BIOGAS FROM SLAUGHTERHOUSE WASTE - DiVA...

61
Digital version: http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-847 ISBN 978-91-87525-85-8 (printed) ISBN 978-91-87525-86-5 (pdf) ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 75 DOCTORAL THESIS Resource Recovery DOCTORAL THESIS The application of anaerobic digestion, from waste streams that currently have no use, can be utilized for bioenergy production. Due to the high protein and fat content, slaughterhouse waste has a high potential for biogas production. However, potential inhibitory compounds can be formed during its degradation making the process prone to failure. One of the ways to overcome these problems is co-digestion with carbohydrate-rich co-substrates i.e., a mixture of agro-wastes. In this study, four different waste fractions, i.e., solid cattle slaughterhouse waste (SB), manure (M), various crops (VC), and the organic fraction of municipal solid waste (MSW) were investigated in mono-digestion and co-digestion processes. Different mixture ratios were prepared, and the methane yield (Y CH4 ), the specific methanogenic activity (SMA), and a kinetic parameter (k 0 ) were determined using the batch digestion assays at thermophilic conditions (55 o C). The SB had a lower degradation rate and lower SMA compared with those of the other samples. In order to investigate the effect of the temperature, a selected mixture ratio was also digested at mesophilic conditions (37 o C), which resulted in a decrease in Y CH4 and in the kinetic parameters, by up to 57% compared to those obtained at the thermophilic conditions. During the next part of the work, a four-factor mixture design was applied aiming to obtain possible synergetic or antagonistic effects. Mixing all four of the substrates resulted in a 31% increase in the Y CH4 compared to the expected yield calculated on the basis of the methane potential of the individual fractions clearly demonstrating a synergistic effect. Nevertheless, antagonistic interactions were also observed for certain mixtures. The impact of the mixture interactions, obtained in the batch operation mode, was also evaluated under semi-continuous co-digestion. Digestion of the SB as the sole substrate failed at an organic loading rate of 0.9 gVS L -1 d -1 , while stable performance with higher loadings was observed for mixtures that displayed synergy earlier during the batch experiments. The combination that showed the antagonistic effects resulted in unstable operation and poor representation of methanogens. It was proved that synergetic or antagonistic effects observed in the batch mode could be correlated to the process performance, as well as to the development of the microbial community structure during the semi-continuous operation. In the last part of the work, the response of the methanogenic biomass to the consecutive feeding applied in the batch assays was evaluated regarding process parameters such as Y CH4 , SMA, and degradation kinetics. The objective was to examine whether there is a possibility to correlate these findings to the expected process performance during the long-term operation. Digestion of the SB alone showed a total inhibition after the second feeding, which is in correlation with the failure observed during the semi-continuous mode. Furthermore, enhanced SMA was observed after the second feeding in those mixtures that showed synergy in the previous batch assays as well as a good process performance during the semi-continuous operation. Keywords: Slaughterhouse waste, Agro-Waste, Co-digestion, Synergistic effects, methanogenic community structure BIOGAS FROM SLAUGHTERHOUSE WASTE Jhosané Pagés Díaz Jhosané Pagés Díaz BIOGAS FROM SLAUGHTERHOUSE WASTE – MIXTURE INTERACTIONS IN CO-DIGESTION This thesis presents work that was done within the Swedish Centre for Resource Recovery (SCRR). Research and education performed within SCRR identifies new and improved methods to convert residuals into value-added products. SCRR covers technical, environmental and social aspects of sustainable resource recovery. Mixture interactions in co-digestion

Transcript of BIOGAS FROM SLAUGHTERHOUSE WASTE - DiVA...

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Digital version: http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-847 ISBN 978-91-87525-85-8 (printed)ISBN 978-91-87525-86-5 (pdf)ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 75

DO

CTO

RA

L TH

ESIS

Reso

urce

Rec

over

yDO

CTO

RA

L THESIS

The application of anaerobic digestion, from waste streams that currently have no use, can be utilized for bioenergy production. Due to the high protein and fat content, slaughterhouse waste has a high potential for biogas production. However, potential inhibitory compounds can be formed during its degradation making the process prone to failure. One of the ways to overcome these problems is co-digestion with carbohydrate-rich co-substrates i.e., a mixture of agro-wastes.

In this study, four different waste fractions, i.e., solid cattle slaughterhouse waste (SB), manure (M), various crops (VC), and the organic fraction of

municipal solid waste (MSW) were investigated in mono-digestion and co-digestion processes. Different mixture ratios were prepared, and the methane yield (YCH4), the specific methanogenic activity (SMA), and a kinetic parameter (k0) were determined using the batch digestion assays at thermophilic conditions (55oC). The SB had a lower degradation rate and lower SMA compared with those of the other samples. In order to investigate the effect of the temperature, a selected mixture ratio was also digested at mesophilic conditions (37oC), which resulted in a decrease in YCH4 and in the kinetic parameters, by up to 57% compared to those obtained at the thermophilic conditions. During the next part of the work, a four-factor mixture design was applied aiming to obtain possible synergetic or antagonistic effects. Mixing all four of the substrates resulted in a 31% increase in the YCH4 compared to the expected yield calculated on the basis of the methane potential of the individual fractions clearly demonstrating a synergistic effect. Nevertheless, antagonistic interactions were also observed for certain mixtures.

The impact of the mixture interactions, obtained in the batch operation mode, was also evaluated under semi-continuous co-digestion. Digestion of the SB as the sole substrate failed at an organic loading rate of 0.9 gVS L-1d-1, while stable performance with higher loadings was observed for mixtures that displayed synergy earlier during the batch experiments. The combination that showed the antagonistic effects resulted in unstable operation and poor representation of methanogens. It was proved that synergetic or antagonistic effects observed in the batch mode could be correlated to the process performance, as well as to the development of the microbial community structure during the semi-continuous operation.

In the last part of the work, the response of the methanogenic biomass to the consecutive feeding applied in the batch assays was evaluated regarding process parameters such as YCH4, SMA, and degradation kinetics. The objective was to examine whether there is a possibility to correlate these findings to the expected process performance during the long-term operation. Digestion of the SB alone showed a total inhibition after the second feeding, which is in correlation with the failure observed during the semi-continuous mode. Furthermore, enhanced SMA was observed after the second feeding in those mixtures that showed synergy in the previous batch assays as well as a good process performance during the semi-continuous operation.

Keywords: Slaughterhouse waste, Agro-Waste, Co-digestion, Synergistic effects, methanogenic community structure

BIOGAS FROM SLAUGHTERHOUSE WASTE

Jhosané Pagés Díaz

Jhosané Pagés D

íaz BIOGAS FROM SLAUGH

TERHOUSE W

ASTE – MIXTURE IN

TERACTIONS IN

CO-DIGESTION

This thesis presents work that was done within the Swedish Centre for Resource Recovery (SCRR).

Research and education performed within SCRR identifies new and improved methods to convert

residuals into value-added products. SCRR covers technical, environmental and social aspects of

sustainable resource recovery.

Mixture interactions in co-digestion

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Biogas from

Slaughterhouse Waste

Mixture Interactions in Co-digestion

Jhosané Pagés Díaz

Thesis for the degree of Doctor of Philosophy at the University of Borås to be publicly defended

on Dec 10th 2015, 10.00 a.m

. in room E310, U

niversity of Borås, Allégatan 1, Borås, Sweden.

Language: English

Faculty Opponent is:

Professor Kornél L. Kovács Departm

ent of Biotechnology U

niversity of Szeged Hungary

PhD thesis available at:

Swedish Centre for Resource Recovery

University of Borås

SE-501 90 Borås, Telephone: +46 (0)33-435 4000

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Th

esis fo

r the

De

gre

e o

f Do

cto

r of P

hilo

sop

hy

Bio

ga

s from

slau

gh

terh

ou

se w

aste

Mixtu

re in

tera

ctio

ns in

co

-dig

estio

n

Jho

san

é P

ag

és D

íaz

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ii Copyright © Jhosané Pagés Díaz

Swedish Centre for Resource Recovery

University of Borås

SE-501 90 Borås, Sweden

Digital version: http://urn.kb.se/resolve?urn=urn:nbn:se:hb:diva-847 ISBN

978-91-87525-85-8 (printed) ISBN

978-91-87525-86-5 (pdf) ISBN

0280-381X Skrifter från Högskolan i Borås, nr. 75 Cover photo: Agriculture biogas plant

Printed in Sweden by Responstryck AB

Borås 2015

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iii Abstract G

lobal environmental concerns connected to the use of fossil fuels have forced the developm

ent of alternative sustainable energy technologies. The application of anaerobic digestion, from

waste stream

s that currently have no use, can be utilized for bioenergy production. D

ue to the high protein and fat content, slaughterhouse w

aste has a high potential for biogas production. How

ever, potential inhibitory compounds

can be formed during the degradation of the proteins and lipids, w

hich can make the process sensitive and

prone to failure. One of the w

ays to overcome these problem

s is co-digestion with carbohydrate-rich co-

substrates i.e., a mixture of agro-w

astes with low

protein/lipid content. This also leads to a better nutritional balance and enhanced m

ethane yield due to the positive mixture interactions.

In this study, four different waste fractions, i.e., solid cattle slaughterhouse w

aste (SB), m

anure (M),

various crops (VC

), and the organic fraction of municipal solid w

aste (MSW

) were investigated in m

ono-digestion and co-digestion processes. D

ifferent mixture ratios w

ere prepared, and the methane yield (Y

CH

4 ), the specific m

ethanogenic activity (SMA

), and a kinetic parameter (k

0 ) were determ

ined using the batch digestion assays at therm

ophilic conditions (55oC

). The SB had a low

er degradation rate and lower SM

A

compared w

ith those of the other samples. In order to investigate the effect of the tem

perature, a selected m

ixture ratio was also digested at m

esophilic conditions (37oC

), which resulted in a decrease in Y

CH

4 and in the kinetic param

eters, specific methane production rate (rsC

H4 ), and k

0 , by up to 57% com

pared to those obtained at the therm

ophilic conditions. During the next part of the w

ork, a four-factor mixture design w

as applied aim

ing to obtain possible synergetic or antagonistic effects. The performance of the process w

as assessed using Y

CH

4 and rsCH

4 as the response variables. Mixing all four of the substrates resulted in a 31%

increase in the Y

CH

4 compared to the expected yield calculated on the basis of the m

ethane potential of the individual fractions and 97%

of the theoretical methane yield, clearly dem

onstrating a synergistic effect. N

evertheless, antagonistic interactions were also observed for certain m

ixtures. In order to maxim

ize both the response variables sim

ultaneously, a response surface method w

as employed to find the optim

al com

bination for the substrate mixture.

The impact of the m

ixture interactions, obtained in the batch operation mode, w

as also evaluated under sem

i-continuous co-digestion. Digestion of the SB

as the sole substrate failed at an organic loading rate of 0.9 gV

S L-1d

-1, while stable perform

ance with higher loadings w

as observed for mixtures that displayed

synergy earlier during the batch experiments. The com

bination that showed the antagonistic effects resulted

in unstable operation and poor representation of methanogens. It w

as proved that synergetic or antagonistic effects observed in the batch m

ode could be correlated to the process performance, as w

ell as to the developm

ent of the microbial com

munity structure during the sem

i-continuous operation.

In the last part of the work, the response of the m

ethanogenic biomass to the consecutive feeding applied in

the batch assays was evaluated regarding process param

eters such as YC

H4 , SM

A, and degradation kinetics.

The objective was to exam

ine whether there is a possibility to correlate these findings to the expected

process performance during the long-term

operation. Digestion of the SB

alone showed a total inhibition

after the second feeding, which is in correlation w

ith the failure observed during the semi-continuous m

ode. Furtherm

ore, enhanced SMA

was observed after the second feeding in those m

ixtures that showed synergy

in the previous batch assays as well as a good process perform

ance during the semi-continuous operation.

Keyw

ords: Slaughterhouse

waste,

Agro-W

aste, C

o-digestion, Synergistic

effects, M

ethanogenic com

munity structure

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iv

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v List of Publications T

he thesis is mainly based on the results presented from

the following original papers:

I. Pagés-D

íaz J, Pereda-Reyes I, Lundin M

and Sárvári Horváth I, C

o-digestion of

different waste m

ixtures from agro-industrial activities: K

inetics evaluation and

synergistic effects. Bioresource Technology 102 (23), 10 834-10 840 (2011).

II. Pagés-D

íaz J, Sárvári Horváth I, Peréz O

lmo J and Pereda-R

eyes, I, Co-digestion of

bovine slaughterhouse wastes, cow

manure, various crops and m

unicipal solid waste at

thermophilic conditions: a com

parison with specific case running at m

esophilic

conditions. Water Science and Technology 67, 989-995 (2013).

III. Pagés-D

íaz J, Pereda-Reyes I, Taherzadeh M

J and Sárvári Horváth I, A

naerobic co-

digestion of

solid slaughterhouse

wastes

with

agro-residues: Synergistic

and

antagonistic interactions determined in batch digestion assays. C

hemical E

ngineering

Journal 245, 89-98 (2014).

IV.

Pagés-Díaz J, W

estman J, Taherzadeh M

J, Pereda-Reyes I and Sárvári H

orváth I,

Semi-continuous co-digestion of solid cattle slaughterhouse w

aste with other w

aste

streams: interactions w

ithin the mixtures and m

ethanogenic comm

unity structure.

Chem

ical Engineering Journal. 273, 28-36 (2015).

V.

Pagés-Díaz J, Sanz J.L., Lundin, M

., Taherzadeh MJ., Sárvári H

orváth I., and Pereda-

Reyes I, C

onsecutive feeding in batch assays: a comparison of process perform

ance

during anaerobic

mono-

and co-digestion

of slaughterhouse

waste

in different

operation modes. Subm

itted

Additional publications during m

y doctoral studies that are not included in this thesis:

VI.

Pereda-Reyes I, Pagés-D

íaz J, Sárvári Horváth, I. A

naerobic biodegradation of solid

substrates from agro-industrial activities: Slaughterhouse w

astes and agro-wastes, In:

Biodegradation, Ed. R

olando Cham

y, ISBN

: 978-953-51-4271-3, InTech, Croatia, In

Press.

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vi Statem

ent of Contributions Paper I: R

esponsible for all the experimental w

ork and the analysis of the data. Responsible

for manuscript preparation and w

riting.

Paper II: Responsible for a m

ajor part of the experimental w

ork and analysis of the data. Perform

ed manuscript preparation and w

riting.

Paper III: Responsible for the idea, experim

ental design, and experimental w

ork. I was

responsible for analyzing the data, manuscript preparation, and w

riting.

Paper IV: R

esponsible for all the experimental w

ork and the analysis of the data. Responsible

for manuscript preparation and w

riting.

Paper V: R

esponsible for part of the idea, all the experimental w

ork, and the analysis of the data. R

esponsible for manuscript preparation and w

riting.

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vii Preface The present study w

as performed w

ithin a special arrangement betw

een the Swedish C

entre

for Resource R

ecovery at the University of B

orås (UB

) in Sweden and the Process

Engineering Center at Instituto Superior Politécnico José A

ntonio Echeverría (Cujae) in

Havana, C

uba. As a “sandw

ich” Ph.D. student from

Cuba, I have been alternating betw

een

these two universities, dividing m

y time at both places, including teaching activities during

my Ph.D

. studies in Cuba. I first cam

e to Borås as an exchange student as part of a Linnaeus-

Palme exchange program

supported by the Swedish International Program

Office in 2010. M

y

intention was to perform

my M

.Sc. thesis project here, and the subject was decided based on

the special interest of the Agricultural M

inistry in Cuba, w

hich suggested AD

technologies for

slaughterhouse waste enterprises for the treatm

ent of their waste. The subject of this first

study, therefore, was to investigate the m

ono- and co-digestion of different waste stream

s

from agro-industrial and agricultural activities. I w

as very grateful that after one year, in 2011,

I had the opportunity to return to continue my research studies w

ithin this field under the

supervision of Dr. Ilona Sárvári H

orváth and Dr. Ileana Pereda R

eyes. I have been registered

as a Ph.D. student at the U

niversity of Borås, w

hich has funded the part of my studies that w

as

performed in Sw

eden. I had the enormous privilege to be a part of the research group of

Resource R

ecovery at the Swedish C

entre for Resource R

ecovery, which is led by Prof.

Moham

mad Taherzadeh.

My first task w

as to carry out a broad literature review in the field of anaerobic co-digestion

of cattle slaughterhouse waste and agro-w

astes. The literature review revealed that even

though anaerobic co-digestion has stood out during the last few years as the m

ost relevant

solution to improving biogas yield and to increasing the econom

ic viability of the biogas

plants, there is still little knowledge available about the m

ixture interactions occurring

between the different substrates. It is very im

portant to study these interactions in detail

because depending on the mixture com

position the process performance, in term

s of methane

yield, biodegradation, and kinetics may be enhanced or attenuated. H

ence, there is a need to

find out these positive or negative interactions between the substrates so that a proper

combination of co-substrates can be selected for the co-digestion process. Since it is know

n

that the digestion of slaughterhouse waste is not an easy task, m

y project focuses on the

mono- and co-digestion of cattle slaughterhouse w

aste (SB) and other w

aste streams from

the

agricultural and municipality activities, such as anim

al manure (M

), various crops (VC

), and

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viii the organic fraction of the m

unicipal solid waste (M

SW). D

uring the first part of the study, the

AD

of slaughterhouse waste and its m

ixtures was investigated using different process

parameters for the evaluation. The m

ixture ratios were defined according to their availability

at the site of the slaughterhouse enterprises in Cuba (Papers I and II). The degradation of the

different mixtures of SB

, M, V

C, and M

SW w

as then evaluated by using two different

models, i.e., the first order kinetic m

odel and the Chapm

an model (Paper II). In order to

quantify the effect of the temperature, a selected m

ixture ratio from a study case in C

uba was

also evaluated at mesophilic conditions. Since so far our results have show

ed positive synergy

effects when applying A

coD, w

e wondered if only positive interactions w

ere occurring, so we

investigated the mixture interactions in m

ore detail. A four-factor m

ixture design was then

applied to define the mixture com

positions of SB, M

, VC

, and MSW

as components, m

aking

possible the statistical evaluations (Paper III). In this study, four individual components as

well as the binary, ternary, and quaternary m

ixture combinations w

ere evaluated with the

objective to explore possible synergistic and antagonistic interactions, as well as to find the

optimal m

ixture composition leading to the highest m

ethane production. The methane yield

and the specific methane production rate therefore w

ere used as response variables. The

results of evaluating eleven mixture com

binations showed that synergetic effects could be

obtained in nine of them; nevertheless, antagonistic effects could also be determ

ined in two of

the studied mixtures. Thus far, w

e had only worked w

ith batch assays and we thought it w

ould

be im

portant to

evaluate these

interaction effects

further during

long-term

operation.

Therefore, four reactors were operated, running as sem

i-continuous processes. We used

mono-digestion

of the

SB

as a

control; furtherm

ore, w

e chose

three other

mixture

combinations representing both synergy and antagonistic effects obtained previously during

the batch assays. Moreover, the effects on the m

ethanogenic comm

unity structure were also

evaluated (Paper IV).

Running sem

i-continuous experiments is a very tim

e consuming m

ethod, but these are

important to verify results obtained through batch assays, as w

ell as to be able to determine

the long-term effects. In the last part of the w

ork, an alternative operation mode therefore w

as

investigated, where consecutive feeding, i.e., second feeding, w

as applied during the batch

assays. The idea was that this m

ethod would m

ake it possible to get a quick prediction of the

process performance, w

hich then can be used when designing the sem

i-continuous operation.

Accordingly, the response of the m

icrobial biomass to an additional second feeding w

as

determined in term

s of the methane yield, degradation kinetics, and the specific m

ethanogenic

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ix activity. A

similar m

ethodology had already been used even in the beginning of the studies,

and the response of the microbial com

munity w

as determined under a substrate lim

itation

(Paper I), while here the second feeding w

as applied under a non-substrate limitation (Paper

V). Finally, the results from

this last part of the study were correlated w

ith the results obtained

from the different operation m

odes during this work, i.e., batch m

ode (Paper III) and semi-

continuous operation (Paper IV), as w

ell as with previous results found in the literature.

Jhosané Pagés Díaz

December 2015

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x

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xi

“M

ystery creates wonder and w

onder is the basis of man’s desire to understand.”

- Neil Arm

strong –

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xii

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xiii Acknow

ledgements

Doing m

y Ph.D. in Sw

eden, while also being a student from

Cuba, has been an exciting opportunity and an extrem

ely challenging journey for me. At this m

oment, I w

ould like to take this opportunity to thank each and every one of you that have m

ade this journey possible with

support, guidance, and love.

First of all, my deepest gratitude and plenty of thanks to m

y dear supervisors Dr. Ilona Sárvári

Horváth and D

r. Ileana Pereda Reyes for providing me this opportunity to grow

in this com

plicated world of research and academ

ics. You have always been there (in person or by

email) to calm

down m

y list of worries and doubts. I have no w

ords to express how m

uch I appreciate you both as persons and as teachers. Tack/Gracias!!

Professor Moham

mad Taherzadeh, m

y examiner, thank you for keeping an eye on m

y progress over these four years, for letting m

e be part of the Resource Recovery group and for being a source of inspiration to m

e. Thank you for all the advice and suggestions that have led to im

provements in m

y research.

A big thanks to my co-supervisor Prof. Claes N

iklasson for all his support concerning the status of m

y research.

I would also like to thank the form

er rector of the University of Borås, Lena Nordholm

, for her interest in m

aintaining the cooperation between the University of Borås and m

y university, Cujae, in Cuba. Thanks to this effort, m

y Ph.D. study w

as possible. Tack så mycket!

I wish to express m

y gratitude to the Research and Education Board of the University of Borås for their financial support.

I would like to thank those people w

ho indirectly made this process easier, especially Susanne

Borg and Sari Sarhamo for keeping in contact w

ith me w

hile I was in Cuba, being alw

ays available at any m

oment w

hen I was in need of docum

ents. Also, my sincere thanks to Louise

Holm

gren, Thomas Södergren, and Jennifer Tydén for their excellent support. I am

also grateful to the D

irector of Ph.D. studies, D

r. Tomas W

ashnström.

I wish to express m

y gratitude to Jonas Hanson and Kristina Laurila for their support and

understanding while I w

as looking for things in the lab. Also, I extend my gratitude to Adib

Kalantar for his support on solving the never-ending problems w

ith my CSTR reactors and D

r. Johan W

estman for his invaluable help w

ith the microbiological experim

ents.

A very special thanks is dedicated to Dr. Elizabeth Feuk Lagerstedt for introducing m

e into the m

icrobiology world but even m

ore for being my friend. Also, I w

ould like to state my

appreciation to Dr. Anita Pettersson and her fam

ily for their friendliness and support during this tim

e.

I would like to express m

y heartfelt thanks to Dr. M

agnus Lundin for his invaluable help, dedication, and patience during our discussions about statistics and kinetic m

odels. I really appreciate your help. Thank you very m

uch!

I cannot thank enough my dear friends M

aryam, Raj, and Solm

az. I always say that I cannot

imagine w

hat my life w

ould have been like here without you. You have fulfilled this journey of

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xiv happiness and am

azing mom

ents that I will never forget. Thank you very m

uch for your friendship!! I w

ould also like to thank Dr. Gergely Forgács for being one of the first ones w

ho taught m

e how to use the GC.

I also want to thank (form

er and current) Ph.D. students from

the Resource Recovery group that I have com

e to know over these years: W

ikan, Supansa, Jorge, Julius, Päivi, Ram, M

ostafa, M

ofoluwake, Alex, Faranak, Regina, Pedro, Veronika, Konstantinos, Patrik, Farzad, Abas, Anna,

Kamran, Behnaz, and Akram

. The master’s student, Chuthathip Siripong and Supusanee

Dulyakasem

, thanks for your hard work in the lab.

I express my sincere gratitude to m

y teachers and colleagues at the Instituto Superior Politénico Jose Antonio Echeverría (Cujae) for giving m

e the education I have today. Your help, advice, and discussions about researching have been invaluable to m

e. Special thanks to Carlos Menéndez,

Miguel D

íaz, Ania, Aimeé, D

eny Oliva, and Peréz Olmo w

ho taught me that I alw

ays need to know

why and w

hy and why?

I have no words to thank Ram

ón Garrote and his family for their priceless help in every sense

and for letting me be part of their fam

ily during my tim

e in Sweden. M

illones de gracias a todos!! Also, I w

ish to thanks Pilar and Juan for their support.

Finally, I would like to thank m

y dear family: especially m

y parents and my sister for their

endless love and support.

Special thanks to Junior Lorenzo “…I don’t care if Septem

ber’s black. October, broken lips, heart attack. N

ovember, never looking back. It’s Decem

ber, I am in love…

Thank you all for making this possible in one w

ay or another!

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xv N

omenclature

AD

A

naerobic Digestion

AcoD

A

naerobic Co-digestion

AD

M1

Anaerobic D

igestion Model N

o.1

ASB

R

Anaerobic Sequential B

atch Reactor

BM

P B

iomethane Potential

EU

European Union

C/N

C

arbon to Nitrogen ratio

CO

D

Chem

ical Oxygen D

emand

CSTR

C

ontinuous Stirred Tank Reactor

HR

T H

ydraulic Retention Tim

e

LCFA

s Long C

hain Fatty Acids

k0

Specific rate constant of the overall process

M

Manure

MSW

M

unicipal Solid Waste

OLR

O

rganic Loading Rate

rsCH

4 Specific m

ethane production rate

Rm

Maxim

um m

ethane production rate

SMA

Specific M

ethanogenic Activity

SRT

Solid Retention Tim

e

SB

Solid cattle slaughterhouse waste

TS Total Solids

UA

SB

Upflow

Anaerobic Sludge B

lanket

VS

Volatile Solids

VFA

s V

olatile Fatty Acids

VC

V

arious Crops

YC

H4

Methane yield

λ

Lag phase

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xvi

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xvii Table of Contents Abstract ................................................................................................................................................................. iii

List of Publications ............................................................................................................................................. v

Statement of Contributions ........................................................................................................................... vi

Preface .................................................................................................................................................................. vii

Acknowledgem

ents ........................................................................................................................................ xiii

Nom

enclature ..................................................................................................................................................... xv

Table of Contents ........................................................................................................................................... xvii

1 Introduction ...................................................................................................................................................... 1

1.1 Outline of the thesis .............................................................................................................................. 2

1.2 Social and ethical reflections ............................................................................................................ 3

2 The anaerobic digestion process .............................................................................................................. 5

2.1 Biogas production – Some rem

arks ............................................................................................... 5

2.2 The basic principles of anaerobic digestion ............................................................................... 7

2.3 Environmental and operational param

eters in AD .............................................................. 10

3 Substrates for biogas production .......................................................................................................... 15

3.1 Animal w

astes ...................................................................................................................................... 15

AD of slaughterhouse w

aste .............................................................................................................. 16

3.2 Residues from agriculture and m

unicipality activities ...................................................... 19

Manure ........................................................................................................................................................ 20

Various crops ........................................................................................................................................... 20

Municipal solid w

aste ........................................................................................................................... 22

4 Assessing the methane potential and the process perform

ance ............................................. 23

4.1 Fermentation assays ......................................................................................................................... 23

Batch operation: Biochemical M

ethane Potential test (BMP) ............................................ 23

Semi-continuous operation ............................................................................................................... 26

Batch operation: Consecutive feeding procedure .................................................................... 28

5 Mono-digestion ............................................................................................................................................. 31

5.1 Biodegradability .................................................................................................................................. 31

5.2 Methane potential .............................................................................................................................. 31

5.3 Specific methanogenic activity ..................................................................................................... 37

5.4 Kinetics .................................................................................................................................................... 39

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xviii 6 Co-digestion .................................................................................................................................................... 45

6.1 Why co-digestion? .............................................................................................................................. 45

6.2 Current scenarios of the AcoD of the slaughterhouse w

aste ........................................... 47

Co-digestion of the slaughterhouse waste and the agro-w

astes ....................................... 47

6.3 Influence of mixture interactions on the process perform

ance and the operation mode ..........53

7 Conclusions ..................................................................................................................................................... 57

8 Future work .................................................................................................................................................... 59

References ........................................................................................................................................................... 61

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1 1 Introduction D

ue to the current energy scenarios and the world’s dependency on fossil fuels, the protection

of the environment has becom

e a major concern. The utilization of fossil fuels has led to

environmental problem

s connected to increased greenhouse gas emissions and clim

ate changes.

Moreover, the deficit of energy in the w

orld and, in particular, in developing countries, has

therefore gained an increased attention; thus, new alternative energy sources m

ust be

introduced. Biogas produced from

organic wastes is a renew

able energy source for future and

sustainable energy supply. In this regard, agriculture is a sector with m

any activities and

necessities that generate a huge amount of biodegradable organic w

aste, which can be utilized

for biogas production. Consequently, it has a unique position to satisfy both food dem

and and

energy supply in farms, rural com

munities, cities, as w

ell as can be applied as fuel within the

transport and industrial sector.

Anim

al by-products and residues from the m

eat industry have high methane potential and are

therefore highly demanded substrates for biogas production on a com

petitive market. D

ue to

the high content of lipids in these residues the methane yield w

ill be high, increasing the

economic feasibility of the biogas plants [1]. H

owever, there are problem

s that may occur in a

full-scale biogas plant treating slaughterhouse waste w

ith significant impacts on process

efficiency, operational costs, and economic profitability. A

mong the operational problem

s, the

formation of foam

can be mentioned, w

hich is usually caused by the high content of lipids and

proteins present in these residues [2]. Another problem

associated with anaerobic digestion of

slaughterhouse wastes is that potential inhibition of the m

icrobial activity may occur, w

ith a

subsequent low m

ethane production, process instability, and finally, failure of the process [3].

The slow degradation rate of the slaughterhouse w

aste will lead to the accum

ulation of

intermediary com

pounds such as long chain fatty acids, volatile fatty acids, amm

onia, and

amm

onium nitrogen, causing inhibition in the system

[4, 5].

One possible solution to solving the above-m

entioned problems is the application of anaerobic

co-digestion (AcoD

). In this system, slaughterhouse w

astes can be treated together with the

other residues generated during the agriculture activities. The main advantages of co-digestion

are related to a balanced nutrient supply, better carbon to nitrogen (C/N

) ratio, the dilution of

inhibitory compounds, as w

ell as to a more efficient utilization of the digester plant by treating

several wastes at the sam

e time.

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2 How

ever, even though there are some results reported in the literature dealing w

ith the AcoD

of

different mixtures of biodegradable w

astes, there is still a lack of investigations to predict the

mixture interactions (i.e., synergy and antagonism

) based on the composition of the w

astes,

biodegradability, and kinetics of the anaerobic process in the long-term operation.

This thesis therefore deals with the evaluation of the anaerobic m

ono- and co-digestion of solid

cattle slaughterhouse waste and other w

aste streams from

the agricultural and agro-industrial

activities. The main objective w

as to evaluate the anaerobic digestion in terms of the

biodegradability of the substrates as well as m

ixture interactions, influencing the performance

of the process.

1.1 Outline of the thesis

A short sum

mary of the chapters com

prised in this thesis are listed as follows:

Chapter 1 introduces the background of the w

ork and the main objectives of the investigations.

Chapter 2 provides general and current inform

ation about the biogas production, the basic

principles of the anaerobic digestion process, as well as the m

ost important environm

ental and

operational parameters.

Chapter 3 presents the substrates evaluated in the present study, i.e., cattle slaughterhouse

waste and residues from

agriculture (animal m

anure, various crops) and municipality activities

(organic fraction of municipal solid w

aste). The current production and the impact of the final

disposal of these waste stream

s are presented, as well as the difficulties associated w

ith the

anaerobic digestion of slaughterhouse waste.

Chapter 4 presents the different ferm

entation assays to assess the methane potential, the

biodegradability of the substrates, and the process performance (Papers I–V

).

Chapter 5 is dedicated to the m

ono-digestion of the slaughterhouse wastes and som

e residues

from the agricultural and m

unicipal activities. The biodegradability of every individual material

is discussed in terms of m

ethane yield, the specific methanogenic activity, and the kinetics of

the process (Papers I, III–V).

Chapter 6 deals w

ith the anaerobic co-digestion process. It gives an overview of the current

scenarios, especially when slaughterhouse w

aste is co-digested with the agro-w

astes in binary,

ternary, and quaternary mixture com

binations. The effect of the mixture interactions, i.e.,

synergy and antagonism, on process perform

ance and operation mode is also discussed (Paper

I–V).

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3 1.2 Social and ethical reflections

During the last few

decades and up until today, there has been a lot of discussions and debates

about ethical, social, and political aspects of biogas production. The debates are based on the

utilization of raw m

aterials, which can also be utilized as food sources, i.e., m

aize, wheat, or

sugarcane, for biogas production. This has resulted in a lot of discussions related to the social

and environmental im

pacts as well as the land and w

ater usage for biofuel production at a time

when there is considerable food dem

ands in developing countries. How

ever, one cannot decide

whether this is w

rong or right without considering the local conditions as w

ell as all the

advantages and disadvantages toward society and environm

ent. If biogas is produced in an

environmentally sustainable w

ay, with no exacerbation of global clim

ate change and in

accordance with the trade principles and hum

an rights, then there is a duty to produce biogas

through the utilization of organic waste fractions. The production of biogas from

raw m

aterials

considered as “wastes,” such as anim

al manure, slaughterhouse w

aste, or crops residues, will

not interfere with hum

an or animal consum

ption but instead have numerous environm

ental and

social benefits. Today, many of these w

aste fractions are still unexploited and by just dumping

them at sites w

ill contribute to environmental dam

age in the rural sectors. Hence, their

utilization will play an im

portant role in the reduction of the greenhouse gas emissions and to

facilitate a sustainable development of energy supply w

hile providing bio-fertilizer to farmers,

especially in the developing countries, like Cuba. A

s these residues are generated from the

agriculture or agro-industrial sector producing the food, their utilization within the sam

e areas

in which they are produced w

ill offer other important benefits as w

ell, like treating wastes close

to their source. Biogas production from

the residues, which cannot be used as food any longer,

will increase the consciousness of people and the public acceptability of the w

aste managem

ent

and at the same tim

e create new job opportunities in the rural areas.

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4

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5 2 T

he anaerobic digestion process 2.1 B

iogas production – Some rem

arks

Today, most of the prim

ary energy supply in the world is covered by fossil fuels such as oil,

coal, and natural gas, which together represent about 81%

of the energy demand of the w

orld

(Figure 2.1) [6]. Current scenarios have show

n that due to the rising price of crude oil, as well

as the negative impacts of fossil fuels on the environm

ent, energy insecurity, and continuous

abuse of the natural resources, the interest has shifted towards renew

able energy sources to

provide a sustainable future energy production. According to the recom

mendations of the

European Union (EU

), about 20% of the total energy supply should com

e from renew

able

resources by the year 2020 [7].

Figure 2.1 W

orld total primary energy supply in 2012 from

fossil fuels, nuclear energy, and renewable sources [6]

Considering the renew

able energy production, biogas production utilizing the municipal,

industrial, and agricultural residues has a potential to play a vital role in the future. Biogas is a

mixture of gases, w

ith the main ingredients of m

ethane (CH

4 ) and carbon dioxide (CO

2 ), which

are produced by the microbial degradation of the organic m

atter under anaerobic conditions.

The energy content of biogas is related to its methane content, due to its com

bustible

characteristic (~10 kWh N

m-3 C

H4 ). The m

ethane content in the produced gas depends on a

number of factors in w

hich process design and substrate characteristics have a major influence.

Figure 2.2 shows an overview

of the biogas industry. Depending on the needs of every society

worldw

ide, i.e., developed or developing countries, different fields of applications can be

Oil

31%

Coal 29%

Natural gas

21%

Nuclear

5%

Hydro

3%

Biofuel and w

aste 10%

Geothermal,

solar, wind, heat

1%

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6 considered. In the European Union (EU

), biogas is mainly used for the co-generation of

electricity and heat, meanw

hile cooking and lighting are the major utilizing form

s in the

developing countries.

Figure 2.2 Overview

of the biogas industry

The primary energy output of biogas heat sales increased in the EU

by 18% and 16%

,

respectively, in 2010 and 2011 [8]. In addition, biogas can also be utilized as a vehicle fuel in

the transport sector if it is upgraded. Sweden has taken the lead in this regard, although other

countries including

Switzerland,

Germ

any, A

ustria, Spain,

and France

have ongoing

development w

ithin this area [9]. On the other hand, G

ermany has a leading role in injecting

upgraded biogas, i.e., biomethane, into the natural gas grids in Europe [8]. These options

represent efficient methods to integrate biogas into the energy sector. M

oreover, biogas

production through AD

offers other significant environmental benefits as w

ell, as the anaerobic

effluent (the digestate residue) can be utilized as a fertilizer, recycling nutrients to the

agricultural land while replacing the chem

ical fertilizers.

Hence, there are strong incentives w

orldwide for the stim

ulation of biogas production using

different biodegradable substrates. According to the European Environm

ental Agency, the

StorageTransport

SeparationSpreading

Solids

Liquids

Compost

Back to digestion or spreading

Gardering

Agriculture

Digestion

Municipality

-Organic fraction of

municipal solid w

aste-Sew

age sludge and urban w

aste water

-Source separated organic w

aste, etc.

Industry

-Food waste

-Industrial effluents-Grease-Slaughterhouse w

aste, etc.

Agriculture

-Animal m

anure-Energy crops, etc..

Biogas U

pgrading

Vehicle fuel

Cooking

Heating

Lighting

CoolingM

unicipalityBiogas

cleaningipality

Biogas utilization

Directburning

Co-generation

Electrical energy

Thermal

energy

Gas grid injectionBiom

ethane

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7 agriculture sector has the greatest potential, w

hich is today still a large unexploited sector.

Currently, there are m

ore than 12,000 biogas plants operating in Europe, among them

about

8,500 are agricultural biogas plants (Table 2.1) using animal m

anure and energy crops as the

main substrates.

Table 2.1. Status of biogas production in several European countries, Brazil and Republic of Korea [10]

n.a: Not available

By the year 2020, it is estim

ated that the number of biogas plants w

ill be more than 45,000 in

Europe [11]. In that sense, the challenges for sustainable biogas production in the future will lie

in the substrates’ availability as well as the governm

ental policies, national strategy plans, and

subventions, in order to be able to compete w

ith the fossil fuels [10].

Com

pared to Europe, in countries in Latin Am

erica and Central A

merica, for exam

ple, Cuba,

Argentina, M

exico, Peru, and Chile, the biogas production at the industrial level is in its initial

phase, and most of the biogas im

plementations are developed on a sm

all-scale level and at

farms.

Finally, we can conclude, that the positive environm

ental impact that a biogas plant provides,

wherever it is located, should be an im

portant issue to take into account.

2.2 The basic principles of anaerobic digestion

Anaerobic digestion is a biological process in w

hich complex organic m

atter (i.e., proteins,

carbohydrates and

lipids) is

broken dow

n through

the action

of different

groups of

microorganism

s (Bacteria and A

rchaea) to produce biogas in the absence of oxygen. The

process takes place through a series of parallel and serial-parallel biochemical reactions, in

Country N

umber of biogas plants unit electricity

generation

Energy production (GW

h/year)

Num

ber of agriculture biogas

plants

Year of the data inform

ation

Germany

9,945 40,970

7,800 2012

United Kingdom

610

10,494 53

2013 Sw

itzerland 600

1,023 89

2012 France

336 1273

105 2012

Austria 336

585 291

2012 N

etherlands 252

n.a 105

2013 Sw

eden 242

1,589 26

2012 Denm

ark 167

1,218 82

2010 N

orway

129 500

4 2010

Republic of Korea 78

1,925 8

2012 Finland

73 569

8 2012

Ireland 30

n.a 8

2012 Brazil

22 697

8 2013

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8 which an active m

icrobial comm

unity has to work together (Figure 2.3). The successful

performance of this biological process is highly dependent on the substrate’s com

position,

microbial com

munity structure, as w

ell as the operational and environmental conditions [12].

The AD

process can be divided into four main degradation steps, i.e., hydrolysis, acidogenesis,

acetogenesis, and methanogenesis, as it is schem

atically represented in Figure 2.3 and briefly

summ

arized as follows:

Figure 2.3 Simplified representation of the anaerobic digestion process (Adapted from

[13])

a. Hydrolysis

In the first stage, the complex organic m

atter, containing proteins, carbohydrates, and lipids

will be hydrolyzed, producing sim

ple monom

ers by the action of the extracellular enzymes,

such as cellulases, proteinases, lipases, etc., excreted by the hydrolytic bacteria [14]. The

presence of the different types of bacteria depends on the variety of substrates, and the secreted

exoenzymes have the function to solubilize particular substrates, leading to products, w

hich, in

turn, will be further transported into the cells to be further degraded by the endoenzym

es and

used as a source of energy. The proteins are first hydrolyzed to amino acids by the proteinases,

and the carbohydrates are hydrolyzed by the cellulases, hemicellulases, and am

ylases to soluble

sugars. On the other hand, lipids are hydrolyzed to m

ainly glycerol and long-chain fatty acids

Complex O

rganic Matter

Proteins, Carbohydrates, Lipids

Soluble Organic M

onomers

Amino acids, Sugars, Long Chain Fatty Acids, etc.

Intermediary products

Volatile fatty acids, Alcohols, etc.

Hydrolysis

Acidogenesis

Acetate(CH

3 COO

-)Hydrogen (H

2 ) Carbon Dioxide (CO

2 )

Methane (CH

4 ) and Carbon Dioxide (CO

2 )

Hydrogenotrophic m

ethanogenesisAcetotrophic

methanogenesis

Acetogenesis

Anaerobic oxidation

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9 (LC

FAs) by exoenzym

es called lipases. Depending on the nature and com

plexity of the

substrates, the process can take hours or days. This stage is usually the rate-limiting step in the

whole anaerobic process w

hen the solid and complex substrates are treated [15, 16].

b. Acidogenesis

The soluble organic monom

ers such as amino acids, sugars, and long chain fatty acids form

ed

during the hydrolysis stage are further converted by facultative bacteria into short-chain organic

compounds containing one to five carbon units. The m

ain products formed during the

acidogenesis steps are volatile fatty acids (VFA

s), like acetic, propionic, valeric, and butyric

acids. Furthermore, depending on the characteristics of the substrates, the environm

ental

conditions and the microorganism

s present in the system alcohols, carbon dioxide, hydrogen,

and amm

onia are also produced [17]. Sugars are converted into VFA

s while am

ino acids are

degraded into acetate, amm

onia, carbon dioxide, and hydrogen sulphide [18]. The LCFA

s are

converted into acetate, hydrogen (H2 ), and propionate through the ß-oxidation pathw

ay. The

accumulation of the interm

ediates, such as ethanol, propionate, butyrate, and benzoate, is

strongly related to the increased hydrogen concentration in the system [19].

c. Acetogenesis

In this stage, the intermediary com

pounds, i.e., acids and alcohols from the acidogenesis step,

are converted into acetate, hydrogen, and carbon dioxide by acetogenic bacteria to be further

utilized in the next step by the methanogens. The anaerobic oxidation of these interm

ediary

compounds is not therm

odynamically possible unless acetogenic bacteria engages in a

syntrophic cooperation with the hydrogen consum

ing methanogens in order to keep a low

H2

partial pressure in the system. A

t a low H

2 partial pressure, the changes in the Gibbs free

energy (ΔG°) is favorable for the A

TP synthesis and bacterial growth [19]. In addition,

homoacetogenic bacteria reduce the H

2 and CO

2 to acetic acid [20, 21]; however, in

environments w

ith increasing concentrations of amm

onia nitrogen, the acetate can also be

oxidized by the syntrophic acetate oxidizing bacteria to H2 and C

O2 [22, 23].

d. Methanogenesis

Methanogenesis is the last step w

ithin the anaerobic digestion process, and it is driven by the

methanogens belonging to the dom

ain of Archaea. In this stage, acetate, H2, and C

O2 produced

during the previous steps are converted into CH

4 and CO

2 , although other methylated

compounds, such as m

ethylamines and m

ethanol, can also be converted into methane [24].

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10 A

lmost 70%

of the produced methane takes place through the acetotrophic pathw

ay (Table

2.2). In this reaction, carried out by the Methanosarcina and M

ethanosaeta, the methyl group

of acetate is reduced to CH

4 , while the carboxylic group is oxidized to C

O2 [25]. The rest (i.e.,

about 30%) of the produced m

ethane is formed through the hydrogenotrophic pathw

ay, in

which H

2 is used to reduce the CO

2 to methane. This group of m

ethanogens has the important

role of consuming the H

2 produced in the previous step, keeping the partial pressure of H2 low

in the system, as discussed above.

Table 2.2: Principal biochemical pathw

ay in methanogenesis [25]

Pathway

Reaction ΔG° (kJ/m

ol)

Acetotrophic CH

3 COO

- + H+

CH4 + CO

2 -32

Hydrogenotrophic CO

2 + 4H2

CH4 + 2H

2 O

-130

2.3 Environm

ental and operational parameters in A

D

a. Temperature

Temperature is an im

portant physical parameter for the anaerobic digestion process, since it is

directly linked to the thermodynam

ics and kinetics of the biochemical reactions, as w

ell as the

growth of the m

icroorganisms [26]. A

D processes can be operated at a w

ide range of

temperatures, i.e., betw

een 10°C and 65°C

; nevertheless, most of the biogas reactors on the

industrial levels operate at mesophilic (35°C

– 38°C) or therm

ophilic (54°C – 56°C

) conditions.

Mesophilic conditions result in higher process stability and low

er energy needs compared to the

thermophilic conditions. H

owever, operating at higher tem

peratures offers several advantages,

like higher efficiency in the biodegradation of organic matter, leading to im

proved gas yield

due to the increased rates in the biochemical and chem

ical reactions, as well as increased

sludge hygienization

and elim

ination of

pathogens [27-29].

The grow

th rate

of the

microorganism

s is also higher at thermophilic conditions com

pared to that at mesophilic

temperatures, m

aking the process faster and more efficient [30, 31]. Today, therm

ophilic AcoD

of waste from

agriculture/agro-industrial activities, especially when slaughterhouse w

aste is

utilized, has gained attention due to its capacity to enhance the hydrolysis rate of this fatty

material, and better destruction of the pathogens.

In the present study, the effect of temperature w

as evaluated during a co-digestion process

using a mixture of SB

, M, and V

C (Paper II). In general, higher tem

peratures have a positive

influence on the activity of the biomass, but on the other hand, the process can be m

ore

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11 sensitive to the accum

ulation of intermediary products, such as am

monia, V

FAs, and LC

FAs

[4, 32, 33].

b. Organic loading rate and retention tim

es

The organic loading rate (OLR

) and the retention times (e.g., hydraulic retention tim

e (HR

T)

and solid retention time (SR

T)) are usually used as design parameters to determ

ine the reactor

size at the industrial level. OLR

expresses the amount of organic m

atter, e.g., total solid (TS),

volatile solid (VS), or chem

ical oxygen demand (C

OD

) fed into the reactor per unit of effective

volume per unit of tim

e. HR

T is defined as the average period of time in w

hich the substrate

remains inside the reactor and is in contact w

ith the microorganism

s present there. Meanw

hile,

SRT is the average tim

e in which the anaerobic biom

ass (microorganism

s) remains inside the

reactor [34]. In continuous stirred tank reactor (CSTR

), SRT and H

RT are the sam

e if there is

no recirculation of biomass back into the reactor.

The extent of a desirable retention time and the am

ount of organic matter fed into the reactor

are related to the reactor configuration and to the characteristics of the materials digested.

Com

plex materials rich in fats and proteins require a longer tim

e at a lower O

LR. For exam

ple,

solid materials such as slaughterhouse w

aste, animal m

anure, and crop residues are normally

digested in completely m

ixed reactors at low O

LRs, i.e., betw

een 1 to 3 kgVS m

-3 d-1 [4, 35,

36]. While liquid substrates such as w

astewaters are norm

ally treated in up-flow anaerobic

sludge blanket (UA

SB) reactors at higher O

LRs betw

een 10 to 20 kg CO

D per m

3 reactor per

day [37]. Both param

eters are very important in order to m

onitor the process and to achieve

stable operation. If too high OLR

is applied, intermediary products can be accum

ulated in the

system causing instability and finally a failure of the reactor.

c. pH, alkalinity, and V

FA

/Alkalinity ratio

The AD

process is strongly affected by the slight changes in the pH. Each group of

microorganism

s works optim

ally in their own optim

um pH

range. Am

ong them, m

ethanogens

are more sensitive to variations in pH

than fermentative bacteria, w

hich are able to function

within a w

ider range of pH, i.e., betw

een 4.8 and 8.5 [38]. The hydrolytic and acidogenic

microorganism

s reach their optimal grow

th at a pH betw

een 5.5 to 6.5, while for m

ethanogens

a pH betw

een 7.8 to 8.2 is optimal [39]. C

onsequently, the optimal pH

for the whole process is

around neutral with values ranging from

6.8 to 7.4 [39].

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12 A

lthough the pH level has a direct influence on the biological activity of the different groups of

microorganism

s, it is not a good indicator for monitoring the perform

ance of the process, since

any slight variation in the pH m

ay lead to stress conditions and failure of the digester. In order

to prevent a drastic decrease in the pH and to keep the digester stability, it is im

portant to have

high alkalinity. Alkalinity m

easures the amount of alkaline substances that neutralize the acids

produced in the anaerobic digestion process. Alkalinity m

ainly consists of the presence of

bicarbonate ions in an equilibrium w

ith CO

2, although protein-rich substrates may also

contribute to the buffer capacity due to the formation of am

monium

bicarbonate [40].

Alkalinity is a proper param

eter to indicate VFA

accumulation, since any increm

ent of VFA

s

will consum

e alkalinity, and it would happen before any greater changes in the pH

can be

detected. Hence, a good indicator and a fast control for the reactor perform

ance is the

VFA

/Alkalinity ratio. If this ratio is kept betw

een 0.1 and 0.35, the process works under stable

conditions [20, 41, 42].

In the present study, the performance of the sem

i-continuous digestion of SB and its co-

digestion with other residues w

as monitored by controlling the V

FA/A

lkalinity ratio (Paper

IV). A

s shown in Figure 2.4, in the case of SB

and SB + V

C, only a sm

all variation in the pH

was observed, w

hile dramatic increases in the V

FA/A

lkalinity ratio occurred making the

process unstable and prone to failure.

Figure 2.4 Variations of the pH and VFA/Alkalinity ratios during the semi-continuous co-digestion studies of: (●)

SB digested alone, (x) SB + M, (■) SB + VC + M

SW, (▲

) SB + VC (Paper IV)

d. Inhibitors

In the AD

process, there are several substances that may be toxic for biom

ass activity at a given

concentration,especially for the methanogens. H

owever, the reported inhibitory levels for som

e

of these substances show a considerable disparity betw

een the different studies [43]. This can

0.0

0.3

0.6

0.9

1.2

1.5

1.8

1 3 5 7 9

010

2030

4050

6070

8090

100

VFA/Alk

pH

Time [days]

Start-up period

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13 be explained by the fact that the inhibitory level can be affected by the com

position of waste,

including the concentration of the inhibitors presented, as well as the origin of the inoculum

and/or the adaptation of the microorganism

s to certain inhibitory compounds, and m

oreover the

environmental and operational conditions. M

echanisms such as antagonism

and synergism

have also shown to have a significant influence on the inhibition phenom

enon [43]. In the case

of slaughterhouse waste, com

pounds such as amm

onia (NH

3 ) and LCFA

s are comm

only

present and develop during the AD

process. Although other com

pounds like sulfides, VFA

s,

metal ions, and other organic com

pounds may also be present at levels causing inhibition.

During the degradation, lipids are broken dow

n into LCFA

s. The adsorption of LCFA

s on the

microbial surface, interfering w

ith the transport of substances and the protective function of the

cells, has previously been described as the mechanism

of the inhibition [44, 45]. How

ever, the

degree of inhibition also depends on the available biomass surface area, the com

plexity of the

carbon chains, as well as on the adaptation of the biom

ass [46]. Oleic (18:1) and palm

itic (16:0)

acids are the most com

mon unsaturated and saturated LC

FAs, respectively, counting up to

~68% in anim

al waste, and they have been reported to inhibit the A

D process [47, 48].

Nevertheless, there are other studies that show

that the inhibition is a reversible process and

that the system m

ay recover after a lag-phase and an adaptation period [5, 49].

On the other hand, during the degradation of proteins, N

H3 and am

monium

(NH

4 +) are released

to the medium

, leading to inhibition and hence, process failure [50]. Depending on the pH

and

the temperature, these are in equilibrium

with each other. A

mong them

, the uncharged NH

3

molecule has show

n to be more inhibitory com

pared to the NH

4 + ion, as it can easily pass

through the cell mem

brane causing proton imbalance and potassium

deficiency within the cells

[51]. A

cetolastic m

ethanogens are

more

sensitive to

amm

onia inhibition

than the

hydrogenotrophic ones [52, 53]. A broad range of total am

monia concentration values, i.e.,

between 1.7 – 14 g L

-1 has been reported to cause inhibition depending on the pH, tem

perature,

and biomass adaptation [43]. H

owever, the interaction that takes place betw

een the free

amm

onia, VFA

s, and pH can lead the process to a so called “inhibited steady state,” in w

hich

the process runs stable but with a low

er methane production [53, 54].

The inhibitory effect of either LCFA

s or NH

3 /NH

4 + might lead to instability and the generation

of intermediates, such as V

FAs, accum

ulated in the system [51, 55]. V

FAs are the m

ain

intermediary com

pounds produced during the degradation of substrates; hence, monitoring the

concentration of VFA

s is an important control param

eter aiming to detect an im

balance among

the acid-producers and acid-consumers [42]. V

FAs are present in the anaerobic digesters, either

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14 in a m

olecular form (un-dissociated) or in an ionic form

(dissociated), depending on the pH and

the temperature. A

t a lower pH

, most of the V

FAs exist in un-dissociated form

s, which are

more hazardous for m

ethanogens than the dissociated forms. This is because the un-dissociated

form can easily enter into the cell affecting its energetic functions [17]. In an anaerobic reactor,

about 85% of the total V

FAs corresponds to acetate, w

hich is the most predom

inant acid

produced [21], although propionate is known to be a better indicator of a process im

balance

[56]. The accumulation of both acetate and propionate are indicators of low

methanogenic

activity, leading to reactor failure [56].

In the present study, semi-continuous co-digestion of SB

with other w

aste streams w

as

monitored by controlling the production of V

FAs (Paper IV

). Figure 2.5 shows the correlation

between the concentration of V

FAs and the m

ethane production when the SB

and VC

were co-

digested. When O

LR of 2 gV

S L-1d

-1 was applied, overloading w

as observed. This could be

concluded from the gradual increase in the V

FAs together w

ith a declined methane production

(Paper IV).

Figure 2.5 The effect of increasing total VFAs production (■) on the methane yield (●

) during the semi-

continuous co-digestion of 50% SB + 50%

VC (Adapted from Paper IV)

0 200

400

600

800

0 5 10 15 20

020

4060

80100

YCH4 [mL gVS -1]

VFA [g L-1]

Time [days]

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15 3 Substrates for biogas production 3.1 A

nimal w

astes

Due to the grow

ing demand on m

eat in the world for hum

an feed, the meat production industry

became a fast increasing industry w

orldwide. A

ccording to the Food and Agriculture

Organization (FA

O) of the U

nited Nations, the global production and consum

ption of meat has

steadily grown both in developed and developing countries during the past decades, and it is

estimated to grow

further in the future (Figure 3.1).

Figure 3.1 Meat production and consum

ption in the World (Adapted from

[57]) From

the bovine animal processing industries, approxim

ately 60% of the cattle anim

al weight

is considered to be waste that is not indented for hum

an or animal consum

ption as shown in

Table 3.1 [58].

Table 3.1 Typical quantity of animal w

aste per species of animals

Species W

eight of the animal

kg M

eat W

astes kg

%

kg %

Cattle

350 140

40 210

60 Pig

70 38.5

55 31.5

45 Sheep/Goat

30 12

40 18

60 Poultry

2 1.12

56 0.88

44 H

ence, the slaughterhouse industry generates a large variety of waste fractions that include both

liquid and solid materials. The anim

al by-products generated from the m

eat producing

industries are rumen, fat, gut fill, blood, stom

ach, or intestinal contents, etc. The uncontrolled

0

100

200

300

400

19801990

20002004

20152030

Meat Production [M

illions of tonnes]

Developing countriesDeveloped countriesW

orld

** projected

0 20 40 60 80

100

120

19801990

20002004

20152030

Meat Consum

ption [kg/person/year]

Developing countriesDeveloped countriesW

orld

** projected

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16 decom

position of theses residuals results in large-scale contamination of land, w

ater, and air as

a result of the organic pollution and microbial pathogenic loads. C

urrently, there are problems

with the disposal of such residues due to the legislative restrictions and high disposal costs. In

the EU, stringent regulations have been adopted to control the handling, safe collection,

transport, storage, processing, use, and disposal of these waste fractions [59]. A

ccording to the

Regulation (EC

) No. 1069/2009 and based on the potential risk to public and anim

al health, as

well as to the environm

ent, the treatment of anim

al wastes is classified into three categories

[59]. Category 1 includes all m

aterials presenting the highest risk of being infected with bovine

spongiform encephalopathy (B

SE) or with pollutant substances such as horm

ones used for

growth. These types of w

aste materials m

ust directly be incinerated or landfilled after a

pressure sterilization treatment [59]. C

ategory 2 includes contaminated anim

als that were sick

or died at the farms in a context or under controlled diseases. These types of fractions also

include manure and digestive tract content and can properly be treated by a biological treatm

ent

such as composting or A

D [59]. C

ategory 3 contains food wastes and all anim

al by-products

originated from healthy anim

als intended for human consum

ption. These materials can be used

for further processing for animal food, biogas production, or com

posting [59].

Taking into account the fact that the meat industry produces large am

ounts of wastes and the

environmental problem

s associated with its activities, it is im

perative that this issue be solved.

In order to diminish such negative environm

ental impacts, several technologies such as

combustion and A

D have been introduced around the w

orld.

AD

of slaughterhouse waste

The proper development of the A

D process is highly dependent on the type and the

composition of the m

aterials to be treated [60]. The composition of anim

al residues differs

slightly, depending on several factors such as weather conditions, anim

al breed, age, and the

quantity/quality of the food. Table 3.2 shows a sum

mary of the characterization of several

animal w

aste fractions suitable for biogas production. As show

n in Table 3.2, the lipid and

proteins content can vary between 15.8 – 86.6%

and 2.14 – 50%, respectively, depending on

the type of the wastes.

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17 Table 3.2 Characteristics of diverse anim

al wastes

n.a: Not available; * based on dry m

atter; TS: Total solid (based on fresh matter); VS: Volatile solid (based on dry m

atter); C/N

: Carbon to nitrogen ratio The theoretical m

ethane yield depends on the content of carbohydrates, proteins, and fats. Due

to their high lipid and protein content, these types of waste fractions have a high m

ethane

potential (e.g., theoretical yield of 980 mLC

H4 gV

S-1, Paper IV

). How

ever, despite the high

methane potential, the A

D treatm

ent is often complicated [65]. The presence of lipids offers the

highest yields, but with low

er kinetic rates, as a consequence of the slow biodegradation of

lipids. Furthermore, the high content of lipids and proteins causes process instability, leading to

several microbiological and operational problem

s when the slaughterhouse w

aste is treated in

mono-digestion. The basic principles related to the degradation of these com

pounds will be

discussed below.

D

egradation of lipids

Lipids are hydrophobic molecules presented m

ainly in fats, oils, and greases, with variable

compositions. They have different characteristics depending on the saturation state, i.e., the

portion of single or double bonds presented in the molecule, the length of the carbon chain,

which determ

ine the melting point. Saturated fats are m

ainly present in the meat and dairy

products, while the unsaturated ones are m

ore comm

on in vegetable oils, in fish, nuts, and corn

oil. During the hydrolysis of lipids, glycerol and LC

FAs are produced by the action of

extracellular lipases. LCFA

s are organic acids that contain long carbon chains of 8 to 18 units.

Raw m

aterials pH

TS (%

) VS (%

)* Total nitrogen (%

)*

Lipids (%

)* Proteins (%

)* Carbohydrates (%

)* C/N

References

Cow rum

en 6.1

14.9 89.4

2.2 n.a

n.a n.a

n.a [61]

Rumen paunch

8.2 13

85 1.2

n.a n.a

n.a 30

[62] Pig paunch w

aste 5.9

31.7 82.7

1.9 n.a

n.a n.a

n.a [61]

Cow blood

7.4 19.8

75.0 15

n.a n.a

n.a n.a

[61] Iberia pig slaughterhouse w

aste

6.2 n.a

n.a n.a

n.a n.a

n.a 4.72

[63]

Solid cattle slaughterhouse w

aste

5.8-6.8 13-26

92-95 4-8

43-67 24-50

0.38 14.4

Papers I, III

Solid cattle meat

and fat n.a

88.5 96.5

0.36 86.1

2.14 n.a

n.a [64]

Solid pig meat and

fat n.a

56.4 98.7

2.44 82.8

14.7 n.a

n.a [64]

Pig stomach

n.a 18.2

98.3 6.75

47.8 36.8

n.a n.a

[64] Rum

en content n.a

11.6 93.1

1.13 15.8

6.72 n.a

n.a [64]

Bovine slaughterhouse w

aste

n.a 53.2

98.8 1.05

86.6 6.57

n.a n.a

[4]

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18 Som

e examples of LC

FA are stearic, palm

itic, oleic, linoleic, and caprylic acids [67]. These

hydrolysis products will then be further converted into H

2 , acetate, and propionate during the

next steps of the degradation process. The degradation of LCFA

s takes place through the ß-oxidation pathw

ay, which is usually reported as the rate-lim

iting step in the AD

because of the

slow grow

th of the LCFA

s utilizing bacteria (i.e., 0.1 – 0.3 d-1) [68].

Anaerobic digestion of lipid-rich w

astes is a challenge, since these types of residues often have

low

alkalinity [69,

70] and

exhibit a

high tendency

to be

toxic for

the anaerobic

microorganism

s [46, 71, 72]. During the conversion of lipids, LC

FAs are released and the

accumulation of these causes inhibition if they are not efficiently rem

oved [36, 52, 73]. It is

well know

n that methanogenic activity can be inhibited by the presence of the LC

FAs. The

mechanism

of inhibition has been attributed to cell damage and to the lim

itation of nutrients

[44]. How

ever, after a lag phase and biomass adaptation, the system

would be able to recover

and efficiently degrade the LCFA

s [49].

Another problem

related to the degradation of lipids is the high tendency to form floating

aggregates and foam in the biogas reactors. Foam

ing is one of the major problem

s that

negatively affects the overall process in the biogas plants digesting the fatty materials [74].

Foaming causes num

erous operational problems, such as blockage of the gas m

ixing devices,

piping and pumping failure, and decrease of the effective w

orking volume in the digester

leading to a drop in the VS reduction, etc. [21]. Several reasons have been identified and

reported in the literature for the causes of foam form

ation, for example, substrate com

position

(i.e., m

ainly fatty

and protein-rich

feedstock), tem

perature fluctuation,

mixing

devices,

overloading, and the presence of specific microorganism

s, among others [2, 21, 75].

Degradation of proteins

Proteins consist of long chains of amino acids, bending together w

ith peptide bonds [21]. Little

is known about the degradation route of the proteins and am

ino acids in the anaerobic reactors

[76, 77]. Nevertheless, during the hydrolysis of protein-rich m

aterials in an anaerobic

environment, proteolytic bacteria from

the genus of Clostridia seem

to be dominant and play an

important role during the subsequent degradation of the am

ino acids [78]. Soluble amino acids

are further converted into a variety of organic acids, together with a release of am

monia and

amm

onium. The increm

ent of amm

onia or amm

onium content in the digester depends on the

prevailing pH and tem

perature conditions as previously discussed in section 2.3. The described

inhibitory effect of amm

onia has been widely discussed in the literature [43, 51].

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19 3.2 R

esidues from agriculture and m

unicipality activities

Agriculture is an econom

ic sector with diverse activities and necessities. There is an abundance

of wastes generated by this sector w

orldwide, w

hich is still unexploited, with an estim

ated

amount of around 140 kg per capita per year [79]. In the EU

, about 1.23 billion tons of organic

wastes are produced every year, in w

hich about 90% of the total organic residue stream

s come

from agriculture [80]. H

owever, in developing countries, m

ost of these types of waste stream

s

are still unused, hence, contributing to environmental pollutions in both urban and rural areas.

Residues from

agriculture consist mainly of grains, w

heat, rice straw as w

ell as the fruit and

vegetable residues generated after each harvesting period as well as the different types of

animal m

anure from cattle, horse, pig, or poultry.

The organic fraction of municipal solid w

aste is another material produced by the com

munities

close to the agricultural activities. These materials are rich in carbohydrates and are very

suitable for biogas production, providing an environmental solution and self-energy supply in

the rural comm

unities. Table 3.3 shows a sum

mary of the characterization of som

e residues

derived from agriculture and the m

unicipality activities suitable for biogas production.

Table 3.3 Characterization data on several agriculture and municipality w

aste fractions

n.a: Not available; * based on dry m

atter; TS: Total solid (based on fresh matter); VS: Volatile solid (based on dry m

atter); C/N

: Carbon to nitrogen ratio D

ue to the relevance to this project, some of these agriculture and m

unicipality waste fractions

will be presented in m

ore detail below.

Raw m

aterials pH

TS (%

) VS* (%

) Total nitrogen (%

)*

Lipids (%

)* Proteins (%

)* Carbohydrates (%

)* C/N

References

Horse manure

n.a 81.5

75.8 2.1

1.9 13.5

60.3 n.a

[81] Cattle m

anure n.a

23 78.6

2.5 1.3

20.8 57.3

n.a [81]

7.7

31 65

3.8 n.a

n.a n.a

39 [62]

Pig manure

n.a 55

63.6 3.2

n.a n.a

n.a 10.2

[82] M

ixture of animal

manure (pig, cow

, horse)

8.36 35

40 1.1

1.1 7.4

51.4 n.a

Papers III-V

Rice straw

6.46 87.8

79.6 n.a

n.a n.a

n.a 43

[83] Various crops

6.9 47

91 4

11 25

n.a 10

Paper I Fruit and vegetable w

astes 4.2

8.3 93

2.1 n.a

n.a n.a

34.2 [84]

Vegetable wastes

n.a 10.8

91 n.a

2.6 22.5

66.7 n.a

[85] M

SW

5.2-5.9 11-18 84-85

2-4 7-14

12-16 43

19.5 Papers I, III

n.a

26 87

n.a 16

16 n.a

18 [86]

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20 M

anure

Liquid and solid animal m

anure is an abundant source of organic material produced daily

within the agricultural industries. The daily production of m

anure is correlated with the anim

al

type. In that sense, 0.08 kg of chicken manure, w

hile 2 kg and 8 kg of pig and cattle manure,

respectively, are produced per animal per day [87]. U

sually, manure is stored at farm

s,

producing spontaneous emissions of m

ethane, carbon dioxide, and amm

onia, thus, contributing

to the greenhouse gas emissions [88, 89]. A

nimal m

anure is a suitable substrate for biogas

production due to the presence of carbohydrates, proteins, and lipids in its composition. In

Sweden, the biogas potential from

animal m

anure is estimated to 2.63×10

8 m3 of m

ethane per

year [90], and the manure is m

ostly treated in centralized biogas plants in co-digestion with

other waste stream

s.

The composition of m

anure from different anim

als differs depending on the species, the breed,

the food quality, the age of the animal, as w

ell as the type and amount of the bedding m

aterial

and the pre-storage conditions [91, 92]. Cow

manure contains a high am

ount of carbohydrates

while pig m

anure is mostly com

posed of proteins, which increase its gas potential, am

ounting

to theoretical yields of 469 and 516 LCH

4 kgVS

-1, respectively [92]. Thus, the methane

potential of manure is linked to its organic com

ponents, as well as to the presence of som

e

recalcitrant organic fibers, such as straw used as bedding m

aterial [93]. Due to the high protein

content in pig manure, an additional alkalinity is provided, as w

ell as essential nutrients and

higher buffer capacity to the system.

In this thesis, cow m

anure was used as a co-substrate in the co-digestion w

ith SB, V

C, and the

organic fraction of MSW

(Papers I and II), meanw

hile a mixture of pig, cow

, and horse

manure w

as also evaluated as an individual substrate as well as in co-digestion w

ithin the

second part of the work (Papers III–V

).

Various crops

Straw

Straw is a lignocellulosic m

aterial mainly derived from

the production of wheat, oat, barley,

rice, rye, triticale, and oil-plants, among others [94]. The w

orld production of rice and wheat

generates about 730 and 530 million tons straw

per year, respectively, which are distributed

around the world [95]. Even though there are several m

ethods available to utilize straw (i.e.,

animal feed, fuel, soil structure, or bedding m

aterial, etc.), there is still a significant amount that

is unused and is burned at open fields, causing environmental and health problem

s [95, 96].

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21 H

owever, effective utilization of straw

in the AD

processes is a challenge due to its recalcitrant

structure, which is resistant to enzym

atic attack [97]. In that sense, the application of a suitable

pretreatment m

ethod is often needed to break down the lignocellulose structure in order to

access the hemicellulose and cellulose fraction from

which m

ethane can be generated. Several

factors, such as the crystallinity of the cellulose, degree of cellulose polymerization, accessible

surface area, and protection by lignin affect the rate of the biological degradation of straw and

lignocelluloses in general [98]. A previous investigation evaluated the biogas production of

straw blended m

anure by the utilization of a pretreatment m

ethod for the straw fraction [81];

others used straw as a co-substrate in the co-digestion processes [92, 99]. In this thesis, 30%

(w/w

) of straw w

as used for the preparation of various crops fraction and the rest consisted of

fruit and vegetable waste.

Fruit and vegetable waste

Fruit and vegetable waste is w

idely produced in large quantities in markets and in agricultural

activities after a harvest season. According to the Food and A

griculture Organization (FA

O) of

the United N

ation, the production of fruit and vegetable waste in the w

orld reached 78 million

tons in 2012 [100]. Nevertheless, a huge am

ount of losses are generated during the post-harvest

chain, i.e., on the way from

the field to the market, due to the inadequate m

anagement and

handling. This results in rotten, injured, and contaminated fruits and vegetables, w

hich do not

meet the quality requirem

ents and that finally end up as waste [101]. In that sense, due to their

high biodegradability, they are also considered as a source of pollution [102]. Using suitable

technologies as part of a secondary processing, might allow

these raw m

aterials to be utilized.

Am

ong others, they can be converted into new com

mercial products, such as food flavors,

animal feed, or can be utilized w

ithin the cosmetic and pharm

aceutical industry [102]. On the

other hand, as alternative utilization concepts, composting and biogas production have also

been widely applied for the treatm

ent of fruit and vegetable waste.

In this regard, these waste fractions are suitable substrates for biogas production as they tend to

have a high organic material content (i.e., m

ore than 95% of dry m

atter), easily degradable, and

have high percentages of moisture (i.e., about 80%

) [103]. Nevertheless, the low

pH and the

fast hydrolysis rate can lead to a rapid acidification within the A

D system

with consequent

inhibition of methanogens. In order to ensure a stable perform

ance process, a two-stage system

[104] or co-digestion process have been suggested [61, 105].

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22 In the present thesis, 70%

(w/w

) of fruit and vegetable waste w

as used for the preparation of

various crops and the rest consisted of straw, as w

as mentioned above. V

arious crops were then

used as an individual fraction and investigated both in mono- and in co-digestion studies

(Papers III–V). The com

position of this various crops fraction was determ

ined considering the

amount of fruit and vegetable w

astes together with the straw

generated in the fields after

harvesting in Cuba.

Municipal solid w

aste

Municipal

solid w

aste (M

SW)

is a

heterogeneous m

aterial generated

by household,

institutional, and comm

ercial activities. The global MSW

generation in 2010 was about 1.3

billion tons, and it is expected to double by 2025 [106]. The generation rate of MSW

is strongly

influenced by the economic developm

ent, the industrialization level, public habits, and the

local climate. U

sually, countries with a higher incom

e rate and urbanization produce a greater

amount of w

astes. Furthermore, the com

position of MSW

is also affected by the climate,

seasonal and regional differences and changes, as well as by the collection and recycling

practices, etc. [107]. The main com

ponents in MSW

are food waste and yard w

aste, while other

components, such as paper, w

ood, plastic, cardboard, metal, textile, and glass w

aste may also

be presented [107].

Separated MSW

, aiming to obtain the organic fraction, is the m

ost suitable fraction for biogas

production, while the rest can be collected and/or recycled. The organic fraction of M

SW has a

high percentage of moisture content. Its V

S content is over 90% of its TS content; m

oreover, it

is highly biodegradable. In Europe, approximately 90%

of the full scale biogas plants use the

the organic fraction of MSW

as a substrate or co-substrate [108]. The application of AD

for the

treatment of this residue has been w

idely studied in the literature [109-112], and the reported

methane yields vary depending on the above-m

entioned factors. In the present research, the

organic fraction of MSW

was investigated as an individual substrate (Papers I and III) and in

co-digestion with the other residues (Papers I–V

).

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23 4 Assessing the m

ethane potential and the process perform

ance 4.1 Ferm

entation assays

Fermentation assays are generally used to evaluate the biogas potential from

the given

substrates. The prediction of the amount of produced gas is a key factor for the econom

ic

calculations to establish future biogas plants since the gas is the energy value carrier. Digestion

tests may be perform

ed in different scales, such as laboratory, pilot, or bank level and different

operation modes, such as batch, continuous, sem

i-continuous, two stage, fed-batch, etc.

Nevertheless, the accuracy of the obtained results w

ill strongly be correlated to the applied test

conditions.

Batch operation: B

iochemical M

ethane Potential test (BM

P)

The first step in evaluating the feasibility of any substrate is the determination of its

biomethane potential by the use of batch anaerobic digestion assays. A

required amount of

fresh substrate to be evaluated is placed in a reactor together with a specific am

ount of

anaerobic biomass, called “inoculum

,” for a period of time until the substrate is totally

degraded. The degradation time w

ill depend on the specific characteristics of the substrate as

well as on the activity of the inoculum

. Determ

ining the feed ratio between the substrate and

the inoculum also plays an im

portant role for a successfully performed B

MP test. N

ormally, a

VS ratio of 1:2 for gV

Ssubstrates : gV

Sinoculum is used to prevent inhibition and guarantee that there

is “enough” microorganism

s to degrade the substrate [113]. In order to maintain strictly

anaerobic conditions, the reactors are flushed with anaerobic atm

osphere, sealed, and placed

under well-controlled conditions at specific optim

al temperature for the inoculum

used. Since

the solid substrates may be quite heterogeneous and the fact that A

D is a com

plex

microbiological process including several steps, as discussed earlier, at least 3 or 4 replicates

are usually performed to facilitate statistical analysis of the obtained data. The m

ethane

volumes are regularly recorded during the test period. O

ne of the advantages of this procedure

is that many tests can be investigated in parallel, to com

pare the different conditions.

In anaerobic fermentation assays, the B

MP test is one of the m

ost used assays for choosing

materials and setting prelim

inary prices, as it has the advantages to determine the possible

biogas yield of the substrates, the biodegradability, estimate the kinetics of the degradation, or

possible inhibitory effects under the investigations [114].

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24 The produced gas is norm

ally presented as accumulated m

ethane/biogas curves in which the

shapes may vary depending on substrate com

positions and experimental factors. Figure 4.1

shows som

e typical shapes for accumulated m

ethane yield obtained during batch fermentation

tests.

Figure 4.1 Typical shapes of gas formation curves during batch ferm

entation tests (Adapted from

Papers I, III, and V) C

urve 1 shows a rapid conversion of the substrate into m

ethane with a steep increase in the

quantity of the produced gas from the start (Paper I). C

omplex substrates w

ith a high content

of fats, such as slaughterhouse wastes, are degraded w

ith difficulty, exhibiting two-stage

degradation (Diauxia) (C

urve 2, Paper III). Curve 3 show

s a strong inhibition resulting in a

negative net gas formation – w

hich appears when the gas form

ation from the sam

ple is less

than that from the inoculum

(blank reactor) (Paper V).

In the present work, batch ferm

entation tests were used to investigate the m

ono-digestion and

the co-digestion process of the SBand different agriculture/agro-industrial residues in term

s of

the methane yield and the degradation rates (Papers I, II, III, and V

).

Mixture experim

ental design

The development of statistical and com

putational methods has been an im

portant contribution

for human activities. A

mong the w

ide applications of statistics, the use of experimental designs

-100 0

100

200

300

400

500

600

700

010

2030

4050

6070

Net methane yield [mL gVS-1]

Time [days]

Inhibition

Diauxia

Retarded degradation

Norm

al

Curve 1 (Paper I) Curve 2 (Paper III)

5060

70Curve 3 (Paper V)

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25 allow

s one to evaluate a studied phenomenon effectively. The selection of an experim

ental

design might help to understand and optim

ize the process, by analyzing several factors and

their impacts on the response variables. W

ithin the experimental designs,

the mixture

experimental designs are particularly useful in m

any research disciplines and industrial

applications, where m

ixtures are involved, such as food, textile, and pharmaceutical processing.

When using these experim

ental designs, the independent factors are proportions of different

components in a blend (e.g., q com

ponents), and the sum is equal to 1 according to the

following equation:

The response variables under investigation are modeled as functions of the relative proportions

of the components in the m

ixture rather than their absolute amounts [115]. If a m

ixture of two

ingredients is investigated, then the factor space for the design lies in a line segment w

ith each

component ranging betw

een 0 and 1. If there are three components, the m

ixture space is a

triangle in three dimensions w

ith corners on the positive coordinate axes. In the case of four

components, the constrained experim

ental region can be represented as a tetrahedron where

each triangular face corresponds to a mixture of three com

ponents; an edge line segment

corresponds to a blend of two com

ponents and the corners correspond to pure blends.

Mathem

atically, this type of region is called a “simplex.” The sim

plex representation of the

design space for the mixture designs w

ith two, three, or four com

ponents is shown in Figure

4.2.

Figure 4.2 Design for tw

o (a), three (b) and four (c) components

Am

ong the mixture experim

ental designs, the simplex centroid design is a particular design in

which centroids are added in the interior of each sim

plex elements of the region (Figure 4.3).

X1 =1

X2 =1 1

1 0

X1

X2

X3 0

1 1

1

X3 =1

X1 =1

X2 =1

X1 + X

2 + X3 =1

X4 =1

X1 =1

X2 =1

X3 =1

X1 + X

2 +X3 X

4 = 1

(a) (b)

(c)

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26

Figure 4.3 Simplex centroid design for q = 4 com

ponents

In this type of design, the experimental points can be determ

ined as w

here q represents

the number of com

ponents in the system, as m

entioned above. Modeling blending surface

predictions can be made as a function of the individual com

ponents. The objective is to fit a

mathem

atical model, w

hich is able to predict and optimize the m

easured response variables as

functions of the proportions of the components in the blend.

In this work, the effect of quaternary m

ixture combinations of SB

, M, V

C,and organic fraction

of MSW

on the methane yield and degradation rates w

as investigated (Paper III). A four-

factor simplex centroid w

as used to allow for the estim

ation of a full cubic model, w

here the

linear coefficients measure the relative effect on the individual substrates, w

hile the quadratic

and cubic terms m

easure the synergistic and antagonistic interactions between the substrates

that take place during the co-digestion process.

Semi-continuous operation

The use of batch fermentation tests has som

e limitations. For exam

ple, no information is

provided regarding process efficiency, stability, or instability under continuous long-term

operation. Furthermore, the m

ethane yield under practical conditions is affected by other

factors as well, i.e., O

LR, H

RT, m

ixing, etc., which cannot be evaluated by the batch m

ethod

[114]. Semi-continuous ferm

entation tests are then generally used to investigate the stability

and performance of the process under practical conditions to sim

ulate the long-term operation

at the industrial level. It has the advantages that a maxim

um grow

th rate can be constantly

achieved at steady state conditions by controlling the loading rate,meanw

hile in batch assays it

is not possible to reach the steady state as the concentrations of the components are changing

along the digestion time [116]. D

epending on the waste com

position, the biodegradation, and

the activity of the biomass, the process can be operated using different retention tim

es. Basic

X4 =1

X1 =1

X2 =1

X3 =1

X14 =0.5

X34 =0.5

X24 =0.5

X13 =0.5

X23 =0.5

X12 =0.5

X1234 =0.25

X234 =0.33

X134 =0.33

X124 =0.33

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27 principles such as the organic loading rate, the hydraulic and solid retention tim

e, the formation

and accumulation of specific m

etabolic intermediates, optim

ization of feed composition, the

microbial com

munity dynam

ics, and structure, etc. may be investigated in a sem

i-continuous

operation. When com

plex solid substrates are treated, the use of a continuously stirred tank

reactor (CSTR

) is recomm

ended [117]. In these cases, usually longer retention times (about 50 ̶

100 days) and lower organic loading rates (0.9 ̶ 4.0 gV

S m-3d

-1) are required.

The effect of mixture interactions (i.e., synergy or antagonism

) found in the batch fermentation

tests was further investigated in this study by perform

ing semi-continuous experim

ents. The

reactor performance, as w

ell as the methanogenic com

munity structure w

as investigated (Paper

IV). Four C

STRs w

ith a working volum

e of 3L (Figure 4.4) were used to evaluate the m

ono-

digestion of the SB and its co-digestion w

ith the other substrates.

Figure 4.4 Schematic diagram

of the CSTRs used for the semi-continuous co-digestion experim

ents

Molecular biology techniques

The study of microbial ecology based on the conventional m

ethods has been inadequate for the

identification of microorganism

s present in the environmental ecosystem

s (e.g., AD

process)

[118]. Traditional methods are based on the isolation and identification of the different species

using pure culturing technique. Nevertheless, in anaerobic reactors, w

here complex m

icrobial

comm

unities are present, the application of these cultivation-based methods is lim

ited. In the

1990s the arisen of new m

olecular biology techniques revolutionized the microbial ecology

research. These new exam

inations allowed the extraction and am

plification of DN

A from

environmental sam

ples with useful genetic inform

ation of the microbial com

munity. G

enerally,

most of the m

olecular biology techniques are based on polymerase chain reaction (PC

R), w

hich

can be classified into qualitative and quantitative methods.

12

3

4

5

6

1.W

ater in2.

Water out

3.Feed / effluent

4.Stirrer

5.Gas outlet

6.Autom

atic gas m

easurement system

(Bioprocess Control, Sw

eden)

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28 A

mong qualitative approaches, clone library sequence and m

olecular profiling such as

denaturant gradient gel electrophoresis (DG

GE) or term

inal restriction fragment length

polymorphism

(T-RFLP) are very popular techniques to study the diversity and the dynam

ic

changes in the microbial com

munity of the A

D reactors [119-122]. C

loning offers a detailed

taxonomical characterization of a m

icrobial consortium; how

ever, it is considered to be a time-

consuming technique w

ith limited application for m

onitoring changes in the larger sets of the

samples from

the natural environment. O

n the other hand, DG

GE is a relatively sim

ple method

where am

plifying the 16S rRN

A genes provides characteristic band patterns by distinguishing

among the D

NA

sequences with the sam

e length. In that sense, it allows a sam

ple profile with

the possibility of sequencing a particular band to get deeper genetic information. This technique

is widely used to study the diversity and abundance shifts [123].

Regarding quantitative m

olecular biology techniques, the use of two m

ethods, fluorescent in

situ hydridization with D

NA

probes (FISH) and real-tim

e PCR

analysis stands out. FISH is

very popular and a widely applied technique w

hen previous knowledge on the m

icrobial

population is available. It makes it possible to identify and quantify m

icroorganisms w

ithout

cultivation at any taxonomical level and m

akes it possible to know if they are active or not in

their natural environment [118, 124]. R

eal-time PC

R is easy to perform

and provides an

accurate and reliable quantification of the target gene sequence allowing analyses of sm

all

amounts of D

NA

. It is based on the same principles as the traditional PC

R m

ethod, but here the

amount of am

plified products can be monitored in real tim

e with the help of using fluorescent

reporter molecules. The increase in the fluorescence intensity is proportional to the increase in

the concentration of the target gene produced with each am

plification cycle. Depending on the

objective of the research, it can be applied to perform relative or absolute quantifications.

In the present study, a relative quantification of the microbial com

munity developed during the

semi-continuous operation w

as performed using the inoculum

samples as a control to com

pare

the relative shift of the methanogenic structure under the co-digestion process of the SB

with

other different co-substrates (Paper IV).

Batch operation: C

onsecutive feeding procedure

A m

ajor drawback w

ith the use of long-term operation tests is that running these experim

ents is

a very time consum

ing task. The use of consecutive feeding in batch assays, e.g., second

feeding, is an intermediary step betw

een batch and semi-continuous ferm

entation tests. So far,

this method has rarely been reported in the literature. N

evertheless, the consecutive feeding

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29 procedure is a good alternative to predicting the response of the m

icrobial comm

unity or the

performance of the reactors by the use of the specific m

ethanogenic activity (SMA

) test [125].

The SMA

test has previously been used to evaluate the methanogenic activity of the granular

sludge from the U

ASB

reactors [126] and the loss in methanogenic activity due to the

inhibitions [125]. Moreover, it w

as also used to measure the activity of

the specific

physiological groups of methanogens consum

ing H2 and acetate, such as hydrogenotrophic and

acetolastic methanogens [127].

In this work, consecutive feeding in batch assays w

as used to investigate the response of the

microbial com

munity to the addition of a second feeding of the SB

co-digested with the other

waste stream

s, in terms of the SM

A, m

ethane yield, and the methane production rate (Paper

V). The aim

was to evaluate the feasibility of using a consecutive feeding to be able to get a

quick prediction on the process performance, w

hich can be correlated to the process

performance under sem

i-continuous operation. The initial experimental set-ups w

ere similar to

that described earlier for the batch fermentation assays using 2L glass bottles. N

evertheless,

when about 80%

of the substrates were consum

ed in the first feeding, an additional feeding was

applied with the sam

e amount of substrates as the initial feed.

Figure 4.4 Schematic diagram

of the consecutive feeding fermentation test

Inoculum + Substrate

Ratio 1:2gVS Substrate /gVS Inoculum

1stFeeding

Day 02

ndFeeding(the sam

e amount of

substrate than in the first feeding)

When about 80%

of the substrate w

ere consum

ed

Consecutive feeding batch

assays

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30

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31 5 M

ono-digestion 5.1 B

iodegradability

Biodegradability of organic substrates is norm

ally referred to as the rate (i.e., the speed of the

substrate utilization) and the extension of conversion. In general, it can be expressed as the

amount of volatile solids converted per am

ount of volatile solids added and can be studied

through biodegradability tests. These tests provide information about the portion of w

aste that

can be used as a substrate in the AD

process; meanw

hile, the indigestible fraction is also

defined. The efficiency of anaerobic biodegradation depends on the coordinated activity of the

different groups of microorganism

s, as discussed previously. These biochemical reactions w

ill,

in turn,

be affected

by the

environmental and

operational conditions,

as w

ell as

the

characteristics and composition of the substrates. H

igh biodegradability implicates that m

ore

methane can be produced per unit of substrate fed into the reactor per hour. H

ence, this can

reduce the required reactor size improving the econom

ics of the biogas plants. Know

ledge

about the biodegradability in terms of the biom

ethane potential and the overall kinetics of the

process, therefore, plays an important role in the developm

ent, operation, process analysis, and

control of the biogas plants.

5.2 Methane potential

Methane yield describes the m

etabolic activity of the methanogenic system

s, and it can be used

to evaluate the biodegradability efficiency of the organic substrates [128]. Methane yield can be

determined as the volum

e of the produced methane m

easured under normal conditions (i.e.,

0°C, 101.325 kPa) [129] per quantity of substrate fed into the reactor (e.g., in term

s of volatile

solid, total solid or fresh matter in the case of solid w

astes). The methane yield basically

depends on the organic content and the composition (e.g., fats, proteins, and carbohydrates) of

the substrate, which is biologically converted into m

ethane under anaerobic conditions.

How

ever, the attainable yield is affected by several other factors as well, such as the particle

size, quality, and adaptation of the inoculum, tem

perature, digestion time, m

ixing, availability

of nutrients, etc.

Methane yield can be determ

ined by theoretical as well as experim

ental methods (i.e., batch

and semi-continuous assays, as previously described in C

hapter 4). Theoretical methods are

usually based on the elemental or the com

ponent composition of the substrate. The B

uswell and

Müller’s [130] equation is based on the elem

ental composition (i.e., the carbon, hydrogen, and

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32 oxygen content) assum

ing the total stoichiometric conversion of the organic m

atter to methane

and carbon dioxide:

YC

H4 Theoretical =

Nevertheless, w

hen protein rich materials are present in the reactor, am

monia and hydrogen

sulphide are also released which m

ust also be taken into consideration, as it is shown by the

modified B

oyle’s [131] equation:

YC

H4 Theoretical =

Where 22.4 is the m

olar volume of a gas in liter per m

ol at standard conditions.

Furthermore, if the proportions of the biodegradable organic fractions (i.e., carbohydrates,

proteins, and lipids) are known, the predicted m

ethane yield can be estimated using the general

chemical form

ulas, e.g., 415, 496, and 1014 mLC

H4 gV

S-1 from

carbohydrates (C6 H

10 O5 ),

proteins (C5 H

7 O2 N

), and lipids (C5 H

104 O6 ), respectively [132].

Solid cattle slaughterhouse waste

In the present research, the methane potential of the solid cattle slaughterhouse w

aste was

investigated in mono-digestion by using the different operation m

odes (Papers I, III–V). There

are only a few studies that report the A

D of residues from

the solid cattle slaughterhouse

activities so far. Most of the studies using the slaughterhouse w

aste deal with pig [63, 133, 134]

and poultry [135, 136] animal by-products, and the processes are investigated under m

esophilic

conditions (Table 5.1). As show

n in Table 5.1, the methane yields of several types of anim

al

wastes from

the pig slaughterhouse activities determined in batch assays range from

230 to 620

mL gV

S-1 [134, 137], w

hile the methane yields from

the different slaughterhouse poultry by-

products including offal, blood, meat, and bone trim

ming w

astes are between 520 and 700 m

L

gVS

-1 [137-139].

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33 Table 5.1 Biodegradation of different anim

al wastes in anaerobic m

ono-digestion

Substrates T(°C)

Operation M

ode and conditions

Methane yield (Y

CH4 ) Degradation efficiency (%

) References

Slaughterhouse waste

Solid cattle 55

Batch (2L) 582-681 (m

L gVS-1 )

n.a Papers I, III, V

55

CSTR (3L), HRT= 25 days, O

LR=0.9 gVS L-1 d

-1 410 (m

L gVS-1)

n.a Paper IV

Solid cattle and pig

35 CSTR (2L), HRT= 30 days

60 (mL gVS

-1) 34.5 (VS red)

[61]

Solid and liquid pig/cattle m

ixtures

35 Batch (1L)

274-302 (mL gCO

D-1)

86-92 (COD red)

[64]

Iberian pig 38

CSRT (2L), HRT=23.5 days

17.84 (m3 m

-3Feed ) 78.59 (CO

D red) [63]

Pig 55

Batch (0.5 and 2L) 230-620 (m

L gVS-1)

50-100 (Y

CH4 /YCH4

Theoretical ) [134]

35

Batch (1.2L)

460 (mL gVS

-1) 76.6 (CO

D red) [137]

Poultry 35

Batch (1.2L)

580 (mL gVS

-1) 55.2 (CO

D red) [137]

35

CSTR (2L), HRT= 50 days, O

LR=0.8 gVS L-1d

-1 520-550 (m

L gVS-1)

n.a [139]

34

CSTR (3L), HRT= 50, 36, 25 days, O

LR=0.9, 1.16, 1.70 gVS L

-1d-1

600-700 (mL gVS

-1) 77-79 (VS red)

[138]

n.a: Not available; CSTR: Continuous stirring tank reactor; HRT: Hydraulic retention tim

e; COD: Chem

ical oxygen demand;

OLR: O

rganic loading rate In this thesis, the m

ethane yield of the solid cattle slaughterhouse waste (SB

) was obtained in

batch (Papers I, III, and V) and sem

i-continuous (Paper IV) operation, and the observed

values ranged between 410 and 681 m

L gVS

-1. A surface response representation from

a four-

factor mixture design show

s that among the individual fractions investigated, the highest

methane yield (i.e., 608 m

L gVS

-1) was obtained from

the SB. From

the plots, it is also shown

that the zones of high yield were located tow

ards the side of the triangle having SB as vertices

(Figure 5.1, in Paper III).

Figure 5.1 Surface response plots of methane yield displayed as a function of different m

ixture ratios of solid cattle slaughterhouse w

aste and other wastes (Paper III)

mLCH4 gVS-1

Manure

11

11

11

Manure

Various Crops

Solid cattle slaughterhouse w

aste

Solid cattle slaughterhouse w

aste

Organic fraction

of MSW

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34 Furtherm

ore, the biodegradability of the SB, in term

s of methane yield, w

as also investigated

using consecutive feedings in the batch assays to assess the response of the microbial

comm

unity under stress (i.e., substrate limitation) (Paper I) and non-stress (i.e., w

ithout

substrate limitation) (Paper V

) conditions. The results show that the stressed system

with a

longer period without feeding w

as able to start-up after an acclimation period of 21 days,

producing a greater amount of m

ethane than that obtained in the first feeding (i.e., 582 vs. 681

mL gV

S-1) (Figure 5.2).

Figure 5.2. Accumulated m

ethane production obtained during the mono-digestion of the solid cattle

slaughterhouse waste using consecutive feedings in cases of substrate lim

itation (▲) (Paper I) or w

ithout

substrate limitation ( ●

) (Paper V)

How

ever, when the sam

e amount of substrate as in the first feeding w

as added before the end

of the exponential phase (e.g., at day 25), strong inhibition was observed (Figure 5.2.). B

acteria

degrading lipids exhibit a slow grow

th rate [68]. This together with the possible form

ation of

the inhibitory compounds (e.g., N

H3 , LC

FAs) during the first feeding resulted in the system

not

being able to handle the second loading.

The AD

of SB during a sem

i-continuous operation for 101 days showed that the m

ono-

digestion failed under thermophilic conditions (55 ± 1°C

) (Paper IV). The average m

ethane

yield observed was 410 m

L gVS

-1 corresponding to 42% of the theoretical m

ethane yield. An

OLR

of only 0.9 gVS L

-1d-1 using H

RT of 25 days could be applied; under these conditions,

large instability in the methane production w

as observed (Figure 5.3). Total inhibition occurred

at day 58, and the methane production w

as completely stopped in agreem

ent with the results

obtained when using the second feeding procedure. A

nother important factor observed during

-100

100

300

500

700

010

2030

4050

6070

80

Methane yield [mL gVS-1 ]

Time [days]

Addition of 2nd feeding

With substrate lim

itation (Paper I)

Without substrate

limitation

(Paper V)

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35 the m

ono-digestion of the SB w

as the formation of foam

, which is highly related to the high

lipid (i.e., 175 g kg-1fresh m

atter ) and protein (i.e., 130 g kg-1fresh m

atter ) content in the feed [2].

Figure 5.3 Obtained m

ethane yield during the thermophilic (55°C) sem

i-continuous mono-digestion of SB

(Paper IV)

A

nimal m

anure, various crops, and the organic fraction of the municipal solid w

aste

The methane yield of anim

al manure (m

ainly cow and pig), crops, and the organic fraction of

the MSW

have been widely studied in the literature either in m

ono-digestion or in co-digestion

processes. Table 5.2 summ

arizes some results of the biodegradability obtained for these

materials during their anaerobic m

ono-digestion.

0 1 2 3

0

200

400

600

800

1 000

020

4060

80100

OLR [gVS L-1 d-1]

Methane yield [mL gVS-1]

Time [days]

Start-up period

HRT=25days 0.9 gVS L-1d

-1

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36 Table 5.2 Biodegradation of different agro-w

astes and municipal solid w

aste in anaerobic mono-digestion

Substrates T(°C)

Operation M

ode and conditions

Methane yield

(YCH4 )

Degradation efficiency (%

) References

Horse manure

55 Batch (118 m

L) 279 (m

L gVS-1)

n.a [81]

Cattle manure

55 Batch (118 m

L) 250 (m

L gVS-1)

n.a [81]

35

Batch (1L) 261(m

L gVS-1)

n.a [140]

35

Batch (1100mL)

148 (mL gVS

-1) 32 (Y

CH4 /YCH4

Theoretical ) [92]

Pig manure

35 Batch (250 m

L) 357 (m

L gVS-1)

n.a [82]

35

Batch (1L) 188 (m

L gVS-1)

n.a [140]

35

Batch (1100mL)

356 (mL gVS

-1) 69 (Y

CH4 /YCH4

Theoretical ) [92]

35

CSTR, HRT=30 days, O

LR=1.2 kgVS m-3d

-1 330 (m

L gVS-1)

36.6 (VS red) [82]

Mixture of anim

al m

anure (pig, cow,

horse)

55 Batch (2L)

384 (mL gVS

-1) n.a

Paper III

Various crops 55

Batch (2L) 422-504 (m

L gVS-1)

n.a Papers I, III

Rice straw

37 Batch (2 L)

226 (mL gVS

-1) 62 (Y

CH4 /YCH4

Theoretical ) [83]

55

Batch (2 L) 281 (m

L gVS-1)

74 (YCH4 /Y

CH4Theoretical )

[83]

55 Batch (118 m

L) 45 (m

L g-1Carbohydrates )

11 (YCH4 /Y

CH4Theoretical )

[141] W

heat straw

35 Batch (1100m

L) 145-195 (m

L gVS-1)

n.a [92]

Tomato processing

waste

38 CSRT (2L), HRT=8 days

5.76 (m3 m

-3Substrate ) 60.76 (CO

D red) [63]

Fruit and vegetable w

astes 35

CSTR ( 2L), HRT=30 days

0.2 (mL gVS

-1) 19.2

[61]

55

ASBR (2L) 278 (m

L gVS-1)

79 (VS red) [84]

Fruit wastes

35 Batch (135m

L) 0.18-0.73 (m

L gVS-1)

n.a [142]

Vegetable wastes

35 Batch (135m

L) 0.19-0.40 (m

L gVS-1)

n.a [142]

Organic fraction of

MSW

55

Batch (2L) 537-596 (m

L gVS-1)

n.a Papers I, III

35

CSTR (2.2m3),

HRT=14.5 days, O

LR=1.7 -3.9 kgVS m

-3d-1

248-403 (mL gVS

-1) 25-71 (VS red)

[143]

35

Batch (1L) 508 (m

L gVS-1)

n.a [144]

n.a: Not available; CSTR: Continuous stirring tank reactor; HRT: Hydraulic retention tim

e; COD: Chem

ical oxygen demand;

OLR: O

rganic loading rate In this thesis, the individual digestion of a m

ixture of animal m

anure, i.e., pig, cow, and horse

(Paper III), various crops (Papers I and III), and the organic fraction of the MSW

(Papers I

and III) was investigated at therm

ophilic conditions. The results showed that the m

anure and

the various crops were the individual fractions that contributed the least to the m

ethane yield

with values ranging from

384 and 422 mL gV

S-1, w

hile when using the organic fraction of

MSW

, 534 mL gV

S-1 m

ethane yield could be achieved (Figure 5.4, Paper III).

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37

Figure 5.4 Surface response plots of methane yield displayed as a function of different m

ixture ratios of manure,

various crops, and the organic fraction of MSW

(Paper III)

The methane potential of various crops and the organic fraction of M

SW w

ere also investigated

using consecutive feeding under stress conditions (substrate limitation, Paper I). The results

showed that after a long adaptation period (i.e., 14 days), increased m

ethane yields were

observed compared to those observed from

the first feeding in both batches. This was ascribed

to the fact that specific groups of microorganism

s were able to adapt to the substrates during

the first feeding, improving its biodegradation efficiency. These substrates play an im

portant

role in the stability of large-scale biogas plants, as they help to improve the buffer capacity of

the system, as w

ell as contribute to a balanced nutrient supply required for the microorganism

s.

The methane yield of the anim

al manure reported in the literature varies to a large extent (i.e.,

140 – 400 mL gV

S-1) since it is affected by several factors, as discussed earlier [81, 82, 92,

140]. Other im

portant aspects are the amount and type of bedding m

aterials used and the

degradation process that occurs under pre-storage conditions [91, 92]. The higher proportions

of carbohydrates and lignin present in the cattle and horse manure cause them

to slowly

degrade in comparison w

ith the pig manure [81].

Meanw

hile, larger proportions of proteins are present in the pig manure, increasing its m

ethane

potential in

comparison

with

the cattle

manure

[92]. M

oreover, pretreatm

ent of

the

lignocellulosic fraction present in the cattle and horse manure can be applied to increase its

anaerobic biodegradability [81].

5.3 Specific methanogenic activity

The specific methanogenic activity (SM

A) is used to estim

ate the capacity of the biomass to

produce methane from

a given substrate during a unit of time.

mLCH4 gVS-1

700

600

500

4001

1

1M

anure

Organic fraction

of MSW

Various Crops

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38 It is expressed as gC

OD

mLC

H4 gV

Sinoculum

-1d-1, and it is calculated from

the slope of the

exponential part of the cumulative m

ethane production curve, according to the following

equation:

where R

is the methane production rate (m

L d-1), determ

ined as a mean slope of the m

aximum

production activity in the methane production curve; C

F is a conversion factor, converting the

produced methane to equivalent C

OD

and is equal to 350 mLC

H4 gC

OD

-1 [145]; V is the

effective volume (m

L) and VS is the concentration of the volatile solids (g m

L-1) of the

inoculum.

The specific methanogenic activity is a good control param

eter for monitoring the stability of

the process, as methane production is an excellent indicator of the activity of the m

ethanogens

[128]. Higher values of SM

A m

ean that the system is able to act on a higher m

ethane

production rate per active biomass in the inoculum

. In this research, the response of the

methanogenic biom

ass to a second feeding (consecutive feeding in batch assays) was evaluated

by the use of SMA

, both under substrate limitation (Paper I) and w

ithout the substrate

limitation (Paper V

).

Slaughterhouse waste, various crops, and the organic fraction of m

unicipal solid waste

In the literature, no values of SMA

for the mono-digestion of SB

, VC

, and the organic fraction

of MSW

were found for com

parison.

Table 5.3 shows the SM

A values as w

ell as the lag phase observed during each feeding. The

results were in accordance w

ith those observed regarding the methane yield, as presented in the

previous section.

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39 Table 5.3 Specific m

ethanogenic activity (gCOD

mLCH4 gVS

inoculum-1 d

-1) a in thermophilic reactors (55ºC) treating the

solid cattle slaughterhouse waste, various crops, and the organic fraction of M

SW using consecutive feeding in

batch assays (Papers I and V)

1

st Feeding 2

nd Feeding Paper

With substrate lim

itation

Slaughterhouse waste

0.06±0.00 0.07±0.00

I O

bserved lag phase (days) 0

21

Various crops 0.11±0.00

0.05±0.01 I

Observed lag phase (days)

0 14

M

unicipal solid waste

0.13±0.01 0.04±0.01

I O

bserved lag phase (days) 0

14

Without substrate lim

itation

Slaughterhouse waste

0.07±0.01 0.00±0.00

V Predicted lag phase b

6.1 n.d

n.d: N

ot determined; a M

ean values from three independent vessels ± standard deviation calculated from

the exponential part of the cum

ulative methane production curve; b Predicted lag phase determ

ined by the modified Gom

pertz model

Furthermore, in the case of SB

, the semi-continuous m

ono-digestion resulted in poor conditions

for the

growth

of hydrogenotrophic

methanogens,

i.e., M

ethanomicrobiales

and

Methanobacteriales w

ith an accumulation of hydrogen in the system

(Paper IV). O

n the other

hand, the biodegradation of VC

and the organic fraction of MSW

showed that the SM

A

decreased by 54% and 69%

, respectively, even though a shorter lag phase (i.e., 14 days, Table

5.3) was observed in com

parison with that obtained w

hen SB w

as investigated.

5.4 Kinetics

Understanding the kinetics of anaerobic degradation for a given m

aterial plays an important

role when designing and operating the biogas plants [16]. The rate of the degradation is based

on the rate of the biochemical reactions in the process, w

hich also depend on both the

environmental and the operational factors. In the literature, a num

ber of relationships can be

found ranging from sim

ple equations [146, 147] to more com

plex models [13, 148] describing

the biological process. Most of the kinetics m

odels reported in the literature focus on the rate of

substrate consumption [16] and the m

aximum

specific growth rate of m

icroorganisms [149].

Nevertheless, the m

ethane production rate is a good indicator to describe the kinetics of the

system in a sim

ple way due to its direct correlation w

ith the substrate consumption.

With the objective to get a quick evaluation of the biodegradation capacity of several different

substrates in the batch assays, mathem

atical models are w

idely used. When solid com

plex

materials are treated, the hydrolysis step is expected to be the rate-lim

iting step in the

degradation process; hence, first order kinetic models are w

idely accepted for obtaining the

kinetic parameters [146, 150, 151].

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40 The first order m

odel provides a simple w

ay to compare the process perform

ance when treating

the different materials under practical conditions. It is based on the availability of the substrate

as a limiting factor and the correlation betw

een the substrate consumption and the biogas

production [151]. The model is expressed as show

n in Equation 2 below:

How

ever, the degradation of certain substrates does not follow this sim

ple first order model

since they are difficult to digest. In these cases, other models like the m

odified Gom

pertz

model can be applied [147, 152, 153]. This m

odel is a type of mathem

atical model based on a

sigmoid function, w

here the degradation profile shows a delay at the start of the gas production,

taking into account a lag phase period. Hence, this m

odel is useful to determine both the

maxim

um m

ethane production rate (Rm ) and the length of the lag phase (λ). The inclusion of

the lag phase as a parameter is im

portant in order to investigate how m

uch time is needed for

the biomass to adapt to the substrate and then to separate this tim

e period from the tim

e needed

for the biodegradation itself. The modified G

ompertz m

odel can be expressed as it is shown in

Equation 3:

Another em

pirical mathem

atical model is the C

hapman m

odel, which predicts the m

ethane

yield (Y), the specific m

ethane production rate (rsCH

4 ) as well as the boundary tim

e in which the

difference in the methane production betw

een the two consecutive days is no m

ore than 1% of

the methane production up to this tim

e, called the Grenz tim

e (tGrenz ) [154]. The integrated form

of the Chapm

an model is expressed according to Equation 4:

The specific methane production rate and the boundary tim

e are expressed by Equations 5 and

6, respectively:

In these models, Y

(t) represents the accumulated m

ethane potential at a given time (m

L gVS

-1);

Ym

ax is the maxim

um m

ethane potential accumulated at an infinite digestion tim

e (mL gV

S-1);

k0 is the specific rate constant of the overall process (often called the hydrolysis constant) given

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41 per day; R

m is the maxim

um m

ethane production rate (mL gV

S-1d

-1), e = 2.7183, λ is the lag

phase (days); t is the digestion time (days); rsC

H4 is the specific m

ethane production rate (mL

gVS

-1d-1); tG

renz is the time during w

hich the daily methane production of tw

o consecutive days

differ less than 1%; and b and c are the m

athematical coefficients obtained from

the fitting

curve.

In this work, the first-order m

odel (Paper I), the Chapm

an model (Papers II and III), and the

Gom

pertz model (Paper V

) were used to evaluate the biodegradability of the SB

and agro-

wastes individually as w

ell as in the co-digestion processes. Although these are very sim

ple

models, they provide useful inform

ation applicable for process design such as reactor size,

OLR

, HR

T, and process performance in term

s of the expected methane yield.

Solid cattle slaughterhouse waste and agro-w

astes

Depending on the tem

perature, quality, and adaptation of the inoculum and the biodegradability

of each individual substrate, the kinetic parameters describing the degradation of the solid

wastes exhibit a w

ide range of variations (Table 5.5). Am

ong the individual waste fractions

studied in the present thesis, the SB displayed a low

er degradation rate compared to that of the

other waste stream

s investigated. The first-order kinetics constant (k0 ) and the specific m

ethane

production rate (rsCH

4 ) showed the low

est values for the SB (i.e., 0.09 d

-1 and 13 mL gV

S-1d

-1)

followed by V

C (i.e., 0.29 d

-1 and 37 mL gV

S-1 d

-1), and the organic fraction of MSW

(i.e.,

0.33 d-1 and 45 m

L gVS

-1d-1) (Table 5.5).