atmosphere (Rittmann, 2008).
A significant number of alternatives have been proposed in order to face those threats,
being the development of renewable energy sources the most common solution to these
problems. It is evident that this search of new energy sources, carbon neutral, cost-
effective, sustainable and environmentally-friendly has become a current challenge for
society, reason why nowadays a vast amount of investigations focus on this matter (Mata
et al, 2010). In this sense, one of the main options extensively studied refers to the use of
bioprocess for producing feedstocks for transportation, power generation, heat, among
others. This alternative essentially includes the production of biomass, biofuels and
biogas derived from a wide variety of sources (Benemann et al, 2011).
Microalgae have become one of the preferred examples of those sources for renewable
energy production. As it can be seen in Figure 1-1, microalgae can be processed by
different routes to obtain a number of replacement products for fossil fuels. From the
thermochemical transformation for producing syngas or biomass, passing by the
extraction of oil for the further production of biodiesel, and concluding with the
biochemical conversion of microalgae, in which ethanol, hydrogen and methane are the
main products.
6 Integration of microalgae culturing within the wastewater
Figure 1-1. Energy feedstocks processing using microalgae.
Particularly, the most well-known route for converting microalgae in energy feedstock is
the biodiesel production. This process has been mainly developed thanks to several
incentives schemes in which biofuels replace fossil fuels in the transportation sector
(Oncel, 2013). Nowadays, biofuels based on vegetal oil and bioethanol are the most
important source of liquid biofuels around the world, but microalgae have key advantages
that make them a latent competitor for this purpose (Cai et al, 2013):
• Microalgae do not compete with crops for arable land and freshwater since they
can be cultivated in brackish water and on non-arable land.
• Microalgae usually grow faster than a crop, and they generate a biomass with high
oil contents (20–50% dry weight basis) without any need of using fertilizers.
• These microorganisms can fix carbon dioxide, thus reducing greenhouse gas
emissions and improving air quality.
• Microalgae culture can be carried out in wastewaters consuming the nutrients
presented in the medium, providing an alternative method for wastewater
treatment.
• Byproducts of microalgae cultivation after lipid extraction, namely algae biomass
residue, can be used as a nitrogen source, such as a protein-rich animal feed or
fertilizer for crops
It has been shown from several studies like presented by (Jayed et al, 2009), (Kurki et al,
2010), (Fargione et al, 2010) or (Cavalett & Ortega, 2010) that the use of agro resources
for generating biofuels generally reduces the negative effects over climate change
potential, but they are highly susceptible to create secondary environmental issues (e.g.
State of the art 7
eutrophication, resource depletion, ecotoxicity). Moreover, the global warming effect

caused by the production of bioenergy from biomass has to be correctly estimated since it
can be sometimes higher than the ones induced by fossil fuel production (Collet et al,
2011).
Besides biodiesel production, microalgal biomass has been employed for additional
purposes, such as a source of valuable chemicals or health foods (Pulz & Gross, 2004);
as source for anaerobic digestion processes (De Schamphelaire & Verstraete, 2009),
(Mussgnug et al, 2010), (Ras et al, 2011), (Zamalloa et al, 2011); also as an efficient
wastewater treatment (Alvarez & Tomás, 1989), (Wang et al, 2010), (Boonchai et al,
2012), (Ji et al, 2013), (Arbib et al, 2014). More recently, microalgal photosynthesis has
been suggested to be an effective mean for reducing the CO2 concentration, major
greenhouse gas in the atmosphere (Martinez, 2009), (Kumar et al, 2011).
This great deal of attention over microalgae as a feedstock can be explained given the
characteristics of these microorganisms. (Schenk et al, 2008) indicate that the main
advantages of second-generation microalgal systems are:
• They have a higher photon conversion efficiency

• Can be harvested batch-wise nearly all-year-round, providing a reliable and
continuous supply of biomass.
• Can utilize salty and wastewater as the growth medium, thus reducing freshwater
use.
• Can couple CO2-neutral fuel production with CO2 sequestration.
• Produce non-toxic and highly biodegradable biofuels. Current limitations exist
mainly in the harvesting process and in the supply of CO2 for high efficiency
production.
Therefore, it can be stated that microalgae constitute a feasible alternative for many
processes, but the recent tendency is focused on processing microalgae for the
production of renewable energy, capture of CO2 and wastewater treatment.
1.2 Microalgae characteristics

Microalgae and cyanobacteria are unicellular species that typically grow suspended in
water performing the same photosynthetic processes than higher plants. Most microalgae
are autotrophs which means they transform sunlight, CO2 and a few nutrients, including
nitrogen and phosphorous, into biomass. Other algae are capable of growing in absence
of light by using sugar or starch (heterotrophic growth) or combining both mechanisms
(mixotrophic growth). Algae are very diverse and found almost everywhere on the planet.
They play an important role in many ecosystems, including providing the foundation for
the aquatic food chains supporting all fisheries in the oceans and inland (Masojídek et al,
2004).
Microalgae contain three principal components: proteins, lipids and carbohydrates.

Depending on the specie and growth conditions, each of them can vary in a large range.
For instance, cyanobacteria have as much as 20% of lipids, while prokaryotic algae can
reach 50% of lipid content (Ruiz, 2011). In terms of the basic constituents, authors like
(Grobbelar, 2004) have proposed an equivalent molecular formula:

The photoautotrophs microalgae are capable of synthesizing organic matter starting from
inorganic constituents and energy from the light thanks to photosynthesis. In this process,
water is decomposed in oxygen and a reducing agent able to transform CO2 into organic
carbon. In fact, some energy is used to strip electrons from suitable substances such as
water, producing oxygen, while two further compounds are generated: reduced
nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate
(ATP) (Caiazzo, 2007). NADPH acts as an energy carrier during the dark phase of
photosynthesis (Calvin cycle) providing the energy to convert carbon dioxide and water
into organic compounds, that is called carbon fixation (Masojídek et al, 2004). The global
stoichiometry of photosynthesis can be seen as:
6 + 6 + ℎ → + 6 (1)
Where hν represent the luminous energy.
One of the most important aspects in photosynthesis is their efficiency and the
photoinhibition phenomenon. The first refers to the efficiency of CO2 conversion per each
photon caught by the cell while the second is a physiological response of the organisms
to high irradiance stress (Martinez, 2009). From the whole electromagnetic spectrum, just
the visible zone (380-710 nm) can be used by the plants to carry out photosynthesis. This
range of light is commonly known as photosynthetically active radiation (PAR) (Masojídek
et al, 2004).
During the absorption process, all photons are caught in the chlorophyll reaction center;
here a maximum of eight photons per mol of CO2 can be storage. In this scenario,
producing a carbohydrate as product of carbon fixation, the maximum photosynthetic
efficiency reaches 34% (Martinez, 2009). However according (Benemann & Tillett, 1993)
there are more aspects that reduce the photosynthetic efficiency like:
• Photons in the visible field, which promote photosynthesis, account for about 43%
of the overall solar radiation at the ground level.
• The fraction of the incident radiation that is adsorbed by the surfaces capable of
promoting photosynthesis can reach a maximum of about 80%.
State of the art 9
• Part of this radiation is used in processes different from photosynthesis (e.g.

transpiration). The available fraction for photosynthesis is never higher than 75%.
• It is possible that cells require more than eight photons per mol of CO2.
Accounting for all this effects, the maximum conversion of solar energy efficiency is 8.5%.
Additionally, it is expected lower values since photons are processed at a lower velocity
than the absorption velocity. This implies that the unmetabolized extra photons are
rejected as heat and fluorescence.
Once the number of rejected photons is such big that there is no enhance in the
photosynthetic activity, photoinhibition phenomenon appears affecting the process. As it
can be seen in Figure 1-2, 3 main zones are distinguished, a light limited zone (zone 1) in
which activity increases with light intensity, a saturated zone (zone 2) that is almost
independent of the amount of light, and a photoinhibition zone (zone 3) where
photosynthetic activity decrease even when light intensity still grow. Constant Ik is
characteristic of each specie and defines the point in which photosynthetic activity suffer a
deceleration and start becoming a saturated system (Masojídek et al, 2004).
Figure 1-2. Photosynthetic activity behavior of microalgae.
1.2.1 Microalgae growth

Microalgae grow very quickly compared to terrestrial crops. They commonly double in
size every 24 hours and during their peak growth phase, some microalgae can double
every 3.5 hours (Chisti, 2007). According to authors like (Barbato, 2009) the growth
process can be split into five steps as Figure 1-3 shows:
• Lag phase. Period in which algae population start adapting to the environmental
conditions of the medium with a negligible grow.
• Exponential phase. During this period algae population grow with an exponential
rate, but only for a short time, due to the little amount of cell presented in the
environment.
• Linear phase. In this step algae grow at an almost linear rate, reaching a high
value at the end of the interval (optimal interval for algae culture).
• Stationary phase. After the linear phase, the growth rate diminishes rapidly until
reaching the maximum value (Limit interval in which saturation occurs).
• Decline phase. In this point algae population suffer a significant reduction due to
the high concentration of algae as well as the nutrient exhaust (Algae die because
of the suspension of the division and metabolic phases).
Figure 1-3. Algae growth phases. Adapted from (Mata et al, 2010)
Notwithstanding, growth rate is highly affected by several abiotic, biotic and operative
factors which define the optimal growth conditions for each particular specie. In this
sense, each variable play an important role as can be stated as follows.
Temperature
Temperature affects the biosynthetic reactions involved in the algae growth increasing
this velocity exponentially till the point of maximum growth rate. Even when each
microalga specie have its range of growth, can be generalized that they growth properly
between 16 and 27°C (Mata et al, 2010). At temperat ures below 16°C the growth rate
becomes such slow that it is almost inhibited. On the other hand, at high temperatures
algae tend to die and then it is necessary to supply less light to the culture or adapt a
cooling system to reduce the negative effects of this phenomenon (Ruiz, 2011).
State of the art 11
pH
The pH of the medium directly influences the equilibrium of several species like CO2 and
also defines how they are presented in the medium. During photosynthetic process pH
increases due to the accumulation of OH- ions, which end up with the nitrogen elimination
by ammonia stripping and precipitation of orthophosphates species (Ruiz, 2011).
According to this, most of algae culture systems operate in the range of pH 7-9 but
optimal value lies typically in the range 8.2-8.7. If pH is not in the proper range, culture
collapses and it is required a compensation by means of addition of CO2 or air depending
on the particular needs.
Oxygen
High dissolved oxygen concentrations can inhibit the CO2 absorption in the enzyme
RuBisCo. This effect is more significant at higher temperatures and radiation rates,
causing the death of algae after 2 or 3 hours if supersaturating conditions (> 120%) are
presented in the culture (Pulz & Gross, 2004). Another consideration regards oxygen is its
effect over the mass transfer diffusion of CO2, where is required a lower concentration of
dissolved oxygen in order to enhance the CO2 flow to the medium. In this sense some
authors (Baquerisse et al, 1999) and (Carvalho et al, 2006) agree that for high cell density
culture, 40 mgO2/L is an admissible limit in which CO2 can be diffused without substantial
interference due to the presence of oxygen.
Light availability
The algae growth is highly influenced by the amount of light they receive, but differently
fom nutrients and CO2, light needs to be added continuously in the culture since radiant
energy cannot be stored (Martinez, 2009). It can be stated that photosynthetic
microorganism’s growth is proportional to the light intensity they get. The optimal light
intensity will vary depending on the specie but it ranges between 30-200 µEs-1m-2
(Uslenghi, 2011). One of the most important parameters to be taken into account in the
design of the reactor is the light penetration distance, which depends on light intensity,
dispersion on the reactor’s walls and attenuation in the culture. In this sense, a suitable
reactor design includes the definition of the optimum culture density in such a way
incident and transmitted light be good enough for the algae growth avoiding
photoinhibition phenomenon (Ruiz, 2011).
As Figure 1-4 shows, microalgal biomass productivity reaches an optimal value

depending on the light intensity and the culture concentration. Taking this into account, in
order to keep high biomass productivity a significant light intensity is needed, but the
system has to distribute this light in a suitable way in order to avoid the photoinhibition
phenomenon. This can be reduced to optimize the light diffusion inside the reactor, and
maintaining a high frequency light-dark cycles by a proper mixing of the culture (Carvalho
et al, 2006).
Figure 1-4. Biomass productivity as function of light intensity
Stirring
A proper stirring mechanism is required in order to enhance the mass transfer between
the gas and liquid phases, for avoiding algae settling inside the reactor and guarantee a
suitable distribution of light and enhance the diffusion of the gaseous species. In terms of
light penetration, a good stirring system allows algae to experience rapid changes
between light and dark zones, promoting better photosynthetic performances since there
is a lower risk of photoinhibition and photo limitation (Coral et al, 2003).
Figure 1-5. Biomass productivity as a function of culture mixing. (*Optimal cell density (g/L);
**air volume/liquid volume/minute).
State of the art 13
On the other hand, this parameter indirectly influences the biomass productivity as it can
be seen in Figure 1-5. Most of the commercial systems employ air streams for this
purpose and this result in an efficient way of providing agitation to the culture at the same
time that CO2 is supplied. Usually a higher ratio Gas/Liquid is guarantee of a well-mixed
culture, however it is known that most of algae species are prone to hydrodynamic stress,
which implies a limitation that has to be considered. Some authors like (Eriksen, 2008)
have suggested that addition of nonionic surfactants can help to microalgae cells to elude
their adhesion to air bubbles and then become more resistance to this stress forces.
Nutrients
• Carbon
External CO2 contribution is practically mandatory when high biomass production rates
are required since its concentration in the atmosphere is not enough for providing carbon
to autotrophs algae (Grobbelar, 2004). Additionally, some species are able to use
bicarbonate ions (HCO3-) by means of the carbonic anhydrase enzyme but this carbon
source is pH dependent (Martinez, 2009). On the other hand, algae tolerance to CO2
varies along the species, but in general concentration has to be kept in a narrow range.
Authors like (Pulz, 2001) indicate that maximum concentrations of 12% are commonly
used in commercial PBR’s, but as (Kumar et al, 2011) summarize, the tolerance can be
as higher as 70-80 % (v/v) under specifically optimized conditions.
CO2 supply is probably after light distribution, the most important aspect for algae growth
since it directly influences the productivity and economy of the process. CO2 needs can
be estimated base on the stoichiometry and algae composition, finding values between
1.8 – 2.0 g CO2/gbiomass (Lardon et al, 2009), (Ruiz, 2011) and (Alcántara et al, 2013).
However, CO2 consumption is dependent on several factors, like CO2 concentration,
temperature, reactor configuration, light intensity, mixing, and algae species. (Ho et al,
2011) Show in their study, a compilation of CO2 needs for several species, finding that it
can vary between 200 and 600 mg CO2/L.day. As it can be seen in Figure 1-6 this range
can be extended in a significant amount, but a higher efficiency fixation it is only
reachable if all variable are properly managed during all steps of the process.
• Nitrogen
After Carbon, Nitrogen is the second most important nutrient for algae. The nitrogen
content inside algae biomass ranges between 7 and 10% and varies depending on
nitrogen availability as well as the type of source (Martinez, 2009). Algae generally take
nitrogen as nitrate (NO3-) and ammonium (NH4+), but there are some species able to use
urea, nitrite (NO2-), nitrogen oxides (NOx) or molecular nitrogen. Notwithstanding
ammonium represent a latent problem for algae growth since at high levels it results toxic
for photosynthetic organisms (Grobbelar, 2004). Ammonium uncouples the electric
transport inside photosystem II and competes with water during oxidation reactions that
produce free O2 (Ruiz, 2011). (Shi et al, 2000) have found that Chlorella protothecodis
can grow with ammonium levels up to 84 mgNH4+/L, while (Yuan et al, 2011) found that
Spirulina is only inhibited with concentrations over 200 mgN-NH3/L.
Figure 1-6. Comparison of microalgal CO2 fixation ability. Adapted from (Ho et al, 2011)
In nitrogen-limited culture systems, some studies reveal that the biosynthesis and
accumulation of lipids are enhanced. Nonetheless, a different behavior can be found in
species like Dunaliella strains where an increment in carbohydrate rather than their lipid
content is the result of nitrogen depletion conditions (Grobbelar, 2004).
• Phosphorus
Even when phosphorus content in algae biomass is limited under 1%, this nutrient plays
and important role during algae growth, since it is essential in cellular processes like the
formation of nucleic acids. The algae usually take it as orthophosphate (H2PO4-, HPO4-2,
PO4-3) and its presence in the culture medium is limited by the dependence of the
equilibrium on the pH. Extreme pH values and absence of ions like K, Na or Mg promote
a lower phosphorus capture (Ruiz, 2011). (Sforza, 2012) suggest that the ratio N:P is
crucial in order to improve the algae growth rate; they clearly indicates that it is always
better to adjust this ratio as closer as possible to the actual composition of the
microorganisms. This does not imply that algae are unable to adapt their selves in order
to take a greater proportion of one of the nutrients. In fact, under absence of nutrients,
algae are fostered to produce more lipids that can be beneficial for production of
secondary products like biodiesel (Khozin-Goldberg & Cohen 2006), (Rodolfi et al, 2008).
It can be stated that the symptoms of phosphorous depletion are similar to those
observed in nitrogen-deficient cultures. Chlorophyll a contents tend to decrease while
carbohydrates rise in eukaryotic and prokaryotic cells (Grobbelar, 2004).
State of the art 15
• Other macro and micronutrients

In addition to C, N and P, algae required macronutrients like Na, K, Mg, Fe and S as well
as micronutrients like B, Cu, Mn, Zn and Se relevant for enzymatic reactions and
biosynthesis of compounds involved in the metabolism (Grobbelar, 2004). Similarly to
phosphorus, these ions are prone to precipitation by significant changes in pH, reason
why EDTA is advised as chelating agent.
Salinity
The salts content in the medium is important for the productivity of lipids and some other
derivate. In general microalgae can collect small molecules as osmoregulatory
substances or osmoticants in response to an increase in salinity or osmotic pressure of
the environment (Grobbelar, 2004). An increase in salinity will promote slight
improvements in the total lipid content of the microorganism.
1.2.2 Culture systems

Photobioreactors (PBR) are a particular kind of reactor in which photoautotrophs
organisms are cultured. It is important to remark that the term PBR even when is widely
used for closed systems, also involves those open system used for the same purpose. In
this sense, the main difference among those reactor designs can be established in the
contact or isolation of the surrounded ambient with the culture. According to (Grobbelaar,
2000), in open systems culture is exposed to the atmosphere while closed system usually
are not.
Open systems
These systems have been widespread since the 50’s and they still cover a considerable
portion of the commercial reactors for algae growth because of their low cost and easy
operation (Borowitzka, 1999). However, these systems are susceptible to several
drawbacks that make them poorly efficient. High land availability requirements, losses due
to evaporation, deficient temperature control, high risk of contamination and limited light
penetration are the main factors that usually affect the algae production in open systems
(Coral et al, 2003). Within the open system can be distinguished the natural and artificial
arrangements, the first are composed by lakes, lagoons and natural ponds, while raceway
ponds and circular systems constitute the most common artificial open systems.
In general, the culture suspension, containing nutrients, is pumped around in a cycle,

being directly illuminated from sunlight. This implies that the raceway depth should not be
higher than 30 cm in order to avoid microalgae to remain into darkness, but also to
increase the CO2 diffusion from the atmosphere (Caiazzo, 2007). This construction design
is the simplest mode for promoting the growth of phototrophic organisms, but due to their
characteristics, open systems only reach limited areal productivity rates (Barbato, 2009).
Furthermore, the energy consumption for pumping is relatively high, as high amounts of
diluted culture have to be processed. Artificial ponds are commonly designed in a

raceway configuration, in which a paddlewheel circulates and mixes the algal cells and
nutrients as can be seen in Figure 1-8.
Figure 1-7. Typical open system configurations.
Usually raceways are made of concrete, steel or plastic materials depending on the size,
or they are dug into the ground and covered with a plastic liner when this option is
feasible. These systems work in a continuous mode, where the fresh feed is added in
front of the paddlewheel, and algal broth is harvested behind the paddlewheel after it has
circulated through the loop (Ruiz, 2011).
Figure 1-8. Typical raceway pond configuration. From (Chisti, 2007)
The other basic design of open systems consists in circular ponds where circulation is
provided by rotating arms, and inclined systems where mixing is reached by means of a
combination of pumping and gravity flow. In both cases, the main characteristics remain
the same as the raceway ponds, except that it is expected that circular ponds can be at
maximum 1000 m2 size in order to keep a suitable mixture of the medium (Caiazzo,
2007).
State of the art 17
Closed systems
Closed PBR´s differ from the open system reactors in the fact that they improve all the
drawbacks presented in the aforementioned, making possible higher algae production
rates. In these systems there is null or negligible contact with the environment,
evaporation losses and temperature can be controlled in an easier way, and CO2 diffusion
is promoted by several mechanism related with the intrinsic reactor design (Pulz, 2001).
One of the main characteristics of the closed photobioreactors is that they are built with
transparent materials (plastic or glass) for providing a better light penetration. In general
those systems are more productive since they use less area for the same feed rate, and
collection of generated biomass result less expensive. Even that, total investment costs
tend to be higher than open systems, as well as operation and maintenance costs
(Martinez, 2009). Table 1-1 summarizes the main differences between open and closed
system.
Table 1-1. Main design characteristics of microalgal bioreactor systems.

Characteristic Open System Closed System
Area/Volume ratio High Low
Population density Low High
Harvesting efficiency Low High
Cultivation period Limited Extended
Pollution control Significant Negligible
Water evaporation Possible Negligible
Light utilization efficiency Poor/fair Fair/Excellent
Gas mass transfer Poor Fair/high
Temperature control None Excellent
Capital investment Low High
PBR´s can be classified according their design and operation mode. Respect to the
design they can be split into cylindrical (vertical- inclined-horizontal), plane (vertical-
inclined-horizontal) and coil type. Respect to the mode of operation they are air driven,
single phase reactors (gas exchange occurs in a separate chamber) and double phase
reactors (gas exchange is carried out along the entire reactor).
• Tubular PBR
These photobioreactors are constituted for several groups of plastic or glass tubes
arranged in parallel or series, located horizontally or vertically as Figure 1-9 shows. In this
section of the reactor microalgae catch light for performing photosynthesis, while, in the
degassing section column produced oxygen is removed in order to prevent high dissolved
oxygen level along the tubes. Unfortunately, the mixing level and therefore the mass
transfer inside these reactors is quite limited, fostering high O2 concentration levels.
Additionally, some photoinhibition problems are common since the poor mixing avoid cells
to reach suitable light-dark cycles (Martinez, 2009).
Figure 1-9. Tubular PBR of parallel runs. (a) Horizontal configuration; (b) Vertical
configuration.
In this configuration, tubes are always oriented North–South and the ground beneath the
solar collector is often painted white, or covered with white sheets of plastic to increase
reflectance, or albedo. Higher reflectance will increase the total light got by the tubes
(Chisti, 2007). Sedimentation in tubes is hinder by adjusting the turbulent flow using either
a mechanical pump or an airlift pump. Mechanical pumps can damage the biomass (but
are easy to design, install and operate, but airlift pumps have been used quite
successfully more recently (Tredici, 2004).
• Flat Plate PBR

The main advantage of this reactor configuration lies in the significant surface exposed to
radiation. In this PBR culture is forced to pass through the channels made by the reactor’s
walls, while CO2 is fed from the bottom and collected in the upper part of the reactor for
recycling or disposal. In Figure 1-10 it can be seen a typical flat plate photobioreactor.
Usually this configuration achieves high productivity levels (cell densities up to 80 g/L)
keeping a simple operation and maintenance (Posten, 2009). However, this design makes
difficult the temperature control and increases the probability of hydrodynamic stress of
cells (Uslenghi, 2011).
Figure 1-10. Flat plate PBR configuration.

State of the art 19
Even when these kind of reactors have been deeply investigated for several authors
(Tredici, 2004), (Hsieh & Wu, 2009), (Valiorgue et al, 2011), among others, in commercial
applications is not wide used due to the great difficulty of scaling and the intensive
material requirements. More recently, flat plate PBR´s have been redesign by using
plastic bags located between two iron frames. In this way, a substantial cost reduction is
achieved, and the system can be set periodically inside water pools in order to control the
temperature (Sierra et al, 2008).
• Bubble columns
This type of reactor is characterized for being compact, low cost and easily of scaling. As
can be observed from Figure 1-11 construction of these reactors is simple and their
geometry allow a good mass transfer between gas and liquid phases, as well as good
controlling of operational conditions. Even when the surface exposed to solar radiation of
column PBR is no the highest respect to another type of photobioreactors, can be
consider an efficient light receptor (Martinez, 2009).
For high volume units, diameter of columns can reach 20cm and more; this leads to a
considerable large dark fraction in the middle of the cylinder. Evidently this becomes a
drawback since this fraction does not contribute to productivity, negative affecting the
growth of microorganisms (Posten, 2009). In order to overcome this effect, annular
reactor configurations have been developed. In that configuration, a concentric tube is
located in the middle of the column as Figure 1-11 (b) shows. The annular section can be
used in different ways depending on the most favorable result over the culture system.
The simplest mode is acting as a wrapped flat plate PBR in which the inner surface
provides additional space for the catching radiation. Since this surface probably does not
contribute significantly to the overall radiation, an external lamp can be fitted in this space
for improving the light capture efficiency of the reactor like in Figure 1-11 (c). Finally, the
annular section can be exploited as a mechanism of mixing the culture for providing
suitable light-dark periods with a small hydrodynamic stress for cells.
This is possible by means of an airlift configuration as shown in Figure 1-11 (d) where the
downcomer is arranged in the coaxial zone, while the riser is composed by the annular
region. As the riser remains most of the time in the dark, the cells flow through it thanks
to the introduction of bubbles in the lower part of the reactor, and then cells go down
receiving the radiation along the downcomer. In this way regular light-dark cycles in the
range of 1–100 milliseconds are promoted, achieving high productivities (Posten, 2009).
Figure 1-11. Bubbling column PBR. (a) simple bubbling; (b) Annular; (c)Internally
illuminated; (d) Airlift.
Design Considerations for PBR’s

A photobioreactor can be considered a three phase system formed by the liquid phase
who acts as a medium, the microalgae cells in solid phase and the gas phase providing
CO2. In general, the design of a PBR requires understanding of the interaction between
the environmental parameters and the biological response involved in the three phase
system as Figure 1-12 depicts.
Figure 1-12. Main interactions accounted for photobioreactors design. From (Posten,
2009)
State of the art 21
Between the most relevant aspects to take into account in the design of any PBR can be
mentioned:
• Light distribution
In photoautotroph microorganisms, culture light availability determines the photosynthesis
velocity and consequently their growth rate. Nevertheless in any culture system, cells that
are closer to PBR walls block light, hindering its penetration to the center of the reactor. In
fact, light cannot penetrate beyond few centimeters when there is a high density culture,
producing a mutual shading effect in those cells placed far away from the surface (Coral
et al, 2003). Clearly, the movement of cells along the PBR stimulates a periodical
exposing of them to light and darkness. This period ranges between 1 millisecond to few
seconds, and it is defined as the time a cell remains in the photic zone. In order to
understand this phenomenon it is essential the definition of the light path length, referred
as the distance a photon has to cover for passing through the PBR.
As long as the light pathway length exceeds the wall thickness, an exponential growth can
be found. Once biomass concentration gets higher values, only linear growth is
presented. This situation does not imply that the efficiency decreases immediately
because the mixing helps to achieve a proper light distribution. However, a fraction of the
total volume remains in the dark not contributing to productivity but to energy cost
(Posten, 2009). As it can be seen in Figure 1-13, the higher the light pathway length, the
higher the volume fraction in dark, due to the mutual shading. This indicates that is
convenient to keep short light pathway lengths (not higher than 3cm) guaranteeing a high
frequency light-dark cycles (Coral et al, 2003).
Figure 1-13. Light pathway and light-darkness cycles. Adapted from (Coral et al, 2003)
• Mixing
Mixing is essential for microalgae growth since a proper culture mixing favors the gas-
liquid mass exchange, prevents the cellular sedimentation, maintains a stable regime
inside the reactor and it is crucial for providing a good light distribution. Actually, light-dark
cycles have a considerable effect on algae growth, as (Posten, 2009) summarizes,
benefits of mixing are not only in the improvement of mass transfer, but also in the
increment of the light-dark cycles frequency.
The major mixing/recirculation systems commercially used are described in Table 1-2. As
it can be seen, they can be split into pumping systems (normally employed when more
than one vessel is present), mechanical stirring (present when only one vessel is used)
and gas mixing (where the injection of CO2 in the culture is exploited for promoting
turbulent mixing and recirculation through the PBR). Even when mixing can be performed
by means of several options, airlift configurations are preferred because of their simplicity
and low mechanical damage caused to cells (Coral et al, 2003).
Table 1-2. Generic description of mixing methods in PBR´s. From (Carvalho et al, 2006)
Type Mixing Gas Hydrodynamic Scale-
Process
reactor efficiency transfer stress up
Pumping mode
Centrifugal fair low medium Easy
Positive
fair low medium Easy
displacement
Peristaltic fair low medium Easy
Diaphragm fair low medium Easy
Lobe fair low medium Easy
Mechanical stirring mode
Stirring with
CSTR Uniform Fair/High High Medium
blades
Gas mixing mode
Air-lift Uniform High Low Medium
Injection of gas
Bubbling Fair Fair Low Medium
• CO2 supply and aeration

Another important aspect that PBR improves is the feeding of CO2 for promoting
microalgal photosynthesis, as well as a suitable removing of the produced oxygen. In this
sense it is particularly important to have a reliable prediction of the CO2 mass transfer for
accurate design, scale-up and operation (Carvalho et al, 2006). It is well known that flux
rate from the gas to the liquid phase depends mainly on the diffusion kinetics, and in a
minor degree in some reactions in the liquid phase occurring in the vicinity of the
interface.
In any case the diffusion of CO2 in the medium can be explained by means of the two-film
theory as depicted in Figure 1-14. The mass transfer start from the bulk of the gas to the
State of the art 23
thin gaseous film at the immediate vicinity of the interface and then take place the
diffusion through this gas film. After this, CO2 passes across the gas/liquid interface and
then diffuses through the adjacent liquid film till arriving at the bulk of the liquid phase
(Carvalho et al, 2006). In that point, carbon dioxide can be metabolized for microalgae.
The overall resistance over the entire path distance can be calculated by adding up the
aforementioned single resistances.
Figure 1-14. CO2 Mass transfer coefficient between gas and liquid phases.
At the same time, the mass transfer rate of this process is proportional to the driving force
for diffusion and the available area for transfer. This proportionality corresponds to the so-
called global volumetric mass transfer coefficient kLa or kGa which is the sum of the
reciprocals of all resistances to transfer and can be established as Eq. (2):
∗

∗

(2)
Where
NCO2: mass transfer rate
kLa, kGa: Global mass transfer coefficient in liquid and gas phase
C*CO2, P*CO2: CO2 concentration and partial pressure in equilibrium from Henry’s law.
CCO2, PCO2: CO2 concentration and partial pressure measured in the bulk of the phases.
In the case of microalgal culture some characteristics of the bubbles affect the value of kL
indirectly, whereas others affect mainly a. Moreover, the amount of metabolites present in
the medium will play an important role since they also affect kLa since metabolites modify
the surface tension of the medium and thus act as an extra barrier to mass transfer
(Molina et al, 1999).
1.2.3 Productivity of microalgae cultures

As it can be seen until this point, there are numerous variables to be considered for
carrying out a microalgal culture. Perhaps the first item to be solved is the selection of the
microalgae strain in the case of monoculture, or the definition of the strains that could
exist in a multiculture system. This selection can be inherent to the environmental
conditions when culture is developed in mediums like natural open systems or
wastewater, where it is advantageous to work with the presented indigenous
microorganisms in the medium. On the other hand, strain selection can be also made
based on the final purpose of the microalgal biomass, as well as the characteristics of
each microorganism (Oncel, 2013).
In any case, one indicator that collects the behavior of the whole system for a particular
strain is the productivity, measured as volumetric biomass productivity (Pvol) or areal
biomass productivity (pArea). These performance indexes are commonly used to define
how much microalgal biomass is produced per unit of reactor volume per day (gbiomass/L
day) and how much biomass is generated per unit of used area per day (gbiomass/m2 day).
This way of measuring the performance of a given culture system result helpful when
comparing different reactor configurations, sizes, operational conditions and strains. This
index is needed since all effects of those conditions are condensed in only one value.
A summary of Pvol for 20 of the most investigated microalgae strains is shown in Figure
1-15. In the figure, the green section corresponds to the most common values for each
strain, while the lines indicate the maximum deviation found within the processed data. As
it can be observed, there are a significant group of microorganisms that independently
from the operational conditions and reactor type, achieve a volumetric productivity always
concentrated in narrow ranges (small green bars). On the contrary, other group like
Spirulina platensis, Schizochytrium limacinum, Porphyridium cruentum, Phaeodactylum
tricornutum and Chlorella protothecoides achieve productivities spread in a wide range.
Evidently, those trends are not only due to the particularities of each strain, but also a
consequence of selected operational conditions, and the type of reactor itself.
Considering this, the summary is only a first compilation of productivities among the
different found alternatives. On the other hand, it is remarkable to note how Pvol is lower
than 1 gbio/L.day in 14 out of 20 strains, being the lowest productivities for Isochrysis
(0.140), Haematococcus pluvialis (0.126), Chlorella vulgaris (0.1248) and chlorella
emersonii (0.0982). Additionally, from those strains who reach higher productivities like
Spirulina platensis, Schizochytrium limacinum, Chlorella protothecoides and Chlorella
pyrenoidosa just the last one has a consistent Pvol around 3.385 gbio/L.day; the remaining
3 strains have a considerable variation from that ranges from 0.1 to 7.
State of the art 25
Figure 1-15. Volumetric productivity of some microalgae strains. Information extracted

from (Eriksen, 2008), (Mata et al, 2010), (Brennan & Owende, 2010), (Chen et al, 2011),
(Ho et al, 2011), (Suali & Sarbatly, 2012), (Lam & Lee, 2012), (Rawat et al, 2013), (Oncel,
2013) and (Bahadar & Bilal Khan, 2013)
These results can be also seen from the reactor design standpoint. As Figure 1-16 shows,
the productivity of different PBR configurations varies significantly. In terms of volumetric
productivity, flat plate PBR’s are in general the most efficient far away from airlift and
tubular configurations. This result is due to the high surface area available for light
catching of the flat plate reactors. However, given the high amount of single units required
for treating a given flow rate, the areal productivity of this reactor is no as higher as the
volumetric one. In this aspect, the simple configuration of bubbling column PBR’s reach
the higher values of Pareal.
Figure 1-16. Mean Pvol and Pareal for different PBR configurations. Information derived from
(Carlozzi, 2008), (Brennan & Owende, 2010), (Ho et al, 2011) and (Oncel, 2013)
1.3 Anaerobic digestion process

The anaerobic digestion (AD) is a complex biological process in which organic raw
materials are transformed into biogas and digestate by means of microorganisms acting
in absence of oxygen. Biogas is composed mainly by methane and carbon dioxide, but it
contains traces of hydrogen, hydrogen sulfide, nitrogen, among others. Meanwhile,
digestate is a sludge rich in nutrients like nitrogen, phosphorus, potassium, calcium, etc.,
characterized for concentrating the fraction of non-degraded organic materials. Since the
biogas stream has a substantial amount of CH4 (see Table 1-3) it becomes a latent
energy source for any application while digestate is commonly used as feedstock for
fertilizer production (Holm-Nielsen et al, 2009).
In effect, it can be stated that biogas covers a variety of markets that include electricity
generation, heat production and vehicle fuels. As Figure 1-17 illustrates, the raw biogas
can be treated by cooling, draining, drying and removing H2S in order to avoid its
corrosive effects for its direct use in boilers. For obtaining a high quality gas, this can be
either upgraded to natural gas standard and compressed (Biomethane 98% CH4), or it
can be employed as feedstock in hydrogen production via methane reforming (AEBIOM,
2009). On the other hand, digestate is usually dried and used as fertilizer or filling material
in construction and sometimes depending on the origin of the sludge, it can be directly
returned to the field as a soil improver. More recently there is an aim for exploiting this
State of the art 27
fraction by means of physicochemical processes like hydrolysis for producing ethanol,

and nutrient extraction for conditioning the remaining biomass as well as obtaining a
nutrient rich biofertilizer (Rigby & Smith, 2011).
Table 1-3. Typical composition of biogas. From (AEBIOM, 2009)

Compound Composition (% v/v)
Methane 50-75
Carbon dioxide 25-45
Water vapor 1-2
Carbon monoxide 0-0.3
Nitrogen 1-5
Hydrogen 0-3
Hydrogen sulfide 0.1-0.5
Oxygen traces
Anaerobic digestion can be carried out starting from a wide variety of feedstocks and its
options for utilization can be equally versatile. A general distinction can be made between
biomass from agriculture like byproducts (manure) or dedicated crops for biogas and
various waste streams. Typical industries with wastewaters suitable for AD are agro food
industries (sugar, potato, starch, yeast, pectin, citric acid, cannery, confectionery, fruit,
vegetable, dairy, bakery), beverage industries (beer, malting, soft drink, fruit juice, wine),
alcohol distilleries (sugarcane juice, sugarcane molasses, sugar beet molasses, grape
wine, grain), pulp and paper industries (recycle paper, mechanical pulp, sulfite pulp,
straw, bagasse), and other fields (chemical, pharmaceutical, sludge liquor, municipal
sewage, landfill leachate, acid mine water, etc.).
Figure 1-17. Typical routes for processing anaerobic digestion products.

From these sources, those with high moisture contents are the ones that reach the lower
yields per unit of treated feedstock, while dry organic feedstocks are commonly highly
productive as can be observed in Figure 1-18. On the other hand, it can be seen how the
methane content in the produced biogas ranges from 50% to 70% depending on the raw
material, but as it will be discussed after, both biogas yield and methane content are also
highly affected by the operational conditions of AD.
In general it can be stated that methane production depends not only on the
biodegradability of the organic waste, but also on other factors such as the operating
temperature, particle size of the waste, pH of the medium, presence of toxic or inhibitory
compounds in the substrate, and oxidation state of the carbon (Asam et al, 2011). In this
sense, variations in feed composition, concentration, or temperature can lead to
imbalances in microbial activity, resulting in changes in pH, gas production and
composition, and efficiency of chemical oxygen demand (COD) removal. Stability is a
primary concern in industrial digesters and may be controlled by addition of alkali as well
as control of feedstock composition and feeding rate or hydraulic retention time (HRT)
(Alvarez, 2003).
Figure 1-18. Biogas yield and methane content of different substrates. From
(Fachagentur, 2006)
1.3.1 Biochemistry of the AD process

Organic matter degradation during anaerobic digestion is based on diverse and complex
interactions between several groups of microorganisms. As a result of the
microorganism’s activity on organic matter, bacterial population grow exploiting the
State of the art 29
presence of an enormous amount of soluble macro and micronutrients derived from the
organic matter decomposition. AD process has been deeply studied from the biochemical
and microbiological standpoint finding that the process evolves in two main phases, a
non-methanogenic phase and the methanogenic stage. Classification of these phases is
commonly carried out based on the intrinsic characteristics of the microorganisms that
develop each step of the process as shown in Figure 1-19.
Figure 1-19. Stages of the anaerobic digestion process.
These microorganisms population are characterized by different growth rates, variable

sensitivity to inhibitory components, and their efficiency is pH dependent. This implies
each step presents a different rate of reaction and the accumulation of intermediates
compounds hinder the development of subsequent stages (Fachagentur, 2006). In
general, the rate of the global process is defined by the limiting step the (slowest one)
which depends on several operational variables as well as the raw organic material
composition. For soluble substrates, the slowest phase is commonly the methanogenesis,
while for solid substrates the limiting step is the hydrolysis.
Hydrolysis
During this step the complex molecules presented in the organic raw material, composed
by proteins, lipids and carbohydrates are broken down into amino acids, free fatty acids
and sugars respectively. This process is developed by exoenzymes released thanks to
the presence of anaerobic facultative bacteria like Enterobacteriacea, Bacilus,
Bacteroides, Micrococcus, Peptostreptococcus, Clostridium, Propionibacterium (Taricksa

et al, 2009).
Acidogenesis
This stage allows soluble organic molecules to be catabolized by a set of microorganisms
like Butyrivibrio, Propionibacterium, Clostridium, Bacteroides, Ruminococcus, Acetivibrio,
Bifidobacterium, Eubacterium, Peptostreptococcus, Peptococcus, Selenomonas,
Lactobacillus, Streptococcus, and members of the Enterobacteriaceae, which are
commonly presented in the medium. During this process microorganisms produce acetic
and formic acid, hydrogen and carbon dioxide that are products able to be used by
methanogens. Moreover, this fermentation step also produces intermediate compounds
like propionic, butyric and valeric acids (VFA) that has to be further oxidized in order to be
ready for the methanogenic step. Under ideal conditions it is expected that acetic acid, H2,
and CO2 are the major constituents, but if those conditions are not achieved, volatile fatty
acid accumulation and H2 production occur (Appels et al, 2008).
Acetogenesis
In this stage the intermediates compounds, mainly VFA and alcohols are converted into
acetic acid, hydrogen and carbon dioxide. This step proceeds taking into account a
division of the microorganisms present in the medium, those that are not obligately
proton-reducing, that is, hydrogen-producing, species and those that do reduce protons to
hydrogen obligately during acetogenesis (Appels et al, 2008). The first group is broad,
including the homoacetogens and species that may direct their metabolisms to proton
reduction in the presence of an efficient hydrogen-removing system. Some of the most
common species are Acetobacterium, Acetoanaerobium, Acetogenium, Butyribacterium,
Clostridium, Eubacterium, and Pelobacter (Massi, 2012). This facultative change in
metabolism has been demonstrated in defined methanogenic co-cultures degrading
alcohols, lactate, pyruvate, cellobiose, glucose, fructose, and cellulose.
On the other hand, obligate proton-reducing acetogenic bacteria can only be grown in an
efficient electron-removing environment, for instance, in monoxenic culture with a
hydrogen-removing or formate-removing species. The simplest mixed culture involving
this type of ‘mutualistic’ interaction is a culture containing the acetogen and a hydrogen-
removing bacterium such as a methanogen. Other obligate proton-reducing acetogens
have been described: Syntrophobacter wolinii degrades propionate, Syntrophomonas
wolfei degrades butyrate, and Syntrophus buswellii degrades benzoate (Massi, 2012).
Methanogenesis
The final step consists in the transformation of acetic acid, hydrogen and carbon dioxide
into methane via acetoclastic methanogenesis, and hydrogenotrophic methanogenesis.
The first is responsible for the conversion of acetic acid into CH4 and CO2, while the
second contribute to the oxidation of hydrogen produced by the secondary fermenting
bacteria and reduction of carbon dioxide to methane (Taricksa et al, 2009). The most
common species of methanogens that use H2 and CO2 as substrate found in anaerobic
State of the art 31
digesters are Methanobacterium bryantii, formicicum, wolfei, thermoautotrophicum,

uliginosum, thermoalcaliphilum, thermoaggregans; Methanobrevibacter arboriphilus,
ruminantium, smithii; Methanothermus fervidus; Methanococcus maripaludis, deltae,
vannielii, voltae, jannaschii, halophilus, thermolithotrophicus, frisius; Methanomicrobium
mobile, paynteri; Methanogenium cariaci, marisnigri, olentangyi, tatii, aggregans,
thermophilicum, bourgense; Methanospirillum hungatei; Methanoplanus limicola among
others. In addition, the most frequent methanogenic species that use acetate as substrate
are Methanosarcina barkeri, mazei, acetivorans and Methanothrix soehngenii, concilii,
among others (Fachagentur, 2006).
1.3.2 Factors affecting AD process

Methane production can be considered one of the most used indexes for measuring the
efficiency of any AD process, but this index is prone to changes due to the variation of
operational conditions like temperature, pH and alkalinity, presence of inhibitor or toxic
component and the content of available nutrients as can be stated as follows.
Temperature
Temperature has an important effect on the physicochemical properties of the
components found in the liquid phase. It also influences the growth rate and metabolisms
of microorganisms and hence the population dynamics in the anaerobic reactor. 3 optimal
ranges for operating an AD reactor can be distinguished which define the growth rate of
the populations: Psychrophilic (0-20 °C), Mesophili c (20-40 °C) and Thermophilic (50-60
°C). As can be seen in Figure 1-20, higher temperat ures favor the development of the
microorganisms, but selection of operative temperature will depend on additional
considerations.
Figure 1-20. Growth rate of microorganisms in each temperature range.

Acetotrophic methanogens are one of the most sensitive groups to increasing

temperatures. Moreover, the temperature has a significant effect on the partial pressure of
H2 in digesters, hence influencing the kinetics of syntrophic metabolism (Taricksa et al,
2009). Thermodynamics show that endergonic reactions (under standard conditions), for
instance, the breakdown of propionate into acetate, CO2, and H2 would become
energetically more favorable at a higher temperature, while reactions that are exergonic
(e.g., hydrogenotrophic methanogenesis) are favored at lower temperatures (Appels et al,
2008).
Increasing temperature has several benefits including a better solubility of the organic
compounds, enhanced biological and chemical reaction rates, and a higher death rate of
pathogens (thermophilic conditions). However, the application of high temperatures
(thermophilic) has counteracting effects: there will be an increase in the fraction of free
ammonia, which plays an inhibiting role for the microorganisms at certain concentrations,
the energy consumption is high and affects the balance between energy supplied to
produced energy, and there is a trend of VFA accumulation even higher than in
Mesophilic conditions (Alvarez, 2003).
pH and alkalinity
pH plays an important role in the monitoring and control of AD because of the inhibitory
effects of low pH on the activity of methanogens. Anaerobic digesters operate optimally in
a pH range of 6.8–7.4, this considering the effect over the acidogens and methanogens
which grow best in a pH range from 5.5-6.5 in the first case and 7.8-8.2 in the second
case (Asam et al, 2011).
pH values lower than 6.2 considerably inhibit the methanogenic activity. In addition, pH
can produce several side effects. It controls the fraction of undissociated VFAs that are
thought to freely permeate the cellular membrane of microorganisms. After permeating
the membrane, the fatty acids internally dissociate, thus lowering the cytoplasmic pH and
affecting bacterial metabolism. Therefore, the occurrence of low pH is the result of a well-
developed imbalance in the anaerobic biomass (Taricksa et al, 2009). A control measure
is usual to reduce the loading rate and employ supplement chemicals to adjust the pH. In
this sense alkaline chemicals such as NaHCO3, NaOH, Na2CO3, quick lime (CaO), slaked
lime (Ca(OH)2), limestone (or softening sludge) CaCO3, and NH3 can be used.
On the other hand, buffer capacity is often referred to as alkalinity in AD, which is the
equilibrium of carbon dioxide and bicarbonate ions that provide resistance to significant
and rapid changes in pH, and the buffering capacity is, therefore, proportional to the
concentration of bicarbonate. Buffer capacity is a reliable method of measuring digester
imbalance. Increasing a low buffering capacity is best accomplished by reducing the
organic loading rate (OLR), although a more rapid approach is the addition of strong
bases or carbonate salts to remove carbon dioxide from the gas space and convert it to
bicarbonate, or alternatively bicarbonate can be added directly. A more sensitive
State of the art 33
parameter for monitoring digesters and measuring process stability is the VFA/alkalinity
ratio: when this ratio is less than 0.35–0.40 (equiv. acetic acid/equiv. CaCO3), the process
is considered to be operating favorably without acidification risk (Appels et al, 2008).
Nutrient content
It is evident that the cellular biomass responsible for the development of the AD process
requires a certain amount of mineral nutrients, carbon and energy for metabolizing them.
Among the most important nutrients, essential for the development of the process are
nitrogen, sulfur, phosphorus, and some micronutrients like iron, nickel, Cobalt, Selenium,
among others. In general, the presence of those nutrients is guaranteed since they are in
a higher proportion than needed for the bacteria populations, and only a small fraction of
the total COD (4-10%) is removed by conversion in biomass (Taricksa et al, 2009).
Inhibitor and toxic substances

A substance may be judged inhibitory when it causes an adverse shift in the microbial
population or inhibition of bacterial growth. Inhibition is usually indicated by a decrease of
the steady-state rate of methane gas production, accumulation of organic acids or
deficiency in the organic matter removal. Those substances can be part of the feed
material or can be generated as by-products in some metabolic conversions. In some
cases microorganisms can overcome the inhibition once they adapt their biological
mechanisms by means of acclimatization, antagonism or synergy. In the first case
microorganism can adapt itself to the inhibitory substance if this latter is presented in the
medium in small concentrations. Antagonism occurs when there exists another secondary
substance that reduces the effect of the inhibitory compound, and synergy is the ability of
microorganisms of exploiting the different conditions in the medium in order to overpass
the limitations imposed by the inhibitor (Bohutskyi & Bouwer, 2013). Between the most
important inhibitor compounds are the free ammonia, sulfur, and some organic
hydrophobic toxics like chlorophenol, aliphatic halides and long chain fatty acids.
HRT, SRT AND OLR

The other set of operational parameters that defines the efficiency of the AD process are
related to the hydraulic residence time (HRT), sludge residence time (SRT) and organic
loading rate (OLR). HRT is related to the average time that a soluble molecule fed into the
reactor remains inside it that is also the time available to microorganisms to degrade this
organic matter. It is defined according Eq. (3) as:
$%&'()*(+
"# ./012 (3)
,&-
On the other hand, SRT quantifies the average time that a particulate (molecule/particles)
fed into the reactor spends inside it, that is also the time available to microorganisms to
remain in the reactor.
4566 78 679:; :< ;:=>6?>@

3"# ./012 (4)
4566 A56?>@ B>@ ;5C
Each time solids are withdrawn from digester, a fraction of the bacterial population is
removed thus implying that the cell growth rate must at least compensate for the cell
removal rate to ensure steady state and avoid process failure (bacteria wash‐out). When
several microorganisms should coexist, the SRT is fixed on the basis of the slowest
growing ones.
The importance of these parameters can be seen from the fact that if HRT is high, the
fraction of organic material degraded increases, but the biogas production will be below of
the optimal value. On the contrary, if HRT is too little biodegradation is incomplete, and a
significant amount of degradable substrate will remain in the digestate. At the same time
HRT has to be fixed high enough for obtaining a proper SRT or the system has to be
conditioned with a solid-liquid separation unit in order to recover the solid fraction for
recirculating it inside the digester.
In this point a third parameter has to be taken into account, the OLR that is defined as:
F+' H=
E" $ G;5C.L
IJ
MN (5)
%&'()*(+
It quantifies the mass of substrate that enters the digester per unit of time and per unit of
digester volume. If OLR is high, it means a large amount of organic material is fed into the
digester and probably there is not neither enough microorganism population nor time for
developing the complete degradation. In that scenario, a VFA accumulation occurs with a
consequence pH drop, causing the stopping of the degradation in the acidogenic step. On
the contrary, if ORL is low, biogas production is not optimal and can be improved.
1.3.3 Potential of microalgae species for producing methane

The preferred way of measuring the ability of a given organic material for producing
biogas via anaerobic digestion is by means of the so call biochemical methane potential
(BMP). This parameter is an index of how much methane can be produced for a given
organic substrate during its disintegration in the AD process. The first approximation is the
theoretical BMP (BMPth) which is a computation based on the stoichiometry of conversion
of the organic compound to CH4, CO2 and NH3 as Eq. (6) states. In this estimation it is
assumed that reaction is completed, there is no biomass synthesis and consequently, all
COD is transformed into methane.
5 O P ; + Q → Q + QR + Q R (6)
BMPth can be computed according to the Buswell and Boruff relationship, in which
coefficients α1 to α4 can be estimated as:
State of the art 35
4 T 2U + 3/ 4 + T 2U 3/
Q ; Q
4 8
4 T + 2U + 3/
QR ; Q /
8
Consequently the BMPth will be given by:
^_`abcbM% M
\ hLij
YZ?[ d
fL g ^
k (7)
H=F+'.]. 5eOePe ; H=IJ
Where Vm is the molar volume of methane at the temperature and pressure conditions
(e.i. 22.14L/mol at 0°C and 1 atm). By means of Eq. (7) it is possible to estimate the
volume of CH4 depending on the amount of volatile solids (VS) available in the substrate
being digested. Nevertheless Eq. (7) overestimates the gas production due to the
assumption of 100% conversion of the volatile solids to biogas, and it does not take into
account the carbon needs for bacterial cell maintenance and anabolism (Ward et al,
2014).
Unfortunately, BMPth can be only used when the elemental composition of the organic
material is known, reason why a second index, based on an experimental assay, is
commonly used for that organic material in which is difficult to estimate their composition.
The BMP experimental (BMPexp) is a laboratory test in which is assessed the potential of
the substrate for producing methane controlling the conditions of the AD (Ward et al,
2014). In Figure 1-21 the BMP of some microalgae strains computed with Eq. (7) based
on average compositions or values found in the literature can be seen.
Besides the convenience of using BMP as guide for measuring the potential of a
feedstock, BMP results an index that only predict the maximum amount of methane that a
given organic material could generate through AD. Since BMP test is carried out under
ideal conditions and considering high HRT, the results can be far away from the situation
found under operative conditions of the anaerobic digester. This means that a better
parameter for assessing the AD process is the methane yield at the operative conditions.
As it can be observed from Figure 1-22, the methane yields for several microalgae strains
was considered based on a compilation of different results. The first conclusion from this
information is that a mean reduction of almost 50% in the methane production is found for
the different species if compared with the BMP of Figure 1-21. Evidently this a case
dependent result since yield can vary substantially depending on the particular operating
conditions. This can be better perceived analyzing the variation that this parameter gets
from 100 until nearly 500 mLCH4/gvs for most of the examined strains.
Figure 1-21. Theoretical Biological methane potential of some microalgae species.

Information derived from (Sialve et al, 2009), (Bohutskyi & Bouwer, 2013), (Ward et al,
2014)
Figure 1-22. Methane yield of some algae strains. Information extracted from (Mussgnug
et al, 2010), (Frigon et al, 2013), (Bohutskyi & Bouwer, 2013), (Lakaniemi et al, 2013) and
(Ward et al, 2014)
State of the art 37
1.4 Integration of microalgae growth with AD

As detailed before, the potential of microalgae for supplying an AD process can be
exploited in several ways. From the large range of configurations that can be used for
integrating these two processes in a WWTP just a few have been studied as a single
system. One of them correspond to the work of (Uttley et al, 2011) in which the modeling
of an integrated process as illustrated in Figure 1-23 is developed. In this case the aim is
the use of microalgae as a tertiary treatment for improving the effluent quality as well as to
produce microalgal biomass able to be used in the AD process of the plant.
Figure 1-23. Scheme of integration AD-microalgae-wastewater proposed by (Uttley et al,

2011)
In this configuration the main characteristic is the use of the treated wastewater for
feeding the PBR, which can be translated in a lower supply of nutrients, since it is
expected that a significant amount of them have been recovered in the former steps of the
treatment. Evidently this arrangement provides a high COD, Nitrogen and phosphorus
removal, but a very low biomass productivity (0.2 g/d m3).
A second alternative investigated by (Yuang et al, 2012) include the combination of the
effluent stream with a portion of centrate derived from the sludge treatment as presented
in Figure 1-24. In this way the algae growth system is fed with more substrates,
increasing the potential of producing microalgal biomass since centrate contains around
15% to 20% of the total N load of the WWTP.
Figure 1-24. Scheme of integration AD-microalgae-wastewater by (Yuang et al, 2012)
One of the disadvantages of this configuration lies in the latent adverse effect of NH4+ on
algae growth, if ammonium concentration reaches levels in which inhibition is
predominant. In that point, the difficulty of controlling conditions of the culture feed, put the
microalgae growing in a unfavorable position. On the other hand, it is essential to assess
the convenience of including all the wastewater effluent or a fraction of it in the algae
culture for guaranteeing the quality of the latter. A possible negative effect due to the
mixture with centrate is the loss of quality at the end of the process.
A similar consideration has been proposed by (Sahu et al, 2013) but considering that
nutrient sources for algae growth can also be extended beyond municipal wastewater. As
it can be seen in Figure 1-25, integration of the processes can be carried out collecting
nutrients from different sources and then use them for algae culture.
State of the art 39
Figure 1-25. Scheme of integration AD-microalgae-wastewater according (Sahu et al,

2013)
Depending on the availability, supplementary sources of nutrients can cover from cattle
manure till fresh human urine which can be properly mixed to achieve a suitable substrate
for the algae culture. This could represent many benefits for the whole process since
uniformity of feed is nearly assured due to the different sources, but as a global result
implementation of this configuration improves the quality of all influents producing more
energy in the CHP unit.
In the present work, the integration has been evaluated considering an intermediate
configuration among the aforementioned. Figure 1-26 presents a block diagram of Bresso
WWTP including the PBR for algae growth. As it can be seen, the integration is made by
means of the use of the centrate stream derived from the dewatering unit. This stream
contains an important load of N, and depending on the influent, amount of phosphorus
can be enough for algae culturing. Furthermore, considering the positive effect of a PBR
in the middle, final centrate is expected to be cleaner than the original one, improving the
quality of the discharge.
Figure 1-26. Proposed scheme of integration in Bresso plant.
An additional consideration that can be assessed is the further integration of processes

for algae harvesting. It can be expected that if solids content of the culture is such that it
can be mixed with the main stream entering the primary settling, this unit can be enough
for separating algae in the primary sludge. Clearly this condition is ideal since avoids the
use of a new separation unit devoted to algae concentration, but it will depend on PBR’s
efficiency of producing microalgal biomass.
2. Mathematical models for algae growth
Mathematical models are representations of physical, chemical, biological, etc.,
phenomena which are usually focused on a set of convenient properties and
characteristics of the latter. Models are essential in almost all engineering process and
they allow us to predict the system behavior based on three different approaches
(Hermanto, 2009):
• First-principles (or white-box) models, which are derived from well-known physical and
chemical relationships, reflecting the underlying principles that govern the process
behavior.
• Data-driven (or black-box) models, which are of empirical nature (e.g. artificial neural
networks, time series).
• Hybrid (gray-box) models: a combination of the above.
In the case of describing a PBR through a mathematical model, the first step is the
identification of the different components involved in the process as stated in Figure 2-1.
Figure 2-1. Operating variables of a PBR for algae growth.
In Figure 2-1, Q and Q0 are the incoming and outgoing liquid flow rate [Ls -1], X0 and X are
the algae concentration [gL-1], Si0 and Si are the substrate concentration of nutrient i at the
inlet and outlet [gL-1], V is the photobioreactor volume capacity [L], I0 the light intensity
[µmol.m-2s-1] and RCo2 the input rate of CO2 from the gas stream [gs-1].
2.1 Mass and energy balances on PBR

Considering the system presented in Figure 2-1 we can perform the mass balances for
each species. In the case of microalgal biomass we can write:
l11 UUlmnopq l11 rns:< l11 rns7t? ± l11 v/mUopq/Uq1mlvopq
/.fx2
y x yx + "f
/o
In which R represents the term of production of biomass (rate of reaction). Moreover,

assuming that the PBR behaves like a perfectly stirred reactor, X can be stated as the
concentration inside the reactor, which lead as to:
/f /x
x +f y x yx + "f
/o /o
The change in volume can be established as the difference of incoming and outgoing flow
/f
y y
/o
Substituting we get:
/x
f y x yx + "f y x + yx
/o
If we consider there is no microalgal biomass in the incoming stream we conclude that:
/x y
" x
/o f
This result indicates that algae concentration depends on the so-called dilution factor
(Q0/V) and the rate of reaction R, which can be expressed in terms of the specific growth
rate µ as:
;z ,}
;?
x {| $
~ (8)
Similarly, we can develop the mass balances for Nitrogen and Phosphorus obtaining:
; ,}
3 3h x (9)
;? $
; ,}
3 3 x (10)
;? $
Where YN and YP are the yield coefficient for nitrogen and phosphorus respectively.
Finally, considering the mass transfer of CO2 given by Eq. (2) we can expect that the
mass balance for this component will be:
Mathematical models for algae growth 43
− 3P − 3P − x
;c ∗ ,}
$ c
;?
(11)
In which YC is the yield coefficient for carbon, kLa the mass transfer coefficient in the
medium, and C*CO2 the equilibrium concentration of CO2 derived from Henry’s law.
On the other hand, a global energy balance in the system can be written in terms of the
main variables affecting the change of energy inside the PBR. According to (Lee & Hui
2004), neglecting the energy as biomass in the inlet stream, it can be expressed as:
o Uℎq pq q0 = q0 T1T/ T0 Tpl11 − q0 pq morns Tpl11
f ;? = −
; ,z

(12)
In which dE is the increase in energy content of the culture per unit of volume [JL-1], A is
the illuminated surface area [m2] and Y is the overall growth yield of algae [gJ-1]
determined in terms of the maximum growth yield YG [gJ -1] and a maintenance coefficient
= +
m [Jg-1s-1] as:
L

(13)
Following the performed analysis of (Hulatt & Thomas 2011) the energy content of the
culture can be written in terms of the low heating value of the microalgal biomass (LHVb)
and its concentration in the medium (X), such that we can obtain from Eq. (12)
LHVb dt = - V {Y + μ ~
dX I0 A QX 1 m
V G
(14)
It is evident that the term dx/dt appears in both mass and energy balances, implying that
final concentration of algae is dependent not just on the specific algae growth rate, but
also on the incident energy over the PBR (I0). This interaction is more complex than
expected, since µ is function of several variables that include nutrients, CO2, pH, light and
temperature and should be properly articulated by means of kinetic models as follows.
2.2 Kinetic models for nutrients

The kinetic expression is the part of the model that relates the growth rate of algae to the
substrate concentration in a culture media. These equations provide an understanding of
biomass production and nutrient consumption rate, both essential for a proper PBR
design as well as a tool for predicting process performance, optimization and control
(Hermanto, 2009). The most commonly used kinetic models in biological, chemical,
pharmacological, and medical processes to describe saturation phenomena are the
Monod and Droop models (Molina et al, 1999). Many studies have been conducted for
finding those two model’s parameters for different species of algae like (Aslan & Kapdan
2006), (de Morais & Costa 2007), (Sasi, 2009), (Xin et al, 2010) and (Chojnacka &
Zielińska 2012). In general the Monod model describes the relationship between
microorganism growth and limiting factor as:
| = |L5
6
) e6
(15)
Where µmax is the maximum specific growth rate achieved at high, non-limiting conditions
and ks is the half-saturation constant defined as the factor concentration at which the
specific growth rate is half of the maximum. On the contrary, Droop model relates growth
rate to the internal nutrient content of a cell rather than the nutrient concentration around
the medium. The Droop model can be written as:
| = | L5 {1 − ~

(16)
Where kq is the limiting cell quota for the limiting nutrient and q is the cell quota for the
limiting substrate. Both kq and q are commonly expressed as the total amount of nutrient
per cell, which is a better index of the nutritional status of the cells than global
concentrations. Nevertheless, the cell quota of individual species cannot be measured
easily under natural conditions, reason why this model results inappropriate for reactor
design.
In the case of kinetic models involving nitrogen effects, a significant amount of

developments have been focus on the use of Monod equation or variations of this model
based on a correction of the inhibition effect at higher substrate concentrations, like the
Haldane model (Zhang et al, 1999). However can be found some other models that
predict the experimental data under particular culture conditions or accounting for different
parameter than µ as can be seen in Table 2-1.
Table 2-1. Kinetic models for nitrogen dependent algae growth.

Model Description Reference
sh
Simple Monod model
μ = μ ¡¢
k ¥,§ + sh ks,N: Half saturation coefficient
µmax: Max. Specific growth rate (Aslan & Kapdan 2006)
(Xin et al, 2010)
s N: Nitrogen concentration
sh
Modified Monod model
R = "L5
k ¥,§ + sh ks,N: Half saturation coefficient
Rmax: Max. nitrogen uptake rate
(Smith, 2002)
Table 2-1 (Continuation). Kinetic models for nitrogen dependent algae growth.
sh
Haldane model
μ = μ ¡¢
1
µmax: Max. Specific growth rate

k ¥,§ + sh + h ks,N: Half saturation coefficient
(Zhang et al, 1999)
:,h ki,N: Inhibition coefficient
,h
Simple Droop model
| = |L5 ©1 − «
q
µmax: Max. Specific growth rate (Goldman & Mccarthy 1978)
Kq,N: Limiting cell quota Nitrogen (Yamaguchi et al, 2008)
q: Cell quota of nitrogen
Similarly, some of the developed models for Phosphorus focus on the basis of Monod
model and its modifications, but a significant concern over Droop model has been posed
from authors like (Flynn, 2002) and (Yao et al, 2011) who have established modification
that fit properly to the experimental data of their studies.
Table 2-2 Summarizes a list of models employed for describing the effect of phosphorus
in algae growth.
Table 2-2. Kinetic models for phosphorus dependent algae growth.

s
Simple Monod model
μ = μ ¡¢
(Aslan &
k ¥,¬ + s
µ max: Max. Specific growth rate
Kapdan 2006)
ks,P: Half saturation coefficient
(Xin et al, 2010)
sP: Phosphorus concentration
s
Haldane model
μ = μ ¡¢
1
k ¥,¬ + s +
:,
(Zhang et al,
ks,P: Half saturation coefficient
1999)
ki,P: Inhibition coefficient
sP: Phosphorus concentration
Simple Droop model
k ,¬
μ = μ ¡¢ ©1 − «
µmax: Max. Specific growth rate (Sommer, 1991)
q
Kq,P: Limiting cell quota (Yamaguchi et
Phosphorus al, 2008)
q: Cell quota of phosphorus
Flynn-Droop model
μ
Qp, Qm , Qh: Actual, minimum and
1 + KyB Q ¯ − Q (Q ± − Q )
= μ ¡¢
maximum phosphorus quota
k + Q¯ − Q
(Flynn, 2002)
Kq: Limiting cell quota
Phosphorus
kq: Fitting parameter
Table 2-2 (Continuation). Kinetic models for phosphorus dependent algae growth.
Yao-Droop model
T
T: Transport rate of phosphorus.
Q´
{1 − ~
km: Half saturation coefficient
s¬ Q
= μ ¡¢ Q k ¯
k p: Fitting coefficient (Yao et al,
s¬ + k Q
{1 − ´ ~ + k the curve
kq: Constant to control the shape of 2011)
Q
Qt, Qm: Total and maximum
phosphorus quota
All kinetic models related to the inorganic carbon concentration have been developed
based on Monod model. Previous work by (Caperon, 1978), (de Morais & Costa, 2007) or
(Tang et al, 2011), were aimed at calculating Monod’s parameters and optimal carbon
concentration for algae growth in carbon limiting cultures. In Table 2-3 is detailed the
description of this model.
Table 2-3. Kinetic models for carbon dependent algae growth.

sµ
Simple Monod model
μ = μ ¡¢
(Caperon, 1978)
k ¥,µ + sµ ks,c: Half saturation coefficient
(de Morais & Costa, 2007)
(Tang et al, 2011)
s c: Carbon concentration
2.3 Kinetic models related to light intensity

For most of the practical operations of photosynthetic cultures, availability and intensity of
light are the most important aspects controlling their productivity (Molina et al, 1999).
Usually, µ increases with increasing irradiance till a point in which µ is maximum (µmax),
and then further increase in irradiance tend to inhibit algae growth (photoinhibition). In this
sense Monod model has been widely used due to its simplicity but only when
photoinhibition is null or neglected. Once this phenomenon becomes important, Monod
model is not able to reproduce experimental results (Merchuk et al, 2007).
Besides the aforementioned models, there exist another set of models that suggest that
algae growth can be accurately expressed in terms of the average irradiance raised to
some power greater than unity, fitting experimental observations with high accuracy
(Martinez et al, 1997). Finally, a vast amount of models correspond to empirical
correlations where the predominant factor is the use of experimental data for fitting
parameters. In Table 2-4 a summary of some of the models employed in the

representation of algae growth by light intensity can be seen.
Table 2-4. Kinetic models related to light intensity and algae growth.
Simple Monod model
(Martinez, 2009)
I
µmax: Max Specific growth
| = |L5
(Hermanto, 2009)
k¶ + I
rate
(Sasi, 2009)
kI: Half saturation
(Chojnacka & Zielińska,
coefficient
2012)
I: Light intensity
Haldane model
I
| = |L5

rate (Aiba, 1982)

k¶ + I +

kI: Half saturation (Merchuk et al, 2007)
coefficient (Hermanto, 2009)
k2: Inhibition coefficient
I: Light intensity
Exponential model
¸
| = |L5 ©1 − ¸]_¹ «
· rate (Vanoorschot, 1995)
I: Light intensity (Martinez et al, 1997)
Imax: Saturation light
intensity
I<
| = |L5
Power law model
< + I<
I: Light intensity (Molina et al, 1999)
n: Fitting coefficient
|
I> 5º I>
{1 − ~{ − ~
Muller-Feuga model
6 6 6
= 2|L5
Iav: Average light intensity
I
I
(Muller-Feuga, 1999)
{1 − > ~ + { 5º − > ~
Is: Saturation light intensity
6 6 6 Ie: Minimum light intensity

(1 + β)
Peeters & Filer’s model
7
| = 2| L5
I: Light intensity

{ ~ + 2¼ + 1
Io: Optimum Irradiance
7 7
(Bouterfas et al, 2002)
intensity
β: Attenuation coefficient
Notwithstanding, one the biggest concerns when using the previous relationships is the
definition of the value for I to be used. It is well known that irradiance will change inside
the PBR even if outdoor incident radiation remains constant, which means cells near
reactor walls will receive more light than the ones in the interior. This situation implies the
specific growth rate varies along position and time. According to some authors (Martinez
et al, 1997), (Fernández et al, 1997), (Masci et al, 2004), the use of an average irradiance
Iav allows to determine µav for a given volume of reactor. Iav can be seen as the light
received as a single cell randomly moving inside the culture, and it depends on the
external irradiance on the surface of the PBR, the reactor geometry, morphology and
concentration of the microalgae and the inherent absorption characteristic of the medium
(Molina et al, 1999).
A first and simple description of the light attenuation condition can be performed in terms
of Beer-Lambert’s law considering that light intensity decreases along the path length it
should cover. This analysis assumes that the direction of the incident radiation does not
change as it crosses through the culture, radiation is monochromatic and scattering effect
due to particles is negligible respect to absorption (Fernández et al, 1997). The simplest
geometry to be analyzed is a flat-plate PBR as shown in Figure 2-2 in which the light
gradient can be expressed with Eq. (17) as:
7t? = 7 ·_z_½' (17)
Where I0 is the irradiance at reactor surface [µmol.m-2.s-1], L is the total path length [m]; ka
is the characteristic extinction coefficient of the medium [m2.kg-1], and Xalg the algae
concentration in the medium [kg.m-3].
Figure 2-2. Irradiance profile along a flat-plate PBR.
In this case the average irradiance can be estimated by integrating along the path length
¿ IÀ e·ÂÃÄÃÅÆ Ç dy
É
of the reactor as:
I¡¾ =
L−0
Finding that:
5º = 1 − ·_z_½'
¸F
_ z_½'
(18)
However, Beer-Lambert’s law approach fails in the sense of considering a monochromatic

light, in which case a correction can be made for using polychromatic light source by
computing an average ka to all wavelengths. Furthermore, the main disadvantage is that
this approach is not appropriate for high biomass concentrations because of the
scattering and selective absorption effects (Masci et al, 2004).
There exist another set of models including scattering and absorption phenomena
separately, like presented by (Aiba, 1982) or (Cornet et al, 1992). The latter, for instance,
uses two fitting parameters Ea and Es for describing both effects independently assuming
an isotropic light field across the culture. The main problem of this approach lies in the
need of sophisticated numerical tools and long calculations. But if culture has a high
concentration, Cornet’s model can be reduced to the monodimensional form, where
scattering is neglected due to the concentration level, finding that:
=
¸ \Ê
¸} > Ë (e\ Ê) ·> bË (·\Ê )
(19)
Where:
5
Q = Ì ; Q = x59= EQ (5 + 6 )
5 + 6
Another important consideration is the computation of the average irradiance in a more

commonly used reactor geometry like a cylinder. In that case light distribution becomes a
function of the total incident radiation at the surface I0, the optical properties of the culture
and the distance from the surface to the point of interest inside the reactor. For any
photobioreactor, the distance traveled by a direct ray from the tube’s surface to a point
within the culture (Pdirect) is calculated based on the position of the Sun, which determines
the point of incidence on the surface of the reactor, as well as the polar coordinates of the
point (ri, ϕ) in a cross-section of the tube as indicated in Figure 2-3.
If only the cross section of the cylinder is considered, some trigonometric relationships
can be used for computing the transverse light path (ai) which is a projection of the real
path Pdirect. Taking this into account we obtain:
;:@>P? = =
5& P76 Í Ò 6:< Ó·@& 6:< Ô
Î Î
P76{ ·ÏÐÑ ~ P76{ ·ÏÐÑ~
(20)

Where parameter ai can be stated as:

Ö cos Ù " cos Ú R sin ε rÖ sin φ

aÖ =
sin Ý cos°ω²
Figure 2-3. Incident radiation and its relationship with a given point inside the culture.
From (Molina et al, 1999)
Moreover, by means of Pythagorean Theorem and using the information of Figure 2-3
(Top view) the path traveled for any disperse ray (Pdisp) can be estimated as:
;:6B â°: 1pq Ù " 1pq Ú² + °: U1 Ù " U1 Ú² (21)
At this point, local irradiance can be calculated by any of the aforementioned approaches
(Eq. 17 and Eq. 19) considering contributions due to direct (IB(r,ϕ)) and disperse (ID(r,ϕ))
radiation in the light path.
The last step is determining the average irradiance along the PBR volume, splitting the
integration between the height and radius of the reactor in order to obtain the average
irradiance of the system (Iav). For this purpose we can expect that:
1
5º ã /f ã°ä + å ²/1
f 3
And splitting the surface integral into two variables we obtain:
1
5º ã ã ° + å ²//Ù
æ" Ò Ô ä
Finally,
5º = è¿Ò ¿Ô ä (, Ù)//Ù + ¿Ò ¿Ô ¿Ó å (, Ù)//Ù/Úé

çÒ ç
(22)
In this point, integration of the average irradiance can be performed numerically in such a
way it can be obtained a radial profile of iso-irradiance values as suggested by (Molina et
al, 1999), or simplifying to a single value that represents the average behavior of the
reactor.
2.4 Multiple factors kinetics

Since µ is affected by all the aforementioned conditions, each of them contribute to the
reduction of the maximum specific growth rate at optimal conditions µopt. Usually, the way
in which this multiple relationship is expressed involves a product function composed by
fractions representing each reduction due to each factor (Bouterfas et al, 2002). In
general we can write that:
|(, , , , #, v) = |7B? "¸ "h " " " ê "Bë (23)
Where RI, RN, RP, RC, RT and RpH are the growth rate reductions by irradiance, nitrogen,
phosphorus, carbon, temperature and pH. Evidently, each of the reductions will have their
form according to the way they affect the specific growth rate as presented in sections 2.2
and 2.3.
Typically, some of the reductions are neglected depending on the particular conditions of
the culture. Factors like temperature and pH are prone to be controlled which always
assure working at optimal conditions. In a lesser extent reductions associated with
substrates are negligible since it depends on the availability of each of them, as well as
their concentration variability on time. For this reason most of the previous works consider
just the reductions that directly affect the analyzed culture like (Zhang et al, 1999) in
which the specific growth rate has been defined in terms of the nutrients and pH effects
as:
S 1
| = |L5 ì îì î
3 [ v
k¥ + S + k ± + +
v [R
Or (Xin et al, 2010) in which the maximum population growth rate is just function of
nitrogen and phosphorus concentration following a double Monod model:
sh s
R = "L5 © «© «
k ¥,§ + sh k ¥,¬ + s
In Table 2-5 it can be seen some of the different values obtained by means of the
aforesaid kinetics models, as well as the main characteristics taken into consideration
during the experimental phase of each study.
Table 2-5. Summary of specific growth rates of previous studies.

µmax (day )
-1
Model Characteristics Reference
Batch culture, Chlorella p., I0 (2022 (Martinez et al,
2.66 Exponential
µmolm-2.s-1) 1997)
Power law Outdoor culture, P. tricornutum, I0 (Molina et al,
1.51
modified (1000 µEm .s )
-2 -1
1999)
Modified Batch culture, Isochrisys g., I0 (200 (Masci et al,
1.7
Droop µmolm-2.s-1) 2004)
Peeters & Batch culture, C. microporum, Iopt (390 (Bouterfas et al,
1.59
Filers µmolm-2.s-1) 2006)
Pilot Plant PBR, Euglena gracilis, I0 (Chae et al,
0.62-0.99 Simple Monod
(150-550 µmolm .s )-2 -1
2006)
Direct Bench scale PBR, Spirulina sp, CO2 (de Morais &
0.44
measurement level (0-12%). Costa 2007)
Semi-continuous culture, Chatonella
(Yamaguchi et
0.70 Simple Droop o., I0 (200 µmolm-2.s-1), NH4+(18.µM),
al, 2008)
NaH2PO4(1.8.µM)
Direct Batch culture, Chlorella p, 10% CO2, I0 (Tang et al,
0.99
measurement (180 µmolm .s )
-2 -1
2011)
Modified Batch culture, Chlamydomonas r., I0 (Tevatia et al,
0.27-1.8
Monod (200 µmolm-2.s -1), NH4+(0-18.7mM) 2012)
3. Experimental phase
In order to identify the main parameters which affect the performance of the microalgae
culture a set of experimental runs has been developed in a pilot plant scale. The aim of
those experiments is to obtain primary information about the behavior of the culturing in
order to correlate it with the parameters that allow the analysis of a complete integrated
mass balance. For this reason experiments were divided into four subsets, in such a way
it was possible to collect information about the dynamic and stable behavior of the PBR,
as well as the harvesting of the produced algae.
On the other hand, for complementing the analysis, a couple of bench scale tests were
performed in order to establish some of the operational parameters in the anaerobic
digestion process. In these experiments a reproduction of the sludge digestion was
considered as a first step, and also as a base for comparing with respect to the co-
digestion with algae. Figure 3-1 contains a short description of the methodology and
dependence of the experimentation process.
Figure 3-1. Methodology employed in the experimental part.

As it can be seen each of the four main experiments have a particular aim, starting with
the batch test in which the establishment of HRT and µ are the main outcomes. After
stabilization of the batch runs it is possible to continue with a steady state operation for a
better understanding of the effects of the multiple variables involved in the culture. In this
stage the removal efficiencies for nitrogen and the biomass yield, linked to the solid
content, are the main results. Thanks to the microalgal biomass produced in the
continuous operation of the PBR it is possible to assess two of the techniques associated
to the concentration of microalgal biomass. In this set of experiments, the mass and
volume efficiency derived of the separation process was determined. Finally, the
concentrated fraction was used as feed for the co-digestion process in which the TVS
efficiency and methane production were established.
3.1 Algae growth in batch operation

Batch runs were carried out as a first approach to the microalgae culturing since it is first
required to determine the growth characteristics of the medium. Beside the information
found in the literature, it is convenient to determine the time microalgae take to grow
under the particular conditions presented in the pilot plant PBR. Additionally, information
in dynamic operation helps to understand intrinsic characteristics of the medium like the
specific growth rate, inhibition phenomenon, among others.
These experiments were run in an 85L (effective volume) cylindrical plastic PBR located
adjacent to Bresso’s CHP as it can be observed in Figure 3-2. A complete description of
the reaction system can be seen in Appendix D, where the equipment diagrams are
explained. Batch runs were performed during the period from March 3 to May 26 2014.
For performing these batch runs the procedure described in Figure 3-3 was employed,
considering as the main monitoring parameters the total and dissolved solid
concentrations, nitrogen and phosphorus contents (nitrogen measured mainly as total
nitrogen and ammoniacal nitrogen) and absorbance of the medium measured at 665nm
following the procedure described by (Uslenghi, 2011). Furthermore, temperature, pH,
conductivity and dissolved oxygen were measured as additional reference parameters.
Total nitrogen and total phosphorus were measured using the persulfate method 4500-P J
of (Standard-Methods, 2005) while N-NH4+ was analyzed following the
spectrophotometric-indophenol methodology of (IRSA-CNR, 2004). Temperature, pH,
conductivity and D.O were measured directly on field by suitable probes, while solid
contents were determined following the methods 2540 B, D and E of (Standard-Methods,
2005) techniques. These analyses were carried out directly by members of the research
group on Bicocca University labs in Milan.
Experimental phase 55
Figure 3-2. Current location and appearance of the PBR pilot plant.
Start
Start
Use
Usethe
theflowmeter
flowmeterfor
for fixing
fixing
the volumetric flow rate IsIsttff achieved?
Let
Letempty
emptythe thecolumn-
column- achieved?
wash
wash ititwith
withwater
water
No
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QQgg adjusted
adjustedand
and Remove
Removecarefully
carefullytop
top
Adjust
Adjustthe
thebubble
bubblesystem
system stable?
stable? covers
coversand
andtake
take500
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putting
puttingititin
inthe
thebottom
bottom sample
sample
Yes
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fieldmeasurements
measurements
Fill
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thecolumn
columnuntil
until90%
90% Take of Trr,,TTamb
amb, Cond,
Cond,pH,
pH,D.O.
D.O.
Takeaa 500
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mLsample
samplefor
fortime
time
of
ofmax
maxcapacity
capacitywith zero and
andPAR
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with zerocharacterization
characterization
fresh
freshcentrate
centrate
Cover
Coverthe
thetop
top of
ofcolumn
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Use
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microalgaesln
sln Cover
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ofcolumn
column with
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and protective
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No
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Open
Openthe
thevalve
valveforfor achieved?
achieved?
supplying
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CO22 stream
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End
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Yes
Figure 3-3. Procedure for operating the pilot plant in batch mode.
In this first experimental stage, four tests were run letting the system to evolve for a period
of 11-16 days in which culture was exposed to the climate changes of the period of
evaluation. As it can be observed in Figure 3-4 both TN and TP maintain a stable

behavior remaining almost constant along the runs. It is evident that this condition is
deviated from the expected since those substrates are consumed in the growing process
for producing biomass, which lead us with a particular situation.
Figure 3-4. Evolution of TN and TP concentration during batch runs. (a) TN, (b) TP.
As observed, there is a parallel tendency in 3 out of 4 experiments, getting similar results

in both substrates in the first 3 batch tests. Batch 4 could be affected by external factors
that make it inappropriate for the analysis due to its deviated behavior, not just in the TN
and TP behavior, but in all determined parameters. Then, considering only the common
trend of the remaining 3 batch runs, the only conclusion we can state, is that there was
something in the analytical procedure followed for determining those parameters that
provide as a result the total content on the medium, including the produced biomass,
remaining in this way a constant value for these parameters along the runs.
The previous result can be better understood when ammonium concentration is analyzed.
In this case, it was found a consistent reduction during the tests, a clear indicator of the
consumption of the substrates. As shown by Figure 3-5, the behavior is the same in the
first 3 batch runs, decreasing at a similar rate until levels of 50-80 mg/L. In this sense, it
can be stated that N-NH4 results a more reliable monitoring parameter than the total
nitrogen content since reflects the reduction of the substrate due to the microalgae
growth.
Furthermore, this reduction has to be correlated to the biomass production. Thus, the
solid content helps us to identify how much biomass is being produced, since
microorganism will be counted as the only source of solids in the medium. Evidently, it is
important to determine which fraction of the total solids corresponds to dissolved and
suspended solids, since each fraction affects in a different way the growing process. In
average, during batch runs TS and TSS have grown at different rates, but there is no a
defined trend among the 4 runs as seen in Figure 3-6. Here, the effects of external
variables like weather are clearer than in the substrate consumption.
Figure 3-5. Evolution of N-NH4 concentration during batch runs.
Figure 3-6. Evolution of solids content during batch test. (a) TSS, (b) TS
In any case the biomass production can also be correlated to the absorbance of the
medium, which at the same time, has a strong link with the solids content. Similarly to was
found with solids, absorbance tends to increase in all batch tests as detailed in Figure 3-7.
This similitude between absorbance and TSS curves, confirming the direct relationship
among these variables. Until this point, we can define thanks to the previous results that
biomass seems to grow continuously till the 10th to 12th day, moment in which there are no
clear trends, since we can observe both further increases and reductions. We can think
this point is a border in which phenomenons like self-shading start to be significant, and
the consequent inhibition of the culture growth is present.
Figure 3-7. Absorbance of the medium during batch runs.
Moreover, based on the ammonium consumption, after the day 12, exhaustion of this
substrate reaches more than the 70% of the initial content, making more difficult the
growing since more microorganisms are presented in the medium, and the higher the
population, the higher the nutrients consumption for a suitable growth. This is more
evident when looking at Figure 3-8, where N-NH4 removal is determined for each batch
test.
Figure 3-8. N-NH4 removal on the different batch tests.

Correlated with the last result, the increase in solid content is also recognizable as
detailed in Figure 3-9. As observed, TSS concentration continuously grows and even
doubles its value after a period of few days in accordance with the expected behavior.
Figure 3-9. TSS concentration change on the batch tests.
However, as explained before, a high biomass concentration inside the photobioreactor

could be adverse for the performance of the culture since inhibitory phenomenons start to
be more relevant. Additionally, if we consider that microalgae growth follows a first kinetic
order, from Eq. (8) we can deduce that:
ï = ï ? ln ï = ln ï + |o
Moreover, taking into account that we can express the biomass concentration x in terms
of any of the measured variables, like absorbance or TSS concentration, we can establish
the mean value for µ in each of the performed runs by means of the previous
linearization. In fact, using the absorbance values for the different batch tests we find the
correlation shown in Figure 3-10, in which it is possible to observe a similar trend in runs
N°1 and N°3 with µ values of 0.387d-1 and 0.43d-1 and good coefficients of correlation. On
the contrary, for batches 2 and 4 the slope was lower (0.05d-1 and 0.109d-1 respectively)
as well as the fitting performance.
Considering the previous results, the expected hydraulic retention time for a continuous
operation, based on batches 1 and 3, would be close to 2.5 days (HRT:1/µ). On the
contrary, considering results of batches 2 and 4, this value would be between 10-20 days.
Then, since there is no a conclusive result, it was decided to fix a ten days period of
growing as HRT of the continuous test, expecting a subsequent reduction in order to
analyze the effectiveness of the culture under different conditions.
Figure 3-10. Linearization of the kinetic growth using absorbance as reference variable.
The obtained values are in a similar range than those found in a previous investigation of
the research group (Uslenghi, 2011) or by researchers like (Tevatia et al, 2012).
Nevertheless, these values are below many other results, when compared to information
reported in Table 2-5, indicating the need of improving the operative conditions of the
culture. Since substrates seem not to be a limiting condition, the main factor to be
exploited is the irradiance caught by the PBR considering that for the tested culture
system, this parameter changes considerably along the day, and even more in
accordance to climatic characteristics of the season. Particularly, for this study
temperature was in the range from 16.2°C in the fir st batch to 23.9°C in the fourth batch,
similar to the range of ambient temperatures achieved during the spring season.
Moreover, the mean pH along the batch was 9.05, 7.64, 9.04 and 6.38 respectively.
3.2 Algae growth in continuous operation

Once the last batch test was concluded, the system was adjusted for changing the
configuration to a continuous operation. For this purpose, the procedure depicted in
Figure 3-11 was followed. Moreover, as stated before, HRT parameter was established
initially in 10 days, which lead us the need of regulating the volumetric flow rate of
centrate in 5.9 mL/min. Similarly to the batch case, collection of information was
performed 2-3 times a week, considering the same monitoring parameters as in the first
case. This set of experiments was carried out within the period from 26 May to 31 July
2014.
Figure 3-11. Procedure for operating the pilot plant in continuous mode.
By operating the PBR in a continuous mode, it was expected to establish the typical
behavior of the culture as well as the productivity of biomass under steady state condition,
correlating the biomass production with the change in the solids content. Considering the
results on the batch experimentation for TN and TP, N-NH4 was taken into account as
monitoring parameter for determining the substrates consumption. For this purpose, along
with the samples collected on the PBR, the feed, composed only by the same centrate
produced in the dewatering section of Bresso plant was similarly characterized.
During the operation in continuous mode, it was possible to observe in a clearer way the
transformation of nutrients and light into biomass. One of the parameters that better
represent this behavior is the solid content as it can be seen in Figure 3-12. In this graph
the difference between inlet (Centrate) and outlet (reactor) conditions is clearly remarked.
Here the change in TS and TSS during the experiment was always positive, and
variations in the second part of the test (After day 30) due to a drastic change in the
added feed, show how important is this condition on the biomass production.
Figure 3-12. Solids content for the continuous operation.
However, the initial solids content was not the only factor that changed in this part of the
experiment, as can be seen in Figure 3-13, a sudden increase of substrates accompanied
the aforementioned rise. However, the final ammonium concentration was stable,
following a similar path that TSS.
Figure 3-13. N-NH4 behavior during continuous operation.
On the other hand, as mentioned for the batch runs, absorbance also reflects the
microalgae grow due to the existing link among TSS and this parameter. In Figure 3-14
we observe how absorbance seems to change erratically along the experiment, ranging
from values close to 1 up to values higher than 2. Surely these changes are more evident
considering that Beer-Lambert law remains valid only in a narrow range from 0.1 to 1,
point in which deviations of its linearity begin affecting the results. In this scenario, it is
better to perform a calibration among absorbance and solid content, in such a way both
parameters can be correlated. For sure, the effect of diluting a sample is less adverse
than measuring a concentrated one, thus getting a more reliable result.
Figure 3-14. Absorbance of the culture medium during continuous operation
Notwithstanding, a simple determination can help us to analyze how deviated from the
linear fitting are TSS and absorbance of the medium. Simply computing the ratio TSS/Abs
we can expect that along the experiment this ratio should remain constant. In Figure 3-15
the correspondence of both parameters in the algae culture is represented. As it can be
seen, we can state that inside the culture media this ratio moves in a narrow range, which
clearly indicate us that even when the split data seem to be not correlated, in fact they
really are.
In conclusion, the continuous operation has lead us to produced microalgal biomass, but
in order to perform the subsequent mass balances, it is important to define the main 3
performance indexes of the PBR. The first is related to the substrate consumption and
can be seen as a change in the substrate concentration, referred to its initial condition.
Thus, for instance, the ammonium yield (YN-NH4) will be a negative value given the
reduction in this substrate content.
Figure 3-15. TSS/Abs correspondence in the microalgae culture.
Similarly, the solid yields (YTSS and YTS) represent the change in the TSS and TS
concentrations respectively, and it is expected that those parameters are positive
according to the expression:
:
(ñ} )
0:äÒ = −1
:
()
In this sense the higher the solid yields, the higher the produced biomass, and
consequently more efficient the PBR is. In Figure 3-16 the behavior of these parameters
along the test is shown. As it can be seen, ammonium removal was considerably
consistent reaching an average of 76.2%, a little higher than the value reached during the
batch experiments.
Regarding the solid yields, it can be seen that there are two regions according to the
change in the feed after day 30. In the first part YTS has reached an average increase of
152.2%, while after the change in the centrate, this yield dropped almost half until 73.6%.
A different scenario is found for YTSS, which has no a defined trend in any moment,
showing extreme changes that range from 400% to 23000%.
Figure 3-16. Estimated yield parameters for the continuous experiments.
3.3 Anaerobic digestion tests

Anaerobic digestion runs were performed in a 2.4L batch reactor, equipped with a water
bath and automatic stirring system. The system was conditioned for operating in a semi-
continuous operation and collecting the produced biogas in a measurement unit fixing the
working temperature at 32°C. The AD system shown in Figure 3-17 was operated
following the procedure depicted in the diagram of Figure 3-18.
Figure 3-17. Sketch of the bench scale anaerobic digestion system.

This system allowed reproducing conditions of the actual AD reactor of Bresso plant.
Information collected is important to verify and complement the operational parameters of
the process. First, after a stabilization period of one month, the system was conditioned
for feeding only the same sludge fed in Bresso’s AD reactor, looking for a line base in
which the idea was to compute the actual TVS removal efficiency, and the methane
production. After concluding this phase, the system was modified for handling the co-
digestion of sludge-microalgal biomass adjusting the HRT, keeping constant the
volumetric flow rate of sludge (30 mL/day).
Figure 3-18. Procedure for operating the AD system in semi-batch mode.
Since the system is naturally batch mode, a 3 days/week frequency for supplying new
feed was implemented, while recovering the produced digestate in the same proportion.
In this way a semi-continuous operation is put in practice, simulating the operation of the
WWTP digester. Analysis made to the digestate consisted in the measurement of TS,
TVS, pH, T, TKN, TN, following the recommended procedures of (Standard-Methods,
2005), and FOS:TAC ratio using the method described by (Lossie & Putz, 2008). The
same parameters were used for characterizing the feed. On the other hand, produced
biogas was conducted through a plastic tube into a graduated cylinder filled with 1M
NaOH solution, as detailed in Appendix D, in order to absorb the CO2 fraction along the
cylinder length in such a way the displaced liquid volume mainly corresponds to CH4.
Results of this experimental run can be analyzed from two standpoints; first the
performance of the AD process when only sludge was fed, and once co-digestion was
implemented. The easiest comparison can be made by means of the produced methane
which was measured with the same frequency of feeding. Theoretically, the expected
result is to achieve a bigger amount of CH4 by co-digestion than only feeding sludge, but
this is not true if operational conditions in the AD do not remain optimized. It is evident
that the co-digestion implies a change in the organic fraction, mainly measured as a
change in OLR, but changes have to be compensated modifying other operative variables
in order to assure a suitable degradation of the new feed.
According to this, co-digestion was split in two phases in which not just the OLR was
changed, but the HRT of the medium was also changed for analyzing the effects on the
process. Then, from Figure 3-19 (a), when both runs are compared, it can be seen that
methane production is higher with only sludge fed reaching a mean absolute production of
7.6mLCH4/h. These results are found working at HRT of 20 days with an effective volume
of 1200 mL. From that plot is also evident that the rate of CH4 production seems to be
quite variable in time due to the semi-continuous operation, but in part (b) when the
complete period is evaluated, it is found the expected linear correlation between methane
volume and time for all runs. This allows us to conclude the convenience of using this
adapted system for reproducing the continuous operation, without losing accuracy.
At the same time, co-digestion was carried out changing the provided load, keeping the
ratio of fed sludge to microalgal biomass (Qs/Qalg) constant in a value of 7. This change
implies that the new feed is composed by 87.5% of sludge and 12.5% of concentrated
microalgae. During the first phase of co-digestion, digester was fed with 240 mL/week of
sludge-microalgae mixture in such a way the expected HRT was 35 days. Under these
conditions, CH4 production reached a mean of 5.8mLCH4/h as detailed in part b of Figure
3-19. After that, volume of feed supplied to digester was changed to 120 mL/week,
reaching an HRT of 70 days that promoted a methane production of 3.1mLCH4/h.
Besides this initial behavior in which it seems that co-digestion is not working as well as
sludge digestion, it is important to state that the absolute methane production is not the
most meaningful indicator, since these values change depending not just on operational
conditions, but depending also on the characteristics of the feed and the provided load. In
fact, we can determine the specific methane production (SMP) as:
lEë^ fë^
3Z g k=
$ l$
Where VCH4 is the volume of produced methane in the evaluated interval of time, and mVS
is the amount of volatile solids added in the same period.
Figure 3-19. Methane production during the AD tests. (a) Punctual values, (b) complete
period performance.
As it can be seen in Figure 3-20 (a), the SMP shows a different behavior than the
absolute methane production. In this case, digestion of only sludge allows an average of
179.2 NmLCH4/gVS (CV 0.3), while the co-digestion phases achieved 151.1NmLCH4/gVS (CV
0.36) and 217.4NmLCH4/gVS (CV 0.18) respectively. From this point of view, along with
changing the load of the digester, the change of HRT has a substantial effect on the
productivity of the process, altering not just the time available for degrading, but
promoting or not the adaptation of the microorganism to the organic matter to be
degraded.
Figure 3-20. (a) Specific methane production and (b) organic loading rate of AD runs.
Furthermore, once we observe the Figure 3-20 (b), we find that the lower absolute
methane production is a direct consequence of the lower OLR in the co-digestion phases,
but at the same time this allows us to conclude that the co-digestion process was able to
almost equalize the sludge digestion SMP, adding a lower amount of organic feed.
On the other hand, we can analyze the AD process from the digestate behavior
standpoint. In that sense, we first find that for only sludge feeding, after a period of
stabilization (almost one month), TVS removal remains in a narrow range surrounding the
27.3 %, considerably close to the mean value found for Bresso AD unit of 25.1%. As it
can be seen in Figure 3-21 (a), TS and TVS in the digestate maintain a similar
comportment with a mean TVS/TS ratio of 59%. On the contrary, once we analyzed the
co-digestion run, we find a slight difference in the removal efficiency, which achieves an
average of 38.2% obtaining a stream of approximately 10 g/L of TVS as Figure 3-21 (b)
details.
Figure 3-21. Feed and digestate solid contents (a) Sludge fed; (b)Co-digestion.
Moreover, it was identified a slight increase in the nitrogen content when co-digestion was
put in practice, changing from 244.9mgN-NH4/L in the sludge fed case to 265.5mgN-NH4/L.
This set of results indicate us that the addition of microalgal biomass, at least in the
previously defined quantities, could have a positive effect on the digestion since
degradability seems to be enhanced by the co-digestion, but in any case, this is only
verified once both processes are compared at similar OLR and HRT, which was not
possible due to the time required for performing each run. By now, the only indicator that
can be involved in this analysis is the individual behavior of the microalgal biomass for
producing methane through a BMP test. This was accomplished once a set of 3 replicates
of microalgal biomass sample were digested during 30 days at 35°C following the BMP
test by pressure monitoring methodology. From the developed BMP test, it was found that
concentrated microalgae have an average of 0.187Nm3/KgVS or 187NmL/gVS. Value
slightly higher than obtained by (Uslenghi, 2011) of 140NmL/gVS but significantly lower
than main results found in other investigations as detailed in chapter 1, 1.3.3, that ranges
from 250 to 350 NmL/gVS. At the same time, the obtained value is almost half of the
sludge BMP that rounds 300 – 350 NmL/gVS.
Finally, using the results of both tests, it was possible to estimate the anaerobic digestion
efficiency as detailed in Table 3-1 considering the average values of each run, as well as
the estimated BMP for the primary sludge and co-digestion feed. Efficiency was
considered in this study as the ratio between the actual SMP, and the maximum expected
3Z>B
methane production as:
ò ñå =
YZ
In which the BMP for co-digestion phases was considered as a weighted average based
on the content of the added feed. As observed, correlated to the higher methane
production, the efficiency of the process is also higher in the sludge fed case, even when
for microalgal biomass the estimated BMP is lower. This is a clear evidence of the need of
optimizing AD parameters for co-digestion, since initial configuration, besides its
effectiveness, results inadequate for the mixture digestion.
Table 3-1. Estimation of anaerobic digestion efficiency.

Parameter (main value) Sludge fed Co-digestion P1 Co-digestion P2
BMP (NmL/gvs) 300.0 285.9 285.9
SMP (NmL/gVS) 179.2 151.1 217.4
HRT (days) 20 35 70
3
OLR (kgVS/m d) 0.757 0.554 0.336
ηAD 0.597 0.528 0.760
3.4 Harvesting of produced algae

In order to identify the efficiency of two solid/liquid separation techniques for providing a
more concentrated microalgal biomass, some experimental runs were developed using
the same samples obtained in the continuous operation tests. It is important to collect and
concentrate microalgae for their further use since they are diluted in the PBR, and those
conditions are deleterious with the development of those further processes. Considering
this, sedimentation by gravity and centrifugation were tested as mechanisms for obtaining
the concentrated biomass.
For the sedimentation the monitoring parameter is typically the settleable solids (STS)
(mLSTS/L) measured in an Imhoff cone, but additionally it is required to determine how is
distributed the solid content in the two generated phases, this is the efficiency of
separation. Figure 3-22 shows the typical behavior of STS during continuous operation;
values range from 5 to 13 but it stabilizes near 8mLSTS/L. This first parameter indicates us
that sedimentation has a volumetric efficiency (ηv=Vconc/Vinitial) below 1%, which lead us to
just a fraction of the total processed volume.
But it is also important to know the TS and TVS content in the concentrated phase since
this is a key factor that affects the further AD process. We can establish here also the
mass efficiency (ηm) as the fraction of TS that ends up in the concentrated phase, or
lPê fP ê
P
òL = =
l:<:?:59
ê f?7? ê
:<:?:59
Where Vtot and Vc are the total volume (initial condition) and volume of the concentrated
phase, CTSinitial and CTSc are the TS concentration in the initial and concentrated solutions
respectively. Then, these two efficiencies clearly identify how much of the TS and TVS
contents can be separated, and how much volume we can get after the process.
Figure 3-22. Settleable solids of produced microalgae during continuous operation.
In the case of centrifugation, samples were put for 10 min at 4000 rpm and then
separated for measuring the same solid contents performed in the sedimentation case.
Since the volume of a single sample (50 mL) was considerably small for providing a
measurable concentrated phase, quantification of the concentrated volume was done only
after the treatment of 1200 mL. This volume was computed as the difference between the
total processed volume (Vtot) and the supernatant volume after separation (VSup):
fP = f?7? − f6tB
Figure 3-23 contains a summary with the results of the 7 run experiments. There, it can
be seen the measured TS and TVS content of the concentrated biomass, as well as the
computed mass and volumetric efficiencies.
It is evident the better performance of centrifugation respect with sedimentation in the

amount of collected solids, represented not only by higher ηm values, but for the
concentration of solid itself. While sedimentation achieves a maximum of 15.1gTS/L
(11.3gTVS/L), centrifugation process is able to achieve until 71gTS/L (56gTVS/L). However,
the mass efficiency is not higher than 16% in sedimentation and 52% for centrifugation,
which means there is still a significant amount of biomass remaining in the supernatant,
which will be lost.
Figure 3-23. Efficiency and solid concentrations after microalgal biomass harvesting tests.
In terms of the volume collected, results evidence a remarkable reduction from Vtot to Vc
with the consequent low volumetric efficiency. In the sedimentation, it is possible to
achieve until 1.9%, but centrifugation allows just an average of 1.2%. This indicator surely
can be enhanced if the initial concentration of microalgal biomass is higher than the
values obtained in this study. Beyond the previous results, it is essential to evaluate the
cost and energy consumption of these separation techniques, because they are factors
that affect the performance of the global plant. Other solutions like flocculation or filtration
can be tested and judged in order to find better results.
4. Integrated Mass Balance
Usually the complete analysis of a WWTP is made by using proper mathematical models
that describe each of the units of the process. The main characteristic those models have
to contemplate is to take into account the components of importance for the whole
process in such a way the entire behavior of the plant can be analyzed (Wentzel et al,
2006). In terms of mass balances, a WWTP can be seen as a set of individual unit
operations connected through a network of flows, which means outputs from upstream
units become inputs for downstream units. Moreover, another big concern is the use of
recycling from downstream to upstream in some of the units since it is commonly used
and affect the behavior of the plant in a significant degree (Ekama, 2009).
Simulation models for WWTP include a wide variety of options that depends on the detail
of the information, as well as the computation mechanisms. In general we can expect
mathematical model can be (Grau Gumbau, 2007):
• Holistic and Reductionist models: A holistic model tries to explain the phenomenon
by means of global parameters and general principles, while a reductionist model
describe the process with a great detail going from the elementary phenomenon to
the global one.
• Internal or external models: The first describe the answer of the system based on
the knowledge of the physical, chemical or biological phenomenon. The latter is
also known as black box model, in which a set of empirical relationships
determines the behavior of the system.
• Dynamic and stationary models: This classification talks about the time
dependence of the model. Meanwhile, dynamics model include the description of
the model during transient, stationary models only reproduce the responses of the
system during steady state conditions.
• Deterministic and stochastic models: In a deterministic model the response of the
system is known with precision given the condition at the inlet. On the other hand
a stochastic model represents the response of the system as a function of
probability, commonly expressed as a probability distribution.
The selection of the model should be strictly related to the aim of the investigation, and in
accordance with the available information for validate the model. Currently the most
effective way of simulating a WWTP is through models derived from the ASM1 (Activated
Sludge Model N°1) developed by (Henze et al, 1987) as well as the ADM1 (Anaerobic
Digestion Model N°1) established by (Batstone et al , 2002). Those models have the
advantage of including all materials that are relevant for the different operation units by
using a reductionist approach, built over a combination of well-known physical, chemical
and biological laws together with empirical relationships that make easier the computation
in some units. In any case this approach implies the deep use of numerical tools due to
the big amount of found interrelationships.
For instance in the original ASM1 the model is able to simulate the organic matter
elimination along with the nitrogen removal considering hydrolysis, organic matter
biodegradation by denitrification and under aerobic conditions, and nitrification of
ammonium species as shown in Figure 4-1.
Figure 4-1. Transformations included in the ASM1. From (Grau Gumbau, 2007)
Similarly, ADM 1 describes the anaerobic digestion through biochemical and physic-
chemical transformations, including the appropriate kinetics expressions and mass
balances according to Figure 4-2.
Integrated Mass Balance 75
Figure 4-2. Transformations included in the ADM1. From (Batstone et al, 2002)
Another set of simpler models for WWTP are based on the global and local mass
balances, in which operations are defined as black boxes using as main parameters the
removal efficiency or production yield of the different components. In this modeling
approach there is no need of advanced numerical tools since almost all expressions are
linear equations that can be solved by fixing some operative conditions (Spellman, 2004).
At the same time, the success of these models lies on the availability of information about
the operation of the real plant, since this information allows to determine the required
efficiency parameters, as well as to fix the recycle ratios and productivities. Only by using
real data from the analyzed WWTP this approach can be properly adjusted for simulating
the system. Evidently beyond the great advantage of a lower computational effort, the
main constraint is to validate the proposed modeling with experimental data and the lower
degree of detail of the simulation, since only can be determined conditions on the
streams, but no inside the process unit.
For the purpose of this work, a black box approach based on efficiencies of each unit was
used and it was adjusted and validated using the compilation of data of Bresso WWTP for
the period 2012-2013. The following segments include the development of the mass
balances in the different sections of Bresso plant and the inclusion of the PBR and its
harvesting unit.
4.1 Wastewater section

From the block diagram of Figure 4-3 it is possible to conclude that Bresso plant can be
split in sections according to the main components of the streams. For instance the
section comprising the wastewater pretreatment, primary and secondary treatment can be
considering a line of process devoted just to the water treatment as shown in Figure 4-5.
Figure 4-3. Proposed scheme of integration inside Bresso plant
As global outcome of this segment we obtain an effluent stream of known characteristics

and a sludge that is usually called primary sludge, which has to be subsequently treated.
Frequently, the characterization of the inlet wastewater is performed not at the very
beginning of the process, but after the pretreatment section (from bar screen to grit
removal) just before entering to primary settling tank. This is convenient since for
municipal wastewater it is avoided accounting for big pieces or residues that are not
representative of the liquid stream (Bonomo, 2008).
Primary
Primary
sludge
sludge
Wastewater
Wastewater In
In
Treated
Treatedwater
water
Biological
Biological stream
stream
Primary
Primary Secondary
Secondary
Bar Screen 1 Bar
Bar Screen
Screen22 Grit
Grit removal
removal oxidation
oxidation
settling
settling clarifier
clarifier
process
process
Fat
Fat && grease
grease
removal
removal
Figure 4-4. Process units composing the wastewater section in Bresso plant.
Taking into account the aforementioned consideration the units commonly analyzed are
the primary settler, the biological oxidation and the secondary clarifier. Since the aim of
this section is the depuration of the wastewater, it is well known that the main parameters
for monitoring the performance of the plant are the suspended solids (TSS), biological
oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), total
phosphorus (TP), ammoniacal nitrogen (NH4+) and microbiological parameters like E coli
(Bonomo, 2008). Those parameters, excluding TP and E. coli, are considered for the
analysis of the section. Furthermore, an extra parameter has been included for
accounting the total solids content of the streams (TS) which is an essential indicator for
the behavior of the subsequent sections.
The analysis of this segment can be started from a global mass balance that includes all
units as can be seen in Figure 4-5. In this section the global mass balance (in which water
is the main component) can be stated as:
ló ¸ = ló 6 + ló = ô¸ y¸ = ô y + ô y (24)
In which mi represent each of the mass flow rates of the streams I (wastewater inlet), S
(primary sludge) and E (Final Effluent), while Qi is the volumetric flow rate of the stream i.
Since the main constituent of the streams is water, and the other component’s
concentrations are always very small in comparison to water content, density of each
stream changes very little, and can be always assumed constant during the process and
equal to pure water density (ρI=ρS=ρE=ρ).
Figure 4-5. Streams of the wastewater section.
At the same time, the global mass balance for each component can be written as:
ló: = ló: + ló: = y¸ : = y : + y :

(¸) () () (¸) () ()
(25)
Where mi(j) is the mass flow rate of component i in stream j, and Ci(j) is the concentration
of component i in stream j.
Similarly, the total and component mass balances in each of the units can be performed,
recalling that global mass balance would be dependent on these unit mass balances, or
the same, that one of the unit balances is a linear combination of the remaining balances
with the global one. Then for the primary settling we have that:
ló ¸ + l@Pó M = ló 6 + l¸¸
ó = y¸ + y@PM = y + y¸¸ (26)
ló: + ló õ(@PM ) = ló: + ló: = y¸ : + y@PM : = y : + y¸¸ :

(¸) () (¸¸) (¸) (@PM ) () (¸¸)
(27)
While for the biological treatment unit:
ó + l@Pó = l¸¸¸
l¸¸ ó = y¸¸ + y@P = y¸¸¸ (28)
ló: + ló õ (@P ) = ló: ± "O? fO? = y¸¸ : + y@P : = y¸¸¸ : ± "O? fO? (29)
(¸¸) (¸¸¸) (¸¸) (@P ) (¸¸¸)
In which the component balance include the term RbtVbt that accounts for the extent of the
reactions involved in the oxidation process. This means the amount of component that
has reacted or has been produced during the biological oxidation. Finally, in the clarifier
we find that:
ó = ló + l@Pó Ê = y¸¸¸ = y + y@PÊ

l¸¸¸ (30)
ló: = ló: + ló õ (@PÊ ) = y¸¸¸ : = y : + y@PÊ : Ê

(¸¸¸) () (¸¸¸) () (@P )
(31)
Additionally to the previous balances, and in order to know the response of the system
under any variation in the inlet stream, we define some operative parameters that allow us
to interconnect the units. The first type of operative parameters is the unit efficiency, or
removal efficiency of the unit (ηu), where the component’s burden removal is evaluated
considering information compiled in Appendix A. For the separation units like the primary
settling and clarifier ηu is defined as:
ó
°÷-² ó
°øù*² °÷-² °øù*²
Lö ·Lö ,÷- & ·,øù*&
ò : ó
°÷-²
°÷-² (32)
Lö ,÷- &
In which ηki is the removal efficiency of the unit k for eliminating component i from the
main stream of the process (In). This means mi(Out) is the remaining load of component i in
the main stream. For the process involving reactions like the biological oxidation ηu can
be defined analogously as:
ó
°÷÷² ó
°÷÷÷² °÷÷² °÷÷÷²
Lö ·Lö ,÷÷ & ·,÷÷÷ &
ò O?
:
ó
°÷÷²
°÷÷² (33)
Lö ,÷÷ &
On the other hand, the additional relationships employed in this section of the plant are
the recycle ratios of the units. They can be specified in concordance to the fresh feed to
the unit as:
= L566 78 8@>6[ 8>>;

L566 78 @>PCP9>
(34)
In this point, it is convenient to use the degree of freedom analysis (D.O.F.) in the section
for establishing a solving procedure as well as for defining the additional parameters
needed for solving the mass balances. The first step is the definition of the total variables
of the analyzed system, divided into mass variables (number of streams) and component
variables (amount of compositions). As it can be seen in Figure 4-5 there are 8 streams
and for each one we need to determine 7 compositions per stream, which means 56
component variables. From the total of 64 variables we can define 2 mass variables
corresponding to streams I and S, and 7 component variables related to compositions in
stream I. This means we have a total of 55 unknown variables.
Then, we can state that for each process unit involved in the section we can write as
much mass balances as components exist in each unit plus a global mass balance. There
are 3 main units (Primary settling, biological treatment and secondary clarifier) which
account for 24 equations and an extra fictitious unit in which stream rc 1 is split into rc 2 and
rc 3 from which we can only write 1 mass balance expression. Accounting for those units
we can write 25 mass balances equations. Furthermore, starting from the assumption of
modeling the units by removal efficiencies, we can specify one efficiency for each
component in each unit, which lead us to 21 efficiencies equations. Until this point we
have 46 independent equations for solving 55 unknowns, which means we need to
establish 9 additional relationships. Two of those additional expressions are related to the
recycle ratio of stream rc3 and the split ratio of stream rc1:
@ = = ; 1@ = L ó =,
L+c
ó M ,+cM L+có ,+c
Ló ÷ ,÷ +c Ê +cÊ
(35)
The remaining 7 additional expressions are related to the very principle of the splitting
unit, in which we can establish that there is no change in concentration of the downstream
respect to the upstream. In other words, concentrations of streams comprised in the
splitting remain unchanged.
Table 4-1. Summary of degree of freedom analysis in wastewater section.

Mass variables Component variables Total Variables
Total variables 8 56 64
Known variables 2 7 9
Unknown variables 6 49 55
Balance equations - - 25
Efficiencies - - 21
Additional rel. - - 9
Total equations - - 55
D.O.F. - - 0
This result indicates it is possible to solve the complete section with the proposed
information. The procedure to achieve this goal can be summarized in few steps as
follows:
• First solve the global mass balance of the section, finding QE.
• By means of the additional relationships rr and sr solve Qrc3, Qrc2 and Qrc1.
• By means of the proper unit mass balances find the remaining streams II and III.
• By using the efficiencies of primary settling unit find the load of each component in
stream II. Similarly, using the efficiency of biological oxidation determine loads of
stream III and then find the final load in the effluent E employing the efficiencies of
the secondary clarifier.
• Solve the remaining component mass balances for determining the loads in the
recycled streams.
• Using the component mass balance in the primary settling determine the
composition in the stream S.
4.2 Sludge treatment section

Following the line of process that contains the primary sludge (S) we find the section
devoted to the treatment of this sludge as seen in Figure 4-6. The main stage is the
anaerobic digestion of the sludge, which produces a gas stream (G) containing mainly
methane and carbon dioxide, and a liquid stream usually called digestate (D) composed
by the non-digested substrates. Then in order to obtain a biosolid stream, with a
concentrated fraction of solids, digestate is treated in the thickening and dewatering units
obtaining streams bs (biosolid) and C (centrate). Due to the integration of the PBR, the
clarified stream C is conducted as feed to the PBR, where it is converted into an algal
diluted solution A0. That stream is going to be fed in the AD unit in co-digestion with the
primary sludge, reason why there exists a mix point before this unit.
The remaining process corresponds to a separation unit which aim is to concentrate A0

for providing a stream Ac with a higher biomass load than the original, producing as a by-
product a secondary centrate Cf composed mainly by water and some traces of the
components.
Figure 4-6. Units and streams composing the sludge treatment section.
This section can be split in 2 subsections containing the sludge treatment and the
microalgae culture as detailed by dash lines in the diagram of Figure 4-6. Given the main
characteristics of those processes, in this section only 4 components have been
considered for monitoring the performance of the treatment, total solids (TS), suspended
solids (TSS), volatile solids (TVS) and total nitrogen (TN). As done in the previous
section, all units are described in terms of the removal efficiency in the case of separation
units, and yields or productivities in the case of reactive units.
Since there is a recycle of stream Ac to the inlet of the anaerobic digestion, there exists a
close loop reference along the mass balances, reason why for solving the section it is
convenient to start from the DOF analysis in order to define the operation parameters that
make possible the solution. Table 4-2 contains a summary of DOF analysis of the section,
recalling that streams FG and FGC are not considered since they are supposed to be
treated independently in the next section.
Table 4-2. Summary of degree of freedom analysis in sludge treatment section.

Mass variables Component variables Total Variables
Total variables 12 44 56
Known variables 1 4 5
Unknown variables 11 40 51
Balance equations - - 27
Efficiencies - - 21
Additional rel. - - 3
Total equations - - 51
D.O.F. - - 0
As it can be seen there are 12 streams in the section, 11 of which contains 4 components,
and stream G whose composition is assumed to be known (See Appendix B). Under
these circumstances, the amount of compositions in the system is 44, entailing 56 total
variables. From the previous section, we can know the complete description of stream S,
obtaining 51 unknown variables. On the other hand can be delineated 3 separation units
and 2 mixing points in which can be performed 5 mass balances each one. Additionally, in
both reactive units (AD and PBR) can be written 2 total unit mass balances completing 27
mass balance equations.
For the main units (apart from mixing points) the description of the system has been
defined in terms of efficiencies per each component, which means they account for 20
additional relationships. At the same time, another efficiency defines the gas production
(stream G) completing a total of 21 new equations. This analysis gives us, as a result, the
need of implementing 3 relationships in order to solve all the units. According to this, the
final result is directly correlated with those 3 relationships, reason why it is essential to
define them based on the information compiled in the real operation of the plant.
First of all, let us define the mass balance expression in the analyzed section. According
to Figure 4-7, for the liquid streams we can expect that a global mass balance satisfies:
ó = lå
lú ó = ló + lO6
ó = yú = yå = y + yO6 (36)
Where the central assumption is the negligible loss of water due to evaporation of dilution
in the gas phase, considering the low content of organic matter.
Figure 4-7. Units of the section dedicated to sludge treatment.
In the meanwhile, for the thickening unit we can write the local and component balances:
ó = låó ¹Ê + låó ¹ = yå = yå¹Ê + yå¹

lå (37)
ló: = ló: + ló õ(å¹ ) = yå : = yå¹Ê : + yå¹ :

(å) (å¹Ê ) (å) (å¹Ê ) (å¹ )
(38)
Likewise, the dewatering section can be described by:
låó ¹Ê = ló + låó ¹M = yå¹Ê = y + yå¹M (39)
ló: ¹Ê = ló: + ló õ(å¹M ) = yå¹Ê : ¹Ê = y : + yå¹M : ¹M

(å ) () (å ) () (å )
(40)
As explained before, the additional relationships than complete this sub-section are the
efficiencies of each unit. In the case of the AD process we can define 2 different
expressions, one that defines the efficiency of the process for producing biogas ηAD:
]Mij
ñP?t59 ë^ g û' ^ k
ò ñå = IJ
]M ij
ä4ü((% g û' ^k
(41)
IJ
Moreover, other which indicates the change in composition of the 4 constituents involved
in the analysis:
ó ó (JÑ) (ý)
ò @: = =
(JÑ) (ý)
Lö ·Lö & ·&
ó
(JÑ) (ý)
&
Lö
(42)
From Eq. (41) we can expect that volumetric flow rate in stream G can be computed as:
y = yú º6 òñå YZ8>>;

ú (þReê (°))
z ij^ þR
(43)
On the other hand, we have the subsequent sub-section in which the microalgae culture
is developed. This portion is composed by two units as can be seen in Figure 4-8, a
photobioreactor for algae growth and a separation unit for concentrating the produced
biomass.
Figure 4-8. Units composing the subsection of microalgae culture.
Considering that flue gas flow through the PBR can be analyzed separately we can
expect that globally:
ló = lñó } = lñó c + ló ü = y = yñ} = yñc + yü (44)
Since the reaction unit can be defined in terms of the efficiency for transforming each of
the components into biomass, we can establish as performance parameter the yield as
the change between the outlet and inlet section of the reactor as previously defined in
chapter 3:
(ó ) ó ( )
0:äÒ = = −1
(i)
Lö } ·Lö & }
ó
(i) &
(i)
Lö
(45)
Where yiPBR is the change in load of the component i given the transformations inside the
PBR. This clearly implies that yiPBR can be positive, negative or zero depending on the
component and the efficiency of the process. It is expected that yTSPBR, for instance, would
be >1 since total solids is a direct indicator of the produced biomass while yTNPBR is
expected to be negative since nitrogen is absorbed for producing microalgal biomass. In
addition, harvesting unit can be described in an analogous way as the previous
separation units, but considering the aim of the unit, which is obtaining a concentrated
stream. In that case we can write the component mass balance:
ló: } = ló: c + ló õ (ü) = yñ} : } = yñc : c + yü :

(ñ ) (ñ ) (ñ ) (ñ ) (ü )
(46)
And the efficiency of the harvesting unit as:
(ó )
ò[: = =
( )
Lö c ,c & c
(ó ) ( )
,} & }
Lö }
(47)
Finally, for defining the 3 required relationships for solving the section we have to
consider the available information of Bresso plant. One of the streams that is commonly
characterized is the final digestate after dewatering (stream bs), particularly in terms of
the total suspended solids (TSS) that has a strong correlation with the TSS load of treated
sludge. According to information collected in Appendix A, it is possible to establish a
linear relationship between stream D and its TSS concentration as:
ê = ló ê +

(O6) (å)
(48)
Where γ and λ are constant derived from the plant performance as shown in Appendix A.
Taking into account that there is no punctual information about the thickening unit, it is
difficult to implement a relationship like Eq. (48), but it can be assumed the relative
behavior of this unit with regards to the dewatering process. From the conceptual point of
view, we know that dewatering allows most of the separation while thickening contributes
in a minor degree. This comprises a latent correlation between TSS concentrations of
streams Dx2 and bs. A first guess can be consider C TSS(Dx2) as a fraction of CTSS(bs) or a
function of the concentration obtained in the dewatering section CTSS(Dx3) as:
ê¹ = r ê ; ê¹ = r ê¹M

(å ) (O6) (å ) (å )
(49)
The last parameter we need to fix is related to the harvesting unit, in which beside the
mass efficiency previously defined, we have to consider the volumetric efficiency (ηv) that
accounts for the fraction of the volume that has the concentrated microalgal biomass and
it is defined as previously shown in chapter 3 as:
òº = ,
, c
(50)
}
Even when the D.O.F. analysis indicates us that the solution of the section is possible
through the definition of the above-mentioned parameters, it does not tells us nothing
about the solving procedure since it is just a global analysis. For this reason, the easier
way to solve the loop system is by means of a trial and error process. This implies an
initial assumption of a set of values that have to be compared at the end of each iteration.
The solving procedure for this section can be summarized as follows:
• Start the algorithm assuming the complete characterization of stream Ac,

considering the experimental results presented in chapter 3.
• Compute the components load in the same stream, mi as Ci(Ac)QAc.
• Using the individual harvesting efficiency ηhi determine the mass load of each
component in stream A0, and by means of ηv estimate QA0.
• Complete the mass balance in the harvesting unit determining the characterization
of stream Cf.
• Using the information of stream S, now it is possible to complete the mass
balances in the mixing point for determining the characterization of stream S’.
• Using the efficiencies related to the AD unit, compute the mass load of each
component in the digestate stream (D), as well as its volumetric flow rate through
the global mass balance of the unit.
• Knowing the burden in the digestate now it is possible to determine the remaining
fraction in stream Dx1 employing the removal efficiency associated to this unit.
• Completing the component mass balances it can be calculated the fraction that
leaves the unit mi(Dx2).
• Similarly, with the mi(Dx1) values it is possible to compute the mass load that will
end up in stream C thanks to the removal efficiency in the dewatering unit.
• Using the mass balance in the dewatering unit now is easy to establish the
removed load that composed stream Dx3, and completing the mass balances in
the mixture point the final biosolid stream (bs) can be characterized.
• Since QD is already known, a global balance in the section covering both
separation units allows us to correlate stream D, C and bs. Additionally, knowing
that Qc is equal to QA0, the only unknown is the volumetric flow rate of biosolids
(Qbs).
• The remaining flow rates QDx1, QDx2 and QDx3, are determined based on the
additional relationships that involved streams Dx2 and bs.
• Once centrate stream (C) is completely characterized, it is possible to determine
the concentrations after the PBR (stream A0).
• It is expected that concentrations computed directly by ηhi and ηv coincide with
calculated in the last step.
• Iteration should continue until finding a set of values that make those
computations coincide.
4.3 Combined Heat and Power section

In this section the produced biogas (G) is conducted to a combined heat and power
station for supplying the same WWTP’s needs. The flue gas produced in the combustion
process is then used as a source of carbon in the microalgae culture, obtaining a stream
with a reduced CO2 content as detailed in the diagram of Figure 4-9.
Figure 4-9. Units and streams composing the CHP section.

Since we cannot determine the real composition of biogas (stream G) as a function of the
feed, because we are not considering the multiple effects of the AD process like HRT,
OLR, temperature, pH, etc., we can assume the average composition of the stream
according to the tests carried out directly on Bresso’s CHP as detailed in Appendix B.
Then, by considering the combustion reaction with air in excess, we can also compute the
composition of the flue gas stream and following the mass balances in the unit, we can
determine the mass flow rate of each of them knowing that:
ó = q ó ¼(1 + Ú)
qñõ@ (51)
Where nAir is the total mole of air used for the combustion process, nG is the total mole of
biogas produced, β is the molar stoichiometric Air/fuel ratio (molAir/molfuel) and ε is the
excess of air defined for the combustion process. The total moles of biogas can be
derived from the volumetric flow rate previously computed and taking into consideration
the assumed composition of the stream, since:
qó = =
L
ó ,
4 4
(52)
In which mG is the total mass flow rate of G, MWG is the average molecular weight of G,
QG the volumetric flow rate of G and ρG the density of stream G at the given temperature.
In addition, we can state for the CHP unit that:
ló + lñõ@
ó = ló (53)
On the other hand, for modeling the response in the reaction unit, we use a single
parameter that indicates how much CO2 is consumed in the process respect to the
biomass produced as microalgae. That parameter has been defined according to the
literature review (view chapter 1, section 1.2.1) and it can be established as:
0 =
H=iø P7<6tL>;
H=J B@7;tP>;
(54)
Considering the change in CO2 due to the absorption in the PBR we can compute both
the mass flow rate of stream FGC and its composition that can be assumed to be only
altered by the carbon dioxide content. In this point it is also assumed that the CO2
dissolved in the PBR’s effluent is negligible regards the consumption in the growing
process, leading a simplification that only includes the change due to the microalgae
growing.
Concluding, based on the information provided of the CHP ordinary operation (see
Appendix A), and considering a mean value for LHVG as detailed in Appendix B, the
energy produced in the unit can be estimated thanks to the efficiency of the plant defined
as:
ò>9 = Ló
(½
ë$
(55)
4.4 Main results of the model

Excel was employed as tool for the implementation of the model. For doing this, the
solution procedures explained in the previous sections were put into practice. A brief
description of this implementation can be seen in Appendix C. Taking into account the
main aim of the model, the analysis is focused on determining whether the inclusion of the
PBR unit promotes or no any improvement in the performance of the plant. For this
reason, it is appropriate to compare the operation of the WWTP with and without the
section of microalgae culture, analyzing the parameters that better represent the
outcomes of the plant.
The first parameter that is expected to be changed is the volumetric flow rate of biogas
(QG) which is a direct indicator of the AD performance. This change has to be directly
related to the biomass production in the PBR, due to the invariable condition of the
primary sludge (stream S). From a conceptual standpoint a simple analysis indicates that
QG is a function of the organic load supplied to the reactor, the efficiency of the process
and the biological methane potential of the feed (see Eq. (43)).
We can start thinking in the new load fed to the digester, considering that it is a weighted
average of both streams feeding the unit, which means that it will vary significantly as the
microalgal biomass increases in volume (QAc increases) and organic content (CTVS(Ac)). In
other words, this factor depends on the productivity of the PBR, but also on the separation
efficiency in the harvesting unit. From results of chapter 3 we can expect that volumetric
efficiency of this concentration unit is as reduced as 10%; this clearly suggests that QAc is
always a subtle fraction of the primary sludge.
Looking at the BMP of the new feed, we could initially assume that there is no a
significant variation considering the ratio Qs:QAc which value is mainly defined by the
volumetric efficiency ηv and should be around 12:1. Notwithstanding, nature of both
streams are quite different and influence synergistically the degradation process. In this
point, a deeper analysis would be possible through experimental estimation of the
mixture’s BMP. Similarly, efficiency of the process will vary depending on the type of load
supplied if all other operative conditions remain unchanged, which lead us to a similar
conclusion as before, AD efficiency can be consider constant when PBR is include in the
model.
This simplified scheme directly gives us as a result a linear dependency of QG with the
additional organic load supplied to the unit (mTVSAc), which at the same time, is a linear
function of the productivity in the PBR and the efficiency in the harvesting unit. In Figure
4-10 it can be seen this linear dependency, considering changes in PBR’s productivity at
a given efficiency on the harvesting unit.
Figure 4-10. Effect of PBR implementation on the produced biogas.
As it can be observed, the increase in the TVS content of the feed, due to the co-digestion
of microalgal biomass, produces a higher biogas production that changes between
seasons mainly because of the intrinsic characteristics of the treated wastewater. While
spring and autumn are characterized by high levels of wastewater (around 66000m3/day),
summer presents the lower volumetric flow rate along the year (36400m3/day approx.),
and winter is closer to 56000m3/day. Furthermore, in terms of the organic matter content,
there is a subtle difference among seasons, but it is not so evident, such that efficiency
parameters can be considered only affected by the first variable. However, the only part of
the year in which there is no a clear tendency in this regard is during winter since is this
season the least effective in terms of pollutant removal.
This effect can be seen directly in the production of primary sludge, which varies from
382m3/d in spring, passing to an average of 337m3/d during summer and 330m3/d in
winter, concluding in 320m3/d in autumn. In conclusion it is expected a lower production of
organic matter available for the AD unit when there is a period in which the WWTP has to
treat higher volumes of wastewater. In other words, the combined effects of dilution of
components and decrease of the efficiency for high volumes, is the key factor that defines
the performance of the plant along the year.
Then, considering the aforementioned aspects, we observed from Figure 4-10 how is
summer the period in which theoretically is expected a better benefit of using the PBR. In
that case an increase of 1.8% in the TVS content of the feed produces a surplus of 1.3%
of produced biogas (considering the main values of efficiency and performance of PBR
and harvesting units). At the same time, during winter the benefit is lower just considering
that an increase of 0.4% of QG is only possible producing an extra of 1.05% of volatile
solids. Evidently, these values are just a representation of the expected behavior under
the conditions obtained in the pilot plant experiments, but PBR performance is still prone
to be improved in several aspects.
In that sense, through the developed mass balance it is possible to compute under ideal
conditions, which should be the maximum biomass production given amount of nutrients
supplied by the primary sludge, and the flue gas. For this purpose, it is convenient to think
in the equivalent chemical formula of the produced microalgae (see chapter 1, section
1.2) and particularly the associated molar ratio N:C (0.151). This means that for each mol
of carbon, we need 0.151 mol of nitrogen. Thus, once the content of nitrogen available for
the culture is define (in the centrate stream), as well as the equivalent content of carbon in
the flue gas (due to the CO2), it is possible to establish which of them is the limiting factor
for the biomass growth. As it is detailed in Table 4-3, nitrogen results the limiting nutrient
of the process, since the N:C ratio remains always below the theoretical ratio.
Table 4-3. Limiting nutrients and maximum expected biogas production.

Increase in
N:C mTVSA0 mTVSAc QG-max
Season (N:C)Act/(N:C)Th 3 biogas
ratio (kgTVS/d) (kgTVS/d) (m /d)
production (%)
Winter 0,026 0,171 1093,2 294,6 3702,6 3,01
Spring 0,068 0,447 2130,1 574,1 2891,4 8,37
Summer 0,056 0,373 1357,2 365,8 2185,4 7,70
Autumn 0,017 0,111 647,7 168,3 3346,8 2,18
Then, in the case all available nitrogen is consumed for producing microalgal biomass,
the system could produce a raw solution with an average of 1307kgtvs/d, able to produce a
surplus of biogas that ranges from 2.2-8.4% respect to the initial scenario in which there is
no PBR implemented. Those values are certainly higher that the experimental one, but
they are a reference in order to optimize the operational parameters of the entire plant.
Additionally, the same behavior exposed before is found in relation to the change of
seasons. Spring and summer count with the most favorable performances almost
doubling the biogas production increase respect to winter and autumn.
Finally another important aspect that can be extracted from the mass balance is the land
requirement for installing the PBR section. In this sense is important to define the
productivity of the culture, as well as the relative dimension of the section based on the
inhabitants which are favored by the WWTP. In the particular case of Bresso, the number
of residents associated to the plant ranges 220113 for 2012. But, taking into account that
productivity cannot be properly estimated based just on the performed experimental runs,
since it only would represent an isolated reactor without any other interaction, it is better
to analyze different scenarios in which productivity varies in range in accordance to
results of previous results, as presented in chapter 1.
Figure 4-11 shows the trend of the required area, measured as the surface needed per
inhabitant according to the expression:
ó ê
l ê
ñ9=
/
l pqℎTpoqo1 pqℎ
@>
=
pqℎ ñ@>59 è êl /é
As the diagram details, the higher the productivity, the lower the required area per
inhabitant. The same behavior could be expected working at the different season
conditions. In that case, if plant would work at summer conditions the required area would
range from 0.042 to 0.084m2/inh depending on the productivity of the system. On the
contrary, the higher requirements are needed when the plant operate at winter and
autumn conditions, in which case the area would range from 0.087 to 0.17m2/inh.
Figure 4-11. Required area for the PBR implementation at different productivities
5. Conclusions and Recommendations
Conclusions
• It was possible to reproduce the behavior of the analyzed WWTP by means of

simple mass balance modeling.
• The implementation of a PBR unit in the WWTP cycle was properly described by
the definition of the associated performance parameters, experimentally
determined.
• Through the experimental part, it was possible to assess the enhancement of the
anaerobic digestion process when a co-digestion of primary sludge and microalgal
biomass is employed.
• The integrated mass balance shows that there exists an improvement in the
production of biogas when a microalgae culture is used as an additional source of
organic matter inside the WWTP.
• From the modeling results, it is expected also an improvement of the quality of the
liquid effluent, since a considerable load is removed by the PBR section, releasing
the centrate mainly from the nitrogen burden.
• The proposed model indicates that under the intrinsic conditions of the Bresso
plant, the nitrogen content is the limiting nutrient for the algae growth.
Recommendations
• It is needed to extend the experimental part related to the microalgae culturing, in

order to involve some variables that critically affect the performance of the
process, like the CO2 effects on the culture (gas diffusion-heat transfer, inhibition
for high levels in the flue gas), and the effect of the irradiance at variable
conditions like presented in this study.
• It would be interesting to design a set of batch test for computing the parameters
that best fit a selected grow kinetic, in order to simulate the culturing behavior as a
dynamic unit, instead of a static unit done in this work.
• It is convenient to complete the characterization of the produced algae for
enhance the knowledge of the AD process. In this sense further BMP test are
essential for understand the degradability of the co-digestion feed, as well as
92 A mass balance assessment approach…
identify the performance of mixtures of different composition on the production of

methane.
• A further effort is needed for improving the harvesting of the biomass, since this
will be a critical factor that will define the feasibility of the energetic and economic
balance of the entire plant.
A. Appendix: Raw operational data
in Bresso WWTP 2012-2013
Since the main assumption of the employed modeling approach is the simulation of the
units by efficiencies, it is required the acquisition of information that provide those
efficiencies, based on the real operation of the units. For this reason, the operational
parameters measured for the period January 2012 to August 2013 in Bresso plant have
been used as a source for the mass balance modeling.
Information provided during this 20 months period corresponds to the monthly reports
Bresso´s staff has to perform for monitoring the performance of the WWTP. Each report
reflects a summary of the monthly activity and a characterization of the streams computed
from at least two measurements. Furthermore, the aforementioned period was chosen
since August 2013 was the last month in which a complete and detailed report was
elaborated. After this month, there is not enough information for describing the entire
performance of the plant continuously.
The most important information for make a good assessment is the characterization of the
streams, components and volumetric flow rates, which are the final result we expect to
determine by means of a mathematical model. As explained in chapter 3, components
involved in the wastewater treatment are limited to 7, but 2 of them (TS and TVS) are not
typically involved since are not clear indicator of the performance of the treatment. Tables
A-1 to A-3 contain the measured concentration of the liquid streams in the section of
waste water treatment for the analyzed period as well as the volumetric flow rate in the
points in which there is an instrument for this purpose.
Table A-1. Inlet wastewater characterization (stream I).

Year Month QS (m 3/month) CCOD (mg/L) CBOD (mg/L) CTSS (mg/L) CT-N (mg/L) CN-NH4 (mg/L)
January N.A. 513 230 200 39,2 39,2
February N.A. 784 335 398 41,9 37,3
March N.A. 588 265 246 33,8 36,2
April N.A. 731 203 218 34,4 28,5
May N.A. 435 192 231 32,3 30
June N.A. 367 166 159 31,1 29,8
2012
July N.A. 332 156 151 26,2 26
August N.A. 379 161 197 26,1 26,5
September N.A. 292 168 127 28,2 25,9
October N.A. 276 178 132 32,2 33,2
November N.A. 306 136 97 21,9 22,3
December N.A. 353 161 159 33,9 32,6
January N.A. 450 141 149 32,4 31,6
February N.A. 282 152 146 27,7 27
March N.A. 381 183 161 33,6 31,9
April N.A. 365 174 153 34,5 35,5
2013
May N.A. 344 168 151 34,3 33,8
June N.A. 315 154 156 29,1 30,2
July N.A. 318 139 158 29,7 29,4
August N.A. 281 98 154 25,8 22,4
Table A-2. 1st settling outlet characterization (stream II).

Year Month QS (m 3/month) CCOD (mg/L) CBOD (mg/L) CTSS (mg/L) CT-N (mg/L) CN-NH4 (mg/L)
January N.A. 330 194 115 45 46,6
February N.A. 207 140 98 37 39
March N.A. 225 162 101 47,5 42,8
April N.A. 245 149 139 42,1 35,8
May N.A. 217 134 104 40 34,9
June N.A. 200 148 87 42 42
2012
July N.A. 196 130 64 34,6 34,6
August N.A. 165 102 51 28 32,6
September N.A. 145 100 54 32,1 32,1
October N.A. 183 140 89 44 35,7
November N.A. 230 145 91 39 40
December N.A. 205 138 93 38,7 38,7
January N.A. 196 116 82 35,3 35,3
February N.A. 264 145 112 49 45
March N.A. 211 133 78 58 40,9
April N.A. 211 105 92 34 31
2013
May N.A. 180 108 82 31,9 31,9
June N.A. 256 135 96 35,2 35,2
July N.A. 177 115 73 29 29
August N.A. 158 70 83 12 12
Appendix: Raw operational data in Bresso WWTP 2012-2013 95
Table A-3. Final effluent outlet characterization (stream E).

Year Month QS (m3/month) CCOD (mg/L) CBOD (mg/L) CTSS (mg/L) CT-N (mg/L) CN-NH4 (mg/L)
January 1611910 26 7 7 27,9 2,3
February 1639072 34 7 12 27,1 0,7
March 1689158 30 6 8 26 0,9
April 1875052 17 5 7 30,9 1,4
May 1877550 22 5 4 23,5 2,2
June 1604532 24 5 6 19,5 2,6
2012
July 1515384 21 5 4 25,7 5
August 1122128 20 5 5 24,6 0,5
September 1771283 24 5 3 24,3 0,5
October 1742726 16 5 3 28,5 0,5
November 1977068 37 6 6 24 0,5
December 1822241 14 5 3 26,9 0,5
January 1791908 24 5 3 25,3 2
February 1671536 19 5 3 24,7 0,5
March 2135918 14 7 3 22,7 0,7
April 1964934 23 5 3 29,8 1,7
2013
May 2407046 24 5 6 27,4 1,8
June 1997069 22 4 3 26,5 1,4
July 1884083 18 3 3 22,4 3,8
August 1597843 18 3 3 20,7 0,8
For complementing this section, it is usually determined for the primary sludge (stream S)
and the secondary clarifier sludge (Stream rc1) the volumetric flow rate and the TSS and
TVS concentrations as detailed in Table A-4. From this information can be computed a
set of removal efficiencies for each component, considering negligible the change in the
total mass flow rate between stream I and E (see how values of Qs are always less than
0.8% of QE).
Evidently these removal efficiencies have a seasonal dependency, which can be seen
from the characterization standpoint, and in terms of the mass of treated wastewater.
From tables A-1 and A-3 we deduced that the global removal efficiency of any of the
components has no a clear tendency between seasons, since the final effluent remain
almost invariable along the year, and even more, the inlet wastewater does not change so
much its characterization.
Taking this into account, the main parameter that allows us to correlate the estimated
efficiencies is the volumetric flow rate of treated wastewater, which changes in a
predictive way from a high value during spring, then going down to the lowest point during
summer, and finally increasing continuously in autumn and winter.
Table A-4. Primary sludge and Secondary sludge characterization (Streams S and rc1).
Primary Sludge Secondary Sludge
3 3
Year Month CTSS (mg/L) CVSS (mg/L) QS (m /month) CTSS (mg/L) CVSS (mg/L) Qrc1 (m /month)
January 28666,7 22818,7 10530 3333,3 2511,1 43963
February 24083,4 19001,7 10844 3000,0 2330,0 45984
March 20785,7 15957,0 12453 3285,7 2519,0 51711
April 21555,6 16406,2 11273 3400,0 2635,0 44142
May 22250,0 16539,1 12370 3333,3 2600,0 47274
June 21846,2 16149,4 10884 3250,0 2470,0 46348
2012
July 23000,0 16928,0 10588 2444,5 1857,8 10309
August 18437,4 13980,3 7370 2750,0 2035,0 20406
September 22315,8 16337,5 7499 3250,0 2275,0 12049
October 28187,5 20939,3 9888 3714,3 2544,3 36242
November 24153,9 18765,6 9682 3777,8 2776,6 25310
December 24000,0 19265,5 9131 4833,3 3600,8 27445
January 30076,9 24038,4 10515 4373,8 3456,3 31265
February 30857,1 24112,6 9120 4500,0 3555,0 27745
March 30545,5 23186,8 10767 3600,0 2808,0 34750
April 28272,7 20819,0 11477 4200,0 3108,0 36545
2013
May 25700,0 18092,8 12073 3600,0 2592,0 39881
June 22555,5 15638,5 12543 3666,7 2640,0 34290
July 22166,7 14574,6 14897 4500,0 3082,5 46447
August 23400,0 15132,0 9295 3571,4 2571,4 26344
Over the section devoted to the sludge treatment, the monitoring is carried out over the
biogas stream (G) and the final digestate after dewatering (stream bs). In the first case,
the main measurements are related to the biogas production (QG) and the total power
produced in the CHP unit (Pe) as it can be observed in Table A-5, while in the latter the
characterization is the same as performed to the sludge streams of Table A-4.
Once again, this set of data can be used for determining the behavior of the units in terms
of efficiencies, particularly considering the relationships between those efficiencies and
the change in the volatile content of the streams. On the other hand, by knowing the
average BMP of the primary sludge as well as its volatile content, it can be determined
the efficiency for the anaerobic digestion in terms of biogas production by means of Eq.
(41) and (43).
Appendix: Raw operational data in Bresso WWTP 2012-2013 97
Table A-5. Biogas and final digestate sludge characterization (streams G and bs).
Biosolid stream Biogas and energy production
3 3
Year MonthCTSS (mg/L) CVSS (mg/L) Qbs (m /month) QG (m /month) Pe (kwh/month)
January 25750,0 16296,1 5124 98312 189680
February 23500,1 14315,4 6351 79051 191520
March 19100,0 12324,5 8763 86323 174800
April 20300,0 12890,5 6503 74353 166400
May 21235,3 13153,4 7705 76885 133040
June 23571,4 15164,3 6341 70232 162080
2012
July 27941,3 17989,7 3898 84665 92510
August 27055,5 16871,0 3551 57896 92510
September 30143,0 17482,9 3077 69167 129120
October 32937,6 19191,7 4148 82635 119840
November 31428,6 17981,6 4669 87020 127120
December 35733,3 20892,1 3854 75560 189680
January 29250,1 17944,8 4890 107650 116880
February 21800,1 13617,7 4222 81590 108265
March 18902,3 13663,1 5200 104775 109360
April 18017,6 13439,8 5684 104007 131500
2013
May 18023,9 15327,8 5101 86573 148960
June 20319,2 15592,9 4780 75233 113120
July 19456,0 16054,6 7106 53286 72640
August 28454,6 16788,3 4567 29654 36880
Then, considering the effect of the total mass of wastewater treated, it was decided to
determine an average value per efficiency for each season. Table A-6 and Table A-7
contain the computed values based on the aforesaid information.
Table A-6. Computed efficiencies for the wastewater section.

Winter Spring
st nd st nd
1 settler Biological T. 2 settler 1 settler Biological T. 2 settler
ηTSS 0,7710 0,8000 0,3150 0,5750 0,89000 0,30000
ηTS 0,8481 0,8000 0,3150 0,5750 0,89000 0,30000
ηT-N -0,1405 0,2940 0,2450 0,0430 0,08050 0,01000
η NH4 -0,0100 0,9410 0,3450 0,1000 0,95500 0,35000
η BOD 0,1620 0,9579 0,1620 0,3500 0,96500 0,48000
η COD 0,3609 0,8794 0,3609 0,4300 0,79560 0,60500
Summer Autumn
ηTSS 0,5350 0,8000 0,7428 0,8400 0,8000 0,2900
ηTS 0,5885 0,8000 0,7428 0,8400 0,8000 0,2900
ηT-N 0,0050 0,0250 0,0750 -0,0700 0,0950 0,1100
η NH4 0,0150 0,9806 0,0350 0,0040 0,9852 0,0100
η BOD 0,3706 0,9232 0,3706 0,2179 0,9551 0,2179
ηCOD 0,5675 0,7238 0,5675 0,3407 0,8696 0,3407
Table A-7. Computed efficiencies for the sludge section.

Winter Spring Summer Autumn
Thickenin Centrifug Thickenin Centrifug Thickenin Centrifug Thickenin Centrifug
g e g e g e g e
η TSS 0,050 0,970 0,050 0,970 0,050 0,970 0,050 0,970
ηTS 0,055 0,970 0,055 0,970 0,055 0,970 0,055 0,970
ηTN 0,350 0,080 0,350 0,080 0,350 0,080 0,350 0,080
η TVS 0,055 0,970 0,055 0,970 0,055 0,970 0,055 0,970
Anaerobic Digestion Unit
Winter Spring Summer Autumn
ηVS 0,540 0,410 0,270 0,530
ηAD 0,755 0,721 0,764 0,833
BMP
3 0,350 0,350 0,360 0,355
(Nm /kgvs)
ηTS 0,480 0,480 0,480 0,480
η TSS 0,370 0,310 0,360 0,395
B. Appendix: Combustion reaction
analysis in CHP unit
Parameters and assumptions employed in the examination of the CHP section are
derived from the analysis made on the combustion of the produced biogas. In fact, the
raw data available for starting this evaluation is the elemental analysis performed during
2012 to the flue gas at Bresso’s CHP outlet shown in Table B-1. As it can be seen,
besides the composition of the stream we find the excess of air used in each case and the
temperature in the point of measurement.
Table B-1. Elemental Analysis of flue gas stream in Bresso plant.

Analysis 1 Analysis 2 Analysis 3
TFG (°C) 463,2 460,2 464,3
ε (%) 26 28 27
Xi (%)
O2 4,34 4,58 4,45
CO2 9,28 9,15 9,22
N2 70,18 70,29 70,24
H20 14,89 14,68 14,78
CO 0,0004810 0,0005210 0,0004790
NO2 0,0000532 0,0000584 0,0000257
NO 0,0010430 0,0007580 0,0010250
H2 0,0000460 0,0000430 0,0000380
We can state that this composition is the result of oxidizing a biogas whose components
are methane, carbon dioxide, hydrogen, nitrogen, oxygen and hydrogen sulfide with an
excess of air defined in terms of parameter ε. Taking this into account, we can go into
deep in the reaction considering an equivalent model in which we analyze the
transformation component by component, computing the atomic mass balance which has
to be in accordance to the raw data of Table B-1. Therefore, we can first establish the
equivalent oxidation reaction as:
ïh ïñ@ ï
+ T + U + / + + r 3 + ¼(1 + Ú) ì + + + î
ï ï ï

ä:7=56
ñ:@
+ + R 3 + +
+ + þ
→
9t> =56
Where coefficients a to f are the molar fraction of the corresponding component in the
biogas stream, β is the stoichiometric air need, ε is the excess air fraction, xi the molar
fraction of the component i in the air stream and parameters z1 to z7 the mol produce of
the corresponding component in the flue gas stream.
Definition of each of the aforementioned coefficients depends essentially on the

characteristics of the biogas, since for air we can expect the typical well-known
characterization. In fact, they are correlated each other thanks to the set of individual
atomic balances of the presented species. Thus, writing each of the balances based on
ï
the equivalent reaction we obtain that:
Tq: + T + ¼(1 + Ú) =
ï
0/q: 4 + 2U + 2r = 2
3mnrm: r = R
ïh
poq: 2/ + 2¼(1 + Ú) = + 2
ï
ïñ@
q: ¼(1 + Ú) =
ï
ï
ï0q: 2T + 2 + ¼(1 + Ú) ©1 + 2 « = 2 + + 2R + + þ
ï
Moreover, completing the set of equations we add a particular condition in which it is

assumed that the only fraction of N2 that is oxidized correspond to the one derived from
the biogas, not from the air. This condition can be written as:
ïh
2¼(1 + Ú) = 2
ï
Molar fractions in flue gases can be directly estimated from z coefficients considering that:

ï =
∑þ: :
In a typical analysis, the composition of fuel is known and it is the set of 7 flue gas molar
fractions the result of solving the system of equations. In this case we use the information
of elemental analysis of flue gas for fitting the main biogas composition, considering some
constraints shown in Table B-2 and minimizing the difference between computed and real
molar fractions in flue gas stream. In other words we expect that:
Appendix: Combustion reaction analysis in CHP unit 101
7O = Zpq ï − x:

:
: Ò>59 7LBt?>;
1. o. ï > ï,L:< ∀
ï < ï,L5 ∀
Table B-2. Constraints for fitting the biogas composition.

Biogas Constrains Air Characteristics
Component ximax ximin Component xi
CH4 0,650 0,69 O2 0,2097
C02 0,200 0,28 N2 0,7808
H2 0,005 0,025 Ar 0,0094
N2 0,010 0,05 C0 2 1E-04
O2 0,001 0,007 - -
H 2S 0,005 0,02 - -
Once this optimization is performed, we find that biogas composition that fits better to the
experimental results is the one presented in Table B-3.
Table B-3. Biogas composition after fitting process.

Component xi
CH 4 0,69
C0 2 0,254
H2 0,025
N2 0,01
O2 0,001
H2S 0,02
Once biogas is characterized, it is possible to define some additional aspects that are
important for the analysis of the CHP unit. First it is needed to compute both the mean
molecular weight MWG and the gas density considering that:
Z = ï: Z:

And
Z
ô
"#
Where PG is the absolute pressure of the biogas, initially assume close to ambient
pressure, R is the universal constant of gases and TG the temperature of the produced
biogas, estimated to be around 40°C.
Another important parameter to be computed is the low heating value (LHV) of the biogas,
which allows us to determine the efficiency of the CHP unit. LHVG can be calculated
considering the combustion reaction of biogas with stoichiometric air, and recalling that
the normal enthalpy of reaction will be given by:
∆Ò = !: Δ8,:

− !: Δ8,:

= Ef
#
B@7;tP?6 @>5P?5<?6
ΔÒ
And the per-mass based LHV will be:
Ef =
Z
Alternatively, considering we are working with a gas stream, the per-volume based LHV
will be:
Ef º = Ef ô
From this expression we obtain that produced biogas has a MW of 23,29 Kg/Kmol, a
density of 0,907 Kg/m3 at 40°C and a LHV of 568,5 KJ/mol, equivalent to 2 4,41 MJ/Kg
and 25,4 MJ/m3.
C. Appendix: Description of the
solution in Excel
Complete mass balance solution of Bresso plant was implemented in Excel due to the
simplicity of expression as well as for the versatility of this software in terms of
computation and graphical visualizations. File containing the mass balances is composed
for 2 main sheets which visualized the main results, and 4 additional sheets that are
devoted to storage the information of appendix A and B, and the performed sensitivity
analysis.
In the sheet “PBR Diag” can be found a brief description of the plant and its units.
Graphically are represented each of the involved streams and the main characteristics of
each one. This is the sheet in which the information for running the model is introduced.
The required information is mainly associated to the characterization of the wastewater at
the inlet and the operational parameters of the PBR section. As observed in Figure C-1,
here is compiled the most representative information of the plant, expecting to provide to
the user a general view of the process trying to use a friendly visual interface. In Figure C-
1 can be also seen the conditions of the plant using the PBR in the winter season as an
example of the generated summary. On the contrary, Figure C-2 contains the same
sketch, but including the results when the WTTP operates without PBR in the same
season.
This sheet is connected with the one called “MB PBR” in which all mass balances
computations are developed. This part of the solution was organized in such a way all
columns represent the streams associated to the plant, while the rows contain the
different parameters and variables related to the streams, like the volumetric flow rates,
concentrations, mass flow rates, etc. In this part, the solution of the section is
implemented, and results are linked to the previous sheet. Figure C-3 shows a part of this
section, which additionally encloses all performance parameters computed in appendix A.
Figure C-1. Sketch of the main page. Results correspond to the use of the PBR unit under
winter conditions.
Figure C-2. Sketch of the main page. Results correspond to the plant operation without
PBR during winter.
Appendix: Description of the solution in Excel 105
Figure C-3. Sketch of the mass balance page inside the software.
Solution of the integrated Sludge treatment-PBR section was implemented by means of

the solver tool of excel. Using this tool it is easy to carry out an iterative process as
described in chapter 4 and determine the values that match constraints instantaneously.
Inside the sheet the parameter to be validated is the set of characteristics of stream A0,
which has to be the same using both computation approaches according to the
explanation in chapter 4.2. Figure C-4 is an example of the appearance of this part of the
solution inside the sheet “MB PBR”.
Remaining sheets are just auxiliary tools for the main purpose of performing the mass
balances. They contain tables with the real data of Bresso plant (sheet “Bresso Data”),
the calculated parameters based on the aforementioned information (sheet “Efficiencies”)
and the implementation of the analysis developed in appendix B (sheet “Flue Gas”). The
last auxiliary sheet is “Sensitivity Analysis” in which a set of data generated with the
model can be used for any purpose, as detailed in chapter 4.4.
Figure C-4. Sketch of the solution with solver for the integrated section.
D. Appendix: Description of the
experimental systems and related
procedures
The following is a brief description of the equipment and procedures employed during the
experimental phase. Figure D-1 represents the equipment diagram for the pilot plant
microalgae culturing. As can be seen beside the reactor, the system is properly
connected to the auxiliary equipment required for storage, supply and control.
Figure D-1. Equipment diagram of the culturing system.
The system consist in a plastic column (150 cm height, 28.9 internal diameter, Vmax 98.4L,
Voperative 85.1L), connected in the inlet section to a feed tank (150L approx.) through a
peristaltic pump (Qmax 100 mL/min at 100% power, 30 mL/min at 20% power). The outlet
section is connected to a storage tank (150L approx.) were produced biomass ends up.
The column is also connected to the outlet line of flue gases coming from the CHP unit,
and after a conditioning system (for avoiding high acid gases) the gas stream is
conducted through a flowmeter before it enters to the bottom of the reactor.
Operation of this system can be split in batch and continuous modes. The following is the
detailed description of both procedures, previously mentioned in chapter 3.
Operation of PBR in batch

• Let empty the column and remove any residual by washing it with abundant water.
• Adjust the bubbling system and put the exit of the gas tube (porous stones) in the
bottom of the column assuring a good distribution of the bubbles.
• By using a bucket fill the column at 90% of its capacity with fresh centrate solution.
Be sure of proceed with caution and using suitable protection element (Wear lab-
coat, glasses, and gloves).
• Proceeding in a similar way fill the remaining 10% of the column volume with fresh
microalgae solution.
• Open the valve that controls the gas flow for supplying the column and adjust the
volumetric flow rate of gas in the desired value.
• After a short period of time, enough for a suitable mixing, take a 500 mL sample of
the mixture for its characterization in terms of T, TS, TSS, TN, N-NH4, N-NO3, TP,
Conductivity, pH, D.O. and absorbance. This would represent conditions at time
zero (t=0).
• Cover the top of the column for isolating it from the surroundings with a plastic
wrap and complete the operation putting a protective mesh above the plastic
cover.
• Define the time period for monitoring the system tf and the maximum time for
performing the run test tmax.
Each tf it is necessary to perform:
• Retire the covers on the top of the column carefully and take a 500 mL sample for
subsequent characterization.
• If necessary perform a cleaning of the column taking care of avoiding any mass
losses.
• Using a suitable probe take the measurements of temperature of the column Tr,
ambient temperature T amb, conductivity, D.O. and PAR directly in the column.
cover.
• Verify the volumetric flow rate of gas and if necessary adjust according to the
defined value.
• Once tmax has been achieved conclude the run test.
For continuous operation
• Define the HRT according to the total volume of the column and the maximum
capacity of the liquid pump.
• Operate in batch mode following the aforementioned instructions until reaching the
HRT.
• Use a barrel for storage the centrate to be fed into the column, be sure of always
keeping a minimum level for supplying the column.
Appendix: Description of the experimental systems and related procedures 109
• Connect the inlet section of the liquid pump to the centrate storage using a proper
plastic tube. Don´t forget to put a filter in the section that remains inside the
centrate barrel for avoiding any obstruction.
• Connect the outlet section of the liquid pump to the inlet of the column. Be sure of
letting the end of the tube in the bottom of the column, far away from the exit
valve.
• Check for any leak and if needed solve it properly before the next step.
• Using a 25 or 50 mL graduated cylinder filled with centrate solution, and a
chronometer, adjust the volumetric flow rate supplied to the PBR to the computed
value based on the defined HRT. Don´t forget to avoid any air bubble inside the
tubes, since they cause wrong values and possible malfunctions of the pump.
• Connect the outlet section of the column with a second barrel for storing the
produced microalgae solution. Be sure of cleaning this container before any use.
• Open the outlet valve at the top of the column allowing the flow from the column to
the microalgae barrel.
• Keep the pump and compressor systems clean and always covered.
• Define the time period for monitoring the system tf and the maximum time for
performing the run test tmax.
Each tf it is necessary to perform:
• Retire the covers on the top of the column carefully and take a 500 mL sample for
subsequent characterization.
• If necessary perform a cleaning of the column taking care of avoiding any mass
losses.
• Using a suitable probe take the measurements of temperature of the column T r,
ambient temperature Tamb, conductivity, D.O. and PAR directly in the column.
cover.
• Verify the volumetric flow rate of gas and if necessary adjust according to the
defined value.
• Verify the volumetric flow rate of centrate and if necessary adjust according to the
computed value.
• Take a 5-10 L sample of the collected microalgae for further tests or as a backup
at least once a week. Be sure of mixing well before taking the sample and
preserve the sample in a refrigerator at 4°C.
• Once tmax has been achieved conclude the run test.
On the other hand, we find in Figure D-2 the diagram for the used AD system. As can be
seen, the system is composed by a 2.4L digester, connected to an automatic stirrer
activated by a circuit breaker. The system is posed inside a water bath in which a couple
of resistances provide the energy for keeping constant the temperature of the medium.
Additionally, one of the outlets of the digester is connected to the system for measuring
the produced methane. This latter comprise a plastic graduated cylinder (1000mL) turned
down inside a container full of a 1M NaOH sln.
Figure D-2. Sketch of the semi-continuous AD system.
The following is the detailed description of the operation procedures required for driving
the AD system.
For batch operation

• Prepare the reaction system by closing In and Out cover, as well as heating the
water bath till 32 + 1°C.
• Add the inoculum into digester doing it in the biggest opening of the reactor.
• Close the system by adjusting the stirring system.
• Using a 1M NaOH solution, completely fill a 1000 mL graduated cylinder. Take
care of using suitable protection elements when manipulating corrosive
substances like NaOH (Wear lab-coat, glasses and gloves).
• Put the cylinder upside down inside a container and fill it with more NaOH solution
in such a way liquid remains inside the cylinder. It is important to keep constant
the liquid level in this container in order to maintain constant the pressure inside
the system. It is easier to perform this operation by using a seal of plastic wrap
over the graduated cylinder.
• Put the gas out stream of digester into the graduated cylinder and fix it always at
the same depth
• Once digester temperature is stable, add the substrate using a syringe according
to the defined proportions using a syringe. Use the In cover for this purpose.
Appendix: Description of the experimental systems and related procedures 111
• If necessary, use the stirring system at 80 rpm.

• Take this point as time zero (t0).
• Take measurements of the cylinder volume displacements with a defined
frequency.
• Once volume displacements are not significant over time, the run test can be
considered finished.
Semi-continuous operation
• Prepare the reaction system by closing In and Out cover, as well as heating the
water bath till 32 + 1°C.
• Add the inoculum into digester doing it in the biggest opening of the reactor.
• Close the system by adjusting the stirring system.
• Using a 1M NaOH solution, completely fill a 1000 mL graduated cylinder. Take
care of using suitable protection elements when manipulating corrosive
substances like NaOH (Wear lab-coat, glasses and gloves).
• Put the cylinder upside down inside a container and fill it with more NaOH solution
in such a way liquid remains inside the cylinder. It is important to keep constant
the liquid level in this container in order to maintain constant the pressure inside
the system. It is easier to perform this operation by using a seal of plastic wrap
over the graduated cylinder.
• It is easier to perform this operation by using a seal of plastic wrap over the
graduated cylinder.
• Put the gas out stream of digester into the graduated cylinder and fix it always at
the same depth cylinder
• Once digester temperature is stable, add the substrate using a syringe according
to the defined proportions using a syringe. Use the In cover for this purpose.
• If necessary, use the stirring system at 80 rpm.
• Define the operation parameters HRT (days) and weekly frequency of feeding (tf).
• Establish the effective feed rate Q’in (mL/day) taking into account that:
f@ /01 pq s (7)

y:< = y:<
ú
= y:<
"# o8 (/01/s)
• Wait a suitable time for the stabilization of the system (temperature, gas
production).
• Depending on the displaced volume in the graduated cylinder, refill it with new
NaOH solution and adjust the liquid level to the starting point. You can use a
vacuum pump directly connected to a plastic tube for this purpose. Be sure of
adjusting the initial volume level V0 in such a way the NaOH Sln displacement can
be handle in the tf period.
Once the tf period has passed carry out the next procedure:
• Take note of the cylinder displaced volume Vf.

• Put out the cylinder the gas outlet stream, you can leave it bubbling water in the
meantime.
• Once stabilized the system (temperature, gas production) collect Q’in mL of the
outlet digestate for further analysis in an Erlenmeyer from Out cover. For this
purpose, you can use a vacuum pump coupled with the Erlenmeyer and the Out
cover. After sampling do not forget to close the outlet section.
• Take a 50 mL sample in a beaker for measuring the temperature and pH of the
medium. Be sure of performing this analysis as faster as possible in order to be
accurate with the temperature of the medium.
• If pH of the medium is out of limits (6.8-7.4) use a basic or acid diluted solution for
adjusting the pH.
• By using a syringe add the same amount previously collected Q’in mL of new
sludge into the inlet section of the digester (In cover). After feeding don’t forget to
close the inlet section.
• Once again adjust the initial volume of NaOH Sln, according to the previous
explanation.
• Take note of the time spent (hours) between each new measurement
• At the same time, collect the outlet sludge in a beaker for analysis collecting it
from Out cover.
• Depending on the gas cylinder collector volume, refill the cylinder with new NaOH
solution.
• Determine de produced methane by means of the variation of volume inside the
graduated cylinder.
• Take note of the time (hours) spent between each new measurement
• Repeat the feeding procedure each tf.
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Integration of Microalgae Culturing Within The Wastewater Scheme

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Integration of microalgae culturing within

the wastewater scheme

Oscar Mauricio Martínez Avila

Keywords: Microalgae, Anaerobic Digestion, WWTP, Mass balance, Integration

List of tables ................................................................................................................... XI

List of Symbols and abbreviations .............................................................................. XII

1. State of the art .......................................................................................................... 5

2. Mathematical models for algae growth ................................................................. 41

3. Experimental phase ............................................................................................... 53

4. Integrated Mass Balance ....................................................................................... 73

5. Conclusions and Recommendations ....................................................................91

A. Appendix: Raw operational data in Bresso WWTP 2012-2013 .............................93

B. Appendix: Combustion reaction analysis in CHP unit .........................................99

C. Appendix: Description of the solution in Excel ..................................................103

D. Appendix: Description of the experimental systems and related procedures .107

Figure 3-7. Absorbance of the medium during batch runs. .............................................. 58

List of Symbols and abbreviations

Symbols with Latin letters

Symbol Definition SI Unit

Symbols with Greek letters

Symbol Definition SI Unit

In terms of wastewater treatment, a widespread amount of techniques have been

In general microalgal biomass production counts with a significant amount of advantages,

Thanks to the synergy presented between these processes, integration of microalgae

In this work, an assessment of the mass balance inside an integrated scheme

1.1 Current expectations on microalgal biomass

Figure 1-1. Energy feedstocks processing using microalgae.

eutrophication, resource depletion, ecotoxicity). Moreover, the global warming effect

• They have a higher photon conversion efficiency

1.2 Microalgae characteristics

Microalgae contain three principal components: proteins, lipids and carbohydrates.

6  + 6 + ℎ →    + 6 (1)

Where hν represent the luminous energy.

• Part of this radiation is used in processes different from photosynthesis (e.g.

Figure 1-2. Photosynthetic activity behavior of microalgae.

1.2.1 Microalgae growth

As Figure 1-4 shows, microalgal biomass productivity reaches an optimal value

Figure 1-4. Biomass productivity as function of light intensity

• Other macro and micronutrients

1.2.2 Culture systems

In general, the culture suspension, containing nutrients, is pumped around in a cycle,

diluted culture have to be processed. Artificial ponds are commonly designed in a

Figure 1-7. Typical open system configurations.

Figure 1-8. Typical raceway pond configuration. From (Chisti, 2007)

Table 1-1. Main design characteristics of microalgal bioreactor systems.

• Flat Plate PBR

Figure 1-10. Flat plate PBR configuration.

Design Considerations for PBR’s

• CO2 supply and aeration

1.2.3 Productivity of microalgae cultures

Figure 1-15. Volumetric productivity of some microalgae strains. Information extracted

1.3 Anaerobic digestion process

fraction by means of physicochemical processes like hydrolysis for producing ethanol,

Table 1-3. Typical composition of biogas. From (AEBIOM, 2009)

Figure 1-17. Typical routes for processing anaerobic digestion products.

1.3.1 Biochemistry of the AD process

Figure 1-19. Stages of the anaerobic digestion process.

These microorganisms population are characterized by different growth rates, variable

Bacteroides, Micrococcus, Peptostreptococcus, Clostridium, Propionibacterium (Taricksa

digesters are Methanobacterium bryantii, formicicum, wolfei, thermoautotrophicum,

1.3.2 Factors affecting AD process

Figure 1-20. Growth rate of microorganisms in each temperature range.

Acetotrophic methanogens are one of the most sensitive groups to increasing

6 + 6 + ℎ → + 6 (1)

l11 UUlmnopq l11 rns:< l11 rns7t? ± l11 v/mUopq/Uq1mlvopq

o Uℎq pq q0 = q0 T1T/ T0 Tpl11 − q0 pq morns Tpl11

7t? = 7 ·_z_½' (17)

Ö cos Ù " cos Ú R sin ε rÖ sin φ

5º = è¿Ò ¿Ô ä (, Ù)//Ù + ¿Ò ¿Ô ¿Ó å (, Ù)//Ù/Úé

ló: = ló: + ló: = y¸ : = y : + y :

ló: + ló õ(@PM ) = ló: + ló: = y¸ : + y@PM : = y : + y¸¸ :

ó = ló + l@Pó Ê = y¸¸¸ = y + y@PÊ

ló: = ló: + ló õ (@PÊ ) = y¸¸¸ : = y : + y@PÊ : Ê

= L566 78 8@>6[ 8>>;