Master’s dissertation submitted in partial fulfillment of the requirements for the joint degree of
International Master of Science
in Environmental Technology and Engineering
an Erasmus+: Erasmus Mundus Master Course jointly organized by
Ghent University, Belgium
University of Chemical Technology, Prague, Czech Republic
UNESCO-IHE Institute for Water Education, Delft, the Netherlands
Academic year 2014 – 2015
Coupling dark fermentation and photofermentation as a way to increase hydrogen
production from glycerol
Host University:
Pontifical Catholic University of Valparaíso, Chile
University of Chemical Technology, Prague, Czech Republic
Rodríguez Muñoz, Yadira
Promoter: Assoc. Prof. Jan Bartáček, MSc., Ph.D.
Co-promoter: Gonzalo Ruiz-Filippi, Ph.D.
This thesis was elaborated and defended at University of Chemical Technology,
Prague, Czech Republic within the framework of the European Erasmus Mundus
Programme “Erasmus Mundus International Master of Science in Environmental
Technology and Engineering " (Course N° 2011-0172)
© 2015, Prague, Yadira Rodríguez Muñoz, Ghent
University, all rights reserved
i
Faculty:
Faculty of Environmental Technology
Department:
Department of Water Technology and Environmental Engineering
Academic Year:
2014/2015
Assignment of Diploma Thesis
Student:
Yadira Rodríguezu Mño z
Study Programme: Environmental Technology and Engineering
Subprogramme:
Environmental Technology and Engineering
Specialisation:
Subject of Diploma Thesis:
Coupling dark fermentation and photo-fermentation as a way to increase
hydrogen production from glycerol
Directions for Elaboration:
Elaborate a literature review on photofermentation by photoheterotrophic bacteria.
Select and cultivate a mixed culture from anaerobic sludges collected from different
wastewater treatment plants.
Study the production of H2 from a glycerol dark fermentation effluent and reference
compounds.
ii
iii
Certification
I hereby declare that this thesis is my own work. Where other sources of information have
been used, they have been acknowledged and referenced in the list of used literature and
other sources.
I have been informed that the rights and obligations implied by Act No. 121/2000 Coll. on
Copyright, Rights Related to Copyright and on the Amendment of Certain Laws (Copyright
Act) apply to my work. In particular, I am aware of the fact that the Institute of Chemical
Technology in Prague has the right to sign a license agreement for the use of this work as
schoolwork under the no 60 paragraph 1 of the Copyright Act. I have also been informed
that in the case that this work will be used by myself or that a license will be granted for its
usage by another entity, the Institute of Chemical Technology in Prague is entitled to require
from me a reasonable contribution to cover the costs incurred in the creation of the work,
according to the circumstances up to the full amount.
This is an unpublished M.Sc. thesis and is not prepared for further distribution. The author
and the promoter give the permission to use this thesis for consultation and to copy parts of
it for personal use. Every other use is subject to the copyright laws. More specifically, the
source must be extensively specified when using results from this thesis.
I agree to the publication of my work in accordance with Act No. 111/1998 Coll. on Higher
Education and the amendment of related laws (Higher Education Act).
The promoter(s):
The author:
Gonzalo Ruiz-Filippi, Ph.D.
Yadira Rodríguez Muñoz
Assoc. Prof. Jan Bartáček, MSc., Ph.D
iv
Acknowledgements
I would like to express my most sincere gratitude to all the people who in so many ways
helped me to make this work a reality.
I am profoundly thankful to my co-promoter Gonzalo Ruiz-Filippi, Ph.D. for hosting me in the
School of Biochemical Engineering at the PUCV in Chile and instructing me through the
course of this work despite the difficult circumstances. I am very grateful for all the shared
moments with him and his family from the very first moment I landed that made me feel
settled quickly.
Thank you to my tutor, Estela Tapia-Venegas, Ph.D., for her contribution in this work and
putting enormous effort in providing me advice whenever needed and the best guidance to
overcome all the challenges that this new and “purple" research line has brought with it.
Thank you to my promoter, Jan Bartáček, Ph.D., for offering me the invaluable chance of
conducting my thesis research at PUCV and for his efforts in helping me from the Czech
Republic.
Thank you to all the members of the LBA work-team, for making my stay in the lab pleasant
and enjoyable. Special thanks to Soraya Salazar for her persistent assistance in the lab; to
Oscar Franchi for his willingness to contribute always with critical thinking; to Eduardo
Ortega for helping me to take my first (and second, and third) steps with the AMPTS II; to
Sean Smith for reaching out to me and reviewing the English in the drafted manuscript; to
Prof. Daniel Undurraga and all the people in the Microalgae lab for finding answers to all my
questions related to illumination; and to María Eugenia Martinez for her time and effort
spent in processing my HPLC samples.
Special thanks to Anita Bravo and Fernando Silva for their friendship and their constant will
to help and find a solution for every problem in the lab.
To my IMETE family, especially to the Florida-Alaska-Gent-Vietnam connection (no matter
the distance and the time difference) for all the joyful and sometimes tough moments we
shared over the past two years.
Thank you to Pablo, for his understanding in this 2-year long distance relationship and his
unconditional support and effort in making life easier during tough moments. I still owe you a
trip to Patagonia as Xoel´s sings.
Last but most importantly, I want to extend my deepest gratitude to my mother Carmen and
my father Manolo, and of course to my grandparents, Lola and Manolo, without whom I
would not have made so far. Thank you to Coral and Manuel, tita Lola y Víctor, family and
friends for their support and encouragement to go always further; and for buying the tickets
to attend the graduation ceremony before the work was done (real proof of trust).
v
Abbreviations
AD
anaerobic digestion
AMPTS II
Automatic Methane Potential Test System
APHA
American Public Health Association
BOD5
biological oxygen demand
CODs
soluble chemical oxygen demand
COD
total chemical oxygen demand
CSTR
continuous stirred tank reactor
DF
dark fermentation
df
dilution factor
GC
gas chromatography
HRT
hydraulic retention time
HPLC
high-pressure liquid chromatography
PHB
polyhydroxybutyrate
PNS
purple non-sulfur bacteria
RC
reaction center
STP
standard temperature and pressure conditions
TCA
tricarboxylic acid
TOD
theoretical oxygen demand
TSS
total suspended solids
VFA
volatile fatty acids
SVFA
synthetic volatile fatty acids
TVFA
total volatile fatty acids
UASB
upflow anaerobic sludge blanket
VSS
volatile suspended solids
WAS
waste activated sludge
WWTP
wastewater treatment plant
vi
Abstract
The potential use of biological processes for the production of hydrogen as an alternative
to physicochemical methods has been extensively reported. For instance, dark
fermentation of organic wastes such as glycerol is seen as an attractive technology for
the production of biofuels such as ethanol and hydrogen due to its simplicity and its low
cost. Nevertheless, its effluent contains high content of metabolites (mainly volatile
organic acids) that can serve as a great bio-refinery platform either for the production of
methane using anaerobic digestion or hydrogen through photo-fermentation processes
achieving a higher efficiency of the overall process. In this study, the biological hydrogen
production through photo-fermentation for the post-treatment of an effluent rich in volatile
fatty acids generated through the dark fermentation of glycerol was studied. For this
purpose, four main experiments were performed sequentially. A photosynthetic bacterial
consortium (purple non-sulfur bacteria (PNS)) was selected using a halogen lamp as light
source, a medium containing malic and glutamic acid, and micro- and macronutrients
and specific operational conditions (anaerobic conditions, 32ºC, 8000 lux) and enriched
from two types of anaerobic sludges (granular and suspended). It came out that a mixed
culture suitable for the production of H2 (no presence of methane) was obtained from
granular sludge. The characterization of this biomass was carried out using a reference
substrate (malic acid (30 mmol L-1)) in two different experiments. Firstly, batch tests were
conducted in triplicate in 500mL-bottles at two pH values (5.5 and 7.0). Secondly, the
degradation kinetics and biomass growth was assessed in a 2.2L reactor in batch mode.
It came out that pH 7.0 led to a higher conversion efficiency (23.4%) than that at pH
5.5 (12.8%) and that the consumption of malic acid obeyed a first order kinetics
(k = 0.011 h-1).
Finally, the study of the bioH2 production of a diluted synthetic dark fermentation effluent
containing succinic acid (5.2 mmol L-1), acetic acid (1.7 mmol L-1), propionic acid
(1.2 mmol L-1) and butyric acid (1.4 mmol L-1) was performed in batch mode
(500 mL-reactors). The results coming out from this investigation show a conversion yield
of the substrates of 3.8 % and the formation of a non-identified product. This suggests
that other metabolic pathways are occurring instead of hydrogen production due to
contamination or non-favorable operational conditions. Thus, a more extensive research
evaluating microbial communities and the degradation of each substrate individually
would be desirable to have a better picture of the process.
vii
Table of Contents
1. INTRODUCTION
1
1.1 RATIONALE OF THE WORK
3
1.2 OBJECTIVES
3
2. LITERATURE REVIEW
4
2.1 BIOHYDROGEN PRODUCTION
4
2.2 BIOHYDROGEN PRODUCTION THROUGH PHOTO-FERMENTATION
4
2.2.1 THE PHOTO-FERMENTATION PROCESS
4
2.2.2 THE PHOTOSYNTHETIC PURPLE NON-SULFUR BACTERIA (PNS)
6
2.2.3 FACTORS INFLUENCING THE PHOTO-FERMENTATION PROCESS
7
2.2.4 SEQUENTIAL PRODUCTION OF H2 THROUGH DARK FERMENTATION AND PHOTOFERMENTATION
11
3. MATERIALS AND METHODS
13
3.1 MATERIALS
13
3.1.1 INOCULUM
13
3.1.2 MINERAL MEDIUM
13
3.1.3 SUBSTRATES: CARBON AND NITROGEN SOURCES
14
3.2 ANALYTICAL METHODOLOGY
15
3.2.1 LIGHT INTENSITY AND SPECTRUM
15
3.2.2 TOTAL (TSS) AND VOLATILE SUSPENDED SOLIDS (VSS)
15
3.2.3 BIOGAS PRODUCTION
16
3.2.4 BIOGAS COMPOSITION
17
3.2.5 SOLUBLE CHEMICAL OXYGEN DEMAND (CODS)
18
3.2.6 DETERMINATION OF VOLATILE FATTY ACIDS (VFA)
19
3.2.7 PH
19
3.3 EXPERIMENTAL SET-UP AND OPERATION
20
3.3.1 EXPERIMENT 1 (E1): SELECTION AND AUGMENTATION OF PHOTO-FERMENTATIVE INOCULUM
FROM DIFFERENT ANAEROBIC SLUDGES
21
3.3.2 EXPERIMENT 2 (E2): INFLUENCE OF PH ON PHOTO-FERMENTATIVE CULTURE GROWN FROM
THE INOCULUM SELECTED IN E1
23
3.3.3 EXPERIMENT 3 (E3): STUDY OF THE KINETICS OF CODS DEPLETION UNDER H2-PRODUCING
CONDITIONS
25
3.3.4 EXPERIMENT 4 (E4): PRODUCTION OF BIO-H2 FROM AN EFFLUENT DERIVED THROUGH THE
DARK FERMENTATION OF GLYCEROL
26
viii
3.4 MATHEMATICAL MODELS
28
4. RESULTS
29
4.1 SELECTION AND AUGMENTATION OF PNS CONSORTIUM FROM ANAEROBIC SLUDGES
29
4.2 CHARACTERIZATION OF THE PNS CONSORTIUM
31
4.2.1 PH INFLUENCE ON BIOHYDROGEN PRODUCTION (E2)
31
4.2.2 KINETICS OF MALIC ACID DEPLETION AND BIOMASS GROWTH IN 2.2L REACTOR (E3)
32
4.2.3 HYDROGEN PRODUCTION AND VFAS DEGRADATION BY PNS CONSORTIUM (E4)
35
5. DISCUSSION
37
5.1 SELECTION AND ENRICHMENT
37
5.2 CHARACTERIZATION OF THE MICROBIAL CONSORTIUM
37
5.3 H2 PRODUCTION FROM A DILUTED DF SYNTHETIC EFFLUENT
39
6. CONCLUSION
40
7. REFERENCES
41
8. ANNEX
45
ix
Figures and tables
FIGURE 2-1 PHOTO-FERMENTATION PROCESS
5
FIGURE 2-2 SPECTRUM OF ABSORPTION OF THE PURPLE NON-SULPHUR BACTERIA.
8
FIGURE 2-3 BACTERIAL GROWTH (☐) H2 PRODUCTION
ACETATE CONCENTRATION (×),
BUTYRATE CONCENTRATION (+) AND PROPIONATE CONCENTRATION
DATA IN MEDIA
CONTAINING (A) MIXTURE OF ACETATE (40 MM), BUTYRATE (10 MM) AND PROPIONATE (5 MM), (B)
MIXTURE OF BUTYRATE (30 MM), PROPIONATE (5 MM) AND ACETATE (10 MM).
10
FIGURE 3-1 AMPTS II: (A) THREE MAIN UNITS OF AMPTS II; (B) FLOW CELLS FOR BIOGAS
MEASUREMENT
17
FIGURE 3-2 SCHEME OF THE EXPERIMENTAL SET-UP
20
FIGURE 3-3 EXPERIMENTAL SET-UP FOR SELECTION AND AUGMENTATION OF PHOTOFERMENTATIVE INOCULUM FROM DIFFERENT ANAEROBIC SLUDGES (A) BATCH TESTS, (B) SCHEME
OF THE EXPERIMENTAL SET-UP
22
FIGURE 3-4 EXPERIMENTAL SET-UP OF BATCH PHOTOBIOREACTORS AT PH 5.5 AND 7.0 (IN
TRIPLICATE)
24
FIGURE 3-5 EXPERIMENTAL SET-UP OF 2.2L PHOTOBIOREACTOR
26
FIGURE 3-6 EXPERIMENTAL SET-UP OF BATCH PHOTOBIOREACTORS CONTAINING DF SYNTHETIC
EFFLUENT (IN TRIPLICATE)
27
FIGURE 4-1 PNS CONSORTIUM (IN RED COLOUR) CULTIVATED FROM: (A) GRANULAR ANAEROBIC
SLUDGE (GAS), (B) SUSPENDED ANAEROBIC SLUDGE (SAS)
29
FIGURE 4-2 COLOUR TONE CHANGE DURING CULTIVATION OF PNS CONSORTIUM (STAGE II).
30
FIGURE 4-3 CUMULATIVE H2 PRODUCTION BY PNS BACTERIA AT PH 5.5 AND PH 7.0 FITTED TO
GOMPERTZ FUNCTION (E2).
31
FIGURE 4-4 CODS AND BIOMASS VS TIME IN 2.2L WORKING VOLUME REACTOR
32
FIGURE 4-5 FIRST-ORDER KINETICS OF MALATE DEPLETION
33
FIGURE 4-6 DETERMINATION OF THE MAXIMUM SPECIFIC GROWTH RATE
33
FIGURE 4-7 CUMULATIVE H2 PRODUCTION BY PNS BACTERIA WITH CONTROLLED PH (E3).
34
FIGURE 4-8 CUMULATIVE H2 PRODUCTION OF PHOTOFERMENTATIVE REACTORS WITH DF
SYNTHETIC EFFLUENT (S3) AND MALIC AND GLUTAMIC ACID (S2)
35
FIGURE 4-9 (A) CENTRIFUGED SAMPLES (4500RPM) OF CONTROL TESTS; (B) MICROSCOPE IMAGE
OF A DILUTED SAMPLE FROM PELLET
36
FIGURE 8-1 SPECTRUM OF (A) HALOGEN LAMP AND (B) INCANDESCENT AND SUBMERGIBLE LAMP
45
FIGURE 8-2 SCHEME OF CSTR USED IN DF OF GLYCEROL
46
FIGURE 8-3 CALIBRATION CURVE FOR (A) HYDROGEN, (B) NITROGEN, (C) METHANE AND (D)
CARBON DIOXIDE GAS
48
x
FIGURE 8-4 CALIBRATION CURVES FOR (A) ACETIC ACID, (B) PROPIONIC ACID (C) BUTYRIC ACID
(D) SUCCINIC ACID (E) MALIC ACID (F) GLUTAMIC ACID
51
FIGURE 8-5 CUMULATIVE METHANE PRODUCTION FROM GRANULAR AND SUSPENDED ANAEROBIC
SLUDGE (STAGE I)
51
FIGURE 8-6 BIOGAS PRODUCTION DURING CULTIVATION OF PNS CONSORTIUM (STAGE II)
52
TABLE 2-1 MECHANISMS FOR BIOHYDROGEN PRODUCTION
4
TABLE 2-2 STUDIES REPORTED IN LITERATURE OF PHOTO-FERMENTATION COUPLED TO DARK
FERMENTATION
12
TABLE 3-1. CHARACTERIZATION OF SLUDGES
13
TABLE 3-2 COMPOSITION OF SYNTHETIC MEDIUM USED FOR CULTURE GROWTH AND HYDROGEN
PRODUCTION BATCH EXPERIMENTS
13
TABLE 3-3 CARBON AND NITROGEN SOURCES
14
TABLE 3-4. INITIAL CONDITIONS OF 2.2L PHOTOBIOREACTOR
25
TABLE 3-5 EXPERIMENTAL SET-UP AND OPERATIONAL CONDITIONS IN PHOTO-FERMENTATION
BATCH TESTS USING REAL AND SYNTHETIC DF EFFLUENT
27
TABLE 4-1 QUALITATIVE RESULTS FROM E1 EXPERIMENT (STAGE I)
29
TABLE 4-2 QUALITATIVE RESULTS FROM E1 EXPERIMENT (STAGE II)
30
TABLE 4-3 PARAMETERS OF MODIFIED GOMPERTZ´S MODEL
31
TABLE 4-4 PARAMETERS OF GOMPERTZ´S MODEL
34
TABLE 4-5 PARAMETERS OF GOMPERTZ´S MODEL FOR CONTROL WITH MALIC ACID (E4)
35
TABLE 4-6 CONVERSION YIELDS OF VFAS MIXTURE AND CONTROL
35
TABLE 4-7 CODS REMOVAL
36
TABLE 8-1 COMPOSITION OF FEEDING SOLUTION IN THE DF OF GLYCEROL
47
TABLE 8-2 CALCULATION OF CARBON CONTRIBUTION IN THE DF EFFLUENT
48
TABLE 8-3 GAS COMPOSITION DURING SELECTION OF PNS CONSORTIUM (STAGE I)
52
TABLE 8-4 GAS COMPOSITION DURING AUGMENTATION OF PNS CONSORTIUM (STAGE II)
53
TABLE 8-5 HPLC AREAS E4 EXPERIMENT SAMPLES.
54
1
1. Introduction
In 2015, worldwide energy demand still relies mostly on the use of finite fossil fuels. Their
extensive use is leading not only to the depletion of reserves, but also to environmental
problems derived through the combustion of these fuels such as greenhouse gases
emissions and ozone layer depletion (Ghimire et al., 2015). Due to this fact, researchers
are putting enormous effort into finding new energy sources that are environmentally
friendly and renewable (Singh and Wahid, 2015). Fuels produced by biological processes
are presented as an attractive alternative since they are usually more cost-effective than
conventional methods, and biomass, which is one of the major renewable energy
sources available, is used as feedstock (Liu et al., 2013).
Among biofuels, hydrogen gas (H2) stands out for having unique features as energy
carrier and alternative fuel. These are its high specific energy content (143 kJ g-1) (Basak
and Das, 2007) and the generation of water vapor as the sole by-product when
combusted in presence of oxygen (Akkerman et al., 2002). The conversion of its
chemical energy into electricity can be conducted with great efficiency in hydrogen fuel
cells through an electrochemical process (Ozmihci and Kargi, 2010).
On an industrial scale, hydrogen is mainly obtained through steam reforming of natural
gas meeting up to 50% of the worldwide hydrogen demand. Other conventional
technologies also used are autothermal reforming, biomass gasification or electrolysis of
water (Chaubey et al., 2013).
In contrast with conventional and mature technologies, research on biological H2producing processes has been conducted mainly at lab- and pilot-scale and largely
remains there, due to low experimental H2 production rates and practical limitations. The
ways in which biohydrogen can be produced are those involving light energy
dependence (photo-production) and anaerobic fermentation (Singh and Wahid, 2015).
The photo-production of H2 consists of the conversion of solar energy through microalgae
or photosynthetic bacteria (bio-photolysis) and photo-fermentation by phototrophic
bacteria into H2, whereas dark fermentation produces H2 based on the incomplete
anaerobic degradation of a carbon source (Elsharnouby et al., 2013).
Within these approaches for obtaining H2, anaerobic fermentation presents several
advantages due to its simplicity and the utilization of a broad range of organic wastes.
Also, it does not require an input of light energy. On the other hand, one of the major
drawbacks arises from a lower production of H2 per mole of substrate than that
theoretically obtainable (Nath and Das, 2009).
2
This incomplete conversion means that effluents derived from hydrogen fermentation
reactors are characterized by their high content in volatile fatty acids (VFAs). Thus, many
authors suggest coupling a second stage to the system with the aim of increasing its
bioenergy recovery. In recent years, technologies such as anaerobic digestion leading to
the co-generation of H2 and CH4 have been widely reported in literature as second
phase. But there are also other promising processes such as photo-fermentation that
might be coupled to dark fermentation. In this process, anoxygenic phototrophs are
capable to achieve high hydrogen conversion yields by depleting organic acids present in
the dark fermentation effluent such as acetic, lactic and butyric acids. Besides that, other
remarkable advantage of the use of this process would be the production of a gas stream
with a sole and more valuable component (H2) (Ghimire et al., 2015).
Among industrial wastes, an interesting substrate for the production of H2 through dark
fermentation is the glycerol that is generated in the biodiesel industry. The market price
of this by-product has substantially decreased in the last years due to the strong increase
of biodiesel manufacturing (Pott et al., 2013) leading to the failure of chemical companies
dedicated to its production (Dharmadi et al., 2006). Given these facts, glycerol is
presented as an attractive feedstock for bio-H2 production. In literature, it has been
proved that facultative anaerobic microorganisms lead to higher hydrogen yields than
those obtained with sugars. Main metabolites of dark fermentation of glycerol reported
are acetate (Akutsu et al., 2010), 1,3-propanediol (Gonzalez et al., 2005; Ito et al., 2005;
Akutsu et al., 2010; Kivistö et al., 2010), ethanol (Ito et al., 2005; Sakai et al., 2007) and
butyrate (Gonzalez et al., 2005).
Based on this background, this work will focus on the integration of photo-fermentation
with dark fermentation for the recovery of the energy remaining in the metabolites
present in the dark fermentation effluent of glycerol.
3
1.1 Rationale of the work
During the last years the scientific output about photo-fermentation and the parameters
influencing such process has increased substantially, but very few investigations have
reported so far the use of anaerobic sludges as seed of the photo-bioreactors unlike
research with pure cultures. Moreover, although the dark fermentation effluent is mostly
composed of VFAs such as acetate, propionate and butyrate, and alcohols, its
composition may vary depending on the substrates that are being processed.
Particularly, the effluent generated through the dark fermentation of the glycerol contains
besides VFAs and ethanol, succinic acid and 1,3-propanediol. In this sense and
considering all the operational parameters attained to the photo-fermentation process,
this work attempts to develop a protocol for the selection and enrichment of a mixed
culture of PNS bacteria from different raw sludges, to characterize the PNS bacterial
consortium in terms of substrate consumption and growth kinetics and to study the
potential of this consortium for the photo-fermentation of the DF effluent.
1.2 Objectives
The overall objective of this research is to evaluate the photo-fermentative hydrogen
production of a selected and enriched PNS mixed culture as a technology for the posttreatment of glycerol dark fermentation effluent
The specific goals of the present work were:

Selection (or isolation) and augmentation of an hydrogen producing bacterial
consortium from different anaerobic sludges

Characterization of the consortium using a reference substrate: sensitivity to pH,
degradation kinetics and biomass growth

Determine the bioH2 production and the VFAs degradation of a synthetic substrate
simulating the effluent from the dark fermentation of glycerol through a photofermentative process
4
2. LITERATURE REVIEW
2.1 Biohydrogen production
In general, the mechanisms for biohydrogen production can be divided into two major
groups. These are those that need the capture of light (biophotolysis and photofermentation) and those that are light independent (dark fermentation) (Ghimire et al.,
2015).
Table 2-1 Mechanisms for biohydrogen production
Mechanism
Microorganisms
Biophotolysis
Algae and
cyanobacteria
(photosynthetic
bacteria)
(Light
dependent)
Photofermentation
Purple non sulfur
bacteria
(Light
dependent)
(phototrophic
bacteria)
Dark
fermentation
(Light
independent)
Anaerobic
bacteria
(heterotrophs)
Carbon and
energy
source
Microorganisms
· CO2
· Light
· Organic acids
· Light
·Carbohydrates
and
fermentation
products
Algae and
cyanobacteria
(photosynthetic
bacteria)
Purple non
sulfur bacteria
(phototrophic
bacteria)
Anaerobic
bacteria
(heterotrophs)
Biogas
composition
H2 and O2
H2 and
CO2
H2 and
CO2
2.2 Biohydrogen production through photo-fermentation
2.2.1 The photo-fermentation process
The photo-fermentation process, which is also known as light fermentation (Argun et al.,
2008), consists in the synthesis of H2 as well as CO2 and new biomass by using light and
organic compounds as energy and carbon source, respectively (Adessi and De
Philippis, 2014). The microorganisms that are capable to carry out this process are the
so-called purple non-sulfur bacteria (PNS).
PNS bacteria contain purple pigments such as carotenoids and bacteriochlorophylls (‘a’
and ‘b’) through which light can be absorbed to carry out photosynthesis (Akkerman et
al., 2002). Unlike other photosynthetic microorganisms, such as cyanobacteria and
algae, PNS bacteria do not use water as electron donor, so O2 is not released during the
photosynthesis (Lee et al., 2010). The anoxygenic photosynthesis is the earliest form of
5
photosynthesis and it is only conducted by green and purple bacteria under anaerobic
conditions.
The
latter
(gammaproteobacteria
is
made
group)
up
and
by
two
purple
major
non-sulfur
classes:
bacteria
purple
bacteria
(alpha-
and
betaproteobacteria groups).
The hydrogen production via photo-fermentation is initiated with the absorption of light in
the reactor center (RC) by the bacteriochorophyll (P840) (Dasgupta et al., 2010). Under an
anaerobic environment, the microorganisms break down organic substrates liberating
electrons through the tricarboxylic acid (TCA) cycle that are taken by different electron
carriers (Figure 2.1). The transport of electrons in the membrane ends up with the
accumulation of protons outside the cell causing an electrostatic gradient, which is used
to evolve ATP by the ATP synthase (Akkerman et al., 2002). Afterwards, in a high energy
demanding process the ferredoxin molecule is reduced. Using the electrons released
from this step and energy in form of ATP, the nitrogenase is able either to reduce protons
into hydrogen gas when no nitrogen gas (N2) is present, or converts the N2 into ammonia
(NH3) under the presence of N2 (Ghimire et al., 2015).
Figure 2-1 Photo-fermentation process (Adapted from: Hallenbeck and Ghosh, 2009;
Adessi and De Philippis, 2014)
During the photo-fermentation process is essential to limit the presence of ammonium
nitrogen (NH4-N) and oxygen to avoid causing inhibition on the nitrogenase enzyme.
Inhibition owing to the presence of O2 is irreversible whereas to the presence of
ammonium is reversible. The influence of NH4-N concentration on the H2 photoproduction was studied by Argun et al. (2008) using a mixture of three strains of
Rhodobacter sphaeroides. Ammonium concentration was found to be inhibitory at 50 mg
6
NH4-N L-1. Regarding the temperature of the process, authors show agreement
establishing an optimum range of 31-36 ºC (Basak and Das, 2007).
2.2.2 The photosynthetic purple non-sulfur bacteria (PNS)
Purple non-sulfur bacteria are a polyphyletic group of microorganisms that stand out for
their versatile metabolism (Basak and Das, 2007). Although there is a preference for
growing in a photoheterotrophic mode, which implies H2 generation, it has been
demonstrated that they also posses the ability to grow photoautotrophically fixing
inorganic carbon from CO2 and using S2, H2 or Fe2+ as electron donors. Moreover, they
can also perform other modes of metabolism that are not light dependent such as
aerobic and anaerobic respiration, and fermentation (Koku et al., 2002).
Apart from organic acids
Their particular color is given by the carotenoids and bacteriochlorophyll pigments. These
pigments are usually red, brown or purple.
The production of biohydrogen by pure cultures of PNS bacteria has drawn the attention
of researchers due to their high conversion yield. The most frequently utilized strains for
biohydrogen production are Rhodobacter sphaeroides, Rhodobacter capsulatus,
Rhodopseudomonas palustris and Rhodospirillum rubrum (Uyar et al., 2009; Lee et al.,
2010; Ozmihci and Kargi, 2010). However, there has been very little research activity in
the use of consortia of purple non-sulfur bacteria for the production of biohydrogen. The
use of mixed bacterial consortium for biological processes is often advantageous in that
they are typically more resilient to overcome changes in their environment, and that they
respond better to variation of feedstock composition (Lazaro et al., 2012).
The equation 2.1 displays the general stoichiometry of H2 production from organic acids
(Pott et al., 2013).
𝐿𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦 𝑦
(2
𝐶𝑥 𝐻𝑦 𝑂𝑧 + (2𝑥 − 𝑧)𝐻2 𝑂 ⇒
+ 2𝑥 − 𝑧)𝐻2 + 𝑥𝐶𝑂2
(Eq. 2.1)
7
Thus, one mole of acetate, lactate, propionate and butyrate yields to the production of 4,
6, 7 and 10 moles of H2, respectively (Eq 2.2).
𝐿𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦
𝐴𝑐𝑒𝑡𝑎𝑡𝑒: 𝐶2 𝐻4 𝑂2 + 2𝐻2 𝑂 ⇒
𝐿𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦
𝐿𝑎𝑐𝑡𝑎𝑡𝑒: 𝐶3 𝐻6 𝑂3 + 3𝐻2 𝑂 ⇒
4𝐻2 + 2𝐶𝑂2
(1)
6𝐻2 + 3𝐶𝑂2
(2)
𝐿𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦
𝑃𝑟𝑜𝑝𝑖𝑜𝑛𝑎𝑡𝑒: 𝐶3 𝐻6 𝑂2 + 4𝐻2 𝑂 ⇒
𝐿𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦
𝐵𝑢𝑡𝑦𝑟𝑎𝑡𝑒: 𝐶4 𝐻8 𝑂2 + 6𝐻2 𝑂 ⇒
7𝐻2 + 3𝐶𝑂2
(3)
10𝐻2 + 4𝐶𝑂2
(4)
(Eq 2.2)
2.2.3 Factors influencing the photo-fermentation process
According to Tawfik et al. (2014), several factors to be taken into consideration for the
optimization of the process, such as lighting conditions, type and concentration of
substrate, inoculum age, composition of nutrient medium, temperature and pH.
a) Lighting conditions

Light source
One particularly key aspect in the photo-fermentation process is the selection of a
suitable light source for the PNS bacteria (Adessi and De Philippis, 2014). Figure 1.1
illustrates the spectrum of absorption of carotenoids and bacteriochlorophylls that are
red-purple pigments present in PNS bacteria and also responsible of the photosynthesis.
Light sources most frequently used in photo-fermentation studies are tungsten lamps
(Fang et al., 2005; Uyar et al., 2009; Tawfik et al., 2014) owing to the broad range of
spectrum covered by these lamps. Although it is also common to find works utilizing
incandescent, fluorescent and halogen lamps. To evaluate the effect of the culture
irradiation on the photo-fermentative hydrogen gas production, Argun and Kargi (2010)
carried out a comparison by using different types of energy sources: tungsten, infrared,
fluorescent and halogen lamp, and sun light. In his study, it came out that under the
same light intensity conditions (270 W m-2), the halogen lamp led to the highest hydrogen
yield (781 mL H2 g-1 VFA which corresponds to 47% of the theoretical value) and specific
hydrogen production rate (17.5 mL H2 g-1 biomass h-1).
8
Figure 2-2 Spectrum of absorption of the purple non-sulfur bacteria. Peaks with single
asterisk: maximum absorbance of carotenoids pigments (450-550 nm); peaks with
double asterisk: maximum absorbance of bacteriochlorophylls (at 850 and 880 nm)
(Source: Adessi and De Philippis, 2014)

Light intensity
Nath and Das (2009) stated that in general terms the higher the light intensity, the more
favorable the growth of biomass, hydrogen yield and productivity, as well as the depletion
of substrates. This is applicable only up to a certain optimum value that ranges from
4000 lux (Uyar et al., 2009) to 10000 (Nath and Das, 2009). According to Basak and Das
(2007), exposure to higher light intensity will not inhibit the process but it will lessen the
light conversion efficiency. In general, different ranges are suggested for biomass growth
(4000 - 6000 lux) and photo-production of hydrogen (6000 - 10000 lux).

Light-dark cycles
Photo-fermentation assays are generally submitted to continuous illumination since the
production of H2 is higher than under the absence of light. However, some studies have
elucidated the effect of light-dark cycles on the performance of the photosynthetic
bacteria. They reveal that an increase of the gas production over the production under
continuous light conditions occurs when the exposure to light is combined with dark
periods, e.g. using cycles of 14 hours of light and 10 h of darkness (Koku et al., 2003) or
cycles of light and darkness every 30 minutes. This evidence indicates that the
performance of bacteria might be greater under outdoor conditions with day-night lighting
cycles.
9

Substrates
Industrial wastes containing organic acids are the most suitable substrates for hydrogen
production through photo-fermentation. Unlike carbohydrates, which are easily found in
nature, organic acids derive mainly from dark fermentation processes (Singh and Wahid,
2015). Studies evaluating individually different organic acids as substrates in terms of
hydrogen production and substrate depletion are widely reported in literature although
little has been published about complex wastes (Koku et al., 2002). Malic acid, which is
100% degradable by PNS (Lazaro et al., 2012), is generally the substrate utilized as
carbon source in photo-fermentation studies to find optimal operational conditions. For
instance, Koku et al. (2003) studied the kinetics of bio-H2 generation by Rhodobacter
sphaeroides using varying parameters such as illumination conditions and the culture
medium composition where vitamins were replaced by yeast. In the same way, the
glutamic acid is commonly used as nitrogen substrate. The relationship between carbon
and nitrogen present in the culture medium plays an important role on the behavior of the
bacteria (Basak and Das, 2007). A high C/N ratio will limit the availability of nitrogen for
the bacteria and will force the production of hydrogen by using the surplus of energy and
reducing power (Uyar et al., 2009). On the contrary, a lower carbon: nitrogen ratio will
promote the biomass growth and will lead to lower hydrogen production. In this sense, for
bacterial growth 7.5 mmol L-1 of malic acid and 10 mmol L-1 of glutamic acid are typically
used (Koku et al., 2003; Uyar et al., 2009) and for hydrogen production 15 and 2 mmol L1
of malic and glutamic acids, respectively (Gilbert et al., 2011). VFAs, as stated
previously, are also suitable substrates for PNS bacteria. In a paper by Uyar et al.
(2009), some organic acids such as malate, acetate, propionate, butyrate and lactate
were evaluated separately and simultaneously using the strain Rhodobacter sphaeroides
(see Table 2-1). In the first case, substrate conversion efficiencies reported from were
50, 33, 31 and 14 % for malate, acetate, propionate and lactate, and butyrate,
respectively, whereas in the second case where the mixture was formed by acetate,
propionate and butyrate there was a considerable increase (47 %). From this study, it
was also inferred the order of consumption of substrates. Acetate and propionate are
more susceptible to be degraded by the PNS bacteria than butyrate (see Figure 2-3).
10
Figure 2-3 Bacterial growth (☐) H2 production (), acetate concentration (×), butyrate
concentration (+) and propionate concentration () data in media containing (a) mixture
of acetate (40 mM), butyrate (10 mM) and propionate (5 mM), (b) mixture of butyrate (30
mM), propionate (5 mM) and acetate (10 mM). Source: Uyar et al., 2009

Photo-bioreactors
Features of photobioreactors must meet some requirements for a proper functioning.
Firstly, the photoreactor should be characterized for being a closed system to avoid any
escape of H2. Secondly, the surface to volume ratio should be high so that availability of
light for the microorganisms will be as uniform as possible. Other important aspects to
consider are the type of mixing as well as control of pH and temperature and factors
mentioned above such as light source and intensity (Adessi and De Philippis, 2014). The
work of Zhang et al. (2015) indicates that the use of magnetic stirring in batch reactors
does not have a positive effect on the cumulative H2 yield which value was 50 % lower
than that in the static one.
Main reactor designs used in lab-scale are: fermenter type (Ozmihci and Kargi, 2010),
flat panel reactor (Gilbert et al., 2011) and tubular reactor (Tawfik et al., 2014). Dasgupta
et al. (2010) reviewed the latest trends concerning photobioreactors design for H2
production.
11
2.2.4 Sequential production of H2 through dark fermentation and photo-fermentation
Unlike combined dark- and photo-fermentation, a sequential configuration allows the
optimization of both stages individually. However, the transfer of the effluent from the
fermenter to the photobioreactor might result in problems that are related to pH and
temperature changes, the lack of several micro- and macronutrients, high content of
VFAs and the presence of potential inhibitors such as ammonium, and that can affect the
well functioning of the second stage (Laurinavichene et al., 2012).
The integration of these two processes has been reported using pure cultures. For
instance, Nath et al. (2005) studied the production of hydrogen from glucose dark
fermentation using the strain Enterobacter cloacae DM11, which was followed by a
photo-fermentation stage with R. Sphaeroides OU001 and an effluent that contained
mostly acetic acid. Thanks to the coupling the system achieved a higher yield than a
single DF stage. The yield was 1.86 moles de H2 per mol de glucose in the first stage
and 1.5 – 1.72 mol H2 per mol of acetic acid in the second stage.
Other examples of studies coupling these two processes are given in Table 2-2.
12
Table 2-2 Studies reported in literature of photo-fermentation coupled to dark fermentation
Type of
microorganism
DF stage
(substrate and/or
yield)
Culture medium
Type of
reactor
T (°C)
Not spc.
(acetate and
Illumination
Reference
Tungsten lamps
(200 W m-2)
Fang et al., 2005
2.5 mol H2/mol
Synthetic effluent
Not spc.
Hydrogen yield
acetate (pH=8);
Batch
32
butyrate)
3.7 mol H2/mol
butyrate (pH=9)
Mixture of
Rhodobacter
1.53 mol H2 mol-1
glucose
1700 mg L-1 TVFA
Batch
30
2.28 mol H2 mol-1
glucose
Fluorescent lamps
36 W (5500 lux)
Argun et al., 2008
Rhodobacter
capsulatus B10 and
Rhodobacter
sphaeroides N7
Starch
Yield 1,4 mol H mol1
hexose
DF effluent
(Acetate and butyrate),
DF = 2
Fed-batch
28
3.9 mol H2 mol-1
hexose
Incandescent light
(30 W m-2)
Laurinavichene et
al., 2012
Rhodobacter
sphaeroides NRRL
– B1727
Ground wheat
starch as substrate
Effluent of DF in
continuous mode (VFAs
concentration
1950 mg L-1)
Semicontinuous
30
185 mL H2 g-1 VFA
Halogen lamp
(5 klux)
Ozmihci and Kargi,
2010
Activated sludge
Starch wastewater
Acetate, propionate,
butyrate, lactate
Continuous
31
3.5 L d.1 (OLR=3.26.4 gCOD L-1 d-1)
Tungsten lamp
(190 W m-2)
Tawfik et al., 2014
Rhodobacter
sphaeroides O.U.
001
Author refers to dark
effluents from
literature
Synthetic effluent
containing acetate (40
mM), butyrate (10 mM)
and propionate (5 mM)
Batch
30-33
2.5 L H2 L-1reactor
Tungsten lamp
(150-200 W m-2)
Uyar et al., 2009
Rhodobacter
sphaeroides O.U.
001
Author refers to dark
effluents from
literature
Synthetic effluent
containing butyrate (40
mM), acetate (10 mM)
and propionate (5 mM)
Batch
30-33
2.2 L H2 L-1reactor
Tungsten lamp
(150-200 W m-2)
Uyar et al., 2009
Rhodobacter
capsulatus (DSM
155)
Miscanthus
hydrolysate as
substrate
Effluent from DF pretreated and diluted.
Sodium glutamate
(2mM), acetate (47mM)
lactate (9.8 mM), fructose
(2 mM)
Batch
30-33
1.37 L H2 L-1reactor
Halogen lamp
(150 W m-2)
4000 lux
Uyar et al., 2009
sphaeroides
(RV, NRLL and
DSMZ)
13
3. MATERIALS AND METHODS
3.1 Materials
3.1.1 Inoculum
In order to study the growth of purple non-sulfur bacteria under photosynthetic
conditions, different sources of inoculum were collected. Inoculum samples were
granular anaerobic sludge from British American an upflow anaerobic sludge blanket
(UASB) reactor of Tobacco (Casablanca, Chile) and non-granular anaerobic sludge from
the wastewater treatment plant (WWTP) of La Farfana (Santiago de Chile, Chile).
Characterization of the sludge was based on the measurements of total suspended
solids (TSS) and volatile suspended solids (VSS) concentration. Values are shown in
Table 3-1.
Table 3-1. Characterization of sludges
Type
TSS (g L-1)
VSS (g L-1)
Granular anaerobic
sludge
35.3  1.0
31.2  1.0
Suspended anaerobic
sludge
28.2  0.2
14.1  0.2
3.1.2 Mineral medium
Mineral medium containing micro and macronutrients was prepared for growth of
photosynthetic bacteria and hydrogen production experiments. The medium, which was
based on the synthetic medium proposed by Fang et al. (2005), is showed in Table 3-2.
Table 3-2 Composition of synthetic medium used for culture growth and hydrogen
production batch experiments
Nutrients
Concentration
(g L-1)
Nutrients
Concentration
(mg L-1)
KH2PO4
0.5
Na2MoO4·2H2O
0.03
MgSO4·7H2O
0.4
ZnSO4·7H2O
0.1
NaCl
0.4
CoCl2·2H2O
0.2
CaCl2·2H2O
0.05
CuCl2·2H2O
0.01
Fe (III) from citrate
3.9 mg L-1
MnCl2·4H2O
0.03
H3BO3
0.3 mg L-1
NiCl2·6H2O
0.02
Vitamin B12
0.04
14
Additionally, NaOH (40 g L-1) and HCl (25M) were used to adjust the pH to the desired
value.
3.1.3 Substrates: carbon and nitrogen sources
Different organic acids were used as substrates depending on the purpose of the assay
being conducted. Malic and glutamic acids were used for Experiments 1, 2 and 3, and
synthetic effluent containing VFAs were used for Experiment 4. Components that make
up the different substrates are shown in Table 3-3.
Table 3-3 Carbon and nitrogen sources
Substrate
Compounds
Experiment
S1
Malic acid (30 mM),
Glutamic acid (4 mM)
1
S2
Malic acid (30 mM)
Glutamic acid (9 mM)
2 and 3
S3 - synthetic effluent
Succinic acid (1.5 g/L)
Acetic acid (0.3 g/L)
Propionic acid (0.2 g/L)
Butyric acid (0.3 g/L)
Glutamic acid (0.4 g/L)
4
15
3.2 Analytical methodology
3.2.1 Light intensity and spectrum
The spectra of two different lamps were studied with a spectroradiometer Go-Spex 500
(EverFine) (see Annex – 8.1) whereas the intensity was determined with a light meter
(Light ProbeMeterTM, EXTECH Instruments) on the surface of the bottles.
3.2.2 Total (TSS) and volatile suspended solids (VSS)
Total and suspended solids contents were analyzed according to an adaptation of the
procedures 2540 D and 2540 E that are described on the Standard Methods for the
Examination of Water and Wastewater (APHA, 2005). Chilean regulation (NCh 2313/3,
Of 95) establishes the application of the same procedure for the determination of TSS.
Total suspended solids represents the organic and mineral non-soluble fraction of solids
contained in a pellet after filtration and drying at 105°C of a sample of known volume.
The adapted procedure consisted of centrifuging a sample for 12min at 4400rpm instead
of filtration as indicated by the APHA. Afterwards, the supernatant was discarded and the
pellet was thoroughly washed three times with 10mL of distilled water. Then, it was
centrifuged again at the same conditions. The resulting pellet was washed with water into
a crucible or aluminum cap and dried in an oven at 105°C for at least 24h.
After drying, samples were allowed to cool down in a desiccator and then they were
weighed. An analytical balance (Radwag AS220/C/2) capable of measuring 0.1mg was
used.
TSS was calculated as follows:
𝑔
𝐵−𝐴
)×
𝑣
𝑇𝑆𝑆 [ 𝐿 ] = (
1000
(Eq.3.1)
where,
A: weight of crucible (g)
B: weight of crucible + weight of dried sample (g)
v: volume of centrifuged sample (mL)
Volatile suspended solids content corresponds to the biomass present in the sample.
VSS was determined by incinerating the sample in a muffle furnace (Vulcan A-1750) for
8 hours at 550°C. After incineration, samples were again placed into the desiccator until
cool, then weighed. The loss in weight during the incineration represents the VSS
contained in the sample.
16
VSS was calculated as follows:
𝑔
𝐵−𝐴
) ∗ 1000
𝑣
𝑉𝑆𝑆 [ 𝐿 ] = (
(Eq.3.2)
where:
A: weight of crucible + weight of incinerated sample (g)
B: weight of crucible + weight of dried sample (g)
v: volume of centrifuged sample (mL)
3.2.3 Biogas production
Biogas production was determined by two different volumetric methods: in the first,
incubation bottles with 500 mL capacities (Simax, Czech Republic) were attached to an
Automatic Methane Potential Test System (AMPTS II, Bioprocess Control). In the second
method, a batch-reactor with a capacity of 2.7 L was connected to a device where the
biogas production was determined by the liquid displacement principle.
The AMPTS (II) (Figure 3-1) is an instrument originally designed for the estimation of
biochemical methane potential (BMP) of organic substrates. It consists of three main
parts: an incubation unit, a CO2-removal unit and a gas collection unit. The incubation
unit is equipped with 15 batch-reactors of 500mL of capacity and includes mechanical
mixing, and speed and run time control. These bottles are connected to the gas
collection unit through the CO2-fixation unit. The latter includes 15 small bottles that
contain a solution of NaOH (3M) and a pH-indicator solution of 0.4% thymolphthalein.
The gas collection unit incorporate 15 flow cells with an embedded webserver that allows
constant monitoring of gas volume and flow measurements in real time and provide data
normalized to standard temperature and pressure (STP) conditions.
resolution of these cells is 10 mL.
Measuring
17
(a)
(b)
Figure 3-1 AMPTS II: (a) Three main units of AMPTS II; (b) Flow cells for biogas
measurement (source: www.bioprocesscontrol.com)
3.2.4 Biogas composition
The composition of the biogas (hydrogen, carbon dioxide and methane) was analyzed by
using gas chromatography (GC) with a thermal conductivity detector (TCD) and helium
as carrier gas.
The chromatograph (Perkin Elmer Clarus 500) was equipped with a packed column of
stainless steel and filled with Hayesep Q (80-100 mesh size). The internal diameter of
the packed column was 1/8” and the column length was 4 m. Temperatures of the oven,
injection port and detector were 80, 80 and 120 °C, respectively. Samples were injected
into the column by using a 5 mL-syringe (©Analyticalscience).
Calibration curves for the determination of the composition of the biogas were
constructed by analyzing different volumes of a standard mixture of 50.1% of H2, 29.8%
CO2, 15% CH4 and 5.1% N2 certified by Linde A.G. The correlation between peak areas
and volumes was found to be linear within a range from 0.7 to 1.8. Then, the
concentration of the gas of interest, H2, was obtained through a rule of three. A volume of
approximately 1mL was taken from the headspace of the bottles in each measurement
and the time of each run was 6 minutes.
Calibration and normalization procedures are detailed in Annex I - 8.3.
18
3.2.5 Soluble chemical oxygen demand (CODs)
The chemical oxygen demand is a measure of the organic matter content that can be
oxidized from a sample of wastewater by a strong oxidant (potassium dichromate). The
determination of the COD was based on a closed reflux-titrimetric method (5220C)
described on the Standard Methods for the Examination of Water and Wastewater
(APHA, 2005).
The procedure consists in adding 1.5 mL of digestion standard solution of potassium
dichromate and, carefully, 3.5 mL of catalytic solution to 2.5 mL of sample. To insure
proper functioning of the method, samples must be filtered or centrifuged and diluted so
that the COD concentration falls within a range of 40-400 mg COD L-1. Once flask tubes
are homogenized with the help of a vortex mixer (Scilogex MX-S), they are placed into an
incubator at 150 ºC for 2 hours. Afterwards, the content of each tube is poured into an
Erlenmeyer flask and two drops of indicator solution of phenanthroline are added. Excess
of dichromate is titrated with standard solution of ferrous ammonium sulphate (FAS). At
the turning point, the color will change from green to reddish-brown. Blanks are also
prepared adding 2.5mL of distilled water instead of sample. COD measurements are
conducted in triplicate. Finally, COD can be determined as follows:
𝐶𝑂𝐷 [
𝑚𝑔 𝑂2
]
𝐿
=
𝑉𝑏𝑙𝑎𝑛𝑘 −𝑉𝑠𝑎𝑚𝑝𝑙𝑒
𝑀
× 8000 × 𝑀𝐹𝐴𝑆 × 𝑑𝑓
where:
Vblank: volume of FAS used in the titration of the blank (mL)
Vsample: volume of FAS used in the titration of the sample (mL)
M: volume of sample (2.5 mL)
MFAS: molarity of FAS
df: dilution factor of the sample
8000: dimensionless constant
(Eq.3.3)
19
3.2.6 Determination of volatile fatty acids (VFA)
Volatile fatty acids were identified and quantified by high-pressure liquid chromatography
(HPLC) using a Biorad HPX-87-H column with sulfuric acid 5mM as the mobile phase.
Flux was set at 0.5 mL min-1, temperature of the oven at 35 ºC and temperature of
detector UV. Samples were filtered with a 0.22 µm-pore filter in order to avoid
interferences and damages in the column. The amount of injected sample into the
column was 20 µL.
Organic acids determined were: acetic acid, propionic acid and butyric acid as VFAs, and
malic, glutamic and succinic acid. Calibration curves for the determination of samples
concentrations were shown in Annex – 8.5 (Figure 8-4).
3.2.7 pH
pH was analyzed with HI 9321 pH-meter (Hanna Instruments). Before measuring,
calibration of the device was conducted by using two buffer solutions at pH 4.01 and
7.01; these values were recommended by the supplier as they lead to more accurate
results for acidic samples.
20
3.3 Experimental set-up and operation
An overall scheme of the experiments that are described in this section is presented
below (Figure 3-2). Four main experiments (E1, E2, E3 and E4) were carried out in this
work sequentially.
Figure 3-2 Scheme of the experimental set-up
21
3.3.1 Experiment 1 (E1): Selection and augmentation of photo-fermentative inoculum
from different anaerobic sludges
Experiments in batch mode were carried out to observe the response of different sludges
in terms of photosynthetic bacteria growth under anaerobic conditions.
In the first stage (Stage I), two sets of experiments were conducted in order to select
purple non-sulfur (PNS) bacteria from anaerobic sludge during a period of one week.
Granular (GAS) and suspended (SAS) anaerobic sludge were used as source of
inoculum for the first and second set, respectively. Sludge inoculum was seeded into
bottles containing the mineral media described above, with additional malic and glutamic
acid as substrates. The pH was initially adjusted at 7.0  0.2 by adding NaOH (40 g L-1).
In order to promote the growth of PNS bacteria, malate (30 mmol·L-1) and sodium
glutamate (4 mmol·L-1) were selected as carbon and nitrogen sources, respectively
(Lazaro et al., 2012).
Samples were conducted in duplicate. For each sample, a blank was also run in parallel
to determine the endogenous production of biogas. Blank consisted of sludge without
any addition of substrates or nutrients, incubated at the same conditions as the other
samples. See Figure 3-3.
Halogen lamp of 150w was placed in such way that the light intensity was of 8000 lux on
average on the surface of the bottles. Temperature was kept at 32  4 ºC by the heat
released by the lamp, so there was no need for a thermostatic water bath or incubator.
Anaerobic conditions were induced by flushing helium gas through the headspace of the
bottles for 5 minutes. After the first three hours of incubation, the pressure of the bottles
was released by opening their valves and closing them afterwards. The start-up time of
the experiments was set at this point.
Assays were all conducted in the AMPTS II. Mechanical stirrers were set to work every 2
minutes. Biogas production was automatically controlled and gas quality was determined
through gas chromatography (GC).
For the enrichment of the bacterial consortium, a second stage (Stage II) was performed.
The purple biomass formed was harvested with a stainless steel spoon and
re-suspended in new bottles under exactly the same conditions as those in the first
stage.
22
Table 3-1. Batch tests for selection and augmentation of photo-fermentative bacteria
from different anaerobic sludges (Stage I)
GAS
SAS
CODs/VSS
0.5
0.5
Inoculum
100 mL
220 mL
Substrates
30 mmol L-1 malic acid
4 mmol L-1 glutamic acid
30 mmol L-1 malic acid
4mmol L-1 glutamic acid
C/N
30
30
Replicates
2 (R1, R2)
2 (R3, R4)
Blank
1 (B1)
1 (B2)
Temperature
324
324
Light intensity
8000 lux
8000 lux
Working volume
500 mL
500 mL
(a)
(b)
Figure 3-3 Experimental set-up for selection and augmentation of photo-fermentative
inoculum from different anaerobic sludges (a) batch tests, (b) scheme of the
experimental set-up
23
3.3.2 Experiment 2 (E2): Influence of pH on photo-fermentative culture grown from the
inoculum selected in E1
In the present section, the hydrogen productivity of the purple non-sulfur (PNS)
photosynthetic bacteria consortium was studied at two different pH values (5.5 and 7.0).
Experiments were conducted in batch mode in bottles of 500 mL working volume over
21 days. Each bioreactor was prepared with 125 mL of mixed culture PNS bacteria
harvested from the previous batch reactors in E1 and 50 mL of stock solution containing
mineral media with malate and glutamate as carbon and nitrogen sources, respectively.
Triplicates were run for both pH values.
The pH 5.5 was selected because it was the original pH of the DF effluent and pH 7.0
was selected because it a typical pH for hydrogen production by these microorganisms.
Concentrations of malate (30 mmol·L-1) and glutamate (9 mmol·L-1) in reactors were set
so that the carbon:nitrogen ratio from the organic substrates was 16:1.
The pH was initially adjusted to the desired value by adding NaOH (40 g L-1) and HCl
(25% (v/v)). Afterwards, bottles were filled with distilled water to 500 mL and closed with
a rubber stopper and the motor support of the AMPTS II. The two openings of the
septum were equipped with tubes and red clamps. Clamps were opened in order to flush
argon gas for 2 minutes to displace all oxygen.
After 5 hours of incubation, pressure in the bottles was released by removing one of the
tubes. Then, reactors were connected to smaller bottles containing alkaline solution for
CO2 absorption and to the gas-measuring device of the AMPTS II.
Mechanical stirrers were configured to work every 2 minutes. Light intensity was 82 klux
on the surface of the bottles, and temperatures were 324 ºC.
Biogas production was continuously monitored through the AMPTS application software
and gas composition was determined through gas chromatography.
24
Figure 3-4 Experimental set-up of batch photobioreactors at pH 5.5 and 7.0 (in triplicate)
Table 3-2. Batch tests for hydrogen production at two different pH values
pH 5.5
pH 7.0
CODs/VSS
1.4
1.4
Inoculum
125 mL of fresh inoculum
grown at E1
125 mL of fresh inoculum
grown at E1
VSS0
3.0  0.1 g L-1
3.0  0.1 g L-1
Substrates
30 mmol L-1 malic acid
9 mmol L-1 glutamic acid
30 mmol L-1 malic acid
9 mmol L-1 glutamic acid
C/N
16
16
Replicates
3 (R1, R2, R3)
3 (R4, R5, R6)
Temperature
324
324
Light intensity
8000 lux
8000 lux
Working volume
500 mL
500 mL
25
3.3.3 Experiment 3 (E3): Study of the kinetics of CODs depletion under H2-producing
conditions
In this study, a photosynthetic bacterial consortium was cultivated in a reactor operated
in batch mode with working and total volumes of 2.2 L and 2.7, respectively (Figure 3-5).
The reactor lid had five drilled outlets, and was connected to a water (pH 7.0)
displacement device for measurement of biogas production, to an ORP and to a pH
sensor. The other two outlets were used for gas and liquid sampling. A magnetic stirrer
(SB162, Stuart) was used at a frequency of 200 rpm to achieve a complete mixing, and a
thermo-circulation bath was used to hold the temperature constant at 37.5 ºC.
The seed inoculum used consisted of 600 mL of mixed culture cultivated at pH 7.0 in E2
experiment and with a VSS content of 4.1 g L-1. The reactor was fed with a mixture of
substrates (malic and glutamic acid) and the solution of nutrients.
Anaerobic conditions were created by passing argon gas through the liquid phase of the
reactor for 2 minutes.
Unlike E1 and E2 experiments, two 150 W halogen lamps were use to illuminate the
reactor with a light intensity of 8000 lux on average.
The experiment was carried out under pH-controlled conditions by manually adjusting
with HCl (25% (v/v)) once a day. Daily procedure also included recording biogas
production and taking a 20 mL sample with a syringe connected to a tube for CODs,
VFAs and solids analyses.
Table 3-4. Initial conditions of 2.2L photobioreactor T = 37.5ºC)
Parameter
Value
CODs/VSS
4.8
Inoculum
600 mL of fresh inoculum
grown at E2
VSS0
1.1 g L-1
Substrates
30 mmol L-1 malic acid
9 mmol L-1 glutamic acid
C/N
16
pH
7.0
Replicates
-
Temperature
324
Light intensity
8000 lux
Working volume
2200 mL
26
Figure 3-5 Experimental set-up of 2.2L photobioreactor
3.3.4 Experiment 4 (E4): Production of bio-H2 from an effluent derived through the
dark fermentation of glycerol
Batch experiments were carried out in the AMPTS II to study the feasibility of the dark
fermentation effluent as substrate for the PNS bacteria. The substrates studied were
synthetic effluent that was prepared based on the composition of an acidogenic reactor
effluent (S3) containing VFAs and succinic acid, the set-up operation of which is detailed
in section 7.1.
Bottles containing synthetic dark fermentation effluent as substrate were prepared and
run in triplicate for a period of 21 days (Figure 3-6). The dilution factor applied to the
effluent was 5, leading to the following initial concentration: succinic acid (5.2 mmol L-1),
acetic acid (1.7 mmol L-1), propionic acid (1.2 mmol L-1) and butyric acid (1.4 mmol L-1).
Glutamic acid (0.6 mmol L-1) was also added as nitrogen source.
Additionally, one blank and two controls with malate and glutamate (S2) were run. Blank
consisted of PNS bacteria as inoculum without any addition of substrates or nutrients,
incubated at the same conditions as the other samples.
In the same way as done in previous experiments, mineral medium was added to each
bottle and pH was adjusted to 7.0 before the addition of the inoculum. Then, bottles were
filled to 500 mL with distilled water and argon was flushed through the headspace of the
reactors to generate anaerobic conditions.
27
Due to fluctuations in room temperature, polystyrene sheets were placed with the aim of
holding the temperature at 30  4 ºC. Stirring was administered every 2 minutes and
halogen lamp of 150w was used.
Main features of both experiments are summed-up in the following table (Table 3-5):
Table 3-5 Experimental set-up and operational conditions in photo-fermentation batch
tests using real and synthetic DF effluent
Synthetic DF effluent
CODs/VSS
0.4
CODs
0.7  0.03 g O2 L-1
C/N
16
TVFA
10.1 mmol L-1
Inoculum
Fresh inoculum grown at E3
(333 mL)
Replicates
3
Blank tests
1 (333 mL of inoculum)
Control tests (S2 as substrate)
2
Temperature
30  4
Light intensity
8000 lux
Initial pH
7.0
Figure 3-6 Experimental set-up of batch photobioreactors containing DF synthetic
effluent (in triplicate)
28
3.4 Mathematical models

Kinetics
Kinetics analysis was conducted in order to determine which model better reproduces the
consumption of substrates by the PNS bacteria.
The integrated equation corresponding to a first-order model is expressed as follows
(Uyar et al., 2009):
𝑆 = 𝑆0 · 𝑒𝑥𝑝(−𝑘1 · 𝑡 )
(Eq. 3.1)
where S0 is the initial concentration of substrates expressed as CODs concentration and
k1 (h-1) is the first-order constant, which represents the consumption rate constant.
Applying logarithms to Eq. 3.1, this can be expressed as a linear function:
𝑆
𝐿𝑛 ( 𝑆0 ) = 𝑘1 · 𝑡

(Eq. 3.2)
Gompertz function
The production of H2 can be fitted to the modified equation of Gompertz, which is a
mathematical model that is used to predict biogas production in batch tests. Using the
Solver add-in for Excel, parameters such as the lag-phase time (λ), the specific hydrogen
production rate constant (Rm), and the cumulative hydrogen yield (Hm) can be calculated
(Argun et al., 2008).
𝑅 ·𝑒
𝐻 = 𝐻𝑚 · 𝑒𝑥𝑝 {−𝑒𝑥𝑝 [ 𝐻𝑚 (𝜆 − 𝑡) + 1]}
𝑚
(Eq. 3.3)
29
4. Results
4.1 Selection and augmentation of PNS consortium from anaerobic sludges
As described in section 3.3.1, two different sludges were evaluated as seeds for growth
of PNS bacteria. Results from the selection phase (stage I) were qualified in accordance
to the following attributes: purple coloring, biogas production, H2 production and CH4
production (Table 4-1).
Table 4-1 Qualitative results from E1 experiment (stage I)
Inoculum
Purple coloring (*)
Biogas
production (**)
H2 production (***)
CH4 production (****)
GAS
+ (2)[1]
+
-
+
SAS
+ (2)[1]
-
-
-
* Purple coloring (positive)
** Biogas production (>10 mmol CH4 L-1 marked positively)
*** H2 generation (positive when H2 (%) > 0 %)
**** CH4 generation (positive when CH4 (%) > 60 %)
[1] The number between the brackets corresponds to the number of reactors in which purple biomass is visible
During this phase, proliferation of purple biomass was observed on the inner surface of
the reactors between the fifth and sixth day of operation. As shown in Figure 4-1 new
bacterial consortium presented different color tones depending on the type of sludge
used.
(a)
(b)
Figure 4-1 PNS consortium (in red color) cultivated from: (a) granular anaerobic
sludge (GAS), (b) suspended anaerobic sludge (SAS)
Data corresponding to the cumulative methane production and the biogas composition
from each type of sludge are attached in the Annex 8.6. Cumulative biogas production
from bottles inoculated with granular sludge were nearly 18-fold higher than those
inoculated with suspended sludge. The blank did not exhibit any biogas production.
30
Similarly, results from the augmentation phase (stage II) were qualified according to the
presence or absence of H2 and CH4 in the biogas produced. Results are shown in Table
4-2.
Table 4-2 Qualitative results from E1 experiment (stage II)
Inoculum
Biogas production(*)
H2 generation(**)
CH4 generation(***)
1 (GAS)
+
+
-
2 (GAS)
-
-
+
3 (SAS)
-
-
+
4 (SAS)
-
-
+
-1
* Biogas production (>10 mmol CH4 L and/or H2 marked positively)
** H2 generation (positive when H2 (%) > 50 %)
*** CH4 generation (positive when CH4 (%) > 0 %)
Based on these results, biomass from bottle 1, in which the concentration of H2 was 83 %
and no CH4 was produced, was selected and afterwards augmented for further
experiments.
The figure 4.2 shows the change of color intensity over time in bottles with harvested
purple biomass inoculated into the fresh malate and glutamate medium.
2
3
(a) 0 h
4
1
2
3
(b) 24 h
4
1
2
3
4
(c) 48 h
Figure 4-2 Color tone change during cultivation of PNS consortium (Stage II). On the left:
1 and 2 corresponds to biomass harvested from R1 and R2. On the right: 3 and 4
corresponds to biomass harvested from B2 and R4
31
4.2 Characterization of the PNS consortium
4.2.1 pH influence on biohydrogen production (E2)
In this study (section 3.3.2), it was observed that the bioreactors suffered a general
increase in their final pH values. Bottles in which the initial pH was lower (pH0 = 5.5)
presented a greater increase (pHf = 8.1) than those with neutral pH (pH0 = 7) where it
varied slightly (pHf = 7.8).
The cumulative production of bio-H2 at pH 5.5 and 7.0 during E2 experiment was plotted
Cumulative hydrogen production
(mmol H2 / Lculture)
in Figure 4-3.
45
40
35
30
25
20
15
10
5
0
0
5
pH 7.0
10
time (d)
Gompertz pH 7.0
pH 5.5
15
20
Gompertz pH 5.5
Figure 4-3 Cumulative H2 production by PNS bacteria at pH 5.5 and pH 7.0 fitted to
Gompertz function (E2).
The parameters resulting from fitting the experimental data to Gompertz model (Eq. 3.3)
are shown in Table 4-3.
Table 4-3 Parameters of modified Gompertz´s model
pH
Hm
Rm
L-1
L-1
(mmol H2
culture)
(mmol H2
λ
culture
d-1)
r2
(d)
5.5
33.1
1.3
2.2
0.993
7.0
44.1
3.7
2.1
0.999
From these results, it came out that the specific production rate at pH 7 is almost three
times higher than that at pH 5.5. The total hydrogen production reached by the end of the
experiment (day 21) is also higher than that at pH 5.5, even though the duration of the
lag phase was practically the same in both cases.
32
The substrate depletion was 42% and 55% for 5.5 and 7.0 pH values, respectively. This
leads to a conversion efficiency of 12.8 % and 23.4 %. Calculations were presented in
Annex 8.7.
The content of H2 in the gas generated was 85 and 76 % for pH 7.0 and pH 5.5,
respectively.
4.2.2 Kinetics of malic acid depletion and biomass growth in 2.2L reactor (E3)
The change of the CODs concentration over time corresponding to the depletion of malic
acid (see section 3.3.3) and the biomass growth are illustrated in Figure 4-4.
6000
2,5
5000
2
4000
1,5
CODs (mg/L) 3000
1
2000
Biomass
(g VSS/L)
0,5
1000
0
0
0
5
10
15
Time (d)
CODs
Biomass concentration
Biomass
Figure 4-4 CODs and biomass vs. time in 2.2L working volume reactor
These values indicate a 96% depletion rate of the malate and glutamate added initially in
the reactor.
CODs data can be converted into a linear function by applying equation 3.2. In Fig. 4.5
logarithms of COD0/COD are plotted against time for the determination of the kinetic
constant.
33
4
Ln(CODs0/CODs)
3,5
y = 0,0102x
R² = 0,9555
3
2,5
2
1,5
1
0,5
0
0
100
200
Time (h)
300
Figure 4-5 First-order kinetics of malate depletion
It can be comfortably asserted that the consumption of these substrates obeys a firstorder model since linear regression of the experimental data was satisfactory with a
correlation coefficient of 0.95. The depletion rate constant obtained was 0.01 h-1.
Regarding the biomass growth, the exponential phase was linearized taking logarithms
and plotting values against time (Figure 4.6). The slope of the curve (Eq 4.1)
corresponds to the maximum specific growth rate (μ).
𝐿𝑛 (𝑋) = 𝐿𝑛(𝑋0 ) + 𝜇𝑚𝑎𝑥 · 𝑡
(Eq. 4.1)
0,6
Ln (X/X0)
0,5
0,4
0,3
y = 0,0053x
R² = 0,9733
0,2
0,1
0
0
20
40
60
Time (h)
80
100
Figure 4-6 Determination of the maximum specific growth rate (μ)
The resulting maximum specific growth rate was 0.005 h-1.
120
34
The cumulative H2 production curve for the run of the 2.2 L photobioreactor is illustrated
in Figure 4-7. These values were calculated assuming a concentration of 85 % H2 in the
biogas that was the average value in the bottles of E2 experiment at pH 7. The
parameters released from fitting the curve to Gompertz model are displayed in
Table 4-4. It is important to note that from day 11 on, one of the two lamps stopped
working so the light intensity decreased abruptly and led to a cease of biogas generation.
Cumulative H2 production
(mmol H2/L culture)
25
20
15
10
5
0
0
5
10
Time (d)
Cumulative H2 production
15
20
Gompertz model
Figure 4-7 Cumulative H2 production by PNS bacteria with controlled pH (E3).
Table 4-4 Parameters of Gompertz´s model
pH
Hm
(mmol H2 L-1culture)
Rm
(mmol H2 L-1culture d-1)
λ
(d)
r2
7.0
20.1
1.95
0.9
0.991
35
4.2.3 Hydrogen production and VFAs degradation by PNS consortium (E4)
The production of H2 from a 5-fold diluted DF that was registered through the course of
the experiment was illustrated in Figure 4-8 together with the production obtained in the
control with malate.
Additionally, to allow comparison with previous experiments (E2 and E3), parameters of
Cumulative H2 production
(mmol H2/L culture)
Gompertz function for the control test were presented in Table 4-5.
12
10
8
6
4
2
0
0
2
4
Control
6
8
Time (d)
10
Gompertz model
12
14
16
VFA average
Figure 4-8 Cumulative H2 production of photofermentative reactors with DF synthetic
effluent (S3) and malic and glutamic acid (S2)
Table 4-5 Parameters of Gompertz´s model for control with malic acid (E4)
Type of
reactor
Hm
(mmol H2 L-1culture)
Rm
(mmol H2 L-1culture d-1)
λ
(d)
r2
Control
9.96
2.9
5.0
0.996
The conversion yields of the mixture of VFAs (acetic, propionic, butyric and succinic acid)
and the control were presented in Table 4.6. The calculations are attached in Annex 8.7.
Table 4-6 Conversion yields of VFAs mixture and control
Type of substrate
𝜼(%)
VFAs (Synthetic DF effluent –S2)
3.8
Control (Malic and glutamic acid – S3)
5.5
36
Degradation of VFAs was evaluated by comparing initial and final CODs values and by
measuring the consumption of each VFA through HPLC. Values from HPLC analysis are
attached in Annex 8.1.
Table 4-5 displays the initial and final CODs concentration values.
Table 4-7 CODs removal
CODs initial
COD final
(g L-1)
(g L-1)
CODs
removal
(%)
VFAs
(average)
670  30 (mg L-1)
652  28 (mg L-1)
3
Control
4.20  0.19
0.68  0.02
89.1
It is also worthy to mention that the presence of a green colored biomass was perceptible
to the eye when centrifuging samples (Fig. 4-9)
(a)
(b)
Figure 4-9 (a) Centrifuged samples (4500rpm) of control tests; (b) Microscope image of
a diluted sample from pellet
37
5. Discussion
5.1 Selection and enrichment
Most of published studies about photoheterotrophic production of H2 were based on the
use of pure cultures. That is, very few studies have to date reported the selection of a
purple non-sulfur consortium from sewage sludges or sediments (Takabatake et al.,
2004; Lazaro et al., 2012). In this work, it was used a granular anaerobic sludge whose
microbial diversity had been previously analyzed by our laboratory group. Results from
massive sequencing of the granular anaerobic sludge used in this work attest the
presence of PNS strains in the sludge in a low/negligible concentration, e.g.:
Rhodobacter sp. (0.023%). Basak and Das (2007) stated that the purple color of these
microorganisms owing to their pigments is observable only under anaerobic conditions.
Based on this evidence and on the results obtained from other qualitative attributes such
as H2 production and no presence of CH4 (Adessi and De Phillipes, 2014), we assume
the dominance of PNS bacteria within the bacterial consortium. Thus, it can be asserted
that under the conditions of our experimental set-up, the selection and enrichment of a
purple non-sulfur consortium from anaerobic sludges is possible. This fact brings several
advantages especially for the treatment of wastes, such as the no need of working under
sterile conditions or the possibility of scaling up the process for bioremediation.
5.2 Characterization of the microbial consortium
Total hydrogen production and the productivity obtained at initial pH 7.0 were higher than
at pH 5.5 (original pH of the DF effluent). This indicates that for the selected PNS
bacterial consortium adjustment of pH of the DF effluent would be necessary prior to be
treated in the photobioreactor. In literature, optimum pH values differ from one species to
others (pH range: 7.0 - 9.0; Li et al., 2009). For instance, research by Nath and Das
(2009) reported that maximum volumetric production values were obtained at pH 6.5 and
7.0 for Rhodobacter sphaeroides O.U. 001. For mixed cultures was reported to fall
between 7.0 and 9.0 (Fang et al., 2005). Thus, it would be desirable to study a wider
range of pH to find the optimal value of this PNS bacterial consortium.
The substrate conversion yields of malic acid at pH 7.0 and pH 5.5 were 23.4 % and
12.8 %, respectively. The former is higher than the yield (15.6 %) achieved by a mixed
culture obtained from granular sludge of an UASB reactor with the same substrate
(Lazaro et al., 2012). In the same way, the yield is comparable with a yield of 36 % on
38
average using Rhodobacter sphaeroides O.U. 001 reported by Koku et al. (2003) at
similar environmental conditions but different light source (tungsten lamp).
In general, the pH increased in those experiments where there was not pH control, either
those in which malic acid (E2) or a mixture of VFAs (E4) was used as substrate. The rise
in pH was expected owing to the consumption of acids and also might be attributed to the
production of poly-β-hydroxybutyrate (PHB), which takes place in the cells when the PNS
bacteria do not follow an H2 production metabolic pathway (Nath et al., 2008).
In trying to examine the importance of the inoculum age, experiments performed
sequentially and with equal initial concentration of malic acid (30 mmol L-1) and initial pH
were compared (E2, E3 and E4 control). Conversion yields of malic acid were 23.4%,
7.5% and 5.5% (biohydrogen production values ranged from 9.8 to 42.1 mmol L-1culture) in
E2, E3 and E4 experiments, respectively. Such a substantial difference might be due to
the age of the inoculum and other factors such as the surface to volume ratio (E3). The
highest value was obtained using fresh inoculum (E2) whereas the lowest one an older
one (E4). Hence, there is a clear trend, the older inoculum the lower yield. This fact could
be explained by changes in the relative abundance of the microbial diversity in the mixed
culture (Tapia-Venegas et al., 2015).
In E3 experiment, the depletion rate of the malic acid (k = 0.011 h-1) was comparable as
well to those values reported in literature that fell from 0.015 to 0.037 h-1 (Koku et al.,
2003). A first order kinetics has been reported also by Uyar et al., (2009) obtaining a
depletion rate constant of k = 0.026 h-1. Thus, it can be asserted that despite the light
limitations in the media due to the thermal jacket surrounding the reactor and a lower
surface to volume ratio (S/V = 0.34) than the AMPTS bottles (S/V = 0.51), the scaling up
to a 2.2 L-photobioreactor was conducted successfully. However, these constraints did
have a negative effect on the productivity, being lower (1.95 mmol H2 L-1 d-1) than those
obtained using 500mL bottles (3.7 mmol H2 L-1 d-1 in E2 and 2.9 mmol H2 L-1 d-1 in E4).
The specific growth rate in the exponential phase in the photobioreactor was 0.005 h-1.
This value is far from those found in literature. Koku et al. (2003) reported values of
0.035 – 0.073 h-1 using the same substrate whereas Nath et al. (2008) obtained using
acetic acid values that fell between 0.04 h-1 and 0.09 h-1. This might be attributed to the
same factors explained above (light limitations) and to the use of an inoculum in nonexponential phase (Basak and Das, 2007).
39
5.3 H2 production from a diluted DF synthetic effluent
The experiment in which the substrates were a mixture of VFAs (9.5 mmol L-1), the
conversion yield was 3.8 %. This value is comparable with those that have been reported
to date in literature using mixed culture. For instance, Takabatake et al. (2004) obtained
a yield of 22 % utilizing a mixture of acetate, propionate and butyrate as substrates and
Zhang et al. (2002) a yield of 12 % for the treatment of synthetic wastewater (acetate,
butyrate and ethanol). On the other hand, using acetate and butyrate as the sole
substrate, Lazaro et al. (2012) achieved yields of 14.5 % and 13.8 %, respectively; and
Ike at el. (1999) reported values from 5.3 % (glycerol) to 27.6 % (lactate). HPLC results
revealed that almost 100% of the organic acids were consumed although the low COD
depletion (3%) and a non-identified peak in the HPLC chromatography suggest that other
metabolites are being formed, e.g. formate which is formed under low light conditions
(back side of the reactors) by the PNS bacteria (Sargsysan et al., 2015). Furthermore,
Lazaro et al. (2012) identified and classified the different groups present in a consortium
of photo-fermentative bacteria obtained from granular sludge. It came out that only 1/3 of
the groups belonged to the α- and β-proteobacteria. Thus, there is a possibility that
competition with other non-photosynthetic bacteria occurred (e.g.: H2-consuming
microorganisms) (Saady, 2013).
40
6. Conclusion

A mixed culture suitable for the production of H2 was selected from granular sludge
collected from a tobacco wastewater treatment plant.

Initial pH has been found to be a key aspect of the design of photobioreactor.
Hydrogen productivity was higher at neutral pH than at the pH of the DF effluent,
indicating that the pH needs to be adjusted prior to being fed into the second stage
fermenter.

There was a clear trend, the older the inoculum, the lower conversion yields of
malic acid (23.4 % (E2) > 7.5 % (E3) > 5.5 % (E4))

Conversion yield of synthetic dark effluent was low although comparable with those
reported in literature for a mixed culture, most probably due to competition with
non-photosynthetic bacteria. Thus, the production of H2 from a DF effluent by a
PNS bacterial consortium is feasible but there is still a lot of room for improvement.
41
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45
8. Annex
8.1 Annex chapter 3 - Selection of light source
Spectra of two different lamps were determined with the spectroradiometer Go-Spex 500
(EverFine) in order to select a lamp suitable for the PNS bacteria.
(a)
(b)
Figure 8-1 Spectrum of (a) halogen lamp and (b) incandescent and submergible lamp
46
8.2 Annex chapter 3 - Dark fermentation of glycerol for the production H2 and
metabolites (VFAs)
Dark fermentation of glycerol was conducted in a continuous stirred tank reactor (CSTR)
of 2 L and 2.7 L of working and total volume, respectively. The temperature of the reactor
was controlled with a thermo-circulating batch at 37 ºC whereas the pH was controlled
through a pH sensor and controller connected to a pump that supplies a solution of
NaOH (0.8M). A magnetic stirrer placed at the bottom of the reactor provided total
mixing. The feeding was daily flushed with N2 (g) to keep anaerobic conditions within the
reactor. The monitoring and the control of the process were carried out by ODIN software
(INRIA, Chile). Steady state was established based on a standard deviation of the
hydrogen yield lower to 30 %. Figure 8.2 shows the scheme of the CSTR
ODIN
PLC
NaOH 0,8M
Biogás
3.12
Gasómetro
Ritter
Efluente
Salida Agua
Caliente
Alimentación
Entrada Agua
Caliente
Agitador Magnético
Figure 8-2 Scheme of CSTR used in DF of glycerol (Source: Silva, 2015)
Sampling of the reactor was conducted over the feeding, the liquid effluent and the
biogas produced.
The inoculum used was waste activated sludge (WAS) taken from the secondary
treatment of the wastewater treatment plant of La Farfana (Santiago de Chile, Chile).
The process consisted of two main stages: 1 - pre-treatment of the inoculum through an
aerobic batch enrichment with 8.2 ppm of dissolved oxygen (Tapia-Venegas, 2014);
2 - anaerobic batch operation during 24h with a feeding of 10 g L-1 of glycerol followed by
continuous operation mode with a feeding of 10 g L-1 of glycerol, pH control at 5.5,
hydraulic retention time (HRT) of 12 h, temperature of 37 ºC, macro and micronutrients.
47
Table 8-1 Composition of feeding solution in the DF of glycerol
Nutrients
Concentration (mg L-1)
Glycerol
10 (g)
NH4Cl
1000
KH2PO4
250
MgSO4·7H2O
100
NaCl
10
NaMoO4· 2H2O
10
CaCl2· 2H2O
10
MnSO4· H2O
9.4
FeCl2
2.78
8.3 Annex Chapter 3 - Calibration of the gas chromatograph
Hydrogen
Nitrogen
4500000
3750000
Area
Area
3000000
2250000
1500000
y = 4E+06x - 2E+06
R² = 0,9947
750000
0
0,7
1,2
1,7
Injected volume (mL)
2,2
350000
300000
250000
200000
150000
100000
50000
0
y = 117073x + 91221
R² = 0,9378
0,7
1,2
Inyected volume (mL)
(b)
(a)
1,7
2,2
48
Carbon dioxide
700000
2100000
600000
1800000
500000
1500000
400000
1200000
Area
Area
Methane
300000
y = 394545x - 65797
R² = 0,9985
200000
100000
y = 1E+06x - 281142
R² = 0,9866
900000
600000
300000
0
0
0,7
1,2
1,7
0,7
2,2
1,2
1,7
2,2
Inyected volume (mL)
Inyected volume (mL)
(c)
(d)
Figure 8-3 Calibration curve for (a) hydrogen, (b) nitrogen, (c) methane and (d) carbon
dioxide gas
8.4 Annex chapter 3 - Addition of glutamic acid as N source
Below, the table 8-2 shows the amount of glutamic acid to be added so that the C/N ratio
will be 16.

S3 – synthetic effluent
Table 8-2 Calculation of carbon contribution in the DF effluent
Substrates
Succinic
acid
Acetic acid
Propionic
acid
Butyric
acid
Glutamic
acid
Molecular
formula
VFAs
conc
effluent
(g/L)
Molecular
weight
(g/mol)
Stock
solution
(g/L)
VFAs
moles
in 250
mL
C
contribution
C4H6O4
1.5455
118.09
3.09
0.0065
0.314
C2H4O2
0.2608
60.05
0.52
0.0021
0.052
C3H6O2
0.22235
74.08
0.44
0.0015
0.054
C4H8O2
0.31975
88.11
0.64
0.0018
0.087
147.13
0.46
C5H9NO4
49
8.5 Calibration curves for determination of organic acids
Standard solutions of six different organic acids were prepared and analyzed by HPLC.
These were acetic, propionic, butyric, succinic, malic and glutamic acid. Admissible
correlation factors (R2 > 0.99) were obtained from all the linear fittings. Peaks of organic
acids were obtained in the following order: glutamic acid (7.3 min), malic acid (11.7 min),
succinic acid (14.5min), acetic acid (18.4min), propionic acid (21.7 min) and butyric acid
Concentration (g/L)
(26.8min). Equations are displays in Figure 8.4.
140 y = 0,1209x - 3,1067
120
R² = 0,9994
100
80
60
40
20
0
0
500
1000
Area (mAU*min)
Acetic acid
1500
Linear (Acetic acid )
Concentration (g/L)
(a)
140
120
100
80
60
40
20
0
y = 0,113x - 1,5863
R² = 0,9999
0
200
400
600
800
Area (mAU*min)
Propionic acid
1000
1200
Linear (Propionic acid)
(b)
Concentration (g/L)
50
70
60
50
40
30
20
10
0
y = 0,1098x - 0,532
R² = 1
0
200
400
Area (mAU*min)
Butyric acid
600
Linear (Butyric acid)
Concentration (g/L)
(c)
10
y = 0,0991x - 0,211
R² = 0,9913
8
6
4
2
0
0
20
40
60
80
Area (mAU*min)
Succinic acid
100
120
Linear (Succinic acid)
Concentration (g/L)
(d)
6
5
4
3
2
1
0
y = 0,3541x - 5,6046
R² = 0,9997
10
15
20
25
Area (mAU*min)
Malic acid
30
Linear (Malic acid)
(e)
35
Concentration (g/L)
51
3
2,5
2
1,5
1
0,5
0
y = 0,2441x + 0,1926
R² = 0,9951
0
5
10
Area (mAU*min)
Glutamic acid
15
Linear (Glutamic acid)
(f)
Figure 8-4 Calibration curves for (a) Acetic acid, (b) Propionic acid (c) Butyric acid (d)
Succinic acid (e) Malic acid (f) Glutamic acid
8.6 Annex Chapter 4 - Cumulative methane production and biogas composition
(E1 experiment)
8.6.1 Stage I:
The cumulative methane production of both types of inoculum under study was
expressed in STP conditions in Figure 8-5. These values correspond to the average
Cumulative methane
production
(mmol CH4/Lreactor)
between duplicates.
50
40
30
20
GAS
10
SAS
0
0
50
100
150
Time (h)
Figure 8-5 Cumulative methane production from granular and suspended anaerobic
sludge (Stage I)
52
Table 8-3 Gas composition during selection of PNS consortium (Stage I)
B1
R1
R2
R3
R4
B2
Time (h)
H2 %)
CH4 (%)
CO2 (%)
24
48
120
24
48
120
22
48
120
24
48
120
24
48
120
24
48
120
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
0,00
9,7
12,0
1,6
75,6
71,2
71,3
74,5
63,7
65,1
13,0
8,2
6,1
8,6
12,6
8,9
6,4
16,3
5,0
7,6
6,9
19,2
23,6
18,8
19,8
22,8
20,1
33,8
37,7
16,1
42,9
45,6
31,1
11,7
15,8
8.6.2 Stage II:
The production of biogas (CH4 and H2) during this stage is shown in Figure 8-6 In this
case, results are not expressed as specific production since the amount of purple
Cumulative H2 and/or CH4
(mmol/Lreactor)
biomass initially inoculated in the bottles was different from each other.
12
10
8
6
4
2
0
0
10
20
30
40
50
time (h)
1 (R1)
2 (R2)
3 (B2)
4 (R4)
Figure 8-6 Biogas production during cultivation of PNS consortium (Stage II)
Furthermore, through the GC analysis (Table 8-4) performed on the second day it was
found that in the reactor 1, the concentration of H2 reached a value higher to 80%
whereas no methane was produced.
53
Table 8-4 Gas composition during augmentation of PNS consortium (Stage II)
H2 %)
CH4 (%)
CO2 (%)
1 (GAS)
83.3
-
9.9
2 (GAS)
-
66.7
23.7
3 (SAS)
-
15.3
53.9
4 (SAS)
18.7
9.5
17.3
8.7 Annex Chapter 4 - Calculation of substrate conversion efficiencies
The calculation of the conversion efficiency resulting from the different experiments was
determined as the fraction between the production of H2 and the theoretical production of
H2 (Eq 8.1). The theoretical production of H2 was estimated by using Eq 2.1
𝑷 (𝒎𝒐𝒍 𝑯𝟐 )
𝒙 𝟏𝟎𝟎
𝒕𝒉 (𝒎𝒐𝒍 𝑯𝟐 )
𝜼(%) = 𝑷

E2 experiment:
𝜂(𝑝𝐻 5.5) =
𝜂(𝑝𝐻 7.0) =

0.0231 𝑚𝑜𝑙 𝐻2
𝑥
30·10−3 (𝑚𝑜𝑙 𝑚𝑎𝑙𝑖𝑐 𝑎𝑐𝑖𝑑)×6(𝑚𝑜𝑙 𝐻2 )
100 = 12.8 %
30 · 10−3
0.0421 𝑚𝑜𝑙 𝐻2
𝑥 100 = 23.4 %
(𝑚𝑜𝑙 𝑚𝑎𝑙𝑖𝑐 𝑎𝑐𝑖𝑑) × 6(𝑚𝑜𝑙 𝐻2 )
40 · 10−3
0.0181 𝑚𝑜𝑙 𝐻2
𝑥 100 = 7.5 %
(𝑚𝑜𝑙 𝑚𝑎𝑙𝑖𝑐 𝑎𝑐𝑖𝑑) × 6(𝑚𝑜𝑙 𝐻2 )
E3 experiment:
𝜂(𝑝𝐻 7.0) =

(Eq. 8.1)
E4 experiment:
𝜂(𝑉𝐹𝐴𝑠) =
𝜂(𝑐𝑜𝑛𝑡𝑟𝑜𝑙) =
9.5 · 10−3
30 ·
10−3
0.0025 𝑚𝑜𝑙 𝐻2
𝑥 100 = 3.8 %
(𝑚𝑜𝑙 𝑇𝑉𝐹𝐴𝑠) × 7(𝑚𝑜𝑙 𝐻2 )
0.00983 𝑚𝑜𝑙 𝐻2
𝑥 100 = 5.5 %
(𝑚𝑜𝑙 𝑚𝑎𝑙𝑖𝑐 𝑎𝑐𝑖𝑑) × 6(𝑚𝑜𝑙 𝐻2 )
54
8.1 Annex chapter 4 - HPLC results from E4
Table 8-5 HPLC areas of E4 samples.
Area (mAU*min)
S3 (10-fold
concentrated)
Final E4
Acetic
acid
Propionic
acid
Butyric
acid
Succinic
acid
Malic
acid
Glutamic
acid
Unknown
VFAs
mixture
18,41
3
4
29
0,025
4
VFA1
0.125
4.933
n.a.
n.a.
n.a.
24.06
VFA2
n.a.
0.034
n.a.
0.035
0.362
0.414
9.543
VFA3
0.085
n.a.
0.042
0.029
0.262
0.398
964
C1
0.022
n.a.
0.055
0.034
0.3
0.234
10.491
C2
3.788
0.149
0.213
0.17
0.527
0.319
78.507
Blank
n.a.
n.a
n.a.
0.029
0.123
0.049
10.522