workshop on abe production and co /h fermentation · 15:00 -15:20 development of a high -pressure...
TRANSCRIPT
Workshop on ABE production and
CO2/H2 fermentation
10th and 11th of November 2016
BOKU University of Natural Resources and Applied Life Science
Vienna, Austria
www.clostridia.boku.ac.at
2
Thursday 10th November 2016
ABE — acetone, butanol and ethanol production
09:00-09:30 Welcome & Introductions Lucy Montgomery, Günther Bochmann, Ullrich Stein
VFA production
09:30-09:50 Evolving Anaerobes by Adaptive and Molecular Methods for Volatile Fatty Acids Production in Lignocellulosic Hydrolysates.
Ivan Baumann
09:50-10:10 Maximizing the production of butyric acid as a precursor for ABE.
Ullrich Stein
10:10-10:30 Quantitative assessment of pure culture dark fermentative biohydrogen production.
Ipek Ergal
10:30 coffee break
ABE Bioprocess
10:50-11:10 Increasing the productivity of ABE fermentation with electrobiotechnology and integrated product removal with solvent impregnated particles.
Nils Tippkötter
11:10-11:40 Downstream processing of ABE fermentation: Separation of acetone, butanol, ethanol and hydrogen from ABE process streams.
Michael Harasek
11:40-12:00 WASTE2FUELS – Sustainable production of next generation biofuels from waste streams.
Walter Wukovits
12:00-12:20 Complete fermentation of pentose and hexose mixtures by catabolite repression mutants of Clostridium acetobutylicum ATCC 824.
Johannes Müller
12:20 Lunch
13:00-14:20 Guided poster session Hans Marx, Stefan Pflügl, Hannes Rußmayer, Kateryna Wöss, Benedikt Kleibl, Maryna Vasylkivska, Marek Drahokoupil, Florian Gattermayr
14:20-14;40 Mathematical modelling supported optimisation of ABE fermentation and improvement of its sustainability.
Sergej Trippel
14:40-14:50 ABE production of different Clostridial strains and influence of supplements.
Seyed Mohsen Abbasi Hosseini
14:50 coffee break
15:20 Guided discussion
17:00 End
3
Friday 11th November 2016
Gas fermentation
09:00-09:30 Welcome & Introductions Lucy Montgomery, Lydia Rachbauer
Acid and solvent production
09:20-10:00 Syngas/waste gas fermentation to ethanol and higher alcohols.
Christian Kennes
10:00-10:30 Selective acetone and isopropanol production using acetogenic bacteria.
Frank Bengelsdorf
10:30 coffee break
10:50-11:10
Moorella thermoacetica, a thermophilic model organism creating value from waste gasses.
Torbjørn Ølshøj Jensen
11:10-11:30 Elevated pressure bioreactors for gas transfer enhancement in gas fermentation.
Rehan Shah
11:30-11:40 Acetate production from CO2 using immobilised homoacetogenic bacteria.
Franziska Steger
Methane production
11:40-12:00 Modelling, qualitative and quantitative analysis of pure-culture biological methane production (BMP) from H2 and CO2.
Simon Rittman
12:00-12:20 Microbial processes in hydrogen exposed underground gas storages - results from lab scale simulation experiments.
Johanna Schritter
12:20-12:40 H2/CO2 (fed-batch) fermentation for biological production of CH4 by (pure culture of) hydrogenotrophic archaea.
Annalisa Abdel Azim
12:40 Lunch
13:20-14:00 Guided poster session Sophie Thallner, Rolf Warthmann, Alba Serna Maza, Charles Banks, Christian Kennes
14:00-14:20 Testing the habitability of Enceladus with Methanothermococcus okinawensis.
Patricia Pappenreiter
14:20-14:40 A new method for indirect quantification of methane production via water production using hydrogenotrophic methanogens.
Ruth-Sophie Taubner
14:40-15:00 Decentralised solutions as part of the energy and nutrient self-sustaining future.
Anni Alitalo
15:00-15:20 Development of a high-pressure process to couple biological hydrogen and methane production.
Lisa-Maria Mauerhofer
15:20-15:40 CO2 conversion to methane by microbial electrosynthesis
Christine Hemmelmair
15:40 coffee break
16:00 Guided discussion
17:30 End
4
Optional evening programme Thursday 10th November 2016
18:00 Guided tour of Vienna
Meet at 17:50 at the Michaelerplatz in front of Raiffeisenbank (Looshaus)
20:00 Dinner
Meet at 19:50 outside restaurant "Zwölf Apostelkeller" Sonnenfelsgasse 3, 1010 Wien
http://www.zwoelf-apostelkeller.at/index_en.html
We will go together after the guided tour or you can meet us there.
A short walking distance from Stephansdom cathedral, metro station Stephansplatz.
5
Optional evening programme Friday 11th November 2016
18:30 Ice stock sport (Bavarian curling)
Meet at the ice stock centre (7) in the Rathaus Christmas market at 18:30. Near Rathaus metro.
20:00 Dinner
Meet at 19:50 outside "Café Einstein" Rathausplatz 4, 1010 Wien, near Rathaus metro station
http://einstein.at/cms/uk/
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Internet access:
Connect to BOKU_Public-Event
Username: h97000_wlan
Password: 0227266280
7
Thursday 10th November 2016, 09:30-09:50
EVOLVING ANAEROBES BY ADAPTIVE AND MOLECULAR METHODS FOR VOLATILE FALLY
ACIDS PRODUCTION IN LIGNOCELLULOSIC HYDROLYSATES
Ivan Baumann, PhD
Department of Sustainable Biotechnology, Institute of Chemistry and Bioscience, Aalborg
University, Copenhagen and University
Biological production of organic acids from conversion of biomass derivatives has received increased
attention among scientists, engineers, and in business because of the attractive properties such as
renewability, sustainability, degradability, and versatility.
The aim of the presentation is to summarize research and development of short chain fatty acids
production by anaerobic fermentation of non-food biomass and to evaluate the status and outlook for a
sustainable industrial production of such bio-chemicals. Volatile fatty acids (VFA) such as acetic acid,
propionic acid, and butyric acid have many industrial applications, they are currently produced from
oil, and are of global economic interest. In industry, VFAs are valuable building blocks for the
production of polymer plastics and coatings and they are also used directly for conservation of food
and feed.
The focus is mainly on the utilization of pretreated lignocellulosic plant biomass as substrate via the
carbohydrate route and development of the bacteria and processes that lead to a high and economically
feasible production of VFA. Microbial conversion of pretreated lignocellulosic biomass hydrolysate is,
however, challenged because of cell growth inhibitors, which are concomitant to the pretreatment
processes. The presentation adresses this fundamental problem and show a strategy for development of
Propionibacterium acidipropionici and Clostridium tyrobutyricum cells with improved capabilities for
biomass conversion and VFA production in wheat straw hydrolysate. The experimental research
followed a two-phase strategy that combined bacterial adaptation and chemical mutagenesis. The
isolated mutant phenotypes tolerated high concentrations of pretreated wheat straw hydrolysate
including high levels of HMF, furfural, and acetate without compromising VFA production. In this
way, the presentation demonstrates a feasible route to development of natural, non-GMO organisms.
References:
Baroi, G.N., Baumann, I., Westermann, P., and Gavala, H.N. (2015). Butyric acid
fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium
tyrobutyricum strain. Microb Biotechnol 8, 874-882.
Baumann, I., and Westermann, P. (2016). Microbial Production of Short Chain Fatty Acids
from Lignocellulosic Biomass: Current Processes and Market. BioMed Research International
2016, 1-15.
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Thursday 10th November 2016, 09:50-10:10
MAXIMIZING THE PRODUCTION OF BUTYRIC ACID FROM FOOD WASTE AS A PRECURSOR
FOR ABE FERMENTATION
Ullrich Heinz Stein1, B. Wimmer
1, M. Ortner
2, W. Fuchs
1 G. Bochmann
1
1University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology,
Vienna, Austria 2Bioenergy 2020+ GmbH, Graz, Austria
The current study reports the maximization of butyric acid concentration from food waste using a
mixed microbial fermentation. The effect of three different pH values (5.5, 7.0 and 9.0), three different
temperatures (37°C, 55°C and 70°C) and two levels of hydraulic retention time (HRT, 2 days and 6
days) on the production of butyric acid were investigated. Overall pH 5.5 showed the lowest butyric
acid concentrations due to reutilization of the produced acids as well as decreased solubilization of the
substrate regardless of the temperature and the HRT. An increase of the temperature from 37°C to
55°C at pH 7 increased the butyric acid concentration significantly by 279.02% and 135.42% for an
HRT of 2 days and 6 days, respectively, whereas a further increase to 70°C showed decreasing butyric
acid production. The prolongation of the HRT from 2 days to 6 days shows an increment of butyric
acid concentration throughout almost all experiments, however the longer fermentation time does not
make up for the decreased production rate. The biggest increase of the butyric acid concentration is
caused by the raise in temperature from mesophilic (37°C) to thermophilic (55°C) conditions at
neutral pH values.
9
Thursday 10th November 2016, 10:10-10:30
QUANTITATIVE ASSESSMENT OF PURE CULTURE DARK FERMENTATIVE BIOHYDROGEN
PRODUCTION
Ipek Ergal1, Werner Fuchs
2, Günther Bochmann
2, Simon K.-M. R. Rittmann
1
1Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems
Biology, University of Vienna 2IFA Tulln, Universität für Bodenkultur Wien
The biological generated molecular hydrogen (biohydrogen) is considered a clean-renewable source of
energy and an ideal environmental friendly substitute for fossil fuels which contribute to emission of
greenhouse gases, ozone layer depletion, global warming, climate change and acid rain. With the high
energy yield (122 kj/g), biohydrogen is an alternative source to replace conventional fossil fuels.
There are three basic biological mechanisms for biohydrogen production: photolysis, photo-
fermentation and dark fermentation. Dark fermentation has some advantages over other biological
processes, such as the high rate of cell growth, non-requirement of light energy, higher hydrogen
evolution rate (Rittmann and Herwig, 2012), no oxygen limitation problems and the potential for cost-
effective hydrogen production (Levin et al., 2004).
The goal of our project is to manage a complete biological conversion of the starting organic matter to
H2 via dark fermentation.
- The first step is a literature overview of microorganisms concerning high hydrogen productivity,
evaluation rate and yield, and bioenergetic and thermodynamic calculations of promising candidates
for both pure and co-culture.
- The next step involves the hydrolysis of biomass and selection of strains, which produce high yields
of H2 and acetate+CO2, and combining the most suitable and syntrophıc microorganisms which
convert acetate to H2 into on effective microbial consortium immobilized in a biofilm.
- The last step is maximizing the production rate of hydrogen. To do so, the structured biofilm will be
coupled with innovative hydrogen removal processes. These "assisted hydrogen production" processes
include "hydrogen milking" with membrane contactors, electrochemistry, and the use of
nanostructured material that specifically adsorb H2.
A literature overview has been done and datasets concerning both pure and co-cultures have been
generated while calculations are still in progress.
References: Rittmann S, Herwig C., 2012, A comprehensive and quantitative review of dark fermentative
biohydrogen production. Microb Cell Factories. 11(1):115.
Levin DB, Pitt L, Love M., 2004, Biohydrogen production: prospects and limitations to practical
application. Int J Hydrog Energy. 29:173–185.
10
Thursday 10th November 2016, 11:40-12:00
WASTE2FUELS – SUSTAINABLE PRODUCTION OF NEXT GENERATION BIOFUELS FROM
WASTE STREAMS
Walter Wukovits, Florian Kirchbacher, Martin Miltner, Sylvia Zibuschka, Anton Friedl
Institute of Chemical Engineering, TU Wien
One of the major challenges Europe will face in the coming decades is to make its energy system
clean, secure and efficient while ensuring EU industrial leadership in low-carbon energy technologies.
In this way, the production of sustainable biofuels that generate a clear and net greenhouse gas (GHG)
saving without negatively impacting on biodiversity and land use is one of the main EU objectives.
WASTE2FUELS aims to produce biobutanol as a sustainable alternative for use as a direct
substitution for virgin fossil fuels, contributing to decentralised energy production towards EU energy
security. It has the potential to significantly reduce the burden on land use for biofuels not only in
Europe but worldwide, along with dramatically improving conversion efficiencies of current biofuel
production technologies.
WASTE2FUELS project aims at:
Mapping & analysing the available agro-food waste (AFW) streams in Europe as a feedstock
for biobutanol production.
Developing novel pretreatment methods for converting unavoidable agrofood waste to an
appropriate feedstock for biobutanol production
Developing methods and technologies that significantly improve the conversion efficiency,
specific productivity and reliability of an integrated ABE fermentation process.
Optimizing and demonstrating novel integrated ethanol to butanol catalytic conversion
processes and valorising post-process waste streams by recovering energy and added value by-
products.
Demonstrating the feasibility of the produced biobutanol to be burned in industrial systems
and to assess its ecotoxicological properties and designing the industrial up-scale of the
technologies.
Performing environmental and economic sustainability assessments through life cycle and cost
analysis (LCA and LCC).
Acknowledgement:
This project has received funding from the European Union’s Horizon 2020 research and innovation
programme under grant agreement No 654623.
11
Thursday 10th November 2016, 12:00-12:20
COMPLETE FERMENTATION OF PENTOSE AND HEXOSE MIXTURES BY CATABOLITE
REPRESSION MUTANTS OF CLOSTRIDIUM ACETOBUTYLICUM ATCC 824
Johannes Müller1, Carlos Pires
2, Nigel Saunders
2, Wolfgang Liebl
1 and Armin Ehrenreich
1
1Technical University of Munich, Chair of microbiology, Freising, Germany
2Brunel University London, Chair of Systems and Synthetic Biology, Uxbridge, United Kingdom
Conventional acetone-butanol-ethanol (ABE) fermentations with C. acetobutylicum using feedstocks
from renewable resources such as starch often compete with food production. Therefore,
hemicellulose containing substrates with a large share of pentoses have recently come into focus. As
these feedstocks always contain glucose that represses pentose utilization, the development of strains
that completely degrade the pentoses in the presence of glucose is essential for efficient butanol
production from such substrates.
Carbon catabolite repression (CCR) is well studied in model organisms such as E. coli or B. subtilis
but only little is known about it in C. acetobutylicum. Therefore, a method for the isolation of mutants
that lack catabolite repression was developed. C. acetobutylicum wild type was cultivated in
continuous cultures in a synthetic medium containing limiting glucose and excess xylose as carbon
sources in order to apply a selective pressure to promote growth of mutants that can use xylose in the
presence of glucose. Also, chemical mutagenesis using ethyl methanesulfonate was done. The
mutagenesis experiments were followed up by a subsequent cultivation of the cells on xylose and the
sugar analogue 2-deoxy-D-glucose, which allowed the isolation of mutants with defects in catabolite
repression. Those mutants showed complete degradation of synthetic media containing a major share
of arabinose and xylose as well as glucose and galactose and significantly increased butanol
production compared to the wild type. Moreover, those mutants showed an increased butanol
production from hemicellulose hydrolysates containing glucose, xylose and arabinose. Genomic
sequencing of the mutants revealed frameshift mutations in the glucose- and mannose-specific
phosphotransferase systems (PTS) as well as in relevant permeases and the sigD/whiG sigma factor
family. Those insights should help directed creation of further strains of solventogenic Clostridia such
as other C. acetobutylicum strains or C. saccharobutyliucm strains with improved substrate utilization
and butanol production. Those strains should be of high biotechnological relevance for new ABE
fermentations from plant biomass.
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Thursday 10th November 2016, 13:00-14:20
THE CD-LABORATORY FOR BIOTECHNOLOGY OF GLYCEROL
Hans Marx, Katharina Lindlbauer, Michael Egermeier, Hannes Rußmayer and Michael Sauer
Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences,
Vienna
The CD-Laboratory for Biotechnology of Glycerol is dedicated to the microbial valorisation of crude
glycerol from biodiesel production. Possible value added products from glycerol are 1,3-Propanediol
(1,3-PDO) and 3-Hydroxypropionic acid (3-HP), which will be produced by the platform organism
Lactobacillus diolivorans. Furthermore, the yeast Yarrowia lipolytica will be characterised for its
potential to produce lipids and sugar alcohols, like mannitol, arabitol and erythritol from raw glycerol.
In addition to these already established platform organisms a clostridial strain shall be added for the
production of butanol from glycerol to the portfolio of the CD-Laboratory for Biotechnology of
Glycerol.
The acetone-butanol-ethanol (ABE) fermentation by certain Clostridia has been an industrially
important microbial production process for already 100 years – based on starch and sugar. A new PhD
position is dedicated to the identification of strains with interesting metabolic traits and a first
characterization of the external parameters triggering butanol formation from glycerol.
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Thursday 10th November 2016, 13:00-14:20
CONTINUOUS BUTANOL PRODUCTION FROM SPENT SULFITE LIQUOR WITH CLOSTRIDIUM
SACCHAROPERBUTYLACETONICUM
Martin Lesniak 1, Michaela Weissgram
1, Sabrina Pober
1, Stefan Pflügl
1, Christoph Herwig
1,2
1 Vienna University of Technology, Institute of Chemical Engineering, Research Division
Biochemical Engineering, Gumpendorferstr. 1a, 1060 Vienna, Austria 2 Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved
Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria [email protected]
ABE fermentation has been known since the early 1900s. Low crude oil prices during the better part of
the 20th century made biotechnological production of acetone, butanol and ethanol economically
unattractive. However, with the prospect of ending crude oil reserves and the need to explore
alternative liquid fuels, research efforts and attempts to optimize ABE fermentation with clostridial
species have been increasing. The goal of this study was to use cheap substrates like spent sulfite
liquor (SSL) as a residual stream during pulp production for conversion into value-added products. To
that end, Clostridium saccharoperbutylacetonicum was used to develop a process for the conversion of
the sugars and the acetate from SSL into acetone, butanol and ethanol. Continuous cultures turned out
to be most efficient way for this transformation step, where a product yield of 0.23 Cmol/Cmol for
butanol was obtained. This shows feasibility to use real industrial residual streams for production of
butanol. With further development and optimization efforts, low product titers and productivities
obtained so far could be improved and the process could eventually be of economic interest for pulp
producers.
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Thursday 10th November 2016, 13:00-14:20
THE PDU MICROCOMPARTMENT FROM LACTOBACILLUS DIOLIVORANS Hannes Rußmayer
1,2, Hans Marx
1,2, Michael Sauer
1,2,3
1 CD Laboratory for Biotechnology of Glycerol, Muthgasse 107, 1190 Vienna, Austria
2 Department of Biotechnology, BOKU – University of Natural Resources and Life Sciences,
Vienna, Austria 3 ACIB – Austrian Centre of Industrial Biotechnology, Vienna, Austria
Clostridia are known for their exceptional capability of converting a variety of material (e.g. cellulose,
glucose, glycerol and gaseous substrates) into industrial valuable products, like higher alcohols. A
prerequisite for an efficient conversion is the optimization of metabolic fluxes through the reactions
leading to the desired product. Therefore, Clostridia developed different strategies to organize
enzymes of specific pathway in higher level structures to allow this optimization. For example
cellulolytic Clostridia use active enzymes complexes called cellulosomes for efficient degradation of
cellulosic material. The assembly of this enzymes complexes are done via interaction of functional
domains of a scaffolding protein and the active enzymes.
Another interesting feature of Clostridia is the ability to convert glycerol to different industrial
relevant production. Especially, 1,3 propanediol as an end product of glycerol metabolism is of
special interest for industry. For Clostridia different biochemical mechanism are described for the
pathway from glycerol to 1,3-propanediol.
In other bacterial species, which a natural producers of 1,3-propanediol, (Enterobacter spp.,
Lactobacillus spp.) was shown that conversion of glycerol to 1,3-propanediol is targeted to another
form of higher level organization, found in bacteria, named Pdu (propanediol utilization)
microcompartment. Microcompartments have a polyhedral shape and are typically built by a protein
shell, which encapsulates enzymes catalysing specific metabolic processes.
In this study we focus on the Pdu microcompartment of Lactobacillus diolivorans, which is a good
natural producer of 1,3-propanediol. For Lactobacillus spp. ( e.g. Lactobacillus reuteri, L. diolivorans)
it is known that within this compartment two different carbon sources 1,2 propanediol and glycerol
are converted with the same set of enzymes in different end product. 1,2-propanediol is converted
to propionate and propanol, whereas under anaerobic condition glycerol is converted to 1,3
propanediol. In this context, the Pdu microcompartment plays an essential role in creating an
individual microenvironment for the encapsulated enzymes to ensure an efficient conversion of both
substrates. Unfortunately, no information about this microcompartment is available for L.
diolivorans. Therefore, it is of special interest for us to understand the underlying metabolic
processes and in this context the function of the Pdu microcompartment in allowing an efficient
conversion of glycerol to 1,3-propanediol.
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Thursday 10th November 2016, 13:00-14:20
INFLUENCE OF INHIBITORS FROM THE SPENT SULPHITE LIQUOR ON THE GROWTH OF
CLOSTRIDIUM SACCHAROPERBUTYLACETONICUM UND IMPROVEMENT OF FERMENTATION
OF THE SPENT SULPHITE LIQUOR BY ITS DETOXIFICATION.
Kateryna Wössa, Hansjörg Weber
b, Hedda K. Weber
a
aKompetenzzentrum Holz GmbH, Altenberger Straße 69, 4040 Linz
bInstitut für Organische Chemie, TU Graz, Stremayrgasse 9, 8010 Graz
One way to produce dissolving pulp is the sulphite process. This process yields large quantities of
spent sulphite liquor (SSL), which contains high amounts of degraded polysaccharides. They represent
an optimal starting substrate for the biofuels production, which does not compete with the food chain
(Olsson and Hahn-Hägerdal, 1996). The production of ethanol from spent sulphite liquor (SSL) was
established a century ago employing yeasts (Saccheromyces cerevisiae). However, the production of
butanol from SSL employing microorganisms is far less explored.
In addition to the desirable high amounts of mono- and oligomeric sugars, SSL also contains other
substances that have inhibitory effects on the microorganisms (Chandel, 2011).
We performed single substance screenings of selected phenolic compounds, organic acids, alcohols
and furan derivatives in order to better understand the inhibitory effects of these substances. Moreover,
we developed a method to directly perform the fermentations in microtiterplates to speed up the
screening.
Certain phenol derivatives show pronounced inhibiting effects on the fermentation. Therefore, the
detoxification techniques addressing aromatic compounds should be more successful than others. In
order to test this hypothesis we performed various detoxification experiments for lignocellulosic
hydrolysates. These included enzymatic treatment, treatment with activated carbon, lignin, and ion
exchange resins as well as ammonium, calcium and magnesium hydroxides. These experiments show,
that the detoxification with hydrogen peroxide and peroxidase as catalyst is the most effective method
for the detoxification of the SSL, the second best method is the detoxification with activated carbon
and lignin. The other techniques are less effective.
References:
Olsson, L., Hahn-Hägerdal B. (1996). Fermentation of lignocellulosic hydrolysates for ethanol
production. Enzyme and Microbial Technology, vol. 18, pp. 312-331.
Chandel, A.K., Silva S.S., Singh O.V. (2011) Detoxification of Lignocellulosic Hydrolysates for
Improved Bioethanol Production. In: Bernardes M.A.S. (ed) Biofuel Production-Recent Developments
and Prospects. In Tech, Rijeka, pp. 225-246.
16
Thursday 10th November 2016, 13:00-14:20
RE-CLASSIFICATION OF CLOSTRIDIUM PASTEURIANUM NRRL B-598 AS CLOSTRIDIUM
BEIJERINCKII NRRL B-598 BASED ON GENOMIC AND PHENOTYPIC TRAITS
M. Vasylkivskaa, K. Sedlář
b, J. Kolek
a, I. Provazník
b, P. Patáková
a
aDepartment of Biotechnology, University of Chemistry and Technology Prague
bDepartment of Biomedical Engineering, Brno University of Technology
The strain Clostridium pasteurianum NRRL B-598 is a spore-forming, mesophilic heterofermentative
bacterium with acetone-butanol fermentation ability. But its phenotypic behaviour differs from other
pasteurianum strains. Our research confirmed its inability of using glycerol as carbon source, which is
well-known phenotypic trait of C. pasteurianum, together with a specific fermentation pattern closer
to C. beijerinckii.
First C. pasteurianum NRRL B-598 genome analysis showed similarity of genes responsible for
solventogenesis to C. beijerinckii. We presented full genome sequence with further in silico
comparison to other Clostridium strains using digital DNA-DNA hybridization. It revealed that
genetic similarity of C. pasteurianum NRRL B-598 to C. beijerinckii strains is 75-78 % while
similarity to C. pasteurianum is only 24-28 %. This value indicates an inaccuracy of the taxonomic
status of strain Clostridium pasteurianum NRRL B-598. Therefore, we suggest its re-classification as
Clostridium beijerinckii NRRL B-598.
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Thursday 10th November 2016, 13:00-14:20
EVALUATION OF VIABILITY, METABOLIC ACTIVITY AND SPORE QUANTITY IN CLOSTRIDIAL
CULTURES DURING ABE FERMENTATION
Marek Drahokoupil, Jan Kolek, Barbora Branska, Petra Patakova
Department of Biotechnology, University of Chemistry and Technology Prague, Technicka 5,
16628 Prague, Czech Republic
Flow cytometry, in combination with fluorescent staining and fluorescent microscopy was used to
evaluate population heterogeneity in acetone-butanol–ethanol fermentation that was carried out with
type strain Clostridium beijerinckii NCIMB 8052 and non-type C. pasteurianum NRRL B-598.
Viability together with physiological changes of both populations were monitored by bis-oxonol
(BOX) and by propidium iodide (PI) in combination with carboxyfluorescein diacetate (CFDA) or
SYTO 9. Common use of CFDA with PI enabled determining mature spores. Using probes CFDA and
PI provided valuable information on the physiological state of clostridia. CFDA and PI double staining
resulted in the best resolution of four distinct subpopulations displaying enzymatically active cells,
doubly stained cells, damaged cells and spores. Occurance of cells in particular sub-regions correlated
with growth characteristics, fermentation parameters such as substrate consumption and product
formation in both species under different cultivation conditions.
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Thursday 10th November 2016, 13:00-14:20
BUTANOL PRODUCTION FROM VOLATILE FEEDSTOCKS. DEVELOPMENT OF AN OPTIMIZED
BIOPROCESS
Florian Gattermayr, Viktoria Leitner, Christoph Herwig
Kompetenzzentrum Holz GmbH Linz
With this study we want to address a major problem of biorefineries using alternative substrates (e.g.
organic residuals): changing feedstock composition and quality.
A typical ABE batch fermentation with Clostridia consists of two characteristic stages occurring in
different phases of growth (with a certain overlap): the acidogenesis (exponential phase) and the
solventogenesis (deceleration to equilibrium phase). Only in the solventogenic phase butanol and
acetone are being produced. Ethanol is formed in small amounts in both phases (Haus et al., 2011;
Millat et al., 2013). Acids built in the acidogenesis are reassimilated in the solventogenesis and then
rebuilt to solvents (Jones and Woods, 1986). A specific process variable to switch between these two
stages is the pH whereas organic acids are known to work as inductor for solventgonesis and raise
product yield (Bahl et al., 1982; Chang, 2010; Chen and Blaschek, 1999; Lee et al., 2008; Matta-El-
Ammouri et al., 1987).
With high feedstock and product flexibility (biorefinery) in mind, these organic acids (mostly acetic
acid and butyric acid) can be derived from other (pre)processes and used as a co-feed in a continuous
fermentation. Overall a lot of research has been done on utilization of various carbon sources for
butanol production (Jones and Woods, 1986; Lee et al., 2008). Within the research project CAFB
(combined agro and forest biorefinery) we follow a novel approach in studying various butanol
producing stems of Clostridia on their suitability for continuous solventogenic phase fermentation of
fluctuating compositions of acids as co-feed along with varying sugar sources. The goal is to
investigate the influence on process stability and butanol production.
The experiments will be performed in 2 L continuously stirred tank reactors. Thereby around five
volume changes are needed to get to a steady state continuous solventogenic culture (C.
acetobutylicum) where it can in principle be kept permanently (Haus et al., 2011). In order to keep the
cultures ability to produce solvents (not losing the megaplasmid pSOL1 in case of C. acetobutylicum)
the media will be limited in phosphate (Cornillot et al., 1997). Furthermore pH and organic acid feed
must be controlled to prevent a so called “acid crash” when concentration of undissociated acids
exceed 57 – 60 mmol/L (Maddox et al., 2000).
We expect to establish stable steady state continuous solventogenic phase fermentation, find optima as
well as limits in fluctuating organic acid and sugar feeds.
References:
19
Bahl, H., Andersch, W., Braun, K., Gottschalk, G., 1982. Effect of pH and butyrate concentration
on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous
culture. European journal of applied microbiology and biotechnology 14, 17–20.
Chang, W.-L., 2010. Acetone-Butanol-Ethanol Fermentation by Engineered Clostridium
beijerinckii and Clostridium tyrobutyricum. The Ohio State University.
Chen, C.-K., Blaschek, H.P., 1999. Acetate enhances solvent production and prevents
degeneration in Clostridium beijerinckii BA101. Appl Microbiol Biotechnol 52, 170–173.
doi:10.1007/s002530051504
Cornillot, E., Nair, R.V., Papoutsakis, E.T., Soucaille, P., 1997. The genes for butanol and acetone
formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to
degeneration of the strain. J Bacteriol 179, 5442–5447.
Haus, S., Jabbari, S., Millat, T., Janssen, H., Fischer, R.-J., Bahl, H., King, J.R., Wolkenhauer, O.,
2011. A systems biology approach to investigate the effect of pH-induced gene regulation on
solvent production by Clostridium acetobutylicum in continuous culture. BMC Systems Biology
5, 10. doi:10.1186/1752-0509-5-10
Jones, D.T., Woods, D.R., 1986. Acetone-butanol fermentation revisited. Microbiol Rev 50, 484–
524.
Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., Jung, K.S., 2008. Fermentative butanol
production by clostridia. Biotechnology and Bioengineering 101, 209–228. doi:10.1002/bit.22003
Maddox, I.S., Steiner, E., Hirsch, S., Wessner, S., Gutierrez, N.A., Gapes, J.R., Schuster, K.C.,
2000. The Cause of“ Acid Crash” and“ Acidogenic Fermentations” During the Batch Acetone-
Butanol-Ethanol(ABE-) Fermentation Process. Journal of molecular microbiology and
biotechnology 2, 95–100.
Matta-El-Ammouri, G., Janati-Idrissi, R., Junelles, A.-M., Petitdemange, H., Gay, R., 1987.
Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum.
Biochimie 69, 109–115. doi:10.1016/0300-9084(87)90242-2
Millat, T., Janssen, H., Bahl, H., Fischer, R.-J., Wolkenhauer, O., 2013. Integrative modelling of
pH-dependent enzyme activity and transcriptomic regulation of the acetone-butanol-ethanol
fermentation of Clostridium acetobutylicum in continuous culture: pH-dependent kinetic and
transcriptomic regulation. Microbial Biotechnology 6, 526–539. doi:10.1111/1751-7915.12033
20
Thursday 10th November 2016, 14:20-14:40
MATHEMATICAL MODELLING SUPPORTED OPTIMISATION OF ABE FERMENTATION AND
IMPROVEMENT OF ITS SUSTAINABILITY
Sergej Trippel, Katja Karstens, Nikola Naschitzki, Peter Götz
Bioprocess Engineering, Biotechnology, Beuth University of Applied Sciences Berlin
In the OPTISOLV project, supported within the frame of the ERA-Net EuroTransBio-7 initiative by
the German Federal Ministry of Education and Research, a new design of a bioreactor system for
ABE-fermentation has been developed. A cascade of 6 bioreactors enables a stable continuous
fermentation process and gives a novel platform for various designs of experiment aiming at better
understanding of the Clostridial metabolism and optimisation of the fermentation process. In this
multistage bioreactor system the two metabolic phases of Clostridium acetobutylicum – acidogenesis
and solventogenesis – are spatially separated. With this strategy, the acidogenic cells can continuously
supply the solventogenic cells with acids for their further conversion to solvents. Under optimized
operating conditions (Dtotal = 0.92 h-1
and pHbioreactor1 = 4.3), we reached a final butanol concentration of
11 gBUT L-1
with a volumetric production rate of 1 gBUT (Lh)-1 in our fermentation system. To get
insights into the dynamics of the differentiation process from acidogenic to solventogenic cells along
the bioreactor cascade we have further developed an agent-based mathematical model. This includes
simulation of the metabolite concentrations in each bioreactor as well as of the metabolic
production/consumption rates of the biomass, represented by 3 different subpopulations, i.e.
acidogenic, intermediate, solventogenic cells (Karstens et al. 2016). We are now using experimental
investigations and mathematical modelling to evaluate different optimisation strategies like addition or
removal of reactor stages, change of residence times in the stages and recirculation or feeding between
the stages.
Collaboration with the “Waste2Fuels” project, funded within the EU programme Horizon2020, may
further improve the economic efficiency of ABE-fermentation. Biomass produced from the continuous
ABE-fermentation can be used as an inexpensive nitrogen and vitamin source for bacteria. Thus a
work package of the “Waste2Fuels” project aims at providing supplementary nutrients for ABE-
fermentations. Hence, the recycling of the biomass can contribute to the sustainability of ABE-
fermentation processes.
References:
Karstens, K. et al., 2016. Modeling Physiological Differences in Cell Populations : Acetone-Butanol-
Ethanol ( ABE ) -Fermentation in a Cascade of Continuous Stirred Tank Reactors. , Chemical
Engineering Transactions, 49, pp.271–276.
21
Thursday 10th November 2016, 14:40-14:50
ABE PRODUCTION OF DIFFERENT CLOSTRIDIAL STRAINS AND INFLUENCE OF
SUPPLEMENTS
Seyed Mohsen Abbasi Hosseini
University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology,
Vienna, Austria
In order to increase butanol production, bacteria strains and culture conditions are two key factors. The
aim of this research is to evaluate ABE production of four native clostridia strains from DSMZ and to
evaluate the addition of supplements with multiple effects on growth and production of ABE.
Clostridium beijerinckii DSMZ 1739 was deficient in butanol production and Clostridium beijerinckii
DSMZ 6422 produces more than Clostridium acetobutylicum DSMZ 792 (ATCC 842) which is the
most promising native strain for butanol production.
22
Friday 11th November 2016, 09:20-10:00
SYNGAS/WASTE GAS (CO, CO2, H2) FERMENTATION TO ETHANOL AND HIGHER ALCOHOLS
Christian Kennes Chemical Engineering Laboratory, Faculty of Sciences, University of La Coruña, Rúa da Fraga 10, E – 15008 – La Coruña, Spain [email protected]
Carbohydrates can be extracted from different feedstocks, such as biomass and agroindustrial wastes.
Subsequently, they can be metabolized by some anaerobic bacteria, mainly clostridia, to produce
alcohols such as ethanol and butanol through the ABE (Acetone-Butanol-Ethanol) fermentation.
Another possible route consists in gasifying such feedstocks, or other carbon-containing ones, into
syngas, which is a gaseous mixture composed mainly of CO, CO2 and H2 (Kennes et al., 2016). Some
industrial waste gases, among others in steel industries, contain also a similar gas mixture. They can
be fermented by few clostridia and other acetogens (van Groenestijn et al., 2013).
The bioconversion of syngas/waste gas takes place through the Wood-Ljungdahl pathway with acetyl-
coA as common intermediate metabolite. Either CO, CO2+H2, or mixtures of those three compounds
are generally all suitable substrates. During the bioconversion process, organic acids appear first,
followed by the production of alcohols. Species such as Clostridium ljungdahlii and C.
autoethanogenum produce acetic acid first which yields some energy and is simultaneous to biomass
growth. This is followed by the conversion of the organic acid into ethanol. Other alcohols such as
2,3-butanediol have sometimes also been detected. In other bacteria, such as C. carboxidivorans, a
mixture of different organic acids has been found as well as higher alcohols, besides ethanol, during
fermentation of the above mentioned gases. In that species, acetic acid, butyric acid, as well as
hexanoic acid appear in a first stage, followed by the production of ethanol, butanol and hexanol.
Contrary to the ABE fermentation, in presence of gaseous substrates no acetone has ever been detected
in wild type acetogens; but, similarly as in the ABE fermentation process, the accumulation of high
concentrations of alcohols as end metabolites inhibits clostridia when grown on gaseous substrates
(Fernández et al., 2016).
References
Fernández-Naveira, A., Abubackar, H.N., Veiga, M.C., Kennes, C. 2016. Carbon monoxide
bioconversion to butanol-ethanol by Clostridium carboxidivorans: kinetics and toxicity of alcohols.
Applied Microbiology and Biotechnology, 100(9): 4231-4240
Kennes, D., Abubackar, H.N., Diaz, M., Veiga, M.C., Kennes, C., 2016. Bioethanol production from
biomass: carbohydrate vs syngas fermentation. Journal of Chemical Technology and Biotechnology,
91(2): 304-317.
Van Groenestijn, J.W., Abubackar, H.N., Veiga, M.C., Kennes, C., 2016. Bioethanol (Chapter 18). In:
Kennes C and Veiga MC (eds), Air Pollution Prevention and Control: Bioreactors and Bioenergy. J.
Wiley & Sons, Chichester, UK, pp. 431-463. ISBN 978-1-119-94331-0.
23
Friday 11th November 2016, 10:00-10:30
SELECTIVE ACETONE AND ISOPROPANOL PRODUCTION USING ACETOGENIC BACTERIA
Frank R. Bengelsdorf, Sabrina Hoffmeister and Peter Dürre
Institut für Mikrobiologie und Biotechnologie, Universität Ulm, Albert-Einstein-Allee 11, D-
89081 Ulm, Germany
Depletion of fossil resources stresses the necessity of developing new alternative routes for the
production of bulk chemicals and fuels beyond petroleum. Gas fermentation (H2 + CO2 or H2 + CO as
substrate) is a microbial process performed by autotrophic acetogenic bacteria that use the Wood-
Ljungdahl pathway. Using these biocatalysts (wild types strains) in the fermentation process, biofuels
such as ethanol or butanol, as well as biocommodities such as acetate, lactate, butyrate, 2,3-butanediol,
can be produced.
Since some products cannot be naturally produced by acetogenic bacteria, selected strains were
metabolically engineered for the production of acetone and isopropanol. Acetone for instance is an
industrial bulk chemical which is currently produced from fossil resources at a global capacity of 6.7
million tons per year (2011).
Different vectors containing the synthetic acetone synthesis operon (ASO) were transformed into wild
type strains of Acetobacterium woodii or Clostridium ljungdahlii. The ASO containing the genes thlA
(encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate
decarboxylase) from C. acetobutylicum were cloned under the control of the thlA promoter into four
vectors having different replicons for Gram-positives. All respective recombinant strains were
cultivated in flask-batch mode and characterized with respect to growth as well as product formation.
Furthermore, recombinant A. woodii strains were cultivated in bioreactors in batch and continuous
mode.
Acetone production using recombinant A. woodii strains was confirmed under heterotrophic as well as
autotrophic growth conditions. Under autotrophic conditions with H2 + CO2, the recombinant strains
produced up to 15 mM acetone in flask-batch mode. Bioreactor-batch fermentations using a
recombinant A. woodii strain revealed that acetate concentration had an effect on acetone production,
due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration
within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was
developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased
from 1.2 mg L-1
h-1
in flask-batch mode up to 26.4 mg L-1
h-1
in continuous gas
fermentation (Hoffmeister, 2016). In addition, formation of isopropanol could be verified in a
recombinant C. ljungdahlii strain that possesses a primary/secondary alcohol dehydrogenase
(Bengelsdorf, 2016).
24
Acetogenic bacteria were successfully engineered to autotrophically produce acetone and isopropanol,
respectively. The use of cheap and abundant carbon sources offers a great potential for industrial
application and reduction of greenhouse gas emissions.
References:
Bengelsdorf, F. R., Poehlein, A., Linder, S., Erz, C., Hummel, T., Hoffmeister, S., Daniel, R., Dürre P.
(2016). Engineering industrial acetogenic biocatalysts: a comparative metabolic and genomic analysis.
Frontiers in Microbiology 7:1036.
Hoffmeister, S., Gerdom, M., Bengelsdorf, F. R., Linder, S., Flüchter, S., Öztürk, H., Wilfried
Blümke, Antje May, Ralf-Jörg Fischer R-J, Hubert Bahl, H, Dürre, P. (2016). Acetone production with
metabolically engineered strains of Acetobacterium woodii. Metabolic Engineering. (in press).
25
Friday 11th November 2016, 10:50-11:10
MOORELLA THERMOACETICA, A THERMOPHILIC MODEL ORGANISM CREATING VALUE
FROM WASTE GASSES.
Torbjørn Ølshøj Jensen, Stephanie a M Redl, and Alex Toftgaard Nielsen
Novonordisk foundation Center for Biosustainability.
The fermentation of waste gas streams to produce high value compounds is an attractive alternative to
traditional biomass hydrolysate fermentation. Industrial waste gasses as well as carbon- and energy-
rich syngas obtained from gasification of organic-residues can serve as substrate for acetogenic
bacteria, but are left unused to date.
The model acetogenic bacterium Moorella thermoacetica is an ideal production organism for gas
fermentation processes. Its ability to grow at elevated temperatures (60°C) provides many advantages,
in particular it allows recovery of chemical compounds that have a low boiling point (such as acetone)
from the vapor phase. However, production of such or other compounds with higher value using
Moorella requires a better understanding of its metabolism, as well as reliable tools that enable genetic
modification. Technology enabling reliable genetic engineering is very limited, and we have focused
on overcoming this challenge through development of selection systems and investigation of the
methylome.
Evaluating the potential of M. thermoacetica as an industrially relevant production strain, we also
assessed the cost-effectiveness of acetone production utilizing M. thermoacetica as production host
The gained expertise on cell-level and on process scale will help to transform the former model
organism into an industrially relevant organism for converting waste gas streams into valuable
compounds.
26
Friday 11th November 2016, 11:10-11:30
ELEVATED PRESSURE BIOREACTORS FOR GAS TRANSFER ENHANCEMENT IN GAS
FERMENTATION
Jasbir Singh , Rehan Shah
HEL Ltd, England
[email protected], [email protected]
Commercial feasibility of many bio-processes can depend on how fast gas transfer takes place. This is
especially true if gas solubility is poor, for example for when working with gases such as hydrogen
and methane in the context of gas fermentation. The engineering approach to this problem is normally
limited to KLa improvements by changing features of sparging and agitation with limited to scope for
improvement. A much more effective alternative is to operate the bio-reactor at pressure and this can
in principle increase gas transfer rate several-fold.
This presentation will present data from mini-bioreactors to illustrate the benefits of working at
elevated pressure. Increases is solubility and max flue will be presented as well as demonstration of
how pressure can be used as a control variable to manipulate dissolved oxygen profile without any
change in the stirring/sparging. Fully automated bio-reactors suited to research and development will
be presented which can be operated at a range of elevated pressures.
27
Friday 11th November 2016, 11:30-11:40
OPTIMISATION OF CONTINUOUS GAS FERMENTATION BY IMMOBILISATION OF ACETATE-
PRODUCING CLOSTRIDIA
Franziska Steger1, Lydia Rachbauer
2, Lucy F. R. Montgomery
3, Günther Bochmann
1
1 Institute of Environmental Biotechnology, BOKU University of Natural Resources and Life
Sciences Vienna, Konrad-Lorenz-Straße 20, 3430 Tulln, Austria 2 Bioenergy 2020+ GmbH, Konrad-Lorenz-Straße 20, Tulln, Austria
3 acib Austrian Center of Industrial Biotechnology, Konrad-Lorenz-Str. 20, 3430 Tulln, Austria
Hydrogen from electrolysis of water is often suggested as a way of storing the excess energy
from wind and solar power plants. However, unlike natural gas, hydrogen is difficult to store
and distribute. One solution is to convert the hydrogen into other fuels or bulk chemicals. In
this study we investigated fermentation in which homoacetogenic Clostridia apply the Wood-
Ljungdahl pathway to generate acetate out of H2 and CO2. Acetate can be used as a bulk
chemical or further transformed. Autotrophic growth with CO2 as the sole carbon source is
much slower compared to heterotrophic growth. That is challenging during continuous
fermentation as bacteria get washed out during product removal. The aim of this work was to
immobilise acetate-producing Clostridia on a suitable membrane material, thus preventing
their wash out. A range of eight membranes was tested in duplicate on the homoacetogenic
strains A. woodii and M. thermoacetica. Tested material included woven, symmetric and
asymmetric membranes. Asymmetric membranes have a relatively dense, thin selective layer
and a much thicker, porous support layer in which bacteria can diffuse while symmetric
membranes are of a uniform structure. On woven membranes immobilisation is by
adherence. Immobilisation was assessed by the decrease in optical density (OD600) and a
microscopic evaluation using scanning electron microscope (SEM). While other materials
dissolved, PET and linen stayed stable in culture conditions. A distinct decrease in OD600 was
achieved with linen and PET for both strains. For A. woodii, acetate production did not
increase with immobilisation while M. thermoacetica showed slightly higher final acetate
concentrations when immobilised on linen and PET.
According to these results linen and PET are very well suited for immobilisation of
homoacetogenic clostridia. Due to its stability and for environmental reasons, linen was
chosen as immobilisation material for the subsequent continuous fermentation. A. woodii was
successfully immobilised while continuously producing acetate. Future work should focus on
optimising fermentation with immobilisation.
28
Friday 11th November 2016, 11:40-12:00
MODELLING, QUALITATIVE AND QUANTITATIVE ANALYSIS OF PURE CULTURE BIOLOGICAL
METHANE PRODUCTION (BMP) FROM H2 AND CO2
Simon K.-M. R. Rittmann1, Arne H. Seifert
2, Sebastien Bernacchi
2 1Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems
Biology, University of Vienna 2Krajete GmbH
One of the possibilities to integrate surplus renewable power storage with CO2 capture and
sequestration technologies is to employ the biological methane production (BMP) process (Bernacchi
et al., 2014; Rachbauer et al., 2016; Rittmann et al., 2012; Seifert et al., 2014). The BMP process is
characterized applying autotrophic and hydrogenotrophic methanogenic archaea (methanogens) for
methane (CH4) production (Rittmann et al., 2015). The BMP process can be operated by using
enrichment cultures as well as pure culture of methanogens (Rachbauer et al., 2016; Rittmann, 2015;
Wise et al., 1978) and benefits from its ability to convert CO2 and molecular hydrogen to CH4 at very
high volumetric methane evolution rates (MERs) in continuous culture (Nishimura et al., 1992; Seifert
et al., 2014). Another advantage is that mild bioprocessing conditions that can be applied in BMP (e.g.
temperatures from approx. 0°C to 122°C (Seifert et al., 2013; Takai et al., 2008; Taubner et al., 2015).
In this presentation the effects of various process parameters on key variables of BMP in pure culture
are presented independent of bioreactor conditions and scale from continuous culture experiments
already published in literature. Data curation procedures for quantitative data substantiation and
quality assessment are applied. Multivariate effects of process parameters are unscrambled applying
principle component analysis and multilinear regression. The limitations of the models will be
discussed, too. Finally, models attained independent of organism and bioreactor type will be compared
to models generated for Methanothermobater marburgensis - the most important model organism in
pure culture BMP. Eventually bioprocess operation windows for BMP in chemostat culture for M.
marburgensis will be presented.
References:
Bernacchi, S., Rittmann, S., H. Seifert, A., Krajete, A., Herwig, C., 2014. Experimental methods for
screening parameters influencing the growth to product yield (Y(x/CH4)) of a biological
methane production (BMP) process performed with Methanothermobacter marburgensis.
AIMS Bioeng. 1, 72–86. doi:10.3934/bioeng.2014.1.72
Nishimura, N., Kitaura, S., Mimura, A., Takahara, Y., 1992. Cultivation of thermophilic methanogen
KN-15 on H2-CO2 under pressurized conditions. J. Ferment. Bioeng. 73, 477–480.
doi:10.1016/0922-338X(92)90141-G
29
Rachbauer, L., Voitl, G., Bochmann, G., Fuchs, W., 2016. Biological biogas upgrading capacity of a
hydrogenotrophic community in a trickle-bed reactor. Appl. Energy 180, 483–490.
doi:10.1016/j.apenergy.2016.07.109
Rittmann, S., Seifert, A., Herwig, C., 2015. Essential prerequisites for successful bioprocess
development of biological CH4 production from CO2 and H2. Crit. Rev. Biotechnol. 35, 141–
151. doi:10.3109/07388551.2013.820685
Rittmann, S., Seifert, A., Herwig, C., 2012. Quantitative analysis of media dilution rate effects on
Methanothermobacter marburgensis grown in continuous culture on H2 and CO2. Biomass
Bioenergy 36, 293–301. doi:10.1016/j.biombioe.2011.10.038
Rittmann, S.K.-M.R., 2015. A Critical Assessment of Microbiological Biogas to Biomethane
Upgrading Systems. Adv. Biochem. Eng. Biotechnol. 151, 117–135. doi:10.1007/978-3-319-
21993-6_5
Seifert, A.H., Rittmann, S., Bernacchi, S., Herwig, C., 2013. Method for assessing the impact of
emission gasses on physiology and productivity in biological methanogenesis. Bioresour.
Technol. 136, 747–751. doi:10.1016/j.biortech.2013.03.119
Seifert, A.H., Rittmann, S., Herwig, C., 2014. Analysis of process related factors to increase
volumetric productivity and quality of biomethane with Methanothermobacter marburgensis.
Appl. Energy 132, 155–162. doi:10.1016/j.apenergy.2014.07.002
Takai, K., Nakamura, K., Toki, T., Tsunogai, U., Miyazaki, M., Miyazaki, J., Hirayama, H.,
Nakagawa, S., Nunoura, T., Horikoshi, K., 2008. Cell proliferation at 122 degrees C and
isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure
cultivation. Proc. Natl. Acad. Sci. U. S. A. 105, 10949–10954. doi:10.1073/pnas.0712334105
Taubner, R.-S., Schleper, C., Firneis, M.G., Rittmann, S.K.-M.R., 2015. Assessing the Ecophysiology
of Methanogens in the Context of Recent Astrobiological and Planetological Studies. Life 5,
1652–1686. doi:10.3390/life5041652
Wise, D.L., Cooney, C.L., Augenstein, D.C., 1978. Biomethanation: Anaerobic fermentation of CO2,
H2 and CO to methane. Biotechnol. Bioeng. 20, 1153–1172. doi:10.1002/bit.260200804
30
Friday 11th November 2016, 12:00-12:20
MICROBIAL PROCESSES IN HYDROGEN EXPOSED UNDERGROUND GAS STORAGES – RESULTS FROM LAB SCALE SIMULATION EXPERIMENTS Johanna Schritter
1, Kerstin Brandstätter-Scherr
1, Robert Komm
1, Diana Backes
1,
Markus Pichler2, Stephan Bauer
2 and Andreas P. Loibner
1
1University of Natural Resources and Life Sciences, Department of Agrobiotechnology,
Institute of Environmental Biotechnology – Geobiotechnology and Environmental Chemistry, Konrad-Lorenz-Strasse 20, 3430 Tulln, Austria; 2RAG, Schwarzenbergplatz 16, 1015 Vienna, Austria;
[email protected], [email protected]
Considering the effects of fossil energy consumption on climate change, it is of utmost importance to
increase the share of energy produced from renewable resources. However, energy produced from
wind or solar power is fluctuating in response to availability of the energy source. Resulting peak
production of electrical power is just partly in line with peak consumption. As a consequence, storage
possibilities for large quantities of energy are required. “Power-to-Gas” (conversion of excess
electricity to hydrogen) is already state of the art but efficient storage facilities for hydrogen are still a
matter of investigation. The Underground Sun Storage Project focuses on the introduction of hydrogen
blended with natural gas into porous underground gas storage facilities. Potentially, hydrogen could be
decreased by biogeochemical transformation processes possibly accompanied by a loss in pressure,
well clogging, acidification and MIC. Therefore, the objective is to study microbial and geochemical
processes associated with the exposure of hydrogen to underground gas storages.
For simulating an underground gas storage facility at lab scale, UGS drilling cores were inoculated
with UGS formation water and then placed in 10 corrosion resistant bioreactors (including two abiotic
controls) and operated at reservoir conditions of the testbed (45°C, 48 bar). In a first step, UGS
conditions were simulated with only methane being stored for 2 months. Following, the cores were
exposed to various gas mixtures (hydrogen 4-10%, carbon dioxide 0,3-2,5%, methane). Prior to and
after hydrogen exposure (6 months), formation water and cores were analysed with respect to
hydrochemical and microbiological characteristics. During hydrogen exposure, the partial pressure of
hydrogen, methane and carbon dioxide was monitored. A loss in pressure and consumption of
hydrogen and carbon dioxide were observed in biotic reactors. Molecular biological analysis revealed
eubacterial and archaeal communities why microbial processes were concluded to be responsible for
hydrogen depletion. Potential microbial hydrogen consumption reactions at UGS conditions comprise
homoacetogenesis, sulphate reduction and methanogenesis with the latter being rather a conversion
than a loss of energy. Data suggest that only in the presence of terminal electron acceptors hydrogen
will be depleted. Therefore the introduction of hydrogen into porous UGS is a promising approach to
integrate renewable energy into state of the art storage techniques.
31
Friday 11th November 2016, 12:20-12:40
H2/CO2 (FED-BATCH) FERMENTATION FOR BIOLOGICAL PRODUCTION OF CH4 BY (PURE
CULTURE OF) HYDROGENOTROPHIC ARCHAEA
Annalisa Abdel Azim1,2
, Christian Pruckner1, Philipp Kolar
1, Debora Fino
2, Guido Saracco
2,
Simon K.-M. R. Rittmann1
1Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems
Biology, University of Wien. 2Department of Applied Science and Technology (DISAT), Politecnico di Torino
With the recent approval of the Paris agreement, we achieved another step in favour of sustainable
energy production, declaring once again that fossil fuels must be replaced by a new generation of fuels
from renewable end recycled sources. However, new processes for producing green energy need to be
competitive and appealing not only at an environmental level but also economic. The biological
methane production (BMP) process owns a great potential due to its ability to convert directly gaseous
substrates simply using the natural metabolism of methanogens which act as bio-catalysts. This study
wants to present results related to CH4 production from H2 and CO2 in exponential fed-batch
fermentations using pure culture of Methanothermobacter marburgensis in a 2L reactors system. By
the support of design of experiment (DoE) methods, different experiments were performed varying the
value of exponential factors of liquid dilution rates, gas feeding rates and H2/CO2 ratio. The
quantitative analysis reveals that by increasing the gas feeding rate and the trace elements dilution rate
the culture performances improved. The highest value of methane evolution rate (MER) achieved here
is the highest ever published among the studies concerning fed-batch fermentation of H2 and CO2 by
Methanothermobacter marburgensis. Furthermore, high trace element (TE) feeding was decisive for
the biomass growth. A second hydrogenotrophic archaeal strain, Methanothermococcus okinawensis,
was also applied for the BMP process and tested in the 2L bioreactors in fed-batch mode. The first
preliminary results are presented here and they show that Methanothermococcus okinawensis was able
to grow and to produce methane, but the conditions in the reactor were limiting the culture growth and
the productivity. On the base of the considerations on the TE importance, Methanothermococcus
okinawensis was also cultivated at increasing concentration of TE in closed batch mode in order to
examine the effects on its performances. It was found that higher TE amount boosted both growth and
CH4 productivity in closed batch cultures compared to standard concentration of TE. Therefore,
varying the TE concentration within the tolerance thresholds could be a turning point for producing
high biomass concentrations and high MER.
References:
Balch, W.E. et al., 1979. Methanogens: reevaluation of a unique biological group. Microbiological
reviews, 43(2), pp.260–96.
32
Bernacchi, S., Rittmann, S., et al., 2014. Experimental methods for screening parameters influencing
the growth to product yield (Y(x/CH4)) of a biological methane production (BMP) process performed
with Methanothermobacter marburgensis. AIMS Bioengineering, 1(2), pp.72–87.
Bernacchi, S., Weissgram, M., et al., 2014. Process efficiency simulation for key process parameters
in biological methanogenesis. AIMS Bioengineering, 1(1), pp.53–71.
Bernacchi, S., Krajete, A. & Herwig, C., 2016. Experimental workflow for developing a feed forward
strategy to control biomass growth and exploit maximum specific methane productivity of
Methanothermobacter marburgensis in a biological methane production process (BMPP). AIMS
Microbiology, 2(3), pp.262–277.
Bernacchi, S., Weissgram, M. & Wukovits, W., 2014. Process efficiency simulation for key process
parameters in biological methanogenesis. AIMS.
Götz, M. et al., 2016. Renewable Power-to-Gas: A technological and economic review. Renewable
Energy, 85, pp.1371–1390.
Groenestijn, J.W. van & Kraakman, N.J.R., 2005. Recent developments in biological waste gas
purification in Europe. Chemical Engineering Journal, 113(2–3), pp.85–91.
Hoekman, S.K. et al., 2010. CO2 recycling by reaction with renewably-generated hydrogen.
International Journal of Greenhouse Gas Control, 4(1), pp.44–50.
Kennes, C., Rene, E.R. & Veiga, M.C., 2009. Bioprocesses for air pollution control. Journal of
Chemical Technology & Biotechnology, 84(10), pp.1419–1436.
Kim, D.-H., Shin, H.-S. & Kim, S.-H., 2012. Enhanced H2 fermentation of organic waste by CO2
sparging. International Journal of Hydrogen Energy, 37(20), pp.15563–15568.
Lee, J.C. et al., 2012. Biological conversion of CO2 to CH4 using hydrogenotrophic methanogen in a
fixed bed reactor. Journal of Chemical Technology & Biotechnology, 87(6), pp.844–847.
Luo, G. & Angelidaki, I., 2012. Integrated biogas upgrading and hydrogen utilization in an anaerobic
reactor containing enriched hydrogenotrophic methanogenic culture. Biotechnology and
bioengineering, 109(11), pp.2729–36.
de Poorter, L.M.I., Geerts, W.J. & Keltjens, J.T., 2007. Coupling of Methanothermobacter
thermautotrophicus Methane Formation and Growth in Fed-Batch and Continuous Cultures under
Different H2 Gassing Regimens. Applied and Environmental Microbiology, 73(3), pp.740–749.
Porqueras, E.M., Rittmann, S. & Herwig, C., 2012. Biofuels and CO 2 neutrality: an opportunity.
Biofuels, 3(4), pp.413–426.
Rittmann, S., Seifert, A. & Herwig, C., 2015. Essential prerequisites for successful bioprocess
development of biological CH4 production from CO2 and H2. Critical reviews in biotechnology,
35(2), pp.141–51.
Rittmann, S., Seifert, A. & Herwig, C., 2012. Quantitative analysis of media dilution rate effects on
Methanothermobacter marburgensis grown in continuous culture on H2 and CO2. Biomass and
Bioenergy, 36, pp.293–301.
Rittmann, S.K.M.R. et al., 2015. One-carbon substrate-based biohydrogen production: Microbes,
mechanism, and productivity. Biotechnology Advances, 33(1), pp.165–177.
Rittmann, S.K.-M.R. et al., 2015. One-carbon substrate-based biohydrogen production: Microbes,
mechanism, and productivity. Biotechnology Advances, 33(1), pp.165–177.
Schonheit, P., Moll, J. & Thauer, R.K., 1980. Growth parameters (Ks, µmax,Ys) of Methanobacterium
thermoautotrophicum. Archives of Microbiology, 127(1), pp.59–65.
33
Friday 11th November 2016, 13:20-14:00
CO2 CONVERSION TO BUTANOL BY MICROBIAL ELECTROSYNTHESIS
Sophie Thallner, Christine Hemmelmair, Silvia Martinek, Wolfgang Schnitzhofer, Marianne
Haberbauer
acib GmbH
The project CO2TRANSFER aims the synthesis of alcohol like butanol by using CO2 and electrons in
a microbial electrosynthesis cell (MES). This new approach addresses two problems: (I) Up to now it
is not possible to effectively store electricity on a large scale. (II) The emission of carbon dioxide
contributes to the greenhouse effect. The new technology offers a possibility of storing electricity from
renewable energies like wind, water and solar energy in an environmentally friendly way. For the
reduction of CO2 to butanol 24 electrons are needed.
In the first project stage the best suited microorganisms will be identified by cultivating them under a
H2 and CO2 atmosphere. Autotrophic bacteria such as Clostridium carboxidivorans as well as
microorganisms such as Clostridium saccharoperbutylacetonicum, which are known for acetone-
butanol-ethanol fermentation will be used. ABE fermentative microorganisms are saccharolytic
clostridia normally grown on glucose or complex carbohydrates. In this project the metabolic pattern
of these microorganisms will be observed when they are provided with CO2 and electrons. Beside pure
also mixed cultures will be studied. In the second project stage suitable microorganisms will be tested
for their ability to grow on electrode materials and take up electrons directly from the cathode instead
of H2. If the direct approach turns out not being possible, it will be tried to produce butanol via an
intermediate using a co-culture. Sporomusa ovata produced acetate via direct electron transfer, which
was proved by Nevin et al, 2010. In a second step acetate will be the source for the production of
alcohol by microorganisms. All experiments are performed in two compartment cells (2 x 250 mL)
with anode and cathode chamber separated by a Nafion membrane, allowing proton transport. A
carbon felt (2.5 x 9 x 0.6 cm) serves as working electrode and a Ag/AgCl (in 3M KCl) electrode is
applied as reference electrode. As counter electrode a DSA is used.
References:
Nevin P.K., Woodard T.L., Franks A.E., Summer Z.M., Lovely D.R., 2010. Microbial
electrosynthesis: feeding microbes electricity to convert carbon dioxide and water to multicarbon
extracellular organic compounds. mBio 1(2):e00103-10. doi:10.1128/mBio.00103-10.
34
Friday 11th November 2016, 13:20-14:00
EXPERIMENTAL INVESTIGATIONS ON IN-SITU METHANANTION IN A LABORATORY
REACTOR - PRELIMINARY RESULTS
Sebastian Schnyder, Rolf Warthmann, Judith Krautwald and Urs Baier
Centre for Environmental Biotechnology, ICBC Institute of Chemistry and Biotechnology,
Zurich University of Applied Sciences, 8820 Wädenswil, Switzerland
Biological in-situ methanation by metered H2 addition seem to be an elegant and easy method for
biogas upgrading in anaerobic digestion (AD) process, such as a biogas plant or a WWTP sludge
digester. Ideally, the product gas formed is a nearly pure biomethane, by what complex downstream
gas purification may not be necessary for gas grid injection. However, the effective operation of the
method is discussed controversial. Thermodynamically, an inhibition of the AD process may be
expected. Our aim was to study experimentally the in-situ methanation process in a controlled
laboratory reactor. Hydrogen was added in a reactor filled with mesophilic municipal digester sludge
and the produced gases were monitored quantitatively and qualitatively by a micro GC. Parameters
such as the stirring rate were adjusted for a good transition of H2 to the liquid phase. First results
showed that H2 gas was consumed up to 94 %, but the product gas was not significantly changed. By
pulsed H2 additions (10 min gassing periods) a methane forming rate of 0.15 [mmol*L-1
*h-1
] was
determined for WWTP digester sludge, which is in fact relatively low. There are indications of an
inhibition of AD, overlapped by the in-situ methanation reaction, which could not be clearly resolved.
Because of the H2 consumption rate was higher than the methane production rate, the formation of
other products than methane such as acetate by homoacetogens cannot be excluded. Further studies
must try to better differentiate the microbial processes which may occur simultaneously.
References: Baier, U.; Krautwald, J. (2016) Biologische Methanisierung: Methanogenese als
mikrobiologische Alternative zur katalytischen Methanisierung. Aqua & Gas, 96, 7/8. 18-23.
35
Friday 11th November 2016, 13:20-14:00
BIOMETHANATION OF H2 IN ANAEROBIC DIGESTERS FOR BIOGAS UPGRADING
Alba Serna-Maza
Faculty of Engineering and the Environment, Highfield Campus, University of Southampton,
The combination of renewable-driven electrolytic H2 production and anaerobic digestion can increase
the CH4 content in biogas to > 95% by biochemical reduction of its CO2. This technology utilises
mainly hydrogenotrophic methanogenesis through H2 injection, in order to maximise the conversion of
the available carbon in waste biomass, increase the CH4 yield by 40% and reduce the CH4 slippage
characteristic of physicochemical upgrading technologies. A range of process configurations is
possible, with carbon supplied from external or internal sources. In all cases, the gas-liquid mass
transfer of hydrogen has been identified as a limiting factor, and presents a significant engineering
challenge. My primary research interest is to maximise the H2 mass transfer from the gas phase into
dissolved form, which will be available to microorganisms. For this purpose, different dissolution
techniques and experimental conditions will be tested with and without microbial activity in an
anaerobic digester.
This hybrid process can increase the overall efficiency of carbon conversion contained in the waste
biomass to a gaseous fuel product and eliminate the need for downstream biogas upgrading, while
improving the process economics.
36
Friday 11th November 2016, 14:20-14:40
A NEW METHOD FOR INDIRECT QUANTIFICATION OF METHANE PRODUCTION VIA WATER
PRODUCTION USING HYDROGENOTROPHIC METHANOGENS
Ruth-Sophie Taubner and Simon K.-M. R. Rittmann
Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems
Biology, University of Vienna, Austria
[email protected]; [email protected]
The broad diversity of microorganisms which are able to tolerate as well as to withstand extreme
environmental conditions leads to the hypothesis that certain organisms could be found in
extraterrestrial habitats (e.g., Taubner et al., 2015). The obligate anaerobic methanogenic archaea
belong to these intriguing microorganisms. In the astrobiological literature, methanogens are described
to be potential candidates to inhabit Mars (e.g., Kral et al., 2011) or icy moons like Enceladus (e.g.,
McKay et al., 2008). Among them, hydrogenotrophic methanogens produce methane (CH4) and water
as a metabolic by-product from molecular hydrogen and organic compounds like carbon dioxide.
There are several ways to quantify growth and/or CH4 production of hydrogenotrophic methanogens,
e.g. by determining optical density or CH4 quantification by using gas chromatography, respectively.
In general, these methods require expensive instruments and/or they are time-consuming. Here, we
present a novel method for indirect quantification of methane evolution rate by measuring water
evolution rate (Taubner & Rittmann, 2016). This method was established in serum bottles for
cultivation of methanogens in closed batch cultivation mode. Water production was determined by
measuring the difference in mass increase in an isobaric setting.
We show that this method is extremely accurate and precise and may be used to rapidly screen
methanogens regarding their CH4 production potential. Moreover, this method can be applied for
examining CH4 production from psychrophilic, mesophilic, thermophilic, and hyperthermophilic
hydrogenotrophic methanogens with astrobiological relevance.
References:
Kral, T.A., Altheide, T.S., Lueders, A.E., Schuerger, A.C., 2011. Low pressure and desiccation effects
on methanogens: Implications for life on Mars. Planetary Space Science, 59, 264–270.
McKay, C.P., Porco Carolyn C., Altheide, T., Davis, W.L., Kral, T.A., 2008. The Possible Origin and
Persistence of Life on Enceladus and Detection of Biomarkers in the Plume. Astrobiology, 8, 909–919.
Taubner, R.-S., Schleper, C., Firneis, M.G., Rittmann, S.K.-M.R., 2015, Methanogenic Life in the
Solar System: an Assessment of Methanogen (Eco-)physiology in the Context of Recent
Astrobiological and Planetological Studies, Life, 5 (4), 1652–1686.
Taubner R.-S. & Rittmann S. K.-M. R., 2016, Method for the indirect quantification of methane
production via water production using hydrogenotrophic methanogens. Frontiers in Microbiology, 7,
532.
37
Friday 11th November 2016, 14:40-15:00
DECENTRALIZED SOLUTIONS AS PART OF THE ENERGY AND NUTRIENT SELF-SUSTAINING
FUTURE
Anni Alitalo, Marko Niskanen, Erkki Aura
Qvidja Kraft AB
The greenhouse effect, climate change, awareness of the continuing depletion of fossil fuel reserves,
increasing energy demands and the environmental impacts of current energy sources have together
stimulated the search for sustainable, alternative energy sources and innovative fuel technologies. The
rapid development of renewable energy technologies and falling prices has created a new situation and
the opportunity for the development and implementation of new solutions.
Decentralized solutions
will increase the energy
self-sufficiency and
improve energy security.
In this a significant role
has the utilization of the
side streams from the
agriculture and forest
sector.
A real scale demonstration
platform will be built in
Qvidja for testing and
demonstration of different
processing options and
application combinations.
38
Friday 11th November 2016, 15:00-15:20
DEVELOPMENT OF A HIGH PRESSURE PROCESS TO COUPLE BIOLOGICAL HYDROGEN AND
METHANE PRODUCTION
Lisa-Maria Mauerhofer, Barbara Reischl, Tilman Schmieder, Simon Rittmann
Archaea Biology and Ecogenomics Division of the Department of Ecogenomics and Systems
Biology, University of Vienna
To ensure a reliable supply of fuels and energy, Europe is expanding proceedings for renewable
electricity production. Partially, generated energy cannot be retained, because of the limiting gird
capacity of the electricity networks. Moreover the society needs fuels for their mobility. The Power-to-
Gas technology can be used for the electrolytic conversion of water to hydrogen (H2) under the
application of excess power to produce fuel by using leftover energy. Through biological methane
production (BMP), H2 and carbon dioxide can be converted to methane.
The BioHyMe project differs in a biological and chemical way from conventional methanisation
procedures, because microorganisms are examined at various pressure levels by using “closed batch”,
“fed batch” and continuous culture. The optimization of process conditions and the selective
prioritization of microorganisms should lead to significant scientific developments in the field of high
pressure biology. Currently, only hydrogentrophic methanogens are used for BMP processing.
However, many microorganisms have not been characterized under high pressure conditions yet.
39
Friday 11th November 2016, 15:20-10:40
CO2 CONVERSION TO METHANE BY MICROBIAL ELECTROSYNTHESIS
C.Hemmelmair, Marianne Haberbauer, Sophie Thallner, Silvia Martinek, Wolfgang Schnithofer
Acib GmbH, Stahlstraße 14, 4020 Linz, Austria
Microbial electrochemical technologies (METs) combine microorganisms and electrodes. Thereby,
electrodes serve as electron acceptor or donor in a microbial metabolism. Microbial electrosynthesis
can produce energy carriers like methane, organic acids and alcohols from electricity and carbon
dioxide. This new approach offers the possibility to store electricity from renewable energy apart from
local solutions like pumped-storage hydropower plants. Furhermore, carbon dioxide, which
contributes to the greenhouse-effect is used as a source chemical. Here we present a first set of
experimental results, which were obtained by running a microbial electrosynthesis cell for the
production of methane. A mixed culture was isolated from digestate of a municipal sewage plant.
After enrichment and adaption with H2/CO2 (80:20), the culture was tested in a two-compartment cell
operated in batch mode. Carbon felt was used as working electrode and a sheet of DSA as counter
electrode. The catholyte solution was a mineral medium and a phosphate buffer (pH = 7) was used as
anolyte solution. At the beginning a potential of – 800 mV (vs. Ag/AgCl) was applied to the MEC and
stopped only for LSV and CV measurements. In addition experiments with a more negative potential
and different operating temperatures were carried out. In sum the chamber was operated for nearly
three years. The results of this long term experiment will be presented. Also a microbial
characterization of the mixed culture was done three times, which showed that the composition of the
culture was very stable.