DR ED VAN NIEL AND SUDHANSHU PAWAR
A new protocol for bioenergy
Dr Ed van Niel and doctorate student Sudhanshu Pawar are modifying a promising bacterium to
maximise its productivity and therefore throughput in manufacturing hydrogen from green sources
As a microbiologist, I recognise that there
is high potential in biological hydrogen
production for energy purposes. However, we
know very little about the physiology behind
hydrogen production from biomass.
We recognised from studies on enzyme kinetics
that C. saccharolyticus is relatively tolerant to
high partial hydrogen pressures, which led us
to test the organism in bioreactor systems with
large contact areas between the liquid and gas
phases that operate without sparging gases.
Our preliminary results were successful, which
brings application a step closer.
How does Caldicellulosiruptor
saccharolyticus produce hydrogen?
What are the existing technologies for
hydrogen production?
This extreme thermophile can use a very broad
spectrum of sugars, including poly-, oligoand mono-saccharides, as substrate. Under
strict anaerobic conditions, it ferments these
sugars to hydrogen with acetic acid and carbon
dioxide as by-products. This happens without
light, hence the name ‘dark fermentation’.
An interesting feature of hydrogen is that there
are many production methods. However, each
possesses its own limitation, thus hampering
commercial application: water splitting
requires more input energy than it outputs;
thermochemical biomass conversions produce
undesirable byproducts such as tars; biological
biomass conversion runs into thermodynamic
limitations and low productivity; and hydrogen
production through photosynthesis is limited by
the requirement for large surface areas and thus
high investment costs for bioreactor materials.
Can you begin by explaining your current
interest in hydrogen production from biomass?
Could you briefly outline the conclusion of your
most recent report on hydrogen production?
As hydrogen gas is poorly soluble in
water, it is relatively easy to extract.
Usually, one applies sparging gas to
actively drive out the hydrogen to prevent
oversaturation, otherwise hydrogen inhibits
its own production through building up a
thermodynamic constraint. The sparging
gas needs to be chosen wisely: it should be
cheap, not affect fermentation, and should
be easily separated from the hydrogen in the
downstream process. None of the available
gases comply with all those criteria.
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INTERNATIONAL INNOVATION
When economically successful, these
technologies will not necessarily compete; each
has its own niche and they can work side by side.
extreme thermophiles and photofermentation
using purple non-sulphurous bacteria which
convert the volatile fatty acids in the effluent
from the dark fermentation into hydrogen and
carbon dioxide.
The process is not yet cost-effective, partly
due to the high costs of nutrients and
materials for the photo-bioreactor, and partly
due to low productivity in both fermentation
processes. Solutions to make it more costeffective exist and need further investigation.
In simple terms, what is the HYVOLUTION
process?
Can you explain the genetic and
physiological modifications required to
improve hydrogen production?
It aims to maximise conversion of sugar
residues in biomass to hydrogen by applying
a combination of dark fermentation with
C. saccharolyticus is naturally able to produce
hydrogen with considerably good yields
but production rate is the real bottleneck.
DR ED VAN NIEL AND SUDHANSHU PAWAR
It is vulnerable to high osmotic pressure so
performance in the presence of the sugar
concentrations required for economic hydrogen
production is weak. Under those conditions it
also produces unwanted byproducts, such as
lactic acid. To circumvent these issues, genetic
engineering is required to obtain improved
mutants by, for example, deleting the genes
that encode for lactic acid synthesis.
How does your knowledge of lactic acid
bacteria (LAB) complement your investigation
into hydrogen-producing thermophiles?
Even though both LAB and these thermophiles
produce different products from sugar
fermentation, they have a common central
carbon metabolism. The metabolic pathways
involved are regulated both at gene level and
at enzyme level. Knowledge of how these
pathways are regulated in one may give insights
into how regulation takes place in the other.
What is the most significant challenge facing
microbiological research at present?
One big challenge is to develop a genetic
protocol, not only for C. saccharolyticus
but also for other thermophilic hydrogen
producers. Without such a protocol, dedicated
mutants cannot be created, which will slow
down deeper understanding of their physiology
and their industrial application. I foresee that,
with perseverance and creativity, a genetic
protocol will be found for C. saccharolyticus,
most probably within this project.
Another challenge is to design a cost-effective
bioreactor configuration that produces
hydrogen with both high productivity and
yields. For a cost-effective process, the
challenge is in finding the right technology.
However, this needs input from reactor
technologists working in close collaboration
with microbiologists.
Power cell
The Swedish Research Council is funding development at Lund
University of a genetic protocol for producing mutant strains of a
heat-loving bacterium that could be the key to establishing costeffective industrial scale hydrogen production without fossil fuels
THE NEED FOR an alternative source to
fossil fuels for powering an energy-hungry
world is urgent for many political, social and
environmental reasons: high oil prices, the
concentration of the largest oil producers in
unstable countries in the Middle East, the
dwindling supply of fossil fuels and harmful
effects of their use on the environment, to name
a few, all combine to make the continuing use of
fossil fuels increasingly unfeasible.
hydrogen is a very clean energy carrier – water
is the only waste product,” states Dr Ed van Niel,
an Associate Professor in applied microbiology
from Lund University. “But today, about 96
per cent of hydrogen is derived directly from
processing fossil fuels, and the remainder is
through electrolysis using electricity generated
with fossil fuels, wind or hydropower. There is
to date hardly any hydrogen produced from
renewable sources.”
Among the competing technologies for
replacing fossil fuels, biomass and hydrogen,
especially hydrogen fuel cells for powering road
vehicles, are promising contenders. However,
each has limitations: the need to utilise land
that could be used for growing food in the case
of land-based biomass; and the fact that there
is at present no cost-effective and sustainable
method of generating sufficient volumes in the
case of hydrogen.
Van Niel and doctorate student Sudhanshu
Pawar are currently working on a project that
aims to determine optimally modified variants
of a bacterium named Caldicellulosiruptor
saccharolyticus to pave the way to large-scale
generation of hydrogen from biomass.
A RENEWABLE ENERGY SOURCE
Though hydrogen powered vehicles are already
available and some states in the US and countries
in Europe have already set up or are in the process
of building networks for distributing hydrogen
fuel cells, the means of production of hydrogen
has so far relied mainly upon fossil fuels: “Where
hydrogen is used, no diffusive carbon dioxide is
produced. From an environmental perspective,
C. SACCHAROLYTICUS
C. saccharolyticus was originally isolated from
a hot spring in New Zealand 25 years ago. It is
an obligate anaerobic extremely thermophilic
bacterium, meaning that it grows best at very
high temperatures – around 70 °C – and does
not require molecular oxygen to grow. In a
suitably oxygen-deprived environment, when
exposed to a mixture of polymeric sugars –
such as pectin and (hemi)cellulose – in organic
material, it triggers fermentation and this gives
rise to hydrogen. C. saccharolyticus can yield
hydrogen from many carbon-based materials,
WWW.RESEARCHMEDIA.EU 13
INTELLIGENCE
DEVELOPMENT OF A GENETIC
SYSTEM FOR THE EXTREME
THERMOPILE CALDICELLULOSIRUPTOR
SACCHAROLYTICUS: THE NEXT
STEP IN IMPROVING HYDROGEN
PRODUCTIVITY
OBJECTIVES
Gábor Rákhely, Department of
Biotechnology, University of Szeged,
Hungary
Van Niel has worked with this bacterium for
more than 10 years, and the more he has
discovered about it the more convinced he
has become that it has immense potential for
industrial scale hydrogen production: “I started
by screening the various thermophilic hydrogen
producers then available and the performance
of C. saccharolyticus really jumped out in the
tests,” he reflects. “C. saccharolyticus was the
first of its genus to have its genome sequenced –
we annotated it in an international consortium.
Knowledge of its genome sequence facilitated
and accelerated insights into its metabolism
and it became our thermophilic hydrogen
producer of choice.”
Pieternel Claassen, Agrotechnology & Food
Sciences Group (DLO-FBR), Wageningen,
The Netherlands
HYVOLUTION: HYDROGEN
PRODUCTION FROM BIOMASS
To further improve efficiency of
C. saccharolyticus hydrogen production so that
the price of hydrogen becomes commercially
viable.
PARTNERS
Karin Willquist, Former PhD student and
postdoc
Ahmad Zeidan, Former PhD student
FUNDING
Swedish Research Council
CONTACT
Dr Ed van Niel
Principal Investigator
Applied Microbiology
Lund University
PO Box 124
SE-221 00 Lund
Sweden
T +46 46 222 9693
E [email protected]
ED VAN NIEL has a doctoral degree in
Microbiology from Technical University
Delft in 1991. Van Niel held different
postdoc positions at Wageningen University,
Groningen University and Lund University.
He currently is senior lecturer in quantitative
microbial physiology at Lund University.
SUDHANSHU PAWAR has a Master’s
degree in Biotechnology (Lund University,
Sweden, 2010). Pawar developed an
interest in academic research during the
Master’s thesis and is currently pursuing a
PhD study partly financed by the Swedish
Energy Agency and partly by the Swedish
Research Council.
14
with high yield per sugar molecule; and can
also tolerate relatively high partial pressures
of hydrogen, which obviates the need to
continuously strip off the hydrogen it produces
during fermentation.
INTERNATIONAL INNOVATION
Van Niel was part of the team on the EU Sixth
Framework Programme-funded HYVOLUTION
project, a five-year exploration of the
production of pure hydrogen from biomass
without the need for additional heating,
through the collaboration of 22 academic and
research institutes and enterprises from 10
European countries, South Africa, Turkey and
Russia. The objective was to develop a blueprint
for decentralised production of 10 to 25 per
cent of the European hydrogen requirement
from locally-produced biomass and thus to
ensure the safety of local energy supply. One
constraint was to produce hydrogen with zero
waste; the biomass used and any byproducts
created needed to be capable of being further
recycled, for example as animal feeds. Another
constraint was that the cost should not exceed
€10 per gigajoule.
The approach that the HYVOLUTION team
explored consisted of a four-stage process. In
the first stage, hemicellulose-rich biomass –
organic waste from other processes, such as
sugar beet detritus from sugar production,
and steamed potato peels produced from
industrial potato chip production – was pretreated to ready it for fermentation. In the
second stage, C. saccharolyticus was exposed
to the biomass to produce hydrogen via
anaerobic thermophilic, or dark fermentation;
the byproducts of this dark fermentation
process were CO2 and organic acids. In the third
stage, the effluent with organic acids produced
from the second stage were converted to
hydrogen and CO2 by phototrophic bacteria
– eg. purple non-sulphurous bacteria – via
a photofermentation process. And in the
last stage the gases produced were cleaned
and if necessary, their quality was upgraded.
The efficacy of fermentations with each
combination of biomass and C. saccharolyticus
or other Caldicellulosiruptor species members
was measured in terms of overall cost and
technological suitability.
The outcome was that processing biomass
with C. saccharolyticus delivered high
quality hydrogen but the productivity per
sugar molecule was lower than needed.
However, van Niel found that, when used in
combination, the synergy of different though
closely-related species of Caldicellulosiruptor
resulted in high efficiency, and better
productivity and yields of hydrogen.
The HYVOLUTION project proved that it
was indeed possible to produce high quality
hydrogen from biomass with the addition
of Caldicellulosiruptor bacteria species,
but there were two issues: under certain
conditions there is creation of lactic acid as
a byproduct during dark fermentation that
actually decreases the hydrogen yield; and
the bacteria did not perform well when the
concentrations of sugar required for economic
reasons were used.
From an environmental
perspective, hydrogen is a very
clean energy carrier – water is the
only waste product
Van Niel and Pawar are now using a plasmid
from C. kristjansonii as a vector to mutate the
genetic makeup of C. saccharolyticus to evolve
several Caldicellulosiruptor variant strains
compatible with the increased hydrogen
production capability required by industry.
They have analysed lactic acid bacteria in
parallel to this work: “Understanding the
operations of the central pathways in these
different, though metabolically similar,
organisms will speed up our understanding
of how to maximise hydrogen production
and yields,” van Niel states. Moreover, he
thinks that the Caldicellulosiruptor species
has further promise in other contexts: “As
well as hydrogen, the species produce various
hydrolases, such as cellulases, pectinases
and xylanases, which could potentially be
exploited as thermozymes in various industrial
processes.”
There is much to be done before sufficient
productivities can be realised in ‘green’ hydrogen
production: “So far, we have just scratched the
surface. There is a long road with challenges
ahead before industrial application is in sight,”
van Niel concludes.