improved understanding of anaerobic digester processes by stable isotope techniques

202 Pages·2015·6.53 MB·English

by  Daniel Girma Mulat

IMPROVED UNDERSTANDING OF ANAEROBIC DIGESTER
PROCESSES BY STABLE ISOTOPE TECHNIQUES
DANIEL GIRMA MULAT
PhD THESIS . SCIENCE AND TECHNOLOGY . 2015

Aarhus University
Department of Engineering
Science and Technology
Hangøvej 2
8200 Aarhus N
Denmark
Preface
This PhD dissertation has been submitted to Aarhus University in partial fulfillment of the
requirements of the degree Doctor of Philosophy at the graduate school of science and technology
(GSST). My main supervisor is Anders Feilberg, Associate Professor at Department of Engineering,
Aarhus University. My co-supervisors are Anders Peter S. Adamsen, Senior Scientist and Alastair
James Ward, Assistant Professor at Department of Engineering, Aarhus University. This study was
conducted from December 1st 2011 until January 29th 2015 at the Department of Engineering,
Aarhus University, located at Research Centre Foulum, Denmark. I also spent about 5 months
abroad between January 2012 and June 2012 at Department of Environmental Microbiology,
Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany in collaboration with
Department of Biochemical Conversion, Deutsches Biomasseforschungszentrum (DBFZ), Leipzig,
Germany. I conducted an experiment at UFZ and DBFZ aimed at investigating the effect of
different operating condition on process performance, isotope signatures, methanogenic pathways
and microbial community composition in lab-scale continuous tank reactor (CSTR) and have gained
hands on experience with operating CSTR, isotope ratio mass spectrometer (IRMS) and some
molecular biology techniques. My immediate supervisors were Dr. Marcell Nikolausz, Scientist at
UFZ and Dr. H. Fabian Jacobi, Head of process monitoring and simulation group at DBFZ.

The primary focus of this Ph.D. project was the development of stable isotope techniques and its
application for better understanding of key intermediates (acetate and hydrogen) and metabolic
pathways involved during conversion of organic material to methane in biogas reactor. This thesis
consists of ten chapters. Chapter 1 is a general introduction about the motivation for conducting this
PhD study and its objectives. A literature review on biogas technology as a source of renewable
energy, biochemistry of anaerobic digestion and operating conditions regulating biogas processes is
presented in chapter 2. General methods that were used and developed in this PhD study are
presented in chapter 3. The key results of the experimental work are presented in chapters 4 to 9 as
published papers, manuscripts under review and manuscripts in preparation. In chapter 10, the
general findings of this thesis are summarized and discussed as well as conclusions and future
perspectives are provided.

Some of the results of this PhD study were presented at two international conferences and one
national conference. I gave oral presentations at International Conference on Biogas Microbiology
ICBM, held on 10th to 12th June 2014 in Uppsala, Sweden and at International Conference on
Anaerobic Digestion, BiogasScience 2014, held on 26th to 30th October 2014 in Vienna, Austria.
Poster was presented at a local conference Energy and Environment for the future-sustainable
energy for a fossil free society and environmental friendly technologies, November 24- 25,
Copenhagen, Denmark. This thesis is based on the following published papers, manuscripts
submitted and manuscripts in preparation.

Peer-Reviewed Journal Articles

1. Mulat, D. G., Ward, A. J., Adamsen, A. P. S., Voigt, N. V., Nielsen, J. L., & Feilberg, A.
(2014). Quantifying contribution of synthrophic acetate oxidation to methane production in
thermophilic anaerobic reactors by membrane inlet mass spectrometry. Environmental
science & technology 48(4), 2505-2511.
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2. Mosbæk, F., Kjeldal, H., Mulat, D. G., Albertsen, M., Ward, A. J., Feilberg, A. & Nielsen,
J. L., (2015). Acetate oxidizing microbial communities during acid accumulation in
anaerobic digestion. In preparation for peer-reviewed journal.
3. Mulat, D. G., Mosbæk, F., Ward, A. J., Polag, D., Greule, M., Keppler, F., Nielsen, J. L., &
Feilberg, A. (2015). Effect of exogenous hydrogen addition on process performance,
methanogenesis and homo-acetogenesis pathways during an in situ biogas upgrading. In
preparation for peer-reviewed journal.
4. Mulat, D. G., & Feilberg, A. (2014). GC/MS method for determining carbon isotope
enrichment and concentration of underivatized short-chain fatty acids by direct aqueous
solution injection of biogas digester samples. Submitted to Talanta. Under review.
5. Mulat, D. G., Jacobi, F., Feilberg, A., Adamsen, A. P. S., & Nikolausz, M. (2015).
Changing feeding regimes to demonstrate flexible biogas production: effects on process
performance, microbial community structure and methanogenesis pathways. In submission
for Bioresource Technology Journal.
6. Mulat, D. G., Feilberg, A., Jacobi, F., & Nikolausz, M. (2015). Stable isotope techniques as
a tool for process monitoring of biogas reactors operating under different condition. In
preparation for peer-reviewed journal.

Conference and poster presentations

7. Mulat, D. G., Ward, A. J., Adamsen, A. P. S., Voigt, N. V., Nielsen, J. L., & Feilberg, A.
(2014). Application of online membrane inlet mass spectrometry (MIMS) combined with
isotope labelling of substrates for quantifying methanogenesis pathway in anaerobic
reactors. Paper presented at International Conference on Biogas Microbiology ICBM, June
10-12, Uppsala, Sweden.
8. Mulat, D. G., Jacobi, F., Feilberg, A., Adamsen, A. P. S., Richnow, H. H., & Nikolausz, M.
(2014). Shifts in methanogenic pathways in response to change in substrate feeding pattern
studied by stable isotope techniques. Conference proceedings for the International
conference on anaerobic digestion, BiogasScience 2014, October 26-30, Vienna, Austria.
9. Mulat, D. G., Ward, A. J., Adamsen, A. P. S., Voigt, N. V., Nielsen, J. L., & Feilberg,
A.(2014). Application of on-line mass spectrometry with stable isotope pairing to study the
pathway of methane production from acetate in anaerobic reactor. Poster session at Energy
and Environment for the future-sustainable energy for a fossil free society and
environmental friendly technologies, November 24- 25, Copenhagen, Denmark.

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Acknowledgements
I would like to thank God for blessing my family, giving me the strength, guidance and resources to
complete my thesis. I am grateful for the numerous people who have helped along my journey here
at Aarhus University and made possible in completing this study. A special thanks goes to my main
PhD supervisor, Associate Professor Anders Feilberg for sharing his experience and helpful
suggestion as well as inspiration and continued encouragement throughout my PhD study. I would
also like to thank my PhD co-supervisors: Senior Scientist Anders Peter S. Adamsen and Assistant
Professor Alastair James Ward for their support and scientific advice.

The project would not have been possible without financial support from the Danish Strategic
Research Council (Grant No. 10-093944). I express gratitude to the external project partners:
Professor Jeppe Lund Nielsen and PhD student Freya Mosbæk for the help with the microbial
community analysis of our samples; Sabine Lindholst for the great help with MIMS and micro-GC
instruments; Dr. Niels Vinther Voigt for the great discussion about MIMS and hydrogen addition
experiment set up; Dr. Daniela Polag, Dr. Markus Greule and Prof. Frank Keppler for analyzing the
isotope composition of our biogas samples. My gratitude also goes to those who supported my
scientific work and assist me with administrative stuffs during my stay at the Department of
Environmental Microbiology, Helmholtz Centre for Environmental Research (UFZ), Leipzig,
Germany in collaboration with Department of Biochemical Conversion, Deutsches
Biomasseforschungszentrum (DBFZ), Leipzig, Germany. In particular, I would like to thank my
immediate supervisors Dr. Marcell Nikolausz and Dr. H. Fabian Jacobi for the fruitful scientific
discussion and great help during my stay in Leipzig; Dr. Sabine Kleinsteuber for allowing me to
join her research group meeting and work in the group’s lab; Dr. Hans-Hermann Richnow for
allowing me to work in his isotope biogeochemistry lab; PhD students Zuopeng Lv and Athaydes
Francisco Leite Junior as well as the lab technicians at UFZ and DBFZ for their invaluable support.
Thanks to the many supports from the current and former lab technicians at the biogas plant in
Foulum: Heidi Grønbæk Christiansen, Britt Amby Malthesen, Claudia Nagy and Patricia De Sousa
as well the Foulum biogas plant manager Mogens Møller Hansen.

Also many thanks to our research group members for creating such a wonderful working and
learning environment. The support from the head and secretary of our section, the PhD partners at
the Graduate School of Science and Technology (GSST) is greatly appreciated and special mention
to Anja Torup Hansen and Morten Dam Rasmussen. I am indebted to all my friends who have been
encouraging along the way and special mention to Setegn for our good friendship and all awesome
dinners we have had together in Foulum.

I want to thank my family. Many thanks to my wife Fre without whose love, encouragement,
understanding and support I would not have completed this work and to my baby girl Yohanna for
being part of our life, source of inspiration and energy.

Lastly, and most importantly, I wish to thank my mother, brothers and close relatives who have
supported me and kept me focused with their love, prayers and words of encouragement. I am
dedicating this thesis to my mom Itenesh who raised me, supported me, taught me and love me.

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Abstract
Anaerobic digestion (AD) of organic matter to methane-rich biogas is carried out by diverse
consortia of anaerobic bacteria and archaea for the purpose of waste management and renewable
energy production. However, the advantages of AD for treating organic wastes have not been fully
realized. Full-scale biogas plants are often operated at suboptimal organic loading rates (OLR) to
avoid process imbalance and failure of the plants. This shows that the process is still far from
optimized due to incomplete process understanding. Developing a comprehensive understanding of
biogas process is the key to employing appropriate strategies that allow stable operation of biogas
plants at optimum OLR, which in turn increases productivity and economy. Therefore, research
aimed at generating in-depth knowledge of the degradation mechanisms of key intermediates and
most important methanogenic pathways for the formation of biogas in AD is highly desired. In this
regard, the main focus of this thesis was the development and application of stable isotope
techniques for better understanding of biogas process. The specific objectives of the PhD study
were: to develop an online membrane inlet quadrupole mass spectrometry (MIMS) methodology for
monitoring of the isotopic distribution of CO and CH in AD to be used in combination with
2 4
isotopically labeled substrates; to quantity the relative contribution of acetoclastic methanogenesis
(AM) and syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis pathway
(SAO-HM) to total CH production from degradation of acetate; to investigate the effect of
4
exogenous hydrogen addition on process performance, methanogenesis, homo-acetogenesis and
microbial community structure; to develop GC/MS method for simultaneous determination of the
concentration of underivatized volatile fatty acid (VFA) and isotope ratio of underivatized acetate
by direct injection of aqueous biogas digester samples; and to investigate possible use of stable
isotope techniques as a tool for monitoring the actual state of biogas process and as an early
warning tool to process instability.
In this study, the experiments involved lab-scale continuous stirred tank reactors (CSTRs) and batch
incubation with 13C labeled tracer substrates and specific inhibitor to acetoclastic methanogens. The
stable isotope composition of products and reactants at natural abundance and 13C enriched
substances were measured and molecular biology techniques and basic analytical methods were
used to support the results of the isotope analysis.
An online MIMS method was developed to trace the incorporation of 13C into the produced CO
2
and CH in real time when incubated with [2-13C]acetate in a thermophilic anaerobic reactor. This
4
novel approach was applied for quantification of the relative contribution of SAO-HM to methane
production from acetate, which was demonstrated to reach a high degree of contribution. Protein
based stable isotope probing (protein-SIP) and metagenome analysis showed that peptides from the
bacteria class Clostridia, the hydrogenotrophs Methanoculleus and the mixotrophic
Methanosarcina were labelled with 13C during degradation of high concentration of 13C labeled
acetate (100 mM), indicating Clostridia possibly oxidizes acetate to CO in syntrophic association
2
with the hydrogenotrophs. Another very simple, accurate, reproducible and rapid method based on
GC/MS was developed for determining both the isotope enrichment of acetate and concentration of
underivatized VFA in a biogas digester sample by direct liquid injection of acidified aqueous
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samples. As an example of application of this method to a biogas process, it was demonstrated that
a stable isotope tracer experiment in combination with tracer-to-tracee ratio (TTR) determination by
the GC/MS method proved that carbon dioxide was reduced to acetate under high H partial
2
pressure, indicating the activity of homoacetogens. The results of exogenous H gas addition for an
2
in situ biogas upgrading showed that all the added H in the presence of stoichiometric amount of
2
CO was almost completely utilized and the methane content of the biogas reached up to 90% with
2
concomitant decrease in the CO content. Unlike the control reactors, the degradation of acetate and
2
other VFA decreased in the H reactors and finally accumulated. Acetate degradation resumed after
2
the concentration of H in the reactor headspace decreased by flushing with helium. The observed
2
lower carbon isotope fractionation between CO and CH in the H reactors is possibly explained by
2 4 2
the differential reversibility concept, indicating exogenous H addition may have led to high H
2 2
concentration within micro-aggregates of methanogens. In addition, it was shown that different
operating conditions (change in feeding interval and continuous increase in OLR) had influenced
the process performance, methanogenesis pathways and bacterial community composition in lab-
scale CSTRs fed with distillers dried grains with solubles (DDGS). Unlike shorter feeding interval
(every 2 hours), longer feeding interval (daily and every 2 days) led to a dynamic process, as
depicted in the short term changes of biogas production rate, biogas composition (CH , CO and
4 2
H ), isotope composition of methane (δ13C-CH and δD-CH ), total VFA, acetate and propionate.
2 4 4
The δ13C-CO remained relatively stable under different feeding intervals during steady state
2
operation but changed slightly during an increase in OLR. Longer feeding interval allows the
flexibility to produce more biogas at times of high energy demand due to the possibility to increase
production shortly after a feeding event whereas feeding did not change the biogas production rate
in the CSTR fed at shorter intervals. Interestingly, the CSTRs fed at longer intervals when
compared to those fed at shorter intervals, demonstrated significantly higher methane yield by about
14% and were less susceptible to stress condition. The bacterial community structure varied
between CSTRs fed under different feeding intervals whereas methanogens remained stable with
higher abundance of the genera Methanosarcina and Methanoculleus. Our observation of the
dominating methanogens community was supported with isotope analysis, indicating HM and AM
contributed almost equally to the produced methane from each feeding event. In addition, among
the studied process monitoring tools, a combination of parameters based on the measurement of the
isotope composition of CH and CO at natural abundance, biogas production rate and biogas
4 2
composition would indicate the actual state and performance of the process as well as process
imbalance at early stage.
In conclusion, the measurement of the stable isotope composition of CH , CO and acetate
4 2
improved our understanding about methanogenesis, homo-acetogenesis and the degradation
mechanisms of key intermediates in AD. The dominating role of SAO-HM and the abundance of
the Methanosarcina in biogas reactors suggested that a re-evaluation of biogas process optimization
and operating conditions should be employed with the consideration of the importance of the SAO-
HM and the Methanosarcina in AD.

v
Dansk Resume
Anaerob nedbrydning af organisk stof til metan-rig biogas foregår ved hjælp af flere grupper af
anaerobe bakterier og arkebakterier for at håndtere biprodukter og producere biogas. Hidtil er
fordelene ved anaerob nedbrydning ikke blevet fuldt ud udnyttet, da fuldskala biogasanlæg ofte
køres lidt under det optimale indfødningsniveau for at undgå procesubalance og reaktornedbrud.
Udvikling af en udtømmende forståelse af biogasprocessen er nøglen til at anvende driftsstrategier
der sikrer en stabil drift ved optimal indfødning, og dermed forøge produktivitet og lønsomhed. Det
betyder at det er vigtigt med forskning der sikrer en dybere forståelse af omsætning af
nøglemellemprodukter og produktion af metan i biogasreaktoren. Hovedformålet med denne
afhandling var at udvikle og anvende stabile isotopteknikker for at få en dybere forståelse af
biogasprocessen. Mere specifikt var formålet at udvikle en online membran-inlet
massespektrometrisk (MIMS) metode til at monitere fordelingen af CO og CH -isotoper i
2 4
biogasprocessen ved anvendelse af substrater mærket med stabile isotoper; at kvantificere de
relative bidrag fra den acetoklastiske metandannelse (AM) og den syntrofe acetatoxidation koblet
med hydrogenotrof metandannelse (SAO-HM) i biogasprocessen; at undersøge effekten af ekstern
tilførsel af hydrogen på metandannelsen og strukturen af de mikrobielle samfund; at udvikle en GC-
MS metode for samtidig bestemmelse af koncentrationer af flygtige fede syrer (VFA) og
isotopforholdet ved direkte injicering af biogasvæske; og at undersøge mulig anvendelse af stabile
isotopteknikker til udvikling af et værktøj til at monitere aktuel tilstand af biogasprocessen og
forudsige kommende procesproblemer.

Denne afhandling beskriver forsøg i laboratorie-skala med kontinuerligt omrørte reaktorer (CSTR)
og batch inkuberet med 13C-mærket substrat og specifikke inhibitorers effekt på acetoklastisk
metandannelse. Fordelingen af stabile isotoper i reaktanter og produkter af naturligt forekommende
og 13C-berigede substrater blev målt og understøttet af molekylærbiologiske teknikker og basale
analytiske metoder. En online MIMS-metode er blevet udviklet til at spore indbygning af 13C i den
producerede CO og CH i realtime ved inkubering med [2-13C]acetat i en termofil anaerob reaktor.
2 4
Denne nye metode blev anvendt til at kvantificere den relative fordeling af SAO-HM i forhold til
produktion fra acetat. Det blev demonstreret at førstnævnte kunne nå en høj andel. En
proteinbaseret stabil isotop-metode (protein-SIP) and metagenom-analyse viste at peptider fra
bakteriegrupper Clostridia, den hydrogenotrofe Methanoculleus og den mixotrofe Methanosarcina
blev mærket med 13C ved omsætning af høje koncentrationer af 13C-mærket acetat (100 mM). Det
indikerer at Clostridia muligvis oxiderer acetat til CO i syntrofisk samarbejde med de
2
hydrogenotrofe bakterier.

En anden enkel, nøjagtig, reproducerbar og hurtig metode baseret på GC-MS blev udviklet for at
bestemme både den isotopiske berigelse af acetat og koncentrationer af uderivatiseret VFA i en
biogas reaktorprøve ved direkte injicering af forsurede væskeprøver. Som et eksempel på
anvendelse af denne metode i en biogasproces blev det demonstreret i en stabil isotop sporstof-
forsøg at CO blev reduceret til acetat under høje H partieltryk, hvilket indikerer homoacetogen
2 2
aktivitet. Tilførsel af ekstern H i en in situ opgradering viste at alt tilsat H ved tilstedeværelse af
2 2
støkiometrisk mængde af CO blev næsten komplet forbrugt, og at metanindholdet i biogassen
2
kunne nå op på en koncentration på 90% med tilsvarende reduktion i CO -indholdet. Sammenlignet
2
med en kontrolreaktor uden tilførsel af ekstern H vistes en lavere omsætning af acetat og andre
2
VFA og som senere akkumuleredes i reaktoren med tilført H . Acetatomsætning blev genoptaget
2
efter at H i gasfasen blev fjernet ved at skylle med helium. Den observerede lave kulstof-isotop
2
fraktionering imellem CO og CH i H -reaktoren kan muligvis forklares med konceptet differentiel
2 4 2
vi
reversibilitet, som indikerer at ekstern tilførsel af H kan have ledt til høje H -koncentrationer inde
2 2
i mikro-aggregater af metanogene bakterier. Det blev også vist at forskellige procesbetingelser
(ændring i indfødningsinterval og kontinuerlig forøgelse af indfødt organisk stof) påvirkede
processen og fordelingen af de mikrobielle samfund i laboratorieskalaforsøg.

En kontinuerlig omrørt reaktor (CSTR) blev fodret med DDGS (tørret kornbærme; distillers dried
grains with solubles). Modsat kortere indfødningsintervaller (hver 2. time) ledte længere
indfødningsintervaller (dagligt eller hver 2. dag) til en mere dynamisk proces påvist ved hurtige
skift i biogasproduktionsrater, fordelingen af CH , CO og H , isotopfordelingen af (δ C-CH og
4 2 2 13 4
δD-CH ) samt total VFA, acetat og propionate. δ C-CO forblev relativt stabilt under forskellige
4 13 2
indfødningsintervaller ved stabile driftsforhold, men ændredes lidt ved højere indfødningsmængder.
Længere indfødningsintervaller tillod en fleksibilitet til at producere mere biogas på tidspunkter
med høj efterspørgsel, da produktionen stiger kort efter en indfødning, hvorimod indfødning med
kortere intervaller ikke ændrede biogasproduktionen i en CSTR-reaktor. CSTR med længere
intervaller mellem indfødning af substrat viste en signifikant højere metanudbytte på 14%
sammenlignet med tilsvarende indfødning med kortere intervaller og var mindre sårbar overfor
stressforhold. Strukturen af bakteriesamfundene varierede imellem CSTR indfødt med forskellige
intervaller, hvorimod de metanogene bakterier forblev stabile med høje mængder af slægterne
Methanosarcina og Methanoculleus. Vores observation af de dominerende metanogene samfund
blev understøttet med isotopanalyser som indikerede, at de hydrogenotrofe og acetatotrofe
metanogene bakterier bidrog næsten ligeligt til den producerede CH fra de enkelte indfødninger.
4
Ud fra de anvendte teknikker vil det være muligt at udvikle en metode til at indikere aktuel status og
forudsige begyndende ustabilitet i biogasprocessen.

Det konkluderes at målinger af sammensætningen af de stabile CH , CO og acetat har forøget
4 2
vores forståelse af dannelse af metan og homo-acetat og omsætningsmekanismer for de vigtigste
mellemprodukter i biogasprocessen. Den dominerende rolle af SAO-HM og tilstedeværelse af
Methanosarcina i biogasreaktorer antyder at en revurdering af metoder til at optimere og køre
biogasreaktorer bør foretages med fokus på betydningen af SAOHM og Methanosarcina i
biogasprocessen.

vii
Lists of abbreviations
AD Anaerobic digestion
AM Acetoclastic methanogenesis
AU Aarhus University
BP Base pair
CF Continuous flow
CRDS Cavity ring-down spectroscopy
CSIA Compound specific isotope analysis
CSTR Continuous stirred tank reactor
DBFZ Deutsches Biomasseforschungszentrum
DDGS Distillers dried grains with solubles
DI Dual-inlet
DNA Deoxyribonucleic acid
DNA-SIP Deoxyribonucleic acid stable isotope probing
EA/IRMS Elemental analyzer isotope ratio mass spectrometry
EI Electron impact
f The fraction of CH produced from the reduction of CO
mc 4 2
GC Gas chromatography
GC/C/IRMS Gas chromatography combustion isotope ratio mass spectrometry
GC/MS Gas chromatography mass spectrometry
GSST Graduate school of science and technology
HM Hydrogenotrophic methanogenesis
HRT Hydraulic retention time
HYCon HYdrogen Control for optimization of methane production from livestock waste
ICBM International Conference on Biogas Microbiology
IRMS Isotope ratio mass spectrometry
KE Kinetic energy
LC-MS/MS Liquid chromatography tandem mass spectrometry
LF Longer feeding interval
mcrA Alpha subunit of methyl coenzyme M reductase gene
MFC Mass flow controller
MIMS Membrane inlet quadrupole mass spectrometry
MRC Methyl coenzyme M reductase
mRNA Messenger ribonucleic acid
nMDS Non-metric multidimensional scaling
OLR Organic loading rate
PCR Polymerase chain reaction
Protein-SIP Protein based stable isotope probing
QMS Quadrupole mass spectrometer
Rd1 Every day fed reactor
Rd2 Every 2 d fed reactor
Rh2 Every 2 h fed reactor
RNA Ribonucleic acid
rRNA Ribosomal ribonucleic acid
SAO Syntrophic acetate oxidation
SAOB Syntrophic acetate-oxidizing bacteria
SBP Specific biogas production
viii
SCFA Short chain fatty acids
SF Shorter feeding interval
SIP Stable isotope probing
SMP Specific methane production
TAN Total ammonia nitrogen
TDLAS Tunable diode laser absorption spectroscopy
TIC Total inorganic carbon
T-RFLP Terminal restriction fragment length polymorphism
T-RFs Terminal restriction fragments
TS Total solid
TTR Tracer-to-tracee ratio
UFZ Helmholtz Centre for Environmental Research
VFA Volatile fatty acids
V-PDB Vienna Pee Dee Belemnite
VS Volatile solid
V-SMOW Vienna Standard Mean Ocean Water
VSR Volatile solid reduction
WP Work package
α Isotope fractionation factor
δ13C-CH 13C isotopic signature of CH
4 4
δ13C-CO 13C isotopic signature of CO
2 2
δD-CH Hydrogen isotope signature of CH
4 4
δ Acetate-derived CH through acetoclastic methanogenesis
ma 4
δ CO -derived CH through hydrogenotrophic methanogenesis
mc 2 4
ε Isotope enrichment

ix