Speciality Chemicals Magazine - June 2009 digital edition

Biotechnology
Speciality Chemicals Magazine June 2009 43
www.specchemonline.com
DSM and Roquette, a French starch and starch-
derivatives company, have combined to develop a
fermentation process for succinic acid that will lead to
an energy savings of 30-40% over the current petro-
chemical route, according to Christophe Rupp-
Dahlem, programme director for natural-based
chemicals at Roquette. Their partnership was estab-
lished in 2006 under the French BioHub programme.
Roquette is applying its expertise in producing
large quantities of gluconic acid via fermentation to
the development of a route to succinic acid. DSM,
meanwhile, contributes its capabilities in white
biotechnology, materials engineering, resin and
polymer technologies and fine and speciality chem-
ical synthesis, which it says are important for the
development of downstream markets.
The fermentation process incorporates develop-
ments from both firms, as well as technology licensed
from two US universities. At Rice University,
Professors Ka-Yiu San and George Bennett, of the
Bioengineering and Biochemistry departments
respectively, have developed a genetically engineered
Escherichia coli strain that produces close to the max-
imum theoretical yield of succinic acid from glucose.
In a typical fermentation process, 1 mole of suc-
cinic acid is produced per mole of glucose and the
process requires 2 moles of NADH, according to
San. The Rice bacteria have been engineered to
produce succinic acid by two separate metabolic
pathways referred to as the glyoxylate and fermen-
tative pathways.
The former requires less NADH, so more succinic
acid can be produced. With the latter, oxaloacetate
is converted to malate, fumarate and then succinate,
a process that requires 2 moles of NADH.
The NADH is generated from glucose and no
more than 2 moles can be produced per mole of
the sugar molecule. Therefore, only if 100% of the
carbon is consumed in the fermentative pathway
can the maximum theoretical yield of 1 mole of suc-
cinic acid/mole of glucose be produced.
In their engineered strain, San and Bennett deacti-
vated enzymes that affect NADH availability for suc-
cinic acid production and activated the glyoxylate
pathway so that it would function simultaneously with
the fermentative pathway under anaerobic conditions.
This strain requires only 1.25 moles NADH/mole of
succinic acid produced, so the overall yield of succinic
acid rises to 1.6 moles/mole of glucose.
The fermentation is a two stage process, with an
aerobic growth phase followed by an anaerobic fer-
mentation step to metabolise the glucose. It oper-
ates efficiently under a CO2 atmosphere at mild
temperatures (37�C).
At the University of Georgia, Elliot Altman,
director of the Centre for Molecular BioEngineering,
Mark Eiteman, professor of engineering and gradu-
ate student Ravi Gokam also worked with E. coli
bacteria. They discovered that the enzyme pyruvate
carboxylase could divert most of the by-products
that stemmed from pyruvic acid, resulting in the
elimination of by-product formation and a dramatic
increase in the production of succinic acid.
Pyruvic acid is produced from phosphoenolpyru-
vic acid (PEP), which is also the precursor to
oxaloacetic acid, the compound that is converted
into succinic acid. Most PEP forms pyruvic acid,
which is metabolised into several materials neces-
sary for cell growth. In the Georgia process, the
pyruvate carboxylase enables more pyruvic acid to
be diverted to oxaloacetic acid and ultimately suc-
cinic acid, with yield improvements of 40%.
Downstream processing is critical for DSM and
Roquette, because many of the potential applica-
tions for bio-based succinic acid will require very
high purity material. Several steps are required to
isolate and purify the succinic acid from the fermen-
tation broth. The firms believe that this situation
presents an opportunity for establishing a competi-
tive advantage over other biobased routes.
The actual process for recovering succinic acid will
depend on the form of the product desired and the
intended downstream application. Succinic acid can
be recovered either as an acid or the salt of an acid,
for example ammoninium succinate or potassium
succinate. Typically, membrane separation technolo-
gies are being considered for the bulk of succinic
acid purification strategies, according to Eiteman.
Knowledge of downstream markets will also be
an important advantage, according to James
Iademarco, VP of bio-based chemicals and biofuels
in DSM's White Biotechnology programme.
"Good technology, while an imperative require-
ment, is not enough on its own. It is equally impor-
tant, once a cost-effective process has been devel-
oped, to identify potential customers and work
closely with them to create products that meet mar-
ket needs," he comments.
Currently Roquette and DSM are producing sev-
eral hundred kilograms/year at a pilot facility at the
former's Lestrem site in France. Customers are being
sampled with material that is of high enough purity
for use in polymer and fine chemical synthesis.
Retrofitting of a demonstration plant at Roquette's
biorefinery at Lestrem is under way. This will produce
several hundred tonnes/year of material. It is expect-
ed to be operational by the end of 2009 and to move
to full-scale commercial production in 2011 or 2012.
Meanwhile, both companies are investigating
other biorenewable products. DSM, according to
Iademarco, has the toolbox to develop a broad port-
folio of bio-based products including other building
blocks and related compounds.
Roquette, again through BioHub, has developed
a process for the production of the diol isosorbide,
which can be polymerised with succinic acid to form
polyols with applications in powder coatings.
Isosorbide is produced by the dehydrogenation of
sorbitol, which is generated from glucose.
DNP Green Technology and Agro-Industrie
Recherches et D�veloppements (ARD), the R&D cen-
tre of the Champagne Ardenne agricultural coopera-
tives that grow cereals, sugar and alfalfa, formed
Bioamber, a 50-50 joint venture, in March 2008. It
has been producing succinic acid on a pilot scale in
80 m3 fermenters and will begin operating a 2,000
tonnes/year demonstration plant at its Pomacle-
Bazancourt biorefinery near Reims later this year.
By 2010, Bioamber expects to have begun licens-
ing its turnkey packages for the production of suc-
cinic acid and derivatives. Its efforts to commercialise
this technology in the global bio-renewable chemi-
cals market won it the 2009 Frost & Sullivan
Technology Innovation Award in March.
The demonstration plant will be able to use a vari-
ety of feedstocks, including wheat, corn, sugar cane,
rice, cellulose and glycerin. All of these and other
alternative materials will be evaluated. "This flexibili-
ty with regard to supply of raw materials provides a
significant advantage," claims Dunuwila.
Bioamber's fermentation process relies on tech-
nology licensed from the DOE and is cost-competi-
tive with petrochemical processes, the company
claims. This strain of E. coli is optimised to produce
succinic acid under anaerobic conditions, using CO2
feed, with limited production of by-products.
Dunuwila adds that Bioamber has achieved
advances in downstream separation and purification
technologies, which "has been a critical part of mak-
ing this route to succinic acid cost competitive". The
demonstration plant will confirm the technology and
provide greater quantities of material for sampling
to customers.
The company is taking a licensing approach,
emphasising its core technologies in the fermenta-
tion and isolation of succinic acid. It does not intend
DSM and Roquette are bringing on a pilot bio-succinic acid plant at Lestrem

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