SOLVENT PRODUCTION
The invention relates to a process and system for the production of a solvent using monophasic
5 solventogenic clostridia in a single stage process. More particularly, the process and system
relate to the use of a culture vessel for culturing monophasic solventogenic clostridia, wherein
cell growth of the clostridium is monitored and optimised by the addition of controlled amounts
of culture media and/or the removal of controlled amounts of solvent. The process and system
may particularly be used to produce solvents such as butanol, acetone and ethanol.
10
The butanol fermentation process utilises renewable bio-based feedstocks and is often referred
to as the acetone, butanol and ethanol (ABE) fermentation after its major chemical products.
The fermentation was first commercialised in the UK in 1916 and spread around the globe
during the 1st and 2nd world wars, mainly to produce acetone for munitions and butanol for
15 paint lacquers. The process fell out of favour in the US and EU in the 1950s when it struggled
to compete with petro-derived equivalents on cost, but persisted in China, Russia and South
Africa until the 1980s. Today, due to higher oil prices, concerns over the supply of oil and
environmental concerns over greenhouse gas (GHG) emission, the ABE fermentation is poised
for re-commercialisation. The fermentation route has the potential to replace petro-derived
20 butanol, acetone and hydrogen with cheaper, more sustainable and environmentally-friendly
chemicals. Indeed, global demand for bio-butanol has been stimulated by investment in new
plant in China. Over $200m has been invested to date, resulting in 0.3M tlyr of installed solvent
capacity with plans to expand to I M tlyr.
25 Traditional batch processes for the fermentation of molasses and/or starch to produce ABE
have been practised for decades (Jones, D.T. and Woods, D.R. (1986) Microbiol. Rev. 50: 484-
524). Typically, batch fermentation produces approximately 18 g/L solvent in 72 hours. The
fermentation is relatively long because it occurs in two distinct stages (biphasic): the first phase
is a growth stage that results in acid production and a drop in pH; the second phase is a survival
30 stage during which the acids are re-assimilated to solvents to neutralise the pH. The cells also
prepare for sporulation. The switch in metabolism is triggered by the acid concentration in the
fermentation broth and/or the drop in pH. The final solvent titres are relatively low in comparison
with yeast ethanol fermentations, and this results in low volumetric productivities (18172 which
equates to approximately 0.25g solventlL/hr). Solvent titres are limited by the relatively low
35 amounts of sugar that can be fermented. Low solvent productivities are a major drawback with
the traditional batch process and many attempts have been made to overcome this limitation.
Variations on batch culture processes including fed-batch processes which attempt to either
increase solvent titre or to reduce the fermentation time have been developed with little success.
Due to the low productivity obtained with batch fermentation processes, several single-stage
5 continuous processes have been proposed (e.g. Jones and Woods (1986), supra) which aim to
provide a continuous flow of feed media in order to achieve a growth rate close to the maximum
growth rate for several weeks, thus offering improving solvent production rates and reduced
downtime. However, in practice, this is difficult to achieve with biphasic solventogenic clostridia
because solvent production is not directly linked to growth and cultures wash out at relatively
10 low dilution rates. Methods of retaining high cell concentrations in the reactor using cellrecycling
and/or immobilisation have been demonstrated at lab-scales, but are difficult to
implement on commercial scales (e.g. Qureshi & Maddox (1987), Enz. Microb. Technol. 9(1 I),
668-67; Maddox (1989) Gen. Eng. Rev., 7, 189-220; Maddox et al. (1993) In: The clostridia and
biotechnology, (eds) D.R. Woods, Butterworth-Heinemann, Boston. 343-369; Gapes et al. (1996)
15 Appl. Env. Microbiol. 62: 321 0-321 9).
Other continuous configurations based on two- or multi-stage vessels have been proposed in an
attempt to separate and control the biphasic fermentation, but none have proved successful due
to difficulties in controlling the flow rates and corresponding dilution rates to maximise solvent
20 productivity (see below). Mixed cell populations of acidogenic and solventogenic cells quickly
build up in both the first and second stage, providing oscillations in growth and solvent formation.
For free cell suspensions if flow rates are not controlled, this typically results in cultures that
wash out if growth slows or perform sub-optimally at low flow rates. In addition, cultures tend to
degenerate quickly losing the ability to produce solvents (Woolley and Norris (1990) J. Appl.
25 Bacteriol. 69: 71 8-728; Jones and Woods, (1 986) supra; Kashket and Cao (1 995), FEMS
Microbiol. Rev., 17, 307-31 5; Afschar ( I 990) DE 3905624 A1 ). Afschar ( I 990) proposed a two
stage molasses fermentation process for the production of butanol and acetone, which is
characterized by a chemostat with substrate limitation at the first stage to produce cells. A twostage
continuous cultivation for clostridia was also proposed by Mutschlechner et al. (J. Mol.
30 Microbiol. Biotechnol. (2000) 2(1):101-105). In this process, the system was designed to mimic
the two phases of batch culture growth by using a first stage to grow the cells acidogenically as
quickly as possible and then transferring cells to the second stage at the 'acid break point'. The
second vessel is larger to provide sufficient residence time to complete solvent production. In
both of these examples, the flow rates into the first and second stage were kept constant and
35 not regulated in response to any growth related signals such as changes in pH or cell density.
Two-stage continuous cultures have also been described with immobilized biomass or cellretention
(e.g. Maddox et al. (1993), supra; Gapes et al. (1996), supra). These authors used a
fixed dilution rate and describe immobilisation methods to retain the microbes in the reactor and
to prevent them from washing out. These cultures can be run at high dilution rates and
5 productivities but the final solvent concentrations tend to be too low for cost-effective recovery.
Also, in both examples, solvent titres and productivity oscillated widely. The main drawback
with immobilisation is the expense and difficulty to scale. Operation over prolonged periods
and/or use of feedstocks with particulates is problematical due to blockages and fouling of the
support matrices. In addition, these systems are prone to contamination and difficult to keep
10 sterile.
A sequential fermentation process has also been commercialised in China wherein a continual
or sequential batch process is used with eight fermentors linked together. The first two vessels
(vessels 1 and 2) are biomass generators and are continuously re-seeded with fresh culture
15 every 24 hours (via a conventional seed train). The biomass generators, once seeded, are fed
continuously with substrate (feedstock) and, when full, the liquid flow goes forward to vessels 3
and 4 (which work in parallel). These two vessels then feed the rest of the fermentation train,
which consists of a sequential series of connected vessels (usually four) giving a total process
residence time of 72 h in the eight fermentation vessels. This complicated process has been
20 designed around the biphasic nature (& limitations) associated with C. acetobutylicum which
generally requires continual re-seeding to avoid microbe degeneration (due to loss of solvent
plasmid). This process is controlled manually, with very little scope to respond quickly to
process fluctuations. The continual Chinese process is described in Ni & Sun (2009), Appl.
Microbiol. Biotechnol., 83, 415-423.
25
To date, fermentation process technology has been developed for biphasic solvent producing
clostridia. The metabolism is characterised by rapid growth and acid formation and then a
transition during which growth slows, acids are re-assimilated and solvent production starts.
This results in relatively long fermentation times.
30
There remains a need, however, to provide a process which provides early onset of solvents,
shorter fermentation times and, if fed continuously, continuous or extended solvent production
for periods in excess of 50 hours.
35 It is one aim of the invention, therefore, to provide a fed batch or continuous fermentation
process which optionally integrates solvent removal with cell and water recycling operations and
which enables high solvent productivity to be maintained for periods in excess of 50 hours. The
inventors have found that such an integrated system can be used to convert high
concentrations of sugar or other carbon feeds into solvents (acetone, butanol and ethanol) at
high yield over a prolonged period of time with minimal substrate inhibition.
5 This invention is based on the surprising discovery that certain solventogenic clostridia are in
fact not biphasic; they are monophasic. In batch fermentations monophasic solvent production
is characterised by simultaneous growth and solvent production with no obvious switch or
change in metabolism from acid to solvent production during the major growth phase. Indeed,
acids and solvents are produced simultaneously and acids do not tend to accumulate in the
10 culture media. In these clostridia, it is not necessary to wait for the second phase before
solvents are produced. For such clostridia, growth and solvent production can occur in a single
vessel. Solvent production can be controlled by monitoring cell growth or a growth-related
feature (e.g. production of gas, sugar utilisation or production of acid) and optimising the
conditions for cell growth, thus obtaining high cell densities and solvent titres. This approach
15 contrasts with previous approaches wherein the culture conditions have been optimised for
either growth in the acidogenic phase or solvent production in the solventogenic second phase.
The batch fermentation profiles for monophasic and biphasic solventogenic clostridia are shown
in Figures 2 and 3.
20
In one embodiment, therefore, the invention provides a single-stage process for the production
of a solvent, the process comprising the steps:
(a) culturing monophasic solventogenic clostridial cells in a liquid culture medium
25 in a culture vessel;
(b) monitoring cell growth of the monophasic solventogenic clostridial cells in the culture
vessel:
30 (c) continuously or semi-continuously introducing fresh culture media and/or nutrients
into the culture vessel:
(d) continuously or semi-continuously removing a stream or portion of liquid culture
medium comprising solvent(s) from the culture vessel and passing said liquid culture
3 5 medium to a solvent remover;
(e) maintaining or increasing cell density within the culture vessel;
wherein cell growth in the culture vessel is regulated and/or optimised by controlling:
(i) the amount or rate of fresh culture medium or nutrients which are introduced into the
culture vessel, and/or
5 (ii) the amount or rate of liquid culture medium comprising solvent(s) which is
removed from the culture vessel.
Preferably, the monophasic clostridium:
i) naturally displays simultaneous growth and solvent production during the major growth
10 phase of batch fermentations;
ii) has been chemically mutated to produce solvents during growth; or
iii) has been genetically modified to produce solvents during growth.
Preferably, the clostridium is a monophasic C. saccharoperbutylacetonicum.
15
Most preferably, the clostridium is a monophasic Clostridium saccharoperbutylacetonicum N1
strain, e.g. Nl-4(HMT) or Nl-504.
Preferably, step (b) comprises monitoring cell growth by one or more of:
20 (i) monitoring production of one or more gases (e.g. hydrogen and/or C02);
(ii) monitoring production of one or more acids;
(iii) monitoring the pH of the culture medium;
(iv) monitoring the utilisation of sugar;
(v) monitoring cell density; and
25 (vi) monitoring the production of one or more solvents.
Preferably, step (d) comprises continuously or semi-continuously removing a stream or portion
of the liquid culture medium from the culture vessel and passing the stream/portion via a cell
separator to a solvent remover, wherein:
3 0 (i) cells are removed from the liquid culture medium in the cell separator and the cells are
returned to the culture vessel (optionally via a cell seeder), and
(ii) wherein solvent is removed from the liquid culture medium in the solvent remover and
the residual liquid culture medium is returned to the culture vessel.
35 Preferably, the cell density is maintained or increased within the culture vessel by:
(i) recycling cells which are removed from the liquid culture medium comprising solvent(s)
back to the culture vessel; and/or
(ii) continuously or semi-continuously feeding cells from a cell seeder into the culture
vessel; and/or
(iii) immobilisation of some or all of the cells within the culture vessel.
5 The process of the invention may be operated in any suitable manner. For example, it may be
operated as a fed-batch process or any form of continuous process or perfusion process; or a
mixture of these types of processes.
In some embodiments, the process is operated in fed-batch mode. In this embodiment, the
10 microorganism is cultured under desired growth conditions in a batch mode for a suitable time,
e.g. about 20 hours until approximately half the sugars are consumed (e.g. 25-30 g/L). The
cells of the microorganism multiply and produce both acids and solvents. The initial volume of
the first batch stage of the process should preferably be about 70% (e.g. 65%-75%) of the total
working volume in the vessel and contain enough sugar (e.g. 55-65 g/L) and nutrients to sustain
15 growth and good solvent yields (e.g. greater than 0.3 g solventslg sugar). The fermentor is fed
with a concentrated sugar solution and nutrients in a volume that equates to about 30% (e.g.
25%-35%) of the culture vessel's working capacity and is fed at a rate designed to last for a
finite number of hours (e.g. between 10-75 hours) The microorganisms are maintained under
conditions that are suitable for them to grow optimally and produce solvents (e.g. temperature,
20 pH, redox).
The process of the invention is preferably operated under continuous culture conditions. As
used herein, the term "continuous culture conditions" refers to a process wherein the culture of
microorganisms in the culture vessel is capable of being maintained with a continuous or
25 substantially continuous flow of feed (nutrients) in steady state conditions (defined by high sugar
uptake rates and solvent productivity) for prolonged periods of time (e.g. >75 hours). Under this
scenario, some bleed may be required to maintain a constant volume in the culture vessel.
Solventogenic clostridia are bacteria that are capable of producing solvents such as acetone,
30 butanol and ethanol. Typically, acids are first produced and then re-assimilated into solvents in
a biphasic fermentation.
The process of the invention is a single stage process. As used herein, the term "single stage"
means that both acid(s) and solvent(s) are produced together in the same culture vessel. This
35 may be contrasted with a two-stage process wherein acids are produced in a first growth stage
and solvents are subsequently produced in a second stage, typically performed in two culture
vessels.
In most solventogenic clostridia (e.g. Clostridium acetobutylicum), fermentation is biphasic: the
first phase is characterised by cell growth, acid production and a fall in culture pH; in the second
phase, the acids are converted to solvents and the culture pH increases. The switch in
5 metabolism is triggered by the acid concentration in the fermentation broth andlor low culture
pH values.
The invention, however, relates to monophasic clostridia. As used herein, the term
"monophasic clostridia" means that the clostridia do not have distinct acid- and solvent-
10 producing phases. Monophasic solvent production is characterised by simultaneous growth
and solvent production; and with no obvious switch or change in metabolism. Acids do not tend
to accumulate in the culture media.
Preferably, the microorganism is a monophasic solventogenic clostridium.
15
Preferably the microorganism is a biphasic clostridium that has been converted to display
monophasic fermentation either by chemical mutagenesis or specific genetic modification or a
combination of both.
20 More preferably, the microorganism naturally displays monophasic metabolism without the need
for modification.
More preferably, the clostridium is a monophasic Clostridium saccharoperbutylacetonicum.
25 Most preferably, the clostridium is a monophasic C. saccharoperbutylacetonicum N1 strain, e.g.
N1-4(HMT) or N1-504, or a variant or derivative thereof. Such variantslderivatives will also be
monophasic solventogenic clostridia. Preferably, the variantslderivatives produce the same or
more acetone, ethanol andlor butanol compared to the clostridia from which they are derived,
under equivalent conditions.
3 0
Such variants may be produced, for example, by random mutagenesis or by recombinant
methods. Recombinant methods include insertional inactivation of genes through use of Type II
introns, e.g. Targetron (Sigma) and Clostron (e.g. WO 20071148091), and integration of new
genes through use of 'allele coupled exchange' (ACE, e.g. WO 20091101400). Random
35 mutagenesis techniques could also be used, as previously demonstrated for generating a more
acetate-tolerant strain (Yang, S., Land, M.L., Klingeman, D.M., Pelletier, D.A., Lu, T.Y., Martin,
S.L., Guo, H.B., Smith, J.C., & Brown, S.D. (2010). Paradigm for industrial strain improvement
identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae.
Proc Natl Acad Sci U S A., 107(23), 10395-400. 2010.), for generating a more ethanol-tolerant
strain (Shao, X., Raman, B., Zhu, M., Mielenz, J.R., Brown, S.D., Guss, A.M., & Lynd, L.R.
(201 1). Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains
5 of Clostridium thermocellum. Appl Microbiol Biotechnol. 92(3), 641-52. 201 1. Also: Brown, S.D.,
Guss, A.M., Karpinets, T.V., Parks, J.M., Smolin, N., Yang, S., Land, M.L., Klingeman, D.M.,
Bhandiwad, A., Rodriquez, M.Jr., Raman, B., Shao, X., Mielenz, J.R., Smith, J.C., Keller, M., &
Lynd, L.R. (201 1). Mutant alcohol dehydrogenase leads to improved ethanol tolerance in
Clostridium thermocellum. Proc Natl Acad Sci U S A., 108(33), 13752-7.201 I .) and for
10 generating a more amylolytic strain (Annous, B.A., & Blaschek, H.P. (1991). Isolation and
characterization of Clostridium acetobufylicum mutants with enhanced amylolytic activity. Appl.
Environ. Microbiol., 57(9), 2544-8. 1991 .)
Such variants may be tested by screening them in small-scale fermentations, i.e. looking for
15 variants that retain monophasic behaviour and produce the same or more acetone, ethanol
and/or butanol compared to the clostridia from which they are derived, under equivalent
conditions.
C. saccharoperbutylacetonicum strain N1-4(HMT) may be obtained from ATCC deposit number
20 27021. C. saccharoperbutylacetonicum strain N1-504 may be obtained from ATCC deposit
number 27022.
In some embodiments of the invention, the clostridium is not C. saccharoperbutylacetonicum
strain N1-4. C. saccharoperbutylacetonicum strain N1-4 was previously available as ATCC
25 deposit number 13564. This deposit is, however, no longer available.
The term "acid-producing" or "acidogenic" refers to the ability of the clostridium to convert a
substrate based on sugars and/or starches into RCOOH (wherein R is as defined below), for
example into acetate and/or butyrate.
30
The term "solvent-producing" or "solventogenic" refers to the ability of the clostridium to convert
a substrate based on sugars and/or starches into a solvent, preferably into one or more of
acetone, ethanol and/or butanol.
35 As used herein, the term "solvent" or "solvents" refers to low boiling point organic solvents or
their azeotropes which are capable of being produced by solventogenic microorganisms in a
liquid fermentation medium. Examples of such solvents include alcohols of formula R-OH,
wherein R is an aliphatic C1-C8 alkyl group or an aliphatic C2-C8 alkenyl group. The R group
may be branched or linear. Preferably, it is linear. The R group may be saturated or
unsaturated. Preferably it is saturated.
5 Preferred examples of alcohols of formula R-OH include methanol, ethanol, 2-methyl-propan-I-
01, 1,3-propanediol, I-butanol, 2-butanol, 2-methyl propan-2-01, pentanol, hexanol, heptanol
and octanol. A further example of a solvent has a formula R-CO including acetone ((CH3)2CO).
Preferably, the solvents comprise ABE solvents, i.e. acetone, I-butanol and ethanol. Most
10 preferably, the solvents comprise I-butanol or substantially I-butanol.
The clostridium may be an aerobic or an anaerobic microorganism. Preferably it is an
anaerobic or aero-tolerant clostridium. Most preferably, it is an aero-tolerant clostridium.
15 In some embodiments, the culture vessel may be inoculated with no special precautions taken
to exclude oxygen or no anaerobic purge. Furthermore, the culture vessel may be operated
with air (of normal atmosphere composition) in the head-space above the culture medium.
In some embodiments of the invention, the process is performed under conditions which are not
20 oxygen-free.
The acid- and solvent-producing clostridia which are used in the culture vessel may be from a
single strain or from a co-culture, preferably from a single strain.
25 In some embodiments, the clostridia are acid-tolerant. The clostridia can preferably tolerate
high concentrations of COOH. In this context, high concentrations of COOH may mean up to
15 glL acetic acid, and/or up to 10gIL lactic acid and/or up to 6 glL formic acid.
In some embodiments, some or all of the clostridial cells may be immobilised in the culture
30 vessel. In other embodiments, the clostridial cells are non-immobilised or are in free
suspension.
The culture vessel may be any form of culture vessel which is suitable for culturing the
clostridial cells in the process of the invention. Preferred types of culture vessel include
35 conventional stirred bioreactors.
As used herein, the term "culture vessel" includes two or more culture vessels which may or
may not be linked in fluid communication. Any multiple culture vessels may be linked in parallel
or in series.
5 In the context of the invention, however, the acid(s) and solvent(s) are both produced together
(i.e. in a mixture) in a single culture vessel. It is not the case that one or more acids are
produced in a first culture vessel and they are then converted to one or more solvents in a
second culture vessel.
10 Suitable culture media are well known in the art. These will be selected according to the
clostridium which is being used. Generally, the culture medium will be an aqueous medium.
In embodiments of the invention which comprise a fed batch process, the fermentation
proceeds in batch mode for approximately 20 hours with approximately 70% of the reactor
15 volume filled. The medium should contain enough sugar and nutrients for the microorganisms
to grow optimally and to produce acids and some solvent(s).
In other embodiments, it is possible initially to fill, and inoculate only about 10% of the reactor
working volume. The remaining about 60% may be filled during cell growth.
20
The medium for the fed-batch process may be a nutrient feed containing higher sugar and/or
nutrient concentrations than those present in the culture vessel during the batch stage. Typical
concentrations of the sugar on the feed may vary between 250 g.L-' and 450 g.LW1T. his may be
fed at a constant flow rate or controlled by a feedback system based on either cell growth (or a
25 related feature), or sugar or solvent levels in the culture vessel. In other embodiments, the
addition of extra nutrient media may commence using an independent trigger than that of the
solvent extraction; this may be, for example, when the sugar concentration has dropped
between a defined level (e.g. below 30 g/L) or when a slow-down in growth rate is detected.
30 The feed rate of the additional sugar solution should preferably keep pace with the uptake rate
of the culture. This may, for example, be about 2-5 g.~-'.h-'through the feed stage in order to
maintain a steady sugar concentration in the culture vessel (e.g. in the range of 5-30 g.L-').
The clostridia in the culture vessel are maintained under conditions which are suitable for them
35 to produce acids and solvents. The conditions will include the provision of nutrient media
comprising appropriate carbon sources, for example assimilable carbohydrates.
Examples of assimilable carbohydrates are sugars such as C5 and C6 monomers, C5 and C6
sugar dimers, sugar oligomers and sugar polymers. Preferred sugars are arabinose, xylose,
mannose, fructose, glucose, galactose, sucrose, lactose, maltose, cellobiose. Preferred
polymers are starch, xylan, pectin, fructan, cellulose and mannitol. Another suitable carbon
5 source is glycerine.
Preferably, sugars are hydrolysates derived from lignocellulosic feedstocks such as agricultural
residues (corn & sugar), woody residues, energy crops or municipal waste.
10 The carbohydrate may also be a glucose-based polysaccharide, e.g. a starch or a starch-based
material. Most preferably, the carbohydrate is starch or a starch-based material, e.g. corn, corn
starch, corn mash, potato, potato starch, potato mash, potato peeling, potato chips, cassava,
cassava starch, cassava chips, sago, sago starch, dextrin or 'soluble starch', e.g. as sold by
Fisher1 Sigma.
15
In some embodiments, the invention allows the use of greater than 200 glL sugar or other
carbon feed with minimal substrate or solvent inhibition, whilst keeping yields of products
greater than 30% on carbon-fed basis.
20 The pH of the culture vessel may be controlled through the automated addition of alkali from a
separate alkali feed or from the addition of feed media that has a pH lower than the culture
vessels (typically 1 -2 pH units).
The pH of the culture vessel is preferably pH 5.5-7.0, more preferably pH 5.5-6.5.
2 5
The pH may also be controlled by a pH auxostat. Preferably, the pH-auxostat has a separate
alkali feed and the pump is linked to the pump controlling the media feed. The cell density in
the culture vessel may be controlled by altering the ratio or relative speed of the alkali-feed and
media-feed pumps.
3 0
The temperature will be selected as being one at which the microorganism grows best. For
example, for mesophilic clostridia the temperature is preferably 30-37 "C, more preferably
31-33 "C, e.g. about 32 "C.
35 In step (b), growth of the monophasic solventogenic clostridial cells in the culture vessel is
monitored. This is done in order to obtain information which can be used to maintain good
growth rates and high cell densities within the culture vessel by the addition of fresh culture
media or nutrients and/or removal of solvents.
Although the cell growth of the clostridia which are in the culture vessel is monitored, the actual
5 monitoring may or may not be done in the culture vessel, e.g. the monitoring may be done
externally or on a sample which is removed from the culture vessel.
Preferably, step (b) comprises monitoring cell growth by one or more of:
(i) monitoring production of one or more gases (e.g. hydrogen and/or C02);
10 (ii) monitoring production of one or more acids;
(iii) monitoring the pH of the culture medium;
(iv) monitoring the utilisation of sugar;
(v) monitoring cell density; and
(vi) monitoring the production of one or more solvents.
15
During the process of the invention, one or more gases may be produced by the clostridia.
These gases may include hydrogen and/or carbon dioxide.
The total amount of gas may be monitored using a flow meter and individual gases may be
20 quantified using specific gas analysers.
The ratio of gases may also be monitored and used to control growth and solvent production by
adjusting the addition of culture media, nutrients and/or removal rate of solvents.
The clostridia convert substrates based on sugars and/or starches into acids (e.g. RCOOH,
25 wherein R is as defined above). Exemplary acids include acetate and/or butyrate. The acid
concentration may be monitored by gas chromatography (GC).
The production of these acids may also be monitored by a change in culture pH. In this case,
the monitoring apparatus may be a pH meter.
3 0
Cell density may be monitored by optical means, for example determining the optical density
(OD) at 600 nm.
In other embodiments, a change in cell density may be linked to a change in the rate of fresh
35 medialnutrient feed or removal of culture media comprising solvent(s), e.g. by using a
turbidostat.
The production of one or more solvents (e.g. ethanol, butanol, acetone) may be monitored by
gas chromatography (GC) and/or high performance liquid chromatography (HPLC).
Preferably, the butanol concentration in the culture vessel should not exceed 10 g butanol L-I.
5
The utilisation of one or more sugars (e.g. glucose, xylose, fructose, arabinose, sucrose,
cellobiose, or others as appropriate depending on the feedstock) may be monitored by HPLC.
Preferably, the sugar concentration in the culture vessel should not exceed 30 g sugar L'".
10
Step (d) comprises continuously or semi-continuously removing a stream or portion of liquid
culture medium comprising solvent(s) from the culture vessel and passing said liquid culture
medium to a solvent remover. In the solvent remover, one or more solvents are preferably
separated from the liquid culture medium, and optionally isolated.
15
Preferably, step (d) comprises continuously or semi-continuously removing a stream or portion
of the liquid culture medium from the culture vessel and passing the streamlportion via a cell
separator to a solvent remover, wherein:
(i) cells are removed from the liquid culture medium in the cell separator and the cells are
20 returned to the culture vessel (optionally via a cell seeder), and
(ii) wherein solvent is removed from the liquid culture medium in the solvent remover and
the residual liquid culture medium is returned to the culture vessel.
The liquid culture medium from the culture vessel may be passed directly or indirectly to the cell
25 separator.
The residual liquid culture medium may be passed directly or indirectly to the culture vessel, e.g.
it may be stored temporarily, e.g. in a reservoir.
30 In order to reduce loss of cells from the culture vessel, a cell separator may be positioned
upstream of the solvent remover. In the cell separator, all or a substantial portion of the cells
are removed from the stream or portion of the liquid culture medium which is passed through
the cell separator. For example, the cell separator may remove at least 50%, preferably at least
70%, and most preferably at least 80% or at least 90% of the cells from the liquid culture
35 medium which is passed through the cell separator.
Examples of cell separators include hollow fibre separators, membrane separators and
centrifugal separators.
After separation from the liquid culture medium, the cells are returned to the culture vessel
5 (preferably with some liquid culture medium), optionally via a cell seeder.
The remaining liquid is then passed to the solvent remover.
In step (d)(ii), solvent is removed from the liquid culture medium by a solvent remover and the
10 residual liquid culture medium (i.e. the liquid culture medium from which some or all solvents
have been removed) is returned to the culture vessel.
The liquid culture medium which is removed from the culture vessel will be enriched in one or
more solvents which have been produced by the solventogenic clostridia.
15
Preferably, liquid culture medium starts to be removed from the culture vessel once the
solvent(s) produced in the culture vessel reach a defined concentration point (e.g. 8-10 g
butanol L-' liquid culture media).
20 The liquid culture medium should preferably be passed to the solvent remover at a rate which
maintains the solvent concentration in the liquid culture medium below the desired solvent
concentration point. This concentration point may, for example, be the toxicity threshold for the
specific solventogenic clostridium used.
25 Generally, the solvent(s) will be recovered from the liquid culture medium (fermentation broth)
by one or more of liquid-liquid extraction, ionic-liquid extraction, gas stripping, vacuum
evaporation, atmospheric or vacuum distillation, pervaporation, ion-exchange adsorption,
counter-current solvent extraction and/or distillation. Alternatively, hydrophobic membranes
may be used, e.g. with air flux or inert gas carrier or vacuum (pervaporation) to aid the
30 separation (preferably in a continuous process).
Preferably, the solvent(s) will be recovered from the liquid culture medium by atmospheric or
vacuum distillation, pervaporation, liquid-liquid extraction and/or gas stripping.
35 Preferably, the solvent extraction is performed continuously.
In some embodiments of the invention, solvent may be produced at a rate of at least 0.8 g.~-'.h-'
for periods in excess of 60 hours or 0.6 g.~-'.h-f'o r over 100 hours.
In some more preferable embodiments, solvent extraction is via continuous atmospheric or
5 vacuum distillation.
Once the solvents are removed from the liquid culture medium, some or all of the residual liquid
culture medium is returned to the culture vessel. This maximizes utilization of nutrients from the
liquid culture medium and minimises water loss.
10
The invention also relates to a solvent which is obtained by a process of the invention.
In step (e), the cell density is preferably maintained or increased within the culture vessel by
one or more of :
15 (i) recycling cells which are removed from the liquid culture medium comprising solvent(s)
back to the culture vessel;
(ii) continuously or semi-continuously feeding cells from a cell seeder into the culture
vessel; and
(iii) immobilising some or all of the cells within the culture vessel.
20
In step (e)(ii), cells are continuously or semi-continuously fed from a cell seeder into the culture
vessel.
In the simplest version of the fermentation process a single batch seed of 5-10% v/v can be
25 used to inoculate the fermentation at the beginning of the fermentation; However it is possible
also to use multiple batch inoculations of 5-1 0% v/v at various time intervals during the
fermentation in order to maintain high cell densities and solvent productivities. Alternatively,
continuous culture can be used to provide a continuous supply of fresh cells to the production
fermentor. The preferred embodiment is a pH-auxostat that is fed from the still bottoms from
30 distillation and self-regulated using a set pH value to control the addition of feed and/or fresh
nutrients and sugars.
In step (e)(iii), some or all of the clostridial cells may be immobilised in the culture vessel.
35 The cells could be immobilized by active immobilization techniques, whereby free suspended
cells are immobilized by covalent attachment to surfaces, cross-linking of cells to surfaces,
entrapment within gels or membrane confinement; or by passive immobilization, exploiting the
natural tendency of cells to adhere to solid porous surfaces due to electrostatic interactions or
by their ability to form films or aggregates around or within a support material.
The preferred methods for immobilizing clostridia in this application are:
5 (i) entrapment of cells within a gel matrix made of naturally occurring polymers (eg
alginates, kappa-carrageenan, collagen, gelatine, cellulose, etc.) or synthetic gels
(polyacrylamide, polymethacrylamide, photo-cross-linkable resin pre-polymers, urethane
pre-polymers, polyethyleneglycol and polyvinyl alcohol, etc.)
(ii) membrane confinement of cells by immobilization behind a barrier. The barriers can
10 be droplets of cell-water suspensions emulsified in organic solvents or semi-permeable
membranes.
In the process of the invention, cell growth (or the rate of cell growth) in the culture vessel is
regulated by controlling:
15 (i) the amount or rate of fresh culture medium or nutrients which are introduced into the
culture vessel, and/or
(ii) the amount or rate of liquid culture medium comprising solvent(s) which is
removed from the culture vessel.
20 The aim of the process of the invention is to maintain cell growth and optimise cell density within
the culture vessel with a view to maximising sugar utilisation and solvent production.
By monitoring cell growth of the clostridia in the culture vessel, sub-optimal growth may be
detected. Optimal growth may then be sought or obtained by the introduction of fresh culture
25 media or nutrients into the culture vessel and/or the removal of some liquid culture media
comprising inhibiting solvents.
Preferably, therefore, the growth of cells in the culture vessel is maintained at an optimal level.
30 In other embodiments, if suboptimal growth of cells is detected, fresh media or nutrients are
introduced into the culture vessel and/or liquid media comprising solvents are removed from the
culture vessel.
In yet other embodiments, the monitored level of cell growth is compared to a reference level
35 and fresh media or nutrients are introduced into the culture vessel and / or liquid media
comprising solvents are removed from the culture vessel if the monitored level is lower than the
reference level.
The invention also provides a system for the production of a solvent, the system (e.g. as shown
in Figure I) comprising:
5 (i) a culture vessel for culturing a solventogenic clostridium in a liquid culture medium;
(ii) monitoring apparatus for monitoring growth of cells in the culture vessel;
(iiia) a cell seeder in fluid communication with the culture vessel and arranged to provide
10 clostridial cells to the culture vessel, and/or (iiib) a cell separator in fluid communication with the
culture vessel and arranged to receive liquid culture medium from the culture vessel
and to separate solventogenic clostridial cells from the liquid culture medium and further
arranged to return separated cells to the culture vessel;
15 (iv) a solvent remover in fluid communication with the culture vessel and arranged to receive
liquid culture medium, from which cells optionally have been removed by the cell separator
further arranged to remove one or more solvents from liquid culture medium, and further
arranged to return residual liquid culture medium to the culture vessel;
20 (v) one or more flow regulators in fluid communication with the culture vessel for controlling the
rate or amount of fresh culture media to be introduced into the culture vessel and/or the rate or
amount of liquid culture medium to be passed to the cell separator;
(vi) one or more controllers for controlling the one or more flow regulators depending on the
25 input received from the monitoring apparatus.
The system may optionally also comprise one or more of the following:
(vii) a beer still where the culture medium at the end of process is led to remove the bulk of the
30 water from the solvents, e.g. through azeotropic distillation;
(viii) one or more rectifying columns to purify the solvents to desired product specifications;
(ix) stillage treatment to lower the CODIBOD (chemical oxygen demand/biochemical oxygen
35 demand), optionally to also recover energy from the final culture medium with anaerobic
fermentation or similar; and
(x) one or more reservoirs of sugar solution and/or nutrients.
In some embodiments of the invention, the culture vessel comprises monophasic solventogenic
clostridia in a liquid culture medium.
The system may additionally comprise a vessel which contains or is adapted to contain an alkali;
and/or a vessel which contains or is adapted to contain fresh culture medium (nutrients). These
latter vessels may be in liquid communication with the first culture vessel.
10 The means for liquid communication preferably comprises a pipe.
The system may additionally comprise one or more additional solvent removers (e.g. one or
more rectifying columns, one or more beer stills, etc.) in fluid communication with the culture
vessel and arranged to remove solvent from liquid culture medium which is removed from the
15 culture vessel.
All of the features of the invention which are discussed above in the context of the process of
the invention apply equally to the above system, mutatis mutandis.
20 BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an example of the integrated fed batch process concept of the invention.
Figure 2 is a batch fermentation profile for a biphasic solventogenic clostridium. The graph
25 shows that solvent production starts when growth slows and the cells enter stationary phase.
The fermentation time is relatively long. The final solvent titres are low and solvent productivity
over the entire fermentation is therefore low (Example I).
t
Figure 3 is a batch fermentation profile for a monophasic solventogenic clostridium. The graph
30 shows that solvent production occurs during growth and slows down when growth slows and the
cells enter stationary phase. The fermentation time is reduced and solvent productivity over the
entire fermentation is higher than in Example 1 (Example 2).
Figure 4 is a batch fermentation profile with a monophasic solventogenic clostridium. The graph
35 shows that sugars are utilised during solvent production and when sugars are exhausted
solvent production stops. The fermentation time is relatively short (Example 3).
Figure 5 is from a fed-batch fermentation with a monophasic solventogenic clostridium. Gas
stripping is used to remove solvents and the sugar feed is molasses. The graph shows the
accumulation in solvents (sum of solvents trapped outside and remaining within the fermentor)
during the course of the fermentation. The final solvent titres obtained are much higher than
5 those obtained in a conventional batch fermentation i.e. Example 3 (Example 4).
Figure 6 is from a fed-batch fermentation with a monophasic solventogenic clostridium. Gas
stripping is used to remove solvents and the sugar feed is molasses. The graph shows a
correlation between solvent productivity and sugar uptake and that peak solvent productivity
10 has been extended by adding additional sugar during the fermentation and by removing
solvents as they are produced (Example 4).
Figure 7 is from a fed-batch fermentation with a monophasic solventogenic clostridium. Gas
stripping is used to remove solvents and the sugar feed is molasses. The graph shows a direct
15 correlation between cell growth and density (measured by optical density) and solvent
productivity (Example 4).
Figure 8 is from a fed-batch fermentation with a monophasic solventogenic clostridium. Gas
stripping was used to remove solvents and the sugar feed was glucose (Example 5). The graph
20 shows the accumulation in solvents during the course of the fermentation.
Figure 9 is from a pilot-scale fed-batch fermentation with a monophasic solventogenic
clostridium. The process contains a membrane filtration unit (cell separator) that removes cells
from the fermentation broth outside the culture vessel. The cells are recycled back into the
25 culture vessel and the culture broth minus the cells is fed to a distillation column (solvent
remover) that separates the solvents from the liquid culture medium. The distillate is returned to
the culture vessel. The sugar feed is glucose. The graph shows that high solvent productivity
can be maintained for over 40 hours and that there is a direct correlation between sugar uptake
and solvent production (Example 6).
30
Figure 10 is from a pilot scale fed-batch fermentation with a monophasic solventogenic
clostridium. The process contains a membrane filtration unit (cell separator) that removes cells
from the fermentation broth outside the culture vessel. The cells are recycled back into the
culture vessel and the culture broth minus the cells is fed to a distillation column (solvent
35 remover) that separates the solvents from the liquid culture medium. The distillate is returned to
the culture vessel. The sugar feed is corn mash. The graph shows that high sugar uptake and
solvent productivity can be maintained for over 40 hours using an industrial corn based
feedstock (Example 7).
Figure 11 is from a pilot scale fed-batch fermentation with a monophasic solventogenic
5 clostridium. The process contains a membrane filtration unit (cell separator) that removes cells
from the fermentation broth outside the culture vessel. The cells are recycled back into the
culture vessel and the culture broth minus the cells is fed to a distillation column (solvent
remover) that separates the solvents from the liquid culture medium. The distillate is returned to
the culture vessel. The sugar feed is molasses. The graph shows the correlation between
10 sugar uptake and solvent productivity during the fermentation (Example 8).
Figure 12 is from a pilot scale fed-batch fermentation with a monophasic solventogenic
clostridium. The process contains a membrane filtration unit (cell separator) that removes cells
from the fermentation broth outside the culture vessel. The cells are recycled back into the
15 culture vessel and the culture broth minus the cells is fed to a distillation column (solvent
remover) that separates the solvents from the liquid culture medium. The distillate is returned to
the culture vessel. The sugar feed is molasses. The graph shows the correlation between gas
production and solvent productivity during the fermentation (Example 8).
20 EXAMPLES
The present invention is further defined in the following Examples, in which parts and
percentages are by weight and degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred embodiments of the invention, are
25 given by way of illustration only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this invention, and without departing from
the spirit and scope thereof, can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, various modifications of the invention in
addition to those shown and described herein will be apparent to those skilled in the art from the
30 foregoing description. Such modifications are also intended to fall within the scope of the
appended claims.
In the lab scale (1 L) examples, gas-stripping is used to maintain a low level of solvents in the
main fermentor. However in the pilot scale (140 L) examples (and the preferred commercial
35 method), solvent removal is performed using atmospheric distillation with nutrient and water
recycle to the main fermentor.
Comparative Example I: Batch fermentation with a biphasic solventogenic clostridium
Obiective:
To demonstrate the biphasic batch fermentation profile obtained using classical biphasic
5 solventogenic clostridia.
Materials and Methods
Bacterial strain
10 A solventogenic Clostridium beijerinckii strain BASIB2 (CSCINCP collection 1510611 945) was
cultured on a standard anaerobic culture medium such as reinforced clostridial medium (RCM -
as in table 1) in 100 mL serum bottles under anaerobic conditions at 32 -t 1 "C for 16-18h.
Table 1. RCM semi-solid medium composition in g.L-'
Lab-Lemco powder 1 10.0
Yeast extract 3.0
I
Soluble starch 1 1.0
Peptone
Glucose
I Sodium chloride 1 5.0 1
10.0
5.0
Cysteine hydrochloride 1 0.5
I
Sodium acetate
I I I
* Adjusted as required to meet performance standards, sterilised by autoclaving at 121 "C.
3.0
Agar
*pH
Fermentation medium
Molasses was the main carbon source for the fermentation medium. Experiments were carried
0.5
6.8 +_ 0.2
out at an initial volume of 0.95 L with starting molasses sugar concentrations of 60 g . ~ -i'n the
20 fermentation vessel and supplemented with 5 g . ~ -c'o rn mash, 5 g.~-Cl aC03 and 2 g.L-'
(NH4)*S04. The initial pH was adjusted with NaOH 20% (wlv) to 6.7 +_ 0.2, thereafter the pH
was not controlled. All mineral salts were laboratory grade (Fisher Scientific).
Culture conditions
25 Fermentations were carried out in 1 L fermentors with initial working volumes of 0.95 L. The
fermentors were equipped with gas exhaust, stirrers, sampling ports, pH and temperature
probes. They were initiated by inoculation withl% vlv seed culture of the final volume (1 L).
The temperature was maintained at 32 + 3 "C.
Analysis of cell qrowth, products and substrates
5 Growth was monitored at 600 nm using a Jenway 6300 spectrophotometer with cuvettes of
1 cm light path. Cultures were diluted if necessary so that the absorbance did not exceed 0.6
units.
Concentrations of acetate, butyrate, ethanol, acetone, and n-butanol were measured by
10 applying the supernatant from centrifuged fermentation samples to gas chromatography on an
Agilent Gas Chromatography system with a network sampler. The equipment was fitted with a
capillary column (Agilent 19091 F115E HP-FFAP) with a column temperature ramp from 80 "C
up to 200 "C. The carrier gas was N2 with a flow rate between 0.8 mL.min-I and 1.3 mL.minW1.
The FID detector temperature (300 "C) was operated with a hydrogen flow at 50 rnL.min-', air
15 flow at 400 mL.min-' and make up flow (N2) at 30 mL.min-'. lsobutanol (99.5%) and isobutyric
acid (99.5%) were used as internal standards, prepared at concentrations of 10 g.L-' each in
HPLC grade water.
The sugar content of the fermentation samples was determined by high pressure liquid
20 chromatography using a Dionex HPLC fitted with an ASlOOauto sampler and a Shodex RI 101
refractive index and UV detector. The separation column was Phenomonex ~ezex-pb2',
operated at 85 "C. HPLC grade water was used as the mobile phase at a flow rate of 0.6
mL/min. The sample injection volumes were 10 PI. The calibration curve was created by
integrating the peak areas from chromatograms generated from solutions of sucrose, maltose,
25 glucose or fructose (as appropriate, according to the particular sugar source used) at
concentrations of 1, 5, 10, 15 and 20 g.~-ol f sugar.
Results
The results are shown in Figure 2. The graph shows that solvent production starts when growth
30 slows and the cells enter stationary phase. The fermentation time is relatively long (55 hours).
The final solvent titres are low and solvent productivity over the entire fermentation is therefore
low.
Comparative Example 2: Batch fermentation with a monophasic solventogenic
clostridium
Obiective:
5 To demonstrate the monophasic batch fermentation profile obtained using a monophasic
solventogenic clostridium.
Materials and Methods
10 Bacterial strain
A solventogenic Clostridium saccharoperbutylacetonicum strain N 1 -4(H MT) was cultured on a
standard anaerobic culture medium such as reinforced clostridial medium (RCM -as in Table 1)
in 100 mL serum bottles under anaerobic conditions at 32 rt 1 "C for 16-1 8 h.
15 Fermentation medium
As in example 1 : Molasses was the main carbon source for the fermentation media.
Experiments were carried at initial volume of 0.95 L with starting molasses sugar concentrations
of 60 g . ~ "in the fermentation vessel and supplemented with 5 g/L corn mash, 5 g/L CaC03 and
2 g/L (NH4)*S04. The initial pH was adjusted with NaOH 20% (wlv) to 6.7 rt 0.2, thereafter the
20 pH was not controlled.
Culture conditions
Similar to Example 1: Fermentations were carried out in 1 L fermentors with initial working
volumes of 0.95 L. The fermentors were equipped with gas exhaust, stirrers, sampling ports,
25 pH and temperature sensors. They were initiated by inoculation with 1 % v/v seed culture of the
final volume (IL) . The temperature was maintained at 32 It: 3 "C.
Analysis of Products and Substrates
As described for Example 1.
3 0
Results
The results are shown in Figure 3. The graph shows that solvent production occurs during
growth and slows down when growth slows and the cells enter stationary phase. The
fermentation time is reduced (45 hours) and solvent productivity over the entire fermentation is
35 higher than in Example I.
Comparative Example 3: Batch fermentation with a monophasic solventogenic
clostridium
Obiective:
5 To demonstrate the relationship between sugar utilisation and ABE production for a batch
fermentation using a monophasic solventogenic clostridium.
Materials and Methods
10 Bacterial strain
A solventogenic Clostridium saccharoperbutylacetonicum strain N 1 -4(H MT) was cultured as
described in example 2.
Fermentation medium
15 Similar to example 2: Molasses was the main carbon source for the fermentation media.
Experiments were carried at an initial volume of 0.95 L with starting sugar concentrations of 55
g.L-I in the fermentation vessel and supplemented with 2.5 g.~'l yeast extract, 2.5 g.L-' tryptone,
0.025 g . ~ "F eS04a nd 0.5 g.LV1( NH4)*S04.
20 Culture conditions
Similar to example 2: Fermentations were carried out in 1 L fermentors with initial working
volumes of 0.8 L. The fermentors were equipped with gas exhaust, stirrers, sampling ports, pH
and temperature sensors. They were initiated by inoculation with 7.5% vlv seed culture of the
final volume (1 L). The temperature was maintained at 32 + 3 "C. 20% wlv NaOH was used to
25 adjust the initial pH to between 6 and 6.5, and when needed during the fermentation to maintain
a pH set point of 5.0-5.5. All mineral salts were laboratory grade (Fisher Scientific).
Analysis of Products and Substrates
As described for Example 1.
3 0
The results are shown in Figure 4. The graph shows that sugars are utilised during solvent
production and when sugars are exhausted solvent production stops. The fermentation time is
relatively short (44 hours).
Example 4: Fed batch fermentation on molasses
0 biective:
To demonstrate a method to cultivate monophasic solventogenic clostridia at high production
5 rates; using a fed batch system focused on the production of an exponentially growing cell
population at or close to its maximum sugar uptake and solvent production rates for an
extended time compared to traditional batch fermentation by keeping low solvent concentration
in the broth (using gas stripping).
10 Materials and Methods
Bacterial strain
A solventogenic Clostridium saccharoperbutylacetonicum strain N1-4(HMT) was cultured as
described in Example 2.
15
Fermentation medium
Molasses was the main carbon source for the fermentation medium. Experiments were carried
out at an initial volume of 1.5 L with starting sugar concentrations of 50 g.L-' in the fermentation
vessel and supplemented 2.5 g.L-' yeast extract, 2.5 g.L-' tryptone, 0.5 g.L-' (NH4)2 SO4, 0.025
20 g.L-' FeS04, and 3 g . ~ -C' aCO3. When needed, the pH was adjusted with 20% w/v of NaOH to
a pH of 6.5 prior to sterilization. All mineral salts were laboratory grade (Fisher Scientific).
Feed medium
500 mL of additional feed of glucose solution at a concentration of 350 g.L-I, with nutrients
25 3.8 g.L-' yeast extract , 3.8 g . ~ -tlr yptone, 0.8 g.L-' (NH4)2S O4, 3 g.L-' CaC03 and 0.05 g.L-'
FeS04.
Culture conditions
Fermentations were carried out in fermentors equipped with gas exhaust, stirrers, sampling
30 ports, pH and temperature sensors. They were initiated by inoculation with 7.5% v/v seed
culture.
The feeding vessel (1 L) was connected to the fermentor immediately after inoculation (Figure
1). After 6 h when the butanol concentration was 1 g/L product removal through gas stripping
35 with nitrogen was commenced at a gas flow rate 2 vvm N2. The fed batch mode operation was
started approximately 15 h after inoculation. The media reservoir pump was linked and
activated once the sugar concentration in the vessel fell below the set value (26 g.~.'). The
medium reservoir supplied fresh sugar and nutrients. The feed pump ran automatically and
intermittently depending on sugar uptake rate and culture growth. A control system was fitted to
maintain temperature at 32 + 3 "C and minimum agitation (50-70 rpm). The pH was not
controlled.
5
Analvsis of Products and Substrates
As described for Example 1.
Results
10 Figure 5 shows the accumulation of solvents (summation of solvents trapped outside and
remaining within the fermentor) during the course of the fermentation. The final solvent titres
obtained are much higher than those obtained in a conventional batch fermentation, i.e.
Example 3.
15 Figure 6 shows the correlation between solvent productivity and sugar uptake, and that peak
solvent productivity has been extended by adding additional sugar during the fermentation and
by removing solvents as they are produced. (The plotted data are cumulative averages.)
Figure 7 shows a direct correlation between cell growth and density (measured by optical
20 density) and solvent productivity. (The plotted data are cumulative averages.)
The solvent concentration value at which the fermentation was controlled was found to have a
significant impact on growth rate and consequently the sugar uptake and solvent production
rates. High solvent production rates were maintained for a prolonged period of time,
25 througough the feeding phase, suggesting the culture was growing at or close to its maximum
growth rate. The fermentation was run for 164 hours, of which 53.5 h was with feeding. The
solvent* productivities were 0.90 glL1h during the feeding period and 0.79 glLlh over the total
fermentation period; the sugar uptake rates were 2.9 and 2.7 gILlh, over the feeding and total
fermentation periods, respectively; and the yields were 0.32 and 0.30 g solvents* I g sugar used,
30 over the feeding and total fermentation periods, respectively. ("Note: 'solvents' data based on
solvents collected - a significant portion of produced solvents were not trapped, so the actual
values of solvent produced would be higher.)
Example 5: Fed batch fermentation on glucose
Objective:
To demonstrate that the above fed batch system (Example 4) for cultivating monophasic
5 solventogenic clostridia at high production rates also works using a different sugar source, e.g.
crystallised glucose.
Materials and Methods
10 Bacterial strain
A solventogenic Clostridium saccharoperbutylacetonicum strain N 1 -4(H MT) was cultured as
described in Example 2.
Fermentation medium
15 Crystallised glucose was the main carbon source for the fermentation medium. Experiments
were carried at an initial volume of 1.55 L with starting sugar concentrations of 53 g.L-' in the
fermentation vessel and supplemented with 2.5 g.~-tlr yptone, 2.5 g.~-yl east extract, 0.5 g.L-'
(NH4)2S040r. 025 g.L-' FeS04and3 g . ~ -C' aC03.
20 Feed medium
650 mL of glucose solution at a concentration of 492 g.L-', with these nutrients: 3.8 g . ~ -y'e ast
extract, 3.8 g.L-' tryptone, 0.8 g.L-' (NH4)2S040r. 05 g.L-' FeS04a nd 3 g.L-' CaC03.
Sugar solutions and nutrient base media were batched and sterilised separately.
2 5
Culture conditions
Fermentations were carried out in 2 L fermentors. The fermentors were equipped with gas
exhaust, stirrers, sampling ports, pH and temperature sensors. They were initiated with the
inoculation of 7.5% vlv seed culture. Inoculation with 7.5% vlv seed culture was repeated every
30 48h.
The feeding vessel (1 L) was connected to the fermentor. Once the initial sugar concentration
dropped below -25g/L, the feed addition (concentrated sugar plus nutrients) was started and
product removal by gas stripping (12 vvm) was also started. The feed pump ran automatically
and intermittently depending on sugar uptake rate and culture growth. A control system was
35 fitted to maintain temperature at 32 + 1 "C and minimum agitation (50-70 rpm).
Analysis of Products and Substrates
As described for Example 1.
Results
5 The results are shown in Figure 8. The graph shows the accumulation of solvents during the
course of the fermentation. The fermentation was run for 21 6.5 hours,. of which 191 h was with
feeding. The solvent* productivities were 0.57 g/L/h during feeding period and 0.53 g/L/h over
the total fermentation period; the sugar uptake rates were 2.1 and 2.0 g/L/h, over the feeding
and total fermentation periods, respectively; and the yields were 0.21 and 0.20 g solvents* / g
10 sugar used, over the feeding and total fermentation periods, respectively. (*Note: 'solvents'
data based on solvents collected - a significant portion of produced solvents were not trapped,
so the actual values of solvent produced would be higher.)
Example 6: Pilot scale fed batch fermentation on glucose
Obiective
To demonstrate that this fed batch fermentation process works at pilot scale, using glucose as
the main sugar source. This example system contains a membrane filtration unit (cell separator)
that removes cells from the fermentation broth outside the culture vessel. The cells are
recycled back into the culture vessel and the culture broth minus the cells is fed to a distillation
column (solvent remover) that separates the solvents from the liquid culture medium. The
distillate is returned to the culture vessel (see Figure 1).
Bacterial strain
25 A solventogenic Clostridium saccharoperbutylacetonicum strain N 1 -4(H MT) was cultured on 38
g . ~D' ifco RCM Medium under anaerobic conditions at 32 + 1 "C for 16-18 h. 150 mL of this
was used to inoculate a 5 L seed carboy containing rich glucose mediumheed (glucose 40 g/L,
yeast extract 5 g/L, meat peptone 5 g/L, (NH4)*S04 2 g/L, FeS04 0.05 g/L and sodium acetate
trihydrate 3 g/L). This was incubated for 5.25 h to generate 5 L of seed.
3 0
Fermentation medium
Glucose was used as the main carbon source for the fermentation medium. 105 L of the
following medium was prepared: glucose 60 g/L, yeast extract 2.5 g/L, meat peptone 2.5 g/L,
(NH4)*S04 2 g/L, FeS04 0.05 g/L, corn oil 0.1% v/v, antifoam, (Suppressor 2343) 0.03 g/L.
35
Feed media
50 L of concentrated sugar feed containing 450 g/L glucose.
16 L of nutrient addition solution comprising: yeast extract 58 g/L, meat peptone 58 g/L,
(NH4)2S04 17 g/L, FeS04 0.4 g/L and corn oil 0.4% v/v.
5 Sugar solutions and nutrient base media were batched and sterilised separately.
Culture conditions
Fermentations were carried out in 140 L fermentors, initially combining 105 L of fermentation
medium with 5 L of seed.
10
Temperature was maintained at 30.5 + 0.3"C. The pH dropped quickly after inoculation until pH
regulation started at pH 5.3 using 10-25% w/v NaOH.
Analysis of Products and Substrates
15 As described for Example 1.
Results
The results are shown in Figure 9. Plotted values are 5 point averages. The graph shows that
high solvent productivity can be maintained for about 50 hours and that there is a direct
20 correlation between sugar uptake and solvent production. The average productivity over whole
102.5 hour fermentation was 0.68 g/L/h, the productivity during 52 h fed-batch phase was 1 .I 1
g/L/h, the sugar consumption over whole 102.5 hour fermentation was 1.91 g/L/h, the sugar
consumption during 52 h fed-batch phase was 2.77 g/L/h, the solvent production based on 120
L final volume was 70 g/L, the ABE yield on sugars consumed was 35.7% and the butanol ratio
25 was 76%.
Example 7: Pilot scale fed batch fermentation on clarified corn mash
Obiective
30 To demonstrate that this fed batch fermentation process works at pilot scale, using clarified corn
mash as the main sugar source. This example system contains a membrane filtration unit (cell
separator) that removes cells from the fermentation broth outside the culture vessel. The cells
are recycled back into the culture vessel and the some of the cell-free culture broth is fed to a
distillation column (solvent remover) that separates the solvents from the liquid culture medium.
35 The stillage is returned to the culture vessel (see Figure 1).
Bacterial strain
A solventogenic Clostridium saccharoperbutylacetonicum strain N1-4(HMT) was cultured on 38
g . ~D' ifco RCM Medium under anaerobic conditions at 32 + 1 "C for 16-18 h. 150 mL of this
was used to inoculate 5 L containing rich clarified corn mash mediumheed (clarified corn mash
5 equivalent to 40 g/L sugars, yeast extract 5 g/L, meat peptone 5 g/L, (NH4)S04 2 g/L, FeS04
0.05 g/L and sodium acetate trihydrate3 g/L). This was incubated for 5.25 h to generate 5 L of
seed.
Fermentation medium
10 Clarified corn mash was used as the main carbon source for the fermentation medium. 105 L of
the following medium was prepared: clarified corn mash equivalent to 60 g/L sugar, yeast
extract 2.5 g/L, (NH4)2S042 g/L and FeS040 .05 g/L.
Feed media
15 75 L of concentrated sugar feed containing clarified corn mash equivalent to 270 g/L sugar.
8L of nutrient addition solution comprising: yeast extract 95 g/L, (NH4)2S046 5 g/L and FeS04
1.7 g/L.
20 Sugar solutions and nutrient base media were batched and sterilised separately.
Culture conditions
Fermentations were carried out in 140 L fermentors, initially combining 100 L of fermentation
medium with 5 L of seed.
25
Temperature was maintained at 30.5 + 0.3 "C. The pH dropped quickly after inoculation until
pH regulation started at pH 5.3 using 10-25% w/v NaOH.
Analvsis of Products and Substrates
30 As described for Example I.
Results
The results are shown in Figure 10. Plotted values are 5 point averages. The graph shows that
high sugar uptake and solvent productivity can be maintained for about 50 hours using an
35 industrial corn based feedstock.
The average productivity over the 110 hour fermentation cycle (97.75 h fermentation + CIP
(cleaning in place) time + turnaround time) was 0.87 g/L/h, the productivity during 52 h fedbatch
phase wasl.51 g/L/h, the sugar consumption over the 11 0 hour fermentation cycle was
2.84 g/L/h, the sugar consumption during 52 h fed-batch phase was 5.25 g/L/h, the solvent
5 production based on 105 L final volume was 96 g/L, the ABE yield on sugars consumed was
30.6% and the butanol ratio was 61.5%.
Example 8: Pilot scale fed batch fermentation on molasses, with gas production
monitoring
10
0 biective
To demonstrate that this fed batch fermentation process works at pilot scale, using molasses as
the main sugar source. This example system contains a membrane filtration unit (cell separator)
that removes cells from the fermentation broth outside the culture vessel. The cells are
15 recycled back into the culture vessel and the culture broth minus the cells is fed to a distillation
column (solvent remover) that separates the solvents from the liquid culture medium. The
distillate is returned to the culture vessel (see Figure 1). Additionally, gases from the
fermentation are monitored.
20 Bacterial strain
A solventogenic Clostridium saccharoperbutylacetonicum strain N1-4(HMT) was cultured on 38
g . ~D' ifco RCM Medium under anaerobic conditions at 32 + 1 "C for 16-18 h. 150 mL of this
was used to inoculate 5 L containing rich molasses mediumlseed (molasses equivalent to 35
g/L sugars, yeast extract 5 g/L, meat peptone 5 g/L, (NH4)2S042 g/L, FeS04 0.05 g/L and
25 sodium acetate trihydrate 3 g/L). This was incubated for 5.25 h to generate 5 L of seed.
Fermentation medium
Sugar cane molasses was used as the main carbon source for the fermentation medium. 105 L
of the following medium was prepared: sugar cane molasses equivalent to 50 g/L sugar, yeast
30 extract 2.5 g/L, meat peptone 2.5 g/L, (NH4),S04 2 g/L, FeS04 0.05 g/L, corn oil 0.1 % v/v and
antifoam (Suppressor 2343) 0.03 g/L.
Feed media
50 L of concentrated sugar feed containing sugar cane molasses equivalent to 350 g/L sugar.
35
16 L of nutrient addition solution comprising: yeast extract 34 g/L, meat peptone 34 g/L,
(NH4)2S042 0 g/L, FeS040 .5 g/L and corn oil 0.6%.
Sugar solutions and nutrient base media were batched and sterilised separately.
Culture conditions
5 Fermentations were carried out in 140 L fermentors, initially combining 105 L of fermentation
medium with 5 L of seed.
Temperature was maintained at 30.5 k 0.3 "C. No calcium carbonate or other buffering agent
was added. The pH dropped quickly after inoculation until pH regulation started at pH 5.3 using
10 10-25% wlv NaOH.
The butanol concentration in the culture vessel was kept below 6 gll, and the sugar
concentration in the culture vessel was maintained between 20 and 30 gIL during the feeding.
15 Analysis of Products and Substrates
As described for Example 1.
Results
The results are shown in Figures 11 and 12. Plotted values are 5 point averages. Figure 11
20 shows the correlation between sugar uptake and solvent productivity during the fermentation,
indicating that high sugar uptake and solvent productivity can be maintained for a prolonged
period using an industrial molasses-based feedstock. Figure 12 shows the correlation between
gas production and solvent productivity during the fermentation.
25 The average productivity over the 67 hour fermentation was 0.60 gILlh, the productivity during
the 30 h fed-batch phase was 0.95 g/L/h, the sugar consumption over the 67 hour fermentation
was 1.67 gILlh, the sugar consumption during the 30 h fed-batch phase was 2.74 glLlh, the
ABE yield on sugars consumed was 35.8% and the butanol ratio was 75%.

AMENDED CLAIMS
CLAIMS
1. A single-stage fed-batch or continuous process for the production of a
solvent, the process comprising the steps:
(a) culturing monophasic solventogenic clostridial cells in a liquid culture
medium in a culture vessel, wherein the cells are from a monophasic
Clostridium saccharoperb utylacetonicum;
(b) monitoring cell growth of the monophasic solventogenic clostridial cells in
the culture vessel;
(c) continuously or semi-continuously introducing fresh culture media andlor
nutrients into the culture vessel;
(d) continuously or semi-continuously removing a stream or portion of liquid
culture medium comprising solvent(s) from the culture vessel and passing
said liquid culture medium to a solvent remover;
(e) maintaining or increasing cell density within the culture vessel;
wherein cell growth in the culture vessel is regulated and/or optimised by controlling:
(i) the amount or rate of fresh culture medium or nutrients which are
introduced into the culture vessel, and/or
(ii) the amount or rate of liquid culture medium comprising solvent(s) which is
removed from the culture vessel.
2. A process as claimed in claim I, wherein step (e) comprises:
e) increasing cell density within the culture vessel;
wherein cell growth in the culture vessel is optimised by controlling:
(i) the amount or rate of fresh culture medium or nutrients which are
introduced into the culture vessel, and/or
(ii) the amount or rate of liquid culture medium comprising solvent(s)
which is removed from the culture vessel,
AMENDED SHEET (ARTICLE 19)
wherein the process maintains cell growth and optimises cell density within the
culture vessel, and maximises sugar utilisation and solvent production.
3. A process as claimed in claim 1 or claim 2, wherein the monophasic
clostridium:
(a) naturally displays simultaneous growth and solvent production during the
major growth phase of batch fermentations;
(b) has been chemically mutated to produce solvents during growth; or
(c) has been genetically modified to produce solvents during growth.
4. A process as claimed in claim 3, wherein the clostridium is a monophasic
Closfridium saccharopenSutylacetonicum N I strain, preferably N 1 -4(HMT) or N 1-504,
or a variant or derivative thereof.
5. A process as claimed in any one of the preceding claims, wherein step (b)
comprises monitoring cell growth by one or more of:
(i) monitoring production of one or more gases;
(ii) monitoring production of one or mQre acids;
(iii) monitoring the pH of the culture medium;
(iv) monitoring the utilisation of sugai;
(v) monitoring cell density; and
(vi) monitoring the production of one or more solvents.
6. A process as claimed in any one of the preceding claims, wherein the monitored
level of cell growth is compared to a reference level and fresh media or nutrients are
introduced into the culture vessel and I or liquid media comprising solvents are
removed from the culture vessel if the monitored level is lower than the reference
level.
7. A process as claimed in any one of the preceding claims, wherein step (d)
comprises continuously or semi-continuously removing a stream or portion of the
liquid culture medium from the culture vessel,,.qnd passing the streamlportion via a
cell separator to a solvent remover, preferably wherein:
(i) cells are removed from the liquid culture medium in the cell separator and
the cells are returned to the culture vessel (optionally via a cell seeder),
and/or
AMENDED SHEET (ARTICLE 19)
(ii) wherein solvent is removed from the liquid culture medium in the solvent
remover and the residual liquid culture medium is returned to the culture
vessel.
, .
8. A process as claimed in any one of the preceding claims, wherein solvent
extraction is performed continuoudy.
9. A process as claimed in any one of the preceding claims, wherein liquid
culture medium comprising solvent(s) is removed from the culture vessel at a rate
which maintains the solvent concentration in the liquid culture below a desired
solvent concentration point.
10. A process as claimed in claim 9, wherein the concentration point is the toxicity
threshold for the solventogenic Clostridium.
11. A process as claimed in claim 9, wherein the defined solvent concentration
point is 8-109 butanolll.
12. A process as claimed in any one of the preceding claims, wherein the butanol
concentration in the culture vessel does not exceed 10gIL.
13. A process as claimed in any one of the preceding claims, wherein the cell
density is maintained or increased within the culture vessel by:
(i) recycling cells which are removed from the liquid culture medium
comprising solvent(s) back to the culture vessel; andlor
(ii) continuously or semi-continuously feeding cells from a cell seeder into the
culture vessel; and/or
(iii) immabilisation of some or all of the cells within the culture vessel.
14. A process as claimed in any one of the preceding claims, wherein the amount
of sugar in the culture vessel is maintained at 5-30g/L.
15. A process as claimed in any one of the preceding claims, wherein the cells
are maintained under redox conditions that are suitable for them to grow optimally
and to produce solvents.
AMENDED SHEET (ARTICLE 19)
16. A process as claimed in any one of the preceding claims, wherein the
monophasic solventogenic clostridial cells are cultured in the liquid culture medium in
the culture vessel with no prior anaerobic purge.
17. A system for the production of a solvent, the system comprising:
(i) a culture vessel for culturing a solventogenic clostridium in a liquid culture
medium;
(ii) monitoring apparatus for monitoring growth of cells in the culture vessel;
(iiia) a cell seeder in fluid communication with the culture vessel and arranged to
provide clostridial cells to the culture vessel, and/or (iiib) a cell separator in fluid
communication with the culture vessel and arranged to receive liquid culture medium
from the culture vessel and to separate solventogenic clostridial cells from the liquid
culture medium and further arranged to return separated cells to the culture vessel;
. " cl
(iv) a solvent remover in fluid communication with the culture vessel and arranged to
receive liquid culture medium, from which cells optionally have been removed by the
cell separator, further arranged to remove one or more solvents from liquid culture
medium, and further arranged to return residual liquid culture medium to the culture
vessel;
(v) one or more flow regulators in fluid communication with the culture vessel for
controlling the rate or amount of fresh culture media to be introduced into the culture
vessel and/or the rate or amount of liquid culture medium to be passed to the cell
separator; and
(vi) one or more controllers for controlling the one or more flow regulators depending
on the input received from the monitoring apparatus.
. . ..,
18. A system as claimed in claim 17, wherein the system additionally comprises
one or more of:
(vii) a beer still where the culture medium at the end of process is led to
remove the bulk of the water from the solvents;
(viii) one or more rectifying columns to purify the solvents to desired product
specifications;
AMENDED SHEET (ARTICLE 19)
(ix) stillage treatment to lower the CODIBOD, optionally to also recover
energy from the final culture medium; and
(x) one or more reservoirs of sugar solution andlor nutrients in fluid
communication with the culture vessel.
19. A system as claimed In claim 17 or claim 18, wherein the culture vessel
comprises monophasic solventogenic clastridial cells in a liquid culture medium.
20. A system as claimed in claim 19, wherein the clostridium is a monophasic
Clostridium saccharoperbutylacetonicum N 1 strain, preferably N1-4(HMT) or N1-504,
or a derivative or variant thereof.